Molecular phylogeny and systematics of Neotropical

Zoologica Scripta Molecular phylogeny and systematics of Neotropical toucanets in the genus Aulacorhynchus (Aves, Ramphastidae) ELISA BONACCORSO, JUAN M. GUAYASAMIN, A. TOWNSEND PETERSON & ADOLFO G. NAVARROSIGUËNZA

Submitted: 13 August 2010 Accepted: 7 February 2011 doi:10.1111/j.1463-6409.2011.00475.x

Bonaccorso, E., Guayasamin, J. M., Peterson, A. T. & Navarro-Siguënza, A. G. (2011). Molecular phylogeny and systematics of Neotropical toucanets in the genus Aulacorhynchus (Aves, Ramphastidae). — Zoologica Scripta, 40, 336–349. We studied the phylogenetic relationships in the genus Aulacorhynchus, an assemblage of Neotropical toucanets distributed from Mexico south to Bolivia. Based on mitochondrial and nuclear DNA characters, we obtained a robust hypothesis of relationships for all recognized species, including good representation of distinct geographic populations. Our results support the monophyly of the genus Aulacorhynchus, but contradict previous taxonomic arrangements. The genus is made up of three major clades: the Aulacorhynchus prasinus complex, Aulacorhynchus huallagae + Aulacorhynchus coeruleicinctis, and Aulacorhynchus haematopygus + Aulacorhynchus sulcatus + Aulacorhynchus derbianus. Andean populations of A. derbianus are more closely related to A. sulcatus than to Pantepuian populations of A. derbianus, rendering A. derbianus paraphyletic. Based on the molecular phylogeny, and information on geographic distributions and morphological and behavioural characters, we review the specific status of these taxa and propose a new taxonomic arrangement within Aulacorhynchus. Corresponding author: Elisa Bonaccorso, Universidad Tecnolo´gica Indoame´rica, Machala y Sabanilla, Cotocollao, Quito, Ecuador. E-mail: [email protected] Elisa Bonaccorso, Universidad Tecnolo´gica Indoame´rica, Machala y Sabanilla, Cotocollao, Quito EC170103, Ecuador; Biodiversity Institute, University of Kansas, 1345 Jayhawk Boulevard, Lawrence, KS 66045-7561, USA. E-mail: [email protected] Juan M. Guayasamin, Universidad Tecnolo´gica Indoame´rica, Machala y Sabanilla, Cotocollao, Quito EC170103, Ecuador. E-mail: [email protected] A. Townsend Peterson, Biodiversity Institute, University of Kansas, 1345 Jayhawk Boulevard, Lawrence, KS 66045-7561, USA. E-mail: [email protected] Adolfo G. Navarro-Siguënza, Museo de Zoologıá ‘‘Alfonso L. Herrera,’’ Facultad de Ciencias, Universidad Nacional Autońoma de Me´xico, Apartado Postal 70-399, Me´xico D.F. 04510, Me´xico. E-mail: [email protected]

Introduction The genus Aulacorhynchus is an assemblage of Neotropical toucanets that inhabit humid montane forests, with isolated populations reaching the upper tropical zone and lowlands in a few areas. They are distributed from Mexico south to Bolivia, including the mountain ranges of northern Venezuela and the Pantepui region of the Guianan Shield (Short & Horne 2002). Although the early taxonomy of the genus experienced certain instability, such as allotment of some species to Pteroglossus (see Cory 1919), modern classifications have recognized only a single genus for this group of green and similarly shaped toucanets. Depending on the authority, numbers of species in Aulacorhynchus may range from 6 to 14. The most accepted 336

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taxonomy (e.g. Short & Horne 2002; Dickinson 2003; Remsen et al. 2010) recognizes six species: Aulacorhynchus prasinus, Aulacorhynchus sulcatus, Aulacorhynchus derbianus, Aulacorhynchus haematopygus, Aulacorhynchus huallagae, and Aulacorhynchus coeruleicinctis. However, A. prasinus, the Emerald Toucanet, is considered to constitute a complex of multiple species (Haffer 1974; Navarro-Siguënza et al. 2001; Ridgely & Greenfield 2001; Clements 2007), a hypothesis that saw considerable support in a recent molecular study (Puebla-Olivares et al. 2008). The authors of the latter study proposed that A. prasinus consists of at least seven morphologically and genetically divergent species, each restricted to specific montane massifs. In that study, two major clades were found within A. prasinus: a

