Species from the genus Paralipsis are obligatory endoparasitoids of root aphids in the Palaearctic. It is known that these species are broadly distributed, parasitizing various aphid hosts and showing great biological and ecological diversity. On the other hand, this group of endoparasitoids is understudied and was thought to be represented by a single species in Europe, viz., Paralipsis enervis (Nees). However, recent description of two new species indicated the possibility of cryptic speciation and recognition of additional Paralipsis species in Europe. In this research, Paralipsis specimens collected during the last 60 years from eight European countries, as well as one sample from Morocco, were subjected to molecular and morphological characterization. Newly designed genus-specific degenerative primers successfully targeted short overlapping fragments of COI of the mitochondrial DNA. Molecular analyses showed clear separation of four independent lineages, two of which are the known species P. enervis and P. tibiator, while two new species are described here, viz., P. brachycaudi Tomanović & Starý, sp. n. and P. rugosa Tomanović & Starý, sp. n. No clear specialization of the taxa to a strict root aphid host has been determined. The recognized mitochondrial lineages were distinct one from another, but with a substantial within-lineage divergence rate, clearly indicating the complexity of this group of parasitoids, on which further research is required in order to clarify the factors triggering their genetic differentiation. We reviewed literature data and new records of Paralipsis enervis aphid host associations and distributions. A key for the identification of all known Paralipsis species is provided and illustrated.

Cryptic speciation, molecular phylogeny, Paralipsis, Paralipsis brachycaudi sp. n., Paralipsis rugosa sp. n.

Parasitoid wasps from the subfamily Aphidiinae (Hymenoptera, Braconidae) attack various aphid phylogenetic lineages, exhibiting several specialized associations with their hosts (Gagić et al. 2016). Few parasitoid wasps are specialized to parasitize only root aphid species (Starý 1961). However, there is no substantial biological knowledge about these obligatory parasitoid species of root aphids, probably as a consequence of them being of little economic importance and difficult to access for sampling. Moreover, it is well known that the parasitoids of root aphids developed obligatory relationships with ants (Starý 1966, Takada and Shiga 1974, Völkl 1992, Völkl et al. 1996). Although, there are many examples of relationships between ants that collected honeydew from aphids and protected the aphid colony and parasitoid wasps, it seems that chemical mimicry plays a more important role in some parasitoids [e.g., Lysiphlebus cardui (Marshall 1896)] than behavioural mimicry (Liepert and Dettner 1993). However, parasitoid wasps from the genus Paralipsis Foerster, 1863 have developed species-specific relationships with ants attending root aphids (Starý 1966, Takada and Shiga 1974, van Achterberg and Ortiz de Zugasti Carrón 2016). The genus Paralipsis is а good example as case of specific obligatory parasitoids of root aphids in Europe and the Palaearctic (Figure 1). Until recently, Paralipsis enervis (Nees) was considered to be the only European species, while P. eikoae (Yasumatsu) was known as a Far Eastern species (Starý and Schlinger 1967). However, after examining two samples of Paralipsis from Spain and the Netherlands, van Achterberg & Ortiz de Zugasti Carrón (2016) described two new species on the basis of morphological characters, viz., P. planus van Achterberg and P. tibiator van Achterberg and Ortiz de Zugasti. It is known that the genus Paralipsis shows great biological and ecological complexity and diversity in view of their acceptance of various aphid hosts and also having a broad geographical distribution.

Figure 1. 

Forda sp. aphids colony on root Dactylis sp. with mummy of P. enervis.

Bearing in mind it was thought that the genus Paralipsis was represented by a single species in Europe, until additional two species were newly described recently, we started this research to address the possibility of cryptic speciation and recognition of additional Paralipsis species in Europe. Since rarely encountered, there is almost a complete lack of knowledge about morphology and reliable characters for diagnostics of these root aphid parasitoids.

A set of wasps collected during the last 60 years from eight European countries and Morocco were initially subjected to morphological characterization. In addition, DNA was extracted from available Paralipsis specimens to perform the amplification and sequencing of the mitochondrial DNA barcoding region of cytochrome c oxidase subunit I (COI). We developed DNA amplification protocol and designed new internal genus-specific degenerative primers in order to retrieve short overlapping COI fragments for molecular characterization of the wasps. Subsequently, we used an integrative approach analyzing the morphological and molecular results to recognize phylogenetic lineages and cryptic species within the analyzed Paralipsis specimens. Two new species in Europe were described. In addition, we reviewed the host aphids and distribution of associations for Paralipsis enervis. A new determination key including all previously known and two newly described species is provided and illustrated.

