PCR Amplification of SSU Was Performed in Four Overlapping Fragments with Primers Described

Electronic supplement file:

The evolution of scarab beetles tracks theconsecutive rise of angiosperms and mammals

Dirk Ahrens†*, Julia Schwarzer*# & Alfried P. Vogler†$

Material and Methods

Sequencing

Nuclear 28S rRNA, containing the variable domains D3–D6, was amplified using primers FF and DD [1]. PCR amplification of 18S was performed in four overlapping fragments with primers described by Shull et al. [2] (primer pairs: 18S5'-18Sb5.0, 18Sai-18Sb0.5, 18Sa1.0-18Sbi and 18Sa2.0-18S3'I). Amplification of partial mitochondrial cox1 and rrnL genes was performed, respectively, with primer pairs C1-J-2183 (Jerry) and TL2-N-3014 (Pat) [3], and LR-N-13398 and LR-J-12887 [3] or LR-J-12961 [4].

Phylogenetic analysis

Convergence of Bayes runs was assessed for all parameters by evaluating stationarity of the Markov chain with Tracer v. 1.5 [5] and by sampling of Bayesian tree topologies using AWTY [6].To assess convergence in the two Bayesian runs with AWTY Bayesian tree files were uploaded to the AWTY website ( and the following plotes were generated: 1) a bivariate plot of the split frequencies for the first and second run; 2) the cumulative split frequency for a number of selected splits; 3) a comparison of the symmetric tree-difference score [7] within and between (among) runs (see Figure below). All three are considered to be a valuable additional tree-based convergence diagnostics [8].

Ancestral feeding behaviour was reconstructed using parsimony in Mesquite by optimising terminal states onto Bayesian tree topology [9].

The protocol of BEAST runs including number of generations, burnin, and resampling for thinning is given in Table 1 of this supplement.

Table 1.Beast run statistics for the different separate runs 1-6 (number of generations, burnin, thinning).

Results

Phylogenetic analysis (Convergence of Bayesian analysis)

Log likelihood values reached stability (stopped improving) within the first 2 and 4 million generations (for Mafft/ Mafft+Aliscore, respectively; see below), and therefore as a rather conservative measure, the first 5 million generations of each run were discarded during burnin. Results of independent runs were similar, so the remaining trees (after burnin) of each run were combined under the allcompat command in MrBayes to a consensus tree. The value of standard deviation of split frequencies of the MrBayes analysis was at the end of Bayesian run 0.012 and 0.006, for the Mafft and the Mafft+Aliscore dataset, respectively.

The bivariate plots of the split frequencies for the first and second run showed a high correlation (B, F) [diagnosing convergence]. The graph of the cumulative split frequency for a number of selected splits showed no clear trend in frequencies (C, G) [which also diagnoses convergence]. The comparison of the symmetric tree-difference score [7] (within and between the runs) resulted in a between-run distance being not or only little separated from the within-run distances [diagnosis of convergence] (D, H).

Convergence of the two Bayesian tree searches (Mafft/ Mafft+Aliscore): Convergence and mixture behavior observed from LnL plots of the two MrBayes runs (A, D), and the results of their analyses with AWTY: (B, F) a bivariate plot of the split frequencies for the first and second run; (C, G) the cumulative split frequency for a number of selected splits; (D, H) comparison of the symmetric tree-difference score [7] within and between (among) runs.

Discussion

The current definition of Scarabaeidae is possibly a residual of traditional scarabaeoid classification (Lawrence & Newton 1982) in course of which more and more monophyletic groups were separated from a remnant that finally constituted the Scarabaeidae (Kohlmann 2006). Browne’s unpublished Ph.D. thesis (1993) provides strong evidence for the monophyly of Scarabaeidae postulating (according to Browne & Scholtz 1995, 1999) 23 synapomorphies that support the monophyly of Scarabaeidae. However, in the subsequent papers with the cladistic analyses they included either only a single terminal branch for Scarabaeidae (Browne & Scholtz 1995) or used only a single outgroup taxon to root the tree of “Scarabaeidae” (Browne & Scholtz 1999). Both approaches made a test for their monophyly impossible.

References

[1] Monaghan, M. T., Inward, D. G., Hunt, T. & Vogler, A. P. 2007 A molecular phylogenetic analysis of the Scarabaeinae (dung beetles). Mol. Phyl. Evol. 45: 674-692.

[2]. Shull V. L., Vogler A. P., Baker M. D., Maddison, D. R. & Hammond, P. M. 2001 Sequence Alignment of 18S Ribosomal RNA and the Basal Relationships of Adephagan Beetles: Evidence for Monophyly of Aquatic Families and the Placement of Trachypachidae. Syst. Biol.50: 945-969.

[3] Simon, C., Frati, F., Beckenbach, A., Crespi, B., Liu, H. & Flook, P. 1994 Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann. Entomol. Soc. Am. 87: 651-701.

[4] Cognato,A. I. & Vogler, A. P. 2001 Exploring Data Interaction and Nucleotide Alignment in a Multiple Gene Analysis of Ips (Coleoptera: Scolytinae). Syst. Biol. 50: 758-780.

[5] Rambaut, A. & Drummond, A. J. 2009 Tracer v1.5, Available from

[6] Wilgenbusch, J. C., Warren, D. L. & Swofford, D. L. 2004. AWTY: A system for graphical exploration of MCMC convergence in Bayesian phylogenetic inference.

[7] Penny, D. & Hendy, M. D. 1985 The use of tree comparison metrics. Syst. Zool.34: 75-82.

[8] Nylander, J. A., Wilgenbusch,J. C., Warren,D. L. & Swofford,D. L. 2008 AWTY (Are We There Yet): a system for graphical exploration of MCMC convergence in Bayesian phylogenetics. Bioinformatics24: 581-583.

[9] Maddison, W. P. & Maddison,D. R. 2011 Mesquite: a modular system for evolutionary analysis. Version 2.75

[10] Lawrence, J.F., & A.F. Newton, Jr. 1982. Evolution and classification of beetles. Ann. Rev. Ecol. Syst. 13: 261-290.

[11] Kohlmann, B. (2006) History of Scarabaeoid Classification. The Coleopterists Bulletin 60 (suppl 5): 19-34.

[12] Browne, D.J. (1993) The significance of the hind wing basal articulation of the Scarabaeoidea (Coleoptera). Ph.D. thesis, University of Pretoria.

[13] Browne, D.J. and Scholtz, C.H. (1995) Phylogeny of the families of Scarabaeoidea (Coleoptera) based on characters of the hindwing articulation, hindwing base and wing venation. Syst.Entomol.20: 145-173.

[14] Browne, J. & Scholtz, C. H. (1999) A phylogeny of the families of Scarabaeoidea. Syst. Entomol.24, 51-84.

1