ELECmOPHORETIC AND CYTOGENETIC STUDY OF SELECTED
MACHAERANfTHFRA SPECIES AND HYBRIDS
by
TERIL BUNDRANT, B.A.. B.S., M.S.
A DISSERTATION
IN
BIOLOGY
Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Approved
December, 1987 i
© 1987 Teril Bundrant ACKNOWLEDGMENTS
I thank Dr. R. C. Jackson for suggesting this study, furnishing the seed and other materials and equipment used, and the guidance and assistance he provided. To all committee members I extend my sincere appreciation, with special thanks to Dr. Ronald Chesser for providing the BIOSYS program and Dr. Charles Werth for critically reading the manuscript. I am grateful to Ken Freiley for reading and commenting on the final draft of this work and helping me with administrative matters while I was enrolled in. absentia. Special thanks goes to Billy Tucker and Dr. Wesley Coffman for use of the word processor and laser printer upon which I prepared this document.
11 CONIENTTS
ACKNOWLEDGMENTS ii TABLES V FIGURES vii INTRODUCTION 1 Taxonomic History of Species Studied 1 Relationships 4 Techniques 5 Purpose 6 MATERIALS AND METHODS 7 Cultivation, Crossing, and Mitotic Analysis 7 Cytogenetic Analysis and Pollen Analysis 8 Electrophoretic Analysis 12 Protocol 12 Subcellular Location 14 Analysis 15 RESULTS 22 Karyotypes 22 Crossing Studies 22 Meiotic Analysis 23 Populations and Species 23 Hybrids 24 Pollen Analysis 25 Electrophoretic Analysis 26 Alleles 26 Measures of Equilibrium 27 Measures of Genetic Variability 27 Genetic Relationships 28 DISCUSSION 112 Chromosome Numbers 112 Crossing Studies 112 Meiotic Analysis 113 Populations and Species 113 Hybrids 114 iii Pollen Analysis 115 Electrophoretic Analysis 116 Alleles 116 Measures of Equilibrium 116 Measures of Genetic Variability 117 Genetic Relationships 118 Synthesis 119 LITERATURE CITED 121 APPENDIX 128
IV TABLES
1. Collection data 19 2. Chromosome numbers of populations 30 3. Cross success 31 4. Configuration distribution of n = 4 diploid populations 32 5. Configuration distribution of n = 5 diploid populations 35 6. Configuration distribution of n = 6 diploid populations 36 7. Configuration distribution of n = 8 tetraploid populations 37 8. Configuration frequency chi-square evaluation of tetraploids 38 9. Configuration distribution of diploid hybrids 42 10. Configuration distribution of triploid hybrids M_- grindelioides var. depressa X M.. gymnocephala 45 11. Chiasma utilization of parents and crosses 46 12. Configuration frequency chi-square evaluation of triploids 51 13. Pollen viability of populations 53 14. Pollen viability of hybrids 54 15. Pollen viability anova of parental populations and crosses 55 16. Allele frequencies 57 17. Alleles present in tetraploid populations 61 18. Chi-square test for deviation from Hardy-Weinberg equilibrium 62 19. Genetic variability measures by population and loci within populations 70 20. Genetic variability at all loci 75 21. Summary of F-statistics at all loci 77 22. Matrix of Nei's (1972) genetic identity and genetic distance 78 23. Matrix of Rogers' (1972) genetic similarity and genetic distance 79 24. Matrix of Cavalli-Sforza and Edwards' (1967) chord distance and arc distance 80 25. Matrix of Prevosti distance (Wnght, 1978) 81 26. Cophenetic matrix of UPGMA cluster analysis using Nei's (1978) unbiased genetic identity coefficients 82 27. Homoplasy matrix of UPGMA cluster analysis using Nei's (1978) unbiased genetic identity coefficients S3 28. Cophenetic matrix of UPGMA cluster analysis using Rogers' (1972) genetic similarity coefficients 84 29. Homoplasy matrix of UPGMA cluster analysis using Rogers' (1972) genetic similarity coefficients 85 30. Cophenetic matrix of UPGMA cluster analysis using Prevosti distance (Wright, 1978) coefficients 86 31. Homoplasy matrix of UPGMA cluster analysis using Prevosti distance (Wright, 1978) coefficients 87 32. Cophenetic matrix of UPGMA cluster analysis using Cavalli- Sforza and Edwards' (1967) chord distance coefficients 88 33. Homoplasy matrix of UPGMA cluster analysis using Cavalli- Sforza and Edwards' (1967) chord distance coefficients 89 34. Patristic distance matrix of distance Wagner tree using Rogers' (1972) genetic distance coefficients 90 35. Homoplasy matrix of distance Wagner tree using Rogers' (1972) genetic distance coefficients 91 36. Patristic distance matrix of distance Wagner tree using Prevosti distance (Wright, 1978) coefficients 92 37. Homoplasy matrix of distance Wagner tree using Prevosti distance (Wright, 1978) coefficients 93 38. Patristic distance matrix of Fitch-Margoliash tree using Rogers' (1972) genetic distance coefficients 94 39. Homoplasy matrix of Fitch-Margoliash tree using Rogers' (1972) genetic distance coefficients 95 40. Patristic distance matrix of Fitch-Margoliash tree using Prevosti distance (Wright, 1978) coefficients 96 41. Homoplasy matrix of Fitch-Margoliash tree using Prevosti distance (Wright, 1978) coefficients 97
VI FIGURES
1. Collection locations 21 2. Karyotype of H. rhizomatous 98 3. Chromosomes of X. tortifolia 99 4. Chromosomes of ML. riparia 100 5. Karyotype of M.- blephariphylla 101 6. Karyotype of M_- grindelioides var. depressa 102 7. Karyotype of M_. grindelioides var. depressa X M^. gymnocephala 103 8. UPGMA phenogram using Nei's (1978) unbiased genetic identity coefficients 104 9. UPGMA phenogram using Rogers' (1972) genetic similarity coefficients 105 10. UPGMA phenogram using Prevosti distance (Wright, 1978) coefficients 106 11. UPGMA phenogram using Cavalli-Sforza and Edwards' (1967) chord distance coefficients 107 12. Distance Wagner phenogram using Rogers' (1972) genetic distance coefficients 108 13. Distance Wagner phenogram using Prevosti distance (Wright, 1978) coefficients 109 14. Fitch-Margoliash phenogram using Rogers' (1972) genetic distance coefficients UO 15. Fitch-Margoliash phenogram using Prevosti distance (Wright, 1978) coefficients 111
vn INTRODUCTION
The genera Machaeranthera. Haplopappus. and Xylorhiza are closely related herbaceous to suffrutescent annuals and perennials of the family Compositae. As a group they are widely distributed in the western United States and Mexico, although individual species may have severely restricted ranges. The genera have been treated taxonomically by several workers, but there has not been unanimity regarding the placement of and relative affinities among the taxa.
Taxonomic History of Species Studied De Candolle (1836) established section Blepharodon in the genus Aplopappus (= Haplopappus'). The monotypic genus Eriocarpum. consisting of E.. grindelioides. was established by Nutall (1840) wherein its distinctness from Aplopappus was emphasized both in the nature of the pappus and in the absence of ray florets. The genus Xylorhiza was also established by Nutall (1840) through descriptions of two species. Torrey and Gray (1842) transferred E. grindelioides to Aplopappus section Aplodiscus which included other eradiate species. They likewise moved Nuttall's two Xylorhiza species to Aster section Orthomeris. Gray (1853, 1884) retained Orthomeris intact within Aster but created section Megalastrum to accommodate two newly-described species. A.- tortifolius (Torrey & Gray) A. Gray and A., wrightii A. Gray. Gray (1879) also placed Aplopappus gymnocephalus in Aster section Machaeranthera. Greene (1894) reinstated Eriocarpum. including within the genus E.. grindelioides. E.. gymnocephalum (formerly A., gymnocephalus^ E.. coloradoensis. and 6 yellow-rayed taxa. With knowledge of additional descriptions, Greene (1896) resurrected Xylorhiza to include a total of seven species from among Orthomeris. Megalastrum. and new descriptions. Hall (1928) placed most members of Greene's Eriocarpum in Haplopappus section Blepharodon (= Sideranthus). Eriocarpum grindelioides (= S_. grindelioides) was renamed H.. nuttalli. presumably to avoid conflict with the South American species already named H_. grindelioides. He suggested that the dwarfing of plants of H.. nuttalli collected from high altitudes may be a
1 hereditary trait but did not propose varietal designation for the form. Hall also reduced U_. blephariphvllus Gray to R. gymnocephalus. stating that he had studied both types and was unable to recognize any significant difference. His declaration of a type species for the section was unclear. At one point in a diagnosis of the sections, under Blepharodon he stated, "Type species: R. gvmnocephalus was first described by De Candolle and is now selected as the type" (Hall, 1928, p. 33). However, in describing individual species of section Blepharodon. under the discussion of H_. nuttalli he stated, "In habit, foliage, development of leaf-bristles, and style-branches, this is very representative of the section Blepharodon. of which it is, indeed, the taxonomic type" (Hall, 1928, p. 70). His section Blepharodon consisted of the following ten species, encompassing yellow-rayed, purple-to reddish-rayed, and eradiate members: Ii. arenarius Benth., K. aureus Gray, H.. brickellioides Blake, H,. gracilis (Nutt.) Gray, H.. gvmnocephalus DC, R. junceus Greene, H.. nuttalli Torr. & Gray, R. phvllocephalus DC, R. spinulosus (Pursh) DC, and R. stenolobus Greene. Machaeranthera corellii was described by Shinners (1949), but he later realized that his new Machaeranthera species already had a place in the synonomy of R. gymnocephalus. He then (Shinners, 1950) moved much of Haplopappus section Blepharodon to Machaeranthera. redefining Machaeranthera to include species having white to purple rays as well as yellow rays. A merger of Machaeranthera and Haplopappus section Blepharodon was necessary, he concluded, since "such keen students as De Candolle, Gray, and Hall have held varying and conflicting opinions regarding species assigned to them," and, "since the only actual distinction is the trivial one of ray color." As described by Shinners (1950), section Blepharodon consisted of M.- arenaria (Benth.) Shinners, M.- aurea (Gray) Shinners, M. australis (Greene) Shinners, M_- blephariphylla (Gray) Shinners, M.- gracilis (Nutt.) Shinners, M.- grindelioides (Nutt.) Shinners, M_. gvmnocephala (DC.) Shinners, M_. havardii (Waterfall) Shinners, M_- juncea (Greene) Shinners, M_. laevis (Woot & Standi.) Shinners, Nl. phyllocephala (DC.) Shinners, M_. phyllocephala var. annua (Rydb.) Shinners, M_- phyllocephala var. mcgacephala (Nash) Shinners, M_. pinnata (Nutt.) Shinners, M_. scabrclla (Greene) Shinners, and Nl. stenoloba (Greene) Shinners. Cronquist and Keck (1957) retained section Blepharodon in Haplopappus. In their taxonomy, white-rayed, purple-rayed, and eradiate members that were included in Machaeranthera section Blepharodon by Shinners, plus M.. coloradoensis (fomerly A., coloradoensis. E_. coloradense. and 2L. coloradensisV were placed in the newly-created section Machaeranthera series Originales. They did not find it necessary to include any yellow-rayed species in Machaeranthera. preferring to retain them in Haplopappus. Thus section Machaeranthera. series Originales consisted of Nl. blephariphvlla (Gray) Shinners, Nl- coloradoensis (Gray) Osterh., M.. grindelioides (Nutt.) Shinners, M.. grindelioides var. depressa (Maguire) Cronq. & Keck, and M. gvmnocephala (DC.) Shinners. The type species of series Originales was M. blephariphylla. Machaeranthera linearis Greene was assigned to section Machaeranthera. series Variabiles. They also reduced Xylorhiza to sectional status in Machaeranthera. explaining that "Xylorhiza and Machaeranthera are so inextricably linked that they must be retained in the same genus." In a paper primarily concerned with Machaeranthera section Psilactis. Turner and Home (1964) stated, "the taxa Blepharodon. Machaeranthera and Psilactis are...more closely related each to the other than any one is to Xylorhiza." They concluded that, "if Xylorhiza is included in Machaeranthera then a case is immediately made for the acceptance of a large collective genus which might include all of the above" (i.e., Psilactis. Machaeranthera. Xylorhiza. and Haplopappus section Blepharodon'). Since publication by the above authors, additional taxa have been described. Turner (1973a, b) described M. gypsophila and M.. restiformis. both of which "presumably belong to the Section Blepharodon of Machaeranthera" (Turner, 1973b). Haplopappus rhizomatous was originally described by Johnston (1961), but R. C. Jackson now feels this yellow-rayed species is more properly a Machaeranthera. citing successful hybridization with M. johnstonii and Nl- restiformis but not with members of Haplopappus (personal communication). Hartman (1976) called it Machaeranthera heterophvlla. placing it in his newly-created Machaeranthera subgenus Sideranthus which was composed of most of the yellow-rayed taxa treated by Hall (1928) in Haplopappus section Blepharodon. Hartman (1976) treated the section Blepharodon as belonging to the genus Machaeranthera instead of Haplopappus. The section consisted of M_. blephariphylla, M.. coloradoensis var. coloradoensis. M. coloradoensis var. brandegei. Nl. crutchfieldii. Nl. grindelioides var. grindelioides. M.. grindelioides var. depressa. M. gvmnocephala. M. gypsophila. M. johnstonii. and M.. restiformis. Among the members of Hartman's section Sideranthus were M^. arenaria. Nl. gracilis, and M.. heterophvlla. Machaeranthera linearis was assigned to section Hesperastrum. Excluded from the study was M. tortifolia (= X_- tortifolia'). Machaeranthera riparia was not addressed. Watson (1977) resurrected the genus Xylorhiza as consisting of eight species, including X_. tortifolia. but excluding X,. coloradoensis and its putative ecotype X_. brandegei.
Relationships Hall (1928) considered R phyllocephalus to be the ancestral type of the series comprising annual taxa of section Blepharodon (i.e., R. gracilis. R. aureus. R stenolobus. and R. phvllocephalus). and R. phvllocephalus var. primitivus Hall, as indicated by the name, to be the most primitive known extant form. R. C. Jackson studied the type of R. phyllocephalus primitivus Hall as well as living material at the type locality and concluded that it is a Machaeranthera. probably M.- gymnocephala (Jackson, 1966). This discrepancy is not as surprising as it may initially seem, for Hall (1928, p. 60), in describing relationships of R. phvllocephalus. stated.
In searching for the most primitive form of this species, one is impressed with a series from central and southern Mexico which have been variously identified as Haplopappus gymnocephalus and as an unknown species of Aster. These are now found to connect very closely with H. phvllocephalus tvpicus and are therefore described as a new subspecies, namely, R. phylocephalus primitivus.
He then acknowledged that there had been some past confusion between R. phyllocephalus var. primitivus and R. gvmnocephalus (= R. gvmnoccphalaV and continued with a description of critical differences between the two as he recognized them. That R. phyllocephalus and the other members of the ohvllocephahis group (R. aureus and R. annus') belong to Haplopappus sect. Isocoma rather than to sect. Blepharodon was demonstrated by Jackson and Dimas (1981). Of the perennial species of Haplopappus section Blepharodon. Hall considered R. gvmnocephalus to be the most primitive, although not necessarily ancestral to that series. Cronquist and Keck (1957) considered an evolutionary line departing from Haplopappus in the section Blepharodon to give rise to Machaeranthera. without disturbing the homogeneity of Haplopappus. Thus, they did not find it necessary to include any yellow-rayed species in Machaeranthera. They hypothesized two lines of evolution from the ancestral type: one leading to Machaeranthera proper, and the other to the Xylorhiza line. They interpreted M. blephariphylla as the most primitive existing species of Machaeranthera. and the one that most nearly approaches Haplopappus.
Techniques Electrophoretic analysis of enzymes and other proteins is an important tool in the investigation of evolutionary and taxonomic problems, coming into widespread use in the late 1960s (Avise, 1975; Gottlieb, 1977). It has been called the most useful procedure yet devised for revealing genetic variation (Hartl, 1980). Information derived from electrophoresis can be used to estimate a host of population genetics parameters and relative taxonomic placements. Some electrophoretic work has been performed on selected members of the three groups (Arnold and Jackson, 1978; Spohn, 1978; Gottlieb, 1981a). Gametic chromosome numbers of 2, 4, 5, 6, 8, and 9 have been reported for various species of the genera Machaeranthera. Haplopappus. and Xylorhiza (Jackson, 1957; Turner cial., 1961; Solbrig £ial., 1964; Turner and Flyr, 1966; Solbrig £iai., 1969; Powell and Sikes, 1970; Turner £lai., 1973; Watson, 1973; Anderson £lii., 1974; Strother, 1976; Brown and Clark, 1981). The basic chromosome number for section Blepharodon is x=4 (Jackson, 1962, 1966). All species of Xylorhiza have a basic chromosome number of x=6 (Watson, 1977). Supernumerary chromosomes have been found in populations of various species of the Blepharodon section (Jackson, 1960a, b; Raven £iai., 1960; Jackson 1962). Polyploidy is known among taxa of Machaeranthera and Haplopappus (Anderson £iai., 1974). Cytological analysis of chromosome pairing in interspecific hybrids provides means of detecting and assessing relationships. A number of artificial and natural hybrids have been reported for these groups (Jackson, 1962; Turner and Sanderson, 1971; Stucky and Jackson, 1975; Stucky, 1978). Cytological evaluation of hybrids can estimate the degree of homology between genomes of a species (Stucky, 1978), and, in the case of polyploids, lead to the determination of basic chromosome numbers (Stucky and Jackson, 1975). Models and equations for quantitative cytogenetic analysis of diploids (Jackson, 1984) and autopolyploids (Jackson and Casey, 1982; Jackson and Hauber, 1982; Jackson, 1984) have been proposed.
Purpose This study utilizes electrophoresis, hybridization studies, and cytological evaluation to estimate the evolutionary relationships among certain members of the genus Machaeranthera and their affinities with Haplopappus rhizomatous and Xylorhiza tortifolia. Taxonomic treatments based on evolutionary relationships are both logical and more widely acceptable than phenetic classifications. MATERIALS AND METHODS
Specimens of the following species and varieties were grown from seed collected at a total of 18 localities by R. C Jackson: Machaeranthera blephariphylla (Gray) Shinners, Nl. grindelioides (Nutt.) Shinners, M. grindelioides var. depressa (Maguire) Cronq. & Keck, Nl- gymnocephala (DC.) Shinners, Nl. gvpsophila Turner, Nl. linearis Greene, Nl- restiformis Turner, M. riparia H.B.K., Haplopappus rhizomatous Johnston, and Xylorhiza tortifolia (T & G) Greene (Table 1 and Figure 1). Special aspects of the collections were (Jackson, personal communication): M.. gypsophila and M.. restiformis were collected from a hybrid area characterized by small, grass-stabilized dunes surrounded by a flatter sandy area. M.- restiformis was growing at the edge of the dunes in small numbers, probably not more than 20 plants total. Along the roadside and in the sand 10 to 15 feet away from the dunes were abundant M.. gypsophila. Seed collected from plants at this locality were identified as to species based upon the appearance and habitat of the parental plants. Plants grown from these seed exhibited more morphological variability than would normally be attributed to intrapopulation variation. Individuals selected for crossing studies and cytological analysis were the best morphological representatives of their respective species. Also collected at the site were seed from plants that appeared to be hybrids of M.- gypsophila and M.- restiformis. These putative hybrids are identified by the collection number 760IF. Plants of M. blephariphvlla population 7559 were found growing only inside shrubs of cat- claw acacia. There they were protected by thorns of the acacia against being eaten by cattle, which apparently prevented the plants from reaching any size elsewhere in the area. At the lime of collection it was thought to possibly represent a new subspecies, primarily because of a larger achene size.
Cultivation. Crossing, and Mitotic Analysis Seeds were selected at random from different heads of each collection or from different parent plants when the mode of collection permitted such identification and germinated in 250 ml Erlenmeyer flasks containing 50-100 ml of 0.1% KNO3 (Copeland, 1976). They were provided with slow, continuous 8 aereation, and the KNO3 was changed twice daily for the first 3 days to remove germination inhibitors and other impurities that might have been leached from the seeds. After germination, 2-3 day old seedlings were transferred to water-expanded Jiffy-7 peat pellets that were then placed in a plastic tray. About 2.5 cm of water was added to provide moisture, and the top was covered with a clear thin plastic sheet to allow light to penetrate. When several root tips about 1 cm long had emerged from the peat pot, they were excised 3 hr. prior to the time of peak mitosis and placed in either a dilute alpha- bromonaphthalene or 0.002 M 8-hydroxyquinoline solution to inhibit spindle fiber formation. At the time of peak mitotic activity, the root tips were fixed in 4:1 95% ethanol/propionic acid for 48-72 hr., then transferred to 70% ethanol for long-term storage in the freezer at approximately -15°C. Four-to six-wk- old seedlings were subsequently transferred to six-in. clay pots containing a mixture of top soil, sand, and peat moss in equal proportions and maintained in the greenhouse. Previously collected and fixed root tips were hydrolyzed in 15% HCl for 15 - 17 min, then gently squashed in FLP orcein stain (Jackson, 1973). Chromosome numbers of representative plants were determined and appropriate photographs taken for karyotyping.
Crosses among representative plants were made by selecting heads of plants from different populations or species in synchronous anthesis and rubbing the heads together during the morning of each of 2-3 consecutive days as new florets opened. A sporophytic incompatibility system prevented virtually all self-pollination, so emasculation of florets was not necessary. Seeds resulting from crosses were allowed to mature on the plant then collected and dried at room temperature for about 2 months. F| seedlings were grown in pans as with the parents. Their chromosomes were then analyzed from root tips, and they were transferred to clay pots as previously described.
