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

a Dipartimento di Biologia, Università “Roma Tre”, Viale Guglielmo Marconi 446, 00146 Roma, Italyb 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 2005Available online 10 May 2005

1. Introduction

The blister beetles (Coleoptera, Tenebrionoidea,Meloidae) include over 2500 species in approximately125 genera. This family is of general biological relevancebecause of its hypermetabolic larval development, theparasitoid biology of the larval phases and the produc-tion of cantharidin. The latter is a defensive and proba-bly aggregative terpenoid, almost exclusive to thisfamily, with a long history of pharmacological use indiVerent countries of the World. The presence of thisdefensive substance may have allowed the evolution ofvery complex and prolonged sexual behaviours in thelargest subfamily Meloinae. Several specialists (e.g.,Cros, 1940; Escherich, 1897; MacSwain, 1956; Selander,1964) repeatedly used ontogeny, larval and adult mor-phology, as well as behaviour, to propose diVerent classi-Wcations of this family. Bologna (1991) summarisedmorphological and biological information on the familyand proposed a classiWcation, which was largely con-Wrmed in the major grouping (subfamilies and tribes) bythe recent phylogenetic studies made by Bologna andPinto (2001). In the latter work, analysing a total of 85genera and subgenera on behavioural and morphologi-cal (adult and larval) characters, relationships of generawithin some tribes (particularly, Lyttini and Mylabrini)remained partly unresolved and/or contrasting. Anundetermined number of characters in the diVerent sub-

* Corresponding author. Fax: +390655176321.E-mail addresses: bologna@uniroma3.it (M.A. Bologna), cervelli@

uniroma3.it (M. Cervelli), marco.oliverio@uniroma1.it (M. Oliverio),mariotpa@uniroma3.it (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 beunder severe adaptive pressures, thus likely homoplastic,in relation to the peculiar biology of meloids. A molecu-lar framework in the phylogenetic studies of this beetlefamily was thus strongly required given these diYcultiesin interpreting the totality of the biological variation.

A molecular approach has never been used to inferphylogeny of Meloidae. Presented here is a molecularstudy of the most speciose meloid tribe, the Mylabrini(subfamily Meloinae). It is distributed only in the OldWorld, and includes more than 650 species belonging to11 genera (Bologna and Pinto, 2002): Actenodia Laportede Castelnau, 1840 (20 spp.), Ceroctis Marseul, 1870 (59spp.), Croscherichia Pardo Alcaide, 1950 (18 spp.), HycleusLatreille, 1817 (ca. 430 spp.), Lydoceras Marseul, 1870 (4spp.), Mimesthes Marseul, 1872 (4 spp.), Mylabris Fabri-cius, 1775 (ca.170 spp.), Paractenodia Péringuey, 1904 (4spp.), Pseudabris Fairmaire, 1894 (4 spp.), SemenoviliaKuzin, 1954 (1 sp.), and Xanthabris Kaszab, 1956 (1 sp.).The two most speciose genera (Hycleus and Mylabris) areoften considered as a single taxonomic unit (Mylabris),particularly in works of applied biology (Greathead, 1963;Paoli, 1932, 1937; Thompson and Simmonds, 1965), inclassical manuals and textbooks (e.g., Resh and Cardé(eds), 2003), and in Weld guides (Chinery, 1997). This posi-tion was also held by one of the few specialists of blisterbeetles, Kaszab (e.g., 1969). Given the relevance of theseinsects also to applied biology, data on their phylogeneticsystematics is of great importance.

Relationships among genera of Mylabrini based onadult morphology, previously proposed by Bologna(1991, 2000), and Bologna and Coco (1991) only partlyoverlapped results emerging from the cladistic analysis

M.A. Bologna et al. / Molecular Phylogenetics and Evolution 37 (2005) 306–311 307

of Bologna and Pinto (2001). The main agreement wasthat Wve genera of Mylabrini (Ceroctis, Croscherichia,Hycleus, Mylabris, and Paractenodia) composed a clearmonophyletic group, mostly based on Wrst instar larvalmorphology (Bologna and Pinto, 2001). A later detailedstudy of the triungulin of Actenodia and Mimesthes(Bologna, unpublished data), supported their member-ship in the tribe, albeit with unclear relationship.

