A revision of European species of the genus Tetrastichus Haliday (Hymenoptera: Eulophidae) using integrative taxonomy

Abstract Background The European species of the genus Tetrastichus (Insecta, Hymenoptera, Eulophidae, Tetrastichinae) are revised with 93 species, including 50 species described as new. The revision was conducted using an integrative taxonomic approach, based on DNA barcoding in combination with morphological characters. The Tetrastichinae are a biologically diverse and species-rich group of parasitoid wasps with numerous complexes of morphologically often very similar species that attack a wide range of hosts in over 100 insect families in 10 different orders. The genus Tetrastichus is, with almost 500 described species, the third largest genus of Tetrastichinae. Although biological information is lacking for most species, current data indicate that Tetrastichus species are gregarious koinobiont endoparasitoids developing on juvenile stages of mainly holometabolous insects. Due to their host specificity, several species of Tetrastichus are used as biological control agents. New information The European species of Tetrastichus Haliday (Hymenoptera: Eulophidae) are revised using a combination of externo-morphological and DNA barcoding data. This is the first integrative approach for any of the large genera of the Tetrastichinae. A total of 93 species are included, of which 50 are described as new: T. agonus sp. n., T. antonjanssoni sp. n., T. argei sp. n., T. argutus sp. n., T. asilis sp. n., T. ballotus sp. n., T. bledius sp. n., T. broncus sp. n., T. calcarius sp. n., T. calmius sp. n., T. clisius sp. n., T. cosidis sp. n., T. cumulus sp. n., T. cyprus sp. n., T. delvarei sp. n., T. doczkali sp. n., T. elanus sp. n., T. elodius sp. n., T. ennis sp. n., T. enodis sp. n., T. erinus sp. n., T. evexus sp. n., T. fadus sp. n., T. fenrisi sp. n., T. flaccius sp. n., T. gredius sp. n., T. iasi sp. n., T. illydris sp. n., T. incanus sp. n., T. inscitus sp. n., T. intruitus sp. n., T. johnnoyesi sp. n., T. lacustrinus sp. n., T. ladrus sp. n., T. lanius sp. n., T. lazius sp. n., T. lixalius sp. n., T. lycus sp. n., T. marcusgrahami sp. n., T. minius sp. n., T. mixtus sp. n., T. nataliedaleskeyae sp. n., T. nymphae sp. n., T. pixius sp. n., T. scardiae sp. n., T. splendens sp. n., T. sti sp. n., T. suecus sp. n., T. tacitus sp. n. and T. tartus sp. n. Two keys for the identification of species are presented, one for females and one for males. Based on DNA barcode sequences for 70 of the species, a Maximum Likelihood tree to assess phylogenetic relationships within the genus is presented. These 70 species are also characterised by a combination of CO1 and morphological data. The remaining 23 species, without a DNA barcode, are characterised by morphological data. Using a combination of data from the morphology and CO1 or morphological data only, the species are separated into three species groups (clito-, hylotomarum-, murcia-groups) with 41 unplaced species outside these groups. Hosts are known for 27 of the species and they are gregarious, koinobiont endoparasitoids on a wide range of immature stages of holometabolous insects and appear to be very host specific. The first host record for Lepidoptera (Tineidae) in Europe is included.


Introduction Tetrastichinae
The Tetrastichinae (Hymenoptera: Eulophidae) are one of the largest groups of parasitoid Hymenoptera. They are, with currently about 1650 species in 91 genera, the largest subfamily of the family Eulophidae, that include 297 genera and about 4500 species (Noyes 2020). Species of tetrastichines are found throughout the world and they occur in most habitats on Earth. The majority of species has been described and recorded from the temperate parts and mainly from Europe, but very little is known about this group from tropical areas. Thus, the recorded number of species is very probably a minor part of the actual number. Apart from species richness, it is also one of the biologically most diverse groups of parasitoids and the range of tetrastichine hosts comprise over 100 insect families in 10 different orders and, in addition, spider eggs and even (through their galls) mites and nematodes are targeted (LaSalle 1994).
The subfamily is a group of particular importance, even within parasitoid wasps that are generally essential for maintaining biological diversity in terrestrial ecosystems (LaSalle and Gauld 1991, LaSalle 1994, LaSalle and Gauld 1993. It is extremely species-rich and has been used in numerous ecological, biological and behavioural studies and several species have been successfully used as biological control agents (see Graham (1987), LaSalle (1994) and references therein). Despite their importance, virtually all genera lack comprehensive taxonomic revisions and the identification of species from most geographic regions is hampered by the lack of identification tools. Furthermore, the classification of Tetrastichinae has been subject to major re-arrangements throughout the history of the subfamily since it was established by A. Förster (Förster 1856) and is treated in further detail below for the genus Tetrastichus.
Taxonomists have largely ignored tetrastichines because they are taxonomically extremely difficult due to a large number of species and their morphological similarity. Both factors have been a major obstacle for taxonomic revisions, based on morphological characteristics. For the present study, we chose an integrative taxonomic approach, based on DNA barcoding combined with features in the external morphology. Barcoding allows for sequencing large numbers of specimens at a reasonable cost and it provides a standardised set of molecular characters that, together and in combination with morphological data, can be used to assess species boundaries. Examination of morphology allows the inclusion of species for which DNA data could not be gathered, for example, museum specimens of rare species that are often very old and pose problems for DNA sequencing.

The genus Tetrastichus
The genus Tetrastichus has been attributed to Haliday (1844), even though it was first described by Walker in 1842, but the Walker name has been suppressed by the International Commission on Zoological Nomenclature (1965). Haliday included a single species (Cirrospilus attalus Walker (= T. miser (Nees)) which then became the type species for Tetrastichus. The subsequent fate of Tetrastichus has been quite turbulent. Burks (1943) gives a detailed account of nomenclatural and classificatory events up to his publication. The history after 1943 is equally turbulent. Graham (1961) synonymised Tetrastichus with Aprostocetus Westwood (priority made Aprostocetus senior to Tetrastichus) and recombined all European species previously in Tetrastichus to Aprostocetus. Domenichini (1966) rejected this action by Graham, resurrected Tetrastichus and recombined all species back to Tetrastichus. Bouček (1977) published a key to all genera of Tetrastichinae and maintained the wide interpretation of Tetrastichus by Domenichini, as did Kostjukov (1977). Graham (1987) revised the classification of European genera in Tetrastichinae and kept Tetrastichus as a valid genus, but now in a very restricted sense, including only species in the so-called miser-group (sensu Graham  Table 1. Tetrastichus hosts conservative approach was followed in that the OTUs were regarded as conspecific until further evidence is available to assess their species status. In these cases, additional genetic markers are needed to disentangle complexes of closely-related species. In several species, the taxonomy has to rely on very few specimens, often with even fewer DNA barcodes and their species status will need to be evaluated as further specimens (and sequence data) become available. Furthermore, the number of species in Europe is probably much higher than currently known and there is, thus, a high probability that species of Tetrastichus that are not included in the present revision will be encountered in the future, in particular with material that is collected in areas other than the main study area in this article and in Graham (1991), i.e. Northern and Central Europe. For these reasons, DNA barcodes, in combination with a thorough morphological examination, will be essential for accurate species level identifications ofTetrastichus.

