Two new species of Erythromelana Townsend, 1919 (Diptera: Tachinidae) from Area de Conservación Guanacaste in northwestern Costa Rica

Abstract Background We describe two new species in the genus Erythromelana Townsend, 1919 from Area de Conservación Guanacaste (ACG) in northwestern Costa Rica. Both species were reared from wild-caughtcaterpillars of Eois spp. (Lepidoptera: Geometridae). We provide a concise description of each species using morphology, life history, molecular data, and photographic documentation. New information Erythromelana jimmychevezi Fleming & Wood sp. nov. Erythromelana glenriverai Fleming & Wood sp. nov.


Introduction
The tachinid genus Erythromelana Townsend, 1919 (Exoristinae: Blondeliini) is a small Neotropical genus in the tribe Blondeliini, occurring from southern Mexico to Bolivia and Brazil. Townsend originally described the genus based on one male and one female collected in Jaen province (Peru) and described as E. jaena Townsend. The genus remained untouched until Wood (1985) revised the entire tribe Blondeliini; in that work, Wood synonymized Erythromelana Townsend with Minthomyia Townsend, Euptilodegeeria Townsend and Myiodoriops Townsend, raising the total number of species within the genus to 6.
In 2013, an in-depth analysis and phylogeny of the genus Erythromelana was provided by Inclan and Stireman (2013), including the description of 11 new species. In addition to the new species descriptions, the authors revived two of the genera synonymized by Wood (1985) i.e. the monotypic genera Euptilodegeeria and Myiodoriops, and treated the nominal species Erythromelana obscurifrons Wulp as a nomen dubium, bringing the total number of recognized species in the genus to 14. Inclan and Stireman (2013) divided the genus into two species groups, the E. jaena group (E. abdominalis (Townsend), E. curvifrons Inclan, E. ecuadoriana Inclan, E. eois Inclan, E. jaena Townsend, E. leptoforceps Inclan and E. nigrithorax (Wulp)), and the E. cryptica group (E. arciforceps Inclan, E. catarina Inclan, E. convexiforceps Inclan, E. cryptica Inclan, E. distincta Inclan, E. napensis Inclan and E. woodi Inclan). Here, we build on this knowledge base by using CO1 (cox1 or cytochrome oxidase 1) gene sequences, or "DNA barcodes", morphology, and life history, to describe two previously undescribed species from Costa Rica, which we place within the E. cryptica species-group. Both species are parasitoids of the small green Eois Hübner (Lepidoptera: Geometridae) caterpillars feeding on Piper L. spp. (Piperales: Piperaceae) within Area de Conservación Guanacaste (ACG) in northwestern Costa Rica. This paper forms part of a larger series of papers describing the parasitoid diversity of ACG (Smith et al. 2006, Fleming et al. 2014a, Fleming et al. 2014b, Fleming et al. 2015c, Fleming et al. 2015a, Fleming et al. 2015b).

Study area and rearing intensity
All flies and rearing information described here were obtained from the 35+ year-old ongoing inventory of the caterpillars, their food plants and their parasitoids of the dry forest, rain forest, cloud forest and intergrades of the 125,000+ ha terrestrial portion of Area de Conservación Guanacaste (ACG) in northwestern Costa Rica (Smith et al. 2006, Smith et al. 2008, Janzen and Hallwachs 2011, Fernandez-Triana et al. 2014, Fleming et al. 2014a. Parasitoid rearing methods are described at http://janzen.bio.upenn.edu/ caterpillars/methodology/how/parasitoid_husbandry.htm.

Imaging and dissections
Descriptions of new species discussed in this paper are deliberately brief, and only include characters commonly used in tachinid fly identification. The descriptions are complemented with color photos, in order to illustrate the readily observed inter-specific differences.
Photographs of the habitus and terminalia were taken using the methods outlined in Fleming et al. (2014a). In brief, raw image files were first processed with Adobe Photoshop CS6, and then digitally stacked to produce a final composite image using the Zerene Stacker software v1.04. Imaging of the male terminalia followed methods described in Fleming et al. 2014a. Dissections of the terminalia followed the methods described in O'Hara (1983).
The morphological terminology and measurements of body parts follow Inclan and Stireman (2013).
Wherever a specimen label has been examined, the information is presented using "/" to indicate the end of a label and the beginning of the next. Labels are presented from the top-most (closest to the specimen) to the bottom-most, with any additional comments given in square brackets" []".

