Phylogeny and biogeography of the amphi-Pacific genus Aphananthe
Yi T-S (2017)
Phylogeny and biogeography of the amphi-Pacific
genus Aphananthe. PLoS ONE 12(2): e0171405.
Phylogeny and biogeography of the amphi- Pacific genus Aphananthe
Mei-Qing Yang 0 1
De-Zhu Li 0 1
Jun Wen 1
Ting-Shuang Yi 0 1
0 Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences , Kunming, Yunnan , China , 2 Baotou Medical College , Baotou, Inner Mongolia, China , 3 Department of Botany , National Museum of Natural History , Smithsonian Institution , Washington DC , United States of America
1 Editor: William Oki Wong, Institute of Botany , CHINA
Aphananthe is a small genus of five species showing an intriguing amphi-Pacific distribution in eastern, southern and southeastern Asia, Australia, and Mexico, also with one species in Madagascar. The phylogenetic relationships of Aphananthe were reconstructed with two nuclear (ITS & ETS) and two plastid (psbA-trnH & trnL-trnF) regions. Clade divergence times were estimated with a Bayesian approach, and the ancestral areas were inferred using the dispersal-extinction-cladogenesis and Bayesian Binary MCMC analyses. Aphananthe was supported to be monophyletic, with the eastern Asian A. aspera resolved as sister to a clade of the remaining four species. Aphananthe was inferred to have originated in the Late Cretaceous (71.5 mya, with 95% HPD: 66.6±81.3 mya), and the crown age of the genus was dated to be in the early Miocene (19.1 mya, with 95% HPD: 12.4±28.9 mya). The fossil record indicates that Aphananthe was present in the high latitude thermophilic forests in the early Tertiary, and experienced extinctions from the middle Tertiary onwards. Aphananthe originated in Europe based on the inference that included fossil and extant species, but eastern Asia was estimated to be the ancestral area of the clade of the extant species of Aphananthe. Both the West Gondwanan vicariance hypothesis and the boreotropics hypothesis could be excluded as explanation for its amphi-Pacific distribution. Long-distance dispersals out of eastern Asia into North America, southern and southeastern Asia and Australia, and Madagascar during the Miocene account for its wide intercontinental disjunct distribution.
The amphi-Pacific tropical disjunction has long been discussed as an important biogeographic
pattern in plants, with more than 100 genera and higher taxa of angiosperms exhibiting this
distribution pattern [1±2]. Two major hypotheses have been proposed to explain
amphiPacific tropical disjunctions. The boreotropics hypothesis postulates a continuous belt of
tropical to subtropical forest at middle to northern latitudes of the Northern Hemisphere, and the
continents were connected by the Bering and North Atlantic land bridges during the early
Cenozoic [3±7]. Thermophilic taxa may have migrated across continents through two land
bridges, especially the North Atlantic land bridges, until the late Eocene [4±5, 8]. The
subsequent gradually cooling and drying climates led to the extinctions of the thermophilic plants
or their southward migration to tropical Americas and Asia, or also to Africa but with
subsequent extinction in Africa. The West Gondwanan vicariance hypothesis postulates a tropical
origin and expansion in southern West Gondwana followed by vicariance from tectonic
separation into South America and Africa at ca. 100 Ma [
]. The descendants of West Gondwanan
taxa showed the amphi-Pacific tropical distribution through migrations from Africa into
tropical Asia followed by extinctions in Africa. In addition, long distance dispersals via birds, wind
or ocean currents were suggested for some of these taxa, as there is evidence for their
migration over water to the Hawaiian or Polynesian islands in the Pacific Basin or to the Mascarene
Islands in the Indian Ocean [
Recent biogeographic studies on pantropical taxa suggested only a few good examples (e.g.,
Strelitziaceae) that have sufficient divergence ages to be explained by the breakup of
]. To our knowledge, the West Gondwanan vicariance hypothesis has not been
applied to any taxa showing the amphi-Pacific tropical distribution pattern. Instead, the
boreotropics hypothesis has been used to explain the amphi-Pacific tropical distribution in multiple
groups (e.g., [10±11]). The estimated divergence times of most other taxa are too young to be
explained by the above two hypotheses. Long-distance dispersals have been proposed to play
an important role in the formation of their amphi-Pacific tropical distributions (reviewed in
]). There are only a limited number of biogeographic studies on taxa that show such an
amphi-Pacific tropical distribution.
