ORIGINAL RESEARCH ARTICLE
published: 03 December 2014
doi: 10.3389/fgene.2014.00362
Patterns of diversification amongst tropical regions
compared: a case study in Sapotaceae
Kate E. Armstrong 1,2,3*, Graham N. Stone 2 , James A. Nicholls 2 , Eugenio Valderrama 2,3 ,
Arne A. Anderberg 4 , Jenny Smedmark 5 , Laurent Gautier 6 , Yamama Naciri 6 , Richard Milne 7 and
James E. Richardson 3,8
1
2
3
4
5
6
7
8
The New York Botanical Garden, Bronx, NY, USA
Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, Scotland
Royal Botanic Garden Edinburgh, Edinburgh, Scotland
Naturhistoriska Riksmuseet, Stockholm, Sweden
University Museum of Bergen, Bergen, Norway
Conservatoire et Jardin botaniques, Genève, Switzerland
Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh, Scotland
Laboratorio de Botánica y Sistemática, Universidad de los Andes, Bogotá DC, Colombia
Edited by:
Marshall Abrams, University of
Alabama at Birmingham, USA
Reviewed by:
Marcial Escudero, Doñana Biological
Station - Consejo Superior de
Investigaciones Científicas, Spain
Ze-Long Nie, Chinese Academy of
Sciences, China
*Correspondence:
Kate E. Armstrong, The New York
Botanical Garden, 2900 Southern
Boulevard, Bronx, NY 10458, USA
e-mail: karmstrong@nybg.org
Species diversity is unequally distributed across the globe, with the greatest concentration
occurring in the tropics. Even within the tropics, there are significant differences in the
numbers of taxa found in each continental region. Manilkara is a pantropical genus of
trees in the Sapotaceae comprising c. 78 species. Its distribution allows for biogeographic
investigation and testing of whether rates of diversification differ amongst tropical
regions. The age and geographical origin of Manilkara are inferred to determine whether
Gondwanan break-up, boreotropical migration or long distance dispersal have shaped
its current disjunct distribution. Diversification rates through time are also analyzed to
determine whether the timing and tempo of speciation on each continent coincides with
geoclimatic events. Bayesian analyses of nuclear (ITS) and plastid (rpl32-trnL, rps16-trnK ,
and trnS-trnFM) sequences were used to reconstruct a species level phylogeny of
Manilkara and related genera in the tribe Mimusopeae. Analyses of the nuclear data using
a fossil-calibrated relaxed molecular clock indicate that Manilkara evolved 32–29 million
years ago (Mya) in Africa. Lineages within the genus dispersed to the Neotropics 26–18
Mya and to Asia 28–15 Mya. Higher speciation rates are found in the Neotropical Manilkara
clade than in either African or Asian clades. Dating of regional diversification correlates
with known palaeoclimatic events. In South America, the divergence between Atlantic
coastal forest and Amazonian clades coincides with the formation of drier Cerrado and
Caatinga habitats between them. In Africa diversification coincides with Tertiary cycles of
aridification and uplift of the east African plateaux. In Southeast Asia dispersal may have
been limited by the relatively recent emergence of land in New Guinea and islands further
east c. 10 Mya.
Keywords: Sapotaceae, Manilkara, pantropical, biogeography, diversification rates
INTRODUCTION
Biodiversity is unevenly distributed across the globe and is most
intensely concentrated in the tropics, particularly in wet tropical
forests, which are the most species-rich biomes on the planet.
Even within the tropics, there are significant differences in the
floristic composition and the numbers of taxa found in each of the
continental regions. It is estimated that there are c. 27,000 species
of flowering plants in tropical Africa (Lebrun, 2001; Lebrun
and Stork, 2003), compared with c. 90,000 for South America
(Thomas, 1999) and c. 50,000 for Southeast Asia (Whitmore,
1998). This uneven species diversity raises the fundamental question of how variation in the pattern and tempo of speciation
and extinction among continents might have driven observed
patterns. Differences in diversity have been attributed to higher
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extinction rates in Africa (Richards, 1973) and faster diversification in the Neotropics (Gentry, 1982). Dated molecular phylogenies suggest speciation in response to recent climatic changes
(such as aridification, e.g., Couvreur et al., 2008; Simon et al.,
2009) or geological phenomena (such as mountain uplift in the
Neotropics, e.g., Richardson et al., 2001; Hughes and Eastwood,
2006).
Intercontinental disjunctions in distribution between tropical
regions of Africa, Asia and South America have been attributed
to Gondwanan break-up (Raven and Axelrod, 1974), and/or
the degradation of the boreotropical flora (e.g., Malpighiacaeae,
Davis et al., 2002b; Meliaceae, Muellner et al., 2006; Moraceae,
Zerega et al., 2005). However, current studies have shown that
many tropical groups are of more recent origin (e.g., Begonia,
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Armstrong et al.
Thomas et al., 2012), and that long distance dispersal has been
an important factor in determining the composition of modern
tropical floras (Pennington et al., 2006; Christenhusz and Chase,
2013). While long-distance dispersal could have occurred at any
time, it was generally believed to be the only viable explanation
for tropical intercontinental disjunctions younger than c. 33 Mya
(although see Zhou et al., 2012).
Pantropically distributed taxa are excellent models for studying the evolution of tropical forests and regional variation in
diversification rates between continents. Manilkara is a genus
of trees in the Sapotaceae comprising c. 78 species distributed
throughout the tropics (30 in South and Central America, 35
in Africa and 13 in Southeast Asia). This even spread and relatively low number of species across major tropical regions
makes Manilkara an excellent candidate for comparison of
regional diversification patterns and testing of hypotheses for
the genesis of pantropical distributions. Here a near specieslevel dated phylogeny of Manilkara is presented. If the distribution of the genus can be explained by Gondwanan break
up, the timing of phylogenetic splits would be expected to
reflect that break up 165–70 Mya (McLoughlin, 2001). Similarly
if splits resulted from the degradation of the boreotropical flora, they would be expected to occur as temperatures
cooled following the Early Eocene Climatic Optimum/Paleocene–
Eocene Thermal Maximum (EECO/PETM), 50–55 Mya (Zachos
et al., 2001). Additionally, a boreotropical origin should leave
a phylogeographic signature in the form of southern lineages being nested within more northern ones. Therefore,
lineages in South America or to the east of Wallace’s Line
would be nested within Laurasian lineages, resulting in the
pattern one would expect from a retreat of the boreotropical flora from the Northern Hemisphere. The onset of glaciation from 33 Mya induced further global cooling (Zachos
et al., 2001) and the disintegration of the boreotropical flora.
Therefore, ages of splits younger than c. 33 Mya would most
likely be explained by long distance dispersal. The prediction
advanced by Gentry (1982) that diversification rates in the
Neotropics have been higher than in other tropical regions is also
tested.
MATERIALS AND METHODS
DNA EXTRACTION, PCR, SEQUENCING, AND ALIGNMENT
Evolutionary relationships were reconstructed using nuclear
(ITS) and plastid (rpl32-trnL, rps16-trnK, and trnS-trnFM)
sequences. Divergence times were calculated using an ITS dataset
with 171 accessions of Sapotaceae. In total 53 of the global total
of 79 Manilkara species (67%) were included in the analysis.
