Our data set consisted of a total of 4,299 total aligned nucleotide sites {1,648 parsimony informative sites [elongation factor-1
; 1,141 total aligned nucleotide sites, 438 parsimony informative sites); RNA polymerase II (889 total aligned nucleotide sites, 300 parsimony informative sites); LW rhodopsin (716 total aligned nucleotide sites, 362 parsimony informative sites); 28S rDNA (772 total aligned nucleotide sites, 448 parsimony informative sites); and 18S rDNA (781 total aligned nucleotide sites, 100 parsimony informative sites)] plus 109 morphological characters (104 parsimony informative characters; see Table 2, which is published as supporting information on the PNAS web site)}. Introns for both EF-1 (two introns) and opsin (three introns) were excluded from the analysis, because alignments were ambiguous.
Parsimony Analyses. DNA data. When the combined five-gene data set was analyzed by equal-weight parsimony, we obtained one tree of 17,284 steps. This tree recovered monophyly of the bees and all bee families excluding Melittidae s.l. (Fig. 1, Table 1). Melittidae s.l. appears as a basal paraphyletic group relative to the other bees (Fig. 1). The basal branch of the bees appears to be the "melittid" subfamily Dasypodainae (Dasypodaidae). Bootstrap analysis indicates that monophyly of several families is well supported by our data, including Megachilidae (100% bootstrap support), Apidae (92% bootstrap support), Andrenidae (99% bootstrap support), Colletidae (100% bootstrap support), and Halictidae (100% bootstrap support). Relationships among the ST bee families (excluding Melittidae s.l.) were also well supported. Stenotritidae is unambiguously sister to Colletidae (99% bootstrap support), Halictidae forms the sister group to Stenotritidae+Colletidae (90% bootstrap support), and Andrenidae forms the sister group to these three families (64% bootstrap support). Overall, the parsimony results support a highly derived position for Colletidae and a basal position for Melittidae s.l. Manipulations of the outgroup topology (e.g., constraining bees to be the sister group to Crabronidae) and exclusion of outgroups did not alter the topology within the bees.
DNA data plus morphology. Addition of morphology to the molecular data set did not alter the relationships among families obtained in the combined molecular data set (
Fig. 1). However, inclusion of morphological data did alter relationships among the subfamilies of Colletidae. Examination of bootstrap values (
Fig. 1,
Table 1) indicates that morphology increases overall levels of bootstrap support in the tree. In particular, morphology adds support to LT bee monophyly.
Bayesian Analyses. Results of the Bayesian analyses were largely congruent with the parsimony results (Fig. 2). Analysis of the data set with the model preferred by MrModelTest Ver. 2.2 [general time reversible (GTR)+SYM[18s] + gamma distribution plus a proportion of invariant sites (I+G)] as well as the most complex model (GTR+I+G with separate gamma distributions and a separate proportion of invariant sites for each gene) yielded well supported trees (Fig. 2). All families (excluding Melittidae s.l.) are supported by posterior probabilities of 100%. Relationships among the families are identical to the parsimony results, with Melittidae s.l. forming a paraphyletic assemblage from which the other bees arose. Most basal nodes in the tree are well supported, although monophyly of the bees, excluding Dasypodaidae, is not strongly supported in the Bayesian analyses (Fig. 2, Table 1). Analyses with alternative models (Table 1) yielded largely congruent results. Our results strongly support the Melittidae-LT basal hypothesis. None of the multiple analyses of the combined data sets or any analyses of the individual gene data sets supported the Colletidae basal hypothesis.
Hypothesis Testing Using Maximum Likelihood and the Bayes Factor. Using the Kishino–Hasegawa (28, 29) and Shimodaira–Hasegawa (30) tests, as implemented in PAUP*, we detected significant support for the Melittidae-LT basal hypothesis over the Colletidae basal hypothesis (Table 3, which is published as supporting information on the PNAS web site). The difference in –ln likelihood for the two tests was 44.04, and the Colletidae-basal hypothesis could be rejected with P
Using the constraint option in MrBayes Ver. 3.1.2, we calculated the harmonic mean of the –ln likelihood values for an unconstrained tree (Melittidae-LT basal) and the tree constrained to Colletidae-basal. The harmonic mean of the –ln likelihood of the last 8,000 trees from the constrained analysis (Colletidae-basal) was –74,052.54. The corresponding harmonic mean for the unconstrained analysis (Melittidae-LT basal) was –74,005.60; twice the difference is 85.64. A value of 6–10 is strong support, and a value of >10 is very strong support for the alternative model (31). Our results provide very strong support for the Melittidae-LT basal hypothesis. This conclusion is consistent with the maximum likelihood results (above).
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