Molecular Ecology (2010) 19, 3421–3443 doi: 10.1111/j.1365-294X.2010.04754.x

Multiple Pleistocene refugia and Holocene range expansion of an abundant southwestern American desert plant species (Melampodium leucanthum, Asteraceae)

CAROLIN A. REBERNIG,* GERALD M. SCHNEEWEISS,†1 KATHARINA E. BARDY,†* PETER SCHO¨ NSWETTER,†‡ JOSE L. VILLASEN˜ OR,– RENATE OBERMAYER,* TOD F. STUESSY* and HANNA WEISS-SCHNEEWEISS* *Department of Systematic and Evolutionary Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria; †Department of Biogeography and Botanical Garden, University of Vienna, Rennweg 14, A-1030 Vienna, Austria; ‡Department of Systematics, Palynology and Geobotany, Institute of Botany, University of Innsbruck, Sternwartestrasse 15, A-6020 Innsbruck, Austria; –Instituto de Biologı´a, Departamento de Bota´nica, Universidad Nacional Auto´noma de Me´xico, Tercer Circuito s ⁄ n, Ciudad Universitaria, Delegacio´n Coyoaca´n, MX-04510 Me´xico D. F., Me´xico

Abstract Pleistocene climatic fluctuations had major impacts on desert biota in southwestern North America. During cooler and wetter periods, drought-adapted species were isolated into refugia, in contrast to expansion of their ranges during the massive aridification in the Holocene. Here, we use Melampodium leucanthum (Asteraceae), a species of the North American desert and semi-desert regions, to investigate the impact of major aridification in southwestern North America on phylogeography and evolution in a widespread and abundant drought-adapted plant species. The evidence for three separate Pleistocene refugia at different time levels suggests that this species responded to the Quaternary climatic oscillations in a cyclic manner. In the Holocene, once differentiated lineages came into secondary contact and intermixed, but these range expansions did not follow the eastwardly progressing aridification, but instead occurred independently out of separate Pleistocene refugia. As found in other desert biota, the Continental Divide has acted as a major migration barrier for M. leucanthum since the Pleistocene. Despite being geographically restricted to the eastern part of the species’ distribution, autotetraploids in M. leucanthum originated multiple times and do not form a genetically cohesive group. Keywords: desert biota, Holocene aridification, Melampodium, phylogeography, polyploidy, refugia Received 24 March 2010; revision received 28 May 2010; accepted 5 June 2010

Abbott & Brochmann 2003; Scho¨nswetter et al. 2005). Introduction The role of these climatic fluctuations in other regions, The impact of Pleistocene climatic fluctuations on however, remains less well understood. This is particu- directly affected areas, such as the Arctic or temperate larly the case for arid regions in northern Mexico and high mountain ranges, has been comparatively well adjacent southwestern United States. Paleoclimatic and investigated phylogeographically in both plants and paleovegetational evidence unambiguously suggests animals (Hewitt 1996, 2001; Brunsfeld et al. 2001; that desert vegetation was strongly restricted during the wetter and cooler pluvial periods (Wells 1966; Van Correspondence: Gerald M. Schneeweiss, Fax: +43 1 4277 9541; Devender & Spaulding 1979; Thompson & Anderson E-mail: [email protected] 1Present Address: Systematic Botany and Mycology, Ludwig- 2000) and confined to refugia in the west and south, Maximilians-University Munich, Menzingerstrasse 67, D-80638 such as the lower Colorado River Basin, the plains Munich, Germany. of Sonora, or the southern Chihuahuan Desert (Van

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Devender 1990; Thompson & Anderson 2000; Hunter tinental Divide (Nason et al. 2002; Clark-Tapia & Moli- et al. 2001). Large-scale aridification of the whole region na-Freaner 2003; Fehlberg & Ranker 2009; Garrick et al. started only after the end of the last glacial maximum 2009; Sosa et al. 2009) or they do not employ molecular (Van Devender & Spaulding 1979; McClaran & Van methods (Hunter et al. 2001; Holmgren et al. 2007). Devender 1995; Bousman 1998; Metcalfe et al. 2000; By affecting the distribution of a species, environmen- Musgrove et al. 2001; Holmgren et al. 2007) and was tal changes will also shape its evolution via, for accompanied by a shift from xeric woodlands, abundant instance, enabling or interrupting gene flow in phases until 8000 years BP (Van Devender 1977), to semidesert of continuous distribution and range disruption, respec- grassland and eventually desert shrubland vegetation tively, or affecting the success of establishment of newly (Neilson 1986). Consequently, drought-adapted species formed polyploids (Husband 2004; Baack & Stanton are expected to have persisted in one or more distinct 2005; Ramsey et al. 2008). The latter is of particular rele- refugia (Nason et al. 2002; Fehlberg & Ranker 2009), vance, because polyploidy is recognized as an impor- from where they reached their current distribution after tant mode of speciation in general and one of the more range expansion within the last 10 000–6000 years (Van likely means of sympatric speciation in particular (Otto Devender & Spaulding 1979; Spaulding 1990; Van & Whitton 2000; Coyne & Orr 2004; Soltis et al. 2007). Devender 1990; Holmgren et al. 2007). While the role of allopolyploidy for speciation has long These range expansions into new arid regions are been recognized (Ramsey & Schemske 1998, 2002; Le- expected to have had major impacts on population itch & Leitch 2008), the rapidly mounting evidence of a structure and genetic diversity, for instance resulting in high frequency of autopolyploids, often in mixed popu- loss of alleles because of bottlenecks and founder lations with their diploid progenitors (Husband 2004; events, or in secondary contact of genetic lineages dif- Suda et al. 2007), has led to a more positive view con- ferentiated in allopatric refugia (Hewitt 2001, 2004). Pa- cerning the evolutionary significance of autopolyploidi- leoclimatic modelling indicates that the aridification zation (Soltis et al. 2007). Despite several recent studies progressed from the Sonoran Desert north- and east- dealing with the dynamics of diploid–autopolyploid wards (Holmgren et al. 2007), and it can be expected complexes (Baack & Stanton 2005; Scho¨nswetter et al. that range expansion of drought-adapted species fol- 2007; Ramsey et al. 2008; Hu¨ lber et al. 2009), their evo- lowed the same general direction (Fehlberg & Ranker lutionary significance and the factors involved in poly- 2009). Additionally, rapid expansion should also be ploid cytotype formation and establishment are still reflected in geographic patterns of genetic diversity, poorly understood (Baack & Stanton 2005). which is expected to be lower in more recently colo- Here we use Melampodium leucanthum (Asteraceae), nized areas because of founder effects (Hewitt 1996). A an abundant taxon of the North American desert and longitudinal migration pattern may, however, be modi- semi-desert regions, to investigate the impact of the fied by the Continental Divide, whose establishment in major aridification in southern North America within the late Tertiary is thought to have caused vicariant the last 10 000 years on phylogeography and evolution, diversification in a number of warm-desert animals including cytotype differentiation, in a drought- (Riddle & Hafner 2006; Castoe et al. 2007). Since then, adapted plant species. This phylogenetically distinct the divide has acted as a formidable migration barrier (Blo¨ch et al. 2009) and morphologically and taxonomi- for desert biota because of the lack of a spatially and cally homogeneous species is particularly well suited temporally continuous connection between the Sonoran to address these questions, because it is distributed and the Chihuahuan Deserts, which currently come over several major arid and semi-arid biogeographic closest at the Derning Plains near the border between regions ranging from the Sonoran and Chihuahuan Arizona and New Mexico (Morafka 1977; Riddle & Haf- Deserts to the Tamaulipan Plain and Southern Plain ner 2006; Castoe et al. 2007). This barrier is expected to region (Stuessy 1972), and it comprises diploid and tet- enhance founder effects in the course of eastward raploid cytotypes, the latter restricted to the eastern migration. Alternatively, if refugia of drought-adapted part of the distribution area (Fig. 1, Table 1; Stuessy species were also located east of the Continental Divide et al. 2004). Our first aim is to analyse the phylogeo- (Hunter et al. 2001; Castoe et al. 2007), this region prob- graphic patterns caused by post-Pleistocene aridifica- ably is the contact zone of western and eastern lineages tion and subsequent migration events. Specifically, we (Castoe et al. 2007). While several of these hypotheses want (i) to determine the locations of the refugia of have been tested in a number of animal groups (Jaeger M. leucanthum and test whether these are congruent et al. 2005; Riddle & Hafner 2006; Castoe et al. 2007; with those suggested by paleoclimatic and phylogeo- Haenel 2007; Fontanella et al. 2008), comparable studies graphic data (Hunter et al. 2001; Castoe et al. 2007; in plants are lacking. The few studies from desert plants Holmgren et al. 2007; Fehlberg & Ranker 2009); (ii) to either investigate species from only one side of the Con- infer the directionality of the range expansion, in

2010 Blackwell Publishing Ltd PHYLOGEOGRAPHY OF NORTH AMERICAN DESERT PLANT 3423

(a) (b)

(a)

(b)

Fig. 1 Physical map of the distribution of the analysed populations of Melampodium leucanthum. The collection area represents the entire distribution range of the species (population numbers as in Table 1).

