ROAD TO RECOVERY: INTRODUCTION OF TWO RARE VERNAL

POOL GRASSES, GREENE’S ()

AND COLUSA GRASS ( colusana)

______

A Thesis

Presented

to the Faculty of

California State University, Chico

______

In Partial Fulfillment

of the Requirements for the Degree

Master of Science

in

Botany

______

by

Erin Gottschalk Fisher

Summer 2013 ROAD TO RECOVERY: INTRODUCTION OF TWO RARE VERNAL

POOL GRASSES, GREENE’S TUCTORIA (Tuctoria greenei)

AND COLUSA GRASS (Neostapfia colusana)

A Thesis

by

Erin Gottschalk Fisher

Summer 2013

APPROVED BY THE DEAN OF GRADUATE STUDIES AND VICE PROVOST FOR RESEARCH:

Eun K. Park, Ph.D.

APPROVED BY THE GRADUATE ADVISORY COMMITTEE:

Colleen A. Hatfield, Ph.D., Chair

Kristina A. Schierenbeck, Ph.D.

F. Thomas Griggs, Ph.D.

Joseph G. Silveira, M.S. ACKNOWLEDGEMENTS

First I would like to thank my advisor, Colleen Hatfield, for despite her very full schedule, always made time to meet with me. If I was struggling, she helped me get out of my head and put things in perspective. Without fail, she knew when to light a fire under me to keep me moving forward. I am also incredibly thankful for her assistance with the hydrology and reference population mapping figures. The rest of my committee, Joe Silveira, Tom Griggs, and Kristina Schierenbeck, were wonderful as well. Joe introduced me to the National Wildlife Refuge system and to a wealth of knowledge on soils, geology, biology, and management. Tom provided generous first-hand information and stories about my study species, and invaluable insight into what would likely work (and not work) for my introduction methods. Kristina was always available and happy to talk to me about my project, including research design and the evolutionary and genetic perspective. A big shout- out to my lab mates, including fellow lovers Melanie Williams, Tim Hanson, and

Rachel Francis, who helped me problem-solve and provided support and friendship in and out of the lab.

CSU Chico state professors outside of my committee were also helpful.

Robert Schlising took me on my first trip to Vina Plains and introduced me to Greene’s tuctoria. He also provided me with a generous amount of literature on Vina Plains and my study species. Nancy Carter was vastly helpful with the statistical approach and tests I used for my research. I am more confident with statistics after working with her iii over many hours. David Brown opened his geology lab to me and helped guide me through the soil salinity analyses.

I am extremely grateful to the U.S. Fish and Wildlife Service for access to

Llano Seco and Colusa NWR; the Nature Conservancy for access to Vina Plains; and the

UC Davis Natural Reserve System and Solano Land Trust for access to Jepson Prairie. I have immense gratitude for Heather Davis, Carol Witham, and John Gerlach for advice and sharing their expertise on vernal pools and associated rare grasses.

I greatly appreciate the funding I received for this research. I was funded in part by Northern Botanists; California Native Plant Society; Grants-In-Aid of

Research from the National Academy of Sciences, administered by Sigma Xi, the

Scientific Research Society; Jim Jokerst Field Botany Award; Garrett Gibson Memorial

Botany Scholarship; and Vesta Holt Field Studies Merit Project Award.

Thank you to my family and friends who have been supportive and cheered me on throughout this process. Especially my Mom and Dad, Ann and Guy Gottschalk, who I am forever grateful for instilling in me a curiosity and love of the natural world, and encouraging me in my educational pursuits. Last but not least, I am eternally thankful to my amazingly supportive, generous, and creative husband, Hyland Fisher.

He was my loyal field assistant and design consultant; hard-working, insightful, and an expert problem-solver. Not to mention that his patience and excellent cooking kept me going along the way. Thank you! I am so happy to have shared this journey with you.

iv TABLE OF CONTENTS

PAGE

Acknowledgements ...... iii

List of Tables ...... vii

List of Figures ...... viii

Abstract ...... xii

CHAPTER

I. Introduction ...... 1

II. Background ...... 9

Vernal Pools ...... 9 Rare Plant Introductions and Reintroductions ...... 16 Study Species ...... 21

III. Methods ...... 44

Study Sites ...... 44 Sampling Design and Data Collection ...... 63 Data Analyses ...... 81

IV. Results ...... 85

Characterizing Study Sites ...... 85 Introductions and Reintroductions ...... 97 Lab ...... 130

V. Discussion ...... 134

Characterizing Study Sites ...... 134 Introductions and Reintroductions ...... 138 Lab Germination ...... 156 Summary, Conclusions, and Implications for Conservation ... 158

v PAGE

References ...... 167

Appendices

A. 2010 Hydrology – Dry Down ...... 183 B. 2010 Reference Populations ...... 205 C. 2011 Hydrology – Dry Down ...... 215 D. 2011 Reference Populations ...... 237

vi LIST OF TABLES

TABLE PAGE

3-1. Length of transects, number of packets, and number of seeds in seed packets for the introduction and reintroduction pools or areas ...... 72

3-2. Sixteen Greene’s tuctoria treatments (n=3 Petri dishes each) and 8 Colusa grass treatments (n=3 Petri dishes each) to test for specific factors affecting seed germination ...... 79

4-1. Approximate final dry-down date for pools at the four study sites ...... 86

4-2. Vegetation community within (on-patch) and outside (off-patch) mapped Greene’s tuctoria areas in five pools at Vina Plains ...... 93

4-3. Vegetation community within (on patch) and outside (off patch) of mapped Colusa grass areas in Olcott Lake at Jepson Prairie ...... 96

4-4. Vegetation community associated with the three introduction pools at Colusa NWR ...... 97

4-5. Statistical results for comparison of Greene’s tuctoria (a) Reintroduction pools at Vina Plains and (b) Introduction pools at Llano Seco ...... 111

4-6. Seed treatment percent germination: Kruskal-Wallis test results for two-way factorial experiment including temperature and substrate ...... 132

4-7. treatment germination/Petri dish: Kruskal-Wallis test results for two-way factorial experiment including temperature and substrate ...... 133

vii

LIST OF FIGURES

FIGURE PAGE

2-1. (a) Colusa grass at Jepson Prairie and (b) Greene’s tuctoria at Vina Plains ...... 22

2-2. Current distribution of Greene’s tuctoria ...... 33

2-3. Current distribution of Colusa grass ...... 41

3-1. Vicinity map and site photos for the Greene’s tuctoria study sites ...... 45

3-2. Vicinity map and site photos for the Colusa grass study sites ...... 46

3-3. Total monthly precipitation for September through June for the three years of the study along with the long-term average for (a) Vina Plains and (b) Llano Seco ...... 47

3-4. Total monthly precipitation for September through June for the three years of the study along with the long-term average for (a) Jepson Prairie and (b) Colusa NWR ...... 48

3-5. Comparison of total and average September through June precipitation for the four study sites ...... 49

3-6. Vina Plains study site and reference pools ...... 52

3-7. Llano Seco study site and created pools ...... 55

3-8. Olcott Lake at Jepson Prairie study site ...... 57

3-9. Colusa NWR study site and restored pools ...... 62

3-10. Packets for (re)introductions: (a) Seed and (b) Inflorescence ...... 68

3-11. Greene’s tuctoria threshing device ...... 76

4-1. Greene’s tuctoria phenology ...... 87

viii FIGURE PAGE

4-2. Three Greene’s tuctoria growing in close proximity as to appear as one plant ...... 89

4-3. Colusa grass phenology...... 90

4-4. Colusa grass juvenile morphology ...... 91

4-5. Introduction (Llano Seco) and reintroduction (Vina Plains) Greene’s tuctoria plants ...... 98

4-6. Comparison of seed packets at reintroduction (Vina Plains) and introduction (Llano Seco) pools (n= 3 pools each) with respect to (a) percent germination, (b) percent survivorship, and (c) vigor...... 100

4-7. Seed packet treatment average number of seeds per inflorescence at Vina Plains reintroduction Pool 35 (n=25 ) and Llano Seco introduction Pool 4 (n= 34 inflorescences) ...... 101

4-8. Comparison of inflorescence packets at reintroduction (Vina Plains) and introduction (Llano Seco) pools (n= 3 pools each) with respect to (a) germination (average number of germinations per packet), (b) percent survivorship, and (c) vigor ...... 102

4-9. Inflorescence packets average number of seeds per inflorescence at Vina Plains reintroduction Pool 35 (n = 25 inflorescences) and Llano Seco introduction Pool 4 (n = 25 inflorescences) ...... 103

4-10 Comparison of reintroduction (Vina Plains) and introduction (Llano Seco) pools (n= 3 pools each) with respect to (a)percent survivorship and (b) vigor ...... 104

4-11. May and July data for seed packet (a) percent germination and (b) percent survivorship to reproduction for Pool 37 (Vina Plains) and Pool 4 (Llano Seco) ...... 105

4-12. May and July data for inflorescence packet (a) average number of germinations per packet and (b) percent survivorship to reproduction for Pool 37 (Vina Plains) and Pool 4 (Llano Seco) ...... 106

ix FIGURE PAGE

4-13. Difference between seed and inflorescence packet treatments (n= 6 pools each) with respect to (a) percent survivorship to reproduction and (b) vigor ...... 107

4-14. Difference in seed and inflorescence packet treatments (n= 2 pools each) with respect to reproductive output ...... 108

4-15. Comparison of wild plants with seed and inflorescence packet treatments (n= 3 pools each) at Vina Plains with respect to vigor ...... 109

4-16. Average number of seeds per inflorescence for seed and inflorescence packets and wild plants at Vina Plains reintroduction Pool 35 (n = 25 inflorescences each) ...... 110

4-17. Comparison of Llano Seco pools seed packets (n= 2 transects each) with respect to percent (a) germination, (b) percent survivorship, and (c) vigor ...... 111

4-18. Comparison of Llano Seco pools inflorescence packets (n= 2 transects each) with respect to (a) percent survivorship and (b) vigor ...... 112

4-19. Linear regression of weight (g) of inflorescence and number of seeds per inflorescence from inflorescences (n= 134) taken from Llano Seco Pool 4 and Vina Plains Pool 35 ...... 113

4-20. Multiple second generation Greene’s tuctoria plants growing out of one first generation inflorescence ...... 115

4-21. Second generation (2012) total number of reproductive plants and total number of culms at Llano Seco introduction pools ...... 116

4-22. Percent population increase from first to second generation at Llano Seco introduction pools ...... 117

4-23. First (2011) and second generation (2012) average number of culms/plant at Llano Seco introduction pools ...... 118

4-24. First generation (2011) total number of reproductive plants collected and remained in introduction pools at Llano Seco ...... 119

x FIGURE PAGE

4-25a,b. Llano Seco (a) Pool 4 and (b) Pool 6 total number of plant material introduced, first generation (total and remained after collection), and second generation ...... 120

4-25c. Llano Seco (c) Pool 11 total number of plant material introduced, first generation (total and remained after collection), and second generation ...... 121

4-26. Introduction (Colusa NWR) Colusa grass plants ...... 122

4-27. Reintroduction (Jepson Prairie) Colusa grass plants ...... 124

4-28. Comparison of reintroduction (Jepson Prairie) (n= 2) and introduction (Colusa NWR) (n= 3) pools with respect to (a) percent germination, (b) percent survivorship, and (c) vigor...... 125

4-29. Comparison of wild plants with seed packet treatment (n= 2 each) at Jepson Prairie, Olcott Lake, with respect to vigor ...... 127

4-30. Percent germination comparison of (a) Jepson Prairie, Olcott Lake, north and south areas and (b) Colusa NWR pools (n = 2 transects each)...... 128

4-31. Soil salinity measured as conductivity in decisiemens/meter (dS/m) from a soil-water mixture from Jepson Priaire, Olcott Lake, and two introduction pools at Colusa NWR (n = 3 soil samples each)...... 129

4-32. Greene’s tuctoria seedlings in lab germination experiment ...... 130

4-33. Greene’s tuctoria seed treatment percent germination in a two-way factorial experiment comparing stratification temperature (n = 12 Petri dishes each) and germination substrate (n = 6 Petri dishes each) ...... 131

4-34. Greene’s tuctoria inflorescence treatment average germinations per Petri dish in a two-way factorial experiment comparing stratification temperature (n = 12 Petri dishes each) and germination substrate (n = 6 Petri dishes each) ...... 132

5-1. Third generation Greene’s tuctoria at Llano Seco introduction Pool 4 ... 166

xi ABSTRACT

ROAD TO RECOVERY: INTRODUCTION OF TWO RARE VERNAL

POOL GRASSES, GREENE’S TUCTORIA (Tuctoria greenei)

AND COLUSA GRASS (Neostapfia colusana)

by

Erin Gottschalk Fisher

Master of Science in Botany

California State University, Chico

Summer 2013

Vernal pool habitats have been significantly reduced by conversion to incompatible agriculture and urbanization. As a result, a number of vernal pool dependent species have become rare, including Colusa grass (Neostapfia colusana) and

Greene’s tuctoria (Tuctoria greenei). This study examined the potential for introductions of the rare grasses into vernal pool habitats. To this end, four study sites were established, two sites for each species – one introduction site with restored or created vernal pools and one reference site with existing populations of the rare grass. The year prior to introductions, pool hydrology and reference populations were monitored and mapped to inform introduction success. Using seed and inflorescence packets, the species were introduced into the restored/created pools and, for comparison, reintroduction into the reference pools. For Greene’s tuctoria, germination and

xii survivorship to reproduction occurred at both introduction and reintroduction pools.

The introduction pools had significantly higher average percent germination (60%) than the reintroduction pools (35%), which may be a result of disturbance to the germination and early seedling stage at the reintroduction pools. Plants from seed packets had significantly higher vigor but showed a trend towards lower reproductive output compared to the plants growing from the higher-density inflorescence packets. In the second year, despite relatively low rainfall and only partial pool filling, the introduction pools supported over 2,000 second generation Greene’s tuctoria plants. For Colusa grass, germination occurred at both the introduction pools (13%) and reintroduction pools (23%); however only one plant survived to reproduce at the introduction pools while reintroduction pools had 40% survivorship to reproduction. Soil testing suggested that the low survivorship may be due to elevated salinity at the introduction pools. The differences between species and packet methods illuminated different paths to potential success in introducing new populations. The results of this research are imperative in informing recovery efforts for Colusa grass and Greene’s tuctoria populations as well as for other rare vernal pool plants.

xiii

CHAPTER I

INTRODUCTION

Worldwide, humans are altering the environment at unprecedented rates, resulting in adverse effects on biodiversity and loss of native species (Chapin et al. 2000;

Pimm et al. 1995; Vitousek et al. 1997). Habitat loss, including and fragmentation, are a leading cause of loss of native plant species (Fahrig 2003; Schemske et al. 1994). Raven (1987, 2000) estimated that approximately 24% of the world-wide vascular plants are currently at risk of extinction. Moreover, plants that are already rare are more likely to be threatened by extinction due to habitat loss because they are already restricted in range, are patchily distributed and have fewer populations or individuals (Giam et al. 2010).

Specifically in the United States, 22% of the vascular plants are currently of conservation concern (Falk 1992). Approximately 7,000 plant species are considered rare or endangered in the United States, out of a total of approximately 17,000 native United

States plant species (Morse 1996). California is known to have large numbers of globally rare vascular plants. While rarity varies geographically, it also varies taxonomically.

For instance, plant families with the largest number of rare plants in the continental

United States include the sunflower family (Asteraceae), the pea family (Fabaceae), the mustard family (Brassicaceae), the figwort family (Scrophulariaceae), and the grass family () (Morse 1996).

1 2 While many habitat types are threatened, wetlands habitats are one of the most endangered in the United States. It is estimated that more than 50% of wetlands have been lost in the lower 48 states since pre-colonial times (Dahl 1990). For example, the California Central Valley was estimated to support 1.6 million hectares of wetlands in the 1850s (Frayer et al. 1989). However, by 1939, the wetland hectares decreased to less than 324,000, and by the mid-1980s the Central Valley supported only 220,000 hectares of wetlands and deep-water habitat. Average net loss in the Valley over a 46- year period was 22,000 wetland hectares per year (Frayer et al. 1989). Wetlands typically support a high diversity of flora and fauna due to the high rate of primary productivity

(USFWS 2005). The destruction of millions of acres of wetlands in the California Central

Valley has, as a result, lead to a marked loss of biodiversity and native species.

Vernal pools are a type of seasonal, freshwater wetland found predominately in Mediterranean regions of the world. Though globally distributed, the vernal pools in

California are unique in that they support an extensive flora endemic to vernal pools

(Keeley and Zelder 1998). In California, vernal pools occur across varied landscapes, from the Modoc Plateau in the northern part of the state to San Diego County along the

Mexico border (Keeler-Wolf et al. 1998). The pools form in depressions in mound-basin landscapes underlain by a water-impermeable layer (e.g., hardpan, claypan, or rock). In

California’s Mediterranean climate (cool, wet winters and dry, hot summers), the pools fill with water in the fall and winter, begin to dry down in the spring, and are completely dry during the summer until rains begin again in the fall. Keeley and Zelder

(1998) identify four stages in an annual vernal pool cycle: wetting phase, aquatic or inundation phase, waterlogged-terrestrial phase, and drought phase. This unique,

3 ephemeral wetland habitat supports an assemblage of species that are not only adapted to this annual cycle but many of which are also endemic to vernal pools.

Vernal pools were once much more prevalent than they are today and are among the most threatened wetland habitats in California (Stone 1990). Due to urbanization and intensive, incompatible agricultural practices, vernal pool habitat loss in the California Central Valley alone has been estimated to be between 60 to 85% prior to 1973 (USFWS 1994; Holland 1978). By 2005, an estimated additional 137,100 acres of vernal pool habitat (13.3% of remaining) had been lost in the Central Valley due to continued land use conversion associated with agricultural activities and urban development (Holland 2009). The largest percentage of vernal pool land use conversion was for “orchards, vineyards, and eucalyptus” (Holland 2009).

Vernal pool habitat loss leads to significant loss of biodiversity. For example,

King (1998) applied a stochastic model to predict the loss of vernal pool crustaceans due to elimination of habitat. He concluded that vernal pool habitat losses ranging between

50 to 85% may have led to extinction of 15 to 33% of the original vernal pool crustacean species in the Central Valley (King 1998).

Vernal pool plants are dominated by native annual forbs, many of which are endemic to California (Keeler-Wolf et al. 1998). Approximately 90% of California’s vernal pool flora is native (Holland 1976), 52 vernal pool taxa are rare (Stone 1990), and

15 of the plants are federally listed as endangered or threatened (USFWS 2005). Seven

(approximately half) of the federally listed vernal pool plant species are grasses from the family Poaceae, tribe Orcuttieae, and from one of the following three genera: Neostapfia,

Orcuttia, and Tuctoria.

4 Due to the native and found in vernal pools and the significant habitat loss, conservation of vernal pool habitats has become a state-wide and national concern. Per the Endangered Species Act of 1973, a recovery plan is required for plant and animal species listed as federally threatened or endangered. The recovery plan

“delineates, justifies, and schedules the research and management actions necessary to support recovery of a species” (USFWS 2005). Conservation of vernal pools is outlined in the Recovery Plan for Vernal Pool Ecosystems of California and Southern Oregon (USFWS

2005). The Recovery Plan presents a recovery strategy including habitat protection; adaptive management, restoration, creation and monitoring; species status surveys; research; and participation and outreach. The Recovery Plan identifies “core areas” throughout the state which are areas determined as primary conservation areas necessary for the recovery and conservation of listed species. These core recovery areas are the initial focus of protection measures in the Recovery Plan. They are distributed throughout 16 California vernal pool regions, originally defined by Keeler-Wolf et al.

(1998) with modifications by the U.S. Fish and Wildlife Service (USFWS 2005).

The Recovery Plan (USFWS 2005) also identified the major threats to vernal pool species which are habitat loss and fragmentation. In addition to direct conversion, habitat loss also encompasses habitat alteration and degradation, which can result from hydrological changes, invasive species, incompatible grazing regimes, infrastructure projects, recreational activities, erosion, climate change, and contaminants (USFWS

2005). Preservation of the intact habitat, habitat restoration and creation are identified as tools to counteract the threat and loss of vernal pool habitat.

5 Habitat restoration, one of the tools to counteract habitat loss, is “the manipulation of the physical, chemical, or biological characteristics of a site with the goal of returning the natural and historic functions to a former or degraded pool”

(USEPA 2013; USFWS 2005). Activities which fall under vernal pool restoration include diverting excess surface runoff, reconstructing the characteristic depth from the overlying soil surface to the impermeable soil layer, managing grazing appropriately, or removing competing species (USFWS 2005). Vernal pool creation, another tool to counteract habitat loss, is “the construction of a vernal pool in an area that was not a vernal pool in the recent past (within the last 100 to 200 years) and that is isolated from existing vernal pools (i.e., not directly adjacent)” (Gwin et al. 1999; USEPA 2013; USFWS

2005).

As part of the restoration effort, the USFWS (2005) recommends conducting species reintroductions and introductions when possible; however, these activities are not to replace conservation of populations in their natural habitat. Reintroductions are seeding or transplanting of a species into a pool from which the species has been extirpated. Introductions are seeding or transplanting a species into a pool where it is not known to have been previously but the pool is “within a vernal pool region, pool type, and set of ecological conditions from which the species was known to occur”

(USFWS 2005).

Two of the listed species in the Recovery Plan are Greene’s tuctoria (Tuctoria greenei) and Colusa grass (Neostapfia colusana). Both species were federally listed in 1997;

Greene’s tuctoria as endangered and Colusa grass as threatened (USFWS 1997). In 1979,

Greene’s tuctoria and Colusa grass were designated as state endangered and rare,

6 respectively (CDFW 2004, 2013). Both species are California Native Plant Society

(CNPS) California Rare Plant Rank 1B.1, which means the plants are seriously endangered in California with over 80% of occurrences threatened (CNPS 2013).

Greene’s tuctoria and Colusa grass are members of the Orcuttieae tribe and

Chloridoideae subfamily of Poaceae. They are herbaceous, wind-pollinated annuals.

Forty-one Greene’s tuctoria occurrences had been documented (USFWS 2005); however,

Gordon et al. (2012) suggest that the number is currently fewer than 22 occurrences.

Historically Colusa grass was known from 60 occurrences in the California Central

Valley from Colusa County to Merced County (Gordon et al. 2012; USFWS 2005).

Today, approximately 42 of these occurrences remain (Gordon et al. 2012; Hogle 2002).

These two species are ideal candidates for research because extant populations are of sufficient size to collect seeds and plants for experimentation, to characterize habitat preferences, and to have sufficient data to inform reintroduction/introduction efforts both in existing vernal pools and newly constructed vernal pools. This is in contrast to working with extremely rare species such as Solano grass () where there is no latitude in manipulating populations, individual plants, or seeds due to its near extirpated status (CNPS 2013; USFWS 2009b).

Though the Recovery Plan recommends conducting species reintroductions and introductions, except for recent efforts by Gerlach (2009a, 2011), establishing populations of vernal pool grasses in restored or created vernal pools has not been documented. Recent studies on genetic diversity and structure of Greene’s tuctoria and

Colusa grass (Gordon et al. 2012) as well as the relationship between the density of populations and seed set for Colusa grass (Davis et al. 2009) have helped inform

7 recovery and management and have also led to recommendations for reintroductions of the two species. Building upon previous research, the goal of this study is to examine the potential for introductions and reintroductions of the two rare grasses into vernal pool habitats. The specific research questions are:

1) How do we choose the appropriate habitat/pools for introduction?

a) Hydrology

b) Reference populations

2) What is the best method for introduction?

a) Seeds

b) Inflorescences (whole plants)

3) What are the measurements of success for Greene’s tuctoria and Colusa grass?

a) Germination

b) Vigor

c) Reproduction

d) Second generation

To address these questions, there are four study sites, two of which support restored or created vernal pools and two reference sites with extant populations of the rare grasses. Information on pool hydrology and reference populations was gathered to compare and inform introduction success. Greene’s tuctoria and Colusa grass were introduced into the restored and created vernal pools and, for comparison, reintroduced into the reference pools. Introductions and reintroductions were monitored and data were collected on germination, survivorship, vigor, and reproduction.

8 The continued destruction of vernal pool habitat is a threat to the diversity and distribution of vernal pools in the California Central Valley. In addition to conservation and preservation of the remaining vernal pools, vernal pool habitat restoration and creation – including rare species reintroductions and introductions - offers a way to re-establish the biodiversity and benefits that are associated with these ecosystems. Results from this research are applicable to other Central Valley vernal pools for the reintroduction and establishment of Greene’s tuctoria and Colusa grass.

Information from this project could also be applied to restoring other vernal pool species, such as the nearly-extirpated Solano grass.

CHAPTER II

BACKGROUND

Vernal Pools

In the spring, portions of the California Central Valley grasslands are alight with circular and wavy patterns of bright purples, yellows, and whites. These colorful designs are painted by in vernal pools, a unique type of wetland in California.

Vernal pools are ephemeral, seasonal wetlands that fill with water in the late fall and winter and dry down in the spring. The life cycle of most of the flora and fauna that live in these pools is tied to the water cycle and the drying of the pools is what triggers the colorful concentric rings of spring blooming flowers. The underlying reason for the standing water is the geology and soils typical of vernal pool landscapes. Together, along with climate and topography, they are the most important physical factors in establishment of vernal pools (USFWS 2005).

Geology

Vernal pools tend to occur in landscapes with level to gently sloping topography on several different geologic landforms found in the Central Valley. These include basin rims, low terraces, high terraces, and volcanic mud and lava flows (Smith and Verrill 1998). Basin rim, low terraces, and high terraces are landforms on which soils are derived from alluvium and range in age from youngest to oldest, respectively.

Basin rim landforms have perched water tables that border the Holocene- period valley

9 10 basins (Smith and Verrill 1998). The largest areas of basin rim vernal pools were bordering the western Yolo and Colusa Basins (Smith and Verrill 1998). The Pleistocene- age low terraces and Pleistocene-Pliocene-age high terraces occur successively higher above the valley basin. The low terraces make up a large portion of the valley floor.

Both of the terraces have more of a “hummocky” micro-topography than the generally lower micro-relief found in the basin rim vernal pools (Holland 1986). The Pleistocene to Miocene-age volcanic mud and lava flow fan out from the Sierra Nevada along the eastern edge of the Central Valley. Pools on these landforms tend to be small with shortened hydrological cycles (Holland 1986).

Soils

Regardless of the geological landform, vernal pool landscapes are similar in that they are generally underlain by a soil layer that restricts the downward flow of water (Holland 1976; Weitkamp et al. 1996). This layer may be (1) an alkaline, sodium- rich clay suspension, (2) claypan, (3) hardpan/duripan, or (4) rock (Thorne 1984; Smith and Verrill 1998). The alkaline clay-suspension soils are found in the basin rim vernal pools (Smith and Verrill 1998). The claypan soils are older with a well-developed clay layer and are saline and/or alkaline with silica-cemented restrictive layer (Holland

1986). According to Holland (1986) these soils are found on basin rims and lower terraces; however, Smith and Verrill (1998) associate them with low and high terrace landforms. Presumably, Holland’s designation of claypan encompasses Smith and

Verrill’s clay-suspension soils of the basin rim. The hardpan soils have an iron-silica cemented hardpan and are nutrient depleted with a low pH (Holland 1986; Solomeshch et al. 2007). Hobson and Dahlgren (1998) studied soil formation in hardpan soils on

11 terrace landforms near Chico, California and determined that the dominant soil processes are ferrolysis (cyclic oxidation and reduction of iron and magnesium), organic matter accumulation, clay formation and translocation, and duripan formation. The last type of restrictive layer is rock which occurs in the mud and lava flow landforms, where vernal pools have shallow soils over restrictive bedrock (Holland 1986; Smith and Verrill

1998).

Hydrology

Geologic and soil features, in conjunction with the Mediterranean climate of

California (cool, wet winters and dry, hot summers), provide an environment for standing water in the pools for two to four months with soil saturation another one to three months (Solomeshch et al. 2007). Pools fill in the winter and dry down in the spring. The subsequent dry phase of the pools may be between five to eight months

(Solomeshch et al. 2007).

Since vernal pools are predominantly precipitation fed, changes in precipitation events can have a large impact on the hydrology in the pools (Bauder

2005). In a dry year, the water in the pools may not reach capacity, while in a wet year the pools may remain full until late in the spring. Also, depending on the timing of rain events, the pools may fill and dry down multiple times during the winter or spring.

Hanes and Stromberg (1998) demonstrated that although precipitation was the primary source of water, overland flow may occur during large storm events, and lateral subsurface flow from the surrounding uplands can buffer water level fluctuations in the late winter and early spring.

12 Hydrologic patterns are different between the different vernal pool types.

Hardpan pools demonstrate the potential for subsurface lateral water movement; however, subsurface water flow in claypan pools is negligible and water loss by evaporation contributes to the higher salinities in these pools (Williamson et al. 2005).

Water in hardpan pools has low conductivity, dissolved salts, and dissolved nitrogen

(Keeley and Zelder 1998; Rains et al. 2006). The conductivity and alkalinity in claypan vernal pools tends to not only be higher, but also increases as the pools dry down

(Barclay and Knight 1984; Williamson et al. 2005). Whereas, the conductivity in hardpan pools does not increase during dry down (Rains et al. 2006).

Communities and Vegetation

The physical characteristics of the pools determine the types of flora and fauna. The prolonged dry phase of the pools precludes fish as well as plants found in perennial or near-perennial marshes and ponds. Species associated with vernal pools must be able to survive the annual hydrological cycle as well as the variation in precipitation from year to year. As a result, the primary plant life forms are annuals that complete their life cycle in one year and survive the dry phase of the pool as seeds

(Stone 1990; Zedler 1990). Moreover, the seeds can remain dormant in the soil for years and germinate when conditions are favorable (Crampton 1959; Griggs 1980; Stone et al.

1988). The perennial species in vernal pools are predominantly found in larger pools that retain water for the longest periods (e.g., spikerush (Eleocharis sp.)). Salt-tolerant species are more common in claypan vernal pools than hardpan pools (Keeler-Wolf et al.

1998). Characteristic species in hardpan pools include two-horned downingia

(Downingia bicornuta), coyote thistle (Eryngium vaseyi), Fremont’s goldfields (Lasthenia

13 fremontii), white meadowfoam (Limnanthes alba), and stalked popcornflower

(Plagiobothrys stipitatus var. micranthus); while in claypan pools characteristic species include alkali weed (Cressa truxillensis), alkali heath (Frankenia salina), California eryngo

(Eryngium aristulatum), and harlequin downingia (Downingia insignis) (Holland 1986).