We have examined almost the same set of generastudied morphologically by Bologna and Pinto (2001),with the addition of Actenodia and Mimesthes. Themonotypic genus Xanthabris Kaszab, 1956, could not beincluded because the type species, X. baluchistana Kas-zab, 1956, is known only from the holotype. The remain-ing genera of Mylabrini (Lydoceras Marseul, 1872;Pseudabris Fairmaire, 1894; Semenovilia Kuzin, 1954,monotypic) are either truly uncommon or originate fromareas presently inaccessible, and it has been impossibleto get living or freshly collected and properly Wxed mate-rial. Anyway, there is strong evidence from morphologythat Lydoceras is very closely related to Hycleus (andpossibly belong in its radiation) (Bologna and Pinto,2002). Thus, the only signiWcant missing taxa are Xan-thabris, Pseudabris, and Semenovilia. Considering theunlikely acquisition of samples of these genera in a rea-sonable time, we decided to proceed with the availablematerial only. This paper is primarily a Wrst attempt todeWne phylogenetic relationships of the blister beetletribe Mylabrini derived from the molecular analysis, andcompare them to those so far obtained from morpholog-ical and biological characters. At this step, we have cho-sen to use partial sequences from the mitochondrial geneencoding for the 16S ribosomal subunit, which hadproven useful in producing reliable phylogenetic hypoth-eses 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 reportedin Table 1. We used one external outgroup (Tenebrio moli-tor, family Tenebrionidae), and Wve additional genera ofMeloidae, belonging to four other tribes of the subfamilyMeloinae: Cerocomini (Cerocoma), Pyrotini (Pyrota), Lyt-tini (Oenas), Eupomphini (Megetra, Phodaga). Voucherspecimens of all species are in the senior author’s collection.

A region of the mitochondrial 16S rDNA, ca. 550 bpencompassing the domains IV and V (Gutell and Fox,1988; Gutell et al., 1993), was ampliWed using the primers16sar-L 5�-CGCCTGTTTATCAAAAACAT-3� and 16sbr-H (5�-CCGGTCTGAACTCAGATCAC-3�) slightly modi-Wed after Palumbi et al. (1991). Nucleotide sequenceswere Wrst aligned by hand, and then the alignment wasprogressively optimised according to secondary struc-ture homology. Folding was predicted for each sequence

on a thermodynamic basis using the software packageRNA Structure version 3.71 available on the Turner LabHomepage http://rna.chem.rochester.edu (Mathewset al., 1999).

The aligned Meloidae sequences were analysed underthe assumptions of maximum parsimony (MP, Farris,1970, equal weighing Tv/Ti, including/excluding gaps)and maximum likelihood (ML, Felsenstein, 1981), by thepackage PAUP* 4b10 (SwoVord, 2002). An optimalmodel of nucleotide evolution for the ML analyses wasdetermined by the software Modeltest 3.06 (Posada andCrandall, 1998). Bayesian analyses were carried out withMrBayes3.0b4 (Huelsenbeck and Ronquist, 2001), whichadopts the Markov chain Monte Carlo method to sam-ple from posterior densities (Larget and Simon, 1999;Yang and Rannala, 1997). After a Wrst run of testing,Bayesian posterior probabilities (BPP) for 110,000 gen-erations were estimated on a 50% majority rule consen-sus 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 16Ssequences caused no problems and all 566 aligned posi-tions were unambiguously homologised. A �2 test ofbase homogeneity, uncorrected for phylogeny, indicatedthat overall base composition was not signiWcantlydiVerent across all sites (P D 0.999). Scatter plots of tran-sitions and transversions against the pairwise ML dis-tance showed evidence of saturation for transitions andtransversions beyond an ML divergence of 0.25 (corre-sponding to pairwise comparisons at the inter-tribelevel). Pairwise ML genetic distance across all sampledtaxa ranged from 0.0210 (Mylabris Xexuosa—M. obso-leta) to 0.6941 (Tenebrio molitor—Pyrota akhurstiana).