Barcoding fragments of COI were successfully recovered from 18 specimens. The material subjected to molecular analyses was of different ages in terms of the time passing between sampling until DNA extraction; several of the oldest had been preserved in collections for nearly 60 years. This probably caused DNA disintegration, which resulted in failed attempts to recover the barcoding region with the LCO1490/HCO2198 standard primer pair. The newly designed Paralipsis-specific primers made it possible through diverse combinations to retrieve short subsequences of different length and position from disintegrated DNA of archival specimens. Prior to molecular analyses, all the barcoding sequences were aligned and trimmed to the same length of 568 bp. Comparison of COI barcodes identified 14 haplotypes (PH1-PH14) distinguished by a total of 83 variable sites, of which 51 were parsimony-informative (Table 3). The phylogenetic relationship was inferred using the MP and ML methods, which resulted in trees sharing identical topology with no substantial differences in bootstrap support (Figure 4).

Figure 4. 

Phylogenetic relationship inferred using the maximum parsimony (MP) method. The consistency index is (0.533333), the retention index is (0.681818), and the composite index is 0.476540 (0.363636) for all sites and parsimony-informative sites. The MP tree was obtained using the subtree-pruning-regrafting (SPR) algorithm with search level 1, in which the initial trees were obtained by the random addition of sequences (10 replicates). The percentage of replicate trees in which >50% of the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches (in red color). Since the topology is identical, the bootstrap support of branches obtained by the maximum likelihood method is presented in black color as well. Barcoding haplotypes of the analyzed archival Paralipsis specimens are designated with codes from PH1 to PH14, species name, host aphid and host plant. Abbreviations for the countries of origin are as follows: GER – Germany; FRA – France; SRB – Serbia; LIT – Lithuania; CR – Czech Republic; MOL – Moldova; MOR – Morocco; and ESP – Spain.

Phylogenetic analysis showed molecular differentiation on the basis of COI barcoding fragments, with recognition of four distinct lineages. The first group includes seven haplotypes: PH1, PH2, PH4, PH5, PH9, PH11, and PH13, which morphologically correspond to the first known species in this genus and in Europe, P. enervis. The specimens were sampled from different aphid hosts (Forda, Aphis, Anuraphis, Dysaphis) in association with different plants originating from Serbia, Germany, France, Lithuania, and the Czech Republic. The average overall divergence rate between the haplotypes within this group was 1%, with distances ranging from 0.4 to 2.5% (Table 4).

The second lineage, a “Mediterranean” clade, includes haplotypes PH12 and PH14 from Spain, and haplotype PH10 from Morocco. The overall divergence rate within this group was 2.8%. Genetic distances show that the haplotype PH12 associated with Forda formicaria is intermediary, diverging from the haplotype PH10 from Aphis rumicis (2.4%) and from the haplotype PH14 associated with Forda formicaria (2%), while the genetic distance between the other two was 4% (Table 4). Haplotype PH14 belongs to the paratype specimen of the newly described species P. tibiator. On the basis of morphological examination, it can be concluded that the haplotypes PH12 and PH10 belong to P. tibiator, although with evident high intraspecific genetic diversity. These three specimens clearly differ from the other congeners in having an elongated flagellomere 1 (F₁) (the ratio between F₁ and F₂ is 1.3–1.4) and a large number of longitudinal placodes on F₁ and F₂ in males (5–6 in P. tibiator versus 0–2 in other Paralipsis).

The third distinct lineage on the phylogenetic tree consists solely of the haplotype PH7, with unknown host data. The single specimen available from Moldova is characterized by having a very rugose and irregularly carinated propodeum. It is described as the new species P. rugosa sp. n., clearly separated genetically, with average distance from the first, second, and fourth lineage of 7.3, 7.7, and 9.6%, respectively.

Three haplotypes (PH3, PH6, and PH8) originating from Brachycaudus sp. and Tetraneura ulmi aphid hosts from Central Europe (Czech Republic and Germany) are grouped within the fourth distinct lineage. The barcoding haplotypes differ in the range of 1.8 to 3.6%, with an average overall interlineage divergence rate of 2.8% (Table 4). Specimens of Paralipsis within this lineage are characterized by having a more elongated petiole and ovipositor sheath in comparison with other congeners, and are described as the new species P. brachycaudi sp. n.

