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2060
American Journal of Botany 91(12): 2060–2068. 2004.
P
OLLINATION AND SEED PRODUCTION IN
XEROPHYLLUM TENAX (M
ELANTHIACEAE
)
IN THE
C
ASCADE
R
ANGE OF CENTRAL
O
REGON
1
N
AN
C. V
ANCE
,
2,5
P
ETER
B
ERNHARDT
,
3
AND
R
ETHA
M. E
DENS
4
2
USDA Forest Service, Pacific Northwest Research Station, 3200 SW Jefferson Way, Corvallis, Oregon 97331 USA;
3
Department of
Biology, Saint Louis University, St. Louis, Missouri 63103 USA; and
4
Department of Educational Studies, Saint Louis University,
St. Louis, Missouri 63103 USA
Xerophyllum tenax is a mass-flowering, nectarless herb in which self-pollination is unavoidable as anthers shed pollen onto the
three, receptive stigmatic ridges attached to each pistil within a few hours after expansion of the perianth. We compared the pollination
system with reproductive success in this species through controlled, hand-pollination experiments. Ovaries of flowers sampled from
unbagged inflorescences were visited by pollen-eating flies (primarily members of the family Syrphidae), beetles (primarilyCosmosalia
and Epicauta spp.), and small bees, and produced normal-sized capsules and mature seeds. Ovaries of flowers from inflorescences
bagged to prevent insect pollination produced small capsules containing undeveloped or no seeds. Epifluorescence analyses suggest
that 0.95 of the uncovered flowers are cross-pollinated by insects with pollen tubes penetrating style and ovary tissue. Flowers show
a ‘‘leaky’’ but early-acting self-incompatibility system. While hundreds of pollen tubes germinate on each stigmatic surface following
self-pollination, few pollen tubes penetrate the stigmatic surface and none penetrate the ovary. In contrast, when stigmas are cross-
pollinated by hand with pollen from a second inflorescence pollen tubes were seen penetrating style and ovary. Self-incompatibility
in X. tenax parallels that of some species of Trillium, a sister genus within the Melanthiaceae.
Key words: beargrass; Melanthiaceae; pollen; pollination; seed production; self-incompatibility; Xerophyllum tenax.
Xerophyllum tenax (Pursh.) Nutt. is an evergreen perennial
monocot of montane forests in the Pacific Northwest with long
fibrous leaves. In openings the long-lived plants often grow
large with multiple shoots of which one or two may produce
an inflorescence. The species is not only a significant com-
ponent of subalpine ecosystems, it is of cultural and commer-
cial importance in the region (Lobb, 1990). Although forest
management practices as well as extensive commercialharvest
may be affecting the species’ reproduction, growth, and sur-
vival (Dimock, 1981; Mosley, 2000), little knowledge exists
of its reproductive ecology (Bradley, 1984). While the floral
phenology and flowering pattern of its inflorescence have been
described (Long, 1981; Utech, 1978; Maule, 1959) its polli-
nation ecology, self-isolation mechanisms (sensu Bernhardt
and Thien, 1987), and rates of fruit and seed set remain largely
anecdotal.
Xerophyllum tenax (beargrass or Indian basket grass) is of
cultural and economic importance in northwestern North
America (Vance et al., 2001). To Native American basket
weavers of California and the Pacific Northwest, X. tenax his-
torically was and continues to be a valued plant (Turner, 1998;
Rentz, 2003). Leaves are carefully selected, gathered and dyed
for use in basketry (Lobb, 1990; Moerman, 1998). Burning
off areas where beargrass grows continues to be a traditional
practice used to produce the pliable and less pigmented leaves
preferred for basketry (Rentz, 2003). The management and
1
Manuscript received 8 January 2004; revision accepted 26 August 2004.
The authors thank Dan Mikowski for his field support, Patrick Vogan for
his technical lab work, John Meyers for his pen and ink illustration, Tammy
Sage for confirming early crossing experiments, Landi Mendoza for her work
on the data, and the USDA Forest Service Willamette National Forest for
providing the sites for the research. This research was supported in part by
USDA Forest Service, Pacific Northwest Research Station contract 43-0453-
0-6028.
5
Author for correspondence (Tel: 541 750-7302; Fax: 541 750-7329;
E-mail: nvance@fs.fed.us).
protection of these resources are supported by the USDA For-
est Service on lands under their jurisdiction (Vance et al.,
2001). In recent years beargrass leaves have grown in com-
mercial value and are harvested for the floral industry. These
leaves are sold to processors for export as raw material to
Asian and European markets for dried floral crafts and deco-
rations. The market potential has been estimated for thousands
of tons of leaves at over $US 1 million (Blatner and Schlosser,
1998). Illegal harvest has been extensive and damaging as
flowering shoots are often destroyed. In one year over 100
tons of illegally harvested leaves were confiscated from the
Willamette National Forest (Mosley, 2000). Interpreting the
floral biology of X. tenax is necessary to understand its con-
servation and to develop appropriate management of this spe-
cies.
Furthermore molecular analyses have changed the morpho-
logically based interpretation of the phylogeny of the mono-
cotyledons in general and Xerophyllum and its allied genera
in particular. Rudall et al. (2000) maintain Xerophyllum within
the Liliales but their strict consensus tree now places this ge-
nus within the family Melanthiaceae. Genera placed within the
Melanthiaceae s.s. are synapomorphic for dorsal composite
vascular bundles in their flowers, a pollen grain with an oper-
culate sulcus and the presence of Veratrum-type alkaloids.