Some genera showing the amphi-Pacific tropical distribution have an extended distribution
to the Mascarene Islands, Madagascar, or even eastern tropical Africa [
]. These taxa should
be good candidates to test the boreotropics hypothesis and the West Gondwanan vicariance
hypothesis involving Africa and/or Madagascar. To our knowledge, there is no biogeographic
study on these taxa. Aphananthe is a small genus in Cannabaceae demonstrating an
amphiPacific tropical disjunction with an expanded distribution range to Madagascar. This genus
contains only five deciduous to semi-deciduous shrubby or tree species [12±14], with three
species from eastern Asia, south and Southeastern Asia and Australia, one in Mexico, and one
in Madagascar. Aphananthe also has abundant fossil records. The earliest fossil is Aphananthe
cretacea Knobl. & Mai reported from the Maastrichtian (66±72.1 mya) of Walbeck, Germany
]. The middle Eocene (48.6±37.2 mya) silicified endocarps of Aphananthe maii Manchester
were identified from the Nut Beds locality of the Clarno Formation of Oregon, USA [16±17].
The fossil species Aphananthe tenuicostata Dorofeev was from the Oligocene (23.0±28.4 mya)
of western Siberia [
]. Miocene (16±18 mya) wood fossils were found from Yamagata
Prefecture, Japan [
]. Pleistocene (0.0±2.6 mya) pollen fossils were found in Queensland, Australia
with a complete pollen record of the last 230 ka from Lynch's Crater, north-eastern Australia.
Aphananthe thus provides a unique opportunity to explore the evolution of the amphi-Pacific
Aphananthe was strongly supported as member of Cannabaceae by recent molecular studies
[20±25]. Aphananthe and Lozanella were weakly supported to be sister to each other .
However most subsequent molecular studies supported Aphananthe to be sister to the rest of
Cannabaceae [21±25]. Nevertheless, the interspecific relationships of Aphananthe remain
poorly understood, because only one to three species were sampled in previous studies [20,
23±25]. The origin and evolution of the intercontinental disjunction of Aphananthe have
never been addressed in previous studies. A fully resolved phylogeny with all species sampled
is needed to infer the biogeographic history of this intriguing genus.
We sampled all Aphananthe species and employed multiple nuclear and plastid markers to
reconstruct their interspecific relationships. Fossil-calibrated divergence time estimates and
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ancestral geographic range analyses were used to test the alternative hypotheses for the current
broad intercontinental disjunctions, with the well confirmed fossils included in the
Materials and methods
Thirteen individuals representing all five recognized Aphananthe species were included in this
study (Table 1). Each species (except for A. sakalava from Madagascar) was sampled with
more than one accession. The study sequenced ITS, ETS, psbA-trnH and trnL-trnF, and
sequences generated from this study are deposited in GenBank (Table 1). Five species from
four genera (Celtis L., Gironniera Gaudichaud-BeaupreÂ, Lozanella Greenm., and Pteroceltis
Maximowicz) representing major clades of Cannabaceae were included as outgroups. More
distantly related taxa from Moraceae and Urtiaceae were not sampled as outgroups because of
the difficulties in aligning matrices of ETS and ITS.
