Revison of Metaplagia Coquillett (Diptera: Tachinidae) with description of five new species from Area de Conservación Guanacaste in northwestern Costa Rica

Abstract Background We revise the genus Metaplagia Coquillett, 1895 and describe five new species from Area de Conservación Guanacaste (ACG) in northwestern Costa Rica. All new species were reared from an ongoing inventory of wild-caught caterpillars spanning a variety of species within the family Sphingidae (Lepidoptera: Sphingidae). Our study provides a concise description of each new species using morphology, life history, molecular data and photographic documentation. In addition to the new species, the authors provide a re-description of the genus and a revised key to the species of Metaplagia. New information The following five new species of Metaplagia are described: Metaplagia leahdennisae Fleming & Wood sp. n., Metaplagia lindarobinsonae Fleming & Wood sp. n., Metaplagia paulinesaribasae Fleming & Wood sp. n., Metaplagia robinsherwoodae Fleming & Wood sp. n. and Metaplagia svetlanakozikae Fleming & Wood sp. n. The following is proposed by Fleming & Wood as new combination of Plagiomima Brauer & Bergenstamm, 1891: Plagiomima latifrons (Reinhard, 1956) comb. n.


Introduction
The tribe Voriini is distributed globally; the vast tachinid fauna of the Neotropical Region and the huge number of genera have proven to be one of the most significant hurdles to understanding the tribal boundaries surrounding the Voriini. Most attempts to understand the synapomorphies or of the tribe have, rather than answered questions, led researchers to even more questions as to what the voriines truly are. Presently, the tribe possesses several diagnostic features which the authors consider as typical to the tribe: conical head profile (longer at level of pedicel than at vibrissa); proclinate, divergent and well-developed ocellar setae; frons wide; proclinate and reclinate orbital setae present in both sexes; facial ridge bare; prosternum bare; anepimeral seta absent or poorly developed appearing hairlike; infrasquamal setae present; apical scutellar setae strong and decussate; dm-m crossvein oblique, making posterior section of M subequal to anterior section; R setulose at least to crossvein r-m and sometimes beyond; middorsal depression of ST1+2 reaching posterior margin; and aedeagus elongate and frequently ribbon-like (Tschorsnig 1985, Cortés andGonzález 1989). Voriini lay flattened membranous incubated eggs directly on the cuticle of the host (Herting 1956, Guimarães 1977 . Unfortunately, some of these diagnostic features can be observed across several other taxa within the Dexiinae and, as such, our recognition of the tribe can be seen as a combination of these features used to exclude other genera and define the Voriini; much variation exists within the catchall tribe and the "gestalt" of the tribe is still somewhat nebulous. The Voriini are a problematic tribe and still the subject of much debate. Recent work has suggested that the Voriini are a vast unifying polyphyletic clade encompassing much of the Dexiinae (Stireman et al. 2019), only further emphasizing the catch-all nature of the tribe; this evidence, however, is still the subject of discussion.
The monotypic genus Metaplagia Coquillett, 1895 (Dexiinae: Voriini) was initially erected for the type species Metaplagia occidentalis Coquillett, 1895( Coquillett 1895. Coquillet based the new species on a single male specimen he collected in San Diego Co.

Voucher specimen management
The management of voucher specimens has been detailed in previous papers in this series (Fleming et al. 2014). In brief, all caterpillars reared from the ACG efforts receive a unique voucher code in the format yy-SRNP-xxxxx. Any parasitoid emerging from a caterpillar receives the same voucher code as a record of the rearing event. If and when the parasitoid is later dealt with individually, it receives a second voucher code unique to it, in the format DHJPARxxxxxxx. These voucher codes, assigned to both host and parasitoids, may be used to obtain the individual rearing record at http://janzen.bio.upenn.edu/ caterpillars/database.lasso.
To date, all DHJPARxxxxxx-coded tachinids have had one leg removed for DNA barcoding at the Biodiversity Institute of Ontario (BIO) in Guelph, ON, Canada. All successful barcodes and collateral data are first deposited in the Barcode of Life Data System (BOLD, www.boldsystems.org) (Ratnasingham and Hebert 2007) and later migrated to GenBank. Each barcoded specimen is also assigned unique accession codes from both the Barcode of Life Data System (BOLD) and GenBank, respectively.
Inventoried Tachinidae were collected under Costa Rican government research permits issued to DHJ and exported from Costa Rica to Philadelphia, en route to their final depository in the Canadian National Insect collection in Ottawa, Canada (CNC). Tachinid identifications for the inventory were done by DHJ in coordination with: a) visual inspection by AJF and DMW, b) DNA barcode sequence examination by MAS and DHJ and c) correlation with host caterpillar identifications by DHJ and WH through the inventory itself. Dates of collection, cited for each ACG specimen, are the dates of eclosion of the fly, not the date of capture of the caterpillar since the fly eclosion date is much more representative of the time when that fly species is on the wing than is the time of capture of the host caterpillar. The collector listed on the label is the parataxonomist who found the caterpillar, rather than the person who retrieved the newly-eclosed fly from its rearing container. The holotypes and paratypes of the species newly-described herein are all deposited at CNC.