Cytogenetic Analysis and Pollen Analvsis Meiotic analyses were performed on buds collected at the time of peak meiotic activity and fixed in 3:1 ethanol/propionic acid for 2-3 days before transfer to 70% ethanol for long-term storage at about -I50C. Anthers removed from individual florets were gently squashed in propiocarminc, and all stages of mciosis from representative plants were examined for evidence ot abnormalities. Configuration and chiasmata frequency data were collected from cells at late diplonema through diakinesis. Following cytogenetic analysis, selected slides were made permanent by immersing them in liquid nitrogen until frozen, then carefully removing the cover slip with a razor blade. Permanent slides were then made using a drop of balsam dissolved in propionic acid as a mounting medium. The following conventions apply to configuration representations in both text and tables herein: Capital Roman numerals indicate chromosome associations, i.e., I = univalent, II = bivalent. III = trivalent, IV = quadrivalent. A circle configuration results when all arms of associated chromosomes are connected by chiasmata, whereas a configuration containing chiasmata- connected chromosomes with two free ends is a chain. A small letter preceeding the Roman numeral indicates the configuration, i.e., o = circle and c = chain. The number of chiasmata in a configuration can be derived from the type of configuration and number of chromosomes involved. For example, oil is a circle of two chromosomes and contains two chiasmata, cll is an arrangement wherein two chromosomes are connected by a single chiasma, and oIV would contain four chromosomes and four chiasmata. An Arabic numeral preceeding the configuration symbol signifies the number of such configurations. Concatenated symbols describe meiotic chromosome configurations within a cell. Thus, 2 oil, 2 cll is a meiocyte containing 2 circle bivalents and 2 chain bivalents, having a total of 6 chiasmata. In the chromosome configuration tables that follow, the column Other contains configurations not expected on the basis of normal pairing. Such configurations contained univalents or univalent-appearing chromosomes. Univalents are usually taken to indicate either pairing failure or crossing over failure. In this study, many univalents undoubtedly resulted when normally paired homologues bearing chiasmata were separated as pressure was applied to the coverslip during preparation of the slide. Evidence for this was numerous observations of bivalents connected by a very thin strand of chromosomal material, and by univalent-appearing pairs of chromosomes which had one or both ends bent toward each other in a manner not expected of true univalents but consistent with chromosomes that had been forced apart after being previously joined. In these instances the chromosomes 10 superficially resembled univalents but were not, and were not counted as such. Other pairs may have lost evidence of being joined, thus being tallied as univalents. Equations of Jackson and Hauber (1982) and Jackson (1984) were used to calculate expected values for each configuration of an autotriploid and autotetraploid of x = 4 having a maximum of 2 chiasmata per bivalent. Any system not fitting the autoploid model was taken to be an alloploid. Chi-square analysis compared those expected values with data from cross-produced triploids and naturally occuring tetraploids to determine if the triploids and tetraploids behaved as expected for autoploids, or if they exhibited alloploid behavior. These models are in fact not different except in the philosophy of their derivation, for they are mathematically equivalent. The equations of Jackson and Hauber (1982) make use of information derived from the data and therefore lose a degree of freedom when analyzed, whereas those of Jackson (1984) do not use data-derived information and do not lose a degree of freedom (Jackson, personal communication). The models of Jackson and Hauber (1982) and Jackson (1984) predict the number of times a particular configuration event will be seen in an autopolyploid—i.e., the number of times a oIV or cIV or some other configuration is expected. If a chi-square table is set up using numbers of configuration events for a polyploid, the total of events observed and events expected will most likely not be equal. The reason can be illustrated by an example involving tetraploids. With the same number of chiasmata a single set of 4 homologous chromosomes can yield 2 different configuration scores if pairing is normal: (1) a score of 1 event (either oIV or cIV), or (2) a score of 2 events (some combination of oil and/or cll). Since one of the assumptions of the chi-square test is that the total of the expected frequencies over all classes equals the total of the observed frequencies over all classes (Dixon and Massey, 1969, p. 238), a chi-square evaluation wherein those totals are not equal may not be valid. Obviously, in a situation wherein the expected number of each configuration closely matches the observed number for that configuration, the number of total events will be very close, and a conclusion of non- significance will not be in error. The other extreme is also true. The validity at the intermediate values is unknown. At present, 1 know of no way of 11 calculating any magnitude of error that may exist, nor of compensating for such differences. A chi-square test that compares chromosomes involved in the various configurations will contain equal totals of expected and observed chromosomes summed over all classes. This is because, to continue with the previous example, 4 chromosomes will still be 4 chromosomes whether they are scored as a unit of 4 chromosomes involved in a oIV or a cIV, or whether they are scored as two sets of 2 chromosomes involved in a oil or cll or combination thereof. The consequent chi-square test is also to be preferred because it is more discriminating. Where appropriate I have determined and listed for comparison both the number of configurations and the number of chromosomes involved in each configuration. I suggest that future investigations also employ comparisons based on the number of chromosomes involved in each configuration. A chi-square test for fit of circle and chain configurations to the binomial distribution was used to test for normality of chiasma distribution. Expected values were obtained by expansion of the binomial (p + q)", where n = the gametic number of chromosomes, p = the proportion of chromosomes observed to be involved in circle configurations, and q = the proportion of chromosomes observed to be involved in chain configurations. Chi-square analysis was also used to compare meiocyte configurations of parents and offspring produced by crossing. Due to the difficulty of finding meiotic material in sufficient quantity from anthers in exactly the proper stage of meiosis, configuration frequency data were not obtained on all plants used as parents in successful crosses. Each cross for which parental data were available was examined by using the offspring as the observed category, and separately comparing it to the maternal parent and the paternal parent used as the expected category. The binomial expansion and chi-square test were performed by a BASIC program I wrote, and executed on an Apple He computer. Pollen analysis was conducted on heads in early anthesis. Fresh pollen from non-dehisced florets was stained with buffalo black NBR dissolved in 45% propionic acid. Pollen was scored as viable or non-viable, with cells having uniformly stained cytoplasm considered viable. 12
Electrophoretic Analysis Protocol Horizontal starch-gel electrophoresis was performed using standard techniques (Selander etal-, 1971; Gottlieb, 1981b) plus special procedures as follows. Material was prepared by crushing 50-100 mg of relatively young healthy leaf tissue in a small plastic weighing boat with an equal amount of polyvinylpolypyrrolidone to absorb phenolic compounds and enough extraction buffer (0.1 M Tris HCl, pH 7.5, used as a solvent for 1 mM disodium EDTA, 10 mM KCl, and 10 mM MgCl2, to which was added 150 microliters 2- mercaptoethanol and 400 microliters Triton X-100 per 100 ml) to produce a thick slurry. A portion of each sample was absorbed onto chromatography paper cut into 4x8 mm wicks. To maintain enzymatic activity, weighing boats containing the specimens were kept on crushed ice before the leaf material was ground, while absorption onto the wicks was taking place, and during the interval required to place the many wicks of a run on each of the 3-4 running trays. Gels were prepared from Electrostarch, lot #371 (Otto Hiller, Madison, Wisconsin) at a concentration of 12.5%. Fifteen enzyme loci were studied. Enzyme systems assayed were aldolase (ALDO, EC 4.1.2.13), catalase (CAT, EC 1.11.1.6), glutamate dehydrogenase (GDH, EC 1.4.1.3), glucose 6-phosphate dehydrogenase (G6PD, EC 1.1.1.49), glutamic oxaloacetic transaminase (GOT, EC 2.6.1.1 [also known as aspartate aminotransferase {AAT}]), glyceraldehyde-3-phosphate dehydrogenase (G3PD, EC 1.2.1.12), glycerate-2-dehydrogenase (G2DH, EC 1.1.1.29), isocitrate dehydrogenase (IDH, EC 1.1.1.42), leucine aminopeptidase (LAP, EC 3.4.11.-), malate dehydrogenase (MDH, EC 1.1.1.37—the NAD-dependent form), malic enzyme (ME, EC 1.1.1.40), phosphoglucose isomerase (PGI, EC 5.3.1.9), phosphoglucomutase (PGM, EC 2.7.5.1), and triose phosphase isomerase (TPI, EC 5.3.1.1). Preliminary studies indicated that the above systems produced consistent results. They provided a mix of glycolytic and non-glycolytic enzymes that contained a variety of catalytic classes which were highly polymorphic with those that were less so. Most of the en/ymes were only monomer or dimer classes, avoiding interpretation difficulties inherent in heterozygous tetramers and were present in one or two active isozymes with 13 the procedures used, again avoiding confounding complexity and possible null alleles. Tetrameric enzymes (ALDO, GDH, and ME) were fixed for one allele in virtually all populations. Only ME in M.. riparia exhibited heterozygotes. Gel systems and running conditions for the various enzymes were as follows: A/B system-ALDO, GDH, GOT, LAP, PGI, PGM, and TPI; fixed current 45 mA, variable potential typically about 197 V initially; 4 hr 30 min. 0.02 M histidine system-G2DH, IDH, and ME; fixed current 55 mA, variable potential typically about 67 V initially; 4 hr 30 min. pH 5.7 histidine system-CAT and G6PD; fixed current 35 mA, variable potential typically about 235 V initially; 3 hr 50 min. TBE system-G3PD and MDH; fixed current 45 mA, variable potential typically about 320 V initially; 3 hr 0 min. Running times were often increased or decreased up to about 10 minutes to allow the bromphenol blue front marker to reach a standard position, thereby yielding very constant migration patterns. Electrode buffer, gel buffer, and stain formulas used are contained in the Appendix. The formulas were for the most part those of Gottlieb (personal communication), and were essentially the same as widely used and reported elsewhere (Scandalios, 1969; Shaw and Prasad, 1970; Selander Slal., 1971; Gottlieb, 1981b). Codominant inheritance was assumed for all allozymes, and the banding patterns were interpreted as genotypes. Whenever specimen availability permitted, at least 40 plants of the parental generation were tested, thus giving a 90% probability of detecting alleles having a frequency of 0.05 or higher at a particular locus. For enzyme systems with two or more isozymes detected, the different isozymes were assigned numbers with the one having the greater electrophoretic mobility (migrating further from the origin) given the higher number. Allozymes were denoted by small letters in a manner similar to isozymes. The slowest allele was thus designated "a," while faster migrating allozymes were designated "b," "c," etc. At least one of the plants used in preliminary studies to establish migration patterns was run concurrently with later unknowns as a control to ensure uniformity of scoring. All enzymes were detected as anodally migrating bands with the buffer systems used. 14 Subcellular Location SubceHular specificity of certain isozymes is well established (Gottlieb, 1977; Newton, 1983; Weeden, 1983). Different fonns of various enzymes have been found in cytoplasm, peroxisomes, glyoxisomes, microbodies, mitochondria, and chloroplasts. For some isozymes the active forms are found in plastids or mitochondria but are encoded for by nuclear genes (Newton, 1983; Weeden, 1983). Assembly can occur at cytosolic ribosomes with the protein becoming active upon transport through the plastid envelope (Weeden, 1983). For enzymes found in both cytoplasm and organelles of plants and animals, substantially less variability is generally observed in the organelle forms, indicating they are evolutionarily highly conserved. Variations in extraction buffer components, substrate, extraction technique, and pH of either buffer, gel, or stain dictate which isozymes will be found. Some idea of the location of isozymes may be gained through breeding studies. In order to unequivocally demonstrate the presence of a particular enzyme or isozyme in chloroplast or mitochondrion, subcellular fractionation studies must be undertaken (Newton, 1983; Weeden, 1983). Based upon my crossing studies and reports by others on the location of various isozymes, my best estimate as to the location of enzymes in this study is as follows (EC numbers given earlier also reflect these locations): ALDO, cytosolic. CAT, cytosolic. GDH, mitochondrial NAD-dependent form; the NADP dependent (EC 1.4.1.4) form would have required a substrate different from the one used. G6PD, cytosolic. GOT-2, cytosolic; both a faster migrating form that was probably mitochondrial, and a slower, probably glyoxosomal form were irregularly seen. G3PD, cytosolic. G2DH, cytosolic. IDH, cytosolic. LAP, cytosolic. MDH-3, nuclear gene, probably cytosolic; a very slow migrating form likely from microbody (mb-MDH) and an intermediate form probably mitochondrial (m-MDH) were occassionally seen, but both are probably under nuclear control (Newton, 1983); an NADP-dependent chloroplast form has been described (Ting and Rocha, 1971), but the substrate used in this study was NAD. ME, cytosolic. PGI, nuclear gene, cytosolic form; also seen with irregularity was a faster migrating form, probably chloroplastic, that exhibited much ie^;s s variability insofar as could be determined. PGM-1, nuclear gene, cytosolic. PGM-2, chloroplast. TPI, cytosolic. 15
