Molecular Phylogenetics and Evolution 60 (2011) 408–415

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Molecular Phylogenetics and Evolution

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The phylogeny of monkey based on mitochondrial and ribosomal RNA genes (Coleoptera: : Hopliini) ⇑ Dirk Ahrens a,b, , Michelle Scott a,c, Alfried P. Vogler a,c a Department of Entomology, Natural History Museum, London SW7 5BD, United Kingdom b Zoologisches Forschungsmuseum Alexander Koenig Bonn, Adenauerallee 160, 53113 Bonn, Germany c Department of Life Sciences, Silwood Park Campus, Imperial College London, Ascot SL7 5PY, United Kingdom article info abstract

Article history: Monkey beetles (Hopliini) are a large clade of flower and leaf feeding species within the Scarabaeidae Received 27 December 2010 (chafers) with greatest diversity in southern Africa. Their internal relationships and sister group affinities Revised 5 April 2011 have not been studied with DNA methods. We used partial gene sequences for 28S rRNA, cytochrome oxi- Accepted 18 April 2011 dase I (cox1) and 16S rRNA (rrnL) for 158 species, representing most recognized subfamilies of Scarabaei- Available online 6 May 2011 dae, including 46 species of Hopliini. Combined analyses using maximum likelihood and Bayesian inference under the two preferred alignment parameters recovered the Hopliini as monophyletic. Hopli- Keywords: ines were inserted at the base of a clade of Cetoniinae + + Dynastinae, being either recovered as Scarabaeidae their immediate sister group, or as part of an expanded set of basal branches that also includes the tribe Classification Macrodactylini which has been classified as part of the Melolonthinae (may chafers). At the level of sub- Activity patterns tribes, we found Hopliina paraphyletic with respect to Pachycnemina which also includes the monophy- Sexual dimorphism letic clade of Heterochelina and Gymnolomina. Trait mapping under parsimony on the preferred tree Species diversification resulted in inferences of three independent origins of sexual dimorphism, which coincided with shifts to ‘flower-embedding’ pollination. In contrast, night active taxa, which are general phyllophages as other pleurostict chafers, never show clear sexual dimorphism. South African lineages include several deep- branching lineages. The exceptional morphological and phylogenetic diversity of the South African fauna may therefore be due to their antiquity, in addition to sexual selection in the day-active lineages. Phylo- genetic studies of the endemic South African plant radiations have demonstrated the repeated evolution- ary shift to pollination, but it remains to be investigated if this is driven by the hopliine pollinators present in the bioregion or by a propensity of the local plant lineages favoring this pollination syndrome. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction 2005; Carrillo-Ruiz and Morón, 2006). Due to this life style they are important pollinators, and several groups of plants have devel- Among the scarab beetles (Scarabaeidae), the tribe Hopliini, or oped specialized features to attract these beetles (Steiner, 1999; monkey beetles, are a diverse group of phytophages comprising Colville et al., 2002; Goldblatt et al., 2005; Goldblatt and Manning, about 1200 species (Lacroix, 1998). They are widely distributed 2006; van Kleunen et al., 2007). Specifically, they have driven the throughout the temperate and tropical regions, but missing from evolution of particular flower types in several genera of Iridaceae: the Neotropics and Australia. Hopliines are particularly diverse in Hopliine-pollinated flowers are often very different in pigmenta- Southern Africa, which harbors up to half of the world’s diversity tion pattern, tepal size and orientation, and floral symmetry from of species and genera, as well as showing great morphological their closest relatives pollinated by bees, moths or hummingbirds diversity, while levels of endemism are also very high in this region (Goldblatt and Manning, 2006). These beetle pollinated flowers (Colville et al., 2002; Colville, 2006). Several species are generalist are typically radially symmetrical, bowl-shaped, and relatively herbivores, while others exhibit close associations with flowers large, and are thus able to accommodate two or more beetles. They as mating sites and as a source of on which most Hopliini often show ‘beetle marks’, which are prominent, contrast-rich feed as adults (Steiner, 1998; Colville et al., 2002; Krenn et al., markings on the vividly pigmented floral tepals (van Kleunen et al., 2007). In addition, pheromones contribute to species-specific aggregation and mate recognition in hopliine beetles (Zhang et al., ⇑ Corresponding author at: Zoologisches Forschungsmuseum Alexander Koenig 2003). Bonn, Adenauerallee 160, 53113 Bonn, Germany. Fax: +49 228 9122212. The unique features of the hopliines raise the question about E-mail addresses: [email protected], [email protected] (D. Ah- their evolutionary origin. Without doubt they are members of rens).