DNA barcoding
DNA barcodes (the standard 5' region of the mitochondrial cytochrome oxidase 1 (CO1) gene) for all ACG inventory specimens were obtained using DNA extractions made from single legs, using a glass fiber protocol (Ivanova et al. 2006). Total genomic DNA was resuspended in 30 μl of dH O, and the 658-bp barcode region near the 5' terminus of the CO1 gene was amplified using standard primers (LepF1-LepR1) and following established 2 protocols (Smith et al. 2006, Smith et al. 2008. All information on the sequences associated with each individual specimen (including GenBank and BOLD accession numbers) can be retrieved from the Barcode of Life Data System (BOLD) (Ratnasingham and Hebert 2007) via the publicly available dataset dx.doi.org/10.5883/DS-ASERYTH. The neighbor-joining (NJ) tree (Saitou and Nei 1987) for the species of Erythromelana from ACG produced by BOLD is presented here as Suppl. material 1.

Voucher specimen management
All caterpillars collected 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, if/and when it is individually processed for DNA barcoding, it then receives a second voucher code unique to it, in the format DHJPARxxxxxxx. These voucher codes, assigned to the host and to any emerging parasitoids, can be looked up at http:// janzen.bio.upenn.edu/caterpillars/database.lasso. To date, all voucher-coded tachinids have had one leg removed for attempted DNA barcoding at the Biodiversity Institute of Ontario (BIO) in Guelph, with all collateral data and all successful barcodes permanently and publicly deposited in the Barcode of Life Data System (BOLD, www.boldsystems.org) (Ratnasingham and Hebert 2007), and later migrated to GenBank as well. The inventory is dynamic and continually growing, with regular additions of new specimens. Erythromelana sequences can be found by searching for "Erythromelana" in BOLD. Each barcoded specimen has been assigned accession codes from the Barcode of Life Data System (BOLD) and GenBank.
The inventoried Tachinidae were collected under Costa Rican government research permits issued to DHJ and likewise exported under permit by DHJ from Costa Rica to Philadelphia, and then to their final depository in the CNC. Tachinid identifications for the inventory were done by DHJ in coordination with a) visual inspection by AJF and DMW, b) DNA barcoding by MAS, and c) correlation with host caterpillar identifications by DHJ and WH through the ACG inventory itself. Dates of capture of each specimen are the dates of eclosion of the flies, and not the dates of capture of the caterpillars since the 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 caterpillar. The collector listed is the parataxonomist who collected the caterpillar, rather than the person who retrieved the newly eclosed fly and processed it by freezing, pinning, labeling and oven-drying. The biologies and parasitization rates of the flies will be the subject of later papers.

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 "Eois Janzen52", where the species epithet is composed of the name of the taxonomist who identified the species and a number. This prevents confusion with already described species while maintaining traceability of each undescribed species within the ACG project.

Description
Erythromelana can be distinguished from other Blondeliiniby the following combination of characters: Head: proclinate orbital setae absent in male, female with 2 setae; 1-2 pairs of reclinate outer orbital setae, lowermost outer orbital seta distinctly longer than uppermost frontal seta; ocellar setae ranging from absent to well developed; eyes haired, of variable density in different species; parafacial bare and extremely narrow, at narrowest point equal to or narrower than the basal width of the palpus; parafacial light gray in ground color, covered with silvery-gold pollinosity, and bare; fronto-orbital plate and vertex black in ground color, covered with a dull silver-gold pollinosity (appearing mostly black (Fig. 2b)), and with faint golden reflections visible only in lateral view (mostly on vertex); lower margin of face level with vibrissa; vibrissa positioned at extreme anteroventral corner of face; facial ridge with a few short hairs on lower third or less; arista black on basal 1/3-1/4, becoming light brownish to orange distally, elongate, and minutely pubescent, thickened only on basal 1/4; palpus light orange to yellow, sometimes darkened basally. Thorax: ground color shiny black; scutum silver pollinose presuturally, postsuturally a polished black in ground color; prosternum setose; postpronotum bearing 2 or, rarely, 3 setae; katepisternum with 2-3 setae; first postsutural supra-alar seta small or sometimes absent; apical scutellar setae lacking; wingvein R setulose at base and vein R setulose or bare; vein M smoothly curved at bend and ending at wing margin anterior to wing tip, separately from vein R ; legs ranging from entirely yellow to entirely black. Abdomen: ground color ranging from yellow-orange to black, without strong banding; mid-dorsal depression not extending to hind margin of T1+2; discal setae only present on T5. Male terminalia: sternite 5 with median cleft either U-or V-shaped; inner margin bearing minute setae; apical lobe rounded or pointed apically with either a single, long, well-developed seta, multiple well developed setae, or bare; pregonite curved anteriorly, with strong setae along posterior margin; postgonite short with curved apex; epiphallus small, usually difficult to see between the pregonites; surstylus with setulae on inner and outer surfaces, or completely bare; surstylus, in lateral view, varied in shape from almost straight to slightly concave on anterior or posterior margins, usually with a broad, rounded apex, occasionally truncate; surstylus and cercus usually subequal in length, sometimes cercus shorter than surstylus; in posterior view, cerci narrowed on apical 1/3.

