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Article type: Original article
RUNNING TITLE
Historical biogeography of Western Ghats Myrtaceae
Western Ghats Myrtaceae are not Gondwana elements but likely dispersed from south-east
Asia
Rajasri Ray1,2, Balaji Chattopadhyay3, Kritika M Garg3, TV Ramachandra1, and Avik Ray2,4*
1 - Center for Ecological Sciences, Indian Institute of Science, Bangalore - 560012, Karnataka,
India
2 - Center for Studies in Ethnobiology, Biodiversity, and Sustainability (CEiBa), Malda, West
Bengal, India
3 - Department of Biological Sciences, National University of Singapore, Singapore - 117543
4 - Ashoka Trust for Research In Ecology and Environment (ATREE), Royal Enclave, Srirampura,
Jakkur Post, Bangalore - 560054, Karnataka, India
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Abstract
Gondwana break-up is one of the key sculptors of the global biogeographic pattern including Indian
subcontinent. Myrtaceae, a Gondwana family, demonstrates a high diversity across Indian Western
Ghats. A rich paleo-records in Deccan Intertrappean bed in India strive to reconcile two contending
hypothesis of origin and diversification of Myrtaceae in India, namely Gondwana elements floated
on peninsular India or lineages invading from south-east Asia. In this study, we have reconstructed
the biogeographic history of Myrtaceae of Western Ghats of India combining fossil-calibrated
phylogeny, ancestral area reconstruction, and comparing various dispersal models.
Phylogenetic reconstructions divided lineages into multiple clades at the tribal level that mostly is
in agreement with previous descriptions. Dated tree indicated a presumable Gondwanan origin of
Myrtaceae (95.7 Ma). The Western Ghats taxa are relatively younger and seem to have arrived
either through long-distance oceanic dispersal in middle Eocene (Rhodomyrtus tomentosa), and
Miocene (Eugenia) or through major overland dispersals throughout Miocene (Syzygium).
However, late Cretaceous fossilsin India lends support to the existence of proto-Myrtaceae on
floating peninsular India. Taken together, we conclude that Myrtaceae lineages on the biotic ferry
were perhaps extirpated by Deccan volcanism leaving a little or no trace of living Gondwanan link,
while the Western Ghats was likely enriched by dispersed elements either from south-east Asia or
from South America.
Keywords
Gondwana, Myrtaceae, Syzygium, Deccan volcanism, Dispersal, Indian flora
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India has a unique yet complex geologic origin as aptly reflected in its rich biological legacy. A
fraction of diversity is attributed to Gondwana elements whereas the rest consisting of dispersed
biota with Sino-Tibetan, Indo-Malayan, or Mediterranean affinities (Mani, 1974). The Western
Ghats, a continuous mountain chain spanning 1500 km2 along the western coast of India, spread
across five states from 8°N to 22°N and 73°E to 77°E. Being a global hotspot, it exhibits rich floral
diversity with more than 8000 taxa of flowering plants, including 7402 species, 117 subspecies, and
476 varieties (Nayar 2014, CEPF 2007). Distinct temperature and rainfall gradients prevail across
south-north and west-east directions of the Ghats. It influences the distribution of biota that is
discernible in species richness and endemicity patterns (Gimaret-Carpentier et al. 2003). The
southern part, presumed to be refugia, is particularly rich in Gondwana flora and could be a home of
ancient lineages (Prasad et al. 2009).
Myrtaceae, the eighth largest flowering plant family of more than 145 genera and 5500 species
(Govaerts et al. 2009), is of Gondwanan origin (Thornhill et al. 2015). It is represented by 17 tribes
mostly in Southern Hemisphere, and quite widespread with centres of diversity in Australia, south
and south-east Asia, tropical to southern temperate America, but has little representation in Africa
(Sytsma et al. 2003). In India, taxa from tribes, Syzygieae and Myrteae, overwhelm Myrtaceae
diversity. There are almost 26 species (1 Meteromyrtus sp., 9 Eugenia spp. and 16 Syzygium spp., 1
Rhodomyrtus) reported in Western Ghats (henceforth WG) region (Pascal & Ramesh, 1997); though
taxonomic discrepancy constrains species delimitation (Byng et al., 2015; Sujanapal and
Kunhikannan 2017). In addition, several species of Syzygium and Eugenia also abound the north-
eastern region of India. Apart from a large number of endemic lineages, a few were introduced and
have been cultivated for various reasons, such as, in afforestation, as fruit-crop or ornamental (e.g.
Eucalyptus, Callistemon, Melaleuca, and Psidium) (Pascal and Ramesh 1997; Nayar et al. 2006).