The unique physical environment of the vernal pools also lessens the ability for non- native species to invade. Although most of the uplands surrounding vernal pools are dominated by non-native species, a high percentage (70- 90%) of the plant species found in undisturbed vernal pools are native (Holland 1976; Holland and Jain 1988; Jokerst

1990; Schlising and Sanders 1982).

Classification

Several ecologists have contributed to the definitions and classification

California vernal pools. Probably the two most frequently referenced in the literature and used in management plans are Holland (1986) and Keeler-Wolf et al. (1998).

Holland (1986) classifies California vernal pools based primarily on their restrictive layer, geographic location, and indicator species. The northern California categories include Northern Hardpan, Northern Claypan, Northern Basalt Flow, and Northern

Volcanic Mudflow. The Northern Hardpan and Claypan Vernal Pools correspond more or less to terrace and basin landforms, respectively (Holland 1986). The classification of vernal pools in the first edition of A Manual of California Vegetation (Sawyer and Keeler-

Wolf 1995) is based on Holland’s definitions.

Keeler-Wolf et al. (1998) further group vernal pools into vernal pool regions.

The geographic regions are delineated using endemic species, geomorphology, and

14 soils. A total of 17 distinct vernal pool regions are described in the text, nine of which occur in northern California. The northern California vernal pool regions are the:

 Modoc Plateau Region,

 Northeastern Region,

 Sierra Valley Region,

 Southeastern Sacramento Valley Region,

 Solano-Colusa Region,

 Santa Rosa Region,

 Lake – Napa Region,

 Mendocino Region, and

 Northwestern Sacramento Valley Region.

Many of these regions are described by pool types defined by Holland (1986).

For example, the Northwestern Sacramento Valley region supports primarily Northern

Hardpan vernal pools and in the adjacent Solano-Colusa region, the predominant vernal pool type is Northern Claypan vernal pools. The regions are modified slightly in the

Recovery Plan for Vernal Pool Ecosystems of California and Southern Oregon (USFWS 2005).

Barbour et al. (2005, 2007) prepared a new classification system based on a state-wide vegetation survey. Rather than a substrate or geographic derived specification, the new classification system defines vegetation associations within pools so that one pool may have more than one association. The hierarchical vegetation classification system is organized into classes, orders, alliances, and associations. This system also links the distribution of rare plant species with the associations. The information could be applied to predicting rare plant occurrence as well as determining

15 suitable habitat for reintroduction of rare plants. The second edition of A Manual of

California Vegetation (Sawyer et al. 2009) adopted the Barbour et al. classification system.

At least 21 vernal pool associations or community types occur in northern California.

Restoration and Creation

Vernal pool restoration and creation are part of species and habitat recovery

(USFWS 2005) but have also been used as mitigation for unavoidable destruction of vernal pools (Sutter and Francisco 1998). Restoration is returning natural or historic functions to a former or degraded pool, while creation is construction of a vernal pool in an area that has not supported vernal pools in the last 100-200 years (USFWS 2005).

Creation of vernal pools to mitigate unavoidable impacts started in the mid-

1980s when isolated wetlands (which vernal pools were considered) became regulated under Section 404 of the Clean Water Act (Sutter and Francisco 1998). As a result, the majority of created vernal pools in the California Central Valley have been as compensatory mitigation. Several studies have evaluated the success of created vernal pools using performance standards and comparing them to their natural counterparts, with mixed results (DeWeese 1998). Review of vernal pool restoration and creation activities in Butte, San Diego, and Santa Barbara Counties showed that created vernal pools have the potential to meet hydrological and vegetation parameters of natural pools; however, results weren’t always conclusive and caution was advised in vernal pool mitigation projects (Black and Zedler 1998; Buck et al. 2007; Ferren et al. 1998).

Recommendations for vernal pool creation and restoration projects include:

(1) pools should be considered in their greater ecosystem context rather than isolated from the larger landscape (Leidy and White 1998); (2) pools should be created in the

16 appropriate geomorphic landform and mirror the edaphic conditions of reference pools

(Wacker and Kelly 2004); (3) reference pools should include a diversity of local pools

(including their diverse species and plant communities) (Barbour et al. 2003); (4) pools should be created to include the spectrum of habitat not only between but within the created pools (create basin, rim, and edge habitat) (Sutter and Francisco 1998); (5) when creating pools for mitigating impacts to certain species, make sure the preferred hydrological habitat for that species is defined (Sutter and Francisco 1998); and (6) consider management and long-term stewardship of the entire site (as well as adjacent upland), including grazing and fire (Sutter and Francisco 1998). These recommendations highlight the trend towards an ecosystem approach to vernal pool creation. Nevertheless, using creation as mitigation for destruction of natural habitat continues to be controversial (Elam 1998; Howald 1996; Hubbart et al. 2001). The preference remains for preservation of natural vernal pools because the long-term success of created pools is uncertain and created habitats can’t replace the inherent

“aesthetic, scientific, and historic value of natural landscapes” (Black and Zedler 1998).

However, when not used for compensatory mitigation but simply as an aid to recovery, vernal pool creation and restoration on degraded habitat provides an opportunity for recovery of species and their habitat (USFWS 2005).

Rare Plant Introductions and Reintroductions

Rare plant species introductions and reintroduction have the ability to play an important role in habitat restoration and species recovery (USFWS 2005).

Reintroductions transplant propagules (i.e., seeds, seedlings) into habitat where the

17 species has been extirpated, while introductions place propagules into new, but potentially suitable habitat. Pavlik (1997) outlines a five step process for conserving rare plants: (1) inventory flora; (2) survey populations; (3) preserve habitat; (4) monitor rare populations; and (5) recovery of rare populations. According to Pavlik, monitoring and recovery (Steps 4 and 5) are less developed in the science of rare plant conservation.

Reintroductions as a tool for recovery (Step 5) are relatively new in an experimental context with “fewer than 20 taxa nationwide” being used as experimental reintroductions towards recovery (Pavlik 1997).

A Review

Pavlik (2004) suggests a three phase approach to reintroductions, which can also be applied to introduction projects. The first phase includes establishing the reintroduced population: selection of the reintroduction site, acquisition of propagules, preliminary ex situ studies, experimental design and installation, demographic monitoring, and evaluation. The second phase applies the results of the first phase to the natural and experimental populations. The third, and last, phase expands monitoring to evaluate population persistence by documenting the effects of natural yearly environmental variation on the populations. Overlooking any of these steps can result in failure for the reintroduced population. When considering site selection, historical and logistical criteria should be evaluated as well as physical and biological (Fielder and

Laven 1996). Genetic, demographic, and horticultural aspects should be considered in propagule selection (Guerrant 1996; McKay et al. 2005). And above all, it is essential to understand the biology and ecology of the reintroduced species (Primack 1996).

18 Godefroid et al (2011) conducted a meta-analysis of plant species reintroductions by reviewing the published literature as well as conducting a survey of organizations that performed reintroductions without publication. Although they used the term “reintroduction” for all of the studies, several of the studies included

“introductions” where species were introduced into habitat where they weren’t known to previously occur but were within the known range of the species. Overall, their evaluation includes 249 reintroductions of 172 taxa in 62 families. Reintroduction locations included Europe, Africa, Australia, and North America. Their analysis showed that survival, flowering, and fruiting rates in reintroductions were, on average, low:

52%, 19%, and 16%, respectively (Godefroid et al. 2011). In a response to Godefroid et al.

(2011), Albrecht et al. (2011) caution making broad conclusions about the usefulness of reintroductions in recovery based on these low percentages. They claim that more information on individual species and life histories is needed in order to understand the analysis. Moreover, it may be inappropriate to use short-term trends to determine long- term success when initial short-term population declines may be expected (Albrecht et al. 2011).

Nevertheless, Godefroid et al. (2011) were able to identify several common factors of both successful and unsuccessful reintroduction projects, and based on these, made recommendations for improvements to reintroduction projects. For unsuccessful projects, survey participants listed “unknown” as the highest reason for failure.

Godefroid et al. (2011) identified experimental design flaws that led to the inability to interpret failed results, such as inadequate monitoring, insufficient knowledge of the reason for decline in existing plant populations, and poorly defined success criteria.

19 Actions of successful projects included: reintroducing into protected sites, using larger number of introduced propagules, mixing propagules from diverse and stable populations, knowledge of the genetic variation of the species, and proper site preparation and management (Godefroid et al. 2011). Based on their meta-analysis they recommend that plant reintroductions as a recovery tool could be improvement by a greater focus on species biology, using a higher number of propagules, and long-term and consistent monitoring which includes information on seed production and recruitment (Godefroid et al. 2011). They also found that reintroductions in the strict sense of the word (i.e., placing propagules in habitat where the species was known to occur) had greater percent survivorship after one and three years than introductions

(i.e., placing propagules in areas within species range but in habitat where it was not known to occur) (Godefroid et al. 2011).

Vernal Pool Species

There are a few documented reintroduction/ introduction attempts and research using vernal pool species. Griggs (1980) introduced slender Orcutt grass

( tenuis) and Greene’s tuctoria (Tuctoria greenei) seed into an artificial vernal pool in Yolo County. The artificial pool was created using a plastic sheet to simulate the hardpan layer. Both of the grasses germinated but only slender Orcutt grass survived to . The poor performance of Greene’s tuctoria was attributed to lack of sufficient soil moisture. Griggs (1980) also introduced the two grasses into two impounded swales north of Bidwell Park in Chico in Butte County. Slender Orcutt grass was seen in subsequent years, but Greene’s tuctoria was not. In Sacramento County at Phoenix

Field, Sacramento Orcutt grass (Orcuttia viscida) seed was introduced into a natural pool

20 200 meters away from the source pool. Sacramento Orcutt grass survived and reproduced and was initially more abundant than the source population (Griggs 1980).

The introduced population has persisted (USFWS 2008b).

In 2008, Gerlach (2009a) reintroduced Colusa grass (Neostapfia colusana) and

Solano grass (Tuctoria mucronata) seeds into restored vernal pools at Grasslands Park in

Yolo County. The seeds were collected from nearby pools at Grasslands Park. Prior to a large storm event, the soil was raked to cover cracks, the seeds were broadcast, and then the soil was raked again to lightly cover the seeds (Gerlach 2009a) Colusa grass was present during the first growing season; however, Solano grass didn’t emerge until two years after the pool was seeded (Gerlach 2011).

Howald (1996) reviewed mitigation related vernal pool rare plant translocations that occurred in the mid 1980s to early 1990s in the Santa Rosa Plain

(Sonoma County). In these projects, vernal pool habitat was created and the rare plants were then relocated to the created pools from pools that were impacted. Species included Burke’s goldfields (Lasthenia burkei), Sonoma sunshine (Blennosperma bakeri), and Sebastopol meadowfoam (Limnanthes vinculans). Evaluation of the vernal pool creation and species translocations were hindered by inadequate documentation, lack of success criteria, short-term monitoring, and lack of significant comparisons to reference sites (Howald 1996). Success typically focused on meeting the same number of plants in the created habitat as had been counted in the impacted natural population (which was usually determined in only one season).

In comparison, in a multi-year introduction research project using reference sites, Collinge (2003) introduced Contra Costa goldfields (Lasthenia conjugens) into 64

21 constructed vernal pools at Travis Air Force Base in Solano County. One-hundred seeds were introduced into permanently marked 0.25-meter squared plots per pool. In the three years of the study, average abundance of Contra Costa goldfields increased in the constructed pools and by the third year was comparable in frequency to reference pools.

Another aspect of the study included testing whether additional introductions over subsequent years had an effect on the abundance of Contra Costa goldfields. In half of the created pools, an additional 100 seeds were introduced into the plots in the second and the third years of the study. In the first and second years, abundance of Contra

Costa goldfields was significantly higher in the plots seeded multiple times than the once-seeded plots; however, by the third year there was no significant difference in abundance. Collinge (2003) concluded that it may be possible to establish viable populations of Contra Costa goldfields in created pools with only one year of seeding.

Study Species

Greene’s tuctoria (Tuctoria greenei (Vasey) Reeder) and Colusa grass

(Neostapfia colusana (Burtt Davy) Burtt Davy) are members of the Orcuttieae tribe and

Chloridoideae subfamily of the grass family Poaceae (Fig. 2-1). They are herbaceous, wind-pollinated annuals that are endemic to vernal pools. Most vernal pool annuals germinate in the late fall and winter with the first precipitation of the season and as the pools are filling; however, these grasses don’t germinate until the pools are drying down in the spring (Griggs 1976; Keeley 1998). Flowering occurs in the late spring through the summer when most of the other vernal pool plants have long since senesced (Griggs 1976). Their basic floral structure is similar to other members of

22

a b a a d d

Figure 2-1. (a) Colusa grass at Jepson Prairie and (b) Greene’s tuctoria at Vina Plains. Photo credit: Erin Gottschalk Fisher.

Poaceae; each floret (or flower) is composed of an ovary and three stamens, with subtending lemma and palea. The is called a caryopsis and includes one seed where the seed coat and pericarp (ovary wall) are fused. In the following text and chapters, the term seed will be used for convenience to refer to the caryopsis enclosed by a lemma and palea.

Greene’s tuctoria and Colusa grass also have unique features that distinguish them from other grasses: their stems are filled with pith (rather than being hollow), their don’t clasp the stem to distinguish between the leaf sheath and blade (stem- clasping leaf characteristic is seen in other grasses), and they lack a ligule (leaf appendage common in grasses) (Keeley 1998). In addition, members of the Orcuttieae produce an often strongly aromatic exudate on their leaves and inflorescences which functions to repel grasshopper herbivory (Griggs 1981). These phenological and anatomical features make Colusa grass and Greene’s tuctoria unique among the other vernal pool flora as well as among other members of Poaceae.

23 Along with being wind-pollinated annuals, the grass species in the

Orcuttieae share other life history traits and habitat conditions. Dispersal of pollen and seed does not likely occur over long distances (Davis et al. 2009; Griggs 1980). Seed dispersal within the pools is by water after the dry inflorescences have fallen apart with fall rains (Crampton 1976; Griggs 1980). Griggs (1980) suggested that historical dispersal of seed between pools may have occurred by waterfowl or native ungulates such as tule elk. The seeds can remain dormant in the soil for several years (Crampton 1976; Griggs

1980) and the soil seed bank may be 50 times greater than the population in any given year (USFWS 2005).

Populations of the grasses in the pools have been shown to vary significantly in response to timing and amount of precipitation (Griggs 1980; Holland 1987).

Although the grasses in the Orcuttieae occur in pools of various sizes, they tend to reside in pools with longer average duration of ponding and greater average depth of ponding

(Sutter and Francisco 1998) that retain standing water through May or June (Crampton

1959; Griggs and Jain 1983). These grasses take advantage of the water in the saturated soil during the early summer (Griggs 1980).

Alexander and Schlising (1997) described the precipitation pattern for 1995, which they considered favorable for vernal pool species: rainfall at regular intervals during the winter and spring, with large amounts in January and March, and late rains that kept pools wet long into the spring. In addition, spring conditions that allow for good soil moisture through dry down of the pool basins are also favorable.

24 Greene’s Tuctoria

Taxonomy and Description. Greene’s tuctoria shares the Tuctoria with two other species: Solano grass (Tuctoria mucronata) which is federally and state endangered and may be near extinction (USFWS 2009b) and Tuctoria fragilis which is found in Baja California, Mexico (Reeder 1982). Greene’s tuctoria was originally named

Orcuttia greenei (Vasey 1891); however, Reeder (1982) segregated Tuctoria from Orcuttia and gave Greene’s tuctoria the new name Tuctoria greenei.

In the Orcuttieae, Tuctoria is considered to be intermediate between Neostapfia and Orcuttia with respect to morphological and photosynthetic adaptations to the aquatic environment (Keeley 1998), as well as arrangement of spikelets, shape of lemma tip, and embryonic features (Reeder 1982). Boykin et al. (2010) analyzed DNA sequence data to determine relationships for all members of the tribe and determined that while

Neostapfia and Orcuttia are monophyletic (include descendents and the most recent common ancestor), the Tuctoria genus is paraphyletic (includes some but not all of the descendents of a common ancestor). Boykin et al.’s Tuctoria results are consistent with

Reeder’s (1982) results on seed proteins which showed that seed proteins of the three

Tuctoria species are more similar to species of Orcuttia than they are to one another.

Boykin et al. (2010) determined that Tuctoria needs taxonomic revision and, based on their molecular data, lean towards creation of a new, monotypic genus for Greene’s tuctoria while keeping the other two Tuctoria species in the genus. However, more molecular work is needed to resolve the relationships (Boykin et al. 2010).

Greene’s tuctoria is darker green than Colusa grass when young, and doesn’t produce as much aromatic viscid secretion as other members of the tribe (Stone et al.

25 1987). In the aquatic stage, Greene’s tuctoria has between one and two leaves (Griggs

1980) to between three and five leaves (Keeley 1998). Unlike members of Orcuttia, it does not produce any floating aquatic leaves. The terrestrial stage is caespitose with all inflorescence-bearing stems (culms) coming from one root system (USFWS 2005). The stems are often fragile and break off at the nodes (Reeder 1982). Stem length is between five and 15 (sometimes up to 30) centimeters. Leaf length is two to three centimeters and less than five millimeters wide (Reeder 2012b). Reeder (1982) noted that generally there is no distinction between leaf sheath and blade in all members of the tribe; however, in Greene’s tuctoria sometimes there is a faint ligule or collar line evident when dry. Each stem produces one inflorescence that is less than eight centimeters in length which, when mature, is exserted beyond the leaves. The spikelets have two glumes subtending the florets, and are flattened and spirally arranged along the inflorescence axis (Reeder 1982). There are less than 40 florets per spikelet (Reeder

2012b). The lemma tips are entire or minutely-tootled usually with a central slightly longer tooth (Reeder 1982). The caryopsis is somewhat laterally flattened, oblong, minutely wrinkled, and two millimeters long (Reeder 1982, 2012b).

Solano grass, the other Tuctoria occurring in California, is distinguishable from Greene’s tuctoria by having an inflorescence that remains partly enclosed by upper leaf sheath, lemma tip that is tapered and sharply-pointed, and caryopsis three millimeters long and smooth (Reeder 2012b). Tuctoria differs from Colusa grass

(Neostapfia) by having laterally-compressed spikelets, narrow lemmas with tips minutely-toothed, caryopsis not viscid, and the visible through the pericarp

(Reeder 1982). Both Neostapfia and Tuctoria differ from Orcuttia by having spirally-

26 arranged spikelets on the inflorescence axis (Orcuttia has a distichous arrangement) and having lemmas that are entire or minutely toothed (Orcuttia lemmas are deeply toothed)

(Reeder 1982).

Keeley (1998) compared leaf morphology in the Orcuttieae and found that for all species, the submerged aquatic leaves were cylindrical and basal. Orcuttia species produced more aquatic leaves and Colusa grass the fewest, with Greene’s tuctoria in between. In comparison to Neostapfia and Tuctoria, the Orcuttia species produced elongated leaves that flattened out and floated when reaching the water surface. Once the pool dried, all genera changed and started to produce foliage with flattened, caulescent leaves.

Germination, Reproduction, and Population Ecology. Griggs (1980) observed

Greene’s tuctoria germination in the field. Germination occurs when the last standing water dries down in the vernal pool. At this time, the plants have one to two leaves, which are fewer than the two to five aquatic leaves that Keeley (1998) observed in his germination experiment.

Griggs (1980) and Keeley (1988, 1998) studied germination of Greene’s tuctoria in the lab or other artificial setting. Griggs (1980) was able to get 90% germination by using whole, intact inflorescences. “Naked” seeds without lemma or palea could not be germinated in the lab. The inflorescences were placed in a petri dish and the petri dish was filled with water and placed in a 12 degree Celsius fridge. After two to four weeks, a black fungus formed around the inflorescence and the seeds began germinating (Griggs 1980). Strauss (2009) was able to germinate hairy Orcutt grass

(Orcuttia pilosa) using similar methods.

27 Keeley (1988) tested germination of Greene’s tuctoria using “naked” seeds in response to the following treatments: anaerobic incubation, cold stratification, light, water extracts from chaff and soil, and fungicide. The seeds were placed in petri dishes between two sheets of filter paper. Deionized water, chaff or soil extracts, or fungicide were added to the petri dish. Dishes were then treated with or without stratification.

Stratification was 5 degrees Celsius for two months. After stratification, dishes were placed in an incubator. Incubator conditions were in light or dark and under aerobic or anaerobic conditions. Overall, the highest percent germination for Greene’s tuctoria occurred in the light and under anaerobic conditions. Stratification did not have an effect under these optimal conditions. However, in the dark and/or in the air, stratification at 5 degrees Celsius increased germination. There was no significant difference between medium treatments (deionized water, extracts, or fungicide) (Keeley

1988). In the treatments without stratification, more than 50% of the seeds had germinated by two weeks. In the stratification treatments, the seeds did not germinate until moved to the incubators (Keeley 1988).

Keeley (1998) compared germination of Greene’s tuctoria, Colusa grass, and two Orcuttia species. The plants were grown outdoors in Los Angeles, CA. Seeds and their source pool substrate were submerged in deionized water in December and exposed to natural precipitation through spring. The pools were allowed to begin drying down in April. Greene’s tuctoria germinated two months after inundation: the

Orcuttia species germinated in late January, Greene’s tuctoria germinated in February, and Colusa grass germinated in March.

28 Greene’s tuctoria flowers from May to September (Reeder 2012b). The flowers are protogynous (the shedding of pollen occurs after the stigma has stopped being receptive). The long stamen filaments position the anther well outside the floret, which helps facilitate outcrossing in the wind pollinated plants. The basal florets all flower at the same time on one inflorescence; therefore, the basal florets are more likely to receive pollen from another plant (Griggs 1980). Conversely, the intermediate and terminal florets are more likely to receive pollen from the same inflorescence (Griggs

1980). The basal florets tend to remain attached to the inflorescence axis; however, the intermediate and terminal florets more easily fall off the inflorescence (Griggs 1980).

Shattering of the inflorescences and seed dispersal begins at the first fall precipitation

(Griggs 1980). The seed is still encompassed in the lemma and palea and either floats on the water or attaches to the wet soil after the first rain. The basal florets are not dispersed far from the parent plant (Griggs 1980).

Griggs (1980) found that average seed weight is characteristic for each population instead of for each species which suggests that seed weight is under genetic control and that there is low rate of gene flow between populations (Boykin et al. 2010).

The number of seeds per plant was highly variable within each population and between years; in some populations individuals failed to produce seed although the plants looked similar to other plants in appearance (Griggs 1980).

The number of stems producing an inflorescence (i.e., culms) changes in response to growing conditions. Under suitable growing conditions, more culms are produced, while under less favorable growing conditions only one culm per plant may occur (Griggs 1980). Moreover, the spikelets exhibit indeterminate growth and the

29 number of florets per spikelet is positively related to the length of suitable growing conditions after the inflorescence has formed (Griggs 1980). Griggs (1980) also demonstrated that in any given year, all the phenological phases of the grass may be present in a pool during one visit. Mature plants with seed may occur at the pool edge while seedlings occur at the center of the pool. These differences are related to the timing of dry down of standing water within the pool and the drying of the pool soil

(Griggs 1980).

Populations of Greene’s tuctoria fluctuate from year to year in the wild.

Griggs (1980) observed these fluctuations in Butte and Tehama Counties during the

1970s, where populations decreased to zero one year but were abundant the next.

Population fluctuations continued in the 1980s and 1990s (USFWS 2005) and Alexander and Schlising (1997) documented large population sizes in Tehama County after a year with favorable rainfall conditions. In Stanislaus County, one population of less than 100 plants fell to two plants and hasn’t been seen since even under favorable precipitation conditions (Griggs 1980; Stone et al. 1988).

Alexander and Schlising (1997) conducted population surveys of Greene’s tuctoria at Vina Plains in 1995. They found Greene’s tuctoria in six pools with a range in density of seven to 133 plants per square meter with an average of 46 plants per square meter. Population estimates per pool ranged from 173,200 to 94 plants with an average of 120,564 plants per pool (Alexander and Schlising 1997). Broyles (1987) surveyed Vina

Plains in the 1980s and estimated that most of the populations of Greene’s tuctoria at

Vina Plains were a few hundred plants per pool but 30,000 plants were observed in “one exceptional pool.”

30 In 1977 and 1978, Griggs (1980) studied population dynamics of two Greene’s tuctoria populations in Butte and the Tehama Counties. Density per decimeter squared was 0.82 and 1.32. Since seed production per individual averaged 111 seeds, seed production per decimeter squared was between 91 and 148. Percent survivorship for the two pools was between 0% and 54% (0% for one year due to grasshopper damage to plants before they set seed) (Griggs 1980). Greene’s tuctoria mortality occurred predominantly in quadrats with higher density, suggesting density dependent mortality

(Griggs 1980).

Griggs (1980) compared the genetic structure between two Greene’s tuctoria pools in Butte and Tehama County using gel electrophoresis comparison of allozymes.

The pools were separated by approximately 35 miles. He found that an average of only

10% of the genetic diversity was shared between the two populations, suggesting a low level of gene flow between the two populations or pools. Within a family of seedlings from a single parent, genetic diversity accounted for an average of 44% of the total genetic variation (Griggs 1980) which indicates a high level of out-crossing for a wind pollinated species (Boykin et al. 2010).

Using microsatellite markers, Gordon et al. (2012) performed a genetic analysis of 317 Greene’s tuctoria plants in 13 pools in Tehama, Butte, and Merced

Counties. They found that there were significant genetic differences between the north and south counties. Except for three pools, there was low genetic structure among the northern Greene’s tuctoria pools in Butte and Tehama Counties. The three pools that had genetic distinction were isolated from other pools by roads, highways, and different land uses. This habitat fragmentation may account for the lack of gene flow (Gorden et

31 al. 2012). The pools at Vina Plains that were not separated by highways showed a high degree of admixture which could be explained by seed movement by cattle (Gordon et al. 2012).

Habitat. Greene’s tuctoria has been found in Northern Basalt Flow,

Northern Claypan, and Northern Hardpan pool types and in the Modoc Plateau,

Northwestern Sacramento Valley, Northeastern Sacramento Valley, ,

Southern Sierra Foothills, and Solano-Colusa Vernal Pool Regions (Keeler-Wolf et al.

1998; USFWS 2005). It has been found on low and high terraces in pools underlain by hardpan, claypan, and rock in basic and saline-alkaline pools (Keeler-Wolf et al. 1998;

Stone et al. 1988; USFWS 2005).

Greene’s tuctoria occurs in shallower pools or shallower margins of deeper pools than other members of Orcuttieae (Alexander and Schlising 1997; Stone et al.

1998). For example, in 1995 at Vina Plains, the pools that supported Greene’s tuctoria dried down in early April to early May, while two pools that dried down in late May to

June supported hairy Orcutt grass (Orcuttia pilosa) but not Greene’s tuctoria (Alexander and Schlising 1997). In addition, at Vina Plains, Greene’s tuctoria occurs in unique areas within the pools, generally not occurring throughout the entire pools or necessarily in the deepest portions of the pools (i.e., occupying only one half, or smaller elliptical or crescent shaped areas) (Alexander and Schlising 1997). These patterns of distribution may be due to small changes in soil within the pools (Alexander and Schlising 1997).

Several vernal pool species are associated with Greene’s tuctoria. At Vina

Plains, Greene’s tuctoria occurs frequently with Great Valley coyote thistle (Eryngium castrense) and hairy waterclover (Marsilea vestita) (Alexander and Schlising 1997; Lazar

32 2006). In other pools in the Central Valley, Greene’s tuctoria occurs with coyote thistle

(Eryngium vaseyi), stalked popcornflower (Plagiobothrys stipitatus), and Pacific foxtail

(Alopecurus saccatus) (USFWS 2005). In some occurrences, Greene’s tuctoria occurs in the same pools with one or more of the following rare plants: hairy Orcutt grass (Orcuttia pilosa), San Joaquin Valley Orcutt grass (Orcuttia inaequalis), slender Orcutt grass

(Orcuttia tenuis), Colusa grass (Neostapfia colusana), Hoover’s spurge (Chamaesyce hooveri) and Boggs Lake hedge hyssop (Gratiola heterosepala) (Alexander and Schlising 1997;

Stone et al. 1988; USFWS 2005).

Distribution. The type specimen of Greene’s tuctoria was collected by E.L.

Greene in 1890 near Chico, presumably in Butte County (Crampton 1959; Hoover 1941;

Reeder 1982). By 1925, Willis Linn Jepson noted that the type locality was extirpated and had been converted to rice agriculture (Jepson 1925). Historically, Greene’s tuctoria had been known from 41 occurrences (Gordon et al. 2012; USFWS 2005). However, a recent survey by Gordon et al. (2012) suggested that the number is currently fewer than

22 occurrences. Greene’s tuctoria has been extirpated from six counties (Glenn, Fresno,

Madera, San Joaquin, Stanislaus, and Tulare) and currently occurs in Shasta, Tehama,

Butte, and Merced Counties (Gordon et al. 2012; USFWS 2005; USFWS 2007b; Fig. 2-2).

The largest concentration of extant occurrences (approximately 50%) occurs in the Vina Plains area in Butte and Tehama Counties (USFWS 2007b). One occurrence occurs in Shasta County on private property surrounded by Lassen National Forest, four occurrences in Butte County on private property (although one is under a conservation easement), and five occurrences in Merced County which are the only remaining populations in the San Joaquin Valley (USFWS 2007b). A single occurrence of Greene’s

33

Figure 2-2. Current distribution of Greene’s tuctoria. Data source from Gordon et al. 2012.

tuctoria was discovered at Sacramento National Wildife Refuge in Glenn County in

1994, the only known population in the Solano-Colusa Vernal Pool Region. However, this population has not been observed in annual surveys since 1996 (USFWS 2007b).

