Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev
Monophyly, divergence times, and evolution of host plant use inferred
from a revised phylogeny of the Drosophila repleta species group
Deodoro C.S.G. Oliveira a, Francisca C. Almeida b, Patrick M. O’Grady c, Miguel A. Armella d,
Rob DeSalle e, William J. Etges f,⇑
a
Departamento de Genética y Microbiología, Universidad Autonóma de Barcelona, Bellaterra BCN 08193, Spain
Departamento de Genètica, Universitat de Barcelona, Barcelona BCN 08071, Spain
Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720, USA
d
Departamento de Biología, Universidad Autónoma Metropolitana-Iztapalapa, Av. Michoacán y la Purísma, Col. Vicentina, 09340 Mexico, D.F., Mexico
e
Division of Invertebrate Zoology, American Museum of Natural History, New York, NY 10024, USA
f
Program in Ecology and Evolutionary Biology, Department of Biological Sciences, SCEN 632, University of Arkansas, Fayetteville, AR 72701, USA
b
c
a r t i c l e
i n f o
Article history:
Received 2 January 2012
Revised 12 May 2012
Accepted 14 May 2012
Available online xxxx
Keywords:
Drosophila repleta species group
Host plants
Molecular phylogeny
Molecular clock
Cactus
Biogeography
a b s t r a c t
We present a revised molecular phylogeny of the Drosophila repleta group including 62 repleta group taxa
and nine outgroup species based on four mitochondrial and six nuclear DNA sequence fragments. With
ca. 100 species endemic to the New World, the repleta species group represents one of the major species
radiations in the genus Drosophila. Most repleta group species are associated with cacti in arid or semiarid
regions. Contrary to previous results, maximum likelihood and Bayesian phylogenies of the 10-gene dataset strongly support the monophyly of the repleta group. Several previously described subdivisions in the
group were also recovered, despite poorly resolved relationships between these clades. Divergence time
estimates suggested that the repleta group split from its sister group about 21 million years ago (Mya),
although diversification of the crown group began ca. 16 Mya. Character mapping of patterns of host
plant use showed that flat leaf Opuntia use is common throughout the phylogeny and that shifts in host
use from Opuntia to the more chemically complex columnar cacti occurred several times independently
during the history of this group. Although some species retained the use of Opuntia after acquiring the use
of columnar cacti, there were multiple, phylogenetically independent instances of columnar cactus specialization with loss of Opuntia as a host. Concordant with our proposed timing of host use shifts, these
dates are consistent with the suggested times when the Opuntioideae originated in South America. We
discuss the generally accepted South American origin of the repleta group.
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
The New World Drosophila repleta species group has proven to
be a valuable ecological and evolutionary model system as one of
the largest species radiations in the genus (Patterson and Stone,
1952; Throckmorton, 1975; Vilela, 1983; Wasserman, 1992;
Markow and O’Grady, 2006). Study of particular species in the
group have revealed general insights into chromosome and genome evolution (Cáceres et al., 1999; Negre et al., 2005), mechanisms of speciation (Coyne and Orr, 1997; Etges and Jackson,
2001; Etges et al., 2010), sperm competition and evolution of
reproductive proteins (Wagstaff and Begun, 2005; Kelleher et al.,
2007; Wagstaff and Begun, 2007; Almeida and DeSalle, 2008,
2009), adaptation to temperature and desiccation stress (Gibbs
and Matzkin, 2001; Gibbs et al., 2003), fly–cactus–yeast/bacteria
interactions (Barker and Starmer, 1982; Barker et al., 1990), and
⇑ Corresponding author.
E-mail address: wetges@uark.edu (W.J. Etges).
ecological genetics and adaptation of host plant use (Ruiz and
Heed, 1988; Etges et al., 1999). Most species are cactophilic, using
fermenting cactus tissues to carry out their life cycles in semiarid
or arid environments (e.g. Ruiz and Heed, 1988; Ruiz et al.,
1990), but some species in the repleta group use a broad array of
different resources and occupy habitats from wet, tropical forests
to temperate environments (Vilela, 1983; Pereira et al., 1983; Vela
and Rafael, 2005; Acurio and Rafael, 2009). Therefore, an accurate
and well-supported phylogeny of the repleta group would help to
place many of these genetic, behavioral, ecological and evolutionary problems into a broader phylogenetic perspective.
Species identifications, taxonomy, and phylogenetic relationships within the repleta group have also proven to be interesting
challenges (e.g. Heed and Grimaldi, 1991; Etges et al., 2001; Diniz
and Sene, 2004). The precise number of species is unclear since
there are taxa that were proposed to be synonymies, and there
are also a number of cryptic species with poorly known species
boundaries (e.g. Oliveira et al., 2005, 2008). Six species subgroups
have been described – mulleri, hydei, mercatorum, repleta, fasciola,
1055-7903/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ympev.2012.05.012
Please cite this article in press as: Oliveira, D.C.S.G., et al. Monophyly, divergence times, and evolution of host plant use inferred from a revised phylogeny
of the Drosophila repleta species group. Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.05.012
2
D.C.S.G. Oliveira et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
and inca – and these subgroups have been further subdivided into
species complexes, clusters and subclusters based on chromosome
banding patterns, male genital morphology and/or ecological associations (Patterson, 1943; Wharton, 1944; Wasserman, 1962;
Rafael and Arcos, 1989). Recently, several molecular studies have
addressed phylogenetic relationships of repleta species at different
phylogenetic levels (e.g. Rodriguez-Trelles et al., 2000; Oliveira
et al., 2005; Silva-Bernardi et al., 2006; Moran and Fontdevila,
2007; Robe et al., 2010). The most inclusive study of the repleta
group so far is that of Durando et al. (2000) in which a phylogenetic
hypothesis for 46 ingroup and six outgroup species was generated.
The overall basal relationships of this tree were poorly resolved
and suggested paraphyly of the repleta and canalinea, dreyfusi and
mesophragmatica species groups, although a few internal taxonomic groups were well resolved.
