Four new species of Philoplitis Nixon (Braconidae, Microgastrinae) with an updated key and illustrations of all described species

Abstract The Microgastrinae genus Philoplitis Nixon is revised and four new species are described: P.keralensissp. n. and P.trifoveatussp. n. authored by Ranjith & Fernandez-Triana, and P.dzangasanghasp. n. and P.margallasp. n. authored by Fernandez-Triana & Ranjith. A key to all nine known species is provided. Philoplitisadustipalpus Ahmad is redescribed and illustrated. Additional specimen records are presented, and the diagnostic value of some morphological characters previously used is discussed. Based on the very few specimens available for study in collections, Philoplitis seems to be restricted to the Old World tropics (Afrotropical and Oriental regions), with most known species found in the Oriental region. The first DNA barcodes for the genus are presented. No host data is currently available, but for one species a mass of five wasp cocoons was found and is illustrated for the first time.


Introduction
Microgastrinae is one of the most diverse and globally ubiquitous subfamilies of braconid parasitoid wasps, commonly encountered as pupae or prepupae encased in white silk cocoons on or near the dead or dying bodies of their host caterpillars. Species are the koinobiont endo-larval parasitoids of Lepidoptera (Quicke 2015). More than 100 species in this group have been used or investigated worldwide in the biological control of lepidopteran pests, and this total is likely to raise . Mason (1981) estimated that worldwide Microgastrinae comprises between 5,000 and 10,000 species; at present Microgastrinae contains 81 extant genera and 2,700+ extant species (Yu et al. 2016, Fernandez-Triana andBoudreault 2018). Rodriguez et al. (2013) estimated the species richness of microgastrines to be 8-10 times that of the ~2,000 species described at that time. The microgastrine fauna of India contains 231 species recorded within 21 genera so far (Gupta and Fernandez-Triana 2014). Recent studies show that there are many new distribution records like Alloplitis Nixon (Ranjith, unpublished) and many more species in both diverse and rare genera (Veena et al. 2014, Ranjith et al. 2015. The genus Philoplitis Nixon was erected by Nixon (1965) and the species can be easily diagnosed by the large scutellum, which is prolonged posteriorly above the propodeum (Mason 1981). Fernandez-Triana and Goulet (2009) revised the genera, described three new species, and provided a key to all known species. The distribution of Philoplitis seems to be restricted to the Afrotropical and Oriental regions (Fernandez-Triana and Goulet 2009, current study). The hosts of Philoplitis species are still unknown.
Here we describe four new species from the Old World tropics, redescribe Philoplitis adustipalpus Ahmad, present additional records for some species, and discuss the diagnostic value of some morphological characters previously used.

Materials and methods
We examined the specimens from various type repositories including the Centre for Biodiversity Genomics, University of Guelph, Canada (BIOUG), the Canadian National Collection of insects, Canada (CNC), the National Museum of Natural History, USA (NMNH), and the collection of the Zoology Department, Aligarh Muslim University, Uttar Pradesh, India (ZDAMU). Morphological terms and wing venation designations follow Huber and Sharkey (1993), Sharkey and Wharton (1997), Karlsson andRonquist (2012), andFernandez-Triana et al. (2014). The abbreviations F2 and F15 refer to antennal flagellomeres 2 and 15; T1, T2, and T3 are used for metasomal mediotergites 1, 2 and 3; and L and W refer to length and width, respectively. Calculation of wing vein ratios follows Fernandez-Triana and Goulet (2009). The terms for integument sculpture follow Harris (1979). Primary types are deposited in Department of Zoology, University of Calicut, Kerala, India (DZUC) and the CNC.
Indian specimens studied by the first author were treated with Hexamethyldisilazane (HMDS) in order to prevent collapsing during mounting. Specimens were imaged using an Leica M205A microscope with automated multiple image capture at preset focal levels using an Leica DFC 500 camera, and image combination using the Leica Application Suite (V4.7) image processing system. All images were edited using Photoshop CS8 (Version 6.1) (Adobe Inc.) for the removal of image artifacts and standardizing the background colour.
All specimens studied from BIOUG and CNC material (African and Oriental regions) were critical point dried, and imaged with a Keyence VHX-1000 Digital Microscope, using lens with a range of 10-130×. Multiple images were taken of a structure through the focal plane and then combined to produce a single in-focus image using the software associated with the Keyence System. Images were corrected using Adobe Photoshop CS4, and plates were prepared using Microsoft PowerPoint 2010 and later saved as .tiff files.
A distribution map of all known species of Philoplitis was made using Simplemappr (https://www.simplemappr.net).

