Pollination Biology of Darlingtonia Californica, the California

POLLINATION BIOLOGY OF DARLINGTONIA CALIFORNICA, THE CALIFORNIA

PITCHER PLANT

George A. Meindl

A Thesis

Presented to

The Faculty of Humboldt State University

In Partial Fulfillment

Of the Requirements for the Degree

Master of Arts

In Natural Resources: Biology

December 2009

POLLINATION BIOLOGY OF DARLINGTONIA CALIFORNICA, THE CALIFORNIA

PITCHER PLANT

George A. Meindl

We certify that we have read this study and that it conforms to acceptable standards of scholarly presentation and is fully acceptable, in scope and quality, as a thesis for the degree of Master of Arts.

Michael R. Mesler, Major Professor Date

John O. Reiss, Committee Member Date

Erik S. Jules, Committee Member Date

Terry W. Henkel, Committee Member Date

Michael R. Mesler, Graduate Coordinator Date

John Lyon, Dean Research & Graduate Studies Date

ABSTRACT

Pollination biology of Darlingtonia californica, the California pitcher plant

George A. Meindl

The flowers of the California pitcher plant, Darlingtonia californica, are unusual and have been the subject of much speculation. Despite the efforts of several workers, the pollination ecology of this plant, including the identities of pollinators, remains enigmatic. Along with determining visitation frequency, this study sought to describe the relative contributions of two documented floral visitors, bees and spiders. Fruit and seed production by emasculated flowers were used to estimate levels of cross-pollination in natural populations of D. californica. Under the assumption that bees predominantly act as cross-pollinators and spiders as self-pollinators, this treatment was used to infer which organisms were responsible for pollen transfer. Levels of pollen-limitation were also assessed at five study sites in northwest California. In order to identify floral visitors, extensive pollinator surveys were conducted. A generalist solitary bee, Andrena nigrihirta, was found to visit D. californica flowers near Scott Mountain and Mt. Eddy,

CA. Despite relatively low visitation rates, individual flowers at all study sites were expected to receive at least one visit by A. nigrihirta. Other regular floral visitors included thrips and several species of spiders. Fruit and seed production by emasculated flowers indicated the occurrence of cross-pollination. Unmanipulated flowers produced more fruits and seeds, on average, compared to emasculated blooms, suggesting that self- pollination contributes to D. californica reproductive success as well. While bees were

iii likely responsible for the majority of cross-pollination, both bees and spiders contributed to autogamous pollen transfer. Following observations of floral visits by A. nigrihirta, it was possible to interpret the functional morphology of D. californica’s flowers. The shape of D. californica’s ovary and petals promote stigma contact both when pollinators enter and exit a flower, contrary to previous thought. This system provides an excellent example of the importance of identifying and observing pollinators in order to truly understand the functional significance of a plant’s floral morphology.

ACKNOWLEDGMENTS

First and foremost, I would like to thank my adviser, Dr. Michael Mesler, whose combination of patience and enthusiasm made this work possible. I am also grateful to the professors who served on my committee: Dr. Erik Jules, Dr. Terry Henkel, and Dr.

John Reiss. Many thanks to Dave Franklin, whose general knowledge contributed much to this thesis. Tim Buonaccorsi and Sasan Hariri-Moghadam were invaluable as research assistants. Without their help I might still be counting Darlingtonia seeds, among other tasks. Figures 2 and 3 were illustrated by Sasan Hariri-Moghadam. Will Goldenberg assisted with video editing. As always, thank you to my family and friends.

TABLE OF CONTENTS

Page

ABSTRACT ...... iii

ACKNOWLEDGMENTS ...... v

LIST OF TABLES ...... vii

INTRODUCTION ...... 1

METHODS AND MATERIALS ...... 5

Study Species ...... 5

Study Sites ...... 6

Pollinator Surveys ...... 9

Analysis of Pollen Loads ...... 10

Pollination Treatments ...... 11

Plant Community Context ...... 12

Statistical Analyses ...... 13

RESULTS ...... 14

Flower Visitors...... 14

Floral Mechanism/ Pollinator Behavior ...... 18

Pollination Treatments ...... 23

Plant/ Insect Community Context ...... 24

DISCUSSION ...... 29

Future Research ...... 33

LITERATURE CITED ...... 35

LIST OF TABLES

Table Page

1 Elevation and geographic coordinates of five study sites...... 7

2 Observed visits to Darlingtonia californica flowers. Aside from the one visit recorded by a European honeybee (Apis mellifera), Andrena nigrihirta was the only bee observed to visit the flowers. Numbers in parentheses indicate the number of visits to different flowers observed during a given time period(s)...... 15

3 Floral visitation rates. The mean number of visits a flower was expected to receive per hour and over its lifetime is presented for each study site. Standard error values for mean visits/hour are given in parentheses for each study site. .... 16

4 Duration of visits to Darlingtonia californica flowers by Andrena nigrihirta. Average visit duration was 2 minutes 8 seconds (SE=12 seconds ...... 17

5 The percentage of examined flowers at each study site that contained one or more of the following: thrips, spiders, and spider webs (either inside or outside the flower). A total of 1125 flowers were individually examined (150 at CL, 225 at SM1, 250 at SM2, 250 at N17, and 250 at DF). “Evidence of Spider” column represents the percentage of examined flowers at each site that had a spider and/or webbing present. Only 3/1125 (0.27%) flowers contained one or more fungus gnats...... 22

6 The percentage of ground covered by Darlingtonia californica and co-flowering plant species. The total percentage of ground covered by angiosperms both within and outside (50m) the seeps are shown in the first two columns. The percentage of ground covered by Darlingtonia californica within the seeps is also presented...... 26

7 Pollinators collected at or near study sites. Order and family is presented for all pollinators (and genus for Hymenopterans). Numbers in parentheses indicate the number of individual pollinators collected from each group ...... 28

vii

LIST OF FIGURES

Figure Page

1 Map of study area. Triangles represent the five study sites. SM1, SM2, and CL were near the summit of Scott Mountain, while N17 and DF were near Mt. Eddy. The gray line separates Siskiyou County and Trinity County...... 8

2 Step by step foraging behavior of A. nigrihirta on a D. californica flower. The bee initially lands on the petals below the windows (a-c) and then enters a window and walks across stigmatic surfaces (d,e). The bee then utilizes the convex portion of one of the five petals to walk onto the ovary and up to collect pollen (f-i). Following pollen collection, the bee uses a petal convexity as before to walk down the ovary, across the stigmas again, and then out one of the windows (j-n). The flower is shown in d-m with one petal removed and half of the two lateral petals removed. F and k show the bee using the convex portion of the petal, which allows the bee to access the stamens...... 20