DNA sequencing
For DNA extraction, whole specimens were sent to the Canadian Centre for DNA Barcoding (CCDB) in Guelph, Canada, for DNA extraction and barcode sequencing and subsequent recovery of vouchers for preparation and morphological study. A complete list of voucher specimens included in the revision is given in Suppl. material S1. DNA extraction, PCR amplification and sequencing were conducted at CCDB, using standardised high-throughput protocols (Ivanova et al. 2006, deWaard et al. 2008. The 658 bp target region, starting from the 5' end of the mitochondrial cytochrome c oxidase I (COI) gene, includes the DNA barcode region of the animal kingdom (Hebert et al. 2003). The DNA extracts are stored at CCDB. Specimens that were successfully sequenced are listed in Suppl. material S1. All specimen data are accessible in BOLD as a single citable dataset (dx.doi.org/10.5883/DS-TTSEUR).
The data include collecting locality, geographic coordinates, elevation, collector, one or more digital images, identifier and voucher depository. Sequence data can be obtained through BOLD and include a detailed LIMS report, primer information and access to trace files. The sequences are also available on GenBank (for accession numbers, see Suppl. material S1).
Due to the expected presence of complexes of cryptic species, i.e. species that are morphologically virtually indistinguishable, all suitable specimens were subjected to DNA barcoding, even at the risk of obtaining a long series of the same species. The final BOLD dataset (DS-TTSEUR) contains 1,128 specimen records, 1,126 of which are associated with a DNA sequence. Amongst the specimens with a barcode sequence, 860 were assigned a Barcode Index Number (BIN) by the BOLD system, resulting in 73 species with one or more BINs. A total of 60 species were represented by a single BIN, 24 species with two BINs and one species (T. sinope) with three BINs (with a single specimen each).
The initial concerns with obtaining multiple sequences of the same species turned out to be unwarranted. A total of 22% of the species were represented by singletons, almost two thirds (63%) by 1-5 specimens and only two species with over 100 specimens each (Fig.  1). The species with the largest number of specimens in our dataset includeTetrastichus atratulus (178 specimens), T. halidayi (155 specimens), followed by T. miser (80 specimens), T. temporalis (62 specimens), T. lyridice (34 specimens) and T. leocrates (31 specimens). The remaining 67 species were represented by at most 25 specimens.

Data analysis
Sequence divergence statistics were calculated using the Kimura two parameter model of sequence evolution (Kimura 1980). Barcode Index Numbers (BINs) were assigned by the BOLD system, representing globally-unique identifiers for clusters of sequences that correspond closely to biological species (Ratnasingham and Hebert 2013). For BIN assignment, a minimum sequence length of 500 bp is required and sequences between 300 and 500 bp can join an existing BIN, but will not create or split BINs. In the present study, BINs were used to delineate Molecular Operational Taxonomic Units (MOTUs) prior to a detailed taxonomic study, based on morphological characters. Sequences were aligned using the BOLD Aligner (amino acid-based hidden Markov models). The analyses are based on sequences with a minimum length of 500 bp and less than 1% ambiguous bases. Genetic distances and summary statistics were calculated using analytical tools in BOLD and are given as mean and maximum pairwise distances for intraspecific variation and as minimum pairwise distances for interspecific variations.
For phylogenetic analyses, the COI sequences were aligned using Muscle (Edgar 2004). Maximum Likelihood (ML) trees were inferred using IQ-TREE, version 2.0.6 (Minh et al. 2020). The best-fit substitution models were inferred separately for the DNA sequence and the gap data partitions using ModelFinder (Kalyaanamoorthy et al. 2017) as implemented in IQ-TREE, prior to tree reconstruction. Branch support was estimated with ultrafast bootstrap estimation (Hoang et al. 2018) using 10,000 bootstrap replicates. The phylogenetic inferences were based on sequences with a minimum length of 500 bp, resulting in a dataset with 748 sequences.

Diagnosis
European species of Tetrastichus can be diagnosed by a combination of three features: submarginal vein in fore wing with one dorsal seta, mid-lobe of mesoscutum with at least three adnotaular setae on each side and propodeum with a ± complete lateral longitudinal carina that splits into two in posterior part (like an inverted "Y", Fig. 2c). In addition, most species have a strong exoskeleton, preventing specimens from collapsing when dried. This last feature is not unique to Tetrastichus, but in combination with the other three features, it helps when diagnosing this group.

Identification keys
Figs 2, 3: The identification keys are based on the two keys in Graham (1991), one for females and one for males, with the new species included and with some modifications of features included and order of couplets slightly changed. The main reason for keeping the sexes apart is that differences between species are frequently based on features unique to each sex (antennal characteristics and length of female gaster).
Since males are unknown for several of the species, the male key is distinctly shorter than the female key.
a b c d Figure 3.

Tetrastichus murcia group Diagnosis
Eyes with long setae (e.g. Fig. 7d); female antenna with relatively short flagellomeres and with a distinct clava (e.g. Fig. 5a); male flagellomeres 1-4 with an externo-dorsal, sub-basal compact whorl of long setae (e.g. Fig. 5d); setae along hind margin of pronotum and adnotaular setae on mesoscutum long and erect (e.g. Fig. 7a); body black non-metallic or with weak metallic tinges; female gaster circular or short ovate. a b c d Figure 6. sheaths reach apex of Gt or slightly beyond. Similar to T. atratulus, but with shorter antennal flagellum and longer ovipositor sheaths.
Colour. Body with weak coppery tinges, entire antenna dark brown, tegulae dark brown, wings hyaline with veins yellowish-white, coxae and femora black, trochanters dark brown, tibiae dark brown to black with apex yellowish-brown, tarsi yellowish-brown or dark brown.

Diagnosis
Female with antennal flagellum long, for example, F1 and F2 both 2.5× and F3 1.9× as long as wide and pedicel+flagellum 1.32× as long as width of mesoscutum; ovipositor sheaths not protruding beyond apex of Gt . Male with scattered setae and without externo-dorsal, sub-basal compact whorls of long dark setae on funiculars. Through the erect setae on vertex and erect adnotaular setae, similar to species in the murciagroup.

