Journal of the Torrey Botanical Society 136(4), 2009, pp. 433–444
In vitro ecology of Calopogon tuberosus var. tuberosus
(Orchidaceae) seedlings from distant populations:
implications for assessing ecotypic differentiation1
Philip J. Kauth2 and Michael E. Kane
Plant Restoration, Conservation, and Propagation Biotechnology Program, Environmental Horticulture
Department, University of Florida, Gainesville, FL 3261
KAUTH, P. J. AND M. E. KANE (Plant Restoration, Conservation, and Propagation Biotechnology
Program, Environmental Horticulture Department, University of Florida, Gainesville, FL 32611). In vitro
ecology among four populations of Calopogon tuberosus var. tuberosus (Orchidaceae): implications for
ecotypic differentiation. J. Torrey Bot. Soc. 136: 000–000. 2009.—In vitro culture techniques can be used to
study the unique growth habits of plants as well as the ecological factors that influence seedling growth and
development (i.e., in vitro ecology) such as adaptation to local environmental conditions. The in vitro
seedling ecology of Calopogon tuberosus var. tuberosus from Michigan, South Carolina, and Florida was
studied with emphasis on timing of corm formation and biomass allocation. In vitro seedling growth and
development were monitored for 20 weeks. Corm formation was most rapid in Michigan seedlings, but was
progressively delayed in southern populations. Similarly, biomass allocation to corms was highest in
Michigan seedlings while south Florida seedlings exhibited the lowest corm biomass allocation. Shoot
senescence in vitro also began earlier in more northern populations. The rapid corm formation and biomass
allocation in seedlings from more northern populations represents an adaptive response to a shorter growing
season. The relative differences in corm formation, biomass allocation, and shoot senescence in C. tuberosus
seedlings suggest that in vitro common garden studies are useful to assess the degree of ecotypic
differentiation among populations for a wide range of ecological factors. Additionally, these in vitro
techniques can be transferred to numerous species worldwide.
Key words: biomass allocation, common garden study, corm, ecotype, orchid.
Introduction. Widely distributed plant species have evolved the ability to survive broad
environmental conditions leading to local
adaptation to biotic and abiotic conditions
(Linhart 1995, Joshi et al. 2001, Sanders and
McGraw 2005). Local adaptation in plants
was first examined using common garden
studies by Turesson (1922), who first used
the term ecotype, and by Clausen et al. (1941)
using reciprocal transplant studies. The importance of using appropriate ecotypes for
conservation and restoration studies has been
recently highlighted. Using locally adapted
plant material for restoration purposes may be
necessary to maintain ecosystem function and
1 We thank Larry Richardson (Wildlife Biologist;
Florida Panther National Wildlife Refuge), Jim
Fowler (South Carolina population), and Kip
Knudson (Michigan population) for collecting
seeds. We also thank Mary Bunch (South Carolina
Heritage Preserve Program). We also thank the U.S.
Fish and Wildlife-Florida Panther National Wildlife
Refuge for assisting with partial financial support.
Brand names are provided as references only and we
do not solely recommend these products.
2 Author for correspondence. E-mail: pkauth@
ufl.edu
Received for publication February 2, 2009, and in
revised form August 27, 2009.
stability since non-locally adapted ecotypes
can reduce plant population fitness (Linhart
and Grant 1996, Hufford and Mazer 2003,
McKay et al. 2005).
Local adaptation has been studied in
numerous species through common garden
and reciprocal transplant experiments (Nuismer and Gandon 2008). Common garden
studies test local adaptation and fitness of
individuals from local or distant habitats in a
common environment. Common garden studies may more efficiently test the genetic
contribution to fitness while minimizing environmental impacts on fitness. Transplant
studies may better estimate environmental
variation since individuals are transplanted
to habitats with environmental conditions not
experienced in the natural habitat (Nuismer
and Gandon, 2008). Local adaptation can be
studied by examining performance of ecotypes
under different photoperiods (Howe et al.
1995, Kurepin et al. 2007), temperatures
(Seneca 1972, Probert et al. 1985), and soil
regimes (Grześ 2007, Sambatti and Rice 2007).