3 Interior view of a D. californica flower with the lower portion of petals removed. Arrows highlight the distance between the petals and the ovary both immediately above a window (shorter arrow) and in between two adjacent windows (larger arrow). More space is provided for A. nigrihirta in between the windows than above them, which encourages the bee to enter a window and then walk across the stigmatic surfaces. The bee then utilizes the convex portion of the petal opposite the window it entered en route to the flower’s stamens...... 21

4 Fruit and seed production by three treatment groups (emasculated, unmanipulated, and hand-pollinated flowers) at each study site. Top: Fruit set (%) of three treatment groups at each field site. Emasculated flowers produced significantly fewer fruits than unmanipulated flowers. No significant difference was found between fruit production of unmanipulated vs. hand-pollinated flowers (χ2=3.50, p=0.0615). Bottom: Average number of seeds produced per capsule from three treatment groups at each field site. Different letters above bars indicate group means are significantly different...... 27

viii

LIST OF APPENDICES

Appendix Page

A Plant species in coincident bloom with Darlingtonia californica near Scott Mountain and Mt. Eddy, CA...... 39

B Results of percent ground cover analyses. Separate tables are presented for ground cover within versus outside seeps at all five study sites...... 43

INTRODUCTION

The study of interactions between plants and their pollinators can provide adaptive explanations for flower morphology (Fenster et al. 2004, Vogel 2006, Hu et al.

2008, Pauw et al. 2008, Crepet and Niklas 2009). Unless a plant’s effective pollinator is determined, however, the adaptive significance of individual floral traits cannot be fully understood. The flowers of the California pitcher plant, Darlingtonia californica, are unusual and have been the subject of much speculation (Debuhr 1973, Schnell 1976).

For example, some have theorized that D. californica’s bell-shaped ovary serves to limit self-pollination (Schnell 1976), but direct observations of flower handling by floral visitors are lacking. Despite the efforts of several workers, the pollination ecology of this plant, including the identities of pollinators, remains enigmatic (Austin 1875-1877, Elder

1997, Nyoka and Ferguson 1999, Nyoka 2000). Until D. californica’s pollinators are identified and observed, we will not be in a position to interpret the functional morphology of the flowers in a meaningful way.

Though pollinator sightings have been rare, fruit set in natural populations of D. californica has been shown to be quite high, indicating an active pollinator community since the flowers do not routinely self-pollinate without an animal pollen vector (Elder

1997, Nyoka 2000). Based on appearances, the flowers of D. californica seem adapted for pollination by bees. They are large, showy, sweetly fragrant, and produce abundant pollen (Debuhr 1973, Nyoka and Ferguson 1999) – all features commonly associated with mellitophily. In addition, D. californica’s closest relatives, Sarracenia and

2 Heliamphora spp., are pollinated predominantly by bumble bees (Thomas and Cameron

1986, Renner 1989, Ne’eman et. al. 2006), suggesting that bee pollination may be primitive for Sarraceniaceae. Nevertheless, bees have seldom been observed as visitors to D. californica flowers (Austin 1875-1877, Elder 1997, Nyoka and Ferguson 1999,

Nyoka 2000). Spiders, in contrast, are relatively commonly observed on the flowers.

While pollination is generally not a service provided by spiders, recent studies suggest that they may be significant pollinators of D. californica flowers (Nyoka and Ferguson

1999, Nyoka 2000). This interpretation seems problematic considering D. californica’s floral morphology, but it is possible that spiders are currently responsible for the majority of pollination even if flower morphology is the result of past selective pressures applied by bees. If D. californica is pollinated exclusively by spiders, this system would provide another example where floral morphology does not necessarily reflect contemporary pollinator type (Fenster et al. 2004, Zhang et al. 2005, Valdivia and Niemeyer 2006).

Certain spider taxa, including members of the families Clubionidae, Salticidae, and Theridiidae, use the flowers of D. californica as hunting grounds and may act as pollen vectors as they move within flowers constructing webs and stalking prey (Nyoka and Ferguson 1999, Nyoka 2000). By experimentally introducing spiders to bagged flowers, Nyoka (2000) showed that spiders can cause autogamous pollen transfer. She also observed pollen-dusted webbing constructed between adjacent flowers, indicating the potential for xenogamous pollen transfer by spiders as well (Susan Nyoka, personal communication).

3 The evidence for spider pollination, however, is far from conclusive. While

Nyoka (2000) demonstrated that spiders can potentially act as pollen vectors, fruit set by open-pollinated flowers (96%, n=25) was much higher than that resulting from spider introductions (41%, n=22) (Nyoka 2000). Of the flowers that matured fruits, far fewer seeds were produced by spider-pollinated flowers (average=32 seeds/capsule) than open- pollinated flowers (average=673 seeds/capsule) (Nyoka 2000). The discrepancy in reproductive success between spider introduced and open-pollinated flowers suggests that another, more effective pollinator is active among D. californica blooms. Though some spiders supplement their diets with nectar and/or pollen (Smith and Mommsen 1984,

Vogelei and Greissl 1989), ultimately they rely on insect prey to sustain themselves. If spiders feed on potentially effective pollinators, they may reduce seed production more than they contribute to it.

This study sought to address the following questions regarding D. californica’s reproductive biology at five study sites in northern California: (1) Do both bees and spiders contribute to overall reproductive success? (2) Are past interpretations of the functional morphology of floral traits correct, i.e., does the shape of D. californica’s ovary limit self-pollination as has been suggested (Schnell 1976)? (3) Is natural pollination sufficient for effective fruit and seed set, or are plants experiencing pollen- limitation? (4) Do cross-pollination and self-pollination each contribute to natural pollination? In order to identify floral visitors, extensive pollinator surveys were conducted. Once a key floral visitor was identified detailed observations of its flower- handling behavior were made to elucidate whether the shape of D. californica’s flowers

4 serves to limit self-pollination. Levels of pollen-limitation were assessed by comparing fruit and seed production by hand-pollinated flowers with fruit and seed production by unmanipulated flowers. The performance of emasculated flowers was used to estimate levels of cross-pollination.

METHODS AND MATERIALS

Study Species

Darlingtonia californica is endemic to western Oregon and northern California, and has a patchy distribution across its range due to its unique habitat requirements. The species is considered an indicator of serpentine soil and is restricted to perennially wet seeps (Whittaker 1954, Schnell 1976, Juniper et al. 1989). A long-lived perennial, D. californica annually produces rosettes of carnivorous leaves from a creeping rhizome.

