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Recent vicariance and the origin of the rare, edaphically specialized Sandhills lily, Lilium pyrophilum (Liliaceae): Evidence from phylogenetic and coalescent analyses


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Establishing the phylogenetic and demographic history of rare plants improves our understanding of mechanisms that have led to their origin and can lead to valuable insights that inform conservation decisions. The Atlantic coastal plain of eastern North America harbours many rare and endemic species, yet their evolution is poorly understood. We investigate the rare Sandhills lily (Lilium pyrophilum), which is endemic to seepage slopes in a restricted area of the Atlantic coastal plain of eastern North America. Using phylogenetic evidence from chloroplast, nuclear internal transcribed spacer and two low-copy nuclear genes, we establish a close relationship between L. pyrophilum and the widespread Turk's cap lily, L. superbum. Isolation-with-migration and coalescent simulation analyses suggest that (i) the divergence between these two species falls in the late Pleistocene or Holocene and almost certainly post-dates the establishment of the edaphic conditions to which L. pyrophilum is presently restricted, (ii) vicariance is responsible for the present range disjunction between the two species, and that subsequent gene flow has been asymmetrical and (iii) L. pyrophilum harbours substantial genetic diversity in spite of its present rarity. This system provides an example of the role of edaphic specialization and climate change in promoting diversification in the Atlantic coastal plain.
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Recent vicariance and the origin of the rare, edaphically
specialized Sandhills lily, Lilium pyrophilum (Liliaceae):
evidence from phylogenetic and coalescent analyses
*Department of Plant Biology, PO Box 7612, North Carolina State University, Raleigh, NC 27695, USA, Directorate of Public
Works, Endangered Species Branch, United States Army, Fort Bragg, NC 28310, USA, US Army Corps of Engineers, Engineer
Research and Development Center, PO Box 9005, Champaign, IL 618262, USA
Establishing the phylogenetic and demographic history of rare plants improves our
understanding of mechanisms that have led to their origin and can lead to valuable
insights that inform conservation decisions. The Atlantic coastal plain of eastern North
America harbours many rare and endemic species, yet their evolution is poorly
understood. We investigate the rare Sandhills lily (Lilium pyrophilum), which is endemic
to seepage slopes in a restricted area of the Atlantic coastal plain of eastern North
America. Using phylogenetic evidence from chloroplast, nuclear internal transcribed
spacer and two low-copy nuclear genes, we establish a close relationship between
L. pyrophilum and the widespread Turk’s cap lily, L. superbum. Isolation-with-migration
and coalescent simulation analyses suggest that (i) the divergence between these two
species falls in the late Pleistocene or Holocene and almost certainly post-dates the
establishment of the edaphic conditions to which L. pyrophilum is presently restricted,
(ii) vicariance is responsible for the present range disjunction between the two species,
and that subsequent gene flow has been asymmetrical and (iii) L. pyrophilum harbours
substantial genetic diversity in spite of its present rarity. This system provides an
example of the role of edaphic specialization and climate change in promoting
diversification in the Atlantic coastal plain.
Keywords: coalescence, divergence, edaphic, Lilium, Pleistocene, rarity
Received 18 November 2010; revision received 28 April 2011; accepted 5 May 2011
Molecular studies of rare plant taxa usually aim to
quantify the level and patterns of genetic diversity in a
particular species (Karron 1987; Hamrick & Godt 1990;
Ellstrand & Elam 1993; Gitzendanner & Soltis 2000).
Phylogeographic studies, on the other hand, often focus
on widespread species and try to discern continental-
scale patterns (Taberlet et al. 1998; Brunsfeld et al. 2001;
Soltis et al. 2006). However, the tools of phylogeogra-
phy, particularly coalescent-based analyses that provide
information about the age and historical demography of
species (Knowles 2009), have only rarely been applied
to investigate the history of rare species (Raduski et al.
2010; Whittall et al. 2010).
Of the ‘seven forms of rarity’ (Rabinowitz 1981), the
most extreme describes taxa that have a narrow geo-
graphic range, require specific habitats and maintain
only small local populations. Many edaphic endemics
(plants restricted to soils with unusual physical or
chemical properties) belong to this category. While the
textbook examples of edaphic endemic plants are
restricted to serpentine, various substrates support
edaphic endemics, including guano, alkali, salt, and
gypsum deposits, limestone, chalk, and granite out-
crops, oligotrophic bogs and deep porous sands (Orn-
duff 1965; Axelrod 1972; Parsons 1976; Kruckeberg &
Correspondence: Norman A. Douglas, Fax: (919) 515 3436;
2011 Blackwell Publishing Ltd
Molecular Ecology (2011) 20, 2901–2915 doi: 10.1111/j.1365-294X.2011.05151.x
Rabinowitz 1985; Kruckeberg 1986; Williamson & Baz-
eer 1997). Many aspects of the origin of edaphic ende-
mic species are poorly understood (Rajakaruna 2004).
For instance, such species often occur in close geo-
graphic proximity to their progenitor lineages (e.g.
Baldwin 2005), yet it is not usually known whether or
how strongly gene flow is interrupted. While taxa dis-
playing edaphic endemic syndromes often show
reduced genetic diversity compared with their close rel-
atives (Godt & Hamrick 1993; Baskauf et al. 1994; Ayres
& Ryan 1999; but see Raduski et al. 2010), this may
reflect genetic drift due to lower population sizes or the
effects of selection. Strong selection imposed by edaphi-
cally challenging soils could be sufficient to foster pop-
ulation divergence (Nosil et al. 2009; Freeland et al.
2010). Some edaphic endemics may represent vicariant
populations isolated in narrow parts of formerly wider
ranges and niches of their progenitors (e.g. Crawford
et al. 1985), which may themselves be able to grow on
the unusual substrate without being restricted to it.
Edaphic specialists (especially in bog and sand habi-
tats, Sorrie & Weakley 2001) are an important compo-
nent of the endemic-rich flora of the coastal plain of
eastern North America. Few coastal plain endemics
have been the subject of molecular analyses. Sand dune
habitats in Florida apparently served as Pleistocene
refugia for the genera Dicerandra and Conradina
(Edwards et al. 2006; Oliveira et al. 2007), and in gen-
eral, Florida has been proposed as a major Pleistocene
refugium for many taxa in eastern North America (Sol-
tis et al. 2006). Yet, recent phylogeographic work indi-
cates that some coastal plain endemic species likely
persisted in northerly latitudes throughout the Pleisto-
cene. For instance, the Atlantic coastal plain endemic
Pyxie Moss, Pyxidanthera (Diapensiaceae), shows long-
term range stasis (Wall et al. 2010).
The Fall-Line Sandhills of North and South Carolina
(which occur at the western boundary of the coastal
plain) provide one of the clearest examples of the
edaphic contribution to the botanical diversity of the
Atlantic coastal plain. This region is comprised of roll-
ing hills of open, fire-maintained longleaf pine (Pinus
palustris) savanna dissected by numerous blackwater
streams and wetlands, providing a diverse matrix of
habitats that support at least eight endemic plants (and
numerous near-endemics, Sorrie & Weakley 2001). In
the core of the Sandhills region in southern North Caro-
lina, the uppermost deposit is the Pinehurst formation,
which is characterized by loose coarse-grained sands
found along ridgetops. This formation was deposited in
a tidal environment (J. Nickerson, North Carolina Geo-
logical Survey, personal communication) and may date
to the Eocene (Cabe et al. 1992). Below the Pinehurst
formation (and exposed along drainages and slopes
throughout the region) lies the Cretaceous Middendorf
formation, which is of deltaic origin and thus has more
abundant clays (Sohl & Owens 1991). At the interface
between these (and similar formations in the Carolinas
and southeastern Virginia) occur Sandhills seep and
streamhead pocosin ecotone communities. When kept
open by frequent fires encroaching from the surround-
ing xeric pine savannas, these wetlands can support
extremely high local species richness, among the high-
est values ever recorded in North America (>102 spe-
cies per 1 100 ha, Schafale & Weakley 1990). The age of
the formations implies that endemic species have poten-
tially had a very long time to adapt to the unusual
edaphic conditions.
In this study, we consider the Sandhills lily, Lilium
pyrophilum (Liliaceae), a striking endemic of the Sand-
hills in the Carolinas and southeastern Virginia. For-
mally described only recently (Skinner & Sorrie 2002),
specimens of this species were previously identified in
herbaria as any of three similar species in the region
(L. superbum,L. michauxii or L. iridollae) that share the
distinctive ‘Turk’s cap’ morphology, in which flowers
are pendent with the tepals reflexed upward. Skinner &
Sorrie (2002) identified three specific plant communities
(Schafale & Weakley 1990; Sorrie et al. 2006) that sup-
port L. pyrophilum, including Sandhills seep and
streamhead pocosin ecotones. The third, small stream
swamps are affected by frequent flooding events in
addition to seepage and rarely support L. pyrophilum
(Sorrie et al. 2006).
Lilium pyrophilum is a very rare species. There are
fewer than 75 historical and extant locations in North
and South Carolina, and Virginia (North Carolina Natu-
ral Heritage Program 2007), and between 2007 and
2009, a survey of all known populations located
<500 stems across 35 populations (W. Wall, unpub-
lished data). Approximately half of the extant popula-
tions and a quarter of the individuals occur on Fort
Bragg Military Reservation in North Carolina, where
prescribed and ordnance-ignited fires maintain appro-
priate habitat.
