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Molecular Ecology (2010) 19, 3421–3443 doi: 10.1111/j.1365-294X.2010.04754.x
Multiple Pleistocene refugia and Holocene rangeexpansion of an abundant southwestern American desertplant species (Melampodium leucanthum, Asteraceae)
CAROLIN A. REBERNIG,* GERALD M. SCHNEEWEISS ,†1 KATHARINA E. BARDY,†* PETER
SCHONSWETTER,†‡ JOSE L. VILLASENOR,– 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 Biologıa, Departamento de Botanica, Universidad Nacional Autonoma de Mexico, Tercer Circuito s ⁄ n,
Ciudad Universitaria, Delegacion Coyoacan, MX-04510 Mexico D. F., Mexico
Corresponde
E-mail: geral1Present Add
Maximilians-
Munich, Ger
� 2010 Black
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
Introduction
The impact of Pleistocene climatic fluctuations on
directly affected areas, such as the Arctic or temperate
high mountain ranges, has been comparatively well
investigated phylogeographically in both plants and
animals (Hewitt 1996, 2001; Brunsfeld et al. 2001;
nce: Gerald M. Schneeweiss, Fax: +43 1 4277 9541;
ress: Systematic Botany and Mycology, Ludwig-
University Munich, Menzingerstrasse 67, D-80638
many.
well Publishing Ltd
Abbott & Brochmann 2003; Schonswetter et al. 2005).
The role of these climatic fluctuations in other regions,
however, remains less well understood. This is particu-
larly the case for arid regions in northern Mexico and
adjacent southwestern United States. Paleoclimatic and
paleovegetational evidence unambiguously suggests
that desert vegetation was strongly restricted during the
wetter and cooler pluvial periods (Wells 1966; Van
Devender & Spaulding 1979; Thompson & Anderson
2000) and confined to refugia in the west and south,
such as the lower Colorado River Basin, the plains
of Sonora, or the southern Chihuahuan Desert (Van
3422 C. A. REBERNIG ET AL.
Devender 1990; Thompson & Anderson 2000; Hunter
et al. 2001). Large-scale aridification of the whole region
started only after the end of the last glacial maximum
(Van Devender & Spaulding 1979; McClaran & Van
Devender 1995; Bousman 1998; Metcalfe et al. 2000;
Musgrove et al. 2001; Holmgren et al. 2007) and was
accompanied by a shift from xeric woodlands, abundant
until 8000 years BP (Van Devender 1977), to semidesert
grassland and eventually desert shrubland vegetation
(Neilson 1986). Consequently, drought-adapted species
are expected to have persisted in one or more distinct
refugia (Nason et al. 2002; Fehlberg & Ranker 2009),
from where they reached their current distribution after
range expansion within the last 10 000–6000 years (Van
Devender & Spaulding 1979; Spaulding 1990; Van
Devender 1990; Holmgren et al. 2007).
These range expansions into new arid regions are
expected to have had major impacts on population
structure and genetic diversity, for instance resulting in
loss of alleles because of bottlenecks and founder
events, or in secondary contact of genetic lineages dif-
ferentiated in allopatric refugia (Hewitt 2001, 2004). Pa-
leoclimatic modelling indicates that the aridification
progressed from the Sonoran Desert north- and east-
wards (Holmgren et al. 2007), and it can be expected
that range expansion of drought-adapted species fol-
lowed the same general direction (Fehlberg & Ranker
2009). Additionally, rapid expansion should also be
reflected in geographic patterns of genetic diversity,
which is expected to be lower in more recently colo-
nized areas because of founder effects (Hewitt 1996). A
longitudinal migration pattern may, however, be modi-
fied by the Continental Divide, whose establishment in
the late Tertiary is thought to have caused vicariant
diversification in a number of warm-desert animals
(Riddle & Hafner 2006; Castoe et al. 2007). Since then,
the divide has acted as a formidable migration barrier
for desert biota because of the lack of a spatially and
temporally continuous connection between the Sonoran
and the Chihuahuan Deserts, which currently come
closest at the Derning Plains near the border between
Arizona and New Mexico (Morafka 1977; Riddle & Haf-
ner 2006; Castoe et al. 2007). This barrier is expected to
enhance founder effects in the course of eastward
migration. Alternatively, if refugia of drought-adapted
species were also located east of the Continental Divide
(Hunter et al. 2001; Castoe et al. 2007), this region prob-
ably is the contact zone of western and eastern lineages
(Castoe et al. 2007). While several of these hypotheses
have been tested in a number of animal groups (Jaeger
et al. 2005; Riddle & Hafner 2006; Castoe et al. 2007;
Haenel 2007; Fontanella et al. 2008), comparable studies
in plants are lacking. The few studies from desert plants
either investigate species from only one side of the Con-
tinental Divide (Nason et al. 2002; Clark-Tapia & Moli-
na-Freaner 2003; Fehlberg & Ranker 2009; Garrick et al.
2009; Sosa et al. 2009) or they do not employ molecular
methods (Hunter et al. 2001; Holmgren et al. 2007).
By affecting the distribution of a species, environmen-
tal changes will also shape its evolution via, for
instance, enabling or interrupting gene flow in phases
of continuous distribution and range disruption, respec-
tively, or affecting the success of establishment of newly
formed polyploids (Husband 2004; Baack & Stanton
2005; Ramsey et al. 2008). The latter is of particular rele-
vance, because polyploidy is recognized as an impor-
tant mode of speciation in general and one of the more
likely means of sympatric speciation in particular (Otto
& Whitton 2000; Coyne & Orr 2004; Soltis et al. 2007).
While the role of allopolyploidy for speciation has long
been recognized (Ramsey & Schemske 1998, 2002; Le-
itch & Leitch 2008), the rapidly mounting evidence of a
high frequency of autopolyploids, often in mixed popu-
lations with their diploid progenitors (Husband 2004;
Suda et al. 2007), has led to a more positive view con-
cerning the evolutionary significance of autopolyploidi-
zation (Soltis et al. 2007). Despite several recent studies
dealing with the dynamics of diploid–autopolyploid
complexes (Baack & Stanton 2005; Schonswetter et al.
2007; Ramsey et al. 2008; Hulber et al. 2009), their evo-
lutionary significance and the factors involved in poly-
ploid cytotype formation and establishment are still
poorly understood (Baack & Stanton 2005).
Here we use Melampodium leucanthum (Asteraceae),
an abundant taxon of the North American desert and
semi-desert regions, to investigate the impact of the
major aridification in southern North America within
the last 10 000 years on phylogeography and evolution,
including cytotype differentiation, in a drought-
adapted plant species. This phylogenetically distinct
(Bloch et al. 2009) and morphologically and taxonomi-
cally homogeneous species is particularly well suited
to address these questions, because it is distributed
over several major arid and semi-arid biogeographic
regions ranging from the Sonoran and Chihuahuan
Deserts to the Tamaulipan Plain and Southern Plain
region (Stuessy 1972), and it comprises diploid and tet-
raploid cytotypes, the latter restricted to the eastern
part of the distribution area (Fig. 1, Table 1; Stuessy
et al. 2004). Our first aim is to analyse the phylogeo-
graphic patterns caused by post-Pleistocene aridifica-
tion and subsequent migration events. Specifically, we
want (i) to determine the locations of the refugia of
M. leucanthum and test whether these are congruent
with those suggested by paleoclimatic and phylogeo-
graphic data (Hunter et al. 2001; Castoe et al. 2007;
Holmgren et al. 2007; Fehlberg & Ranker 2009); (ii) to
infer the directionality of the range expansion, in
� 2010 Blackwell Publishing Ltd
(a)
(a)
(b)
(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).
PHYLOGEO GRAPHY OF NORTH AMERICAN D ESERT PLANT 3423
particular, whether it was essentially unidirectional fol-
lowing the north- and eastwardly progressing aridifica-
tion (Holmgren et al. 2007); and (iii) to test whether
inferred range expansions fit the time frame predicted
by paleoclimatic data (Holmgren et al. 2007; Holliday
et al. 2008). A second aim is to infer origin and evolu-
tion of the polyploids. Specifically, we want to test (i)
whether the polyploids originated once, as suggested
by their compact and restricted distribution, or recur-
rently, as observed in many species including the clo-
sely related M. cinereum (Rebernig et al. 2010) and (ii)
whether they form a genetically cohesive group clearly
separated from the diploids, as has been found in
M. cinereum (Rebernig et al. 2010). To this end, we
generated amplified fragment length polymorphism
(AFLP) and cpDNA sequence data from several hun-
dred individuals, whose ploidy level was determined
flow cytometrically from 92 populations over the whole
distribution area. These data were analysed using,
among others, a coalescent-based Bayesian approach
for hypothesis testing and molecular dating, comple-
mented by ecological niche modelling for inferring
putative paleodistributions.
� 2010 Blackwell Publishing Ltd
Materials and methods
Study species
Melampodium leucanthum (blackfoot daisy) is a drought-
tolerant, summer-flowering perennial subshrub grow-
ing on calcareous soils between 500 and 2590 m a.s.l.
and is abundant in its distribution area encompassing
northern Mexico and the southwestern United States
from Arizona to eastern Texas, northwards extending
into Oklahoma and Colorado (Fig. 1; Stuessy 1972).
