Phylogenetic analysis and a time tree for a large drosophilid data set (Diptera:Drosophilidae)

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Zoological Journal of the Linnean Society, 2013, 169, 765–775. With 2 figures

Phylogenetic analysis and a time tree for a large drosophilid data set (Diptera: Drosophilidae) CLAUDIA A. M. RUSSO*, BEATRIZ MELLO, ANNELISE FRAZÃO and CAROLINA M. VOLOCH Genetics Department, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Received 9 February 2013; revised 21 May 2013; accepted for publication 6 June 2013

Drosophila is the genus responsible for the birth of experimental genetics, but the taxonomy of drosophilids is difficult because of the overwhelming diversity of the group. In this study, we assembled sequences for 358 species (14 genera, eight subgenera, 57 species groups, and 65 subgroups) to generate a maximum-likelihood topology and a Bayesian timescale. In addition to sampling an unprecedented diversity of Drosophila lineages, our analyses incorporated a geographical perspective because of the high levels of endemism. In our topology, Drosophila funebris (Fabricius, 1787) (the type species of Drosophila) is tightly clustered with the pinicola subgroup in a North American clade within subgenus Drosophila. The type species of other drosophilid genera fall within the Drosophila radiation, presenting interesting prospects for the phylogenetic taxonomy of the group. Our timescale suggests that a few drosophilid lineages survived the Cretaceous–Palaeogene (K-Pg) extinction. The drosophilid diversification began during the Palaeocene in Eurasia, but peaked during the Miocene, an epoch of drastic climatic changes. The most recent common ancestor of the clades corresponding to subgenera Sophophora and Drosophila lived approximately 56 Mya. Additionally, Hawaiian drosophilids diverged from an East Asian lineage approximately 26 Mya, which is similar to the age of the oldest emerging atoll in the Hawaiian–Emperor Chain. Interestingly, the time estimates for major geographical splits (New World versus Asia and Africa versus Asia) were highly similar for independent lineages. These results suggest that vicariance played a significant role in the radiation of fruit flies. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013, 169, 765–775. doi: 10.1111/zoj.12062

ADDITIONAL KEYWORDS: biogeography – Drosophila – molecular phylogeny – phylogenetic taxonomy –

timescale.

INTRODUCTION When Thomas Hunt Morgan created the Fly Room at Columbia University, he could not have foreseen the upheaval that those Drosophila bottles would cause in the life sciences. Today, many databanks and stock centres are dedicated to Drosophila diversity, demonstrating the singular importance of the group responsible for the birth of experimental genetics. Because of its overwhelming diversity, however, Drosophila has proven to have a particularly difficult taxonomy (O’Grady & Markow, 2009).

*Corresponding author. E-mail: [email protected]

Exclusive taxonomic ranks have been proposed to accommodate this diversity (Bächli, 2013), but many of these ranks do not represent evolutionary lineages when tested (Russo, Takezaki & Nei, 1995; O’Grady & Markow, 2009; van der Linde et al., 2010). Various fractions of drosophilid diversity have been recurrently analysed in a molecular framework (e.g. O’Grady & DeSalle, 2008; Robe, Valente & Loreto, 2010; Gao et al., 2011; Morales-Hojas & Vieira, 2012), but more comprehensive phylogenetic studies are rare (Russo et al., 1995; Van der Linde et al., 2009, 2010; Morales-Hojas & Vieira, 2012; Yassin, 2013). In this study, we gathered molecular sequences to obtain the most comprehensive primary data set used to date in drosophilid phylogeny. We compiled

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sequences for 358 drosophilid species, representing 14 genera, eight subgenera, 57 species groups, and 65 subgroups (Table S1) to test the monophyly of the major taxonomic ranks, and to date the major diversification events. Because of the high rate of endemism in this group, we also analysed the geographical distributions of recent and ancestral drosophilid lineages.

