Genetic Risk Assessment of a Threatened Remnant Population of Hairy Prairie-Clover (Dalea villosa var. villosa) in the Canadian Prairie

Transcription

1 Diverity 011, 3, ; doi: /d OPEN ACCESS diverity ISSN Article Geetic Rik Aemet of a Threateed Remat Populatio of Hairy Prairie-Clover (Dalea villoa var. villoa) i the Caadia Prairie Yog-Bi Fu *, Gregory W. Petero ad Ke W. Richard Plat Gee Reource of Caada, Sakatoo Reearch Cetre, Agriculture ad Agri-Food Caada, Sakatoo, SK S7N 0X, Caada; Gregory.Petero@agr.gc.ca (G.W.P.); Ke.Richard@agr.gc.ca (K.W.R.) * Author to whom correpodece hould be addreed; Yog-Bi.Fu@agr.gc.ca; Tel.: ; Fax: Received: 4 Jue 011; i revied form: 13 July 011 / Accepted: 8 July 011 / Publihed: 8 Augut 011 Abtract: Hairy prairie-clover [Dalea villoa (Nutt.) Spreg. var. villoa] i a threateed Caadia wildflower. To facilitate the effort of coervig thi threateed plat, amplified fragmet legth polymorphim (AFLP) techique wa applied to ae geetic diverity i a remat hairy prairie-clover populatio i the Caadia Prairie. Three AFLP primer pair were employed to geotype 610 idividual plat from the populatio ad 15 plat from a North Dakota compoite populatio, ad 100 polymorphic AFLP bad were aalyzed. The aayed plat diplayed 3% AFLP variatio preet betwee the remat populatio ad the North Dakota compoite populatio, but maitaied a high level (91%) of AFLP variatio withi patche of the remat populatio. The idividual geetic ditictivee meaured by average AFLP diimilarity wa poitively aociated with latitude ad egatively with elevatio. The amog-patch AFLP variatio wa igificatly related to iter-patch ditace, idicatig local geetic differetiatio withi the remat populatio. However, the proportio of withi-patch AFLP variatio were ot aociated with ay patch characteritic aeed (i.e., patch ize, perimeter, earet eighbor ditace, mea iter-patch ditace). No fie-cale geetic tructure wa foud withi three large patche, uggetig little geetic correlatio preet for plat five meter apart. Some geetically ditictive ad divere patche were alo idetified. Thee fidig idicate that the geetic rik of the remat hairy prairie-clover populatio i the Caadia Prairie i low.