1055-7903/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2011.04.011 D. Ahrens et al. / Molecular Phylogenetics and Evolution 60 (2011) 408–415 409 the ‘pleurostict’ scarab beetles, a huge radiation of almost exclu- study, e. g. Palearctic species not included resemble the large genus sively herbivorous species, of which only a small proportion are . Other pleurostict chafers included here represent all princi- flower feeding. However, the precise relationships of Hopliini with pal lineages (subfamilies) of Scarabaeidae, plus two species each of other major scarab lineages, and hence the first appearance of this Hybosoridae and Glaphyridae, largely following Ahrens and Vogler specialized life style, remain unclear. Some authors considered (2008) but with the number of Sericini reduced (Supplementary Hopliini as a lineage that is phylogenetically separated from the Table S1). All trees were rooted with Hybosorus illigeri. DNA was main scarab clades of ‘rutelines’ and ‘melolonthines’ based on extracted from thoracic leg muscle tissue using Promega WizardSV striking diagnostic features, specifically the single metatarsal claw extraction plates. Following DNA extraction, beetles were dry that distinguishes them from all related groups (e.g. Mulsant, mounted. Vouchers will be deposited at ZFMK Bonn. 1842; Reitter, 1902; Peringuey, 1902; Janssens, 1949; Medvedev, Mitochondrial gene regions included cytochrome oxidase sub- 1976; Iablokoff-Khnzorian, 1977). Others considered Hopliini as unit 1 (cox1) and 16S ribosomal RNA (rrnL) with the adjacent re- part of the Melolonthinae, which has also been referred to as Mel- gions NAD dehydrogenase subunit 1 (nad1) and tRNA leucine olonthides (Lacordaire, 1856), Melolonthini (Gemminger and von (trnLeu), hereafter referred to as rrnL. PCR and sequencing was per- Harold, 1869), Melolonthinae (e.g. Dalla-Torre, 1913; Evans, formed using primers Pat and Jerry for cox1 and 16Sar and ND1A 2003; Smith, 2006), or Melolonthidae (Bates, 1888; Balthasar, for rrnL (Simon et al., 1994). For a few specimens only a shorter 1963). Yet others placed the Hopliini within ‘Rutelidae’ (=Ruteli- fragment could be obtained using the primer pair 16Sar and nae) (Burmeister, 1844; Mulsant and Rey, 1871; Paulian, 1959; 16SB2 (Simon et al., 1994). Nuclear 28S rRNA containing the vari- Paulian and Baraud, 1982). Alternatively, Lacroix (1998) consid- able domains D3–D6 was amplified using primers FF and DD ered Hopliini as an independent group at the family level, but he (Monaghan et al., 2007). Sequencing was performed on both did not comment on its relationships with other groups of Scarab- strands using BigDye v. 2.1 and an ABI3730 automated sequencer. aeoidea. The discussion about the position of Hopliini among either Sequences were edited manually using Sequencher v. 4.8 (Gene- rutelines or melolonthines focused on the retractable metatarsal codes Corp., Ann Arbor, MI, USA). GenBank accession numbers claws found in both Hopliini and Rutelinae (Burmeister, 1844). are given Supplementary Table S1. Only Hopliini and some species of the distinct tribe Macrodactylini have a single claw, as opposed to two claws in all other groups 2.2. Phylogenetic analysis using multiple alignment (Katovic, 2008). The close relationship of the latter with Hopliini was also suggested in a study using various morphological charac- Evolutionary dynamics of mitochondrial and nuclear rRNA ters (Carrillo-Ruiz and Morón, 2006). However, recent studies have genes differ (e.g. Vogler and Pearson, 1996; Terry and Whiting, shown that establishing the systematic position of Hopliini is com- 2005), and hence different alignment parameters and algorithms plicated because the Melolonthinae, the largest