Three new species in the genus Wilkinsonellus (Braconidae, Microgastrinae) from the Neotropics, and the first host record for the genus

Abstract The genus Wilkinsonellus Mason is a poorly sampled but widely distributed tropical genus of Microgastrinae (Braconidae), parasitoid wasps that exclusively attack caterpillars (Lepidoptera). Currently, species of Wilkinsonellus have been described only from the Palaeotropics, but the genus was known to occur in the Neotropics. Here we describe the first three species from Central and South America: Wilkinsonellus alexsmithi sp. n., Wilkinsonellus kogui sp. n.,and Wilkinsonellus panamaensis sp. n. These species descriptions confirm that Wilkinsonellus is a Pantropical genus. A dichotomous key for the three new Neotropical species is given. The first recorded host for the genus, Microthyris prolongalis (Crambidae), is also reported, for Wilkinsonellus alexsmithi.


introduction
The genus Wilkinsonellus was erected by Mason (1981) to accommodate four Palaeotropical species that Nixon (1965) included in the Apanteles henicopus and Apanteles daira groups. The former of those two species-groups contained three species, Apanteles henicopus ( de Saeger 1944) from Kenya and Rwanda, A. iphitus (Nixon 1965), and A. thyone (Nixon 1965) both from the Philippines, and the daira-group is monotypic, with A. daira (Nixon 1965) being from Papua New Guinea. In both species-groups, tergite I exhibits a distinctively narrow petiole, which is constricted medially and also with a deeply median groove (Nixon 1965).
After its origin as a recognized genus, the frequency of additional new Wilkinsonellus species descriptions has been spasmodic. Eleven years after its acceptance as a new genus, three new species were described from Papua New Guinea and Australia (Austin and Dangerfield 1992); the latter country was added as a new continental record of distribution. In the early twenty-first century, two more species were described, but this time from Northwest Vietnam (Long and van Achterberg 2003). Between 2005Between -2007, two additional species were described, one from India (Ahmad et al. 2005) and a further one from Vietnam (Long 2007). The most recent descriptions are from Taiwan and Vietnam (Long and van Achterberg 2011), when another four new species were added. Thus, the genus currently contains 15 described species, mainly from the Palaeotropics: Africa, south and Southeast Asia, Indonesia and Australia. The presence of Wilkinsonellus in the Neotropics was reported more than a decade ago during the elaboration of a key to Microgastrinae (Whitfield 1997), published in the Manual of the New World Genera of the family Braconidae. However, no Neotropical species were formally described at that time.
Currently, there is no information available about which families of Lepidoptera are used by these wasps as hosts, except that newly reported here. However, they are assumed to be koinobiont endoparasitoids of caterpillars (larvae of Lepidoptera), as are all genera of Microgastrinae. Only one of the previously described species, W. daira, has natural history data associated. It was labeled as bred from the plant Hibiscus, Malvaceae (Nixon 1965), which is obviously incomplete without an insect host.
Three undetermined species of Wilkinsonellus were reported in Kalimantan (Indonesia: Borneo island) during a study that assessed braconid parasitoid wasps diversity after the reforestation of degraded Imperata grassland (Imperata cylindrica, Poaceae) with Acacia mangium, Fabaceae (Maeto et al. 2009). In that study, one undetermined species was reported in matured Acacia plantations (aged 5-12 years), and two other unidentified species were found in old secondary logged dipterocarp forests.
The phylogenetic position of the genus within Microgastrinae is unclear. However, some authors have used comparative morphology to suggest a close relationship with Diolcogaster Ashmead. Two Diolcogaster species-groups have been proposed as close relatives. One of them is the xanthaspis-group (Austin and Dangerfield 1992). As with Wilkinsonellus, this species-group is characterized by its narrow petiole of tergite I, but the petiole has more or less parallel sides, while that of Wilkinsonellus (Nixon 1965) is constricted laterally. The other species-group is the fasciipennis-group (Mason 1981), which differs from xanthaspis only in that tergites II and III show no delimited median area (Nixon 1965). At the present time, the phylogenetic position of Wilkinsonellus within Microgastrinae remains an open question, largely due to little effort having been expended in representing all relevant groups in phylogenetic analyses.
