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Genetic diversity in North American populations of carex viridula michx. (cyperaceae)

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Title:
Genetic diversity in North American populations of carex viridula michx. (cyperaceae)
Creator:
Kuchel, Shannon D
Publication Date:
Language:
English
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ix, 45 leaves : ; 28 cm

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Subjects / Keywords:
Cyperaceae -- Genetics -- Colorado ( lcsh )
Plant genetics -- Research ( lcsh )
Plant diversity -- North America ( lcsh )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 39-45).
General Note:
Department of Integrative Biology
Statement of Responsibility:
by Shannon D. Kuchel.

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|University of Colorado Denver
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|Auraria Library
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
43923053 ( OCLC )
ocm43923053
Classification:
LD1190.L45 1999m .K83 ( lcc )

Full Text
GENETIC DIVERSITY IN
NORTH AMERICAN POPULATIONS OF
CAREX VIRIDULA
MICHX. (CYPERACEAE)
Shann
by
on D. Kuchel
B.S., Colorado Christian University, 1996
A thesis submitted to the
University of
Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Mas :er of Arts
Etiology
1999


This thesis ror the Master of Arts
degree by
Shannon D. Kuchel
has been approved
2,Dfc££At
Date


Kuchel, Shannon D. (M.A., Biology)
Genetic diversity in North American pc pulations of Car ex viridula Michx.
(Cyperaceae)
Thesis directed by Associate Professor Leo P. Bruederle
ABSTRACT
Carex viridula Michx. (Cyperaceae), the green sedge, occurs in wetland habitats
alpine wetlands in Colorado, where it is
Colorado were investigated using starch
iral North America. Its distribution also
distributed throughout northern and ceni
extends to the southern Rocky Mountain region in several disjunct sites, including
rare. Populations of C. viridula from
gel electrophoresis of soluble enzymatic
proteins coupled with substrate specific staining in order to describe genetic diversity
and structure. The objective was to determine if Colorado populations exhibited the
reduced genetic diversity expected of marginal populations when compared to other
populations from North America and Europe. Genotypic data were collected for 15
enzyme systems encoded by 21 putative
loci in 350 individuals from seven
in


populations in Colorado and in 179 individuals from eight populations from
elsewhere throughout the range in North America. No variation, either within or
among North American populations, we.s detected at any of the loci. However, North
American populations were genetically differentiated from European populations,
with significantly more diversity maintained by European populations. The surprising
lack of genetic diversity in North Ameri
can populations is probably the combined
result of high levels of selfing and inbreeding, restricted ecological amplitude, and
genetic drift. Genetic bottlenecks are presumed to have occurred as a result of climate
changes associated with Pleistocene glaciation or founding events associated with
colonization of North America by propo
sed ancestral European populations.
This abstract accurately represents the content of the candidates thesis. I recommend
its publication.
Signe
M.
Leo P. Bruederle
IV


DEDICATION
I dedicate this thesis to my family Mo
m and Dad, Warren, and Michelle and Russ -
for their incredible love, support, assistance, and encouragement during this project.


ACKNOWLEDGEMENTS
I would like to thank Leo P. Bruederle
his excellence in instruction, and for his
or his guidance throughout this project, for
endeavoring to give me numerous
opportunities to gain experience. I am grateful to the following people and
organizations for their assistance with collections: Peter Ball, University of Toronto;
Bill Crins, Ontario Ministry of Natural Resources; Allison Cusick, Ohio State
Department of Natural Resources; John
Sanderson, Colorado Natural Heritage
Program; Fred Weinmann; Sue Komarek; Bill Gordon; and Don Hart. This work was
supported, in part, by grants from the Sigma Xi Grants-in-Aid of Research Program
and the Colorado Native Plant Society.


CO
NTENTS
Figures
Tables.
CHAPTER
1. INTRODUCTION......
2. MATERIALS AND METHO
3. RESULTS............
4. DISCUSSION.........
APPENDIX
A. HABITAT DESCRIPTION..
LITERATURE CITED..........
DS
viii
..ix
..1
..9
16
23
36
39
Vll


FIGURES
Figure
1 Car ex viridula.................................................
2 Circumboreal distribution of Car ex nridula.....................
3 Distribution of North American Carix viridula populations sampled
.3
.5
10
vni


TABLES
Table
1 Locations of Car ex viridula populations sampled
2 Allele frequencies.................
3 Summary of genetic diversity.......
4 Summary of allozyme literature...
11
17
19
25
IX


CHAPTER 1
INTRODUCTION
Since 1986, starch gel electrophoresis and allozyme analysis have been used
to study genetic diversity in no fewer thiin 43 species representing nine sections of the
genus Car ex. These studies have been useful not only in elucidating systematic
relationships (e.g., Whitkus 1992; Ford ot al. 1998), but also in providing indirect
estimates of mating systems (e.g., Waterway 1990), identifying hybrid origins (e.g.,
Standley 1990), and revealing correlations between genetic diversity and structure
and certain life-history traits (e.g., Jonsson et al. 1996). However, few studies have
examined genetic diversity in broadly distributed species of Carex.
Car ex section Ceratocystis Dumort. (Cyperaceae) comprises seven species
worldwide, which collectively occur throughout much of the northern hemisphere,
particularly in boreal latitudes and the subalpine (Crins and Ball 1988). Carex
viridula Michx. ssp. viridula var. viridula, the green sedge, is the only representative
of section Ceratocystis in Colorado.
1