DNA extraction, PCR amplification and sequencing
Two nuclear (ETS and ITS) and two plastid (psbA-trnH and trnL-trnF) markers were
employed in this study. The following primers were used for both amplification and
sequencing: ªN-nc18S10º and ªC26Aº  for the entire ITS region, or ªITS2º and ªITS5º, ªITS3º and
ªITS4º  for two separate fragments of the ITS region, respectively; ``18S-IGSº and ``ETS1Fº
GenBank accession number
psbA-trnH trnL-trnF ETS ITS
KR086759 KR086777 KR086725 KR086741
KR086760 KR086778 KR086726 KR086742
KR086763 KR086779 KR086729 KR086745
KR086764 KR086780 KR086730 KR086746
KR086765 KR086781 KR086731 KR086747
KR086766 JN040357 KR086732 KR086748
KR086767 KR086782 KR086733 KR086749
KR086768 KR086783 - KR086750
KR086770 KR086784 - KR086752
KR086753 KR086771 KR086719 KR086735
KR086754 KR086772 KR086720 KR086736
KR086755 KR086773 KR086721 KR086737
KR086756 KR086774 KR086722 KR086738
KR086757 KR086775 KR086723 KR086739
KR086758 KR086776 KR086724 KR086740
KR086761 JN040375 KR086727 KR086743
KR086762 JN040379 KR086728 KR086744
KR086769 JN040385 KR086734 KR086751
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[28±29] for the ETS region; ``psbA-Fº and ``trnH-Rº [
] for the psbA-trnH region; primers
``cº and ``fº for the trnL-trnF region, or primers ``cº and ``dº, ``eº and ``fº for two separate
fragments of the trnL-trnF region, respectively [
Total DNA was obtained from silica-gel-dried leaf fragments or herbarium specimens
using the CTAB protocol of Doyle and Doyle [
]. Polymerase chain reaction (PCR)
amplifications and the sequencing procedure followed those of Yang et al. [
Sequence alignment and phylogenetic analyses
Sequences were initially aligned using Geneious ver. 4.8.2 [
] followed by manual
adjustments using Se-Al ver. 2.0 [
]. Phylogenetic analyses were conducted on cpDNA, nrDNA, as
well as the combined cpDNA and nrDNA data set, respectively. The data matrices are available
at Data Dryad (http://dx.doi.org/10.5061/dryad.9082p).
Topological incongruence between ITS and ETS data sets, as well as between the nrDNA
and cpDNA data sets were tested using the incongruence length difference (ILD) test [
implemented in PAUP ver. 4.0b10 [
]. For the ILD test, 1000 heuristic searches were carried
out after the removal of all invariable characters from the data set [
]. The ILD test between
the ITS and ETS partitions estimated a p value of 0.71, they were thus combined in the
analyses. The phylogenies of the cpDNA and the nrDNA data sets were largely congruent (see S1
and S2 Figs), and the ILD test estimated a p value of 0.06 between these two partitions. The
nuclear and plastid data sets were thus combined in the following analyses.
Phylogenetic analyses were conducted on the cpDNA data set, the nrDNA data set, and the
combined cpDNA and nrDNA data set. For the combined data set, we divided it into three
partitions: psbA-trnH, trnL-trnF and ETS+ITS, and substitution model for each partition was
TVM+G, which were identified by the PartitionFinder ver. 1.1.1 [
]. The estimated
substitution models were applied in phylogenetic reconstruction using maximum likelihood (ML) and
Bayesian inference (BI), and divergence time estimation used a Bayesian approach.
Phylogenetic relationships were inferred with maximum parsimony (MP) as implemented
in PAUP ver.4.0b10 [
], maximum likelihood (ML) as implemented in GARLI ver. 2.0 [
and Bayesian inference (BI) with MrBayes ver. 3.1.2 [
The MP analyses used heuristic searches with 1000 random addition sequence replicates,
tree bisection reconnection (TBR) branch swapping, and MULTREES on. All character states
were treated as unordered and equally weighted, with gaps as missing data. To evaluate the
relative robustness of clades in the MP trees, the bootstrap analysis was performed with 1000
replicates using the same options as above.
For the ML analysis [
], the previously determined partitioning scheme and substitution
model for each partition were applied. To obtain statistical support, 1000 bootstrap replicates
were conducted with a rapid bootstrapping and subsequent ML search.
