Taxonomic revision of Australian Amobia Robineau-Desvoidy, 1830 (Sarcophagidae: Miltogramminae): integrating morphology and genetics finds a new species and tackles old problems

A taxonomic revision of the Australian species of Amobia Robineau-Desvoidy, 1830 (Diptera: Sarcophagidae: Miltogramminae) is completed using an integrated approach combining four molecular loci (three mitochondrial, COI, ND4 and CYTB; one nuclear, EF1α) and morphological data. A new species, Amobia (s. str.) serpenta sp. nov., endemic to Australia, is described, and Amobia auriceps (Baranov, 1935) and Amobia burnsi (Malloch, 1930) are re-described. Molecular data are used to reconstruct inter-specific and generic relationships and support morphological species hypotheses. Phylogenetic analysis places all three Australian Amobia species together with Amobia signata (Meigen, 1824) (a Palaearctic species) in a single clade sister to Senotainia Macquart, 1846 (in part), which is in agreement with previous phylogenetic studies of the Miltogramminae. In addition to the description of species and molecular phylogenetics, general host associations for the Australian species of Amobia are discussed and evidence for the synonymisation of A. pelopei (Rondani, 1859) and A. auriceps is refuted.

In an attempt to better understand the diversity of Australian sarcophagids, there has been a recent targeted effort to systematically revise the Australian Miltogramminae and place their constituent genera and species within a global phylogenetic context. This research has been targeted at specific genera, and revisions of Aenigmetopia and Macronychia have already been published (Johnston et al. 2020a(Johnston et al. , 2020b. This paper focuses on the genus Amobia, within the larger aim of a full treatment of the Australian Miltogramminae. The genus Amobia is characterised morphologically by 1) the autapomorphic configuration of the anterior margin of the anepimeron, which shows a distinct swelling below the ampulla (Shewell 1987;Pape 1996), and 2) the reduction of the epiphallus, which most likely is apomorphic at the genus level (Pape 1996;Buenaventura et al. 2020). Additional diagnostic features, which are also found in other miltogrammine genera, are the relatively high number (> 5) of fine, hair-like proclinate fronto-orbital setae; the antennal insertion at or below the midpoint of the eye, and the claws longer than the fifth tarsomere in both sexes (Pape 1996). Similarly, species of Amobia can be characterised by the hosts of their inquilinous larvae, which are predominantly mud daubers and potter wasps (Hymenoptera: Sphecidae, Crabronidae; Vespidae: Eumeninae) (Itino 1988;Downing 1996;Coville 2000;Fateryga & Kovblyuk 2014).
The Australian fauna of Amobia has so far received little attention. Until now, the genus was represented in Australia by two species: Amobia auriceps (Baranov, 1935) (which is also distributed throughout the broader Australasian/Oceanian and Oriental regions) and the endemic Amobia burnsi (Malloch, 1930). This paper adds to this body of knowledge through the taxonomic revision of Australian Amobia, describing a new endemic species, providing new characters for the separation of species, and providing extensive photographic documentation of the adult morphology and terminalia. Molecular data (four loci: COI, CYTB, ND4 and EF1α) are utilized to support morphological species hypotheses, allow further insight into the taxonomic distinctiveness of this genus and, through phylogenetics, place the Australian Amobia within the framework provided by Piwczyński et al. (2017).

