The family Sarcophagidae is a highly diverse group, being one of the largest insect radiations among the living organisms (1). The present study corroborates previous findings regarding the monophyly of sarcophagids (14,17–19,27,33–37) and its three subfamilies (6,14,15,18,27,38). Several studies have challenged the traditional classification and questioned subfamily-level relationships (18,34,39,40), but more evidence is accumulating in support of Paramacronychiinae as sister to Miltogramminae (14,15,17,27,38). Regarding the phylogenetic relationships within Miltogramminae and Paramacronychiinae, the relationships recovered here are consistent with those estimated using other molecular datasets. For example, Eumacronychia Townsend is sister to the remaining Miltogramminae (14) and the genera Dexagria Rohdendorf, Paramacronychia Brauer & Bergenstamm, and Brachicoma Rondani are early-branching lineages within Paramacronychiinae (15).
The relationships within the subfamily Sarcophaginae have historically been a challenge for phylogeneticists. Previous morphology-based studies suggested phylogenetic hypotheses for the genera of this subfamily (7,40), which are not fully supported by Sanger-based phylogenies (16–18). Most of these studies have important differences in taxon sampling and molecular markers, and many of them received weak statistical support. More recent next generation sequencing (NGS)-based phylogenies using transcriptomes (41), anchored hybrid enrichment (15), and UCEs (27) received stronger statistical support, but had limited taxon sampling for genera. In the present study, the use of UCE data augmented by improved taxon sampling, results in a much better resolved phylogeny, with most nodes receiving maximum support (Fig. 2).
The new phylogeny does not generally contradict well-supported clades in the previous NGS-based studies but does uncover many novel relationships that are revealed by the inclusion of a wide sampling of genera. These novel relationships include 1) the placement of the clade A or (Lepidodexia (Emdenimyia (Boettcheria + Tripanurga))) as sister to the remaining sarcophagids, 2) the sister-group relationship between Microcerella and the clade C or (Ravinia ((Oxyvinia + Oxysarcodexia) + ((Cistudinomyia + Tricharaea) + (Dexosarcophaga (Nephochaetopteryx + Sarcofahrtiopsis)))), 3) the sister-group relationship between Cistudinomyia and Tricharaea, 4) the sister-group relationship between Dexosarcophaga and (Nephochaetopteryx + Sarcofahrtiopsis), 5) the sister-group relationship between Argoravinia Townsend and (Malacophagomyia Lopes (Titanogrypa + Rafaelia Townsend + Tulaeopoda Townsend)) in clade D, 6) the sister-group relationship between (Comasarcophaga (Mecynocorpus + Fletcherimyia)) and (Emblemasoma Aldrich (Spirobolomyia Townsend + Blaesoxipha)), 7) the sister-group relationship between Helicobia and ((Chrysagria (Peckiamyia + Retrocitomyia)) + (Villegasia ((Peckia (Pattonella) Enderlein (Peckia (Engelimyia + Lipoptilocnema))) + (Sarcophaga)))), and 8) the sister-group relationship between (Peckia (Pattonella) (Peckia (Engelimyia + Lipoptilocnema))) and Sarcophaga. These results partially agree with phylogenetic relationships for Sarcophaginae based on morphological and molecular characters. For example, the ‘Blaesoxipha clade’ of Buenaventura & Pape (7) is represented here by Blaesoxipha, Comasarcophaga, Fletcherimyia, Mecynocorpus, and Spirobolomyia and is recovered as a well-supported clade, although the clade recovered here also includes Emblemasoma. The internal relationships support those previously recovered by morphology (40) and UCEs (27) with Blaesoxipha as sister to Spirobolomyia and Comasarcophaga closely related to Fletcherimyia. The results partially support the ‘Sarcophaga clade’ of Buenaventura & Pape (7), which includes genera Chrysagria, Engelimyia, Helicobia, Lipoptilocnema, Peckia, and Sarcophaga, although the clade recovered here also includes Peckiamyia, Retrocitomyia, and Villegasia. The present study also affirms the existence of clades that previously had only weak or ambiguous support such as a Boettcheria + Tripanurga (16) and Peckiamyia + Retrocitomyia (7,42).