MATERIAL AND METHODS TAXON SAMPLING AND MARKERS Our multi-locus data set included sequences from 358 drosophilid species [plus two Tephritidae out-groups: Bactrocera oleae (Rossi, 1790) and Ceratitis capitata (Wiedemann, 1824)], nearly doubling the maximum number of drosophilids analysed to date using primary data (Van der Linde et al., 2010; Yassin, 2013). Coding-strand shifts, which are common in arthropods, are a shortcoming of mitochondrial genome markers because these shifts result in evolutionary model reversals (Hassanin, Léger & Deutsch, 2005). Ribosomal genes and introns were also discarded because of alignment and paralogy issues. Thus, only widely sequenced nuclear markers with highly reliable alignments were included in our data set. The final alignment contained 9917 bp from the coding regions of the nuclear genes Adh, Amyrel, Ddc, Gpdh, Sod, and Xdh, but not all genes were included for all species (Table S1). We adopted the taxonomic nomenclature proposed in the March 2013 TaxoDros release (Bächli, 2013). For species not included in TaxoDros, we considered the taxonomic assignments given in GenBank and other sources (Wang et al., 2006, Yassin et al. 2008, Bisby et al., 2012). Furthermore, we incorporated the modifications proposed in a novel review of drosophilid taxonomy (Yassin, 2013). These modifications include the expansion of subgenus Siphlodora and the group status of ananassae and montium, which were previously considered melanogaster subgroups. We also adopted the generic assignment of Idiomyia for the Hawaiian Drosophila, as proposed in Grimaldi’s (1990) morphological monograph, although this designation is rare in molecular studies (for support, see Bächli 2013; Yassin 2013). The species names, genes, and GenBank accession numbers are listed in Table S1. The geographical distributions and taxonomic assignments of all species are listed in Table S2. The geographical distributions follow the stock-centre listings of the University of California at San Diego website (https:// stockcenter.ucsd.edu/info/welcome.php). For other species, the curated ZipcodeZoo databank was used to evaluate the geographical range (http://zipcodezoo .com/default.asp).

ALIGNMENT

AND PHYLOGENETIC ANALYSIS

Alignments were conducted separately for each nuclear marker using CLUSTALW with amino-acid translation performed using the MEGA 4 platform (Tamura et al., 2007). Individual gene alignments were concatenated using SEAVIEW (Gouy, Guindon & Gascuel, 2010). Gaps, missing and ambiguous data were retained. The final alignment contained 9917 bp and is available upon request. The maximum-likelihood (ML) method was used to reconstruct the phylogenetic tree shown in Figure 1. The ML analysis was performed using RAxML 7.0.4, with maximum-parsimony starting trees and the rapid hill-climbing algorithm (Stamatakis, 2006). The ML phylogenetic search was computationally exhaustive. Our final tree was selected from topologies generated by 800 separate RaxML runs, each with a separate maximum-parsimony starting tree. The best-fitting substitution model for each individual gene alignment was determined using the HYPHY package (Pond, Frost & Muse, 2005), based on Akaike’s information criterion (Akaike, 1974). For most individual gene alignments, the GTR + G + I model (Rodríguez et al., 1990) was selected. Thus, this model was used for the concatenated analysis. Branch-support values for the interior branches in the final ML topology were estimated using 1000 pseudo-replicates of the conservative bootstrap (BP) test (Russo, 1997) in RAxML. Ancestral geographical distributions were reconstructed by tracing ancestral characters using a likelihood approach in MESQUITE (Maddison & Maddison, 2011).

DIVERGENCE

TIMES

Divergence-time estimates were computed using the relaxed molecular clock method, which permits evolutionary rate variation among lineages, as implemented in BEAST 1.6.1 (Drummond & Rambaut, 2007). In this case, lineage rates vary according to a specified probability distribution. An uncorrelated lognormal rate evolution model was assumed for descendant lineages, and the Bayesian Markov chain Monte Carlo method (MCMC) was used to estimate the parameters. The alignment and evolutionary models were the same as those used in phylogenetic tree reconstruction. A Yule prior was applied for the speciation process, and the topology shown in Figure 1 was provided as a starting tree. This topology was fixed over the MCMC sampling so that it was retained for the time estimates, which greatly increased the computational time. Four MCMC chains of 100 million generations each were sampled every 1000th generation. We collected 2500 post burn-in samples from each of the four independent runs to obtain the posterior distributions of the divergence times. Thus, all ESS

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Figure 1. Timescale for drosophilids based on a maximum-likelihood (ML) analysis using a concatenated alignment (9917 bp) of six protein-coding nuclear genes. Several monophyletic branches have been collapsed, indicating that all taxa within that taxonomic rank form a cluster. Support values above branches are bootstrap proportions performed on the ML tree; values less than 50 are not shown. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013, 169, 765–775

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(effective sample size) values were high (greater than 250) when checked in TRACER 1.5 (Drummond & Rambaut, 2007). The time priors included two fossil calibrations from Dominican Republic amber records (Grimaldi, 1987): Scaptomyza dominicana Grimaldi, 1987 and Chymomyza primaeva Grimaldi, 1987 (see Table S3). The time priors also included radiometric estimates for Hawaiian island formation (Hardy, 1974; Clague & Dalrymple, 1987). Because of the high levels of endemism, island formation dates are useful to calibrate divergence times in drosophilids. The fossil and radiometric calibrations were assumed to be normally distributed time priors with means and standard deviations as shown in Table S3.