In describing L. pyrophilum (Skinner & Sorrie 2002),
the authors outlined three phylogenetic hypotheses con-
cerning the origin of the species. First, they speculated
that L. pyrophilum may represent a peripheral isolate of
the Turk’s cap lily, L. superbum, which it most resem-
bles morphologically (albeit with significant differences,
Skinner & Sorrie 2002). Lilium superbum is distributed
throughout much of eastern North America (Fig. 1),
and in contrast to the edaphically specialized L. pyro-
philum, it is a generalist, occurring in rich woods and
oligotrophic wetlands from high elevation to sea level.
Especially in northern parts of its range (e.g. the Pine
Barrens of New Jersey), it can be found in saturated
2011 Blackwell Publishing Ltd
sandy habitats not unlike those preferred by L. pyrophi-
lum, but it is not restricted to them. However, it is
essentially absent from the Piedmont and Atlantic
coastal plain from the Carolinas southward. Thus, it is
disjunct from L. pyrophilum by at least 150 km every-
where except in southeastern Virginia (Fig. 1) where
the coastal plain narrows.
Second, they speculated that L. pyrophilum may repre-
sent a hybrid species, with the widespread Carolina lily
(L. michauxii) and L. superbum as progenitors. Homop-
loid hybrid speciation has been implicated in the origin
of other edaphic specialists, e.g. Helianthus paradoxus
(Rieseberg et al. 1990) and Hawaiian Scaevola (Howarth
& Baum 2005). Of the three potentially related species,
L. pyrophilum resembles L. michauxii least, differing in
leaf shape and producing fragrant flowers (Skinner
2002). While the range of L. michauxii does overlap the
range of L. pyrophilum (Fig. 1), they occur in contrast-
ing habitats, with L. michauxii favouring much drier
sites. Notably, L. michauxii and L. superbum co-occur
throughout much of their ranges (Fig. 1), yet natural
hybrids are apparently rare (Skinner 2002).
Finally, Skinner and Sorrie suggested the possibility
that L. pyrophilum may represent a disjunct population
of the Pot-o’-gold or Panhandle lily (L. iridollae), a nar-
row endemic of wet pine savannas in northwestern
Florida (where it is listed as endangered) and adjacent
Alabama. This hypothesis emphasizes similar habitat
requirements of the two species, but downplays consis-
tent morphological differences (e.g. details of rhizome
structure, Skinner 2002; Skinner & Sorrie 2002) and a
range separation of over 700 km (Fig. 1).
In this study, we report the results of a molecular
study focused on L. pyrophilum and its close relatives.
First, we investigated the phylogeny of the eastern pen-
dent species of Lilium to address whether L. pyrophilum
represents a peripheral isolate of L. superbum, a hybrid
between L. superbum and L. michauxii, or a disjunct
population of L. iridollae. Second, we analysed the dis-
tribution of genetic variation within and among the taxa
thought to be closely related to L. pyrophilum and used
coalescent-based methods to explicitly evaluate the pos-
sible timing of the divergence of L. pyrophilum. Our
results are interpreted in the context of the evolution of
rare, edaphically specialized lineages in the Atlantic
coastal plain.
Materials and methods
Sampling and molecular data
Samples were obtained from 50 populations spanning
the geographic range of each of the four focal species
(Fig. 1). We also sampled two populations of Lilium
20 40
Fort Bragg
L. iridollae
L. michauxii
L. superbum
L. pyrophilum
0150 300
Fig. 1 Distribution of populations included in this study and geographic ranges of the four focal species.
2011 Blackwell Publishing Ltd
canadense, another pendant species that lacks the Turk’s
cap morphology. Sampling information is provided in
Table S1 (Supporting Information). Populations were
located in the field based on documented occurrences
from herbarium specimens, element occurrence records
from state Natural Heritage Programs and communica-
tion with local botanists. We endeavoured to sample a
similar number of populations of L. superbum and
L. michauxii spanning the geographic range of each spe-
cies. Our sampling of the rare L. iridollae was limited to
two populations. In general, one individual was taken
to represent each population. Genomic DNA was iso-
lated from fresh or frozen leaves, using the CTAB
method (Doyle & Doyle 1987). Nuclear ribosomal inter-
nal transcribed spacer (‘ITS’) sequences were obtained
with primers ITS4 and ITS5a (White et al. 1990; Stan-
ford et al. 2000). This locus was sequenced to facilitate
comparison with abundant existing data available in
GenBank to determine whether the species in this study
form a monophyletic group. We screened eight chloro-
plast markers from Shaw et al. (2007); of these, three
(the atpI-atpH,psbD-trnT and rpl32-trnL intergenic spac-
ers) consistently amplified and contained variable sites.
As the chloroplast behaves as a single nonrecombining
locus, sequences of these three regions were concate-
nated, and this marker is hereafter referred to as ‘CP’.
We developed single-copy nuclear markers for Lilium.
In general, we screened EST or complete CDS
sequences from Lilium against the Oryza sativa genomic
sequence at GenBank using SPIDEY (Wheelan et al.
2001) with the ‘divergent sequences’ and ‘use large
intron sizes’ options. Candidate sequences were down-
loaded and manually aligned in Se-Al (Rambaut 1996)
using amino acid translations. Homologous sequences
from GenBank were incorporated into the alignments.
When we were confident of the positions of the introns
in the rice genome, we then designed primers using Pri-
mer3 (Rozen & Skaletsky 2000), which were screened
against DNA extracted from L. longiflorum and an Asi-
atic hybrid cultivar (which served as positive controls
because nearly all of our candidate regions were based
on sequences from these cultivated lilies) and the four
taxa in our study. We were able to obtain single ampli-
cons for relatively few of these regions even after exten-
sive PCR optimization; it was often the case that
primers would amplify nontarget regions or that introns
would be small, invariant or missing entirely. The clo-
sely related L. canadense has a phenomenally large gen-
ome (1C = 47.90 pg, 46.9 Gbp; Zonneveld et al. 2005;
Peruzzi et al. 2009), which may have contributed to the
difficulty we encountered in obtaining single-copy
nuclear sequences. However, we were able to design
primers that amplified two novel regions. The first
includes two introns between exons 8 and 10 of the
L. longiflorum alkaline phytase gene, LlAlp (‘AP’, prim-
involved in phytic acid metabolism (Mehta et al. 2006).
While GenBank contains sequences for two isoforms of
this gene, our PCR experiments are consistent with
these representing splice variants of a single locus. The
second region corresponds to a region between exons 5
and 10 of the AKT1-like potassium channel LilKT1
CAACTTTCATTCC). This locus was more difficult to
amplify, and we were unable to generate sequences for
L. iridollae. Primers and PCR conditions for ITS and the
chloroplast loci followed White et al. (1990) and Shaw
et al. (2007). For AP and AKT, PCR contained 2.5 lL
10·PCR buffer, 1%BSA, 200 lMdNTPs, 2.5 mMMgCl
4lMof each primer and 0.5 U Taq DNA polymerase.
Cycling conditions were 95 C for 4 min, followed by
35 cycles of 95 C for 30 s, 58 C for 30 s, 72 C for
2.5 min, and a final extension step of 72 C for 4 min.
Amplicons were cleaned with Antarctic Phosphatase
and Exonuclease I (New England Biolabs, Ipswich, MA,
USA). Sequencing was performed on an Applied Bio-
systems 3730 capillary sequencer (Foster City, CA,
USA) using Big Dye chemistry. Chromatograms were
edited in Sequencher 4.1.2 (Gene Codes Corporation,
Ann Arbor, MI, USA). Heterozygous bases were easily
identified in the chromatograms for the three nuclear
regions and coded with standard IUPAC notation.
Because of the low levels of divergence among our
sequences, alignment was trivial and performed manu-
ally in Se-Al. The most likely haplotypic phases of AP
and AKT genotype sequences were ascertained with a
combination of cloning and the program PHASE 2.1
(Stephens et al. 2001; Stephens & Donnelly 2003) called
by the ‘Open Unphase genotype’ option in DnaSP v. 5
(Librado & Rozas 2009); the inferred alleles form the
basis for all further analyses involving these loci. The
preferred model of sequence evolution for each locus
(ITS: TIM3ef + I + G; CP: K81uf + I; AP: TVM + I; AKT:
TVM + I + G) was determined according to Akaike
Information Criterion (AIC) in jModelTest (Posada
2008). Sampling details, genotype information and Gen-
Bank accession numbers are provided in Tables S1 and
S2 (Supporting Information).
