Revision of Paranastatus Masi (Eupelmidae, Eupelminae) with descriptions of four new species

Abstract Paranastatus Masi, 1917 (Eupelmidae, Eupelminae) was originally described based on two species from Seychelles: Paranastatus egregius and Paranastatus violaceus. Eady (1956) subsequently described Paranastatus nigriscutellatus and Paranastatus verticalis from Fiji. Here, four new species of Paranastatus are described: Paranastatus bellus Scallion, sp. n. and Paranastatus pilosus Scallion, sp. n. from Indonesia, and Paranastatus halko Scallion, sp. n. and Paranastatus parkeri Scallion, sp. n. from Fiji. A key to all Paranastatus species based on females is included and lectotypes are designated for Paranastatus egregius and Paranastatus violaceus. Finally, previously unobserved colour variation from newly collected material of Paranastatus verticalis, distribution patterns of species, and possibilities for future research are discussed.


Introduction
Paranastatus Masi, 1917 (Eupelmidae, Eupelminae) is one of 33 currently recognized genera within Eupelminae (Gibson 1995). This genus was initially established for two species based primarily on the distinctive triangular shape of the head of females. Four species have been described to date: P. egregius Masi, 1917 andP. violaceus Masi, 1917 from Seychelles, and P. verticalis Eady, 1956 andP. nigriscutellatus Eady, 1956 from Fiji (Masi 1917, Eady 1956. No new specimens of either P. egregius or P. violaceus have been reported since their original description and their biology remains unknown. However, O'Conner et al. (1955) and Rapp (1995) subsequently reared P. nigriscutellatus and P. verticalis from the eggs of the walking stick, Graeffea crouanii Le Guillou (Phasmatodea: Phasmatidae). Males are known only for P. egregius, P. nigriscutellatus, and P. verticalis. A key to these males was provided by Eady (1956).
Eupelmidae is likely a grade-level taxon (Gibson 1989) rather than being monophyletic (Heraty et al. 2013), though Eupelminae is supported as monophyletic (Gibson 1989). The subfamily is characterized in part by its extreme sexual dimorphism, and species and higher level taxonomy is based primarily on female morphology. Eupelmines are parasitoids or predators of eggs and primary or hyperparasitoids of other immature stages of various arthropods, including Blattaria, Diptera, Hemiptera, Hymenoptera, Lepidoptera, Mantodea, Orthoptera and Phasmida, as well as Araneae and even Pseudoscorpionida (Gibson 1995, Austin et al. 1998). Gibson (1995) hypothesized that Paranastatus and five other genera, including Anastatus Motschulsky, formed a monophyletic clade, though with unresolved relationships and with Paranastatus possibly rendering Anastatus paraphyletic. Like known Paranastatus, members of Anastatus are mostly egg parasitoids and have been recorded as endoparasitoids of the eggs of Phasmida (Gibson 1995).
More recent collections from the South Pacific revealed new specimens of Paranastatus, including what appeared to be undescribed species. The purpose of this study was to differentiate and describe these new species and provide observations on variation observed among new P. verticalis material. An illustrated key to the world species of female Paranastatus is also included.