Zoologica Scripta ª 2011 The Norwegian Academy of Science and Letters, 40, 4, July 2011, pp 336–349

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Phylogenetics of Aulacorhynchus

Fig. 1 Geographic distribution of species in Aulacorhynchus. Subspecies correspond to numbers (see Table 1), as follows: 1, 3 A. prasinus prasinus; 2 A. p. wagleri; 4 A. p. caeruleogularis; 5–9 A. p. albivitta; 10–12 A. p. griseigularis; 13 A. p. atrogularis; 14–17 A. sulcatus sulcatus; 18–23 A. s. calorhynchus; 24, 25 A. s. sulcatus · A. s. calorhynchus?; 26–31 A. s. erythrognathus; 32–42 A. derbianus derbianus; 43–45 A. d. duidae; 46–49 A. d. whitelianus; 50–52 A. d. osgoodi; 53 A. haematopygus haematopygus; 54–66 A. h. sexnotatus.

Mesoamerican clade including four species-level taxa (Aulacorhynchus cognatus, Aulacorhynchus caeruleogularis, A. prasinus, and Aulacorhynchus wagleri) and a South American clade composed of at least another four (Aulacorhynchus albivitta, Aulacorhynchus griseigularis, Aulacorhynchus lautus, and Aulacorhynchus atrogularis). Although most attention has been paid to the A. prasinus complex, Aulacorhynchus includes other broadly distributed species consisting of multiple geographically distinct populations (Fig. 1). For example, A. sulcatus, the Groove-billed Toucanet, inhabits the western and central (A. s. sulcatus), and eastern (A. s. erythrognathus) mountain ranges of northern Venezuela, and the Andes of Venezuela, Sierra de Perija´, and Sierra de Santa Marta (A. s. calorhynchus). The subspecies A. s. calorhynchus has been treated as a separate species (Cory 1919; Peters 1948; Phelps & Phelps 1950; Meyer de Shauensee 1970; see also Hilty 2003), but Schwartz (1972) considered it a synonym of A. sulcatus, based on acoustic evidence and the presence of populations with intermediate-coloured bills, in what seem to be areas of contact in central and northwestern Venezuela. Aulacorhynchus derbianus, the Chestnut-tipped Toucanet, is represented by four subspecies: one inhabits the eastern slope of the Andes, from northern Ecuador to central Bolivia (A. d. derbianus; we will use the simpler ‘A. derbianus [Andes]’, hereinafter), whereas the other three range along the highlands of the Guianan Shield, from Venezuela east to Surinam (A. d. duidae, A. d. whitelianus, and A. d. osgoodi; A. derbianus [Pantepui], hereinafter). Aulacoª 2011 The Authors

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rhynchus haematopygus, the Crimson-rumped Toucanet, consists of two subspecies, one distributed from extreme western Venezuela to western Colombia (A. h. haematopygus), and another from southwestern Colombia to southwestern Ecuador (A. h. sexnotatus). Whether they intergrade in western Colombia is unclear, since specimens from that area appear intermediate, but morphologically closer to A. h. sexnotatus (Chapman 1926). The remaining two species, A. coeruleicinctis, the Bluebanded Toucanet, distributed from central Peru to central Bolivia, and A. huallagae, the Yellow-browed Toucanet, from a restricted area in the eastern Andes of central Peru, are monotypic and considered more closely related to each other than to other species (Short & Horne 2002). Although the two species are morphologically similar, A. huallagae has yellow undertail coverts, a character not found in any other Aulacorhynchus. Given its morphological distinctiveness, A. huallagae has been considered a full species since its description (Carriker 1933), and has been treated as Endangered (BirdLife International 2009), based on its restricted range and ongoing habitat transformation in the region. Haffer (1974) presented the most thorough revision of the genus Aulacorhynchus as a whole. Based on patterns of plumage variation and geographic distributions, he distinguished three species groups: the A. prasinus species complex, the A. sulcatus superspecies (consisting of A. sulcatus and A. derbianus), and the A. haematopygus superspecies (consisting of A. haematopygus, A. coeruleicinctis, and


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A. huallagae). Regardless of the dearth of quantitative studies with which to test this arrangement, Haffer’s species groups have remained broadly accepted hypotheses of relationships (Fjeldsa˚ & Krabbe 1990; Short & Horne 2002; Remsen et al. 2010). Herein, we analyze sequences of two mitochondrial and two nuclear genes to produce a phylogenetic hypothesis of relationships of the species and differentiated populations within Aulacorhynchus. Based on this hypothesis, geographic distributions, and published information on morphological and behavioural characters, we review the specific status of taxa and propose a taxonomic arrangement that is consistent with the evolutionary history of Aulacorhynchus.

