Hemisphere-scale differences in conifer
evolutionary dynamics
Andrew B. Lesliea,1, Jeremy M. Beaulieub, Hardeep S. Raic, Peter R. Cranea, Michael J. Donoghueb,1,
and Sarah Mathewsd
a
School of Forestry and Environmental Studies, Yale University, New Haven, CT 06511; bDepartment of Ecology and Evolutionary Biology, Yale University,
New Haven, CT 06520; cWildland Resources Department, Utah State University, Logan, UT 84322; and dArnold Arboretum of Harvard University, Boston,
MA 02131
Fundamental differences in the distribution of oceans and landmasses in the Northern and Southern Hemispheres potentially
impact patterns of biological diversity in the two areas. The evolutionary history of conifers provides an opportunity to explore
these dynamics, because the majority of extant conifer species
belong to lineages that have been broadly confined to the Northern or Southern Hemisphere during the Cenozoic. Incorporating
genetic information with a critical review of fossil evidence, we
developed an age-calibrated phylogeny sampling ∼80% of living
conifer species. Most extant conifer species diverged recently during the Neogene within clades that generally were established
during the later Mesozoic, but lineages that diversified mainly in
the Southern Hemisphere show a significantly older distribution of
divergence ages than their counterparts in the Northern Hemisphere. Our tree topology and divergence times also are best fit
by diversification models in which Northern Hemisphere conifer
lineages have higher rates of species turnover than Southern
Hemisphere lineages. The abundance of recent divergences in
northern clades may reflect complex patterns of migration and
range shifts during climatic cycles over the later Neogene leading
to elevated rates of speciation and extinction, whereas the scattered persistence of mild, wetter habitats in the Southern Hemisphere may have favored the survival of older lineages.
biogeography
| chronogram | climate change
A
ttempts to understand the global drivers of terrestrial species
diversity and patterns of diversification often have focused on
latitudinal gradients and differences between tropical and temperate environments (e.g., 1–3). However, the specific disposition
of the Earth’s landmasses undoubtedly also has influenced the
origin, persistence, and distribution of species at continental scales.
In particular, there are fundamental geographic differences between the Northern and Southern Hemispheres in terms of land
and ocean area, and these differences might be expected to influence broad evolutionary patterns among the organisms in these
regions. The impact of these major geographic differences on the
evolutionary history of animals and plants of the Northern and
Southern Hemispheres has received relatively little attention (4,
5), perhaps because the complex histories of lineages and the
landscapes themselves obscure the deepest imprints of large-scale
geographic patterns.
Conifers provide an opportunity to evaluate whether such continental-scale differences are reflected in broad evolutionary patterns because the group is diverse (over 600 living species) and
has an extensive and well-documented fossil record, and because
∼90% of extant conifer diversity is contained in major clades that
generally are confined to one of the two hemispheres. Extant
Pinaceae and the diverse Cupressoideae within the Cupressaceae
are found primarily in temperate and subtropical regions of the
Northern Hemisphere (6, 7), whereas extant species of Araucariaceae, Podocarpaceae, and the Callitroideae (the sister group to
Cupressoideae) are found primarily in the Southern Hemisphere
(6–8; see SI Appendix for a full discussion of geographic ranges).
www.pnas.org/cgi/doi/10.1073/pnas.1213621109
Notably, these strong biogeographic distinctions appear to have
been maintained since the end of the Cretaceous. Fossil evidence
suggests that Araucariaceae were restricted to the Southern
Hemisphere throughout the Cenozoic, as they are today (9).
Podocarpaceae were primarily Southern Hemisphere as well (10),
although pollen evidence suggests that some members extended
farther north during warm time intervals (11).
To explore the impact of these biogeographic differences on the
evolutionary history of conifers, we constructed a dated phylogeny
that samples 489 conifer species, ∼80% of extant diversity. This
phylogeny is based on a molecular dataset including two nuclear
genes (18S and a phytochrome gene, PHYP) and two chloroplast
genes (matK and rbcL). A maximum-likelihood (ML) tree topology, which agrees well with previously published phylogenetic
studies of conifers (e.g., 12–16), was used as a constraint topology
for inferring the dated phylogeny. Divergence times were estimated using a Bayesian implementation of an uncorrelated lognormal relaxed clock model with fossil calibration points used as
minimum age constraints (Materials and Methods). These analyses
demonstrate significant differences in the diversification of conifer
clades inhabiting the Northern and Southern Hemispheres over
the last 65 million years (My), suggesting that a broad geographic
signature is imprinted in the evolutionary history of conifers.
Results
To date the nodes of the phylogeny, we focused on calibration
fossils that possessed unambiguous, derived characters shared
with extant clades, usually at the generic level or above. For example, the characteristic vegetative shoots of Phyllocladus, which
consist of flattened photosynthetic branches, are not seen in any
other extant genus or species within the Podocarpaceae, including
Lepidothamnus, the sister taxon of Phyllocladus. Therefore, the
first fossil appearance of such shoots can be used to set a minimum age constraint for the divergence of the Phyllocladus lineage
from other conifers. Similar criteria were applied across the conifer tree, because all of the major clades in this study possess
a fossil record and contain at least one fossil calibration point (see
SI Appendix for a full discussion of calibration points). Within this
framework we performed two sets of analyses. Each used the
same 16 fossil calibrations but differed in the width of the prior
age distributions linked with each calibration point (Materials and
Methods). Absolute and relative ages among clades differed little
between the analyses (SI Appendix, Table S1); here we present
Author contributions: A.B.L., J.M.B., P.R.C., M.J.D., and S.M. designed research; A.B.L.,
J.M.B., P.R.C., M.J.D., and S.M. performed research; A.B.L., J.M.B., H.S.R., P.R.C., M.J.D.,
and S.M. analyzed data; and A.B.L., J.M.B., P.R.C., M.J.D., and S.M. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. JX559887–JX560092).
1
To whom correspondence may be addressed. E-mail: [email protected] or michael.
[email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1213621109/-/DCSupplemental.
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EVOLUTION
Contributed by Michael J. Donoghue, August 16, 2012 (sent for review April 23, 2012)
(5.2 and 8.7 My, respectively). These differences appear to be robust to sampling; although Southern Hemisphere lineages are
more poorly sampled than Northern Hemisphere lineages in our
dataset, median node ages remain significantly younger in Northern Hemisphere lineages even after species sampling is reduced to
50% (SI Appendix, Table S1). Raw molecular branch lengths also
are consistent with results from dating analyses; median molecular
branch length in combined Southern Hemisphere clades is more
than twice that of Northern Hemisphere clades (Fig. 1C) and also
is greater in individual clades (SI Appendix, Fig. S3).
These broad differences among northern and southern conifers
are driven by the major individual conifer clades. Predominantly
Southern Hemisphere lineages such as Araucariaceae, Callitroideae, and Podocarpaceae have significantly different age distributions (P < 0.01 using Mann–Whitney U test) and older median
node ages than the Pinaceae and Cupressoideae, the major lineages in the Northern Hemisphere (Fig. 1D), even after accounting
for differences in sampling (SI Appendix, Table S1 and Fig. S1).
Within the major Northern Hemisphere clades, the abundance of
results from the more conservative analysis using wider prior
age distributions.
The dated phylogeny shows that most extant conifer species
diverged in the Neogene but belong to major lineages that generally diverged much earlier in the Mesozoic (Fig. 1A; see also the
expanded version in SI Appendix, Fig. S4), a pattern that is similar
to those found in analyses of other nonangiosperm seed plant
lineages (17–19) and is broadly consistent with the paleobotanical
record. However, our analyses indicate that the evolutionary dynamics of the conifer lineages inhabiting mainly Southern Hemisphere environments, including both temperate and tropical
habitats, differ from those in the Northern Hemisphere lineages.
Specifically, Northern Hemisphere lineages have a greater proportion of very recent divergence times (i.e., within the past 5 My)
and fewer deep divergences than Southern Hemisphere clades
(Fig. 1B; distributions are significantly different at P < 0.01 using
Mann–Whitney U test). Median node age, which we use to summarize these distributions, for combined Northern Hemisphere
clades is nearly half that of combined Southern Hemisphere clades
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Fig. 1. (A) Dated phylogeny for 489 extant conifer species with cycads as an outgroup. Dashed circles with dates in millions of years ago (Ma) indicate
boundaries between the Paleozoic/Mesozoic and Mesozoic/Cenozoic. The 95% confidence intervals on node ages are colored according to the geographic
range of extant descendant species. Gray indicates a node that joins extant taxa with both Northern and Southern Hemisphere distributions, red indicates
a node joining only extant Southern Hemisphere taxa, and blue indicates a node joining only extant Northern Hemisphere taxa. (B) Scaled density distributions
of node ages (in million years; My) for all Northern and Southern Hemisphere clades. (C) Median molecular branch lengths in Northern Hemisphere clades,
Southern Hemisphere clades, and Northern Hemisphere clades that were resampled to 50% species sampling; error bars represent 95% confidence intervals
based on median node ages for 10,000 resampled trees. (D) Observed median node ages for major Northern and Southern Hemisphere conifer clades.
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Leslie et al.
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Fig. 2. Diversification dynamics of Northern and Southern Hemisphere conifer clades. (A) Lineage accumulation over the Cenozoic in Northern Hemisphere clades (Pinaceae, Cupressoideae) and Southern Hemisphere clades
(Araucariaceae, Podocarpaceae, and Callitroideae). (B) Estimated turnover
rates for Northern and Southern Hemisphere clades. Error bars represent 95%
confidence intervals derived from fitting a constant diversity with turnover
model to 1,000 different dated trees sampling the posterior age distributions
from the BEAST analysis.
Leslie et al.
clades may cause the apparent slowdown in lineage accumulation
in the last 5 My in Podocarpaceae and Callitroideae (although
not in the better-sampled Araucariaceae), but this effect does not
change the general difference between Southern Hemisphere
and Northern Hemisphere clades over the Cenozoic. The rapid
recent accumulation of lineages in Araucariaceae (and their
relatively young divergence ages) primarily reflects a recent burst
of speciation within Araucaria localized to New Caledonia (21).
To examine further the differences in evolutionary dynamics, tree
topology and divergence time estimates in each focal clade were fit
to five diversification models reflecting various origination, extinction, and diversification scenarios using a coalescence approach
that allows extinction and origination rates to vary through time
(22). All focal clades (Northern Hemisphere Cupressoideae and
Pinaceae and Southern Hemisphere Podocarpaceae, Araucariaceae, and Callitroideae) were best fit by a model in which species
diversity remained roughly constant through time, but species
turnover rates in Northern Hemisphere clades were high, whereas
those of Southern Hemisphere clades were variable but often lower
(Fig. 2B; see also SI Appendix, Table S3).
Discussion
Our estimated divergence ages generally agree with results from
published studies focusing on Podocarpaceae (15) and Pinaceae
(23; in the conservative version of their calibrations), although
they are considerably younger than a recently published study of
Cupressaceae (24). The discrepancy in age estimates most likely
results from differences in the implementation of prior age distributions, because both studies used a similar set of fossils as
minimum age constraints. Mao et al. (24) generally used extremely wide uniform priors on age distributions associated with
each fossil, potentially biasing posterior densities toward older
divergence ages (25). Our conservative use of fossils as stem taxa
with narrower lognormal prior age distributions potentially may
bias estimates toward younger ages, but the marked relative
differences between Northern and Southern Hemisphere clades
that are the primary focus of this study remain unaffected.