However, while Xerophyllum spp. bear many small, whitish
flowers on a much elongated, racemose-paniculate inflores-
cence, this genus is not a sister of Veratrum and Zigadenus
with similar modes of floral presentation. Instead, Xerophyllum
is a sister genus of Paris and Trillium. In these two genera the
short flowering shoot usually terminates in a large but solitary
and often sessile flower. Recent work by Sage et al. (2000)
has also found an early acting, stigmatic self-incompatibility
in Trillium spp. This self-incompatibility mechanism is rare in
the monocotyledons in general and is currently restricted to
lineages in only four other families. Consequently additional
December 2004] 2061V
ANCE ET AL
.—P
OLLINATION OF
X
EROPHYLLUM TENAX
work on the floral biology of Xerophyllum is also required to
compare phenotypic characters between this taxon and its sis-
ter genera to determine the divergence of pollination mecha-
nisms and breeding systems within this lineage.
MATERIALS AND METHODS
Study species—Xerophyllum tenax (Melanthiaceae) is a perennial, acaules-
cent, monocot that grows in cool, coniferous forest communities in the sub-
alpine zone of northwestern North America. In the western hemlock zone of
the Cascade Mountains it is often a dominant component of the understory.
There it grows on rocky shallow soils in association with hemlock and true
firs where the forb layer may often be depauperate (Franklin and Dyrness,
1973). Xerophyllum tenax consists of a short rhizome and tightly connected
shoots. Each vegetative shoot of X. tenax arises from a basal meristem, pro-
ducing grass-like, keeled, rigid leaves with a life span of several years (Brad-
ley, 1984). In openings the long-lived plants may become large with multiple
shoots of which one or two may produce an inflorescence. The onset or length
of flowering at different locations varies with differences in seasonal temper-
atures, aspect, and elevation (Long, 1981). In the Oregon Cascades flowering
may be initiated as early as April and continue into August. Flowering is
often most prevalent in plants growing in forest openings created by distur-
bance such as wildfire; however, flowering becomes less frequent and may
disappear altogether as the forest canopy recloses (Maule, 1959; Simpson,
1990).
Floral morphology—The inflorescense is a mass-flowering terminal raceme
of small, white to cream-colored flowers on pedicels 2–5 cm long maturing
successively from the bottom to the top (Maule, 1959). The flowering phase
is initiated when an inflorescence elongates on an axis that may extend as
high as 15 dm. The lower flowers have distinctive bracts that become reduced
in length and adnate to the pedicels of the upper flowers. The flower pedicels
become elongate and erect at maturity, so that midway through the flowering
season, the raceme appears as a conical or cylindrical cluster of opened flow-
ers topped by unopened floral buds. Each flower consists of six oblong to
ovate tepals without basal glands. We detected a distinct odor (see Floral
attractants and rewards under RESULTS) when the majority of the flowers in
the raceme were open. The gynoecium is tricarpellate with three free, recurved
styles. The carpels are unilocular, each carpel having a maximum of four
ovules (Utech, 1978). The capsule is ovoid, acute, and about 5–7 mm long
and it undergoes loculicidal dehiscence when seeds mature in late summer.
After the capsule dehisces and seed has been released, the shoot senesces
(Hitchcock and Cronquist, 1978; Utech, 1978).
Study sites and design—Two similar but geographically separated sites
were selected for the experimental pollination study series and for the obser-
vation/collection of insect pollinators. A third site not used for experimenta-
tion was used only to collect pollinating insects. These three sites are in the
central Cascade Range of Oregon on the USDA Forest Service, Willamette
National Forest. The plant association at these sites is Abies amabilis/Rho-
dodendron macrocarpum/Xerophyllum tenax. At all sites flowering occurs
usually from late May through early July. The first experimental study site,
at about 1020 m elevation, is on a shallow slope of the Hackleman Creek
drainage within 100 m of the road. The coniferous stand had been logged on
part of the site in the early 1990s and trees were scattered in clumps on grassy
openings. The second experimental site is situated on a shallow slope of Camp
Creek drainage at about 1120 m elevation and also logged in the early 1990s.
Removal of a part of the overstory at both sites created sufficiently large
openings that favored X. tenax flowering. These two sites were about 13 km
apart in order to ensure that separate populations were being sampled. The
third site, used exclusively for insect collection, was Browder Ridge at about
1350 m elevation where the coniferous overstory was also removed in the
early 1990s.
The controlled pollination study was carried out on the two widely sepa-
rated sites noted above. Four treatments were applied to 24 independent plants
selected at each site and randomly assigned. Treatment descriptions follow.
(1) Self: stigmas of tagged flowers on re-bagged inflorescences received no
additional pollen. (2) Open: stigmas of tagged flowers that opened that morn-
ing on unbagged inflorescences were exposed to insect pollinators for 24 h.
(3) Short-distance hand cross-pollination (SDP): stigmas of bagged inflores-
cences received pollen from a solitary inflorescence of a neighboring plant
within the same population. (4) Long-distance hand cross-pollination (LDP):
stigmas of bagged inflorescences received pollen from a solitary inflorescence
of a plant from a disjunctive population at the other site (13 km).