Conservation. Greene’s tuctoria is federally endangered, state rare, and designated California Native Plant Society (CNPS) California Rare Plant Rank 1B.1

(CDFW 2013; CNPS 2013; USFWS 1997). In the Recovery Plan, the recovery step outlined for Greene’s tuctoria is downlisting with 80% of known occurrences protected in nine core areas. These nine core areas are: Western Modoc Plateau, Oroville,

Richvale, Vina Plains, Sacramento National Wildlife Refuge (NWR), Fresno, Madera,

34 Merced, and Waterford which are distributed among four vernal pool regions: Southern

Sierra Foothills, Northeastern Sacramento Valley, Solano-Colusa, and Modoc Plateau

(USFWS 2005). Other recovery measures include protection of habitat, status surveys, monitoring and adaptive management, seed banking, and research. No reintroductions are recommended in the Recovery Plan; however, introductions are recommended in the

Southern Sierra Foothills Region (USFWS 2005). A reintroduction attempt by Griggs

(1980) in Butte County (described above) was unsuccessful.

The northern and southern-most occurrences of Greene’s tuctoria are not currently protected. The northern-most occurrence in Shasta County population is on private land and only one of the southern occurrences in Merced County is protected under the Drayer Ranch Conservation Bank (USFWS 2007b). There are five protected occurrences at Vina Plains Preserve in Tehama County. Factors leading to extirpation of populations include the following land use changes: irrigated agriculture, irrigated pasture, disking, hydrologic alteration, urbanization, and heavy grazing (Stone et al.

1987). Other causes of extirpation include competition with non-native weed and native species (Stone et al. 1987). Greene’s tuctoria is the most susceptible of the Orcuttieae to grazing impacts because it often occurs in shallower pools and edges than the other grasses (Stone et al. 1987). Threats continue to occur to extant populations of Greene’s tuctoria and include hydrological alteration, cattle grazing, weed and native species competition, and grasshopper damage (Alexander and Schlising 1997; Griggs 1980;

Stone et al. 1987). Species competition has been reported with swamp picklegrass

( schoneoides), Italian rye grass (Festuca perennis), hood canarygrass (Phalaris paradoxa), common spikerush (Eleocharis palustris), creeping spikerush (Eleocharis

35 macrostachya), and coyote thistle (Eryngium vaseyi) (Alexander and Schlising 1997; Stone et al. 1988). Small population sizes are potential threats to populations in Butte and

Shasta Counties where the populations have numbers fewer than 100 (USFWS 2005).

Smaller populations of Greene’s tuctoria have a greater threat of extirpation due to stochastic events (Gordon et al. 2012; USFWS 2005).

Based on their surveys at Vina Plains Preserve in 1995, Alexander and

Schlising (1997) provide management recommendations applicable to Greene’s tuctoria.

These recommendations include, but are not limited to: (1) whole landscapes must be taken into consideration and include preserves with diverse pool types; (2) information from extremely dry and wet years must be collected to fully understand population dynamics; (3) must recognize and allow for the exchange of propagules between different vernal pool populations to support metapopulations and genetic diversity; and

(4) removal of exotic, invasive species must be part of management activities (Alexander and Schlising 1997).

Along with population and distribution information, genetic studies have highlighted the significance of conservation of multiple populations of Greene’s tuctoria

(Davis et al. 2009; Gordon et al. 2012; Griggs 1980). Based on their genetic analysis described above, Gordon et al. (2012) recommend that the north (Tehama and Butte) and south (Merced) regions should be separate conservation units. There is particular conservation concern in Merced County since all of the occurrences are on private property (Gordon et al. 2012). In Butte and Tehama Counties, there may be a need for facilitated out-crossing based on the three pools that showed genetic distinction possibly due to human-caused habitat fragmentation (Gordon et al. 2012). For vernal pool

36 creation, introduction, and reintroduction, they recommended collecting seeds from the most geographically nearby pool(s), collecting an excess of 70 maternal families to capture the rare alleles, and establishing a high density for the initial population to maximize seed set (Davis et al. 2009). Since temporal variation in genetic structure between years was small but significant, they recommended collecting seeds over multiple years if possible (Gordon et al. 2012).

Colusa Grass

Taxonomy and Description. Colusa grass was first collected and described by Burtt Davy (1898) as Stapfia colusana. It has also been known as Anthochloa colusana

(Scribner 1899). In 1899 Burtt Davy gave Colusa grass its current name Neostapfia colusana, which Hoover (1940) concluded was distinct from Antochloa and more closely related to Orcuttia. Neostapfia is a monotypic genus (Reeder 1982) in the Orcuttieae tribe and is considered ancestral due to fewer morphological and photosynthetic adaptations to aquatic environments (Keeley 1998) and DNA sequence data (Boykin et al. 2010).

When young, Colusa grass is pale green (Burtt Davy 1898), turning brown, sticky, and glandular with age (Reeder 2012a). The aquatic seedlings have one to two leaves (Kelley 1998). The terrestrial stage is caespitose, with multiple ascending, inflorescence-bearing stems (or culms) (Reeder 1982). Stem length is between 10 and 30 centimeters and leaf blades range between five and 12 centimeters wide (Reeder 2012a).

Each stem produces one dense, cylindrical inflorescence ranging between two and eight centimeters long and eight and 12 millimeters wide (Reeder 2012a). Crampton (1976) observed Colusa grass stems as having a characteristic zigzag growth form. The spikelets lack glumes and are arranged spirally along the inflorescence axis. The axis

37 may extend beyond the spikelets with or without small scales (Reeder 2012a). The floret lemma is flat and fan-shaped and strongly seven to 11 veined and the lemma is approximately the same size as the lemma. The seed is flattened, obovate, and generally

2.5 millimeters long (Reeder 1982, 2012a). Colusa grass differs from Tuctoria species by having a cylindrical inflorescence, dorsally compressed spikelets, broad lemmas, caryopsis covered in the viscid exudate, and embryo hidden by a thicker pericarp

(Reeder 1982).

Germination, Reproduction, and Population Ecology. Gerlach (2011) studied germination and seedling development of Colusa grass in the wild. He observed a floating-leaf seedling stage which can last two to three weeks when the water is approximately five to 10 centimeters deep. In addition, a few lab or greenhouse germination studies have evaluated the germination of Colusa grass. In a germination experiment described above under Greene’s tuctoria (Keeley 1998), Colusa grass germinated the latest of the studied Orcuttieae species: three months after inundation.

Davis et al. (2009) conducted greenhouse experiments on Colusa grass to determine self-compatibility. Colusa grass plants were grown from seed and transferred to pots for the experiments. To germinate Colusa grass, Davis (2009, 2010 personal communication) filled trays with vernal pool soil, sprinkling on seed (not removing lemma and palea) and filled the trays with filtered tap water so that the mud was submerged. The trays were placed in a refrigerator (1.7-3.3 degrees Celsius) for six to eight weeks and the soil was kept submerged. Once out of the refrigerator, the trays were placed in an east facing greenhouse window. The water was allowed to evaporate

38 so that the surface of the soil was damp but not submerged. The seeds started to germinate within a couple weeks.

Gerlach (2009b) also conducted a germination experiment on Colusa grass.

For his methods, he created a 10 X 10 germination grid of tulle fabric with one seed

(“naked” – caryopsis only without lemma or palea) per cell. The bottom part of the germination grid consisted of two layers (one sheet of tulle and a sheet of wax paper) and the top part was two sheets of tulle. The seeds were between the bottom and top parts. The grids were placed in containers of vernal pool soil at a depth of 3 centimeters and then covered with water. Treatments included containers with and without added litter from Colusa grass. The containers were placed in a 1.5 degree Celsius refrigerator for one or 2.5 months. After stratification, the grids were placed in growth chambers or allowed to dry down while exposed to normal late-May temperatures. Only a single seed of Colusa grass (out of 800) germinated though the seeds were viable because seeds from the same seed source germinated during reintroduction into restored pools

(Gerlach 2009b).

Colusa grass blooms from May to August (Reeder 2012a) and generally sets seed in mid to late summer (Davis et al. 2009; personal observation). In a study on reproductive ecology in wild populations over a three year period, Colusa grass at

Jepson Prairie set fewer seeds when the population density was low, which suggests some reliance on outcrossing for seed set (Davis 2009). The number of neighboring inflorescences within 0.5 meter was the best predictor of seed set, which has implications for size and density of populations and their reproductive output (Davis 2009). Colusa

39 grass has the ability to self fertilize as field and greenhouse trials demonstrated that the species is self compatible (Davis 2009).

Populations of Colusa grass in the wild can be relatively stable or vary greatly with marked variation often seen between years. In Merced County, a population was absent for two consecutive years but then had over 2,000 plants the third year (USFWS 2005). The Colusa grass population at Olcott Lake has been monitored annually since 1989 (USFWS 2005); the population is stable with fluctuations due to climatic factors (USFWS 2008a).

Using microsatellite markers, Gordon et al. (2012) performed a genetic analysis of 240 Colusa grass plants in eight pools in Yolo, Solano, Stanislaus, and Merced

Counties to determine the genetic diversity between and within populations within the

Central Valley. Their results indicated that there was little to no gene flow between the north (Yolo and Solano) and south (Stanislaus and Merced) populations of Colusa grass.

Moreover, they found support for genetic distinction between each of the two counties within the two north/south regions (Davis et al. 2009; Gordon et al. 2012).

Habitat. Colusa grass has been found on Northern Hardpan and Northern

Claypan vernal pool types and in the Solano-Colusa, San Joaquin Valley, and Southern

Sierra Foothills vernal pool regions (Keeler-Wolf et al. 1998). It has been found in alkaline basin rim vernal pools in the Sacramento and San Joaquin Valleys, as well as the basic vernal pools on terraces of the San Joaquin Valley and adjacent foothills (Stone et al. 1988). Most often, Colusa grass grows in the deepest portion of the pool (Crampton

1959) but it may also grow along the pool margins (Hoover 1937).

40 Hogle (2002) found an increase in distribution and fitness of Colusa grass in microtopographic depressions that were less than one centimeter to over three centimeters deeper than the average pool depth. She also found that pools containing

Colusa grass had slightly higher percent sodium than adjacent pools that did not contain

Colusa grass; however, Colusa grass fitness was inversely correlated with percent sodium. This suggests that the increased salinity in Colusa grass pools may not be beneficial to the plant but simply be due to the fact that the pools retain water longer.

Crampton (1959) noted that Colusa grass often forms monoculture stands in the vernal pools. In saline-alkaline pools, associated species include saltgrass (Distichlis spicata) and alkali heath (Frankenia salina) (Stone et al. 1988) as well as alkali weed (Cressa truxillensis), swamp picklegrass (Crypsis schoenoides), California eryngo (Eryngium aristulatum), and woolly marbles (Psilocarphus brevissimus) (Lazar 2006). In acidic soil pools, Colusa grass grows with coyote thistle (Eryngium sp.), turkey mullein

(Eremocarpus setigerus), and stalked popcornflower (Plagiobothrys stipitatus) (Stone et al.

1988). Rare plants that have been seen growing in the same pools at Colusa grass include San Joaquin Valley Orcutt grass (Orcuttia inaequalis), hairy Orcutt grass (Orcuttia pilosa), Solano grass (Tuctoria mucronata), Greene’s tuctoria (Tuctoria greenei), Hoover’s spurge (Chamaesyce hooveri), Sacramento saltbush (Atriplex persistens), and alkali milk vetch (Astragalus tener var. tener) (USFWS 2005).

Distribution. The type specimen of Colusa grass was collected by Burtt Davy

(1898) near the town of Princeton in Colusa County. Historically Colusa grass was known from 60 occurrences in the California Central Valley from Colusa County to

Merced County (Gordon et al. 2012; USFWS 2005). Today no more than 42 of these

41 occurrences remain, with the majority of them in Stanislaus and Merced Counties

(Gordon et al. 2012; Fig, 2-3), including at the Flying M Ranch, The Ichord Ranches, and

Figure 2-3. Current distribution of Colusa grass. Data source from Gordon et al. 2012.

the Virginia Smith Trust site (USFWS 2008a). Only three occurrences are currently known in the Solano-Colusa Vernal Pool Region, of which one is possibly extirpated

(USFWS 2008a). The extant populations occur in Solano County at Olcott Lake in Jepson

Prairie and in Yolo County at Grasslands Regional Park (Davis Communications Annex site) (USFWS 2008a). Colusa grass is currently extirpated from Colusa County (USFWS

2005, 2008a).

Eighty-five percent of the Colusa grass occurrences are on private land

(USFWS 2008a). Three occurrences are on federal land: Davis Communications

42 Annex/Grasslands Regional Park in Yolo County and Arena Plains Unit of the Merced

National Wildlife Refuge in Merced County (Silveira 2000). The population in Olcott

Lake at Jepson Prairie Reserve in Solano County is managed and permanently protected by the Nature Conservancy (USFWS 2005, 2008a).

Conservation. Colusa grass is federally threatened, state endangered, and has the California Native Plant Society (CNPS) California Rare Plant Rank 1B.1 (CDFW

2013; CNPS 2013; USFWS 1997). In the Recovery Plan, the recovery step outlined for

Colusa grass is delisting with 90% of known occurrences protected in eight core areas

(USFWS 2005). These eight core areas are: Grasslands Ecological Area, Davis

Communications Annex, Jepson Prairie, Farmington, Madera, Merced, Turlock, and

Waterford which are distributed among three vernal pool regions: San Joaquin Valley,

Solano-Colusa, and Southern Sierra Foothills (USFWS 2005). Other recovery measures include protection of habitat, status surveys, monitoring and adaptive management, seed banking, and research. Introductions are proposed for areas in the San Joaquin

Valley Vernal Pool Region and reintroductions in the Colusa County portion of the

Solano-Colusa Vernal Pool Region (USFWS 2005).

Threats to the species include habitat loss and fragmentation, competition with nonnative species, incompatible grazing regimes, altered hydrology, and increased nutrient load (Hogle 2002; Stone et al. 1987; USFWS 2008a). In a comprehensive survey of Orcuttieae grasses in 1986, Colusa grass had the highest number of extirpated populations, with over half of them being lost to irrigated agriculture (Stone et al. 1987).

The next two highest reasons for extirpation were heavy grazing and irrigated pasture, but also included disking and competition with non-native and native species (Stone et

43 al. 1987). According to the U.S. Fish and Wildlife Service five-year review for Colusa grass, agricultural conversion is the largest threat (especially in Stanislaus County) with urbanization being the second largest threat (especially in eastern Merced County)

(USFWS 2008a).

Based on their genetic research, Gordon et al. (2012) suggested that each of the four subregions in Yolo, Solano, Stanislaus, and Merced should be considered evolutionary significant units and managed likewise for conservation. They recommended against facilitated outcrossing and genetic rescue across the north/south populations and, if possible, between each of the subregions (Gordon et al. 2012).

General recommendations for vernal pool creation, reintroduction and introduction of populations are similar to those described above for Greene’s tuctoria.

CHAPTER III

METHODS

Study Sites

The goal of this research was to examine the potential for introductions of two rare grasses, Greene’s tuctoria (Tuctoria greenei) and Colusa grass (Neostapfia colusana), into vernal pool habitats. To this end, four study sites were established, two sites with restored or created vernal pools and two reference sites with extant populations of the rare grasses. Introductions occurred within the restored/ created vernal pools while reintroductions occurred at the reference pools for comparison and to test the methods. Although the reintroductions occurred in pools with extant populations and not in pools with extirpated populations (as is typically the case), the term reintroduction was used for purposes of this study to distinguish from introductions by making clear that the propagules were returned to their source pools during the course of the experiments.

Greene’s tuctoria was introduced into newly created vernal pools in Tract 17 at Llano Seco Unit of the North Valley Wildlife Management Area (Llano Seco) of the

Sacramento National Wildlife Refuge Complex in Butte County (Fig. 3-1).

Reintroduction of Greene’s tuctoria occurred at the reference site Vina Plains Preserve

(Vina Plains) in Tehama County, where six vernal pools support Greene’s tuctoria.

Olcott Lake at Jepson Prairie Reserve (Jepson Prairie) in Solano County, which currently

44 45

Figure 3-1. Vicinity map and site photos for the Greene’s tuctoria study sites: Vina Plains reference site and Llano Seco introduction site.

supports a population of Colusa grass, was the reference (and reintroduction) site and

Colusa National Wildlife Refuge (Colusa NWR) in Colusa County served as the introduction site for Colusa grass (Fig. 3-2).

All four study sites are within the California Great Central Valley and, in particular, the Sacramento Valley and Sacramento River watershed (Figs. 3-1 and 3-2).

The climate within the Sacramento Valley is characteristic of Mediterranean regions with cool, wet winters and hot, dry summers. The September to June average monthly precipitation for the three years of this study and the long term averages for each study site are shown in Figures 3-3, 3-4, and 3-5 (UCIPM 2013).

46

Figure 3-2. Vicinity map and site photos for the Colusa grass study sites: Jepson Prairie reference site and Colusa NWR introduction site.

Greene’s Tuctoria Study Sites

Vina Plains. The Vina Plains landscape is a mosaic of vernal pools and seasonal drainages within level to gently rolling grassland and is one of the few remaining vernal pool preserves in the northern Sacramento Valley. Vina Plains supports populations of Greene’s tuctoria in six vernal pools (Alexander and Schlising

1997; Schlising 2009 personal communication). The original Vina Plains Preserve

(approximately 620 hectares), excluding the Wurlitzer Ranch and other additions west of

Highway 99 (approximately 1,257 hectares), was the study location for the Greene’s tuctoria reference pools and reintroductions.

47

Vina Plains a a d

200

150 2009-2010

2010-2011 100

2011-2012 Precipitation (mm) Precipitation 50 Long-term Average (30 year) 0

Llano Seco b a d

200

2009-2010 150

2010-2011 100

2011-2012

Precipitation (mm) Precipitation 50

Long-term Average 0 (30 year)

Figure 3-3. Total monthly precipitation for September through June for the three years of the study along with the long-term average for (a) Vina Plains and (b) Llano Seco. The climate stations used for each site are Gerber (CIMIS #8) and Durham (CIMIS #12), respectively (UCIPM 2013).

48

Jepson Prairie a a d

200

150 2009-2010

2010-2011 100

2011-2012

Precipitation (mm) Precipitation 50 Long-term Average 0 (19 year)

Colusa NWR b a d

200

2009-2010 150

2010-2011 100

2011-2012

Precipitation (mm) Precipitation 50

Long-term Average 0 (29 year)

Figure 3-4. Total monthly precipitation for September through June for the three years of the study along with the long-term average for (a) Jepson Prairie and (b) Colusa NWR. The climate stations used for each site are Dixon (CIMIS #121) and Colusa (CIMIS #32), respectively (UCIPM 2013).

49

700

600

500

June (mm) June

- 400 Vina Plains Llano Seco 300 Jepson Prairie 200 Colusa NWR

100 Precipitation Sept Sept Precipitation

0 2009-2010 2010-2011 2011-2012 Long-term Average

Figure 3-5. Comparison of total and average September through June precipitation for the four study sites. The climate stations used for each site are Gerber (CIMIS #8), Durham (CIMIS #12), Dixon (CIMIS #121), Colusa (CIMIS #32), respectively (UCIPM 2013).

Vina Plains is located approximately 24 kilometers northwest of Chico, east of Highway 99, south of Lassen Road, in the southern part of Tehama County (portions of Sections 20, 21, 22, 27, 28, 29, and 34 of Township 24 north and Range 1 west on the 7

½ minute Richardson Springs NW USGS quadrangle and portion of Sections 20 and 29 on the 7 ½ minute Vina USGS quadrangle). The site is located on an upper terrace of the

Sacramento Valley at elevations ranging between approximately 60 to 70 meters above sea level. The site gently slopes to the south and a few intermittent drainages, including

Singer Creek Ditch and Sheep Camp Ditch, flow across Vina Plains in a southerly direction towards Singer Creek. Singer Creek runs 500 meters south-southeast of Vina

Plains and ultimately drains into the Sacramento River.

50 Vina Plains is located within the Northeastern Sacramento Valley Vernal Pool

Region and is comprised of Northern Hardpan vernal pools which are on alluvial soils derived from volcanic formations (Keeler-Wolf et al. 1998). Vina Plains is underlain predominately by the Red Bluff Formation (coarse red gravel, sand, and silt) along with smaller inclusive areas of Modesto Formation (alluvium), both from the Pleistocene, and basin deposits (alluvium) from the Holocene (CDC 1992). The Red Bluff alluvial formation that underlies Vina Plains was derived from the older volcanic Tuscan

Formation that occurs to the east in the Cascade foothills. The Red Bluff formation was fashioned from the Tuscan formation as it was carved by streams and sediment deposited into the valley (Conlin 2012 personal communication). At Vina Plains, alluvium cemented one to two million years ago creating the hardpan impermeable layer underlying the landscape. Over 100,000 years ago, weathering of the hardpan began which created the vernal pool basins and eventually the basins were filled with clay (Broyles 1987).

The soil series mapped onsite include Tuscan Loam and Clay Loam, Keefers

Loam Moderately Deep, Anita Clay and Anita Clay Moderately Deep, and Barrendos

Clay Hardpan Substratum (USDA NRCS 2011). All of these soils are underlain by impermeable hardpan. The majority of the site and grassland areas support Tuscan

Loam. Tuscan Clay Loam, Barrendos Clay Hardpan Stratum, and Keefers Loam are associated with some of the onsite drainages and swales. Five of the six pools that contain Greene’s tuctoria are associated with Anita Clay. The sixth pool (near Lassen

Road) is mapped as Tuscan Loam. The depth to hardpan is the shallowest for Anita

Clay: between 30 to 51 centimeters. Anita Clay is poorly drained, with slow to very slow

51 permeability. Cracks in the soil one to three centimeters wide extend from the surface to the hardpan from July to October. The surface commonly has hoof prints at a depth of

15 centimeters (Soil Survey Staff USDA NRCS 2007).

The majority of the vernal pools that support Greene’s tuctoria are located in the south-central and eastern portion of Vina Plains (Pools 14, 22, 35, 36, and 37). The exception is one pool (Pool 21) near Lassen Road in the northern portion of the site (Fig.

3-6). Plant species in these pools include common vernal pool plants such as double- horned downingia (Downingia bicornuta), stipitate popcorn-flower (Plagiobothrys stipitatus var. micranthus), dwarf woolly marbles (Psilocarphus brevissimus), Fremont’s goldfields (Lasthenia fremontii), and hairy water clover (Marsilea vestita ssp. vestita)

(Alexander and Schlising 1997; personal observation). Other rare plant species occur in the same pools as Greene’s tuctoria: hairy Orcutt grass (Orcuttia pilosa) and Hoover’s spurge (Chamaesyce hooveri) are located in Pool 35, but generally in the deeper portions of the pool than Greene’s tuctoria. In 2011, one individual of hairy Orcutt grass was observed in Pool 36. Boggs Lake hedge hyssop (Gratiola heterosepala) occurs in Pool 37, but in shallower areas than Greene’s tuctoria (personal observation). The grassland surrounding the six pools is dominated by non-native annuals, such as long beaked filaree (Erodium botrys), ripgut brome (Bromus diandrus), soft chess (Bromus hordeaceus),

Italian ryegrass (Festuca perennis), and medusa head (Elymus caput-medusae), but also includes perennials and native species such as narrow-leaved milkweed (Asclepias fascicularis), various brodiaeas (Brodiaea spp.), and yellow mariposa-lily (Calochortus luteus). The native annual hogwallow starfish (Hesperevax caulescens) is found near a couple of the reference pools.

52

Figure 3-6. Vina Plains study site and reference pools. Reintroduction pools (Pools 22, 35, and 37) are represented in bold font.

53 Vina Plains is owned and managed by the Nature Conservancy, which purchased the property in 1982 (Vina Plains Preserve Docent Committee 1994). The site is leased for cattle grazing and cattle are rotated through four pastures typically from fall to late spring. Control burns are also conducted at the preserve by the California

Department of Forestry and Fire. In 2011, a prescribed burn was conducted in the “Big

Pool Unit” pasture which includes Pool 14. In 2012 a prescribed burn occurred in the

“Safe Unit” pasture which includes Pools 21 and 22.

Llano Seco. Llano Seco is located approximately 16 kilometers southwest of

Chico, south of Ord Ferry Road and west of Seven Mile Lane (portions of Llano Seco

Section of Township 20 north and Range 1 west on the 7 ½ minute Llano Seco USGS quadrangle). Angel Slough runs through the western boundary of Llano Seco and the site is seven kilometers east of the Sacramento River. Onsite elevation is approximately

32 meters above sea level, and topographic relief varies by less than three meters from east at Seven Mile Lane to west at Angel Slough. Llano Seco is divided into two sanctuaries –Llano Seco Unit Sanctuary I (Llano Seco I) which borders Angel Slough to the west and Llano Seco Unit Sanctuary II (Llano Seco II) which borders Seven Mile

Lane to the east.

Llano Seco II is underlain by natural levee and channel deposits and basin deposits from the Holocene (CDC 1992). The soil series mapped at Llano Seco II include

Moda Taxadjunct-Arbuckle Complex, 0 to 2 percent slopes, Dodgeland Silty Clay Loam,

0 to 5 percent slopes, both occasionally and frequently flooded, Ordferry Silty Clay, 0 to

1 percent slopes, occasionally flooded, Lofgren-Bravo complex, 0 to 1 percent slopes, and

Esquon-Neerdobe Complex, 0 to 1 percent slopes (USDA NRCS 2009a). These soils are

54 moderately to poor drained and typically associated with floodplains, channels, basins, and/or vernal pools. The created vernal pools where Greene’s tuctoria was introduced are within the area mapped as Ordferry Silty Clay. The Ordferry soils consist of moderately deep, poorly drained soils that formed in alluvium from mixed rocks.

Depth to duripan for these soils ranges between one-half to one meter (USDA NRCS

2009a).

Llano Seco Unit (Llano Seco I and II) of the North Central Valley Wildlife

Management Area is part of the Sacramento National Wildlife Refuge Complex and is managed by the U.S. Fish and Wildlife Service (USFWS). Prior to being purchased by the USFWS, much of Llano Seco was leveled, intensely farmed, and/or used for pasture

(Oswald and Ahart 1996). Tract 17, where the Greene’s tuctoria introductions occurred, was an old irrigated pasture and had been managed for annual grassland for over 27 years (Silveira 2007). Today, natural vegetation at Llano Seco still exists including various riparian willow scrubs and herblands, riparian and floodplain cottonwood and mixed riparian forests valley oak woodlands, valley oak and elderberry savannas, perennial and annual grasslands, vernal pools and freshwater marshes associated with the Sacramento River, Angel Slough, Little Chico Creek floodplains (Silveira et al. 2003).

Llano Seco is located within the Northeastern Sacramento Valley Vernal Pool Region

(Keeler-Wolf et al. 1998). Vernal pools are characterized as northern claypan pools which have a lime-silica (clay) restrictive layer (Holland 1978).

Greene’s tuctoria was introduced into three newly created vernal pools

(Pools 4, 6, and 11) in Llano Seco II Tract 17 (Fig. 3-7). Natural vernal pools at the adjacent Llano Seco Tract 15 and Llano Seco Rancho were used as reference pools to

55

Figure 3-7. Llano Seco study site and created pools. Introduction pools (Pools 4, 6, and 11) are represented in bold font.

56 develop the topographical restoration plans for Tract 17 (Silveira 2007). The topographic restoration of a total of eleven Llano Seco Tract 17 vernal pools (2.7 hectares), including the three introduction pools, was completed in summer 2009 (USFWS 2007d). These 2.7 hectares of vernal pools occur in a 58-hectare annual grassland, which also includes 24 hectares of seasonally flooded annual grassland (USFWS 2007c). The grassland in Tract

17 is dominated by non-native annuals grasses, invasive yellow star-thistle (Centaurea solstitialis), and willow-leaved and prickly lettuce (Lactuca saligna and L. serriola). Native annual grassland plants at Tract 17 include Fitch’s spikeweed (Centromadia fitchii), hayfield tarweed (Hemizonia congesta spp. luzulifolia), meadow barley (Hordeum brachyantherum spp. brachyantherum), and Ferris’ milk vetch (Astragalus tener var. ferrisiae) (Oswald and Ahart 1996). After the first cycle of inundation and evaporation, hyssop loosestrife (Lythrum hyssopifolium), bird's foot trefoil (Lotus corniculatus), field bindweed (Convolvulus arvensis), English plantain (Plantago lanceolata), and purslane speedwell (Veronica peregrina ssp. xalapensis) were observed germinating and growing in the created pool beds.

Colusa Grass Study Sites

Jepson Prairie. Jepson Prairie Reserve is an approximately 630-hectare grassland and vernal pool landscape. It is one of the few remaining natural prairies in the southern Sacramento Valley and home to some of the best remaining vernal pools in the state. Many endemic and rare species inhabit the vernal pools (Davis et al. 2009;

Witham and Mawdsley 2012). A large vernal pool, Olcott Lake (Fig. 3-8), supports an abundant population of Colusa grass, and is the reference site for introductions at

Colusa NWR. Jepson Prairie is located approximately 19 kilometers south of downtown

57

Figure 3-8. Olcott Lake at Jepson Prairie study site. Reference population and reintroductions occurred in Olcott Lake east of Cook Lane.

58 City of Dixon in Solano County. Jepson Prairie is located in portions of Sections 13, 14,

23, and 24 of Township 5 north and Range 1 east on the 7 ½ minute Dozier USGS quadrangle. Cook Lane bisects a portion of the property and State Highway 113 borders portions of the north and eastern edges of the site. Power lines cross the property from the northwest to southeast and an abandoned railroad bed borders the western edge of the site. Barker Slough and Calhoun Cut run through the northeastern and southwestern corners of Jepson Prairie. Water from these features ultimately drains into the Sacramento River before entering Suisun Bay. Onsite elevations range between three and eight meters above sea level with the site gently sloping to the east.

Jepson Prairie is underlain by older alluvium of the Quaternary period (CDC

1981). The soil series mapped onsite include San Ysidro sandy loam, 0 to 2 percent slopes, Antioch-San Ysidro complex, 0 to 2 percent slopes, Pescadero clay loam, Solano loam, and Pescadero clay (USDA NRCS 2007). The majority of Jepson Prairie supports the two San Ysidro soils, which are associated with the upland grassland habitats.

Solano loam occurs along swales and Barker Slough. Pescadero clay loam underlies many of the depressions and vernal pools. Olcott Lake, the largest vernal pool onsite and the location of reference population of Colusa grass, is underlain by Pescadero clay.

Pescadero soils are deep, poorly drained or ponded soils that formed in alluvium from sedimentary rocks. Depth to restrictive layer is approximately two meters. They are very slightly saline to moderately saline soils (Soil Survey Staff USDA NRCS 1997).

