.. ”an" n.-...‘.... .. u. ..---.. walnut .. V'Xl [ ll nllrl THESIS 4IllfillllillllllllllilillliilllilllifillL 3 1293 01050 3195 This is to certify that the dissertation entitled Genetic Heterogeneity and Differentiation Resulting From Sichuan Pheasant (Phasianus colchicus strauchi) Introductions in Southern Michigan presented by Catherine Simpson Flegel has been accepted towards fulfillment of the requirements for Doctoral of Philosophy degree in Fish. & Wildl. Major professor Date August 27, 1996 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Michigan State University PLACE It RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE MSU le An Affirmative Action/Ewe] Opportunity lnetltuion WM! GENETIC I-IETEROGENEITY AND DIFFERENTIATION RESULTING FROM SICHUAN PI-IEASANT (Phasianus colchicus strauchi) INTRODUCTIONS IN SOUTHERN MICHIGAN By Catherine Simpson F legel A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Fisheries and Wildlife 1 996 ABSTRACT GENETIC I-IETEROGENEITY AND DIFFERENTIATION RESULTING FROM SICHUAN PHEASANT (Phasiatms cholchus strauchi) INTRODUCTIONS IN SOUTHERN MICHIGAN By Catherine Simpson F legel Levels of genetic heterogeneity and difi‘erentiation were estimated among four populations of common pheasants (Phasianus cholchus) in southern Michigan collected from 1991-1993 using starch gel electrophoresis. The pure Sichuan (P. c. strauchi) (n = 37) gene pool was evaluated by sampling the captive breeding stock housed at the Michigan Department of Natural Resources, Mason Wildlife Facility. Free-ranging ring- necked (P. c. torquatus) (n = 48), and populations that received Sichuan (Sichuan Release, n = 60) or a mixture of Sichaun x ring-necked hybrids (Mixed Release, n = 45) constituted the remaining three populations. Estimates of observed (171,.) and expected (Hm) heterozygosity and inbreeding coeflicient, F13, were calculated. Nei’s (1978) unbiased (DN), Rogers (1972) (DR), Rogers (1972) as modified by Wright (1978) (Dw) and Cavalli- Sforza and Edwards (1967) chord (DC) distances were calculated. Unweighted pair-group method with arithmetic averaging branching diagrams were constructed using all distance measures, and a branching diagram using Dw was generated using the distance Wagner procedure. Allelic and genotypic data were compared to morphological (neck ring) data. Thirty enzymes were examined for polymorphism using liver tissue. No unique alleles were detected in the ring-necked (n = 4) or Sichuan (n = 8) individuals used in the initial screening process. Hob, ranged from 0.019 in the Sichuan Release population to 0.029 in the Captive Sichuan population. Hm, values were higher and ranged from 0.035 to 0.042 in the Sichuan Release and Captive Sichuan populations, respectively. The deficiency of heterozygotes was also reflected in FIs (-O.114). The majority of loci in the fi'ee-ranging populations were not in Hardy-Weinberg equilibrium, while all loci in the Captive Sichuans were in equilibrium. The low proportion of heterozygotes may have resulted fi'om assortative mating, small population size, and/or biased sampling. Neighborhood size was estimated at 3234 pheasants. Given current pheasant density estimates, an area equivalent to 2 townships should have been sampled compared to the 4 that were, suggesting more than one breeding unit was sampled. The deficiency in heterozygotes could have resulted from a Walilund efl‘ect. Gene flow from the captive Sichuan into the release populations appeared to be substantial as evidenced by the genetic identity measures. Sichuan and Mixed Release populations were intermediate to the Captive Sichuan and ting-necked populations. Diagrams incorporating the morphological data showed a difi‘erent pattern. FST averaged 0.298 suggesting that 70% of the total genetic variation was within populations, while 30% was distributed among populations. To my late husband, Thomas Dale Flegel ACKNOWLEDGMENTS This research reflects the efl‘orts of many. My major advisor, Dr. Harold H. Prince, Department of Fisheries and Wildlife, is acknowledged for his role as a mentor and friend without whom I could not have succeeded. From the Department of Horticulture, gratitude is extended to Dr. James Hancock for his invaluable assistance in providing the needed laboratory space and in the interpretation of the results. Dr. Don Straney, Department of Zoology, is recognized for his comments concerning phylogeny and speciation. Dr. Scott R. “finterstein, Department of Fisheries and Wildlife, was the fourth committee member. Dr. John Epifanio, Department of Fisheries and Wildlife, is also acknowledged for his genetic advice. Student research assistants that aided in the laboratory include Lee Flore and Curtis Wright. Pete Calow and Colleen Mulinix were horticulture technicians that provided invaluable assistance in the laboratory. Pete Squibb’s role as the liaison from the Michigan Department of Natural Resources was appreciated. Michigan Department of Natural Resources personnel, Dave Dorn, Brad Johnson, and Bruce Warren from the Mason Wildlife Facility are acknowledged for their role in assisting in the collection of captive pheasants. Dave Luukkonen and Glenn Belyea from the Rose Lake Wildlife Research Station are recognized for their assistance in collections of free ranging birds from Livingston and Huron counties. Pheasant Forever Chapter presidents (by county) that assisted in the collection of harvested birds include Mark Sabin (Montcalm), Steven Hepker (Hillsdale), and Karen Schefl‘er (Huron). On a more personal note, appreciation is extended to John Niewoonder and Linda Briggs for their role as agreeable and helpful ofice mates. A final note of recognition is extended to my late husband, Thomas Flegel. He supported my professional desires and had unwavering faith in me. TABLE OF CONTENTS List of Tables ....................................................................................................... ix List of Figures ...................................................................................................... xi Introduction .......................................................................................................... l Taxonomy of Pheasants and History of Early Introductions ....................... 2 Background of Michigan’s Sichuan Pheasant Release Program ................. 7 Genetics of Successfirl Introductions and Objectives of this Study ............. 9 Isozyme Variability in Harvested Ring-necked and Sichuan Pheasants .................. 11 Introduction ............................................................................................. 11 Electrophoretic Methodology ................................................................... 12 Electrophoretic Results and Discussion ..................................................... l4 Isozyme Variability in Pure and Mixed Pheasant Populations in Michigan ............. 18 Introduction ............................................................................................. 18 Methods .................................................................................................. 18 Stocks .......................................................................................... 18 Collection of Pheasants for Electrophoresis .................................. 18 Pure Sichuans .................................................................... 18 Michigan’s Local Ring-necked Pheasants .......................... 19 Sichuan Release Population .............................................. 19 Mixed Release Population ................................................. 21 Statistical Estimates ..................................................................... 23 Genetic Heterogeneity ...................................................... 25 Hardy-Weinberg Equilibrium ............................................ 25 Population Differentiation ................................................. 25 Results ..................................................................................................... 30 Genetic Variability ....................................................................... 30 Genetic Distance .......................................................................... 37 Discussion .............................................................................................. 43 Genetic Heterogeneity .................................................................. 44 Population Differentiation ............................................................ 49 Conclusions .................................................................................. 54 vii Appendices Appendix A Appendix B Appendix C Appendix D Literature Cited ........ Electrophoretic Methodology and Techniques ................... 55 Allele Frequencies by Year ................................................ 66 Genetic Heterogeneity in the Class Aves ............................ 68 Genetic Distance and F 81- Values in the Class Aves ............ 78 ........................................................................................... 83 viii Table 1. Table 2. Table 3. Table 4. Table 5. Table 6. Table 7. Table 8. LIST OF TABLES Systematic list of common pheasants Phasianus colchicus (fi'om Delacour 1977 and Johnsgard 1986) .......................................... Neck ring characteristics of some common pheasants introduced into North America ..................................................................... Enzymes evaluated for genetic analysis of common pheasants in southern Michigan ...................................................................... Allele and Genotypic fiequencies, at polymorphic loci, of Sichuan and ring-necked pheasants used in the evaluation of enzymes for genetic analysis of common pheasants in Michigan. Sample sizes, by loci, for 4 populations of common pheasants in southern Michigan used for genetic analysis. Captive pheasants were collected fiom 1990-92, while flee-ranging pheasants were collected from 1991-1993 ........................................................... Allele frequencies of common pheasants collected for genetic analysis fi'om 4 populations in southern Michigan. Captive pheasants were collected fi'om 1990-92, while tree-ranging pheasants were collected fi'om 1991-1993 ................................... G values generated fi'om the analysis of homogeneity in the number of individuals carrying each allele, by loci, for each population (df = 1) and combined populations (df = 3) of common pheasants collected for genetic analysis in southern Michigan. .................................................................................. Genetic variability (3: SE) in 6 loci of 4 populations of common pheasants found in southern Michigan ........................................ 16 17 32 33 34 34 Table 9. Table 10. Table 1 1. Table 12. Table 13. Table 14. Table 15. Genotypic frequencies of common pheasants collected for genetic analysis fiom 4 populations in southern Michigan. Captive pheasants were collected from 1990-1992, while fiee- ranging pheasants were collected from 1991-1993 ...................... G values, (df = 1), generated from testing for homogeneity between the observed number of genotypes and the Hardy- Weinberg expected number of genotypes, for the 4 populations of common pheasants collected in southern Michigan for genetic analysis ....................................................................................... Summary of hierarchical F-statistics (Wright 1965, 1978) at all polymorphic loci in southern Michigan common pheasants. Significance levels are associated with Chi-square tests of (1) Ho: FIs = O, and (2) Ho: FST = O; * P < 0.05, *“ P < 0.01 .............. Genetic distance matrix summarizing the genetic variance found in 4 populations of common pheasants in southern Michigan. Above the diagonal are Rogers (1972) genetic distances (DR); below the diagonal are Rogers (1972) distances as modified by Wright (1978) (1),) .................................................................... Genetic distance matrix summarizing the genetic variance found in common pheasants in southern Michigan. Above the diagonal are Neis (1978) unbiased genetic distances (DN); below the diagonal are Cavalli-Sforza and Edwards (1967) chord distances (DC) ............................................................................................ Average neck ring width : SE and percent neck ring closure of males harvested in the fall for genetic analysis fi'om 4 populations of pheasants in southern Michigan ............................ A summary of literature FIs values in the Class Aves ................... 