particular, whether it was essentially unidirectional fol- Materials and methods lowing the north- and eastwardly progressing aridifica- tion (Holmgren et al. 2007); and (iii) to test whether Study species inferred range expansions fit the time frame predicted by paleoclimatic data (Holmgren et al. 2007; Holliday Melampodium leucanthum (blackfoot daisy) is a drought- et al. 2008). A second aim is to infer origin and evolu- tolerant, summer-flowering perennial subshrub grow- tion of the polyploids. Specifically, we want to test (i) ing on calcareous soils between 500 and 2590 m a.s.l. whether the polyploids originated once, as suggested and is abundant in its distribution area encompassing by their compact and restricted distribution, or recur- northern Mexico and the southwestern United States rently, as observed in many species including the clo- from Arizona to eastern Texas, northwards extending sely related M. cinereum (Rebernig et al. 2010) and (ii) into Oklahoma and Colorado (Fig. 1; Stuessy 1972). whether they form a genetically cohesive group clearly Pollen ⁄ ovule ratios (Cruden 1977) indicate that separated from the diploids, as has been found in M. leucanthum is outcrossing (data not shown). No M. cinereum (Rebernig et al. 2010). To this end, we population differentiation concerning morphology, generated amplified fragment length polymorphism flowering time or breeding system has ever been (AFLP) and cpDNA sequence data from several hun- reported (Stuessy 1972). Melampodium leucanthum com- dred individuals, whose ploidy level was determined prises diploid and tetraploid cytotypes (with occa- flow cytometrically from 92 populations over the whole sional triploid individuals in diploid populations; distribution area. These data were analysed using, Stuessy et al. 2004), which are morphologically indis- among others, a coalescent-based Bayesian approach tinguishable (Stuessy 1971, 1972). The two cytotypes for hypothesis testing and molecular dating, comple- occur mostly parapatrically, and tetraploids occupy a mented by ecological niche modelling for inferring compact area in eastern Texas to the near exclusion of putative paleodistributions. diploids (Stuessy et al. 2004).

2010 Blackwell Publishing Ltd 3424 C. A. REBERNIG ET AL.

lations with at least three individuals in ARLEQUIN 3.10 Plant material (Excoffier et al. 2005). Geographic patterns in AFLP de- Plant material was collected from 92 populations of scriptors were tested using multiple linear regressions M. leucanthum covering the entire distribution of the in Excel 2007 (Microsoft, Redmond, CA, USA). Principal species (Fig. 1). Samples were dried and stored in silica coordinate analysis (PCO) was conducted using gel until DNA isolation. Herbarium vouchers are NTSYSPC 2.20e (Rohlf 2007) with the default settings deposited in the herbarium of the University of Vienna both on the whole data set as well as on a reduced data (WU; voucher numbers given in Table 1). set after exclusion of the genetically distinct western populations (pops. 1–17; see Results). Neighbour-nets of the same two data sets were constructed with SPLITSTREE Ploidy level determination and molecular methods 4.8 (Huson & Bryant 2006) using Nei-Li distances (Nei Measurements of DNA ploidy levels (Suda et al. 2006) & Li 1979) calculated with FAMD 1.108 (Schlu¨ ter & were conducted as described in Rebernig et al. (2010). Harris 2006). Three-level hierarchical analyses of molec- Correct interpretation of DNA ploidy levels was con- ular variance (AMOVA) using the groups suggested by firmed by chromosome numbers determined for PCO (see Results) were conducted on both data sets selected individuals using standard Feulgen staining as with ARLEQUIN 3.10 (Excoffier et al. 2005), estimating the described by Weiss-Schneeweiss et al. (2007). significance of variance components from 10 000 per- Total genomic DNA was extracted as described in Re- mutations. bernig et al. (2010). AFLP fingerprint profiles were gen- Genetically homogeneous groups of diploid individu- erated for 1–5 individuals per population totalling 377 als were identified using genetic mixture analysis individuals (Table 1) following the protocol described implemented in STRUCTURE 2.2 (Pritchard et al. 2000; Fa- in Dixon et al. (2008). Two negative controls were lush et al. 2007) as described in Rebernig et al. (2010) included in each PCR, and 6.25% of the samples were with minor modifications (see Supporting materials). replicated. After initial screening of 33 selective primer Tetraploid individuals were excluded, because the mod- combinations with three to four selective nucleotides, els implemented in STRUCTURE are not suited for analy- the following five primer combinations were selected sing polyploids (Pritchard et al. 2000). for the final analyses (fluorescent dyes in parentheses): Assignment tests to determine the most likely source EcoRI-ACA ⁄ MseI-CAT (FAM), EcoRI-ACG ⁄ MseI-CAA populations for the tetraploid individuals were per- (VIC), EcoRI-ACC ⁄ MseI-CAG (NED), EcoRI-ACT ⁄ MseI- formed using AFLPOP 1.1 (Duchesne & Bernatchez CAC (FAM), EcoRI-AGG ⁄ MseI-CAA (VIC). Purification 2002) with the default settings. All diploid populations of selective PCR products, their electrophoretic separa- were considered as potential source populations, and tion and subsequent alignment as well as their scoring allocation was tested using two levels (0 or 2) of mini- (bands in the size range of 100–500 bp) were carried mal log-likelihood differences (as recommended by out as described in Rebernig et al. (2010). Nonreproduc- Duchesne & Bernatchez 2002) with frequency values of ible bands identified by comparisons among replicated zero replaced by 1 ⁄ (sample size + 1). individuals were excluded from further analyses. The following three noncoding chloroplast DNA cpDNA. Prior to all analyses, inversions in the plastid spacer regions were amplified and sequenced as sequence data were re-inverted to avoid introducing described in Rebernig et al. (2010) for one to three indi- substitutional mutations, which in fact are the result of viduals per population (Table 1), totalling 228 individu- structural mutations (Lo¨hne & Borsch 2005). This data als: psbA-trnH, rpl32-trnL, and ndhF-rpl32. Sequences set was used only for the BEAST analysis, and for the were assembled using SEQMAN II 5.05 (DNAStar, Madi- other analyses, indels longer than 1 base pair and inver- son, WI, USA) and manually aligned using BIOEDIT 7.0 sions were additionally recoded as single characters, (Hall 1999). Sequences are deposited in GenBank and mononucleotide repeats were removed because of (Table 1). their high degree of homoplasy at larger geographic scales (Ingvarsson et al. 2003). Group and population differentiation was assessed Data analyses via a spatial analysis of molecular variance (SAMOVA), AFLP. AFLP data descriptors include the total number which allows defining population groups that are genet- of fragments (Fragtot), the percentage of polymorphic ically differentiated from each other and occur in a fragments (Fragpoly), the number of private fragments geographically homogeneous area (Dupanloup et al.

(Fragpriv) and the index of average differences within 2002). This analysis was conducted with the program populations (AWD; Kosman 2003) calculated for popu- SAMOVA 1.0 (available from http://web.unife.it/progetti/

2010 Blackwell Publishing Ltd Table 1 Population numbers, sampling location coordinates, voucher information, number of analysed individuals, AFLP data descriptors, DNA ploidy level and GenBank 00BakelPbihn Ltd Publishing Blackwell 2010 accession numbers for Melampodium leucanthum

pop Sample N GenBank * † ‡ § – nr. Location (voucher nr.) AFLP ⁄ cpDNA Fragtot Fragpoly% Fragpriv AWD ± StdDev Ploidy accession numbers

1 N 35.522, W 113.455 (18819) 3 ⁄ 2 239 17.56 0 0.113 ± 0.250 2x FJ846219, FJ846220, FJ846017; FJ846018, FJ846421, FJ846422 2 N 35.122, W 113.666 (18820) 3 ⁄ 3 213 15.18 0 0.098 ± 0.236 2x FJ846221, FJ846222, FJ846223; FJ846019, FJ846020, FJ846021; FJ846423, FJ846424, FJ846425 3 N 34.755, W 112.091 (18814) 3 ⁄ 2 231 15.18 0 0.098 ± 0.236 2x FJ846213, FJ846214, FJ846011; FJ846012, FJ846415, FJ846416 4 N 34.730, W 111.969 (18813) 3 ⁄ 2 244 19.64 0 0.127 ± 0.262 2x FJ846211, FJ846212, FJ846009; FJ846010, FJ846413, FJ846414 PLANT DESERT AMERICAN NORTH OF PHYLOGEOGRAPHY 5 N 34.712, W 111.881 (18812) 5 ⁄ 2 279 29.76 0 0.140 ± 0.226 2x FJ846209, FJ846210, FJ846007; FJ846008, FJ846411, FJ846412 6 N 34.826, W 111.779 (18816) 3 ⁄ 2 229 17.26 0 0.111 ± 0.249 2x FJ846215, FJ846216, FJ846013; FJ846014, FJ846417, FJ846418 7 N 34.757, W 111.765 (18817) 5 ⁄ 2 271 24.40 0 0.110 ± 0.204 2x FJ846217, FJ846218, FJ846015; FJ846016, FJ846419, FJ846420 8 N 34.606, W 111.858 (18808) 4 ⁄ 2 255 19.19 0 0.102 ± 0.215 2x FJ846200, FJ846201, FJ846998; FJ846999, FJ846402, FJ846403 9 N 34.618, W 111.843 (18809) 5 ⁄ 2 271 24.26 0 0.112 ± 0.208 2x FJ846202, FJ846203, FJ846000; FJ846001, FJ846404, FJ846405 10 N 34.639, W 111.808 (18811) 5 ⁄ 2 260 24.26 0 0.115 ± 0.213 2x FJ846207, FJ846208, FJ846005; FJ846006, FJ846409, FJ846410 11 N 34.650, W 111.760 (18810) 5 ⁄ 3 270 28.81 0 0.113 ± 0.211 2x FJ846202, FJ846203, FJ846204; FJ846002, FJ846003, FJ846004; FJ846406, FJ846407, FJ846408 12 N 34.349, W 112.184 (18807) 4 ⁄ 2 246 18.60 0 0.096 ± 0.206 2x FJ846198, FJ846199, FJ846996; FJ846997, FJ846400, FJ846401 13 N 33.963, W 111.863 (18805) 3 ⁄ 2 223 13.48 0 0.089 ± 0.227 2x FJ846196, FJ846197, FJ846994; FJ846995, FJ846398, FJ846399 14 N 34.002, W 111.314 (18801) 5 ⁄ 2 262 22.02 0 0.104 ± 0.206 2x FJ846192, FJ846193, FJ846990; FJ846991, FJ846394, FJ846395 15 N 33.480, W 111.443 (18802) 4 ⁄ 2 234 14.73 0 0.077 ± 0.192 2x FJ846194, FJ846195, FJ846992; FJ846993, FJ846396, FJ846397 16 N 32.602, W 110.745 (18822) 5 ⁄ 2 241 18.30 1 0.087 ± 0.192 2x FJ846224, FJ846225, FJ846022; FJ846023, FJ846426, FJ846427 17 N 33.239, W 110.253 (18800) 3 ⁄ 3 250 18.60 0 0.120 ± 0.256 2x FJ846189, FJ846190, FJ846191; FJ846987, FJ846988, FJ846989; FJ846391, FJ846392, FJ846393 18 N 32.786, W 108.139 (20005) 5 ⁄ 2 282 29.76 0 0.138 ± 0.223 2x FJ846236, FJ846237, FJ846034; FJ846035, FJ846438, FJ846439 3425 3426 Table 1 (Continued) .A REBERNIG A. C. pop Sample N GenBank * † ‡ § – nr. Location (voucher nr.) AFLP ⁄ cpDNA Fragtot Fragpoly% Fragpriv AWD ± StdDev Ploidy accession numbers