RESULTS Our phylogenetic tree (Fig. 1) recovers the family Drosophilidae as a monophyletic clade with a high support value, although many of the other support values are comparatively low. The diversity of the genera Chymomyza, Leucophenga, Phortica, and Scaptodrosophila clearly falls outside the cluster containing the Drosophila lineages (Drosophila radiation, Fig. 1). In contrast, the type species of Idiomyia, Liodrosophila, Mycodrosophila, and Scaptomyza, and certain species of Dichaetophora, Hirtodrosophila, Lordiphosa, Samoaia, and Zaprionus, are included in the Drosophila radiation. The monophyly of Leucophenga, Liodrosophila, Phortica, and Samoaia remains untested because only one species from each of these genera was analysed. However, the status of the remaining genera is discussed below. Our drosophilid timescale suggests that only two extant lineages survived the Cretaceous–Palaeogene extinction (K-Pg; Fig. 1). The topology indicates that Scaptodrosophila latifasciaeformis (Duda, 1940) is sister to all remaining drosophilids. Two other Scaptodrosophila lineages diverged sequentially, one of which also includes species of Chymomyza, Leucophenga, and Phortica. Phortica and Leucophenga are members of the subfamily Steganinae, but Chymomyza and Scaptodrosophila belong to Drosophilinae. Our results indicate that this initial drosophilid diversification occurred during the Eocene, compatible with the age of the oldest fossil drosophilid, Electrophortica, from Eocene strata (Hennig, 1960). Because no sequence is available for the type species of Scaptodrosophila, Scaptodrosophila scaptomyzoidea Duda, 1923, the generic status of this group remains to be clarified in future analyses. The general branching pattern, however, suggests that these early drosophilid lineages share generalist feeding habits (Throckmorton, 1975). Furthermore, prescutellar acrostical setulae, observed in most Scap-

todrosophila species, also occur within Steganinae (Grimaldi, 1990; Markow & O’Grady, 2006), potentially providing independent morphological support for our topology. The ancestral reconstructions of the geographical distributions of these lineages indicate a Eurasian origin (Fig. 2), but S. latifasciaeformis shows a disjunct distribution in Africa and the Americas (Table S2).

LORDIPHOSA–SOPHOPHORA

CLADE

Following these early Palaeogene divergences, the Drosophila radiation shows a clear Eurasian origin (Figs 1, 2). The ancestor lineage splits to form the Lordiphosa–Sophophora clade and the major Drosophila clade in the middle Palaeocene (56 Mya), considerably earlier than previous estimates (40 Mya, Russo et al., 1995; 25–40 Mya, Obbard et al., 2012). According to our topology, New World Sophophora share an ancestor with Eurasian Lordiphosa from Japan, Korea, and Russia, rather than with Eurasian Sophophora. Previous molecular studies have reported a close relationship between Lordiphosa and Drosophila (Gao et al., 2011; Yassin, 2013). This pattern is morphologically reasonable because Lordiphosa was originally described as a subgenus of Drosophila Basden, 1961 (for further morphological evidence, see Okada, 1963; Lastovka & Máca, 1978, Fig. 2; Hu & Toda, 2001). Our time estimate for the split between Lordiphosa and New World Sophophora is near the Eocene–Oligocene boundary (36 Mya). The New World Sophophora groups (saltans and willistoni) are monophyletic and form a clade. The monophyly of the willistoni group is now firmly established by morphological and molecular data, including the deletion of an Adh intron (Van der Linde & Houle 2008, but see also O’Grady and Kidwell 2002). The willistoni subgroup also appears to be monophyletic, but is clustered within bocainensis with low support. In the saltans group, the saltans and sturtevanti subgroups are monophyletic with higher support values. Only one species was analysed for each of the cordata and elliptica subgroups, and these were clustered in our topology. The Old World Sophophora groups (ananassae, melanogaster, montium, and obscura) also form a clade. Our timescale indicates that the protomelanogaster ancestor (sensu Throckmorton, 1975) radiated in Eurasia during the late Eocene (Figs 1, 2), but three African lineages emerged during the Miocene. The first African radiation took place during the middle Miocene, giving rise to the entire melanogaster subgroup. The second, during the late Miocene, occurred within the montium group, and the third African lineage (Drosophila lachaisei Tsacas, 1984) diverged during the early Miocene within the ananassae group.

