3. was. w. ‘ A l§¢31 9.... 2 . ,2. . Hm“... . . .mma. A . . . a. .Mu’s wanna... _ .2 x L. 1 .. .. 3r . . . Kazlifu i n .31; fine fik... . 5.. 1.. u. ugh... fianfififihh =2: .4”... 11:29.8... inwfll.3‘flul.u. 3. .4 . 4...... I 1....» . on ~v~t 51“ :- .. .||: «:5 I... .II l. t“ 5:). {.1 I \. 7-. t... . 5. 5.2315219. (103...! . . . x? .3 0.1.1.131». \ 5. y... . I. . 20 4.! 5? 1L 33$. g... . . 2...... , .22... 3:2! :0. . . ha... 4.. .. z .. . .2 :i . .u‘! f .. .: 2v. 13:95 ‘32)- LIBRARY Michigan State University This is to certify that the thesis entitled THE REPRODUCTIVE BIOLOGY AND TAXONOMIC ORIGIN OF MICHIGAN MONKEY-FLOWER MIMULUS GLABRATUS VAR. MICHIGANENSIS presented by Amanda L. Posto has been accepted towards fulfillment of the requirements for M. S. Botany & Plant degree in Pathology Wfi Majouprofessor Date . L M Q 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution PLACE IN RErURN Box to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 541404 i a” 6 0&8 {ii—280? LL55 '9’?- 1g§ta° 8 {mm 11202909 6/01 cJCIRC/DateDue.p65p.15 THE REPRODUCTIVE BIOLOGY AND TAXONOMIC ORIGIN OF MICHIGAN MONKEY-FLOWER, MIM UL US GLABRA TUS VAR. MICHIGANENSIS By Amanda L. Posto A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Botany and Plant Pathology 2001 ABSTRACT THE REPRODUCTIVE BIOLOGY AND TAXONOMIC ORIGIN OF MICHIGAN MONKEY-F LOWER, MIM UL US GLABRA TUS VAR. MICHIGANENSIS By Amanda L. Posto Michigan monkey-flower, Mimulus glabratus var. michiganensis, is a rare plant endemic to Michigan. Because this taxon is federally endangered and very rare there is much interest in learning more about its biology. Research on its reproductive biology focused on mating system parameters, pollen viability and seed germination. Michigan monkey-flower is self-compatible and plants from the Maple River population are capable of self-pollination. There is considerable variation (27-52%) in pollen viability between individuals of the Maple River population, which has implications for mating, selection, and gene flow within this population. The Reese’s Swamp population, which had not been previously studied, has 0% viable pollen similar to other populations studied with the exception of the Maple River population. Among four seed germination regimes tested, the highest germination rates (67%) were observed at ~23°C with exposure to light. Molecular markers were used in the taxonomic analysis to test between alternative hypotheses of hybrid origin from James’ monkey-flower, M. glabratus var. jamesii, and Common monkey-flower, M. guttatus, versus divergence from one or the other. Michigan monkey-flower is not of recent origin and, based on genetic similarity between taxa, is most likely diverged from James’ monkey-flower. Michigan monkey-flower is genetically distinct from the other taxa and has relatively high intraspecific identity compared to the more widespread J ames’ monkey-flower and Common monkey-flower. ACKNOWLEDGEMENTS First and foremost, I would like to thank Alan Prather for his guidance, instruction, patience and prodding. I would like to thank my committee members, Tao Sang and J eff Conner, for providing constructive criticism on this manuscript and my original research proposal. Mike Penskar and Phyllis Higman were instrumental in developing and inspiring this work. The Michigan Department of Natural Resources Non-Game Division and the Michigan Chapter of The Nature Conservancy provided funding to support this research. The Department of Botany and Plant Pathology provided assistantships to support me during my education. Robert K. Vickery Jr. at the University of Utah and Susan Trull with the National Forest Service for providing plant material that was necessary to conduct the RAPD analysis. Jan Szyren helped to maintain the greenhouse plants and Michael Chamberland provided critical comments on grant proposals. I would like to thank Anna Monfils and Rachel Williams for providing assistance with manuscripts, teaching, coursework and labwork. Also thank you Rachel for fiequent coffee trips to Beaners. In addition, I would like to thank Amanda Rinehart and Anita Davelos for providing statistical assistance and, more importantly, for their lasting friendships. Lastly I would like to thank Garry Milius who put up with more fiom me than I could ever ask of anyone. iii TABLE OF CONTENTS LIST OF TABLES ................................................................................... vi LIST OF FIGURES ................................................................................ vii CHAPTER 1 INTRODUCTION .................................................................................... 1 CHAPTER 2 THE REPRODUCTIVE BIOLOGY OF MICHIGAN MONKEY-FLOWER Introduction ................................................................................... 4 Materials and Methods Pollen Stainability ................................................................... 5 Mating System ....................................................................... 6 Seed Germination ................................................................... 9 Results Pollen Stainability ................................................................... 9 Mating System ..................................................................... 10 Seed Germination .................................................................... 12 Discussion Pollen Stainability ................................................................. 12 Mating System ..................................................................... 13 Seed Germination .................................................................. 14 Conclusions ........................................................................ 14 CHAPTER 3 THE TAXONOMIC ORIGIN OF MICHIGAN MONKEY-F LOWER Introduction .................................................................................. 16 Materials and Methods Sampling ............................................................................ 22 DNA Isolation ..................................................................... 23 DNA Amplification and Visualization ......................................... 25 RAPD Analysis ..................................................................... 25 Results RAPD Primers ..................................................................... 27 RAPD (Banding) Patterns ........................................................ 28 Similarity Coefficients and UPGMA ........................................... 29 Discussion .................................................................................... 33 CHAPTER 4 CONCLUSION ....................................................................................... 36 iv APPENDICES ....................................................................................... 39 APPENDIX A Data Matrix ........................................................................ 40 APPENDIX B Similarity Matrix .................................................................. 56 CITED LITERATURE ............................................................................. 63 LIST OF TABLES Table 1: Experimental treatments for determining the mating system of Michigan monkey-flower. The outcome determined by each treatment is indicated with positive cells ..................................................................................................... 7 Table 2: Pollen stainability of Michigan monkey-flower individuals sampled from five populations (n >500 pollen grains for all samples) .............................................. 9 Table 3: Percent fruit set in crosses between plants of the Maple River population. Total that did not set fruit in cross-pollinated treatments shown in parentheses. . . . . . . . l 0 Table 4: Percentage fruit set between crosses of Maple River individuals and pollen-sterile individuals ............................................................................ 10 Table 5: Percent germination for four treatments ............................................... 12 Table 6: Identity of Mimulus samples in the RAPD analysis .................................. 25 Table 7: Primers used in the RAPD analysis ..................................................... 27 Table 8. Bands shared between taxa. Michigan monkey-flower is “MICH”, James’ monkey-flower is “JAMS”, Utah monkey-flower is “UTAH”, and Common monkey- flower is “GUTI‘”. Fixed bands are found in all individuals of taxa in the lefi column, polymorphic bands are found in one or more, but not all, individuals. Moving down the table, bands shared between taxa are not cumulative. That is, the bands shared between all four taxa (MICH-JAMS-GUTT-UTAH) are not found in any other rows in the table. The same is true for all other rows ............................................................... 