For the BI inference, one cold and three incrementally heated Markov chain Monte Carlo
(MCMC) chains were run for 20,000,000 generations. Trees were sampled every 100
generations. The previously determined partitioning scheme and substitution model for each
partition were applied. MCMC runs were repeated twice to avoid spurious results. Stationarity of
the Markov chain was ascertained by plotting and interpreting likelihood values against
number of generations in Tracer ver. 1.3 [
]. The first 50,000 trees before stationarity were
discarded as burn-in, and the remaining trees were used to construct the majority-rule consensus
trees. The average standard deviation of split frequencies between the two runs was 0.001862
and ESS values as computed by Tracer ver. 1.3 [
] were above 200 for two individual MCMC
runs. The posterior probabilities (PP) 0.95 were considered to be significant probability for
a clade following that of Alfaro et al. [
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Estimation of divergence times
To estimate divergence times between lineages, we employed the combined data matrix. Rate
constancy was tested with a likelihood ratio test [
]. Because the data matrix departed from
the clock-like evolution, a Bayesian approach as implemented in BEAST ver. 1.7.5 [
applied under a log-normal relaxed molecular clock [
] and a Yule pure birth model of
speciation to estimate the times of divergence and their credibility intervals. The previously
determined partitioning scheme and substitution model for each partition were applied. Posterior
distributions of parameters were approximated with two independent MCMC analyses of
20,000,000 generations with a 25% burn-in. Sample from the two runs (which yielded similar
results) were combined and convergence of the chains was checked with the program Tracer
]. A maximum clade credibility (MCC) tree was produced with Tree Annotator ver.
Two confident macrofossils were applied as constraints. The oldest fossil species of
Aphananthe, A. cretacea with relatively small endocarps reported from the Late Cretaceous
(Maastrichtian: 66±72.1 mya) of Germany. The stem age of Aphananthe (node A) was thus
constrained at 66 mya based on the low boundary age of Maastrichtian. Prior settings for node
A were: offset of 66 mya, a log mean of 1.0 (log stdev of 0.5), yielding a prior age distribution
with a median age of 68.7 mya and a 97.5th percentile of 73.2 mya. Based on the oldest fossil
species of Celtis, Celtis aspera (Newberry) Manchester, Akhmetiev & Kodrulhas foliage and
endocarps from Paleocene (56±64 mya) in eastern Russia as well as the United States and
], the stem age of Celtis (node B) was constrained to 56 mya (the low boundary age of
the Paleocene). Prior settings for node node B were: offset of 56 mya, a log mean of 1.0 (log
stdev of 0.5), yielding a prior age distribution with a median age of 58.7 mya and a 97.5th
percentile of 63.2 mya.
We reconstructed the ancestral areas of Aphananthe by applying the
dispersal-extinctioncladogenesis (DEC) analysis implemented in LAGRANGE [47±48] and the Bayesian Binary
MCMC (BBM) analysis implemented in RASP ver. 2.0 [
]. In order to avoid any analytical
biases caused by the incomplete sampling of outgroup taxa, we excluded all outgroups. We
pruned the maximum clade credibility (MCC) tree of BEAST to include only one individual
of each species. To integrate fossil ranges into the reconstructions, three fossil species were
placed manually into the newick tree chronogram according to their age. Each fossil was
given a short branch length (0.5 Ma), simulating an extinct range. Other fossils were not
used in the ancestral area reconstructions (AARs) because they were too young to be
assigned to the relevant stem lineage. Six areas of endemism were defined for the
biogeographic analyses based on the distribution and phylogeny of the extant and extinct
Aphananthe species: A, eastern Asia (including China, Japan and Korea); B, southern and
southeastern Asia (including India, Indochina, Indonesia, Malaysia and the Philippines); C,
North America; D, Australia (including New Guinea); E, Madagascar and F, Europe. For the
DEC analysis, we used default settings without imposing any additional expansion or time
constraints. For the BBM analysis, we used 1000 MCC trees from the BEAST output. The
MCMC chains were run simultaneously for 5,000,000 generations. The state was sampled
every 100 generations. Fixed JC + G (Jukes-Cantor + Gamma) were used for the BBM
analysis with a null root distribution. The maximum number of areas for this analysis was kept as
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The ILD test revealed congruence between ITS and ETS data sets (p = 0.71), as well as between
the cpDNA and nrDNA data sets (p = 0.06). Furthermore, there was no well-supported
conflict between cpDNA and nrDNA phylogenies (S1 and S2 Figs). The combined cpDNA and
nrDNA data set was applied to reconstruct interspecific relationships of Aphananthe.