The presence of disjunct Myrtaceae flora across WG and north-eastern India is intriguing. It evokes
two alternate routes in terms of their origin and evolution: the first route posits that proto-Myrtaceae
remained on the Indian raft after Gondwana vicariance (biotic ferry or Noah’s ark model) (Conti et
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al. 2002) and colonised WG later (figure 1a - c). Had they persisted, these lineages would be
expected to emerge from deeper phylogenetic nodes. The abundance of fossils in Deccan
Intertrappean Bed and elsewhere in India provides support to this Gondwana lineage hypothesis
(Srivastava, 2011 and references therein; table S1). In contrast, the alternate hypothesis posits floral
dispersals from south-east Asia (originally from the part of Gondwana that now makes up
Australia) which eventually led Myrtaceae lineages to reach WG (Mani, 1974). This late arrival via
south-east Asia ascribes it to relatively younger taxa (figure 1d).
However, none of these hypotheses have been rigorously tested within a phylogenetics driven
biogeography framework and the origin of Indian Myrtaceae remains unsolved. We have sought to
address this question by generating sequence data from a host of Indian Myrtaceae species and
tested the following hypotheses: i) whether the extant lineages stem from Gondwana, or, ii) they
represent dispersed elements, or, iii) both vicariance and dispersal have co-contributed to the
generation of modern Myrtaceae diversity in WG. We performed fossil calibrated divergence
dating, compared various dispersal-vicariance scenarios to reconstruct the history of Western Ghats
Myrtaceae.
1. Materials and methods
1.1 Taxon sampling, DNA extraction, amplification, and sequencing
Relatively fewer taxonomic studies on Indian Myrtaceae impeded exhaustive taxon sampling.
Moreover, inadequate information on species distribution (e.g. S. agasthyamalayanum, S.
beddomei, S. microphyllum and many others) coupled with erroneous identification owing to a few
characters were other obstacles (Sujanapaul and Kunhikannan 2017). Hence, in order to circumvent
this problem, we sampled fifteen species of Myrtaceae from the WG of India(13 species of
Syzygium, 1 species each of Eugenia and Rhodomyrtus sp) with little or no taxonomic disputes.
Additionally, we supplemented our dataset with seventy additional species from GenBank; so that
the final dataset consisted of a total of 81 species of Myrtaceae sensu lato (Wilson et al. 2005) as
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ingroup and four species (Crypteronia paniculata, Alzatea verticillata, Olinia ventosa, Penaea
mucronata) from Crypteroniaceae, and Peneaceae as outgroup following Sytsma et al. (2003).
Voucher specimens information and corresponding GenBank accession numbers of the existing and
the new taxa are enlisted in table-S2-3.
We extracted DNA following modified Doyle and Doyle (1987), amplified the regions of
chloroplast (matk), mitochondria (ndhF), and nucleus (ITS) using the standard protocol, and
sequenced (for primer sequences, see refs. Biffin et al. 2006 and table S4). We aligned the
sequences using ClustalX version 1.83 (65) and adjusted manually in MEGA 5.2 (Tamura et al.
2011).
1.2 Dated Molecular Phylogeny and fossil calibration
a) Molecular phylogeny: Sequences were concatenated using Sequence Matrix v1.7.8 (Vaidya et
al., 2011) and data partition analyses were performed in Partition Finder v1.1.1 (Lanfear et al.,
2012) using AIC method for model selection. We used unlinked branch lengths and greedy search
scheme. Seven partitions for the data, consisting of a single partition for ITS and 3 partitions each
for both matk and ndhF based on codon position, were defined; and finally, the best partition
scheme determined by partition finder used to define data partitions for all concatenated
phylogenetic reconstructions. We performed phylogenetic tree reconstructions with concatenated
data using maximum likelihood method in RaxML GUI v1.5 (Silvestro and Michalak, 2012) and
Bayesian reconstruction in MrBayes. We used the most complex model of sequence evolution
(GTR + gamma) in RaxML. After performing a hundred runs implementing a thorough bootstrap
algorithm with 1000 replicates, finally, we have obtained a maximum likelihood (ML) tree. In
MrBayes, we performed two independent runs each for 2,000,000 generations with four chains
keeping the swapping frequency and temperature at default values. We sampled the trees at every
500th generation and obtained diagnostics at every 5,000th generation. In course, we ensured that
the standard deviation of the split frequency has reached below 0.01 by the end of each run. Finally,
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we checked the parameter convergence in Tracer v1.6 (Rambaut and Drummond 2007) and
obtained the consensus trees from MrBayes after discarding 25 % of the trees as burn-in.
