Phylogenetic analysis of the tribe Dufouriini (Diptera: Tachinidae) using a total evidence approach based on adult and immature stages

Dufouriini are a worldwide distributed tachinid tribe comprised of 51 species in 13 genera, made up of parasitoids of adult Coleoptera. The systematic positioning of Dufouriini has been controversial. Currently, it is placed within Dexiinae, but was previously placed in Phasiinae and Voriinae, and has even had the status of subfamily. Delimitation and composition of Dufouriini has also been debated, whether it is a single tribe or divided into two (Dufouriini and Freraeini) or three (Dufouriini, Oestrophasiini and Freraeini) tribes. Herein, we present the first phylogenetic analysis of Dufouriini based on total evidence using morphological data from adult and immature stages. The taxonomic sampling included all genera in Dufouriini (including Oestrophasiini) and also the genus belonging to Freraeini, a historically related tribe. Data matrix comprised 35 species and 22 genera in the ingroup, and 185 characters constructed from eggs, first instar larvae, puparia and adults, including female and male terminalia and spermathecae. The phylogenetic analysis recovered Dexiinae as paraphyletic in relation to Phasiinae, since the clade (Freraeini (Dufouriini + Oestrophasiini)) is more closely related to Phasiinae than Dexiinae. Dufouriini, Oestrophasiini and Freraeini are recovered as separate monophyletic tribes, strongly supported by a number of synapomorphies. Oestrophasiini is revalidated. A new synonymy is proposed: Comyopsis Townsend syn. nov. of Ebenia Macquart. Accordingly, Ebenia fumata (Townsend, 1919) is nomen preoccupatum by Ebenia fumata (Wulp, 1891), thus we change its specific epithet by designation of the new replacement name Ebenia neofumata Santis and Nihei [nomen novum]. The genera Mesnilana and Rhinophoroides are removed from Dufouriini and tentatively placed into Palpostomatini. Finally, Cenosoma stat. rev., previously a subgenus of Oestrophasia, is revalidated as genus.


Introduction
Tachinidae is one of the largest Diptera families, with 8547 described species worldwide (O'Hara et al. 2020). Four subfamilies have traditionally been recognized in Tachinidae: Exoristinae, Phasiinae, Tachininae and Dexiinae (Herting and Dely-Draskovits 1993;Tschorsnig and Richter 1998;O'Hara and Wood 2004; monotypic tribe, separated from Dufouriini by its type genus Freraea. The resulting tree showed a polyphyletic Dufouriini split into two groups: 1) with Microsoma forming a clade with Freraeini, sister group of some Palpostomatini genera; and 2) the other genera of Dufouriini (Oestrophasia, Rondania, Dufouria and Ebenia) forming a clade nested with some clades of Voriini and Telothyriini. The authors stressed that the tribal classification of Dexiinae requires major revision and that the phylogenetic resolution was unsatisfying in several parts of the tree.
The current concept of Dufouriini (Table 1) (= after Herting 1984;Tschorsnig 1985;Cantrell and Burwell 2010;O'Hara and Wood 2004;Cerretti et. al. 2014;Stireman et al. 2019;O'Hara et al. 2020), called "sensu lato" herein, includes the genera that were historically recognized in the tribe, as well as the genera from Oestrophasiini, and Eugymnopeza, Microsoma and Pandelleia. Although most of the Palaearctic Dufouriini and Neotropical genera belonging to former Oestrophasiini are well delimited and revised, their phylogenetic relationships are poorly resolved and suprageneric delimitations are unclear. For all recorded species, members of Dufouriini are characterized as parasitoids of adult beetles. Most genera present modified ovipositors with diverse forms (Herting 1957) to parasitize their hosts through different strategies, e.g., perforating the epithelium to introduce larvae in natural openings as in Microsoma; using its ovipositor to inject first instar larvae directly into the mouth of its host (Fluiter and Blijdorp 1935) as in Rondania; and depositing microtype eggs into leaves that are swallowed by the host as in Oestrophasia (Cenosoma) sp. (Grillo and Alvarez 1984).
In Tachinidae systematics, adult morphology (excluding male or female terminalia) had initially been used as the primary, and in most cases, unique criterium for their classification (e.g., Villeneuve 1924;Mesnil 1939). Later, male terminalia had its taxonomic value accepted and progressively added, becoming ever since one of the most important character sources in Tachinidae (e.g., Verbeke 1962Verbeke , 1963Tschorsnig 1985), as has occurred for many other insect groups (Song and Bucheli 2010). The use of different character sources other than adult morphology and male terminalia has been revealed and encouraged by a number of authors over time for Tachinidae systematics. The relevance of larval morphology as a valuable source of data for the classification of tachinids was discussed in several articles by Thompson (1914Thompson ( -1967. While Herting (1957) was the first to emphasize the importance of female terminalia and eggs. Considering the importance of Thompson and Herting's discoveries, Mesnil (1966) recognized that an appropriate classification of Tachinidae would only be possible using other data sources, and then revised his early classification using characters from larvae, and male and female terminalia. Later, Herting (1983) discussed the main groups of Tachinidae and concluded that (p. 2 therein): "The most reliable indicators of phylogenetic relationships appear to be the biologically-adaptive characteristics that are pronounced in the female ovipositor, in the structure of the egg membrane and the morphology of the first ínstar larva". In a comprehensive study, Ziegler (1998) described and discussed the phylogenetic significance of characters from puparia and larval cephaloskeleton for 261 tachinid species, defining putative synapomorphies for the family and, whenever possible, for tribes. Barraclough (1992Barraclough ( : p.1149, reinforced these viewpoints by stating: "This broad-based approach is preferable, since it is particularly unwise in the Tachinidae to give undue weighting to particular characters or suites of characters.". In the present study, we carried out a phylogenetic analysis including a complete sampling of all genera belonging to Dufouriini and all genera belonging to Freraeini, a tribe that has historically been related to and controversial for Dufouriini, in order to clarify the internal relationships and monophyly of the tribe Dufouriini and its supra-tribal relationships. Our phylogenetic analysis was based on Hennig's concept of holomorphology (Hennig 1966), i.e., the integration of data from different life cycle stages (semaphoronts), embodied in the light of the 'requirement of total evidence'. This requires that all relevant evidence be used for an appropriate inductive or abductive inference (Fitzhugh 2006). Therefore, the higher the number and more sources of characters, the greater the degree of being a natural group, i.e., ontologically realistic taxa (Rieppel 2005). We examined a large number of morphological characters from adult (external morphology, male and female terminalia, spermathecae) and immature stages (e.g., eggs, larvae, puparia). Herein, morphology from the puparia is included in a phylogenetic analysis of Tachinidae for the first time.