The median-joining network recognized the same four distinct groups of mitochondrial haplotypes with a confidence limit of 95%: group 1 (P. enervis) – haplotypes PH1, PH2, PH4, PH5, PH9, PH11, and PH13; group 2 (P. tibiator) – haplotypes PH10, PH12, and PH14; group 3 (P. rugosa sp. n.) – the single haplotype PH7 from Moldova; and group 4 (P. brachycaudi sp. n.) - haplotypes PH3, PH6, and PH8 (Figure 5). Using maximum parsimony calculation, we determined that all haplotypes are connected with no ambiguities, and the median vectors representing either unsampled or extinct haplotypes. A significant number of mutational steps (up to 40) connecting the groups confirms clear separation of the lineages, which corresponds with high divergence rates between the groups (group 1 and group 2 – 5.2%; group 1 and group 3 – 7.3%; group 1 and group 4 – 5.9%; group 2 and group 3 – 7.7%; group 2 and group 4 – 7.5%; and group 3 and group 4 – 9.6%).

Figure 5. 

Median-joining network obtained for 14 Paralipsis COI barcoding haplotypes. Green circles represent group 1 (P. enervis), with haplotypes PH1, PH2, PH4, PH5, PH9, PH11, and PH13; yellow circles represent group 2 (P. tibiator), with haplotypes PH10, PH12, and PH14; the black circle represents the single haplotype PH7 from Moldova within group 3 (P. rugosa sp. n.); blue circles represent group 4 (P. brachycaudi sp. n.), consisting of haplotypes PH3, PH6, and PH8. Circle size reflects the number of individuals with that haplotype (not to scale). Red dots are median vectors. Black dots are mutational steps.

Descriptions of new species in Europe

On the basis of morphological examination of our available material from across Europe and the Mediterranean and using the COI mitochondrial barcoding marker, we confirmed the existence of the recently described Paralipsis species P. tibiator. In addition, two new Paralipsis species are described below.

Paralipsis brachycaudi Tomanović & Starý, sp. n.

http://zoobank.org/E2918E28-9DA3-41C1-8ABA-3546CF423793

Figures 6–14

Material

Holotype ♀, Czech Republic, Čejč, 28.V.1963, reared from Brachycaudus mordvilkoi Hille Ris Lambers on Echium vulgare L., leg. J Holman; deposited in the IECR collection, slide mounted.

Paratypes 2♀♀, Czech Republic, Prague, 26.IX.1960, reared from Brachycaudus ballotae (Passerini) on Ballota nigra L., leg. J Holman; deposited in the FBS collection, slide mounted. Germany, Erlangen, Nordbayern, reared from Tetraneura ulmi (L.) on Avena sativa L., leg. H. Zwölfer; deposited in the IECR collection, slide mounted.

Diagnosis

The new species morphologically resembles P. enervis in petiole shape, absence of longitudinal placodes from flagellomeres 1 (F₁) and 2 (F₂), and fore wing venation pattern. Paralipsis brachycaudi sp. n. differs from P. enervis in having a longer petiole (Figure 13), the ratio between petiole length and width at the spiracle level is 1.50-1.60 in P. brachycaudi sp. n., while in P. enervis it is 1.30–1.40; somewhat shorter F₁ and F₂ (Figure 7) (the ratio between length and width of F₁ and F₂ is approximately 2.00 in P. brachycaudi sp. n., as opposed to 2.20–2.30 in P. enervis); and a propodeum that is smooth with just a few rugosities at the side (Figure 9), while the propodeum in P. enervis sometimes possess rugosities in the central parts which indicate for the presence of a central areola. Additionally, F₁ and F₂ are light-brown to yellow in P. brachycaudi sp. n., while in P. enervis only half of flagellomere 1 is yellow and the remaining parts of the flagellomeres are brown. The ovipositor sheath in P. brachycaudi sp. n. (Figure 14) is more elongated than in P. enervis.

Figures 6–14. 

Paralipsis brachycaudi sp. n., female 6 head, anterior view 7 antennae, lateral view 8 scutellum, dorsal view 9 propodeum, dorsal view 10 second-fourth segments of fore tarsus, dorsal view 11 hind leg, lateral view 12 fore wing 13 petiole, dorsal view 14 ovipositor sheath, lateral view.