Pollen ⁄ ovule ratios (Cruden 1977) indicate that
M. leucanthum is outcrossing (data not shown). No
population differentiation concerning morphology,
flowering time or breeding system has ever been
reported (Stuessy 1972). Melampodium leucanthum com-
prises diploid and tetraploid cytotypes (with occa-
sional triploid individuals in diploid populations;
Stuessy et al. 2004), which are morphologically indis-
tinguishable (Stuessy 1971, 1972). The two cytotypes
occur mostly parapatrically, and tetraploids occupy a
compact area in eastern Texas to the near exclusion of
diploids (Stuessy et al. 2004).
3424 C. A. REBERNIG ET AL.
Plant material
Plant material was collected from 92 populations of
M. leucanthum covering the entire distribution of the
species (Fig. 1). Samples were dried and stored in silica
gel until DNA isolation. Herbarium vouchers are
deposited in the herbarium of the University of Vienna
(WU; voucher numbers given in Table 1).
Ploidy level determination and molecular methods
Measurements of DNA ploidy levels (Suda et al. 2006)
were conducted as described in Rebernig et al. (2010).
Correct interpretation of DNA ploidy levels was con-
firmed by chromosome numbers determined for
selected individuals using standard Feulgen staining as
described by Weiss-Schneeweiss et al. (2007).
Total genomic DNA was extracted as described in Re-
bernig et al. (2010). AFLP fingerprint profiles were gen-
erated for 1–5 individuals per population totalling 377
individuals (Table 1) following the protocol described
in Dixon et al. (2008). Two negative controls were
included in each PCR, and 6.25% of the samples were
replicated. After initial screening of 33 selective primer
combinations with three to four selective nucleotides,
the following five primer combinations were selected
for the final analyses (fluorescent dyes in parentheses):
EcoRI-ACA ⁄ MseI-CAT (FAM), EcoRI-ACG ⁄ MseI-CAA
(VIC), EcoRI-ACC ⁄ MseI-CAG (NED), EcoRI-ACT ⁄ MseI-CAC (FAM), EcoRI-AGG ⁄ MseI-CAA (VIC). Purification
of selective PCR products, their electrophoretic separa-
tion and subsequent alignment as well as their scoring
(bands in the size range of 100–500 bp) were carried
out as described in Rebernig et al. (2010). Nonreproduc-
ible bands identified by comparisons among replicated
individuals were excluded from further analyses.
The following three noncoding chloroplast DNA
spacer regions were amplified and sequenced as
described in Rebernig et al. (2010) for one to three indi-
viduals per population (Table 1), totalling 228 individu-
als: psbA-trnH, rpl32-trnL, and ndhF-rpl32. Sequences
were assembled using SEQMAN II 5.05 (DNAStar, Madi-
son, WI, USA) and manually aligned using BIOEDIT 7.0
(Hall 1999). Sequences are deposited in GenBank
(Table 1).
Data analyses
AFLP. AFLP data descriptors include the total number
of fragments (Fragtot), the percentage of polymorphic
fragments (Fragpoly), the number of private fragments
(Fragpriv) and the index of average differences within
populations (AWD; Kosman 2003) calculated for popu-
lations with at least three individuals in ARLEQUIN 3.10
(Excoffier et al. 2005). Geographic patterns in AFLP de-
scriptors were tested using multiple linear regressions
in Excel 2007 (Microsoft, Redmond, CA, USA). Principal
coordinate analysis (PCO) was conducted using
NTSYSPC 2.20e (Rohlf 2007) with the default settings
both on the whole data set as well as on a reduced data
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
4.8 (Huson & Bryant 2006) using Nei-Li distances (Nei
& Li 1979) calculated with FAMD 1.108 (Schluter &
Harris 2006). Three-level hierarchical analyses of molec-
ular variance (AMOVA) using the groups suggested by
PCO (see Results) were conducted on both data sets
with ARLEQUIN 3.10 (Excoffier et al. 2005), estimating the
significance of variance components from 10 000 per-
mutations.
Genetically homogeneous groups of diploid individu-
als were identified using genetic mixture analysis
implemented in STRUCTURE 2.2 (Pritchard et al. 2000; Fa-
lush et al. 2007) as described in Rebernig et al. (2010)
with minor modifications (see Supporting materials).
Tetraploid individuals were excluded, because the mod-
els implemented in STRUCTURE are not suited for analy-
sing polyploids (Pritchard et al. 2000).
Assignment tests to determine the most likely source
populations for the tetraploid individuals were per-
formed using AFLPOP 1.1 (Duchesne & Bernatchez
2002) with the default settings. All diploid populations
were considered as potential source populations, and
allocation was tested using two levels (0 or 2) of mini-
mal log-likelihood differences (as recommended by
Duchesne & Bernatchez 2002) with frequency values of
zero replaced by 1 ⁄ (sample size + 1).
cpDNA. Prior to all analyses, inversions in the plastid
sequence data were re-inverted to avoid introducing
substitutional mutations, which in fact are the result of
structural mutations (Lohne & Borsch 2005). This data
set was used only for the BEAST analysis, and for the
other analyses, indels longer than 1 base pair and inver-
sions were additionally recoded as single characters,
and mononucleotide repeats were removed because of
their high degree of homoplasy at larger geographic
scales (Ingvarsson et al. 2003).
Group and population differentiation was assessed
via a spatial analysis of molecular variance (SAMOVA),
which allows defining population groups that are genet-
ically differentiated from each other and occur in a
geographically homogeneous area (Dupanloup et al.
2002). This analysis was conducted with the program
SAMOVA 1.0 (available from http://web.unife.it/progetti/
� 2010 Blackwell Publishing Ltd
Tab
le1
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pu
lati
on
nu
mb
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sam
pli
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nco
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es,
vo
uch
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aly
sed
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s,A
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des
crip
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idy
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um
ber
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podi
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leu
can
thu
m
po
p
nr.
Lo
cati
on
(vo
uch
ern
r.)
Sam
ple
N
AF
LP
⁄cp
DN
AF
rag
tot*
Fra
gp
oly%
†F
rag
pri
v‡
AW
D±
Std
Dev
Plo
idy
§
Gen
Ban
k
acce
ssio
nn
um
ber
s–
1N
35.5
22,
W11
3.45
5(1
8819
)3
⁄223
917
.56
00.
113
±0.
250
2x
FJ8
4621
9,F
J846
220,
FJ8
4601
7;
FJ8
4601
8,F
J846
421,
FJ8
4642
2
2N
35.1
22,
W11
3.66
6(1
8820
)3
⁄321
315
.18
00.
098
±0.
236
2x
FJ8
4622
1,F
J846
222,
FJ8
4622
3;
FJ8
4601
9,F
J846
020,
FJ8
4602
1;
FJ8
4642
3,F
J846
424,
FJ8
4642
5
3N
34.7
55,
W11
2.09
1(1
8814
)3
⁄223
115
.18
00.
098
±0.
236
2x
FJ8
4621
3,F
J846
214,
FJ8
4601
1;
FJ8
4601
2,F
J846
415,
FJ8
4641
6
4N
34.7
30,
W11
1.96
9(1
8813
)3
⁄224
419
.64
00.
127
±0.
262
2x
FJ8
4621
1,F
J846
212,
FJ8
4600
9;
FJ8
4601
0,F
J846
413,
FJ8
4641
4
5N
34.7
12,
W11
1.88
1(1
8812
)5
⁄227
929
.76
00.
140
±0.
226
2x
FJ8
4620
9,F
J846
210,
FJ8
4600
7;
FJ8
4600
8,F
J846
411,
FJ8
4641
2
6N
34.8
26,
W11
1.77
9(1
8816
)3
⁄222
917
.26
00.
111
±0.
249
2x
FJ8
4621
5,F
J846
216,
FJ8
4601
3;
FJ8
4601
4,F
J846
417,
FJ8
4641
8
7N
34.7
57,
W11
1.76
5(1
8817
)5
⁄227
124
.40
00.
110
±0.
204
2x
FJ8
4621
7,F
J846
218,
FJ8
4601
5;
FJ8
4601
6,F
J846
419,
FJ8
4642
0
8N
34.6
06,
W11
1.85
8(1
8808
)4
⁄225
519
.19
00.
102
±0.
215
2x
FJ8
4620
0,F
J846
201,
FJ8
4699
8;
FJ8
4699
9,F
J846
402,
FJ8
4640
3
9N
34.6
18,
W11
1.84
3(1
8809
)5
⁄227
124
.26
00.
112
±0.
208
2x
FJ8
4620
2,F
J846
203,
FJ8
4600
0;
FJ8
4600
1,F
J846
404,
FJ8
4640
5
10N
34.6
39,
W11
1.80
8(1
8811
)5
⁄226
024
.26
00.
115
±0.
213
2x
FJ8
4620
7,F
J846
208,
FJ8
4600
5;
FJ8
4600
6,F
J846
409,
FJ8
4641
0
11N
34.6
50,
W11
1.76
0(1
8810
)5
⁄327
028
.81
00.
113
±0.
