Braconid parasitoids of ants (Hymenoptera, Braconidae, Euphorinae, Neoneurini) from Baltic amber with a discussion of records of fossil larvae parasitizing ant workers

A new record of the fossil genus Elasmosomites Brues, 1933 from Baltic amber with an illustrated description of a new species, E. arkadyleleji sp. nov. and the first description of a male of E. primordialis Brues, 1933 are provided. A key to currently known Neoneurini genera is compiled. A new record of fossil braconid larva emerging from an ant worker of Lasius schiefferdeckeri Mayr, 1868 is discussed and a correction to previous determination of ant hosts from amber is included.


Introduction
The subfamily Euphorinae is a very diverse group of mostly koinobiont braconid wasps featuring a wide spectrum of host orders and their immature stages used for infestation and development of both larval and adult stages within the host (Tobias 1965(Tobias , 1966Shaw 1985). The members of this group are relatively common in nature and this subfamily has almost cosmopolitan distribution.
Two interesting discoveries from Baltic amber include extinct taxa with unknown biologies (Tobias 1987). Oncometeorus aduncus Tobias, 1987 belonging to the tribe Oncometeorini Tobias, 1987 has an abnormally hook-like ovipositor. The tribe Prosyntretini Tobias, 1987 for Prosyntretus calcaratus Tobias, 1987 is only superficially similar to the tribe Syntretini, parasitoids of hymenopterous aculeates. Both these monotypic tribes are found exclusively in the Eocene.
This article provides an illustrated description of the female of a new species of Elasmosomites and the first description of the male of E. primordialis Brues from Baltic amber. It also gives details of the second known record of an amber inclusion of a braconid larva emerging from an ant worker, and provides the correct determination of its host.

Material and method
Baltic amber is the most famous and most diverse deposit of fossil insects of the Late Eocene age (33.9±0.1-37.2±0.1 Mya) (Rasnitsyn and Quicke 2002). The source of the resin for these fossils is believed to be mixed pine forests that covered the Scandinavian territory in the Eocene, with most of the collected material deposited along the eastern part of the south shore of the Baltic Sea.
During the present study, braconid specimens were examined using an Olympus SZ51 stereomicroscope. Photographs were obtained using a Canon EOS 70D digital camera mounted on an Olympus SZX10 microscope (Zoological Institute RAS, St Petersburg). A Leica M205C stereo microscope was used for photography and morphological analysis of the worker ant with an emerged larva. Subsequent image processing was performed using Helicon Focus Pro 7 software and Krita 4.4.2. Final plates were prepared in Adobe Photoshop CS6 and Inkscape 1.0.2. The X-ray micro-CT study of the ant with the emerged braconid larva was conducted at St. Petersburg University using a SkyScan1172 system. The specimen was CT scanned at 40 kV and 0.21 mA, generating a resolution of 1.99 μm pixel size and output of 2800 × 2800 pixels per slice. CT scan data were imported to the software Amira 8.0 (FEI-VSG Company) for interactive segmentation and 3D visualization.
The terminology employed for morphological features, sculpture and body measurements follows Belokobylskij and Maetô (2009). Wing venation nomenclature also follows Belokobylskij and Maetô (2009), with the terminology of van Achterberg (1993) shown in parentheses.
The material used for this study is deposited in the collection of the Kaliningrad Museum of Amber (KAM; Russia), except for the specimen with the parasitoid larva (MCZ:ENT:PALE-43618), which will be stored in the Museum of Comparative Zoology, Harvard University (MCZ, USA). Elasmosomites is a monotypic fossil genus of the euphorine tribe Neoneurini. The genus and type species were described by Brues (1933) on the basis of two female specimens from Baltic amber.
Key to genera of the tribe Neoneurini (Updated after van Achterberg and Argaman 1993, in which morphological illustrations for the all extant genera are present).