Differences in biomass allocation have also
been proposed as an important aspect of
ecotypic differentiation. Northern ecotypes of
Spartina alterniflora allocated more biomass
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to underground organs including roots and
rhizomes (Gallagher 1983, Gallagher and
Howarth 1987, Gross et al. 1991). Greater
biomass allocation to underground organs in
northern ecotypes of several species was due to
a shorter growing season (Potvin 1986, Sawada et al. 1994, Kane et al. 2000, Liancourt
and Tielbörger 2009) and a higher allocation
of carbohydrate reserves to overwintering
structures (Mooney and Billings 1960). Biomass allocation has also been correlated with
various reproductive strategies in ecotypes.
Ecotypes found in fields or areas of younger
succession allocated more biomass to reproductive organs than those in wooded habitats
that allocated more biomass to vegetative
structures (Abrahamson 1975, 1979). Marsh
plants that occupied areas of greater disturbance allocated more biomass and carbohydrate reserves to underground storage organs
(Sun et al. 2001, Peñas-Fronteras et al. 2009).
Common garden and transplant studies can
be performed in greenhouses, growth chambers, natural habitats, and outdoor plots
(Gallagher et al. 1988, Howe et al. 1995,
Majerowicz et al. 2000, Suzuki 2008), but
obtaining permits to collect and transplant
protected, rare, threatened, or endangered
species, as many orchids are, is difficult. Seeds
can be used to produce mature plants for
common garden and transplant studies. While
this may be an effective method for quickgrowing species, orchids often require four or
more years to flower from initial seed germination (Stoutamire 1964). Additionally, in situ
orchid seed germination is difficult and time
consuming since germination is often low
(Brundrett et al. 2003, Zettler et al. 2005, Diez
2007). Alternatively, in vitro techniques can be
used to study environmental requirements for
orchid seed germination (Kauth et al. 2008) as
well as seedling growth and development (Dijk
and Eck 1995).
Many in vitro culture techniques can be
grouped under the discipline of in vitro
ecology. In vitro ecology has been previously
defined to include environmental and exogenous factors (i.e., temperature, light, gas
phase, culture media) that affect in vitro
growth and development (Hughes 1981, Williams 2007). Here, we further define in vitro
ecology to include the evaluation and use of in
vitro culture techniques to identify, propagate,
evaluate, and select plant genotypes and
ecotypes for ecological purposes. Specifically,
[VOL. 136
in vitro ecology studies can be used to
correlate environmental and genetic variables
that affect plant growth and development in
vitro with ecological factors affecting growth
and development in situ. In vitro ecology
could also be used to assess ecotypic differentiation for habitat restoration and plant
reintroduction programs by conducting in
vitro common garden studies under controlled
environmental conditions. Since this use of in
vitro ecology is a new area of research its
validity must be verified.
Calopogon tuberosus var. tuberosus (L.)
Britton, Sterns, & Poggenberg is a terrestrial
orchid native to eastern North America, and
occupies diverse habitats such as wet prairies,
pine flatwoods, roadsides, fens, and sphagnum
bogs. Based on morphological variation,
Goldman et al. (2004) defined three specific
geographic areas for C. tuberosus: northern
plants in glaciated areas, southwest plants west
of the Mississippi Embayment, and southeast
plants east of the Mississippi River and south
of the glaciated zone. However, Goldman et
al. (2004) did not classify C. tuberosus
ecotypes, but stated that variation in C.
tuberosus could be caused by environmental
conditions. Further ecotypic differentiation
has not been previously explored in C.
tuberosus. Additionally, little information exists on ecotypic differentiation of orchids.
Although morphological and genetic variation
exists in C. tuberosus, all plants throughout its
range form corms. Differences in biomass
allocation among C. tuberosus populations
have been previously reported (Kauth et al.
2008). However, a detailed timecourse comparison for C. tuberosus seedling development
has not been reported, and little information
exists regarding the influence of storage organ
biomass allocation on ecotypic differentiation.
Evaluation of in vitro seedling development
from several Calopogon tuberosus populations
from diverse geographic sources might clarify
the extent of ecotypic differentiation across its
range. In this study, the in vitro ecology of C.
tuberosus seedlings was studied in relation to
corm formation, biomass allocation, and
geographic source. Additionally, our goal is
to confirm the effectiveness of using an in vitro
common garden study to aid in differentiating
C. tuberosus ecotypes.
Materials and Methods. SEED COLLECTION.