The dataset includes representatives of the tribe Mimusopeae as
well as multiple representatives of the tribes Isonandreae and
Sideroxyleae, which also belong to the subfamily Sapotoideae,
in order to accommodate calibration of fossils related to those
groups. The tree was rooted using Sarcosperma, shown in previous studies to be sister to the rest of the family (Anderberg
and Swenson, 2003). The plastid dataset comprised 95 accessions of subtribe Manilkarinae, as well as outgroups in subtribe Mimusopinae, plus Northia, Inhambanella, Eberhardtia,
and Sarcosperma, which provided the root for the tree. See
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Diversification amongst tropical regions compared
Supplementary Table 1 for the list of taxa with voucher specimen
information and GenBank accession numbers.
Total DNA was extracted from herbarium specimens and silica gel-dried leaf samples using the Qiagen Plant DNeasy Mini Kit
following the manufacturer’s instructions. Amplifications of the
ITS region were performed using the ITS5p/ITS8p/ITS2g/ITS3p
(Möller and Cronk, 1997) and ITS1/ITS4 (White et al., 1990)
primer pairs. Polymerase chain reaction (PCR) was carried out
in 25-µL volume reactions containing 1 µL of genomic DNA,
5.75 µL sterile distilled water, 2.5 µL 2 mM dNTPs, 2.5 µL 10x
NH4 reaction buffer, 1.25 µL 25 mM MgCl2 , 0.75 µL of each
10 µM primer, 10 µL 5 M betaine, 0.25 µL BSA and 0.25 µL of
5 u/µL Biotaq DNA polymerase buffer. The thermal cycling profile consisted of 5 min denaturation at 95◦ C, followed by 35 cycles
of 30 s at 95◦ C for denaturation, 50◦ C for 30 s for annealing
and 72◦ C for 1 min and 30 s for extension with a final extension period of 8 min at 72◦ C on a Tetrad2 BioRad DNA Engine.
Extraction from herbarium specimens often yielded low amounts
of degraded DNA and required nested PCR to amplify quantities sufficient for sequencing. In nested PCR the ITS5/ITS8
primer pair was used in the first reaction. 1 µl of this PCR
product was then used in a second PCR with the ITS1/ITS4
primer pair and the same thermocycling profile. Further internal primers, ITS2g and ITS3p, were used in place of ITS1 and
ITS4 when amplification using the latter primers was unsuccessful. Plastid markers were amplified using rpl32-trnL (Shaw et al.,
2007), rps16-trnK (Shaw et al., 2007), and trnS-trnFM (Demesure
et al., 1995) primer pairs as well as Manilkara-specific internal primers designed for this study (Supplementary Table 2).
PCR was carried out in 25 µL volume reactions containing 1 µL
of genomic DNA, 15.25 µL sterile distilled water, 2.5 µL 2 mM
dNTPs, 2.5 µL 10x NH4 reaction buffer, 1.25 µL 25 mM MgCl2 ,
0.75 µL of each 10 µM primer, 0.8 µL BSA and 0.2 µL of 5 u/µL
Biotaq DNA polymerase buffer. All plastid regions were amplified
using the rpl16 program of Shaw et al. (2005). Nested PCR was
also performed on selected accessions using self-designed internal
primers (Supplementary Table 2). PCR products were purified
using Exo-SAP (GE Healthcare) according to the manufacturer’s
instructions.
Sequencing PCRs were carried out using the BigDye
Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems) and
were purified and sequenced on an ABI 3730 sequencer at the
University of Edinburgh’s GenePool facility. Forward and reverse
sequences were assembled into contiguous sequences (contigs)
and edited using the alignment software Sequencher ver. 4.7.
Edited contigs were assembled and aligned by eye in MacClade
ver. 4.08 (Maddison and Maddison, 2008) and later in BioEdit
ver. 7.0.5 (Hall, 2005).
Potentially informative indels in the plastid dataset were coded
according to the simple indel coding method of Simmons and
Ochoterena (2000). Ambiguous alignment regions 113–118 and
380–459 in rps16-trnK were excluded. Indel events in ITS were
so frequent that their coding as additional characters was deemed
to be too ambiguous. Gaps were treated as missing data and all
characters were equally weighted.
The ITS dataset was partitioned into three segments: ITS1
(372 bp), 5.8 s (167 bp), and ITS2 (339 bp). Plastid regions and
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Armstrong et al.
their indels were retained as separate partitions: rpl32-trnL
(1130 bp + 26 indels), rps16-trnK (1134 bp + 21 indels), and
trnS-trnFM (999 bp + 13 indels).
PHYLOGENETIC ANALYSIS
Bayesian analyses were carried out using MrBayes 3.1
(Huelsenbeck and Ronquist, 2001). Two independent runs
of four Metropolis Coupled Monte Carlo Markov Chains
(MCMCMC) each (three heated and one cold) were run with
a temperature setting of 0.10 for 8,000,000 generations, which
was found to provide sufficient mixing between chains and
convergence between runs. Trees were sampled every 8000
generations and a 10% burn-in was removed from the sampled
set of trees leaving a final sample of 900 trees, which were used to
produce a majority rule consensus tree. Convergence of models
was determined to have occurred when the standard deviation
of split frequencies for two runs reached 0.01 (Ronquist et al.,
2005). Appropriate burn-in and model convergence were checked
by visual confirmation of parameter convergence of traces in
Tracer v.1.5 (Rambaut and Drummond, 2009). Clade support
values are posterior probabilities (pp); pp values of 100–95% are
taken to indicate strong support, values of 94–90% moderate
support, and values between 89 and 55% weak support for nodes,
respectively. The output tree files were visualized in FigTree
v.1.3.1. The majority rule consensus tree was used to determine
the monophyly of key clades used to define calibration points in
the dating analysis.
Plastid data were not included in the subsequent BEAST analysis because they were not informative enough to discern between
alternative hypotheses and because fewer taxa were sampled.
Additionally, hard incongruence was demonstrated between the
topologies reconstructed in MrBayes from the nuclear and plastid
datasets (see Supplementary Material Section on chloroplast capture, and Supplementary Figure 1). Therefore, the two datasets
were not combined and only nuclear data was used for divergence
time analysis.
FOSSIL CALIBRATION
Sideroxyleae pollen from the Ypresian (47.8–56 Mya) of England
(Gruas-Cavagnetto, 1976) was used to constrain the minimum
age of the Sideroxyleae stem node (node B in Figure 1). A log
normal prior was used to constrain the age of this node (offset: 52.2 Ma, mean: 0.001). A mean of 0.001 was chosen so that
95% of the probability is contained in an interval between the
midpoint and the upper boundary of the Ypresian (52.2–55.6
Mya). A Mid-Eocene (37.2–48.6 Mya) Tetracolporpollenites pollen
grain from the Isle of Wight was used to constrain the minimum age of the node for the tribe Mimusopeae. This pollen grain
was described by Harley (1991) and determined to closely resemble Tieghemella heckelii (a monotypic genus in the Mimusopeae).
Harley suggested (pers. comm. 2010) that it would be appropriate to err on the side of caution with the identification and use
the fossil to constrain the age of the tribe Mimusopeae rather
than the genus itself. This fossil was, therefore, used to constrain
the age of the crown node of Mimusopeae (node D in Figure 1:
offset: 42.9 Mya, mean: 0.095). A mean of 0.095 was chosen so
that 95% of the probability was contained in an interval between
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Diversification amongst tropical regions compared
the midpoint (42.9) and the upper boundary of the mid Eocene
(42.9–48.6 Mya). The final calibration point is based on a series
of Oligocene (23–33.9 Mya) fossil leaves from Ethiopia (Jacobs
et al., 2005). Pan described these specimens as Sapoteae sp. and
suggested possible placement in either Manilkara or Tieghemella
(pers. comm. 2010) based on the occurrence of stoma surrounded
by fimbricate periclinal rings, a character present in these genera, but absent from the related genera Autranella and Mimusops.