The distribution and abundance of recently diverged species in
Northern Hemisphere lineages is not related directly to latitude or
specific habitat type within the Northern Hemisphere (SI Appendix,
Fig. S2); they are broadly concentrated in regions of high conifer
diversity such as mountainous areas of western North America
and southern China. Southern Hemisphere conifers likewise show
similar age patterns across wide geographic and environmental
ranges; species inhabiting open, dry habitats in Australia and
Argentina, temperate rainforests in New Zealand, and tropical
montane forests in New Guinea all have generally older estimated
divergence times than species in Northern Hemisphere clades.
The process or processes underlying these differences therefore
appear to operate on regional and continental scales, and an explanation for these patterns must encompass a wide variety of
specific habitats and environments.
In the Northern Hemisphere, falling global temperatures over
the Cenozoic are associated with a general shift in the middleand high-latitude landmasses from warm subtropical and temperate climates in the Eocene to colder, drier, and more strongly
seasonal climates from the Oligocene onwards (26–30), especially in the later Neogene with the onset of extensive Northern
Hemisphere ice cover and glaciations (31–33). Increasingly extreme climatic shifts through time, especially in the later Neogene, may have favored the replacement of older lineages with
those better adapted to cooler and/or drier conditions, resulting
in high turnover rates and the disproportionate loss of ancient
lineages. In addition, climatic and landscape history may have
contributed to the abundance of very recent species divergences
in Northern Hemisphere clades. Repeated instances of species
migration, range contraction, and range expansion in response to
glacial cycles can lead to isolated populations and speciation,
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young divergences is largely the result of several unrelated speciesrich subclades that have diversified recently. For example, ∼45% of
Northern Hemisphere diversity is contained in just two clades, Pinus
and Juniperus, whose median node ages are under 3.5 My. Other
large Northern Hemisphere subclades such as Abies, Picea, and
Cupressus sensu lato show similarly young ages. Although large
radiations are not absent in the Southern Hemisphere, they often
show older divergences times. For example, Northern Hemisphere
Pinus and Southern Hemisphere Podocarpus are comparable in
terms of species diversity, but the observed median node age of
Podocarpus is older than that of Pinus (5.96 versus 3.21 My), even
though the initial divergence of Podocarpus from other conifers is
more recent based on dating analyses and fossil evidence (10, 13, 20).
The different evolutionary dynamics of Northern Hemisphere
clades and Southern Hemisphere clades also are reflected in lineage
accumulation plots, which show a sharp increase in the number of
Northern Hemisphere lineages that diverge in the last 20 My
compared with Southern Hemisphere Callitroideae and Podocarpaceae (Fig. 2A). Sparser sampling in Southern Hemisphere
especially in the types of mountainous environments where conifer diversity is high (34–36). In a general sense, the structure
of the Northern Hemisphere conifer clades may record the complex history of climate-driven turnover and range shifts taking
place within the large midlatitude land areas of the Northern
Hemisphere.
In contrast, southern landmasses with high conifer diversity,
such as Australia, New Zealand, and South America, have drifted
apart and northwards from the Cretaceous to the recent (37, 38).
These movements have led to the scattered persistence of relatively warm or wet habitats, often moderated by oceanic climates
(4), which appear to be reflected in aspects of modern vegetation. Trees living in Southern Hemisphere temperate forests are
not as cold tolerant as many Northern Hemisphere temperate
species (4, 39), and most southern conifers prefer relatively wet
environments (10, but also see ref. 40). The Southern Hemisphere clearly experienced major climatic shifts over the Cenozoic, particularly the spread of open, dry environments in the late
Neogene (41–43). However, temperate rainforests and broadleaf
evergreen forests were common before the Pleistocene (44, 45)
and still are present in New Zealand and in parts of Australia
and South America. These now fragmented habitats are among
those in which older conifer lineages adapted to warmer or
wetter climates have survived at high diversity.
The evolutionary history of conifers undoubtedly reflects complex
interactions among a suite of factors ranging from global-scale climatic and geographic patterns to smaller-scale attributes of specific
lineages and their environmental settings. Nevertheless, this study
suggests that there are large-scale and consistent differences between the diversification of southern and northern conifer clades.
General differences in climatic and landscape history resulting from
the dispositions of landmasses in the hemispheres appear to have
left a distinct imprint on conifer evolutionary history. More work
is needed to understand how these factors may have affected the
biogeographic history of other clades.
Materials and Methods
We inferred a large DNA sequence-based phylogeny for conifers using the
PHyLogeny Assembly with Databases pipeline (PHLAWD, http://code.google.
com/p/phlawd/) (46). PHLAWD uses a “baited” sequence-comparison approach in which a small subset of sequences for a clade of interest provided
by the user is used to filter GenBank sequences and to determine whether
these sequences are homologous to the gene regions of interest. The program then conducts saturation analyses on these sequences by comparing
uncorrected genetic distances with corrected distances based on a Jukes–
Cantor (JC) substitution model. If a gene alignment is saturated, the alignment is broken up according to the National Center for Biotechnology
Information classification system, and the blocks are aligned separately.
We used this approach to search GenBank for three genes that generally
have been used to construct phylogenetic relationships both within and
among the major conifer clades, including two regions of chloroplast genome
DNA, matK (sampled for 446 taxa in our dataset) and rbcL (437 taxa), and
one nuclear coding region, 18S (97 taxa). We then added newly generated
sequences from another nuclear gene, PHYP (212 taxa), which were contributed by S.M. Our total species list was compared with conservative taxonomic treatments (6–8, 47), especially for Northern Hemisphere conifers, to
avoid biasing our results by oversplitting particular clades. Species varieties
generally were excluded from the analysis unless recent phylogenetic studies
suggest they should be considered as separate species [e.g, in Hesperocyparis
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BEAST. We ran four independent MCMC runs of 50 million generations,
sampling every 1,000 generations. To ensure that the posterior distribution
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10 million generations discarded as burn-in for each run. Post burn-in samples from the marginal posterior distribution were combined using LogCombiner v. 1.6.1, and trees were summarized with TreeAnnotator. The
results represent the maximum clade credibility trees with the consensus
ages being the median estimate.
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Taylor Feild, and Jianhua Li for providing tissues and DNA samples and Todd
Dawson, Susana Magallón, and Scott Wing for helpful comments and suggestions that improved the manuscript. This work was supported by National
Science Foundation (NSF) Grant 0629890 (to S.M.) and by the iPlant Tree of
Life (iPToL) program within the NSF-funded iPlant Collaborative (J.M.B.).
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SUPPLEMENTARY MATERIALS AND METHODS
Geographic Ranges of Extant Conifers
The Araucariaceae, Podocarpaceae, and Callitroideae are generally referred to as
Southern Hemisphere conifers because most of their species diversity (~75%) is found
south of the Equator (numbers based on midpoint species latitude ranges compiled
primarily from monographs (1-4)). However, about 34% of Podocarpaceae species are
found in Northern Hemisphere tropical environments (and very occasionally warm
temperate forests), especially in Central America and Southeast Asia. While most
species diversity in Araucariaceae and Podocarpaceae is found in southern tropical
latitudes, Podocarpaceae includes a significant number of Southern temperate species
(around 20% of species diversity), including a large temperate clade (the prumnopityoid
clade of (5), also found in this analysis). Callitroideae are also primarily temperate, with
80% of species diversity found south of the Tropic of Capricorn. Among Northern
Hemisphere conifer clades, most Pinaceae and Cupressoideae species diversity is found
in mountainous temperate and warm temperate regions of western North American and
southern and southwestern China. A few species of Pinaceae and Cupressoideae extend
into lowland tropical environments, and only a single species of Juniperus (J. procera)
extends below the Equator.
Calibration approach
In general, we did not place fossils in subclades within extant genera, because
such placement often depends on the correct systematic assessment of detailed
morphological features like cuticle characters or continuous variables such as the size of
seed cones. Since the systematic patterns in such features have rarely been explored in an
explicit phylogenetic framework, it is often difficult to confidently determine which
states are pleisiomorphic and which synapomorphic. The inability to reliably assign most
fossils to specific groups within modern genera, combined with relatively poor
knowledge of even the most completely understood fossils, means that many fossils
cannot be assigned securely to the crown group of the genus (the group that includes all
living members) and could instead belong to the stem group (the group that includes all
living species and some extinct species which may lack all the defining features of the
living genus). We therefore conservatively place fossils as stem members unless they
possess extremely distinctive morphological features shared only with particular extant
clades within genera.
For all calibration points we developed an associated prior age distribution, which
represents the initial probability distribution of ages for a calibrated node used in BEAST
analyses. In most cases, we used priors with a lognormal probability distribution where
the minimum age was set by the age of the fossil constraint and 95% confidence intervals
of the probability distribution extend either 20 or 40 million years (My) earlier than this
minimum age. We chose these two different distributions in order to explore the effect of
prior width on divergence age estimates, although both numbers are somewhat arbitrary.
In effect, we make an initial hypothesis that the actual date of a given divergence had a
1
95% probability of occurring within 20 or 40 My of the first fossil evidence, which can
be subsequently altered by the analysis based on information from the sequence data. If
fossil age is known to be within a general geologic time interval but its exact age has not
been resolved, we used the younger boundary of the time period to set the minimum age.
Throughout this study, dates were based on the 2009 geologic timescale published by the
Geological Society of America (primarily based on 6). We used lognormal prior age
distributions with either 20 or 40 My 95% confidence intervals for all calibration points
unless there was fossil evidence suggesting the actual divergence may significantly
predate the first unambiguous fossil appearance. For example, we extended the 95%
confidence intervals beyond the typical 20 My (or 40 My in the alternate analysis) for the
divergence of Tsuga because their seed cones first occur in the Eocene, but there are
reports of their distinctive pollen grains from the Late Cretaceous (Turonian). We
therefore extended the 95% confidence intervals of the prior to reflect the possibility of
their earlier divergence time (see Discussion of Calibration Points and Priors for more
detail). The divergence between conifers and cycads (the outgroup used in this study)
was constrained between 275-350 My using a uniform prior, where any divergence age
between these dates is equally probable. We used a uniform prior for this date because
we are confident that the divergence did not occur before 350 My based on substantial
fossil evidence, even though the relationships among putative early conifers, extant
conifers, and cycads, as well as the timing of their respective appearances during this
interval, have not been resolved unambiguously (see Discussion of Calibration Points and
Priors).
Evaluating the Impact of Different Priors
Within the focal clades, there were only minor differences in divergence age
estimates when using fossil calibrations with associated prior age distributions of 20 My
or 40 My 95% confidence intervals (Table S1). Surprisingly, median node ages were
slightly younger within some focal clades using the calibration scheme with wider prior
age distributions. Because the differences between analyses were minimal, both in terms
of absolute and relative ages, we primarily illustrate results from the analysis using 40
My prior age distributions. This analysis uses wider, more conservative prior age
distributions. In this analysis, the distributions of divergence times in specific focal
clades are broadly similar to overall Northern Hemisphere (NH) and Southern
Hemisphere (SH) conifer distributions shown in the main text. Cupressoideae and
Pinaceae distributions are skewed towards more recent divergence times than
Callitroideae and Podocarpaceae, and also show proportionally fewer deep divergences
(Fig. S1A).
