Crossing effects on seed viability and experimental germination of the federal threatened Platanthera leucophaea (Orchidaceae)

17 Pages·2002·8.4 MB·English

by  M L Bowles

RIIODORA.
Vol. 104, No. 917, 14-30. 2002
pp.
ON AND
CROSSING EFFECTS SEED VIABILITY
EXPERIMENTAL GERMINATION OF THE FEDERAL
THREATENED PLATANTHERA LEUCOFHAEA
(ORCHIDACEAE)
Marlin Bowlhs' and Karel A. Jacobs
L.
The Morton Arboretum. 4100 Route IL 60532
Illinois 53, Lisle,
[email protected]"
'e-niail:
Lawrhnce W. Zettler and Tonya Wieson Deeaney
Dcparlmenl of Biology. The College, 1101 West CoUege Ave
Illinois
62650
Jacksonville, IL
We
ABSTRACT. conclucted experimental pollination and seed germination
trials with PlatcnUhera Icucophaea. a threatened north temperate grassland
orchid species for which propagation and restoration are important conser-
Our how
vation needs. objectives were to determine the species' breeding
modes
system and of pollination interact to affect prtKluction of viable seed
how
and seed germination, and seed germination responds
to stratification
and to inoculation by mycorrhizal fungi. Outcrossing by hand pollination
produced a higher percentage of viable seed than did natural pollination, as
between
did outcrossing populations in C(Miiparison to outcrossing or selling
within populations, indicating a facultative outcrossing breeding system. Out-
crossing also enhanced percent germination, which was positi\ely correlated
A
with the percentage, but not number, of viable seeds. 16 wk.
stralilicatit)n
period resulted significantly higher perceni germination than 8 wk.
in strati-
hcation or no stratification. Cjcrmination was further enhanced by inoculation
with a mycorrhi/al fungus (Ceratorhiza sp.) isolated from P, leucophcicd.
These results indicate that the breeding system of P, lencophcwci allows for
numbers when
greater of viable seeds with greater germination rates plants
are outcrosscd. Thus, hand pollination and outcrossing can enhance propa-
when
gation and restoration efforts, especially coupled with scarification and
maximi/e
treatments eermination
stratification that rales.
Key
Words: Orchidaceae, seed germination, breeding
PlcitcifUhcrci, s}'stem,
conservation
pollination,
Gaining a better understanding of the propagation and resto-
become
ration reciuirements of terrestrial orchids has erueial to
Rasmussen
conservation (Johansen and
their 1992; Zettler
1996a), and an important conservation objective for the Federal
is
Threatened eastern prairie fringed orchid Plafanfhcra leucophaea
(Nutt,) Lindl. (Bowles and Bell 1999; U.S. Fish and Wildlife
Service 1999). This orchid occurs in tallgrass prairie reninants
14
—
Bowles germination
-Flatanthera 15
2002] et
al.
and wetlands eastern Iowa, southern Wisconsin, and northern
in
meadows,
and and sedge
Illinois Indiana, in shoreline prairies,
bogs, and fens eastward Maine (Bowles 1983; Sheviak 1974;
to
70%
Sheviak and Bowles 1986). has declined by more than
It
from original county records due to habitat conversion to agri-
culture. Remaining populations are often small and continue to
woody
be threatened by succession vegetation, competition
to
from exotic species, collecting, and drainage (Bowles 1983;
illicit
we
and on
U.S. Fish Wildlife Service 1999). In this study, report
on and
of experimental pollination seed production,
effects
its
effects of seed treatment and fungal inoculation on seed germi-
nation.
Showy
Pkitanthera are thought to have facultative outcrossing
known
breeding systems (Gregg 1990), a well strategy for avoid-
Willson
ance of deleterious effects of inbreeding 1983).