Morphology
Specimens were collected during several research expeditions across Australia during the summer months of 2016-2018. All specimens were collected individually using a hand net, killed in ethyl acetate vapour and either preserved in ethanol the same day or pinned for taxonomic identification. Photodocumentation of the adult morphology and male terminalia of A. auriceps and A. burnsi was completed using a BK lab system (Visionary Digital, VA, USA), with photographic stacking performed with Zerene Stacker (Zerene Systems, LLC, Richland, WA, USA). Photographs of dissected male terminalia were taken from preparations in glycerol. Photo-documentation of A. serpenta sp. nov. was completed using a Leica DFC295 digital camera (3M pixel) mounted on a Leica MZ16a stereo-microscope and analysed using Leica Application Suite ver. 3.8.0 (Leica Microsystems Limited, Germany).
For preparation of SEM images, male terminalia in glycerol were rinsed in demineralised water, dehydrated in 96% ethanol, air-dried, mounted on adhesive electrical tape attached to aluminium stubs, coated with ~100 nm of platinum/palladium and studied using a JEOL JSM-6335 F scanning electron microscope (JEOL, Tokyo, Japan). Due to the availability of only two male specimens of A. serpenta sp. nov., it was decided to avoid dissection of this species and to document and describe the relevant structures based on extended terminalia of the holotype.
For documentation of adult morphology, specimens were studied under a Leica MZ75 microscope (Leica Microsystems Limited) in conjunction with the photographs and SEM images. Terminology for morphological characters follows Cumming & Wood (2017), with additional characters relevant to Miltogramminae obtained from Pape (1996). For label data, Australian states and territories are denoted by standard abbreviations (ACT: Australian Capital Territory, NSW: New South Wales, NT: Northern Territory, QLD: Queensland, SA: South Australia, WA: Western Australia), additional information provided verbatim in square brackets after the label data, and repository by institutional abbreviation in parentheses.

AM
= Australian Museum, Sydney, Australia ANIC = Australian National Insect Collection, Canberra, Australia BM = Bishop Museum, Hawaii, USA NMNH = National Museum of Natural History, Washington, D.C., USA QM = Queensland Museum, Brisbane, Australia

DNA extraction, amplification and sequencing
Molecular analysis was employed to support the morphological species hypotheses and phylogenetically place the Australian Amobia among the global Miltogramminae. DNA extraction, amplification and sequencing methods followed Meiklejohn et al. (2011) and Piwczyński et al. (2017). In short: the total genomic DNA was extracted from a single leg of four specimens of A. auriceps, two of A. burnsi and a single specimen of A. serpenta sp. nov. using the salting-out method described by Aljanabi & Martinez (1997) (see Supplementary file 1 for a full list of taxa and specimens used in the molecular analysis, including accession numbers for genetic data originally sequenced by Piwczyński et al. 2017) and stored at 4°C prior to amplification. The four genes used by Piwczyński et al. (2017) for their global miltogrammine phylogeny, COI, CYTB, ND4 and EF1α, were also amplified for our Amobia specimens. Amplification success was confirmed by resolving PCR products through agarose gel electrophoresis. Prior to sequencing, 5 µl aliquots of samples were treated with 2 µl of 2x diluted ExoSAPIT Express ® (Applied Biosystems, Foster City, CA, USA), following the manufacturer's instructions, in order to digest unincorporated dNTPs and primers. ExoSAPIT Express ® treated products were then sequenced using ABIPRISM ® BigDye TM Terminator ver. 3.1 Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The sequencing products were separated using an ABI 3130xl Genetic Analyser (Applied Biosystems, Foster City, CA, USA).
Sequence electropherograms were edited using ChromasPro version 1.33 (Technelysium, South Brisbane, QLD, Australia; available online at www.technelysium.com.au/wp/chromaspro) and confirmed to be of miltogrammine origin by cross-checking species identity by submission to the Basic Local Alignment Search Tool (BLAST; National Center for Biotechnology Information, Bethesda, MD, USA; available online at blast.ncbi.nlm.nih.gov).

Automatic barcode gap discovery for species delimitation
Prior to phylogenetic analysis, COI sequences for all extracted Amobia (seven in total: four A. auriceps, two A. burnsi and a single A. serpenta sp. nov) were aligned using MUSCLE (Edgar 2004 (Kimura 1980; default parameters). The number of input sequences used was lower than recommended for ABGD (three-five sequences per species); the developers (Puillandre et al. 2012) note that having less than the recommended number of sequences can result in the inability to accurately detect the "barcode gap". As such, this study did not use the prior intraspecific divergence (barcode gap) as the basis for species delimitation, but rather the K2P distance matrix generated by ABGD was used to calculate inter-and intraspecific distance between species. The distance between COI barcodes was then used to support morphological species hypotheses. All priors for the analysis remained at their default settings. To confirm run success and effective sampling of the priors, MCMC convergence was confirmed by examining effective sample size (ESS) and potential scale reduction factors (PRSF) (which were confirmed to be greater than 100 and ~1, respectively). A consensus tree was then prepared from the resultant trees with the first 25% of all trees discarded as burn-in. Paritionfinder2, RAxML and MrBayes analyses were performed within the online CIPRES platform (Miller et al. 2010).