Phylogenetic analyses and descriptive population
For the ITS analysis, 44 new sequences were aligned
with 49 from GenBank to create a matrix of 93
sequences. Included were the four species in this study,
plus 37 other taxa including the pendent eastern North
2011 Blackwell Publishing Ltd
American species, L. michiganense,L. canadense and
L. grayi, and eight others from Lilium section Pseudoliri-
um, the monophyletic group of North American species
(Nishikawa et al. 1999) to which all taxa in this study
belong. Unweighted parsimony analysis for the ITS
locus was accomplished using PAUP* 4.0b10 (Swofford
2002) using 100 random-addition sequence replicates
with TBR branch swapping; owing to overall low
sequence divergence, parsimony bootstrapping was
conducted with 10
‘fast’ stepwise addition sequences
(Soltis & Soltis 2003). Maximum-likelihood (ML) analy-
sis for this locus was conducted in GARLI v. 1.0
(Zwickl 2006). Likelihood bootstrap values were
obtained with 1000 replicate searches. The statistical
parsimony haplotype network was computed for com-
plete sequences of the three chloroplast regions, atpI-
atpH,psbD-trnT and rpl32-trnL (38 sequences), using
TCS (Clement et al. 2000). The nuclear loci (AP: 82
haplotypes; AKT: 62 haplotypes) have a more compli-
cated evolutionary history than chloroplast sequences;
thus, network analyses for the two were conducted
using the geodesically pruned quasi-median network
algorithm (Ayling & Brown 2008) as implemented in
SplitsTree4 (Huson & Bryant 2006), which produces
pruned networks that connect all sequences (including
multistate characters) by at least one shortest path. ML
trees (not shown) were inferred for these sequences as
well; they were poorly resolved and showed few sup-
ported nodes. However, neither nuclear locus showed
phylogenetic evidence of paralogy. For L. michauxii,
L. superbum and L. pyrophilum, Arlequin v. 3.5 (Excof-
fier & Lischer 2010) was used to estimate haplotype
richness, number of segregating sites, nucleotide diver-
sity p(Nei 1987) and Watterson’s (1975) population
mutation parameter h, for the chloroplast and single-
copy nuclear loci.
Testing divergence between L. michauxii,
L. pyrophilum and L. superbum
As our data include a single individual per ‘popula-
tion’, we treated species as the main hierarchical level
for the purposes of these analyses. Pairwise F
(Weir &
Cockerham 1984) and the exact test of population dif-
ferentiation (Raymond & Rousset 1995; Goudet et al.
1996) between L. michauxii,L. superbum and L. pyrophi-
lum were calculated in Arlequin v. 3.5 (Excoffier & Li-
scher 2010), with individuals and species used as the
hierarchical groupings. Significance was assessed with
permutations (F
Markov chain steps
(exact test).
The nature of the divergence between L. superbum
and L. pyrophilum was further investigated using the
isolation-with-migration model (Nielsen & Wakeley
2001), implemented in IMa2 (Hey & Nielsen 2007). The
full model in the two-population case includes six
parameters (divergence time, hfor the ancestral and
two descendent populations and migration rates
between the descendent populations). This model
assumes no recombination within loci and free recombi-
nation between loci and that markers are selectively
neutral. Thus, several recombination detection methods
available in the program RDP3 (beta 40; Martin et al.
2005) were used to search for recombinant alleles. As
selection or demographic changes can cause departures
from neutral expectations, DnaSP v. 5 (Librado & Rozas
2009) was used to perform three different tests of neu-
trality: Tajima’s D(Tajima 1989), Fay and Wu’s H(Fay
& Wu 2000) and R
(Ramos-Onsins & Rozas 2002). Crit-
ical values for these statistics were obtained using 10
coalescent simulations. The chloroplast data set showed
no evidence of recombination; the AP and AKT data
sets were filtered with IMgc Online (Woerner et al.
2007) to create data sets that were free of detectable
recombination and infinite sites violations. Maximum
priors for the IMa2 analysis were based on recom-
mended starting values given in the program documen-
tation and refined after preliminary exploratory runs.
Priors ultimately selected were population mutation
rates (for L. pyrophilum,L. superbum and ancestral pop-
ulation) h
and h
= 47, splitting time parameter
t= 3 and population migration rate m
and m
= 10.
Mutation rate priors (CP: 1.5 ·10
, AP & AKT:
6.03 ·10
) were specified based on values given by
Gaut (1998). Seventy geometrically heated chains (using
the heating parameters ha= 0.98, hb= 0.50) were run
for 750 000 generations beyond a 150 000 generation
burn-in and trees were sampled every 75 generations.
This process was repeated 10 times using different ran-
dom number seeds.
Because results from each replicate were similar, 10
trees were concatenated into a single run in load-trees
mode and the ‘test nested models’ option was activated.
This option evaluates the likelihood of 24 models sim-
pler than the full isolation-with-migration model by
constraining parameters (other than divergence time)
and rejecting those that are significantly worse than the
full model based on a likelihood ratio test. We also
compared models using an information-theoretic
method (Carstens et al. 2009), which allows the relative
performance of nested and non-nested models to be
compared using AIC. Compared with a hypothesis-test-
ing approach, which simply identifies models that are
rejected as significantly worse than the full model, the
information-theoretic approach provides model weights
that allow the relative performance of each of a given
set of models, including the full model, to be com-
pared directly with others given the data (Burnham &
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Anderson 2002). We used the full model posterior prob-
ability and the 2(log-likelihood ratio) values, which
IMa2 estimates for each model under the assumption
that the model’s posterior probability is proportional to
its likelihood, to calculate the AIC for the full model
and each nested model. Subsequently, Akaike weights
and evidence ratios were calculated (Burnham &
Anderson 2002; Carstens et al. 2009).
Conversion of the IMa2 parameter estimates from
coalescent to demographic units was accomplished
assuming a generation time of 20 years. This is arbitrary
but conservative, based on what little is known about
the natural history of these species. Germination and
establishment is slow, taking two seasons, and plants
need 7 years to reach flowering size. Year-to-year survi-
vorship is relatively high (>0.95, Wade Wall, unpub-
lished data). Using the equation T=a+[s(1 )s)],
where T= generation time, a= age of first reproduction
and s= adult survivorship (Lande et al. 2003), we
obtain a value of 26 years. Although estimates of survi-
vorship could be too high, the Lande equation does not
account for the fact that older plants are typically larger
and more fecund than younger ones. In either case, our
generation time should be considered a minimum esti-
Because isolation is implicit in the isolation-with-
migration framework, we tested this assumption with a
series of coalescent simulations. Briefly, we estimated
for each locus using BEAST (Drummond & Rambaut
2007). Because only L. pyrophilum and L. superbum
sequences were included, simpler ML models were uti-
lized (CP: HKY, AP: TnN + I + G, AKT: K81uf + I). We
then used Mesquite v. 2.73 (Maddison & Maddison
2010) to simulate 1000 data sets under each of several
simple divergence models (using estimated substitution
models for each locus). We treated each species as a
population such that L. superbum had a N
3·that of
L. pyrophilum (the total N
corresponding to the value
from BEAST). The two populations coalesced at times cor-
responding to 2.58 Ma (earliest Pleistocene), 126 ka
(upper Pleistocene) or 18 ka (last glacial maximum). We
then conducted parsimony searches using PAUP* 4.10b
(Swofford 2002) on each simulated data set saving 1000
consensus trees. Slatkin and Maddison’s s(i.e. the num-
ber of parsimony steps implied by a given topology
treating source population as a character, Slatkin &
Maddison 1989) was computed for each tree to create a
null distribution for each locus and divergence time.
This was compared with the value of sfor the empirical
data. When minimum empirical values for swere
higher than 95%of the simulated values, we rejected
the scenario. To evaluate the effect of the level of migra-
tion inferred by IMa2, we duplicated these analyses,
but allowing migration. Because Mesquite only allows
symmetrical migration, we specified a rate of 9.8 ·10
migrants per individual per generation, which corre-
sponds to the estimated value of the parameter under
the ‘equal migration rate’ nested model in IMa2.
Finally, following Gugger et al. (2010), we evaluated the
no-divergence scenario by simulating 1000 data sets per
locus under a single population scenario. The resulting
parsimony consensus trees were contained within the
two-population model described previously, and the
null distributions of swere calculated. In this case, the
scenario was rejected if the maximum empirical values
of swere lower than 95%of the simulated values. As
coalescent parameter estimates based on single loci are
highly sensitive to stochastic error (Edwards & Beerli
2000), these simulations were conducted for both the
upper and lower 90%HPD estimates of N
from BEAST.
Table 1 Genetic diversity and results of neutrality tests
Species locus
Lilium michauxii Lilium pyrophilum Lilium superbum
8 (8) 5 (10) 7 (14) 15 (15) 13 (26) 18 (36) 13 (13) 12 (24) 15 (30)
Aligned length (bp) 2361 1428 453 2360 1428 453 2361 1428 453
Segregating sites 7 10 13 7 24 8 9 30 18
Observed haplotypes 5 7 9 4 16 9 7 17 12
Nucleotide diversity p0.0010 0.0033 0.0098 0.0008 0.0024 0.0016 0.0009 0.0040 0.0053
Watterson’s theta h0.0011 0.0025 0.0090 0.0009 0.0044 0.0043 0.0012 0.0061 0.0100
Tajima’s D)0.4150 0.0487 0.3349 )0.4468 )1.7637* )1.8536** )1.0835 )1.2142 )1.6319*
Fay and Wu’s H1.7857 0.8000 2.2418 1.3429 )8.8862* )2.8794* )1.9615 )4.8333 0.6437
0.1577 0.2091 0.1597 0.1301 0.0625** 0.0495*** 0.1105* 0.0828 0.0692*
Sampling represents the number of individuals and the number of haplotypes (for phased nuclear loci). Significance of neutrality
tests was assessed with 10
coalescent simulations in DnaSP v. 5.1 (*P< 0.05, **P< 0.01, ***P< 0.001).
