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Supporting Information Supporting Information Marek and Bond 10.1073/pnas.0810408106 SI Text them in color analyses testing mimetic resemblance. We did not Sampling Strategy. Specimens were collected during mid to late estimate phylogeny, conduct NCA, or calculate ␪. Support peak activity periods for apheloriines: August 16–20, 2006; and indices for all species clades were greater than 0.96 probability September 26 to October 4, 2006. Collection effort, speed, and for Bayesian posterior probabilities (except for the species B. time spent sampling were constant across sites. Overall, we mendota, with 0.73 posterior clade probability). Aligned species collected 398 millipedes at 29 sites. At each 50-m2 site, 2 datasets lacked signatures of complex indel events and varied sampling strategies were implemented to ensure precise popu- lengthwise on average by 6 bp. lation sampling: leaf raking (with a millipede rake) and boulder/ log turning (with a rock hammer). One strategy was assigned to NCA and Estimation of Demographic Processes. We conducted each collector for 30 min and then exchanged for another 30 min. phylogeographic analyses by using NCA to test for an association Collection effort and speed (raking and turning) were therefore between DNA haplotype variation and geographical variation. constant across sites. Live specimens captured during these Nested haplotype networks were analyzed by using a permuta- surveys were brought back to the laboratory for color measure- tion contingency test in the program GEODIS, version 2.5 (5), ment and DNA sequencing. Table S1 summarizes sites and their to calculate the statistics Dc (geographical range of a clade) and respective species, haplotypes, color morphs, and abundances Dn (geographical distribution of a clade relative to its closest (species and site totals). sister). We used the program ANeCA, version 1.0 (6), to automate nesting and statistical calculations and to confirm DNA Sequencing, Alignment, and Phylogeny Estimation. Left legs manual inferences drawn from the NCA key (version 11, No- (segments 8–19) were removed, immersed in RNAlater (Qia- vember 2005). Hierarchical pattern was congruent, and the gen), and archived in an ultracold freezer. Protocols and primers network root nodes (determined by using a frequency-based used to amplify a region of the mitochondrial genome spanning estimate) were consistent with the Bayesian trees. We used the ribosomal RNA genes are described in detail previously (1). Templeton, Crandall, Sing (TCS) statistical parsimony (7) and a Initial cycle sequencing reactions consisted of 1 primer (LR-J- 95% connection limit to estimate haplotype networks in the 12887dip2) to prescreen for redundant mitochondrial sequences, program TCS, version 1.21 (8). Species haplotype networks were and a subsequent reaction with a second primer (LR-J-APHE1, similar to their estimated phylogenies. The networks for the which sequences an overlapping fragment) to screen for the species A. clade A, B. cedra, and B. mendota have unconnected unique 16S rDNA mitochondrial sequences reported herein. See clades with 3, 2, and 5 separate haplotype groups, respectively. Marek and Bond (1) for details on multiple sequence alignment. By using GEODIS contingency tests, we observed a number of First, sequences were aligned and analyzed within the context significant inferences favoring restricted gene flow with isolation of the phylogeny of Apheloriini (2) to categorize sequences into by distance at a lower nesting level (between 1-step and 3-step species. We estimated the phylogeny by using maximum likeli- clades) and allopatric fragmentation at a higher nesting level hood and Bayesian inference with the programs GARLI version (between 4-step and higher clades). Detailed results of the 0.951 (3) and MrBayes version 3.1.2 (4) using the settings nested clade analyses among the 7 codistributed species are described in Marek and Bond (2). In the phylogenetic analysis, available upon request from the corresponding author. some species did not fit preexisting taxonomy. Individuals iden- tified as Apheloria virginiensis corrugata, Apheloria montana, and Species Population Sizes, Estimation of ␪, and Abundance. We used an undescribed species did not occur as monophyletic lineages on the program LAMARC, version 2.1.2 (9), to estimate the the phylogeny. Overall, there were 2 Apheloria clades, designated population parameter ␪. LAMARC employs a Markov chain clades ‘‘A’’ and ‘‘B.’’ The species Brachoria dentata and B. Monte Carlo sampling method to first estimate genealogies, and mendota are paraphyletic with respect to 2 new species and then it generates a maximum-likelihood estimation of popula- Brachoria evides. However, in this study, B. dentata and B. tion parameters. We estimated ␪ only for those clades in the 7 mendota are treated as a separate species because nested lin- endemic species connected at 95% statistical parsimony (10) and eages are considerably disjunct geographically (species in B. with Ն8 haplotypes (11), subsequently treating them as popu- dentata occur on the other side of Cumberland Mountain in lations in the analysis. The parameter ␪ is not directly related to Kentucky, and B. evides in Jefferson County, Tennessee), have Nc because we must factor in mutation rate, breeding ratio, and extensively different male genitalia, and represent independent overlapping generations—items that may affect Ne (12). How- genetic lineages. ever, we expect the magnitude of the values to be strongly related Subsequently, each cluster of sequences that represents a because ␪ is predominately governed by Ne, mutation rate is species in the Apheloriini phylogeny was then realigned on its assumed constant, and breeding and generational strategies are own to construct population-level phylogenies by using MrBayes. equal. To test the relationship between ␪ and Nc, we used linear Fig. S1 shows the 7 codistributed species population phylogenies regression of census abundance as a predictor of ␪ by using the and their location in the apheloriine phylogeny. The sequences statistical package JMP, version 7 (SAS). We found a strong 2 are available for download from the National Center for Bio- positive linear correlation of ␪ with Nc (R ϭ 0.64, ln- technology Information (accession nos. FJ667003–186). By using transformed Nc; Fig. S2). Because Nc is an accurate predictor of random population sampling, we collected 5 additional species ␪, we expect Nc to reflect the ancestral frequency-dependent representing populations at the edge of their distributions in the conditions under which mimicry evolved, provided ␪ has not study area that were uncommon (Appalachioria species ‘‘n,’’ changed substantially since that time. Brachoria species ‘‘e,’’ B. hoffmani, and Rudiloria kleinpeteri; Table 1). Because we did not comprehensively survey these Color Measurement. We measured millipede color patterns by additional species throughout their known ranges (as we did with using a USB4000-VIS-NIR fiber optic spectrometer (blazed at the 7 endemic species: A. clade A, A. clade B, B. cedra, B. dentata, 500 nm) attached to an LS-1 tungsten halogen light source with B. insolita, B. mendota, and B. species ‘‘n’’), we only included a QR400-7-VIS-NIR bifurcated probe with an acceptance angle Marek and Bond www.pnas.org/cgi/content/short/0810408106 1of35 of 24.8° (Ocean Optics). No color changes occurred between color patches were analyzed by using the segment classification measurements taken in the field versus the lab, based on a method (SCM) (14) without calculating differences between time-series color analysis using Munsell color chips (13). Seven segments of the spectrum to estimate opponency. This method surface regions were measured on intact living millipedes (on the examines the unprocessed physical properties of color spectra eighth metatergite—1: right paranotal spot; 2: median metater- and does not base a comparison on a receiver model, because gal spot; and 3: metatergal background; and on the 11th metater- apheloriine predators have not been identified. The SCM is gite—4: left paranotal spot; 5: median metatergal spot; 6: appropriate because it preserves the general features of animal metatergal background; and 7: on the legs, apheloriines curl in visual processing in which spectra are divided into regions that a defensive ball with legs tightly appressed). Supplementary have a variable probability of exciting different photoreceptor measurements were made, when needed, to sample proportion- classes. In the SCM, brightness and color are decoupled for ally to additional color spots if present (on the eighth proter- separate analysis, as in the case for animal visual modalities that gite—8: median protergal spot; on the 11th protergite—9: process intensity, or brightness, separately from hue. We divided median protergal spot). Ambient habitat light, or irradiance, was radiance spectra from millipede body patches into 4 equally measured at 2 old-growth mixed mesophytic Appalachian forests spaced segments (B: 400–475 nm; G: 475–550 nm; Y: 550–625 near the area (Joyce Kilmer Memorial Forest, Swain County, nm; and R: 625–700 nm) and calculated each segment’s total North Carolina, and Blanton Woods, Harlan County, Kentucky; intensity by integrating between segmental limits; in practice, by forest shade canopies, mostly closed but with a few small gaps) summation of radiant photon flux across discrete wavelength during the same time of day (12:01 PM) and under similar intervals because the spectral formula is unknown. We divided
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