211
2x
FJ8
4620
2,F
J846
203,
FJ8
4620
4;
FJ8
4600
2,F
J846
003,
FJ8
4600
4;
FJ8
4640
6,F
J846
407,
FJ8
4640
8
12N
34.3
49,
W11
2.18
4(1
8807
)4
⁄224
618
.60
00.
096
±0.
206
2x
FJ8
4619
8,F
J846
199,
FJ8
4699
6;
FJ8
4699
7,F
J846
400,
FJ8
4640
1
13N
33.9
63,
W11
1.86
3(1
8805
)3
⁄222
313
.48
00.
089
±0.
227
2x
FJ8
4619
6,F
J846
197,
FJ8
4699
4;
FJ8
4699
5,F
J846
398,
FJ8
4639
9
14N
34.0
02,
W11
1.31
4(1
8801
)5
⁄226
222
.02
00.
104
±0.
206
2x
FJ8
4619
2,F
J846
193,
FJ8
4699
0;
FJ8
4699
1,F
J846
394,
FJ8
4639
5
15N
33.4
80,
W11
1.44
3(1
8802
)4
⁄223
414
.73
00.
077
±0.
192
2x
FJ8
4619
4,F
J846
195,
FJ8
4699
2;
FJ8
4699
3,F
J846
396,
FJ8
4639
7
16N
32.6
02,
W11
0.74
5(1
8822
)5
⁄224
118
.30
10.
087
±0.
192
2x
FJ8
4622
4,F
J846
225,
FJ8
4602
2;
FJ8
4602
3,F
J846
426,
FJ8
4642
7
17N
33.2
39,
W11
0.25
3(1
8800
)3
⁄325
018
.60
00.
120
±0.
256
2 xF
J846
189,
FJ8
4619
0,F
J846
191;
FJ8
4698
7,F
J846
988,
FJ8
4698
9;
FJ8
4639
1,F
J846
392,
FJ8
4639
3
18N
32.7
86,
W10
8.13
9(2
0005
)5
⁄228
229
.76
00.
138
±0.
223
2x
FJ8
4623
6,F
J846
237,
FJ8
4603
4;
FJ8
4603
5,F
J846
438,
FJ8
4643
9
PHYLOGEO GRAPHY OF NORTH AMERICAN D ESERT PLANT 3425
� 2010 Blackwell Publishing Ltd
Ta
ble
1(C
on
tin
ued
)
po
p
nr.
Lo
cati
on
(vo
uch
ern
r.)
Sam
ple
N
AF
LP
⁄cp
DN
AF
rag
tot*
Fra
gp
oly%
†F
rag
pri
v‡
AW
D±
Std
Dev
Plo
idy
§
Gen
Ban
k
acce
ssio
nn
um
ber
s–
19N
32.6
40,
W10
7.95
5(2
0003
)4
⁄225
922
.62
00.
121
±0.
231
2xF
J846
234,
FJ8
4623
5,F
J846
032;
FJ8
4603
3,F
J846
436,
FJ8
4643
7
20N
31.9
54,
W10
7.67
6(2
0002
)5
⁄328
730
.06
10.
146
±0.
234
2xF
J846
231,
FJ8
4623
2,F
J846
233;
FJ8
4602
9,F
J846
030,
FJ8
4603
1;
FJ8
4643
3,F
J846
434,
FJ8
4643
5
21N
32.2
63,
W10
7.23
3(2
0000
)3
⁄223
318
.15
00.
117
±0.
254
2xF
J846
229,
FJ8
4623
0,F
J846
027;
FJ8
4602
8,F
J846
431,
FJ8
4643
2
22N
32.3
98,
W10
6.61
4(2
0046
)5
⁄225
118
.60
00.
090
±0.
196
2xF
J846
287,
FJ8
4628
8,F
J846
085;
FJ8
4608
6,F
J846
489,
FJ8
4649
0
23N
32.9
53,
W10
7.49
0(2
0007
)5
⁄226
229
.02
10.
125
±0.
214
2xF
J846
238,
FJ8
4623
9,F
J846
036;
FJ8
4603
7,F
J846
440,
FJ8
4644
1
24N
33.2
75,
W10
7.28
2(2
0010
)5
⁄227
227
.98
00.
133
±0.
225
2xF
J846
240,
FJ8
4624
1,F
J846
038;
FJ8
4603
9,F
J846
442,
FJ8
4644
3
25N
34.1
46,
W10
6.90
8(2
0011
)5
⁄226
926
.93
00.
129
±0.
223
2xF
J846
242,
FJ8
4624
3,F
J846
040;
FJ8
4604
1,F
J846
444,
FJ8
4644
5
26N
33.7
94,
W10
6.27
4(2
0014
)4
⁄228
228
.72
00.
152
±0.
247
2xF
J846
244,
FJ8
4624
5,F
J846
042;
FJ8
4604
3,F
J846
446,
FJ8
4644
7
27N
34.0
10,
W10
5.94
2(2
0016
)5
⁄227
627
.23
00.
128
±0.
220
2xF
J846
246,
FJ8
4624
7,F
J846
044;
FJ8
4604
5,F
J846
448,
FJ8
4644
9
28N
34.9
46,
W10
6.19
1(2
0017
)5
⁄327
628
.57
10.
135
±0.
224
2xF
J846
248,
FJ8
4624
9,F
J846
250;
FJ8
4604
6,F
J846
047,
FJ8
4604
8;
FJ8
4645
0,F
J846
451,
FJ8
4645
2
29N
35.2
88,
W10
6.21
6(2
0021
)5
⁄326
325
.00
00.
119
±0.
216
2xF
J846
251,
FJ8
4625
2,F
J846
253;
FJ8
4604
9,F
J846
050,
FJ8
4605
1;
FJ8
4645
3,F
J846
454,
FJ8
4645
5
30N
34.0
36,
W10
4.74
7(2
0032
)5
⁄228
928
.57
00.
135
±0.
224
2xF
J846
261,
FJ8
4626
2,F
J845
059;
FJ8
4506
0,F
J846
463,
FJ8
4646
4
31N
34.8
98,
W10
4.71
8(2
0031
)5
⁄226
924
.40
00.
115
±0.
212
2xF
J846
259,
FJ8
4626
0,F
J845
057;
FJ8
4505
8,F
J846
461,
FJ8
4646
2
32N
35.3
96,
W10
4.18
0(2
0030
)4
⁄228
727
.08
00.
142
±0.
242
2xF
J846
257,
FJ8
4625
8,F
J845
055;
FJ8
4505
6,F
J846
459,
FJ8
4646
0
33N
35.2
30,
W10
3.76
7(2
0029
)4
⁄228
025
.30
00.
133
±0.
236
2xF
J846
254,
FJ8
4625
5,F
J846
256
FJ8
4505
2,F
J845
053,
FJ8
4505
4;
FJ8
4645
6,F
J846
457,
FJ8
4645
8
34N
31.3
16,
W10
6.07
8(2
0045
)5
⁄228
126
.39
00.
125
±0.
215
2xF
J846
285,
FJ8
4628
6,F
J846
083;
FJ8
4608
4,F
J846
487,
FJ8
4648
8
35N
31.0
05,
W10
4.82
5(1
8726
)3
⁄338
220
.09
00.
130
±0.
264
2xF
J846
120,
FJ8
4612
1,F
J846
122;
FJ8
4591
8,F
J845
919,
FJ8
4592
0;
FJ8
4632
2,F
J846
323,
FJ8
4632
4
3426 C. A. REBERNIG ET AL.
� 2010 Blackwell Publishing Ltd
Ta
ble
1(C
on
tin
ued
)
po
p
nr.
Lo
cati
on
(vo
uch
ern
r.)
Sam
ple
N
AF
LP
⁄cp
DN
AF
rag
tot*
Fra
gp
oly%
†F
rag
pri
v‡
AW
D±
Std
Dev
Plo
idy
§
Gen
Ban
k
acce
ssio
nn
um
ber
s–
36N
31.0
04,
W10
4.82
5(2
0043
)4
⁄230
028
.42
00.
147
±0.
242
2xF
J846
280,
FJ8
4628
1,F
J846
078;
FJ8
4607
9,F
J846
482,
FJ8
4648
3
37N
30.7
89,
W10
4.03
3(1
8725
)5
⁄233
833
.33
10.
159
±0.
237
2xF
J846
118,
FJ8
4611
9,F
J845
916;
FJ8
4591
7,F
J846
329,
FJ8
4632
1
38N
30.9
99,
W10
3.75
6(1
8727
)5
⁄233
331
.99
00.
152
±0.
233
2x*
FJ8
4612
3,F
J846
124,
FJ8
4592
1;
FJ8
4592
2,F
J846
325,
FJ8
4632
6
39N
31.3
52,
W10
3.57
9(1
8729
)2
⁄225
513
.99
00.
135
±0.
342
2xF
J846
125,
FJ8
4612
6,F
J845
923;
FJ8
4592
4,F
J846
327,
FJ8
4632
8
40N
31.6
11,
W10
4.85
7(2
0044
)5
⁄331
434
.97
10.
163
±0.