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Figure 2. Phylogenetic tree showing the reconstructed ancestral geographical distributions for extant and ancestral drosophilids estimated by the maximum-likelihood algorithm. Extant geographical distributions were retrieved from the Drosophila Stock Center or from the ZipcodeZoo database. See Table S2 for geographical distributions.

In the melanogaster group, the elegans, melanogaster, and pseudotakahashii subgroups appear to be monophyletic, but the suzuki and ficusphila subgroups do not (Fig. 1). Although there are several Australian Drosophila lineages (e.g. Drosophila flavohirta Malloch, 1924; Drosophila pseudotakahashii Mather, 1957), no melanogaster radiation occurred on that

continent. Based on morphological and ecological data, Throckmorton (1975) has suggested that such a radiation was prevented by a major Scaptodrosophila diversification when the melanogaster ancestor arrived in Australia. Our timescale marginally supports this hypothesis, but a larger sample of Australian Scaptodrosophila species is needed to verify this conclusion.

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The radiation of the obscura species group apparently began during the middle Miocene (Fig. 1). Most lineages within this group are Eurasian, except for a small African clade. Apart from those, two subgroups are endemic to the New World: affinis and pseudoobscura. These New World subgroups are clustered within a clade that also includes the incertae sedis Drosophila helvetica Burla, 1948. Drosophila helvetica is from Asia and is weakly clustered with the New World pseudoobscura, but the New World affinis is strongly linked to this group.

ZAPRIONUS

CLADE

The early branches within the major Drosophila clade indicate a Eurasian origin (Fig. 2). Nevertheless, most early branches of this clade are restricted to Southeast Asia and the Pacific (SEA), strongly suggesting that the clade originated in the latter region (Table S2). The cosmopolitan Drosophila busckii Coquillett, 1901, which is sister to all remaining species, is the type species of the SEA subgenus Dorsilopha (see also Grimaldi, 1990; Robe et al., 2010). Samoaia leonensis Wheeler & Kambysellis, 1966, a member of the Pacific genus Samoaia, is the next species to diverge from the remaining lineages. Geographical consistency and morphological data indicate that Samoaia is monophyletic, but the type species, Samoaia ocellaris Malloch, 1934, was unavailable. Therefore, the status of this genus remains to be ascertained (Grimaldi, 1990; O’Grady & DeSalle, 2008). The remaining lineage divides to form the Zaprionus clade and the main Drosophila cluster (Fig. 1). The Zaprionus clade includes the SEA Liodrosophila aerea Okada, 1956, which joins the clade consisting of the SEA Drosophila repletoides (Carson & Okada, 1980) plus Zaprionus. The repletoides species group is currently unassigned to higher rankings (Yassin, 2013), but our topology suggests that it belongs to the Zaprionus clade (see also Van der Linde et al., 2010). Zaprionus and Samoaia are currently assigned to the Zaprionus genus group (Grimaldi, 1990; Yassin et al., 2010), but our results suggest that Samoaia is sister to a much larger group that includes Hirtodrosophila, Dichaetophora, Mycodrosophila, the Hawaiian Drosophilid clade, the subgenera Siphlodora and Drosophila, and the Zaprionus clade. Because the type species of Zaprionus (Zaprionus vittiger Coquillett, 1901) and Liodrosophila (L. aerea) were sampled, our results indicate that these genera belong to the Zaprionus clade (see also O’Grady & Markow, 2009). Our timescale shows that during the early Oligocene, an Old World Zaprionus ancestor diverged to form the subgenera Anaprionus (Eurasia) and Zap-