2
Median Node Ages (My)
Clade
Observed
(20 My Priors)
Observed
(40 My Priors)
50% Resampling
(40 My Priors)
Araucariaceae (SH)
Podocarpaceae (SH)
Podocarpus (SH)
Callitroideae (SH)
All Southern
Cupressoideae (NH)
Pinaceae (NH)
Pinus (NH)
All Northern
6.13
8.66
5.67
16.34
8.45
4.34
3.67
3.09
4.39
6.07
8.72
5.96
16.75
8.72
4.15
3.77
3.21
5.21
4.28 (5.60) 7.82
4.37 (5.04) 5.78
3.52 (4.52) 5.09
5.03 (5.53) 6.17
Table S1. Median node ages (in millions of years; My) of Southern Hemisphere (SH)
and Northern Hemisphere (NH) focal clades using different calibration schemes.
Resampled values in parentheses represent the median value of 10,000 median node ages
calculated from a resampling routine where NH clades were reduced to 50% species
sampling (see discussion in Supplementary Materials). Values outside of the parentheses
give the lower and upper boundaries of the 95% confidence intervals for this distribution.
Note that while the upper limits of the 95% confidence intervals in Cupressoideae and
Pinaceae are older than observed median node age in Araucariaceae, this clade is sampled
nearly as well as NH clades and its observed median node age can be directly compared
with observed values in NH clades.
3
A
Cupressoideae
Callitroideae
Density
Pinaceae
Podocarpaceae
0
10
20
30
40
50
60
0
Node Age (My)
10
20
30
40
50
60
Node Age (My)
Frequency
B
Callitroideae
Median Node Age
Podocarpaceae
Median Node Age
2
4
6
8
Pinaceae Resampled
Median Node Age (My)
10
0
5
10
15
20
Cupressoideae Resampled
Median Node Age (My)
Fig. S1. (A) Distribution of divergence times (in millions of years; My) in Northern
Hemisphere and Southern Hemisphere focal clades. A density function was calculated
from the age distributions and then scaled to facilitate comparisons between clades with
different numbers of extant species. Density values are arbitrary and are not shown on the
axes. (B) Distribution of 10,000 median node ages for Pinaceae and Cupressoideae
generated through an iterative resampling procedure (see discussion in Supplementary
Materials). Red arrows indicate the value of the observed median node age in
Podocarpaceae and Callitroideae.
4
Evaluating Potential Sample Bias
Southern Hemisphere clades are not as well sampled in our dataset as NH clades, which
could potentially bias our results towards finding relatively older ages in SH groups. In
order to assess this, we performed an iterative resampling analysis where NH clades were
randomly degraded to 50% sampling of extant species and the median node age was
recalculated. This was repeated 10,000 times to form a distribution of median node ages.
This process gives conservative estimates, since neither Callitroidea nor Podocarpaceae
are that poorly sampled in our dataset (61% and 66% of extant species are present
respectively) and Araucariaceae (81% species sampling) is sampled nearly as well as NH
clades (84% and 95% species sampling for Pinaceae and Cupressoideae respectively).
After resampling, median node ages for NH clades remained significantly older than SH
clades (Table S1, Fig. S1B)
Southern Hemisphere conifer clades also contain a greater proportion of tropical
taxa than NH clades, which could potentially result in differences in divergence times
based on broad latitude patterns rather than landmass configuration. However, estimated
divergence ages for individual species do not show a strong latitudinal gradient in either
hemisphere (Fig. S2). In the Northern Hemisphere, species with extremely recent
estimated divergence ages (e.g., 1.0 My or less) occur across a very broad range of
latitudes. These species tend to occur in large regions of high conifer diversity, including
western North America and southern and southwestern China. In the Southern
Hemisphere, species with extremely recent divergence ages are largely absent from all
latitudes, although there is a weak positive relationship between high southern latitudes
and older species divergence age (R2 = 0.11), which primarily reflects deep divergence
times in a subset of extant temperate species of Podocarpaceae.
To test whether differences between Northern and Southern Hemisphere clades
resulted from a bias in our divergence time estimates, we also examined raw molecular
branch lengths calculated using RAxML in NH and SH focal clades. As discussed in the
main text, these results are consistent with our estimated divergence times; median
molecular branch lengths are smaller in NH clades than SH clades (Table S2). In
particular, NH clades have relatively few long branches and an abundance of extremely
short branches compared to SH clades (Fig. S3A,B; focal clades significantly different at
p<0.01 using Mann-Whitney U-test). These differences do not appear to result from
biased genetic sampling; NH clades consistently have a greater proportion of short branch
lengths when calculated using rbcL sequences (Fig. S3C) and matK sequences alone
(Fig. S3D). In both cases, branch length distributions between the focal clades are
significantly different at p<0.01 using Mann-Whitney U-test).
5
Northern Clades
Southern Clades
Species Divergence Age (My)
104
103
102
100
10-1
-60
-40
-20
0
20
40
60
Midpoint Species Latitude
Fig. S2. Relationship between latitude and estimated divergence age (in million of years;
My) for individual living conifer species. Species latitudes were determined based on the
midpoint of present day geographical range (primarily found in refs. 1-4).
Median Molecular Branch Lengths
Clade
Araucariaceae (SH)
Podocarpaceae (SH)
Callitroidea (SH)
Cupressoidea (NH)
Pinaceae (NH)
Observed
50% species resampling
9.1×10-4
1.2×10-3
3.4×10-3
4.3×10-4
4.0×10-4
4.5×10-4- (7.8×10-4) -1.1×10-3
4.2×10-4- (5.0×10-4) - 6.1×10-4
Table S2. Median branch lengths for major Southern (SH) and Northern Hemisphere
(NH) clades. Branch lengths are given in substitutions per site. Resampled values in
parentheses represent the median of 10,000 median branch lengths calculated from a
resampling routine where NH clades were reduced to 50% species sampling (see
discussion in Supplementary Materials). Values outside of the parentheses give the lower
and upper boundaries of the 95% confidence intervals for this distribution.
6
B
Cupressoideae
Callitroideae
Density
A
Cupressoideae
Pinaceae
Podocarpaceae
Density
Callitroideae
0.00
0.01
0.02
C
0.03
0.04
0.05
Density
Cupressoideae
Callitroideae
Podocarpaceae
Density
Pinaceae
Podocarpaceae
Araucariaceae
0.00
0.01
0.02
D
0.03
0.04
0.05
Density
Cupressoideae
Callitroideae
Pinaceae
Podocarpaceae
Density
Pinaceae
0.00
0.03
0.01
0.02
0.03
0.04
0.05
Log Molecular Branch Length
Fig. S3. (A) Molecular phylogeny based rbcL, matK, 18S and PHYP sequences with
branch lengths representing raw molecular substitutions per site. Focal clades are labeled
and indicated in red (Southern Hemisphere) and blue (Northern Hemisphere) (B)
Comparison of molecular branch length distributions in focal clades based on rbcL,
matK, 18S and PHYP. Lines represent density functions calculated from histograms of
branch lengths, which were scaled for comparison. Density values are arbitrary and are
not shown on the axes (C) Comparison of molecular branch length distributions based on
an analysis of rbcL sequences only. (D) Comparison of molecular branch length
distributions based on an analysis matK sequences only.
7
Diversity Patterns Through Time
We constructed lineage-through-time (LTT) plots for each of the major clades as
a way to visualize the timing of speciation events. In addition, to better understand the
dynamics of these originations we also assessed the fit of various lineage-specific
diversification models. We used recently developed coalescent-based methods that work
back from the present to the root of a tree to approximate the likelihood of observing the
distribution of internode distances under various diversification scenarios ("backwards"
approach; see 7). The coalescent-based approach can readily accommodates incomplete
species sampling and is known to outperform “time-forward” methods in both its
robustness and its ability to accommodate time-varying rates (7).
We assessed the fit of five diversification scenarios (Table S3). The first
corresponds to a hypothesis that diversity is saturated, and is maintained by a turnover
process such that an extinction event is immediately followed by a speciation event
(Time-constant turnover model). The second model is a generalization of the first, in that
the turnover rate decays through time (Time-varying turnover model). The remaining
three scenarios assume that diversity is expanding by independent speciation and
extinction events that vary in fundamentally different ways. In one scenario, extinction is
assumed to be zero, and speciation is allowed to vary (Yule with time-varying
speciation). In the remaining two scenarios, the extinction rate is constant, and speciation
rates are allowed to vary through time (Birth-Death with time-varying speciation), or
speciation rate is assumed to be constant and extinction rate is allowed to vary (BirthDeath with time-varying extinction).
Saturated Diversity
Expanding Diversity
Clade
Timeconstant
Turnover
Timevarying
Turnover
Yule
Timevarying
Speciation
BD
Timevarying
Speciation
BD
Timevarying
Extinction
Araucariaceae
Podocarpaceae
Callitroideae
Cupressoideae
Pinaceae
0.336 (522)
0.021 (0)
0.097 (20)
0.018 (0)
0.131 (0)
0.548 (393)
0.979 (1000)
0.435 (889)
0.944 (997)
0.869 (1000)
0.091 (85)
<0.01 (0)
0.321 (91)
0.011 (1)
<0.01 (0)
<0.01 (0)
<0.01 (0)
0.083 (0)
<0.01 (1)
<0.01 (0)
0.024 (0)
<0.01 (0)
0.063 (0)
<0.024 (1)
<0.01 (0)
Table S3. Average AIC weights across a set of 1000 trees for different diversification
models in each of the major conifer clades under study. The numbers in parentheses are
the frequencies that each model had the highest AIC weight. Time-varying turnover
models were best fit in most clades, with a very slight decrease in turnover rate over time.
BD refers to birth-death models with varying speciation and extinction parameters.
8
A maximum-likelihood based approach was used to obtain parameter estimates
and assess the model fit across 1000 trees randomly chosen from the posterior
distribution of dated trees. Initial exploratory analyses revealed that the likelihood
function was “noisy”, often converging to several different local maximum rather than
consistent and potentially global one. To avoid this situation, we implemented an initial
“coarse” grid search prior to the full likelihood search to find the set of starting
conditions that ensured finding the global maximum (i.e., the highest negative loglikelihood). The AIC weight (wi), which represents the relative likelihood that model i is
the best model given a set of models (8), was calculated and averaged for all the models
across the 1000 trees. The model with the highest average AIC weight was considered the
model with highest probability. All analyses were carried out using scripts written for R
(R Development Core Team, 2011).
Time-calibrated Conifer Phylogeny
Fig. S4. A detailed version of the dated phylogenetic tree used in the primary text is
given on the following pages, where it has been broken into separate panels showing
Cupressaceae and Taxaceae (A), Podocarpaceae and Araucariaceae (B), and Pinaceae (C)
respectively. Calibration points are labeled on the tree numerically, and are described in
more detail in the following sections. Dashed lines indicate the Paleozoic-Mesozoic
boundary (251 My), the Cretaceous-Paleogene boundary (65 My), and the PaleogeneNeogene boundary (23 My; dates from 5). Bars at each node represent 95% confidence
intervals for the distribution of estimated divergence times, and the median value of this
distribution is illustrated as the divergence time. Blue bars indicate age distributions for
nodes whose extant descendant members have a Northern Hemisphere distribution, while
red bars indicate nodes where all extant descendant taxa have a Southern Hemisphere or
tropical distribution. Gray bars are associated with nodes that have both northern and
southern descendant taxa.
