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Eastern North American plant biogeography has traditionally focused on two primary issues: (i) the location of temperate Pleistocene refugia and their proximity to the southern margin of the ice sheet during the last glacial maximum, and (ii) the origin of the temperate element of northern Latin America. While numerous population genetic and phylogeographical studies have focused on the first issue, few (if any) have considered the second. We addressed these issues by surveying 117 individuals from 24 populations of Liquidambar styraciflua (American sweetgum; Altingiaceae) across the southeastern USA, eastern Mexico, and Guatemala, using more than 2200 bp of chloroplast DNA sequence data. To specifically address the issue of timing, we estimated intraspecific divergence times on the basis of multiple fossil-based calibration points, using taxa from Altingiaceae (Liquidambar and Altingia) and Hammamelidaceae (Hamamelis) as outgroups. More than half of the sampled localities exhibited multiple haplotypes. Remarkably, the greatest variation was observed within the USA, with Mexico and Guatemala sharing widespread haplotypes with Texas, Mississippi, Kentucky, Ohio, and northern Virginia. This lack of differentiation suggests shared ancestral polymorphisms, and that the genetic signal we observed is older than the disjunction itself. Our data provide support for previously proposed hypotheses of Pleistocene refugia in peninsular Florida and along the eastern Atlantic, but also for deeper divergences (approximately 8 million years ago) within the USA. These patterns reflect a dynamic biogeographical history for eastern North American trees, and emphasize the importance of the inclusion of a temporal component in any phylogeographical study.
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Molecular Ecology (2008) 17, 3889–3900 doi: 10.1111/j.1365-294X.2008.03875.x
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd
Blackwell Publishing Ltd
Phylogeographical structure and temporal complexity in
American sweetgum (Liquidambar styraciflua; Altingiaceae)
*Department of Biology, University of South Alabama, Mobile, AL 36688, USA, Department of Biology and Wildlife, University of
Alaska, Fairbanks, AK 99775, USA, Department of Botany, University of Florida, Gainesville, FL 32611, USA, §Florida Museum of
Natural History, University of Florida, Gainesville, FL 32611, USA
Eastern North American plant biogeography has traditionally focused on two primary
issues: (i) the location of temperate Pleistocene refugia and their proximity to the southern
margin of the ice sheet during the last glacial maximum, and (ii) the origin of the temperate
element of northern Latin America. While numerous population genetic and phylogeo-
graphical studies have focused on the first issue, few (if any) have considered the second.
We addressed these issues by surveying 117 individuals from 24 populations of Liquidambar
styraciflua (American sweetgum; Altingiaceae) across the southeastern USA, eastern Mexico,
and Guatemala, using more than 2200 bp of chloroplast DNA sequence data. To specifically
address the issue of timing, we estimated intraspecific divergence times on the basis of
multiple fossil-based calibration points, using taxa from Altingiaceae (Liquidambar and
Altingia) and Hammamelidaceae (Hamamelis) as outgroups. More than half of the sampled
localities exhibited multiple haplotypes. Remarkably, the greatest variation was observed
within the USA, with Mexico and Guatemala sharing widespread haplotypes with Texas,
Mississippi, Kentucky, Ohio, and northern Virginia. This lack of differentiation suggests
shared ancestral polymorphisms, and that the genetic signal we observed is older than the
disjunction itself. Our data provide support for previously proposed hypotheses of Pleistocene
refugia in peninsular Florida and along the eastern Atlantic, but also for deeper divergences
(~8 million years ago) within the USA. These patterns reflect a dynamic biogeographical
history for eastern North American trees, and emphasize the importance of the inclusion of
a temporal component in any phylogeographical study.
Keywords: divergence time estimation, fossil calibration, Latin America, phylogeography, tem-
perate disjunction
Received 27 March 2008; revision received 13 June 2008; accepted 25 June 2008
A rapidly growing database of phylogeographical literature
for eastern North American plants and animals indicates
greater spatial and temporal complexity than previously
suggested (Soltis et al. 2006). In the absence of divergence
time estimates, most of these studies assume Pleistocene
glaciation as the primary causal factor associated with
observed phylogeographical breaks. Numerous researchers
have indicated the potential for interpretative pitfalls in the
absence of temporal data (Bermingham & Avise 1986;
Cunningham & Collins 1994; Avise 2000; Donoghue et al.
2001; Xiang & Soltis 2001; Donoghue & Moore 2003) yet
few phylogeographical studies have dealt with this issue
(but see Church et al. 2003; Sota & Hayashi 2007). The
possibility of convergent patterns being derived from
different evolutionary processes at different times, often
referred to as pseudocongruence (Cunningham & Collins
1994), has become an increasingly important consideration
in phylogeographical studies. In fact, several recent studies
suggest that observed phylogeographical breaks may
predate the last glacial maximum (LGM), or even the
Pleistocene (Klicka & Zink 1997; Austin et al. 2002; Church
et al. 2003; Zamudio & Savage 2003; Near & Keck 2005;
Howes et al. 2006). Divergence time estimation using fossil
Correspondence: Ashley B. Morris, Fax: 251-414-8220; Email:
3890 A. B. MORRIS ET AL.
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd
constraints of phylogenetic trees is becoming a standard
analytical tool for many biogeographical studies of
plants, and has provided a great deal of evidence for
pseudocongruent patterns among codistributed plant
genera in the Northern Hemisphere (Donoghue et al. 2001;
Xiang & Soltis 2001). The same theory and methods used
in higher-level taxa should be extended to intraspecific
phylogeographical data to formally assess the commonly
cited hypothesis of Pleistocene-driven divergence.
As many as 50 species of plants exhibit a disjunction
between the temperate floras of the eastern USA and the
mountains of eastern Mexico (Graham 1973, 1999). Oddly,
this pattern is only seen in a few animal taxa (Martin &
Harrell 1957), including the flying squirrel (Glaucomys
volans, Arbogast 2007), the red-bellied snake (Storeria
occipitomaculata), and certain Pselaphid beetle genera (Carlton
1990). Deevey (1949) suggested that this disjunction is the
result of Pleistocene cooling in the southeastern USA, forcing
temperate taxa into the southern reaches of the region,
including peninsular Florida and the mountains of eastern
Mexico. Braun (1950) countered that these elements arrived
in Mexico as early as the mid-Cenozoic, and were later
isolated by more arid conditions during the Pliocene. Fossil
pollen data support Braun’s hypothesis of early arrival,
indicating the occurrence of 10 temperate woody genera
(including Liquidambar) in the Mexican palynoflora as early
as the mid-Pliocene, approximately 5.2 to 1.6 million years
ago (Graham 1973, 1999). Graham (1999) further noted that
the appearance of these temperate woody taxa is consistent
with a major temperature decline in the mid-Miocene,
before which very few of these elements are found in the
northern Mexican palynofloras. He suggested that the
absence of temperate pollen from earlier Mexican records,
and the presence of temperate pollen in the Eocene record
of the southeastern USA, provide evidence for a southeastern
USA origin for the Mexican temperate element. However,
Axelrod (1975) argued that the Mexican temperate element
represents ‘specialized outliers’ of what was once a con-
tinentally extensive Paleogene temperate rainforest, and not
the result of a southward migration from the southeastern
For each of the taxa with a disjunct biogeographical
distribution documented by Graham (1999), the extent of
the disjunction varies (as does the degree of morphological
variation). This might be expected given differences in life
histories, habitat requirements, and responses to environ-
mental change. The taxonomic rank of taxa involved varies
as well, with some Mexican disjunct lineages considered
conspecific to their US counterparts (e.g. Carpinus caroliniana,
Fagus grandifolia, and Nyssa sylvatica), and others considered
sister species to their US counterparts (e.g. Illicium floridanum/
mexicanum, Taxodium distichum/mucronatum, and Liquidambar
styraciflua/macrophylla; Graham 1973). While these differences
are likely to result in some variability in phylogeographical
structure, the overall expectation is one of phylogeographical
convergence, given some common underlying biogeo-
graphical history. However, as mentioned above, shared
phylogeographical patterns can also be the result of
pseudocongruence, and it is essential that timing be taken
into consideration. Divergence time estimation based on
molecular sequences should provide some resolution to
this issue.
The objectives of this study were to use L. styraciflua
(American sweetgum; Altingiaceae) to (i) assess the degree
of genetic divergence between populations from eastern
North America and northern Latin America, and (ii) test
hypotheses related to the location of Pleistocene refugia for
temperate taxa. The integration of chloroplast DNA (cpDNA)
sequence data and fossil-based calibrations for the estimation
of intraspecific divergence times should shed new light on
this topic.
Materials and methods
Liquidambar styraciflua is considered a bottom-land species
and is most abundant in the Lower Mississippi Valley
(LMV). Its modern-day distribution is primarily in the
southeastern USA, extending from Connecticut to central
Florida and west to eastern Texas (Fig. 1). Liquidambar
styraciflua (including Liquidambar macrophylla as a synonym
of L. styraciflua) occurs frequently throughout eastern and
central Mexico, and extends as far south as Nicaragua
(Little 1971; Kormanik 1990). It is a relatively fast-growing
pioneer species, has an average lifespan of 200 years, and
reaches reproductive maturity at 20–30 years of age. It is a
monoecious species, with wind-pollinated flower production
in mid- to late spring and fruit production in late fall.
