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Systematics of Carex section Scirpinae tuck. (Cyperaceae), with insight into the origins of edaphic endemics

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Title:
Systematics of Carex section Scirpinae tuck. (Cyperaceae), with insight into the origins of edaphic endemics
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Pembrook, James W. ( author )
Place of Publication:
Denver, CO
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University of Colorado Denver
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English
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1 electronic file (117 pages). : ;

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Carex -- Speciation ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Carex section Scirpinae sensu lato (Cyperaceae) provides a model for the study of edaphic speciation. This widespread group of predominantly North American sedges comprises six taxa of which three are narrowly distributed edaphic endemics. To better understand the evolutionary relationships among the members of this section, we sequenced highly variable nuclear (nrDNA: ETS, ITS) and chloroplast (cpDNA: atpF, rpS16) DNA regions and constructed phylogenetic hypotheses using maximum parsimony analysis for at least two individuals representing each taxon. Carex section Scirpinae s.l. forms a monophyletic group with strong bootstrap support (100%, combined nr- and cpDNA phylogeny). Carex section Scirpinae sensu stricto is also monophyletic (100%) in the combined phylogeny, as is C. scabriuscula (100%), which Dunlop (1990) excluded from the section. However, phylogenetic relationships within Carex section Scirpinae s.s. are unresolved, which may be explained by low phylogenetic information, a recent evolutionary history with incomplete lineage sorting or hybridization obscuring phylogenetic resolution. These preliminary data coupled with other taxonomic evidence support subspecific status for each of the remaining taxa, potentially including the reclassification of C. curatorum as a subspecies of C. scirpoidea. In an attempt to shed some light on the lack of resolution, we used starch gel electrophoresis coupled with allozyme analysis to describe population genetic diversity and structure in C. scirpoidea subsp. convoluta.In comparison to the widespread C. scirpoidea subsp. scirpoidea, our data reveal relatively high levels of genetic diversity for all parameters analyzed (e.g. expected heterozygosity), with no significant differences between means for the two subspecies. As expected for an obligately outcrossing dioecious species, most loci for most populations were in Hardy-Weinberg Equilibrium. Population differentiation was significantly higher in C. scirpoidea subsp. scirpoidea (FST= 0.468) than C. scirpoidea subsp. convoluta ( FST= 0.185), suggesting that populations of the narrow endemic are much more similar genetically than are populations of the widespread congener. Taken together, the morphologically distinct ecotypes of C. scirpoidea, as exemplified by C. scirpoidea subsp. convoluta, have not yet diverged genetically, with fixation of neutral loci lagging behind morphological differentiation
Thesis:
Thesis (M.S.)--University of Colorado Denver. Biology
Bibliography:
Includes bibliographic references.
General Note:
Department of Integrative Biology
Statement of Responsibility:
by James W. Pembrook III.

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|Auraria Library
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904611964 ( OCLC )
ocn904611964

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SYSTEMATICS OF CAREX SECTION SCIRPINAE TUCK. (CYPERACEAE), WITH INSIGHT INTO THE ORIGINS OF EDAPHIC ENDEMICS by JAMES W. PEMBROOK III B.S. University of Colorado Denver, 2011 A t hesis submitted to the faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Master of Science Biology 201 4

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ii This thesis for the Master of Science d egree by James Wesley Pembrook III h as been approved for the Biology Program by Leo P. Bruederle, Chair Jennifer Ramp Neale John Swallow November 20, 2014

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iii Pembrook, James W. III (M.S., Biology) Systematics of Carex Section Scirpinae (Cyperaceae) with Insight into the Origins of Edaphic Endemics Thesis directed by Associate Professor Leo P. Bruederle ABSTRACT Carex section Scirpinae sensu lato (Cyperaceae) provides a model for the study of edaphic speciation. This widespread group o f predominantly North American sedges comprises six taxa of which three are narrowly distributed edaphic endemics. To better understand the evolutionary relationships among the members of this section, we sequenced highly variable nuclear (nrDNA: ETS, ITS) and chloroplast (cpDNA: atpF rpS16 ) DNA regions and constructed phylogenetic hypotheses using maximum parsimony analysis for at least two individuals representing each taxon. Carex section Scirpinae s.l. forms a monophyletic group with strong bootstrap s upport (100%, combined nr and cpDNA phylogeny). Carex section Scirpinae sensu stricto is also monophyletic (100%) in the combined phylogeny, as is C. scabriuscula (100%), which Dunlop (1990) excluded from the section. However, phylogenetic relationships w ithin Carex section Scirpinae s.s. are unresolved, which may be explained by low phylogenetic information, a recent evolutionary history with incomplete lineage sorting or hybridization obscuring phylogenetic resolution These preliminary data coupled with other taxonomic evidence support subspecific status for each of the remaining taxa, potentially including the reclassification of C. curatorum as a subspecies of C. scirpoidea In an attempt to shed some light on the lack of resolution, we used starch gel electrophoresis coupled with allozyme analysis to describe population genetic diversity and structure in C. scirpoidea subsp. convoluta In comparison to the widespread C. scirpoidea subsp. scirpoidea our data reveal relatively high levels of genetic div ersity for all parameters analyzed (e.g. expected heterozygosity), with no significant differences between means for the two subspecies. As expected for an obligately outcrossing dioecious species, most loci for most popu lations were in Hardy Weinberg E qui librium. Population differentiation was

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iv significantly higher in C. scirpoidea subsp. scirpoidea ( F ST = 0.468) than C. scirpoidea subsp. convoluta ( F ST = 0.185), suggesting that populations of the narrow endemic are much more similar genetically than are populations of the widespread congener Taken together, the morphologically distinct ecotypes of C. scirpoidea as exemplified by C. scirpoidea subsp. convoluta have not yet diverged genetically, with fixation of neutral loci lagging behind morphological differentiation. The form and content of this abstract are approved. I recommend its publication Appro ved: L e o P. Bruederle

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v DEDICATION To my parents, who have always encouraged my pursuit of knowledge.

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vi ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Leo P. Bruederle, for his research guidance and ability for critical, logical thinking which he most enthusiastically expresses while teaching. I would like to thank Dr. Aimee Bernard for her mentorship throughout my a cademic career, her passion for education, and for being the best, most fun boss I have ever had. I would like to thank my fellow students and friends for their continual moral and intellectual support. I would also like to thank Tayvia Bourret and Dr. Richard Yeatts for all of their help in the laboratory. Finally, I would like to thank the Un iversity of Colorado Denver Department of Integrative Biology for the remarkable community and facilities within which to conduct research.

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vii TABLE OF CONTENTS Ch apter 1. .. 1 2. Method 7 2.1. Phylogenetics 7 2.1.1 Sampling 2.1.2 Laboratory Methods 7 2.2. Population Genet ics 8 2.2.1 2.2.2 Samp 2.2. 3 Laboratory Methods 3. Results 2 3.1 Phylogenetics 12 3.2 Population Genetics 1 4 4. Discussion 4.1 Phylogenetics ...17 4.2 Population Genetics 2 1 7 References Appendix A. Summary of morphological and environmental characters of Carex section Scirpinae 55 B. Phylogenetic collection info 57 C. PCR Templates 6 2 D. Electrophoresis systems and enzymes 6 5 E. GenePop input file 6 6 F. Allele frequencies . 99

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viii LIST OF TABLES Table 1 Outgroup sequences utilized in the phylogenetic analysis of Carex section Scirpinae s.l. Tuck. (Cyperaceae). Sequences were obtained from one of two sources: the Applied Systematics Laboratory at the University of Colorado Denver (unpublished data) and Gen Bank. GenBank accession numbers and dates accessed are listed 2. General sequence information for phylogenetic analysis of Carex section Scirpinae s.l. Tuck (Cyperaceae). describing seven populations of the narrowly distributed edaphic endemic Carex scirpoidea subsp. convoluta (Kkenthal) Dunlop (Cyperaceae) and ten populations of the widespread C. scirpoidea subsp. scirpoidea Michaux Abbreviations for p opulations of C. sc irpoidea subsp. convoluta are as follows : BSC = Big Shoal Cove, MI; BI = Burnt Island, ON; MaxP = Maxton Plains, MI; MurP = Murphy Point, ON; PI = Presque Isle, MI; HSB = Horseshoe Bay, MI; BLR = Brevort Lake Road, MI. Carex scirpoidea subsp. scirpoidea po pulations are: AV = Arctic Valley, AK; CC = Campbell Creek, AK; FR = Fort Richardson, AK; HCFS = High Creek Fen South, CO; HCFN = High Creek Fen North, CO; BCF = Beaver Creek Fen, CO; GPC = Geneva Park Creek, CO; HSM = Horseshoe Mountain, CO; ESC = Escanab a River, MI; BB = Beaver Bay, MN. 4. Descriptive population genetic statistics in the edaphic endemic Carex scirpoidea subsp convoluta (Kkenthal) Dunlop (Cyperaceae) and its widespread congener C. scirpoidea subsp. scirpoidea Michaux, where N = number of individuals sampled, P = percent polymorphic loci, A P = mean number of alleles per polymorphic locus, H O = observed heterozygosity, H E = expected heterozygosity, and F IS Mean descriptive statistics for each re gion of Carex scirpoidea subsp. scirpoidea sampled as well as means for both subspecies are summarized below. P values comparing the means of the two subspecies are tests (when data are normally distributed) or Wilcoxon rank sum tests (when data are not normally distributed) 5 Heterozygote deficiencies by locus and population for seven populations of the rare edaphic endemic C. scirpoidea subsp. convoluta (Kkenthal) Dunlop (Cyperaceae) and eight populations of its widespread congener C. scirpoidea subsp. scirpoidea Michaux Significace (p < 0.05) is indicated by *. Population abbreviations are as follows: BSC = Big Shoal Cove, MI; BI = Burnt Island, ON; MaxP = Maxton Plains, MI; MurP = Murphy Point, ON; PI = Presque Isle, MI; HSB = Horseshoe Bay, MI; BLR = Brevort Lake Road, MI. Carex scirpoidea subsp. scirpoidea populations are: AV = Arctic Valley, AK; CC = Campbell Creek, AK; FR = Fort Richardson, AK; HCFS = High Creek Fen South, CO; HCFN = High Creek Fen North, CO; BCF = Bea ver Creek Fen, CO; GPC = Geneva Park Creek, CO; HSM = Horseshoe Mountain, CO; ESC = Escanaba River, MI; BB = Beaver Bay, MN.

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ix 6 Pairwise F ST describing seven populations of the narrowly distributed edaphic endemic C. scirpoidea subsp. convoluta (Kkenthal) Dunlop (Cyperaceae) and eight populations of its widespread congener C. scirpoidea subsp. scirpoidea Michaux. Population abbreviations are as follows: BSC = Big Shoal Cove, MI; BI = Burnt Island, ON; MaxP = Maxton Plains, MI; MurP = Murphy Poi nt, ON; PI = Presque Isle, MI; HSB = Horseshoe Bay, MI; BLR = Brevort Lake Road, MI. Carex scirpoidea subsp. scirpoidea populations are: AV = Arctic Valley, AK; CC = Campbell Creek, AK; FR = Fort Richardson, AK; HCFS = High Creek Fen South, CO; HCFN = High Creek Fen North, CO; BCF = Beaver Creek Fen, CO; GPC = Geneva Park Creek, CO; HSM = Horseshoe Mountain, CO; ESC = Escanaba River, MI; BB = Beaver Bay, MN.

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x LIST OF FIGURES Figure 1 Distribution of all individuals sampled for phylogenetic analysis of Carex section Scirpinae s.l. Tuckerman (Cyperaceae) across North America, Russia, and Greenland. Colors represent taxa and are as follows: green = Carex curatorum brown = C. scirpoidea subsp. scirpoidea red = C. scirpoidea subsp. convoluta purple = C. scirpoidea subsp. pseudoscirpoidea blue = C. scirpoidea subsp. stenochlaena and yellow = C. scabriuscula Note that the single Norwegian sample of C. scirpoidea subsp. scirpoidea is not shown. 2 Sampled populations of Carex scirpoidea subsp. scirpoidea Michaux (Cyperaceae), represented by circles on the large map, and C. scirpoidea subsp. convoluta (Kkenthal) Dunlop, represented by circles in the inset of the Great Lakes region, for soluble protein extraction and electrophoresis. A single circle is use d to represent all three Alaskan and Coloradan populations of C. scirpoidea subsp. scirpoidea due to geographic proximity of populations; similarl, for C. scirpoidea subsp. convoluta Shaded region represents potential range for C. scirpoidea subsp. scirpo idea. 3 Chloroplast DNA marker atpF phylogenetic hypothesis of Carex section Scirpinae s.l. Tuck. (Cyperaceae), where scir = Carex scirpoidea subsp. scirpoidea conv = C. scirpoidea subsp. convoluta pseu = C. scirpoidea subsp. pseudoscirpoidea sten = C scirpoidea subsp. stenochlaena cura = C. curatorum scab = C. scabriuscula Cama_25 = C. magellanica (outgroup) and C. bigelow = C. bigelowii (outgroup). Numbers at nodes represent bootstrap values, numbers within sample names are internal IDs and do no t represent taxa nor geography. Each sample ID is followed by the two letter postal code of the state (United States), province or territory (Canada), or country (Greenland, Norway, and Russia) of origin The origin of sample 97 ( C. scirpoidea subsp. scirp oidea ) is unknown (un). 4 Chloroplast DNA marker matK phylogenetic hypothesis of Carex section Scirpinae s.l. Tuck. (Cyperaceae), where scir = Carex scirpoidea subsp. scirpoidea conv = C. scirpoidea subsp. convoluta pseu = C. scirpoidea subsp. pseudoscirpoidea sten = C. scirpoidea subsp. stenochlaena cura = C. curatorum scab = C. scabriuscula Cama_25 = C. magellanica (outgroup) and C. bigelow = C. bigelowii (outgroup). Numbers at nodes represent bootstrap values, numbers within sample names are internal IDs and do not represent taxa nor geography. Each sample ID is followed by the two letter postal code of the state (United States), province or territory (Canada), or country (Greenland, Norway, and Russia) of origin. The origin of sample 97 ( C. scirpoidea subsp. scirpoidea ) is unknown (un). 5 Chloroplast DNA marker rpS16 phylogenetic hypothesis of Carex section Scirpinae s.l. Tuck. (Cyperaceae), where scir = Carex scirpoidea subsp. scirpoidea conv = C. scirpoidea subsp. convoluta pseu = C. scirpoidea subsp. pseudoscirpoidea sten = C. scirpoidea subsp. stenochlaena cura = C. curatorum scab = C. scabriuscula Cama_25 = C. magellanica (outgroup) and C. bigelow = C. bigelowii (outgroup). Numbers at nodes

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xi represent bootstrap values, numbers within sample names are internal IDs and do not represent taxa nor geography. Each sample ID is followed by the two letter postal code of the state (United States), province or territory (Ca nada), or country (Greenland, Norway, and Russia) of origin. The origin of sample 97 ( C. scirpoidea subsp. scirpoidea ) is unknown (un). 6 Concatenated chloroplast DNA phylogenetic hypothesis of Carex section Scirpinae s.l. Tuck. (Cyperaceae) using the at pF matK and rpS16 regions, where scir = Carex scirpoidea subsp. scirpoidea conv = C. scirpoidea subsp. convoluta pseu = C. scirpoidea subsp. pseudoscirpoidea sten = C. scirpoidea subsp. stenochlaena cura = C. curatorum scab = C. scabriuscula Cama_2 5 = C. magellanica (outgroup) and C. bigelow = C. bigelowii (outgroup). Numbers at nodes represent bootstrap values, numbers within sample names are internal IDs and do not represent taxa nor geography. Each sample ID is followed by the two letter postal c ode of the state (United States), province or territory (Canada), or country (Greenland, Norway, and Russia) of origin. The origin of sample 97 ( C. scirpoidea subsp. scirpoidea ) is unknown (un). 7 Nuclear DNA marker ETS phylogenetic hypothesis of Carex section Scirpinae s.l. Tuck. (Cyperaceae), where scir = Carex scirpoidea subsp. scirpoidea conv = C. scirpoidea subsp. convoluta pseu = C. scirpoidea subsp. pseudoscirpoidea sten = C. scirpoidea subsp. stenochlaena cura = C. curatorum scab = C. scabriuscula Cama_25 = C. magellanica (outgroup) and C. bigelow = C. bigelowii (outgroup). Numbers at nodes represent bootstrap values, numbers within sample names are internal IDs and do not represent taxa nor geography. Each sample ID is followed by the two letter postal code of the state (United States), province or territory (Canada), or country (Greenland, Norway, and Russia) of origin. The origin of sample 97 ( C. scirpoidea subsp. scirpoidea ) is unknown (un). 8 Nuclear DNA marker ITS phylogen etic hypothesis of Carex section Scirpinae s.l. Tuck. (Cyperaceae), where scir = Carex scirpoidea subsp. scirpoidea conv = C. scirpoidea subsp. convoluta pseu = C. scirpoidea subsp. pseudoscirpoidea sten = C. scirpoidea subsp. stenochlaena cura = C. cu ratorum scab = C. scabriuscula Cama_25 = C. magellanica (outgroup) and C. bigelow = C. bigelowii (outgroup). Numbers at nodes represent bootstrap values, numbers within sample names are internal IDs and do not represent taxa nor geography. Each sample ID is followed by the two letter postal code of the state (United States), province or territory (Canada), or country (Greenland, Norway, and Russia) of origin. The origin of sample 97 ( C. scirpoidea subsp. scirpoidea ) is unknown (un). 9 Concatenated nucle ar DNA marker phylogenetic hypothesis of Carex section Scirpinae s.l. Tuck. (Cyperaceae) using ETS and ITS sequences, where scir = Carex scirpoidea subsp. scirpoidea conv = C. scirpoidea subsp. convoluta pseu = C. scirpoidea subsp. pseudoscirpoidea sten = C. scirpoidea subsp. stenochlaena cura = C. curatorum scab = C. scabriuscula Cama_25 = C. magellanica (outgroup) and C. bigelow = C. bigelowii (outgroup). Numbers at nodes represent bootstrap values, numbers within sample names are internal IDs and d o not represent taxa nor geography. Each sample ID is followed by

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xii the two letter postal code of the state (United States), province or territory (Canada), or country (Greenland, Norway, and Russia) of origin. The origin of sample 97 ( C. scirpoidea subsp. s cirpoidea ) is unknown (un). 10 Concatenated nr and cpDNA ( atpF matK rpS16 ETS, and ITS) phylogenetic hypothesis of Carex section Scirpinae s.l. Tuck. (Cyperaceae), where scir = Carex scirpoidea subsp. scirpoidea conv = C. scirpoidea subsp. convoluta pseu = C. scirpoidea subsp. pseudoscirpoidea sten = C. scirpoidea subsp. stenochlaena cura = C. curatorum scab = C. scabriuscula Cama_25 = C. magellanica (outgroup) and C. bigelow = C. bigelowii (outgroup). Numbers at nodes represent bootstrap values, numbers within sample names are internal IDs and do not represent taxa nor geography. Each sample ID is followed by the two letter postal code of the state (United States), province or territory (Ca nada), or country (Greenland, Norway, and Russia) of origin. The origin of sample 97 ( C. scirpoidea subsp. scirpoidea ) is unknown (un).

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xiii LIST OF ABBREVIATIONS AAT = aspartate aminotransferase ADH = Alcohol dehydrogenase ALD = aldalose A P = mean number of alleles per polymorphic locus AV = Arctic Valley, AK BB = Beaver Bay, MN BCF = Beaver Creek Fen, CO BI = Burnt Island, ON BLR = Brevort Lake Road, MI BSC = Big Shoal Cove, MI C.bigelow = C. bigelowii Cama = C. magellanica CC = Campbell Creek, AK CMN = Canadian Museum of Nature conv = C. scirpoidea subsp. scirpoidea cpDNA = chloroplast DNA cura = C. curatorum D DIA = diaphorase ESC = Escanaba River, MI F IS = inbreed ing coefficient FR = Fort Richardson, AK F ST = fixation index G3PDH = gyceraldehyde 3 phosphate dehydrogenase HB = Horseshoe Bay, MI HCFN = High Creek Fen North, CO HCFS = High Creek Fen South, CO H E = expected heterozygosity H O = observed heterozygosity H WE = Hardy Weinberg equilibrium I IDH = isocitrate dehydrogenase KHD = Kathryn Kalmbach Herbarium L. = Linnaeus MaxP = Maxton Plains, MI MDH = malate dehydrogenase ME = malic enzyme MNR = menadione reductase MP = maximum parsimony MurP = Murphy Point, ON N = number of individuals per population NHA = Hodgdon Herbarium, University of New Hampshire N M = mean number of migrants per generation

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xiv nrDNA = nuclear DNA P = percent polymorphic loci PCR = polymerase chain reaction PGD = 6 phosphogluconate PGI = phosphoglucose isomerase PGM = phosphoglucomutase PI = Presque Isle, MI pseu = C. scirpoidea subsp. scirpoidea s.l. = sensu lato (in the broad sense) s.s. = sensu stricto (in the strict sense) scab = C. scabriuscula scir = C scirpoidea subsp. scirpoidea SDH = shikimic dehydrogenase SOD = superoxide dismutase sten = C. scirpoidea subsp. scirpoidea subsp. = subspecies TPI = triosephosphate isomerase Tuck. = Tuckerma n

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1 1. Introduction Speciation is the process by which one or more new species diverge from a progenitor due to the accumulation of genetic changes over evolutionary time. Regardless of which of the four traditional geographic models of speciation (e.g. allopatric, sympatric) a specie s evolve s by novel species invariably arise involving pre and/ or post zygotic isolating mechanisms due to reproductive isolation and the cessation of the exchange of genetic information. This process is usually a slow continuum with various subspecific r anks sometimes applied to taxa undergoing this process, including subspecies and varieties (Lowry 2012) In plants, t he principal evolutionary force that initiates speciation is natural selection in response to environment al conditions often climate and e daphic factors (i.e. properties of the soil such as chemical composition and texture ) ( Rajakaruna 2004 ) Edaphic endemics are restricted to chemically harsh substrates that place extreme selective pressure on plants growing in these habitats Examples of these substrates include: guano deposits, limestone including alvar, gypsum, granite, volcanic or ultramafic substrates including serpentine, and soils rich in h eavy metals such as mine tailings. Selective pressures imposed by these substrates can be expected to reduce genetic diversity in edaphic endemics. Additionally, since most edaphic endemics have a narrow geographic distribution and often occur as small, isolated populations, random genetic drift may also severely reduce genetic diversity within populations and may lead to fixed genetic differences among populations in the absence of gene flow. Hence, natural selection and random genetic drift are the principle factors that drive edaphic speciation (Kruckeberg 1951, Rajakaruna 2004, Anacke r 2014).