29 Table 9: Genetic distance between Michigan monkey-flower, James’ monkey-flower and Common monkey-flower. Except for within taxon comparisons, distance measures are calculated from the similarity between taxa and ignores similarity within taxa. (i.e. comparisons between michiganensis and guttatus are based only on the similarity between individuals of michiganensis and guttatus and not the similarity between individuals of michiganensis) ...................................................................... 30 vi LIST OF FIGURES Figure 1: Assignment of mating system treatments to stolons of potted individuals. Each different coded circle represents one of the four treatments applied to one flower on each of five stolons: Unmanipulated, 2) Emasculated, 3) Self-pollinated and 4) Cross- pollinated. Each plant produced many stolons and only treated stolons are depicted in the figure ................................................................................................... 8 Figure 2: Image from a light microscope of pollen stained with aceto-carmine jelly. A stained pollen grain can be seen in the upper right corner of the slide. The other four grains are not stained ................................................................................ 10 Figure 3: Distribution of Michigan monkey-flower (solid line), James’ monkey-flower (dashed line) and Common monkey-flower (dotted line and filled circle in the western Upper Peninsula of Michigan) in North America. .............................................. 19 Figure 4: Flowchart of the similarity analysis. The gel image (upper left corner) is scored and the data is recorded in the data matrix. The similarity matrix is constructed using J accard’s coefficient and a phenogram is constructed by clustering the similarity matrix using UPGMA ....................................................................................... 26 Figure 5: Image for thirteen individuals amplified with primer A07. Lanes 1 and 10 are molecular weight standards. Lanes 2-9 are Michigan monkey-flower, lanes 11-12 are Common monkey-flower and lanes 13-15 are James’ monkey-flower. Band A is present in all taxa. Bands B, E and F are unique to Common monkey-flower. Band C is unique to Michigan monkey-flower. Bands D and G are shared by Michigan monkey-flower and J ames' monkey-flower .............................................................................. 27 Figure 6: UPGMA phenogram of 42 Mimulus samples genotyped by RAPD banding patterns. Taxon abbreviations follow those used in Table 3. Location abbreviations are as following: (CA = California, MX = Mexico, M1 = Michigan, NE = Nebraska, OK = Oklahoma, QE = Quebec, UT = Utah), and ID number (refer to Table 6). The scale represents the similarity coefficient .......................................................... 31-32 vii CHAPTER 1 INTRODUCTION Michigan monkey-flower, Mimulus glabratus var. michiganensis (Pennell) F assett, is the only plant taxon endemic to Michigan. It is rare in Michigan and is known from only 15 extant localities and 3 historical sites all within the Mackinac Straits (Charlevoix, Cheboygan, Emmet and Mackinac counties) and Grand Traverse (Benzie and Leelanau counties) regions of Michigan (U SF WS, 1997). It is protected under the Federal Endangered Species Act (U .S. Department of Interior, 1990) and in Michigan (Michigan Department of Natural Resources, 1991) and is most likely endangered because of shoreline development (USFWS, 1997). It is an aquatic perennial in a widespread complex of yellow monkey-flowers that are mainly distributed in the western United States. Because this taxon is federally listed and very rare there is much interest in learning more about its biology. In this study I explore important questions about the reproductive biology and taxonomic origin of Michigan monkey-flower. My research priorities were chosen by consulting the recovery plan for this taxon (U SFWS, 1997) and conferring with Michigan Natural Features Inventory staff. The investigation of the reproductive biology of Michigan monkey-flower will involve assessment of its mating system biology, pollen viability and seed germination. An understanding of the basic reproductive biology is essential for understanding the causes and consequences of rarity and will provide the foundation and focus for further studies. A study of the reproductive biology will provide information relevant to the biology of Michigan monkey-flower in the following areas: . Mating system biology. Persistence of rare species depends largely on their reproductive ability (Heunneke, 1991; Barret and Kohn 1991). Furthermore, low rates of sexual reproduction are often associated with rarity, sometimes as a cause and sometimes as a consequence. Because sexual reproduction has an impact on population persistence and on the maintenance of genetic diversity, testing for self- compatibility will provide baseline data needed for management plans for this endangered species. . Pollen viability. Michigan monkey-flower has very low pollen viability, less than 1%, in all but one population studied by Bliss (1983, 1986). The remaining population, the Maple River population, was reported to have 30% pollen viability. In the Maple River population pollen viability may differ considerably between individuals, but Bliss sampled only three individuals, one anther from each, per population, thus possible variation could have gone undetected. Differences in pollen viability between individuals may have important consequences for reproduction and individual fitnesses in the population. . Seed germination. Fruit set is low in most populations and capsules were never full of seeds (Bliss 1986). This low level of seed production can also have important consequences for sexual reproduction. Quantifying rates of seed germination may give us insight into one of the causes of rarity of this species and may provide information critical to management planning. Experiments to investigate the mating system of Michigan monkey-flower were conducted using individuals of the Maple River population, the only population known to have significant pollen viability and fi'uit set (Bliss, 1986). The taxonomic origin of Michigan monkey-flower was investigated using molecular markers to test between the alternative hypotheses of hybrid origin fi'om J ames’ monkey-flower, M. glabratus var. jamesii, and Common monkey-flower, M. guttatus, versus divergence from one or the other. Bliss (1983, 1986) proposed three hypotheses to explain its origin: (1) that Michigan monkey-flower originated from a hybridization event between James’ monkey-flower and the Common monkey-flower, (2) that Michigan monkey-flower originated from a chromosomal rearrangement of J arnes’ monkey-flower, or (3) that Michigan monkey-flower originated as a disjunct aneuploid (gain or loss of a chromosome) of the Common monkey-flower. The RAPD (Random Amplified Polymorphic DNA) technique (Welsh and McClelland 1990; Williams et a1. 1990) was used to compare Michigan monkey-flower to its putative parental species, James’ monkey-flower and Common monkey-flower. Understanding the genetic relationship of Michigan monkey-flower to J arnes’ monkey-flower and Common monkey-flower is important because these relationships may offer insight into the appropriate taxonomic rank for the taxon, which in turn will have implications for its level of protection. Investigation of the reproductive biology and taxonomic origin will contribute greatly to our understanding of the biology of Michigan monkey-flower, provide baseline knowledge for prioritizing conservation programs, and lay the groundwork for future studies. CHAPTER 2 THE REPRODUCTIVE BIOLOGY OF MICHIGAN MONKEY-FLOWER Introduction Michigan monkey-flower is an emergent aquatic restricted to cool, alkaline springs and streams usually associated with Northern white-cedar (T huja occidentalis) swamps along current and post-glacial Great Lakes shorelines (U SFWS 1997). It is a prostrate to erect herb usually found growing in muck or sandy soils and the stems often root at the lower nodes to produce numerous shoots via stolons. It typically flowers from mid-June to late August. Bliss (1986) conducted a reproductive study of Michigan monkey-flower focusing on pollen viability and fi'uit set. She documented less than one percent pollen viability in seven populations and thirty percent pollen viability in the population at Maple River. Three plants were sampled per population and intrapopulational variation in pollen viability, if present, was not reported. Bliss observed 75% pollen viability in 14 populations of J ames' monkey-flower, which is more typical of angiosperms (Keams and Inouye, 1993). Michigan monkey-flower is reported to be self-incompatible (Bliss, 