Review of the Swedish Enicospilus ( Hymenoptera ; Ichneumonidae ; Ophioninae ) with description of three new species and an illustrated key to species

The Swedish species of Enicospilus are reviewed. Three species are described from Swedish material; Enicospilus cederbergi sp. nov., Enicospilus intermedius sp. nov. and Enicospilus ryrholmi sp. nov. Four species: Enicospilus cerebrator Aubert, 1966, Enicospilus combustus (Gravenhorst, 1829), Enicospilus merdarius (Gravenhorst, 1829) and Enicospilus myricae Broad & Shaw, 2016, are reported from Sweden for the first time. An illustrated key to the Swedish species of Enicospilus is provided. Validity of the new species is supported by DNA barcoding.


Introduction
Enicospilus Stephens, 1835 is a genus within the subfamily Ophioninae which consists of large, testaceus nocturnal Ichneumonidae with large ocelli and long antennae.Enicospilus species in Northern Europe are distinguished from other Ophioninae by the narrow twisted mandibles and a large glabrous area in the fore wing discosubmarginal cell, often displaying one or two sclerites.The known hosts of European Enicospilus are Lasiocampidae and Noctuidae, feeding on shrubs and herbs (Broad & Shaw 2016).
In Sweden, a majority of the species of Enicospilus belong to the monophyletic Enicospilus ramidulus (Linnaeus, 1758) species group.This species group is defined by the combination of having both the proximal and central sclerites present and a longitudinal setose groove on the mandible.The presence of two pigmented sclerites in the fore wing is a feature also shared by the primarily tropical E. antefurcalis species group (Gauld & Mitchell 1981;Gauld 1988;Broad & Shaw 2016;Shimizu 2017).Due to some unfortunate misidentifications and synonymizations, the species within the E. ramidulus species group have been partly misinterpreted historically.Broad & Shaw (2016) revised the British fauna and resolved some of the taxonomic confusion, thereby preparing a foundation for further revisions.This paper, which treats the Swedish species of Enicospilus, including the description of three new species of the E. ramidulus group from Sweden, adds another piece to the Western Palaearctic puzzle.

Material and methods
Material of Swedish Enicospilus was mainly obtained from private butterfly collectors, the vast and longlasting survey with MV-light traps conducted by Nils Ryrholm and Clas Källander, and the Swedish Malaise trap project (SMTP).The author also studied specimens deposited in Biologiska Museet in Lund (MZLU) and Naturhistoriska Riksmuseet in Stockholm (NHRS).The material was sorted and clustered into morphospecies which were then compared to available and relevant type material.DNA barcoding of representatives for each morphospecies focused on the sequencing of a standardized 658 base pairs segment of the mitochondrial cytochrome c oxidase I gene (COI).This method has been proposed for species discovery, identification and delimitation (Hebert et al. 2003).The combination of classical morphometrics and barcoding has been shown to be a very accurate method in species delimitation (Schwarzfeld & Sperling 2014) as COI analysis alone is not sufficient to exclude distorting factors such as hybridization and high intraspecific variation (Funk & Omland 2003;Klopfstein et al. 2016).The barcodes are stored in BOLD; BIN-codes, as well as specimen codes (STI:NJBC"X") are given in the text.Morphological terminology (Fig. 1) follows Gauld (1988Gauld ( , 1991) ) and Broad & Shaw (2016).Photos of the habitus lateral and head in anterior and lateral view were taken by the author using an Olympus OMD M10 and an Olympus M. Zuiko 60mm/f.2.8 macro lens.All other photos were taken by Krister Hall using a Canon eos 5dr with a Mitutoyo 5x Plan Apo Infinity corrected/Mitutoyo 10x Plan Apo Infinity corrected lens (microscopic pictures) and a Canon mp-e 65mm f/2.8 1-5x for pictures of the wing details and stacked using Zerene Stacker, except Fig. 2A

Diagnosis
Enicospilus cederbergi sp.nov.(Fig. 2A-B) can be distinguished from other members of the E. ramidulus species group based on the relatively stout habitus and legs, the short but numerous flagellomeres, the slightly sinuate Rs&M-vein, the infuscate and thickened veins in the fore wings and the infuscate margins of the pterostigma.Specimens with an infuscate tip of the metasoma are most likely to be confused with E. ramidulus Linnaeus, 1758, while the thicker antennal segments are reminiscent of E. cerebrator Aubert, 1966.Also similar and probably closely related to E. intermedius sp.nov.but distinguishable from that species on the stouter and less numerous flagellomeres.