1
Antenna of female robust, with 12 segments (that of male with 14-15 segments). Scape shorter than pedicel; third antennal segment shorter than fourth segment. Nervulus (cu-a) and metacarp (1-SRl) of fore wing absent. Malar space short, about 0.3 times eye height. Face slightly convex, without facial tubercles and spines, its minimum width 0.8 times medial height with clypeus, 0.7 times eye height. Clypeus distinctly curvedly emarginated on lower margin, short, its width about 6.0 times maximum height medially, almost equal to width of face. Mandible and palps not visible. Face seemingly entirely smooth.
Antenna: Slightly thickened, 14-segmented, with short and rather sparse dark setosity, about 0.7 times as long as body. Scape 1.8 times longer than maximum width, about 2.2 times longer than pedicel. Length of first flagellar segment 3.7 times its maximum apical width, 1.3 times length of scape, 1.3 times length of second segment. Length of penultimate segment 1.9 times its width, 0.7 times length of acuminate apical segment.
Mesosoma: Length of mesosoma 1.6 times its maximum width. Mesoscutum 1.4 times wider than its median length, seemingly densely punctate with submedian longitudinal carina in posterior third. Prescutellar depression (scutal sulcus) relatively short, with at least two distinct submedian carinae and slightly sparsely and finely crenulate. Scutellum slightly convex, about as long as wide anteriorly, seemingly slightly punctate. Mesopleuron not visible. Prepectal carina seemingly absent. Mesosoma densely and finely granulate below.
Legs: Fore coxa subglobular. Trochantelli short and distinctly separated from femora on all legs. Fore femur rather wide, almost 3.0 times longer than wide, without tooth. Fore tibia without protuberances or processes, distinctly widened towards apex, its length 4.7 times maximum width. Fore tarsus slightly longer than fore tibia. Fore tibial spur almost straight, about 0.8 times as long as fore basitarsus. Tarsal segments of middle leg elongate. Hind femur wide, elongate-oval, about 2.8 times longer than its maximum width. Hind tibia distinctly widened towards apex, about 6.0 times longer than maximum width, 1.1 times longer than femur, 0.9 times as long as hind tarsus. Both spurs of hind tibia acute apically, longest spur about as long as hind basitarsus. Hind tarsus relatively slender, all its segments of similar width and not narrowed towards apex.
Metasoma: Almost twice longer than maximum width, 1.3 times longer than meso soma; its sculpture and pubescence not visible (dorsally covered by wings). First tergite distinctly widened towards apex; its median length about 0.8 times maximum distal width. Segments behind third one distinctly projected; any sutures, if present, not visible. Sternites with distinct median keel. Ovipositor relatively short, its sheath with dense short setae, 0.4 times as long as hind tibia, about 0.2 times as long as metasoma.
Colour: Body reddish brown, with a few dark spots, metasoma in posterior half pale brown. Fore and middle legs yellowish brown, hind leg reddish brown. Wings faintly and evenly infuscate; pterostigma dark brown medially and paler basally and apically.
Male. Unknown. Comparative diagnosis. The new species differs from the type species E. primordialis Brues, 1933, by having the body mainly reddish brown and metasoma in posterior half pale brown (entirely black in E. primordialis), antenna basally distinctly pale (entirely black in E. primordialis), fore and middle legs yellowish brown (almost black or dark brown in E. primordialis), length of first flagellar segment 3.7 times its maximum width (2.8 times in E. primordialis), recurrent vein (m-cu) of fore wing longer, about 1.5 times longer than second medial abscissa (2-SR+M) (shorter, not longer than second medial abscissa (2-SR+M) in E. primordialis).
Etymology. This species is named in honour of Professor Arkady Stepanovich Lelej, the well-known Russian hymenopterist, dedicated in celebration of his 75 th birthday.
Remarks. Most extant species of the tribe Neoneurini are predominantly black in colour, only sometimes legs entirely pale or with a contrasting black and white pattern. Elasmosomites arkadyleleji sp. nov. is the first species of the Neoneurini with mainly reddish brown body. Brues, 1933Figs 2, 3 Brues 1933Poinar and Miller 2002: 42;Yu et al. 2016.
