Immature stages of Palearctic Mecinus species (Coleoptera, Curculionidae, Curculioninae): morphological characters diagnostic at genus and species levels

Abstract The immature stages of ten Mecinus species are described for the first time and those of two other species are redescribed, adding important chaetotaxy characters that were missing from previous descriptions. These species belong to six of the nine assemblages of Mecinus species previously established according to a phylogenetic analysis. All these groupings are confirmed on the basis of several characters of mature larvae and pupae. Moreover, all the species show several characters that are useful for distinguishing them from each other, including cryptic species that previously had few differential characters. Some characters that may be useful for separating Mecinus from other genera in the tribe are suggested. To confirm the taxonomic identification of some larvae, the mtCOII gene was obtained and compared with sequences from identified adult specimens. The most important characters for separating the immature stages of the genera and species groups in Mecinus are the number of palpomeres of the labial palpi (1 or 2), the number of air tubes of the thoracic and abdominal spiracles (unicameral or bicameral), and the number of epipharyngeal setae. The species studied herein were compared with those known from other genera in the tribe Mecinini. Two keys, one to the described larvae and the other to the pupae, are provided. Detailed biological data, several of which are new, on some species are reported.


Introduction
The genus Mecinus Germar, 1821 belongs to the tribe Mecinini (Curculionidae, Curculioninae) and includes approximately 50 Palearctic species (Alonso-Zarazaga et al. 2017). Adults of this tribe were recently subjected to morphological revision (Caldara and Fogato 2013) and phylogenetic analysis . Based on this analysis, seven species groups and two "complexes" of species were recognised. Moreover, a phylogenetic study on the tribe Mecinini, based on morphological characters, suggests that Mecinus is the sister group of the remaining Mecinini like Gymnetron Schoenherr, 1825 and Rhinusa Stephens, 1829 (Caldara 2001). Preliminary molecular studies seem to confirm the systematic separation of these genera (I. Toševski, unpublished data).
All known Mecinus species live on angiosperms belonging to the tribes Plantagineae and Antirrhineae of the family Plantaginaceae as recently defined (Olmstead et al. 2001;Albach et al. 2005;APG 2016). The larvae develop inside the ovaries, stems, or roots of the host plants and are sometimes able to induce the formation of galls (Hoffmann 1958;Caldara 2001;Toševski et al. 2011). Several species of the genus have been the subject of detailed ecological studies (De Clerck-Floate and Harris 2002;De Clerck-Floate and Miller 2002;McClay and De Clerck-Floate 2002;Sing et al. 2005;Toševski et al. 2011) as potential biological control agents for some species of toadflax (Linaria spp.) that were introduced into North America and have since become invasive (Vujnovic and Wein 1997).
To date, larvae of only approximately 30 Mecinini species have been described, while descriptions of pupae have been made for 15 Mecinini species (see Skuhrovec et al. 2018 for complete references). However, there are only a few detailed descriptions of larvae and pupae that can be used for an adequate taxonomic comparison; these include immatures of three species of Gymnetron (Jiang and Zhang 2015), two species of Rhinusa (Gosik 2010;Ścibior and Łętowski 2018), five species of Cleopomiarus (Skuhrovec et al. 2018;Szwaj et al. 2018) and three species of Miarus (Skuhrovec et al. 2018). In fact, the comparison of approximately ten previously described immatures of mecinines, including two Mecinus species, M. heydenii Wencker, 1866 (Emden 1938) and M. janthinus Germar, 1821 (Scherf 1964), is somewhat problematic due to the absence of important details of the chaetotaxy and/or the absence of quality drawings.
Therefore, the aims of this study were to describe several larvae and pupae of Mecinus for the first time, to find characters that are diagnostic at genus and species levels, and finally to compare the characters on immature stages of this genus with other genera of the same tribe that might be phylogenetically informative.

Insect collection
The material for this study was collected mainly from June to August 2017, in localities of Serbia, Macedonia, Bulgaria and France. The immature stages, i.e., L3 larvae and pupae from every studied species, were collected from their host plants and subsequently preserved in 2 ml screw-cap micro tubes (Sarstad, Germany) filled with 96% ethanol at 4-6 °C.

Molecular analysis
In specific cases, when two species inhabit the same host plant and larval development occurs in the same host niche, the taxonomic identity of collected larvae and pupae was confirmed by molecular methods. Since it is known that the immature specimens are unavoidably damaged by these procedures, before their sequencing the specimens were compared with the others used for the morphological study in order to be sure on their conspecificity. Total DNA was extracted using the QIAGEN Dneasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The mitochondrial cytochrome oxidase subunit II gene (mtCOII gene) was amplified using the primers TL2-J-3038 (5'-TAATATGGCAGATTAGTGCATTGGA-3') (Emerson et al. 2000) and TK-N 3782 (5'-GAGACCATTACTTGCTTTCAGT-CATCT-3') (EVA-Harrison Laboratory, Cornell University, Ithaca, NY, USA). The polymerase chain reactions (PCRs) contained NH4 buffer (19), 5 mM MgCl2, 0.8 mM of each dNTP, 0.75 μM of each primer and 0.75 U of Taq polymerase (Fermentas) in a 20 μL final volume. PCR cycles were carried out in a Mastercycler EP Gradient S (Eppendorf, Germany) with the following thermal steps: 95 °C for 5 min (initial denaturation), 40 cycles at 95 °C for 1 min, 1 min at 45 °C (annealing), 72 °C for 2 min and a final extension at 72 °C for 10 min. The amplified products of the COII gene were sequenced with the forward primer only. The sequencing was performed on an ABI Prism 3700 automated sequencer using the commercial services of Macrogen Inc. (Seoul, South Korea). In addition, adult specimens of all species, whose larvae and pupae were described in this study, were identified by two of the authors (RC and IT), based on morphology. Subsequently, specimens were sequenced for the mitochondrial COII gene. The taxonomic identity of the larvae and pupae was done by comparing their sequences with the adult ones. Pairwise distances using the p-distance model were analysed using MEGA5 software (Tamura et al. 2011). The obtained sequences were deposited in the GenBank database under accession numbers MN991999-MN992012.

Confirmation of taxonomic status using molecular tools
Molecular analysis confirmed the taxonomic identity of the larval and pupal stages of M. labilis and M. pascuorum which occur together developing in pyxidia of Plantago lanceolata L., and also helped to discriminate between the immature stages of M. pirazzolii and M. ictericus (Gyllenhal, 1838), which sometimes co-occur associated with P. arenaria Waldst. & Kit. All Mecinus taxa whose immature stages are described in this study were sequenced for the mtCOII gene. Sequences were edited with FinchTV v.1.4.0 (http://www.geospiza.com) and aligned with ClustalW integrated in the Mega5 software (Tamura et al. 2011). Aligned sequences were truncated to 655 bp from the 3' end prior to calculating the pairwise distances among the taxa. The recorded divergences among the analysed taxa ranged from 1.5 and 23.4% between M. janthinus-M. janthiniformis and M. collaris-M. heydenii, respectively (Table 1). The complete mtCOII gene showed different lengths across Mecinus species, ranging from a 678 bp (M. janthinus) group to 696 bp. in M. pyraster.

Morphological descriptions
Part of the larval and pupal material was preserved in Pampel fixation liquid (see Skuhrovec and Bogusch 2016) and used for the morphological descriptions. To prepare the slides, we followed May (1994): a larva was decapitated, and the head was cleared in a 10% potassium hydroxide (KOH) solution and then rinsed in distilled water. After clearing, the mouthparts were separated from the head capsule, and the head capsule and all mouthparts were mounted on permanent microscope slides in Euparal. All other body parts were mounted on temporary microscope slides in 10% glycerine.
The observations and measurements were conducted using a light microscope with calibrated ocular lenses (Olympus BX 40 and Nikon Eclipse 80i). The following characters were measured for each larva: head width, body length (larvae fixed in a C-shape were measured in segments), and body width in the widest place (i.e., metathorax or abdominal segments I-IV). For the pupae, the length and width at the widest place were measured. All results of the measurements are given in Table 2 (mature larva) and in Table 3 (pupa). The lengths of all setae are visible in the figures. table 1. Mitochondrial DNA cytochrome oxidase subunit II (COII) divergence based on pairwise analysis (p-distance method) among Mecinus species elaborated in this study. Numbers in brackets represent complete length of the COII gene.  Drawings were created with a drawing tube on a light microscope and processed by a computer (Adobe Photoshop, Corel Photo-Paint 11, GIMP 2). The numbers of setae in bilateral structures are given for one side.
We used the terms and abbreviations for the setae of the mature larvae and pupae found in Scherf (1964), May (1977May ( , 1994, and Marvaldi (1998Marvaldi ( , 1999. The sequence of the species followed that proposed by Caldara and Fogato (2013) and .

