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Genetic diversity in a rare North American endemic

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Genetic diversity in a rare North American endemic
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DePrenger-Levin, Michelle Emily
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English
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xii, 64 leaves : illustrations ; 28 cm

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Northern singlespike sedge -- Genetics ( lcsh )
Northern singlespike sedge -- Variation ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Bibliography:
Includes bibliographical references (leaves 59-64).
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Department of Integrative Biology
Statement of Responsibility:
by Michelle Emily DePrenger-Levin.

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Auraria Library
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Full Text
GENETIC DIVERSITY IN A RARE NORTH AMERICAN ENDEMIC,
CAREXSCIRPOIDEA SSP. CONVOLUTA
by
Michelle Emily DePrenger-Levin
B.A., Macalester College, 1999
A thesis submitted to the
University of Colorado at Denver
and Health Sciences Center
in partial fulfillment
of the requirements for the degree of
Masters of Science,
Biology
2007


This thesis for the Master of Science
degree by
Michelle Emily DePrenger-Levin
has been approved by
Leo P. Bruederle
2oo^~
Date


DePrenger-Levin, Michelle Emily (M.S., Biology)
Genetic diversity in a rare North American endemic, Carex scirpoidea ssp. convoluta
Thesis directed by Associate Professor Leo P. Bruederle
ABSTRACT
This research quantifies genetic diversity in Carex scirpoidea ssp. convoluta
(Kiikenthal) Dunlop, interpreting these data within the context of life history,
geographic distribution, and recent evolutionary history. To control for factors that
affect genetic diversity, a comparison is made between the rare, narrow endemic
subspecies and its more widespread conspecific C. scirpoidea Michx. ssp. scirpoidea
using starch gel electrophoresis coupled with allozyme analysis. Carex scirpoidea
ssp. convoluta is a Great Lakes endemic found in Michigan and Ontario. This taxon is
threatened, being restricted in distribution to prairie pavement barrens in globally rare
alvar communities occupying exposed limestone bedrock. Five populations of C.
scirpoidea ssp. convoluta were sampled from the Lake Huron shoreline on
Drummond Island, the lower peninsula of Michigan, and Manitoulin Island, Ontario.
Despite factors that are expected to lead to decreased levels of genetic diversity, C.
scirpoidea ssp. convoluta maintains a high or similar level of genetic diversity when
compared to its widespread conspecific, C. scirpoidea ssp. scirpoidea, as well as
other taxa with similar life history traits. Fourteen enzymes comprising 19 loci were
resolved for this study. Carex scirpoidea ssp. convoluta exhibits greater genetic
diversity than its widespread conspecific, C. scirpoidea ssp. scirpoidea, with 27.37%
loci polymorphic and 2.32 alleles per polymorphic locus compared to 20.00%
polymorphic loci, with an average 2.29 alleles per polymorphic locus in C. scirpoidea
ssp. scirpoidea. Less heterozygosity was observed in the rare, narrow endemic than


would be expected with Hardy-Weinberg equilibrium (Ho=0.089, He=0.097), while C.
scirpoidea ssp. scirpoidea had greater observed heterozygosity than expected. Carex
scirpoidea ssp. convoluta has relatively low population differentiation (Fst= 0.242)
implying greater gene flow, although it has approximately 1.4 times more population
differentiation than other narrow endemic outcrossing species.
While phylogenetic analyses provided support for the subspecies as distinct, the
relationship among taxa in Carex section Scirpinae is not clear. Despite its caespitose
habit, limited range and recent post-glacial evolutionary history, obligate outcrossing
appears to be maintaining genetic diversity in this threatened subspecies.
This abstract accurately represents the content of the candidates thesis. I recommend
its publication.
Signed
Leo P. Bruederle


DEDICATION
I wish to dedicate this thesis to my husband, Zeth, who has supported me and allowed
me to devote time away from our home and beautiful child, Ella Sage, to complete
this degree. Without their love and support I would not have completed this degree.


ACKNOWLEDGEMENT
I thank my advisor, Dr. Leo P. Bruederle, for his guidance and patience to help me
reach my goals and further my botanical knowledge. I would also like to thank my
committee for their support and insight.


TABLE OF CONTENTS
Figures...................................................................viii
Tables....................................................................ix
Chapter
1. Introduction........................................................12
2. Research Objectives.................................................17
2.1 Dioecy..............................................................18
2.2 Biogeography........................................................19
2.3 Endemism............................................................20
3. Materials and Methods...............................................22
3.1 Allozyme Analysis...................................................22
3.2 DNA sequencing......................................................23
4. Results.............................................................26
4.1 Population Genetics.................................................26
4.2 Phylogeny...........................................................29
5. Discussion..........................................................31
Appendix
A. Genomic DNA Protocol and Solution Recipes..........................49
B. Allele Frequencies of Polymorphic Loci.............................51
C. Genotypes of the Five Carex scirpoidea Michx. ssp. convoluta
(Kiikenth.) Dunlop Populations.....................................52
vii


References
58
viii


LIST OF FIGURES
Figures
1. Distribution of (a) Carex scirpoidea Michx. ssp. convoluta (Kiikenth.).38
Dunlop on the shores of Lake Huron in Michigan and Ontario
and (b) Carex scirpoidea Michx. ssp. scirpoidea in North America
and Beringia. Populations sampled for allozyme analysis shown in red.
Range maps adapted from Dunlop and Crow (1999).
2. Neighbor-joining tree based on Neis identity (1978)..................46
and coancestry distances (GDA: Lewis and Zaykin, 2001)
3. Consensus phylogeny of 1000 trees from nrDNA ITS-1..................47
sequence. Parsimony bootstrap values are listed at each node.
C. donnell-smithii is used as the outgroup.
* sequences from GenBank
4. Consensus phylogeny of 1000 trees from nrDNA ETS sequence...........48
Parsimony bootstrap values are listed at each node. C. stylosa
is used as the outgroup. sequences from GenBank
5. Consensus phylogeny of 1000 trees from the combined.................49
nrDNA regions, ETS and ITS. Parsimony bootstrap values
are listed at each node. C. donnell-smithii is used as the outgroup.
* sequences from GenBank
tx


LIST OF TABLES
Tables
1. Habitat, distribution, morphological characteristics, habit,..............39
and state ranks for each of five taxa in
Car ex section Scirpinae (Cyperaceae)
2. Locations for five Carex scirpoidea Michx. ssp. convoluta................40
(Kiikenth.) Dunlop (Cyperaceae) populations sampled for
allozyme analysis, where N equals the number of individuals sampled.
3. Location for populations sampled for sequence analysis,........................41
Location for populations sampled for sequence analysis from
Carex section Scirpinae (Cyperaceae):
C. scirpoidea Michx. ssp. scirpoidea (CaScSc),
C. scirpoidea Michx ssp. pseudoscirpoidea (Rydb.) Dunlop (CaScPs),
C. scirpoidea Michx. ssp. stenochlaena (CaScSt),
C. scirpoidea Michx. ssp. convoluta (Kiikenth.) Dunlop (CaScCo),
and C. curatorum Stacey (CaCu). Carex scirpoidea ssp. stenochlaena
samples are from herbarium specimens deposited at the
University of Washington, Seattle herbarium (WTU).
4. Starch gel and electrode buffer systems and substrate-specific.............42
stains used in allozyme analysis conducted on
Carex scirpoidea Michx. ssp. convoluta (Kiikenth.) Dunlop
5. Genetic Diversity of Carex scirpoidea ssp. convoluta and....................43
Comparison of population level genetic diversity statistics of the
narrow endemic, Carex scirpoidea Michx. ssp. convoluta (Kiikenth.) Dunlop and its
five populations: Presque Isle, Maxton Plains, Big Shoal Cover, Burnt Island, and
Murphy Point, the widespread, Carex scirpoidea Michx. ssp. scirpoidea, caespitose,
and rhizomatous carices. Comparative data for C. scirpoidea ssp. scirpoidea,
caespitose and rhizomatous carices taken from Yarbrough (2000). P = percent of loci
polymorphic, Ap = number of alleles per polymorphic locus, Ho = observed
heterozygosity, He = expected heterozygosity, and/= fixation index. Pj, Ap.y, Hos,
Hes, and fs are species estimates.
x


6. Variable loci, deviations from Hardy-Weinberg Equilibrium..............44
Variable loci, deviations from Hardy-Weinberg Equilibrium if),
and significance value (p) for five populations of
Carex scirpoidea Michx. ssp. convoluta (Kukenth.) Dunlop.
Values of -1 0 indicate heterozygotic excess.
Values from 0-1 indicate a loss of heterozygosity.
95% confidence intervals from bootstrap analysis are
listed for population level inbreeding coefficients.
Standard errors (SE) listed below each population.
Significant deviations indicated in bold (a = 0.05).
7. Comparison of F statistics for.........................................45
Carex scirpoidea Michx. ssp. convoluta (Kukenth.) Dunlop,
C. scirpoidea ssp. scirpoidea, caespitose carices,
rhizomatous carices, and all carices.
Data taken from Bruederle et al, in print.
xi


1. Introduction
Patterns of plant diversity, range, and resilience have changed over time. Evolving
methods to examine diversity have also changed our perception and ability to detect
diversity at varying levels. A perception that has remained constant is that diversity
translates to evolutionary potential and resilience. An ecosystem with diverse species
and functional groups provides niches for wildlife, protection from invasion of
noxious weeds, and increased plant productivity (Pokomy et al., 2005). Genetic
diversity in a species allows the potential for range expansion and resilience against
changes in the environment.
Determining genetic variation present in a population can be useful for the
conservation status of a rare species. The amount of genetic variability and its
apportionment can determine a species ability to adapt to changing selection
pressures (Maki and Asada, 1998; Batista et al., 2001). High genetic diversity
indicates the ability to survive stochastic events. Herein, I am testing how life traits
and range affect genetic diversity by comparing a rare species to its widespread
conspecific. As the number of rare species grows and threats increase, it is
exceedingly important to understand what factors influence rarity and endemism and
how narrow endemic species are able to respond to rapid changes in their
environments.
12


The genus Carex L., commonly referred to as the sedges, is one of the largest
genera of plants, with as many as 2,000 species (Ball et al., 2002). Sedges are
distributed worldwide and found in many different habitats such as the arctic tundra,
grasslands, and forests, although they are generally associated with moist to wet
substrates (Ball et al., 2002). All carices possess unisexual flowers, with the pistilate
flower enclosed within a sac-like structure called a perigynium; this structure forms
much of the basis for classification in this complex genus (Starr et al., 1999).
Although most Carex species are monoecious, containing both male and females
flowers on the same plant, some species are dioecious, with male and female flowers
on separate plants. Dioecy, which can be expected to affect the genetic structure of a
species, is rare among angiosperms and tends to occur in derived portions of
angiosperm clades (Dorken and Barrett, 2004). Only a few sections of Carex,
including section Scirpinae Tuckerman, contain dioecious species (Ball et al., 2002).
It is likely that dioecy evolved independently in each of these sections by selection
favoring outcrossing, which avoids inbreeding and optimizes resource allocation
(Mitchell and Diggle, 2005). Inbreeding has been shown to be common in Carex,
particularly in species with a caespitose habit (Bruederle, Yarbrough, and Kuchel, in
press).
13