35 36 36 38 39 42 45 Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. LIST OF FIGURES Distribution of Phasianus in Asia. The introduced range in Europe (cross-hatched) is of birds of varied racial origins (fiom J ohnsgard 1986) ......................................................................... Counties sampled to evaluate free-ranging pheasant populations. Three free-ranging populations, the local ring-necked, the Mixed Release, and the Sichuan Release were sampled in 1991-1993. Pure Sichuan pheasants were assessed by sampling the MDNR’s captive breeding population at the Mason Wildlife Facility (denoted by star) ......................................................................... Townships (shaded) and number of free-ranging common pheasant males that were harvested in the fall for genetic analysis (1991-1993). The number of pure Sichuans released (1986- 1992), and the townships (shaded and/or unshaded) into which they were released, are also noted ............................................... Townships (shaded) and the number of free-ranging common pheasant males harvested in the fall for genetic analysis (1991- 1993). The number of birds of mixed heritage released (1986- 1993 ), and the townships (shaded and/or unshaded) into which they were released, are also noted ............................................... UPGMA branching diagram based on Rogers (1972) genetic distance (DR) representing the variation between the 4 populations of common pheasants found in southern Michigan, F = 9.306, T.» = 0.878 .................................................................... UPGMA branching diagram based on Roger’s (1972) distance as modified by Wright (1978) (Dw) representing the variation between the 4 populations of common pheasants in southern Michigan, F = 6.405, rcc = 0.950 ................................................. xi 20 22 24 38 38 Figure 7. Figure 8. Figure 9. Figure 10. Figure 11. Figure 12. Figure 13. UPGMA branching diagram based on Nei (1978) unbiased genetic distance (DN) representing the variation between the 4 populations of common pheasants found in southern Michigan, F = 15.313, r“ = 0.921 .................................................................. UPGMA branching diagram based on Cavalli-Sforza and Edwards (1967) chord distance (DC) representing the variation between the 4 populations of common pheasants in southern Michigan, F = 4.503, rcc = 0.960 ................................................. Distance Wagner tree showing the association of 4 populations of common pheasants in southern Michigan generated from Rogers (1972) distance as modified by Wright (1978). Total length of tree = 0.263, F = 0.917, r“= = 0.998 .............................. Relative genetic distances of the 4 populations of common pheasants found in southern Michigan, plotted by the first 2 principle coordinates. Distance measure used was Rogers (1972) distance as modified by Wright (1978) ............................. UPGMA cluster analysis using standardized morphological data fi'om the 4 populations of common pheasants in southern Michigan .................................................................................... Cluster analysis, using UPGMA methodology on standardized morphological, allelic, and genotypic frequencies for the 4 population of common pheasants in southern Michigan ............... Means (I) and ranges (—) of observed genetic distances for members of the Class Aves between local populations of the same species (upper graph) and between subspecies (lower graph). Values are Nei (1978) unbiased genetic distances. Numbers in parentheses number of populations or subspecies compared, and reference number from Appendix D ..................... xii 39 39 40 40 42 43 53 INTRODUCTION In 1925, Michigan held its first ring-necked pheasant (Phasianus colchicus) hunting season (MacMullan 1957) and this species soon became the most popular upland game bird in the state (McCabe et a1. 1956). The success of common pheasants in Michigan is owed in part to the state-sponsored introduction of ring-necked pheasants that began in 1918 (McCabe et a1. 1956, MacMullan 1957). State and Midwest pheasant populations increased to record numbers during the mid-1940s. Populations have since declined, sometimes rapidly, over the past four decades. Individual factors believed to be associated with declines in pheasant populations include changes in agricultural land use patterns, predation, stochastic climatic events, changes in agricultural chemical applications, and loss of genetic diversity from state sponsored and private propagation and release programs. As farmers shifted from small grains and forage crops to row crop production, optimal pheasant nesting and brood cover declined (Leedy and Dustman 1947, Warner 1979, Warner et a]. 1984). Winter cover, as fence rows and small herbaceous wetlands, declined when the agricultural community began practicing clean farming, which promotes use of all available land, and fall plowing (Labisky 1976, Warner and David 1982). Loss of adequate cover led to increased 2 vulnerability of pheasants to predators (Dumke and Pils 1973, Petersen et al. 1988) and mortality due to exposure and/or starvation during harsh winters (McClure 1948, Kopischke and Chesness 1967). The negative relationship of biocides on survival and reproduction is well documented (Adams and Prince 1972, Stromborg 1977, 1979, Bennett and Prince 1981). Unfortunately, awareness of the individual factors that negatively impact pheasant populations has not enabled state agencies to restore pheasants populations to the levels of the 1940's. Taxonomy of Pheasants and History of Early Introductions Pheasants belong in the Tribe Phasianini. While only one species, the ring-necked pheasant, Phasianus colchicus, is currently recognized in North America by the American Omithologists’ Union (1982), the genus as a whole is very diverse fi'om a world wide perspective. Delacour (1977) recognized 17 genera and 124 races of pheasants distributed across Asia that belong to the Subfamily Phasianinae. True pheasants of the genus Phasianus consist of 2 species; P. colchicus, or the common pheasant and P. versicolor, the green pheasant (Delacour 1977, Johnsgard 1986). Thirty races of common pheasants (P. colchicus) are geographically distributed across Asia into 4 groups; the black-necked (5 races), white-winged (7 races), olive-rumped (1 race), and the grey- rumped (17 races) group (Delacour 1977, Johnsgard 1986) (Table 1, Fig. 1). These 30 races replace one another geographically and have been termed a “superspecies” because of their ability to readily breed and produce fertile hybrids (Delacour 1977). Introduced populations, representing combinations of several races, exist in Europe from the British Isles and southern Norway, through Sweden, Germany, and Greece, south to Bulgaria (Johnsgard 1986) (Fig. 1). A pure black-necked type from 3 Table 1. Systematic list of common pheasants Phasianus colchicus (from Delacour 1977 and Johnsgard 1986). Group Scientific Name Common Name Black-necked P. c. colchicus Linne Southern Caucasian pheasant pheasants P. c. septenm'onalt's Lorenz Northern Caucasian pheasant P. c. talischenst‘s Lorenz Talisch Caucasian pheasant P. c. persicus Severtzov Persian pheasant P. c. principalis Sclater Prince of Wales’ pheasant White-winged P. c. zarudnyt' Buturlin Zamdny’s pheasant pheasants P. c. bianchii Buturlin Bianchi’s pheasant P. c. chrysomelas Severtzov Khivan pheasant P. c. zerajlrcham‘cus Tarnovski Zerafshan pheasant P. c. shawi Elliot Yarkand pheasant P. c. turcestam'cus Lorenz Syr Daria pheasant P. c. mango/iota Brandt Kirghiz pheasant Olive-rumped pheasant P. c. tan‘mensis Pleske Tarim pheasant Grey-amped P. c. hagenbecla' Rothschild Kobdo ring-necked pheasant pm” P. c. pallasi Rothschild Manchurian ring-necked P. c. kamowi Buturlin Korean ring-necked pheasant P. c. kiangsuensis Buhn'lin Shansi pheasant P. c. alascham'cus Alpheralty & Bianchi Alashan pheasant P. c. edzinenst's Suchkin Gobi ring-necked pheasant P. c. satscheuensis Pleske Satchu ring-necked pheasant P. c. vlangalt'i Przevalski Zaidan pheasant P. c. strauchi Przevalski Strauch’s pheasant P. c. sohokhotensis Buturlin Sohokhoto pheasant P. c. suehschanem‘s Bianchi Sungpan pheasant P. c. elegant Elliot Stone’s pheasant P. c. rothscht'ldt' La Touche Rothschild’s pheasant P. c. decollatus Swinhoe Kweichow pheasant P. c. takatsukasae Delacour Tonkin ring-necked pheasant P. c. torquatus Gmelin Chinese ring-necked pheasant P. c. formasanus Elliot Taiwan ring-necked pheasant one vane? :55 ans. 85% 2593 e 8:42.823 825 a use 892.23 2:. .32 5 3.35.3.8 8:355 a sari. 3.8.43. 5:38.: . 883:: 523. . Benin: 325 . 889?: 52: . i=5 . 4.8% . 32.203. . 88.9935. 5.3 . “.232 . £35 . name—5 . =63. . =53 . stoiééo . 333i 8%: . as; . Ems. . £5 5 . .5293 . .q 955 Bagel—o 8.55 _o 8s: . E53 335-323 be 3:54 5V5.— . =05 BQEE->P_M mo £85 9.5..» Exogiom—n Lo 2 53:6 .2: . 5:330 .5582 . A 53830 Eofiaow . a ‘ .5 he so. 5 Persia dominated in England until ring-necked pheasants from the grey-rumped group were imported from the Orient in the eighteenth century (MacPherson 1896). A diverse mix of at least 3 to 4 races existed in Europe by the late nineteenth century (W ayre 1969, Bohl and Bump 1970) and it was fi'om this heterogenous gene-pool that introductions were made into the US. in the late 1980's and early 1900's (Prince et al. 1988). Michigan began a successfiil pheasant introduction program in 1918 (Allen 1956). By 1925, populations of pheasants had reached huntable levels (MacMullen 1957). Michigan game farm ring-necks exhibit a mixed heritage including Chinese ring-necked (P. c. torquatt's), Korean ring-necked (P. c. karpowi), English black-necked (P. c. colchicus) and Mongolian or Kirghiz ring-necked (P. c. mongolicus) (Prince et al. 1988). Males difi‘er in plumage (Table 2) while females have a brown spotted mantle and an under body which is not mottled (J ohnsgard 1986). Population sizes gradually increased with fluctuations until 1935, and then dramatically declined in the 1940's with a peak harvest of 1,404,076 males in 1944 (MacMullen 1957). Spring estimates of hens in Michigan fell from 713,600 in 1961 to 145,500 in 1986 (Dahlgren 1988). This decline was similar to those seen throughout the ring-neck’s Midwest range (Dahlgren 1988). In the 19603, interest in introductions of exotic game birds renewed (Prince et al. 1988). Allen (1956) suggested that releases in America of races of pheasants living in remote parts of Asia, and not yet released in America, might be usefiil. This suggestion lead to the formation of “The Foreign Game Introduction Program” (FGIP) of the US. Fish and Wildlife Service in the mid-19505 (Prince et al. 1988). The program was designed to limit unwise introductions, while, promoting trial introductions of previously unavailable pheasants into vacant habitats. Five races were subsequently imported into the 6 Table 2. Neck ring characteristics of some common pheasants introduced into North America. Group Neck Ring Scientific Name Common Name Present Width at Front Black- necked No White- winged Yes Grey- Rumped Yes - P. c. colchicus wide P. c. mongolt'cus wide P. c. karpowi Southern Caucasian or European Blackneck pheasant Kirghiz or Mongolian pheasant Korean ring-necked pheasant Yes narrow P. c. torquams Chinese ring-necked pheasant No - P. c. strauchi Strauchi’s or Sichuan pheasant U. S. including 2 races of black-necks (P. c. talischensis, Talish Caucasian and P. c. persicus, the Persian pheasant), 1 white-winged (P. c. bianchi, Bianchi’s pheasant) and 1 grey-rumped (P. c. karpawi, Korean ring-necked pheasant). In addition, the Northern green pheasant, P. versicolor robustt'pes, was imported from Japan (Prince et al. 1988). Work by Warner et al. (1988) identified regional difi‘erences in genotype among wild pheasants established fi'om releases in Illinois. These difl'erences could be attributed to differences between founding populations and/or selection following release. Prince et al. (1988) hypothesized that the establishment of pheasant populations, or the revitalization of declining pheasant populations is, to a large part, a firnction of genotype. The release of newly imported races, along with selective breeding of genotypes, was 7 proposed to be critical to future pheasant management programs. Background of Michigan’s Sichuan Pheasant Release Program The Michigan Department of Natural Resources (MDNR) embarked on a new program in 1983 to bolster pheasant populations in Michigan using the Strauch’s, pheasant (P. c. stranchi), a race of common pheasant within the grey-rumped group found in the Zhenja District of the northeastern region of Sichuan Province, People’s Republic of China (Squibb 1985, Prince et al. 1988). This race has been given the honorary appellation of Sichuan pheasant in recognition of the generosity of The People of Sichuan Province for providing birds (Prince et al. 1988). In 1983, the improved political climate between the United States and the People’s Republic of China offered the WNR an opportunity to acquire a subspecies of pheasant from Guangyuan County not yet introduced on the North American continent. Climatic difi‘erences between Guangyuan County, based on 3 years of weather records for Chengdu, Sichuan Province (adjusted for the 970 m elevation difl‘erence) and Ingham County, Michigan, are minor. Late winter and early spring mean temperatures in Lansing, Michigan (Sommers 1977) are slightly cooler by 3.7° and 29° C for January and April, respectively, compared with Guangyuan County. Annual rainfall for both areas averages 81.2 - 86.4 cm. However, 80% of the annual rainfall in Guangyuan occurs from July to September (Chen 1970), while annual rainfall in Michigan is more evenly distributed. Chinese officials offered Michigan 200 wild pheasant chicks. Approximately 300 eggs of Sichuan pheasants were collected in the Zhenja District of Sichuan Province in the spring of 1984. Eggs were hatched and chicks were reared by the Sichuan Forestry Department. Rearing problems were encountered and only 30 chicks survived to be 8 shipped to Hawaii where they were quarantined for at least one month. In February of 1985, Michigan finally received 24 Sichuan pheasants, 9 males and 15 hens. In the spring of 1985, an American delegation traveled to Zhenja District, in the northeastern part of Sichuan Province, People’s Republic of China, on a mission to collect Sichuan pheasant eggs. Approximately 23 00 eggs were obtained from more than 500 nests. Eggs were subsequently shipped to Michigan from which 550 chicks were pedigree hatched by family unit at the Rose Lake Wildlife Research Station. The chicks were transferred to the Mason Wildlife Facility following the quarantine period. By February 1986, a second group of P1 Sichuans (n = 420) were available for captive breeding. Michigan biologists returned to Zhenja District in the spring of 1988 for a second collection of eggs. More than 1,300 eggs were collected and shipped to Michigan. These efforts resulted