Although they are both members of the Tribe Mimusopeae,
Manilkara and Tieghemella are not sister taxa, and placing the fossil at the node of the most recent common ancestor (the entire
Tribe Mimusopeae) seemed illogical for such a young date, when
a 45 Mya fossil pollen grain of cf. Tieghemella was a better fit for
the same node. Instead, the fossil was alternatively placed at the
Manilkara crown node (node Q in Figure 1) and on the node of
the split between Tieghemella and Autranella (node I in Figure 1),
in order to determine whether placement on either genus made a
significant difference to age estimates using a prior age estimate
with an offset of 28 Mya, mean: 0.1. A mean of 0.1 was chosen so that 95% of the probability was contained in an interval
between the midpoint and the upper boundary of the Oligocene
at (28-33.9 Mya).
DATING ANALYSIS
The software package BEAST v.1.7.5 (Drummond and Rambaut,
2007) was used to analyze divergence times in the ITS dataset. An
xml input file was created in BEAUti v.1.7.5. Substitution models were unlinked across partitions, but clock models and tree
topologies were kept on the linked default setting. Four taxon sets
per analysis were generated in order to define nodes for placement
of fossil calibration points. They were based on known monophyletic clades from previous analyses and were constrained to be
monophyletic.
The GTR + I + G model was applied to each partition. The
mean substitution rate was not fixed and base frequencies were
estimated. Following support for a molecular clock in these data
using MrBayes, an uncorrelated log-normal model was selected to
allow for relaxed clock rates and rate heterogeneity between lineages. A speciation: birth-death process tree prior was used with
a randomly generated starting tree. The most recent common
ancestor (MRCA) node age priors were set to define calibration
points using taxon sets. All other priors were left at default settings
that were either uniform or gamma-distributed. Posterior distributions for each parameter were estimated using MCMCMC run
for 40,000,000 generations, with parameters logged every 5000
generations, giving 8000 samples per run. The BEAUti xml file
was executed in BEAST v.1.7.5. Two separate analyses were run
and the output log files were reviewed in Tracer v.1.5 (Rambaut
and Drummond, 2009) to check for convergence between runs
and adequate effective sampling sizes (ESS) of >200 (Drummond
et al., 2007). The tree files from the two runs were combined
in LogCombiner v.1.7.5 (Drummond and Rambaut, 2007) with
a conservative burn-in of 4000 generations. The combined tree
files were input into TreeAnnotator v.1.5.3 (Drummond and
Rambaut, 2007). The Maximum Clade Credibility (MCC) tree
was selected with mean node heights; this option summarizes
the tree node height statistics from the posterior sample with
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Armstrong et al.
Diversification amongst tropical regions compared
Labramia costata
Labramia louvelii
Labramia mayottensis
Labramia ankaranaensis
81%
Labourdonnaisia calophylloides
30
Labourdonnaisia revoluta
L
10
Faucherea manongarivensis
O
Faucherea parvifolia
Labourdonnaisia madagascariensis
28
Faucherea thouvenotii
N
Faucherea sp
Manilkara dissecta
91%
15
Manilkara fasciculata
P
Manilkara udoido
16
Manilkara triflora
- 15
6 Ma
Ma
Manilkara staminodella
Ma
4M
26-18
T1
27-23 Ma
15
Manilkara zapota
5M a
a
9-4 M
15 Ma
Manilkara pleeana
82
T
a
22-9 M
a
Manilkara mayarensis
32-30 Ma
T2
T3 Manilkara sideroxylon
Manilkara chicle
Manilkara jamiqui
Manilkara gonavensis
18
Manilkara valenzuelana
S
Manilkara bidentata (a)
North America (Central America & Caribbean)
Manilkara bidentata (b)
71%
32
Manilkara bidentata (c)
South America
K
10 U1
Manilkara inundata
Africa
Manilkara cavalcantei (a)
96%
Manilkara huberi (a)
Madagascar
Manilkara huberi (b)
12
86%
Manilkara paraensis
U
Seychelles
Manilkara decrescens
26
R
Middle East
Manilkara rufula
10 U2
Manilkara maxima
South Asia
Manilkara elata
East Asia (including Indochina)
Manilkara cavalcantei (b)
Manilkara salzmanni
Sunda shelf (Malesia west of Wallace’s Line)
Manilkara bella
Manilkara subsericea
Sahul shelf (Malesia east of Wallace’s Line & Pacific Islands)
Manilkara longifolia
8
Manilkara obovata (b)
21
Manilkara lacera (a)
V
6
Manilkara concolor
Manilkara mochisia (a)
97%
Manilkara letouzei
Manilkara bequaertii
Manilkara capuronii
29
X3
Manilkara bovinii
Q
4
Manilkara perrieri
96%
Manilkara suarezensis
X1
Manilkara zenkeri
39
G
Manilkara multinervis
Manilkara pelligriniana
99%
Manilkara lososiana
Letestua durissima
15
Manilkara fouillayana
X
Manilkara mabokeensis
Manilkara welwitschii
99%
Manilkara koechlinii
Manilkara dawei
Manilkara discolor
X2
Manilkara sansibarensis
27
5
Manilkara sahafarensis
W
Manilkara butugi (b)
X4
97%
Manilkara cuneifolia
Manilkara hoshinoi
Manilkara vitiensis
Y2
Manilkara smithiana
Manilkara sp 1
23
43
Manilkara kauki
Y
D
Manilkara littoralis
Y1
52%
99%
Manilkara hexandra
Manilkara sp 2
31
Tieghemella heckelii
I
Autranella congolensis
Mimusops kummel
35
Mimusops obovata
H
Mimusops elengi
6
Mimusops comorensis
99%
J2 J3
22
Mimusops zeyheri
J
Mimusops caffra
C
97%
Mimusops coriacea
9
Mimusops sp
99%
Mimusops lecomtei
J1
Mimusops perrieri
Mimusops membranacea
Baillonella toxisperma
31
Vitellaria paradoxa
E
26
Vitellariopsis cuneata
Vitellariopsis dispar
2
Vitellariopsis marginata
F
Vitellariopsis kirkii
Isonandreae & Inhambanella
Capurodendron androyense
Northia seychellana
Xantolis/Englerophytum
62
Sideroxyleae
B
107
Eberhardtia tonkinensis
A
Eberhardtia aurata
Sarcosperma laurinum
6
75
70
Cretaceous
65
60
Paleocene
55
50
45
40
35
Eocene
FIGURE 1 | Maximum clade credibility chronogram of the ITS dataset.
Dashed lines indicate branches which lead to nodes with a posterior
probability of <0.95. Mean ages are given for profiled nodes. Node bars
indicate 95% HPD age ranges. Lettered nodes are discussed in the text.
Stars indicate the placement of fossils. Lineages are colored according to
their distribution: Yellow, Africa; Green, Madagascar; Blue, Asia; Pink,
South America; Orange, Central America and the Caribbean. Geological
Frontiers in Genetics | Evolutionary and Population Genetics
30
25
Oligocene
20
15
Miocene
10
M
5
0 MYA
Plio Ple
epochs are indicated in a scale at the bottom of the chronogram.