Diagnosis
Female with antennal clava (incl. spicule) 2.8× as long as wide; POL/OOL 2.0; distance between SMG 1.7× distance SMG to SLG; ovipositor sheaths not protruding beyond apex of Gt .

Notes
Holotype deposited in MZLU, paratypes in MZLU. Colour. Similar to female, but antennal scape dark brown.

Diagnosis
Female with distance between posterior ocelli relatively long, POL/OOL= 2.4; F1 1.7× as long as wide; distance between SMG 1.5× distance SMG to SLG; ovipositor sheaths reaching to, but not protruding beyond, apex of Gt . Male with antennal clava 5.3×, F4 2.3× and scape 3.2× as long as wide.

Diagnosis
Female gaster with ovipositor sheaths projecting distinctly beyond apex of Gt , length of projecting part about equal to length of hind basitarsus; setae on eyes very long, 0.8× OD; distance between posterior ocelli relatively short, POL/OOL= 1.5-1.6.

Diagnosis
Female with antennal funicle stout, F1 and F2 both 1.1-1.25× as long as wide, pedicel 1.35-1.5× as long as F1; mid-lobe of mesoscutum without a median groove or with the groove indicated only near mesoscutellum.

Material examined
Type material: holotype ♀ (NHM, type no. 5.3629).  Colour. Similar to female but scape always dark brown and tibiae brown in some specimens.

Notes
Holotype deposited in NHM.

Diagnosis
Body bright metallic and shiny; ovipositor sheaths retracted in dry specimens and do not reach apex of Gt (as in Fig. 2b); female gaster circular to short ovate; male flagellomeres 1-4 without externo-dorsal, sub-basal compact whorls of long setae (e.g. Fig. 15c); usually relatively large specimens.  Colour. As in female.

Diagnosis
Tibiae yellowish-brown; female with malar space 0.8× eye height, scape 3.5× as long as wide, antennal clava 3.4× as long as wide; mesoscutellum with ratio distance between submedian grooves to distance between submedian and sublateral grooves 1.4.

Distribution
Sweden.

Host
Gregarious endoparasitoid on Arge ustulata (L.) (Hymenoptera: Argidae), 14♀ and 3♂ have been reared from the same host specimen. The label information does not specify which stage of the sawfly that was parasitised. Another Tetrastichus species, T. Colour. Body metallic bluish-green, scape yellowish-brown, pedicel and flagellum pale brown, tegulae dark brown with metallic tinges, wing venation yellowish-brown, coxae and femora concolorous with body, trochanters dark brown, tibiae and tarsi yellowishbrown, T4 pale brown.

Notes
Holotype deposited in ZSM. a b Figure 16.

Host
Unknown.

Distribution
Sweden.

Notes
Holotype deposited in MZLU.

Host
Unknown.

Host
Unknown.

Diagnosis
Mouth opening very wide, 1.8-2.0 × malar space; body bright metallic blue to bluishpurple; ovipositor does not reach apex of Gt ; male funiculars with short whorled setae, 0.5-0.7 × as long as funicular attached to.

Distribution
France, Germany, Hungary, Italy, The Netherlands, United Kingdom (Graham 1991); Norway and Sweden (new records).

Diagnosis
Hind coxa with a strong, sharp and complete carina along posterior margin; malar space 0.9× eye height; antennal clava 3.9× as long as wide; mesoscutellum with ratio  distance between SMG/distance between SMG and SLG 1.4, length/width of enclosed space between submedian grooves 2.6.

Distribution
Sweden.

Notes
Holotype deposited in MZLU.

Host
Unknown.
Tetrastichus cumulus, sp. n. Colour. Body metallic blue-green, scape dark brown with base dark yellowish-brown, pedicel and flagellum dark brown, tegulae black with metallic tinge, wings hyaline with veins yellowish-brown, coxae and femora concolorous with body, trochanters black, tibiae yellowish-brown, fore tarsus dark brown, mid and hind tarsi yellowish-brown with T4 brown.
Variation. Colour of scape varies from completely dark brown to yellowish-brown with apicodorsal ⅓ dark brown, but most specimens with colour as holotype. Paratypes with body metallic blue or blue-green.  Colour. As in female, but scape completely dark brown. Body metallic blue-green or purple.

Diagnosis
Mid and hind tibiae yellowish-brown; mesoscutellum with distance between submedian grooves short, distance between SMG/distance between SMG and SLG 1.2; female with malar space 0.9× height of eye, antenna with F2 2.0×, F3 1.8× and clava 3.8× as long as wide; male antenna with scape 2.2× and clava 5.0× as long as wide and scape length 1.0× height of eye.

Distribution
Russia and Sweden.

Host
Unknown.
Colour. Body with metallic greenish-blue, entire antenna dark brown, tegulae black, wings hyaline with veins yellowish-brown to brown, coxae concolorous with body, trochanters dark brown, fore and mid femora dark brown, hind femur concolorous with body, tibiae and tarsi yellowish-brown.

Diagnosis
Similar to T. halidayi with the wide mouth and large and oddly-shaped mandibles (as in Figs. 2a and 65d). Differs from T. halidayi in having female gaster short ovate, 1.3× as long as wide, ovipositor sheaths short and not visible in dorsal view, cerci on gaster placed ventrally and not visible in dorsal view, setae on vertex shorter, 0.45× OD and body strongly metallic.

Distribution
Cyprus.

Notes
The specimens in the type series are not in pristine condition, all specimens being more or less broken and dirty. The holotype is missing right wing pair and right pedicel+flagellum is broken off and glued separately to the card.

Host
Reared from an unidentified Coleoptera and from an unidentified leaf miner on broad bean (Vicia faba L.).
Colour. Body with golden-green, scape yellowish-brown with apical ⅓ brown, pedicel and flagellum dark brown, tegulae black with metallic tinge, wings hyaline with veins brown to fuscous, coxae and femora concolorous with body, trochanters dark brown, tibiae yellowish-brown, fore tarsus brown, mid and hind tarsi yellowish-white with T4 brownish.
Variation. Body blue-green in paratype.

Distribution
Sweden.

Notes
Holotype deposited in MZLU, paratypes in SMTP.

Host
Unknown.
Colour. Body metallic blue-green, scape yellowish-brown with dorsal edge darker, pedicel and flagellum dark brown, tegulae black with metallic tinge, wings hyaline with veins yellowish-white, coxae concolorous with body, trochanters dark brown, fore and mid femora dark brown with metallic tinges and with apical ⅓ yellowish-brown, hind femur concolorous with body, tibiae yellowish-brown, fore tarsus dark brown, mid and hind tarsi yellowish-brown with T4 dark brown.