Morphological study and terminology
To study adult morphology, dried and pinned specimens were examined under a Leica EZ4 stereomicroscope. A Leica DM2500 optical microscope was used to analyze the postabdomen, first instar larvae and spermathecae.
To study the male postabdomen, the specimens were carefully dissected from the fifth segment to avoid damaging the sixth tergite and to maintain the integrity of the abdomen as much as possible. To study the female abdomen and obtain the spermathecae, first instar larvae and/or eggs, the abdomen was dissected from the fourth segment and rarely in the third. The male terminalia were bleached in 10% potassium hydroxide solution (KОН) for four minutes in boiling water, neutralized with 5% acetic acid solution and washed with water. The female terminalia, larvae, spermathecae and eggs were subjected to a similar procedure, except they were heated for 10 minutes in 10% KOH solution. At the end of the procedure, the material was preserved in glycerin, packed in microplastic vials and pinned with the respective specimen.
The terminology of adult and spermathecae morphology follows Cumming and Wood (2017). The terminology used for the wing structure and trace of M 2 vein is taken from Crosskey (1976). For male terminalia, we follow Tschorsnig (1985). The terminology of the first instar larva follows Thompson (1963), with some modifications discussed by Cantrell (1988). The term "cephaloskeleton" from Courtney et al. (2000) was used. The terminology for the puparium follows Ziegler (1998) and that for the eggs follows Gaponov (2003).

Selection of taxa
To select the terminals of the ingroup, three premises were considered: (1) the availability of adult specimens for morphological study; (2) the availability of immature stage material (e.g., first instar larvae); and (3) differences in geographic distribution and morphology. All 13 genera included in Dufouriini (sensu Herting 1984) (Table 1) were studied, and 11 were sampled, including those genera from the formerly valid Oestrophasiini. Additionally, the sole genus currently assigned to Freraeini (Freraea) (O'Hara et al. 2020) was included, as it has historically been related to Dufouriini. The ingroup included 26 species from 13 genera. In light of the results of Cerretti et al. (2014) (where Dexiinae is paraphyletic in relation to Phasiinae) and Stireman et al. (2019) (Dufouriini polyphyletic, split into two lineages), some additional representative tribes were chosen as outgroup taxa. The basis of paraphyly of Dexiinae derived from historically problematic taxa: Strongygastrini, Imitomyiini and Catharosiini, besides other Phasiinae (Cylindromyia) were included. We also included species of Dexiini and Voriini concerning Dufouriini monophyly and relationships. Xanthozona (Tachinini) was selected as the root for the analyses. The outgroup included a total of nine species from Phasiinae, Dexiinae and Tachininae. Supplementary file 1 shows the terminals included in the cladistic analysis with geographical distribution, data source and observed structures (whether personal observation or literature data).

Phylogenetic analysis and character coding
The study of phylogenetic relationships was based on morphological characters of adults (including female and male genitalia and spermathecae), first instar larva, egg and puparium, which was based on parsimony as the optimality criterion. Whenever possible, the characters were constructed according to the proposal of Sereno (2007), with preference for the contingent coding (Forey and Kitching 2000). Characters are scored with "-" in case of inapplicability (usually taxon lacking the character-bearing structure), and with "?" in case of lacking observation. The data and putative synapomorphies of the male terminalia presented by Tschorsnig (1985) were reanalyzed and included within a cladistic framework. Characters from the literature, e.g., Cerretti et al. (2014), have been properly indicated in the character list. The polarization was conducted using the method of outgroup comparison (Nixon and Carpenter 1993). The matrix of characters was built with Mesquite 3.04 software (Madison and Madison 2015). For the parsimony analysis using equal and implied weighing, the TNT 1.1 software (Goloboff et al. 2008) and the strategies of the New Search Technology (Ratchet, Drift, Tree Fusion and Sectorial Searches) were used. The analysis was performed according to the following parameters: random seed = 1; number of replicates = 10,000; number of trees saved per replication = 10. The software Winclada 1.00.08 (Nixon 2002) was used to display the trees with the transformation series of each character, in addition to its optimization. For the MP tree under equal weights, we provide the total length (L), the consistency index (CI) (Kluge and Farris 1969) and the retention index (RI) (Farris 1989), calculated from all characters.
The parsimony criterion of Fitch (1971), which treats the characters as unordered (or non-additive), was used in this study. Autapomorphic characters of single terminals were maintained in the analysis because they are part of cladistic results (Yeates 1992). Implied weighting (Goloboff 1993) was used to observe how the characters behave in different weighing schemes, based on the fit measure of each character and its overall fit of the topology. The k-values of 1, 2, 3, 5 and 10 were tested. Branch support was checked using Bremer support (1994), with the "Bremer.run" script provided in the TNT Software Wiki (http://phylo.wdfiles.com).
Character optimization is often performed following the proposal of De Pinna (1991), which argues that ACCTRAN is preferable to DELTRAN because it preserves more primary homology hypotheses of. However, Agnarsson and Miller (2008) argue that they do not see theoretical components that make ACCTRAN more preferable than DELTRAN. Amorim (2002) argues that it is more reasonable to analyze the evolution of the characters case by case and to explicitly explain the reason for using ACCTRAN or DELTRAN rather than using only one optimization for all characters. Thus, in some cases (e.g., when there are terminals with non-observable or inapplicable state) ACCTRAN would consider it a spurious synapomorphy, whereas DELTRAN does not perform this transformation, considering an apomorphy for the taxa that have the given state only. Thus, it is safer to adopt the latter. The preference of each optimization was explicitly indicated in the character list.

