Introduction

Oestroidea comprise a diverse clade of true flies comprising some of the most familiar insects, such as blow flies and flesh flies. The group accounts for about 15,000 known species [1], but estimates suggest that the true number may be at least twice as many [2, 3]. As holometabolous insects, their larval stage is morphologically and functionally entirely different from the adult stage, and whereas adults are often flower visitors, oestroid larvae can be general scavengers; vertebrate or invertebrate necrophages; vertebrate coprophages; vertebrate parasites; invertebrate parasitoids; predators of frog spawn, molluscs, earthworms, termites, grasshopper eggs or spider eggs; and even mycophages and palynophages [2, 4, 5].

Reconstructing the phylogenetic relationships among oestroid lineages has long represented a major challenge. Morphology has provided sparse and conflicting evidence [6, 7], and molecular studies have differed, mainly relating to gene-choice and taxon sampling [8–10]. However, consensus on the oestroid backbone is now emerging through phylogenomic and phylotranscriptomic approaches [11–13]. In-depth phylogenetic studies aiming at the reconstruction of relationships within particular clades have also been performed [13 (Calliphoridae); 14, 15 (Oestridae); 12, 16 (Sarcophagidae); 17 (Mesembrinellidae); 18 (Tachinidae)]. The present paper focuses on two oestroid subclades for which molecular genus-level phylogenies are still largely lacking, namely the Rhinophorinae and the Polleniidae.

Polleniidae (Fig 1)—also known as cluster flies due to the tendency of adults of some species to cluster indoors for overwintering—is a family of earthworm parasitoids [19–23]. The polleniids have been treated either as a subfamily (Polleniinae) or tribe (Polleniini) within the Calliphoridae [24–27] or given family rank [28]. Polleniids have recently become well-established as the extant sister taxon of the megadiverse parasitoid clade Tachinidae [11, 12, 18, 29–33], even though morphology or other character systems so far have provided few clues in support of this relationship. Studies of the phylogenetic relationships among polleniid genera are limited and include only sparse taxon sampling [28, 34, 35]. Currently the family contains some 150 named species in eight genera, with the bulk of diversity in the Palaearctic Region [36]. Polleniids are also widespread and abundant in the Oriental and Australasian regions, but native species are confined to smaller areas in the Nearctic (West Coast of the USA) and the Afrotropics (southern Africa), and are entirely absent from the Neotropics [36, 37]. A few species have become widely distributed, possibly due to individuals diapausing in shipping containers and with the widespread establishment of introduced populations of their host earthworms [38].

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Fig 1. Polleniidae species included in the present analyses; adult habitus.

A Melanodexia glabricula (Bigot, 1887). B Melanodexia grandis (Shannon, 1926). C Melanodexia tristina (Hall, 1948). D Melanodexia tristis Williston, 1893. E Pollenia rudis (Fabricius, 1794). F Pollenia nr. stolida Malloch. [A–D, F = male; E = female].

https://doi.org/10.1371/journal.pone.0285855.g001

Rhinophorinae (Calliphoridae) (Fig 2) is a small clade of woodlouse parasitoids, which, as for the cluster flies, have also been bouncing around within the Oestroidea. The group was long considered a subfamily, either under Calliphoridae [22, 39–42] or Tachinidae [43–48], or treated at family rank [6, 7, 12, 49–61]. Recently, however, Yan et al. [13], based on transcriptomes and a limited taxon sampling, reclassified part of the oestroids proposing woodlouse flies as a subfamily of Calliphoridae with a sister-group relationship to the Ameniinae (including the Helicoboscinae) [11, 13]. Regardless of taxonomic ranking, the monophyly of woodlouse flies has never been questioned, although the lack of unique adult synapomorphies and only scattered information on immature stages and natural history has caused several genera to shift either into or out of the taxon [61]. At present, rhinophorines number 180 species in 33 genera, and their species diversity peaks in the Mediterranean area [61, 62]. Woodlouse flies are widespread except for a notable absence from temperate North America, where they are only represented by a few species recently introduced from the Palaearctic Region [63, 64]. This peculiar distribution may be due to the paucity of native Nearctic terrestrial isopod species [65–68], or to low host population densities [69–71].