2011 Blackwell Publishing Ltd
Phylogenetic analyses
In the analysis of ITS data, overall support is quite weak
at the level of intra- and interspecific relationships, with
no significant (70%) bootstrap support for the mono-
phyly of the North American section Pseudolirium or the
eastern pendent-flowered species (Fig. 2). However,
there is a relatively high level of support for the branch
uniting two accessions of Lilium iridollae, for that uniting
the eight samples of L. michauxii, and, finally, for the
branch leading to the 32 samples of L. pyrophilum and
L. superbum. Little divergence is evident among the
Fig. 2 Maximum-likelihood (ML) Phylogram of internal transcribed spacer sequences. Support values are ML bootstrap Bayesian
posterior probability.
2011 Blackwell Publishing Ltd
accessions of each species (with the exception of the
GenBank sequences for L. superbum,L. canadense and
L. michiganense). The statistical parsimony network
(Fig. 3) computed for the chloroplast data revealed a
common haplotype (1) that was found in all four species,
plus 11 less common types. Overall, four of the six non-
singleton haplotypes occur in multiple species. Quasi-
median networks produced for the AKT and AP loci
(Fig. 4) showed that, while AP haplotype 8 is one muta-
tional step from the nearest L. michauxii haplotype
(m4a), most L. michauxii (and L. iridollae in AP) haplo-
types are separated from a cloud of L. pyrophilum and
L. superbum haplotypes, which are thoroughly inter-
mixed and frequently shared. No haplotypes were
shared between L. pyrophilum and L. michauxii.
Genetic diversity
Haplotype richness h, segregating sites S, nucleotide
diversity pand Watterson’s hare given in Table 1.
Nucleotide diversity is relatively low, with values
between 0.0008 and 0.00978 substitutions per site, and
average values for AP and AKT are nearly five times
the value for the chloroplast data set.
Tests of neutrality
Departures from neutrality were detected in the
nuclear data sets in L. pyrophilum and L. superbum,
where there were significant negative estimates of
Tajima’s Dand R
. Fay and Wu’s His significant in
L. pyrophilum only. Tajima’s Dis sensitive to both
demographic expansion and selection, and R
designed to detect population expansion (Ramos-
Onsins & Rozas 2002). While Fay and Wu’s His most
sensitive to recent positive selection, it may be sensi-
tive to particular demographic conditions involving
structured populations (Fay & Wu 2000). We believe
these loci are unlikely to be under positive selection,
because there is no obvious reason two loci should
deviate from neutrality more strongly in L. pyrophilum
than in the other two taxa. The chloroplast data also
show some demographic expansion in L. superbum
(weakly significant R
) without a significantly negative
D. Thus, while we cannot eliminate the possibility of
some background selection in the nuclear data sets
(which does not violate the assumptions of IMa2), it is
more likely that demographic factors explain the signif-
icant values for these statistics.
2: i3,i1,m8,s14
8: p3,p8,p11,p12,p13,s4,s16
L. iridollae
L. michauxii
L. superbum
L. pyrophilum
6: p21
3: m1
10: s11,s3,s15
12: s12
5: m5,m9
11: s5
9: p15 7: p1
4: m4,s6
1: i2,m7,m6,p16,p17,p18,
Fig. 3 Chloroplast haplotype network. Statistical parsimony network for CP haplotypes. Chart area reflects the frequency of the hap-
lotype; each slice reflects the frequency at which each haplotype was found in each species. Haplotype numbers (bold) and sample
abbreviations correspond to those in Tables S1 and S2 (Supporting Information). Edges represent mutations, black dots unsampled
2011 Blackwell Publishing Ltd
Differentiation of L. michauxii
Pairwise F
values (Table 2) revealed that L. michauxii
was significantly divergent from L. pyrophilum and
L. superbum for the AKT and AP data sets, whereas dif-
ferentiation between L. pyrophilum and L. superbum was
minimal and only significant in the AKT data set. No
significant differentiation was detected among any of
the three species for the CP data set. Conversely, all
pairwise exact differentiation tests (Raymond & Rousset
1995) were significant for the two nuclear loci; for the
cpDNA, a significant result was only obtained between
L. pyrophilum and L. michauxii.
Divergence between L. pyrophilum and
L. superbum
Under the isolation-with-migration model, estimates of
the mutation parameter theta (h) were L. pyrophilum:
3.736; L. superbum: 10.79; and ancestral population:
1.292, corresponding to effective population sizes (95%
highest posterior density interval, abbreviated ‘95%
HPD’) of 11 400 (2800–29 700), 32 900 (12 800–86 900)
and 3900 (0–14 400), respectively (Fig. 5a). The splitting
time between L. pyrophilum and L. superbum was esti-
mated as 0.7725 coalescent units, with the 95%HPD
being 0.3435–2.405 (Fig. 5b). This estimate corresponds
to a divergence time of 188 ka (95%HPD 84–586 ka)
with the assumed mutation rates and generation time.
The posterior distribution of splitting time did not reach
zero (nor did it for much higher prior values in preli-
minary runs), so 95%HPD intervals should be inter-
preted with caution. The coalescent migration rate m
from L. superbum into L. pyrophilum was highest at
zero, while the converse was 1.915. Thus, population
migration rates (2 NM, Hey & Nielsen 2004) are asym-
metrical and quite high from L. pyrophilum into L. su-
perbum (2 NM = 9.98, Fig. 5c). The model selection
procedure (Table 3) preferred a model that holds the
two species’ population sizes equal and the migration
rate from L. superbum to L. pyrophilum at zero (model
weight w= 0.32). The next best model (w= 0.22) also
fixed the L. superbum L. pyrophilum migration rate at
zero but allowed the population sizes to vary. The full
model (w= 0.19) had the next highest weight, and the
next three models differed in that they fixed the
population sizes as above (model 4), held migration
rates equal (model 5) and held the L. pyrophilum
L. superbum migration rate at zero (model 6). The six
best models are assigned 95.6%of the total weight. The
remaining 19 models had some combination of zero
migration, and one or both of the population sizes
Fig. 4 Quasi-median joining networks for the nuclear loci AP and AKT. Network representations of the relationships between
nuclear haplotypes (bold numbers and sample abbreviations correspond to Tables S1 and S2, in Supporting Information). In quasi-
median-joining networks, each haplotype is connected to the others by at least one shortest path. Mutational steps are indicated by
edges, and black dots represent potential unsampled haplotypes.
2011 Blackwell Publishing Ltd
equal to the ancestral population size. For the sake of
comparison, likelihood ratio tests comparing each
nested model to the full model rejected 20 of 24 nested
models. The four that were not rejected, combined
with the full model, represent 94.2%of the cumula-
tive model weight from the information-theoretic anal-
ysis. Coalescent simulations under both the earliest
Pleistocene (129 000 generations, 2.58 Ma) and upper
Pleistocene (6300 generations, 126 ka) divergence sce-
narios were rejected (Table 4). However, divergence
during the last glacial maximum (900 generations,
18 ka) was not rejected, and neither was the single
population scenario under either the highest or lowest
credible estimates for N
. Inclusion of migration in
these simulations did not qualitatively change the
Three hypotheses
Our results do not favour two of the three hypotheses
concerning the relationships of Lilium pyrophilum
advanced by Skinner & Sorrie (2002). First, it is unlikely
that L. pyrophilum simply represents a disjunct popula-
tion of L. iridollae: the ITS phylogeny unambiguously
allies L. pyrophilum with L. superbum, whereas L. iridollae
is most closely related to L. michauxii. That L. pyrophilum
and L. iridollae are independent only heightens the con-
servation concern of each of these rare species.
Second, the hypothesis that the species originated as
a hybrid between L. michauxii and L. superbum is not
supported by network analyses (Fig. 4). If L. pyrophilum
represented a recent hybrid, single-copy nuclear loci
should be related to both parental species. Instead, most
L. pyrophilum and L. superbum haplotypes are closely
related to each other (and many are shared), while they
show less similarity to L. michauxii. The phylogenetic
analysis of ITS sequences placed the L. pyrophilum sam-
ples with L. superbum sequences only, to the exclusion
of the L. michauxii sequences.
Lilium pyrophilum appears to be a peripheral isolate
of L. superbum. Our results indicate that the overall
magnitude of divergence between the two lily species
is very low and that the origin of L. pyrophilum is
likely to have been very recent, i.e. during the latter
Pleistocene or Holocene. Our estimated divergence
date from the IMa2 analysis of 188 ka (Fig. 5b) would
fall within the Illinoian glacial period. The minimum
credible divergence time of 84 ka would seem to indi-
cate that L. pyrophilum is in fact isolated from L. super-
bum. In spite of low F
values (Table 2), zero
probability is assigned to the most recent divergence
times in this analysis. The results of the simulation
Table 2 Pairwise Fst and exact test of
population differentiation
Lilium michauxii Lilium pyrophilum Lilium superbum
L. michauxii 0.109 0.393*** 0.625*** 0.046 0.328*** 0.567***
L. pyrophilum **** *** 0.007 0.021 0.057*
L. superbum *** ** ** *
Loci: CP AP AKT. Above diagonal, pairwise F
; below diagonal, exact test of
differentiation (Goudet et al. 1996; Raymond & Rousset 1995). Significance assessed in
Arlequin by either 10
permutations (F
Markov chain steps (exact test);
*P< 0.05, **P< 0.01, ***P< 0.001.