Diagnosis
Tibiae yellowish-brown; female with malar space 1.0× eye height; scape 3.6× and antennal clava 3.9× as long as wide; mesoscutellum with ratio distance between submedian grooves to distance between submedian and sublateral grooves 1.4. Similar to T. helviscapus, but with longer antennal clava in female, 3.9× as long as wide.

Distribution
Sweden.

Notes
Holotype deposited in MZLU, paratypes in MZLU and NHM.

Host
Unknown.

Distribution
Sweden.

Notes
Holotype deposited in SMTP.

Host
Unknown.

Material examined
Holotype

Diagnosis
Tibiae yellowish-brown; female with malar space 0.7× eye height; scape 4.0× and antennal clava 3.0× as long as wide; mesoscutellum with ratio distance between submedian grooves to between submedian and sublateral grooves 1.4 and enclosed space between submedian grooves 2.7× as long as wide.

Host
Unknown.

Diagnosis
Female antenna: F3 1.0-1.5× as long as wide, clava 2.6-3.0× as long as wide; male antenna: funiculars without an externo-dorsal, sub-basal compact whorl of long setae, F1 1.1-1.4× as long as wide and about as long as pedicel, distinctly shorter than F2; both sexes with mid and hind tibiae broadly infuscate, sometimes mainly black; body bright metallic green.

Distribution
Bulgaria, (former) Czechoslovakia, France, Germany, Hungary, The Netherlands, Russia, Sweden and United Kingdom (Graham 1991); Italy and Romania (new records). a b c Figure 28.
Colour. Scape dark brown. Otherwise similar to female.

Diagnosis
Mesoscutellum with submedian grooves diverging towards posterior part; female antenna with clava 3.3× as long as wide.

Distribution
Romania.

Notes
Holotype and paratype deposited in NHM.

Host
Unknown.

Diagnosis
Mesoscutellum 0.8× as long as wide, length/width of enclosed space between submedian grooves 2.6; mesoscutum bluish and mesoscutellum greenish; antennal clava 3.3× as long as wide.

Distribution
Germany and Romania.

Host
Unknown.

Diagnosis
Antenna with F1 0.8× as long as F2 and only very slightly longer than the pedicel, F3 1.9× as long as wide; tibiae yellowish-brown; body bright metallic green.

Distribution
United Kingdom (Graham 1991), France and Sweden (new records).
Variation. Paratypes with body metallic blue or blue-green. Colour. Similar to female, but with scape dark brown. Body metallic blue-green.

Diagnosis
Mid and hind tibiae yellowish-brown; mesoscutellum with distance between submedian grooves 1.6× distance between submedian and sublateral grooves; female with malar space 0.9× height of eye, antenna with F2 2.5×, F3 2.2× and clava 3.8× as long as wide; male antenna with scape 2.7× and clava 4.9× as long as wide and scape length 1.0× height of eye.

Distribution
Sweden.

Host
Unknown.

Diagnosis
Mesoscutellum with ratio length/width of enclosed space between submedian grooves 3.3; antennal clava 3.2× as long as wide.

Etymology
Named after John S. Noyes (NHM), collector of the type specimens.

Distribution
France and Romania.

Notes
Holotype and paratypes deposited NHM.

Diagnosis
Mandibles as in T. halidayi, i.e. mandibles very large with outer tooth falcate and separated by a wide gap from the two small inner teeth (as in Figs. 2a and 65d); mouth opening large, 1.9× as wide as malar space. Differs from T. halidayi in having a shorter female gaster with ovipositor retracted and apex not reaching apical margin of gaster. Similar also to T. cyprus, from which it differs in antennal characters as indicated in the key.

Etymology
Named after collector of type specimen, Marcus William Robert de Vere Graham. 7 7

Distribution
United Kingdom.

Notes
The head of the holotype is detached and glued separately on a card and the right antennal flagellum is missing (deposited in NHM).

Host
Unknown. green tinges and with apex yellowish-brown, fore tarsus dark yellowish-brown, mid and hind tarsi yellowish-brown with T4 dark brown.

Etymology
Named for the shiny appearance, from the Latin splendens = shiny.

Distribution
Sweden.

Host
Unknown.

Etymology
Named after acronym STI = Swedish Taxonomy Initiative, the major funding source for this project.

Distribution
Romania, Sweden and United Kingdom.

Notes
Holotype deposited in SMTP, paratypes in MZLU, SMTP and NHM.

Host
Unknown.
Variation. Three paratypes with entire antennal scape dark brown. Colour of body blue, blue-green or green-blue and small specimens with weaker metallic tinges on body.

Distribution
Sweden.

Host
Unknown. a b c Figure 37.

Tetrastichus clito group Diagnosis
Frons with a more or less distinct median longitudinal carina extending from between the toruli to near the median ocellus, the sutures which define the scrobal area laterally tend to diverge ventrally, away from the median carina (Fig. 3b); hind coxae usually finely reticulate on the externo-dorsal surface (Fig. 3d).

Diagnosis
Ratio POL/OOL 2.3; mesoscutellum with distance between SMG/distance between SMG and SLG 1.9; female gaster ovate, 1.7× as long as wide. See also key to distinguish from similar species.

Romania and Sweden.
Host: Unknown.

Notes
Holotype deposited in NHM, paratypes in MZLU, NHM and ZSM.  Colour. Body with weak metallic blue-green tinges, entire antenna dark brown, tegulae dark brown, wings hyaline with veins yellowish-white, coxae and trochanters dark brown, femora concolorous with body, fore tibia yellowish-brown, mid and hind tibiae dark brown, tarsi brownish.

Diagnosis
Body weakly metallic; scape, mid and hind tibiae dark brown.

Notes
Holotype deposited in ZSM. A second specimen of the species from the same locality is severely damaged and, therefore, excluded as a paratype. Colour similar to female, but body with weak metallic blue tinges.

Diagnosis
Female with antennal clava long, 4.2× as long as wide; gaster ovate, 1.4× as long as wide; male with scape 1.1× as long as eye and 2.1× as long as wide. See key for separation from similar species.

Notes
Holotype deposited in NHM, paratype in NHM.

Diagnosis
Female antenna short, at most 1.1× as long as width of mesoscutum; mesoscutellum with distance between submedian grooves 1.5× the distance between submedian and sublateral grooves; female gaster short ovate, 1.5× as long as wide.

Diagnosis
Antenna with F1 1.6× as long and 1.7× as wide as pedicel; mesoscutellum with distance between submedian grooves 1.6× the distance between submedian and sublateral grooves and with enclosed area between submedian grooves 1.9× as long as wide; female gaster elongate-acuminate, 2.2× as long as wide, ovipositor sheaths reach beyond apex of Gt .