1983). However, Bliss used only plants from populations with less than 1% pollen viability in her crossing experiments. Mating crosses conducted using individuals from Maple River, the only population known to produce viable pollen (Bliss, 1983), may provide a more accurate view of the mating system of this group. Other varieties of Mimulus glabratus are known to be self-compatible and readily self-pollinating (Vickery, 1991). Bliss observed, but did not quantify, seed set. She found 100% of shoots with developing fi'uit in the Maple River population and only 3.4% of shoots with developing fi'uit in the remaining populations; she noted that capsules were never full in any population. These results (Bliss, 1983, 1986) suggest that reproductive biology is an important factor in the rarity of Michigan monkey-flower. While the plants reproduce clonally, their rates of sexual reproduction are apparently very low. In order to gain a greater understanding of the reproductive biology of Michigan monkey-flower, I measured pollen viability in multiple individuals of the Maple River population and several other populations, including individuals from Reese’s Swamp, a vigorous population that had not been previously studied. In addition, I investigated the mating system of Maple River individuals and tested four regimes for measuring seed germination. Materials and Methods Pollen Stainability Pollen stainability, as an estimate of pollen viability, was measured using the aceto-carmine jelly staining technique (Radford et al., 1974). Aceto-carmine jelly is a semi-permanent mounting medium in which pollen grains containing cytoplasm stain purple and are assumed to be viable. Pollen grains lacking cytoplasm are assumed to be inviable and do not stain. This method probably overestimates pollen viability (Peters et al., 1990; Keams and Inouye, 1993). Pollen was sampled from 17 individuals of five populations of Michigan monkey- flower: one individual from Burt Lake, five individuals from Carp Creek, four fiom Glen Lake, five from Maple River and two from Reese’s Swamp. Bliss (1983, 1986) did not fine statistical differences in pollen viability, estimated as pollen stainability, between anthers of a single flower therefore, one undehisced anther was sampled from a single flower per individual. The anther was removed from a bud of each individual and macerated in a drop of aceto-carmine jelly. Pollen grains lacking cytoplasm tend to float to the edges of a coverslip, thus stained pollen may be over-represented in microscope fields that are not near the edge, while unstained pollen may be over-represented near the edge. Therefore, systematic sampling may lead to a bias, depending on the ratio of edge vs. non-edge fields that are counted. To insure that unbiased, accurate measures of pollen stainability were recorded, total counts of pollen stainability were made counting all pollen grains per slide for 5 slides. This provided a total measure of pollen viability, which was evaluated with three different methods of sampling: 1) randomized vertical coordinates (corresponding to the width of the coverslip), 2) randomized horizontal coordinates (corresponding to the length of the coverslip) and 3) randomized mixed (corresponding to randomized combinations of vertical and horizontal coordinates). For each sampling method, a minimum of 500 pollen grains was counted per slide. Pollen stainability was calculated as the percentage of viable pollen grains of total pollen grains counted. The methods were compared for statistical differences using the GLM procedure in SAS software version 8.1 (SAS Institute Inc., 2001). An ANOVA was conducted on normal data recorded as the proportion of viable pollen. Mating system Mating system studies were conducted to test for asexual seed production, self- compatibility, self-pollination, and cross pollination in the Maple River population, which is the population with the highest level of fertile pollen and seed set. For these tests, twenty-two five-inch stolons, presumed to represent different genets, were collected from the Maple River population and grown in the greenhouse under a 16-18 day length at 70°C. The stolons exhibited vigorous growth in the greenhouse and were contained in 8-inch pots in flats of water. They were watered daily with fertilized water and treated with pesticides and fungicides as needed. The following treatments were applied to ten randomly chosen plants: 1) unmanipulated, 2) emasculated, 3) self-pollinated and 4) cross-pollinated (Table 1). Self- compatibility is required for self-pollination but to understand the breeding system, the two must be distinguished. Seed set after treatments 1 and 3 both suggest self- compatibility. Seed set after treatment one further suggests the ability to self-pollinate. Table 1: Experimental treatments for determining the mating system of Michigan monkey-flower. The outcome determined by each treatment is indicated with positive cells. Treatment 5.6m. self: . asexual CIOSS: pollination compatibility reproduction pollination l) unmanipulated + + - - 2) emasculated - - + - 3) self-pollinated - + - - 4) cross-pollinated - - - + Experimental plants were enclosed within a fine mesh to exclude pollinators. Each treatment was randomly applied to one flower on each of five haphazardly chosen stolons per plant (Figure 1), thus a total of 5 flowers per treatment per plant and 50 flowers per treatment were manipulated. All treated flowers were marked for later identification. Unmanipulated flowers (treatment 1) were marked and not manipulated. Emasculated and cross-pollinated flowers were emasculated prior to floral opening. Self- pollinated flowers were not emasculated. For self-pollinations and cross-pollinations, pollen was applied with a toothpick to the stigma of the experimental flower. Pollen was removed from the anthers of two or three flowers of non-experimental stolons per plant for self and cross pollen donors (two plants per supposed genet). Five randomly chosen plants, which were not experimental plants, were chosen as cross pollen donors and a mixture of pollen from the five plants was used in all cross-pollinations. The presence or absence of hit set was recorded. 0 Figure 1: Assignment of mating system treatments to stolons of potted individuals. Each different coded circle represents one of the four treatments applied to one flower on each of five stolons: 1) Unmanipulated, 2) Emasculated, 3) Self-pollinated and 4) Cross- pollinated. Each plant produced many stolons and only treated stolons are depicted in the figure. Interpopulation cross pollinations were conducted between Maple River individuals and pollen-sterile individuals to determine the ability of pollen-sterile plants to set seed. Two plants from each of four pollen-sterile populations (Burt Lake, Carp Creek, Glen Lake, Reese’s Swamp) were randomly chosen as experimental plants. Four randomly chosen Maple River plants, which were not experimental plants or pollen donors for the intrapopulation cross experiments, were chosen as pollen donors. Two to five flowers per plant were pollinated with a mixture of pollen from the four Maple River plants using a toothpick applied to the stigma. The presence or absence of mu set was recorded. Seed Germination Fifteen seeds per treatment were allowed to germinate under different light and temperature conditions for 4 treatments: ~23°C light/dark, ~23°C dark, 8°C light/dark and 8°C dark. These temperatures were chosen because they were readily available (room temperature and refiigerator) and they encompass the range of water temperatures at which Michigan monkey-flower is known to grow in nature. Seeds were collected and pooled from five individuals in the greenhouse for germination experiments. Seeds were placed in sealed petri plates on moistened filter paper. Petri plates were maintained at room temperature on a lab bench near a window (approximately 23°C) or in a refiigerator (8°C) with a clear, glass door to allow light exposure. For light/dark treatments, seeds were exposed to approximately 16 hours light per day. For dark treatments, petri plates were contained in closed boxes and did not receive any light. The percent germination was measured for each treatment over a 30 day period. Results Pollen Stainability Total pollen stainability did not differ between the three methods of sampling pollen stainability (p=0.6756). An image taken from a light microscope is shown in Figure 2. Pollen stainability of all individuals from Burt Lake, Carp Creek, Glen Lake and Reese’s Swamp was 0% (Table 2). Pollen stainability varied among the five Maple River individuals fi‘om 27-52% (Table 2). . Stained .5, . \f x ‘13 pollen gain Figure 2: Image from a light microscope of pollen stained with aceto-carmine jelly. A stained pollen gain can be seen