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Fig 2. Rhinophorinae species included in the present analyses; adult habitus.

A Aporeomyia elaphocera Gisondi, Pape, Shima & Cerretti, 2020. B Axinia arenaria Colless, 1994. C Baniassa pennata Gisondi, Pape, Shima & Cerretti, 2020. D Bezzimyia yepezi Pape & Arnaud, 2001. E Bixinia winkleri Cerretti, Lo Giudice & Pape, 2014. F Melanophora roralis (Linnaeus, 1758). G Oplisa tergestina (Schiner, 1861). H Paykullia partenopea (Rondani, 1861). I Paykullia nr. nubilipennis. L Rhinophora lepida (Meigen, 1824). M Stevenia deceptoria (Loew, 1847). N Tromodesia angustifrons Kugler, 1978. [A–N = male].

https://doi.org/10.1371/journal.pone.0285855.g002

The present paper provides a comprehensive phylogeny of the Rhinophorinae and the Polleniidae, involving an extensive taxon sampling and employing two nuclear protein-coding genes, CAD and MCS, previously evaluated as having high phylogenetic informativeness [31, 72].

Materials and methods

Ethanol-preserved material was obtained for Polleniidae (3 out of 8 currently recognized genera) and Rhinophorinae (19 out of 33 currently recognized genera), with a complete biogeographical coverage, and a set of outgroup taxa (49 genera) representing Calliphoridae (Ameniinae, Bengaliinae, Calliphorinae [including calliphorine taxa formerly in Melanomyinae and Toxotarsinae], Chrysomyinae, Luciliinae, Phumosiinae), Mesembrinellidae, Oestridae, Sarcophagidae, Tachinidae (Dexiinae, Exoristinae, Phasiinae, Tachininae) and Ulurumyiidae (see S1 Table). GenBank sequences were included from Winkler et al. [31] and for Musca domestica Linnaeus for outgroup rooting. Extractions and amplifications were carried out at the GeoGenetics Lab at University of Copenhagen, Denmark, while sequencing was outsourced to Macrogen Europe (Amsterdam, the Netherlands).

CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) and MCS (molybdenum cofactor sulfurase) were chosen for their phylogenetic information and reliability for Mesozoic-Cenozoic-aged explosively radiated groups such as the Oestroidea [72], as well as for ease of comparisons with results and integration of sequences from analyses of other available datasets.

Three legs were removed from ethanol-preserved specimens and stored in ethanol until extraction. Extractions were performed using the DNeasy Blood and Tissue Kit (Quiagen, Venlo, the Netherlands) with the following modifications of the manufacturer’s protocol: legs were placed entire in the digestion buffer and Buffer ATL was replaced with a digestion buffer as described by Gilbert et al. [73] but modified to consist of 10 mM Tris-HCl (pH 8), 10 mM NaCl, 5 mM CaCl₂, 2.5 mM EDTA, 1% sodium dodecyl sulphate (SDS), 250 μg/mL proteinase K, and 40 mM dithiotreitol (DTT) (final concentrations).

PCR amplification reactions (total volume 25 μL) were composed of 18 μL deionized water, 4 μL of 5X HOT FIREPol Blend Master Mix (Solis BioDyne), 0.5 μL of each primer (final concentration of 0.2 μM), and 2 μL of DNA solution. The most effective program among all the experimental variations was a PCR protocol consisting of an initial denaturation stage of 12 min at 95°C; 35 cycles of 95°C for 30 sec, variable annealing temperature (depending on the primers used, see Table 1) for 1 min, 72°C for 2 min, and a final extension time of 10 min at 72°C. After visualization on a 2% Agarose gel, PCR products were sent to Macrogen Europe (Amsterdam, the Netherlands) for PCR product cleanup and sequencing.