Jepson Prairie is located within the Solano-Colusa Vernal Pool Region and includes Northern Claypan vernal pools on the basin rim, where the restrictive layer is a densely compacted silica-cemented clay layer (Keeler-Wolf et al. 1998). The pools are

59 typically alkaline and are sometimes large with shallow topographical relief, often resembling playas more than vernal pools. The reference population of Colusa grass at

Jepson Prairie is located in a large (39-hectare) playa-like vernal pool, Olcott Lake (Fig. 3-

8). Cook Lane bisects Olcott Lake and the population of Colusa grass is located east of

Cook Lane. Common plant species occurring in Olcott Lake include alkali mallow

(Malvella leprosa), alkali heath (Frankenia salina), lippia (Phyla nodiflora), and alkali weed

(Cressa truxillensis). No other rare plants are known to occur with Colusa grass in the eastern part of Olcott Lake. Olcott Lake, west of Cook Lane, supported a population of

Solano grass (Tuctoria mucronata); however, it hasn’t been observed since the mid 1990s.

The grassland habitat adjacent to Olcott Lake includes native species such as saltgrass

(Distichlis spicata), and non-native species such as Italian ryegrass (Festuca perennis) and foxtail barley (Hordeum murinum).

Jepson Prairie was purchased by the Nature Conservancy in 1980 and was transferred to the Solano Land Trust in 1997. The site is also part of the University of

California Natural Reserve System. Sheep seasonally graze Jepson Prairie; however, a fence around the eastern portion of Olcott Lake prevents them from grazing in this portion of pool.

Colusa National Wildlife Refuge. The 1,896 hectare Colusa NWR lies in the

Colusa Basin, approximately two kilometers southwest of the town of Colusa in Colusa

County. Prior to conversion to agriculture, the area north of and including Colusa

NWR was part of the Colusa Plains, which included alkali meadows and vernal pools.

Currently, Colusa NWR supports a network of seasonal wetlands, ponds, alkali meadows, and vernal pools managed by the U.S. Fish and Wildlife Service (USFWS

60 2009a). Colusa NWR is located approximately nine kilometers east of Interstate 5 and

State Highway 20 borders the site to the north (portions of Sections 1, 2, 11, 12, 13, 14, 23,

24, 25, 26, 34, and 35 of Townships 15 and 16 north and Range 2 west on the 7 ½ minute

Colusa USGS quadrangle; and portions of Sections 25 and 26 of Township 16 north and

Range 2 east on the 7 ½ minute Arbuckle USGS quadrangle). Colusa NWR is bisected by the Colusa Basin Drain (Colusa Trough) and Powell Slough, which drain the Basin southeast to the Sacramento River. Onsite elevations range between approximately 12 and 14 meters above mean sea level.

Colusa NWR is underlain by basin deposits (alluvium) (CDC 1960). The basin rim soils mapped at Colusa NWR include several soil series: Capay clay loam,

Willows silty clay, Scribner silt loam, Westfan loam, Clear Lake clay, Mallard clay loam,

Colusa loam, and Alcapay clay (USDA NRCS 2009b). The Colusa grass introduction pools are mapped as Willows silty clay, 0 to 1 percent slopes. These are deep, non-saline to moderately saline, poorly drained soils with a restricted feature greater than two meters deep (USDA NRCS 2009b).

Colusa NWR is part of the Sacramento National Wildlife Refuge Complex and managed by the USFWS. The refuge was established in 1945 as a “refuge and breeding ground for migratory birds and other wildlife and to reduce damage to agricultural crops caused by waterfowl” (USFWS 2009a). Colusa NWR is currently divided up into 59 management units of managed and unmanaged wetlands, alkali meadows, vernal pools, grasslands, and riparian habitats (USFWS 2009a). The native vernal pool landscape of the Colusa Basin is almost inconceivable and the habitat conversion has been nearly complete. Prior to European settlement, a relatively dense

61 aggregation of large intermittent lakes, vernal pools, mima mounds, and alkali sinks and flats covered the “Colusa Plains” – these lakes were recorded on early soil survey maps

(Holmes and Nelson 1915)– and they were the habitat of Colusa grass. In normal to wet years, these natural wetlands and the surrounding annual grasses provided habitat for massive “hordes” of wild geese followed by a vast springtime parade of wildflowers “of every color of the rainbow” (Hall 1975; Hanson 1944; Silveira 2000, 2001).

Today, the natural vegetation at Colusa NWR is characterized as claypan or saline vernal pools of the Solano–Colusa Vernal Pool Region (Barbour et al. 2003;

Holland 1978; Keeler-Wolf et al. 1998) and alkali meadow plant communities (Griggs et al. 1992; Holland 1986). A majority of the vernal pool and alkali meadow forbs are natives, while most of the grasses are non-natives (Oswald and Silveira 1995; Wight

2000). Colusa NWR is within the historic habitat range for Colusa grass (USFWS 2005).

Colusa grass was introduced into three restored pools in Tracts 24.12, 24.13, and 25 (east) (Fig. 3-9). This includes approximately 12 hectares of restored vernal pools in 92 hectares of vernal pool–alkali meadow habitat (USFWS 2007a). The restored pools were designed after referencing historic air photos, soil surveys, and U.S. Geologic

Survey topographical maps of pools that originally occurred in Tracts 24 and 25 prior to agricultural conversion (Silveira 2007). For the purposes of this study, pools at Colusa

NWR are considered restored since they were modeled after previously existing natural pools in the same location. This is in comparison to the pools at Llano Seco which were not modeled after vernal pools at the site but after nearby vernal pools. Therefore, for purposes of this study, the pools at Llano Seco are considered created.

62

Figure 3-9. Colusa NWR study site and restored pools. Introduction pools (Tracts 24.12, 24.13, and 25 (east)) are represented in bold font.

Topographic restoration of Colusa NWR Tract 24.13 was completed in 2001.

The east half of Tract 25 was completed in 2004. Native plants have been slow to colonize Tracts 24.13 and 25: species include native alkali weed (Cressa truxillensis), a few individuals of harlequin downingia (Downingia insignis) and Fremont’s goldfields

(Lasthenia fremontii), and more abundant, non-native redscale (Atriplex rosea), five horn bassia (Bassia hyssopifolia), swamp picklegrass (Crypsis schoenoides) and African picklegrass (C. vaginiflora). Topographic restoration of Colusa NWR Tract 24.12 was completed summer 2008. Restored pool baseline conditions are characterized by relative open soil. Plant species include swamp picklegrass (Crypsis schoenoides), five horn bassia

63 (Bassia hyssopifolia), redscale (Atriplex rosea), and alkali weed (Cressa truxillensis). Plants in the surrounding alkali meadows include native pappose tarweed (Centromadia parryi ssp. rudis), horned sea-blight (Suaeda calceoliformis), bush seepweed (Suaeda moquinii), alkali heath (Frankenia salina), and saltgrass (Distichlis spicata), and non-natives such as yellow star-thistle (Centaurea solstitialis), common burclover (Medicago polymorpha),

Mediterranean mustard (Hirschfeldia incana), and ripgut brome (Bromus diandrus)

(Oswald and Silveira 1995).

Sampling Design and Data Collection

Characterizing Study Sites

During spring and summer 2010, environmental data at both the reference and introduction sites was collected to compare and inform the species introductions.

Hydrology (spring dry down) was mapped at all four study sites, locations of the extant populations at reference sites were mapped, and vegetation surveys were performed to determine the vegetation associates of the two rare grasses and of the study site pools.

Dry down and extant populations in 2011 were also mapped to note any changes between the two years.

Hydrology. Starting in April 2010 for Vina Plains and Llano Seco, and May

2010 for Jepson Prairie and Colusa NWR, the extent of standing water in the pools was mapped every one to two weeks using a GPS unit (Trimble GPS XH, NAD 83 Zone 10, accuracy potential < 1 meter). The edge of standing water was generally clearly delineated except for Vina Plains. Due to the deep cow prints that would retain water for longer periods of time, the edge of the standing water in the Vina Plains pools was sometimes irregular. As a rule, these cow-print areas were mapped when at least 50% of

64 the area was covered in water. If there were a few isolated cow prints with standing water, they would not be included in the polygon. Spring hydrology (dry down) was studied instead of fall and winter hydrology because unlike many of the vernal pool species that germinate in the fall, the study grasses germinate in the spring as the pools are drying down. In 2011, dry down mapping commenced in April at all four study sites. Data was collected every one to two (occasionally three) weeks through June.

Reference Populations. In order to compare the location of reference populations in relation to topography and dry down within a pool, the extent of the reference populations were mapped. In June and July 2010, within each reference pool, the location of Greene’s tuctoria at Vina Plains and Colusa grass at Jepson Prairie was mapped using the Trimble GPS unit. First, the pools were thoroughly walked to find all

Greene’s tuctoria or Colusa grass individuals. Since the grasses are relatively short and difficult to see from a distance, individuals or groups of plants were marked with pin flags, and then the flagged perimeter was mapped with the GPS as a polygon. Some of the pools had more than one polygon mapped within each pool, such as at Olcott Lake at Jepson Prairie where the areas of Colusa grass were not contiguous. Separate polygons were mapped within pools if the groups of plants were greater than three to five meters apart. Notes were also taken on where the densest areas of the plants occurred within the pool. In 2011, mapping of the Greene’s tuctoria and Colusa grass populations occurred later, in September and November, due to more time-sensitive data collection occurring during the summer. In both 2010 and 2011, general phenology observations, such as when Greene’s tuctoria and Colusa grass germinated, flowered, and set seed, were also recorded.

65 Vegetation. In 2010, plant communities were characterized at the study sites to assess potential similarities in vegetation associates at reference and introduction sites, and to determine the plant taxa most commonly associated with Greene’s tuctoria or Colusa grass. At Vina Plains and Jepson Prairie reference pools, vegetation was surveyed both in areas mapped with Greene’s tuctoria or Colusa grass (on patch) and in areas without these two rare grasses (off patch). At Colusa NWR, vegetation was surveyed in the three introduction pools. Since the Llano Seco pools were constructed in

2009, formal vegetation surveys were not conducted due to the minimal vegetation in the introduction pools and overall immature and likely highly dynamic vegetation community.

Vegetation surveys were conducted in August at Vina Plains and Colusa

NWR and in early September at Jepson Prairie. Vegetation surveys in the late summer are not ideal for identifying the spring-flowering plants which dominate the vernal pools. However, due to late-season phenology typical of Greene’s tuctoria and Colusa grass, and also that the populations had to be mapped prior to the on and off patch vegetation surveys, the surveys occurred when most of the plants had senesced. Every plant was identified to genus and, when possible, effort was made to identify plants to species. Scientific names follow The Jepson Manual, Second Edition (Baldwin et al.

2012).

Depending on the size of the pool, between 10 and 14 quadrats were placed on patch and between 10 and 14 quadrats off patch at each of the reference pools at Vina

Plains. At Jepson Prairie, 30 quadrats were placed both on and off patch in Olcott Lake.

At Colusa NWR, 10 quadrats were placed in each of the three introduction pools. The

66 location of the quadrat placement was determined using a stratified random design. A trial run of the quadrat placement resulted in many of the quadrats concentrated in one or two areas of the patch. Therefore, a stratified random design was applied for quadrat placement. First the patch was divided into relatively equal areas and a corresponding equal number of quadrats were assigned to that area. The quadrat was positioned by standing in the center of an area and using a random number table to select a compass bearing and number of paces. If the randomly selected number of paces or compass bearing resulted in traveling outside the extent of the area, a new number of paces or compass bearing was chosen. A 0.5 meter squared quadrat was placed on the ground at the end of the paced line. Absolute cover of all observed plants was recorded, along with cover of bare ground and rock. The following 13 categories for cover classes were used: 1, 2-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, and 100%.

In addition, for the on patch areas, the abundance of Greene’s tuctoria or Colusa grass plants rooted within the quadrat was recorded.

Introductions and Reintroductions

The second phase of the research was to use the information gathered characterizing and observing the study sites in 2010 to make informed decisions for

Greene’s tuctoria and Colusa grass introductions at Llano Seco and Colusa NWR, respectively.

Seed Collection. In order to harvest seed and inflorescences for the introduction experiment, entire Greene’s tuctoria and Colusa grass plants were collected from the reference sites in 2010. First, throughout the spring and summer of 2010, the phenology of Greene’s tuctoria at Vina Plains and Colusa grass at Jepson Prairie was

67 observed in order to collect the plants after the seeds matured but before the inflorescences had shattered. Greene’s tuctoria was collected randomly from five pools at Vina Plains on September 8, 2010: 30 plants each from Pools 14, 22, and 35, and 20 plants each from Pools 36 and 37. A total of 105 Colusa grass plants were collected randomly from Olcott Lake on September 17, 2010. Plants collected were limited to no more than five percent of the plants growing at any of the pools. Since there were thousands of plants in most of the pools, the percent of total collected was minimal.

For all plants collected, a single plant was placed in a brown paper lunch bag.

After the Colusa grass plants were collected, the inflorescences shattered in the bags; however, the Greene’s tuctoria inflorescences remained relatively intact. Bags of plants were stored in a cool, dry location in cardboard boxes until the packet construction in

December 2010.

Seed and Inflorescence Packets. It was necessary to keep track of the introductions in the pools for monitoring, so instead of broadcasting the plant material into the pool, packets were made to hold the seeds and inflorescences. The general design and construction of the packets was modeled after Gerlach’s (2009a,b) 10 X 10 germination grids; however, the packets for this study were smaller, used different types of tulle, and contained no wax paper. The packets for this study consisted of two layers of tulle (see tulle description below) with the plant material (individual seed or piece of inflorescence) between the two layers. The external dimensions of one packet were 10 x

7.5 centimeters. The internal cell, which contained the seeds or inflorescence piece, measured six by six centimeters. For the seed packets, the internal cell was further divided into nine separate cells with one seed (with lemma and palea) in each of the

68 nine cells (Fig. 3-10a). The inflorescence packets contained a one-centimeter piece of inflorescence in the internal cell, with no further cell divisions (Fig. 3-10b). The cell structure of the packets was created with a hot glue gun.

a a d

b a d

Figure 3-10. Packets for (re)introductions: (a) Seed and (b) Inflorescence.

An important aspect of the packets was to make sure that the holes in the tulle were large enough to allow for germination but small enough so that the seeds would not fall out of the packets. Since Greene’s tuctoria seeds are approximately one millimeter wide, the tulle used for the Greene’s tuctoria packets had holes just under one millimeter in diameter. Colusa grass seeds are larger (two millimeters wide), so the tulle used for these packets had holes just under two millimeters in diameter. In addition, the packets were designed to prevent other plants from germinating underneath and growing up through the packets. To prevent this, the bottom layer of tulle had smaller holes than the top layer. The smaller hole sizes (between 0.25-0.75 millimeter) on the

69 packet bottom still allowed Greene’s tuctoria and Colusa grass to root as the plant roots are generally smaller in diameter than the shoots.

It was important to have fertile seeds with lemma and palea for the introduction experiments (Davis et al. 2009; Griggs 1980). In order to do this, seeds were separated from the inflorescence by hand using a microscope and forceps. Fertile seeds were identified as being plump, round, and full of endosperm. Approximately 2,300

Greene’s tuctoria seeds, 2,200 Colusa grass seeds, and 250 one-centimeter Greene’s tuctoria inflorescences were separated from the plants. To prevent potential genetic contamination of the natural populations at the reference pools, packet reintroductions contained only seeds and inflorescences from plants collected from that pool. For example, only plant material from plants collected from Vina Plains Pool 35 was reintroduced back into Pool 35. For the introductions sites, packets contained a mix of seeds or inflorescences from the five Vina Plains pools (for Greene’s tuctoria) or throughout Olcott Lake (for Colusa grass). Due to the shattering of the Colusa grass inflorescences after collection, it was impossible to create inflorescence packets for

Colusa grass. Therefore, inflorescence packets were only produced for Greene’s tuctoria.

To create the packets, an approximately 1.3 meter by 0.4 meter piece of plywood was covered with aluminum foil. An outline was drawn on the foil showing

48 (3 x 16) packets with cell boundaries. To construct the packets, the bottom layer of tulle was first stretched across the wood and secured. Next, the top layer of tulle was placed over the bottom, secured on one end, and folded back off the bottom layer. Then the seeds (caryopsis with lemma and palea) or pieces of inflorescences were placed on

70 the bottom layer of tulle in the correct locations using the drawing on the foil as a guide.

When the plant material was in place, the top tulle layer was folded back on top.

Finally, using the outline on the foil as a guide, a glue gun traced the edges of the cells with enough pressure so the glue would go through both pieces of tulle and secure them together. Once the glue dried, the individual packets were cut. A total of 750 packets were created for the introductions and reintroductions.

Pools and Transects. To optimize successful introductions, introduction pools were chosen at Llano Seco and Colusa NWR based on habitat characterization comparisons between reference and restored/created pools. The final date of dry down for the pool as well as the rate of dry down for the extant populations was taken into consideration when choosing the introduction pools (refer to Appendices A and B for the 2010 dry down and reference population figures). Pools with similar dry down patterns to the reference pools were chosen at Llano Seco and Colusa NWR. Based on the similarities, Pools 4, 6, and 11 were chosen at Llano Seco and Tracts 24.12, 24.13, and

25 (east) at Colusa NWR. In addition, for comparison, reintroduction pools were chosen at Vina Plains (Pools 22, 35, and 37) and at two locations within Olcott Lake.

Introduction transects were established at each of the study site pools. The major considerations for the location of the introduction transects at Llano Seco and

Colusa NWR were depth and topography. Observations in 2010 of the extant populations showed that the grasses not only grow in the deeper parts of the pools, but also at the pool edges. Therefore, the location, position, and length of the introduction transects crossed a range of pool depths, including the deeper areas of the pool and, if possible, the pool edges. For the reintroduction transects at Vina Plains and Jepson

71 Prairie, the placement occurred within areas that in 2010 had supported areas of

Greene’s tuctoria or Colusa grass. The purpose of placing the packets within known suitable habitat for Greene’s tuctoria and Colusa grass was not only to compare with the introduction sites, but to also to identify the possible treatment effect of the packets.

Two transects were located at each introduction or reintroduction pool. At

Vina Plains and Llano Seco, since both seed and inflorescence packets were created for

Greene’s tuctoria, each transect consisted of two parallel rows, one of seed packets and the other of inflorescence packets. The rows of seed and inflorescence packets were one meter a part (0.50 meter from transect centerline) and packets were placed every meter.

The second transect in the pool was placed within three meters from centerline of the first transect. Three meters was chosen so that the transects were close enough to capture similar topography within the pool but far enough apart that walking between them would not create significant disturbance to the packets. At Jepson Prairie and

Colusa NWR, only seed packets were created for Colusa grass. Therefore, the packets were placed on transect centerline every meter. Landscape pins were used at two ends of the packets to secure all packets into the soil. Refer to Appendices C and D figures for the transect locations in the pools.

Introductions of the two grasses at the introduction pools and reintroductions at the reference pools occurred January 1, 2, and 3, 2011. At Vina Plains, the length of the transects varied between pools in order to capture the variety of pool topography and area of extant population (Table 3-1). For example, in larger pools (Pool

35) or pools with larger areas of Greene’s tuctoria (Pool 22), the transects were longer, and the smaller pool (Pool 37) had shorter transects (Appendix D, Figs. D-3, D-4, and D-

72 Table 3-1. Length of transects, number of packets, and number of seeds in seed packets for the introduction and reintroduction pools or areas. Site Pool or Area Transect Packets Totals Length per (m) transect Seed Packets Infl Packets Seeds packets Vina Plains Pool 22 25/27 26/28 54 486 54 Pool 35 22 23 46 414 46 Pool 37 14/15 15/16 31 279 31 Llano Seco Pool 4 29 30 60 540 60 Pool 6 14 15 30 270 30 Pool 11 15 16 32 288 32 Jepson North 30 31 62 558 Prairie South 17 18 36 324 Colusa Tract 24.12 26 27 54 486 NWR Tract 24.13 23 24 48 432 Tract 25 21 22 44 396 (east)

6). Pool 22 transects were positioned from the pool edge and continued into the deeper area of the pool. Pool 35 transects started at the pool edge and continued into the deeper portions of the pool. Pool 37 transects occurred in the deepest area of the pool. The two transects within Pools 22 and 37 were different lengths due to human error.

Since the pools at Llano Seco were relatively smaller than those at Vina

Plains, the transects spanned the widths of the pools, from pool edge to pool edge

(Appendix C, Figs. C-9, C-10, and C-11). Using micro-topography provided by the U.S.

Fish and Wildlife Service and dry down information from our habitat characterizations,

Llano Seco transects were positioned so that they traversed the areas of the pool that contained standing water for longer periods of time. The transect lengths varied between pools because the widths of the pools varied (Table 3-1).

At Jepson Prairie, reintroduction transects were located in both the northern and southern portion of Olcott Lake (Appendix C, Fig. C-12). Each area had two

73 transects for a total of four transects at Olcott Lake (Table 3-1). The northern transects started at the edge of the pool and continued towards the deeper area of the pool. The southern transects were positioned near an upland island that had supported large

Colusa grass plants in 2010 (Appendix B, Fig. B-6). At Colusa NWR, the transects were positioned so that they traversed the deeper areas of longest standing water and continued towards the pool edge (Appendix C, Figs. C-13, C-14, and C-15).

Monitoring and Data Collection. The reintroductions and introductions were monitored in order to compare introduction success within a pool, between pools, and between sites. Monitoring of the introductions and reintroductions began in February

2011 and notes were taken when the first germinations were observed in April; however; data collection for the entire pool did not commence until there was no standing water and data could be collected for the entire pool at one time. At Vina Plains and Llano

Seco, at the shallowest pools where standing water dried down the soonest (Vina Plains

Pool 37 and Llano Seco Pool 4; Appendix C, Table C-1), the first data collection for the entire pool occurred in May. For the remaining pools, the initial data collection for the entire pools occurred in July (while data was collected a second time in the two shallow pools, Pools 37 and 4, in July). The last data collection occurred in October for all Vina

Plains and Llano Seco pools. At Jepson Prairie (Olcott Lake) and Colusa NWR, the initial data collection for the entire pool occurred between May and August depending on when the pool dried down and the last data collected occurred in November.

For the first data collections, the following was recorded for each of the nine cells in the seed packets: germination, survivorship (whether the plant had died), and reproduction (whether the plant had produced an inflorescence). For the inflorescence

74 packets, germination, survivorship, and reproduction were recorded. In addition, an estimate of the total number of plants was noted. Since many of the plants in the inflorescence packets were growing tightly together, teasing them a part to count individuals could have caused stress or mortality.

During the last data collections, all of the plants had senesced. For both the seed and inflorescence packets, the number of flowering culms (inflorescences) for each plant was recorded. Since the plants were dead, at this time the number of plants in the inflorescence packets were counted and recorded.

Inflorescences were also collected from the packets during the last day of data collection at Greene’s tuctoria study sites in order to determine the number of seeds per inflorescence as a measure of reproductive output. Three plants were chosen from every other packet along the transects and all inflorescences from the three plants were collected. For the three by three (nine cell) seed packets, to capture any variability for the location of plants within a packet, plants were collected from the center cell and one random edge and corner using a random number table. For the inflorescence packets, a comparable center, corner, and edge plant was selected. The center plant was bordered on all sides by another plant, the edge plant on three sides, and the corner plant on two.

At every fifth packet for both seed and inflorescence packet transects, all the plants and their inflorescences were collected. In addition to the packet collections, the nearest wild

Greene’s tuctoria plant to the packet was collected. For all seed, inflorescence, and wild collected plants, each inflorescence was placed in a separate coin envelope and all the coin envelopes from one plant were put in a paper lunch bag. The inflorescences were placed in a cardboard box and stored in a cool, dark location.

75 The introduction sites were also monitored and surveyed during the spring and summer of 2012. Greene’s tuctoria second generation plants at the Llano Seco introduction pools were surveyed: each reproductive plant and the number of culms were recorded.

Inflorescence Processing. Due to the minimal success of the Colusa grass plants at the introduction site (see Results Chapter), only Greene’s tuctoria inflorescences were collected and processed to remove seeds. Unlike isolating the seeds for the introductions, the purpose here was to measure reproductive output and only count the number of fertile seeds; it wasn’t necessary to keep the seed with the lemma and palea attached. Therefore, a more mechanical threshing process was used for seed removal. Due to Greene’s tuctoria’s small seeds and the difficulty removing some of the seeds from the inflorescence (in particular the basal seeds in the spikelets), seeds were harvested from the inflorescence in a two to three-step process. First, to isolate the seeds from the florets and spikelets, the inflorescences were agitated and crushed using a rubber-lined tray and hand tool. The tray was constructed by gluing a rubber mat to the bottom of a 23 by 33 centimeter cooking tray. The rubber mat had a network of small ridges approximately 0.25 millimeter high. The hand tool was a modified hand sander with the same rubber mat attached to its surface (Fig. 3-11). After processing an inflorescence using circular movements with the hand tool, if seeds were still stuck in the basal florets, one of two second steps were utilized. Either forceps were used to tease the seeds out of the bottom florets or a second tray was used. The second tray was the same dimensions at the rubber-lined tray but instead the bottom was covered in sandpaper. With the use of a hand sander, the inflorescence would be lightly sanded in

76

Figure 3-11. Greene’s tuctoria threshing device: rubber – lined tray and hand tool.

the tray so that the glumes, lemma, and palea would disintegrate and free the seeds.

After the seeds were removed from the inflorescence, the material was passed through a one-millimeter sieve (U.S.A. Standard Test Sieve No. 13) which separated the majority of the chaff from the seeds. The fertile seeds were then counted. Although the seeds were small, it was fairly easy to distinguish between fertile and unfertile seeds. Fertile seed were plump and full of endosperm (and easily rolled between thumb and index finger) while infertile seeds were flat.

Due to the significant amount of time to process and collect the data on one inflorescence (approximately 10-20 minutes), a subset of the collected inflorescences was processed to track trends and inform future research. For the subset, 25-34 inflorescences were selected each from the seed packet, inflorescence packet, and wild

Greene’s tuctoria plants from Pool 35 at Vina Plains and from seed and inflorescence packets from Pool 4 at Llano Seco. The inflorescences were chosen to make sure the

77 representative sample included inflorescences along the entire transect and from center, edge, and corner positions within the packet. In total, 134 Greene’s tuctoria inflorescences were processed to remove and count seed, and measure length

(millimeters), weight (grams), and number of spikelets. Prior to processing the inflorescences to remove the seeds, the flowering culm was cut below the lowest spikelet and the weight, length, and number of spikelets were recorded for each inflorescence.

The spikelet information was collected in order to assess a possible correlation with number of seeds.

Soil Salinity Analysis. Due to the evidence of salt crystals on the soil surface in 2011 within the restoration pools at Colusa NWR, conductivity measurements were taken on soils at Colusa NWR and Jepson Prairie to compare the salinity levels between the reference and introduction sites for Colusa grass. Soils were collected at three locations along one introduction transect at both Colusa NWR Tracts 24.12 and 24.13

(the deepest and shallowest of the three introduction pools; Appendix C, Fig. C-13) and one reintroduction transect at Jepson Prairie Olcott Lake. The soil samples were taken at the center of each transect and three meters in from each end of the transect, at a depth up to three centimeters. Samples were air-dried, ground and passed through a two millimeter sieve (U.S.A. Standard Test Sieve No. 10).

The conductivity measurements methods were modeled after the U.S.

Department of Agriculture guidelines (USDA 1954). For a 1:1 mixture, 40 milliliters of deionized water was added to 40 grams of soil. The soil was a composite of the three soil samples along each transect (approximately 13.3 grams from each sampling point).

The soil and water mixture was stirred vigorously in 100 milliliter beakers with a glass

78 stirring rod for one minute, four times at 30 minute intervals. After the stirring session, the mixture was poured through a funnel fitted with filter paper (Whatman #1). Liquid was collected in 40 milliliter beaker and then poured into the conductivity meter (Beta digital pH, conductivity, and temperature tester- model DIG PCT/PH COMP) receptacle for reading. The experiment was repeated three times for each transect using the composite soils. Conductivity measurements were measured in microsiemens per centimeter (uS/cm) and converted to decisiemens per meter (dS/m) for comparison.

Before use, the conductivity meter was calibrated with 1413 uS/cm conductivity standard and rinsed with deionized water. Temperature was calibrated to the room temperature of 23 degrees Celsius.

Lab Germination

To examine the germination requirements of Greene’s tuctoria and Colusa grass in the lab, a factorial design was implemented using two or three separate treatments: temperature of cold stratification, germination substrate, and seeds or inflorescences (for Greene’s tuctoria only) (Table 3-2). The design and treatments were modeled after previous successful germination experiments of Greene’s tuctoria and

Colusa grass (Davis 2009, 2010 personal communication; Davis et al. 2009; Griggs 1980;

Keeley 1988). Each of the treatments had three replicates of 10 Greene’s tuctoria or

Colusa grass seeds (with lemma and palea) or one centimeter piece of Greene’s tuctoria inflorescence placed in a nine centimeter glass Pyrex Petri dish. The seeds and inflorescences were collected in September 2010 and stored in a cool, dry indoor location until the germination trials in early May 2011. Similar to the introductions, since the seeds required a lemma and palea, the seeds were separated for the lab germination

79 Table 3-2. Sixteen Greene’s tuctoria treatments (n=3 Petri dishes each) and 8 Colusa grass treatments (n=3 Petri dishes each) to test for specific factors affecting seed germination. Species Seeds / Cold Stratification Substrate Inflorescence Greene’s tuctoria Seeds 4 degrees Celsius Reintro. pool soil Greene’s tuctoria Seeds 4 degrees Celsius Intro. pool soil Greene’s tuctoria Seeds 4 degrees Celsius Mixed soil Greene’s tuctoria Seeds 4 degrees Celsius No soil Greene’s tuctoria Seeds 9 degrees Celsius Reintro. pool soil Greene’s tuctoria Seeds 9 degrees Celsius Intro. pool soil Greene’s tuctoria Seeds 9 degrees Celsius Mixed soil Greene’s tuctoria Seeds 9 degrees Celsius No soil Greene’s tuctoria Inflorescence 4 degrees Celsius Reintro. pool soil Greene’s tuctoria Inflorescence 4 degrees Celsius Intro. pool soil Greene’s tuctoria Inflorescence 4 degrees Celsius Mixed soil Greene’s tuctoria Inflorescence 4 degrees Celsius No soil Greene’s tuctoria Inflorescence 9 degrees Celsius Reintro. pool soil Greene’s tuctoria Inflorescence 9 degrees Celsius Intro. pool soil Greene’s tuctoria Inflorescence 9 degrees Celsius Mixed soil Greene’s tuctoria Inflorescence 9 degrees Celsius No soil Colusa grass Seeds 4 degrees Celsius Reintro. pool soil Colusa grass Seeds 4 degrees Celsius Intro. pool soil Colusa grass Seeds 4 degrees Celsius Mixed soil Colusa grass Seeds 4 degrees Celsius No soil Colusa grass Seeds 9 degrees Celsius Reintro. pool soil Colusa grass Seeds 9 degrees Celsius Intro. pool soil Colusa grass Seeds 9 degrees Celsius Mixed soil Colusa grass Seeds 9 degrees Celsius No soil

experiment using a microscope and forceps. A total of 240 Greene’s tuctoria seeds, 240

Colusa grass seeds, and 24 one-centimeter piece of Greene’s tuctoria inflorescence were used for the lab germination experiment.