Insect identification and observations of floral development and scent oc-
curred during two flowering seasons from 2001 through 2002. The hand cross-
and self-pollination study occurred during flowering season of 2002. Micro-
graphs were taken from collections made in 2001 and 2002.
Flower and floral forager observations—Individual flowers on inflores-
cences were labeled with jeweler’s tags and observed while wearing optical
glass magnifiers (Opti Visor). Insect foraging behavior was recorded for 2 d
in early July 2000, and 3 d in late June 2001 and 2002, representing approx-
imately 22 h of observation. Optical glass magnifiers made it possible for the
observer to note whether the forager contacted the stigmas and the anthers
while foraging.
To determine whether visitors carried significant quantities of pollen of X.
tenax (.25 grains/insect) insects observed foraging on flowers and/or manip-
ulating floral organs with their legs or mouth parts were collected in butterfly
nets and killed in jars poisoned with fumes of ethyl acetate on each obser-
vation day. Removal, staining, mounting, identification, counting and record-
ing of individual pollen grains carried on insect bodies followed Bernhardt
and Weston (1996). We used Calberla’s fluid (Ogden et al., 1974) to stain the
exine of the pollen walls with basic fuchsin. Insect body length was recorded
by measuring the body from its labrum to the apex of the abdomen prior to
pinning. Pinned specimens were sent for identification and vouchers were
deposited in respective collections: Coleoptera (J. Chemsak, Division of Insect
Biology, University of California, Berkeley, California, USA), Diptera (F. C.
Thompson, Entomology Division, Smithsonian, Washington, D.C., USA) and
Hymenoptera (C. D. Michener; Snow Entomological Museum, Kansas State
University, Lawrence, Kansas, USA).
Pollination experiments—Twenty-four flowering plants were randomly as-
signed to the Open, Self, short-distance cross-pollination (SDP), and long-
distance hand cross-pollination (LDP) treatments at each site in late May–
early June 2002. For all treatments except the Open treatment, an inflores-
cence on each plant was isolated in a nylon mesh bag before the flower buds
opened.
For determining natural rates of successful pollination Open-treated (un-
bagged) flowers (each sampled within 24 h of anthesis) were collected from
inflorescences at both experimental sites every second day over a 10-d period
during mid-June (at peak flowering). For the Self, SDP, and LDP treatments,
bags were removed every day and 3–6 flowers were sampled from each in-
florescence on their first day of anthesis, hand-pollinated and then labeled
with jeweler’s tags. Hand-pollinations were made at this time with the aid of
an optical glass binocular magnifier, but pollen was applied to the stigmatic
surfaces until it was visible to the naked eye. These treatments required re-
bagging the inflorescence for an additional 24 h prior to harvesting, fixing,
and preserving whole flowers. Pollination success was determined by the
numbers of pollen tubes in pistils that penetrated the micropyles of ovules.
All flowers were too small to emasculate prior to hand-pollination so the SDP
and LDP treatments represent ‘‘cross-pollen enhancements’’ as in Lipow et
al. (2002).
Collected flowers were fixed ina3:1glacial acetic acid : 95% ethanol
solution for 2 h before decanting the fixative and storing flowers in 70%
ethanol. Individual flowers were softened in a 5% aqueous solution of sodium
sulphite at room temperature for 24 h. Each pistil was then excised and taken
through three consecutive baths of distilled water. Each washed pistil was
placed on its own glass slide, the styles were teased apart with forceps or
dissecting probes, and then the entire organ was spread and squashed in 2–3
drops of decolorized aniline blue, labeled, and refrigerated a minimum of 7
d. To view pollen tubes in the pistil under epifluorescence we used a Carl
2062 [Vol. 91A
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OURNAL OF
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OTANY
Fig. 1. Illustration shows Xerophyllum tenax styles as they move from upright (A) to the fully recurved (C) position exposing the stigmatic ridges to insects
as the pistil ages. The stigmatic ridges are outlined with dark stippling.
Zeiss Incident Fluorescence Microscope with a violet exciter filter as in Bern-
hardt et al. (1980). Viewing techniques and micrography followed Goldblatt
and Bernhardt (1990).
To determine the number of germinated pollen grains thathad grown pollen
tubes two pollen tube counts were taken for each pistil. Because the ovary of
each pistil bears three separate styles, the number of pollen tubes observed
to penetrate the transmission tissue in each style was counted. The total counts
from each style were pooled and pollen tubes per style calculated. The number
of pollen tubes penetrating ovary tissue was similarly determined and record-
ed separately.
Open-pollination vs. self-pollination effects on seed set—Bagged stalks
bearing infructescences that received no cross-pollination and stalks with
open-pollinated flowers were collected in August when capsules were fully
developed but had not yet dehisced. At this stage the capsules were suffi-
ciently mature to dissect each ovary to see if they contained seeds. In the
laboratory, each infructescence of 11 flowering stalks from the Camp Creek
site (the covering of one infructescence was too damaged to use in analysis)
and 12 from the Hackleman Creek site were measured to the nearest milli-
meter and divided into three equal sections. Sampled capsules were dissected,
and ovules were observed under a dissecting microscope. The basal, medial,
and axial portions were analyzed separately to determine the phenology of
flowering in which the flowers basally located on the inflorescence reached
anthesis earlier than the axial flowers. The number of flowers was inferred
based on counting each pedicel and bract. The capsules were also counted so
that ratio of capsules to flowers could be determined. In each (basal, medial,
and axial) section of the infructescence, 20 capsules were randomly selected
and the length of each measured to the nearest millimeter; seeds were ex-
tracted, counted, and the length determined to the nearest millimeter. Total
number of seeds and average number of seeds per capsule were determined
for the capsules collected from each infructescence of the bagged and
unbagged flowering stalks.