Plants often occur in dense patches, which are likely the result of clonal spread by rhizomes and stolons (Schnell 1976).

The solitary flowers begin as upright buds, but become pendant when mature

(Debuhr 1973). The flower-bearing scape generally ranges from 40-60 cm, but may grow as tall as 90 cm (Debuhr 1973). On average, each scape bears nine bracts, all of which have nectar secreting glands (Macfarland 1908). Unlike some confamilial

Sarracenia spp. (Ne’eman et al. 2006), the flowers of D. californica produce no nectar

(Debuhr 1973). Abundant pollen is the only likely reward for pollinators, though a sugar- rich stigmatic exudate may also attract visitors (Nyoka 2000). Five lanceolate-ovate, yellow-green sepals hang loosely around five crimson petals. The five petals almost completely enclose the reproductive whorls, except for windows formed by notches in adjacent petals, which allow access to the flower’s interior. The windows are level with the five stigmatic lobes, a feature that may promote the deposition of outcrossed pollen as

5 6 pollinators initially enter a flower. Twelve-fifteen stamens are located at the base of the ovary. The bell-shaped ovary is flared towards the stigmas, which has been postulated to function to guide pollinators away from the stigmas as they exit a flower and thus limit self-pollination (Schnell 1976). Flowers mature into upright capsules capable of producing around 2000 seeds (Debuhr 1973). The flowers of D. californica are self- compatible, but do not self-pollinate autonomously (Elder 1997, Nyoka 2000).

Study Sites

Five seeps located near Scott Mountain and Mt. Eddy, CA were used in this study

(Figure 1, Table 1). The five study sites will hereafter be referred to as SM1, SM2, CL,

N17, and DF. Distance between sites ranged from ~0.1 to 14.5 km. Near the border of

Trinity and Siskiyou Counties, this portion of the Klamath Bioregion represents the

center of D. californica’s range (Debuhr 1973). Common woody associates adjacent to

seeps include Jeffrey pine (Pinus jeffreyi), huckleberry oak (Quercus vaccinifolia),

western azalea (Rhododendron occidentale), white fir (Abies concolor), red fir (Abies

magnifica) and incense cedar (Calocedrus decurrens). Within the seeps, common

associates include white rushlily (Hastingsia alba), California bog asphodel (Narthecium californicum), Sierra shootingstar (Dodecatheon jeffreyi), marsh marigold (Caltha

leptosepala var. biflora), and Bigelow’s sneezeweed (Helenium bigelovii). Flowering

occurred at all study populations between June 12, 2008 and June 22, 2008, except for

CL where flowering started earlier (June 6, 2008). Study sites were chosen based on

accessibility and population size.

7 Table 1. Elevation and geographic coordinates of five study sites.

Site Elevation Spatial Coordinates

SM1 1635 m. 41° 16’ 25.00” N; 122° 41’ 58.21” W SM2 1630 m. 41° 16’ 38.57” N; 122° 41’ 57.54” W CL 1693 m. 41° 18’ 01.49” N; 122° 40’ 59.90” W N17 1945 m. 41° 20‘ 08.05“ N; 122° 31‘ 41.53“ W DF 2001 m. 41° 20’ 09.13” N; 122° 31‘ 11.39“ W

Figure 1. Map of study area. Triangles represent the five study sites. SM1, SM2, and CL were near the summit of Scott Mountain, while N17 and DF were near Mt. Eddy. The gray line separates Siskiyou County and Trinity County.

Pollinator Surveys

Three observation points were established in each seep in order to monitor

pollinator activity. At these points a series of timed (15 minute) surveys were conducted,

focusing on 13-17 flowers. I sat quietly and motionless while observing the flowers in an

effort not to disturb active pollinators or alter their behavior. Ten 15-minute surveys (2.5

hours total) were conducted during each trip to a field site. Most surveys were made

between 10:00 a.m. and 6:00 p.m., but several were made in the early morning and late

evening. In total, 57.5 hours were devoted to surveys between June 6, 2008 and July 3,

2008. A subset of pollinator visits was timed and filmed with a digital camera. Timing

began when a pollinator entered one of the windows, and ended when it exited a window.

Videos were used to provide detailed accounts of insect visitation.

Mean flower visitation rates (visits/flower/hour) were calculated for each study site. The expected number of visits a flower received over its lifetime was estimated by

multiplying the flowering period (in days) by the number of hours in a day pollinators

were active (six hrs.) by the visits/hour calculated for each site. D. californica pollinators

were considered to be active for six hours a day because all visits occurred between 10:30

a.m. and 4:30 p.m. Flower lifespan was determined by monitoring the development of 30 tagged buds at each study site. The flowers were synchronously bisexual (i.e., stigma

receptivity and anther dehiscence occurred simultaneously). Flowers were deemed

mature when stigmas were receptive (exudates present) and anthers were dehiscing

10 pollen and were considered past maturity when stigmas withered and ovaries began to swell and turn upright. By July 1, 2008, flowers were developing fruits at all sites.

Tagged flowers were considered receptive at CL between June 6, 2008 and June 22, 2008

(17 days), while tagged flowers were receptive at SM1, SM2, N17, and DF between June

12, 2008 and June 22, 2008 (11 days). Visitation rates were calculated for each site independently.

Following each 15 minute census period, five flowers were carefully examined by spreading apart the sepals and petals to check for animals already present within the flowers. A total of 1125 flowers were inspected in this way for spiders, spider webs

(either inside or outside the flower), fungus gnats, and thrips. Insects were captured by aerial netting or by hand, and identified.

Pollinator surveys were also conducted on concurrently flowering plant species.

Approximately 10 hours were devoted to collecting and describing the local pollinator community. Surveys were conducted by walking in and around seeps and stopping periodically to observe and collect pollinators on concurrently blooming plants.

Analysis of Pollen Loads

Each insect collected during surveys that carried a visible pollen load was

systematically dabbed with a small cube of glycerin jelly containing basic fuchsin stain

(Kearns and Inouye 1993). Following pollen removal, the jelly was placed on a

microscope slide, melted and covered with a cover slip for analysis. Pollen grains were

identified by comparing them to a series of slides prepared from flowers at each site.

