ARTICLE IN PRESS Biol. Lett. 1. INTRODUCTION doi:10.1098/rsbl.2008.0589 is a fascinating evolutionary pro- 1 cess that has generated much biodiversity. Although 65 2 Evolutionary biology several mechanisms may be responsible for such 66 3 diversification, the ‘ecological theory’ holds that it is 67 4 the outcome of divergent natural selection between 68 5 Convergent evolution of environments (Schluter 2000). Whether adaptive 69 6 radiations result chiefly from such ecological speciation, 70 7 ‘creepers’ in the Hawaiian however, remains unclear (Schluter 2001). Convergent 71 8 evolution is often considered powerful evidence for the 72 9 honeycreeper radiation role of adaptive forces in the speciation process 73 (Futuyma 1998), and thus documenting cases where it 10 Dawn M. Reding1,2,*, Jeffrey T. Foster1,3, 74 has occurred is important in understanding the link 11 Helen F. James4, H. Douglas Pratt5 75 12 between natural selection and adaptive radiation. 76 and Robert C. Fleischer1 13 The more than 50 of Hawaiian honeycree- 77 1 14 Center for Conservation and Evolutionary Genetics, National pers (subfamily Drepanidinae) are a spectacular 78 Zoological Park and National Museum of Natural History, 15 Smithsonian Institution, Washington, DC 20008, USA example of adaptive radiation and an interesting 79 16 2Department of Zoology, University of , Honolulu, system to test for convergence, which has been 80 17 HI 96822, USA suspected among the nuthatch-like ‘creeper’ eco- 81 3Center for Microbial Genetics & Genomics, Northern Arizona 18 morph on the islands (figure 1). The Hawaii creeper 82 University, Flagstaff, AZ 86011, USA 19 4Division of , National Museum of Natural History, (Oreomystis mana) has traditionally been allied with 83 20 Washington, DC 20013, USA the creeper (Oreomystis bairdi ), either as con- 84 5 21 North Carolina State Museum of Natural Sciences, 11 West Jones specific (Amadon 1950) or as a congener (Henshaw 85 Street, Raleigh, NC 27601, USA 22 *Author and address for correspondence: Department of Ecology, 1902; Pratt 1992). The two birds share many 86 23 Evolution, and Organismal Biology, Iowa State University, similarities including bill shape, foraging and mob- 87 24 Ames, IA 50011, USA ([email protected]). bing behaviours, and juvenile plumage pattern (Pratt 88 25 Natural selection plays a fundamental role in the 1992; Foster et al. 2000; Lepson & Woodworth 89 26 ecological theory of adaptive radiation. A pre- 2001). Their juvenile begging calls are nearly identical 90 27 diction of this theory is the convergent evolution and distinct from those known for other honeycree- 91 28 of traits in lineages experiencing similar pers (Pratt 2001). Both species possess a narrow, 92 29 environments. The Hawaiian honeycreepers are simple notch-tipped tongue very different from the 93 30 a spectacular example of adaptive radiation tubular tongues of most other honeycreepers (Pratt 94 31 and may demonstrate convergence, but uncer- 1992), including akepas () and amakihis (Hemi- 95 32 tainty about phylogenetic relationships within gnathus in part). But despite these apparent synapo- 96 the group has made it difficult to assess such 33 