Molecular Phylogenetics and Evolution 108 (2017) 34–48
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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier .com/locate /ympev
Biogeographical patterns of Myrcia s.l. (Myrtaceae) and their correlation
with geological and climatic history in the Neotropics
http://dx.doi.org/10.1016/j.ympev.2017.01.012
1055-7903/� 2017 Elsevier Inc. All rights reserved.

⇑ Corresponding author at: Universidade Federal de São Carlos, Campus Sorocaba,
Departamento de Biologia, Rod. João Leme dos Santos (SP 264), km 110, 18052-780
Sorocaba, SP, Brazil.

E-mail addresses: matheus_fs@yahoo.com.br (M.F. Santos), e.lucas@kew.org
(E. Lucas), ptsano@usp.br (P.T. Sano), s.buerki@nhm.ac.uk (S. Buerki), v.staggemeier
@gmail.com (V.G. Staggemeier), f.forest@kew.org (F. Forest).
Matheus Fortes Santos a,b,⇑, Eve Lucas c, Paulo Takeo Sano a, Sven Buerki d,
Vanessa Graziele Staggemeier e, Félix Forest b

aUniversidade de São Paulo, Instituto de Biociências, Depto. Botânica, Lab. Sistemática Vegetal, R. do Matão 277, 05508-090 São Paulo, SP, Brazil
b Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, United Kingdom
cHerbarium, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, United Kingdom
dDepartment of Life Sciences, Natural History Museum, Cromwell Road, London SW7 5BD, United Kingdom
eUniversidade Estadual Paulista Júlio de Mesquita Filho, Instituto de Biociências de Rio Claro, Av. 24A 1515, Caixa Postal 131, 13506-900 Rio Claro, SP, Brazil

a r t i c l e i n f o
Article history:
Received 13 July 2016
Revised 24 November 2016
Accepted 19 January 2017
Available online 1 February 2017

Keywords:
Amazon Forest, Atlantic Forest
Calyptranthes
Caribbean
Guiana Shield
Plant diversity
a b s t r a c t

Many recent studies discuss the influence of climatic and geological events in the evolution of
Neotropical biota by correlating these events with dated phylogenetic hypotheses. Myrtaceae is one of
the most diverse Neotropical groups and it therefore a good proxy of plant diversity in the region.
However, biogeographic studies on Neotropical Myrtaceae are still very limited. Myrcia s.l. is an informal
group comprising three accepted genera (Calyptranthes, Marlierea and Myrcia) making up the second lar-
gest Neotropical group of Myrtaceae, totalling about 700 species distributed in nine subgroups.
Exclusively Neotropical, the group occurs along the whole of the Neotropics with diversity centres in
the Caribbean, the Guiana Highlands and the central-eastern Brazil. This study aims to identify the time
and place of divergence of Myrcia s.l. lineages, to examine the correlation in light of geological and cli-
matic events in the Neotropics, and to explore relationships among Neotropical biogeographic areas. A
dated phylogenetic hypothesis was produced using BEAST and calibrated by placing Paleomyrtinaea
princetonensis (56 Ma) at the root of the tree; biogeographic analysis used the DEC model with dispersal
probabilities between areas based on distance and floristic affinities.Myrcia s.l. originated in the Montane
Atlantic Forest between the end of Eocene and early Miocene and this region acted as a secondary cradle
for several lineages during the evolution of this group. The Caribbean region was important in the diver-
sification of the Calyptranthes clade while the Guayana shield appears as ancestral area for an older sub-
group of Myrcia s.l. The Amazon Forest has relatively low diversity of Myrcia s.l. species but appears to
have been important in the initial biogeographic history of old lineages. Lowland Atlantic Forest has high
species diversity but species rich lineages did not originate in the area. Diversification of most subgroups
of Myrcia s.l. occurred throughout the Miocene, as reported for other Neotropical taxa. During the
Miocene, geological events may have influenced the evolution of the Caribbean and Amazon forest lin-
eages, but other regions were geological stable and climate changes were the most likely drivers of diver-
sification. The evolution of many lineages in montane areas suggests that Myrcia s.l. may be particularly
adapted to such environments.

� 2017 Elsevier Inc. All rights reserved.
1. Introduction

The Neotropical region encompasses a wide range of geological
and geomorphological formations, including young landscapes
such as the Amazon basin (Neogene; Hoorn et al., 2010a), as well
as old landscapes such as the Espinhaço Range (Proterozoic;
Saadi, 1995). As a result of the large range of latitude (ca. 76�)
and elevation (ca. 7000 m), Neotropical climatic diversity is exten-
sive (Köppen, 1948). Plant biodiversity is congruent with such
environmental diversity; the Neotropics boasts enormous varia-
tion in vegetation types (Cabrera and Willink, 1980) and corre-
spondingly high taxonomic diversity totalling between 90,000
and 110,000 species (Antonelli and Sanmartín, 2011). Recent stud-
ies have evaluated the influence of geological and climatic events

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mailto:matheus_fs@yahoo.com.br
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mailto:ptsano@usp.br
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mailto:v.staggemeier @gmail.com
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mailto:f.forest@kew.org
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M.F. Santos et al. /Molecular Phylogenetics and Evolution 108 (2017) 34–48 35
in the generation of the Neotropical plant diversity. Congruence
between these events and time and place of lineage origin has been
tested through dated phylogenies and biogeographic analyses
(Antonelli et al., 2009; Perret et al., 2013; Fine et al., 2014).

The main geological events influencing Neotropical biogeogra-
phy were the isolation of South America, Andean uplift and the clo-
sure of the Isthmus of Panama (Burnham and Graham, 1999). The
connection between South America, Antarctica and Australia and
their subsequent separation after the isolation of Antarctica influ-
enced the early diversification of Neotropical groups, including
Myrtaceae (Sytsma et al., 2004; Lucas et al., 2007; Buerki et al.,
2011; Labiak et al., 2014). The Andean uplift had multiple conse-
quences for the Neotropical landscape such as the formation of
the Amazon basin, the Pebas system and the creation of new habi-
tats (e.g., white sand and flooded soils), which influenced the
diversification of different groups (Antonelli et al., 2009; Hoorn
et al., 2010a, 2010b; Roncal et al., 2013; Fine et al., 2014). Closure
of the Isthmus of Panama also had impact on the evolutionary his-
tory of plant taxa, allowing intense floristic exchange between
South America and Mesoamerica (Richardson et al., 2001; Bacon
et al., 2013).

Global transition to colder climates during the Paleogene and
Neogene (Zachos et al., 2001; Graham, 2010) influenced the diver-
sification of groups in different biomes (Antonelli et al., 2010;
Hughes et al., 2013). The two highly species diverse biomes of
Central-Eastern Brazil (Atlantic Forest and Cerrado) are located in
an area that has undergone little geological change but significant
climatic changes during the Cenozoic. Pleistocene climate changes
in the Atlantic Forest (Carnaval and Moritz, 2008) are mentioned as
important events that drove the evolution of recent lineages of
plants (Martins, 2011). Climatic fluctuations of the Neogene and
Quaternary connected the Northern Atlantic and Amazon forests,
resulting in conflicting biogeographic relationships between the
Amazon forest and the northern and southern parts of the Atlantic
forest (Perret et al., 2006; Santos et al., 2007; Fiaschi and Pirani,
2009). Diversification of Cerrado taxa is hypothesized to be linked
to climatic changes (low precipitation and high climatic seasonal-
ity), expansion of C4 grass and fire events (Simon et al., 2009;
Simon and Pennington, 2012).