The ecology and biogeography of the repleta group has also provided insights into its origins and history. The adoption of cacti as
breeding and feeding sites by many repleta group species is certainly one of the most extensive and successful ecological transitions in the genus, resulting in about 100 known species. The
‘‘virilis-repleta’’ radiation forms a basal lineage within the subgenus
Drosophila appearing 25–36 million years ago (Mya; Throckmorton,
1975; Powell and DeSalle, 1995; Russo et al., 1995). Since the D.
repleta group is confined to the New World (apart from human
influences), it is likely the group arose in South America well after
the origins of many of the major cactus groups when the interior
of the continent became warmer and drier due to the Andean uplift
ca. 17 Mya (Mauseth, 1990; Nyffeler, 2002). The centers of radiation
of the major cactus groups are located in Peru-Bolivia, the chaco
and caatinga of eastern South America, and possibly the Caribbean.
Based on the current distribution of the most generally ancestral
genera within the subfamilies Opuntoideae, Cactoideae, and Pereskioideae, the arid lands in Peru and Bolivia may be the centers of
origin for all cacti (Edwards et al., 2005).
Here we present molecular phylogenetic analyses based on four
mitochondrial and six nuclear gene regions from 62 ingroup and
nine outgroup taxa belonging to the virilis–repleta radiation
(Throckmorton, 1975; Tatarenkov and Ayala, 2001). Sampling included five of the six proposed subgroups (only the inca subgroup
was missing) and ca. 60% of the described species. We used the
resulting phylogenetic hypothesis to address several outstanding
systematic and evolutionary problems: (1) with a monophyletic
repleta species group recovered, this phylogenetic hypothesis provided groundwork for further systematic analysis of some subgroups and species complexes, (2) we present the first global
dating of species divergence within the repleta group, (3) we
mapped host cactus use onto the tree and show that there have
been a number of phylogenetically independent host transitions
from Opuntia to the more chemically specialized columnar cacti,
and (4) we mapped current species geographical locations onto
the tree but were unable to resolve a clear historical biogeography
of these species. We discuss the evolution of host use and the geographic origins of the repleta group.
2. Materials and methods
2.1. Samples for molecular analyses
Data and specimens of ingroup and outgroup taxa included in
this study (Table S1) were deposited in the Ambrose Monell Cryo
Collection at the American Museum of Natural History, New York.
Most ingroup species were represented by one sample with a few
exceptions. Drosophila canapalpa has been questioned as a valid
species and its potential synonym, D. neorepleta, was also included
in the ingroup species (Vilela, 1983; Wasserman, 1992). Further,
four species were represented by two taxa each considered to be
different subspecies: these are D. mojavensis baja, D. meridiana rioensis, D. fulvimacula flavorepleta, and D. mercatorum pararepleta.
2.2. Molecular methods
Four mitochondrial and six nuclear primer pairs were used to
generate characters for phylogenetic analyses: primer sequences
were previously published (see references below). One of the mitochondrial regions includes partial sequences of both the small ribosomal RNA (srRNA) and large ribosomal RNA (lrRNA) and the
complete tRNA-Val gene (srRNA–lrRNA; Oliveira et al., 2005). A second region is part of the mitochondrial Cytochrome c oxidase subunit I (COI; Oliveira et al., 2005). A third region includes the
complete mitochondrial Cytochrome c oxidase subunit II gene and
partial sequences of flanking tRNA-Leu and tRNA-Lys genes (COII;
Beckenbach et al., 1993). A fourth region is part of the mitochondrial NADH-ubiquinone oxidoreductase chain 2 (ND2; Oliveira
et al., 2005). Partial sequences of the following six nuclear genes
were also included: bride of sevenless (boss), sans fille (snf), Mitochondrial assembly regulatory factor (Marf), seven in absentia (sina),
fork head (fkh), and wee. Primer sequences for these nuclear regions
were reported in Bonacum et al. (2001).
Template DNA was extracted from 1 to 5 flies using a DNeasy
Extraction Kit (Qiagen, Valencia, CA), and loci of interest were amplified using standard PCR protocols. Direct sequencing from purified
PCR products was performed on an ABI 3700 sequencer (PE Applied
Biosystems, Foster City, CA, USA). Sequences were corrected and
compiled using Sequencer 4.7 (Gene Codes Corporation, Ann Arbor,
MI, USA). All sequences generated for this study were deposited in
GenBank under accession numbers JF736018–JF736503 (Table S1).
2.3. Phylogenetic analyses
Multiple sequence alignments were first adjusted by eye, and
highly variable regions for which positional homology could not
be determined were manually excluded using MacClade 4.08
(Maddison and Maddison, 2005). These regions were the introns
of boss, snf, and Marf, a region of lrRNA, and most of the tRNA-Leu
along with an intergenic spacer. The program Gblocks (Castresana,
2000) was used to further trim gapped regions, removing another
5% of the nucleotide sites (parameters used were: minimum number of sequences for a conserved position = 36, maximum nonconserved positions = 8, and minimum length of a block = 10). This
matrix, 3957 bp in length and with 1204 parsimony-informative
characters (Table S2), was analyzed by maximum likelihood (ML)
based approaches for the estimation of the best data partitioning
scheme. Four alternative partition schemes were tested; (I) no partitioning of the data, (II) partitioning by gene (10 partitions), (III)
partitioning codon positions 1 + 2 and 3 of mitochondrial and nuclear protein-coding regions separately, plus an additional partition
for the non-protein-coding mitochondrial sequences (5 partitions),
and (IV) the same as before but separating codon positions 1 and 2
in different partitions, nuclear and mitochondrial genes separately
(7 partitions). These analyses were performed with Treefinder (Jobb
et al., 2004), and the GTR+C substitution model was generally used
for all partitions. The best scheme according to both Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC; as
in Sullivan and Joyce, 2005) was partition scheme IV (Table S3).