rionus (Africa). Our time estimates imply that the Oligocene Zaprionus radiation did not prevent the Miocene radiation of the melanogaster subgroup, but may have impeded the radiation of the African ananassae and montium lineages, which remained undiversified (Fig. 2). The difference in the melanogaster subgroup may be related to distinct ancestral ecological requirements or to sampling error; these alternatives remain to be tested using additional data. In a recent Bayesian molecular analysis, one Zaprionus species (Zaprionus K1) clustered with high support in a clade that included Drosophila funebris (Fabricius, 1787) (Yassin et al., 2010). In our tree, however, Zaprionus is monophyletic, and D. funebris clusters elsewhere. Furthermore, Bayesian analyses are known to inflate clade support, and their statistical support may not hold if more consistent statistical tests are performed (Suzuki et al. 2002). Even so, because this undescribed Zaprionus species was not included in our data set, the current diagnosis for Zaprionus may not represent a true clade. In a recent review of African Zaprionus, Yassin & David (2010) proposed a new definition of the species groups within Zaprionus based on morphological and molecular data. Their classification agrees well with our major Zaprionus clades (Fig. 2), except that Zaprionus tsacasi Yassin, 2008 (from the inermis group) is tightly clustered with Zaprionus taronus Chassagnard & Tsacas, 1993 (from the vittiger group), well inside the vittiger clade.

MAIN DROSOPHILA

CLUSTER

The main Drosophila cluster splits into clades A and B (Fig. 1). Clade A includes three lineages. The first is from the Far East (Japan and Korea), and includes several Hirtodrosophila lineages and a monophyletic Dichaetophora. The second is the Hawaiian drosophilid clade (HD), which includes the Drosophila annulipes Duda, 1924 (SEA) plus Drosophila maculinotata Okada, 1956 (Japan) cluster. The African Drosophila adamsi Wheeler, 1959 (unclassified) is weakly linked to the remaining members of the second lineage. Finally, the third lineage within clade A is the newly diagnosed subgenus Siphlodora (sensu Yassin, 2013). This association between the HD clade and a Far East Asian cluster (D. annulipes and D. maculinotata) is particularly interesting. Drosophila maculinotata is currently not assigned to any species group (Bächli, 2013), but Okada’s original description placed it in the funebris group (i.e. within our subgenus Drosophila clade; Okada, 1956). Later, Okada argued that D. maculinotata actually belongs to the virilis– repleta radiation (i.e. our Siphlodora subgenus clade; Okada, 1988): this hypothesis is more concordant

© 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013, 169, 765–775

DROSOPHILID TIMESCALE with our findings (see also Katoh et al., 2007). The other member of the cluster that is sister to the HD clade is D. annulipes, which is currently assigned to the immigrans group within subgenus Drosophila. Nevertheless, morphological and karyotypic data indicate that D. annulipes differs from other immigrans species (Wakahama et al., 1983; Zhang & Toda, 1992). Thus, despite the geographical support for this association, further morphological analyses are necessary. The monophyly of the HD clade is far from novel (see O’Grady & DeSalle, 2008; O’Grady et al., 2011). Nevertheless, the inclusion of the type species of Scaptomyza, Scaptomyza graminum (Fallén, 1823), confirms the taxonomic status of this genus (see also Yassin, 2013). The HD clade includes a monophyletic Scaptomyza, which clusters with the Hawaiian Idiomyia. The monophyly of Scaptomyza is strongly supported; among lower ranks, however, only subgenus Bunostoma receives strong support. The largest drosophilids in the world are grouped in the Hawaiian Idiomyia, which was originally described by Grimaldi (for support, see Yassin 2013). In Idiomyia, the antopocerus and modified-tarsus species groups are joined to form the AMC clade (see O’Grady & DeSalle, 2008), but the support for this grouping is relatively weak. Also, most of the Hawaiian picture-winged species (the adiastola, grimshawi, and planitibia groups) form a cluster, except for Idiomyia gymnobasis (Hardy & Kaneshiro, 1971) (grimshawi species group), which is sister to all other Hawaiian Idiomyia in our tree. This result is surprising because the Hawaiian picture-winged clade appears to be highly stable (see O’Grady & DeSalle, 2008, and references therein, but also see Van der Linde et al., 2010). Our data set, however, includes only the Ddc sequence for this species; thus, it may not be directly comparable with many other picture-winged species. In a BLAST search, the I. gymnobasis Ddc sequence most closely matches other sequences from the grimshawi species group, as expected. Therefore, the anomalous placement of I. gymnobasis appears to be an artefact related to the missing data in our matrix or to the ML algorithm. Morphological data support the inclusion of the unclassified Drosophila fluvialis Toda & Peng, 1989 in the Siphlodora clade (see Grimaldi, 1990). Although the species of the robusta group are united in our topology, the monophyletic quadrisetata group is clustered within the robusta group with strong support. Similar results have been reported in a molecular phylogeny using mitochondrial genes (Wang et al., 2006). Altogether, these results strongly suggest that the quadrisetata and robusta groups form a single clade.