Car ex viridula is a short-lived perennial with a densely caespitose habit. It is
monoecious, characterized by a single terminal staminate spike and several sessile
pistillate spikes (Fig. 1). It has been suggested that C. viridula is a dispersal
generalist, with possible transport by biotic, e.g., birds and mammals, and abiotic
agents, e.g., wind and water (Schmid IS 84a; Crins and Ball 1989). While there are no
apparent impediments to outcrossing, the breeding system is predominantly selfing.
In a study by Schmid (1984a) examining the life history of C. viridula, tests for self-
compatibility in the field and in experimental gardens using fine mesh bags to control
pollination were positive. Additionally, inflorescences from which the staminate
spike had been removed had maximum seed sets of only 10% when growing in the
immediate vicinity of other fertile plants
. Similarly, Bruederle and Jensen (1991)
heterozygosity) and deviations from Hat
attributed low genetic diversity (e.g., proportion of loci polymorphic and observed
dy-Weinberg equilibrium in West European
populations of C. viridula to selfing. Genetic diversity was apportioned among
populations with relatively little variation found within populations.
Ecologically, C. viridula is characterized by rapid growth and development,
small size, short life-span, early reproduction, large reproductive effort, and small
2


3


population size (Schmid 1984a, 1984ty
. As such, C. viridula is an early successions!,
r-selected species, or in Grimes (1979) classification, a ruderal species. Although C.
viridula typically occupies moist, early
successional sites characterized by fluctuating
and unpredictable water levels, these ctn vary from calcareous, acidic, sandy, or
organic shorelines; runnels in limestone
of streams, ponds, and lakes; and fens. This species success in colonizing is likely
due to its tolerance of diverse and fluctuating environments, high phenotypic
plasticity, and ability to reproduce quiddy and profusely (Schmid 1984a, 1984b;
Crins and Ball 1989).
Geographically, C. viridula is thi
Ceratocystis, with a near circumboreal
barriers; wet meadows; marshes; on borders
e most widespread taxon in section
distribution. It is common throughout northern
Europe, and much of northern and central North America; it is also scattered across
the central and eastern parts of temperate Asia to the Pacific Ocean (Fig. 2). In North
America, its range extends south in the Rocky Mountain region to several disjunct
sites in Colorado, Wyoming, Utah, and
viridula occupies an uncommon habitat.
Mevada. In the Southern Rocky Mountains, C.
alpine wetlands, with habitat specificity
contributing to rarity in this region (Rabinowitz 1981). The most significant threat to
4




these rare populations may be habitat alteration and loss, as a result of peat mining
and the draining of wetlands for irrigat .on of surrounding ranchlands and diversion to
municipal drinking water supplies. Although C. viridula has been assigned a state
ranking of SI, indicating that it is critically imperiled in Colorado, it has received a
global ranking of G5, indicating that it is demonstrably secure globally (Spackman et
al. 1997).
Car ex viridula is putatively one
of the most recently derived members of
section Ceratocystis, although it is not entirely clear when it diverged from its closest
relative, C. viridula ssp. oedocarpa (N.T. Anderson) B. Schmid (Schmid 1984b; Crins
and Ball 1989; Bruederle and Jensen IS 91). It has been hypothesized that their
common ancestor differentiated in West Europe. Thereafter, C. viridula colonized the
remainder of the temperate and boreal northern hemisphere, perhaps before
Pleistocene glaciation, by way of the Bering land bridge involving seed dispersal by
birds (Crins and Ball 1989).
Extant Colorado populations of
C. viridula are geographically marginal,
North American populations, in general
occurring at the edge of the species distribution in North America. Furthermore,
are peripheral relative to West Europe, the
6


putative center of diversity and origin for C. viridula. Genetic theory predicts
differentiation of marginal populations with respect to central populations, with
reduced levels of genetic variation and greater population differentiation (Bruederle
1999). While marginal populations are e xpected to maintain a subset of the genetic
variation observed in central populations as a result of reduced gene flow (Yeh and
Layton 1979), both random genetic drift
and selection may cause the fixation of
alleles that are rare in central populations (Blows and Hoffman 1993).
However, other factors in addition to distribution may influence levels and
apportionment of genetic diversity in species. Geographical range, successional
status, population size, life form, breeding system, and seed dispersal mechanism
have all been demonstrated to have significant effects on genetic diversity and
structure (Brown 1979; Hamrick et al. 1979; Loveless and Hamrick 1984; Karron
1987; Hamrick and Godt 1989; Hamrick
et al. 1991; Barrett and Kohn 1991).
Furthermore, levels and apportionment c f genetic variation could be the consequence
not only of life-history characteristics, but also of historical and evolutionary events
such as genetic bottlenecks resulting from founder effect, glaciation, migration, and
speciation (Lewis and Crawford 1995).
7