in a third group of P1 pure Sichuans (n = 363) available for captive breeding in February of 1989. The Sichuan pheasant propagation program was designed to maintain genetic heterogeneity in a breeding and rearing environment that would facilitate release. The captive breeding program focused on holding breeders for as many seasons as possible to reduce inbreeding, selection, and genetic drift within the captive population (Prince et al. 1988). The propagation program was adaptive in the sense that space and habitat modifications in breeding and rearing areas were made as the program continued. The captive Sichuan population for this study included F1 progeny of Sichuan breeders originally obtained by the MDNR from China. In its native range, the Sichuan pheasant inhabits brushy, agricultural and mountainous pine (Pinus spp.) and oak (Quercus spp.) forests, a habitat type difi‘erent 9 fi'om perceived bush, grass and agricultural habitat preferences of the introduced races, primarily the Chinese ring-necked pheasant (P. c. torquatus), that were released in North America fi'om the late 1800's until the present. Sichuan pheasants had not been subjected to any captive propagation programs and for this reason were thought to represent a unique gene-pool. Genetics of Successful Introductions and Objectives of this Study A key to the success of the Sichuan introduction program will be the maintenance of genetic variability, to afford the populations with the maximal chance of adaptation. Genetic variability in populations can decay through selection, inbreeding, or random drift especially if the population size becomes small. A reduction in genetic heterozygosity in Michigan’s existing ring-necked compared to the Sichuan pheasant would be expected if local pheasant populations in Michigan have undergone recent population bottlenecks or are suffering from inbreeding depression resulting from generations of captive breeding. While propagation and release programs that mix stocks can result in the dilution of co-adapted gene complexes, the infusion of new genetic material into a breeding population may prove advantageous in offsetting the loss of genetic variability by increasing heterozygosity. Increased levels of heterozygosity lead theoretically to increased fitness, decreased frequency of deleterious alleles, and reduced inbreeding depression. The introduction of yet another race of pheasant in Michigan offered an opportunity to measure the genetic difl‘erences in common pheasants, and to generate hypotheses associated with the introduction of a new race. Direct sampling of the original races fiom Asia, although desirable, was beyond the scope of this project. However, it 10 was possible to compare the genetic composition of a race (ring-necked) that has been subjected to the continuous anthropomorphic influences of captive propagation with one that has not (Sichuan). Michigan’s current free-ranging ring-necks were used to represent populations of mixed racial heritage established via releases from traditional game farms. P, and F, breeders from Michigan’s captive Sichuan pheasant program were used to represent populations that were new to the captive breeding environment and considered to be a pure race. Introductions of Sichuan pheasants into free-ranging populations led to the following predictions. First, a reduction in genetic heterozygosity in Michigan’s existing local ring-necked pheasant populations compared to the captive Sichuan breeding stock would be expected if local ring-necks had undergone recent population bottlenecks or were sufi‘ering from inbreeding depression as a consequence of generations of captive breeding. Secondly, the infiision of new genetic material into a free-ranging breeding population may prove advantageous in offsetting the loss of genetic variability by increasing heterozygosity. Increased levels of heterozygosity lead, theoretically, to increased fitness. Finally, since these two races freely interbreed in captivity and produce fertile hybrids, levels of genetic heterozygosity and the distribution of genetic variance in flee-ranging populations should vary since the infusion of the Sichuan genetic component was controlled by the number and genetic heritage of birds released fi'om the captive breeding program. An assessment of phenological traits in differentiating subspecies or populations was also possible by comparing neck-ring characteristics of male pheasants with their biochemical profile. ISOZYME VARIABILITY IN HARVESTED RING-NECKED AND SICHUAN PI-IEASANT S INTRODUCTION Starch gel electrophoresis provides a tool to assess the biochemical genetic variability of captive and wild pheasants. The use of plumage or other traits to describe genetic variation has been insuflicient (Trautrnan 1982) and often the phenotypic expression of meristic and morphometric traits are too variable to be reliable (Ihssen et al. 1981). Visible genetic variation affecting phenotypic traits are often influenced by many genes, as well as by the efi‘ects of the environment, so phenotypic differences for such traits can rarely be traced to the efl‘ects of particular genes (Hartel 1987). Since the advent of starch gel electrophoresis in 1959, the technique of electrophoresis has been used to provide useful information on variability patterns in a wide range of biological situations (Richardson et al. 1986). The banding patterns fiom separation of proteins afier exposing the medium to histochemical specific stains, can be related to frequencies of various alleles at single loci, and each individual can be assigned a genotype. Estimation of heterozygosity, genetic distance, and the calculation of the among and within components of genetic variation is possible. 11 12 Early electrophoretic analyses indicated difl‘erences in pheasant genotypes in North America. Brandt et al. (1952), Sandness (1954), and Baker et al. (1966) used proteins from eggs and sera to describe genetic difl‘erences between pheasants and their hybrids. Blood group factors indicated regional differences in pheasants from Iowa (Vohs 1966). An east-west gradient in the frequency of fast-binding forms of blood protein was found in pheasants from Illinois, Iowa, and Kansas (Baker et al. 1966). Warner et al. (1988) identified a north-south cline for wild pheasants in Illinois using isozyme methods. The genetic heritage of North America’s ring-necked pheasant (P. colchr'cus) is, at best, a blend of races dominated by P. c. torquatus (Chinese ring-necked pheasant) that originated in Asia. The use of electrophoretic procedures have identified difl’erences in wild pheasant populations resulting fi'om range expansions from releases in the 1930's through the early 1950's (Warner et al. 1988). There is no clear explanation of how these difl‘erences emerged and several factors must be considered including initial stock differences, founder effects, and selection in response to environmental gradients. It is clear, however, that starch electrophoresis is a useful tool for describing patterns of variability in wild pheasant populations, and where race specific markers exist, it can be used to measure the integration of a new race of pheasant into existing populations. This methodology was used in this study to assess the genetic structure of 4 populations of common pheasants in Michigan. ELECTROPHORETIC METHODOLOGY An initial electrophoretic screening was performed using Sichuan and ring-necked breeders housed at MDNR’s Mason Wildlife Facility. All ring-necks were wild trapped birds obtained from various locations throughout Michigan in the winter of 1989 in an l3 attempt by the MDNR to improve their captive ring-necked stock. Tissue samples were obtained from birds within 4 hours of death. Leg band numbers were recorded for comparison of pedigrees. A detailed description of starch electrophoretic techniques, including grinding buffers and gel preparation are presented in Appendix A. Individuals were initially screened for variation at 30 biochemical loci using 15 electrode bufl‘er systems. Staining was also done for enzymes found to be polymorphic in other galliforrnes (Baker and Manwell 1975, Gutierrez et al. 1983, Gyllensten 1985, link et al. 1987, Warner et al. 1988, Scribner et al. 1989, and Randi et al. 1991, 1992). The objective of this process was to refine laboratory procedures and find enzymes that were polymorphic in which clear banding patterns could be replicated. The liver was removed from all birds and stored at -70° C at MSU for the duration of this study. Care was taken to insure an airtight condition to prevent dehydration. Four milligrams of liver tissue was homogenized in a grinding bufi‘er on ice using a pestle and mortar the day before an electrOphoretic run. Contamination of the sample was reduced by avoiding connective and adipose tissue. Paper wicks were saturated with the resulting supernatant, placed in individual wells in ELISA trays, double wrapped in plastic and fi'ozen at -70°C for use the next day. Starch gels were prepared 12 hours before each electrophoretic run. Once the liquid starch had solidified and cooled, the gels were covered with plastic wrap to prevent desiccation. Gels were kept at room temperature until 1 hour before the start of the run, when they were cooled to 4°C. Gels were continuously cooled over an ice bath for the duration of each run. 14 Wicks were removed 20 minutes after the beginning of the run that lasted 6 V2 hours. Completed gels were sliced horizontally, placed in enzyme stains, and incubated in the dark. Acetate was used to halt the staining process. Gels were fixed using 50% ethanol, placed in air tight plastic storage bags, labeled, and stored at 4°C. Gels were scored immediately after staining. When more than one putative locus was observed for a particular enzyme, they were numbered sequentially, beginning with the most anodal. Alleles at variable loci were coded by letters beginning with “a” for the most anodal. All genotypes were scored for all the loci. ELECTROPHORETIC RESULTS AND DISCUSSION Although thirty enzymes were examined (Table 3), only 5 loci, representing 4 enzymes; alkaline phosphatase (AP), acid phosphatase (ACP), aconitase (ACON 1 and 2), and isocitrate dehydrogenase (IDH 1); were consistently scoreable and polymorphic. Staining for enzyme activity followed methods outlined by Richardson et al. (1986). Esterase appeared to be polymorphic and sub-banding was excessive which confounded scoring. While the enzymes found to be polymorphic in other galliforrnes were systematically stained in all our free-ranging pheasant collections, none proved to be polymorphic except AP, ACP, ACON and IDH. ACON, IDH, AP, and ACP were found to be polymorphic in many other galliforrnes (Baker and Manwell 1975, Gutierrez et al. 1983, Gyllensten 1985, link et al. 1987, Scribner et al. 1989, Randi et a1. 1992,) but not in all (Warner et al. 1988, Randi et al. 1991). Difi‘erences between studies may be attributable, in part, to the composition of tissues used and laboratory conditions. Since an enzyme’s expression varies between tissues, the ability to detect polymorphisms may have been limited by using only liver 15 tissue. Barrowclough and Corbin (1978) found ACP and IDH were most commonly found in liver tissue. Scribner et al. (1989) found AP to be polymorphic in ring-necked pheasants using tissues from the liver, heart and kidney. ACON was polymorphic in a homogenous mix of heart, liver, kidney, and muscle in California quail (Callipepla califomica) (Zink et al. 1987). No unique alleles were detected in the ring-necked or Sichuan individuals used in the initial screening process (Table 4). This is surprising as different, but closely related species of animals typically show fixed difl'erences, or almost fixed differences, for at least some of their electrophoretic loci (Ayala 1975, Richardson et al. 1986). However, bird species are commonly indistinguishable at allozyme loci (Avise et al. 1982). Why birds are different from other vertebrates is unknown, but it is generally accepted that birds at all taxonomic levels exhibit less genetic divergence than do many of their counterparts in other vertebrate classes (Avise and Aquardro 1982, Barrowlclough et al. 1985, Prager et al. 1974). It is likely that markers between ring-necked and Sichuan pheasants can be found since they are recognizable at the subspecies level. However, it may require a different type of molecular analysis to identify species or subspecies specific alleles. 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Exam. ...z .0... .80 Sat—£2 30558 a. 3:88.... ago» we «6x38 0.38;. 33256 gum .m 0.9.... 