Seeds were collected throughout summer 2007
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Elevational data from Google Earth.
Photoperiod and temperature data from Weather Underground, Inc.
Data from the 2007 Climatological Data Annual Summary.
d
Growing season length is the number of days between the last spring frost (above 0uC) and first fall frost (below 0uC).
c
b
a
Michigan (Escanaba)
South Carolina (Greenville)
North Central Florida (Ocala)
435
from the following locations (Table 1): upper
peninsular Michigan (Menominee County,
Michigan), Blue Ridge Escarpment (Greenville County, South Carolina), north central
Florida (Levy County, Florida), and Florida
Panther National Wildlife Refuge (Collier
County, Florida). Seed capsules from at least
three parent plants in each population were
collected before complete dehiscence and
stored at 23uC over silica gel for two weeks.
Seeds were then removed from capsules,
pooled by geographic source, and stored in
complete darkness at 211uC until used.
740
4.9
240
Northern fen
125
15.7
8.8
24.4 (Jul.) 212.8 (Dec.)
800
16.5
500
Cataract bog
210
14.5
9.8
34.2 (Jun.)
0.3 (Jan.)
1400
21.7
7.8 (Jan.)
12
Mesic roadside
270
14.1
10.3
33.4 (Jul.)
22.1
10.5
13.8
365
4
Wet prairie
26u109060 N
81u219510 W
29u09180 N
82u37120 W
35u049280 N
82u369190 W
45u349470 N
87u399380 W
South Florida (Naples)
Elev. (m)
Habitat
Population (weather center)
32.1 (Jun.)
11.9 (Jan.)
Total precipitationc
(mm)
Mean
Min
Max
Min
Max
Average monthly air temperatureb (uC)
Day lengthb (hrs)
Growing
seasonc,,d
a
Population
Coordinates
Location, habitat, and environmental conditions of Calopogon tuberosus var. tuberosus seed sources used in the present study.
Table 1.
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SEED AND GERMINATION MEDIUM PREPARASeeds were surface disinfected in sterile
scintillation vials for 3 minutes in a solution of
5 ml absolute ethanol, 5 ml 6% NaOCl, and
90 ml sterile distilled-deionized (dd) water.
Seeds were rinsed with sterile dd water after
surface sterilization, and solutions were removed with sterile Pasteur pipettes.
Seeds were transferred with a sterile inoculating loop to BM-1 Terrestrial Orchid Medium (PhytoTechnology Laboratories, Shawnee
Mission, KS, USA) in 100 3 15 mm Petri
plates (Fisher Scientific, Pittsburgh, PA,
USA). BM-1 medium was selected based on
prior Calopogon tuberosus germination and
seedling development performance (Kauth et
al. 2008b). The medium was supplemented
with 1% activated charcoal. Medium pH was
adjusted to 5.7 with 0.1 N KOH prior to
autoclaving for 40 minutes at 117.7 kPa and
121uC. Ten replicate Petri plates with 30 ml
medium each were used for each seed source
with approximately 100 seeds per plate. Cultures were placed in an environmental growth
chamber (#I-35LL; Percival Scientific, Perry,
IA, USA) under cool-white fluorescent lights
in a 12/12 hr photoperiod at 24.2 6 0.2uC and
a light level of 40 mmol m22 s21.
TION.
SEEDLING TRANSFER AND DATA COLLECTION.
After 6 weeks culture seedlings were transferred from Petri plates to PhytoTech Culture
Boxes (PhytoTechnology Laboratories) containing 100 ml of BM-1 medium. Medium was
prepared as described previously. Uniformsized seedlings with developing leaves were
then transferred to individual culture boxes.
Three PhytoTech Culture Boxes with nine
seedlings each were prepared per seed source
for each week. A total of 21 PhytoTech
Culture Boxes were prepared per seed source.
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[VOL. 136
FIG. 1. In vitro seedling development of Calopogon tuberosus from distinct geographic sources. Note the
delayed corm formation in southern populations. Shoot die-back was characterized by yellowing and
browning of leaves. A–D. Seedlings after 8 weeks culture. E–H. Seedlings after 12 weeks culture. I–L.
Seedlings after 16 weeks culture. M–P. Seedlings after 20 weeks culture. A, E, I, M. Michigan seedlings. Scale
bars 5 1 cm.