Methods Taxon and gene sampling Samples were obtained from vouchered tissue collections (see Acknowledgements) and from our own fieldwork in Guyana, El Salvador, Mexico, and Venezuela (Table 1). Our sampling strategy focused on analyzing all traditionally recognized species in Aulacorhynchus, as well as all recognized subspecies within A. sulcatus, A. derbianus, and A. haematopygus; within A. sulcatus, we included two specimens from putative hybrid populations (A. s. sulcatus · A. s. calorhynchus) that show intermediate bill colours (sensu Schwartz 1972). Within the A. prasinus complex, we included representatives of all putative evolutionary species proposed by Puebla-Olivares et al. (2008), with the exception of A. p. cognatus. Following Moyle (2004) and Weckstein (2005), we polarized character states by analyzing samples from all other genera in the family Ramphastidae (Andigena, Selenidera, Pteroglossus, and Ramphastos). For combined analyses (see below), nuclear sequences of Andigena hypoglauca were concatenated with mitochondrial sequences of Andigena cucullata published elsewhere (Table 1). Although these combined sequences are technically chimeric, their appropriateness to root the trees is justified because sequences from both taxa are, in all probability, more closely related to one another than to those of other species in the combined dataset (i.e. we assume that Andigena is monophyletic). Gene sampling consisted of sequences for the mitochondrial genes NADH dehydrogenase subunit 2 (ND2) for 76 individuals and cytochrome b (cytb) for 69 individuals of Aulacorhynchus. To obtain independent estimates of relationships among taxa, two nuclear loci, the b-fibrinogen intron 7 (bfb7) and the transforming growth factor beta 2 intron 5 (TGFb2.5), were sequenced for 34 and 36 individuals, respectively. Differences in numbers of samples analyzed for each gene resulted from difficulties in 338

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sequencing several relatively poorly preserved tissue samples or samples with seemingly high concentrations of PCR inhibitors. DNA extraction, PCR, and alignment The DNA from preserved tissue samples was extracted with the DNeasy Tissue extraction kit (Qiagen Inc., Valencia, CA, USA) or a modified salt precipitation method (M. Fujita, unpublished data). For A. huallagae (LSUMZ B-48608), DNA was extracted from toe pads clipped from dried study skin material by R. Fleischer in the laboratories of the National Museum of Natural History and National Zoological Park, using established ancient DNA protocols (Fleischer et al. 2000, 2001). PCR amplification was completed using the following primer pairs: L5216 or L5143, and H6313 for ND2 (Sorenson et al. 1999); L14990 (Kocher et al. 1989) and H16065 (T. Birt, unpublished data) for cytb; FIB-B17U and FIB-B17L for bfb7 (Prychitko & Moore 1997); and TGFb2.5F and TGFb2.6R for TGFb2.5 (Sorenson et al. 2004). Mitochondrial genes were amplified using a standard PCR protocol (94 C ⁄ 5 min; 35 cycles of 93 C ⁄ 1 min, 52 C ⁄ 1 min, 72 C ⁄ 2 min; and 72 C ⁄ 10 min), whereas nuclear genes were amplified using a touchdown protocol (i.e. 94 C ⁄ 3 min; 5 cycles of 94 C ⁄ 30 s, 60 C ⁄ 30 s, 72 C ⁄ 40 s; 5 cycles of 94 C ⁄ 30 s, 56 C ⁄ 30 s, 72 C ⁄ 40; 35 cycles of 96 C ⁄ 30 s, 52 C ⁄ 30 s, 72 C ⁄ 40 s; 72 C ⁄ 10 min; R. Moyle, personal communication). ND2 amplification for A. huallagae was achieved by combining primer L5143 and internal primers developed for this study (i.e. 218H-GAGAGTAGGATGAYAGCTGARG, 163L-GCCTCAATTAAATAYTTCCTAGTAC, and 515H-TGGTTGAGRCCTATTCAG). To avoid cross contamination with other samples, amplification for this sample was carried out in a separate lab facility, using proper controls and fresh reagents, and where no toucanet material had been amplified previously. The PCR products were treated with ExoSAP-IT (Affymetrix Inc., Santa Clara, CA, USA) to degrade unincorporated primers and dNTP’s. Cycle sequencing was completed with the corresponding PCR primers and BigDye Terminator 3.1 chemistry (Applied Biosystems, Foster City, CA, USA). Sequencing reaction products were purified with CleanSEQ magnetic beads (Agencourt Bioscience, Beverly, MA, USA) and resolved on an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Raw chromatograms from heavy and light strands were assembled and edited with Sequencher 4.8 (Gene Codes Corporation. 2007). Alignments were obtained in CLUSTAL X (Thompson et al. 1997), and inspected and translated to proteins (mitochondrial genes) in MacClade ver. 4.07 (Maddison & Maddison 2007).