Outgroups have been reduced to gray bars at the base of the
chronogram. Ten regions were coded in the ancestral area reconstruction
as illustrated in the map and legend. Pie charts represent the percentage
likelihood of the ancestral state at the selected node. Map inset depicts
the timing and direction of long-distance dispersal events reflected in the
chronogram.
December 2014 | Volume 5 | Article 362 | 4
Armstrong et al.
the maximum sum of posterior probabilities. The output file was
visualized in FigTree v.1.3.1.
ANCESTRAL AREA RECONSTRUCTION IN RASP
Ancestral area states were reconstructed in RASP (Reconstruct
Ancestral State in Phylogenies; http://mnh.scu.edu.cn/soft/blog/
RASP) software that implements Bayesian Binary MCMC (BBM)
time-events curve analysis (Yu et al., 2011) and allows multiple
states to be assigned to terminals. BBM suggests possible ancestral ranges at each node and also calculates probabilities of each
ancestral range at nodes. The analysis was performed using the
MCC tree generated in BEAST as an input file, with 5,000,000
cycles, 10 chains, sampling every 100 cycles, with a temperature
setting of 0.1 and with the maximum number of areas set to four
for all nodes. The root node was defined a priori as Asian; because
the Asian taxa Sarcosperma and Eberhardtia form a grade within
which the rest of the family is nested, this is the most likely state
for the crown node of the family.
Areas are coded according to continent, based predominantly
on tectonic plate margins and then on floristic regions (Figure 1).
In Southeast Asia, the Sahul and Sunda Shelves (which mark the
boundary between continental Asia and Australia-New Guinea)
were coded as separate states within the Malesia floristic region,
which stretches from the Isthmus of Kra on the Malay Peninsula
to Fiji (Takhtajan, 1986; Van Welzen et al., 2005). East Asia is
defined as being east of the Himalayas and south as far as the
Malay Peninsula, with a predominantly Indo-Chinese flora. South
Asia is delineated by the margin of the Indian subcontinent.
The countries of Iran, Turkey and the Arabian Peninsula support a drier Irano-Turanian flora (Takhtajan, 1986) and were,
therefore, designated as being part of the Middle-Eastern region.
The remaining regions (the Seychelles, Madagascar, Africa and
North and South America) are all on separate continental tectonic plates and are floristically unique from one another (see
Supplementary Table 1 for species-specific area codes).
DIVERSIFICATION RATE METHODS
A separate ITS lineage through time (LTT) plot dataset (hereafter referred to as ITS LTT) was used to compare diversification rates within Manilkara. Because the genus was found to
be paraphyletic, with the Southeast Asian M. fasciculata clade
(P in Figure 1) being more closely related to Labourdonnaisia
and Faucherea, this small clade was excluded, leaving only the
monophyletic lineage of Manilkara s.s. (clade Q in Figure 1)
for analysis. Additionally, only one individual per species was
included. The simple diversification rate estimators of Kendall
(1949) and Moran (1951) were calculated for the African,
Neotropical and Asian clades, where the speciation rate lnSR =
[ln(N)−ln(N0 )]/T (N = standing diversity, N0 = initial diversity,
here taken as = 1, and T = inferred clade age). This is a pure-birth
model of diversification with a constant rate and no extinction (Magallón and Sanderson, 2001). Another model that does
not assume constant rates of speciation and extinction through
time within lineages was applied using BAMM (Bayesian Analysis
of Macroevolutionary Mixtures; Rabosky, 2014). BAMM uses
a reversible-jump Markov Chain Monte Carlo to explore shifts
between macroevolutionary regimes, assuming they occur across
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Diversification amongst tropical regions compared
the branches of a phylogenetic tree under a compound Poisson
process. Each regime consists of a time-varying speciation rate
(modeled with an exponential change function) and a constant
rate of extinction. The BAMM analysis used the BEAST MCC
tree, but because not all species were sampled, it was necessary
to specify to which lineage each of the missing taxa belonged (i.e.,
to which species it was most closely related based on morphological similarity). The results of the analysis with adjustments to
account for missing taxa were not different from those assuming complete taxon sampling. Two MCMC simulations were run
with 5,000,000 generations, sampling every 1000, and discarding
the first 10% as burn-in. Appropriate priors for the ITS LTT phylogeny, convergence of the runs and effective sampling size were
each estimated using the BAMMtools (Rabosky, 2014) package in
R (R development team).
LTT plots were generated using phytools (Revell, 2012) in R
for 1000 trees sampled through the post-burn-in (20%) posterior distribution generated by BEAST (see above for details). The
median and 95% highest posterior density (HPD) were estimated
for the ages of each number of lineages in each plot. To compare the observed LTT plots with the predictions of a model with
constant diversification rates, 1000 trees were simulated using
the mean speciation and extinction rates estimated by BAMM
in TreeSim (Stadler, 2011). Simulations used the age of the most
recent common ancestor of each of the 1000 observed trees and
the current number of species per plot. LTT plots were drawn
for the trees including all species of Manilkara s.s. and to examine region-specific patterns for pruned lineages that included only
those species from each of Africa, the Neotropics and Asia.
RESULTS
NODE AGES
Mean ages with 95% HPD confidence intervals for key nodes
are reported in Table 1. The MCC tree from the BEAST analysis
(Figure 1) resolves the mean crown age of the tribe Mimusopeae
as 43 Mya (HPD 44–42 Mya; node D), in the Mid Eocene. The
mean age of subtribe Manilkarinae is estimated to be 32 Mya
(HPD 36–29 Mya; node K) and the genus Manilkara is resolved
as 29 Mya (HPD 32–28 Mya; node Q), both having originated
during the Oligocene. Results also reveal that cladogenesis and
inter-continental dispersal (see below and Figures 1, 3) within
Manilkara occurred from the Oligocene through the Miocene—
and most intensively from the mid-late Miocene.
ANCESTRAL AREA RECONSTRUCTION AND INTERCONTINENTAL
DISPERSAL EVENTS
Ancestral area inferences and likelihood support are given in
Table 1 and Figure 1, which also indicates the age and direction of inferred dispersal events. The tribe Mimusopeae, subtribe Manilkarinae and the genera Manilkara, Labramia, and
Faucherea/Labourdonnaisia are all inferred to have African ancestry (Figure 1).
Following its origin in Africa during the Oligocene 32
Mya (HPD 36–29; node K) and subsequent diversification 29
Mya (HPD 32–28 Mya; node Q), Manilkara s.s. spread via
long distance dispersal to Madagascar twice, Asia once and
the Neotropics once during the Oligocene–Miocene. Both the
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Armstrong et al.
Diversification amongst tropical regions compared
Table 1 | Summary of clade support values, node ages and ancestral areas from Figure 1.