After a brief mention of Dr. Wilkinson's contribution to the knowledge of Microgastrinae, the first three Neotropical species of Wilkinsonellus are described. A Pantropical distribution for the genus is confirmed, along with the first host data for the genus, and we offer a key for the three new species.

Douglas Shipton Wilkinson (1890-1941)
In 1981, William R. M. Mason named the genus in honor of D. S. Wilkinson, a renowned British entomologist at the Natural History Museum in London -then known as the British Museum (Natural History), who dedicated his entire career to the study of Microgastrinae. Wilkinson was a significant contributor to Microgastrinae taxonomy. He concentrated his efforts in understanding the morphological variability of Apanteles not only regionally, but also on a global scale. His vast knowledge of Apanteles helped him to design a morphology-based classification (Papp 1976). He proposed six groups; each one was named with arbitrarily chosen letters [A, F, G, S, U, & M] (first developed in Wilkinson 1932). This system of letter-designated groups was adopted, modified and extended from the previous four sections proposed by Marshall (1885) for the British Apanteles fauna (Nixon 1965). He was the first European entomologist to recognize the necessity of critically reviewing the classification of the Palaearctic Apanteles species. Wilkinson enlisted in the navy during World War II and was killed at sea in 1941, terminating his intention of attaining a world classification of Microgastrinae (Papp 1976, Whitfield et al. 2002).
Wilkinson's later work on the Palaearctic fauna was published after his death, with the aid of Gilbert Nixon, who became his successor in studying the group (Wilkinson 1945). In this monumental work he re-described 58 European Apanteles species in a highly detailed way, and included nomenclatural comments and extensive information on natural history. The detailed critical analysis of host-ranges was due to his proficient collaborator Richard Laurence Edward Ford, who could replicate in the laboratory the conditions of rearing parasitoids and their hosts.

Methods
Specimens used by this revision were obtained on loan from the following institutions, which are identified in the text by their acronyms: The specimens from the IAvH-E collection are the result of the project "Insect Survey of a Megadiverse Country Phase I and II: Colombia" conducted from 2002 to 2006. More than 25 natural protected areas managed by the Colombian government were sampled (Arias-Penna 2007). Specimens from the DHJWH collection are the result of "the caterpillar and parasitoid inventory of the Área de Conservación en Guanacaste (ACG)" Costa Rica ), a large-scale on-going rearing project. Caterpillars were collected directly in the field and subsequently reared in laboratory conditions. Information about taxonomic identification for caterpillar, host plant and parasitoids as well as data of parasitoid eclosion is available. Each caterpillar is tagged with a voucher code: YY-SRNP-XXXX. The prefix refers to the last two digits of the year that caterpillar was discovered in the field. SRNP stands for Santa Rosa National Park, and the suffix is a unique number assigned within the year. When a parasitoid emerged from its host, the same caterpillar voucher code is assigned, but also a unique DNA wasp voucher code is assigned: DHJPARxxxxxxxx ).

Morphology and taxonomic characters
Initial identification to genus level followed the key to the Neotropical microgastrine genera (Whitfield 1997). The original Wilkinsonellus species descriptions from Papua New Guinea (Nixon 1965), Australasian Region (Austin and Dangerfield 1992), Vietnam (Long andAchterberg 2003, 2011), India (Ahmad et al. 2005) and China (Zeng et al. 2011) were consulted to confirm that the new species matched the generic aspects of those descriptions. The cuticular sculpturing terminology utilized in this revision follows Harris (1979). Morphological terms for body structures as well as venation are a variation of the Comstock-Needham system that was used by Sharkey and Wharton (1997, Fig. 15). Photos were taken with a Leica DFC425 digital microscope camera mounted on a Leica M205 stereomicroscope, (Wetzlar, Germany). The LAS (Leica Application Suite) multifocus module integrated within the Leica microscope was used for taking the pictures. The stack of images at different focus positions was processed with Zerene Stacker version 1.04 (http://zerenesystems.com/cms/stacker).