Within the largest radiation of Sarcophaginae, phylogenetic relationships in the hyperdiverse genus Sarcophaga closely match those recovered in previous molecular phylogenies, with the Nearctic subgenus Neobellieria as one of the earliest divergences (27,38,43). An early-branching clade including subgenera Heteronychia Brauer & Bergenstamm and Brasia Strand is also supported by data from an anchored hybrid enrichment analysis (15). The clade (Sarcophaga + Thyrsocnema) has also been supported in previous molecular phylogenies (15,27,38,43), as well as its close relationship to Myorhina (15,43). For the first time the Nearctic endemic subgenus Neosarcophaga is included in a phylogenetic analysis and it emerges in clade H as the sister to the Palaearctic clade (Myorhina (Sarcophaga + Thyrsocnema)). The relationships within clade J with subgenera Bellieriomima, Helicophagella, Mauritiella Verves, and Asceloctella Enderlein (among others) and clade K with subgenera Pandelleana Rohdendorf and Sarcorohdendorfia Baranov (among others) highly resemble those recovered in previous NGS-based phylogenies (15,27). Within clade K, the sister-group relationship between Stackelbergeola Rohdendorf and Rohdendorfisca Grunin is also consistent with previous Sanger- and NGS-based phylogenies (15,27,44). A close relationship between Bercaea and Liopygia has been suggested in previous phylogenies (15), which is supported here with Liopygia rendered paraphyletic by Bercaea. Similarly, a close relationship between subgenera Liosarcophaga, Parasarcophaga, Robineauella, and Rosellea is supported by previous molecular data (43) and it is here confirmed with clade M that has (Robineauella (Parasarcophaga (Liosarcophaga + Rosellea))). Most of the similar phylogenetic results of the present study and those of previous NGS-based phylogenies are due to taxon sampling compatibility and not to data duplication or data similarity, as the loci studied in these studies are not compatible but they give a significantly consistent phylogenetic signal.
Evolution of life habits
Flesh flies successfully feed on a breadth of live or dead hosts (vertebrates, invertebrates, feces), yet these vary tremendously in accessibility, volatile profile, attractiveness to potential competitors, as well as the host of behavioral, chemical, and morphological specializations needed for the gravid females and the first instar larvae to survive interactions with their hosts. A significant distinction can usually be made between a community of predatory species and a community of decomposer species living as sarcosaprophages, corprophages, or kleptoparasitic flies. There is a general fidelity in the choice of larval feeding habits, with some flesh-fly genera adapted to behave as predators on invertebrates (Blaesoxipha, Boettcheria, Chrysagria, Cistudinomyia, Emblemasoma, Emdenimyia, Lepidodexia, Malacophagomyia, Rafaelia, Spirobolomyia, Tripanurga, and several subgenera of Sarcophaga) and a group of genera of a generally smaller size adapted as decomposers of organic matter (sarcosaprograges and coprophages) either on invertebrates or vertebrates (Engelimyia, Lipoptilocnema, Microcerella, Nephochaetopteryx, Peckia, Peckiamyia, Oxysarcodexia, Oxyvinia, Ravinia, Retrocitomyia, Sarcofahrtiopsis, Tricharaea, and Villegasia). Very few Sarcophagidae taxa can be considered generalists, as usually only one or two species in particular genera are able to feed in different trophic substrates according to their availability. However, it has historically been unclear how this manifold ecology of predators, sarcosaprograges, coprophages, and kleptoparasites evolved within the flesh flies.
The question of whether the MRCA of all sarcophagids was a predator or a decomposer, or in association to which host have sarcophagids evolved, has thus always piqued the curiosity of flesh fly specialists. However, most of this curiosity has often been hindered by the impossibility of having a well-supported phylogeny of Sarcophagidae and its sister family to trace live habits and also by the scarcity of information on the biology of the group. The possibility of having a robust phylogeny for Sarcophagidae has improved over the years with the advance of phylogenetic methods and the efficiency in NGS-based molecular techniques and morphological methods for accessing and collecting different sources of data for phylogenetic reconstruction. The knowledge on the biology of the group, especially of some genera, continues to be a limitation, which can be mitigated with the use of algorithms for ancestral character state reconstruction that allow for missing data. However, this comes with costs in the uncertainty of the estimations.
Earlier hypotheses suggested that at least the subfamily Sarcophaginae had a coprophage or saprophage ancestor, and was undergoing the change from coprophagous and saprophagous nutrition to pure parasitic nutrition (45). Regarding subfamily Miltogramminae, a recent hypothesis suggests that larvae of ancient miltogrammines were sarcosaprophagous (14). An ancestral sarcosaprophagous Sarcophagidae fly feeding on invertebrates as primary food source was recently supported by the first formal analysis of this question, and the ancestors of the subfamilies Miltogramminae and Paramacronychiinae were further reconstructed as sarcosaprophagous feeding on both vertebrates and invertebrates (41). These hypotheses are only partially supported by the data presented here. The results presented here support kleptoparasitism (or inquilinism) of Miltogramminae arising from a predator (i.e., parasitic) ancestor (46,47). In contrast with previous hypotheses, the MRCA of all sarcophagids is here estimated to have been a predator on invertebrates, which is consistently supported by both the Mesquite-based (including the sister-family Oestridae, among other outgroups) and the corHMM-based (including only Sarcophagidae) ancestral character state reconstructions. This same combination of larval feeding habit and larval food resource is also supported for the MRCA of each of the three subfamilies.
