Western North American Naturalist

Volume 63 Number 2 Article 12

4-30-2003

Inter-simple sequence repeat (ISSR) variation in three populations of neomexicana ssp. coloradensis (), F.E. Warren Air Force Base, Cheyenne, Wyoming

Dorothy E. Tuthill University of Wyoming, Laramie

Gregory K. Brown University of Wyoming, Laramie

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Recommended Citation Tuthill, Dorothy E. and Brown, Gregory K. (2003) "Inter-simple sequence repeat (ISSR) variation in three populations of Gaura neomexicana ssp. coloradensis (Onagraceae), F.E. Warren Air Force Base, Cheyenne, Wyoming," Western North American Naturalist: Vol. 63 : No. 2 , Article 12. Available at: https://scholarsarchive.byu.edu/wnan/vol63/iss2/12

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INTER-SIMPLE SEQUENCE REPEAT (ISSR) VARIATION IN THREE POPULATIONS OF GAURA NEOMEXICANA SSP. COWRADENSIS (ONAGRACEAE), F.E. WARREN AIR FORCE BASE, CHEYENNE, WYOMING

Dorothy E. Tuthillt and Gregory K. Brown1

ABSTRAGr.-We investigated genetic variation within and between 3 populations of Gaura neomexicana ssp. 0010­ rademis, a federally listed threatened species, using inter-simple sequence repeats. The data matrix included 24 indi­ viduals scored for 88 bands, with 3.4% missing data. Cluster analysis showed that members ofthe 3 populations are very similar and are intermixed in the phenogram. Principal coordinate analysis indicated that members of the Crow Creek population are different from the Diamond Creek and "unnamed drainage" populations. This could be the result ofhis­ toric differences, current changes in vegetation at the Crow Creek site, or an artifact of limited sampling. In any case, for management purposes it is best to preserve the dwindling population at Crow Creek as well as the thriVing popula­ tions at the other 2 sites.

Key words: Gaura neomexicana ssp. coloradensis, inter-simple sequence repeats, ISSR, populations, variation, manage­ ment, olw>ter analysis, principal oovrdinate analysis, DNA quality.

Colorado butterfly (Gaura neomexil;ana (Brown 1999, 2000), and demographic structure Wooton ssp. coloradensis [Rydb.] Raven and and survivorship (Floyd 1995b, Floyd and Gregory) is a federally listed threatened species Ranker 1998) ofthe Colorado butterfly plant. endemic to north central Colorado, southeast­ The 3 populations selected for study are ern Wyoming, and a small, adjacent portion of located on, and designated as, Crow Creek (Ce), western Nebraska. Currently, 18 extant popula­ Diamond Creek (DC; a tributary of Crow tions are known, a decrease from 26 locations Creek), and "unnamed drainage" (DD; Fig. 1). recorded in the past (Fertig 2001). Two of the The first 2 populations are nearly contiguous largest populations are within the EE. Warren at the confluence of the 2 streams, while the Air Force Base (WAFB) in Cheyenne, Wyoming, 3rd site contains an ephemeral stream that and are managed within the Colorado Butter­ drains into Crow Creek, somewhat downstream fly Plant Research Natural Area. ofthe other 2 populations. Diamond Creek and The subspecies is restricted to drainage bot­ "unnamed drainage" populations were sampled toms, low alluvial terraces, and abandoned and numbered from west to east along the stream channels with short, sparse vegetative drainages, but the Crow Creek population was cover. The do not thrive where shrubs sampled from just a single site, the only appar­ such as Salix spp. or noxious weeds like Cir­ ent remaining population in the Crow Creek sium arvense (L.) Scop. or Euphorbia esulo L. area. The 3 populations combined extend approx­ have become dominant. Annual surveys of the imately 3 km from uorth to south and 2 km Colorado butterfly plant have been conducted east-west and may represent remnants of a on WAFB since 1984, so that distribution, size, single continuous population that existed before and trends within the populations there are settlement. This area, and Crow Creek in well understood (Fertig 2001 and citations with­ particular, are also within the range of Preble's in). In addition, the u.s. Air Force has spon­ jumping mouse (Zapus hw:lsonius preblei), sored research on weed management (Floyd another federally listed threatened species. 1995a, Hollingsworth 1996, Munk 1999, Heim­ The purpose of this study was to determine stra and Fertig 2000), population genetics the extent of inter- and intra-site variation in

IDepartment ofBotany, University ofWyoming, Laramie, WY 82071.