Carex section Scirpinae is distributed across northern North America, from
Greenland west to Alaska, south into northeastern United States, and west to Oregon
through the Rocky Mountains; it is also known from East Asia and Scandinavia
(Dunlop, 1990). As currently described, this section is composed of two species:
Carex scirpoidea Michx. and C. curatoum Stacey. Furthermore, C. scirpoidea
comprises four subspecies: C. scirpoidea Michx. ssp. scirpoidea, C. scirpoidea
Michx. ssp. pseudoscirpoidea (Rydberg) Dunlop, C. scirpoidea ssp. convoluta
(Kukenthal) Dunlop, and C. scirpoidea Michx. ssp. stenochlaena (Holm) Love and
Love. Although these taxa are relatively well defined based upon vegetative and
reproductive structures, distribution, and habitat, their phylogenetic relationships are
poorly understood.
Most recently, Dunlop (1990) circumscribed this section based on variation in
morphology, anatomy, achene, and perigynium micromorphology, chromosome
number, ecology, breeding relationships, and distribution patterns. Dunlop found
evidence to exclude two taxa that are often listed as synonymous and, in most
previous studies, included in section Scirpinae', these are C. gigas (Holm) Mackenzie
and C. scabriuscula Mackenzie (Dunlop, 1997). As currently defined by Dunlop
(1990), all taxa in section Scirpinae have unisexual inflorescences on unispicate
culms. Although C. scirpoidea is considered secure throughout its range (G5), it is
14


rare in several states (Table 1) and locally rare in the periphery of its range. However,
certain taxa within Carex section Scirpinae are extremely rare throughout their entire
ranges, such as C. scirpoidea ssp. convoluta, which is the focus of this research.
Many taxa in this section are found in unusual and rare habitats within wet
substrates and high levels of calcium. For instance, C. scirpoidea ssp. convoluta is an
edaphic endemic, restricted to threatened alvar communities of shallow soil and
exposed bedrock, and known only from the northern shores of Lake Huron. Globally,
alvars are primarily found in the Baltic regions of Sweden and Estonia and the Great
Lakes region in North America (Kaeli et al., 2003). These rare habitats have open
areas of exposed limestone or dolomitic bedrock with patches of thin soil and are
often demarcated from other such habitats by forest or woodlands with distinct breaks
between the two (Kaeli et al., 2003). Carex scirpoidea ssp. convoluta is rare and
typically isolated from other taxa in this section.
The locations of Pleistocene glacial refugia for this section are unknown (Dunlop,
1990). Populations of C. scirpoidea could have survived below the glacial maximum
in the eastern United States subsequently colonizing prairie pavements in the Great
Lakes region; alternatively, refugia could have been located in the western United
States or Alaska, with populations spreading to the Great Lakes region, where C.
scirpoidea ssp. convoluta differentiated as it adapted to this harsh environment. The
15


Laurentide ice sheet retreated from this region approximately 13,000 years BP and
was followed by a time of isostatic rebound, retreating shorelines due to lowered
drainage outlets, and finally the development of soil horizons between 7,700 and
3,300 years BP (Delcourt et al., 2002). As such, C. scirpoidea ssp. convoluta has had
a seemingly short evolutionary history and might have lacked the time to expand its
range. Range expansion can also be limited by poor or short distance dispersal of
seeds, inhospitable soil moisture and surface characteristics, predation, competition
with other species, or any combination of these (Maschinski et al, 2004).
16


2. Research Objectives
The objectives of this research are to describe the genetic diversity and structure
in C. scirpoidea ssp. convoluta within the context of its unique combination of
breeding system, evolutionary history, and narrow endemism. I hypothesize that C.
scirpoidea ssp. convoluta will have lower genetic diversity than its widespread
conspecific because of its narrow endemism, rarity, and recent evolutionary history
and will have similar genetic structure, as both are obligate outcrossers.
Studying the genetic variation present in a population can be useful for
determining the conservation needs of a rare species, such as C. scirpoidea ssp.
convoluta. The amount of genetic variability and its apportionment can determine a
species ability to adapt to changing selection pressures (Maki and Asada, 1998;
Batista et al., 2001). Genetic diversity, which can be apportioned within- and among-
populations, is influenced by evolutionary history and life history traits, such as
breeding system and habit, which can be expected to alter rates of gene flow
(Hamrick, 1982; Hamrick and Godt, 1996; Procaccini et al., 2001). Numerous factors
can be expected to influence the level and apportionment of genetic diversity in C.
scirpoidea ssp. convoluta, often with contradictory outcomes. These include dioecy,
narrow endemism, and rarity as they relate to evolutionary history.
17


Finally, I propose framing this comparison within a phylogenetic context. I
hypothesize that C. scirpoidea ssp. convoluta has a recent evolutionary origin and
will thus be located towards the derived ends of the sectional phylogeny. This study
will present a phylogenetic hypothesis of Carex section Scirpinae based on nuclear
ribosomal sequence data, which will clarify the relationship of C. scirpoidea ssp.
convoluta to its widespread conspecific, C. scirpoidea spp. scirpoidea, and will begin
to test the hypotheses proposed by Dunlop (1990) as to the location of glacial refugia
of this section.
2.1 Dioecy
Dioecy in Carex, as in many flowering taxa, is the result of the repeated evolution
of an adaptation that serves to avoid inbreeding and allocate resources (Mitchell and
Diggle, 2005). Through obligate outcrossing, dioecy can affect the genetic makeup of
a population and species in several ways. Dioecious species often exhibit
heterozygote excess and a trend towards greater genetic diversity within populations
relative to closely related monoecious species (Sole et al., 2004). Consequently,
dioecy can be expected to reduce population differentiation regardless of range or life
history traits (Hamrick and Godt, 1996).
Overall, dioecy should increase or maintain variation within a population by
reducing the likelihood of inbreeding and decreasing genetic structure within and
among populations through increased gene flow. Theory holds that only a low rate of
18


gene movement is needed to prevent population differentiation (Loveless, 1984).
Hamrick and Godt (1996) found that species with less inter-population differentiation
were associated with greater genetic diversity (higher Ps: percentage of polymorphic
loci within a species and Hes: expected herterozygosity for a species). This correlation
should hold true for dioecious species with an expectation of high levels of genetic
diversity due to obligate outcrossing and little genetic structure due to greater gene
flow.
2.2 Biogeography
Carex scirpoidea ssp. convoluta is restricted to rare alvar communities. Bedrock
of geological series from the Devonian, Silurian, or Ordovician underlie alvar habitats
and influence conditions and vegetation. The underlying series have different rates of
erosion and weathering, which creates varying depths and types of soil. Alvar
communities are characterized by a mix of boreal and prairie species from both the
cold post-glacial environment and the warmer, drier period that followed (Reschke et
al., 1999). However, although most of the United States was drying and warming
during the mid to late Holocene (6,000 3,000 yrs BP) (Shin et al., 2006), the
microclimate of the Great Lakes region received greater precipitation due to the lake
effect approximately 5,000 yrs BP (Delcourt et al., 2002).
Within the North American range of Carex section Scirpinae from Alaska to the
northeastern United States and as far south as the Southern Rocky Mountains, Dunlop
19


(1990) found the greatest morphological diversity in western North America, with the
exception of Carex scirpoidea ssp. convoluta, which is the only member of this
section not found in the west. Furthermore, this taxon is found only north of the
glacial boundary in eastern North America. The range of C. scirpoidea ssp.
scirpoidea extends south through the Rocky Mountains into Colorado. The last
glacial maximum did not reach the populations in Colorado, although global climate
changes approximately 6,000 yrs BP affected the range of wetland species with ebbs
and advances of alpine ice sheets (Benson et al., 2005, Shin et al., 2006).
2.3 Endemism
Endemism simply implies a restriction in distribution. As such, species can be
broad endemics, such as C. scirpoidea ssp. stenochlaena found in western North
America from Washington and Montana north to Alaska, or narrow endemics such as
Carex scirpoidea ssp. convoluta. This latter is an edaphic endemic that is restricted to
a certain soil substrate. Edaphic endemics are believed to be restricted either as the
result of a reduction in their range associated with climate change or due to recent
speciation associated with adaptation to a new substrate (Baldwin, 2005). Ecological
selection and genetic drift can cause rapid divergence among populations found in
isolation on a unique peripheral habitat (Baldwin, 2005). The harsh environment of
alvars, with fluctuations in moisture and small pockets of soil, can be expected to lead
to high selection rates. The limestone and dolomite bedrocks erode slowly leading to
20


shallow soil layers, which prevent surrounding forest species encroachment into
alvar communities (Kaeli et al., 2003; Schaefer and Larson, 1997). Natural selection
acts to eliminate maladapted traits and can be expected to reduce genetic diversity as
a consequence. Furthermore, range restricted species tend to maintain less genetic
diversity than more widespread species (Godt et al., 2004). However, a species
distribution is highly influenced by historical origins and past reductions in
population size (Loveless, 1984; Gitendanner and Soltis, 2000; Godt et al., 2004).
The history of a species might be more influential in shaping its genetic
apportionment and diversity than current range.
This study examines the genetic diversity of this rare, narrow endemic within the
context of its closest relatives to provide perspective for the relative level of genetic
diversity. Allozymes are a quick and effective way to characterize and quantify the
genetic diversity within and among populations and examine departures from Hardy-
Weinberg equilibrium (Ayres and Ryan, 1999).
21


3. Materials and Methods
3.1 Allozyme analysis
Ramets, comprising at least one fertile and one vegetative culm, were collected
from five populations of C. scirpoidea spp. convoluta from the Lake Huron shoreline
on Drummond Island, the lower peninsula of Michigan, and from Manitoulin Island,
Ontario (Table 2). Soluble enzymatic proteins were extracted from approximately 1
cm2 of young leaf tissue for each of 25 to 50 individuals per populations following
Kuchel and Bruederle (2001). Extracts were absorbed onto wicks cut from No. 17
chromatography paper and kept at -70C until electrophoresis. Wicks were subjected
to horizontal 11 % starch gel electrophoresis by means of three gel-electrode buffer
systems: lithium-borate (pH 7.6/8.0), histidine-HCL (pH 7.0), and tris-citrate (pH 7.5)
(Yarbrough, 2000). Fifteen substrate-specific stains were used to visualize loci
following eletrophoretic separation (Table 4). Data were collected as genotypes and
subjected to statistical analysis.
The Genetic Data Analysis (GDA) software of Lewis and Zaykin (2001) was
used to calculate genetic diversity statistics including the percent of loci that were
polymorphic (P), number of alleles per polymorphic locus (Ap), expected
heterozygosity (He), observed heterozygosity (Ho), and the inbreeding coefficient (f),
22


as well as the F-statistics, which describe genetic apportionment. Specifically, the F-
statistics F\$ and Fn were used to determine the difference between the observed and
expected levels of heterozygosity, while Fst was used to estimate the reduction of
heterozygosity due to genetic differences among populations. Neis (1978) genetic
identity coefficient was used to quantify similarity of allele frequencies among
populations based upon allele frequency. Unique genotypes were counted by hand
and Arlequin version 3.11 (Excoffier et al., 2005) was used to determine the
partitioning of genetic variation within and among populations through an analysis of
molecular variance (AMOVA). A two-sided Mann-Whitney U-test was used to
determine any significant differences between genetic diversity statistics of C.
scirpoidea ssp. convoluta and C. scirpoidea ssp. scirpoidea.
3.2 DNA Sequencing
Eight populations representing four subspecies of C. scirpoidea and one
population of C. curatorum were sampled for sequence analysis (Table 3). From each
population, leaf tissue was harvested haphazardly from 3-50 individuals and either
dried and stored in silica gel or stored at 6C until DNA extraction. Additional leaf
tissue was collected from herbarium specimens at the University of Washington,
Seattle herbarium (WTU) for C. scirpoidea ssp. stenochlaena. Vouchers are
deposited at the Kathryn Kalmbach Herbarium of Denver Botanic Gardens (KHD).
23