Plant evolution of other highly diverse Neotropical regions also
show biogeographical patterns. Taxa from the Guiana Shield
emerge from deep nodes in phylogenetic hypothesis and the
Guayana region has been found as an ancestral area in some groups
(Givnish et al., 2004, 2011; Struwe et al., 2009; Roncal et al., 2013).
Biogeographic relationships between Guayana and Brazilian mon-
tane taxa and correlation with the separation of the Guayana and
Brazilian shields has been suggested (Givnish et al., 2011). Origin
of Caribbean taxa has been explained both by a Northern origin
or a Southern origin through long-dispersal, for different groups
(Santiago-Valentín and Olmstead, 2004; Fine et al., 2014).

Several plant groups have been the focus of biogeographic stud-
ies but many remain poorly studied such as Myrtaceae and most of
its genera, including the large Myrcia s.l. (700 spp.; Govaerts et al.,
2016). Lucas et al. (2007) studied the biogeography of Myrteae
using a Dispersal vicariance analysis (DIVA; Ronquist, 1997), but
did not find an unequivocal ancestral area for Myrcia s.l. and inter-
nal relationships were not discussed. Biffin et al. (2010) suggested
a rapid diversification of Myrcia s.l. in the Early Miocene, but again
internal relationships were not considered. Staggemeier et al.
(2015) studied Myrcia sect. Aulomyrcia (clade 9 of Lucas et al.,
2011) and found lower extinction rates and accumulation of spe-
cies inside Pleistocene forest refugia of the Atlantic Forest. Lucas
et al. (2011) speculated that after the colonization of the southern-
most parts of South America by Myrteae ancestors, Myrcia s.l.
ancestors migrated north and east through tropical forests and
subsequently dispersed northward. Andean uplift and its
consequences in the continental vegetation would have been con-
comitant with the early Miocene diversification of Myrcia s.l. in the
Atlantic and northwest Amazon forests, as well as in the grasslands
between them; the formation of the Isthmus of Panama would
have allowed the migration of Caribbean taxa (Lucas et al., 2011).

Groups with wide distribution and high species diversity in the
Neotropical region allow the examination of an overall relationship
between different biogeographic regions. The high diversity of
Myrcia s.l. suggests that it is a potential proxy of the plant biogeog-
raphy in the region, as the entire Myrtaceae in Australasia (e.g.,
Ladiges et al., 2003). Therefore, the aims of the study are: (1) to
identify the time and place of divergence in Myrcia s.l. lineages
focusing in the subgroups diagnosed by Lucas et al. (2011); (2) to
examine the spatial and temporal relationship between biogeo-
graphic patterns of Myrcia s.l. and geological and climatic events
during the Cenozoic in Neotropics; (3) to explore relationship
among biogeographic areas of Neotropics.
2. Materials and methods

2.1. Taxonomy and diversity of Myrcia s.l

Myrcia s.l. (Fig. 1) is an informal group proposed by Lucas et al.
(2007; as the Myrcia group) in a phylogenetic study of tribe Myr-
teae (sensu Wilson et al., 2005). Myrcia s.l. is the second largest
Neotropical group of Myrtaceae (Govaerts et al., 2016). Exclusively
Neotropical, Myrcia s.l. is distributed along the whole region with
diversity centres in the Caribbean, the Guiana Highlands and the
central-eastern Brazil (McVaugh, 1968, 1969). It includes three
accepted genera: Calyptranthes Sw., Marlierea Cambess. and Myrcia
DC. (Govaerts et al., 2016). Lucas et al. (2011) conducted a phyloge-
netic study with these genera and diagnosed nine clades, showing
that Marlierea and Myrcia are polyphyletic, while Calyptranthes is
monophyletic but embedded within the former two. These nine
clades of Myrcia s.l. form the basis of an infrageneric classification
with nine sections that will include Calyptranthes and Marlierea
under Myrcia. These nine clades are referred from here forward
as Myrcia s.l. subgroups.

The diversity of the nine clades of Lucas et al. (2011) is not
equally divided among the diversity centres of Myrcia s.l. Clade 1
(similar to Calyptranthes) contains about 280 spp. with its main
diversity in the Atlantic Forest, Guiana Highlands and Caribbean
(McVaugh, 1969; Lucas et al., 2011; Govaerts et al., 2016). Clades
2 (ca. 20 spp.) and 3 (ca. 60 spp., similar to the genus Gomidesia
O.Berg) occur mainly in the Atlantic Forest (Lucas et al., 2011;
Govaerts et al., 2016; Sobral et al., 2016). Clade 6 (ca. 20 spp.)
has its species concentrated in the Atlantic Forest and associated
montane open vegetation (Lucas et al., 2011). Clade 7 (=Myrcia
sect. Sympodiomyrcia; ca. 20 spp.) is distributed in montane areas
of Atlantic Forest and Campo Rupestre vegetation with three dis-
junct species in the Guayana Region (Santos et al., 2016). Clade 9
(=Myrcia sect. Aulomyrcia; ca. 130 spp.) has its diversity divided
between the Atlantic Forest, Amazon Forest and Guiana Highland
(Lucas et al., 2016). Clades 4 (ca. 50 spp.), 5 (ca. 100 spp.) and 8
(ca. 10 spp.) have their diversities equally spread throughout the
distribution of Myrcia s.l. (Lucas et al., 2011).
2.2. Sampling and data collection

Molecular sequences were accessed from previous phylogenetic
studies in Myrteae and Myrcia s.l. (Lucas et al., 2007, 2011;
Staggemeier et al., 2015; Santos et al., 2016; Wilson et al., 2016).
One nuclear and four chloroplast DNA markers were used (supple-
mentary Table 1 of Appendix A). These respectively, were ITS, ndhF,
psbA-trnH, trnL-trnF and trnQ-50rps16. The sample of Myrcia s.l.



Fig. 1. Morphological features of Myrcia s.l. A, habit of Myrcia eriocalyx DC.; B, bark of Myrcia megaphylla M.F.Santos & Sobral with papyraceous exfoliation; C, dichotomic
branching of Calyptranthes pulchella DC.; D, pubescent leaves with decussate phyllotaxy of Myrcia lasiantha DC.; E, inflorescence with flower buds of Myrcia sp.; F,
inflorescence with flower buds and open flowers of Myrcia mutabilis (O.Berg) N. Silveira; G, fruits of Myrcia stricta (O.Berg) Kiaersk; H, reddish leafy embryo of Myrcia
ferruginosa Mazine.

36 M.F. Santos et al. /Molecular Phylogenetics and Evolution 108 (2017) 34–48
species used is the largest assembled to date; all available
sequences of Myrcia s.l. were considered. In total, 182 samples
(173 species) were used including taxa from all nine subgroups
and 13 outgroup taxa for improved accuracy of molecular dating
(supplementary Table 2 of Appendix A). Sampling covers around
25% of Myrcia s.l. species diversity, which is acceptable representa-
tion because all clades and biogeographic regions are represented
in the phylogenetic analysis. Sampling covers around 50% of the
central-eastern Brazil diversity centre (areas D-G and I, see Sec-
tion 2.4), ca. 15% for the Caribbean, Mesoamerica and NW South
America; ca. 20% for Amazon forest; ca. 30% for Guayana region
(percentages based on the databases of Govaerts et al., 2016 and
Sobral et al., 2016).
2.3. Data edition and phylogenetic analysis

Sequences were aligned using the default parameters of Muscle
(Edgar, 2004) conducted on Geneious 6.1.6 (Drummond et al.,
2013). Final alignments were visually checked to minimise number
and size of gaps. Models of nucleotide substitution were calculated
for each marker using MrModeltest2 version 2.2 (Nylander, 2004)
implemented using PAUP⁄ 4.0b2 (Swofford, 2002). Selected mod-
els under the Akaike Information Criterion were: GTR + I + G for
ITS, psbA-trnH and trnQ-50rps16; and GTR + G for ndhF and trnL-
trnF.