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Based on morphological, ecological, and geographical data, Throckmorton (1975) suggested a primary radiation in Eurasia, with secondary radiations in the New World for what he called the virilis–repleta radiation (i.e. our subgenus Siphlodora). Most of the species in this group breed in rotting plant matter (Markow & O’Grady, 2006). In our tree, species groups within the subgenus Siphlodora are assembled according to larger geographical areas, following Throckmorton’s proposal. One cluster contains monophyletic Old World species groups (angor, melanica, and the quadrisetata plus robusta clade), whereas the other cluster includes species groups that mostly inhabit the New World (annulimana, bromelia, canalinea, mesophragmatica, nannoptera, and repleta). Clade B includes the remaining Hirtodrosophila lineages, Mycodrosophila, and the subgenus Drosophila. The Mycodrosophila cluster does not include the type species of the genus, M. poecilogastra, suggesting the need for a taxonomic reassessment of its generic status. In contrast, Throckmorton (1975) grouped Hirtodrosophila and Mycodrosophila with the Hawaiian drosophilids (see also Grimaldi, 1990). Our tree suggests that certain Hirtodrosophila lineages are closer to the Hawaiian drosophilids and subgenus Siphlodora, whereas others are connected to Mycodrosophila and subgenus Drosophila. Unfortunately, our data set did not include the type species of Hirtodrosophila, Hirtodrosophila latifrontata (Frota-Pessoa, 1945). Therefore, additional data are needed to verify the status of this genus. The strong bootstrap support for the position of the cosmopolitan type species of Drosophila, D. funebris, is an important finding. The type species of Drosophila belongs to the small funebris group, so that an uncertain phylogenetic placement of this group would preclude a formal review of the genus (O’Grady & Markow, 2009). Throckmorton considered this species group to be basal within the Drosophila radiation, but our results provide further statistical support for the funebris plus pinicola clade (see also van der Linde et al., 2010; Yassin, 2013). Our results indicate a consistent position for the funebris group, which is closely linked to Drosophila pinicola Sturtevant, 1942 within a North American clade of subgenus Drosophila. Recently, the pinicola group has been assigned to subgenus Siphlodora (Yassin, 2013). According to our results, however, the pinicola group clusters with the type species of (sub)genus Drosophila with strong support (92 BP). The critical position of the type species, D. funebris, is now more firmly established, and a phylogenetic taxonomy for most drosophilids is taking shape (Fig. 1; Yassin, 2013).

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DISCUSSION Our timescale provides solid evidence for a Palaeogene Eurasian drosophilid radiation, with important Neogene diversifications in the New World, Africa, and Hawaii. The New World–Eurasian splits ranged from the late Eocene to the Holocene. The Sophophora– Lordiphosa, immigrans–tripunctata, Siphlodora (Drosophila flexa Loew, 1866 and virilis–repleta) divergences all occurred around the boundary between the late Eocene and early Oligocene (Table 1). A recent study using mitochondrial markers (Gao et al., 2011) estimated that these splits occurred earlier, well into the Eocene (46 Mya), but the analysis employed an indirect calibration point. Our concordant time estimates for the same geographical splits indicate a vicariant event (see also Gao et al., 2011). Many early Eocene mammalian genera are found in both European and North American fossil records, strongly suggesting a temporary land connection between these landmasses: the North Atlantic land bridge (NALB; McKenna, 1975). After the middle Eocene, however, fossil mammalian faunas began to

diverge, indicating the closure of the passage, at least to mammals (McKenna, 1975; but see Thierstein & Berger 1978 for a more recent Eocene–Oligocene boundary). Nevertheless, a few portions of the NALB, such as the Greenland–Iceland–Faroe ridge, may have remained emergent until the Miocene (Eldholm & Thiede, 1979). For organisms with greater dispersal abilities, such as drosophilids, these remnants would have been sufficient to maintain gene flow between landmasses until more recently. Even for strong dispersers, however, temperature resistance would have been a key factor preventing high-latitude crossings (Raymo & Ruddiman, 1992; Milne, 2006), as has been reported in ants (Archibald et al. 2006). Given these variables, the NALB break ranges widely from the Eocene (for mammals; McKenna, 1975) to the Pliocene (for plants; Denk, Grímsson & Zetter, 2010). This wide temporal range obviously fits our estimates for the New World–Eurasian splits, but does not add much information about this event. Endemic geographical distributions, however, may tell a more detailed story (Fig. 2; Table S2). For example, Lordiphosa is endemic to the Far East,