Cultures were completely randomized within
the growth chamber under the same conditions previously described.
Data were collected bi-weekly on three
replicate PhytoTech Culture Boxes containing
nine seedlings each per seed source. Data were
taken on 27 seedlings per seed source each
week. Data for week 10 South Carolina
seedlings were collected on two replications
due to contamination of one replicate. The
following data were collected: shoot length,
root number, root length, corm diameter, and
dry weight. Shoot, root, and corm dry weights
were measured after tissues were dried for 24 h
at 60uC. Seedling percent biomass allocation
was determined by dividing corm, root, and
shoot weights by the total seedling weight.
Shoot length, root number, root length, corm
diameter, and biomass data were statistically
analyzed using general linear procedures,
ANOVA, and Tukey’s HSD test at a 5 0.05
in SAS 9.1 (SAS Institute 2003). Regression
and Pearson’s correlation analyses were performed on corm biomass allocation and
growing season length reported in Table 1.
Corm biomass allocation data were arcsine
transformed prior to regression analysis.
Results. CORM FORMATION. Corm formation
differed significantly by population (F 5
73.86, P , 0.0001), week (F 5 70.54, P ,
0.0001), as well as population by week (F 5
15.12, P , 0.0001). Corm formation on
Michigan seedlings was evident by week 8,
week 10 on South Carolina seedlings, week 14
on north central Florida seedlings, and week
18 on south Florida seedlings (Fig. 1, Table 2). Initial mean corm diameter on Michigan and South Carolina seedlings was similar
until week 16 (Table 2). Mean corm diameter
on Michigan seedlings did not change significantly after week 14. Mean corm diameter
was similar in Michigan and south Florida
seedlings, but south Florida seedlings continued to grow after week 20 while Michigan
seedlings were fully dormant (pers. obs). Mean
corm diameter was largest on South Carolina
and north central Florida seedlings at week 20.
SHOOT LENGTH. Shoot lengths were significantly different among populations (F 5
340.78, P , 0.0001), week (F 5 340.78, P ,
0.0001), and population by week (F 5 45.76, P
, 0.0001). Initial shoot lengths on Michigan
and South Carolina seedlings were larger than
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4.62c
3.69b
3.16b
1.43b
2.07a
1.54a
10.6 6 0.69b
E
22.4 6 1.32b
DE
40.0 6 2.99b
D
74.4 6 5.01a
C
111.1 6 6.2a
B
137.2 6 8.85a
A
131.9 6 9.13a
A
0.55a
2.34 6 0.16c
A
2.59 6 0.22b
A
0
0
2.05 6 0.16b
D
3.59 6 0.24a
C
4.49 6 0.23a
B
5.48 6 0.23a
A
0
0
0
0
1.78 6 0.08b
D
2.18 6 0.09a
CD
2.75 6 0.15a
C
3.90 6 0.12a
B
4.18 6 0.15a
AB
4.76 6 0.16a
A
Week 20
Week 18
Week 16
Week 14
Week 12
Week 10
Week 8
1.42 6 0.07a
D
1.73 6 0.09a
CD
2.11 6 0.08a
BC
2.37 6 0.09a
AB
2.60 6 0.10b
A
2.72 6 0.12b
A
2.77 6 0.16b
A
0
0
0
13.4 6 0.88a
A
14.4 6 0.95c
A
14.7 6 0.91c
A
14.9 6 1.06d
A
11.7 6 1.22c
A
6.0 6 1.49c
B
2.2 6 1.66d
C
13.8 6
C
29.0 6
B
5A0.2 6
A
52.2 6
A
50.0 6
A
49.9 6
A
48.0 6
A
South Florida
North Central Florida
Shoot length (mm)
South Carolina
Michigan
South Florida
North Central Florida
Corm diameter (mm)
South Carolina
Michigan
Table 2. Mean (6 SE) corm diameter and shoot length measurements of Calopogon tuberosus var. tuberosus seedlings from four populations after 20 weeks in vitro
culture. Means with the same letter (lowercase 5 among population comparisons; uppercase 5 weekly comparison within populations) are not significantly different
according to Tukey’s HSD test at a 5 0.05.