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Table 1. Primers used and their annealing temperatures.

https://doi.org/10.1371/journal.pone.0285855.t001

Sequencing output files were assembled and trimmed using Geneious 9.1.8 (Biomatters Ltd., Auckland, New Zealand). FASTA files of sequences were aligned using MAFFT (v.7.017) with the G-INS-i algorithm using the default parameters [74, 75] (see S1 Dataset). The resulting alignments were checked for accuracy by looking for stop codons and spurious gaps once the alignments were translated into proteins. The single-gene alignments were then concatenated using the “Concatenate alignments” tool in Geneious (see S1 Dataset).

PartitionFinder v2 [76, 77] was used to find the best-fitting partitioning scheme and to select substitution models for each partition without overparameterization, evaluated by the information-theoretic metric BIC (Bayesian Information Criterion). The initial 6 data blocks were the first, second, and third codon positions of each of CAD and MCS, and the program was set to perform a greedy search to compare all possible partitioning schemes. The best-fit scheme grouped all data blocks in one single partition. The model GTR+I+G was selected as the substitution model for this partition.

Likelihood analyses were conducted using RAxML version 8.2.12 [78] on XSEDE (Extreme Science and Engineering Discovery Environment) through the CIPRES (Cyberinfrastructure for Phylogenetic Research) Science Gateway [79]. The tree of highest likelihood from 100 replicate runs was selected for plotting the bootstrap values from 250 ML rapid bootstrap replicates obtained through a GTR+G+I approximation. Trees from all analyses were visualized using FigTree [80].

Results

Analysis of the concatenated matrix from CAD (1794 bp) and MCS (2078 bp) resulted in a well-resolved ML topology, although with some branches having low bootstrap support values (henceforth b.v.) (Fig 3).

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Fig 3. ML tree with bootstrap values mapped.

https://doi.org/10.1371/journal.pone.0285855.g003

The ML tree (Fig 3) shows overall strong support for shallow nodes within families, but somewhat lower support for many of the deeper branches representing relationships among families and other major clades. Relationships among the genera within the target cluster flies and woodlouse flies are generally robust and well-resolved. DNA sequence data for Aporeomyia Pape & Shima, Baniassa Kugler, Malayia Malloch, Macrotarsina longimana (Egger), Marshallicona Cerretti & Pape, Shannoniella Townsend, Trypetidomima Townsend and Melanodexia Williston are here obtained for the first time.

The recently recircumscribed Calliphoridae are divided into a grade of four clades as follows: Bengaliinae (including Auchmeromyiini and Bengaliini) + Rhiniinae (b.v. = 43); Luciliinae + Calliphorinae (b.v. = 50); and Ameniinae + (Chrysomyinae + Phumosiinae) (b.v. = 20). Our analysis reconstructed the clade Tachinidae + Polleniidae (b.v. = 80) nested within the calliphorid grade and sister to the core Calliphoridae (i.e., excluding the Bengaliinae + Rhiniinae clade), but overall statistical support is low. The sister group to Rhinophorinae is a clade composed of [Ameniinae (b.v. = 38) + [Chrysomyinae (b.v. = 100) + Phumosiinae (b.v. = 98)]]; however, these relationships are only weakly supported.

Sarcophagidae form a low-supported clade (b.v. = 44), which is sister taxon to a low-supported Oestridae (b.v. = 38), while Mesembrinellidae are retrieved as well supported (b.v. = 100) monophyletic group sister to Ulurumyiidae (b.v. = 92).