growth, herbaceous habit, ruderal strate
confer low levels of within-population
Collectively, the aforementioned factors lead to three specific predictions
regarding population genetic diversity characteristics, such as self-compatibility, restricted habitat, short-lived perennial
gy, and small population sizes, are expected to
genetic variation (Schmid 1984a, 1984b; Crins
and Ball 1988,1989; Bruederle and Jensen 1991). Deviations from Hardy-Weinberg
equilibrium and heterozygote deficiency are expected as a result of the caespitose
habit, which has been correlated with h: gh levels of inbreeding and genetic
substructuring. Finally, pronounced difJ
a result of reduced gene flow, isolation,
American populations, in general, will
European populations. North American
'erentiation among populations is expected as
inbreeding, and genetic drift.
The purpose of this research is to describe genetic diversity and structure in
populations of C. viridula from Colorac o relative to other North American and West
European populations. It is hypothesize that Colorado populations will be
genetically differentiated from other Ncrth American populations, and that North
>e genetically differentiated from West
populations are further expected to maintain
low levels of diversity within and high levels of differentiation among populations.
8


CHAPTER 2
MATERIALS AND METHODS
Fifteen populations of C. viridula were sampled during the summers of 1998
and 1999 from the Pacific Northwest, Rocky Mountain, and Great Lakes regions of
the United States and Canada (Fig. 3, Table 1). Sites were typically peatlands or other
wetlands, with C. viridula occupying early successional microsites along the shores
of streams, springs, ponds, creeks, and swamps; or along roadsides, ditches, and ruts
(Appendix A). Population samples ranged in size from six to 50 individuals. Because
C. viridula is caespitose, samples obtained from discrete, well-spaced clumps were
assumed to represent different individuals. At each site, whole vegetative culms were
harvested, placed in separate plastic bags with moist paper towels, and kept
refrigerated until extraction of soluble enzymatic proteins. Extraction followed
Bruederle and Fairbrothers (1986). Leav
ss were homogenized in 0.1 M Tris-HCl
extraction buffer, pH 7.5, modified by the addition of 20% polyvinylpyrrolidone
(molecular mass 40,000) and 0.1% 2-mercaptoethanol. Sea sand was added to
facilitate homogenization. Enzyme extra :ts were absorbed onto filter paper
9


Fig. 3. Map of North America showing the locations of 15 Car ex viridula ssp. viridula var. viridulct
populations sampled for allozyme analysis. Detailed locations are provided for seven Colorado
populations. Population numbers correspond to those in Table 1.


Table 1. Locations of 15 North American Car ex viridula Michx. ssp. viridula var. viridula populations sampled for
allozyme analysis, as well as three West European populations (Bruederle and Jensen 1991).
Pop.
No.
Country,
State, and
Province
Sample
Size
1 CO, USA 50
2 CO, USA 50
3 CO, USA 50
4 CO. 50
USA
5 CO, USA 50
6 CO, USA 50
7 CO, USA 50
8 OH, USA 28
9 MI, USA 27
Location
Park Co., Colorado, High Creek Fen, 13 km (8 m) S of Fairplay on U.S.
Rte. 285
Park Co., Colorado, Sweet Water Ranch, 18 km (11 m) S of Fairplay on
U.S. Rte. 285
Park Co., Colorado, Warm Springs Ranch, 5 km (3 m) S of Fairplay on
U.S. Rte. 285
Jackson Co.. Colorado. Lone Pine. 2;
Rd.16
Jackson Co., Colorado, Bear Creek, 24 km (15 m) W of Walden on Co.
Rd. 16
Grand Co., Colorado, Haystack Mountain, 16 km (10 m) N of
Silverthorne on St. Rte. 9
San Juan Co., Colorado, Andrew's Lake, 10 km (6 m) S of Silverton on
U.S. Rte. 550
Ottawa Co., Ohio, Quarry Rd., 1 km (0.6 m) SW of Lakeside on St. Rte.
163
Iosco Co., Michigan, 6 km (4 m) W of U.S. Rte. 23, N of Alabaster Rd.
Latitude Longitude
3905'N 10558'W
3903'N 10558'W
3909'N 10603'W
4044'N _106341W-
4045'N 10635'W
3955'N 10619'W
3743'N 10742'W
4r31'N 8245'W
4412'N 8337'W


Table 1. Continued.
Pop. No. Country, State, or Province Sample Size Location Latitude Longitude
10 MI, USA 25 Mackinac Co., Michigan, 13 km (8 m) N of U.S. Rte. 2, W of Borgstrom Rd. 4612'N 8521'W
11 WI, USA 27 Waushara Co., Wisconsin, Hills Lake, 6 km (4 m) E of St. Rte. 22, S of Co. Rd. H 4409'N 8909W
12 WA, USA 28 King Co., Washington, Snoqualmie Bog, 21 km (13 m) E of St. Rte. 203, N of N. Fork Co. Rd. 4740'N 12137W
13 ONT, CAN 25 Norfolk Co., Ontario, Long Point on Lake Erie, 48 km (30 m) S of Hwy. 401 4234'N 8025W
14 ONT, CAN 6 Peterborough Co., Ontario, Belmont Lake, 24 km (15 m) N of Hwy. 7 4440'N 7880W
15 ONT, CAN 13 Nipissing Dist., Ontario, Radiant Lake, Algonquin Provincial Park, 16 km (10 m) N of Rte. 60 4560'N 7830W
16 AUS 3 Trunnahiitte, Austria, 3.8 km (2.4 m) SSW of Trin 4730'N 1170'E
17 SWE 26 Asa, Sweden, 0.9 km (0.56 m) WSW 5865'N 1180'E
18 SWE 28 Skanor, Sweden, 1 km (0.62 m) E 5610'N 1290'E