Methods
Type material of P. verticalis, P. nigriscutellatus, P. egregius, and P. violaceus was examined as part of a loan from The Natural History Museum, London, England (BMNH). Paratypes of one female of P. nigriscutellatus and a male and female of P. verticalis were also examined on loan from the U.S. National Museum of Natural History, Washington, DC, USA (USNM). Other material was borrowed from the Canadian National Collection of Insects, Arachnids and Nematodes, Ottawa, ON, Canada (CNC). Some of the latter material was collected in projects requiring primary type material to be returned to the Bernice P. Bishop Museum, Honolulu, HI, USA (BPBM).
Two systems were used to image specimens, a Nikon D5200 camera mounted on an Olympus SZX16 stereomicroscope, and a Canon DSLR 7D Mark II camera with a MP-E 65mm macro lens attached to a motorized rail. Images were taken at multiple levels of focus, and stacked into a single image using the program Helicon Focus 6 (Helicon Soft Ltd, 2014). Images were processed and enhanced using Adobe Photoshop CC 2014. Scanning electron microscope images were obtained using a Hitachi Tabletop Microscope TM-1000. Measurements were taken using a Motic SMZ-168 microscope with an Olympus G10x micrometre eyepiece. Body length was measured in millimetres a total of three times and averaged. Excluding primary types, imaged specimens are labelled with a "JBWM Photo 2015-X" specimen number label. This is cited with other label data given for the respective specimen, in the Suppl. material 1: "Paranastatus Label Data", and in the figure captions.
Structure and sculpture terminology follows Gibson et al. (1997), but additional clarification on sculpture terminology is provided below. Images are provided for clarity where necessary in the keys and descriptions. Alutaceous and coriaceous are similar in that they both mean leather-like (Harris 1979). Here, alutaceous refers to sculpturing where fine grooves create elongated cells, whereas coriaceous refers to sculpturing where the fine grooves create more square but irregularly-shaped cells. Coriaceous-imbricate refers to cells that appear overlapping. Reticulate refers to cells that are delineated by ridges. Pustulate refers to a bumpy texture, whereas granulate refers to many fine bumps, like granules. Rugose means wrinkled, whereas rugulose means very finely wrinkled.
Facial structuring can be divided into the lower face (region below toruli to clypeus and between malar sulci), scrobes (depressions rising above toruli and joining anterior to frontovertex), and interantennal area (region between scrobes and toruli). Gena refers to the region delineated by the malar sulcus and occipital margin, and extends to halfway along posterior margin of the eye. The vertex lies between the eyes from the frontovertex to the posterior margin of the eyes, where the temple begins. The temple runs between the posterior occipital margin and eyes to the genae.
Colouration of the antennomeres is often a quick identifier to species because females of four species have unique antennal colouration, though females of two species have overlapping colour patterns and two have the same colour pattern. The sculpture of the mesoscutum in combination with the extent of its concavity can be used to separate species with similar antennal colouration.
Due to the number of specimens examined, paratype and other material label data has been condensed for a few species to include localities, dates collected, and collector. A number in brackets corresponds to the number of specimens from each locality. For verbatim label data, see the Suppl. material 1: "Paranastatus Label Data". A double line, ǁ, represents a new line on the label, and ++ represents a separate label.

Key to world species of Paranastatus Masi based on females
Note: When viewing mandible dentition, a dorsolateral view with the teeth directed forward is best for visualizing dentition (see Fig. 3).
Metasoma. Entirely coriaceous with white setae ventrally, the setae very sparse dorsally and long at apex of gaster.
Male. Unknown. Etymology. From the Latin bellus, meaning handsome, in memory of Melanie Scallion's dog Beau (French: handsome). This is an adjective in the nominative singular.