Fruits, known colloquially as gumballs, open to release
wind-dispersed seeds, which are also eaten (and ultimately
dispersed) by birds, squirrels, and chipmunks. The
relationship between North American Liquidambar and its
Asian and European relatives is complex, but molecular
data suggest that divergence between L. styraciflua and its
sister, Liquidambar orientalis, occurred between the Oligocene
and Miocene (Ickert-Bond & Wen 2006), during which time
the North Atlantic Land Bridge is hypothesized to have
been available for migration between these major areas
(Donoghue et al. 2001). Post-glacial fossil pollen recon-
structions for eastern North American Liquidambar support
a western Gulf Coast refugium, with subsequent northeastern
spread (Williams et al. 2004).
Data collection
arcview 3.2 (ESRI 1992–2000) was used to update the
boundaries of Little’s (1971) species distribution map for
L. styraciflua using occurrence data acquired from North
American herbarium records online (Fig. 1). Target sampling
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd
localities were evenly distributed throughout the species
range by superimposing a grid (cell size approximately
241 km2) on the distribution map and identifying localities
near the vertices of each cell. Actual sampled localities
depended on the ability to identify suitable sites at or near
those vertices, such that distances between sites varied
(Fig. 1; Table 1). Leaf material was obtained from a
maximum of 30 individuals at each locality and preserved
in silica gel desiccant.
DNA extraction, polymerase chain reaction amplification,
and DNA sequencing
Total genomic DNA was extracted from silica-dried material
using a modified cetyltrimethyl ammonium bromide
(CTAB) protocol (Doyle & Doyle 1987). Four plastid regions
were surveyed for genetic variation within and among
populations of L. styraciflua: atpB-rbcL (Hodges & Arnold
1994), psbA-trnH (Sang et al. 1997; Tate & Simpson 2003),
psbE-petL, and the trnL intron (Taberlet et al. 1991). For
psbE-petL, primers used for initial amplification were
taken from an unpublished source. Because those primers
resulted in differential amplification success, Liquidambar-
specific primers were designed for this study: PSBE1 5-
3. Final concentrations of polymerase chain reaction (PCR)
components were as follows: 1× PCR buffer, 3 mm MgCl2,
200 nm dNTPs, 200 nm forward primer, 200 nm reverse
primer, 1 m Betaine, and 1.25 U Ta q polymerase. The PCR
profile followed that of Taberlet et al. (1991). PCR products
were cleaned with Exo-Sap and were sequenced at a com-
mercial sequencing facility (High-Throughput Genomics Unit,
Department of Genome Sciences, University of Washington).
Sequence alignment and haplotype network construction
All sequences were aligned using clustal_x version 1.83
(Thompson et al. 1997) and were manually checked for
corrections using se-al Sequence Alignment Editor version
2.0a11, available for download from Andrew Rambaut
( Haplotype
networks were constructed for L. styraciflua intraspecific
Table 1 Collection locality information and cpDNA haplotypes recovered for Liquidambar styraciflua
Locality State code Latitude Longitude NHaplotype(s)†
Hackneyville, AL AL 33.07 –85.88 3 K Q R
Pinnacle Mountain State Park, AR AR1 34.84 –92.48 12 K T V
Blaylock Mountain, AR AR2 36.09 –94.37 1 S
Apalachicola Bluffs and Ravines Preserve, FL FL1 30.49 –84.98 10 K L
San Felasco Hammock State Park, FL FL2 29.70 –82.46 4 E
Wekiva Springs State Park, FL FL3 28.71 –81.49 4 E G
George L. Smith State Park, GA GA1 32.54 –82.12 4 E F
Tallulah Gorge State Park, GA GA2 34.74 –83.39 2 A
Green River Lake State Park, KY KY 37.25 –85.34 4 M P
Chemin-a-Haute State Park, LA LA1 32.91 –91.85 4 H I T
Louisiana State Arboretum, LA LA2 30.80 –92.28 4 K N
Ragland Hills, MS MS1 31.20 –89.18 3 P
Starkeville, MS MS2 33.46 –88.79 3 J T W
Ev-Henwood Nature Preserve, NC NC 34.16 –78.12 9 A C D K
Hueston Woods, OH OH 39.51 –84.75 2 P
Columbia, SC SC 34.00 –81.04 4 T
Great Smoky Mountains National Park, TN TN1 35.65 –83.51 8 T U
Paris Landing State Park, TN TN2 36.43 –88.08 4 T
Sewanee, TN TN3 35.20 –85.92 4 T
Big Thicket National Preserve, TX TX 30.43 –94.10 4 P
Chesapeake, VA VA1 36.58 –76.15 3 A B
Pace Estate, VA VA2 37.92 –78.53 3 P
Mesa de la Yerba, Veracruz MEX‡ 19.56 –97.01 11 O P
San Pedro Carcha, Alta Verapaz GUA§ 15.54 –90.24 7 P
†Haplotype codes are from haplotype network in Fig. 1 and maximum likelihood topology in Fig. 2;
‡single collecting locality from Mexico; §single collecting locality from Guatemala.
3892 A. B. MORRIS ET AL.
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd
data only. A haplotype network was constructed using
statistical parsimony as implemented in tcs 1.21 (Clement
et al. 2000) with gaps treated as a fifth character state. While
it is more desirable to treat indels as additional coded
characters than as a fifth character state, tcs does not
currently allow for the inclusion of symbols in the data
matrix. All gaps were the result of length variation in
mononucleotide repeat regions. While we acknowledge
the potential for homoplasy in such regions (reviewed in
Kelchner 2000), the observed patterns among individuals
of L. styraciflua were largely congruent with those exhibited
by substitution data, warranting the consideration of these
repeats as potentially informative characters.
Phylogenetic analyses
Maximum parsimony (MP), maximum-likelihood (ML),
and Bayesian analyses were conducted using paup * (Swofford
2002) and beast version 1.43 (Drummond & Rambaut
2007). Analyses were performed on the combined data,
Fig. 1 Chloroplast haplotype distribution for Liquidambar styraciflua sampled in this study. The haplotype network constructed using
statistical parsimony is given on the left side of the figure. Each circle represents a unique haplotype, with circle size reflecting frequency
of that haplotype. Lines between circles indicate single mutational steps, while hash marks indicate unsampled or extinct haplotypes.
Dotted boxes indicate the two major intraspecific clades recovered in phylogenetic analyses. Haplotype codes (A–W) correspond to those
given in all other Tables and Figures. Colours correspond to major clades of interest as identified by maximum-likelihood analysis (Fig. 3),
and are consistent throughout Figs 2 and 3. For the distribution map given on the right of the figure, the current species range is indicated
y black outline. Pie charts provide haplotype frequency data for sampled localities, with colours being consistent with those given in the
haplotype network. MR, Mississippi River; AR, Apalachicola/Chattahoochee River.
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd
collapsing all individuals with the same haplotype to a
single representative (see Supplementary material). To
allow for fossil-based calibrations of our intraspecific data,
we included accessions of Liquidambar acalycina (EU595851,
EU595860, EU595869, DQ352216), Liquidambar formosana
(EU595852, EU595861, EU595870, DQ352221), L. orientalis
(EU595846, EU595855, EU595864, DQ352222), Altingia
chinensis (EU595847, EU595856, EU595865, DQ352203),
Altingia excelsa (EU595850, EU595859, EU595868, DQ352226),
Altingia obovata (EU595853, EU595862, EU595871, DQ352208),
Altingia poilanei (EU595849, EU595858, EU595867, DQ352210),
and Altingia yunnanensis (EU595848, EU595857, EU595866,
DQ352211), as well as Hamamelis virginiana (Hamamelidaceae;
EU595854, EU595863, EU595872, DQ352196), the latter
serving as an outgroup to Altingiaceae.
MP analyses were performed both with gaps treated as
missing and with gaps coded as additional characters
following the simple indel-coding method of Simmons &
Ochoterena (2000). For each analysis, we performed a heu-
ristic search with 1000 random addition replicates (holding
one tree each step), tree-bisection–reconnection (TBR) branch
swapping, and multrees in effect. All characters were
treated as unordered and equally weighted. Support for
recovered nodes was inferred by bootstrap analysis (1000
replicates, search parameters same as above).
Recent work by Posada & Buckley (2004) suggests that
while the hierarchical likelihood-ratio tests (HLRT) are the
most commonly employed model selection strategy, the
Akaike information criterion (AIC) is a superior approach.
We therefore used the AIC as implemented in modeltest
version 3.6 (Posada & Crandall 1998) to determine an
appropriate model of evolution for use in ML analyses. The
best-fit model for the combined cpDNA data was TVM + G,
with base frequencies of A = 0.3096, C = 0.1533, G = 0.1512,
and T = 0.3858, and a gamma distribution shape parameter
equal to 0.1785. A heuristic search was performed using
1000 random addition sequence replicates, holding one
tree at each step, with multrees in effect, and saving all
trees, and TBR branch swapping. Internal support was
inferred from 100 bootstrap replicates, using the full heuristic
search option and the same search strategy as above.
Estimating divergence times using fossil calibrations
The ML topology recovered above was used to test for rate
constancy. Log-likelihood scores were generated with (+cl)
and without (–cl) a molecular clock enforced. A likelihood-
ratio test (LRT) was performed to determine if there was a
significant difference in evolutionary rates among lineages
(Felsenstein 1988). The hypothesis of a molecular clock was
rejected (P value = 0.015). Divergence time estimation was
performed under a Bayesian approach as implemented in
beast version 1.43 (Drummond & Rambaut 2007). For
readers unfamiliar with beast version 1.43, two substitution
models [HKY (Hasegawa–Kishino–Yano) or GTR (general
time reversible)], and three site heterogeneity models (gamma,
invariant sites, or gamma + invariant sites) are available.