Male (first description). Body length 2.5 mm, fore wing length 1.7 mm. Head: Short and transverse in dorsal view. Occipital carina laterally present but fine, below reaching lower margin of head, perhaps independently from hypostomal carina. Ocelli slightly enlarged, arranged in obtuse triangle with base about 1.5 times its sides. POL about 2.0 times OD. Eyes enlarged, slightly converging ventrally. Malar suture absent. Malar space short, about 0.5 times basal width of mandible. Face convex, without facial tubercles and bristles. Mandible rather long, narrow, slightly twisted, with two pointed teeth, upper tooth distinctly longer than lower tooth. Maxillary palpus rather long and slender, 6-segmented, its second segment slightly thickened. Labial palpus short, perhaps 3-segmented, but third segment very short and subglobular. Vertex and frons with distinct and dense transverse and slightly curved striae. Temple relatively wide and distinctly punctate.
Antenna: Relatively long, slightly thickened, 16-segmented, with short and very dense pale setosity, about 0.6 times as long as body. Scape longer than pedicel. Length of first flagellar segment 2.7 times its maximum apical width, 1.4 times length of second segment. Length of penultimate segment 1.7 times its width, 0.8 times length of acuminate apical segment. Mesosoma: Mesoscutum about 1.2 times wider than its median length, distinctly and densely punctate with very short and fine submedian longitudinal carina posteriorly. Prescutellar depression (scutal sulcus) relatively short, with several sparse crenulae. Scutellum slightly convex, weakly and sparsely punctate. Mesopleuron slightly convex, mainly smooth in the middle, with fine and sparse marginal punctation. Prepectal carina present and distinct laterally, absent ventrally. Prepectus distinctly rugulose-reticulate. Subalar depression slightly concave, narrow, areolate with oblique transverse striae. Posterior mesopleural suture shallow, narrow and distinctly crenulate. Mesosternal sulcus narrow but distinct and rugose. Metapleuron anteriorly with subvertical crenulate depression, mainly rugose-areolate and with additional reticulation; metapleural lower process short, wide and nearly round distally.
Legs: Fore coxa subglobular. Short trochantellus distinctly separated in all legs. Fore femur relatively narrow, 2.8 times longer than wide, apically without tooth. Fore tibia without protuberances or processes, distinctly widened towards apex, its length 5.0 times maximum width in lateral view. Fore tarsus about as long as fore tibia. Fore tibial spur slightly curved, 0.7 times as long as fore basitarsus and 0.2 times as long as fore tibia. Tarsal segments of middle leg elongate. Hind coxa dorsally slightly concave submedially, distinctly and densely punctate, evenly convex anteriorly. Hind femur wide, elongate-oval, 3.3 times longer than its maximum width. Hind tibia distinctly widened towards apex, 4.5 times longer than maximum apical width, 1.1 times longer than femur, about as long as hind tarsus. Both spurs of hind tibia acute apically, longest spur 0.8 times as long as hind basitarsus. Hind tarsus slender, all its segments of similar width and not narrowed towards apex. All tarsal claws slender and simple.
Metasoma: About 3.0 times longer than maximum width, almost as long as mesosoma and head combined, seemingly entirely smooth and bare, only with sparse short setae in posterior thirds of third and following tergites, with spiracles situated almost at the middle of lateral parts of tergites, without separated laterotergites. First tergite widened towards apex. Suture between second and third tergites absent. Median length of second and third tergites combined about 1.3 times distal width of third tergite. Segments behind third one distinctly projected.
Remarks. The differences between both known species of the genus Elasmosomites are given previously after the description of the new species.  Brues, 1933 (male, Baltic amber, # BX 9/21.2) A fore wing B hind wing C posterior margin of hind wing.