Paranastatus egregius
Distribution. Mahé Island, Seychelles. Biology. Unknown. Remarks. Masi (1917) established P. egregius based on one female and two males, but without designating a holotype. Of the three specimens, the BMNH only has the female and one of the males in its collection (Dale-Skey, pers. comm.). Here we designate the female as lectotype and the male as paralectotype, and have labelled the specimens accordingly. The location of the second male is presently unknown. Diagnosis. Females of P. halko are differentiated by the following combination of features: vertex granulate between ocelli and smooth posterior to ocelli; temple smooth; scape and pedicel blue (Fig. 8); mandible tridentate ( Fig. 1); mesoscutum smooth or very slightly rugulose.
Head. In lateral view, vertex distinctly convex between eyes, and temple sloping toward occiput to create a strongly obtuse angle (Fig. 8); vertex granulate between ocelli and smooth posterior to ocelli, sometimes appearing pustulate due to setae; temple smooth; gena coriaceous-imbricate to reticulate along malar sulcus (Figs 2, 8); face reticulate; frontovertex usually with blunt teeth projecting posteriorly towards vertex. Mandible tridentate, possibly appearing quadridentate in some views due to slight bump on ventral edge of large middle tooth (Fig. 1). Vertex and temple with evenly dispersed light brown setae; gena with brown setae, but with a patch of thick white setae on upper part of gena below eye; parascrobal region and region between toruli and clypeus with thick white setae; eye with short white setae; face otherwise with thinner brown setae.
Male. Unknown. Etymology. Named in honour of Ed and Eliz Halko from Winnipeg, Manitoba, Canada. Their daughter, Gail Halko, also from Winnipeg, has made a donation to the Wallis-Roughley Museum of Entomology at the University of Manitoba to honour her parents, who both celebrated their 85 th birthdays in 2015. This is a noun in apposition to retain integrity of the name Halko in the species name.
Distribution. Viti Levu, Fiji. Biology. Unknown. Remarks. Vertex may appear pustulate under a stereomicroscope due to the setae. Care should be taken when using antennal colouration as a guide to species since flagellomere 7 is sometimes completely brown instead of white at apex, thus resembling the antennae of P. verticalis. Other material. Females (11), dry pinned, deposited in BPBM and CNC. Collecting data for all specimens examined are listed below. However, date ranges are provided when multiple specimens were collected from the same locality with the full label data for each specimen listed in Suppl. material 1: "Paranastatus Label Data".
Distribution. Islands of Fiji, Tonga, Western Samoa (Noyes 2015 Diagnosis. The unique female of P. parkeri is differentiated by the following combination of features: vertex and temple smooth; frontovertex smooth with a few small bumps; face smooth to alutaceous; mandible quadridentate; mesoscutum smooth except faintly coriaceous in posteromedial concavity. Description. Female. Length: 2.2 mm. Colour. Head with vertex coppery between ocelli, metallic green to blue-purple posterior to ocelli; temple shining metallic green-purple dorsally to metallic blue-purple laterally; gena shining metallic coppery-green; entire face metallic dark purple-brown; frontovertex blue-green with brown centrally. Antenna with scape lightly shining green; pedicel, anellus (flagellomere 1), and flagellomeres 2-6 brown, 7, 8 and club white. Pronotum coppery-green; mesoscutum purplish-coppery, slightly bluish-green posteriorly; scutellar-axillar complex dull black; mesopleuron purple-coppery. Legs with procoxa dark brown, protrochanter light brown; mesocoxa light yellow-brown; metacoxa brown basally and white apically; remaining leg segments straw-yellow. Fore wing very lightly infuscate with hyaline band below distal half of submarginal vein; hind wing hyaline. Gaster green apically, tergites otherwise dark coppery-green and sternites brown. Colour of setae on various body regions discussed in appropriate sections below.  Head. Vertex and temple smooth; gena smooth to alutaceous along occipital margin; lower face smooth to alutaceous centrally, scrobes smooth to weakly alutaceous, interantennal area alutaceous; occipital margin straight in dorsal view; frontovertex smooth with a few small bumps. Mandible quadridentate. Entire head with sparse brown setae; eyes with sparse, very short white setae.
Metasoma. Entirely coriaceous with long, brown setae sparsely distributed. Male. Unknown. Etymology. Named in honour of Parker Brant, nephew of Barb Sharanowski, born November 2, 2012 in Australia to Julie and Billy Brant. This is a noun in the genitive case.

Remarks.
Abdomen was broken and lost after description and imaging had been completed. Antennae cannot be used as an identifying character in this species because the antennal colouration is the same as that of P. nigriscutellatus. Diagnosis. Females of P. pilosus are differentiated by the following combination of features: vertex granulate between ocelli, reticulate posterior to ocelli (Fig. 11); temple reticulate (Fig. 11); antenna mostly white except scape brown basally and club lightly darkened apically (Fig. 22); mandible tridentate; mesoscutum blue-purple medially, brown laterally, and reticulate (Fig. 9).
Colour. Head with vertex dull black-brown, sometimes purple-brown posterior to ocelli; temple dark blue-purple; gena blue-purple (Fig. 7); lower face mostly blue-purple but brown centrally below toruli; scrobes and interantennal area green or copperygreen; frontovertex dull black-brown or with blue centrally. Antenna white, except basal half of scape brown and very tip of club slightly darkened, and sometimes club completely white (Fig. 22). Pronotum metallic purple-blue, sometimes purple-brown laterally; mesoscutum blue-purple medially, brown laterally; scutellar-axillar complex dull black; mesopleuron purple. Legs with profemur white; mesofemur white with darkened posterior apical edge; metafemur white becoming yellow-brown apically; rest of legs white. Fore wing lightly infuscate in apical half, hyaline in basal half with small infuscate patch at base; hind wing hyaline. Gastral tergites 1-2 white, rest dark brown; gastral sternites 1-4 white, remainder purplish-brown. Colour of setae on various body regions discussed in appropriate sections below.
Metasoma. Entirely coriaceous with white setae evenly distributed ventrally, setae sparser and shorter dorsally, and longer at apex of gaster.
Male. Unknown. Etymology. From Latin pilosus-hairy, in reference to the females having noticeably more setae than the other species. This is an adjective in the nominative case.
Distribution. Seram Island, Indonesia. Biology. Unknown. Diagnosis. Females of P. verticalis are differentiated by the following combination of features: vertex raised between eyes, and temple flat such that temple and occiput form almost a right angle (Fig. 5); vertex granulate; lower face with fringe of setae below toruli (Fig. 6); mandible tridentate; mesoscutum smooth and convex or flat, not concave (Fig. 16). Males of P. verticalis are differentiated by the following combina-tion of features: vertex granulate; mandible tridentate; in dorsal view occipital margin deeply excavate; colouration darker than that of females.