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2 Th e knowledge of a species phylogenetic h istory coupled with population genetic diversity and structure may be essential for unders tanding speciation as well as determining conservation status and implementing conservation plans for a taxon (May er and Soltis 1994, Baldwin 2005, Burge and Manos 2011) However, a ccurately representing evolutionary relati onships within large groups is often a challenge, and the genus Carex (Cyperaceae) is particularly challenging. Subtle characters, phenotypic plast icity, broad and often disjunct distributions, parallel evolution, frequent hybridization, holocentric chromosomes, and aneu ploidy all variously complicate elucidating systematic relationships within and among sedges ( Dunlop 1990, Roalson et al. 2001, Hipp 2007 King and Roalson 2009, Rothrock et al. 2009, Starr and Ford 2009, Starr et al. 2009, Hipp et al. 2010 Gehrke et al. 2010) On the other hand, there is a large body of literature describing population genetic diversity and structure in carices with more than 50 species described representing at least 15 sections ( reviewed in Bruederle et al. 2001). The amount of genetic diversity and its apportionment within and among population s can affect including the threats of global climate change wi th high diversity indicating evolutionary potential, resilience, and the ability to survive such stochastic events (Soltis and Gitzendanner 1999, Gitzendanner and Solits 2000, Derieg et al. 2008) Howeve r, t heory suggests that edaphic endemics should have low levels of genetic diversity due to the high selection pressures imposed by unusual substrates Consistent with this, many narrow endemics including edaphic endemics, have been found to maintain reduced genetic diversity relative to their widespread congener s ( see reviews by Hamrick and Godt 1989, Gitzendanner and Soltis 2000, and Cole 2003 ). However, other factors may confound

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3 this expectation and several other narrow endemics have been found to have similar or greater levels of genetic diversity than their widespread relatives (e.g. Karron et al. 1988, Bruederle 1999, Standley et al. 20 01, Song and Mitchell Olds 2007 ) Carex section Scirpinae Tuc kerman provides a model system for the study of s peciation of edaphic endemic s The section is a small, predominately North American group recently transferred from subgenus Primocarex Kk. to subgenus Carex L. (Roalson et al. 2001). Carex s ection Scirpinae is distinguished from other sections within the genus by the presence of a dioecious breeding system, unispicate inflorescence s and pubescent perigynia (Dunlop 1990). definitive treatments of Carex section Scirpinae sensu stricto (1990, 1997 ; Dunlop and Crow 1999 ) recognize d five t axa comprising two species; these are: Carex curatorum Stacey and Carex scirpoidea Michaux, with the latter including s ub sp. scirpoidea s ub sp. convoluta (Kkenthal) Dunlop, s ub sp. pseudoscirpoidea (Rydberg) Dunlop, and s ub sp. stenochlaena (Holm) Lve & Lve. With the exception of the type subspecies C. scirpoidea subsp. scirpoidea all of the subspecies of C. scirpoidea are considered regional ecotypes with each being associated with a different substrate ( Appendix A ). Additionally, Carex section Scir pinae sensu lato includes a sixth taxon, Carex scabriuscula Mack., into which Dunlop (1990, 1997) subsumed C. gigas (Holm) Mack. due to morphological overlap between the two taxa. C arex scabriuscula was excluded from Carex section Scirpinae based on several diagnostic characters: While the taxa comprising Carex section Scirpinae s.s. are predominately dioecious, unispicate, and possess densely pubescent perigynia with many long hairs, C. scabriuscula is as often monoecious as dioecious as well as being as o ften multispicate as unispicate whi l e possessing scarcely

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4 pubescent perigynia with few short hairs. Additionally, haploid chromosome counts of Carex section Scirpinae s.s. are typically n = 31 (less commonly n = 29 + III [trivalents], 30, 32, and 34), while those for C. scabriuscula are n = 29 (and less commonly 30). Despite these key differences, C. scabriuscula has recently been found to segregate with C. scirpoidea subsp. scirpoidea in phylogenies of subgenus C arex with strong bootstrap support (99 and 100%; Roalson et al. 2001 and Hendrichs et al. 2004, respectively). Interestingly Carex scabriuscula is also an edaphic endemic, being restricted to serpentine soils in northern California and southwestern Oregon which represents what is perhaps the most well Rajakaruna 2004, Anacker 2014). Herein we utilize molecular systematics to develop a phylogenetic hypothesis for Carex section Scirpin ae s.l. Specifically we ask the questions: i ) i s Carex section Scirpinae s.l. monophyletic (i.e. should Carex scabriuscula be included within the section)? ii ) i s Carex section Scirpinae s.s. monophyletic? i ii ) a re Carex curatorum and C. scirpoidea well s upported, monophyletic species? i v ) d o the subspecies of Carex scirpoidea segregate independently (i.e. do our phylogenies support subspecific or specific status for these taxa, utilizing the phylogenetic species concept [Baum 1992] ) ? Within the context of our phyloge ne tic hypotheses, we further describe the population genetic diversity and structure observed in the rare, edaphic endemic caespitose sedge Carex scirpoidea subsp. convoluta relative to its widespread congener C. scirpoidea subsp. scirpoidea. Carex scirpoidea subsp. convoluta was chosen for analysis because it is the most narrowly distributed edaphic endemic subspecies of C. scirpoidea restricted to alvar substrates also known as calcareous shores or limestone pavements, around Lake

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5 Huron an d the northern shores of Lake Michigan (Dunlop 1990, Reznicek et al. 2011) (Fig. 1 and 2 ) Alvar substrates, spe cifically those of the Great Lakes, are stressful for plants in several respects including high calcium concentrations but severe deficiency of almost all other nutrients, seasonal flooding and drought, and desiccating winds off of the Great Lakes ( Stephenson and Herendeen 1986, Schaefer and Larson 1997, Reschke et al. 1999). Due to its narrow endemism, C scirpoidea subsp. convoluta is the most threatened subspecies of C. scirpoidea NatureServe Explorer (2013) ranks C. scirpoidea subsp. convoluta as G5 T3, with state rankings of SNR (unranked) in Michigan and S3, or vulnerable, in Ontario (NatureServe Explorer 2013). Additionally, alvar ecosyste ms are rare and imperiled globally (G2) and in the state of Michigan (S2) (NatureServe Explorer 2013, Shackleford 2003). North American alvar communities, including pavements, grasslands, and savannahs, are particularly worthy of conservation due to their unique species composition that blends glacial relicts with species that evolved in the warmer, drier period that followed the last glacial maximum, effectively amalgamating boreal, southern, and prairie taxa. Additionally many of these species are either highly disjunct or endemic. Threats to this habitat and its biodiversity include water and soil pollution, livestock grazing, logging, introduction of non native invasive s off road vehicle use (both recreational and commercial), trampling, dumping, and sh oreline development, including quarry and residential development ( Catling and Brownell 1995, Schaefer and Larson 1997, Reschke et al. 1999, Shackleford 2003). Carex scirpoidea subsp. scirpoidea is the most widespread taxon in Carex section Scirpinae occurring on calcareous soils from Alaska to Greenland and as far south as Nevada, Utah, and Colorado in the west, Michigan and Minnesota in the Midwest and New

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6 and Chuk chi Peninsulas and three highly disjunct populations in Norway (Fig. 1) (Dunlop 1990, Dunlop and Crow 1999). Given its wide distribution the taxon is ranked as G5, or globally secure, but is S2 in several states with disjunct or marginal populations such as Colorado and New York (NatureServe Explorer 2013) Previously, DePrenger Levin (2007) found that genetic diversity was higher in the narrow edaphic endemic C. scirpoidea subsp. convoluta than its widespread congener C. scirpoidea subsp. scirpoidea Howev er, the sample of C. scirpoidea subsp. scirpoidea (Yarbrough 2000) that she used as a comparative baseline was limited to only five populations from one county (Park County, Colorado) at the southern margin of the er Levin 2007). Genetic diversity in marginal populations of the widespread taxon would therefore be expected to be significantly lower relative to the core of its distribution (Yarbrough 2000, Bruederle et al. 2001, DePrenger Levin 2007). This study furth er examines DePrenger genetic diversity in the narrow edaphic endemic C. scirpoidea subsp. convoluta relative to its widespread consepecific and presumed progenitor C. scirpoidea subsp. scirpoidea Specifically, I will be t esting the following three hypotheses: ( i ) C. scirpoidea subsp. convoluta will have low levels of genetic diversity relative to the widespread C. scirpoidea subsp. scirpoidea as a result of its narrow distribution, isolated populations, and harsh substrate ( ii ) o bligate outcrossing will limit inbreeding and result in higher than expected genetic diversity in populations of both subspecies relative to other caespitose carices; and ( iii ) h abitat fragmentation and small population size will contribute to genetic structure across the range of C. scirpoidea subsp. convoluta

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7 2. Methods 2.1. Phylogenetic s 2.1.1. Sampling: Air dried leaf tissue collected from individuals representing nearly the entire range of each taxon comprising Carex section Scirpinae s.l. was obtained for Dunlop and Crow 1999) voucher specimens deposited in the Hodgdon Herbarium (NHA); t he Canadian M useum of Nature Herbarium (CMN); and collections of the University of Colorado Denver Applied Sy stematics Laboratory deposited at the Kathryn Kalmbach Herbarium (KHD) (Appendix B ; Fig. 1). 2.1.2. Laboratory Methods: Total genomic DNA was ext racted from approximately 20.0 mg silica gel dried leaf tissue as described by Starr et al. (2009) with modifications made by Alexander et al. (2007) Extracted DNA was stored at 4C until PCR amplification. Nuclear (nrDNA; ITS, ETS) and chloroplast (cpDNA; atpF rps16 matK ) regions were amplified following a protocol modified from Starr et al. (2003). These regions were chosen because they have been shown to be useful for both broad and narrow taxonomic analyses in Carex (e.g. Roalson et al. 2001, Hendrichs et al. 2004, Star r and Ford 2009). See Appendix C for PCR mixes and thermocycler programs. Amplification success was verified on 1.5% agarose gels using 0.25 mg/ml ethidium bromide (EtBr) and visualized under ultraviolet light using a Fotodyne CFW 1312C camera and FOTO/Analyst PC Image Version 10.30 imaging program (FOTODYNE, Inc.; Hartland, WI, USA). PCR products were purified following the ExoSAP IT protocol (Affymetrix; Santa Clara, CA USA). Sequencing was performed by Macrogen Inc. (Seoul, South Korea). Forward and reverse sequences were manually edited and aligned in BioEdit version 4.7.0 (BioEdit,

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8 Manchester, UK). Maximum parsimony (MP) phylo genies were constructed for each region and combinations of regions using TnT 1.1 (Goloboff et al. 2000). Model tests were performed using jModeltest 2.1.3 (Posada, in press). Outgroup sequences were obtained from GenBank and previous studies in the Applie d Systematics Laboratory at the University of Colorado Denver (Table 1 ). 2.2. Population Genetics 2.2.1. Model System: Reiterating, Carex scirpoidea subsp. convoluta was chosen for population genetic analysis because it is the most narrowly distributed subspecies of C. scirpoidea being restricted to alvar ecosystems around Lake Huron and the no rthern shores of Lake Michigan (Dunlop 1 990, Reznicek et al. 2011) (Fig. 2 ). Populations of this caespitose sedge are typically small, though large populations of thousands of individuals exist (e.g. Maxton Plains, MI). Additionally, due to the rarity and patchiness of suitable alvar substrate, some populations of C. scirpoidea subsp. convoluta are quite isolate d from one another (Shackleford 2003). Anthropogenic habitat fragmentation may be further exacerbating the isolation of populations. Carex scirpoidea subsp. scirpoidea on the other hand, is the most widespread taxon in Carex section Scirpinae s.l. rangin g from the Kamchatka and Chukchi Peninsulas in Russia across boreal and arctic North America to Greenland, with marginal or disjunct populations as far south as Utah and Colorado in the western United States, Michigan and Minnesota in the Midwest, and New York and New Hampshire in the east, and three highly disjunct sites in Norway (Dunlop 1990, Westergaard, pers. comm. ). This subspecies typically occurs on calcareous substrates in wet habitats, such as fens and stream banks,

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9 though it appears to have a greater edaphic amplitude than C. scirpoidea subsp. convoluta (Dunlop 1990). 2.2.2. Sampling: Seven populations of C. scirpoidea subsp. convoluta were sampled from the Upper Peninsula of Michigan across the Manitoulin Islands including Drummond Island, re presenting the majority of its range. Eight populations of C. scirpoidea subsp. scirpoidea were sampled from three distinct regions of its extensive range: three populations from the Chugach Mountains of southern Alaska, three populations from the Mosquito Range in the Southern Rocky Mountains of Colorado, and one population each from escarpments near Beaver B ay, MN and Escanaba, MI (Fig. 2 ). Although limited, we believe that it is satisfactory for a baseline comparison of the taxon of interest, C. scirpoid ea subsp. convoluta Population sample size ranged from eight to 50 individuals per site, with a mean sample of 40.8 individuals per site. Individuals were sampled every 3 10 m along linear transects 5 m apart in an effort to avoid repeated collection of genetically identical clones. One reproductive culm and one vegetative culm representing each individual were collected, from which leaf tissue was harvested for the extraction of soluble enzymatic proteins and silica gel drying, after which the specimen was pressed. Vouchers were deposited at the Kathryn Kalmbach Herbarium (KHD) at the Denver Botanic Gardens. 2.2.3. Laboratory Methods: Methods for soluble enzymatic protein extraction, horizontal starch gel electrophoresis, and allozyme analysis follow ed B ruederle and Fairbrothers (1986) and Bruederle and Jensen (1991) with modifications made by Yarbrough (2000) and DePrenger Levin (2007).

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10 Samples were electrophesed on each of four 10.5% starch (Starch Art Co., Smithville, TX) gel and electrode buffer syst ems: lithium borate, pH 7.6/8.0 (Soltis et al. 1983); tris citrate, pH 7.5 (Soltis et al. 1983); histidine HCl, pH 7.0 (Gottlieb, 1981); and histidine citrate, pH 6.5 (Shields et al. 1983). Genotypic data were collected subsequent to staining with 14 subs trate specific stains encoding 19 loci in some cases with slight modifications (Appendix D ). Lithium borate gels were stained for alcohol dehydrogenase (ADH; Soltis et al. 1983), diaphorase (DIA), malic enzyme (ME), menadione reductase (MNR; Conkle et al. 1982) superoxide dismutase (SOD), and triosephosphate isomerase (TPI); Tris citrate gels were stained for aspartate aminotransferase (AAT), glyceraldehyde 3 phosphate dehydrogenase ([NADP dependent] (G3PDH; Soltis et al. 1983), and shikimic dehydrogenase (SDH); histidine HCl gels were stained for isocitrate dehydrogenase (IDH; Soltis et al. 1983), malate dehydrogenase (MDH), MNR, 6 phosphogluconate (PGD), phosphoglucose isomerase (PGI;), phosphoglucomutase (PGM), and SDH; histidine citrate gels were stain ed for aldalose (ALD; Soltis et al. 1983), IDH, PGI, and PGM. Estimates of population genetic diversity and structure were calculated from genotypic data for several parameters using GenePop ver. 4.2 (Raymond and Rousset 1995) and FSTAT ver. 2.9.3.2 (Goud et 2002), including percent polymorphic loci ( P ), number of alleles per polymorphic locus ( A p ), and expected heterozygosity ( H e ). Population structure was estimated using F statistics ( F IT F IS F ST ) for taxonomic groups in addition to calculating F IS for populations I ) was estimated using the program Populations ver. 1.2.30.

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11 In order to assess the significance of the differences among populations of the narrow and widespread taxa, RStudio (2014) was used to implement Wi lcoxon rank sum tests (non tests (normal distribution) were performed using R ver. 0.98.932, with the two taxa representing the paired values. Gitzendanner and Soltis (2000) suggest ed that a Wilcoxon signed ran k test be used, since this nonparametric test does not assume that the data for the congener s are identically distributed despite being independent (Felsenstein 1985). However, we used a Wilcoxon rank sum test since only two parameters were used rather tha n the three parameters recommended for the Wilcoxon signed rank test.

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12 3. Results 3.1. Phylogenetics Phylogenetic hypotheses were constructed for nr (ETS, ITS) and cpDNA ( atpF rps16 matK ) and concatenations of these regions ( Table 2, Figs. 3 10 ). Conc atenated phylogenetic hypotheses (Fig. 10 ) reveal a monophyletic Carex section Scirpinae including C. scabriuscula ( sensu lato ) with strong bootstrap support (100%). Carex scabriuscula also forms a well supported (100%) monophyletic clade, as does section Scirpinae s.s. (100%), resulting predominately due to variation in chloroplast sequences. Furthermore, there is a well defined second clade within C. scabriuscula (100%) that originates from variation in ETS that does not appear to be correlated with previous taxanomic treatments of these species. Relationships within Carex section Scirpinae s.s. are unresolved in the concatenated phylogeny, although a clade compri sing Russia and Nunavut samples of C. scirpoidea subsp. scirpoidea segregate with 71% bootstrap support. Chloroplast phylogenies ( atpF rpS16 matK ) vary similarly (Fig. 3 4 and 5 respectively), revealing a monophyletic Carex section Scirpinae s.l. (100 %, 95%, and 100%, respectively), a monophyletic C. scabriuscula (100%, 62%, and 65%, respectively), and a monophyletic Carex section Scirpinae s.s. (100%, 87%, and 65%, respectively). Again, systematic relationships within Carex section Scirpinae s.s. are unresolved. Concatenating the three chloroplast loci yields similar results (Fig. 6 ). Concatenated ETS and ITS phylogenies are relatively uninformative. They reveal a well supported monophyletic Carex section Scirpinae s.l. (98%), which shows that the pol yphyly observed in the ETS phylogeny does not occur with strong signal and is nullified by variation in ITS sequences. All taxa within Carex section Scirpinae s.l. are unresolved,

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13 including C. scabriuscula However, three C. scabriuscula individuals segreg ate together with strong bootstrap support (96%) while the Russian and Nunavut samples segregate together with weak support (51% and 53%) (Fig. 9 ). The ETS phylogeny is unusual in that it do es not yield a monophyletic Carex section Scirpinae s.l. the outgroup Carex magellanica segregates within the main polytomy of the section. Relationships within the section are unresolved. However, three samples of C. scabriuscula segregate together with moderately strong bootstrap support (88%), forming the cla de that is also observed in the concatenated nr and cpDNA phylogeny, as well as one of the two Russian samples segregating with the Nunavut sample with weak support (52%) (Fig. 7 ). The ITS phylogeny produces a well supported monophyletic Carex section Sci rpinae s.l. (97%). Carex scabriuscula is unresolved in the section but five samples segregate together with weak bootstrap support (65%). Additionally, two samples of C. scirpoidea subsp. scirpoidea from the Yukon segregate together with weak bootstrap sup port (61%), and one sample of C. curaturom segregates with a C. scirpoidea subsp. stenochlaena with m oderate bootstrap support (79%) despite being separated by half a continent and presumably, evolutionary time. Perhaps most interesting is the cluster of all of the samples of C. scirpoidea subsp. scirpoidea east of the Rocky Mountains with C. scirpoidea subsp. convoluta the only taxon in the section that is restricted east of the Rockies albeit with weak bootstrap support (63%) (Fig. 8 ). In other words, all samples east of the Rocky Mountains segregate together weakly. Using genotypic data, I ) reveals little genetic differentiation between C. scirpoidea subsp. convoluta and subsp. scirpoidea based on our sampled populations (Table 3 ). Most pairwise comparisons between the two subspecies are