Illustration
Most characters were illustrated using photographs and line drawings to facilitate identification of different character states. The photographs were taken with a Leica DFC420 digital camera coupled to a Leica MZ16 stereomicroscope. The images were obtained through the software LAS V4.1, then stacked in the software Helicon Focus 5.3.14 and edited in the software Adobe Photoshop CS6 and Adobe Illustrator CS6. The microphotographs of eggs and puparia were processed in Balzers CPD 030, and later were metallized in the Balzers SCD 050 for analysis using the scanning electron microscope, Zeiss DSM 940. In addition, drawings were made using the Leica DM2500 optical microscope with its coupled camera. Subsequently, these drawings were vectored and edited in Adobe Illustrator CS6 software.

List of characters used in the cladistic analysis
A total of 185 characters were constructed, 5 of the egg, 22 of the first instar larva, 7 of the puparium (posterior spiracle), 67 of the external morphology (except terminalia) 53 of the male terminalia, 23 of the female terminalia, and 8 of the spermatheca. The optimizations of the ambiguous characters will be discussed. When relevant, comments will be made for some characters. The characters from literature will be properly referenced with the statement of the author and/or first observer. phologically and functionally defined (Gaponov 2003) and traditionally used for delineation of some groups, e.g., macrotype in Phasiinae and Exoristini and microtype in Goniini (Herting 1960;Gaponov 2003). Following the ideas of Townsend (1934) and Mesnil (1966), for the first time, microtype eggs was shown to be found outside the Exoristinae (Goniini and some Blondeliini), in the tribe Oestrophasiini, and this character is resolved as a synapomorphy for this tribe, confirming the importance of eggs for the classification of Tachinidae. -L = 2; CI = 100; RI = 100. 2. Microtype egg, stalk with hooks: absent (0); present ( Fig. 3C)

LARVA (1 st instar)
6. Short rod-shaped sensorium, dorsally: absent (0); present (1) (Fig. 5C). -Character after Thompson (1954); this structure is found only in Strongygaster. -L = 1; non-informative. (2). -The type of dermal cuticle comprises important biological characteristics in relation to form of host infection. State 0 is found in Xanthozona and in species that perform the sit-and-wait strategy to find the host, which is often a Lepidopteran larva, and as soon as this larva moves, the Lepidopteran larva is infected; in addition, during this time the first instar larvae does not suffer desiccation while waiting for its host because the larva has these dermal plates. State 1 is found in Billaea, Dexia and Prophorostoma, and is characteristic of Dexiini (clade 2), which actively seek their host, mostly larvae of beetles and such granular scales help in this search, providing friction against the substrate, which may be the ground or within trunks of plants. State 2 is found in many other tachinids, where larvae do not undergo major morphological modifications, possessing several other forms of host infection. Ambiguous character, however ACCTRAN or DELTRAN optimization are shown to be equal in this case, i.e., state 2 is synapomorphic for clade 4 and (Voria (Dexia (Billaea + Prophorostoma))). We chose DELTRAN in this case. -L = 2; CI = 100; RI = 100.

PUPARIUM (posterior spiracle)
28. Peritreme, paired structure divided into two parts, i.e., two ventrally and two dorsally: absent  of Pandelleia is unknown. In DELTRAN, this state is a synapomorphy for the clade of Freraea, Microsoma and Eugymnopeza representing the codification for this character, therefore being preferred.

Antennae
-In his dichotomous key of the male terminalia, Tschorsnig (1985) reported that almost no member of Dufouriini s.l. possess these lateral lobes. Here, this characteristic was recovered as a synapomor-  16D) (1). -Character after Tschorsnig (1985). The elongated central plate of the hypandrium was the only putative synapomorphy for Phasiinae found by Tschorsnig (1985).
127. Phallapodeme, length, relative to hypandrium: equal length (0); larger than hypandrium (Fig. 17A)  17E) (1). -Verbeke (1962-Verbeke ( , 1963 was the first to recognize the systematic value of this character, which separated his subfamilies Dexiinae, Voriinae and Dufouriinae from the other tachinids by the presence of a membranous connection between basiphallus and distiphallus. Described as "indirect and mobile" (Type II). Tschorsnig (1985) recognized this character as a putative synapomorphy of Dexiinae, which contained the tribes Dexiini, Voriini, and Dufouriini sensu lato. Based on this character, Wood (1987) and subsequent authors, considered Dexiinae as a possible monophyletic group within Tachinidae. However, in the first cladistic analysis of the family (Cerretti et al. 2014), it was recovered as a reversal in Phasiinae, not confirming the monophyly of Dexiinae. This putative synapomorphy of Dexiinae was also not found herein, appearing in Dexiinae and in Dufouriini s.l., with a reversion in Phasinae. Thus, confirming that it is a homoplastic character. -L = 2; CI = 50; RI = 66.
146. Ejaculatory apodeme, shape: narrow (0); fanshaped ( Fig. 18A) (1). -L = 1; CI = 100; RI = 100.  Table 1) only for taxa that possess the fused pregonites, thus excluding genera traditionally considered in the tribe, such as Freraea and Microsoma. This character was analyzed and redefined to include one more state: whether the fusion is complete (Oestrophasiini synapomorphy) or incomplete (Dufouriini s.s. synapomorphy). This is an ambiguous character, since there are no missing or inapplicable data, and the two optimizations do not provide spurious results. Both forms being considered, thus, in ACCTRAN, state 2 is a synapomorphy for Oestrophasiini and Dufouriini s.s., with a reversion to state 1 in Dufouriini s.s. In DELTRAN, state 1 is a synapomorphy for Dufouriini s.s. and state 2 is a synapomorphy for Oestrophasiini. -L = 2; CI = 100; RI = 100.    (1); completely fused ( Fig. 19B) (2). -L = 3; CI = 66; RI = 85. 160. Tergite 6, direction: anterior (bent forward) (0); posterior (1). -Ambiguous character. In ACCTRAN optimization, state 1 is a synapomorphy for Oestrophasiini and Dufouriini s.l., but they are inapplicable for this character; thus that synapomorphy is spurious. In DELTRAN, this state becomes a synapomorphy for Rondania, representing the codification for that character, so it was used. (1). -Ambiguous character. In ACCTRAN optimization, state 1 is a synapomorphy for Jamacaria and Cenosoma, but as this character is inapplicable in Jamacaria, the indicated synapomorphy becomes spurious. In DELTRAN, this state becomes a synapomorphy for Cenosoma, representing the correct transformation for that character, so it was used.