Paranastatus verticalis
Distribution. Islands of Fiji, Tonga, Western Samoa (Noyes 2015). Biology. Parasitoids of Graeffea crouanii eggs. Variation. The two specimens collected in 2004 differ in several features compared to the type series described by Eady (1956). The specimen from Taveuni Island has a body length of 2.85 mm, whereas all specimens in the type series range from 2.4-2.5mm. Unfortunately, an accurate measurement of body length was not possible in the specimen from Vanua Levu because the body is contorted. The scutellar-axillar complex in the new material is dark black-brown (Fig. 18) not light orange-brown (Fig. 16), and the legs are darker than in the type series (Fig. 17). Other slight variations in colour include: temple green-purple laterally, not blue-purple; lower face metallic blue-green, not coppery-green; and pronotum purple-blue dorsally, not purple-coppery. The new material has gastral tergite 1 white, tergite 6 light orange-brown, and remaining tergites dark brown, not gastral tergite 1 white and remaining tergites light brown, or gastral tergite 1 white, tergites 2 and 3 dark brown, and remaining tergites grading to light brown at gastral apex. Masi, 1917 Figs 13, 25 Paranastatus violaceus Masi, 1917: 166-167. Material examined. Lectotype female, here designated; dry pinned, deposited in BMNH (Hym Type 5.1,036). Label data: "Silhouette, '08. Seychelles Exp. Percy Sladen Trust Exped. B.M. 1913-170." Diagnosis. Females of P. violaceus are differentiated by the following combination of features: vertex and temple coriaceous; antenna brown except flagellomeres 7 and 8 light yellow-brown (Fig. 25); mandible quadridentate; fore wing evenly infuscate (Fig.  25). Males unknown.

Paranastatus violaceus
Distribution. Silhouette Island, Seychelles. Biology. Unknown. Remarks. Masi (1917) established P. violaceus based on three females, one of which was stated as lacking its gaster. Of the three females, the BMNH only has one complete specimen in its collection (Dale-Skey, pers. comm.). We here designate this female as lectotype and have labelled it accordingly. The location of the other two females is presently unknown.