Based on the results of AIC model selection above, we
used a GTR + G model of sequence evolution, under an
uncorrelated lognormal relaxed clock model. We con-
strained several groups to be monophyletic: L. styraciflua;
L. styraciflua +L. orientalis; and A. obovata +L. acalycina +
L. formosana (see below for details). Because the tree priors
available in the current version of beast are not designed
to model mixed inter- and intraspecific data (A. Drummond,
personal communication.), we performed a sensitivity
analysis comparing two approaches to modelling the tree
prior: (i) the Yule speciation process, and (ii) a coalescent
model assuming logistic population growth. Fossil calibration
points were used to determine specific node priors (see
discussion in Ickert-Bond & Wen 2006), and lognormal
distributions were used for all priors to approximate
minimum ages while allowing nodes to be slightly younger
or considerably older. Microaltingia Zhou, Crepet, and
Nixon from the Late Cretaceous of New Jersey was used to
calibrate the root node, approximating a median age of 90
million years [lognormal mean 4.5, SD 0.2, zero offset 0;
range of 60–133 million years (Myr)]. In a morphological
study comparing Liquidambar changii Pigg, Ickert-Bond,
and Wen from the Middle Miocene of eastern Washington
to all other recognized species of Liquidambar, L. changii
was found to most closely resemble L. acalycina (Pigg et al.
2004; Ickert-Bond & Wen 2006). The original survey of L.
changii recovered the fossil as sister to all other Liquidambar
species, which have since been shown by molecular data to
be nested within Altingia (Shi et al. 2001; Ickert-Bond & Wen
2006). However, Ickert-Bond et al. (2007) recently published
a more extensive morphological survey of Altingiaceae
and found strong support for Altingia and Liquidambar as
mutually exclusive sister clades, citing a complex relationship
between morphological convergence and evolutionary
diversification rates.
Previous molecular work recovered a clade containing
L. formosana, A. obovata, and L. acalycina, the latter of which
(as described above) is most similar to L. changii, leading
Ickert-Bond & Wen (2006) to use L. changii to constrain the
age of this clade, which we do here as well (approximate
median age of 15.6 Myr; lognormal mean 2.71, SD 0.5, zero
offset 0; range of 15–40 Myr). For the L. styraciflua crown
group, we set a median age of 3 Myr (lognormal mean 1.1,
SD 1.0, zero offset 0; range of 3–21 Myr) on the basis of fossil
material from the Citronelle formation of southern Alabama,
which appears to be identical to modern L. styraciflua (B.
Axsmith, personal communication, unpublished data).
We compared the estimated mean ages and 95% confidence
intervals for individual fossil calibrations (i.e. just Microaltingia;
Microaltingia and L. changii; Microaltingia and L. styraciflua)
as well as for the three calibration points combined.
3894 A. B. MORRIS ET AL.
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Journal compilation © 2008 Blackwell Publishing Ltd
Haplotype network
The aligned haplotype data set for Liquidambar styraciflua
(excluding all other taxa) was 2165 bp and included two
variable mononucleotide repeat regions. We initially
sampled a maximum of four individuals from each of 24
populations for a total of 96 individuals (Fig. 1). Twenty-
three haplotypes were recovered with these samples, with
13 of 25 localities exhibiting more than one haplotype
(including five populations with three haplotypes each).
Samples from Guatemala and Mexico shared one of the
most frequently recovered haplotypes (P, shown in dark
red, Figs 1–3), present in samples from Texas, southern
Mississippi, Kentucky, Ohio, and northern Virginia. Given
the higher than expected haplotype diversity recovered
from our initial sampling, we increased the sample sizes for
six populations: Pinnacle Mountain State Park (AR2);
Apalachicola Bluffs and Ravines Preserve (FL1); Ev-
Henwood Nature Preserve (NC); Great Smoky Mountains
National Park (TN1); Mesa de la Yerba, Mexico (MEX); and
San Pedro Carcha, Guatemala (GUA). For each of these
populations, we sequenced an additional six to eight
individuals, bringing the total number sampled to 117. No
additional unique haplotypes were recovered (within or
among populations), but one haplotype (K, shown in
dark red, Figs 1–3) was recovered in a new locality (North
Fig. 2 Maximum-likelihood topology for all inter- and intraspecific cpDNA sequences collected for Altingiaceae. Thicker branches
correspond to bootstrap values of 93% or greater from all maximum-likelihood and maximum parsimony analyses. Additional limited
support (55–74%) was recovered within each of the major clades (data not shown). State codes within the ENA/CA clade (Liquidambar
styraciflua) correspond to those given in Table 1; haplotype codes (A–W) correspond to those in Table 1 and Fig. 1. Colour-coding indicates
major clusters of interest and is used throughout all figures. ENA/CA, Eastern North America/Central America; EAS, Eastern Asia;
WAS, Weste rn Asia .
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd
Carolina). Haplotype distribution and GenBank Accession
nos are provided in Appendix S1, Supplementary
material. Descriptions of unique haplotypes defined by
polymorphic sites are provided in Appendix S2, Supple-
mentary material.
Phylogenetic analyses
The aligned Liquidambar/Altingia data matrix included 33
taxa and 2297 bp of data. Several indels, ranging in length
from 5 to 20 bp, were required to align the data. Eight
Fig. 3 Chronogram for Altingiaceae based on a coalescent-based Bayesian approach assuming logistic population growth. Calibrated nodes
are indicated by numbers (1–3): (1) Microaltingia was used to calibrate the root; (2) Liquidambar changii was used to calibrate the clade
including Liquidambar formosana, Liquidambar acalycina, and Altingia obovata. All calibration points were calibrated using lognormal priors
to approximate minimal ages (see methods for details). Gray bars indicate 95% HPD intervals for nodes of particular interest, with ages and
95% HPD given (in millions of years) above the bars. These nodes all have posterior probabilities of 0.99–1.00 (Table 2). Haplotype letter
codes (A–W) and colour codes correspond to those in all other Tables and Figures. Divergence time estimates are further summarized in
Table 2. ENA/CA, Eastern North America/Central America; EAS, Eastern Asia; WAS, Western Asia.
3896 A. B. MORRIS ET AL.
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synapomorphic indels were coded for parsimony analyses.
MP analyses with gaps treated as missing recovered 26
most parsimonious trees (MPT) with a shortest length of
187 (48 parsimony-informative characters; CI = 0.7761;
HI = 0.2239; RC = 0.8515), while analyses with gaps coded
as additional characters recovered 52 MPTs with a shortest
length of 197 (56 parsimony-informative characters;
CI = 0.7792; HI = 0.2208; RC = 0.8476). While all gaps were
phylogenetically informative, their inclusion decreased
bootstrap support for some nodes (data not shown).
Relationships among the species of Liquidambar and Altingia
are largely congruent with previously published data
(Ickert-Bond & Wen 2006), with the exception of the position
of Liquidambar orientalis. This species has been shown to be
sister to L. styraciflua in almost every other study of the
group (Hoey & Parks 1994; Li et al. 1997; Li & Donoghue
1999; Shi et al. 2001; Ickert-Bond & Wen 2006), but its
position is somewhat unresolved here. The strict consensus
constructed from MPTs with gaps treated as missing
recovered L. orientalis as sister to all other members of the
family, while the strict consensus constructed from MPTs
with gaps coded as additional characters recovered the L.
styraciflua sister relationship, as previously published.
However, neither position is strongly supported (< 50%
and 67% bootstrap support, respectively). Short branch
lengths across the tree indicate limited divergence within
L. styraciflua and among members of otherwise well-
supported clades (i.e. the Indochina and Eastern Asian
clades). ML analyses recovered one tree with limited
intraspecific resolution; major clades were congruent with
those recovered using parsimony (Fig. 3).
Divergence time estimation
Results from divergence time estimation using the two tree
prior alternatives (Yule speciation process and coalescent
assuming logistic population growth) are summarized in
Table 2. Implementation of individual fossil calibration
points relative to an approach with three calibration points
result in considerably older nodes with much wider 95%
higher posterior density intervals (HPD; data not shown).
For the combined calibration approach, the coalescent model
assuming logistic population growth yielded considerably
younger mean ages and narrower 95% HPD intervals than
did the Yule speciation prior. Of particular interest are
those nodes within L. styraciflua. Under the coalescent model,
the two major intraspecific clades diverged approximately
8.34 million years ago (95% HPD = 3.14–15.97) relative to
19.21 million years ago (95% HPD = 8.52–39.31) under
the Yule speciation model. Additionally, the Carolina
coastal clade (haplotypes A–D, shown in greens and blues,
Figs 1–3) diverged during the Pleistocene (mean 1.89, 95%
HPD = 0.27–2.66) under the coalescent model, relative to
the Miocene age estimated under the Yule speciation model
(mean 5.48, 95% HPD = 0.85–9.34). Given the differences in
the two approaches, and the predominantly intraspecific
nature of the data, the coalescent tree prior appears to be a
better fit to the data.
The patterns revealed in Liquidambar styraciflua reflect
the dynamic phylogeographical history of eastern North
American taxa, and emphasize the importance of the
integration of a temporal component into any phylo-
geographical study. The greatest divergence observed
within the species was not between the disjunct US and
eastern Mexican populations, as may have been expected
due to the great geographical distance between extant
populations, but was instead within the US populations
(Figs 1–3). Our estimates attribute this break to a pre-
Pleistocene event, roughly 8 million years ago, with
Yule speciation prior Coalescent prior
PP Age (95% CI) PP Age (95% CI)
Root 1.00 101.78 (50.35, 107.50) 1.00 93.11 (64.81, 149.61)
EAS/ENA* 0.90 43.55 (19.45, 72.45) 0.99 22.16 (9.74, 44.59)
IND/EAS† 0.98 28.29 (10.41, 51.36) 0.99 19.20 (6.39, 30.75)
LORIE/LS‡ 1.00 35.85 (13.18, 58.80) 1.00 14.44 (6.51, 31.64)
LS major clades§ 1.00 19.21 (8.52, 39.31) 1.00 8.34 (3.14, 15.97)
LS Carolina clade¶ 0.99 5.48 (0.85, 9.34) 0.99 1.89 (0.27, 2.66)
*Split between the Eastern Asian + Indochina and Eastern North American clades of
†Split between the Eastern Asian and Indochina clades of Altingiaceae.