in the upper right comer of the slide. The other four gains are not stained. Table 2: Pollen stainability of Michigan monkey-flower individuals sampled from five populations (n >500 pollen gains for all samples). No. Individuals Population Sampled P011? Stainability (N 0. Flowers) (A stained) Burt Lake 1 (1) 0 Carp Creek 5 (1) 0 Glen Lake 4 (1) 0 Maple River 5 (1) 27.4, 36.6, 46.0, 49.9, 51.6 Reese’s Swamp 2 (1) 0 Mating System Data for the intrapopulation Maple River crosses are reported in Table 3. A total of 50 flowers per treatment were pollinated, however, not all flowers survived to fruiting because of fungal disease and insect infestation in some plants. All surviving unmanipulated and self-pollinated flowers set fruit. No fruit set was observed in the emasculated flowers. Of 35 surviving cross-pollinated flowers, three did not set fruit. For the interpopulational crosses between pollen-sterile individuals from other sites and Maple River individuals, 24 treated flowers representing all 4 pollen-sterile populations in the study survived to fi'uiting and all 24 set fruit (Table 4). Table 3: Numbers of flowers and percent fruit set in crosses between plants from the Maple River population. Total that did not set fruit in cross-pollinated treatments shown in parentheses. Plant Total Total Total Total Treated Emasculated Unmanipulated Self-Pollinated Cross-Pollinated 07A 4 3 3 4 09B 4 3 5 4 10A 5 4 4 4 12A 2 l 2 2 14A 3 4 2 3 28A 4 4 4 4 29A 5 5 5 5 (1) 31B 2 1 1 2 33B 4 4 4 4 (1) 34B 4 3 4 4 (1) Total 37 32 34 36 (1:3me 0% 100% 100% 91.7% Table 4: Percentage fruit set between crosses of Maple River pollen donors and pollen- sterile pollen recipients. Number Flowers % Setting Population Treated Fruit Burt Lake 6 100% Carp Creek 6 100% Glen Lake 6 100% Reese’s Swamp 6 100% 11 Seed Germination Percent germination for each treatment is shown in Table 5. After 7 days, ten of fifteen seeds had germinated at ~23°C in the light/dark treatment, one of fifteen seeds had germinated at ~23°C in the dark treatment, and no seeds germinated at 8°C in both the light/dark and dark treatments. No further germination was observed over the 30 day period. Table 5: Percent germination for four treatments. Treatment Number Seeds % Germination Treated ~23°C light/dark 15 66.7 ~23°C dark 15 6.7 8°C light/dark 15 0 8°C dark 15 0 Discussion Pollen Stainability Earlier studies by Bliss (1983, 1986) have shown that pollen viability (measured as stainability) of Michigan monkey-flower is low in all populations compared to typical plant species. These results were consistent with those of Bliss. The Reese’s Swamp population, which had not been tested for pollen viability prior to this study, had 0% viable pollen. Therefore, the only population known to have viable pollen is the Maple River population. Among the five individuals of the Maple River population tested, 1 found a two-fold variation in the levels of viability ranging from 27 to 52 percent. 12 Mating System These results clearly show that individuals of Michigan monkey-flower are self- compatible, contrary to the results of Bliss (1983). Bliss initiated crossing experiments simultaneously with pollen measurements and crosses were conducted with pollen-sterile individuals only; she had no a priori knowledge that they were pollen-sterile. This result is consistent with reports of self-compatibility in other varieties of Mimulus glabratus (V ickery, 1991). Individuals of Michigan monkey-flower do not produce seed asexually but those from the Maple River site are self-pollinating when gown in the geenhouse. Based on observations at the Maple River site (Bliss, 1986; A. Posto, pers. obs.), the amount of fruit set, based on the number of fruits per inflorescence per plant, due to self-pollination is higher in geenhouse gown Maple River plants than total observed fruit set in the field. Fruit set in nature may be limited by resource competition or, alternatively, handling the inflorescences in the geenhouse while labeling flowers or hand-pollinating other flowers on the same inflorescence could have caused pollen to be transferred to the pistil. However fruit set and seed set was observed on Maple River plants in the geenhouse that had not been handled, indicating that self-pollination is occurring. Reduced fruit set in cross-pollinated treatments is likely due to damage to the pistil during emasculation. The three flowers that did not set fruit were the first subjects of cross-pollination. In interpopulational crosses in which Maple River individuals served as pollen donors for pollen-sterile individuals, 100% fruit set (as indicated by swelling of ovary and calyx) was observed, suggesting that the ovules are viable and will set seed. 13 However, in studies of synthesized F I hybrids of Mimulus guttatus and M. luteus, Roberts (1964) found that self-pollinated semisterile plants (1-26%, 12-31%, and 2-20% pollen viability) exhibited enlargement of the capsule and calyx, but no seed set. The same effect was observed in backcrosses between the hybrids and parents, but with a geater degee of enlargement. Roberts suggests this may be due to hormone action following pollination. Thus it is unclear whether fi'uit set in crosses between Maple River and pollen-sterile plants is due to seed development or possibly, hormone action. Seed germination There are no prior data on requirements for seed germination for Michigan monkey-flower. Although our results are preliminary, and from only a few individuals, germinating the seeds in light at room temperature (approximately 23°C) clearly produced the best results. This is notable because water temperatures at Michigan monkey-flower sites are considerably cooler than 23°C and full sunlight is generally lacking in their environment. Bliss (1983) found that water temperatures at eight populations ranged from 11-18°C (average is 14°C). My results suggest that natural seed germination in Michigan monkey-flower may be dependent on variability in water temperatures. Further support from additional experiments conducted across Michigan monkey-flower’s natural temperature range is necessary. Conclusions Michigan monkey-flower is self-compatible and plants from the Maple River population are capable of self-pollination and regularly set selfed fruits in the geenhouse. There is considerable variation (27-52%) in pollen viability levels between individuals of the Maple River population, which has implications for mating, selection, and gene flow 14 within the Maple River population. The Reese’s Swamp population, which had never before been studied, has 0% viable pollen, similar to other populations studied with the exception of the population at Maple River. Among four seed germination regimes tested, the highest rates of germination were observed at room temperature (approximately 23°C) under the light/dark treatment. 15 CHAPTER 3 THE TAXONOMIC ORIGIN OF MICHIGAN MONKEY-FLOWER Introduction Michigan monkey-flower, Mimulus glabratus var. michiganensis (Pennell) F assett, is endemic to Michigan and is found within the Mackinac Straits and Grand Traverse regions of Michigan (Figure 3). It is a diploid perennial in a widespread and morphologically diverse complex of yellow monkey-flowers most commonly found in the western United States. Pennell (193 5) originally described it as a subspecies of M. glabratus. F assett (1939) proposed a change in rank to variety. Current workers treat the taxon as a variety (Bliss, 1983, 1986; Crispin and Penskar, 1989; Mine, 1989; Vickery, 1991; Voss, 1996). Michigan monkey-flower is of interest taxonomically because it has been suggested that it may have originated via hybridization (Bliss, 1983, 1986) and that it may merit promotion to specific status (Vickery, 1991). Michigan monkey-flower is characterized by having a yellow, bilabiate corolla with an irregularly red-spotted lower lip. The leaves are opposite, ovate to broadly rounded with dentate margins and the lower leaves tend to have a well-developed petiole. The calyx is cup shaped with unequal teeth and becomes inflated at maturity. The fruit is a dehiscent capsule and the seeds are ovate with longitudinal striations. Mimulus glabratus is the most widely distributed species in the genus Mimulus (Grant, 1924) and seven varieties of M. glabratus have been recogrized (Grant, 1924; Pennell, 1935; Fassett, 1939; Skottsberg, 1953; Bliss, 1983). Mimulus glabratus var. jamesii (Benth.) A. Gray, James’ monkey-flower, is the most broadly distributed variety 16 in the species ranging fi'om western Quebec Province to Saskatchewan Province and south to Mexico. This variety is broadly distributed throughout in North America and is found throughout Michigan (Figure 3). Two other varieties occur in N. America: M. glabratus var. utahensis Pennell occurs from Colorado to Nevada