11 Pollination Treatments

In order to test the assumption that fruit and seed set in natural populations of D. californica are high, 30 unmanipulated flowers at each site were monitored through their development. Because flowers do not autonomously self-pollinate (Elder 1997, Nyoka

2000), pollinator activity can be estimated based on the reproductive success of unmanipulated flowers. Fruit and seed set resulting from unmanipulated flowers were also compared against that of an equal number of hand-pollinated flowers to determine if plants were experiencing pollen-limitation. If there is no difference in fruit and seed set between these two treatment groups then we can conclude that natural pollination is sufficient, i.e., plants were not pollen-limited. Following the appearance of stigmatic exudates, supplemental pollen was applied twice (separated by one week) to 30 flowers at each site by rubbing two-three mature anthers directly against stigmatic surfaces. Pollen used for hand-pollinations was collected from flowers at least five m away in the same population.

To gauge the relative importance of spiders versus bees as pollinators, 30 flowers in each seep were emasculated prior to maturity. Bees, which tend to visit multiple flowers on any given foraging bout, would likely promote xenogamous pollen transfer for

D. californica (though depending on their foraging behavior and flower handling, bees may cause some level of autogamy as well). If past interpretations of the functional morphology of flowers hold true (i.e., if ovary shape limits insect mediated self- pollination) then bees may contribute minimally to autogamous pollen transfer. Spiders, however, tend to remain resident within single flowers for longer periods of time and

12 would likely contribute primarily to autogamous pollen transfer. Any seeds produced in the emasculated treatment group must be the result of cross-pollination, thus levels of cross-pollination and self-pollination can be estimated by comparing the seed set of the emasculated flowers with the seed set of unmanipulated flowers. Under the assumption that bees would act mainly as cross-pollinators and spiders as self-pollinators, this comparison was also intended to show what organisms were likely responsible for pollen transfer.

A total of 450 flowers were used for fruit and seed set experiments, with 150 flowers in each of the three treatments: hand-pollinated, emasculated, and unmanipulated.

These treatments were spread equally across the five study sites (i.e., 90 flowers at each site in three treatment groups of 30). Once fruit maturation began, all treatment flowers were bagged with Reemay® to ensure seeds were not lost when capsules began to dehisce. This proved to be an unnecessary precaution since all fruits were collected prior to dehiscence. Fruit set was determined for each site, as well as the number of seeds produced by each capsule.

Plant Community Context

To characterize floral resources available to pollinators that were active during the blooming period of D. californica, all concurrently blooming plant species were identified in and around the field sites (within 150 m). Due to the close proximity of

SM1 and SM2, all coincidentally blooming plants discovered at either site were

13 considered to be present at both sites. In order to quantify floral resources provided by these plants, percent ground cover was determined in eight m x eight m plots both within the seeps, as well as 50 m outside each seep in the surrounding uplands. Five eight m transects, separated by two m from each other, were laid down to establish the plots.

Plots within the seeps were chosen haphazardly, while the corresponding upland plots were placed 50 m outside each seep at a right angle to the center of the plot within the seep. Within each eight m x eight m plot, 25 0.5 m x 0.5 m subplots were used to describe percent ground cover of all coincidentally blooming species. A frame constructed of PVC pipe with a wire grid (100 squares) was laid over the vegetation and used to estimate ground cover on a scale of 0-100% for each species in bloom.

Statistical Analyses

Log linear analysis was used to compare the fruit set of the three experimental

treatment groups, with treatment, site and the interaction term included in the model. A

two-way ANOVA was used to compare seed set across all sites, with treatment and site

as the independent variables. Due to a significant interaction term from the two-way

ANOVA (p=0.041), separate one-way ANOVAs were run for each site independently

using treatment as the independent variable. A multiple comparison of means was also

completed, using a Bonferroni adjustment. A Kruskal-Wallis one-way analysis of

variance was used to compare visitation rates across all sites. All statistical analyses

were performed using NCSS (Hintze 2004).

RESULTS

Flower Visitors

Over 57.5 hours of surveys, only 38 visits by flying pollinators were observed: 37

by a solitary bee, Andrena nigrihirta, and one by a European honeybee (Apis mellifera)

(Table 2). Estimated visit rates varied from 0.077 (SE=0.025) visits/flower/hour at N17 to 0.016 (SE=0.029) visits/flower/hour at CL, which correspond to 5.08 and 1.60 visits/flower’s lifetime, respectively (Table 3). Visit rates, however, were not significantly different across all five sites (Kruskal-Wallis χ2=5.72, p=0.22). A. nigrihirta

stayed in individual flowers for extended time periods. Out of 14 timed visits, A.

nigrihirta foraged on a single D. californica flower for an average of approximately two

minutes and eight seconds (128 seconds +/- 12 seconds; Table 4). Eight individuals were

collected immediately following visits, and all carried D. californica pollen on their scopae. Of these, six carried D. californica pollen exclusively while two carried heterospecific pollen as well (Asteraceae). A. nigrihirta was the only pollinator collected found to carry the pollen of D. californica.

Spiders were common on flowers at all five study sites, and were active at all hours of the day. Whereas 26.2% of all flowers examined contained a spider, 64.8% showed evidence of spider occupancy (webbing and/or spider present). Thrips were also present in large numbers at all five sites: 48.4% of flowers contained thrips actively foraging for pollen (Table 5). Fungus gnats, while frequently encountered in the seeps, were only observed within D. californica flowers three times.

14 15

Table 2. Observed visits to Darlingtonia californica flowers. Aside from the one visit recorded by a European honeybee (Apis mellifera), Andrena nigrihirta was the only bee observed to visit the flowers. Numbers in parentheses indicate the number of visits to different flowers observed during a given time period(s).

Visitor Site Date Time Period(s) Observed Apis mellifera SM2 6/12/08 4:34-4:49 p.m. Andrena nigrihirta DF 6/13/08 12:07-12:22 p.m. A. nigrihirta (4) N17 6/13/08 1:58-2:13 p.m. A. nigrihirta (3) SM2 6/14/08 10:30-10:45 a.m., 10:48-11:03 a.m. A. nigrihirta (2) CL 6/14/08 2:24-2:39 p.m. A. nigrihirta (3) DF 6/15/08 11:20-11:35 a.m., 11:41-11:56 a.m. A. nigrihirta (5) SM2 6/19/08 12:32-12:47 p.m., 1:45-2:00 p.m. A. nigrihirta (2) SM1 6/19/08 4:24-4:39 p.m. A. nigrihirta (5) N17 6/20/08 11:18-11:33 a.m., 12:12-12:27 p.m., 12:28-12:43 p.m. A. nigrihirta DF 6/20/08 2:20-2:35 p.m. A. nigrihirta (2) SM2 6/21/08 3:34-3:49 p.m., 3:50-4:05 p.m. A. nigrihirta (5) DF 6/22/08 10:51-11:06 a.m., 11:57-12:12 p.m., 12:13-12:28 p.m. A. nigrihirta (2) N17 6/22/08 1:17-1:32 p.m., 1:49-2:04 p.m. A. nigrihirta N17 7/2/08 11:33-11:48 a.m. A. nigrihirta SM1 7/3/08 11:55-12:10 p.m.