‡Split between Liquidambar orientalis and Liquidambar styraciflua.
§Split between the two major clades within L. styraciflua.
¶Split between L. styraciflua clade containing haplotypes A–D and all other clades.
Table 2 Summary of divergence time
estimation results using two alternative
Bayesian tree priors using multiple fossil
calibration points. Estimates are given
as mean ages (in millions of years) with
95% confidence intervals in parentheses.
See methods for explanation of fossil
calibrations and a discussion of models.
Posterior probabilities (PP) are given for
each model
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd
admittedly broad confidence intervals (Table 2; Fig. 3). The
family Altingiaceae has a long and diverse fossil record in
the Northern Hemisphere that spans the Late Cretaceous
(Microaltingia) into the Recent in North America, Europe,
and Asia. There are North American Miocene reports of
Liquidambar from Alaska (Seldovian point), Idaho (Clarkia),
Georgia, Kansas, and eastern Mexico, but the relationships
of these fossil taxa to extant L. styraciflua are poorly known.
Given this information, the two clades of L. styraciflua
recovered here may represent ancient relicts of a once
widespread Liquidambar species complex. A lack of differen-
tiation among Latin American and North American
populations may actually reflect the retention of ancestral
haplotypes, as these widespread types are found in the
interior of the haplotype network (Fig. 1).
Phylogeographical structure in L. styraciflua
Previous allozyme work by Hoey & Parks (1994) uncovered
lower than average genetic diversity in L. styraciflua when
compared to other wind-pollinated taxa. From their data,
they inferred two closely allied population centres (i.e. the
southeastern US and eastern Mexico) representing one
species. Hoey & Parks (1994) further suggested that the
Mexican populations must be relatively old, consistent with
fossil data supporting a Miocene arrival of the temperate
element into Mexico, while the disjunction itself is relatively
recent. They found greater structure among Mexican
populations than among US populations, and suggested
that any observable structure in the US would have been
erased by persistent glacial expansion and contraction
during the Pleistocene, whereas Mexican populations
would have remained relatively unaffected. The data
presented here are consistent with this hypothesis, in that
only ancestral, widespread haplotypes were recovered in
Latin American populations sampled for this study
(Fig. 1). Unlike Hoey & Parks (1994), we recovered limited
genetic differentiation within and among Latin American
populations. Differences in our results and those of the
previous study could be attributed to variation in pollen-
and seed-mediated gene flow. Nuclear markers, such as
allozymes, are bi-parentally inherited, reflecting a combination
of both pollen and seed movement. Chloroplast markers,
which are maternally inherited in most plants, reflect only
seed movement. Liquidambar styraciflua is wind-pollinated
and its seeds are largely wind-dispersed, although they are
also known to be eaten by birds, squirrels, and chipmunks
(Kormanik 1990). In a study of seed dispersal in bottomland
hardwood forests, Nuttle & Haefner (2005) found that the
majority of L. styraciflua seeds fell within 50 to 100 m of the
parent tree, with many gumballs containing as many as 50
seeds falling directly below the tree. Such limited dispersal
would likely result in greater localized structure for chloro-
plast markers, while nuclear markers should exhibit a
more homogeneous signal, due to wind pollination, which
is the inverse of the patterns seen here. It may be more likely
that the cpDNA regions used here evolve more slowly than
the allozyme markers used by Hoey & Parks (1994), such
that the resulting patterns reflect different timescales.
In the present study, we recovered only two haplotypes
from Mexico and Guatemala (haplotypes O and P, shown
in dark red on Figs 1–3). Furthermore, haplotype O, which
was shared by both Latin American localities, was one of
the two most common types recovered within and among
US populations. Graham’s (1999) palynological data indicate
the presence of Liquidambar in eastern Mexico as early as
the Pliocene, and there are additional macrofossil reports
of Liquidambar from this region as early as the late Miocene
(Berry 1923). Graham (1999) suggested that the arrival of
temperate taxa such as Liquidambar into eastern Mexico
was the result of range expansion from the southeastern
USA in response to climatic cooling, such that the observed
modern disjunction would be a consequence of repeated
range expansion and contraction in response to dynamic
climate change over the last 3–6 Myr. From an evolutionary
perspective, it is possible that frequent population extinction
and recolonization of ephemeral sinks from a stable source
(within Mexico) could have prevented population subdivision
by drift, and resulted in relatively short coalescence times
among populations (Avise 2000). Alternatively, the genetic
signal we observed may be older than the disjunction itself,
reflecting a time of admixture among continuously distributed
stands of Liquidambar. Additional genetic markers (e.g.
nuclear DNA sequences and microsatellites) will be needed
to provide additional resolution to this question.
Comparisons with other ENA taxa
Some phylogeographical structuring in L. styraciflua is
consistent with previously published biogeographical
patterns for eastern North America (reviewed in Soltis et al.
2006). While phylogenetic support was limited, there
appears to be a trend towards an ‘out of Florida’ track, with
haplotypes from peninsular Florida, eastern and northern
Georgia, and coastal North Carolina and Virginia clustering
together in the haplotype network (haplotypes A–G, shown
in blues and greens, Figs 1–3). Based on our estimates, the
age of this group corresponds with the Pleistocene (Table 2;
Fig. 3), which is consistent with hypotheses that peninsular
Florida served as an ice-age refugium to many temperate
plants and animals. Alternatively, some authors have
suggested that the coastal areas of the Carolinas may have
also played a role as temperate refugia, based in part on
modern species diversity and endemism (Estill & Cruzan
2001; Sorrie & Weakley 2001), and in part on the presence
of early Pleistocene fossils for temperate species (including
Liquidambar; Whitehead 1983). Most recently, in a phylo-
geographical survey of the eastern tiger salamander
3898 A. B. MORRIS ET AL.
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd
(Ambystoma tigrinum), Church et al. (2003) found evidence
for two independent refugia along the mid-Atlantic Coastal
Plain, one of which is centred on the Carolina coast, which
is consistent with what we have found here. Given this
information, it is possible that this primarily coastal clade
recovered for L. styraciflua may represent multiple refugia
(Florida refugium in blue, Carolina refugium in green),
although additional data are needed to assess this
Caveats to divergence time estimation
While our study represents one of only a few examples of
intraspecific dating in plants, there are numerous caveats
to the analyses presented here. First, limited phylogenetic
resolution within L. styraciflua likely dramatically increased
confidence intervals associated with estimated mean ages.
Future work including more rapidly evolving sequence
data may provide a solution to this issue. Second, generally
speaking, fossil position is somewhat subjective when
not resolved phylogenetically through inclusion in a
morphological matrix. In this case, previously published
morphological studies on Liquidambar (Pigg et al. 2004;
Ickert-Bond et al. 2005), Altingia (Ickert-Bond et al. 2007),
and Altingioid relatives (Zhou et al. 2001; Ickert-Bond &
Wen 2006) provided strong rationale for our choices for
fossil placement. Third, theoretical models for divergence
time estimation currently do not explicitly allow for the
analysis of mixed inter- and intraspecific data. Recent studies
have indicated the potential for decreasing substitution
rates with increasing calibration depth, which may ultimately
overestimate intraspecific divergence times (Ho et al. 2005,
2007, 2008). Approaches to rectify this issue are in the early
stages of development, and will ultimately serve as powerful
tools for estimating divergence times in such cases. Finally,
divergence time estimates are based on gene trees, and
may not reflect species trees. Particularly in the case of
more recently diverged taxa (both intra- and interspecific),
gene divergence times are likely to precede species
divergence, such that the latter may be overestimated in
attempts to estimate divergences within phylogeographical
studies (Jennings & Edwards 2005). Comparison of multiple
gene trees from independent loci coupled with coalescent
approaches my aid in the resolution of this issue (Jennings
& Edwards 2005).
Given these caveats, the work presented here still pro-
vides an important lesson. In the absence of any divergence
time estimates, a standard phylogeographical approach to
data interpretation for L. styraciflua would be to assume
two primary Pleistocene refugia on the basis of two
intraspecific clades, one potentially originating along the
Gulf Coast (shown in red, blue, and green, Figs 1–3), and
one from the highlands of the Cumberland Plateau and the
Southern Appalachians (shown in orange and yellow,
Figs 1–3). Our divergence time estimates indicate that such
inferences would be misleading, given that those two
primary clades appear to have diverged at least 8 million
years ago. Regardless of the potential challenges associated
with intraspecific divergence time estimation, the importance
of such attempts is obvious, as has been seen in other tree
species (Dick et al. 2003; Magri et al. 2007).
Importance of recognizing pseudocongruence
Pleistocene glaciation is often presumed to be the primary
factor resulting in observed phylogeographical breaks, but
studies rarely include data that provide support for temporal
associations. It is obvious from comparing molecular
topologies of codistributed organisms that while major clades
may be congruent, there tends to be great heterogeneity
among mutation rates of these codistributed species (reviewed
in Avise 2000). Such heterogeneity does not preclude the
assumption of a shared vicariant event, but it does suggest
the potential for other possibilities (Avise 2000). In particular,
relatively frequent climatic oscillations during the course
of the last three million years likely resulted in numerous
cycles of species range expansions and contractions,
overlaying multiple evolutionary signals on the landscape
of the genome. To better evaluate the extent to which this
occurred, it will be necessary to approach phylogeographical
studies much the same way phylogeneticists are approaching
the reconstruction of relationships among species and
genera, which is with the integration of fossil data (e.g.