Both ML and Bayesian (BI) analyses were accomplished using
the best partition scheme and partition-wise parameters estimated
from the data. ML searches were done in 20 independent runs
using RAxML 7.2.6 (Stamatakis, 2006) and the GTR+C model was
used for each partition. Statistical support for nodes was obtained
with 200 bootstrap replicates and plotted on the best of the 20
trees obtained in the independent runs. BI searches were
Please cite this article in press as: Oliveira, D.C.S.G., et al. Monophyly, divergence times, and evolution of host plant use inferred from a revised phylogeny
of the Drosophila repleta species group. Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.05.012
D.C.S.G. Oliveira et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
performed with MrBayes (Huelsenbeck and Ronquist, 2005), using
2 independent runs of 10,000,000 generations each, with trees
sampled every 1000 generations. Convergence was checked with
Tracer v1.5 (Rambaut and Drummond, 2003) and with the online
program AWTY (Wilgenbusch et al., 2004). The first 8000 sampled
trees were discarded as the burn in. The difference between ML
and BI trees was evaluated with the SH test (Shimodaira and
Hasegawa, 1999) as implemented in RAxML 7.2.6 (Stamatakis,
2006). Maximum Parsimony (MP) analyses were done using PAUP
4.0b10 with random, stepwise addition and 1000 replicates
(Swofford, 2002). For parsimony trees, clade support was assessed
with bootstrap (1000 replicates, Felsenstein, 1985, 1988) and
decay indices (Bremer, 1988), which were calculated using
TreeRot.v3 (Sorenson, 1999). Incongruence between the phylogenetic signal of the mitochondrial and nuclear sequence sets was
tested using the Incongruence Length Difference test (Farris
et al., 1995).
2.4. Divergence time estimation and biogeographical analysis
A Bayesian approach was used to date the nodes of the repleta
group phylogeny with Multidistribute (Thorne and Kishino, 2002).
This method employs a relaxed clock allowing independent rates
among branches, and lower and upper hard bounds for calibration
dates. Following the manual by Rutschmann (2004), parameters for
the F84 sequence evolution model were estimated for the BI topology with the program baseml from PAML v 4.4 (Yang, 2007). These
parameters and the BI topology were used for estimating the
branch lengths with estbranches and the node ages with multidivtime, using 5 million generations sampled every 100 generations.
Since there is no reliable time calibration point available within
the repleta group, we used previously inferred divergence times to
calibrate a molecular clock. Two internal calibration points were
defined as follows. First, assuming that the Russo et al. (1995)
dates were underestimated as suggested by Tamura et al. (2004),
3
we set their lower bound of the estimated time for the divergence
of D. mettleri and D. mulleri (15.9 Mya) as a constraint for this split.
The second internal point was set using information from Russo
et al. (1995) and Matzkin and Eanes (2003) on the divergence of
D. mojavensis and D. arizonae, ca. 1.2–4.2 Mya (lower and upper
bounds, respectively). Additionally, this dating method requires a
prior for the root of age. Since divergence between the virilis and
the repleta species groups was not included in either of the most
cited Drosophila divergence time estimates (Russo et al., 1995;
Tamura et al., 2004), we had to choose a date based on available
evidence. Divergence between the repleta group and the Hawaiian
Drosophila was estimated to have occurred 32 Mya, suggesting that
the virilis-repleta split occurred most likely after that (Russo et al.,
1995). Spicer and Bell (2002) estimated a divergence date between
D. arizonae (repleta group) and the species of the virilis group of
approximately 20 Mya. Combining this information and using
D. virilis and the nannoptera species as outgroups, we set a prior
on the base of the tree of 26 ± 6 Mya.
In order to place these divergence estimates into a biogeographic context, we categorized available species distribution data
for all species in our ML phylogenetic reconstruction. We categorized all species into North America, Caribbean, or South America
distributions plus all combinations of these locations and used
DIVA (Dispersal Vicariance Analysis; Ronquist, 1996, 1997) to assess whether there was evidence supporting a North American or
South American origin of the D. repleta group. We used the ML tree
with maxarea = 2. Consistent with recent phylogenetic and biogeographic analyses (Griffith and Porter, 2009; Nyffeler and Eggli,
2010), we hypothesized a priori that South American species
should constitute the majority of taxa at the base of our tree.
2.5. Fly host use
Published and unpublished records of Drosophila host use, i.e.
breeding substrates, were gathered for the species used in
Fig. 1. Map of Mexico and the southern USA showing the sites sampled for Drosophila species in this study.
Please cite this article in press as: Oliveira, D.C.S.G., et al. Monophyly, divergence times, and evolution of host plant use inferred from a revised phylogeny
of the Drosophila repleta species group. Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.05.012
4
D.C.S.G. Oliveira et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
constructing our repleta group phylogeny, all ingroup and outgroup
taxa, and a few additional species not included in the phylogeny,
for a total of 75 species/subspecies (Table S4). For North American
species, we collected Drosophila in 33 locations throughout Mexico,
southern California, and Arizona (Fig. 1). Adult flies were collected
over fermenting bananas in the field; additionally, fermenting
cactus tissues, rots, were returned to the laboratory and all
emerging imagoes were recorded and identified to species. Comparison of species that were baited with those emerging from rots
allowed us to determine the degree to which cacti were being used
as hosts by Drosophila at each site. There were no apparent differences in species collected by baiting and those reared from
fermenting cactus rots at most of our collecting sites, other than
the presence of non-cactophilic species attracted to baits (Etges,
unpubl. data).
Host use was mapped onto the repleta group phylogeny using
MacClade ver. 4.08 (Maddison and Maddison, 2005). Character
states were unweighted and considered unordered. Host use was
coded as Opuntia (1), columnar cactus (2), ‘‘polymorphic’’ (1 and
2), soil (3), or ‘‘other’’ (4) to determine the number of ecological
transitions over the course of their history. ‘‘Other’’ included fermenting fruit, flowers, and sap. We did not specify individual species of Opuntia due to difficulties with field identifications, known
hybrids, and the decision that as a genus, species designations
were likely to be less ecologically important to the flies than for
the more chemically complex columnar cacti. Enumerating the
various tribes and subtribes of columnar cacti added little more
resolution to the patterns of host switching (results not shown).