and M. glabratus var. oklahomensis F assett is found in Oklahoma, Nebraska and Kansas. M. glabratus HBK var. glabratus occurs throughout Mexico and Guatemala. The remaining two varieties are only found in S. America. M. glabratus var. parviflorus (Lindl.) Grant is found from Peru to Chile and Argentina and M. glabratus var. externus (Skottsb.) Skottsb. occurs only in the Juan Fernandez Islands. Alam and Vickery (1973) and Vickery (1978) studied the interfertility of the M. glabratus complex. They focused on crosses between taxa with differing ploidy levels. There are three diploid varieties (vars. michiganensis, oklahomensis, and utahensis), one aneuploid tetraploid (var. glabratus), and two hexaploids (vars. parviflorus and externus). Mimulus glabratus var. jamesii has been reported to have both diploid and tetraploid individuals. There is complete reproductive isolation between all heteroploid levels (diploid, tetraploid, aneuploid tetraploid, and hexaploid; Alam and Vickery, 1973). Vickery (1978) found that none of the North American diploid varieties were completely reproductively isolated, based on their inter-fertility when crossed. His study of the inter- fertility M. glabratus varieties excluded Michigan monkey-flower, M. glabratus var. michiganensis. Mimulus glabratus is included in section Simiolus of Mimulus. This section of approximately 20 species (Grant, 1924) includes M. guttatus DC, the Common monkey- flower, which is distributed from the Aleutian Islands of Alaska south to Mexico and in 17 the western US from California to the Rocky Mountains. Common monkey-flower is also found in Michigan (Figure 3). It was discovered in 1987 and is found at only one known location in the western Upper Peninsula, an area known for the occurrence of disjuncts of western species (Voss, 1996; USFWS, 1997). There is considerable morphological similarity between Michigan monkey- flower, James’ monkey-flower and Common monkey-flower. James’ monkey-flower is characterized by having a smaller corolla than Michigan monkey-flower, few or no red spots on the lower lip, and a low, creeping habit. Common monkey-flower is characterized by having a larger corolla, a strongly red-spotted lower lip and an upright gowth habit. The similarities between these two taxa and Michigan monkey-flower prompted two quantitative morphological studies, one by Bliss (1983, 1986) and the other by Minc (1989). Bliss (1983, 1986) investigated the morphological similarity of Michigan monkey-flower and J arnes’ monkey-flower. Michigan monkey-flower was sigrificantly larger for 22 of 25 quantitative floral and vegetative characters, with little overlap between taxa in floral measurements. In a study of all three taxa, Mine (1989) showed that Michigan monkey-flower is morphologically intermediate. In an analysis of seven floral characters, Michigan monkey-flower was sigrificantly larger than James’ monkey- flower for all characters and sigrificantly smaller than Common monkey-flower for six characters. Minc found little overlap between Michigan monkey-flower and James’ monkey-flower, but some overlap between Michigan monkey-flower and Common monkey-flower due to extensive variability within Common monkey-flower. There was a clear separation of the three taxa based on a canonical discriminant function analysis of 18 O - ~ ~ I s \ .. ° , l ..l r r o, 0.. r 0.. ' . ‘0‘ ...o o c . - °. '6 I a 0.. ' a. r , I ’. ‘. I " °. '. I S M .0 .0 I . , ‘ 0.. \ 0. Figure 3: Distribution of Michigan monkey-flower (solid line), James’ monkey-flower (dashed line) and Common monkey-flower (dotted line and filled circle in the western Upper Peninsula of Michigan) in North America. two variates in which the first variate reflected differences in floral size and the second reflected the relative differences in ovary size. Thus, Bliss and Mine demonstrated the morphological distinctiveness of Michigan monkey-flower from James’ monkey-flower and Common monkey-flower. Furthermore, crossing experiments performed by Vickery (1991), using pollen-fertile individuals of Michigan monkey-flower, showed that Michigan monkey-flower was 19 completely reproductively isolated from James’ monkey-flower and Utah monkey- flower, M. glabratus var. utahensis. In addition an allozyme analysis (V ickery, 1990) showed Michigan monkey-flower to be as distinct from the diploid as they are from the tetraploid and hexaploid forms of M. glabratus (Vickery, 1991). Based primarily on morphological and cytological information, Bliss (1983, 1986) proposed three possible origins for Michigan monkey-flower: (1) Michigan monkey-flower (n=l4,15) originated from a hybridization event between James’ monkey- flower (n=14) and the Common monkey-flower (n=15), (2) Michigan monkey-flower originated from a chromosomal rearrangement of James’ monkey-flower, or (3) Michigan monkey-flower originated as a disjunct aneuploid of the Common monkey- flower. A number of factors are important when considering the likelihood of any of the possible hypotheses of origin for Michigan monkey-flower. The first hypothesis, that Michigan monkey-flower is a hybrid between James’ monkey-flower and Common monkey-flower, is supported by intermediate morphology and low pollen viability in Michigan monkey-flower, characteristics usually associated with hybridization (Grant, 1981; Avise, 1994). However, hybridization between James’ monkey-flower and Common monkey-flower seems unlikely for the following reasons: James’ monkey- flower and the Common monkey-flower are not currently sympatric in Michigan and pollen flow over long distances seems unlikely. Furthermore, James’ monkey-flower is primarily selfing and hybridization could only occur via pollination by animals. Because of the large difference in floral size between J arnes’ monkey-flower and Common monkey-flower, it is unlikely that the same pollinator species could effectively transfer 20 pollen between species. Also, Common monkey-flower may be a recent introduction to Michigan (Voss, 1996; USFWS, 1997). Arguments pro and con can likewise be made for the hypothesis that Michigan monkey-flower arose from a chromosomal rearrangement of James’ monkey-flower. Chromosomal rearrangements usually result in low fertility and morphological changes (Avise, 1994; Rieseberg, 1997), as seen in Michigan monkey-flower. Cytological abnormalities cited by Tai and Vickery (1970, 1972) and Vickery (197 8) for the M. glabratus complex corroborate the possibility of this origin (USFWS, 1997). On the other hand, the intermediate morphology of Michigan monkey-flower is unexpected if Michigan monkey-flower is derived solely from James’ monkey-flower. Arguments pro and con also exist for the third hypothesis, that Michigan monkey- flower originated as a disjunct aneuploid of the Common monkey-flower. Aneuploidy is known among monkey-flowers (Vickery et al., 1968) and the presence of Common monkey-flower in Michigan, which was undocumented when Bliss first proposed these hypotheses (Bliss, 1983), suggests that the disjunction event would not have had to occur across long distances. Neither low fertility nor intermediate morphology is expected with aneuploidy, and it is highly improbable that both occurred simultaneously by chance. To address these hypotheses, the random amplified polymorphic DNA (RAPD) technique (Welsh and McClelland, 1990; Williams et al., 1990) was used to investigate the origin of Michigan monkey-flower. RAPD is based on the polymerase chain reaction (PCR) and utilizes random 10 base-pair primers to amplify fragnents of total genomic DNA. The amplified DNA fragments are separated by gel electrophoresis and viewed as bands, which are scored for presence and absence. RAPD bands are genetic markers that 21 exhibit dominant inheritance, meaning band absence indicates a homozygous recessive genotype, but band presence does not distinguish between a homozygous or heterozygous genotype for band presence. Patterns of band sharing between the taxa can be examined in order to discriminate between the hypotheses proposed by Bliss (1986). If Michigan monkey- flower originated via hybridization, the RAPD pattern should show an additive banding pattern for genetic markers of James’ monkey-flower and Common monkey-flower. Likewise, if Michigan monkey-flower originated from James’ monkey-flower or from Common monkey-flower, it should share bands with only the respective parent or bands fi'om one parent and few unique bands. If Michigan monkey-flower is a derivative of either James’ monkey-flower or Common monkey-flower it should not share any bands with the non-parental species unless the band is common to all three taxa. RAPD is a useful technique because it offers an essentially unlimited number of markers for study, requires minimal amounts of DNA, is not limited to functional