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14 above 0.900 the only values that are not are pairwise comparisons with isolated, marginal Colorado populations of C. scirpoidea subsp. scirpoidea all of which are above 0 .700, many of w hich are above 0.8 00. 3.2. Population Genetics Patterns of allozyme variability were determined for seven populations of the rare, alvar restricted edaphic endemic Carex scirpoidea subsp. convoluta representing nearly its entire range and eight populations of C. scirpoidea subsp. scirpoidea representing three geographically disparate regions of its extensive range, two of which were marginal (Colorado and the Upper Midwest) and one more central (Al aska). Sixteen of the 19 (84.2%) loci sampled were polymorphic in one or both subspecies. Carex scirpoidea subsp. convoluta had a mean percent polymorphic loci ( P ) of 27.7% (range = 26.0 32.0%) while C. scirpoidea subsp. scirpoidea had a mean of 36. 4% ( range = 16.0 63.0%). Although the mean was slightly higher for C. scirpoidea subsp. scirpoidea there was no statistical difference between means (Wilcoxon rank sum test P value = 0.4059) despite obvious heterogeneity in the data. C entrally located Alask an populations maintained a much higher mean percent polymorphic loci ( P = 48.0%, range = 53.0 63.0%) than marginal populations of C. scirpoidea subsp. scirpoidea from Colorado ( P = 28.3%, range = 16.0 37.0% ) and the Upper Midwest ( P = 16.0%, range = 1 6.0%) as well as C. scirpoidea subsp. convoluta ; some Alaskan populations maintain ed as many as three times more polymorphic loci per population (Table 4 ) Mean number of alleles per polymorphic ( A P ) locus was 2.29 for C. scirpoidea subsp. convoluta (ran ge = 2.00 2.60) and 2.21 for C. scirpoidea subsp. scirpoidea (range = 2.00 2.42), again with no statistical difference between means of both subspecies (Welch two

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15 sample t test P value = 0.8045). Nine loci had more than two alleles, including seven loci with three alleles and two loci with five alleles ( MNR 2 and PGI ) among the two subspecies. P rivate alleles were identified in geographic r egion s such as MNR 1 a Colorado populations of C. scirpoidea subsp. scirpoidea and TPI po pulations of C. scirpoidea subsp. scirpoidea However, the frequency of private alleles per population wa s relatively low (0.085 and 0.052 for C. scirpoidea subsp. convoluta and subsp. scirpoidea respectively). Similarly, mean expected heterozygosity ( H E ) did not differ significantly between the two subspecies of C. scirpoidea with subsp. convoluta having a mean H E of 0.090 (range = 0.04 0.13) and subsp. scirpoidea having a mean H E of 0.077 (range = 0.03 0.12) (Welch two sample t test P value = 0.654). Alaskan populations maintained higher levels of expected heterozygosity ( H Emean = 0.117, range = 0.11 0.12) than the marginal populations of C. scirpoidea subsp. scirpoidea from Colorado ( H Emean = 0.057, range = 0.05 0.07 ) and the Upper Midwest ( H Emean = 0.045, range = 0.03 0.06). M ost loci were in Hardy Weinberg Equilibrium (HWE) in most p opulati ons. However s urprising for an obligately outcrossing dioecious species, all loci that were not in HWE had a positive F IS indicating heterozygote d eficiency ( Table 5 ). f or F IS ) wa s similar in C. scirpoidea subsp. convoluta ( F IS = 0.13 9 ) relative to subsp. scirpoidea ( F IS = 0.14 0 ), indicating that inbreeding was low in both subspecies again with no si gnificant difference between means for both subspecies Finally, genetic differentiation among populations ( F ST ) in C. scirpoidea subsp. convoluta ( F ST = 0.1 85 ) wa s much lower than subsp. scirpoidea ( F ST = 0.44 6 ). Pairwise F ST values we re low for all population pair s of C. scirpoidea subsp. convoluta most below

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16 0.20 and many below 0.10 Interestingly, one of the westernmost populations, Horseshoe Bay MI ha d the highest pairwise F ST values, each above 0.25 and most above 0.30 Conversely, pairwise F ST values reveal ed clear and extensive population structuring in C. scirpoidea subsp. scirpoidea across its range. Although Alaskan populations we re clearly genetically similar to one another (pairwise F STmean = 0.038, range = 0.022 0.070), most populations of C. scirpo idea subsp. scirpoidea maintain ed genetic variation even within regions for example, mean F ST for Colorado populations was 0.468 ( range = 0.397 0.578) (Table 6 ) Gene flow was low in C. scirpoidea subsp. convoluta ( N M = 0.792) but high in C. scirpoidea subsp. scirpoidea ( N M = 1.647). However, the large N M value found for C. scirpoidea subsp. scirpoidea is misleading as all eight populations were sampled from three distinct and very scattered regions across its broad distribution. Indeed, when populations from each region we re analyzed separately, it wa s found that Alaskan populations we re inflating the overall N M value because they maintai n very high gene flow ( N M Alaska = 2.52 5 ; F ST = 0.036). Surprisingly Colorado populations we re exchanging very few genes ( N M Colorado = 0.06 7 ; F ST = 0.473) despite being in the same watershed ; nor we re the two populations from the Upper Midwest ( N M Upper Midwest = 0.141; F ST = 0.392). Alternatively, this wa s also evident when N M is calculated tre ating all of the populations of C. scirpoidea subsp. scirpoidea from a region as one entity (e.g. all three Colorado populations being d an overall Nm value of 0.138 for all three 1.647, and an overall F ST of 0.3921, suggesting that there wa s very little gene flow between Alaskan, Coloradan, and Upper Midwest populations of C. scirpoidea subsp. scirpoidea

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17 4. Discussion 4.1. Phylogenetics The concatenated nr and cpDNA phylogeny reveals a well supported, monophyletic Carex section Scirpinae s.l (95 100% bootstrap support) Indeed, the only phylogenetic hypothesis that does not yield a well supported Carex section Scirpinae s.l. is ETS, which was variable but obviously had weak signal relative to the other regions (Table 1) Carex section Scirpinae s.l including C. scabriuscula is therefore a natural group of populations with a recent shared evolutionary history Chloroplast and concatenated nr and cpDNA phylogenies further reveal a well supported monophyletic Carex scabriuscula (100%) sister to a monophyletic Carex section Scirpinae s.s. (i.e. C. curatorum and C. scirpoidea ) (99%), although C. scabriuscula does not segregate in dependently in nuclear phylogenies. This further supports a very recent, common ancestry between C. scabriuscula and the rest of Section Scirpinae with chloroplast regions perhaps mutating at an accelerated rate relative to nuclear regions. Although other systematic characters separate C. scabriuscula from the section, the most prominent being the fact that this taxon is 50% unispicate, 50% dioecious, and has a lower chromosome count than Carex section Scirpinae s.s. this may actually support the conclusi on that it retains ancestral traits shared by the progenitor of Carex section Scirpinae s.l. This is b ecause dioecy is considered to have evolved from monoecy, unispicate sedges are considered to have evolved from multispicate sedges, and lower chromosome counts are expected to be ancestral to higher chromosome counts in Carex (Dunlop 1990, 1997; Dunlop and Crow 1999). These data show that there is no reason to exclude C. scabriuscula from Carex section Scirpinae

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18 Carex curatorum one of only two recognize d species in Carex section Scirpinae s.s. does not segregate independently from C. scirpoidea in any of our phylogenies. Although morphological data and substrate type clearly separate C. curatorum from C. scirpoidea (Dunlop 1990 ) our phylogenies do not support C. curatorum as a distinct species if applying the phylogenetic species concept (Baum 1992). Furthermore, none of the subspecies comprising C. scirpoidea segregate in any phylogenies (Fig. 3 10 ). However, ITS phylogenies ( Fig. 8 ) show a general biogeographical trend with all samples east of the Rocky Mountains segregating with weak support (63%) apart from samples west of or in the Rocky Mountains; this includes populations of the widespread C. scirpoidea subsp. scirpoidea As such, Carex scirpoidea subsp. convoluta segregates with eastern samples of C. scirpoidea subsp. scirpoidea in the ITS phylogeny. This suggests that there were different refugia during the Pleistocene or that there was geographic barrier that separated eastern and western populations in the more recent past (e.g., prairie formation) and that C. scirpoidea subsp. convoluta evolved from eastern populations of C. Scirpoidea subsp. scirpoidea Possible explanations for the lack of resolution in our phylogen etic hypotheses include incomplete lineage sorting and hybridization. Incomplete lineage sorting is a phenomenon in which currently diverging taxa retain shared, neutral, ancestral polymorphisms. Incomplete lineage sorting is indicative of a recent evoluti onary history in which neither taxon has yet to become fixed for one allele or another suggesting that our markers specifically nuclear markers, were not variable for phylogenetic resolution of this recently diverged group (Feliner and Rossello 2007). Al ternatively, hybridization will introduce novel alleles from one population or taxon (e.g., subspecies) into the gene pool of another,

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19 effectively reducing the genetic distance between the two groups. Although these two hypotheses are not mutually exclusiv e, incomplete lineage sorting is more probable given key for ITS (more of a guideline for interpretation than a strict key), it was determined that a recent evoluti onary history and incomplete lineage sorting was the cause of our phylogenetic lack of resolution. However, it has also been shown that the subspecies of C. scirpoidea are highly interfertile in greenhouse experiments (Dunlop 1990), though reports of co oc curring populations and natural hybridi zation are limited. Excep tions include C. scirpoidea subsp. stenochlaena and subsp. scirpoidea which may hybridize in the wild (Dunlop and Crow 1999, Dunlop 2008). Additionally, one field site (Brevort Lake Road, MI) visited by JWP and LPB in the summer of 2013 contained a previously unreported mixed population of C. scirpoidea subsp. scirpoidea and C. scirpoidea subsp. convoluta as well as potential hybrids. Further research is necessary to determine the amount of i nterfertility and hybridization of natural populations of C. scirpoidea Clearly, both morphological (Dunlop 1990, 1997, 1999) and molecular data suggest that the ecotypic subspecies of Carex scirpoidea appear to be recently derived from within C. scirpoid ea This recent radiation is most likely a result of colonization of different recognition of su bspecies as ecotypes of C. scirpoidea ( C. scirpoidea subsp. convoluta subsp. pseudoscirpoidea and subsp. stenochlaena ) based on her definition of subspecies: C. scirpoidea are geographically based ecotypes that share a common chromosome number, possess similar achene micromorphology and leaf anatomy, interbreed in greenhouse experiments, and have the majority of morphological characters falling within the normal

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20 range for C scirpoidea (Dunlop 1997). Although the mining of online databases such as SEINET show that the potential ranges of C. curatorum and C. scirpoidea overlap slightly, there is nothing in the literature t hat suggests that they co occur or that natural populations currently hybridize (not to say that they did not once co occur nor interbreed in the recent or distant past). Additional field surveys are required to verify this. Although Dunlop (1990) performe d greenhouse cross fertilization experiments for the section, no data were available for C. curatorum crossing, since most wild collected individuals did not survive in the greenhouse. Additionally, the first treatment of the taxon was at the species level (Stacey 1937), although Cronquist (1977) later treated the taxon as a variety of C. scirpoidea ( C. scirpoidea var. curatorum ), after which Dunlop (1990) elevated the variety back to species (Dunlop 1990). Our results indicate subspecific status, if follow ing the phylogenetic species concept (Baum 1992). Given the morphological and phylogenetic data available, it is highly probable that C. curatorum has evolved recently from southwestern populations of C. scirpoidea though additional research is needed to determine the taxonomic status of C. curatorum I ), estimated from allozyme data from seven populations of the narrowly distributed C. scirpoidea subsp. convoluta and ten populations of its widespread co nsepecific C. scirpoidea subsp. scirpoidea from three distinct regions of its range, support the hypothesis of a recent origin of this subspecies from within Carex scirpoidea Mean genetic identity obtained from pairwise comparisons of the two subspecies i s 0.918, just above th e 0.900 threshold used typically to distinguish subspecies (Nei 1972, Gottlieb 1981). Additionally, I suggests that marginal Colorado populations have been isolated longer, perhaps finding refugia in the American West or

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21 Rocky Mountai ns because they display the lowest genetic identity values observed for any population pairs, many below the 0.900 range. Conversely, marginal and relict populations of C. scirpoidea subsp. scirpoidea from the Upper Midwest are genetically similar to Alas kan populations ( I mean = 0.958). Also, C. scirpoidea subsp. convoluta is very similar to Alaskan and nearby Upper Midwest populations of subsp. scirpoidea ( I mean = 0.958 and 0.963, respectively), indicating a recent origin of this edaphic endemic, and presumably the other subspecies of C. scirpoidea given the phylogenetic data (Table 3) Our results reveal that Carex section Scirpinae s.l. is a monophyletic group that should not ex clude C. scabriuscula despite the fact that it does not consistently po ssess the defining characteristics of Carex section Scirpinae s.s. specifically being dioecious and unispicate. Additionally, Carex section Scirpinae s.s. is a monophyletic group based on our chloroplast and concatena ted phylogenies. Additionally, our phy logenies may suggest that C. curatorum be again demoted to subspecific status within C. scirpoidea Finally, none of the subspecies within Carex scirpoidea segregate independently, reaffirming the conclusion that this group is of recent origin and that sub species of C. scirpoidea are morphologically distinct ecotypes of C. scirpoidea subsp. scirpoidea that are currently diverging as a result of natural selection imparted by soil chemistry, with fixation of neutral loci lagging behind morphological different iation. 4.2. Population Genetics C arex scirpoidea harbors high genetic diversit y at the species level (e.g., P = 84.2%), judging from our sample of two of the four subspecies. However, o nly a fraction of this variation is maintained by either subspecies ( P = 27.71% and 36.38% for C. scirpoidea subsp. convoluta and subsp. scirpoidea respectively). Contrary to expectations, t here wa s

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22 no statistica l l y significant difference between the two subspecies of C. scirpoidea for mean P A P and H E indicating that t he narrow endemic harbors levels of diversity that are similar to its widespread congener. However, this is an artifact of our heterogenous sample of populations. Although C. scirpoidea subsp. convoluta maintains a level of genetic diversity considerably l ower than the highly polymorphic Alaskan populations of subsp. scirpoidea (e.g., P = 58.00%), genetic diversity in this narrow endemic is similar to slightly higher than that observed in marginal populations (e.g., P = 28.33% and 16.00% for Colorado and Up per Midwest populations, respectively). While this is most pronounced for P it is true for A P and H E as well (e.g., H E = 0.09 in C. scirpoidea subsp. convoluta and 0.08 in C. scirpoidea subsp. scirpoidea with H E = 0.12, 0.06, and 0.05 in Alaskan, Colorad an, and Upper Midwest populations, respectively) supporting DePrenger conclusions of levels of genetic diversity in C. scirpoidea subsp. convoluta similar to marginal populations of C. scirpoidea subsp. scirpoidea While the two subspecies are genetically similar subspecies share the mo st common allele at most loci there is a substantial amount of genetic diversity that may be attributed to differences between the two subspecies. For example, three alleles were f ound exclusively in C. scirpoidea subsp. convoluta and 12 more were found only in C. scirpoidea subsp. scirpoidea (Appendi ces E and F ). High levels of genetic diversity were not unexpected in Carex scirpoidea Strict dioecy is expected to increase genetic diversity relative to monoecious taxa as a result of obligate outcrossing, preventing self fertilization and limiting inbreeding due to consanguineous matings. While few studies have analyzed population genet ic diversity in strictly dioecious species (e.g., Populus [Jelinski and Cheliak 1992]), o bligate outcrossing acts as a

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23 stabilizing force that preserves rare alleles in the population by maintaining a stable frequency of heterozygotes when in Hardy Weinberg equilibrium (HWE) (Bawa 1980, Bruederle et al. 2001) Habit has been shown to also have a great effect on population genetic diversity and structure. Supporting the predictions of Stebbins (1950) concerning grasses, caespitose carices have been observed to harbor less genetic diversity while exhibit ing higher levels of heterozygote deficiency in comparison to rhizomatous carices. This is due to frequent self compatibility among caespitose sedges as well as the clump forming habit, which facilitates self fertilization (Stebbins 1950, Bruederle et al. 1987). Obviously, this is not expected for an obligate outcrossing caespitose taxon; strict dioecy is expected to be correlated with an increase in genetic diversity regardless of habit, as is in evidence here in which genetic diversity in C. scirpoidea is much higher than expected for ca espitose carices ( P = 14.2% ; Bruederle et al. 2008 ) for all population genetic diversity parameters analyzed, including P A P and H E (Bruederle et al. 2008). C arex scirpoidea maintains a moderately high level of genetic diversity relati ve to other sedges and all plant taxa particularly when considering the centrally located Alaskan populations. These levels are lower than reported for other dioecious plant species ( H E = 0.297 ; Yarbrough 2000 ) and other wind pollinated obligately outcrossing species (0.148 [ Hamrick and Godt 1990 ] ) However genetic diversity is significantly higher than the surveyed mean for other caespitose carices ( H E = 0.043 [ Bruederle et al. 2001 ] ) presumab ly due to dioecy Mean expected heterozygosity is very similar to all plant taxa ( H E = 0.113 [ Hamrick and Godt 1990 ] ), with central Alaskan populations higher and marginal populations lower. Despite wind pollination and obligate outcrossing, both

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24 subspecies of C. scirpoidea maintain levels of genetic diversity that are lower than expected even given their caespitose habit. This is most likely due to sampling H E is expec ted to be higher for other centrally located Alaskan and Canadian populations of C. scirpoidea subsp. scirpoidea as well as consanguineous matings within small populations sampled, including marginal populations of C. scirpoidea subsp. scirpoidea and som e populations of subsp. convoluta F IS values provide e vidence for consanguineous matings ; these are positive for every population except ing two populations of C. scirpoidea subsp. scirpoidea that had inbreeding coefficient s of 0.000 (Table 4 ) L oci that were not in HWE all show heterozygote deficiency and inbreeding with positive F IS values (Table 5 ). This is surprising as heterozygote excess was expected among populations of both subspecies due to obligate outcrossing. F IS wa s, on average, higher in C. scirpoidea subsp. convoluta than subsp. scirpoidea indicating s omewhat more inbreeding in the highly restricted edaphic endemic. C ontrary to expectations that habitat fragmentation and small population size would contribute to genetic structure in C. sci rpoidea subsp. convoluta differentiation among populations ( F ST ) of the edaphic endemic ( F ST = 0.1845) was much lower than C. scirpoidea subsp. scirpoidea ( F ST = 0.4456), suggesting that popu lations of C. scirpoidea subsp. convoluta are genetically simila r though still harboring moderate diversity (Table 6 ). Interestingly, the westernmost and most isolated population of C. scirpoidea subsp. convoluta Horseshoe Bay MI, was also the most differentiated in comparison to all other populations of C. scirpoidea subsp. convoluta ( F ST = 0.272 0.439). Conversely, pairwise F ST values demonstrate clear population structuring in C. scirpoidea subsp. scirpoidea

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25 with the most pronounced structure evident among regions (e.g., comparing Alaska to Colorado), t hough some structuring also occurs within regions (e.g. within Colorado). Additionally while Alaskan populations are most similar to one another, they are also interestingly similar to Beaver Bay, the lone Minnesota population (pairwise F STmean = 0.130, range = 0.114 0.148) and C. scirpoidea subsp. convoluta (data not shown). Moderate gene flow was observed in C. scirpoidea subsp. convoluta ( N M = 0.792), or less than one migrant per generation. Together, low F ST ( F ST convoluta = 0.1845; data not shown) and moderate gene flow may suggest a recent evolutionary history and a genetic bottleneck including a founder effect, in C. sciroidea subsp. convoluta High gene flow was reported for C. scirpoidea subsp. scirpoidea though this value is inflated by very high gene flow among Alaskan populations ( N M = 2.525) while Colorado and U pper Midwest populations undergo little gene flow within regions ( N M = 0.666 and 0.141 respectively). The fact that Alaskan populations are experiencing high gene flow amo ng each ot her is not surprising given their geographic proximity and continuity of suitable habitat but the same was expected for Colorado populations since they are in the same watershed. As expected, there was little gene flow between the three regions of C. scir poidea subsp. scirpoidea sampled due to the large distance between these regions. Two non mutually exclusive hypotheses have been proposed to account for the high proportion of disjunct and endemic taxa found in North American al var communities. The Perig lacial Hypothesis states that these populations may be relicts of spruce parkland that existed along the southern edge of the Laurentide Ice Sheet approximately 11,000 ybp. The Prairie Expansion Contraction Hypothesis states that alvar disjuncts are relict s from a prairie expansion that took place during the Hipsothermal warming approximately 5,000

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26 ybp and were later isolated when the climate cooled and the prairie retreated (Catling and Brownell 1995, Hamilton and Eckert 2007). Although this study does not provide evidence for either hypothes i s with respect to Carex section Scirpinae our data provide support for a genetic bottleneck including a founder effect, in C. scirpoidea subsp. convoluta as well as other marginal populations of C. scirpoidea subsp. scirpoidea Carex scirpoidea subsp. convoluta does not fit the expectation of low genetic diversity and high structure expected for a narrow edaphic endemic. C ontrary to expectations, population genetic diversity was similar relative to its widesp read congener C. scirpoidea subsp. scirpoidea when comparing means; however, the narrow endemic maintains lower genetic diversity than centrally located Alaskan populations but higher than marginal populations of C. scirpoidea subsp. scirpoidea Unexpecte dly, genetic structure ( F ST ) was not strong in C. scirpoidea subsp. convoluta but was rather low and gene flow moderate This is almost certainly due to obligate outcrossing, and wind pollination resulting in moderate gene flow. These results should have implications for conservation of both subspecies, particularly the edaphic endemic C. scirpoidea sub s p. convoluta and the isolated, marginal populations of C. scirpoidea subsp. scirpoidea including very rare Upper Midwest populations.