Other species included
Based on synapomorphieswithin the male terminalia of Erythromelana described by Inclan and Stireman (2013), the two newly described species belong to the E. cryptica species-group and as such were compared to the other species belonging to that group.
Head: (Fig. 2b, e) eye haired, with long ommatrichia about as long as combined width of 4-5 eye facets; eye approximately 0.9x head height; vertex 0.12-0.14x head width in male, 0.21-0.23x head width in female; frontal vitta 0.33-0.35x vertex width in male, 0.32-0.34x vertex width in female; first flagellomere black, reaching facial margin; fronto-orbital plate with 2 reclinate inner orbital setae, the lowermost large and well developed in contrast to the uppermost, which is small or reduced, almost hair-like (especially in the female); ocellar setae proclinate; facial ridge bare; palpus yellowish, with dark umber base, distally haired and almost uniform in width.
Abdomen: dorsal surface of abdomen mostly black in ground color; ventral margins of T1+2 and T3 yellow in ground color, the yellow extending to 3/4 of lateral surface of these tergites; T4 yellow in ground color on 1/3-1/2 of lateral surface; in dorsal view, both T3 and T4 black in ground color medially and yellow laterally; ground color of T5 entirely black; transverse bands of sparse white pollinosisty present on anterior 1/3-1/4 of T3 and T4 and on anterior 2/3 of T5; one pair of median marginal setae on T1+2 and 1 4+5 T3; T4 and T5 with a full row of marginal setae; mid-dorsal depression of T1+2 not reaching median marginal setae.
Male terminalia: (Fig. 3) sternite 5 with median cleft V-shaped; apical lobes broadly pointed, each with 2-3 long, well-developed setae, the longest seta at least 2x as long as second longest; surstylus with small hairs on inner and outer surfaces; surstylus, in lateral view, almost straight on basal 1/2 and convex on apical 1/2 of anterior margin, and very slightly concave along posterior margin; surstylus and cercus almost equal in length in lateral view (Fig. 3b); cercus, in lateral view, bent, digitate, not strongly concave on anterior surface, very slightly concave on postero-apical margin, and with a rounded tip; in posterior view, cerci narrowed inapical 1/3, length of upper lobes almost equal to medial section and longer than the apical cleft; apical cleft well defined with rounded tips directed slightly medially, basal section 2/3 the length of apical section.

Diagnosis
This species is included in the E. cryptica species group (Inclan and Stireman 2013) because of its morphological similarity to other members of the group. Erythromelana jimmychevezi can bedistinguished from E. arciforceps by the following character states: light brown wings; multiple, strong apical setae on the lobes of sternite 5; more pointed appearance to the apices of the lobes of sternite 5. It is worth noting that E. Neighbor-Joining (NJ -Saitou and Nei 1987) tree comparing the two species of Erythromelana present in ACG and the GenBank sequences of Erythromelana cryptica and E. napensis. Tree based on Kimura 2-parameter distances (K2P -Kimura 1980) made using MEGA6 (Tamura et al. 2013). Tip labels include species name, individual specimen ID (accession numbers for the Inclan and Stireman (2013) specimens, and voucher codes for the ACG specimens) and, for the two new species described herein, host species and the image of a male in lateral view. This phenogram demonstrates the low intra-specific and high interspecific variation in CO1 barcode sequences.
jimmychevezi may overlap in range with E. arciforceps, which has been collected from as far north as Monteverde, Costa Rica.