251 252 WESTERN NORTH AMERICAN NATURALIST [Volume 63

appropriate for PCR amplification (-200­ 2000 bp), a product is formed that consists of the 2 terminal microsatellites and the inter­ vening DNA, all of which may harbor length polymorphism.

METHODS AND MATERIALS F.E. Warren AFB Previous experience (Brown 1999) had shown the procurement of PCR-quality DNA from Gaura to be problematic, due to the 1 mile presence ofmucilage that co-precipitated with the DNA. Therefore, the initial portion of this study focused on ways to eliminate or reduce the amount of mucilage through modification !II Crow creek of tissue-collection methods, DNA-extraction ~ Diamond Creek methods, and post-extraction cleanup. All leaf OJ Unnamed Drainage samples for this preliminary study were col­ lected midday from the "unnamed drainage" site, 1 September 1999. Tables 1 and 2 contain complete descriptions ofthe method modifica­ tions. Once the trials were complete, we collected samples for the major part of the study. For each of the 3 populations, we collected 50 leaf samples, each from a different plant, on 7 Sep­ Fig. L Location of Colorado butterfly plant populations tember 1999. Leaves were immediately placed in RE. Warren Air Force Base. Diamond Creek and in silica gel, stored at room temperature for 24 "unnamed drainage" populations were sampled from west hours, then frozen (option 1, Table 1). DNA to east along the drainages. The Crow Creek population was sampled at only a single site, marked with *. extraction followed the standard 2X CTAB protocol (Doyle and Doyle 1987), followed by cleanup with Qiagen DNeasy plant kit (Qiagen, Inc., Valencia, CAl. Cleanup was repeated as the Colorado butterfly plant to facilitate man­ necessary until no more mucilage was apparent agement decisions that will affect maintenance in the sample. Typically this required 1 or 2 ofboth threatened taxa. applications. Among the number of PCR-based proce­ The 25 ~L PCR reactions consisted of IX dures that have been developed in recent buffer (Sigma; 10 mM Tris-HCI, pH 8.3, 50 years to assess molecular variation, inter-simple mM KCI, 1.5 mM MgClz, 0.0001% gelatin), sequence repeats (ISSRs) have many advantages additional 50 nmol MgClz, 8 nmol dNTPs, 6 in terms of speed, number of polymorphisms nmol primer, 10 ~g BSA, 5 ~L saturated be­ detected, and reproducibility (Zi~tkiewicz et taine, 1 U Taq polymerase, and 2 ~L DNA. al. 1994). They have been used successfully to Thermal cycling parameters were initial dena­ assess variation in a variety of plants (e.g., God­ turing for 2 minutes at 96°C, followed by 40 win et al. 1997, Leroy et al. 2000, Li and Ge cycles of 30 seconds at 96°C, 45 seconds at 2001, Meekins et al. 2001). Although ISSRs rely 44°C, and 90 seconds at 72°C, with a final on the presence of simple sequence repeats elongation at 72°C for 10 minutes. Primers (SSRs), no knowledge of the surrounding se­ were purchased from the University of British quence is required, as is the case for micro­ Columbia, Nucleic Acid-Protein Service Unit. satellite markers. ISSR primers are designed We tried many primers and selected 12 based to complement the SSRs themselves and may on the number of scorable bands generated possess ODe or more bases at either end to (Table 3). A number of the primers, including anchor the primer to the end of the SSR. When all based on repeats of AT, failed to produce 2 SSRs are present in the genome at a distance any bands. 2003] ISSR VARIATION IN GAURA 253

TABLE 1. Leaf tissue-collection methods attempted for Gaura neomexicana ssp. coloradensis. All collection trials used plants from the "unnamed drainage" population, RE. Warren AFB. Only healthy, undamaged leaves were used. Each trial was replicated twice. All leaves were collected midday, 1 September 1999.