Phylogenetic analysis
Our study included 35 species and 22 genera, with 26 species and 13 genera in the ingroup. All genera of Dufou-riini (including Oestrophasiini and excluding Mesnilana and Rinophoroides, see more in Discussion) and Freraeini were sampled. Our holomorphological analysis included a total of 185 characters from the egg (5 characters), first instar larva (22), puparium (7), adult external morphology (67, excl. terminalia), female terminalia (23), male terminalia (53) and spermatheca (8). The data matrix is provided in Supplementary file 2. Cladistic analysis with equal weights resulted in a single, most parsimonious tree (L = 400; CI = 61; RI = 83) (Fig. 23). The implied weighting analysis resulted in a single tree with the same length and topology as the equal weighted analysis, but with differences in the optimization of some characters. The single most parsimonious tree with equal weighting will be used in the discussion with unambiguous characters optimized and clades numbered (Fig. 24). Cladograms with ACCTRAN and DEL-TRAN character optimization, in addition to the Bremer support of each clade, are provided in Supplementary file 3.
The Phasiinae was recovered as sister group (clade 4) of the tribes Dufouriini s.s., Freraeini and Oestrophasiini (clade 5), being supported by six synapomorphies and two homoplasies.

Dufouriini, Oestrophasiini and Freraeini as separate tribes
Dufouriini was recovered as paraphyletic, confirming earlier results by Ziegler (1998), Barraclough ( 2005)  Oestrophasiini) and Freraeini separately. The former was sampled with five genera (Oestrophasia, Rondania, Microsoma, Dufouria and Ebenia), while the latter with one (Freraea). Their recovered Dufouriini (with four genera of Dufouriini and Oestrophasiini) and Freraeini (with two genera) as not closely related, but instead intergraded by Telothyriini and by small clades of Voriini and Palpostomatini.
Although our analysis was based on a complete generic sampling of Dufouriini, Oestrophasiini and Freraeini and considered a comprehensive and detailed morphological study of adult and immatures stages (totaling 185 characters), our results might be limited, especially concerning supratribal relationships. On one hand, those three tribes were strongly supported by comprehensive morphological evidence and based on thorough sampling of each tribe. On the other hand, to obtain a reliable intertribal relationship, a more comprehensive sampling of other tribes of Dexiinae (and perhaps Phasiinae) is recommended and desired. Our outgroup sampling was composed of taxa of Phasiinae, that were found to be closely related to Dufouriini (Cerretti et al. 2014), and Dexiinae, wherein Dufouriini are considered to belong, so it is expected that these closely related taxa to Dufouriini present the greatest potential to access the robustness of its monophyly (Grant 2019). Thus, we believe this sampling was sufficient for establishing the monophyly of Dufouriini, Oestrophasiini and Freraeini, as we are not inferring its placement within Tachinidae. Finally, we are confident that our choice of outgroups provides a crucial test of the ingroup topology -by evaluating the ingroup character-state transformations (Grant 2019) -as it can reliably answer our question within this paper, i.e., what are the relationships among the genera and the tribes Dufouriini, Oestrophasiini and Freraeini.
Given the size, diversity and distribution of Tachinidae, taxonomic sampling in Stireman et al. (2019) was far from complete, but was enough to shed light on several questions. In this sense, their findings were a step forward since they indicated that the delimitation and relationships of Dexiinae groupings remains unclear and puzzling. Now, as it will be discussed, our study adds some more evidence to the classification of Tachinidae by supporting that Dufouriini is not a single tribe, but, in fact, three separate tribes. Herting (1957Herting ( , 1960 grouped the taxa with modified ovipositor (syntergite 9 + 10) in Dufouriini s.l., composed of the following Palaearctic genera: Chetoptilia, Dufouria, Eugymnopeza, Freraea, Microsoma, Pandelleia and Rondania. However, the three synapomorphies and one homoplasy for the tribe found herein (clade 13) were not from the female terminalia. Besides, the homology among their ovipositors was not conclusively demonstrated (O'Hara and Wood 2004), and in the present analysis some structures were considered non-homologous. For example, Rondania has a posteriorly directed tube-shaped ovipositor, completely fused syntergosternite 6 + 7 and lacks sternite 9, while in Freraea and Eugymnopeza the tube-shaped ovipositor is directed anteriorly, has a partially fused syntergosternite 6 + 7 and well-developed sternite 9.