Descriptions and imaging
Species accounts and descriptions are deliberately brief and concise, complemented by a series of color photos of every species, used to illustrate the morphological differences amongst them. The morphological terminology used follows Cumming and Wood (2017). All dissections and photography were carried out following the methods detailed in Fleming et al. (2014). If only one male were available, it was designated as the holotype and not subjected to dissection.

Interim names of undescribed host species
Names of undescribed host species follow a standardized, interim naming system used for taxonomic units considered as distinct species and identified by DNA barcodes. The interim names are given in the format "Manduca sextaDHJ02" or "Manduca sextaDHJ03", where the "species epithet" is either composed of the name of the taxonomist who identified the species and a number or the name of a species-group, followed by a code. This prevents confusion with already-described species, while maintaining traceability of each undescribed species within the ACG project.

DNA barcoding
We generated DNA extracts from single legs using a glass fibre protocol (Ivanova et al. 2006), using the standard DNA barcode region (5' cytochrome c oxidase I (COI) gene) for all specimens of ACG Metaplagia. The DNA barcodes (658 bp near the 5' terminus of the COI gene) were amplified using general insect primers, using standard protocols for both production and quality control , Smith et al. 2007, Smith et al. 2008, Smith 2012. DNA sequences, trace files and accessions were deposited in the Barcode of Life Data System (BOLD) (Ratnasingham and Hebert 2007). Metadata (including GenBank accession codes) associated with each sequence can be consulted on BOLD by using the persistent DOI (https://doi.org/10.5883/DS-ASMETAPL).
The phylogeny ( Fig. 1) was created using the Maximum Likelihood method, based on the Tamura 3-parameter model (Tamura 1992) created using holo-and paratype DNA barcode sequences. Aligned with each tip of the tree (species name|DHJPARxxxxxxx accession) is a pictorial representation of the DNA barcode of that specimen and the lateral image of the holotype for that species. The best nucleotide substitution model and the phylogeny itself were created using MEGA X (Kumar et al. 2018) and the figure created using the R (R Core Team 2019) package ggtree (Yu et al. 2017).

Figure 1.
A phylogeny of the type series of Metaplagia species described from Area de Conservacion Guanacaste. Tip photos are the holotypes of the species.

Description
Male, head: frontal vitta wide, 1/3-1/6th width of front-orbital plate; with 2-3 proclinate orbital setae and 1-2 reclinate orbital setae; ocellar setae proclinate slightly divergent; eye bare, not descending beyond the level of the vibrissa; fronto-orbital plate coloration ranging from shining silver to gold; fronto-orbital plate with short black setulae interspersed amongst frontal setae; fronto-orbital setae not extending below lower margin of pedicel, with fronto-orbital setulae sometimes extending below lower margin of pedicel; pedicel orange, with a black-dark brown post pedicel; arista bare and subequal to slightly shorter than postpedicel, distinctly-thickened on basal 1/2-2/3, ranging from dark orange to dark brown-black; lower margin of face almost level with vibrissa, not visible in profile; facial ridge bare, but with setulae along parafacial sometimes so close to facial ridge as to be confused with facial ridge setulae; palps either straight or with a slight club at apex, sparsely setulose. Thorax: tomentosity ranging from pale beige-gray to dark grey or silver over a black ground color; thorax black ground color tomentum of thorax ranging from pale brassy to silver grey; prosternum bare; 3-4 postpronotal setae arranged in a straight line; supra-alar setae 1-2:3; intra-alar setae 2-3:3; dorsocentral setae 3:3-4; acrostichal setae 3:3; katepisternum with 3 setae. Scutellum black ground color, with tomentum ranging from gray to pale brassy; with five pairs marginal setae; apical scutellar setae 1/2-1/3 as long as subapical scutellars, sub-erect, slightly above marginal plane; basal scutellar subequal in length to subapical setae; subapical setae straight, ranging from divergent to convergent. Legs: dark reddish-brown to black ground color; tarsal claws and pulvilli ranging from shorter than to longer than last tarsomere. Wings: slightly longer than abdomen; translucent slightly hyaline; R and R can be setulose, setulae of R ranging from node to crossvein r-m or beyond. Abdomen: ground color black, pale silver tomentum in varying degrees on T3-T5, ST1+2 typically glabrous; middorsal depression on ST1+2 reaching to hind margin of tergite; median marginal setae present on T3 and complete rows on T4 and T5; median discal setae absent on all tergites; sex patch absent.
Terminalia: posterior margin of sternite 5 with a deeply excavated and wide U-shaped (sometimes sculptured) median cleft; lateral lobes of sternite apically rounded, often with setae along caudal margin; basal section 1/5 the length of apical lobes. Epandrium often with 3-4 long, strong setae along anal edge. Cerci, in posterior view, medially separated, but parallel and often touching so as to appear fused, with a few short setae on basal half. In lateral view, bowed and sharply tapered apically. Surstylus welldeveloped, stout basally in lateral view, like a stout broadly rounded triangle terminating in a small knob, appearing hooked or slightly beaked apically; in posterior view, basally enlarged and apically straight.
Female as in male, except in the following aspects: head: tomentum of fronto-orbital plate and parafacial can sometimes differ from that of the conspecific male; frontoorbital plate and parafacial up to 0.5x wider than in males.

