S Brunsfeld* Steven 583.99 J Nllpga Preliminary 1994 eenetic analysis of C i rsiun loneistyluni {Lone- styled thisti ) 1 ^ ' ' 583.99 MONTANA STATE LIP mil II III n nil III! - ' Nllpga II' _ ^ ^^^^ 3 0864 0014 0441' b STATS B8CUMCTTS COLLECTION HELENA. MONTANA 59620 PRELIMINARY GENETIC ANALYSIS OF CIRSIUM LONGISTYLUM (Long-styled thistle), A CANDIDATE THREATENED SPECIES Prepared by: Steven J. Brunsfeld and Calib T. Baldwin Wildland Plant Ecogenetics Cooperative University of Idaho in cooperation with: Montana Natural Heritage Program State Library 1515 E. 5th Ave. Helena, MT 59620 A Section 6 study prepared for: U.S. Fish and Wildlife Service - Region 4 P.O. Box 25486, Denver Federal Center Denver, CO 8 0225 S3 . ' ^m i p ';• *•; !," May 1994 DATE DUE r^ ■ •-. - -. ' nnR DEMCO 38-301 I ® 1994 Montana Natural Heritage Program This report should be cited as follows: Brunsfeld, S. J. and C. T. Baldwin. 1994. Preliminary genetic alalysis of Cirsium longistvlum (Long-styled thistle) , a candidate threatened species. Unpublished report to the U.S. Fish and Wildlife Service. Wildland Plant Ecogenetics Cooperative, University of Idaho, in cooperation with Montana Natural Heritage Program, Helena. 20 pp. plus appendices. EXECUTIVE SUMMARY A genetic analysis of Cirsium longistylum (Long-styled thistle) , a candidate threatened plant species, was initiated in 1993 to investigate hypothesized hybridization with C^ hookerianum. The objective of the analysis was to determine if C^ longistylum is a genetically unique entity, and to assess the importance of introgression in the evolution of the species. Enzyme electrophoresis was performed on 150 individuals from 12 populations of westcentral Montana. Cirsium longistylum was found to be genetically distinct from Montana populations of C^ hookerianum and C^ scariosum. Putative hybrids of C^ longistylum were found to be most similar to "pure" C^ longistylum plants, a finding consistent with either a hypothesis of hybridity or high natural variability within C^ longistylum. A preliminary analysis of randomly amplified polymorphic DNA (RAPD) was initiated to gain better resolution of genetic relationships among C^ longistylum, putative hybrids, and closely related Cirsium species. Twentyone samples representing C^ longistylum, C. hookerianum, C. scariosum, and putative hybrids were analyzed, including four samples of C^ hookerianum and C^. scariosum from outside the study area in westcentral Montana. The RAPD data was congruent with electrophoretic data in identifying Cirsium longistylum as genetically distinct. Cirsium hookerianum and C^ scariosum sampled outside the region were found to be genetically distant from samples of the same species in westcentral Montana. These data suggest that introgression historically may have occurred among all three species of the region. In order to document introgression using RAPD markers, a thorough understanding of the genetic composition of "pure" C^ hookerianum and C^ scariosum from outside the region is required. In addition, phylogenetic and additional morphological analyses are proposed as a basis for future conservation and management decisions. TABLE OF CONTENTS Introduction ..... 1 Methods ...... 2 Results ...... 5 Discussion ..... 16 Literature Cited .... 19 Appendix I. Allele frequencies and genetic variability measures by population. Appendix II. Allele frequencies and genetic variability measures by species. Appendix III. Summary of Principal Component Analysis. TABLES AND FIGURES Table 1. Sample Sites. Table 2. Key to electrophoretic by population. Table 3. Allele frequencies by locus and population. Table 4. Genetic variability by populations. Table 5. Nei (1978) unbiased genetic identity by population. Table 6. Key to electrophoretic analysis by species. Table 7. Allele frequency by locus and species. Table 8. Genetic variability by species. Table 9. Nei (1978) unbiased genetic identity by species. Figure 1. UPGMA cluster analysis of electrophoretic data by population, using Nei (1978) unbiased genetic identity. Figure 2. UPGMA cluster analysis of electrophoretic data by species, using Nei (1978) unbiased genetic identity. Figure 3. PCA of Cirsium RAPD data. Figure 4. PCA of Cirsium RAPD data. INTRODUCTION Cirsium lonaistvlum Moore & Frankton (Long-styled thistle) is a species endemic to four "island" mountain ranges of westcentral Montana, currently placed in Category 2 by the U.S. Fish and Wildlife Service as a candidate for federal listing as threatened (58 FR51144). Cirsium lonqistvlum is one of six closely related species of thistle distributed in the Rocky Mountains. Cirsium hookerianum Nutt. is primarily distributed in British Columbia and Alberta, but extends southward into westcentral and western Montana where it occurs sympatrically with C^ lonqistylum in the Big Belt and Little Belt Mountain Ranges. Two additional species, Cirsium scariosum Nutt. and C. tweedyi (Rydb.) Petrak, approach the range of C^ lonqistvlum from the southwest. Cirsium scopulorum (Greene) Cockerell and C. eatonii (A. Gray) Robinson (Moore and Frankton 1965) occur south of Montana in the southern Rocky Mountains. Survey and monitoring work has been conducted to characterize distribution and life history of Cirsium lonqistvlum (Schassberger 1991, Schassberger and Achuff 1991, Roe 1992, Poole and Heidel 1993, Heidel 1994). At many sites, plants with bract characteristics deviating from the taxonomic circumscription of Moore and Frankton (1963) were noted. One of the three monitoring sites (Neihart) was interpreted as consisting primarily of Cirsium hookerianum. possibly with C. hookerianum x C. lonqistvlum hybrids present (Cronquist pers. commun. as cited in Roe 1992) . Monitoring work of the following year included an expanded morphological investigation (Poole and Heidel 1993). This investigation revealed that some populations contained plants with the diagnostic bract characteristics of both C. lonqistvlum and C^ hookerianum. Based on morphology it was concluded that Cirsium lonqistylum appeared to be a distinct species that hybridizes