2 Diverity 011, Keyword: Dalea villoa; edagered plat pecie; patch heterogeeity; geetic diverity; geetic rik; AFLP; coervatio geetic 1. Itroductio The lat decade ha ee a ubtatial icreae i the umber of threateed plat pecie worldwide [1]. Coiderable effort have bee made to protect the remat populatio of threateed plat pecie [,3]. However, iufficiet effort have bee made to ae the geetic rik of the threateed plat pecie [4,5]. Moder molecular techique make a marker-baed geetic diverity aalyi more feaible tha before to poorly kow plat pecie [6]. The marker-baed aalyi ca accurately meaure the extet ad correlative chage of geetic diverity with ifluecig evirometal ad habitat factor i mall plat populatio ad ha bee uccefully applied to may threateed plat pecie [4,7,8]. Thu, a iformative aemet o geetic rik of threateed remat plat populatio i poible. Udertadig geetic variatio of plat pecie i importat for predictig the evolutioary potetial of the pecie ad for formulatig the effective trategie of coervig remat plat populatio [,9]. Remat populatio ca be theoretically predicted to have greater chace of deterioratig i geetic variatio ad becomig icreaigly differetiated due to fouder effect at the time of fragmetatio, icreaed radom geetic drift, elevated ibreedig, ad reduced iterpopulatio gee flow [10-1]. I the log term, thee mall populatio may have reduced ability to adapt to chagig eviromet ad potetially become extict [4,13,14]. Coequetly, remat populatio have gaied attetio i coervatio ad bee ubjected to reearch. However, empirical tudie of mall, iolated populatio have yielded variable reult [9,1]. Cae have bee reported i which fragmeted populatio of ome widepread plat pecie promoted gee flow ad were ot detrimetal [15-17]. Several factor repoible for uch dicrepacy have bee propoed uch a iterpecific logevity differetial, pre-fragmetatio abudace, differet matig ytem, the preece of eed bak, ad iteractio of aimal populatio ad eed diperal [1]. The ifluece of thee factor o the extet ad chage of exitig geetic diverity durig habitat fragmetatio are, however, le well udertood [18,]. Further reearch effort are warrated to ae variable geetic effect of habitat lo o edagered plat [0,]. Hairy prairie-clover [Dalea villoa (Nutt.) Spreg. var. villoa] i a member of the Fabaceae or pea family ad ha become a threateed Caadia wildflower (Global Rak: G5T5; COSEWIC tatu: threateed; Sakatchewa Provicial Rak: S1, ad Provicial Protectio Statu: edagered). It i foud i cetral North America, from Maitoba ad Sakatchewa outh to New Mexico ad Texa, ad from Colorado eat to Michiga [], ad become rare i Sakatchewa, Maitoba, Motaa, Iowa ad Wyomig. Thi pecie i retricted to ady ite or ad hill blowout. Survival of the pecie i primarily threateed by the procee of ad egatively ifluecig factor for ad due tabilizatio []. A due cotiue to become vegetated via ivaive weed pecie like leafy purge (Euphorbia eula L.), the cotiuou lo of habitat for thi pecie i expected.

3 Diverity 011, The hairy prairie-clover plat i a pereial with a woody taproot ad caudex ad reproduce by eed. I the Caadia Prairie, the plat flower from late July to late Augut ad et eed i September [3]. The total Sakatchewa populatio wa etimated to be i the low to mid-hudred [3]. Coervatio effort have bee iitiated ad a recovery trategy i beig developed to protect thi pecie i the Caadia Prairie by field aemet, coervatio trategy formulatio, ad critical habitat reearch [4]. However, little i kow about the biology of thi pecie ad the geetic rik of mall remat patche []. To facilitate the effort to coerve the remat hairy prairie-clover populatio, we coducted a geetic rik aemet of a threateed populatio of hairy prairie-clover i the Caadia Prairie uig amplified fragmet legth polymorphim (AFLP) techique. The AFLP marker [5], although cored domiatly (i.e., without ditictio betwee homozygote ad heterozygote) ad ot alway homologou [6], ha bee widely applied to ae geetic variability i may plat pecie ad i a robut, highly effective tool for poorly kow plat [7-9]. The pecific objective of thi tudy were: (1) to ae geetic variatio ad patial tructure withi the remat hairy prairie-clover populatio ad () to evaluate the aociatio of geetic variatio with patch characteritic uch a patch ize ad perimeter.. Material ad Method.1. Plat Samplig ad Tiue Collectio The remat hairy prairie-clover populatio tudied here wa located i the Caadia Prairie ad maaged by the Agri-Evirometal Service Brach, Agriculture ad Agri-Food Caada (AAFC) (Figure 1). The populatio ha paed over a area of approximately 13 quare kilometer. Patche of variable ize ragig from a few to hudred of plat are cattered over parely-vegetated active ad due or ad hill blowout i a moaic of foret, hrublad, gralad ad barre ad patche. Some commoly aociated pecie iclude it cloe relative D. purpurea Vet., Stipa comata Tri. & Rupr., Calamovilfa logifolia (Hook.) Scrib., ad Roa woodii Lidl. [3]. Two amplig trategie were applied i thi tudy. Ditributio-focu amplig elected plat from a may mall patche a poible to icreae the coverage of the exitig plat ditributio acro the remat populatio. Thi amplig yielded a collectio of 188 plat from more tha 70% of the previouly idetified patche, particularly thoe of ize a few plat oly. Patch-focu amplig icreaed amplig iteity withi the elected patche of variable ize to ehace the aemet of geetic variability ad patchy characteritic. Thi amplig yielded a collectio of 4 plat from defied patche (i.e., thoe patche with clear boudary ad iolatio; Figure 1). I either amplig trategy, plat at leat 5 m apart from each other were radomly elected to miimize the amplig of cloe geetic relative ad the GIS iformatio for each elected plat wa recorded. Youg ad growig leaf tiue from 4 cm log brach tip of elected plat were idividually collected i paper evelope ad immediately tored i cool cotaier with dry ice. O the ame day, the collected tiue were traported to ad re-tored at 80 C at Plat Gee Reource of Caada, Sakatoo Reearch Cetre, Agriculture ad Agri-Food Caada. The collected ample were later freeze-dried ad kept at 0 C for AFLP aalyi.