0 20 40 60 80 100 120 140
0.0 0.1 0.2 0.3 0.4 0.5
Effective pop. size (Ne),
L. pyrophilum
L. superbum
(a) (b) (c)
0 200 400 600
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
Divergence time (ka)
0 20406080
0.00 0.05 0.10 0.15 0.20 0.25
Migration (2NM)
L. pyrophilum
L. pyrophilum
L. superbum
L. superbum
Fig. 5 Posterior probability distributions for IMa2 model parameters under the full model. (a) Effective population size for both spe-
cies and the ancestral population. Both descendent taxa are inferred to have larger effective population size in this analysis. Esti-
mated values for N
are Lilium pyrophilum, 11 400 (95%HPD 2800–29 700); L. superbum, 32 900 (12 800–86 900); and ancestral, 3900
(0–14 400). (b) Divergence time. No probability is found for divergence times near zero; however, the distribution fails to reach zero
at the upper end. The peak corresponds to a value of 188 (84–586) ka. (c) Migration rate. Highest probability for migration from
L. superbum into L. pyrophilum is zero; there is, however, a higher probability of migration in the opposite direction (2 NM = 9.98).
2011 Blackwell Publishing Ltd
analysis lead us to interpret the IMa2 results with cau-
tion, however, because they reject divergence >6300
generations (126 ka) ago for each locus and fail to
reject the scenarios with divergence at 900 generations
(18 ka) and with no divergence (Table 4). The models
tested in this approach, however, were simplified with
respect to the full IMa2 model and treat each locus
separately rather than jointly. Regardless of whether
the IMa2 results or the coalescent simulation results
are preferred, the isolation between the two taxa is not
ancient. Mid- to late Pleistocene divergence times have
been found in surprisingly few studies of plants (e.g.
Strasburg & Rieseberg 2008; Bittkau & Comes 2009;
Cooper et al. 2010).
Our results provide insight into the demographic pat-
terns that have affected the two species. Deviations
from neutral expectation indicate population expansion
in both taxa (e.g. the average value for Tajima’s D
across three loci: L. pyrophilum =)1.35, L. super-
bum =)1.31, Table 1). This result is corroborated by the
IMa2 analysis, which demonstrates modern effective
population sizes higher than the ancestral, with the
widespread L. superbum having a larger value (N
times that of the endemic L. pyrophilum, Fig. 5a). It is
worth noting that the effective population size of
L. pyrophilum (11 000 individuals) is surprisingly high
considering the very small range of the species; in fact,
our estimate of N
is well in excess of the current cen-
sus population size estimated by a recent inventory.
Two factors may explain this discrepancy. First, our
estimated generation time may be too low, which
would cause us to overestimate effective population
size (and underestimate divergence time). Second, agri-
culture, timber harvesting and fire suppression have
Table 3 IMa2 analysis of nested models
Model description log(P) Terms AIC DAIC
weight d.f. 2LLR
h(pyrophilum)=h(superbum), mzero from superbum
to pyrophilum
)4.442 3 14.884 0 0.301 0.301 2 2.986 0.2247
mzero from superbum to pyrophilum )3.825 4 15.65 0.766 0.2052 0.5062 1 1.752 0.1856
Full IM model )2.949 5 15.898 1.014 0.1813 0.6875
h(pyrophilum)=h(superbum))3.972 4 15.944 1.06 0.1772 0.8647 1 2.045 0.1527
Symmetrical migration )4.803 4 17.606 2.722 0.0772 0.9419 1 3.707 0.0542
mzero from pyrophilum to superbum )6.29 4 20.58 5.696 0.0174 0.9593 1 6.681 0.0097
h(pyrophilum)=h(ancestral), mzero from superbum to
)7.985 3 21.97 7.086 0.0087 0.968 2 10.07 0.0065
h(pyrophilum)=h(ancestral), mzero from pyrophilum to
)8.116 3 22.232 7.348 0.0076 0.9757 2 10.33 0.0057
h(pyrophilum)=h(superbum), symmetrical migration )8.408 3 22.816 7.932 0.0057 0.9814 2 10.92 0.0043
h(pyrophilum)=h(ancestral), symmetrical migration )8.424 3 22.848 7.964 0.0056 0.987 2 10.95 0.0042
All hequal, mzero from superbum to pyrophilum )9.858 2 23.716 8.832 0.0036 0.9906 3 13.82 0.0032
h(pyrophilum)=h(ancestral) )7.899 4 23.798 8.914 0.0035 0.9941 1 9.9 0.0017
h(superbum)=h(ancestral), mzero from superbum to
)9.192 3 24.384 9.5 0.0026 0.9967 2 12.49 0.0019
All hequal )9.858 3 25.716 10.832 0.0013 0.9981 2 13.82 0.001
h(superbum)=h(ancestral) )9.192 4 26.384 11.5 0.001 0.999 1 12.49 0.0004
h(pyrophilum)=h(superbum), mzero from pyrophilum
to superbum
)10.63 3 27.26 12.376 0.0006 0.9997 2 15.36 0.0005
h(superbum)=h(ancestral), symmetrical migration )12.1 3 30.2 15.316 0.0001 0.9998 2 18.3 0.0001
All hequal, symmetrical migration )13.4 2 30.8 15.916 0.0001 0.9999 3 20.9 0.0001
h(superbum)=h(ancestral), zero migration )14.26 2 32.52 17.636 0 0.9999 3 22.63 0
Zero migration )13.35 3 32.7 17.816 0 1 2 20.8 0
h(superbum)=h(ancestral), mzero from pyrophilum to
)14.26 3 34.52 19.636 0 1 2 22.63 0
All hequal, mzero from pyrophilum to superbum )18.52 2 41.04 26.156 0 1 3 31.13 0
h(pyrophilum)=h(superbum), zero migration )24.86 2 53.72 38.836 0 1 3 43.83 0
h(pyrophilum)=h(ancestral), zero migration )29.23 2 62.46 47.576 0 1 3 52.57 0
All hequal, zero migration )30.93 1 63.86 48.976 0 1 4 55.97 0
Models include the full IM model and 24 simpler nested models for the two-population case. Information-theoretic statistics, based
on log(P), follow Burnham & Anderson (2002) and have been sorted by model weight. Models not rejected under traditional-
likelihood ratio tests (LRT) are included in the 95%confidence set of models selected by AIC.
2011 Blackwell Publishing Ltd
dramatically transformed much of the landscape of the
Sandhills over the past few hundred years, which may
well have extirpated many populations. As these plants
are long-lived outcrossers, too few generations may
have elapsed for the impact of the current bottleneck to
be fully reflected in the estimated N
(Lande & Bar-
rowclough 1987). Although our results suggest that the
existing population has apparently been greatly
reduced recently, much of the original genetic diversity
remains and could be conserved, minimizing the impact
of the present-day population bottleneck.
Gene flow is inferred from L. pyrophilum to L. super-
bum. Models including symmetrical migration are not
weighted heavily compared with models that have zero
or nearly zero gene flow from L. superbum to L. pyrophi-
lum (Table 3). Presently, the two species are disjunct.
However, the plants are visited by strong-flying pollina-
tors, such as swallowtail butterflies and hummingbirds
(Skinner 2002), and the seeds are adapted for wind dis-
persal. Why migration would be asymmetrical is
unknown, but this could be explained by pollinator
behaviour, dispersal or intrinsic barriers to gene flow.
Edaphic endemism in the Sandhills
The Sandhills pre-date the Pleistocene and may be sub-
stantially older, raising the possibility that some ende-
mic taxa may have originated in the Pliocene or earlier
and maintained populations in the region continuously.
How might Pleistocene climate changes have affected
the distribution of Lilium spp. in the coastal plain and
effected the isolation of L. pyrophilum? While periods of
severe climate change may eliminate edaphic endemics
that are unable to migrate to areas with a suitable cli-
mate and substrate, edaphic endemics may in fact be
likely to endure climate change in their geographic
ranges. As their niches are defined more by soils than
climate, they are likely to remain the best competitors
on restrictive soils under a wide range of conditions. In
fact, the degree of edaphic restriction exhibited by a
species often varies with climate: populations may be
widespread in environments with low competition and
edaphically restricted in more favourable climates
(Brooks 1987; Harrison et al. 2009).
The edaphic conditions that currently support popu-
lations of L. pyrophilum have probably been relatively
stable, because the erosional process has no doubt con-
tinually exposed the interface between permeable and
impermeable soils, creating seeps. Yet, the divergence
between L. pyrophilum and L. superbum is compara-
tively recent. Genetic diversity of L. pyrophilum, while
lower than that of L. superbum, is still high, making a
vicariant scenario likely. Thus, the phenotypic diver-
gence described by Skinner & Sorrie (2002) probably
occurred in the context of large populations and sub-
stantial gene flow.