morphies, recent osteological and mitochondrial 97 evolutionary patterns. We examine the phyloge- 34 netic relationships of the Hawaii creeper DNA (mtDNA) evidence (Fleischer et al. 2001; 98 35 (Oreomystis mana), a that in a suite of James 2004) suggests these two species are evolution- 99 36 morphological, ecological and behavioural arily quite distant, and instead points to a close 100 37 traits closely resembles the Kauai creeper relationship between O. mana and the cross-billed 101 38 (Oreomystis bairdi ), but whose mitochondrial akepas or curve-billed amakihis. Pratt (2001) 102 39 DNA (mtDNA) and osteology suggest a relation- remarked that if the Kauai and Hawaii creepers 103 40 ship with the amakihis ( in part) derived their diverse similarities by convergence, they 104 41 and akepas (Loxops). We analysed nuclear DNA would present the most dramatic example of conver- 105 42 sequence data from 11 relevant honeycreeper gent evolution yet discovered in birds. 106 taxa and one outgroup to test whether the 43 As an alternative explanation for the conflicting 107 character contradiction results from historical 44 hybridization and mtDNA introgression, or con- phylogenetic evidence, Pratt (2001) suggested a past 108 45 Q1 vergent evolution. We found no evidence of past hybridization event. Under this scenario, hybrids 109 46 hybridization, a phenomenon that remains would have involved male Oreomystis mating with 110 47 undocumented in Hawaiian honeycreepers, and female akepa or amakihi. If the Hawaii creeper 111 48 confirmed mtDNA and osteological evidence descended from such offspring, it could have retained 112 49 that the Hawaii creeper is the most closely most of the phenotypic characteristics of Oreomystis 113 50 related species to the amakihis and akepas. but possess mtDNA from the introgressing species. 114 51 Thus, the morphological, ecological and beha- Hybridization can result in a significantly different 115 52 vioural similarities between the evolutionarily genealogy for mtDNA from that of most genes in a 116 distant Hawaii and Kauai creepers represent an 53 species, as mtDNA is more likely to introgress than 117 extreme example of convergent evolution and 54 demonstrate how natural selection can lead to nuclear DNA (nucDNA; Ballard & Whitlock 2004). 118 55 repeatable evolutionary outcomes. Indeed, mtDNA from one taxon can completely replace 119 56 that in another, with little or no evidence of nuclear 120 57 Keywords: ecological convergence; introgression or morphological change (Bernatchez 121 58 convergent evolution; Hawaiian honeycreepers; et al.1995). No unequivocal cases of hybridization have 122 59 mitochondrial DNA introgression; hybridization; yetbeendocumentedinHawaiianhoneycreepers.If 123 60 adaptive radiation past introgression is responsible for the incongruence 124 61 between mitochondrial sequences and some striking 125 62 phenotypic characters, nucDNA markers should reveal 126 63 Electronic supplementary material is available at http://dx.doi.org/ the contribution of the Oreomystis lineage to the genetic 127 64 10.1098/rsbl.2008.0589 or via http://journals.royalsociety.org. signal of the Hawaii creeper. 128