Manos et al. 2007). While this will not be a viable option for
many population-level studies due to lack of appropriate
fossils, a ‘hybrid phylogeographical’ approach aimed at
both inter- and intraspecific resolution (e.g. Liston et al. 2007)
will allow for the integration of a temporal element in groups
with good fossil histories. Additionally, such an approach
would better allow for the inclusion of fossil DNA, providing
potentially more accurate divergence time estimates as the
relative position of the fossil in the topology becomes more
certain. Another approach that is gaining attention for testing
phylogeographical hypotheses is that of niche modelling of
paleodistributions (Hugall et al. 2002; Carstens & Richards
2007). Because these models are entirely dependent on modern
collection records and historical climate reconstructions,
they may provide an additional line of evidence towards
the resolution of biogeographical histories.
This work was funded by the Canon National Parks Science Scholars
Program through a fellowship awarded to AB Morris. Additional
support was provided by the Botanical Society of America Karling
Award, Society of Systematic Biologists Graduate Student Award,
and Deep Time NSF RCN DEB-0090283. All analyses were completed
on the University of Florida Phyloinformatics Cluster for High
Performance Computing in the Life Sciences, funded by NSF grant
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd
DEB-0083659/0196412. The authors thank the many individuals
who aided in the collection of leaf material, particularly S. Church,
T. Cross, J. Evans, M. Fishbein, E. Hardcastle, B. McMillan, P.
Manos, B. Mattingly, M. Mendez, J. Nelson, S. Rosso, L. Thien,
K. Tobiason, and R. Zipp. Thanks to A. Bigger and Z. Damji for
laboratory assistance, and J. Clayton, A. Drummond, S. Stutsman,
and M. Gitzendanner for discussions regarding analyses. Finally,
thanks to R. Petit and three anonymous reviewers for comments on
previous versions of this manuscript.
Arbogast BS (2007) A brief history of the new world flying squir-
rels: phylogeny, biogeography, and conservation genetics.
Journal of Mammalogy, 88, 840–849.
Austin JD, Lougheed SC, Neidrauer L, Chek AA, Boag PT (2002)
Cryptic lineages in a small frog: the post-glacial history of the
spring peeper, Pseudacris crucifer (Anura: Hylidae). Molecular
Phylogenetics and Evolution, 25, 316–329.
Avise JC (2000) Phylogeography: The History and Formation of Species.
Harvard University Press, Cambridge, Massachusetts.
Axelrod D (1975) Evolution and biogeography of Madrean-
Tethyan sclerophyll vegetation. Annals of the Missouri Botanical
Garden, 62, 280–334.
Bermingham E, Avise JC (1986) Molecular zoogeography of
fresh-water fishes in the southeastern United States. Genetics,
113, 939–965.
Berry EW (1923) Miocene fossil plants from southeastern Mexico.
Proceedings of the US National Museum, 62, 1–27.
Braun EL (1950) Deciduous Forests of Eastern North America.
Blakiston, Philadelphia.
Carlton CE (1990) Biogeographic affinities of Pselaphid beetles of
the Eastern United States. Florida Entomologist, 73, 570–579.
Carstens BC, Richards CL (2007) Integrating coalescent and
ecological niche modeling in comparative phylogeography.
Evolution, 61, 1439–1454.
Church SA, Kraus JM, Mitchell JC, Church DR, Taylor DR (2003)
Evidence for multiple pleistocene refugia in the postglacial
expansion of the eastern tiger salamander, Ambystoma tigrinum
tigrinum. Evolution, 57, 372–383.
Clement M, Posada D, Crandall KA (2000) tcs: a computer pro-
gram to estimate gene genealogies. Molecular Ecology, 9, 1657–
Cunningham C, Collins T (1994) Developing model systems for
molecular biogeography: vicariance and interchange in marine
invertebrates. In: Molecular Ecology and Evolution: Approaches and
Applications (eds Schierwater B, Streit B, Wagner P, DeSalle R),
pp. 405–433. Birkhauser Verlag, Basel, Switzerland.
Deevey ES (1949) Biogeography of the Pleistocene. Bulletin of the
Geological Society of America, 60, 1315–1416.
Dick CW, Abdul-Salim K, Bermingham E (2003) Molecular sys-
tematic analysis reveals cryptic tertiary diversification of a
widespread tropical rain forest tree. American Naturalist, 162,
Donoghue MJ, Bell CD, Li J (2001) Phylogenetic patterns in north-
ern hemisphere plant geography. International Journal of Plant
Sciences, 162, S41–S52.
Donoghue MJ, Moore BR (2003) Toward an integrative historical
biogeography. Integrative and Comparative Biology, 43, 261–270.
Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure from
small quantities of fresh leaf tissues. Phytochemical Bulletin, 19,
Drummond A, Rambaut A (2007) beast: Bayesian evolutionary
analysis by sampling trees. BMC Evolutionary Biology, 7, 214.
Estill JC, Cruzan MB (2001) Phytogeography of rare plant species
endemic to the southeastern United States. Castanea, 66, 3–23.
Felsenstein J (1988) Phylogenies from molecular sequences: infer-
ence and reliability. Annual Review of Genetics, 22, 521–565.
Graham A (1973) History of the arborescent temperate element in
the northern Latin American Biota. In: Vegetation and Vegetational
History of Northern Latin America (ed. Graham A), pp. 301–314.
Elsevier Scientific Publishing Co., New York.
Graham A (1999) The tertiary history of the northern temperate
element in the northern Latin American biota. American Journal
of Botany, 86, 32–38.
Ho SYW, Phillips MJ, Cooper A, Drummond AJ (2005) Time
dependency of molecular rate estimates and systematic overesti-
mation of recent divergence times. Molecular Biology and Evolution,
22, 1561–1568.
Ho SYW, Shapiro B, Phillips MJ, Cooper A, Drummond AJ (2007)
Evidence for time dependency of molecular rate estimates.
Systematic Biology, 56, 515–522.
Ho SYW, Saarma U, Barnett R, Haile J, Shapiro B (2008) The effect
of inappropriate calibration: three case studies in molecular
ecology. PLoS ONE, 3, e1615.
Hodges SA, Arnold ML (1994) Columbines: a geographically
widespread species flock. Proceedings of the National Academy of
Sciences, USA, 91, 5129–5132.
Hoey MT, Parks CR (1994) Genetic divergence in Liquidambar
styraciflua, L. formosana, and L. acalycina (Hamamelidaceae).
Systematic Botany, 19, 308–316.
Howes BJ, Lindsay B, Lougheed SC (2006) Range-wide phylog-
eography of a temperate lizard, the five-lined skink (Eumeces
fasciatus). Molecular Phylogenetics and Evolution, 40, 183–194.
Hugall A, Moritz C, Moussalli A, Stanisic J (2002) Reconciling
paleodistribution models and comparative phylogeography in
the wet tropics rainforest land snail Gnarosophia bellendenkerensis
(Brazier 1875). Proceedings of the National Academy of Sciences,
USA, 99, 6112–6117.
Ickert-Bond SM, Wen J (2006) Phylogeny and biogeography of
Altingiaceae: evidence from combined analysis of five non-
coding chloroplast regions. Molecular Phylogenetics and Evolution,
39, 512–528.
Ickert-Bond SM, Pigg KB, Wen J (2005) Comparative infructescence
morphology in Liquidambar (Altingiaceae) and its evolutionary
significance. American Journal of Botany, 92, 1234–1255.
Ickert-Bond SM, Pigg KB, Wen J (2007) Comparative infructescence
morphology in Altingia (Altingiaceae) and discordance between
morphological and molecular phylogenies. American Journal of
Botany, 94, 1094–1115.
Jennings BW, Edwards SV (2005) Speciational history of Australian
grass finches (Poephila) inferred from thirty gene trees. Evolution,
59, 2033–2047.
Kelchner S (2000) The evolution of non-coding chloroplast DNA
and its application in plant systematics. Annals of the Missouri
Botanical Garden, 87, 482–498.
Klicka J, Zink RM (1997) The importance of recent ice ages in
speciation: a failed paradigm. Science, 277, 1666–1669.
Kormanik PP (1990) Liquidambar styraciflua L. In: Silvics of North
America: 2. Hardwoods (eds Burns RM, Honkala BH). US Depart-
ment of Agriculture, Washington, DC.
Li JH, Donoghue MJ (1999) More molecular evidence for inter-
specific relationships in Liquidambar (Hamamelidaceae). Rhodora,
101, 87–91.
3900 A. B. MORRIS ET AL.
© 2008 The Authors
Journal compilation © 2008 Blackwell Publishing Ltd
Li JH, Bogle AL, Klein AS (1997) Interspecific relationships and
genetic divergence of the disjunct genus Liquidambar (Hamamel-
idaceae) inferred from DNA sequences of plastid gene matK.
Rhodora, 99, 229–240.
Liston A, Parker-Defeniks M, Syring JV, Willyard A, Cronn R
(2007) Interspecific phylogenetic analysis enhances intraspecific
phylogeographic inference: a case study in Pinus lambertiana.
Molecular Ecology, 16, 3926–3938.