9
Juniperus_pinchotii
Juniperus_angosturana
Juniperus_comitana
Juniperus_monosperma
Juniperus_coahuilensis
Juniperus_deppeana
Juniperus_gamboana
Juniperus_durangensis
Juniperus_saltillensis
Juniperus_californica
Juniperus_ashei
Juniperus_osteosperma
Juniperus_occidentalis
Juniperus_monticola
Juniperus_flaccida
Juniperus_przewalskii
Juniperus_saltuaria
Juniperus_pingii
Juniperus_indica
Juniperus_squamata
Juniperus_recurva
Juniperus_komarovii
Juniperus_convallium
Juniperus_tibetica
Juniperus_pseudosabina
Juniperus_procumbens
Juniperus_chinensis
Juniperus_thurifera
Juniperus_procera
Juniperus_scopulorum
Juniperus_horizontalis
Juniperus_saxicola
Juniperus_barbadensis
Juniperus_gracilior
Juniperus_bermudiana
Juniperus_semiglobosa
Juniperus_sabina
Juniperus_blancoi
Juniperus_microsperma
Juniperus_virginiana
Juniperus_excelsa
Juniperus_phoenicea
Juniperus_communis
Juniperus_formosana
Juniperus_taxifolia
Juniperus_rigida
Juniperus_oxycedrus
Juniperus_drupacea
Hesperocyparis_montana
Hesperocyparis_lusitanica
Hesperocyparis_forbesii
Hesperocyparis_glabra
Hesperocyparis_stephensonii
Hesperocyparis_guadalupensis
Hesperocyparis_benthamii
Hesperocyparis_arizonica
Hesperocyparis_macrocarpa
Hesperocyparis_sargentii
Hesperocyparis_goveniana
Hesperocyparis_nevadensis
Hesperocyparis_macnabiana
Hesperocyparis_bakeri
Callitropsis_nootkatensis
Xanthocyparis_vietnamensis
Cupressus_torulosa
Cupressus_cashmeriana
Cupressus_duclouxiana
Cupressus_dupreziana
Cupressus_chengiana
Cupressus_sempervirens
Cupressus_funebris
Microbiota_decussata
Platycladus_orientalis
Tetraclinis_articulata
Calocedrus_formosana
Calocedrus_macrolepis
Calocedrus_decurrens
Chamaecyparis_pisifera
Chamaecyparis_formosensis
Chamaecyparis_thyoides
Chamaecyparis_lawsoniana
Chamaecyparis_obtusa
Fokienia_hodginsii
Thuja_koraiensis
Thuja_sutchuensis
Thuja_standishii
Thuja_plicata
Thuja_occidentalis
Thujopsis_dolabrata
Actinostrobus_arenarius
Actinostrobus_pyramidalis
Neocallitropsis_pancheri
Callitris_macleayana
Callitris_endlicheri
Callitris_rhomboidea
Callitris_preissii
Actinostrobus_acuminatus
Widdringtonia_schwarzii
Widdringtonia_nodiflora
Widdringtonia_cedarbergensis
Fitzroya_cupressoides
Diselma_archeri
Libocedrus_austrocaledonica
Libocedrus_yateensis
Libocedrus_bidwillii
Pilgerodendron_uviferum
Libocedrus_plumosa
Austrocedrus_chilensis
Papuacedrus_papuana
Taxodium_mucronatum
Taxodium_distichum
Glyptostrobus_pensilis
Cryptomeria_japonica
Sequoiadendron_giganteum
Sequoia_sempervirens
Metasequoia_glyptostroboides
Athrotaxis_laxifolia
Athrotaxis_selaginoides
Athrotaxis_cupressoides
Taiwania_cryptomerioides
Cunninghamia_lanceolata
Cunninghamia_lanceolata_var__konishii
Cephalotaxus_lanceolata
Cephalotaxus_fortunei
Cephalotaxus_sinensis
Cephalotaxus_latifolia
Cephalotaxus_griffithii
Cephalotaxus_hainanensis
Cephalotaxus_mannii
Cephalotaxus_wilsoniana
Cephalotaxus_harringtonia
Cephalotaxus_oliveri
Cephalotaxus_koreana
Torreya_grandis
Torreya_nucifera
Torreya_californica
Torreya_taxifolia
Torreya_jackii
Amentotaxus_poilanei
Amentotaxus_formosana
Amentotaxus_yunnanensis
Amentotaxus_argotaenia
Taxus_baccata
Taxus_fuana
Taxus_canadensis
Taxus_cuspidata
Taxus_chinensis
Taxus_sumatrana
Taxus_wallichiana
Taxus_floridana
Taxus_globosa
Taxus_brevifolia
Pseudotaxus_chienii
Austrotaxus_spicata
Sciadopitys_verticillata
A
13
12
11
10
9
8
7
continued
on next page
Dev
400
Carb
350
Perm
300
Tri
Jur
250
200
150
Millions of Years Ago
Cret
100
Pal
50
Cupressoideae
Callitroideae
Early-Diverging
Cupressaceae
“Taxodiaceae”
Taxaceae
Sciadopityaceae
N
0
10
Podocarpus_chinensis
Podocarpus_neriifolius
Podocarpus_nakaii
Podocarpus_fasciculus
Podocarpus_chingianus
Podocarpus_macrophyllus
Podocarpus_annamiensis
Podocarpus_salomoniensis
Podocarpus_spathoides
Podocarpus_subtropicalis
Podocarpus_costalis
Podocarpus_brevifolius
Podocarpus_rubens
Podocarpus_pilgeri
Podocarpus_archboldii
Podocarpus_bracteatus
Podocarpus_affinis
Podocarpus_rotundus
Podocarpus_brassii
Podocarpus_elatus
Podocarpus_polystachyus
Podocarpus_grayae
Podocarpus_rumphii
Podocarpus_polyspermus
Podocarpus_sylvestris
Podocarpus_lucienii
Podocarpus_novae_caledoniae
Podocarpus_longifoliolatus
Podocarpus_spinulosus
Podocarpus_drouynianus
Podocarpus_dispermus
Podocarpus_coriaceus
Podocarpus_trinitensis
Podocarpus_costaricensis
Podocarpus_sellowii
Podocarpus_guatemalensis
Podocarpus_matudae
Podocarpus_angustifolius
Podocarpus_aristulatus
Podocarpus_purdieanus
Podocarpus_urbanii
Podocarpus_hispaniolensis
Podocarpus_oleifolius
Podocarpus_latifolius
Podocarpus_milanjianus
Podocarpus_elongatus
Podocarpus_henkelii
Podocarpus_salignus
Podocarpus_nubigenus
Podocarpus_parlatorei
Podocarpus_lambertii
Podocarpus_acutifolius
Podocarpus_alpinus
Podocarpus_cunninghamii
Podocarpus_totara
Podocarpus_lawrencii
Podocarpus_gnidioides
Podocarpus_nivalis
Nageia_formosensis
Nageia_nagi
Nageia_fleuryi
Nageia_wallichiana
Afrocarpus_mannii
Afrocarpus_usambarensis
Afrocarpus_gracilior
Afrocarpus_falcatus
Retrophyllum_comptonii
Retrophyllum_minor
Retrophyllum_vitiense
Retrophyllum_rospigliosii
Dacrydium_nidulum
Dacrydium_nausoriense
Dacrydium_balansae
Dacrydium_guillauminii
Dacrydium_araucarioides
Dacrydium_lycopodioides
Dacrydium_xanthandrum
Dacrydium_magnum
Dacrydium_elatum
Dacrydium_beccarii
Dacrydium_cupressinum
Falcatifolium_taxoides
Falcatifolium_falciforme
Dacrycarpus_kinabaluensis
Dacrycarpus_cinctus
Dacrycarpus_compactus
Dacrycarpus_dacrydioides
Dacrycarpus_imbricatus
Dacrycarpus_veillardii
Acmopyle_pancheri
Acmopyle_sahniana
Microstrobos_fitzgeraldii
Microstrobos_niphophilus
Microcachrys_tetragona
Saxegothaea_conspicua
Phyllocladus_toatoa
Phyllocladus_hypophyllus
Phyllocladus_asplenifolius
Phyllocladus_alpinus
Phyllocladus_trichomanoides
Lepidothamnus_laxifolius
Lepidothamnus_fonkii
Halocarpus_bidwillii
Halocarpus_kirkii
Halocarpus_biformis
Prumnopitys_ferruginoides
Prumnopitys_ferruginea
Prumnopitys_ladei
Prumnopitys_taxifolia
Prumnopitys_andina
Sundacarpus_amarus
Manoao_colensoi
Lagarostrobos_franklinii
Parasitaxus_usta
Araucaria_humboldtensis
Araucaria_laubenfelsii
Araucaria_rulei
Araucaria_luxurians
Araucaria_nemorosa
Araucaria_columnaris
Araucaria_schmidii
Araucaria_subulata
Araucaria_bernieri
Araucaria_scopulorum
Araucaria_montana
Araucaria_biramulata
Araucaria_muelleri
Araucaria_heterophylla
Araucaria_cunninghamii
Araucaria_araucana
Araucaria_angustifolia
Araucaria_hunsteinii
Araucaria_bidwillii
Agathis_dammara
Agathis_borneensis
Agathis_macrophylla
Agathis_silbae
Agathis_atropurpurea
Agathis_microstachya
Agathis_robusta
Agathis_corbassonii
Agathis_montana
Agathis_ovata
Agathis_moorei
Agathis_lanceolata
Agathis_australis
Wollemia_nobilis
continued
on previous
page
B
6
4
5
2
3
continued
on next page
Dev
400
Carb
350
Perm
300
Tri
Jur
250
200
150
Millions of Years Ago
Cret
100
Pal
50
Podocarpaceae
Araucariaceae
N
0
7
11
Pinus_palustris
Pinus_echinata
Pinus_occidentalis
Pinus_elliottii
Pinus_herrerae
Pinus_glabra
Pinus_pungens
Pinus_taeda
Pinus_rigida
Pinus_serotina
Pinus_praetermissa
Pinus_caribaea_var__caribaea
Pinus_oocarpa
Pinus_pringlei
Pinus_caribaea_var__hondurensis
Pinus_caribaea_var__bahamensis
Pinus_lawsonii
Pinus_cubensis
Pinus_leiophylla
Pinus_lumholtzii
Pinus_greggii
Pinus_teocote
Pinus_radiata
Pinus_attenuata
Pinus_muricata
Pinus_patula
Pinus_sabiniana
Pinus_torreyana
Pinus_ponderosa
Pinus_douglasiana
Pinus_coulteri
Pinus_durangensis
Pinus_jeffreyi
Pinus_devoniana
Pinus_engelmannii
Pinus_arizonica
Pinus_hartwegii
Pinus_pseudostrobus
Pinus_montezumae
Pinus_virginiana
Pinus_clausa
Pinus_contorta
Pinus_tabuliformis
Pinus_yunnanensis
Pinus_luchuensis
Pinus_densata
Pinus_kesiya
Pinus_taiwanensis
Pinus_sylvestris
Pinus_densiflora
Pinus_nigra
Pinus_uncinata
Pinus_mugo
Pinus_massoniana
Pinus_hwangshanensis
Pinus_tropicalis
Pinus_resinosa
Pinus_merkusii
Pinus_halepensis
Pinus_brutia
Pinus_roxburghii
Pinus_pinea
Pinus_pinaster
Pinus_canariensis
Pinus_heldreichii
Pinus_armandii
Pinus_morrisonicola
Pinus_fenzeliana
Pinus_bhutanica
Pinus_pumila
Pinus_dalatensis
Pinus_wallichiana
Pinus_sibirica
Pinus_koraiensis
Pinus_lambertiana
Pinus_albicaulis
Pinus_parviflora
Pinus_monticola
Pinus_strobus
Pinus_ayacahuite
Pinus_flexilis
Pinus_strobiformis
Pinus_chiapensis
Pinus_peuce
Pinus_krempfii
Pinus_bungeana
Pinus_gerardiana
Pinus_squamata
Pinus_cembroides
Pinus_remota
Pinus_culminicola
Pinus_johannis
Pinus_edulis
Pinus_rzedowskii
Pinus_pinceana
Pinus_maximartinezii
Pinus_monophylla
Pinus_quadrifolia
Pinus_aristata
Pinus_longaeva
Pinus_balfouriana
Pinus_nelsonii
Picea_jezoensis
Picea_crassifolia
Picea_koraiensis
Picea_torano
Picea_meyeri
Picea_retroflexa
Picea_asperata
Picea_obovata
Picea_abies
Picea_koyamae
Picea_omorika
Picea_mariana
Picea_rubens
Picea_glehnii
Picea_wilsonii
Picea_purpurea
Picea_morrisonicola
Picea_brachytyla
Picea_spinulosa
Picea_farreri
Picea_likiangensis
Picea_schrenkiana
Picea_chihuahuana
Picea_alcoquiana
Picea_orientalis
Picea_maximowiczii
Picea_smithiana
Picea_pungens
Picea_engelmannii
Picea_glauca
Picea_sitchensis
Picea_breweriana
Cathaya_argyrophylla
Larix_occidentalis
Larix_decidua
Larix_gmelinii
Larix_griffithiana
Larix_kaempferi
Larix_laricina
Larix_potaninii
Pseudotsuga_menziesii
Pseudotsuga_sinensis
Pseudotsuga_japonica
Pseudotsuga_macrocarpa
Abies_fraseri
Abies_lasiocarpa
Abies_koreana
Abies_nephrolepis
Abies_fabri
Abies_fargesii
Abies_holophylla
Abies_kawakamii
Abies_sibirica
Abies_nordmanniana
Abies_alba
Abies_numidica
Abies_pinsapo
Abies_nebrodensis
Abies_grandis
Abies_concolor
Abies_amabilis
Abies_procera
Abies_magnifica
Abies_homolepis
Abies_veitchii
Abies_sachalinensis
Abies_firma
Abies_mariesii
Abies_bracteata
Keteleeria_davidiana
Keteleeria_evelyniana
Tsuga_dumosa
Tsuga_forrestii
Tsuga_sieboldii
Tsuga_chinensis
Tsuga_caroliniana
Tsuga_diversifolia
Tsuga_canadensis
Tsuga_heterophylla
Tsuga_mertensiana
Nothotsuga_longibracteata
Pseudolarix_amabilis
Cedrus_atlantica
Cedrus_deodara
Cedrus_libani
Zamia
Encephalartos
Cycas
continued
on previous
page
C
1
14
15
16
Dev
400
Carb
350
Perm
300
Tri
Jur
250
200
150
Millions of Years Ago
Cret
100
Pal
50
Pinaceae
Cycads
N
0
12
Summary of Calibration Points and Priors
The age ranges (in My) shown represent the 95% confidence intervals for the 20
My lognormal prior. Figures given in parentheses are for the 40 My prior. See text
below for more detailed information concerning these age ranges, particularly for the
divergences (2. Araucariaceae – Podocarpaceae, 6. Podocarpus – Retrophyllum, 16.