Statistical analysis—Tests were conducted using Statgraphics Plus statis-
tical software version 5.1. (Manugistics, 2000). The pollen tube data were
tested for equal variances and distribution departures from normality (Kol-
mogorov-Smirnov [KS] test for goodness of fit). The Student’s ttest and KS-
test Dstatistic were used on the pollen tube data to determine significant
differences among treatments without assumptions of equal variances. For the
seed set analysis, potential site differences were assessed in a two-way anal-
ysis of variance (ANOVA); we found no significant differences in the vari-
ables tested. Because there were no interactions, each site was treated sepa-
rately. Significant differences in means were assessed by Tukey’s Honestly
Significant Difference (HSD). Levene’s test of variance was used. Where pa-
rameters were not met for the test of variance, the nonparametric Kruskal
Wallis test was used. Regression analysis was used to relate capsule length
to number of filled seed.
RESULTS
Inflorescence and floral presentation—Inflorescences have
a gradient in development at anthesis so that the terminal flow-
er buds opening at the end of the season either produced un-
dersized, malformed flowers or never opened at all (open de-
velopment). Because perianths on different flowers typically
overlapped and their stiff, erect, elongated stamens were al-
ways held well above the shallow, salverform perianths, the
entire inflorescence had an unusually dense, brush-like ap-
pearance.
As the perianth expanded in an opening bud, 1–2 of the six
anthers were dehiscent, while the three, often appressed, styles
curve upwards in suberect positions (Fig. 1A). Within the next
two-four hours all remaining anthers dehisced and the style
arms began to curve downwards (Fig. 1B). The receptive stig-
matic surface on each of the three styles ran from the blunted
tip of the style down to its base where the style ‘‘arm’’ con-
nected to the top of the ovary forming a narrow ridge. As the
pistil aged each style arm coiled, beginning at the tip, exposing
a now upwardly curved segment of the stigmatic surface while
concealing more terminal portions of the stigmatic surface
within the coil (Fig. 1C). While an individual flower’s recep-
tivity is measured in days, each inflorescence may bear from
150 to 400 flowers. Anthesis occurs successively along the
December 2004] 2063V
ANCE ET AL
.—P
OLLINATION OF
X
EROPHYLLUM TENAX
T
ABLE
1. Pollen load analyses of insects collected on flowers of Xe-
rophyllum tenax.
Insect taxon
Pollen load
X. tenax only X. tenax
1
other
species No pollen
Coleoptera
Cermbycidae
Anastrangalia laetifica
Cosmosalia chrysocoma
Leptaura propinqua
2
18
1
1
7
0
0
0
0
Cleridae
Trichodes ornatus 230
Meloidae
Epicauta sp. 12 0 1
Scarabaeidae
Dichelonyx backi
Diplotaxis sp. 2
10
00
0
Subtotals 38 11 1
Diptera
Acroceridae
Eulonchus sp. 1 0 0
Asilidae
Laphria sp. 0 0 1
Bombyllidae
Conophorus sp. 1 0 0
Calliphoridae
Calliphora vomitoria 100
Tachinidae
Tachina sp. 0 1 0
Syrphidae
Cheilosia hoodiana
Chrysotoxum fasciatum
Eriozona laxa
Eupeodes aberrantis
E. lapponicus
20
4
3
1
2
0
0
0
0
1
1
1
0
1
0
Hadromyia crawfordi
H. pulcher
Melangyna triangulifera
Parasyrphus relictus
Sericomyia chalopyga
1
1
1
18
1
0
0
0
1
0
0
0
0
0
0
Syritta pipiens
Syrphus opinator
S. ribesii
1
5
3
0
1
2
0
0
0
Subtotals 61 5 3
Hymenoptera
Andrenidae
Andrena nivalis
A. vicina 0
11
10
0
Apidae
Apis mellifera
Bombus fernaldi 1
00
10
0
Halictidae
Halictus rubicundus sp.
Lasioglossum athabascense
L. sp. 1 (Dialictus)
L. sp. 2 (Dialictus)
L. sp. (Evylaeus)
0
1
1
2
1
1
1
0
0
1
0
0
0
0
0
Megachilidae
Coelioxys sp.
Megachile vidua 1
00
10
0
Subtotals 8 6 0
Grand totals 110 23 5
peduncle, beginning with flowers in the basal portion of the
raceme and progressing acropetally so that the same inflores-
cence may have receptive flowers for up to 2 wk.
The ovary swells within 3–4 d following the withering of
the perianth and the androecium. These ovaries remained on
the infructescence whether or not they contained seeds. As the
ovaries matured into fruits, the capsules of the nonpollinated
flowers that presumably had no developing seed appeared ap-
preciably smaller in size than those containing seeds (see sec-
tion Fruit and seed set below).
Floral attractants and rewards—Perianth segments and
pistils were white to the human eye, while dehiscent anthers
released yellowish, cream-colored pollen. Ovaries commonly
turned pink-burgundy purple after the perianth and androeci-
um withered or when the outer whorls were dried by excessive
heat.