(e.g.,
kmcophaea and
Pkitanthera has the largest flowers nectar spurs
North American and
of eastern Pkitanlliera, large floral dis-
its
play and lack of vegetative reproduction indicate a strong in-
vestment toward pollination and seed production (Bowles 1983,
1985; Sheviak and Bowles 1986). Pollination by hawkmoths.
is
The orchid pollinarium, which comprises a pollinium (pollen
hawkmoth's
mass), caudicle, and viscidium, adheres to a probos-
movement
by the viscidium. Caudicle (taxis) positions the
cis
40
pollinium for contact with the stigmatic surface after about
seconds, thereby promoting outcrossing (Bowles 1985). Pollen
grains are then removed from the pollinium as they adhere to the
may when
Selfmg geitonogamy occur moths
plant's stigma. or
flowers or inflorescences, especially small orchid pop-
revisit in
and could potentially influence production of viable
ulations,
As
seeds this species affected by inbreeding depression. in
if is
&
praeckira Sheviak M. L. Bowles (Sieg and King 1995),
P.
median number
most flower once and of flowering
plants the
seasons than Seedling establishment therefore an
is less three. is
and and
important history stage for this species, pollination
life
(Bowles
seed production are factors population viability
critical in
and
Bell 1999).
propagate due physiolog-
Terrestrial orchids are difficult to to
dormancy
seed and the need for mycorrhizal fungi for suc-
ical
development (Rasmussen
cessful seed germination and seedling
1995; Stoutamire 1974; Zettler 1996b; Zetller and Mclnnis 1992).
Experimental propagation with mycorrhi/al fungi has been
re-
Rhodora
16 104
[Vol.
ported for a few species of the widespread north teiiiperate orchid
genus Platanthera, including Luer
P. intci^rilahia (Correll) (Zct-
and Mclnnis (Michx.) Luer and
tler 1992), P. clavellalci (Zettler
Hofer 1998), and P. leiicophaca (Zettler et 2001). Seed ger-
al.
was among
mination highly variable seed sources in these studies,
was
and facilitated by, but not dependent upon, the presence of
may many
Such
mycorrhizal fungi. variation be influenced by
factors, including sensitivity to inbreeding and levels of genetic
diversity within populations of different sizes Fenster and
(e.g.,
Dudash
1994; Weller 1994), and differing germination require-
ments or different experimental methods used by researchers
(Rasmussen
1995; Zettler 1996b).
Seed germination and mycorrhizal fungi of Platanthera
leu-
who
cophaea were
investigated by Curtis (1939), isolated
first
(now
species of the fungus Rhiz.octonia Ceratarhiza; Ander-
soil
and Rasmussen from
sen 1996) P. leucopliaea roots in different
was
habitats. Curtis unable to germinate seeds inoculated with
these fungi, perhaps due to failure to properly scarify or stratify
seeds. Stoulamire (1996) increased asymbiotic seed germination
rates by increasing scarification time diluted NaOCl, and rec-
in
ommended
two more months
or of cold Stoutamire
stratification.
(1996) also germinated leucopliaea seeds 35 Nitex
P. in bolt
nifx
Whigham
cloth (following Rasmussen and 1993) buried
in prairie
sod that contained soil fungi, but neither mycorrhi/ae nor further
development my-
seedling occurred. Zettler (2001) achieved
et
al.
cotrophic germination of leucopliaea seeds using Ceratorhiza
P, a
species isolated from roots of this orchid, with development of
leaf-bearing seedlings occurring after a second cold treatment.
More optimum
information needed and
^ibout pollination
is
germination requirements of Platanthera
leucopliaea, factors that
will lead to a better understandhig of reproductive biology,
its
population demographic processes, and restoration requirements.
Our we how
study had two related objectives. assessed
First,
modes
different of pollination natural versus hand pollina-
(i.e.,
tion, selfing, outcrossing within, and outcrossing between popu-
lations) affect the percentage of viable seeds and the germinabil-
ity oi seeds. Because of the breeding system of this species, we
expected outcrossing would enhance seed by reduc-
that viability
we
ing inbreeding depression. SeccMid, using scarified seed, tested
how
periods and symbiotic versus asymbiotic
stratification cc^n-
—
Bowles
2002 Plataiitliera termination 17
et al.
Table Seed sources, sample sizes, and seed colleclion dates for ex-
1.
=
number
perimental crossing and germination o^ Plcitcuithcra lencophaecr, n
sampled
of plants for seed.
Sampling Dale and Sample Size
by Experiment
Germination Crossing
Experiment Expcrimenl
Location
Site
Aug
Abbott Lake Co., 1996 Sep 1997
III.
Park
(n 3) (n 3)
Sep 1998
(n
1)
Wadsworth Lake Aug 1995 Sep 1997
Co.,
Ill
Prairie (n 111) (n 3)
Sep 1996 Sep 1998
(n=
30) (n 18)
Lyons Lake Sep 1996
Co.,
III.