4 Diverity 011, Figure 1. Locatio of the remat populatio of hairy prairie-clover i the Caadia Prairie ad ditributio of defied patche with clear boudary ad iolatio. To compare the geetic diverity of thi remat populatio with other populatio, a compoite populatio of multiple ource repreetig the wide ditributio of hairy prairie-clover plat i North Dakota wa obtaied from USDA-NRCS Plat Material Cetre, Bimarch, North Dakota. Seed were grow i a greehoue at the Sakatoo Reearch Cetre ad youg leaf tiue were idividually collected from 15 growig eedlig for thi tudy... AFLP Aalyi Geomic DNA wa extracted from 50 mg of freeze-dried leaf tiue uig the E.Z.N.A. Plat DNA mii-kit (Omega Bio-Tek, Doraville, GA, USA) pi method. Extracted DNA wa quatified fluormetrically with Hoecht 358 dye (Sigma Life Sciece, Oakville, ON, Caada) agait kow cocetratio tadard ad diluted to 5 g µl 1 with water. AFLP aalyi wa coducted accordig to Vo et al. [5] uig retrictio ezyme Eco RI ad Me I (New Eglad Biolab, Pickerig, ON, Caada). Selective amplificatio wa preformed with primer pair: E+AGG/M+CTG, E+ACC/M+CAG, ad E+ACG/M+CTG with the Eco RI primer radiolabelled with γ- 33 P-dATP (Perki-Elmer, Waltham, MA, USA) ad eparated o 35 cm 50 cm 0.4 mm, 5% (:1) acrylamide:biacrylamide, 7 M urea, deaturig polyacrylamide gel. Gel were traferred to Whatma 3MM blottig paper, dried, ad expoed agait Kodak BioMax MR film for oe to four day at 80 C. Three additioal radomly elected ample were duplicated acro all the gel a cotrol to miimize techique-related ad corig error. Data from the autoradiogram were cored maually.