Node
Posterior
Clade
probability
Mean age and
Ancestral Area
95% HPD in Mya
(likelihood %)
East Asia 99
Epoch
A
1
Sapotaceae
107 (126−88)
B
1
Sideroxyleae
62 (73−52)
Africa 58
Cretaceous
Cretaceous-Paleocene
C
0.99
Isonandreae/Inhambanella/Mimusopeae
52 (58−48)
Africa 99
Paleocene-Eocene
Mimusopeae
43 (44−42)
Africa 99
Eocene
Baillonella/Vitellaria/Vitellariopsis
31 (39−23)
Africa 99
Eocene-Oligocene
Pliocene
D
1
E
0.99
F
0.99
Vitellariopsis
2 (4−0.5)
Africa 99
G
0.85
Mimusopeae subclade 1
39 (43−35)
Africa 99
Eocene
H
0.67
Mimusops/ Tieghemella/Autranella
35 (40−30)
Africa 99
Eocene-Oligocene
I
0.68
Tieghemella/Autranella
31 (38−23)
Africa 99
Eocene-Oligocene
J
0.99
Mimusops
22 (28−17)
Africa 97
Miocene
K
0.99
Manilkarinae
32 (36−29)
Africa 96
Eocene-Oligocene
L
0.44
Labr./Fauch./Labourd./ sm. Asian Manilkara
30 (35−26)
Madagascar 81
Eocene-Oligocene
M
0.99
Labramia
6 (10−3)
Madagascar 99
Miocene-Pliocene
N
0.92
Faucherea/Labourdonnaisia/Manilkara
28 (33−23)
Madagascar 91
Oligocene
O
0.99
Faucherea/Labourdonnaisia
10 (14−7)
Madagascar 99
Miocene-Pliocene
P
0.99
Small Asian Manilkara
15 (20−10)
Sahul Shelf 90
Miocene
Q
1
Manilkara s.s.
29 (32−28)
Africa 96
Oligocene
R
0.98
Manilkara s.s. subclade 1
26 (30−22)
Africa 86
Oligocene-Miocene
S
0.99
Neotropical Manilkara
18 (22−14)
South America 71
Miocene
T
0.90
Central American and Caribbean Manilkara
15 (20−13)
North America 95
Miocene
U
0.99
South American Manilkara s.s.
12 (16−9)
South America 93
Miocene
V
0.77
Small African Manilkara
21 (27−15)
Africa 97
Oligocene
W
0.99
Manilkara s.s. subclade 2
27 (30−23)
Africa 97
Oligocene
X
0.99
Large African Manilkara
15 (18−11)
Africa 99
Miocene
Y
0.99
Asian Manilkara s.s.
23 (27−19)
Sahul Shelf 52
Oligocene-Miocene
Faucherea/Labourdonnaisia/Manilkara clade (N) (28 Mya; HPD
33–23 Mya) and the genus Mimusops (clade J) (22 Mya; HPD
28–17 Mya) also exhibit a similar pattern, having originated in
Africa and later dispersed to both Madagascar and Asia during
the Miocene.
Long-distance dispersal from Africa to Madagascar and the
surrounding islands has occurred on multiple occasions in the
tribe Mimusopeae: twice in Manilkara s.s. (X3 and X4, 8–4 Mya);
at least once for the clade comprising Labramia, Faucherea, and
Labourdonnaisia between 32 Mya (HPD 36–29; node K) and 30
Mya (HPD 35–26 Mya; node L); and twice in Mimusops between
22 Mya (HPD 28–17 Mya; node J) and 9 Mya (HPD 13–5 Mya;
node J1), as well as 5 Mya (HPD 2–6 Mya; node J3).
The Neotropical Manilkara clade (S) is also derived from an
African ancestor, which dispersed to South America during the
Oligocene–Miocene between 26 Mya (HPD 30–22 Mya; node R)
and 18 Mya (HPD 22–14 Mya; node S). From South America,
further dispersal occurred to Central America 16–15 Mya and
throughout the Caribbean islands starting from 15 to 10 Mya.
Asia was reached by three independent dispersal events within
the tribe Mimusopeae. Manilkara s.s. reached Asia from Africa
between 27 Mya (HPD 30–23 Mya; node W) and 23 Mya (HPD
27–19 Mya; node Y), while Mimusops did the same 8–6 Mya
(node J2). The Manilkara fasciculata clade reached Asia from
Madagascar between 28 (HPD 33–23 Mya; node N) and 15 Mya
(HPD 20–10 Mya; node P).
Frontiers in Genetics | Evolutionary and Population Genetics
DIVERSIFICATION RATES
Net diversification rates (lnSR) differed somewhat between
regions, ranging from a lowest mean value of 0.06 (0.05–0.07) for
the Asian lineage, through 0.10 (0.09–0.10) for the African lineage
to a maximum of 0.15 (0.12–0.19) for the Neotropical lineage.
Despite sampling models with up to five different macroevolutionary regimes, BAMM analysis selected models without shifts
between macroevolutionary regimes along the Manilkara s.s. phylogeny, with the highest posterior probability obtained for zero
shifts models, i.e., a single, constantly varying net diversification
rate throughout the history of the genus (Figure 2). Bayes Factor
comparison, following the criteria of Kass and Raftery (1995)
provided unsubstantial support (1.68) for the zero shifts models over the models including shifts between macroevolutionary
regimes.
LTT plots are presented in Figure 3, for all regions (Figure 3D)
and for the pruned African, Asian and Neotropical lineages
(Figures 3A–C respectively). The figure shows both observed
rates, and rates predicted for the same numbers of lineages
evolving under a constant net diversification rate process (i.e.,
constant speciation and extinction rates, estimated using BAMM
for the genus s.s.). None of the observed LTT patterns diverge
significantly from those predicted assuming a constant diversification rate. The analyses including all Manilkara s.s. lineages
(Figure 3D) and only the Neotropical lineage (Figure 3C) both
show a good fit between observed patterns and those predicted
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Diversification amongst tropical regions compared
0.6
0.4
0.0
0.2
Posterior probability
0.8
1.0
Armstrong et al.
0
1
2
3
4
5
# Shifts between macroevolutionary regimes
FIGURE 2 | Posterior probability of models with different number of
shifts between macroevolutionary regimes considered in BAMM. The
best models for Manilkara s.s. indicate no significant shifts in diversification.
under a constant diversification rate. In contrast, African lineages
(Figure 3A) show a trend toward reduced diversification rates
from 25 to 12 Mya, followed by an increase in diversification rates
to levels matching those in the Neotropics from 12 Mya to the
present. The Asian lineage shows low and decreasing diversification rates toward the present. While the Asian pattern is derived
from just eight species, and thus any observed pattern must be
interpreted with caution, it is striking that Asia produced no new
lineages during the last 7 Mya, at a time when Africa and the
Neotropics were both showing rapid diversification.
DISCUSSION
ORIGIN OF MANILKARA
The tribe Mimusopeae evolved ∼52 Mya (HPD 58–48 Mya; node
C) and began to diversify 43 Mya (HPD 44–32 Mya; node D)
during the Eocene when global climates were warmer and wetter
and a megathermal flora occupied the northern hemisphere. This
age estimate also coincides with the first occurrence of putative
Mimusopeae fossils recorded from North America and Europe,
e.g., Tetracolporpollenites brevis (Taylor, 1989), and Manilkara
pollen (Frederiksen, 1980) in addition to the Tetracolporpollenites
sp., pollen grain (Harley, 1991), used in this study, which give
further weight to the hypothesis that the tribe Mimusopeae
was present in the boreotropics and may have originated there.
Previous studies (Smedmark and Anderberg, 2007) implicate the
break-up of the boreotropics in creating intercontinental disjunctions in the tribe Sideroxyleae and data from the present study
are consistent with this hypothesis. Smedmark and Anderberg’s
(2007) estimate for the age of Sideroxyleae was 68 Mya and in
this study the crown node age is reconstructed as being 62 Mya
(HPD 73–52 Mya; node B).
