Appendix two
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INSERT X PAGES FOR TABLE TWO page 1INSERT X PAGES FOR TABLE TWO page 2 INSERT X PAGES FOR TABLE TWO page 3 INSERT X PAGES FOR TABLE TWO page 4 INSERT X PAGES FOR TABLE TWO page 5 For each specimen, DNA from three gene regions was amplified and sequenced: 2335 bp of the mitochondrial cytochrome oxidase subunits I and II (COI-COII), 1260 bp of the Elongation Factor-1gene (EF-1) and 382 bp of the wingless (wg) gene. Primers for COI-COII include those listed in Brower & Jeansonne (2004), and Table 3; for EF-1 from Cho et al. (1995) and Table 3, and for wingless from Brower & DeSalle (1998). Table 3. New PCR and sequencing primers employed in this study. Additional primers are listed in the references cited in the Materials and Methods section. “Strand” refers to the sense/antisense strand of the target gene region.
1 primer positions based on Drosophila yakuba sequence (Clary & Wolstenholme 1985) 2 primer positions based on the Drosophila melanogaster sequence (Hovemann et al., 1988) PCR amplifications were performed in a 50 l reaction volume, with 3.1 l template, 6 l 25 M MgCl2, 5 l 10x buffer (0.1M Tris HCl, 0.1M KCl, 1% Triton X-100, pH 8.3), 1 l 10 M dNTPs, 0.4l Taq polymerase, 1 l of each primer (10 mM) (2 l of each for the nuclear genes) and 32.5 l H2O (30.5 l for nuclear genes). The cycling profile for COI-COII was initial annealing at 95°C for 3 minutes, then 33 cycles of (95°C for 1 minute, 45°C for 1 minute, 72°C for 90 seconds) and a final extension period of 72°C for 5 minutes. The cycling profile for wg was the same as that for COI-COII, except the annealing temperature was 62°C. The cycling profile for EF-1 was 94°C for 2 minutes, followed by 37 cycles of (94°C for 1 minute, 62°C for 1 minute, 72°C for 90 seconds) and a final extension period of 72°C for 10 minutes. For all three gene regions, the PCR primers and additional internal primers were used for sequencing. Amplified DNA fragments were gene-cleaned with silica beads (Bio101) or Qiaquick PCR purification kits (Qiagen) , cycle-sequenced in a MJ Research DNA Engine, and run on an ABI 373 automated sequencer from sense and anti-sense strands. Some of the sequencing was outsourced to a commercial firm (Macrogen). Automated sequence outputs were edited manually and aligned by eye. The aligned data matrix is available on the web at (http://wwwsciencet.oregonstate.edu/systematics/browera/datasets/pubdata/xxx.html), and individual sequences have been submitted to GenBank (see Table 2 for accession codes). Phylogenetic Analysis The data were concatenated and analyzed as a single matrix under the parsimony criterion using PAUP*4.0b10 (Swofford 2002). Characters were treated as unordered and weighted equally, with inferred gaps encoded as “missing.” A TBR heuristic search with 1000 random addition sequences was performed, and a single most parsimonious cladogram was discovered. The tree was rooted with Tellervo, and other non-ithomiine taxa were included in the ingroup to test monophyly of Ithomiini. The structure of the data was explored with separate analyses of each gene region, using the same procedures as the simultaneous analysis. Branch support (BS; Bremer, 1988; 1994) and partitioned branch support parameters (PBS; Baker & DeSalle, 1997; Baker et al., 1998) were calculated by the anticonstraint method with 10 random addition sequences per node. Fractional PBS values were rounded to two decimal places. Incongruence among gene regions was assessed at each node by comparison of PBS values. Because a BS value of “1” is a summary that does not reveal conflict among partitions, a new parameter, the partition congruence index (PCI; Brower and Gatesy, in prep.), was used, to measure of the degree of conflict among partitions relative to PBS. The PCI for a given branch is calculated as follows:
PCI = BS – (PBSi – BS)/BS) i = 1 In words, the partition congruence index for a given branch equals the branch support minus (the difference between the sum of the absolute values of the branch support for each partition and the branch support, divided by the branch support). PCI is always equal to or less than BS for a given branch. It is equal to BS for a branch that is not contradicted by any partition, and becomes negative for nodes with low BS and one or more partitions with strongly negative PBS. Branches with a negative PCI can be considered weakly supported, and branches with highly negative values, indicating low overall support underlain by partitions with strong disagreement, should be reexamined. The PCI is discussed further in Brower (submitted). The classification of Ithomiini discussed below is based on the results of the cladistic analysis. The nomenclatorial philosophy is that all named superspecific taxa should be monophyletic. We provide names for subtribes representing monophyletic groups of genera, and recommend that a few small or monotypic genera should be synonymized (because recognizing them results in paraphyly of a larger genus). To preserve clarity with respect to current literature (e. g., Lamas, 2004), we do not formally alter generic affiliations here. Results The aligned data matrix consists of 3957 characters, of which 2241 are invariant, and 1291 are parsimony-informative. Some sequences are incomplete or missing for a few taxa (see Table 2), but these apparently do not impact the ability of the data to provide relatively unambiguous resolution. The analysis of the entire dataset yielded a single most parsimonious tree with a length of 10421 steps, which is presented in Fig. 2. Separate analyses of the three gene regions were conducted to investigate incongruence (Mickevich & Farris 1981; Farris et al. 1994). Although the data set as a whole is highly homoplastic, almost none of this (<2%) is due to incongruence among gene regions (Table 4). |