234
2xF
J846
282,
FJ8
4628
3,F
J846
284;
FJ8
4608
0,F
J846
081,
FJ8
4608
2;
FJ8
4648
4,F
J846
485,
FJ8
4648
6
41N
32.4
90,
W10
4.34
8(2
0033
)5
⁄326
225
.74
30.
112
±0.
199
2xF
J846
263,
FJ8
4626
4,F
J846
265;
FJ8
4506
1,F
J845
062,
FJ8
4506
3;
FJ8
4646
5,F
J846
466,
FJ8
4646
7
42N
32.5
29,
W10
3.80
2(2
0034
)3
⁄325
921
.28
00.
137
±0.
270
2xF
J846
266,
FJ8
4626
7,F
J846
268;
FJ8
4506
4,F
J845
065,
FJ8
4506
6;
FJ8
4646
8,F
J846
469,
FJ8
4647
0
43N
32.5
07,
W10
3.12
7(2
0035
)4
⁄325
924
.12
00.
130
±0.
239
2xF
J846
269,
FJ8
4627
0,F
J846
271;
FJ8
4506
7,F
J845
068,
FJ8
4506
9;
FJ8
4647
1,F
J846
472,
FJ8
4647
3
44N
32.2
88,
W10
2.61
1(1
8737
)3
⁄228
921
.28
00.
137
±0.
270
2xF
J846
139,
FJ8
4614
0,F
J845
937;
FJ8
4593
8,F
J846
341,
FJ8
4634
2
45N
31.8
52,
W10
3.11
4(1
8738
)3
⁄229
620
.04
00.
159
±0.
365
2xF
J846
141,
FJ8
4614
2,F
J845
939;
FJ8
4594
0,F
J846
343,
FJ8
4634
4
46N
31.6
40,
W10
2.63
4(1
8730
)5
⁄231
630
.95
00.
147
±0.
231
2xF
J846
127,
FJ8
4612
8,F
J845
925;
FJ8
4592
6,F
J846
329,
FJ8
4633
0
47N
31.6
98,
W10
2.57
2(1
8731
)5
⁄231
729
.02
00.
134
±0.
220
2x⁄3
xF
J846
129,
FJ8
4613
0,F
J845
927;
FJ8
4592
8,F
J846
331,
FJ8
4633
2
48N
30.7
48,
W10
2.90
7(1
8722
)5
⁄236
940
.92
00.
188
±0.
240
2xF
J846
116,
FJ8
4611
7,F
J845
914;
FJ8
4591
5,F
J846
318,
FJ8
4631
9
49N
30.2
39,
W10
3.38
0(2
0038
)5
⁄231
532
.59
00.
149
±0.
226
2xF
J846
272,
FJ8
4627
3,F
J846
070;
FJ8
4607
1,F
J846
474,
FJ8
4647
5
50N
29.7
85,
W10
3.17
7(2
0039
)5
⁄327
624
.70
00.
119
±0.
218
2xF
J846
274,
FJ8
4627
5,F
J846
276;
FJ8
4607
2,F
J846
073,
FJ8
4607
4;
FJ8
4647
6,F
J846
477,
FJ8
4647
8
51N
29.5
16,
W10
3.40
3(2
0040
)3
⁄325
318
.30
10.
118
±0.
255
2xF
J846
277,
FJ8
4627
8,F
J846
279;
FJ8
4607
5,F
J846
076,
FJ8
4607
7;
FJ8
4647
9,F
J846
480,
FJ8
4648
1
52N
31.9
34,
W10
1.86
6(1
8732
)1
⁄120
60
0⁄
2xF
J846
131,
FJ8
4592
9,F
J846
333
PHYLOGEO GRAPHY OF NORTH AMERICAN D ESERT PLANT 3427
� 2010 Blackwell Publishing Ltd
Ta
ble
1(C
on
tin
ued
)
po
p
nr.
Lo
cati
on
(vo
uch
ern
r.)
Sam
ple
N
AF
LP
⁄cp
DN
AF
rag
tot*
Fra
gp
oly%
†F
rag
pri
v‡
AW
D±
Std
Dev
Plo
idy
§
Gen
Ban
k
acce
ssio
nn
um
ber
s–
53N
31.8
71,
W10
1.64
6(1
8733
)5
⁄233
332
.39
00.
156
±0.
238
2xF
J846
132,
FJ8
4613
3,F
J845
930;
FJ8
4593
1,F
J846
334,
FJ8
4633
5
54N
32.9
64,
W10
2.01
2(1
8735
)5
⁄233
233
.33
10.
156
±0.
233
2xF
J846
137,
FJ8
4613
8,F
J845
935;
FJ8
4593
6,F
J846
339,
FJ8
4634
0
55N
35.3
26,
W10
2.37
0(1
8753
)3
⁄229
423
.51
00.
152
±0.
280
2xF
J846
143,
FJ8
4614
4,F
J845
941;
FJ8
4594
2,F
J846
345,
FJ8
4634
6
56N
35.0
03,
W10
1.91
9(1
8756
)4
⁄230
328
.27
00.
149
±0.
246
2xF
J846
145,
FJ8
4614
6,F
J845
943;
FJ8
4594
4,F
J846
347,
FJ8
4634
8
57N
34.9
86,
W10
1.71
7(1
8757
)3
⁄228
623
.51
00.
152
±0.
280
2xF
J846
147,
FJ8
4614
8,F
J845
945;
FJ8
4594
6,F
J846
349,
FJ8
4635
0
58N
36.2
21,
W10
1.33
4(1
8758
)5
⁄232
020
.21
20.
140
±0.
225
2xF
J846
149,
FJ8
4615
0,F
J845
947;
FJ8
4594
8,F
J846
351,
FJ8
4635
2
59N
36.4
49,
W10
0.37
2(1
8760
)4
⁄329
826
.04
00.
138
±0.
240
2xF
J846
151,
FJ8
4615
2,F
J846
153;
FJ8
4594
9,F
J845
950,
FJ8
4595
1;
FJ8
4635
3,F
J846
354,
FJ8
4635
5
60N
36.4
27,
W99
.883
(187
61)
4⁄2
298
20.8
70
0.15
2±
0.24
82x
⁄3x
FJ8
4615
4,F
J846
155,
FJ8
4595
2;
FJ8
4595
3,F
J846
356,
FJ8
4635
7
61N
35.8
45,
W10
0.39
7(1
8762
)3
⁄325
718
.90
00.
122
±0.
278
2xF
J846
156,
FJ8
4615
7,F
J846
158;
FJ8
4595
4,F
J845
955,
FJ8
4595
6;
FJ8
4635
8,F
J846
359,
FJ8
4636
0
62N
35.4
32,
W10
0.77
0(1
8764
)3
⁄227
319
.64
00.
127
±0.
262
2xF
J846
159,
FJ8
4616
0,F
J845
957;
FJ8
4595
8,F
J846
361,
FJ8
4636
2
63N
35.0
09,
W10
0.89
6(1
8772
)4
⁄228
420
.98
10.
111
±0.
222
2x⁄3
xF
J846
168,
FJ8
4616
9,F
J845
966;
FJ8
4596
7,F
J846
370,
FJ8
4637
1
64N
34.7
88,
W10
0.89
8(1
8771
)1
⁄120
40
0⁄
2xF
J846
167,
FJ8
4596
5,F
J846
369
65N
34.3
80,
W10
1.11
1(1
8770
)4
⁄229
333
.44
00.
152
±0.
231
2xF
J846
165,
FJ8
4616
6,F
J845
963;
FJ8
4596
4,F
J846
367,
FJ8
4636
8
66N
34.2
19,
W10
0.88
8(1
8769
)3
⁄130
929
.02
00.
187
±0.
230
2xF
J846
163,
FJ8
4616
4,F
J845
961;
FJ8
4596
2,F
J846
365,
FJ8
4636
6
67N
33.8
60,
W10
0.85
2(1
8768
)3
⁄227
023
.10
00.
149
±0.
278
2xF
J846
161,
FJ8
4616
2,F
J845
959;
FJ8
4596
0,F
J846
363,
FJ8
4636
4
68N
31.9
00,
W10
0.71
7(1
8734
)5
⁄ 331
326
.79
00.
127
±0.
221
2xF
J846
134,
FJ8
4613
5,F
J846
136;
FJ8
4593
2,F
J845
933,
FJ8
4593
4;
FJ8
4633
6,F
J846
337,
FJ8
4633
8
69N
29.7
18,
W10
1.36
1(1
8720
)4
⁄329
829
.32
10.
159
±0.
256
4xF
J846
113,
FJ8
4611
4,F
J846
115;
FJ8
4591
1,F
J845
912,
FJ8
4591
3;
FJ8
4631
5,F
J846
316,
FJ8
4631
7
70N
28.2
28,
W10
1.05
6(1
9056
)3
⁄322
918
.75
00.
121
±0.
267
2xF
J846
226,
FJ8
4622
7,F
J846
228;
FJ8
4502
4,F
J845
025,
FJ8
4502
6;
FJ8
4642
8,F
J846
429,
FJ8
4643
0
3428 C. A. REBERNIG ET AL.
� 2010 Blackwell Publishing Ltd
Ta
ble
1(C
on
tin
ued
)
po
p
nr.
Lo
cati
on
(vo
uch
ern
r.)