Description Female
Body length 18-20 mm.Fore wing length 13-15 mm.Number of flagellomeres 58-61 (mean 59.5).Mandible strongly twisted with upper tooth two times as long as lower tooth.First flagellomere relatively stout, about 3.5-4.0times as long as apically wide.Mid-and subapical flagellomeres about 1.5 times as long as wide (Fig. 3C-D), slightly longer than average in E. cerebrator.Head in dorsal view always with distinct gap of about 0.2 times ocellar diameter between inner orbit of compound eye and lateral ocellus (Fig. 5B).Temples buccate, in dorsal view curved, rounded immediately behind eye, usually distinctly wider than in E. adustus (Haller, 1885) and in lateral view about 0.7 times width of compound eye.Occipital carina conspicuously curved before indicated junction with hypostomal carina.Indicated angle between occipital carina and hypostomal carina slightly acute or right angled (as in Fig. 6A).Clypeus apically truncate, moderately convex, in lateral view almost right angled, sparsely punctate, interstices shining.Mesopleuron closely punctate on a polished background, centrally becoming more rugose, intermixed with transverse striae as in typical specimens of E. adustus but interstices between punctures normally wider, about equal to diameter of punctures.Epicnemial carina ventrally complete, sinuate, often indistinct or absent dorsally well before indicated joint with propleuron.Mesoscutum with notauli weakly indicated anteriorly, entirely closely punctate.Scutellum with lateral carinae, its surface with dense punctures, punctures larger than on mesoscutum.Sides of scutellum rather parallel,  apically and proximally wider than in E. ramidulus.Sclerites in fore wing (Fig. 4B) reminiscent of other species in the E. ramidulus group.Proximal sclerite entirely pigmented, approximately triangular with rounded anterior angle, central sclerite semi-circular, pigmented distally, fading to unpigmented proximally.Distal sclerite generally more prominent than in other Swedish species of the E. ramidulus group, represented by a relatively distinctly pigmented crescent along distal margin of fenestra.Fore wing veins thickened, black or dark brown.Pterostigma centrally pale brownish with margins more or less infuscate.Vein Rs&M distinctly sinuate, conspicuously bent at least in lower third (Fig. 4B).Vein Rs+2r strongly thickened, distinctly sinuate.Propodeum with anterior transverse carina strong, anterior of carina rather densely punctate, posterior entirely reticulate-rugose often with faint longitudinal striae centrally.Legs usually thicker than in other members of E. ramidulus group.Hind femur about 7-8 times as long as wide.Hind metatarsus about 10 times as long as wide.Hind claws short, more strongly curved than in E. adustus but significantly less than in E. ramidulus.

Male
Size, structure and colour as in female but generally with more flagellomeres (63-64) and striation on mesopleuron feebler in studied specimens, basically absent medio-ventrally.Parameres long, in lateral view reminiscent of E. adustus (Fig. 7B).
Based on the limited material of males and the fact that the parameres often are slightly deformed due to storage and chemical treatment, no detailed description can be made of the shape of the genitalia at this stage.

Colour
Uniformly reddish brown.Inner and outer eye margins yellowish; mandibular teeth black.Metasoma usually with infuscation from 5th tergite onwards (Fig. 2), posterior tergites usually totally infuscate, black or dark brown.Ovipositor sheath of same colour as posterior metasomal segments.In some specimens infuscation of metasomal tip is partly reduced or absent.Antennae darker in apical half, terminal segment paler.

DNA barcode
The full DNA barcode sequences of five specimens of the Swedish E. cederbergi sp.nov.specimens are available at the BOLD systems database (www.boldsystems.org,BIN; BOLD:AAI5191).