Redefining the tribe Dufouriini
The configuration of genera recovered here highly agrees with Verbeke (1962), with the Dufouria group within his Dufouriines, containing the Palaearctic genera Chetoptilia, Dufouria and Rondania. Accordingly, herein, Rondania is sister group to the genera Chetoptilia, Dufouria, Comyops and Ebenia. The last two genera mentioned, Comyops (including Comyopsis) and Ebenia, pertained to the Neotropical tribe "Ebeniini". This tribe, currently invalid and formerly composed of 11 genera, was an assemblage of many unrelated taxa that was put together by Townsend (1936). The remaining genera of the former tribe Ebeniini of Townsend (1936) are currently placed in different tribes, like Voriini, and even subfamilies, like Palpostomatini in Tachininae (O'Hara et al. 2020). However, when better studied, some of the "Ebeniini", i.e., Ebenia, Comyops and Comyopsis, showed affinities with the Dufouriini as discussed by Thompson (1963) and placed formally in Dufouriini by O'Hara et al. (2020). Thompson (1963) argued for a probable relationship between Comyops, Comyopsis and Ebenia with Dufouria based on larval anatomy and cephaloskeleton (similar to Dufouria chalybeata Meigen), as well as male terminalia (Comyopsis resembling Dufouria occlusa (Robineau-Desvoidy, 1863)). This relationship was confirmed here with Comyops and Ebenia as sister group to Dufouria (clade 15). Additionally, Comyopsis is considered a junior synonym of Ebenia herein (see below). Mesnil (1975) delimited Dufouriini into three subtribes: Dufouriina with Dufouria; Campogastrina with Chetoptilia, Pandelleia, Rondania and Microsoma; and Freraeina with Eugymnopeza and Freraea. His classification was not recovered herein, with some genera of Campogastrina placed in Dufouriini (Chetoptilia, Rondania) and others in Freraeini (Pandelleia and Microsoma). Ziegler (1998) Mesnil, 1953 in Dufouriini. This genus, known from two species from Myanmar, was originally assigned to Dufouriinae (Dufouriini, in part) by Mesnil (1953). However, later he changed his mind (Mesnil 1966) and placed this genus within his subtribe Ptilopsinina near Macquartini and Leskiini in Tachininae, only to be placed again in Dufouriini by Crosskey (1976) -by relying only on the similar external adult facies with other taxa placed in this tribe by him, including the Macquartini (Tachininae) genus, Anthomyiopsis Townsend, 1916. Verbeke (1962 was the first author who examined the male terminalia of Kambaitimyia (K. carbonata Mesnil, 1953), and concluded that the presence of a reduced distiphallus inserted on an U-shaped basiphallus is very close to the Strongygaster group and it would be best placed in a group including genera like Imitomyia, Strongygaster and Rondaniooestrus (all currently placed in Phasiinae). Later, Tschorsnig (1985) confirmed Verbeke's (1962) conclusion and placed Kambaitimyia in Strongygastrini (Phasiinae). Herein, by examining and dissecting a male of K. carbonata from NHMUK, we further confirm the peculiar and strong resemblance of the male terminalia of members of the genus Strongygaster and confirm the conclusion of Verbeke (1962) and Tschorsnig (1985) that Kambaitimyia is conclusively not a Dufouriini and is probably best placed in Strongygastrini.
The results of Cerretti et al. (2014) with Dufouriini s.l. as paraphyletic and closely related to Phasiinae was partially confirmed herein. Our analysis confirms the close relationship between Dufouriini s.l. and Phasiinae, but both were monophyletic and sister groups. The clade with Dufouriini s.l. + Phasiinae was supported by a single homoplasy (character 45:0 of Cerretti et al. 2014): presutural acrostichal seta absent; which is not a reliable character, since it appears independently in several other taxa within Tachinidae and other muscoestroid families. Besides, the genera that were restricted to Dufouriini did not group together. Stireman et al. (2019) recovered part of Dufouriini s.l. forming a clade with (Ebenia (Dufouria (Rondania + Oestrophasia), which was confirmed here, but included a less comprehensive sampling. We support both Dufouriini and Oestrophasiini as monophyletic and sister group to each other (clade 9).