freely with C. hookerianum, producing swarms of morphologically variable individuals. This provisional interpretation warranted genetic documentation, and the present study was initiated to test the discreetness and persistence of a recognizable Cirsium lonqistylum genome. METHODS Sampling Procedures Leaf samples of Cirsium longistvlum were taken from seven study sites representing major population centers and a full range of morphological variation (Table 1) . Collections were made of green leaf material on flowering stems, using the freshest available intact leaves, usually upper stem leaves. A minimum of two leaves were collected, sealed between damp paper towels in zip-loc plastic bags, labelled with an unique sequential number, stored on ice, and mailed in overnight mail to the University of Idaho. The bract category was recorded for each sampled individual as delimited in Poole and Heidel (1993). At all sites of Cirsium lonqistylum, collections were made within a circular plot of 10 m radius, except for Russian Creek, with a 20 m radius. The sample areas corresponded to circular demographic monitoring plots at four of the seven sample sites (Heidel 1994). Sample size was a minimum of 20 plants per site, and included all flowering plants in the plot. Actual sample size ranged from 24 (Kings Hill #2-USFS) to 38 (S. Fk. Deadman Cr.) per site, though in most cases fewer than 15 plants were used per set for preliminary genetic analysis. Six of the seven sample sets from the Little and Big Belt Mountain ranges represent discrete population at least 2 airmiles (3.2 km) apart. The Kings Hill pair of samples were collected from two adjoining segments of the population in monitoring plots from contrasting habitats. Leaf size and condition varied between sites and within sites. The degree of inflorescence branching varied widely between plants, and upper stem leaf size was significantly smaller on the more highly-branched plants. Many stem leaves remained green through August in the exceptionally cool and moist season of 1993, but herbivory and rapid decay in storage markedly reduced usable leaf material. Leaf samples of Cirsium hookerianum were collected at two sites over 30 miles (48 km) beyond the known range of Cirsium lonqistylum at Flesher Pass (Schassberger #464 iCronquist) and Lewis and Clark Pass in the Blackfoot Range. Leaf samples of Cirsium scariosum were collected at one site over 50 miles (80 km) beyond the known range of Cirsium lonqistylum at Warren Pass in the Anaconda Range. These samples were collected in the first week of September when only basal rosette material retained living tissue. DNA from herbarium specimens of Cirsium hookerianum from Pondera Co., MT (Hitchcock #18177) and Williams Lake, British Columbia (Calden #17953) and of C. scariosum from Nye Co., Nevada (0 0) 4J •H a) 6 ^1 M X o j: 0) 4J XI (0 u U ti Ml 03 C o •H 4J D •H u o CO 0) m B Q) 1^ o o w c ■p w 0) o -p u c W •H 0) C o o (C U) 0) -p > •H +J w c 3 • O +J o s CQ (0 n 0) •H CO to •H c o H in o^ (N U (1) w u CO ^-1 0) s 0) 0 0) 0 rH OQ c +J (0 +J Q) u •H iH ^ 4J • -P 4J f-i •H s rH J CO CM u W W CO 2 2 CM (N rH H X! 0) CQ -P -P I O n rH I (N ^ H rH in VO H tn u 0) w w o CM o 0) CO w CTi u o w w CO « ec; « tf 2 H 2 2 n 0) £! T! ■P C n3 •H -H O 3 (0 (0 f^ ca u w rH 0) ca rH -p ■p •rH (0 ■p a -P ■p o U u 4-1 rH 0) 03 -P ■P I I I I CM CM U (1) w w CO 2 iH 0) 4-> rH 0) CQ -P CO n CM o U W W Di CiJ 2 0) 4J (T3 o m u 0) CQ U o ■p -P 2 rH (1) (0 DQ C 0) CTrH -H 0) CQ E 0^ _^ K w 2 fe • EH CO V^ 1 D 1 u to rH (N c to =«= =**! X (0 (T3 "^ ^~^ (1) 0) 73 &i r-{ rH ^1 y. (0 X r-A rA u u (1) Q) •H ■M (0 Q 0) X X c V Oi ^ (0 u • U m to •H (0 0) ^ en D1 to J3 to 6h X c c to ■H 0 0 •H -H 3 QJ 0 • 3 « « a 2 s to Q (N (N o CO o rH VO O O rg o H rH o O O o o O O o c Id •H u 0) >: o o 4J 4J to to Q) 0) M ^-1 0 O b fc^ • • rH r-i -P -P (C (T3 2 2 rO (t3 C C Q) (U r-i rH 0) Q) K X t-» r) rj , , U 0 0) Q) CO CO S s «D r» • • « Di 2 2 'a- VO -p o o (w J^ tJ (0 i-i CQ to to (0 u 0) to 0) -p o o < (1) 2 O U Q) 2 CO o o =«= ■p to •H 3 cr c o u u ■p U) Q) »H O CC4 4J (C 2 n) a> c ■H o Q) O O U -P n3 e (0 o CO n C T3 r-i CQ g g g 3 3 3 C c c ra (0 fC •H •H •H Jh Ih u rH > rH > +J -P -P to to to -H •H •H D D Cr C c: C 0 0 0 r-i r-t rH g g g 3 3 3 ■rH •H •rH to to to U ^H U •H •H •H u u 0 I I I XXX (Cronquist #11040) and Klamath Co., Oregon (Baldwin #380) were utilized for the RAPD analysis. Electrophoretic Analysis Electrophoretic protocols employed were primarily those described in Brunsfeld et al. (1991) and Soltis et al. (1983) . Leaf tissue was ground in Tris-HCL grinding buffer, absorbed onto filter paper wicks, loaded into starch gels, and electrophoresed for approximately six hours. In preliminary tests seventeen enzymes were stained: aconitase (ACN) , alcohol dehydrogenase (ADH) , acid phosphatase (APH) , aspartate aminotransferase (AAT) , esterase (EST) , glutamate dehydrogenase (GDH) , hexokinase (HK) , isocitrate dehydrogenase (IDH) , leucine aminopeptidase (LAP) , malate dehydrogenase (MDH) , malic enzyme (ME) , menadione reductase (MNR) , phosphoglucoisomerase (PGI) , phosphoglucomutase (PGM), 6-phosphogluconate dehydrogenase (6-PGD), shikimate dehydrogenase (SKDH) , and triosephosphate isomerase (TPI) . Of the enzymes tested, eleven (AAT, ADH, APH, IDH, LAP, MDH, PGI, PGM, 6-PGD, SKDH, and TPI) produced enzyme activity and were analyzed in all subsequent gel runs. The genetic basis of enzyme banding patterns was inferred from observed segregation patterns in light of typical subunit structure and subcellular compartmentalization (Gottlieb 1981, Weeden and Wendel 1989). For enzymes with more than one locus (TPI) , the isozymes were numbered sequentially, with the most anodal isozyme designated 1. Allozymes were labeled alphabetically starting with the fastest allozyme. Enzyme data were analyzed using BIOSYS-1 (Swofford and Selander 1981) . Three measures of genetic diversity were calculated: mean number of alleles per locus (A) , percentage of loci polymorphic (P) , and mean expected heterozygosity (HJ . Genetic divergence among