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Article On the Phylogenetic Position of the Weevil Tribe Acentrusini Alonso-Zarazaga, 2005 (Coleoptera: Curculionidae: Curculioninae)

Michael Košt’ál 1,* and Peter Vd’aˇcný 2

1 Stˇrelecká 459, 500 02 Hradec Králové, Czech Republic 2 Department of Zoology, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská dolina B-1, Ilkoviˇcova6, 842 15 Bratislava, Slovakia; [email protected] * Correspondence: [email protected]; Tel.: +420-606-729-022

Received: 26 March 2018; Accepted: 4 May 2018; Published: 7 May 2018

Abstract: Based on intrinsic morphological and extrinsic bionomic characters, the systematic position of the weevil tribe Acentrusini Alonso-Zarazaga, 2005 (Coleoptera: Curculionidae: Curculioninae) was determined. Maximum parsimony and Bayesian inference as well as nonmetric multi-dimensional scaling were used to analyze 34 morphological characters of adults, complemented by four host plant characters associated with particular weevil tribes. Sixteen species belonging to two subfamilies (Brachycerinae, Curculionidae) and seven tribes (Acentrusini, Anthonomini, Ellescini, Erirhinini, Smicronychini, Storeini, Styphlini) of the family Curculionidae and one outgroup species (Attelabidae) were studied. Phylogenetic and multi-dimensional analyses revealed the tribe Smicronychini as most closely related to Acentrusini. Of the tribes of Curculioninae studied, Styphlini, Anthonomini and Ellescini showed a certain degree of phylogenetic relation to Acentrusini, whereas Storeini were found to be least related.

Keywords: Coleoptera; Curculionidae; Curculioninae; Acentrusini; phylogeny

1. Introduction The tribe Acentrusini was described quite recently by Alonso-Zarazaga [1]. Until the description of two additional taxa [2], the tribe was monotypical and contained only Cryptorhynchus histrio Boheman, 1837, the type species of the genus Acentrus. Acentrus histrio (Boheman, 1837), A. boroveci (Košt’ál, 2014) and A. zarathustra (Košt’ál, 2014) are morphologically highly similar, apparently forming a monophyletic group [2]. Hence, the tribe is morphologically uniform, showing no subgeneric divergence and was characterized by Alonso-Zarazaga [1] by a body covered with densely arranged light scales; normal, horizontally movable mandibles with teeth on the inner side; antennae with seven funicular segments; eyes more close to each other on the ventral than on the dorsal part of the head; a lower rostrum margin in the lateral view directed to the middle of eye; postocular lobes on the anterior margin of the pronotum; prosternum with emargination; a precoxal distance twice as long as the metacoxal distance; ventrite 2 longer than ventrites 3–4 combined; the distance between metacoxae larger than the metacoxal width; free claws; and other, presumably apomorphic characters. The phylogenetic relation of Acentrusini has not yet been studied. Only Alsonso-Zarazaga [1] suggested a tentative hypothesis of their possible close relationship to Styphlini, without identifying any shared tribal characters. Later, some characters typical of Acentrusini, but also of other tribes, like ventrally contiguous eyes, were reported as descriptive characters without drawing phylogenetic afﬁnities to Acentrusini [3]. The distribution of Acentrusini extends from the Iberian Peninsula and North Africa through the Mediterranean to southern Ukraine, Caucasus, the Middle East, Iran and

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Turkmenistan. All known host plants belong to the family Papaveraceae. Alonso-Zarazaga [1] suspected that Acentrusini is most closely related to Styphlini, however noting the necessity of more detailed analysis of the phylogenetic relationships to this tribe.