Received 20 October 2008 Accepted 26 November 2008 1 This journal is q 2008 The Royal Society RSBL 20080589—5/12/2008—16:41—CHANDRAN—318457—XML – pp. 1–5 ARTICLE IN PRESS 2 D. M. Reding et al. Convergence in Hawaiian honeycreepers

129 193 (a) (b) 130 194 131 195 132 196 133 197 134 198 135 199 136 200 137 201 138 202 139 203 140 204 141 205 142 206 143 207 144 208 145 Figure 1. Photographic comparison of the (a) Kauai creeper and (b) Hawaii creeper. Photographs by Jack Jeffrey. 209 146 210 147 Here, we examine nucDNA sequences from several for placement of Hawaii creeper with the akepas and 211 148 honeycreeper species to test whether convergence or amakihis, as noted by a high posterior probability 212 149 introgression characterizes the relationship between value of 1.0 for this clade (figure 2). Trees con- 213 150 the Hawaii and Kauai creepers. structed using mtDNA sequences from the specimens 214 151 in this analysis (figure S3, in the electronic supple- 215 152 mentary material) were nearly identical to the trees 216 2. MATERIAL AND METHODS 153 from the nuclear gene analyses (figure 2)and 217 We analysed 19 individuals belonging to 11 relevant honeycreeper 154 taxa and one outgroup. See appendix S1, electronic supplementary generally matched previous trees based on more taxa, 218 155 material, for provenance of samples. Five distinct gene regions, especially with regards to the position of the Hawaii 219 156 including four nuclear introns and one exon (see electronic creeper (Fleischer et al. 2001). 220 supplementary material), generated ca 2500 BP of sequence data. 157 We analysed the intron and exon datasets both separately and Constraining the topology such that the Hawaii 221 158 concatenated. We conducted a homogeneity partition test (ILD, and Kauai creepers formed a monophyletic group 222 159 Farris et al. 1995) with heuristic search, as implemented in the increased the length of the MP tree by eight steps and 223 program PAUPÃ v. 4.0.b10 (Swofford 2002), to evaluate the 160 lowered the score of the ML tree by 28.6. Likelihood 224 congruence of phylogenetic signal between the two sequence sets. 161 We reconstructed phylogenies using three approaches: maximum scores of the constrained trees were significantly 225 162 parsimony (MP); maximum likelihood (ML); and Bayesian infer- worse ( pZ0.007, S–H test) than that of figure 2. 226 163 ence (see electronic supplementary material). To evaluate the 227 strength of the evidence for the placement of the Hawaii creeper 164 with akepa/amakihi versus Kauai creeper, we subsequently applied 228 165 constraints in the MP and ML methods to force the alternative 4. DISCUSSION 229 166 topology, and compared the resulting ML trees with the uncon- By corroborating the phylogeny obtained from 230 strained trees using the S–H test (Shimodaira & Hasegawa 1999; 167 231 see electronic supplementary material). In addition to the nucDNA mtDNA and osteology, the results from nucDNA 168 data, we obtained and analysed 1254 bp of mtDNA sequences from markers indicate that creepers in the Hawaiian Islands 232 169 the same specimens (see electronic supplementary material for are an astounding and strongly supported example of 233 170 methods and genes used). All sequences have been deposited in ecological convergence. The two species apparently 234 GenBank (FJ266094–FJ266312). 171 evolved independently to fill similar niches on separ- 235 172 ate islands, and morphological and behavioural traits 236 173 3. RESULTS converged as a result of similar selective pressures. 237 174 The partition-homogeneity test was non-significant Although a detailed osteological analysis supports the 238 175 ( pZ0.10), indicating congruence between the different molecular result (James 2004), most other phenotypic 239 176 gene regions and justifying the concatenation of the characters support the hypothesis of a close relation- 240 177 nucDNA sequences into a single data matrix, but we ship between the Hawaii and Kauai creepers (Pratt 241 178 also analysed the introns and exon separately. The 2001). Characters such as song and plumage, which 242 179 results of the separate analyses were concordant with may be subjected to natural or sexual selection, 243 180 those from the concatenated analysis (see electronic exhibit high levels of evolutionary lability and homo- 244 181 supplementary material), so we present only the results plasy in birds (e.g. Price et al. 2007). Tongue 245 182 from the combined dataset. morphology may also be under selection for different 246 183 MP analysis of the combined dataset resulted in foraging strategies, with a simple forked tongue being 247 184 120 equally parsimonious trees of length 113, and better adapted to picking up insects than the brush- 248 185 ML analysis resulted in four equally likely trees with tipped tubular tongue of most other honeycreepers 249 186 a Kln likelihood score of 4195.1. All of the MP and (Richards & Bock 1973). It is, however, surprising 250 187 ML trees placed the Hawaii creeper in a clade with that a behavioural trait not clearly related to foraging, 251 188 the akepas and amakihis (figure 2). Bootstrap values such as juvenile begging calls, would be convergent. 252 189 at this node for both MP and ML analyses were Overall, the observed level of discord between 253 190 moderately high (higher than 75%). Bayesian infer- evolutionary relatedness and behavioural and 254 191 ence also produced a consensus topology similar to morphological traits is unprecedented among Hawaiian 255 192 the MP and ML results and showed strong support honeycreepers, and surprising among birds generally. 256

Biol. Lett.

