Phylogeny and evolutionary history of the blister beetles (Coleoptera, Meloidae)

15
Phylogeny and evolutionary history of the blister beetles (Coleoptera, Meloidae) Marco A. Bologna a, * , Marco Oliverio b , Monica Pitzalis a , Paolo Mariottini a a Dipartimento di Biologia, ‘‘Roma Tre’’ University, Viale G. Marconi 446, 00146 Roma, Italy b Dipartimento di Biologia animale e dell’Uomo, ‘‘La Sapienza” University, Viale dell’Università 32, 00187 Roma, Italy article info Article history: Received 15 November 2007 Revised 11 March 2008 Accepted 9 April 2008 Available online 22 April 2008 Keywords: Phylogeny Meloidae 16S rDNA ITS2 rDNA Secondary structure Evolutionary history abstract Meloid beetles are well characterised by both morphological and biological features. Previous phyloge- netic hypotheses based on morphological characters assumed the repeated parallel evolution of complex biological novelties. In this work relationships among several taxa of the four subfamilies and almost all tribes representing meloid diversity are examined by using mitochondrial (16S) and nuclear (ITS2) DNA sequences, in 25 genera (using Anthicidae as outgroup). Secondary structure of 16S and ITS2 rRNAs were modelled. ITS2 structure represents a synapomorphic condition for the family and informative characters at the tribal level. Phylogenetic hypotheses based on separate and combined analysis of the 16S and ITS2 rDNA sequences, and morpho-biological characters were tested, and compared with previous morpho- logical classifications. Molecular dating allowed an outline of the main steps of the evolutionary history of Meloidae, which evolved during Early Cretaceous and then radiated considerably with the adoption of hypermetaboly and parasitic behaviour, and with repeated, parallel evolution of larval phoresy on its hosts. Ó 2008 Elsevier Inc. All rights reserved. 1. Introduction Meloidae, commonly called blister beetles, is a family of Coleop- tera Tenebrionoidea which includes almost 3000 species in approximately 125 genera. The family is relatively well known in the biological literature due to its hypermetabolic larval develop- ment, parasitoid biology of its larval phases and production of can- tharidin (see Bologna, 1991, for a review). Hypermetaboly is common to all Meloidae, with the probable exception of the sup- posedly primitive subfamily Eleticinae, which may have general- ized predatory rather than parasitoid larvae (Pinto et al., 1996). Meloid hypermetaboly includes seven larval instars often divided into five (i–v) morphologically and biologically distinct phases: (i) the ‘‘triungulin” phase, adapted to reach its host, in several cases by phoresy; (ii) the second phase includes the feeding larval instars II–V; (iii) the hypnotheca, a quiescent sixth instar; (iv) the post- hypnotechal instar VII; (v) the pupa. Meloids primarily parasitize Hymenoptera, usually bees (superfamily Apoidea), rarely other Aculeata; two speciose tribes of Meloinae (Mylabrini, Epicautini) attack Acridoidea (Orthoptera). Cantharidin is a defensive and probably aggregative terpenoid, known also in the Oedemeridae (e.g. Carrel et al., 1986; Frenzel and Dettner, 1994), a related fam- ily, and used by some Pyrochroidae, another family of Tenebrionoi- dea, as a sexual attractive (Holz et al., 1994). Cantharidin has a long history of pharmacological use in different countries of the World, as a sexual stimulant as well as an anti-hydrophobic, anti-inflam- matory substance, etc. The presence of this defensive substance in Meloidae is linked to complex and prolonged sexual behaviour in Meloinae, the largest subfamily (Selander, 1964; Bologna, 1991). Ontogeny, larval and adult morphology, cleaning and sexual behaviour have been used in the classification of the family (e.g. Escherich, 1897; Cros, 1940; MacSwain, 1956; Selander, 1964,1991; Kaszab, 1969; Bologna, 1991). Bologna and Pinto (2001) attempted to define phylogenetic relationships of meloid genera by utilizing numerous morphological and biological fea- tures in a large set of taxa. This cladistic analysis supported the monophyly of Meloidae and its division into four subfamilies (Ele- ticinae, Meloinae, Tetraonycinae, Nemognathinae), as well as the recognition of several of the traditional tribes. Eleticinae is hypoth- esized as the most primitive subfamily based on both larval and adult features: first instar larva orthosomatic (Fig. 1a, g and j); probable non-hypermetabolic and non-parasitic larval develop- ment; adult abdominal integument highly sclerotized and with a basal depression to receive the metacoxae; presence of an extrusi- ble female ovipositor; probably oviposition in plant tissues. Meloi- nae is a subfamily traditionally divided into seven tribes with complex and differentiated sexual behaviour, correlated with mor- phological dimorphism and hooked male genitalia. Their campode- iform larvae (Fig. 1b, c, f, h, i, k and l) show different biological specialisations related to parasitism primarily on Apoidea, but also on Acridoidea or other Aculeata. The female lacks an ovipositor and 1055-7903/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2008.04.019 * Corresponding author. Fax: +39 0657336321. E-mail addresses: [email protected] (M.A. Bologna), marco.oliverio@ uniroma1.it (M. Oliverio), [email protected] (M. Pitzalis), [email protected] (P. Mariottini). Molecular Phylogenetics and Evolution 48 (2008) 679–693 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev

Transcript of Phylogeny and evolutionary history of the blister beetles (Coleoptera, Meloidae)

Page 1: Phylogeny and evolutionary history of the blister beetles (Coleoptera, Meloidae)

Phylogeny and evolutionary history of the blister beetles (Coleoptera, Meloidae)

Marco A. Bologna a,*, Marco Oliverio b, Monica Pitzalis a, Paolo Mariottini a

a Dipartimento di Biologia, ‘‘Roma Tre’’ University, Viale G. Marconi 446, 00146 Roma, Italyb Dipartimento di Biologia animale e dell’Uomo, ‘‘La Sapienza” University, Viale dell’Università 32, 00187 Roma, Italy

a r t i c l e i n f o

Article history:Received 15 November 2007Revised 11 March 2008Accepted 9 April 2008Available online 22 April 2008

Keywords:PhylogenyMeloidae16S rDNAITS2 rDNASecondary structureEvolutionary history

a b s t r a c t

Meloid beetles are well characterised by both morphological and biological features. Previous phyloge-netic hypotheses based on morphological characters assumed the repeated parallel evolution of complexbiological novelties. In this work relationships among several taxa of the four subfamilies and almost alltribes representing meloid diversity are examined by using mitochondrial (16S) and nuclear (ITS2) DNAsequences, in 25 genera (using Anthicidae as outgroup). Secondary structure of 16S and ITS2 rRNAs weremodelled. ITS2 structure represents a synapomorphic condition for the family and informative charactersat the tribal level. Phylogenetic hypotheses based on separate and combined analysis of the 16S and ITS2rDNA sequences, and morpho-biological characters were tested, and compared with previous morpho-logical classifications. Molecular dating allowed an outline of the main steps of the evolutionary historyof Meloidae, which evolved during Early Cretaceous and then radiated considerably with the adoption ofhypermetaboly and parasitic behaviour, and with repeated, parallel evolution of larval phoresy on itshosts.

� 2008 Elsevier Inc. All rights reserved.

1. Introduction

Meloidae, commonly called blister beetles, is a family of Coleop-tera Tenebrionoidea which includes almost 3000 species inapproximately 125 genera. The family is relatively well known inthe biological literature due to its hypermetabolic larval develop-ment, parasitoid biology of its larval phases and production of can-tharidin (see Bologna, 1991, for a review). Hypermetaboly iscommon to all Meloidae, with the probable exception of the sup-posedly primitive subfamily Eleticinae, which may have general-ized predatory rather than parasitoid larvae (Pinto et al., 1996).Meloid hypermetaboly includes seven larval instars often dividedinto five (i–v) morphologically and biologically distinct phases:(i) the ‘‘triungulin” phase, adapted to reach its host, in several casesby phoresy; (ii) the second phase includes the feeding larval instarsII–V; (iii) the hypnotheca, a quiescent sixth instar; (iv) the post-hypnotechal instar VII; (v) the pupa. Meloids primarily parasitizeHymenoptera, usually bees (superfamily Apoidea), rarely otherAculeata; two speciose tribes of Meloinae (Mylabrini, Epicautini)attack Acridoidea (Orthoptera). Cantharidin is a defensive andprobably aggregative terpenoid, known also in the Oedemeridae(e.g. Carrel et al., 1986; Frenzel and Dettner, 1994), a related fam-ily, and used by some Pyrochroidae, another family of Tenebrionoi-

dea, as a sexual attractive (Holz et al., 1994). Cantharidin has a longhistory of pharmacological use in different countries of the World,as a sexual stimulant as well as an anti-hydrophobic, anti-inflam-matory substance, etc. The presence of this defensive substancein Meloidae is linked to complex and prolonged sexual behaviourin Meloinae, the largest subfamily (Selander, 1964; Bologna, 1991).

