On the Affinities and Systematic Position of Lachnaeus Schoenherr and Rhinocyllus Germar in the Tribe Lixini (Coleoptera: Curculionidae: Lixinae) Based on the Morphological Characters of the Immature Stages

Simple Summary The detailed descriptions of immature stages of Lachnaeus crinitus Schoenherr, 1826, Rhinocyllus alpinus Gültekin, Diotti and Caldara, 2019 and R. conicus (Frölich, 1792), belonging to the Lixini (Curculionidae: Lixinae), are very important for an increased understanding of the relationship between the species and of the taxonomic and phylogenetic value of the tribes and genera in the Lixinae. The complex of these new data has allowed us to support that (1) Lachnaeus and Rhinocyllus are two valid genera that are different from Larinus, (2) Rhinocyllini is not a tribe different from Lixini, and (3) the separation of Rhinocyllus into two subgenera is rational. In the tribe Lixini, these new data may have an important role, because Rhinocyllus conicus and several other species of the genera Lixus and Larinus have a practical or at least potential use as biological control agents against invasive and noxious weeds. Abstract Mature larvae and pupae of Lachnaeus crinitus Schoenherr, 1826 and Rhinocyllus alpinus Gültekin, Diotti and Caldara, 2019 and pupae of R. conicus (Frölich, 1792), belonging to the Lixini (Curculionidae: Lixinae), are morphologically described for the first time. They possess all the characters considered distinctive in the immature stages of this tribe and are distinguishable from all the related genera by a combination of some characters (e.g., presence of endocarina, shape of premental sclerite; the number of pds on the abdominal segments; size and presence of urogomphi). It is emphasized that the controversial tribe Rhinocyllini is not supported by the characters of the larvae and pupae of Rhinocyllus and that the two subgenera of this genus, Rhinocyllus s. str. and Rhinolarinus, are separable from each other not only by characters of the adult but also distinctive characters of the larvae and pupae. These results confirm that the morphology of the immature stages, which is usually overlooked, can be very important for the purpose of identifying new characters that are useful for clarifying taxonomical and phylogenetic complex situations based only on the study of the imagoes.


Introduction
The cosmopolitan subfamily Lixinae of Curculionidae includes approximately 90 genera and 1500 species [1,2], with the largest number of taxa present in the Palaearctic and Slide preparation followed by May [24]. The larvae selected for study were cleared in 10% potassium hydroxide (KOH), rinsed in distilled water and dissected under the microscope. After clearing, the head, mouthparts, and body (thoracic and abdominal segments) were separated and mounted on permanent microscope slides in Faure-Berlese fluid (50 g of gum arabic and 45 g of chloral hydrate dissolved in 80 g of dissolved water and 60 cm 3 of glycerol) [25].
Photos were taken using an Olympus BX63 microscope and processed in Olympus cellSens Dimension software (Olympus Inc. Tokyo, Japan). The larvae selected for pictures using SEM (scanning electron microscope) were first dried in absolute ethyl alcohol (99.8%), rinsed in acetone, treated by CPD (critical point drying), and then gold-plated. A TESCAN Vega 3 SEM (Tescan, Brno, Czech Republic) was used for the examination of selected structures.