Botanical taxonomy
For families and subfamilies, we complied with APG (2016) whereas for the complex situation concerning the nomenclature of some common species of Plantago, we followed the proposals by Applequist (2006) and Dowel and Shipunov (2017).
Body distinctly white to yellow. Body curved, slender, rounded in cross section. Setae on body thin, in different colouration, distinctly different in length; piliform, integument often with some asperities. Prothorax slightly smaller than meso-and metathorax. Spiracle placed between the pro-and mesothorax (see e.g., Gosik et al. 2016). Abdominal segments I-III(VI) of almost equal length, next abdominal segments decreasing gradually to the terminal parts of the body. Abdominal segment X reduced to three or four anal lobes of unequal size. Anus located terminally. Thoracic spiracles uni-or bicameral, eight abdominal spiracles unicameral, all spiracles functional, close to anterior margin of segment. Prothorax with eight to eleven prns; two ps; and one eus. Mesothorax with one prs, two or three pds; one or two as; three ss; one eps; one ps; and one eus. Chaetotaxy of metathorax almost identical to that of mesothorax. Each pedal area of thoracic segments well separated, with three to six pda. Abdominal segments I-VIII with one prs; three or four pds; two or three ss; two long eps; one ps; one lsts; and two eus. Abdominal segment IX with two to four ds; one or two ps; and two sts. Abdominal segment X without or with up to two minute ts.
Head capsule yellow to pale brown, rounded or flattened laterally, endocarinal line distinct, half or more than half the length of frons. Frontal sutures extended to antennae. One or two stemmata (st), anterior stemma in the form of a pigmented spot with convex cornea. Dorsum of the epicranium with five setae; des 1 located in the central part of epicranium, des 2 lateral, des 3 located anteriorly on epicranium close to frontal suture, des 4 often medially, des 5 located anterolaterally. Frons with three to five fs, fs 1 sometimes absent, fs 2 absent except one exception; fs 4 and fs 5 subequal. Head with two les, one or two ves, and one to five pes. Antennae located at the end of the frontal suture on each side, membranous and distinctly convex basal article bearing three or four sensilla and a conical sensorium, the later elongated, narrow. Clypeus trapezium-shaped, with one or two cls, and one sensillum (clss); all very close to margin with frons. Labrum with three lms; anterior margin bisinuated; lrs 1 placed posteromedially, lrs 2 anteromedially, lrs 3 posterolaterally. Epipharynx with three finger-like als; with two or three ams; and one or two mes; labral rods (lr) distinct, kidney-shaped. Mandibles distinctly broad, bifid, teeth of unequal height; slightly truncate; both setae piliform. Maxilla stipes with one stps, two pfs and one short to minute mbs; mala with six or seven finger-like dms; four or five vms; all vms distinctly shorter than dms. Maxillary palpi with two palpomeres; basal palpomere with one short mxps and two sensilla; distal palpomere with one sensillum and a group of microcuticular apical processes. Prelabium various in shape, with one prms; ligula with sinuate margin and two or three ligs; premental sclerite well sclerotised but without anterior and posterior extensions, U-shaped or cup-like. Labial palpi with one or two palpomeres; each of the palpomeres with one sensillum, distal palpomere with cuticular apical processes. Postlabium with three pms: pms 1 usually the shortest, placed anteromedially or anterolaterally, pms 2 the longest, placed laterally, and pms 3 short or medium, placed posterolaterally.
Body stout or elongate; normally white, but sometimes yellowish; cuticle smooth. Rostrum various in length, from two to five times as long as wide. Antennae short or elongate. Pronotum 1.1-2.2 times as wide as long. Meso-and metanotum often equal in length. Abdominal segments I-(IV)VII of equal length; segment VIII almost semicircle, segment IX distinctly reduced. Spiracles on abdominal segments placed dorsolaterally; on segments I-V functional, on segment VI atrophied on next ones invisible. Urogomphi (ur) short or elongate.
Chaetotaxy often well developed, but sometimes almost invisible. Head capsule without or with one vs, without or with up to two sos, without or with up to two os. Rostrum without or with up to two rs, and without or with one pas. Pronotum without or with up to two as, without or with one ds, one or two sls, without or with up to two ls, and two to four pls. Dorsal parts of meso-and metathorax with two or three setae. Apex of femora normally with one short fes. Abdominal segments I-VIII without or with up to two setae laterally and without or with up to three setae ventrally. Dorsal parts of abdominal segments I-VII with three to seven setae; abdominal segment VIII with three to six setae dorsally. Abdominal segment IX without or with up to four micro-setae ventrally.
Remarks and comparative notes. The adults of this assemblage of several taxa are mostly very similar to each other, but, lacking synapomorphies, they were treated by  as a "complex" of species. Overall, they are characterised by small size (length shorter than 2.5 mm), usually with short, oval elytra, and with integument, at least in part, reddish. The larvae also have a combination of characters that distinguish them from those of the other groups, although with no clear autapomorphies. In contrast, the pupae are unique in having abdominal segments I-VII with 2-3 setae dorsally and three minute setae ventrally. Therefore, we can consider these species as an informal group like the other species groups.  Figure 1A-D) white, slender, curved. Chaetotaxy of thoracic segments relatively well developed, setae capilliform, different in length, light yellow, on thoracic segments elongated or medium, on abdominal segments very short. Prothorax (Figure 1B) with eight prns of almost equal length, two ps and one eus. Meso-and metathorax ( Figure 1B) with one medium prs, three medium pds, equal in length; one medium as, three medium ss, equal in length; one long eps, one long ps and one long eus. Pedal area with five long, equal in length pda. Abdominal segments I-VIII ( Figure 1C, D) with one very short prs, four short pds (arranged along the posterior margin), two minute ss, two short eps, one short ps, one short lsts and two short eus. Abdominal segment IX ( Figure 1D) with three medium ds, all located close to the posterior margin, one short ps and two short sts. Each of anal lobes with two minute ts.
Chaetotaxy well visible, all setae (except those on rostrum and ventral part of abdomen) almost equal in length, medium. Head with one os. Rostrum with one minute rs ( Figure 3A). Pronotum with two as placed beside protuberances, two ls, one ds and three pls. Dorsal parts of meso-and metathorax with two setae placed laterally. Abdominal segments I-VIII with three setae situated posteriorly, two elongated setae laterally and three short setae ventrally (median setae distinctly bigger than others). Abdominal segment IX with two micro-setae ventrally.
Biological notes. This species lives on Plantago lanceolata L. In spring, the female lays one egg per developing pyxidium, and each larva consumes the contents of a pyxidium, usually two seeds, without causing externally visible modification. Pupation takes place within the same pyxidium. Adults emerge from the beginning of summer until September. They overwinter in the soil (Hoffmann 1958;Scherf 1964;Dickason 1968;Nieminen and Vikberg 2015).
Remarks and comparative notes. This species is one of the most common species in the genus, with a very large range of distribution: Europe, the Caucasian states, the Middle East, central Asia, and Algeria (Caldara and Fogato 2013). It has been imported to North America, Australia and New Zealand (O'Brien and Wibmer 1982;Debinski and Holt 2000) and recently collected in South Africa (Caldara et al. 2009). Morphologically, this species is more closely related to other species of the group, i.e., M. latiusculus (Jacquelin du Val, 1855) and M. ictericus than to M. labilis studied herein, and this seems also to be corroborated by the preliminary molecular data (I. Toševski, unpublished data). However, the relationships among the immatures of these species are closer than their relationships with all the other species currently known. Body ( Figure 4A-D) white, moderately slender, slightly curved. Chaetotaxy of thoracic segments relatively well developed, setae capilliform, different in length, light yellow, on abdominal segments I-VII very short, on segments VIII and IX of medium length. Prothorax ( Figure 4B) with eleven long prns of almost equal length, two long ps and one long eus. Meso-and metathorax ( Figure 4B) with one short prs, three pds (pds 1 short, pds 2-3 long), one long as, three ss different in length (one minute and two medium), one long eps, one long ps and one long eus. Pedal area with four pda (two long and two medium). Abdominal segments I-VIII ( Figure 4C, D) with one short prs, four short pds arranged along posterior margin, two minute ss, two short eps, one short ps, one short lsts and two short eus. Abdominal segment IX ( Figure 4D) with three medium ds, all located close to posterior margin, one medium ps and two rather short sts. Each of anal lobes with one minute seta.
Body rather stout, slightly curved, white. Rostrum slender, moderately short, about 2.0 times as long as wide, reaching procoxae. Antennae short. Pronotum 2.2 times as wide as long. Urogomorpi (ur) very short, conical, only slightly reaching outline of the body ( Figure 6A-C).
Biological notes. Larvae feed on Plantago lanceolata L. in galled pyxidia, where they pupate in the collar without causing externally visible modifications (Hoffmann 1958).
Remarks and comparative notes. This species is widely distributed in Europe, the Caucasian states, and Turkey. Concerning the adults, the pattern of the elytral integument (reddish with two black oblique bands from interstria 1 to 7) and the shape of the rostrum (in lateral view moderately curved in basal half then straight to apex) allow us to separate these two species from all the others. With regard to the immatures, the differences from the other studied species of the group, M. pascuorum, are several and are reported in the key. Molecular data also do not show a close relationship between these two species (I. Toševski, unpublished data).
Remarks and comparative notes. The very short, conical and in lateral view straight rostrum, and the protibiae with apical third distinctly enlarged, sometimes with outer margin and apex bearing stout denticles, are truly noteworthy and unique in Mecinini. Both characters are oddly similar to those of a mole, and the tibiae are similar to those of Scarabaeidae. Since nothing was known about their biology except for their host plants, Caldara and Fogato (2013) speculated on the possibility that the species of this group deposit eggs in plant roots. The new biological data on M. pirazzolii below reported exclude this hypothesis and suggest that most likely the female is able to approach as close as possible to the pistil of the flower and deposit the egg thanks to the shape of its protibiae, since it is regularly found deeply stuck between Plantago inflorescences. This group might be related to the M. collaris group on the basis of the morphological characters of the adults , whereas it seems more related to the M. circulatus group according to the preliminary molecular data (I. Toševski, unpublished data). Unfortunately, the study of immatures did not clarify this situation. In fact, the presence of one palpomere on the labial palpi and of all spiracles unicameral contradicts this hypothesis, and the same combination of these two characters is found only in M. pascuorum and M. heydenii groups, with the former of which the M. simus group might have major similarities. However, the immatures of the M. simus group have some autapomorphies, such as a smooth body cuticle and prominent pedal lobes in larvae and abdominal segments I-VII with three setae dorsally and without setae ventrally, apart from an obvious extraordinarily short rostrum tapering to the apex in pupae. Body ( Figure 7A-D) white, slender, curved. Chaetotaxy of thoracic segments relatively well developed, setae capilliform, different in length, light yellow; on abdominal segments almost invisible (except dorsal parts of abdominal segments IX and X). Prothorax ( Figure 7B) with eight prns of unequal length (seven relatively long and one medium), two relatively long ps and one short eus. Meso-and metathorax ( Figure 7B) with one medium prs, two long pds, equal in length, one long as, three ss different in length (two relatively long, one short), one long eps, one long ps and one short eus. Pedal area with five long pda, equal in length. Abdominal segments I-VIII ( Figure 7C, D) with one very short prs, three short pds (on segment VIII medium, equal in length), arranged along posterior margin, two minutess, one short eps, one short ps, one short lsts and two short eus. Abdominal segment IX ( Figure 7D) with three medium ds, all located close to posterior margin, one short ps and two short sts. Each of anal lobes with one minute seta.
short. Epipharynx ( Figure 8D) with three finger-like als of almost equal length; two medium finger-like ams; two short finger-like mes; surface smooth; labral rods very short, rounded. Mandibles ( Figure 8E) conical, rather wide, with a small protuberance in the middle of the cutting edge; both mds capilliform, medium, equal in length, placed transversely. Maxilla ( Figure 8F) with one stps and two pfs of equal length; mbs short; mala with six long rod-like dms of almost equal size, five vms different in length. Maxillary palpi: basal palpomere distinctly wider and longer than distal. Prelabium ( Figure 8F) cup-like with one long prms; ligula with two short ligs; premental sclerite clearly visible, cup-shaped, posterior extension with acute apex. Postlabium ( Figure 8F) with medium pms 1 , medium pms 2 , and short pms 3 .
Body stout, slightly curved, white. Rostrum slender, very short, tapering to its top. Antennae moderately elongated. Pronotum 2.0 times as wide as long. Urogomorpi (ur) very short, conical, not reaching outline of the body ( Figure 9A-C).
Chaetotaxy almost invisible, all setae minute, possible to observation only under higher magnification. Head with one os. Rostrum with one rs placed medially (Figure 9B). Pronotum with two as, one ls, one ds and three pls. Dorsal parts of meso-and metathorax with two setae placed laterally. Dorsal parts of abdominal segments I-VIII with three setae situated posterolaterally and one seta laterally. Abdominal segment IX without setae.
Biological notes. This species is associated with the annual plant Plantago arenaria Waldst. & Kit. The adult aggregation on plants is followed by the appearance of flowering stems with spikes in late spring. The females lay one egg onto the base of the pistil or the initialised seed. The act of oviposition is followed by proliferative growth of the ovarian tissue in the form of gall but without changes in the external morphology of the pyxidium. During development, the larvae consume all the tissue inside the pyxidium, leaving only the fruit shell intact. The larvae pupate inside the fruit shell, from which adults emerge after being completely sclerotised. Overwintering takes place in the soil litter near the host plant (I. Toševski, pers. obs.). Sympatric occurrence with M. ictericus is common (Caldara and Fogato 2013).
Remarks and comparative notes. This species is distributed in eastern Central Europe, southeastern Europe and Turkey. In our keys, this species is closer to the species of the M. pascuorum group than to others, as already discussed in the Remarks for the group.
(1) body elongated or very elongated; (2) urogomphi slender, short or medium, reaching outline of the body, directed downward; (3) rostrum moderately elongated; (4) setae minute or medium; (5) head with one vs, one or two sos, one or two os; (6) rostrum with one pas and one rs; (7) pronotum with one or two as, without or with one ds, two sls, without or up to two ls, two or three pls; (8) meso-and metanotum with two setae; (9) abdominal segments I-VII with three or five setae dorsally.
Remarks and comparative notes. The adults of this group are characterised by body elongate, subcylindrical, elytral integument reddish and black to completely black, protibiae with apical part of ventral surface distinctly directed outward. On the basis of these characters, this group might be related to M. collaris and especially to the M. simus group . The study of the immatures does not support this latter relationship. The immatures of this group lack autapomorphies. However, the larvae possess the unique combination of one palpomere + thoracic spiracle bicameral and abdominal spiracles unicameral, which do not share with the species of the M. simus group, M. pirazzolii, that we have studied. Body ( Figure 10A-D), light yellow, slender, curved ( Figure 10B). Thoracic segments larger than abdominal segment I. Abdominal segments I-VI of almost equal length; segments VII-IX decreasing gradually to the terminal body part; segment X reduced to three anal lobes of those lateral are the largest, and dorsal the smallest (sometimes absent). Chaetotaxy weakly developed, setae short, transparent, difficult to observe ( Figure 10B). Prothorax ( Figure 10B) with eight prns (six medium and two very short); two medium ps and one very short eus. Meso-and metathorax (Figure 10B) with one very short prs, two pds (one very short, one medium), one medium as, three ss (two medium and one very short), one medium eps, one medium ps and one very short eus. Pedal area with three pda (one medium and two very short). Abdominal segments I-VIII ( Figure 10C, D) with one very short prs, three pds arranged along the posterior margin (order: very short, medium and very short), two ss different in length, Figure 10. Mecinus circulatus mature larva, habitus and chaetotaxy A habitus of the body and frontal view of the head B lateral view of thoracic segments C lateral view of abdominal segment I D lateral view of abdominal segments VII-X. Abbreviations: Th. I-III -number of thoracic segments, Abd. I-X -number of abominal segments, setae: as -alar, ds -dorsal, eps -epipleural, eus -eusternal, lsts -laterosternal, pdapedal, pds -postdorsal, prns -pronotal, prs -prodorsal, ps -pleural, ss -spiracular, sts -sternal, ts -terminal. two eps different in length, one medium ps, one medium lsts and two very short eus. Abdominal segment IX ( Figure 10D) with two ds (one medium and one very short), all located close to the posterior margin, one very short ps and two very short sts. Each lateral anal lobe (abd. seg. X) with one minute seta.