19 N 32.640, W 107.955 (20003) 4 ⁄ 2 259 22.62 0 0.121 ± 0.231 2x FJ846234, FJ846235, FJ846032; FJ846033, FJ846436, FJ846437 20 N 31.954, W 107.676 (20002) 5 ⁄ 3 287 30.06 1 0.146 ± 0.234 2x FJ846231, FJ846232, FJ846233; FJ846029, FJ846030, FJ846031; FJ846433, FJ846434, FJ846435 TAL. ET 21 N 32.263, W 107.233 (20000) 3 ⁄ 2 233 18.15 0 0.117 ± 0.254 2x FJ846229, FJ846230, FJ846027; FJ846028, FJ846431, FJ846432 22 N 32.398, W 106.614 (20046) 5 ⁄ 2 251 18.60 0 0.090 ± 0.196 2x FJ846287, FJ846288, FJ846085; FJ846086, FJ846489, FJ846490 23 N 32.953, W 107.490 (20007) 5 ⁄ 2 262 29.02 1 0.125 ± 0.214 2x FJ846238, FJ846239, FJ846036; FJ846037, FJ846440, FJ846441 24 N 33.275, W 107.282 (20010) 5 ⁄ 2 272 27.98 0 0.133 ± 0.225 2x FJ846240, FJ846241, FJ846038; FJ846039, FJ846442, FJ846443 25 N 34.146, W 106.908 (20011) 5 ⁄ 2 269 26.93 0 0.129 ± 0.223 2x FJ846242, FJ846243, FJ846040; FJ846041, FJ846444, FJ846445 26 N 33.794, W 106.274 (20014) 4 ⁄ 2 282 28.72 0 0.152 ± 0.247 2x FJ846244, FJ846245, FJ846042; FJ846043, FJ846446, FJ846447 27 N 34.010, W 105.942 (20016) 5 ⁄ 2 276 27.23 0 0.128 ± 0.220 2x FJ846246, FJ846247, FJ846044; FJ846045, FJ846448, FJ846449 28 N 34.946, W 106.191 (20017) 5 ⁄ 3 276 28.57 1 0.135 ± 0.224 2x FJ846248, FJ846249, FJ846250; FJ846046, FJ846047, FJ846048; FJ846450, FJ846451, FJ846452 29 N 35.288, W 106.216 (20021) 5 ⁄ 3 263 25.00 0 0.119 ± 0.216 2x FJ846251, FJ846252, FJ846253; FJ846049, FJ846050, FJ846051; FJ846453, FJ846454, FJ846455 30 N 34.036, W 104.747 (20032) 5 ⁄ 2 289 28.57 0 0.135 ± 0.224 2x FJ846261, FJ846262, FJ845059; FJ845060, FJ846463, FJ846464 31 N 34.898, W 104.718 (20031) 5 ⁄ 2 269 24.40 0 0.115 ± 0.212 2x FJ846259, FJ846260, FJ845057; FJ845058, FJ846461, FJ846462 32 N 35.396, W 104.180 (20030) 4 ⁄ 2 287 27.08 0 0.142 ± 0.242 2x FJ846257, FJ846258, FJ845055; 00BakelPbihn Ltd Publishing Blackwell 2010 FJ845056, FJ846459, FJ846460 33 N 35.230, W 103.767 (20029) 4 ⁄ 2 280 25.30 0 0.133 ± 0.236 2x FJ846254, FJ846255, FJ846256 FJ845052, FJ845053, FJ845054; FJ846456, FJ846457, FJ846458 34 N 31.316, W 106.078 (20045) 5 ⁄ 2 281 26.39 0 0.125 ± 0.215 2x FJ846285, FJ846286, FJ846083; FJ846084, FJ846487, FJ846488 35 N 31.005, W 104.825 (18726) 3 ⁄ 3 382 20.09 0 0.130 ± 0.264 2x FJ846120, FJ846121, FJ846122; FJ845918, FJ845919, FJ845920; FJ846322, FJ846323, FJ846324

00BakelPbihn Ltd Publishing Blackwell 2010 Table 1 (Continued)

pop Sample N GenBank * † ‡ § – nr. Location (voucher nr.) AFLP ⁄ cpDNA Fragtot Fragpoly% Fragpriv AWD ± StdDev Ploidy accession numbers

36 N 31.004, W 104.825 (20043) 4 ⁄ 2 300 28.42 0 0.147 ± 0.242 2x FJ846280, FJ846281, FJ846078; FJ846079, FJ846482, FJ846483 37 N 30.789, W 104.033 (18725) 5 ⁄ 2 338 33.33 1 0.159 ± 0.237 2x FJ846118, FJ846119, FJ845916; FJ845917, FJ846329, FJ846321 38 N 30.999, W 103.756 (18727) 5 ⁄ 2 333 31.99 0 0.152 ± 0.233 2x* FJ846123, FJ846124, FJ845921; FJ845922, FJ846325, FJ846326 39 N 31.352, W 103.579 (18729) 2 ⁄ 2 255 13.99 0 0.135 ± 0.342 2x FJ846125, FJ846126, FJ845923; FJ845924, FJ846327, FJ846328

40 N 31.611, W 104.857 (20044) 5 ⁄ 3 314 34.97 1 0.163 ± 0.234 2x FJ846282, FJ846283, FJ846284; PLANT DESERT AMERICAN NORTH OF PHYLOGEOGRAPHY FJ846080, FJ846081, FJ846082; FJ846484, FJ846485, FJ846486 41 N 32.490, W 104.348 (20033) 5 ⁄ 3 262 25.74 3 0.112 ± 0.199 2x FJ846263, FJ846264, FJ846265; FJ845061, FJ845062, FJ845063; FJ846465, FJ846466, FJ846467 42 N 32.529, W 103.802 (20034) 3 ⁄ 3 259 21.28 0 0.137 ± 0.270 2x FJ846266, FJ846267, FJ846268; FJ845064, FJ845065, FJ845066; FJ846468, FJ846469, FJ846470 43 N 32.507, W 103.127 (20035) 4 ⁄ 3 259 24.12 0 0.130 ± 0.239 2x FJ846269, FJ846270, FJ846271; FJ845067, FJ845068, FJ845069; FJ846471, FJ846472, FJ846473 44 N 32.288, W 102.611 (18737) 3 ⁄ 2 289 21.28 0 0.137 ± 0.270 2x FJ846139, FJ846140, FJ845937; FJ845938, FJ846341, FJ846342 45 N 31.852, W 103.114 (18738) 3 ⁄ 2 296 20.04 0 0.159 ± 0.365 2x FJ846141, FJ846142, FJ845939; FJ845940, FJ846343, FJ846344 46 N 31.640, W 102.634 (18730) 5 ⁄ 2 316 30.95 0 0.147 ± 0.231 2x FJ846127, FJ846128, FJ845925; FJ845926, FJ846329, FJ846330 47 N 31.698, W 102.572 (18731) 5 ⁄ 2 317 29.02 0 0.134 ± 0.220 2x ⁄ 3x FJ846129, FJ846130, FJ845927; FJ845928, FJ846331, FJ846332 48 N 30.748, W 102.907 (18722) 5 ⁄ 2 369 40.92 0 0.188 ± 0.240 2x FJ846116, FJ846117, FJ845914; FJ845915, FJ846318, FJ846319 49 N 30.239, W 103.380 (20038) 5 ⁄ 2 315 32.59 0 0.149 ± 0.226 2x FJ846272, FJ846273, FJ846070; FJ846071, FJ846474, FJ846475 50 N 29.785, W 103.177 (20039) 5 ⁄ 3 276 24.70 0 0.119 ± 0.218 2x FJ846274, FJ846275, FJ846276; FJ846072, FJ846073, FJ846074; FJ846476, FJ846477, FJ846478 51 N 29.516, W 103.403 (20040) 3 ⁄ 3 253 18.30 1 0.118 ± 0.255 2x FJ846277, FJ846278, FJ846279; FJ846075, FJ846076, FJ846077; FJ846479, FJ846480, FJ846481