Table 1. Bayesian time estimates with 95% ranges for African–Asian and New World–Eurasian splits in drosophilids Splits African–Asian Drosophila adamsi Zaprionus bogoriensis Drosophila lachaisei Drosophila pruinosa Drosophila melanogaster Scaptodrosophila Drosophila varians Drosophila iri obscura group montium group Drosophila nasuta NewWorld–Eurasian Lordiphosa–north-west Sophophora immigrans–tripunctata group Drosophila flexa virilis–repleta group immigrans–tripunctata group Hirtodrosophila melanica group obscura group quinaria group robusta group virilis 1 group virilis 2 group Scaptodrosophila

Mya

Timescale

Support

95% range

33.3 29.4 23.8 21.2 17.6 12.4 12.1 12.0 9.6 6.0 1.3

Late Eocene Early Oligocene Late Oligocene Early Miocene Early Miocene Middle Miocene Middle Miocene Middle Miocene Late Miocene Late Miocene Pleistocene

70 94 100 30 100 100 100 100 34 100 83

28.0–38.7 23.3–36.8 18.2–30.1 14.2–27.9 14.1–21.1 6.4–20.7 8.8–16.8 8.0–16.3 7.4–11.5 4.5–7.7 0.8–2.1

35.9 34.5 31.3 27.1 23.5 17.5 12.9 7.7 7.7 4.7 4.6 2.0 0.7

Late Eocene Late Eocene Early Oligocene Middle Oligocene Late Oligocene Early Miocene Middle Miocene Late Miocene Late Miocene Early Pliocene Early Pliocene Late Pliocene Holocene

99 30 39 73 23 99 100 99 89 96 99 100 73

28.6–44.0 29.4–40.8 26.8–37.0 22.9–31.7 18.8–29.4 11.2–24.3 9.0–17.5 5.9–9.6 5.4–10.5 2.5–7.7 3.3–5.9 1.2–3.1 0.0–0.8

Values of support are bootstrap proportions in our maximum-likelihood tree. They are given for the actual geographical split (regular) or for deeper branches (underlined), provided that they support independent (dispersal or vicariant) events. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013, 169, 765–775

DROSOPHILID TIMESCALE reported in Japan, Korea, and Russia. Thus, based on geographical data, it appears likely that an eastbound land bridge enabled the Asian Lordiphosa– Sophophora ancestor to colonize the New World. In this case, the principal land connection between these landmasses would have been the Bering land bridge. The break of this bridge has been accurately dated using the known fossil distribution of the marine mollusc Astarte, which migrated into the North Pacific Ocean in the middle Pliocene (Gladenkov et al., 2002). We would expect a marine-based estimate to be earlier than the land-dweller drosophilid split, but the break was probably too late to account for our drosophilid divergences around the Eocene– Oligocene boundary (Table 1). Furthermore, in the case of Lordiphosa–Sophophora, the geographical distribution of the sophophoran groups (willistoni and saltans) is currently in Central and South America, with a few dispersed Mexican lineages, whereas the Bering land bridge connected North America to East Asia. Sampling error, asymmetric extinctions, and other geological events may explain these results (see also Milne, 2006). On the other hand, the Bering land bridge hypothesis seems compatible with the remaining set of New World and Eurasian geographical splits in our tree (Fig. 2; Table 1), which involve the obscura and quinaria groups (late Miocene) and the robusta and virilis groups (early Pliocene). For the obscura group, for instance, the geographical distribution is also more concordant with a Bering land bridge setting, as the obscura and the pseudoobscura subgroups are mainly distributed in North America. In this case, the closure of the bridge would have restricted the D. helvetica ancestor to Asia, whereas the New World ancestor gave rise to the obscura and pseudoobscura subgroups before the Bering land bridge first flooded during the Pliocene. Our results also support three nearly concurrent geographical splits between Africa and Eurasia before the middle Miocene. These splits involve Scaptodrosophila finitima (Lamb, 1914) (African) and Scaptodrosophila bryani (Malloch, 1923) (Australian), the ananassae subgroup lineages of Drosophila varians Duda, 1922 (SEA) and the clade endemic to the East African Islands, and Drosophila hirtipes Lamb, 1914 (African) and the remaining members of the polychaeta species group (SEA). Because these estimates are all by the middle Miocene, they further endorse a vicariance-driven diversification for drosophilids (Table 1). A major vicariant event between the African and Eurasian landmasses was the opening of the Tethys Seaway during most of the Palaeogene (Harzhauser et al., 2007). At that time, the collision between the African and Eurasian plates created the Gomphoth-