proteins, does not require sequence knowledge, and is relatively inexpensive (Hadrys et al., 1992; Williams et al., 1993). Furthermore, RAPD markers were appropriate for this study because they require very little plant material, which is a consideration when studying endangered species. Materials and Methods Sampling Forty-two samples were included in the analysis including thirty-three samples of Mimulus glabratus and nine samples of Mimulus guttatus (Table 6). Sampling within 22 Mimulus glabratus included 19 samples of Michigan monkey-flower (Mimulus glabratus var. michiganensis) from 10 of 15 known populations. These samples were collected from across its range in Michigan. Sampling of other M. glabratus included 13 samples of James’ monkey-flower (Mimulus glabratus var. jamesii) and one sample of Utah monkey-flower (Mimulus glabratus var. utahensis). Sampling was concentrated in James’ monkey-flower because it is the only other variety of M. glabratus found in Michigan. Samples of James’ monkey-flower are included from Michigan, Quebec, Oklahoma, Nebraska, Texas and Mexico. Samples of Common monkey-flower cover its geogaphic range and are from Michigan, California, Utah and Mexico. Sampling of Michigan populations of James’ and Common monkey-flower concentrated on Michigan populations because local populations are more likely to have been important in the origin of Michigan monkey-flower. DNA Isolation Genomic DNA was isolated using the method of Doyle & Doyle (1987) as modified by Loockerman and Jansen (1996) for small amounts of plant tissue. DNA was isolated from fresh tissue or tissue preserved in silica or liquid nitrogen. Approximately 20 mg of fresh tissue or 1 mg dried tissue was gound in 0.4 mL extraction buffer consisting of 2% hexadeclytrimethyl-ammonium bromide (CTAB), 1.4 M NaCl, 25 mM EDTA , 100 mM Tris-HCl, pH 8.0, 0.5% B-mercaptoethanol and 4% polyvinyl-pyrrolidone (PVP—40). An additional 0.4 mL extraction buffer was added to the homogenate and incubated at 60°C for 20-30 minutes. DNA was extracted from the homogenate with the addition of two—thirds volume chloroformzoctanol (24:1) followed by high-speed centrifirgation. The resulting supernatant was transferred to a clean tube 23 Table 6: Identity of Mimulus samples in the RAPD analysis. Posto, Prather and Trull vouchers housed at MSC. RSA vouchers housed at RSA. Vickery vouchers housed at UT. Taxa ID No. Location Voucher M. glabratus var. mich l, 2 Benzie Co , MI A.L. Posto 8 michiganensis mich 3, 4 Charlevoix Co., MI A.L. Posto 11 mich 5, 6 Cheboygan Co., MI A.L. Posto 4 mich 7, 8 Cheboygan Co., MI A.L. Posto 7 mich 9, 10 Emmett Co., MI A.L. Posto 18 mich 11, 12 Emmett Co., MI A.L. Posto l3 mich 13, 14 Leelanau Co., MI A.L. Posto 6 mich 15, 16 Leelanau Co., MI A.L. Posto 9 mich 17 Leelanau Co., MI A.L. Posto 5 mich 18, 19 Mackinac Co., MI A.L. Posto 12 M. glabratus var. jamle 1, 2, 3 Ontonagon Co., MI S. J. Trull 336 jamesii jamle 4, 5, 6 Ostego Co., MI A.L. Posto 1 jamle 7 Ostego Co., Ml A.L. Posto 10 jamsMX l Chihuahua, Mexico R.K. Vickery Jr. 12183 jamsMX 2 Guanajuato, Mexico R.K. Vickery Jr. 6201 jamsNE Custer Co., NE R.K. Vickery Jr. 7135 jamsOK Woodward Co., OK R.K. Vickery Jr. 7132 jamsQE Quebec, Canada R.K. Vickery Jr. 10226 jamsTX TX L.A. Prather 1805 M‘ glabrat'fs m" utah Wayne Co., UT R.K. Vickery Jr. 5265 utahenszs M. guttatus guttCA 1 San Bemardino Co., CA RSA 20448 guttCA 2 Los Angeles Co., CA RSA 19998 guttCA 3 Contra Costalo Co., CA R.K. Vickery Jr. 5052 guttMI l, 2 & 3 Ontonagon Co., MI 8.1. Trull 332 guttMX Chihuahau, Mexico R.K. Vickery Jr. 12180 guttUT l, 2 UT A.L. Posto 3 24 and DNA was precipitated with two-thirds volume ice-cold isopropanol and stored at —20°C overnight. Precipitated DNA was condensed to a pellet in a centrifuge at high speed and washed with 0.8 mL 76% EtOH/0.01 M NH40Ac. DNA was resuspended in water. DNA was quantified following extraction and was not cleaned prior to amplification. DNA Amplification and Visualization Samples were screened with 50 RAPD primers from sets A, B, and C from Operon Technologies (Alameda, CA). Primers that were easily amplified and which maximized the number of bands per primer were chosen. Reactions were carried out in 25 ul consisting of IX T aq DNA polymerase buffer, 2 mM MgC12, 0.2 mM of each dATP, dCTP, dGTP and dTTP (Boehringer Mannheim), 0.4 uM of primer (Operon), 1 unit of T aq DNA polymerase (Promega) and 25 ng of DNA. Amplifications were performed in an MJ Research Progarnmable Thermal Controller with the following PCR profile: 5 minutes at 94°C; 45 cycles of 1 minute at 94°C, 1 minute at 75°C, 2 minutes at 35°C; 5 minutes at 72°C; 15°C soak. Each sample was amplified twice with each primer to demonstrate repeatability. The bands were resolved by electrophoresis on a 2% agarose gel in 1X TAE (Tris-Acetate-Borate) buffer, stained with ethidium bromide and visualized with UV illumination. Images were recorded digitally using Alphalmager 2000 software (Alpha Innotech). RAPD Analysis A schematic diagam of the similarity analysis is shown in Figure 4. RAPD images were scored for band presence and absence using Pro-RF LP Molecular Weight software (DNA Pro-Scan). Only reproducible bands were scored and reproducibility was 25 tested by scoring a minimum of two amplifications of each DNA sample with primer. A sample by sample similarity matrix (Appendix B) was constructed from a sample by marker data matrix (Appendix A) using J accard’s coefficient. J accard’s coefficient estimates similarity for all pairwise comparisons based on the number of shared traits and omits negative matches (matches based on the absence of a marker) (Sokal and Sneath, 1963). UPGMA clustering analysis was used to analyze the similarity matrix to examine relationships between the samples. NTSYSpc version 2.1 (Applied Biostatistics, Inc.) was used for similarity and clustering analyses. Data Matrix W X Y Z llllllll TaxonWXYZ Band — — — 300nt _ _ 200m 300m 1 0 1 1 ——> 200m 1 l 0 0 _ _ _ 100m 100m 1 ll 1 0 l l Jaccard’s Coefficient UPGMA Similarity Matrix W X Y Z JW I Ix W 1.00 I z x 0.67 1.0 Y ‘—_ Y 0.33 0.0 1.0 028 ' ' 038 h 0.27 ' ' 0'57" ' 067 Z 0.67 0.3 0.5 1.00 Coefficient Figure 4: Similarity analysis flowchart. The gel image (upper left comer) is scored and the data is recorded in the data matrix. The similarity matrix is constructed using J accard’s coefficient and a phenogarn is constructed by clustering the similarity matrix using UPGMA. 26 Results RAPD Primers Six primers were chosen and a total of 99 amplified products were scored in the analysis (Table 7). The number of amplified products ranged from 8-22 products per primer. A representative image is shown in Figure 5. Table 7: Primers used in the RAPD analysis. Primer Nucleotide Sequence Number Amplified Fragment Size Identification (5’ to 3’) Products Range (nt) CPA-07 GAAACGGGTG 1 9 500-1 760 OPA-l l CAATCGCCGT 22 350-1 100 OPA-12 TCGGCGATAG 14 650-1550 OPE-05 TGCGCCCTTC 8 750-1325 OPB- 1 0 CTGCTGGGAC 1 7 43 0-2000 OPC-02 GTGAGGCGTC l 9 3 50-1 325 12345 6 789101112131415 Figure 5: Image for thirteen individuals amplified with primer A07. Lanes 1 and 10 are molecular weight standards. Lanes 2-9 are Michigan monkey-flower, lanes 1 1-12 are Common monkey-flower and lanes 13-15 are James’ monkey-flower. Band A is present in all taxa. Bands B, E and F are unique to Common monkey-flower. Band C is unique to Michigan monkey-flower. Bands D and G are shared by Michigan monkey-flower and James' monkey-flower. 27 RAPD Banding Patterns Of the 99 amplified products, 70 were specific to individuals of one of the four taxa in the analysis (Table 8). The remaining twenty-nine bands are shared between samples of the four taxa. Within Common monkey-flower, five polymorphic bands were found only in Michigan individuals, and 4 polymorphic bands were shared between individuals from Michigan and other localities. Of the three bands Michigan and James’ monkey-flower shared, one band was found in all individuals of Michigan monkey-flower and three of seven individuals of James’ monkey-flower fiom Michigan populations. The second was found in all individuals of Michigan monkey-flower and all individuals of James’ monkey-flower from Michigan and individuals from Texas and Quebec. The third was found in all individuals of Michigan monkey-flower and individuals of James’ monkey-flower fi'om all populations sampled (Mexico, Oklahoma, Nebraska, Texas and Canada). Of the three bands Michigan -and Common monkey-flower shared, the first was shared between individuals of Michigan and Common monkey-flower fi'om Michigan, Utah and California. The remaining two bands were shared between individuals of Michigan and Common monkey-flower from Michigan only. The band shared between J arnes’ and Utah monkey-flower was found in individuals from all localities of