52 N 31.934, W 101.866 (18732) 1 ⁄ 1 206 0 0 ⁄ 2x FJ846131, FJ845929, FJ846333 3427 Table 1 (Continued) 3428

pop Sample N GenBank REBERNIG A. C. * † ‡ § – nr. Location (voucher nr.) AFLP ⁄ cpDNA Fragtot Fragpoly% Fragpriv AWD ± StdDev Ploidy accession numbers

53 N 31.871, W 101.646 (18733) 5 ⁄ 2 333 32.39 0 0.156 ± 0.238 2x FJ846132, FJ846133, FJ845930; FJ845931, FJ846334, FJ846335 54 N 32.964, W 102.012 (18735) 5 ⁄ 2 332 33.33 1 0.156 ± 0.233 2x FJ846137, FJ846138, FJ845935; FJ845936, FJ846339, FJ846340 55 N 35.326, W 102.370 (18753) 3 ⁄ 2 294 23.51 0 0.152 ± 0.280 2x FJ846143, FJ846144, FJ845941;

FJ845942, FJ846345, FJ846346 AL. ET 56 N 35.003, W 101.919 (18756) 4 ⁄ 2 303 28.27 0 0.149 ± 0.246 2x FJ846145, FJ846146, FJ845943; FJ845944, FJ846347, FJ846348 57 N 34.986, W 101.717 (18757) 3 ⁄ 2 286 23.51 0 0.152 ± 0.280 2x FJ846147, FJ846148, FJ845945; FJ845946, FJ846349, FJ846350 58 N 36.221, W 101.334 (18758) 5 ⁄ 2 320 20.21 2 0.140 ± 0.225 2x FJ846149, FJ846150, FJ845947; FJ845948, FJ846351, FJ846352 59 N 36.449, W 100.372 (18760) 4 ⁄ 3 298 26.04 0 0.138 ± 0.240 2x FJ846151, FJ846152, FJ846153; FJ845949, FJ845950, FJ845951; FJ846353, FJ846354, FJ846355 60 N 36.427, W 99.883 (18761) 4 ⁄ 2 298 20.87 0 0.152 ± 0.248 2x ⁄ 3x FJ846154, FJ846155, FJ845952; FJ845953, FJ846356, FJ846357 61 N 35.845, W 100.397 (18762) 3 ⁄ 3 257 18.90 0 0.122 ± 0.278 2x FJ846156, FJ846157, FJ846158; FJ845954, FJ845955, FJ845956; FJ846358, FJ846359, FJ846360 62 N 35.432, W 100.770 (18764) 3 ⁄ 2 273 19.64 0 0.127 ± 0.262 2x FJ846159, FJ846160, FJ845957; FJ845958, FJ846361, FJ846362 63 N 35.009, W 100.896 (18772) 4 ⁄ 2 284 20.98 1 0.111 ± 0.222 2x ⁄ 3x FJ846168, FJ846169, FJ845966; FJ845967, FJ846370, FJ846371 64 N 34.788, W 100.898 (18771) 1 ⁄ 1 204 0 0 ⁄ 2x FJ846167, FJ845965, FJ846369 65 N 34.380, W 101.111 (18770) 4 ⁄ 2 293 33.44 0 0.152 ± 0.231 2x FJ846165, FJ846166, FJ845963; FJ845964, FJ846367, FJ846368 66 N 34.219, W 100.888 (18769) 3 ⁄ 1 309 29.02 0 0.187 ± 0.230 2x FJ846163, FJ846164, FJ845961; FJ845962, FJ846365, FJ846366 ⁄ 67 N 33.860, W 100.852 (18768) 3 2 270 23.10 0 0.149 ± 0.278 2x FJ846161, FJ846162, FJ845959;

00BakelPbihn Ltd Publishing Blackwell 2010 FJ845960, FJ846363, FJ846364 68 N 31.900, W 100.717 (18734) 5 ⁄ 3 313 26.79 0 0.127 ± 0.221 2x FJ846134, FJ846135, FJ846136; FJ845932, FJ845933, FJ845934; FJ846336, FJ846337, FJ846338 69 N 29.718, W 101.361 (18720) 4 ⁄ 3 298 29.32 1 0.159 ± 0.256 4x FJ846113, FJ846114, FJ846115; FJ845911, FJ845912, FJ845913; FJ846315, FJ846316, FJ846317 70 N 28.228, W 101.056 (19056) 3 ⁄ 3 229 18.75 0 0.121 ± 0.267 2x FJ846226, FJ846227, FJ846228; FJ845024, FJ845025, FJ845026; FJ846428, FJ846429, FJ846430

00BakelPbihn Ltd Publishing Blackwell 2010 Table 1 (Continued)

pop Sample N GenBank * † ‡ § – nr. Location (voucher nr.) AFLP ⁄ cpDNA Fragtot Fragpoly% Fragpriv AWD ± StdDev Ploidy accession numbers

71 N 31.226, W 99.757 (18774) 5 ⁄ 2 300 25.15 0 0.119 ± 0.215 2x ⁄ 3x FJ846170, FJ846171, FJ845968; FJ845969, FJ846372, FJ846373 72 N 31.781, W 99.779 (18776) 4 ⁄ 2 278 17.41 0 0.089 ± 0.206 4x FJ846172, FJ846173, FJ845970; FJ845971, FJ846374, FJ846375 73 N 31.761, W 98.899 (18778) 4 ⁄ 2 281 17.86 0 0.095 ± 0.210 4x FJ846174, FJ846175, FJ845972; FJ845973, FJ846376, FJ846377 74 N 31.689, W 98.814 (18779) 4 ⁄ 2 292 23.17 0 0.126 ± 0.234 4x FJ846176, FJ846168, FJ845974; FJ845975, FJ846378, FJ846379

75 N 31.793, W 98.228 (18787) 4 ⁄ 2 252 12.28 1 0.114 ± 0.226 4x FJ846187, FJ846188, FJ845985; PLANT DESERT AMERICAN NORTH OF PHYLOGEOGRAPHY FJ845986, FJ846389, FJ846390 76 N 31.575, W 97.834 (18786) 4 ⁄ 2 248 20.98 0 0.112 ± 0.224 4x FJ846183, FJ846184, FJ845983; FJ845984, FJ846387, FJ846388 77 N 31.358, W 98.129 (18781) 5 ⁄ 2 255 24.55 0 0.118 ± 0.217 4x FJ846178, FJ846179, FJ845976; FJ845977, FJ846380, FJ846381 78 N 31.170, W 98.183 (18782) 5 ⁄ 2 237 22.17 0 0.103 ± 0.201 4x FJ846180, FJ846181, FJ845978; FJ845979, FJ846382, FJ846383 79 N 30.928, W 98.002 (18783) 1 ⁄ 1 171 0 0 ⁄ 4x FJ846182, FJ845980, FJ846384 80 N 31.064, W 97.572 (18785) 4 ⁄ 2 246 20.09 0 0.108 ± 0.222 4x FJ846183, FJ846184, FJ845981; FJ845982, FJ846385, FJ846386 81 N 30.616, W 97.860 (18709) 5 ⁄ 2 248 20.39 0 0.095 ± 0.197 4x* FJ846103, FJ846104, FJ845901; FJ845902, FJ846305, FJ846306 82 N 30.694, W 97.981 (18710) 3 ⁄ 2 224 15.33 0 0.099 ± 0.237 4x FJ846105, FJ846106, FJ845803; FJ845804, FJ846307, FJ846308 83 N 30.696, W 98.254 (18711) 5 ⁄ 2 252 21.58 0 0.101 ± 0.202 4x FJ846107, FJ846108, FJ845905; FJ845906, FJ846309, FJ846310 84 N 30.671, W 98.256 (18712) 5 ⁄ 2 248 21.43 0 0.102 ± 0.205 4x FJ846109, FJ846110, FJ845907; FJ845908, FJ846311, FJ846312 85 N 30.280, W 98.907 (18681) 5 ⁄ 2 306 33.78 0 0.15 ± 0.229 2x FJ846093, FJ846094, FJ845891; FJ845892, FJ846295, FJ846296 86 N 30.170, W 98.849 (18687) 3 ⁄ 2 246 23.81 0 0.134 ± 0.281 4x FJ846101, FJ846102, FJ845899; FJ845900, FJ846303, FJ846304 87 N 30.170, W 98.907 (18682) 5 ⁄ 2 289 29.02 1 0.137 ± 0.225 2x ⁄ 3x FJ846095, FJ846096, FJ845893; FJ845894, FJ846297, FJ846298 88 N 29.616, W 98.757 (18686) 3 ⁄ 2 237 18.75 0 0.121 ± 0.257 4x FJ846099, FJ846100, FJ845897; FJ845898, FJ846301, FJ846302 89 N 29.891, W 98.408 (18683) 5 ⁄ 2 295 32.89 0 0.160 ± 0.241 4x FJ846097, FJ846098, FJ845895; FJ845896, FJ846299, FJ846300 90 N 30.194, W 98.478 (18676) 0 ⁄ 2 ⁄⁄ ⁄⁄ 2x FJ846087, FJ846088, FJ846289;

FJ846290, FJ845885, FJ845886 3429 3430 C. A. REBERNIG ET AL.