The combined chloroplast and nuclear data set included 2579 aligned positions with 349
parsimony-informative characters. The parsimony analysis yielded one most parsimonious
tree with 870 steps (CI = 0.856; RI = 0.863, Table 2). The ML analysis and BI yielded similar
topologies to the MP tree (Fig 1). Each species was monophyletic with high support except that
only one accession of A. sakalva was sampled. Aphananthe was strongly supported as
monophyletic (maximum parsimony bootstrap support (MPBS) = 100%; posterior probabilities
(PP) = 1.0; maximum likelihood bootstrap support (MLBS) = 100%). The eastern Asian species
(A. aspera) was sister to the clade of the rest of the genus, and the next diverged clade was the
Mexican species A. monoica (MPBS = 56%; PP = 1.00; MLBS = 61%). The clade of A.
philippinensis and A. sakalava was moderately supported (MPBS = 61%; PP = 1.00; MLBS = 89%) and
was sister to A. cuspidata (MPBS = 75%; PP = 1.00; MLBS = 86%).
Bootstrap values (%) of MP, the PP values of BI analysis, and bootstrap values of ML
analysis are shown above the branch (MPBS/PP/MLBS, asterisks indicate 100% support by MPBS/
Divergence time estimations and biogeographic inference
The stem age of Aphananthe was estimated to be 71.5mya (95% HPD: 66.6±81.3 mya); and the
crown age was estimated to be 19.1 mya (95% HPD: 12.4±28.9 mya) (Fig 2).
The results of the DEC and BBM analyses were largely similar (Fig 3), which suggested
Europe as the ancestral area for Aphananthe, and eastern Asia for extant clade of Aphananthe
species. Aphananthe migrated from eastern Asia to North America at 18.1 mya (95% HPD:
11.7±26.6 mya) and southern and southeastern Asia at 13.4 mya (95% HPD: 8.6±19.3 mya),
and from southern and southeastern Asia to Australia and Madagascar at 10.8 mya (95%
HPD: 6.7±15.7 mya).
The most likely ancestral ranges of nodes with frequency of occurrence inferred by the
Bayesian Binary MCMC analysis are shown above branches, those by the DEC analysis below
branches. For results of the DEC analysis, a slash indicates the split of areas in two daughter
lineages, i.e., left/right, where ªleftº and ªrightº are the ranges inherited by each descendant
branch. The distribution ranges were divided into six areas for both analyses: A, eastern Asia
(including China, Japan and Korea); B, southern and southeastern Asia (including India,
Indochina, Indonesia, Malaysia and the Philippines); C, North America; D, Australia (including
New Guinea), E, Madagascar; and F, Europe. Stars indicate the fossil records, the pink star
indicates late Cretaceous and Oligocene fossils in Europe, and the purple star indicates the
middle Eocene fossil in North America. The background map was downloaded from http://
www.naturalearthdata.com/; the figure is similar to the original one but not identical to it, and
therefore it is for illustrative purposes only.
Phylogenetic relationships in Aphananthe
Our phylogenetic analyses strongly support the monophyly of Aphananthe (Fig 1). All
Aphananthe species share a few indels in the trnL-trnF data: three deletions and two insertions in
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trnL-trnF (a 5-bp deletion at position 110±114bp; an 88-bp deletion at position 664±751bp; a
7-bp insertion at position 752±758bp; and an 82-bp insertion at position 795±876bp), which
further support the monophyly of Aphananthe. Aphananthe was considered to be isolated
from other members of Cannabaceae based on morphology and molecular evidence.