populations and species was analyzed using Nei's (1978) unbiased genetic identity measures calculated by BIOSYS-1, and a UPGMA cluster analysis of identity values was performed. DNA Analysis To gain better resolution of putative hybridization and genetic relationships among Cirsium species, we conducted a genetic analysis called "Randomly Amplified Polymorphic DNA" (RAPD) , following the general methods of Williams et al. (1990) and Welsh and McClelland (1990) . This technique involves amplifying numerous random portions of the plant genome using the polymerase chain reaction (PCR) . In PCR, a heat-stable enzyme repeatedly replicates regions of the plant DNA, specifically where the enzyme is "primed" by small pieces of synthetic DNA (primers) . The amplified DNA products are separated in a gel. stained with a dye that fluoresces in ultraviolet light, and photographed . Total DNA was extracted from each leaf sample using a modified CTAB method previously described (Brunsfeld et al. 1992) . Purified DNA was next quantified and diluted to a uniform concentration (10 ng/ul) . One hundred and twenty 10-mer primers of random sequence (Operon Technologies) were tested using a subset of Cirsium DNA samples. RAPD products whose presence or absence was unambiguous were scored. Population samples were scored from at least three different amplification and electrophoretic runs, ensuring the repeatability of RAPD products. Phenetic segregation of populations based upon presence (1) and absence (0) of RAPD markers was conducted using Principal component analysis (PCA) (SAS Institute Inc., Gary, NC) . RESULTS Enzyme Electrophoresis Seven loci (AAT, APH, LAP, PGI, PGM, TPI-1, and TPI-2) were resolved for 150 individuals from twelve Cirsium populations from westcentral Montana. Data from five enzymes could not be used because of inconsistent staining or uninterpretable banding patterns. Data were sorted and analyzed two ways: by population/locality, and by species. Population/Locality Analysis - For this analysis each locality was considered a single biological population and the tentative field identification of individuals was ignored. Table 2 identifies the names of the 12 populations or localities used in this analysis. Table 3 lists allele frequencies and the number of individuals sampled (N) at each locality. Sample sizes are small for some populations because of poor enzyme activity in many of the late season leaf samples. Levels of genetic variation differ considerably among the 12 populations sampled (Table 4 and Appendix 1) . Values of A ranged from 1.1 to 1.6; P varied from 14.3 to 4 2.9 percent; and H^ ranged from 0.006 to 0.110. Nei's (1978) unbiased genetic identity values were calculated for pairwise comparisons of all 12 populations (Table 5) . Table 2. Key to electrophoretic analysis by population. Original Pop, . no. on pop. no. printout Population name FP 1 FLESHER PASS DC 2 DUCK CREEK PASS DM 3 DEADMAN CREEK MP 4 MOOSE PARK NH 5 NIEHART RC 6 RUSSIAN CREEK KH 7 KINGS HILL SH 8 SHEEP CREEK LC 9 LEWIS & CLARK AC 10 ALICE CREEK WP 11 WARREN PASS SL 12 SLAUGHTERHOUSE c o ■r-t 4-> (0 rH a, o a, a\ CO VD IT) (N o o O O O O O O O O O o O (Tl rH TOO ■* O '!■ O r^ (N H O O (N O O O (N O H O O O O rH O rH O Oi O C\ O ON O O f\) CO o o o o o o o o fN) CO o o o (N T in o n o in o o o o n o 00 o fN T in o o o rH O (Tl O H O o o o o H O o H o en o CO o o o o o o o r^ o o o o N to O o o o in in o o o r^ o in 'T o o o \o o H CO H O o o H o cr\ o iH O O CO H o H O o H rH CD O O O iH CJ^ O o O O O O o O vo ■«*• o O O 'T r- og o rH O o o in m o rH O t^ O n ^r in o O O rH O 0^ O iH O rH O CT\ O O H O O H n V£) o o o n o o o o o o ■TOO o o o o TOO o o o o o n o o o o o o o o o T O O O O O o t^ T O CM o vD n o o n o o o T O o o o o o CO o o o T O o o r- o H O n o o o o o o o o o o o o T O O O o o o o o o o o o o o o o o o o o o o o o o o o o o o o o in o o o in o in o o o o in o in o in o o o o o o o o o o en rH o o o o o o o o o o o o o o o o o o o o o o o in o o o in o CN o in o in in o in o in o o o o o H o^ o o o r- o CM o o o o o o o o o o o o o o o o o o o o o o in o o o in o in o o o o in o in o in o o o o o H CTi O o o in in o o o o o o r>- n VD T O o O VD T O o O O O O CO rH CO CO in T o CD O CO o in T O CO o CO o n o o o H CTi o H o en o H O H o in T O H O H O H O O O r>j O O r) n n o o H en o o o O O O O O O n n n in o n o n vo o o o 00 o in CO CN o O O n O CTi o H o H o r^ (Ni o r>j o rH O f>j o o^ O w rH rH (N H H rH H p u o M S'< CO IX, 1 -^ H 2 < CQ U a. -— Eh 1 ^ M 2; < 1 -- Oi 2 < CQ U Q 3^ 1 -^ xz < < 1 — H 2 < S 2 < PQ U O ■— able 4. Genetic variability by populations (standard errors in parentheses). Population Mean sample size per Locus Mean no. of alleles per locus Percentage of loci polymorphic* Mean heterozygosity Direct- HdyWbg count expected** 1. FLESHER PASS 20.3 (2.2) 1.6 (0.3) 42.9 .075 (.037) .098 [.058) 2. DUCK CREEK PASS 17.3 (0.7) 1.4 (0.2) 42.9 .087 (.048) .110 (.071) 3 . DEADMAN CREEK 5.0 (0.0) 1.3 (0.2) 28.6 .114 (.086) .108 [.080) 4. MOOSE PARK 4.6 (0.4) 1.3 (0.2) 28.6 .100 (.072) .100 [.072) 5. NIEHART 5.0 (0.0) 1.1 (0.1) 14.3 .029 (.029) .029 [.029) 6. RUSSIAN CREEK 15.3 (2.1) 1.6 (0.4) 28.6 .089 (.058) .122 [.081) 7, KINGS HILL 4.0 (0.0) 1.1 (0.1) 14.3 .036 (.036) .036 [.036) 8. SHEEP CREEK 3.0 (0.0) 1.1 (0.1) 14.3 .095 (.095) .076 [.076) 9. LEWIS & CLARK 11.0 (0.8) 1.4 (0.2) 42.9 .046 (.023) .101 '.065) 10. ALICE CREEK 8.9 (0.7) 1.4 (0.2) 42.9 .040 (.027) ( .096 '.052) 11. WARREN PASS 10.3 (1.1) 1.3 (0.2) 28.6 .024 (.015) ( .024 .015) 12. SLAUGHTERHOUSE 17.1 (2.5) 1.1 (0.1) 14.3 .006 (.006) ( .006 .006) * A locus is considered polymorphic if more than one allele was detected. ** Unbiased estimate (see Nei, 1978) . ( ible 5. Nei (1978) unbiased genetic identity by population. Population 1 2 3 4 5 6 7 8~ 1 FLESHER PASS ***** 2 DUCK CREEK PASS .996 ***** 3 DEADMAN CREEK .998 1.000 ***** 4 MOOSE PARK 1.000 .988 .988 ***** 5 NIEHART .998 .984 .982 1.000 ***** 6 RUSSIAN CREEK .998 .999 1.000 .995 .988 ***** 7 KINGS HILL .947 .975 .986 .919 .915 .961 ***** 8 SHEEP CREEK ,982 1.000 1.000 .964 .960 .990 1.000 ***** 9 LEWIS & CLARK .985 .959 .957 .986 .985 .966 .882 .930 10 ALICE CREEK 1.000 .984 .983 1.000 .999 .988 .918 .962 11 WARREN PASS .992 .972 .968 1.000 1.000 .978 .890 .940 12 SLAUGHTERHOUSE .991 .972 .968 1.000 1.000 .977 .891 .941 Population 9 10 11 12 9 LEWIS & CLARK ***** 10 ALICE CREEK 1.000 ***** 11 WARREN PASS .989 .998 ***** 12 SLAUGHTERHOUSE .985 .996 1.000 ***** The UPGMA cluster analysis of the Nei identity values is shown in Figure 1. Populations cluster into two major groups with an overall similarity of 0.96; one group contains C. lonqistylum (L) and putative hybrids (LX, X, Table 1) , the second group contains C^^ scariosum (S) , C^ hookerianum (H) , and putative hybrids. The three pure or largely pure populations of C. lonqistylum (Duck Creek Pass, Deadman Creek, Russian Creek) form a distinct subcluster with a similarity of approximately 0.98. The remaining two populations in the group (Kings Hill [USFS] and Sheep Creek) are represented by small samples consisting of a mixture of L, LX, X, and a single H. In the second large cluster, one "pure" C_^ hookerianum population (Lewis and Clark) is the most distinct, and two other "pure" C_i. hookerianum populations (Flesher Pass and Alice Creek) cluster a small sample of hybrids (Moose Park) . In addition, two "pure" C_i. scariosum populations cluster with another small group of putative hybrids. No "pure" C^ lonqistylum individuals were part of the sample in any of the 6 populations in this cluster. Species Analysis - In this analysis individuals were grouped by their field identification, i.e. L, H, S, LX, and X. To increase sample sizes individuals for different populations were grouped together. Table 6 identifies the names of the species used in this analysis. Table 7 lists allele frequencies and the number of individuals sampled (N) of each species or hybrid class. Genetic variation statistics for each group are presented in Table 8 and Appendix II. Values of A ranged from 1.3 to 1.7; P varied from 28.6 to 57.1 percent; and H^ ranged from 0.019 to 0.123. Nei's (1978) unbiased genetic identity values were calculated for pairwise comparisons of all 5 species or hybrid groups (Table 9) . A UPGMA cluster analysis of this data matrix (Figure 2) has two major clusters: one containing C_^ lonqistylum individuals and those considered hybrids of C^ lonqistylum and C. hookerianum; and a second cluster of "pure" C^ hookerianum and C. scariosum, which have similarity of 0.99 based on these isozyme loci . DNA Analysis A preliminary analysis of twentyone Cirsium samples was conducted. These included: 6 "pure" C^ lonqistylum. 5 C. hookerianum from Montana, 1 C^ hookerianum from British Columbia, 4 C^ scariosum from Montana, 2 C^ scariosum from Nevada and Oregon, and three hybrids (HX, LX, X) . Fortyseven RAPD loci were scored for each sample. The first 3 principal components accounted for 47% of the variance in the data set (Appendix III) . Figures 3 and 4 display the distribution of samples in principal component space. 10 Table 6. Key to electrophoretic analysis by species, Original Pop. no. on pop. no. printout Population name sc 1 SCARIOSUM HO 2 HOOKERIANUM X 3 HOOKxLONG (X) LX 4 LONGxHOOK (LX) LO 5 LONGISTYLUM Table 7. Allele frequency by locus and species, Population Locus 1 2 3 4 5 PGI-1 (N) 23 44 5 12 14 A 1 .000 1.000 .900 .958 .964 B .000 .000 .100 .042 .036 TPI-1 (N) 36 58 5 12 30 A .042 .043 .000 .083 .133 B .958 .940 1 .000 .917 .833 C .000 .017 .000 .000 .033 TPI-2 (N) 37 40 5 12 36 A 1 .000 .988 1 .000 1.000 1.000 B .000 .013 .000 .000 .000 LAP-1 (N) 15 35 4 11 35 A .000 .000 .000 .000 .014 B 1 .000 .814 .375 .545 .600 C .000 .186 .625 .409 .386 0 .000 .000 .000 .045 .000 APH-1 (N) 23 45 5 9 36 A 1 .000 1.000 1 .000 1.000 1.000 AAT-1 (N) 22 35 5 11 35 A 1 .000 1.000 1 .000 1.000 1.000 PGM-1 (N) 36 48 5 11 31 A .014 .188 .000 .000 .000 B .972 .813 1 .000 1.000 1.000 C .014 .000 .000 .000 .000 ( Table 8. Genetic variability by species (standard errors in parentheses) Mean heterozygosity Mean sample Mean no. Percentage size per of alleles of loci Direct- HdyWbg Locus per locus polymorphic* count expected** Population 1. SCARIOSUM 2. HOOKERIANUM 3. HOOKxLONG(X) 4. LONGxHOOK(LX) 5. LONGISTYLUM 27.4 (3.3) 43.6 (3.0) 4.9 (0.1) 11.1 (0.4) 31.0 (3.0) 1.4 (0.3) 1.7 (0.3) 1.3 (0.2) 1.6 (0.3) 1.7 (0.4) 28.6 57.1 28.6 42.9 42.9 .020 .019 (.013) (.013) .065 .108 (.028) (.054) .064 .105 (.042) (.077) .140 .114 (.101) (.078) .098 .123 (.055) (.074) * A locus is considered polymorphic if more than one allele was detected. ** Unbiased estimate (see Nei, 1978). Table 9. Nei (1978) unbiased genetic identity by species, Population 1 SCARIOSUM 2 HOOKERIANUM ^ 3 HOOKxLONG(X) 4 LONGxHOOK(LX) 5 LONGISTYLUM ***** .008 ***** .055 .031 ***** .026 .013 .000 ***** .023 .013 .005 .000 ***** Figure 1. UPGMA cluster analysis of electrophoretic data by population, using Nei (1978) unbiased genetic identity. .93 .95 .97 .98 1.00 + + + + + + + + + * FLESHER PASS ( Hooker ianum) * *** MOOSE PARK (3X:2LX) * * * * ALICE CREEK (Hookerianum) * ****** * * * NIEHART (2X:3H) * * * ****************** *** WARREN PASS (Scariosum) * * * * * * SLAUGHTERHOUSE (Scariosum) * * * ******** LEWIS & CLARK (Hookerianum) * * DUCK CREEK PASS (13L:4LX:1X) * * * ********** DEADMAN CREEK (3L:2LX) * * * **************** * RUSSIAN CREEK (Longistylum) * * * KINGS HILL (2L:1LX:1X) ^ ********** * SHEEP CREEK (1L:1LX:1H) .93 .95 .97 .98 1.00 Similarity Figure 2. UPGMA cluster analysis of electrophoretic data by species, using Nei (1978) unbiased genetic identity. .93 .95 .97 .98 1.00 ****** SCARIOSUM * *********** * ****** HOOKERIANUM * * HOOKxLONG(X) * * * * * k * * * LONGxHOOK(LX) *************** * * * LONGISTYLUM + + + + + + + + + .93 .95 .97 .98 1.00 Similarity I prin: 2: -1 -3 © C. scariosum (NV) C. scariosum (OR) C. h ■C. scariosum (MT) -T — I — I — I — I— -5 -3 -2 -1 0 1 PRIN1 Figure 3. PCA of RAPD data from C^ lonqistvlum (L) EO number 02 0, Oil & 006, C^ hookerianum from southwestern Montana (H) , C. hookerianum from northern Montana and British Columbia (I) , C. scariosum