We also used Mesquite, ver. 2.6 (Maddison and Maddison, 2009)
to examine the evolution of host plant use within the repleta species group. We traced host use to reconstruct ancestral character
states in a maximum parsimony framework. We performed 1000
randomizations using the following options: Analysis: New Bar
and Line Chart for Trees: Randomly modify current tree: reshuffle
current taxa: steps in character. Attempts to assess the evolution of
host cactus use in a maximum likelihood context failed because
polymorphic or missing host use data are not currently supported
by categorical data likelihood calculations in Mesquite.
3. Results
A dataset of four mitochondrial and six nuclear DNA sequences
was generated to examine the phylogenetic relationships of 58
species of the Drosophila repleta species group in greater detail.
In addition, our taxon sampling included four subspecies, as previously described (see Material and Methods), and nine outgroup
belonging to the virilis–repleta radiation (sensu Throckmorton,
1975) for a total of 71 terminal taxa. The phylogenetic inference
methods (ML, BI, and MP) recovered similar topologies and were
congruent with respect to the most well-supported lineages recovered by all three methods (Fig. 2). BI and ML trees were not statistically different (SH test, D(LH) = 17.3 ± 16.5).
A well-supported, monophyletic repleta group was recovered
from the ML and BI analyses, but not the MP analysis. Most major
repleta group lineages, subgroups, and species complexes previously defined by shared chromosomal inversions (Wasserman,
1992) were also recovered. Consistent resolution and/or support
for basal nodes, i.e. the overall relationships amongst species
complexes and subgroups of the repleta group, were less clear
and were the primary source of topological incongruence among
trees based on different inference methods (Fig. 2).
The lack of support at some hypothesized basal nodes is a common observation in molecular phylogenies. Saturation in third base
positions and consequent long-branch attraction are common
explanations, especially when mitochondrial genes account for a
large proportion of informative characters (Durando et al., 2000;
Bull et al., 2003; Bergsten, 2005). Despite using six nuclear genes,
67% of the informative characters in our data came from mtDNA
(Table S2). MP inferences are more prone to be confounded by
homoplasic molecular characters. The partition scheme and GTR+C
model used for ML and BI methods appears to have sufficiently corrected the saturation problem. The number of changes per distance
unit for the combined matrix did not show signs of a plateau for
either transitions or transversions (Fig. S1). Further, incongruence
between mitochondrial and nuclear data partitions was significant
(ILD test, p = 0.004) and may also have accounted for the weak basal support. However, this test was done only in a MP framework
and so was probably influenced by saturation and long-branch
attraction as suggested earlier.
3.1. Monophyly of the repleta species group
The branch leading to the repleta group was highly supported
by model-based inference methods (ML bootstrap = 98%, BI posterior probability = 1), contrary to a previous MP phylogeny that
failed to recover a monophyletic repleta group and suggested paraphyly in relation to the canalinea, mesophragmatica, and dreyfusi
groups (Durando et al., 2000). In the MP tree presented here,
D. pegasa, a Mexican species assigned to the repleta group based
on chromosomal inversions (Wasserman, 1992), clustered with
D. canalinea, the only representative canalinea group species we
could access. The poor resolution obtained by Durando et al.
(2000) appeared to be caused by saturation of third positions of
mitochondrial protein-coding genes leading to long branch attraction. This problem is likely still affecting our MP tree, in spite of the
extended dataset. Our MP tree, nevertheless, provided much better
resolution and separated the repleta group from the mesophragmatica and dreyfusi groups. Therefore, we conclude that the present
data provided strong corroboration for monophyly of the repleta
group (Fig. 2).
3.2. Monophyly and relationships among subgroups and species
complexes
The relatedness of several species of previously defined lineages
within the repleta group was consistently corroborated (Wasserman,
1992; Fig. 2). All analyses showed strong support (MP bootstrap > 98%, ML bootstrap = 100%, BI posterior probability = 1) for
monophyly of three of the five subgroups investigated, i.e. the
fasciola, hydei, and mercatorum subgroups. The repleta subgroup
was not well supported due to intermingling with the mercatorum
subgroup. The affinity of the mercatorum and repleta subgroups
was consistent across analyses and well supported (MP bootstrap
90%, ML bootstrap = 97%, BI posterior probability = 1), providing
further evidence for a previously implied relationship between
these two subgroups (Wasserman, 1982, 1992; Tatarenkov and
Ayala, 2001). The large mulleri subgroup remained polyphyletic,
consistent with Wasserman’s (1982, 1992) conclusions.
Species complexes in the mulleri subgroup were analyzed separately to refine their systematic relationships. A monophyletic mulleri subsection, including the mulleri, longicornis, and buzzatii
complexes matched the results obtained by Durando et al.
(2000). In our trees, the meridiana complex joined that subsection
with good support. Monophyly of the mulleri, buzzatii, and meridiana complexes was corroborated (MP bootstrap 100%, ML bootstrap = 100%, BI posterior probability = 1), but not that of the
longicornis complex (Oliveira et al., 2005). The anceps (ML bootstrap = 79%, BI posterior probability = 0.98) and the eremophila
(MP bootstrap 90%, ML bootstrap = 97%, BI posterior probability = 1) complexes were placed outside this monophyletic mulleri
subsection. Two unaffiliated species, D. nigricruria and D. pegasa,
Please cite this article in press as: Oliveira, D.C.S.G., et al. Monophyly, divergence times, and evolution of host plant use inferred from a revised phylogeny
of the Drosophila repleta species group. Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.05.012
D.C.S.G. Oliveira et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
5
Fig. 2. Molecular phylogenetic hypotheses for the Drosophila repleta species group. Yellow boxes delimit complexes of the mulleri subgroup, while green boxes delimit other
repleta subgroups. (A) The tree obtained by ML searches, with the GTR+C substitution model, and parameters estimated for each of the 7 data partitions (see text and Table S2
for details). Numbers on the node: top left ML bootstrap values > 50%, top right BI posterior probability > 0.7, bottom left MP bootstrap > 50%, bottom right, Bremer decay
values > 1. (B) The tree obtained by Bayesian Inference. (C) Strict consensus of the nine most parsimonious trees (steps = 7628, CI = 0.30, RC = 0.16). (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: Oliveira, D.C.S.G., et al. Monophyly, divergence times, and evolution of host plant use inferred from a revised phylogeny
of the Drosophila repleta species group. Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.05.012
6
D.C.S.G. Oliveira et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
had uncertain positions consistent with previous chromosomal
analyses (Wasserman, 1992).