Results
With the four new species described below, the total number of known Philoplitis almost doubles, from five (Fernandez-Triana and Goulet 2009) to nine in this paper. It is likely that a few more species are found in the future, especially when the fauna of the Afrotropical and Oriental regions is more comprehensively collected and studied. Still, this genus is not likely to be very specious, and the specimens seem to be very rarely collected. Indeed, most of the species are known from one or very few specimens in collections (Table 1), despite of hundreds of microgastrinae specimens collected in some of the localities where Philoplitis specimens have been found so far. Table 1. Described species of Philoplitis with their known geographical distribution (by biogeographical region and country), and the number of specimens in collections. Data from Nixon (1965), Mason (1981), He (1983), Ahmad et al. (2005), Fernandez-Triana and Goulet (2009), and present paper.

Philoplitis species
Known distribution as Biogeographical region: Countries Number of known specimens P. adustipalpus Ahmad, 2005 Oriental: India 2 female P. coniferens Nixon, 1965 Oriental Based on current data, the genus seems to be restricted to the Old World tropics, with the majority of the species being found in the Oriental region and two species in the Afrotropics (Fig. 1). No host data is known at present but for one species, P. keralensis Ranjith & Fernandez-Triana, sp. n., a mass of five wasp cocoons was found associated with an unidentified lepidopteran larvae and is here illustrated for the first time (Fig. 8). We suspect other species of Philoplitis may be gregarious as well.
Seven sequences (DNA barcodes) are available in the Barcode of Life Data System (BOLD), representing three of the nine described species (Suppl. material 1, Supplementary Appendix 1). Those sequences are grouped in four Barcode Index Numbers (for details on the BIN system see Ratnasingham and Hebert 2013). The current BINs representing Philoplitis are: BOLD:AAW0954, BOLD:AAZ9051, BOLD:ACA6591, and BOLD:ACP5254 (http://www.boldsystems.org/index.php/Public_BINSearch?qu ery=Philoplitis&searchBIN=Search+BINs). The DNA barcodes of Philoplitis are very distinct, and cluster fairly separated from 35,000+ sequences of other microgastrines available in BOLD.
Future work should focus on better morphological characterization of the species, as the majority have been described from and/or are currently known from very few specimens (Table 1). Fernandez-Triana and Goulet (2009) put too much emphasis on the relative proportions of the fore wing veins near the areolet, but those proportions may be found to vary when more specimens become available. We have found that color of palpi and metatibia spurs, sculpture of head near occipital carina, and metafemur L/W ratio seem to have more diagnostic value than vein proportions in the fore wing. But we also caution that if/when more specimens become available, the value of those characters may also need to be reassessed.  Redescription. Head distinctly rugose, frons transversely striated. Occipital carina strongly defined and crenulate. Moderately smooth and shiny area centrally between posterior ocelli and occipital carina. Antennae long, L of F2 3.00 × its W, L of F15 1.90 × its W. Mesosoma mostly covered with silver setae. Anteromesoscutum coarsely rugose. Notauli faintly impressed with impressed postero-lateral area above tegula. Scutellar disc L/W ratio 1.20 ×, and its length 0.80 × that of anteromesoscutum. Mes-opleuron rugose anteriorly, smooth to sparsely punctate medially, rugose posteriorly with a median, smooth area centrally. Metapleuron rugose. Propodeum rugose with complete, medial longitudinal carina, lateral carinae forming crenulations. Tarsal claws with one tooth and with arolium subequal to claw length. Metafemur L 3.47 × its maximum width. Inner spur of metatibia 0.39 × L of first metatarsomere. T1 finely striate, slightly emarginate medially with smooth triangular area medio-apically, T1 with shallow, median longitudinal groove extending beyond half of tergite length, T1 L 2.20 × its W at posterior margin. T2 smooth, medial zone outlined by divergent carinae on either side, T2 medial L 0.47× its W at posterior margin. T3+ smooth, sparsely setose.