In addition, we also performed species tree reconstruction under Bayesian inference in *BEAST
v1.8.2 (Drummond et al., 2012) considering each locus as a separate partition. For each locus, the
substitution model was determined through jModelTest v 2.1.7 (Guindon and Gascuel, 2003;
Darriba et al., 2012), and a separate relaxed molecular clock model for each locus was adopted to
estimate relative clock rates. We implemented Yule speciation process with random starting gene
trees and conducted two independent runs (of 100 million generations each and sampling every
100th generation) followed by a checking of parameter convergence of both the runs in Tracer
(Rambaut et al., 2014). We combined the two runs were combined in Log Combiner 1.8.2 with a
50% burn-in while resampling at a frequency of 1000 generations. Subsequently, a maximum clade
credibility tree was constructed using median heights in Tree Annotator 1.8.3 while allowing for a
further 20% burn-in using a posterior probability limit of 0.95.
b) Fossil calibration and divergence dating: In addition to phylogenetic reconstruction, we also
used the partitioned dataset of species tree reconstruction to obtain dates of divergences of various
nodes in BEAST v1.8.3 (Drummond et al. 2012). We performed two independent runs of 50 million
generations each, with sampling at every 1000th generation. We used the Birth-Death process of
speciation with random starting trees and four time points for calibration of divergence dates based
on previous studies of Biffin et al. (2010), Shukla et al. (2012) and Thornhill et al. (2015) (table -
1).
Despite a continued effort to unravel historical biogeography of Myrtaceae, the results tended to
vary greatly partly due to different fossil dates (Vasconcelos et al. 2017). To avoid this, we largely
followed the fossil dates in the previous studies by Biffin et al. (2010) and Thornhill et al. (2015).
Biffin et al. (2010) used the pollen taxon Myrtaceidites lisamae recorded from fossils obtained from
multiple places, e.g. Gabon (Herngreen, 1975; Boltenhagen, 1976 Muller, 1981), Borneo (Muller,
1968), and from Colombia (van der Hammen, 1954) and that provided the earliest estimate of
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Myrtaceae radiation. Hence, we calibrated the Myrtaceae stem following this estimate at 92 Ma (±
3.5).
We calibrated Myrtaceae [85 (± 3.5) ma] and Myrtoideae [71.5 (± 3)] crown dates adhering to the
large-scale phylogenetic study by Thornhill et al. (2015). They obtained 85 Ma (75.0–93.2 Ma) as
the crown date of Myrtaceae when the two subfamilies diverged in the Late Cretaceous and
Myrtoideae crown dates as 71.5 Ma (65.4-78.3) when tribes Xanthostemoneae, Osbornieae, and
Lophostemoneae split from the rest of the Myrtoideae.
Lastly, a Eucalypt fossil, Eucalyptus ghughuensis, found in Deccan Intertrappean Bed (henceforth
DIB) from India was used to calibrate the Eucalypteae stem in our phylogeny (Shukla et al.
2012).DIB of India has luxuriant records of Myrtaceae fossils preserved over a long period from
Danian-Maastrichtian. However, the xylotomical features of the fossil, e.g. characteristic solitary
vessels, paratracheal and apotracheal parenchyma, vasicentric tracheids, fine rays highlighted its
morphological similarity with both Fagaceae and Myrtaceae. However, a characteristic echelon
arrangement of the vessels has suggested a Myrtaceae affinity, ascribing to Eucalyptus, Melaleuca,
or Xanthostemon. The final inclusion into the genus Eucalyptus was based on semi-ring porous
wood which is absent in other two genera but present in a few species of Eucalyptus. It is the oldest
known Myrtaceae fossil not only from India perhaps in the world.
For all calibrations, priors were provided with the normal distribution. Parameter convergence of
each run was confirmed in Tracer and both the runs were combined in Log Combiner 1.8.3 with a
50% burn-in. Further, a maximum clade credibility tree was constructed keeping target heights in
Tree Annotator 1.8.3 while allowing for a further 20% burn-in using a posterior probability limit of
0.95.
1.3 Ancestral area reconstruction
We have sought to understand the area of origin given their dispersal probabilities. We have
reconstructed the ancestral area using a maximum likelihood method under the divergence–
extinction–cladogenesis model (DEC) implemented in the software Lagrange build 20091004 (Ree
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& Smith, 2008). We have generated python scripts using the online configurator
(http://www.reelab.net/lagrange) and run using python launcher.
Five areas were demarcated for examination of ancestral areas and the choice was based on the
distribution of included taxa as described in Wilson (2011): A, Australia; B, Africa, and
Madagascar; C, India; D, south-east Asia; E, South America (figure - 4). In addition, we have also
retrieved the distribution data from other sources e.g. web-resource like Global Biodiversity
Information Facility (GBIF), Encyclopaedia of Life (EOL), South African National Biodiversity
Institute (SANBI), India Biodiversity Portal, to assign each genus to one or more areas (table-S5).