(Whatman No. 17) wicks that were stored at -76C until electrophoresis. Voucher
specimens for each population have been deposited in the herbarium at the University
of Colorado at Denver.
Three gel-buffer systems and 15
putative loci involving 10.5% starch gels (Sigma-Aldrich, Inc.). Electrophoresis and
staining followed Bruederle and Fairbrcthers (1986) and Bruederle and Jensen
(1991). System 1 (Gottlieb 1981) was a
(SkDH). System 2 (Soltis et al. 1983) w
0.223M Tris/0.086M citric acid monoh)
tris, was diluted to 1 liter to form the ge'.
glyceradehyde-3-phosphate dehydrogen
enzyme stains were used to resolve 21
discontinuous histidine-HCl system using a
0.02M L-histidine-HCl gel buffer, titrated to pH 7.0 with NaOH, and a 0.4M
trisodium citrate electrode buffer, titrated to pH 7.0 with HC1. These gels were run for
7 hr at 140mA, and stained for isocitrate dehydrogenase (IDH), malate
dehydrogenase (MDH), 6-phosphogluconate dehydrogenase (6PGD), phosphoglucose
isomerase (PGI), phosphoglucomutase (PGM), and shikimic acid dehydrogenase
as a tris-citrate system; thirty-five ml of a
drate electrode buffer, titrated to pH 7.5 with
buffer. These gels were run for 9 hr at
60mA, and stained for aspartate aminotransferase (AAT), acid phoshatase (ACP), and
ase (G3PDH). System 3 (Soltis et al. 1983)
13


was a discontinuous lithium-borate system using a 0.042M Tris/0.007M citric
acid/0.004M lithium hydroxide/0.0251V[ boric acid gel buffer, titrated to pH 7.6 with
HC1, and a 0.263M boric acid/0.039M
(MNR), superoxide dismutase (SOD), £
ithium hydroxide electrode buffer, titrated to
pH 8.0 with NaOH. These gels were ru:i for 11 hr at 230V, and stained for alcohol
dehydrogenase (ADH), diaphorase (DIA), malic enzyme (ME), menadione reductase
nd triose phosphate isomerase (TPI). Staining
followed Soltis et al. (1983) with minor modifications for IDH, MDH, PGI, PGM,
SkDH, ACP, G3PDH, ME, and TPI; Gottlieb (1973) for PGD and ADH; and Cardy et
al. (1981) for AAT.
Electrophoretic allozyme pheno types were interpreted genetically on the basis
of segregation patterns, known substructure and intracellular compartmentalization of
enzymes, and previously observed electrophoretic patterns (e.g., Bruederle and
Fairbrothers 1986). Data were collected as individual genotypes; these data have been
deposited in the Department of Biology
and are available from L.P. Bruederle upon
request. For enzymes with more than one locus, isozymes were labeled sequentially
with the most rapidly migrating locus designated 1. Alleles were labeled similarly
with the most rapidly migrating allele designated a. Standards representing most of
14


the electrophoretic variants for the secti
on were incorporated into each of the gels to
facilitate allele identification.
YS-1 (Swofford and Selander 1981) to obtain
Data were analyzed using BIOS
common measures of genetic diversity including proportion of loci polymorphic (P),
mean number of alleles per locus (A) ar d per polymorphic locus (Ap), observed
heterozygosity (H0), and expected heterozygosity (He). The distribution of genetic
variation within and among populations
diversity statistics and GENESTAT-PC
differences in genetic diversity, data from North American populations were
compared with data previously reported
using a t-test (Table 1; Bruederle and Jensen 1991).
was calculated using Neis (1973) gene
(Lewis and Whitkus 1989). In order to assess
for European populations of this species
15


CHAPTER 3
The fifteen enzymes assayed for
RESULTS
C. viridula are encoded by 21 putative loci:
AAT-1, AAT-2, ACP-1, ADH, DIA-1, DIA-3, G3PDH, IDH-1, IDH-2, MDH-1, MDH-
2, ME, MNR, 6PGD, PGI-2, PGM-1, PGM-2, SkDH, SOD, TPI-1, and TP I-2. Four
additional loci (MDH-3, PGI-1, ACP-2,
analysis, because they did not exhibit co
and ACP-3) were not included in the
nsistent activity or clearly interpretable
banding patterns.
North American C. viridula maintains no genetic diversity based upon this
sample of 15 populations and 21 loci. None of the loci examined were polymorphic.
At every locus assayed, each of the 529 individuals was homozygous for the same
allele; no heterozygosity or allozyme va nation was observed (Table 2). As such, for
all populations, the proportion of loci polymorphic (P), observed heterozygosity (H0),
and expected heterozygosity (He) were all zero. Similarly, the number of alleles per
locus (A) was one, the minimum value far this statistic (Table 3).
16


