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_ , AGEE A OGA FINAL REPORT RESTORATION AND RECOVERY OF MASON'S LILAEOPSIS: PHASE I Submitted by: Peggy L. Fiedler & Randy K. Zebell Biology Department San Francisco State University San Francisco, California 94132 Submitted to: Ms. Tish Sprague Shell Oil Spill Litigation Settlement Trustee Committee C/o California Environmental Trust Hearst Building, Room 612 5 Third Street San Francisco, CA 94103 and Ms. Sandra Morey California Department of Fish & Game Endangered Plant Program 1416 Ninth Street, P.O. Box 94409 Sacramento, California 95814-2090 October 28, 1993 • .. • . • .. • . ......• • • • • .. • . • . • . • . • .. • .. • .. • . IA EO ESOAIO A ECOEY O MASOS IAEOSIS: ASE I Submitted by: Peggy L. Fiedler & Randy K. Zebell Biology Department San Francisco State University San Francisco, California 94132 Submitted to: Ms. Tish Sprague Shell Oil Spill Litigation Settlement Trustee Committee C/o California Environmental Trust Hearst Building, Room 612 5 Third Street San Francisco, CA 94103 and Ms. Sandra Morey California Department of Fish & Game Endangered Plant Program 1416 Ninth Street, P.O. Box 94409 Sacramento, California 95814-2090 October 28, 1993 FINAL REPORT RESTORATION AND RECOVERY OF MASON'S LILAEOPSIS: PHASE I I. EECUIE SUMMAY Field reconnaissance of the littoral zones of the Sacramento-San Joaquin Delta and the Napa River estuary, and greenhouse and genetic studies were initiated to investigate the geographic distribution, ecophysiology, and molecular genetics of Lilaeopsis masonii Mathias & Constance (Mason's Lilaeopsis). The report summarizes briefly the results this research, conducted from June 15, 1992 to October 28, 1993. The geographic distribution of Lilaeopsis masonii has expanded significantly with additional field reconnaissance. As of October 1993, 114 element occurrences are on record in the California Natural Diversity Data Base. Additional new geographic records not yet submitted to the California Natural Diversity Data Base include extensive populations in Suisun Marsh. Greenhouse experiments have demonstrated a significant morphological response of decreasing growth with increasing levels of water salinity, in part, confirming the earlier studies performed by Golden (1992). Seed germination trials demonstrated a strong negative correlation between water salinity and germination success, -- i.e., as salinity levels increased, both total germination success and rapidity of germination decreased. Seeds of Lilaeopsis masonii germinated in salinities of up to 12 parts per thousand, unlike the results of Golden (1992). These results suggest that increased water salinity levels in the Sacramento-San Joaquin Delta would probably adversely affect growth and establishment of L. masonii. Molecular studies of the ITS2 region of the Lilaeopsis genome did not find any differences among ouaios of L. masonii, nor between L. masonii and L. occidentalis. It is possible that additional molecular analyses may find genetic differences. Many significant populations of Lilaeopsis masonii coiue to be at risk, largely from human disturbance. Documented forms of disturbance include trampling from fishing and other human activities, erosion by wave and wake action, disintegration of pilings, and potentially by sea level rising and water quality degradation. The type locality of L. masonii presently remains overgrown by invasive species and may be threatened by loss of shoreline. Water hyacinth (Eichornia crassipes) appears to be a serious threat invading the western portion of the range of L. masonii. For Phase II, we suggest that additional molecular genetic studies be implemented for Lilaeopsis masonii and for additional geographically and taxonomically proximate taxa. We believe that performing a molecular technique known as RAPDs is likely to be the most illustrative of intraspecific and intrapopulational genetic differences. We also propose additional field studies involving reciprocal transplants, breeding biology, and plant community analyses. Final L masonii 28 October 1993 TABLE OF CONTENTS I. EXECUTIVE SUMMARY TABLE OF CONTENTS H. INTRODUCTION AND PROJECT OBJECTIVES 1 II.A. Purpose of Project 1 II.A.1. History of the Spill 1 H.A.2. Settlement Objective for the Restoration and Recovery of Lilaeopsis masonii 3 II.A.3. Research Completed in Phase I 3 II.A.3.a. Field Surveys 3 II.A.3.b. Ramet Ecophysiology Study 4 II.A.3.c. Seed Ecophysiology Study 4 ILA.3.d. Population Genetics Study 4 III. MATERIALS AND METHODS 5 IH.A. Field Research 5 IH.A.1. Geographic Distribution 5 III.A.2. Field Collection of Lilaeopsis 5 III.B. Greenhouse Studies 7 III.B.1. Ramet Growth Experiment 7 III.B.1.a. Ramet Propagule Preparation 9 IH.B.1.b. Ramet Experimental Setup 9 Ramet Experimental Run 10 HI.B.1.d. Ramet Data Collection and Analysis 10 IH.B.2. Seed Germination Physiology Experiment 11 Germination Experimental Setup 11 III.B.2.b. Germination Experimental Run and Data Collection 11 HI.C. Molecular Genetics Studies 12 III.C.1. General Background of Molecular Techniques 12 III.C.2. Sequencing Methods 13 IV. RESULTS AND DISCUSSION 15 IV.A. Background