At the frontier of marine species
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Colour pattern as a single trait driving speciation in Hypoplectrus coral reef fishes? Electronic Supplementary Material Oscar Puebla, Eldredge Bermingham, Frédéric Guichard and Elizabeth Whiteman Supplementary Methods Genetics analyses Identification, catalogue number, collection sites and collection dates of all samples are detailed in Supplementary Table 3. Total DNA was extracted using column DNeasy® Tissue Kits (Quiagen). An enriched Hypoplectrus microsatellite library was built following Kandpal et al. (1994) and 5 microsatellite markers were developed (GenBank accession no. DQ517318, DQ517319, DQ517320, DQ517321, DQ517322, Supplementary Table 4). These markers as well as 5 previously existing ones (McCartney et al. 2003) were tested and optimized on 84 individuals from 7 colour morphs (Supplementary Table 4). PCR amplifications were performed on PTC-200 Peltier Thermal Cyclers (MJ Research) in a 12-ml final volume containing 10-100 ng DNA, 0.2 mM each dNTP, 0.01 mM primer F, 0.4 mM primer R, 0.15 mM primer M13, 1x PCR Buffer II with no MgCl2 (Applied Biosystems, unknown composition), 2 mM MgCl2, and 0.25 U of AmpliTaq® DNA polymerase (Applied Biosystems). All primers F contained a 5’ M13 extension and the additional M13 primer (5’CACGACGTTGTAAAACGAC3’) was fluorescently labelled at 5’ with 6-FAM, VIC, NED, or PET (Applied Biosystems; Supplementary Table 4). PCR cycling parameters were as follow: 40s of initial denaturing at 92°C; 30 cycles of 40s at 92°C, 40s at annealing temperature (Supplementary Table 4), 40s at 72°C; and a final extension of 3 min at 72°C. Amplified fragments were separated by capillary electrophoresis on an ABI PRISM® 3130xl automated genetic analyser (Applied Biosystems) using 500 LIZ® as size standard and analysed with GENESCAN and GENOTYPER software packages (Applied Biosystems). 1 Statistical analyses of genetic data Linkage disequilibrium between loci was tested with genotype randomizations overall samples between all pairs of loci (4,500 permutations) using the log-likelihood ratio G-statistic with FSTAT version 2.9.3 (Goudet 1995). Hardy-Weinberg equilibrium was tested with allele randomizations within samples (1,000 permutations per test) and overall samples (10,000 permutations) with FSTAT. The statistics used for these tests were Fis and Fit for within-samples an overall test respectively, both estimated following Weir & Cockerham (1984). An allele size permutation test (Hardy et al. 2003) was performed using SPAGeDi version1.2 (Hardy & Vekemans 2002) to test whether stepwise-like mutations contributed significantly to genetic differentiation (10,000 allele size permutations per locus). Stepwise-like mutations did not contribute significantly to genetic differentiation in 9 loci out of the 10 analysed (p-values between 0.156 and 0.973). Fst rather than Rst (Slatkin 1995) were therefore considered for the following analyses. Fst between sympatric colour morphs was estimated following Weir & Cockerham (1984) with FSTAT. Genetic differentiation was tested with 10,000 allele permutations using the G loglikelihood statistic with FSTAT as this test proved to be more efficient than Fst-estimator based tests when Fst values are low (Goudet et al. 1996). 