Scent was variable. In three inflorescences sampled in the
field, the floral odor was sweet and agreeable; to the second
author it was reminiscent of cultivated lilacs (Syringa). In the
remaining 12 inflorescences the odor was musty-acrid. Flow-
ers were removed from eight inflorescences at random and
placed in a clean, capped glass vial. The lid was removed and
the accumulated odor was described at 5-, 10-, and 30-min
intervals. Musty-acrid notes dominated with undertones of
sweet notes. The odor of these bottled flowers most resembled
cultivated privet (Ligustrum). Floral nectar was not detected
at any stage in the floral life-span.
Foraging insects and their pollen loads—Prospective pol-
linators represented three insect orders and varied considerably
in size, yet almost all foragers examined carried the pollen of
X. tenax (Table 1). Taxa within the order Diptera (true flies)
provided the most numerous and diverse group of foragers.
Flies represented six families, but the majority of identified
taxa (0.91) belonged to the family of hover flies (Syrphidae).
Hover flies were observed probing the stamens and style arms
with their probosces while they foraged. These insects re-
mained on an inflorescence for only a few seconds before fly-
ing to a second inflorescence. A total of 17 fly specimens were
measured with lengths varying from 8 mm (e.g., Parasyrphus
relictus)to17mm(Laphria sp.). The male-to-female sex ratio
of collected hover flies visiting X. tenax was low. Of 69 hover
flies evaluated, 0.277 were males. Only one specimen of Chei-
losia hoodiana (Syrphidae), the most commonly collected fly,
was a male.
Flower-visiting beetles (Coleoptera) were represented by
four families (Table 1). Beetles carried the pollen of X. tenax
as they grazed on whole anthers but were also observed to
place their heads into the shallow floral cup touching the style
arms with their legs and mouth parts. Cosmosalia chrysocoma
(Cerambycidae) was the most commonly observed species at
both sites and was observed flying from inflorescence to in-
florescence. However, individual beetles, particularly C. chry-
socoma, would remain on the same inflorescence for .1h.
On 21 June, 2001, at the Browder Ridge site, we observed
Epicauta spp. flying from X. tenax to flowers of Ceanothus
velutinus to drink nectar. A total of 33 beetles were measured,
ranging in length from 9 mm (e.g., Anastranglia laetifica)to
16 mm (e.g., Cosmosalia chrysocoma). Of 50 beetle speci-
mens in which gender was identified, 0.64 were males but only
two of the 25 specimens of Cosmosalia chrysocoma were fe-
males.
2064 [Vol. 91A
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OURNAL OF
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Fig. 2. (A) Germination of pollen grains and pollen tube penetration of the style in an insect-pollinated (Open) flower of X. tenax (note that at least four
pollen tubes have penetrated the stigmatic surface and are now growing down through the transmission tissue). 12.53. (B) Early-acting self-incompatibility
response on the stigmatic ridge of a bagged, self-pollinated (Self) flower. Note the short pollen tubes on the stigmatic surface and the rare, solitary tube that
penetrated the style but grew into a triangular configuration. 253. (C) Skein of pollen tubes growing from the bases of the styles into the top of the ovary
following a long-distance pollination (note the tubes penetrating ovules). 253. (D) Stigmatic surface following an enhanced, long-distance pollination showing
far more pollen tubes penetrating the stigma and growing down through the transmission tissue then in either the insect-pollinated flower (A) or the bagged,
self-pollination (B). 253.
Bees (Hymenoptera) on X. tenax represented four families
but were the least frequently observed of all visitors. A total
of 14 bee specimens were measured ranging in size from 11
mm (e.g., Coelioxys sp.) to 18 mm (Bombus fernaldi). A sol-
itary specimen of Megachile vidua was the only male cap-
tured. All females, excluding Bombus fernaldi, were observed
clutching and scraping anthers for pollen. The ventral portions
of the bees’ bodies bounced against the style arms while they
scraped or vibrated anthers. The solitary specimen of Bombus
fernaldi was observed to extend its tongue into the inflores-
cence as if it were attempting to take nectar from the pedicels,
but this is a brood-parasitic species (syn. Psithyrus fernaldi)
that never collects pollen for its offspring. The collected spec-
imen represented the only observation of this species on X.
tenax for the duration of the study.
Of the 138 insect foragers collected on flowers of X. tenax
110 specimens (0.797) carried pollen of X. tenax exclusively
(Table 1). Insects bearing mixed loads carried a maximum of
two extra pollen types mixed with the pollen of X. tenax. The
most common pollen grains, in order of frequencies, were
identified as Ceanothus velutinus, general rosiid-type (Ame-
lanchier alnifolia,Fragaria spp., Rubus spp.—all flowering at
the three sites), and Rhododendron macrophylum. These spe-
cies are common co-flowering associates of X. tenax in the
Western Cascades (Ross and Chambers, 1988).
Pollen-pistil interactions in unbagged controls—The stig-
matic surfaces of all styles (N5243) of the unbagged, nat-
urally pollinated pistils (Open) bore hydrated pollen grains,
but the amount of pollen per stigmatic ridge varied from 14
to 790 grains. Pollen grains were deposited from the blunted,
circular, stigma tip down to the base of the stigmatic ridge
above the ovary (Fig. 2A). However, the vast majorityof these
grains either failed to germinate and were covered in callose
crusts, or produced short, irregular tubes, lacking callose plugs
that failed to penetrate the stigma surface.