of Lilaeopsis masonii 15 IV.A.1. Taxonomy 15 IV.A.2. Autecology 18 IV.B. Field Research 20 IV.B.1. Geographic Distribution 20 IV.B.2. Summary of Findings 22 IV.C. Greenhouse Studies 22 IV.C.1. Ramet Experiment 22 IV.C.1.a. Summary of Findings 28 IV.C.2. Seed Germination Experiment 28 IV.C.2.a. Summary of Findings 32 IV.D. Molecular Genetics Studies 32 IV.D.1. ITS1 32 IV.D.2. ITS2 and Carrot 32 IV.D.3. ITS2 Variation Between Lilaeopsis masonii and L. occidentalis 37 IV.D.4. ITS2 Variation Within Lilaeopsis masonii 37 ia n 28 Ocoe IV.D.5. Summary of Findings 41 V. CONCLUSIONS 41 V.A. Relationship of Lilaeopsis masonii to L. occidentalis 41 V.B. Ecophysiology and Genetics of Lilaeopsis masonii 41 V.C. Recommendations for Phase II: Recovery and Restoration 42 V.C.1. Recommended Research 42 V.C.1.a. Molecular Genetics 42 V.C.1.b. Breeding Biology 43 V.C.1.c. Reciprocal Transplant Experiments 43 V.C.1.d. Community Analyses 44 V.C.2. Time Line 44 V.C.3. Proposed Budget 45 VI. REFERENCES CITED 46 LIST OF APPENDICES Appendix A. Color Plates of Lilaeopsis masonii. LIST OF FIGURES Figure 1. Site map of collection locations and permanent sampling sites used in this study for Lilaeopsis masonii in the Sacramento-San Joaquin Delta and the Napa River, California. Figure 2. Repeat unit of 18-25S nuclear ribosomal DNA, minus intergenic spacer. Figure 3. Line drawing of Lilaeopsis masonii (Source: Affolter 1985). Figure 4. Line drawing of Lilaeopsis occidentalis (Source: Affolter 1985). Figure 5. Map of the known locations of Lilaeopsis masonii. Figure 6. Gram fresh weigh increase of Lilaeopsis masonii for six salinity treatments from four locations. Data are final - initial weights. Figure 7. Gram fresh weigh increase of Lilaeopsis masonii for six salinity treatments from four locations. Data are (final - initial)/initial weights. Figure 8. Germination counts of seeds of Lilaeopsis masonii from Cache Slough in six salinities over time in days since start of experiment. Figure 9. Germination counts of seeds of Lilaeopsis masonii from Cache Slough in six salinities for 10, 20, 32, and 43 days since start of experiment. Figure 10. Germination vs salinity for seeds of Lilaeopsis masonii collected from Cache Slough. Figure 11. Regression of salinity vs. numbers of germinations of Lilaeopsis masonii seed ia masonii Ocoe 1993 collected from Cache Slough. igue 2. Photoautoradiograph of sequencing gel from which nucleotide sequences of Lilaeopsis masonii are obtained. igue . Photoautoradiograph of sequencing gel from which nucleotide sequences of Lilaeopsis masonii are obtained. IS O AES ae . Field collection sites and permanent field monitoring sites for Lilaeopsis masonii. ae 2. Actual mean salinities for each treatment and replication of Lilaeopsis masonii growth experiment. ae . Mean weight gain of Lilaeopsis masonii for each location and salinity regime of ramet growth experiment. Means are final - initial weight measured as grams fresh weight, n = 18 for each, ± standard error. ae 4. Mean weight gain of Lilaeopsis masonii for each location and salinity regime of ramet growth experiment. Means are (final - initial)/initial weight measured as grams fresh weight, n = 18 for each, ± standard error. ae . F-ratio values and levels of significance from a two-way ANOVA (salinity tolerance vs location with two nested replications) using Lilaeopsis nulsonii growth experiment data, final - initial weights. ae 6. F-ratio values and levels of significance from a two-way ANOVA (salinity tolerance vs location with two nested replications) using Lilaeopsis masonii growth experiment data, (final - initial)/initial weights. ae . Significantly different salinity and location groups of final - initial weights data for Lilaeopsis masonii growth experiment as revealed by Student-Newman-Keuls' multiple range tests. ae 8. Significantly different salinity and location groups of (final - initial)/weights weights data for Lilaeopsis masonii growth experiment as revealed by Student-Newman-Keuls' multiple range tests. ae . Germination counts for seeds of Lilaeopsis masonii collected from Cache Slough for selected days. ae 0. Aligned DNA sequences of the ITS2 Region in 18-25S nuclear ribosomal DNA from nine populations of Lilaeopsis masonii, one population of L. occidentalis, and Daucus carota (carrot; from Yukihiko et al. 1989). ia . masonii Ocoe 1993 iv II. INTRODUCTION AND PROJECT OBJECTIVES ILA. uose o oec As part of the legal settlement of the 1988 Shell Oil Refinery spill at Martinez, California, a cooperative agreement was entered involving the Shell Oil Spill Litigation Settlement Trustee Committee, San Francisco State University, and the California Department of Fish and Game. The purpose of the cooperative agreement was to begin the restoration and recovery of Mason's lilaeopsis (Lilaeopsis masonii). Several populations of this state-listed rare plant were oiled during the 1988 oil spill from Shell Oil Company's Martinez refinery, and as part of the settlement, various aspects of the geographic distribution, ecophysiology, and molecular genetics of Mason's lilaeopsis were examined.
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