Wilkinsonellus Mason, 1981
http://species-id.net/wiki/Wilkinsonellus Type species: Apanteles iphitus, Nixon 1965 Diagnosis. Wilkinsonellus can be differentiated from other Microgastrinae genera by the combination of the following characters: body coloration largely yellowish ( (Whitfield 1997). . General body pale yellow, except posterior half of hind coxa with an infuscated ventral band (Fig. 1I). Flagellum, trochanter, trochantellus, apex of both femur and tibia brown, hind tarsi, and tarsal claws of all legs completely brown. Scape and pedicel yellow-brown. Eyes silver mottled with gray, ocelli silver (Figs 1A-C, L). Membrane and microtrichiae of both fore and hind wings light brown (Figs 1A, L).
Legs (Figs 1A, I, L, O-R). Hind coxa surpassing apex of tergite III (Figs 1A, L, Q-R), outer dorsal surface of hind coxa delimiting an area surrounded by a strong longitudinal carina running from base to apex, but last third apically the carina turns inward (Fig. 1Q); that area with rugulose punctuations and with an extra strong basal carina inclined and reaching only the first third basally; hind tibia with outer spur half as long as inner spur (0.34:0.66 mm); inner spur more than half as long as hind basitarsus (0.66:0.90 mm) (Fig. 1P); hind tibia and hind tarsi both with spines throughout, hind tarsal claw with a short comb (Fig. 1O).
Etymology. This species is named in honor of Dr. M. Alex Smith of the University of Guelph, Canada, in recognition of his decade of deep intellectual, laboratory and logistic support for the DNA barcoding of the parasitoid wasps and flies of ACG.
Distribution. The species is only known from the original rain forest collection site, Sector Rincon Rain Forest, in Área de Conservación Guanacaste in northwestern Costa Rica. In 1999, ACG was inscribed as a UNESCO World Heritage site containing the best-preserved and regenerating dry forest habitats from Central America to northern Mexico.
Host. Wilkinsonellus alexsmithi has been reared from the leaf-roller Microthyris prolongalis, Crambidae (Figs 2A, C-D) three times, while feeding on the rain forest leaves of Ipomoea phillomega or sweet potatoes I. batatas (Convolvulaceae) (http://janzen. bio.upenn.edu/caterpillars/database.lasso). The larva of M. prolongalis lives inside of the leaf roll that it constructs, eating leaf tissue there. It is therefore likely that oviposition takes place through the leaf into the moth larva. The wasp cocoon (Fig. 2B) is lightly silked to the inner wall of the leaf roll and the larva dies at about the time that the wasp larva exits the cadaver.
Comments. The last three antennal segments are missing from the holotype. W. alexsmithi is a parasitoid of a crambid leaf roller larva, Microthyris prolongalis (Crambidae). In ACG, this moth larva feeds only on Convolvulaceae (410 rearing records, Janzen & Hallwachs 2009a). Within the subfamily Microgastrinae besides Wilkinsonellus, members of two other genera, Apanteles and Diolcogaster, are parasitoids only on this species of moth. The taxonomic range of insect parasitoids that use M. prolongalis as a host entails two insect orders, Hymenoptera and Diptera. Within Hymenoptera the chalcidoid family Encyrtidae (genus not reported), and two additional subfamilies of Braconidae, Orgilinae (Stantonia) and Agathidinae (Alabagrus maya) were reported; for the Diptera parasitoids, two genera of Tachinidae, Actia and Argyrophylax also parasitize this caterpillar   (Fig. 3E). Axillary trough of metanotum with a few striated grooves defined at least posteriorly (Fig. 3G). Body longer than fore wing (Fig. 3A).
