SI Methods ‘‘Phytomyza Main Lineage (PML Clade)’’ of Phytomyza

Supporting Information Winkler et al. 10.1073/pnas.0904852106 SI Methods ‘‘Phytomyza main lineage (PML clade)’’ of Phytomyza. To obtain Datasets and Phylogeny Estimation. The across-Agromyzidae data- meaningful estimates, these nodes with strong previous support set of Scheffer et al. (1) totaled 2,965 base pairs from 86 were also constrained as monophyletic. exemplars and three genes: cytochrome oxidase I (COI), 28S ribosomal RNA, and the nuclear protein-coding CAD, or rudi- Host Effects on Diversification. We tested for host effects on mentary. It was augmented by COI and partial CAD sequences diversification by comparing independent clades of Phytomyza for an additional 13 species of Phytomyza reported in ref. 2, plus associated with Ranunculaceae (n ϭ 5) and asterids (n ϭ 5). We 28S data from these same species, newly generated following the chose the most inclusive clades available that (i) fed entirely or methods of ref. 1. The unpartitioned dataset was analyzed with predominantly on one or the other host clade, (ii) had strong maximum likelihood (ML) in GARLI v.0.951 (3) using the support on the molecular phylogeny, and (iii) supported credible GTRϩIϩG model. Monophyly for the Phytomyza s.str. clade (2), estimates of both crown group age (from the PL analysis) and not initially recovered, was enforced in subsequent analyses, minimum species diversity (from ref. 2) from our sampling. Each because this grouping was strongly established by previous of the asterid-feeding clades represents an independent shift results (1, 2). A bootstrap analysis (500 replicates) performed in from Ranunculaceae feeding with the possible exception of the GARLI to gauge support for monophyly of Phytomyza sensu lato, nigra clade. In total, these 10 clades represent Ͼ440 species, 69% as newly defined (ref. 2; including Napomyza, Ptochomyza, and of the known diversity of Phytomyza s.str. Our selection criteria Chromatomyia), resulted in strong (90%) support for this node, probably bias the test against the predicted trend, since many of which defines the ingroup for the present study. The maximum the taxa excluded belong to apparently species-poor clades likelihood tree of ref. 2 was based on 3,065 base pairs from COI, feeding on Ranunculaceae (see Fig. 2). CAD, and PGD (phosphogluconate dehydrogenase), sequenced The geiger package (12) was used to calculate diversification from 108 species of Phytomyza and 5 outgroup taxa. rates for Phytomyza overall and for each of the 10 focal clades given observed present diversities and clade crown ages from the Divergence Time Calibration. For penalized likelihood analysis of PL analysis. Expected clade sizes, given this overall rate, were divergence times in r8s (4), the logarithmic penalty was used. then calculated for the focal clades (equation 5 of ref. 13), and Identification of the optimal smoothing parameter (s) by cross probabilities of observed clade sizes were calcuated in geiger. validation analysis was not straightforward because calculations For comparison, absolute diversification rates were also esti- failed for some values of s. However, as the remaining calculations mated for each clade. The minimum diversity of each clade was implied an optimal value near 103, suggested in the r8s manual as estimated by summing the numbers of described species in each an upper bound for s in usual cases, the smoothing parameter was component species group as listed in ref. 2. A likelihood ratio test set to 1,000. Penalized likelihood (PL) analysis of the Phytomyza was performed to compare a model with separate speciation only phylogeny used the same parameters. rates for the two groups vs. a model with a single combined rate. The BEAST (5) analysis was performed using the GTRϩIϩG For this test, log likelihoods for individual clades were computed model (partitioned by gene), with a Yule (pure birth) model using equation A18 of ref. 14 (the corresponding equation 1b in prior on speciation rates, implementing the uncorrelated log- ref. 13 is missing one term) and summed over each clade to normal relaxed clock (6) and using an output tree from r8s as a obtain an overall likelihood score (15, 16). An iterative script in starting topology. The weights of several parameter operators R was used to maximize this likelihood score by varying the rate were increased from default values to increase mixing of the parameter r in this equation separately for each group of clades, Yule prior and the frequency of topology changes (swap oper- then under a single combined rate. The ratio of observed to ator on branch rate categories and wide exchange and Wilson– expected clade size was also compared for the two host groups Balding operators to 100; uniform operator on internal node by using a Mann–Whitney U test (one-tailed). Both the Mann– heights and narrow exchange operator to 50). To reach station- Whitney U test and the likelihood ratio tests were repeated for arity, it was also necessary to constrain the following groups as