Distribution
Enicospilus cederbergi sp.nov. is so far only known from Sweden.In the BOLD database there are also specimens from Germany and Israel that seem to share the genotype, but these specimens have not been studied and might refer to E. intermedius sp.nov.In Sweden it seems to be rare but widespread and known from the southern and central parts of the country (Skåne, Småland, Öland, Uppland).

Phenology
The dates on the labels of the type series indicate that the species occurs mainly during July.

Ecology
No detailed information on the biology of E. cederbergi sp.nov. is known.The habitat of known localities ranges from coniferous to deciduous forests in coastal areas, as well as an inland pine bog and semi-open calcareous heathland.

Diagnosis
Enicospilus intermedius sp.nov.(Fig. 8) is superficially similar to E. myricae Broad & Shaw, 2016, but it is separated from that species by the larger size, the more numerous flagellomeres and the wider head in frontal view.Also very similar to E. adustus but with face wider and head more buccate behind the eyes.The face is usually entirely testaceous without yellowish areas.The only barcoded specimen shows very little genetic differentiation from E. cederbergi sp.nov.(Fig. 10) and the two species share the same BIN.Besides the differences in colour between typical specimens, the two species are distinguishable by the shape and number of the flagellomeres.

Etymology
The species is morphologically intermediate in relation to E. adustus and E. myricae.Mandible strongly twisted with upper tooth about two times as long as lower tooth.First flagellomere slender, about 4.5-5.0times as long as apically wide.Mid-and preapical flagellomeres about 1.7 times as long as wide, shape of flagellomeres similar to E. adustus.Head in dorsal view usually with small gap of about 0.1 times ocellar diameter between inner orbit of compound eye and lateral ocellus, distinctly narrower than in E. myricae.Ocelli larger than in E. myricae.Temples buccate, in dorsal view curved, rounded immediately behind eye, distinctly wider than in E. adustus and in lateral view about 0.7 times width of compound eye (Fig. 9C).Occipital carina conspicuously curved before indicated junction with hypostomal carina.Indicated angle between occipital carina and hypostomal carina slightly acute or right angled (as in Fig. 6A).Clypeus apically truncate, moderately convex, in lateral view almost right angled, sparsely punctate, interstices shining.Mesopleuron closely punctate on a polished background, centrally becoming more rugose, intermixed with transverse striae.Epicnemial carina ventrally complete, sinuate, often indistinct or absent dorsally well before indicated joint with propleuron.Mesoscutum with notauli weakly indicated anteriorly, entirely closely punctate.Scutellum with lateral carinae, its surface with dense punctures, punctures larger than on mesoscutum.Sclerites in fore wing identical to E. adustus (Fig. 4D) and E. myricae.Proximal sclerite entirely pigmented, approximately triangular with rounded anterior angle, central sclerite semi-circular, pigmented distally, fading to unpigmented proximally.Distal sclerite quite strong and elongate.Fore wing veins and pterostigma testaceous.Propodeum with anterior transverse carina strong, anterior of the carina rather densely punctate, posterior entirely reticulate-rugose often with faint longitudinal striae centrally.Legs of same proportions as in E. adustus.

Male
Size, structure and colour as in female.Antenna with slightly more numerous flagellomeres than in the female (65-67).Parameres long, in lateral view reminiscent of E. adustus (as in Fig. 7B).Based on the limited material of males and the fact that the parameres are often slightly deformed due to storage and chemical treatment, no detailed description can be made of the shape of the genitalia at this stage.

Colour
Uniformly testaceous.Inner and outer orbits usually only slightly paler than face (one male from Croatia with the undulation of the inner margin of compound eye and outer margin yellow); mandibular teeth black.Apical metasomal segments sometimes slightly infuscate.Ovipositor sheath usually darker than apical metasomal segments.Antennae slightly darker in apical half, but not as distinct as in typical specimens of E. myricae.

DNA barcode
The full DNA barcode sequence of one Swedish ♀ of E. intermedius sp.nov. is available at the BOLD systems database (www.boldsystems.org,BIN; BOLD:AAI5191).

Phenology
The dates on the labels of the type series range from late May to August.The main flight period is probably during June and July.In Southern Europe the species seems to be active during April and May.