Analysis The FORTRAN IV computer program BIOSYS-1 (Swofford and Selander, 1981) installed on an IBM 4361 computer was used to calculate the following: allele frequency, percentage of loci polymorphic, average number of alleles per locus, average heterozygosity, test of Hardy-Weinberg equilibrium, Wright's fixation index, various coefficients of genetic similarity and genetic distance, genetic differentiation by F-statistics, unweighted pair-group method phenogram, and distance Wagner tree. Data were input to BIOSYS-1 as genotype frequency data. Allele frequency determinations were by direct count. Phylogenetic trees were also constructed using the program FITCH in the Phylogeny Inference Package (PHYLIP), version 2.9 (Felsenstein, 1986). Programs of PHYLIP were written and distributed in a standard subset of Pascal. The programs were compiled and run on a COMPAQTM DESKPRO'^^ computer (Compaq Computer Corporation) installed with MS^^-DOS version 2, using Turbo Pascal (Borland International), version 3.0. Measures of genetic variation. The percentage of polymorphic loci is a rough guide to the level of genetic variation in a sample (Brown and Weir, 1983). This statistic was calculated using three different criteria for polymorphism: (1) the frequency of the most common allele being 0.95 or above, (2) the frequency of the most common allele being 0.99 or above, and (3) any variation observed in the population (termed "no criterion"). Allelic richness is addressed by the average number of alleles per locus, and is sensitive to sample size. Average heterozygosity is a measure of evenness of allele frequencies. It is relatively insensitive to sample size. Average heterozygosity was caclucated in three ways: (1) by direct count, (2) an estimate based on Hardy-Weinberg expectations, and (3) an unbiased estimate based on conditional expectations (Levene, 1949; Nei, 1978). Measures of equilibrium. Chi-square goodness-of-fit tests of the hypothesis that each population analyzed was in Hardy-Weinberg equilibrium at each variable locus were performed using observed genotype frequencies and those expected under Hardy-Weinberg equilibrium. The formula of Levene (1949) for small sample size was used in the calculations, with the exact significance probability option of BlOSYS-1 invoked. Wright's fixation index 16 (F) is another measure of conformance to equilibrium conditions (Wright, 1965). It can be interpreted as the proportional increase or reduction in heterozygosity as compared to panmictic expectations. F ranges from -1.0 to -1-1.0. Negative values indicate an excess of heterozygotes; positive values indicate a deficit of heterozygotes. SubpoDulation structure, F-statistics (Wright, 1965) were originally formulated in terms of correlation between uniting gametes, but they may also be viewed as relative reductions in heterozygosity compared with panmictic expectations. Fjs is the inbreeding coefficient of an individual (I) relative to its subpopulation (S); it is also the probability that two alleles at a locus in an individual are identical by descent. FIT is the inbreeding coefficient of an individual (I) relative to the total population (T). It may also be seen as the average reduction in individual heterozygosity relative to the total sample. EST is the probability that two alleles chosen at random from within the same subpopulation are identical by descent. F-statistics were calculated by BIOSYS- 1 using the fonnulas of Wright (1965, 1978) and Nei (1977). Coefficients of relationship. Measures of genetic distance and similarity calculated by BIOSYS-1 were those of: Nei (1972) genetic identity and genetic distance, Rogers (1972) genetic similarity and genetic distance, Nei (1978) unbiased genetic distance and unbiased genetic identity, Cavalli-Sforza and Edwards (1967) chord distance and arc distance, and Prevosti distance (Wright, 1978). Dendrograms. Three widely-used methods of estimating phylogenetic trees from distance matrices were used: (1) a hierarchical cluster analysis using the unweighted pair-group method with arithmetic averaging (UPGMA, Sneath and Sokal, 1973), (2) the distance Wagner procedure (Farris, 1972), and (3) the Fitch-Margoliash method (Fitch and Margoliash, 1967). These methods have general acceptance and application, represent fundamentally different approaches of arriving at trees, and have different dependencies regarding constancy of genetic divergence across phyletic lines. Dendrograms derived from UPGMA cannot be considered representations of actual evolutionary trees unless rates of genetic divergence are constant across phyletic lines (Avise, 1975; Swofford, 1981). The distance Wagner method does not assume rate constancy (Farris, 1981). Whether the Fitch-Margoliash method makes 17 the assumption of rate constancy depends on the zeal with which possible rearrangements are investigated (Farris, 1981). Input to the UPGMA cluster analysis were the above matrices of Nei (1978) unbiased genetic distance and unbiased genetic identity, Nei (1972) genetic identity, Rogers (1972) genetic similarity and genetic distance, Prevosti distance (Wright, 1978), and Cavalli-Sforza and Edwards (1967) arc distance and chord distance. Generated by the program for each phenogram were goodness of fit statistics (f of Farris [1972], F of Prager and Wilson [1976], percent standard deviation [Fitch and Margoliash, 1967], and a cophenetic correlation), a cophenetic matrix, and a homoplasy matrix. Prevosti distance (Wright, 1978) and Rogers (1972) genetic distance were used as input to the distance Wagner procedure since those measures conform to a metric (obey the triangle inequality [Farris, 1981]). The root of each tree was placed midway between the two terminal taxa separated by the greatest patristic distance (Farris, 1972). FITCH produced unrooted trees, the objective being to find that tree which minimized the sum of squares of distances between points on the tree. Rogers' (1972) genetic distance and Prevosti distance (Wright, 1978) were used as input. Prior to compilation, I modified the source code as follows to allow DOS redirection from the keyboard: input and output buffers were specified as a new first line by using {$G2048, P8192}; all references to "infile" were replaced with "INPUT", and "outfile" with "OUTPUT"; and the "var" statements were commented out. Additionally, the variables "maxsp" and "maxsp2" of FITCH were changed to increase the maximum number of populations from 16 to 20, and the CONTML variable "maxchr" was increased to 40 to accomodate the total number of alleles in the present study. Since the order of input into PHYLIP programs may affect the fit of the output topology (Felsenstein, 1986), the input order was optimized in the following manner: BIOSYS-1 was used initially on five fundamentally different orders of data to determine only allele frequencies and Rogers' genetic distance. Next, the five arrangements of the Rogers' genetic distance matrix were individually input to FITCH. The order of data that produced the Fitch-Margoliash tree having the lowest sum of squares was considered the optimum order. That optimum order of data was then used for BIOSYS-1 input to produce the full output run of the BIOSYS-1 18 program; it was also used as the order of populations presented in tables herein. The optimum sequence of both Rogers' genetic distance and Prevosti distance was submitted to FITCH with the Global option of PHYLIP invoked to produce the final Fitch-Margoliash trees. Global rearranged the data by removing each possible subtree from the main tree, then adding it back in all possible places in an iterative fashion until all subtrees could be removed and added again without any improvement in the tree. Initial determination of the optimum order of data followed by application of Global was designed to investigate enough rearrangements to overcome any assumption of rate constancy. The Fitch-Margoliash dendrograms shown are not in the same form that was output by the program. Although the topology has not been altered, I manually restructured the branch layout so as to coincide as closely as possible with the BIOSYS-1 trees for ease of comparison. The physical distance of the branch lengths were also drawn more nearly proportional to the numerical branch length produced by the program. I hand-calculated the patristic distance matrix and homoplasy matrix for Fitch-Margoliash trees since PHYLIP did not provide them. 19 oo o .—1 oo CS ^ ON vo ON CN cs CaO\ TT 1—1 o «n lO ^ »n c »n >o >o >r> «o vo »n VO o t^ r- r^ r^ r-* t^ r^ r^ c G a c c c c C 3 c o o O o o O 00 o00 WJ Vo3 c/o3 0o0 00 0o0 C/3 •i< .:;^ ^ .^ .1^ .^ .i^ J
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I Jua n count y 3 Pim a county . 3n g busines s U 3 X 0 X -3 u 03 ^ E ^ H a> CJ d d 00 00 CJ E O :s 3 3 0 s „ 1X 3 u CJ o 3 X 3 U 0 tah . o tah . 0 rizo rizo 3: 0 'C ::s ::§ p D D z < S < Cfl Z .0 3 c/3 c« c/3 c/3 3 0 «J t> 0 ^i^ 03 00 T3 •0 -0 •0 3 F—. • MM • MM •UI J -^ efl O •-" '0 cn '0 0 0 CJ x: >^« 00 • MM • MM c/3 D 0 0 0 'kZ (.-1 -0 • c/3 -a T3 03 03 00 0 0 3 CJ 3 3 3 i) O O c/3 3 • —. k. k. k. 3 3 o > CJ i- •0 CJ 61 i \- W 61 0/ 6/ — JO o CJ a, H 00 sl sl § si :^'l si si s ^„ 21 Figure 1. Collection locations. RESULTS Karvotvpes Chromosome numbers for the populations studied are shown in Table 2. They were initially determined from mitotic root tip preparations as previously described and confirmed by subsequent examination of meiotic material from anthers. Representative karyotypes are contained in Figures 2 through 6 wherein conventional practice has been followed in assigning numbers to each chromosome. Karyotypes of the x = 4 species studied were identical in morphology. Each basic complement contained four chromosomes. The longest, and presumably the one containing the most DNA, was acrocentric. The remaining 3 were somewhat shorter and all about the same length but were distinguishable by centromere location and presence or absence of a nucleolar organizing region and accompanying satellite. Crossing Studies Success of germination, growth, and flowering of hybrids is shown in Table 3. Plants used as the maternal parent are listed by collection number down the left side of the table; those used as the paternal parent are across the top. Numbered comments at the intersection indicate the highest level of success with each cross as follows: (0) Seed did not germinate. Achenes were typically shrivelled and apparently did not contain a viable embryo. (1) At least some seed from the cross germinated, but seedlings did not survive and grow to the size required for transfer to peat pellets. From the entire head, usually only a few seed germinated. (2) Some seedlings survived after being transferred to peat pellets, but none survived until flowering. Most seedlings in this group exhibited a general failure to thrive; they appeared weak, were often pale, and usually grew slowly. A few endured long enough to be transplanted to clay pots in the greenhouse, but most expired before attaining that size. (3) Plants grew and flowered; pollen counts and meiotic material were obtained. Electrophoretic studies demonstrated the hybrid nature of these plants in instances wherein parents and offspring possessed informative alleles. 22 23 Most of the successful crosses were between n = 4 populations of the same or different species of the genus Machaeranthera. Of 15 attempted M_. gymnocephala intraspecific crosses, two-thirds produced offspring, of which only 8 reached maturity. None of the crosses among the different site collections of M.. linearis reached maturity. Crosses between M.- gymnocephala and M.. blephariphylla gave greater than 60% seed set and were the most successful of the interspecific crosses. The only intergeneric cross that produced offspring that eventually flowered was U_- rhizomatous (5071) X M_. gymnocephala (7559). A triploid was produced in several replicates by crossing the ray less M.. grindelioides var. depressa (7631, n = 8) and a purple- rayed variety of NL. gvmnocephala (7528, n = 4). Two different plants served as maternal parent. One plant was used as the paternal parent, since it was the only one of that population in anthesis at the time the particular crossing sequence was made. The resultant triploids had the general growth appearance of M_. grindelioides var. depressa. Heads were small like the maternal parent, but a few much-reduced white ligules were present unlike either parent. The karyotype of the triploid is at Figure 7. Not reflected in Table 3 is a putative successful cross between the tetraploid population 7632 of M. grindelioides and the diploid population 7633 of M.- grindelioides. Seed collected from 7632 produced a tetraploid; seed from 7633 did not germinate. Since the parental plants did not have informative alleles, electrophoretic studies to determine parentage were inconclusive. The offspring could have been the result of either selfing or fertilization by unreduced gametes. Meiotic Analysis Populations and Species In all diploid and tetraploid populations, a maximum of 2 chiasmata was observed per bivalent. Chiasma distribution results for diploid populations are summarized in Tables 4 through 6. All configurations expected to be produced by normal pairing appear as column headings in the tables. These configurations were seen, although not all were necessarily present in each plant. Chiasma distribution results for tetraploid populations are in Table 7. Of normal configurations, only those observed appear as column headings. 24 Additional normal configurations were possible but were not observed. Table 8 contains a chi-square evaluation of observed values and those expected under the non-random tetraploid model of Jackson and Hauber (1982) and Jackson (1984). Both the number of configuration events and the number of chromosomes involved therein are examined; the statistical outcome was the same for the two comparison forms. The random model, which predicted trivalents and univalents did not apply to this system and is not illustrated. A fairly substantial number of quadrivalents was predicted for an autotetraploid, but only a few were observed. Of the 42 n = 4 diploids, 28 (0.667) showed no significant difference between circle and chain bivalents observed and those expected by the binomial expansion. For the 3 plants analyzed in each of the n = 5 and n = 6 diploid populations, 2 (0.667) of each population showed no significant difference between observed and expected oil and cll. A similar test employing multivalents and bivalents in the binomial expansion was conducted for the tetraploid populations. In order for observed data to approximate the distribution assumed by chi-square tables, the sample size must be sufficiently large for none of the theoretical frequencies to be less than 1, and for not more than 20% of the expected frequencies to be less than 5 (Dixon and Massey, 1969, p. 238). Due to the low proportion of multivalents encountered, the sample size of 50 meiocytes was not large enough for many of the classes to have the requisite number in the expected category. When classes were combined to meet the criteria for the expected category, all degrees of freedom were lost, so the tetraploid populations could not be tested. Hybrids Configuration data for diploid hybrids are contained in Table 9. Chromosome associations of diploid hybrids were very regular. Only one diploid hybrid (M. gymnocephala 7528 X M_- gymnocephala 7542) exhibited an unusual number of univalents or an abnormally low chiasma frequency. The paternal plant of that cross was used in other intra-M_. gymnocephala crosses as either the paternal parent or the maternal parent without unusual results. The maternal parent was not used in any other successful cross nor was 25 meiotic material from it examined. Other plants of the maternal population were crossed and scored with unremarkable outcomes. Table 10 contains configuration data for triploid hybrids produced by crossing M.. grindelioides var. depressa with M.. gvmnocephala. Artifactual univalents were likely present in the triploids, but were not individually distinguishable from univalent-containing configurations expected to be produced by an autotriploid. Satisfactory meiotic material was extremely difficult to obtain from that hybrid. Meiocytes seemed to almost invariably be either in early stages of prophase I, or in the tetrad of spores stage. Each head contained only 25-44 florets (seldom as high as 35), making it difficult to find a slightly better stage by "shopping around." For these reasons only 20 meiocytes were scored for each triploid hybrid. Table 11 compares utilization of chiasmata in parents and their progeny, both diploid and triploid. Since only 20 cells were tabulated in the triploids only the first 20 of the 50 cells tabulated for the paternal parent were used in the comparison; the maternal parent was not scored. Those first 20 paternal cells consisted of 35 bivalents having 2 chiasmata and 45 bivalents having 1 chiasma. Alternatively, 20/50 of the total number of paternal configurations observed could have been used with essentially no difference—there would have been 34 bivalents with 2 chiasmata and 46 bivalents with 1 chiasma. Table 12 is a chi-square comparison of observed configurations with those predicted by the model of Jackson and Hauber (1982) and Jackson (1984). As with the tetraploid data, both the number of configuration events and the number of chromosomes involved therein are examined. Unlike the tetraploid data, however, the two types of comparisons did not provide consistent outcomes. The configuration events comparison showed no significant difference between observed and expected for all three plants. The more conservative method of comparing chromosomes involved in the various configurations showed a significant difference between observed and expected for two of the three plants. Pollen Analvsis Tables 13 and 14 show percent pollen viability of populations and crosses, respectively. Anova was performed on arcsine transformed percent pollen viability data. Comparisons were made between each of the two 26 parental populations of successful crosses, and between those pooled populations and their hybrids. Table 15. Electrophoretic Analysis Alleles Allele frequencies for all enzymes examined in the diploid populations are contained in Table 16. Although electrophoresis was performed on the tetraploid populations and results were recorded, the techniques employed did not permit identification of the number of representations seen for each allele. The assumption was that tetraploid plants had four genes for each nuclear-coded enzyme. When two or three different alleles were seen in the same plant it was impossible to identify which alleles were present in multiples. Consequently, allele frequencies could not be calculated. Table 17 lists alleles seen within each tetraploid population. Most tetraploid plants exhibited no more bands than would be expected of a diploid, even when three or more alleles were found within the population. Two plants of population 7633 had a phenotype of 3 simultaneous alleles for the enzyme PGM-1. Only in GOT-2 of population 7632 were four representations of different alleles seen in the same plant, and then only once. Since allele frequencies for the tetraploids could not be calculated, discussion, tables, and figures that follow will refer only to diploid populations studied unless otherwise stated. Loci for ALDO, G6PD, and IDH were fixed for the same allele throughout all populations, assuming equal electrophoretic mobility equates to the same protein structure. K. rhizomatous possessed private alleles GDH-a and PGI-a, the slowest migrating form seen for each enzyme. GDH-a was fixed in H.. rhizomatous whereas GDH-b was fixed in all other populations. PGI-a was not the only allele for PGI in H.. rhizomatous for it also had PGI-c which was present in a single population of Nl. gymnocephala. G2DH-b was fixed in X^. tortifolia. and present in only one other population, then in low frequency. The CAT-c allele was exclusive to X_. tortifolia. and PGl-e was found only in M. blephariphylla 7616, although in neither case was the allele fixed. ME allele c was found only in M.