Mecinus circulatus
Head capsule ( Figures 10A, 11A-F) pale brown, narrowed bilaterally. Frontal suture poorly visible. Des 1-3 very long, equal in length, des 4 four times shorter than des 1 , des 5 slightly shorter than des 1 . Fs 1 short; fs 3 short, fs 4,5 long. Les 1 and les 2 equal in length, slightly shorter than des 1 ; both ves short, and five short pes ( Figure 11A). Antennae ( Figure 11B) with conical sensorium (Se) four times as long as wide, and three sensilla basiconica. Clypeus ( Figure 11C) trapezium-shaped, anterior margin concave; cls 1-2 relatively short; clss placed close to cls 2 . Labrum ( Figure 11C) with distinctly sinuate anterior margin; lrs 1 very long, lrs 2 slightly shorter than lrs 1 , lrs 3 two times shorter than lrs 1 . Epipharynx ( Figure 11D) with three medium, finger-shaped als of almost equal length; two rod-like ams, equal in length; one finger-like mes of medium length; surface smooth; labral rods close to kidney-shaped. Mandibles ( Figure 11E) conical, wide, with a small protuberance in the middle of the cutting edge; both mds capilliform, medium, equal in length, placed mediolaterally. Maxilla ( Figure 11F) with one stps and two pfs equal length; mbs very short; mala with six finger-like dms of almost equal size; four vms different in length. Maxillary palpi: basal palpomere distinctly wider and slightly longer than distal. Prelabium ( Figure  11F) cup-like with one relatively short prms; ligula with two ligs different in length; premental sclerite well visible, cup-shaped. Postlabium ( Figure 11F) with medium pms 1 , long pms 2 , and medium pms 3 .
Body moderately elongated, white. Rostrum rather short, about 3.2 times as long as wide, reaching up to mesocoxae. Antennae slender and elongated. Pronotum 1.25 times as wide as long. Mesonotum slightly shorter than metanotum. Urogomphi (ur) short, slender, conical, with sclerotised, sharp apex, slightly reaching outline of the body, directed downward ( Figure 12A-C).
Chaetotaxy very sparse, setae short or minute. Head with one vs, one os and one sos. Rostrum with one rs and one pas. Setae on head and rostrum straight, as long as those on prothorax ( Figure 12A). Pronotum with one as, one ls, two sls, and two pls. Dorsal parts of meso-and metathorax with two setae placed medially. Apex of femora with one minute fes ( Figure 12A-C). Dorsal parts of each abdominal segments I-VIII with three setae placed posteromedially along margins of each segments. Abdominal segment IX with two micro-setae ventrally.
Remarks and comparative notes. This is a common species in western, central and southern Europe, northern Africa and the Middle East. By the colour of the elytral integument, with black and reddish vittae, the adults differ from M. pyraster, whose integument is completely black. However, both the study of the morphological characters in adults and immatures and the preliminary molecular study (I. Toševski, unpublished data) agree with the hypothesis of close relationships between these two species.
Larvae are easily separable from those of M. pyraster: the pronotum has eight prns instead of 11, the pedal lobes has three pda instead of five, the anal lobes with one ts instead of two, the head with five pes instead of four, the mandible with two mds instead of one, the mala with four vms instead of five, and the prms are shorter.
Pupae differ from those of M. pyraster by the head with one sos and one os instead of two, the pronotum with a different number of setae in all positions, and the abdominal segments I-VII with three setae dorsally instead of five. Body ( Figure 13A-D) yellowish, slender, curved, densely covered with asperities. Metathorax as large as abdominal segment I. Abdominal segments I-VI of almost equal length, abdominal segments VII-IX decreasing gradually to the terminal body part, segment X reduced to three anal lobes of those lateral are the largest, and dorsal the smallest (sometimes absent). Chaetotaxy weakly developed, setae short or medium. Prothorax ( Figure 13B) with eleven prns (eight medium and three very short); two medium ps and one very short eus. Meso-and metathorax ( Figure 13B) with one very short prs, two very short pds, one very short as, three ss (two medium and one very short), one medium eps, one medium ps and one very short eus. Pedal area with five pda (three medium and two very short). Abdominal segments I-VIII ( Figure 13C, D) with one very short prs, three very short pds arranged along the posterior margin, two very short ss, two eps different in length, one medium ps, one medium lsts and two very short eus. Abdominal segment IX ( Figure 13D) with three ds (one medium and two very short), all located close to the posterior margin, one medium ps and two very short sts. Each lateral anal lobe with two minute setae.
Body elongated, white. Rostrum rather slender, about three times as long as wide, reaching almost up to mesocoxae. Antennae slender and elongated. Pronotum 1.8 times as wide as long. Urogomphi (ur) slender and rather elongated, conical, with sclerotised apex, reaching outline of the body, directed downward ( Figure 14A-C).
Chaetotaxy well developed, setae rather short. Head capsule with one vs, two sos equal in length, two os equal in length. Rostrum with one rs and one pas ( Figure 14A). Pronotum with two as, one ds, two sls, two ls, and three pls ( Figure 14A-C); equal in length ( Figure 14B). Setae on head and rostrum as long as those on prothorax. Dorsal parts of meso-and metathorax with two setae different in length placed medially. Abdominal segments I-VIII with two medium setae laterally and three medium setae ventrally, distributed in regular lines. Dorsal parts of abdominal segments I-VII with five setae (d 1 placed anteromedially, d 2-4 posteromedially and d 5 located posterolaterally); abdominal segment VIII with only four very long setae dorsally. Abdominal segment IX with two micro-setae ventrally.
Biological notes. This species is associated with some Plantago species (P. lanceolata L., P. lagopus L., P. media L.) (Hoffmann 1958;Sprick 2001). In west Palearctic larvae are most frequently found in the roots of P. lanceolata, boring channels in upper part of the root crown. Larger roots may inhabit several larvae. Pupation takes place during early summer in the pupal chamber situated in the upper part of larval channel. After emergence, adults overwinter in the soil litter nearby host plant.
Remarks and comparative notes. This species is very common and widespread in the Palearctic region. It was also reported in North America (O'Brien and Wibmer 1982). The adult is distinctly variable in the size and shape of the body and vestiture within the same population. It differs from M. circulatus by the black integument and ventrite 5 in the male bearing a median tuft of hair. In larvae, the pronotum has eleven prns instead of eight, the pedal lobes have five pda instead of three, the anal lobes with two ts instead of one, the head with four pes instead of five, the mandible with one mds instead of two, the mala with five vms instead of four, and the prms are longer. The pupae also differ from those of M. circulatus by a different number of setae on head, pronotum and abdominal segments I-VII (see key to the pupae). However, morphological and molecular studies (I. Toševski, unpublished data) demonstrate a clear relationship between M. pyraster and M. circulatus.
Remarks and comparative notes. The adults of this monobasic group are easily distinguishable from all other species of Mecinus by several autapomorphies, such as rostrum short and wide, straight in lateral view, scrobe not reaching anterior margin of eye, elytra elongate, broad scales densely covering base of pronotum, epimera and episterna. In contrast, immatures have few autapomorphies, i.e., larvae are slightly pressed dorsoventrally, with a densely tuberculate cuticle, whereas premental sclerite, pedal lobes and spiracular area of meso-and metathorax are dark pigmented; the pupae have abdominal segments I-IV lacking setae dorsally, whereas segments V-VII dorsally possess five growing setae.
Presently, it is unclear to which species M. collaris is more closely related. The other species with short and straight rostrum, such as those of the M. simus group, do not apparently share other synapomorphies with M. collaris. In contrast, the larvae of the latter share the number of palpomeres of the labial palpi (two) and the shape of the thoracic spiracle (bicameral) and abdominal spiracles (unicameral) with the M. janthinus group. The pupae of M. collaris differ from all the others studied here by the dorsal setae of the abdominal segments because segments I-IV are without setae and segments V-VII have setae growing gradually.  Figure 16A-D) light yellow, slender, curved, slightly pressed dorso-ventrally. Premental sclerite, pedal lobes and spiracular area of meso-and metathorax dark pigmented. Chaetotaxy of thoracic segments relatively well developed, setae capilliform, variable in length, light yellow, on thoracic segments medium or relatively long, on abdominal segments I-IX short or medium. Prothorax ( Figure 16B) with eleven prns (six long and one short placed on premental sclerite), next three close to spiracle; two medium ps and one short eus. Meso-and metathorax ( Figure 16B) with one medium prs, three medium pds of equal length, one medium as, three medium ss, equal in length, one long eps, one long ps and one short eus. Pedal area with three pda, long or medium. Abdominal segments I-VIII ( Figure 16C, D) with one short prs, four short pds arranged along the posterior margin, two minutess, one short eps, one short ps, one short lsts and two short eus. Abdominal segment IX ( Figure 16D) with three short ds, all located close to posterior margin, one short ps and two rather short sts. Anal lobes without setae.
Body moderately elongated, light yellowish. Rostrum moderately stout, about 2.1 times as long as wide, reaching up to mesocoxae. Antennae relatively short. Pronotum 1.6 times as wide as long. Mesonotum distinctly shorter than metanotum. Urogomphi (ur) short, conical, with sclerotised, sharp apex, slightly reaching outline of the body, directed downward ( Figure 18A-C).
Biological notes. Larvae feed on various species of Plantago, but mainly on P. media L. and P. maritima L. Plantago lanceolata, P. coronopus L., and P. major L. are also known as host plants. The adults are active from mid-spring following the growth of the flowering stems of the host plant. The female oviposits inside the upper parts of the flowering stem that are covered with floral spikes, which induces clearly visible oblong galls. Very often, several larvae develop in a single flowering shoot. The larvae pupate inside the galls and the adults emerge during summer. Overwintering takes place in the soil litter near the host plant.
Remarks. This species, which is widely distributed in the Palearctic region except in North Africa (Alonso-Zarazaga et al. 2017), is unique in Mecinus, being characterised by long elytra and whitish to orange, wide scales covering the base of the pronotum, the epimera and the episterna. For the differences from the immatures of the other species, see the remarks for the group.
(1) body very slender and elongated; (2) urogomphi rather elongated, distinctly reaching outline of the body, directed outside; (3) rostrum elongated and slender; (4) setae more or less elongated; (5) head with one vs, two sos, two os; (6) rostrum with one or two pas and without or with one rs; (7) pronotum with two as, one ds, two sls, two ls, three or four pls; (8) meso-and metanotum with two or three setae; (9) abdominal segments I-VII dorsally with six or seven elongated, growing setae.
Remarks and comparative notes. The adult of this species is characterised by elytra distinctly elongate, dorsal integument black or blue, sometimes with metallic reflections.
The shape of the body together with the colour of the dorsal integument are characters that this group shares only with M. heydenii. These two groups include the species of Mecinus not living on Plantago. Nevertheless, they seem to be not closely related on the basis of both a phylogenetic study of the adults and of molecular data as well as the examination of the immatures. The adults of the species related to M. janthinus are distinguishable from those related to M. heydenii by the less curved rostrum in lateral view, the shape of the penis, the distinctly longer flagellum and the completely unusual shape of the spermatheca that is reminiscent of the Cionini. The larvae and pupae differ in a series of characters in the chaetotaxy. Moreover, the immatures of this group possess some autapomorphies, i.e., in larvae four pds and usually three ss on the abdominal segments I-VIII and the surface of postlabium densely covered with asperities, and in pupae, the more or less elongated setae on the body and the abdominal segments I-VII dorsally with six or seven elongated, growing setae. Body ( Figure 19A-D) yellowish, very slender, densely covered with asperities. Prothorax smaller than meso-and metathorax. Abdominal segments I-V of almost equal length; segments VI-IX decreasing gradually to the terminal body part; segment X reduced to three anal lobes of those lateral are the largest, and dorsal the smallest (sometimes absent). Chaetotaxy well developed, setae capilliform, variable in length, greyish or yellow. Prothorax ( Figure 19B) with eight long prns of equal length; two long ps and one short eus. Meso-and metathorax ( Figure 19B) with one very short prs, three pds, variable in length (pds 1 short, pds 2-3 medium), one short as, three short ss, one long eps, one long ps and one long eus. Pedal area with five long pda. Abdominal segments I-VIII ( Figure 19C, D) with one very short prs, four pds of different length (on segments I-V: pds 1-2 short, pds 3-4 long; on segments VI-VIII all pds very long, almost equal in length) and arranged along posterior margin; one minute and two medium ss, one short and one long eps, one long ps, one long lsts and two medium eus. Abdominal segment IX ( Figure 19D) with four very long ds, all located close to the posterior margin, two long ps and two short sts. Each of lateral anal lobe with two minute setae.
Body elongated, white. Rostrum slender, about 3.4 times as long as wide, reaching almost up to mesocoxae. Antennae slender and elongated. Pronotum 1.1 times as wide as long. Mesonotum slightly shorter than metanotum. Urogomphi (ur) slender and elongated, conical, with sclerotised apex, distinctly reaching outline of the body, directed outside ( Figure 21A-C).
Chaetotaxy well developed, setae short or medium long. Head with one vs, two sos, two os and two pas. Rostrum with one rs placed medially. All setae of head equal in length ( Figure 21A, B). Pronotum with two as, one ds, two sls, two ls, and three pls ( Figure 21B, C). All setae on pronotum elongated, equal in length ( Figure 21C). Dorsal parts of meso-and metathorax with two setae placed medially. Abdominal segments I-VIII with two setae laterally and three medium long setae ventrally. Dorsal parts of abdominal segments I-VII with six setae (d 1 placed anteromedially, d 2-4 placed posteromedially, d 5-6 posterolaterally); segment VIII with five very long setae dorsally. Abdominal segment IX with two micro-setae ventrally.
Biological notes. The host plant of M. janthinus is the yellow toadflax, Linaria vulgaris Mill. This species is distributed in temperate regions of the eastern Palearctic region, inhabiting lowlands and hilly slopes up to 500 m altitude. From beginning of the 1990s, M. janthinus was introduced as biological control agent for the control of invasive toadflaxes in North America ). The adults emerge in early March and feed intensively on the newly growing shoots of the host plant. Oviposition occurs on actively growing shoots, and the preferred oviposition site is the widest part of the stem. Females lay one or, rarely, two eggs per shoot. This species is a true stem borer with larval feeding and mining in the central part of the stem. The adults overwinter in the stems of the host plant inside an elongated pupal chamber built by the last instar larva prior to pupation.
Remarks and comparative notes. Mecinus janthinus is largely distributed in northern, central and southeastern Europe, Russia from the western borders to southern central Siberia, the Caucasian states, and Turkey. This species was introduced in North America for the biological control of toadflaxes in 1991-1999 (Wilson et al. 2005). The adults can be easily confused with M. janthiniformis, both sympatric in part of their range of distribution, since the differences between them are few and subtle. In contrast, the larvae of these two species show numerous differences in the number of setae in many parts of the body, such as the head, antenna, pronotum and thoracic segments (see key). Body ( Figure 22A-D) yellowish. Prothorax smaller than meso-and metathorax. Abdominal segments I-VII of almost equal length; segments VIII and IX decreasing gradually to the terminal body part; segment X reduced to three anal lobes of those lateral are the largest, and dorsal the smallest (sometimes absent). Dorsum of abdominal segments I-VI divided into three lobes; on seventh into two lobes. Chaetotaxy well developed, setae various in length. Prothorax ( Figure 22B) with eleven long prns; two medium ps and one medium eus. Meso-and metathorax ( Figure 22B) with one short prs, three pds (pds 1 short, pds 2-3 medium), one medium as, three medium ss, one medium eps, one medium ps and one medium eus. Pedal area with six pda of different length (four of them placed on well isolated pedal sclerite). Abdominal segments I-VIII ( Figure 22C, D) with one short prs, four pds (on segments I-V: pds 1,3 medium, pds 2,4 short; on segments VI-VIII all pds very long, equal in length), always arranged along the posterior margin, one minute and two long ss, one short and one medium eps, one medium ps, one medium lsts and two medium eus. Abdominal segment IX ( Figure 22D) with four long ds located close to posterior margin, two ps different in length, and two short sts. Each of anal lobe (abd. segment X) with two minute setae.
dms of almost equal size, five vms different in length. Maxillary palpi: basal palpomere wider than distal, both of almost equal length. Prelabium ( Figure 23F) cup-shaped with one very long prms; ligula with two relatively long ligs; premental sclerite clearly visible, cup-like. Labial palpi two-segmented; basal palpomere distinctly wider than distal, both almost equal in length. Postlabium ( Figure 23F) with three medium pms.
Body elongated, white. Rostrum slender, about four times as long as wide, reaching almost up to mesocoxae. Antennae slender and elongated. Pronotum 1.3 times as wide as long. Urogomphi (ur) slender and elongated, conical, with sclerotised apex, distinctly reaching outline of the body, directed outside ( Figure 24A, C).
Chaetotaxy well developed, setae medium long or elongated, unequal length. Head with one long vs, two sos different in length, two os different in length and two pas different in length. Rostrum with one rs ( Figure 24A). Pronotum with two as, one ds, two sls, two ls, and three pls ( Figure 24B, C). All setae on prothorax elongated, equal in length ( Figure 24C). Setae on head and rostrum shorter than those on prothorax. Dorsal parts of meso-and metathorax with three setae placed medially. Abdominal segments I-VIII with two setae placed laterally and three medium long setae ventrally. Dorsal parts of abdominal segments I-VII with six setae (d 1-4 placed posteromedially, d 5-6 posterolaterally); segment VIII with five very long setae dorsally. Abdominal segment IX with two micro-setae ventrally.
Biological notes. The host plants of M. janthiniformis are Linaria genistifolia (L.) Mill. and L. dalmatica (L.) Mill., as well as all variable forms and hypothetical hybrids between these two plant species. Mecinus janthiniformis inhabits stands from lowlands to mountain pastures and meadows up to 1500 m. At the beginning of the 1990s, this species was introduced as a biological control agent for the control of invasive toadflaxes in North America . Adults emerge in early spring and feed on the apical part of newly growing shoots. The females lay eggs over the next three months on the upper part of the main stem, including the lateral branches of the plant. Oviposition and larval development induce a slightly elongate gall in which the larvae pupate. The adults of this species overwinter inside the main stem of the host plant or inside induced galls on lateral branches (Toševski et al. 2011).
Remarks and comparative notes. The distribution of M. janthiniformis follows that of the two host plants, L. genistifolia (L. ) Mill. and L. dalmatica (eastern part of central and southeastern Europe to southern central Siberia, the northern Caucasian states and Turkey). Its separation from M. janthinus at the species level was clearly shown based on very careful biological and genetic studies (Toševski et al. 2011), but unfortunately, easy identification is only possible by collecting the specimens together with their host plants. Usually, in M. janthiniformis, the body is larger (length 3.2-6.0 mm), the apical part of the rostrum in females in lateral view is more curved, the punctures of the pronotum are slightly smaller and more densely adpressed, and the scales of the elytral interstriae are denser, arranged in two rows on part of several interstriae. The larvae of these two species show numerous differences in the number of setae in many parts of the body, whereas the differences are few in the pupae (see keys). Body ( Figure 25A-D) yellowish, slender. All thoracic segments almost equal in length. Abdominal segments I-V of almost equal length; segments VI-IX decreasing gradually to the terminal body part; segment X reduced to three anal lobes of those lateral are the largest, and dorsal the smallest (sometimes absent). Chaetotaxy weakly developed, setae capilliform, variable in length, yellow. Prothorax ( Figure 25B) with ten prns of unequal length (seven medium length, three short); two medium ps and one medium eus. Meso-and metathorax ( Figure 25B) with one very short prs, three pds, different in length (pds 1,3 very short, pds 2 medium); one short as, three short ss, one medium long eps, one medium long ps and one medium eus. Pedal area with five pda, different in length. Abdominal segments I-VIII ( Figure 25C, D) with one short prs, four pds of different length (pds 1,2,4 short, pds 3 medium; all pds on segments VI-VIII very long, equal in length) arranged along the posterior margin, one short and one medium ss, two medium eps, one medium ps, one medium lsts and two relatively long eus. Abdominal segment IX ( Figure 25D) with three very long ds, all located close to the posterior margin, one medium ps and two medium sts. Each of lateral anal lobe with two minute setae.