three values of relative extinction rate: ␧ ϭ 0, ␧ ϭ 0.9, and ␧ ϭ monophyletic: Agromyzinae, Phytomyzinae, and (Phytobia ϩ 0.99. Amauromyza ϩ Phytoliriomyza robiniae ϩ Phytomyza group). While the simple birth-death model is widespread in studies of The final analysis was run for 100 million generations, sampling diversification, the temporal constancy of speciation and extinction the chain every 1,000 generations after the initial burn-in period. rates it assumes has recently been rejected for many groups, in part The prior was unstable in this analysis, not reaching stationarity, because the correlation it implies between (log) size and age of but inspection of log files in TRACER v.1.4 (7) showed that age clades is only rarely observed (17). Instead, diversification may estimates for Phytomyza and its component clades were station- often be density dependent or otherwise limited by ecological ary and not affected by this anomaly. opportunity (17, 18). Inspection of the plot in Fig. 3 suggests that Two secondary fossil calibrations were used as minimum ages for our 10 focus clades, the relationship of diversity to age, even for for corresponding stem groups: Palaeophytobia prunorum (Ͼ48 clades on the same host, is neither simple nor obvious. We Ma) (8, 9), a trace fossil in Eocene wood identical to traces of the performed rank correlation analysis on clade age and log of species extant agromyzid genus Phytobia, and the Florissant fossil ‘‘Agro- number, for all 10 clades together as well as separately for asterid myza’’ praecursor (34 Ma) (10, 11), which exhibits the expanded and Ranunculaceae feeders. The results are summarized in Table third antennal flagellomere diagnostic of Cerodontha subgenus S4. The correlation coefficient was positive in every case, but never Dizygomyza. To facilitate comparison of estimates between approached statistical significance. While small sample size may be analyses, the root height (fixed at 64.4 Ma in PL analysis) was part of the explanation, it seems plausible that constant diversifi- tightly constrained in the BEAST analysis with a tight normally- cation does not accurately describe clade dynamics for Phytomyza distributed prior (mean ϭ 64.4 Ma and standard deviation ϭ 0.5 species groups. Ma). In addition to nodes used for calibration, divergence times Given our small sample sizes and the strong host-related were estimated for the Phytomyza group of genera and for the apparent rate heterogeneity, we did not attempt to directly Winkler et al. www.pnas.org/cgi/content/short/0904852106 1of8 compare the fit of alternative, time-dependent diversification (tm for the second comparison is greater than the crown group models. As a test of the sensitivity of our conclusions to model age, so k3 ϭ 0). In each case, single-rate vs. two-rate models were assumptions, however, we conducted a ‘‘model-independent’’ compared by the Akaike information criterion (AIC) and by comparison of net diversification on the two hosts, consisting of likelihood ratio tests. a Mann–Whitney U test on clade sizes uncorrected for age. This We implemented the method of Rabosky (20) using the procedure is justified by the closely similar age distributions of LASER package v.2.1 (21) to compare single-rate vs. three-rate the two host-related sets of clades, as seen in Table S4. This test, Yule models by using the AIC and a likelihood ratio test. All like those based on the simple birth-death model, showed times were again adjusted using t0 ϭ 19 Ma, and shift times were significantly greater diversities for asterid-feeding clades (P ϭ constrained to 33 Ma and 24 Ma as in the previous analysis by 0.016, one-tailed; Table S4). Thus, while uncertainty about the modifying the yule3rate function of LASER. Because the shift model hinders identification of the underlying mechanism (see times were not estimated from the data (as they are generally by Discussion), and precludes strong inferences regarding actual using this function in LASER), we adjusted the AIC accordingly diversification rates during host-associated radiations, the fun- to incorporate only three free parameters. damental conclusion of greater net diversification by asterid An assumption of the branching time tests is complete sam- feeders seems robust to model details. An analogous tradeoff of pling of lineages up to 19 Ma. We believe this assumption to be detail for robustness is implicit in the often used replicated sister approximately true for our data, we know of no definite excep- group approach to diversification patterns. tions. However, some excluded taxa may belong to unidentified, early-branching lineages. A rough estimate of the possible Temporal Shifts in Diversification Rate. For tests of temporal shifts number of such taxa follows, derived from ref. 2. Our initial in diversification rate across major climatic transitions, the phylogenetic study left ϳ150 of 700 Phytomyza species unplaced equations of Paradis (19) were modified to model only periods to species group.

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