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Table 1 List of tissue samples and GenBank accession numbers for sequences of species included in the present study

Species

No.

Museum and collection number

Locality information

ND2

cytb

bfib7

TGFb2.5

A. prasinus prasinus A. prasinus wagleri A. prasinus prasinus A. prasinus caeruleogularis A. prasinus albivitta A. prasinus albivitta A. prasinus albivitta A. prasinus albivitta A. prasinus albivitta A. prasinus griseogularis A. prasinus griseogularis A. prasinus griseogularis A. prasinus atrogularis A. sulcatus sulcatus A. sulcatus sulcatus A. sulcatus sulcatus A. sulcatus sulcatus A. sulcatus calorhynchus A. sulcatus calorhynchus A. sulcatus calorhynchus A. sulcatus calorhynchus A. sulcatus calorhynchus A. sulcatus calorhynchus A. sulcatus sulcatus · calorhynchus? A. sulcatus sulcatus · calorhynchus? A. sulcatus erythrognathus A. sulcatus erythrognathus A. sulcatus erythrognathus A. sulcatus erythrognathus A. sulcatus erythrognathus A. sulcatus erythrognathus A. derbianus derbianus A. derbianus derbianus A. derbianus derbianus A. derbianus derbianus A. derbianus derbianus A. derbianus derbianus A. derbianus derbianus A. derbianus derbianus A. derbianus derbianus A. derbianus derbianus A. derbianus derbianus A. derbianus duidae A. derbianus duidae A. derbianus duidae A. derbianus whitelianus A. derbianus whitelianus A. derbianus whitelianus A. derbianus whitelianus A. derbianus osgoodi A. derbianus osgoodi A. derbianus osgoodi A. haematopygus haematopygus A. haematopygus sexnotatus A. haematopygus sexnotatus A. haematopygus sexnotatus

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

MZFC-HGUSLP132 MZFC-OMVP705 KUNHM-4932 FMNH-393039 COP-81127 COP-81128 COP-81129 KUNHM-111216 IAvH-CT1752 IAvH-CT2611 IAvH-CT1696 IAvH-CT4003 FMNH-433258 KUNHM-111219 EB05-No voucher EBGR-12237 EBGR-400 KUNHM-111218 EBGR-12234 ULA-565 COP-81124 COP-81125 COP-81126 KUNHM-111221