RSBL 20080589—5/12/2008—16:41—CHANDRAN—318457—XML – pp. 1–5 ARTICLE IN PRESS Convergence in Hawaiian honeycreepers D. M. Reding et al. 3

257 akepa, Loxops coccineus (SOL245) 321 258 support values 322 (M) akepa, Loxops coccineus (SOL769) 259 (node) MP/ML/BA 323 260 akepa, Loxops coccineus (SOL242) 324 (A) 80/64/98 261 (L) akekee, Loxops caeruleirostris (RCF2681) 325 262 (B) 64/63/61 (J) 326 akekee, Loxops caeruleirostris (MVZ178405) 263 (C) 100/100/100 327 Hawaii creeper, Oreomystis mana (SOL1639) 264 (D) 85/88/100 328 265 (I) (K) 329 (E) 81/80/100 Hawaii creeper, Oreomystis mana (SOL1251) 266 330 (H) Hawaii creeper, Oreomystis mana (SOL1068) 267 (F) 66/62/92 331 268 (G) 88/86/100 Hawaii amakihi, Hemignathus virens (BRY003) 332 269 (H) 85/76/100 (D) Kauai amakihi, Hemignathus kauaiensis (168010331) 333 270 334 (I) -/53/76 Iiwi, Vestiaria coccinea (PUU023) 271 (G) 335 (J) -/-/53 272 (E) apapane, sanguinea (CJR057) 336 (K) 97/97/100 (B) 273 (F) , bailleui (178429) 337 274 (L) 51/-/82 338 Laysan , cantans (738) 275 (M) 50/52/84 339 276 (C) Kauai creeper, Oreomystis bairdi (178406) 340 277 Kauai creeper, Oreomystis bairdi (PFC203) 341 278 342 (A) alauahio, montana (H119) 279 343 280 Maui alauahio, Paroreomyza montana (H116) 344 281 House finch, Carpodacus mexicanus (93011782) 345 282 346 283 347 284 348 0.008 285 349 286 Figure 2. Bayesian consensus topology obtained from the analysis of sequences from five introns and one exon, partitioned 350 ! 287 into two parts. Bootstrap values from MP and ML analyses (1000 replicates each), and posterior probability values ( 100) 351 288 from Bayesian analysis (15 000 trees sampled) are provided for each node. A dash indicates less than 50% support. 352 Collapsed nodes had low bootstrap and posterior probability values (i.e. less than 50%). The Hawaii creeper/akepa/amakihi 289 353 clade (node H) is shown in bold. The scale at the bottom indicates 0.8% divergence along a branch. 290 354 291 Based on our and previous results, we recommend lizards: Losos et al. 1998; cichlid fishes: Ru¨ber et al. 355 292 the removal of the Hawaii creeper from the genus 1999; Hawaiian Tetragnatha spiders: Gillespie 2004). 356 293 Oreomystis. The statistical polytomy among the Such deterministic patterns of evolution underscore 357 294 Hawaii creeper, amakihis and akepas (figure 2), the important role of natural selection in shaping 358 295 however, makes the relationships among those three adaptive radiations, and call into question the view 359 296 groups unclear. Further research will be needed that evolution is inherently contingent and unpredict- 360 297 before we can say whether the Hawaii creeper able (Gould 1989). 361 298 deserves a monotypic genus (which would require a 362 299 new name) or can be classified within an existing one. Q2 We thank T. Pratt, C. Atkinson, S. Conant, A. McClung, 363 300 364 But because of the rapidity of the radiation and the P. Hart, D. Duffy and B. Woodworth for providing samples 301 365 large number of (Fleischer et al. 1998; for analysis, and M. Haynie, C. McIntosh and the EEOB 302 Department at Iowa State University for logistic support. 366 James 2004), phylogenetic study of the Hawaiian 303 NSF DEB-0083944, DEB-0643291 and the Smithsonian 367 honeycreepers remains challenging. 304 Institution’s Center for Conservation and Evolutionary 368 Genetics provided funding. 305 The convergence of creepers contrasts with the 369 306 pattern of the few other honeycreeper ecomorphs that 370 307 have been examined, which apparently evolved only 371 308 once and dispersed among the islands (Fleischer et al. 372 309 2001). These analyses, however, included only extant Amadon, D. 1950 The Hawaiian honeycreepers (Aves, 373 310 taxa. In addition, in the absence of convincing Drepaniidae). Bull. Am. Mus. Nat. Hist. 95, 151–262. 374 311 evidence of hybridization among them, Hawaiian Ballard, J. W. & Whitlock, M. C. 2004 The incomplete 375 312 honeycreepers contrast sharply with the other iconic natural history of mitochondria. Mol. Ecol. 13, 729–744. 376 313 avian island radiation, Darwin’s finches, in which (doi:10.1046/j.1365-294X.2003.02063.x) 377 Bernatchez, L., Gle´met, H., Wilson, C. C. & Danzmann, 314 hybridization and introgression are known to play a 378 R. G. 1995 Introgression and fixation of Arctic char 315 major evolutionary role (Grant & Grant 2008). None- 379 (Salvelinus alpinus) mitochondrial genome in an allopa- theless, the discovery of this previously unknown 316 tric population of brook trout (Salvelinus fontinalis). Can. 380 317 example of convergence in Hawaiian honeycreepers J. Fish. Aquat. Sci. 52, 179–185. (doi:10.1139/f95-018) 381 318 adds to the growing list of adaptive radiations Farris, J. S., Kallersjo, M., Kluge, A. G. & Bult, C. 1995 382 319 demonstrating repeated evolution of similar eco- Testing significance of incongruence. Cladistics 10, 383 320 morphs in comparable environments (e.g. Anolis 315–319. (doi:10.1111/j.1096-0031.1994.tb00181.x) 384