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27 5. Conclusions Our data reveal that Carex section Scirpinae s.l. is a monophyletic group composed of Carex scabriuscula and the taxa comprising Carex section Scirpinae s.s. suggesting that there is a very recent shared evolutionary history involving C. scabriuscula and Carex section Scirpinae s.s with C. scabriuscula retaining many of the ancestral character states of the recent common ancestor of it and the taxa comprising Carex section Scirpinae s.s. ; this is also supported by previously published phylogenies of subgen us Carex (Roalson et al. 2001 and Hendrichs et al. 2004). There seems to be no geographic signature within C. scabriuscula supporting recognition of C. gigas which was purportedly restricted in distribution to California Within the monophyletic Carex se ction Scirpinae s.l. is a monophyletic Carex section Scirpinae s.s. sister to C. scabriuscula However, there is little resolution within this group,. Indeed, this study brings into question the taxonomic status of C. curatorum is it a distinct species or, as it was previous ly treated, a subspecies of C. scirpoidea ? Applying the phylogenetic species concept, specific status does not appear to be merited Future research is required to answer this question. Some of the only phylogenetic resolut ion within Carex section Scirpinae s.s. is the repeated segregation of one or both of our Russian samples with one of the Nunavut samples with weak bootstrap support, perhaps demonstrating an ancient split within C. scirpoidea subsp. scirpoidea from when R ussian individuals were isolated from North America following the formation of the Bering Strait. Additionally, there is a biogeographical signal in ITS phylogenies in which all samples east of the Rocky Mountains segregate together with weak bootstrap sup port (63%). This includes every

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28 individual representing C. scirpoidea subsp. convoluta the only taxon found exclusively east of the Rockies, and eastern individuals of C. scirpoidea subsp. scirpoidea This may be used in conjunction with future research t o determine the radiation of the group following the LGM, specifically identifying refugia and the path of recolonization. Otherwise, the lack of resolution in our phylogenies supports subspecific status for each subspecies of C. scirpoidea including C. c uratorum High levels of population genetic diversity were detected in Carex scirpoidea based on overall percent polymorphic loci (84.2%) found in one or both subspecies. The major contributor to genetic diversity in both species was P rather than A P or surprisingly for a wind pollinated obligate outcrosser H E Unexpectedly, the narrow edaphic endemic C. scirpoidea subsp. convoluta maintained similar mean P A P and H E as its widespread congener C. scirpoidea subsp. scirpoidea More a ccurately our da ta suggest that C. scirpoidea subsp. convoluta maintains higher levels of population genetic diversity than marginal or disjunct populations of C. scirpoidea subsp. scirpoidea (e.g., Colorado and even nearby Upper Midwest) but lower variation than central or core populations (e.g., Alaska). High diversity is presumably a consequence of dioecy. Contrary to expectations that a rare taxon experiencing habitat fragmentation will h ave pronounced genetic structure across its range, F ST among populations of C. scirpoidea subsp. convoluta was minimal while much higher in the widespread C. scirpoidea subsp. scirpoidea ; however, this was due to the sample of the latter. While low gene fl ow was observed in C. scirpoidea subsp. convoluta despite low F ST values, very high gene flow was observed among the three Alaskan populations of the widespread taxon but not among the same number of Colorado populations despite being within one watershed. These

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29 results should have conservation implications for populations of both subspecies, particular the rare edaphic endemic and locally rare, marginal or disjunct populations of the widespread taxon. Future phylogenetic research should includes extendin g our model tests to maximum likelihood and Bayesian inference phylogenies to give a mo re robust phylogenetic hypothesi s of Carex section Scirpinae s.l. Also, a molecular clock technique, perhaps using BEAST analysis, may shed light on the timeframe of evo lution of the group, particularly the split between C. scabriuscula and Carex section Scirpinae s.s. specifically whether the spit occurred before the LGM, in which case C. scabriuscula may have been isolated south of the ice near its present range while C. scirpoidea was north of the ice in Beringia or south of the ice in the Rocky Mountains Additionally, SNPs are being developed by K. Westergaard ( pers. comm.) for C. scirpoidea in an effort to determine the origin of highly disjunct Norwegian populatio ns of subsp. scirpoidea These markers may have other utilities within Carex section Scirpinae including detecting any differences between populations of C. scabriuscula that were once labeled as C. gigas assessing the taxonomic status of C. curatorum and detecting biogeographical trends within the section. Future directions for conservation genetic research of the taxa within Carex section Scirpinae should include widening the allozyme sample of C. scirpoidea subsp. scirpoidea as a comparative baseli ne, primarily core Alaskan and Canadian populations. Additionally, a combination of techniques may be used to determine the pattern of post glacial colonization of this group as well as elucidating genetic diversity and relationships of any of the rare, ed aphic endemics within Care section Scirpinae s.l

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30 REFERENCES Alexander, P. J., G. Rajanikanth, C. D. Bacon, and C. D. Bailey. 2007. Recovery of Plant DNA Using a Reciprocating Saw and Silica Based Columns. Molecular Ecology Notes 7 : 5 9. Anacker, B. 2014. The Nature of Serpentine Endemism. American Journal of Botany 101 : 1 6. Baldwin, B. G. 2005. Origin of the Serpentine Endemic Herb Layia discoidea from the Widespread L. glandulosa (Compositae). Evolution 59 : 2473 2479. Baum, D. 1992. Phylogenetic Species Concepts. Trends in Ecology 7 : 1 2. Bawa, K. S. 1980. Evolution of Dioecy in Flowering Plants. Annual Review of Ecology and Systematics 11 : 15 39. Bruederle, L. P., and D. E. Fairbrothers. 1986. Allozyme Variation in Populations of The Carex crinita Complex (Cyperaceae). Systematic Botany 66 : 583 594. Bruederle, L. P., and U. Jensen. 1991 Genetic Differentiation of Carex flava and Carex viridula in West Europe (Cyperaceae). Systematic Botany 16 : 41 49. Bruederle, L. P. 1999. Genetic Differentiation of Geographically Marginal Populations in Carex michelliana (Cyperaceae): Implications of Conservation. Journal of the Torrey Botanical Society 126 : 1 8. Bruederle, L. P., S. L. Yarbrough, and S. D. Fehlberg. 2001. Allozyme Variation in the Genus Carex 2001. Sedges: Uses, Diversity, and Systematics of the Cyperaceae, Missouri Botanic Garden Press, St. Louis, MO. Burge, D. O. and P S. Manos. 2011. Edaphic Ecology and Genetics of the Gabbro Endemic Shrub Ceanothus roderickii (Rhamnaceae). Madrono 58 : 1 21. Catling, P. M., and V. R. Brownell. 1995. A Review of the Alvars of the Great Lakes Region: Distribution, Floristic Composition Biogeography, and Protection. Canadian Field Naturalist 109 : 143 171. Co le, C. T. 200 3. Genetic Variation in Rare and Common Plants. Annual Review of Ecology, Evolution, and Systematics 34 : 213 237. Conkle, M. T., P. D. Hodgskiss, L. B. Nunnally, and S. C. Hunter. 1982. Starch Gel Electrophoresis of Conifer Seeds: A Laboratory Manual. General Technical Report PSW 64, prepared for the United States Department of Agriculture, Forest Service.

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31 DePrenger Levine, M. E. 2007. Genetic Diversity in a Rare North American Endemic Carex scirpoidea ssp. convoluta Denver, Denver. Derieg, N. J., A. Sangaumphai, and L. P. Bruederle. 2008. Genetic Diversity and Endemism in North American Carex Section Ceratosystis (Cyperaceae). American Journal of Botany 95 : 1287 1296. Dunlop, D. A. 1990. The Biosystematics of Carex Section Scirpinae (Cyperaceae). Ph.D. Dissertation. University of New Hampshire, Durham. Dunlop, D. A. 19 97. Taxonomic Changes in Carex (Section Scirpinae Cyperaceae). Novon 7 : 355 356. Dunlop, D. A. 2008. Carex section Scirpinae For: Flora of North America; eFloras.org. 30 vols. New York and Oxford. Vol. 23. Website http://floranorthamerica.org/ [accessed 2012 Dec 11]. Dunlop, D. A. and G. E. Crow. 1999. The Taxonomy of Carex section Scirpinae (Cyperaceae). Rhodora 101 : 163 199. Feliner, G. N. and J. A. Rossello. 2007. B etter the devil you know? Guidelines for insightful utilization of nrDNA ITS in species level evolutionary study in plants. Molecular Phylogenetics and Evolution 44 : 911 919. Felsenstein, J. 1985. Phylogenies and the Comparative Method. American Naturali st 125 : 1 15. Gehrke, B., S. Martin Bravo, M. Muasya, and M. Luceo. 2010. Monophyly, Phylogenetic Position and the Role of Hybridization in Schoenoxiphium Nees (Cariceae, Cyperaceae). Molecular Phylogenetics and Evolution 56 : 380 392. Gitzendanner, M. A ., and P. S. Soltis. 2000. Patterns of Genetic Variation in Rare and Widespread Plant Congeners. American Journal of Botany 87 : 783 792. Goloboff, P., S. Farris, and K. Nixon. 2000. TNT (Tree analysis using New Technology) (BETA) ver. 1.1 Published by the authors, Tucum n, Argentina. Gottlieb, L. D. 1981. Electrophoretic Evidence and Plant Populations. Progress in Phytochemistry 7 : 1 46. Goudet J. 1995. FSTAT Version 1.2: a computer program to calculate F statistics. J. Heredity 86 : 485 486.

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32 Hamilton, J. A., and C. G. Eckert. 2007. Population Genetic Consequences of Geographic Disjunction: A Prairie Plant Isolated on Great Lakes Alvars. Molecular Ecology 16 : 1649 1660. Hamrick, J. L., and M. J. W. Godt. 1989. Allozyme Diversity in Plant Spec ies. In A. H. D. Brown, M. T. Clegg, A. L. Kahler, and B. S. Weir [eds.], Plant Population Genetics, Breeding, and Genetic Resources, 43 63. Sinauer, Sunderland, Massachusetts, USA. Hendrichs, M., F. Oberwinkler, D. Begerow, and R. Bauer. 2004. Carex sub genus Carex (Cyperaceae) A Phylogenetic Approach. Plant Systematics and Evolution 246 : 89 107. Hipp, A. L. 2007. Nonuniform Processes of Chromosome Evolution in Sedges ( Carex : Cyperaceae). Evolution 61 : 2175 2194. Hipp, A. L., P. E. Rothrock, and E. H. Roalson. 2010. The Evolution of Chromosome Arrangements in Carex (Cyperaceae). Botanical Review 75 : 96 109. Mayer, M. S. and P. S. Soltis. 1994.The Evolution of Serpentine Endemics: A Chloroplast DNA Phylogeny of the Streptanthus glandulosus Complex (Cruc iferae). Systematic Botany 19 : 557 574. Karron, J. D., Y. B. Linhart, C. A. Chaulk, and C. A. Robertson. 1988. Genetic Structure of Populations of Geographically Restricted and Widespread Species of Astragalus (Fabaceae). American Journal of Botany 75 : 11 14 1119. King, M. G. and E. H. Roalson. 2009b. Isolation and Characterization of 11 Microsatellite Loci from Carex macrocephala (Cyperaceae). Conservation Genetics 10 : 531 533. Kruckeberg, A. 1951. Intraspecific Variability in the Response of Certain Native Plant Species to Serpentine Soil. American Journal of Botany 38 : 408 419. Kuchel, S. D. 1999. Genetic Diversity and Structure in Geographically Marginal Populations of Carex viridula Colorado at Denver, Denver. Kkenthal, G. 1909. Cyperaceae Caricoidea. In: Engler A. (ed.) Das Pflan zenreich. Engelmann, Leipzig: 1 824. Lowry, D. B. 2012. Ecotypes and the Controversy Over St ages in the Formation of New Species. Biological Journal of the Linnean Society 106 : 241 257. Mayer, M. S. and P. S. Soltis. 1994.The Evolution of Serpentine Endemics: A Chloroplast DNA Phylogeny of the Streptanthus glandulosus Complex (Cruciferae). Syste matic Botany 19 : 557 574.

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33 NatureServe Explorer Online [Internet]. 2013. Website http://www.natureserve.org/explorer/ [accessed 2013 March 28]. Nei, M. 1972. Genetic Distance between Populations. The American Naturalist 106 : 283 292. Rajakaruna, N. 2004. The Edaphic Factor in the Origin of Plant Species. International Geology Review 46 : 471 478. Raymond M. and F. Rousset. 1995. GENEPOP (version 1.2): Population Genetics Software for Exact Tests and Ecumenicism. J. Heredity, 86:248 249 Reschke, C., R. Reid, J. Jones, T. Feeney, and H. Potter. 1999. Conserving Great Lakes Alvars: Final Technical Report of the International Alvar Conservation Initi ative. Alvar Working Group, The Nature Conservancy, Chicago, Illinois, USA. Reznicek, A.A, E.G. Voss, and B.S. Walters. 2011. Michigan Flora Online. University of Michigan Website http://w ww.michiganflora.net/species.aspx?id=1045 [accessed 2013 Feb 22]. Roalson, E. H., J. T. Columbus, and E. A. Friar. 2001. Phylogenetic Relationships in Cariceae (Cyperaceae) Based on ITS (nrDNA) and trnT L F (cpDNA) Region Sequences: Assessment of Subgeneri c and Sectional Relationships in Carex with Emphasis on Section Acrocystis Systematic Botany 26 : 318 341. Rothrock, P. E., A. A. Reznicek, and A. L. Hipp. 2009. Taxonomic Study of the Carex tenera Group (Cyperaceae). Systematic Botany 34 :297 311. RStudio (2014). RStudio: Integrated Development Environment for R (Version 0.98.932) [ Computer Software]. Boston, MA. Retrieved August 18, 2014. Schaefer, C. A., and D. W. Larson. 1997. Vegetation, Environmental Characteristics and Ideas on the Maintenanc e of Alvars on the Bruce Peninsula, Canada. Journal of Vegetation Science 8 : 797 810. Shackleford, R. 2003. Conservation Assessment for Bulrush Sedge ( Carex scirpoidea Michx.): Subspecies scirpoidea and convoluta (Kkenth.) Dunlop. Prepared for the USDA F orest Service, Eastern Region of the Forest Service, Milwaukee, Wisconsin, USA. Shields, C. R., T. J. Orton, and C. W. Stuber. 1983. An Outline of General Resource Needs and Procedures for the Electrophoretic Separation of Active Enzymes from Plant Tissu e. Isozymes in Plant Geentics and Breeding, Part A, Elsevier Science Publishers B.V., Amsterdam: 443 468.

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34 Soltis, D. E., C. H. Haufler, D. C. Darrow, and G. J. Gastony. 1983. Starch Gel Electrophoresis of Ferns: A Compilation of Grinding Buffers, Gel and Electrode Buffers, and Staining Schedules. American Fern Journal 73 : 9 27. Soltis, P. S. and M. A. Gitzendanner. 1999. Molecular Systematics and the Conservation of Rare Species. Conservation Biology 13 : 471 483. Song, B. and T. Mitchell Olds. 2007. Hig h Genetic Diversity and Population Differentiation in Boechera fecunda a Rare Relative of Arabidopsis Molecular Ecology 16 : 4079 4088. Standley, L. A., J. L. Dudley, and L. P. Bruederle. 1991. Electrophoretic Variability in the Rare Sedge, Carex polymorpha (Cyperaceae). Bulletin of the Torrey Botanical Club 118 : 444 450. Intergenic Spacer (IGS) of rDNA in the Cyperaceae: New Sequences for Lower Level Phylogenies in Sedges with an Example from Uncinia Pers. International Journal of P lant Sciences 164 : 213 227. Starr, J. R. and B. A. Ford. 2009. Phylogeny and Evolution in Cariceae (Cyperaceae): Current Knowledge and Future Directions. Botanical Review 75 : 110 137. Starr, J. R., R. F. Naczi, and B. N. Chouinard. 2009. Plant DNA Barc odes and Species Resolution in Sedges ( Carex Cyperaceae). Molecular Ecology Resources 9 : 151 163. Stebbins, G. L. 1950. Variation and Evolution in Plants. Columbia University Press, New York, NY. Stephenson, S. N., and P. S. Herendeen. 1986. Short term Drought Effects on the Alvar Communities of Drummond Island, Michigan. Journal of the Michigan Botanical Club 25 : 16 27. USDA, NRCS. 2013. The PLANTS Database. National Plant Data Team, Greensboro, NC 27401 4901 USA. Website http://plants.usda.gov [accessed 15 February 2013]. Yarbrough, S. L. 2000. Effects of Dioecy on Population Genetic Structure in Carex scirpoidea Michaux ssp. scirpoidea Denver, Denver

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35 Table 1 Outgroup sequences utilized in the phylogenetic analysis of Carex section Scirpinae s.l. Tuck. (Cyperaceae). Sequences were obtained from one of two sources: the Applied Systematics Laboratory at the University of Colorado Denver (unpublished data) and GenBank. GenBank accession numbers and dates accessed are listed Taxa Assession # Regio n Date Accesse d Carex bigelowii AM085624. 1 rps16 4/02/13 Carex bigelowii FJ548390.1 atpF 4/02/13 Carex bigelowii JN895168.1 matK 5/14/14 Carex bigelowii CQ223498.1 ETS 4/02/13 Carex bigelowii AY770469.1 ITS 4/02/13 Carex magellanica CAMA_25 All *Carex magellanica Sequences obtained from prior research in the Applied Systematics Lab at UCD (unpublished); CAMA_25

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36 Table 2 General sequence information for phylogenetic analysis of Carex section Scirpinae s.l. Tuck (Cyperaceae). atpF ETS ITS matK rpS16 Aligned length (bp) 555 538 493 729 763 G C content mean (%) 27.6 54.8 61.3 28.9 26.7 Number of indels 15 10 5 0 4 Number of potentially informative characters 16 33 30 2 6

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37 Table 3. seven populations of the narrowly distributed edaphic endemic Carex scirpoidea subsp. convoluta (Kkenthal) Dunlop (Cyperaceae) and ten populations of the widespread C. scirpoidea subsp. scirpoidea Michaux. Abbreviations for populations of C. scirpoidea subsp. convoluta are as follows: BSC = Big Shoal Cove, MI; BI = Burnt Island, ON; MaxP = Maxton Plains, MI; MurP = Murphy Point, ON; PI = Presque Isle, MI; HSB = Horseshoe Bay, MI; BLR = Brevort Lake Road, MI. Carex scirpoidea subsp. scirpoidea populations are: AV = Arctic Valley, AK; CC = Campbell Creek, AK; FR = Fort Richardson, AK; HCFS = High Creek Fen South, CO; HCFN = High Creek Fen North, CO; BCF = Beaver Creek Fen, CO; GPC = Geneva Park Creek, CO; HSM = Horseshoe Mountain, CO; ESC = Escanaba River, MI; BB = Beaver Bay, MN. AV CC FR HCFS HCF N BCF GPC HSM ESC BB BSC BI MAX P MUR P PI HSB BLR AV ---0.01 0.00 5 0.06 0 0.13 7 0.21 4 0.08 2 0.08 3 0.06 7 0.02 6 0.04 3 0.05 8 0.035 0.024 0.03 3 0.06 2 0.02 1 CC 0.98 8 ---0.00 4 0.05 3 0.12 9 0.20 3 0.06 7 0.07 7 0.05 9 0.02 1 0.05 5 0.07 3 0.046 0.039 0.03 9 0.05 4 0.02 8 FR 0.99 5 0.99 6 ---0.05 2 0.13 2 0.20 8 0.06 9 0.07 7 0.06 2 0.02 1 0.04 8 0.06 2 0.038 0.028 0.03 6 0.05 7 0.02 0 HCFS 0.94 2 0.94 9 0.94 9 ---0.05 9 0.11 9 0.03 2 0.03 6 0.11 7 0.06 6 0.10 1 0.10 8 0.067 0.084 0.08 6 0.09 9 0.05 7 HCFN 0.87 2 0.87 9 0.87 6 0.94 3 ---0.05 0 0.11 1 0.09 4 0.18 6 0.14 2 0.17 6 0.18 7 0.150 0.159 0.16 3 0.17 6 0.13 6 BCF 0.80 7 0.81 6 0.81 2 0.88 8 0.95 2 ---0.12 4 0.10 6 0.15 2 0.16 6 0.19 0 0.19 3 0.192 0.215 0.22 3 0.14 7 0.18 0 GPC 0.91 9 0.93 5 0.93 3 0.96 8 0.89 5 0.88 4 ---0.03 4 0.07 2 0.07 2 0.09 8 0.09 1 0.068 0.102 0.10 1 0.06 1 0.06 6 HSM 0.92 1 0.92 6 0.92 6 0.96 5 0.91 0 0.89 9 0.96 7 ---0.08 9 0.08 9 0.10 0 0.10 1 0.083 0.110 0.11 7 0.08 0 0.08 0 ESC 0.93 5 0.94 3 0.94 0 0.89 5 0.83 0 0.85 9 0.93 0 0.91 5 ---0.01 9 0.03 7 0.04 5 0.054 0.054 0.05 6 0.00 2 0.04 2 BB 0.97 4 0.97 9 0.97 9 0.93 6 0.86 8 0.84 7 0.93 1 0.91 5 0.98 1 ---0.03 4 0.05 0 0.037 0.036 0.04 4 0.02 0 0.01 3 BSC 0.95 8 0.94 6 0.95 3 0.90 4 0.83 8 0.82 7 0.90 6 0.90 5 0.96 4 0.96 7 ---0.01 1 0.020 0.014 0.02 4 0.03 6 0.01 9 BI 0.94 3 0.93 0 0.94 0 0.89 8 0.82 9 0.82 4 0.71 3 0.90 4 0.95 6 0.95 1 0.98 9 ---0.026 0.012 0.02 4 0.04 0 0.02 9 MAX P 0.96 6 0.95 5 0.96 3 0.93 6 0.86 0 0.82 6 0.93 4 0.92 0 0.94 7 0.96 4 0.98 0 0.97 4 ---0.021 0.03 2 0.05 0 0.02 2 MUR P 0.97 6 0.96 2 0.97 2 0.92 0 0.85 3 0.80 7 0.90 3 0.89 6 0.94 7 0.96 5 0.98 6 0.98 8 0.979 ---0.01 1 0.04 9 0.01 5 PI 0.96 7 0.96 2 0.96 5 0.91 7 0.84 9 0.80 0 0.90 4 0.89 0 0.94 5 0.95 7 0.97 6 0.97 6 0.969 0.989 ---0.04 8 0.03 0 HSB 0.93 9 0.94 7 0.94 4 0.90 6 0.83 9 0.86 3 0.94 1 0.82 3 0.99 8 0.98 1 0.96 4 0.96 1 0.952 0.952 0.95 3 ---0.04 1 BLR 0.97 9 0.97 3 0.98 0 0.94 4 0.87 3 0.83 6 0.93 6 0.92 3 0.95 9 0.98 7 0.98 1 0.97 2 0.978 0.985 0.97 1 0.96 0 ---