Holotype male. Body length 4.30 (4.30-4.55 mm), fore wing length 4.15mm, hind wing length 3.59 mm. Coloration (Figs 3A-L). General body dark yellow; all legs yellow, except hind leg: coxa infuscated at the apex forming a ventral, wide brown band; apex of trochanter, and trochantellus, base of tibia and tarsi brown (Fig. 3A). Scape and pedicel brown both with thin apical yellow ring. Flagellum dark brown. Eyes and ocelli silver (Figs 3A-D, F.) Tergite IV and beyond mostly brown, but subapically and subbasally with a transverse yellow band (Figs 3I, L). Membrane and microtrichiae of fore and hind wing infusctate (Figs 3J-K).
Legs (Figs 3A,H,L). Hind coxa very long, reaching apex of tergite III (Fig. 3H), outer dorsal surface of hind coxa delimited by a strong carina, area coarsely rugulose and with a short, strong basal carina (Fig. 3H); hind tibia with outer spur more than half as long as inner spur (0.40:0.66 mm), inner more than half as long as hind basitarsus (0.66:0.88 mm) (Fig. 3A), hindtibia and tarsi with spines throughout.
Female. Unknown Etymology. From Kogui = jaguar in the Kogui language. The Kogui are indigenous in the Colombia Caribbean coast at the foot of the Sierra Nevada de Santa Marta, the highest coastal mountains in the world and not directly attached to the Andean mountain range.
Distribution. Colombia, from PNN Tayrona and PNN Utría, both being marine ecosystems protected by the Colombian government and belonging to the National Natural systems. Tayrona is located on the Caribbean coast in Magdalena Department, whereas Utría is located on Colombia's Pacific coast, in Chocó Department.
Host. Unknown Comments. Holotype lacks the last antennal flagellomeres. The specimens from Utría with antennae length = 4.8 mm, body length 4.3 mm. Last antennal flagellomere length = 0.35 mm, penultimate flagellomere antennae length = 0.30 mm. Male from Chocó shows hind legs with the same pattern of coloration but darker and Tergite VI and beyond with brown spots (Fig. 3L). Diagnosis. Eyes and ocelli appearing reddish in preserved specimens (Figs 4A-D). Metasoma curve (Fig. 4J). Fore wing longer than body length. Vein 2M as long as (Rs+M)b. Scutellar sulcus with seven carinated foveae heterogeneous in size (Fig. 4G). Axillary trough of scutellum and axillary trough of metanotum both with complete parallel carinae (Fig. 4H).
Maxillary palps longer than labial palps (Fig. 4D). Distance between lateral ocellus and adjacent compound eye margin sub-equal in length to the diameter of the lateral ocellus (0.09:0.10 mm), distance between lateral ocelli shorter than diameter of lateral ocellus (0.06:0.10 mm) (Fig. 4D). Vertex narrow, medially with a smooth area, but laterally with small and sparse punctuations. Occiput slightly concaved with a short grove medially.
Mesosoma (Figs 4A-B, G-H). Mesosoma dorsoventrally convex (Figs 4A-B). Pronotum shiny, smooth, but curvature of pronotum with a deep grove. Mesopleuron convex, extended smooth except margins lateral and ventro-lateral that form a L-shaped region that possesses small, dense and homogeneous punctuations (Fig. 4B); mesopleuron with a deep dent just above of L-shaped area, demarcating the border of the area with elongate foveae (Fig. 4B). Mesosternum slightly flat with a median row of foveae. Metepisternum and metepimeron separated by a groove with several deep foveae throughout (Fig. 4B), metepisternum smooth and narrower than metepimeron, apical margin metepisternum just above hindcoxa outlined by a wide, flat carina (Fig. 4B). Mesoscutum as wide as head, with small, sparse and homogenous punctures. Notauli clearly impressed, but not reaching the transscutal articulation (Fig. 4G). Scutellar sulcus with seven deep, carinated foveae of heterogeneous size (Fig. 4G). Scutellum shiny with fine, sparse punctures and delimited by carina. Axillary trough of scutellum and axillary trough of metanotum both with vertical parallel carinae (Fig. 4H); space among ATM carinae wider than ATS carinae (Fig. 4H). Lunule of scutellum and medioposterior band of scutellum smooth and shiny (Fig. 4H). Medioposterior band of metanotum and medioanterior pit of metanotum forming a pentagonal-shaped delimited by carinae (Fig. 4H). Posterior rim of metanotum thin and smooth (Fig. 4H). Propodeum with a complete medianlongitudinal carina dividing the propodeum in two halves, each half with one additional carina that does not branch basally at the same point than median-longitudinal carina (Figs 4H-I); space between median and an additional carina wider as they become more distant from propodeal foramen (Figs 4H-I), and all three carinae crossed by transverse semicircular carinae, although apically less transverse carinae than basally.