Tsuga – Nothotsuga) in which the typical 20 and 40 My prior age distributions were
modified.
1. Conifer divergence. Uniform prior: 275-350 My
2. Araucariaceae - Podocarpaceae divergence. Lognormal prior: 176-230 My
3. Araucaria – (Wollemia + Agathis) divergence. Lognormal prior: 165-185 (205) My
4. Dacrycarpus – (Falcatifolium+Dacrydium) divergence. Lognormal prior: 51.9-71.9
(91.9) My
5. Phyllocladus – Lepidothamnus divergence. Lognormal prior: 48-68 (88) My
6. Podocarpus – Retrophyllum divergence. Lognormal prior: 28-60 (80) My
7. Taxaceae – Cupressaceae divergence. Lognormal prior: 197- 217 (237) My
8. Metasequoia – (Sequoia+Sequoiadendron) divergence. Lognormal prior: 55-75 (95)
My
9. Taxodium – Glyptostrobus divergence. Lognormal prior: 65-85 (105) My
10. Papuacedrus – (other Callitroids) divergence. Lognormal prior: 51.9-71.9 (91.9) My
11. Thuja – Thujopsis divergence. Lognormal prior: 58-78 (98) My
12. Tetraclinis – (Platycladus+Microbiota) divergence. Lognormal prior: 28-48 (68) My
13. Juniperus – Cupressus sensu lato divergence. Lognormal prior: 33-53 (73) My
14. Picea- Cathaya divergence. Lognormal prior: 133-153 (173) My
15. Larix – Pseudotsuga divergence. Lognormal prior: 41-61 (81) My
16. Tsuga – Nothotsuga divergence. Lognormal prior: 41-90 (100) My
13
Details and Discussion of Calibration Points and Priors
1. Conifer divergence
Fossils: Upper bound based on megasporophylls that are morphologically similar to
extant Cycas from the uppermost Lower Permian of China (Crossozamia spp. ~275 My;
9). Lower bound based on the appearance of ovules with a fully fused integument similar
to that of all living seed plant groups in the Lower Carboniferous (350 My; 10).
Prior: Uniform; 275-350 My
Discussion: Extant cycads are often thought to be related to medullosan seed ferns (11),
suggesting that the first appearance of medullosans in the fossil record (Quaestora; Late
Mississippian ~320 My; 12) could be used as a minimum age for the cycad-conifer split.
However, recent cladistic analyses have not generally supported a close relationship,
suggesting instead that lyginopterid and medullosan seed ferns form an early grade of
seed plants no more closely related to living cycads than other extant groups (13-15). In
this context, the first appearance of megasporophylls that are morphologically similar to
those of extant Cycas (Early Permian ~275 My; 9) are here used as the minimum age for
the conifer – cycad divergence. The first appearance of conifers or conifer-like plants in
the Pennsylvanian is not used as the minimum age because pollination and fertilization in
these plants is unlike modern conifers and is more similar to primitive seed plant groups
(for example, they were most likely zooidogamous). This may suggest that these plants
are not closely related to modern conifers (see 13), even though Paleozoic conifers have
been linked with living conifers based on other shared aspects of their vegetative and
reproductive morphology as well as in more recent phylogenetic analyses (14,15). The
first appearance of derived conifer or conifer-like traits could potentially be used as the
minimum age for the divergence of living conifers from other groups of extant seed
plants, but they fall within the uniform prior employed here. Saccate pollen grains
similar those produced by younger Paleozoic conifer and conifer-like macrofossils
(Florinites, Potoniesporites) enter the fossil record in the Serpukovian (~326 My; 16).
Diverse saccate pollen, including bisaccate forms characteristic of extant conifers but also
many other groups of extinct gymnosperms, are known from the early Bashkirian (~317
My; 17) and may suggest early conifers were present for some time before their
recognition as macrofossils.
In light of the uncertainty regarding the first appearance of early conifers and their
exact phylogenetic placement, we extend the maximum age of the cycad-conifer
divergence to the earliest evidence of seed plants with a fused integument, a trait shared
by all living groups but that is not present in the earliest seed plants. This feature appears
as early as the Tournasian in the lowermost Carboniferous (~350 My; see 10), and was
also used by Crisp and Cook (18) to set the lower boundary of the divergence of extant
seed plant groups. We use this date so that we do not potentially bias our analyses
toward younger dates for this divergence, but there is no fossil evidence from either
palynology or reproductive morphology that suggests early members belonging to the
five living seed plant groups were present before the Serpukovian (~326 My). Early
14
ovules with a fully fused integument also retain morphological features suggesting a
more primitive pollination biology that is not shared with any living gymnosperm,
including tetrahedral division of the megaspore mother cell, an elaborate nucellar pollen
chamber for receiving pollen, and the hydrasperman-type pollination biology
characteristic of primitive seed plants (see 15).
2. Araucariaceae – Podocarpaceae divergence
Fossils: Presence of Araucariacites pollen grains in conjunction with the earliest
macrofossils putatively assigned to Araucariaceae (Araucarites rudicula; Late Carnian,
~230 My; 19). Unambiguous evidence of Araucariaceae by the Aalenian (172-176 My)
based on Araucaria-like ovulate cones and scales (Araucarites phillipsii) as well as
pollen cones (attached to Brachyphyllum mammilare foliage) containing pollen
comparable to Araucariacites and living Araucariaceae (20).
Prior: Lognormal; 172-230 My (95% confidence intervals)
Discussion: Several Middle Triassic fossils have been considered to represent early
Podocarpaceae, and have been used to date the split between this family and the
Araucariaceae (see excluded fossils below). However, fossils with ovulate cones that are
comparable to those of living Podocarpaceae do not enter the record until the Middle
Jurassic (20) and the earliest appearance of unambiguous Podocarpaceae is not until the
Early Cretaceous (e.g., 21), well after the earliest appearance of unambiguous
Araucariaceae. Therefore, we use the more definitive early record of the Araucariaceae
to constrain the divergence of these two families.
Pollen grains similar to those of modern Araucariaceae (Araucariacites) are first
described from the Early Triassic (22), and are also known from the Middle-Late Triassic
of Patagonia (23) and the Late Triassic of Europe (24). Although Araucariacites and
similar palynomorphs therefore have a substantial Triassic record, so far they have not
been found associated with reproductive structures until the Middle Jurassic. In general,
Araucariacites (and the potential araucarian grain Inaperturopollenites, which is also
applied to pollen grains of Cupressaceae sensu lato) are similar to the pollen of living
Araucariaceae in that they are relatively large, non-saccate, and have a granular surface
ornamentation. However, these are not sufficiently definitive characters to securely infer
the presence of either stem or crown group Araucariaceae.
The earliest potential macrofossils of Araucariaceae consists of ovulate cones and
isolated scales preserved as compression or impression fossils in several Late Triassic
macrofloras (19, 25, 26). However, due to their poor preservation, it is difficult to assign
them unambiguously to the Araucariaceae. The isolated scales are usually broadly
triangular and bear a single large ovule or seed, although they lack evidence of an ovule
scale, or ligule, which is characteristic of the extant genus Araucaria (although not the
extant genera Wollemia or Agathis). The earliest dated occurrence of these of triangular,
non-ligulate scales (Araucarites rudicula; 19) is used to define the lower 95% confidence
interval on our lognormal prior age distribution in order to allow for the possibility that
15
these specimens are related to extant Araucariaceae. These fossils have been dated to the
late Carnian– middle Norian (~230-213 My) by palynology and detrital zircons (27, 28).
However, we do not consider scales of this kind to be unambiguous evidence of the
family because they differ in their overall morphology from unambiguous Middle
Jurassic Araucariaceae (a ligule is lacking and they have relatively small ovules with a
highly constricted scale base), their anatomy is not preserved, and they lack co-occurring
foliage or palynomorphs that would help support placement in the family.