from southwestern Montana (S) , C^ scariosum from Nevada (T) , C_^ scariosum from Oregon (U) , and putative hybrids HX (X) , LX (Y) and X (Z) . PRIN3 Figure 4. PCA of RAPD data from Cj_ longistvlum (L) EO number 020, Oil & 006, C^ hookerianum from southwestern Montana (H) , C. hookerianum from northern Montana and British Columbia (I) , C. scariosum from southwestern Montana (S) , C^ scariosum from Nevada (T) , C^ scariosum from Oregon (U) , and putative hybrids HX (X) , LX (Y) and X (Z) . Cirsium lonqistylum is strongly separated from both C. scariosum and C_^ hookerianum. Cirsium scariosum from Montana is distinct from the samples from Nevada and Oregon. Similarly, C. hookerianum samples from British Columbia and northern Montana separate from westcentral Montana samples. Hybrids as a group are scarcely distinguishable from C_^ lonqistylum and C. hookerianum. DISCUSSION Enzyme Electrophoresis Analysis - The Cirsium samples we analyzed contain levels of enzyme variability that would be expected, based on data from other species with similar life history characteristics (Hamrick & Godt 1989) . Whether data is sorted and analyzed by taxonomic classification or population/locality, similar results are obtained. Plants identified in the field as Cirsium longistylum are genetically distinct from plants identified as C^ hookerianum and C. scariosum. Indeed, of the three species sampled from westcentral Montana, C^ lonqistylum appears to be the most genetically distinct. Three populations that are considered to be pure or nearly pure C_^ lonqistylum cluster together in our analysis, and are very distinct from populations of Cj_ hookerianum and C. scariosum, which appear more closely related. All plants classified as hybrids of C^;. lonqistylum cluster with pure C. lonqistylum. whereas populations composed of a mixture of parents and putative hybrids are heterogeneous - two cluster with C. lonqistylum, one clusters with C^ hookerianum. and one clusters with C^ scariosum. While these results are consistent with the hypothesis that hybridization is occurring between C^ lonqistylum and another species, enzyme electrophoresis does not provide enough diagnostic genetic markers to rule out alternative explanations. DNA Analysis - Preliminary results of the analysis of RAPD data are congruent with electrophoretic results, and also provide additional insights into Cirsium genetics. Plants identified as Cirsium lonqistylum are separable from all others, but exhibit more variability than other species within westcentral Montana. Several of the C^ lonqistylum samples are among the most genetically distinct plants that we analyzed, suggesting that C. lonqistylum represents a well-differentiated evolutionary lineage. Whether the variability within C^ lonqistylum is attributable to hybridization requires more detailed investigation. Two widely separated northern samples of C^ hookerianum. obtained from herbarium specimens, are similar to each other, but differentiated from westcentral Montana samples. Based on morphology, Cronquist (1964) considered northern C_^ hookerianum 16 populations to be the purest form of the species. A better genetic understanding of "pure" northern C_^ hookerianum is needed to interpret variability in westcentral Montana C^. hookerianum and C^ lonqistylum. Distant samples of C_^ scariosum from Nevada and Oregon are also genetically distinct from westcentral Montana C. scariosum. Furthermore, the relative genetic similarity of C. scariosum and C^. hookerianum from westcentral Montana raises the question of whether introgression has affected all of the species in this region. In the PCA analysis of RAPD data, putative hybrid plants are placed near or intermediate between C_^ lonqistylum. C. hookerianum, and C_^ scariosum plants, consistent with the hypothesis that they arose by hybridization. However, until the genetic composition of "pure" C^ lonqistylum, C. hookerianum, and C. scariosum is understood, through rangewide sampling of the species, it will not be possible to distinguish between natural variability in each species and variability induced by hybridization/ introgression. Hypothesized Recent Evolutionary History of Cirsivun lonqistyl\im The evolutionary history of Cirsium species in Montana can be hypothesized in light of existing genetic, morphologic, and geographic information. Our preliminary DNA analysis suggests that Cirsium lonqistylum, C. hookerianum. and C^ scariosum are well-differentiated entities in the allopatric parts of their ranges, apparently representing separate evolutionary lineages. During glacial times, the northern species of the group, C. hookerianum, was likely forced southward into sympatry with C. lonqistylum in westcentral and southern Montana. Cirsium scariosum also colonized the region, most likely from its principal range to the southwest. Gene flow via wind-dispersed seed and nondiscriminating pollinators probably occurred among many populations in westcentral Montana, resulting in regional introgression of the three species. Cirsium lonqistylum genes have introgressed into C^ hookerianum and C_^ scariosum producing distinct westcentral Montana races of these species. Because genes from all three species are likely widespread throughout westcentral Montana, distinguishing between introgression and natural variation within each species is not possible without understanding genetic patterns and variability outside of westcentral Montana. A phylogenetic analysis of all Cirsium species in the region is needed to test the evolutionary hypotheses presented above. Knowledge of phylogenetic relationships and processes in Cirsium would allow managers to judge: the biological significance of C. lonqistylum. whether human activity or natural processes have produced the current biological situation, and whether any further management action is warranted. 