- gvpsophila and M_- restiformis. and was fixed therein. Also found, but not fixed, in M_. restiformis was GOT-2 allele a, making it the only population and species to exhibit all 3 GOT forms. That allele would also 27 be expected in Nl. gvpsophila. but small sample size may have precluded its detection. Several alleles appeared to be fixed within one species yet variable in others. For species in which more than one population was sampled, fixed alleles not previously discussed as private were: M.. gypsophila-CAT-a. MDH- 3-b, ME-b, PGM-2-b, TPI-b; M. bleDhariphvlla-MDH-3-h. ME-b; M. grindelioides (including tetraploids)-CAT-a, ME-b, PGI-d, PGM-l-c, PGM-2-c, TPI-b. The enzymes PGI and PGM are typically highly polymorphic and were found to be so in this study, too. Measures of Equilibrium For polymorphic loci, chi-square tests for deviation from Hardy- Weinberg equilibrium expectations were performed (Table 18). Although results of the chi-square test generated by BIOSYS-1 are reproduced herein, it should be noted that many are not valid due to small sample sizes in concert with apparent allele frequencies that led to low expected numbers within some classes. When classes were combined to meet the previously discussed chi-square criteria, the only populations and enzymes that continued to show significant differences were M.. riparia (7550) G3PD, ME, and PGM-1; M. gvmnocephala (7521) G3PD; and Nl. gymnocephala (7528) LAP. The fixation index (F), another measure of equilibrium, is listed in Table 19 for each population. Fixation indices varied to both extremes overall, from -1 (only heterozygotes observed) to +\ (no heterozygotes observed), although well over one half of the polymorphic loci exhibited reasonably small deviations (between -0.2 and -1-0.2). Predictably the same loci that showed significant deviation from Hardy-Weinberg equilibrium also had F values of large magnitude. Measures of Genetic Variability Genetic variability measures over all loci, tabulated by population, are in Table 19. Both direct count mean heterozygosity and an unbiased estimate of Hardy-Weinberg expected mean heterozygosity (Nei, 1978) are shown. Measures of genetic variability by locus within each population are in Table 20. 28 The proportion of polymorphic loci exhibited large ranges both overall and within species. M_. gvmnocephala 7542, which exhibited virtual fixation for all loci, had the lowest rate of polymorphism (0.0%). Nl. restiformis followed fairly closely by Nl- gvpsophila exhibited by far the highest (53.3% and 46.7%, respectively). M.. blephariphylla 7559 was considerably lower than the general range of polymorphic loci for all populations, whereas M. blephariphylla 7616 was somewhat higher than the general range. Table 21 summarizes Fjs, FIT, and FsT at all loci. Although individual loci exhibited considerable variation, overall FsT was the substantially greater contributor to FIT. Extreme heterozygosity for ME, indicated by an Fis of -1, is correlated with its F value of -1. Both were due to the fact that only in M. riparia were different alleles seen, and then only heterozygotes were observed. Fairly large excess homozygosity was present for G2DH and LAP. Other loci exhibited slight to moderate excess homozygosity or heterozygosity. Since GDH had two identified alleles it was considered a polymorphic locus. The fact that one population was fixed for one allele (H.. rhizomatous for GDH- a), and all other populations were fixed for the alternate allele yielded a 100% probability that two alleles chosen at random from a given population would be identical by descent. Genetic Relationships Matrices of genetic similarity and genetic distance based on the methods of Nei (1978), Rogers (1972), Cavalli-Sforza and Edwards (1967), and the Prevosti distance of Wright (1978) are in Tables 22, 23, 24, and 25, respectively. The coefficients of these tables were used as input for construction of the various dendrograms that follow. UPGMA phenograms using various genetic distance and similarity coefficients, each followed by its cophenetic matrix and homoplasy matrix, comprise the next 4 sets of figures and tables. In order, they are based upon Nei's (1978) unbiased genetic identity, Figure 8, Tables 26 and 27; Rogers' (1972) genetic similarity. Figure 9, Tables 28 and 29; Prevosti distance (Wright. 1978), Figure 10, Tables 30 and 31; and Cavalli-Sforza and Edwards' (1967) chord distance. Figure 11, Tables 32 and 33. Goodness of fit statistics are also listed for each phenogram. 29 Distance Wagner trees based on Rogers' (1972) genetic distance and Prevosti distance (Wright, 1978) are shown in Figures 12 and 13 respectively. Each is followed by its patristic distance and homoplasy matrix. Tables 34 through 37. FITCH examined a total of 2499 trees over all cycles to optimize a fit to the Rogers matrix; 816 trees were examined on the last cycle to fit the final population into the framework of the previous 15. For the Prevosti coefficients, 2501 total trees were examined, 816 on the final cycle. The Fitch- Margoliash dendrograms are in Figures 14 (Rogers' genetic distance) and 15 (Prevosti distance). Tables 38 through 41 contain the respective patristic distance and homoplasy matrices. Virtually all distance matrices and dendrograms, regardless of method, indicated the following relationships among the populations and species tested: M.. blephariphylla and Nl. gymnocephala were formed into a large assemblage with no particular distinction made between the two species. Population 7644 of M.. gymnocephala was uniformly separated from the rest of the assemblage and joined directly to the ancestral line. All populations of M. linearis segregated together. Nl- linearis and the M_. blephariphylla/M. gvmnocephala complex were depicted as derived from common ancestry. M. gypsophila and Nl. restiformis were closely related; Nei's genetic distance of 0.077 between the two suggested that only about 8 detectable changes per 100 loci have ocurred during the separate evolution of the two species from a common ancestor. Distance matrices depicted Nl. riparia and M.- grindelioides as closely related, with most dendrograms showing them as having shared a most recent common ancestor. Both K- tortifolia and H_. rhizomatous were distant from the Machaeranthera species. Xylorhiza was somewhat closer to Machaeranthera than Haplopappus. The greatest patristic distance between any two species was that of X.- tortifolia and H. rhizomatous. Table 2. Chromosome numbers of populations. 30 Species Collection 2n Number of Chromosomes H_. rhizomatous Jackson 5071 8 X. tortifolia Jackson 7598 12 M. riparia Jackson 7550 10 M. gymnocephala Jackson 7521 8 Jackson 7528 8 Jackson 7539 8 Jackson 7542 8 Jackson 7644 8 M. blephariphylla Jackson 7559 8 Jackson 7616 8 M. gypsophila Jackson 760IG 8 M. restiformis Jackson 760IR 8 M. grindelioides var. depressa Jackson 7631 16 M. grindelioides Jackson 7632 16 Jackson 7633 8 Jackson 7636 16 M. linearis Jackson 7602 8 Jackson 7641 8 Jackson 7642 8 Tj- o o o o 31 vo r- w—H ^ o o o o o O O (N ^ VO r- vo CN O O O O cn o ^ vo r- cn cn O —' o o o o cs o o vo r- ..oM (U co 3- •3 E O cn cn o ^ o o CJ •3 00 3 c O X) 1— o cn cn o o o o o o o sful . lets . o flo w c/3 1— u lUi ^-u^ CJ (X .c CJ "3 00 oo o ocncnoo 3 3 o cs o C/] •»-C3« O 3 § k. 3 u JZ CJ ex CA > o o o cn o o o o C/3 > .-• •3 o o cn o (S o Ui 0 ki 3 T3 > U c/3 CJ t-i > ,.. ^^ ki k. flj O CJ 3 (_l c OOO o CJ ooo —I o o ooo ooo ooo x; o X> T3 r~- cs Tt ^ Tt —. 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Xi O CA sz 3 QJ QJ t^ r^ r-« E 03 3 Qi U CA •3 CO XXX 03 3 x: O 3 Ul 3 Tt Tt Tt T3 ^ QJ OJ O O —' > ca E o CO "3 CO .—I 3 .T -3^ 3, . «J o QJ 3 u. cn cn cn 3 O OJ NO vo vo U 3 OJ. r- r- o- >—^ 3 Xi QJ CU CT OJ CX . o C/i Si 03 CA Xi 46 QJ CJ 3 • • oJ CO —, 3 * * * • • * 3 • 3 OO W-3 Os vo vo cs OO CS OO Os X cn Tt —^_ cn C3S cs o OS w-3 Tr~t- o CA ^Q^J C/3 k- QJ Xi 4-1 3 E QJ f-4 o O 3 3 > z 3 •3 ^ 3Oj 3 OJ O E .—1 CA OJ OJ k- 3 wn Tt cn Tt wn r^ oo ON *3 wn cn cn oo -H 1^3 op W-) IM u 3 ,_i o u 4—* Oj 3 ea QJ UI S 3> • v^ .2 PQ W3 wn wn w-3 cs cs ^ '3 Tt Tt OO o vo vo f-H oo Tt cs CA QJ CA CA o QJ XJ BU I 3 Z ia s el l o oo Tt OO CS Tt CS CTv o 03 3 x: Os cs CS cs NO OO OJ O u w^ w-3 Tt wn Tt CA QJ k« (4H QJ 4-c1 s O CU QJ k- ea CX OJ ea 3 00 3 '> 00 x; 00 cs cn vo x: 3 3 3 3 4.H Ul .— 3 OOO 3 k. QJ OJ 03 u. CX CX O cx N CO CA 03 CA ooo 03 o > SZ 3 OJ 3, ON Os E oo oo CX3 QJ wn wn > CS CS CS CO W3 CA w-3 wn w-3 wn CA OJ O ca r^ r- r^ 3l :=: > > 3 E o 4-> ^ si oo .-• . . CA Si Os .. 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X Oj QJ -3 T3 o 3 u vo k- QJ <1) ,.—\ /*—s ^'^s. •-^ CJ CJ *•*** O y—S /"-^s y—^V •n OO .-I ^—•^ u w-^-3\ w-3 O T3 QJ OJ •3 03 OJ > wn w-3 »n wn vn > vo ..^ wn wo WO CA 3 Pale r CJ 03 U S OJ QJ CU cs 3 CX wn ^ s OJ c/3 si w^ "^ X) QJ 48 CJ 3 ea o CA (A 3* $ I 3* 3 CO o- r^ cs cn vo OO X CD o CS vo NO wn CJ CO ^Q^J C/3 Ul CJ Xi C QJ O -H O -H B3 va l • •^ z 3 "3 D 3OJ 3 ea O E •f—• CO ea 2 u3 r^ o oo r- o w-3 '3 NO cs Tt vo W-3 Tt op Tt vo t*H u 3 ,_ O U ^a ea 3 ;ui s 03 al e > • ^4 2 CQ Os cn Os cs cn Os cn wn 3 w-3 cn w~i cn U cn r^ Tt w-3 OJ QJ Xi OJ E CA 2 QJ oo Tt VO 3 3 U Tt OO OJ U w-3 «J QJ CU 03 cn Tt cn Tt 03 SZ o o Tt 3i 00 00 o o 00 QJ 3 wn wn 3 wn wn CJ o O Ul c O 'k- 'ui CX CX 3 OJ 3 CA CA cx cs OJ cs cs CA o E 03 > w-> wn W3 w-3 OS 04 w^n OJ cn CA wn ^3 X X CO X X 3 (A OJ * * o 3 00 CO CS OO cs Ol cn r-~ cn X No c^ vo wn ^ w-3 oC O ••—1 QJ to u Qqu i J en t cs o o ^ 3 3 > z •8 ^ ^ •3 3 D OJ 3 OJ o E CA •*^ca u 2 3 NO r^ cs vo OO '3 Tt OJ 00 cs W-) Tt 'CZ u 3 o ,__, u ^-^ OJ 3 ca QJ UI S 3 > .2 Tt I-H OO Tt —' •CQ ^ 3 w-3 CS u cn cn w-3 cs QJ BU I Xui n ) Z el l ia s o o 3 Si Oj u QJ U Ul wn wn C4H QJ s O Cu OJ OJ OO f—1 vo 00 f-H sz O QJ 3 o • ^^ o 3. o c 1 1 r—\ ^ ' '"^ (-1 'f 1 QJ /•"N XOiJ /—N O 3 /-»» exc/3 . 03 cx o o o o CA ^-^ 3 t: ^ 3 3 oo oo > o oo E cs cs 03 OJ > > CO wn wn y—\ 3 CO w-3 CJ O/i OJ > r~- r~- o 3 > r-- 3 3 <-H OO 3 X X oo 3 ^ X 03 X cs QJ 3 73 3 cs QJ 3 3 3 3 •3 wn Ul CJ QJ /~v QJ /"^ y^ W-) k- QJ QJ /•—s QJ /*-S O •3 CJ CA 03 o- ea o Tt CJ CO 3 QJ CX ec t CJ pa r er v (0 4 CO CO cx O v^ (0 4 cx lec t O -o CO f-H CA CO cx r-H 3. ko. 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QJ Q> 4J NO O vo O o o o o O QJ CJ CX Xi X B 000 " W 3 .00 0 .00 0 z .00 0 C/2 T3 ^ cn OJ cs NO NO 3 CN cn oo Os W-) w-3 wn w-3 *^ ea 3 cx O ea Ul .33 2 .99 7 3 ,33 2 .99 5 OO CN NO vo OO o Ov o r- • ^^ CN ^ VO Tt cn cs c Tt CM o OO 3 •^ OS O 03 .-H 3 u ^•^ .66 8 .00 3 ea c .66 8 .00 5 > o Os (S r^ oo oo oo rt NO QJ 00 OO ON f-H W-3 w-3 .i •3 nvo l nvo l CT eve n QJ eve n QJ QJ •3 T3 T3 •3 •3 X2 T3 "3 "o • —^ 31 3 kn 3 •3 X QJ QJ CJ 3 QJ QJ CO (*5 4-1 C/3 3 03 OJ 3 > *-• vo QJ Ct e rv e E k>- QJ k- o rv e U>l CA O o o OJ QJ QJ •^ QJ QJ QJ QJ 3 X QJ CA QJ QJ o X •^ CO E CO E CA CA OJ CX CA CX OJ CX CX QJ o •—s o .2 Xi u. Ul Xi X CO Xi X u Xi X CO -O X 4-1 -a w Pi 3 o o Ui O O ca O 00 W O w c QJ o w ig u k- Oj o E E 3 E T3 OO UH o oo <4H o QJ u •1—» cs 3 Ul cs 3 C HH w-3 O Si wn o Si o r^ CJ 3 c/3 U U u O J4 "3 CO X X U o CJ CJ OJ CO •3 CO O Tt Tt Ul o CN O CJ o CJ o .—3 I cn cn CA CO 03 C NO NO QJ QJ li k- o- TD O K, Oil Xi QJ ca O CX H e C/D Xi QJ 52 O 3 OJ o CO •3 QJ 3* •3 3 3 QJ OO 3 3- CX CO _o CO OJ CO 3 3 W-3 w-3 CJ 03 cs O CA Tt CS •3 X CS 3 03 (4H 3 ^- O .—1 c •^ X 03 QJ ko- u ^—t a 3 3- CO 3 O o QJ O o o QJ CM CO "ea c rat i u . QJ [01 ] o OJ QJ CO X CO CA Ul CO r—1 3 CA Si y-^ cx o cx X) 00 Xi X Xi X CJ CJ II T3 "o CJ W E OJ X) o CM O Ui o O w QJ cx 3 CJ CJ o N*-f-H ^ 3 Ul E E 03 o oo O Si (4H •3 3 o E f—1 o o 3 cs CJ CJ o •3 u 3 tD c QJ f-H CJ w-3 O QJ Xi o r^ o o CO CJ ^^ > u i.— 03 3 o cd QJ SZ QJ "^ •3 CO c O X X) CM O QJ A A ^^ 3 CO o Ta QJ o -a -3 CA ,_^ E w-3 04 OH 3 CA D 3 •_) o O Tt Species Average Standard Range Population Number Viability Deviation Low High H.. rhizomatous 5071 80.61% 8.15% 72.00% 88.20% K. tortifolia 7598 88.90% 5.95% 79.88% 96.05% M. riparia 7550 87.34% 11.90% 70.69% 96.84% M.. gvmnocephala 7521 98.43% 1.71% 96.60% 99.90% 7528 96.00% 2.66% 93.74% 98.94% 7539 94.28% 4.31% 89.76% 98.35% 7542 93.84% 3.81% 89.92% 99.02% 7644 92.57% 5.57% 84.96% 98.66% M. blephariphylla 7559 95.29% 4.63% 88.43% 97.25% 7616 97.25% 0.35% 97.00% 97.50% M.. gypsophila 7601G 76.12% 12.59% 63.85% 89.00% M. restiformis 7601R 68.38% 6.97% 61.32% 78.40% Nj- grindelioides 7631 74.62% 6.12% 69.92% 84.79% 7632 80.55% 6.65% 71.60% 89.40% 7633 90.30% 2.33% 87.40% 92.70% 7636 69.38% 11.80% 62.15% 83.00% Nl. linearis 7602 92.64% 6.28% 89.90% 98.32% 7641 93.81% 3.04% 91.73% 97.30% 7642 93.34% 5.13% 88.35% 99.00% Table 14. Pollen viability of hybrids. 54 Species Crossed Average Standard Range Population Nu mbers Crossed Viability Deviation Low High H. rhizomatous X M. blephariphvlla 5071 X 7559 59.16% 6.20% 49.20% 66.00% M. gvmnocephala X M. gvmnocephala 7521 X 7528 61.89% 4.84% 57.93% 68.80% 7521 X 7542 61.38% 7.63% 53.50% 68.74% 7528 X 7521 60.19% 9.99% 49.24% 68.82% 7528 X 7542 58.61% 6.86% 49.43% 65.60% 7528 X 7644 66.45% 7.99% 60.80% 72.10% 7539 X 7644 66.93% 8.51% 59.56% 76.24% 7542 X 7521 49.50% 15.06% 34.00% 66.20% 7644 X 7539 59.15% 8.05% 49.73% 66.82% M. gvmnocephala X M. blephariphvll:a . 7521 X 7616 33.26% 11.96% 20.20% 50.20% 7539 X 7616 55.31% 5.60% 49.50% 61.84% M. blephariphvlla X M. gvmnocephala 7559 X 7539 59.96 5.94% 53.37% 64.90% 7616 X 7521 30.70% 8.06% 25.00% 36.40']'c M. gypsophila X M.- restiformis 7601G X 7601R 77.79% 11.10% 66.80% 89.00% M-. restiformis X R. gypsophJla 7601R X 7601G 70.05% 6.21% 63.65% 78.42% Natural hybrid of M_. gvpsophila and M.- restiformis 7601F 74.51% 8.59% 68.30% 89.00% M.. grindelioides X M.- gvmnocephala 7631 X 7528 11.72% 4.13% 5.20% 16.20% Table 15. Pollen viability anova of parental populations and crosses. 55 Species Crossed Degrees of Significance Populations Freedom H.. rhizomatous X M_. blephariphylla 5071 v/s 7559 1,5 10.311 Pooled 5071 & 7559 v/s 5071 X 7559 1, 10 24.964 * * • M. gvmnocephala X M.. gvmnocephala 7521 v/s 7528 1,4 1.664 n.s. Pooled 7521 & 7528 v/s 7521 X 7528 1,8 109.384 * * * 7521 v/s 7542 1,5 3.523 n.s. Pooled 7521 & 7542 v/s 7521 X 7542 1.8 46.478 * * * 7528 v/s 7521 1.4 1.664 n.s. Pooled 7528 & 7521 v/s 7528 X 7521 1,7 66.247 * * * 7528 v/s 7542 1,5 0.450 n.s. Pooled 7528 & 7542 v/s 7528 X 7542 1,9 90.554 * * * 7528 v/s 7644 1.6 0.753 n.s. Pooled 7528 & 7644 v/s 7528 X 7644 1,8 23.144 * • 7539 v/s 7644 1,6 0.129 n.s. Pooled 7539 & 7644 v/s 7539 X 7644 1.9 28.335 * * • 7542 v/s 7521 1.5 3.523 n.s. *** Pooled 7542 & 7521 v/s 7542 X 7521 1, 10 64.712 7644 v/s 7539 1,6 0.129 n.s. Pooled 7644 & 7539 v/s 7644 X 7539 1, 10 55.804 ** • M_. gvmnocephala X M_. hlephariohvlla 1,3 0.912 n.s. 7521 v/s 7616 *** Pooled 7521 & 7616 v/s 7521 X 7616 1, 11 175.135 7539 v/s 7616 1,3 0.717 n.s. • • * Pooled 7539 & 7616 v/s 7539 X 7616 1.7 128.509 M_. blephariphvlla X M.- gymnocephala 1.5 0.120 n.s. 7559 v/s 7539 * * + Pooled 7559 & 7539 v/s 7559 X 7539 1, 8 64.809 1, 3 0.912 n.s. 7616 v/s 7521 * • » Pooled 7616 & 7521 v/s 7616 X 7521 1,5 186.094 Table 15. continued. 56 Species Crossed Degrees of Fs Significance Populations Freedom M. gypsophila X M.. restiformis 7601G v/s 7601R 1. 