group of pleurost- were applied to various partitions. As we used the new data for ict scarabs, has been found to be paraphyletic (Ahrens, 2006; Hopliini in the context of an existing taxon sampling of pleurostict Ahrens and Vogler, 2008). Consequently, inferring the position of scarabs of Ahrens and Vogler (2008), the best parameters Hopliini will require the resolution of basal relationships of pleu- established in that study were applied here. The choice of combi- rostict chafers more widely, based on broad taxon sampling be- nation of aligned gene matrices was made according to the yond what is currently available (e.g. Browne and Scholtz, 1998; preferred combinations established in Ahrens and Vogler (2008) Hunt et al., 2007). based on congruence of phylogenetic signal in a hundred In addition, the internal relationships of Hopliini are of interest combinations of different primary alignments made from various for studying what drives the morphological, behavioral and ecolog- alignment procedures and parameters with various software pack- ical diversity in this lineage (Midgeley, 1992; Colville et al., 2002; age. Here we only used the two best performing combinations Colville, 2006). In particular, a phylogenetic framework is needed using MAFFT (vers. 5.8; Katoh et al., 2002, 2005) and MUSCLE vers. to explore the evolution of major morphological tendencies and 3.6 (Edgar, 2004) for rrnL (both with default parameters), and life traits (e.g. feeding behavior, diel activity pattern, sexual dimor- Clustal X (Thompson et al., 1997) for 28S (optimized parameters phism) and the underlying processes of trait selection (Midgeley, gap opening cost 10 and gap extension cost 6.66). These align- 1992) and niche partitioning (Picker and Midgeley, 1996). As a ments were combined for phylogenetic analysis together with framework for investigating the evolution of species richness in cox1, referred to as AL1 (cox1 + rrnLMAFFT + 28SClustalX) and AL2 the Hopliini and their mutual relations with flowering plants we (cox1 + rrnLMuscle + 28SClustalX). study the phylogenetic relationships of the tribe using DNA se- The concatenated data matrices were subjected to parsimony quences from one nuclear and two mitochondrial genes. We are tree searches using TNT (Goloboff et al., 2004) with 10 ratchet iter- specifically interested in the extent to which the pollinator guilds, ations, 10 cycles of tree drifting and three rounds of tree fusing for as defined by Picker and Midgeley (1996), are phylogenetically re- each of 200 random addition sequences. Gaps were treated as a lated to lineages with different life styles and how the origin of fifth character state. Model-based phylogenetic analyses under flower feeding might be correlated with the occurrence of sexual Maximum Likelihood (ML) were performed in PhyML (Guidon dimorphism as a possible mechanism for increased diversification and Gascuel, 2003) using a GTR model (as selected by Modeltest rates via sexual selection. using AIC; Posada and Crandall, 1998; Akaike, 1974) with all parameters estimated from the data and four substitution rate cat- egories. Bayesian analysis was conducted using parallel MrBayes 2. Material and methods 3.2 (Huelsenbeck and Ronquist, 2001), conducting MCMC runs (Yang and Rannala, 1997) after partitioning (Nylander et al., 2.1. Taxon sampling, DNA extraction, and DNA sequencing 2004; Brandley et al., 2005) the data for rrnL, 28S, and three codon position of cox1. Based on the preferred alignment combination, we A representative sample of 46 species was collected from vari- used a GTR + I + G model as determined by Modeltest with five par- ous biogeographic regions with focus on South Africa and Mada- titions referring to rrnL, 28S, and