2 Supplementary Table 3. Identification, catalogue number, collection sites, and collection dates of all samples considered in this study. Species Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella Accession # Site Reef Latittude N [o ' ''] Longitude W [o ' ''] Collection date [dd/mm/yy] 19363 19367 19378 19379 19383 19853 19051 19055 19056 19057 19059 19060 19062 19064 19065 19071 19072 19073 19074 19075 19079 19084 19088 19089 19090 19095 19096 19097 19082 19083 19856 19100 19417 19419 19430 19433 19437 19851 19864 19077 19087 19442 19446 19447 19448 19450 19353 19355 19357 19358 19359 19360 19361 19364 19365 19368 19369 19370 19371 19372 19373 19374 19377 19380 19382 19384 19385 19387 19388 19389 19390 19391 19393 19394 Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Batt's Reef Batt's Reef Batt's Reef Batt's Reef Batt's Reef Batt's Reef Batt's Reef Batt's Reef Batt's Reef Batt's Reef Batt's Reef Batt's Reef Batt's Reef Batt's Reef Batt's Reef Batt's Reef Batt's Reef Batt's Reef Batt's Reef Batt's Reef Batt's Reef Batt's Reef Sandy Lane Sandy Lane Sandy Lane T ropicana T ropicana T ropicana T ropicana T ropicana T ropicana T ropicana T ropicana T ropicana Bank T ropicana Bank T ropicana Bank T ropicana Bank T ropicana Bank T ropicana Bank T ropicana Bank Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Alleyne's Reef Sandy Lane Sandy Lane 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 03/06/05 04/06/05 04/06/05 04/06/05 04/06/05 16/06/05 10/06/05 08/06/05 08/06/05 08/06/05 08/06/05 08/06/05 08/06/05 08/06/05 08/06/05 10/06/05 10/06/05 10/06/05 10/06/05 10/06/05 10/06/05 10/06/05 10/06/05 10/06/05 10/06/05 10/06/05 10/06/05 10/06/05 12/06/05 12/06/05 12/06/05 13/06/05 06/06/05 06/06/05 06/06/05 06/06/05 06/06/05 13/06/05 13/06/05 09/06/05 09/06/05 07/06/05 07/06/05 07/06/05 07/06/05 09/06/05 03/06/05 03/06/05 03/06/05 03/06/05 03/06/05 03/06/05 03/06/05 03/06/05 04/06/05 04/06/05 04/06/05 04/06/05 04/06/05 04/06/05 04/06/05 04/06/05 04/06/05 04/06/05 04/06/05 04/06/05 04/06/05 04/06/05 04/06/05 04/06/05 04/06/05 04/06/05 05/06/05 05/06/05 3 11 11 11 11 11 11 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 10 10 10 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 10 10 56 56 56 56 56 56 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 21 21 21 25 25 25 25 25 25 25 25 34 34 34 34 34 34 34 56 56 56 56 56 56 56 56 56 56 56 56 56 56 56 56 56 56 56 56 56 56 56 56 56 56 21 21 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 39 39 39 39 39 39 39 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 55 55 55 55 55 55 26 26 26 26 26 26 26 26 26 26 26 26 26 26 26 26 26 26 26 26 26 26 48 48 48 42 42 42 42 42 42 42 42 10 10 10 10 10 10 10 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 48 48 Continued Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans puella puella puella puella puella puella puella puella 19396 19399 19400 19401 19402 19403 19404 19406 19407 19408 19409 19410 19414 19415 19421 19422 19423 19424 19426 19427 19428 19429 18262 18049 18155 18156 18158 18159 18223 18224 18233 18472 18474 18481 18482 18483 18487 18490 18185 18187 18188 18189 18190 18191 18192 18193 18194 18196 18229 18241 18243 18245 18252 18255 17994 18186 18199 18269 18480 18491 18494 18151 18153 18162 18165 18171 18182 18282 18285 18289 18291 18293 18256 18258 18259 18260 18264 18265 18266 18268 Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Barbados Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Sandy Lane Sandy Lane Sandy Lane Sandy Lane Sandy Lane Sandy Lane Sandy Lane Sandy Lane Sandy Lane Sandy Lane Sandy Lane Sandy Lane T ropicana T ropicana T ropicana T ropicana T ropicana T ropicana T ropicana T ropicana T ropicana T ropicana Blue Ground Range Carrie Bow Cay Buoy Carrie Bow Cay Buoy Carrie Bow Cay Buoy Carrie Bow Cay Buoy Carrie Bow Cay