Diagnosis
Metaplagia can be distinguished by the following combination of traits: head distinctly conical; males with upper frontal setae reclinate; proclinate orbital setae in both sexes; frontal setae descending below level of pedicel; both sexes with well-developed lateral vertical setae; eye bare; parafacial setulose, but not with strong stout setae, only hairlike setulae; genal dilation very slightly developed; prementum shorter than height of head with an enlarged labellum; prosternum bare; three postsutural supra-alar setae, the anteriormost reduced and much weaker than first postsutural dorsocentral seta; scutellum with four pairs of marginal setae and one pair of erect to semi-erect apical setae; one or two pairs of sub-erect discal setae on scutellum, in line with subapical setae; vein M ending separately in wing margin; anepimeral seta short, not extending beyond edge of lower calypter; wing vein R setulose.

Distribution
From Manitoba, Canada east to Newfoundland and south to Costa Rica.

Ecology
Within the ACG inventory, Metaplagia has only been reared from the Lepidoptera family Sphingidae: Lepidoptera throughout the diverse ecosystems of the research area.

Diagnosis
Metaplagia leahdennisae sp. n. can be distinguished from all other Metaplagia by the following combination of traits: fronto-orbital plate at least 50% gold, with a silver parafacial and setulae on vein R not extending beyond crossvein r-m. Metaplagia leahdennisae sp. n. can be separated from Metaplagia occidentalis by the presence of gold on the fronto-orbital plate. Metaplagia leahdennisae sp. n. is clearly distinguished by its COI sequence clustered within the Barcode Identification Number (BIN) BOLD:AAE2876.
Terminalia (Fig. 6): posterior margin of sternite 5 with a deep and narrow excavated Ushaped median cleft (Fig. 6d); lateral lobes of sternite apically rounded, with a vestiture of short setae along disc, 3x as long along caudal margin; basal section 1/3 the length of apical lobes. Cerci, in posterior view, medially separated and parallel not touching medially, with a few short setae on basal half (Fig. 6a); in lateral view, bowed and sharply tapered to a point apically (Fig. 6b, c). Surstylus well-developed, stout basally in lateral view, rounded basally and appearing hooked or slightly beaked apically; in posterior view, basally enlarged and apically straight.

Etymology
Metaplagia lindarobinsonae sp. n. is named in honor of Linda Robinson for her many years of coordinating and administrating a plethora of problems and events for the undergraduate biology teaching laboratories of the Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania.

Diagnosis
Metaplagia paulinesaribasae sp. n. can be easily distinguished from all other Metaplagia by the following combination of traits: both fronto-orbital and parafacial gold and setulae on vein R not extending beyond crossvein r-m. It is distinguishable from all other congeners by the presence of an entirely gold fronto-orbital plate and parafacial. Metaplagia paulinesaribasae sp. n. is clearly distinguished by its COI sequence clustered within the Barcode Identification Number (BIN) BOLD:AAX4230.

Etymology
Metaplagia paulinesaribasae sp. n. is named in honor of Pauline Saribas for her many years of coordinating and administrating a plethora of problems and events in the Academic Office of the Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania.

Diagnosis
Metaplagia svetlanakozikae sp. n. can be distinguished from all other Metaplagia by the following combination of traits: frontal vitta indistinct around ocellar triangle, infrasquamal setae present, fronto-orbital plate at pale gray or silver, with a silver parafacial and setulae on vein R not extending beyond crossvein r-m, surstylus rounded at tip. Metaplagia svetlanakozikae sp. n. can be separated from Metaplagia occidentalis by the presence of infrasquamal setae and from Metaplagia lindarobinsonae sp. n. by the presence of a small circular formation of setulae on the parafacial and not distinct row of setulae along the facial ridge. Metaplagia svetlanakozikae sp. n. is clearly distinguished by its COI sequence clustered within the Barcode Identification Number (BIN) BOLD:AAD5456.

Ecology
Metaplagia svetlanakozikae sp. n. has been reared seven times from one species of Lepidoptera in the family Sphingidae: Agrius cingulata, in dry forest.

Identification keys
Key to the Metaplagia of North and Mesoamerica