Tribe Freraeini
Our results diverge from Herting (1957Herting ( , 1960Herting ( , 1984, who brought together Eugymnopeza and Freraea in Dufouriini based on the structure of the ovipositor, thereby invalidating Freraeini. Herein, Freraeini (clade 6) was supported by two ovipositor characters: one synapomorphy (tergite 8 fused with sternites 8 and 9 (170:1)) and one homoplasy (sternite 8 elongated (172:2)). On the other hand, Freraeini defined herein agrees partially with Verbeke (1962) and his Freraea-group (containing Freraea, Litophasia and Microsoma) and Pandelleia-group (with Pandelleia). Verbeke based these groups on male terminalia: the former group has thin and elongated distiphallus and the latter a reduced and subrectangular basiphallus. We obtained two homoplasies from the male terminalia supporting Freraeini: tergite 6 fused but with visible suture (median dividing line present) in segment 7 + 8 (103:2) and long basiphallus (133:0). Excluding Litophasia (see below), Verbeke's (1962) proposal to leave these genera outside the Dufouria-group was accurate according to the present study, because Dufouriini (clade 13) does not in-clude Freraea, Eugymnopeza, Pandelleia and Microsoma. These three genera in turn belong to Freraeini (clade 6), similar to his Pandelleia-group plus Freraea-group (except for Eugymnopeza, which Verbeke did not study). Mesnil (1975) considered his subtribe Freraeina with the same genera as Townsend (1936), with Freraea and Eugymnopeza only. Herein, this subtribe was monophyletic (clade 8). He commented that Microsoma is very closely related to Freraeina, and we confirm here Microsoma as sister group of Freraea + Eugymnopeza (clade 7). O'Hara and Wood (2004) transferred Freraea from Dufouriini to Freraeini. Later Eugymnopeza was too placed in Freraeini by O'Hara et al. (2009), agreeing with Townsend (1936 and Mesnil (1975), a relationship that was confirmed in the present study; however, in clear contrast, O'Hara et al. (2020) changed the placement of this genus one more time, and returned it to Dufouriini. Furthermore, the character used for this transfer, presence of fused pregonite (our character 150:1), was confirmed as a synapomorphy for clade 9 (Oestrophasiini + Dufouriini). Cerretti et al. (2014) recovered a clade with most Freraeini genera ((Pandelleia + Rondania) (Microsoma (Eugymnopeza + Freraea)))) supported by one character from the female terminalia, tergite 6 long and tubular (Cerretti et al. 2014, character 128:1). However, when scrutinized, this character shows differences among these genera. Although both Pandelleia and Rondania have a long and tubular tergite 6, it is anteriorly directed (160:0) in Pandelleia, while it is posteriorly directed (160:1) in Rondania. Additionally, only Rondania possesses fully telescoped terminalia. The relationships found by these authors is nearly identical to those found herein, differing only by the presence of Rondania, which was placed in Dufouriini herein (clade 13). Finally, the presence of the six unique synapomorphies of the first instar larva (as listed in Results), in addition to the unique synapomorphy found on the female terminalia -tergite 8 fused with sternites 8 and 9 (170:1) -are compeling evidence for the unique habit of host infection that evolved in Freraeini. As this tribe, in the same way of Oestrophasiini and Dufouriini, attacks adult Coleoptera, the functional solution to overcome this challenge was developed by some of its members (Pandelleia, Eugymnopeza and Freraea). Thus, they place the eggs inside the beetles with their terminalia in order to infect them; Microsoma, distinctively, avoided this problem by piercing the sclerite of the beetle with its sharp terminalia. This strategy, even if functionally equivalent to some Dufouriini (Chaetoptilia, Dufouria, Ebenia and Comyops), is morphological different in Microsoma, particularly the larva and the female terminalia, as it happens to the other members of Freraeini. It differs considerably from those genera of Dufouriini as pointed by the synapomorphies above, and clearly indicate a unique solution to infect their hosts. Thus, our preference to maintain this tribe as unique and separate from Dufouriini (clade 13).
Litophasia is a very special case, as it has been considered in Catharosiini (Phasiinae) (Cerretti et al. 2014) Guimarães (1971) considered the Neotropical genera of Glaurocarini sensu Townsend (1936) as the new tribe Oestrophasiini. Moreover, Mesnil (1973) mentioned that Townsend (1936) erroneously classified Oestrophasia and Cenosoma in Glaurocarini and these genera are related to Dufouria, with a connection with his subtribe Campogastrina near Chetoptilia. Our results partially agree with Mesnil's (1973) since members of his Campogastrina, namely, Chetoptilia, Pandelleia and Rondania, but not Microsoma, are placed in Dufouriini and sister group to Oestrophasiini (clade 9). Guimarães (1977), in his revision of Oestrophasiini, discussed a likely relationship of this tribe with the Old World Dufouriini based on Verbeke's (1962: pl. X) genitalia drawings of Chetoptilia, Dufouria and Rondania. Tschorsnig (1985) formally considered Oestrophasiini as belonging to Dufouriini. O'Hara and Wood (2004) agreed with Tschorsnig (1985) based on the presence of a fused pregonite. This character was used here (character 150:1) and appeared to be an ambiguous homoplasy grouping Oestrophasiini and Dufouriini. Despite the importance of the pregonite, a relevant synapomorphy for Oestrophasiini is the presence of microtype eggs (character 1:2). Thus far, this feature had only been considered to be present in Goniini and some Blondeliini (Gaponov 2003), however we found and characterized it as present in Oestrophasiini based on the evidence provided by Gaponov (2003) and Salked (1980) for the eggs, the internal morphology of the female and the larva by Thompson (1924Thompson ( , 1963. This is so because these eggs are very small in size (less than 0.4 mm in length); are placed on leaves and are accidentally ingested by the host, which are thus infected (Grillo and Alvarez 1984); are present in high quantity (between 2,000 and 3,000); the female ovary have more than 100 ovarioles (Grillo and Alvarez 1984); while the larvae have extremely reduced antennae and posterior spiracles; transparent and colourless cuticle, with rows of spines at the posterior end of the first two thoracic segments; segment I extremely well-developed and pigmented, with the rest of the body without spines.

Tribe Oestrophasiini revalidated
Accordingly, the important biological significance of the presence of microtype eggs in Oestrophasiini, which indicates a very specific and complex adaptation to host infection (Gaponov 2003;Thompson 1963), in addition to the posterior spiracles of the puparia, with the peritreme completely fused (character 30:1) -constituting a unique characteristic within Tachinidae, unknown elsewhere in the family (Ferrar 1987;Greene 1921;Ziegler 1998)confirm that this tribe is best ranked as a separate tribe from Dufouriini. Moreover, an additional 17 unambiguous synapomorphies are shared by Oestrophasiini and separate them from Dufouriini.
Still within Oestrophasiini, Wood (1987) synonymized Cenosoma with Oestrophasia, an act that was maintained by O'Hara and Wood (1998Wood ( , 2004. Here the synonymy was not supported, with Oestrophasia monophyletic and supported by four autapomorphies and two homoplasies, and sister group of Cenosoma, Euoestrophasia and Jamacaria (clade 11). Based on this evidence, Cenosoma and Oestrophasia are considered as distinct genera herein. Jamacaria is a monotypic genus that is sister group to Cenosoma. Finally, our analysis did not support the placement of Cenosoma thompsoni as unplaced species of Oestrophasia (sensu O'Hara et al. 2020) as done by O'Hara et al. (2020). Contrarily, our phylogenetic analysis places Cenosoma thompsoni conclusively within Cenosoma as proposed by Guimarães (1977).