genetica/isabelle/samova.html) employing 500 repli- cates and testing K = 2–8, the final number of groups

being chosen based on the highest FCT value, which – describes the proportion of total genetic variance because of differences between groups of populations (Dupanloup et al. 2002). A haplotype network was con- structed using statistical parsimony as implemented in TCS 1.21 (Clement et al. 2000). The recoded gaps were FJ846089, FJ846090, FJ845887; FJ845888, FJ846291, FJ846292 FJ846091, FJ846092, FJ845889; FJ845890, FJ846293, FJ846294 FJ846111, FJ846112, FJ845909; FJ845910, FJ846313, FJ846314 GenBank accession numbers treated as a fifth character state, and the connection level was set to 95%. Demographic histories, especially population expan- § sions, were tested in several ways. We used Tajima’s D

x x x (1996) and Fu’s FS (1997), where negative values indi-

cate population expansion, and the R2 statistic, where small values indicate population expansion (Ramos-On- sins & Rozas 2002). All three statistics and their signifi- cance, assessed using 10 000 samples simulated under a model of constant population size, were calculated with

DNASP 5.10 (Rozas et al. 2003). Values for FS were con- sidered significant at P £ 0.02 (Fu 1997). As an alterna- AWD ± StdDev Ploidy tive approach, we used mismatch distribution (the distribution of pairwise differences among individuals), ‡ where a unimodal distribution indicates population priv expansion (Rogers & Harpending 1992), as described in

Frag Rebernig et al. (2010) with minor modifications (see Supporting Material). All these analyses were con-

† ducted for the whole data set as well as for the three % population groups identified by the haplotype network poly and by the SAMOVA (see Results), applying in case of

Frag multiple comparisons P-value correction via sequential Bonferroni correction (Rice 1989). As another way to test for population expansions, we used the method *

tot implemented in BEAST 1.4.8 (Drummond & Rambaut 2007), which allows divergence times to be estimated simultaneously, as described in Rebernig et al. (2010) with some modifications (see Supporting Material). Migration directionality was tested using BEAST 1.4.8 employing the best demographic history identified in N cpDNA Frag ⁄ the previous step. Each of the three main haplotype 2 300 27.68 0 0.145 ± 0.243 2 22 289 319 33.63 25.60 0 0 0.156 ± 0.231 0.122 ± 0.213 2 4 ⁄ ⁄ ⁄ groups (see Results) was considered in return as repre- AFLP Sample senting the source for range expansion of the whole species. Using BEAST, these hypotheses can only be implemented via topological constraints, specifically by constraining the nonrefugial populations to be mono- L; different individuals are separated by semicolons.

trn phyletic. By doing so, we have to assume that gene lin-

32- eages within the nonrefugial populations coalesced

rpl before they coalesced with those from the refugial pop-

32, ulations. Besides, we also tested whether polyploid rpl populations of M. leucanthum originated once (consti- F- tute a monophyletic group) as suggested by the com- ndh pact distribution area parapatric to that of the diploids. H, (Continued)

trn All hypotheses testing in BEAST employed Bayes fac-

A- tors (BF; Suchard et al. 2001, 2005). Marginal likelihoods psb percentage of polymorphic AFLP fragments. number of private AFLP fragments. asterisks indicate DNA ploidy levels confirmed by chromosome counts. number of total AFLP fragments. Table 1 91 N 30.216, W 98.478 (18677) 4 * † ‡ § – pop nr. Location (voucher nr.) 9293 N 30.227, W 98.382 (18679) N 30.387, W 98.366 (18714) 5 5 (including their Monte Carlo error: Suchard et al. 2003;

2010 Blackwell Publishing Ltd PHYLOGEOGRAPHY OF NORTH AMERICAN DESERT PLANT 3431

Redelings & Suchard 2005) and BFs were calculated System Model version 3 (CCSM3: Collins et al. 2006) with Tracer 1.4 (available from http://evolve.zoo.ox.a- and the Model for Interdisciplinary Research on Cli- c.uk/). As test statistic, we used the widely applied mate version 3.2 (MIROC3.2: Hasumi & Emori 2004).

2 · lnBF, considering 2 · lnBFmodel 1 vs. model 2 >10as The list of localities of M. leucanthum (Table 1) was strong support for model 1. The BEAST input files augmented with data from our own collections and pre- (xml-files) are available as Supporting online material. viously published data (Stuessy et al. 2004), resulting in As a different approach for assessing geographic loca- a final data set of 164 entries. Distribution modelling tions of ancestors and migration directionalities of con- was performed using MAXENT 3.2.19 (available from tinuously distributed species, we used the method http://www.cs.princeton.edu/~schapire/maxent/), implemented in PHYLOMAPPER 1.b1 (Lemmon & Lemmon which uses the maximum entropy method. Using pres- 2008). Briefly, using a spatial random walk model of ence-only data, it estimates a target probability distribu- migration, it calculates the likelihood of geographic coor- tion by finding the maximum entropy probability dinates of clade ancestors (i.e., specified internal nodes) distribution with the constraint that the expected value and the mean per-generation dispersal distance (which of each feature should match its empirical average may be treated as a nuisance parameter), given the geo- (Phillips et al. 2006). The model for the current distribu- graphic coordinates of the sampled individuals (i.e., tree tion was calculated using all 19 bioclimatic variables terminals) and assuming tree topology and branch and was in the following applied to the bioclimatic lengths to be known without error (Lemmon & Lemmon variables of CCSM and MIROC, respectively. Perfor- 2008). We took the maximum clade posterior probability mance of this model was evaluated by the area under tree (determined with the TreeAnnotator module of the receiver operating characteristic (ROC) curve BEAST) from the BEAST analysis with the best sup- (AUC), which ranges from 0.5 (random prediction) to 1 ported demographic model (see Results), complemented (maximum prediction), and a binomial test of omission ) with dummy outgroup sequences (required for defini- with the default convergence threshold (10 5) and the tion of clades). If populations included individuals with maximum number of iterations set to 500, using 25% of identical haplotypes, only one individual was retained. localities for model training (Phillips et al. 2006). The We estimated likelihood surfaces for the parameter of relative contribution of each variable was assessed via interest, i.e., geographic location of the ancestor, using 1 the increase in gain (a measure of model fit) of the steps on a geographic grid with a longitudinal extension model for a given environmental variable in the train- from 116 to 95 E and a latitudinal extension from 14 to ing set. An alternative test for determining which vari- 40 N (resulting in 594 grid points), thus safely covering ables are the most important ones employs a jackknife the current as well as the putative paleodistribution of procedure and compares models with single variables M. leucanthum (see Results). These analyses were per- (assessing the model gain from one variable) and mod- formed on the whole data set as well as on the three els with all variables except one to the full model main haplotype groups (see Results). As the western (assessing the decrease of model gain when not consid- haplotype group is not mono- but paraphyletic (see ering one variable), again using the training set. Model Results), we pruned sequences of the other haplotype predictions were visualized in ARCMAP 9.3 (ESRI, Red- groups prior to the analysis. All analyses included 1000 lands, CA, USA). optimization iterations. Results Ecological niche modelling DNA ploidy level To model the ecological niche and geographic distribu- tion of Melampodium leucanthum, spatially interpolated DNA ploidy level analyses with flow cytometry of all climate data on grids with a resolution of 2.5 arc-min molecularly investigated individuals showed the pres- were obtained from the WorldClim database (Hijmans ence of diploid and tetraploid cytotypes (Table 1), their et al. 2005; available from http://www.worldclim.org/), ploidy levels being confirmed by chromosome counts of which is based on data from the PMIP2-project (http:// selected populations (Weiss-Schneeweiss et al. 2009; data pmip2.lsce.ipsl.fr) and consists of 19 bioclimatic vari- not shown). In only three diploid populations, intrapop- ables (Table S1). For reconstruction of the geographic ulational cytotype mixture with triploids was found. distribution during the last glacial maximum (LGM; c. 21 000 years BP), two coupled climate models, which AFLP have been successfully used in the past (e.g., Carstens & Richards 2007; Waltari et al. 2007; Cordellier & The five AFLP primer combinations chosen for the anal- Pfenninger 2009), were used: the Community Climate ysis generated 691 unambiguously scorable fragments:

2010 Blackwell Publishing Ltd 3432 C. A. REBERNIG ET AL.

EcoRI-ACA ⁄ MseI-CAT (FAM), 162; EcoRI-ACG ⁄ MseI- tern (P-values >0.05) with the exception of AWD, CAA (VIC), 141; EcoRI-ACC ⁄ MseI-CAG (NED), 84; which showed a weak yet significant positive relation- EcoRI-ACT ⁄ MseI-CAC (FAM), 155; EcoRI-AGG ⁄ MseI- ship with longitude (slope 0.0016, P = 0.0068), i.e., CAA (VIC), 149. All 377 individuals investigated had a AWD values were higher in the east than in the west. unique AFLP profile. The error rate based on pheno- PCO conducted on the whole data set resulted in a typic comparisons among replicated individuals (Bonin clear separation of a western group comprising the et al. 2004) amounted to 4%. populations west of the Continental Divide (pops. 1– The total number of AFLP fragments per population 17) from the remaining ones (Fig. 2a). After exclusion ranged from 171 to 382 (mean ± SD 273.7 ± 35.1), with of these western populations, a group of several, yet 0–40.9% (mean ± SD 23.4 ± 7.1) being polymorphic not all, tetraploid populations (pops. 75–84) was sepa- and 0–3 (median 0) private bands. The distribution of rated from the rest (Fig. 2b). The remaining tetraploid the genetic diversity estimated with AWD (mean ± SD) individuals grouped together with diploids in one big ranged from 0.077 ± 0.192 (population 15) to group, which was weakly differentiated into a central 0.188 ± 0.240 (population 48; Table 1). None of these and an eastern subgroup (Fig. 2b). Results from the descriptors suggested any significant geographic pat- neighbour-net network (Fig. 2c) are highly congruent

(a) (b)

(c) (d)

Fig. 2 Genetic structure of Melampodium leucanthum inferred from AFLP data. (a) Principal Coordinate Analysis (PCO) of the whole data set and (b) of one excluding the western populations; (c) Neighbour-net analysis of the whole data set; (d) STRUCTURE analysis of a data set excluding the tetraploid populations and those of unknown ploidy level (indicated by small black dots). Colours refer to the western (red), central (blue), eastern (green) and tetraploid group (grey). In (c), tetraploids are indicated by thick lines (and lighter green in case of the eastern group). Inserts in (d) as in Figure 1.