Diagnosis
The males of this species can be distinguished from the other two Australian species of Amobia by the combination of dull yellow microtomentum present on the fronto-orbital and parafacial plates, brown basicosta and bare proepisternum (the latter feature, however, is variable in the two other Australian species and therefore not by itself fully diagnostic).

Male
Head (Fig. 2B, D, G). With dull yellow microtomentum on fronto-orbital and parafacial plates; frontal stripe dark brown and concave; fronto-orbital plate setose with a single distinct row (at least 15 setae) and additional unordered fine fronto-orbital setulae; third aristomere tapered, fine and hair-like distally, black and twice as long as first flagellomere; pedicel and scape setose; first flagellomere black and 1.5 × the length of pedicel; gena and postgena grey with black setae; genal groove present, dark brown; vibrissa located midway between tip of first flagellomere and mouth margin; numerous supra-vibrissal setae; vibrissal setae and supra-vibrissal setae present in two rows, inner row strong and ordered, outer row weak and unordered.
Thorax ( Fig. 2A, C). Grey with dull yellow-brown microtomentum (visible only at certain angles); one major median and two major lateral stripes; major stripes ending at anterior margin of scutellum; scutellum with some darkening on lateral margin; two notopleural setae with additional finer setae on entire surface; proepisternum bare; katepisternal setae 1+1.
Abdomen ( Fig. 2A, C, E). T1+2 dark brown; setose and with two distinct median marginal setae; T3 setose, with two distinct median marginal setae and additional finer setae along entire posterior margin; T4-T5: setose with marginal setae on entire posterior margin; black ground colour most clearly visible towards the posterior margin; dull grey microtomentum present (occupying the majority of each tergite); three distinct black spots (two lateral and one median) on T3 and T4; T5 with only median black spot.
Male terminalia (Fig. 3A-B). Cercus sickle-shaped in lateral view and narrowest in middle portion (middle portion ~20% of cercus length); setose dorsally; cerci straight in posterior view, separated by a small gap between tips of cercal prongs; surstylus straight in lateral view, shorter than cercus; curved medially towards cercus in posterior view; phallus blunt with small spines on distal lobe.

Female
As male except microtomentum generally paler, especially on fronto-orbital plate where it appears silvery grey. The authors have chosen not to redescribe the female genitalia.

Biology
Label data confirm an association with 'mudwasps', probably family Sphecidae.

Remarks
There has been some contention as to the validity of A. auriceps. Kurahashi (1974) considered A. auriceps to be conspecific with Amobia pelopei (Rondani, 1859), a species known only from the western Palaearctic region. He did, however, note differences in the shape of the cerci and surstyli, as well as in the colouration of the basicosta. Verves (1979) and Pape (1996), by comparison, considered A. pelopei and A. auriceps as distinct species, separated through the morphology of the terminalia and by the colouration of the fronto-orbital plates. This study is in agreement with Verves (1979) and Pape (1996). It should be noted that perhaps the most reliable character for the separation of these species is the shape of the cercus in lateral view, which is gradually tapering in A. pelopei (Kurahashi, 1974: fig. 1) and distinctly narrowed in the middle in A. auriceps (Fig. 3B). (Malloch, 1930) Figs 4-5

Diagnosis
Males of this species can be distinguished from the other two Australian species of Amobia by the combination of distinct bright golden microtomentum on the fronto-orbital as well as parafacial plates and yellow-brown basicosta.