1. Leaves were placed in a clean 50-mL plastic, screw­ 6. Leaves were placed in a small, sandwich-sized zip~ top centrifuge tube. Thbe was filled with 200-mesh type plastic bag. The bag was closed and placed on silica gel (desiccant), lid was firmly attached, and dry ice for transport back to the lab. Immediately tube placed in a styrofoam ice chest to keep cool and upon return to lab, leaves were extracted for DNA. out ofdirect sunlight. After leaves were fully dried 7. Leaves were placed in a small, sandwich-sized zip­ (ca. 24 hours), tubes were placed in a lab freezer type plastic bag. The bag was closed and placed on (-20°C) and held there until extraction. dry ice for transport back to the lab. Immediately 2. Leaves were placed in a clean 50~mL plastic, screw­ upon return to lab, bags were transferred to a lab top centrifuge tube. Tube was filled with 95% ethanol freezer (-20°C) and held there until extraction. containing 100 roM EDTA, lid was firmly attached, 8. Leaves were ground to a fine powder in liquid nitro­ and tube placed on wet ice in ice chest. Immediately gen while in the field, and the powder transferred to upon return to lab, tubes were transferred to a lab a clean 50-mL plastic, screw-top tube containing 10 freezer (-20°C) and held there tmtil extraction. mL of2X crAB extraction buffer. Tube was immedi­ 3. Leaves were placed in a field press while in the field. ately placed on dry ice for transport back to the lab. Immediately upon return to lab, press was placed on Immediately upon retum to lab, the extraction was plant drier and kept there until leaves were fully dry completed. (ca. 36 hours). Dried leaves were kept at room tem~ 9. Leaves were ground to a fine powder in liquid nitro­ perature in a lab cabinet until extracted. gen while in the field, and the powder transferred to 4. Leaves were placed in a small, sandwich-sized zip­ a clean 50-mL plastic, screw-top tube containing 20 type plastic bag. The bag was closed and placed on mL of2X CTAB extraction buffer. Tube was immedi­ wet ice for transport back to the lab. Immediately ately placed on dry ice for transport back to the lab. upon return to lab, leaves were extracted for DNA. Immediately upon retum to lab, the extraction was completed. 5. Leaves were placed in a small, sandwich~sized zip­ type plastic bag. The bag was closed and placed on wet ice for transport back to the lab. Immediately upon return to lab, bags were transferred to a lab freezer (-2O°C),and held thereuntil extraction.

Initially, all 150 samples were subjected to RESULTS PCR with 3 different primers. Because the level of variation seen in these analyses was Of the 9 tissue-collection methods tested, more or less uniform across the 3 populations, none successfully eliminated the mucilage. In and because of logistic problems associated fact, methods that included leaf grinding in with analysis of that number of samples, we the field (options 8, 9, Table I) resulted in decided that a random sample of 8 individuals increased mucilage levels. The method that from each population would adequately sample resulted in the least amount ofmucilage being variation. The reduced sample size allowed us released involved placing each leaf into a 50­ to evaluate more primers, and therefore more mL screw-top tube, filling the tube with des­ polymorphism, in the given time frame. iccated silica gel, capping the tube, storing it ISSR reactions were visualized follOwing at room temperature until dry followed by electrophoresis on 1.5% agarose stained with storage at -20°C until DNA extraction. Like­ ethidium bromide and photographed on a UV wise, modifications of the basic 2X CTAB ex­ transilluminator. We scored bands directly from traction protocol were not successful. The sys­ the gel photos as present (I) or absent (0) for tem that worked best was the standard protocol each sample (data matrix available from the followed by cleanup with the Qiagen DNeasy authors). Missing data points were scored as 9. plant kit. NTSYS-pc 2.0 (Rohlf 1997) was used for clus­ We scored a total of 88 bands, with the ter analysis with Dice similarity coefficient number ofbands per primer ranging from 5 to and principal coordinate analysis (PCO), also 14 (Table 3). Failed reactions resulted in miss­ with Dice similarity. ing data for only 72 of 2Il2 matrix cells (3.4%). 254 WESTERN NORTH AMERICAN NATURALIST [Volume 63