Notes
Holotype deposited in MZLU. Colour. Body with weak metallic blue tinges, entire antenna dark brown, tegulae black, wings hyaline with venation yellowish-white, coxae and femora concolorous with body, trochanters dark brown, fore tibia yellowish-brown, mid and hind tibiae dark brown, tarsi brownish.

Diagnosis
Female gaster ovate, 1.6× as long as wide. See key for separation from similar species.

Distribution
Romania and Russia.

Notes
Holotype deposited in NHM, paratype in MZLU and NHM.

Diagnosis
Very similar to T. clito , female differs in having a longer and more acute gaster; hosts are different (Graham 1991).

Diagnosis
Mesoscutellum with distance between submedian groove 1.0× the distance between submedian and sublateral grooves and median part with weaker reticulation and more shiny than lateral parts; mesoscutum black with weak metallic tinges, mesoscutellum dark brown, dorsellum pale brown; fore tibia infuscate, mid and hind tibiae dark brown, fore tarsus dark brown, mid and hind tarsi dark brown with T1 yellowish-white.

Notes
Holotype deposited in SMTP. Colour. Body with weak metallic greenish-blue tinges, entire antenna dark brown, tegulae dark brown, wings hyaline with venation brown to fuscous, coxae and femora concolorous with body, trochanters dark brown, tibiae brownish, fore tarsus brownish, mid and hind tarsi yellowish-brown with T4 brown.

Diagnosis
Female gaster ovate, 1.4× as long as wide. See key for separation from similar species.

Notes
Holotype deposited in MZLU.
Tetrastichus lazius, sp. n. Colour. Body metallic blue, antenna dark brown, tegulae black, wings hyaline with venation yellowish-brown, coxae and hind femur concolorous with body, trochanters and fore and mid femora dark brown, tibiae pale yellowish-brown, fore tarsus pale brown, mid and hind tarsi yellowish-white with T4 pale brown.

Diagnosis
Mesosoma distinctly blue metallic. See key for separation from similar species.

Distribution
Italy.

Notes
Holotype deposited in UCRC.

Distribution
Sweden.

Notes
Holotype deposited in MZLU, paratypes in MZLU and SMTP.
Tetrastichus lycus, sp. n.  Colour. Body with weak golden-green tinges, entire antenna dark brown, tegulae dark brown, wings hyaline with venation yellowish-brown to brown, coxae and femora concolorous with body, trochanters dark brown, tibiae pale brown, fore tarsus brown, mid and hind tarsi yellowish-brown with T4 brown.

Diagnosis
Female gaster ovate, 1.5× as long as wide. See key for separation from similar species.
Host: Unknown.  Colour. Similar to female.

Remarks:
The type specimens are in poor condition, shrivelled and with parts missing: the holotype lacks antennal clava on both sides and left mid tibia and tarsus.

Diagnosis
Antenna with F1 1.3× as long as and 1.3× as wide as pedicel; mesoscutellum with distance between submedian grooves 1.7-1.9× the distance between submedian and sublateral grooves; female gaster short ovate, 1.4× as long as wide, with ovipositor sheaths reaching beyond apex of Gt .
Colour. Body with weak metallic blue tinges, entire antenna dark brown, tegulae dark brown, wings hyaline with venation yellowish-white, coxae and femora concolorous with body, trochanters dark brown, fore tibia yellowish-brown, mid and hind tibiae brownish, fore tarsus brown, mid and hind tarsi yellowish-brown with T3-4 brown.

Diagnosis
Female gaster ovate, 1.3× as long as wide. See key for separation from similar species.

Notes
Holotype deposited in MZLU, paratypes in MZLU and SMTP.

Taxon discussion
The following species could not be assigned to any of the existing species groups.

Description
See Graham (1991). The male is unknown.

Diagnosis
Gaster 2× as long as wide with Gt 1× as long as width at base. Similar to T. leocrates, but with longer gaster and longer antenna, as mentioned in the key.

Diagnosis
Very similar to T. miser, but the male differs in having whorled setae of F1-F4 reaching slightly beyond the tip of funicular attached to; female tentatively separated through characters mentioned in the key.

Distribution
Colour. Body with golden-green tinges, entire antenna dark brown, tegulae black, wings hyaline with venation dark yellowish-brown, coxae and femora concolorous with body, trochanters black, fore tibia dark yellowish-brown, mid and hind tibiae pale brown, fore tarsus dark brown, mid and hind tarsi yellowish-brown with T3-4 dark brown.

Diagnosis
Very similar to T. miser, differs morphologically in having a longer distance between lateral ocelli and eyes and in having a longer antennal clava.
Colour. Body with weak metallic bluish-green tinges, scape pale brown with dorsal edge dark brown, pedicel and flagellum dark brown, tegulae dark brown, wing venation yellowish-white, coxae concolorous with body, trochanters dark brown, fore and mid femora dark brown with apex yellowish-brown, hind femur concolorous with body, fore tibia yellowish-brown, mid tibia pale brown with apex yellowish-brown, hind tibia dark brown with apex yellowish-brown, tarsi with T1-3 yellowish-brown and T4 brown.

Diagnosis
Similar to T. agrilocidus, but differs in having a shorter gaster, 1.4× as long as length of mesosoma and with antennal flagellomeres shorter and clava longer.

Etymology
Named after the collector of holotype, Gerard Delvare (CIRAD).

Notes
Holotype deposited in NHM, paratype in ZSM.
Colour. Head and mesosoma black with golden-green tinges, gaster dark brown with metallic tinges, scape yellowish-brown with dorsal edge pale brown, pedicel brown with dorsal part dark brown, flagellum dark brown, tegulae dark brown, wing venation yellowish-white, fore and mid coxae dark brown and hind coxa concolorous with body, trochanters and femora dark brown, tibiae yellowish-brown, tibiae yellowish-white.

Diagnosis
Similar to T. agrilocidus, but differs in having a longer distance between posterior ocelli and with shorter F1 and F2.

Etymology
Named after the collector of the species, Dieter Doczkal (ZSM).

Notes
Holotype deposited in ZSM.

Diagnosis
Very similar to T. julis, female differs morphologically in having a longer distance between lateral ocelli and eyes and in having a shorter marginal vein compared to length of stigmal vein (see key).
Colour. Body dark brown to black with weak metallic tinges, antenna pale brown, tegulae dark brown, wings hyaline with veins pale yellowish-brown, coxae black with weak metallic tinges, femora and trochanters dark brown, fore tibia and tarsus infuscate, mid and hind tibiae pale yellowish-brown, mid and and hind tarsi white with T4 pale brown.

Diagnosis
Frons with interscrobal area as a wide carina reaching almost to median ocellus; propodeum smooth and shiny.

Distribution
France.

Remarks:
The left femora, tibia and tarsus of mid and hind leg are detached and glued to a separate card.