Our analyses recover a monophyletic Polleniidae with strong support (b.v. = 100). Within this clade, Pollenia nr. stolida Malloch from Australia is reconstructed as sister to the remaining polleniids; however, the latter clade has low support (b.v. = 45). The remaining species of Pollenia Robineau-Desvoidy form a well-supported clade (b.v. = 100), sister to the Melanodexia–Morinia clade composed of a monophyletic Melanodexia (b.v. = 98) reconstructed as sister to Morinia doronici (Scopoli), and with a subordinate Morinia sp. from South African rendering Morinia Robineau-Desvoidy paraphyletic.

Rhinophorinae are retrieved as monophyletic (b.v. = 98) as are the two subgroups, each of which is characterized by a highly derived larval morphology and locomotory behaviour (see further below), i.e., Phytonini (b.v. = 53) and Rhinophorini (b.v. = 75). Among the included non-monotypic genera for which we included more than one species, Axinia Colless, Bixinia Cerretti, Lo Giudice & Pape, Paykullia Robineau-Desvoidy, Phyto Robineau-Desvoidy and Stevenia Robineau-Desvoidy emerged as monophyletic with strong support (b.v. = 100), whereas the species traditionally assigned to genus Rhinomorinia Brauer & Bergenstamm separated into two geographically disjunct groups: an Afrotropical clade (henceforth as Oxytachina Brauer & Bergenstamm stat. rev., b.v. = 100) and a Palaearctic clade (Rhinomorinia), both belonging to the Rhinophorini. Oxytachina is reconstructed as sister to the remaining Rhinophorini. Within this tribe, the Afrotropical genus Ventrops Crosskey is reconstructed in a nested position within a clade of Neotropical endemic taxa (Bezzimyia Townsend, Marshallicona Cerretti & Pape, Shannoniella Townsend and Trypetidomima Townsend), but overall support is weak. The clade composed of the Palaearctic Macrotarsina Schiner, Rhinomorinia, Oplisa Rondani and Stevenia received moderate support (b.v. = 74). Within the Phytonini, deeper branches have low or moderate support (b.v. = 38–85). Bixinia spp. emerge as sister to the remaining Phytonini, with Baniassa pennata Gisondi, Pape, Shima & Cerretti as the next most basal branching. Sister to Baniassa is a weakly supported clade (b.v. = 47) composed of the Australasian Aporeomyia + Axinia clade (b.v. = 56) and the Oriental/Palaearctic [Malaya + Phyto] (b.v. = 38) + [Paykullia + Melanophora] (b.v. = 85) clade (b.v. = 39).

Discussion

A fully resolved and well supported phylogeny of oestroid flies has proved difficult to attain through both morphological and Sanger-generated molecular data. However, a consensus on the topology of the backbone is now emerging through phylogenomic and phylotranscriptomic approaches [11–13]. Conflicts in the deeper splits, i.e., in the position of families and subfamilies between the present study and the more recent phylogenomic studies are here considered as most likely resulting from our use of data from only two nuclear loci. Many deep nodes received low statistical support values, and they are not discussed further.