Bayesian analysis was conducted in BEAST 1.8.0 (Drummond
and Rambaut, 2007) using the combined data set. Site and clock



M.F. Santos et al. /Molecular Phylogenetics and Evolution 108 (2017) 34–48 37
models were implemented independently for each region. The
prior substitution rate was defined as uniform and molecular clock
model as lognormal relaxed clock (uncorrelated) for all partitions.
Tree priors were set as a Yule speciation process. Myrtus communis
was assigned as the outgroup taxon and the internal group was set
as monophyletic.

Calibration of phylogenetic trees is a subjective process that
varies depending on the practitioner. In this study the tree was cal-
ibrated using Paleomyrtinaea princetonensis Pigg, Stockey & Max-
well (Pigg et al., 1993). The study followed Staggemeier et al.
(2015) and placed this fossil at the root of the tree, using a lognor-
mal distribution with median age of 56 Ma, offset to 55 Ma with a
standard deviation of 1.0. An older fossil assigned to Myrteae
(Myrceugenelloxylon antarcticus Poole, Hunt & Cantrill), from ca.
72–66 Ma (Maastrichtian age – Upper Cretaceous; Poole et al.,
2003) was also considered. However, the absence of diagnostic
characters of Myrtaceae wood, such as presence of vestured pits
(Oskolski, pers. comm.), led us to disregard this fossil. The fossil
wood of Myrceugenia chubutense Ragonese (1980) was not used
because its radially extended vessel-ray pits that are said to be
diagnostic for Myrceugenia have been found in other Myrtaceae
genera from temperate parts of South America such as Luma
(InsideWood, 2004-onwards) meaning that the fossil would need
to be placed at the root of Myrteae; such placement produces sim-
ilar nodes ages to those found here (Vasconcelos et al., 2017).
Murillo-A et al. (2016) use Myrceugenia chubutense to calibrate
Myrceugenia and Myrceugenelloxylon antarcticus to calibrate the
node from which Luma arises, thereby greatly increasing node ages
beyond those returned by any other study. Fossil pollen employed
by Thornhill et al. (2012) is not used here as those authors state
that using macrofossil or macrofossil plus pollen fossil produces
congruent results. Further justification for the calibration used
here is from Vasconcelos et al. (2017) who present a comprehen-
sive comparative review of the use of fossils for calibration in
Myrteae and find similar results when conservatively using macro-
fossils and pollen; those authors find node dates that match those
presented here.

Bayesian analysis was conducted with 400 million generations
of Monte Carlo Markov chains and sampling frequency of 1000.
The file with matrix and parameters was assembled in BEAUti
Fig. 2. Biogeographic areas defined for this study. Border
1.8.0 (Drummond and Rambaut, 2007). Analysis in BEAST was
run on the CIPRES portal (Miller et al., 2010). Outcomes of Bayesian
analysis were visualized in Tracer 1.4.1 (Rambaut et al., 2013) to
examine convergence and sufficiency of effective sample size
(i.e., higher than 200), ensuring good representation of the poste-
rior distributions. A maximum clade credibility tree with median
branch lengths and a 95% highest posterior density interval on
nodes was generated in TreeAnnotator 1.8.0 (Drummond and
Rambaut, 2007) using the standard burn-in of 10% (40,000 trees
in this case).

2.4. Delimitation of biogeographic areas

The biogeographic areas proposed by Cabrera and Willink
(1980) and Morrone (2006, 2014) are the basis of the areas delim-
ited here (Fig. 2). These were modified to adjust area limits to the
diversity of Myrcia s.l., to incorporate evidence of biogeographic
patterns from other studies, and to minimise the number of areas,
due to restrictions of the DEC analysis (see Ree and Smith, 2008).
All areas include different types of vegetation, but the discussion
is focused in the main type (in terms of area) that occurs in each
area unless clearly stated. The concept of Neotropical region fol-
lows Morrone (2006).

A. Caribbean. This area is formed by the Antilles area of
Morrone (2014) that includes the islands of the Caribbean Sea.

B. Amazon Forest. This area includes the South-eastern Amazo-
nia, the South-western Amazonia and part of the Northern Amazo-
nia (except the upper part of Rio Negro basin) of Morrone (2014).
The fusion of Amazon areas is due to the low level of endemism
in Myrcia s.l. (McVaugh, 1968, 1969).

C. Guayana Region. This area is composed of the Guiana High-
lands and surrounding lowland areas including the upper part of
Rio Negro basin and North-eastern Venezuela, Guiana, Suriname
and part of French Guiana. This delimitation follows the Guayana
Region of Berry et al. (1995) and covers most of the Guiana Shield.
The unit includes Venezuela, removed from North-western South
America and the Rio Negro basin removed from Northern Amazo-
nia (Morrone, 2014). Guayana region was proposed as an indepen-
dent area due to high levels of Myrcia s.l. endemism in the region
(McVaugh, 1968, 1969).
s of South and Central America countries are shown.



38 M.F. Santos et al. /Molecular Phylogenetics and Evolution 108 (2017) 34–48
D. Cerrado. This area includes only the savanna vegetation of
the Chaco (sensu Morrone, 2014). Chaco vegetation was excluded
as none of the species in the analysis occurs in this vegetation type
(Govaerts et al., 2016; Sobral et al., 2016).

E. Campo Rupestre. This area is composed of Campo Rupestre
vegetation (Giulietti and Pirani, 1988; Harley, 1995; Rapini et al.,
2008; Silveira et al., 2016) that occurs in the Chaco (sensu
Morrone, 2014). This vegetation is found in quartzitic montane
areas of Central-eastern Brazil (Giulietti and Pirani, 1988). Despite
similarity between Campo Rupestre and Cerrado (both open areas,
i.e., without canopy), environmental and floristic differences
(Harley, 1995; Rapini et al., 2008) support their consideration as
distinct areas. There is significant difference in Myrcia s.l. species
composition between these two vegetation types (e.g., Clade 7;
Santos et al., 2016).

F. Montane Atlantic Forest (MAF). This area includes the
southern part of the Paraná unit of Morrone (2014), from the
Mixed Rain Forest (the Araucaria angustifolia Forest Province of
Morrone, 2006) to the region around the mouth of the Rio Doce.
Unlike the northern part of Atlantic forest that is a narrow strip
along coastal areas (Fiaschi and Pirani, 2009), the southern part
of that Atlantic Forest encompasses large areas of inland semi-
deciduous forest (Oliveira-Filho and Fontes, 2000; Oliveira-Filho
et al., 2006). This unit includes most montane Atlantic Forest
although some Northern montane areas are included in the Low-
land Atlantic Forest unit as available surveys suggest a higher
floristic similarity with the surrounding lowland forests
(Oliveira-Filho et al., 2006; Santos et al., 2007).