Description of pupa
Body elongated, white. Rostrum moderately slender, about 3.5 times as long as wide, reaching up to mesocoxae. Antennae elongated. Pronotum 1.8 times as wide as long. Urogomphi (ur) slender, conical, with sclerotised apex, both directed outside, distinctly reaching outline of the body (Figure 27A-C).
Chaetotaxy setae medium or elongated. Head with one vs, two sos and two os. Rostrum with one pas. Setae on head and rostrum straight, as long as those on prothorax ( Figure 27A). Pronotum with two as, two sls, two ls, one ds and four pls. Dorsal parts of meso-and metathorax with three setae equal in length setae placed medially. Abdominal segments I-VIII with three very short setae ventrally and two setae laterally. Dorsal parts of abdominal segments I-VII with six setae growing gradually from segment I to VII (d 1 placed anteromedially, d 2-5 placed posteromedially, d 6 posterolaterally); segment VIII with five elongated setae dorsally. Abdominal segment IX with two micro-setae ventrally.
Biological notes. The host plant of this species, at least in Côte d'Azur, is Antirrhinum latifolium Mill. As reported by Caldara and Fogato (2013), larvae feed on the larger stems of the plant and dig tunnels, causing at most very small lateral deformations. They pupate in summer, and adults stay inside the plant until the spring of the following year. Before pupation, however, the mature larvae leave the main tunnel, which runs longitudinally, and produce a small oblique tunnel that ends just in proximity of the external cuticle of the stem. Therefore, when leaving their cells, adults have only to bore a thin layer, although in the meantime, the plant has become dry and hard.
Remarks and comparative notes. The adults of this rare species, with a narrow range of distribution -in fact, it is known in a few localities of southeastern Spain, eastern and southern France, and north-western Italy -differ from the other species studied here by the black elytra instead of blue. Due to this character, this species may be superficially confused with M. pyraster, from which it is easily distinguishable by the elytral vestiture composed of scales that are uniformly arranged and are all recumbent. The rostrum (in dorsal view) is distinctly wider, and the pronotum has sides slightly more rounded and is usually widest towards the middle. Finally, ventrite 5 of the male lacks a tuft of hairs, and the shape of the penis is different.
The larvae of this species differ from the others of the group by the abdominal segments I-VIII with two ss (instead of three) and asperities covering only the posterior part of postlabium, whereas pupae differ in having the rostrum with only one pas (instead of two) and without rs and the pronotum with four pls (instead of three).
(1) body very slender and elongated; (2) urogomphi short, only slightly reaching outline of the body, directed downward; (3) rostrum slender and elongated; (4) setae minute; (5) head with one os; (6) rostrum with without or with one pas and one rs; (7) pronotum with without or with up to two as, one ds, without or with one sls, without or with one ls, three pls; (8) meso-and metanotum with three setae; (9) abdominal segments I-VII with three or five setae dorsally.
Remarks and comparative notes. The adults of this group are characterised by the rostrum in the basal half strongly and abruptly curved, the elytra distinctly elongate, and the dorsal integument black or blue, usually with metallic reflections apart from several characters of the male and female genitalia. In immatures, the autapomorphies seem limited to a U-shaped premental sclerite in larvae.
This group seems more closely related to the M. janthinus group than to other groups of Mecinus in both morphological characters (shape of body and colour of dorsal integument) and biology (hosts in Plantaginaceae other than Plantago). However, the species of the M. heydenii group clearly differ from those of the M. janthinus group by the rostrum being strongly curved in the basal half and by the shape of the penis and spermatheca. The study of immatures also did not show close relationships between these two unique groups living on Antirrhineae, since the species of the M. heydenii group have one palpomere on the labial palpi instead of two, and all spiracles are unicameral. The pupae also differ somewhat in the shape of the urogomphi, which are shorter, only slightly reaching the outline of the body, and directed downward. The setae of the head and pronotum are also shorter, and the dorsal setae of abdominal segments I-VII are less numerous.  -alar, ds -dorsal, eps -epipleural, eus -eusternal, lsts -laterosternal, pdapedal, pds -postdorsal, prns -pronotal, prs -prodorsal, ps -pleural, ss -spiracular, sts -sternal, ts -terminal. 28B) with one short prs, three short pds, one very short as, three minute ss, one very short eps, one very short ps and one very short eus. Pedal area with four very short pda. Abdominal segments I-VIII ( Figure 28C, D) with one very short prs, three very short pds arranged along the posterior margin, two minute ss, two very short eps, one very short ps, one very short lsts and two very short eus. Abdominal segment IX ( Figure 28D) with two ds (one medium and one very short), all located close to the posterior margin, one medium ps and two very short sts. Each lateral anal lobe with two minute setae.

