Molecular phylogenetic studies of the Mylabrini blister beetles (Coleoptera, Meloidae)

Molecular Phylogenetics and Evolution 37 (2005) 306–311 www.elsevier.com/locate/ympev

Short communication

Molecular phylogenetic studies of the Mylabrini blister beetles (Coleoptera, Meloidae) Marco A. Bologna a,¤, Benedetta D’Inzillo a, Manuela Cervelli a, Marco Oliverio b, Paolo Mariottini a b

a Dipartimento di Biologia, Università “Roma Tre”, Viale Guglielmo Marconi 446, 00146 Roma, Italy Dipartimento di Biologia animale e dell’uomo, Università di Roma “La Sapienza”, Viale dell’Università 32, 00187 Roma, Italy

Received 21 September 2004; revised 25 February 2005 Available online 10 May 2005

1. Introduction The blister beetles (Coleoptera, Tenebrionoidea, Meloidae) include over 2500 species in approximately 125 genera. This family is of general biological relevance because of its hypermetabolic larval development, the parasitoid biology of the larval phases and the production of cantharidin. The latter is a defensive and probably aggregative terpenoid, almost exclusive to this family, with a long history of pharmacological use in diVerent countries of the World. The presence of this defensive substance may have allowed the evolution of very complex and prolonged sexual behaviours in the largest subfamily Meloinae. Several specialists (e.g., Cros, 1940; Escherich, 1897; MacSwain, 1956; Selander, 1964) repeatedly used ontogeny, larval and adult morphology, as well as behaviour, to propose diVerent classiWcations of this family. Bologna (1991) summarised morphological and biological information on the family and proposed a classiWcation, which was largely conWrmed in the major grouping (subfamilies and tribes) by the recent phylogenetic studies made by Bologna and Pinto (2001). In the latter work, analysing a total of 85 genera and subgenera on behavioural and morphological (adult and larval) characters, relationships of genera within some tribes (particularly, Lyttini and Mylabrini) remained partly unresolved and/or contrasting. An undetermined number of characters in the diVerent sub*

Corresponding author. Fax: +390655176321. E-mail addresses: [email protected] (M.A. Bologna), cervelli@ uniroma3.it (M. Cervelli), [email protected] (M. Oliverio), [email protected] (P. Mariottini). 1055-7903/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2005.03.034

sets employed (larval, adult, ethological, etc.) could be under severe adaptive pressures, thus likely homoplastic, in relation to the peculiar biology of meloids. A molecular framework in the phylogenetic studies of this beetle family was thus strongly required given these diYculties in interpreting the totality of the biological variation. A molecular approach has never been used to infer phylogeny of Meloidae. Presented here is a molecular study of the most speciose meloid tribe, the Mylabrini (subfamily Meloinae). It is distributed only in the Old World, and includes more than 650 species belonging to 11 genera (Bologna and Pinto, 2002): Actenodia Laporte de Castelnau, 1840 (20 spp.), Ceroctis Marseul, 1870 (59 spp.), Croscherichia Pardo Alcaide, 1950 (18 spp.), Hycleus Latreille, 1817 (ca. 430 spp.), Lydoceras Marseul, 1870 (4 spp.), Mimesthes Marseul, 1872 (4 spp.), Mylabris Fabricius, 1775 (ca.170 spp.), Paractenodia Péringuey, 1904 (4 spp.), Pseudabris Fairmaire, 1894 (4 spp.), Semenovilia Kuzin, 1954 (1 sp.), and Xanthabris Kaszab, 1956 (1 sp.). The two most speciose genera (Hycleus and Mylabris) are often considered as a single taxonomic unit (Mylabris), particularly in works of applied biology (Greathead, 1963; Paoli, 1932, 1937; Thompson and Simmonds, 1965), in classical manuals and textbooks (e.g., Resh and Cardé (eds), 2003), and in Weld guides (Chinery, 1997). This position was also held by one of the few specialists of blister beetles, Kaszab (e.g., 1969). Given the relevance of these insects also to applied biology, data on their phylogenetic systematics is of great importance. Relationships among genera of Mylabrini based on adult morphology, previously proposed by Bologna (1991, 2000), and Bologna and Coco (1991) only partly overlapped results emerging from the cladistic analysis