TABLE 2. Trial modifications of the standard 2X crAB DNA extraction protoool (Doyle and Doyle 1987) to improve the quality of DNA yield in Gaura neomexicana ssp. coloradensis. Judgment on improved DNA quality was based on results visible in gel photograph, compared with DNA extraction using unmodified 2X CTAB protocol. Modified step Improved DNA quality 1 Increase crAB detergent from 2% to 6%. None observed 2 Reduce ratio oftissue to extraction buffer volume by 50%. None observed 3 Grind leaftissue in ice-cold extraction buffer, not in liquid nitrogen. None observed 4 Omit freezing ofextraction buffer/ground leafslurry prior to incubation. None observed 5 Insert DNAwl Esa purification treatment of DNA-mucilage pellet None observed after first DNA precipitation. 6 Soak DNA-mucilage pellet in 8mM NaOH for 24 hours; process supernate. None observed 7 Insert a 2nd chloroform-isoamyl alcohol partition ofaqueous phase. None observed 8 Substitute ammonium acetate for sodium acetate in the 2nd precipitation. None observed 9 Replace entire crAB protocol with TIiReagentb extraction protocol. None observed 10 After standard CTAB extraction, clean DNA-mucilage sample with Positive, but not DNeasy plant kite, following instructions. universally so aMoleclllar Research Genre!; Inc., Cincinnati, 08 bSigma Chemical Co., St. 1.oll;$, MO "Qiagen, Inc., Valencia, CA

TABLE 3. ISSR primers producing data analyzed in this study. Any of the nucleotides together in parentheses may occupy that position. Primer number 5' to 3 ; sequence Repeat motif # ofbands scored 816 CT=crCTCfCIT -cr- 5 829 TGTGTGTGTGTGTGTGC -TG- 7 834 AGAGAGAGAGAGACAG(CI}T -AG- 8 841 CACACACAGAGACAGA(CI}C -GA- 13 855 ACACACACACACACAC(CT)T -AT- 5 860 TGTGTGTGTGTGTGTG(AG)A -TC- 7 864 ATGATGATGATGATGATG -ATG- 7 866 crccrccrCcrCcrCCTC -crc- 5 868 GAAGAAGAAGAAGAAGAA -CAA- 6 880 CCAGACGAGACGAGA -GGAGA- 14 884 (ACT)(CGT)(ACI}AGAGACACAGAGAC -AG- 6 885 (CGT)(ACT)(CGT)GACACAGAGAGAGA -GA- 5

•• Only 3 samples (UD41, UD42, UD50) had •• missing data points, and most of these (46 of :• 72) were from individual UD50. • Cluster analysis indicated a high degree of • i. :• similarity across the 3 populatious (Fig. 2). All -c::! samples, with the single exception of UD50, ~ were at least 80% similar, and in many cases -[ •* 0049 individuals were more similar to members ofa different population than to members of their I ~, own population. UD50 is very differeut; this "'~ •• o. may be a reflection of the many missing data Dice Similarity Coefficient points or an indication of true differences in the genome. Disregarding the 46 missing data Fig. 2. Cluster analysis based on presence/absence of88 points, this individual was unique in 9 of the ISSR bands, using Dice similarity coefficient. Matrix cor­ remaining 42 characters. relation = 0.956.• denotes individuals from Crow Creek population; * denotes individuals from Diamond Creek Principal coordinate analysis (Fig. 3) pro­ population; _ denotes individuals from "unnamed drain­ vided a different picture of variation among age" population. 2003] ISSR VARIATION IN GAURA 255

o,'~ • • • • .* '.$ • • .UD50 • UD41 • • • • Axis 2 O.Q~ • • *0019• • 4" • • •

• CC10 ••• 4" -11.53 ••• -II.D7 0.1$ Axis 1

Fig. 3. Principal coordinate analysis based on Dice similarity. Variation accounted for by axis 1 = 23.7% and by axis 2 = 12.5%.• denotes individuals from Crow Creek population; * denotes individuals from Diamond Creek population; • denotes individuals from "unnamed drainage" population.