The proposed secondary structure for the 3� half por-tion of the meloid 16S rRNA (model available onrequest from the authors) conforms to the canonicalarchitecture (Gutell and Fox, 1988; Gutell et al., 1993).Both domains IV and V show a high conservation infolding when compared to the common secondary struc-ture shared by taxa as distant as molluscs (Lydeard et al.,2000) and mammals (Horovitz and Meyer, 1995). Withthe exception of the very small L7 loop (Vawter andBrown, 1993) in domain V, all the remaining stems,bulges and loops are structurally conserved allowing asequence alignment without uncertainty.

MP analyses on the 16S dataset recovered two trees(gaps as Wfth base: 267 variable positions, of which 179parsimony informative) and one tree (gaps as missing:254 variable positions, of which 171 parsimony informa-tive) with mostly the same topology. The Mylabrini weremonophyletic, Ceroctis and Paractenodia were sistertaxa belonging in the same lineage as a monophyletic

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

Hycleus radiation, Actenodia (monophyletic) positionedbasally in the Mylabrini. The main diVerence was in theposition of Croscherichia. It was with Mimesthes in aclade, sister to a Mylabris–Hycleus clade (includinggaps); with gaps as missing character it was the sister topart of the species of an otherwise monophyletic Myla-bris. 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 likelihoodsettings in a heuristic search. The 50% majority rule con-sensus on 10,000 trees from a MCMC sampling procedurewith the same settings, provided the posterior probabilitiesfor the clades of interest in the Bayesian analysis. The MLtopology recovered (Fig. 1: ¡Ln likelihoodD4798.78744),diVered in some respects from the parsimony topologies.Mimesthes was the sister to Actenodia, and this clade was

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

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

Species Sample data 16S rDNA

Accession No. bp

Mylabris (Eumylabris) calida Greece, Lamia prov., 10 km N of Lamia, Stenà Fouskas,39°00�N–22°33�E, 06.1999, M. Bologna et al., leg.

AJ633652 548

Mylabris (Eumylabris) cincta Greece, Trikala prov., 5 km N junction with Egnatia road,39°47�N–21°31�E, 06.1999, M. Bologna et al., leg.

AJ633653 548

Mylabris (Mesosulcata) hieracii Spain, Segovia prov., Pradales, 06.2002, M. Garcia Paris leg. AJ633654 547Mylabris (Micrabris) Xexuosa Italy, Abruzzo, Pescara prov., Majella N. P., Caramanico

Monte Cavallo, 06.2002, L. Facchinelli leg.AJ633655 548

Mylabris (Micrabris) obsoleta Italy, Basilicata, Potenza prov., Episcopia, Sinni River, 06.06.2002,M. Bologna leg.

AJ633656 548

Mylabris (Mylabris) quadripunctata Greece, Tripolis prov., 7 km E Langadia, 37°31�N–22°04�E, 06.1999,M. Bologna et al., leg.,

AJ633657 547

Mylabris (Mylabris) variabilis Greece, Trikala prov., 5 km N junction with Egnatia road,39°47�N–21°31�E, 06.1999, M. Bologna et al., leg.

AJ633658 547

Hycleus (Mesogorbatus) dentatusa 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.

AJ633659 546

Hycleus (Mesoscutatus) brevicollisa Morocco, Fôret de Mamora, 34°01�31.2�N–6°35�53.7�E, 02.05.2003,A. Venchi and C. Settanni leg.

AJ633660 549

Hycleus (Mesoscutatus) zebraeusa Greece, Larissa prov., Spilià env., 39°49�N–22°39�E, 06.1999,M. Bologna et al., leg.

AJ633661 546

Hycleus (Mesotaeniatus) deserticolusa 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.