Table 3. Floral visitation rates. The mean number of visits a flower was expected to receive per hour and over its lifetime is presented for each study site. Standard error values for mean visits/hour are given in parentheses for each study site.

Site Visits/Hour Visits/Lifetime CL 0.016 (0.029) 1.60

SM1 0.041 (0.042) 2.71

SM2 0.073 (0.025) 4.84

N17 0.077 (0.025) 5.08

DF 0.067 (0.025) 4.42

Table 4. Duration of visits to Darlingtonia californica flowers by Andrena nigrihirta. Average visit duration was 2 minutes 8 seconds (SE=12 seconds

Site Date Time Period Observed Duration of Visit CL 6/14/08 2:24-2:39 p.m. 1 min. 54 sec. DF 6/15/08 11:41-11:56 a.m. 2 min. 40 sec. SM2 6/19/08 12:32-12:47 p.m. 1 min. 15 sec. SM2 6/19/08 1:45-2:00 p.m. 2 min. 20 sec. SM1 6/19/08 4:24-4:39 p.m. 3 min. 4 sec. N17 6/20/08 11:18-11:33 a.m. 2 min. 15 sec. N17 6/20/08 12:12-12:27 p.m. 1 min. 49 sec. DF 6/20/08 2:20-2:35 p.m. 2 min. 33 sec. SM2 6/21/08 3:34-3:49 p.m. 1 min. 25 sec. DF 6/22/08 10:51-11:06 a.m. 3 min. 40 sec. DF 6/22/08 10:51-11:06 a.m. 2 min. 15 sec. DF 6/22/08 11:57-12:12 p.m. 2 min. 24 sec. N17 6/22/08 1:17-1:32 p.m. 1 min. 30 sec. N17 6/22/08 1:49-2:04 p.m. 50 sec.

18 Floral Mechanism/ Pollinator Behavior

Detailed observations of visits by A. nigrihirta revealed that the ovary shape of D. californica promotes stigma contact by bees both as they enter and exit a flower (Figure

2). Immediately above the windows (towards the morphological base of the pendant flower), the petals overlap and the underlying petal is appressed to the flared portion of the ovary, which limits the ability of a pollinator the size of A. nigrihirta to enter a window and crawl directly up onto the ovary on its way to collect pollen (Figure 3). In between the windows, however, the petals bulge outward (Figure 3), and it is this space that A. nigrihirta utilized to ascend the ovary to reach the stamens. This convex portion of each of the five petals is located directly opposite each of the five windows, such that a pollinator enters a window and walks in a straight line across the stigmas and then onto the ovary (directed by the convex portion of the petal). The shape of D. californica’s ovary has previously been thought to guide an insect pollinator away from the receptive stigmatic surfaces as it exits the flower, thus preventing self-pollination. However, in exiting the flower, the pollinator was observed to leave in the same fashion as it entered

(guided by petal convexities across the stigmas and out one of the windows, thus likely effecting autogamy). This behavior was exhibited by multiple individuals, and was consistent at all sites.

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i j k l

m n o p

20 Figure 2. Step by step foraging behavior of A. nigrihirta on a D. californica flower. The bee initially lands on the petals below the windows (a-c) and then enters a window and walks across stigmatic surfaces (d,e). The bee then utilizes the convex portion of one of the five petals to walk onto the ovary and up to collect pollen (f-i). Following pollen collection, the bee uses a petal convexity as before to walk down the ovary, across the stigmas again, and then out one of the windows (j-n). The flower is shown in d-m with one petal removed and half of the two lateral petals removed. F and k show the bee using the convex portion of the petal, which allows the bee to access the stamens.

Figure 3. Interior view of a D. californica flower with the lower portion of petals removed. Arrows highlight the distance between the petals and the ovary both immediately above a window (shorter arrow) and in between two adjacent windows (larger arrow). More space is provided for A. nigrihirta in between the windows than above them, which encourages the bee to enter a window and then walk across the stigmatic surfaces. The bee then utilizes the convex portion of the petal opposite the window it entered en route to the flower’s stamens.

22 Table 5. The percentage of examined flowers at each study site that contained one or more of the following: thrips, spiders, and spider webs (either inside or outside the flower). A total of 1125 flowers were individually examined (150 at CL, 225 at SM1, 250 at SM2, 250 at N17, and 250 at DF). “Evidence of Spider” column represents the percentage of examined flowers at each site that had a spider and/or webbing present. Only 3/1125 (0.27%) flowers contained one or more fungus gnats.

Site Web Outside Flw. Web Inside Flw. Spider Present Evidence of Spider Thrips

CL 38.7 13.3 16.7 48.7 25.3

SM1 48.9 39.6 20 61.8 40.9

SM2 47.2 30.4 24.8 57.2 31.6

N17 69.6 20 34 74 75.6

DF 68.8 30.8 31.2 75.6 58.4

TOTAL 56.2 27.7 26.2 64.8 48.4

23 Pollination Treatments

Both fruit and seed production varied depending on treatment. Fruit set was

significantly affected by floral treatment (χ2=37.69, p<<0.01) and treatment effects were

similar across all sites (χ2=0.85, p=0.99). Differences in mean seed production between the three treatment groups were also evident at all five sites (SM1: F2, 63=65.35, p<<0.01;

SM2: F2, 65=53.76, p<<0.01; CL: F2, 59=52.49, p<<0.01; N17: F2, 58=46.31, p<<0.01; DF:

F2, 57=24.19, p<<0.01; Fig. 3).

Fruit set of D. californica populations near Scott Mountain and Mt. Eddy, CA was

high. Fruit production by unmanipulated flowers (76%) was marginally comparable to

that of hand-pollinated flowers (96%) (χ2=3.50, p=0.0615). Though fruit set by

unmanipulated flowers did not indicate significant pollen-limitation, seed production did.

Hand-pollinated flowers produced significantly more seeds than unmanipulated flowers

at each of the five study sites (Fig. 3), indicating that plants in all five populations

experienced pollen-limitation. On average, unmanipulated flowers produced 674

(n=114) seeds per capsule, versus 1422 (n=144) by hand-pollinated flowers and 439

(n=59) by emasculated flowers.