Aphananthe possesses asymmetrical ovules (symmetrical ovules in other Cannabaceae) and flavonol
production (the only genus producing flavonol in Cannabaceae) [
]. Aphananthe is the only
genus of Cannabaceae which has x = 13 [
]. A distinct pattern of diversity in seed coat
morphology, as well as the occurrence of a unique derived seed coat feature (i.e., the raised middle
part of exotestal cells in Aphananthe aspera), may also suggest that Aphananthe is in an isolated
lineage in Cannabaceae [
The monophyly of each species was strongly supported and their relationships were
resolved and well supported (Fig 1). Aphananthe species are also easily distinguishable by
morphological characters: A. aspera possesses trinerved venation (rather than pinnate venation in
other species); A. cuspidata is characterized by its large leaf blade (5-)10-15 × (2-) 3±5 (-7) cm,
usually with entire leaf margin, and large fruits 1.3±2 × 0.7±1.2 cm; A. philippinensis can be
distinguished from other species by its relatively small leaves (4±6 cm × 1.5±3 cm vs. 5±15
cm × 3±7 cm in other species), and its sharply toothed leaf margin; A. sakalava is characterized
by its cymose female inflorescences of 2±3 axillary flowers; and the leaves of A. monoica have
the highest number of secondary veins (18±20 pairs vs. 6±14 pairs in other species).
The early diversification in the Northern Hemisphere
Previous studies on the diversification of lineages have shown that a long stem leading to a
crown of short branches may indicate major early extinctions [53±54]. Multiple taxa including
Orontioideae of Araceae [
], Hamamelis L. [
], and Hedyosmum Sw. [
] have shown such a
phylogenetic pattern. A similar pattern was detected in Aphananthe, which contains a
remarkable temporal difference (52.4 million years) between the stem lineage age (71.5 mya, with 95%
HPD: 66.6±81.3 mya) and the crown age of the extant species (19.1 mya, with 95% HPD: 12.4±
28.9 mya). Furthermore, the fossil records indicate that Aphananthe was widely distributed in
the middle to high latitudes of the Northern Hemisphere including Europe, western Asia and
North America during the early Tertiary until the Oligocene [15±16, 18]. Climates were warm
enough to support the thermophilic vegetation at the high latitudes in the Northern
Hemisphere during the Eocene [4±5], and many fossils of thermophilic taxa were recovered across
this region [3, 5, 58±59]. The phylogenetic and dating results as well as fossil data suggest that
Aphananthe was part of the high latitude thermophilic elements during the early Tertiary, and
it subsequently experienced extinctions from Europe and North America in the middle
Tertiary when a dramatic cooling of climates extirpated many evergreen plant lineages that were
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Fig 1. Phylogram obtained from Bayesian inference using the combined cpDNA and nrDNA data set.
once part of the Holarctic boreotropical flora [
]. Many Northern Hemisphere lineages
experienced similar histories [
56, 60, 61
Formation of amphi-Pacific tropical distribution
The West Gondwanan vicariance hypothesis could be confidently excluded as an explanation
for the amphi-Pacific tropical distribution of Aphananthe. Our ancestral range analyses
strongly support a Northern Hemisphere boreotropical origin for Aphananthe, and eastern
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Fig 2. The maximum clade credibility chronogram of Aphananthe and its relatives inferred by the BEAST analysis. Node A was constrained to 66
mya and node B was constrained to 56 mya.
Asia as the ancestral area for the extant species. Although Aphananthe has a relatively old stem
age (71.5 mya), the estimated crown age (19.1 mya) of the extant species is much younger than
that expected for a West Gondwanan origin (> c. 84 Ma). The North American and
Madagascan lineages were inferred to have migrated from eastern Asia, southern and southeastern Asia
instead of their Northern Hemisphere regions. Furthermore, both migrations occurred during
late Tertiary, which were too young to be explained by the boreotropics hypothesis.
Long9 / 18
Fig 3. Results of the biogeographic reconstruction for Aphananthe estimated using Bayesian Binary MCMC and
the DEC analyses.
10 / 18
distance dispersals from eastern Asia during Miocene may be the best hypothesis to account
for the current amphi-Pacific tropical distribution of this genus.
This genus migrated into North America by the early Miocene (18.1 mya, 95% HPD: 11.7±
26.6 mya). This migration into North America was perhaps through the Bering land bridge
. Furthermore, we could not exclude the possibility of a long-distance dispersal via birds.