G. Lowland Atlantic Forest (LAF). This area corresponds to the
northern part of the Paraná unit of Morrone (2014) mostly includ-
ing lowland Atlantic Forest (a rainforest usually referred as Tab-
uleiro Forest) that occurs from the North of Rio de Janeiro State
(Brazil) to the northernmost limit of Atlantic Forest (Thomas and
Barbosa, 2008; Fiaschi and Pirani, 2009). The North/South Atlantic
Forest division has been highlighted by previous studies
(Mori et al., 1981; Thomas et al., 1998; Perret et al., 2006;
Murray-Smith et al., 2008). The northernmost Atlantic Forest is
also considered a distinct centre of endemism (the Pernambuco-
Alagoas Centre; Thomas et al., 1998) but is not separated here
due to low levels of Myrcia s.l. diversity and endemism.

H. Mesoamerica and NW South America. This area unites
Mesoamerica and the North-Western South America units of
Morrone (2014). Both areas have low diversity of Myrcia s.l.
(Govaerts et al., 2016; Sobral et al., 2016) and are included here
to accommodate few non-endemic species. The biogeographic
affinity of these areas was documented by Morrone (2014).

I. Caatinga. This area includes only the Caatinga vegetation of
the Chaco (sensuMorrone, 2014). Like the previous region, Caatinga
boasts few species of Myrcia s.l. (Govaerts et al., 2016; Sobral et al.,
2016) and is included to house non-endemics.
Table 1
Dispersal constraints used in the model with selected parameters.

Caribbean Amazon F. Guayana R. Cerrado

Caribbean – 0.1 0.5 0.001
Amazon F. – – 1.0 1.0
Guayana R. – – – 1.0b

Cerrado – – – –
CR – – – –
MAF – – – –
LAF – – – –
Meso. & NW SA – – – –
Caatinga – – – –

F.: Forest; R.: Region; CR: Campo Rupestre; MAF: Montane Atlantic Forest; LAF: Lowland A
Extra-Neotropical.

a Non-adjacent biogeographic areas, but with floristic connections.
b Non-adjacent biogeographic areas, but distance <2000 km from each other.
J. Extra-Neotropics. Luma apiculata,Myrceugenia ovata,Myrceu-
genia planipes and Myrtus communis are outgroup taxa with extra-
Neotropical distributions that occur in other biogeographic
regions. The first three taxa occur in Central Chile while Myrtus
communis is endemic to the Mediterranean. Placing them all in this
single generic category minimises the total number of biogeo-
graphic units required.

2.5. Biogeographic analysis

The dispersal-extinction-cladogenesis model (DEC; Ree and
Smith, 2008) was used to reconstruct the biogeographic history
of Myrcia s.l. over the phylogenetic hypothesis. A binary matrix
of species presence/absence per biogeographic area was compiled
(supplementary Table 2 of Appendix A) using data from Sobral
et al. (2016), Govaerts et al. (2016), CRIA (2016) and herbarium
collections.

DEC analysis was carried out using RASP 3.2 (Yu et al., 2015).
Two models were used, one with default parameters (MDP) and
other with selected parameters (MSP); in doing this, we aimed to
analyse the influence of parameter choice in the analysis. The num-
ber of ancestral areas (‘‘maximum range size”) was implemented
as two, preventing high numbers that might result in slower anal-
ysis and less resolution. Ancestral areas were estimated for all
nodes. In the MSP, the range constraints limited ancestors to
inhabiting only the combination of biogeographic units with dis-
persal constraints equal or bigger than 0.5 (Table 1).

Dispersal constraints were based primarily on area proximity
with evidence of floristic connections as auxiliary criteria (Table 1).
Dispersal values between adjacent units or non-adjacent
units with past or present floristic connections (i.e., Amazon
Forest �MAF and LAF) were set up as 1.0 with exceptions for the
Guayana Region and Campo Rupestre whose dispersal values were
set at 0.5 despite the floristic connection (Giulietti and Pirani,
1988), due to substantial geographic separation. Non-adjacent
areas with small geographic distances (<2000 km, e.g., Amazon
Forest � Campo Rupestre, Guayana Region � Cerrado) were also
assigned dispersal values of 1.0. No dispersal value was assigned
as zero due to reports of Neotropical long-distance dispersals
(e.g., Givnish et al., 2004). Probabilities of areas being ancestral
are represented as pie charts on the resulting chronogram; these
indicate the areas with probability higher than 0.5 and unite the
others into a single category.

Time slices were not implemented in the biogeographic analysis
as each area has likely existed with similar connections during the
period covered by the molecular dating (Eocene to the Holocene).
For example, the Caribbean was connected to South America dur-
ing the Paleogene and Neogene (Gentry, 1982; Iturralde-Vinent
and MacPhee, 1999; Graham, 2010) making it difficult to establish
different parameters throughout the period. Despite geological
CR MAF LAF Meso. & NW SA Caatinga EN

0.001 0.001 0.001 1.0 0.001 0.001
1.0b 1.0a 1.0a 1.0 1.0 0.001
0.5a 0.001 0.1 1.0 0.1 0.001
1.0 1.0 1.0 0.001 1.0 0.001
– 1.0 1.0 0.001 1.0 0.001
– – 1.0 0.001 1.0 0.001
– – – 0.001 1.0 0.001
– – – – 0.001 0.001
– – – – – 0.001

tlantic Forest; Meso.: Mesoamerica; NW SA: Northern-Western South America; EN:



M.F. Santos et al. /Molecular Phylogenetics and Evolution 108 (2017) 34–48 39
changes in the Amazon Forest during the Neogene (Hoorn et al.,
2010b), forest areas were present in the region since the Paleocene
(Jaramillo et al., 2010; Graham, 2012). The Guayana Region has
undergone geomorphological changes during the Neogene but at
least some regions have been exposed since the Paleogene
(Wesselingh et al., 2010). Simon et al. (2009) suggest the expansion
of C4 grasses around 4 Ma was the main cause of Cerrado emer-
gence, but other evidence suggests similar vegetation in central
South America considerably earlier (Van der Hammen, 1983). Pale-
oenvironmental records for Campo Rupestre are scanty, but sur-
faces in the Espinhaço range, the core area of this vegetation
type, have been exposed since the Mesozoic (Saadi, 1995;
Almeida-Abreu and Renger, 2002). Little data exists concerning
MAF and LAF, but the tropical forest is believed to have been pre-
sent in these areas since the Paleogene (Morley, 2000; Burnham
and Johnson, 2004).
3. Results

3.1. Phylogenetic analysis

The ESS of all markers in the Bayesian analysis exceeded 200,
indicating they had a good statistical sampling of the posterior dis-
tributions of the data. The cladogram resulting from Bayesian anal-
ysis (Fig. 3) shows Myrcia s.l. to have been recovered as
monophyletic and sister to the Plinia Group. Myrcia s.l. subgroups
were recovered with PP higher than 0.9, except clade 9, which
was separated in two groups (Fig. 3). A clade formed by Myrcia
aff. maximiliana O.Berg and Myrcia robusta Sobral (PP 1.0) did not
show affinity with any clade proposed by Lucas et al. (2011). Other
clades receiving PP higher than 0.9, include a large clade composed
by the clades 1, 2, 3, 4, 6, 7 and 8.

3.2. Molecular dating of nodes

Intervals of 95% Highest Posterior Density (HPD) of divergence
for nodes with PP higher than 0.9 are shown in Fig. 3; those dis-
cussed are summarised in Table 2. The 95% HPD intervals are wide,
likely due to the use of a single fossil for calibration. The crown
node of Myrcia s.l. has an age of 36.1–21.1 Ma (95% HPD), indicat-
ing an origin between end of Eocene and early Miocene. Ages of the
Myrcia s.l. subgroups varied with the oldest reported for part of
clade 9 (9b) at 29.0–15.9 Ma (95% HPD; end of Oligocene to middle
Miocene) and the youngest, clade 8 at 9.8–2.5 Ma (95% HPD; end of
Miocene to end of Pliocene).