Differences in conclusions between previous hypotheses and the present analyses are probably due to a) differences in taxon sampling and b) differences in the topologies used for ACR. The MRCA of Miltogramminae subfamily is estimated as sarcosaprophagous when species of only this subfamily are analyzed (e.g., 14). However, when the scope of the analysis includes other subfamilies and outgroups of other Oestroidea families, such as Oestridae, i.e., the sister-group of Sarcophagidae, the MRCA of Miltogramminae is reconstructed as a predator on invertebrates. Differences in topologies, especially regarding the earliest divergences, have an important impact on the ACR. For example, the inclusion of the genus Galopagomyia Bischof as sister to the remaining Paramacronychiinae in the supertree of Yan et al. (41) dramatically affects the estimated ancestral life history for the subfamilies Miltogramminae and Paramacronychiinae, giving sarcosaprophagy on invertebrates or vertebrates as the ancestral character state of the MRCA of these subfamilies. The phylogenetic position of Galopagomyia has only once been evaluated in a phylogenetic analysis including only Paramacronychiinae species and two Sarcophaginae species as outgroup (39). The sister-group relationship of Galopagomyia to the remaining Paramacronychiinae is supported by two character states, i.e., the color of the tegula with respect to the basicosta and the shape of the posterior margin of the ST5 (39), both of which can vary greatly across taxa of the family and do not constitute synapomorphies for Galopagomyia. For example, a black tegula contrasting with yellowish or light brown basicosta as observed in Galopagomyia is also found in many non-related genera of Sarcophaginae (7). Similarly, many non-related genera of Sarcophaginae share a distinctly emarginated and either broadly U-shaped or distinctly V-shaped posterior margin of the ST5 with Galopagomyia. The evaluation of these characters in the broad context of the family would most likely not support the position of Galopagomyia as sister to the remaining Paramacronychiinae. Therefore, the position of Galopagomyia is considered uncertain. This taxon was manually added to the supertree used for ACR in Yan et al. (41), and therefore its phylogenetic position continues to be uncertain. Without Galopagomyia, the supertree used for ACR in Yan et al. (41) would have genus Agria Robineau-Desvoidy, a predator on invertebrates, as sister to the remaining Paramacronychiinae. Thus, as in Yan et al. (41) the MRCA of Miltogramminae is estimated as a predator or sarcosaprophage on vertebrates or invertebrates, then an ACR with an adjusted topology (without Galopagomyia) would most likely estimate the MRCA of both Miltogramminae and Paramacronychiinae as a predator on invertebrates, which would be consistent with results presented here. Another topology effect in the estimations of Yan et al. (41) is related to the phylogenetic taxa populating the early divergences within Sarcophaginae. The MRCA of Sarcophaginae is estimated as a sarcosaprophage on invertebrates in Yan et al.’s (41) supertree, as it has Tricharaea and Sarcofahrtiopsis, which are sarcosaprohages/coprophages and sarcosaprohages, respectively, as laddered sister-groups to the remaining Sarcophaginae. In contrast, the present ACR estimates the MRCA of Sarcophaginae as a predator on invertebrates based on an UCE-based phylogeny providing robust support for (Lepidodexia (Emdenimyia (Boettcheria + Tripanurga))) or clade A as sister to the remaining sarcophagids (Figs. 3 and 4). Not only the basal position of this clade of invertebrate predators determines the ACR estimation for the MRCA of Sarcophaginae, but the subsequent divergence of another taxon, Udamopyga Hall, which is coded as predator or sarcosaprophage on invertebrates.