The germination substrate treatment was used to determine whether the plants germinated better on soils from reintroduction pools, introduction pools, a mixture of the two, or no soil. The soils were collected in January 2011 and stored in a cool, dry location until use in the germination trials. Greene’s tuctoria reintroduction

80 pool soil was from Vina Plains Pool 35 and introduction pool soil was from Llano Seco

Pool 6. Colusa grass reintroduction pool soil was from Olcott Lake and introduction pool soil was from Colusa NWR Tract 25 (east).

The purpose of the two cold stratification temperatures was to determine optimum cold stratification for germination. The two temperatures were 4 and 9 degrees Celsius and were based on different stratification temperatures in previous germination experiments (Davis et al. 2009; Griggs 1980; Keeley 1988; Strauss 2009).

Reintroduction pool soil control treatments (two replicates each) did not contain added seeds or inflorescences and were used to determine whether any Greene’s tuctoria or

Colusa grass germinated from the reintroduction pool soil. For the no soil treatments, the Petri dishes only included water along with the seeds or inflorescences.

To assemble the Petri dishes, 15 milliliters of soil was added to a Petri dish and the seeds or inflorescence was placed on top of the soil. Next, 45 milliliters of deionized water was added to each Petri dish and the Petri dish was covered with the glass Petri dish lid. After assembled, the Petri dishes were randomly placed in one of two cold stratification fridges and cold stratified for 11 weeks. While in the fridges, the

Petri dishes were regularly checked for germination since previous experiments demonstrated germination in the fridge while under cold temperatures (Griggs 1980).

On July 19, 2011, the Petri dishes were removed from the fridges and placed indoor near an east-facing window. The temperature in the lab ranged between 22 and 28 degrees

Celsius. They were checked regularly for germination and rotated in the lab with respect to adjacency to the window. Water was added as needed to maintain standing

81 water over the soil and seeds. The number of germinating seeds declined by August 1, and the experiment ended after one month on August 19.

Data Analyses

Data analysis was performed with Minitab 16 statistical software.

Assumptions of individual statistical tests were tested prior to analysis. Data-sets were tested for normality using an Anderson-Darling test. Test for equal variances were performed using Bartlett’s test, Levene’s test, or F test. Significance levels for all analyses was α = 0.05. Standard error of means are reported in text and in figures using error bars. Pool dry down (extent of standing water) and reference population locations were depicted using ESRI ArcGIS 9.3.

Percent germination, percent survivorship to reproduction (percentage of germinated plants that survived to reproduce), vigor (average number of culms per plant), and reproductive output (average number of seeds per inflorescence) were the dependent variables compared in the introduction and reintroduction study. The number of culms (stems supporting the reproductive structure/inflorescence) per plant was used as an estimation of plant vigor because these two rare grasses continue to produce culms in response to favorable growing conditions (Griggs 1980).

Pools were considered the experimental units (and therefore, replicates) since the pools received the treatments (created/restored or reference and, therefore, introduction or reintroduction). Packets and transects represented sampling replicates per pool (30 to 62 packets along two transects). Packets were removed from the data set if they were: (1) removed by animals, wave action, or wind, (2) completely covered in soil or (3) undercut and not in contact with the soil surface. Data were combined over

82 an entire pool to get one number for the pool. For instance, percent survivorship for the pool was calculated as the total number of plants that survived to produce an inflorescence over the total number of plants that germinated. Due to time and budget constraints and the desire to choose the most suitable pools for introduction, there were a limited number of experimental replicates (i.e., pools) for this study.

There were three experimental units (i.e., study pools) at Vina Plains, Llano

Seco, and Colusa NWR. Unlike the other study sites, Jepson Prairie reintroductions occurred in only one pool (Olcott Lake). However, the reintroductions occurred in two separate areas (north and south) of the large pool and, following dry down of the pool, were hydrologically divided for a large portion of the Colusa grass growing season.

Therefore, for purposes of the statistical analyses, the two areas were considered separate experimental units.

For comparison of the two pool treatments (created/restored and reference and, therefore, introduction and reintroduction), the mean and variance were calculated using the experimental units (pools) at one site. Statistical significance was analyzed using a two sample t-test or Mann-Whitney test when data did not meet the conditions for a parametric test. For comparison of the two packet treatments (seed and inflorescence) for Greene’s tuctoria, a paired analysis was required because both packet treatments were located within one experimental unit. Therefore, a paired t-test was used to determine statistical significance, to test if the mean difference in the dependent variable was zero for the seed and inflorescence packets by pool. Dependent variables

(i.e., percent germination, survivorship, vigor) were combined for each packet treatment over the entire pool or experimental unit. Comparison of mean vigor between wild

83 plants and the two packet treatments was evaluated similarly since all treatments (seed, packet, wild) occurred within one experimental unit (i.e., pool). Comparisons were made using a single-factor repeated measures ANOVA model and post-hoc Tukey-

Kramer multiple comparison. Pools were blocked to remove the pool to pool variability from the packet treatment variability.

Comparing experimental units (in this study, the pools) within a site using inferential statistics could be considered “pseudoreplication” (Hurlbert 1984). However, a comparison of the pools provided insight into the variability within and between the pools. It was important to determine which introduction pools provided the most suitable habitat for germination and reproduction in order to better understand introduction success. To compare pools within a site, the two parallel transects of packets within each pool were used as replicates in one-way ANOVA or Kruskal-Wallis when data did not meet the conditions for a parametric test. Post-hoc Tukey-Kramer multiple comparisons were calculated for factors contributing significant differences in the ANOVA model. At Vina Plains Pool 37 and Llano Seco Pool 4, percent germination and survivorship from the earlier data-set (collected in May) were also calculated from an average of the two transects. The data-set was compared with the later data-set

(collected in July) using descriptive statistics; however, inferential statistics were not conducted due to the low number of replicates and sample size.

When comparing the plant vigor between the first (2011) and second (2012) generation for the three introduction pools at Llano Seco, pool averages were calculated over all plants in the pool instead of an average of two transects (n= total number of plants per pool). Since the second-generation data set was not organized by transect or

84 packet treatments, all plants for second generation were pooled to get one average for the pool. The same was done for the first generation for comparison using descriptive statistics. Significant differences in mean plant vigor between generations were not analyzed using inferential statistics.

For comparison of salinity at two Colusa NWR introduction pools and Olcott

Lake reference pool at Jepson Prairie, differences in mean conductivity were compared in a one-way ANOVA (n = 3 composite soil samples/pool). Post-hoc Tukey-Kramer multiple comparisons were calculated for factors contributing significant differences to the model.

Analysis of the lab germination included comparison of stratification temperature and substrate between average percent germination and for seed treatment and average number of germinations per Petri dish for the inflorescence treatment. The data-sets were compared using non-parametric Kruskal-Wallis tests since they did not meet the assumption of normality.

CHAPTER IV

RESULTS

Characterizing Study Sites

The goal of this research was to examine the potential for introductions of rare grasses into vernal pools. Prior to introductions and reintroductions, it was important to gather biological and environmental information about the pools at the four study sites to compare and inform introduction success. It was assumed that the introduction pools that were the most similar to the reference pools with respect to hydrology (timing and pattern of dry down) would have the potential for the highest success for introductions. Not only was the hydrology of the pools compared but the specific hydrology where the target species occurred was used to inform introduction pool selection and within pool introduction locations.

Hydrology

Pools were selected for introduction that had similar timing of dry down as the pools at the reference sites. The extent of standing water in the study pools during specific 2010 survey dates is illustrated in Appendix A. The first survey date when no standing water was observed in the pools (“final dry down date”) is shown in Table 4-1.

The six Vina Plains pools that support Greene’s tuctoria are shown in

Appendix A, Figure A-1. Pools 36 and 37 dried down the fastest of the six reference pools, with no standing water by May 18th (Table 4-1). On this same day, Pools 14, 22, 85

86 Table 4-1. Approximate final dry-down date (first survey date with no standing water) for pools at the four study sites. Pools marked with an asterisk (*) are introduction or reintroduction pools. All six Vina Plains pools support Greene’s tuctoria. Site Pool Approximate Final Dry-Down Date 2010 Vina Plains Pool 14 May 30 Pool 21 May 30 Pool 22* ~June 5 Pool 35* May 30 Pool 36 May 18 Pool 37* May 18 Llano Seco Pools 1, 2, 3, 7, 8, 9 ,10 May 8 Pool 4* May 26 Pool 5 June 23 Pool 6* June 17 Pool 11* June 4 Jepson Prairie Olcott Lake* June 24-July 1 Colusa NWR Tract 24.12* June 23 Tract 24.13* May 19 Tract 25 east* June 18

and 35 maintained large areas of shallow, standing water (Appendix A, Figs. A-3, A-5, and A-6), while Pool 21 had only a few small areas of standing water (Appendix A, Fig.

A-4). By May 30th all pools except Pool 22 had dried down (Table 4-1). Pool 22 had a few small areas of standing water on May 30 (Appendix A, Fig. A-5) and dried down by early June (Table 4-1).

At Llano Seco, the last standing water in the majority of the created pools was observed in mid April (Appendix A, Fig. A-9). These pools were dry by early May

(Table 4-1). The exceptions were Pool 5 and the three introduction pools: Pools 4 maintained standing water through mid May, Pool 11 by end of May, and Pool 6 through early June (Appendix A, Figs. A-10, A-11, and A-12).

87 At Olcott Lake at Jepson Prairie, the majority of the pool maintained standing water through mid June with smaller areas maintaining water through June 24th

(Appendix A, Fig. A-13). Olcott Lake was dry by July 1st (Table 4-1). At Colusa NWR,

Tract 24.13 contained standing water through mid May, Tract 25 (east) through early

June, and Tract 24.12 through mid June (Appendix A, Figs. A-14, A-15, and A-16).

Reference Populations

Greene’s Tuctoria. Timing of phenology (Fig. 4-1) was dependent on dry down of the pools. Plants germinated and began their life cycle earlier at the pool edges

a b c a a a d d d

d e f a a d d

Figure 4-1. Greene’s tuctoria phenology: (a) young seedling, (b) older seedling with initiation of multiple culms, (c) plant with flowering inflorescences, (d) dead plant after seed set, (e) shattering inflorescences in August, and (f) shattered inflorescences collecting in pool depressions. Photo credit: Erin Gottschalk Fisher.

and in pools that dried down first. In 2010 at Vina Plains, Greene’s tuctoria were germinating by early May at the pool edges, nearly a month before the pools were dry.

88 By mid-May the seedlings had several leaves and by mid June, two weeks after the standing water was gone in some of the pools, the plants had produced inflorescences and were flowering (Fig. 4-1c). The seeds developed and matured in July and August

(Fig. 4-1d). Some inflorescences partially shattered (terminal florets) in August and

September, prior to fall precipitation (Fig. 4-1e). Griggs (1980) observed that shattering of the inflorescences began with first fall precipitation; however, Griggs also observed inflorescences that shattered soon after maturing in some populations of slender Orcutt grass (Orcuttia tenuis). In many instances, the shattered inflorescences collected in small depressions (Fig. 4-1f).

The spatial distribution of Greene’s tuctoria in the pools illustrated that the rare grass did not always occur in the deepest parts of the reference pools and often occurred at the shallower edges of the pools. Appendix B figures show the locations of the reference populations at Vina Plains. As was obvious during the vegetation surveys in August, some Greene’s tuctoria plants grew extremely close together. Without pulling up the plants (after they’ve died) to examine the root system, it was impossible to identify separate plants (Fig. 4-2).

Colusa Grass. In mid-June 2010 in Olcott Lake at Jepson Prairie, the first

Colusa grass seedlings were observed near the receding water line at mid-elevations within the pool; however, germination had likely occurred earlier in some of the shallower portions of Olcott Lake. By the end of June, mature flowering plants were present at the edges of Olcott Lake (Fig. 4-3c) while recently germinated seedlings occurred at the water’s edge. The variation in phenology within the pool reflected the different timing since dry down. The majority of Colusa grass plants were flowering in

89

Figure 4-2. Three Greene’s tuctoria plants growing in close proximity as to appear as one plant. Photo credit: Erin Gottschalk Fisher.

July. In August and September the plants were setting seed. In September the inflorescences began to shatter prior to fall rains as individual spikelets, florets, or the entire spike (inflorescence) (Fig. 4-3e,f). With the first rains, the florets would often stick to the soil surface (Fig. 4-3g).

As shown in Appendix B, Figure B-6, the spatial distribution of Colusa grass at Jepson Prairie illustrated that the rare grass occurred in the deepest as well as the shallowest portions of Olcott Lake. Moreover, by the end of the season, the largest plants (with multiple culms; Fig. 4-3c) were often at the edges of the pool while the deepest areas of the pool typically supported the smallest, often one culm plants (Fig. 4-

3d).

90

a b a a d d

c d a a d d

e f a a d d

g a d

Figure 4-3. Colusa grass phenology: (a) young seedling, (b) older seedling with initiation of multiple culms at pool edge, (c) plant at pool edge with multiple culms, (d) plants at pool center with one culm, (e) shattered inflorescences, (f) inflorescence stuck to soil after precipitation; and (g) individual florets stuck to soil after precipitation. Photo credit: Erin Gottschalk Fisher.

91 As observed by Gerlach (2009b) but unlike prior descriptions of Colusa grass which state that the species does not have elongated, floating juvenile leaves (Keeley

1988, 1998; Reeder 1982), long first leaves of Colusa grass were observed floating on the surface of the water (Fig. 4-4a). These long juvenile leaves often remained as the pool

a b a a d d

c d a a d d

e f a a d d

Figure 4-4. Colusa grass juvenile leaf morphology: (a) long juvenile leaves floating on top of water, (b) – (d) long juvenile leaves, (e) shorter juvenile leaf in seedling in shallow water, and (f) shorter juvenile leaf. Photo credit: Erin Gottschalk Fisher.

92 dried down and were up to six centimeters long (Fig. 4-4b-d); however, not all of the seedlings appeared to have the long initial floating leaves (Fig. 4-4e,f). The trait may be plastic and dependent on whether the seed germinates under sufficient standing water.

Vegetation

The vegetation surveys conducted in August and September were not ideal for identifying spring-flowering plants in the vernal pools; however, it was necessary to wait until later in the season after the summer-flowering Greene’s tuctoria and Colusa grass had mostly completed their life cycle and the populations were mapped. As a result, at the time of the vegetation survey, a few of the species had senesced to the point where they were only identified to genus. This occurred predominantly at Vina Plains where the vegetation is dominated by spring-flowering annuals. The taxa identified to genera for the Vina Plains vegetation surveys represented a few different possible species based on personal observations earlier in the season and floras for Vina Plains

(Broyles 1987; Vina Plains Preserve Docent Committee 1994): Popcorn flower species

(Plagiobothrys spp.) included a few possible species such as stalked popcornflowers (P. stipitatus var. micranthus and P. s. var. stipitatus), and less commonly finebranched popcorn flower (P. leptocladus) and Greene’s popcorn flower (P. greenei); Spike primrose species (Epilobium spp.) were selfing willowherb (E. cleistogamum), denseflower spike primrose (E. densiflorum), and smooth spike primrose (E. pygmaeum); Downingia species

(Downingia spp.) were two-horned downingia (D. bicornuta), folded downingia (D. ornatissima), and less commonly toothed downingia (D. cuspidata); Goldfields species

(Lasthenia spp.) were Fremont’s goldfields (L. fremontii), alkali goldfields (L. platycarpha), and smooth goldfields (L. glaberrima) (Table 4-2). African picklegrass (Crypsis vaginiflora)

93 Table 4-2. Vegetation community within (on-patch) and outside (off-patch) mapped Greene’s tuctoria areas in five pools at Vina Plains. Frequency represents the percent of quadrats in which each taxonomic class occurred. Average cover represents the mean percent cover in quadrats where the taxonomic class was present. Average number of Greene’s tuctoria (Tuctoria greenei) per quadrat represents quadrats in which the species occurred. California non-native taxa are in bold. Perennial taxa are indicated by an asterisk (*). Taxon with average cover without SE occurred in only one quadrat. Nomenclature follows Baldwin et al. (2012). Number of 0.5 meter squared quadrats 58 each on/off patch Total plant taxa 16 on/ 18 off (19 total) Average (±SE) Tuctoria greenei per 20.1 (±3.7) quadrat On patch Off patch Taxonomic class Frequency Average Frequency Average (%) Cover (%) Cover (%)(±SE) (%)(±SE) Bare ground 100.0 42.9(±2.0) 100.0 27.1(±2.4) Rock 6.9 14.6 (±4.4) 8.6 29.5 (±11.8) Forbs Plagiobothrys spp. 89.7 28.1 (±2.6) 96.6 49.3 (±3.4) Eryngium castrense* 84.5 16.7 (±1.8) 69.0 20.2 (±2.7) Epilobium spp. 77.6 9.3 (±1.1) 44.8 6.5 (±0.9) Marsilea vestita ssp. vestita* 44.8 12.3 (±3.2) 32.8 15.1 (±2.5) Downingia spp. 41.4 9.0 (±1.4) 25.9 10.8 (±1.3) Psilocarphus brevissimus var. 32.8 9.2 (±1.6) 70.7 22.7 (±2.8) brevissimus Croton setigerus 10.3 16.4 (±5.0) 8.6 9.7 (±2.3) Lasthenia spp. 10.3 9.9 (±3.6) 22.4 19.1 (±5.1) Navarretia leucocephala ssp. 6.9 14.0 (±1.9) 46.6 16.0 (±1.9) leucocephala Asclepias fascicularis* 0.0 0.0 3.4 4.25 (±3.25) Chamaesyce hooveri 0.0 0.0 1.7 7.5 Graminoids Tuctoria greenei 81.0 22.3 (±2.7) 0.0 0.0 Alopecurus saccatus 31.0 11.3 (±1.7) 41.4 11.8 (±1.4) Crypsis spp. 15.5 10.9 (±2.7) 8.6 12.4 (±5.2) Orcuttia pilosa 6.9 3.3 (±1.5) 3.4 26.3 (±18.8) Deschampsia danthonioides 5.2 10.0 (±2.5) 5.1 15.8 (±5.1) Hordeum marinum ssp. 1.7 3.5 3.4 26.3 (±18.8) gussoneanum Festuca perennis* 1.7 1.0 3.4 5.5 (±2.0) Eleocharis macrostachya* 0.0 0.0 3.4 20.0 (±5.0)

94 and swamp picklegrass (C. schoenoides) were grouped into one taxon (Crypsis spp.) for all sites.

Both on and off patch, Vina Plains pools were dominated by native annuals and perennials (Table 4-2). The vegetation within these pools falls within the Lasthenia fremontii-Downingia (bicornuta) Herbaceous Alliance (Fremont’s goldfields-Downingia vernal pools) and Lasthenia glaberrima Herbaceous Alliance (Smooth goldfields vernal pool bottoms) (Sawyer et al. 2009). The only non-native species within the surveyed

Vina Plains pools were three graminoids with relative low frequency (Table 4-2). The following species had the highest frequency of occurrence (above 40%) on patch: downingia species (Downingia spp.) (41.4%), spike primrose species (Epilobium spp.)

(77.6%), Great Valley coyote thistle (Eryngium castrense) (84.5%), hairy waterclover

(Marsilea vestita ssp. vestita) (44.8%), and popcorn flower species (Plagiobothrys spp.)

(89.7%). A higher average percent cover of bare ground was found on patch (42.9%) compared to off patch (27.1%). A few taxa showed a trend towards greater occurrence on patch compared to off patch, including Great Valley coyote thistle (Eryngium castrense) (84.5% on verses 69.5% off), spike primrose species (Epilobium spp.) (77.6% on verses 44.8% off), hairy waterclover (Marsilea vestita ssp. vestita) (44.8% on verses 32.8% off), and downingia species (Downingia spp.) (41.1% on verses 25.9% off). A couple species occurred off patch at a much higher frequency than on patch, such as white headed navarretia (Navarretia leucocephala ssp. leucocephala) (6.9% on verses 46.6% off) and woolly marbles (Psilocarphus brevissimus var. brevissimus) (32.8% on verses 70.7% off). These off patch species are typically located at the edges of the pools and at shallower pool depths.

95 The vegetation communities within the Greene’s tuctoria created and reference pools were markedly different. Although not surveyed in detail, Llano Seco pools were recently created and, therefore, were largely populated by non-native species from the surrounding upland including hyssop loosestrife (Lythrum hyssopifolium), bird’s foot trefoil (Lotus corniculatus), field bindweed (Convolvulus arvensis), English plantain

(Plantago lanceolata), and purslane speedwell (Veronica peregrina ssp. xalapensis). This species composition was notably different than the mostly native species that dominated the natural pools at Vina Plains (Table 4-2).

Olcott Lake at Jepson Prairie vegetation was dominated by native perennials

(Table 4-3). The species with the highest frequencies of occurrence on patch (20% or higher) were alkali weed (Cressa truxillensis) (76.7%), alkali heath (Frankenia salina)

(43.3%), salt grass (Distichlis spicata) (20.0%), alkali mallow (Malvella leprosa) (26.7%), and lippia (Phyla nodiflora) (33.3%). On and off patch frequencies and abundances were relatively similar for most species. A couple of taxa showed a trend towards greater occurrence on patch compared to off patch, including lippia (Phyla nodiflora) (33.3% on verses 13.3% off) and alkali mallow (Malvella leprosa) (26.7% on verses 16.7% off). The largest difference between on and off patch was creeping spikerush (Eleocharis macrostachya) that occurred primarily off patch: 10.0% frequency and 12% cover on patch verses 46.6% frequency and 70.7% cover off patch. The vegetation within Olcott Lake as well as the Colusa NWR introduction pools falls within the Cressa truxillensis-Distichlis spicata Herbaceous Alliance (Alkali weed-Salt grass playas and sinks) (Sawyer et al.

2009). However, unlike Olcott Lake, Colusa NWR pools have a higher percentage of

96 non-native annuals (Table 4-4). Only two taxa were found at both sites (picklegrass

(Crypsis sp). and alkali weed (Cressa truxillensis)).

Table 4-3. Vegetation community within (on patch) and outside (off patch) of mapped Colusa grass areas in Olcott Lake at Jepson Prairie. Frequency represents the percent of quadrats in which each taxonomic class occurred. Average cover represents the mean percent cover in quadrats where the taxonomic class was present. Average number of Colusa grass (Neostapfia colusana) per quadrat represents quadrats in which the species occurred. California non-native taxa are in bold. Perennial taxa are indicated by an asterisk (*). Taxa with average cover without SE occurred in only one quadrat. Nomenclature follows Baldwin et al. (2012). Number of 0.5 meter squared quadrats 30 each on/off Total plant taxa 10 on/ 9 off (12 total) Average number Neostapfia colusana per 6.7 (±1.7) quadrat On Patch Off Patch Taxonomic class Frequency Average Frequency Average (%) Cover (%) (%) Cover (±SE) (%)(±SE) Bare ground 100.0 57.0 (±2.5) 100.0 46.6 (±4.5) Forbs Cressa truxillensis* 76.7 20.8 (±2.1) 96.7 20.4 (±2.7) Frankenia salina* 43.3 25.2 (±2.1) 40.0 22.7 (±3.4) Phyla nodiflora* 33.3 21.5 (±4.0) 13.3 16.3 (±5.1) Malvella leprosa* 26.7 27.5 (±2.5) 16.7 21.0 (±2.4) Eryngium aristulatum* 13.3 19.6 (±6.7) 13.3 17.1 (±6.5) Psilocarphuys brevissimus var. 3.3 7.5 6.7 11.25 (±3.8) brevissimus Symphotrichum subulatum var. 3.3 65.0 0.0 0.0 parviflorum* Epilobium cleistogamum 0.0 0.0 3.3 7.5 Graminoids Neostapfia colusana 73.3 15.3 (±2.2) 0.0 0.0 Distichlis spicata* 20.0 27.1 (±5.1) 23.3 24.8 (±4.0) Crypsis spp. 13.3 1.0 (±0.0) 0.0 0.0 Eleocharis macrostachya* 10.0 12.0 (±6.6) 66.7 44.1 (±6.2) Alopecurus saccatus 0.0 0.0 3.3 35.0

97 Table 4-4. Vegetation community associated with the three introduction pools at Colusa NWR. Frequency represents the percent of quadrats in which each taxonomic class occurred. Average cover represents the mean percent cover in quadrats where the taxonomic class was present. California non-native taxa are in bold. Perennial taxa are indicated by an asterisk (*). Nomenclature follows Baldwin et al. (2012). Number of 0.5 meter squared quadrats 30 Total plant taxa 6 Taxonomic class Frequency Average Cover (%) (%)(±SE) Bare ground 100.0 62.0 (±4.0) Forbs Atriplex rosea 46.7 15.6 (±3.5) Cressa truxillensis* 40.0 35.4 (±6.7) Bassia hyssopifolia 23.3 26.2 (±7.2) Spergularia rubra 6.7 25.0 (±0.0) Xanthium strumarium 6.7 11.3 (±3.8) Graminoids Crypsis spp. 73.3 30.8 (±5.2)

Introductions and Reintroductions

Greene’s Tuctoria

Introductions at the Llano Seco created pools and reintroductions at Vina

Plains reference pools occurred at the beginning of January 2011 (Fig. 4-5). At both introduction and reference pools, germination occurred and the first seedlings observed between the end of April (in the shallower pools) and the first of May (in the deeper pools). Young, recently germinated seedlings (with one to three leaves) were observed under a range of hydrological conditions from damp soil to two centimeters of standing water. At Vina Plains, inflorescences began appearing at the end of May. At Llano Seco pools, the plants also started producing inflorescences at the end of May in Pool 4

(shallower pool) and the middle of June in Pools 6 and 11 (deeper pools).

98

a b a a d d

c d e a a a d d d

Figure 4-5. Introduction (Llano Seco) and reintroduction (Vina Plains) Greene’s tuctoria plants: (a) Reintroduction inflorescence packet, (b) Reintroduction reproductive plants, (c) Introduction seed packet, (d) Introduction inflorescence packet, and (e) Introduction reproductive plants. Photo credit: Erin Gottschalk Fisher.

Appendix C (Figs. C-1 – C-11) shows the dry down hydrology at both Vina

Plains and Llano Seco in 2011. Germination and phenology, triggered as the pools dried down, resulted in Greene’s tuctoria plants in a range of phenological stages within each pool and between pools. For instance, at Vina Plains reintroduction Pool 37, which was more shallow, plants germinated, produced inflorescences and died while the plants in

Pools 22 and 35 were past flowering but still green and setting seed. Likewise, in the beginning of May at Llano Seco Pool 4, edges of the pool supported large plants with multiple culms while the center of the pool contained small seedlings. In the deeper pools (such as Pool 11), flowering occurred at the edges of the pool in early June with recently germinated seedlings at the center of the pool.

99 Germination in 2011 was also triggered by late spring precipitation. In Llano

Seco Pool 4, germination occurred after the pool dried down when above-average precipitation fell in June (Fig. 3-3 in Methods Chapter). For example, 26 plants germinated in June or early July and 14 survived to reproduce. Another interesting observation in Pool 4 was the pattern in plant vigor within the pool. Plants with the highest vigor (greatest number of culms per plant) occurred not in the deepest or shallowest portions of the pool but at middle range in elevation at both ends of each transect. Presumably this range corresponded with the optimum pool depth (and hydrology) for Greene’s tuctoria for that particular year in Pool 4.

Appendix D (Figs. D-1 – D-6) depicts the reference populations at Vina Plains in 2011. Compared to 2010, the extent of the Greene’s tuctoria wild populations at Vina

Plains reference pools had either stayed relatively the same (e.g., Pools 36 and 37;

Appendix D, Figs. D-5 and D-6) or increased (e.g., Pools 14, 22, and 35; Appendix D,

Figs. D-1, D-3, and D-4) and in at least one pool the wild plants were notably more vigorous (greater number of culms) (e.g., Pool 22). Moreover, Greene’s tuctoria occurred in Pool 21 in 2011 (Appendix D, Fig. D-2) when there were no plants in 2010.

Comparison of Introduction and Reintroduction Pools. For the seed packets, comparison of introductions at Llano Seco created pools and reintroductions at Vina

Plains reference pools evaluated the dependent variables of percent germination, percent survivorship, and vigor (average number of culms per plant). Percent germination was measured as the percentage of introduced or reintroduced seeds that germinated. Percent survivorship was a measure of survivorship to reproduction: the percentage of germinated plants that survived to produce at least one inflorescence.

100 Average number of culms per plant was the average of the number of flowering inflorescences produced by the plant and a measurement of plant vigor. Of these three variables (percent germination, percent survivorship, and vigor), only percent germination showed a significant difference between Vina Plains and Llano Seco in a two-sample t-test where germination was significantly higher at Llano Seco compared to

Vina Plains (t = 3.63, P =0.022, Fig. 4-6).

Germination a Survivorship b a a 100 100 a d a d

80 80

b 60 60 a

40 40

Percent Percent 20 20 0 0 Vina Plains Llano Seco Vina Plains Llano Seco

Vigor c a 6 a d 5 4 a 3 2

Culms/Plant 1 0 Vina Plains Llano Seco

Figure 4-6. Comparison of seed packets at reintroduction (Vina Plains) and introduction (Llano Seco) pools (n= 3 pools each) with respect to (a) percent germination, (b) percent survivorship, and (c) vigor (average culms/plant). A difference in lowercase letters indicates a significant difference (P < 0.05) between the treatments. Values are means ± SE.