The Miocene was an important period for the migration of plant taxa between the eastern
Asian and North America [4, 7, 62±65].
The region of eastern Asia to Australia harbors three of the five Aphananthe species and is
the diversification center of the genus. Aphananthe aspera has the northernmost distribution
in eastern Asia (China, Japan, Korea, to the northern Vietnam). Aphananthe cuspidata has a
more southern distribution in southern and southeastern Asia (from southern China to India,
Malaysia, to Indonesia). Aphananthe philippinensis is distributed in the Philippines, New
Guinea and Australia. Aphananthe aspera was resolved to be the first diverged clade of the
genus at 19.1 mya (95% HPD: 12.4±28.9 mya, Fig 2). Aphananthe cuspidata represents the
second diverged clade (13.4 mya, 95% HPD: 8.6±19.3 mya) within this region, and A. philippinens
is the third diverged lineage (10.8 mya, 95% HPD: 6.7±15.7 mya) within this region.
Aphananthe seems to have experienced species diversification following the southward and
southeastward dispersal from eastern Asia to India, the Malesian region, and Indochina (i.e., the
regions extends from southern Thailand through Malaysia, Singapore, Indonesia, eastern
Timor and the Philippines, to Papua New Guinea and the Solomon Islands via southeastern
Asia, or the mountain ranges of Taiwan and the Philippines), and Australia. Immigration of
the continental Asian taxa is the major resources of flora of Malesia [66±68]. Recent molecular
phylogenetic and biogeographic studies also supported the major north-to-southeast dispersal
of taxa in this region [69±74].
Species of Aphananthe most likely dispersed eastwards across the Wallace's line into the
Wallacea, New Guinea and Australia in the middle Miocene. The ªwest-to-eastº dispersal was
inferred from other plant taxa of different families, e.g., Pseuduvaria Miq. (Annonaceae) [
Alocasia (Schott) G. Don (Araceae) [
], Begonia L. (Begoniaceae) [
] and tribe Millettieae
]. This west-to-eastward dispersal is consistent with the geologic history of the
region. The land of the western Malesia remained emerged throughout the Cenozoic, while
the emergence of the land masses east of the Wallace's line including the Wallacea, Sulawesi,
New Guinea and a series of volcanic islands along the Sunda Arc, the Banda Arc, and the
Halmahera Arc connecting these region from the late Miocene onwards, supplying a potential
channel for dispersal by island-hopping between western and eastern Malesia [78±79]. The
niche pre-emption following the formation of new landmass in the eastern Malesia [80±81]
may have facilitated the similar west-to-east dispersal pattern observed in various taxa with
different life forms, generation times and dispersal capabilities [
76, 79, 82
Biogeographic disjunction between Asia and Madagascar
Four major hypotheses have been proposed to explain the disjunctions across paleotropical
regions around the Indian Ocean Basin: (1) the ªout-of-Indiaº hypothesis, i.e., rafting of biota
of Gondwanan origin by the Indian plate and subsequent dispersal to Asia during the
Cenozoic [83±85]; (2) the ªboreotropical migrationº hypothesis, i.e., dispersal through a northern
mid-latitude corridor of extensive boreotropical forests during the Paleocene and Eocene [3,
86±87]; (3) the overland migration across Arabia during a warm phase in the early to middle
]; and (4) the transoceanic long-distance dispersal [89±90]. Aphananthe sakalava
from Madagascar was inferred to be dispersed from southern and southeastern Asia at around
10.8 mya (6.7±15.7 mya) or less. The Indian landmass separated from Madagascar during the
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mid-late Cretaceous (90±85 mya), and the collision of the Indian subcontinent with the
Eurasian continent was estimated at ca. 50 mya [
] to 35 mya [
], which is much earlier than
the estimated dispersal age of Aphananthe from Asia to Madagascar. Furthermore, the
dispersal direction is inferred from southern and southeastern Asia to Madagascar instead of the
reverse. The ªout-of-Indiaº hypothesis thus can not be applied to explain the disjunction
between Asia to Madagascar. The ªboreotropical migrationº hypothesis also seems unlikely,
because this corridor was disrupted following climatic deterioration in the late Eocene [3, 86±
87, 93]. The estimated age is too young to be consistent with this hypothesis. Overland
migration between Africa and Eurasia through Arabia has been hypothesized for some tropical plant
]. Africa and Eurasia were connected during the early to middle Miocene [
which coincided with a warm phase with the peak at the Middle Miocene Climatic Optimum
during 17±15 mya [
]. The estimated dispersal time is later than this climate optimum, and
the extant Aphananthe species are absent from Arabia and continental Africa. There are no
reported fossils there, and extinctions from both regions are required to apply this hypothesis
to explain this disjunction, making this hypothesis less likely. The long-distance dispersal
seems the most likely hypothesis for this disjunct pattern in Aphananthe. There are no reports
of the adaptation of Aphananthe or other Cannabaceae fruits or seeds to hydrochory, and
Aphananthe species are absent from sea shores. Aphananthe thus most likely arrived in
Madagascar through the long-distance dispersal by birds, as fruits of Aphananthe are eaten and
dispersed by many kinds of birds [96±98]. This dispersal may have been promoted by two factors.