We have restricted the number of ancestral areas were restricted to three given a wider cross-
continental distribution of the family and to avoid added complexity of the analyses due to many
areas. Our basic assumption was that the that past distributions of members of this group were
similar to the present distribution. Given the medium dispersal ability of the extant members
(mostly dispersal takes place through birds and small mammals like bats) of this group, we assumed
that dispersal was possible only between adjacent areas (i.e. no dispersal was allowed between
widely-separated areas e.g. A and B or E, and between C and E). Hence, the final permissible
ancestral areas were ABC, ABD, AC, ACD, AD, BC, BCD, BCE, BD, BDE, BE, and CD. The
relative position and connectivity among the five geographic areas underwent major change over
the considered time period (110-0 Ma) i.e. post-Gondwana fragmentation and hence major events
e.g. floatation of Indian landmass and its position relative to other landmasses, position of Afro-
Madagascar and formation of transient land-bridges, and final collision into India were taken into
account. Thus, we assigned dispersal probabilities between all adjacent areas within all time-slice
based on geological history, climate history (Morley, 2000, 2003; Tiffney & Manchester, 2001;
Zachos et al., 2001, Scotese, 2001), and general dispersal ecology of Myrtaceae, e.g. small to
medium sized seeds means moderate level of dispersal, and grossly followed previous studies
(Couvreur et al. 2011; Mao et al. 2012). After several exploratory runs with a range of dispersal
probabilities and time-slices, we selected five dispersal probability category (low or no dispersal =
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0.01; low dispersal = 0.25; medium dispersal = 0.5; high dispersal = 0.75; areas adjacent or very
close = 1), and five time-slices (110-90, 90-65, 65-45, 45-30, and 30-0 Ma) for final model testing.
Differences between models were assessed by directly comparing their respective log-likelihoods
where the conventional cut-off value of two log-likelihood units considered to assess the statistical
significance of likelihood differences (Ree et al. 2005). We evaluated the goodness of fit of the data
for three alternative biogeographic models in an ML framework: model 0 was unconstrained with
dispersal events between all adjacent areas possible during the whole period considered (110–0 Ma)
which was considered as the null model. To account for Gondwana relict hypothesis model 1 was
constrained in the following manner: all dispersal out of India were allowed with varied dispersal
probability as predicted from past geologic events but restricting dispersal into India throughout
110Ma. Model 2: dispersal out of India was not allowed during 110 Ma as well as dispersal into
India was not allowed till 45 Ma, but dispersal from south-east Asia during 45-0 Ma was allowed to
emulate south-east Asian affinity (table-S6). We incorporated prior information on range evolution
as well as probabilities between areas given discrete periods. Finally, we estimated the ancestral
areas for all nodes relevant to this study.
2 Results
2.1 Phylogeny, molecular dating, and timing of divergence
A total of 45 sequences were generated for this study 15 each from three loci. Post-editing, the
aligned dataset of 85 taxa consisted of 1824 sites with 923 variable, 583 parsimony informative, and
339 singleton sites. We scrutinised the resulting trees for weak nodal supports and collapsed such
nodes (50% cutoff for bootstrapped nodes in RAxML and 0.9 cutoff for posterior probabilities in
Bayesian reconstructions) in Tree Graph 2.11.1 (Stover and Muller 2010). All three methods of
phylogenetic reconstructions revealed almost similar evolutionary relationships of major clades. In
general, Myrtaceae sensu lato comprised two well-differentiated clades of subfamilies,
Psiloxyloideae and Myrtoideae; subsequently, Myrtoideae split into tribes i.e. Lophostemoneae,
bioRxiv preprint doi: https://doi.org/10.1101/2020.04.12.037960; this version posted April 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Xanthostemoneae, Eucalypteae, Syncarpieae, Chamelaucieae, Leptospermeae, Syzygieae,
Backhousieae, Metrosidereae, Tristanieae and Myrteae (figure-2a-b). However, the nodal supports
for the lower division were low in the concatenated reconstructions of the partitioned data and could
collapsed into a broad polytomy at which point topological incongruences between concatenation
and species tree methods would disappear (figure-S1). Likewise, for shallower nodes, the
partitioned concatenated reconstructions revealed better support and hence depicted monophyletic
origin of the tribal clades e.g. Syzygieae, Myrteae, Eucalypteae, Leptospermeae, Chamelaucieae; of
which Syzygieae and Myrteae were non-monophyletic in species tree. Whereas, in all three
reconstructions, tribes Melaleuceae and Backhousieae portrayed monophyly. Within concatenated
tree, Bayesian and ML tree revealed near similar topology. The minor differences were mostly due
to comparatively lower nodal supports in the maximum likelihood tree compared to the Bayesian
tree e.g. Metrosidereae tribe. Our tree demonstrated tribe-wise clades that was mostly in agreement
with the previously published tress by Wilson et al. (2005), Biffin et al. (2010), and Thronhill et al.