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38 Table 4. Descriptive population genetic statistics in the edaphic endemic Carex scirpoidea subsp convoluta (Kkenthal) Dunlop (Cyperaceae) and its widespread congener C. scirpoidea subsp. scirpoidea Michaux where N = number of individuals sampled, P = percent polymorphic loci, A P = mean number of alleles per polymorphic locus, H O = observed heterozygosity, H E = expected heterozygosity, and F IS coefficient. Mean descriptive statistics for each region of Carex scirpoidea subsp. sc irpoidea sampled as well as means for both subspecies are summarized below. P values comparing the means of the two subspecies are either tests ( when data are normal ly distributed ) or Wilco x on rank sum tests ( when data are not normal ly distribut ed ) Carex scirpoidea Population State / Province N P Ap Ho He Fis subsp. convoluta Big Shoal Cove (BSC) MI 50 26.00 2.60 0.08 0.11 0.28 Brevort Lake Road (BLR) MI 18 26.00 2.20 0.08 0.10 0.17 Horseshoe Bay (HB) MI 25 26.00 2.00 0.03 0.04 0.16 Maxton Plains (MaxP) MI 50 32.00 2.50 0.11 0.13 0.18 Presque Isle (PI) MI 50 32.00 2.33 0.09 0.10 0.03 Burnt Island (BI) ON 50 26.00 2.20 0.08 0.09 0.03 Murphy Point (MurP) ON 25 26.00 2.20 0.07 0.07 0.07 subsp. scirpoidea Arctic Valley (AV) AK 50 58.00 2.09 0.12 0.12 0.02 Campbell Creek (CC) AK 50 63.00 2.42 0.08 0.11 0.23 Fort Richardson (FR) AK 50 53.00 2.30 0.11 0.12 0.09 Beaver Creek Fen (BCF) CO 50 16.00 2.33 0.04 0.05 0.18 High Creek Fen South (HCFS) CO 50 37.00 2.29 0.07 0.07 0.11 High Creek Fen North (HCFN) CO 50 32.00 2.17 0.05 0.05 0.00 Beaver Bay (BB) MN 8 16.00 2.00 0.03 0.06 0.49 Escanaba River (ESC) MI 36 16.00 2.33 0.03 0.03 0.00 Means Alaska subsp. scirpoidea AK 150 58.00 2.27 0.10 0.12 0.11 Colorado subsp. scirpoidea CO 150 28.33 2.26 0.05 0.06 0.10 Upper Midwest subsp. scirpoidea MN / MI 48 16.00 2.17 0.03 0.05 0.25 C. scirpoidea subsp. scirpoidea 348 36.38 2.24 0.07 0.08 0.14 C. scirpoidea subsp. convoluta 268 27.71 2.29 0.08 0.09 0.13 Significance P value 0.41 0.80 0.54 0.65 0.70

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39 Table 5 Heterozygote deficiencies by locus and population for seven populations of the rare edaphic endemic C. scirpoidea subsp. convoluta (Kkenthal) Dunlop (Cyperaceae) and eight populations of its widespread congener C. scirpoidea subsp. scirpoidea Michaux. Significace (p < 0.05) is indicated by *. Population abbreviations are as follows: BSC = Big Shoal Cove, MI; BI = Burnt Island, ON; MaxP = Maxton Plains, MI; MurP = Murp hy Point, ON; PI = Presque Isle, MI; HSB = Horseshoe Bay, MI; BLR = Brevort Lake Road, MI. Carex scirpoidea subsp. scirpoidea populations are: AV = Arctic Valley, AK; CC = Campbell Creek, AK; FR = Fort Richardson, AK; HCFS = High Creek Fen South, CO; HCFN = High Creek Fen North, CO; BCF = Beaver Creek Fen, CO; GPC = Geneva Park Creek, CO; HSM = Horseshoe Mountain, CO; ESC = Escanaba River, MI; BB = Beaver Bay, MN. C. scirpoidea subsp. convoluta BSC BI MaxP MurP PI HB BLR DIA 1 ------------1.0000 ME --------------TPI 1 --------------TPI 2 --------------ADH --------------SOD --------------AAT 2 0.4902 0.6536 0.3187 0.6239 0.5082 0.2883 0.8302 SDH --------------PGD ----*0.0361 1.0000 ------PGM 1 --0.8057 0.9821 ----0.1204 1.0000 PGM 2 --------0.0668 ----IDH *0.000 0.8054 0.6227 1.0000 --1.0000 *0.0244 MDH 3 --------0.8702 1.0000 --MNR 1 --------------MNR 2 0.4966 *0.0362 *0.0037 *0.0061 0.4489 ----PGI *0.0003 0.8054 *0.0000 0.6288 0.5677 1.0000 0.5267 C. scirpoidea subsp scirpoidea HCFS HCFN BCF AV CC FR ESC BB DIA 1 ----------------ME ------0.1650 0.1842 *0.0066 ----TPI 1 1.0000 ------1.0000 0.3207 ----TPI 2 --1.0000 --1.0000 1.0000 0.2013 ----ADH --------1.0000 1.0000 ----SOD ------0.2489 1.0000 1.0000 ----AAT 2 ----0.1115 ------1.0000 *0.049 SDH 1.0000 ----0.0620 *0.0001 0.1261 ----PGD ------0.1727 --1.0000 ----PGM 1 *0.0000 ------1.0000 1.0000 ----

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40 PGM 2 1.0000 --1.0000 ----------IDH ------1.0000 0.3614 0.3086 0.4063 0.4406 MDH 3 --1.0000 ----1.0000 ------MNR 1 1.0000 0.4422 ------------MNR 2 --------*0.0000 1.0000 1.0000 --PGI 0.2849 0.4360 *0.0394 0.9031 1.0000 0.7642 ----* = significant, P value < 0.05

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41 Table 6 Pairwise F ST describing seven populations of the narrowly distributed edaphic endemic C. scirpoidea subsp. convoluta (Kkenthal) Dunlop (Cyperaceae) and eight populations of its widespread congener C. scirpoidea subsp. scirpoidea Michaux Population abbreviations are a s follows: BSC = Big Shoal Cove, MI; BI = Burnt Island, ON; MaxP = Maxton Plains, MI; MurP = Murphy Point, ON; PI = Presque Isle, MI; HSB = Horseshoe Bay, MI; BLR = Brevort Lake Road, MI. Carex scirpoidea subsp. scirpoidea populations are: AV = Arctic Valley, AK; CC = Campbell Creek, AK; FR = Fort Richardson, AK; HCFS = High Creek Fen South, CO; HCFN = High Creek Fen North, CO; BCF = Beaver Creek Fen, CO; GPC = Geneva Park Creek, CO; HSM = Horseshoe Mountain, CO; ESC = Esca naba River, MI; BB = Beaver Bay, MN. Carex scirpoidea Population BSC BI MaxP MurP PI HB BLR subsp. convoluta BSC --------------BI 0.086 ------------MaxP 0.118 0.168 ----------MurP 0.104 0.103 0.131 ------PI 0.164 0.185 0.188 0.087 ----HB 0.272 0.341 0.303 0.439 0.356 --BLR 0.098 0.187 0.099 0.098 0.166 0.320 --Population HCFS HCFN BCF AV CC FR ESC BB subsp. scirpoidea HCFS ----------------HCFN 0.429 --------------BCF 0.578 0.397 ------------AV 0.357 0.615 0.717 ----------CC 0.340 0.606 0.695 0.073 --------FR 0.316 0.582 0.673 0.022 0.023 ------ESC 0.609 0.808 0.795 0.458 0.414 0.397 ----BB 0.409 0.706 0.747 0.148 0.127 0.114 0.320 --

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42 Fig ure 1 Distribution of all individuals sampled for phylogenetic analysis of Carex section Scirpinae s.l. Tuckerman (Cyperaceae) across North America, Russia, and Greenland. Note : the Norwegian sample of C. scirpoidea subsp. scirpoidea is not shown.

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43 Figure 2 Sampled populations of Carex scirpoidea subsp. scirpoidea Michaux (Cyperaceae), represented by circles on the large map, and C. scirpoidea subsp. convoluta (Kkenthal) Dunlop represented by circles in the inset of the Great Lakes region, for soluble protein extraction and electrophoresis. A single circle is used to represent all three Alaskan and Coloradan populations of C. scirpoidea subsp. scirpoidea due to geographic proxi mity of populations; similarl, for C. scirpoidea subsp. convoluta Shaded region represents potential range for C. scirpoidea subsp. scirpoidea.

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44

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45 Figure 3 Chloroplast DNA marker atpF phylogenetic hypothesis of Carex section Scirpinae s.l. Tuck. (Cyperace ae), where scir = Carex scirpoidea subsp. scirpoidea conv = C. scirpoidea subsp. convoluta pseu = C. scirpoidea subsp. pseudoscirpoidea sten = C. scirpoidea subsp. stenochlaena cura = C. curatorum scab = C. scabriuscula Cama_25 = C. magellanica (outgroup) and C. bigelow = C. bigelowii (outgroup). Numbers at nodes represent bootstrap values, numbers within sample names are internal IDs and do not represent taxa nor geography. Each sample ID is followed by the two letter postal code of the state ( United States), province or territory (Canada), or country (Greenland, Norway, and Russia) of origin The origin of sample 97 ( C. scirpoidea subsp. scirpoidea ) is unknown (un).

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46 Fig ure 4 Chloroplast DNA marker matK phylogenetic hypothesis of Carex secti on Scirpinae s.l. Tuck. (Cyperaceae), where scir = Carex scirpoidea subsp. scirpoidea conv = C. scirpoidea subsp. convoluta pseu = C. scirpoidea subsp. pseudoscirpoidea sten = C. scirpoidea subsp. stenochlaena cura = C. curatorum scab = C. scabriuscula Cama_25 = C. magellanica (outgroup) and C. bigelow = C. bigelowii (outgroup). Numbers at nodes represent bootstrap values, numbers within sample names are internal IDs and do not represent taxa nor geography. Each sample ID is followed by the two letter postal code of the state (United States), province or territory (Canada), or country (Greenland, Norway, and Russia) of origin The origin of sample 97 ( C. scirpoidea subsp. scirpoidea ) is unknown (un).

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47 Figure 5 Chloroplast DNA marke r rpS16 phylogenetic hypothesis of Carex section Scirpinae s.l. Tuck. (Cyperaceae), where scir = Carex scirpoidea subsp. scirpoidea conv = C. scirpoidea subsp. convoluta pseu = C. scirpoidea subsp. pseudoscirpoidea sten = C. scirpoidea subsp. stenochlae na cura = C. curatorum scab = C. scabriuscula Cama_25 = C. magellanica (outgroup) and C. bigelow = C. bigelowii (outgroup). Numbers at nodes represent bootstrap values, numbers within sample names are internal IDs and do not represent taxa nor geography. Each sample ID is followed by the two letter postal code of the state (United States), province or territory (Ca nada), or country (Greenland, Norway, and Russia) of origin The origin of sample 97 ( C. scirpoidea subsp. scirpoidea ) is unknown (un).

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48 Figure 6 Concatenated chloroplast DNA phylogenetic hypothesis of Carex section Scirpinae s.l. Tuck. (Cyperaceae) usi ng the atpF matK and rpS16 regions, where scir = Carex scirpoidea subsp. scirpoidea conv = C. scirpoidea subsp. convoluta pseu = C. scirpoidea subsp. pseudoscirpoidea sten = C. scirpoidea subsp. stenochlaena cura = C. curatorum scab = C. scabriuscul a Cama_25 = C. magellanica (outgroup) and C. bigelow = C. bigelowii (outgroup). Numbers at nodes represent bootstrap values, numbers within sample names are internal IDs and do not represent taxa nor geography. Each sample ID is followed by the two letter postal code of the state (United States), province or territory (Canada), or country (Greenland, Norway, and Russia) of origin The origin of sample 97 ( C. scirpoidea subsp. scirpoidea ) is unknown (un).

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49 Fig ure 7 Nuclear DNA marker ETS phylogenetic hypothesis of Carex section Scirpinae s.l. Tuck. (Cyperaceae), where scir = Carex scirpoidea subsp. scirpoidea conv = C. scirpoidea subsp. convoluta pseu = C. scirpoidea subsp. pseudoscirpoidea sten = C. scirpoidea su bsp. stenochlaena cura = C. curatorum scab = C. scabriuscula Cama_25 = C. magellanica (outgroup) and C. bigelow = C. bigelowii (outgroup). Numbers at nodes represent bootstrap values, numbers within sample names are internal IDs and do not represent tax a nor geography. Each sample ID is followed by the two letter postal code of the state (United States), province or territory (Canada), or country (Greenland, Norway,

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50 and Russia) of origin The origin of sample 97 ( C. scirpoidea subsp. scirpoidea ) is unkno wn (un). Figure 8 Nuclear DNA marker ITS phylogenetic hypothesis of Carex section Scirpinae s.l. Tuck. (Cyperaceae), where scir = Carex scirpoidea subsp. scirpoidea conv = C.

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51 scirpoidea subsp. convoluta pseu = C. scirpoidea subsp. pseudoscirpoidea sten = C. scirpoidea subsp. stenochlaena cura = C. curatorum scab = C. scabriuscula Cama_25 = C. magellanica (outgroup) and C. bigelow = C. bigelowii (outgroup). Numbers at nodes represent bootstrap values, numbers within sample names are internal IDs a nd do not represent taxa nor geography. Each sample ID is followed by the two letter postal code of the state (United States), province or territory (Canada), or country (Greenland, Norway, and Russia) of origin The origin of sample 97 ( C. scirpoidea subs p. scirpoidea ) is unknown (un).

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52 Figure 9 Concatenated nuclear DNA marker phylogenetic hypothesis of Carex section Scirpinae s.l. Tuck. (Cyperaceae) using ETS and ITS sequences, where scir = Carex scirpoidea subsp. scirpoidea conv = C. scirpoidea subsp. convoluta pseu = C. scirpoidea subsp. pseudoscirpoidea sten = C. scirpoidea subsp. stenochlaena cura = C. curatorum scab = C. scabriuscula Cama_25 = C. magellanica (outgroup) and C. bigelow = C. bigelowii (outgroup). Numbers at nodes represent bootstrap values, numbers within sample names are internal IDs and do not represent taxa nor geography. Each sample ID is followed by the two letter postal code of the state (United States), province or territory (Canada), or country (Greenland, Norway, a nd Russia) of origin The origin of sample 97 ( C. scirpoidea subsp. scirpoidea ) is unknown (un).

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53 Figure 10 Concatenated nr and cpDNA ( atpF matK rpS16 ETS, and ITS) phylogenetic hypothesis of Carex section Scirpinae s.l. Tuck. (Cyperaceae), where scir = Carex scirpoidea subsp. scirpoidea conv = C. scirpoidea subsp. convoluta pseu = C. scirpoidea subsp. pseudoscirpoidea sten = C. scirpoidea subsp. stenochlaena cura = C. curatorum scab = C. scabriuscula Cama_25 = C. magellanica (outgroup) and C bigelow = C. bigelowii (outgroup). Numbers at nodes represent bootstrap values, numbers within sample names are internal IDs and do not represent taxa nor geography. Each sample ID is followed by the two letter postal code of the state (United States), p rovince or territory (Canada), or country (Greenland, Norway, and Russia) of origin The origin of sample 97 ( C. scirpoidea subsp. scirpoidea ) is unknown (un).

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54 Appendices A Summary of distribution, habitat, growth form, morphological characteristics, a nd chromosome counts among taxa within Carex section Scirpinae s.l Tuck. (Cyperaceae) (Data obtained from Dunlop 1990, 1997, 2008 and Dunlop and Crow 1999). B. Collection information describing all individuals sampled for DNA sequence analysis of Carex section Scirpinae (Tuck.) sensu lato Sources are either the Canadian Museum of or collections from the Applied Systematics lab at University of Colorado Denver, deposited a t the Kathryn Kalmbach Herbarium (KHD). Internal IDs are for the purposes of this study only. If available, the United States county or Canadian province of origin for each sample are reported, as well as the complete collection information provided with t he sample. C PCR templates of each locus analyzed for the generation of a .) nuclear (nrDNA) and b .) chloroplast (cpDNA) phylogenetic hypotheses of Carex section Scirpinae (Tuck.) sensu lato including PCR master mixes as well as thermocycler programs. D Starch gel systems, enzymes, and run voltage / amperage with authorities for allozyme analysis of the edaphic endemic C. scirpoidea subsp. convoluta (Kkenthal) Dunlop and its widespread congener C. scirpoidea subsp. scirpoidea Michaux (Cyperaceae). E Example of one of the input file for the program GenePop ver. 4.2 (Raymond and Rousset 1995) used to analyze raw data, collected as individual genotypes, of the narrow edaphic endemic C. scirpoidea subsp. convoluta (Kkenthal) Dunlop and its widespread congener C. scirpoidea subsp. scirpoidea Michaux (Cyperaceae). Abbreviations for C. scirpoidea subsp. convoluta are as follows: BSC = Big Shoal Cove, MI; BLR_C = Brevort Lake Road, MI; BI = Burnt Island, ON; HB = Horseshoe Bay, MI; MaxP = Maxton Plains, MI ; MurP = Murphy Point, ON; PI = Presque Isle, MI. Abbreviations for C. scirpoidea subsp. scirpoidea are: HCFS = High Creek Fen South, CO; HCFN = High Creek Fen North, CO; BCF = Beaver Creek Fen, CO; AV = Arctic Valley, AK; CC = Campbell Creek, AK; FR = Fo rt Richardson, AK; ESC = Escanaba River, MI; BB = Beaver Bay, MN. F Allele frequencies per population per locus generated from the program FSTAT ver. 2.9.3.2 (Goudet 2002) and modified in Microsoft Excel for a C. scirpoidea subsp. convoluta (Kkenthal) Dunlop and b C. scirpoidea subsp. scirpoidea Michaux (Cyperaceae).