Male
Head (Fig. 4B, D, G). With golden microtomentum on fronto-orbital and parafacial plates; frontal stripe dark brown and concave; fronto-orbital plate setose with two distinct rows of setae, one row of frontal setae (> 10 setae) and one row of proclinate fronto-orbital setae (> 10 setae) and additional fine frontoorbital setulae, setal rows ending before lunule; third aristomere broad at base and tapering to fine hairlike tip distally, black and twice as long as first flagellomere; pedicel and scape setose; first flagellomere black and slightly longer than pedicel; parafacial plate with fine setae near facial ridge in multiple disordered rows; gena and postgena grey with black setae; genal groove present, dark brown; vibrissa located midway between tip of first flagellomere and mouth margin; numerous supra-vibrissal setae present in two rows, inner row strong and ordered, outer row weak and unordered.
Thorax (Fig. 4A, C). Grey with some dull yellow microtomentum anteriorly; one major median, two major lateral and two faint minor lateral stripes (located mediad to lateral major stripes and approximately one quarter of their width); major stripes ending at anterior margin of scutellum; minor stripes ending just anterior to suture; scutellum with some darkening on lateral margins; two notopleural setae, with numerous additional fine setae; proepisternum setose; katepisternal setae 1+1. Wing (Fig. 4F). Hyaline; vein R 1 bare; vein R 4+5 with several setae at base; cell r 4+5 open; tegula black; basicosta light yellow brown. Legs (Fig. 4A). Black; claws of fore tarsus slightly longer than tarsomere 5; pulvilli slightly shorter than fore tarsal claws; mid tibia with one anteroventral and one anterodorsal seta.
Abdomen (Fig. 4A, C, E). T1+2 black; setose, with two distinct median marginal setae; T3-T5: setose, with two distinct median marginal setae and additional finer setae along entire posterior margin of each tergite; black ground colour most clearly visible towards posterior margin; dull grey microtomentum present (occupying almost the entirety of each tergite); three distinct black spots (two lateral and one median) on T3 and T4; T5 with only one median black spot.
Male terminalia (Fig. 5A-D). Cercus sickle-shaped in lateral view, broad at base and pointed apically; densely setose dorsally; cerci diverging in posterior view with a weak curve away from one another; surstylus straight in lateral view, shorter than the cerci; in posterior view, surstyli slightly curved medially towards cerci; phallus blunt with small spines on distal lobe, swollen apically.

Female
As male except with generally paler microtomentum, especially on head where it appears dull grey. The authors have chosen not to redescribe the female genitalia.

Remarks
Amobia burnsi was described in the monotypic genus Austrometopia Malloch, 1930. This classification has since been rejected as it rendered the genus Amobia paraphyletic (Kurahashi 1970;Pape 1996). Pape (1996) specified that, in the genus Amobia, the fore-tarsal claws are longer than the fifth tarsomere in both sexes; however, this was not observed in females of A. burnsi examined in this study, and as such may not be a character of all Amobia.
It should be noted that proepisternal setosity has been observed to be variable in this species and as such cannot be used to diagnose it.

Etymology
The species epithet 'serpenta', which should be treated as a noun in apposition, refers to Serpentine Gorge, the type locality of this species.