Total genomic DNA was extracted following lab procedures in preparation for
amplification of two nuclear ribosomal regions (Appendix A). Reaction mix for PCR
amplification of nrDNA regions consisted of: 2.5pl 10X reaction buffer; 1.5pl 25 mM
MgCh; l.Opl 25 mM dNTPs, equimolar ratio; 0.25pl lU/pl Taq\ 1.5pl 10 pM
forward primer; 1.5pl 10 pM reverse primer; lpl of approximately 10 ng/pl sample
DNA; and 15.75pl water (total of 25pl). Primers for the internal transcribed spacer
regions (ITS-1 and ITS-2) and the intervening 5.8S ribosomal subunit were taken
from Roalson et al. (2001) as ITS 4i and ITS 5i; primers for a portion of the five
prime end of the intergenic spacer (ETS If) were taken from Starr et al. (2003) as
18Sr and ETS If. The reaction profile for both regions was the same: an initial
denaturation at 95C for two minutes; 32 cycles of 95C for one minute, 55C for one
minute, and 72C for one minute, followed by a final extension at 72C for ten
minutes. Amplification products were purified using ExoSAP-IT (USB, Cleveland,
OH, USA), following a modification of the manufacturers protocol involving
reduction in enzyme concentration and increase in incubation time. Sequencing of
both forward and reverse strands was performed at the Rocky Mountain Center for
Conservation Genetics and Systematics (University of Denver, Denver, Colorado,
USA) on a Beckman Coulter CEQ 8000 Genetic Analysis System following cycle-
sequencing with the GenomeLab DTCS Quick Start Kit for Dye Terminator Cycle
Sequencing (Beckman Coulter, Fullerton, California, USA).
24


Sequences were aligned by visual proof reading in Sequencher and then analyzed
using Phylip for creating phylogenetic trees. Additional sequences were obtained
from the open source National Institute of Health genetic sequence database,
GenBank (Figures 3-5). The phylogeny of Carex section Scirpinae was rooted with
the closely related Carex stylosa C.A. Mey. and Carex donnell-smithii L. H. Bailey
(Hendrichs et al., 2004a).
25


4. Results
4.1 Population Genetics
Fourteen enzymes encoded by 19 loci were resolved for this study (Table 4).
Three loci (PGI-1, PGM-1, and IDH-1) were excluded due to poor or inconsistent
resolution. Averaged across populations, 27.37% of the loci were polymorphic for C.
scirpoidea ssp. convoluta, with 2.32 alleles per polymorphic locus. In contrast, C.
scirpoidea ssp. scirpoidea averaged only 20.00% of loci that were polymorphic, with
2.29 alleles per polymorphic locus (Yarbrough, 2000). Car ex scirpoidea ssp.
convoluta does not have significantly greater percent of polymorphic loci nor alleles
per polymorphic locus (W=l 8.5, p=0.2477 and W=19,/)=0.2059 respectively).
Percent of loci polymorphic at the species level was 42.11% for C. scirpoidea ssp.
convoluta, with 2.63 alleles per polymorphic locus. In comparison, C. scirpoidea ssp.
scirpoidea had 29.41% loci polymorphic and 2.29 alleles per polymorphic locus
(Yarbrough, 2000).
Mean observed heterozygosity (H0) for C. scirpoidea ssp. convoluta was 0.089
(SE 0.011) with 0.097 (SE 0.011) expected heterozygosity (He). Expected
heterozygosity at the species level was 0.092 (SE 0.096). Less heterozygosity was
observed in the rare, narrow endemic than expected from Hardy-Weinberg
26


(Ho=0.089, He=0.097), while C. scirpoidea ssp. scirpoidea had more observed
heterozygosity than expected (Table 5). Observed heterozygosity for C. scirpoidea
ssp. convoluta for the species was also lower than expected heterozygosity
(HOi=0.092, Hei=0.126). Carex scirpoidea ssp. convoluta had a trend towards greater
expected heterozygosity although the difference was not significant (W=20,p=0.07).
Averaged across populations, C. scirpoidea ssp. convoluta exhibits a slight
reduction in heterozygosity (/=0.067, SE 0.074), while the species average shows a
greater reduction (^=0.275, SE 0.072) (Table 5). Only two populations, Maxton Plains
and Big Shoal Cove, both from Drummond Island, had significant deviations from
Hardy-Weinberg equilibrium, each indicating a loss of heterozygosity (Table 6). Only
four significant deviations at a single locus occurred (p < 0.05) and three of these
deviations indicated a loss of heterozygosity (Table 6).
The estimate of inbreeding in individuals relative to each population (Fis) was
slightly positive, but not significantly different from zero for C. scirpoidea ssp.
convoluta (Fis=0.091), while the estimate of inbreeding in individuals compared to all
populations as a whole (Fit) was even greater (Frr=0.311) and differed significantly
from zero. Both indicate a reduction of heterozygosity from expectations. Genetic
apportionment and differentiation among populations was relatively low, but
significantly different from zero in C. scirpoidea ssp. convoluta (Fst=0.232).
27


However, population differentiation was lower in the widespread conspecific, C.
scirpoidea ssp. scirpoidea (Fst=0.123).
Precautions were taken in the field to prevent multiple collections of a single
clone. Among The percentage of unique genotypes ranged from 48% in Burnt Island
to 84% in Big Shoal Cove. Some genotypes were found across populations: Maxton
Plains and Murphy Point, Big Shoal Cove and Murphy Point each shared one;
Maxton Plains and Burnt Island, Big Shoal Cove and Burnt Island each shared two;
Presque Isle and Burnt Island shared three genotypes; Maxton Plains and Big Shoal
Cove shared four; and Burnt Island and Murphy Point shared seven genotypes.
Populations of C. scirpoidea ssp. convoluta were genetically very similar. Neis
genetic identity (1978) among populations was very high, with a range of 0.94 to
0.99. The greatest identity (I) was found between the two Manitoulin Island
populations, Burnt Island and Murphy Point (I = 0.99). A phenogram of the
populations based on Neis identity and coancestry distances cluster the two
Manitoulin Island populations as one monophyletic group and the Drummond Island
populations with the mainland Presque Isle population as another (Figure 2).
Significant variation among all populations, indicated by AMOVA, produced an
amount similar to the Fst value calculated by GDA. Although the Manitoulin Island
populations diverged from the Drummond Island and mainland populations in the
phenogram, only 1.92% of variation was explained by differences between these two
28


groups (p<0.05). There is a significant difference between the four island populations
and the one mainland population, Presque Isle. The AMOVA explained 24.93% of
the differences among populations by the comparison of the island populations to the
mainland population (p<0.05) with 12.94% of the variation found within the island
group. In a comparison between populations of C. scirpoidea ssp. scirpoidea and C.
scirpoidea ssp. convoluta, 55.22% of the variation is explained by differences
between populations of the two taxa (p<0.05), with 18.76% of the differences among
populations within each group (p<0.05), and 26.01% of the variation within
populations (p=0.01).
4.2 Phylogeny
Phylogenies created from the internal transcribed spacer (ITS-1) region and the
external transcribed spacer (ETS) region were not congruent. The ITS-1 consensus
phylogeny provides evidence for placing C. scirpoidea ssp. stenochlaena basal in a
monophyletic group comprising Carex section Scirpinae and the previously excluded
C. gigas (Figure 3). However, confirmation of the correct identification of the latter
and other GenBank samples was not possible. ITS-1 provides weak support to resolve
C. scirpoidea ssp. convoluta as a derived taxon. Carex curatorum, an Arizona and
Utah endemic, is also derived in the ITS-1 phylogenetic tree. However, according to
the external transcribed spacer (ETS) consensus phylogeny, C. curatorum is basal in
the monophyletic group containing taxa from Carex section Scirpinae. Carex
29


scirpoidea ssp. convoluta is sister to the group comprising Washington C. scirpoidea
ssp. stenochlaena and Alaskan C. scirpoidea ssp. scirpoidea (Figure 4). Car ex
scirpoidea ssp. convoluta is weakly supported as a monophyletic group with C.
scirpoidea ssp. scirpoidea providing support to make this an appropriate comparison
of levels of genetic diversity. Alhtough the two nuclear ribosomal regions were non-
congruent, the datasets were combined to create a consensus tree with C. donnell-
smithii as the outgroup (Figure 5). This final analysis indicates that C. scirpoidea is
derived from C. curatorum and that C. scirpoidea ssp. convoluta is sister to the
remaining two subspecies, C. scirpoidea ssp. stenochlaena and C. scirpoidea ssp.
scirpoidea. Carex scirpoidea ssp. pseudoscirpoidea was poorly resolved in ETS and
thus excluded from both analyses. The combined analysis provides very strong
support for each subspecies division with bootstrap values from 994 to 1000,
although only weak support for the relationship among these taxa.
30


5. Discussion
Despite factors that should lead to decreased levels of genetic diversity, C.
scirpoidea ssp. convoluta maintains a high or similar amount of genetic diversity both
when compared to its widespread conspecific, C. scirpoidea ssp. scirpoidea, and
when compared to other taxa with similar traits (Hamrick and Godt, 1996). Narrow
endemism is expected to decrease genetic diversity. If C. scirpoidea ssp. convoluta is
a narrow endemic because it is adapted to prairie pavement habitats, then extreme
selection for survival in this harsh environment could be expected to affect levels of
genetic diversity. However, life history traits can be variously influential on levels of
genetic diversity. Endemic species that are obligate outcrossers have a relatively high
level of expected heterozygosity (Hes=0.142) compared to self compatible endemic
species (Hes=0.034) (Hamrick and Godt, 1996; Bruederle, Yarbrough, and Kuchel, in
press). Carex scirpoidea ssp. convoluta has nearly four times the expected diversity
of self compatible endemic species and nearly the same amount as outcrossing
endemics. The dioecy of C. scirpoidea ssp. convoluta may serve to maintain greater
levels of genetic diversity despite its restricted range. Hamrick and Godt (1996) found
that outcrossing species for which seed dispersal involved epizoochory (attachment to
animals), gravity, or ingestion had an average expected heterozygosity of H6J=0.180.
31