5.04 6 0.35c
D
9.56 6 1.05d
D
22.8 6 2.71c
C
29.3 6 2.23c
C
41.8 6 3.66b
BC
52.4 6 4.42b
B
90.7 6 7.25b
A
KAUTH AND KANE: IN VITRO ECOLOGY OF CALOPOGON TUBEROSUS
2009]
437
both Florida populations (Table 2). After
week 12, mean shoot length on Michigan
seedlings were the shortest of all seedlings.
Shoot growth on Michigan seedlings did not
significantly increase from week 8 to 16, but
did decrease significantly there after. Similarly,
shoot growth did not increase significantly on
South Carolina seedlings from week 12 to 20.
Shoots on north central Florida seedlings were
the largest by week 14, and growth continued
to increase until week 18. Shoot growth on
south Florida seedlings was initially small, and
only north central Florida seedlings exceeded
mean shoot length of south Florida seedlings
at week 20.
Shoot senescence, characterized by yellowing and browning of leaves, began on Michigan seedlings after 16 weeks culture, and by
week 20 almost 100% of shoots were senesced
(Fig. 1M). Shoot senescence was delayed in
southern populations. Shoot senescence on
South Carolina seedlings did not occur until
24 weeks culture, 32 weeks culture on north
central Florida seedlings, and 38 weeks on
south Florida seedlings (pers. obs.).
ROOT LENGTH AND NUMBER. Root length
was significantly influenced by population (F
5 161.91, P , 0.0001), week (F 5 89.62, P ,
0.0001), and population by week (F 5 12.61, P
, 0.0001). Root elongation was similar in
Michigan, South Carolina, and north central
Florida seedlings after 8 weeks culture (Table 3). By week 14, mean root length was
longest on north central Florida seedlings,
while few differences were observed in south
Florida and South Carolina roots. Mean root
length on Michigan seedlings was generally the
shortest. Root length increased in south
Florida and north central Florida seedlings
throughout the experiment. Mean root length
decreased on Michigan seedlings after week 18
due to root die-back, which was characterized
by shriveling and browning of roots. After 20
weeks culture, roots were longest on north
central Florida seedlings.
Population (F 5 238.57, P , 0.0001), week
(F 5 59.24, P , 0.0001), and population by
week (F 5 18.34, P , 0.0001) all significantly
influenced root number. Root number on
north central Florida seedlings increased
significantly, and by week 16 they contained
the highest number of roots (Table 3). Root
number on Michigan seedlings was initially
similar to South Carolina and north central
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0.33 6 0.09b
C
0.56 6 0.11c
C
1.37 6 0.15b
B
2.15 6 0.17b
A
2.22 6 0.10c
A
2.63 6 0.22b
A
2.59 6 0.13b
A
1.04 6 0.06a
E
2.33 6 0.12b
D
2.56 6 0.13a
D
3.00 6 0.16a
CD
3.85 6 0.27a
BC
4.85 6 0.38a
A
4.11 6 0.30a
AB
1.33 6 0.09a
B
2.83 6 0.09a
A
2.52 6 0.10a
A
2.74 6 0.13a
A
2.89 6 0.15b
A
2.85 6 0.13b
A
2.67 6 0.15b
A
1.22 6 0.10a
AB
0.96 6 0.11c
AB
1.33 6 0.11b
A
1.07 6 0.12c
AB
1.04 6 0.14d
AB
0.81 6 0.12c
BC
0.33 6 0.12c
C
1.3 6 0.37b
D
5.4 6 1.55b
D
21.7 6 2.34ab
C
19.1 6 1.58c
C
31.0 6 2.63b
B
30.9 6 2.99b
B
43.9 6 3.64b
A
10.0 6 0.85a
DE
23.3 6 1.70a
D
29.1 6 2.32a
D
42.4 6 1.90a
C
52.4 6 2.82a
BC
58.5 6 3.41a
A
65.5 6 3.54a
A
10.7 6 0.60a
D
19.2 6 1.48a
CD
27.1 6 1.26a
BC
33.6 6 1.07b
AB
38.6 6 2.39b
A
39.7 6 2.59b
A
41.1 6 3.99b
A
Week 20
Week 18
Week 16
Week 14
Week 12
Week 10
10.0 6 0.93a
A
9.6 6 1.30b
A
16.8 6 2.21b
A
12.9 6 2.13c
A
13.1 6 1.91c
A
17.3 6 4.89c
A
7.3 6 3.43c
A
Week 8
North Central Florida
South Carolina
North Central Florida
South Carolina
Root Length (mm)
South Florida
Michigan
Root Number
South Florida
JOURNAL OF THE TORREY BOTANICAL SOCIETY
Michigan
Table 3. Mean (6 SE) root length and root number on Calopogon tuberosus var. tuberosus seedlings from four populations after 20 weeks in vitro culture. Means
with the same letter (lowercase 5 among population comparisons by week; uppercase 5 weekly comparison within populations) are not significantly different
according to Tukey’s HSD test at a 5 0.05.