Discussion
The actual evidence of parasitic lifestyle of Hymenoptera is quite rare in the fossil record despite the rather numerous discoveries of the parasitoid adults. The first record of a hymenopterous parasitoid larva emerging from the metasoma of a worker ant (the phenomenon of imagobiosis : Shaw 2004) in Baltic amber was published by Poinar and Miller (2002). However, it is especially noteworthy that the emergence of an ant parasitoid from its host is a rarely observable phenomenon even among extant taxa (Shaw 1993;Gómez Durán and van Achterberg 2011). The previous study of the fossil parasitoid larva in general and its cephalic structure recognized this larva as a member of the genus Elasmosomites, perhaps E. primordialis, emerging from the host, a worker of probably Lasius schiefferdeckeri Mayr, 1868 (Formicinae), because this is one of the most common ants in Baltic amber (Poinar and Miller 2002).
However, our re-examination of the ant fossil presented in the publication by Poinar and Miller (2002) allowed us to re-determine the actual taxonomic status of this ant host. Based on morphological characters in the present photos and drawings (e.g., the general shape of the mesosoma, the shape of the propodeum, the short antennal scape and the shape of the clypeus) we state that this ant specimen belongs to Ctenobethylus goepperti (Mayr, 1868) of the subfamily Dolichoderinae. This is the most abundant ant species in the Late Eocene European amber, whereas L. schiefferdeckeri is also very common, although less than C. goepperti (Dlussky and Rasnitsyn 2009).
Almost 20 years after the publication of the article (Poinar and Miller 2002) with the first finding of an ant adult with a parasitoid larva emerging from it, Artur Michalski from Poland made a new find, which he kindly provided to us for study. In this amber inclusion the ant host undoubtedly belongs to Lasius schiefferdeckeri. This piece of Baltic amber includes the ant worker with an emerging hymenopterous larva with more than the half of its body visible from the lower posterior part of the ant's metasoma (Fig. 4). In this specimen, it is possible to study the larval body that is distinctly thickened submedially with six to eight visible segments and the head of the parasitoid larva clearly visible on the top of its most narrow distant part. Unfortunately, it is impossible to study the cephalic structure of this larva because this part of its body is poorly preserved in the amber, but based on the larva from the previous article (Poinar and Miller 2002) we suggest that it is also a member of Elasmosomites. However, species identification of the larva is very complicated because there are at least two species (if not more) of this genus.
Extant members of the tribe Neoneurini are mainly known as parasitoids of worker ants of the genus Formica Linnaeus, 1758, and more rarely of Camponotus Mayr, 1861, Cataglyphis Foerster, 1850, Lasius Fabricius, 1804 and Polyergus Latreille, 1804 (all from the subfamily Formicinae), whereas for fossil members of the genus Elasmosomites, hosts belonging to the genera Ctenobethylus Brues 1939 and Lasius are now known (subfamilies Dolichoderinae and Formicinae, respectively). This is the first record of the ant subfamily Dolichoderinae as the host of a Neoneurini braconid species.
We don't know much about hosts of this tribe, and the discoveries in fossils that allow us to establish a host-parasitoid relationship of extinct taxa are extremely rare. However, based on the existing two findings, and the available data on the hosts of extant species, it is possible to assume a wider range of hosts for the genus Elasmosomites during the Late Eocene. In addition to the two common species mentioned above, it could also be quite numerous in European Late Eocene amber representatives (as potential hosts) of the genera Formica (Formicinae) and Yantaromyrmex Dlussky & Dubovikoff, 2013 (Dolichoderinae), such as F. flori Mayr, 1868 andY. geinitzi (Mayr, 1868).
(Stockholm, Sweden) and Dr. Vladimir E. Gokhman (Moscow, Russia) for their very useful suggestions for the first version of the manuscript.
The photographs and micro-CT data of the fossil ant with a braconid larva were taken using the equipment of the Science Park of St. Petersburg State University (Resource Centers "Microscopy and Microanalysis" (project No. 112-11405) and "Centre for X-ray Diffraction Studies" (projects No. 103-21316 and 103-11527). The article was made with support from the Ministry of Science and Higher Education of the Russian Federation in accordance with agreement No. 075-15-2020-922 date 16.11.2020 on providing a grant in the form of subsidies from the Federal Budget of the Russian Federation. The grant was provided for state support for the creation and development of a World-class Scientific Center "Agrotechnologies for the Future" for DAD.