the first, second and third codon gascar representing almost 20% of the 108 genera of Hopliini. postion of cox1. Tracer 1.3 (Rambaut and Drummond, 2003) was Together, these regions harbor almost 70% of species and >80% of used to graphically determine stationarity and convergence of genera of hopliines in the world. Missing genera from elsewhere runs. Tree searches were conducted for 13 and 25 Â 106 genera- are likely to be phylogenetically close to those included in the tions, using a random starting tree and two runs of three heated 410 D. Ahrens et al. / Molecular Phylogenetics and Evolution 60 (2011) 408–415 and one cold Markov chains (heating of 0.1). Chains were sampled Table 2 every 1000 generations and 106 generations were discarded as Comparison of tree topologies from the different analyses regarding the phylogenetic resolution for major clades. Topologies differing from the preferred tree are burn-in based on the average standard deviation of split frequen- highlighted bold (M – monophyletic, P – paraphyletic; Ã – under inclusion of cies as well as by plotting Àln L against generation time. Bayes fac- Diphycerini; ? – uncertain polytomy; § – Cetoniinae + Rutelinae [including ‘Dynas- tors (Kass and Raftery, 1995) were calculated for both the MAFFT tinae’]; § – including the tribes Automolini, Liparetrini, Scitalini, Maechidiini, and the MUSCLE alignments in order to determine significant dif- Heteronycini). ferences in the marginal likelihood of initial unconstrained com- MrBayes PhyML Parsimony pared to analyses using a constraint topology for the monophyly AL1 AL2 AL1 AL2 AL1 AL2 AL2 (5th = ?) of Hopliini with a single metatarsal claw. Pleurosticti M M M M M – – Aph + Scar M M M M M M M 3. Results Glaphyridae M M M M M M M Hop + Macr – – – – – – – Hop + MM––––– Sequences (n = 158) for three loci (cox1, rrnL, 28S) were aligned Dyn + Ano M M M – – – M and concatenated to result in two alignments produced with Dyn + Ado – – – – M – – MAFFT (AL1) and MUSCLE (AL2), to produce matrices of 2491 Ser + Abl M M – – – – – § and 2487 nucleotide positions, respectively (Table 1). Parsimony Cet + Rut MM––––– ratchet searches on the combined matrices using gaps as 5th char- Aphodiinae M M M M M M M Dynastinae M M M M P P M acter state returned two overall shortest trees of 21,621 and 22,037 Cetoniinae M M PPPPP steps for alignment AL1 and AL2. These analyses performed poorly Melolonthinae P P P P P P P with regard to recovery and position of well established monophy- Rutelinae P P P P P P P letic groups (Supplementary Fig. S3), for example placing the dung Scarabaeinae M M M M M M M Adoretini M M M M M M M beetles (Scarabaeinae) among the pleurostict clades under AL2, Anomalini M M M M M M M and the macrodactyline genus Plectris nested within the Hopliini Cetoniini M M M M M M M in the AL1 alignment. Only the AL2 alignment recovered the Hopli- Diplotaxini M M M M M P M ini as monophyletic in the strict consensus, as sister to a clade that Enariini M M M M M M M comprised Rutelinae and Dynastinae. The poor performance of par- Geniatini M M M M M M M Hopliini M M M M P M M simony was not improved when gaps were treated as missing data, Macrodactylini P P MÃ MÃ P MÃ P resulting in two best trees of 20,625 steps (AL2) or in four best Melolonthini M M M M M M M trees of 20,597 steps (AL1) (see Supplementary Fig. S3). While in Rhizotrogini P P?P P P PP AL2 dung beetles (Scarabaeinae) were nested again within pleu- Rutelini M M M M P P P Sericini M M M P P P P rosticts as sister to the monophyletic Hopliini (Supplementary SW Melolonthines$ MMMMMMM Fig. S3), the strict consensus tree of the AL1 was only poorly re- solved but recovered most major clades including the Hopliini as monophyletic (tree not shown). rRNA gene alone (Supplementary Fig. S4). At the basal node, a Model based analyses performed with PhyML and MrBayes group of Southern Hemisphere melolonthines (node 5 in Fig. 1) separating functional data partitions produced trees that were sim- was found sister to all other Pleurosticti. This group had been rec- ilar for AL1 and AL2 (Tables 1 and 2). All analyses recovered the ognized in Ahrens and Vogler (2008) as a deeply separated lineage, Hopliini as monophyletic (Fig. 1, node 1), and while posterior as sister to Sericini + Ablaberini (node 6). The Macrodactylini probabilities for this node was always 1 in the Bayesian analysis, (nodes 2 and 3), the possible sister group of Hopliini (Carrillo-Ruiz bootstrap support for the Hopliini was low under ML (18%). The and Morón, 2006), was not monophyletic. In the Bayesian analysis tree (Fig. 1) also provides an update on the phylogeny of pleurost- the three species (node 3 in Fig. 1) were widely separated ict scarabs. Most of the major clades including the principal scarab from Plectris, the fourth representative of this tribe in this study. In lineages as well as Hopliini, however, seem to be particularly well both PhyML trees Macrodactylini were monophyletic but only un- supported mainly by the mtDNA since they did not appear mono- der inclusion of the genus Diphycerus which has been treated as phyletic in model based searches (PhyML, MrBayes) on the 28S separate tribe since the work of Medvedev (1952) (see Smith, 2006). However, in all analyses both lineages of Macrodactylini Table 1 were closely associated to the cetonine–ruteline–dynastine clade Comparison of alignments and tree scores under various tree search methods. All (node 4). Several rutelines and dynastines added here resulted in likelihood and Bayesian analyses were conducted with the GTR + I + G model. The last a placement of the monophyletic Dynastinae either as sister to row shows the results from a Bayesian analysis constrained for monophyly of taxa with a single metatarsal claw. Anomalini in the Bayesian analysis, or as sister to Adoretin- i + Anomalini (AL2) or to Adoretini (AL1) with PhyML (Supplemen- AL1 AL2 tary Fig. S1). The Rutelini + Geniatini always diverged at the base of Alignment lenth the dynastine–ruteline clade. The South African genus Isoplia cox1 826 bp 826 bp which strongly resembles the Hopliini in external appearance, in rrnL 884 bp 880 bp 28S rRNA 781 bp 781 bp particular the hairy species of Eriesthis, was always recovered as sister of Anomalini, albeit with low branch support (Fig. 1 and Sup- Informative characters cox1 420 420 plementary Fig. S1). Hence, these similarities are due to conver- rrnL 451 (486)a 456 (477)a gence, as initially proposed by Peringuey (1902), who treated this 28S rRNA 84 (100) 84 (100) group as a separate tribe (Isopliini) of the Rutelinae. Tree scores The critical question about the sister group of Hopliini remained TNT (tree length) 21,621 22,037 incompletely resolved. The Bayesian analyses identified the PhyML À81909.37 À81858.39 macrodactyline genus Plectris (node 2) as sister to the Hopliini, MrBayes À78161.03 À78033.34 but the relevance of this finding is unclear because of the non- MrBayes constr. À78166.27 À78039.82 monophyly of the Macrodactylini in these trees. Under ML, in the a Values in parentheses when gaps were treated as 5th character. AL1 tree Macrodactylini + Ablaberini were sister to the Hopliini D. Ahrens et al. / Molecular Phylogenetics and Evolution 60 (2011) 408–415 411

Fig. 1. Majority-rule consensus-tree of the Pleurosticti from Bayesian analysis of the AL1 alignment. Posterior probabilities are given below the branches. Key nodes discussed in the text are marked by numbers 1–7.