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Florida seedlings, but by week 14 Michigan
seedlings had the lowest root number.
BIOMASS ALLOCATION. ANOVA results revealed that percent biomass allocation to
shoots, corms, and roots differed significantly
among populations (Table 4). Corm biomass
allocation was inversely related to latitude
with the highest allocation being observed on
Michigan seedlings. Approximately 97% biomass was allocated to corms in Michigan
seedlings by week 20, which was significantly
higher than the 77% to corms in South
Carolina seedlings, 53% to corms in north
central Florida seedlings, and 7% to corms in
south Florida seedlings (Fig. 2). Greater corm
biomass allocation was evident on Michigan
seedlings by week 8, and continued throughout the experiment (Fig. 2C). Corm biomass
allocation on South Carolina seedlings was
significantly greater than both Florida populations, and north central Florida greater than
south Florida with the exception of week 10
and 12 when corms were not present (Fig. 2C).
Percent shoot biomass allocation generally
declined among populations throughout the
experiment (Fig. 2A). However, shoot biomass allocation was significantly higher on
south Florida seedlings than all other populations. South Florida seedlings allocated more
biomass to shoots compared to roots and
corms over the 20 wk period. Shoot and root
biomass allocation of Michigan seedlings
decreased simultaneously. After week 10,
shoot and root biomass allocation in South
Carolina and north central Florida seedlings
followed the same trend. Root biomass
allocation was significantly higher on south
Florida seedlings compared to all other
populations, while root biomass was lowest
on Michigan seedlings (Fig. 2B).
Correlation analysis revealed a strong negative correlation between corm biomass allocation and growing season length so that as
percent corm biomass allocation increased the
length of growing season decreased. Growing
season was considered the number of days
between the first spring and last fall frost.
Pearson’s correlation coefficients (all P values
, 0.0001) were as follows: 20.73 (all weeks),
20.67 (week 8); 20.81 (week 10); 20.87 (week
12); 20.93 (week 14); 20.96 (week 16); 20.91
(week 18); 20.95 (week 20). Regression
analysis also revealed a negative trend for all
weeks (Fig. 3). With the exception of week 8
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Table 4. ANOVA results showing main effects and interactions contributing to variation in percent
shoot, root, and corm biomass allocation of Calopogon tuberosus seedlings over a 20 week period.
Shoot
Corm
Root
Source of variation
df
F
P
df
F
P
df
F
P
Population
Week
Population 3 week
3
6
18
642.6
236.1
6.72
, 0.0001
, 0.0001
, 0.0001
3
6
18
166.8
31.7
35.3
, 0.0001
, 0.0001
, 0.0001
3
6
18
2154.7
527.9
54.4
, 0.0001
, 0.0001
, 0.0001
and 10, regression models accounted for much
of the data variance with strong r2 values over
0.75 (Fig. 3). Due to the lack of corm
formation in week 8 and 10 data, r2 values
were not as strong (Fig. 3). When weekly data
FIG. 2. Shoot, root, and corm biomass allocation of Calopogon tuberosus seedlings over 20 weeks
in vitro culture. Each data point represents the mean
of three replications 6 1 standard error. Data points
with the same letter are not significantly different
according to Tukey’s HSD test at a 5 0.05.
were combined the r2 was 0.54, but the model
was significant.