2010 Blackwell Publishing Ltd PHYLOGEOGRAPHY OF NORTH AMERICAN DESERT PLANT 3433 with those from the PCO. Specifically, the western Genetic assignment tests congruently suggested six populations are clearly distinct from the remaining populations (pops. 48, 49, 71, 85, 87, 92) as the most populations, and those tetraploids, which were sepa- likely sources for the majority of tetraploid individuals. rated in the second PCO analysis, constitute two well- Qualitatively similar results were obtained when using separated subgroups. All triploid individuals included the more stringent cut-off level of two, which led to a in the analysis fall within their diploid source popula- higher proportion of individuals remaining unassigned tions. The second analysis conducted on the reduced (Table 2). Four of the potential source populations data set did not reveal any new structure (data not (pops. 71, 85, 87, 92) are in close proximity to the tetra- shown). In an AMOVA on the whole data set with four ploid populations, whereas two (pops. 48, 49) are groups identified in the PCO, 22.37% of the variance located further to the west (Fig. 1). The genetic differ- was accounted for by variation among groups, 23.27% entiation of the tetraploid populations (Fig. 2b, c) is not among populations within the groups and 54.36% reflected in their assignments to diploid putative source within populations (P < 0.001 in all cases). Excluding populations. the western populations (using only three groups) gave similar results with 17.37% of the variance cpDNA data accounting for among-group variation, 25.59% for vari- ation among populations within a group and 57.04% Combining psbA-trnH (359–427 bps), rpl32-trnL (679– for variation within populations (P < 0.001 in all 1057 bp) and ndhF-rpl32 (795–1020 bp) resulted in an cases). aligned data matrix of 2584 characters, of which, after Nonhierarchical clustering of only diploid individuals conversion of microinversions, 44 were variable. After using STRUCTURE suggested K = 3 clusters as the optimal recoding indels and inversions as single characters and solution (Fig. S1), irrespective of whether an admixture removal of mononucleotide repeats, the alignment of or a no-admixture model was used. The geographic dis- 2000 bp included 42 variable characters, of which 30 tribution of these three groups, which are concordant were parsimony-informative. with the ones found in the PCO (excluding the tetra- The SAMOVA suggested K = 6 groups, whose distribu- ploid populations), is shown in Fig. 2d. Whereas popu- tion is shown in Fig. 3a. Using these six groups, lations of the western cluster showed no or negligible 75.97% of the genetic variation is found among groups, admixture, populations in the southern part of the con- 12.33% among populations within groups and 11.70% tact zone between the other two clusters showed clear within populations (all P < 0.0001). A similar apportion- signs of admixture (Fig. 2d). ment is obtained with K = 3 groups (Fig. 3a), i.e., the

Table 2 Number of individuals of tetraploid populations (in rows; numbers as in Table 1) assigned to diploid populations (numbers as in Table 1) or remaining unassigned (columns) using a cut-off level of two or, in parentheses, of 0

48 49 71 85 87 92 Unassigned

69 4 (4) 0 72 5 (5) 0 73 3 (4) 1 (0) 74 4 (4) 0 75 1 (1) 2 (2) 0 (1) 1 (0) 76 2 (2) 0 (1) 1 (1) 1 (0) 77 2 (5) 3 (0) 78 1 (3) 1 (2) 3 (0) 79 1 (1) 4 (4) 0 80 0 (3) 1 (1) 3 (0) 81 1 (2) 1 (2) 0 (1) 3 (0) 82 1 (3) 2 (0) 83 1 (5) 4 (0) 84 3 (3) 0 (1) 1 (1) 1 (0) 86 1 (2) 0 (1) 2 (0) 88 2 (2) 0 (1) 1 (0) 89 1 (3) 1 (2) 3 (0) 93 1 (4) 0 (1) 4 (0)

Sum 15 (27) 6 (15) 12 (13) 7 (11) 1 (4) 5 (8) 32 (0)

2010 Blackwell Publishing Ltd 3434 C. A. REBERNIG ET AL.

(a) (c)

(b)

Fig. 3 Genetic structure of Melampodium leucanthum inferred from plastid sequence data. (a) spatial analysis of molecular variance (SAMOVA); (b) statistical parsimony network (unsampled haplotypes indicated by ticks); (c) relaxed clock Bayesian analysis with a demographic model of constant population size (node heights correspond to median ages; clades have posterior probabilities of 0.99 or more unless noted otherwise, their size being proportional to the height of the triangles; terminals are populations numbered as in Table 1; scale bar with increments of 0.25 million years). The three main groups are indicated by colours. In (a), additionally the cir- cumscription of the groups with K = 6, which has the highest FCT value, is indicated by different shades of red (western group) and green (eastern group). Populations harbouring haplotypes belonging to two different groups are highlighted in (a) by an outline col- our corresponding to the second group involved or in (c) by a larger font of the population numbers. Inserts in (a) as in Figure 1.

number of groups identified with network and tree separated along a longitudinal gradient (Fig. 3a). Nota- methods, with 72.91% of the genetic variation among bly, the western haplotype group crosses the Continen- groups, 16.03% among populations within groups and tal Divide and extends east of the upper Rio Grande. 11.03% within populations (all P < 0.0001). The majority of populations are monomorphic, whereas Using statistical parsimony, all 78 haplotypes are one-third of populations comprise haplotypes separated joined in a single network (Fig. 3b). Of those, 38 were by single steps only or haplotypes separated by several found in more than one individual, whereas the mutational steps but still belonging to the same haplo- remaining ones are singletons (Fig. S2). The haplotype type groups (Fig. 3a). Three populations possess haplo- network falls into three haplotype groups, which corre- types belonging to two different haplotype groups spond to the ones identified in a SAMOVA with K = 3, dif- (pops. 40, 47, 56; Fig. 3a). ferences concerning those populations, which harbour For population expansion tests, we used the whole haplotypes from two different haplotype groups. These data set, the three haplotype groups suggested by the three groups are separated from each other by at least statistical parsimony network, and the haplotype four mutational steps (Fig. 3b) and are geographically groups delimited by a SAMOVA with K = 3, because the

2010 Blackwell Publishing Ltd PHYLOGEOGRAPHY OF NORTH AMERICAN DESERT PLANT 3435

only slightly better solution of K = 6 resulted in three ‡ small and genetically homogeneous groups not amena- ble to mismatch distribution analysis. Population expan- % sion in the whole data set is supported by Fu’s FS and

by the sum of squared differences test of the mismatch , results signifi- s distribution (Table 3), although the plot of the observed

pairwise differences was multimodal (Fig. S3), as Long generation time expected for a structured population (Schneider & Ex- ‡

coffier 1999). Whereas there was congruent strong evi- * dence for population expansion in the central group, no population expansion was inferred for the western group (Table 3). Evidence for population expansion in the eastern group is ambiguous and supported only by

Fu’s FS and a unimodal plot of the observed pairwise differences (Fig. S3), but not by the sum of squared dif- Populations expansion time in kyrconfidence (95 interval) ferences test (Table 3). Estimates for the time of expan- sion varied considerably depending on the generation time used, ranging from 77 to 193 kyr for the whole data set (confidence limits 9–581 kyr). If the central and eastern subgroups are considered separately, their expansion times are largely congruent, ranging from 32 to 34.5 kyr with long generation time to c. 75–86 kyr confidence interval) Short generation time with short generation time, again with wide confidence % (95 2.410 (0.805–4.074) 75.3 (25.2–127.3) 30.1 (10.1–50.9) 2.621 (0.609–4.781) 81.9 (19.0–149.4) 32.8 (7.6–59.8) 6.176 (0.727–18.602) 193.0 (22.7–581.3) 77.2 (9.1–232.5) intervals from 0.5 to 173 kyr (Table 3). s Bayesian skyline plots with different group intervals (m = 20, 30, 40) gave similar results (the absolute value

of 2 · lnBF <2.6) with no obvious indications for popu- P lation size changes through time and were indeed rejected in favour of the simpler model of constant pop- † 0.0055 0.5389 0.0047 0.7214 ulation size through time (2 · lnBF <)11). Likewise, a SSD model of different constant population sizes for each haplotype group was rejected in favour of a model of one constant population size (2 · lnBF -6.8). Under a P model of constant population size, the diversification age of the whole species (given as mean ⁄ median and, statistic Mismatch distribution 2 2 0.05640.0561 0.1115 0.1137 0.0121 0.0106 0.0148 0.0141 2.758 (2.150–3.377) 2.732 (2.158–3.328) 86.2 (67.2–105.5) 85.4 (67.4–104.0) 34.5 (26.9–42.2) 34.2 (27.0–41.6) 0.0462 0.0238 0.0749 0.4405 R in parentheses, its 95% highest posterior density inter- R val) is estimated to be 2.22 ⁄ 1.43 (0.24–7.11) million years statistic and mismatch distribution with population expansion times derived from the moment estimator ago (that is more than twice as old as inferred under 2 P