5 Diverity 011, Data Aalyi The preece/abece AFLP data for all the ample were aalyzed for the level of polymorphim by coutig the total umber of bad ad the umber of polymorphic bad, calculatig the proportio of polymorphic bad, ad geeratig mea bad frequecie with repect to primer ad populatio. Thi wa doe uig a SAS program writte i SAS IML [30]. To ae AFLP variace compoet ad tet their tatitical igificace level, aalyi of molecular variace (AMOVA) wa performed uig Arlequi 3.0 [31] with repect to group (populatio or patch). Thi aalyi allow for partitioig of the total AFLP variatio ito withi- ad amog-group compoet, meaurig iter-group ditace a a proportio of the total AFLP variatio reidig betwee ay two group (Phi tatitic), ad etimatig group-pecific Ft []. Four model of geetic tructurig were made: (1) remat v compoite populatio; () ditributio-focu v. patchfocu ample; (3) all defied patche; ad (4) defied patche of ize 10 or larger. The igificace of variace compoet ad iter-group ditace wa teted with 10,100 radom permutatio. The optimal geetic tructure wa iferred uig a model-baed Bayeia method available i the BAPS oftware [33]. Cluterig of idividual plat wa doe uig the model for o-liked marker ad 0 replicate ru of the algorithm with the upper-boud value (K) for the umber of cluter ragig betwee ad 30. Recetly, the BAPS aalyi wa foud to diplay more advatage i iferrig geetic tructure over the STRUCTURE [34,35] aalyi (e.g., ee [36-38]). The advatage largely reflect it power of etimatig optimal cluter umber ad admixture coefficiet with much le computatio time. To ae the geetic aociatio of the 65 idividual plat, a eighbor-joiig aalyi wa alo coducted uig PAUP* [39] ad a radiatio tree wa diplayed uig MEGA 3.01 [40]. The idividual geetic diimilarity wa etimated uig the imple matchig coefficiet [41] ad followig Fu [4] a the average AFLP diimilarity of a ample agait the remaiig ample. A higher average diimilarity obtaied from uliked marker mea that the ample ha a geetic backgroud more ditict from the other ample [4]. Thi aemet wa doe uig a SAS program writte i SAS IML [30]. Liear regreio were eparately performed of idividual geetic ditictivee etimate over idividual poitio characteritic uch a latitude, logitude, ad elevatio. To ae the geetic aociatio of plat amog the aayed patche, a dedrogram wa geerated from the iter-patch geetic ditace matrix uig the uweighted pair-group method, arithmetic average (UPGMA) cluterig procedure i NTSYS-PC.01 [43]. The Matel tet wa carried out to ae the correlatio betwee patch geetic ditace ad patch differece i ditace, elevatio, ad ize. Simple liear regreio were eparately made of withi-patch AFLP variatio over patch characteritic uch a patch ize (i term of polygo area), patch perimeter, earet eighbour ditace, ad mea iter-patch ditace. Some of thee patch characteritic were provided by Dr. Darcy Hedero, Eviromet Caada. Fie-cale patial geetic tructure withi a patch wa aeed uig GeAlEx v6 oftware [44] for three defied patche of the larget ample ize ad geetic ditace at idividual level were geerated. Matel tet wa applied to ae the aociatio of idividual geetic ditace with idividual poitio characteritic uch a latitude, logitude ad elevatio.

6 Diverity 011, Reult ad Dicuio 3.1. AFLP Variatio The three AFLP primer combiatio detected a total of 131 DNA fragmet amog the 65 idividual, of which 18 (97.7%) fragmet were polymorphic ad cored. However, oly 100 polymorphic bad diplayed a coitecy of preece or abece i all the cotrol ample acro all the gel ad thu were ued for further data aalyi (Table 1). The primer pair E+ACC/M+CAG had the highet umber of polymorphic bad (35), while the primer pair E+AGG/M+CTG had the highet mea bad frequecy of The frequecie of all the polymorphic bad raged from 0.00 to ad averaged A ubtatial proportio of the polymorphic bad (i.e., 45%) had frequecie maller tha Thee frequecy ditributio ugget that the aayed primer combiatio ampled variou part of hairy prairie-clover geome. The fidig of the high level (97.7%) of AFLP polymorphim idicate that thi pecie i geetically highly heterogeeou ad i coitet with the obervatio of the outcroig ature of thi pecie by bee-polliatio, although the extet of outcroig remai ukow. Two cloely related pecie D. purpurea Vet. ad D. cadida Michx. ex Willd. are both highly outcroig, beig polliated by a diverity of bee ad fly pecie [45]. Such a high level of AFLP polymorphim i expected for a outcroig plat pecie [46] ad i compatible with thoe AFLP fidig reported i other outcroig plat pecie ative to North America [7-9,47,48]. Table 1. Patter of amplified fragmet legth polymorphim (AFLP) i the remat populatio of hairy prairie-clover with repect to AFLP primer pair. Primer Pair Percetage of polymorphic bad oberved Number of polymorphic bad ued Frequecy of polymorphic bad Mea Rage E+AGG/M+CTG E+ACC/M+CAG E+ACG/M+CTG All primer Populatio Variatio Aalyi of AFLP variatio revealed 3.3% AFLP variatio preet betwee the remat populatio i Sakatchewa ad the compoite populatio from North Dakota (Table ). The populatio-pecific proportioal variatio (Ft) wa i the remat populatio ad i the compoite populatio. Thee populatio-pecific Ft value meaure the extet to which each populatio differ geetically from the combied populatio a a whole [49], thu uggetig a little more diticte for the Sakatchewa threateed populatio tha for the North Dakota compoite populatio i the cetre ditributio of the pecie. Such AFLP fidig i omehow expected, a the plat from the compoite populatio may repreet variou eed ource i North Dakota. Withi the remat populatio, the plat collected from ditributio-focu ad patch-focu amplig diplayed o-igificat differetiatio i AFLP variatio (Table ). Thi ugget that the two amplig