We set minimum age for the Podocarpaceae – Araucariaceae split by the first
appearance of fossils we consider to be unambiguous evidence of stem or crown
Araucariaceae (Araucarites phillipsii cone and associated Brachyphyllum mammilare
foliage with attached pollen cones) from the Aalenian (172-176 My). Harris (20)
considered these fossils to belong to the same plant based on their common cooccurrence and similar cuticular morphology, and both could independently be assigned
to the Araucariaceae based on their morphology. Pollen cones attached to
Brachyphyllum mammilare foliage produced relatively large, non-saccate pollen
comparable to modern Araucaria (29) and the foliage also contains oval sclereids similar
to those found in extant Araucaria cunninghamiii. Araucarites phillipsii seed cones
consist of broad cone scales in which the ovule scale appears to be fused to the bract for
most of its length but is separated at the tip. The material is not permineralized but the
ovule or seed is interpreted as being covered by cone scale tissue as in extant
Araucariaceae. Due to the preservation, it is not possible to definitely assign this species
to a living section of Araucaria, but it has been linked to Section Eutacta (20; 30). Using
the minimum age of 172 My, in this instance we use a lognormal prior of 58 My to
accommodate the possibility that the late Carnian – middle Norian fossils are related to
extant Araucariaceae (see above).
The Brachphyllum – Araucarites phillipsii plant may represent definitive
evidence of the divergence of Araucaria based on the wingless seed (Agathis and
Wollemia seeds are winged) and the presence of a free ligule tip (Agathis and Wollemia
lack the ligule tip and their ovule scale is completely fused to the bract). However, since
these fossils are not anatomically preserved and because the exact sequence of character
changes occurring in ovulate scale evolution in early Araucariaceae are not entirely clear
(see also below), we do not use it to date the Araucaria – (Agathis+Wollemia)
divergence.
3. Araucaria – (Wollemia + Agathis) divergence
Fossils: Araucaria sphaerocarpa (31) from the Inferior Oolite at Bruton Station,
Somerset, UK. At this locality, the Inferior Oolite has yielded ammonites suggesting a
Middle Bathonian age, although they do not appear to have been recovered from the
limestone where this species was found. Deposition of the Upper Inferior Oolite in
southern England ends at the very beginning of the Bathonian (32), so it is likely that the
deposits yielding A. sphaerocarpa span the Bajocian-Bathonian boundary (33). We
therefore set the minimum age at the end of the Bathonian (165 My).
16
Prior: Lognormal; 165-185 (205) My (95% confidence intervals)
Discussion: Araucaria sphaerocarpa is known from a large cone embedded in marine
limestone. It is assigned to Araucariaceae based on its large size, its expanded cone axis,
cone scales with single ovules, and the presence of branched zig-zag sclereids in the seed
sclerotesta. Assignment to Araucaria is based on the wingless seed and a well-developed
ligule on the cone scale. Assignment to Section Bunya of extant Araucaria (containing
the living species A. bidwillii) is based on the woody winged bract, the complex
vascularization of the ovule scale (especially the complex vascular “plexus” at the
chalazal end of the ovule), and the separate vascular strands that supply the ovule scale
and the bract (all other living sections of Aracuaria have a single vascular trace for both).
Very similar characteristics of the ovulate scale are found in the slightly younger and
better preserved Araucaria mirabilis cones from Patagonia (34), although the errors
associated with dating the Patagonian localities (157 +/- 10 My) suggest these fossils
could be more or less contemporaneous with A. sphaerocarpa (35). The placement of
both A. sphaerocarpa and A. mirabilis in Section Bunya is strongly suggested by their
ovulate scale morphology, although molecular evidence does not place living Section
Bunya as sister to other Araucaria or to Araucariaceae, and the foliage and seedlings
associated with these early Araucariaceae (e.g., A. mirabilis) are not similar to those of
modern Section Bunya (30). The fossils could therefore be interpreted in two ways:
either Section Bunya has been present since the Middle Jurassic while evidence of its
sister taxon (Section Intermedia; Araucaria hunsteinii) only enters fossil record much
later in the Late Cretaceous (30), or that extant Section Bunya is a younger taxon that
preserves the pleisiomorphic ovulate features of Araucaria that have been lost
independently in the other living sections.
In an earlier version of this study, we implemented these interpretations and
explored their effects on our results. In one analysis, ages within the Araucariaceae were
not constrained (referred to as No Araucaria, No Bunya analysis) while in a second, A.
sphaerocarpa was used to date the divergence between extant Araucaria and its sister
clade consisting of extant Agathis and Wollemia (the Araucaria, No Bunya analysis).
Finally, A. sphaerocarpa was used to set the minimum age for the divergence between
extant Section Bunya (which includes the living species A. bidwillii) and its sister taxon,
Section Intermedia (which includes the living species A. hunsteinii). These different
calibration schemes had little overall impact on estimated divergences times beyond the
small clades directly affected (Table S4). Even within Araucariaceae, dating a crown
group divergence (the Section Intermedia and Section Bunya split) to the Middle Jurassic
did not greatly alter the median node age for the clade, as the other divergence time
estimates within it remained relatively unchanged. In this study, we chose to use A.
sphaerocarpa to date the first appearance of the Araucaria genus.
17
Median Node Age in Araucaria Calibration Schemes
Clade
Araucariaceae (SH)
Podocarpaceae (SH)
Callitroidea (SH)
Cupressoidea (NH)
Pinaceae (NH)
No Araucaria
No Bunya
Araucaria
No Bunya
Bunya
4.58
7.04
14.62
3.04 (4.34)
2.69 (4.17)
5.04
7.35
14.97
3.31 (4.92)
2.95 (4.40)
5.05
7.85
15.63
3.43 (5.13)
3.04 (4.73)
Table S4. Median node ages (in millions of years; My) for major Southern Hemisphere
(SH) and Northern Hemisphere (NH) clades using different calibration schemes based on
the interpretation of the Araucariaceae fossil record. Ages are derived from an earlier
version of the analyses whose results are not otherwise presented in this study. Ages in
parentheses represent the median value of 10,000 median node ages derived from a
resampling routine where NH clades were reduced to 50% species sampling.
4. Dacrycarpus – (Falcatifolium+Dacrydium) divergence
Fossils: Dacrycarpus puertae from the Laguna del Hunco Flora of Argentina (36),
radiometrically dated to the Early Eocene (51.9 My).
Prior: Lognormal; 51.9-71.9 (91.9) My (95% confidence intervals)
Discussion: This species is very similar in reproductive and vegetative morphology to
living Dacrycarpus, especially D. imbricatus. It includes foliage with attached pollen
cones and ovulate structures, which also exhibit the warty receptacle characteristic of the
genus. The fossil material differs from extant D. imbricatus only in a few continuous
variables, such as the size of bracts subtending the cones, but was assigned to a separate
species because not all aspects of the whole plant are known. We therefore use this fossil
conservatively to date the appearance of crown Dacrycarpus, rather than the divergence
between D. imbricatus and its sister species (a clade of D. kinabaluensis and D. cinctus).
5. Phyllocladus – Lepidothamnus divergence
Fossils: Phylloclades are described from the mid-Late Eocene (~37 My; 37), as well as
from the Early Eocene (48-55 My) of Tasmania (mentioned in Hill and Brodbribb (38).
Prior: Lognormal; 48-68 (88) My (95% confidence intervals)
Discussion: Phyllocladus has extremely distinctive, flattened photosynthetic shoots that
are unlike any other living conifer. Thus there is little doubt about the assignment of
fossils with similar foliage to the Phyllocladus lineage, and they are readily identifiable
without detailed cuticular or anatomical analysis. However, we treat these fossils as stem
taxa and do not attempt to place them within the crown because other features of these
18
plants are unknown and their relationship to particular extant species of Phyllocladus is
unclear.
6. Podocarpus – Retrophyllum divergence
Fossils: Retrophyllum australe from the West Dale Flora of southwestern Australia (39),
which is dated to the Middle Eocene to Oligocene (28-48 My).
Prior: Lognormal; 28-60 (80) My (95% confidence intervals)
Discussion: Retrophyllum has distinctive heterofacially flattened foliage, where leaves on
opposite sides of the branch axis twist in opposite directions; therefore the upper surface
of the leafy shoot is formed by the abaxial side of one leaf rank and the adaxial side of
the other. The same kind of heterofacially flattened foliage also occurs in Nageia and
Afrocarpus (which belong to the clade containing Retrophyllum), but these taxa lack the
prominent midvein seen in Retrophyllum. Retrophyllum australe is assigned to extant
Retrophyllum based on the presence of a midvein, its heterofacially flattened foliage, and
its cuticular morphology, which consists of straight epidermal cells, stomata on both sides
of the leaf, and prominent Florin rings. However, we do not use R. australe as
unambiguous evidence of Retrophyllum due to uncertainty in placing these fossils in the
crown group of the genus. Since Podocarpus also has a prominent midvein, this feature
most likely represents the pleisiomorphic condition in the
Retrophyllum/Afrocarpus/Nageia clade. However, we use R. australe to indicate the
minimum age for the appearance of the extant clade characterized by heterofacially
flattened foliage (the Retrophyllum+Afrocarpus+Nageia clade). Given the very poor
constraint on the age of this fossil we used wider priors than in most other calibrations
(32 and 52 My respectively).
7. Taxaceae – Cupressaceae divergence
Fossils: Palaeotaxus rediviva from the Upper coal bed of the Skromberga Colliery in
Scania, Sweden (40). This deposit has been dated to the Lowermost Jurassic (Hettangian,
201-197 My).
Prior: Lognormal; 197- 217 (237) My (95% confidence intervals)
Discussion: Palaeotaxus rediviva is similar to modern Taxaceae in having an axillary
short shoot bearing sterile scales that terminates in a single ovule. According to Florin
(40) the ovule is also surrounded by an aril, although this is difficult to see in the figured
specimens (see 41). Regardless of the presence or absence of the aril, the position and
form of the ovule strongly suggest this fossil belongs to the lineage that includes extant
Taxaceae, because no other conifer family produces reduced axillary ovulate cones with
single terminal ovules. Sterile scales on P. rediviva seed cones are helically arranged,
similar to extant Austrotaxus and Taxus (whereas Amentotaxus and Torreya have
decussate scales) and the leafy shoots of Palaeotaxus are also broadly similar to those of
some modern Taxaceae in having spirally arranged, bifacially flattened leaves with a
19
single midvein that form distichous rows on the shoot. Florin hesitated to include
Palaeotaxus within the modern family due to its unusual cuticular morphology,
particularly the wavy epidermal cell walls that are unlike any living genus. We therefore
treat Palaeotaxus as a minimum age estimate for the divergence between Taxaceae sensu
lato (including Cephalotaxus) and Cupressaceae sensu lato.
Depending on the topology of the tree, Palaeotaxus could be used to date the
Cephalotaxaceae – Taxaceae divergence, since its reduced ovulate cones would appear to
place it securely along the Taxaceae lineage. However, this interpretation requires that
the sister group relationship of Cephalotaxaceae and Taxaceae is secure and some
molecular data (including ours), suggest that Cephalotaxus is nested within genera
traditionally included in Taxaceae. Specifically, it is resolved as sister to a Torreya –
Amentotaxus clade in some analyses whereas in others it is resolved as sister to the Taxus
clade (42). In both these cases, the cone morphology of Cephalotaxus is interpreted
either a derived elaboration of an ancestrally reduced axis or a retention of the
pleisiomorphic condition which requires two separate clades (the Taxus clade and the
Torreya clade) have independently reduced the ovulate cone. Given the uncertainty in
the interpretation of the aril in Palaeotaxus, combined with the uncertainty of the
phylogenetic relationships between Cephalotaxus and other Taxaceae, it is not prudent to
use Palaeotaxus to date the Cephalotaxus/Taxaceae split. However, its extremely simple
ovulate cone, consisting of a single ovule, does at least provide a minimum age for the
divergence between Taxaceae broadly considered from the Cupressaceae sensu lato
clade.