Woods
(n 82)
Sandusky Oct 1995
Pickerel Co.,
Ohio
Creek
(n 10)
We
germination. expected longer
ditions affected that stratifica-
and symbiotic conditions wcuild enhance germination.
tion
AND MHTHODS
MATRRIALS
Seed and fungus sources. Pkitanthera leiicophaea seeds
were from one Ohio and
obtained three sites in Illinois
site in
(Table The Pickerel Creek, Ohio, contains one of the
site
1).
known
where occupy
largest P. leiicopluiea populations, plants
wethmd Lake
succcssional habitat of the Erie lake plain (U.S.
The Lake
Fish and Wildlife Service 1999). Illinois sites are in
County, in the Chicago region of northeastern Illinois, and are
km
from one The Wadsworth Lyons Woods,
15 other. Prairie,
among
and Abbott Park populations are the largest in Illinois,
meadow
with plants occurring in successional prairie and sedge
(U.S. Fish and Wildlife Service 1999). All seeds were collected
from mature capsules prior to dehiscence in late August or early
October, and were stored 5°C a desiccator (containing
at in
Rhodora
18 [Vol. 104
CaS04) sowing. The mycorrhi/al fungus used germi-
prior to in
nation treatments has been tentatively identified as a species of
W. and was from
Ceratorliizxi (L. Zettler, pers. obs.), isolated the
roots a mature P. leucopluiea specimen obtained from Abbott
ot"
Park and Morton Arboretum.
1995. cultured the
in at
Crossing on
effects seed viability* This study investigated
whether manually placing entire pollinia on stigmatic surfaces (=
hand
pollination) yielded a greater percentage of viable seeds than
Hand
natural pollination. pollination ccMisisted of crosses between
plants within populations, while natural pollination could also
have included selfing through geitonogamy. Pollinated plants
were not bagged to exclude subsequent natural pollination be-
cause placement of pollinium on stigma excludes
the entire the
were conducted
additional pollen deposition. Pollinations 1998,
in
with one or two mature capsules sampled per plant from ten nat-
urally pollinated plants and from nine hand-pollinated plants (Ta-
bic vSeeds were pooled from capsules within plants, and ap-
I).
proximately 200 seeds were sampled per Seeds were
plant. briefly
NaOCI, mo.
surface disinfested in dilute moist stratified for
1 1
(SDW)
sown
by suspending deionized water 6°C, and
in sterile
at
X cm cm
on a 4 filter paper strip in a 9 diameter petri dish
1
containing 20 ml of modified oats medium (Dixon 1987). The
dishes were then examined with dissecting microscope count
a to
nimibers of apparently viable and non-viable seeds based on the
presence or absence rounded and hyaline embryos
o[^ dislii^ct,
(Zcltler et 2001). Viable seed numbers were expressed as a
al.
percent of seeds each sample. These percentages were
the
total in
arcsine-transfbrmed (Zar 1974), and tested against the null hy-
pothesis that hand-pollinated capsules did not contain a greater
We
percentage oi viable seeds. used a one-tailed based on
t-test
our expectation of more viable seeds with hand pollinaticMi be-
maximizes
caLise pollen availability.
it
A
second study examined crossing effects on production of
Wads
viable seed using the worth Prairie and Abbott Park popu-
We
compared Wadsworth
and
lations. self-pollination plant
(1
1
Abbott plant), outcrossing within populations Abbott plant and
(1
3 Wadsworth plants), and reciprocal outcrossing between popu-
>
These
lations plants). pollinations included 5 (lowers per
(2
inflorescence, and were conducted 1997 (Table Seeds
in col-
1).
lected from mature capsules were pooled within each plant, dis-
—
20021 Be:)wles Platanthera izcrmination 19
et al.
NaOCl
by shaking 0.5% one and
infested, scarified for
in
hr.,
SDW.
wk. 5°C Seed suspension samples were
stratified for 8 at in
removed from with an eyedropper. Each sample con-
stratification
^
As
tained about 100 seeds, with 10 samples per cross. de-
numbers
scribed above, the of seeds containing round
distinct
embryos were counted with a dissecting microscope and ex-
One
pressed as a percent of the total seeds. of the seffed plants
0%
produce mature
did not capsules seeds, resulting
w^ith in vi-
able seeds for this cross. Differences between crossing treatments
were analyzed by inspection because the non-normal distribution
of data and unbalanced design prevented appropriate
statistical
testin^g oW.f t..h,eW hW.i^e.ra..r.c^IhWiVc..a.l. n..e^s.,tUi..nlg^ of seed sources within treat-
ments.