Mecinus heydenii
Head capsule (Figures 28A, 29A-F) pale yellow, distinctly narrowed bilaterally. Frontal suture poorly visible. Des 1-3,5 very long, equal in length; des 4 four times shorter than other des 1 . Fs 1 and fs 2 absent, fs 3 very short, fs 4 and fs 5 long. Les 1 shorter than les 2 ; two ves and four pes short ( Figure 29A). Antennae ( Figure 29B) with sensorium (Se) conical, thrice as long as wide, and three sensilla of different types: one sa and two sb. Clypeus ( Figure 29C) trapezium-shaped, anterior margin slightly concave; cls 2 relatively long; clss clearly visible. Labrum ( Figure 29C) with sinuate anterior margin; lrs 1 long, lrs 2 slightly shorter than lrs 1 , lrs 3 two times shorter than lrs 1 . Epipharynx ( Figure  29D) with three medium, finger-shaped als of almost equal length; two rod-like, different in length ams; two finger-like mes of medium length; surface smooth; labral rods close to kidney-shaped. Mandibles ( Figure 29E) conical, wide, with a small protuberance in the middle of the cutting edge; both mds capilliform, medium, equal in length, placed mediolaterally. Maxilla ( Figure 29F) with one stps and two pfs long, of equal length; mbs very short; mala with six finger-like dms different in length (dms 1,2 medium, dms 3-6 long to very long), five vms different in length. Maxillary palpi: basal palpomere distinctly wider than distal, both of almost equal length. Prelabium (Figure 29F) almost rounded with one very long prms; ligula with two ligs different in length; premental sclerite clearly visible, U-shaped. Postlabium ( Figure 29F) with short pms 1 , long pms 2 , and short pms 3 .
Body elongated, white. Rostrum slender, about five times as long as wide, but reaching up only to procoxae. Antennae slender and elongated. Pronotum 1.4 times as wide as long. Urogomphi (ur) very short, conical, with sclerotised apex, reaching outline of the body, directed downward ( Figure 30A-C).
Chaetotaxy sparse, setae short, unequal length. Head with one os. Rostrum with one rs placed medially. Setae on head and rostrum straight, as long as those on prothorax ( Figure 30A). Pronotum with two as, one ds and three pls. Dorsal parts of meso-and metathorax with three setae placed medially. Abdominal segments I-VIII with one seta laterally and three very short setae ventrally. Dorsal parts of abdominal segments I-VII with five setae (d 1 placed anteromedially, d 2-4 posteromedially, d 5 posterolaterally); segment VIII with four setae dorsally. Abdominal segment IX with two micro-setae ventrally.
Biological notes. This monophagous species is associated with yellow toadflax, Linaria vulgaris Mill. The adults are active from early spring, following the appearance of the first growing shoots of its host plant. The adults exhibit extreme cryptic behavior, which makes them difficult to collect. Oviposition occurs on actively growing young shoots, usually in the top or middle part of the stem. Females often lay several eggs distributed along the host plant shoot. Oviposition provokes primi- Figure 29. Mecinus heydenii mature larva, head and mouth parts A head, frontal view B antenna C clypeus and labrum, dorsal view D epipharynx e left mandible F maxillolabial complex, ventral aspect. Abbreviations: at -antenna, clss -clypeal sensorium, des -dorsal epicranial, lr -labral rods, sa -sensillum ampullaceum, sb -sensillum basiconicum, Se -sensorium, st -stemmata, setae: als -anterolateral, amsanteromedial, cls -clypeal, dms -dorsal malar, fs -frontal, ligs -ligular, lrs -labral, ls -lateral epicranial, mbs -malar basiventral, mds -mandibular, mes -median, mxps -maxillary palp, pes -postepicranial, ves -ventral, pfs -palpiferal, pms -postlabial, prms -prelabial, stps -stipal, vms -ventral malar.  ur -urogomphi, setae: as -apical, d -dorsal, ds -discal, fes -femoral, l, ls -lateral, os -orbital, plsposterolateral, rs -rostral. tive shoot swelling and hypertrophy that leads to the formation of a pseudo-gall of the young shoot. Larval development occurs inside this pseudo-gall, and pupation takes place in larval chambers prepared very close to the stem surface. Emerged adults stay inside the stem until August, when all adults leave their host plant within a two-week period. Overwintering takes place in the soil litter near the host plant.
Remarks and comparative notes. This species is widely distributed in Europe and is the only one of its group present in northern Europe, from Germany to Sweden. The adult is distinguishable by the rostrum very strongly curved from base to apex, especially before antennal insertion, in both sexes. However, it is somewhat difficult to morphologically separate this taxon from the two cryptic species M. peterharrisi and M. laeviceps. They are well distinguishable, however, by molecular and biological data (Toševski et al. 2014).
The study of the immatures allowed us to add numerous other interesting differences: larvae of M. heydenii differ from those of M. laeviceps by the pronotum with eight prns (instead of nine), the thoracic segments with three pds (instead of two), each pedal lobe with four pda (instead of three), pds of abdominal segments I-VIII distinctly smaller, and the head with four pes (instead of one). Both species differ from M. peterharrisi by fs 1 and fs 2 absent and the antennae with two sb (instead of four).
The pupae of the three species are also slightly different in the presence or lack of some setae on the rostrum and pronotum (without pas and ls in M. heydenii and M. peterharrisi, respectively) and femora (with fes in M. heydenii and M. laeviceps), and their number in the abdominal segments.
Body elongated, white. Rostrum slender, about four times as long as wide, reaching up to mesocoxae. Antennae slender and elongated. Pronotum 1.5 times as wide as long. Urogomphi (ur) very short, conical, with sclerotised apex, only slightly reaching outline of the body, directed downward ( Figure 33B, C).
Chaetotaxy sparse, setae short, unequal length. Head with one os. Rostrum with one rs and one pas. Setae on head and rostrum straight, as long as those on prothorax ( Figure 33A). Pronotum with one as, one ls, one ds and three pls. Dorsal parts of mesoand metathorax with three setae placed medially. Abdominal segments I-VIII with three very short setae ventrally, distributed in regular lines. Dorsal parts of abdominal segments I-VII with five setae (d 1 placed anteromedially, d 2-4 posteromedially, d 5 posterolaterally); segment VIII with four setae dorsally. Abdominal segment IX with two micro-setae ventrally.
Biological notes. This is a monophagous species associated with broomleaf toadflax, Linaria genistifolia (L.) Mill. The adults are active from early spring, following the appearance of the growing shoots of its host plant. The adults feed intensively on shoot points and apical leaves. Females oviposit batches of 3-6 eggs into the lower to middle part of the young growing shoots. Larval development usually induces stunted growth in the young shoot. The larvae develop in the central part of the stem, forming a relatively short tunnel and the formation of a pseudo-gall in which pupation takes place in the larval chamber very close to the stem surface. Like M. heydenii, the adults stay inside the stem until mid-August, when all adults leave their host plants. Adults overwinter in the soil close to the host plant.
Remarks and comparative notes. This species, found only in the Balkans (Macedonia, Greece, Montenegro) as above reported, is very similar to M. laeviceps, from which it differs by the body being more robust and usually longer, the integument of the pronotum more distinctly bluish, and the penis with longer tip. Moreover, the vestiture is usually formed by slightly broader scales and is therefore generally more distinct. As reported in the remarks on M. heydenii and in the keys, the study of the immatures has revealed other interesting differences between these three species that are very useful for their separation.