Despite the relatively large variation, all nodes fall in the Neo-
gene except the upper 95% HPD limit for clades 1 and 9. Node ages
within the Myrcia s.l. subgroups are also from the Neogene, with
rare diversifications in the Quaternary. Node ages inside the Qua-
ternary are found mainly in clades originating from most recent
nodes with low statistical support.

3.3. Comparison of model parameters

The biogeographic hypotheses generated by the MDP (supple-
mentary Figs. 1 and 2 of Appendix A) and MSP (Fig. 4) return the
same overall patterns but with meaningful differences. Likelihood
of MSP was higher than likelihood of MDP (�lnL = 453.4 and
–lnL = 491.8, respectively). Under MSP, constraining range and dis-
persal resulted in fewer ancestral areas per node, allowing clearer
hypothesis of ancestral areas. The constraints also decreased the
probability of unlikely combinations of ancestral areas (e.g., Carib-
bean plus MAF). Section 3.4 and the discussion of this work are
based on the biogeographic hypothesis generated using MSP as it
incorporates more realistic biogeographic scenarios.
3.4. Biogeographic reconstruction

The biogeographic hypothesis generated from the MSP indi-
cates many nodes with high probability of ancestral area(s)
(Fig. 4; Table 2). A major finding is that MAF is highly likely to
be the ancestral area for most Myrcia s.l. subgroups (clades 2, 3,
6 and 7) (Figs. 3 and 4; Table 2). Some inclusive clades (e.g., node
261; Table 2) also have MAF as the most likely ancestral area.

The most likely ancestral area of clade 9b is the Guayana Region
plus the Amazon Forest. The Amazon Forest and Caribbean appears
to have been important areas during the diversification of older
lineages of Calyptranthes (Fig. 4; Table 2). Campo Rupestre emerges
as ancestral areas of some internal clades of the groups of Myrcia s.
l. (e.g., nodes 295 and 213, respectively), while the Cerrado appar-
ently did not have major importance in the diversification of Myr-
cia s.l. (Fig. 4; Table 2). The oldest lineages of Myrcia s.l. tend to
have less resolved ancestral areas, a shortcoming reported in other
biogeographic analyses using the DEC model (Buerki et al., 2011).
According to the analysis, the main process driving the biogeo-
graphic history is dispersal (global cost = 240), while vicariance
and extinction had small influences (global cost 29 and one,
respectively).
4. Discussion

4.1. Phylogenetic analysis

Results broadly support those presented in previous papers
(Lucas et al., 2011; Staggemeier et al., 2015). Low support for clade
9 and its division into two clades (clades 9a and 9b) was highly
supported; this agrees with previous studies (Staggemeier et al.,
2015; Santos et al., 2016) who also found low support for clade 9
(PP 0.5); however, Lucas et al. (2011) reported a PP higher than
0.9. Clade 9 is morphologically consistent and supported by a diag-
nostic combination of characters (Lucas et al., 2016); further inves-
tigation is required to understand whether clade 9 is a single
lineage or two lineages with independent evolutionary history.
Inclusion of more taxa and markers provided higher support for
phylogenetic structure within the Myrcia s.l. subgroups and for
relationships between them.
4.2. The southern origin of Myrcia s.l. and rapid expansion to northern
South America

Tribe Myrteae originated as part of the temperate flora of Aus-
tralasia during the Paleocene (Sytsma et al., 2004; Lucas et al.,
2007) and the ‘‘Antarctica route” was the likely path to the Amer-
icas (Pennington and Dick, 2004; Buerki et al., 2011; Labiak et al.,
2014). After the opening of the Drake Passage and with the pro-
gressive isolation of Antarctica, Neotropical Myrtaceae were sepa-
rated from the Australasian group (Sytsma et al., 2004; Lucas et al.,
2007). The biogeographic hypothesis presented here shows MAF
with the highest probability (0.50; Table 2) of being the site of ini-
tial diversification of Myrcia s.l. The southern position of Atlantic
Forest is congruent with the migration of Neotropical Myrteae
ancestors through Antarctica. The ancestral area of the clade com-
prising most Myrcia s.l. subgroups (node 261) is MAF, while clades
1, 5, 9a and 9b apparently have more northern origins (Fig. 4;
Table 2). The most likely subsequent scenario is diversification in
southeast South America concurrent with an expansion to the
north. Other Neotropical groups have followed a similar path, such
as Alstroemeria (Alstroemeriaceae), Bignonieae (Bignoniaceae),
Escallonia (Escalloniaceae), Rhipsalis (Cactaceae) and Weinmannia
(Cunnoniaceae) (Fiaschi and Pirani, 2009; Calvente et al., 2011;
Lohmann et al., 2013). The results are congruent with the rapid



Fig. 3. Majority rule consensus cladogram of Bayesian analysis with five molecular markers run in BEAST. Posterior Probabilities above 0.5 are shown in the nodes, as well as
the 95% High Posterior Density (HPD). The fossil used for calibration, Paleomyrtinaea princetonensis, was placed at the root of the tree. Subgroups of Myrcia s.l. diagnosed by
Lucas et al. (2011) are highlighted; clade 9 was divided in clade 9a and 9b. Full circles indicate crown nodes of these groups. Pre. = Prevost; Stagg. = Staggemeier.

40 M.F. Santos et al. /Molecular Phylogenetics and Evolution 108 (2017) 34–48
diversification of Myrcia s.l. proposed by Biffin et al. (2010) and the
biogeographic scenario proposed by Lucas et al. (2011).

Vegetation records from the Atlantic Forest during the
Paleogene and Neogene are scanty. Forests are suggested to have
covered MAF during the period encompassed by the crown node
age of Myrcia s.l. (95% HPD, 36–21 Ma) (Morley, 2000; Burnham
and Johnson, 2004), and the ancestor of Myrcia s.l. might have
inhabited them. Pollen and macrofossils indeed indicate a wet



Fig. 3 (continued)

M.F. Santos et al. /Molecular Phylogenetics and Evolution 108 (2017) 34–48 41



Table 2
High Posterior Density (95% HPD) age and the most probable ancestral area
(probability in parenthesis) of nodes discussed in the text. Clades 1–8 are delimited
according to Lucas et al. (2011); the clade 9 was divided in clade 9a and clade 9b.
HAP: highest ancestral area probability. A. Caribbean; B. Amazon Forest; C. Guayana
Region; D. Cerrado (savanna vegetation); E. Campo Rupestre; F. Montane Atlantic
Forest; G. Lowland Atlantic Forest.