17 Future Work To gain a complete understanding of C^ lonqistylum, the following additional work is needed: 1) A restriction site analysis of chloroplast DNA should be performed on C^^ lonqistylum and all closely related species. The phylogeny derived from these data will be the cornerstone of our understanding of the genetic significance of the C_^ lonqistylum lineage and the evolutionary events that occurred in Montana. 2) Gain a better understanding hybridization and introgression in Montana by obtaining essential information on the variability of C_^ hookerianum and C_^ scariosum throughout their geographic range. 3) Study the morphology of genetically pure C^ lonqistylum, C. hookerianum. and C^ scariosum so that botanists and managers can more easily interpret plants encountered in the field. 18 (# LITERATURE CITED Brunsfeld, S.J., D.E. Soltis, and P.S. Soltis. 1991. Patterns of genetic variation in Salix sect. Longif oliae (Salicaceae) . American Journal of Botany 78:855-869. Brunsfeld, S.J., D.E. Soltis, and P.S. Soltis. 1992. Evolutionary- patterns and processes in Salix sect. Longif oliae; evidence from chloroplast DNA. Systematic Botany 17(2): 239-256. Gardner, R.C. 1974. Systematics of Cirsium (Compositae) in Wyoming. Madrono 22:239-265. Gottlieb, L.D. 1981. Electrophoretic evidence and plant populations. Progress in Phytochemistry 7:1-45. Hamrick, J.L. and M.J.W. Godt. 1989. Allozyme diversity in plant species. In A.H.D. Brown, M.T. Clegg, A.L. Kahler, and S.B. Weir [eds.]. Plant population genetics, breeding, and genetic resources, 43-63. Sinauer, Sunderland, MA. Heidel, B.L. 1994. Monitoring of Cirsium longistvlum (Long- styled thistle). Report to U.S. Fish and Wildlife Service - Region 4. Hitchcock, C. , A. Cronquist, M. Ownbey and J.W. Thompson. 1964. Vascular Plants of the Pacific Northwest, Vol. 5. University of Washington Press, Seattle. Mathews, S. 1990. Cirsium longistylum project: summary report, (chromosome counts) . Prepared for Montana Natural Heritage Program. Montana State University, Bozeman. 3 pp. Moore, R.J. and C. Frankton. 1963. Cytotaxonomic notes on some Cirsium species of the western United States. Can. J. Bot. 41:1553-1567. Moore, R. J. and C. Frankton. 1965. Cytotaxonomy of Cirsium hookerianum and related species. Can. J. Bot. 43:597-613. Moore, R. J. and C. Frankton. 1974. The thistles of Canada. Canadian Dept. of Agriculture, Research Branch. Monograph No. 10. Ottawa, Ontario. Nei, M. 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89:583-590. Ownbey, G. B. and Y. Hsi. 1963. Chromosome numbers in some North American species of the genus Cirsium. Rhodora 65:339-354. 19 I Poole, J. M. and B.L. Heidel. 1993. A taxonomic assessment and monitoring study of the long-styled thistle (Cirsium longistylum) . Montana Natural Heritage Program, Helena. 97 pp. Roe, L. S. 1992. Taxonomic and demographic studies of Cirsium longistylum in the Little Belt Mountains, Montana. Montana Natural Heritage Program, Helena. 23 pp. Schassberger, L. A. 1991. Report on the conservation status of Cirsium longistylum, a candidate threatened species. Unpublished report for U.S. Fish and Wildlife Service. Montana Natural Heritage Program, Helena. 92 pp. Schassberger, L. A. and P. L. Achuff. 1991. Status review of Cirsium longistylum. Unpublished report for Lewis and Clark National Forest. Montana Natural Heritage Program, Helena. 78 pp. Soltis, D.E., C. Haufler, D. Darrow, and G.J. Gastony. 1983. Starch gel electrophoresis of ferns. Am. Fern J. 73: 9-27. Swofford, D.L. and R.B. Selander. 1981. BIOSYS-1. University of Illinois, Urbana. t^ Weeden, N.F., and J.F. Wendel. 1989. Genetics of plant isozymes. * In D.E. Soltis and P.S. Soltis [eds.]. Isozymes in plant biology, 46-72. Dioscorides Press, Portland, OR. Welsh, J. and M. McClelland. 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Research 18:7213 - 7218. Williams, J.G.K., A.R. Kubelik, K.J. Livak, J. A. Rafalski, and S.V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18:6531-6535. 20 X APPENDIX I Allele frequencies and genetic variability measures of electrophoretic data ^^ by population. Population: FLESHER PASS (FP) Locus and sample size PGI-1 TPI-1 TPI-2 LAF >-l APH-1 AAT-1 PGM-1 Allele 21 30 15 13 20 18 25 A 1.000 .033 1.000 .000 1.000 1.000 .080 B .000 .933 .000 .731 .000 .000 .920 C .000 .033 .000 .269 .000 .000 .000 H .000 .127 .000 .393 .000 .000 .147 H(unb) .000 .129 .000 .409 .000 .000 .150 H(D.C.) .000 .133 .000 .231 .000 .000 .160 Mean heterozygosity per locus (biased estimate) = .095 (S.E. .055) Mean heterozygosity per locus (unbiased estimate) = .098 (S.E. .058) Mean heterozygosity per locus (direct-count estimate) = .075 (S.E. .037) Mean number of alleles per locus = 1.57 (S.E. .30) Percentage of loci polymorphic (0.95 criterion) = 42.86 Percentage of loci polymorphic (0.99 criterion) = 42.86 .Percentage of loci polymorphic (no criterion) = 42.86 Population: DUCK CREEK PASS (DC) Locus and sample size PGI-1 TPI-1 Allele 18 18 A .917 .056 B .083 .944 C .000 .000 H .153 .105 H(unb) .157 .108 H(D.C.) .167 .111 TPI-2 LAP-1 18 18 APH-1 18 AAT-1 18 PGM-1 13 000 000 000 000 000 000 000 556 444 494 508 333 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 000 107 (S.E. .069) (S.E. .07 087 (S.E. .048) Kean heterozygosity per locus (biased estimate) = ean heterozygosity per locus (unbiased estimate) = .110 (S.E. .071) Mean heterozygosity per locus (direct-count estimate) = Mean number of alleles per locus = 1.43 (S.E. .20) Percentage of loci polymorphic (0.95 criterion) = 42.86 Percentage of loci polymorphic (0.99 criterion) = 42.86 Percentage of loci polymorphic (no criterion) = 42.86 Population: DEADMAN CREEK (DM) Locus and sample size t PGI-1 TPI-1 TPI-2 LAP-1 APH-1 AAT-1 PGM-1 llele 5 5 5 5 5 5 5 A 1.000 .100 1.000 ,000 1.000 1. 000 .000 B .000 .900 .000 .500 .000 .000 1.000 C .000 .000 .000 .500 .000 .000 .000 H .000 .180 .000 .500 .000 .000 .000 H(unb) .000 .200 .000 .556 .000 .000 .000 H(D.C.) .000 .200 .000 .600 .000 .000 .000 Mean heterozygosity per locus (biased estimate) = .097 (S.E. .072) Mean heterozygosity per locus (unbiased estimate) = .108 (S.E. .080) Mean heterozygosity per locus (direct-count estimate) = .114 (S.E. .086) Mean number of alleles per locus = 1.29 (S.E. .18) Percentage of loci polymorphic (0.95 criterion) = 28.57 Percentage of loci polymorphic (0.99 criterion) = 28.57 Percentage of loci polymorphic (no criterion) = 28.57 Population: MOOSE PARK (MP) Locus and samp I ,e size PGI-1 TPI-1 TPI-2 LAI '-1 APH-1 AAT-1 PGM-1 Allele 5 5 5 2 5 5 5 A 1.000 .100 1.000 .000 1.000 1.000 .000 B .000 .900 .000 .750 .000 .000 1.000 C .000 .000 .000 .000 .000 .000 .000 D .000 .000 .000 .250 .000 .000 .000 H . 