2. Materials and Methods

2.1. Taxonomic and Morphological Methods Sixteen weevil species from the family Curculionidae s. l., belonging to seven tribes, and one outgroup species from the family Attelabidae, tribe Rhynchitini (Rhynchites bacchus (Linnaeus, 1758)), were studied. Tribes of the subfamily Curculioninae from the Palaearctic region included in this study were selected based on published (Styphlini) [1] and unpublished assumed phylogenetic relationships to Acentrusini with respect to the morphological similarity to Acentrusini. Characters reported in the detailed redescription of Acentrusini as presumable apomorphies [1] were used as the tribe selection guideline. These characters include scales on the body (head and rostrum), dentation of the mandibles, the number of antennal funicle segments, the dorsal vs. ventral distance of eyes, the direction of the lower rostrum margin in relation to the eye, the presence or absence of postocular lobes and prosternal impression, the ratio of the precoxal and postcoxal distance and the medial length of ventrite 2 and ventrites 3–4 combined, and claw connation at the base [1]. As an additional important character, we consider here the venation of the hind wings. Of the 21 currently reported Palaearctic tribes of Curculioninae [4], only six meet to some extent a substantial part of the characters listed above. Those tribes that are generally not consistent with Acentrusini in most characters of higher taxonomic weight were not included. To support the validity of the phylogenetic tree, we also included three species of the subfamily Brachycerinae, tribe Erirhinini, which might remotely resemble Acentrusini in several plesiomorphies. The taxonomy follows the latest higher taxonomical classification of Curculionoidea by Zarazaga et al. [4]. We included two subfamilies of Curculionidae, Brachycerinae Billberg, 1820 and Curculioninae Latreille, 1802, into the phylogenetic and multi-dimensional phenetic analyses. Brachycerinae were represented by the tribe Erirhinini Schoenherr, 1825, with the following species listed in alphabetical order: Notaris scirpi (Fabricius, 1772), Thryogenes fiorii Zumpt, 1928, and T. scirrhosus (Gyllenhal, 1835). Curculioninae were represented by the following tribes and species reported in brackets, both listed in alphabetical order: Acentrusini Alonso–Zarazaga, 2005 (Acentrus histrio (Boheman, 1837), A. zarathustra Košt’ál, 2014), Anthonomini C.G. Thomson, 1859 (Anthonomus behnei Košt’ál, 2014, Bradybatus seriesetosus Petri, 1912), Ellescini C.G. Thomson, 1859 (Dorytomus taeniatus (Fabricius, 1781), Ellescus bipunctatus (Linnaeus, 1758)), Smicronychini Seidlitz, 1891 (Smicronyx jungermanniae (Reich, 1797), S. reichii (Gyllenhal, 1835), Sharpia sp.), Storeini Lacordaire, 1863 (Pachytychius hordei hordei (Brullé, 1832), P. sparsutus (Olivier, 1807)), Styphlini Jekel, 1861 (Pseudostyphlus pillumus (Gyllenhal, 1835), Trachystyphlus beigerae (Smreczy´nski,1975)). We studied both sexes of well-preserved, mature adult specimens. External characters were always examined in male specimens. All measurements were made under a stereomicroscope (Intraco Micro NSZ-810) using an ocular micrometer. Dissection of genitalia was carried out in both sexes after at least 24 h incubation in a wet chamber. Male genital structures were dissected and treated for ﬁve days in 10% KOH, which was then transferred to water and observed in glycerol. Female genitalia were studied, embedded in Solakryl BMX on a transparent plastic board. The hind wings of A. histrio were photographed embedded in Solakryl BMX, using a high-resolution camera (Canon EOS 50D) under the stereomicroscope in transmitted light. The spiculum ventrale (female eighth sternite) was mounted on a transparent board in Solakryl BMX and photographed under a laboratory microscope (Intraco Micro LMI T PC). Multilayer pictures were processed using the software Combine ZP. The morphologic nomenclature was used according to the latest interpretation [5], following updates of the online glossary of weevil characters proposed in the International Weevils Community Website (18 February 2018) (http:/weevil.info/glossary-weevil-characters) (accessed 14 March 2018). Diversity 2018, 10, x FOR PEER REVIEW 3 of 11

DiversityCommunity2018, 10 Website, 34 (18 February 2018) (http:/weevil.info/glossary-weevil-characters) (accessed3 of 14 11 March 2018). TheThe followingfollowing abbreviationsabbreviations areare used:used: Char.Char. == character, nn == not applicable.

2.2. Characters Used for Phylogenetic and Multi-DimensionalMulti-Dimensional PheneticPhenetic AnalysesAnalyses

2.2.1. Morphological Characters Morphological characterscharacters were were selected selectedde de novo novoaccording according to the to theaforementioned aforementioned criteria criteria and theyand werethey were complemented complemented by hind by hind wing wing venation venation characters. characters The. The habitus habitus of Acentrus of Acentrus histrio histriois shown is shown in Figurein Figure1. 1.

Figure 1. Acentrus histrio. Male. According to [2]. (Not to scale). Copyright confirmed by M. Jäch Figure 1. Acentrus histrio. Male. According to [2]. (Not to scale). Copyright conﬁrmed by M. Jäch (Koleopterologische Rundschau), 30 April 2018. (Koleopterologische Rundschau), 30 April 2018. Char. 1: (0) eyes small to medium large, situated exclusively on lateral part of head; (1) eyes large,Char. situated 1: (0) either eyes smallonly on to mediumlateral part large, of head situated or also exclusively partially on on lateral dorsal part part of of head; head, (1) or eyes med large,ium situatedlarge situated either partially only on lateralon dorsal part part of headof head. or also partially on dorsal part of head, or medium large situated partially on dorsal part of head.