(Supplementary Fig. S1) while in the AL2 tree Macrodactylini was while in AL2 Eriesthis was monophyletic in a polytomy with again sister (under inclusion of Pachydemini, i.e. the genus Buet- Pachycnema and Lepithrix (Fig. 2, node A). tikeria) to the cetonine–ruteline–dynastine clade which together were the sister of Hopliini. 3.1. Evolutionary trends and biogeography Moving now to the relationships within Hopliini (Fig. 2), the tree topology was very similar among various model-based The analysis established a phylogenetic framework for investi- searches. We found the Hopliina, one of the currently recognized gating the evolution of Hopliini and its interaction with flowering subtribes (Smith, 2006), paraphyletic with respect to the Pachy- plants. We found that, although the monophyly of Hopliini was cnemina (clade A in Fig. 2), as well other clades such as clade B well supported, the tree topology was not consistent with a poten- (corresponding to Heterochelina Burmeister, 1844) and clade C, tial key synapomorphy, the single metatarsal claw, contradicting corresponding to the genus Gymnoloma (=Gymnolomina Burmei- Carrillo-Ruiz and Morón (2006) (see Fig. 2). According to their phy- ster, 1844). The latter two groups have already been synonymized logenetic hypothesis, a group of ‘Hopliini’ with two metatarsal with Hopliina (Smith, 2006). In all model-based analyses Hoplia claws, which were not included in their study, were expected to was monophyletic, as sister to a clade of night active species from be the sister of all single-clawed Hopliini. This concerns only two Madagascar (Amorphochelus, Michaeloplia) and South Africa (Con- hopliine genera, Cylichnus Burmeister (1844) and Nanaga Peri- gella, Microplus). Only five taxa changed positions slightly in the nguey (1902) that have been classified as Hopliini by Peringuey two Bayesian searches (Fig. 2). These were Scelophysa and Echyra (1902). In our analysis the double-clawed genus Cylinchus was which switched their positions close to the ‘Clania’-Eriesthis and nested deeply within the single-clawed clade and never branched the Hoplia-Congella clade. In AL1 Eriesthis was paraphyletic in the off at the base. Therefore, we performed the Bayesian MCMC con- MrBayes and ML trees since Pachycnema and Lepithrix were nested strained for the monophyly of hopliine taxa with single metatarsal between Eriesthis cf. rhodesiana and all other Eriesthis species, claw and found the Bayes factors (B10)  1 for AL1 and AL2 (see 412 D. Ahrens et al. / Molecular Phylogenetics and Evolution 60 (2011) 408–415

Fig. 2. Majority-rule consensus trees of Hopliini from Bayesian inference for AL1 (left panel) and AL2 (right). Posterior probabilities are given below the branches. Gray and dark shading indicates taxa with moderate and severe sexual dimorphism, respectively. Branch color on tree on the left indicates one (red) or two (black) two metatarsal claws. Branch color on tree on left indicates day active (black) and night active (blue) species. Colored dots refer to various zoogeographic regions: red, southern Africa; blue, Madagascar; yellow, Asia; white, Europe. Arrows mark the taxa whose position differ in both trees. A, B, C refer to clades of Pachycnemina (A), Heterochelina (B) and Gymnolomina (C). The habitus images from top-left to right bottom side: Scelophysa endroedyyoungai, Congella tesselatula, Echyra umbrina, Clania macgregori, Eriesthis hypocrita, Paramorphochelus agricola, Ceratoscelis spSA2, Heterochelus vulpinus, Hoplia farinosa, Cylinchus vanus, Pachycnema calviniana, Lepithrix cf. forsteri, Omocrates sp., Gymnoloma nigra. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 1), i.e. the present data would not reject the hypothesis that of Fig. 2), and specifically only among ‘embedding’ pollinators that the single metatarsal claw had a single origin. Reversals to a second persist on a single flower large enough to harbor multiple individ- claw may not be implausible because rudiments of it are evident in uals. In contrast, night active taxa, all of which are general phyllo- many species of Hoplia, indicating a labile trait. phages, never show clear sexual dimorphism. Due to the poorly Further morphological and ecological features were mapped on supported basal relationships of Hopliini the ancestral state for diel the preferred tree, to assess their role in clade diversification and in activity pattern (day or night active) remains unresolved. Interest- the evolution of the beetle-flower mutualism. Many taxa exhibit ingly, sexual dimorphism is found predominantly in South African sexual dimorphism (gray shading in the left tree, Fig. 2), whereby lineages although flower feeding may occur also in European and males exhibit strongly developed hind legs and claws (Scelophysi- Asian