Male
Head (Fig. 6B, D, G). Grey with silver microtomentum on fronto-orbital and parafacial plates, and golden microtomentum on upper mouth edge, gena and postgena; frontal stripe black and distinctly concave; fronto-orbital plate setose with one distinct row of fronto-orbital setae (> 10 setae) and additional unordered fine fronto-orbital setulae; setal rows ending at height of lunule; third aristomere broad in basal third then tapering to a fine hair-like tip distally, black and 2 × length of the first flagellomere; pedicel and scape setose; first flagellomere black and slightly longer than pedicel; parafacial plate with fine setae near facial ridge, in multiple disordered rows; gena yellow and postgena light grey with black setae; genal groove present, dark brown; vibrissa located midway between tip of first flagellomere and mouth margin; numerous supra-vibrissal setae.
Thorax (Fig. 6A, C). Grey with golden microtomentum, particularly antero-dorsally and forming distinct patch on notopleuron; one major median, two major lateral and two faint minor lateral stripes (minor stripes placed medially to major stripes and approximately one quarter of their width); major stripes continuing full length of scutellum, minor stripes ending just anterior to suture; two notopleural setae, with numerous additional fine setae on entire notopleural surface; proepisternum bare or setose; katepisternal setae 1+1.
Legs (Fig. 6A). Black; claws of fore tarsus slightly longer than tarsomere 5; pulvilli as long as fore tarsal claws; mid tibia with one anteroventral and one anterodorsal seta.
Abdomen (Fig. 6A, C, E). T1+2: black; setose and with two distinct median marginal setae; T3-T5: setose, each with one pair of distinct median marginal setae; T5 with additional marginal setae along entire posterior surface; black ground colour most clearly visible towards posterior margin; dull grey microtomentum present, occupying entirety of tergites; three distinct black stripes (two lateral and one median) on T3; T4 with median stripe and two lateral spots; T5 with only one median black spot.

Female
As male except for slightly darker microtomentum on the fronto-orbital plate, appearing dull yellow. Parafacial plate, gena and post gena remain golden and distinctly different to fronto-orbital plate. The authors have chosen not to describe the female genitalia.

Biology
The holotype of this species was collected on a hilltop, perching on an exposed rock. Paratype label data indicate an association with vespid wasps (Vespidae: Eumeninae).

Remarks
Amobia serpenta sp. nov. is placed within the subgenus Amobia s. str. due to the brown coloured terminalia and more than 10 proclinate fronto-orbital setae (Zumpt 1961;Pape 1996). Due to the limited number of male specimens (two), it was decided to avoid dissection of the male terminalia and rather to describe the extended male terminalia of the pinned holotype. For this reason, and as the phallus was broken off completely in the holotype, only the cerci and surstyli could be accurately described.

Molecular analysis
BLAST searches of COI sequences from A. auriceps, A. burnsi and A. serpenta sp. nov. all showed the closest matches with a previously submitted sequence erroneously identified as a Protomiltogramma sp. (> 96% similarity; JN964698). Fortunately, the original voucher specimen could be retrieved from the University of Wollongong Diptera collection and re-identified as A. auriceps. The sequences of Amobia were also a good match with a previously identified Amobia signata originally sequenced for the phylogeny of Piwczyński et al. (2017) (> 92% similarity; KY749709).
Representative COI sequences from each species were analysed using the ABGD online tool. Preliminary analysis of COI data supported the morphological species hypothesis, despite the low number of input sequences. All three species were molecularly distinct, with mean inter-and intraspecific distance calculated as 4.00% and 0.24%, respectively (calculated using Kimura two parameters model; Kimura 1980). The greatest between-species distance was observed between A. burnsi and A. auriceps (mean interspecific distance = 4.85%, n = 8). The next greatest distance was between A. auriceps and A. serpenta sp. nov. (mean interspecific distance = 4.64%, n = 2). The smallest distance was between A. burnsi and A. serpenta sp. nov. (mean interspecific distance = 2.08%, n = 4), this distance is ~10 × higher than the mean intraspecific distance for all Amobia species (0.24%) and 13 × higher than the mean intraspecific distance calculated for A. burnsi (0.15%).

Phylogenetic reconstruction
A Maximum Likelihood phylogeny was reconstructed based on the new sequences obtained from Australian Amobia (three concatenated sequences, one for each of the three species) and the sequence data of Piwczyński et al. (2017) (Fig. 1). The Bayesian inference phylogeny was generally concordant with the Maximum Likelihood phylogeny ( Fig. 1; only support values shown).

