Cotesiacassina sp. nov. from southwestern Colombia: a new gregarious microgastrine wasp (Hymenoptera, Braconidae) reared from the pest species Opsiphanescassina Felder & Felder (Lepidoptera, Nymphalidae) feeding on Elaeis oil palm trees (Arecaceae)

Abstract A new species of microgastrine wasp, Cotesiacassina Salgado-Neto, Vásquez & Whitfield, sp. nov., is described from southwestern Colombia in Tumaco, Nariño. This species is a koinobiont gregarious larval endoparasitoid, and spins a common mass of cocoons underneath the host caterpillars of Opsiphanescassina (Felder & Felder) (Lepidoptera, Nymphalidae), feeding on oil palm trees (interspecific hybrid Elaeisoleifera × E.guineensis) (Arecaceae). While superficially similar, both morphologically and biologically, to C.invirae Salgado-Neto & Whitfield from southern Brazil, the two species are distinct based on DNA barcodes, host species, geographical range and morphological characters.

Cotesia is easily recognizable morphologically among microgastrine braconids, although the huge variety of species can be difficult to distinguish from each other ), especially those without host data. The wasps have a koinobiont habit (Kankare and Shaw 2004) and both solitary and gregarious species are known. Cotesia (Braconidae, Microgastrinae) currently contains roughly 300-400 described species (Fernandez-Triana et al. 2020), but this number will certainly increase dramatically, as world estimates range from 1000-2000 species (Mason 1981;Michel-Salzat and Whitfield 2004;Whitfield et al. 2018;Fernandez-Triana et al. 2020), and a relatively small number of studies recording Neotropical species of Cotesia and their biology are available so far (Whitfield 1997;Whitfield et al. 2018), particularly in South America.
As Cotesia species appear to be highly host specialized (Kankare and Shaw 2004), with many cryptic species and geographically restricted distributions (Fiaboe et al. 2017), the use of an integrative taxonomic approach (combining morphological, molecular, biological and geographical data) is critical for recognizing and distinguishing these parasitoid wasps (Smith et al. 2008;Kaiser et al. 2017).
Using such an integrative taxonomic approach, this paper provides a description of a new species of Cotesia, whose brood was produced from caterpillars of Opsiphanes cassina (Felder & Felder) (Lepidoptera, Nymphalidae) ( Fig. 1) feeding on palm trees (interspecific hybrid Elaeis oleifera × E. guineensis) (Arecaceae) in Tumaco, Nariño, south-west Colombia. We compare it with the other described species of Opsiphanes that have been formally recorded from the Neotropical region, two of which have been well characterized and two of which are of uncertain identity.

Materials and methods
Between April 2018 and March 2019; we collected 35 larvae of Opsiphanes cassina as part of a survey carried out on exotic palms in the Palmeiras plantation A.S., 58 km from San Andrés de Tumaco, Nariño, Colombia (1°47'28.0"N, 78°47'33.9"W, 28 m elev.see Fig. 1A, B). The larvae of O. cassina were found on the interspecific hybrid Elaeis oleifera × E. guineensis (Arecaceae). Upon collecting, larvae were kept in the laboratory (25 ± 1 °C; 70% RH; photoperiod of 14 hours of light) and observed daily until the emergence of the butterflies or parasitoids, which were then preserved in 70% ethanol.
Photographs of the caterpillar and parasitoid cocoons (Fig. 1C, D) were taken in the field by CANV. Morphological photographs of the Cotesia ( Fig. 2A-F) were taken by DSM at the University of Illinois, USA using a Leica M205 C stereo microscope (467 nm resolution) fitted with a 5 megapixel Leica DFC 425 digital microscope camera. Images were stacked using a motor drive on the microscope and Zerene Stacker software. Morphological terms and measurements of structures are mostly those used by Salgado-Neto et al. (2019).
To characterize and compare the new species at the molecular level, the mitochondrial (DNA barcode) gene cytochrome oxidase I (COI) was analyzed. For the amplification of a fragment of approximately 460 bp of this gene, we used the following primer pair: COI-F (5'-GATTTTTTGGKCAYCCMGAAG-3') and COI-R (5'CRAATACRGCTCCTATWGATAAWAC-3') (Gusmão et al. 2010). DNA extraction of one specimen was performed with the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich) and followed the manufacturer's protocol. The product was amplified via Polymerase Chain Reaction (PCR) according to the following schedule: 94 °C for 2 minutes, 40 cycles of 94 °C for 30 seconds, 54 °C for 30 seconds, 72 °C for 40 seconds and 72 °C for 4 minutes. Then the PCR product was purified using polyethylene glycol precipitation (PEG; Schmitz and Riesner 2006). These samples were sequenced using the Big Dye 3.1 reagent (Life Technologies) and a 3500 XL automatic sequencer (Life Technologies).