Buoy Carrie Bow Cay Buoy Carrie Bow Cay Buoy Carrie Bow Cay Buoy Carrie Bow Cay Buoy Carrie Bow Cay Buoy Carrie Bow Cay Buoy Carrie Bow Cay Buoy Carrie Bow Cay Buoy Carrie Bow Cay Buoy Carrie Bow Cay Buoy Carrie Bow Cay Forereef Carrie Bow Cay Forereef Carrie Bow Cay Forereef Carrie Bow Cay Forereef Carrie Bow Cay Forereef Carrie Bow Cay Forereef Carrie Bow Cay Forereef Carrie Bow Cay Forereef Carrie Bow Cay Forereef Carrie Bow Cay Forereef Curlew Cay Curlew Cay Curlew Cay Curlew Cay Curlew Cay Curlew Cay Pelican Cays Pelican Cays Pelican Cays T obacco Cay T obacco Cay T obacco Cay T obacco Cay Entrance W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay Blue Ground Range Blue Ground Range Blue Ground Range Blue Ground Range Blue Ground Range Blue Ground Range Blue Ground Range Blue Ground Range 4 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 10 10 10 10 10 10 10 10 10 10 10 10 13 13 13 13 13 13 13 13 13 13 49 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 48 47 47 47 47 47 47 39 39 39 53 53 53 53 45 45 45 45 45 45 45 45 45 45 45 49 49 49 49 49 49 49 49 21 21 21 21 21 21 21 21 21 21 21 21 25 25 25 25 25 25 25 25 25 25 04 03 03 03 03 03 03 03 03 03 03 03 03 03 03 03 21 21 21 21 21 21 21 21 21 21 02 02 02 02 02 02 56 56 56 38 38 38 19 55 55 55 55 55 55 55 55 55 55 55 04 04 04 04 04 04 04 04 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 059 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 08 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 04 11 11 11 03 03 03 03 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 48 48 48 48 48 48 48 48 48 48 48 48 42 42 42 42 42 42 42 42 42 42 54 44 44 44 44 44 44 44 44 44 44 44 44 44 44 44 45 45 45 45 45 45 45 45 45 45 36 36 36 36 36 36 37 37 37 29 29 29 57 39 39 39 39 39 39 39 39 39 39 39 54 54 54 54 54 54 54 54 05/06/05 05/06/05 05/06/05 05/06/05 05/06/05 05/06/05 05/06/05 05/06/05 05/06/05 05/06/05 05/06/05 05/06/05 06/06/05 06/06/05 06/06/05 06/06/05 06/06/05 06/06/05 06/06/05 06/06/05 06/06/05 06/06/05 24/07/04 27/07/04 25/07/04 25/07/04 25/07/04 25/07/04 27/07/04 27/07/04 27/07/04 23/07/04 23/07/04 23/07/04 23/07/04 23/07/04 23/07/04 23/07/04 26/07/04 26/07/04 26/07/04 26/07/04 26/07/04 26/07/04 26/07/04 26/07/04 26/07/04 26/07/04 27/07/04 27/07/04 27/07/04 27/07/04 23/07/04 23/07/04 26/07/04 26/07/04 26/07/04 24/07/04 24/07/04 24/07/04 24/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 24/07/04 24/07/04 24/07/04 24/07/04 24/07/04 24/07/04 24/07/04 24/07/04 Continued Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans 18271 18273 18275 18294 18296 18297 18300 17995 18232 18161 18228 18230 18231 18234 18253 18254 18198 18200 18152 18154 18166 18167 18169 18170 18172 18174 18175 18176 18178 18179 18180 18181 18184 18272 18277 18279 18280 18281 18283 18284 18286 18292 18424 18428 18323 18326 18328 18330 18331 18332 18333 18335 18340 18342 18418 18436 18301 18308 18309 18311 18312 18317 18319 18320 18322 18362 18366 18368 18371 18375 18376 18380 18381 18383 18387 18388 18392 18393 18394 18395 Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Belize Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas del del del del del del del del del del del del del del del del del del del del del del del del del del del del del del del del del del del del del del T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Blue Ground Range Blue Ground Range Blue Ground Range Blue Ground Range Blue Ground Range Blue Ground Range Blue Ground Range Carrie Bow Cay Buoy Carrie Bow Cay Buoy Carrie Bow Cay Forereef Curlew Cay Curlew Cay Curlew Cay Curlew Cay