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Char. 2: (0) head between eyes broad, of more than half of rostrum width at base; (1) head between eyes narrow, of at most half of rostrum width at base. Char. 3: (0) distance between eyes larger or equal on ventral side of head than on forehead; (1) distance between eyes smaller on ventral side of head than on forehead. Char. 4: (0) lower rostrum margin directed to inferior part of eye or below eye; (1) lower rostrum margin directed to middle of eye. Char. 5: (0) antennal funicle with six or less segments or not differentiated from scape; (1) antennal funicle with seven segments. Char. 6: (0) head and rostrum base dorsally bare or sparsely to semidensely, not conﬂuently covered with hairs or scales, integument at least partially visible; (1) head and rostrum base covered with conﬂuently densely arranged scales fully covering integument. Char. 7: (0) antennal segment 1 bare or sparsely covered with hairs or seta-like scales; (1) antennal segment 1, at least in distal part densely covered with shortly elongated scales. Char. 8: (0) lateral margin of mandibles with one or more large teeth; (1) lateral margin of mandibles without or with one small tooth or tubercle. Char. 9: (0) lateral anterior margin of pronotum without postocular lobes; (1) lateral anterior margin of pronotum with postocular lobes. Char. 10: (0) anterior margin of prosternum with no or shallow emargination, of less than 1/3 of the medial prosternal length; (1) anterior margin of prosternum with deep emargination, of at least 1/3 of the medial prosternal length. Char. 11: (0) prosternum in medial part without impression along its whole medial length; (1) prosternum in medial part with impression along its whole medial length. Char. 12: (0) mesoventral process markedly longer than wide at base; (1) mesoventral process at most as long as wide at base or slightly longer. Char. 13: (0) distance between metacoxal apices less than twice as long as distance between precoxal apices; (1) distance between metacoxal apices twice as long or more as distance between precoxal apices. Char. 14: (0) medial length of ventrite 1 shorter or equal to medial length of ventrite 2; (1) medial length of ventrite 1 longer than medial length of ventrite 2. Char. 15: (0) medial length of ventrite 2 shorter or of the same length as medial length of ventrites 3–4 combined; (1) medial length of ventrite 2 longer than medial length of ventrites 3–4 combined. Char. 16: (0) claws free; (1) claws at least at base connate. Char. 17: (0) parameres absent, rudimentary in form of tubercles, “brush-like”, transformed to plates or connate in their whole length; (1) parameres present, separated or connate only in basal part. Char. 18: (0) tegmen oval to markedly elongated, always closed; (1) tegmen round, subround or moderately elongated, closed or open [6]. Char. 19: (0) manubrium tegmeni short to medium length, at most as long as longitudinal diameter of tegmen; (1) manubrium tegmeni long, longer than longitudinal diameter of tegmen [6]. Char. 20: (0) temones longer than median lobe; (1) temones shorter or equally as long as median lobe or rudimentary [6]. Char. 21: (0) intertemonal sclerites absent; (1) intertemonal sclerites present. Char. 22: (0) saccus internus in median lobe without sclerites or sclerotized ductus; (1) saccus internus in median lobe with sclerites or sclerotized ductus. Char. 23: (0) spiculum ventrale without arch-like arms; (1) spiculum ventrale with two arch-like arms and developed apodeme (Figure2). Diversity 2018, 10, x FOR PEER REVIEW 5 of 11