17 Table 4. Allele and genotypic frequencies, at polymmphic loci, of Sichuan and ring-necked pheasants used in the evaluation of enzymes for genetic analysis of commogheasants in Michigan. Allele Frequencies Genotypic Frequencies Locus/Allele Sichuan Wild-trapped Genotype Sichuan Wild trapped Ring-neck Ring-neck ACP 0.875 0.750 aa 0.875 0.750 0.125 0.250 ab 0.0 0.0 8 4 bb 0.125 0.250 AP 0.938 0.750 a 0.875 0.750 0.063 0.250 ab 0.125 0.0 8 4 bb 0.0 0.250 IDH-1 0.813 0.750 as 0.750 0.750 0.188 0.250 ab 0.125 0.0 8 4 bb 0.125 0.250 ACON-1 0.938 1.0 aa 0.875 1.0 0.063 0.0 ab 0.125 0 8 3 bb 0.0 0 ACON-2 0.750 1.0 a 0.625 1.0 0.250 0.0 ab 0.250 0 8 3 bb 0.125 0 ISOZYME VARIABILITY IN PURE AND MIXED PHEASANT POPULATIONS IN MICHIGAN INTRODUCTION Releases of Sichuan pheasants into southern Michigan provided an opportunity to evaluate the impact of an introduction on existing ring-necked populations and to measure the genetic difl‘erences in common pheasants. Refinement of electrophoretic methods provided the tool to investigate levels of genetic heterogeneity and the distribution of genetic variance within and between populations. METHODS Stocks Two populations were sampled to represent “pure” P. c. strauchi (Sichuan) and P. cholchus (North American ring-necked pheasant): 1) breeders from Michigan’s captive Sichuan propagation program, and 2) samples obtained from hunter harvest of free- ranging, local common pheasants. Two additional free-ranging populations were sampled including one that received a mixture of Sichuan, ring-neck, and hybrid releases, and another which other received only pure Sichuan releases. Collection of Pheasants for Electrophoresis: Burg Sichugs: Livers of both male and female, adult pure P1 and F1 Sichuan 18 l9 breeders, from the MDNR’s Mason Wildlife Facility were collected during 1990-92. This sample included the birds used in the initial electrophoretic screening process plus birds that died when a mink(s), (Mustela vison), gained access into several outside flight pens and killed dozens of 2+ year old breeders during the winter of 1992. Carcasses were partially thawed to allow for the removal of the liver at a later date. Michigan’s Local Ring-necked Pheasants: Samples from Montcalm and Hillsdale counties were used to assess the genetic composition of free-ranging wild ring-necked pheasants in Michigan (Fig. 2). The free-ranging wild ring-neck population included local pheasants in areas thought to have strong remnant ring-neck numbers and habitats not suitable for Sichuan releases. Samples of free-ranging male pheasants from Montcalm and Hillsdale counties were collected with the assistance of local Pheasant Forever Chapters. Local hunters were supplied with a collection bag before the regular season. The liver was removed within 4 hours of death. Width of the neck ring at its widest point was marked along the edge of each collection bag and measured to the nearest mm at a later date. Hunters were asked to estimate the percent closure of the neck ring in V4 intervals and indicate the location of each kill. Materials from each individual bird were placed in a corresponding collection bag and stored in a home freezer until the end of the season. All samples were then stored at -70°C at MSU for the duration of this study Sichuan Release Population: Samples from Livingston and surrounding counties were used to measure the influence of Sichuan releases on local ring-necked populations (hereafter referred to as the Sichuan Release Population) (Fig. 2). All Sichuan releases were F , progeny of breeders obtained from China in 1985 and 1988 as eggs. Release 20 Pheasant Populations I Ring-necked - no release Mixed Release Sichuan Release E Captive Sichuan - no release { l Huron Montcalm Cl H Clinton 1 A \\ Eton Livingston Jackson Washtenaw / mm. Figure 2. Counties sampled to evaluate free-ranging pheasant populations. Three free- ranging populations, the local ring-necked, the Mixed Release, and the Sichuan Release were sampled in 1991-1993. Pure Sichuan pheasants were assessed by sampling the MDNR’s captive breeding population at the Mason Wildlife Facility (denoted by star). 21 efl‘orts by the MDNR focused on establishing pheasants with Sichuan heritage in habitats in Michigan that were void, or nearly void, of wild pheasants and to maximize founding populations. Potential release sites were evaluated based on the structure of vegetation similar to that in Sichuan Province. Groups of 50-60 pheasants were released and hunting was restricted to minimize mortality and facilitate dispersal (Prince et al. 1988). From 1986 to 1993, approximately 20,517 pure Sichuan pheasants were released in Livingston and surrounding counties (Fig. 3). Livingston and Jackson received similar numbers of releases (3 0% and 39% of all releases, respectively). Since the objective of this study was to examine the introgression of the Sichuan genetic material into local common pheasant populations, Livingston County remained the “core” of the Sichuan release population for all releases occurred during 1986-1990 (n=6251). Males harvested during 1991-1993 would reflect the introgression of Sichuan genes into the population. Samples of free-ranging males from Livingston and surrounding counties were collected with the aid of MDNR personnel. Collection materials were distributed to biologists before the regular season. On opening day, individual hunters were actively sought in the field by MDNR personnel. If successfiil, hunters were asked to donate the liver from their harvested bird(s). Ring neck width and closure were recorded by MDNR personnel. All materials were placed in a frozen state (-70°C) within 4 hours of death. Mixed Release Population: Samples from Huron county were used to measure the efl‘ect of releases with varied racial heritage on the local remnant ring-necked population (hereafter referred to as the Mixed Release Population) (Fig.2). An effort to improve the captive breeding program was initiated in 1989 by the inclusion of winter trapped “wild” ring-necked birds Michigan into the MDNR’s captive propagation program. Subsequent 22 880: 83 Pa 60332 20? >05 .833 as 83% 888 EEE age 05 Ba Aaaeg c 8322 888% 2% co saga 2:. .38 3%: egg onoaom .8.“ :8 ea B 8883.3 803 35 838 5883 8858 waneéoc mo Hon—ES 85 @353 320% .m 2:me a: - S: - a: - 82 - $2.883 uu3-~< acafiomm. 93% _I.Fl n n ........................ 2.3.3 asenemneee 3.3.3.8 3<2mEm<3 in 759.3... >52... 88.863828 “3me Emma 33.8%: HE “swam 8.8.88.8; v: EL. .an 3.93832 1 mm .5 3.33.83: . 3.22.832 1'. 8% m. smemaemmefié masses I 32%.: s smegmeezée l 8% 2882—24 33.8 I; 23$: $.33-m:-§ m . . H 2352. 28.3. 883 820% - massacraéa ZOE—AU 23 hybrid, ring-neck, and back-cross releases were derived from crosses between the pure Sichuan and “wild” ring-necked captive breeding stocks. Releases in Huron county included pure Sichuans, captive reared wild-trapped ring-necks from Michigan, Iowa, and North Dakota, Fl hybrids (Sichuan x wild-trapped ring-neck), and various back-crosses. Locations of harvested and released birds were mapped by township for the Mixed release population (Fig. 4). Harvest locations for this study occurred in townships that had, or were next to, townships that had previously received release pheasants. The heritage of released pheasants in Huron county varied and most of the releases occurred on the western side of the county. Since 1986, 2,681 pheasants of Sichuan and/or ring-necked heritage have been released in 11 townships in Huron County. Pheasants were harvested in 11 townships, within which 7 (64%) had, or were adjacent to, townships that had previously received pheasant releases. The MDNR in an attempt to reduce mortality, closed areas surrounding release sites to hunting making it dificult to sample release areas directly. Samples of flee-ranging males pheasants from Huron County were collected as described above with the assistance of local Pheasant Forever Chapters. Statistical Estimates The allelic variation revealed by electrophoretic examination was analyzed using BIOSYS (Swofl‘ord and Selander 1989). The non-parametric Mann-Whitney U and Kniskal Wallis tests (Siegel 1956) were used to determine whether it was valid to increase sample sizes within the same site by pooling samples taken between pairs of years and within all years, respectively. 24 .880: 8? 93 60322 803 >05 :03? 85 8035.8— 585 838 33839 05 Ba .9833: e883. amazon E8 ho was co 3&3 one .28 _- so: ages 309% .88 ma 05 E 88on 838 388:8 :oEEOo mangéoc mo Hogan: 05 85 8893 333308. .v 0:85 ZOKB— 8-8.8.8888 8.8.8-N ~ m. .8 — K c.3823 ~ 8um8-NnT8.8 I 88888.88 8878.88-88 8.8.8.8888 mini 873.836 8.8888788 3322 aam + is: 383.5 a 533m : n .8. 3563 :8 +82 .5 aéaaarmaagaé + 85»: 8*.er x 5.65 r. x: n 82 38:3: :2. .522: $3 + is»: 3% u fiaflm E 3: u 8.: 63»: 335 x 5:55 E n 82 83>: Bissau a .356 E u $.82 fi 32 $83 - 32 -883 88.883 au3u~uz 25 W Allele and genotypic frequencies for each population were determined from the banding pattern on the gels. Mean observed, or direct count heterozygosity (Hm) , was calculated by determining the number of individuals heterozygous at a particular loci and dividing by the total number of individuals examined for that loci. This process was repeated for other loci and a mean estimate was obtained by averaging values over all loci. Mean expected heterozygosity (Flap) from the allele frequencies as if the population was in equilibrium was calculated as 1 - :15} where 16, is the frequency of the ith allele at a locus, with n alleles (Nei 1975). An unbiased estimate of Hap, based on conditional expectations, was corrected for small sample size afier Levene (1949) and Nei (197 8). The percentage of polymorphic loci was calculated using the 0.99 and 0.95 criterion for the fi’equency of the most common allele. The proportion of polymorphic loci in the populations was calculated counting the number of polymorphic loci and then dividing by the total number of loci examined. Homogeneity in the number individuals carrying each allele in samples obtained fi'om difl‘erent sites was tested using the log- likelihood G-test (Sokal and Rohlf 1981). Hardy-Weinberg Equilibrium: For each polymorphic locus in each population, observed and expected genotypic fi'equencies were compared by the log-likelihood G-test to evaluate departure from Hardy- Weinberg equilibrium. Levene’s (1949) correction for small samples size was used to calculate expected values. Population Differentiation The pattern of variation in allelic frequencies among populations was evaluated 26 using hierarchical F-statistics (Wright 1978). These statistics, F13, F ST and FH , are related by the equation (1 - Frle - PST) = (1 - F”) (Wright 1965, 1978). The effects of population subdivision were measured by FsT , the fixation index which varies from 0 to l, and is the reduction in heterozygosity of a subpopulation due to random genetic drift. The inbreeding coeflicient, F5, is the measure of reduction in heterozygosity of an individual due to nonrandom mating within a population. When positive, F 13 indicates matings between relatives occurs more often than would be expected if random, while a negative value indicates an avoidance of matings with relatives. Frr is the most inclusive measure of inbreeding that takes into account both the effects of nonrandom mating within subpopulations and the efl‘ects of population differentiation (Hartel 1987). Four methodologies were used to calculate genetic distance between pairs of populations: (1) Rogers (1972) distance (DR), (2) Rogers (1972) distance as modified by Wright (1978) (Dw), (3) Nei (1978) unbiased genetic distance (DN), and (4) Cavalli-Sforza and Edwards (1967) chord distance (DC). Rogers DR was used because it is equivalent in principle to Mahalanobis’ distance for morphological characters, so distances calculated fi'om morphological and allelic data could be compared. It is also a simple observational measure with no assumptions. DR is calculated using allele frequency data with one axis being used for each allele at the locus. When DR equals zero, the two populations being compared are genetically identical, whereas if DR equals unity, then the populations are fixed for difl‘erent alleles. Therefore, the larger the value of DR, the less ‘related’ are the populations. DR is afi‘ected by the number of alleles. If multiple alleles are found in both populations, but none are held in common, then a distance that is a little less than 1 will be obtained. To calculate DR over 27 several loci, the aritlunetic mean of the DR value at each locus is normally used. Wright (197 8) suggested it might be better to calculate the Euclidean distance over all loci by adding a dimension for every allele at every locus, (Dw), instead of taking an arithmetic mean as Rogers (1972) did. This gives less weight to loci in which the difference in allelic frequencies are small. To be an accurate estimate of distance in Euclidean hyperspace, each axis must be independent and on the same ‘scale’. With genetic data, the ‘scale’ criteria is met, however the independent criteria is not for the fi'equency of all alleles at a locus must add up to unity. The failure to meet this assumption is routinely ignored. Nei (197 7) proposed a genetic distance measure based on a totally difl‘erent concept to Rogers distance and begins with Nei (1972) standard distance (D). It is derived from the probability that 2 alleles, one drawn from each population unit being compared, are the same. The probability of picking the same allele from each population unit depends of the frequency of that allele in the 2 populations (i.e. px x p,). Ifthere are several alleles at a locus, then the chance of picking identical alleles is the sum of the probabilities of picking 2 copies of allele 1 plus the probability of picking 2 copies of alleles 2. The arithmetic mean is then taken over all loci. But, the probability of 2 alleles being identical when taken from the same population will be < 1 if there is polymorphism at the locus. Therefore a measure of distance between populations must take into account the amount of divergence within each of the populations. Nei gives a ‘biological’ meaning for D as an estimate of the number of DNA base differences per locus between populations. Expanding on this concept, Nei (1978) suggested that genetic identity (I) could be 28 estimated as the normalized probability that 2 alleles, one taken from each population, are identical. It provides a measure of similarity in frequency of each alleles, summed over all alleles and the relationship between Nei’s I and D is D = -log, I. Nei (1978) also noted that estimates of genetic distance are systematically biased when sample sizes are small. To accommodate this, he replaced population gene identities with sample gene identities resulting in an unbiased estimate of genetic distance (DN). The difference between biased (D) and unbiased (DN) estimators of Nei’s genetic distances is very small when the number of individuals is large (> 50). Cavalli-Sforza and Edwards (1967) developed a measure of genetic distance which is Pythagorean in a Euclidian hyperspace, but which difl‘ers fiom Rogers (1972) concept in that it takes the square root of the allelic frequencies as the coordinates of the points representing the populations, instead of the fi'equencies themselves (Wright 197 8). This locates all populations on the surface of a hyperspace such that all coordinates are non- negative. They all fall on the portion of this surface in which the coordinates of the point are non-negative. The chordal distance (DC) is 0.9003 for populations with no allele in common. In detemtining chordal distances from multiple loci, the authors locate the population distances in a hyperspace with a dimension for each locus. The population coordinates along these are then equal to the chordal distances and thus not terminating on a hyperspace. Patterns of population relatedness were examined by the unweighted pair-group method with arithmetic averaging (UPGMA) cluster analysis on the matrices of genetic distance for all methodologies, and Farris’s (1972) distance Wagner network, optimized according to Swofford (1981) using Roger’s (1972) distance as modified by Wright 29 (1978). Genetic distances using the methodology of Nei (1978) (D...) was provided for comparison with the literature. Branching diagrams using DR and DC were provided for comparison with DN. The “fits” of distances implied by the branching diagrams generated from genetic distances were evaluated by the F statistic of Prager and Wilson (1978). Their statistic is F = 100 :1 II, - 0,.