Stratification and symbiotic culture on germina-
effects
we
tion. In these experiments, tested effects of duration of
moist stratification, seed age (storage time), and presence or
cib-
sence of fungal inoculant on seed germination. Seeds were
dis-
We
NaOCl
and
infested scarified in ().59f for used seedling
hr.
1
development stages as defined by Hadley (1983), where Stage
1
germination achieved by rupturing o^ the testa (seed coat) by
is
the enlarging embryo, and Stage 2 germination coincides with
enlargement of the protocorm beyond the original seed size and
development Our
of rhi/oids. observations suggest that scarifi-
may
promote
cation Stage germination by water
facilitating
1
imbibition and rupture o[ the testa by the enlarging embryo. Tn
contrast, other studies Zettler and Hofer 1998; Zettler
(e.g., et
al.
2001) using unscarified seed describe germination Stage
to as
1
production of rhizoids, and Stage 2 as rupture of the In
testa. this
may
unscarified seeds germination by producing
situation, initiate
rhizoids that help imbibe water and then cause rupture of the
testa.
The
effect of stratification period on germination to Stage
1
was tested on seeds collected in 1996 (Table After scarifica-
1).
SDW
seeds were plated on paper moistened with
tion, sterile
filter
and given treatments of either no stratification (Abbott popula-
tion), or moist stratification in darkness at 4°C for 8 wk. (all sites)
wk. (Wadsworth and Lyons Seed numbers
or 16 populations).
ranged from 450 140 per population. Seeds were plated onto
to
1
medium
modified oats in petri dishes, with six to nine replicates
30-160
per treatment and seeds per dishes were
dish. Petri
Rhodora
20 [Vol. 104
wrapped and incubated 25°C darkness, and germi-
in foil at in
was monitored biweekly mo. with dissecting mi-
nation for a
1 I
croscope. Additional germination that might have occurred with
a second cold treatment was not considered in this experiment.
As described above, seed viability counts were based on the pres-
ence of round embryos. Counts of Stage germinated
distinct
1
among
were pooled within each treatment
seeds replicate plates
numbers
and by Chi-square analysis for differences of
tested in
among
germinated and ungerminated seeds periods,
stratification
and between Wadsworth and Lyons seed sources.
the
on
Effects of inoculant, hcati(Mi, and seed storage time
strati
germination Stace 2 were tested between the 1995 seed batch
to
—
(n 3621 seeds), which was stored for 18 mo., and the 1996
=
seed batch 1315 seeds), which was stored for 6 mo. (Table
(n
only seeds were used, and seeds were
Tn this study, scarified
1).
among
pooled seed sources. To whether a fungal inoculant
test
and stratification resulted in ^rcater ^germination than either treat-
^.v,^.vw»
ment 1995 and 1996 seed
separately, replicate plates for the
batches were given treatments of 16 wk. stratification, 16 wk.
As
stratification plus inoculant, or inoculant with Ceratorhizo sp.
medium
above, seeds were plated onto modihed oats and seed
viability counts were based on presence of distinct embryos. Ger-
The number
mination was monitored biweekly mo. of
for
1
1
was
Stage 2 seedlings on each plate expressed as a percent of the
number
on
of viable seeds originally present the plate.
ANOVA
A
was used compare germination
two-factorial to
treatment and seed storage time effects on percent germination.
Exchision of contaminated plates resulted in an unbalanced ex-
4-16 we
perimental design (replicates ranged from plates), which
General Linear Model.
tested using a Prior to analysis, all per-
We
centages were arcsine transformed (Zar 1974). also tested for
a correlation between the percentage of viable seeds and the per-
centage of those seeds reaching Stage To determine whether
2.
seedling development (and seed viability) was independent of
we
between percentage
seed density, tested for a correlation the
number
of viable seeds reaching Stage 2 and the total of seeds
and non- each
(both viable viable) in plate.
RESULTS
Crossing effects on percent viable seed. Seed viability var-
made
ied anions pollination crossing treatments in 1997 and in
—
Bowles
2002 PlaUuithera germination 21
et
al.
Seed
Crossing on Percent
Effect
Viability
Cross
Population
1.0
n
Between
\'--
1
Within
Self
0.8
0.5
03
CO 0.3
0.0
T^-^
2 4
2 2 3
1 1
Seed
Source
mean
Figure 1. DifTcrcnccs in percent viable seed produced by selfing,
between
crossing within, or crossing populations of Plataniliera leiicopliaea.