6 1.424 n.s. Pooled 7601G & 7601R v/s 7601G X 7601R 1, 9 0.939 n.s. M. restiformis X M_. gypsophila 7601R v/s 7601G 1, 6 1.424 n.s. Pooled 7601R & 7601G v/s 7601R X 7601G 1, 10 0.083 n.s. M. gvpsophila. M . restiformis. and their natural hybrid 7601G X 7601R v/s 7601R X 7601G 1. 5 1.487 n.s. Pooled 7601G & 7601R v/s 7601F 1. 9 0.102 n.s. Pooled 7601G X 7601R & 7601R X 7601G v/s 7601F 1, 10 0.061 n.s. M. grindelioides X Nl gvmnocephala 7631 v/s 7528 1, 6 36.610 * * • Pooled 7631 & 7528 v/s 7631 X 7528 1, 11 87.873 * • * n.s. P>0.05 * 0.05 > P > 0.01 ** 0.01 >P> 0.001 *** P< 0.001 cs o 0 Tt 0 0 0 0 Tt O 0 0 CD 0 CD .00 0 Tt vo Tt Tt .91 2 .08 7 ^ CD cd d d 0 0 o 0 0 OS Tt CN 0 CS 0 cn cn NO CD 0 0 , , VO cn o cn cn 0 0 cn 0 CD —' cd CD d ^ ^ 0 CN 0 0 Tt VO 00 0 00 0 00 cn NC o cn o cn 0 CD 0 CD 0 cn CD Os 0 vo cn —' CD O cn 0 ON f-H o r-~ wn 0 CS 0- o o 00 Ci r- CD CD r- VO f-H CO 3 OJ CJ 0 0 o ^ HJ H Z X z 303 Q Z 03 < ^ 03 Xi o Q ^ 03 JD ea Xi HJ ^ CJ O H < o 58 OJ o o o cn Ci CD OJ o 2 NO .87 0 .13 0 ^ ^ s -H* d 0 0 Tt O O 0* — o o o NC Tt Tt Ol — oc r^ O C3S VO <^ s ^ .90 5 .09 5 cn o cn o cn C4-I CD CD 0 0 o c; J. d cs o o o cn r- o Ov oc 2 vo cn O cn '>° ]C^ X » S ^ 0.94 7 0.05 3 cn O ON C4-3 O CD o o cn o-H* d o cn cCSs cs •^ s ^ .95 5 .04 5 ^ .00 0 .16 7 «~^ s .00 0 vo f-H O O —' -^ 0 0 -H d d O o o .-H o o o~ O o t^ CD r- o r- r~- .00 0 ,14 3 ,85 7 O .00 0 VO ^ d »—» d ^ 0 0 000 * vo o 0 C?N 0 f-H ^ CD CD o 2 .91 2 VO Tt .08 7 cn Tt .02 5 .97 5 rH* d 0 0 ^ 0 0 3 C?v o wn wn o w^ W-3 O wn CD CD wn o CD .00 0 .00 0 .00 0 w-3 .00 0 ca '- d f-H d ^ d ^ "3 CX 0 000 * Tt 0 o Tt 00 0 33 3 o vo CD vo NO VO 66 7 VO CD 0 o OH f-H d ,-4 O wn o —' o o cs 0 000 * 0 cs Tt Tt 0 CD 0 wn f-H ;=: CD CD •-^ o — c f-H d -J d '^ d -- 0 00 cs o Tt so Os 0 ^ o cn Tt .00 0 Tt Tt 2 .0 2 wn crj 00 -M ^ 0 0 d d d 0 r- CS 00 ^ Os o cs 0 0 0 00 CJ 0 ^ ^ ^ Tt 0 Os wn C<3 CTN CD cn o f-H d d d d d CS 00 r~- cn o o 0 0 c^ vo cn 0 W-) w-i cs cn OS CD u-3 wn -r o 2 Tt 0 CTN wn cn o o- d d JC^ O; Oj d d o 0 dvo Tdt cs 00 cs 0 cs o- (S NO cn wn CD pc^ w-^ Tt 01 w-3 Tt CD Tt d d d 0 d 0 00 0 o o 0 Os 00 00 wn cs CD 0 «» S ^ OJ o OJ O (S CD 0 f-H —' T3 d (NJ CD vo CO 3 CJ X o Q o Q o CU V'. Xi O Q Z CU ^ OH I NO 00 CS 0 0 ^ vo cn 0 0 0 Tt O O CD O- CS O .00 0 Tt vo Tt .00 0 0 ^ 0 ^ 0 CD CD CD CD CD CD 3 Ov 0 0 O O wn 0 W-3 0 w-3 <=> w-3 0 0 0 0 0 .—I wn ^ CD CD CD CD CD CD 03 0 —' 0 ^ 0 CD CD 0 0 O cs 0 0 Tt 0 Tt Tt "=> f-H 0 0 0 0 0 wn f-X CD CD CD CD CD Ci 0 ^ 0 ^ 0 CD S CD '-^ CD (D 0 -H cn NO Os CD OJ 0 cs 0 fvj cn cn cn cn 0 CD CD 0 CD wn Tt d Tt CD CD vo cn CD CD d d CD d> CD CD CD CD 0 0 O 00 0 0 0 0 CS <=> cs 0 0 0 0 0 wn Tt Tt C*^ o o o o o o 0 r-H 0 0 — CD CD CD ^ CD CD 0 0 O 0 0 cs 0 0 0 wn Tt 0 0 Tt 0 0 ^ CD CD O CD CD CD 0 I-H 0 ^ 0 d> CD d ^ CD Tt VO 0 o- cn 00 00 wn Tt 00 0 00 *^ "^ Os wn Tt 0 0 0 wn CS fS cs O cn CD w-3 NO o o c^ o w-1 w-3 Ov Tt w-3 o Tt CD C?N O o NO o c^ o o cn .02 5 .97 5 o o o o d d o o o d 3 Os o o o O wn wn o w-3 o wn <=> C3N o o o CS o cs o ^ o cn CD CD wn Tt CD CD CD CD Tt CD Tt O CD CD d .-H d d d f-H d d ^ d oo o o cs o o O o o o CD CD Tt CD CD o <=> wn Tt CD d f-H d d d d •5- o o o • • • o O O O -H o OJ o o o CD CD CD CD CD O CD o wn Tt Tt o <=> o- d d d d '-' d " •g- o o o oo o • • • o cs O r-l O 1 C3S o CS o W-) CD CD Tt CD CD o O wn Tt wn Tt OJ O d d d d d d ^ CD CD CD O d o'-< " d 00 o OO OO O oo o c^ CD O CD CD CJ O wn cs o o o o cs cs o o o d ^ d d ^ d QJ 3 o o NO Tt 3 ^^ o o ^ r~ cs o OJ Os O O o cs o o o o OJ o o 3 w-3 O -^ o o O O —' O d d d O vo CO 3 CS o CJ O HJ i z « Z ea o 03 -O o "3 03 Xi O o, ^ 03 JD O CU CU Table 17. Alleles present in tetraploid populations. 61 Locus Population and Alleles Seen 7631 7632 7636 ALDO a a a CAT a a a GDH b b b GOT-2 b, c a, b, c, d^ b, c G2DH a a a G3PD a, b a, b a, b G6PD a IDH a a LAP a, b a, b a, b MDH-3 a, b a a, b ME b b b PGI c, d, e c, d d PGM-1 b, c b, c a, b, cb PGM-2 a, b b b TPI b b a, b a. The phenotype abed was seen in one plant. Other plants exhibited no more than 2 alleles of either a, b, c, or d. b. The phenotype abc was seen in two plants. Other plants exhibited no more than 2 alleles of either a, b, or c. Table 18. Chi-square test for deviation from Hardy-Weinberg equilibrium. 62 Species Locus Class Observed Expected Significance^ Population Frequency Frequency H. rhizomatous 5071 CAT aa 20 19.024 ab 0 1.951 bb 1 0.024 41.026 NA GOT-2 bb 17 17.146 be 4 3.707 CC 0 0.146 0.171 n.s. G3PD aa 19 19.024 ab 2 1.951 bb 0 0.024 0.026 n.s. PGI aa 2 2.220 ac 10 9.561 CC 9 9.220 0.047 n.s. TPI aa 20 20.000 ab 1 1.000 bb 0 0.000 0.000 n.s. X. tortifolia 7598 CAT bb 27 26.053 be 1 2.895 cc 1 0.053 18.327 NA MDH-3 aa 9 8.455 ab 13 14.091 bb 6 5.455 0.174 n.s. PGI bb 5 3.455 bd 10 13.091 dd 13 11.455 1.630 n.s. Table 18. continued. 63 Species Locus Class Observed Expected Significance^ Population Frequency Frequency M. riparia 7550 G3PD aa 3 10.649 ab 35 19.701 bb 1 8.649 24.139 *** LAP aa 28 24.289 ab 8 15.422 bb 6 2.289 10.154 NA ME aa 0 10.373 ab 42 21.253 bb 0 10.373 41.000 *** PGM-1 CC 8 14.519 cd 33 19.963 dd 0 6.519 17.959 *** M. gymnocephala 7521 GOT-2 bb 33 31.456 be 5 8.089 cc 2 0.456 6.489 NA G2DH aa 37 36.039 ab 1 2.922 bb 1 0.039 24.996 NA G3PD aa 26 19.971 ab 1 13.058 bb 8 1.971 31.396 NA LAP aa 1 0.076 ab 2 3.848 bb 37 36.076 12.154 NA Table 18. continued. 64 Species Locus Class Observed Expected X^ Significance*^ Population Frequency Frequency 7528 GOT-2 bb 30 27.987 be 7 11.025 cc 3 0.987 5.717 NA G3PD aa 31 31.141 ab 5 4.718 bb 3 0.141 0.158 n.s. LAP aa 3 0.266 ab 1 6.468 bb 36 33.266 32.971 NA 7539 GOT-2 bb 30 29.940 be 11 11.120 cc 1 0.940 0.005 n.s. G3PD aa 28 28.493 ab 9 8.014 bb 0 0.493 0.623 n.s. LAP aa 0 0.012 ab 2 1.975 bb 39 39.012 0.013 n.s. PGI cc 16 16.602 cd 21 17.880 CF 0 1.916 dd 2 4.554 df 3 1.012 ff 0 0.036 7.856 NA 7542 GOT-2 bb 13 13.000 be 1 1.000 cc 0 0.000 0.000 n.s. 7644 LAP aa 4 2.545 ab 0 2.909 bb 2 0.545 7.619 NA PGM-1 bb 0 1.364 be 6 3.273 cc 0 1.364 5.000 NA Table 18. continued. 65 Species Locus Class Observed Expected Significance*^ Population Frequency Frequency M. blephariphylla 7559 GOT-2 bb 13 13.034 be 2 1.931 cc 0 0.034 0.037 n.s. 7616 CAT aa 38 38.013 ab 2 1.975 bb 0 0.013 0.013 n.s. GOT-2 bb 34 31.456 be 3 8.089 cc 3 0.456 17.613 NA G3PD aa 33 33.266 ab 7 6.468 bb 0 0.266 0.312 n.s. LAP aa 0 0.013 ab 2 1.975 bb 38 38.013 0.013 n.s. PGI dd 30 24.111 de 3 14.778 ee 8 2.111 27.252 NA PGM-1 bb 36 36.076 be 4 3.848 cc 0 0.076 0.082 n.s. PGM-2 bb 37 35.078 be 0 3.844 cc 2 0.078 51.361 NA TPI aa 0 0.013 ab 2 1.975 bb 38 38.013 0.013 n.s. Table 18. continued. 66 Species Locus Class Observed Expected Significance*^ Population Frequency Frequency M.- gvpsophila 7601G CAT aa 6 5.077 ab 0 1.846 bb 1 0.077 13.091 NA GOT-2 bb 0 1.154 be 6 3.692 cc 1 2.154 3.124 n.s. LAP aa 1 0.077 ab 0 1.846 bb 6 5.077 13.091 NA PGI dd 5 5.077 df 2 1.846 ff 0 0.077 0.091 n.s. PGM-1 bb 4 4.231 be 3 2.538 cc 0 0.231 0.327 n.s. PGM-2 bb 1 0.769 be 3 3.462 cc 3 2.769 0.150 n.s. TPI bb 5 5.077 be 2 1.846 cc 0 0.077 0.091 n.s. M. restiformis 7601R CAT aa 6 5.440 ab 5 6.120 bb 2 1.440 0.480 n.s. GOT-2 aa 0 0.913 ab 0 0.304 ac 7 4.870 bb 0 0.000 be 1 0.696 cc 4 5.217 2.567 n.s. LAP aa 1 0.261 ab 2 3.478 bb 9 8.261 2.789 n.s. Table 18. continued. 67 Species Locus Class Observed Expected Significance' Population Frequency Frequency 7601R (continued) MDH-3 aa 10 8.261 ab 0 3.478 bb 2 0.261 15.439 NA PGI dd 1 0.913 df 5 5.174 ff 6 5.913 0.015 NA PGM-1 aa 7 3.957 ab 0 4.870 ac 0 1.217 bb 3 1.217 be 2 0.696 ec 0 0.043 13.527 NA PGM-2 bb 5 5.913 be 7 5.174 cc 0 0.913 1.699 n.s. TPI aa 0 0.043 ab 2 1.739 ac 0 0.174 bb 8 8.261 be 2 1.739 cc 0 0.043 0.347 n.s. M.. grindelioides 7633 GOT-2 bb 19 19.070 be 3 2.860 ec 0 0.070 0.077 n.s. G3PD aa 21 20.023 ab 0 1.953 bb 1 0.023 43.024 NA LAP aa 3 0.651 ab 2 6.698 bb 17 14.651 12.144 N.\ Table 18. continued. 68 Species Locus Class Observed Expected X^ Significance^ Population Frequency Frequency M. linearis 7602 GOT-2 bb 33 33.133 be 5 4.733 cc 0 0.133 0.149 n.s. G3PD aa 17 17.027 ab 2 1.946 bb 0 0.027 0.029 n.s. LAP aa 2 0.080 ab 0 3.840 bb 36 34.080 50.028 NA MDH-3 aa 0 0.000 ab 1 1.000 bb 33 33.000 0.000 n.s. 7641 GOT-2 bb 29 29.092 be 4 3.815 cc 0 0.092 0.102 n.s. G3PD aa 17 17.146 ab 4 3.707 bb 0 0.146 0.171 n.s. LAP aa 0 0.000 ab 1 1.000 bb 31 31.000 0.000 n.s. 7642 GOT-2 bb 33 33.266 be 7 6.468 cc 0 0.266 0.312 n.s. G3PD aa 21 20.396 ab 5 6.208 bb 1 0.396 1.173 n.s. Table 18. continued. 69 n.s. P > 0.05 * 0.05 > P > 0.01 •• 0.01 > P > 0.001 *** P < 0.001 NA Not applicable. The expected frequency of one or more classes is not large enough. See text for discussion. a. Degrees of freedom = 1 for all except the following wherein d.f. = 3: 7539 PGI; 7601R GOT-2, PGM-1, TPI. 70 3 3 O O O w-3 NO oo Tt Tt r- r- o o wn o c3s c?N r^ Tt cn vo wn OS c^ o o o O —' o Tt o O Tt cn oo .-H o oo QJ o o d o d d d d d d —' o CO O •3 OO QJ >-. 1/3 cn r- cn wn oo N O ^n 00 w-3 O- NC O- O 03 C3s r~ c^ w-3 Tt O O vo O NO o oo .5 CD -4 o Tt o -H wn TJ- w-3 cn w-3 Tt 3 o o o d QJ D o o o d d odd X •3 -^ cs —' Tt vo oo Tt OS C3S cn o -H QJ CO ON r- Ov Tt Tt C?N C3S w-3 c^ vo o oo 03 O -^ CD Tt .H 03 cx o cx 3 3 [I. 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K Q ^ I 3 H g OH „ OH S o H o b Q OH ^ O Jn < O cn < O O ol cn < O O HJ o < o HJ O O H4 OH o HJ OH o O O O HJ 3 3 03 O "ca CJ 3 x: O 3 QJ ca CJ CTs (S Tt 3 o 00 Os Tt Tt CO CX 3 cs OJ cn QJ o E w-1 wn w-3 wn NO •5 OH > o- r^ r^ Xi QJ CUi 03 CX H C/i si 72 3 3 O U ownwnocnooo cn wnr^r-wnr-oow-3 o r- o NO o o NO CJ cn oo — oo—'OO o W-, o oo cs cs <» QJ ooooodoo CD oc O CN Tt Tt cs d o d d o o o CO O OO •3 >% QJ c/3 cys C7NCSCSOSOVOOSC> N Tt r- Tt Tt cn w-3 Tt 03 cs TtOvOTtvOC7sC^•^ vo cs NO NO vo Cs NO o cs w-3 cs cs cn Tt cs k-l ".S ocs — ocnooo QJ d o o d o o o .—* 3 ddcDddddd QJ D X T3 Tt c:^ooovvow-3r-c^ wn O w-3 w-3 t^ OS w^ QJ Tt c^ Tt Tt cn W-) Tt CO cs TtONOTtWnOsOvTt cs Tt cs cs cn -t cs 03 ocs — ocnooo « dddddddd d d d d d d d 3 tin O o o o t^ cn r^ o> .—H vOTtvoNOTtcnovo ^ CSCSOVCSONW-JOCS o w-1 o NO r- vo vo <-* X o o r~ o — cs o —I 03 Q> ovooor--ocDO X •3 3 ^ d ^ d d d d UH dddddd — d •3 QJ mONOowncjocjOTtwn NO cs vo vo oo cs NO 4H o-oovor~r~-TtTto- Tt OS Tt Tt cn NO -^ CA o cn ONOTtONO»OOOOOv oo vo oo oo w-3 Tt oo QJ QJ CX c> 4-4 4-HOONOHHTtcncn^H f-H cn .-H f-H cs cn ^H o X 60 W o l-l QJ '«H -3 QJ QJ > X I— CJ cs oJcnr-cscnTtocs O vo O cs cn cn cs CA Xi O HH OJ CS — OJ CA cs cs I I I 3 c- f^ Q HH S S H H Ou HH S S CJ H H t^ a. OOO OH < O < OOO CU o O < O ^ OH OH OH H HJ O UOO OH OH 0. U O HJ QJ 3 3 03 t— 3 ^-M O > CJ 3 -3 03 O 3 1 '«H^ •k —. X 03 03 3 (TV O o "3 x: c^ vo CO CA CX 3 w-) ^H D o QJ O VO > CJ o wn vo "G OH X) r~ r~ Xi CJ 03 CX c/3 Si 73 3 3 O U wnt^o-ot^r~cncn VO O — ooNovoo-Hvooocn cn o OS o cnNO-HOTtt-HU-3cn '-; Ci CD QJ oooooooo odd CO O (30 •3 >> QJ CO '-lC^oo•-Hunf-HTt O OS Tt N 03 o-<»c7NO\cnvocno cn oo o o TtTtoJOJTtwnTtcn -^ CD rn IH 'X) 3 QJ oooodddd d d o* QJ o X "3 QJ cnc^oooocncscncs r^ f^ oo CO w-^Nor^r^-HH^t'-'CJv cs oo c^ Tt Tt cs OJ Tt w-3 Tt cs -H o cs 03 oooodddd "« odd §^ ocsoooocscscn cn o Tt W-3CSOOO<7NHHTt r~ o c^ ca QJ — Tt Tt CD O vo Tt HH o o vo X T3 E .S odo—'dooo d ^ d T3 QJ 4-H ooooooTtcn-«toJ o cn oo cso-t^o-r~-ooo-w-3 vo w-3 CTv CA o oo ON NO QJ QJ »^ooTtTt^r^^vo 4H CX vdwncncnw3vow^cn cs' .-H* vo o X 00 Ui N o k- QJ T3 QJ QJ > X Ul QJ wnoocsowncsr^Tt cn o CS CA Xi O cn CO cs cs cs 3 o OH o < 9 O O O OH O cn < HJ u o 2 OH OH OH H O O HJ QJ 3 C 3 CO O CJ o 3 CA o 'E •f-» k- ON 03 QJ 3 o T3 cn CA CX o 3 cn QJ o (A so QJ vo k. 'o OH QJ Di QJ 03 CX C/i Si Si 74 3 3 O O cs w-3 O o ^^ •^^ ^^ w-3 w-> cn ol cs cn o^-H o cs r- oo o '—' o o — HH O f-H ^- QJ o o o o ooo o o CA o -3 CO CJ CA W-) cs —c a\ NO vo — (N O 03 O O OJ — — cn VO cn o cs HH f-H O k- -O ' '— —1 o — OJ QJ 3 QJ o o o o ooo o o X D •3 OS H^ T^ — QJ cn O o o vo CA CN O o CN — — cn NO OJ 03 ^^ —' —• O —1 — o -^ OJ CQ o o o o ooo o o 3 [JU O o vo o w-3 W3 wn NO NO OS t~- wn o f-H NO o f-H o r~ —' —' X ^ o o o o o o o X -Q O O —' o ooo o o -3 QJ cn vo o o wn o- o oo oo O f-H CA Tt Tt vo QJ cn o o o o CJ r-> Ov oo o OO r-~ o Tt cs .-4 ex o X Tt -H cn — cn cn ^H vo NO 00 Ui o k- CJ -3 QJ QJ X > QJ •«t Tt CO wn cs o — r- w^ Xi O CO cs cs cs 3 X (-4 Q a O HSO. Q t! CU o O cn < OC cOHn ^<^ O cn O O HJ '>^ O O HJ O O QJ 3 3 3 O CJ 3 O ca OS 3 cs ,-H cs CO CX o Tt Tt QJ o so NO NO o O OH r~ r^ O 03 CX H c/3 si Table 20. Genetic variability at all loci. 75 Population Mean Sample Mean Number Percentage Mean Heterozygosity Size Per of Alleles of Loci Locus^ Per Locus^ Polymorphic^ Direct H-W Count^ Expectcd^'^ H. rhizomatous 5071 20.8 1.3 13.3 0.054 0.058 ( 0.1) ( 0.1) (0.033) (0.031) X. tortifolia 7598 28.1 1.2 20.0 0.057 0.071 ( 0.1) ( 0.1) (0.038) (0.044) M. riparia 7550 41.5 1.3 26.7 0.193 0.124 ( 0.3) ( 0.1) (0.096) (0.056) M. gvmnocephala 7521 39.4 1.3 20.0 0.015 0.050 ( 0.4) ( 0.1) (0.009) (0.027) 7528 39.0 1.2 20.0 0.023 0.038 ( 0.6) ( 0.1) (0.014) (0.022) 7539 41.3 1.3 20.0 0.075 0.068 ( 0.3) ( 0.2) (0.042) (0.037) 7542 13.3 1.1 0.0 0.005 0.005 ( 0.4) ( 0.1) (0.005) (0.005) 7644 5.9 1.1 13.3 0.067 0.069 ( 0.1) ( 0.1) (0.067) (0.047) M . blephariphvlla 7559 14.9 1.1 6.7 0.009 0.009 ( 0.1) ( 0.1) (0.009) (0.009) 7616 39.9 1.5 33.3 0.038 0.071 ( 0.1) ( 0.1) (0.013) (0.026) Table 20. continued. 76 Population Mean Sample Mean Number Percentage Mean Heterozygosity Size Per of Alleles of Loci Locus^ Per Locus^ Polymorphic" Direct H-W Count^ Expeeted^''^ M_. gvpsophila 7601G 7.0 1.5 46.7 0.152 0.163 ( 0.0) ( 0.1) (0.066) (0.051) Nl. restiformis 7601R 12.1 1.7 53.3 0.181 0.218 ( 0.1) ( 0.2) (0.061) (0.058) M.. grindelioides 7633 21.8 1.2 13.3 0.015 0.035 ( 0.1) ( 0.1) (0.011) (0.022) M_. linearis 7602 36.3 1.3 20.0 0.018 0.024 ( 1.3) ( 0.1) (0.011) (0.012) 7641 31.1 1.2 13.3 0.023 0.022 ( 0.8) ( 0.1) (0.014) (0.014) 7642 38.9 1.1 13.3 0.024 0.026 ( 0.9) ( 0.1) (0.016) (0.018) a. Standard errors are in parenthesis. b. A locus is considered polymorphic if the frequency of the most common allele does not exceed 0.95. c. Unbiased estimate (Nei, 1978). Table 21. Summary of F-statistics at all loci. 77 Locus Fis FsT FiT CAT 0.498 0.669 0.834 GDH ... 1.000 1.000 GOT-2 -0.122 0.364 0.286 G2DH 0.653 0.962 0.987 G3PD -0.007 0.182 0.176 LAP 0.690 0.586 0.872 MDH-3 0.384 0.888 0.931 ME -1.000 0.914 0.829 PGI 0.094 0.557 0.599 PGM-1 -0.279 0.767 0.702 PGM-2 -0.044 0.859 0.853 TPI -0.134 0.741 0.706 MEAN 0.064 0.722 0.740 CS 78 Tt 86 3 88 0 86 3 87 0 77 7 86 0 69 3 99 9 00 0 .60 1 .62 1 .77 6 .86 0 vo .86 1 .83 0 3 QJ o- o o o o o o o o o o o o o o — T3 OJ Tt o. 86 4 88 3 86 4 87 0 77 6 86 1 00 0 •c- 00 0 .60 4 .62 3 .77 7 NO .86 0 .86 2 .83 1 O o- o o o o o o o o o o o o o — o C o cs o 86 4 88 6 86 4 87 0 78 0 69 5 86 4 00 0 00 1 .61 1 .62 4 QJ NO .78 0 .85 9 .86 2 .83 0 k- 03 o o o o o o o o o o o o o o o "ea cn 3 cn 14 7 15 0 15 1 86 0 82 7 86 2 89 6 86 1 82 9 86 0 77 5 .62 3 O vo .69 6 .93 3 .85 7 fcO r- o o o o o o o o o o o o o o o 03 Pi CD 75 2 74 9 74 3 72 0 25 5 36 4 36 9 O 74 6 75 4 92 6 36 7 .57 3 VO .68 4 .74 1 .73 9 x: o o o o o o o o o o o o o o o .-H r- CJ > o O o 18 7 79 4 80 3 77 3 79 8 76 5 80 0 80 0 24 9 25 3 07 7 25 3 X3 NO .65 6 .71 5 .79 8 03 o o o o o o o o o o o o o o o c« QJ vo 13 9 15 0 13 9 13 9 80 1 28 2 99 4 99 5 97 6 99 6 22 3 99 5 95 1 "ca VO .61 9 .76 1 > o o o o o o o o o o o o o o o Ov w-3 14 6 14 7 CJ 14 9 14 6 80 0 22 3 60 5 76 5 99 6 29 4 CJ wn 99 9 97 0 00 0 95 2 00 4 3 r- o o o o o o — o o o o o o o o C3 ^c^« oo Tt OJ •-^ Tt 'O 12 5 11 0 12 1 32 9 87 6 26 8 NO 63 8 70 9 95 0 95 7 92 0 95 2 04 9 05 1 o r- o o o o o o o o o o o o o o o 4—> QJ 3 cs o Tt 22 6 80 1 76 6 96 9 04 9 00 0 00 5 60 6 99 6 99 8 w-3 .29 7 .14 9 .14 6 .14 7 .14 7 r- o o o o o o o o o o o o o o o t3 3 ea W-) .03 1 .02 4 .25 7 .28 9 .19 0 .18 6 .18 6 .18 6 4-I .-« . r- o o o o o o o o o o o o o o o *0 c^ oo • ^M ^J 00 5 80 8 00 2 04 4 00 1 99 8 03 0 cs 61 0 75 9 C/3 w^ .21 9 .28 6 .15 0 .14 9 .14 9 .14 9 r- o o o o o o o o o o o o o o o .1^y^ -o w ,, QJ — cs 81 2 00 4 05 1 00 4 00 2 03 2 60 1 76 0 00 QJ wn .00 6 .23 1 .30 2 .15 5 .15 2 .15 1 .15 0 3 o- o o o o o o o o o o o o o o o ^^ QJ CS 60 93 3 r^ ON D o 13 2 w-1 22 1 29 9 22 3 22 2 »-H «_ 20 8 21 3 26 1 63 5 69 8 ^-^ 03 wn .06 9 .24 8 .25 2 .25 3 t^ o o o o o o o o o o o o o o o Vi -H ."- 03 O c oo Os 33 6 27 3 34 4 26 8 26 7 27 6 29 9 35 9 27 4 46 1 .47 2 .47 6 ^ o .37 9 .36 2 .47 3 60 w-3 VHH ca r- o o o o o o o o o o o o o o o 0--B X . f-H .47 4 .49 2 .50 3 .50 9 .55 8 .42 1 .48 0 .44 9 .50 3 .50 2 .44 2 .49 4 .51 0 .77 4 .45 4 03 •^ 50 7 Ss p o11 < • QJ 3 CN Xi O CS .