An interesting transition from the predatory habits on invertebrates observed in the early divergences of Sarcophaginae towards non-predatory habits is observed within the clade B (Figs. 3 and 4). The MRCA of clade B is estimated as a predator on invertebrates, while the MRCA of Microcerella is estimated as a sarcosaprophage on vertebrates and the MRCA of genera in its sister clade C is estimated as a coprophagous. Within this clade, other interesting results are related to the habitats occupied by closely related taxa and their use of available resources. Thus, the sister-group relationship between Tricharaea and Cistudinomyia tells the story of taxa living on beaches (although Tricharaea can also be found in other environments), which are adapted to predate on a specific host like turtles in the case of Cistudinomyia, and a more plastic taxon like Tricharaea using both dead invertebrates and vertebrates as well as feces to feed on. Another switch from predatory habits to sarcosaprophagy occurs in the MRCA of the clade containing genera Engelimyia, Lipoptilocnema, and Peckia, a few species of genera Blaesoxipha and Argoravinia, and within the genus Sarcophaga with the MRCA of (Sarcosolomonia Baranov + Sarcorohdendorfia). In contrast, there are no switches from sarcosaprophagy or coprophagy to predatory habits, but clearly overall sarcosaprophagy and coprophagy have evolved in a nonrandom fashion in Sarcophaginae. No switches from an exclusively sarcosaprophagous habit back to predation are supported (although exceptions could occur in Peckia and some uncertainty remains regarding Helicobia). This may indicate benefits of an adaptation to sarcosaprophagy that prevent the reversal to a predatory lifestyle. These results also indicate that the origin of sarcosaprophagy would have taken place in the context of an existing ecology of an ancestor with a predator-host established relationship. This would also support a hypothesis of a gradual transition from predation to sarcosaprophagy or coprophagy, with predator flesh flies gradually attacking not only healthy hosts, but also injured or weakened hosts, or even dead hosts. A switch from predation to sarcosaprophagy or coprophagy would imply less risk for a predator flesh fly, while the opposite could mean a sarcosaprophagous flesh fly attacking a healthy host that could eventually counterattack. More detailed species-level analyses estimating ancestral larval feeding habits within genera including ‘generalist species’ (in genera such as Argoravinia, Peckia, Sarcophaga) are necessary, which in turn could lead to slightly different conclusions at the genus level.
The observation of non-reversals from predation to sarcosaprophagy or coprophagy could also be used in future research using a different approach to model the parameter process (transitions between the different rate classes) in a hidden Markov model to assume that sarcosaprophagy and coprophagy are not lost once they evolve, which contrasts with the assumption for the models used here where all transitions among the specified number of rate classes are the same (ER) or all transition rates are allowed and are independently estimated (ARD). Such a different approach would allow the inference of a biologically relevant, but unmeasured ‘hidden’ character that could have influenced the evolution of the observed characters here.
Evolution of male terminalia traits
Male terminalia traits evolved almost equally convergently and non-convergently in multiple lineages across all three main clades of Sarcophagidae, as it has been found in other studies (15), although there is a slight dominance of non-convergent traits here. The six convergent traits were the shape of posterior margin of the abdominal ST5, outline of dorsal surface of cercal prong, connection between basi- and distiphallus, shape of connection between basi- and distiphallus, harpes, and capitis, while the seven non-convergent traits were the vesica, phallotrema configuration, phallotrema position with regard to phallic tube, acrophallic levers, number of styli, median process, and juxta.
Regarding convergent traits, there are degrees in convergence with characters states evolving twice in the phylogeny, while other characters have multiple independent origins across the tree. For example, a straight posterior margin of the abdominal ST5 evolves twice independently in the MRCA of (Miltogramminae + Paramacronychiinae) and in the MRCA of the clade containing genera Cistudinomyia, Dexosarcophaga, Nephochaetopteryx, Oxysarcodexia, Oxyvinia, Sarcofahrtiopsis, and Tricharaea. An absent or reduced capitis also evolves twice in the subfamily Miltogramminae and the genus Peckia. Whereas characters like the outline of dorsal surface of cercal prong and the harpes have multiple independent origins and few reversals across the sarcophagid tree. The multiple origins of the harpes and possibly also the loss of capitis could be explained by a homology definition problem, as at least the harpes are difficult to delimitate and could be confused with other accessory appendages of the phallus.
An interesting case of convergence is observed for the traits related to the connection and shape of connection between basi- and distiphallus. In general a continuous connection between basi- and distiphallus evolves twice in the family, once in the MRCA of a smaller clade of (Miltogramminae + Paramacronychiinae) and another time in the MRCA of the larger clade C (Additional file 5). A second character, which looks into the details of this connection, shows that there is a transition in the degree of sclerotization originating the continuous connection between basi- and distiphallus in the larger clade (Fig. 5). Thus, the MRCA of clade B has a distinct hinge between basi- and distiphallus, while the MRCA of genus Microcerella has an intermediate character state between a distinct hinge and a fully sclerotized connection, and the MRCA of clade C has a fully sclerotized connection. The intermediate character state of the genus Microcerella consists of a hinge on the dorsal side of the phallus and a sclerotized, paler, rigid and tubular ventral area between basi- and distiphallus. This sclerotized, paler, rigid and tubular ventral area between basi- and distiphallus had been described before (48) but not analyzed in a broader phylogenetic context of the family. Such transitions in the degree of sclerotization have not been reported before in the morphological evolution of Sarcophagidae and it has not been reported for lineages within Miltogramminae or Paramacronychiinae.