Carex scirpoidea ssp. convoluta, which is likely dispersed by one or more of these
methods, has an expected heterozygosity of Hes=0.126.
Dioecy can explain the maintenance of high genetic diversity in C. scirpoidea
ssp. convoluta compared to other rare, endemic species, many of which are not
dioecious. This does not explain the comparison to C. scirpoidea ssp. scirpoidea,
which is both widespread and an obligate outcrosser. Populations of C. scirpoidea
ssp. scirpoidea in Colorado are at the periphery of their range, which may lead to
lower genetic diversity than would be expected in the center of its distribution
(Yarbrough, 2000). However, C. scirpoidea ssp. scirpoidea tends to maintain large
populations, even in the periphery of its range (Shackleford, 2003). The southern
Rocky Mountain region was periodically covered in discontinuous glacial sheets
during the last glaciation, allowing some gene flow among populations persisting in
glacial refugia (DeChaine and Martin, 2005). Correspondingly, Colorado populations
of C. scirpoidea ssp. scirpoidea have very low population differentiation, potentially
as a result of continuous gene migration among these populations, which are all found
within one watershed.
Unlike most rare and endemic species (Gitzendanner and Soltis, 2000), C.
scirpoidea ssp. convoluta has relatively low population differentiation (Fst= 0.242)
implying greater gene flow. However, compared to other outcrossing species with
similar seed dispersal methods (average Fst= 0.175) or with similarly narrow ranges
32


(Fst~ 0.179), C. scirpoidea spp. convoluta has approximately 1.4 times more
population differentiation (Hamrick and Godt, 1996). A negative value of FIS is
expected for an obligate outcrosser indicating heterozygotic excess. However, C.
scirpoidea ssp. convoluta has a slightly positive FiS indicating some, though not a
significant, loss of heterozygosity. This can be explained by cryptic structuring within
the population; a departure from panmixia resulting from the Walhund effect due to
subpopulation structuring. Heterozygotes are lost either through direct inbreeding or
remote inbreeding. Species with a dioecious breeding system are obligate outcrossers
and thus cannot self. However, if a population is small, there is still the likelihood that
close relatives will mate, leading to remote inbreeding and a reduction of
heterozygosity. When closely related species continuously mate, the likelihood of
identity by decent increases where an individual at a particular locus will be
homozygous with two copies of the same allele from one ancestor. The F statistics
indicate that there is some subpopulation structuring present and that closely related
individuals are likely to mate and pass along two copies of an allele that is identical
by decent. This increased chance of homozygosity accordingly leads to a loss of
heterozygosity due to remote inbreeding.
In populations of C. scirpoidea ssp. convoluta, Fu was greater than Fis indicating
some loss of heterozygosity due to genetic differentiation among populations. The
widespread conspecific, C. scirpoidea ssp. scirpoidea, has a negative value for Fis,
33


only slightly larger Fit, and very few differences among populations (Fst = 0.123).
This pattern is more common for an obligate outcrosser whose breeding system
increases gene flow. Lowered gene flow among C. scirpoidea ssp. convoluta
populations could be caused by the restricted range and edaphic conditions. The open
alvar communities and surrounding forested areas tend to represent distinct plant
communities with little overlap in either the seed bank or above ground vegetation
(Kaeli et al., 2003). Despite a few carices that frequently inhabit both ecotypes (Kaeli
et al., 2003), there is little chance that C. scirpoidea ssp. convoluta inhabits any land
between populations, even in ephemeral pockets, that would facilitate gene flow
among populations. The evolutionary history of C. scirpoidea ssp. convoluta
examined through the phylogeny of the section suggests recent divergence. If this
narrow endemic is recently derived, the founder effect could play a role in larger
levels of population differentiation. However, the combined regions phylogeny does
not support C. scirpoidea ssp. convoluta as the most recently derived.
Carex section Scirpinae consists of two relatively well-supported species, C.
curatorum and C. scirpoidea. Based upon the combined analyses of the ITS and ETS
nuclear ribosomal regions, each subspecies (C. scirpoidea ssp. convoluta, C.
scirpoidea ssp. stenochlaena, and C. scirpoidea ssp. scirpoidea) appears as a highly
resolved taxon, although the relationship among these taxa is weakly supported
(Figure 5). Carex scirpoidea ssp. convoluta, in particular, is highly resolved with
34


100% of the most parsimonious trees placing the two C. scirpoidea ssp. convoluta
samples as one clade. However, the individual analyses do not agree.
In general, nuclear ribosomal regions follow concerted evolution with little to no
intraspecific variation. However, it is common for ITS and ETS regions to not
coalesce, because the ITS region can possess intraspecific polymorphisms that lead to
false phylogenetic hypotheses (Roalson and Friar, 2004). The ETS region does not
add support to resolve most of the taxa in this section except for moderate resolution
for C. scirpoidea ssp. convoluta (Figure 4). Non-coalesence of the ETS and ITS
regions could be due to insufficient data and thus random sampling error (Roalson
and Friar, 2004) or could represent a real difference in the evolution of these two
genes (Planet, 2006). Among those who debate when and if datasets should be
combined to create consensus trees, Planet (2006) suggests combining when non-
congruence can be tested and determined to be due to more than random error.
Because the ITS-1 and ETS regions are not congruent, and because further testing of
this phylogenetic hypothesis was not within the scope of this study, results from both
the individual and combined trees should be taken as suggestive, not conclusive,
evidence of this phylogenetic hypothesis. Additional sequencing should lend further
credence to test this phylogenetic hypothesis.
The ITS region, for which a C. gigas sequence was obtained through GenBank,
places this previously excluded taxon in a group with section Scirpinae. However,
35


correct identification of the C. gigas sample could not be confirmed and, thus, the
possibility exists that this finding is purely due to an identification error. Carex
scirpoidea ssp. stenochlaena from Washington appears basal to the group providing
evidence to support Dunlops (1990) hypothesis that glacial refugia might have
survived below the ice in western North America. If this were true, post glacial
migration could have been influenced by migratory birds transporting seed. Viable
Carex seeds have been found in the digestive tract of some ducks and sedges can
tolerate mammal browsal (Leek and Schiitz, 2005). These taxa are likely not used
heavily for forage, but epizoochory involvoing water fowl might move seed.
Past allozyme studies of Carex species has shown less genetic diversity than
reported for other flowering plants (Bruederle et al., in press), a pattern of high inter-
population differentiation and low intra-population differentiation (Ford et al., 1991).
Although more sequence data is needed to help resolve this phylogeny, it appears that
C. scirpoidea ssp. convoluta has a combination of adaptations that help maintain
genetic diversity and thus the evolutionary potential to withstand stochastic events.
Alvar communities could have been formed south of the last glacial maximum and
have become isolated as the ice sheet retreatws around 11,000 BP or as the climate
warmed and prairies expanded around 5,000 BP (Hamilton and Eckert, 2007). Both
proposed mechanism of formation of these unique and rare communities have
implications for the genetic differentiation of their component species. Evidence from
36


C. scirpoidea ssp. convoluta does not support the assumption that populations in alvar
communities should have less genetic diversity and more population differentiation
than those in the center of their range (Hamilton and Eckert, 2007). However, future
study should include populations of C. scirpoidea ssp. scirpoidea from the Great
Lakes region. The high level of genetic diversity within populations of C. scirpoidea
ssp. convoluta could either indicate the formation of alvar communities was a
postglacial expansion of prairie habitat with only slight loss of genetic variation from
its widespread conspecific or could be due to the general adaptability and diversity of
the genus Car ex and the dioecious breeding system of section Scirpinae..
37


Figure 1: Distribution of (a) Carex scirpoidea Michx. ssp. convoluta (Kiikenth.) Dunlop
on the shores of Lake Huron in Michigan and Ontario and (b) Carex scirpoidea Michx.
ssp. scirpoidea (Cyperaceae) in North America and Beringia. Populations sampled for
allozyme analysis shown in red. Range maps adapted from Dunlop and Crow (1999).
38


Table 1: Habitat, distribution, morphological characteristics, habit, and state ranks for
each of five taxa in Carex section Scirpinae (Cyperaceae).____________________________________
Taxa Habitat Distribution Morphology/Habit The Nature Conservancy Rank
Carex scirpoidea ssp. scirpoidea Wet substrates, high level of calcium Northern United States, Rocky Mountains, Canada, Greenland. Disjuncts in Norway and the Arctic Caespitose, new shoots diverge variously from short rhizomes, shoots of previous year die back completely to rhizome. Ligules 0.2 to 3mm. Perigynium pubescence white to light tan. Imperiled (S2): Maine, Nova Scotia, Washington, Wyoming
Carex scirpoidea ssp. pseudoscirpoidea Dry ridges, alpine fellfields, non- calcareous soils Higher elevations of Rocky Mountains, west into Oregon, Washington, British Columbia Rhizomatous, shoots arise linearly. Intemodes of rhizomes greater than 1 cm. Culms from second year shoots, leaves shorter and wider than ssp. scirpoidea. Ligules 0.2 to 3mm. Perigynium pubescence white to light tan. Critically Imperiled (SI): California Vulnerable (S3): Wyoming
Limestone Ontario, Caespitose, new shoots diverge variously from
pavement Michigan short rhizomes, shoots of previous year die back
§ 3 where gypsum completely to rhizome. CO 00
.§-"3 and shale has Ligules 0.2 to 3mm.
* § eroded §
3 8 Narrow leaf variety of Carex scirpoidea, V- 2 o *n
q d- C £ shaped ^ £ 3 C
> o
Weakly acidic Mountains of Caespitose, new shoots diverge variously from
soils, high Washington and short rhizomes, shoots of previous year die back
levels of Montana, Coastal completely to rhizome.
d. in magnesium, range southern Ligules 0.2 to 3mm. O0
low levels of British Columbia "2
1 calcium. and Alaska. Flowering spikes have a clavate shape, scales are C
'5 § longer and more numerous at the spike apex, 1> O.
Sr o -5 spikes droop E
-s x % Perigynium pubescence white to light tan, >>
lanceolate, longer than 3mm, over 2.5 times long o o ri W>
U ^5 as wide u. u o
Riparian and Southern Utah, Caespitose, leaves longer than C. scirpoidea,
hanging northern Arizona sparsely pilose adaxial leaf surface
garden Achenes not tightly enveloped by perigynia,
s communities subtending scales light in color and narrower than 1 5
O along the San perigynia. Perigynia and achenes disarticulate § >>
a ** Juan and from spikes. Pistillate spikelets poses a rachilla E W
8 CullU cull) rivers, Ligules 1mm to 5mm, generally more than 3mm = Cd O cd e -r
rQ sandstone and 2 § g.
u limestone a'-* m
39


Table 2: Locations for five Carex scirpoidea Michx. ssp. convoluta (Kiikenth.) Dunlop
(Cyperaceae) populations sampled for allozyme analysis, where N equals the number of
individuals sampled.
Population N County/Island State
Presque Isle 50 Presque Isle County MI
Maxton Plains 50 Chippewa County MI
Big Shoal Cove 50 Chippewa County MI
Burnt Island 50 Manitoulin Island ONT
Murphy Point 25 Manitoulin Island ONT
40