Ontogeny, larval and adult morphology, cleaning and sexualbehaviour have been used in the classification of the family (e.g.Escherich, 1897; Cros, 1940; MacSwain, 1956; Selander,1964,1991; Kaszab, 1969; Bologna, 1991). Bologna and Pinto(2001) attempted to define phylogenetic relationships of meloidgenera by utilizing numerous morphological and biological fea-tures in a large set of taxa. This cladistic analysis supported themonophyly of Meloidae and its division into four subfamilies (Ele-ticinae, Meloinae, Tetraonycinae, Nemognathinae), as well as therecognition of several of the traditional tribes. Eleticinae is hypoth-esized as the most primitive subfamily based on both larval andadult features: first instar larva orthosomatic (Fig. 1a, g and j);probable non-hypermetabolic and non-parasitic larval develop-ment; adult abdominal integument highly sclerotized and with abasal depression to receive the metacoxae; presence of an extrusi-ble female ovipositor; probably oviposition in plant tissues. Meloi-nae is a subfamily traditionally divided into seven tribes withcomplex and differentiated sexual behaviour, correlated with mor-phological dimorphism and hooked male genitalia. Their campode-iform larvae (Fig. 1b, c, f, h, i, k and l) show different biologicalspecialisations related to parasitism primarily on Apoidea, but alsoon Acridoidea or other Aculeata. The female lacks an ovipositor and

1055-7903/$ - see front matter � 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.ympev.2008.04.019

* Corresponding author. Fax: +39 0657336321.E-mail addresses: [email protected] (M.A. Bologna), marco.oliverio@

uniroma1.it (M. Oliverio), [email protected] (M. Pitzalis), [email protected](P. Mariottini).

Molecular Phylogenetics and Evolution 48 (2008) 679–693

Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution

journal homepage: www.elsevier .com/locate /ympev

Page 2: Phylogeny and evolutionary history of the blister beetles (Coleoptera, Meloidae)

lays its eggs in holes dug in the ground. Finally, phoresy on bees(and marginally on carabid beetles) seems to have occurred severaltimes independently. The Tetraonycinae have several adult mor-phological traits similar to those of Meloinae, but the first instarlarva is highly specialized for phoresy (Fig. 1e), yet distinct fromthe phoretic larvae of the other subfamilies. Females also lack anovipositor and lay eggs on flowers. Nemognathinae is a cosmopol-itan subfamily (the only meloids in Australia and Pacific Islands),with a simple sexual behaviour. The male genitalia are unarmedand oviposition is either on flowers or inside (or close to) the host

nest. With the exception of the Stenoderini, the first larva is highlyadapted for phoresy (Fig. 1d), and each larval instar II–VII remainswithin the exuvia of the previous instar. Within subfamily, how-ever, certain relationships are questionable and the monophylyof tribes such as Lyttini and Pyrotini in the subfamily Meloinae,and Nemognathini in the subfamily Nemognathinae, remain unre-solved or uncertain. Recognition of the subfamily Tetraonycinaecontrasted with previous classifications of Selander (1966, 1991)and Kaszab (1969). There was instead clear evidence that thestrong adaptive pressures operating on adults and larvae (mostlyrelated to hypermetaboly and phoresy) could hamper the reliableuse of morphological features for deriving phylogenetichypotheses.

The first molecular data of a blister beetle were published in ageneral study of holometabolous insect phylogeny based on thenuclear 18S and 28S ribosomal RNA (rRNA) genes (Chalwatziset al., 1995), in which one species of Meloe Linnaeus, 1758 (M. pro-scarabaeus Linnaeus, 1758) was examined. More recently, relation-ships among genera of the tribe Mylabrini were clarified by using16S mitochondrial DNA (mtDNA) sequences by Bologna et al.(2005). More detailed molecular studies focused on relationshipsamong species of the genera Actenodia Laporte de Castelnau,1840 (Bologna et al., 2008) and Iselma Haag-Rutenberg, 1879 (Pitz-alis, 2007) and a phylogeographic study was carried out on Apen-nine species of the genus Mylabris Fabricius, 1775 using COIhaplotypes (Ketmayer et al., 2008).

This paper revisits phylogenetic relationships of the Meloidae,at the subfamily and tribal levels. The possibility of combininganalyses of different DNA sequences and a morpho-biological data-set, seemed to us a promising approach to the phylogenetics of thisbeetle family. In particular, this paper aims at: (a) defining the phy-logenetic relationships of the blister beetle subfamilies and tribesusing molecular, morphological (both adult and larval) and biolog-ical characters; (b) testing the hypothesis (Bologna and Pinto,2001) that phoresy evolved independently several times in thefamily (and particularly in the subfamily Meloinae); (c) evaluatinghow the 16S mitochondrial and ITS2 nuclear DNA sequences areinformative for dating deep nodes in the meloid tree; (d) describein a biogeographical framework, the evolutionary steps in the his-tory of the family.

2. Materials and methods

2.1. Taxon sampling and DNA sequencing

Species employed in the molecular analyses are reported inTable 1, with locality data and specimen acronyms used in thepaper, along with data on larvae used to produce new morpholog-ical data. They represented all four traditional subfamilies and 12of the 16 tribes recognised by previous morphological studies(see Bologna, 1991; Pinto and Bologna, 1999; Bologna and Pinto,2001): Eleticinae (Derideini), Meloinae (Pyrotini, Lyttini, Eupom-phini, Cerocomini, Epicautini, Mylabrini, Meloini), Tetraonycinae(Tetraonycini), Nemognathinae (Stenoderini, Nemognathini, Sita-rini). Unavailable for study were four tribes of the subfamily Eletic-inae (Protomeloini, Spasticini, Morphozonitini and Eleticini), andthe Horiini, which presumably belong into the subfamily Nemo-gnathinae; all of these tribes include very uncommon species.Specimens of these groups suitable for DNA extraction were notavailable, but considering the unlikely acquisition of samples in areasonable time, we decided to proceed with the available mate-rial. Anthicus antherinus (Linnaeus, 1761), family Anthicidae, wasemployed as outgroup for both the molecular and the morpholog-ical analyses. Anthicidae are commonly considered the most likelysister group to Meloidae (Selander, 1966) and for this reason was

Fig. 1. First instar larval morphology (SEM) of selected meloid taxa. (a) Primitivemorphology of the deridein Iselma pallidipennis Haag-Rutenberg, 1879. (b and c)Campodeiform non-phoretic morphology of (b) the ‘‘lyttin” Alosimus chalybaeus (-Tauscher, 1812) and (c) the mylabrin Mylabris fabricii Soumakov, 1929. (d–f) Ca-mpodeiform phoretic morphology of (d) the nemognathin Euzonitis rubida(Ménétriés, 1832), (e) the tetraonycin Tetraonyx fulvus LeConte, 1853 and (f) themeloin Epispasta abbreviata (Klug, 1825). (g, l) Primitive antennal (g) and leg (l)morphology of Iselma pallidipennis. (h and m) Intermediate antennal (h) and leg (m)morphology of the hypermetabolic non-phoretic ‘‘lyttin” Teratolytta gentilis (Friv-aldszky, 1877). (i and n) Derived antennal (i) and leg (n) morphology of the hype-rmetabolic phoretic meloin Meloe variegatus Donovan, 1793.