Mexico: Hidalgo; Pisaflores, El Coyol Mexico: Oaxaca; Putla, Sta Ana del Progreso El Salvador: Morazań; Cerro Cacahuatique Costa Rica: San Jose´; Rio Tiribi, 2 km SW Rancho Redondo Venezuela: Zulia; Sierra de las Lajas, Serranıá de Perija´ Venezuela: Zulia; Sierra de las Lajas, Serranıá de Perija´ Venezuela: Zulia; Sierra de las Lajas, Serranıá de Perija´ Venezuela: Me´rida; La Mucuy, Parque Nacional Sierra Nevada Colombia: Norte de Santander; Carrizal, Cucutilla Colombia: Caldas; El Laurel, Aranzazu Colombia: Rizalda; La Cumbre, Pueblo Rico Colombia: Valle del Cauca; Chicoral, La Cumbre Peru: Cuzco; Consuelo, 15.9 km SW Pilcopata Venezuela: Aragua; Estacioń Biolo´gica Rancho Grande Venezuela: Aragua; Estacioń Biolo´gica Rancho Grande Venezuela: Aragua; Estacioń Biolo´gica Rancho Grande Venezuela: Aragua; Estacioń Biolo´gica Rancho Grande Venezuela: Lara; El Hacha, Parque Nacional Yacambu´ Venezuela: Lara; El Hacha, Parque Nacional Yacambu´ Venezuela: Me´rida; San Luı´s Venezuela: Sucre; Sierra de las Lajas, Serranıá de Perija´ Venezuela: Sucre; Sierra de las Lajas, Serranıá de Perija´ Venezuela: Sucre; Sierra de las Lajas, Serranıá de Perija´ Venezuela: Gua´rico; Hacienda Picachito, Cerro Platilloń

JF424372 JF424373 JF424374 JF424375 JF424376 JF424377 JF424378 JF424379 JF424380 JF424381 JF424382 JF424383 JF424384 JF424385 JF424386 JF424387 JF424388 JF424389 JF424390 JF424391 JF424392 JF424393 JF424394 JF424395

JF424451 JF424452 JF424453 — JF424454 JF424455 JF424456 JF424457 — JF424458 JF424459 JF424460 — JF424461 JF424462 JF424463 JF424464 JF424465 JF424466 JF424467 JF424468 JF424469 JF424470 JF424471

JF424519 JF424520 JF424521 — JF424522 — — — — — — — — JF424523 JF424524 JF424525 — JF424526 — JF424527 JF424528 — — —

JF424557 JF424558 JF424559 — JF424560 — — JF424561 — — — — JF424562 JF424563 JF424564 JF424565 — JF424566 — JF424567 JF424568 — — —

25

EBGR-12233

Venezuela: Gua´rico; Hacienda Picachito, Cerro Platilloń

JF424396

JF424472

JF424529

JF424569

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

KUNHM-111217 EBGR-12235 EBGR-12236 KUNHM-111220 EB17-no voucher EBGR-12232 LSUMNS-6094 LSUMNS-33091 LSUMNS-33119 LSUMNS-39971 LSUMNS-40372 LSUMNS-40322 LSUMNS-8140 FMNH-433259 LSUMNS-22592 LSUMNS-22718 LSUMNS-22825 LSUMNS-7589 LSUMNS-7592 AMNH-RWD 17119 FMNH-339643 ANSP-7465 ANSP-7522 ANSP-8115 KUNHM-3964 KUNHM-4080 USNM-B10604 IAvH-CT 799 IAvH-CT 2168 IAvH-CT 4915 IAvH-CT 4535

Venezuela: Sucre; Las Melenas, Penıńsula de Paria Venezuela: Sucre; Las Melenas, Penıńsula de Paria Venezuela: Sucre; Las Melenas, Penıńsula de Paria Venezuela; Sucre; La Medianıá Venezuela; Sucre; La Medianıá Venezuela; Sucre; La Medianıá Ecuador: Morona Santiago; Cordillera del Cutucu´ Peru: Cajamarca; Ca 3km NNE San Jose´ de Lourdes Peru: Cajamarca; Ca 3km NNE San Jose´ de Lourdes Peru: Loreto; Ca. 86 km SE Juanjui on E bank upper Rıó Pauya Peru: Loreto; Ca. 86 km SE Juanjui on E bank upper Rıó Pauya Peru: Loreto; Ca. 86 km SE Juanjui on E bank upper Rıó Pauya Peru: Pasco; Cushi Peru: Cuzco; Consuelo, 15.9 km SW Pilcopata Bolivia: La Paz, 83 km by road E Charazani, Cerro Asunta Pata Bolivia: La Paz, 83 km by road E Charazani, Cerro Asunta Pata Bolivia: La Paz, 83 km by road E Charazani, Cerro Asunta Pata Venezuela: Amazonas; Cerro Neblina Venezuela: Amazonas; Cerro Neblina Venezuela: Amazonas; Rıó Mawarinumo Venezuela: Bolı´var; Santa Elena Highway, Km 118 Guyana: Potaro-Siparuni; Iwokrama Mountains Guyana: Potaro-Siparuni; Iwokrama Mountains Guyana: Potaro-Siparuni; Iwokrama Mountains Guyana: East Berbice-Corentyne; Acari Mountains, N side Guyana: East Berbice-Corentyne; Acari Mountains, N side Guyana: East Berbice-Corentyne; Acari Mountains, N side Colombia: Caqueta´; La Esmeralda, San Jose´ de Fragua Colombia: Antioquia; Amalfi, Las Animas, Amalfi Colombia: Antioquia; Amalfi, El Encanto Colombia: Antioquia; Anori, El Llano