(2015) albeit some topological incongruences. Firstly, the position of the few tribes, e.g.,
Xanthostemoneae and Lophostemoneae that were sister to the rest of Myrtoideae, and Osbornieae
and Melaleuceae that were sister to the rest of Myrtoideae except Xanthostemoneae, and
Lophostemoneae, was other point of discordance. Secondly, other topological difference was in the
position of Leptospermeae-Chamelaucieae clade that was sister to Eucalypteae in previous studies.
These instances of discordance could be largely due to lack of power in out reconstructions since
our reconstruction consisted of a subset of all taxa used in previous studies. However, our study
focuses on the phylogenetic history of the Myrtaceae of WG of India and we are confident that
these discordances would not affect our inferences.
The Fossil calibrated timetree portrayed that the oldest split between Myratceae sensu lato and
outgroups occurred in the Cretaceous (Cenomanian -Turonian) c.a 95.7 Ma (95% HPD: 88.82 -
101.9 Ma) (figure-3, table - 2). Following this, two subfamilies of Myratceae s.l.i.e. Psiloxyloideae
and Myrtoideae emerged in Santonian i.e. around 84.3 Ma (95% HPD: 77.93 - 90.15 Ma) with the
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subsequent origin of the most recent common ancestor (henceforth MRCA) of Myrtoideae
happening in early Maastrichtian (68.83 Ma, 95% HPD: 63.11 - 73.07 Ma). Thereafter, clades of
Myrtoideae continued rapid diversification in middle to late Paleocene (55.4 - 60.28 Ma). Most of
the lower level cladogenesis were very recent, ranging from early to middle Miocene. Indian extant
lineages appeared to be quite a young group born mostly during Miocene except Rhodomyrtus
originating in Oligocene.
2.2 Ancestral area reconstruction
The reconstruction of biogeographic history revealed that the unconstrained model 0 (where
dispersal to and from India was allowed throughout 110 Ma) outperformed other two models (table-
3). Neither the Gondwana relict nor the south-east Asian dispersal model was a supported scenario.
In summary, the best selected model suggested that the ancestors of outgroup taxa and Myrtaceae
s.l. clade perhaps originated in the African landmass (a similar centre of origin was also depicted
for the MRCA of Myrtaceae s.l. clade) (figure- 4). Our analyses further indicated that the Austro-
Africa-Indian landmass was the putative ancestral area for the Myrtoideae subfamily from where all
the major genera have evolved. Afterwards, multiple dispersal events to Asia, South America, India
and neighbouring island groups continued mostly during Eocene-Miocene. Origin of ancestors of
Syzygium spp owed to Australia and dispersal to India was presumably mediated through the Asian
mainland. Likewise, the Indian species of Eugenia portrayed a long distance dispersal from
neotropics in Miocene. In contrast, Rhodomyrtus demonstrated origin in floating Indian landmass in
Oligocene. However, it is noteworthy that many of the internal lower nodes lack strong posterior
support thereby restricting us from making strong assumptions regarding their historical
biogeography and future studies consisting of extensive taxon sampling as well as sequencing of
more molecular markers may provide a better resolution in this regard.
3 Discussion
Myrtaceae, a Gondwana family, is a key element of the south and south-east Asian tropical forests.
The Western Ghats, a refuge of several taxa Myrtaceae, have been little explored to elucidate its bioRxiv preprint doi: https://doi.org/10.1101/2020.04.12.037960; this version posted April 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
history. Our central question on the origin of Myrtaceae diversity in the Western Ghats primarily
sought to test two major hypotheses in historical biogeography, namely vicariance or dispersal. In
the light of the interpretation of the evolutionary signatures of our molecular data, the extant flora
seemed to be dispersed elements and not a derivative of Gondwana vicariance. However, our results
do not entirely refute Gondwanan link as the vibrant fossil records in India are perhaps reminiscent
of an extirpated Gondwanan flora.
3.1 Origin of the extant Myrtaceae lineages in WG
The shallow branches and recent dates of major clades of the sampled taxa indicated younger
lineages of Syzygium and Eugenia. They presumably arrived via dispersal from south-east Asia or
South America. On the other hand, Rhodomyrtus tomentosa appeared relatively older taxa, splitting
early from rest of the Myrteae. It perhaps originated in floating Indian landmass and reached India
via transoceanic dispersal.