680 M.A. Bologna et al. / Molecular Phylogenetics and Evolution 48 (2008) 679–693

Page 3: Phylogeny and evolutionary history of the blister beetles (Coleoptera, Meloidae)

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used as outgroup by Bologna and Pinto (2001). In the molecularanalyses we have also used as outgroup the less related tenebrio-nid Tenebrio molitor Lineaus, 1758 with identical results (data notshown). We report here only the results using A. antherinus in anal-ogy with Bologna and Pinto (2001), primarily because of thenumerous difficulties in coding homologous morpho-biologicalcharacters in other taxa.

With reference to traditional taxonomy, we adopt here the fol-lowing convention for the common names of the tribes and sub-families: we use the ending —ine(s) when referring to taxatraditionally ranked as subfamilies (as e.g. meloine(s) referring toMeloinae), and—to avoid confusion—the desinence –in(s) whenreferring to taxa traditionally ranked as tribes (as e.g. meloin(s)referring to Meloini).

Total genomic DNA was extracted from metasternal muscles,dissected in freshly collected and alcohol fixed specimens, or, ina few cases, in dried collection specimens. DNA was extractedand purified either by standard phenol-chloroform-ethanol meth-ods (Hillis et al., 1990) with slight modification as previously de-scribed by Oliverio and Mariottini for molluscs (2001), or by the‘‘DNeasy Blood and Tissue Kit” (Qiagen, Hilden, Germany) follow-ing manufacturer procedures.

Partial sequences of the 16S rDNA mitochondrial region wereamplified by PCR with primers 16sar-L and 16sbr-H as defined inBologna et al. (2005). A portion of the nuclear ribosomal cluster,including the 30 end of the 5.8S, the entire ITS2 spacer, and the 50

end of the 28S, was amplified by the primers its-3d and its-4r, com-plementary to conserved regions of the 5.8S and 28S rDNA se-quences (Oliverio and Mariottini, 2001). PCR amplified productswere either sequenced directly or (for some ITS2 amplicons) clonedusing the pGEMT-easy Vector System (Promega), following theexperimental procedures provided by the manufacturers. PCRproducts and plasmid DNA from positive clones were sequencedby an automated sequencer (Hitachi 3100 Genetic Analyzer) atthe ENEA laboratories (La Casaccia, Rome), and at Macrogen Inc.(Seoul). All sequences have been deposited at EMBL (see Table 1for accession numbers).

2.2. Sequence and secondary structure analysis

Sequences for each gene were initially aligned by ClustalX 1.83(Thompson et al., 1997). Then, the alignment was progressivelyoptimised according to secondary structure homology. Foldingwas predicted for each sequence on a thermodynamic basis usingthe software package RNA Structure version 3.71 available on theTurner Lab Homepage http://rna.chem.rochester.edu (Mathewset al., 1999). Thermodynamic and comparative approaches werecombined to derive the best-supported folding models and themaximised structural homologies were retained and checked forthe presence of compensatory changes. Notwithstanding, someportions of the ITS2 dataset were too highly divergent to allowreliable alignment. These portions (396 positions; partition:‘‘ambiguous”) were therefore excluded from the phylogeneticanalyses. The complete alignments are available from the authorson request.

The partial 16S rRNA structure (the portion corresponding todomains IV and V) was used as reference for the alignment; theITS2 sequence of Iselma brunneipes Haag-Rutenberg, 1879 (hypoth-esized as a primitive taxon among the examined blister beetles),was used as reference for both the alignment and the structuralcomparison among meloid tribes (Fig. 2) and for other beetle fam-ilies (Fig. 3), such as Byturidae (Byturus affinis Reitter, 1874; acces-sion number AJ409080), Tenebrionidae (Tenebrio molitor Linnaeus,1758; accession number AJ635266), Chrysomelidae (Timarcha gen-iculata Germar, 1824; Accession No. AJ512579) and Meloidae(Cerocoma schreberi Fabricius, 1781).

Analysis of the nucleotide sequences were performed by usingMEGA version 3.1 (Kumar et al., 2004). Mutational saturationwas examined for the 16S and ITS2 datasets, by plotting uncor-rected ‘‘p” pairwise, Tv and Ti distances against the estimated MLdistances (Philippe et al., 1994; Nichols, 2005) in DAMBE (Xia,2000; Xia and Xie, 2001).

2.3. Morphological and biological datasets

The cladistic analysis of the genera of Meloidae by Bologna andPinto (2001) was based on a set of 126 morphological and eco-ethological characters of both larvae and adults. For the presentwork we wanted to include a morphological dataset includingthe same 126 characters (see Bologna and Pinto, 2001: AppendixA, for the matrix of characters), for the species used in the molec-ular analyses. The matrix of 126 characters (91 parsimony informa-tive) for the 30 ingroup taxa (plus the outgroup Anthicusantherinus) is reported in Appendix A. All characters are treatedas unordered and equally weighted. Compared to Bologna and Pin-to (2001), the present morphological dataset differed in the follow-ing aspects. Four genera, namely Euzonitis Semenov, 1893,Actenodia Laporte de Castelnau, 1840, Mimesthes Marseul, 1872,Diaphorocera Heyden, 1863 were added (several others were notincluded, lacking the relevant molecular data); larval charactersunavailable for Diaphorocera hemprichi were inferred from thestudy of the closely related D. chrysoprasis Fairmaire, 1863. Ceroco-ma graeca Maran, 1944 was added in the datasets. Some species re-placed other of the same genus: Stenodera caucasica (Pallas, 1782),Ceroctis angolensis (Harold, 1870), Hycleus brevicollis (Baudi, 1878),H. deserticolus (Wellmann, 1908), Mylabris flexuosa Olivier, 1811,Megetra vittata (LeConte, 1853), Oenas crassicornis (Illiger, 1800).In total, new data on larval morphology for 12 species of two sub-families have been added.

For some other species used in the molecular analyses, informa-tion on larval morphology was missing and impossible to obtain:in these few cases, larval characters of other congeneric and closelyrelated species (after Bologna and Pinto, 2001) were utilized: Epic-auta albovittata (Gestro, 1878), E. bispinosa Werner, 1949, E. pruin-osa (LeConte, 1866); Hycleus burmeisteri (Bertoloni, 1850), H.humerosus (Escherich, 1899), H. phaleratus (Pallas, 1781), H. poly-morphus (Pallas, 1771); Iselma pallidipennis Haag-Rutenberg,1879; Pyrota postica LeConte, 1866, P. sinuata (Olivier, 1795), P. ter-minata LeConte, 1866, P. wagneri Denier, 1934.

2.4. Phylogenetic analyses

Three distinct datasets were analysed: (a) the 16S sequences;(b) the ITS2 sequences; (c) the morphological and eco-ethologicalcharacters. Then the respective matrices were joined in a combinedmolecular dataset (16S+ITS2) and a combined molecular + mor-pho-biological dataset.

The aligned sequences were analysed under the assumptions ofmaximum parsimony (MP: Farris, 1970) and maximum likelihood(ML: Felsenstein, 1981), and by Bayesian inferences, using thepackages PAUP* v. 4b10 (Swofford, 2002), Modeltest v. 3.7 (Posadaand Crandall, 1998), MrModeltest v. 2.2 (Nylander, 2004), Treefind-er (Jobb, 2007), MrBayes v. 3.1.2 (Ronquist and Huelsenbeck,2003). Each molecular marker (16S and ITS2) was first analysedseparately, then combined, and finally, the two loci and the mor-pho-biological dataset were analysed simultaneously.

Maximum parsimony (MP) analyses were performed by PAUP*

(ACCTRAN optimization, gaps alternatively coded as a new state oras missing, TBR branch swapping), using heuristic searches. Boot-strap support for MP trees was calculated using 10000 bootstrapreplicates. In MP analyses of the morpho-biological dataset allcharacters were treated as unordered and equally weighted; re-

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sults did not employ successive weighting. As in Bologna and Pinto(2001), a set of 12 characters (partition: ‘‘quantitative”) was re-ferred to traits which were continuously variable and difficult tocode objectively. As already experienced by Bologna and Pinto(2001) these characters displayed considerable homoplasy (lowconsistency indices CI) and were alternatively included and ex-cluded in the analyses. The same alternative inclusion/exclusionwas performed for the set of 10 characters related to phoresy (par-tition: ‘‘phoresy-related”. See Bologna and Pinto, 2001).