M.A. Bologna et al. / Molecular Phylogenetics and Evolution 37 (2005) 306–311

of Bologna and Pinto (2001). The main agreement was that Wve genera of Mylabrini (Ceroctis, Croscherichia, Hycleus, Mylabris, and Paractenodia) composed a clear monophyletic group, mostly based on Wrst instar larval morphology (Bologna and Pinto, 2001). A later detailed study of the triungulin of Actenodia and Mimesthes (Bologna, unpublished data), supported their membership in the tribe, albeit with unclear relationship. We have examined almost the same set of genera studied morphologically by Bologna and Pinto (2001), with the addition of Actenodia and Mimesthes. The monotypic genus Xanthabris Kaszab, 1956, could not be included because the type species, X. baluchistana Kaszab, 1956, is known only from the holotype. The remaining genera of Mylabrini (Lydoceras Marseul, 1872; Pseudabris Fairmaire, 1894; Semenovilia Kuzin, 1954, monotypic) are either truly uncommon or originate from areas presently inaccessible, and it has been impossible to get living or freshly collected and properly Wxed material. Anyway, there is strong evidence from morphology that Lydoceras is very closely related to Hycleus (and possibly belong in its radiation) (Bologna and Pinto, 2002). Thus, the only signiWcant missing taxa are Xanthabris, Pseudabris, and Semenovilia. Considering the unlikely acquisition of samples of these genera in a reasonable time, we decided to proceed with the available material only. This paper is primarily a Wrst attempt to deWne phylogenetic relationships of the blister beetle tribe Mylabrini derived from the molecular analysis, and compare them to those so far obtained from morphological and biological characters. At this step, we have chosen to use partial sequences from the mitochondrial gene encoding for the 16S ribosomal subunit, which had proven useful in producing reliable phylogenetic hypotheses below the family level in coleopterans (see e.g., Ribera et al., 2004; Satoh et al., 2004; Simon et al., 1994).

2. Material and methods Specimens and source of sampling species are reported in Table 1. We used one external outgroup (Tenebrio molitor, family Tenebrionidae), and Wve additional genera of Meloidae, belonging to four other tribes of the subfamily Meloinae: Cerocomini (Cerocoma), Pyrotini (Pyrota), Lyttini (Oenas), Eupomphini (Megetra, Phodaga). Voucher specimens of all species are in the senior author’s collection. A region of the mitochondrial 16S rDNA, ca. 550 bp encompassing the domains IV and V (Gutell and Fox, 1988; Gutell et al., 1993), was ampliWed using the primers 16sar-L 5⬘-CGCCTGTTTATCAAAAACAT-3⬘ and 16sbrH (5⬘-CCGGTCTGAACTCAGATCAC-3⬘) slightly modiWed after Palumbi et al. (1991). Nucleotide sequences were Wrst aligned by hand, and then the alignment was progressively optimised according to secondary structure homology. Folding was predicted for each sequence

307

on a thermodynamic basis using the software package RNA Structure version 3.71 available on the Turner Lab Homepage http://rna.chem.rochester.edu (Mathews et al., 1999). The aligned Meloidae sequences were analysed under the assumptions of maximum parsimony (MP, Farris, 1970, equal weighing Tv/Ti, including/excluding gaps) and maximum likelihood (ML, Felsenstein, 1981), by the package PAUP* 4b10 (SwoVord, 2002). An optimal model of nucleotide evolution for the ML analyses was determined by the software Modeltest 3.06 (Posada and Crandall, 1998). Bayesian analyses were carried out with MrBayes3.0b4 (Huelsenbeck and Ronquist, 2001), which adopts the Markov chain Monte Carlo method to sample from posterior densities (Larget and Simon, 1999; Yang and Rannala, 1997). After a Wrst run of testing, Bayesian posterior probabilities (BPP) for 110,000 generations were estimated on a 50% majority rule consensus tree of 10,000 trees remaining after burn-in.