the 3 Gaura populations: UD4I and UD50, responsible for a significant decrease in the again, are unique, but the remaining individu­ size of the Gaura population at that site. In als fall into 2 groups, 1 containing all members fact, from 1986 (the 1st year of monitoring) of the Crow Creek population plus DC49 and through 1990, the Crow Creek site population the other containing the rest of the "unnamed accounted for approximately 40% of the total drainage" and Diamond Creek members. Gaura count for the 3 sites; in 2001 it accounted for only 12% of the total (Heidel 2002). There DISCUSSION may be some strong selective pressures on plants establishing at that site, compared to The high degree of similarity displayed in plants recruited into the other 2 sites. This the cluster phenogram is not surprising, given may account for the observed genetic diver­ the spatial proximity and presumably close gence of the Crow Creek population from genetic relationship ofthe populations at the 3 the Diamond Creek and "unnamed drainage" sampling sites. Both analyses indicate that, populations. genetically, individuals from the Diamond Creek However, historic differences between the and "unnamed drainage" populations are sites could also be responsible for the separa­ indistinguishable, although both populations tion. The soil is moister at the Crow Creek contain "genetic outliers" (DC49, UD41, UD50). site, and plants there have been observed to The evidence suggests that these 2 populations flower and set seeds later in the season than are members ofa single gene pool, which could elsewhere (W Fertig personal communication). result from contemporary cross-pollination or This temporal difference in flowering could be an artifact, if these populations are rem­ lead to the distinct genetic structure. In fact, nants ofa once larger, continuous population. we may be seeing only the remains of a once The PCO separation of the Crow Creek greater distinction, if the Crow Creek popula­ population from the other 2 populations is tion is currently interbreeding with the now­ intriguing, since this site is currently nearly adjacent Diamond Creek population. contiguous with the Diamond Creek site. How­ Finally, the separation could be an artifact ever, the Crow Creek site has recently become of the limited sample size. A larger sample dominated by willow (particularly Salix exigua may reveal genotypes that bridge the gap be­ Nutl. and S. eriocephala Michx. var. ligulifolia tween Crow Creek and the other sites. One [Ball] Dorn; Fertig 2001), and this has been Diamond Creek plant, DC49, is genetically 256 WESTERN NORTH AMERICAN NATURALIST [Volume 63 more like Crow Creek individuals than others Management Office, EE. Warren Air Force ofDiamond Creek or the"unnamed drainage" Base, Cheyenne, Wyoming. (Fig. 3). It is possible that more individuals within the Diamond Creek population are LITERATURE CITED genetically related to DC49. From a management perspective, it will be BROWN, G.K. 1999. Exploratory molecular characterization of Gaum neomexicana ssp. coloradensis-variability necessary to preserve at least 2 of the 3 popu­ in the RE. Warren Air Force Base populations. Report lations, 1 of which must be Crow Creek, to submitted to Environmental Management Office, maintain maximum diversity within the taxon. RE. Warren AFB. The Diamond Creek and "unnamed drainage" ---'C":' 2000. Inter-simple sequence repeat (ISSR) varia­ populations are currently thriving, and there is tion in three populations of Gaura neomaxicana ssp. coloradensw, RE. Warren Air Force Base, Cheyenne, no apparent need to choose between them. Wyoming. Report submitted to Environmental Man­ However, the "unnamed drainage" population agement Office, FE. Warren AFB. harbors some unique alleles (in UD41 and DARK-SMILEY, D., AND D.A. KEINATH. 2002. Survey for UD50) that are not present in the other 2 pop­ Preble's meadow jumping mice on RE. Warren Air Force Base, Wyoming, 2001. Report prepared for the ulations. For this reason the "unnamed drain­ U.S, Air Force by the Wyoming Natural Diversity age" population is an obvious candidate for Database, Laramie. preselVation. DOYLE, J.J., AND ].L. DOYLE. 1987. A rapid DNA isolation The Crow Creek population represents a procedure for small quantities of fresh leaf tissue. genetic composition shared apparently by only Phytochemical Bulletin 19:11-15. FERTIG, W, 2001. 