Comyopsis as synonym of Ebenia
Herein, Comyopsis Townsend, 1919 is conclusively transferred from the former tribe Ebeniini to Dufouriini, confirming the proposal of Thompson (1963), and most recently by Stireman et al. (2019) andO'Hara et al. (2020). Additionally, following our phylogeny, we also propose Comyopsis as a junior synonym of Ebenia Macquart, 1846. Furthermore, our work does not confirm the proposition of O'Hara et al. (2020) that, oddly, placed Comyopsis in Voriini. In our analysis however, Comyopsis fumata is sister group of E. claripennis + Ebenia sp. 1 (within clade 16). Unlike Townsend's (1927: 234) key, C. fumata does have a costal spine and vein R 4 + 5 with setulae reaching crossvein r-m, as well as Ebenia species. In Thompson's key (1963: 342), the couplet separating Ebenia and Comyopsis uses the length of the costal spine (long in Comyopsis, short in Ebenia) and wing membrane pigmentation (smoky in Comyopsis, and totally hyaline in Ebenia). After examining some species of Ebenia, we found that the only characteristic distinguishing these genera is the setulose prosternum in Ebenia. We considered this character as very unsubstantial to justify generic separation. Besides, there is no significant difference between their male terminalia, therefore, we propose a synonymy between Comyopsis and Ebenia. The only species of Comyopsis, C. fumata Townsend, 1919 (type-locality: Nicaragua, Chinandega) is consequently transferred to Ebenia. However, when O'Hara et al. (2020) placed the previously unplaced species of "Ebeniini" (Guimarães 1971), Ebenia fumata (van der Wulp, 1891) in Ebenia, our new synonymy, E. fumata (Townsend, 1919), constitutes a junior secondary homonomy. In order to resolve this issue, we herein propose a new name for this new combination: Ebenia neofumata Santis and Nihei nomen novum for Ebenia fumata (Townsend, 1919) [nomen preoccupatum].

Systematic placement of Mesnilana and Rhinophoroides
The Afrotropical genera Mesnilana, with one single species M. bevisi Emden, 1945, and Rhinophoroides, also with one single species R. minutus Barraclough, 2005, were originally included in Dufouriini. Emden (1945) Crosskey (1980Crosskey ( , 1984 maintained Mesnilana in Dufouriini, but in the subfamily Dufouriinae. Barraclough (2005) Barraclough (2005), we found that the observed features do not correspond to the Dufouriini as redefined herein, nor with any of the related tribes, Freraeini and Oestrophasiini. Some of these characters include the bare facial ridge, three katepisternal setae and anepimeron with a well-developed seta. In addition to external morphology, more evidence seems to provide an important biological insight: both genera were collected in light traps, suggesting nocturnal hosts (Barraclough 2005). This is not known from other members of Dufouriini and is uncommon in Tachinidae (occurring, for instance, in the cricket parasitoid tribe Ormiini, Tachininae). Some of the characters found in both Mesnilana and Rhinophoroides are the small and tongue-shaped lower calyptra that diverges from the scutellum and the parafacial with several setulae; these traits are also found in the coleopteran parasitoid tribe Palpostomatini. Besides these traits, the general appearance (abdominal chaetotaxy and head proportions) is very similar to some Palpostomatini, mainly the ge-nus Palpostoma (e.g., Palpostoma subsessile Malloch, 1931). Based on these observations, Mesnilana and Rhinophoroides are removed from Dufouriini and tentatively considered as Palpostomatini, until additional evidence becomes available. In Stireman et al. (2019), Palpostomatini was a polyphyletic group, with one part forming a clade with Imitomyiini and sister to all other Dexiinae + Phasiinae, and another part as sister to Freraeini.

Dufouriini or Dufouriinae?
For a long time, Dufouriini was considered a tribe or subtribe of Phasiinae. It was initially allocated as a subtribe of Phasiini by Mesnil (1939), and then as tribe of Phasiinae by Emden (1945Emden ( , 1950 based mainly on chaetotaxy. Verbeke (1962Verbeke ( , 1963 considered it as a new subfamily: Dufouriinae, including two tribes, Dufouriini and Macquartiini (the latter currently in Tachininae), based mainly on postgonites of the intermediate type (in relation to the sensory and the connective Type II) and distiphallus DEG subtype. Verbeke also noted similarities in the male postabdomen shared by Dufouriinae and Phasiinae and was the first to suggest a close relationship between Dufouriinae and Phasiinae. Finally, the specializations of the female terminalia which allow Dufouriini to parasitize adult Coleoptera, as well as Phasiinae to parasitize adult Heteroptera, support the proximity between the two groups (Verbeke 1962). In contrast, in Dexiini hosts are actively sought out by first instar larvae deposited by females near the host and females possess a simple and short terminalia, with larvae completing their development in the host (Barraclough 1992). Later, Crosskey (1976Crosskey ( , 1980 also recognized the subfamily Dufouriinae with the tribes Imitomyiini and Dufouriini, as these two would be excluded from Phasiinae and Dexiinae, respectively.
Following Herting (1984), Tschorsnig (1985) considered Dufouriini as a tribe of Dexiinae, with this subfamily as probably monophyletic, being supported by characters of the male terminalia; however, he recognized it as very inconsistent considering its biology and adult external characters. As discussed previously, the main putative synapomorphy discussed by Tschorsnig (1985) -aedeagus with basiphallus and distiphallus articulated to each other -was not recovered as a synapomorphic character herein, agreeing with Cerretti et al. (2014). The state 1 of character 130 is a synapomorphy shared by clade 1 (Dexiini + Voriini) and clade 4 ((Freraeini (Oestrophasiini + Dufouriini)) + Phasiinae) but undergoes a reversal in Phasiinae. Cerretti et al. (2014) proposed the paraphyly of Dufouriini s.l. in relation to Phasiinae, providing more evidence for a close phylogenetic relationship between these groups. Furthermore, Tschorsnig (1985) also recognized a number of similarities between the male terminalia of Dufouriini and Phasiinae, reporting that only the pregonite and phallus would place Dufouriini near Dexiinae. Considering his dichotomous key of the male terminalia of Tachinidae (Tschorsnig 1985), several shared characteristics can be found in the couplet of Dufouriini and Phasiinae: sternite 5 without lobes and without lateral membranous line; membranous connection between sternites 5 and 6; tergite 6 fused to segment 7 + 8. In the same line, Cantrell (1988: 147) stated: "The affinities of the Dufouriinae appear to be intermediate between those of the Phasiinae and Dexiinae and deserve further study." Barraclough (1992) reported that the Palaearctic Dufouriini would not belong to Dexiinae, considering modifications in the female terminalia (elongated tergite 8 forming dorsal lamellae). He then affirmed: "[T]he Dufouriini belong in neither the Phasiinae nor Dexiinae." (1992Dexiinae." ( : 1152.
Our phylogenetic results support the proximity between the clade (Freraeini (Oestrophasiini + Dufouriini) and Phasiinae, as previously suggested by Verbeke (1962Verbeke ( , 1963, Crosskey (1976Crosskey ( , 1980 and Cerretti et al. (2014). Furthermore, Verbeke (1962Verbeke ( , 1963, Crosskey (1976Crosskey ( , 1980, Cantrell (1988) and Barraclough (1992) indeed argued for Dufouriini as a separate subfamily, i.e., Dufouriinae. Despite our results, other relevant phylogenetic results (Cerretti et al. 2014;Stireman et al. 2019) were not conclusive in supporting (or rejecting) the ideas of a clade formed by Dufouriini, Oestrophasiini and Freraeini or a close relationship between this clade and Phasiinae. Our taxonomic sampling, with five out of 12 Dexiine tribes (Dexiini, Voriini, Dufouriini, Freraeini and Oestrophasiini), does not allow any conclusions at the subfamily level. Hence, we included a comprehensive sampling for Dufouriini, Oestrophasiini and Freraeini, but a reduced and critical sampling of other Dexiine tribes. Cerretti et al. (2014) sampled five tribes (Dexiini, Dufouriini, Eutherini, Freraeini, Voriini) and Stireman et al. (2019) included representatives from all Dexiine tribes, and Dufouriini + Oestrophasiini was not closely related to Freraeini, nor was it close to Phasiinae. However, many tribes were not monophyletic (namely Dexiini, Voriini, Palpostomatini, and Dufouriini), perhaps indicating the need for more information (e.g., phylogenomic approaches and a detailed morphological analysis; and/or the need for better sampling of each tribe). This matter is completely open to debate with Dexiinae deserving further studies to reach a better conclusion about the systematic ranking and placement of Dufouriini, Oestrophasiini and Freraeini. Only time and more empirical data will tell whether these three tribes should be better elevated to subfamily level (the Dufouriinae of Verbeke 1962Verbeke , 1963.