Char. 27: (0) cornu of spermatheca long, of more than maximal diameter of corpus; (1) cornu of spermatheca short, not longer than maximal diameter of corpus. Char. 28: (0) hind wings absent or brachypterous; (1) hind wings fully developed. Char. 29: (0) radial tending zone of hind wings long, of at least 0.45 wing length; (1) radial tending zone of hind wings short, of less than 0.45 wing length (Figure 3). Char. 30: (0) hind wings with well-developed RP2 (radius posterior); (1) hind wings with no or indistinct RP2 (Figure 3). Char. 31: (0) radial cell (RC) of hind wings developed; (1) radial cell (RC) of hind wings absent (Figure 3). Char. 32: (0) central field of hind wings with no or indistinct lateral sclerotized plate (SPl); (1) centralDiversity field 2018, of10 , hindx FOR wingsPEER REVIEW with well -developed SPl (Figure 3). 5 of 11 Char. 33: (0) R–M loop of hind wings feebly visible or missing, r4 indistinct or missing; (1) R–M Char. 27: (0) cornu of spermatheca long, of more than maximal diameter of corpus; (1) cornu of loop of hind wings well developed, r4 clearly visible (Figure 3). spermatheca short, not longer than maximal diameter of corpus. Char. 34: (0) apical blood sinus (“pterostigma”) clearly visible; (1) apical blood sinus DiversityChar.2018, 28:10, 34(0) hind wings absent or brachypterous; (1) hind wings fully developed. 5 of 11 (“pterostigma”) absent or indistinct (Figure 3). Char. 29: (0) radial tending zone of hind wings long, of at least 0.45 wing length; (1) radial tending zone of hind wings short, of less than 0.45 wing length (Figure 3). Char. 30: (0) hind wings with well-developed RP2 (radius posterior); (1) hind wings with no or indistinct RP2 (Figure 3). Char. 31: (0) radial cell (RC) of hind wings developed; (1) radial cell (RC) of hind wings absent (Figure 3). Char. 32: (0) central field of hind wings with no or indistinct lateral sclerotized plate (SPl); (1) central field of hind wings with well-developed SPl (Figure 3). Char. 33: (0) R–M loop of hind wings feebly visible or missing, r4 indistinct or missing; (1) R–M loop of hind wings well developed, r4 clearly visible (Figure 3). Char. 34: (0) apical blood sinus (“pterostigma”) clearly visible; (1) apical blood sinus (“pterostigma”) absent or indistinct (Figure 3).

Figure 2. 2.Spiculum Spiculum ventrale ventrale (female (female eighth eigh sternite)th sternite) of Acentrus of Acentrus histrio. “Arch”-like histrio. “Arch” arms (a-like), apodeme arms ( (ab),). (Notapodeme to scale). (b). (Not to scale).

Char. 24: (0) spermatheca simple, strongly sclerotized, not transparent, U-shaped, with almost undistinguishable corpus and cornu; (1) spermatheca more or less differentiated, moderately to slightly sclerotized, semi-transparent, with distinguishable corpus and cornu. Char. 25: (0) ramus of spermatheca absent; (1) ramus of spermatheca present. Char. 26: (0) nodulus of spermatheca present; (1) nodulus of spermatheca absent. Char. 27: (0) cornu of spermatheca long, of more than maximal diameter of corpus; (1) cornu of spermatheca short, not longer than maximal diameter of corpus. Char. 28: (0) hind wings absent or brachypterous; (1) hind wings fully developed. Char.Figure 29: 2. (0)Spiculum radial tending ventrale zone (female of hind eigh wingsth sternite) long, of of at Acentrus least 0.45 histrio wing. length;“Arch”-like (1) radial arms tending (a), zone ofapodeme hind wings (b). (Not short, to scale). of less than 0.45 wing length (Figure3).

Figure 3. Hind wing of Acentrus histrio. Lateral sclerotized plate (SPl), radio-medial loop (R–M loop), radius posterior 2 (RP2), r4 vein (r4). (Not to scale).

2.2.2. Host Plant Characters Char. 35: (0) host plants not Eudicots [7]; (1) host plants Eudicots. Char. 36: (0) host plants not Ranunculares [7]; (1) host plants Ranunculares. Char. 37: (0) host plants Superrosids [7]; (1) host plants not Superrosids. Char. 38: (0) host plants Superasterids [7]; (1) host plants not Superasterids.

FigureFigure 3. 3.Hind Hind wingwing ofofAcentrus Acentrus histrio histrio.. LateralLateral sclerotizedsclerotized plateplate (SPl),(SPl), radio-medial radio-medial looploop (R–M(R–M loop),loop), radiusradius posterior posterior 2 2 (RP2), (RP2), r4 r4 vein vein (r4). (r4). (Not (Not to to scale). scale).