a b
c d e f
Head: (Fig. 4b, e) eye haired, with long ommatrichia about as long as combined width of 4-5 eye facets; eye approximately 0.9x head height; vertex width 0.13-0.15x head width in male, 0.22-0.24x head width in female; frontal vitta width 0.43-0.46x vertex width in male, 0.32-0.34x vertex width in female; first flagellomere black, reaching facial margin; fronto-orbital plate with 2 reclinate inner orbital setae of subequal length in male, whereas in the female the uppermost is large and well developed, 2x the length of the lowermost; ocellar setae small, poorly developed and proclinate; facial ridge bare; palpus yellowish, with dark brown-yellow base; distally haired; almost uniform in width.
Thorax: (Fig. 4a, c, d, f) dorsal vittae faintly visible presuturally as slightly darker stripes, virtually invisible postsuturally; postpronotum with 2 setae, usually with one additional small seta; two postsutural supra-alar setae (rarely only 1); katepisternum with 3 setae, ventromedial seta poorly developed, almost hair-like; scutellar discal setae absent; legs black; wing smoky gray, vein R dorsally bare, vein R dorsally with 3-4 setulae at base in male, and 5-6 setulae at base in female.   Abdomen: ground color of dorsal surface mostly black; T1+2 all black in ground color in dorsal view, and of yellow ground color ventrally in lateral view; T3 mostly yellow on anterior 3/4, black posteriorly; coloration of T3, in dorsal view, appearing as a black triangle on a yellow background; T4 yellow in ground color on 1/3-1/2 of lateral surface, appearing as entirely black in dorsal view; T5 entirely black; transverse bands of sparse white pollinosity present on anterior 1/3-1/4 of T3 and T4, and on anterior 2/3 of T5; one pair of median marginal setae on T1+2 and T3; T4 and T5 with a full row of marginal setae; mid-dorsal depression of T1+2 not reaching median marginal setae.
Male terminalia: (Fig. 5) sternite 5 with median cleft V-shaped, apical lobes bearing rounded apices, each with 1 long, well-developed seta, the longest seta at least 4x as long as the second longest; anterior margin of basal plate slightly concave; surstylus with small hairs on inner and outer surfaces; in lateral view, surstylus almost straight on basal 1/2 and convex on apical 1/2 of anterior margin, and very slightly concave along posterior margin; in lateral view, surstylus and cercus almost equal in length; cercus in lateral view bent, digitate, strongly concave on anterior surface, very slightly concave on postero-apical margin, and with a rounded tip; cercus truncate in lateral view, appearing like a slightly bent thumb; in posterior view, cerci narrowed on apical 1/3, length of upper lobes almost equal to medial section and longer than the apical cleft, basal section 1/3 longer than apical section; apical cleft well defined, with rounded tips directed medially.

Diagnosis
This species is included in the E. cryptica species group (Inclan and Stireman 2013) because of its morphological similarity to other members of the group. Erythromelana glenriverai can bedistinguished from E. cryptica by the following characters: consistently having only two postsutural supra-alar setae; a less developed medial katepisternal seta; a more spatulate surstylus (visible in both lateral and posterior views); and a more curved, narrower anterior section of cercus (in posterior view). Additionally, the posterior section of the cercus in E. glenriverai is almost equal in length to the length of both the anterior and medial sections combined. Erythromelana cryptica was one of the few species for which a barcode was available among the previously described species; its sequence was also different from that of E. glenriverai (Fig. 1).

Etymology
Erythromelana glenriverai is named in recognition of Glen Rivera Chaves for his contributions to the accounting team for Area de Conservación Guanacaste, the forest this fly lives in.

Analysis
The DNA barcode sequences recovered from the new Erythromelana species from ACG display the characteristic strong AT bias of insect mitochondrial DNA (mean percent GC content 32.89%, SE 0.08) and no insertions or deletions. Within-species variation was low (mean distance of 0.28%) compared to between-species variation (mean distance 5.18%). All values of DNA barcode variation were calculated within BOLD and can be re-calculated in the future as more specimens are recovered from the ACG inventory and added to the DNA library. Fig. 1 presents a neighbor-joining tree for the Erythromelana specimens reared and DNA barcoded by this inventory to date.