7 Diverity 011, trategie yielded little differece i amplig AFLP variatio. The patter of patch AFLP variatio preeted ad dicued i the later ectio hould be repreetative for the whole remat populatio. Table. The reult for the aalyi of molecular variace of 65 aayed plat of hairy prairie-clover with four model of geetic tructurig. Model ad ource of variatio df Variace compoet % variatio Remat v compoite populatio Betwee populatio **** Withi populatio Ditributio- v patch-focu ample Betwee group Withi group All defied patche Amog patche **** Withi patche Defied patche (of ize >9) Amog patche **** Withi patche Note: i for o-igificace at P > 0.05; **** for tatitical igificace at P < The model-baed Bayeia iferece of the geetic tructure amog the 65 plat ampled from the remat ad compoite populatio revealed five optimal cluter with the highet log likelihood of 13,578.6 (Figure A). Oly three plat (0.5%) had multiple memberhip of variou cluter. The cluter ize raged from 18 for cluter 5 to for cluter 3. The mallet, cluter 5, coited of 15 idividual from the North Dakota compoite populatio ad three plat from the remat populatio ad wa the mot diverget cluter (Figure B). The memberhip compoitio of other four cluter wa ot uique with repect to plat locatio ad patch. Overall, the geetic tructure preeted i the four optimal cluter for the remat Sakatchewa populatio wa weak (Figure B). The aemet of idividual geetic aociatio from both remat ad compoite populatio revealed ot oly the heterogeeou ature of thi pecie, but alo little diticte of the hairy prairie-clover plat i the North Dakota compoite populatio from the remat Sakatchewa populatio (Figure 3). The aayed plat were looely clutered ito five cluter, imilar to the model-baed tructure iferece (Figure ). Thee cluter did ot eem to be aociated with the plat locatio, or with the defied patche. More iteretigly, the plat from the North Dakota compoite populatio did ot form their ow cluter, differig lightly from the BAPS iferece (Figure A), but eemed to be derived from a ubgroup of the Sakatchewa plat. It i poible that the compoite plat aayed may ot truly reflect the origial ource populatio, a the 15 ampled eed may be offprig of the origial compoite populatio with ome level of geetic chage. Alo, a expected, there wa o clear eparatio amog the plat either from patch-focued or ditributio-focu amplig.

8 15 Diverity 011, 3 38 Figure. Geetic tructure of 65 aayed hairy prairie-clover plat iferred uig the BAPS oftware. (A) five optimal cluter; (B) geetic divergece amog five optimal cluter. A: Five optimal cluter B: Geetic divergece amog five optimal cluter Nei miimum ditace Figure 3. AFLP-baed eighbor-joiig dedrogram of 65 aayed plat of hairy prairie-clover. The plat of defied patche are labeled with patch umber, from the ditributio-focu amplig with letter, ad of the North Dakota compoite populatio with ad highlight