The subtribe Manilkarinae evolved 39 Mya (HPD 43–35
Mya; node G), consistent with the hypothesis that it arose late
during the existence of the boreotropics. Diversification began
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32 Mya (HPD 36–29 Mya; node K), around the time that
global cooling and the widening Atlantic were breaking up the
boreotropics. Hence migration toward the equator as the climate in the northern hemisphere cooled might have caused
or promoted diversification. This transition from the northern
hemisphere to equatorial latitudes is also reflected in the putative
Manilkarinae fossil record, where during the Oligocene, there is
still a strong representation of northern fossils [e.g., Isle of Wight,
UK (Machin, 1971), Vermont, USA (Traverse and Barghoorn,
1953; Traverse, 1955) and Czechoslovakia (Prakash et al., 1974)],
but fossils also begin to appear in Africa (e.g., Sapoteae sp.
leaves in Ethiopia, Jacobs et al., 2005). Further cooling and aridification during the Oligocene coincides with diversification of
Manilkarinae into genera and may have been a causal factor in this
diversification. Alternatively, Manilkarinae may have originated
in Africa, as suggested by the ancestral area analysis. However,
the analysis cannot account for southward climate shifts and the
modern absence of the group from higher latitudes.
Manilkara is nested within a grade of other representatives
of the tribe Mimusopeae, which is predominantly composed
of African taxa (Mimusops, Tieghemella, Autranella, Baillonella,
Vitellaria, and Vitellariopsis) and this suggests that the genus may
have had its origin there. In the ancestral area reconstruction both
Manilkara and the subtribe Manilkarinae are resolved as having a
96% likelihood of an African origin, and the tribe Mimusopeae is
reconstructed as having a 99% likelihood of originating in Africa.
As such, there is very strong support for an African ancestry for
the genus Manilkara, the subtribe Manilkarinae and the tribe
Mimusopeae.
THE ORIGIN OF MANILKARA’S PANTROPICAL DISTRIBUTION
Intercontinental disjunctions in Manilkara are too young (27–4
Mya) to have been caused by Gondwanan break-up, which would
have had to occur before 70 Mya. Manilkara is also too young for
its pantropical distribution to be the result of migration through
the boreotropics, which would have had to occur between 65 and
45 Mya, after which the climate would have been too cool for tropical taxa to cross the North Atlantic Land Bridge, even though
this might have persisted until ∼33 MYA (Milne and Abbott,
2002). The most likely period for migration of tropical taxa by
this route was during the PETM/EECO, 55–50 Mya (Zachos et al.,
2001). Furthermore, a boreotropical origin should leave a phylogeographic signature in the form of southern lineages being
nested within more northern ones. However, South American
lineages are not nested within Central American lineages, and
neither are those southeast of Wallace’s line nested within those
to the northwest. With these vicariance-based explanations not
supported, Manilkara’s disjunct pantropical distribution could
only have resulted from long-distance dispersal from Africa to
Madagascar, Asia and the Neotropics. This has been demonstrated for numerous other groups distributed across the tropics,
e.g., Begonia (Thomas et al., 2012) and Renealmia (Särkinen et al.,
2007).
Manilkara has fleshy, sweet fruit ranging in size from 1.5 to
10 cm, which are consumed by a wide variety of animals. With
seeds that are too bulky for wind dispersion, it is more likely
that long distance dispersal could have been achieved through
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Africa
20
Asia
5
N
10
10
1
1
2
2
5
N
Neotropics
15
10
5
0
35
50
C
20
Time
D
30
All lineages
15
10
5
0
15
10
5
0
Time
20
50
25
1
1
2
2
5
5
N
N
10
10
20
Number of lineages
B
20
A
50
Diversification amongst tropical regions compared
50
Armstrong et al.
35
30
10
Time
5
0
35
Time (Million years before present)
FIGURE 3 | LTT plots for lineages that included only those species
from each of Africa (A), Asia (B), the Neotropics (C), and all species
of Manilkara s.s. (D). Each plot shows the median and 95% HPD of the
ages for each number of lineages in solid and dashed lines, respectively.
The lines for observed trees are shown in blue and for the trees
transport in the gut-contents of birds or by transoceanic rafting in
large mats of vegetation. Houle’s (1998) study demonstrated that
during the Miocene, intercontinental rafting could have occurred
in less than 2 weeks on the North and South Equatorial currents.
REGIONAL DIVERSIFICATION IN MANILKARA
Within the Neotropics, Manilkara first colonized South America,
as indicated in the reconstruction of the ancestral distribution
of clade S. The South American clade (U) is divided into two
subclades, which correspond to contrasting regional ecologies,
with one clade (U1) comprised of Amazonian species and the
other (U2) of Atlantic coastal forest species. The only inconsistency in this geographic pattern is the second accession of
Manilkara cavalcantei (b), an Amazonian species that the analysis
places in the Atlantic coastal forest clade. However, in the plastid tree (Supplementary Figure 1) this accession is resolved in a
strongly supported (0.99 pp) Amazonian clade with M. bidentata,
M. huberi, and M. paraensis. The phylogenetic split between these
two regions occurred during the Mid-Miocene (12–10 Mya),
when the Andes were being elevated (Gregory-Wodzicki, 2000;
Graham, 2009) and drainage systems in the Amazon basin began
to shift eastwards.
Atlantic coastal species in clade U2 and Amazonian species
in clade U1 are geographically separated by the dry biomes of
Frontiers in Genetics | Evolutionary and Population Genetics
25
20
Time
simulated under a constant diversification process in red. The thinner
blue lines correspond to each of the 1000 observed trees. The 95%
HPD intervals show major overlap in all plots but non-significant patterns
suggest lower diversification rates in part of the histories of African and
Asian lineages.
the Cerrado and the Caatinga, as well as the higher relief of the
Brazilian shield. Simon et al. (2009) and Fritsch et al. (2004)
found that the origin of dry-adapted Cerrado Leguminosae and
Melastomataceae lineages span the Late Miocene to the Pliocene
(from 9.8 to 0.4 Mya), broadly coinciding with the expansion of
C4 grass-dominated savanna biomes. However, it is likely that a
dry environment would have been present just prior to this time
to allow for adaptation of these groups to the new biome. Such
timing is exhibited by the Microlicieae (Melastomataceae), where
the crown node is 9.8 Mya, and the stem node is 17 Mya (Fritsch
et al., 2004). Manihot (Euphorbiacae) species of this biome began
to diversify from 6.6 Mya (Chacón et al., 2008). Likewise, a phylogenetic study of Coursetia (Leguminosae) (Lavin, 2006) reveals
that species which inhabit the dry forest of the Brazilian Caatinga
are 5–10 My old. This suggests that the Cerrado and Caatinga
could have been in existence, at least in part, by the time the South
American Manilkara subclades U1 and U2 diverged ca.12 Mya,
and their development may have driven the geographical split in
this South American lineage of Manilkara.
The Central American/Caribbean clade (T) originated following dispersal from South America 16–15 Ma, and then split
geographically into a Central American subclade (T1, 6 Ma), and
a Caribbean subclade (T2, 11 Ma). The only exception to this
geographical structure is the single Central American species,
December 2014 | Volume 5 | Article 362 | 8
Armstrong et al.