2.2.2.Char. Host 30: Plant (0) Characters hind wings with well-developed RP2 (radius posterior); (1) hind wings with no or indistinctChar. RP2 35: (Figure(0) host3 ).plants not Eudicots [7]; (1) host plants Eudicots. Char.Char. 31:36: (0)(0) radialhost plants cell (RC) not ofRanunculares hind wings [7]; developed; (1) host plants (1) radial Ranunculares. cell (RC) of hind wings absent (FigureChar.3). 37: (0) host plants Superrosids [7]; (1) host plants not Superrosids. Char.Char. 38: 32: (0) (0) ho centralst plants ﬁeld Superasterids of hind wings [7]; with(1) host no plants or indistinct not Superasterids. lateral sclerotized plate (SPl); (1) central ﬁeld of hind wings with well-developed SPl (Figure3). Char. 33: (0) R–M loop of hind wings feebly visible or missing, r4 indistinct or missing; (1) R–M loop of hind wings well developed, r4 clearly visible (Figure3). Char. 34: (0) apical blood sinus (“pterostigma”) clearly visible; (1) apical blood sinus (“pterostigma”) absent or indistinct (Figure3). Diversity 2018, 10, 34 6 of 11

2.3. Phylogenetic and Multi-Dimensional Phenetic Methods Phylogenetic analyses were conducted on the data set (Table1) comprising 34 morphological characters (characters 1–34) and four extrinsic characters concerning the host plants and their phylogenetic relationships [7]. All character states were unordered and unweighted except for characters 7, 14–16, 30, 32, 33, which were double-weighted, characters 2, 8, 11, 13, 18, 21, 23, 29, 36, which were triple-weighted and characters 1, 3, 4, 12, 17, 24, 31, which had a weight of 5. The weighting of the characters reflected their assumed taxonomic importance. Phylogenetic trees were computed in maximum parsimony and Bayesian frameworks. The most parsimonious tree was found in PAUP* ver. 4.0b8 [8] using a heuristic search and 10 random addition species replicates. The accelerated transformation (ACCTRAN) optimization algorithm as well as the three bisection–reconnection (TBR) branch-swapping algorithm were applied. The reliability of its branching pattern was assessed using the bootstrap method with 100 replicates. Phylogenetic relationships were reconstructed also using Bayesian inference in the computer program MrBayes ver. 3.2.1 [9]. Bayesian analyses were conducted with the standard discrete evolutionary model and symmetric Dirichlet distribution for state rate variation among characters. The standard discrete model is analogous to the Jukes–Cantor evolutionary model in that any particular change from one state to another is equally probable, but has a variable number of states as it is in the one-parameter Markov k-state model. Two parallel runs with four chains were performed as part of the Markov chain Monte Carlo simulations. Posterior probabilities of the branching pattern were estimated from one million generations and trees sampled every 100 generations. The first 25% of sampled trees were discarded before constructing the 50% majority-rule consensus tree and calculating its posterior probabilities. Stationarity in the Bayesian analyses was confirmed in that the average standard deviation of the split frequencies was well below 0.01, the potential scale reduction factor approached 1, and no obvious trends were in the plots of generation vs. log probability. The evolutionary history of all characters was reconstructed using the most parsimonious tree inferred from the matrix in Table1 and the parsimony ancestral character state reconstruction method implemented in Mesquite ver. 3.40 [10]. The similarity of the analyzed taxa was assessed by nonmetric multi-dimensional scaling, as implemented in the scikit-learn ver. 0.19.1 package in Python [11]. When a character was not applicable to at least one taxon, it was excluded from the analysis. The SMACOF algorithm was run with 1000 initializations, each run had 20,000 iterations, and ε was set to 1 × 10−8 to declare convergence. Diversity 2018, 10, 34 7 of 11

Table 1. Character matrix of tribes of the family Curculionidae ordered alphabetically and one outgroup species of the family Attelabidae.

Character No. Family Subfamily Species Tribe 1 2 3 12345678901234567890123456789012345678 Attalabidae Rhynchitinae Rhynchitini Rhynchites bacchus 0000000000000100000000000111000011100 1 Curculionidae Brachycerinae Erirhinini Notaris scirpi 1000100010000110000000011111011111001 1 Curculionidae Brachycerinae Erirhinini Thryogenes ﬁorii 1000100000000110000000111110nnnnnn001 1 Curculionidae Brachycerinae Erirhinini Thryogenes scirrhosus 1000100000000110000000111111011111001 1 Curculionidae Curculioninae Acentrusini Acentrus histrio 1011111111111110111111111111111111111 1 Curculionidae Curculioninae Acentrusini Acentrus zarathustra 1011111111111110111111111111111111111 1 Curculionidae Curculioninae Anthonomini Anthonomus behnei 1000000100010100011100010101001100100 1 Curculionidae Curculioninae Anthonomini Bradybatus seriesetosus 1000000101010101011100010101001100100 1 Curculionidae Curculioninae Ellescini Dorytomus taeniatus 1100100100010100111001011111101100100 1 Curculionidae Curculioninae Ellescini Ellescus bipunctatus 1100100101110100111101011001101101100 1 Curculionidae Curculioninae Smicronychini Sharpia sp. 1011110101011111111101011101111110100 1 Curculionidae Curculioninae Smicronychini Smicronyx jungermanniae 1011100111111111111100010111101111101 0 Curculionidae Curculioninae Smicronychini Smicronyx reichii 1011100111111111111000010101101111101 0 Curculionidae Curculioninae Storeini Pachytychius sparsutus 1000100101000110110100011101011110100 1 Curculionidae Curculioninae Storeini Pachytychius hordei 1000100000000110111100011000nnnnnn001 1 Curculionidae Curculioninae Styphlini Trachystyphlus beigerae 1010100111001100011110011100nnnnnn101 0 Curculionidae Curculioninae Styphlini Pseudostyphlus pillumus 1010110101000100011010011101111111101 0 Diversity 2018, 10, x FOR PEER REVIEW 8 of 11