The diversification of the subfamily Sarcophaginae, which includes 2/3 of the diversity of the family, is marked by the concerted evolution of a set of phallic traits that were found to be non-convergent. Some of these phallic traits involve complex structures like the juxta, vesica, and a complex acrophallus (with various styli), which have only isolated reversals or losses. The loss of complex structures as irreversible over time is a concept known as Dollo’s law (49). Although this evolutionary principle is still commonly accepted, a number of cases where it is apparently violated have been proposed. Here I found that most of the complex phallic structures (e.g., juxta, vesica, a complex acrophallus with more than one styli) are rarely lost once they have evolved, and only the harpes seem to be the exception. The juxta originates most probably in the ancestor of all sarcophagids, and became a more complex structure separate from the rest of the phallus by a hinge in the clade A (Lepidodexia (Emdenimyia (Boettcheria + Tripanurga))) (Additional file 13), which constitutes the first branching within Sarcophaginae. The phallotrema placed in a ventral position with regard to phallic tube follows the same evolutionary pattern as the juxta (Additional file 9). The vesica and folding of the phallotrema evolve in the MRCA of Sarcophaginae (Additional file 7), while the acrophallic levers evolve in the MRCA of clade C (Additional file 10). There might be some correlation between morphological characters, such as the concerted evolution of a phallotrema placed in a ventral position with regard to phallic tube and the origin of the juxta. Similarly, a concerted evolution is observed between the origin of the vesica and the folding of the phallotrema. Interestingly, the vesica is particularly ornamented and complex in the genera Cistudinomyia, Dexosarcophaga, Nephochaetopteryx, Oxysarcodexia, Oxyvinia, Sarcofahrtiopsis, and Tricharaea, and it seems to be functionally related to an extrusion of the styli during mating, which is mediated by the acrophallic levers (7), another trait showing a non-convergent evolution.
Regarding the relation between traits and diversification rate, the only trait having an evolutionary pattern close to the increased rate of diversification identified along the branch leading to genus Sarcophaga (Fig. 6B) is the number of styli (Fig. 6A). The MRCA of Sarcophaginae was estimated to have had three styli, which switches to two styli in the MRCA of the large clade F that contains Sarcophaga and other genera (Fig. 2). There is no evident explanation on how the reduction in number of styli could have been related to the massive radiation within the genus Sarcophaga.
Diversification of Sarcophagidae
The family Sarcophagidae is one of the most species-rich and diverse lineages of flies in the world. A majority of this diversity is represented by the subfamily Sarcophaginae, which contains three of the largest, most species-rich genera within the Sarcophagidae: Blaesoxipha, Lepidodexia, and Sarcophaga. One diversification rate shift was inferred by the present study and this shift is associated unsurprisingly with the genus Sarcophaga, which is also among the geographically most widespread taxa within Sarcophagidae. A moderate diversification rate increase is further estimated for Blaesoxipha, but with no distinct shift receiving strong support. The genus Lepidodexia is not associated with any diversification rate shift. One explanation is that Lepidodexia is estimated with a not so young age (9.1 Ma), an estimate that could have been influenced by incomplete taxon sampling in our dating analyses and by the underestimated number of species of Lepidodexia due to this genus is the least studied among the most species-rich genera. The accuracy of BAMM in estimating diversification rates and rate shifts under certain circumstances has been debated, especially regarding the sensitivity of BAMM analyses to the selected rate shift prior (50,51). Additional criticism has been raised regarding a tendency to overestimate diversification rates in smaller clades, thereby resulting in a potential underestimation of rate shifts overall (52). The results presented here are most likely not affected by potential erroneous diversification rate estimates, given that I recovered only one statistically significant shift for the most species-rich genus within Sacophagidae only — a result that essentially confirms observations based on taxonomic species diversity. Our results are concordant with studies showing lineages within Sarcophagidae (14,15,17,38) as the dominant fast-evolving groups of Oestroidea. Furthermore, our results support a super-radiation within the genus Sarcophaga, as recent studies suggest (15,17,27,38,43).