Curlew Cay Curlew Cay Pelican Cays Pelican Cays W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay W eewee Cay Adriana Cay East Adriana Cay East Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Conch Point Conch Point Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Solarte Cay South Solarte Cay South Solarte Cay South Solarte Cay South ST RI Point ST RI Point ST RI Point ST RI Point ST RI Point ST RI Point ST RI Point ST RI Point ST RI Point ST RI Point ST RI Point 5 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 16 10 10 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 49 49 49 49 49 49 49 48 48 48 47 47 47 47 47 47 39 39 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 14 14 18 18 18 18 18 18 18 18 18 18 22 22 19 19 19 19 19 19 19 19 19 19 19 19 19 20 20 20 20 20 20 20 20 20 20 20 04 04 04 04 04 04 04 03 03 21 02 02 02 02 02 02 56 56 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 21 21 41 41 41 41 41 41 41 41 41 41 39 39 58 58 58 58 58 58 58 58 58 33 33 33 33 53 53 53 53 53 53 53 53 53 53 53 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 088 083 083 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 08 08 08 08 08 08 08 04 04 04 04 04 04 04 04 04 11 11 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 08 10 10 12 12 12 12 12 12 12 12 12 12 18 18 15 15 15 15 15 15 15 15 15 13 13 13 13 15 15 15 15 15 15 15 15 15 15 15 54 54 54 54 54 54 54 44 44 45 36 36 36 36 36 36 37 37 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 23 23 49 49 49 49 49 49 49 49 49 49 14 14 17 17 17 17 17 17 17 17 17 12 12 12 12 48 48 48 48 48 48 48 48 48 48 48 24/07/04 24/07/04 24/07/04 24/07/04 24/07/04 24/07/04 24/07/04 27/07/04 27/07/04 26/07/04 27/07/04 27/07/04 27/07/04 27/07/04 23/07/04 23/07/04 26/07/04 26/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 25/07/04 12/05/04 12/05/04 08/03/04 08/03/04 08/03/04 08/03/04 08/03/04 09/03/04 09/03/04 09/03/04 09/03/04 09/03/04 12/05/04 12/05/04 06/03/04 06/03/04 06/03/04 07/03/04 07/03/04 07/03/04 07/03/04 07/03/04 07/03/04 12/03/04 12/03/04 12/03/04 12/03/04 13/03/04 13/03/04 13/03/04 13/03/04 13/03/04 13/03/04 14/03/04 14/03/04 14/03/04 14/03/04 14/03/04 Continued Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans nigricans puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella 18396 18397 18401 18407 18408 18409 18410 18412 18414 18415 18416 18417 18419 18324 18325 18327 18334 18336 18338 18339 18341 18957 18958 18965 18978 18979 18982 18995 18996 18997 18999 19000 18934 18912 18915 18917 18919 18922 18924 18928 18954 18981 18984 18989 18991 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del del del T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, T oro, Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama ST RI Point ST RI Point ST RI Point ST RI Point ST RI Point ST RI Point ST RI Point ST RI Point ST RI Point ST RI Point ST RI Point ST RI Point Adriana Cay East Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Crawl Key Cristobal Island, Punta Cristobal Island, Punta Cristobal Island, Punta Cristobal Island, Punta Cristobal Island, Punta Cristobal Island, Punta Cristobal Island, Punta Cristobal Island, Punta Cristobal Island, Punta Cristobal Island, Punta Cristobal Island, Punta Cristobal Island, Punta Cristobal Island, Punta Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Solarte Cay North Solarte Cay North Solarte Cay North Solarte Cay North Solarte Cay North Solarte Cay North Solarte Cay North Solarte Cay North Solarte Cay North Solarte Cay North Solarte Cay South Solarte Cay South Solarte Cay South