Comparison with immature stages of known Mecinini
The present detailed descriptions of the immature stages of 12 species of Mecinus constitute a good sample, comprising approximately 20% of the known species of this genus, allowing a comparison with other genera within the tribe Mecinini. Unfortunately, some descriptions previously published on immature stages of species belonging to other genera of Mecinini are somewhat problematic because of missing details about the chaetotaxy and/or the absence of quality drawings (see Skuhrovec et al. 2018), making such comparisons still difficult. Only the recent descriptions of six Cleopomiarus and three Miarus species (Skuhrovec et al. 2018;Szwaj et al. 2018), three Gymnetron species (Jiang and Zhang 2015), and two Rhinusa species (Gosik 2010;Ścibior and Łętowski 2018) were sufficiently complete for such a comparison. Skuhrovec et al. (2018) emphasised that the taxonomical interpretation of some characters (e.g., thoracic and abdominal dorsal setae) in the papers above is very disputable. This can cause an incorrect differential diagnosis and preclude the composition of a key to the tribe. Our new data might be able to resolve some of these uncertainties.
The number of palpomeres of the labial palpi was shown to be one of the most important morphological characters of larvae in this tribe (Skuhrovec et al. 2018). Some Gymnetron species have only one palpomere (May 1993;Jiang and Zhang 2015), but the basal state in weevils is the presence of two palpomeres on labial palpi (Marvaldi 1997). Mecinus species can be clearly separated into two groups based on the presence of both states. Some species groups have the plesiomorphic condition, with two palpomeres on the labial palpi, but other species groups have only one palpomere, such as Gymnetron species. A completely different situation has been observed in some Cleopomiarus species (Skuhrovec et al. 2018), where there is not a distinct separation of the basal palpomere from the labium, and it can appear to be only one palpomere. This state in Cleopomiarus and partially in Miarus could be an intermediate stage in the reduction to Gymnetron species. This should also be compared with a higher number of Rhinusa species, and only then the evolutionary history of this character in the whole tribe can be discussed. Skuhrovec et al. (2018) suggested that the number of air tubes of the thoracic and abdominal spiracles is the next crucial genus-specific character in Mecinini larvae. Larvae of Mecinus species have two states of this character: (1) all spiracles unicameral, as in Gymnetron species (Jiang and Zhang 2015), or (2) thoracic spiracle bicameral and abdominal spiracles unicameral, as in some Rhinusa (Anderson 1973;May 1993). A completely unique situation is seen in all known larvae of Cleopomiarus and Miarus species, which have bicameral spiracles on the thorax and abdomen.
Another disputable state is the number of epipharyngeal setae (especially ams and mes), which is not yet completely resolved in Curculionidae and was also discussed several times for different groups, e.g., Lixinae (Gosik and Skuhrovec 2011;Stejskal et al. 2014;Trnka et al. 2015). In our view, the final decision of the number of each seta is important, but not crucial, and the comparison between groups/genera should be made together for all three of these epipharyngeal setae in order to make fewer mistakes in the creation of a differential diagnosis for genera in the tribe.
The last important characteristic observed within the Mecinini tribe is the integument of the body with distinct asperities (Skuhrovec et al. 2018). This feature is very variable within each genus (Mecinus, Cleopomiarus, Miarus), probably due to specific environmental conditions within plant tissues. This feature may be discussed after other detailed descriptions are made within the Mecinini tribe.