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erium land bridge, rupturing Tethys, but providing an early Miocene passage to Asia for African proboscideans. By the middle Miocene, however, climate warming restored Tethys, as demonstrated by marine fossil records (Zachos et al., 2001). This event re-isolated the African and Asian terrestrial faunas, and may have caused several of the African–Asian geographical splits in our topology. Finally, the finding that the sister group of the Hawaiian drosophilids is an SEA lineage (Fig. 2) provides a more robust picture of this important diversification, compared with previous studies (Russo et al., 1995; O’Grady et al., 2011). The Hawaiian– Emperor Chain is a series of islands and eroded seamounts resulting from a hotspot of volcanic activity that accurately records the speed and direction of the moving Pacific plate. Hence, each island of the chain is sequentially older than that lying to the south-east (Cowie & Holland, 2008). During the Cretaceous, intense volcanism at the hotspot formed the Meiji Seamount (Heads, 2010), which now lies near the Kamchatka Peninsula in continental Asia. According to our timescale, the split between drosophilid lineages occurred during the early Oligocene (26 Mya). This date is compatible with one of the estimates (i.e. model A2) reported in a recent Hawaiian divergence analysis (Obbard et al., 2012; see also O’Grady and Markow, 2009), but is younger than other previous estimates (32.2 Mya, Russo et al., 1995; 43 Mya, Tamura et al. 2004). Our estimate matches the age of the oldest still-emergent islands in the Hawaiian–Emperor Chain: the Kure Atoll (29 Mya; see Heads, 2010; Table 1). Our results also imply that isolation between lineages occurred when the islands were still near the continent. Curiously, older seamounts north-west of Kure Atoll are generally smaller and more sparsely distributed than those on the Emperor side of the chain. This observation suggests that a break in gene flow might have occurred as a result of increased erosion when the hotspot was located beneath that area. Altogether, our results indicate that drosophilid diversification was initially influenced by the radiation of the angiosperms, most likely exploiting their newly evolved, appealing fruits (Dilcher 2000). Our time estimates also suggest that the radiation peaked during the Miocene, an epoch of drastic climatic changes (Zachos et al., 2001). This finding is also consistent with a vicariant setting for drosophilid radiation, which is supported by our similar time estimates for parallel geographical splits. The drosophilid radiation was apparently fuelled by the flies’ exploitation of the newly diversified fleshy fruits of angiosperms, which contributed to the specialization and ecological success of fruit flies.

© 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013, 169, 765–775

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ACKNOWLEDGEMENTS The authors are indebted to Carlos Eduardo Guerra Schrago, Ismar Carvalho, Franklin Rumjanek, Suzana Casaccia Vaz, and Wilson Costa for their insightful suggestions on earlier versions of the article. Carlos Schrago helped in merging Bayesian runs to infer the timescale. The authors thank FAPERJ (Rio de Janeiro State Research Foundation) and CNPq (Brazilian National Research Council) for the research grants that enabled this study. This article was reviewed by a professional science editor and by a native Englishspeaking copy editor to improve the readability.

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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Table S1. GenBank accession numbers for the six nuclear protein-coding markers and the 358 drosophilids used in this analysis. Table S2. Taxonomic assignments and geographical distribution data for the 358 drosophilids analysed in this paper. Table S3. Calibrations used in the divergence time estimates based on fossil records and on Hawaiian archipelago formation for endemic drosophilids lineages. © 2013 The Linnean Society of London, Zoological Journal of the Linnean Society, 2013, 169, 765–775