Discussion. This study represents the application of in vitro ecology to assess the extent
of ecotypic differentiation of a latitudinally
widespread orchid species. Although information connecting the timing of biomass allocation to ecotypic development is scarce (Gallagher 1983, Gallagher and Howarth 1987,
Gross et al. 1991, Seliksar et al. 2002, Yoshie
2007), timing of corm formation is an
important factor in the ecotypic development
of Calopogon tuberosus. Few published articles
exist that utilize in vitro techniques to correlate
ecotypic life history traits with in vitro growth
strategies of orchids (Dijk and Eck 1995,
Kauth et al. 2008). The present results also
indicate the potential use of in vitro common
garden studies to detect unique growth strategies. In particular biomass allocation in C.
tuberosus ecotypes is influenced by growing
season length.
Biomass allocation dynamics and storage
organ function have been previously described
in situ for single orchid populations (Whigham
1984, Snow and Whigham 1989, Zimmerman
and Whigham 1992, Tissue et al. 1995, Øien
and Pederson 2003, 2005). However, biomass
allocation in orchids has not been explored
with respect to ecotypic differentiation. In the
present study, Calopogon tuberosus biomass
allocation to corms ranged from 7% to 97%,
depending on seed source. Whigham (1984)
reported nearly 80% of biomass in a single
Tipularia discolor population was allocated to
underground storage organs. Zimmerman and
Whigham (1992) reported that 61% and 66%
of the total non-structural carbohydrates were
allocated to the youngest corms in vegetative
and dormant plants, respectively. In a detailed
analysis of biomass allocation in T. discolor,
66% of the total biomass was allocated to
corms during fruit maturation and 80% during
leaf senescence (Tissue et al. 1995). These data
are comparable to C. tuberosus since more
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FIG. 3. Correlation of growing season length and percent corm biomass allocation represented as mg of
dry weight per total dry weight. Each value point represents the mean response of three replications with nine
seedlings each. Corm biomass percentages were arcsine transformed prior to regression analysis. Regression
analysis was performed for each week as well as pooled data combing all weekly data. Note that N 5 99 in
week 10 due to contamination of one South Carolina replication.
biomass was allocated to corms just prior to
and during leaf senescence. Although carbohydrate analysis of C. tuberosus was not
investigated, reallocation of carbohydrates
from leaves to corms might explain increased
corm biomass allocation in C. tuberosus as was
similarly reported for Dactylorhiza lapponica
tubers (Øien and Pederson 2005).
Regardless of orchid species, storage organs
such as corms represent ecological adaptations
to ensure survival during unfavorable growing
conditions. In Tipularia discolor corms are vital
to support growth and reproduction (Zimmerman and Whigham 1992, Tissue et al. 1995),
and serve as sinks for nutrient reserves (Whigham 1984). Corms may also aid in long term
survival by protecting the shoot meristem
during periods of stress (Whigham 1984).
Greater and faster biomass allocation to
underground organs in northern Calopogon
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tuberosus ecotypes followed a similar trend to
ecotypes of Spartina alterniflora (Gallagher
1983, Gallagher and Howarth 1987, Gross et
al. 1991) and Sagittaria latifolia (Kane et al.
2000, Kane et al. 2003). The faster biomass
allocation to corms in C. tuberosus is likely a
selection pressure favored by the shorter
growing season at northern latitudes as
reported with ecotypes of S. alterniflora
(Seliksar et al. 2002), Plantago asiatica (Sawada et al. 1994), grass species (Potvin 1986,
Liancourt and Tielbörger 2009), and Eriophorum vaginatum (Fetcher and Shaver 1990).
Northern ecotypes of Calopogon tuberosus
may allocate larger carbohydrate reserves in
storage organs to survive winter conditions,
and subsequently reallocate those carbohydrates to rapid growth the following spring
(Seliksar et al. 2002). Greater corm biomass in
northern C. tuberosus ecotypes could be
influenced by faster reallocation of carbohydrates from shoots to corms leading to faster
shoot senescence compared to southern ecotypes (Mooney and Billings 1960). Further
investigation may also determine whether
northern ecotypes are more tolerant to freezing temperatures due to higher corm carbohydrate reserves.
A short life cycle from initial shoot production to shoot senescence as well as low
temperature tolerance is an adaptation to
northern environments where the growing
season is short (Potvin 1986). Even under the
same environmental conditions in vitro, northern Calopogon tuberosus ecotypes expressed a
shorter growth cycle and faster corm biomass
allocation. Since seeds were collected directly
from wild populations, pre-conditioned environmental carry-over effects may have explained this adaptation. A long-term genetic
adaptation to shorter growing seasons may
also explain the differences in growth (Shaver
et al. 1986), and plants from northern latitudes
may always express the shorter life cycle and
greater corm biomass allocation regardless of
environmental conditions. The adaptation
may also be a consequence of primary
productivity where plants from northern
latitudes are not able to take advantage of
increased temperatures or constant growing
conditions (Fetcher and Shaver 1990).