9 Diverity 011, The aemet of idividual geetic ditictivee with plat locatio characteritic revealed that the plat i thi remat populatio were igificatly aociated with latitude ad elevatio readig, but ot with logitude readig (Figure 4). Thee aociatio, although explaiig relatively mall proportio of variatio, might igal that more gee flow had occurred i the outh tha the orth ide of the populatio, ad imilarly, more gee flow for the plat o the hill tha o the bottom of ad due. Thee patter of variatio could be geerated due to retricted eed diperal, limited bee polliatio, or directed ad movemet. Figure 4. Correlatio aalyi amog the geetic ditictivee etimate of idividual hairy prairie-clover plat, ite latitude (A) ad elevatio (B). Idividual geetic ditictivee A: Y= X R =0.013, P= Latitude (UTM ) Idividual geetic ditictivee B: Y= X R =0.016, P= Elevatio (meter) 3.3. Patch Variatio A aalyi of AFLP variatio amog the defied patche wa made. Thee patche had ample ize ragig from 3 to 40 ad averagig 13. (Figure 5). Nie patche had a ample ize maller tha 10 ad three patche had a ample ize larger tha 0. The AFLP aalyi howed that 90.5% of the total AFLP variatio reided withi defied patche ad oly 9.5% exited amog variou defied patche (Table ). Removig patche of ize le tha 10 geerated imilar reult with 91.5% of the total AFLP variatio reidig withi the patche (Table ). Thu, a patch of coiderable ize i thi populatio maitaied a relatively high level of geetic diverity. Thi may alo help to explai the

10 Diverity 011, weak geetic tructure oberved at the idividual plat level (Figure 3), a ome plat hared more geetic backgroud with plat from differet patche tha thoe withi patche. Etimate of patch-pecific proportioal AFLP variatio (Ft) raged from to ad averaged (Figure 5). The aemet of thee patch-pecific Ft value with patch characteritic revealed o-igificat aociatio of patch geetic variatio with patch ize (i term of polygo area), patch perimeter, earet eighbour ditace, ad mea iter-patch ditace (reult ot how). Thee patter of o-igificat aociatio remai the ame whe oly 3 patche of ize greater tha 9 were coidered. Thee fidig idicate that thee patch characteritic may ot directly cotribute to the chage i withi-patch AFLP variatio if ay. A patch of everal plat hould maitai coiderable geetic variatio [48]. Figure 5. AFLP-baed UPGMA dedrogram of the aayed plat i the defied patche withi the remat populatio of hairy prairie-clover, alog with patch ample ize (P) ad patch-pecific proportioal AFLP variatio (Ft). A patch i labelled with a letter P followed by a umber. Geetic ditace (Phi tatitic) Patch P Ft The correlatio aalyi of patch geetic ditace with patch differece i ditace, elevatio, ad ize revealed a igificat aociatio with geographic ditace (Figure 6), ot with patch elevatio ad ize. A patch of plat were more geetically ditictive to a patch far away tha to a patch earby. The aemet of patch geetic aociatio howed the mot geetically ditictive patch wa patch#4, followed by patch#5, #7, #3, ad #1 (Figure 5). Idetificatio of thee geetically ditictive patche i ueful for the effective i itu maagemet of the remat populatio ad for amplig