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55 Appendix A Summary of distribution, habitat, growth form, morphological characteristics, and chromosome counts among taxa within Carex section Scirpinae s.l Tuck. (Cyperaceae) (Data obtained from Dunlop 1990, 1997, 2008 and Dunlop and Crow 1999). C. scirpoidea subsp. scirpoidea Michx. C. scirpoidea subsp. conovolut a (Kk.) Dunlop C. scirpoidea subsp. pseudoscirpoide a (Rydb.) Dunlop C. scirpoidea subsp. stenochlaen a (Holm) .Lve & D.Lve C. curatorum Stacey C. scabriuscul a Mack. Range Widesprea d across N. Amer., with populations in Greenland, Russia, and Norway Shores of Lake Huron and northern Lake Michigan Western N. America, from British Columbia to California and west to Colorado Western N. America, from Montana to Alaska and the Yukon Hanging gardens and riparian areas of Utah and Arizona Klamath, Coastal, and northern Sierra Nevada mtn. ranges of SW Oregon and NW California Ele vationa l Range 0 1200 m 100 200 m 3300 3900 m 1600 2600 m 900 1300 m 1000 2000 m Substrate Typically calcareous Alvar Noncalcareous, gravelly Weakly acidic, high Mg, low Ca Navajo Sandstone Serpentine Dioecy ~95% ~95% ~95% ~95% Always ~50% Habit Caespitose to short rhizomes Caespitose Rhizomatous Caespitose Caespitose to short rhizomes Caespitose Culms Vegetative culms seasonally die back to the rhizome, which only have buds for the next year. Vegetative culms seasonally die back to the rhizome, which only have buds for the next year. Vegetative culms arise from nodes within the vegetative shoots. Vegetative culms seasonally die back to the rhizome, which only have buds for the next year. Vegetative culms seasonally di e back to the rhizome, which only have buds for the next year. Vegetative culms seasonally die back to the rhizome, which only have buds for the next year. Involucral bract Varies, but rarely exceeds the top of the spike. Varies, but rarely exceeds the top of the spike. Naked, or a short scale like bract. Varies, but rarely exceeds the top of the spike. Varies, but rarely exceeds the top of the spike. Exceeds the top of the spike; wider and longer than all other taxa. Ligule length 0.2 3.0 mm 0.2 3.0 mm 0.2 3.0 mm 0.2 3.0 mm 1.0 5.0 mm N/A; no data found

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56 Leaf length & width 31 cm x 2.5 mm 23 cm x 1.8 mm 21 cm x 3.0 mm 28 cm x 2.5 mm curatorum is distinguishe d from C. scirpoidea by its longer (Dunlop 2008) N/A; no data found N/A; no data found Unispicate Yes, though rarely to occasionall y a small, sessile lateral spike is present. Yes, though rarely to occasionall y a small, sessile lateral spike is present. Yes, though rarely to occasionally a small, sessile lateral spike is present. Yes, though rarely to occasionally a small, sessile lateral spike is present. Yes, with the presence of a small, sessile lateral spike being more frequent than C. scirpoidea ~ 50% multispicate Inflores cence Linear Linear Linear Clavate Ellipsoid Longer and wider than C. scirpoidea Pistillate spikes Obovate to ovate Obovate to ovate Obovate to ovate Lanceolate Obovate to ovate Ellipsoid Perigynia shape Ovate Ovate Ovate Lanceolate Ovate Lanceolate Perigynia length (mm) 1.5 3 1.5 3 1.5 3 2.8 5 1.5 3 2.5 4 Perigynia length : width ratio < 2.5 < 2.5 < 2.5 > 2.5 < 2.5 <2.5 but may be >2.5 Perigynia beak Perigynia ends abruptly in the beak Perigynia ends abruptly in the beak Perigynia ends abruptly in the beak Perigynia gradually tapers to beak Perigynia ends abruptly in the beak Perigynia ends abruptly in the beak Achene overlap Tightly enveloped Tightly enveloped Tightly enveloped Achene filled full width but only of length by perigynia Achene filled < width and to of length by perigynia Achene filled 1/3 2/3 width and 1/3 length by perigynia Rachilla Low % present Low % present Low % present Low % present High % present Absent Smut infection Common Common Common Common Free Common Haploid chromo some count (n) 31 (29 + III = trivalent, 30, 32, & 34 exist) 31 31 31 31 & 30 31 29; 29 30

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57 Appendix B Collection information describing all individuals sampled for DNA sequence analysis of Carex section Scirpinae (Tuck.) sensu lato Sources are either the Canadian Herbarium (NHA) or collections from the Applied Systematics lab at University of Colorado Denver, deposited at the Kathryn Kalmbach Herbarium (KH D ) Internal IDs are for the purposes of this study only. If available, the United States county or Canadian province of origin for each sample are reported, as well as the complete collection information provided with the sample. Taxon Herbarium Accession Number / Applied Systematics Collection Number Herbariu m Internal ID USA State / CAN Province Carex scirpoidea subsp. scirpoidea 2003e 18 NHA CSC17 Vermont Carex scirpoidea subsp. scirpoidea 2414a 15 NHA CSC18 New York Carex scabriuscula 3009b 13 NHA CSC19 Oregon Carex scabriuscula 3063 8 NHA CSC20 California Carex scabriuscula 3044 3 NHA CSC21 California Carex scabriuscula 3026c 1 NHA CSC22 California Carex scirpoidea subsp. scirpoidea 2140 10 NHA CSC23 Nevada Carex scirpoidea subsp. scirpoidea 2522 5 NHA CSC24 Newfoundland Carex scirpoidea subsp. scirpoidea 2505 1 NHA CSC25 Newfoundland Carex scirpoidea subsp. scirpoidea 2440a 6 NHA CSC26 Newfoundland Carex scirpoidea subsp. scirpoidea 2394 14 NHA CSC27 New Hampshire Carex scirpoidea subsp. scirpoidea 1965 13 NHA CSC28 New Hampshire Carex scirpoidea subsp. scirpoidea 2100a 16 NHA CSC29 Utah Carex scirpoidea subsp. scirpoidea 2025 7 NHA CSC30 Colorado Carex scirpoidea subsp. scirpoidea 2256 8 NHA CSC31 Colorado Carex curatorum 2087 24 NHA CSC32 Utah

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58 Carex scirpoidea subsp. convoluta CMN 334612 CMN CSC33 Michigan Carex scirpoidea subsp. stenochlaena 2272d 22 NHA CSC34 Montana Carex scabriuscula 3036 7 NHA CSC35 California Carex scabriuscula 3018 6 NHA CSC36 California Carex scabriuscula 3065 5 NHA CSC37 California Carex scabriuscula 3047 4 NHA CSC38 California Carex scabriuscula 3041 2 NHA CSC39 California Carex scabriuscula 3049b 9 NHA CSC40 California Carex scabriuscula 3054 10 NHA CSC41 California Carex scabriuscula 3056d 11 NHA CSC42 California Carex scabriuscula 3012 12 NHA CSC43 Oregon Carex scabriuscula 3000a 14 NHA CSC44 Oregon Carex scirpoidea subsp. scirpoidea 1964 12 NHA CSC45 New Hampshire Carex scirpoidea subsp. scirpoidea 2510 4 NHA CSC46 Newfoundland Carex scirpoidea subsp. scirpoidea 2480 3 NHA CSC47 Newfoundland Carex scirpoidea subsp. scirpoidea 2473 2 NHA CSC48 Newfoundland Carex scirpoidea subsp. scirpoidea 2264 9 NHA CSC49 Colorado Carex scirpoidea subsp. scirpoidea 2100d 17 NHA CSC50 Utah Carex scirpoidea subsp. scirpoidea 2130 11 NHA CSC51 Nevada Carex scirpoidea subsp. pseudoscirpoidea 2075 20 NHA CSC52 Utah Carex scirpoidea subsp. pseudoscirpoidea 2158 19 NHA CSC53 California Carex scirpoidea subsp. pseudoscirpoidea 2285 21 NHA CSC54 Montana

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59 Carex scirpoidea subsp. stenochlaena 2241h 23 NHA CSC55 Unknown Carex scirpoidea subsp. pseudoscirpoidea CMN 304880 CMN CSC56 Wyoming Carex scirpoidea subsp. pseudoscirpoidea CMN 246483 CMN CSC57 Montana Carex scirpoidea subsp. pseudoscirpoidea CMN 550927 CMN CSC58 Utah Carex scirpoidea subsp. scirpoidea CMN 311390 CMN CSC59 Greenland Carex scirpoidea subsp. scirpoidea CMN 318197 CMN CSC60 Greenland Carex scirpoidea subsp. stenochlaena CMN 270031 CMN CSC61 Alaska Carex scirpoidea subsp. stenochlaena CMN 21303 CMN CSC62 Alberta Carex scirpoidea subsp. stenochlaena CMN 21324 CMN CSC63 British Colombia Carex scirpoidea subsp. convoluta CMN 341016 CMN CSC64 Ontario Carex scirpoidea subsp. convoluta CMN 392029 CMN CSC65 Ontario Carex scirpoidea subsp. convoluta CMN 391916 CMN CSC66 Ontario Carex scirpoidea subsp. scirpoidea CMN 266099 CMN CSC67 Alberta Carex scirpoidea subsp. scirpoidea CMN 372400 CMN CSC68 British Colombia Carex scirpoidea subsp. scirpoidea CMN 469821 CMN CSC69 British Colombia Carex scirpoidea subsp. scirpoidea CMN 369284 CMN CSC70 Alberta Carex scirpoidea subsp. scirpoidea CMN 369193 CMN CSC71 Alberta Carex scirpoidea subsp. scirpoidea CMN 282719 CMN CSC72 Saskatchewan Carex scirpoidea subsp. scirpoidea CMN 525570 CMN CSC73 Saskatchewan Carex scirpoidea subsp. scirpoidea CMN 247340 CMN CSC74 Manitoba Carex scirpoidea subsp. scirpoidea CMN 447183 CMN CSC75 Manitoba Carex scirpoidea subsp. scirpoidea CMN 360412 CMN CSC76 Ontario

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60 Carex scirpoidea subsp. scirpoidea CMN 265052 CMN CSC77 Ontario Carex scirpoidea subsp. scirpoidea CMN 440780 CMN CSC78 Quebec Carex scirpoidea subsp. scirpoidea CMN 374459 CMN CSC79 Quebec Carex scirpoidea subsp. scirpoidea CMN 582243 CMN CSC80 Newfoundland Carex scirpoidea subsp. scirpoidea CMN 389526 CMN CSC81 Newfoundland Carex scirpoidea subsp. scirpoidea CMN 313110 CMN CSC82 Yukon Carex scirpoidea subsp. scirpoidea CMN 383773 CMN CSC83 Yukon Carex scirpoidea subsp. scirpoidea CMN 399326 CMN CSC84 Northwest Territories Carex scirpoidea subsp. scirpoidea CMN 479684 CMN CSC85 Northwest Territories Carex scirpoidea subsp. scirpoidea CMN 565228 CMN CSC86 Nunavut Carex scirpoidea subsp. scirpoidea CMN 518385 CMN CSC87 Nunavut Carex scirpoidea subsp. scirpoidea CMN 239015 CMN CSC88 Norway Carex scirpoidea subsp. scirpoidea CMN 408319 CMN CSC89 Russia Carex scirpoidea subsp. scirpoidea CMN 386548 CMN CSC90 Russia Carex curatorum 04260601 01 KHD CSC91 Utah Carex curatorum 0719201001 01 KHD CSC92 Arizona Carex scirpoidea subsp. convoluta 07070601 04 KHD CSC93 Michigan Carex scirpoidea subsp. convoluta 06250503 01 KHD CSC94 Michigan Carex scirpoidea subsp. convoluta 06240502 31 KHD CSC95 Michigan Carex scirpoidea subsp. convoluta 06250501 01 KHD CSC96 Michigan Carex scirpoidea subsp. pseudoscirpoidea 07080501 18 KHD CSC97 Unknown Carex scirpoidea subsp. pseudoscirpoidea 07090501 21 KHD CSC98 Unknown

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61 Carex scirpoidea subsp. scirpoidea 0704021105 07 KHD CSC99 Alaska Carex scirpoidea subsp. scirpoidea 2 KHD CSC100 Wyoming Carex scirpoidea subsp. scirpoidea 0707201301 04 KHD CSC101 Minnesota Carex scirpoidea subsp. scirpoidea 0809200801 26 KHD CSC102 Alaska Carex scirpoidea subsp. scirpoidea 0708201301 08 KHD CSC103 Minnesota Carex scirpoidea subsp. scirpoidea 0711201301 20 KHD CSC104 Michigan Carex scirpoidea subsp. scirpoidea 0821200801 30 KHD CSC105 Alaska Carex scirpoidea subsp. scirpoidea 23 KHD CSC106 Alaska

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62 Appendix C PCR templates of each locus analyzed for the generation of a .) nuclear (nrDNA) and b .) chloroplast (cpDNA) phylogenetic hypotheses of Carex section Scirpinae (Tuck.) sensu lato including PCR master mixes as well as thermocycler programs. a nrDNA ETS DNA H 2 O 5.79 ul Primers: ETSf 10X buffer 1.50 ul ETS 18Sr MgC l 2 (25mM) 1.50 ul dNTPs (10mM) 0.45 ul primer F (10mM) 0.600 ul primer R (10mM) 0.600 ul Betaine 3.00 ul Taq (1U/ul) 0.06 ul template DNA (10ng/ul) 1.50 ul total reax. vol. 15.00 ul Thermal Cycler: Cycle number: 38 temp (C) time pre treatment 94 1 min denaturing 94 30 sec annealing 48 30 sec extension 72 2 mins post treatment 72 7 mins final 10 indefinite ITS DNA H 2 O 5.73 ul Primers: ITS 5i 10X buffer 1.50 ul ITS 4i MgC l 2 (25mM) 1.50 ul dNTPs (10mM) 0.45 ul primer F (10mM) 0.600 ul primer R (10mM) 0.600 ul Betaine 3.00 ul Taq (1U/ul) 0.12 ul template DNA (10ng/ul) 1.50 ul total reax. vol. 15.00 ul Thermal Cycler: Cycle number: 30 temp (C) time denaturing 95 1 minute annealing 55 1 minute

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63 extension 72 2 minutes final 72 7 minutes b cpDNA atpF DNA H 2 O 9.990 ul Primers: atpF 10X buffer 1.500 ul atpH MgC l 2 (25mM) 0.900 ul dNTPs (10mM) 0.300 ul primer F (10mM) 0.375 ul primer R (10mM) 0.375 ul Taq (1U/ul) 0.060 ul template DNA (10ng/ul) 1.500 ul total reax. vol. 15.000 ul Thermal Cycler: Cycle number: 35 temp (C) time initial 94 3 mins denaturing 94 30 secs annealing 55 30 secs extension 72 1 min 30 sec final 72 5 mins matk DNA H 2 O 8.290 ul Primers: matK 2.1f_J 10X buffer 1.500 ul matK 5r_J MgC l 2 (25mM) 1.500 ul dNTPs (10mM) 0.300 ul primer F (10mM) 0.375 ul primer R (10mM) 0.375 ul Plus BSA 1.100 ul Taq (1U/ul) 0.060 ul template DNA (10ng/ul) 1.500 ul total reax. vol. 15.000 ul Thermal Cycler: Cycle number: 40 temp (C) time initial 94 3 mins denaturing 94 30 secs annealing 46 1 min 15 sec

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64 extension 72 1 min 30 sec final 72 7 mins PCR: rpS16 DNA H 2 O 9.990 ul Primers: rpS16 F 10X buffer 1.500 ul rpS16 R MgC l 2 (25mM) 0.900 ul dNTPs (10mM) 0.300 ul primer F (10mM) 0.375 ul primer R (10mM) 0.375 ul Taq (1U/ul) 0.060 ul template DNA (10ng/ul) 1.500 ul total reax. vol. 15.000 ul Thermal Cycler: Cycle number: 35 temp (C) time initial 94 2 mins denaturing 94 1 min annealing 46 1 min extension 72 4 mins final 72 7 mins

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65 Appendix D Starch g el systems, enzymes, and run voltage / amperage with authorities for allozyme analysis of the edaphic endemic C. scirpoidea subsp. convoluta (Kkenthal) Dunlop and its widespread congener C. scirpoidea subsp. scirpoidea Michaux (Cyperaceae). System Substr ate Specific Stain Authority Lithium borate; pH 7.6/8.0 Alchohol dehydrogenase (ADH) Soltis et al. 1983 Diaphorase (DIA 1) Mod. from Yeh pers. comm. Malic enzyme (ME) Soltis et al. 1983 Menadione reductase (MNR)** Mod. Conkle et al. 1982 Superoxide dismutase (SOD) Vallejos 1983 Triose phosphate isomerase (TPI) Soltis et al. 1983 Tris citrate; pH 7.5 Aspartate aminotransferase (AAT) Cardy et al. 1981 Acid phosphatase (ACP)* Soltis et al. 1983 Glyceraldehyde 3 phosphate dehydrogenase Mod. from Soltis et (G3PDH) al. 1983 Shikimatic dehydrogenase (SDH)* Soltis et al. 1983 Histidine HCl; pH 7.0 Isocitrate dehydrogenase (IDH) Soltis et al. 1983 Malate dehydrogenase (MDH) Soltis et al. 1983, mod. Shaw and Presod 1970 Menadione reduct ase (MNR)* Mod. Conkle et al. 1982 6 phosphogluconate dehydrogenase (PGD) Soltis et al. 1983 Phosphoglucose isomerase (PGI) Soltis et al. 1983 Phosphogucomutase (PGM) ** Soltis et al. 1983 Shikimatic dehydrogenase (SDH)** Soltis et al. 1983 Histidin e HCl; pH 7.0 Aldalose (ALD)* Soltis et al. 1983 Only in Yarbrough (2000) ** Only in DePrenger Levin (2007)

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66 Appendix E Example of one of the input file for the program GenePop ver. 4.2 (Raymond and Rousset 1995) used to analyze raw data, collected as individual genotypes, of the narrow edaphic endemic C. scirpoidea subsp. convoluta (Kkenthal) Dunlop and its widespread co ngener C. scirpoidea subsp. scirpoidea Michaux (Cyperaceae). Abbreviations for C. scirpoidea subsp. convoluta are as follows: BSC = Big Shoal Cove, MI; BLR_C = Brevort Lake Road, MI; BI = Burnt Island, ON; HB = Horseshoe Bay, MI; MaxP = Maxton Plains, MI; MurP = Murphy Point, ON; PI = Presque Isle, MI. Abbreviations for C. scirpoidea subsp. scirpoidea are: HCFS = High Creek Fen South, CO; HCFN = High Creek Fen North, CO; BCF = Beaver Creek Fen, CO; AV = Arctic Valley, AK; CC = Campbell Creek, AK; FR = Fort Richardson, AK; ESC = Escanaba River, MI; BB = Beaver Bay, MN. Lithium Borate Tris Citrate Histidine HCl Population Individual DIA 1 ME TPI 1 TPI 2 ADH SOD AAT 1 AAT 2 G3PDH SDH PGD PGM 1 PGM 2 IDH MDH 2 MDH 3 MNR 1 MNR 2 PGI HCFS ind_1, 0202 0101 0202 0101 0202 0000 0000 0101 0000 0202 0101 0202 0202 0000 0101 0202 0202 0404 0405 ind_2, 0202 0101 0202 0101 0202 0000 0000 0101 0000 0202 0101 0202 0202 0000 0101 0202 0102 0404 0304 ind_3, 0202 0000 0202 0101 0202 0000 0000 0101 0000 0202 0101 0202 0202 0000 0000 0000 0202 0404 0304 ind_4, 0202 0101 0202 0101 0202 0000 0000 0101 0000 0000 0101 0000 0202 0000 0000 0000 0202 0404 0404 ind_5, 0202 0101 0202 0101 0202 0000 0000 0101 0000 0000 0101 0000 0203 0000 0101 0202 0102 0404 0404 ind_6, 0202 0101 0202 0101 0202 0000 0000 0101 0000 0202 0101 0000 0202 0000 0101 0202 0102 0404 0405 ind_7, 0202 0101 0202 0101 0202 0000 0000 0101 0000 0202 0101 0102 0202 0000 0101 0202 0202 0404 0404 ind_8, 0202 0000 0202 0101 0202 0000 0000 0101 0000 0102 0101 0102 0202 0000 0101 0202 0202 0404 0404 ind_9, 0202 0000 0202 0101 0202 0000 0000 0101 0000 0202 0101 0202 0202 0000 0101 0202 0102 0404 0000 ind_10, 0202 0101 0202 0101 0202 0000 0000 0101 0000 0202 0101 0102 0202 0000 0101 0202 0202 0404 0404 ind_11, 0202 0101 0202 0101 0202 0000 0000 0101 0000 0102 0101 0102 0202 0000 0101 0202 0202 0404 0404