|/ :11, where for n pair wise comparisons of populations, 1 and 0 are input values of the original matrix and the output values of the tree, respectively. Smaller values of F indicate greater congruence, but any F < 0.10 implies a good fit (Avise et al. 1982). The cophenetic correlation (rm) was also used to evaluate how well the resultant branching diagram represents the original distance matrix. Cluster analysis using the UPGMA algorithm, was also performed first on the morphological data, (standardized neck ring closure and width), and then on the combination of standardized morphological and genetic (allele and genotype fi'equencies) of harvested males using PROC CLUSTER in PC-SAS (SAS Institute, Inc. 1993). An alternative multivariate technique, principle component analysis, was used to examine the relationship among the populations. The purpose of principal component analysis is to derive a small number of linear combinations (principle components) of a set of variables that retain as much of the information in the original variables as possible (Rao 1964). Roger’s (1972) distance as modified by Wright (1978) was used for comparison with the distance Wagner procedure. An extensive literature review of protein electrophoresis studies dealing with avian species was performed. Observed (direct count) and expected (N ei’s (1978) unbiased estimate) heterozygosity (Appendix C), and Nei (197 8) unbiased genetic distance and Wright’s (1978) FST (Appendix D) were recorded for comparison with this study. 30 RESULTS Genetic Variability Captive F , Sichuans, obtained either from the breeding stock directly or from individuals selected for release, showed similar allele frequencies (Mann-Whitney U, z = - 0.484, P = 0.613, df = 1) (Appendix B). Allele frequencies were similar between pheasants from Hillsdale and Montcalm counties in 1991 (Appendix B) (Mann-Whitney U, z = -0.721, P = 0.471, df= l) and there was no significant difference among years within the ring-necked individuals (Kruskal-Wallis, T = 0.061, P = 0.970, df= 2) (Appendix B). Allele frequencies (Appendix B) were similar among years within mixed release (KW, T = 0.143, P = 0.931, df= 2 and the Sichuan release population (KW, T = 0.319, P = 0.853, df= 2). Since no significant differences were noted between sites and among years, data on the 192 individuals were pooled for the 6 loci within the Captive Sichuan, ring-necked, Pure Sichuan Release, and Mixed Release populations (Table 5). Allele frequencies were determined for all 4 combined populations (Table 6). Allele frequencies of ACON-2 were similar between the Captive Sichuan, Mixed Release, and Sichuan release populations, which were collectively different from the ring-necked population. Log-likelihood G values indicated a significant difference in the number of individuals carrying each allele for all loci except ACP (Table 7). For each population, direct counts were made of the proportion of heterozygous individuals per locus. When averaged across the 30 assayed loci, the resulting heterozygosity (Pick) ranged from 0.019 in the Sichuan Release population to 0.029 in the Captive Sichuan population (Table 8). 171m, ranged from 0.035 to 0.042 for the Sichuan Release and Captive Sichuan populations, respectively. Heterozygosity levels observed in 31 this study were slightly lower than those published for other Phasianus (1710,, = 0.041 : 0.007 SE, n = 5, range 0.026 - 0.066) and other galliforrnes (PIN, = 0.040, n = 18, range 0.000 to 0.083) (Appendix C). Percentage of loci polymorphic was similar across all 4 populations. Genotypic frequencies varied between populations (Table 9). G values (Table 10) generated fiom testing for homogeneity between the observed and the expected number of genotypes indicated that the captive Sichuan population was in Hardy-Weinberg equilibrium, whereas the three release populations were not. Average FIs was -0.114 across populations and all loci except ACP were negative indicating an avoidance of matings with relatives (Table 11). An average F S, of 0.298 (0.000 to 0.718) was observed suggesting that 70% of the total genetic variation is found within populations, while 30% is distributed among populations (Table 11). 32 Table 5. Sample sizes, by loci, for 4 populations of common pheasants in southern Michigan used for genetic analysis. Captive pheasants were collected from 1990-92 while free-ranging pheasants were collected from 1991-1993. Loci Captive' Free-ranging Total w/o releases with releases Sichuan Ring-necked” Mixedc Sichuan‘I ACP 37 49 60 45 l 91 AP 3 5 46 5 9 45 1 85 IDH-1 35 47 59 45 186 IDH-2 3 5 48 60 45 l 88 ACON-l 35 48 61 45 189 ACON-2 37 48 62 45 l 92' ' sanplee fi'orn MDNR’I captive breeding stock of Sichuan pheasants at the Mason Wildlife Facility (1990-92) ' samples fiom Hillsdale (1991-93) and Montcalm (1991) counties ‘ samples from Huron County (1991-93) ‘ sampleefi'om livingston(l991-93), Jackaon(l991), Ingham(l991), Washtenaw(l991), Barry (1992-93), Clinton (1992), & Eaton (l993)countiee ' maidrmnnmnnberofindividuals sampled 33 Table 6. Allele frequencies of common pheasants collected for genetic analysis from 4 populations in southern Michigan. Captive pheasants were collected from 1990-92, while free-ranging pheasants were collected from 1991 -1 993. Captive Free-ranging Loci Allele w/o releases w/releases S ichuan‘ Ring-necked” Mixed‘ Sichuan“ ACP a 0.892 0.827 0.902 0.891 b 0.108 0.173 0.098 0.109 AP a 0.643 0.772 0.517 0.859 b 0.357 0.228 0.483 0.141 IDH-l a 0.843 0.750 0.692 0.913 b 0.157 0.250 0.308 0.087 IDH-2 a 0.971 1.000 0.852 1.000 b 0.029 0.000 0.148 0.000 ACON-1 a 0.886 0.260 0.379 0.337 b 0.1 14 0.740 0.621 0.663 ACON-2 a 0.947 0.1 17 0.881 0.989 b 0.054 0.883 0.119 0.01 l ' sumles from MDNR‘s captive breeding stock of Sichuan pheasants at the Mason Wildlife Facility (1990-92) 'samplesfi'omHillsdale(l991-93)andMontcahn(l991)counties ‘samplesfromfluronComrtyu991-93) ‘ samples fi'om Livingston (1991-93), Jackson (1991), Ingham(1991), Washtenaw(199l), Barry (1992-93), Clinton (1992), & Eaton (1993) cormties 34 Table 7. G values generated from the analysis of homogeneity in the number of individuals carrying each allele, by loci, for each population (df = 1) and combined populations (df = 3) of common pheasants collected for genetic analysis in southern Michigan. Locus Captive F rec-ranging Combined w/o release with release Sichuan Ring-necked Mixed Sichuan ACP 0.803 0.983 0.403 0.108 2.99 AP 0.312 1.622 7.551” 7.270” 33.51'” [DH-l 1.014 0.467 3.248 5.037' 19.54'” IDH-2 0.484 5.191' 7.576" 4.974' 36.45“" ACON-l 31.690" 6.084' 0.715 1.733 80.45'” ACON-2 1 1.604'” 79.137'” 8.277” 24.050'” 246.14'” 'P 5 0.05, " P 5 0.01, ’” P 5 0.005 Table 8. Genetic variability (1- SE) in 6 loci for populations of common pheasants collected for genetic analysis in southern Michigan. mean no. percentage of loci of alleles polymorphic mean heterozygosity Population per locus .95' .99" DCc unbiasedd Captive Sichuan 1.19 16.13 19.35 0.029 0.042 (0.07) (0.014) (0.019) Free-ranging Ring-necked 1.16 16.13 16.13 0.020 0.052 (0.07) (0.009) (0.022) Mixed Releases 1.19 19.35 19.35 0.023 0.066 (0.07) (0.01 1) (0.027) Sichuan Releases 1.16 12.90 16.13 0.019 0.035 (0.07) (0.009) (0.018) ‘tl'refi'equencyoftl'remostcomrrronalleleisO.95;"thefiequencyofthemostcormnonalleleiso.99;‘DC=directcount;‘unbiased estimateofNei(l978) 35 Table 9. Genotypic frequencies of common pheasants collected for genetic analysis from 4 populations in southern Michigan Captive pheasants were collected from 1990-1992, while free-ranging pheasants were collected from 1991-1993. Loci Genotype Captive Free-ranging w/o releases with releases Sichuan' Ring-necked” Mixedc Sichuan‘l ACP an 0.84 0.78 0.84 0.83 ab 0.11 0.10 0.13 0.13 bb 0.05 0.12 0.03 0.04 AP as 0.49 0.74 0.45 0.80 ab 0.31 0.06 0.13 0.11 bb 0.20 0.20 0.42 0.09 IDH-l a 0.74 0.64 0.58 0.85 ab 0.20 0.21 0.22 0.13 bb 0.06 0.15 0.20 0.02 IDH-2 an 0.97 1.00 0.85 1.00 ab 0.00 0.00 0.00 0.00 bb 0.03 0.00 0.15 0.00 ACON-l a 0.80 0.19 0.27 0.24 ab 0.17 0.14 0.21 0.20 bb 0.03 0.67 0.52 0.56 ACON-2 an 0.92 0.06 0.87 0.98 ab 0.05 0.1 1 0.02 0.02 bb 0.03 0.83 0.1 l 0.00 ' samples fi’om MDNR’s captive breeding stock of Sichuan pheasants at the Mason Wildlife Facility (1990.92) " samples from Hillsdale (1991-93) and Montcalm (1991) counties ‘ samples from Huron Corny (1991-93) ° samples from Livingston (1991-93), Jackson (1991), Ingham (1991), Washtenaw(l991), Ban-y (1992-93), Clinton (1992), & Eaton (1993) courlies 36 Table 10. G values, (df = 1), generated fiom testing for homogeneity between the observed number of genotypes and the Hardy-Weinberg expected number of genotypes, for the 4 populations of common pheasants collected in southern Michigan for genetic analysis. Locus Captive Free-ranging w/o releases with releases Sichuan Ring-necked Mixed Sichuan ACP 1.36 1615'" 0.80 0.99 AP 3.45 28.38.” 36.00'” 9.49'” IDH-1 1.16 8.67" 14.12'” -1.04 IDH-2 1 .97 fixed 51 . 10'” fixed ACON-1 -0.90 17.37'" 19.61 ... 14.54‘” ACON-2 -0.74 4.81 35.00'” -0.01 ' P5005.“ P_<_o.01, '"PS0.001;for1-tailtest Table 1 1. Summary of hierarchial F -statistics (Wright 1965, 1978) at all polymorphic loci in southern Michigan common pheasants. Significance levels are associated with Chi-square tests of ( 1) H,: FB = 0, and (2) Ho: Fgr = 0', ' P < 0.05, ” P <0.01. Locus FE: Fr, Pg,” ACP 0.001 0.001 0.000 AP 0079 0.069 0.137" IDH-1 0048 0.038 0.082“ IDH-2 -0.046 0.080 0.120" ACON-1 0151‘ 0.236 0.336” ACON-2 -0.268" 0.651 0.718" Combined 0114 0.218 0.298" ' Chi-square a FB’N(k-l), df = [k(k-1)]/2 (Waples 1987), whereN is the total number of individuals sampled from populations polymorphic for thelocubemgtenedarsdkisthemmberofallelesatthatlocm ’Chi-square= 2NF,.(k-l).df=(k-1)(s-1)(Waplesl987),whereNandkaredefimduabove,mdsisthemberofpoptdstiom polymorphicforthelocusbeingtested. 37 Genetic Distance Genetic distances between the 4 populations of common pheasants in southern Michigan were small. Rogers (1972) (DR) and Roger’s distance as modified by Wright (1978) (Dw) (Table 12), along with Nei’s (1978) unbiased genetic distance (DN) (Table 13) are presented for comparison to other studies of this type. Average distances ranged from 0.019 (DN, 0006-0038, n = 6 comparisons) to 0.132 (Dw, 0081-0189, n = 6). Wright’s modification of Rogers (1972) distance resulted in a larger estimate. Cavalli- Sforza and Edwards distances (Dc = 0.101, 0076-0137, n = 6) (Table 13) were intermediate. In all cases, individuals from Captive Sichuan and free-ranging ring-necked populations were identified as having the greatest genetic distance. Free-ranging Sichuan and Mixed Release populations were intermediate. The branching diagrams for all distance measures indicate similar patterns regardless of the genetic distance index used (Figs. 5-9). The distance Wagner tree, generated from Roger’s (1972) distance as modifed by Wright (1978) (Fig. 9) showed the best fit (F = 0.917, f... = 0.998) compared to the other diagrams. Although the branching diagram based on Nei’s (1978) unbiased genetic distance (Fig. 7) had a high F value (F = 15.313), the cophenetic correlation indicates the diagram represents the original distance matrix (I... = 0.921). Relative distances between populations generated from the first 2 principle coordinates (Fig. 10) indicates a pattern similar to those seen in the UPGMA branching diagrams using genetic distances. 38 Distance .10 .08 .07 .05 .03 .02 .00 4 4 4 4 4 4 4 4 4 4 4 4 4 aaaeaeaaetaaaaaaaaa cgprzvg granny" ttfiitttttttt a a aaaaaaaaaeaeasaea gzcgufiu 3333‘s; a it a asaaaaeaeaaeaaaae “1x39 333333; a are.eaaaaaaaaaaaeaaateetaaetaa RING-NICKID 4 4 A. A A L A J. J. T T T T T T T .10 .00 .07 .05 .03 .02 .00 4 4 4 4 Figure 5. UPGMA branching diagram based on Rogers (1972) genetic distance (DR) representing the variation between the 4 populations of common pheasants found in southern Michigan, F = 9.306, r,c = 0.878. Table 12. Genetic distance matrix summarizing the genetic variance found in 4 populations of common pheasants in southern Michigan. Above the diagonal Rogers (1972) genetic distances (DR )', below the diagonal are Rogers (1972) genetic distances as modified by Wright (1978) (Dw). Population 1 2 3 4 l Captive Sichuan --- 0.057 0.032 0.029 2 Ring-necked 0.189 --- 0.046 0.041 3 Mixed Release 0.101 0.150 --- 0.028 4 Sichuan Release 0.107 0.161 0.081 «- Distance .20 .17 .13 .10 .07 .03 .oo +----+----+----+----+----+- 4 4 4 4 4 4 4 cease.aaaaaaaaaaaeaasaaaaaastate cgprxv; gxcgugns asassaaacessaaaaaeee e a aaaaaaaaeaaaaaaaaaaeaaaaa 31c3u33 333333; a aaaeaaae a eaaaaaaaaaaaeaaaaaaaaaaaa “Iggy gnggsg a eeaaaaaaaeeeateaeaeaeeaaaaaaaaaaaaaaaaasaeaaestates RING-NBCKID J. .L A A. A. A A. A. J. L J. T T T T T T T T T T T 4 ‘L T .20 .17 .13 .10 .07 .03 .00 Figure 6. UPGMA branching diagram based on Rogers (1972) distance as modified by Wright (1978) (Dw) representing the variation between the 4 populations of common pheasants in southern Michigan, F = 6.405, r,c = 0.950. 