ML analyses were performed by PAUP*, using a heuristic searchstrategy. A model of evolution was chosen (for each gene and forthe combined molecular dataset) after evaluation by Modeltestusing likelihood ratio tests (LRT) and the Akaike information meth-od (AIC). The analyses were all replicated with the different optionsproposed by the two methods with virtually the same results.Therefore, only results obtained by AIC method are shown. Thesame AIC-selected models have been used to estimate ML dis-tances employed for the mutational saturation analysis. Each anal-ysis was replicated by Treefinder to check consistency of theresults. Bootstrap support was calculated for ML trees using 100(PAUP*: heuristic search, stepwise addition, 10 random-sequence

addition replicates, TBR branch swapping) and 10000 (Treefinder)bootstrap replicates under the same assumptions.

A partitioned Bayesian analysis (BI) was performed to obtainBayesian posterior probabilities (bpp) of nodes using the softwareMrBayes that adopts the Markov Chain Monte Carlo method tosample from posterior densities (Larget and Simon, 1999; Yangand Rannala, 1997). Also in this case, separate analyses were firstperformed on each dataset (16S, ITS2 and morphology), and thena single analysis was performed on a combined dataset. The mod-els of evolution were the ones chosen by MrModeltest for eachDNA dataset using the AIC method. Base frequencies, the relativerates of the substitution types and model parameters were esti-mated during the analysis (unlinked in MrBayes), separately foreach DNA region. For the morphological dataset the model isessentially based on Lewis (2001), adopting an analogue to aJukes-Cantor model for DNA, but with a variable number ofstates. A four chain metropolis-coupled Monte Carlo analysiswas run twice in parallel for 107 generations, and trees were sam-pled every 100 generations, starting after a burn-in of 2.5 � 106

generations. Stationarity was considered to be reached whenthe average standard deviation of split frequencies shown in

Fig. 2. Secondary structure model of the ITS2 rRNA spacer of the meloid Iselma brunneipes. DI-VI indicate stem–loop domains. The core region is in dark and conserved singlechain nucleotide positions are indicated. Schematic hairpin outlines are reported for each tribe.

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MrBayes was less than 0,01 (Ronquist and Huelsenbeck, 2003).Bayesian posterior probabilities (bpp) were estimated on a 50%majority rule consensus tree of the sampled trees (with the com-

mand ‘‘sumt”, after burn-in). Four searches were preliminarily runon 106 generations to check the burn-in levels and the conver-gence topology.

a

c d

b

Fig. 3. Secondary structure model of the ITS2 rRNA spacer of selected Polyphaga genera: (a) Byturus affinis (Byturidae); (b) Tenebrio molitor (Tenebrionidae); (c) Timarchageniculata (Chrysomelidae); (d) Cerocoma schreberi (Meloidae).

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2.5. Divergence rates and time estimation

Our preferred phylogram (that which we believe better por-trayed the evolutionary history of the family) was used for theestimation of the rates and ages of the nodes. A Likelihood ratiotest (Huelsenbeck and Rannala, 1997) was performed usingModeltest in ‘‘LRT calculator mode’’, to test the null hypothesisthat there was no difference in evolutionary rate among differ-ent lineages (molecular clock). We also performed likelihood ra-tio tests of whether the two lineages descending from eachnode evolved at the same constant rate (as implemented inr8s, see below).

The LRT rejected the null hypothesis, suggesting the need toestimate node ages by methods which allow for different ratesamong lineages. To estimate the ages of the nodes and the ratesof the species lineages, we thus did not apply the Langley–Fitchmethod (LF; Langley and Fitch, 1973, 1974), which assumes amolecular clock, but rather the nonparametric rate smoothing(NPRS: Sanderson, 1997) and the semiparametric penalized likeli-hood (PL: Sanderson, 2002b) methods. NPRS uses a least squaressmoothing, which allows limited lineage-specific rate variationwithin a constrained rate space by using an optimality criterion(log likelihood—a roughness penalty), according to a smoothingparameter. We calculated divergence times using this method inthe program r8s v 1.71 (Sanderson, 2003). We determined the opti-mal smoothing value for each dataset by using cross-validation(Sanderson, 2002a), and then estimated divergence times usingthe truncated Newton (TN) algorithm. The branch lengths esti-mated on each optimal tree found in the 1000 ML bootstrap repli-cates, were used by r8s to summarize variation in divergence timesfor these phylograms.

We have selected the ‘‘root” of the meloid tree (i.e. the mostrecent common ancestor to the Meloidae) as either a fixed agenode or a constrained node. The origin of the family in Gondw-ana (Bologna, 1991; Pinto and Bologna, 1999; Bologna and Pin-to, 2002) is congruent with the supposedly most primitivesubfamily Eleticinae, being distributed only in the southernHemisphere. With reference to a Gondwanan origin of theMeloidae and to their absence during the early stages of meloidevolution in Australia (see Discussion), we imposed a date of125 Mya (as either fixed or constrained maximum age) for theorigin of the family. This is congruent with the commonassumptions (Crowson, 1960; Kirejtshuk, 2003; Watt, 1974) onthe origins of the Coleoptera Polyphaga (ca 190–200 Mya), thePolyphaga Cucujiformia (ca 180–190 Mya), the CucujiformiaTenebrionoidea (ca 135–140 Mya), and with the notions onthe bioeogeography of Gondwana (Sammartín and Ronquist,2004, with references therein). We have also imposed fixedages of 80–90 Mya to nodes arguably related to the completeseparation of Africa and northern South America, to check thereliability of the deeper dates. Finally, we have imposed a fixedage of 5 Mya for the initial radiation of the genus Iselma (seePitzalis, 2007).

3. Results

3.1. 16S and ITS2 rDNA sequence variation and secondary structures

A total of 31 sequences of 16S rDNA were obtained, rangingfrom 544 bp in Euzonitis rubida to 551 bp in Oenas crassicornis. Av2 test of base homogeneity, uncorrected for phylogeny, indicatedthat overall base composition was not significantly different acrossall sites (p = 0.999). Scatter plots of transitions and transversionsagainst the pairwise ML distance showed evidence of saturationfor transitions and transversions beyond an ML divergence of

0.25 (corresponding to pairwise comparisons over the inter-triballevel). The alignment of the 16S sequences caused no problemsand all positions were unambiguously homologized.

The proposed secondary structure for the 30 half portion of themeloid 16S rRNA conforms to the canonical architecture (Gutelland Fox, 1988; Gutell et al., 1993). Both domains IV and V showa high conservation in folding when compared to the common sec-ondary structure shared by taxa as distant as molluscs (Lydeardet al., 2000) and mammals (Horovitz and Meyer, 1995). With theexception of the very small L7 loop (Vawter and Brown, 1993) indomain V, all the remaining stems, bulges and loops were structur-ally conserved allowing a sequence alignment without uncertainty.

Thirty-one sequences of ITS2 rDNA from the same samples usedfor the 16S rDNA sequencing were used in the phylogenetic study(a total of 45 ITS2 sequences were obtained for comparisons of thesecondary structures: data not shown in details). The sequences in-cluded a portion of the 5.8S rDNA (128 bp) at the 50 end, and a por-tion of the 28S rDNA (67 bp) at the 30 end. The ITS2 sequencesranged from 252 (Oenas crassicornis, 447 bp including the ribo-somal ends) to 614 bp (Iselma brunnipes, 809 bp including the ribo-somal ends). All sequences were heavily A + T skewed, particularlywhen bearing large insertions of short AT rich repeats. A v2 test ofbase homogeneity, uncorrected for phylogeny, indicated that over-all base composition was significantly different across all sites(p = 0.000). Also for ITS2 sequences, scatter plots of transitionsand transversions against the pairwise ML distance showed evi-dence of saturation for transitions and transversions beyond aML divergence of 0.18 (corresponding to pairwise comparisonsover the inter-tribal level). All 45 sequences were used for second-ary structure reconstructions, while only 31 sequences were incor-porated in the phylogenetic dataset.