Despite recent study, the phylogenetic relationships among polleniid genera are still tentative. Employing a combination of morphological characters and fragments of three nuclear markers (CAD, MCS, MAC) on a selection of Morinia species, one Pollenia and the monotypic Alvamaja Rognes, Cerretti et al. [28], reconstructed Morinia as monophyletic and sister to Alvamaja, this clade being sister to Pollenia. Recently, Johnston et al. [35] presented a mitogenomic analysis of 21 polleniid taxa, including a broad representation of West Palaearctic Pollenia and one species each of Melanodexia, Morinia and Dexopollenia Townsend. The study retrieved Dexopollenia as sister to a clade composed of Morinia and Melanodexia, with this clade in turn sister to Pollenia. Johnston et al. [35] performed further analyses by using COI sequences (i.e., not the entire mitogenome) of Xanthotryxus mongol Aldrich and an additional species of Morinia, and recovered Dexopollenia + Xanthotryxus Aldrich as sister to the remaining Polleniidae, and the latter resolving as Pollenia being sister to Morinia + Melanodexia. These relationships come with low support, which is partly obscured by Johnston et al. [35] incorrectly using support values for particular nodes to indicate support for the basal dichotomy. However, the present ML topology is largely consistent with the results in Johnston et al. [35] (except for retrieving Pollenia as paraphyletic), but the limited taxon sampling does not allow for testing the phylogenetic position of Dexopollenia and Xanthotryxus. The sparse morphological evidence tends to support a Dexopollenia–Xanthotryxus–Pollenia-clade. All species of Xanthotryxus and most species of Dexopollenia and Pollenia share the presence of golden, wavy, hair-like setae on parts of the body, and the morphologically very similar Pollenia and Xanthotryxus also share a subcostal sclerite with a bundle of long, black or yellow setae among the micropubescence. However, comparative morphology of polleniids needs much more study, and differing phylogenetic topologies obscure interpretations of character state polarities. For instance, the Australian Pollenia nr. stolida examined here differs from the Palaearctic and New Zealand species of Pollenia by lacking the first presutural intra-alar seta and by the three preapical setae of the hind tibia (anterodorsal, dorsal and posterodorsal) being subequal in size; both these character states are shared with Morinia and Melanodexia and may represent plesiomorphic conditions. Interestingly, we found this species taking up the position of sister taxon to all other polleniids included in the analyses, although with weak support (Fig 3).

The Morinia–Melanodexia clade is supported by the following putative morphological autapomorphies: i) narrow, tongue-shaped, lower calypter, ii) posterior spiracle with reduced posterior lappet (rhinophorine-like) and iii) node at base of R₄₊₅ bare. Morinia is here represented by the type species M. doronici (Scopoli) (Palaearctic) and by an undescribed species from South Africa [19, 29]. Our phylogeny reconstructed the two included species of Morinia as paraphyletic with respect to Melanodexia. Indeed, our careful examination of Morinia from both Palaearctic and Afrotropical regions has not revealed any strong evidence supporting their monophyly, except for sharing a slim, narrow, body shape, which contrasts with the stouter body characterizing the other polleniids, except Alvamaja. Despite this, we consider it premature to lump species currently assigned to Morinia and Melanodexia under the same genus-group name as long as there is inconclusive data on the phylogenetic position of Alvamaja, Anthracomyza Malloch, Dexopollenia Townsend, Nesodexia Villeneuve and Xanthotryxus, all of which may belong in the Polleniidae [36]. Alvamaja presents a unique combination of character states and could belong to the Morinia–Melanodexia clade based on its rhinophorine-like (i.e., non-operculate) posterior spiracle and narrow lower calypter. This relationship is supported by the morphological evidence presented in Cerretti et al. [28]. Dexopollenia and Xanthotryxus share several, derived character states with Pollenia, including the golden, wavy hair-like setae particularly abundant on the thorax, and the cluster of long black or yellow setae on the subcostal sclerite. No progress has been made so far in resolving the phylogenetic placement of Anthracomyza and Nesodexia. These are both monotypic genera and no molecular sequence data have been obtained from them. Although several polleniid nominal genus-group taxa remain to be included in a phylogenetic analysis, we are here proposing to apply a subfamily classification with the Polleniidae composed of two subfamilies, as follows:

1. Moriniinae Townsend: including Morinia, Melanodexia, and Alvamaja;
2. Polleniinae Brauer & Bergenstamm: including Pollenia, Dexopollenia, and Xanthotryxus.

We here treat Anthracomyza and Nesodexia as Oestroidea incertae sedis pending further study.