3. Results and Discussion The weighted maximum parsimony analysis revealed only a single most parsimonious tree with a length of 161 steps, a consistency index of 0.56 and a retention index of 0.77. The 50% majority-rule consensus tree inferred by Bayesian inference had a branching pattern identical to the most parsimonious tree and therefore posterior probabilities were mapped onto it along with maximum parsimony bootstrap values (Figure 4). Morphological character changes were also Diversitymapped2018, 10 on, 34 the most parsimonious tree (Figure 5). Interestingly, although multi-dimensional scaling8 of 11 is a phenetic method based only on similarity, the results were fully consistent with clades inferred by phylogenetic techniques (Figure 6). Taking into account the results of both phylogenetic and 3. Resultsphenetic and analyses, Discussion we assumed that the similarity of the weevil tribes in terms of the studied characters also reflected their phylogenetic relatedness. The weighted maximum parsimony analysis revealed only a single most parsimonious tree Smicronychini were recognized as the most closely phylogenetically-related tribe to withAcentrusini. a length of The 161 monophyletic steps, a consistency origin of the index Smicronychin of 0.56 andi–Acentrusini a retention group index was ofsupported 0.77. The by 50% majority-rule98% maximum consensus parsimony tree bootstrap inferred and by Bayesiana posterior inferenceprobability had of 1.00 a branching in the Bayesian pattern tree identical(Figure to the most4). Likewise, parsimonious Acentrusini tree and and therefore Smicronychini posterior formed probabilities the most distinct were cluster mapped in onto the ordination it along with maximumdiagram parsimony of nonmetric bootstrap multi-dimensional values (Figure scaling4). Morphological(Figure 6). Smicronychini character, represented changes were in this also study mapped on theby most two species parsimonious of Smicronyx tree and (Figure one species5). Interestingly, of Sharpia, were although however multi-dimensional depicted as a paraphyletic scaling is a phenetictribe methodencompassing based Acentrusini only on similarity, in the phylogenetic the results trees. were Specifically fully consistent, Smicronyx with was clades classified inferred as a by phylogeneticsister taxon techniques of Acentrus (Figure with medium6). Taking (71% into bootstrap) account to the poor results (posterior of both probability phylogenetic 0.86) andstatistical phenetic analyses,support we (Figure assumed 4). thatOn the the other similarity hand, ofafter the nonmetric weevil tribes, multi in-dimensional terms of the scaling, studied Smicronychini characters also and Acentrusini each formed a homogenous group, corroborating the validity and distinctness of reﬂected their phylogenetic relatedness. both tribes (Figure 6).

Figure 4. Weighted most parsimonious tree inferred from 38 characters of 16 species belonging to Figure 4. Weighted most parsimonious tree inferred from 38 characters of 16 species belonging to seven tribes of the family Curculionidae and one outgroup species (Rhynchites bacchus). The tree was seven tribes of the family Curculionidae and one outgroup species (Rhynchites bacchus). The tree was constructed with the maximum parsimony method using PAUP*. Nodal supports are indicated as constructed with the maximum parsimony method using PAUP*. Nodal supports are indicated as maximum parsimony (MP) bootstrap values in % and posterior probabilities for the Bayesian maximuminference parsimony (BI). A dash (MP) indicates bootstrap support values below in % and 0.50. posterior Nodes without probabilities statistical for the support Bayesian were inference not (BI). Arecognized dash indicates in Bayesian support and MP below bootstrap 0.50. Nodesanalyses. without statistical support were not recognized in Bayesian and MP bootstrap analyses.