Biol. Lett.

RSBL 20080589—5/12/2008—16:41—CHANDRAN—318457—XML – pp. 1–5 ARTICLE IN PRESS 4 D. M. Reding et al. Convergence in Hawaiian honeycreepers

385 Fleischer, R. C., McIntosh, C. E. & Tarr, C. L. 1998 Evolution Losos, J. B., Jackman, T. R., Larson, A., de Queiroz, K. & 449 386 on a volcanic conveyor belt: using phylogeographic recon- Rodriguez-Schettino, L. 1998 Contingency and determin- 450 387 structions and K–Ar-based ages of the Hawaiian Islands to ism in replicated adaptive radiations of island lizards. Science 451 388 estimate molecular evolutionary rates. Mol. Ecol. 7, 279, 2115–2118. (doi:10.1126/science.279.5359.2115) 452 533–545. (doi:10.1046/j.1365-294x.1998.00364.x) 389 Pratt, H. D. 1992 Systematics of the Hawaiian “creepers” 453 Fleischer, R. C., Tarr, C. L., James, H. F., Slikas, B. & 390 Oreomystis and Paroreomyza. Condor 94, 836–846. 454 McIntosh, C. E. 2001 Phylogenetic placement of the (doi:10.2307/1369281) 391 455 Poouli, Melamprosops phaeosoma, based on mitochondrial Pratt, H. D. 2001 Why the Hawaii Creeper is an Oreomystis: 392 456 DNA sequence and osteological characters. Stud. Avian what phenotypic characters reveal about the phylogeny 393 Biol. 22, 98–103. of Hawaiian honeycreepers. Stud. Avian Biol. 22, 81–97. 457 394 Foster, J. T., Scott, J. M. & Sykes Jr, P. W. 2000 Akikiki Price, J. J., Friedman, N. R. & Omland, K. E. 2007 Song 458 395 (Oreomystis bairdi ). In The birds of North America, no. 552 and plumage evolution in the new world orioles (Icterus) 459 396 (eds A. Poole & F. Gill), Philadelphia, PA: The Birds of show similar lability and convergence in patterns. 460 Q3 397 North America, Inc. Evolution 61, 850–863. (doi:10.1111/j.1558-5646.2007. 461 398 Futuyma, D. J. 1998 Evolutionary biology. Sunderland, MA: 00082.x) 462 Sinauer Associates. 399 Richards, L. P. & Bock, W. J. 1973 Functional anatomy and 463 Gillespie, R. 2004 Community assembly through adaptive 400 adaptive evolution of the feeding apparatus in the 464 radiation in Hawaiian spiders. Science 303, 356–359. genus Loxops (Drepanididae). 401 (doi:10.1126/science.1091875) 465 Ornith. Monog. 15, 1–173. 402 Gould, S. J. 1989 Wonderful life: the Burgess Shale and the 466 Ruber, L., Verheyen, E. & Meyer, A. 1999 Replicated 403 nature of history. New York, NY: W.W. Norton. ¨ 467 404 Grant, P. R. & Grant, B. R. 2008 How and why species evolution of trophic specializations in an endemic cichlid 468 405 multiply: the radiation of Darwin’s finches. Princeton, NJ: fish lineage from Lake Tanganyika. Proc. Natl Acad. Sci. 469 406 Princeton University Press. USA 96,10230–10235.