Solarte Cay South Solarte Cay South Solarte Cay South Solarte Cay South Solarte Cay South Solarte Cay South Solarte Cay South Solarte Cay South Solarte Cay South Solarte Cay South 6 Juan Juan Juan Juan Juan Juan Juan Juan Juan Juan Juan Juan Juan 09 09 09 09 09 09 09 09 09 09 09 09 10 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 09 20 20 20 20 20 20 20 20 20 20 20 20 14 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18 15 17 17 17 17 17 17 17 17 17 17 17 17 17 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 53 53 53 53 53 53 53 53 53 53 53 53 21 41 41 41 41 41 41 41 41 41 41 41 41 41 41 41 41 41 41 41 03 54 54 54 54 54 54 54 54 54 54 54 54 54 58 58 58 58 58 58 58 58 58 58 58 51 51 51 51 51 51 51 51 51 51 33 33 33 33 33 33 33 33 33 33 33 33 33 082 082 082 082 082 082 082 082 082 082 082 082 083 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 082 15 15 15 15 15 15 15 15 15 15 15 15 10 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 07 17 17 17 17 17 17 17 17 17 17 17 17 17 15 15 15 15 15 15 15 15 15 15 15 12 12 12 12 12 12 12 12 12 12 13 13 13 13 13 13 13 13 13 13 13 13 13 48 48 48 48 48 48 48 48 48 48 48 48 23 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 49 50 22 22 22 22 22 22 22 22 22 22 22 22 22 17 17 17 17 17 17 17 17 17 17 17 52 52 52 52 52 52 52 52 52 52 12 12 12 12 12 12 12 12 12 12 12 12 12 14/03/04 14/03/04 14/03/04 16/03/04 16/03/04 16/03/04 16/03/04 16/03/04 16/03/04 16/03/04 16/03/04 16/03/04 12/05/04 08/03/04 08/03/04 08/03/04 09/03/04 09/03/04 09/03/04 09/03/04 09/03/04 23/03/05 23/03/05 23/03/05 23/03/05 23/03/05 23/03/05 23/03/05 23/03/05 23/03/05 23/03/05 23/03/05 27/03/05 25/03/05 25/03/05 25/03/05 25/03/05 25/03/05 25/03/05 25/03/05 25/03/05 25/03/05 25/03/05 25/03/05 25/03/05 25/03/05 06/03/04 06/03/04 06/03/04 06/03/04 07/03/04 07/03/04 07/03/04 07/03/04 07/03/04 07/03/04 07/03/04 22/03/05 22/03/05 22/03/05 22/03/05 22/03/05 22/03/05 22/03/05 22/03/05 22/03/05 22/03/05 11/03/04 11/03/04 11/03/04 11/03/04 11/03/04 11/03/04 11/03/04 11/03/04 11/03/04 11/03/04 11/03/04 12/03/04 12/03/04 Continued Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus Hypoplectrus puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella puella unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor unicolor 18361 18363 18364 18367 18369 18370 18374 18377 18378 18379 18384 18386 18389 18391 18399 18400 18405 18420 18464 18469 18475 18329 18337 18976 18456 18457 18447 18442 18443 18458 18461 18462 18463 18465 18467 18930 18933 18956 18303 18435 18437 18439 18440 18441 18444 18445 18446 18448 18450 18455 18459 18460 18470 18473 18373 18406 18454 Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas Bocas del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, del Toro, Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Panama Solarte Cay South Solarte Cay South Solarte Cay South Solarte Cay South Solarte Cay South Solarte Cay South STRI Point STRI Point STRI Point STRI Point STRI Point STRI Point STRI Point STRI Point STRI Point STRI Point STRI Point Adriana Cay East Buttonwood Cay South Buttonwood Cay South Buttonwood Cay South Buttonwood Cay West Buttonwood Cay West Buttonwood Cay West Conch Point Conch Point Cristobal Island Notheast Cristobal Island, Punta Juan Cristobal Island, Punta Juan Cristobal Island, Punta Juan Cristobal Island, Punta Juan Cristobal Island, Punta Juan Cristobal Island, Punta Juan Cristobal Island, Punta Juan Cristobal Island, Punta Juan Cristobal Island, Punta Juan Cristobal Island, Punta Juan Cristobal Island, Punta Juan Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn Mangrove Inn STRI Point STRI Point STRI Point 7 09 19 33 09 19 33 09 19 33 09 19 33 09 19 33 09 19 33 09 20 53 09 20 53 09 20 53 09 20 53 09 20 53 09 20 53 09 20 53 09 20 53 09 20 53 09 20 53 09 20 53 10 14 21 09 17 54 09 17 54 09 17 54 09 18 41 09 18 41 09 18 41 09 22 39 09 22 39 09 17 22 09 17 54 09 17 54 09 17 54 09 17 54 09 17 54 09 17 54 09 17 54 09 17 54 09 17 54 09 17 54 09 17 54 09 19 58 09 19 58 09 19 58 09 19 58 09 19 58 09 19 58 09 19 58 09 19 58 09 19 58 09 19 58 09 19 58 09 19 58 09 19 58 09 19 58 09 19 58 09 19 58 09 20 53 09 20 53 09 20 53 082 13 12 082 13 12 082 13 12 082 13 12 082 13 12 082 13 12 082 15 48 082 15 48 082 15 48 082 15 48 082 15 48 082 15 48 082 15 48 082 15 48 082 15 48 082 15 48 082 15 48 83 10 23 082 12 28 082 12 28 082 12 28 082 12 49 082 12 49 082 12 49 082 18 14 082 18 14 082 15 32 082 17 22 082 17 22 082 17 22 082 17 22 082 17 22 082 17 22 082 17 22 082 17 22 082 17 22 082 17 22 082 17 22 082 15 17 082 15 17 082 15 17 082 15 17 082 15 17 082 15 17 082 15 17 082 15 17 082 15 17 082 15 17 082 15 17 082 15 17 082 15 17 082 15 17 082 15 17 082 15 17 082 15 48 082 15 48 082 15 48 12/03/04 12/03/04 12/03/04 12/03/04 12/03/04 12/03/04 13/03/04 13/03/04 13/03/04 13/03/04 13/03/04 13/03/04 14/03/04 14/03/04 14/03/04 14/03/04 16/03/04 12/05/04 12/07/04 09/07/04 12/07/04 08/03/04 09/03/04 23/03/05 03/07/04 03/07/04 09/07/04 08/07/04 08/07/04 08/07/04 09/07/04 09/07/04 09/07/04 08/07/04 09/07/04 29/03/05 29/03/05 25/03/05 06/03/04 23/06/04 29/06/04 25/06/04 25/06/04 25/06/04 28/06/04 28/06/04 29/06/04 28/06/04 25/06/04 07/07/04 07/07/04 07/07/04 10/07/04 10/07/04 13/03/04 16/03/04 30/06/04 Supplementary Table 4. Description of 10 microsatellite markers tested on 84 individuals from 7 Hypoplectrus colour morphs. n=12 per colour morph, gene diversities estimated following Nei (1987). GenBank Locus Repeat array in cloned allele Primer sequence (5' to 3') accession no. Pam013 1 - - F: CACGACGTTGTAAAACGACGATCTGCCAGATGGAAAGAC Number of alleles / gene diversity Ta Label Allele size o ( C) used range (bp) H. aberrans H. chlorurus H. guttavarius 55 VIC 106 - 160 13 / 0.93 17 / 0.97 55 6-FAM 109 - 167 14 / 0.94 49 6-FAM 145 - 173 49 NED 49 H. unicolor all 17 / 0.97 16 / 0.96 28 6 / 0.75 13 / 0.96 11 / 0.90 24 6 / 0.64 6 / 0.60 6 / 0.78 4 / 0.63 11 10 / 0.90 8 / 0.77 9 / 0.83 11 / 0.86 10 / 0.85 22 13 / 0.90 12 / 0.89 9 / 0.86 11 / 0.91 15 / 0.91 11 / 0.91 24 7 / 0.87 8 / 0.87 6 / 0.83 7 / 0.78 7 / 0.88 5 / 0.80 8 / 0.85 9 230 - 285 12 / 0.96 10 / 0.91 11 / 0.91 8 / 0.79 11 / 0.76 13 / 0.94 12 / 0.92 27 NED 126 - 138 4 / 0.56 3 / 0.41 4 / 0.61 2 / 0.17 3 / 0.64 4 / 0.43 5 / 0.72 7 55 NED 176 - 234 12 / 0.92 15 / 0.93 11 / 0.85 10 / 0.86 13 / 0.94 10 / 0.90 13 / 0.95 27 55 PET 186 - 202 3 / 0.67 6 / 0.77 3 / 0.66 5 / 0.60 3 / 0.51 3 / 0.59 3 / 0.59 7 H. indigo H. nigricans H. puella 15 / 0.96 12 / 0.92 14 / 0.96 12 / 0.90 9 / 0.84 10 / 0.81 7 / 0.71 6 / 0.68 5 / 0.61 203 - 263 15 / 0.93 12 / 0.88 VIC 200 - 248 9 / 0.85 55 6-FAM 221 - 237 55 VIC 55 R: CCACTCACTGGTGCAGAAA Gag010 2 AF183141 (GT)37 F: CACGACGTTGTAAAACGACCTAGAGGATCATTTGACAATGTAG R: CCTGACTAATCCACAGTAATTGC E2 3 AY316531 (TC)3(CA)12 F: CACGACGTTGTAAAACGACAACATTTGTGGCCAAAGAGTGGC R: TCTGCTACGGTTACAGTATGCGG G2 3 AY316532 (CA)26 F: CACGACGTTGTAAAACGACCTGACTAGCCATGTCCTTCCTAC R: AGTCAGGCTGTCTTCCTTCAGGC H24 3 AY316530 (GT)16 F: CACGACGTTGTAAAACGACCACAACCTTCACACCTGCATGC R: CTCAGCCGCCTGCGTCAGTGCA Hyp001 4 DQ517318 (TG)11 … (TG)7 F: CACGACGTTGTAAAACGACGCGCAGAGAGTATGTAGA R: TATCTGCGTCCTCAGTCTTAG Hyp008b 4 DQ517319 (CA)3…(CA)13…(CA)4…(GC)8 