Results
General features: body ( Figure 1) stout, C-shaped, rounded in cross-section. Cuticle of the body densely covered with asperities ( Figure 1).
Coloration. Dorsal parts of all pronotal and abdominal segments I-IX yellowish pigmented, rest of the body greyish ( Figure 1). Head light yellow (Figure 1), frons dark yellow pigmented.
Body prothorax prominent. Meso-and metathorax almost equal in size, each divided dorsally into two lobes (prodorsal lobes medium, postdorsal lobes prominent). Pedal lobes of thoracic segments conical (Figure 2A). Abdominal segments I-II of similar size, the largest segments IIII-IX tapering towards the posterior body end ( Figure 2B,C). Abdominal segments I-VII, each with a relatively narrow prodorsal lobe and always well developed, divided into two equal postdorsal lobes. Dorsal part of abdominal segment VIII undivided. Epipleural, pleural, and laterosternal lobes of segments I-VIII conical, well-isolated. Abdominal segment IX divided into large dorsal, large pleural, and relatively small sternal lobes ( Figure 2C). Abdominal segment X divided into four lobes of various sizes (dorsal the largest, ventral the smallest, and lateral lobes equal in size). Anus situated ventrally ( Figure 2C). The spiracles of thorax look vestigial and on high magnification appear bicameral ( Figure 3A), placed latero-ventrally on prothorax; abdominal spiracles on abdominal segments I-VIII vestigial ( Figure 3B), placed medio-laterally. Chaetotaxy of thoracic and abdominal segments well-developed. Prothorax (Figure 2A) with 20 medium prns, almost equal in length (17 placed on pronotal sclerite, next three above the spiracle); two medium ps and one medium eus. Meso-and metathorax each with three short prs; seven medium pds, equal in length; alar area with two medium as; three ss of various lengths (two short and one minute); one medium eps; one medium ps and one medium eus. Pedal areas of thoracic segments each with eight pda of various lengths. Abdomen ( Figure 2B,C): setae on abdomen seem generally shorter than those on thorax. Abdominal segments I-VIII with three minute prs; six pds (pds 1 , pds 3 and pds 5 medium, pds 2 , pds 4 and pds 6 minute); two ss (ss 1 minute, ss 2 medium; segment VIII with only medium ss 1 ); two minute eps; two minute ps; one minute lsts and two minute eus. Abdominal segment IX with four minute ds; two minute ps and two minute sts. Abdominal segment X with one minute ts on each of the lobes ( Figure 2C). Head capsule rounded ( Figure 4A,B). Endocarinal line absent. Frontal sutures distinct in entire length up to antennae. A single pair of stemma (st) in the form of prominent dark pigmented spots with convex corneas, placed anterolaterally ( Figure 4A). Hypopharyngeal bracon without median sclerome. Setae of head of various lengths, elongated to minute. Des 1 elongated, placed medially; des 2 elongated, placed posterolaterally; des 3 elongated, placed above frontal suture; des 4 medium, placed anteromedially; des 5 elongated placed anterolaterally, and des 6 elongated, placed above stemma. Fs 1 medium, placed posteriorly; fs 2 medium, placed mediolaterally; fs 3 medium, placed medially; fs 4 elongated, placed anteromedially; and fs 5 elongated, placed anterolaterally, close to epistome. Les 1 and les 2 slightly shorter than des 1 . Postepicranial area with four minute pes ( Figure 4B). Antennae ( Figure 5A,B) with oblique positions on each side at anterior margins of the head; membranous basal segment convex, semi-spherical, bearing conical, relatively elongated sensorium and four sensilla ampullaceum (sa). Clypeus ( Figure 6A-C) approximately 3.7 × wider than long, cls 1 and cls 2 medium, equal in length, elongated, sensillum (clss) placed between them; anterior margin of clypeus rounded to inside. Labrum ( Figure 6A-C) approximately 1.7 × wider than long, anterior margin strongly sinuated; lrs 1 and lrs 2 placed anteromedially, lrs 3 placed anterolaterally, all lrs elongated, equal in length. Epipharynx ( Figure 6A,B) with three elongated, finger-like als, of various lengths; three finger-like ams: ams 1 short, ams 2 medium, ams 3 relatively elongated; mes 1 distinctly smaller than mes 2 both placed anteriorly; labral rods (lr) kidney-shaped, placed parallel; a single pair of sensillae pores (snp) arranged in the posterior part; surface smooth. Mandibles ( Figure 6D) with two apical teeth of unequal height, the inner one subapical and much smaller; an additional protruding protuberance on the cutting edge between the apex and the mid-dle of the mandible; mds 1 minute, mds 2 medium; both placed laterally in shallow holes. Maxillolabial complex ( Figure 7A-D) on stipes with one elongated stps, two elongated pfs and one minute mbs. Mala with a row of six elongated, finger-like dms of various lengths and four vms (two medium and two short) ( Figure 7A-D and Figure 8A). Maxillary palpi with two palpomeres; basal palpomeres, some wider than distal; length ratio of basal and distal palpomeres almost 1:1; basal palpomere with one medium mps and two pores, distal palpomere with one pore, one digitiform sensillum (ds) and a group of nine apical sensillae (four ampullaceae (sa) and five basiconicae (sb)) in terminal receptive area (tra) ( Figure 8B). Dorsal parts of mala partially covered with fine asperities. Labium with prementum almost triangular shaped, with one elongated prms placed medially. Ligula concave, semicircular at margin, with a single minute ligs. Labial palpi two-segmented; basal palpomere wider than distal; length ratio of basal and distal palpomeres almost 1:1. Each palpomera with a single pore, distal palpomerae with a group of 14 apical sensillae (five ampullaceae and nine basiconicae) in terminal receptive area ( Figure 8C). Premental sclerite U-shaped; postmentum prominent, membranous, triangular, with three pms of various lengths: pms 1 elongated, located posterolaterally; pms 2 elongated, situated mediolaterally and pms 3 medium, placed anterolaterally; lateral and posterolateral parts covered with minute asperities.