3. Results A total of 25 16S rRNA sequences were obtained, ranging from 546 to 558 bp. The alignment of the 16S sequences caused no problems and all 566 aligned positions were unambiguously homologised. A
2 test of base homogeneity, uncorrected for phylogeny, indicated that overall base composition was not signiWcantly diVerent across all sites (P D 0.999). Scatter plots of transitions and transversions against the pairwise ML distance showed evidence of saturation for transitions and transversions beyond an ML divergence of 0.25 (corresponding to pairwise comparisons at the inter-tribe level). Pairwise ML genetic distance across all sampled taxa ranged from 0.0210 (Mylabris Xexuosa—M. obsoleta) to 0.6941 (Tenebrio molitor—Pyrota akhurstiana). The proposed secondary structure for the 3⬘ half portion of the meloid 16S rRNA (model available on request from the authors) conforms to the canonical architecture (Gutell and Fox, 1988; Gutell et al., 1993). Both domains IV and V show a high conservation in folding when compared to the common secondary structure shared by taxa as distant as molluscs (Lydeard et al., 2000) and mammals (Horovitz and Meyer, 1995). With the exception of the very small L7 loop (Vawter and Brown, 1993) in domain V, all the remaining stems, bulges and loops are structurally conserved allowing a sequence alignment without uncertainty. MP analyses on the 16S dataset recovered two trees (gaps as Wfth base: 267 variable positions, of which 179 parsimony informative) and one tree (gaps as missing: 254 variable positions, of which 171 parsimony informative) with mostly the same topology. The Mylabrini were monophyletic, Ceroctis and Paractenodia were sister taxa belonging in the same lineage as a monophyletic

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M.A. Bologna et al. / Molecular Phylogenetics and Evolution 37 (2005) 306–311

Table 1 Species and sample data, along with GenBank accession numbers and sequence length Species

Mylabris (Eumylabris) calida Mylabris (Eumylabris) cincta Mylabris (Mesosulcata) hieracii Mylabris (Micrabris) Xexuosa Mylabris (Micrabris) obsoleta Mylabris (Mylabris) quadripunctata Mylabris (Mylabris) variabilis Hycleus (Mesogorbatus) dentatusa Hycleus (Mesoscutatus) brevicollisa Hycleus (Mesoscutatus) zebraeusa Hycleus (Mesotaeniatus) deserticolusa Ceroctis phalerata Paractenodia glabra Actenodia chrysomelina Actenodia distincta Mimesthes maculicollis Croscherichia paykulli Pyrota akhurstiana Pyrota postica Phodaga marmorata Megetra vittata Cerocoma graeca Cerocoma prevezaensis Oenas crassicornis Tenebrio molitor

Sample data

16S rDNA

Greece, Lamia prov., 10 km N of Lamia, Stenà Fouskas, 39°00⬘N–22°33⬘E, 06.1999, M. Bologna et al., leg. Greece, Trikala prov., 5 km N junction with Egnatia road, 39°47⬘N–21°31⬘E, 06.1999, M. Bologna et al., leg. Spain, Segovia prov., Pradales, 06.2002, M. Garcia Paris leg. Italy, Abruzzo, Pescara prov., Majella N. P., Caramanico Monte Cavallo, 06.2002, L. Facchinelli leg. Italy, Basilicata, Potenza prov., Episcopia, Sinni River, 06.06.2002, M. Bologna leg. Greece, Tripolis prov., 7 km E Langadia, 37°31⬘N–22°04⬘E, 06.1999, M. Bologna et al., leg., Greece, Trikala prov., 5 km N junction with Egnatia road, 39°47⬘N–21°31⬘E, 06.1999, M. Bologna et al., leg. Namibia, 3–8 km S of Omaruru on C23 road, 21°29.69⬘S–15°58.28°E, 26.02.2001 M. Bologna and P. Bombi leg. Morocco, Fôret de Mamora, 34°01⬘31.2⬙N–6°35⬘53.7⬙E, 02.05.2003, A. Venchi and C. Settanni leg. Greece, Larissa prov., Spilià env., 39°49⬘N–22°39⬘E, 06.1999, M. Bologna et al., leg. Namibia, 16 km S of Karibib on B2 road, 21°56.15⬘S–15°42.39⬘E, 03.03.2001, M. Bologna and P. Bombi leg. Namibia, 10 km N Aais on C20, 22°58.45⬘S–18°43.68⬘E, 21.02.2001, M. Bologna and P. Bombi leg. Namibia, W side of Brandeberg Mt, Messum Valley 21.30483’S–14.65736’E, 11.4.2004, M. Bologna et al., leg. Namibia, 16 km S Gobabis on C20 road, 22°57⬘S–18°56.53⬘E, 21.02.2001, M. Bologna and P.Bombi leg. Morocco, Fôret de Mamora, 34°01⬘31.2⬙N–6°35⬘53.7⬙E, 02.05.2003, A. Venchi and C. Settanni leg. South Africa, Northern Cape, 5 km W of N7 Hwy on road to Hondeklipbaai, 29°47⬘S–17°48⬘E, 01.09.1999, M. Bologna leg. Morocco, Fôret de Mamora, 34°01⬘31.2⬙N–6°35⬘53.7⬙E, 02.05.2003, A. Venchi and C. Settanni leg. USA, Arizona, Willcox, 24.08.2000, M. Bologna and J.D. Pinto leg. USA, Arizona, Cochise Country, Douglas airport env., 17.08.1990, M. Bologna and J.D. Pinto leg. USA, Arizona, 2 miles S of Kansas Settlement, Chiricahua, 24.08.2000, M. Bologna and J.D. Pinto leg. USA, New Mexico, Sandoval County, 12 miles S of Cuba on road 197, 18.08.2000, C. Pierce and G. Pratt leg. Greece, Trikala prov., 5 km N junction with Egnatia road, 39°47⬘N–21°31⬘E, 06.1999, M. Bologna et al., leg. Greece, Préveza prov., Kanali 18 km N Préveza, 39°03⬘N–20°41⬘E, 06.1999, M. Bologna et al., leg. Greece, Atikì prov., between Erythrés and Thiva, 38°13⬘E–23°20⬘E, 06.1999, M. Bologna et al., leg. Larvae from breeding stocks