Diagnosis.
As discussed above, Cotesia is a huge worldwide genus of hundreds of species, with many morphologically similar species. While useful world identification keys are not available, it is currently possible to successfully diagnose species regionally, especially combined with molecular and host data. The closest described species, morphologically, biologically and within the region, is Cotesia invirae from southern Brazil, which also parasitizes Opsiphanes on palms (different species). The table below provides a diagnostic comparison between the two species.
Cotesia alia (Muesebeck), also recorded from Opsiphanes, resembles these two species but has a relatively longer first metasomal tergite (see illustration in ). Like C. cassina, the second tergite has the medial part of the second tergite longer than the lateral portions, and the tergites tend to both be blackish (tending to be mostly orangish in C. invirae). The other two named Cotesia species recorded from Opsiphanes, C. biezankoi (Blanchard) and C. opsiphanis (Schrottky), are both very poorly characterized in their descriptions and their type locations are unknown (Fernandez-Triana et al. 2020), so they are not compared here. There is a possibility that C. invirae might prove to be a junior synonym of C. biezankoi, based on shared host and geographic region, if the holotype of the latter were to resurface and be examined. Our understanding of the correct nomenclature for the entire complex would benefit from a full review of the named and putative unnamed species across all of Central and South America, especially if all the types could eventually be located. In the meantime, it is possible to characterize the relationships among the species for which we can clearly establish the identity.
Description. Female. Body length 3.1-3.3 mm; fore wing length 2.9-3.1 mm. Coloration ( Fig. 2A-F). General body coloration black except: scape shading from light to dark brown, palps pale yellow, tegulae brown, fore legs all yellowish, middle legs all yellowish, hind legs all yellowish except distal end of femur brown/black dorsally; distal end of tibia brown, coxae translucent yellowish, laterotergites yellowish ventrally, shading to brown dorsally; sternites and hypopygium translucent yellowish. Head ( Fig. 2A, E). Facial sculpture weakly punctate; vertex sculpture smooth to very weakly punctate; distance between posterior ocellae nearly identical with distance from outer ocelli to compound eyes. Mesosoma ( Fig. 2A, B, F). Pronotum with both dorsal and ventral grooves present, ventral groove crenulate. Mesoscutum fully and distinctly but shallowly punctate; scutoscutellar scrobe slightly sunken groove and formed by 8 pits. Scutellum shield-shaped to subtriangular (anteriorly straight and posteriorly rounded) and weakly convex, weakly punctate. Mesopeuron smooth and polished throughout. Propodeum generally finely rugose/punctate with indistinct longitudinal medial carina. Legs ( Fig. 2A-C). Hind coxa mostly smooth with faint sculpture on dorsal face; inner hind tibial spurs slightly longer than outer. Wings (Fig. 2B, C). Fore wing hyaline with dark brownish vein pigmentation; stigma more than 2× as long as broad, without obvious pale spot at proximal end. Metacarp extending 0.60-0.70 to end of 3Rs fold along wing edge; r approximately same length as 2RS vein and meeting it at a distinct shallow angle; vannal lobe edge roughly semicircular with distal end slightly flattened; vannal lobe fringe even and dense. Metasoma (Fig. 2B-D, F). Tergite 1 roughly as long as broad, evenly widening from anterior margin then rounding over posterior half, mostly rugulose; tergite 2 very weakly rugulose peripherally, mostly smooth and slightly raised centrally, roughly twice as broad as long, subrectangular with posterior margin slightly longer medially than laterally. Hypopygium with angled but blunt tip, not extending past dorsal end of metasoma; ovipositor with very sparse setae at tip. Male. Similar to female except with slightly narrower metasoma. Molecular data. COI barcode deposited in GenBank (MW405620). Using the identification tools in the Barcode of Life Database (Ratnasingham and Hebert 2007), C. cassina is closest to C. salebrosa (Marshall), a primarily Eurasian species attacking geometrid larvae, at a similarity level of 97.4%. Interestingly, C. invirae appears closest (97.02% similarity) to Cotesia Whitfield78 and Cotesia Whitfield20, two apparently conspecific sets of rearings of an undescribed species from the Lepidoptera Inventory of the Guanacaste Conservation Area (ACG) in northwest Costa Rica (Janzen et al. 2009); these rearings are from another species of Opsiphanes. It thus appears that there is a complex of at least four closely related species attacking different Opsiphanes species in a variety of geographically dispersed Neotropical habitats, as suggested by Salgado-Neto et al. (2019). BOLD and NCBI use slightly different criteria to make cutoffs in sequence comparisons, and to calculate % similarity. They also contain different sets of sequences. We checked the BOLD investigations of related species by using BLASTn (Altschul et al. 1990) to query the NCBI nucleotide database (NCBI 1988). The same most closely related species to Cotesia cassina and C. invirae, respectively, were recovered, with the exception that for C. cassina, C. melitaearum (Wilkinson) and C. koebelei (Riley), both attacking other genera of Nymphalidae but in the Holarctic region, joined C. salebrosa as closest, at roughly 94.6-95.6% similarity for all of them. In neither the BOLD nor the NCBI search did C. cassina come within 2.5% similarity of any other known Cotesia species.
Host. Opsiphanes cassina (Felder & Felder) (Lepidoptera, Nymphalidae) (Fig. 1C, D). Biology/ecology. Cotesia cassina is a gregarious parasitoid wasp that occurs mainly in the wet season (March-May); however, their host, O. cassina, occurs throughout the year, mainly in the rainy season (March-July). Cotesia cassina larvae kill the host larva before the end of the last instar and form their cocoons in a regular mass of dirty whitish cocoons, regularly arranged disposed under the host (Fig. 1B). The larvae of this gregarious species all emerge from the host in a short time through many different holes in the host cuticle and spin a common woolly cocoon mass within which the individual cocoons can be distinguished.
Etymology. The specific epithet cassina, is a reference to Opsiphanes cassina (Felder & Felder) (Lepidoptera, Nymphalidae), the host caterpillar name. The word cassina is the feminine of cassino which in Italian means playhouse.