Self- and cross-pollination both contribute to D. californica reproductive success.

Emasculated flowers produced fruit and seed at all five sites, indicating that cross-

pollination occurred. However, overall fruit set of emasculated flowers (39%) was

significantly lower than that of unmanipulated flowers (76%) (χ2=17.79, p<0.001),

highlighting the importance of autogamous pollen transfer for fruit production.

24 Unmanipulated flowers produced significantly more seeds, on average, than emasculated flowers at SM1 and SM2, but there was no significant difference found between these two treatment groups at the remaining three sites (Fig. 3). Average seed production by unmanipulated flowers was always higher than that of emasculated flowers, regardless of statistical significance, suggesting that cross-pollination cannot account for all of the seeds that were produced by unmanipulated flowers. Therefore, fruit and seed production of naturally pollinated flowers were the result of both autogamous and xenogamous pollen transfer.

Plant/ Insect Community Context

Plant communities in and around D. californica seeps were diverse. A total of 51

angiosperm species, all with blooming periods that at least partially overlapped that of D.

californica, were found at the study sites (Appendix 1). The plant communities at all five

sites were similar, with approximately 1/3 of all observed taxa found at every site. 42

concurrently blooming species were found at SM1 and SM2 combined, 35 at DF, 32 at

N17, and 27 at CL.

Species richness and percent ground cover were greater in the mesic areas within

the seeps compared to adjacent xeric habitats (Table 6, Appendix 2). In the seeps, D.

californica covered 31.7% of the ground and concurrently blooming plants covered

34.1% of the ground. In the more xeric areas surrounding the seeps, an average of 14.5%

of the ground was occupied by coflowering species. As judged by cover values, more

floral resources were available within the seeps than outside them.

25 Eighty eight pollinators (including the eight A. nigrihirta individuals collected after visits to D. californica flowers) were collected from the community, representing four orders of insects (Coleoptera, Diptera, Hymenoptera, and Lepidoptera) (Table 8).

Hymenopterans were the most abundant insect floral visitors, representing 62% of all collected insect pollinators (n=49). Mason bees (Osmia spp.) were the most frequently collected pollinator (n=23), followed by andrenid bees (Andrena spp., n=17) and bumblebees (Bombus spp., n=9). A. nigrihirta was not collected on any plant other than

D. californica, but one individual was collected in flight (i.e., not on a flower) that carried

D. californica pollen.

26 Table 6. The percentage of ground covered by Darlingtonia californica and coflowering plant species. The total percentage of ground covered by angiosperms both within and outside (50m) the seeps are shown in the first two columns. The percentage of ground covered by Darlingtonia californica within the seeps is also presented.

Site % cover within % cover outside % D. californica cover within

SM1 49.4 35.9 17.8

SM2 78.2 8.8 38.3

CL 63.6 5.4 34.7

DF 66.6 7.2 42.4

N17 71.2 15.2 25.4

TOTAL 65.8 14.5 31.7

Emasculated Unmanipulated Hand-Pollinated

1 0.9 0.8 0.7 ) 0.6 0.5 0.4 Fruit set(% Fruit 0.3 0.2 0.1 0 SM1 SM2 N17 CL DF

Emas c ulated Unmanipulated Hand-Pollinated 1800 b c 1500 b c b

1200

b 900 b a a a a a 600 a a a

300 Average # of seeds per fruit

0 SM1 SM2 N17 CL DF

Figure 4. Fruit and seed production by three treatment groups (emasculated, unmanipulated, and hand-pollinated flowers) at each study site. Top: Fruit set (%) of three treatment groups at each field site. Emasculated flowers produced significantly fewer fruits than unmanipulated flowers. No significant difference was found between fruit production of unmanipulated vs. hand- pollinated flowers (χ2=3.50, p=0.0615). Bottom: Average number of seeds produced per capsule from three treatment groups at each field site. Different letters above bars indicate group means are significantly different.

28 Table 7. Pollinators collected at or near study sites. Order and family is presented for all pollinators (and genus for Hymenopterans). Numbers in parentheses indicate the number of individual pollinators collected from each group

Diptera (18) Anthomyiidae (1) Asilidae (1) Bombyliidae (6) Callophoridae (1) Muscidae (1) Syrphidae (8) Colleoptera (5) Hymenoptera (58) Andrenidae Andrena nigrihirta (9) Other Andrena spp. (7) Apidae Apis (1) Bombus (9) Colletidae Hylaeus (1) Halictidae Agapostemon (2) Lasioglossum (4) Megachilidae Osmia (23) Vespidae (2) Lepidoptera (7)

DISCUSSION

Near the summits of Scott Mountain and Mount Eddy, CA, populations of

Darlingtonia californica are pollinated by the solitary bee Andrena nigrihirta, with additional pollination likely provided by spiders and small insects. Direct observations of floral visits and the results of pollination treatments suggest pollination by both bees and spiders. The success of emasculated flowers indicated that cross-pollination occurred at all sites, implicating bees as pollen vectors. However, considering that average fruit and seed production was always higher in unmanipulated versus emasculated flowers, some level of autogamy must have occurred at all sites as well. Based on observations of visits by bees and spiders, both organisms have the potential to effect self-pollination. Because both self-pollination and cross-pollination contributed to fruit and seed production, both spiders and bees likely contributed to D. californica reproductive success.

Both fruit and seed production by emasculated flowers implicate bees as pollen vectors. However, because bees contact stigmatic surfaces when they exit a flower, they have the ability to effect self-pollination and likely contributed to autogamous pollen transfer as well. In light of this, the great majority of pollination may be mediated exclusively by A. nigrihirta. Spiders, which were highly abundant on flowers and have already been shown to effect autogamy in D. californica (Nyoka 2000), likely further contributed to fruit and seed production via self-pollination. Spiders were constantly seen patrolling and building webs on flowers, and in several instances webs were

29 30 constructed within flowers linking anthers and stigmas and were completely dusted with pollen, indicating spider facilitated pollen movement within flowers. Thrips, which also tend to remain resident within single flowers, were also present in large numbers and were observed within D. californica flowers at all sites. Though fungus gnats do not appear to be major pollinators near Scott Mountain and Mt. Eddy, CA, Nyoka and

Ferguson (1999) collected fungus gnats in southwestern Oregon that carried the pollen of

D. californica. Repeated floral visits by small insects like thrips and fungus gnats have been shown to contribute substantially to fruit set in other angiosperms (Mesler et al.