Little E (1971) Atlas of United States Trees, Vol 1: Conifers and Impor-
tant Hardwoods. US Department of Agriculture, Forest Service,
Was hing ton, D C.
Magri D, Fineschi S, Bellarosa R et al. (2007) The distribution of
Quercus suber chloroplast haplotypes matches the palaeogeo-
graphical history of the western Mediterranean. Molecular
Ecology, 16, 5259–5266.
Martin PS, Harrell BE (1957) The Pleistocene history of temperate
biotas in Mexico and eastern United States. Ecology, 38, 468–
Manos PS, Soltis PS, Soltis DE et al. (2007) Phylogeny of extant and
fossil Juglandaceae inferred from the integration of molecular
and morphological data sets. Systematic Biology, 56, 412–430.
Near TJ, Keck BP (2005) Dispersal, vicariance, and timing of
diversification in Nothonotus darters. Molecular Ecology, 14,
Nuttle T, Haefner JW (2005) Seed dispersal in heterogeneous
environments: bridging the gap between mechanistic dispersal
and forest dynamics models. American Naturalist, 165, 336–349.
Pigg KB, Ickert-Bond SM, Wen J (2004) Anatomically preserved
Liquidambar (Altingiaceae) from the Middle Miocene of Yakima
Canyon, Washington State, USA and its biogeographic implica-
tions. American Journal of Botany, 91, 499–509.
Posada D, Buckley TR (2004) Model selection and model averaging
in phylogenetics: advantages of akaike information criterion and
Bayesian approaches over likelihood ratio tests. Systematic
Biology, 53, 793–808.
Posada D, Crandall KA (1998) modeltest: testing the model of
DNA substitution. Bioinformatics, 14, 817–818.
Sang T, Crawford DJ, Stuessy TF (1997) Chloroplast DNA phylogeny,
reticulate evolution, and biogeography of Paeonia (Paeoniaceae).
American Journal of Botany, 84, 1120–1136.
Shi S, Huang Y, Zhong Y et al. (2001) Phylogeny of the Altingiaceae
based on cpDNA matK, PY-IGS and nrDNA ITS sequences.
Plant Systematics and Evolution, 230, 13–24.
Simmons MP, Ochoterena H (2000) Gaps as characters in sequence-
based phylogenetic analyses. Systematic Biology, 49, 369–381.
Soltis DE, Morris AB, McLachlan JS, Manos PS, Soltis PS (2006)
Comparative phylogeography of unglaciated eastern North
America. Molecular Ecology, 15, 4261–4293.
Sorrie BA, Weakley AS (2001) Coastal Plain vascular plant endemics:
phytogeographic patterns. Castanea, 66, 50–82.
Sota T, Hayashi M (2007) Comparative historical biogeography of
Plateumaris leaf beetles (Coleoptera: Chrysomelidae) in Japan:
interplay between fossil and molecular data. Journal of Bio-
geography, 34, 977–993.
Swofford DL (2002) PAUP*. Phylogenetic Analysis Using Parsimony
(* and Other Methods). Sinauer & Associates, Sunderland,
Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers
for amplification of three non-coding regions of chloroplast
DNA. Plant Molecular Biology, 17, 1105–1109.
Tate JA, Simpson BB (2003) Paraphyly of Ta r a s a (Malvaceae) and
diverse origins of the polyploid species. Systematic Botany, 28,
Thompson J, Gibson T, Plewniak F, Jeanmougin F, Higgins D
(1997) The clustal_x windows interface: flexible strategies for
multiple sequence alignments aided by quality analysis tools.
Nucleic Acids Research, 24, 4876–4882.
Whitehead DR (1983) Pollen analysis of the peat member from the
Lee Creek mine. Smithson Contributions to Paleobiology, 53, 265–285.
Williams JW, Shuman BN, Webb T, Bartlein PJ, Leduc PL (2004)
Late-quaternary vegetation dynamics in North America: scaling
from taxa to biomes. Ecological Monographs, 74, 309–334.
Xiang Q-Y, Soltis DE (2001) Dispersal-vicariance analyses of inter-
continental disjunctions: historical biogeographical implications
for angiosperms in the northern hemisphere. International Journal
of Plant Science, 162, S29–S39.
Zamudio KR, Savage WK (2003) Historical isolation, range
expansion, and secondary contact of two highly divergent
mitochondrial lineages in spotted salamanders (Ambystoma
maculatum). Evolution, 57, 1631–1652.
Zhou ZK, Crepet WL, Nixon KC (2001) The earliest fossil evidence
of the Hamamelidaceae: late Cretaceous (Turonian) inflores-
cences and fruits of Altingioideae. American Journal of Botany, 88,
Ashley Morris is interested in phylogeography of disjunct
populations. This work was part of her PhD at the University of
Florida. Steffi Ickert-Bond is an expert on biography and character
evolution of Altingiaceae. Burke Brunson is a Master’s student
in the Morris lab. Pam and Doug Soltis are interested in
phylogeography, systematics, and polyploidy in angiosperms.
Supplementary material
The following supplementary material is available for this article:
Appendix S1 Haplotype distribution and GenBank Accession
nos for Liquidambar styraciflua. Haplotype codes correspond to
those in Figs 2–4 and Table 1. Sample ID, GenBank isolate name;
localities, all sampling localities in which a given haplotype was
Appendix S2 Polymorphic sites among cpDNA haplotypes
recovered within Liquidambar styraciflua. Numbers indicate base
position; ‘.’, same as previous sample; and ‘–’, an indel
This material is available as part of the online article from:
(This link will take you to the article abstract).
Please note: Blackwell Publishing are not responsible for the con-
tent or functionality of any supplementary materials supplied by
the authors. Any queries (other than missing material) should be
directed to the corresponding author for the article.
... Ma), though this subclade was weakly supported. Such intraspecific divergence events predating the Pleistocene have been recovered for several ENA taxa (Lyman and Edwards, 2022), including plants such as Liquidambar styraciflua L. (Morris et al., 2008), Campanulastrum americanum (L.) Small (=Campanula americana L.; Barnard-Kubow et al., 2015), and Symplocarpus foetidus (L.) Salisb. ex W.P.C. Barton (Kim et al., 2018). ...
Full-text available
Premise Glacial/interglacial cycles and topographic complexity are both considered to have shaped today's diverse phylogeographic patterns of taxa from unglaciated eastern North America (ENA). However, few studies have focused on the phylogeography and population dynamics of wide‐ranging ENA herbaceous species occurring in forest understory habitat. We examined the phylogeographic pattern and evolutionary history of Podophyllum peltatum L., a widely distributed herb inhabiting deciduous forests of ENA. Methods Using chloroplast DNA (cpDNA) sequences and nuclear microsatellite loci, we investigated the population structure and genetic diversity of the species. Molecular dating, demographic history analyses, and ecological niche modeling were also performed to illustrate the phylogeographic patterns. Results Our cpDNA results identified three main groups that are largely congruent with boundaries along the Appalachian Mountains and the Mississippi River, two major geographic barriers in ENA. Populations located to the east of the Appalachians and along the central Appalachians exhibited relatively higher levels of genetic diversity. Extant lineages may have diverged during the late Miocene, and range expansions of different groups may have happened during the Pleistocene glacial/interglacial cycles. Conclusions Our findings indicate that geographic barriers may have started to facilitate the population divergence in P. peltatum before the Pleistocene. Persistence in multiple refugia, including areas around the central Appalachians during the Quaternary glacial period, and subsequent expansions under hospitable climatic condition, especially westward expansion, are likely responsible for the species’ contemporary genetic structure and phylogeographic pattern.
... Chloroplast genomes are more conserved and shorter in length, and they are widely used to determine evolutionary patterns (Jansen et al. 2007) and phylogenetic analysis (Moore et al. 2010). Several studies have successfully used chloroplast sequences to infer relationships at all taxonomic levels, from the deepest level relationships between land plants and Angiosperms (Hilu et al. 2003), through intermediate taxonomic levels of orders and family (Chin et al. 2014;Potter et al. 2007), to relationships among closely related species or populations (Morris et al. 2008;Shaw and Small 2004). Molecular trees based on chloroplast genomes have been used as phylogenetic frames to examine and discuss similarities, and dissimilarities of profiles of secondary metabolites (Wink and Mohammed, 2003). ...
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The current study examined the phylogenetic pattern of medicinal species of the family Apiaceae based on favonoid groups production, as well as the overall mechanism of the key genes involved in favonol and favone production. Thirteen species of the family Apiaceae were used, including Eryngium campestre from the subfamily Saniculoideae, as well as Cuminum cyminum, Carum carvi, Coriandrum sativum, Apium graveolens, Petroselinum crispum, Pimpinella anisum, Anethum graveolens, Foeniculum vulgare, Daucus carota, Ammi majus, Torilis arvensis, and Deverra tortuosa from the subfamily Apioideae. The seeds were cultivated, and the leaves were collected to estimate favonoids and their groups, physiological factors, transcription levels of favonol and favone production-related genes. The phylogenetic relationship between the studied species was established using the L-ribosomal 16 (rpl16) chloroplast gene. The results revealed that the studied species were divided into two patterns: six plant species, E. campestre, C. carvi, C. sativum, P. anisum, An. graveolens, and D. carota, contained low content of favonoids, while the other seven species had high content. This pattern of favonoids production coincided with the phylogenetic relationships between the studied species. In contrast, the phylogeny of the favonol and favone synthase genes was incompatible with the quantitative production of their products. The study concluded that the increment in the production of favonol depends on the high expression of chalcone synthase, chalcone isomerase, favanone 3 hydroxylase, favonol synthase, the increase of Abscisic acid, sucrose, and phenyl ammonia lyase, while favone mainly depends on evolution and on the high expression of the favone synthase gene.