101 After the first generation introduction plants had set seed and died, a sample of inflorescences from the seed packet (n =25 inflorescences from Vina Plains Pool 35 and n=34 inflorescences from Llano Seco Pool 4) was processed to count the total number of seeds. Average number of seeds per inflorescence, as a measure of reproductive output, was compared between introduction and reintroduction pools. An average of 6.5 (±0.7) and 7.4 (±0.7) seeds per inflorescence were counted at Vina Plains

Pool 35 and Llano Seco Pool 4, respectively (Fig. 4-7). Since data were collected for one pool per treatment, statistical analysis was not performed; however, there was no clear trend in differences between the two pool treatments.

Reproductive Output

10

5

0 Vina Plains Llano Seco

Seeds/Inflorescence Pool 35 Pool 4

Figure 4-7. Seed packet treatment average number of seeds per inflorescence at Vina Plains reintroduction Pool 35 (n=25 inflorescences) and Llano Seco introduction Pool 4 (n= 34 inflorescences). Values are means ±SE.

For inflorescence packets, comparison of reintroduction and introduction pools included average number of germinations per packet instead of percent germination since it was unknown exactly how many seeds were in each one-centimeter

102 piece of inflorescence. No significant differences occurred between Vina Plains and

Llano Seco for any of the dependent variables in two-sample t-tests (Fig. 4-8) though the average percent survivorship was noticeably lower and both percent survivorship and vigor (average number of culms/plant) were quite variable for Llano Seco.

Germination a Survivorship b a a a 6 a a 100 d a d 5 80 4 60 3 40 2 Percent 1 20 0 0

Germinations/Packet Vina Plains Llano Seco Vina Plains Llano Seco

Vigor c a 6 a d

4 a

2 Culms/Plant 0 Vina Plains Llano Seco

Figure 4-8. Comparison of inflorescence packets at reintroduction (Vina Plains) and introduction (Llano Seco) pools (n= 3 pools each) with respect to (a) germination (average number of germinations per packet), (b) percent survivorship, and (c) vigor (average number of culms per plant). A difference in lowercase letters indicates a significant difference (P < 0.05) between the treatments. Values are means ± SE.

A sample of inflorescences was processed from the first generation inflorescence packet plants (n = 25 inflorescences each from Vina Plains Pool 35 and

Llano Seco Pool 4) to count the total number of seeds per inflorescence. Average number of seeds per inflorescence, as a measure of reproductive output, was compared

103 between introduction and reintroduction pools. An average of 9.2 (±0.8) and 9.6 (±1.3) seeds per inflorescence was counted at Vina Plains and Llano Seco, respectively (Fig. 4-

9). There was no trend in differences between the two pool treatments.

Reproductive Output

10 8 6 4 2 0

Vina Plains Llano Seco Seeds/Inflorescence Pool 35 Pool 4

Figure 4-9. Inflorescence packets average number of seeds per inflorescence at Vina Plains reintroduction Pool 35 (n = 25 inflorescences) and Llano Seco introduction Pool 4 (n = 25 inflorescences). Values are means ±SE.

Although there were no significant differences in percent survivorship and vigor in both seed and inflorescence packets, the data trended toward Vina Plains plants having higher survivorship and Llano Seco plants having higher vigor (Fig. 4-10).

Moreover, the variation among pools was greater at Llano Seco (Llano Seco standard error two to ten times greater than Vina Plains standard error) (Fig. 4-10).

In addition to the data collected above in July, data were collected for Llano

Seco Pool 4 and Vina Plains Pool 37 in May since these pools were shallower and dried down sooner than the other study pools. Overall, comparing percent germination between the May and July visits demonstrated that the May data had a higher percent

104

Survivorship a Vigor b a a a a

100 a 6 a

a a d 5 d

80 4 60 a a 3 40

Percent 2 20

Culms/Plant 1 0 0 Seed Infl Seed Infl Seed Infl Seed Infl Vina Plains Llano Seco Vina Plains Llano Seco

Figure 4-10. Comparison of reintroduction (Vina Plains) and introduction (Llano Seco) pools (n= 3 pools each) with respect to (a) percent survivorship and (b) vigor (average number of culms per plant). A difference in lowercase letters indicates a significant difference (P < 0.05) between the pool treatments. Values are means ± SE.

germination (Figs. 4-11 and 4-12). Due to the greater number of observed germinations in May, the percentage of plants that survived to reproduce is comparably lower for

May than July (Figs. 4-11 and 4-12). Comparing introduction and reintroduction pools, the difference between May and July is greater at Vina Plains Pool 37 than Llano Seco

Pool 4. In the seed packets, the difference at Vina Plains Pool 37 between May and July is nearly half for percent germination per packet and twofold for percent survivorship

(Fig. 4-11).

Comparison of Seed and Inflorescence Packets. Comparison of the seed and inflorescence packet treatments was possible for percent survivorship and vigor

(average number of culms per plant). Comparing germination was not possible because the number of seeds in the inflorescence packets was not known. Over both introduction and reintroduction pools, the mean difference in vigor was significant in a paired t-test (t = 3.40, P =0.019, Fig. 4-13b) but not significant for survivorship (t = -0.67,

P =0.535, Fig. 4-13a) though there was a trend for the inflorescence packets to have

105 higher survivorship. Comparing introduction and reintroduction pools separately (n = 3 pools each) the differences in survivorship and vigor were not significant in a paired t- test for both Vina Plains (survivorship: t = -2.53, P =0.127 and vigor: t = 2.77, P =0.109) and Llano Seco (survivorship: t = 0.79, P =0.511 and vigor: t = 3.02, P =0.094) pools.

Germination a a 100 d

80

60

40 May Percent July 20

0 Vina Plains Llano Seco Pool 37 Pool 4

Survivorship b a 100 d

80

60

40 May Percent 20 July 0 Vina Plains Llano Seco Pool 37 Pool 4

Figure 4-11. May and July data for seed packet (a) percent germination and (b) percent survivorship to reproduction for Pool 37 (Vina Plains) and Pool 4 (Llano Seco). Values are means ± SE.

106

10 a Germination a 8 d 6

4 May July

2 Germinations/Packet 0 Vina Plains Llano Seco Pool 37 Pool 4

Survivorship b 100

80

60

40 May Percent 20 July 0 Vina Plains Llano Seco Pool 37 Pool 4

Figure 4-12. May and July data for inflorescence packet (a) average number of germinations per packet and (b) percent survivorship to reproduction for Pool 37 (Vina Plains) and Pool 4 (Llano Seco). Values are means ± SE.

Average number of seeds per inflorescence was compared between seed and inflorescence packet treatments across both the reintroduction and introduction pools

(Vina Plains Pool 35 and Llano Seco Pool 4). Data show a trend with greater number of seeds per inflorescence in plants from inflorescence packets in both pools (Fig. 4-14).

107

Survivorship a a 20 d

15

10 5

0

Inflorescence

- Pool 22 Pool 35 Pool 37 Pool 4 Pool 6 Pool 11

Survivorship; Survivorship; -5 Seed Seed

Difference in Percent Percent in Difference Vina Plains Llano Seco -10 -15 -20

Vigor b a 1.4

d 1.2 1.0 0.8

0.6

Inflorescence

-

0.4 Difference in Vigor; Vigor; in Difference Seed Seed 0.2 0.0 Pool 22 Pool 35 Pool 37 Pool 4 Pool 6 Pool 11 Vina Plains Llano Seco

Figure 4-13. Difference between seed and inflorescence packet treatments (n= 6 pools each) with respect to (a) percent survivorship to reproduction and (b) vigor (average number of culms per plant). The average difference in percent survivorship between the seed and inflorescence packets was not significant in a paired t-test (P < 0.05); however, the average difference in vigor is significant (not zero) for seed and inflorescence packets in a paired t-test, with seed packet plants being significantly greater (P < 0.05).

108

Reproductive Output 0 Pool 35 Pool 4 -0.5 Vina Plains Llano Seco

-1

-1.5

Inflorescence

Output; Output; - -2

Seed Seed -2.5 Difference in Reproductive Reproductive in Difference -3

Figure 4-14. Difference in seed and inflorescence packet treatments (n= 2 pools each treatment; one reintroduction pool and one introduction pool) with respect to reproductive output (average number of seeds per inflorescence). The average difference in reproductive output between the seed and inflorescence packets was not significant in a paired t- test (P < 0.05).

Although the purpose was to track trends, a paired t-test was used to compare treatments. The mean difference between treatments was not significant in the paired t- test (t = -9.80, P =0.065, Fig. 4-14).

Comparison of Packet Treatments with Wild Plants. In order to determine whether there was a packet treatment effect, vigor (average number of culms per plant) for the seed and inflorescence packet plants was compared to nearby wild Greene’s tuctoria plants at the reintroduction pools at Vina Plains. The three types were evaluated using a single-factor repeated measures design ANOVA model and post-hoc

Tukey-Kramer multiple comparison. Wild plants were shown to have a significantly greater vigor in the three study pools at Vina Plains (F = 355.41, P =<0.001, Fig. 4-15).

Although vigor was significantly greater in seed packets over all study pools (n = 6),

109 vigor was not significantly different between seed and inflorescence packets at Vina

Plains reintroduction pools only (n = 3) (Fig. 4-15).

Vigor 8

b 6

4 a a

2 Culms/Plant

0 Seed Infl Wild

Figure 4-15. Comparison of wild plants with seed and inflorescence packet treatments (n= 3 pools each) at Vina Plains with respect to vigor (average number of culms per plant). A difference in lowercase letters indicates a significant difference (P < 0.05) between the treatments. Values are means ± SE.

Average number of seeds per inflorescence was evaluated for a subset (n= 25) of wild inflorescences in Vina Plains Pool 35. Wild and inflorescence packets were similar whereas the seed packet plants were approximately a quarter lower in average number of seeds per inflorescence (Fig. 4-16). No statistical analyses could be performed due to observation at only one pool.

Comparison of Pools within Sites. Though perhaps not statistically independent and thus results need to be interpreted with caution, a comparison of the pools provided insight into the variability within and between pools at a site. This comparison established which introduction pools provided the most suitable habitat for

110

Reproductive Output

12

10 8 6 4

Seeds/Inflorescence 2 0 Seed Infl Wild Vina Plains Pool 35

Figure 4-16. Average number of seeds per inflorescence for seed and inflorescence packets and wild plants at Vina Plains reintroduction Pool 35 (n = 25 inflorescences each). Values are means ± SE.

germination and reproduction and thus provided a better understanding of introduction success. No significant differences were found between the three Vina Plains reintroduction pools (Table 4-5). At Llano Seco, significant differences occurred between pools for all dependent variables for seed and inflorescence packets except for inflorescence packets average number of germinations per packet (Table 4-5). For seed packets, percent germination and percent survivorship were significantly greater at Pool

4 than Pool 6 (Table 4-5, Fig. 4-17a,b). Vigor was also significantly different between all pools, with Pool 4 and Pool 6 again being the highest and lowest, respectively (Table 4-5,

Fig. 4-17c).

For inflorescence packets, Pool 4 had the highest percent survivorship which was significantly greater than Pool 6 but not significantly different from Pool 11 (Table

4-5, Fig. 4-18a). Vigor was also greater in Pool 4 than the other two pools (Table 4-5, Fig.

111

Table 4-5. Statistical results for comparison of Greene’s tuctoria (a) Reintroduction pools at Vina Plains and (b) Introduction pools at Llano Seco. Results are for a one-way ANOVA (F statistic) or Kruskal-Wallis test (H statistic) (n = 3 pools each). An asterisk (*) indicates a significant difference (P < 0.05) between the pools. Seed Packets Inflorescence Packets Germination Survivorship Vigor Germination Survivorship Vigor (a) H = 3.43 H = 3.71 F = 0.88 F = 0.95 F = 8.06 F = 0.69 Vina P = 0.180 P = 0.156 P = P = 0.479 P = 0.062 P = Plains 0.501 0.569 (b) F = 14.52 F = 12.48 F = H = 3.43 F = 20.21 F = Llano P = 0.029* P = 0.035* 781.81 P = 0.180 P = 0.018* 49.66 Seco P = P = 0.000* 0.005*

Germination a Survivorship b 100 a 100 a a

d a, b d

80 a a, b 80 60 b 60 b

40 40

Percent Percent 20 20 0 0 Pool 4 Pool 6 Pool 11 Pool 4 Pool 6 Pool 11

Vigor c a 8 a d 6 4 c b

2 Culms/Plant 0 Pool 4 Pool 6 Pool 11

Figure 4-17. Comparison of Llano Seco pools seed packets (n= 2 transects per pool) with respect to percent (a) germination, (b) percent survivorship, and (c) vigor (average number of culms per plant). A difference in lowercase letters indicates a significant difference (P < 0.05) between the treatments. Values are means ± SE.

112

Survivorship a Vigor b a a 100 a 8 a d a d

80 6 60 b 4 b

40 b Percent

20 2 Culms/Plant 0 0 Pool 4 Pool 6 Pool 11 Pool 4 Pool 6 Pool 11

Figure 4-18. Comparison of Llano Seco pools inflorescence packets (n= 2 transects each) with respect to (a) percent survivorship and (b) vigor (average number of culms per plant). A difference in lowercase letters indicates a significant difference (P < 0.05) between the treatments. Values are means ± SE.

4-18b). As shown across both seed and inflorescence packets, the data suggests that Pool

4 had the best introduction success, Pool 6 the lowest, and Pool 11 generally falling somewhere in between.

Relationship between Inflorescence Characteristics and Reproductive Output.

A total of 134 Greene’s tuctoria inflorescences were processed to count the total number of seeds per inflorescence as a measure of reproductive output. The inflorescences were selected from seed and inflorescence packets and wild plants from Vina Plains Pool 35 and seed and inflorescence packets from Llano Seco Pool 4. The potential strength of the linear relationship between the number of seeds and weight (grams), length

(millimeters), and number of spikelets was determined across all 134 inflorescences. The highest coefficient of determination (R2) was inflorescence weight (Fig. 4-19). Forty percent of the variation in the number of seeds per inflorescence was accounted for in the model using inflorescence weight (Fig. 4-19).

Length of inflorescence (R2 = 0.2682) and number of spikelets per inflorescence (R2 = 0.2744) were less informative. There was a lot of variability in the

113

40

35 30 y = 196.14x + 2.5791 25 R² = 0.4008 20 15 10

Seeds/Inflorescence 5 0 0.000 0.050 0.100 0.150 Weight (g) of Inflorescence

Figure 4-19. Linear regression of weight (g) of inflorescence and number of seeds per inflorescence from inflorescences (n= 134) taken from Llano Seco Pool 4 and Vina Plains Pool 35.

length of inflorescence and corresponding number of seeds. Smaller inflorescences didn’t always have fewer seeds. For example, the smallest inflorescences were nine millimeters and they had five or six seeds while inflorescences with only one seed were between 10 and 15 millimeters long. The largest inflorescences (above 40 millimeters) ranged in number of seeds from eight to 22; for instance, the longest inflorescence measured 53 millimeters and had only 13 seeds. However, inflorescences with the most seeds (above 20) were found in the larger inflorescences (≥34 millimeters); the plant with the most seeds (35) was 39 millimeters long. A similar trend was found with number of spikelets: spikelet number did not determine number of seeds; however, a greater number of seeds typically came from inflorescences with higher spikelet numbers.

Second Generation. Precipitation between September and June for the second year of the introduction (Figs. 3-3 and 3-5; 2011-2012) was approximately half of both the 30 year average and the precipitation for the first year of the introduction (2010-

114 2011). However, despite the low precipitation and partial pool filling, second generation plants germinated and survived to reproduce in the three Llano Seco introduction pools.

In early March 2012, all three of the Llano Seco introduction pools were dry and no Greene’s tuctoria had germinated. By the end of April, after a few weeks of precipitation, Pools 4 and 11 had evidence of recent standing water (e.g., algal crust and cracked soil) and Pool 6 had standing water in the region of the introduction transects.

Greene’s tuctoria seedlings were present in all three pools. The second generation plants were flowering by June, and by September and October they had set seed.

Plump, apparently viable seeds were observed in the spikelets. The second generation plant dispersal was limited; for the most part the second generation plants were located directly adjacent to first generation plants, dispersing <0.25 meters (unless the packets were moved by animals). Several of the second generation plants were growing directly out of a first generation inflorescence that was lying flat on the soil. As many as 19 plants were observed coming from a single inflorescence (Fig. 4-20). These plants all went on to produce at least one culm (Fig. 4-20). An unknown portion of these plants may have germinated from the introduction seeds that didn’t germinate in 2011; however, for purposes of this study, all 2012 plants are considered the “second generation population.”

Comparatively, reference pools at Vina Plains contained standing water and

Greene’s tuctoria seedlings in early March 2012. By early May, four of the six pools with

Greene’s tuctoria supported standing water. In one of the reintroduction pools, Pool 22, the pool had dried down by early May, a few weeks earlier than it had in 2011

(Appendix C, Fig. C-5). Likely in response to the shorter duration of standing water, the

115

a b a a d d

Figure 4-20. Multiple second generation Greene’s tuctoria plants growing out of one first generation inflorescence. (a) Side and (b) underneath views. Photo credit: Erin Gottschalk Fisher.

Greene’s tuctoria plants in Pool 22 appeared to have fewer culms (less vigor) in 2012 than 2011. By the summer, the extent of Greene’s tuctoria within the reference pools was comparable to 2011 (Appendix D, Figs. D-1, D-3 - D-6). This included Pool 14 whose pasture received a prescribed burn in early July 2011. The fire went through the pool and some Greene’s tuctoria plants were burned; however, in 2012 the vernal pool vegetation and the Greene’s tuctoria population were thriving.

Differences from 2011 were also noted in Pool 21 near Lassen Road which supported vernal pool flora (e.g., popcorn flower species (Plagiobothrys spp.) and downingia species (Downingia spp.)) and Greene’s tuctoria seedlings in early May of

2012; however, by August there was oddly next to no vegetation remaining in the pool.

It is unclear what contributed to the plant death, as it didn’t appear to be grasshopper herbivory or the prescribed burn that occurred in this pasture in early June 2012 (which did not burn through the pool). Some of the vegetation in May looked sickly (i.e., plant death at shoot tips) and may be an early indication of the lack of plants in this pool by

August. Pool 21 has had a cyclic pattern of vernal pool vegetation over the past few

116 years. In 2009 there was dense vegetation and large Greene’s tuctoria plants; however, in 2010 the pool was devoid of vegetation. In 2011 Greene’s tuctoria plants survived to reproduce and the population was mapped (Appendix D, Fig. D-2).

The second generation populations at Llano Seco were surveyed in order to better monitor the introductions. In each introduction pool, the total number of reproductive plants and number of culms per plant were counted in 2012 (Fig. 4-21).

Figure 4-22 shows the percent population increase by pool between 2011 and 2012 with a wide range of 9 to 900%. Pool 4 by far supported the greatest number of second generation plants. Although Pool 6 had the fewest second generation plants, it had the highest number of culms/plant (almost double Pool 11) (Fig.4-21).

3500

3000

2500

2000 Reproductive 1500 Plants Total number Total 1000 Culms 500

0 Pool 4 Pool 6 Pool 11

Figure 4-21. Second generation (2012) total number of reproductive plants and total number of culms at Llano Seco introduction pools.

117

1000

900 800 700 600 500 400 300 200 Percent Population Increase Population Percent 100 0 Pool 4 Pool 6 Pool 11

Figure 4-22. Percent population increase from first to second generation at Llano Seco introduction pools. The percent population increase was calculated using the remaining first generation plants to determine the second generation population increase.

Figure 4-23 compares plant vigor (average number of culms per plant) between first and second generation populations at Llano Seco. In Pools 4 and 11, the vigor of the plants decreased in the second generation, likely due to the low rainfall and partial pool filling. However, the plants in the deepest pool which retained water the longest (Pool 6) not only increased in vigor in the second generation but had the highest vigor out of all three introduction pools in 2012. From a restoration and management perspective, it is important to know how much introduction material as well as first generation plants ultimately produced these second generation plants. However, for this experiment, a portion of first generation plants and their culms were removed from the pools for processing and counting the seeds. As outlined in the Methods Chapter, three plants were collected from every other packet and every fifth packet all plants were collected. The result is that up to 75% of the plants were removed from the

118

Vigor 8 7

6 5 4 First Gen.

3 Second Gen. Culms/Plant 2 1 0 Pool 4 Pool 11 Pool 6

Figure 4-23. First (2011) and second generation (2012) average number of culms/plant at Llano Seco introduction pools. Values are means ± SE.

introduction pools. The percentage of plants removed was higher for some pools because there may have been only three (or less) plants in a packet. Therefore, often all or close to all of the plants were collected from that packet. So in pools with fewer plants (fewer plants/packet), there was a greater percentage of plants collected.

The removed plant material was not returned to the pools during this study to avoid introducing a confounding factor (i.e., removal and reintroduction) into the experiment. However, typically an introduction project would not remove a significant number of first generation plants for analysis of reproductive output.

Figure 4-24 shows the total number of first generation plants and culms for each introduction pool at Llano Seco, as well as the numbers that were collected and remained in the pool. For example, in Pool 4, there were a total of 508 reproductive plants of which 62.0% were collected. Pool 11 had a total of 239 first generation reproductive plants of which 57.3% were collected. The highest percentage of plants

119

3500

3000

2500

2000

1500 Collected Remained Total Number Total 1000

500

0 Plants Culms Plants Culms Plants Culms Pool 4 Pool 6 Pool 11

Figure 4-24. First generation (2011) total number of reproductive plants collected and remained in introduction pools at Llano Seco.

was collected from Pool 6: 75.0% of the 88 plants. Note that the greater number of first generation plants in Pool 4 is due in part to the fact that a greater number of seed and inflorescence packets were introduced into Pool 4 in 2011. Pool 4 is wider than Pool 6 or

11, therefore, the introduction transects were longer to cover the width of the pool.

To summarize the chronology for each pool at Llano Seco, Figure 4-25a, b and c shows the amount of plant material introduced (collected from 130 maternal families), total numbers of first generation plants and culms and those that remained after collection, and second generation plants and culms. Despite the removal of first generation plants, Pool 4 second generation plant numbers were greater than total first generation numbers (Figure 4-25a). The numbers of culms were relatively similar for total first and second generation indicating that in Pool 4 second generation plants were greater in numbers but smaller (fewer culms/plant) than first generation plants. Pool 6

120

3500 a 3000 a

d 2500 2000 1500

Total Number Total 1000 500 0 Total Remained Total Remained Seeds Infl Plants Culms Plants Culms Pieces Introduced First Generation Second Generation

300 b a 250

d 200

150

100 Total Number Total 50

0 Total Remained Total Remained Seeds Infl Plants Culms Plants Culms Pieces Introduced First Generation Second Generation

Figure 4-25a,b. Llano Seco (a) Pool 4 and (b) Pool 6 total number of plant material introduced, first generation (total and remained after collection), and second generation. Bars are shaded to indicate that the number introduced in January 2011 produced the total number of first generation plants and culms (2011), while it was those that remained after collection that produced the second generation populations (2012). Note for comparison purposes the differences in the y-axis scales between Figure 4-25a, b, and c.

121

800 c 700 a

600 d 500 400 300

Total Number Total 200 100 0 Total Remained Total Remained Seeds Infl Plants Culms Plants Culms Pieces Introduced First Generation Second Generation

Figure 4-25c. Llano Seco (c) Pool 11 total number of plant material introduced, first generation (total and remained after collection), and second generation. Bars are shaded to indicate that the number introduced in January 2011 produced the total number of first generation plants and culms (2011), while it was those that remained after collection that produced the second generation populations (2012). Note for comparison purposes the differences in the y-axis scales between Figure 4-25a, b, and c.

and 11 both had fewer plants and culms in the second generation than the total first generation which is presumably due in part to the removal of first generation plants

(Figure 4-25b and c). Comparing second generation with the first generation that remained, Pool 6 increased in numbers of plants and culms (Figure 4-25b), while Pool 11 had a small increase in number of plants but decreased in number of culms (Figure 4-

25c).

Colusa Grass

Introductions at Colusa NWR restored pools and reintroductions at Jepson

Prairie, Olcott Lake reference pool occurred in the beginning of January 2011. At Colusa

122 NWR, the shallowest pool, Tract 24.13, had dried down by mid May (Appendix C, Table

C-1, Fig. C-13). Colusa grass seedlings were first observed in mid-May with a range of between one and five leaves (Fig. 4-26a). Some of the seedlings had already died and the leaves on a few of plants were turning brown and dying (Fig. 4-26b). Most of the plants

a a d

b c a a d d

d e a a d d

Figure 4-26. Introduction (Colusa NWR) Colusa grass plants: (a) Young seedlings in Tract 24.13, (b) Dead seedling in Tract 24.13, (c) Introduction packet with salt crystals, (d) and (e) Plant that survived to reproduce in Tract 24.13. Photo credit: Erin Gottschalk Fisher.

123 were alive; however, by the first of June (two weeks later) only one plant had survived

(Fig. 4-26d). By mid June the single plant had three culms and all were producing an inflorescence. The end of the June the plant was flowering with anthers extending beyond the spikelets (Fig. 4-26e). The plant was collected in November to determine whether there were any apparently viable (i.e., plump) seeds. The plant had mostly senesced with shattered inflorescences and only two of the three culms remained. Of the two culms, there were three to seven viable seeds near the top of each inflorescence.

There were likely additional seeds that had fallen off the inflorescences prior to collection.

The Colusa grass plants at Tracts 24.12 and 25 (east) died after producing the coleoptiles or first leaf, likely shortly before the pools dried down in mid July and late

June, respectively. Dense salt crystals were observed in all three introduction pools, including on and around the introduction packets (Fig. 4-26c). These dense salt crystals were not observed at Jepson Prairie, Olcott Lake.

At Jepson Prairie, Olcott Lake, Colusa grass seedlings (including individuals in standing water with floating leaves) were observed in the wild population at the edges of the pool in early June. By the end of June, germinations were observed in the reintroduction packets that were above the water line. By late July, most plants within the reintroduction packets as well as in the wild population were flowering (Fig. 4-27).

The eastern half of Olcott Lake (where the reintroductions took place) is fenced and sheep are typically kept out. However, in early August sheep accidentally made their way into the fenced-out area. Although some minimal trampling occurred, the sheep did not appear to be eating the Colusa grass. Overall the wild Colusa grass population

124

a b a a d d

Figure 4-27. Reintroduction (Jepson Prairie) Colusa grass plants: (a) Reintroduction packet, and (b) Close-up of flowering reintroduction packet plant. Photo credit: Erin Gottschalk Fisher.

was smaller in extent in 2011 than 2010 (Appendix B, Fig. B-6; Appendix D, Fig. D-7).

The north and south reintroduction transects were placed in an area where Colusa grass occurred in 2010. However, in 2011 Colusa grass was largely absent in the area around the southern transects (Appendix D, Fig. D-7). Moreover, the Colusa grass plants throughout the lake appeared to be smaller (with respect to number of culms and height) compared to 2010. Appendix C (Figs. C-12 – C-15) and Appendix D (Fig. D-7) contain figures showing the 2011 dry down hydrology at both Olcott Lake (Jepson

Prairie) and Colusa NWR and the reference population in Olcott Lake.

Comparison of Introduction and Reintroduction Pools. Percent germination at Jepson Prairie, Olcott Lake, varied greatly between the north and south transects areas. Both were located within areas that supported Colusa grass the previous season

(2010; Appendix B, Fig. B-6). In 2011, the north area again supported Colusa grass

(Appendix D, Fig. D-7); however, the south area only had a few Colusa grass plants.

This difference was also reflected in the reintroduction packets: the northern area packets had 43.4% germination and the southern packets had 2.0% germination (with a

125 reintroduction pool average of 22.7% (±20.7%)) (Fig. 4-28a). Only three plants germinated in the south transect packets. Germination of Colusa grass at Colusa NWR

Germination a Survivorship b a a 50 a 50 a

d d

40 40 30 30 a

20 20

Percent Percent 10 10 a 0 0 Jepson Prairie Colusa NWR Jepson Prairie Colusa NWR

Vigor c a

4 d 3 2 1

Culms/Plant 0 Jepson Prairie Colusa NWR (one plant)

Figure 4-28. Comparison of reintroduction (Jepson Prairie) (n= 2) and introduction (Colusa NWR) (n= 3) pools with respect to (a) percent germination, (b) percent survivorship, and (c) vigor (average number of culms per plant). A difference in lowercase letters indicates a significant difference (P < 0.05) between the treatments. Values are means ± SE, except for vigor at Colusa NWR.

was less varied though generally low with 7.7%, 11.9%, and 19.4% at the three introduction pools (with an introduction pool average of 13.0% (±3.4%)). There was no significant difference between introduction and reintroduction percent germination in a two-sample t-test test (Fig. 4-28a) or Kruskal-Wallis test. Data met normality assumptions but was borderline for the test for equal variances (met with F-test and not with Levene’s test).

126 At Colusa NWR, only one plant survived to reproduce (Fig. 4-28b). The plants that germinated in two of the pools (Tracts 24.12 and 25 east) died after emergence of the coleoptile or first leaf. Most of the plants that germinated in Tract 24.13 survived to the two to five leaf stage before dying. The plant that reproduced occurred in Tract 24.13 (resulting in 1.2% survivorship for that pool). At Jepson Prairie, Olcott

Lake, of the three plants that germinated in the south transects, one survived to reproduce (33.3% survivorship). The north transects had 45.3% survivorship (Fig. 4-

28b). In total, there was an average of 39.3% (±6.0%) survivorship at Jepson Prairie and

0.4% (±0.4%) survivorship at Colusa NWR. Although the data suggest a trend in differences at the two study sites, there was no significant difference between introduction and reintroduction percent survivorship in a Welch’s t-test (t-test assuming unequal variances; t = -6.47, P =0.098; Fig. 4-28b) or Kruskal-Wallis test (H = 3.00, P =

0.083).

The one Colusa grass plant at Colusa NWR produced three culms (Fig. 4-

28c). The one reproductive plant at Jepson Prairie, Olcott Lake, south transects produced two culms, while the plants in the north transects produced an average of 1.5 culms per plant. Overall, the average number of culms per plant was 1.8 (±0.25) for

Jepson Prairie (Fig. 4-28c).

Comparison of Seed Packets with Wild Plants. In order to determine whether there was a packet treatment effect, seed packets were compared to nearby wild

Colusa grass plants in Olcott Lake with respect to vigor (average number of culms per plant) (Fig. 4-29). Vigor did not vary significantly in a one-way ANOVA.