Notes
Holotype deposited in MZLU.

Diagnosis
Mouth opening very wide, 2.0-2.2× malar space; mandibles very large, with outer tooth falcate and separated by a wide gap from the two small inner teeth, which are subacute and closely approximated; female gaster relatively long, 1.8× as long as wide with ovipositor sheaths protruding beyond apex of Gt .

Description
See Graham (1991) and Fig. 67. a b c Figure 67.

Diagnosis
Female with antennal spicule 0.5× as long as C3 and scape longer than eye; mouth opening 1.14× malar space in both sexes.   (Fig. 74).

Diagnosis
Antenna with scape and pedicel yellowish-brown; frons with a median longitudinal carina that reaches above the middle of frons (as in Fig. 3b); thoracic dorsum strongly shiny with very weak reticulation; propodeal plicae missing in anterior half.

Diagnosis
The female is similar to the female of T. miser and difficult to separate by morphology; male antenna is without whorls of setae on F1-F4, which separates it from males of the very similar T. miser, in which males have these whorls.

Diagnosis
Eyes relatively large, separated by a distance of 1.1-1.3× the length of an eye and with narrow temples; antenna with clava 0.7-0.9× as long as F2+F3.

Distribution
The Netherlands and United Kingdom (Graham 1991).

Diagnosis
Fore wing with costal cell very narrow, 13-17× as long as broad; antenna with claval spine about 0.5× length of C3; scape yellow; body bright green to blue. Colour. Body dark brown to black, partly with weak metallic tinges, antenna dark brown, tegulae black with metallic tinges, wings hyaline with venation dark yellowish-brown, coxae, trochanters and femora concolorous with body, tibiae and tarsi dark yellowishbrown.

Diagnosis
Mouth opening 1.4× malar space; female flagellum short, for example, F3 1.1× as long as wide and clava 2.2× as long as wide; body dark brown black with metallic tinges. Similar to T. polyporinus, see key for characters to separate.

Notes
Holotype deposited in NHM, paratype in NHM.

Diagnosis
Similar to T. leocrates, but with gaster and Gt shorter; also similar to T. sinope, but with longer distance between submedian grooves on mesoscutellum and to T. ballotus, but with shorter antennal clava and shorter marginal vein in fore wing.

Etymology
Named after Natalie Dale-Skey, curator of the Hymenoptera section at the NHM, who collected one type specimen and for having been a great help with logistics at the NHM.

Distribution
Romania and United Kingdom.

Notes
Holotype and parytype deposited in NHM.

Description
See Graham (1991). a b c d Figure 83.

Diagnosis
Antennal scape, tibiae and wing veins dark brown to black; gaster lanceolate 2.4-2.9× as long as wide with Gt 1.1-1.5× as long as wide.

Distribution
France and United Kingdom (Graham 1991).

Diagnosis
Mouth opening 1.4× malar space; antenna with scape 1.2× as long as an eye, funiculars 1.5-1.6× as long as wide.

Tetrastichus polyporinus
MALE. There are four males in the type series, but all specimens are badly damaged and fragmented and all specimens lack pedicel+flagellum. The antenna holds several diagnostic characters and without them and, as the specimens are broken, it is not  possible to give a useful description. However, the male shares the same diagnostic features as the female, except characters in the antenna and it should be possible to recognise the characters through these.

Diagnosis
Posterior ocelli close, POL/OOL= 0.9; eyes small and malar space large, length of eye/ malar space = 0.9 in female, 1.0 in male; female antenna short, length pedicel+flagellum/width of mesosoma = 0.9, with F1 and F2 ± merged in most specimens (incl. holotype, only two of the paratypes have these flagellomeres separated); submedian grooves on mesoscutellum distinctly converging towards posterior part; mesoscutum and mesoscutellum with weak reticulation and shiny.

Distribution
Finland.
Host: Scardia boletella (Fabricius) (Lepidoptera: Tineidae). There is no information if all specimens in the type series are from the same host specimen, but as the identical barcode in all specimens indicates that they are from the same clutch (including 20♀ and 4♂), this is probably the case. Thus, this species is a gregarious endoparasitoid.

Diagnosis
Scape in both sexes with numerous (about ten) setae along frontal edge; female with a long antennal flagellum, pedicel+flagellum 1.6× as long as width of mesoscutum; ovipositor sheaths do not reach apex of Gt ; male antenna with scape and pedicel dark brown almost black, flagellum pale brown.

Host
Unknown.

Diagnosis
Female gaster very long, 2.9-5.0× as long as wide with Gt 1.6-2.5× as long as wide.
The only other species with this long gaster is T. legionarius, that differs from T. telon in having the body bright metallic bluish-green to green, antennal scape longer than eye and a longer flagellum, about 2.8× the length of scape (about 2.6× in T. telon) and F1 about 4× as long as wide (about 2.8× in T. telon).

Diagnosis
Female gaster long, 1.9-2.3× as long as wide, with Gt 0.9-1.0× as long as wide; female antenna with F1 2.6-2.9×, F2 2.1-2.8×, F3 1.9-2.4× as long as wide; male scape with ventral plaque 0.6-0.7× length of scape, whorled setae of funiculars reaching the tips of funicular attached to or beyond tips; eye height 1.3× malar space in both sexes; both sexes with relatively bright metallic green (usually) or blue (more seldom) colour.

Distribution
Sweden, United Kingdom (Graham 1991) and France (new record).

Description
See Graham (1991). The male is unknown.

Diagnosis
Similar to T. agrilocidus, but with female antenna shorter and stouter (Fig. 95). a b Figure 94.

Identification keys to females and males of European species of Tetrastichus
1 Females see Table 2 -Males see Table 3 1.
Frons without a narrow median longitudinal carina below the median ocellus (Fig. 3a); hind coxae strongly reticulate to rugulose on the externo-dorsal surface (Fig. 3c) 2 -Frons with a very narrow and distinct median longitudinal carina extending from between the toruli to near the median ocellus (Fig. 3b); hind coxae usually finely reticulate on the externo-dorsal surface ( Fig.  3d)

75
2. Head with mouth opening 2.0-2.2× malar space; mandibles (Fig. 3a) very large, with outer tooth falcate and separated by a wide gap from the two small inner teeth 3 -Mouth opening 1.8-2.0× malar space in coeruleus, which has mandibles less large, head and mesosoma dark to bright blue or bluish-purple; in all the other species the mouth is at most 1.5× the malar space (usually hardly wider than the malar space) 5
-Eye usually as long as or longer than malar space; submedian grooves on mesoscutellum usually ± parallel; antenna with distinct constriction between F1 and F2 6 Table 2.
Key to females of European species of Tetrastichus.