Philoplitis coniferens
Diagnosis. Philoplitis dzangasangha sp. n. differs from all other species by its generally lighter coloration with palpi, pro-and mesocoxae yellow, and flagellomeres light brown.
Female. Unknown, but see Notes below. Distribution. Central Africa Republic, Dzanga Sangha Reserve. Etymology. Named after the type locality, part of the important Dzanga Sangha Protected Area Complex, as recognition of the value that complex has to protect the biodiversity of central Africa.
Notes. Among the specimens we studied there was a female collected on the same place and date than the paratypes; however, some morphological characters are different. With so few specimens available it is not possible at the time to conclude if the female belongs to the same species or a different one. For the time being we prefer to keep within the species, although we did not include it as a paratype. Paratype. One male, same data than holotype, except 21.v.2013, AP Ranjith leg. Diagnosis. Philoplitis keralensis sp. n. is similar to P. trifoveatus sp. n., but it can be distinguished from the latter by having different ratios of fore wing veins 2RS/2M 1.13 × (1.30 × in P. trifoveatus sp. n.), eyes dark brown (eyes dark yellow in P. trifoveatus sp. n.), and fore wing with a small, brownish patch beneath vein 1-CU1 (without small brownish patch beneath 1-CU1 in P. trifoveatus sp. n.).
Colour. Head and mesosoma black, scape and pedicel reddish brown, flagellomeres brown, ocelli brown, profemur mostly brown, yellow basally and apically, protibiae and protarsomeres dark brown, mesofemur and metatibia black, mesotarsomeres yellowish brown, hind leg dark brown, metatibial spurs yellowish brown, wing veins and pterostigma brown, except for junction of veins 2CU1, m-cu and 3CU1 which is white, fore wing vein 1SR-M white, with a brownish cloud beneath pterostigma that extends to the posterior margin of the wing, small brownish patch beneath 1-CU1, Figure 6. Philoplitis keralensis, female holotype A habitus, lateral view B head, frontal view C head and mesosoma, lateral view D mesosoma, dorsal view E metasomal tergite I, dorsal view F metasomal tergites 2 and 3, dorsal view. fore wing faintly infuscated apically. T1 apically and T2 entirely yellow. Laterotergites 1-3 yellowish-white.
Male. Same as female.

Distribution. India (Kerala).
Etymology. This new species is named after the Indian state where type locality is found.    Diagnosis. Philoplitis margalla sp. n. is the only known species of Philoplitis with sexual dimorphism in the color of the metatibial spurs, which is light yellow in females, dark brown in males.
Colour. Head and mesosoma black; scape and pedicel orange-yellow; flagellomeres brown to light brown; palpi mostly dark brown to black (only two apical maxillary palpi yellow); all coxae, metafemur, metatibia and metatarsus black; pro-and mesofemorae and pro-and metatibiae mostly dark brown (at most with small yellow spot apically); pro-and mesotarsi yellow; metatibial spurs light yellow; most wing veins and pterostigma brown to light yellow; area beneath stigma light brown, clearly darker than rest of wing which is hyaline; metasoma dorsally mostly dark brown, except for T1 black on anterior 0.8, and for posterior 0.2 of T1 and entire T2 yellow-white; laterotergites 1-3 white, rest dark brown.
Male. As female, but with darker body color: palpi, scape, pedicel, most of legs (including metatibial spurs) dark brown to black; metasoma dorsally and laterally darker.
Distribution. Pakistan, Islamabad, Margalla Hills National Park. Etymology. The name, to be treated as a name in apposition, refers to the Margalla Hills National Park, a 12,600+ ha protected area in Islamabad Capital Territory, and where the type locality (Shakarparian Park) is located. Despite its small size, the fauna and flora of the Margalla Hills are quite diverse and mostly tropical and constitute a transitional zone between the high mountains to the north and plain areas to the south, providing a corridor for many Himalayan species to disperse south. Fig. 10 Philoplitis masneri Fernandez-Triana & Goulet, 2009: 292, figs 1, 4, 7, 10, 14 (original description).

Philoplitis punctatus Fernandez-Triana & Goulet, 2009
Notes. This species was described and diagnosed by Fernandez-Triana and Goulet (2009). Here we only provide new images of the species, including the first illustrations of a female specimen.
Notes. This species was described and diagnosed by Fernandez-Triana and Goulet (2009 Diagnosis. Philoplitis trifoveatus sp. n. differs from all other species by having the occiput medially with three pits. This new species can be separated from P. keralensis sp. n. by having scutellar disc L/W ratio 1.10 ×, and its L 0.90 × that of anteromesoscutum (scutellar disc L/W ratio 1.27×, its length 1.10 × that of anteromesoscutum in Figure 10. Philoplitis masneri, male holotype A habitus, lateral view B mesosoma, dorsal view C metasomal tergites 1-3 (partially), dorsal view D head, frontal view E body, dorsal view.  P. keralensis sp. n.), and by the ratio of fore wing veins r/r-m (2.50 ×in trifoveatus sp. n., 2.11 × in keralensis sp. n.).

Distribution. India (Karnataka).
Etymology. This species name alludes to the distinctive three pits present in the occiput.