The derived ITS2 rRNA sequences have been folded taking intoaccount the recently proposed secondary structures for Chrysome-lidae (Gómez-Zurita et al., 2000) and Byturidae beetles (Mallochet al., 2001), and Drosophila flies (Young and Coleman, 2003). Themeloid ITS2 rRNA conforms very well to the general eukaryotemodel (Coleman, 2003 and references therein), but it reveals agreater complexity in secondary structure due to the presence oftwo extra hairpins (Da and Db) and/or stem length variability (Figs.2 and 3). The meloid consensus ITS2 rRNA folding shows four mainstructural stem–loop domains (DI-IV), which branch out of a highlyconserved core structure.

This consists of the proximal portions of stems DI, DII, DIII, DIVand Db domains, that show typical compensatory base changes,and few unpaired nucleotides. From these secondary structuralfeatures, consensus folding models can be derived for all the Meloi-nae tribes. The most conserved domains are DI and DII. On the con-trary, DIII and DIV show a high degree of variability in both lengthand nucleotide composition; in particular, DIII and DIV are verylong in Eupomphini and Mylabrini, respectively (Fig. 2). The extraDa stem branches out from the DII domain and is also very variablein both length and nucleotide composition. Furthermore, it is lack-ing in Epicautini, Lyttini, Mylabrini, Nemognathini, Sitarini andStenoderini. On the contrary, the extra Db stem is very conservedand present in all the analyzed tribes.

Furthermore, within the core in the DI proximal portion, the se-quence 50-G/GUCG-30 forms an extremely conserved bulge, as wellas the bulge produced by the sequence 50-AA/AA-30 located be-tween DIII and DIV. The conserved unpaired bases AG and U lo-cated between DI and DII, and DIII and DIV, respectively,represent further consensus sequence of the core (Fig. 2). Searchingfor a putative CAGAC motif, consensus of the metazoan 8S rRNAcleavage site (Joseph et al., 1999; Michot et al., 1999; Oliverioet al., 2002) was not positive.

Given the high divergence and the large amount of indels, wecould not acceptably align the entire ITS2 sequences. For the phy-

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logenetic analysis of the same 31 taxa as in the 16S dataset, a ma-trix of 779 unambiguously aligned positions (195 of the 5.8S and28S ends, and 584 of the ITS2) was used, by deleting all regionswith uncertain alignment (mostly corresponding to large indelsin loop regions). For this reduced dataset the v2 test of base homo-geneity indicated that overall base composition was not signifi-cantly different across all sites (p = 0.778).

The best fits models, selected by Modeltest 3.7 and MrModel-test 2.1 for the various datasets, under the hLRT and the AIC criteriaare reported in Table 2.

3.2. Phylogenetic analyses

3.2.1. Morphological and biological datasetsMP and BI analyses of all morphological and eco-ethological

characters recovered topologies, all with biologically implausiblegroupings and are not shown nor discussed here (e.g. part of thepolyphyletic Nemognathinae associated with Tetraonyx and onemeloine). MP analysis after removal of the 22 ‘‘quantitative” and‘‘phoresy-related” characters (heuristic search; 10,000 bootstrapreplicates) produced 1023 equally parsimonious trees. In the con-sensus tree (Fig. 4a), the Eleticinae were the sister to the rest of themeloids in all trees (100% bs). Then, a clade included always Tetra-onyx fulvus and a monophyletic Nemognathinae. This clade was thesister to the meloines. The Meloinae included a basal Pyrota akhur-stiana, and several lineages with unresolved relationships: Ceroco-mini (1.0 bpp), Meloini (0.92 bpp) plus part of polyphyletic Lyttini,Epicautini (1.0 bpp), Eupomphini (1.0 bpp) + Mylabrini (0.84 bpp).

The Bayesian analysis of the same dataset (Fig. 4b) recoveredthe best posterior support for nearly the same nodes. Again a basalposition of the Eleticinae, then a trichotomy including: (i) a cladewith Tetraonyx fulvus and a monophyletic (0.81 bpp) Nemognathi-nae; (ii) Pyrota akhurstiana, and (iii) the remaining Meloinae. Thelatter include four lineages with unresolved relationships: Ceroco-mini (1.0 bpp); Meloini (0.92 bpp) plus paraphyletic Lyttini; Epi-cautini (1.0 bpp); Eupomphini (1.0 bpp)+Mylabrini (0.84 bpp).

3.2.2. Molecular datasetsMP analysis of either 16S or ITS2 dataset with gaps coded as a

fifth state produced highly improbable reconstructions (notshown), contradicting all other available evidence for meloid evo-lution, supporting nodes only for very closely related taxa, whiledisrupting monophyly of tribes clearly diagnosed by morphology.Excluding indels from the analyses resulted in more plausiblereconstructions, thus we report here only results from gap-ex-cluded analyses.

MP analysis of the combined molecular (ITS2+16S) datasetrecovered two most parsimonious trees (Fig. 5a). The Eleticinaewere basal and the sister group to the rest of the meloids. Then,four unresolved lines emerged: (1) the Nemognathinae, (2) theMeloini, (3) the Cerocomini and (4) a clade with the remaining

groups (e.g. a monophyletic Lyttini and lineage with Tetraon-yx + Pyrota + Epicautini). MP analyses of the single datasets (ITS2,16S: not shown) recovered always the Eleticinae as the sister groupto the rest of the meloids. Major differences with the combinedanalysis, were in the Nemognathinae not monophyletic (ITS2), orincluded within the Meloinae (16S), and the Meloini as paraphylet-ic (16S).

ML analysis on the combined molecular dataset resulted in atree (�ln likelihood = 12380.80378) (Fig. 5b), where the Eleticinaewere basal, followed by a monophyletic Nemognathinae (includingthe stenoderin Stenodera caucasica) as sister to the Meloinae. Thelatter included a clade with Tetraonyx fulvus and the Cerocomini,followed by the Meloini which preceded the Pyrota akhursti-ana + Epicautini lineage. The final clade had the paraphyletic lyt-tins and the Eupomphini as the sister to the Mylabrini. MLanalysis of the single ITS2 dataset (not shown) did not recovermonophyly of Nemognathinae, Eupomphini and Mylabrini. MLanalysis of the 16S dataset (not shown), although recovered wellsupported monophyly of all tribes (except Lyttini), showed theNemognathinae as the sister to the rest of the meloids, and the Ele-ticinae in a well derived ‘‘meloine” clade.

Results of the Bayesian analysis on the combined moleculardataset are summarised in the tree of Fig. 5c (consensus of7.5 � 106 trees after burn-in) with mostly the same topology asthe ML one. BI analysis of the single datasets (ITS2, 16S: not shown)recovered topologies less resolved, and particularly failing to showmonophyly of most subfamilies.

3.2.3. Combined molecular + morpho-biological datasetsA BI analysis performed on the combined molecular + morpho-

biological dataset yielded very good support to the ML topology(Fig. 5d: on 7.5 � 106 trees; results were nearly identical whether‘‘quantitative” and/or ‘‘phoresy-related” morphological characterswere included or excluded, and bpp values for crucial nodes werealways very high). The Eleticinae were always (1.0 bpp) at the baseof the meloid tree. The Nemognathinae (1.0 bpp; including thestenoderin Stenodera caucasica) was monophyletic and sister to aclade including all remaining taxa (1.0 bpp). In the latter the posi-tion of Tetraonyx fulvus with respect to the Meloinae was not re-solved. It consisted of a basal trichotomy with Tetraonyx fulvus,the Cerocomini (1.0 bpp) and all remaining taxa (0.99 bpp). Thelatter lineage had the Meloini as sister to the other groups, fol-lowed by the Pyrota akhurstiana+Epicautini lineage (0.98 bpp),the paraphyletic lyttins, and a final clade (0.88 bpp) with theEupomphini (1.0 bpp) as the sister to the Mylabrini (1.0 bpp).

3.3. Divergence rates and time estimation

A Likelihood ratio test rejected the molecular clock hypothesis(D = 2 � [L1 � L0] = 192.00586, DF = 30, p = 0.000), suggesting theuse of methods of estimate of the node ages which allowed for dif-

Table 2Length of PCR products and of the respective alignments, along with the best-fit models selected by hLRT and AIC in Modeltest 3.7 (for ML analyses) and MrModeltest 2.1 (for BYanalyses)

16S ITS2 Combined

PCR product length 544–550 441–725 —Alignment length 569 1176 (779 unambiguous) 1745 (1348 unambiguous)Variable chrs 283 398 (unamb.) 681Parsimony inf. chrs 217 132 (unamb.) 349ML model

hLRT TVM+I+G TVM+I+G TVM+I+GAIC K81uf+I+G TVM+I+G GTR+I+G

BI modelhLRT GTR+I+G GTR+I+G —AIC HKY+I+G GTR+I+G —

In the Bayesian analysis of the combined dataset, all parameters for the two partitions were unlinked.