Table 3: Location for populations sampled for sequence analysis from Carex section
Scirpinae (Cyperaceae): Carex scirpoidea Michx. ssp. scirpoidea (CaScSc), Carex
scirpoidea Michx. ssp. pseudoscirpoidea (Rydb.) Dunlop (CaScPs), C. scirpoidea Michx.
ssp. stenochlaena (CaScSt), Carex scirpoidea Michx. ssp. convoluta (Kukenth.) Dunlop
(CaScCo), and Carex curatorum Stacey (CaCu). Carex scirpoidea ssp. stenochlaena samples
are from herbarium specimens deposited at the University of Washington, Seattle herbarium
(WTU).
Population Species County State
Coyote Gulch, Glen Canyon CaCu Kane County UT
Ft. Richardson CaScSc Anchorage AK
Arctic Valley CaScSc Anchorage AK
Whisky Gap CaScPs Carbon County WY
Union Peak CaScPs Sublette County WY
High Creek Fen CaScSc Park County CO
Beaver Creek CaScSc Park County CO
Big Sandy Opening CaScSc Sublette County WY
Avalanche Chute CaScSt Skagit County WA
Twin Sisters Mountain CaScSt Whatcom County WA
Big Shoal Cove CaScCo Chippewa County MI
Maxton Plains CaScCo Chippewa County MI
41


Table 4: Starch gel and electrode buffer systems and substrate-specific stains used in
allozyme analysis conducted on Carex scirpoidea Michx. ssp. convoluta (Kiikenth.) Dunlop.
System Substrate-Specific Stain Loci Polymorphic
o 1 no
cc diaphorase (DLA) menadione reductase 2/1 yes
X (MNR) 3/2 yes
Qm o malic enzyme (ME) 1 no
cd u O triose-phosphate isomerase (TPI) 1 no
i e 2 jS i-t 2 no
alchohol dehydrogenase (ADH) 1 no
superoxidase dismutase (SOD) 1 no
m aminotransferase (AAT) 1 no
X
2 yes
ed s- -4< *o glyceraldehyde-3 -phosphate dehydrogenase (G3PDH) 1 no
CO n H shikimate dehvdrnfrenase tSDHt 1 no
2 no
6-phosphogluconate dehydrogenase (PGD) 1 yes
phosphogluco-isomerase (PGI) 1 unresolved
K a. 2 yes
U K 1 phosphoglucomutase (PGM) 1 unresolved
2 yes
isocitrate dehydrogenase (IDH) 1 unresolved
to s 2 yes
malate dehydrogenase (MDH) 1 yes
2 Yes
42


Table 5: Comparison of population level genetic diversity statistics for five populations of
the narrow endemic, Carex scirpoidea Michx. ssp. convoluta (Kiikenth.) Dunlop
(Cyperaceae): Presque Isle, Maxton Plains, Big Shoal Cover, Burnt Island, and Murphy
Point, the widespread, Carex scirpoidea Michx. ssp. scirpoidea, caespitose, and rhizomatous
carices. Comparative data for C. scirpoidea ssp. scirpoidea, caespitose and rhizomatous
carices taken from Yarbrough (2000). P = percent of loci polymorphic, Ap = number of
alleles per polymorphic locus, Ho = observed heterozygosity, He = expected heterozygosity,
and/= fixation index. Ps, Aps, Hos, Hes, and fs are species estimates.
Genetic Diversity Population Mean P Ap Ho He /
Carex scirpoidea ssp. convoluta 27.37% (0.21-0.37) 2.32 (2.14-2.60) 0.089 (0.094-0.104) 0.097 (0.122-0.144) 0.08 (-0.122-0.254)
Presque Isle 0.368 2.143 0.118 0.132 -0.122
Maxton Plains 0.263 2.600 0.111 0.086 0.224
Big Shoal Cove 0.263 2.400 0.116 0.087 0.254
Burnt Island 0.211 2.250 0.073 0.072 0.019
Murphy Point 0.263 2.200 0.069 0.071 -0.039
Carex scirpoidea ssp. scirpoidea 20.00% 2.17 0.074 0.068 na
caespitose carices 14.15% 2.06 na 0.043 na
rhizomatous carices 41.93% 2.23 na 0.179 na
Genetic Diversity Species Ps Ap.5 Ho.s' Hes fs
Carex scirpoidea ssp. convoluta 42.11% 2.63 (2-3) 0.091 (0-0.549) 0.126 (0-0.600) 0.275 (-0.033-0.625)
Carex scirpoidea ssp. scirpoidea 29.41% 2.29 na 0.08 na
43


Table 6: Variable loci, deviations from Hardy-Weinberg Equilibrium (/), and significance
value (p) for five populations of Carex scirpoidea Michx. ssp. convoluta (Kiikenth.) Dunlop.
Values of -1 0 indicate heterozygotic excess. Values from 0 1 indicate a loss of
heterozygosity. 95% confidence intervals from bootstrap analysis are listed for population
level inbreeding coefficients. Standard errors (SE) listed below each population. Significant
Locus Presque Isle Maxton Plains Big Shoal Cove Burnt Island Mur phy Point
P- value / P- value / P- value / P- value / P- value f
DIA-2 (MNR-l) 0.338 -0.175 ...
DIA-3 (MNR-2) 0.625 0.085 0.000 0.568 1.000 0.050 0.056 0.295 1.000 0.000
AAT-2 1.000 -0.031 0.462 0.104 0.384 0.175 1.000 -0.011 1.000 0.013
PGI-2 0.000 -0.356 0.004 0.276 0.239 0.092 0.550 -0.129 1.000 0.028
PGD 0.263 0.202 1.000 -0.029
PGM-2 0.140 0.246 1.000 -0.089
MDH-2 0.236 -0.174
IDH-2 1.000 0.000 1.000 -0.001 0.000 0.747 1.000 -0.053 0.545 -0.211
Overall: (bootstrap) (SE) -0.1217 (-0.26- 0.08) (0.0744) 0.2245 (0.05- 0.46) (0.0965) 0.2539 (0.02- 0.57) (0.1444) 0.0189 (-0.13- 0.29) (0.0929) -0.0395 (-0.19- 0.03) (0.0437)
44


Table 7: Comparison of F statistics for Carex scirpoidea Michx. ssp. convoluta (Kukenth.)
Dunlop, C. scirpoidea ssp. scirpoidea, caespitose carices, rhizomatous carices, and all
carices. Data taken from Bruederle et al, in print.
Fis Fit Fst / Gst*
Carex scirpoidea ssp. convoluta 0.091 (-0.014-0.208) 0.311 (0.133-0.536) 0.232 (0.106-0.448)
Carex scirpoidea ssp. scirpoidea -0.097 0.023 0.123
Caespitose (N = 28) 0.412* (0.000-0.967)
Rhizomatous (N = 17) 0.159* (0.025-0.553)
All carices (N = 45) 0.344* (0.000-0.967)
45


Burnt Island
6
-------------- Murphy Point
9
Big Shoal Cove
8
7
Presque Isle
Maxton Plains
Distance Matrix Presque Isle Maxton Plains Big Shoal Cove Burnt Island Murphy Point
Presque Isle 0.9458 0.9437 0.9532 0.9505
Maxton Plains 0.2948 0.9801 0.9607 0.9623
Big Shoal Cove 0.2974 0.1321 0.9848 0.9826
Burnt Island 0.3094 0.2790 0.1275 0.9908
Murphy Point 0.3073 0.2590 0.1331 0.1058
Figure 2: Neighbor-joining tree based on Neis identity (1978) above and coancestry
distances below (GDA: Lewis and Zaykin, 2001)
46


Location
Species
doimdl-sm C. donnell- smithii* unknown
stjtaAY C. stylosa* Canada
ciinita AY C. crinita* Quebec
meitensii C. mertensii* Washingto n
aquatls C. aquatilis* Finland
bigdowii C. bigelowii* Finland
ecbiiioclilo C. echinochloe* Kenya
grayiAY7? C. grayi* Illinois
steno WC51 C. scirpoidea ssp. stenochlaena Washingto n
steuo SK52 C. scirpoidea spp. stenochlaena Washingto n
gj$asAF285 C. gigas* unknown
sciiAY?57? C. scirpoidea ssp. scirpoidea* Canada
conv BSC45 C. scirpoidea ssp. convoluta Michigan
conv Max50 C. scirpoidea ssp. convoluta Michigan
psuedo 4" C. scirpoidea ssp. psuedoscirpoidea Wyoming
sciip AV19 C. scirpoidea. ssp. scirpoidea Alaska
sciip FR48 C. scirpoidea. ssp. scirpoidea Alaska
sciiAF2850 C. scirpoidea. ssp. scirpoidea* unknown
ciuator 16 C. curatorum Utah
Figure 3: Consensus phylogeny of 1000 trees from nrDNA ITS-1 sequence. Parsimony
bootstrap values are listed at each node. C. donnell-smithii is used as the outgroup.
* sequences from GenBank
47


Species__________Location
stylosa Wa C. stylosa* Canada
uieitensu C. mertensii* Washington
grayi AY" 5 C. grayi Illinois
line cans AY c- baccans* Taiwan: Wu Lai
ediinochlo C. echinochloe* Kenya
cnnitaAY" C. crinita Quebec, CA
limosa AY7 C. limosa Quebec, CA
aquatilisA C. aquatilis* C. lenticularis Finland unknown
lenticular var. impressa
curator 46 C. curatorum Utah
C. scirpoidea ssp. Canada
scu Stan scirpoidea*
scu 48 C. scirpoidea. ssp. scirpoidea Alaska
coiivol 45 C. scirpoidea ssp. convoluta Michigan
couvo 50 C. scirpoidea ssp. convoluta Michigan
steno 52 C. scirpoidea ssp. stenochlaena Washington
scu 49 C. scirpoidea. ssp. scirpoidea Alaska
steno 51 C. scirpoidea spp. stenochlaena Washington
Figure 4: Consensus phylogeny of 1000 trees from nrDNA ETS sequence. Parsimony
bootstrap values are listed at each node. C. stylosa is used as the outgroup, ^sequences from
GenBank
48


Species
Location
1000
658
1000
999

453
994

domidl-sm C. donnell- smithii Unknown
echinoclilo C. echinochloe* Kenya
curator 46 C. curatorum Utah
convol 4? C. scirpoidea ssp. convoluta Michigan
couvo 50 C. scirpoidea. ssp. convoluta Michigan
steno 51 C. scirpoidea ssp. stenochlaena Washington
steno 52 C. scirpoidea ssp. stenochlaena Washington
scir 49 C. scirpoidea. ssp. scirpoidea Alaska
scir 48 C. scirpoidea spp. scirpoidea Alaska
Figure 5: Consensus phylogeny of 1000 trees from the combined nrDNA regions, ETS and
ITS. Parsimony bootstrap values are listed at each node. C. donnell-smithii is used as the
outgroup. *sequences from GenBank
49