The phylogenetic relationships among these strongly supported
repleta group lineages described above, as well as the order of early
branching in the repleta phylogeny, remain unresolved. Besides the
methodological differences mentioned above, this result is actually
in agreement with morphological and chromosomal data (Vilela,
1983; Wasserman, 1992). The paucity of informative characters
may be an indication of rapid diversification early on in the evolutionary history of the repleta group (Throckmorton, 1975).
Fig. 3. Results of DIVA (Dispersal Vicariance Analysis) with current geographic locality (-ies) of D. repleta group species and outgroups mapped onto the ML tree. See the text
for details.
Please cite this article in press as: Oliveira, D.C.S.G., et al. Monophyly, divergence times, and evolution of host plant use inferred from a revised phylogeny
of the Drosophila repleta species group. Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.05.012
D.C.S.G. Oliveira et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
7
Fig. 4. Host use and divergence times for the D. repleta group plotted onto the BI tree. Numbers by the nodes are the time estimates and the bars represent their 95%
confidence intervals. Host substrates are color coded. ‘‘Soil’’ refers to cactus exudate-soaked soils, and ‘‘other’’ refers to other substrates, but not cactus. The pictures illustrate
typical Opuntia and columnar cactus growth forms.
Please cite this article in press as: Oliveira, D.C.S.G., et al. Monophyly, divergence times, and evolution of host plant use inferred from a revised phylogeny
of the Drosophila repleta species group. Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.05.012
8
D.C.S.G. Oliveira et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
3.3. Divergence times and biogeography of the Drosophila repleta
group
Our global divergence time estimates for the repleta group revealed that the split between the ancestor of the repleta group
and those of related species groups was approximately 20.9 Mya,
with the ‘‘crown’’ repleta group ca. 16.3 Mya old. Based on Bayesian
methodology with calibration points taken from the literature
(Russo et al., 1995; Spicer and Bell, 2002; Matzkin and Eanes,
2003; Tamura et al., 2004), this is the first estimate made for divergence dating in this group. By the mid-Miocene (12–15 Mya), all
major repleta lineages, including the subgroups and species complexes described above, had already emerged. More closely related
species, often morphocryptic, diverged during the Pleistocene, often less than 1 Mya, e.g. the Caribbean species triad of D. mayaguana, D. straubae, and D. parisiena (Heed and Grimaldi, 1991).
Ancestral area construction revealed poor resolution of basal
taxa in our ML tree (Fig. 3). We tried to resolve the locations of
poorly known species, and categorized several species that are
now human commensals, i.e. D. virilis, D. repleta, and D. hydei, or
have been transported around the world with their host plants,
i.e. D. mercatorum and D. buzzatii based on locations of natural populations and of their closest relatives. We then tried to use Mesquite ver. 2.6 (Maddison and Maddison, 2009) with both the MP
and ML trees, but there no improvement in the resolution of ancestral localities (results not shown) because of the number of equivocal nodes due to the widespread distributions of some species
(Fig. 3). Also, incomplete sampling of South American (SA) species
in our outgroups, e.g. one species each in the canalinea and mesophragmatica groups, and species at the base of this tree, e.g. in
the fasciola group, probably inhibited clearer biogeographic resolution. However, most of these groups are either restricted to SA (D.
pavani of the mesophragmatica group) or are distributed in both SA
and North America (NA), or in the case of the 14 known species in
the canalinea group (Stensmyr et al., 2008), in all three areas. Thus,
current locations of the representatives of the more basal lineages
in this MP tree suggest that dispersal between SA, NA, and the
Caribbean was widespread early in the diversification of the
D. repleta group.
parts of their species range (Fellows and Heed, 1972; Heed,
1982; Ruiz and Heed, 1988). Others, such as D. buzzatii, use one
host predominantly, but have been found repeatedly using alternate hosts in low frequency. Opuntia is the main host for D. buzzatii
throughout its range in South America and the world where it has
been introduced (Carson and Wasserman, 1965), yet a small
(3–6%), but repeatable percentage of flies have been reared from
Echinopsis terschekii in Argentina (Table S4) where they are sympatric with D. koepferae (Hasson et al., 1992; Fanara et al., 1999).
Including both Opuntia and columnar cacti as hosts for D. buzzatii
produced the results presented in Fig. 4 with all South American
buzzatii complex species having gained the use of columnar cacti
only once, including four columnar specialists, D. borborema,
D. serido, D. gouveai and D. uniseta (Fig. 4, Table S4).
Since current phylogenetic consensus suggests that the Opuntioideae are monophyletic (Griffith and Porter, 2009) and are sister
to the Cactoideae, including columnar cacti (Nyffeler, 2002; Griffith,
2004), we hypothesized that the switch to Opuntia use is ancestral,
with the use of columnar cacti for breeding representing the derived state, among extant cactus-breeding members of the repleta
group. Mapping host use onto the BI phylogeny revealed that
Opuntia is generally the ancestral host and columnar cactus use is
a derived condition based on available data (Fig. 4). Further, the
phylogeny revealed multiple independent transitions, at least 10,
from Opuntia to columnar cactus (Fig. 4). Results of ancestral character state reconstruction in Mesquite (Maddison and Maddison,
2009) indicated no phylogenetic structure in host use (P > 0.05).
