2007American Journal of Botany 89(12): 2007–2016. 2002.GLACIAL HISTORY OF THE ALPINE HERBRUMEX NIVALIS(POLYGONACEAE):A COMPARISON OF COMMONPHYLOGEOGRAPHIC METHODS WITHNESTED CLADE ANALYSIS1IVANASTEHLIK2Institute of Systematic Botany, University of Zurich, Zollikerstrasse 107, CH-8008 Zurich, SwitzerlandThe glacial history of the alpine herb Rumex nivalis was investigated using amplified fragment length polymorphisms (AFLPs) andrestriction fragment length polymorphisms with polymerase chain reaction (PCR-RFLPs) of cpDNA. Both traditional statistical methodswidely applied in phylogeographic research and nested clade analysis were used. The AFLPs indicated little geographic structureprobably due to the wind-pollinated reproductive system of the dioecious R. nivalis. Because cpDNA haplotypes exhibited distinctdistributional patterns, correlation between AFLPs and PCR-RFLPs was low. The results of common statistical methods and of nestedclade analysis were largely congruent. Both supported in situ survival of one group of common haplotypes in the Central Alps. Foranother group of common haplotypes, classical phylogeographic analyses gave strong evidence for survival in peripheral refugia atthe northern alpine border, whereas this conclusion was not as clearly supported in the nested clade analysis. Nested clade analysisprovided several detailed insights on past and ongoing populational demographic processes. Thus, it is a valuable tool in the phylo-geographical analysis of haplotype data, but it should preferably be combined with other statistical analyses. In situations with lowgenetic variation in cpDNA, classical phylogeographic analytical tools on nuclear DNA will still be the methods of choice.Key words: AFLP; glacial survival; methodological comparison; migration; nested clade analysis; nunataks; PCR-RFLPs ofcpDNA; Pleistocene glaciation.Nested clade analysis was developed to reconstruct the evo-lutionary history of populations by combining a cladogram ofthe genetic relationships among haplotypes within these pop-ulations and their geographic distribution (Templeton, Boer-winkle, and Sing, 1987). Population structure thereby can beseparated from population history, if it is rigorously and ob-jectively assessed (Templeton, Routman, and Phillips, 1995;Templeton, 1998). Thereby, nested clade analysis can discrim-inate between phylogeographic associations due to recurrentbut restricted gene flow vs. historical events operating at thepopulation level, such as past fragmentatian, colonization, orrange expansion (Templeton, 1998). Haplotypes are first linkedin a cladogram that portrays the evolutionary steps connectingthem to one another. The accuracy of the following analysisdepends on the absence of recombination in case of nucleargenomes and the reliability of the estimated cladogram topol-ogy (Templeton and Sing, 1993). A program for nested cladeanalysis of the geographic distribution of haplotypes has re-cently become available (GEODIS 2.0; Posada, Crandall, andTempleton, 2000; program available at http://bioag.byu.edu/zoology/crandallplab/geodis.htm). This has stimulated phylo-geographic investigations of mitochondrial DNA in animals(mtDNA; e.g., Creer et al., 2001; Mardulyn, 2001; Seddon etal., 2001; Turgeon and Bernatchez, 2001) and of ribosomalDNA in fungi (James et al., 2001), whereas studies of cpDNA1Manuscript received 12 March 2002; revision accepted 27 June 2002.The author thanks Rolf Holderegger for his help in the field and KonradBachmann, Elena Conti, Rolf Holderegger, Peter Linder, and Jakob Schnellerfor fruitful discussions and comments on the manuscript. I would like toexpress my gratitude to Alan Templeton for his help to nest the haplotypesof Rumex nivalis. This research was supported by the Swiss National ScienceFoundation (SNF, grant number 31-55390.98) to J.J. Schneller and grants fromthe Swiss Alpine Club SAC and the Georges and Antoine Claraz Schenkungboth to I. Stehlik.2E-mail: [email protected] plants using nested clade analysis are still scarce (e.g., Mas-kas and Cruzan, 2000). Currently, much research is aimed toelucidate specific phylogeographic processes that have shapedthe genetic pattern of alpine plants of the European Alps andof the Arctic (Nordal and Jonsell, 1998; Widmer and Baltis-berger, 1999; Gugerli and Holderegger, 2001; Hagen, Giese,and Brochmann, 2001; Stehlik, Schneller, and Bachmann,2001, 2002; Stehlik, Tribsch, and Scho¨nswetter, 2001; Zhang,Comes, and Kadereit, 2001; Holderegger, Stehlik, and Abbott,2002). A broad array of ‘‘traditional’’ statistical approaches toanalyze molecular genetic data has been used in these studies.Among the more frequently applied are cluster analyses orordination methods (neighbor joining, UPGMA [unweightedpair group method using arithmetic averages], correspondenceanalysis), Mantel tests of the relationships of different classi-fications of individuals to geographic subunits (e.g., in popu-lations, in regions), AMOVA (analysis of molecular variance),census of rare alleles in specific geographic subunits within ataxon, or methods to detect isolation by distance. Nested cladeanalysis seems to be very promising in that it unifies manycharacteristics of these traditional methods and, at the sametime, appears to be more powerful in disentangling older frommore recent population level processes (Posada, Crandall, andTempleton, 2000). Many phylogeographic investigations willprobably use nested clade analysis in the near future. It istherefore of interest to apply nested clade analysis and moretraditional methods to the same data and to see whether theywill come to equivalent or complementary phylogeographicconclusions. Therefore, I subjected molecular genetic data onpopulations of the European endemic Rumex nivalis He-getschw. (Polygonaceae) to both an array of classical statisticsas used, e.g., in Stehlik, Schneller, and Bachmann (2001,2002), and to nested clade analysis.In phylogeographic investigations of the European alpineplants Eritrichium nanum (Boraginaceae) and Erinus alpinus2008 [Vol. 89AMERICANJOURNAL OFBOTANYFig. 1. The 23 populations sampled of Rumex nivalis in the Alps and the species’ total distribution according to Meusel, Ja¨ger, and Weinert (1965; inset).Twenty-two populations were sampled in the western part of the species’ distribution, and one population from the eastern distribution limit was included forcomparison. Rumex nivalis also occurs in Montenegro (Wagenitz,
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