Table 2. Allele frequencies at 21 loci for 18 populations of Carex viridula Michx. ssp. viridula var. viridula.
Population numbers correspond to those in Table 1. See text for allozyme nomenclature.
North American Populations
European Populations
Locus
IDH-1
IDH-2
MDH-1
MDH-2
6PGD
PGI-2
PGM-1
PGM-2
SkDH
AAT-1
AAT-2
ACP-1
G3PDH
ADH
Allele 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
b 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
b 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
h 0.00 0.00 0.00 0.00 0,00 0.00 0.00 0.00 0.00 0.00 0.00 0.00_0.00_QmjQ-QQ_
c 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
b 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
c 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
a 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
b 1.00 1.00 1.00 1.00 LOO 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
b 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
16 17 18
1.00 1.00 1.00
1.00 1.00 1.00
1.00 1.00 1.00
1.00 1.00 1.00
1.00 1.00 1.00
-QJXL 0.00- 0.036
1.00 1.00 0.964
1.00 1.00 1.00
1.00 1.00 1.00
1.00 1.00 1.00
1.00 1.00 1.00
1.00 1.00 1.00
0.00 0.058 0.071
1.00 0.942 0.929
1.00 1.00 1.00
DIA-1


Table 2. Continued.
Locus Allele North American Populations European Populations
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
MNR a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
ME a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
SOD a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.00 1.00 0.964
b 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.667 0.00 0.00
d 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.333 0.00 0.036
TPI-1 a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
TPI-2 a 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00


Table 3. Summary of genetic diversity for 18 populations of Carex
viridula Michx. ssp. viridula var. viridula : sample size (N), mean number of
alleles per locus (A) and per polymorphic locus (Ap), proportion of loci
polymorphic (P), observed heterozygosity (H0), and expected heterozygosity
(HJ. Population numbers corresponc' to those in Table 1. A locus was
considered polymorphic if the frequency of the most common allele did not
exceed 0.95.
Pop. No. State, Province, or Country N A A. p H H,
1 CO, USA 50 1.0 - 0.0 0.000 0.000
2 CO, USA 50 1.0 - 0.0 0.000 0.000
3 CO, USA 50 1.0 - 0.0 0.000 0.000
4 CO, USA 50 1.0 - 0.0 0.000 0.000
5 CO, USA 50 1.0 - 0.0 0.000 0.000
6 CO, USA 50 1.0 - 0.0 0.000 0.000
7 CO, USA 50 1.0 - 0.0 0.000 0.000
8 OH, USA 28 1.0 - 0.0 0.000 0.000
9 MI, USA 27 1.0 - 0.0 0.000 0.000
10 MI, USA 25 1.0 - 0.0 0.000 0.000
11 WI, USA 27 1.0 - 0.0 0.000 0.000
12 WA, USA 28 1.0 - 0.0 0.000 0.000
13 ONT, CAN 25 1.0 - 0.0 0.000 0.000
14 ONT, CAN 6 1.0 - 0.0 0.000 0.000
15 ONT, CAN 13 1.0 - 0.0 0.000 0.000
Mean North America 529 1.0 _ 0.0 0.000 0.000
19


Table 3. Continued.
Pop. No. State, Province, or Country N A A p H H,
16 AUS 3 1.15 2.0 15.0 0.017 0.070
17 SWE 26 1.05 2.0 5.0 0.006 0.006
18 SWE 28 1.25 2.0 10.0 0.018 0.042
Mean West Europe 57 1.15 2.0 10.0 0.014 0.039
Mean Species 586 1.03 2.0 r.7 0.002 0.007
20


number of alleles per polymorphic locn,
and expected heterozygosity was 0.039
While low, West European pop jlations exhibited higher levels of genetic
diversity than North American populations (Bruederle and Jensen 1991). On average,
two of the 20 loci examined for West European populations (10.0%) were
polymorphic (Table 2). Mean number of alleles per locus was 1.15, while mean
s was 2.0. Observed heterozygosity was 0.014
(Table 3). Genetic diversity in West European
populations was significantly higher thiin that in North American populations when
compared using a two sample t-test assuming unequal variances for proportion of loci
polymorphic (p<0.10), mean number of alleles per locus (p<0.05), observed
heterozygosity (p<0.05), and expected heterozygosity (p<0.10).
Levels of genetic diversity for the species across its sample range in North
ely low. The mean proportion of loci
America and West Europe were extrem
polymorphic was 1.7%. Mean number of alleles per locus was 1.03, while mean
number of alleles per polymorphic locus was 2.0. Observed heterozygosity was 0.002
and expected heterozygosity was 0.007
Despite the differences in levels
North American populations are, in fact,
(Table 3).
of genetic diversity, West European and
, very similar. Mean genetic identity obtained
21


from pairwise comparisons of populations from North America and West Europe was
0.988. Mean genetic identity among North American populations was 1.000, and
among European populations was 0.977
, ranging from 0.964 to 1.000 for the latter.
Of the small amount of diversity maintained by populations of C. viridula, the
majority (Gst=0.650) was due to differences among populations, both among West
European and between North American
and West European populations.
22