The timing of MRCA of Crypteroniaceae, Peneaceae and Myrtaceae s.l.(95.7 Ma) roughly
coincided with the completion of Gondwana break-up which echoed earlier inference (Thornhill et
al. 2015). The dates of two oldest nodes (Myrtaceae stem and crown) in our study were very close
to Biffin et al. (2010) (Myrtaceae stem (94 Ma) and crown (86 Ma)), and Thornhill et al. (2015)
(Myrtaceae stem (91.6 Ma) and crown (85 Ma)) but different from Berger et al. (2016) who
suggested relatively older dates for the Myrtaceae stem and crown while deciphering Myrtales
biogeographic history. The different dates of Berger et al. (2016) possibly occurred due to
dissimilar fossil calibration or incorporation of a large section of other taxa. The Eucalypteae stem
node which was calibrated with an Indian fossil deviated widely from other studies (MRCA the
tribe Eucalypteae showed 33.82 Ma in comparison to 40 Ma in Biffin et al. (2010) and 55.2 Ma in
Thornhill et al. (2015).
Our sampled taxa mostly belonged to two tribes, i.e., Syzygieae and Myrteae. Myrteae, mostly a
South American tribe, radiated during the Eocene to Miocene. According to our dated tree, the
origin of Myrteae crown group happened in Oligocene (31.73 Ma) when Indian plate had collided
bioRxiv preprint doi: https://doi.org/10.1101/2020.04.12.037960; this version posted April 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
with Asia and was staying far away from Africa-Australia-Antarctica. In contrast, early Myrteae
diversification was shown to be 50.7 Ma (Thornhill et al. 2015), and 65.55 Ma (Vasconcelos et al.
2017 using macrofossil dates); however, the timing of Australian and South American Myrteae
crown remained very close to our findings i.e. between 34.7 and 38.4 Ma (Thornhill et al. 2015).
Near similar results were obtained by Biffin et al. 2010 (34 Ma) and by Vasconcelos et al (2017)
(40.76 Ma) implementing microfossil dates. Within this clade, Indian species of Rhodomyrtus
demonstrated an early split in Eocene-Oligocene transition perhaps while floating on Indian
landmass. It is contradictory to south-east Asian origin of Rhodomyrtus macrocarpa which likely
occurred in Oligocene-Miocene (Thornhill et al. 2015; Vasconcelos et al. 2017). It can be attributed
to insufficient taxon sampling of Rhodomyrtus which is not a monophyletic genus with 18 species
disjunctly distributed in southern China, eastern Australia and Caledonia (Snow et al. 2011, Wilson
2011). Majority of the species of Eugenia super-group is pantropical in distribution. Vasconcelos et
al. (2017) have estimated the origin of the crown group of Eugenia in Oligocene with subsequent
rapid radiation in Miocene. Most of such events had occurred in South and central America.
Likewise, Eugenia portrayed an origin in late Miocene (10.42 Ma) in the clade populated with
South American lineages probably suggesting a recent dispersal from neotropics. In Myrtaceae,
many tribes including Myrteae produce succulent fruits which are devoured by birds and bats
(Banack, 1998; Travest et al. 2001). Doing so, they may carry the seeds and promote long-distance
dispersal and establishment of many taxa (henceforth LDDE).
The biogeographic evolution of Syzygium, a predominantly Australasian genus, appeared
unequivocal from the past studies (Thornhill et al. 2015). LDDE has been cited as a likely cause of
divergence between south-east Asian, Indian and Australian Syzygium (Thornhill et al. 2015). It is
possible that multiple stepping stone colonisation from Australia via south-east Asia took place in
the middle to late Miocene. A growing body studies support similar kind of dispersals from
Australia to Asia (Barker et al., 2007; Renner et al., 2010). Those were facilitated by tectonic
movements exerted by the collision of Australian plate with the Philippine and Asian plate.
bioRxiv preprint doi: https://doi.org/10.1101/2020.04.12.037960; this version posted April 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Dispersals from Australasia to Sunda over Oligocene-Miocene were likely mediated through i)
island hop for well-dispersed taxa, ii) chance dispersals, and iii) dispersals of montane taxa (Morley
2003). A sudden increased abundance of Myrtaceae pollen around 17 Ma ascribed to the island
hopping (Muller 1972). Many other plants, e.g., Podocarpaceae followed similar dispersal route to
reach south-east Asia. Presumably, the dominance and the survival of rain-forest in Australia almost
throughout its geologic history and its extension to the neighbouring island groups fuelled biotic
migration (Morley, 2003). The dates of origin of Indian Syzygium fell appropriately in the time
frame to support the view of overland dispersal from south-east Asia, probably via north-east Indian
corridor. We can trace multiple such dispersals during the middle to late Miocene, but insufficient
sampling and low resolvability of the markers have not deciphered the finer details.
Pertinent to say that Indian biological diversity owes significantly to the ‘into-India’ dispersal
(Mani, 1974). Floral massing from south-east Asia by Syzygium seems to reiterate this emerging
pattern. However, a general absence of empirical works impeded us to delineate its biogeographic
significance and to mark it as a common dispersal route.