6.
Antenna (Fig. 88b): clava with a very long, slightly tapering spicule, the clava itself much longer than F2+3; flagellum stout; funiculars subequal in length or differing only slightly, all less than twice as long as broad; scape virtually as long as an eye and reaching level of vertex; mouth opening 1.4-1.5× malar space

T. setifer Thomson
-Antennal clava usually with a relatively short spicule, if approaching the length seen in T. setifer, then clava is not or hardly longer than F2+3 and either the flagellum is less stout or the scape is longer than an eye and reaches above the vertex; width of mouth opening variable 7 7. Width of mouth opening 1.3-2.0× malar space .8 -Width of mouth opening equal to or only slightly wider (at most 1.14×) than malar space 18 8. Gaster 2.4-2.5× as long as wide (Fig. 65) T. gredius sp. n.
-Gaster at most 2.1× as long as wide 9

T. brachyopae Graham
-Submedian grooves of mesoscutellum nearer to sublateral grooves than to each other; gaster with ovipositor sheaths not or hardly projecting beyond apex of gaster; antennal funicle less stout, funiculars on average longer, F1 1.7-2.5×, F2 1.7-2.5× as long as broad (antenna of T. antonjanssoni short, but with distance between submedian grooves 1.7× the distance between submedian and sublateral grooves)

29.
Ovipositor sheaths retracted and do not reach apex of gaster (as in Fig. 2b); gaster at most 1.6× as long as broad, usually subcircular sometimes short ovate; not longer, usually broader than mesosoma; eyes relatively small, usually separated by 1.6-1.8× their own length; antennal scape virtually as long as, or even slightly longer than an eye (in specimens having undistorted head the scape sometimes reaches slightly above the vertex); most species are strongly metallic (e.g. Fig.  27)

30
-Ovipositor sheaths reach apex of gaster or even extending slightly beyond it, so that their tips are visible in dorsal view (as in Fig. 2a); gaster variable in shape, rarely subcircular, usually ovate to lanceolate; eyes usually relatively larger; antennal scape usually shorter than an eye and not reaching above the vertex; species not as strongly metallic as in alternate 50 30. Antenna ( Fig. 31a) with F1 0.8× as long as F2 and only very slightly longer than the pedicel

T. inaequalis Graham
-Antennae with F1 0.9-1.2× as long as F2 and at least slightly longer than the pedicel 31 31. Antennal flagellum very long and narrow (Fig. 91e), 1.6× as long as width of mesoscutum; ventral margin of scape with more than ten setae

T. tachos (Walker)
-Antennal flagellum 1.0-1.4× as long as width of mesoscutum; ventral margin of scape with fewer setae, usually at most six 32

32.
Mid and hind tibiae predominantly to completely dark brown to black (Fig. 28a, b)

T. hylotomarum (Bouché)
-Mid and hind tibiae predominantly to completely yellowish-brown to pale brown 33

T. brevicalcar Graham
-Antennal clava shorter and more stout (Fig. 36a), 4.4× as long as wide, with a strong constriction between C1 and C2 T. sti sp. n.

T. telon (Graham)
-Gaster with last tergite very rarely 1.9× as long as broad, usually at most as long as broad, but if long and the gaster itself is almost as elongate as in telon, then either the body is brighter green to blue or the antennal clava is only about twice as long as broad 51

51.
Gaster lanceolate, at least slightly longer than head+mesosoma (Figs 73b, 93b); antennae with scape as long as or longer than an eye, in specimens with undistorted head reaching at least slightly above the vertex; pedicel 2.1-2.5× as long as broad; flagellum slender, at most slightly stouter than the pedicel; F3 1.9-2.8× as long as broad; clava (incl. spicule) 3.6-4× as long as broad, usually at least slightly stouter than F2+F3; malar space 0.8-1.0× length of eye; head at most 2.2× as broad as long; temples distinct, rounded; body relatively bright metallic green to blue (Figs 73, 93)

52
-Either the gaster is ovate and about as long as mesosoma, or the antennal scape is slightly shorter than an eye or does not reach vertex, the pedicel is relatively shorter and the flagellum is less slender; antennal clava (incl. spicule) usually at most 3× as long as broad; malar space usually shorter; head usually more strongly transverse and with temples very short; body colour sometimes with different colour/shine 53 52. Gaster (Fig. 73b) 2.6-3.3× as long as broad and nearly twice the length of mesosoma, last tergite 1.5-1.9× as long as broad; antenna ( Fig. 73a) with scape somewhat longer than an eye; funiculars more elongate, F1 4.1-4.3×, F2 3.2-3.9×, F3 2.8-3.1× as long as wide; head, pronotum, mesoscutum and mesoscutellum duller, their sculpture slightly stronger; body colour blue (Fig. 73b)

T. theoi Graham
-Not with above combination of characters: either head more transverse with temples shorter or submedian grooves of mesoscutellum less far apart or costal cell of fore wing broader or antennal scape mainly to wholly black or propodeum distinctly reticulate 66 66. Spicule on antennal clava 0.5× as long as C3 (Fig. 69d), scape 1.06× as long as an eye, reaching nearly to level of vertex, clava (incl. spicule) as long as F2+F3

T. melasomae Graham
-Fore wing with costal cell broader; antenna with claval spicule relatively shorter, at most 0.4× length of C3, scape often black or partly infuscate; body sometimes dark blue or olive-greenish 68.
Antenna (Fig. 77e) with pedicel+flagellum 1.3-1.4× width of mesoscutum; setae of flagellum long and standing out at a greater angle; clava with a distinct constriction between C1 and C2 and 0.9× as long as F2+F3; scape more or less testaceous; gaster (Fig. 77a, c) long-ovate, 1.6-2.0× as long as broad, usually slightly longer than mesosoma; last tergite usually as long as broad, occasionally slightly transverse

T. lyridice (Walker)
-Antennae with pedicel+flagellum 1.1-1.3× width of mesoscutum; setae of flagellum standing out less strongly and tending to be shorter; clava either with a very weak constriction between C1and C2 or else at least as long as F2+F3; scape often black/dark brown; gaster subcircular to ovate, 1.2-1.9× as long as broad, shorter than to slightly longer than mesosoma; last tergite usually at least slightly broader than long 69 69. Gaster 1.8-1.9× as long as wide, nearly or just as long as head+mesosoma, strongly acute at apex, Gt 0.8-1.0× as long as broad

70
-Gaster not more than 1.6× as long as broad, not longer than mesosoma, except in some sinope which has gaster only slightly longer than mesosoma and Gt 0.4-0.6× as long as wide)
-Antenna either with ventral plaque of scape relatively longer or, if short (0.5× length of scape), then scape not longer than an eye or pedicel+flagellum at most 1.9× width of mesoscutum; setae on front edge of scape often shorter; head usually with temples very short or extremely short, rarely 0.15× length of eyes; the head itself usually more transverse 8 7. Antenna ( Fig. 73d) with whorled setae of funiculars not reaching the tip of the segment attached to; length of longest setae on front edge of scape tending to be slightly greater than width of scape