James’ monkey-flower (Michigan, Texas, Nebraska, Oklahoma, Quebec and Mexico). For one polymorphic band shared between all four taxa, the band was absent in all samples of the Michigan populations of James’ monkey-flower and Common monkey- 28 flower. Thus, all samples of Michigan monkey-flower shared this band with samples of James’ monkey-flower from Nebraska, Oklahoma and Texas, with samples of Common monkey-flower from Utah, California and Mexico, and with Utah monkey-flower and not with local populations. Table 8. Bands shared between taxa. Michigan monkey-flower is “MICH”, James’ monkey-flower is “JAMS”, Utah monkey-flower is “UTAH”, and Common monkey- flower is “GUTI‘”. Fixed bands are found in all individuals of taxa in the left column, polymorphic bands are found in one or more, but not all, individuals. Moving down the table, bands shared between taxa are not cumulative. That is, the bands shared between all four taxa (MICH-JAMS-GUTT-UTAH) are not found in any other rows in the table. The same is true for all other rows. Taxa (No. Individuals) Fixegand F’gjggmhic Total MICH-JAMS-GUTT-UTAH (42) 1 8 9 MICH-JAMS-GUTT (41) 0 3 3 MICH-JAMS-UTAH (33) 2 3 5 MICH-UTAH-GU'IT (29) 0 1 1 JAMS-UTAH-GUTT (23) 0 1 1 MICH-JAMS (32) 0 3 3 MICH-GU'I'I‘ (28) 0 3 3 JAMS-GUTT (22) 0 3 3 JAMS-UTAH (14) 0 1 1 MICH (19) 3 10 13 JAMS (13) 0 14 14 UTAH (1) NA 3 3 ourr (9) 0 40 40 Similarity Coefficients and UPGMA Among the 19 samples of Michigan monkey-flower, eight different multilocus genotypes were represented (Figure 6). The average similarity coefficient among all Michigan monkey-flower individuals was 0.92, indicating high genetic similarity among 29 the samples (Table 9). Based on the UPGMA phenogam (Figure 6), all individuals of Michigan monkey-flower are distinct from the other taxa. Michigan monkey-flower exhibits much higher genetic similarity to James’ monkey-flower (0.52) than it does to Common monkey-flower (0.24) (Table 9). Among the 13 samples of James’ monkey-flower, nine multilocus genotypes were represented. Utah monkey-flower falls within James’ monkey-flower in the UPGMA phenogam and all individuals of M. glabratus (Michigan monkey-flower, James’ monkey-flower and Utah monkey-flower) are distinct from Common monkey-flower, M. guttatus (Figure 6). Among the nine Common monkey-flower samples eight multilocus genotypes were represented. All three individuals of Common monkey-flower from Michigan form a cluster with the samples of Mimulus glabratus and exhibit low genetic similarity to other individuals of Common monkey-flower (Figure 6). Table 9: Genetic distance between Michigan monkey-flower, James’ monkey-flower and Common monkey-flower. Except for within taxon comparisons, distance measures are calculated from the similarity between taxa and ignores similarity within taxa. (i.e. comparisons between michiganensis and guttatus are based only on the similarity between individuals of michiganensis and guttatus and not the similarity between individuals of michiganensis) Michigan James’ Common monkey-flower monkey-flower monkey-flower Michigan 0.92 monkey-flower (0.77-1 .000) James’ 0.52 0.66 monkey-flower (0.33-0.68) (0.38-1.000) Common 0.24 0.20 0.30 monkey-flower (0075-034) (0033-028) (0.10-1.00) 30 456508 55:86 65 3:8898 28m 2:. .G 638. 9 8.3: e383: 9 can .383 u PD .8925 0 m0 £80530 0 Va .3356 Z n mZ .cewEBZ n :2 .8382 0 X2 .m_Eo.:_eU .I. <8 “wEBozota mm 0.8 323389? 5:83 .m 2an E wow: omofi 3o=£ £28305? coxfl. .25th wEeSfi an; .3 womboeow 338% 335.5 5. mo Emcwoeona 52055 no Semi 31 o3 32 Discussion The data presented in this analysis suggest that Michigan monkey-flower is not a recent hybrid of James’ monkey-flower and Common monkey-flower. It is expected that a recent hybrid species should show additivity of parental marker alleles, but few if any unique alleles (Gallez and Gottlieb, 1982; Rieseberg et al., 1990; Wolfe and Elisens, 1995; Morrell and Rieseberg, 1998). Michigan monkey-flower exhibits an additive banding pattern for its putative parents because it shares as many bands with Common monkey-flower (that are not also shared with any other taxa) as it does with James’ monkey-flower (Table 8). However, the number of shared bands is a small fraction of the total markers in the analysis and it is likely the sampling of other M. glabratus and M. guttatus in this analysis is insufficient to detect all possible genetic markers that might be shared between taxa. Thus the band sharing data is difficult to interpret. In addition, Michigan monkey-flower possesses many unique genetic markers not found in any other taxa in the analysis (Table 8). In fact, it has nearly as many as found in the widespread James’ monkey-flower (Table 8). Michigan monkey-flower is also unlikely to be of recent origin because it shares genetic markers with western populations of James’ monkey-flower and Common monkey-flower that it does not share with more local, Michigan populations. Furthermore, Michigan monkey-flower is genetically distinct from other M. glabratus and M. guttatus in this study. It exhibits low genetic similarity to these other taxa and has a high intraspecific identity compared to the more widespread James’ monkey-flower and Common monkey-flower (Michigan individuals of James’ monkey-flower do not form a distinct cluster). 33 The relatively low genetic diversity between individuals of Michigan monkey- flower (average genetic similarity 0.92; range 0.77-1.0) does not contradict an ancient origin for this group. Speciation events are often associated with genetic bottlenecks (Grant, 1981; Mayr, 1954, 1963; Avise, 1994). Genetic diversity in Michigan monkey- flower is also be expected to be low because asexual reproduction appears to predominate in all but one population (Bliss, 1983, 1986). Evidence to support an ancient hybrid origin comes from the considerable genetic similarity between Michigan monkey-flower and Michigan populations of James’ monkey-flower and Common monkey-flower. The average genetic similarity between Michigan monkey-flower and Michigan populations of James’ monkey-flower is 0.76 versus 0.52 between Michigan monkey-flower to all samples of James’ monkey-flower. The average genetic similarity between Michigan monkey-flower and Michigan populations of Common monkey-flower is 0.77 versus 0.24 between Michigan monkey- flower to all samples of Common monkey-flower. In addition, considerable divergence between Michigan populations and western populations of Common monkey-flower (Figure 6) suggest that Common monkey-flower in Michigan may be a natural population, and not a recent introduction. The potential existence of Common monkey- flower in Michigan for a long period of time bolsters the ancient hybridization hypothesis. Based on the genetic distance between taxa it is likely that Michigan monkey- flower originated from James’ monkey-flower. Michigan monkey-flower and James monkey-flower are much more similar to each other (0.52) than Michigan monkey-flower is to Common monkey-flower (0.24). Additionally James’ monkey-flower and Common 34 monkey-flower are greatly differentiated (0.20). Based on these findings it seems more probable that Michigan monkey-flower originated from James’ monkey-flower than Common monkey-flower or as a hybrid of James’ and Common monkey-flower. 35 CHAPTER 4 CONCLUSION My research on the biology of Michigan monkey-flower focused on its reproductive biology and taxonomic origin. An investigation of its reproductive biology specifically focused on its mating system parameters, pollen viability and seed germination. Experiments to determine the mating system of Michigan monkey-flower were conducted using individuals of the Maple River population, the only population known to have significant pollen viability and fruit set (Bliss, 1986). The results show that Michigan monkey-flower is self-compatible and plants from the Maple River population are capable of self-pollination and regularly set selfed fruits in the greenhouse. Pollen viability was examined in five populations including the Maple River and Reese’s Swamp populations. This study found considerable variation (27-52%) in pollen viability between individuals of the Maple River population. Maple River was reported to have 30% pollen viability (Bliss, 1986), but Bliss sampled only three individuals, one anther from each, from this population, thus variation between individuals, if detected, was not reported. Differences in pollen viability between individuals may have important consequences for reproduction and fitness among individuals of the population. These consequences in turn will have important implications for mating, selection, and gene flow within the Maple River population. The Reese’s Swamp population, which had not been previously studied, lacks viable pollen. Pollen viability was examined