Our study is the first attempt at resolving the phylogenetic relationships within the woodlouse flies using molecular data. Analyses support both the monophyly of the subfamily and its division into two subclades, which we propose here as tribes: Rhinophorini and Phytonini (i.e., the Stevenia group and the Phyto group of Pape & Arnaud [81], respectively). By integrating our results (Fig 3) with those from previous phylogenetic reconstructions, deduced from morphological data and a larger taxon sampling [61, and literature therein], the two recognized tribes are composed as follows (an asterisk indicates taxa which have not been placed based on molecular data):

1. Rhinophorini: Acompomintho Townsend*, Apomorphyto Cerretti, Lo Giudice & Pape*, Azaisia Villeneuve*, Bezzimyia, Macrotarsina, Maurinophora Cerretti & Pape*, Melanomyiodes Crosskey*, Marshallicona, Metoplisa Kugler*, Neotarsina Cerretti & Pape*, Oplisa, Oxytachina stat. rev., Queximyia Crosskey*, Rhinomorinia, Rhinophora Robineau-Desvoidy, Shannoniella, Stevenia, Tricogena Rondani*, Tromodesia Rondani*, Trypetidomima, Ventrops;
2. Phytonini: Aporeomyia, Axinia, Baniassa, Bixinia, Comoromyia Crosskey*, Kinabalumyia Cerretti & Pape*, Malayia, Melanophora Meigen, Parazamimus Verbeke*, Paykullia, Phyto, Rhinodonia Cerretti, Lo Giudice & Pape* and Rhinopeza Cerretti, Lo Giudice & Pape*.

Within Rhinophorini, all the included non-monotypic genera emerged as monophyletic except for “Rhinomorinia”. This nominal genus was retrieved as polyphyletic, being divided into two well-supported lineages, which are characterized by distinctive morphological features [60–62; see also the key below]. One clade comprises exclusively Afrotropical species (here placed in the resurrected nominal genus Oxytachina stat. rev., see below) and was retrieved as sister to the remaining Rhinophorini with moderate support. The other clade comprises two Palaearctic/western Oriental species, of which Rhinomorinia sarcophagina (Schiner) (type species of the genus) was included and clustered within the Rhinophorini as sister to Oplisa with strong support. Under ML, Rhinophora lepida (Meigen) was recovered as sister to all Rhinophoriini except Oxytachina, differing from the morphology-based phylogeny of Cerretti et al. [61] that retrieved Rhinophora joining a primarily Palaearctic subclade composed of Rhinomorinia, Macrotarsina, Oplisa and Stevenia, which also contained some Afrotropical and Oriental species for which molecular data are not available. The position of the Afrotropical genus Ventrops within an otherwise Neotropical clade is biogeographically challenging, but the low support indicates that this hypothesis needs further testing. Support values for the genus-level reconstruction within this clade were weak to moderate. Interestingly, the morphology-based phylogeny of Cerretti et al. [61] had Ventrops as the sister taxon of a clade containing all the Neotropical taxa and the Australian genus Bixinia. Analyses of morphological data separated the genus Bixinia widely from the other Australasian taxa [61], while the present molecular analysis of Axinia and Bixinia places these genera within the same tribe but separated by multiple intervening genera (Fig 3).

Conclusions

Until recently, the taxonomic boundaries and phylogenetic affinities of Polleniidae and Rhinophorinae–two key groups of parasitoids of soil-dwelling organisms–remained controversial. Our analysis, despite being limited to two protein-coding nuclear genes, confirmed previous hypotheses on the relationships between the two groups and provided new insights into their internal phylogenetic relationships. These results allowed us to formally propose a subfamilial and tribal classification for the polleniids and rhinophorines, respectively, and to resurrect the genus Oxytachina to include five Afrotropical species previously assigned to the genus Rhinomorinia, which thereby is restricted to two Palaearctic species.

Supporting information

Acknowledgments

The authors are thankful to Martin Hauser and Steve Gaimari (California Department of Food and Agriculture, California, USA) and Juan Manuel Perilla Lopez (Wright State University, Ohio, USA) for sending ethanol-preserved specimens. SG and PC are grateful to Emanuele Berrilli (University of L’Aquila, Italy) for sequencing Macrotarsina longimana.