Therefore, there was no evidence that the Opuntia to columnar cactus switch has a phylogenetic component.
In some clades, however, many species retained the ancestral
state of Opuntia use in parts of their species ranges or have not
completely specialized to columnar cacti. Loss of Opuntia use has
occurred six times in the repleta group. Outside of the repleta species group, D. pavani and the nannoptera group independently acquired the trait of being strict columnar breeders (Ward et al.,
1975; Heed, 1982; Pitnick and Heed, 1994; Etges et al., 1999; Table
S4, Fig. 4).
4. Discussion
3.4. Host use and host shifts
4.1. Molecular systematics of the Drosophila repleta group
Together with our collections of wild Drosophila (Fig. 1) and
available published records, a detailed record of host use for most
of the repleta group and some related outgroup species was assembled (Table S4). In total, host use data was compiled for 63 species
of repleta and 10 species belonging to other species groups. Five
repleta group species, D. antonietae, D. desertorum, D. gouveai,
D. seriema, and D. nigrohydei, and one species of the mesophragmatica group, D. gaucha, were not available for molecular analysis (Table S4). Comparing Opuntia to columnar breeders, 33.3% (21/63)
used only Opuntia species, 17.5% (11/63) of these repleta group species used only columnar cacti as hosts, and at least 25.4% (16/63) of
these species used both types of hosts. Therefore, 50.8% of repleta
group species are host specialists, at least with respect to this
broad ecological division between flat leaf Opuntia and columnar
cacti. The use of soil refers to oviposition in fermenting cactus exudate-soaked soils that has evolved in all members of the D. eremophila complex (3/63). The remaining repleta species used other
substrates (4/63) or breeding sites are unknown (8/63).
Polymorphism in character states can increase the uncertainty
of mapping host use evolution. A recurring problem in this analysis
for some Drosophila species was deciding when to add additional
hosts based on rearing records, particularly for those species that
are relatively unstudied. Some species, e.g. D. mojavensis, are well
known to be oligophagous, using different host cacti in different
A previous combined molecular and chromosomal MP phylogeny generated for the repleta group was characterized by a wide
basal polytomy (Durando et al., 2000). This assortment encompassed the anceps and eremophila complexes of the mulleri subgroup, the fasciola, hydei and mercatorum subgroups, and several
non-repleta species from three other species groups, i.e. the canalinea, dreyfusi, and mesophragmatica groups, suggesting that repleta
group was not monophyletic. They concluded that the lack of resolution was caused by saturation of third positions in mitochondrial protein coding genes and suggested that better taxon
sampling, increased numbers of parsimony informative characters,
and more characters sampled from slower evolving nuclear gene
regions should improve phylogenetic resolution (Durando et al.,
2000). To overcome this problem, we (1) increased the number
of parsimony informative characters from 501 to 1204, including
the addition of six slow evolving nuclear genes, (2) increased sampling from 54 to 71 taxa, and (3) used model-based inference
methods.
The extended molecular phylogeny (Fig. 2) is largely concordant
with previous analyses of morphology, biogeography, chromosomal
gene arrangements and molecular data for the repleta group (Vilela,
1983; Wasserman, 1992; Durando et al., 2000) suggesting that we
are moving closer to estimating the phylogenetic relationships of
Please cite this article in press as: Oliveira, D.C.S.G., et al. Monophyly, divergence times, and evolution of host plant use inferred from a revised phylogeny
of the Drosophila repleta species group. Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.05.012
D.C.S.G. Oliveira et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
this large Drosophila radiation. Our phylogenetic hypothesis is better resolved and supported (including the MP tree) than previous
attempts, and has allowed us to address several important evolutionary problems for reconstructing a phylogeny of the repleta group
despite the persistent lack of support for some basal nodes. Thus, the
present study has strengthened support for monophyly of the repleta group due to the BI and ML inferred phylogenies that robustly
supported the repleta group separating it from its closest sister
groups.
The hydei and fasciola subgroups and the eremophila complex
were recovered as the most basal repleta lineages (ML and BI trees;
Fig. 2). The poorly studied fasciola subgroup was unfortunately
underrepresented here due to difficulties in obtaining species.
Twenty-one species have been described for this subgroup from
tropical and cloud forests from Brazil to North America (reviewed
in Silva-Bernardi et al. (2006)). Wasserman (1992) proposed that
the fasciola subgroup arose from a cytological ancestor of the mulleri complex based on sharing of a single chromosome inversion.
However, Diniz and Sene (2004) showed that this gene arrangement was a different inversion with breakpoints very close to those
of the one found in the mulleri complex. Chromosome (Diniz and
Sene, 2004) and mtDNA COI gene-based (Silva-Bernardi et al.,
2006) phylogenies have resolved some of the relationships among
smaller subsets of fasciola subgroup species but many of these taxa
are poorly known. Inclusion of the understudied and presumably
basal inca subgroup (Rafael and Arcos, 1989) and a larger sampling
of fasciola subgroup species may help to resolve the proper placement of these basal repleta group lineages.
The evolutionary association of the repleta and mercatorum subgroups was apparent from chromosome inversions (Wasserman,
1982, 1992) and DNA phylogenies (Tatarenkov and Ayala, 2001).
All our analyses supported the clustering of the mercatorum and
repleta subgroups (Fig. 2). Nevertheless, relationships within this
clade were highly variable, including the placement of D. peninsularis that has been considered to belong to either the mercatorum
subgroup (Vilela, 1983) or the repleta subgroup (Wasserman,
1992). Therefore, merging the repleta and mercatorum subgroups
may be phylogenetically justifiable.