Of 103 pistils examined only five (0.048) lacked pollen
tubes penetrating style and ovary tissue. A mean of approxi-
mately 8 pollen tubes was found in the pooled, 24-h-old styles
on each pistil with a mean of approximately 2 tubes actually
penetrating each ovary. We reject the null hypothesis thatflow-
ers that were never bagged contain the same number of pollen
tubes/pistil as those that were bagged throughout the flowering
period (Table 2).
Pollen-pistil interactions in bagged flowers—As described
for unbagged flowers above, all stigmatic surfaces were coated
with pollen grains regardless of treatment. In all treatments,
hydrated grains that failed to produce tubes penetrating trans-
mission tissue outnumbered hydrated grains with penetrating
tubes. The continued presence of heavy deposits of pollen on
Self–treated stigmas, despite no hand-pollination, indicated
that mechanical self-pollination (autogamy) occurred consis-
tently in this species. The mean number of pollen tubes de-
tected in styles of bagged flowers (Self treatment) was ap-
December 2004] 2065V
ANCE ET AL
.—P
OLLINATION OF
X
EROPHYLLUM TENAX
T
ABLE
2. Mean number of pollen tubes and 95% confidence intervals
(CI) in style and ovary of self-pollinated Xerophyllum tenax flowers
(Self), open-pollinated flowers (Open), flowers mechanically cross-
pollinated with flowers from nearby plants (Short-distance cross),
and flowers mechanically cross-pollinated with flowers from a dif-
ferent population separated by 13 km (Long-distance cross). Dif-
ferent letters denote significant differences between treatments
(Kolmogorov-Smirnov test P
#
0.01).
Treatment NMean CI
Style
Self
Open
Short-distance cross
Long-distance cross
62
106
51
55
3.79
a
8.21
b
24.00
c
16.58
c
[2.17, 5.37]
[6.10, 10.32]
[18.73, 29.25]
[11.03, 16.14]
Ovary
Self
1
Open
Short-distance cross
Long-distance cross
62
106
51
55
1.52
a
19.49
b
14.96
b
[0.72, 2.32]
[14.70, 24.20]
[10.64, 19.29]
1
No analysis performed as there were no pollen tubes detected in
ovary.
T
ABLE
3. Differences between treatments of bagged (Self) and unbagged for natural insect pollination (Open) inflorescences collected from
Xerophyllum tenax. Significant differences between treatments in mean inflorescence length and mean number of flowers per inflorescence by
Tukey’s HSD; the Kruskal-Wallis test was also used for detection of significant difference between treatments in mean capsules per flower at
P
5
0.05. Significant difference between treatments is indicated by different letters. Data are presented as means (
6
1 SD).
Site
Inflorescence length (cm)
Open Self
No. flowers per inflorescence
Open Self
No. capsules per flower
Open Self
Camp Creek
N
5
11
Hackleman Creek
N
5
12
51.3
a
(10.3)
61.5
a
(13.4)
35.4
b
(12.8)
36.8
b
(11.2)
276.8
a
(82.2)
296.3
a
(87.9)
240.4
a
(93.3)
282.5
a
(69.3)
0.952
a
(0.070)
0.907
a
(0.146)
0.686
a
(0.259)
0.883
a
(0.168)
proximately 4 tubes and no tubes were found in their ovaries
(Table 2). Furthermore, the majority of these pollen tubes
showed some form of aberrant growth in their styles, either
developing excessive deposits of callose at their tips and/or
forming short ‘‘corkscrews’’ or tubes that grew backwards
forming geometric patterns (Fig. 2B). We reject the null hy-
pothesis that pistils of flowers that were bagged but not hand-
pollinated contained the same number of tubes in their styles
and ovaries as those pistils that were bagged and hand cross-
pollinated (Table 2).
There was a greater number of penetrating pollen tubes in
the stigmas and ovules of pistils that received SDP and LDP
(Fig. 2C, D) than those counted in Open or Self treatments
(Fig. 2A, B). In styles and ovaries of pistils that received the
SDP treatment, the mean number of penetrating pollen tubes
were approximately 24 and 20 tubes, respectively. In styles
and ovaries of pistils that received the LDP treatment the mean
numbers of penetrating pollen tubes were approximately 17
and 15 tubes, respectively. No significant difference was de-
tected in mean number of pollen tubes counted in styles or
ovaries between the SDP- and the LDP-treated pistils (Table
2).
Fruit and seed set—Observations of the infructescences
and fruits at both sites showed distinct differences between
those that developed from flowers that were naturally polli-
nated (Open) and those bagged (Self). Although there was no
significant difference in the number of flowers per inflores-
cence, we detected a significant difference in mean length be-
tween bagged and unbagged infructescences at both sites (Ta-
ble 3). The size of infructescence may be a function of capsule
development as the open pollinated flowers on the (Open)
treated inflorescences produced larger, but not significantly
more capsules (Table 3). We observed that development was
arrested in capsules sampled from bagged inflorescences.
Those found to be #1 mm in length were not included in any
capsule analysis as they were obviously undeveloped capsules.
Even with this exclusion of undeveloped capsules, the analysis
indicated that there was no significant difference detected in
the mean capsule-to-flower ratio among the bagged and un-
bagged flowers (Table 3).