3.2 Alternate route: floatation on biotic ferry, colonization, and extinction
Although our molecular data supported relatively recent Neogene ‘into-India’ dispersal of extant
lineages, it failed to reconcile with the abundance of Eocene to Miocene fossils in India. The fossil
woods resembling Eucalyptus (E. dharmendrae, E. ghughuensis, Eucalyptoxylon eocenicus) of
Maastrichtian-Danian in Deccan Intertrappean Bed strongly argue in favour of Myrtaceous presence
on Indian Ferry (table - S1). Whereas, the oldest fossil of Australian Eucalyptus appeared in Eocene
which is much younger compared Indian counterpart (Hermsen et al. 2010; Shukla et al. 2012).
The prevalence Cenozoic fossils invoked an ancient origin tracing an alternate route after
Gondwana break-up (figure - 1a-c).Perhaps, proto-Myrtaceae swam on the Indian plate, and/or
arrived via early transoceanic exchange between landmasses via transient land bridges (Chatterjee
& Scotese 1999; Briggs, 2003) e.g. in the mid-Early Cretaceous, when India was nearer to
Australia-Antarctica (Birkenmajer and Zastawniak, 1986; Ali & Aichinson, 2008); and by
bioRxiv preprint doi: https://doi.org/10.1101/2020.04.12.037960; this version posted April 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Cenomanian, Turonian, and Early Coniacian, between African and Indian plate when Madagascar
laid very close (Morley, 2003). Palynological records have provided strong support for such
exchanges especially in plant families like Sapindaceae, Palmae, Myrtaceae (Srivastava 1987/88).
This biotic flux gradually declined with India’s movement away from Madagascar towards
mainland Asia (54-36 Ma). Verdant tropical rain forests during the drifting towards Asia created a
conducive environment for the survival of several angiosperm families on Deccan plate e.g.
persistence of Crypteroniaceae on Indian plate and subsequent out-of-India dispersal (Morley,
2000; Conti et al. 2002; Srivastava, 2011). It also draws further support from the rich dicot
megafossils uncovered across a vast region of central and western India, which forms the Deccan
Intertrappean Bed (Srivastava 2011). The flourishing tropical forest hinted at a climatic condition
with heavy precipitation throughout the year quite similar to present day Western Ghats climate.
The effect of tropical climate was also reflected in wood anatomical characters e.g. lack of growth
rings, and ring porosity due to non-seasonality, simple perforated vessel elements, an absence of
scalariform perforation plates owing to high rates of transpiration (Baas, 1982; Srivastava 2011).
However, the coupled effect of climate change in concert with Deccan volcanism had played a
central role in Cretaceous-Paleogene mass extinction, including the disappearance tropical forest
(Keller et al. 2009; Bajpai, 2009; Renne et al. 2015). The members of Myrtaceae along with several
other angiosperm families presumably faced extirpation from Indian land except for the southern
WG which served as refugia (Prasad et al. 2009). Similar extinction events had been quite common
that tended to obscure robust interpretations. But, rich fossil records, despite an apparent absence of
extant taxa, often invoked as the witness of existence in the past, e.g. fossils of marsupials,
Rhynchocephalia, and plant families such as Araucariaceae and many Podocarpaceae in Northern
Hemisphere (Crisp et al. 2011).
However, an absence of relevant information on various fronts e.g. the span and the intensity of
climatic change, the spatial and temporal severity of the volcanism, and post-volcanism floral
assemblage, constrained our inference. Several remaining questions can be elucidated with species-
bioRxiv preprint doi: https://doi.org/10.1101/2020.04.12.037960; this version posted April 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
level well-resolved phylogeny. Thus, future research based on exhaustive sampling from India as
well as from south-east Asia would allow delineating probable migration route(s), timing, and rate
and potential driver(s) of the lineage diversification.
Data Availability
All the sequence generated in this study deposited in the Genbank (e.g.
https://www.ncbi.nlm.nih.gov/nuccore/KT936455) and the corresponding accession numbers
(KT936455, KT907476-77, KT936449-60, KT907475, KT907475, KT990096, KU049645-47,
KU060784-86, KU060791-93, KU060787-90, KT970714-19, KT982668-76) are added in the
supplementary information.
Acknowledgements
We gratefully acknowledge the suggestions and assistance of the following persons: Vishnu Mukhri
and Srikanth in field sampling; Kerala and Karnataka Forest Department for permitting collection
of specimens. The project was primarily funded by SERB-fast track grant for young researchers
(grant no. SERC/LS-158/2011). BC acknowledges fellowship and computational support from
SEABIG grant (WBS R-154-000-648-646 and WBS R-154-000-648-733). Authors declare no
competing interests.
bioRxiv preprint doi: https://doi.org/10.1101/2020.04.12.037960; this version posted April 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
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TABLES
Table -1: Fossil calibrations used for molecular dating of major nodes of Myrtaceae. All priors are
normally distributed.