1980, Zamora 1999), and may contribute to seed set in D. californica as well.

The lifespan of D. californica flowers is much longer than most temperate zone flowering plants, and near sea level D. californica populations may bloom for as long as

48 days (Debuhr 1973). Assuming an active pollinator community, extended flower longevity will result in an increase in pollinator visits (Primack 1985, Ashman and

Schoen 1994, Rathke 2003). The estimated visitation rates from this study show that visits to D. californica flowers by its main insect pollinator, A. nigrihirta, are relatively few. Despite low visit rates, flowers are still likely to receive at least one visit by A. nigrihirta due to the flowers’ prolonged lifespans. A long blooming period not only promotes visits by effective cross pollinators like bees, but also allows for repeated visits by less effective pollinators like spiders and thrips. The longevity of D. californica flowers may be an adaptation to deal with an unpredictable pollinator community and favors pollination by several groups of organisms.

31 Plant species with overlapping flowering periods may either facilitate each other’s pollination (Gross et al. 2000, Bruno et al. 2003, Moeller 2004) or inhibit it through competition for pollinator services (Vamosi et al. 2006). Darlingtonia californica plants at all five sites showed evidence of pollen-limitation of seed production. Due to the abundance of heterospecific taxa with overlapping flowering times and the scarcity of large bodied pollinators found to carry the pollen of D. californica, it is likely that these populations are experiencing a reduction in seed set due to competition for floral visitors.

Other than spiders and thrips, A. nigrihirta was the only pollinator seen to consistently visit D. californica flowers, and was the only insect taxon collected found to carry the pollen of D. californica.

How close is the relationship between D. californica and A. nigrihirta? Though many species of Andrena are specialists (Larsson 2005, Diamond et al. 2006), only utilizing the pollen from particular groups of angiosperms, many are also generalist foragers using a wide range of pollen hosts (Nyoka 2000). Across its range, which spans

North America and greatly exceeds that of D. californica, A. nigrihirta is a generalist that has been observed to visit flowers from a diverse array of plants, including members of

Portulacaceae (Motten et al. 1982), Fabaceae (Tepedino et al. 1995), and Ericaceae

(Barry Rice, personal communication), along with D. californica. Three individuals of A. nigrihirta were collected that carried both D. californica and Asteraceae pollen, indicating that A. nigrihirta is utilizing floral resources from multiple species of flowering plants. As the general biology and life history of A. nigrihirta is still poorly described, future studies are needed to identify what floral resources are used by A.

32 nigrihirta and how much these bees rely on resources provided by D. californica in the

Pacific Northwest. Though D. californica is not considered threatened or endangered by state or federal government, the California Native Plant Society has placed it on their watch list of species with limited distributions (Skinner and Pavlik 1994). Because D. californica may rely on A. nigrihirta for reproductive success, both organisms should be considered in management efforts aimed at conserving present D. californica populations.

Pollinator communities have been known to fluctuate in composition and abundance from year to year (Wolfe 1988, Price et al. 2005), and past researchers trying to determine the pollinators of D. californica may have inadvertently chosen time periods when bees were largely absent. Others have witnessed visitation of D. californica by A. nigrihirta as well, but only on a single day over the course of nine years observing the flowers (Barry Rice, personal communication). Whether there is synchronicity between the emergence of A. nigrihirta and the flowering period of D. californica throughout its range needs to be determined. For D. californica populations near sea level, A. nigrihirta may only be active for a fraction of the time D. californica is in bloom. Also, there may be geographic regions where D. californica populations exist, but those of A. nigrihirta do not. Either of these possibilities could potentially hinder efforts to witness floral visitation. Alternatively, the foraging behavior of A. nigrihirta on D. californica flowers may have limited prior pollinator sightings. After entering a D. californica flower the bee is out of sight until emerging up to four minutes later. Therefore, if the initial

33 approach of the bee is not witnessed, an observer may never realize a flower had been entered.

The bell-shaped ovary of D. californica was previously thought to prevent self- pollination from occurring by guiding an insect pollinator away from stigmatic surfaces as it exits a flower. However, following observations of visits by A. nigrihirta, it was determined that the shape of the ovary actually promotes stigma contact both when bees enter and exit flowers. This system provides an excellent example of the importance of identifying and observing pollinators in order to truly understand the functional significance of a plant’s floral morphology.

Future Research

While the present study sought to estimate the relative contributions of spiders vs.

other pollinators to D. californica seed set, these contributions were not directly

quantified. To better understand the importance of spiders as pollinators, future studies

should develop spider exclusion treatments that would prevent spiders from colonizing

flowers. By comparing the seed set of open-pollinated flowers with those that exclude the presence of spiders, the contribution of spiders to seed and fruit production would become evident. Spider exclusions may prove to be difficult, however. Not only can some spiders jump relatively great distances (e.g. salticid spiders), but smaller spiders can also “fly” by releasing a silken strand into the wind, which may then carry them to a new location (potentially a D. californica flower). Therefore, simply coating the scape with a

34 sticky substance to limit cursorial colonization of flowers may not provide an adequate barrier to all spiders. Further development of this method is needed.

There are a number of interesting ecological questions that have yet to be addressed regarding D. californica pollination. For instance, how do spiders occupying

D. californica flowers interact with bees? Does the presence of spiders within a flower deter visitation by bees, or do bees frequently fall victim to lurking spiders, and what bearing does this have on D. californica reproductive success? Over the course of floral observations conducted in this study, A. nigrihirta was seen “buzzing” flowers, i.e. approaching flowers but not entering them, more frequently than entering flowers (37 flowers visited, 50 flowers buzzed). This behavior could be the result of floral marking by bees, which may be done to alert future visitors of resource availability (Schmitt and

Bertsch 1990, Goulson et al. 2001), but may also be the result of altered foraging behavior due to the presence of flower-occupying spiders (Bruce et al. 2005, Goncalves-

Souza 2008). Also, do the same floral traits that promote pollen deposition on stigmatic surfaces by bees (shape of ovary, etc.) also promote pollen deposition by spiders, or should we expect divergence of floral morphology in D. californica populations that occur in areas where A. nigrihirta is absent over time? As we seek to explain the adaptive significance of D. californica’s floral traits, we need to understand, in greater detail, the effects of these multi-species interactions on trait selection.

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Appendix A. Plant species in coincident bloom with Darlingtonia californica near Scott Mountain and Mt. Eddy, CA.