Description

Female : Head (Figure 6) rounded, narrower than mesosoma at tegulae, bearing sparse setae (Figure 6). Head 1.1 times wider than long medially. Eyes oval, small with scarse and long setae. Tentorial index approximately 0.95. Clypeus with 15–20 long setae. Maxillary and labial palpi with one palpomere each. Ocular-ocelar line: diameter of posterior ocellus: Postocelar line=12:4:14. Malar space: height of eye =20:26. Antenna 16-segmented, filiform (Figure 7). Scapus widened at the tip, vase shaped at lateral view. Pedicel subspherical. F₁ equal to F₂ and F₃ and 2.0–2.1 times as long as its maximum width at the middle. Penultimate flagellomera 1.6 times as long as wide. F₁, F₂ and F₃ without and F₄ with one short longitudinal placode (Figure 7). Flagellomeres covered uniformly with short appressed and semi-erect setae.

Mesosoma: Mesoscutum smooth, and only moderately sculptured within small central area, usually with four rows of setae along its dorsolateral part. Mesoscutum 1.4 times as long as wide. Scutellum (Figure 8) smooth elongated, bearing 20–30 long setae in the central part. Propodeum (Figure 9) smooth, sometimes with rugosities laterally. Upper and lower parts of propodeum with 3–5 and 15–20 long setae on each side (Figure 9). Fore wing (Figure 12) densely pubescent, with long marginal setae, longer than those on fore wing surface. Vein 2-1A sclerotized (Figure 12). Pterostigma triangular, 1.7–1.9 times as long as its width (Figure 12). Secondfourth segments of fore tarsus in dorsal view (Figure 10) almost as long as wide (1.1–1.2 times as long as wide) and medium sized of apical bristles. Hind tibia medially and femur subbasally parallel-sided (Figure 11).

Metasoma: Petiole (Figure 13) smooth, with prominent spiracular tubercles, its length 1.50–1.60 times its width at spiracles and maximum width at level of spiracles 0.7 times distance between spiracle and apex of tergite 1; 10–15 setae positioned on posterior dorsolateral margin on each side. Ovipositor sheath (Figure 14) elongated, dorsally straight, narrowed toward tip, bearing 2–6 long setae on the ventral and dorsal surface. Length of ovipositor sheath 2.25–2.87 times its maximum width.

Length: body 1.5–2.0 mm; fore wing 1.3–1.7 mm.

Coloration: General body color light-brown to brown. Head brown with light-brown mouthparts. Scape and pedicel yellow to light-brown. Flagellomere 1 and 2 yellow, remaining parts of antennae brown. Mesosoma brown. Legs yellow to light-brown. Propodeum yellow. Metasoma brown. Petiole yellow. Ovipositor sheath dark-brown.

Male: unknown.

Etymology

The name of the new species is derived from that of its aphid host.

Distribution

Czech Republic, Germany.

Paralipsis rugosa Tomanović & Starý, sp. n.

http://zoobank.org/8BA5B5C2-F16E-4006-AAC6-001662AC26B3

Figures 15–21

Material

Holotype female, Moldova, Kišinev, 26.VI.1960, unknown aphid host and host plant, leg. Adaškevič; deposited in the IECR collection, slide mounted.

Diagnosis

The new species differs clearly from all known Paralipsis species in having a strongly rugose propodeum (Figure 18) and scutellum (Figure 17) that are irregularly deep carinated, while other Paralipsis species are characterized by a smooth propodeum, sometimes with moderately expressed rugosities. Also, P. rugosa sp. n. is with F₁ longer than F₂ (the ratio between F₁ and F₂ is approximately 1.15) (Figure 16), while P. enervis, P. brachycaudi sp. n., and P. planus have F₁ equal or subequal to F₂. An exception is P. tibiator, which has much longer F₁ than F₂ (the ratio of F₁ and F₂ is about 1.4). Further, F₁ and F₂ are very short (proportion of length and width of F₁ and F₂ are 1.76 and 1.50, respectively) (Figure 16).

Figures 15–21. 

Paralipsis rugosa sp. n., female 15 head, anterior view 16 antennae, lateral view 17 scutellum, dorsal view 18 propodeum, dorsal view 19 second-fourth segments of fore tarsus, dorsal view 20 hind leg, lateral view 21 fore wing.