The combination of long-term persistence and recent
divergence of L. pyrophilum indicates that this species
descends from locally adapted populations that were
stranded in the Sandhills as L. superbum retreated to
higher elevations. It is not clear why the intervening
Piedmont region supports neither taxon; however,
many groups show a similar disjunction (Braun 1955;
Sorrie & Weakley 2001). This study indicates that for
these lilies, at least, the disjunction coincided with Pleis-
tocene climate oscillations; this may apply to other taxa
that share similar distributions. More in-depth studies
of the L. pyrophilum L. superbum system, using micro-
satellite markers, will quantify genetic structure within
L. pyrophilum, and gene flow within and between L. py-
rophilum and L. superbum. These more detailed analyses
will improve estimates of divergence time and gene
flow and identify populations of high conservation pri-
ority. Better understanding of this group will provide
further insight into the role of edaphic specialization,
possibly brought on by climate change, in promoting
We thank Fort Bragg Military Reservation and the Endangered
Species Branch for logistic support and the Construction Engi-
neering Research Laboratory (US Army Corps of Engineers
Agreement #W9132T-07-2-0019) for funding. We also thank
Xiang Liu, David Thomas, Patrick Zhou, Esther Ichugo, Matt
Table 4 Results of coalescent simulation study
Simulation model
Divergence time (in generations) without migration
129 000 0.000 0.000 0.000
6300 0.023 0.008 0.036
900 0.992 0.379 0.394
Divergence time (in generations) with migration
129 000 0.001 0.001 0.006
6300 0.028 0.013 0.034
900 0.839 0.438 0.422
No divergence
High N
0.122 0.148 0.546
Low N
0.065 0.181 0.235
P-value for each model was obtained by comparison of either
minimum (divergence) or maximum (no divergence) empirical
svalue (Slatkin & Maddison 1989) with simulated distributions
of sunder coalescent scenarios to test whether observed data
were consistent with divergence times discussed in text.
Simulations were based on assumed 20-year generation time.
2011 Blackwell Publishing Ltd
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N.A.D. is an evolutionary biologist who focuses on phylo-
genetics and phylogeography of arid-adapted and edaphic
endemic plants. W.A.W. is a plant ecologist interested in the
interplay between population genetics and population dyna-
mics of rare and endemic species. Q.Y.X.’s research explores the
mechanisms underlying biodiversity patterns and morphologi-
cal variation, with emphasis on the Cornales. W.A.H. is inter-
ested in ecology and conservation of savanna ecosystems.
T.R.W. is a vegetation scientist with research interests in vegeta-
tion environment relationships and biological diversity. J.B.G.
is a botanist working on conservation of rare plants in Coastal
Plain habitats. M.G.H. uses diverse approaches to inform rare
species conservation.
Data accessibility
Sample and haplotype information is found in Table S1 (Sup-
porting Information). DNA sequences: GenBank accessions
JF829316–JF829423 (Table S2, in Supporting Information). ITS
data and phylogenetic tree available at
Supporting information
Additional supporting information may be found in the online
version of this article.
Table S1 Sampling. Haplotype numbers correspond to sample
labels in Figs 3 and 4, and to names accessioned in GenBank
(Table S2, in Supporting Information). For AP and AKT
sequences, integers identify haplotypes recovered more than
once in this study and other identifiers refer to unique haplo-
Table S2 GenBank accession numbers. Haplotype names corre-
spond to samples in Table S1 (Supporting Information)
Please note: Wiley-Blackwell are not responsible for the content
or functionality of any supporting information supplied by the
authors. Any queries (other than missing material) should be
directed to the corresponding author for the article.
2011 Blackwell Publishing Ltd
... The spatial distributions of cpDNA haplotype H1 and the star-like networks (see Fig. 2) plausibly reflect the range expansion of their ancestor, which occurred before the speciation events. Such vicariant separation scenarios have been hypothesized for the evolution of other serpentine plants (Kropf et al. 2008;Michl et al. 2010;Douglas et al. 2011;Moore et al. 2013). Our results, together with those of previous studies, highlight the relative role of Pleistocene climatic oscillations in the evolution of serpentine plants. ...
... Such asymmetrical interspecific gene flow across soil boundaries was also reported in other edaphic endemic pairs, and was explained by pollinator behavior, dispersal ability or transfer of adaptive alleles (e.g. Douglas et al. 2011;Kay et al. 2018;Arnold et al. 2016). Although a thorough investigation of the explanations for asymmetrical migration is beyond the scope of this study, we conceived several potential explanations. ...
Climatic perturbation during the Pleistocene era has played a major role in plant evolutionary history by altering species distribution range. However, the relative roles of climatic and geographic factors in the distribution dynamics remain poorly understood; in particular, the edaphic endemics. In this paper, we examine the evolutionary history of two ultramafic primroses, Primula hidakana and Primula takedana. These species are ecologically and morphologically distinct with disjunct distributions on Hokkaido Island, Japan. Primula hidakana is found on various rocks in southern Hokkaido and P. takedana in serpentine areas in northern Hokkaido. We performed population genetics analyses on nuclear and chloroplast data sets and tested alternative phylogenetic models of divergence using approximate Bayesian computation (ABC) analyses. Nuclear microsatellite loci clearly distinguished the two sister taxa. In contrast, chloroplast sequence variations were shared between P. takedana and P. hidakana. ABC analyses based on nuclear data supported a secondary contact scenario involving asymmetrical gene flow from P. hidakana to P. takedana. Paleodistribution modeling also supported the divergence model, and predicted their latitudinal range shifts leading to past secondary contact. Our findings highlight the importance of the distribution dynamics during the Pleistocene climatic oscillations in the evolution of serpentine plants, and demonstrate that tight species cohesion between serpentine and nonserpentine sister taxa has been maintained despite past interspecific gene flow across soil boundaries.
... Therefore, we cannot necessarily rely on these divergent ITS copies to predict the phylogenetic implications of Cardiocrinum. To better resolve the phylogenetic relationships within Cardiocrinum, three LCNGs that are universally amplifiable markers for phylogenetic reconstructions of closely related plant species (Douglas et al., 2011;Day et al., 2014), AP (alkaline phytase gene), AT103 (putative magnesium-protoporphyrin monomethyl ester cyclase, exon 3) and XDH (xanthine dehydrogenase gene), were amplified for all samples. Primer sequences and amplification conditions are listed in Table S2. ...
... Generally, conservation action has greatly benefited rare or endangered plant species, and abundant studies continue to pay attention to these high-profile species (Douglas et al., 2011;Tomasello et al., 2015). However, little attention has been given to some common and widespread plant species that have a narrow habitat tolerance. ...
... Therefore, we cannot necessarily rely on these divergent ITS copies to predict the phylogenetic implications of Cardiocrinum. To better resolve the phylogenetic relationships within Cardiocrinum, three LCNGs that are universally amplifiable markers for phylogenetic reconstructions of closely related plant species (Douglas et al., 2011;Day et al., 2014), AP (alkaline phytase gene), AT103 (putative magnesium-protoporphyrin monomethyl ester cyclase, exon 3) and XDH (xanthine dehydrogenase gene), were amplified for all samples. Primer sequences and amplification conditions are listed in Table S2. ...
... Generally, conservation action has greatly benefited rare or endangered plant species, and abundant studies continue to pay attention to these high-profile species (Douglas et al., 2011;Tomasello et al., 2015). However, little attention has been given to some common and widespread plant species that have a narrow habitat tolerance. ...
Full-text available
Background and Aims The patterns of evolutionary assembly in the Sino-Japanese floristic region (SJFR) remain largely unknown due to a lack of integrative multidimensional studies throughout the region. To address this issue, we elucidated the evolutionary history of Cardiocrinum (Liliaceae), a genus containing four taxa distributed across the SJFR. Methods Fifty-four populations were sampled throughout the geographical range of Cardiocrinum to assess genetic structure, analyse phylogenetic relationships and reconstruct ancestral area based on six chloroplast DNA (cpDNA) fragments and three low copy nuclear genes (LCNG). Ecological niche modelling was used to examine the potential range shifts of Cardiocrinum in response to climatic change. Key Results The molecular data showed high genetic similarity in the cpDNA (98·37 %) and LCNG (94·53 %) sequences. The biogeographical analyses revealed that the ancestor of Cardiocrinum diversified during the late Miocene (approx. 7·32 Mya) in Central China. The ancestor of the C. giganteum lineage dispersed westward to the Himalayas and south-west China with the split between C. giganteum and C. giganteum var. yunnanense occurring around 4·11 Mya consistent with the period of orogeny of the Hengduan Mountains. Some populations of the C. cathayanum lineage dispersed eastward to south Japan via the land bridge approx. 4·97 Mya, providing opportunities for allopatric speciation of C. cordatum. The predicted suitable habitats of Cardiocrinum have become smaller and more fragmented since the Last Glacial Maximum. Conclusions Our study provides evidence of a biogeographical pattern of dispersal from Central China to the Himalayas in the west and Japan in the east for genera distributed across the SJFR, and highlights that the orogeny of the Hengduan Mountains and fluctuations of the sea level of the East China Sea played important roles in promoting species divergence.
... Edaphic endemics (Kruckeberg and Rabinowitz 1985) contribute to the increased biodiversity of a flora (Ferreira and Boldrini 2011). Edaphic endemics are also vulnerable to extinction via climate change where migration to new, suitable habitats is difficult (Caha et al. 1998;Damschen et al. 2010;Douglas et al. 2011;Damschen et al. 2012), and to pressures from human encroachment (Zhang et al. 2011;Frey et al. 2012;Fenu et al. 2013). Most studies on plants exhibiting edaphic endemism have focused on those adapted to serpentine soils (Brady et al. 2005;Damschen et al. 2012). ...