chapter 4
DISCUSSION
As expected, North American pc pulations of C. viridula do maintain
extremely low levels of genetic diver sit}' within populations. However, due to the low
levels of genetic diversity within populations and subsequent lack of allozyme
markers, no genetic differentiation was observed among populations in Colorado or
North America. Nevertheless, North Ami
erican populations were genetically
differentiated from West European populations, with significantly more diversity
maintained by West European populatio ns. A number of factors could have
contributed to the paucity of genetic diversity observed in populations of this species.
cals strong associations between genetic
The plant allozyme literature revi
diversity and breeding system, with those species characterized by outcrossed
breeding systems having significantly higher levels of genetic diversity apportioned
among individuals within populations. Significant correlations have also been
reported between genetic diversity and s: ze of range, with widespread species having
significantly higher levels of genetic diversity, both within and among populations
23


(Brown 1979; Hamrick et al. 1979; Loveless and Hamrick 1984; Hamrick and Godt
1989; Hamrick et al. 1991).
Car ex viridula has been shown lo exhibit population genetic structure and
seed set suggestive of selfing (Schmid 1984a; Bruederle and Jensen 1991). The
extremely low levels of genetic variation found in North American populations of C.
viridula in this study may be the result, in part, of such selfing. High levels of
inbreeding attributable to selfing are expected to result in homozygosity and
decreased genetic variability. On average, species that have selfing breeding systems
maintain significantly lower levels of genetic diversity, including proportion of loci
polymorphic, number of alleles per locus and per polymorphic locus, and observed
heterozygosity, when compared to predominantly outcrossed species (Hamrick and
Godt 1989). Interestingly, of the large number of species of vascular plants that have
been examined similarly, only 13 other taxa have been reported to maintain no
detectable allozyme diversity (Table 4).
also show substantial levels of selfing.
Overwhelmingly, almost all of these taxa
In graminoids, high levels of inb -eeding have also been correlated with the
caespitose growth form. Stebbins (1950'
proposed a relationship between growth
24


Table 4. Summary of allozyme literature for those species for which there is no detectable allozyme variation.
Species
Breeding System
Geographic Range /
Ecological Amplitude
Inferred historical mechanisms
Carex viridula selfing
Bensoniella selfing
oregona
(Abrams &
Bacig) Morton
narrow distribution Genetic drift: inbreeding and genetic
ecologically bottleneck associated with founding
events and/or climate changes during
glaciation
Rapid colonizer
Disturbed habitats
narrow distribution Genetic drift: inbreeding and genetic
geographically bottleneck associated with range
restrictions during glaciation
Clonal growth
____________________Small population sizes-------------
Chrysosplenium selfing
iowense Rydb.
narrow distribution
geographically
and ecologically
Genetic drift: inbreeding and genetic
bottleneck associated with climate
changes during glaciation
Clonal growth
Small population sizes
Howellia approaches
aquatilis Gray obligate
selfing
narrow distribution Genetic drift: inbreeding and genetic
geographically bottleneck associated with range
and ecologically restrictions during glaciation
Age of populations, not enough time to
accumulate variability and
heterozygosity
Reference
Soltis et al. 1992
Schwartz 1985
Lesica et al.
1988


Table 4. Continued.
Species
Breeding System
Geographic Range /
Ecological Amplitude
Inferred historical mechanisms
Lespedeza selfing
leptostachya
Engelm.
narrow distribution
geographically
Genetic drift: inbreeding and genetic
bottleneck associated with range
restrictions during glaciation
Oenothera selfing
hookeri
Torr. and Gray
nairow distribution Genetic drift: inbreeding
geographically Age of populations, not enough time
to accumulate variability and
heterozygosity
____________________Rapidcolonizer---------------------
Pedicularis furbishiae S. Wats pollinator required for pollination, but possibly self- compatible nairow distribution geographically and ecologically Genetic drift: inbreeding and genetic bottleneck associated with range restrictions during glaciation and/or founding events Local population extinctions Disturbed habitats
Pirns resinosa Ait. highly self- compatible widespread geographically Genetic drift: inbreeding and genetic bottleneck associated with range restrictions during glaciation Age of populations, not enough time to accumulate variability and heterozygosity
Reference
Cole and
Biesboer 1992
Levy and Levin
1975
Waller et al. 1987
Fowler and
Morris 1977;
Allendorf et al.
1982; Simon et al.
1986; Mosseler et
al. 1991


Table 4. Continued.
Species Breeding System Geographic Range / Ecological Amplitude Inferred historical mechanisms Reference
Senecio mohavensis Gray obligate selfing narrow distribution geographically Genetic drift: genetic bottleneck associated with founding events (recent colonization of North America) and/or climate changes during glaciation Rapid colonizer Liston etal. 1989
Sullvantia oregana S. Wats. selfing narrow distribution geographically and ecologically Genetic drift: inbreeding and genetic bottleneck associated with range restrictions during glaciation Solits 1982
Taraxacum obliquum (Fr.) Dahlst. selfing widespread geographically Genetic drift: inbreeding and genetic bottleneck associated with range restrictions during glaciation Selection in more severe and less diverse environments Rapid colonizer Van Oostrum et al. 1985
Thuja plicata Donn ex D. Don self-compatible narrow distribution geographically No explanation given Genetic bottleneck associated with range restrictions unlikely; no evidence of physical barriers and associated species show abundant genetic variation Copes 1981