the Bayesian Skyline Plot model; data not shown). The ), R S maximum clade posterior probability tree as well as a S 50% majority rule consensus tree revealed the same 11.264 <0.001 10.010 0.0011 14.191 <0.001 14.283 <0.001 0.0440 0.0158 0.0029 0.5814 17.977 0.0015 0.0850.102 0.5285 0.5307 0.0948 0.0937 0.4214 0.3962 0.1290 0.1303 0.0395 0.0420 2.584 (0.188–5.459) 2.574 (0.043–5.541) – – – – S ) ) ) ) ) ) ) three groups as the TCS network with eastern and cen- F tral groups as monophyletic clades (mean ⁄ median ages of 0.64 ⁄ 0.37 and 0.65 ⁄ 0.37, respectively, with 95% high- est posterior density intervals of 0.05–2.12 and 0.03– P 2.21, respectively) and the western group as a paraphy- letic grade (Fig. 3c). Explicit testing the locations of the 1.2971 0.0840 1.2992 0.0801 1.7249 0.0158 1.7613 0.0129 source for the whole species range expansions provides 0.3770 0.4167 0.38160.3992 0.4075 0.3938 ) ) ) ) ) ) ) D Tajima’s D Fu’s F negligible to positive evidence for a central refugium compared with an eastern (2 · lnBF 1.822) or a western refugium (2 · lnBF 5.098). Monophyly of the polyploids Neutrality tests (Tajima’s D and Fu’s F is clearly rejected (2 · lnBF -64.942). Results of the maximum likelihood approach imple- TCS SAMOVA SAMOVA TCS SAMOVA TCS sum of squared deviations. generation time of 2 and 5 years, respectively. calculated only for groups with evidence for population expansion. Eastern haplotype group Central haplotype group Western haplotype group Table 3 All populations * † ‡ mented in PHYLOMAPPER for inferring ancestral locations cantly supporting population expansion indicated in bold (see text for details)

2010 Blackwell Publishing Ltd 3436 C. A. REBERNIG ET AL. were unstable, and different runs of 1000 optimizations on the same tree resulted in sometimes largely different geographic coordinates (data not shown). This behav- iour was not restricted to the maximum clade posterior probability tree but also occurred in other posterior trees tested (data not shown). Ancestral location likeli- hood surfaces were flat over large parts of the covered geographic range (Fig. S4) and only small areas (0.5% of grid points for the whole species, 4.7% to 8.6% of grid points for the three haplotypes groups) could be rejected as ancestral locations using a doubled likeli- hood difference of two or more. In the whole species data set, 72.4% of grid points had better likelihood scores (up to 0.173 log units) than the maximum likeli- hood locality identified after optimizations and used to obtain the dispersal parameter values. Similar values were obtained for the western and the central haplotype group (24.9% and 35.2% of grid points with likelihood scores better up to 0.011 and 0.053 log units, respec- tively). Consequently, inference of the ancestral loca- tions for these three data sets was not sensibly possible. For the eastern haplotype group, only 1.7% of grid points had better likelihood scores (up to only 0.004 log units), and a region of unlikely ancestral location was inferred for an area between 25–34 N and 101–108 W, thus covering major parts of the Chihuahuan Desert (Fig. 4). Fig. 4 Contour graph of the likelihood surface of ancestral locations for the eastern haplotype group of Melampodium leu- Ecological niche modelling canthum. Darker colours indicate higher doubled log-likelihood differences, i.e., less likely ancestral locations. Pixels represent Model predictive performance of the bioclimatic model 1·1 cells centred at the sampled points, which cover an area was high with AUC values of 0.98 for the training data between 116–95 E and 14–40 N. Coastline shown for the last and 0.96 for the test data and a highly significant bino- glacial maximum. mial test of omission (P < 0.001) and a consequently high fit of the modelled and the actually observed cur- rent distribution (Fig. 5a). The most important environ- the CCSM3 model, regions with highest predicted spe- mental variables, assessed with the heuristic estimates cies occurrence are found east of 102W, thus mostly of relative contributions, were mean temperature of falling into the Tamaulipan Plains, while under the MI- wettest quarter (bio08), the mean temperature of coldest ROC3.2 model, the less compact region of highest pre- quarter (bio11) and the precipitation of warmest quarter dicted species occurrence is found mostly west of (bio18; 20.9 20.8 and 20.6, respectively). Jackknife tests 101W, thus being essentially restricted to the current of variable importance on different sets and test statis- Chihuahuan Desert (Fig. 5b, c). tics congruently suggested the precipitation of warmest quarter (bio18), the mean temperature of coldest quarter Discussion (bio11) and the precipitation of wettest quarter (bio16) as the most important variables (Fig. S5). Southwestern North America faced dramatic changes in Both climatic models used, CCSM3 and MIROC3.2, the Holocene with a shift from woodlands to the cur- indicate two distinct areas of environmental suitability rently widespread desert vegetation, and this was (Fig. 5b, c), but these differ in their extent and their accompanied by range expansions of drought-adapted precise locations. Whereas in the western area this species from their refugia into the newly forming habi- mostly affects the latitudinal extent with otherwise simi- tats (Van Devender 1977; Van Devender & Spaulding lar distribution of highly suitable regions in the north- 1979; Hunter et al. 2001; Jaeger et al. 2005; Haenel ern Sonora, in the eastern area it affects both latitudinal 2007). Based on fossil and paleoclimatic data as well extension and longitudinal position. Specifically, under as on phylogeographic inferences, refugia have been

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(a) Fig. 5 Ecological niche modelling of Melampodium leucanthum for (a) the present and (b, c) the last glacial maximum (c. 21 000 years BP). The paleodistribution was modelled using (b) the CCSM3 and (c) the MIROC3.2 climatic models (see text for details). White dots represent the 164 localities of M. leucant- hum used for ecological niche modelling and cover the entire range of the species. Darker colours indicate higher climatic suitability. In (b) and (c), the coastline is that of the last glacial maximum.

suggested in the western and southern parts of the cur- rent deserts, such as the lower Colorado Basin, the east- ern Sonoran or the southern Chihuahuan Desert (Van Devender 1977; Van Devender & Spaulding 1979; Hun- ter et al. 2001; Riddle & Hafner 2006; Castoe et al. 2007; Holmgren et al. 2007; Fehlberg & Ranker 2009; Sosa et al. 2009). The presence of three genetically clearly separated (more than 70% of genetic variance are explained by (b) among-group variation) and longitudinally arranged haplotype groups in M. leucanthum (Fig. 3), which lar- gely correspond to three major AFLP groups identified by PCO and STRUCTURE analyses (Fig. 2), suggests three separate refugia. Ecological niche modelling supports two major geographically separate refugia west and east of the Continental Divide (Fig. 5), but these were prob- ably not homogeneous, but rather consisted of multiple refugia at least east of the Continental Divide (Castoe et al. 2007; Haenel 2007). As suggested by the location of ecologically suitable areas at the LGM (Fig. 5b, c), the refugium for the western haplotype group is proba- bly tied to the lower Colorado basin refugium identified previously (Hunter et al. 2001; Jaeger et al. 2005; Castoe et al. 2007; Fehlberg & Ranker 2009). Inferences for M. leucanthum east of the Continental Divide are more difficult, because the circumscription of ecologically (c) suitable areas at the LGM differs considerably between the two climatic models used (Fig. 5b, c). Both models congruently support a refugium in the central to south- ern Chihuahuan Desert, which has been repeatedly identified for desert plants and animals (Hunter et al. 2001; Riddle & Hafner 2006; Castoe et al. 2007) and probably harboured the refugium for the central haplo- type group. Although the eastern haplotype group might also be connected to the Chihuahuan refugium, a more easterly refugium around the Tamaulipan Plains (Castoe et al. 2007; Rebernig et al. 2010) remains plausi- ble and finds support from the CCSM3 climatic model (Fig. 5b). A refugium east of the Chihuahuan Desert is also supported by the ancestral location inferred with PHYLOMAPPER (Fig. 4). For the other groups, this method gave, however, noninterpretable results (Fig. S4). Rea- sons for this weak performance may include insufficient signal in our data or deficiencies in the underlying models, such as the single spatially and temporarily