Discussion
During the last 100 years, Paranastatus has been recorded throughout the South Pacific and from one location in the Indian Ocean (Masi 1917, Eady 1956, Rapp 1995, O'Connor 1955. Paranastatus egregius and P. violaceus were described from Seychelles and additional specimens have not been reported since. This may be because these two species are extremely rare, now extinct, or most likely collecting efforts have been insufficient to recover them. Differentiating between these possible reasons would require more intensive sampling of biodiversity, an issue that is important worldwide because of climate change, habitat destruction, and species extinctions. Paranastatus nigriscutellatus and P. verticalis have been recorded subsequent to their description, mostly from the islands of Fiji, but also from Tonga (Rapp 1995) and only through rearing rather than collecting. Most of the new material described here was obtained through passive collecting by Malaise traps. This and other passive collecting methods may provide the best way to obtain specimens of Paranastatus other than through rearing.
Graeffea crouanii, the coconut stick insect, is a pest of coconut palms and is found on many islands throughout the South West Pacific, including Fiji (Deesh et al. 2013). Deesh et al. (2013) hypothesized that G. crouanii dispersed in one of three ways: (1) by eggs that fell into canoes from overhanging palms on the beach; (2) by eggs floating across the ocean to other islands because they are saline-tolerant; or (3) simply by the adults being carried on coconut leaves by humans to new locations. The dispersal of eggs of G. crouanii could account for the dispersal of P. verticalis and P. nigriscutellatus, suggesting that wherever this stick insect is found, these two species of Paranastatus could be found as well. Gibson (1995) proposed a hypothesis to explain dispersal ability of females in Eupelminae. Because of structural modifications to improve jumping ability, females appear to have reduced flight capabilities that reduce their ability to disperse. He used this hypothesis to explain why better known eupelmine species often have several to numerous hosts, it being advantageous to develop on a wide array of hosts within a limited dispersal area for survival of the parasitoid population. This suggests that species of Paranastatus could have a wider host range than is currently recorded.
The current known distribution of Paranastatus is perplexing because there are large distances between localities, which leaves the question of how the wasps dispersed through time. One hypothesis that could explain Paranastatus distribution is wind dispersal as aerial plankton. Insects have been collected far from land in both the Indian and South Pacific Ocean through aerial netting (Holzapfel and Harrel 1968), and since Paranastatus wasps are very small it is possible that they were carried across the ocean on the wind. Another hypothesis is that the wasps dispersed passively through their hosts, such as what may have occurred with parasitized Graeffea crouanii eggs (Deesh et al. 2013). If other hosts are discovered similar dispersal mechanisms might also be discovered. It is also possible that the true distribution of this genus has yet to be discovered. Because two of the new species as well as P. nigriscutellatus and P. verticalis are from Fiji, and the other two new species are from Indonesia, it is conceivable that more species of Paranastatus exist in other regions of the South Pacific and Indian Ocean.
Another possible explanation for the distribution of Paranastatus is that it was once larger than it is now. Paranastatus species are basically confined to geographical clusters: the Fijian species have not been found in Indonesia and vice versa. Paranastatus nigriscutellatus and P. verticalis have been found on several other South Pacific islands, but only east of Fiji. The two Seychelles species (P. egregius and P. violaceus) have never been found since their original capture. It may be that species of Paranastatus did exist in other regions, but have since become extinct. Extinction, if it has happened, may have occurred through habitat fragmentation that reduced the genus to its current number of species and localities. Fragmented habitats can lead to extinction by decreasing available habitat and causing smaller population sizes, and parasitoids tend to be more sensitive to habitat fragmentation than other trophic levels (Kruess and Tscharntke 2000).
It is interesting that the newly collected female specimens of P. verticalis are darker in colour than the original specimens collected in 1954. One possibility for this is that the type specimens have faded over the course of 50 years; however, this does not seem likely as the specimens still conform to Eady's (1956) description. The two newly collected specimens of P. verticalis are from localities east and west of the original type locality. The slightly more western specimen is from Bua province on Vanua Levu, whereas the eastern specimen is from Cakaudrove province, Taveuni Island. The type specimens were from Savusavu, Vanua Levu, which is centered between Bua province and Taveuni Island. It is possible that continual colour variation would be found across the entire distribution of this species, if sampled, or that females vary in colour pattern based on a specific niche, host, environment or some other difference affecting development.
One complication that arises when studying the taxonomy of Eupelminae is their extreme sexual dimorphism. Non-chalcidologists are likely to identify male eupelmines as Pteromalidae rather than Eupelmidae, and most identification keys to species of eupelmine genera are based only on females, which makes it difficult to correctly identify males unless they are reared together with females. There is a chance that Paranastatus males have been collected before, but were misidentified or unidentified. This may account for the lack of males recorded in this genus.
Future work on Paranastatus could include a closer examination of the biogeography of the different species. The disparate distribution between P. egregius and P. violaceus from Seychelles, and the remaining species from islands in the South Pacific, suggests that locations between these regions may have additional species of Paranastatus. Therefore, it could be worthwhile collecting in regions between Seychelles and Indonesia to improve knowledge of the distribution of the genus or to discover new species.
ting up with all sorts of questions from MLS. We are also grateful to the two reviewers for their valuable comments. This research was funded by the NSERC Discovery program from a grant awarded to BJS and a NSERC Undergraduate Student Research Award to MLS.