Sam
ple
N
AF
LP
⁄cp
DN
AF
rag
tot*
Fra
gp
oly%
†F
rag
pri
v‡
AW
D±
Std
Dev
Plo
idy
§
Gen
Ban
k
acce
ssio
nn
um
ber
s–
71N
31.2
26,
W99
.757
(187
74)
5⁄2
300
25.1
50
0.11
9±
0.21
52x
⁄3x
FJ8
4617
0,F
J846
171,
FJ8
4596
8;
FJ8
4596
9,F
J846
372,
FJ8
4637
3
72N
31.7
81,
W99
.779
(187
76)
4⁄2
278
17.4
10
0.08
9±
0.20
64x
FJ8
4617
2,F
J846
173,
FJ8
4597
0;
FJ8
4597
1,F
J846
374,
FJ8
4637
5
73N
31.7
61,
W98
.899
(187
78)
4⁄2
281
17.8
60
0.09
5±
0.21
04x
FJ8
4617
4,F
J846
175,
FJ8
4597
2;
FJ8
4597
3,F
J846
376,
FJ8
4637
7
74N
31.6
89,
W98
.814
(187
79)
4⁄2
292
23.1
70
0.12
6±
0.23
44x
FJ8
4617
6,F
J846
168,
FJ8
4597
4;
FJ8
4597
5,F
J846
378,
FJ8
4637
9
75N
31.7
93,
W98
.228
(187
87)
4⁄2
252
12.2
81
0.11
4±
0.22
64x
FJ8
4618
7,F
J846
188,
FJ8
4598
5;
FJ8
4598
6,F
J846
389,
FJ8
4639
0
76N
31.5
75,
W97
.834
(187
86)
4⁄2
248
20.9
80
0.11
2±
0.22
44x
FJ8
4618
3,F
J846
184,
FJ8
4598
3;
FJ8
4598
4,F
J846
387,
FJ8
4638
8
77N
31.3
58,
W98
.129
(187
81)
5⁄2
255
24.5
50
0.11
8±
0.21
74x
FJ8
4617
8,F
J846
179,
FJ8
4597
6;
FJ8
4597
7,F
J846
380,
FJ8
4638
1
78N
31.1
70,
W98
.183
(187
82)
5⁄2
237
22.1
70
0.10
3±
0.20
14x
FJ8
4618
0,F
J846
181,
FJ8
4597
8;
FJ8
4597
9,F
J846
382,
FJ8
4638
3
79N
30.9
28,
W98
.002
(187
83)
1⁄1
171
00
⁄4x
FJ8
4618
2,F
J845
980,
FJ8
4638
4
80N
31.0
64,
W97
.572
(187
85)
4⁄2
246
20.0
90
0.10
8±
0.22
24x
FJ8
4618
3,F
J846
184,
FJ8
4598
1;
FJ8
4598
2,F
J846
385,
FJ8
4638
6
81N
30.6
16,
W97
.860
(187
09)
5⁄2
248
20.3
90
0.09
5±
0.19
74x
*F
J846
103,
FJ8
4610
4,F
J845
901;
FJ8
4590
2,F
J846
305,
FJ8
4630
6
82N
30.6
94,
W97
.981
(187
10)
3⁄2
224
15.3
30
0.09
9±
0.23
74x
FJ8
4610
5,F
J846
106,
FJ8
4580
3;
FJ8
4580
4,F
J846
307,
FJ8
4630
8
83N
30.6
96,
W98
.254
(187
11)
5⁄2
252
21.5
80
0.10
1±
0.20
24x
FJ8
4610
7,F
J846
108,
FJ8
4590
5;
FJ8
4590
6,F
J846
309,
FJ8
4631
0
84N
30.6
71,
W98
.256
(187
12)
5⁄2
248
21.4
30
0.10
2±
0.20
54x
FJ8
4610
9,F
J846
110,
FJ8
4590
7;
FJ8
4590
8,F
J846
311,
FJ8
4631
2
85N
30.2
80,
W98
.907
(186
81)
5⁄2
306
33.7
80
0.15
±0.
229
2xF
J846
093,
FJ8
4609
4,F
J845
891;
FJ8
4589
2,F
J846
295,
FJ8
4629
6
86N
30.1
70,
W98
.849
(186
87)
3⁄2
246
23.8
10
0.13
4±
0.28
14x
FJ8
4610
1,F
J846
102,
FJ8
4589
9;
FJ8
4590
0,F
J846
303,
FJ8
4630
4
87N
30.1
70,
W98
.907
(186
82)
5⁄2
289
29.0
21
0.13
7±
0.22
52x
⁄3x
FJ8
4609
5,F
J846
096,
FJ8
4589
3;
FJ8
4589
4,F
J846
297,
FJ8
4629
8
88N
29.6
16,
W98
.757
(186
86)
3⁄2
237
18.7
50
0.12
1±
0.25
74x
FJ8
4609
9,F
J846
100,
FJ8
4589
7;
FJ8
4589
8,F
J846
301,
FJ8
4630
2
89N
29.8
91,
W98
.408
(186
83)
5⁄2
295
32.8
90
0.16
0±
0.24
14x
FJ8
4609
7,F
J846
098,
FJ8
4589
5;
FJ8
4589
6,F
J846
299,
FJ8
4630
0
90N
30.1
94,
W98
.478
(186
76)
0⁄2
⁄⁄
⁄⁄
2xF
J846
087,
FJ8
4608
8,F
J846
289;
FJ8
4629
0,F
J845
885,
FJ8
4588
6
PHYLOGEO GRAPHY OF NORTH AMERICAN D ESERT PLANT 3429
� 2010 Blackwell Publishing Ltd
Ta
ble
1(C
on
tin
ued
)
po
p
nr.
Lo
cati
on
(vo
uch
ern
r.)
Sam
ple
N
AF
LP
⁄cp
DN
AF
rag
tot*
Fra
gp
oly%
†F
rag
pri
v‡
AW
D±
Std
Dev
Plo
idy
§
Gen
Ban
k
acce
ssio
nn
um
ber
s–
91N
30.2
16,
W98
.478
(186
77)
4⁄2
300
27.6
80
0.14
5±
0.24
32x
FJ8
4608
9,F
J846
090,
FJ8
4588
7;
FJ8
4588
8,F
J846
291,
FJ8
4629
2
92N
30.2
27,
W98
.382
(186
79)
5⁄2
289
33.6
30
0.15
6±
0.23
12x
FJ8
4609
1,F
J846
092,
FJ8
4588
9;
FJ8
4589
0,F
J846
293,
FJ8
4629
4
93N
30.3
87,
W98
.366
(187
14)
5⁄2
319
25.6
00
0.12
2±
0.21
34x
FJ8
4611
1,F
J846
112,
FJ8
4590
9;
FJ8
4591
0,F
J846
313,
FJ8
4631
4
* nu
mb
ero
fto
tal
AF
LP
frag
men
ts.
†p
erce
nta
ge
of
po
lym
orp
hic
AF
LP
frag
men
ts.
‡n
um
ber
of
pri
vat
eA
FL
Pfr
agm
ents
.§as
teri
sks
ind
icat
eD
NA
plo
idy
lev
els
con
firm
edb
ych
rom
oso
me
cou
nts
.–ps
bA-t
rnH
,n
dhF
-rpl
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;d
iffe
ren
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div
idu
als
are
sep
arat
edb
yse
mic
olo
ns.
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
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
(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-
tive approach, we used mismatch distribution (the
distribution of pairwise differences among individuals),
where a unimodal distribution indicates population
expansion (Rogers & Harpending 1992), as described in
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
and by the SAMOVA (see Results), applying in case of
multiple comparisons P-value correction via sequential
Bonferroni correction (Rice 1989). As another way to
test for population expansions, we used the method
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
the previous step. Each of the three main haplotype
groups (see Results) was considered in return as repre-
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-
phyletic. By doing so, we have to assume that gene lin-
eages within the nonrefugial populations coalesced
before they coalesced with those from the refugial pop-
ulations. Besides, we also tested whether polyploid
populations of M. leucanthum originated once (consti-
tute a monophyletic group) as suggested by the com-
pact distribution area parapatric to that of the diploids.
All hypotheses testing in BEAST employed Bayes fac-
tors (BF; Suchard et al. 2001, 2005). Marginal likelihoods
(including their Monte Carlo error: Suchard et al. 2003;
� 2010 Blackwell Publishing Ltd
PHYLOGEO GRAPHY OF NORTH AMERICAN D ESERT PLANT 3431
Redelings & Suchard 2005) and BFs were calculated
with Tracer 1.4 (available from http://evolve.zoo.ox.a-
c.uk/). As test statistic, we used the widely applied
2 · lnBF, considering 2 · lnBFmodel 1 vs. model 2 > 10 as
strong support for model 1. The BEAST input files
(xml-files) are available as Supporting online material.