Description

Female: Head rounded, smooth, narrower than mesosoma at tegulae, bearing dense setae (Figure 15). Head 1.1 times wider than long medially. Eyes oval, small, with scarse and long setae. Tentorial index 0.67. Clypeus with ten long setae (Figure 15). Maxillary and labial palpi with one palpomere each. Ocular-ocelar line: diameter of posterior ocellus: Postocelar line =12:4:14. Malar space: height of eye =11:13. Antenna 15-segmented, slightly thickened at apex (Figure 16). Scapus subapically with subparallel side at lateral view. Pedicel subspherical. F₁ (Figure 16) longer than F₂ and about 1.76 times as long as its maximum width at the middle, and F₂ and F₃ about 1.50 times as long as its maximum width at the middle. F₁ and F₂ without longitudinal placodes. Penultimate flagellomera 1.6 times as long as wide. F_1, F₂ and F₃ without, and F₄ with one short longitudinal placode. Flagellomeres covered uniformly with short appressed and semi-erect setae (Figure 16).

Mesosoma: Mesoscutum smooth, sculptured laterally, with very dense setae laterally. Mesoscutum 1.4 times as long as wide. Scutellum subspherical, strongly rugose with about 15 setae (Figure 17). Propodeum (Figure 18) extremely rugose. Upper and lower parts of propodeum with 5–6 long setae on each side. Fore wing (Figure 21) densely pubescent, with long lower marginal setae, longer than those on fore wing surface. Pterostigma triangular, 1.62 times as long as its width. Vein 2-1A sclerotized. Metacarpus absent. Secondfourth segments (Figure 19) of fore tarsus in dorsal view distinctly longer than wide (1.4–1.8 times as long as wide) and medium sized of apical bristles. Hind tibia medially and femur subbasally parallel-sided (Figure 20).

Metasoma: damaged.

Length: head and mesosoma combined about 1 mm; fore wing about 1.7 mm.

Coloration: General body color brown. Head brown. Mouthparts light-brown. Scape and pedicel brown with small yellow terminal part. F₁ and F₂ yellow, remaining parts of antennae brown. Mesosoma brown to light-brown. Legs yellow to light-brown.

Male: unknown.

Etymology

The name of the new species refers to the very rugose propodeum and scutellum.

Distribution

Moldova.

Key to the species of the genus Paralipsis Foerster on the basis of females

1	Propodeum and scutellum with strong and deep rugosities (Figs 17, 18); F1 and F2 stout, 1.7 and 1.5 times as long as wide, respectively (Fig. 16); pterostigma triangular, about 1.6 times as long as wide (Fig. 21)	P. rugosa sp. n.
–	Propodeum and scutellum smooth or with moderate expressed rugosities (Figs 8, 9); F1 and F2 elongate, 2.0–2.2 and 1.8–2.1 times as long as wide, respectively (Fig. 7); pterostigma 1.8–2.0 times as long as wide (Fig. 12)	2
2	Second-fourth segments of fore tarsus distinctly longer than wide in dorsal view (Fig. 22); hind tibia medially and femur subbasally widened (Fig. 23); flagellar segments narrowed basally	3
–	Second-fourth segments of fore tarsus approximately as long as wide in dorsal view (Fig. 19); hind tibia medially and femur subbasally almost parallel sided (Fig. 20); flagellar segments parallel-sided	4
3	Mesoscutum and scutellum smooth and densely setous; flagellomere 1 distinctly longer than F2 (1.3–1.4 times as long as wide); pterostigma triangular, approx. 1.8 times as long as wide	P. tibiator
–	Mesoscutum and scutellum moderate rugose and setous; F1 subequal to F2 (about 1.1 times as long as wide); pterostigma twice as long as wide	P. planus
4	Forewing 2-1A vein absent; Japan and Far East	P. eikoae
–	Forewing 2-1A vein present, partly or completely sclerotized; Europe	5
5	Petiole 1.50-1.60 times as long as wide at spiracles level; F1 about 2.0 times as long as wide; F1 and F2 yellow	P. brachycaudi sp. n.
–	Petiole 1.30–1.40 times as long as wide at spiracles level; flagellomere 1, 2.0–2.2 times as long as wide; basal third of F1 yellow till light brown and remaining part F1 and whole F2 brown	P. enervis

Figures 22, 23. 

Paralipsis tibiator, female 22 second-fourth segments of fore tarsus, dorsal view 23 hind tibia and femur, lateral view.