Clade/node 95% HPD (Ma) HAP

Node 172 (Clade 6) 19.1–6.1 EF (0.64)
Node 175 (Clade 8) 9.8–2.5 F (0.64)
Node 184 (Clade 2) 17.1–8.2 F (1.00)
Node 186 15.7–6.4 G (0.56)
Node 197 (Clade 3) 18.3–9.0 F (0.82)
Node 205 9.1–2.2 D (0.70)
Node 209 (Clade 4) 15.7–6.9 DF (0.44)
Node 213 21.2–8.7 B (0.46)
Node 215 15.7–5.6 A (1.00)
Node 219 18.5–8.8 AB (0.77)
Node 226 18.3–8.8 B (0.36)
Node 230 16.1–6.1 A (1.00)
Node 231 22.8–12.6 AB (0.84)
Node 232 (Clade 1) 26.9–14.9 B (0.61)
Node 244 4.1–1.0 E (1.00)
Node 258 (Clade 7) 16.0–7.3 F (0.58)
Node 261 32.2–18.6 F (0.85)
Node 279 (Clade 9a) 24.2–12.6 FG (0.53)
Node 293 (Clade 5) 22.6–11.9 BF (0.53)
Node 295 7.7–2.0 E (0.81)
Node 300 9.4–3.2 G (0.72)
Node 305 24.4–11.6 B (0.21)
Node 316 (Clade 9b) 29.0–15.9 BC (0.33)
Node 319 (Myrcia s.l.) 36.1–21.1 F (0.50)

42 M.F. Santos et al. /Molecular Phylogenetics and Evolution 108 (2017) 34–48
and warm forest, but also with evidences of dry periods with open
vegetation (Lima, 1991; Biagolini et al., 2013). Connection of south-
ern and northern South American forests may have occurred via
different routes. Records of forests in Patagonia (Ortiz-
Jaureguizar and Cladera, 2006) indicate that the climate of South
America was wet and warm throughout most of its range during
the Oligocene-Middle Miocene, the diversification period of the
first lineages ofMyrcia s.l. This climate would have promoted forest
development across the continent allowing migration to northern
forests, a scenario proposed by Lucas et al. (2011).

A montane migration may have been via the Andes Mountains,
which reached current elevations only after the late Miocene
(Graham, 2011; Hughes et al., 2013). Before this period, the region
was covered by montane forests (Graham, 2010). Before reaching
their present elevations and influencing the current dry environments
on their eastern side (Ortiz-Jaureguizar and Cladera, 2006; Graham,
2010), the Andes may have been covered by montane forests linking
MAF to the northern part of South America. Andean uplift and forma-
tion of the Amazon basin probably influenced the initial diversifica-
tion of lineages in northern South America. However, it is necessary
to improve the resolution of the relationships at the deepest nodes
of Myrcia s.l. for a better understanding of its initial diversification.

4.3. Montane Atlantic Forest (MAF): origins and main centre of
diversification

The Montane Atlantic Forest region is the most important area
in the biogeographic history of Myrcia s.l., as it is the most likely
Fig. 4. Phylogenetic hypothesis of Myrcia s.l. with ancestral areas at nodes reconstru
demonstrates the relationships inside node 261. Probabilities of ancestral areas are into p
analysis. Only ancestral areas with probability higher than 0.05 were kept distinct, whil
ancestral areas with probability higher than 0.5, the area with highest probability was k
beside the pie chart. The number inside the pie chart corresponds to the node number c
name. Each division in the horizontal bar below the cladogram corresponds to five millio
on Stratigraphy (Cohen et al., 2013). A. Caribbean; B. Amazon Forest; C. Guayana Region
Lowland Atlantic Forest; H. Mesoamerica and NW South America; I. Caatinga; J. Extra-
⁄⁄ = Prevost 4716.
area of origin and main centre of diversification. The clade encom-
passing all Myrcia s.l. subgroups except clades 5 and 9 (node 261)
has MAF as its ancestral area. Inside this group, the origin of most
Myrcia s.l. subgroups is also linked to MAF, frequently with high
probability (e.g., clades 2, 3, 6 and 7; Figs. 3 and 4; Table 2).

Calyptranthes is positioned in the clade delimited by node 261
but the oldest Calyptranthes lineages are associated with the Carib-
bean and Amazon Forest (see subsection 4.4). The terminal posi-
tion of MAF lineages of Calyptranthes indicate that the group
returned to diversify in the south; a clade close to the MAF lineage
(node 219; Fig. 4; Table 2) has as ancestral area Amazon Forest and
Caribbean suggesting an intermediate step to this expansion. Ulti-
mately, node 225 delimits the MAF group of Calyptranthes (Fig. 4;
Table 2). However, further sampling is required to confirm that
no Calyptranthes from deeper nodes occur in the MAF.

The principle period of origin of the subgroup lineages was the
Miocene. Pollen records (Lima, 1991; Biagolini et al., 2013) suggest
forest vegetation in the MAF at that time. The Serra do Mar (main
area of MAF) was already montane in the Miocene (Almeida and
Carneiro, 1998), and it is likely that a wide area of montane forest
already occurred along it (Morley, 2000; Burnham and Johnson,
2004). Miocene climate changes to colder climates may have had
a central role in the simultaneous origin of MAF taxa. Perret et al.
(2013) found that diversification of Atlantic Forest Gesneriaceae
initiated around 30 Ma and was influenced by the uplift of the
Serra do Mar during the Oligocene, but before diversification of
most subgroups of Myrcia s.l. The influence of tectonic events in
the origin and evolution of Serra do Mar is still controversial
(Hiruma et al., 2010). More data on ages, dates and elevation is
necessary for accurate understanding of the role of geology in
the diversification of Myrcia s.l. in MAF.

Recent studies have associated climate fluctuations with forest
refugia and lineage diversification in MAF (Carnaval and Moritz,
2008; Martins, 2011). However results presented here suggest that
most cladogenesis took place before the Quaternary. Nodes with
Quaternary age usually do not have high statistical support; phylo-
geographic studies with selected taxa are encouraged to properly
address this question.

The results presented here indicate that lineages of Myrcia s.l.
from MAF have closer relationships with lineages from LAF; there
is not a clear relationship between LAF and Amazon Forest, as indi-
cated for other groups (Mori et al., 1981; Thomas et al., 1998). The
coalescence of Amazon and Atlantic forests during the climatic
fluctuations of the Neogene and Quaternary (Santos et al., 2007)
apparently did not influence the diversification of Myrcia s.l.
4.4. The Caribbean lineage

The Caribbean has an important role in the diversification of
Calyptranthes (=clade 1). While the crown node and the first lin-
eage of Calyptranthes in this analysis appear to have originated in
the Amazon Forest (see subsection 4.5), the Caribbean is the
unique ancestral area of the subsequent two lineages (nodes 215
and 230; Fig. 4; Table 2). The stem nodes leading to these lineages
(nodes 228 and 231) have ambiguous ancestral areas (Caribbean
and Amazon Forest; Fig. 4; Table 2) preventing confirmation that
cted from the DEC analysis (model with selected parameters). Lower cladogram
ie charts, which are just shown at nodes with PP higher than 0.5 in the phylogenetic
e ancestral areas with lower probability were mixed (black slices); if there was not
ept distinct. The acronym of the ancestral area with higher probability is indicated
ited in the text. Terminal distribution is indicated in brackets in front of the species
n years; geological periods are indicated according to the International Commission
; D. Cerrado (savanna vegetation); E. Campo Rupestre; F. Montane Atlantic Forest; G.
Neotropics. Plio. = Pliocene; Quat. = Quaternary. Stagg. = Staggemeier. ⁄ = Lucas 72;

"



M.F. Santos et al. /Molecular Phylogenetics and Evolution 108 (2017) 34–48 43



Fig. 4 (continued)

44 M.F. Santos et al. /Molecular Phylogenetics and Evolution 108 (2017) 34–48



M.F. Santos et al. /Molecular Phylogenetics and Evolution 108 (2017) 34–48 45
these lineages were the result of a single or two independent
events. Nevertheless, the high diversity of Calyptranthes in the Car-
ibbean (ca. 130 spp.; Govaerts et al., 2016) and the association of
old lineages of the genus with the Caribbean highlight the impor-
tance of the region in its diversification.