Accession No.

bp

AJ633652

548

AJ633653

548

AJ633654 AJ633655

547 548

AJ633656

548

AJ633657

547

AJ633658

547

AJ633659

546

AJ633660

549

AJ633661

546

AJ633662

546

AJ633663

546

AJ842954

547

AJ633664

549

AJ633665

558

AJ633666

550

AJ633667

548

AJ633668 AJ842956

547 548

AJ633669

549

AJ633670

550

AJ633671

549

AJ842955

548

AJ633672

551

AJ633673

550

a

Subgeneric notation used for Hycleus species, refers to the “sections” as proposed by Pardo Alcaide (1950, 1955, 1958), with no intentions by us to give them actual subgeneric value.

Hycleus radiation, Actenodia (monophyletic) positioned basally in the Mylabrini. The main diVerence was in the position of Croscherichia. It was with Mimesthes in a clade, sister to a Mylabris–Hycleus clade (including gaps); with gaps as missing character it was the sister to part of the species of an otherwise monophyletic Mylabris. In either case the bootstrap support was very low. The other meloine tribes were clearly identiWed, although with variable relative placement.

Modeltest 3.06 selected TVM + G model of evolution (by both hLRT and AIC) that was used for the likelihood settings in a heuristic search. The 50% majority rule consensus on 10,000 trees from a MCMC sampling procedure with the same settings, provided the posterior probabilities for the clades of interest in the Bayesian analysis. The ML topology recovered (Fig. 1: ¡Ln likelihood D 4798.78744), diVered in some respects from the parsimony topologies. Mimesthes was the sister to Actenodia, and this clade was

M.A. Bologna et al. / Molecular Phylogenetics and Evolution 37 (2005) 306–311

309

Fig. 1. Preferred cladogram for the meloid examined taxa. This topology was recovered under ML analysis of the 16S dataset (¡Ln likelihood D 4798.78744). Numbers at the branch represent bootstrap support in MP analyses (gap included and gaps as missing), and posterior probabilities in Bayesian analysis (only values higher that 65% are reported).

the most basal in the Mylabrini. Mylabris was monophyletic and its sister group was Hycleus and the clade Ceroctis–Paractenodia. Croscherichia was the sister to the Hycleus Mylabris lineage. Monophyly of Mylabrini was relatively well supported (bpp 0.90). The basal position of the Mimesthes–Actenodia clade and their monophyly were supported with a low posterior probability (bpp < 0.75). The inclusion of Ceroctis and Paractenodia in the same clade as a Hycleus radiation was supported (bpp 0.96).