39 DlltIDCB .10 .00 .07 .05 .03 .02 .00 racist-s CAPTIVI arcnuau ittfiiitttti. * t s.... arcauan nsnsass * i... * ..... urxln assess; . aaaaaaaaaaaaaaaaeaa BIRD-NICKID .10 .00 .07 .05 .03 .02 .00 Figure 7. UPGMA branching diagrams based on Nei (1978) unbiased genetic distance (DN) representing the variation between the 4 populations of common pheasants found in southern Michigan, F = 15.313, rac = 0.921. Table 13. Genetic distance matrix summarizing the genetic variance found in common pheasants in southern Michigan. Above the diagonal are Neis (1978) unbiased genetic distances (DN); below the diagonal are Cavalli-Sforza and Edwards (1967) chord distances (DC). Population 1 2 3 4 1 Captive Sichuan m 0.038 0.010 0.011 2 Ring-necked 0.137 --- 0.023 0.027 3 Mixed Release 0.075 0.1 13 --- 0.006 4 Sichuan Release 0.079 0.125 0.076 --- Distance .20 .17 .13 .10 .07 .03 .00 4 J. J. .L J. J. T T T T T 4 4 4 4 4 4 4 aaaaeaaaaaaaeaaaaaaaaaaa CAPTIVI SICHUAN a ti...iiftifit.tflttiliiiiitittiiiifiitlt exam ”I“: i i i .Qittititttfltiiitfititifit mm m I it...Qittit.ittfiitifiiiitiitifiiiifiitfit. RING-m .L A _L. .I. A .L T T T T T T T .20 .17 .13 .10 .07 .03 .00 + I I I I + I I I I + I I I I + I I 41- '41- 4. Figure 8. UPGMA branching diagram based on Cavalli-Sforza and Edwards (1967) chord distance (Dc) representing the variation between the 4 populations of common pheasants in southern Michigan, F = 4.503, r,c = 0.960. 40 Distance from root: .00 .02 .03 .05 .06 .08 .09 J. .I. J. J. J. J. J. J. .1. A .L A J. T T T T T T T T T aaaeaaeeaeaaeaeeeaaeeaaaaaaaataeaaeaaaeae cgp¢1v3 gzcaugu teases aaaaaaaaeaeaaaaa aaaaaeaaaaaeaeaeeaaaaaaeaaaaaaaaaaaaaaaaa 31c3u5n 333333; a a a ataaerateaaaeaaseaaaaaaaeaaaaaaaaaaeeaaeaaaaaa “1x39 RILIABI a asaaaseaeaaaaaaasaaeeaaatasteseasaaaaaaaaaaaaeaaaeeaeaeastate RING-NICKID J. .L J. J. J. J. J. .L J. A T T T T T T T T T T J. A J. T T T .00 .02 .03 .05 .06 .00 .09 Figure 9. Distance Wagner tree showing the association of 4 populations of common pheasants in southern Michigan generated from Rogers (1972) distance as modifed by Wright (1978). Total length of tree = 0.263, F = 0.917, r,c = 0.998. PRINCIPLI COMPONINT 2 0.10 + CAPTIVI SICHUAN l l I l I l l I 0.05 + I l l l I RING-NICKID I I a 0.00 4 4 l I I MIXID RILIABI l I * I -0.05 +SICEUIN RILIASI l I * I I I I I -0.10 + | I I 4 4 ------- + ----------- + ---------- 4 4 4 -0.10 -0.05 0.00 0.05 0.10 0.15 0.20 Figure 10. Relative genetic distances of the 4 populations of common pheasants found in southern Michigan, plotted by the first 2 principle coordinates. Distance measure used was Rogers (1972) distance as modified by Wright (1978). 41 Neck ring width and percent closure data of sarnpled males are presented in Table 14. Captive Sichuans did not possess a neck ring. Neck rings of hybrid males were intermediate between the races and ranged from white spots to a narrow white ring. Although neck ring width was the greatest in the ring-necked population, it was not significantly difi‘erent fiom the Mixed (Mann-Whitney U test, 2 = 0.3328, P = 0.7393) or the Sichuan release (2 = -0.9784, P = 0.3279) populations. Closure of the neck ring was similar between the ring-necked and Mixed populations (2 = 0.2835, P = 0.7768). However, neck ring closure in the Pure Sichuan Release population was significantly more open than the ring-necked (z = 3.9337, P = 0.001) and Mixed release (2 = 3.8291, P = 0.001) populations. UPGMA clustering using standardized neck ring width and closure separated the captive Sichuan population from the others (Fig. 11). Separation remained using UPGMA clustering on morphological, allelic, and genotypic data resulted in a pattern similar to that seen with the genetic distance meaures where Captive Sichuan and release populations clustered together (Fig. 12). 42 Table 14. Average neck ring width 1 SE and percent neck ring closure of males harvested in the fall for genetic analysis from 4 populations of pheasants in southern Michigan. Neck ring Captive Free-Ranging w/o releases w/releases Sichuan‘ _ . . Ring-necked” Mixedc Srchuan‘l % of indivs. showing a 0 100 100 95 ring width (mm) NA 20.83 18.87 18.94 1- SE 1.0 0.07 1.1 n 40 47 39 % closure NA .90 0.92 0.80 1 SE 0.7 1.0 0.0 n 49 52 39 ' sanples from MDNR's carnive breedingstock ofSichuan pheasarns stthe Mason Wildlife Facility(1990-92)(n = 30) “ samples from Hillsdale (1991-93) and Montca1m(l991) couraies ‘ samples from Huron county (1991-93) ‘ sanmles from livingston(1991-92), Jackson (1991), lngham(1991), Washtenaw(l991), Barry (1992-93), Clir:on(1992) and Eaton (1993) counties NA = not applicable Average Instance Between Clusters 1.0 0.9 0.0 0.0 0.6 0.5 0.4 0.3 0.2 0.1 0 4. ..... 4... ¢ ¢ ¢ ¢ ¢ ¢ ¢ -_-+ ..... + ifitfitifiiitfiitttitiiiiifiitittiittiitttififlfittfiitttttiiiiitii * ** KIXID name: i t if. t i i t i t in cream name i t 0 aaeaaaaaaasassaaasaaaaasaaaaaaaaaaaaaaaaaseasaaaaaaaaaaaaass CAPT!“ 31m Figtu'e 1 l. UPGMA cluster analysis using standardized morphological data from the 4 populations of common pheasants in southern Michigan. 43 Average Distance Between Clusters 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 + ------ + ------ +---- 4 4 4 4 4 4 4 asaasaaaaaaaaaaaaeaaaaasaaaaaeaaaa cgprrv; SICHUAN a assasasaaaaaaaaa a a a aaaaaaasseassassaaeeaaaaeaaaaaaaea “Iggy gnghgg a aaaasaaaaasaaaas a aaaastatsasaassessaacetateaaaaaasaaaaaaaaaaaaaaas gzcgugn gnghsg i i i i i t it.flitiiittitiit.i.tit*ititfifittttiitittfififittiittifitttitt**i*i*t RING-“ICKID Figure 12. Cluster analysis, using UPGMA methodology on standardized morphological, allelic, and genotypic frequencies for the 4 populations of common pheasants in southern Michigan. DISCUSSION Levels of genetic heterogeneity and differentiation were estimated between populations of common pheasants in southern Michigan that areSichuan, ring-necked and a mixture of Sichuan and ring-necked hybrids. The free-ranging ring-necked population represents the pheasant gene pool that remains in Michigan from more than 40 years of releases by the Michigan DNR (1919 to 1950's). Also included in this gene pool were continuous, unmonitored introductions by release and/or escape of game farm ling-necked pheasants each year by private individuals and organizations. The flee-ranging Sichuan gene pool was from the captive breeding population of Sichuan pheasants. Electrophoretic screening of samples of individuals provided allele frequencies, from which estimates of genetic structure could be made, which are concordant with those based on demographic modeling (Barrowclough 1980a). The biochemical methods also yielded information on the magnitude and distribution of genetic heterozygosity and polymorphism (Barrowclough 1983). 44 Genetic Heterogeneity Little difference was noted in the levels of polymorphism in the Captive Sichuan and free-ranging populations. However, the polymorphism is an imprecise measure of genetic variability as a slightly polymorphic locus has as much weight as a very widely polymorphic one, and the accuracy of this estimate depends on the number of loci examined (minimum of 14, 20 recommended) and the number of individuals (minimum of 30, recommended 100) (Evans 1987). This survey, as well as those of other wild galliformes (Gutierrez et al. 1983, Warner et al. 1988, Scribner et al. 1989) often survey more than the suggested number of loci, but fail to survey the recommended number of individuals. Probably the most widespread measure of genetic variation used to describe genetic variability in populations is the level of heterozygosity (Evans 1987). Although average observed heterozygosity (Ho...) in the ring-necked and captive Sichuan populations in this study were similar (2% and 2.9%, respectively), both were slightly lower than values reported in the literature for other avian species (Appendix C). Average observed variability in five other Phasianus studies was 4.1%. Within the subfamily Phasianinae, values averaged 4.8% (0-8.3%, n = 18 studies). Many avian surveys typically have very low levels of within species genetic variation. (Barrowclough 1980a). The deficiency of heterozygotes in common pheasant populations in Michigan is also reflected in the negative values observed for the inbreeding coemcient, F13. Negative Fls values are thought to indicate a general avoidance of matings with relatives. Values of FIS in this study averaged -0.114 (-0.238 to 0.001) and were similar to those found in wintering populations of brant (Branta berm’cla hrota) ( -0. 126 to -0.012) (N ovak et al. 45 1989), and Florida wood storks (Mycteria americana) (-0.091) (Stangel et al. 1990). Generally, avian populations show a tendency toward matings with relatives (Table 15). The majority of the loci in the free-ranging pheasant populations in Michigan were not in Hardy-Weinberg equilibrium, while all loci in the Captive Sichuans were in equilibrium. However, there were three loci, (ACP, IDH-1, and ACON-2) in equilibrium in the Sichuan Release population, compared with only one loci (ACP) in the Mixed Release population and none in the non-release ring-necked population. It is possible that the pure releases had positively influenced levels of heterozygosity in the free-ranging Table 15. A summary of literature Fuvalues in the Class Aves. Common Name Scientific Name F18 Source yellow-rumped warbler Dendroica coronata 0.08 Barrowclough 1980b northern oriole Icterus galbula 0.312 Corbin et al. 1979 piping plover Charadrius melodus 0.049 Haig and Oring 1988 brant Branta bemicla hrota -0.126 to -0.012 Novak et al. 1989 American wigeon Anas americana 0.046 Rhodes et al. 1993 starling Strunus vulgaris 0.040 Ross 1983 willow flycatcher Empidonax traillii -0.025 Seutin and Simon 1988 alder flycatcher E. alnorum 0.063 Seutin and Simon 1988 white headed gulls Lam: spp. 0.081 Snell 1991 Florida wood stork Mycteria americana -0.09l Stangel et a1. 1990 California quail Callipepla californica 0.130 Zink et al. 1987 ring-necked pheasant Phasianus colchicus 0.130 Scribner et al. 1989 ring-necked pheasant P. colchicus -0.1 14 This study 46 populations, and pure Sichuan releases had the greatest impact since they were in Hardy- Weinberg equilibrium. In a non-equilibrium population, the observed heterozygosity may not accurately reflect the amount of genetic variation in the population, and to deal with this problem, it is advised that the expected heterozygosity be calculated (Evans 1987). The difl‘erence between observed and expected heterozygosity was 2 times greater for populations not in equilibrium compared with those at or near equilibrium. Average expected heterozygosity in common pheasants in southern Michigan (0.035-0.066) was similar to values reported within the Subfamily Phasinidae (Hexp = 0.048 j; 0.02, n = 2) (Appendix C). The low proportion of heterozygotes found in common pheasants in Michigan may have resulted from a variety of factors including assortative mating, small population sizes, and/or biased sampling. The pheasant is a polygynous species and males will mate with a limited number of females within a defined tenitory (Taber 1949). Research in Michigan indicates that average summer home range size of Sichuan and ring-necked males released in Livingston County, Michigan, ranged from 67.4 to 100.2 ha, respectively (Campa 1989). Aggressive behavior between males of both races was observed in the field (Campa et al. 1987) suggesting an overlap of territories. Grahn et al. (1993) estimated harem size at 1.3 i 1.0 SD hens per day for pheasants in Sweden under controlled conditions and DNA fingerprinting revealed that males sired an average of 6.0 i 1.01 SD chicks from a total of 17 broods (approximately 102 chicks). The lack of males in the fall harvest without neck-rings in this study suggests almost no breeding between pure free- ranging Sichuan males and females. It is likely that hens had the opportunity to select between males in the field. 