Comparison of the immature stages of Mecinus species groups
Before this study, larvae of only four Mecinus species had been described -M. pascuorum, M. pyraster, M. heydenii, and M. janthinus (Emden 1938;Scherf 1964;Anderson 1973), while a description of pupae was available for only three of these species (Scherf 1964;Anderson 1973). Unfortunately, these descriptions did not include the chaetotaxy, with a few exceptions, and included only general characteristics, such as the number of teeth on the mandible or the colouration of the head and body. Therefore, a detailed description of all four species has been necessary for their incorporation into our key.
The main differential characters in larvae and pupae among known species are presented in the diagnosis of species groups and in the keys. The 12 species described here belong to five groups and one complex of the seven groups and two complexes detected by  on the basis of morphological and biological apomorphies. It should be recalled that these authors defined their complexes as an assemblage of species formed by several taxa, which are mostly very similar to each other but lacking synapomorphies in contrast with the species forming the groups. Therefore, we now have the opportunity to assess whether the differences found in the immatures are able to confirm or refute the conclusions obtained after a phylogenetic study of the imagoes.
Based on several unique characters, we can confirm that all six assemblages of species obtained in the present study completely agree with those based on adult characters. Moreover, we found distinctive characters in the previously considered "complex" of M. pascuorum, which can now be considered a "true" group.
The main differential characters in larvae among the known species include the following: (1) the number of palpomeres of the labial palpi, (2) the number of air tubes of the thoracic and abdominal spiracles, and (3) the shape of the head and the number of stemmata on the head. The combination of the states of these three characters can easily separate all species groups. Only future studies of the whole tribe together with adult morphology and biological information may identify the values of each character and verify its effect on evolution within this tribe. Skuhrovec et al. (2018) reported that fewer genus-specific character states in larvae than in pupae, which are more conservative in chaetotaxy, were also shown in another tribe of the Curculioninae (Tychiini) with regard to the genera Tychius Germar, 1817 and Sibinia Germar, 1817 (see Skuhrovec et al. 2014Skuhrovec et al. , 2015Gosik et al. 2017).
According to the two above-mentioned main characters of larvae, the groups of Mecinus could be assembled as follows: (1)  Species of the genus Mecinus feed on different genera of host plants in two different tribes belonging to the family Plantaginaceae: Plantago (Plantagineae) and Linaria, Antirrhinum and Anarrhinum (Antirrhineae). The M. heydenii group and the M. janthinus group include all the species of Mecinus living on Antirrhineae. Until now, this ecological character was the unique putative synapomorphy that allows the assemblage of these two groups, although these species are clearly similar overall, and only these groups include species with blue elytral integument. This character is not possessed by any other Mecinini, and the other species of these groups have black elytral integument. In contrast, most species living on Plantago, at least in part, have reddish integument.  did not find other consistent synapomorphies that allow the separation of the M. janthinus + M. heydenii groups feeding on Antirrhineae from all other species living on Plantago. Similarly, a molecular study did not relate these two groups which possibly evolved in parallel (I. Toševski, unpublished data).
Unfortunately, the morphology of the immatures does not seem to shed more light on this situation. In fact, the two groups living on Antirrhineae share only the following two characters in larvae: (1) head brown, distinctly narrowed bilaterally, with two pairs of stemmata and (2) endocarina reaching 4/5 of the frons. The characters of the head are also possessed by the M. circulatus group, which, in contrast, has the endocarina reaching only half of the frons. With regard to the pupae, the relationship of these three groups might be suggested by abdominal segments I-VII dorsally with setae. However, if the available data are assembled, one could assume that the M. janthinus + M. heydenii groups are not monophyletic sharing only a few homoplasies and suggesting that the switch from Plantaginaceae s. str. to Antirrhinae occurred independently in both the M. heydenii and M. janthinus species groups. It is noteworthy that the species of Mecinus that share an unusual elongated body, i.e., M. circulatus, M. janthinus and M. heydenii, are generally stem borers, with larval feeding and mining in the central part of the stem producing no externally visible damage or small external galllike deformations -except for M. dorsalis Aubé, 1850 of the M. heydenii group, which produces globose galls -suggesting that the elongated body is an adaptative character for their ecological niche. At present, the only known gall-inducing species living on Plantago is M. collaris, which appears morphologically distinct from all these species.