4. Discussion The 16S rRNA secondary structure was highly conserved in the examined tribes, and allows no inferences on

the phylogeny of Meloinae. While MP supported monophyly of several clade at diVerent levels, ML and Bayesian analyses provided a critical test for a relatively robust phylogenetic hypothesis. The relationships among Meloinae tribes are only partially overlapping with the results emerging from morphological and biological features (Bologna and Pinto, 2001), evidently due to the very high number of taxa (96) morphologically examined, comparing with the reduced molecular dataset. Thus, we do not discuss this issue in details here. We only take notice (for future testing) that in our analyses there was a good indication of a closer relationship of Pyrotini with Mylabrini than with any other examined tribes. Furthermore, there was a support to a basal position (among the examined lineages) of Eupomphini and Lyttini.

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In all trees the Mylabrini were monophyletic and supported by good bootstrap and posterior probability. Monophyly of Mylabrini is also strongly supported by several derived morphological characters, particularly of larvae (Bologna, 1991; Bologna and Pinto, 2001). Present work provided insights on the relationships among genera of Mylabrini (Fig. 1). Separation of Mylabris and Hycleus received strong support in all analyses (and it is supported by the analysis of the faster evolving gene ITS2 in a larger taxonomic dataset: Bologna et al., unpublished). Given their relevance to applied biology, the likelihood that they represent two distinct evolutionary lineages is of great importance. Particularly, in several analyses, Hycleus species represent a holophyletic lineage only with the inclusion of Ceroctis and Paractenodia. They have in common mesosternal and aedeagal features, but are diVerentiated by antennal shape and palpal structure of Wrst instar larvae. Present results support the inclusion of both Ceroctis and Paractenodia within Hycleus, as suggested by Bologna (1991, 2000) and Bologna and Coco (1991), contrasting the classiWcation proposed by Kaszab (1969). Hycleus is the most speciose genus of blister beetle, and its internal systematics deserves a deeper insight also with respects to the “sections”, morphologically proposed by Pardo Alcaide (1950, 1955, 1958). Topology of the Mylabris species is congruent with the current subgeneric taxonomy. (Mylabris Mesosulcata) and particularly M. (Eumylabris) are more derived morphologically. The more speciose and widespread M. (Mylabris) and particularly M. (Micrabris) have more primitive morphological features. Monophyly of the genera with more than a single species assayed was always supported with good bootstrap and bpp, in agreement with morphological and biological features of adults (Bologna, 1991, 2000; Bologna and Coco, 1991), and of Wrst instar larvae (see for a review Bologna and Pinto, 2001). The identiWcation of the most basal oV-shoot (either Croscherichia or Actenodia / Mimesthes) in the radiation of Mylabrini could beneWt from the indication of morphology. Croscherichia shares several adult morphological features with Mylabris, and closer aYnities with the Mylabris/Hycleus lineage seem reasonable. The extremely distinct larvae of Croscherichia could represent the adaptive results of undiscovered phoretic behaviour (Bologna and Pinto, 2001). The molecular relationships between Actenodia (with a disjoint distribution in the Afrotropical and Mediterranean regions) and Mimesthes (endemic to Namaqualand, Karoo and Namib deserts), are also supported by several morphological features. This study oVers the Wrst attempt to provide an independent phylogenetic framework for studies on the evolution of Meloidae. This Wrst step—admittedly still based on a short fragment of sequence—indicates that this approach is promising and should be extended to

other tribes (Lyttini could be the next target). We expect also that the addition of other genes will improve the resolution. The availability of good phylogenetic hypotheses, will allow integration with morphological and behavioural data, helping to get a full understanding of the evolution of blister beetles.

Acknowledgments We wish to thank the following students, fellowships and colleagues who collaborated on the collection of specimens during several Weld researches made by one of us (M.A.B.) in the Mediterranean Basin and in Southern Africa: Valentina Amore, Pierluigi Bombi, Paola De Salvo, Andrea Di Giulio, Simone Fattorini, Luca Facchinelli, Carla Marangoni, Francesca Montalto, John D. Pinto, George Pratt, Monica Pitzalis, Chiara Settanni, Federica Turco, Alberto Venchi, Marzio Zapparoli. A special thank to Mario Garcia Paris (Madrid), who supplied a specimen of Mylabris hieracii, and to Maria Vittoria Modica, and Annarita Wirz, for helping with laboratory work. This research was supported by grants of the Italian Ministry of University (99C271884-007) and Roma Tre University (Faculty grant).

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