Molecular phylogenetics and evolution of host plant use in the Neotropical rolled leaf ‘hispine’ beetle genus Cephaloleia (Chevrolat) (Chrysomelidae: Cassidinae)IntroductionMaterials and methodsTaxaTaxon sampling and specimen identificationOutgroup choiceLaboratory proceduresDNA extraction, amplification, and sequencingDNA sequence dataAlignment of nucleotide sequences and preliminary sequence analysisPhylogenetic analysesResultsSequence alignmentMaximum parsimony phylogenetic analysesModel selection and Bayesian inference of phylogenyML phylogenetic analyses implemented in PAUPPatterns of host tissue and host taxon usageDiscussionCephaloleia phylogenyEvolution of host taxon and host tissue usePhylogenetic relationships among Cephaloleia species and implications for taxonomyAcknowledgmentsReferencesMolecular Phylogenetics and Evolution 37 (2005) 117–131www.elsevier.com/locate/ympev1055-7903/$ - see front matter  2005 Elsevier Inc. All rights reserved.doi:10.1016/j.ympev.2005.06.011Molecular phylogenetics and evolution of host plant use in the Neotropical rolled leaf ‘hispine’ beetle genus Cephaloleia (Chevrolat) (Chrysomelidae: Cassidinae)Duane D. McKenna¤, Brian D. FarrellHarvard University, Museum of Comparative Zoology, 26 Oxford Street, Cambridge, MA 02138, USAReceived 22 October 2004; revised 20 May 2005Available online 27 July 2005AbstractHere, we report the results of a species level phylogenetic study of Cephaloleia beetles designed to clarify relationships and pat-terns of host plant taxon and tissue use among species. Our study is based on up to 2088 bp of mtDNA sequence data. Maximumparsimony, maximum likelihood, and Bayesian methods of phylogenetic inference consistently recover a monophyletic Cephaloleiaoutside of a basal clade of primarily palm feeding species (the ‘Arecaceae-feeding clade’), and C. irregularis. In all three analyses, the‘Arecaceae-feeding clade’ includes Cephaloleia spp. with unusual morphological features, and a few species currently placed in othercassidine genera and tribes. All three analyses also recover a clade that includes all Zingiberales feeding Cephaloleia and most Cepha-loleia species (the ‘Zingiberales-feeding clade’). Two notable clades are found within the ‘Zingiberales-feeding clade.’ One is com-prised of beetles that normally feed only on the young rolled leaves of plants in the families Heliconiaceae and Marantaceae (the‘Heliconiaceae & Marantaceae-feeding clade’). The other is comprised of relative host tissue generalist, primarily Zingiberales feed-ing species (the ‘generalist-feeding clade’). A few species in the ‘generalist-feeding clade’ utilize Cyperaceae or Poaceae as hosts. Over-all, relatively basal Cephaloleia (e.g., the ‘Arecaceae clade’) feed on relatively basal monocots (e.g., Cyclanthaceae and Arecaceae),and relatively derived Cephaloleia (e.g., the ‘Zingiberales-feeding clade’) feed on relatively derived monocots (mostly in the orderZingiberales). Zingiberales feeding and specialization on young rolled Zingiberales leaves have each apparently evolved just once inCephaloleia. 2005 Elsevier Inc. All rights reserved.Keywords: Cassidinae; Cephaloleia; Chrysomelidae; Hispinae; mtDNA; Zingiberales1. Introduction“The plant herbivore “interface” may be the majorzone of interaction responsible for generating terrestrialorganic diversity (Ehrlich and Raven, 1964).” Severalecological hypotheses have been proposed to explainhow plant/insect interactions aVect diversiWcation (e.g.,Berenbaum, 1983; Futuyma and Moreno, 1988; Thomp-son, 1994), for example, as a function of the physicalenvironment, spatial distribution of resources, competi-tion for resources, or limitations to dispersal. The adventof modern (especially molecular) phylogenetic studieshas occasioned complementary, explicitly historicalapproaches to the study of plant/insect diversiWcation(Farrell, 1998, 1999; Mitter et al., 1988; Page, 1994). Nev-ertheless, most such studies have focused on temperateinsects, especially those feeding on conifers and dicots(Farrell et al., 2001; Jordal et al., 2000; Kelley and Far-rell, 1998; Kelley et al., 2000; Normark et al., 1999;Sequeira et al., 2000; Sequeira and Farrell, 2001), withcomparatively little study of tropical insect herbivores ormonocot associates. Here, we address this deWciency by*Corresponding author. Fax: +1 617 495 5667.E-mail address: [email protected] (D.D. McKenna).118 D.D. McKenna, B.D. Farrell / Molecular Phylogenetics and Evolution 37 (2005) 117–131examining the phylogenetic relationships among mono-cot feeding Neotropical beetles in the genus Cephaloleia(Chevrolat) (Chrysomelidae: Cassidinae).With more than 200 described species, Cephaloleia isone of the most species rich genera of monocot feedingleaf beetles (Staines, 1996, 2004). Members of the genushave been the subjects of ecological study for more than25 years (Johnson, 2004; Seifert and Seifert, 1976; Strong,1977a,b, 1982). Cephaloleia feed only on Monocotyledo-nae, especially the young rolled leaves of plants in theorder Zingiberales, and the young folded leaves of vari-ous Arecaceae. Other Cephaloleia host plants includeBromeliaceae, Cyclanthaceae, Cyperaceae, Orchidaceae,and Poaceae (Table 1) (D. McKenna, unpublished data;D. Windsor pers. comm., 2002; Staines, 1996, 2004). Fos-sil evidence suggests that Cephaloleia-like beetles havemaintained specialized interactions with their Zingibe-rales host plants for more than 66 Ma (Wilf et al., 2000).Cephaloleia are known from a diversity of tropical andsubtropical New World plant communities from Mexicoand Cuba to Argentina. All life stages and most behavior,including mating, take place on host plants, and for manyZingiberales feeders, almost entirely in rolled leaves(Auerbach and Strong, 1981; Morrison and Strong, 1981;Seifert and Seifert, 1976; Strong, 1977a,b, 1982).Cephaloleia has traditionally been placed in the tribeCephaloleiini Baly in the subfamily Hispinae (leaf-min-ing beetles) (Hincks, 1952), but recent studies haveshown that the Hispinae are polyphyletic, and the divi-sion of the Cryptostoma into the subfamilies Cassidinaeand Hispinae is unnatural (Borowiec, 1995). Here, thesubfamily name Cassidinae is used following Staines(2002). The subfamily Cassidinae belongs to the excep-tionally diverse family Chrysomelidae (leaf or plant bee-tles). Despite recent advances in subfamily-leveltaxonomy, the tribal-level taxonomy of