11 Diverity 011, germplam for ex itu coervatio. Figure 6. Correlatio aalyi betwee geetic ad geographic ditace for the defied patche withi the remat populatio of hairy prairie-clover. Geetic ditace (Phi tatitic) Y= X R =0.1169, P= Geographic ditace (meter) 3.4. Spatial Variatio withi Patche Fie-cale patial geetic variatio wa examied withi three large patche (i.e., patch#, #, # with ample ize 40, 3, 8, repectively). The phyical area for thee three patche raged from 50 to 100 m log ad 0 to 40 m wide ad were etimated i the polygo area of 8668, 3955, ad 68 m, repectively. However, o patial geetic tructure wa foud i ay patche, a oe of the etimated autocorrelatio coefficiet were tatitically igificat at P < 0.05 (reult ot how). Thi wa further cofirmed by the Matel tet fidig o-igificat idividual geetic aociatio with idividual ampled locatio withi the patche (reult ot how). Thee fidig idicate the preece of little geetic correlatio amog plat five meter apart withi each aayed patch. The lack of fie-cale geetic tructure may reflect the amplig bia of excludig plat withi five meter. It i poible that ome fie-cale geetic tructure exit withi five meter ditace due to limited eed diperal [50]. Alo, relatively dee plat (i.e., roughly 4 plat/m ) growig early cotiuouly withi each patch may igal ome geetic relatede preet amog earby plat Implicatio for Hairy Prairie-Clover Coervatio The patter of AFLP variatio preeted here are ueful to the maagemet ad coervatio of hairy prairie-clover i the Caadia Prairie. Firt, the aayed patche appear to be maitaiig large geetic diverity, implyig that the geetic impact of radom drift ad ibreedig wa mall (if ay) i thee patche. Thi implicatio wa alo upported by aother two fidig that the patch AFLP variatio wa ot igificatly aociated with patch ize ad the hairy prairie-clover plat from patch-focu ad ditributio-focu amplig diplayed little AFLP differetiatio acro the remat populatio. Secod, the fidig of large AFLP variatio ad o-igificat patial variatio with a patch alo imply little geetic rik for thi remat populatio. The coervatio effort may be emphaized le

12 Diverity 011, o geetic factor ad more o reproductive biology ad ecological attribute of the populatio. The maiteace of active bee polliatio icludig uitable habitat for etig appear to be crucial to avoid poible geetic rik. Further tudie o plat regeeratio ability aociated with variou ecological ad eviromet factor may yield ueful iformatio for log-term coervatio of thi populatio. Third, the igificat aociatio of patch variatio with iter-patch ditace implie that the geetic maagemet may be focued o patche that are far apart, particularly thoe patche i the orth ide of the remat populatio. More attetio hould be paid to the mot geetically ditictive patche uch a patch #4, #5, #7, #3, ad #1. Gee flow may be relatively limited for plat i the orth ide of the populatio ad located o the bottom of ad due. Fourth, the revealed patter of geetic variatio withi the populatio are ueful for amplig hairy prairie-clover plat for patch retoratio. The etimated geetic ditictivee ad geetic ditictive patche hould provide ome guide o the effective amplig of geetic diverity either for retoratio or ex itu coervatio. The patche i the outh ide of the populatio teded to have more AFLP variatio ad the plat i the bottom of ad due may ot ecearily have more AFLP variatio. Alo, eed from a few patche hould capture coiderable geetic variatio harbored i thi populatio. Fifth, it remai ukow how geeral thee patter of AFLP variatio are with repect to other hairy prairie-clover populatio i the Caadia Prairie or other ad due pecialit pecie. Give the fidig of large AFLP variatio ad the ature of outcroig, however, it i ot difficult to reao that the geetic rik may be relatively low for the other remat populatio i the Caadia Prairie [48]. Further geetic ad ecologic tudie with more populatio i the orther rage of the pecie ditributio hould yield more ueful iformatio to idetify the potetial rik of the remat populatio. 4. Cocluio Thi diverity aalyi revealed large AFLP variatio ad o-igificat patial variatio with patche of hairy prairie-clover, uggetig low geetic rik for thi remat hairy prairie-clover populatio. The revealed patter of AFLP variatio were iformative to the maagemet ad coervatio of hairy prairie-clover i the Caadia Prairie. Ackowledgemet Thi reearch wa part of a iterdepartmetal recovery effort for the threateed hairy prairie-clover led by Darcy Hedero, Eviromet Caada, ad partially fuded by the Iterdepartmetal Recovery Fud of Caada. The author would like to thak Darcy Hedero for hi leaderhip i the effort, advice i the amplig, ad aitace i the decriptio of patch ditributio (Figure 1) ad characteritic; Bill Houto for hi upport for the reearch; Sarah Lowe ad Hüeyi Uyal for their techical aitace i field amplig ad AFLP geotypig; Waye L. Duckwitz for providig the eed of the North Dakota compoite populatio; ad three aoymou reviewer for their helpful commet o a early verio of thi maucript.

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