T. legionarius Giraud
-Antenna ( Fig. 93c) with whorled setae of funiculars reaching level with the tip of the segment attached to or slightly beyond tip; length of longest setae on front edge of scape equal to width of scape

T. coeruleus (Nees)
-Antenna with length of whorled setae on funiculars at least 1.2× the length of the segment attached to; body not so bright metallic 10 10.
Length of whorled setae on funiculars at least 1.4× the length of the segment attached to (Fig. 86c); F4 2.0× as long as wide (Fig. 86c)

T. polyporinus Askew
-Length of whorled setae on funiculars about 1.0-1.2× the length of the segment attached to; F4 1.6-1.8× as long as wide -Antenna ( Fig. 88f) shorter, 1.4× as long as width of mesoscutum and F1 1.5× as long as wide

12.
Setae on vertex long (as in Fig. 5c), 0.8-1.0× OD; eyes in most species with relatively long and conspicuous pubescence (as in Fig.  7d), length of the setae 0.3-0.6× OD; body black with very weak bronze or bluish tints (Figs 4-14) 13 -Setae on vertex short, 0.3× OD; eyes with short or very short pubescence, length of the setae at most 0.25× OD; body sometimes with brighter metallic tints 20 13. Antenna with pedicel 1.25× as long as F1, the latter only 1.5× as long as broad

T. solvae Graham
-Antennae with pedicel about as long as or shorter than F1; F1 1.4-2.0× as long as broad 14 14.
Submedian grooves of mesoscutellum equidistant from each other and from sublateral grooves (as in Fig. 6a)

21.
Antenna with length of longest seta on front edge of scape 1.1-1.5× width of scape; C1and C2 each at most 1.7× as long as broad

22
-Antenna with length of longest seta on front edge of scape less than or hardly as long as width of scape; C1and C2 often relatively longer 23. Eyes small, separated by 1.6-1.85× their length; antennal scape as long as or somewhat longer than an eye, reaching above level of vertex -Eyes larger, separated by 1.35-1.5× their length; antennal scape either not quite as long as an eye or, if as long, then not reaching above level of vertex 25

24.
Antenna with scape about 1.25× length of eye; the whorled dark setae of each funicular segment reach to level of tip of the segment attached to; scape about 2.8× as long as broad; eyes separated by 1.85× their length

T. perkinsorum Graham
-Antenna ( Fig. 69e) with scape 1.14× length to an eye; the whorled setae of each funicular segment reach slightly beyond the tip of the segment attached to; scape 2.4-2.5× as long as broad; eyes separated by 1.6-1.65× their length T. ilithyia (Walker) 25.
Antenna with whorled setae of funiculars at most just reaching the tip of funicular attached to, scape 2.7-3.2× as long as broad

26
-Antenna with whorled setae of funiculars reaching slightly beyond the tip of funicular attached to, scape 2.4-2.8× as long as broad 27 26. Antenna ( Fig. 81d) with F1 not or hardly shorter than F2; clava with short spicule

Phylogenetic analysis
Of the 93 species, 70 were represented by sequences of at least 500 bp, and the sequences were used for assessing phylogenetic relationships within the genus. The Maximum Likelihood analysis resulted in trees with several distinct clades (Fig. 96), most of which were supported by bootstrap support values of 80% or higher, but only three of them can be defined unequivocally by morphological characters. These are the Tetrastichus hylotomarum group, the T. murcia group and -albeit with low bootstrap support, the T. clito group (Fig. 96).
Species of the T. clito group differ from otherTetrastichus species in having the frons with a more or less distinct median longitudinal carina that is extending from between the toruli to near the median ocellus, the sutures that define the scrobal area laterally tending to diverge ventrally and the hind coxa having relatively weak sculpture on the outer surface. These characters are shared with other species of the subfamily Tetrastichinae, for example, by species of Aprostocetus. Within the clito group, there are species with a protruding or a retracted ovipositor, with the latter also occurring in otherTetrastichus species, suggesting that it is homoplasious and that the "T. clito group" is nonmonophyletic. Species of the T. hylotomarum group are regarded as monophyletic, despite the disparate placement of T. brevicalcar in the tree, because it came out as monophyletic in other tree reconstructions. The group is morphologically characterised by the retracted ovipositor sheaths that do not not reach the apex of the gaster, gaster subcircular to short ovate and colour usually strongly metallic. Species of the T. murcia group can be recognised by the eyes usually with long, conspicuous pubescence, setae on hind margin of pronotum and adnotaular setae relatively long and suberect, gaster short ovate to subcircular and body black non-metallic or with weak metallic tinges.
The remaining 41, or about half of the 93 species recognised in the current study, were regarded as unplaced with respect to species groups, due to the absence of supporting morphological characters. We refrained from establishing species groups, based on molecular characters. Despite high bootstrap support values of a clade, based on mitochondrial COI providing support for its monophyly, additional markers, in particular from nuclear genes, are needed for the reliable assessment of phylogenetic relationships within the genus Tetrastichus. Maximum Likelihood tree, based on COI sequences of 70 species of Tetrastichus, with Diglyphus isaea as an outgroup taxon. Numbers above branches represent bootstrap support values (only values > 50% are shown). Triangles at terminal branches indicate the presence of two or more specimens for a species. The three species groups that are recognised, based on morphological characters (T. clito, T. hylotomarum and T. murcia groups), are highlighted by different colours.
The present analysis includes only species of the subfamily Tetrastichinae that were traditionally regarded as species of Tetrastichus, based on morphological characters. This genus is morphologically much more readily definable than some other genera of Tetrastichinae, in particular compared to the genus Aprostocetus that has been used as a "dump" for species that did not seem to fit elsewhere. European species of Tetrastichus can, amongst other characters, be recognised by having a single seta on the submarginal vein and by having branched (Y-shaped) carinae laterally on the propodeum. The morphologically-close genera Quadrastichus and Oomyzus have also one seta on the submarginal vein, but they lack the Y-shaped carina on the propodeum (but may have a simple longitudinal carina laterally). Females of Oomyzus differ from Quadrastichus by their antennae, with flagellomeres being short in Oomyzus and comparatively long in Quadrastichus. In Tetrastichus, there are species with females having short or long flagellomeres.
Whereas 46 of the 50 (92%) species described as new are associated (and can be identified) with a DNA barcode, from one third (14) of the 43 described species, no sequences could be obtained and their identification had to rely on morphological characters.