for individuals from Burt Lake, Carp Creek and Glen Lake and the results were similar to Bliss’s results for these same populations. I 36 found that these populations lack viable pollen; Bliss found less than 1% viable pollen (1986). Thus the population at Maple River is remarkable for its production of viable pollen. Among four seed germination regimes tested, the highest germination rates were observed at approximately 23°C with exposure to light. A significant decrease in germination was observed at approximately 23°C in the absence of light and no germination was observed at 8°C. Water temperatures at Michigan monkey-flower sites are considerably cooler than 23°C (Bliss, 1983) and full sunlight is generally lacking, therefore a reduction in germination at cooler temperatures and in the absence of light may have important consequences for recruitment of individuals fi'om seed into the population. Molecular markers were used in the taxonomic analysis to test between alternative hypotheses of hybrid origin from James’ monkey-flower, M. glabratus var. jamesii, and Common monkey-flower, M. guttatus, versus divergence from one or the other. The presence of unique genetic markers in Michigan monkey-flower and its low genetic similarity to other M. glabratus and M. guttatus are inconsistent with a recent origin of Michigan monkey-flower. At this time it seems most likely that Michigan monkey-flower diverged from James’ monkey-flower due to greater genetic similarity to it than to other taxa in the analysis. Among 19 samples of Michigan monkey-flower included in the taxonomic analysis, eight different multilocus genotypes were represented. Michigan monkey-flower is genetically distinct from the other taxa in the study and has relatively high intraspecific identity compared to the more widespread James’ monkey-flower and Common monkey-flower. 37 Future work to clarify the origin of Michigan monkey-flower can be conducted through phylogenetic work and pollination crosses. The construction of a sectional phylogeny of Simiolus using representatives of other sections in Mimulus as well as groups outside of Mimulus as outgroups, may clear up relationship issues between M. glabratus and M. guttatus and the other species of the section. This data could be estimated fi'om sequence data of nuclear and chloroplast genomes. Such a phylogeny is underway in Richard Olmstead’s lab at the University of Washington, Seattle, WA. Pollination crosses between Common monkey-flower and James’ monkey-flower will determine if two species are interfertile and whether viable and/or fertile F1 hybrids are produced. If hybrids could be formed it would be possible to determine morphological similarity of F. hybrids to Michigan monkey-flower by making quantitative observations of the F1 hybrid morphology and conducting a comparative study to Michigan monkey-flower based on quantitative characters studied by Bliss (1983, 1986) and Minc (1989). Though extant populations of James’ and Common monkey-flower may have diverged from ancient populations that could have contributed to a hybridization event leading to Speciation, this information may provide evidence to support an ancient hybrid origin for Michigan monkey-flower. However, the inability to produce F1 hybrids between crosses of James’ monkey-flower and Common monkey- flower will not rule out an ancient hybrid origin. 38 APPENDICES 39 APPENDIX A DATA MATRIX 4o 3.2-2.... 8:-:<, COCO CCCC CCCC COCO CCCC CCCC CCCC F-I CCCCC ~ fl — F! l l 8922 .. .r-’ lllll'lllillllillyl! mmn$o< ocm$o< .1. own$o< on??? u—no—‘OOOOO 85-53‘ "“J? -———4 "fly - - ov>$o< CCCCCv— __ -4? ow??? CCCCCC CCCCCC _ CCCCO'C u—t CCCCCC T. I l l l l i CCCCCCF' p—I CCCCCC—‘CCCC mob$o< — 2.5-22 c CCC oo—Eoooooo—oooo "CC I— f o 1.1.! l '1'! 1. I irlJ... _ 1 EC C F“: E . 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L :..? 83 83 283 83 83 88W? ..-... 388.»... m 83 83 83 83 83 2888 8 ‘ 1H I L. 83 83 83 28.. 8881 ,- , 3 L: iiiy H 83 .283 83 28W. L L 8 H 3 -:-- M L 83 83 S888 2285.. 3288.. :58.» 2588 N288 .288 288 L 228 T 3888 2888 888 . 288 2288 $88 m X—DZm—mm< 57 $2 $2 22 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $68» $2 $2 22 22 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 228% $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 88. $2 $2 82 82 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 228% $2 $2 $2 $2 22 22 $2 $2 $2 $2 $2 $2 $2 $2 moms: $2 $2 82 82 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 23 $2 $2 82 82 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 «<98» $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 :68» $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $088.: $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 mzmafi $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $28MW $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 :28» $2 $2 $2 $2 $2 $2 :2 :2 :2 :2 :2 :2 :2 :2 :288 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 2293 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 2288.2 $2 $2 $2 $2 $2 :2 $2 $2 $2 $2 $2 $2 $2 $2 2225.. 82 82 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 :288.. $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $42 $2 $2 $88.. $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 .$2 $228 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $228 $2 $2 $2 $2 82 82 $2 $2 $2 $2 $2 $2 :$2 $2 $328 222.. 22.88.: :58,» .582 N328 :38 M $88 3L $28 $328 $328 $88 288 2.8.8. $28 2.82 m $522.? 58 Fwd mood and owwd mood nmod $2028 o8.— amwd ”who :56 anwd ohmd mania o8; omod mmod o8; 2.06 $_8o88 So.— Sad omod mmcd n 30:: coo; mmod $06 2&8 coo.— owed _8088 o8; $_$4888$ mazauan «azaaah NH$288u _ Stu—w N328 __guuu $8088 $8088 ¢_ao:8 m 39:: $2088 29.8 o_8o=8 anoi— mmeamdnun ~_$2288_ __2428a_ Vaganuaa :.8088 0328 w 328 £28 328 030.2: m 328 :goau _8088 $_$4aua_ 35:03 m 592mm: 59 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 208» 82 82 $2 82 $2 $2 $2 $2 :2 $2 $2 $2 $2 $2 2225.2 $2 $2 $2 $2 $2 $2 $2 $2 :2 $2 $2 :2 $2 $2 $8 $2 $2 $2 :2 82 $2 $2 $2 $2 82 $2 $2 82 $2 $295.: $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 moms.»H $2 $2 $2 $2 $2 :2 :2 $2 $2 $2 :2 $2 $2 $2 $288 $2 $2 22 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 298$ 22 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 $2 298» 28.2 $2 $2 $2 $2 22 22 $2 :2 $2 22 $2 $2 $2 $0.88.. 82 $2 $2 $2 $2 .22 22 $2 :2 $2 22 $2 $2 $2 $888..“ 22 £2 :2 $2 $2 $2 22 $2 $2 $2 $2 $2 $2 $2 :23 $2 m $2 $2 :2 82 $2 $2 $2 $2 82 $2 $2 82 $2 :28» $2 2 $2 $2 $2 $2 $2 $2 :2 $2 $2 82 $2 $2 $2 :22~ $2 $2 82 82 $2 $2 $2 $2 $2 $2 $2 :2 $2 $2 :EEE 83 83 $2 82 $2 $2 $2 $2 82 :2 $2 $2 $2 $2 228% 83 $2 82 $2 $2 $2 $2 82 $2 $2 $2 $2 $2 $388.: 83 $2 82 $2 $2 $2 $2 82 $2 $2 82 82 _382 83 $2 $2 $2 $2 82 $2 $2 $2 $2 $2 x53 83 $2 $2 $2|imm$d 83 $2 $2 83 $2 :58 83 $2 $2 $22.21 $2 $2 $2 $2 $2 $8.?) 83 $2 $2 $2 $2 $2 $2 $2 $328 ngrszgq 2225 528.. :58 $228 $388 $28 $88 $288 $388 $28 388 £228.H 2.88; 596.5. 60 203$ 23% n8: 222i 32.. x23» $6.3m 203$ gone: mzaa: $2..» 323$ :25» :53: 2.32; m “REM—._.? 61 111-1 11! 1 111-I :8; £3 ...-..ooo. _ Ed a 8.1.51. .1085 mde N_N.o otd omNd omNd -aocd Nmod GONd OONd n26 End :56 owe. .-o11 mvmd1. ...cll 11 :..-1111111.]! .11 295» .- l1|111|11 11A .qu,?..§.H coo; coo. _ oom111. o1 F11 111.111. $9.3m. NXZmEmm _ 11111311111! .11.. I.. 111111.... .:.-v1lll1ll 1:515. $3488: 1 1 1 F _T cos.— _ 83 82 and 23 $3 $3 23 was $3 $2. and 2:3 _ awa- 111 --:.1- - 83 MES 23 £3 8:. 23 33 $3 2.8 8.3 $3. . m .:.-.mxzmaa 1- - - 83 ~25 £3 35¢ 33 an; 33 23 23 Rom-fl-.. 33% 1111 .11 11.11711- - 83 :2 83 :3 55 SS :3 23 $51.... 2% - -.1- ...V- 11 83 w: .o 83 £3 93 83 83 so. o1H1--..-~<93w 1- 1 -1-1- .11 - 1-11-11, 83 $3 $3 23 83 83 m: .o _ 20.5w oo.1.11m c 1 ———-1—- __ 1 1 1 1 1 1 fiflff,‘_r-, 1 1 I 1 .» ..r - 1 I I .11-1 111 1 111 111 11. . 11 111111 ..1+11.111 v 1- . T1111 1 .1...- w—uv 1 1 1 1 1 1 1 1 -. . .1-1. 1 .. .1 1+ 1...! 1.1111- .11 . . 08aq x53» 563% 295» ..111.H111.1... .11! ...F1ill: .111) ...-.11 I. .1 $1111.11 1.101.111- N36 N26 .. .. 11 1 11111111111 22%» 1 I1 I..-1.111 1.1111?! 1 3:6 1111 .11. 1 1 11 -_711 1111-1 ? _ coo.— _1:. 11-1 11- - 11-1141.-- 1, 1- ,.1 11- 1.1.711 - 1111-1-11.-. -... 11....r..-1--11 11-11 1“?! 1 -1111! 111. $1.: _. 1 -:_T- 11 11.1.1111, -111-1:..-111.11“-1:-. 1 ._- - - 1 1 _-1 1 .11 ....... M - 11 .1. 1111 1111 111.111.4111 11 1|P1..11.11 11 11+ ..1 11 1 1111* 11, 11111111.“..- ” _ 1 1 1 .1 11111 1111 11110.1 1 1111M111 1 1 + 11 , m n _ _ m 1 1 L1 1-111 1 1 .1... v 11111.- 4 =an 22-5“ 2225.. .Al’1lyll' j -.NHEmama 1XHmES. 528a; w _ + $525:- _1mZman-HM. 22:an fizz—gm 3.83 m 594%.? 3:25. 62 REFERENCES 63 CITED LITERATURE Alam, M. T. and R. K.Vickery Jr. 1973. Crossing relationships of the Mimulus glabratus heteroploid complex. American Midland Naturalist 90: 449-454. 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