Prior mean ± SD Reference
Myrtaceae stem 92 ± 3.5 Biffin et al. (2010)
Myrtaceae crown 85 ± 3.5 Thornhill et al. (2015)
Myrtoideae crown 71.5 ± 3 Thornhill et al. (2015)
Eucalypteae stem 65 ± 7 Shukla et al. (2012)
Table-2: Posterior age distributions (with 95% HPD) of major nodes of Myrtaceae
bioRxiv preprint doi: https://doi.org/10.1101/2020.04.12.037960; this version posted April 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Split between outgroup taxa and Myrtaceae 95.7 Ma, 95% HPD: 88.82 - 101.9 Ma sensu lato
MRCA of Myrtaceae 84.3 Ma, 95% HPD: 77.93 - 90.15 Ma
MRCA of Myrtoideae 68.33 Ma, 95% HPD: 63.12 - 73.07 Ma
Table-3: Results of ancestral area reconstruction using three different models of geographic range
evolution (details of the models described in the text).
Model - loglikelihood Dispersal Extinction Ln(L)
Model 0 (unconstrained dispersal) 146.0 0.022 0.0007
Model 1 (Gondwana element) 152.6 0.021 0.0007
Model 2 (south-east Asian affinity) 152.7 0.022 0.0008
bioRxiv preprint doi: https://doi.org/10.1101/2020.04.12.037960; this version posted April 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Figures
Figure 1: Evolutionary trajectory of Indian Myratceae from a. Late Jurassic: showing putative
distribution of Proto-Myrtaceae, b. Late Cretaceous and c. K/T boundary: Movement of Indian
peninsular mass and biotic exchange with Afro-Madagascar, Austrlia, d. Middle Miocene: India
sutured to mainland Asia and overland dispersal from Australia and south-east Asia (redrawn after
Scotese 2001)
Figure 2: (a) Bayesian and (b) Maximum likelihood tree comprising 81-taxon of Myrtaceae and
four outgroup taxa. Strongly supported nodes (posterior probability > 90%) are marked by stars in
Bayesian tree. The closed squares at the nodes indicate high bootstrap support (>90%) in ML tree.
Indian taxa are highlighted in light green.
Figure 3: Bayesian maximum clade credibility tree of 85 taxa with four outgroup obtained from
BEAST analyses. Nodes with blue node bars representing 95% highest posterior density intervals
(Table 2). The numbers 1-4 represent four calibration points. Bayesian posterior probabilities < 0.9
are indicated by closed circles. A geological time scale is shown at the bottom with geological
epoch abbreviations: Pli, Pliocene; Ple, Pleistocene.
Figure 4: LAGRANGE derived cladogram of ancestral distributions in Myrtean lineages. Letters at
nodes represent putative ancestral-area of the major nodes. The underscore separates the ancestral
area reconstructed for the lower branch (left letter) from the one reconstructed for the upper branch
(right letter) arising from the node. The letters at the node denote landmasses as following: A,
Australia; B, Africa; C, India; D, Southeast Asia (without India); E, South America. Single-area
letters indicate an ancestor restricted to a single area; two-letters indicate an ancestor restricted to
two areas. The colours of the edges denote their current distribution and the same corresponds to the
inset map: Light red - Australia, Light ash - Africa, Sky blue - South America, Light green - India, bioRxiv preprint doi: https://doi.org/10.1101/2020.04.12.037960; this version posted April 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Yellow - south-east Asia. A geological time scale is shown at the bottom with geological epoch
abbreviations: Pli, Pliocene; Ple, Pleistocene.
SUPPLEMENTARY INFORMATION
Table S1: A list of Myrtaceae fossils discovered in India
Table S2 - Genbank accessions of the specimens used this study. * marked specimens are collected
in this study.
Table S3 - Voucher number of the specimens used this study
Table S4 - ITS primer sequences used in this study
Table S5: Distribution matrix used in this study
Table S6: Dispersal matrix used to test various scenarios in LAGRANGE analyses. The letters
represent as following: A, Australia; B, Africa; C, India; D, Southeast Asia (without India); E,
South America. Probabilities of dispersal: 0.01, none or low; 0.25, medium-low; 0.5, medium; 0.75,
medium-high; 1, high.
Figure S1: Bayesian species tree comprising 81-taxon of Myrtaceae and four outgroup taxa.
Strongly supported nodes (posterior probability > 90%) are marked by triangles in Bayesian tree.
bioRxiv preprint doi: https://doi.org/10.1101/2020.04.12.037960; this version posted April 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.12.037960; this version posted April 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.12.037960; this version posted April 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.12.037960; this version posted April 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.12.037960; this version posted April 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.