000 .180 .000 .375 .000 .000 .000 H(unb) .000 .200 .000 .500 .000 .000 .000 H(D.C.) .000 .200 .000 .500 .000 .000 .000 Mean heterozygosity per locus (biased estimate) = .079 (S.E. .055) Kean heterozygosity per locus (unbiased estimate) = .100 (S.E. .072) ean heterozygosity per locus (direct-count estimate) = .100 (S.E. .072) Mean number of alleles per locus = 1.29 (S.E. .18) Percentage of loci polymorphic (0.95 criterion) = 28.57 Percentage of loci polymorphic (0.99 criterion) = 28.57 Percentage of loci polymorphic (no criterion) = 28.57 Population: NIEHART (NH) Locus and sample size PGI-1 TPI-1 aiele 5 5 TPI-2 LAP-1 APH-1 AAT-1 PGM-1 5 5 5 5 5 A B C H H(unb) H(D.C. ) 000 000 000 000 000 000 000 1.000 .000 1.000 000 .000 .900 .000 000 .000 .100 .000 000 .000 .180 .000 000 .000 .200 .000 000 .000 .200 .000 000 000 000 000 000 000 000 000 000 000 000 000 Mean heterozygosity per locus (biased estimate) = .026 (S.E. .026) Mean heterozygosity per locus (unbiased estimate) = .029 (S.E. .029) Mean heterozygosity per locus (direct-count estimate) = .029 (S.E. .029) Mean number of alleles per locus = 1.14 (S.E. .14) Percentage of loci polymorphic (0.95 criterion) = 14.29 Percentage of loci polymorphic (0.99 criterion) = 14.29 Percentage of loci polymorphic (no criterion) = 14.29 Population: RUSSIAN CREEK (RC) Locus and sampl e size PGI-1 TPI-1 TPI-2 LAI '-1 APH-1 AAT-1 PGM-1 Allele 4 17 17 22 17 17 13 A 1.000 .147 1.000 .023 1.000 1.000 .000 B .000 .794 .000 .614 .000 .000 1.000 C .000 .059 .000 .364 .000 .000 .000 H .000 .344 .000 .491 .000 .000 .000 H(unb) .000 .355 .000 .502 .000 .000 .000 H(D.C.) .000 .353 .000 .273 .000 .000 .000 Mean heterozygosity per locus (biased estimate) = .119 (S.E. .079) K'rtean heterozygosity per locus (unbiased estimate) = .122 (S.E. .081) ^^ean heterozygosity per locus (direct-count estimate) = .089 (S.E. .058) Mean number of alleles per locus = 1.57 (S.E. .37) Percentage of loci polymorphic (0.95 criterion) = 28.57 Percentage of loci polymorphic (0.99 criterion) = 28.57 Percentage of loci polymorphic (no criterion) = 28.57 Population: KINGS HILL (KH) Locus and sample size PGI-1 TPI-1 TPI-2 LAP-1 APH-1 AAT-1 PGM-1 allele 4 4 4 4 4 4 4 A 1.000 .000 1.000 .000 1.000 1.000 .000 B .000 1.000 .000 .125 .000 .000 1.000 C .000 .000 .000 .875 .000 .000 .000 H .000 .000 .000 .219 .000 .000 .000 H(unb) .000 .000 .000 .250 .000 .000 .000 H(D.C.) .000 .000 .000 .250 .000 .000 .000 Mean heterozygosity per locus (biased estimate) = .031 (S.E. .031) Mean heterozygosity per locus (unbiased estimate) = .036 (S.E. .036) Mean heterozygosity per locus (direct-count estimate) = .036 (S.E. .036) Mean number of alleles per locus = 1.14 (S.E. .14) Percentage of loci polymorphic (0.95 criterion) = 14.29 Percentage of loci polymorphic (0.99 criterion) = 14.29 Percentage of loci polymorphic (no criterion) = 14.29 Population: SHEEP CREEK (SH) Locus and sample size PGI-1 TPI-1 TPI-2 LAP-1 APH-1 AAT-1 PGM-1 Allele 3 3 3 3 3 3 3 A 1.000 .000 1.000 .000 1.000 1.000 .000 B .000 1.000 .000 .333 .000 .000 1.000 C .000 .000 .000 .667 .000 .000 .000 H .000 .000 .000 .444 .000 .000 .000 H(unb) .000 .000 .000 .533 .000 .000 .000 H(D.C.) .000 .000 .000 .667 .000 .000 .000 Mean heterozygosity per locus (biased estimate) = .063 (S.E. .063) lean heterozygosity per locus (unbiased estimate) = .076 (S.E. .076) lean heterozygosity per locus (direct-count estimate) = .095 (S.E. .095) Mean number of alleles per locus = 1.14 (S.E. .14) Percentage of loci polymorphic (0.95 criterion) = 14.29 Percentage of loci polymorphic (0.99 criterion) = 14.29 Percentage of loci polymorphic (no criterion) = 14.29 Population: LEWIS & CLARK (LC) Locus and samp] .e size 1 PGI-1 TPI-1 TPI-2 LAP '-1 APH-1 AAT-1 PGM -1 Rllele 11 14 11 10 11 7 13 A 1.000 .071 1.000 .000 1.000 1.000 .346 B .000 .929 .000 .950 .000 .000 .654 C .000 .000 .000 .050 .000 .000 .000 H .000 .133 .000 .095 .000 .000 .453 H(unb) .000 .138 .000 .100 .000 .000 .471 H(D.C.) .000 .143 .000 .100 .000 .000 .077 Mean heterozygosity per locus (biased estimate) = .097 (S.E. .063) Mean heterozygosity per locus (unbiased estimate) = .101 (S.E. .065) Mean heterozygosity per locus (direct-count estimate) = .046 (S.E. .023) Mean number of alleles per locus = 1.43 (S.E. .20) Percentage of loci polymorphic (0.95 criterion) = 42.86 Percentage of loci polymorphic (0.99 criterion) = 42.86 Percentage of loci polymorphic (no criterion) = 42.86 Population: ALICE CREEK (AC) Locus and sampl e size PGI-1 TPI-1 TPI-2 LAP >-l APH-1 AAT-1 PGM -1 Allele 8 10 10 7 10 6 11 A 1.000 .050 1.000 .000 1.000 1.000 .182 B .000 .950 .000 .857 .000 .000 .818 C .000 .000 .000 .143 .000 .000 .000 H .000 .095 .000 .245 .000 .000 .298 H(unb) .000 .100 .000 .264 .000 .000 .312 H(D.C.) .000 .100 .000 .000 .000 .000 .182 m Mean heterozygosity per locus (biased estimate) = .091 (S.E. .049) ean heterozygosity per locus (unbiased estimate) = .096 (S.E. .052) ean heterozygosity per locus (direct-count estimate) = .040 (S.E. .027) Mean number of alleles per locus = 1.43 (S.E. .20) Percentage of loci polymorphic (0.95 criterion) =42.86 Percentage of loci polymorphic (0.99 criterion) = 42.86 Percentage of loci polymorphic (no criterion) = 42.86 Population: WARREN PASS (WP) Locus and sample size PGI-1 TPI-1 TPI-2 LAP-1 APH-1 AAT-1 PGM-1 Lllele 9 12 13 5 13 8 12 A 1.000 .042 1 .000 .000 1 .000 1 .000 .042 B .000 .958 .000 1.000 .000 .000 .958 H .000 .080 .000 .000 .000 .000 .080 H(unb) .000 .083 .000 .000 .000 .000 .083 H(D.C.) .000 .083 .000 .000 .000 .000 .083 Mean heterozygosity per locus (biased estimate) = .023 (S.E. .015) Mean heterozygosity per locus (unbiased estimate) = .024 (S.E. .015) Mean heterozygosity per locus (direct-count estimate) = .024 (S.E. .015) Mean number of alleles per locus = 1.29 (S.E. .18) Percentage of loci polymorphic (0.95 criterion) = .00 Percentage of loci polymorphic (0.99 criterion) = 28.57 Percentage of loci polymorphic (no criterion) = 28.57 Population: SLAUGHTERHOUSE (SL) Locus and sampl e size PGI-1 TPI-1 TPI-2 LAP-1 APH-1 AAT-1 PGM -1 Allele 14 24 24 10 10 14 24 A 1.000 .000 1.000 .000 1.000 1.000 .000 B .000 1.000 .000 1.000 .000 .000 .979 C .000 .000 .000 .000 .000 .000 .021 H .000 .000 .000 .000 .000 .000 .041 H(unb) .000 .000 .000 .000 .000 .000 .042 H(D.C.) .000 .000 .000 .000 .000 .000 .042 ^m. Mean heterozygosity per locus (biased estimate) = .006 (S.E. .006) ean heterozygosity per locus (unbiased estimate) = .006 (S.E. .006) ean heterozygosity per locus (direct-count estimate) = .006 (S.E. .006) Mean number of alleles per locus = 1.14 (S.E. .14) Percentage of loci polymorphic (0.95 criterion) = .00 Percentage of loci polymorphic (0.99 criterion) = 14.29 Percentage of loci polymorphic (no criterion) = 14.29 . . APPENDIX II Allele frequencies and genetic variability measures of electrophoretic data species. Population: SCARIOSUM (SC) Locus and samp] .e size PGI-1 TPI-1 TPI-2 LAP-1 APH-1 AAT-1 PGM -1 Allele 23 36 37 15 23 22 36 A 1.000 .042 1.000 .000 1.000 1.000 .014 B .000 .958 .000 1.000 .000 .000 .972 C .000 .000 .000 .000 .000 .000 .014 H .000 .080 .000 .000 .000 .000 .054 H(unb) .000 .081 .000 .000 .000 .000 .055 H(D.C.) .000 .083 .000 .000 .000 .000 .056 Mean heterozygosity per locus (biased estimate) = .019 (S.E. .013) Mean heterozygosity per locus (unbiased estimate) = .019 (S.E. .013) Mean heterozygosity per locus (direct-count estimate) = .020 (S.E. .013) Mean number of alleles per locus = 1.43 (S.E. .30) Percentage of loci polymorphic (0.95 criterion) = .00 Percentage of loci polymorphic (0.99 criterion) = 28.57 Percentage of loci polymorphic (no criterion) = 28.57 Population: HOOKERIANUM (HO) Locus and sampl e size PGI-1 TPI-1 TPI-2 LAP '-1 APH-1 AAT-1 PGM -1 Allele 44 58 40 35 45 35 48 A 1.000 .043 .988 .000 1.000 1.000 .188 B .000 .940 .013 .814 .000 .000 .813 C .000 .017 .000 .186 .000 .000 .000 H .000 .115 .025 .302 .000 .000 .305 H(unb) .000 .116 .025 .307 .000 .000 .308 H(D.C.) .000 .121 .025 .143 .000 .000 .167 m Mean heterozygosity per locus (biased estimate) = .107 (S.E. .053) ean heterozygosity per locus (unbiased estimate) = .108 (S.E. .054) ean heterozygosity per locus (direct-count estimate) = Mean number of alleles per locus = 1.71 (S.E. .29) Percentage of loci polymorphic (0.95 criterion) = 42.86 Percentage of loci polymorphic (0.99 criterion) = 57.14 Percentage of loci polymorphic (no criterion) = 57.14 065 (S.E, 028) Population: HOOKxLONG (X) Locus and sample size PGI-1 TPI-1 TPI-2 LAP-1 APH-1 AAT-1 PGM-1 Allele 5 5 5 4 5 5 5 A .900 .000 1.000 .000 1.000 1.000 .000 B .100 1.000 .000 .375 .000 .000 1.000 C .000 .000 .000 .625 .000 .000 .000 H .180 .000 .000 .469 .000 .000 .000 H(unb) .200 .000 .000 .536 .000 .000 .000 H(D.C.) .200 .000 .000 .250 .000 .000 .000 Mean heterozygosity per locus (biased estimate) = .093 (S.E. .068) Mean heterozygosity per locus (unbiased estimate) = .105 (S.E. .077) Mean heterozygosity per locus (direct-count estimate) = .064 (S.E. .042) Mean number of alleles per locus = 1.29 (S.E. .18) Percentage of loci polymorphic (0.95 criterion) = 28.57 Percentage of loci polymorphic (0.99 criterion) = 28.57 Percentage of loci polymorphic (no criterion) = 28.57 Population: LONGxHOOK (LX) Locus and sample size PGI-1 TPI-1 Allele 12 12 TPI-2 LAP-1 APH-1 AAT-1 PGM-1 12 11 9 11 11 A .958 .083 1.000 .000 1.000 1.000 .000 B .042 .917 .000 .545 .000 .000 1.000 C .000 .000 .000 .409 .000 .000 .000 D .000 .000 .000 .045 .000 .000 .000 H .080 .153 .000 .533 .000 .000 .000 H(unb) .083 .159 .000 .558 .000 .000 .000 H(D.C.) .083 .167 .000 .727 .000 .000 .000 Mean heterozygosity per locus (biased estimate) = .109 (S.E. .074) Mean heterozygosity per locus (unbiased estimate) = .114 (S.E. .078) Mean heterozygosity per locus (direct-count estimate) = .140 (S.E. .101) Mean number of alleles per locus = 1.57 (S.E. .30) Percentage of loci polymorphic (0.95 criterion) = 28.57 'ercentage of loci polymorphic (0.99 criterion) = 42.86 Percentage of loci polymorphic (no criterion) = 42.86 Population: LONGISTYUM (LO) Locus and sample size PGI-1 TPI-1 TPI-2 LAP-1 APH-1 AAT-1 PGM-1 Allele 14 30 36 35 36 35 31 A .964 .133 1.000 .014 1.000 1.000 .000 B .036 .833 .000 .600 .000 .000 1.000 C .000 .033 .000 .386 ,000 .000 .000 H .069 .287 .000 .491 .000 .000 .000 H(unb) .071 .292 .000 .498 .000 .000 .000 H(D.C.) .071 .300 .000 .314 .000 .000 .000 Mean heterozygosity per locus (biased estimate) = .121 (S.E. .073) Mean heterozygosity per locus (unbiased estimate) = .123 (S.E. .074) Mean heterozygosity per locus (direct-count estimate) = .098 (S.E. .055) Mean number of alleles per locus = 1.71 (S.E. .36) Percentage of loci polymorphic (0.95 criterion) = 28.57 Percentage of loci polymorphic (0.99 criterion) =42.86 Percentage of loci polymorphic (no criterion) = 42.86 APPENDIX III Summary of Principal Component Analysis. PRINl PRIN2 PRIN3 Eigenvalues of the Correlation Matrix Eigenvalue Difference Proportion 8.95259 4.57832 3.97412 4.37428 0.60420 0.241962 0.123738 0.107409 Cumulative 0.241962 0.365700 0.473109 ID PRINl PRIN2 PRIN3 L L L L L L H H H H I I S S s s T U X Y Z 5, 5, 5, 2. 2. 1. -0, 0, -0, 1. -2. -2. -2. -2. ■3. ■1. •4. •3. 0. .32762 .66437 ,30231 ,87641 ,26257 ,03792 ,71977 ,75144 ,59977 ,06330 ,59933 ,56822 59528 31521 16861 47966 60801 38484 93016 •1.45439 0.27699 ■1, ■0, 1, 0, 2, 42814 81522 20822 70227 08623 1.92393 0.81299 35082 04879 14517 17734 95166 90440 85768 21038 54100 94440 32185 94578 25429 85662 1. 1. 1, 0. ■2. -2. •1. 1. 1. 24829 94642 36906 12751 81516 94642 94595 23620 19420 1.23879 1.86975 12984 75579 06297 15217 89198 79246 18387 14927 53418 13705 H C. hooker ianum (Southern Montana) I C. hookerianum (Northern Montana & British Columbia) longistylum scariosum (Montana) scariosum (Nevada) scariosum (Oregon) L C. S C. T C. U C. X HX Y LX Z X W^'