APPENDIX A
Genomic DNA Protocol and Solution Recipes
1. Collect leaf or flower tissue (~0.1 g tissue) in a ceramic mortar
2. Grind in liquid nitrogen and let evaporate
3. Add 375 pi of DNA extraction buffer (with fresh, less than 1 month old, B-Me
at 7pi per lOmL of extraction buffer) and grind tissue with pestle
4. Add 375pl more DNA extraction buffer and grind with pestle
5. Transfer into an Eppendorf tube
6. Vortex and incubate in 65C waterbath for 10 minutes
7. Add 150pl solution III
8. Invert several times
9. Tubes on ice for 20 minutes
10. Label a second set of tubes and add 750pl of isoproanol to each tube
11. Centrifuge samples for 5 minutes at maximum speed
12. Transfer 750pl of sample to the second set of labeled tubes
13. Invert mixture several times
14. Centrifuge samples for 2 minutes at maximum speed
15. Remove liquid and leave the pellet
16. CAREFULLY add 500pl of 80% Ethanol
17. Swirl, remove liquid and leave the pellet
18. Let the pellet air dry overnight or at least 30 minutes in 37C incubator
19. Resuspend DNA in 50-200pl sterile water
50


DNA Extraction Buffer (100 ml)
50mM Tris, pH 8
lOmM EDTA, pH 8
lOOmMNaCl
1% SDS
Add fresh less than 1 month old
lOmM b-Mercaptoethanol
Solution III
5 M KOAc (potassium acetate)
Glacial Acetic Acid
H20
5ml 1 M stock
2ml 0.5 M stock
2ml 5 M stock
10ml 10% stock
70 pi of pure reagent
100ml 500ml
60ml 147.225g
11.5ml 57.5ml
28.5ml 142.5m
51


APPENDIX B
Allele Frequencies of Polymorphic Loci
Loci & Alleles Big Shoal Cove Burnt Island Maxton Plains Murphy Point Presque Isle
DIA-2/MNR-1 N= 100 N= 100 N = 100 N =48 N = 96
a 1.000 1.000 1.000 1.000 0.844
b 0.000 0.000 0.000 0.000 0.156
DIA-3 / MNR-2 N = 100 N= 100 N = 100 N = 48 N = 98
a 0.000 0.190 0.000 0.021 0.827
b 0.770 0.810 0.240 0.979 0.153
c 0.230 0.000 0.760 0.000 0.000
AAT-2 N = 92 N = 98 N= 100 N = 48 N = 78
a 0.446 1.000 0.620 0.708 0.756
b 0.554 2.063 0.360 0.292 0.244
c 0.000 0.000 0.020 0.000 0.000
PGI-2 N= 100 N= 100 N = 100 N = 50 N = 98
a 0.360 0.780 0.420 0.720 0.531
b 0.190 0.200 0.270 0.280 0.306
c 0.450 0.020 0.310 0.000 0.163
PGD N= 100 II o N= 100 N = 50 N = 96
a 1.000 1.000 0.920 0.940 1.000
b 0.000 0.000 0.070 0.040 0.000
c 0.000 0.000 0.010 0.020 0.000
PGM-2 N= 100 N= 100 N= 100 N = 48 N = 94
a 0.090 0.000 0.000 0.000 0.128
b 0.910 1.000 1.000 1.000 0.872
MDH-2 N= 100 N= 100 N = 100 N =48 N = 96
a 1.000 1.000 1.000 1.000 0.646
b 0.000 0.000 0.000 0.000 0.354
IDH-2 N= 100 N= 100 N= 100 N = 48 N = 98
a 0.260 0.170 0.320 0.188 0.010
b 0.610 0.830 0.680 0.813 0.990
c 0.130 0.000 0.000 0.000 0.000
52


APPENDIX C
Genotypes of the Five Carex scirpoidea Michx. ssp. convoluta (Kukenth.) Dunlop Populations
DIA-2 DIA-3
/ /
ADH TPI- 1 TPI- 2 SOD DIA- 1 MNR- 1 MNR- 2 ME AAT- 1 AAT- 2 G3PDH SDH- 1 SDH- 2 PGI- 2 PGD PGM- 2 MDH- 1 MDH- 2 IDH- 2
Presque Isle: indivj Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk NB Nk B/B Nk Nk B/B
indiv_2 ?/? ?/? ?/? ?/? ?/? ?/? Nk ?/? ?/? NB Nk Nk ?/? kIB ?/? ?/? 'll? ?/? ?/?
indiv_3 klk Nk Nk Nk Nk Nk NB Nk Nk NB Nk Nk Nk NB Nk B/B Nk NB B/B
indiv_4 klk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk A/B Nk BIB Nk Nk BIB
indiv_5 klk Nk Nk Nk Nk Nk NB Nk Nk NB Nk Nk Nk NB Nk BIB Nk NB BIB
indiv_6 klk Nk Nk Nk Nk Nk Nk Nk Nk kIB Nk Nk Nk NB Nk BIB Nk NB BIB
indiv_7 klk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk NB Nk BIB Nk NB BIB
indiv_8 klk Nk Nk Nk Nk Nk NB Nk Nk NB Nk Nk Nk NB Nk BIB Nk BIB BIB
indiv_9 klk Nk Nk Nk Nk Nk Nk Nk Nk NB Nk Nk Nk NB Nk BIB Nk NB BIB
indivj 0 klk Nk Nk Nk Nk Nk Nk Nk Nk BIB Nk Nk Nk A/B Nk BIB Nk NB BIB
indivj 1 klk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk A/B Nk BIB Nk Nk BIB
indivj 2 Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk A/B Nk ?/? Nk Nk BIB
indivj 3 Nk Nk Nk Nk Nk Nk Nk Nk Nk NB Nk Nk Nk A/B Nk B/B Nk NB BIB
indivj 4 Nk Nk Nk Nk Nk Nk Nk Nk Nk NB Nk Nk Nk NB Nk BIB Nk NB BIB
indivj 5 Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk NB Nk NB Nk NB BIB
indivj 6 Nk Nk Nk Nk Nk Nk NB Nk Nk NB Nk Nk Nk NB Nk BIB Nk NB BIB
indivj 7 Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk NB Nk BIB Nk Nk BIB
indivj 8 Nk Nk Nk Nk Nk Nk NB Nk Nk NB Nk Nk Nk NB Nk B/B Nk Nk BIB
indivj 9 Nk Nk Nk Nk Nk Nk Nk Nk Nk NB Nk Nk Nk NB Nk BIB Nk NB BIB
indiv_20 Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk NB Nk NB Nk Nk BIB
indiv_21 Nk Nk Nk Nk Nk Nk BIB Nk Nk Nk Nk Nk Nk NB Nk BIB Nk NB BIB
indiv_22 Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk NB Nk BIB Nk NB BIB
indiv_23 Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk NB Nk BIB Nk NB BIB
indiv_24 Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk Nk NB Nk NB Nk NB BIB
indiv_25 Nk Nk Nk Nk Nk Nk NB Nk Nk BIB Nk Nk Nk NB Nk BIB Nk NB BIB
indiv_26 Nk Nk Nk Nk Nk NB Nk Nk Nk ?/? Nk Nk Nk C/C Nk NB Nk Nk BIB
indiv_27 Nk Nk Nk Nk Nk NB Nk Nk Nk ?/? Nk Nk Nk A/C Nk BIB Nk Nk BIB
indiv_28 Nk Nk Nk Nk Nk Nk Nk Nk Nk ?/? Nk Nk Nk Nk Nk BIB Nk BIB BIB
indiv_29 Nk Nk Nk Nk Nk NB Nk Nk ?/? ?/? Nk Nk Nk A/C Nk B/B Nk Nk BIB
indiv_30 Nk ?/? ?/? ?/? ?/? ?/? ?/? ?/? ?/? ?/? ?/? Nk Nk ?/? ?/? ?/? ?/? ?/? BIB
indivj 1 Nk Nk Nk Nk Nk Nk Nk Nk Nk NB Nk Nk Nk Nk Nk A/B Nk Nk BIB


indiv_32 A/A A/A A/A A/A A/A A/B A/A
indiv_33 A/A A/A A/A A/A A/A A/B A/A
indiv_34 A/A A/A A/A A/A A/A A/B A/B
indiv_35 A/A A/A A/A A/A A/A A/A A/B
indiv_36 A/A A/A A/A A/A A/A A/B A/A
indiv_37 A/A A/A A/A A/A A/A A/A A/B
indiv_38 A/A A/A A/A A/A A/A A/B A/A
indiv_39 A/A A/A A/A A/A A/A A/B A/A
indiv_40 A/A A/A A/A A/A A/A A/A A/B
indiv_41 A/A A/A A/A A/A A/A A/B A/A
indiv_42 A/A A/A A/A A/A A/A A/B A/A
indiv_43 A/A A/A A/A A/A A/A A/A A/B
indiv_44 A/A A/A A/A A/A A/A A/B A/A
indiv_45 A/A A/A A/A A/A A/A A/B A/A
indiv_46 A/A A/A A/A A/A A/A A/B A/A
indiv_47 A/A A/A A/A A/A A/A A/A A/B
indiv_48 A/A A/A A/A A/A A/A A/A A/B
indiv_49 A/A A/A A/A A/A A/A A/A B/B
indiv_50 A/A A/A A/A A/A A/A A/B A/A
Maxton Plains:
indivj A/A A/A A/A A/A A/A A/A C/C
indiv_2 A/A A/A A/A A/A A/A A/A C/C
indiv_3 A/A A/A A/A A/A A/A A/A C/C
indiv_4 A/A A/A A/A A/A A/A A/A C/C
indiv_5 A/A A/A A/A A/A A/A A/A C/C
indiv_6 A/A A/A A/A A/A A/A A/A C/C
indiv_7 A/A A/A A/A A/A A/A A/A C/C
indiv_8 A/A A/A A/A A/A A/A A/A C/C
indiv_9 A/A A/A A/A A/A A/A A/A C/C
indivj 0 A/A A/A A/A A/A A/A A/A C/C
indivj 1 A/A A/A A/A A/A A/A A/A B/C
indivj 2 A/A A/A A/A A/A A/A A/A B/B
indivj 3 A/A A/A A/A A/A A/A A/A B/B
indivJ4 A/A A/A A/A A/A A/A A/A C/C
indivj 5 A/A A/A A/A A/A A/A A/A C/C
indivj 6 A/A A/A A/A A/A A/A A/A C/C
indivj 7 A/A A/A A/A A/A A/A A/A C/C
indivj 8 A/A A/A A/A A/A A/A A/A C/C
indivj 9 A/A A/A A/A A/A A/A A/A C/C
indiv_20 A/A A/A A/A A/A A/A A/A C/C
indiv_21 A/A A/A A/A A/A A/A A/A C/C
indiv_22 A/A A/A A/A A/A A/A A/A C/C
indiv_23 A/A A/A A/A A/A A/A A/A C/C
indiv_24 A/A A/A A/A A/A A/A A/A C/C
indiv_25 A/A A/A A/A A/A A/A A/A C/C
A/A A/A A/A A/A A/A A/A A/A A/A B/B A/A A/B B/B
A/A A/A A/A A/A A/A A/A A/B A/A B/B A/A A/A B/B
A/A A/A A/A A/A A/A A/A C/C A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/C A/A A/A A/A B/B B/B
A/A A/A A/A A/A A/A A/A A/C A/A B/B A/A B/B B/B
A/A A/A A/A A/A A/A A/A A/A A/A B/B A/A A/A B/B
A/A A/A ?/? A/A A/A A/A A/C A/A A/A A/A A/A B/B
A/A A/A ?/? A/A A/A A/A A/C A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/B A/A B/B A/A A/B B/B
A/A A/A A/A A/A A/A A/A A/C A/A A/B A/A A/B B/B
A/A A/A A/A A/A A/A A/A A/B A/A A/B A/A A/B B/B
A/A A/A A/B A/A A/A A/A A/A A/A B/B A/A A/A B/B
A/A A/A A/A A/A A/A A/A A/A A/A B/B A/A A/A B/B
A/A A/A ?/? A/A A/A A/A C/C A/A B/B A/A A/B B/B
A/A A/A ?/? A/A A/A A/A A/C A/A B/B A/A A/B B/B
A/A A/A ?/? A/A A/A A/A A/C A/A A/B A/A A/B B/B
A/A A/A A/A A/A A/A A/A A/C A/A B/B A/A A/B A/B
A/A A/A ?/? A/A A/A A/A A/B A/A B/B A/A A/B B/B
A/A A/A A/A A/A A/A A/A A/B A/A B/B A/A A/B B/B,
A/A A/A A/B A/A A/A A/A C/C A/A B/B A/A A/A A/B
A/A A/A A/B A/A A/A A/A A/C A/A B/B A/A A/A A/B
A/A A/A A/A A/A A/A A/A A/A A/A B/B A/A A/A A/B
A/A A/A A/A A/A A/A A/A A/A A/B B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/C A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/A A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/A C/C A/A B/B A/A A/A A/A
A/A A/A A/A A/A A/A A/A C/C A/A B/B A/A A/A B/B
A/A A/A A/A A/A A/A A/A A/C A/A B/B A/A A/A A/B
A/A A/A A/B A/A A/A A/A A/C A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A C/C A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/B A/A B/B A/A A/A A/B
A/A A/A A/A A/A A/A A/A B/B A/A B/B A/A A/A A/B
A/A A/A B/B A/A A/A A/A A/A A/A B/B A/A A/A A/B
A/A A/A A/B A/A A/A A/A A/C A/A B/B A/A A/A B/B
A/A A/A A/A A/A A/A A/A A/C A/B B/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A A/B B/B B/B A/A A/A A/B
A/A A/A A/B A/A A/A A/A A/B A/A B/B A/A A/A A/B
A/A A/A A/A A/A A/A A/A B/B A/B B/B A/A A/A B/B
A/A A/A A/A A/A A/A A/A C/C A/A B/B A/A A/A A/B
A/A A/A A/B A/A A/A A/A A/B A/A B/B A/A A/A B/B
A/A A/A A/A A/A A/A A/A A/C A/A B/B A/A A/A A/B
A/A A/A B/B A/A A/A A/A C/C A/B B/B A/A A/A A/A
A/A A/A A/A A/A A/A A/A A/C A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/C A/A B/B A/A A/A B/B