—» cn ca C"? Pi ,—1 oo o f-H oo Os Ol Tt OS vo •—> cn cs —' ri W-3 Tt Tt S -5 pu l r- CjN wn o 85 8 84 6 85 9 85 0 71 3 62 8 85 2 99 3 .60 2 .59 6 C .72 7 .84 9 VO .85 3 .81 5 60 o o o o o o o o o o o o o o o uQJ. ca ca O 08 3 f-H c» O 15 1 70 8 73 6 70 3 73 8 74 2 23 0 QJ 28 7 28 9 .61 5 VO .64 6 .71 4 .72 5 .74 5 o o o o o o o o o o o o o o o > VO 16 8 15 4 15 2 96 6 15 0 94 7 89 2 96 2 96 4 25 8 31 5 NO .61 5 .72 2 .74 0 .96 0 QJ r- o o o o o o o o o o o o o o o CJ 3 03 ON wn 14 9 14 1 14 1 14 3 97 5 98 3 94 1 99 8 91 8 26 2 32 7 03 6 w-3 .60 1 .73 7 .75 5 o- o o o o o o o o o o o o o o o •^ Tt cn CJ Tt 10 8 14 0 15 0 15 4 15 9 91 3 86 2 92 0 34 6 29 7 oo 08 2 3 VO .60 8 .66 3 .89 9 QJ r- o o o o o o o o o o o o o o o 60 cs Tt 15 1 14 3 14 2 14 5 97 3 98 1 93 9 26 4 32 9 08 0 00 2 03 8 an d .59 9 .73 9 .75 7 ance . wn 4—• r-- o o o o o o o o o o o o o o o 36 3 •c ^ Os ca cn 13 8 19 0 19 8 18 3 18 5 63 5 94 2 94 8 32 4 06 1 05 9 05 3 7Z ^ wn .70 7 .71 6 r^ o o o o o o o o o o o o o o o '55 c 90 9 QJ oo oo CN Tt 14 4 14 7 14 8 25 5 72 0 75 9 98 0 05 2 01 9 08 7 03 4 32 0 01 7 •-H W-1 ^Q^J QJ t— o o o o o o o o o o o o o o o Dc cba 60 f-H 10 1 16 1 15 3 14 8 CN 15 1 27 5 34 0 76 9 02 0 05 8 02 7 02 5 04 0 .-V « 59 3 71 6 CN C wn t^ o o- o o o o o o o o o o o o o o o ON 5P r-i ca 98 3 ^^"O o wn 13 2 16 9 24 5 26 0 34 0 27 2 27 3 27 3 23 1 24 1 28 4 24 3 59 8 64 1 c/3 .x wn U. QJ r~ o o o o o o o o o o o o o o o QJ x: 60 •^ o oo Oi ^ Os 37 4 33 7 29 3 26 1 26 4 35 9 28 4 28 0 40 3 40 4 40 7 45 1 o wn .27 8 .35 4 .33 8 <*H — r- o o o o o o o o o o o o o o o o w X) X f-H •JH (/3 r- .38 5 .38 5 .47 0 .38 4 .39 2 .39 2 .39 9 .39 8 .40 0 .39 4 .36 5 .40 1 .40 7 .40 2 •13 CJ o .54 9 03 3 w-3 l^-^> . • •• 3 cn >% O CN .ti k_ *H 01 QJ i2 O oi f-H f-H Tt Os Ol • • ^H 3 oo o oo C3S ca .61 6 .60 7 .44 0 vo .36 9 .36 7 O- o o o o o o o o o o o o o o o QJ x: CS o 39 9 37 0 31 8 37 0 32 7 35 3 49 7 43 6 03 0 04 9 .60 1 .60 3 .42 5 QJ vo .37 0 .36 7 > o- o o o o o o o o o o o o o o o o X) ca cn cn 37 4 30 6 37 4 35 7 40 2 38 1 32 0 33 3 33 7 c/3 45 1 .59 7 .54 8 vo .22 3 .37 2 .36 7 QJ o o o o o o o o o o o o o o c ^ > o 23 9 47 2 48 4 46 8 45 1 47 7 42 3 46 7 47 6 .60 5 .53 7 vo .49 9 .46 8 .45 7 .46 6 r~ o o o o o o o o o o o o o o o QJ O O c 03 o 23 4 35 7 43 7 44 7 43 5 42 0 40 6 41 7 41 9 .56 6 .50 8 vo .43 5 .43 7 .42 7 .45 2 o o o o o o o o o o o o o o o CJ «H vo 03 11 6 10 8 18 3 12 1 11 9 20 2 30 4 38 7 32 9 30 3 30 4 42 4 VO .58 9 .48 3 .42 0 T3 o o o o o o o o o o o o o o o C 03 se e QJ w-3 17 1 11 9 07 1 20 8 33 4 33 4 01 2 33 3 40 2 44 0 O wn .62 5 .48 2 .44 5 .09 9 3 r- o o o o o o o o o o o o o o o 03 Tt Tt 18 7 19 9 35 5 52 1 20 6 25 8 20 6 28 3 29 5 30 3 31 4 20 1 41 4 45 4 VO .59 4 o o o o o o o o o o o o o o o ° 6 se e CS -g ^ Tt o 17 3 19 9 12 1 33 9 33 4 33 4 62 5 07 6 48 1 44 4 f-H 01 2 40 4 44 4 ^.^ ca wn r^ c« t^ o o o o o o o o o o o o o o o NO •'•' ON "^ HH Ov cn 17 6 15 9 16 8 16 6 16 3 36 6 36 4 36 3 36 3 25 0 57 5 wn 51 0 45 3 41 9 43 9 "(A ca o o o o o o o o o o o o o o o TS kn u ca UH oo ^ ca cs 15 4 18 3 10 7 33 1 39 4 33 1 33 1 33 3 07 5 07 0 43 1 60 5 05 5 42 1 •n wn 48 9 W ca o- o o o o o o o o o o o o o o o 3 TJ O 3 60 03 10 0 11 5 cs 15 7 33 4 33 6 33 3 33 3 20 2 09 8 44 2 ca 05 5 40 5 • wM 61 2 wn 47 6 41 8 ca •o o o o o o o o o o o o o o o o N IH QJ X3 a •H o w-1 38 7 38 4 00 42 0 40 9 59 7 53 7 w-3 .33 1 .41 0 .39 2 .41 0 .46 7 .21 9 .39 5 .40 6 .41 3 ^ o o o o o o o o o o o o o o o ca .—I > QJ 03 X^ oo CJs 44 7 46 6 43 8 72 3 43 8 49 5 u .47 9 .43 9 .44 4 .47 4 .49 8 .49 9 .54 7 .55 1 .55 2 on wn <*H QJ o o o o o o o o o o o o o o o o ^ 54 1 56 4 56 5 52 4 54 9 54 4 55 5 65 6 .56 4 4—• o .53 7 .51 8 .56 0 .54 2 .54 6 .55 7 ^ es w-i o o o o o o o o o o o o o o o c 03 3 O (^•*N l^ 03 QJ T3 O Oi —' u. . 1 oo o .—1 oo Os Ol Tt Os NO .—1 cn (-~i —" C J W-1 Tt XJ o CX r- o w-3 Ol cs cn Tt Tt •—• o o cn o rr 03 o w-1 w-1 w-3 wn w-3 w-3 NO w-3 NO NO NO vo NO NO NO sz o w-1 o- r^ r- r^ O- r^ o- O- r- r^ r- r- r-- o- r- CJ OH CS 81 NO wn Tt NO o cs o o NO o o t^ o cn cs oo cs cn wn vo Tt Tt 0^ o 30 3 vo 38 2 38 8 39 0 o o o o o f—H 15 8 23 0 o 28 0 28 7 28 9 vo o o o o o NO 16 8 15 0 15 4 15 2 vo 26 1 32 9 r^ o o o o o o se e Os cn Tt 14 9 14 1 14 1 wn 26 2 wn 03 6 f-H t^ o o o o o o o 38 0 Tt Tt 10 8 15 0 14 0 15 4 15 9 vo 29 7 35 9 r- o o o o o o o o cs Tt 15 1 14 3 14 2 14 5 34 0 26 4 08 0 03 8 wn .00 2 r- o o o o o o o o o ^-v 39 0 oo ON 13 9 19 9 19 1 r- cn 18 6 18 4 29 5 33 6 c^ wn 06 0 06 0 f-H r^ o o o o o o o o o o ^ 4—> t't' l x: oo 60 14 8 14 7 14 8 cs 25 5 33 0 05 3 01 9 08 7 01 7 03 4 IH wn t^ o o o o o o o o o o o ^ oo QJ NO w-1 Tt 10 1 cs 15 3 27 5 35 0 06 0 02 7 02 5 04 0 CJ 02 0 c wn t^ o o o o o o o o o o o o 4—ea> • cm^« Ta o 17 1 25 4 27 2 29 4 25 1 27 3 27 3 24 9 wn 24 0 .26 8 .30 1 .37 5 .13 2 HH wn C/5 t^ o o o o o o o o o o o o o o > se e 26 5 28 2 26 3 28 2 30 1 40 4 40 5 40 9 28 5 36 9 Pr e .35 8 .39 3 .34 0 59 8 o o o o o o o o o o o o o (4H r^ o O oo c^ X ^ NO —' r^ wn Tt .40 2 .49 8 .39 1 .40 7 .40 0 .39 9 .40 6 .40 8 .40 9 .40 0 .36 7 .40 2 •c o .41 5 ta wn ^ 3 w-i O cs ',""! 03 QJ — O a 3 f—a oo o f-H oo CTN OJ • (HCH r^ QJ O cs o o o CJ o o o o vo O o o >^ t^ ^1^ ^^ cn O CN CN CN o cn cn cn de n cn o oo oo oo srs -H O o QJ o o o vo vo vo vo c o o vo t^ —' o 60 O o o- t~- o o cn vo vo VO vo QJ o o. t^ c/3 vo ca ^ o IS vo o o- cs cn cn cn c O vo NO cn vo 3 f-H vo vo OO VO o r- oo 03 oo oo -H o ON o NO O- t— CS cn cn cn wn o Ov vo .vo cn NO vo vo yi wn o OS r^ t^ oo oo OO OO t^ 'QJ — o Tt o o O t^ cs cn cn cn W-1 vo 60 Tt O w-1 vo cn vo vo vo vo O ON C2s r- oo oo oo oo t^ -H O 3 OOO vo r~ t^ cs cn cn c« CN Tt o wn o c^ vo vo cn vo vo vo O CTv O Ov r^ oo oo oo oo wn —' o C OJ o o OCNCSCNCNCSCNCNf-^t^TtCNCNCS wn Ocncncncncncncnvovocncncncn X wn OOOOOOOOOOOOOC»t^t^ONOOOOOO • ^H t^ IH '-^dcDddddddddddd ea oo E c^ ooooooooooooooo CJ w^ 0^^^^^^^.-H^HH—l.-4^_l H-* Ot^r^f^r^r^r-t^r^r^t^f^t^r^r~- QJ ••^ddddcDCDddcDdddd 3 VO O CN 03 O Pi 3 f-H oo o f-H oo Ov 3 O Tf 'G NO o CD d d QJ c CN CJ O NO o CD CD d d c cn cn o Ov QJ cn cn cn CN T3 NO cp ooo t^ d CJ PC QJ cs oo cs — 3 o .-, NO r- r~- CJ vo O O CD C3 o 60 o o o o o I O c» C50 «n w^ QJ c« o NO '-H HH HH ea vo o cz> o o o o *-o t^ O C3 o' C=> CD C3 c fill 3 VO OO OO ON OO OO C30 cn o cs o O O OO VO CO O CO o O O O d d c=i d d d d Ov I I I I I OS OO l^ O cs cs 'H wn cn '-H cn o O O wn C3C30CDOOOO 'o t^ OOOOOCDOO Z. I I • I I Tt w-3TtNO^^r^r^cn^H OOOTtvOCSCSCS 60 Tt NC oooocpoooo C t^ '< r-- 4-4 oooooooooooooo ea I I oo .-Hf-H0sCSv0'-HV0CSOCScnvOf--00 B c^ — w-iTtcnw-30w-3wnHHCs-Hoooooo >s wn OOOOOOOOOOOCDCDCDCZ) C/5 CDCDCDCDCDCDOCDOcdoOOOO 03 I I I I I I I • CX wnONVOTtvO'-'Tt'-'CSTt^t^incsw-3 o •«a-csoocnocnOHHuncn.-Hooo o O—'OOOOOOOOOCI)000 0_ s wn CJcdoOOCDOCDOOOOOOOO o 3 CN o 03 0 Oti o f-H "3 (—) 00 0 .—. 00 OS cs •^ c^ VO m (S —• cs cx w-1 OS w-1 OJ cs cn Tt Tt w-3 ^~^ 0 0 cn 0 't Tt 03 O V/-3 W-1 w-1 w-3 w-3 wn w-3 VO W-3 NO NO NO vo NO NO NO t^ f^ 0- t-~ f^ t^ 1^ 0- OH f^ r- r^ r~ r- r- r^ r^ CN 84 Tt VO t^ .00 0 ^ Tt VO 00 t~- .00 0 .99 5 C —' o O CN O VO .00 0 .99 1 .99 1 CJ O T-H o o CJ cn >N cn 4-^ vo r>- .00 0 .79 8 .79 8 .79 8 03 —' o o o Pi 1 o '(A vo .00 0 .69 5 .69 5 .69 5 .69 5 o -' o o o o O o f-H 3 QJ o 60 vo .00 0 .84 9 .69 5 .69 5 .69 5 .69 5 —' o o o o o CN VO ON VO .00 0 .69 5 .69 5 .79 8 .84 6 .84 6 ,84 6 -' o o o o o o in IH wn QJ wn .00 0 .96 3 .69 5 .69 5 .79 8 .84 6 .84 6 .84 6 60 — O o o o o o o o 0< Tt 60 Tt 3 vo .00 0 .90 1 .90 1 .69 5 .69 5 .79 8 .84 6 .84 6 .84 6 . —H t^ -^ o o o o o o o o 3 CN 00 Tt wn .00 0 .90 1 .99 8 .96 3 .69 5 .69 5 .79 8 .84 6 .84 6 .84 6 t^ '- "ea o o o o o o o o o 3 ea c^ cn wn .00 0 .94 3 .90 1 .94 3 .94 3 .69 5 .69 5 .79 8 .84 6 .84 6 .84 6 QJ t~- —' o o o o o o o o o o 00 3 oo CN wn .00 0 .94 3 .98 2 .90 1 .98 2 .96 3 .69 5 .69 5 .79 8 .84 6 .84 6 .84 6 < v—t o o o o o o o o o o o o OH cs wn .00 0 .79 6 .94 3 .97 6 .90 1 .97 6 .96 3 .69 5 .69 5 .79 8 .84 6 .84 6 .84 6 t^ — o o o o o o o o o o o o ^H O o wn >< wn ,00 0 .79 8 .79 8 .79 8 .79 8 .79 8 .79 8 .79 8 .69 5 .69 5 .86 8 .79 8 .79 8 .79 8 t^ 4-H -- o o o o o o o o o o o o o ea oo 6 c^ wn ,00 0 .66 9 .66 9 .66 9 .66 9 .66 9 .66 9 .66 9 .66 9 .66 9 .66 9 .66 9 .66 9 .66 9 .66 9 o t^ 4—* -^ o o o o o o o o o o o o o o QJ 3 O CN QJ O C?v x: o wn .59 2 .59 2 .59 2 .59 2 .59 2 .59 2 .59 2 .59 2 .59 2 o .59 2 .59 2 cx wn .59 2 .59 2 .59 2 o ^ o U 3 oo O • —H CN •-H 03 QJ O Pi ,-^ oo o -H oo Os (N Tt ON vo _ ^^ cn CN —• Ol X5 cx r- ON wn Ol OJ cn Tt Tt wn •— o o cn o Tt •^ 03 o o wn wn w-1 wn wn wn VO w-1 NO vo vo vo NO NO sC OH wn r^ r^ o- r^ o- r^ r- f~- r- r- r^ r- o- o- r~- OJ 85 Tt VO CD o> d Tt VO CD O t^ O 3 d CJ OJ CN CS o NO o o CJ CD O CD o O O* CD CJ cn I cn —I Tt O vo VO W-3 W-1 t^ CD O O O CD CD CD CO Pi I I I oo —' r~. (jv I o —' vo vo NO 'c/3 vo O O CN VO t^ O Tt Tt CS Tt HH t-O O O cys vo CD O O O O O O o o o o o o o I I II 00 c^ ^ cn cs Tt Ol cs .-4 IH wn o Tt cs wn ^ -H o QJ wn CDOOOOOO-4 60 CDcdcDCDCDCDcdcd O II I I I I Tt t^ c^ OO (S (S Tt w-3 60 Tt -H o o Tt vo o o VO COOCDOCDOOCDCD 'Vi ooooooooo 3 I I I < CN Os -HOTtCSO^OO Tt —I OTtoiwn^^o c/3 w-3 OOOCDOCDOOOO t^ OoOOOOOcOOO 13 I I I I I I 3 ea Os TtOOCS-»tCSOTtvOCSO cn Ocnoo—icsocncncn wn 0 ..H OS C/3 O w-1 o > w-1 .03 7 .31 3 .31 3 .20 5 .15 4 .15 4 .15 4 QJ r^ o o o o o o o o IH CLH Tt 60 Tt o G NO .10 0 .10 0 .31 3 ,31 3 .20 5 ,15 4 .15 4 ,15 4 t^ o o o o o o o o o 1/3 3 CS c/3 Tt o C<3 wn ,10 0 .00 2 .03 7 .31 3 .31 3 .20 5 .15 4 .15 4 ,15 4 >^ r^ o o o o o o o o o o ca G ca Os cn o IH wn .05 9 .10 0 .05 9 .05 9 .31 3 .31 3 .20 5 .15 4 .15 4 .15 4 QJ t^ o o o o o o o o o o o 4-H C/3 3 oo O cs O wn .05 9 .01 8 .10 0 .01 8 .03 7 .31 3 .31 3 .20 5 .15 4 .15 4 .15 4 < t-- o o o o o o o o o o o o :s r—l CS o(IH o w-1 .02 4 .05 9 .02 4 .10 0 .02 4 .03 7 .31 3 .31 3 .20 5 .15 4 .15 4 .15 4 ^ o o o o o o o o o o o o o (4-1 o o w-1 o X w-3 ,20 5 ,20 5 .20 5 .20 5 .20 5 .20 5 .20 5 .31 3 .31 3 .13 2 .20 5 .20 5 .20 5 IH r~~ o o o o o o o o o o o o o o 4.H see * see * see * see * see * see * see ' see ' see ' see ' see ' 03 se e wn se e B oo cn OS o cn CJ wn t^ o o o o o o o o o o o 4—1 o o o o QJ 3 c^ Os Os c^ C?v c^ CTN c^ C?v c^ Os ON Os CTv CTN QJ .-H t-.- -3 O Tt Tt Tt Tt Tt Tt Tt Tt Tt Tt Tt Tt Tt Tt Tt CX o O wn OOO O o o o o o o o o o o O o u 3 o O cn 4—• c3 Pi QJ ^^ O 3 f—l oo o f-H oo c^ cs Tt o vO ,—> cn OJ -—' cs Xi CX o- Os w-1 Ol OJ cn Tt Tt w-1 r—l o o cn o •^ TT 03 o o W-3 wn w-3 w-1 w-1 W-3 NO wn VO vo NO NO NO VO VO H OH wn O- r^ r- r~~ r-~ r^ r~- r~- r- r~- r^ r- t^ r-- r- cs 87 Tt vo o f^ CD ^^ Tt VO CO o • c/3 r»- cd 3 d O cs Ol OJ o o o *G vo CD t~~- o o o cd o cd o 1 o cn cn cn NO wn w-3 ^~v vo CD CO CD o oo r-^ CD O Crs- o o f—H a O oo W-3 o NO o- W vo o O o o o x: r^ • ' • 60 o o o o* o •c 1 ' ' of-H Tt cn vo w-1 ^ o OO cn OJ (S '^-^ NO o o o o o o QJ t~- o o o o o o O vO cs NO r>- Tt Ol f-H ta n •—> w-1 cn o o 00 vo o o o o o o o -5 r^ o o o o o o o 1 «^ c^ _ _ W'3 r- cs cn o wn o W3 cs w-3 <—l f-H f-H w-3 o o o o o CD o o CD revo s o o o o o OH o o• Tt r^ o vo vo w-3 Tt w-3 60 -^ —H o r—l Tt vo o o vo O t^ o o o o o o o o 00 o o o o o O o o o 3 1 1 1 OJ Ov ^H (Ts f-^ Tt o ^-4 oo 00 Tt r-H o Tt Ol w-3 f—l .—H o *^ W-3 o O o o o o o o o o ^ t-- o O o o o o o o o o 03 1 1 3 rt o cn c^ .-H —1 00 cn NO f~~ cn f-H cn . o cn o o f-H cs o cn cn cn 1- W3 o o o o o o o o o o o QJ t^ o o o o o o o o o o o H-OO* 1 1 1 1 1 1 1 1 3 OO vo -H cs -H cn Os t^ oo o r^ vo O cs o o f-H o o w-3 f-H w-1 f—l o o wn o o o o o o o o o o o o r^ o o o o o o o o o o o o 1 ^ Tt v-M cn f-H ,..H cn OS r^ Tt ^ (S w-1 cs o o o o o o cn cn Tt o o o wn o o o o o o o o o o o o o UPGM A t-- o o o o o o o o o o o o o 1 1 1 1 1 1 (HH O o Tt Tt Os vo Tt OO cn cs OJ r^ r>~ oo wn cn Tt OO Tt cn Tt vo —H NO NO vo vo X w-3 o O o o o o o o o o o o O o 'C t^ o O o o o o o o o o o o o o rt 1 1 1 1 1 1 1 ' • 1 OO Tt o cn Tt cs cn o cn cn t~~ Tt OS o cn e Os cn w-1 w-3 cn r~ o f^ wn cs wn o vo r^ r-« w-3 O >-N o o o o o o o o o o o o o o 00 O d r^ o o o o o o o o o o o o o o 1 ^ oo O Tt oo cn .-H c^ cn OS oo o oo O cn W-) .11 6 .45 6 .45 6 .39 6 ,36 1 .36 1 ,36 1 > o o o o o o o o ca Tt u Tt vo 60 o G vo .21 1 .21 1 .45 6 .45 6 ,39 6 ,36 1 cn ,36 1 o o o o o o o o o Eo 3 cs 00 Tt o .21 1 .01 2 .11 6 .45 6 .45 6 .39 6 .36 1 .36 1 .36 1 00 wn t^ o o o o o o o o o o "rt C Os rt cn O vn .17 0 .21 1 .17 0 .17 0 .45 6 .45 6 ,39 6 .36 1 ,36 1 .36 1 QJ o o o o o o o o o o o 00 3 oo cs o wn .17 0 .08 7 .21 1 .08 7 .11 6 .45 6 .45 6 ,39 6 .36 1 ,36 1 ,36 1 < o o o o o o o o o o o o o cs OH o wn .05 5 .17 0 .08 7 .21 1 .08 7 .11 6 .45 6 .45 6 .39 6 .36 1 .36 1 .36 1 D o o o o o o o o o o o o o o wn o X wn .39 6 .39 6 .39 6 .39 6 .39 6 .39 6 .39 6 .45 6 .45 6 .22 3 .39 6 .39 6 .39 6 •c o o o o o o o o o o o o o o ^rt^ oo oo s C3N cs o wn O w-1 ,52 8 .52 8 .52 8 .52 8 .52 8 .52 8 .52 8 .52 8 .52 8 .52 8 .52 8 .52 8 .52 8 O O o o o o o o o o o o o o o «C^J 3 o o o o o o o o o o QJ o o o o o J= t^ NO NO NO NO NO vo NO NO NO NO so CX o O vo vo vo vo o wn ooo O o o o o o o o o o o o O U . 00 HH 3 3 OJ o O cn CJ 03 o <4• ^_ ^ O Pi »4-, 3 f-H oo o •-H OO OS 00 Tt 3 VO CD t^ .00 5 QJ O* o CS QJ o O vo wn .06 1 .14 1 .05 9 .05 3 .44 0 .49 8 ,21 2 ,22 2 ,22 8 ,23 3 t^ QJ o o o o o o o o o o o O 3 oo a cs 4—1 o 19 1 wn .05 7 .01 9 .09 9 .01 7 .03 4 .39 8 ,45 5 ,17 0 ,17 9 ,18 6 o o o o o o o o o o o o (4-1 O cs o wn .02 0 .06 5 .02 7 .10 7 .02 5 .04 3 .40 6 .46 4 .17 8 .18 8 ,19 4 ,19 9 o o o o o o o o o o o o o rt E o W-I o wn .30 9 .30 0 .34 2 .28 1 .27 1 .28 3 .31 9 .28 6 .34 3 .13 2 ,27 2 .27 9 ,28 3 QJ o o o o o o o o o o o o o o cCJ LLP- see * a SIS ' 4—1 oo Tt 00 c^ o .-H wn o cn .50 7 .54 9 .48 8 .49 0 .52 6 .35 4 .41 1 .47 9 .48 5 .49 0 o o o o o o o o o o o o o o o CJ CJs Os 00 Tt cn o wn Tt .59 4 .53 3 .55 2 .53 5 o .56 0 w^ 1.52 2 1.53 5 1.57 1 1.44 0 1.49 8 1.38 4 1.52 4 1.53 1 OH 3 Tt O cn .-H rt QJ O Pi 3 _ oo o -H oo c^ Ol Tt Ov NO ^.^ ^ cn Ol — OJ X5 OH r~ CTv w-3 Ol Ol cn Tt Tt w-3 .-H o O cn O ^ TJ- C3 O o wn wn w-1 wn w-1 wn NO wn vo vo NO vo vO NC NO H OH w^ r- f^ r~ r- r~- r^ o- r- t^ r^ r^ t^ r~- r^ r~ cs 91 Tt NO o f^ cd ,_, Tt NO CO o r^ cd o* . cs CO o 3 NO CO CO CD QJ f^ o CD cd •U ^^ (HH (*H cn QJ cn O vo CD CO o o CJ r-- cd cd o d QJ Pi O f-H cs cs w-3 G o VO VO VO 4—a4 vo CD CO CD O CO 00 r^ CD cd CO O* O* • ^^ T3 O f-H o o cn CJ O Os Os O H-* vo CD CD CO CD CD CD QJ (^ G cd CD cd cd cd . 3 O wn • w^ cn 4~-t ca QJ O Pi .—1 Ol • vo .14 2 .14 8 ,15 3 r~- ., cd o o o x: Pi 60 o Ul o vo .31 3 .38 2 .38 8 ,39 3 ^ r~ o o o o o •<—• O »-H oo QJ wn O O o 3 vo .23 0 .29 9 .30 5 .31 0 a t^ o o o o o o 00 vo o VO .34 6 .42 9 .18 9 ,19 9 ,20 5 ,21 0 c/3 o o o o o o o O > QJ c^ w-3 wn o t-- o o .03 6 o .31 0 o ,39 3 o .15 3 o .16 3 o .16 9 o ,17 4 60 G Tt f-H Vi Tt NO 3 o VO .08 2 .11 8 .29 7 ,38 0 .14 0 ,15 0 .15 6 QJ o o o o o o o o o QJ 300 ' cs Tt o QJ wn .08 0 .03 8 .30 8 ,39 1 ,15 1 ,16 1 ,16 7 ,17 2 3 t~- o o o o o o o o o o 60 cA cn o wn .06 2 .14 2 .06 0 .06 0 .37 0 ,45 3 .21 3 .22 3 .22 9 ,23 4 QJ o o o o o o o o o o o O C f:a oo 4-> OJ oo o wn .05 8 .01 9 .09 9 .01 7 .03 4 .32 6 ,41 0 ,17 0 .17 9 .18 6 ,19 1 o o o o o o o o o o o o (4-1 see ' o cs o wn .02 0 .06 6 .02 7 .10 7 .02 5 .04 3 .41 8 .17 8 .18 8 .19 4 .19 9 t^ o o o o o o o o o o o o o ca E o wn o wn .30 9 ,30 0 .34 3 .28 1 .27 1 .28 3 .31 9 .36 0 .44 3 .13 2 .27 2 .27 9 ,28 3 QJ CJ o o o o o o o o o o o o o o c oo Os O Tt wn .44 5 .43 7 .48 0 .41 8 .40 7 .42 0 .45 6 .35 8 .44 1 .34 0 .40 9 .41 5 .42 0 o o o o o o o o o o o o o o o (J oo cs 00 NO cs .47 1 .54 1 .45 9 .50 7 .46 7 .48 8 .53 1 .47 1 o .49 7 o wn w^ 1.46 9 1.45 8 1.39 1 1.46 0 rt w-1 OH 3 vo O cn O Pi o ,-H OJ ^ OO O OO <7v Tt ON vO ^H ,-H cn OJ ^ cs 3. r~- o wn CS OJ cn Tt Tt W-1 — o O cn O "^ Tt a o o wn wn wn wn wn W-3 NO w-3 vo NO NO NC vO VO vO H OH w-3 r- o- r~ r~ f^ r- f^ t^ r^ r^ [^ t^ o- t-- r~ cs 93 Tt NO o o* Tt vo CD o cd CD c/3 Ol 3 o O >o o CD o t^ CO CD o" cn oQJ cn CJ vo o o t^ CD CD CO o* cd o' OO Pi ON CD VO o o o .01 0 ,00 3 r^ o o o o o O 60 o vo o o o .01 8 .01 8 ,02 1 t^ o o o o o o QJ VO O 3 VO o rt t~- .08 5 .10 0 .02 0 .04 5 ,05 5 ,05 8 o o o o o o o 00 • —^ c^ w-3 o o wn .04 7 .05 5 .00 4 .02 1 ,02 8 ,03 0 00 O t~- o o o o o o o o > QJ Tt Tt o o o o CO vo .01 0 .02 1 .00 3 ,00 2 60 o o o o o o o o o G CS ISO ' 3 Tt o o o o o wn .04 3 .01 7 .02 5 ,02 6 QJ o o o o o o o o o o (•3 c^ cn o o o o QJ w-1 .00 3 .07 4 .11 6 .01 4 .03 2 .04 3 .04 9 C t~- o o o o o o o o o o o 60 rt oo cs o o o o wn .00 6 .01 2 .07 2 .07 9 .02 2 .03 5 .03 9 .04 2 QJ o o o o o o o o o o o o O G rt HH cs 00 o o o o w-3 .00 7 .00 7 .00 2 .06 0 .06 7 .01 7 .03 5 .04 3 .05 0 r-- o o o o o o o o o o o o o O o ISO " wn X o o o w-3 .06 9 .04 9 .03 0 .09 9 .03 0 .05 2 .05 9 .06 8 .00 6 ,01 0 'C o o o o o o o o o o o o o o la oo E c^ o O HH o o o >^ wn .16 0 .15 5 .17 9 .15 5 .06 9 .15 5 .17 4 .04 9 .00 4 .01 0 00 t^ O o o o o o o o o o o o o o o ca o cn t~- o o o -- o 06 4 E o .08 2 .08 7 .16 5 .06 1 .05 9 .06 5 .10 7 .05 6 .04 2 .06 1 ,06 1 wn o o o o o o o o o o o o o ooo c X 3 O cn H-* 03 CJ O Pi 3 _l oo o f—l oo c^ cs Tt c^ NO cn (N .