47 Allele frequencies under complete positive assortative mating will not change from generation to generation, but the genotypic proportions will change considerably (Li 1955). If H, is the proportion of heterozygotes in a population at time n, and p is the frequency of the A allele (which will be the same for both parental and ofi’spring; see Li (1955) pg 233), then heterozygotes in the next generation (HM) under complete positive assortative mating will be reduced by: H, ,1 = [2pH,] / [2p + 11,] (Li 195 5). If assortative mating continues for n generations, and H, and p are the values from the parental generation, then: H, = [2pH,] / [2p + nH,] and H, approaches zero as it increases. It would take 0.6 to 4.4 generations to achieve the 0.1% reduction in observed heterozygosity based on the average observed heterozygosity (Hm) estimates from the ring-necked (H, = 0.020) and Sichuan Release (H, = 0.019) populations. Estimates range fi'om 2.2 to 15.4 generations if expected heterozygosity (171”) is used. These estimates assume that the existing remnant ring-necked population within the Sichuan release population was substantial and genetically similar to our sampled ring-necked population, and complete positive assortative mating occurred between existing and released birds. The lower observed heterozygosity estimates of free-ranging populations in this study (0.019 to 0.023) may also depend on neighborhood size. If population dispersion is unifomr, neighborhood size (N,) can be estimated by N, = 4 1:602 where 0 is the number of breeding individuals/unit area, and o2 is the amount of dispersion between an individual’s birth place and that of its offspring. Luukkoneon (1991) estimated wintering population densities of pheasants at 16.9 birds/ km2 in Livingston County (1574 pheasants / township). Dispersion estimates between a breeding individual’s birth place and that of 48 its 033ng are not available for pheasants, however they can be approximated by using the distances females disperse from release to nesting sites and between nesting attempts (mean = 3.9 km) (Prince et al. 1986, Campa et al. 1987, Rabe et al. 1988, Campa 1989) . Substituting the winter population estimates for 0 and dispersal distances for 02, results in a neighborhood size for pheasants in Livingston County of 3234 pheasants (41: * 16.9 birds/km2 * 15.2 kmz). prheasant densities remained at 16.9 birds/km2 throughout the sampling period, an area equivalent to 2 townships should have been sampled. Since 4 townships were sampled in Livingston County, the possibility of sampling more than one neighborhood exists. This could lead to disequilibrium condition if allele differences existed between neighborhoods. The deficiency in heterozygosity, resulting from combining within a single sample, individuals fiom more than one genetically distinct population is known as the Wahlund efl‘ect (W ahlund 1928) and the outcome is similar to inbreeding (Li 1955). The Wahlund effect, for a particular allele at some locus, is the expected deficiency of heterozygotes in a non-interbreeding mixture of two populations. It is expressed as: Hog, - H“, = -2f,f2 (pl - p92, where H“, and H“, are the observed and expected fi'equencies of heterozygotes, f, and f2 are the proportional contributions of populations 1 and 2 to some mixture (f, +f2 = 1), and p1 and p22 are the frequencies of the alleles in populations 1 and 2, respectively. The difl‘erence between observed and expected heterozygosity will be 5 zero and will equal zero only if allele frequencies in the two populations are identical (pl = p2). The difference is maximized when the two populations present in the mixture are in equal proportions (fl =f2 = 0.5) (Ryman and Utter 1987). If local populations with different allelic frequencies are sampled, then the mixture of individuals fi'om the various 49 populations could result in an apparent overall excess of homozygotes even if each local population is in equilibrium. The effect of assortative mating and small neighborhood size on levels of heterozygosity in Michigan pheasants may have overridden the selective benefit of heterozygosity Niewoonder (1995) documented in the survival of hybrid females. Sichuan x ring-necked hybrid females in southern Michigan had slightly higher survival and produced 2 to 4 times more chicks/hen/ season compared with Sichuan and ring-necked females, respectively. Heterosis in F1 crosses is possible if allele frequencies difl‘er between the crossed lines, however hybrid vigor is expected to halve in the F 2 progeny (Falconer 1989). Population Differentiation Gene flow from the captive Sichuan into the release populations appeared to be substantial as evidenced by the genetic identity measures. In all indices, the release populations were intermediate to the pure Captive Sichaun and free-ranging ring-necked populations. This is supported by other evidence that assortative mating between ring- necked and Sichuan pheasant is not complete. Prince et a1. (1991) found evidence of positive assortative mating when wild-trapped Michigan ring-necked hens were given the opportunity to select between ring-necked and Sichuan males under controlled conditions. Ring-necked hens mated more frequently with ring-necked (80%) compared with Sichaun males (20%). In a reciprocal experiment, Sichuan females mated with ring-necked and Sichuan males at a similar frequency (48% and 52%, respectively). The biochemical markers were more usefill in distinguishing the various populations than the morphological marker. Pure Sichuans had no ring, but both the pure SO and mixed flee-ranging populations had rings similar to the ring-necked population. Plumage patterns are the product of regulatory genes and may not mirror patterns found in structural genes that code for proteins. Pleiotropy or complex genetic-environmental interactions during ontogeny are reduced by biochemical methods. Therefore, biochemical characters may be “cleaner” than are phenotypic ones (Barrowclough 1983). Difl‘erences between branching diagrams based on genetic distances to those based on phenotypic variables suggest caution in the interpretation of the relationship between groups based on phenotypic characters alone. The utility of phenological traits in difierentiating species and subspecies has been highly variable. Barrowclough (1980b) did find sumciently strong phenotypic difi‘erentiation within a zone of hybridization between Denrocia c. coronata and D. c. auduboni where the two forms were originally thought to be separate species. However, his analysis of the genetic data indicated no difl‘erentiation among populations (DN = 0.006 1 0.002). Johnson and Marten (1992) found considerable concordance of morphometric (body size) and genetic patterns in a subspecies of sage sparrow (Amphispiza belli). However, Lougheed and Handford (1992) found no discernable relationship between the pattern of trill rate variation (dialects) and genetic population structure in rufous-collared sparrows (Zonotrichica capensis) in northwestern Argentina, and link (1982) measured 40 skeletal characters in the rufous-collared sparrow and 4 other congeners and found genetic divergence without concomitant morphological change. While documenting patterns of phenotypic variation is useful because they may provide general indications of evolutionary trends, genic evolution can occur without concomitant morphological change (Gorman and Kim 1977, Highton and Larson 1979), and organisms with different 51 morphologies may be even be genetically similar (Avise et al. 1975, King and Wilson 1975, Yang and Patton 1981). While the biochemical markers were more useful than size of the neck-ring in distinguishing the populations of pheasants in Michigan, levels of differentiation were much lower than most other animal species. They were, however, in the range generally found for avian species. Literature values of Nei’s unbiased genetic distance for between subspecies and between local populations averaged 0.013 1 0.016 (n = 10) and 0.002 1 0.016 (n = 6), respectively (Appendix D). The difl‘erences for Michigan pheasants ranged from 0.006 to 0.038. The values of DN measured in this study were more similar to those reported for comparisons between avian subspecies than for local populations of the same species (Fig. 13). In spite of their high levels of genetic identity, the populations of common pheasants in Michigan showed substantial differentiation using Wright’s (1965, 1978) hierarchical F-statistics. Wright (197 8) defines F ST as the correlation between alleles of gametes sampled at random from two subdivisions of a population, with the distribution of alleles within the entire population sampled. Therefore, F ST reflects the extent of local difl‘erentiation into subpopulations or demos and is always positive. Wright (1978) described four ordinal levels of F ST, 1) little genetic difi‘erentiation (F ST = 0 to 0.05), 2) moderate (0.05 - 0.15), 3) great (0.15 - 0.25), and 4) very great (> 0.25). The average FST of 0.298 (0.000 to 0.718)in the common pheasants of Michigan indicates a large amount of differentiation between populations. Distributions of interpopulational values of FST have been summarized for several groups of organisms (Barrowclough 1983, Corbin 1983, 1987). For vertebrates, 52 interdemic FST values range between 0.0 to 0.91, with the largest values being found among salamander populations. The value of FST for populations of common pheasants in southern Michigan is 10 fold greater than that previously described among avian populations where the largest interdemic F ST values have been reported between 0.029 to 0.039 (Corbin 1987). In other pheasant work, Scribner et al. (1989) found in Texas Panhandle populations (n = 10) derived from P c. bianchi, P. c. torquatus, and P. c. colchicus, that 91% of the genetic variance was found within populations (F ST = 0.086). The patchy distribution of playa basin habitat in the Texas Panhandle, coupled with large interplaya distances were suggested causes for spatial structuring over a short post-introduction period. The average F ST value for populations of common pheasants in Michigan (0.298) is 3 ‘/2 fold greater than that seen by Scribner et al. (1989). Differences in stocking history and population densities could account for part of this difference, as the heritage of pheasants in Texas was similar to that of Michigan’s current ring-necked population. Introductions of the Sichuan pheasant, a subspecies new to North America, contributed to the increase in difl‘erentiation seen in Michigan populations. Pheasant populations in Texas were considered quite large and averaged 40 birds/km2 (Guthery and Whiteside 1984), a density 2 '/2 that of Michigan populations (16.9 birds/kmz) (Luukkoneon 1991). The effect of genetic drift is greater in smaller populations and may result in a greater degree of differentiation between populations. .9 among... 80.... .35.... 855.8. 23 60.3868 8.8833 .5 828380.“ .20 .38.... 2862 88.2.88 E 33:52 82.82.. 2.25m .8839. 835 8 Z 2.. 82.5 .383 .0308 8.00385 52.33 new 393 8&3 88on 083. 05 80 8283808 .80. 803.3 8.2. 830 05 e0 menace. .08 8052.. 2.8% 3.530 80 TV momma. 2... Q 382 .2 cam... 53 ... .28 .833 a-.. .228 62.52. om2~tonEm . a. a... .23... are sea... as. 2.22.38."— . .222 .83... a-.. .395... 3:33.... 822.89....8 . 5:2 .95... a-.. gees. om2omao_oom . E :2 .83... «ta ego.e 3...... 322.898,...8 . 5 28 .833 N... es... .853. 2.252923 . LL. ta. LL. LL. LL. Ia. qnsuccmcg sounds as... a... .823 N-.. sass... 8.8.8. 82.8.3.3 .m a is a-.. 6:5. 895.33... om2EBEm .m a... .8 to. .2-.. cats. e893... 82:...zm .m m a." .22 ....t.e gamers... 82.84 .m m .8 a .2 .nue ..._a «.523. 3254 .m .wl .e .22 .2-.. decades. 822.8205 .8 m. .3 :2 are 3%. 32:28.5 .m We .2 .22 a-.. .3... 83:3. 82:28.3 .8 m. a :2 .ete .53. p.955. 8285. .m w. .. .22 f... ea..a=0a2§:< .m m an... .2 .2-.. 98.883 8.8.... 2.2.320 .m 48 .4.” 85.23 02.080 .0 8.0 No.8 8.8 _ . . . ...a..-.AJ...J.—...._....2...._.q.._.. .. n. 111. .T. e hill]. 54 Conclusions Repeated releases over many years were ofien necessary before pheasants established self-maintaining populations within what is recognized as the range of common pheasants in North America (Prince et al. 1988). Releases of the Sichuan pheasant, P. c. strauchi, into Michigan habitats not traditionally used by ring-necked pheasants, P. c. colchicus, were made over a 10 year period beginning in 1986. Starch electrophoresis was used to examine levels of genetic heterogeneity and the introgression of Sichuan genes into existing ring-necked pheasant populations from 1991-1993. Results indicate Sichuan pheasants readily crossed with ring-necks as evidenced by the high rate of gene flow into the free-ranging populations. It is likely that several neighborhoods were sampled for each free-ranging population generating a Hardy-Weinberg disequilibrium condition that resulted in an excess in homozygotes, or Wahlund effect. Under such conditions, the expected average heterozygosity may be a more appropriate measure of genetic variation than the observed average heterozygosity. Southern Michigan populations of pheasants currently show low levels of genetic heterogeneity. However, the expected average heterozygosity levels found in populations of pheasants from southern Michigan are similar to levels of other galliformes. This suggests that introduced pheasants in Michigan have experienced evolutionary forces similar to those of other populations of galliformes. The adaptation of genotypes to local environmental conditions is possible only if genetic heterogeneity exists. However, the persistence and maintenance of these levels will be dependent on the nature and extent of future evolutionary forces. APPENDIX A Electrophoretic Methodology and Techniques APPENDIX A Electrophoretic Methodology and Techniques Sample Preparation Liver tissue was homogenized 1-2 days prior to the electrophoretic rtm. Grinding procedures were as follows: On the day prior to minding: prepare tissue grinding buffer (see below) place ceramic mortars on ice and leave in ultra—cold freezer overnight On the day of grinding: place liver samples on ice place tissue grinding buffer on ice place 4 mg of liver tissue in grinding well (avoid adipose and connective tissue) add 34 drops of tissue grinding bufl'er homogenize tissue with pestle place a 40 micron screen over homogenized tissue place paper wicks on top of screen an allow them absorb the supernatant place ELIZA trays on ice fill each well of the ELIZA tray with one wick double wrap the ELIZA tray with plastic label and store ELIZA tray with wicks in the ultra cold fi‘eezer Tissue Grinding Bufler W 1.21 g Tris 50 ml distilled H10 adjust pH w/ 4M HCL bring to volume with distilled H20 store at 34 C Starch Gels 6 n_1n_) horizontal gel 10 mm horizontal gel 22 g potato starch 33 g potato starch 5 ml electrode bufl‘er 8 ml electrode buffer 195 ml distilled H20 292 ml distilled H20 heat above solution in a side arm flask over an open flame until boiling deaerate solution, pour liquid gel solution into gel tray, remove any air bubbles afier gel has cooled, cover with plastic wrap to prevent desiccation store at room temperatme overnight cool gel at 4 C for 1 how prior to setting wicks 55 56 Electrode Bufl'er System and Running Conditions Mgmholm' e Citrate (Clayton and Tretiak, 1972) ACON, AP, and IDH at pH 6.1 AP at pH 5.2 1 liter stock solution of Morpholine Citrate 8.4 g/l citric acid, monohydrate 500 ml distilled H20 store at 4 C overnight pH with N-(3-amino propyl) morphline bring to volume with distilled H20 store at 4 C 6 mm gels: run at 30 mAMPS (approx. 