Species diagnosis
This study is the first to attempt to integrate morphological and molecular data to establish a strongly supported species hypothesis for the Australian Amobia. Morphology supported a three-species hypothesis, with the description of a new species, A. serpenta sp. nov., and the re-description of both A. auriceps and A. burnsi. The most diagnostic characters for the Australasian species of Amobia were colouration of the fronto-orbital plate and colouration of the basicosta, characters also known to be diagnostic for species of this genus in the Oriental, Nearctic and Palaearctic regions (Kurahashi 1974;Pape 1996). Unlike other regions, the presence and colouration of the microtomentum on the notopleuron was also found to be diagnostic in Australian Amobia.

DNA barcoding and molecular phylogenetics
Despite the low number of input sequences, preliminary ABGD analysis provided evidence supporting all three Australian species of Amobia identified by morphology. Between-species genetic distance was ~17 × higher than within-species genetic distance (4.00% compared to 0.23%). The most closely related COI barcodes were observed between A. burnsi and A. serpenta sp. nov. (2.08% interspecific distance) however this was still ~10 × higher than the mean intraspecific distance calculated for all species.
Both the Maximum Likelihood and Bayesian phylogenetic analyses clustered all three Australian species of Amobia together with A. signata. Within this Amobia clade A. serpenta sp. nov. + A. auriceps is sister to A. burnsi. Other than providing insight into the taxonomy and phylogeny of the Australian fauna of Amobia, our results agree with the intergeneric relationships presented by Piwczyński et al. (2017); Amobia is sister to Senotainia (in part; S. tricuspis and S. albifrons) then Amobia + Senotainia (in part) is sister to Macronychia. Thus, the addition of additional Amobia species in this analysis provides additional support to the previous phylogenetic hypotheses for the subfamily Miltogramminae.

Rejection of synonymisation of A. auriceps (Baranov, 1935) and A. pelopei (Rondani, 1859)
This revision supports the separation of A. auriceps and A. pelopei as distinct species. Verves (1979) and Pape (1996) separated A. pelopei and A. auriceps through the colouration of the fronto-orbital plates (A. pelopei with silver microtomentum, A. auriceps with yellow gold microtomentum). Verves (1979) additionally suggested that the terminalia of A. auriceps could be distinguished from those of A. pelopei by the terminally angular cerci and an apically dilated phallus. We follow Verves (1979) and Pape (1996) in considering A. auriceps and A. pelopei as separate species and confirm the dull yellow colouration of the fronto-orbital plate in A. auriceps as an important character for the separation of these species.
In addition to the morphological evidence for separation, the barcode library of Australian Amobia prepared in this study will help to either support or refute the separation of A. auriceps and A. pelopei once molecular data have been obtained for A. pelopei.
All Australian representatives identified by Meiklejohn et al. (2012) as A. pelopei were examined as part of this study and are re-identified as A. burnsi through the diagnostic combination of golden frontoorbitals and male terminalia. The COI barcode fragment of these specimens also matched to A. burnsi.

Wasp associations and the distribution of species of Amobia
Label data from Australasian species provides evidence for the association of A. burnsi and A. serpenta sp. nov. with vespid wasps (recorded from the nests, or closely following 'potter' wasps; subfamily Eumeninae), and A. auriceps and A. burnsi with sphecid wasps (recorded from 'mud' wasps; family Sphecidae). This is generally consistent with previously recorded host associations for this genus in other parts of the world. Species of Amobia have been recorded as kleptoparasites of sphecid and vespid wasps in the Nearctic, Oriental and Palaearctic regions (Fye 1965;Itino 1988;Wcislo 1996;Fateryga & Kovblyuk 2014) and of vespids and crabronid wasps in the Neotropical region (Freeman & Jayasingh 1975;Buys 2007;Nascimento & Garófalo 2014). No records are available that associate any Australasian species with wasps of the family Crabronidae.
While known from locations hundreds of kilometres from the nearest city or town (e.g., Litchfield NP, Northern Territory), Australia's species of Amobia also appear to occur with their hosts in urban environments. Amobia burnsi and A. auriceps species have been observed usurping wasp nests located in tunnels, overpasses and attached to buildings (pers. obs.). Australian Amobia should be expected to occur wherever suitable wasp hosts are found.