indiv_26 A/A A/A A/A A/A A/A A/A C/C
indiv_27 A/A A/A A/A A/A A/A A/A B/C
indiv_28 A/A A/A A/A A/A A/A A/A B/C
indiv_29 A/A A/A A/A A/A A/A A/A B/B
indiv_30 A/A A/A A/A A/A A/A A/A BIB
indiv_31 A/A A/A A/A A/A A/A A/A C/C
indiv_32 A/A A/A A/A A/A A/A A/A B/C
indiv_33 A/A A/A A/A A/A A/A A/A C/C
indiv_34 A/A A/A A/A A/A A/A A/A C/C
indiv_35 A/A A/A A/A A/A A/A A/A B/B
indiv_36 A/A A/A A/A A/A A/A A/A C/C
indiv_37 A/A A/A A/A A/A A/A A/A C/C
indiv_38 A/A A/A A/A A/A A/A A/A C/C
indiv_39 A/A A/A A/A A/A A/A A/A B/C
indiv_40 A/A A/A A/A A/A A/A A/A B/C
indiv_41 A/A A/A A/A A/A A/A A/A B/C
indiv_42 A/A A/A A/A A/A A/A A/A C/C
indiv_43 A/A A/A A/A A/A A/A A/A B/B
indiv_44 A/A A/A A/A A/A A/A A/A B/C
indiv_45 A/A A/A A/A A/A A/A A/A C/C
indiv_46 A/A A/A A/A A/A A/A A/A C/C
indiv_47 A/A A/A A/A A/A A/A A/A B/B
indiv_48 A/A A/A A/A A/A A/A A/A C/C
indiv_49 A/A A/A A/A A/A A/A A/A C/C
indiv_50 A/A A/A A/A A/A A/A A/A B/B
Big Shoal Cove:
indivj A/A A/A A/A A/A A/A A/A B/B
indiv_2 A/A A/A A/A A/A A/A A/A C/C
indiv_3 A/A A/A A/A A/A A/A A/A B/B
indiv_4 A/A A/A A/A A/A A/A A/A B/C
indiv_5 A/A A/A A/A A/A A/A A/A B/B
indiv_6 A/A A/A A/A A/A A/A A/A B/C
indiv_7 A/A A/A A/A A/A A/A A/A B/C
indiv_8 A/A A/A A/A A/A A/A A/A B/C
indiv_9 A/A A/A A/A A/A A/A A/A B/C
indiv_10 A/A A/A A/A A/A A/A A/A B/B
indivj1 A/A A/A A/A A/A A/A A/A B/B
indivj 2 A/A A/A A/A A/A A/A A/A B/B
indivj 3 A/A A/A A/A A/A A/A A/A B/B
indiv J4 A/A A/A A/A A/A A/A A/A B/B
indivj 5 A/A A/A A/A A/A A/A A/A B/B
indiv_16 A/A A/A A/A A/A A/A A/A B/C
indivj 7 A/A A/A A/A A/A A/A A/A B/B
indivj 8 A/A A/A A/A A/A A/A A/A B/C
indivj 9 A/A A/A A/A A/A A/A A/A B/B
A/A A/A A/A A/A A/A A/A A/B A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/A A/A A/B B/B A/A A/A A/B
A/A A/A A/B A/A A/A A/A B/B A/A B/B A/A A/A B/B
A/A A/A A/A A/A A/A A/A B/B A/A B/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A B/B A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/B A/A B/B A/A A/A B/B
A/A A/A A/A A/A A/A A/A B/B A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/A B/B A/C B/B A/A A/A A/B
A/A A/A A/B A/A A/A A/A A/A A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/B A/A B/B A/A A/A A/B
A/A A/A A/A A/A A/A A/A A/A A/A B/B A/A A/A A/B
A/A A/A A/A A/A A/A A/A A/B A/A B/B A/A A/A A/B
A/A A/A B/B A/A A/A A/A A/B A/A B/B A/A A/A B/B
A/A A/A A/A A/A A/A A/A B/B A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/A A/A A/A B/B A/A A/A A/A
A/A A/A A/C A/A A/A A/A A/C A/A B/B A/A A/A A/B
A/A A/A A/B A/A A/A A/A A/A A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/A A/A B/B A/A A/A B/B
A/A A/A A/A A/A A/A A/A A/C A/A B/B A/A A/A B/B
A/A A/A A/C A/A A/A A/A A/C A/A B/B A/A A/A A/B
A/A A/A B/B A/A A/A A/A C/C A/A B/B A/A A/A A/B
A/A A/A A/A A/A A/A A/A A/C A/A B/B A/A A/A A/B
A/A A/A A/A A/A A/A A/A C/C A/A B/B A/A A/A A/B
A/A A/A A/A A/A A/A A/A B/C A/A B/B A/A A/A A/B
A/A A/A A/B A/A A/A A/A B/C A/A B/B A/A A/A B/B,
A/A A/A ?/? A/A A/A A/A A/C A/A B/B A/A A/A C/C
A/A A/A ?/? A/A A/A A/A A/C A/A B/B A/A A/A C/C
A/A A/A ?/? A/A A/A A/A A/C A/A B/B A/A A/A B/B
A/A A/A ?/? A/A A/A A/A A/C A/A A/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A C/C A/A B/B A/A A/A B/B
A/A A/A A/A A/A A/A A/A A/C A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/C A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A C/C A/A B/B A/A A/A A/A
A/A A/A A/A A/A A/A A/A C/C A/A A/B A/A A/A B/B
A/A A/A B/B A/A A/A A/A B/B A/A B/B A/A A/A B/B
A/A A/A A/A A/A A/A A/A C/C A/A A/B A/A A/A A/B
A/A A/A B/B A/A A/A A/A C/C A/A B/B A/A A/A A/A
A/A A/A B/B A/A A/A A/A B/B A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/A A/A A/A B/B A/A A/A B/B
A/A A/A A/A A/A A/A A/A A/B A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/A A/B A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/A A/B A/A A/B A/A A/A C/C
A/A A/A B/B A/A A/A A/A C/C A/A B/B A/A A/A B/C
A/A A/A A/A A/A A/A A/A A/B A/A B/B A/A A/A B/B