Greater biomass to corms may represent a
successful survival strategy. The populations
used in the present study from Michigan and
South Carolina have long periods of water
441
availability in the form of ground water
(Nelson 1986, Cohen and Kost 2008), while
populations in Florida experience distinct dry
seasons (Davis 1943). Ecotypes in areas prone
to flooding allocated more biomass and
carbohydrates to corms and tubers indicating
a vegetative growth strategy (Li et al. 2001,
Sun et al. 2001, Peñas-Fronteras et al. 2009).
Higher biomass to underground storage organs
may be a response to prolonged flooding when
plants would need a readily available source of
carbohydrates (Peñas-Fronteras et al. 2009).
Growth differences may be related to reproductive strategy as well. Florida populations in
the present study produce more flowers and
seed capsules then the plants in Michigan and
South Carolina, which may lead to higher seed
production (Peñas-Fronteras et al. 2009).
Higher seed production may be necessary in
order to colonize areas of earlier succession
such as prairies and non-wooded areas in south
Florida (Abrahamson 1975, 1979).
Differences in root number, length, and
biomass of Calopogon tuberosus ecotypes may
be related to soil nutrient and water availability. Biomass allocation to roots was greater in
several annual plant species and Populus
davidiana ecotypes under low nutrient and
water stressed soils (McConnaughay and
Coleman 1999, Zhang et al. 2005). Massachusetts ecotypes of Spartina alterniflora were
found to have shorter roots due to the shallow,
organic soils compared to the deeper sandbased soils in Georgia (Seliksar et al. 2002).
Longer or deeper roots on southern C.
tuberosus ecotypes may be an adaptation to
water-stressed environments where the upper
soil layers have poor water availability
(Kondo et al. 2003).
Shoot biomass as well as shoot length on
Calopogon tuberosus was highest in Florida
populations that experience higher growing
temperatures. The larger shoots on Florida C.
tuberosus seedlings may be a selection pressure
to maximize photosynthesis to outcompete
vegetation during a longer growing season
(Gallagher and Howarth 1987). A higher
shoot biomass may be a requirement to reach
reproductive size to set seed before adverse
environmental conditions are experienced
(Rice et al. 1992). Faster shoot growth in
Michigan seedlings may be due to earlier
carbohydrate allocation.
Common garden studies are useful tools to
detect local adaptation influenced by genetics,
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but often diminish the impact of environmental conditions in situ (Nuismer and Gandon
2008). Transplant and reciprocal transplant
studies better indicate environmental effects
on local adaptation (Nuismer and Gandon
2008). Conditions in vitro can be controlled to
represent in situ conditions by controlling
environmental conditions experienced across
a species’ distribution such as photoperiod,
temperature, and humidity. Using a captive
generation of seeds may be necessary to
further investigate the role of environment
and genetics on local adaptation. Growing
Calopogon tuberosus seedlings in vitro under
different temperatures or photoperiods may
lead to different results. However, biomass
allocation in the South Carolina and Florida
populations were unaffected by different
photoperiods after 16 weeks culture. Michigan
seedlings did allocate more biomass to shoots
in short days, while neutral and long days
promoted higher corm biomass allocation
(Kauth et al. 2008).
Screening for ecotypic differentiation is an
exciting application for in vitro culture, and in
vitro common garden studies can be effective
at detecting different growth strategies. This
study along with Kauth et al. (2008) provides
strong evidence that, based on the differences
in biomass allocation, Calopogon tuberosus
ecotypes exist. The adaptations presented in
this study are likely influenced by environmental conditions at each site such as photoperiod, temperature, and growing season
length. Rapid corm formation in seedlings
from northern populations may be due to
shorter growing seasons. Conversely, slower
corm formation in seedlings from southern
populations is a possible adaptation to a
longer growing season. While in vitro studies
may provide be appropriate to exam local
adaptation, combining in situ and in vitro
studies may provide better insight into ecotypic differentiation of C. tuberosus.
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