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ferent rates among lineages. Likelihood ratio tests, performed ateach node, of whether the two lineages descending from that par-ticular node evolved at the same constant rate, rejected a clock-likebehaviour for every lineage, with the exception of two clades: thegenus Mylabris and the tribe Eupomphini.

We have used the ML tree of Fig. 5b as the base for the analysesin r8s (summarized in Fig. 6). NPRS produced estimates differentfrom PL. Cross-validation analysis to find the optimal smoothingvalue (on a range of values from 0.1 to 104) produced the best scorefor the lowest values. This indicates the need for a smoothing valuethat would allow the existence of abrupt disparities in rates amongclose lineages. We have thus used a value of 0.1; values up to 1,produced nearly identical results (data not shown).

4. Discussion

The objective of this paper was to define the phylogenetic rela-tionships in blister beetles (Meloidae) based on molecular data,and to explore their evolutionary history by integrating molecularand morpho-biological information. For these targets we have usedone external outgroup of the related family Anthicidae (Anthicusantherinus) and an ingroup including 30 species belonging to 23genera of 12 meloid tribes out of 16 currently recognized, repre-senting all the major groups. Consequently, even if three tribes ofthe Eleticinae (Morphozonitini, Spasticini, Eleticini) and one ofthe Nemognathinae (Horiini) were not included, this study allowsa first estimate of phylogenetic relationships of the major lineageswithin this important family of the Coleoptera using moleculardata. We have combined the two gene partitions and the mor-

pho-biological characters in a single dataset, producing the firstmolecular phylogeny for the Meloidae, which is highly congruentwith the available biological knowledge and allows a discussionof biogeographic hypotheses for the blister beetles.

Based on these results, we propose a phylogenetic hypothesisfor the blister beetle subfamilies and tribes, describe and discussthe evolutionary biology and biogeography of the Meloidae withinthis phylogenetic framework, and test the hypothesis (Bologna andPinto, 2001) that phoresy evolved several times in the family inde-pendently and is a homoplastic biological feature in the Meloidae.

4.1. Phylogenetic relationships

Our preferred topology (Fig. 5c) is derived from the ML analysisof the molecular dataset, noting that the Bayesian analysis on thecombined molecular + morpho-biological dataset did not differ sig-nificantly, except for the lack of resolution of the position of Tetra-onyx fulvus with respect to the Meloinae.

The subfamily Eleticinae, represented by the genus Iselma (tribeDerideini) always emerged as the most basal group (very shortbranches in both molecular and morphological analyses), sisterto the rest of the Meloidae. This is perfectly congruent with thepossession of primitive morphological and biological features (lar-val and adult).

Interestingly, our study places the Nemognathinae alone, as ba-sal to all other non-eleticine meloids. The traditional literature(MacSwain, 1956; Kaszab, 1969; Bologna, 1991; Selander, 1991)has often treated the subfamily as the most derived meloid group.More recently Bologna and Pinto (2001) proposed a sister group

Zonitis flavaEuzonitis rubida60

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Anthicus antherinusIselma brunneipesIselma planidorsis

Berberomeloe majalis

Oenas crassicornis

Hycleus brevicollisHycleus deserticolusHycleus dentatusParactenodia parvaCeroctis angolensis

Mylabris calidaMylabris variabilisMylabris flexuosaActenodia chrysomelinaMimesthes maculicollisPhodaga marmorataMegetra vittata

Pyrota akhurstiana

Epicauta atrataEpicauta pensylvanica

Meloe proscarabeusPhysomeloe corallifer

Tetraonyx fulvus

Diaphorocera hemprichi

Cerocoma schreberiCerocoma graeca

Stenodera caucasicaSitaris muralis

Croscherichia paykulli

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Paractenodia parvaCeroctis angolensisCroscherichia paykulliMylabris calidaMylabrys variabilisMylabrys flexuosaPhodaga marmorata

Cerocoma schreberi

Physomeloe coralliferEpicauta atrata

Oenas crassicornisBerberomeloe majalis

Tetraonyx fulvus

Megetra vittata

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Hycleus deserticolus

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Fig. 4. Cladograms from the analyses of the morphological-biological dataset. (a) MP analysis after excluding ‘‘quantitative” and ‘‘phoresy-related” partitions. Consensustopology of 1023 trees [length = 170, on 79 parsimony-informative characters; Consistency index (CI) = 0.6412; Homoplasy index (HI) = 0.3588; CI excluding uninformativecharacters = 0.6039; HI excluding uninformative characters = 0.3961; Retention index (RI) = 0.7845; Rescaled consistency index (RC) = 0.5030]. Numbers are the bootstrapsupport (10000 reps.) of the relevant node. (b) Bayesian analysis: numbers are bpp (7.5 � 106 generations). EL: Eleticinae, Derideini; LY: Meloinae, Lyttini; ME: Meloinae,Meloini; EP: Meloinae, Epicautini; MY: Meloinae, Mylabrini; EU: Meloinae, Eupomphini; CE: Meloinae, Cerocomini; PY: Meloinae, Pyrotini; TE: Meloinae, Tetraonycini; ST:Nemognathinae, Stenoderini; SI: Nemognathinae, Sitarini; NE: Nemognathinae, Nemognathini.

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relationship between nemognathines and the Tetraonycinae. Insupport of earlier studies by Selander (1991), Bologna and Pinto(2001), and Bologna et al. (2002), the subfamily as defined here

clearly included the generalized Stenoderini (Stenodera) as a basalbranch relative the Sitarini (Sitaris) and Nemognathini (Zonitis andEuzonitis).

EU

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Actenodia chrysomelina

Mylabris calida

Paractenodia parvaHycleus deserticolus

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Phodaga marmorata

Diaphorocera hemprichiCerocoma graecaCerocoma schreberi

Mimesthes maculicollis

Croscherichia paykulli

Oenas crassicornisBerberomeloe majalis

Epicauta pensylvanica

Meloe proscarabaeusPhysomeloe corallifer

Hycleus dentatus

Anthicus antherinus

Zonitis flavaEuzonitis rubida

Iselma brunneipesIselma planidorsis

Tetraonyx fulvus

Epicauta atrataPyrota akhurstiana

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Hycleus dentatusParactenodia parva

Ceroctis angolensisMylabris calida

Mylabris variabilisMylabris flexuosa

Actenodia chrysomelinaMimesthes maculicollis

Phodaga marmorataMegetra vittata

Pyrota akhurstianaEpicauta atrata

Epicauta pensylvanicaMeloe proscarabeus

Physomeloe coralliferTetraonyx fulvusDiaphorocera hemprichi

Cerocoma schreberiCerocoma graeca

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Zonitis flavaEuzonitis rubida

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Paractenodia parvaCeroctis angolensis

Mylabris calidaMylabrys variabilis

Mylabrys flexuosa

Croscherichia paykulli

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Cerocoma graecaCerocoma schreberiDiaphorocera hemprichi

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Hycleus dentatusHycleus deserticolus

Oenas crassicornisBerberomeloe majalis

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Mylabris calidaMylabrys variabilisMylabrys flexuosa

Croscherichia paykulliActenodia chrysomelina

Cerocoma graecaCerocoma schreberi

Diaphorocera hemprichi

Phodaga marmorataMegetra vittata

Epicauta atrata

Hycleus dentatusHycleus deserticolus

Oenas crassicornisBerberomeloe majalis

Epicauta pensylvanica

Meloe proscarabaeus

Pyrota akhurstianaPhysomeloe corallifer

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Fig. 5. Cladograms from the analyses of the molecular and molecular + morpho-biological datasets. (a) MP analysis of the combined molecular (16S+ITS2) dataset. Numbersare the bootstrap supports of the nodes (10.000 reps.). (b) ML phylogram from the analysis of the combined molecular dataset (16S+ITS2). Numbers are the bootstrap supportof the nodes (1000 reps.). (c) Bayesian analysis of the combined molecular dataset (16S+ITS2). Numbers are bpp (7.5 � 106 generations). (d) Bayesian analysis of the combinedmolecular+morpho-biological dataset. Numbers are the bpp (7.5 � 106 generations). EL: Eleticinae, Derideini; LY: Meloinae, Lyttini; ME: Meloinae, Meloini; EP: Meloinae,Epicautini; MY: Meloinae, Mylabrini; EU: Meloinae, Eupomphini; CE: Meloinae, Cerocomini; PY: Meloinae, Pyrotini; TE: Meloinae, Tetraonycini; ST: Nemognathinae,Stenoderini; SI: Nemognathinae, Sitarini; NE: Nemognathinae, Nemognathini.