New classification proposal
We propose a new classification for Dufouriini based on our phylogenetic results (see Supplementary file 4). The tribe Dufouriini is redefined and restricted now to five genera only: Chetoptilia, Comyops, Dufouria, Ebenia and Rondania. Comyopsis is proposed as a junior synonym of Ebenia, and Ebenia neofumata Santis and Nihei nom. nov. is transferred from Comyopsis to Ebenia. The other genera formerly recognized in Dufouriini are allocated to Freraeini and Oestrophasiini. The tribe Freraeini is redefined and broadened to include Microsoma, Eugymnopeza and Pandelleia, along with the type genus, Freraea.
The tribe Oestrophasiini sensu Guimarães (1977) is revalidated, including four genera: Cenosoma, Jamacaria, Oestrophasia and Euoestrophasia, all removed from Dufouriini. Cenosoma stat. rev., previously a subgenus of Oestrophasia is revalidated as genus. Finally, although not included in the phylogenetic analysis, Mesnilana and Rhinophoroides are removed from Dufouriini and are tentatively transferred to Palpostomatini.

Conclusions
This is the first phylogenetic study to include all genera of Dufouriini s.l. (Dufouriini, Oestrophasiini) and Freraeini. Our study supported the monophyly and taxonomic validity of Dufouriini, Oestrophasiini and Freraeini, each defined by several synapomorphies. Furthermore, the three tribes formed a sister group clade to Phasiinae sharing six synapomorphies. Despite the most recent efforts, phylogenetically supported definitions of tachinid groupings remain uncertain at all levels. At the subfamily level, morphological data only recovered Phasiinae as monophyletic (Cerretti et al. 2014), whereas molecular data recovered Phasiinae and Exoristinae (Stireman 2002;Tachi and Shima 2010;Blaschke et al. 2018;Stireman et al. 2019), in addition to Dexiinae more recently (Stireman et al. 2019).
The present study carried out a holomorphological phylogenetic analysis based on total evidence of morphological characters from eggs, puparium, larvae and adults (including male and female terminalia, and spermathecae). Morphological characters of adults along with male terminalia are traditionally used as main character sources in Tachinidae systematics and this study demonstrated that characters from eggs, larvae, puparia, female terminalia and spermathecae have great systematic importance, as they mutually supported clades and resulted in important synapomorphies for several taxonomic levels. The clade grouping Dufouriini, Oestrophasiini and Freraeini was supported by three unambiguous synapomorphies from adult external morphology, male terminalia and spermathecae, and one homoplasy from female terminalia. The eight unambiguous synapomorphies supporting Freraeini were from first instar larvae (six synapomorphies), adult external morphology (1) and female terminalia (1). Oestrophasiini is a separate case, being supported by characters from all sources of evidence, with synapomorphies from the egg (1), first instar larva (4), puparium (3), adult external morphology (10), male terminalia (5), female terminalia (5) and spermatheca (2). The use of other character sources to infer phylogenetic relationships besides the traditional adult external morphology and male terminalia has been discussed and emphasized by a number of authors that dealt withTachinidae classification (e.g., Thompson 1954Thompson , 1960Thompson , 1961Thompson , 1963Herting 1957Herting , 1983Mesnil 1966;Richter 1987;Ferrar 1987;Barraclough 1992;Ziegler 1998;Cerretti et al. 2014), and our study is a confirmation of their views.
We hope that, besides contributing to the phylogeny and classification of Dufouriini, Oestrophasiini and Freraeini, our study also highlights the need for more detailed morphological studies of Tachinidae taxa. Our study demonstrates that little is known about the basic morphology and biology of this group. For example, microtype eggs were previously described and recognized only in Goniini and some Blondeliini (Gaponov 2003), being a synapomorphy for Goniini (Cerretti et al. 2014), but herein were also recognized in Oestrophasiini. Therefore, we wonder how many trivial discoveries are still hidden inside the drawers just waiting for our curiosity.