AJ633662 546

Ceroctis phalerata Namibia, 10 km N Aais on C20, 22°58.45�S–18°43.68�E, 21.02.2001,M. Bologna and P. Bombi leg.

AJ633663 546

Paractenodia glabra Namibia, W side of Brandeberg Mt, Messum Valley21.30483’S–14.65736’E, 11.4.2004, M. Bologna et al., leg.

AJ842954 547

Actenodia chrysomelina Namibia, 16 km S Gobabis on C20 road, 22°57�S–18°56.53�E,21.02.2001, M. Bologna and P.Bombi leg.

AJ633664 549

Actenodia distincta Morocco, Fôret de Mamora, 34°01�31.2�N–6°35�53.7�E,02.05.2003, A. Venchi and C. Settanni leg.

AJ633665 558

Mimesthes maculicollis South Africa, Northern Cape, 5 km W of N7 Hwy on roadto Hondeklipbaai, 29°47�S–17°48�E, 01.09.1999, M. Bologna leg.

AJ633666 550

Croscherichia paykulli Morocco, Fôret de Mamora, 34°01�31.2�N–6°35�53.7�E, 02.05.2003,A. Venchi and C. Settanni leg.

AJ633667 548

Pyrota akhurstiana USA, Arizona, Willcox, 24.08.2000, M. Bologna and J.D. Pinto leg. AJ633668 547Pyrota postica USA, Arizona, Cochise Country, Douglas airport env., 17.08.1990,

M. Bologna and J.D. Pinto leg.AJ842956 548

Phodaga marmorata USA, Arizona, 2 miles S of Kansas Settlement, Chiricahua, 24.08.2000,M. Bologna and J.D. Pinto leg.

AJ633669 549

Megetra vittata USA, New Mexico, Sandoval County, 12 miles S of Cuba on road 197,18.08.2000, C. Pierce and G. Pratt leg.

AJ633670 550

Cerocoma graeca Greece, Trikala prov., 5 km N junction with Egnatia road,39°47�N–21°31�E, 06.1999, M. Bologna et al., leg.

AJ633671 549

Cerocoma prevezaensis Greece, Préveza prov., Kanali 18 km N Préveza,39°03�N–20°41�E, 06.1999, M. Bologna et al., leg.

AJ842955 548

Oenas crassicornis Greece, Atikì prov., between Erythrés and Thiva,38°13�E–23°20�E, 06.1999, M. Bologna et al., leg.

AJ633672 551

Tenebrio molitor Larvae from breeding stocks AJ633673 550

M.A. Bologna et al. / Molecular Phylogenetics and Evolution 37 (2005) 306–311 309

the most basal in the Mylabrini. Mylabris was monophy-letic and its sister group was Hycleus and the clade Ceroc-tis–Paractenodia. Croscherichia was the sister to theHycleus Mylabris lineage. Monophyly of Mylabrini wasrelatively well supported (bpp 0.90). The basal position ofthe Mimesthes–Actenodia clade and their monophyly weresupported with a low posterior probability (bpp<0.75).The inclusion of Ceroctis and Paractenodia in the sameclade as a Hycleus radiation was supported (bpp 0.96).

4. Discussion

The 16S rRNA secondary structure was highly con-served in the examined tribes, and allows no inferences on

the phylogeny of Meloinae. While MP supported mono-phyly of several clade at diVerent levels, ML and Bayes-ian analyses provided a critical test for a relatively robustphylogenetic hypothesis. The relationships among Meloi-nae tribes are only partially overlapping with the resultsemerging from morphological and biological features(Bologna and Pinto, 2001), evidently due to the very highnumber of taxa (96) morphologically examined, compar-ing with the reduced molecular dataset. Thus, we do notdiscuss this issue in details here. We only take notice (forfuture testing) that in our analyses there was a good indi-cation of a closer relationship of Pyrotini with Mylabrinithan with any other examined tribes. Furthermore, therewas a support to a basal position (among the examinedlineages) of Eupomphini and Lyttini.