127

Vigor

5 a

4 3 a 2

Culms/Plant 1 0 Seed Wild

Figure 4-29. Comparison of wild plants with seed packet treatment (n= 2 each) at Jepson Prairie, Olcott Lake, with respect to vigor (average number of culms per plant). A difference in lowercase letters indicates a significant difference (P < 0.05) between the treatments. Values are means ± SE.

Comparison of Pools within Sites. Although comparing pools (i.e., experimental units) can be considered artificial, it was helpful in interpreting results and determining introduction success for this project. At Jepson Prairie, the transects in the northern portion of Olcott Lake averaged 43.4%(±1.0%) germination while the southern transects averaged 1.5% (±1.5%) (Fig. 4-30a). The southern transects had a high standard error because the three plants that germinated occurred in only one transect. The north area of Olcott Lake supported a significantly higher percent germination in a one-way

ANOVA (F = 508.16, P =0.002, Fig. 4-30a). Percent reproduction and average number of culms per plant could not be compared between north and south because only one transect in the south supported germinated plants.

128

Jepson Prairie: Germination a Colusa NWR: Germination b a a 50 a 50

d d

40 40 30 30 a 20 a a

Percent 20 Percent 10 10 b 0 0 Tract Tract Tract 25 North South 24.12 24.13 east

Figure 4-30. Percent germination comparison of (a) Jepson Prairie, Olcott Lake, north and south areas and (b) Colusa NWR pools (n = 2 transects each). A difference in lowercase letters indicates a significant difference (P < 0.05). Values are means ± SE.

At Colusa NWR, the pools ranged from 19.3% (±2.4%) germination at Tract

24.13 to 7.4% (±3.7%) at Tract 25 east (Fig. 4-30b). Differences were not significant in a one-way ANOVA (F = 4.55, P =0.124). Percent reproduction and average number of culms per plant were not analyzed because only one plant at Colusa NWR survived to reproduce.

Soil Salinity. The presence of dense salt crystals on the soils surface of the introduction pools at Colusa NWR (Fig. 4-26a-c) was an indication of marked salinity differences between Colusa NWR pools and Olcott Lake at Jepson Prairie. Colusa NWR

Tracts 24.12 and 24.13 pools had between two and nine times greater salinity as measured as conductivity (dS/m) from soil water extracts and were significantly different in a one-way ANOVA (F = 7007.81, P =0.000 , Fig. 4-31). The Colusa NWR pool with the lowest salinity (Tract 24.13) was the pool that supported the one plant that survived to reproduce. These measured differences in salinity support the hypothesis that the low survivorship at Colusa NWR was a result of higher salinity.

129

20 a 18 16 14 12 10 8 6 b Conductivity (dS/m) Conductivity 4 c 2 0 Olcott Lake Tract 24.12 Tract 24.13 Jepson Prairie Colusa NWR

Figure 4-31. Soil salinity measured as conductivity in decisiemens/meter (dS/m) from a soil-water mixture from Jepson Priaire, Olcott Lake, and two introduction pools at Colusa NWR (n = 3 soil samples each). A difference in lowercase letters indicates a significant difference (P < 0.05). Values are means ± SE.

Second Generation. Only one plant survived to reproduce in the second generation of Colusa grass plants at Colusa NWR. It was located in the same pool and transect as the first generation plant (Tract 24.13) and produced one culm. The plant germinated off packet but it is unclear whether this plant is truly second generation from the one first generation plant that produced seeds or whether it germinated from one of the introduction seeds that fell out of a decomposing packet. The plant produced one culm which reached anthesis (flowering). The plant was collected in July after it had died. Two apparently viable (i.e., plump) seeds were present in the lower half of the inflorescence.

130 Lab Germination

Greene’s Tuctoria

Seed germination was low across all treatments in a factorial design experiment with percent germination averaging 8.3%. The mean number of germinated seeds per Petri dish was lower in the seed treatment than then inflorescence treatment

(0.8 (±0.8) and 7.1 (±2.4), respectively), but this may be due to more seeds occurring in the inflorescence treatments. Since there were an unknown number of seeds in the inflorescence treatment, the seed and inflorescence treatments were analyzed separately.

Photos from the seed germination experiment are shown in Fig. 4-32.

a b a a d d

c d a a d d

Figure 4-32. Greene’s tuctoria seedlings in lab germination experiment (a) no soil treatment with black fungal sheath on inflorescence, (b) no soil treatment with clear fungal sheath on inflorescence, (c) introduction soil treatment, and (d) reintroduction soil treatment. All from 4 degree Celsius fridge. Photo credit: Erin Gottschalk Fisher.

131 For the seed treatment, percent germination varied from 0% to 26.7% across all stratification and substrate treatments (Fig.4-33). The 4 degree Celsius treatment showed significantly higher percent germination than the 9 degree Celsius treatment in a Kruskal-Wallis Test (H = 7.36, P = 0.007, Table 4-6). However, germination substrate did not contribute statistically significant differences in percent germination (H = 0.49, P

= 0.9222, Table. 4-6).

For the inflorescence treatment, average number of germinations per Petri dish varied from 0 to 19.3 (Fig. 4-34). Similar to the seed treatment, the number of germinations in the 4 degree Celsius treatment was significantly greater than the 9 degree Celsius treatment in a Kruskal-Wallis Test (H = 10.08, P = 0.001, Table 4-7).

Germination substrate did not have a significant effect on seed germination of Greene’s tuctoria (H = 6.69, P = 0.082, Table 4-7).

40

30

20

10

Percent Germination Percent 0

No Soil No Soil No

Intro. Soil Intro. Soil Intro.

Mixed Soil Mixed Soil Mixed

Reintro. Soil Reintro. Soil Reintro. 4 degrees Celsius 9 degrees Celsius

Figure 4-33. Greene’s tuctoria seed treatment percent germination in a two-way factorial experiment comparing stratification temperature (n = 12 Petri dishes each) and germination substrate (n = 6 Petri dishes each). Values are means ± SE.

132 Table 4-6. Seed treatment percent germination: Kruskal-Wallis test results for two- way factorial experiment including temperature and substrate. A difference in lowercase letters indicates a significant difference (P < 0.05) between factor levels of significant treatments. *P <0.05 Treatment n Median Average Rank Z H DF P Temperature 7.36 1 0.007* 4 degree 12 1.00000E+01 16.4 2.71 Celsius 9 degree 12 0.000000000 8.6 -2.71 Celsius Overall 24 12.5 Substrate 0.49 3 0.922 Mixed Soil 6 0.000000000 11.2 -0.53 No Soil 6 5.000000000 12.4 -0.03 Reintro. 6 5.000000000 12.4 -0.03 Soil Intro. Soil 6 5.000000000 14.0 0.60 Overall 24 12.5

30 25 20 15 10 5

0

Germinations/Petri dish Germinations/Petri

No Soil No Soil No

Intro. Soil Intro. Soil Intro.

Mixed Soil Mixed Soil Mixed

Reintro. Soil Reintro. Soil Reintro. 4 degrees Celsius 9 degrees Celsius

Figure 4-34. Greene’s tuctoria inflorescence treatment average germinations per Petri dish in a two-way factorial experiment comparing stratification temperature (n = 12 Petri dishes each) and germination substrate (n = 6 Petri dishes each). Values are means ± SE.

133 Table 4-7. Inflorescence treatment germination/Petri dish: Kruskal-Wallis test results for two-way factorial experiment including temperature and substrate. A difference in lowercase letters indicates a significant difference (P < 0.05) between factor levels of significant treatments. *P <0.05 Treatment n Median Average Z H DF P Rank Temperature 10.08 1 0.001* 4 degree 12 12.500 17.1 3.18 Celsius 9 degree 12 2.000 7.9 -3.18 Celsius Overall 24 12.5 Substrate 6.69 3 0.082 Mixed Soil 6 9.000 14.9 0.97 No Soil 6 2.000 8.5 -1.60 Reintro. 6 12.000 17.3 1.93 Soil Intro. Soil 6 2.500 9.3 -1.30 Overall 24 12.5

Colusa Grass

No germination occurred in any of the Colusa grass treatments. In order to try to get the seeds to germinate, the Colusa grass treatments were subjected to another period of cold stratification; however, the seeds still did not germinate.

CHAPTER V

DISCUSSION

Introduction and reintroduction of species requires knowledge of the species’ biology, natural populations and habitat, the environmental parameters of the introduction habitat compared to natural habitat, as well as careful monitoring of the populations after introductions occur. Results of this research demonstrate that all of these factors are important in the ultimate success of introduction and reintroduction projects. Greene’s tuctoria and Colusa grass responded differently to introduction and reintroduction efforts and thus illuminate different paths to potential success in introducing new populations of rare vernal pool grasses.

Characterizing Study Sites

Hydrology and Reference Populations

The dry down and reference population maps (Appendices A – D), in combination with the precipitation (Figs. 3-3, 3-4, and 3-5), demonstrate the interannual variability in vernal pools and the associated reference populations. During the first two seasons of this study (2009-2010 and 2010-2011), the total September to June precipitation was similar but in 2011 a larger portion of precipitation fell in May and

June (Figs. 3-3 and 3-4). While Colusa grass markedly decreased in extent within Olcott

Lake from 2010 to 2011, Greene’s tuctoria increased in extent in most of the reference

134

135 pools (Pools 14, 21, 22, and 35) and essentially remained the same in others (Pools 36 and

37; Appendices B and D). In the following season, 2011-2012, approximately half of the average precipitation occurred September through June (Fig. 3-3). Although not mapped, the extent of the Colusa grass population decreased further from the 2011 extent while the extent of the Greene’s tuctoria populations remained relatively the same. (The exception to the Vina Plains pattern is Pool 21 which supported reproducing populations of Greene’s tucotoria every other year since 2009.) The Colusa grass population at Olcott Lake responded considerably more to dry years and higher than average late spring (May and June) precipitation. The variability between years and within and between species demonstrated the challenges of vernal pool research, especially with short term projects. The introduction and reintroduction transects within the pools were designed to capture some of this variability; however, variation within the results would still be expected.

Surveying the pools at Llano Seco and Colusa NWR the year prior to introduction was beneficial in identifying the pools whose hydrology would be most similar to the reference pools and therefore potentially more suitable to introductions.

The selection of pools with a variety of pool depths was also important. The three pools selected at Llano Seco represented a diversity of hydrological conditions that were suitable under varying climate conditions. The other eight created pools at Llano Seco would have likely been unsuitable. For example, when precipitation was approximately half average (as in 2012), the shallower pools at Llano Seco (shallower than introduction

Pool 4) did not support standing water and would likely not have supported Greene’s tuctoria germination. While in an average or above-average rainfall year, the one deeper

136 pool at Llano Seco (deeper than introduction Pool 6) retained water throughout most of the pool into July which is longer than reference pools at Vina Plains. Therefore, the variety of hydrological conditions at introduction pools at Llano Seco was similar to reference pools at Vina Plains and suitable for Greene’s tuctoria germination, growth, and reproduction. On the other hand, similar hydrological conditions between Colusa

NWR introduction pools and the reference pool (Olcott Lake) were not sufficient for

Colusa grass which was potentially limited by the increased salinity in the introduction pools.

Vegetation

The species associated with Greene’s tuctoria at the five Vina Plains reference pools were similar to other documented species associates (Alexander and Schlising

1997; Lazar 2006). In this study, hairy waterclover (Marsilea vestita ssp. vestita) (44.8%) and Great Valley coyote thistle (Eryngium castrense) (84.5%) were highly associated with

Greene’s tuctoria at five pools at Vina Plains. In a survey of one vernal pool also in

Tehama County in 2002, the highest species co-occurring with Greene’s tuctoria were also coyote thistle (Eryngium sp.) and hairy waterclover (Marsilea vestita) (88% and 75% absolute constancy, respectively) (Lazar 2006). Although survey methods were different

(relevés in 2002 and random quadrats in 2010), the coyote thistle (Eryngium sp.) frequencies were notably similar between the 2002 and 2010 surveys.

Greene’s tuctoria introduction pools at Llano Seco were not surveyed in detail for vegetation; however, the general species composition was very different than

Vina Plains and consisted primarily of non-native species from the surrounding uplands. Nevertheless, the difference in vegetation composition did not reflect

137 unsuitable habitat for Greene’s tuctoria at Llano Seco, instead, it reflected the fact that the Llano Seco pools were recently created and were not treated with natural vernal pool soil and thus lacked a ready source of vernal pool associated species.

The species associated with Colusa grass in Olcott Lake are similar to other documented species associates (Lazar 2006; Stone et al. 1988), most of which are halophytes (e.g., salt-tolerant or avoidant species). In a 2005 study of Olcott Lake, the associated species with the total absolute constancy (i.e., frequency) above 50% were swamp picklegrass (Crypsis schoneoides) (100%) and woolly marbles (Psilocarphus brevissimus var. brevissimus) (63%) (Lazar 2006). In this study’s 2010 vegetation survey at Olcott Lake, swamp picklegrass (Crypsis schoneoides) and woolly marbles (Psilocarphus brevissimus var. brevissimus) occurred with Colusa grass but at much lower frequencies

(13.3% and 3.3%) than recorded in 2005. Although vegetation data were not collected using the same method (relevés in 2005 and random quadrats in 2010), in general, in

2010 there was a higher frequency of perennials and a lower frequency of vernal pool

“edge” species, which might suggest a possibly wetter year in 2009-2010. However, this is not the case. Historical precipitation records show that not only was March to June precipitation similar in 2005 and 2010 (123 and 124 millimeters, respectively), there was less total precipitation between September to June in 2009-2010 compared to 2004-2005

(UCIPM 2013). Therefore, the differences in species with highest co-occurrence with

Colusa grass in Olcott Lake may be attributable to differences in sampling methods or, less likely, a general trend in the overall vegetation composition in Olcott Lake.

Although pool vegetation at both Colusa NWR and Jepson Prairie is within the Cressa truxillensis-Distichlis spicata Herbaceous Alliance (Alkali weed-Salt grass

138 playas and sinks) (Sawyer et al. 2009), the vegetation composition differences between the two sites reflect the higher saline environment at Colusa NWR. Overall species richness (12 at Jepson Prairie Olcott Lake and six at Colusa NWR pools) and species assemblages were different between the reintroduction and introduction pools with only two taxa (non-native Crypsis spp. and native Cressa truxillensis) shared between the pools at the two sites. Of the 12 taxa surveyed in Olcott Lake, only one (Crypsis spp.) was non- native (Table 4-3) whereas at Colusa NWR four of the six taxa surveyed were non-native

(Table 4-4). Also at Colusa NWR, the non-native Crypsis spp. had the highest frequency while native Cressa truxillensis had the highest frequency at Olcott Lake. The percent bare ground was also consistently higher at Colusa NWR introduction pools than at

Olcott Lake. Unlike the newly created introduction pools at Llano Seco, the pools at

Colusa NWR are older, restored in 2001 and 2004, therefore, the pools have had time more time for species colonization and community development. The differences in species richness, native and non-native species, and cover of bare ground likely reflect the higher saline soils at Colusa NWR.

Introductions and Reintroductions

Greene’s Tuctoria

Comparison of Introduction and Reintroduction Pools. Overall, it is noteworthy that there was largely no significant difference between introduction and reintroduction pools for Greene’s tuctoria for the duration of this study. However, when there was a significant difference, the results favored Llano Seco; percent germination was significantly higher in the introduction pools. This suggests that for

139 Greene’s tuctoria, when hydrology matches reference pools, introduction of the species into created vernal pools is possible.

Seed Packets

Percent Germination

The germination at the introduction site is the first step in the possible successful introduction of a population and the results of this study demonstrate that germination at the introduction pools is at least comparable to, if not better than, the reintroduction pools. Percent germination in the seed packets was significantly higher at the introduction pools than the reintroduction pools. Although this is counter to what may be expected, when considering the site conditions in early and late spring when

Greene’s tuctoria is germinating and in the early seedling stage, there are disturbance factors at Vina Plains that may contribute to the lower percent germination.

Seventy-five seed packets or 57.3% were removed from the Vina Plains data set due to being significantly disturbed by wave action, soil movement, or cattle (i.e., covered in soil, undercut, or trampled), compared to only two seed packets removed from the data set at Llano Seco. Moreover, while bendable, the packets work best on relatively even terrain. The bottom of the Llano Seco pools was smooth while the bottom of the Vina Plains pools was generally uneven due to soil movement and cow prints.

In addition to directly affecting germination, the disturbances may have masked evidence of germination by contributing to disintegration of dead seedlings.

There is also the potential that cattle and soil movement could have contributed to the stress if not loss of young seedlings. For all pools, the germination data were collected

140 in July, approximately one to two months after some of the seed germinated (based on pool dry down). If the seed germinated and died, it was often still present as a dead seedling, as observed at Llano Seco. The disturbance and uneven substrate at Vina had a greater potential to mask early germinations and contribute to the loss of young seedlings.

One of the potential disturbance factors is cattle trampling. Since purchased by the Nature Conservancy, there has been a history of cattle grazing at Vina Plains from 1982-1987 and 1995-present (Griggs 2000). Cattle grazing has been shown to be beneficial to the diversity of upland habitat surrounding the vernal pools as well as to vernal pool edges (Marty 2005). However, timing of cattle grazing can be important in management of vernal pool landscapes (Griggs 2000; Keever 2006). Impacts from cattle grazing have been documented for grasses in the Orcuttieae (Griggs 2000; Stone et al.

1987; USFWS 2005) and Greene’s tuctoria appears to be one of the more susceptible rare vernal pool grasses due to its presence near the edges of some of the vernal pools (Stone et al. 1987). The populations of Greene’s tuctoria at Vina Plains are among the largest known populations of the species and the large seed bank likely provides a buffer to some mortality associated with cattle disturbance. However, cattle have historically been shown to damage the Vina Plains populations in some years (Griggs 2000).

During this study, the cattle were removed from Vina Plains in June, thus cattle were present after the pools dried down and during Greene’s tuctoria seedling development. In July at Pool 37, evidence of cattle disturbance (including cow prints and flag munching) was throughout the pool. The seed packet percent germination data collected from this pool and Llano Seco Pool 4 lend support to the hypothesis of lower

141 germination due to cattle disturbance at Vina Plains. For example, for Pool 37, there was

56.6% germination in May and 31.1% germination in July. The difference was not as great at Pool 4 at Llano Seco: 69.6% germination in May and 64.8% germination in July

(Fig. 4-11). The larger difference between May and July percent germination at Vina

Plains suggests that cattle disturbance may have contributed to the lower observed July germination in the reintroduction pools.

This marked difference between May and July data collection likely only occurred for Pool 37. The other two pools at Vina Plains (Pool 22 and Pool 35) were less impacted by cattle than Pool 37. In 2011, Pool 35 dried down in mid-June, which coincided with the removal of the cattle. Pool 22 straddles two pastures with the fence separating the pastures running down the center of the pool. The southern portion of the Pool is surrounded by a dilapidated fence. The reintroduction transects straddled the fence but in the northern portion were in the deeper area of the pool (and therefore less likely to be affected by cattle). Evidence of cattle occurred in both Pools 22 and 35; but to a much less degree than in Pool 37. There were tracks from a few cows that crossed Pool 22 and at the edge of Pool 35, but not throughout as in Pool 37.

Furthermore, of the packet data that were removed due to cattle disturbance (clear evidence of trampling), 27 seed packet cells (16%) were removed from Pool 37 and 14

(7%) from Pool 35, while none were removed from the Pool 22 data set. Due to their later dry down and consequently later Greene’s tuctoria germination along with the less cow disturbance, loss of early germination data due to cattle is likely less at Pools 22 and

35.

142 A second disturbance factor, soil movement in the pools, could be another potential reason for the lower observed germination at Vina Plains. The study pools at

Vina Plains are 0.5 to 1.5 hectares, while the study pools at Llano Seco are 0.1 to 0.2 hectares. In the larger pools at Vina Plains, the northerly winds blow across the pools which contribute to the pools’ dry down and create waves that churn up the soil in the pools. The wind and waves can create foam and uproot seedlings which collect at the edges of the drying pools (personal observation). Since Greene’s tuctoria was observed germinating in a couple centimeters of water, wave action and soil movement when these seedlings are young could uproot or kill them. The majority of the packet data set removed due to disturbance was likely attributable to the soil movement; between approximately 75 to 90% of the packet data removed was due to being covered in soil or undercut (the remaining removal was attributed to obvious cattle disturbance).

In addition to disturbance factors, the number of sampling replicates could be a contributing factor in the differences between introduction and reintroduction pools. As mentioned in the Methods Chapter, the length of the transects was not the same between the pools or between the sites and was based on the size of the pools and the area of the reference populations. In total, originally there were 132 seed packets introduced at Vina Plains and 122 seed packets at Llano Seco. There were a greater number of seed packets removed from the Vina Plains data set due to disturbance. In the end, data were recorded from 56 seed packets at Vina Plains and 120 seed packets at

Llano Seco. As a result, by chance alone the expectation may be for higher percent germination at Llano Seco since a greater number of seeds were used in the analysis.

143 Percent Survivorship to Reproduction

Though not statistically significant, percent survivorship to reproduction (the percentage of the germinated plants that survived to produce an inflorescence) was higher at Vina Plains. If disturbance masked early germination and led to the potential under representation of germination at Vina Plains, this would in turn lead to an over representation of the percent survivorship to reproduction. However, if the observed lower germination at Vina Plains is an actual reflection of lower germination and early seedling mortality at Vina Plains, then the higher percent survivorship at Vina Plains represents survivorship after these early life history stages. Therefore, this suggests that the critical life history stages for Greene’s tuctoria at Vina Plains are germination and young seedling development.

Vigor

Comparing the measurement of vigor (average number of culms per plant), there was no significant difference between reintroduction and introduction pools; however, vigor on average was higher at Llano Seco. This higher average as well as the high variance between pools at Llano Seco (Fig. 4-8c) can be attributed to the significantly greater vigor of Pool 4 compared to the other two pools at Llano Seco (Fig.

4-17c). Without Pool 4, the other two pools are more similar to the vigor of Vina Plains reintroduction plants. The unique attributes of Pool 4 are discussed below under

“Comparison of Pools within Sites.”

Interestingly, a greenhouse study by Strauss (2009) looking at the importance of soil biota in vernal pool restoration determined that biomass and reproduction of hairy Orcutt grass (Orcuttia pilosa) was lower in “live” natural vernal pool soil, despite

144 plants having the highest level of AMF root colonization in this soil treatment. In fact, biomass was higher in all other treatments, including sterile natural vernal pool soil, live created vernal pool soil, and commercial arbuscular mycorrhizal fungi (AMF) soil whereas reproduction was higher in the live created vernal pool soil (Strauss 2009).

Strauss suggested that the AMF from vernal pools may be an antagonistic or parasitic rather than mutualistic relationship with some vernal pool plants. Although unknown, one possible explanation for these results is that initially AMF may have deleterious effects; however, as the pools dry down the AMF may aid the plant with drought tolerance during the harsh summer conditions (Strauss 2009). This result of the AMF study provides a possible hypothesis for the trend in higher average vigor in the introduction pools at Llano Seco.

Inflorescence Packets

There were no significant differences between introduction and reintroduction pools for inflorescence packets average number of germinations per packet, percent reproduction, or average number of culms per plant. Trends were similar to the seed packets except for germination, where in the inflorescence packets the average number of germinations per packet was slightly higher for Vina Plains. Unlike the seed packets, the number of seeds in the inflorescence packets is unknown.

Collecting data on germination was sometimes difficult because it was nearly impossible to tease apart individual plants as they were growing without potentially damaging the plants. As a result, the number of germinations per packet may have been underestimated. This is especially the case for packets with higher germination, as was generally the case at Llano Seco. Therefore, percent germination may be underestimated

145 and percent survivorship overestimated since it was based on germination results. This was not an issue for average number of culms per plant because these data were collected at the end of the season when the plants were dead, so it was possible to pull up the packet and tease apart the plants.

Disturbance factors at Vina Plains also affected inflorescence packets. Sixty- four inflorescence packets (48.9%) were removed from Vina Plains data set due to being significantly disturbed by wave action, soil movement, or cattle (i.e., covered in soil, undercut, or trampled), compared to only six inflorescence packets removed from the data set at Llano Seco. These disturbance factors could also contribute to and mask germination data with resulting higher percent survivorship to reproduction at Vina

Plains.

Comparison of Seed and Inflorescence Packets. There was a significant difference in vigor between packet treatments. Over both introduction and reintroduction pools, seed packets had a significantly greater average number of culms per plant. As the plants in the inflorescence packets were closer together than the seed packets, often growing within millimeters of one another from the same inflorescence, the difference in vigor between the two packet treatments is likely due to higher density and greater intraspecific competition in the inflorescence packet treatment.

Intraspecific competition has been observed in vernal pool annuals (Linhart

1988). As plants grow closer together, they are competing more for resources such as water availability and sunlight. Increased density may result in a decrease in overall dry matter of a population (Donald 1951; Harper 1977). Moreover, it has been shown that as

146 plants grow closer to one another, they are increasingly more affected and stressed by the proximity (Harper 1977; Ross and Harper 1972).

Griggs and Jain (1983) suggested that Greene’s tuctoria was regulated by density dependent mortality because survivorship was lower in areas with higher density. However, a decline in percent survivorship to reproduction in the higher density inflorescence packets was not suggested by the data in this study. The reason there might not be a significant trend in greater seedling mortality in the inflorescence packets may be due to the fact that it was somewhat difficult to capture seedling germination and therefore mortality in the inflorescence packets. In this study, the comparison between packet treatments is more appropriately made with vigor (average number of culms per plant).

Vigor of wild plants was compared to the packet treatments at Vina Plains to determine potential treatment effects. Wild plants had significantly greater vigor (5.8

(±0.3) culms/plant) compared to seed (3.0 (±0.3) culms/plant) and inflorescence (2.6

(±0.2) culms/plant) packet treatments. This suggests that the plants from the packets may have been compromised by growing inside the packets. However, the wild plant vigor at Vina Plains was comparable to the vigor at Llano Seco Pool 4 (average of 6.4 culms per plant for both seed and inflorescence packets), which suggests that it is possible to have higher vigor of plants growing in the packets. The high vigor in Pool 4 may be attributed to optimal hydrologic and soil moisture conditions and is discussed below under “Comparison of Pools within Sites.” These optimal conditions in combination with the lack of disturbance present at Vina Plains may explain why plants from Pool 4 had comparable vigor to wild plants at Vina Plains.

147 This study showed a trend in the number of seeds per inflorescence (as a measure of reproductive output) between the various treatments. Average number of seeds per inflorescence at Vina Plains was similar between wild plants and inflorescence packet plants (9.0 (±1.6) and 9.2 (±0.7), respectively) and higher than seed packet plants

(6.5 (±0.8)). Interestingly, the trend in reproductive output is opposite of vigor in the packet treatments. Seed packet plants showed an increased vigor (number of culms/plant), but trended towards fewer number of seeds per inflorescence. It is important to note that this study measured the number of seeds per inflorescence which is a measure of reproductive output, but it is not a direct measure of seed set

(seed/floret). The following are a couple possible explanations for the difference in reproductive output in the packet treatments.

One possible explanation is pollen limitation. Based on genetic structure and floral morphology, Orcuttieae are thought to be primarily out-crossing species (Gordon et al. 2012; Griggs 1980). However, although not directly studied in Greene’s tuctoria,

Davis et al. (2009) looked at the effects of pollen limitation due to plant density on individual reproductive success of Colusa grass. They demonstrated that increased density of Colusa grass resulted in an increase in seed set (seeds per floret) per inflorescence. In a preliminary observation at Olcott Lake plants at high densities

(“grouped close together”) set nearly twice as much seed as those from low-densities

(“relatively sparse”) (Davis et al. 2009). In a later experiment looking at seed set they found that the number of inflorescences within 0.5 meter directly upwind of the focal plant were the best able to predict seed set in that plant (second best was 0.5 meter in any direction). Therefore, a greater number of plants within 0.5 meter resulted in an

148 increase in seed set. The number of inflorescences (i.e., culms) on the focal plant itself was not significant. The proximity of plant density that Davis et al. (2009) found to be significant is larger than the distances experienced by the plants in the seed or inflorescence packets. Plants within both packet treatments are well within 0.5 meter of one another. Therefore, it is unlikely that the lower reproductive output in the seed packets could be explained solely by pollen limitation.

A second possible explanation is allocation of resources. As the plants in the inflorescence packets had significantly less average number of culms per plant and were smaller plants (less biomass), resources may have been allocated to production of seeds rather than biomass. Likewise, the larger plants in the seed packets may have been allocating more resources to growth of culms and less to seed production. Sugiyama and Bazzaz (1998) showed that under varying nutrient levels and competition there is typically a positive relationship between vegetative growth and reproduction; however, plants from different families demonstrated a negative relationship between vegetative biomass and reproductive biomass when grown under the same conditions. Since this study mixed families across all treatments and did not look at affects of different plant families, the results of this study are not readily explainable. It is unclear whether pollen limitation, allocation of resources, or other mechanisms are responsible for the trends observed in reproductive output between seed and inflorescence packets. Since the trends observed in seed and inflorescence packets were from only two sites, further study is required.

Comparison of Pools within Sites. In natural pools at Vina Plains where

Greene’s tuctoria has been growing for possibly thousands of years or more (Broyles

149 1987; Crampton 1959), there were no significant differences in germination, survivorship, and vigor between the three reintroduction pools (Table 4-5). However, there was greater variation between the newly created introduction pools at Llano Seco where differences between pools were apparent for both seed and inflorescence packets with respect to germination, survivorship, and vigor. These differences may be attributable to the differences in pool depth and therefore duration of standing water. In increasing order of depth and retention of standing water the Llano Seco introduction pools are as follows: Pool 4, Pool 11, and Pool 6 (Appendices A and C). The shallowest pool, Pool 4, had the greatest success with respect to germination, survivorship, and vigor of plants, and the deepest pool, Pool 6, had the least success (Fig. 4-17). The largest difference between the pools was in the vigor of Pool 4 plants which was over

50% greater than the other two introduction pools (Fig. 4-17c). This suggests that suitable growing conditions were present in Pool 4 for a longer duration than the other two introduction pools.

Greene’s tuctoria germinated along the leading edge of the dry down until the pool was dry. The clay soils in vernal pools retained moisture at least during the early stages of seedling development. When the shallower Pool 4 started to dry down and the plants germinated, it was earlier in the season when temperatures were cooler.