(doi:10.1073/pnas.96.18.10230) 470 Schluter, D. 2000 The ecology of adaptive radiation. 407 Henshaw, H. W. 1902 Birds of the Hawaiian Islands being a 471 New York, NY: Oxford University Press. 408 complete list of the birds of the Hawaiian possessions with 472 Schluter, D. 2001 Ecology and the origin of species. Trends 409 notes on their habits. Honolulu, HI: Thos. G. Thrum. 473 James, H. F. 2004 The osteology and phylogeny of the Ecol. Evol. 16, 372–380. (doi:10.1016/S0169-5347(01) 410 474 Hawaiian finch radiation (Fringillidae: ), 02198-X) 411 475 including extinct taxa. Zool. J. Linn. Soc. 141, 207–255. Shimodaira, H. & Hasegawa, M. 1999 Multiple compari- 412 (doi:10.1111/j.1096-3642.2004.00117.x) sons of log-likelihoods with applications to phylogenetic 476 413 Q4 Lepson, J. K. & Woodworth, B. L. 2001 Hawaii Creeper inference. Mol. Biol. Evol. 16, 1114–1116. 477 414 (Oreomystis mana). In The birds of North America, no. 680 Swofford, D. L. 2002 PAUPÃ: Phylogenetic analysis using 478 415 (eds A. Poole & F. Gill), Philadelphia, PA: The Birds of parsimony (Ãand other methods), v. 4.0. Sunderland, MA: 479 416 North America, Inc. Sinauer Associates. 480 417 481 418 482 419 483 420 484 421 485 422 486 423 487 424 488 425 489 426 490 427 491 428 492 429 493 430 494 431 495 432 496 433 497 434 498 435 499 436 500 437 501 438 502 439 503 440 504 441 505 442 506 443 507 444 508 445 509 446 510 447 511 448 512

Biol. Lett.

RSBL 20080589—5/12/2008—16:41—CHANDRAN—318457—XML – pp. 1–5 ARTICLE IN PRESS Convergence in Hawaiian honeycreepers D. M. Reding et al. 5

513 Author Queries 577 514 578 JOB NUMBER: 20080589 515 579 516 JOURNAL: RSBL 580 517 581 518 Q1 Please check the edit of the sentence ‘We found no 582 519 evidence.’ 583 520 Q2 Please provide an ethics statement relevant to the 584 521 guidelines of the country where the research took 585 522 place. 586 523 Q3 Please provide page range details for the reference 587 524 Foster et al. (2000). 588 525 Q4 Please provide page range details for the reference 589 526 590 Lepson & Woodworth (2001). 527 591 528 592 529 593 530 594 531 595 532 596 533 597 534 598 535 599 536 600 537 601 538 602 539 603 540 604 541 605 542 606 543 607 544 608 545 609 546 610 547 611 548 612 549 613 550 614 551 615 552 616 553 617 554 618 555 619 556 620 557 621 558 622 559 623 560 624 561 625 562 626 563 627 564 628 565 629 566 630 567 631 568 632 569 633 570 634 571 635 572 636 573 637 574 638 575 639 576 640

Biol. Lett.

RSBL 20080589—5/12/2008—16:41—CHANDRAN—318457—XML – pp. 1–5