Description of Pupa
Measurements (in mm): body length: 5.00 to 6.50 (mean 6.00). Body width at the widest part: 3.00 to 3.50 (mean 3.40). All results of measurements are given in Table 1.
Body stout, slightly curved ( Figure 9A-C). Dorsal part dark brown, rest of the body

Description of Pupa
Measurements (in mm): body length: 5.00 to 6.50 (mean 6.00). Body width at the widest part: 3.00 to 3.50 (mean 3.40). All results of measurements are given in Table 1.
Body stout, slightly curved ( Figure 9A-C). Dorsal part dark brown, rest of the body yellowish; cuticle covered with fine asperities, smooth only on head and pronotum ( Figure 10D,E). Rostrum short, as long as wide, reaching procoxae. Pronotum twice as wide as long, trapezium-shaped. Mesonotum and metanotum almost equal in size. Abdominal segments I-III of equal length, segments IV-VI tapered gradually towards end of the body, segment VII prominent, segments VIII and IX very small. Spiracles placed dorsolaterally on abdominal segments I-VI; those on segments I-V functional, and that on segment VI vestigial ( Figure 10A-C). Chaetotaxy well-visible, with setae of various lengths from medium to short on the dorsal part of the body placed on prominent protuberances, especially on prothorax and abdominal segments VI and VII. Head with one pas, two os and two sos, rostrum without setae. All setae on head equal in length, short ( Figure 10A-C). Pronotum with three as, three ls, two sls, three ds, and seven pls equal in length ( Figure 10D). Each, meso-and metathorax with nine minute setae (three placed posteromedially, six along anterior margin). Each femora with two medium, hair-like setae ( Figure 10A-C). Abdominal segments I-V with 11 minute setae (three placed posteromedially, eight placed along anterior margin of each of the segments) ( Figure 10E). Abdominal segment VI with 11 setae (three minute placed posteromedially, eight short, placed on protuberances along anterior margin of the segment). Abdominal segment VIII with 10 setae: three minute placed posteromedially, four medium placed anteromedially and three short placed anterolaterally, d 4 -d 7 placed on prominent protuberances ( Figure 10F). Abdominal segment VIII with four medium setae, placed anteromedially on small protuberances.
Each lateral part of abdominal segments I-VIII with two minute setae. Ventral parts of abdominal segments I-VIII without setae. Abdominal segment IX with a pair of short, conical urogomphia ( Figure 10G).  Table 1.
General features: body stout, C-shaped, rounded in cross-section ( Figure 11). Cuticle of the body densely covered with asperities ( Figure 11). Coloration: only pronotal sclerites are brownish; the rest of the thoracic and all abdominal segments are white or greyish ( Figure 11). Head is brown, covered with irregular dark brown stripes.
Vestiture: Setae on body of various lengths from medium to minute, but all of hair form.
Body prothorax is small, with slightly pigmented pronotal shield. Meso-and metathorax are almost equal in size, each divided dorsally into two lobes (prodorsal lobes small, postdorsal lobes prominent). Pedal lobes of thoracic segments are well isolated, conical ( Figure 12A). Abdominal segments I-III of similar size, the largest, segments IV-IX tapering towards posterior body end. Abdominal segments I-VIII with a relatively narrow, prodorsal lobe, and always well-developed, divided into two equal-sized parts of postdorsal lobes ( Figure 12B,C). Epipleural, pleural and laterosternal lobes of segments I-VIII conical, well-isolated. Abdominal segment IX divided into large dorsal folds and relatively small pleural and sternal lobes ( Figure 12C). Abdominal segment X divided into four lobes of various sizes (dorsal the largest, ventral the smallest, and lateral lobes equal in size). Anus situated terminally ( Figure 12C). All spiracles bicameral (with two branches); thoracic ( Figure 13A) placed latero-ventrally on prothorax; abdominal spiracles ( Figure 13B) placed medio-laterally on segments I-VIII. Chaetotaxy of thoracic and abdominal segments welldeveloped. Prothorax ( Figure 12A) with 10 medium and one short prns (eight placed on pronotal sclerite, next three above the spiracle); two medium ps and one medium eus. Mesoand metathorax with one minute prs; four medium pds, equal in length; alar area with one medium as; three ss of various lengths (two short and one minute); one medium eps; one medium ps and one medium eus. Pedal areas of thoracic segments with five pda of various lengths. Abdominal segments I-VIII ( Figure 12B,C) with one minute prs; five pds (pds 1 , pds 3 and pds 5 medium, pds 2 and pds 4 short), two ss (ss 1 minute, ss 2 medium; segment VIII with one medium ss 1 ); two eps (one medium and one minute); two ps (one medium and one minute); one medium lsts and two medium eus. Abdominal segment IX with four medium ds; two ps (one medium and one minute) and two medium sts. Abdominal segment X with two ts equal in size on each of the lobes ( Figure 12C).   