First, the Indian winter monsoon winds blow from the Indian subcontinent towards the
Madagascar region [99±100]. Second, sea level has fluctuated in the recent geologic past, and a
chain of islands stretched between the granitic Seychelles, Mascarenes and India. Such
stepping-stones may have provided a channel for communication of plants and animals between
Asia and Madagascar [101±102]. This scenario has been applied to explain dispersals from
Asia to Madagascar in a few recently studied taxa [90, 103±104]. Recent molecular studies
overwhelmingly favor long-distance dispersals from southeastern Asia to Africa [88, 105±108],
rather than the rare dispersals from Africa to Asia [
Our analyses largely resolve the phylogenetic relationships among Aphananthe species. The
temporal and spatial diversification patterns of Aphananthe were explored with respect to the
paleoenvironmental changes. A long stem (52.4 million years) of Aphananthe indicated the
ancient origin and relatively recent diversification of extant species. Fossil records indicate
that Aphananthe was widely distributed in Northern Hemisphere during the early Tertiary
until the Oligocene, and experienced major extinctions from western Eurasia and North
America during the middle Tertiary. The oldest fossil of Aphananthe was from Europe, and
eastern Asia was inferred to be ancestral region of the extant clade of Aphananthe species. The
broad intercontinental disjunction across eastern Asia, southern and southeastern Asia,
Australia, North America and Madagascar is due to migration via the Bering land bridge and the
long-distance dispersals via birds during the Miocene. Significant climatic and geologic
changes have shaped the species diversification and distribution pattern of this
biogeographically unique genus Aphananthe.
S1 Fig. Phylogram obtained from Bayesian inference using the combined cpDNA data set.
Bootstrap values (%) of MP, the PP values of BI analysis, and bootstrap values of ML analysis
are shown above the branch (MPBS/PP/MLBS, and asterisks indicate 100% MPBS/PP/MLBS
12 / 18
values, dashes indicate the MPBS/PP/MLBS values are less than 50%).
S2 Fig. Phylogram obtained from Bayesian inference using the combined nrDNA data set.
Bootstrap values (%) of MP, the PP values of BI analysis, and bootstrap values of ML analysis
are shown above the branch (MPBS/PP/MLBS, and asterisks indicate 100% MPBS/PP/MLBS
values, dashes indicate the MPBS/PP/MLBS values are less than 50%).
We thank Pan Li (Zhejiang University) and Kyong-Sook Chung (Jungwon University) for
sample collection, and the staff in the Key Laboratory of the Southwest China Germplasm
Bank of Wild Species, Kunming Institute of Botany, the Chinese Academy of Sciences for
Data curation: MY.
Formal analysis: MY.
Funding acquisition: TY DL.
Investigation: MY TY.
Methodology: MY TY JW.
Project administration: TY JW.
Resources: TY DL.
Supervision: TY JW DL.
Writing ± original draft: MY TY.
Writing ± review & editing: MY TY DL.
13 / 18
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