As a result, soil moisture persisted longer in Pool 4, leading to longer availability of water to the plants while they were growing and creating culms (as suggested by Griggs

1980). In contrast, by the time the deeper Pool 6 dried down (no more standing water) and plants were germinating in June, it was warmer and presumably the soil dried faster and soil moisture was available to the plants for a shorter duration. Furthermore,

150 greater than average early and late spring rains in 2011 kept the pools full for longer into the spring (Fig. 3-3) which may have contributed to the success in Pool 4 as it extended the growing season into late spring but may have also contributed to the lower performance in Pool 6 as it retained water and extended dry down into the warm early summer months. The hydrologic and soil moisture conditions at Pool 4 in 2011 resulted in high plant vigor for both seed and inflorescence packets.

Second Generation. Despite approximately half the amount of rainfall (Fig.

3-5) and only partial filling of the introduction pools at Llano Seco in 2012, there was a population increase in each of the three introduction pools in the second generation (Fig.

4-22). This demonstrated that the first generation plants produced viable seeds and the created pools at Llano Seco provided suitable habitat for a population to persist for at least two generations in both average and below average rainfall years. At least in the short term of this study, the implications are that the introduction pools selected at

Llano Seco have the ability to support persistent populations of Greene’s tuctoria in varying climate years. An unknown portion of the second generation plants may have germinated from the introduction seeds that didn’t germinate in 2011; however, irrespective of the source and even with interannual variability, conditions in the introduction pools were conducive to persistence and population growth.

The variation between pools observed in the first generation at Llano Seco persisted for the second generation in 2012. This variation was a result of both first generation reproductive output as well as the suitability of pool hydrological conditions during the second generation. The shallower Pool 4 had a considerably higher population increase (900%) than Pools 6 (132%) and 11 (9%). Based on the assumption

151 that a greater number of culms generally represent a greater number of seeds (Griggs

1980), the significantly greater number of first generation culms per plant in Pool 4 contributed to the larger population and population increase in the second generation.

Moreover, the variation between pools was also apparent in the vigor of the second generation plants. Due to the partial pool filling in 2012, there was an overall decrease in the second generation vigor in the introduction pools 4 and 11 compared to

2011 (i.e. more plants but fewer culms) (Fig. 4-23). Pool 6, the deepest pool that retained water the longest and had plants with the lowest vigor in 2011 (an average rainfall year), contained the plants with the greatest vigor in 2012 (a dry year). Presumably soil moisture during culm formation was optimal in deeper Pool 6 in the dry 2012 year

(while it was optimal in shallower Pool 4 in the average rainfall year in 2011). This illustrates that the variation between pools and years at Llano Seco is a factor of both rainfall and pool depth.

From a persistence perspective, smaller populations of Orcuttieae (less than

50-100) are often more susceptible to extirpation (Stone et al. 1987; USFWS 2005).

Moreover, smaller populations are prone to genetic drift and inbreeding depression, which may be especially pertinent in new populations without a seed bank (Elam 1998).

According to Pavlik (1993, 1996), the minimal viable population for annual species is around 1,500 – 2,500. Based on this estimation of minimum viable population size and the second generation data at Llano Seco, the population at Pool 4 (2,010 reproductive plants) is more likely to persist than the populations at Pools 6 (51 reproductive plants) and Pool 11 (111 reproductive plants).

152 The results from this first and second generation study highlight the importance and interaction of pool depth, precipitation, and initial population numbers in introduced populations. For introduced and reintroduced populations, the most suitable pools may differ depending on precipitation amounts and patterns.

Introduction of additional seeds and plant material may provide a buffer to the varying responses to precipitation variation. Collinge (2003) demonstrated that initial introduction numbers had an effect on the abundance of Contra Costa goldfields

(Lasthenia conjugens) introduction populations. However, by the third year there was no significant difference in abundance. A similar trend may play out at Llano Seco, where in the first couple years the larger amount of propagules introduced at Pool 4 factor in to the larger initial abundance. However, over the years, with annual changes in rainfall, the abundances in the Llano Seco introduction pools may not be significantly different.

Colusa Grass

Colusa grass introduction results demonstrated the species ability to germinate in restored pools at Colusa NWR that have similar hydrological patterns as the reference pool (Olcott Lake, Jepson Prairie). However, there was still a trend towards greater germination, survivorship, and reproduction at the reference

(reintroduction) pool. The trend was particularly clear for percent survivorship (39.3% at Olcott Lake, Jepson Prairie and 0.4% at Colusa NWR).

At the Colusa NWR introduction pools, one possible explanation for the lack of survivorship was the higher soil salinity measured as conductivity from a soil-water extract. The pools are mapped as Willows silty clay which is non-saline to moderately saline (2.0 to 15.0 dS/m) (USDA NRCS 2009b). The salinity measurements for this study

153 were within this range at Tract 24.13 (2.9 dS/m) but significantly higher at Tract 24.12

(18.4 dS/m). The reintroduction pool, Olcott Lake, is mapped as Pescadero clay which is very slightly saline to moderately saline (4.0 to 16.0 dS/m) (USDA NRCS 2007). The salinity measurement for Olcott Lake soil (1.9 dS/m) was lower than the soil series range, but is similar to salinity measured at nearby existing pools at Grasslands Park (1.1 to 2.1 dS/m) (Gerlach 2009b, Appendix B).

The negative effects of increased soil salinity have been documented for annual plants in various habitats (Choudhouri 1968; Houle et al. 2001; Noe and Zelder

2000). Increased salinity in soils affects germination and plant growth by (1) increasing osmotic pressure in the soil solution and therefore preventing uptake of water (and essential nutrients) by the seed or seedling and/or (2) increasing salts and ions in plant tissues that may be toxic to the embryo or seedling (Choudhuri 1968; USDA 1954). A soil is considered to be saline if it has an electric conductivity from a saturated soil paste of 4 dS/cm or more (USDA 1954). However, even at 2 dS/cm the USDA handbook for saline and alkaline soils notes that “yields of some sensitive crops may be restricted”

(USDA 1954) and above 16 dS/cm (i.e., Tract 24.12) “only a few very tolerant crops yield satisfactorily” (USDA 1954).

The alkalinity and conductivity in claypan vernal pools along basin rims tend to increase as the pools dry down (Barclay and Knight 1984; Williamson et al. 2005).

Since Colusa grass generally germinates in standing water, the conductivity at the time of germination may be lower than when the pool has dried down and the seedlings are young. This increase may be significant. Conductivity of Olcott Lake water has been measured at 0.33 dS/cm in April (Gerlach 2009b). Assuming the soil salinity at dry

154 down is at a range between 1 to 2 dS/cm (based on this study and results from nearby pools at Grasslands Park), this represents over a doubling of the salinity. Assuming there was a similar increase in salinity at Colusa NWR, an increase in salinity after germination may account for the death of the seedlings in Tracts 24.12 and 25 (east).

The differences in Colusa grass in response to salinity levels were noticeable not only between the reintroduction and introduction pools but also between the two salinity tested introduction pools at Colusa NWR. The pool with the higher salinity

(Tract 24.12) had lower average percent germination and none of the plants survived past production of the coleoptiles or first leaf. However, the pool with the lower salinity

(Tract 24.13) had higher average percent germination, the seedlings survived longer and grew bigger, and it supported the one plant that survived to reproduce. Moreover, the difference between conductivity of Tract 24.13 and Olcott Lake was only 1.0 dS/m, which has less potential to be biologically significant (USDA 1954).

Collinge et al. (2003) conducted an experiment on the germination and growth of the rare vernal pool annual Lasthenia conjugens (Contra Costa goldfields) in response to salinity. They found that an increase in salinity (from 5 ppt to 20 ppt synthetic sea salt in tap water) reduced percent germination in two of the three seed source populations. In the third source population, salinity showed no significant effect.

Plant growth rate and floral initiation from all three populations decreased in treatments with increasing higher salinity (Collinge et al. 2003). In a study of Colusa grass, Hogle

(2002) showed that Colusa grass vigor (number of culms per plant) was inversely correlated with percent sodium in six vernal pools in three vernal pool regions: Solano-

Colusa, San Joaquin, and Southern Sierra Foothills. These other studies support the

155 hypothesis that the high salinity at Colusa NWR affected the survivorship of introduction plants.

The importance of suitable soil moisture in the spring has been proposed as important for Orcuttieae as well as other vernal pool plants (Alexander and Schlising

1997; Collinge et al. 2003; Griggs 1980). Soil moisture was also discussed above as a contributing factor in the variability in Greene’s tuctoria vigor between years and pools at the Llano Seco. Further evidence for the importance of soil moisture in the vigor of

Orcuttieae was observed in Olcott Lake in 2010. Typically Colusa grass plants were the largest (with multiple culms) at the edges of the pool while the deepest areas of the pool typically supported the smallest, often one culm plants (Fig. 4-3). Presumably the plants at the pool edge experienced longer periods of soil moisture since they germinated earlier in the spring under cooler conditions. In the deeper portions of the pool, the plants germinated in the early summer months where warmer, drier conditions may have contributed to less amount and duration of soil moisture.

For reintroduction efforts for Colusa grass, it is important to note that there was high interannual variation between the two areas sampled (north and south transects) at Jepson Prairie. For example, the reintroduction transects were placed in areas that supported Colusa grass in 2010. However, in 2011, while there were still

Colusa grass plants in the area of the northern transects , there were very few Colusa grass plants in the area of the southern transects (Appendix D). The scarcity of Colusa grass in the area of the southern transects was also reflected in the reintroduction packets. There was a significant difference in germination between north and south transects (Fig. 4-30). The low germination and survivorship in the southern transect

156 packets, also reflected in the wild population, suggests that the differences between the two transect areas were due to unsuitable environmental conditions.

Lab Germination

There were no significant differences in Greene’s tuctoria germination between the four treatment substrates in either the seed or inflorescence treatments.

This was similar to Keeley’s (1988) study which also showed no significant differences in germination substrates. It was also consistent with the introduction experiment since

Greene’s tuctoria germinated readily in both the reintroduction and introduction pools.

This suggested that germination of Greene’s tuctoria is not dependent on native vernal pool soil, or soil at all, considering that Greene’s tuctoria also germinated in Petri dishes with water only.

No previous germination studies on Greene’s tuctoria specifically tested differences in stratification temperature. In this study, the Petri dishes were stratified in a refrigerator before placing in a lab near an east facing window. The Petri dishes stratified in the colder 4 degree Celsius treatment had significantly greater germination than the warmer 9 degree Celsius treatment in both seed and inflorescence treatments.

Griggs (1980; 2013 personal communication) germinated Orcuttieae in a “mostly dark” refrigerator at 10 degrees Celsius. However, the germination rate increased when exposed to the light. Strauss (2009) germinated hairy Orcutt grass (Orcuttia pilosa) in an open-air refrigerator at 12 degrees Celsius. Neither germination treatments had prior stratification at colder temperatures. In Keeley’s factorial experiment (1988), he stratified seeds at 5 degrees Celsius prior to placing into an incubator. He found that in

157 the light and under anaerobic conditions, stratification did not have an effect on germination. However, in the dark or in the air, stratification increased germination

(Keeley 1988). Moreover, Keeley’s stratified seeds did not germinate until transferred to incubators. Similarly, in this study, germination did not occur until the Petri dishes were removed from the dark refrigerators and placed near an east-facing window. It is unclear whether it was the light or warmer temperature (or both) that triggered germination. Nevertheless, these results support the idea that Greene’s tuctoria germination is inhibited by the dark and cold temperatures. However, when stratified in the dark, cooler temperatures (4 degrees Celsius) result in higher germination once exposed to the light.

Griggs posited that “naked” seeds (without lemma and palea) could not be germinated and that the seeds germinated after being encompassed by a fungal sheath

(Griggs 1980). Conversely, Keeley (1988) was able to germinate naked seeds. Although this study did not test “naked” seeds, a fungal sheath was observed in most instances prior to germination (Fig. 4-32). If soil fungi spores from the reference pool soil can be transported on inflorescences or simply the lemma and palea, then it is possible that the fungi growing on seeds and inflorescences during germination may be from the reference pool soil.

Percent germination in the lab experiment (8.3% over all treatments) was lower than in the introduction (59.1%) and reintroduction (32.9%) experiments using the same seed. Percent germination in this lab experiment was also lower than in the anaerobic treatments (44.7% over all anaerobic treatments) in Keeley’s study, but approximated percent germination in Keeley’s air treatments (5.8% over all air

158 treatments). Keeley and Busch (1984) suggested that water in the pools was probably not anaerobic, while sub-surface soil conditions may be (Keeley 1988). Therefore, since the seeds were either directly on top of the soil or in the water column, the treatments in this germination experiment did not experience anaerobic conditions. Future studies could continue to test different stratification temperatures as well as test anaerobic conditions such as burying the seed at different depths in the soil.

Summary, Conclusions, and Implications for Conservation

Differences between the Two Study Species

This study demonstrated that germination was possible in introduced

Greene’s tuctoria and Colusa grass populations when introduction pools were selected to match the hydrological dry down patterns of reference pools. However, that’s largely where the similarities ended for the introduction efforts of the two grasses. Although these grasses are members of the same tribe (Orcuttieae) in the grass family (Poaceae), they have unique environmental requirements. Previous studies have also demonstrated the differences between Orcuttieae populations with regard to population dynamics and in response to timing and amount of precipitation (Griggs 1980; Holland

1987).

Differences in the two species were noticeable in the area extent of populations at the reference sites from 2010-2012. The Colusa grass population at Olcott

Lake responded considerably more to dry years and higher than average late spring

(May and June) precipitation by decreasing extent of the population. These differences

159 may suggest that the Colusa grass reference population is more sensitive to climatic fluctuations compared to Greene’s tuctoria reference populations at Vina Plains.

In addition to the reference population trends, Greene’s tuctoria fared significantly better than Colusa grass in the lab germination and introduction studies.

While Colusa grass germinated in both reintroduction and introduction pools, suitable germination conditions were not replicated in the lab. Greene’s tuctoria proved less tricky in the lab and showed no significant differences in germination on different substrates and germinated with two different stratification temperatures. Moreover, at the study sites, percent germination was in fact higher not in the natural habitat

(reintroduction pools) but in the created habitat (introduction pools). The differences between the two species was perhaps most apparent in their differences in survivorship to reproduction. The high survivorship and reproduction of the Greene’s tuctoria introduction populations provide the possibility of population persistence. The lone

Colusa grass plant that survived to reproduce highlights the lack of suitable habitat at

Colusa NWR for seedling survivorship in the first generation let alone for population persistence. Results of this study support the idea that salinity was a limiting factor for

Colusa grass while disturbance (e.g., soil movement and cattle trampling) during the germination and early seedling stage may be a limiting factor for Greene’s tuctoria.

Greene’s Tuctoria

An implication for Greene’s tuctoria introductions or reintroductions is that a significant number of propagules may be lost when exposed to disturbances such as cattle trampling or in large pools (and therefore greater wave action and corresponding soil movement). Particularly in new populations without the buffer of a large seed

160 bank, disturbance to the populations may have a large effect. As a result, when taking these disturbance factors into account, additional propagules may need to be introduced to establish a desired population size.

Long term population monitoring should be implemented at Vina Plains to track trends in population numbers as a result of management practices. This study also supports the idea that cattle should be removed in early May to prevent unnecessary trampling of Greene’s tuctoria seedlings (which was also recommended by Griggs

(2000)). Alternatively or in addition, fencing could be erected around the shallower pools that support rare species (such as Pool 37). Although the established seed bank may act as a buffer to loss of germination due to cattle trampling at Vina Plains, impacts due to management are important to monitor and take into consideration. Especially in pools without an established seed bank (such as in introduced populations), pools should be fenced in cattle grazed pastures to prevent trampling of the rare species including Greene’s tuctoria.

Both seed and inflorescence packet plants had high germination and survivorship to reproduction. Differences in the seed and inflorescence packet treatments for Greene’s tuctoria demonstrate potential benefits of both treatments.

While significantly greater vigor was observed in the seed packets, a trend towards larger reproductive output was shown in the inflorescence packets. Although the cause of the greater reproductive output in the inflorescence packets is unclear, the likely reason for the higher vigor in the seed packets is the lower density and less intraspecific competition.

161 Observations of both treatments (i.e., “strategies”), either germinating separate from or attached to the maternal inflorescence, was observed under natural conditions and in wild populations. During the processing of the inflorescences to remove and count the seeds, the seeds in the terminal florets were relatively easy to remove while the seeds in the basal florets were extremely difficult to remove. As seen at the second generation at Llano Seco, these basal seeds will germinate directly from the maternal plant’s inflorescence (Fig. 4-20). As a result, they grow very close together.

However, plants were also observed growing solitarily at greater distances from the maternal plant. At the reference pools, plants grew solitarily as well as very close together as to appear as one plant (Fig. 4-2). Some Greene’s tuctoria inflorescences were observed shattering (primarily the terminal florets and spikelets) prior to fall precipitation (Fig. 4-1e). This study showed that plants from inflorescence packets

(higher density) trended towards greater reproductive output and the plants in the seed packets (lower density) had higher vigor. In having an inflorescence structure and mechanism that easily sheds some seeds while retaining others, the plant may be maximizing the benefits of both strategies for future generations.

These results suggest the benefit of using both packet treatment strategies in an introduction or reintroduction effort. Due to the time involved in constructing the seed and inflorescence packets, habitat managers may not choose to use packets in introduction or reintroduction efforts. While using whole plants may be preferential with regard to efficiency, the down-side is that it is difficult to track individual plants and calculate percent germination. However, if using whole plants (and not packets), the propagules collected for introduction should include intact inflorescences to ensure

162 collection of both the basal florets that will remain close to the inflorescence as well as the terminal florets that may be the first to shatter. In other words, break up some propagule material and keep some intact to ensure varying plant densities. Doing so will allow for both “strategies” in the introduced population. Future studies could address and help tease a part the mechanism(s) behind the differences in vigor and reproductive output at different densities.

Colusa Grass

The results imply that introduction and reintroduction of Colusa grass in the

Colusa Basin may be hindered in areas of increases soil salinity. Future studies and introduction/reintroduction projects should consider possible limitations in germination, growth, and survivorship of Colusa grass. With this study suggesting that salinity is a strong factor, future introduction efforts should measure soil salinity and other associated soil chemical properties such as alkalinity prior to introduction or reintroduction. Differences in the type of salts (such as sodium chloride (NaCl), sodium sulfite (Na2SO3), and calcium sufate (CaSO4)) may also affect plant response and should be evaluated (USDA 1954). Hydrological patterns alone may not be sufficient in determining suitable habitat in basin rim habitats. Since playa pools in basin rim landscapes are characterized by having elevated salinity and when taking into consideration former agricultural areas which may have elevated salinity (Allison 1964;

Reitz and Haynes 2003; Schoups et al. 2005), it becomes increasingly important to test soil characteristics prior to introducing populations. The potential for introduction of native vernal pool species, including rare grasses, may be limited at Tracts 24 and 25 at

Colusa NWR; however, efforts should continue to be made to reintroduce Colusa grass

163 into Colusa County. Potential opportunities occur at Colusa NWR (excluding Tracts 24 and 25), Delevan NWR, and Sacramento NWR. Prior to reintroductions, hydrology and salinity comparison should be made with reference pools. Hydrologic patterns should match and salinity concentrations should not be greater than the reference pools.

Interannual Variability

Interannual variability is a part of vernal pool habitats (Griggs 1980; Holland

1987). Fluctuations in population numbers and locations within pools are not uncommon and have been documented in the literature (Alexander and Schlising 1997;

Bauder 2005). This study highlighted the ways that variability affects introduction and reintroduction efforts. Reference population locations may vary from year to year within a pool (Appendices B and D). This has implications for introductions because suitable habitat information gathered from reference populations may change from year to year. Moreover, a reference population may be present in a pool one year but not the next (as observed in Vina Plains Pool 21). Interannual variability was also observed in the introduction pools at Llano Seco, where the annual differences in precipitation resulted in differences in the vigor of plants within the introduction pools. These results imply that the ideal pools and location within pools for introduction and reintroductions may vary annually. Ideally, introduction efforts should take variability into consideration by introducing propagules into more than one location within a pool and more than one pool within hydrologically suitable parameters at a site. Furthermore, introducing propagules across a topographic gradient would ensure that they occur within areas of potentially suitable habitat for that year. Future studies should evaluate the trends in interannual variability, including the spatial extent of Colusa grass at

164 Jepson Prairie and the vigor of the introduction plants at Llano Seco with respect to pool depth and hydrology.

Not only does this study have conservation and management implications for the two study species, this study has relevance for recovery of other vernal pool grasses. For example, similar conservation measures could be applied to Solano grass

(Tuctoria mucronata) recovery and reintroduction into Olcott Lake. The above introduction recommendations based on interannual variability could be valuable in being able to capture the suitable hydrology and habitat for Solano grass in the given year of a reintroduction project. Likewise, as suggested for Colusa grass, soil salinity at

Solano grass reintroduction and introduction sites should be tested to assure comparable soil chemistry to reference pools.

Monitoring and Recovery

Implications of the Colusa grass population fluctuations in Olcott Lake at

Jepson Prairie suggest the importance of multi-year evaluation of reference sites prior to introduction or reintroduction efforts. Populations should be monitored before and after introduction/reintroduction for at least two years to capture the interannual variability. Determining the most suitable locations within a pool may take at least a couple years of observing and monitoring reference populations as well as potential target pools.

Determination of successful introductions will require long term monitoring to establish whether the population can withstand not only climatic fluctuations but also changes in vegetation composition and species dynamics. This is particularly relevant for Llano Seco where the pools are relatively young and the vegetation community is

165 likely still in transition. In addition, understanding the species’ life history and how it responds to these changes are critical to making management decisions (Schemske et al.

1994; USFWS 2005). Future studies should include population viability analyses including seed bank studies. Without a seed bank, new populations of vernal pool grasses may be more prone to extinction. Due to the inherent risk associated with introduction of populations into variable habitats, introductions should not be a substitution for conservation. However, in tandem with conservation and as a tool for recovery, introductions and reintroductions are necessary to reestablish extirpated populations of Greene’s tuctoria and Colusa grass in the Sacramento and San Joaquin

Valleys.

The most exciting implication for recovery was the two year successful introduction of Greene’s tuctoria at Llano Seco, the first documented occurrence of

Greene’s tuctoria introduction or reintroduction. Monitoring should continue at Llano

Seco to determine whether the populations continue to reproduce and, if not, implement any necessary management changes. Monitoring was continued in 2013. From

November 2012 to February 2013, cattle were introduced into Tract 17 after fences had been erected to prevent the cattle from entering the introduction pools (USFWS 2012).

The cattle significantly reduced the thatch in the upland areas surrounding the pools.

All three introduction pools filled with water during early spring 2013. An April 2013 visit showed third generation seedlings in all three introduction pools. In Pool 4, the

Greene’s tuctoria plants were starting to produce inflorescences (Fig. 5-1). Based on the results of this study, cattle fences should continue to be placed around the introduction pools at Llano Seco to prevent trampling. Success requires long term persistence and a

166 third year at Llano Seco adds to the evidence that the populations at Llano Seco have a good chance to persist.

Figure 5-1. Third generation Greene’s tuctoria at Llano Seco introduction Pool 4. Photo credit: Erin Gottschalk Fisher.

REFERENCES

REFERENCES

Literature Cited

Albrecht, M.A., E.O. Guerrant, J. Maschinski, K.L. Kennedy. 2011. A long-term view of rare plant introduction. Biological Conservation. Letters to the Editor. 144: 2557- 2558.

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U.S. Fish and Wildlife Service (USFWS). 2007b. Greene’s tuctoria (Tuctoria greenei) Five- year Review: Summary and Evaluation. Sacramento Fish and Wildlife Office, Sacramento, CA.

U.S. Fish and Wildlife Service (USFWS). 2007c. North Central Valley Wildlife Management Area, Llano Seco Unit 2006–2007 Habitat Management Plan. Sacramento National Wildlife Refuge Complex, Willows, CA.

181 U.S. Fish and Wildlife Service (USFWS). 2007d. North Central Valley Wildlife Management Area, Llano Seco Unit, Tract 17 Vernal Pool Restoration. Proposal for CPV Conservation Program/CVPIA Habitat Restoration Program. Sacramento National Wildlife Refuge Complex, Willows, CA.

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Vitousek, P.M., H.A. Mooney, J. Lubchenco, and J.M. Melillo. 1997. Human domination of Earth’s ecosystems. Science 277: 494–499.

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APPENDIX A

Appendix A contains figures showing the 2010 dry down hydrology at the four study sites. Starting in April 2010 for Vina Plains and Llano Seco, and May 2010 for

Jepson Prairie and Colusa NWR, the extent of standing water in the pools was mapped every one to two weeks using a Trimble GPS unit (accuracy potential < 1 meter).

The figures in Appendix A include:

 Vina Plains

o Figure A-1: Study Site

o Figure A-2: Reintroduction Pools 22, 35, and 37

o Figure A-3: Pool 14

o Figure A-4: Pool 21

o Figure A-5: Pool 22

o Figure A-6: Pool 35

o Figure A-7: Pool 36

o Figure A-8: Pool 37

 Llano Seco

o Figure A-9: Study Site

o Figure A-10: Pool 4

o Figure A-11: Pool 6

o Figure A-12: Pool 11

 Jepson Prairie

o Figure A-13: Olcott Lake

 Colusa NWR

o Figure A-14: Study Site

o Figure A-15: Tract 24.12

o Figure A-16: Tract 25 (east)

184

VINA PLAINS

2010

HYDROLOGY

DRY DOWN

186

Figure A-1

187

Figure A-2

188

Figure A-3

189

Figure A-4

190

Figure A-5

191

Figure A-6

192

Figure A-7

193

Figure A-8

LLANO SECO

2010

HYDROLOGY

DRY DOWN

195

Figure A-9

196

Figure A-10

197

Figure A-11

198

Figure A-12

JEPSON PRAIRIE

2010

HYDROLOGY

DRY DOWN

200

Figure A-13

COLUSA NWR

2010

HYDROLOGY

DRY DOWN

202

Figure A-14

203

Figure A-15

204

Figure A-16

APPENDIX B

Appendix B contains figures showing the 2010 reference populations at Vina

Plains and Jepson Prairie. In June and July 2010, within each reference pool, the location of Greene’s tuctoria at Vina Plains and Colusa grass at Jepson Prairie was mapped using a Trimble GPS unit (accuracy potential < 1 meter).

The figures in Appendix B include:

 Vina Plains

o Figure B-1: Pool 14

o Figure B-2: Pool 22

o Figure B-3: Pool 35

o Figure B-4: Pool 36

o Figure B-5: Pool 37

 Jepson Prairie

o Figure B-6: Olcott Lake

206

VINA PLAINS

2010

REFERENCE POPULATIONS

208

Figure B-1

209

Figure B-2

210

Figure B-3

211

Figure B-4

212

Figure B-5

JEPSON PRAIRIE

2010

REFERENCE POPULATIONS

214

Figure B-6

APPENDIX C

Appendix C contains figures showing the 2011 dry down hydrology at the four study sites. Dry down mapping commenced in April at all four study sites. Data was collected every one to two (occasionally three) weeks through June using a Trimble

GPS unit (accuracy potential < 1 meter).

The figures and table in Appendix C include:

o Table C-1: Comparison of 2010 and 2011 approximate final dry down date

 Vina Plains

o Figure C-1: Study Site

o Figure C-2: Reintroduction Pools 22, 35, and 37

o Figure C-3: Pool 14

o Figure C-4: Pool 21

o Figure C-5: Pool 22

o Figure C-6: Pool 35

o Figure C-7: Pool 36

 Llano Seco

o Figure C-8: Study Site

o Figure C-9: Pool 4

o Figure C-10: Pool 6

o Figure C-11: Pool 11

 Jepson Prairie

o Figure C-12: Olcott Lake

 Colusa NWR

o Figure C-13: Study Site

o Figure C-14: Tract 24.12

o Figure C-15: Tract 25 (east)

216 217 Table C-1. Comparison of 2010 and 2011: approximate final dry-down date (first survey date with no standing water) for pools at the four study sites. Pools marked with an asterisk (*) are introduction or reintroduction pools. All six Vina Plains pools support Greene’s tuctoria. Site Pool Approximate Final Dry-Down Date 2010 2011 Vina Plains Pool 14 May 30 May 29 Pool 21 May 30 June 12 Pool 22* ~June 5 May 29 Pool 35* May 30 June 14 Pool 36 May 18 May 3 Pool 37* May 18 April 24 Llano Seco Pools 1, 2, 3, 7, 8, 9 ,10 May 8 < April 22 (Pool 2 April 27) Pool 4* May 26 May 6 Pool 5 June 23 July 9 Pool 6* June 17 ~June 30 Pool 11* June 4 May 31 Jepson Prairie Olcott Lake* June 24-July 1 ~July 3 Colusa NWR Tract 24.12* June 23 July 11 Tract 24.13* May 19 May 13 Tract 25 east* June 18 June 21

VINA PLAINS

2011

HYDROLOGY

DRY DOWN

219

Figure C-1

220

Figure C-2

221

Figure C-3

222

Figure C-4

223

Figure C-5

224

Figure C-6

225

Figure C-7

LLANO SECO

2011

HYDROLOGY

DRY DOWN

227

Figure C-8

228

Figure C-9

229

Figure C-10

230

Figure C-11

JEPSON PRAIRIE

2011

HYDROLOGY

DRY DOWN

232

Figure C-12

COLUSA NWR

2011

HYDROLOGY

DRY DOWN

234

Figure C-13

235

Figure C-14

236

Figure C-15

APPENDIX D

Appendix D contains figures showing the 2011 reference populations at Vina

Plains and Jepson Prairie. In September and November 2011, within each reference pool, the location of Greene’s tuctoria at Vina Plains and Colusa grass at Jepson Prairie was mapped using a Trimble GPS unit (accuracy potential < 1 meter).

The figures in Appendix D include:

 Vina Plains

o Figure D-1: Pool 14

o Figure D-2: Pool 21

o Figure D-3: Pool 22

o Figure D-4: Pool 35

o Figure D-5: Pool 36

o Figure D-6: Pool 37

 Jepson Prairie

o Figure D-7: Olcott Lake

238

VINA PLAINS

2011

REFERENCE POPULATIONS

240

Figure D-1

241

Figure D-2

242

Figure D-3

243

Figure D-4

244

Figure D-5

245

Figure D-6

JEPSON PRAIRIE

2011

REFERENCE POPULATIONS

247

Figure D-7