Table 4. Continued.
Species Breeding System Geographic Range / Ecological Amplitude Inferred historical mechanisms Reference
Tragopogon pratensis Ownbey selfing widespread geographically Genetic drift: inbreeding Rapid colonizer Roose and Gottlieb 1976
Typha domingensis Pers. selfing widespread geographically and ecologically Genetic drift: inbreeding Clonal growth Rapid colonizer Disturbed habitats Mashburn et al. 1978; Sharitz et al. 1980


contrast, the caespitose habit results in a
form and breeding system among grasses, suggesting that species with a rhizomatous
growth form are predominantly outcrossing due to the intermingling of genets. In
growth form in which the nearest neighbor
of a flowering culm is another culm from the same plant (e.g., ramet), thus promoting
inbreeding, and specifically, selfing. Genetic evidence substantiating this
phenomenon in the graminoid genus Cai
ex was first reported by Bruederle and
Fairbrothers (1986) and Bruederle (1987). A survey of the population genetic
literature for the genus Carex revealed data for 29 taxa including six rhizomatous and
23 caespitose carices (Kuchel, unpub. data). On average, populations of rhizomatous
species harbor high levels of genetic diversity, e.g., Aj=2.26 0.12, P=44.5 4.08%,
and He=0.171 0.038, while caespitose s pecies have significantly less, e.g., Ap=2.03
0.09 (p<0.05), P=13.4 12.0% (p<0.001), and H =0.042 0.04 (p<0.001).
Furthermore, whereas populations of rhizomatous species are poorly differentiated
(Gst=0.159 0.053), caespitose species a:-e well-differentiated with nearly half of all
genetic diversity attributable to differences among populations (Gst=0.462 0.272).
Although exceptions exist (Ford et al. 1998), it would appear that rhizomatous species
maintain more variation within and less differentiation among populations,
29


presumably due to outcrossing. Conversely, caespitose species have less variation
within and more differentiation among populations, presumably due to inbreeding. As
such, the extremely low levels of genetic variation found in North American
populations of C. viridula in this study may be the result, in part, of the caespitose
growth form.
As previously mentioned, narrowly distributed plant species tend to maintain
lower levels of genetic variation than more widespread species (Karron 1987; Karron
et al. 1988; Hamrick and Godt 1989). The lower levels of genetic variation observed
in narrowly distributed species may be c ue to changes in allele frequencies due to
chance (genetic drift and founder effect) or strong, directional selection toward
genetic uniformity in a limited habitat t>
pe (Karron 1987). Almost all of the
aforementioned genetically invariable taxa are narrowly distributed (Table 4).
Waller et al. (1987) hypothesized that the lack of diversity in one of these
taxa, Pedicularis furbishiae, could be due to its narrow distribution resulting from a
narrow ecological amplitude. This species is confined to a chronically disturbed, early
successional riparian habitat where individuals and populations are subject to
considerable turnover and population reestablishment by one or a few seedlings. The
30


observed paucity of genetic diversity in
narrow habitat requirements, founding events, small population sizes, and possible
selection pressures experienced in such an environment could have contributed to the
P. jurbishiae.
Even though C. viridula is distributed throughout boreal North America, its
ecological amplitude also appears to be narrow. Car ex viridula is a habitat specialist,
occurring only in highly disjunct wetland habitats. Additionally, C. viridula is
confined to early successional environments, which tend to be small, ephemeral,
highly variable, and subject to repeated local extinction and colonization. As in P.
jurbishiae, it is possible that the extremely low levels of genetic variation found in
populations of C. viridula in this study may be, in part, the result of this narrow
ecological amplitude.
Thus, low levels of genetic diversity found within populations of C. viridula
may be attributed in part to effective breeding system and narrow ecological
amplitude. However, populations of this
species in North America show substantially
examined were monomorphic. The most
lower levels of genetic variability, even when compared to those means reported for
selfing or narrowly distributed species :.t should be reiterated that all putative loci
likely explanation for such low levels of
31


all or most of the allozyme polymorphism.
polymorphism and heterozygosity is genetic drift. It is possible that a genetic
bottleneck occurred at some point in the history of these populations that eliminated
One possibility is that a genetic
bottleneck resulted from the founding of
North American populations. During m
igration and dispersal, new populations may
be formed by a small number of initial colonists. The genetic material of such
populations is limited to those alleles introduced by these few founders and may not
be representative of the species as a whole (Schwaegerle and Schaal 1979). Since
European populations are the proposed progenitors of North American populations
(Crins and Ball 1989), it would be expected that the allozymes present in North
American populations would largely comprise a subset of those alleles present in
European populations (Crawford 1983;
American populations are genetically di
Cole and Biesboer 1992). Indeed, North
ancestral European populations founded
'ferentiated from West European populations,
with West European populations of C. viridula harboring higher levels of genetic
diversity, and North American populations harboring a subset of that genetic
diversity. These data suggest that a small number of individuals from the putatively
North American populations. Limited gene
32