2000 census of Colorado butterfly plant a small number of Diamond Creek individuals (Gaura neomexicana ssp. coloradensw) on F.E. War­ and not at all by members of the "unnamed ren Air Force Base. Report prepared for the u.s. Air drainage" population. Unfortunately, this pop­ Force by the Wyoming Natural Diversity Database, ulation is threatened by encroachment of wil­ Laramie. FLOYD, S.K. 1995a. Experimental control of Canada this­ low and the need to manage for Preble's jump­ tle within the Gaum neomexicana ssp. coloradensis ing mouse, which prefers such densely vege­ Research Natural Area on F.E. Warren Air Force tated riparian areas (Dark-Smiley and Keinath Base and recommendations for continued monitor· 2002). However, the conflicting habitat prefer­ ing of the Gaura population. Unpublished report ences of the 2 threatened taxa may not be a prepared for the Wyoming Nature Conservancy. _--,-' 1995b. Population structure, dynamics, and genet­ management problem. Population segments of ics of Gaura neomexicana ssp. coloradensis (Dna­ the Colorado butterfly plant persist in patches graceae), a rare, semelparous perennial. Master's thesis, as small as 20 m2 (Heidel 2002), and jumping Department ofEnvironmental, Population and Organ­ mice home ranges are several orders ofmagni­ ismic Biology, University ofColorado, Boulder. FLOYD, S. K, AND T. RANKER. 1998. Analysis ofa transition tude larger than that and often encompass matrix model for Gaura neomexicana ssp. coloraden­ many patches of open riparian habitat (Dark­ sis (Onagraceae) reveals spatial and temporal demo­ Smiley and Keinath 2002). Therefore, it should graphic variability. International Journal of Plant be possible to manage several small patches of Sciences 159:853-863. Colorado butterfly plant habitat in the Crow GODWIN, J.D., E. AITKEN, AND L.w. SMITH. 1997. Applica­ tion of inter-simple sequence repeat (ISSR) markers Creek area without substantially affecting to plant genetics. Electrophoresis 18:1524-1528. habitat quality for the other threatened taxou. HEIDEL, B. 2002. 2001 census of Colorado butterfly plant (Gaura neomexicana ssp. coloradensis) on FE. Warren ACKNOWLEDGMENTS Air Force Base, Wyoming. Report prepared for the U.S. Air Force by the Wyoming Natural Diversity Database, Laramie. The authors wish to thank Walter Fertig, HEIMSTRA, C., AND w: FERTIG. 2000. The distribution of Wyoming Natural Diversity Database, for assis­ noxious weeds on F.E. Warren Air Force Base. Report tance in the field, for sharing his knowledge of prepared for the U.S. Air Force by the Wyoming the Colorado butterfly plant, and for comments Natural Diversity Database, Laramie. HOLLINGSWORTH, C. 1996. Canada thistle (Cirsium arvense) on the manuscript. Dr. Gary Beauvais and and leafy spurge (Euphorbia esula) biological control Bonnie Heidel, also of the Wyoming Natural program as proposed for the Colorado Butterfly Plant Diversity Database, were generous with their Research Natural Area on FE. Warren Air Force knowledge. In addition, the authors thank 2 Base. Report prepared for the Wyoming Nature anonymous reviewers whose comments led to Conservancy. LEROY, X.J., K. LEON, AND M. BRANCHARD. 2000. Charac­ improvements ofthe manuscript. This work was terisation of Brassica okracea L. by microsatellite funded by a contract from the Environmental primers. Plant Systematics and Evolution 225:235-240. 2003] ISSR VARlATlON IN GAUIlA 'lE7

Lr, A., AND S. CE. 2001. Genetic variation and clonal di­ ROHLF, F.J. 1997. NTSYS-pc: numerical and mul­ versity of Psamnwchka oiUasa (Poaceae) detected by tivariate analysis system. Version 2.0. Exeter Software, ISSR markers. Annals of Botany, London 87:.585-590. Setauket, NY, MEEKINS, J.F., H,E. BALLARD. JR., AND B.C. McCARTHY. ZI~TKJEW1CZ, E., A. RAFALSKI, AND D. LABUDA. 1994. 2001. Cenetic variation and molecular biogeography Genome fingerprinting by simple sequence repeat of a North American invasive plant species (Alliaria (SSR}-anchored polymerase chain reaction amplifi· peticlaJa, Brassicaceae). International Journal of cation. Genomics 20:17&-183. Plant Sciences 162:161-169. MUNK, L. 1999. Colorado butterfly plant regeneration with Recriv

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