Wasserman (1982, 1992) considered the mulleri subgroup as a
number of independent lineages with unclear evolutionary relationships. According to the phylogenetic hypothesis presented
here, a monophyletic mulleri subgroup would include four species
complexes (Fig. 2). The meridiana complex is the basal lineage of
this clade. The buzzatii complex is the next one to branch off, while
the mulleri and the longicornis complexes are sister taxa and the
most recently diverged in the mulleri subgroup; the latter are predominantly North American with a few exceptions. The proposed
longicornis complex does not appear to be monophyletic because
D. huckinsi and D. huichole clustered with the mulleri complex, in
agreement with previous results (Oliveira et al., 2005; Fig. 2).
Two other mulleri species complexes, the eremophila and anceps
complexes, and two additional species, D. nigricruria, and D. pegasa
all occupy more basal relationships in our phylogeny with unclear
affinity to other mulleri complexes.
4.2. Evolution of host use
Use of fermenting cactus tissues as breeding substrates is the
hallmark of the repleta group (Carson, 1971, 2001; Heed, 1978).
The evolution of the repleta group seems to be closely associated
with the transition from the use of fermenting fruits of non-cactus
plants in moist forests to arid-adapted fleshy-stemmed desert
plants like Opuntia and other cacti. Interestingly, our divergence
time dating suggested that the diversification of the main repleta
group lineages occurred from 16 to 12 Mya (Fig. 4), which is close
to the estimate of the appearance of the Opuntioideae (15 Mya;
9
Nyffeler and Eggli, 2010). The use of forest fruits is still observed
in species representing the immediate outgroups of the repleta
group, i.e. D. camargoi, and D. canalinea; unfortunately, the ecology of the annulimana group remains poorly characterized. The
overall ecology of fasciola subgroup species is not well characterized, but some have been reported to use fruits, flowers, and fungi
(Wasserman, 1992). One member of this subgroup, D. onca, not
included here, is known to breed in Rhipsalis, a forest dwelling
epiphytic cactus in Brazil (Pereira et al., 1983). Further, study of
communities of yeasts responsible for tissue fermentation associated with fasciola group species D. carolinae, D. coroica, D. onca,
and D. fascioloides revealed significant species differences from
other forest dwelling drosophilids and more similar to yeast communities from known cactophilic Drosophila serido (Morais et al.,
1995). Nyffeler and Eggli (2010) consider the 54 species in Rhipsalideae to be allied with the Cactoideae, so the humid-forest
dweller condition of the fasciola subgroup could be either a derived, reversed adaptation or the ancestral state for the entire
repleta group (Wasserman, 1962; Throckmorton, 1975; Vilela,
1983; Diniz and Sene, 2004; Silva-Bernardi et al., 2006).
The use of Opuntia cactus species is common throughout the
repleta phylogeny (Fig. 4), excluding the fasciola subgroup. Ecology
of two of the three species in the eremophila complex, D. eremophila
and D. micromettleri, is understudied, but they have been associated
with Opuntia exudate-soaked soils (Fogleman and Williams, 1987;
Heed, 1989; Table S4). Therefore, once the transition to the use of
Opuntia cactus evolved, almost all species retained host breeding
affinities for cactus use. Since many repleta group species have been
observed using seasonal cactus fruits for feeding and breeding
(Etges and Heed, unpubl. data), this seems an obvious ecological
bridge from fruit use to fermenting pads and stem tissues. The hydei, mercatorum, and repleta subgroups species are restricted to
Opuntia cactus with the exception of D. eohydei and D. repleta. As
D. repleta has become cosmopolitan and commensal with humans,
it has likely expanded its ancestral breeding site repertoire.
The use of the more chemically complex columnar cacti, as
reconstructed in our phylogeny, is derived relative to Opuntia. Most
columnar cacti studied are characterized by significant concentrations of a variety of different secondary chemicals including alkaloids, triterpene glycosides, fatty acids, and phytosterols while
Opuntia species typically lack appreciable amounts of these compounds (Fogleman and Abril, 1990; Fogleman and Danielson,
2001; Kircher, 1982). The transition to columnar cactus specialization has thus been a gradual one: the sister groups of almost all
columnar cactus specialists are either restricted to Opuntia or can
use both. Columnar cactus use has evolved in the mulleri-longicornis lineage 6 times: in D. aldrichi, in the D. mojavensis cluster, in D.
longicornis, in D. huaylasi, in D. spenceri and D. hexastigma, and in
the mayaguana subcluster. The latter 3 cases have been accompanied by the loss of Opuntia use by D. huaylasi, D. parisiena, and
the D. spenceri/D. hexastigma pair. Drosophila huaylasi has now been
moved from the D. mojavensis cluster (Durando et al., 2000) to the
mulleri cluster, and this species is known only from a few collections in Peru reared only from smaller columnar-like cacti in the
genera Armatocereus and Neoraimondia (Table S4). In addition, populations of D. aldrichi in Texas and central-northern Mexico use
Opuntia exclusively (Patterson, 1943), but we discovered ‘‘D. aldrichi’’ using Myrtillocactus geometrizans in Tehuacan, Puebla and
Pachycereus weberi in Cañón Zopilote, Guerrero, Mexico (Table
S4). In South America, ‘‘D. aldrichi’’ has also been reared from
Armatocereus sp. (Suyo and Pilares, 1987). These disparate populations of ’’D. aldrichi’’ are likely be different species given the degree
of reproductive isolation among some of them (Wasserman, 1992;
Krebs and Barker, 1994), significant genetic differentiation of eastern and western Mexican populations (Beckenbach et al., 2008;
Oliveira et al., 2008), and differentiation in patterns of host use.