The placement of Calyptranthes in an inclusive Atlantic Forest
clade but with its own origin in the Amazon suggests initial migra-
tion of a Calyptranthes ancestor to the north of South America and
then to the Caribbean. The routes of this Calyptranthes ancestor
may be similar to those proposed for Myrcia s.l. in subsection 4.2.
Migration from the continent to the Caribbean islands may have
been via various routes. At the time of the initial diversification
of Calyptranthes (95% HPD, 28.9–16.4 Ma), the Lesser Antilles had
already emerged (Iturralde-Vinent and MacPhee, 1999; Graham,
2010), offering connection between South America and the Greater
Antilles, where Calyptranthes has its main Caribbean diversity
(McVaugh, 1969; Govaerts et al., 2016). Another possibility is
through the Isthmus of Panama, whose appearance has been sug-
gested to date from the end of the Oligocene (Montes et al.,
2012). The latter scenario agrees with the proposal of Lucas et al.
(2011) but at an earlier time. Bacon et al. (2013) proposed that
some Arecaceae taxa used this route during a similar period. The
South American origin of Caribbean Calyptranthes is congruent
with other Caribbean taxa, such as Bactris (Arecaceae), Goetzeoidea
(Solanaceae) and Protieae (Burseraceae) (Santiago-Valentín and
Olmstead, 2004; Fine et al., 2014). Migration from the Caribbean
to South America may have used the same routes (see subsection
4.3), still present during the diversification of Calyptranthes in the
Atlantic Forest (95% HPD, 18.3–8.8 Ma).

4.5. Early nodes in the Guayana region

The Guayana Region plus the Amazon is recovered as the ances-
tral area of clade 9b (0.32; Fig. 4; Table 2), one of the first diverging
lineages of Myrcia s.l. This arrangement agrees with the pattern
found in Rapateaceae (Givnish et al., 2004), Bromeliaceae
(Givnish et al., 2004, 2011), Gentianaceae (Helieae; Struwe et al.,
2009), Eriocaulaceae (Trovó et al., 2013) and Astrocaryum (Roncal
et al., 2013). However, the region is not the area of origin of Myrcia
s.l. Low statistical support at the deepest nodes of Myrcia s.l. pre-
vents the confirmation of the exact relationship with other areas.
One of the main lineages of clade 9b is almost restricted to the
Guayana Region (node 314; Fig. 4), showing that endemism in
the region is also found at the lineage level.

Node 295 of clade 9b (Fig. 4; Table 2) originated in the Campo
Rupestre, suggesting links between these two montane areas
(Giulietti and Pirani, 1988). However, reconstruction of ancestral
areas inside clade 9b is inconclusive, preventing firm conclusions
about relationships between these areas. It is not clear if dispersal
to the Campo Rupestre occurred through short steps or by long-
distance dispersal. Clade 7 is mainly distributed in Montane Atlan-
tic Forest and Campo Rupestre with three species in the Guayana
Region (not included in the analysis), reinforcing the connection
between these areas.

The ‘Roraima group’ is a geological formation bearing the typi-
cal habitat of vegetation of the region (Berry et al., 1995; Huber,
1995; Berry and Riina, 2005) and being the main source of sedi-
ment to the Rio Negro basin (Hoorn et al., 2010c). The surface of
the Roraima group was at least partially exposed during the Pale-
ogene and Neogene (Huber, 1995; Hoorn et al., 2010b) suggesting
that geomorphological conditions similar to today existed there
during the diversification of clade 9b. It is less likely that climate
changes during the Miocene (Zachos et al., 2001; Graham, 2011)
had an impact on the region due to its equatorial position. Thus,
it is not clear if geological or climatic changes have influenced
diversification of Myrcia s.l. in this region. Such relative stability
of the region is invoked as the reason for the old lineages that
are found there and that later colonized other areas (Roncal
et al., 2013).

4.6. The intriguing role Lowland Rainforests in the Myrcia s.l
biogeography

Lowland Forests areas have a counterintuitive role in the diver-
sification of Myrcia s.l. lineages, since the high and low species
diversity in LAF and Amazon, respectively, is not coincident with
the origin of species rich lineage in each area. The Amazon Forest
is not a main centre of endemism of Myrtaceae (McVaugh, 1968,
1969) however, initial diversification of some clades occurred
there; e.g., it was recovered as the most probable ancestral area
for Calyptranthes. The Amazon Forest appears relevant to the initial
diversification of clade 5, together with MAF, and clade 9b,
together with the Guayana region. Other Amazonian species were
included in the analysis, but these are scattered in different lin-
eages suggesting origins from sporadic events. The diversification
age of these clades corresponds to the period of the Pebas System
(Ortiz-Jaureguizar and Cladera, 2006; Hoorn et al., 2010a, 2010b;
Antonelli and Sanmartín, 2011), indicating no impediment to the
diversification of these clades. Roncal et al. (2013) suggest that
the diversification of Astrocaryum (Arecaceae) only took place after
the drainage of Pebas system, the same pattern found in Rubiaceae
(Antonelli et al., 2009).

Lowland Atlantic Forest boasts high Myrcia s.l. species diversity
and endemism (Mori et al., 1983; Murray-Smith et al., 2008) but
surprisingly it is only the ancestral area of clade 9a, shared with
MAF (Fig. 4; Table 2). Besides the crown node of clade 9a and inter-
nal lineages, only small clades (node 186, clade 3; node 300, clade
9b) have LAF as its ancestral area. Together with low diversity in
Amazon Forest, the few lineages with origin in LAF highlight the
fact that montane environments are the main areas of diversifica-
tion of Myrcia s.l.

High levels of diversity of Myrcia s.l. in lowland areas of Atlantic
Forest (Tabuleiro Forest) may be related to their proximity to mon-
tane areas, allowing punctual speciation rather than development
of species rich lineages. High species diversity in this area is
embedded in clades that have their origin in other areas; this is
proposed by Pennington and Dick (2010) regarding speciation of
Andean montane taxa in surrounding lowland Amazon Forest.
These patterns are further analogous to patterns suggested by
Simon and Pennington (2012) for forest groups with punctual spe-
ciation in the adjacent Cerrado.

Tabuleiro Forests occur on sediments of the Barreiras formation,
deposited during the Miocene (Thomas et al., 1998; Arai, 2006;
Thomas and Barbosa, 2008). Evolution of clade 9a peaked during
the Miocene (95% HPD, 24.2–12.6 Ma), corresponding to the
appearance of the new geology. The occurrence of lowland rain for-
ests in the region is almost exclusive to this geological formation
and it is likely that these forests only developed in the region after
the deposition of the Barreiras formation. It is the first time that
such relationship is observed. Biogeographic studies in other taxa
are necessary to further investigate links between the deposition
of the Barreiras formation and regional plant species diversifica-
tion. Further paleo-vegetation data in the region is also required
to accurately infer past landscapes.

4.7. Lineages from open areas: Cerrado and Campo Rupestre

The biogeographic analysis presented here suggests that the
Cerrado has not played a major role in the evolution of Myrcia s.l.
DespiteMyrcia s.l. being one of the most species-rich groups in Cer-
rado species surveys (Oliveira-Filho and Fontes, 2000; Lucas et al.,
2011), the number of endemic species is not high (about 30



46 M.F. Santos et al. /Molecular Phylogenetics and Evolution 108 (2017) 34–48
species; Sobral et al., 2016). Clade 4 contains many typical Cerrado
species but most occur in other areas and ancestral area recon-
struction is inconclusive. Only node 204 (Figs. 3 and 4; Table 2)
has Cerrado as ancestral area, but statistical support for this node
is low.