... In phylogeographical studies, chloroplast DNA sequences (cpDNA) have been used successfully to infer relationships among closely related species or populations of plants [17][18][19][20]. However, cpDNA, like any other single gene (e.g., LEAFY), is subject to stochastic processes. ...
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The evolutionary histories of ornamental plants have been receiving only limited attention. We examined the origin and divergence processes of an East Asian endemic ornamental plant, Conandron ramondioides. C. ramondioides is an understory herb occurring in primary forests, which has been grouped into two varieties. We reconstructed the evolutionary and population demography history of C. ramondioides to infer its divergence process. Nuclear and chloroplast DNA sequences were obtained from 21 Conandron populations on both sides of the East China Sea (ECS) to explore its genetic diversity, structure, and population differentiation. Interestingly, the reconstructed phylogeny indicated that the populations should be classified into three clades corresponding to geographical regions: the Japan (Honshu+Shikoku) clade, the Taiwan–Iriomote clade, and the Southeast China clade. Lineage divergence between the Japan clade and the Taiwan–Iriomote and Southeast China clades occured 1.14 MYA (95% HPD: 0.82–3.86), followed by divergence between the Taiwan–Iriomote and Southeast China clades approximately 0.75 MYA (95% HPD: 0.45–1.3). Furthermore, corolla traits (floral lobe length to tube length ratios) correlated with geographical distributions. Moreover, restricted gene flow was detected among clades. Lastly, the lack of potential dispersal routes across an exposed ECS seafloor during the last glacial maximum suggests that migration among the Conandron clades was unlikely. In summary, the extant Conandron exhibits a disjunct distribution pattern as a result of vicariance rather than long-distance dispersal. We propose that allopatric divergence has occurred in C. ramondioides since the Pleistocene. Our findings highlight the critical influence of species’ biological characteristics on shaping lineage diversification of East Asian relic herb species during climate oscillations since the Quaternary.
... Current genetic structure patterns are produced by evolutionary and demographic processes at different temporal scales 47 . Plant palaeoecology reconstruction provides fundamental guidance for testable phylogeographic hypotheses, but it cannot indicate the detailed population history 48 . ...
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Gynostemma pentaphyllum (Thunb.) Makino is a perennial creeping herbaceous plant in the family Cucurbitaceae, which has great medicinal value and commercial potential, but urgent conservation efforts are needed due to the gradual decreases and fragmented distribution of its wild populations. Here, we report the high-quality diploid chromosome-level genome of G. pentaphyllum obtained using a combination of next-generation sequencing short reads, Nanopore long reads, and Hi-C sequencing technologies. The genome is anchored to 11 pseudo-chromosomes with a total size of 608.95 Mb and 26,588 predicted genes. Comparative genomic analyses indicate that G. pentaphyllum is estimated to have diverged from Momordica charantia 60.7 million years ago, with no recent whole-genome duplication event. Genomic population analyses based on genotyping-by-sequencing and ecological niche analyses indicated low genetic diversity but a strong population structure within the species, which could classify 32 G. pentaphyllum populations into three geographical groups shaped jointly by geographic and climate factors. Furthermore, comparative transcriptome analyses showed that the genes encoding enzyme involved in gypenoside biosynthesis had higher expression levels in the leaves and tendrils. Overall, the findings obtained in this study provide an effective molecular basis for further studies of demographic genetics, ecological adaption, and systematic evolution in Cucurbitaceae species, as well as contributing to molecular breeding, and the biosynthesis and biotransformation of gypenoside.
... (20 %). Ambos géneros presentan polinización anemófila y se considera que las plantas con este tipo de polinización producen polen de baja calidad alimenticia para los abejorros de acuerdo a la cantidad de proteínas y lípidos (Stanley y Linskens, 1974;Morris et al., 2008). ...
... Genetic structure patterns are produced by evolutionary and demographic processes at different temporal scales (Morris et al., 2008). In the present study, STRUCTURE analysis indicated the existence of two genetic structures (K = 2; Figure 1A) that roughly corresponded to western and eastern China. ...
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Oak trees (Quercus L.) are important models for estimating abiotic impacts on the population structure and demography of long life span tree species. In this study, we generated genetic data for 17 nuclear microsatellite loci in 29 natural populations of Quercus fabri to estimate the population genetic structure. We also integrated approximate Bayesian computation (ABC) and ecological niche analysis to infer the population differentiation processes and demographic history of this oak species. The genetic analyses indicated two genetic clusters across the 29 populations collected, where most approximately corresponded to the intraspecific differentiation among populations from western and eastern China, whereas admixed populations were mainly found in central mountains of China. The best model obtained from hierarchical ABC simulations suggested that the initial intraspecific divergence of Q. fabri potentially occurred during the late Pliocene (ca. 3.99 Ma) to form the two genetic clusters, and the admixed population group might have been generated by genetic admixture of the two differentiated groups at ca. 53.76 ka. Ecological analyses demonstrated clear differentiation among the Q. fabri population structures, and association estimations also indicated significant correlations between geography and climate with the genetic variation in this oak species. Our results suggest abiotic influences, including past climatic changes and ecological factors, might have affected the genetic differentiation and demographic history of Q. fabri in subtropical China.
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Small and isolated populations face several intrinsic risks, such as genetic drift, inbreeding depression and reduced gene flow, patterns of genetic diversity and differentiation have become an important focus of conservation genetics research. The golden snub-nosed monkey Rhinopithecus roxellana, an endangered species endemic to China, has experienced rapid reduction in population size and severe population fragmentation over the past few decades. We measured the patterns of genetic diversity and population differentiation using both neutral microsatellites and adaptive major histocompatibility complex (MHC) genes in two R. roxellana populations (DPY and GNG) distributed on the northern and southern slopes of the Qinling Mountains, respectively. Eight MHC linked haplotypes formed by five DQA1 alleles, five DQB1 alleles, five DRB1 alleles and four DRB2 alleles were detected in the two populations. The larger GNG population showed higher genetic variation for both MHC and microsatellites than the smaller DPY population, suggesting an effect of genetic drift on genetic variation. Genetic differentiation index (FST) outlier analyses, principal coordinate analysis (PCoA) and inferred population genetic structure showed lower genetic differentiation in the MHC variations than microsatellites, suggesting that pathogen-mediated balancing selection, rather than local adaptation, homogenized the MHC genes of both populations. This study indicates that both balancing selection and genetic drift may shape genetic variation and differentiation in small and fragmented populations.
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In a landmark comparative phylogeographic study, “Comparative phylogeography of unglaciated eastern North America,” Soltis et al. (Molecular Ecology, 2006, 15, 4261) identified geographic discontinuities in genetic variation shared across taxa occupying unglaciated eastern North America and proposed several common biogeographical discontinuities related to past climate fluctuations and geographic barriers. Since 2006, researchers have published many phylogeographical studies and achieved many advances in genotyping and analytical techniques; however, it is unknown how this work has changed our understanding of the factors shaping the phylogeography of eastern North American taxa. We analyzed 184 phylogeographical studies of eastern North American taxa published between 2007 and 2019 to evaluate: (1) the taxonomic focus of studies and whether a previously detected taxonomic bias towards studies focused on vertebrates has changed over time, (2) the extent to which studies have adopted genotyping technologies that improve the resolution of genetic groups (i.e., NGS DNA sequencing) and analytical approaches that facilitate hypothesis‐testing (i.e., divergence time estimation and niche modeling), and (3) whether new studies support the hypothesized biogeographic discontinuities proposed by Soltis et al. (Molecular Ecology, 2006, 15, 4261) or instead support new, previously undetected discontinuities. We observed little change in taxonomic focus over time, with studies still biased toward vertebrates. Although many technological and analytical advances became available during the period, uptake was slow and they were employed in only a small proportion of studies. We found variable support for previously identified discontinuities and identified one new recurrent discontinuity. However, the limited resolution and taxonomic breadth of many studies hindered our ability to clarify the most important climatological or geographical factors affecting taxa in the region. Broadening the taxonomic focus to include more non‐vertebrate taxa, employing technologies that improve genetic resolution, and using analytical approaches that improve hypothesis testing are necessary to strengthen our inference of the forces shaping the phylogeography of eastern North America. We surveyed the phylogeographical literature published between 2006 and 2019 and evaluated how both the field of phylogeography and our understanding of the major phylogeographical forces shaping taxa in unglaciated eastern North America has changed over time. Our review is important in that it provides not only an updated understanding of the historical factors shaping the phylogeography of species in the region but also because it highlights the fact that studies suffer from a notable lack of taxonomic breadth and that only a small proportion are employing technologies that can help improve resolution and hypothesis testing ability of phylogeographic studies. Our article highlights the need for improved taxonomic diversity in phylogeographical studies to broaden our inference of the generality of the forces shaping the geographical patterns of genetic variation across species. We also conclude that it is imperative that future studies begin to employ technologies and analytical approaches that will improve their hypothesis‐testing ability.