Figure 14A). Hypopharyngeal bracon without median sclerome. Setae of head of various lengths, medium to minute. Des 1 placed medially; des 2 placed posterolaterally; des 3 placed above frontal suture; des 4 placed anteromedially; des 5 much shorter than the other des, placed anterolaterally above the stemma. Fs 1 short, placed posteriorly; fs 2 short, placed mediolaterally; fs 3 short, placed medially; fs 4 medium, placed anteromedially, and fs 5 medium, placed anterolaterally, close to epistome. Les 1 and les 2 slightly shorter than des 1 . Postepicranial area with five minute pes ( Figure 14B). Antennae ( Figure 15) with oblique positions on each side at anterior margins of head; membranous basal segment convex, semi-spherical, bearing conical, relatively short sensorium and nine sensilla: five basiconica (sb), one styloconica (ss) and three ampullaceum (sa). Clypeus ( Figure 16A-C) approximately 2 × wider than long, cls 1 short, cls 2 elongated, both placed posterolaterally, sensillum (clss) placed close to cls 2 ; anterior margin of clypeus slightly rounded to inside. Labrum ( Figure 16A-C) approximately 2 × wider than long, anterior margin sinuated; lrs 1 and lrs 2 elongated, both placed anteromedially, lrs 3 short, placed laterally. Epipharynx ( Figure 16A,B) with three finger-like als of various lengths; three finger-like ams: ams 1 medium, ams 2 thin, ams 3 short and thick; mes 1-2 equal in length, thick; labral rods(lr) elongated, more sclerotized at the apex, almost parallel; a single pair of sensillae pores (snp) arranged in the posterior part; surface smooth. Mandibles ( Figure 16D) with two apical teeth of unequal height; single medium mds placed laterally in shallow holes. Maxillolabial complex ( Figure 17A-D) on stipes with one medium stps, two medium pfs and one minute mbs plus sensillum. Mala with a row of seven finger-like dms of various lengths and five vms (three medium and two short) ( Figure 17A-D and Figure 18A). Maxillary palpi with two palpomeres; basal palpomere much wider than distal; length ratio of basal and distal palpomeres almost 1:1; basal palpomere with one short mps and two pores, distal palpomere ( Figure 18B) with one pore, one digitiform sensillum (ds) and a group of 12 apical sensillae (ampullaceae) in the terminal receptive area (tra). Dorsal parts of mala partially covered with fine asperities. Labium with a prementum cup shape, with one medium prms placed medially. Ligula concave, semicircular at margin, with single minute ligs. Labial palpi two-segmented; basal palpomere wider than distal; length ratio of basal and distal palpomeres almost 1:0.7; each palpomerae with a single pore, distal palpomerae with a group of 13 apical sensillae (ampullaceae) in terminal receptive area ( Figure 18C). Premental sclerite trident-shaped (median branch well-sclerotized), posterior extension with elongated, blunt apex; postmentum prominent, membranous, triangular, with three pms of various lengths: pms 1 medium, located posterolaterally, pms 2 elongated, situated mediolaterally and pms 3 short, placed anterolaterally; lateral and posterolateral parts covered with minute asperities ( Figure 18A). digitiform sensillum (ds) and a group of 12 apical sensillae (ampullaceae) in the terminal receptive area (tra). Dorsal parts of mala partially covered with fine asperities. Labium with a prementum cup shape, with one medium prms placed medially. Ligula concave, semicircular at margin, with single minute ligs. Labial palpi two-segmented; basal palpomere wider than distal; length ratio of basal and distal palpomeres almost 1:0.7; each palpomerae with a single pore, distal palpomerae with a group of 13 apical sensillae (ampullaceae) in terminal receptive area ( Figure 18C). Premental sclerite trident-shaped (median branch well-sclerotized), posterior extension with elongated, blunt apex; postmentum prominent, membranous, triangular, with three pms of various lengths: pms1 medium, located posterolaterally, pms2 elongated, situated mediolaterally and pms3 short, placed anterolaterally; lateral and posterolateral parts covered with minute asperities ( Figure 18A).