Ecology
No detailed information on the biology of E. intermedius sp.nov. is known.The habitat in Sweden seems to mainly consist of semi-open xerothermic pine forests.

Etymology
The name ryrholmi refers to the lepidopterologist Nils Ryrholm who, by sorting out and donating a large portion of Ophioninae from decades of sampling with MV-light traps, has contributed greatly to the taxonomy and knowledge of Scandinavian Enicospilus.Öland, Mörbylånga, Karlsro, 56.579° N, 16.421° E, 13 Jul. 1978, sweepnet in daylight, S. Johansson leg. (MZLU Type no. 06123:10).

Description Female
Body length 16-17 mm, fore wing length 12-13 mm.Number of flagellomeres 51-56 (mean 54).Mandible strongly twisted with upper tooth distinctly longer than lower tooth, usually slightly more than two times as long as lower tooth.First flagellomere very slender, about 5 times as long as apically wide.Central-and subapical flagellomeres slender, 2.0-2.1 times as long as wide (Fig. 3G-H).Head in dorsal view with small gap of about 0.1 times ocellar diameter between inner margin of compound eye and lateral ocellus.Temples in dorsal view narrowed behind eye as in typical specimens of E. adustus and in lateral view about 0.5 times the width of compound eye.Occipital carina only slightly curved before junction with hypostomal carina (Fig. 6B), joint sometimes indistinct or absent.Indicated angle between occipital carina and hypostomal carina acute, about 45 degrees.Clypeus apically truncate, convex in lateral view, very sparsely punctate.Mesopleuron punctate, weakly sculptured or polished, central punctures intermixed with vague transverse striae.Distance between punctures about two times diameter of punctures.Speculum without punctures, strongly polished.Epicnemial carina between pleurosternal angles and sternal part almost straight (Fig. 12A).Mesoscutum with notauli faintly indicated anteriorly, very sparsely and vaguely punctate.Sides of scutellum strongly converging posteriorly.Proximal sclerite entirely pigmented, approximately triangular with rounded anterior angle.Central sclerite varying from almost unpigmented, reminiscent in shape of E. merdarius (Gravenhorst, 1829) but narrowly pigmented distally to more distinctly sclerotized and elongate.Distance between the proximal and central sclerites often smaller than in E. adustus and E. cerebrator (Fig. 4C).Fore wing veins thin, pale brownish.Pterostigma pale.Propodeum with anterior transverse carina strong, anterior of this rather densely punctate, posterior to this entirely reticulate-rugose often with faint longitudinal striae centrally.Proportion of legs as in E. adustus.Hind femur about 10 times as long as wide.Hind metatarsus about 12 times as long as wide.

Male
Size, structure and colour as in female.Parameres in lateral view short and obtuse as in E. ramidulus (Fig. 7A).

DNA barcode
The full DNA barcode sequences of four of the Swedish E. ryrholmi sp.nov.specimens are available at the BOLD systems database (www.boldsystems.org,BIN;BOLD:ADF8803).

Distribution
Enicospilus ryrholmi sp.nov. is so far only known from Sweden where it seems to be rare but widespread at least in the southern part of the country, including the Baltic islands Öland and Gotland (Skåne, Blekinge, Småland, Öland, Gotland).It can, however, be expected to occur over a wider geographical range.

Phenology
The species occurs in late summer.The documented flight period is July to August.

Ecology
No detailed information on the biology of E. ryrholmi sp.nov. is known.The habitat consists mainly of semi-open areas ranging from alvar to rocky slopes, sandy heaths, industrial landscapes and gardens.The known localities might suggest a host connected to plants depending on open sandy or rocky grounds.