indiv_20 A/A A/A A/A A/A A/A A/A B/B
indiv_21 A/A A/A A/A A/A A/A A/A BIB
indiv_22 A/A A/A A/A A/A A/A A/A BIB
indiv_23 A/A A/A A/A A/A A/A A/A BIB
indiv_24 A/A A/A A/A A/A A/A A/A BIB
indiv_25 A/A A/A A/A A/A A/A A/A BIB
indiv_26 A/A A/A A/A A/A A/A A/A C/C
indiv_27 A/A A/A A/A A/A A/A A/A B/C
indiv_28 A/A A/A A/A A/A A/A A/A B/B
indiv_29 A/A A/A A/A A/A A/A A/A B/B
indiv_30 A/A A/A A/A A/A A/A A/A BIB
indiv_31 A/A A/A A/A A/A A/A A/A B/C
indiv_32 A/A A/A A/A A/A A/A A/A B/C
indiv_33 A/A A/A A/A A/A A/A A/A B/C
indiv_34 A/A A/A A/A A/A A/A A/A B/B
indiv_35 A/A A/A A/A A/A A/A A/A B/B
indiv_36 A/A A/A A/A A/A A/A A/A B/B
indiv_37 A/A A/A A/A A/A A/A A/A B/B
indiv_38 A/A A/A A/A A/A A/A A/A B/B
indiv_39 A/A A/A A/A A/A A/A A/A B/C
indiv_40 A/A A/A A/A A/A A/A A/A B/B
indiv_41 A/A A/A A/A A/A A/A A/A B/B
indiv_42 A/A A/A A/A A/A A/A A/A B/C
indiv_43 A/A A/A A/A A/A A/A A/A B/B
indiv_44 A/A A/A A/A A/A A/A A/A B/C
indiv_45 A/A A/A A/A A/A A/A A/A C/C
indiv_46 A/A A/A A/A A/A A/A A/A B/C
indiv_47 A/A A/A A/A A/A A/A A/A B/B
indiv_48 A/A A/A A/A A/A A/A A/A B/C
indiv_49 A/A A/A A/A A/A A/A A/A B/B
indiv_50 A/A A/A A/A A/A A/A A/A B/C
Bumt_lsland:
indiv_1 A/A A/A A/A A/A A/A A/A B/B
indiv_2 A/A A/A A/A A/A A/A A/A B/B
indiv_3 A/A A/A A/A A/A A/A A/A A/B
indiv_4 A/A A/A A/A A/A A/A A/A B/B
indiv_5 A/A A/A A/A A/A A/A A/A B/B
indiv_6 A/A A/A A/A A/A A/A A/A B/B
indiv_7 A/A A/A A/A A/A A/A A/A B/B
indiv_8 A/A A/A A/A A/A A/A A/A B/B
indiv_9 A/A A/A A/A A/A A/A A/A A/B
indiv_10 A/A A/A A/A A/A A/A A/A B/B
indiv_11 A/A A/A A/A A/A A/A A/A A/B
indiv_12 A/A A/A A/A A/A A/A A/A A/B
indiv_13 A/A A/A A/A A/A A/A A/A B/B
A/A A/A A/A A/A A/A A/A A/A A/A A/B A/A A/A B/C
A/A A/A B/B A/A A/A A/A A/A A/A B/B A/A A/A B/C
A/A A/A A/A A/A A/A A/A A/C A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/A A/C A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/A A/C A/A B/B A/A A/A C/C
A/A A/A A/A A/A A/A A/A A/C A/A A/B A/A A/A C/C
A/A A/A A/B A/A A/A A/A B/C A/A B/B A/A A/A B/B
A/A A/A A/A A/A A/A A/A B/B A/A B/B A/A A/A A/B
A/A A/A A/B A/A A/A A/A B/C A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/A B/C A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/C A/A B/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A C/C A/A A/B A/A A/A A/A
A/A A/A B/B A/A A/A A/A A/A A/A B/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A C/C A/A B/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A A/A A/A A/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/A A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/C A/A B/B A/A A/A A/B
A/A A/A B/B A/A A/A A/A A/C A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A B/C A/A A/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A B/C A/A B/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A B/C A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/A B/B A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A B/C A/A B/B A/A A/A B/B
A/A A/A A/A A/A A/A A/A C/C A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/C A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/A A/C A/A B/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A C/C A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/A A/A B/B A/A A/A A/A
A/A A/A B/B A/A A/A A/A A/C A/A B/B A/A A/A A/A
A/A A/A A/A A/A A/A A/A A/C A/A B/B A/A A/A A/B
A/A A/A A/B A/A A/A A/A A/C A/A B/B A/A A/A B/B,
A/A A/A A/B A/A A/A A/A A/A A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/A A/B A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/A A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/A A/A B/B A/A A/A A/B
A/A A/A A/A A/A A/A A/A A/A A/A B/B A/A A/A A/B
A/A A/A B/B A/A A/A A/A A/B A/A B/B A/A A/A A/B
A/A A/A A/B A/A A/A A/A A/A A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/A A/A A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/A A/B A/A B/B A/A A/A A/B
A/A A/A B/B A/A A/A A/A A/A A/A B/B A/A A/A A/B
A/A A/A A/B A/A A/A A/A A/A A/A B/B A/A A/A A/B
A/A A/A B/B A/A A/A A/A A/C A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/A A/A A/A B/B A/A A/A B/B


indiv_14 A/A A/A A/A A/A A/A A/A B/B
indiv_15 A/A A/A A/A A/A A/A A/A A/A
indiv_16 A/A A/A A/A A/A A/A A/A B/B
indiv _17 A/A A/A A/A A/A A/A A/A B/B
indiv_18 A/A A/A A/A A/A A/A A/A A/B
indiv_19 A/A A/A A/A A/A A/A A/A B/B
indiv_20 A/A A/A A/A A/A A/A A/A B/B
indiv_21 A/A A/A A/A A/A A/A A/A B/B
indiv_22 A/A A/A A/A A/A A/A A/A A/A
indiv_23 A/A A/A A/A A/A A/A A/A A/B
indiv_24 A/A A/A A/A A/A A/A A/A B/B
indiv_25 A/A A/A A/A A/A A/A A/A B/B
indiv_26 A/A A/A A/A A/A A/A A/A A/B
indiv_27 A/A A/A A/A A/A A/A A/A B/B
indiv_28 A/A A/A A/A A/A A/A A/A A/A
indiv_29 A/A A/A A/A A/A A/A A/A B/B
indiv_30 A/A A/A A/A A/A A/A A/A BIB
indiv_31 A/A A/A A/A A/A A/A A/A A/B
indiv_32 A/A A/A A/A A/A A/A A/A B/B
indiv_33 A/A A/A A/A A/A A/A A/A B/B
indiv_34 A/A A/A A/A A/A A/A A/A A/A
indiv_35 A/A A/A A/A A/A A/A A/A B/B
indiv_36 A/A A/A A/A A/A A/A A/A B/B
indiv_37 A/A A/A A/A A/A A/A A/A B/B
indiv_38 A/A A/A A/A A/A A/A A/A B/B
indiv_39 A/A A/A A/A A/A A/A A/A B/B
indiv_40 A/A A/A A/A A/A A/A A/A A/B
indiv_41 A/A A/A A/A A/A A/A A/A B/B
indiv_42 A/A A/A A/A A/A A/A A/A B/B
indiv_43 A/A A/A A/A A/A A/A A/A B/B
indiv_44 A/A A/A A/A A/A A/A A/A B/B
indiv_45 A/A A/A A/A A/A A/A A/A B/B
indiv_46 A/A A/A A/A A/A A/A A/A B/B
indiv_47 A/A A/A A/A A/A A/A A/A B/B
indiv_48 A/A A/A A/A A/A A/A A/A B/B
indiv_49 A/A A/A A/A A/A A/A A/A A/B
indiv_50 A/A A/A A/A A/A A/A A/A A/B
Murphy_Point:
indiv_1 A/A A/A A/A A/A A/A A/A B/B
indiv_2 A/A A/A A/A A/A A/A A/A B/B
indiv_3 A/A A/A A/A A/A A/A A/A B/B
indiv_4 A/A A/A A/A A/A A/A A/A B/B
indiv_5 A/A A/A A/A A/A A/A A/A B/B
indiv_6 A/A A/A A/A A/A A/A A/A B/B
indiv_7 A/A A/A A/A A/A A/A A/A B/B
A/A A/A A/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A
A/A A/A B/B A/A A/A A/A
A/A A/A B/B A/A A/A A/A
A/A A/A B/B A/A A/A A/A
A/A A/A A/A A/A A/A A/A
A/A A/A A/B A/A A/A A/A
A/A A/A B/B A/A A/A A/A
A/A A/A A/A A/A A/A A/A
A/A A/A A/B A/A A/A A/A
A/A A/A B/B A/A A/A A/A
A/A A/A B/B A/A A/A A/A
A/A A/A B/B A/A A/A A/A
A/A A/A A/A A/A A/A A/A
A/A A/A A/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A
A/A A/A B/B A/A A/A A/A
A/A A/A B/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A
A/A A/A B/B A/A A/A A/A
A/A A/A B/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A
A/A A/A B/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A
A/A A/A B/B A/A A/A A/A
A/A A/A B/B A/A A/A A/A
A/A A/A B/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A
A/A A/A A/B A/A A/A A/A
A/A ?/? ?/? A/A A/A A/A
A/A A/A A/A A/A A/A A/A
A/A A/A A/B A/A A/A A/A
A/A A/A A/A A/A A/A A/A
A/A A/A B/B A/A A/A A/A
A/A A/A B/B A/A A/A A/A
A/A A/A A/A A/A A/A A/A
A/A A/A A/B A/A A/A A/A
A/A A/A A/A A/A A/A A/A
A/B A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A B/B
A/B A/A B/B A/A A/A A/B
A/B A/A B/B A/A A/A A/B
A/A A/A B/B A/A A/A B/B
A/B A/A B/B A/A A/A A/B
A/A A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/B
A/A A/A B/B A/A A/A B/B
A/C A/A B/B A/A A/A A/B
A/B A/A B/B A/A A/A B/B
A/B A/A B/B A/A A/A B/B
B/B A/A B/B A/A A/A A/B
A/A A/A B/B A/A A/A B/B
A/B A/A B/B A/A A/A B/B
A/B A/A B/B A/A A/A B/B
A/B A/A B/B A/A A/A A/B
A/A A/A B/B A/A A/A A/A
A/A A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A B/B
A/B A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A B/B
A/B A/A B/B A/A A/A B/B
A/B A/A B/B A/A A/A B/B
A/B A/A B/B A/A A/A B/B
A/B A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A B/B
A/B A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/B
A/A A/A B/B A/A A/A A/B
A/A A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A B/B,
A/A A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/B
A/B A/A B/B A/A A/A B/B
A/B A/B B/B A/A A/A B/B
A/A A/A B/B A/A A/A B/B
A/A A/A B/B A/A A/A A/B
A/B A/A B/B A/A A/A B/B


indiv_8 A/A A/A A/A A/A A/A A/A B/B
indiv_9 A/A A/A A/A A/A A/A A/A B/B
indiv_10 A/A A/A A/A A/A A/A A/A B/B
indivj 1 A/A A/A A/A A/A A/A A/A B/B
indiv_12 A/A A/A A/A A/A A/A A/A B/B
indiv_13 A/A A/A A/A A/A A/A A/A B/B
indiv_14 A/A A/A A/A A/A A/A A/A B/B
indivj 5 A/A A/A A/A A/A ?/? ?/? ?/?
indivj 6 A/A A/A A/A A/A A/A A/A B/B
indivj 7 A/A A/A A/A A/A A/A A/A B/B
indivj 8 A/A A/A A/A A/A A/A A/A B/B
indivj 9 A/A A/A A/A A/A A/A A/A B/B
indiv_20 A/A A/A A/A A/A A/A A/A B/B
indiv_21 A/A A/A A/A A/A A/A A/A B/B
indiv_22 A/A A/A A/A A/A A/A A/A B/B
indiv_23 A/A A/A A/A A/A A/A A/A B/B
indiv_24 A/A A/A A/A A/A A/A A/A A/B
indiv_25 A/A A/A A/A A/A A/A A/A B/B
00
A/A A/A A/B A/A A/A A/A A/B A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/B A/A B/B A/A A/A B/B
A/A A/A A/A A/A A/A A/A A/B A/A B/B A/A A/A A/B
A/A A/A A/A A/A A/A A/A A/A A/A B/B A/A A/A B/B
A/A A/A A/A A/A A/A A/A A/A A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/B A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/A A/B B/B A/A A/A B/B
A/A ?/? ?/? A/A ?/? ?/? B/B A/A ?/? ?/? ?/? ?/?
A/A A/A A/A A/A A/A A/A A/B A/A B/B A/A A/A A/B
A/A A/A A/A A/A A/A A/A A/A A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A A/A A/A B/B A/A A/A B/B
A/A A/A A/A A/A A/A A/A A/A A/A B/B A/A A/A A/B
A/A A/A A/A A/A A/A A/A A/B A/A B/B A/A A/A A/B
A/A A/A A/B A/A A/A A/A A/A A/A B/B A/A A/A A/B
A/A A/A A/A A/A A/A A/A A/A A/A B/B A/A A/A B/B
A/A A/A A/B A/A A/A A/A B/B A/A B/B A/A A/A A/B
A/A A/A A/B A/A A/A A/A A/A A/C B/B A/A A/A A/B
A/A A/A A/A A/A A/A A/A A/B A/A B/B A/A A/A B/B,


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