Differences between immatures at the species level
All the studied immatures have characters that allow us to distinguish them from each other. Whereas it was expected that very characteristic species such as M. collaris or M. labilis belong to different groups on the basis of the morphology of the adult, it is also true for species within the same group. Moreover, it is even more noteworthy that cryptic species, such as M. janthinus and M. janthiniformis and M. heydenii, M. peterharrisi and M. laeviceps, are clearly distinguishable by the morphological characters of the immatures. Differences were found in the lack or presence and, in the latter case, the number of setae both in larvae (head, pronotum, thoracic and abdominal segments) and pupae (rostrum, pronotum, abdomen).

Biological and evolutionary considerations
It appears probable that all Mecinus species live on Plantaginaceae. The majority of them feed on species of Plantago, whereas a quarter of Mecinus species live on Antirrhineae, especially Linaria and occasionally Antirrhinum and Anarrhinum. As reported above, neither the study on adults by  nor ours on the immature stages have found consistent synapomorphies that allow the separation of the M. janthinus + M. heydenii groups feeding on Antirrhineae from all other species living on Plantago. It is noteworthy that no mecinines other than Mecinus live on Plantago , whereas several species belonging to Rhinusa, the sister-group of Mecinus, live on Antirrhineae (Caldara et al. 2010). These data as a whole tend to suggest that Mecinini, during their evolution, switched more than once from Plantagineae to Antirrhineae or vice versa, and that this switch could easily have occurred independently in both the M. heydenii and M. janthinus species groups.
The larvae of closely related species of Mecinus seem to differ in their modes of parasitism, although less significantly than Rhinusa larvae (see Caldara et al. 2010). Concerning the species living on Plantago, the apparently more primitive species belonging to the M. pascuorum group feed on pyxidia without producing externally visible damage. In contrast, Mecinus circulatus and M. pyraster of the M. circulatus group are stem borers with larval feeding and mining in the central part of the stem, producing no externally visible damage or small external gall-like deformations. At present, the only known gall-inducing species living on Plantago is M. collaris, which appears morphologically distinct from all other species. The situation of the M. simus group is very interesting: whereas M. pirazzolii feed on seeds, M. comosus Boheman, 1845 tunnels into the central axis of the spike of Plantago maritima (Prena and Caldara 2017). With regard to the species living on Antirrhineae, the same variability in biological habits is found in the M. heydenii group: from M. heydenii producing weak deformation of the stems to M. dorsalis producing globose galls. In contrast, in the M. janthinus group, there are true stem borers that produce no visible damage (M. janthinus and M. barbarus) or only small gall-like deformations (M. janthiniformis, M. sicardi). One thing seems clear: all the larvae of Mecinus species with elongated bodies develop along stems or, as in M. comosus, along spikes. It is worth noting that the genus Mecinus was created for these elongated species and that the other species with short bodies were described and considered Gymnetron for many years, until Caldara (2001) transferred them from Gymnetron to Mecinus on the basis of several synapomorphies in the male genitalia. It is also noteworthy that it was very simple to identify the species of Mecinus as they were formerly defined, since there are very few Curculioninae with similar elongate bodies. If we now consider the very probable polyphyly of species with long bodies on the basis of morphological, biological and molecular characters, we can speculate that the elongated body is an adaptive character for the particular ecological niche, i.e., the somewhat narrow stems of Plantago and Linaria or the spikes of Plantago. It is noteworthy that stems are not used as niches for the growth of larvae of other Curculioninae, except for Rhinusa asellus (Gravenhorst, 1807) and R. tenuirostris (Stierlin, 1888) (Doğanlar and Üremiş 2014), which are the more elongated species in the R. tetra group that usually feed on the seeds of Verbascum spp.

Conclusions
Our detailed descriptions of the immature stages of the Mecinini species demonstrate their importance for the taxonomy and further study of the phylogenetic relationships within the genera of the tribe Mecinini, although the number of described immatures is still low in comparison with the total number of Mecinus species. This is our second paper about the Mecinini, after that of Miarus and Cleopomiarus. We are confident that the description of immatures of the genera Rhinusa and Gymnetron, which are currently under investigation, will provide an interesting final arrangement to the taxonomy of the tribe.