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67 ind_12, 0202 0101 0202 0101 0202 0000 0000 0101 0000 0202 0101 0202 0203 0000 0101 0202 0202 0404 0404 ind_13, 0202 0101 0202 0101 0202 0000 0000 0101 0000 0102 0101 0202 0202 0000 0101 0202 0202 0404 0404 ind_14, 0202 0101 0202 0101 0202 0000 0000 0101 0000 0102 0101 0202 0202 0000 0101 0202 0202 0404 0404 ind_15, 0202 0101 0102 0101 0202 0000 0000 0101 0000 0202 0101 0000 0202 0000 0101 0202 0202 0404 0404 ind_16, 0202 0101 0202 0101 0202 0000 0000 0101 0000 0102 0101 0102 0202 0000 0101 0202 0202 0404 0405 ind_17, 0202 0101 0202 0101 0202 0000 0000 0101 0000 0102 0101 0102 0202 0000 0101 0202 0202 0404 0404 ind_18, 0202 0101 0102 0101 0202 0000 0000 0101 0000 0102 0101 0102 0202 0000 0101 0202 0202 0404 0303 ind_19, 0202 0101 0202 0101 0202 0000 0000 0101 0000 0202 0101 0102 0202 0000 0101 0202 0202 0404 0404 ind_20, 0202 0101 0202 0101 0202 0000 0000 0101 0000 0202 0101 0102 0202 0000 0101 0202 0202 0404 0404 ind_21, 0202 0101 0202 0101 0202 0000 0000 0101 0000 0202 0101 0000 0000 0000 0101 0202 0202 0404 0303 ind_22, 0202 0101 0202 0101 0202 0000 0000 0101 0000 0202 0101 0102 0202 0000 0101 0202 0102 0404 0404 ind_23, 0202 0101 0202 0101 0202 0000 0000 0101 0000 0202 0101 0102 0202 0000 0101 0202 0202 0404 0404 ind_24, 0000 0101 0202 0101 0000 0000 0000 0101 0000 0202 0101 0102 0202 0000 0101 0202 0202 0404 0404 ind_25, 0000 0101 0202 0101 0000 0000 0000 0101 0000 0202 0000 0303 0202 0000 0000 0000 0202 0404 0000 ind_26, 0202 0101 0202 0101 0202 0000 0000 0000 0101 0202 0101 0101 0202 0000 0101 0202 0202 0404 0404 ind_27, 0202 0101 0202 0101 0202 0000 0000 0000 0101 0202 0101 0101 0202 0000 0101 0202 0202 0404 0404 ind_28, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0000 0101 0101 0202 0000 0101 0202 0202 0404 0404 ind_29, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0102 0101 0101 0202 0000 0101 0202 0202 0404 0304

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68 ind_30, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0202 0101 0101 0202 0000 0101 0202 0202 0404 0304 ind_31, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0202 0101 0101 0203 0000 0101 0202 0202 0404 0404 ind_32, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0202 0101 0101 0202 0000 0101 0202 0102 0404 0303 ind_33, 0202 0101 0102 0101 0202 0101 0000 0000 0101 0202 0101 0101 0202 0000 0101 0202 0202 0404 0304 ind_34, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0000 0101 0101 0202 0000 0101 0202 0202 0404 0404 ind_35, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0202 0101 0101 0202 0000 0101 0202 0102 0404 0304 ind_36, 0202 0101 0202 0101 0000 0101 0000 0000 0101 0102 0101 0101 0202 0000 0101 0202 0202 0404 0304 ind_37, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0202 0101 0101 0203 0000 0101 0102 0202 0404 0304 ind_38, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0102 0101 0101 0203 0000 0101 0202 0202 0404 0404 ind_39, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0202 0101 0101 0203 0000 0101 0202 0202 0404 0404 ind_40, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0202 0101 0101 0202 0000 0101 0202 0202 0404 0304 ind_41, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0202 0101 0101 0202 0000 0101 0202 0202 0404 0404 ind_42, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0202 0101 0101 0203 0000 0101 0202 0202 0404 0304 ind_43, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0202 0101 0101 0202 0000 0101 0202 0202 0404 0404 ind_44, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0202 0101 0101 0202 0000 0101 0202 0202 0404 0405 ind_45, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0202 0101 0101 0202 0000 0101 0202 0102 0404 0405 ind_46, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0202 0101 0101 0202 0000 0101 0202 0102 0404 0404 ind_47, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0202 0101 0101 0202 0000 0101 0202 0202 0404 0304

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69 ind_48, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0202 0101 0101 0202 0000 0101 0202 0202 0404 0405 ind_49, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0202 0101 0101 0202 0000 0101 0202 0202 0404 0405 ind_50, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0202 0101 0101 0202 0000 0101 0202 0202 0404 0404 HCFN ind_1, 0202 0101 0202 0102 0202 0101 0101 0101 0101 0101 0101 0101 0202 0000 0101 0202 0202 0000 0405 ind_2, 0202 0101 0102 0102 0202 0101 0101 0101 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0405 ind_3, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0101 0101 0101 0202 0000 0101 0202 0102 0404 0405 ind_4, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0101 0101 0101 0202 0000 0101 0202 0102 0404 0404 ind_5, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0101 0101 0101 0202 0000 0101 0202 0102 0404 0405 ind_6, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0101 0101 0101 0202 0000 0101 0202 0102 0404 0404 ind_7, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0101 0101 0101 0202 0000 0101 0202 0102 0404 0405 ind_8, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0404 ind_9, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0405 ind_10, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0304 ind_11, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0404 ind_12, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0404 ind_13, 0000 0101 0000 0000 0202 0101 0101 0101 0101 0101 0101 0000 0202 0000 0101 0202 0202 0404 0404 ind_14, 0202 0101 0202 0102 0202 0101 0101 0101 0101 0101 0101 0101 0202 0000 0101 0202 0102 0404 0404 ind_15, 0000 0000 0000 0000 0000 0000 0101 0101 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0304

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70 ind_16, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0101 0101 0101 0202 0000 0101 0202 0102 0404 0404 ind_17, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0101 0101 0101 0202 0000 0101 0102 0202 0404 0405 ind_18, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0000 0202 0000 0101 0202 0202 0404 0405 ind_19, 0000 0000 0000 0000 0202 0000 0101 0101 0101 0101 0101 0101 0202 0000 0101 0102 0202 0404 0404 ind_20, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0304 ind_21, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0404 ind_22, 0202 0101 0000 0000 0202 0101 0101 0101 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0304 ind_23, 0202 0101 0000 0000 0202 0000 0101 0101 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0404 ind_24, 0202 0101 0202 0101 0202 0000 0101 0101 0101 0000 0101 0101 0202 0000 0101 0202 0202 0404 0405 ind_25, 0000 0000 0000 0000 0000 0000 0000 0000 0101 0000 0000 0000 0202 0000 0101 0000 0000 0000 0000 ind_26, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0000 0101 0202 0000 0101 0202 0000 0404 0404 ind_27, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0404 ind_28, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0102 0404 0404 ind_29, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0404 ind_30, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0102 0404 0404 ind_31, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0203 0000 0101 0202 0102 0404 0404 ind_32, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0102 0404 0404 ind_33, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0102 0202 0404 0304

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71 ind_34, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0101 0404 0405 ind_35, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0404 ind_36, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0304 ind_37, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0304 ind_38, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0304 ind_39, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0405 ind_40, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0405 ind_41, 0000 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0102 0101 0404 0404 ind_42, 0000 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0303 ind_43, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0404 ind_44, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0404 ind_45, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0404 ind_46, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0102 0404 0303 ind_47, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0404 ind_48, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0102 0404 0405 ind_49, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0405 ind_50, 0202 0101 0202 0101 0202 0000 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0202 0404 0404 BCF ind_1, 0000 0101 0202 0101 0000 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404

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72 ind_2, 0000 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_3, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0203 0000 0101 0202 0000 0404 0404 ind_4, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_5, 0202 0101 0202 0101 0202 0000 0000 0102 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0304 ind_6, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0304 ind_7, 0202 0101 0202 0101 0202 0000 0000 0101 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0304 ind_8, 0202 0101 0202 0101 0202 0000 0000 0102 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_9, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_10, 0202 0101 0202 0101 0202 0000 0000 0101 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_11, 0202 0101 0202 0101 0202 0000 0000 0102 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0405 ind_12, 0202 0101 0202 0101 0202 0000 0000 0102 0101 0101 0101 0101 0203 0000 0101 0202 0000 0404 0404 ind_13, 0202 0101 0202 0101 0202 0000 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0405 ind_14, 0202 0101 0202 0101 0202 0000 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0405 ind_15, 0202 0101 0202 0101 0202 0000 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0405 ind_16, 0202 0101 0202 0101 0202 0000 0000 0101 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0304 ind_17, 0202 0101 0202 0101 0202 0000 0000 0102 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_18, 0202 0101 0202 0101 0202 0000 0000 0102 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_19, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404

PAGE 87

73 ind_20, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_21, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_22, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0203 0000 0101 0202 0000 0404 0405 ind_23, 0000 0101 0202 0101 0202 0000 0000 0102 0101 0101 0101 0101 0203 0000 0101 0202 0000 0404 0404 ind_24, 0000 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0303 ind_25, 0000 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0203 0000 0101 0000 0000 0404 0404 ind_26, 0202 0000 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_27, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_28, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_29, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_30, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_31, 0000 0101 0000 0000 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_32, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_33, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_34, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_35, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0405 ind_36, 0202 0101 0202 0101 0202 0000 0000 0102 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0304 ind_37, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404

PAGE 88

74 ind_38, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_39, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0203 0000 0101 0202 0000 0404 0303 ind_40, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_41, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_42, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0303 ind_43, 0202 0101 0202 0101 0202 0000 0000 0000 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0000 ind_44, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_45, 0202 0101 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_46, 0202 0000 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_47, 0202 0000 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0405 ind_48, 0202 0000 0202 0101 0202 0000 0000 0202 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0405 ind_49, 0202 0101 0202 0101 0202 0000 0000 0102 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 ind_50, 0202 0101 0202 0101 0202 0000 0000 0102 0101 0101 0101 0101 0202 0000 0101 0202 0000 0404 0404 BSC ind_1, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0303 0101 0202 0202 0404 0204 ind_2, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0303 0101 0202 0202 0505 0204 ind_3, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0205 ind_4, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0405 0204 ind_5, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404

PAGE 89

75 ind_6, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0405 0204 ind_7, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0405 0203 ind_8, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0101 0101 0202 0202 0405 0505 ind_9, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0405 0505 ind_10, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0303 ind_11, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0505 ind_12, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0101 0101 0202 0202 0404 0505 ind_13, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0303 ind_14, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_15, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_16, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0405 0204 ind_17, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0303 0101 0202 0202 0404 0204 ind_18, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0203 0101 0202 0202 0405 0304 ind_19, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_20, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0203 0101 0202 0202 0404 0202 ind_21, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0203 0101 0202 0202 0404 0202 ind_22, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_23, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204

PAGE 90

76 ind_24, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0303 0101 0202 0202 0404 0204 ind_25, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0303 0101 0202 0202 0404 0204 ind_26, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0505 0205 ind_27, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0405 0202 ind_28, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_29, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0205 ind_30, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0101 0101 0202 0202 0404 0202 ind_31, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0101 0101 0202 0202 0405 0505 ind_32, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0101 0101 0202 0202 0405 0202 ind_33, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0101 0101 0202 0202 0405 0404 ind_34, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_35, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_36, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0204 ind_37, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0205 ind_38, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0101 0101 0202 0202 0404 0204 ind_39, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0101 0101 0202 0202 0405 0205 ind_40, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0205 ind_41, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202

PAGE 91

77 ind_42, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0405 0205 ind_43, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0505 ind_44, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0405 0205 ind_45, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0101 0101 0202 0202 0505 0205 ind_46, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0405 0505 ind_47, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0101 0101 0202 0202 0404 0202 ind_48, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0101 0101 0202 0202 0405 0505 ind_49, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0205 ind_50, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0202 0101 0202 0202 0405 0204 BI ind_1, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_2, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0203 0202 0202 0101 0202 0202 0404 0204 ind_3, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0202 0101 0202 0202 0404 0202 ind_4, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0102 0101 0202 0202 0404 0202 ind_5, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0202 ind_6, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0203 0202 0102 0101 0202 0202 0404 0204 ind_7, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_8, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_9, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0202 0202 0102 0101 0202 0202 0104 0204

PAGE 92

78 ind_10, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0202 ind_11, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0102 0101 0202 0202 0104 0202 ind_12, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0203 0202 0202 0101 0202 0202 0104 0205 ind_13, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0203 0202 0202 0101 0202 0202 0404 0202 ind_14, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0202 0101 0202 0202 0404 0204 ind_15, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0101 0202 ind_16, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_17, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0203 0202 0102 0101 0202 0202 0404 0204 ind_18, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0102 0101 0202 0202 0104 0204 ind_19, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_20, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0102 0101 0202 0202 0404 0204 ind_21, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0203 0202 0202 0101 0202 0202 0404 0202 ind_22, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0203 0202 0102 0101 0202 0202 0101 0202 ind_23, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0104 0202 ind_24, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0203 0202 0102 0101 0202 0202 0404 0205 ind_25, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_26, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0104 0202 ind_27, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0204

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79 ind_28, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0202 0101 0202 0202 0101 0202 ind_29, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0202 0101 0202 0202 0404 0204 ind_30, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_31, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0102 0101 0202 0202 0104 0204 ind_32, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0101 0101 0202 0202 0404 0202 ind_33, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_34, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0101 0202 ind_35, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_36, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_37, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_38, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_39, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_40, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0104 0404 ind_41, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_42, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_43, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_44, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0202 ind_45, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0202

PAGE 94

80 ind_46, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_47, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_48, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_49, 0202 0000 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0104 0202 ind_50, 0202 0101 0202 0101 0202 0101 0000 0000 0101 0202 0101 0303 0202 0202 0101 0202 0202 0104 0202 MaxP ind_1, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0102 0101 0202 0202 0404 0303 ind_2, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0103 ind_3, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0101 ind_4, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0103 0303 0202 0202 0101 0202 0202 0404 0101 ind_5, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0202 0101 0202 0202 0404 0103 ind_6, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0202 0101 0202 0202 0404 0101 ind_7, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0101 0101 0202 0202 0404 0303 ind_8, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0303 ind_9, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0103 ind_10, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0103 ind_11, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0204 0303 ind_12, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0202 0202 0102 0101 0202 0202 0202 0102 ind_13, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0202 0202

PAGE 95

81 ind_14, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0203 0202 0102 0101 0202 0202 0404 0101 ind_15, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0103 ind_16, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0103 0303 0202 0101 0101 0202 0202 0404 0103 ind_17, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0303 0303 0202 0102 0101 0202 0202 0404 0102 ind_18, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0102 ind_19, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0303 0303 0202 0202 0101 0202 0202 0404 0202 ind_20, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0303 ind_21, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0202 0101 0202 0202 0404 0102 ind_22, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0103 ind_23, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0103 0303 0202 0101 0101 0202 0202 0404 0303 ind_24, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0103 ind_25, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0202 0101 0202 0202 0404 0103 ind_26, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0304 ind_27, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0103 0203 0202 0102 0101 0202 0202 0204 0303 ind_28, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0202 0101 0202 0202 0204 0404 ind_29, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0203 0202 0101 0101 0202 0202 0404 0404 ind_30, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0202 0101 0202 0202 0404 0202 ind_31, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0202 0101 0202 0202 0404 0203

PAGE 96

82 ind_32, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0203 0202 0202 0101 0202 0202 0204 0202 ind_33, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0103 0203 0202 0102 0101 0202 0202 0404 0303 ind_34, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0202 0101 0202 0202 0404 0202 ind_35, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0102 0101 0202 0202 0202 0103 ind_36, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0203 0202 0102 0101 0202 0202 0404 0202 ind_37, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0203 0202 0102 0101 0202 0202 0404 0103 ind_38, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0203 0202 0202 0101 0202 0202 0404 0103 ind_39, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0203 0202 0202 0101 0202 0202 0204 0404 ind_40, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0203 0202 0101 0101 0202 0202 0204 0101 ind_41, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0102 0101 0202 0202 0204 0104 ind_42, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_43, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_44, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0203 0202 0202 0101 0202 0202 0204 0103 ind_45, 0202 0101 0202 0101 0202 0101 0101 0103 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0104 ind_46, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_47, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0202 0204 ind_48, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0203 0202 0102 0101 0202 0202 0404 0304 ind_49, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0303

PAGE 97

83 ind_50, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0202 0303 MurP ind_1, 0202 0101 0202 0101 0000 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_2, 0202 0101 0202 0101 0000 0101 0101 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0202 ind_3, 0202 0101 0202 0101 0000 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0101 0204 ind_4, 0202 0101 0202 0101 0000 0101 0101 0202 0101 0202 0102 0303 0202 0202 0101 0202 0202 0404 0204 ind_5, 0202 0101 0202 0101 0000 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_6, 0202 0101 0202 0101 0000 0101 0101 0102 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0202 ind_7, 0202 0101 0202 0101 0000 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_8, 0202 0101 0202 0101 0000 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_9, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_10, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0204 ind_11, 0202 0101 0202 0101 0000 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_12, 0202 0101 0202 0101 0000 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_13, 0202 0101 0202 0101 0000 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_14, 0202 0101 0202 0101 0000 0101 0101 0102 0101 0202 0102 0303 0202 0202 0101 0202 0202 0404 0202 ind_15, 0000 0000 0202 0101 0000 0101 0101 0101 0101 0202 0101 0303 0202 0000 0101 0202 0202 0101 0404 ind_16, 0202 0101 0202 0101 0000 0101 0101 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0204 ind_17, 0202 0101 0202 0101 0000 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202

PAGE 98

84 ind_18, 0202 0101 0202 0101 0000 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_19, 0202 0101 0202 0101 0000 0101 0101 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0202 ind_20, 0202 0101 0202 0101 0000 0101 0101 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0204 ind_21, 0202 0101 0202 0101 0000 0101 0101 0102 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0202 ind_22, 0202 0101 0202 0101 0000 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_23, 0202 0101 0202 0101 0000 0101 0101 0102 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_24, 0202 0101 0202 0101 0000 0101 0101 0102 0101 0202 0103 0303 0202 0102 0101 0202 0202 0104 0202 ind_25, 0202 0101 0202 0101 0000 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 PI ind_1, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0102 ind_2, 0000 0000 0000 0000 0000 0000 0101 0102 0101 0000 0101 0000 0000 0000 0101 0000 0000 0000 0000 ind_3, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0203 0202 0405 0202 ind_4, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_5, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0203 0202 0405 0204 ind_6, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0203 0202 0404 0204 ind_7, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0203 0202 0404 0404 ind_8, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0303 0202 0405 0202 ind_9, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0203 0202 0404 0404 ind_10, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0203 0202 0404 0202

PAGE 99

85 ind_11, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_12, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0000 0202 0101 0202 0202 0404 0202 ind_13, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_14, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0203 0202 0404 0202 ind_15, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0203 0202 0404 0204 ind_16, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0203 0202 0405 0202 ind_17, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_18, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0405 0204 ind_19, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0203 0202 0404 0202 ind_20, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0102 0202 0101 0202 0202 0404 0404 ind_21, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0203 0202 0505 0404 ind_22, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0203 0202 0404 0202 ind_23, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0203 0202 0404 0202 ind_24, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0102 0202 0101 0203 0202 0404 0202 ind_25, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0203 0202 0405 0202 ind_26, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0102 0202 0101 0202 0202 0404 0404 ind_27, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_28, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0303 0202 0404 0202

PAGE 100

86 ind_29, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_30, 0000 0000 0000 0000 0202 0000 0101 0101 0101 0202 0101 0000 0000 0202 0101 0000 0000 0000 0000 ind_31, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0102 0202 0101 0202 0202 0404 0202 ind_32, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0203 0202 0404 0202 ind_33, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_34, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0405 0404 ind_35, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0101 0202 0101 0303 0202 0405 0204 ind_36, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0303 0202 0404 0204 ind_37, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0405 0202 ind_38, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0101 0202 0101 0202 0202 0404 0204 ind_39, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_40, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0405 0304 ind_41, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0203 0202 0404 0204 ind_42, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0102 0202 0101 0203 0202 0404 0204 ind_43, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0405 0202 ind_44, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_45, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0203 0202 0404 0104 ind_46, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0203 0202 0404 0204

PAGE 101

87 ind_47, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0102 0202 0101 0203 0202 0405 0204 ind_48, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0102 0101 0203 0202 0405 0204 ind_49, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0203 0202 0505 0204 ind_50, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0203 0202 0404 0204 AV ind_1, 0202 0101 0202 0102 0202 0101 0000 0101 0101 0303 0303 0303 0202 0202 0101 0202 0202 0404 0204 ind_2, 0202 0101 0202 0102 0202 0101 0000 0101 0101 0303 0303 0303 0202 0202 0101 0202 0202 0404 0204 ind_3, 0202 0102 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0405 ind_4, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0103 0303 0202 0102 0101 0202 0202 0404 0204 ind_5, 0202 0102 0202 0101 0202 0102 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0205 ind_6, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0204 ind_7, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0103 0303 0202 0202 0101 0202 0202 0404 0404 ind_8, 0202 0101 0000 0000 0202 0000 0000 0101 0101 0000 0000 0303 0202 0000 0101 0202 0000 0000 0000 ind_9, 0202 0202 0202 0101 0202 0101 0000 0101 0101 0202 0103 0303 0202 0202 0101 0202 0202 0404 0404 ind_10, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_11, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0103 0303 0202 0202 0101 0202 0202 0404 0404 ind_12, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_13, 0202 0101 0203 0101 0202 0102 0000 0101 0101 0202 0101 0203 0202 0202 0101 0202 0202 0404 0405 ind_14, 0202 0101 0202 0102 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404