As a different approach for assessing geographic loca-
tions of ancestors and migration directionalities of con-
tinuously distributed species, we used the method
implemented in PHYLOMAPPER 1.b1 (Lemmon & Lemmon
2008). Briefly, using a spatial random walk model of
migration, it calculates the likelihood of geographic coor-
dinates of clade ancestors (i.e., specified internal nodes)
and the mean per-generation dispersal distance (which
may be treated as a nuisance parameter), given the geo-
graphic coordinates of the sampled individuals (i.e., tree
terminals) and assuming tree topology and branch
lengths to be known without error (Lemmon & Lemmon
2008). We took the maximum clade posterior probability
tree (determined with the TreeAnnotator module of
BEAST) from the BEAST analysis with the best sup-
ported demographic model (see Results), complemented
with dummy outgroup sequences (required for defini-
tion of clades). If populations included individuals with
identical haplotypes, only one individual was retained.
We estimated likelihood surfaces for the parameter of
interest, i.e., geographic location of the ancestor, using 1�steps on a geographic grid with a longitudinal extension
from 116 to 95� E and a latitudinal extension from 14 to
40� N (resulting in 594 grid points), thus safely covering
the current as well as the putative paleodistribution of
M. leucanthum (see Results). These analyses were per-
formed on the whole data set as well as on the three
main haplotype groups (see Results). As the western
haplotype group is not mono- but paraphyletic (see
Results), we pruned sequences of the other haplotype
groups prior to the analysis. All analyses included 1000
optimization iterations.
Ecological niche modelling
To model the ecological niche and geographic distribu-
tion of Melampodium leucanthum, spatially interpolated
climate data on grids with a resolution of 2.5 arc-min
were obtained from the WorldClim database (Hijmans
et al. 2005; available from http://www.worldclim.org/),
which is based on data from the PMIP2-project (http://
pmip2.lsce.ipsl.fr) and consists of 19 bioclimatic vari-
ables (Table S1). For reconstruction of the geographic
distribution during the last glacial maximum (LGM;
c. 21 000 years BP), two coupled climate models, which
have been successfully used in the past (e.g., Carstens
& Richards 2007; Waltari et al. 2007; Cordellier &
Pfenninger 2009), were used: the Community Climate
� 2010 Blackwell Publishing Ltd
System Model version 3 (CCSM3: Collins et al. 2006)
and the Model for Interdisciplinary Research on Cli-
mate version 3.2 (MIROC3.2: Hasumi & Emori 2004).
The list of localities of M. leucanthum (Table 1) was
augmented with data from our own collections and pre-
viously published data (Stuessy et al. 2004), resulting in
a final data set of 164 entries. Distribution modelling
was performed using MAXENT 3.2.19 (available from
http://www.cs.princeton.edu/~schapire/maxent/),
which uses the maximum entropy method. Using pres-
ence-only data, it estimates a target probability distribu-
tion by finding the maximum entropy probability
distribution with the constraint that the expected value
of each feature should match its empirical average
(Phillips et al. 2006). The model for the current distribu-
tion was calculated using all 19 bioclimatic variables
and was in the following applied to the bioclimatic
variables of CCSM and MIROC, respectively. Perfor-
mance of this model was evaluated by the area under
the receiver operating characteristic (ROC) curve
(AUC), which ranges from 0.5 (random prediction) to 1
(maximum prediction), and a binomial test of omission
with the default convergence threshold (10)5) and the
maximum number of iterations set to 500, using 25% of
localities for model training (Phillips et al. 2006). The
relative contribution of each variable was assessed via
the increase in gain (a measure of model fit) of the
model for a given environmental variable in the train-
ing set. An alternative test for determining which vari-
ables are the most important ones employs a jackknife
procedure and compares models with single variables
(assessing the model gain from one variable) and mod-
els with all variables except one to the full model
(assessing the decrease of model gain when not consid-
ering one variable), again using the training set. Model
predictions were visualized in ARCMAP 9.3 (ESRI, Red-
lands, CA, USA).
Results
DNA ploidy level
DNA ploidy level analyses with flow cytometry of all
molecularly investigated individuals showed the pres-
ence of diploid and tetraploid cytotypes (Table 1), their
ploidy levels being confirmed by chromosome counts of
selected populations (Weiss-Schneeweiss et al. 2009; data
not shown). In only three diploid populations, intrapop-
ulational cytotype mixture with triploids was found.
AFLP
The five AFLP primer combinations chosen for the anal-
ysis generated 691 unambiguously scorable fragments:
3432 C. A. REBERNIG ET AL.
EcoRI-ACA ⁄ MseI-CAT (FAM), 162; EcoRI-ACG ⁄ MseI-CAA (VIC), 141; EcoRI-ACC ⁄ MseI-CAG (NED), 84;
EcoRI-ACT ⁄ MseI-CAC (FAM), 155; EcoRI-AGG ⁄ MseI-CAA (VIC), 149. All 377 individuals investigated had a
unique AFLP profile. The error rate based on pheno-
typic comparisons among replicated individuals (Bonin
et al. 2004) amounted to 4%.
The total number of AFLP fragments per population
ranged from 171 to 382 (mean ± SD 273.7 ± 35.1), with
0–40.9% (mean ± SD 23.4 ± 7.1) being polymorphic
and 0–3 (median 0) private bands. The distribution of
the genetic diversity estimated with AWD (mean ± SD)
ranged from 0.077 ± 0.192 (population 15) to
0.188 ± 0.240 (population 48; Table 1). None of these
descriptors suggested any significant geographic pat-
(a) (b
(c) (d)
Fig. 2 Genetic structure of Melampodium leucanthum inferred from A
data set and (b) of one excluding the western populations; (c) Neighb
a data set excluding the tetraploid populations and those of unknow
the western (red), central (blue), eastern (green) and tetraploid gro
lighter green in case of the eastern group). Inserts in (d) as in Figure
tern (P-values >0.05) with the exception of AWD,
which showed a weak yet significant positive relation-
ship with longitude (slope 0.0016, P = 0.0068), i.e.,
AWD values were higher in the east than in the west.
PCO conducted on the whole data set resulted in a
clear separation of a western group comprising the
populations west of the Continental Divide (pops. 1–
17) from the remaining ones (Fig. 2a). After exclusion
of these western populations, a group of several, yet
not all, tetraploid populations (pops. 75–84) was sepa-
rated from the rest (Fig. 2b). The remaining tetraploid
individuals grouped together with diploids in one big
group, which was weakly differentiated into a central
and an eastern subgroup (Fig. 2b). Results from the
neighbour-net network (Fig. 2c) are highly congruent
)
FLP data. (a) Principal Coordinate Analysis (PCO) of the whole
our-net analysis of the whole data set; (d) STRUCTURE analysis of
n ploidy level (indicated by small black dots). Colours refer to
up (grey). In (c), tetraploids are indicated by thick lines (and
1.
� 2010 Blackwell Publishing Ltd
PHYLOGEO GRAPHY OF NORTH AMERICAN D ESERT PLANT 3433
with those from the PCO. Specifically, the western
populations are clearly distinct from the remaining
populations, and those tetraploids, which were sepa-
rated in the second PCO analysis, constitute two well-
separated subgroups. All triploid individuals included
in the analysis fall within their diploid source popula-
tions. The second analysis conducted on the reduced
data set did not reveal any new structure (data not
shown). In an AMOVA on the whole data set with four
groups identified in the PCO, 22.37% of the variance
was accounted for by variation among groups, 23.27%
among populations within the groups and 54.36%
within populations (P < 0.001 in all cases). Excluding
the western populations (using only three groups)
gave similar results with 17.37% of the variance
accounting for among-group variation, 25.59% for vari-
ation among populations within a group and 57.04%
for variation within populations (P < 0.001 in all
cases).
Nonhierarchical clustering of only diploid individuals
using STRUCTURE suggested K = 3 clusters as the optimal
solution (Fig. S1), irrespective of whether an admixture
or a no-admixture model was used. The geographic dis-
tribution of these three groups, which are concordant
with the ones found in the PCO (excluding the tetra-
ploid populations), is shown in Fig. 2d. Whereas popu-
lations of the western cluster showed no or negligible
admixture, populations in the southern part of the con-
tact zone between the other two clusters showed clear
signs of admixture (Fig. 2d).
Table 2 Number of individuals of tetraploid populations (in rows; nu
as in Table 1) or remaining unassigned (columns) using a cut-off leve
48 49 71
69 4 (4)
72 5 (5)
73 3 (4)
74 4 (4)
75 1 (1) 2 (2)
76 2 (2) 0 (1)
77 2 (5)
78 1 (3) 1 (2)
79
80 0 (3)
81 1 (2) 1 (2)
82 1 (3)
83 1 (5)
84 3 (3)
86
88
89
93 1 (4)
Sum 15 (27) 6 (15) 12 (13)
� 2010 Blackwell Publishing Ltd
Genetic assignment tests congruently suggested six
populations (pops. 48, 49, 71, 85, 87, 92) as the most
likely sources for the majority of tetraploid individuals.