... Most studies on plants exhibiting edaphic endemism have focused on those adapted to serpentine soils (Brady et al. 2005;Damschen et al. 2012). However, several studies have focused on psammophytes, plants adapted to sand dunes or deep sand, mostly with an emphasis on coastal dune systems (Caha et al. 1998;Tepedino et al. 2007;Rosenthal et al. 2010;Douglas et al. 2011;Zhang et al. 2011;Monserrat et al. 2012;Á lvarez-Molina et al. 2013). These plants may have increased drought tolerance, which would be beneficial in a warming environment, but their restriction to sand dunes or deep sand results in a discontinuous distribution (Rosenthal et al. 2010;De Queiroz et al. 2012;Á lvarez-Molina et al. 2013). ...
Full-text available
Penstemon albomarginatus is a psammophytic endemic of the Mojave Desert, found only in deep sand and dune habitats of San Bernardino County, California, Mohave County, Arizona, and Clark and Nye Counties in Nevada. We used six microsatellite loci to assess genetic differentiation and diversity for 228 individuals across the 12 known populations of this rare species. A slight heterozygote deficiency was found in two populations, but most populations show no signs of inbreeding. Results show a geographic pattern of northern populations being more closely related to one another compared to all other geographic regions. Genetic diversity was greatest in the southern populations, with decreasing amounts of diversity observed with latitude. In general, the geographic pattern of genetic diversity among all populations suggests a post-glacial dispersal from south-to-north. Our results are discussed in the framework of anthropogenic pressures on deep sand habitats of the Mojave Desert.
... Astragalus michauxii is a leguminous perennial herb endemic to the Sandhills of North Carolina, South Carolina, and Georgia, where it occurs in xeric sandhill scrub and pine-scrub-oak sandhill communities (Schafale and Weakley 1990, Sorrie et al. 2006, Wall et al. 2012, Weakley 2015. Lilium pyrophilum is a dormancy-prone perennial monocot restricted to sandhill seeps, streamhead wetlands (known locally as pocosin) ecotones, and more rarely small stream swamps in the Sandhills of North and South Carolina (Schafale and Weakley 1990, Skinner and Sorrie 2002, Douglas et al. 2011. Lysimachia asperulifolia is a rhizomatous pseudo-annual that primarily occurs within the Coastal Plain of North and South Carolina, with isolated populations in the Sandhills (Sorrie et al. 2006, Kunz et al. 2014). ...
Rare species reintroductions are an increasingly common conservation strategy, but often result in poor survival of reintroduced individuals. Reintroduction sites are chosen primarily based on historical occupancy and/or abiotic properties of the site, with much less consideration given to properties of the larger biotic community. However, ecological niche theory suggests that the ability to coexist with other species is determined in part by the degree of functional similarity between species. The degree to which functional similarity affects the survival of reintroduced plants is poorly understood, but has important implications for the allocation of limited conservation resources. We collected a suite of abiotic, biotic and functional trait variables centered on outplanted individuals from four reintroduced rare plant species and used logistic regression and model selection to assess their influence on individual survival. We show that higher functional similarity between reintroduced individuals and the local community, measured by differences between their multivariate functional traits and the community weighted mean traits of their immediate neighbors, increases survival and is a stronger predictor of survival than local variation in abiotic factors, suggesting that the functional composition of the biotic community should be incorporated into site selection to improve reintroduction success.
... This set the stage for the divergence of edaphically specialized species via allopatric speciation. A similar scenario is presented for the evolution of the edaphically specialized Lilium pyrophilum (Douglas et al., 2011). ...
Full-text available
Plants adapted to special soil types are ideal for investigating evolutionary processes, including maintenance of intraspecific variation, adaptation, reproductive isolation, ecotypic differentiation, and the tempo and mode of speciation. Common garden and reciprocal transplant approaches show that both local adaptation and phenotypic plasticity contribute to edaphic (soil-related) specialization. Edaphic specialists evolve rapidly and repeatedly in some lineages, offering opportunities to investigate parallel evolution, a process less commonly documented in plants than in animals. Adaptations to soil features are often under the control of major genes and they frequently have direct or indirect effects on genes that contribute to reproductive isolation. Both reduced competitiveness and greater susceptibility to herbivory have been documented among some edaphic specialists when grown in ‘normal’ soils, suggesting that a high physiological cost of tolerance may result in strong divergent selection across soil boundaries. Interactions with microbes, herbivores, and pollinators influence soil specialization either by directly enhancing tolerance to extremes in soil conditions or by reducing gene flow between divergent populations. Climate change may further restrict the distribution of edaphic specialists due to increased competition from other taxa or, expand their ranges, if preadaptations to drought or other abiotic stressors render them more competitive under a novel climate.
... It was assumed that a strict molecular clock was the best choice for intraspecific data (as suggested in the user guide of BEAST). A common strict molecular clock with a mean evolution rate of 1.5x10 -9 per site per year used by (Douglas et al., 2011) on another geophyte species, the rare Sandhills lily, Lilium pyrophilum (Liliaceae) (mutation rate specified in Gaut, 1998). However in absence of fossil record, or of a better estimation of mutation rate, the results have to be to discuss with caution, and taking into account the limit of molecular dating. ...
Full-text available
Diversity patterns represent a temporary state in a dynamic continuum of ecological and evolutionary changes. Thus, conservation policies have to integrate this dynamics to ensure long term conservation of biodiversity. Conservation priorities have to be oriented towards the processes which generate and maintain diversity. An original approach is to assess the capacity of phylogeography as a method to integrate processes of diversification and persistence into conservation. Indeed, the phylogeography allows providing indices for diversification zonation and can be used to delineate the units (e.g. evolutionary significant units) that support diversification at intraspecific level. The main objective of this PhD thesis is to evaluate, in terms of targets and surrogates, the role of phylogeography for conservation of the Mediterranean flora. We based our analyses on two study areas harbouring two endemic plant species with restricted distributions: (i) the coastal ranges of the Maritime Alps where the endemic Acis nicaeensis grows, (ii) the calcareous Provence where the endemic Arenaria provincialis is found. At a sub-regional scale, spatial diversity of Arenaria provincialis showed a spatial distribution of persistence and divergence which reveal the potentials of research on the use of the evolutionary legacy, as a surrogate for biodiversity. Our study highlights the critical role of phylogeography to delineate and assess conservation efficiency of the protected areas as well as in the search for optimal criteria for the defining the conservation strategies. The marked differences in populations of Acis nicaeensis in terms of isolation, size, genetic origin, and ecology but also in terms of its vulnerability to urbanization are highlighted in a local scale. The results demonstrate the important vulnerability of coastal population, whose originality necessitates conservation actions designed for small areas to avoid the loss of Acis nicaeensis evolutionary legacy.
... We expected that strictly bifurcating trees may not completely describe the evolutionary relationships within Lilium-Nomocharis, because hybridization in Lilium-Nomocharis has been postulated [13,17,57] and incomplete lineage sorting has been detected in many plant lineages [40]. Therefore, we used the statistical parsimony network approach implemented in TCS v.1.21 ...
Full-text available
Several previous studies have shown that some morphologically distinctive, small genera of vascular plants that are endemic to the Qinghai-Tibetan Plateau and adjacent Hengduan Mountains appear to have unexpected and complex phylogenetic relationships with their putative sisters, which are typically more widespread and more species rich. In particular, the endemic genera may form one or more poorly resolved paraphyletic clades within the sister group despite distinctive morphology. Plausible explanations for this evolutionary and biogeographic pattern include extreme habitat specialization and hybridization. One genus consistent with this pattern is Nomocharis Franchet. Nomocharis comprises 7-15 species bearing showy-flowers that are endemic to the H-D Mountains. Nomocharis has long been treated as sister to Lilium L., which is comprised of more than 120 species distributed throughout the temperate Northern Hemisphere. Although Nomocharis appears morphologically distinctive, recent molecular studies have shown that it is nested within Lilium, from which is exhibits very little sequence divergence. In this study, we have used a dated molecular phylogenetic framework to gain insight into the timing of morphological and ecological divergence in Lilium-Nomocharis and to preliminarily explore possible hybridization events. We accomplished our objectives using dated phylogenies reconstructed from nuclear internal transcribed spacers (ITS) and six chloroplast markers. Our phylogenetic reconstruction revealed several Lilium species nested within a clade of Nomocharis, which evolved ca. 12 million years ago and is itself nested within the rest of Lilium. Flat/open and horizon oriented flowers are ancestral in Nomocharis. Species of Lilium nested within Nomocharis diverged from Nomocharis ca. 6.5 million years ago. These Lilium evolved recurved and campanifolium flowers as well as the nodding habit by at least 3.5 million years ago. Nomocharis and the nested Lilium species had relatively low elevation ancestors (<1000 m) and underwent diversification into new, higher elevational habitats 3.5 and 5.5 million years ago, respectively. Our phylogeny reveals signatures of hybridization including incongruence between the plastid and nuclear gene trees, geographic clustering of the maternal (i.e., plastid) lineages, and divergence ages of the nuclear gene trees consistent with speciation and secondary contact, respectively. The timing of speciation and ecological and morphological evolutionary events in Nomocharis are temporally consistent with uplift in the Qinghai-Tibetan Plateau and of the Hengduan Mountains 7 and 3-4 million years ago, respectively. Thus, we speculate that the mountain building may have provided new habitats that led to specialization of morphological and ecological features in Nomocharis and the nested Lilium along ecological gradients. Additionally, we suspect that the mountain building may have led to secondary contact events that enabled hybridization in Lilium-Nomocharis. Both the habitat specialization and hybridization have probably played a role in generating the striking morphological differences between Lilium and Nomocharis.