flow between populations would likely maintain the lower levels of genetic diversity
al. 1988; Cole and Biesboer 1992). Clim
atic changes during the Pleistocene,
particularly the Xerothermic period about 8,500 to 3,000 years B.P., have been
observed in North American populations.
Another possibility is that a genetic bottleneck occurred at some point after
the founding of North American populations. This bottleneck could have resulted in
genetic uniformity in an original population or populations with a reduced geographic
distribution, followed by spread of this species to the range now occupied (Lesica et
suggested as a possible cause for genetic
bottlenecks in a number of other North
American species exhibiting extremely low levels of genetic diversity (Table 4), as
well as in other species of Car ex (Waterway 1990). Additionally, it has been
suggested that these populations have not had enough time to accumulate variation
and differentiate since these events (Levy and Levin 1975; Ledig and Conkle 1983;
Lesica et al. 1988; Liston et al. 1989).
It is interesting to note that a nun: ber of species reported to maintain no
olonizers of disturbed habitats (Table 4).
detectable allozyme diversity are rapid c
Carex viridula has also been described as a rapid colonizer and ruderal species with
33


rapid growth and development, small size, short life-span, early reproduction, large
reproductive effort, and small populatio n size (Schmid 1984a, 1984b). In Switzerland,
C. viridula often occupies newly disturbed sites, with many small and isolated
populations. It is a pioneer in open, wet
habitats, but is quickly excluded
successionally (Schmid 1986). Not supiisingly, most populations sampled for this
study appeal- to occupy early successior al microsites, growing along pond shores,
stream banks, roadsides, ditches, and rub, comprising a part of larger, later
successional communities (Appendix A). Schmid (1984b) hypothesized that such
ruderal species with early successional populations would have high genetic
variability between, but low genetic variability and high plasticity within populations,
as a result of small population size, genetic drift, and directional selection. This
hypothesis is supported by the ecology of C. viridula (Schmid 1984b), as well as the
present study of genetic diversity.
It has been suggested that allozy ne variation detectable by electrophoresis
may not provide a complete measure of genetic diversity in the genome (Mosseler et
al. 1991,1992). A more direct analysis of variation in DNA, e.g., RAPDs, may
provide the genetic markers necessary tc
infer genetic diversity and structure in C.
34


viridula. Data from the present study do
indicate that C. viridula is genetically
depauperate over a large portion of its range. However, additional analyses of
populations lying at the extreme northwestern, northeastern, and eastern edges of the
distribution in North America should be
carried out to confirm this. Analyses of
additional Eurasian populations would also contribute to a reconstruction of the
biogeography of C. viridula.
35


APPENDIX A
HABITAT DESCRIPTION
Site information for 15 Carex viridula Michx. ssp. viridula var. viridula
(Cyperaceae) populations from North America sampled for allozyme analysis
36


Appendix A.
Location Stae ^ Habitat and Microhabitat Description
, High Creek Fen
Park Co., CO, USA
1000 scattered throughout well-developed peatland;
colonizing alongside the banks of a few streams
9 Sweet Water Ranch
z Park Co., CO, USA
scattered throughout well-developed peatland;
1200 colonizing alongside large, deep ditch dug
through peatland
o Warm Springs Ranch
J Park Co., CO, USA
2qq growing on shore of spring and on moist areas
adjacent to spring, alongside shore of pond
7Lone Pine
Jackson Co., CO, USA
^ shore of pond, colonizing edge of one bank and on
top of a few hummocks
c Bear Creek
Jackson Co., CO, USA
50
shore of creek, colonizing along fallen log and
within adjacent rut
Haystack Mountain
0 Grand Co., CO, USA
near outlet of small alkaline spring, some peat
75 accumulation, growing on top of small
hummocks
7 Andrew's Lake
San Juan Co., CO, USA
jqqq scattered throughout well-developed peatland;
colonizing shores of a few ponds
Ecological Status of
Microhabitat
early successional
early successional
early successional
early successional
early successional
early successional
early successional


Appendix A. Continued.
Location ^Size^ Habitat and Microhabitat Description
o Quarry Rd.
6 Ottawa Co., OH, USA
1000 scattered trough moists areas in floor of old
limestone quarry
n Alabaster Rd.
Iosco Co., MI, USA
500 adjacent to swamp, colonizing borrow pit
, n Borgstrom Rd.
Mackinac Co., MI, USA
50
well-drained edge of sandy road
t Hills Lake
^Waushara Co^WI, USA
100
shore of lake; successive, patallel colonizations
19 Snoqualmie Bog
1 King Co., WA, USA
1000 scattered throughout large peatland system
i ^ Long Point
Norfolk Co., ONT, CAN
jqq colonizing middle and edges of raised vehicular
track
Belmont Lake
14 Peterborough Co., ONT,
CAN
Radiant Lake
15 Nipissing Dist., ONT,
CAN
100
shore of lake
Ecological Status of
Microhabitat
early successional
early successional
early successional
early successional
early successional
early successional
early successional
100
shore of lake
early successional


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