Qualitatively similar results were obtained when using
the more stringent cut-off level of two, which led to a
higher proportion of individuals remaining unassigned
(Table 2). Four of the potential source populations
(pops. 71, 85, 87, 92) are in close proximity to the tetra-
ploid populations, whereas two (pops. 48, 49) are
located further to the west (Fig. 1). The genetic differ-
entiation of the tetraploid populations (Fig. 2b, c) is not
reflected in their assignments to diploid putative source
populations.
cpDNA data
Combining psbA-trnH (359–427 bps), rpl32-trnL (679–
1057 bp) and ndhF-rpl32 (795–1020 bp) resulted in an
aligned data matrix of 2584 characters, of which, after
conversion of microinversions, 44 were variable. After
recoding indels and inversions as single characters and
removal of mononucleotide repeats, the alignment of
2000 bp included 42 variable characters, of which 30
were parsimony-informative.
The SAMOVA suggested K = 6 groups, whose distribu-
tion is shown in Fig. 3a. Using these six groups,
75.97% of the genetic variation is found among groups,
12.33% among populations within groups and 11.70%
within populations (all P < 0.0001). A similar apportion-
ment is obtained with K = 3 groups (Fig. 3a), i.e., the
mbers as in Table 1) assigned to diploid populations (numbers
l of two or, in parentheses, of 0
85 87 92 Unassigned
0
0
1 (0)
0
0 (1) 1 (0)
1 (1) 1 (0)
3 (0)
3 (0)
1 (1) 4 (4) 0
1 (1) 3 (0)
0 (1) 3 (0)
2 (0)
4 (0)
0 (1) 1 (1) 1 (0)
1 (2) 0 (1) 2 (0)
2 (2) 0 (1) 1 (0)
1 (3) 1 (2) 3 (0)
0 (1) 4 (0)
7 (11) 1 (4) 5 (8) 32 (0)
(a)
(b)
(c)
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.
3434 C. A. REBERNIG ET AL.
number of groups identified with network and tree
methods, with 72.91% of the genetic variation among
groups, 16.03% among populations within groups and
11.03% within populations (all P < 0.0001).
Using statistical parsimony, all 78 haplotypes are
joined in a single network (Fig. 3b). Of those, 38 were
found in more than one individual, whereas the
remaining ones are singletons (Fig. S2). The haplotype
network falls into three haplotype groups, which corre-
spond to the ones identified in a SAMOVA with K = 3, dif-
ferences concerning those populations, which harbour
haplotypes from two different haplotype groups. These
three groups are separated from each other by at least
four mutational steps (Fig. 3b) and are geographically
separated along a longitudinal gradient (Fig. 3a). Nota-
bly, the western haplotype group crosses the Continen-
tal Divide and extends east of the upper Rio Grande.
The majority of populations are monomorphic, whereas
one-third of populations comprise haplotypes separated
by single steps only or haplotypes separated by several
mutational steps but still belonging to the same haplo-
type groups (Fig. 3a). Three populations possess haplo-
types belonging to two different haplotype groups
(pops. 40, 47, 56; Fig. 3a).
For population expansion tests, we used the whole
data set, the three haplotype groups suggested by the
statistical parsimony network, and the haplotype
groups delimited by a SAMOVA with K = 3, because the
� 2010 Blackwell Publishing Ltd
Ta
ble
3N
eutr
alit
yte
sts
(Taj
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sD
and
Fu
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atch
dis
trib
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wit
hp
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nti
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der
ived
fro
mth
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or
s,re
sult
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gn
ifi-
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tly
sup
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gp
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(see
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tic
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mat
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(95%
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PHYLOGEO GRAPHY OF NORTH AMERICAN D ESERT 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
distribution (Table 3), although the plot of the observed
pairwise differences was multimodal (Fig. S3), as
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-
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
with short generation time, again with wide confidence
intervals from 0.5 to 173 kyr (Table 3).
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-
lation size changes through time and were indeed
rejected in favour of the simpler model of constant pop-
ulation size through time (2 · lnBF <)11). Likewise, a
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
model of constant population size, the diversification
age of the whole species (given as mean ⁄ median and,
in parentheses, its 95% highest posterior density inter-
val) is estimated to be 2.22 ⁄ 1.43 (0.24–7.11) million years
ago (that is more than twice as old as inferred under
the Bayesian Skyline Plot model; data not shown). The
maximum clade posterior probability tree as well as a
50% majority rule consensus tree revealed the same
three groups as the TCS network with eastern and cen-
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–
2.21, respectively) and the western group as a paraphy-
letic grade (Fig. 3c). Explicit testing the locations of the
source for the whole species range expansions provides
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
is clearly rejected (2 · lnBF -64.942).
Results of the maximum likelihood approach imple-
mented in PHYLOMAPPER for inferring ancestral locations
� 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 ancestrallocations for the eastern haplotype group of Melampodium leu-
canthum. Darker colours indicate higher doubled log-likelihood
differences, i.e., less likely ancestral locations. Pixels represent
1�·1� cells centred at the sampled points, which cover an area
between 116–95� E and 14–40� N. Coastline shown for the last
glacial maximum.
Ecological niche modelling
Model predictive performance of the bioclimatic model
was high with AUC values of 0.98 for the training data
and 0.96 for the test data and a highly significant bino-
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-
mental variables, assessed with the heuristic estimates
of relative contributions, were mean temperature of
wettest quarter (bio08), the mean temperature of coldest
quarter (bio11) and the precipitation of warmest quarter
(bio18; 20.9 20.8 and 20.6, respectively). Jackknife tests
of variable importance on different sets and test statis-
tics congruently suggested the precipitation of warmest
quarter (bio18), the mean temperature of coldest quarter
(bio11) and the precipitation of wettest quarter (bio16)
as the most important variables (Fig. S5).
Both climatic models used, CCSM3 and MIROC3.2,
indicate two distinct areas of environmental suitability
(Fig. 5b, c), but these differ in their extent and their
precise locations. Whereas in the western area this
mostly affects the latitudinal extent with otherwise simi-
lar distribution of highly suitable regions in the north-
ern Sonora, in the eastern area it affects both latitudinal
extension and longitudinal position. Specifically, under
the CCSM3 model, regions with highest predicted spe-
cies occurrence are found east of 102�W, thus mostly
falling into the Tamaulipan Plains, while under the MI-
ROC3.2 model, the less compact region of highest pre-
dicted species occurrence is found mostly west of
101�W, thus being essentially restricted to the current
Chihuahuan Desert (Fig. 5b, c).
Discussion
Southwestern North America faced dramatic changes in
the Holocene with a shift from woodlands to the cur-
rently widespread desert vegetation, and this was
accompanied by range expansions of drought-adapted
species from their refugia into the newly forming habi-
tats (Van Devender 1977; Van Devender & Spaulding
1979; Hunter et al. 2001; Jaeger et al. 2005; Haenel
2007). Based on fossil and paleoclimatic data as well
as on phylogeographic inferences, refugia have been
� 2010 Blackwell Publishing Ltd
(a)
(b)
(c)
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.
PHYLOGEO GRAPHY OF NORTH AMERICAN D ESERT PLANT 3437
� 2010 Blackwell Publishing Ltd
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
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
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
3438 C. A. REBERNIG ET AL.
constant dispersal parameter. A more detailed assess-
ment of these issues is, however, beyond the scope of
this paper and will require more extensive simulation
studies. In summary, the refugia inferred for M. leu-
canthum are congruent with those previously suggested
by paleoclimatic and phylogeographic data for both
plants and animals (Hunter et al. 2001; Jaeger et al.
2005; Riddle & Hafner 2006; Castoe et al. 2007; Haenel
2007; Holmgren et al. 2007; Fehlberg & Ranker 2009).
Plastid and AFLP data are congruent with respect to
number and location of refugia, but because the fast-
evolving AFLPs probably trace more recent events
(Kropf et al. 2009) than plastid sequences, they probably
show the signal of different time levels. Although no
direct age estimates can be obtained from AFLP data
(the presence of an AFLP clock suggested by Kropf
et al. 2009 is still contentious: Ehrich et al. 2009), their
signal probably reflects Late Quaternary differentiation
possibly as recent as the last glacial maximum. In con-
trast, lineage differentiation identified in the plastid
data is deeply within the Pleistocene and might date
back to the Tertiary (Fig. 3c). Unless M. leucanthum
remained restricted to these refugia over an extended
period of time, the evidence for refugia at different time
levels suggests that this species responded to the Qua-
ternary climatic oscillations in a cyclic manner (Stewart
et al. 2010).
Paleoclimatic data suggest that the Holocene aridifica-
tion progressed from the west to the east (Holmgren
et al. 2007) with desert species occurring earlier in the
Sonoran Desert (Van Devender 1990; McAuliffe & Van
Devender 1998). Thus, it may be expected that range
expansion of drought-adapted species would follow the
same general direction. For M. leucanthum, this hypoth-
esis finds, however, no support from our data.
Although the topology from the Bayesian analysis is in
line with the hypothesis of an eastward migration start-
ing from an ancestral western lineage (Fig. 3c), explicit
hypothesis testing provides evidence for a central or
eastern origin instead. Furthermore, there is no evi-
dence for eastwardly decreasing times of range expan-
sion (Table 3), a loss of average within-population
diversities inferred from AFLP data or the number of
polymorphic or private AFLP fragments (Table 1), as
would be expected if colonization progressed east-
wards. Instead, both plastid and AFLP data (Figs. 2, 3)
indicate that independent range shifts occurred out of
several Pleistocene refugia, a widespread pattern in
southern North American desert biota (Hunter et al.