At both sites, the fruit that developed from the bagged flow-
ers, (autogamy) were significantly smaller in size (based on
measured capsule length) than those of the open-pollinated
flowers. The capsules from the open-pollinated flowers had a
significantly higher rate of filled seed compared to those from
the bagged inflorescences (Table 3). The number of filled seed
per capsule was related to capsule length which ranged from
1.6 to 7.2 mm. Capsules appeared to expand to accommodate
the number of developing seed, which was high as 12 seeds.
Length was directly related to number of filled seed (adjusted
R
2
50. 65, P,0.001), where the model (Y53.75 10.248X)
predicted that capsule ,4.0 mm would have a high probability
of no or few filled seed. The mean number of filled seed per
capsule was 7.06 and 0.94 seeds at the Hackleman Creek site,
and 6.63 and 0.18 seeds at the Camp Creek site for Open- and
Self-treated flowers, respectively (Fig. 3). The mean filled seed
per capsule of the bagged flowers was an order of magnitude
lower than that of the open-pollinated flowers indicating al-
most complete lack of ovule development in flowers prohib-
ited from cross-pollination. The effect was not absolute as a
few capsules developed from flowers that were inside the ny-
lon bags, and contained filled seeds. Excision of those capsules
revealed seeds that were fewer in number, smaller in size, and
predominantly empty. The mean number of empty seeds per
capsule was 0.03 and 0.30 seeds at the Hackleman Creek site,
and 0.08 and 0.23 seeds at the Camp Creek site for Open- and
Self-treated flowers, respectively (Fig. 3). In the capsules of
the open-pollinated flowers, empty seeds contributed to ,1%
of the total number of seeds, whereas in capsules of the flow-
ers receiving no cross-pollination, empty seeds contributed to
$50% or more of the total. Either total lack of any pollen
reaching the anthers or autogamy was possible for flowers that
had been bagged. It is likely no seed (or capsule) development
occurred if there was no pollination and aborted development
occurred with autogamy.
DISCUSSION
In the absence of floral nectar, X. tenax is restricted to guilds
of pollinators that consume pollen as a primary reward, but it
2066 [Vol. 91A
MERICAN
J
OURNAL OF
B
OTANY
Fig. 3. Differences between treatments in (A) capsule length, (B) filled
seeds per capsule, and (C) empty seeds per capsule of 60 capsules sampled
from bagged (Self) and insect cross-pollinated (Open) inflorescences of Xe-
rophyllum tenax at the Camp Creek (N511) and Hackleman Creek (N5
12) sites in the central Cascades of Oregon. Differences between treatments
indicated are by different letters (P,0.01). Data are means 61 SD.
is not a pollinator-limited herb. Results here show that some
orders containing pollen-eating species can be both highly di-
verse and some may represent extensive populations in the
Cascade Mountains of Oregon. Floral presentation and insect
activity on X. tenax suggest a generalist mode of pollination
(sensu Waser et al., 1996), but direct observation, insect col-
lections, and pollen load analyses indicate that flies, beetles
and bees do not appear to contribute equally to the cross-
pollination of these flowers. Although female bees carried the
greatest numbers of pollen grains on their bodies and in their
pollen baskets (P. Bernhardt, unpublished data), they were the
least frequent foragers over two seasons. We observed medi-
um-large beetles on inflorescences every day we were in the
field and noted they moved sluggishly, at best, among neigh-
boring inflorescences of X. tenax. Flies probably effected the
majority of cross-pollinations. Among these three sites, species
within the family Syrphidae were the most frequent and
‘‘faithful’’ pollen vectors. Note that 0.88 of the hover flies
collected on X. tenax carried only the pollen of X.tenax.
The diversity of flies on the flowers of montane plants in
North America is usually explained on the basis of elevation
and prevailing climate. Bees are less common and become less
active under cooler, wetter regimes. Consequently plants dis-
tributed at higher elevations show shifts towards fly-pollina-
tion. Our own unpublished observations, though, suggest that
a wide variety of solitary bees and eusocial Bombus spp. for-
aged daily on C. velutinus,Fragaria spp., R. macrophyllum,
and Pedicularis racemosa. Despite the mass-flowering mode
of presentation, X. tenax is probably unattractive to the ma-
jority of common, montane bees as the light colors, disagree-
able odors and nectarless condition of its flowers fails to reflect
the suite of characters most often associated with bee-polli-
nation. Floral presentation in X. tenax is simply much closer
to the classic descriptions of fly-pollinated syndromes (Faegri
and van der Pijl, 1979) or the mass-flowering, ‘‘brush mode’’
of beetle-pollination (see review in Bernhardt, 2000).
There is precedence for the skewed sex ratios of some insect
foragers. The greater proportion of foraging female bees is
predictable. Male bees do not, as a rule, collect pollen for the
offspring they sire and are probably not attracted to the nec-
tarless flowers of X. tenax. Flowers that have abundant pollen
may be more attractive to some female hover flies as a source
of lipids and amino acids to invest in eggs; to male flies not
having that use for pollen, nectarless flowers would be less
attractive. In contrast the higher ratio of males of the beetle
C. chrysocoma on inflorescences followed a pattern noted by
Dafni et al. (1990) and Goldblatt et al. (1998) for hairy, flower-
visiting scarabs. Male flower beetles often assemble and wait
for the arrival of unfertilized female beetles on preferredflow-
ers (Bernhardt, 2000), which suggests that an attribute not re-
lated directly to floral food may influence the sex ratio ofsome
beetles found on the flowers.