PAGE 102

88 ind_15, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0204 ind_16, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0405 ind_17, 0202 0101 0202 0101 0202 0102 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0405 ind_18, 0202 0101 0202 0102 0202 0101 0000 0101 0101 0203 0103 0303 0202 0202 0101 0202 0202 0204 0202 ind_19, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0303 0303 0202 0102 0101 0202 0202 0404 0204 ind_20, 0202 0102 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0405 ind_21, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0103 0303 0202 0202 0101 0202 0202 0404 0404 ind_22, 0202 0101 0202 0101 0202 0202 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0405 ind_23, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0405 ind_24, 0202 0101 0202 0101 0202 0102 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0405 ind_25, 0202 0101 0202 0102 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_26, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0203 0101 0303 0202 0102 0101 0202 0202 0404 0405 ind_27, 0202 0101 0202 0101 0102 0101 0000 0101 0101 0202 0103 0303 0202 0102 0101 0202 0202 0404 0204 ind_28, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0203 0103 0303 0202 0102 0101 0202 0202 0404 0404 ind_29, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0203 0101 0303 0202 0102 0101 0202 0202 0404 0204 ind_30, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0204 CC ind_1, 0202 0102 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0405 0404 ind_2, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404

PAGE 103

89 ind_3, 0202 0102 0202 0102 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_4, 0202 0101 0203 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_5, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0102 0202 0405 0204 ind_6, 0202 0102 0202 0101 0202 0102 0000 0101 0101 0202 0101 0303 0202 0101 0101 0202 0202 0204 0404 ind_7, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0405 0404 ind_8, 0202 0000 0203 0101 0202 0101 0000 0101 0101 0303 0101 0303 0202 0202 0101 0102 0202 0404 0204 ind_9, 0202 0102 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_10, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0303 0101 0203 0202 0101 0101 0202 0202 0404 0204 ind_11, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0203 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_12, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_13, 0202 0102 0202 0101 0202 0101 0000 0101 0101 0303 0101 0203 0202 0101 0101 0202 0202 0404 0404 ind_14, 0202 0102 0202 0101 0202 0101 0000 0101 0101 0203 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_15, 0202 0102 0203 0101 0202 0101 0000 0101 0101 0202 0101 0203 0202 0202 0101 0202 0202 0104 0404 ind_16, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0203 0202 0404 0404 ind_17, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0505 0405 ind_18, 0202 0101 0203 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_19, 0202 0101 0203 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0505 0404 ind_20, 0202 0101 0203 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404

PAGE 104

90 ind_21, 0202 0000 0203 0101 0202 0102 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_22, 0202 0102 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_23, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0203 0101 0303 0202 0202 0101 0102 0202 0404 0404 ind_24, 0202 0101 0203 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_25, 0202 0101 0203 0101 0202 0101 0000 0101 0101 0303 0101 0203 0202 0102 0101 0202 0202 0404 0404 ind_26, 0202 0102 0202 0101 0102 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_27, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0303 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_28, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_29, 0202 0101 0203 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_30, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_31, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_32, 0202 0101 0202 0101 0102 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0204 0404 ind_33, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0203 0202 0202 0101 0202 0202 0202 0404 ind_34, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_35, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0505 0404 ind_36, 0202 0202 0202 0101 0202 0101 0000 0101 0101 0202 0101 0203 0202 0102 0101 0202 0202 0404 0404 ind_37, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0203 0202 0202 0101 0202 0202 0505 0404 ind_38, 0202 0101 0202 0102 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404

PAGE 105

91 ind_39, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0505 0404 ind_40, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0405 ind_41, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0505 0404 ind_42, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0203 0101 0202 0202 0404 0404 ind_43, 0202 0202 0202 0101 0202 0101 0000 0101 0101 0202 0103 0303 0202 0102 0101 0202 0202 0404 0404 ind_44, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0203 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_45, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_46, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_47, 0202 0101 0203 0101 0202 0101 0000 0101 0101 0202 0101 0203 0202 0203 0101 0202 0202 0404 0404 ind_48, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_49, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0203 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_50, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0303 0101 0303 0202 0202 0101 0202 0202 0404 0404 FR ind_1, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_2, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_3, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0203 0202 0102 0101 0202 0202 0404 0404 ind_4, 0202 0102 0203 0101 0102 0101 0000 0101 0101 0202 0103 0303 0202 0102 0101 0202 0202 0404 0404 ind_5, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_6, 0202 0102 0202 0101 0202 0101 0000 0101 0101 0203 0101 0303 0202 0202 0101 0202 0202 0404 0404

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92 ind_7, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0103 0303 0202 0202 0101 0202 0202 0404 0404 ind_8, 0202 0101 0202 0102 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_9, 0202 0102 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_10, 0202 0101 0203 0101 0102 0101 0000 0101 0101 0202 0103 0303 0202 0101 0101 0202 0202 0404 0204 ind_11, 0202 0202 0203 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_12, 0202 0101 0202 0101 0102 0101 0000 0101 0101 0202 0103 0303 0202 0102 0101 0202 0202 0404 0404 ind_13, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0203 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_14, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0303 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_15, 0202 0101 0203 0202 0202 0101 0000 0101 0101 0203 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_16, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0203 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_17, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0303 0101 0303 0202 0101 0101 0202 0202 0404 0404 ind_18, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0205 ind_19, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_20, 0202 0202 0202 0102 0202 0102 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_21, 0202 0101 0203 0101 0202 0102 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_22, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_23, 0202 0101 0202 0102 0202 0101 0000 0101 0101 0203 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_24, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0203 0101 0303 0202 0102 0101 0202 0202 0404 0404

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93 ind_25, 0202 0102 0202 0101 0202 0101 0000 0101 0101 0202 0103 0303 0202 0202 0101 0202 0202 0404 0404 ind_26, 0202 0102 0202 0101 0102 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0205 ind_27, 0202 0101 0203 0101 0202 0101 0000 0101 0101 0202 0103 0203 0202 0202 0101 0202 0202 0204 0404 ind_28, 0202 0102 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0405 ind_29, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_30, 0202 0101 0203 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_31, 0202 0202 0203 0102 0202 0101 0000 0101 0101 0202 0103 0303 0202 0102 0101 0202 0202 0204 0204 ind_32, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_33, 0202 0101 0202 0101 0102 0101 0000 0101 0101 0202 0101 0303 0202 0101 0101 0202 0202 0404 0205 ind_34, 0202 0101 0203 0101 0202 0101 0000 0101 0101 0202 0101 0203 0202 0202 0101 0202 0202 0204 0404 ind_35, 0202 0101 0203 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_36, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0204 ind_37, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0204 ind_38, 0202 0101 0203 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0101 0101 0202 0202 0404 0405 ind_39, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0203 0202 0102 0101 0202 0202 0404 0205 ind_40, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_41, 0202 0101 0203 0101 0102 0101 0000 0101 0101 0202 0103 0303 0202 0202 0101 0202 0202 0404 0204 ind_42, 0202 0101 0203 0101 0202 0101 0000 0101 0101 0203 0101 0303 0202 0202 0101 0202 0202 0404 0204

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94 ind_43, 0202 0102 0202 0101 0202 0101 0000 0101 0101 0202 0102 0303 0202 0202 0101 0202 0202 0404 0404 ind_44, 0202 0101 0203 0102 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_45, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_46, 0202 0101 0203 0101 0202 0101 0000 0101 0101 0202 0101 0203 0202 0102 0101 0202 0202 0404 0204 ind_47, 0202 0202 0203 0101 0202 0101 0000 0101 0101 0202 0101 0203 0202 0101 0101 0202 0202 0404 0404 ind_48, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_49, 0202 0101 0203 0101 0202 0101 0000 0101 0101 0202 0101 0203 0202 0102 0101 0202 0202 0404 0205 ind_50, 0202 0101 0202 0101 0202 0101 0000 0101 0101 0203 0101 0203 0202 0102 0101 0202 0202 0404 0205 HB ind_1, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_2, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_3, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_4, 0102 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_5, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_6, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_7, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_8, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0203 0202 0404 0404 ind_9, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_10, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0203 0202 0202 0101 0202 0202 0404 0404

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95 ind_11, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_12, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_13, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0203 0202 0404 0404 ind_14, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_15, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_16, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_17, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0202 0202 0202 0101 0202 0202 0404 0404 ind_18, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0203 0202 0202 0101 0202 0202 0404 0404 ind_19, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_20, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_21, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_22, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0203 0202 0404 0204 ind_23, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_24, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_25, 0202 0101 0202 0101 0202 0000 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ESC ind_1, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_2, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_3, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404

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96 ind_4, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_5, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0203 0101 0202 0202 0404 0404 ind_6, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0203 0101 0202 0202 0404 0404 ind_7, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0101 0101 0202 0202 0304 0404 ind_8, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_9, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_10, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0203 0101 0202 0202 0304 0404 ind_11, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_12, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_13, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_14, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_15, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_16, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_17, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_18, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0203 0101 0202 0202 0404 0404 ind_19, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_20, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_21, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404

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97 ind_22, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0304 0404 ind_23, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_24, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_25, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_26, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_27, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_28, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_29, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_30, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_31, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_32, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_33, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_34, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_35, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0102 0101 0202 0202 0304 0404 ind_36, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0202 0101 0202 0202 0304 0404 BLR_C ind_1, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0204 ind_2, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_3, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0101 0101 0202 0202 0404 0204

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98 ind_4, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0202 ind_5, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0202 0101 0202 0202 0404 0404 ind_6, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_13, 0102 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0204 ind_14, 0102 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0103 0101 0202 0202 0404 0404 ind_17, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0101 0101 0202 0202 0404 0204 ind_18, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_12, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0203 0202 0303 0101 0202 0202 0404 0404 ind_15, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0103 0101 0202 0202 0404 0204 BB ind_1, 0102 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0101 0101 0202 0202 0404 0404 ind_2, 0202 0101 0202 0101 0202 0101 0101 0102 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_3, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0101 0101 0202 0202 0404 0404 ind_4, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_5, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_6, 0202 0101 0202 0101 0202 0101 0101 0101 0101 0202 0101 0303 0202 0202 0101 0202 0202 0404 0404 ind_7, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404 ind_8, 0202 0101 0202 0101 0202 0101 0101 0202 0101 0202 0101 0303 0202 0102 0101 0202 0202 0404 0404

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99 Appendix F Allele frequencies per population per locus generated from the program FSTAT ver. 2.9.3.2 (Goudet 2002) and modified in Microsoft Excel for a C. scirpoidea subsp. convoluta (Kkenthal) Dunlop and b C. scirpoidea subsp. scirpoidea Michaux (Cyperaceae). a. Allele frequencies per locus per population for C. scirpoidea subsp. convoluta BSC BI MaxP MurpP PI HSB BRL All_W All_UW Locus: DIA 1 N 50 50 50 24 48 25 12 p: 1 0 0 0 0 0 0.02 0.083 0.006 0.015 p: 2 1 1 1 1 1 0.98 0.917 0.994 0.985 Locus: ME N 50 49 50 24 48 25 12 p: 1 1 1 1 1 1 1 1 1 1 p: 2 0 0 0 0 0 0 0 0 0 Locus: TPI 1 N 50 50 50 25 48 25 12 p: 1 0 0 0 0 0 0 0 0 0 p: 2 1 1 1 1 1 1 1 1 1 p: 3 0 0 0 0 0 0 0 0 0 Locus: TPI 2 N 50 50 50 25 48 25 12 p: 1 1 1 1 1 1 1 1 1 1 p: 2 0 0 0 0 0 0 0 0 0 Locus: ADH N 50 50 50 2 49 25 12 p: 1 0 0 0 0 0 0 0 0 0 p: 2 1 1 1 1 1 1 1 1 1 Locus: SOD N 50 50 50 25 48 24 12 p: 1 1 1 1 1 1 1 1 1 1 p: 2 0 0 0 0 0 0 0 0 0 Locus: AAT 1 N 50 49 50 25 50 25 12 p: 1 1 1 1 1 1 1 1 1 1 Locus: AAT 2 N 50 49 50 25 50 25 12 p: 1 0.39 0.327 0.62 0.72 0.73 0.12 0.667 0.506 0.51 p: 2 0.61 0.673 0.37 0.28 0.27 0.88 0.333 0.492 0.488 p: 3 0 0 0.01 0 0 0 0 0.002 0.001 Locus: G3PDH N 50 50 50 25 50 25 12

PAGE 114

100 p: 1 1 1 1 1 1 1 1 1 1 Locus: SDH N 50 50 50 25 49 25 12 p: 1 0 0 0 0 0 0 0 0 0 p: 2 1 1 1 1 1 1 1 1 1 Locus: PGD N 50 50 50 25 50 25 12 p: 1 1 1 0.91 0.94 1 1 1 0.977 0.979 p: 2 0 0 0 0.04 0 0 0 0.004 0.006 p: 3 0 0 0.09 0.02 0 0 0 0.019 0.016 Locus: PGM 1 N 50 50 50 25 48 25 12 p: 1 0 0 0 0 0 0 0 0 0 p: 2 0.01 0.17 0.25 0 0 0.08 0.125 0.096 0.091 p: 3 0.99 0.83 0.75 1 1 0.92 0.875 0.904 0.909 Locus: PGM 2 N 50 50 50 25 47 25 12 p: 1 0 0 0 0 0.106 0 0 0.019 0.015 p: 2 1 1 1 1 0.894 1 1 0.981 0.985 p: 3 0 0 0 0 0 0 0 0 0 Locus: IDH N 50 50 50 24 49 25 12 p: 1 0.26 0.17 0.32 0.188 0.01 0.06 0.333 0.185 0.192 p: 2 0.61 0.83 0.68 0.813 0.99 0.94 0.5 0.783 0.766 p: 3 0.13 0 0 0 0 0 0.167 0.033 0.042 Locus: MDH 2 N 50 50 50 25 50 25 12 p: 1 1 1 1 1 1 1 1 1 1 Locus: MDH 3 N 50 50 50 25 48 25 12 p: 1 0 0 0 0 0 0 0 0 0 p: 2 1 1 1 1 0.667 0.94 1 0.933 0.944 p: 3 0 0 0 0 0.333 0.06 0 0.067 0.056 Locus: MNR 1 N 50 50 50 25 48 25 12 p: 1 0 0 0 0 0 0 0 0 0 p: 2 1 1 1 1 1 1 1 1 1 Locus: MNR 2 N 50 50 50 25 48 25 12 p: 1 0 0.18 0 0.1 0 0 0 0.044 0.04 p: 2 0 0 0.18 0 0 0 0 0.035 0.026 p: 3 0 0 0 0 0 0 0 0 0 p: 4 0.77 0.82 0.82 0.9 0.823 1 1 0.844 0.876

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101 p: 5 0.23 0 0 0 0.177 0 0 0.077 0.058 Locus: PGI N 50 50 50 25 48 25 12 p: 1 0 0 0.29 0 0.021 0 0 0.06 0.044 p: 2 0.48 0.81 0.22 0.72 0.604 0.04 0.375 0.492 0.464 p: 3 0.06 0 0.36 0 0.01 0 0 0.083 0.061 p: 4 0.2 0.17 0.13 0.28 0.365 0.96 0.625 0.312 0.39 p: 5 0.26 0.02 0 0 0 0 0 0.054 0.04 b Allele frequencies per locus per population for C. scirpoidea subsp. scirpoidea HCFS HCFN BCF GPC HSM AV CC FR ESC BLR BB All_W All_UW Locus: DIA 1 N 48 44 44 25 50 30 50 50 36 4 8 p: 1 0 0 0 0 0 0 0 0 0 0.125 0.063 0.003 0.017 p: 2 1 1 1 1 1 1 1 1 1 0.875 0.938 0.997 0.983 Locus: ME N 47 47 46 49 50 30 48 50 36 4 8 p: 1 1 1 1 1 1 0.917 0.865 0.85 1 1 1 0.96 0.966 p: 2 0 0 0 0 0 0.083 0.135 0.15 0 0 0 0.04 0.034 Locus: TPI 1 N 50 44 49 50 50 29 50 50 36 4 8 p: 1 0.03 0.011 0 0 0 0 0 0 0 0 0 0.005 0.004 p: 2 0.97 0.989 1 1 1 0.983 0.89 0.83 1 1 1 0.961 0.969 p: 3 0 0 0 0 0 0.017 0.11 0.17 0 0 0 0.035 0.027 Locus: TPI 2 N 50 44 49 50 50 29 50 50 36 4 8 p: 1 1 0.966 1 1 1 0.914 0.98 0.93 1 1 1 0.98 0.981 p: 2 0 0.034 0 0 0 0.086 0.02 0.07 0 0 0 0.02 0.019 Locus: ADH N 47 48 49 50 48 30 50 50 36 4 8 p: 1 0 0 0 0 0 0.017 0.02 0.06 0 0 0 0.011 0.009 p: 2 1 1 1 1 1 0.983 0.98 0.94 1 1 1 0.989 0.991 Locus: SOD N 23 44 0 50 50 29 50 50 36 4 8 p: 1 1 1 NA 1 1 0.897 0.98 0.98 1 1 1 0.985 0.986 p: 2 0 0 NA 0 0 0.103 0.02 0.02 0 0 0 0.015 0.014 Locus: AAT 1 N 0 23 0 0 0 0 0 0 36 4 8 p: 1 NA 1 NA NA NA NA NA NA 1 1 1 1 1 Locus: AAT 2 N 25 23 46 0 0 30 50 50 36 4 8 p: 1 1 1 0.174 NA NA 1 1 1 0.069 0.625 0.563 0.719 0.715

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102 p: 2 0 0 0.826 NA NA 0 0 0 0.931 0.375 0.438 0.281 0.285 p: 3 0 0 0 0 0 0 0 0 0 0 0 0 0 Locus: G3PDH N 25 50 50 50 49 30 50 50 36 4 8 p: 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Locus: SDH N 46 48 50 46 50 29 50 50 36 4 8 p: 1 0.109 1 1 0.217 0 0 0 0 0 0 0 0.271 0.211 p: 2 0.891 0 0 0.783 0.5 0.862 0.83 0.88 1 1 1 0.625 0.704 p: 3 0 0 0 0 0.5 0.138 0.17 0.12 0 0 0 0.104 0.084 Locus: PGD N 49 48 50 48 50 29 50 50 36 4 8 p: 1 1 1 1 1 1 0.759 0.99 0.91 1 1 1 0.972 0.969 p: 2 0 0 0 0 0 0 0 0.01 0 0 0 0.001 0.001 p: 3 0 0 0 0 0 0.241 0.01 0.08 0 0 0 0.027 0.03 Locus: PGM 1 N 45 47 50 25 24 30 50 50 36 4 8 p: 1 0.689 1 1 0.06 0.292 0 0 0 0 0 0 0.37 0.276 p: 2 0.289 0 0 0.94 0.708 0.017 0.08 0.08 0 0.125 0 0.169 0.204 p: 3 0.022 0 0 0 0 0.983 0.92 0.92 1 0.875 1 0.461 0.52 Locus: PGM 2 N 49 50 50 49 50 30 50 50 36 4 8 p: 1 0 0 0 0 0 0 0 0 0 0 0 0 0 p: 2 0.929 0.99 0.94 0.929 0.99 1 1 1 1 1 1 0.974 0.98 p: 3 0.071 0.01 0.06 0.071 0.01 0 0 0 0 0 0 0.026 0.02 Locus: IDH N 0 0 0 0 0 29 50 50 36 4 8 p: 1 NA NA NA NA NA 0.276 0.21 0.28 0.111 0.625 0.438 0.24 0.323 p: 2 NA NA NA NA NA 0.724 0.77 0.72 0.833 0.125 0.563 0.737 0.622 p: 3 NA NA NA NA NA 0 0.02 0 0.056 0.25 0 0.023 0.054 Locus: MDH 2 N 47 50 50 50 50 30 50 50 36 4 8 p: 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Locus: MDH 3 N 47 49 49 36 50 30 50 50 36 4 8 p: 1 0.011 0.041 0 0.194 0 0 0.03 0 0 0 0 0.027 0.025 p: 2 0.989 0.959 1 0.806 1 1 0.96 1 1 1 1 0.972 0.974 p: 3 0 0 0 0 0 0 0.01 0 0 0 0 0.001 0.001 Locus: MNR 1 N 50 48 0 50 47 29 50 50 36 4 8 p: 1 0.09 0.177 NA 0 0.319 0 0 0 0 0 0 0.075 0.059 p: 2 0.91 0.823 NA 1 0.681 1 1 1 1 1 1 0.925 0.941 Locus: MNR 2

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103 N 50 48 50 50 50 29 50 50 36 4 8 p: 1 0 0 0 0 0 0 0.01 0 0 0 0 0.001 0.001 p: 2 0 0 0 0 0 0.017 0.04 0.03 0 0 0 0.009 0.008 p: 3 0 0 0 0 0 0 0 0 0.069 0 0 0.006 0.006 p: 4 1 1 1 1 1 0.983 0.8 0.97 0.931 1 1 0.966 0.971 p: 5 0 0 0 0 0 0 0.15 0 0 0 0 0.018 0.014 Locus: PGI N 48 49 49 49 50 29 50 50 36 4 8 p: 1 0 0 0 0 0 0 0 0 0 0 0 0 0 p: 2 0 0 0 0 0 0.207 0.04 0.16 0 0.5 0 0.043 0.082 p: 3 0.177 0.122 0.112 0.173 0 0 0 0 0 0 0 0.068 0.053 p: 4 0.75 0.735 0.806 0.796 0.61 0.621 0.94 0.76 1 0.5 1 0.782 0.774 p: 5 0.073 0.143 0.082 0.031 0.39 0.172 0.02 0.08 0 0 0 0.108 0.09