—, OJ CX r~ o wn cs cs cn Tt Tt w-3 ••^ o o r^, o Tt Tt rt O O w-3 w-1 w-1 w-1 w-1 wn NO wn NO NC ^ NO o o vo o- wn r- r- r- r-- t^ r^ f~- t^ t*- f-- o- r- r~~ r^ r- CS 94 "* NO CD t^ cd 00 wn 3 Tt o O NO o o t^ CN r» f-H o o O -4 o vo CJ CO o o cd 6 CJ cn 3 t^ vo o cn vo VO t^ rt NO CO 00 t~- o Oi CN Tt -"f oo CD O Tt Tt Tt VO QJ t-- o CS cn cn cn 3 CD CD cd CD cd QJ O 60 —' —4 cn cn f^ o wn cn oo oo oo o ^ cs cs Ol cs CN vo ON CD cd O CD CD CD NO *-H t^ OO O CS HH u-i VO r- cn NO NO NO vo tA O cs cn HH f-H f-H f-H b- rg o cn ONr.-t--cnooc?v-H 00 wnw-iOCSHHHHcnOvOw-ivOTtvovOO IH 4—1 o r~cnc3Noocsoooof^oc7NW-iw-300^H in wn OTtTtcncnTtcncncnTtcnTtcnTtTtTt OH cdooooooooooooooo 3 OC3 o cn 4~( ta Pi QJ O f-H ON Tt C3s •—3 • . , oo o oo cs vo ^-, en cs .—' cs XJ CX r-- Ov wn CS Ol cn Tt Tt w-1 —' o o cn o -r Tt cri o m W^ wn wn wn wn w-3 NO w-1 vo NO NO vo vo o vo t^ t~- t^ H OH w-1 r- r~- t-~ t~- t^ r-~ r- r~ e' r- r- r^ cs 95 Tt vo CD t^ o ^^ Tt NO CO CD r~ cd o* 00 cs o en t vo o o o • ^^ t^ • • • CJ o o o ' ne t r^ CD CD cd CD o CD QJ 1 60 vo c^ cn oo OO _ cn ^.^ r-H •—1 Ol o o f-H f-H CS VO o CO o CD CD CD CD r^ cd o* cd CD CD CD C3N d Os "^ NO ^^ O wn o o o CD w-3 o "oo o o o o o o o VH t^ o QJ o o o1 o1 1 o1 o1 o 60 O Tt Ol cs t^ w^ cn wn o ,—i 0< Tt o o cn f-H ^^ f_ _ oo cs cn vo Tt rg o cs o o o o o o o o rt wn o o o o o o o o o o o o r» o o o o o o o o o o o o 1 1 1 1 1 1 1 JG ^ CJ cn vo Os cn cs r~ ,-H f-H . —H cs o o o o o UH wn o o o o o o o o o o o o o r- o o o o o o o o o o o o o (HH 1 1 1 1 1 O o f-H o cs ^ w-> W3 <7N wn cs wn vo OJ wn o f—4 cs cs wn cs f-H Ol cn OJ OJ cs X wn o o o o o o o o o o o o o o t^ 4—1 o o o o o o o o o o o o o o rt 1 1 1 1 1 1 ' 1 1 E oo oo vo cs ON vo oo f-H cn cn oo oo ^H cs ^^ OS o VO vo 00 cn cn cn oo o cn vo oo oo oo >N wn o o o o o o o o o o o o o o o 00 r- o o o o o o o o o o o o o o o rt 1 1 1 1 1 1 «-H Tt O r~- Ol vo o C3S o wn o Tt o Tt oo o r-> f-H f-H r~ cn f-H f-H w-3 Ol o OJ •—> cn o —1 CD CD mop ] o ooo o o o o o o o o o o o o w-3 o o o o o o o o o CD o o o o o o X 1 1 1 1 1 1 1 1 3 C3s O cn o 'ca Pi 3 f-H oo O f-H oo c^ vo .15 8 .23 6 .28 3 .28 2 .28 6 rt t^ tA o o o o o o vo VO o tA .27 8 .35 2 .16 0 .16 2 ,16 2 ,16 6 o o o o o o o o > QJ C?N wn wn o .03 6 .25 6 .33 0 ,13 9 ,14 0 ,14 0 ,14 4 60 o o o o o o o o G t't'l ' tA Tt Tf 3 o vo .08 4 .10 6 .21 6 .29 0 ,09 9 ,14 4 ,14 8 QJ o o o o o o o o o QJ cs 300 ' Tt Xi o wn .08 6 .03 8 .25 8 ,33 2 .14 1 ,14 2 ,14 2 ,14 6 tA o o o o o o o o o o ea cn cn o oo wn .06 0 .12 8 .05 8 .06 0 .30 0 .37 4 .18 4 .18 4 ,18 8 60 t~- o o o o o o o o o o o IH rtI Xi oo CJ cs 4-H o ,—4 wn .05 5 .01 9 .08 7 .01 7 .03 2 .25 9 .33 3 .14 2 .14 3 .14 3 ,14 7 UH o o o o o o o o o o o o o cs w-> o X wn .02 0 .06 2 .02 7 .09 5 .02 5 ,04 0 .26 7 ,34 1 .15 0 ,15 1 ,15 5 t~- 4-H o o o o o o o o o o o o o ca E o wn o QJ wn .23 6 .22 8 .26 9 .22 8 .23 0 .22 6 .24 7 .32 2 .39 6 .13 2 .25 2 .25 2 ,25 6 U o o o o o o o o o o o o o o G rt 4-H oo cn 00 o vo o cn wn .30 8 .30 0 .34 1 .29 9 .30 1 .29 7 .31 8 .35 8 .43 2 .27 7 .32 4 .32 4 .32 8 "5 t~- o o o o o o o o o o o o o o o o H^ oo cn OO oo Wl O Tt Tt o ,39 7 .38 9 .43 0 .38 8 .39 0 .38 7 .40 8 .41 1 .48 5 .36 6 .41 3 .41 3 .41 7 C3 wn ooo o o o o o o o o o o o o o OH 3 O O Tt rt O Pi _ oo o f-H oo o (N Tt OS vo .—. —I cn cs 1— ri CX r^ Os w-1 Ol OJ cn Tt Tt w-3 —I O O cn O -r Tt ca Wl vO vO vO o W-3 wn wn w-3 wn vo w-3 NO vO NO NC o w-1 t^ f^ c~ r- f^ t-~- r^ r- r^ r~- H OH r- r~- r^ r~ r- CS 97 Tt VO 0 0 Tt vo 0 0 t~^ 0 0 tA CS 'S o QJ vo 0 0 0 *o 0 0 0 (4-1 QJ cn o cn CJ 01 7 vo 0 01 7 r- .02 4 0 0 0 0 Pi 00 LOO'O (7N f-H o vo 0. 0 -0.02 5 x: O -0.03 2 -0.03 0 60 o vo t^ 0. 0 0. 0 0.00 6 0.00 3 -0.00 5 -0.00 3 QJ VO CJ 3 VO 0. 0 0.02 3 0.01 7 0.00 8 0.01 2 0.01 4 tA 0.00 8 Os NO 00 O HH 1—I HH Wl O O HH o o o wn CDeOOOOOOCD tA o OOOCDOOcdo > I I I I I QJ Tt CSCSI-HCTV^HNOOHH Tt OOOOVOTtO^H^ vo CDCDOOOOOOO 60 t^ cdocdcDOCDCDcdcd 3 I I I I I I I 00 CS NO NO 00 O 1-H •-> 3 Tt o cn o 1-H o o w-1 QJ t~~ ocpoocDoooocp QJ CDOcdoOCDOOOO I I I <3N CSHHCS OONOt^^CSTt cn OHHO cnnHooo SZ OOOOOCOCOOOOO tA w-3 rt ooooooooooo III III "o 00 cs CS-^CnNOnHTti-H 60 OJ o 0000000 w^ OOOOOOCOCDOOOO IH OOOcdcDcdcDOOcicDCD I I I I I cs NO 00 ON HH cs r-- I OJ O O O O r^ o o X5 w-3 OOOOOOOOOOOCOCD CJ UH ooooooooooooo I I I I I o o Hfi-HW3CnCTvOO<-Hi-H^H oi-Ht~~ w-3 ocscsoiwncscscsoj CSCS^H X wn ooooooooocpoooo oocdoooooooocDcJo "^ till II III NOcnooovot^cSNO CTvcno^H.—I E 00 Os ooJ.-HTtcncncncn cnNOoooooo >N w-3 oooooooo oocooooo 00 ca ooooooooooooooo I I I I I I "a. OTtoccnm-HOOoooscnw-3Ttr~-Ov o QQfr^^f-HNOCS-HCSOOi^CS—'OO t~- E o OOOOOpoOOCOCDOcOCiCDC) o wn oodo'cdooooooooooo X 3 O --1- «J , , ,—. 01 1—1 3 CC 0 -^ oo c^ cs •^ OS NO ^^ cn OJ cx r- OS u-i 01 01 cn Tt TT w-1 —< 0 0 cn 0 Tt Tt ca o .—3 w-1 w-1 w^ w-1 wn w-3 NO w-1 0 vo NO NO NO 0 ^ a. w-3 r~ r-~ r^ r-- t-~ r~ t-- t^ r- r^ r- r^ t^ r~ f~- 98 n Figure 2. Karyotype of H.. rhiy,omatQUS. 99 Ji' Figure 3. Chromosomes of X- tortifolia. 100 Figure 4. Chromosomes of M-. riparia- 101 n u Figure 5. Karyotype of M.. Mfiphariphvlla. 102 Figure 6. Karyotype of R. grindgjifiidcs var. dgprcssa- 103 ' r , « •»v '*\^ < •». -*» "* \j^ J ^ Figure 7. Karyotype of M-. grindelioides var. dcprcssa X M.. gvmnocephala. 104 ca rt (/i rt rt la ZZ a rt 1/3 u w rt rt > > w m 3 'U J= sz JZ sz sz -C x: rt M O o O p p, P, P O, Q rt OJ QJ QJ e> c> •' •• E rt •— • — rt « o on •f— o E ha r s ° de l sz fol i no c no c no c 1- no c c ari s ari s w o rt c O <4- N p (-> ^ u P •— E E E V o c E c P 1/) !->>> — > > .7: t- lin e > S tort i B4 o4 e4 e4 .^ S b4 e4 — (4 ^ Il >^i :sl si ^1 ^1 si d :i| d si si si ^ si si c u -o •3 o <-H00OC«^HH00CSO\voOTtCS-HCSf-HHH r-C3NW-3COCSCSTtW3HHC<^TtO'*TtoO o OW-i>rivow-3W-lW-l>nvOW-lvovOvOvOvOvO O o QJ w-ir-r^r~t~-r~r~t~r^r^r^r~r.«r-t^t~~ O o ^ + CJ 3 vo Q) C3N Cfs T3 O *-^ o Ol 3 cs QJ C?v 60 "O O oo rt Lo 00 00 3 oo 00 3 oo OJ t-- cn CTN w-3 Tt Tt OO tl o QJ vo Z Os 60 o o 3 oo 00 ,—4 Xi OO (A 3 rt E "o rt vo t-i 60 %-, o- 60 ea -; + O CJN 3 -3 QJ r- 3 Xi Oj" cx Si OJ < CJ vo U4 UH o CD PH OJ y^ 3 f-H \Q o 4-* oi S: .2 3 00 00 *> NO vo QJ _o oo 'HH "3 (/3 3 ca O O o VI •3 IJ t-, 3 t-> 60 •3 o rt CS 3 Tt H-l rt vo SO (/3 Vi -^ QJ 3 3 QJ 3 d -3 O O O 105 rt «l Ui ci rt rt rt w « v> ii rt rt rt > > rt rt D 'U J= SZ x: SZ -C x: x: rt C/l P P p. Pl P p, p '^-ot o SU B rt o CJ Q) • — 01 u SU B -^ E rt O o O o o fol i no c no c ari s I-. T3 c 1 )ha r c g.SEEESoEEc " ..: •- > > > — — > >.n lin e lin e restifo r gvpsop l tort i rhizom a l-l (>4b4(^c4.^.^ &4o4-~ _E Kl xl sl :^ Si Si Si sl sl Si i:^ sl sl sl Si Si E C/5 3 -H 00 C*1-H00CSC?N>OC7\TtCSi-HCS-.— o ^-c^^^c^cscSTtlr3l-Hf^^TtOTtT3•oo CD Ow-3vr3V£>inw-iv-iw-iNOw-iv£>vovo>ONCNO O CD w-ic-o-r~t^r~r-t-~t--r~r~t^o-r-r-r~ o o + • o LLI o Tt •«t rt C3s 1 C?N 1 c« CJ 00 00 QJ 00 S QJ o 60 + CN ol cs 00 00 + * o d cn c« IH QJ W3 60 NO vo O o- -t- 1^. ^ o r^ 60 vo 3 c^ .-H ^^ t/3 o 3 sz o t~ tA E ca ea o k- 60 60 Ul O ei C Tt o s vo vo f-H xz Tt -3 cx Tt 3 d ea cn < SZ II o wn 00 00 w^ o tin £ .9 5 W3 = o r- y—\ 3 o VO O II cs r~ •t^ c> cn OS ea 3 ^H • »•« o > .-H C>J QJ Ii Q> 4~* CS w-3 t— "3 W3 c/• i• 3 a 3 o z O QJ ; + o 60 ^H l4H C/l -3 IH 4-1 . 1 kH ^ UH CO 13 o , ^ •^ •3 CJ c3 CS 3 NO rt NO c/3 Tt 4—1 TT an d eti c ;i9 7 ^ HH 3 tH 3 Q> CO QJ QJ i^H SZ O cx er e o ar r ra g (/3 UH OH OH c/3 o QJ u o 3 Tt •a o o O 106 rtj «] u> .2 rt rt ^ ca rt Ui 4> rt rt « > > rt w O 'U JC JC x: sz x: x: sz rt| M o O P P p P P p p *^ rt 4; V u • w^ • ^^ (I) (1) E rt CJ 0 0 u> 0 0 0 k- P. 0 ha r de l fol i no c no c 0 VHI u c »ha r c ari s ari s rt S.5EEE5SEE*> 0 « o u o •J lin e tort i ^ ^ e4 e>i c4 Xl Si c4c4—. c rhizom a e4 k- « c =:xiislslslsl:^slsl:^^slsl^slsl re O Ct. — 00Ocn^00tSC7\vOC>\TtCSHHCS^^ 3 t~-0\w-icncScSTtwii-HcnTtOTt^OC QJ OW-3W-lvOiriW3W3W3>0«nvovo>Ovo>cvC w-ir--t^r^t^t--t^r-r~-r.-t~-r~r-t~>r-r- o LU CJ Iff CX5 vo O vo c^ o xz 60 CS Ol -: -H d O oc ea 00 -t- CD o C^ 00 tA 00 o vd > Tt Tt QJ cs CM 60 3 vo C3S 'c/3 o o 3 cn cn JS tA E CS ea k- "o 60 60 O k. 3 rt NO vo QJ c^ cn cn .3 o CX •3 3 < cs' ea ja O CJ ,_ cs Ol Tt DH Tt UH CD OS •—N 3 VO VO .2 II Tt t~- cn .2 3 f-H > o 00 00 Q> •;2 II "3 rt ^ + o f/3 3 0 0 : "H3H **U>l rt •3 o CJ rt CS Tt H.» CO -3 S CJ w-1 w-3 c^ 3 C/3 "S rt QJ CD ti <= tA o 3 QJ u! 60 QJ r- O ut ed rt Ul ^i ^ O CO UH OH o CO vO O o O vo 3 OH U -; + ^ o •3 O O O 107 rtl ed IO rt Z2 c' al a al a "rt *> > rt al a 'U x: x: x: x: x: j= sz ca to o P P P P Pl 0 c M 03 Q) 4; •— •— «> (1) F 0 (O CO CO rt 0 CJ •— 0 0 0 u u u 9. 0 ha r OO 00 O w XZ o o o NO vj OS >o vo -: + d tA •a kn Tt Tt ea CJ cs Ui CTN -a t^ G VO ea 01 CM 0C5 ea cn cn N k- o CHH 00 vo ea c^ O O Tt Tt > d ea t/3 rt u 60 o G 00 tA ea 00 00 Tt Tt 3 n^ 0 cn £ OJ cs CTN kH an d W-3 60 O sz vo IO S o E rt E o E o o N CJ CJ c « c » Xl H. o 3 - 2 .2 o O Q r Q ." t- .2 o o — o o cn + in Oi + f^ d o d CJ QJ CJ 3 rt o- cs CM 00 CjN IC a o Tt Tt to 3 CM ts O 60 CS Qi, f-H o vo CM « t-- + ^ d' o tA p U. o QJ to 60 O o P rt CD 00 > P 00 C< f-H cn d Si d 60 Si 3 II o c^ /—\ HH 3 o IO r~ O Wl so VO w-1 O HH E c^ + "—' d O ' d rt •-* k. »> V 60 sz •3 O tA + 3 ea c Qi u . o CU ; cs .3 60 cs CX Ul f-H rt ^ ! 0. 1 d QJ r^ s 3 wn cn + 60 f-H -3 cn rt 3 vo vd ea f~ 0\ Ol o SZ o II QJ Tt O 3 rt UH (F i .9 4 - o NO y-^\ 3 o VO o II >o vo 4H CD r^ CD o f^ 3 9 + vi a (1 9 o d II QJ 'HH CS .. •3 C3 CO 3 o ; O QJ -H Ct^ CO "3 Ui HH Ul Ui cn C/3 — ,—" rt o o ^,^ o Ul H-» S -3 o a cs U 3 c/3 O 60 Ov •3 3 Sta n HH •"^ rt QJ 'u^ 4-H iHH ''^'^ 3 Ul 3 er e o ra g arr : d + c/3 UH OH OH CO QJ u 3 -3 O O O 109 E o E c u 4J U 3 * C * 3 QJ rt -^ 2 o .2 o .2 o O rs J~ Q ." (4.H CJ 00 o CM 00 + CJ CJ d oo Ov VO CS NO CM XZ 60 cn CM cn CM QJ CJ 3 rt c O O CS CM V3 O > cn QJ t^ ka OJ OH f-H 1-H CS d d 60 II 00 y^'-S 3 t^ NO Tt •'t E c^ 1-H rt f-H u. d d 60 Si • O tA CS 3 QJ o Xi 60 CX k. ca CJ Tt s 3 OO 60 Tt "3 rt 3 rt df-H CTN CTN -3 o o CJ QJ II H-» r~ CJ 3 UH (F i .9 6 rt = o iO o ,.—\ 3 NO VO O I ^ o II I o wn t^ HH o Tt + —! II cn devi a atio n c/3 n (1 9 o - o Q> Ct-( v> -3 Ul H-» . 1 kf Ui CO ^ O cr, ^ rt o '^ T3 CJ O =3 rt H-* CJ CO •3 97 2 Sta n l"H H-l 3 rt QJ .—1 CHH ' 3 kH QJ CO 3 E E o uo ClH ti CJ CO CJ 3 X c t; C5 c '-' o ca *^ 00 to o rt Ui o o 3 E u Q .^ o Q QJ *o o o cn + cn CHH + QJ d O CJ t -H QJ CJ t^ o 3 cs W'3 cs rt 4-H tA i>^ CJ Tt Tt ^ OO CM QJ cs cjN ca 3 + d QJ 60 o p NO t^ rt CN "rt X: edi c^ (C«A tuto 1 CM cs p — c/l • ^1 « rr u u d o o > u— rt rt d o sz rt 9J 4> W3 UJ c C p W^ c o C •—( • ^rt o t- rt S ;3l •—:3M| + QJ to rt M > « rt P SZ JC x: 60 rt p Si Si 00 OO > Pl p . P x: o w-3 f-H U 4J P i> «> CJ Pi CJ o Sl-^f O QO d o o CJ -H cs si rt c CM Tt Tt c c ^ x: o 60 to E E tys c O NO NO cn :^ E NO 3 o 4J > > E f-4 T3 >n >e4 r^ '^ o4 01 > vn o NO 1^ W 3 t^ vo ^sl vO Si Si d VO d t-» OS E Tt rt u CM CM I C3ON CM • 3 o + d <4H flj d + o a QJ CO fS^ QJ 3 -3 O O O 111 c c o ••J S 'n c - •HI O 5 Z to o 3 CJ Q =" o cn + QJ d d o OC cs ts CTN d 60 Tt TT CM CM O O 3 M to rt CO CM *^^ • ^* CM u ta sd C/) n Ci O d d u c C rt c • »-• ••^ rt ^« . rtl x: 00 -— rt P ^sl O 00 o 00 > f-H > rt o QJ SZ x: o HH Chromosome Numbers Chromosome numbers are consistent with those previously reported for H.. rhizQm^tQus (Johnston, 1961; Hartman, 1976), Xylorhiza (Watson, 1977), and Machaeranthera species (Turner, £tai., 1961; Anderson, eiai-, 1974; Hartman, 1976; Turner, 1973a, b; Stucky and Jackson, 1975). Machaeranthera blephariphylla has not been reported from the area around Camargo, Mexico (Hartman, 1976; Jackson, personal communication). Crossing Studies A variety of non-genetic factors are generally understood to cause failure of artificial crosses (timing of pollination with respect to viability of pollen or receptivity of stigmas, mechanical injury of the pistil during pollination, temperature excesses, inappropriate humidity or soil moisture), so negative observed results of crossing attempts do not necessarily affect ideas of phylogenetic affinities. Incompatability alleles may have been shared by otherwise compatible populations, and could have contributed to some hybridization failures. Determination of shared incompatibility alleles among conspecific and congeneric populations may be beneficial. Bud pollinations, which were not attempted in this study, may overcome that form of genetically determined incompatibility. Formation of hybrid offspring between some collections of M. blephariphylla and M-. gymnocephala. and between M_. grindelioides var. depressa and Nl. gymnocephala does indicate a degree of relationship between those different species. Hartman (1976, p. 110) reported a single specimen of M. grindelioides which possessed white ray florets but was otherwise typical of other specimens from the particular area of collection. He dismissed the possibility of the plant being a hybrid, stating, "the only known sympatric species of the genus is the yellow-rayed M_- pinnatifida which has very different leaf morphology." Whether these two species hybridize is unknown, but the triploid hybrid of the rayless M.- grindelioides var. dcprcssa and the pink rayed variety of M.- gymnocephala did produce a hybrid that could have 112 113 been described morphologically as an M_- grindelioides var. depressa having white ray florets. Presence/absence of ray florets was found to be controlled by a single dominant/recessive gene in members of the H_. phyllocephalus complex, although length was a quantitative trait (Jackson and Dimas, 1981). The trait for ray flowers may have been present in low frequency in that population. Undoubtedly, the triploid offspring of the above cross inherited the gene for presence of ray florets from the paternal N£. gymnocephala. The successful cross between H_. rhizomatous and M.- gymnocephala was important. R. C. Jackson has crossed H.. rhizomatous with Nl. johnstonii and with M.. restiformis. both of Machaeranthera section Blepharodon as is M. gymnocephala (Jackson, personal communication). These successful crosses of H.. rhizomatous with members of Machaeranthera section Blepharodon indicate it has a close affinity with the section. Meiotic Analvsis Populations and Species That two-thirds of the diploid plants showed no significant difference between circle and chain bivalents observed and those expected by the binomial expansion indicated (1) lack of bias, and (2) equal participation by all chromosomes and chromosome arms in formation of chiasmata. The latter is a basic assumption of the models used herein (Jackson and Hauber, 1982; Jackson, 1984). Tetraploid populations of Nl. grindelioides behaved as allotetraploids. Strict alloploidy would preclude observation of multivalents, but alloploid pairing forms a continuum from autoploid type to strict or genomic alloploids. A few multivalents were observed, the number varying with the population and plant. The variation may be attributable to the presence of different alleles of pairing control genes operating at different levels within each plant, or it may be a function of variability and the sample size used. There were no differences in statistical outcome between the tests of configuration events and those concerning chromosomes involved in the configuration events. As previously discussed, this lack of difference was to be anticipated in any situation wherein the observed outcome closely matched the expected. 