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

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

In all trees the Mylabrini were monophyletic and sup-ported by good bootstrap and posterior probability.Monophyly of Mylabrini is also strongly supported byseveral derived morphological characters, particularly oflarvae (Bologna, 1991; Bologna and Pinto, 2001).

Present work provided insights on the relationshipsamong genera of Mylabrini (Fig. 1). Separation of Myla-bris and Hycleus received strong support in all analyses(and it is supported by the analysis of the faster evolvinggene ITS2 in a larger taxonomic dataset: Bologna et al.,unpublished). Given their relevance to applied biology,the likelihood that they represent two distinct evolution-ary lineages is of great importance. Particularly, in sev-eral analyses, Hycleus species represent a holophyleticlineage only with the inclusion of Ceroctis and Paracte-nodia. They have in common mesosternal and aedeagalfeatures, but are diVerentiated by antennal shape andpalpal structure of Wrst instar larvae. Present results sup-port the inclusion of both Ceroctis and Paractenodiawithin Hycleus, as suggested by Bologna (1991, 2000)and Bologna and Coco (1991), contrasting the classiWca-tion proposed by Kaszab (1969). Hycleus is the mostspeciose genus of blister beetle, and its internal systemat-ics deserves a deeper insight also with respects to the“sections”, morphologically proposed by Pardo Alcaide(1950, 1955, 1958). Topology of the Mylabris species iscongruent with the current subgeneric taxonomy. (Myla-bris Mesosulcata) and particularly M. (Eumylabris) aremore derived morphologically. The more speciose andwidespread M. (Mylabris) and particularly M. (Micra-bris) have more primitive morphological features.Monophyly of the genera with more than a single speciesassayed was always supported with good bootstrap andbpp, in agreement with morphological and biologicalfeatures of adults (Bologna, 1991, 2000; Bologna andCoco, 1991), and of Wrst instar larvae (see for a reviewBologna and Pinto, 2001).

The identiWcation of the most basal oV-shoot (eitherCroscherichia or Actenodia / Mimesthes) in the radiationof Mylabrini could beneWt from the indication of mor-phology. Croscherichia shares several adult morphologi-cal features with Mylabris, and closer aYnities with theMylabris/Hycleus lineage seem reasonable. Theextremely distinct larvae of Croscherichia could repre-sent the adaptive results of undiscovered phoretic behav-iour (Bologna and Pinto, 2001). The molecularrelationships between Actenodia (with a disjoint distri-bution in the Afrotropical and Mediterranean regions)and Mimesthes (endemic to Namaqualand, Karoo andNamib deserts), are also supported by several morpho-logical features.

This study oVers the Wrst attempt to provide an inde-pendent phylogenetic framework for studies on the evo-lution of Meloidae. This Wrst step—admittedly stillbased on a short fragment of sequence—indicates thatthis approach is promising and should be extended to

other tribes (Lyttini could be the next target). We expectalso that the addition of other genes will improve theresolution. The availability of good phylogenetichypotheses, will allow integration with morphologicaland behavioural data, helping to get a full understandingof the evolution of blister beetles.

Acknowledgments

We wish to thank the following students, fellowshipsand colleagues who collaborated on the collection ofspecimens during several Weld researches made by one ofus (M.A.B.) in the Mediterranean Basin and in SouthernAfrica: Valentina Amore, Pierluigi Bombi, Paola DeSalvo, Andrea Di Giulio, Simone Fattorini, Luca Fac-chinelli, Carla Marangoni, Francesca Montalto, John D.Pinto, George Pratt, Monica Pitzalis, Chiara Settanni,Federica Turco, Alberto Venchi, Marzio Zapparoli. Aspecial thank to Mario Garcia Paris (Madrid), who sup-plied a specimen of Mylabris hieracii, and to Maria Vit-toria Modica, and Annarita Wirz, for helping withlaboratory work. This research was supported by grantsof the Italian Ministry of University (99C271884-007)and Roma Tre University (Faculty grant).

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