Hopliini, while night activity occurs in lineages from Asia, na, Pachycnemina) or bear strong spines on the hind legs and differ South Africa and Madagascar. in the color of the pygidium (often the only part of the beetle that is visible when embedded in the flower bottom) from females (Heterochelina). Based on inferred character changes on the tree, 4. Discussion sexual dimorphism has originated three times given the present taxon sampling, although only two nodes defining these transi- Using the dataset of Ahrens and Vogler (2008) as a backbone, tions are strongly supported. The dimorphism is only found in and applying the same alignment and tree search procedures day active flower visiting taxa (black branches in tree on the right found to be preferable, we now modified the sampling scheme to D. Ahrens et al. / Molecular Phylogenetics and Evolution 60 (2011) 408–415 413 focus on the systematic position of Hopliini within the major her- melolonthines focused on the retractable metatarsal claws found bivore scarab lineages. Taxon sampling of Hopliini was incomplete in both Hopliini and Rutelinae (Burmeister, 1844) that are pres- but likely included a large proportion of major clades, as the basis ent also in some Macrodactylini (Katovic, 2008). Given Burmei- for a framework of phylogenetic relationships and major evolu- ster’s (1844) classification grouping Hopliini within Rutelinae, tionary trends within the tribe. The strategy for alignment was this character might be a synapomorphy for the Rutelinae plus based on the notion that homology at the nucleotide level can be Hopliini, if the single-clawed genus Cylichnus represents the established, first, relative to a phylogenetic tree that recovers ancestral state for the Hoplini. The inferred trees (Fig. 1, Supple- ‘known’ groups whose monophyly is not in doubt based on existing mentary Fig. S1) contradict this possibility, although a topology knowledge and, second, using an overall measure of character required under this scenario that places Cylichnus as sister to all homology at the nucleotide level (taxonomic congruence and char- other Hopliini could not be excluded with high statistical support. acter congruence sensu Wheeler, 1995). Ahrens and Vogler (2008) Metatarsal claws are of high adaptive value for clinging to leaves combined both measures of congruence into a single value to se- and other plant parts (see Fig. 1) and seem to be subject to selec- lect the best alignment. All alignments generated in this study tion for reduction and asymmetry of both claws, leading to con- were based on progressive methods of pairwise alignment follow- vergent evolution. The evolutionary plasticity of this trait is also ing a guide tree, implemented initially in the Clustal software and evident in some species of Hoplia that only show a trace of a sec- in more recent methods that use an additional refinement step. ond metatarsal claw, with some species even showing intraspe- The latter would be expected to improve homology statements cific polymorphism. over the simple Clustal procedure by realignment of several closely Although the functional significance of the reduction or elimi- related sequences from the initial pairwise alignment, but this was nation of the second metatarsal claw is unclear, these changes not the case throughout, as apparently the theoretical improve- are found not only in the obligatory pollen feeders. The latter show ments of the refinement do not necessarily provide an increase some apparent adaptations for pollination shared among all day- of homology across the entire data set, given the criteria used (e. active and floricolous species. These include dense and long pilos- g. the ensemble RI as a measure of total synapomorphy). This ity of the body that makes hopliine beetles more effective pollen seems to be confirmed by recent studies showing that no single carrier even than bees (Mayer et al., 2006). In addition, hopliines method was capable of producing high quality alignments for all often exhibit striking coloration conferred by setae that have been types of data (Aniba et al., 2010). Here we accepted the preferred transformed to colored scales. These are likely to be relevant in alignment parameters of the earlier study under the assumption mate recognition and also produce mimetic patterns, e.g. to resem- that in the current data set new taxa are added to existing deep ble bumblebees. Finally, flower visiting species show great diver- branches already present on the tree. Hence, by adding these taxa, sity in the morphology of mouth parts (Peringuey, 1902; Nel and homology assignment at the nucleotide level would mainly affect Scholtz, 1990; Carrillo-Ruiz and Morón, 2006), including the pres- the sampling density of indels (and nucleotide