M. chicle (T3), which is nested in the Caribbean clade, suggesting a Pliocene dispersal (2 Ma) back to the continent. These
age estimates place the New World spread of Manilkara prior to
the estimated age of the closing of the Isthmus of Panama ∼3.5
Ma (Coates and Obando, 1996), although recent studies (Farris
et al., 2011) indicate that the Isthmus may have closed much earlier, in which case Manilkara may have taken an overland route.
Overwater dispersal between Central and South America has been
demonstrated in numerous other plant taxa (Cody et al., 2010).
African Manilkara species are resolved in two clades, both of
which are Oligo-Miocene in age. The main African/Madagascan
clade (X) is estimated to be 15 My old (HPD 18–11 Mya), and
the smaller clade (V) is 21 My old (HPD 27–15 Mya). Africa
has been affected by widespread aridification during the Tertiary
(Coetzee, 1993; Morley, 2000). The response by Manilkara to
this changing climate could have been migration, adaptation
or extinction. A study of the rain forest genera Isolona and
Monodora (Annonaceae) found that throughout climatic cycles,
taxa remained in remnant pockets of wet forest (Couvreur et al.,
2008). They are, therefore, an example of a group that migrated
or changed its distribution to track wetter climates. Another study
of the genus Acridocarpus (Malpighiaceae) (Davis et al., 2002a)
indicated an east African dry forest adapted lineage nested within
a wet forest lineage. The dry adapted lineage was dated to periods of Oligo-Miocene aridification, and is, therefore, an example
of a wet forest lineage, which has adapted to changing environmental conditions rather than becoming restricted to areas
of favorable climate. The timing of diversification and evolution of dry-adapted species vs. wet-restricted species in the three
African Manilkara clades suggests a combination of both scenarios. The split between the African clades occurred between 29
Mya (HPD 32–28 Mya; node Q) and 26 Mya (HPD 30–22 Mya;
node R), during a period of dramatic continent-wide cooling,
which fragmented the Eocene coast to coast rain forest, potentially isolating the three lineages. A second wave of diversification
within the main African/Madagascan clade (X) coincides with the
Mid-Miocene climatic optimum 17–15 Mya, when global temperatures warmed (Zachos et al., 2001). During the same period the
collision of the African and Eurasian plates closed the Tethys Sea,
instigating further aridification. The resulting drier and warmer
climates caused the spread of savannas and the retraction of rain
forest, as evidenced by an increase in grass pollen during this
period (Morley, 2000; Jacobs, 2004). Nonetheless, cladogenesis
in the main African/Madagascan clade (X) gained pace from the
Mid-Miocene onwards. In particular, a third wave of diversification from rain forest into drier shrubland environments in eastern
and southern Africa occurred subsequent to the main uplift of the
Tanganyikan plateau in the East African Rift System ca. 10 Mya,
which had a significant impact on further regional aridification
(Lovett and Wasser, 1993; Sepulchre et al., 2006) (Table 1).
Clade X is predominantly composed of Guineo-Congolian
rain forest species. This is almost exclusively the case in subclade
X1, aside from the Madagascan taxa, which are also rain forest
species. However, within subclade X2, there is a transition from
wet to dry environments. The sole Madagascan taxon in this lineage (M. sahafarensis) is a dry, deciduous forest species. The four
dry, eastern-southern African taxa in subclade X2 (M. discolor,
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Diversification amongst tropical regions compared
M. sansibarensis, M. butugi, M. cuneifolia) all evolved between 8
and 5 Mya subsequent to the main uplift of the East African Rift
System. The ancestor of the smaller African clade composed of
M. mochisia and M. concolor also diversified into these two dryadapted eastern/southern species at the same time 6 Mya (HPD
10–2 Mya). Hence, some African Manilkara lineages adapted to a
drying climate, while others remained in their ancestral rain forest
habitat.
Within the main Asian clade of the plastid phylogeny (Yc1,
Supplementary Figure 1), the Indian species Manilkara roxburghiana is sister to the other species and the two Fijian species
are among the most derived, consistent with the hypothesis that
the founding dispersal event was from Africa to India with subsequent spread eastward into Malesia. However, ancestral area
reconstruction of the ITS data (node Y, Figure 1) suggests that
migration within Asia was from east to west (Sahul Shelf to Sunda
Shelf) 23 Mya (HPD 27–19 Mya). Dated phylogenies also indicate that many other angiosperm groups have crossed Wallace’s
Line from the late Miocene onwards: Pseuduvaria (Annonaceae)
(Su and Saunders, 2009), Aglaieae (Meliaceae) (Muellner et al.,
2008), at least four separate lineages of Begonia (Begoniaceae)
(Thomas et al., 2012) and Cyrtandra (Gesneriaceae) (Cronk et al.,
2005). In Sapotaceae four lineages of Isonandreae have migrated
from west to east across Wallace’s Line (Richardson et al., 2014),
whereas evidence from the tribe Chrysophylloideae suggests
recent movement in the opposite direction, from Sahul to Sunda
Shelf (Swenson et al., 2008). The two youngest (9 Mya) Asian
species (M. vitiensis and M. smithiana) are both Fijian. The oldest land available for colonization in Fiji is between 14 and 5 Mya
(Johnson, 1991; Heads, 2006) hence, the age of these two Fijian
taxa coincides with the first emergence of land in the archipelago.
DIVERSIFICATION RATES OF MANILKARA IN DIFFERENT PARTS OF THE
TROPICS
The BAMM analysis did not support significant rate variation
among lineages or regions in Manilkara s.s. Despite apparent variation in regional patterns revealed by LTT plots (Figure 3), the
data most strongly support a model with a single net diversification rate throughout the genus. Trends within the data for specific
regions only suggest departure from a constant rate model in Asia
and Africa. Given that observed patterns do not exceed the 95%
confidence intervals for the constant rate model for either region,
these trends must be considered with caution. This is particularly
true for Asia, for which the pattern was derived from only eight
species. Because sensitivity and statistical power of methods for
detection of shifts in diversification rates may correlate positively
with the number of species in the clade (Silvestro et al., 2011),
rate shifts in clades with a small number of species (as in Asia for
Manilkara s.s.) may not have been detected by the methods used
here (a potential type two error). A simulation study would be
required to examine the impact of taxon number on type two
error rates in these analyses. Similarly, small numbers of taxa
may be more likely to generate apparent trends through stochastic effects, and these could also generate the apparent two-phase
pattern of low, and then rapid, diversification in African lineages.
Taken at face value, net diversification rates and LTT plots
both suggest a trend for more rapid diversification in Neotropical
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Armstrong et al.
and African lineages than in Asian ones. The timing of rapid
Neotropical diversification falls within the time frame of Andean
uplift (i.e., from the late Miocene onwards), proposed as a diversification engine in many taxa (e.g., Richardson et al., 2001).
However, because many South American Manilkara species
are native to the Atlantic Forest, on the opposite side of the
continent from the Andes, Andean uplift may be considered
unlikely to directly explain high diversification rates region-wide.
Interestingly, the rapid diversification of the African lineage coincided with periods of regional aridification. The slowest diversification rate, in the Southeast Asian lineage, includes species that
are mostly to the east of Wallace’s Line. This may be explained
by the fact that the mountainous topography of much of this
region (dominated by New Guinea) limits the habitat available for
lineages such as Manilkara that are largely restricted to lowland
rain forest that covers a greater area of Africa or the Neotropics.