Discussion
This study recognizes 11 species of Enicospilus occurring in Sweden (Fig. 10).Nine of the species belong to the E. ramidulus species group.The barcoding in combination with the morphological analysis indicates that E. merdarius is to be regarded of a member of the E. ramidulus species group despite the unpigmented central sclerite.The barcoding results within the aggregates consisting of E. myricae, E. adustus, E. combustus and E. ramidulus on one hand and E. cederbergi sp.nov.and E. intermedius sp.nov. on the other, seem to display very small genetic differentiation despite the species being morphologically as well as ecologically distinct (Fig. 10) and several species seem to share the same BIN.These results could indicate that the chosen sequencing method might not be suitable for detecting and genetically defining closely related species in this genus, but might also be caused by distorting factors such as hybridization or low intraspecific genetic variation in the markers chosen.A more extended barcoding project including more specimens from a wider geographical range and a deeper analysis of the barcoding methods will probably shed more light on the detailed relation between the species involved.Apart from the three species described new to science, four species -Enicospilus myricae, E. combustus, E. merdarius and E. cerebrator -are reported as new to Sweden.
When it comes to the species and their abundance and distribution in Sweden, E. cerebrator seems to be a very rare species with predominately southern distribution and confined to open or semi-open, often calcareous grasslands.Enicospilus combustus prefers similar habitats as E. cerebrator, but is slightly more common, especially on the Baltic islands Öland and Gotland.Enicospilus myricae is also rather rare, but slightly more widespread in Southern and Central Sweden and seems to occur in several different habitats ranging from semi-open calcareous grasslands to semi-open pine dominated forests.This shows that the species seems to have a much wider habitat preference than referred to in the original description (Broad & Shaw 2016) and probably can be expected to occur wherever the known host, the noctuid moth Orthosia gracilis, is present.Enicospilus merdarius is also a rather rare and local species known from a few coastal localities in Southern Sweden and on the Baltic island of Gotland.
Among the species previously recorded from Sweden, the distribution pattern for E. inflexus indicates that it is a rare but widespread species in Southern and Central Sweden.It shall hereby be noted that there are no Swedish records of the closely related E. undulatus (Gravenhorst, 1829).This rare parasitoid of Lasiocampa trifolii is known from Denmark and the Baltic countries and can be expected to occur in coastal heaths in Western Sweden where the host is quite common.Enicospilus repentinus is a widespread species in Southern and Central Sweden but rather rarely encountered.The remaining species, E. adustus and E. ramidulus, seem to be the most common, occurring all over Southern and Central Sweden in a wide variety of open and semi-open environments.
The dominance of the E. ramidulus species group in Northern Europe is interesting and might indicate that this is a predominately Palaearctic species complex.This is also indicated by the fact that the aggregate still holds several undescribed species in Central and Southern Europe.An ongoing wider revision of the Enicospilus of the Western Palaearctic will contribute to the knowledge of European Enicospilus and most likely reveal a significantly larger diversity than known today.
4. Antenna with central and apical flagellomeres very slender with central and apical segments more than 2 times as long as wide (Fig. 3G-H); number of flagellomeres 53-56; occipital carina only slightly curved before indicated junction with hypostomal carina (Fig. 6B); epicnemial carina between mesopleural angles and sternal part almost straight (Fig. 12A); central sclerite in glabrous area in fore wing usually more circular in shape, largely unpigmented; central and proximal sclerite closer to each other (Fig. 4C).Small species, fore wing length 12-13 mm ....... E. ryrholmi sp.nov.-Antenna with central and apical flagellomeres at most 1.8 times as long as wide (Fig. 3A-F); occipital carina distinctly curved before indicated junction with hypostomal carina, (Fig. 6A); epicnemial carina between mesopleural angles and sternal part sinuous (Fig. 12B); central sclerite in glabrous area in fore wing more semi-ovoid in shape, often elongate; central and proximal sclerites further apart (Fig. 4A-B

Fig. 1 .
Fig. 1.Morphological terminology referred to in the text.

Fig. 10 .
Fig. 10.Relationships among Swedish species of the genus Enicospilus according to their DNAbarcodes.The neighbour-joining phylogenetic tree was constructed on the basis of Kimura 2-parameter distances.All samples are from Sweden.The scale line indicates 1% sequence divergence.
Enicospilus ryrholmi sp.nov.can be distinguished from other members of the E. ramidulus species group by the position and shape of the central sclerite; the smaller size on average; the limited number of flagellomeres; the elongate central flagellomeres; the more or less straight mesopleural part of the epicnemial carina and the less curved lower part of the occipital carina.It is most likely to be confused with small specimens of E. adustus.