8. Metasequoia – (Sequoia+Sequoiadendron) divergence
Fossils: Metasequoia occidentalis from the Wuyun Formation in China (43), dated to the
Paleocene (44)
Prior: Lognormal; 55-75 (95) My (95% confidence intervals)
Discussion: Metasequoia occidentalis is a whole plant reconstruction that is very similar
to modern Metasequoia in all major morphological features. Its leaves are arranged on
determinate short shoots with fascicled scale leaves at the base, as in modern
Metasequoia. Pollen cones are axillary on specialized fertile branches, subtended by
single bract, as in living Metasequoia. In contrast, Sequoia and Sequoiadendron produce
pollen cones terminally (or subterminally) on vegetative shoots. Additionally, in both
fossil and extant Metasequoia the seed cones have decussate cone scales, while the sister
clade comprised of Sequoia and Sequoiadendron have cones with helically arranged
scales as in other members of early-diverging Cupressaceae.
9. Taxodium – Glyptostrobus divergence
Fossils: Taxodium wallissii from the Horseshoe Canyon Formation, Alberta (45). These
deposits are dated to the Maastrichtian (70-65 My).
20
Prior: Lognormal; 65-85 (105) My (95% confidence intervals)
Discussion: Taxodium wallissii is a whole plant reconstruction that is similar to modern
Taxodium in all major morphological features. Pollen cones were produced in axillary
clusters on branches (which also terminate in a cone), similar to the fertile branches in
extant Taxodium. In contrast, Glyptostrobus (sister genus to Taxodium) has pollen cones
that are borne terminally on vegetative shoots. Taxodium wallissii is also similar to
modern Taxodium in its seeds, which are unwinged and prominently three angled; a
morphology that is unique to Taxodium among living conifers.
10. Papuacedrus – (other Callitroids) divergence
Fossils: Papuacedrus prechilensis from the Laguna del Hunco and Rio Pichileufu floras,
Argentina (46), radiometrically dated to the Early to Middle Eocene (51.9-47.5 My).
Prior: Lognormal; 51.9-71.9 (91.9) My (95% confidence interval)
Discussion: Papuacedrus prechilensis has leafy shoots bearing foliage indistinguishable
from the characteristic dimorphic transitional foliage of extant Papuacedrus. The foliage
leaves also have stomata in separate rows that are not confined to medial strips, which is
also characteristic of Papuacedrus to the exclusion of related genera such as
Austrocedrus and Libocedrus. The seed cone of P. prechilensis is also similar to extant
Papuacedrus, with two pairs of highly dimorphic valvate scales. Characteristically, the
bract apex is at the center of a long shield-like scale. Among related genera, Libocedrus
has a much longer pointed bract apex and the apex of Austrocedrus is at the margin of the
scale.
11. Thuja – Thujopsis divergence
Fossils: Thuja polaris from the Strand Bay Formation - Iceberg Bay Formation of the
Eureka Sound Group on Ellesmere Island, dated to the Middle Paleocene (47).
Prior: Lognormal; 58-78 (98) My (95% confidence intervals)
Discussion: Thuja polaris has leaflets that are broad and flattened in regular splays,
unlike closely related Thujopsis which has similar, but irregular splays. Cones of T.
polaris are similar to those of extant Thuja in having long thin scales with reflexed
umbos, and a central columella-like structure formed from two connate apical scales. In
contrast, Thujopsis cones scales are more robust and lack the columella-like structure.
We therefore interpret T. polaris as evidence for a minimum age of the Thuja-Thujopsis
divergence.
12. Tetraclinis – (Platycladus+Microbiota) divergence
Fossils: Tetraclinis salicornoides from the Lost Creek Locality of the Bridge Creek Flora
(48), which is considered to be early Oligocene in age (28-33 My; 49).
21
Prior: Lognormal; 28-48 (68) My (95% confidence intervals)
Discussion: Tetraclinis salicornoides is known from fossil foliage, seeds, and seed cones.
Its seed cones are extremely similar to extant Tetraclinis, and have two pairs of
dimorphic, valvate scales with cordate bases. Fossil seeds also have large symmetric
wings, similar to living Tetraclinis. Tetraclinis foliage (T. brachyodon) is described from
European sediments as far back as the Eocene, although floras with preserved cones are
not known until the Oligocene (48). We therefore conservatively consider the Oligocene
to be the earliest unambiguous appearance for the stem group of extant Tetraclinis.
13. Juniperus – Cupressus sensu lato divergence
Fossils: Juniperus pauli from the Ústí Formation, Czech Republic, dated to the
Eocene/Oligocene boundary (~33 My; 50).
Prior: Lognormal; 33-53 (73) My (95% confidence intervals)
Discussion: Juniperus pauli has the characteristic fibrous spherical cones and the
relatively large, wingless seeds with resin scars that are typical of extant Juniperus.
Kvaček (50) assigns J. paulii to extant section Sabina based on its cuticular characters,
which include weakly cutinized Florin rings and indistinct or absent distal papillae on the
adaxial leaf side. However, a more comprehensive analysis of the evolution of cuticular
features Juniperus would be necessary in order to determine whether they can be used to
place this fossil within crown Juniperus, or whether it represents a stem member. We
therefore consider this fossil as unambiguous evidence of Juniperus, but do not place it
within crown Juniperus or use it to date sectional splits within the genus.
14. Picea- Cathaya divergence
Fossils: Picea burtonii from the Apple Bay locality, Vancouver Island, British Columbia
(51), dated to the Valanginian Stage of the Early Cretaceous (~140-133 My)
Prior: Lognormal; 133-153 (173) My (95% confidence intervals)
Discussion: This seed cone specimen shares a number of morphological and anatomical
characteristics with extant Picea, especially in regards to the precise distribution and
branching pattern of resin canals in the ovule scale. A cladistic analysis including fossil
and living seed cone specimens resolved this fossil as more closely related to extant
Picea than Cathaya (51), leading the authors to suggest that this fossil can be used to date
the divergence of these two lineages. Based on their analysis, we follow this
interpretation.
22
15. Larix – Pseudotsuga divergence
Fossils: Larix altoborealis from the Buchanan Lake formation, Axel Heiberg Island (52),
dated to the Middle Eocene (41-47 My).
Prior: Lognormal; 41-61 (81) My (95% confidence intervals)
Discussion: Fossil Larix altoborealis is known from foliage, shoots, and attached seed
cones that are virtually identical to modern Larix. As in extant species in the genus, L.
altoborealis branches have short shoots bearing leaves and upright cones. Closely related
Pseudotsuga does not produce short shoots and has ovulate scales with much longer
bracts.
16. Tsuga – Nothotsuga divergence
Fossils: Tsuga swedaea from the Buchanan Lake formation, Axel Heiberg Island (53),
dated to the Middle Eocene (41-47 My).
Prior: Lognormal; 41-100 My (95% confidence intervals)
Discussion: Tsuga swedaea is known from well-preserved mummified seed cones.
These seed cones are not distinguishable from Nothotsuga based on morphology, since
both taxa have numerous scales and well-developed bracts. However, non-saccate pollen
similar to extant Tsuga canadensis is known from this formation (53). Since Nothotsuga
pollen has more developed sacci than most Tsuga species (except T. mertensiana), the
presence of Tsuga-like pollen suggests that the split between Nothotsuga and Tsuga had
occurred by the Middle Eocene. This pollen morphology (Tsugapollenites) is also known
earlier in the fossil record, from Paleocene and perhaps even Late Cretaceous deposits
(see summary in 53). These records suggest Tsuga may have a deeper history, but these
grains are not associated with any diagnostic foliar or cone macrofossils and we therefore
regard them as insufficient for an unambiguous indicator of the presence of Tsuga clade.
Therefore, we use T. swedaea to set the minimum age for the calibration, but extend the
95% confidence intervals of the prior age distribution to the beginning of the Late
Cretaceous (~100 My) to include Late Cretaceous Tsuga-like pollen grains that may have
been produced by members of the Tsuga lineage.
Fossils not used in our analyses
In this section, we discuss fossils that were initially considered for their potential
to date divergences within conifers but were ultimately not used. Generally, these fossils
were excluded because we felt there was too much uncertainty regarding their
phylogenetic placement, particularly in terms of whether or not the fossil taxa represented
stem or crown members of the clades of interest. However, these fossils merit discussion
because many have been used in previous analyses to date divergences, or because they
could be particularly useful for dating divergences if more were known about them or
their systematic placement. The use of these fossils may result in older divergence time
23
estimates in some cases, because they often represent ambiguous early occurrences of
clades or particular traits. This is not universally true, however; the first unambiguous
fossil appearance of cone morphology indicative of Cupressaceae sensu stricto
(Widdringtonia americana at ~95 My; 54) postdates the estimated divergence time for
the clade when this fossil information is not used in the analysis.
Wollemia-like foliage and pollen
Fossils: Dilwynites pollen is known from the Turonian of Australia (89-93 My) and
leaves very similar to modern Wollemia are known from the Cenomanian (93-99 My)
Winton flora from Queensland (55).
Potential calibration node: Wollemia – Agathis divergence
Discussion: Fossil Dilwynites pollen is similar to that of modern Wollemia in the
thickness of the exine and the pronounced granular ornamentation, whereas modern
Araucaria and Agathis pollen have a thinner exine and a less granular surface. While
some fossil leaves assigned to Wollemia are very similar to the extant genus, they show a
range of variation that exceeds that of extant Wollemia. The seed cone scales of both
Agathis and Wollemia are somewhat similar in their lack of a ligule, making the
assignment of associated ovulate scales to either lineage difficult. The Winton flora also
contains Wollemia-like pollen cones, although these are also not diagnostic for the genus.
Given the uncertainties in the interpretation of these fossils and their disarticulated
nature, it is not unambiguously clear whether they represent crown members of the
Wollemia – Agathis clade or stem members predating the divergence between the extant
genera. While the pollen evidence is suggestive that the Wollemia lineage had diverged
by the Turonian, we decided not to base a calibration point on this evidence because the
systematic and taxonomic importance of the characters in which Wollemia and other
Araucariaceae differ are not clear.
Notophytum krauselii
Fossils: Stems and attached leaves from the Fremouw Formation, Antartica (56), which is
dated to the Early Middle Triassic (Anisian; 241-245 My)
Potential calibration node: Podocarpaceae-Araucariaceae divergence
Discussion: Notophytum krauselii consists of multiveined leaves attached to stems that
are referred Podocarpaceae on the basis of the abundant sclereids, the position of
transfusion tissue, and the presence of resin canals below the vascular bundle. Large
multi-vein leaves of this kind are characteristic of some extant Podocarpaceae (as well as
Agathis in the Araucariaceae), and this fossil has been used to date the Podocarpaceae Araucariaceae divergence (57). However, among extant Podocarpaceae multi-vein
leaves are characteristic of derived taxa with a relatively recent divergence (Nageia; see
58) and their presence in Early Triassic members seems unlikely. Furthermore, recent
studies have linked Notophytum foliage with Parasciadopitys cones (58), which are not
24
similar morphologically to known members of Podocarpaceae. Notophytum foliage may
represent the permineralized form of Heidiphyllum compression foliage, a widespread
Gondwanan leaf that is associated with a compression seed cone (Telemachus) also
unlike modern Podocarpaceae (58). Notophytum fossils therefore may belong to an
extinct conifer group whose relationships have not been currently resolved. We therefore
did not use these fossils to calibrate the Podocarpaceae – Araucariaceae split.