Description of Pupa
Measurements (in mm): body length: 5.00 to 6.50 (mean 6.00). Body width at the widest part: 3.00 to 3.50 (mean 3.40). All results of measurements are given in Table 1.
Body yellowish, rather stout, cuticle covered with fine asperities, smooth only on head and pronotum ( Figure 19A-C). Rostrum 1.2 × longer than wide, reaching procoxae. Pronotum twice as wide as long, sinuated laterally. Mesonotum smaller than metanotum. Abdominal segments I-V of equal length, segments VI-VII tapered gradually towards end of the body, segment VIII semicircular, segment IX very small. Spiracles placed dorsolaterally on abdominal segments I-VI; those on segments I-V functional, and that on segment VI vestigial.

Description of Pupa
Measurements (in mm): body length: 5.00 to 6.50 (mean 6.00). Body width at the widest part: 3.00 to 3.50 (mean 3.40). All results of measurements are given in Table 1.
Body yellowish, rather stout, cuticle covered with fine asperities, smooth only on head and pronotum ( Figure 19A-C). Rostrum 1.2 × longer than wide, reaching procoxae. Pronotum twice as wide as long, sinuated laterally. Mesonotum smaller than metanotum. Abdominal segments I-V of equal length, segments VI-VII tapered gradually towards end of the body, segment VIII semicircular, segment IX very small. Spiracles placed dorsolaterally on abdominal segments I-VI; those on segments I-V functional, and that on segment VI vestigial.   Chaetotaxy visible, with setae of various lengths from medium to short on abdominal segments VI-VIII placed on small (slightly growing on segment VIII) protuberances. Head with three pas, two os and single sos, rostrum with two rs. All setae on head and rostrum equal in length and medium ( Figure 20A-C). Pronotum with one ls, one sls, two ds, and five pls equal in size. Meso-and metathorax with three short setae placed medially on dorsum. Each femora with two medium, hair-like setae ( Figure 20A). Abdominal segments I-V with eight minute setae (all placed along anterior margins of each of the segments). Abdominal segments VI and VII with eight short setae placed on thorn-like protuberances. Abdominal segment VIII with seven medium setae placed on thorn-like protuberances ( Figure 20D). Abdominal segment IX with four minute setae placed ventrally. Each lateral part of abdominal segments I-VIII with two minute setae. Ventral parts of abdominal segments I-VIII without setae. Urogomphia invisible ( Figure 20E).

Description of Pupa
Measurements (in mm): body length: 4.50 to 7.00 (mean 5.50). Body width at the widest part: 2.10 to 3.40 (mean 2.90). All results of measurements are given in Table 1.
Body greyish or yellowish, slightly elongated, cuticle covered with fine asperities, smooth only on the head and pronotum ( Figure 21A-C). Rostrum almost as wide as long, reaching procoxae. Pronotum 2.25 × wider than long, rounded laterally. Mesonotum and metanotum equal in size. Abdominal segments I-IV of equal length, segments V-VII tapered gradually towards the end of the body, segment VIII semicircular, segment IX small. Spiracles placed dorsolaterally on abdominal segments I-VI; those on segments I-V functional, and that on segment VI vestigial. Chaetotaxy well-visible, with setae of various lengths from medium to short on abdominal segments VII and VIII placed on small protuberances. Head with three pas, two os and three sos, rostrum without setae. All setae on head equal in length, medium ( Figure 22A-C). Pronotum with two ls, three sls, two ds, and four pls equal in size. Meso-and metathorax with three medium setae placed medially on dorsum. Each femora with two medium, hair-like setae ( Figure 22A). Abdominal segments I-VII with seven (segment VIII with six) medium setae (the first placed medially, the next placed along anterior margins of each of the segments). Setae on abdominal segments VII and VIII placed on protuberances ( Figure 22D). Abdominal segment IX with four minute setae placed ventrally. Each lateral part of abdominal segments I-VIII with two minute setae. Ventral parts of abdominal segments I-VIII with three setae. Urogomphia invisible ( Figure 22E).