We have demonstrated here a progress in methodology of DNA amplification by designing Paralipsis-specific degenerative primers to retrieve disintegrated DNA fragments from archived museum specimen collections of which can be considered as biobanks and used to discover new species (Yeates et al. 2016). Sequencing of the COI barcoding region of 18 specimens collected across the Western Palaearctic over a long period of time did not determine any clear specialization of taxa to a strict root aphid host. There is no geographical structuring of genetic variation between specimens associated with the same aphid host within the lineages P. enervis, P. tibiator, nor P. brachycaudi sp. n. However, it confirmed the existence of Paralipsis tibiator, a species recently described by van Achterberg and Ortiz de Zugasti Carrón (2016). Although a second recently described species, viz., P. planus (van Achterberg and Ortiz de Zugasti Carrón 2016), was not available for molecular analysis, the general morphological description (petiole shape, wing venation pattern, antennae) indicates that it is close to P. enervis, so we suppose it belongs to the P. enervis lineage. However, since P. planus was described on the basis of a single specimen, it is necessary to further explore the morphological and genetic variability of this species in order to finally confirm its taxonomic status. In addition, the present study revealed two new Paralipsis species, P. rugosa sp. n. and P. brachycaudi sp. n. Four separate phylogenetic lineages showed clear distinction, but with significant intralineage genetic variation between the haplotypes associated with different aphid/host associations. All phyletic lineages share aphid hosts from the subfamilies Aphidinae and Eriosomatinae. Many Eriosomatinae are specialized gall-producing aphids, but only on primary host plants, while this is not the case on secondary host plants (grasses), where they are parasitized by Paralipsis wasps and other specialized root aphid parasitoids.

It is necessary to examine in detail all known records of P. enervis in the light of the diagnosis given for the new Paralipsis species described in the present paper. Probably, P. enervis represents a complex of cryptic species, which is a common case among aphid parasitoids (Mitrovski-Bogdanović et al. 2013, Derocles et al. 2016). Although our molecular analyses were restricted to only 18 COI barcoding sequences retrieved from dry Paralipsis specimens, we recognized four phyletic lineages on the phylogenetic tree with a sister position of P. brachycaudi sp. n. in relation to P. enervis + P. rugosa sp. n. + P. tibiator lineages. The strong rugosities of the propodeum and scutellum in P. rugosa sp. n. represent an autapomorphic character state, while its possession of very short flagellomeres is a plesiomorphic character state. We recognize the elongated ovipositor sheath and petiole in P. brachycaudi sp. n. as apomorphic characters. We did not find any strong support for the existence of Paralipsis host-specific lineages. Brachycaudus aphid hosts were found only in the P. brachycaudi sp. n. lineage, while other aphid hosts are mainly shared between P. enervis and P. tibiator. Although Forda root aphids are distributed throughout the whole of Europe, P. tibiator attacked them only in Mediterranean-type habitats. It is known that the distribution of parasitoids is usually narrower than that of their aphid hosts, due to the more specific habitat and microhabitat of parasitoids (Starý 1970). The records of P. enervis associated with Brachycaudus aphid hosts should be carefully examined, as they may be referable to P. brachycaudi sp. n. All findings of P. tibiator are from Mediterranean areas.

Although most of our samples originated from central and southern Europe, Paralipsis species are distributed in several European countries, including ones in the northern part of the continent (van Achterberg 2012, Staverløkk and Ødegaard 2016). However, no Paralipsis species have been recorded in more than half of the countries of Europe (van Achterberg 2012). In the present study, we have not explored the relationships between ants and Paralipsis wasps. However, future research should reveal existing relationships of the two newly described species with ants and result in new knowledge about the biology and ecology of these Paralipsis wasps.

The authors are grateful to G Remaudière (Paris, France) (deceased) and J Danilov (Lithuania) for supplying valuable material that contributed significantly to the presented research. We thank Frédérique Bakker (Collection Manager Hymenoptera, Naturalis Biodiversity Center, Leiden, The Netherlands) for loaning us the P. tibiator paratype. This study was supported by the Ministry of Education, Science, and Technological Development of the Republic of Serbia (III43001). P Starý was supported by the Institute of Entomology, Biology Centre of the Czech Academy of Sciences. The sampling of radicicolous aphids on the Iberian Peninsula was conducted in the context of Project CGL2015-68188-P, funded by “Ministerio de Economia, Industria y Competitividad” of Spain (MIMECO).