Species Name Family Habitat Flw. First Obs. Fruit First Obs. Sites Found CL, SM1, Lomatium sp. Apiaceae xeric 6/22/2008 7/12/2008 SM2 SM1, SM2, Achillea CL, DF, millefolium Asteraceae mesic 6/19/2008 7/12/2008 N17 SM1,SM2, N17,DF Aster alpigenus Asteraceae mesic 6/20/2008 7/1/2008 Eriophyllum lanatum Asteraceae xeric 6/22/2008 7/12/2008 N17, DF Helenium SM1, SM2, bigelovii Asteraceae mesic 6/22/2008 7/12/2008 CL Senecio integerrimus SM1,SM2, var.exaltatus Asteraceae xeric 6/6/2008 7/1/2008 CL, N17,DF

Senecio SM1,SM2, triangularis Asteraceae xeric 6/19/2008 7/1/2008 CL, N17,DF SM1,SM2, Taraxacum CL, N17, officinale Asteraceae mesic 6/6/2008 7/1/2008 DF Arenaria congesta Caryophyllaceae xeric 6/22/2008 7/12/2008 N17 Pseudostellaria SM1, SM2, jamesiana Caryophyllaceae xeric 6/22/2008 7/12/2008 DF, N17 SM1, SM2, Silene lemmonii Caryophyllaceae xeric 6/22/2008 7/12/2008 CL N17, DF SM1,SM2, Eriophorum CL, N17, criniger Cyperaceae mesic 6/21/2008 7/11/2008 DF Arctostaphylos SM1, SM2, sp. Ericaceae xeric 6/6/2008 7/1/2008 CL Rhododendron occidentale Ericaceae xeric 6/6/2008 7/12/2008 SM1, SM2 Lupinus croceus Fabaceae xeric 6/22/2008 7/12/2008 N17,DF SM1,SM2, Lotus pinnatus Fabaceae mesic 6/22/2008 7/12/2008 CL, N17,DF

39 40

Appendix A. Plant species in coincident bloom with Darlingtonia californica near Scott Mountain and Mt. Eddy, CA.

Species Name Family Habitat Flw. First Obs. Fruit First Obs. Sites Found Trifolium SM1,SM2, longipes Fabaceae mesic 6/19/2008 7/1/2008 CL, N17,DF SM1,SM2, Ribes roezlii Grossulariaceae xeric 6/19/2008 7/1/2008 N17 Sisyrinchium idahoense SM1,SM2, ssp.occidentale Iridaceae mesic 6/6/2008 7/4/1998 CL, N17, DF Prunella vulgaris var. SM1, SM2, lanceolata Lamiaceae mesic 6/22/2008 7/12/2008 DF Allium validum Liliaceae mesic 6/22/2008 7/12/2008 N17 Calochortus SM1,SM2,CL, nudus Liliaceae xeric 6/19/2008 7/11/2008 N17, DF Calochortus SM1, SM2, tolmiei Liliaceae xeric 6/19/2008 7/11/2008 CL, N17, DF Dichelostemma multiflorum Liliaceae xeric 6/22/2008 7/12/2008 SM1, SM2 Hastingsia SM1,SM2,CL, alba Liliaceae mesic 6/22/2008 7/12/2008 N17, DF Lilium pardalinum SM1,SM2, ssp. shastense Liliaceae xeric 6/22/2008 CL, N17, DF Narthecium californicum Liliaceae mesic 6/22/2008 7/12/2008 SM1, SM2 Xeropyhylum tenax Liliaceae xeric 6/19/2008 7/1/2008 SM1, SM2 Sidalcea SM1, SM2, glaucescens Malvaceae xeric 6/22/2008 7/12/2008 N17 Sidalcea SM1, SM2, oregana Malvaceae mesic 6/22/2008 7/12/2008 N17, DF Epilobium ciliatum ssp. SM1, SM2, ciliatum Onagraceae mesic 6/22/2008 7/12/2008 DF Corallorhiza maculata Orchidaceae xeric 6/22/2008 SM1, SM2

Appendix A. Plant species in coincident bloom with Darlingtonia californica near Scott Mountain and Mt. Eddy, CA.

Species Name Family Habitat Flw. First Obs. Fruit First Obs. Sites Found Platanthera SM1, SM2, leucostachys Orchidaceae mesic 6/19/2008 N17, DF Platanthera sparsiflora Orchidaceae mesic 6/22/2008 DF Polygonium SM1, SM2, bistortoides Polygonaceae mesic 6/22/2008 7/11/2008 N17, DF Dodecatheon N17,SM1,SM2, jeffreyi Primulaceae mesic 6/6/2008 7/12/2008 CL, DF Aquilegia SM1,SM2, formosa Ranunculaceae xeric 6/19/2008 7/12/2008 CL, N17, DF Caltha leptosepala var. biflora Ranunculaceae mesic 6/6/2008 7/12/2008 CL , N17, DF Delphinium SM1, SM2, glaucum Ranunculaceae xeric 6/21/2008 7/12/2008 CL, N17, DF

Amelanchier utahensis Rosaceae xeric 6/6/2008 7/12/2008 SM1,SM2 Potentilla drummondii SM1,SM2, ssp. breweri Rosaceae mesic 6/20/2008 7/4/2008 CL, N17, DF Rosa woodsii var. ultramontana Rosaceae xeric 6/22/2008 7/12/2008 SM1, SM2 Spiraea densiflora Rosaceae mesic 6/22/2008 7/12/2008 SM1,SM2 Castilleja sp. Scrophulariaceae xeric 6/19/2008 7/12/2008 SM1,SM2 Mimulus SM1,SM2, guttatus Scrophulariaceae mesic 6/22/2008 7/12/2008 CL, N17, DF Mimulus SM1,SM2, primuloides Scrophulariaceae mesic 6/6/2008 7/4/2008 CL, N17, DF Penstemon speciosus Scrophulariaceae xeric 6/22/2008 7/12/2008 SM1, SM2 Viola glabella Violaceae mesic 6/22/2008 7/11/2008 DF

Appendix A. Plant species in coincident bloom with Darlingtonia californica near Scott Mountain and Mt. Eddy, CA.

Species Name Family Habitat Flw. First Obs. Fruit First Obs. Sites Found Viola lobata SM1,SM2, ssp. lobata Violaceae mesic 6/6/2008 7/4/2008 CL, N17,DF Viola sororia ssp. affinis Violaceae mesic 6/6/2008 7/4/2008 SM1,SM2 Viola maclosky Violaceae mesic 6/6/2008 7/4/2008 SM1, SM2