Therefore, floral presentation has diverged significantly
within the lineage that includes Paris,Trillium, and Xero-
phyllum. The first two genera produce much larger flowers
and, in most cases, a single flower terminates each peduncle.
Little is known of pollination systems in Paris but pollination
in Trillium appears to alternate between species pollinated by
flies vs. those pollinated by bees (including queens of Bombus
spp.) and several Trillium spp. are known to secrete nectar
(see review by Irwin, 2000). The trend within this lineage
(sensu Rudall et al., 2000) suggests a bifurcation in floral pre-
sentation. The mass-flowering of many small, nectarless flow-
ers of X. tenax attracts a broader range of potential pollen
vectors than the much larger (often nectariferous) flowers of
Trillium spp. that may attract a less diverse, but more spe-
cialized, spectrum of pollinators belonging to a single order.
More significantly, several species within this lineage retain
the same, early-acting self-incompatibility response. The stig-
matic ridge of X. tenax recognizes and rejects pollen produced
by the same plant as do the stigmas of some Trillium spp.
(Sage et al., 2000). While this self-incompatibility response is
atypical for monocotyledons in general, it may be more con-
servative within these allied genera than their more variable
modes of insect-pollination as Trillium populations show ei-
ther early-acting SI or are self-compatible (Irwin, 2000; Sage
December 2004] 2067V
ANCE ET AL
.—P
OLLINATION OF
X
EROPHYLLUM TENAX
et al., 2000). As a few bagged flowers of X. tenax set seeds
in the absence of cross-pollen, early-acting SI may be ‘‘leaky,’’
a feature common to other, unrelated taxa with early-acting
systems (Richards, 1997). A less likely possibility is that these
two populations of X. tenax show a small but persistent rate
of agamospermy.
This means that while short-tongue flies and beetles are in-
dicative of a ‘‘mess and soil’’ mode of pollination as described
by Faegri and van der Pijl (1979), our pollen tube analyses
suggest at least that some members of these orders are fairly
efficient agents of cross-pollination. Most vectors must transfer
a minimum of 1–8 viable grains of pollen from one plant to
a minimum of one flower on a second genet within 24 h after
the flower buds first open as almost 95% of all insect-polli-
nated pistils contained at least eight normal, penetrating tubes.
While less than two tubes penetrated ovules within 24 h, we
must remind ourselves that the tubes in the styles showed nor-
mal development and probably represented more than one in-
sect visit several hours apart. These tubes would have probably
reached the ovary had each one been allowed to grow the full
24 h as in the case of both sets of single-deposition, manually
manipulated cross-pollinations. As all flowers were harvested
24 h after the perianth expanded our results reflect insect vis-
itations limited to the first 25–33% of the actual floral life-
span.
Technically, X. tenax cannot be labeled as a ‘‘pollen-limit-
ed’’ species either, because even the stigmatic ridges of bagged
flowers remain liberally coated with grains due to mechanical
or wind-driven self-pollination. However, due to the early-act-
ing self-incompatibility system relatively few adhering grains
produce tubes that penetrate pistil tissue. Although insects
transport pollen between compatible genets, pollen tube and
fruit and seed set analyses show clearly that they don’t deposit
enough compatible grains to fertilize every ovule in the same
ovary. This may also be because some insect pollinators are
likely to transfer additional incompatible grains as they visit
more than one flower in succession on the same inflorescence.
Therefore this geitonogamous transfer of grains may decrease
the effectiveness of an insect’s cross-pollination service.
Cross-pollinations made by hand from single sires selected
from presumably different genets result in far more pollen
tubes penetrating ovules than the vast majority of cross-pol-
linations perpetuated by insects. Consequently this species
may be described as ‘‘compatible-pollen limited’’ for, although
this plant is a copious pollen producer and attracts many vec-
tors that cross-pollinate, in 95% of all flowers seed set remains
relatively low.
The species is further limited by a fairly substantial light
requirement to produce the large racemes. It is adapted to the
variation and unpredictability of climate in late May and early
June in its montane habitat by producing an inflorescence with
many flowers that open sequentially over the span of several
weeks. Despite that flowering trait, we observed at one site
almost all flowers damaged by a late frost. In addition, flowers
are browsed presumably by large ungulates (Young et al.,
1939; Simpson, 1990). As long as a population remains intact,
plants within a population maintain their genetic structure
through rhizomatous regeneration and growth. However, if a
population is reduced in number by severe fire (Bradley,1984)
or other disturbances that would displace whole plants, this
study suggests that demographic recovery through outcrossing
may be slow. Seeds do not germinate readily in cultivation
and have long stratification requirements (Smart and Minore,
1977), so it is unlikely that hand-planting seedlings currently
represents a feasible technology. The rhizomatous habit may
maintain individual genets particularly when environmental
conditions do not favor flowering. However, after natural dis-
turbance such as fire, X. tenax with an incompatibility system
and copious pollen production as an attractant can ensure suf-
ficient outcrossing to effect gene flow and remixing of alleles.
In addition, by providing food that attracts numerous and di-
verse pollinators, X. tenax is assuming an important functional
role in a montane pollinator system.
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