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The monophyly of the subfamily Meloinae (including Tetraonyx)is strongly supported in most of our analyses (only ITS2 did not re-flect this clade). Bologna and Pinto (2001) could not completelyelucidate the relationships among the meloine tribes. Particularlyproblematic were taxa traditionally placed in the tribe Lyttini.The present analysis, although supporting the monophyly of al-most all previously recognized tribes of Meloinae, also failed toshow phylogenetic support for the Lyttini.

The relationship of Tetraonyx to other genera has been variouslyinterpreted. Originally considered as meloids, they were ranked asa distinct family by Böving and Craighead (1931). Kaszab (1969)included the group in the Meloinae, while Selander (1991), empha-sizing larval and oviposition features, placed the genus in theNemognathinae. Its treatment as a subfamily, Tetraonycinae, andallied to the Meloinae was proposed by some authors (e.g.: Mac-Swain, 1956; Bologna, 1991), whereas, as already indicated, Bolo-gna and Pinto, 2001 most recently placed the subfamily as sisterto the Nemognathinae. All of our analyses indicated a position of

Tetraonyx closer to the Meloinae than to the Nemognathinae (assupported by the similarities in adult morphology), yet in a basalposition within a ‘‘meloine” clade. The possibility of a relationshipto the Cerocomini (Diaphorocera, Cerocoma), which emerged fromthe molecular dataset, is surprising from the morpho-biologicalpoint of view and may deserve further study. We may argue thatthis result is biased by the effects of mutational saturation overthe inter-tribal level.

Another relatively basal clade immediately apical to Tetraonyx-Cerocomini, was the tribe Meloini (Physomeloe and Meloe), a clearlymonophyletic group in all of our analyses. This supported the rela-tionship suggested by Bologna and Pinto (2001) and was counter tothe earlier placement of Physomeloe in the Lyttini (Bologna andAloisi, 1994).

Pyrotini (Pyrota) and Epicautini (Epicauta) also form a clade.While the relatively basal position of Pyrotini among meloineswas already suggested by Bologna and Pinto (2001), its associationwith the Epicautini did not have previous support.

125

ANTHICIDAE

ELETICINAE

“Lyttini”

“Lyttini”

Eupomphini

Mylabrini

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Fig. 6. Our preferred phylogenetic hypothesis for meloid lineages represented in this study. This chronogram is substantially derived from the phylogram of Fig. 5b. The timescale on the bottom is in Million years. Numbers at nodes are estimated dates (Mya) for the origin of the subtended lineage. The star marks the adoption of hypermetabolyand parasitic habit. The circles mark the arising of larval phoresy. Heights of terminal branches are roughly proportional to species richness of the tribe.

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Bologna and Pinto (2001) stressed how ‘‘the definition of Lyttin-i. . .. remains inadequately supported” even on a morphological ba-sis. Berberomeloe and Oenas, both traditionally included in the tribeLyttini (Bologna, 1989, 1991) did not form a monophyletic clade.Instead the molecular data argues strongly for the polyphyly ofthe Lyttini as traditionally conceived. This should stimulate furtherresearch with wider taxonomic coverage to delineate the bordersof the tribe if it can be justified at all, and to clarify the relation-ships of all included genera.

The remaining tribes, Eupomphini (Phodaga, Megetra) andMylabrini formed a weakly supported monophyletic group (basedon both bootstrap or Bayesian posterior probabilities). Molecularrelationships among the genera of the Mylabrini were already dis-cussed by Bologna et al. (2005), and the present study confirmedtheir conclusions.

Different levels of phylogenetic information can be obtainedfrom the analysis of RNA secondary structure. The 16S rRNA struc-ture is highly conserved and allows few inferences on the phylog-eny of Meloidae. On the contrary, some exclusive derived featuresof the ITS2 rRNA structure support the monophyly of the wholefamily and is greatly congruent with the present classification ofthe recognised subfamilies and tribes. First of all, the ITS2 foldingmodel here proposed for Meloidae and other beetle families con-forms to the universal eukaryote secondary structure (Young andColeman, 2003), but shows peculiar structural features characteris-tic at family to tribal levels (Fig. 2). The extra Da and Db hairpinsare lacking in other Coleoptera, such as Byturidae, Tenebrionidae,Chrysomelidae (Fig. 3), all included with the Meloidae in the sub-order Polyphaga.

At the moment, our results support the notion that the extra Dbdomain represents a synapomorphy of the Meloidae. It is presentin all subfamilies, both primitive (Eleticinae) and derived (Nemo-gnathinae, Meloinae). Also, the extra Da is present in the basal Ele-ticinae and in most other meloid tribes; it is thus another possiblesynapomorphy of Meloidae. Its absence is likely to be a derivedcondition of Nemognathinae (Stenoderini, Sitarini, Nemognathini)and of some Meloinae (‘‘lyttins”, Epicautini, Mylabrini). The corestructure shows paired and single chain regions that are very con-served in nucleotide sequences and position. This could reflectsome possible structural/functional constrains operating on it. Inspite of the fact that there is no indication of any potential alterna-tive or degenerated CAGAC sequence, we observed the presence ofthe extremely conserved GUCG sequence, as a bulge in the DI prox-imal portion, which could represent an alternative processing site.The Eupomphini show, as a clearly derived condition, a very longstructure of both extra Da hairpin and DIII domain; Mylabrini havethe autapomorphic condition of the DIV domain much longer thanin other tribes.

4.2. Evolutionary biology of Meloidae

The lack of more than one reliable date to correlate to relevantbasal nodes in our phylogenetic tree hampered the production ofrobust dating at each node. However, some congruent informationemerged from our molecular analyses (Fig. 6).

The Coleoptera Polyphaga may have originated ca 270 Mya, theCucujiformia (to which tenebrionoideans belong) ca 236 Mya, andthe Tenebrionoidea in the Late Triassic, according to Hunt et al.(2007). The latter work significantly predated previous datings(e.g. 190–200 Mya for the Polyphaga and ca 180–190 Mya for theCucujiformia: Crowson, 1960; Kirejtshuk, 2003; the Late Jurassic,i.e. around 135–140 Mya, for the Tenebrionoidea: Watt, 1974).An Early Cretaceous origin of the Meloidae (125–135 Mya) is con-gruent with all these notions (particularly, with Hunt et al., 2007,fig. 3) and with the current knowledge on the Gondwana biogeog-raphy (Sanmartín and Ronquist, 2004). It is noteworthy that this is

the period of flowering plant radiation (Heimhofera et al., 2007)which allowed the diversification of most floricolous insect groups.

The molecular dating indicates a diversification at the subfamilylevel having occurred during the Cretaceous, between 125 and ca100 Mya. The lineages corresponding to the tribe level in tradi-tional taxonomy, would have diversified between 90 and 65 Mya,thus having been derived by the end of Cretaceous. We refrainfrom using further subdivisions until a more complete coveragebelow the tribe level is accomplished.