Rissikia plant
Fossils: Leafs, shoots, and associated ovulate and pollen cones from the Late Triassic
(Carnian; 228-235 My) Molteno Formation in South Africa (59, 60)
Potential calibration prior: Podocarpaceae – Araucariaceae divergence
Discussion: The reconstructed Rissikia plant consists of several species that include
associated foliage (Rissikia), pollen cones (Rissikianthus), and ovulate cones
(Rissikistrobus) that are believed to belong to the same living plant based on shared
cuticular features, attachment, and association evidence. Taken separately, any particular
feature or organ is not diagnostic of Podocarpaceae, but the plant has been assigned to the
family when considered as a whole.
Rissikia leaves are similar to some extant Podocarpaceae in that they are single
veined, bifacially flattened, and rotated into a plane, although some other conifer groups
such as Taxaceae also show these features. Rissikia foliage also has a short shoot/long
shoot arrangement that is similar to the extant Podocarpaceae genera Acmopyle and
Dacrycarpus. Rissikianthus pollen cones are attached to branches with scale leaves and
have two unfused microsporangia containing bisaccate pollen grains. This is morphology
is similar to extant Podocarpaceae, although these features are not strictly diagnostic of
the family. Several living and extinct conifer groups produce bisaccate pollen and have
pollen cones with two microsporangia per microsporophyll. The pollen grains
themselves are also faintly striate, a feature not seen in any extant group of conifers.
The ovulate cone structure is significantly different than that of extant
Podocarpaceae. Townrow (59) interpreted the ovules are being covered by an
epimatium, but based on the figured specimens the evidence for this structure is not
completely clear. The basic unit of the Rissikia cones consists of two ovules borne on a
small scale that is subtended by subtended unfused bract; these units are then often
clustered groups of three which are helically arranged around an axis. Since these
ovulate cones are very different from modern Podocarpaceae (as well as extant
Araucariaceae and extant conifers in general) we do not consider these fossils to
represent unambiguous evidence of the appearance of Podocarpaceae by the Late
Triassic.
25
Parasciadopitys aequata
Fossils: Permineralized cone from Fremouw Formation, Antartica (61), which is dated to
the Early Middle Triassic (Anisian; 241-245 My)
Potential calibration node: Cupressaceae sensu lato – Taxaceae divergence
Discussion: Parasciadopitys aequata was originally assigned to the Cupressaceae sensu
lato (“Taxodiaceae”) based on its general similarity in morphology and vasculature to
Sciadopitys (which was considered to be included in Cupressaceae sensu lato at the time).
However, the cone is not readily distinguishable from other fossil ovulate cones
considered to belong to the more primitive voltzialean conifer group – such as
Aethophyllum, Telemachus, and Swedenborgia. More recent work (58) compares
Parasciadopitys cones to those of Telemachus, which was previously considered to be an
early member of Podocarpaceae. This suggests these fossils may belong to an extinct
group with combinations of features seen in several different extant groups. For
example, Parasciadopitys and Telemachus, that have ovule scales comparable to those of
Cupressaceae, are associated with multiveined “podocarpaceous” leaves (Notophytum
and Heidiphyllum respectively; see above discussion of Notophytum). Without additional
evidence from pollen or pollen cones we cannot assign these species definitively to
Cupressaceae.
Widdringtonia americana
Fossils: Widdringtonia americana from the Tuscaloosa Formation in Alabama (54),
dated to the Cenomanian (~95 My).
Prior: Cupressaceae sensu stricto – Taxodioid clade of Cupressaceae sensu lato
divergence
Discussion: McIver (54) assigns leafy shoots and attached cones to extant Widdringtonia
based on overall similarity in the seed cones. While it is true that the fossil has 4
decussate, valvate cone scales as in extant Widdringtonia, this is also seen in several
unrelated genera of Cupressaceae, including Callitropsis, Diselma, and Tetraclinis. In
addition, the fossil cones of W. americana do appear more similar to Widdringtonia than
other extant genera, but they are significantly smaller than living species and also lack
their characteristic warty protuberances. Molecular evidence from this and other studies
also suggests Widdringtonia is a relatively recently derived genus nested within the
callitroid clade and the modern geographical range in southern Africa, where there are
four species, is far removed from these North American localities. Together, the lack of
diagnostic synapomorphies, the disjunction in geographical range between fossil and
living species, and the much greater age for the Cupressaceae as a whole implied by a
derived taxon such as Widdringtonia appearing at 95 My, suggests this fossil may not
belong to the modern genus. However, its decussate scale leaves and 4-valved cone do
provide strong evidence of a conifer that clearly belongs within the lineage of extant
Cupressaceae sensu stricto. However, in light of the older age for the Cupressaceae
26
sensu stricto found by Mao et al. (62) compared to our initial analyses in which we used
W. americana to calibrate this divergence, we chose not to use this calibration point in
order to more directly compare results between the two studies.
Chamaecyparis corpulenta
Fossils: Leafy shoots with attached seed cones from the Late Cretaceous (Santonian; 8385 My) Comox Formation, Vancouver Island, British Columbia (63)
Potential calibration node: Chamaecyparis – Fokienia divergence
Discussion: Chamaecyparis corpulenta was assigned to extant Chamaecyparis based on
very small, spherical seed cones with pairs of peltate, interlocking decussate scales.
However, seed cones of modern Chamaecyparis species have more than the four scales
present in these fossils. Although extremely small seed cones are unique to
Chamaecyparis among extant Cupressaceae, the architecture of the leafy shoots of this
fossil species differs from that of extant Chamaecyparis (or any other Northern
Hemisphere species of Cupressaceae) in that is has both alternate and opposite branches.
Opposite branching is seen today only in some Southern Hemisphere genera such as
Papuacedrus. This difference in branching architecture raises the possibility that C.
corpulenta is not within the crown Chamaecyparis–Fokienia clade, although the
importance of opposite versus alternate branching as a phylogenetic character in
Cupressaceae requires more study. Because of the uncertainties regarding the diagnostic
value of the foliage characters and because C. corpulenta also lacks strongly diagnostic
seed cone features (other than its small size), further studies are needed before the
relationships of this species to extant Chamaecyparis can be demonstrated securely.
Fokienia ravenscragensis
Fossils: Leafy shoots with attached seed cones from the Early Paleocene (65.5-61.7 My)
Ravenscrag Formation, Saskatchewan, Canada (64).
Potential calibration node: Chamaecyparis – Fokienia
Discussion: Fossil Fokienia ravenscragensis was assigned to extant Fokienia based on
the structure of their ovulate seed cones, which are similar to those of the extant genus
although they are smaller and bear fewer scales. As is also true of Chamaecyparis
corpulenta, the opposite branching of this species also differs from extant Fokienia and
other Northern Hemisphere Cupressaceae. While the ovulate cones of F.
ravenscragensis are similar to those of Fokienia, they are also generally similar to seed
cones of many Cupressaceae and lack strongly diagnostic features. Some phylogenetic
analyses (although not those conducted in this study) suggest extant Fokienia is nested
within Chamaecyparis, and therefore that this fossil (in conjunction with Chamaecyparis
corpulenta) could be used to date the presence of crown Chamaecyparis in the fossil
record (as in 62). However, given the lack of unique diagnostic features in Fokienia
ravenscragensis, their relatively small size compared to those of modern taxa, and the
27
differences in branching between the fossils and living taxa, we think it is premature to
use F. ravenscragensis to calibrate the Fokienia-Chamaecyparis split until more is
known about it.
Pseudolarix erensis
Fossils: Shoots and cone scales from Mongolia, dated to the Late Jurassic or Early
Cretaceous (65)
Potential calibration node: Pseudolarix – Tsuga divergence
Discussion: This fossil material includes short shoots, leaves, and scales that appear
similar to extant Pseudolarix (65, 66). They were also used by Gernandt et al. (67) as
one of two potential calibration schemes for the first appearance of the Pinaceae.
However, the stratigraphy, and therefore the dating, of these localities is not clear.
Krassilov (65) thought they were similar in age to the Tsagan Tsab Formation, which he
considered Early Cretaceous but which has been radiometrically dated to the Oxfordian,
~156 My at some localities (68). However, Krassilov provides no direct evidence that
that deposits containing Pseudolarix are coeval with the Tsagan Tsab. These deposits
also contain apparent angiosperm seeds and foliage, raising further questions about a Late
Jurassic date for these deposits. Therefore, these fossils were not used in the analysis
even though their morphology suggests they are similar to extant Pseudolarix.
Pinus belgica
Fossils: Permineralized seed cone, probably from the Early Cretaceous (145-89 My)
Wealden Formation, Belgium (69)
Potential calibration node: Pinus – (Picea+Cathaya) divergence
Discussion: The morphology and anatomy of this specimen unambiguously places it
within Pinus. However, its provenance is uncertain and it is attributed to the Wealden
Formation based on adhering particles and its general type of preservation (69). This is
especially problematic because the Belgian Wealden has a wide range of possible dates,
spanning 127-89 My (67, 70). It is therefore possible that this cone is actually similar in
age to the first appearance of Pinus that is well placed stratigraphically – Pinus mutoi in
the Coniacian (71). Due to the uncertainties in the provenance of the cone and its age, it
was not used in the analyses.
Pinus hokkaidoensis
Fossils: Permineralized needles from the Upper Yezo Group, Hokkaido (72) dated to the
Late Cretaceous (Santonian, 83-85 My).
Potential calibration node: Pinus Subsection Strobus – Pinus Subsection Pinus
divergence
28
Discussion: Pinus hokkaidoensis possesses needles with two vascular strands, a feature
unique to Pinus Subsection Pinus among extant Pinaceae, and could be used to indicate
divergence of the two major Pinus subsections had occurred by the Late Cretaceous. In
addition to this fossil, needles with two vascular bundles are known from SantonianConiacian of Massachusetts (73) and wood with features indicative of Pinus subsection
Strobus (nondentate ray tracheids) also occurs by the Santonian (74). However, in the
Eocene Princeton Chert, cones of Pinus arnoldii show with a mixture of Subsection
Pinus characters (pronounced umbos, inflated scales, and sclerified outer cortex in the
cone axis) and Subsection Strobus characters (cylindrical cones). These cones are
strongly associated with single-bundled needles, suggesting to Miller (75) that the
Pinus/Strobus divergence had not occurred by the Eocene. More recently, the P. arnoldii
plant has been fully reconstructed (76), further strengthening the relationship between
subsection Pinus-like cones and Strobus-like leaves. Because the fossil evidence does
not resolve when the split between these groups occurred, and because fossil taxa exist
with intermediate character combinations, Pinus hokkaidoensis was not used to calibrate
the Pinus/Strobus split.
Abies deweyensis
Fossils: Twigs and seeds from the Thunder Mountain Flora, Idaho (77), radiometrically
dated to the Middle Eocene (45.5 My).
Potential calibration node: Abies – Keteleeria divergence
Discussion: Abies deweyensis twigs are similar to extant Abies in their characteristic
circular leaf scars and leaves with expanded bases, although circular leaf scars are also
found in closely related extant Keteleeria. Seeds present in the Thunder Mountain Flora
are also similar to extant Abies, in the slight marginal lobe of the seed wing where it
connects to the seed. This material was used by Gernandt et al. (67) to date the Abies
divergence. However, given the fragmentary nature of the material generally and the
lack of articulated cones we do not use it to date this split.
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