Legs (Figs 4A, F, I-J). Hind coxa reaching apex of tergite III (Fig. 4I), outer dorsal surface of hind coxa with an area delimited by a strong longitudinal carina running from base to apex, but last third apically the carina turns inward (Fig. 4I); area surrounded by the carina with rugulose punctuations that are more visible in dorsal view, and with an additional basal carina which splits and runs only the first third basally (Fig. 4I); hind tibia with outer spur half as long as inner spur (0.36:0.72 mm); inner spur more than half as long as hind basitarsus (0.72:0.92 mm) (Fig. 4F); outer dorsal side of hind tibia moderately spinose (Fig. 4F).
Distribution. The species is only known from the original collecting site in Panamá. Host. Unknown.

Conclusions
Neotropical Wilkinsonellus range from 4.0 to 4.8 mm in length, excluding antennae, and all specimens were collected in lowland tropical rain forest 500 m.a.s.l. or lower in elevation. Palaeotropical Wilkinsonellus range from 2.5 to 4.8 mm in length, and occur at altitudes up to 1700 m.a.s.l. Wilkinsonellus has not been the only genus within Microgastrinae that was initially believed to be confined to a specific zoogeographical region. This is also true for Austrocotesia Austin and Dangerfield and Parapanteles Ashmead, each of which has turned out to have a much wider distribution. Austrocotesia was erected as a new genus in 1992. In that time, it was considered restricted to Papua New Guinea and the adjacent Australian region of North Queensland (Austin and Dangerfield 1992). However, the first two species from South America-Colombia and Ecuador-were described thirteen years later . Equally, Parapanteles was originally recorded only from the Australian and American continents. Ashmead proposed the genus in 1900. However, after little more than a century, Parapanteles was reported in South Africa -Western Cape province, Cederberg . Austrocotesia was also reported from the Neotropics at the same time as Wilkinsonellus in a chapter on Microgastrinae (Whitfield 1997) included in the Manual of the New World genera of the family Braconidae (Hymenoptera). Another genus mentioned in that manual was the Afrotropical genus Beyarslania, formerly known as Xenogaster (Koçak and Kemal 2009). However, those undescribed Neotropical Beyarslania species possibly belong to Mariapanteles, a genus recently erected (Whitfield et al. 2012). Thus, Beyarslania is still restricted to the Afrotropical region. In brief, all the new records point suggest that Wilkinsonellus as well as Austrocotesia and Parapanteles have a more extensively Gondwanan distribution.
The new distribution of Wilkinsonellus has been discovered thanks to large-scale rearing projects as well as arthropod diversity surveys undertaken in recent decades in the Neotropical region. The importance of rearing projects lies in the fact the associations across more than two trophic levels are possible. The identification of parasitized larval hosts implies the use of external morphological characteristics present on the larvae combined with the food plant and microlocation, because the caterpillar host is often destroyed when the larval parasitoid emerges. In contrast, specimens collected by malaise trap contribute mainly to inventory of biological diversity, except that most ecological information is lacking. Notwithstanding the current efforts, the extreme richness of the Neotropics means that both taxonomic and biological records continue to be highly incomplete (Whitfield et al. 2002) and more studies are considered necessary in order to improve understanding of the distribution patterns of the Neotropical Microgastrinae fauna. This situation also applies to other critical areas in the planet (i.e., Wallacea, New Guinea, Solomon Islands) that are still unexplored; consequently the understanding of the global distribution patterns remains incomplete.