114 Hybrids The regularity of chromosomal pairing attests to a high degree of chromosomal homology among the species analyzed. Only one diploid hybrid exhibited an unusual number of univalents (M. gymnocephala 7528 X M. gymnocephala 7542). As previously discussed the same plants or other plants from the same populations were crossed and scored with unremarkable outcomes. The high number of univalents and consequent low chiasma frequency was therefore probably due to a pairing control mutation in the population. Of considerable importance is the outcome of the cross between H.. rhizomatous (5071) and M.. blephariphylla (7559). Only one univalent pair was observed, which was no more than commonly seen in non-hybrid plants. The mean number of chiasmata per cell in the hybrid was higher than that of either parent [5.28 for the hybrid versus 4.90 for H_. rhizomatous 5071(02) and 4.94 for M-- blephariphylla 7559(01)]. Consequently, chiasmata utilization for the hybrid was significantly different from that of either parent, but not because it was reduced as a result of biochemical or genetic differences. It was significantly increased, perhaps due to heterosis. R. C. Jackson has obtained F2 plants without difficulty from a cross of H. rhizomatous and Nl- johnstonii which is another member of the section Blepharodon. sensu Hartman (1976). Cytologically, there was a translocation difference between the two (Jackson, unpublished). There is not good agreement of the triploid hybrid with configuration events predicted by the autoploid model. When analyzed using the chromosomes involved method, this more conservative approach indicated statistically significant differences in two out of the three cases. One of the hybrids therefore behaved like an autotriploid; the other two behaved like allotriploids. This may have been caused by mutational differences for pairing control genes, and allelic variation for those genes in the tetraploid population. Such variation has been suggested in the tetraploid grass Alopccurus (Murray £iai., 1984). Comparison of observed and expected results 115 reveals in all cases a deficiency of both cIII and cII,I with an excess of observed oII,I. A deficiency of observed cIII would be expected with pairing control genes. Pollen Analysis Pollen viability within most of the diploid populations was generally as high as is usually found for most related species. Haplopappus rhizomatous. and particularly Nl. gypsophila and Nl. restiformis had low stainability. Viability of tetraploid pollen was substantially lower than that of most diploids, an occurrence that has been previously noted (Hauber, 1986). Hybrid pollen viability was significantly lowered over that of the parents in every instance except that of crosses involving M.. gypsophila and M. restiformis. These greenhouse-produced hybrids exhibited no statistically significant difference between either parents or naturally occurring hybrids, but pollen viability of the parents was initially much lower than other species. Turner (1973b) did report reduced fertility in putative natural hybrids as judged by pollen stainability. Plants in the present study identified as either M. restiformis or M.. gypsophila apparently possessed appropriate morphological genes but were actually hybrid, thus giving them a reduced pollen viability. This would be consistent with the fact that there was a great deal of variability in appearance of plants grown from seed taken from parents identified as one species or the other as previously discussed. Turner may have sampled from a locality wherein the same degree of hybridization did not occur. In view of the apparent normality of late diplotene and of diakinesis stages examined, reduced pollen viability seen in hybrids does not appear to be due to any major chromosomal differences between the parent populations. Earlier stages of prophase I were not carefully scrutinized, but an overall survey did not identify any chromosomal aberrations. The reduced pollen viability of hybrids may have been due to small cryptic differences (Jackson, 1985). 116 Electrophoretic Analysis Alleles Populations of all species analyzed in this study exhibited comparable values for mean number of alleles per locus, averaging about 1.3. This is within the general range of about 1.1 to 3.6 reported for angiosperms in general (Bayer and Crawford, 1986; Gottlieb, 1977; Soltis and Bloom, 1986), but somewhat below the 2.26 average of 28 taxa of selfers reported by Gastony and Gottlieb (1985). Measures of Equilibrium All populations except NL. riparia were in overall equilibrium for the loci tested, as determined by both chi-square analysis of Hardy-Weinberg expectations and by fixation indices. Machaeranthera riparia was not in equilibrium for any genes that were not fixed. It is unlikely that sample size was causative, for 42 plants were tested. ME alleles a and b had an apparent frequency of 0.5 each, but only heterozygotes were found. Other unusual traits regarding allele frequencies were also found, as will be discussed subsequently. Two explanations concerning the absence of ME homozygotes are possible: (1) a homozygous lethal gene closely linked to the ME locus, and (2) gene duplication. In duplications, "the key initial observation is that all individuals of one species display an electrophoretic pattern for an enzyme system which is shown only by heterozygous individuals in closely related species" (Gottlieb, 1981b). Both G3PD and PGM-1 exhibited mostly heterozygotes. Of the 4 enzymes not fixed within the population, only LAP did not show extreme excess heterozygosity. If the LAP class bb is combined with the next smaller class so as to give only classes wherein the expected frequency is greater than 5, the resultant chi- square is not significant. If a homozygous lethal gene were responsible for the appearance of only ME heterozygotes, the loci for G3PD and PGM may likewise be linked but at a sufficient distance to allow for some crossing over. If gene duplication were responsible, G3PD and PGM-1 may also be duplicated, with G3PD and PGM-1 duplicates flanking the more centrally located duplicates of ME a and b. 117 Measure?^ of Genetic Variability Among 21 taxa of outcrossers the mean proportion of loci polymorphic was found to be 51%, whereas among 28 taxa of selfers it was 18.3% (Gastony and Gottlieb, 1985). In this respect most of the populations in the present study more resembled selfers than the outcrossers they actually were. Machaeranthera gvpsophila and Nl. restiformis exhibited a substantially higher proportion of polymorphic loci than the other populations, presumably because they shared a common gene pool that provided a rich source of variation. Although the smaller value for 7559 may have been due to sample size, this was the population found growing among cat-claw acacia and thought by R. C. Jackson at the time of collection to possibly represent a different subspecies. Small heterozygote deficiencies are commonly observed in outbreeding plant populations (Brown, 1979). Factors most commonly responsible are partial selfing, population structure due to consanguineous matings, and the Wahlund effect. Small sample size undoubtedly affected F values. Comparison of the chi-square tests of Table 18 and the fixation index of Table 19 show a fairly good correlation between those chi-square results that were of doubtful validity due to low frequency of expected classes and fixation indices that vary greatly from zero. Self-fertilization is not a likely explanation for heterozygote deficiencies, since experience over a number of years' of crossing experiments in the greenhouse has shown these and related groups to be almost entirely self-incompatible (Jackson, personal communication). Also, partial selfing would be expected to affect the entire genome, a condition that was not observed. Subdivision of the populations into genetically divergent subpopulations may be the most likely explanation for excess homozygosity. Gene dispersal through both pollen and seeds can be extremely limited in plant populations (Levin and Kerster, 1974). Whereas subpopulations may be panmictic within themselves, if more than one subpopulation were sampled, and if they were divergent at one or more loci, then an overall heterozygote deficiency would be observed (Wahlund effect). The overall major component of FIT was FsT. with the Fis component being rather small. The low mean Fis (0.064) indicates only a small amount of inbreeding among the sampled populations. This suggests that there is no 118 overall deviation from random mating within populations and is consistent with the measures of equilibrium previously discussed. Accompanying the hierarchical step from subpopulation (Fis) to population (FIT) was a dramatic increase in heterozygote deficiency (FIT = 0.740). That large increase was due to differentiation among populations, measured by FsT (mean = 0.722). Genetic Relationships Conspecific plant populations reportedly show a very high degree of genetic similarity with mean genetic identity values ranging from about 0.87 to 1.00; most are in the range of 0.95 to 1.00 (Crawford, 1983; Gottlieb, 1977). Similarly high values were found in this study among different populations of the same species which exhibited ranges from 0.92 to 1.00 with Nei's genetic identity and 0.86 to 0.99 with Rogers' genetic similarity. This was to be expected and affirms Gottlieb's (1977) statement that electrophoretic evidence from one or a few populations very often constitutes an adequate sample of an entire species. Machaeranthera blephariphvlla and Nl. gymnocephala were not distinguished from each other, exhibiting values of 0.95 to 1.00 with Nei's identity and 0.89 to 0.99 with Rogers' similarity. These two species appear to be closely related, exhibiting an average of only 4 or 5 detectable changes per 100 loci between any two populations regardless of species. They do, however, have very different ranges and habitat preferences. It is interesting to note that the electrophoretic data are in agreement with Hall (1928, p. 68) in that he was unable to recognize any significant difference between the two species. The relationship of Nl. gypsophila and Nl. rggtifgrmig was within the conspecific range. Surprisingly, Nl. grindelioides and M.. riparia exhibited genetic identity values in the conspecific range and consequently were identified as very closely related on subsequent dendrograms. This was certainly questionable-the two species are very much dissimilar in morphology, habitat, range, and chromosome number. Gottlieb (1977, 1981b) and Crawford (1983) reviewed genetic identities among congeneric species. The general range was about 0.4 to 0.9, with values most commonly being from around 0.6 to 0.8. Under this criterion H.. rhizomatous and K- tortifolia would not be congeneric with respect lo each 119 other, but each would be congeneric with all other populations examined. A number of congeneric species were reported by Gottlieb (1977) to have very high genetic identities within the range of those characterisUc of conspecific populations. Some of those were known to be related as progenitor and derivative with the derivative being of relauvely recent origin. Synthesis Affinities based purely on overall electrophoretic data supported the generic classificauons already ascribed, ie., all the Machaeranthera were more closely related to each other than to the other two groups, and Xylorhiza and Haplopappus were at a distance from each other and from Machaeranthera. Sectional classification sensu Hartman (1976) was not affirmed by the electrophoretic data, for M.. linearis of his section Hesperastrum was more closely related to the NL. blephariphylla/M. gymnocephala complex of section Blepharodon than was that complex to any other examined member of section Blepharodon. Reduction of Xylorhiza to sectional status in Machaeranthera (after Cronquist and Keck, 1957) could be justified since Nei's identity values between Xylorhiza and Machaeranthera were within the congeneric range. But that would also mean the inclusion of H- rhizomatous. for it was also within the congeneric range with Machaeranthera. Thus, the biochemical data were in agreement with the morphologic data with respect to the assertion of Turner and Home (1964) that inclusion of Xylorhiza in Machaeranthera would make a case for a large collective genus composed of Machaeranthera. Xylorhiza. and Haplopappus section Blepharodon. 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APPENDIX Buffer formulas A/B system Buffer A Lithium hydroxide 4.8 grams Boric acid 34.2 grams Distilled water to volume 3.0 liters Adjust pH to 8.3 Buffer B Tris 18.6 grams Citric acid (anhydrous) 3.3 grams Distilled water to volume 3.0 liters Adjust pH to 8.3 Electrode buffer Buffer A only Gel buffer Buffer A 1.0 part Buffer B 9.0 parts 0.02 M histidine system Electrode buffer Sodium citrate, 0.4 M 352.92 grams Distilled water to volume 3.0 liters Adjust pH to 7.0 with HCl Gel buffer Histidine HCl, 0.02 M 11.5 grams Distilled water to volume 3.0 liters Adjust pH to 7.0 with 4 M NaOH pH 5.7 histidine system Electrode buffer L-histidine (free base), 0.065 M 30.3 grams Distilled water to volume 3.0 liters Adjust pH to 5.7 using citric acid Gel buffer Electrode buffer 1.0 part Distilled water 6.0 parts Readjust pH to 5.7 128 129 TBE system (Tris-borate-EDTA) Stock buffer Tris, 0.9 M 327.0 grams EDTA (free acid), 0.02 M 22.8 grams Boric acid, 0.5 M 92.7 grams Distilled water to volume 3.0 liters Adjust pH to 8.6 Electrode buffer Stock buffer 1.0 part Distilled water 4.0 parts Gel buffer Stock buffer 1.0 Distilled water part 19.0 parts Stain formulas Aldolase (ALDO) 0.1 MTris HCl, pH 8.0 100 ml Fructose-1,6-diphosphate 250 mg Arsenic acid 300 mg Glyceraldehyde-3-phosphate dehydrogenase 200 U Beta-NAD 40 mg MTT (dissolve in N,N dimethylformamide) 8 mg PMS 2 mg Catalase (CAT) Incubate for 15 minutes in 0.1 M phosphate, pH 7.0 100 ml 30% hydrogen peroxide 0.5 ml 0.06 M sodium sulfite 66 ml Rinse and immerse in 0.09 M potassium iodide 50 ml Glacial acetic acid 2 ml Distilled water 50 ml Glutamate dehydrogenase (GDH) 0.1 MTris HCl, pH 8.5 100 ml 1 M sodium glutamate in 0.5 M Tris HCl, pH 8.5 20 ml Beta NAD 40 mg MTT (dissolve in N,N dimethylformamide) 15 mg PMS 2 mg Glucose-6-phosphate dehydrogenase (G6PD) 0.1 MTris HCl, pH 8.0 100 ml Glucose-6-phosphate 75 mg NADP 5 mg MTT (dissolve in N,N dimethylformamide) 8 mg PMS 2 mg 0.1 M magnesium chloride 5 ml 130 Glutamic oxaloacetic transaminase (GOT) 0.2 M Tris HCl, pH 8.0 50 ml Alpha aspartic acid 200 mg Alpha ketoglutaric acid 100 mg Fast blue BB 150 mg Pyridoxal-5'-phosphate 0.5 mg Dissolve using stir plate and beaker protected from light. Glyceraldehyde-3-phosphate dehydrogenase (G3PD) Incubate 30 at 37^0 in 0.1 M Tris, pH 7.7 100 ml Fructose-1,6-diphosphate 140 mg Aldolase 10 U Add to above without rinsing Arsenic acid 75 mg Tetrasodium EDTA 114 mg 0.1 M magnesium chloride 5 ml MTT (dissolve in N,N dimethylformamide) 8 mg PMS 2 mg NADP 10 mg Glycerate-2-dehydrogenase (G2DH) 0.1 MTris HCl, pH 8.0 100 ml DL glyceric acid 150 mg Beta NAD 40 mg MTT (dissolve in N,N dimethylformamide) 8 mg PMS 2 mg Isocitrate dehydrogenase (IDH) 0.1 MTris HCl, pH 7.2 100 ml DL-isocitric acid, trisodium salt 200 mg 0.1 M magnesium chloride 5 ml NADP 10 mg MTT (dissolve in N,N dimethylformamide) 8 mg PMS 2 mg Leucine aminopeptidase (LAP) Incubate 30 minutes in 0.1 M phosphate, pH 7.0 100 ml 0.1 M magnesium chloride 2 ml Alpha-leucyl-beta-naphthylamide HCl 30 mg Pour above into flask, add the following and stir to dissolve Black K salt 40 mg Restain with above solution until bands appear. 131 Malate dehydrogenase (MDH) 0.1 M Tris HCl, pH 7.2 100 ml L-malate 42o mg Tetrasodium EDTA 38 mg NAD 30 nig MTT (dissolve in N,N-dimethylformamide) 8 mg PMS 2 mg Titrate to pH 7.2 with 4 M sodium hydroxide Malic enzyme (ME) 0.1 M Tris HCl, pH 7.2 100 ml L-malate 420 mg NADP 10 mg 0.1 M magnesium chloride 5 ml MTT (dissolve in N,N-dimethylformamide) 8 mg PMS 2 mg Titrate to pH 7.2 with 4 M sodium hydroxide Phosphoglucose isomerase (PGI) 0.1 M Tris HCl, pH 8.0 90 ml 0.1 M magnesium chloride 5 ml Fructose-6-phosphate 35 mg Glucose-6-phospliate dehydrogenase 40 U NADP 5 mg MTT (dissolve in N,N dimethylformamide) 8 mg PMS 2 mg Phosphoglucomutase (PGM) 0.05 M Tris HCl, pH 8.0 90 ml Disodium alpha-D-glucose-1-phosphate 75 mg Dipotassium aIpha-D-glucose-l,6-diphosphate,0.00017 M 5 ml 0.1 M magnesium chloride 5 ml Glucose-6-phosphate dehydrogenase 40 U NADP 5 mg MTT (dissolve in N,N dimethylformamide) 8 mg PMS 2 mg Triose phosphate isomerase (TPI) 0.1 MTris HCl, pH 8.0 100 ml Dihydroxyacetone phosphate 10 mg EDTA, tetrasodium salt 38 mg Beta NAD 30 mg Sodium arsenate 460 mg Glyceraldehyde-3-phosphate dehydrogenase 300 U MTT (dissolve in N,N dimethylformamide) 8 mg PMS 2 mg n m in in in in m in >n in VO ^ o G in »n m > r- r^ r- r~ r~- r- r- r- r^ r^ r^ r~- Ol r- r~ r- OJ ex H C/D Kl si i s -3 V3 CJ Tt cs oo oo Tt OJ vo cs oo oo cs vo o OJ cs 2: .2 OS vo Tt -H 0\ U r>- cn r~ vo oo cn oo •^ in cn Xi Tt m in Tt in Tt k. in Tt in wn in Tt in in vo 2 O OLCHJ -. 0 •3 o X5 CA •3 Q> 0 Q> 3 Tt •3 6 3 oJ QJ 0 2i S • 2 3 _• — I-H 3t : O CJ CS O o 3 CJ ^ Q> •3 Q> i_r •-. rt flj >• 5 000 •- OJ 03 x: CJ 000 000 CJ m m wn m m m Q> •-- cn OJ m wn wn •3 •s *^ CJ CA 3 CJ z S w I-H CJ CJ CA o 4J O 00 3 o o .-• o •3 3 J^ -3 •3 o O, 03 « 3 C c 3 ^ x: 'c3 •3 -3 03 CJ c O \0 C?v Ov 3 3 tA CJ Tt w-^ Os cs Tt r- 3 O o 00 — -3 6 u o o 000 00 — \^^ N^^ ^*-^ (X c c4 —/• v-' >—' 04 CS OJ 01 CS CS ^H .—I .—I 3 03 Tt Tt Tt •^ "o CJ o CA QJ 000 Tt Tt Tt e OJ \0 vo vO Q> NO vO vo \0 "^ ^^ O o r- o- r~ r~- t^ r~- i3 O r- r~ t^ O QJ 03 X3 03 3. 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