2001; Jaeger et al. 2005; Castoe et al. 2007; Fehlberg &
Ranker 2009; Garrick et al. 2009; Rebernig et al. 2010).
A major obstacle for longitudinal migration is the
Continental Divide. Since its initial formation because
of the uplift of the Colorado Plateaus and the Sierra
Madre Occidental in the late Miocene to early Pliocene
(Morafka 1977), it was an effective barrier for exchange
between desert biota in this region (Morafka 1977; Mor-
afka et al. 1992; Hunter et al. 2001; Jaeger et al. 2005;
Riddle & Hafner 2006; Castoe et al. 2007). The effective-
ness of this barrier explains the strong phylogeographic
split between populations on both sides of the Conti-
nental Divide seen in the AFLP data (Fig. 2). It has
been suggested that Pleistocene climatic fluctuations in
combination with a relatively broad ecological ampli-
tude allowed migrations between western and eastern
deserts (Jaeger et al. 2005; Riddle & Hafner 2006; Castoe
et al. 2007). This appears also to be the case for M. leu-
canthum, whose migration across the Continental
Divide, albeit only in an easterly direction, is evident
from the extension of the western haplotype group
across this barrier (Fig. 3a). The discrepancy between
plastid and AFLP data is likely due to the rapid homog-
enization of AFLPs (because of repeated backcrossing of
hybrids between resident and immigrant genotypes
with the resident ones, Zhou et al. 2005), which, in con-
trast to plastid data, will quickly erase traces of gene
flow across the Continental Divide.
Reduced migration possibilities across the Continental
Divide probably contribute to the contrasting range
dynamics of populations west and east of it. In the
west, ecologically suitable areas at the LGM were geo-
graphically close and of comparable extent to the cur-
rent distribution area in this region (Fig. 5). Supported
by the lack of signal for range expansion (Table 3), this
indicates that in this region postglacial range shifts and
population size changes were of limited magnitude
(Castoe et al. 2007). In contrast, east of the Continental
Divide, the presumptive refugial areas were much
smaller than the current distribution area (Fig. 5),
implying major postglacial range expansion. This is
supported by signals for range expansion in both the
central and the eastern haplotype group (Table 3). The
stronger signal for range expansion in the central haplo-
type group, which is also reflected in the rather star-like
structure in the haplotype network (Fig. 3b), might be
because of a more rapid colonization or more strongly
reduced population sizes in a smaller refugium
(Fig. 5b, c), but further data are necessary to distin-
guish among these hypotheses.
In the course of range expansions from separate refu-
gia, lineages, which differentiated in isolation, came
into secondary contact and started to intermix. This is
evident from the co-occurrence of plastid haplotypes of
different haplotype groups within the same populations
(Fig. 3) as well as the strong signal for genetic admix-
ture (Fig. 2) in some populations east of the Continental
Divide as the result of extensive interpopulational
gene flow (M. leucanthum is an obligate outcrosser).
� 2010 Blackwell Publishing Ltd
PHYLOGEO GRAPHY OF NORTH AMERICAN D ESERT PLANT 3439
Secondary contact zones are well known from other
regions, where Pleistocene climatic fluctuations caused
major range shifts (Taberlet et al. 1998; Soltis et al. 2006;
Castoe et al. 2007; Fehlberg & Ranker 2009). The
secondary contact of differentiated lineages evidenced
by the AFLP data is probably connected to range
expansions during the Holocene aridification (Van
Devender 1977; Webb 1977; Van Devender & Spaulding
1979; Holmgren et al. 2007; Holliday et al. 2008), but
this cannot be directly tested with the AFLP data.
Range expansions for the central and eastern haplotype
groups (there is no evidence for range expansion in the
western haplotype group) are dated to the late Pleisto-
cene (Table 3), but these estimates are burdened with
wide confidence intervals partly extending to the Holo-
cene, although only under an assumed generation time
of 5 years. These time estimates might be biased
towards older ages because of the time dependency of
molecular rates (Ho et al. 2005, 2007), although its effect
appears to be of smaller magnitude than initially antici-
pated (Debruyne & Poinar 2009). Consequently, it
remains uncertain whether the range expansions
inferred from the plastid data are connected to the
Holocene aridification or to earlier phases of warmer
and drier climate (Allen & Anderson 2000).
The distribution pattern of tetraploids in M. leucant-
hum is conspicuous, because this cytotype is found
exclusively in the eastern part of range of the species
(Stuessy et al. 2004; Fig. 2d). Despite this geographic
distinctness, tetraploids do not form a genetically cohe-
sive group (Fig. 2), which contrasts with the pattern
observed in the closely related M. cinereum (Rebernig
et al. 2010). Instead, a monophyletic origin of polyp-
loids is clearly rejected by the plastid data (2 · lnBF
<)64.942). Furthermore, AFLP data detect both distinct
polyploid lineages as well as polyploids that usually
intermix with those diploid populations they were
assigned to (Table 2), thus supporting the hypothesis of
multiple origins. The presence of triploid individuals in
diploid populations (Table S2; Stuessy et al. 2004; R.
Obermayer et al. unpublished) suggests that the polyp-
loids formed via unreduced gametes. Despite their
reduced viability (triploids show a higher amount of
aborted pollen, data not shown), the number of resul-
tant triploids could be sufficient to act as triploid bridge
(Ramsey & Schemske 1998, 2002; Leitch & Leitch 2008).
The alternative, not mutually exclusive, hypothesis for
explaining the genetic heterogeneity of tetraploids is
gene flow between cytotypes (Gauthier et al. 1998),
which may be facilitated by the lack of any ecological
or breeding system differentiation among cytotypes but
is counteracted by their geographic cohesiveness. The
restriction of tetraploids to the eastern edge of the dis-
tribution of the species might be attributed to establish-
� 2010 Blackwell Publishing Ltd
ment during geographic isolation in phases of climatic
deterioration, as suggested for M. cinereum (Rebernig
et al. 2010), but further data on the actual dynamics at
the contact zone are necessary to test this or alternative
hypotheses.
The Holocene aridification in southwestern North
America undoubtedly had a major impact on the phy-
logeography and population history of drought-adapted
species (Van Devender & Spaulding 1979; Spaulding
1990; Van Devender 1990; Castoe et al. 2007; Holmgren
et al. 2007). In M. leucanthum, phases of restriction to
multiple refugia, which enhanced lineage differentiation
and possibly also polyploid establishment, alternated
with phases of range expansions and secondary contact,
the Continental Divide currently being the only major
migration barrier. These dynamics resulted in a com-
plex phylogeographic history in this seemingly homoge-
neously distributed species.
Acknowledgements
The authors thank Michael Lenko (University of Vienna, Aus-
tria), Enrique Ortiz (UNAM, Mexico), Monique Reed and Hugh
Wilson (Texas A&M University, U.S.A.), and Donovan Bailey
and Patrick Alexander (New Mexico State University, U.S.A.)
for help with material collection. We thank Gudrun Kohl (Uni-
versity of Vienna, Austria) for technical assistance and Sabine
Jakob (IPK Gatersleben, Germany) for help with the bioclimatic
data. We thank two anonymous reviewers for helpful criti-
cisms. The study was financially supported by Austrian Science
Fund (FWF) grants no. P18201-B03 (to TFS) and Hertha-Firn-
berg postdoctoral fellowship T-218 (to HWS).
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This study is part of the PhD thesis of C.A.R., dedicated to the
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of series Leucantha of Melampodium (Asteraceae). This work
was supervised by T.F.S. and H.W.-S., both interested in plant
evolution (including chromosomal and genome evolution) and
speciation. G.M.S. is interested in different aspects of plant
evolution, including genome evolution of parasitic plants, phy-
logeography, polyploid evolution and speciation. K.E.B. is
interested in phylogeography, polyploid evolution and hybrid-
ization of plants on the Balkan Peninsula. P.S. is interested in
polyploid evolution and in the spatio-temporal diversification
of European alpine plants. J.L.V. works on the flora of Mexico
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tion.
Supporting information
Additional supporting information may be found in the online
version of this article.
Table S1 Bioclimatic variables used to calculate the probability
of geographic distribution of Melampodium leucanthum.
Table S2 Relative fluorescent intensity and derived DNA
ploidy level of all investigated individuals of Melampodium leu-
canthum.
Fig. S1 Summary of the STRUCTURE analysis of AFLP data of
diploid individuals of Melampodium leucanthum.
Fig. S2 Distribution of plastid haplotypes sampled in Melampo-
dium leucanthum.
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PHYLOGEO GRAPHY OF NORTH AMERICAN D ESERT PLANT 3443
Fig. S3 Mismatch distributions for all populations as well as
each haplotype group found in Melampodium leucanthum.
Fig. S4 Likelihood surfaces for ancestral locations estimated
with PHYLOMAPPER in Melampodium leucanthum.
Fig. S5 Jackknife tests of variable importance in ecological
niche modelling in Melampodium leucanthum.
Supporting File 1 Details of data analyses.
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Supporting File 2–8 BEAST input files, their names indicating
the used demographic model (files 1–3) or topological hypothe-
sis tested (files 4–7; see text for details).
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