The genus Cryptopone Emery contains 25 species of litter and soil ants, 5 of which occur in the Americas. Cryptopone gilva (Roger) occurs in the southeastern United States and cloud forests of Mesoamerica, exhibiting an uncommon biogeographic disjunction observed most often in plants. We used phylogenomic data from ultraconserved elements (UCEs), as well as mitogenomes and legacy markers, to investigate phylogenetic relationships, species boundaries, and divergence dates among New World Cryptopone. Species delimitation was conducted using a standard approach and then tested using model-based molecular methods (SNAPP, BPP, SODA, and bPTP). We found that Cryptopone as currently constituted is polyphyletic, and that all the South American species belong to Wadeura Weber, a separate genus unrelated to Cryptopone. A single clade of true Cryptopone occurs in the Americas, restricted to North and Central America. This clade is composed of four species that originated ~4.2 million years ago. One species from the mountains of Guatemala is sister to the other three, favoring a vicariance hypothesis of diversification. The taxonomy of the New World Cryptopone and Wadeura is revised. Taxonomic changes are as follows: Wadeura Weber is resurrected, with new combinations W. guianensis Weber, W. holmgreni (Wheeler), and W. pauli (Fernandes & Delabie); C. guatemalensis (Forel) (rev. stat.) is raised to species and includes C. obsoleta (Menozzi) (syn. nov.). The following new species are described: Cryptopone gilvagrande, C. gilvatumida, and Wadeura holmgrenita. Cryptopone hartwigi Arnold is transferred to Fisheropone Schmidt and Shattuck (n. comb.). Cryptopone mirabilis (Mackay & Mackay 2010) is a junior synonym of Centromyrmex brachycola (Roger) (syn. nov.).
A pollen analytical study of the peat horizon exposed in the Lee Creek phosphate mine indicates that it was deposited in a freshwater environment during interglacial time (probably Sangamon). The freshwater nature of the deposit is suggested by the high percentage of sedge and grass pollen; the presence of Potamogeton , Brasenia, Nuphar, Mynophyllum scabratum, M. heterophyllum, Pontedena, Sagittana, Nymphaea , Typha-Sparganium , and Isoetes ; the occurrence of Botryococcus, Pediastrum boryanum , and Tetraedron ; the low percentage of chenopod-amaranth pollen; and the absence of brackish indicators, such as Ruppia and Iva . The interglacial age (rather than interstadial) is suggested by the general absence of “boreal indicators,” the similarity of the tree pollen frequencies to those from interglacial deposits both to the north and south, and the general similarity of the fossil spectrum to modern pollen assemblages from eastern North Carolina.
Restriction fragment length polymorphisms in mitochondrial DNA (mtDNA) were used to reconstruct evolutionary relationships of conspecific populations in four species of freshwater fish—Amia calva, Lepomis punctatus, L. gulosus, and L. microlophus. A suite of 14-17 endonucleases was employed to assay mtDNAs from 305 specimens collected from 14 river drainages extending from South Carolina to Louisiana. Extensive mtDNA polymorphism was observed within each assayed species. In both phenograms and Wagner parsimony networks, mtDNA clones that were closely related genetically were usually geographically contiguous. Within each species, major mtDNA phylogenetic breaks also distinguished populations from separate geographic regions, demonstrating that dispersal and gene flow have not been sufficient to override geographic influences on population subdivision.—Importantly, there were strong patterns of congruence across species in the geographic placements of the mtDNA phylogenetic breaks. Three major boundary regions were characterized by concentrations of phylogenetic discontinuities, and these zones agree well with previously described zoogeographic boundaries identified by a different kind of data base—distributional limits of species—suggesting that a common set of historical factors may account for both phenomena. Repeated episodes of eustatic sea level change along a relatively static continental morphology are the likely causes of several patterns of drainage isolation and coalescence, and these are discussed in relation to the genetic data.—Overall, results exemplify the positive role that intraspecific genetic analyses may play in historical zoogeographic reconstruction. They also point out the potential inadequacies of any interpretations of population genetic structure that fail to consider the influences of history in shaping that structure.
Seed dispersal is an important determinant of vegetation composition. We present a mechanistic model of seed dispersal by wind that incorporates heterogeneous vegetation structure. Vegetation affects wind speeds, a primary determinant of dispersal distance. Existing models combine wind speed and fall velocity of seeds. We expand on them by allowing vegetation, and thus wind profiles, to vary along seed trajectories, making the model applicable to any wind‐dispersed plant in any community. Using seed trap data on seeds dispersing from forests into adjacent sites of two distinct vegetation structures, we show that our model was unbiased and accurate, even though dispersal patterns differed greatly between the two structures. Our spatially heterogeneous model performed better than models that assumed homogeneous vegetation for the same system. Its sensitivity to vegetation structure and ability to predict seed arrival when vegetation structure was incorporated demonstrates the model’s utility for providing realistic estimates of seed arrival in realistic landscapes. Thus, we begin to bridge mechanistic seed dispersal and forest dynamics models. We discuss the merits of our model for incorporation into forest simulators, applications where such incorporation has been or is likely to be especially fruitful, and future model refinements to increase understanding of seed dispersal by wind.
Sequence data of the chloroplast matK gene generated a phylogeny of Liquidambar containing two robust clades. One clade consisted of the Chinese species L. acalycina and L. formosana, while the other was composed of L. orientalis from Turkey and the North American L. styraciflua. The data support a close relationship between the western Asian and North American species, but not the division of Liquidambar into section Cathayambar (L. formosana) and section Euliquidambar (L. acalycina, L. orientalis and L. styraciflua). Sequence divergence of the matK gene ranged from 0.1 to 1.0% among Liquidambar species and the estimated divergence times of the disjunct species in the genus were 45-90 mya, which agrees with the fossil record.
We investigated the patterns of genetic divergence in three Liquidambar species, L. styraciflua from the eastern United States and Mexico and L. acalycina and L. formosana from China. Estimates of genetic diversity for each species were similar to averages for plants as a whole but lower than expected for wind-pollinated, outcrossing woody plants. For L. styraciflua, almost all of the genetic variation was partitioned within populations (GST = 0.106) and genetic identity among populations was high (I = 0.981). On a regional basis, the Mexican populations were more differentiated than the United States populations (GST = 0.23 and I = 0.94 for the Mexican vs. GST = 0.027 and I = 0.99 for the U.S.). High genetic identities between the Mexican and United States populations indicate recent contact between the two distribution centers. Genetic variation in both L. formosana and L. acalycina was found mainly within populations (GST = 0.098 and 0.118 respectively). Estimates of intraspecific genetic identity were 0.976 for L. formosana and 0.939 for L. acalycina. The genetic identity between L. formosana and L. acalycina was 0.82. The divergence patterns in each of the three species have been influenced by past climatic events.
The family Pselaphidae in eastern North America consists of 67 genera and approximately 352 species. Thirty genera are endemic to the region. Sixteen genera belong to two generalized tracks of Laurasian ancestry. One of these is a poorly defined Holarctic Track, the other a well defined Eastern Nearctic-European Track. Thirteen genera belong to two Nearctic-Neotropical Tracks. The first extends from eastern North America through Mexico to Central America. The second overlaps the first and also extends from Central America and Mexico up the West Coast of the United States to southwestern Canada. Biogeographic affinities were not determined for 34 genera, primarily because of a lack of information about sister group relationships at either generic or specific levels. /// La familia Pseláfida del este de Norte América consiste de 67 géneros y aproximadamente 352 especies. Treinta géneros son endémicos a la región. Dieciseis génros pertenecen a dos trazas generalizadas de antecedentes Laurasianos. Uno de estos es una traza Holártica pobremente definida, el otro es una traza del este Neártico-Europeo muy bien definida. Trecce géneros pertenecen a dos trazas Neárticas-Neotropicales. La primera se extiende del este de Norte América a traves de México hacia Centro América. La segunda sobrelapa la primera y también se extiende desde centro América y México hacia la costa occidental de los Estados Unidos y el suroeste de Canadá. No se determinaron las afinidades biogeográficas de 34 géneros principalmente por falta de información sobre relaciones de grupos hermanos a niveles genéricos o específicos.
Broadleaved evergreen sclerophyllous taxa occupied a subhumid belt across much of North America-Eurasia by the middle Eocene. They originated from alliances in older laurophyllous forests that adapted to spreading dry climate. Since the continued trend to aridity finally restricted sclerophyllous vegetation to subhumid areas separated by drier tracts, it now occurs in areas with summer rain as well as in summer-dry mediterranean climates. Taxa of chaparral and macchia habit are common undershrubs in sclerophyll woodlands, to which they are seral. Shrublands spread only recently, though the adaptive structural features of the taxa are ancient and probably not pyrogenic. The history of Madrean-Tethyan sclerophyll vegetation illuminates three biogeographic problems. First, related taxa that link the Mediterranean-California areas are part of the larger problem of ties between these areas and those of summer rainfall, of taxa now in summer-rain areas that were in presently summer-dry areas into the early Pleistocene, and of the more numerous taxa that linked sclerophyllous vegetation of the Madrean-Tethyan regions during the Tertiary. The ties between summer-dry and summer-wet areas are relicts of the Neogene; taxa now in mediterranean-climate areas adapted functionally to these new climates during the Pleistocene; and most trans-Atlantic links owe to migration across a narrower ocean with more numerous islands, to a broader zone of subhumid climate, and to a more easterly trending Appalachian axis with numerous dry edaphic sites. Second, by the mid-Oligocene spreading dry climate had confined a formerly continuous temperate rainforest to southern Mexico, the West coast and the Appalachian area. Winter cold and summer drought exterminated it in the West, whereas in the East winter cold eliminated most evergreen dicots, leaving a dominantly deciduous hardwood forest there. The temperate "Appalachian disjuncts" in southern Mexico are therefore ancient, and did not migrate south to enter a forest previously without deciduous hardwoods, as others maintain. Third, the Canarian laurel forest derived its taxa from those in laurophyllous forests that covered northern Africa into the middle Miocene, not by southward migration from southern Europe in the Pliocene. Since many shrubs in the surviving laurel forest also contribute to macchia on bordering slopes, the ancient origin of their typical adaptive structural features is clearly implied.