=
Seed Wadsworth X Wadsworth X Wads-
source replicates: Abbott, 2
1
= X = One Wadsworth
worth. 3 Abbott Abbott. 4 Abbott selied plant
self.
produced no viable seed. Lines represent standard errors.
50%
About from hand
1998. of the seeds capsules obtained
in
made
cross-pollinations 1998 contained viable embryos, almost
in
—
twice the percentage from naturally pollinated plants —1.785,
(t
P ^
made
0.046). Within-population outcrosses 1997 also av-
in
50%
eraged about viable seed, but between-population crosses
70%
among
averaged almost with wide variation means (Figure
One
self-pollinated plant produced no capsules with viable
1).
15%
while second averaged
seeds, the viable seeds.
rm
Effects of stratification period on Stage
1
tion. Stratification period, but not seed source, significantly af-
fected germination Stage with percent germination increas-
to
1,
ing with increasing period across seed sources
stratification
all
< 5%
was
(Figure Overall, germination for unslratified seeds,
2).
Rhodora
22
104
[Vol.
and Seed Source
Period
Stratification
Seed
on Germination
Effects
No
stratification
50
1
weeks
8
stratification
45-
40-
w
6 eeks
stratif ication
1
35-
o
30-
ro
25-
£
20-
0)
15-
10-
CO
5-
L E b
Lyons
Abbott V\fedsw Rx)led
orth
Seed Source
Figure Longer period increases percent seed germination
2. stratitieation
among
of rUiUtfUhcrci U'ucni?h(U'a, wilh similar effects seed sources. Chi-
=
— P <
sqnare: Stratification period 275.76, 0.001), Seed source
(x"
(x"^
r -
0.945, 0.332).
a 30% wk
10 20 vr cifter 8 wk. strati ficalion and after the 16
stratilication.
and
Effects of seed storage time, germination treatment,
No
seed on germination Stage
viability to 2. significant ef-
of seed storage time or germination treatment were found
fects
for Stage germination. However, Stage 2 germination (rhizoid
1
production) was significantly higher for seeds from the 1996 seed
batch than for seeds collected in 1995 and stored for an additional
12 (Figure Moreover, Stage 2 germination both seed
luo, in
3).
batches was higher for stratified seeds that were also germinated
symbiotically with Cenitorhiz.ci than for either treatment alone
Aniong
(Figure the 1996 seeds, percent germination to Stage
3).
was
2 also significantly correlated with percentage of viable seeds
(Figure Percent germination was not, however, significantly
4).
number ^ P
correlated with seed per plate 0.003, 0.85)
total
(r
—
Bowles
2002 Platanihera germination 23
et al.
Age
Treatment and Seed on Germination
Effect
Treatment
80.0
(3 Inoculated
noculated/stratified
Stratified
60.0
o
TO
C
£
40.0
CNi
(D
O)
TO
o
20.0
03
0.0
mo
6
18 mo.
Seed Age
The mean
Figure percentage of viable Phitcuitliera h-ncopJiaca seeds
3.
"erininatinii to vStaiie 2 is lower i'ov older seeds and Lzreater for stratified seeds
ANOVA:
inoculated with Ceraforln'za sp. isolated from P, leitcophaeci. Treat-
P X
ment (F 6-97, Aizc (F 20.74. O.OOOI), Treatment
().()03), I
=
Age P
(F 1.78, 0.1846). Lines represent standard errors.
^
P
nor with numt>cr of viable seeds per plate 0.008,
total
(r^
0.755).
DISCUSSION
Crossing effects on seed viability. As suggested for showy
Platcuithera (Gre gg 1990) our pollination experiments indicate
that P. leKcopliaea has a facultative outcrossing breeding system.
Because system allows mixed mating, apparently vul-
this
is
it
nerable inbreeding depression, which can be expressed
to at dif-
Dudash Du-
Carr and
ferent plant life-history stages 1996;
(e.g.,
Dudash
dash 1990; Fenster and 1994). Tn P. leucophaea, inbreed-
ing depression appears to have cascading effects liy decreasing
the percentage of capsules formed, the percentage ot viable seeds
within capsules, and the percent germination of those seeds. For
may
outcrossing species, process be alleviated larger pop-
this in
ulations that maintain hi^h levels of tienetic diversity (Schaal et