Unlike other Neotropical groups such as Andira and Mimosa
(Fabaceae) and Eriotheca (Malvaceae) (Simon and Pennington,
2012), transition to savanna vegetation seems to be relatively
uncommon in Myrcia s.l. Species of Myrcia s.l. rarely possess mor-
phological features typical of Cerrado taxa such as thick corky bark
and enlarged underground xylopodia (Simon et al., 2009; Simon
and Pennington, 2012) and the absence of these features may have
impeded colonization of Myrcia s.l. in the Cerrado. AmongMyrcia s.
l. subgroups, only species of clades 4 and 5 regularly show xylopo-
dia (e.g., Myrcia pinifolia – clade 4; Myrcia microphylla, Myrcia
suffruticosa – clade 5). Clade 5 also may present the forest-
cerrado transition proposed by Simon et al. (2009) as it mostly
comprises forest taxa but also some Cerrado endemic species.
Future studies in these clades are important for better understand-
ing of the role of Cerrado in Myrcia s.l. evolution. No significant
relationship was found between Cerrado and Campo Rupestre taxa
despite their geographical proximity.

As in the Cerrado, Myrcia s.l. species richness in Campo Rupestre
is often high (e.g., Pirani et al., 2003) but endemism is low (about
10 species; based on Sobral et al., 2016). Results presented here
suggest that Myrcia s.l. lineages did not expand significantly in
the Campo Rupestre and the region is here suggested to have been
the ancestral area of only two lineages in clades 7 and 9b that have
low diversity (nodes 244 and 295, respectively; Fig. 4; Table 2). The
latter lineages have relatively recent origins (95% HPD, 4.1–1.0
and 7.7–2.0 Ma, respectively) as reported for other taxa in the
biome (Microlicieae [Melastomataceae] – Fritsch et al., 2004;
Hoffmannseggella [Orchidaceae] – Antonelli et al., 2010). Climate
change during Miocene cooling and appearance of fire four Ma
ago are suggested to be drivers of diversification in this region
(Antonelli et al., 2010; Fritsch et al., 2004).

Campo Rupestre environments share similarities to those of the
Guiana Highland (Giulietti and Pirani, 1988), but while the former
has lowMyrcia s.l. endemism, endemism is high in the latter. Other
Neotropical taxa have high species diversity in both regions (e.g.,
Eriocaulaceae – Trovó et al., 2013; Microlicia – Berry and Riina,
2005; Velloziaceae – Mello-Silva et al., 2011). A plausible cause
for such difference is the continually wet climatic regime of the
Guiana Highland, the conditions whereMyrcia s.l. has higher diver-
sity, while Campo Rupestre is more seasonal.
5. Conclusions

The analysis indicates that the origin of Myrcia s.l. took place in
the Atlantic Forest, probably in forest vegetation according to
paleo-vegetation records (Morley, 2000; Burnham and Johnson,
2004). Part of the group remained and diversified in the region,
currently its main centre of diversity both in morphological varia-
tion and number of species. However, three old lineages (clades 5,
9a and 9b) have their origin in northern areas, indicating that their
ancestors moved relatively quickly to northern South America after
the origin of Myrcia s.l. Some routes available during this period
may have facilitated this migration (see section 4.3). Unfortu-
nately, phylogenetic and geographical relationships between these
lineages are not statistically supported. The Guayana Region and
the Caribbean were also important for the evolution of major lin-
eages (Myrcia s.l. subgroups). Despite low species diversity of Myr-
cia s.l. in the Amazon Forest, the region appears to be important in
the initial diversification of old lineages, while LAF has high species
diversity but is the area of origin of only one species rich lineage.
Geological events appear to have had an important role in the evo-
lution of the Caribbean and Amazon forest lineages (Iturralde-
Vinent and MacPhee, 1999; Hoorn et al., 2010a, 2010b), but other
regions, especially the Atlantic Forest, were geologically stable. In
these regions, climate changes during the Neogene were the most
likely abiotic drivers of diversification. Diversification of Myrcia s.l.
subgroups took place during the Miocene, between 22.1 Ma and
5.6 Ma, a pattern found for other Neotropical taxa (Rull, 2011;
Hughes et al., 2013). The range of reported periods of lineage origin
is large, possibly due to the availability of only a single calibration
point. Unquestionable Neogene fossils of Myrcia s.l. are still lack-
ing; their discovery will be essential to improve lineages diver-
gence times.

The high number of species-rich lineages (including the crown
node of subgroups) in montane areas (usually above 800 m) sug-
gests that Myrcia s.l. may be particularly adapted to such environ-
ments. If lineages arising directly from the oldest Myrteae nodes
evolved in more temperate habitats, today’s montane regions are
more environmentally similar to ancestral areas and less change
is required to occupy them than more tropical environments. Glo-
bal climatic cooling during the Neogene may have facilitated this
temperate to tropical transition. Fewer apparent transitions to
lower altitudes and the low number of species rich lineages in such
areas suggest the effect of Phylogenetic Niche Conservatism (Crisp
and Cook, 2012). A better biogeographical understanding of tribe
Myrteae will demonstrate how many times this transition
occurred and allow inference of the occupation of temperate or
tropical areas by stem lineages of Myrcia s.l. At this point, it is
important to highlight the need for biological and field-based stud-
ies of physiological features, phenological traits, ecological interac-
tions, among others. These data are essential to ground the analysis
and for interpretation of model-based biogeography. Only holistic
knowledge of the physiology, biology and ecology of Myrcia s.l.
taxa will allow detailed, accurate investigation of the biogeo-
graphic process involved in its diversification.

Acknowledgements

M.F.S. acknowledges FAPESP for a PhD fellowship (grant#
2010/09473-0 and grant# 2012/14914-1), V.G.S. acknowledges
FAPESP for a PD fellowship (grant# 2016/02312-8), and P.T.S.
acknowledges the financial support of CNPq (process number
308300/2012-2). We are grateful to Dion Devey, Edith Kapinos,
Jim Clarkson, Laszlo Csiba, Laura Martinez-Suz and Thomas Booth
for help in the laboratory at RBG Kew; to the colleagues of the Lab-
oratório de Sistemática Vegetal for discussion about Biogeography;
to Alexei Oskolski for information about Myrteae fossils.
Appendix A. Supplementary material

Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ympev.2017.01.
012.

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	Biogeographical patterns of Myrcia s.l. (Myrtaceae) and their correlation with geological and climatic history in the Neotropics
	1 Introduction
	2 Materials and methods
	2.1 Taxonomy and diversity of Myrcia s.l
	2.2 Sampling and data collection
	2.3 Data edition and phylogenetic analysis
	2.4 Delimitation of biogeographic areas
	2.5 Biogeographic analysis

	3 Results
	3.1 Phylogenetic analysis
	3.2 Molecular dating of nodes
	3.3 Comparison of model parameters
	3.4 Biogeographic reconstruction

	4 Discussion
	4.1 Phylogenetic analysis
	4.2 The southern origin of Myrcia s.l. and rapid expansion to northern South America
	4.3 Montane Atlantic Forest (MAF): origins and main centre of diversification
	4.4 The Caribbean lineage
	4.5 Early nodes in the Guayana region
	4.6 The intriguing role Lowland Rainforests in the Myrcia s.l biogeography
	4.7 Lineages from open areas: Cerrado and Campo Rupestre

	5 Conclusions
	Acknowledgements
	Appendix A Supplementary material
	References