190 volts) for the first hour, 35-40 mAMPS (approx. 210 volts) for the remaining 5 hours 10 mm gels: nm at 55 mAMPS (240 volts) for the first hour, 55-65 mAMPS(approx. 250 volts) for the remaining 5 hours Histochemical Enzyme Specific Stains Used in This Study ACOE (Richardson et al. 1986) EH (Richardson et al. 1986) 100 ml 0.1 M Tris-HCL pH 8.0 95 ml 0.1M Tris-HCL pH 8.0 1 ml cis-aconitic acid, pH 8.0 8 ml 1.0 M MgCl2 5 ml 0.1M MgCl2 25 mg NADP 15 mg NADP 30 mg MTT 15 mg MT T 2 mg PMS 2 mg PMS 60 mg isocitrate dehydrogenase ACE (Richardson et al. 1986) AB (Richardson et al. 1986) 100 ml 0.1M tris-HCl, pH 8.6 100 ml 0.05 M Na-acetate, pH 5.0 100 mg B—napthyl acid phosphate 100 mg a-napthyl acid phosphate 100 mg Fast Blue RR salt 100 mg Fast Garnet GBC salt 6 ml 0.1 M MgCl2 Stains were allowed to develop at room temperature in the dark. Staining time varied with enzyme, but ranged from 15 to 60 minutes. H istochemical Enzyme Stain Buffers 7.4 g trisma base 0.68 g sodium acetate 6.1 g trizma Hcl 100 ml distilled H20 100 ml distilled H20 adjust pH with 1N HCl adjust pH w/ lN HCl cis-aconiti acid H 8.0 W, 50 mg cis-aconitic acid 9.5 g magnesium chloride 100 ml distilled H20 100 ml distilled H20 adjust pH w/ 4M NaOH 57 Fixing Gels Staining process was halted using 1% acetic acid Gels were allowed to sit an fix in 50% ETOH for 1 hour Gels were placed in ziplock bags for final storage Chemical List (Sigma Chemical Co. 1995) WWW A-6283 A-3412 A-9028 C-71 29 285-8 F 0500 F-8761 H-7020 H 877 M- 1028 M-21 28 N-7000 N-7375 N-95 1 l P-9625 S-87 50 S-5881 T-1503 T-3253 acetic acid, glacial cis-aconitic acid N-(3-amino propyl) morpholine citric acid, monohydrate ethanol fixative (ETOH) Fast Blue RR salt Fast Garnet GBC salt, sulfate salt hydrochloric acid (HCl) isocitrate dehydrogenase magnesium chloride (MgClz) 3-[4,5—dimethylthiazol-2-yl]-2,5-diaphenyletrazolium bromide, (MTT); thiazolyl blue a-napthyl acid phosphate, monosodium salt fl-napthyl acid phosphate, monosodium salt B-nicotinamide adenine dinucleotide phosphate (NADP) phenazine methosulfate (PMS); N-methyldibenzopyrazine methyl suflate salt sodium acetate, anhydrous sodium hydroxide (N aOI-I) trizma base trim-hydrochloride (tris-HCI) Electrode Buffers used in the Screening Process A. Morpholine-citrate (Clayton and Tretiak 1972) pH 6.1 or 5.2 0.04 M citric acid - monohydrate adjust pH using N-(3-aminopropyl) morpholine Gel: 1:19 parts, electrode bufler to distilled H20 B. Lithium-berate (Scandolis 1969) pH 8.3 0.19 M boric acid 0.04M lithium hydroxide adjust pH with dry ingredients Gel: 1:10 parts, electrode buffer to 0.05 M tris - 0.007 M citric acid, pH 8.3 C. Tris-citrate (Meizel and Markert 1967) pH 7.0 0.155 M tris 0.043 M citric acid - monohydrate adjust pH with dry ingredients Gel: 1:14 parts, electrode buffer to distilled H20 58 D. Tris-citrate (Shaw and Prasad 1970) pH 8.0 0.687 tris 0.157 M citric acid - monohydrate adjust pH with dry ingredients Gel: 0.023 M tris , 0.001 M citric acid, monohydrate, distilled H20 pH with dry ingredients E. Tris-versene-borate (Selander et al. 1971) pH 8.0 0.5 M tris 0.5 M boric acid 0.016 M Na2EDTA adjust pH with dry ingredients Gel: 1 :10 parts, electrode bufl'er to distilled H20 F. Sodium borate (Poulik 1957) pH 8.0 0.30 M boric acid adjust pH with NaOH Gel: 0.076 M tris, pH with citric acid G. Phosphate (Richardson et a1. 1986) pH 7.0 1 1.6 mM Na,HPO., anhydrous 8.4 mM NaHzPO. Gel: 1:10 parts, electrode buffer to distilled H20 H. Phosphate-citrate (Shaw and Prasad 1970) pH 8.0 0.214 M K,HPO. 0.027 M citric acid, monohydrate pH with dry ingredients Gel: 1.16 mM K,HPO., 0.2 mM citric acid, monohydrate, distilled H20 1. Tris-maleate-EDTA-MgCl2 (Richardson et a1. 1986) pH 7.8 0.05 M tris 1 mM NaEDTA 1 mM MgCl2 20 mM maleic acid Gel: 1 :10 parts, electrode buffer to distilled H20 .1. Sodium-borate (Ayala et al. 1972) pH 8.65 0.3 M boric acid 0.06 M NaOH Gel: 0.076 M tris, 0.005 M citric acid, distilled H20 K Tris-borate (Shaw and Prasad 1970) pH 7.5 0.0546 M tris 0.2354 boric acid Gel: 0.198 mM tris, 5.5 mM boric acid L. 0.5 M Phosphate (Shaw and Prasad 1970) pH 7.0 87.0 g/l K,HPO. 68.0 g/l KH1p04 Gel: l :10 parts, electrode bufi‘er to distilled H20 M. Tris-maleate (Richardson et al. 1986) pH 7.8 50 mM trizrna 20 mM maleic acid Gel: 1:10 parts, electrode buffer to distilled H20 N. Tris-EDTA-borate—MgCl2 (Richardson et al. 1986) pH 7.8 0.015 M tris 0.005 M Na2EDTA 0.01 M MgCl2 0.05 M boric acid Gel: 1:10 parts, electrode buffer to distilled H20 0. Citrate-phosphate (Richardson et al. 1986) pH 6.4 2.5 mM citric acid 10 mM NaQHPO. Gel: 1 :10 parts, electrode bufi'er to distilled H20 60 H istochemical Stains used in the Screening Process ACON - aconitase hydratase - E.C. # 4.2.1.3 (Richardson et al. 1986) 0.1Mtris-HCL,pH8.0 100ml cis-aconitic acid, pH 8.0 (50 mg/ml) 1 ml NADP 25 mg MgCl, 8 mg MIT 30 mg PMS 2 mg isocitrate dehydrogenase 40 units ALD - aldolase - E.C. # 4.1.2.13 (Wendel and Wwden 1989, Richardson et al. 1986) also known as fructose-bisphosphate aldolase (FBA) 0.1 M tris-HCL ph 7.4 100 ml arsenate Na salt (6 rug/ml) 75 mg fructose 1,6 diphosphate (Na,) 200 mg NAD 30 mg MTT 30 mg PMS 5 mg glyceraldehyde-3-phosphate dehydrogenase 100 units triose phosphate isomerase 100 units ADH - alcohol dehydrogenase - E.C. # 1.1.1.1 (Shaw and Prasad 1970, Richardson et al. 1986) 0.1 M tris-HCL pH 8.0 89 ml ethanol (95%) 6 ml 0.1 M NaCN 5 ml PMS 4 mg MTT 20 mg NAD 20 mg AP - alkaline phosphatase - E.C. # 3.1.3.1 (Ayala et al. 1972, Shaw and Prasad 1970, Richardson et al. 1986) 0.1 M tris-HCL pH 8.6 100 ml Fast Blue R or Fast Blue BB salts 100 mg B-napthyl acid phosphate 100 mg MgClz 60 mg MnCl2 60 mg AK - adenylate kinase - E.C. # 2.7.4.3 (Wendel and Weeden, 1989, Ayala et al. 1972, Richardson et al. 1986) 0.1 M tris-HCL, pH 8.0 100 ml glucose 90 mg MgCl2 20 mg NADP 30 mg ADP 40 mg PMS 4 mg MIT 30 mg glucose-6-phosphate dehydrogenase 80 units ACP - acid phosphatase - E.C. # 3.1.3.2 (Richardson et a1. 1986) 0.05 M Na-acetate, pH 5.0 100 ml a-Na-napthyl acid phosphate 100 mg Fast Garnet GBC salt 100 mg MgCl, 10 mg 61 AAT- aspartate aminotransferase - E.C. # 2.6.1.1 (O’Malley et al. 1980, Richardson et a1. 1986) Also known as glutamic oxaloacetate transaminase (GOT) 0.1 M tris-HCL pH 8.0 L-aspartic acid Fast Blue BB, Fast Garnet GBC, or Fast Violet B salt 100ml 200 mg 150 mg CK - creatine kinase - E.C. # 2.7.3.2 (Shaw and Prasad 1970, Richardson et al. 1986) 0.1 M tris-HCL pH 8.0 creatine phosphate glucose ADP MgCl2 NADP MT T PMS hexokinase glucose-6-phosphate dehydrogenase 100 ml 731 mg 90 mg 75 mg 21 mg 25 mg 20 mg 5 mg 160 units 80 units EST- esterase - E.C. # 3.1.1.1 (Wendel and Weeden 1989, Richardson et al. 1986) 0.1 M tris-maleate, pH 6.5 a or B-napthyl acetate (in 2 ml acetone) Fast Blue RR salt or Fast Garnet GBC salt 100ml 50mg 100mg FDP - fructose 1.6 diphosphate - E.C. # 3.1.3.11 (O’Malley et a1. 1980, Wendel and Weeden 1989, Richardson et a1. 1986) also known as fructose-bisphosphatase (FBP) 0.1 M tris-HCL pH 8.0 MgCl2 NADP MTT PMS fi'uctose 1.6 diphosphate phosphoglucose isomerase g1ucose-6-phosphate dehydrogenase 100 ml 100 mg 20 mg 20 mg 20 mg 120 units 80 units 80 units FUM- fumarase hydratase - E.C. # 4.2.1.2 (Wendel and Weeden 1989, Richardson et al. 1986) 0.1 M tris-HCL pH 8.0 NAD MTT PMS fumaric acid, Na salt malate dehydrogenase 100ml 20 mg 20mg 2mg 200 mg 200 units GDH - glutamate dehydrogenase - E.C. # 1.4.1.3 (Wendel and Wwden 1989, Richardson et al. 1986) 0.1 M tris-HCL pH 8.0 glutamate (glutamic acid) NAD MTT PMS 80 ml 100 mg 20 mg 10 mg 2 mg 62 aGPDH - a-glycerol-3-phosphate dehydrogenase - E.C. # 1.1.1.8 (Shaw and Prasad 1970, Richardson et a1. 1986) 0.1 M tris-HCL pH 7.0 0.1 M NaCN l M Na-a-glycerophosphate, pH 7.0 NAD MTT PMS 100ml 10ml 10ml 50mg 30 mg 2mg G3PDH - glyceraldehyde-3-phosphate dehydrogenase - E.C. # 1.2.1.12 (Ayalya et a1. 1972, Richardson et al. 1986) also known as triosephosphate dehydrogenase 0.1 M tris-HCL pH 8.0 NAD MTT PMS arsenic acid, Na salt aldolase glyceraldehyde-3-phosphate 100 ml 50 mg 30 mg 4 mg 150 mg 100 units 200 units G6PDH - glucose-6-phosphate dehydrogenase - E.C. # 1.1.1.49 (Richardson et a1. 1986) 0.1 M tris-HCL pH 8.0 NADP MgCl, MTT PMS D-glucose-6-phosphate 100 ml 20 mg 20 mg 20 mg 5 mg 200 units max - hexokinase - E.C. # 2.7.1.1 (Richardson et al. 1986) 0.1 M tris-HCL pH 8.6 NADP MgCl2 MTT PMS ATP D-glucose g1ucose-6-phosphate dehydrogenase 100 ml 20 mg 20 mg 20 mg 2 mg 6 mg 20 mg 10 units IDH - isocitrate dehydrogenase - E.C. # 1.1.1.42 (Wendel and Weeden 1989, Richardson et a1. 1986) 0.1 M tris-HCL pH 8.0 isocitric acid (N a3) MgCl2 NADP MTT PMS 100ml 100mg 5mg 15mg 15 mg 2mg LDH - lactate dehydrogenase - EC. # 1.1.1.27 (Selander et al. 1971, Richardson et a1. 1986) 0.1 M tris-HCL pH 8.0 0.5 M lithium DL lactate NAD MTT PMS 100ml 800 mg 40 mg 8mg 16mg 63 MDH - malate dehydrogenase - EC. 1.1.1.37 (Richardson et a1. 1986) 0.1 M tris-HCL pH 8.0 0.2 M Na-malate buffer, pH 8.0 NAD MTT PMS 0.2 M Na-malate buffer DL- malic acid NaOH adjust pH with 2 N NaOH 50 ml 50 ml 30 mg 20 mg 5 mg 26.88g/1 16 g/l MP1 - mannose phosphate isomerase - E.C. # 5.3.1.8 (Nichols and Ruddle 1973, Richardson et a1. 1986 ) 0.1 M tris-HCL pH 8.0 mannose-6-phosphate NADP MTT PMS MgCl2 glucose-6-phosphate isomerase g1ucose-6-phosphate dehydrogenase 100ml 34 mg 35 mg 35 mg 7 mg 40 mg 100 units 100 units ME-malicenzyme-E.C. # 1.1.1.40 (Ayalaetal. 1972, Richardsonetal. 1986) 0.1 M tris-HCL pH 7.4 MgCl2 NADP MTT PMS malic acid 100 ml 25 mg 25 mg 20 mg 5 mg 50 mg PGI - phosphoglucoisomerase - EC. 5.3.1.9 (Selander et a1. 1971, Richardson et a1. 1986) also known as g1uoose-6-phosphate isomerase (GPI) 0.1 M tris-HCL pH 8.0 MgCl, NADP MTT PMS disodiurn fructose-6-phosphate g1ucose-6-phosphate dehydrogenase 100 ml 20 mg 20 mg 20 mg 4 mg 50 mg 20 units PGM - phosphoglucomutase - E.C. # 2.7.5.1 (Wendel and Weeden 1989, Richardson eta1.1986) 0.1 M tris-HCL pH 8.0 MgCl2 NADP MTT PMS glucose-l-phosphate, Na2 salt glucose-6-phosphate dehydrogenase 100ml 10mg 10 mg 10 mg 2 mg 50 mg 40 units 64 6PGD - phophoglucconate dehydrogenase - EC. # 1.1.1.44 (Wendel and Weeden 1989, Richardson et a1. 1986) 0.1 M tris-HCL pH 8.0 MgCl2 NADP MTT PMS 6-phophogluconic acid (Na or Ba salt) PEP - peptidase - E.C. # 3.4.11 (Richardson et a1. 1986) 0.1 M tris-HCL pH 8.0 peroxidase o—dianisidine (di-HCL salt) peptide (see below) L-amino acid oxidase (snake venom) MgCl2 PEP - A = valine-leueine PEP - B = leucine-glucine-glucine PEP - C =1ysine-leucine PEP - D = phenylalanine-proline SDH - sorbitol dehydrogenase - EC. 1.1.1.14 (Shaw and Prasad 1970, Richardson et a1. 1986) also known as L-iditol dehydrogenase (SORH) 0.1 M tris-HCL pH 8.0 sorbitol NAD MTT PMS SOD - superoxide dismutase - E.C. # 1.15.1.1 (Wendel and Weeden 1989, Richardson et a1. 1986) 0.1 M tris-HCL pH 8.0 riboflavin EDTA MTT PMS 100ml 10 mg 10 mg 10 mg 5mg 20 mg 100ml 20 mg 10 mg 10 mg 10 mg 20 mg 100ml 500 mg 10 mg 15 mg 2mg 100ml 4mg 2mg 20 mg 5mg TPI - triose phosphate isomerase - E.C. # 5.3.1.1 (Richardson et al. 1986) 0.1 M tris-HCL pH 8.0 DL—a-glycerophosphate (DHAP) pyruvic acid NAD MTT PMS arsenate glycerophosphate dehydrogenase lactate dehydrogenase 100ml 2 g 1.1 g 50 mg 30 mg 5 mg 50mg 200 units 200 units 65 XDH - xanthine dehydrogenase - EC. # 1.1.1.204 (Richardson et a1. 1986) 0.1 M tris-HCL pH 8.0 100 ml hypoxanthine 50 mg NAD 20 mg MTT 20 mg PMS 10 mg APPENDIX B Allele Frequencies by Year APPENDIX B Allele Frequencies by Year Allele frequencies of common pheasants in southern Michigan harvested in Montcalm and Hillsdale counties during 1991. Locus Allele Montcalm Hillsdale ACP 1 0.80 0.88 2 0.20 0.12 n 15 20 AP 1 0.71 0.90 2 0.29 0.10 n 12 20 IDH-1 1 0.46 0.98 2 0.54 0.02 n 14 20 IDH-2 1 1.00 1.00 2 0.00 0.00 n 14 20 ACON-l 1 0.14 0.28 2 0.86 0.72 n 14 20 ACON-2 1 0.04 0.00 2 0.96 1.00 n 14 20 66 67 Allele frequencies, by year for 3 populations of free-ranging common pheasants harvested in southern Michigan. free-ranging Locus Allele w/o releases w/releases ring-necked mixed Sichuan 1991 1992 1993 1991 1992 1993 1991 1992 199 3 ACP l 0.84 1.00 0.50 0.92 0.89 0.88 0.84 1.00 1.00 2 0.16 0.00 0.50 0.08 0.11 0.12 0.16 0.00 0.00 n 35 8 6 20 33 7 32 3 10 AP 1 0.83 0.50 0.83 0.72 0.44 0.31 0.86 0.88 0.85 2 0.17 0.50 0.17 0.28 0.56 0.69 0.14 0.12 0.15 n 32 8 6 20 32 7 32 3 10 IDH-1 1 0.77 0.81 0.58 0.55 0.80 0.32 0.89 1.00 0.95 2 0.23 0.19 0.42 0.45 0.20 0.38 0.11 0.00 0.05 n 34 8 5 20 32 7 32 3 10 IDH-1 l 1.00 1.00 1.00 0.55 1.00 1.00 1.00 1.00 1.0 2 0.00 0.00 0.00 0.45 0.00 0.00 0.00 0.00 0.0 n 34 8 6 20 33 7 32 3 10 ACON 1 0.22 0.12 0.67 0.60 0.32 0.06 0.36 0.25 0.30 2 0.78 0.88 0.33 0.40 0.682 0.94 0.64 0.75 0.70 n 34 8 6 21 33 7 32 3 10 A00“ 1 0.02 0.44 0.30 1.00 0.77 1.00 1.00 0.88 1.00 2 0.98 0.56 0.70 0.00 0.23 0.00 0.00 0.12 0.00 n 34 8 6 22 33 7 32 3 10 APPENDIX C Genetic Heterogeneity in the Class Aves 68 avg 35 o 3012... 9595qu 3985: . om - 15.0 mm o 2. 3.3.: 4 5283a :92 a. - 80.0 a n Be .383 4 52533 30;: 8 - 23.0 a 0 SN 3&3 2.894 Eggs 32:: 2 - 80.0 R _ 2 36583~umu§§§am§mu no 3929 9395 .389. mm 83 $3 2 m 2. 3.38 see»? 3% 2 - ~86 R _ 2 538 foe»? 535 2 - 39° 8 a 2 38:38 a .m 588.3 56326 .m an - mm 0.0 8 a 2 2233 o m 388% ”.285 «m - 9.3 8 a 2 33.32 a m .5823 88.35. _. $3 83 8 v «2 3.2.38 access .5823 38.3% R 48.0 R 0.0 3 a E 33.38 assuage 35.3% 33:99:. 2 - 83 R _ 2 32.38 3.83am .fiafia 28395 85555“ .mm 5628;,“ ..._ 853530 .o a“ - 38° 8 .: 30 283.332.. 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