Although there is no statistical support for significant diversification rate variation in Manilkara s.s., the causes highlighted here
should have similar impacts on other lowland rainforest taxa—a
prediction that can be tested in future studies utilizing phylogenies of more species rich taxa and meta-analyses of multiple
unrelated lineages.
AUTHOR CONTRIBUTIONS
This paper is a result of Kate E. Armstrong’s Ph.D. thesis
research at the Royal Botanic Garden Edinburgh and University
of Edinburgh. Kate E. Armstrong and James E. Richardson conceived the study and Kate E. Armstrong carried out the research
and wrote the manuscript apart from the diversification rate analysis, which was conducted and written by Eugenio Valderrama.
James E. Richardson, Graham N. Stone, and Richard Milne
supervised the Ph.D. project. Graham N. Stone and James E.
Richardson edited the manuscript. James A. Nicholls assisted
with phylogenetic analyses. Arne A. Anderberg, Jenny Smedmark,
Laurent Gautier, and Yamama Naciri contributed DNA sequence
data to the study. All authors have reviewed the manuscript.
ACKNOWLEDGMENTS
This doctoral research was made possible through a scholarship from the Torrance Bequest at the University of Edinburgh.
Grants for fieldwork from the Royal Geographical Society, the
Carnegie Trust, the Systematics Association, and the Davis
Expedition Fund are also gratefully acknowledged. D. Ndiade
Bourobou (CENAREST, IRAF) is thanked for a DNA aliquot of
Baillonella toxisperma, and Jerome Chave (CNRS) is thanked for
ITS sequences of Manilkara bidentata and M. huberi. Thanks to
members of the Stone Lab at the University of Edinburgh for
comments on an earlier draft of the manuscript.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: http://www.frontiersin.org/journal/10.3389/fgene.
2014.00362/abstract
INCONGRUENCE BETWEEN NUCLEAR AND PLASTID TREES
Phylogenies generated with nuclear (Figure 1) and plastid data
(Supplementary Figure 1) showed high topological congruence.
However, there are a couple examples of hard incongruence
Frontiers in Genetics | Evolutionary and Population Genetics
Diversification amongst tropical regions compared
(strongly supported clades which conflict in their placement
between the two datasets), both of which have biogeographic
implications. The first is in the placement of the two Asian species
Manilkara hexandra and M. littoralis, and the two African species
M. mochisia and M. concolor. In the ITS phylogeny M. hexandra and M. littoralis are resolved in the Asian clade Y, while M.
mochisia and M. concolor are resolved in the small African clade
V. In contrast, in the plastid phylogeny, these four species form a
strongly supported clade (posterior probability 1), marked in
Supplementary Figure 1.
A second hard incongruence is apparent in the placement
of the three taxa Manilkara yangambensis, M. triflora, and M.
suarezensis. In the plastid phylogeny these form a monophyletic
clade Z (Supplementary Figure 1). In contrast, in the ITS analysis, the Brazilian M. triflora was poorly resolved at the base
of clade T, whereas the Madagascan M. suarezensis was resolved
within the main African clade (X). The Congolese species
M. yangambiensis was not included in the ITS analysis due to
difficulties in amplifying its DNA from herbarium specimens.
These discrepancies between the nuclear and plastid trees may
be the result of either ancestral polymorphism with incomplete
lineage sorting or chloroplast capture (introgression) following
dispersal.
EVIDENCE FOR CHLOROPLAST CAPTURE?
In the dated nuclear phylogeny, the Asian species M. hexandra
(Sri Lanka) and M. littoralis (Myanmar) (clade Y1) are placed
with other Asian species (clade Y2), whereas in the plastid phylogeny, they are resolved in clade with two African species M.
mochisia (Zambia) and M. concolor (South Africa) (from clade
V in ITS). This suggests hybridization of taxa across the Indian
Ocean possibly resulting in chloroplast capture. Intercontinental
chloroplast capture may also be implicated in the case of clade
Z, which is resolved in the plastid analyses but not in the ITS
analyses and is composed of M. suarezensis (Madagascar), M. triflora (Brazil), and M. yangambiensis (Congo). The ITS analysis
did not include M. yangambiensis, but placed M. triflora with
other Neotropical species in clade S, and M. suarezensis with other
Madagascan species within a larger clade of African species (clade
X). Therefore, ITS resolved at least two of the clade Z species with
species from the same landmass, but cpDNA did not, and resolved
them together instead. Clade Z is strongly supported (pp 0.99) in
the plastid analysis. Assuming that the correct species level relationships are resolved, clade Z presents a case of long distance
dispersal and chloroplast capture more remarkable than the clade
V/Y1 scenario, because it involves species from three landmasses,
and hence two dispersal events.
Hybridization and chloroplast capture across long distances such as ocean barriers has been indicated previously in
Sapotaceae. The species Chrysophyllum cuneifolium is inferred
to have originated from an intercontinental hybridization event
where the chloroplast is South American and the nuclear genome
is African (Särkinen et al., 2007). Likewise, the Pacific genus
Nesoluma is hypothesized to have arisen as a result of intercontinental hybridization in the boreotropical region during the
Eocene (Smedmark and Anderberg, 2007). Nesoluma presents
the opposite pattern to Chrysophyllum, where the chloroplast is
December 2014 | Volume 5 | Article 362 | 10
Armstrong et al.
African and the nuclear genome is Neotropical. Hybridization
between New and Old World lineages has also been demonstrated in the pantropical genus Gossypium (Malvaceae; Wendel
et al., 1995) and intercontinental chloroplast capture is hypothesized to have also occurred in Thuja (Cupressaceae; Peng and
Wang, 2008). Additionally, both hybridization and introgression
events are inferred to have occurred between distantly related
species in Ilex (Aquifoliaceae; Manen et al., 2010). What is
abundantly clear is that long distance dispersal has played a
crucial role in the establishment of the modern distribution of
Manilkara.
Supplementary Figure 1 | Bayesian majority rule consensus tree of the
chloroplast dataset. Posterior probability values are indicated above
branches. Nodes with letters/symbols are discussed in the text.
Supplementary Figure 2 | Phylogenetic tree used in the BAMM analysis
showing the nodes for which a proportion of sampled species was
calculated, as shown in Supplementary Table 3.
Supplementary Table 1 | Herbarium specimen data, GenBank accession
number and ancestral area coding for taxa included in the analyses.
Accessions of newly generated sequences are emboldened.
Supplementary Table 2 | Chloroplast primers designed for this study.
Supplementary Table 3 | Lineage specific correction used to take into
account incomplete taxon sampling in the BAMM analysis. The
unsampled species were assigned to the more recent node including
the species with the most similar morphology. The proportion of
sampled over total taxa was calculated for the nodes shown in
Supplementary Figure 2.
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Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
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Diversification amongst tropical regions compared
Received: 18 July 2014; accepted: 29 September 2014; published online: 03 December
2014.
Citation: Armstrong KE, Stone GN, Nicholls JA, Valderrama E, Anderberg AA,
Smedmark J, Gautier L, Naciri Y, Milne R and Richardson JE (2014) Patterns of diversification amongst tropical regions compared: a case study in Sapotaceae. Front. Genet.
5:362. doi: 10.3389/fgene.2014.00362
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journal Frontiers in Genetics.
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