... The chances of primers annealing to a region that has a single copy within such a vast genome is likely to be much lower than for repetitive regions such as the nrITS, which can have thousands of copies per genome (Álvarez and Wendel, 2003). Indeed, it has been suggested that a very large genome size contributed to difficulties in developing low-copy nuclear gene regions in the Sandhills lily, Lilium pyrophilum (Douglas et al., 2011). Efforts to develop LCNG regions for Fritillaria are ongoing (Kelly et al., unpublished results), and future phylogenetic analyses of such loci will be important in helping to resolve the outstanding question of the relationship between subgenus Liliorhiza and the remaining subgenera of Fritillaria. ...
Premise of the study: Investigations of recently derived and edaphically (soil) defined plant systems have provided insight into important mechanisms of ecological divergence. We investigated the impact of edaphic adaptation on recent divergence between two Colorado Plateau endemics: the gypsum facultative Oreocarya revealii (Boraginaceae) and its more generalist sister species O. paradoxa. We assessed morphological stability, genetic identity, and soil chemistry to determine whether O. revealii is a distinct lineage edaphically adapted from O. paradoxa, as has been described in the literature. Methods: We genotyped 21 populations throughout the ranges of both species using 11 microsatellite markers and three plastid regions (trnL-F, trnT-L, trnQ-rps16) for haplotype analysis. We compared these data with soil chemistry (Ca and S concentrations, indicating gypsum levels), location, and morphological identity of populations. Key results: Soil chemistry failed to explain genetic or morphological identity in either taxon. Haplotype analysis suggests ancestral variation in the more geographically restricted O. revealii, along with regional geographic isolation. A discontinuity was identified between the morphological and genetic identity in several populations, suggesting incomplete lineage sorting and the nonfixation of identifying morphological traits. Conclusions: Oreocarya revealii is unlikely to have arisen via edaphic selection, because soil chemistry of population sites, morphology of individuals, and genetic identity are not strongly correlated. The nonfixation of identifying traits is likely a result of recent divergence in this system, and the potentiality of such discrepancies should be considered when investigating recently diversified gypsum-associated groups.
Designing PCR and sequencing primers are essential activities for molecular biologists around the world. This chapter assumes acquaintance with the principles and practice of PCR, as outlined in, for example, refs. 1, 2, 3, 4.
Positive selection can be inferred from its effect on linked neutral variation. In the restrictive case when there is no recombination, all linked variation is removed. If recombination is present but rare, both deterministic and stochastic models of positive selection show that linked variation hitchhikes to either low or high frequencies. While the frequency distribution of variation can be influenced by a number of evolutionary processes, an excess of derived variants at high frequency is a unique pattern produced by hitchhiking (derived refers to the nonancestral state as determined from an outgroup). We adopt a statistic, H, to measure an excess of high compared to intermediate frequency variants. Only a few high-frequency variants are needed to detect hitchhiking since not many are expected under neutrality. This is of particular utility in regions of low recombination where there is not much variation and in regions of normal or high recombination, where the hitchhiking effect can be limited to a small (<1 kb) region. Application of the H test to published surveys of Drosophila variation reveals an excess of high frequency variants that are likely to have been influenced by positive selection.
A method for estimating the average level of gene flow among populations is introduced. The method provides an estimate of Nm, where N is the size of each local population in an island model and m is the migration rate. This method depends on knowing the phylogeny of the nonrecombining segments of DNA that are sampled. Given the phylogeny, the geographic location from which each sample is drawn is treated as multistate character with one state for each geographic location. A parsimony criterion applied to the evolution of this character on the phylogeny provides the minimum number of migration events consistent with the phylogeny. Extensive simulations show that the distribution of this minimum number is a simple function of Nm. Assuming the phylogeny is accurately estimated, this method provides an estimate of Nm that is as nearly as accurate as estimates obtained using FST and other statistics when Nm is moderate. Two examples of the use of this method with mitochondrial DNA data are presented.
What is the minimum viable population (MVP) of a particular species? Besides the obvious implications for conservation, especially of endangered species, this question raises important issues in population biology. MVP obviously varies with demographic, life history and environmental factors, but also depends upon genetic load and genetic variability. This book addresses the most recent research in the rapidly developing integration of conservation biology with population biology. Chapters consider the roles of demographic and environmental variability; the effects of latitude, body size, patchiness and metapopulation structure; the implications of catastrophes; and the relevance of effective population size on inbreeding and natural selection. Other topics addressed include the role of decision theory in clarifying management alternatives for endangered species, and the opportunities for improved co-operation between agencies responsible for management. The book concludes with a forward-looking and plain-speaking summary on future research and its application for conservation practice.
Tradescantia hirsuticaulis, the hairy-stemmed spiderwort, is an insect-pollinated perennial plant species found primarily on rock outcrops in Georgia, South Carolina, and Alabama. Although populations of T. hirsuticaulis are rare and scattered, local populations are frequently large. Levels of genetic variation were assessed for 13 populations representing the species' range in these three states. Despite the disjunct distribution of this habitat specialist and apparent lack of specialized seed and pollen dispersal mechanisms, exceptionally high levels of genetic variation are maintained within the species, with a moderate level of variation (18%) found among populations. Twenty-nine of the 33 loci resolved (88%) were polymorphic within the species; the mean number of loci polymorphic within populations was 54%. The mean number of alleles per polymorphic locus was 3.24 across all populations and averaged 2.37 within populations. Genetic diversity was 0.206 for the species, whereas mean population genetic diversity was 0.157, both much higher than the average for other short-lived herbaceous perennials. Estimated levels of gene flow were moderate (Nm = 0.95), and a significant association between geographic distance and genetic distance between populations was found (r = 0.68; P < 0.0001). Habitat destruction is the major threat to this genetically diverse species. Since gene flow among its highly dispersed populations is limited, diminution or extinction of local populations could jeopardize the long-term evolutionary potential of this species.
Enzyme electrophoresis was employed to examine genetic variation at 20 loci in 16 populations of Lasthenia minor and 18 populations of its presumed derivative species L. maritima. The purposes of the study were to ascertain levels of genetic variation in each species, to assess how the variation at enzyme-coding genes is apportioned within and among populations of each species, and to determine the level of divergence between the two species. The two species are both diploid annuals, similar morphologically, and produce fertile F1 hybrids when crossed. Lasthenia minor is self-incompatible and restricted to mainland California, whereas L. maritima is self-compatible and probably largely autogamous; it occurs on seabird rocks from central California to British Columbia. Mean genetic identities for pair-wise comparisons of populations of the two species are similar to values for populations of the same species, indicating they have not diverged at the 20 genes coding for soluble enzymes. Despite its more extensive geographical range, L. maritima exhibits only 50% of the genetic diversity of L. minor. The latter species apportions a greater amount of its diversity within populations, whereas the former harbors more diversity among populations than within them. This is probably a reflection of the different breeding systems of the two species. Six unique alleles were detected in L. minor, whereas only one novel allele was found in a single individual of L. maritima. The electrophoretic data are concordant with the suggestion that L. maritima is relatively recently derived from L. minor. The switch from outcrossing to selfing and selection of genotypes adapted to the chemically and physically unusual substrate on the seabird rocks are considered the critical steps in the evolution of L. maritima.
Enzyme electrophoresis and restriction-fragment analysis of chloroplast DNA (cpDNA) and nuclear ribosomal DNA (rDNA) were used to test the hypothesis that both Helianthus neglectus and H. paradoxus are stabilized hybrid derivatives of H. annuus and H. petiolaris. The four species are annuals, diploid outcrossers, and have the same chromosome number. Helianthus annuus and H. petiolaris had the same allele in highest frequency for 16 of the 18 isozyme loci examined and had different majority alleles for only 6-Pgd3 and Pgi2. The two species had divergent rDNAs that could be distinguished by seven diagnostic restriction site mutations and three length mutations, and their cpDNAs could be differentiated by three diagnostic restriction site mutations. The alleles observed in H. neglectus were not a combination of those observed in H. annuus and H. petiolaris. Although H. neglectus had only one unique allele, it possessed none of the three alleles specific to H. annuus. In contrast, it had four of the seven alleles specific to H. petiolaris. Furthermore, H. neglectus had the same rDNA type as H. petiolaris and had the same cpDNA as that found in two populations of H. petiolaris ssp. fallax. These data allowed us to speculate that H. neglectus may be a recent derivative of H. petiolaris ssp. fallax, rather than a stabilized hybrid derivative as originally proposed. In contrast, H. paradoxus combined the alleles of H. annuus and H. petiolaris and had no unique alleles. At Adh2, H. paradoxus was monomorphic for an allele found only in H. petiolaris ssp. fallax, whereas at 6-Pgd3 and Pgi2, it was monomorphic for high frequency H. annuus alleles. Furthermore, H. paradoxus combined the rDNA repeat types of both proposed parents and had the chloroplast genome of H. annuus. These data provide compelling evidence that H. paradoxus, in contrast to H. neglectus, was derived via hybridization.