changes) on the ence of either toothed maxilla or untoothed to membranous max- tree, but their variation is the result of the same mutational steps illa. These appear to be correlated with functional aspects of the and therefore can be approximated by the same alignment param- feeding on pollen vs. nectar, respectively, and may be important eters. While specific tests might be required to establish this more for partitioning the food resources. Detailed analysis of various firmly, we accepted the preferred settings from the earlier study on character states and homology assignment of various mouth parts the higher hierarchical level, not least to maintain the basal rela- are required to corroborate the link of mouthpart morphology and tionships found there (i.e. the outgroups for the Hopliini). feeding choice. The test of homology was based on parsimony analysis, and un- It is well known that morphological diversity of Hopliini is like the current implementations of likelihood and Bayesian anal- greatest in the South African lineages (Peringuey, 1902). This might yses, parsimony searches permit the use of indel variation for be due to a greater lineage age of the flower dwelling hopliines in tree building by coding ‘gaps’ as a fifth character state. Parsimony South Africa, or to evolutionary pressures that differ from other re- analysis therefore is an important heuristic tool for assessing the gions in the world. The latter hypothesis would predict that South data set. However, phylogenetic relationships inferred with this African lineages are more diverse than their sister taxa elsewhere, method were unsatisfactory in particular for deep nodes. Both but this is not the case for at least one clade composed of herbiv- types of model-based analyses performed better in this regard, orous South African (Congella) and Malagasy taxa that is the sister indicating that the complex character variation is better described to the species rich genus Hoplia (Fig. 2). On the contrary, the South by these models. Problems with the tree are therefore mainly due African fauna appears to be a composite of several basal lineages to the difficulties with character reconstruction in the parsimony whose greater clade age results in higher species richness overall analysis, in particular affecting basal relationships, rather than compared to other regions. However, the support at basal nodes the alignment. However, although the results from an unparti- is weak and the reconstruction of ancestral geographic areas tioned likelihood analysis and a partitioned Bayesian analysis were (South Africa vs. Asia/Holarctic) remains uncertain (Fig. 2). In addi- largely consistent, both using the GTR model, differences among tion, lineages associated with flower feeding may not have exhib- the two alignments still show the dominant effect of alignment ited this life style ancestrally, as it requires co-radiation with choice on the tree topology and the difficulty of selecting one align- plants. Studies of the evolution of pollinator syndromes in Irida- ment over another. ceae (genus Babiana; Schnitzler et al., in press) demonstrated that With regard to the taxonomic conclusions, we confirm the hopliine beetle pollination has arisen multiple times as a recently consistent recovery of a monophyletic cetonine–ruteline–dynas- derived trait. Moreover, host data of Babiana species (Goldblatt tine clade (node 4 in Fig. 1), and the association of Macrodactylini and Manning, 2007) indicate that various lineages have recruited and Hopliini with that clade. This is broadly congruent with the pollinators independently from different genera, arguing against topology proposed by Carrillo-Ruiz and Morón (2006) based on ancestral relationships with pollinators or co-evolution. Vice versa, a morphological character set. Whereas the evidence for the closely related lineages of pollinators are associated with several monophyly of Macrodactylini and their phylogenetic position rel- Babiana species, suggesting that these lineages of hopliines existed ative to other major groups including the Hopliini remains some- already before a shift to feeding on certain flowers. The age of what unclear, these findings resolve the reasons for the lineages therefore seems to be less crucial for the flower-beetle controversy about the placement of Hopliini in the past. In partic- mutualism and possible effects on diversification rate. It remains ular, the discussion about their position among either rutelines or to be investigated if the repeated shifts to beetle pollination is 414 D. Ahrens et al. / Molecular Phylogenetics and Evolution 60 (2011) 408–415 driven by the pollinator environment or due to traits of the ende- Evans, A.V., 2003. 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