F: CACGACGTTGTAAAACGACTTGGGTTGCAGCTGTATCTT R: GTCTCAAATAATGCGTAAAA Hyp015 4 DQ517320 (AC)6 F: CACGACGTTGTAAAACGACTGACATGGCAGTTTCTAACC R: AGCCTTCCATCTGCTACTTT Hyp016 4 DQ517321 (TG)18 F: CACGACGTTGTAAAACGACAGTTACAGCAGGAAGTTCAG R: GTTAGCGTGCTGTCATTAG Hyp018 4 DQ517322 (CA)8…(AC)5…(TG)3 F: CACGACGTTGTAAAACGACCCGGAAAACACACATGCATA R: GCTGGAGCCCATGCATTAT 1 Ball et al (unpublished), 2Chapman et al (1999), 3McCartney et al (2003), 4this study, Ta: annealing temperature 8 Supplementary Table 5. Fis estimate (Weir & Cockerham 1984), heterozygote deficit test p-value and heterozygote excess test p-value per locus and sample. All loci are at Hardy-Weinberg equilibrium at all samples. Locus H. nigricans Barbados 2005 n=46 H. nigricans Belize 2004 n=50 H. nigricans Panama 2004 n=50 H. puella Barbados 2005 n=50 H. puella Belize 2004 n=50 H. puella Panama 2004 n=50 H. puella Panama 2005 n=35 H. unicolor Panama 2004 n=40 E2 0.127 (0.134/0.934) 0.076 (0.219/0.870) -0.068 (0.839/0.295) -0.048 (0.786/0.362) -0.053 (0.779/0.353) 0.100 (0.146/0.922) -0.055 (0.773/0.397) 0.124 (0.158/0.925) Gag010 0.028 (0.386/0.762) -0.029 (0.792/0.375) 0.073 (0.122/0.948) 0.037 (0.270/0.864) -0.003 (0.616/0.598) 0.022 (0.427/0.770) -0.012 (0.708/0.565) -0.018 (0.721/0.506) Hyp001 -0.027 (0.734/0.434) 0.110 (0.079/0.963) 0.127 (0.044/0.981) 0.046 (0.280/0.831) 0.100 (0.074/0.965) 0.003 (0.542/0.617) -0.008 (0.621/0.573) 0.075 (0.197/0.896) Hyp015 0.022 (0.571/0.690) 0.040 (0.416/0.708) 0.125 (0.143/0.922) -0.040 (0.720/0.407) 0.157 (0.088/0.954) 0.174 (0.067/0.968) -0.147 (0.943/0.152) 0.148 (0.093/0.956) G2 -0.025 (0.724/0.440) 0.020 (0.425/0.739) -0.090 (0.957/0.102) -0.060 (0.955/0.133) -0.055 (0.936/0.171) -0.058 (0.935/0.166) 0.022 (0.429/0.776) -0.030 (0.811/0.392) H24 0.069 (0.141/0.935) 0.039 (0.229/0.894) -0.055 (0.904/0.212) 0.001 (0.583/0.654) -0.001 (0.601/0.639) 0.025 (0.341/0.824) 0.058 (0.192/0.921) -0.038 (0.870/0.326) Hyp008 0.146 (0.054/0.977) 0.124 (0.015/0.996) -0.088 (0.970/0.086) 0.013 (0.453/0.729) -0.076 (0.992/0.050) 0.036 (0.262/0.867) -0.108 (0.993/0.051) -0.020 (0.773/0.477) Hyp016 0.009 (0.519/0.655) -0.025 (0.894/0.354) -0.016 (0.743/0.477) -0.030 (0.918/0.296) -0.026 (0.901/0.336) -0.032 (0.847/0.331) -0.051 (0.916/0.261) 0.052 (0.167/0.940) Hyp018 0.072 (0.282/0.825) 0.001 (0.556/0.588) 0.058 (0.343/0.792) -0.011 (0.608/0.521) -0.161 (0.979/0.049) -0.024 (0.655/0.497) 0.127 (0.164/0.918) -0.028 (0.670/0.477) Pam013 -0.054 (0.969/0.143) -0.016 (0.772/0.477) 0.089 (0.033/0.990) -0.005 (0.659/0.691) 0.009 (0.474/0.754) 0.034 (0.218/0.912) -0.012 (0.718/0.567) -0.053 (1.000/0.123) n: number of individuals analysed per sample. Supplementary Discussion Colour pattern Seven colour morphs (H. nigricans, H. puella, H. unicolor, H. indigo, H. aberrans, H. guttavarius, and tan hamlet) were observed in Belize, 6 (H. puella, H. nigricans, H. unicolor, H. aberrans, H. gummigutta, and tan hamlet) in Bocas del Toro, 6 (H. puella, H. nigricans, H. unicolor, H. aberrans, H. indigo, and tan hamlet) in Kuna Yala, and 4 (H. puella, H. nigricans, H. chlorurus, and H. guttavarius) in Barbados. H. puella and H. nigricans were the only colour morphs present at all study sites. Apart from the rare occurrence of “intermediate” individuals, colour pattern was clearly discrete at each site and most samples could be attributed to one colour morph confidently. That sympatric colour morphs constitute discrete phenotypic entities is generally not disputed in the Hypoplectrus 9 literature, even by authors who regard colour morphs as polymorphs of a single species (e.g. Graves & Rosenblatt 1980). Note that the Kuna indigenous people from Panama recognize and differentiate the 5 colour morphs present in Kuna Yala as well. Genetics The 10 microsatellite markers amplified successfully and were polymorphic in the 7 colour morphs tested (Supplementary Table 4). The 371 samples considered in this study were genotyped at the 10 microsatellite loci with less than 1.4% missing data (locus poorly or not amplifying for a given sample) overall. A total of 231 alleles were identified, 138 of which where shared by the 3 colour morphs examined. Forty-two alleles were observed in one colour morph exclusively yet all were rare, with a frequency smaller than 1% overall samples for the locus considered. There was no evidence of linkage disequilibrium between any pair of loci at the 0.05 level after sequencial Bonferroni correction for multiples tests (Rice 1989). All loci were at Hardy-Weinberg equilibrium within all samples after sequencial Bonferroni correction for multiple tests (Supplementary Table 5). Even without corrections for multiple tests, only 5 departures from HardyWeinberg would be significant (3 heterozygote deficits and 2 excesses) out of the 160 tests performed (heterozygote deficit and excess on 10 loci x 8 samples, Supplementary Table 5). In contrast 6 loci out of the 10 analysed presented a deficit in heterozygotes overall samples (p-values between 0.0008 and 0.0372) and there was a global deficit in heterozygotes overall samples and loci (Fit estimate=0.033, p-value<0.0001), suggesting reduced gene flow between samples. 10 Supplementary References Chapman, R.W., Sedberry, G.R., Koenig, C.C. & Eleby B.M. 1999 Stock identification of gag, Mycteroperca microlepis, along the southeast coast of the United States. Marine Biotechnol. 1, 137-146. Goudet, J. 1995 FSTAT (version 1.2): a computer program to calculate F-statistics. J. Hered. 86, 485-486. Hardy, O. J. & Vekemans X. 2002 SPAGeDi: a versatile computer program to analyse spatial genetic structure at the individual or population levels. Mol. Ecol. Notes 2, 618-620. Hardy, O. J., Charbonne N., Fréville, H. & Heuertz, M. 2003 Microsatellite allele sizes: a simple test to assess their significance on genetic differentiation. Genetics 163, 1467-1482. Kandpal, R. P., Kandpal, G. & Weissman, S. M. 1994 Construction of libraries enriched for sequence repeats and jumping clones, and hybridization selection for region-specific markers. Proc. Natl. Acad. Sci. USA 91, 88-92. Nei, M. 1987 Molecular Evolutionary Genetics. Columbia University Press: New York. Rice, W.R. 1989 Analyzing tables of statistical tests. Evolution 43, 223-225. Slatkin, M. 1995 A mesure of population subdivision based on microsatellite allele frequencies. Genetics 139, 457-462. 11 Supplementary Video Legends Supplementary Video Sequence 1. Illustration of the behavioural pattern considered as “tracking” in the present study. Putative mimic H. unicolor presents clear changes in speed and/or direction in order to stay within approximately 30 cm of two C. capistratus putative models. This sequence would be considered as 24 seconds of “tracking” in our study. Supplementary Video Sequence 2. Illustration of the behavioural pattern considered as “predatory strike” in the present study. H. unicolor performs a clear, sharp, and long acceleration. Note that the strike is performed close to a C. capistratus and that the prey, a small fish, is visible in this sequence. This sequence would be considered as one “predatory strike” in the presence of C. capistratus in our study. 12