2010 Blackwell Publishing Ltd 3438 C. A. REBERNIG ET AL. constant dispersal parameter. A more detailed assess- Madre Occidental in the late Miocene to early Pliocene ment of these issues is, however, beyond the scope of (Morafka 1977), it was an effective barrier for exchange this paper and will require more extensive simulation between desert biota in this region (Morafka 1977; Mor- studies. In summary, the refugia inferred for M. leu- afka et al. 1992; Hunter et al. 2001; Jaeger et al. 2005; canthum are congruent with those previously suggested Riddle & Hafner 2006; Castoe et al. 2007). The effective- by paleoclimatic and phylogeographic data for both ness of this barrier explains the strong phylogeographic plants and animals (Hunter et al. 2001; Jaeger et al. split between populations on both sides of the Conti- 2005; Riddle & Hafner 2006; Castoe et al. 2007; Haenel nental Divide seen in the AFLP data (Fig. 2). It has 2007; Holmgren et al. 2007; Fehlberg & Ranker 2009). been suggested that Pleistocene climatic fluctuations in Plastid and AFLP data are congruent with respect to combination with a relatively broad ecological ampli- number and location of refugia, but because the fast- tude allowed migrations between western and eastern evolving AFLPs probably trace more recent events deserts (Jaeger et al. 2005; Riddle & Hafner 2006; Castoe (Kropf et al. 2009) than plastid sequences, they probably et al. 2007). This appears also to be the case for M. leu- show the signal of different time levels. Although no canthum, whose migration across the Continental direct age estimates can be obtained from AFLP data Divide, albeit only in an easterly direction, is evident (the presence of an AFLP clock suggested by Kropf from the extension of the western haplotype group et al. 2009 is still contentious: Ehrich et al. 2009), their across this barrier (Fig. 3a). The discrepancy between signal probably reflects Late Quaternary differentiation plastid and AFLP data is likely due to the rapid homog- possibly as recent as the last glacial maximum. In con- enization of AFLPs (because of repeated backcrossing of trast, lineage differentiation identified in the plastid hybrids between resident and immigrant genotypes data is deeply within the Pleistocene and might date with the resident ones, Zhou et al. 2005), which, in con- back to the Tertiary (Fig. 3c). Unless M. leucanthum trast to plastid data, will quickly erase traces of gene remained restricted to these refugia over an extended flow across the Continental Divide. period of time, the evidence for refugia at different time Reduced migration possibilities across the Continental levels suggests that this species responded to the Qua- Divide probably contribute to the contrasting range ternary climatic oscillations in a cyclic manner (Stewart dynamics of populations west and east of it. In the et al. 2010). west, ecologically suitable areas at the LGM were geo- Paleoclimatic data suggest that the Holocene aridifica- graphically close and of comparable extent to the cur- tion progressed from the west to the east (Holmgren rent distribution area in this region (Fig. 5). Supported et al. 2007) with desert species occurring earlier in the by the lack of signal for range expansion (Table 3), this Sonoran Desert (Van Devender 1990; McAuliffe & Van indicates that in this region postglacial range shifts and Devender 1998). Thus, it may be expected that range population size changes were of limited magnitude expansion of drought-adapted species would follow the (Castoe et al. 2007). In contrast, east of the Continental same general direction. For M. leucanthum, this hypoth- Divide, the presumptive refugial areas were much esis finds, however, no support from our data. smaller than the current distribution area (Fig. 5), Although the topology from the Bayesian analysis is in implying major postglacial range expansion. This is line with the hypothesis of an eastward migration start- supported by signals for range expansion in both the ing from an ancestral western lineage (Fig. 3c), explicit central and the eastern haplotype group (Table 3). The hypothesis testing provides evidence for a central or stronger signal for range expansion in the central haplo- eastern origin instead. Furthermore, there is no evi- type group, which is also reflected in the rather star-like dence for eastwardly decreasing times of range expan- structure in the haplotype network (Fig. 3b), might be sion (Table 3), a loss of average within-population because of a more rapid colonization or more strongly diversities inferred from AFLP data or the number of reduced population sizes in a smaller refugium polymorphic or private AFLP fragments (Table 1), as (Fig. 5b, c), but further data are necessary to distin- would be expected if colonization progressed east- guish among these hypotheses. wards. Instead, both plastid and AFLP data (Figs. 2, 3) In the course of range expansions from separate refu- indicate that independent range shifts occurred out of gia, lineages, which differentiated in isolation, came several Pleistocene refugia, a widespread pattern in into secondary contact and started to intermix. This is southern North American desert biota (Hunter et al. evident from the co-occurrence of plastid haplotypes of 2001; Jaeger et al. 2005; Castoe et al. 2007; Fehlberg & different haplotype groups within the same populations Ranker 2009; Garrick et al. 2009; Rebernig et al. 2010). (Fig. 3) as well as the strong signal for genetic admix- A major obstacle for longitudinal migration is the ture (Fig. 2) in some populations east of the Continental Continental Divide. Since its initial formation because Divide as the result of extensive interpopulational of the uplift of the Colorado Plateaus and the Sierra gene flow (M. leucanthum is an obligate outcrosser).

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Secondary contact zones are well known from other ment during geographic isolation in phases of climatic regions, where Pleistocene climatic fluctuations caused deterioration, as suggested for M. cinereum (Rebernig major range shifts (Taberlet et al. 1998; Soltis et al. 2006; et al. 2010), but further data on the actual dynamics at Castoe et al. 2007; Fehlberg & Ranker 2009). The the contact zone are necessary to test this or alternative secondary contact of differentiated lineages evidenced hypotheses. by the AFLP data is probably connected to range The Holocene aridification in southwestern North expansions during the Holocene aridification (Van America undoubtedly had a major impact on the phy- Devender 1977; Webb 1977; Van Devender & Spaulding logeography and population history of drought-adapted 1979; Holmgren et al. 2007; Holliday et al. 2008), but species (Van Devender & Spaulding 1979; Spaulding this cannot be directly tested with the AFLP data. 1990; Van Devender 1990; Castoe et al. 2007; Holmgren Range expansions for the central and eastern haplotype et al. 2007). In M. leucanthum, phases of restriction to groups (there is no evidence for range expansion in the multiple refugia, which enhanced lineage differentiation western haplotype group) are dated to the late Pleisto- and possibly also polyploid establishment, alternated cene (Table 3), but these estimates are burdened with with phases of range expansions and secondary contact, wide confidence intervals partly extending to the Holo- the Continental Divide currently being the only major cene, although only under an assumed generation time migration barrier. These dynamics resulted in a com- of 5 years. These time estimates might be biased plex phylogeographic history in this seemingly homoge- towards older ages because of the time dependency of neously distributed species. molecular rates (Ho et al. 2005, 2007), although its effect appears to be of smaller magnitude than initially antici- Acknowledgements pated (Debruyne & Poinar 2009). Consequently, it remains uncertain whether the range expansions The authors thank Michael Lenko (University of Vienna, Aus- inferred from the plastid data are connected to the tria), Enrique Ortiz (UNAM, Mexico), Monique Reed and Hugh Holocene aridification or to earlier phases of warmer Wilson (Texas A&M University, U.S.A.), and Donovan Bailey and Patrick Alexander (New Mexico State University, U.S.A.) and drier climate (Allen & Anderson 2000). for help with material collection. We thank Gudrun Kohl (Uni- The distribution pattern of tetraploids in M. leucant- versity of Vienna, Austria) for technical assistance and Sabine hum is conspicuous, because this cytotype is found Jakob (IPK Gatersleben, Germany) for help with the bioclimatic exclusively in the eastern part of range of the species data. We thank two anonymous reviewers for helpful criti- (Stuessy et al. 2004; Fig. 2d). Despite this geographic cisms. The study was financially supported by Austrian Science distinctness, tetraploids do not form a genetically cohe- Fund (FWF) grants no. P18201-B03 (to TFS) and Hertha-Firn- sive group (Fig. 2), which contrasts with the pattern berg postdoctoral fellowship T-218 (to HWS). observed in the closely related M. cinereum (Rebernig et al. 2010). Instead, a monophyletic origin of polyp- References loids is clearly rejected by the plastid data (2 · lnBF <)64.942). Furthermore, AFLP data detect both distinct Abbott RJ, Brochmann C (2003) History and evolution of the ´ Molecular Ecology polyploid lineages as well as polyploids that usually arctic flora: in the footsteps of Eric Hulten. , 12, 299–313. intermix with those diploid populations they were Allen BD, Anderson RY (2000) A continuous, high-resolution assigned to (Table 2), thus supporting the hypothesis of record of late Pleistocene climate variability from the multiple origins. 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Taberlet P, Fumagalli L, Wust-Saucy AG, Cosson JF (1998) Supporting information Comparative phylogeography and postglacial colonization Additional supporting information may be found in the online routes in Europe. Molecular Ecology, 7, 453–464. version of this article. Tajima F (1996) The amount of DNA polymorphism maintained in a finite population when the neutral mutation Table S1 Bioclimatic variables used to calculate the probability rate varies among sites. Genetics, 143, 1457–1465. of geographic distribution of Melampodium leucanthum. Thompson JN, Anderson KH (2000) Biomes of western North America at 18 000, 6000 and 0 14C yr BP reconstructed from Table S2 Relative fluorescent intensity and derived DNA pollen and packrat midden data. Journal of Biogeography, 27, ploidy level of all investigated individuals of Melampodium leu- 555–584. canthum. Van Devender TR (1977) Holocene woodlands in the Fig. S1 Summary of the STRUCTURE analysis of AFLP data of Southwestern deserts. Science, 198, 189–192. diploid individuals of Melampodium leucanthum. Van Devender TR (1990) Late Quaternary vegetation and climate of the Chihuahuan Desert, United States and Mexico. Fig. S2 Distribution of plastid haplotypes sampled in Melampo- In: Packrat Middens: The Last 40 000 years of Biotic Change (eds dium leucanthum.

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Fig. S3 Mismatch distributions for all populations as well as Supporting File 2–8 BEAST input files, their names indicating each haplotype group found in Melampodium leucanthum. the used demographic model (files 1–3) or topological hypothe- sis tested (files 4–7; see text for details). Fig. S4 Likelihood surfaces for ancestral locations estimated with PHYLOMAPPER in Melampodium leucanthum. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the Fig. S5 Jackknife tests of variable importance in ecological authors. Any queries (other than missing material) should be niche modelling in Melampodium leucanthum. directed to the corresponding author for the article. Supporting File 1 Details of data analyses.

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