Remarks on Lachnaeus
Within the tribe Lixini, the immature stages of Lachnaeus crinitus have three unique morphological features: (1) an endocarina is missing (vs. Larinus and Lixus species with endocarina); (2) the spiracle of thorax looks vestigial, and on high magnification, appears bicameral (vs. the spiracle of the thorax is distinctly bicameral and without the need to control it under high resolution); and (3) the entire dorsal parts of the bodies of the larvae and pupae are distinctly pigmented (vs. the dorsal regions of the larvae and pupae, which not pigmented and sometimes have small dark sclerites). According to the morphological features of immature stages, Lachaneus crinitus has a more noticeable affinity with the genus Larinus than with the genera close to the genus Lixus, as also suggested by adult morphology. Larinus species and Lachnaeus crinitus share two unique morphological features: (1) a Ushaped premental sclerite (vs. Lixus species, which have a trident-shaped premental sclerite similar to Rhinocyllus species and some Cleonini species); and (2) abdominal segments I-VII with three folds (vs. four folds).

Remarks on Rhinocyllus
The immature stages of R. conicus and R. alpinus have several specific features, the combination of which is completely unique within the Lixini tribe: (1) a trident-shaped premental sclerite (identical to Lixus species, but other genera have a U-shaped premental sclerite); (2) abdominal segments on larva with five or six pds (vs. more pds on abdominal segments); (3) meso-and metanotum on pupa with only three d (vs. more than five d); and finally, (4) pupa "invisible" urogomphi (vs. distinct or sometimes very small indistinct). Based on our data on Rhinocyllus, the type genus of the tribe Rhinocyllini, although without information on Bangasternus and Microlarinus, we can conclude that this tribe is not supported by unique character states (apomorphies) in the immature stages. The pupae of both species of Rhinocyllus seem to show a closer relationship with the genus Lixus than to Larinus, in contrast to what is suggested by the characters of the adults, because Rhinocyllus species do have "invisible" urogomphi, while Lixus species do not have urogomphi, or they are very small and almost indistinct, unlike the pupae of Larinus and Lachnaeus, which always have quite noticeable urogomphi. Yet, Rhinocyllus is similar to Larinus obtusus and Lachnaeus, having only five or six pds on abdominal segments instead of more pds on abdominal segments, similar to other "large" species of Larinus and Lixus. In this case, however, it is possible that this character might only be related to the shrinking of the body and may not be phylogenetically important, as we have observed in other unrelated tribes, such as the Tychiini of the subfamily Curculioninae [42].
The primary differences between the larvae and pupae of Rhinocyllus alpinus and R. conicus currently are those that distinguish the two subgenera Rhinocyllus and Rhinolarinus and are as follows: (1) des 5 very short (vs. R. conicus with medium des 5 ), (2) des 5 close to des 3 and des 4 (vs. des 5 separate), (3) lrs 2 as long as lrs 1 (vs. lrs 2 twice as long as lrs 1 ), (4) head of the pupa with one sos (vs. with three sos and one vs), (5) rostrum of the pupa with two rs (vs. without rs), and (6) dorsum of the pupal abdomen with eight minute d (vs. with seven short d).

Conclusions
The complex of these new data have allowed us to support that (1) Lachnaeus and Rhinocyllus are two valid genera that are different from Larinus, (2) Rhinocyllini is not a tribe different from Lixini, and (3) the separation of Rhinocyllus into two subgenera is rational. Therefore, it seems obvious why it will be important to increase the exiguous number of species of this tribe with known immature stages in order to address other unresolved questions, as already shown in other tribes or subfamilies (see Otiorhynchinini [43], Cionini [44], Mecinini [45,46], Smicronychini [47], Tychiini [42] and Cossoninae [48]). It is clearly confirmed that a detailed description of immature stages is a very important component for a better understanding of generic taxonomy but also tribal taxonomic and phylogenetic relationships within Lixinae, as well as within other subfamilies (see above). It is not to be neglected that in the Lixini, these new data may have particular relevance, because Rhinocyllus conicus and several other species of the genera Lixus and Larinus have a practical or at least potential use as biological control agents against invasive and noxious weeds (e.g., Carduus, Cirsium, Tanacetum).