Please cite this article in press as: Oliveira, D.C.S.G., et al. Monophyly, divergence times, and evolution of host plant use inferred from a revised phylogeny
of the Drosophila repleta species group. Mol. Phylogenet. Evol. (2012), http://dx.doi.org/10.1016/j.ympev.2012.05.012
10
D.C.S.G. Oliveira et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
The transition to columnar cacti has also occurred in the buzzatii
complex and in the anceps complex. Subsequent switches away
from Opuntia use has occurred twice in the South American buzzatti complex, in D. uniseta within the northern martensis species
cluster and in D. borborema, D. serido, and D. gouveai (Table S4) in
the southern buzzatii cluster. Loss of Opuntia use has also occurred
in the D. anceps/D. nigrospiracula species pair (Heed, 1982). Overall,
columnar specialization has evolved at least seven times involving
11 of the 65 species in the repleta group: D. huaylasi, D. parisiena,
D. spenceri, D. hexastigma, D. borborema, D. serido, D. gouveai,
D. uniseta, D. nigrospiracula, D. anceps, and D. mettleri (the latter
species is included because it is associated with columnar cacti,
although it breeds in fermented cactus exudate-soaked soils at
the base of the plants). Therefore, ecological transitions to columnar cacti are common, having occurred multiple times in different
clades from North and South America within the D. repleta group.
4.3. Origin and dispersal patterns of the Drosophila repleta group
Throckmorton (1975) proposed that Drosophila colonized the
New World from Asia and went through a series of major species
radiations during the Miocene. In general, rapid species radiations
should occur soon after the origin of a group, likely related to ecological innovation and invasion of new niches (Kocher, 2004; Rokas
et al., 2005; Hallstrom and Janke, 2008). The Mexican Trans-Volcanic Region had been considered the center of diversification for the
repleta group (Patterson and Stone, 1952; Throckmorton, 1975),
where most D. repleta species were known due to earlier, more
intensive collecting efforts in the USA and Mexico starting in the
1940s. Patterson and Stone (1952) pointed out that central and
southern Mexico (app. 19–22° N. latitude) ‘‘is of most interest for
Drosophila distribution, for here are to be found 89 of the 391 species known to occur in the Americas and Neotropical regions’’. The
D. repleta group has about half its known members in this zone’’.
However, the diversity of D. repleta group species and relatives
has become apparent in South America with increased collecting
efforts (e.g. Brncic, 1957; Pereira et al., 1983; Vilela, 1983; Ruiz
et al., 2000; Vela and Rafael, 2005; Acurio and Rafael, 2010). More
species continue to be described, e.g. the inca subgroup with three
described (Rafael and Arcos, 1989; Rafael and Vela, 2003; Vela and
Rafael, 2005) and additional undescribed species from Peru and
Ecuador (Andrea Acurio pers. comm.).
Similarly, host plant information has also been heavily biased
towards samples from USA and Mexico so far preventing definitive
biogeographical analysis of host use. Nevertheless, recent phylogenetic and systematic analyses of cacti in the Opuntioideae have
suggested a South American origin and that only late-diverging lineages are represented in North America (Griffith and Porter, 2009;
Nyffeler and Eggli, 2010). Given the elevated species diversity and
endemism of Mexican columnar cacti, particularly in southern
Mexico, this region likely represents a more recent center of radiation of endemic columar cactus species, such as those in the genera Carnegiea, Escontria, Lophocereus, Myrtillocactus, Pachycereus,
Polaskia, and Stenocereus, all of which are used by various Drosophila species.
Despite the lack of a clear historical biogeography, the repleta
group seems to be marked by repeated interchanges between South
and North America, since most subgroups have representative species in both continents (Fig. 3). Mapping species geographic distributions on our phylogeny suggested multiple dispersal events
across the Isthmus of Panama in both directions (Fig. 3). At least
some of these intercontinental dispersal events may have occurred
long before the Panama Isthmus joined North and South America
ca. 3 Mya, creating a land bridge for the Great American Biotic Interchange (Stehli and Webb, 1985; Marshal, 1988). In one case, the
split between the predominately South American buzzatii complex
and the mostly North American mulleri complexes was dated to
approximately 11.3 Mya. Furthermore, the phylogeny presented
here suggests that the islands of the Caribbean were colonized independently by repleta species at least five times as evidenced by the
geographic distributions of D. mulleri, the closely related triad of
D. mayaguana, D. straubae and D. parisiena, D. stalkeri and D. richardsoni, D. peninsularis, and D. micromettleri. This history is, however,
incomplete because we did not have access to the complete D. repleta fauna in the Caribbean, e.g., D. paraguttata of the fasciola subgroup, a species known only from a single strain from Jamaica
(Wasserman, 1992). This distribution of sister species suggests that
dispersal into the Caribbean may have originated from both North
(e.g. D. mayaguana, D. straubae, and D. parisiena) and South America
(e.g. the stalkeri subcluster and the canalinea group), followed by
local inter- and intra-island diversification (Heed and Grimaldi,
1991).
The origin of the repleta group cactus hosts and the discovery of
a broader South American drosophilid species diversity suggest a
South American origin for the repleta group. Taken together, the
mass of historical and biogeographical data suggest that during
the mid-Miocene, in an isolated and drier South America, the repleta group originated and quickly radiated along with its cactus
hosts. As South America moved northward, diversification of both
cacti and the D. repleta group allowed colonization of North
America and invasion of the Caribbean islands. During this process,
breeding site shifts from tropical, ephemeral fruits and flowers to
epiphytic cacti and the widespread and abundant flat leaf Opuntia
allowed further host plant shifts and specialization in many repleta
species to the more chemically complex columnar cacti.
Acknowledgments
We are grateful to A. Alverson, A. Acurio, and D. Vela and two
anonymous reviewers for comments on the manuscript. Financial
assistance was provided by grants from the National Science Foundation DEB 01-29105 to R. DeSalle and P.M. O’Grady, INT-9724790
to W.J. Etges and W.B. Heed, and CONACyT and the Universidad
Autónoma Metropolitana-Iztapalapa to M.A. Armella. F.C. Almeida
was funded by a Juan de la Cierva fellowship, Ministerio de Ciencia
y Innovación, Spain. Molecular data were generated while D.C. Oliveira was an Ambrose Monell Research Fellow at the AMNH. This
paper is dedicated to Bill Heed who inspired the study of repleta
group breeding sites, provided unpublished host use data, and
helped identify species for this study.
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.
2012.05.012.
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