Smicronychini were recognized as the most closely phylogenetically-related tribe to Acentrusini. The monophyletic origin of the Smicronychini–Acentrusini group was supported by 98% maximum parsimony bootstrap and a posterior probability of 1.00 in the Bayesian tree (Figure4). Likewise, Acentrusini and Smicronychini formed the most distinct cluster in the ordination diagram of nonmetric multi-dimensional scaling (Figure6). Smicronychini, represented in this study by two species of Smicronyx and one species of Sharpia, were however depicted as a paraphyletic tribe encompassing Acentrusini in the phylogenetic trees. Speciﬁcally, Smicronyx was classiﬁed as a sister taxon of Acentrus with medium (71% bootstrap) to poor (posterior probability 0.86) statistical support (Figure4). On the other hand, after nonmetric, multi-dimensional scaling, Smicronychini and Acentrusini each formed a homogenous group, corroborating the validity and distinctness of both tribes (Figure6). Diversity 2018, 10, 34 9 of 11 Diversity 2018, 10, x FOR PEER REVIEW 9 of 11

Figure 5. Evolution of character states in seven tribes of the family Curculionidae and one outgroup Figure 5. Evolution of character states in seven tribes of the family Curculionidae and one outgroup species (Rhynchites bacchus). Changes in character states were mapped on the most parsimonious tree species (Rhynchites bacchus). Changes in character states were mapped on the most parsimonious tree shown in Figure 4, using the parsimony reconstruction method implemented in Mesquite. Black dots shownindicate in Figure unique4, using character the parsimonychanges, while reconstruction white dots indicate method homoplastic implemented changes. in Mesquite. Black dots indicate unique character changes, while white dots indicate homoplastic changes. In accordance with the current taxonomic concept, all other tribes of Curculioninae, except for InStoreini, accordance were revealed with the to current be monophyletic taxonomic, usually concept, with all strong other statistical tribes of support. Curculioninae, Thus, only except the for Storeini,node were separating revealed members to be of monophyletic, Storeini was very usually poorly withstatistically strong supported statistical (51% support. MP boots Thus,trap onlyand the 0.86 posterior probability). Hence, the monophyly of Pachytychius species could also not be excluded. node separating members of Storeini was very poorly statistically supported (51% MP bootstrap and The monophyletic origin of the subfamily Curculioninae was well corroborated, while the subfamily 0.86 posterior probability). Hence, the monophyly of Pachytychius species could also not be excluded. Brachycerinae, represented here by the tribe Erirhinini, was only weakly statistically supported (58% The monophyleticMP bootstrap originand posterior of the subfamily probability Curculioninae below 0.50) (Figure was well 4). The corroborated, clusters recognizable while the in subfamily the Brachycerinae,ordination represented diagram were here also by basically the tribe consistent Erirhinini, with was and only supported weakly statistically the current supported taxonomic (58% MP bootstrapconcept (Figure and posterior6). probability below 0.50) (Figure4). The clusters recognizable in the ordinationThe diagram close phylogenetic were alsobasically relationship consistent of Acentrusini with andand supportedStyphlini proposed the current by Alonso taxonomic-Zarazaga concept (Figure[1]6 ). was only partially confirmed by the present phylogenetic and multi-dimensional phenetic Theanalyses. close Similarly,phylogenetic the otherrelationship supposedly of Acentrusini related tribe and Storeini Styphlini is even proposed less related by Alonso-Zarazaga to Acentrusini [1] was onlythan partially Styphlini.conﬁrmed Moreover, by Storeini the present are presently phylogenetic regarded and as multi-dimensionala disputable tribe. Two phenetic Palaearctic analyses. genera, Pachytychius, included in this study, and Aubeonymus Jaquelin du Val, 1855, are considered Similarly, the other supposedly related tribe Storeini is even less related to Acentrusini than Styphlini. genera incertae sedis [3]. According to the newest higher taxonomy concept, the tribe is treated as Moreover,“sensu Storeini lato” [4]. are The presently paraphyly regarded of this tribe, as a disputable revealed in tribe. this study Two Palaearctic(Figure 4), also genera, reflectsPachytychius these , includeddoubts in thisabout study, the common and Aubeonymus origin of Storeini.Jaquelin On du the Val, other 1855, hand, are the considered grouping generaof Acentrusincertaeini and sedis [3]. AccordingSmicronychini to the newest was consistently higher taxonomy recognized concept, in all theour tribeanalyses. is treated Their asmost “sensu important lato”[ apomorphy4]. The paraphyly is of thisthe tribe, lower revealed rostrum margin in this directed study (Figure to the 4middle), also of reﬂects the eye these (char. doubts 4). Styphlini about is thepart common of the clade origin of Storeini.that unit Ones Acentrusini the other hand, and Smicronychini, the grouping with of Acentrusinia smaller ventral and Smicronychini interocular distance was (char. consistently 3). recognizedHowever, in the all difference our analyses. between Their the dorsal most and important the ventral apomorphy interocular isdistance the lower is not rostrumso strikingly margin directedexpressed to the in middle Styphlini of as the it is eye in Acentrusini (char. 4). and Styphlini Smicronychini. is part Despite of the cladethe relatively that unites small Acentrusininumber and Smicronychini,of apomorphies inwith the a cladogram smaller ventral (Figure interocular 5), at least one distance apomorphy (char. was 3). found However, for almost the difference each clade. For instance, the feebly visible R-M loop and r4 (char. 33) is synapomorphic for the tribes between the dorsal and the ventral interocular distance is not so strikingly expressed in Styphlini as Anthonomini and Ellescini. The latter tribe is, in addition, characterized by an unequivocal it is inapomorphy, Acentrusini with and a narrow Smicronychini. head between Despite eyes (char. the relatively 2). small number of apomorphies in the cladogram (Figure5), at least one apomorphy was found for almost each clade. For instance, the feebly visible R-M loop and r4 (char. 33) is synapomorphic for the tribes Anthonomini and Ellescini. The latter tribe is, in addition, characterized by an unequivocal apomorphy, with a narrow head between eyes (char. 2). Diversity 2018, 10, 34 10 of 11 Diversity 2018, 10, x FOR PEER REVIEW 10 of 11

