Article Type: Original Article

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

* [email protected]

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

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

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

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,

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

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

& 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 =

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,

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

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

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.

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

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-

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.

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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

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

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.