The antiquity of the Eleticinae is strongly supported by bothmorphology and DNA. Their complete absence in Australia, Mada-gascar and New Zealand, suggests an origin in the western part ofthe Gondwana, probably after the separation of Eastern and Wes-tern Gondwana (which started ca. 135–125 Mya). Remarkably,the few eleticine species (genus Eletica Dejean, 1834) presentlyknown from the Indian subcontinent and Indochina, are very likelyto have recently arrived there (Late Tertiary) from Eastern Africathrough an Arabo-Sindian connection. Conversely, the Eleticinaehave radiated in both Africa and South America, with an old diver-sification at the tribal level which is probably the result of a longevolutionary history, yet maintaining their plesiomorphic Bauplanof both larva and adult. According to known larval morphology,eleticines (for which a very scanty knowledge on biology is avail-able) are hypothesized to be neither hypermetabolic nor parasitic.With the different distributional patterns shown by the varioustribes (Spasticini endemic to South America, Derideini commonto Africa and Chile, Morphozonitini endemic to eastern and centralAfrica, Eleticini common to Africa and Oriental Region), this is cer-tainly a very promising group for biogeographic studies of Gon-dwanan history.

The early history of Meloidae seems to have been characterisedby a relatively short period of evolutionary stasis. In fact, the line-age leading to the other two subfamilies (Nemognatinae andMeloinae), which could have originated shortly later (112 Mya),has been marked by two exceptional (and likely correlated) evolu-tionary events: the adoption of hypermetaboly and parasitism onother insects (primarily bees). The ancestor of this lineage wouldhave evolved a campodeiform first instar larva (usually called tri-ungulin), adapted for host searching. The triungulin Bauplan mayhave provided the possibility for the repeated evolution of phoreticspecializations of the larva. Bologna and Pinto (2001) suggested,after cladistic analyses of morpho-biological characters, that larvalphoresy had evolved several times independently in the blisterbeetles. Yet, the complicated morphological and eco-ethologicallarval modifications required to exploit this strategy, render thishypothesis very unparsimonious. Our study provided strongmolecular support for the polyphyly of meloid phoresy, whichwould have evolved independently at least six times. In the Meloi-nae alone, the five occurrences of phoretic larvae were scored asindependent events. It is probable that the strong advantages pro-vided by being carried directly to the host nest at the larval stage,overcame the need for a repeated evolution of similar (homoplas-tic) adaptations. This points to the difficulties of using morpholog-ical (and eco-ethological) characters in phylogenetic analyses ofthis group.

The Nemognathinae include a basal tribe (Stenoderini) with acampodeiform larva, and several plesiomorphic traits in the adult.Antiquity (an estimated origin ca 84 Mya) is also supported by thepresent disjunct distribution of the single living genus Stenodera inthe eastern Mediterranean and in the Far East of the Palearctic.Then, the level of divergence between Sitaris and the two Nemog-nathini (Zonitis and Euzonitis) supports their inclusion in a singletaxon at the tribal level. This is in agreement the morphologicalevidence (Bologna and Pinto, 2001). Furthermore, the origin of thislineage has been accompanied by the evolution of striking phoreticbehaviour and morphology. It is probably worthy of an insight the

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hypothesis that in the Nemognatini, phoresy has prompted aremarkable radiation when adopted, resulting in higher speciesrichness and number of genera: Sitarini + Nemognatini total ca300 spp. and well over 28 genera, vs. one genus with 7 species inthe non-phoretic Stenoderini. The presence in Australia of Nemog-nathini is likely due to a relatively old colonization by phoresy(probably from South East Asia) rather than an ancient pan-Gondwanian distribution. The Australian radiation, resulting inremarkable morphological diversification, is currently under studyby one of us (MAB).

The last subfamily (Meloinae) is the most diversified, both interms of species and higher hierarchical levels (Bologna, 1991).This is also reflected in their considerable biological diversification.Furthermore, meloines include both widely distributed tribes(Meloini, Epicautini, Mylabrini) and lineages endemic to more re-stricted areas (Tetraonycini, Cerocomini, Pyrotini, Eupomphini).

The position of Tetraonyx proposed here differs significantlyfrom anything previously published. Although the adult morphol-ogy does suggest relationship to the Meloinae, several biologicaltraits (courtship, copulation, and oviposition) are more characteris-tic of Nemognathinae. A basal position within a meloine clade isnot implausible especially considering the plesiomorphic repro-ductive traits of Tetraonyx (Selander, 1964), and the lack of a cam-podeiform larva, which we assumed to be a secondary loss (seeIntroduction) The present restriction of the Tetraonycini nearlyexclusively to the Neotropical Region suggests an origin thereand an age no older than ca 90 Mya, when South American landsseparated from Africa. Noteworthy, are the few living species inthe Nearctic, probably linked to an old colonization as indicatedby the fossil Tetraonyx minuscula Wickham, 1920 described fromthe Oligocene (23–36 Mya) of Florissant, Colorado.This is anothergroup which exploited phoresy and admittedly, their larval biolog-ical and morphological modifications (highlighted by longbranches in the morphological analyses) can largely be driven byadaptive pressure. The proposed sister group to the Tetraonycini,the Cerocomini, probably originated in xeric areas of eastern Africa,from where they subsequently dispersed to desert and steppeareas of southern Palearctic. The Meloini, apparently with very de-rived adult morphology, are relatively basal within the Meloinae.Their higher diversity is presently centered in the Holarctic Region(a single South American genus—Epispasta Selander, 1960—couldbe a meloine, according to larval morphology). Of the two recog-nized genera, the apparently more primitive one (Physomeloe, Reit-ter, 1911, with non phoretic triungulin) is endemic to the Iberianpeninsula. The other genus (Meloe Linnaeus, 1758, with phoreticlarvae) is more widely distributed in the Holarctic and northern,eastern and southern Africa, as the result of recent radiation and

dispersal. We hypothesize its origin in the northernmost lands ofAfrican Gondwana, with a possible dispersal to the Iberian Laurasiaduring the middle Cenozoic and a remarkable recent (Plio-Pleisto-cene) diversification and spread of the genus Meloe.

The stem of the pyrotin-epicautin clade should have originatedin western Gondwana. The origin of the Pyrotini (which is dated atabout 68 Mya) was plausibly in South America, where they aremost diverse (10 genera). The presence of a single pyrotin genus(Pyrota) in the Nearctic Region is probably due to dispersal eventsthrough the Panama isthmus during the Late Tertiary. Their sistergroup (Epicautini) is sub-cosmopolitan (with the obvious excep-tion of Australasia) and shows a remarkable plasticity in larval par-asitism: mostly predators on grasshopper eggs (Orthoptera)(Epicauta Redtenbacher, 1845, Linlseya MacSwin, 1951), and onegenus (Cyaneolytta Péringuey, 1909) phoretic on ground beetlesand possibly parasitic on termites (Di Giulio et al., 2003). The originof the Eupomphini seems older (ca 67 Mya) than expected, giventheir endemicity to a very restricted area, the relatively young de-serts of western North America. Their biogeographic history is hardto reconstruct. It is possible that they originated as the sister groupof the Mylabrini, in southern American lands, then dispersed to thenorth where they survived subsequent southern extinction(s). TheMylabrini, probably originated in Africa, and may have dispersed inboth southern Palaearctic and Oriental regions with significantpost-Eocene radiations.

Acknowledgments

We thank the following peoples who collaborated in the col-lection of specimens during several field researches made by oneof us (MAB) in the Mediterranean area and in Southern Africa:Valentina Amore, Pierluigi Bombi, Paola De Salvo, Andrea Di Giu-lio, Simone Fattorini, Luca Facchinelli, Emanuela Lorenzetti, CarlaMarangoni, Francesca Montalto, John D. Pinto, George Pratt, Mas-similiano Rosolino, Chiara Settanni, Federica Turco, Alberto Ven-chi, Marzio Zapparoli. A special thank to Mario García París(Madrid, Spain) Hemant Ghate (Pune, India), and Daniel Marsc-halek (Madison, USA), for having provided meloid specimens,and to Andrea Di Giulio who arranged SEM pictures. ManuelaCervelli, Benedetta D’Inzillo, Maria Vittoria Modica, Federica Tur-co and Annarita Wirz, helped us with laboratory work. AlessioDe Biase and Paolo Audisio friendly supported the Ph.D. labora-tory training of Monica Pitzalis. Special thanks are due to John D.Pinto (Riverside, USA), who kindly revised the English and pro-vided interesting suggestions.

This research was supported by grants of the Italian Ministry ofUniversity and Research (No. 2004057217 to MAB).

M.A. Bologna et al. / Molecular Phylogenetics and Evolution 48 (2008) 679–693 691

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