Figure 6. Nonmetric multi-dimensional scaling of 16 species belonging to seven tribes of the family Figure 6. Nonmetric multi-dimensional scaling of 16 species belonging to seven tribes of the family Curculionidae and one outgroup species (Rhynchites bacchus). The ordination diagram was Curculionidae and one outgroup species (Rhynchites bacchus). The ordination diagram was constructed constructed from 32 characters, using the scikit-learn package in Python. from 32 characters, using the scikit-learn package in Python. 4. Conclusions 4. Conclusions The weighted cladistic and multi-dimensional phenetic analyses showed that Smicronychini is Thethe most weighted closely cladistic related tribe and to multi-dimensional Acentrusini. On the phenetic other han analysesd, Styphlini showed and Storeini that Smicronychini were shown is the mostto be closely relatively related unrelated tribe to to Acentrusini. Acentrusini. On the other hand, Styphlini and Storeini were shown to be relativelyAuthor Contributions: unrelated to Both Acentrusini. authors contributed equally to the design, analysis and writing of the paper. AuthorFunding Contributions:: This researchBoth received authors no contributed external funding. equally to the design, analysis and writing of the paper. Funding:Acknowledgments:This research received We are thankful no external to R. funding. Caldara (Milano) for pointing out the taxonomical disputability of the tribe Storeini, to J. Kukalová-Peck (Ottawa) for worthy comments on the terminology of hind wing Acknowledgments: We are thankful to R. Caldara (Milano) for pointing out the taxonomical disputability of the venation, to R. Borovec (Sloupno) and J. Hájek (Prague) for providing us with additional material of Styphlini. tribe Storeini, to J. Kukalová-Peck (Ottawa) for worthy comments on the terminology of hind wing venation, to R. BorovecConflicts (Sloupno) of Interest: and The J. authors Hájek (Prague)declare no for conflicts providing of interest. us with additional material of Styphlini. Conﬂicts of Interest: The authors declare no conﬂicts of interest. References References1. Alonso-Zarazaga, M.A. Diagnosis preliminares de nuevos táxones de Curculionidae (Coleoptera). Bol. SEA 2005, 37, 89–93. 1. Alonso-Zarazaga,2. Košťál, M. Revision M.A. Diagnosis of the genus preliminares Acentrus Desmarest, de nuevos 1839 táxones (Coleoptera: de Curculionidae Curculionidae). (Coleoptera). Koleopt. Rdsch.Bol. SEA 2005, 372014, 89–93., 84, 329–336. 2. Košt’ ál, M. Revision of the genus Acentrus Desmarest, 1839 (Coleoptera: Curculionidae). Koleopt. Rdsch. 2014, 84, 329–336. Diversity 2018, 10, 34 11 of 11

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