JF424397 JF424398 JF424399 JF424400 JF424401 JF424402 JF424403 JF424404 JF424405 JF424406 JF424407 JF424408 JF424409 JF424410 JF424411 JF424412 JF424413 JF424414 JF424415 JF424416 JF424417 JF424418 JF424419 JF424420 JF424421 JF424422 JF424423 JF424424 JF424425 JF424426 JF424427

JF424473 JF424474 JF424475 JF424476 JF424477 JF424478 JF424479 JF424480 JF424481 JF424482 JF424483 JF424484 JF424485 JF424486 JF424487 JF424488 JF424489 JF424490 JF424491 JF424492 JF424493 — — JF424494 JF424495 JF424496 JF424497 JF424498 — — JF424499

JF424530 JF424531 — — JF424532 — JF424533 — — — — — — JF424534 — JF424535 — JF424536 JF424537 JF424538 JF424539 — — JF424540 JF424541 JF424542 JF424543 — — JF424544 JF424545

JF424570 JF424571 — — JF424572 — JF424573 — — — — — — JF424574 — JF424575 — JF424576 JF424577 JF424578 JF424579 — — JF424580 JF424581 JF424582 JF424583 — — JF424584 JF424585

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Table 1 (Continued)

Species

No.

Museum and collection number

Locality information

ND2

cytb

bfib7

TGFb2.5

A. haematopygus sexnotatus A. haematopygus sexnotatus A. haematopygus sexnotatus A. haematopygus sexnotatus A. haematopygus sexnotatus A. haematopygus sexnotatus A. haematopygus sexnotatus A. haematopygus sexnotatus A. haematopygus sexnotatus A. haematopygus sexnotatus A. huallagae A. coeruleicinctis A. coeruleicinctis A. coeruleicinctis A. coeruleicinctis A. coeruleicinctis A. coeruleicinctis A. coeruleicinctis A. coeruleicinctis A. coeruleicinctis Outgroup Andigena cucullata Andigena hypoglauca Selenidera gouldii Pteroglossus azara Ramphastos sulfuratus

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76

ANSP-11854 ANSP-11958 ANSP-12105 ANSP-12109 ANSP-12136 ANSP-12072 ANSP-2912 ANSP-3159 ANSP-3400 ANSP-7850 LSUMNS-B48608 LSUMNS-1616 LSUMNS-1647 FMNH-429942 FMNH-429943 FMNH-429944 AMNH-OMZ 131 LSUMNS-34164 LSUMNS-39644 LSUMNS-39649

Ecuador: Esmeraldas; El Placer Ecuador: Esmeraldas; El Placer Ecuador: Pichincha; Mindo Ecuador: Pichincha; Mindo Ecuador: Pichincha; Mindo Ecuador: Pichincha; Mindo Ecuador: El Oro; Machalilla, Cerro San Sebastiań Ecuador: El Oro; Machalilla, Cerro San Sebastiań Ecuador: El Oro; Machalilla, Cerro San Sebastiań Ecuador: El Oro; 9.5 km W Pinãs Peru: La Libertad; Cumpang Peru: Pasco; Santa Cruz, about 9 km SSE Oxapampa Peru: Pasco; Santa Cruz, about 9 km SSE Oxapampa Peru: Cuzco; Pillahuata Peru: Cuzco; Pillahuata Peru: Cuzco; Pillahuata Bolivia: La Paz; Parque Nacional Apolobamba, Franz Tamayo Bolivia: Santa Cruz; Chuchial, Ca 37km, SE Samaipata Bolivia: Santa Cruz; La Pajcha, Ca 28km S Samaipata Bolivia: Santa Cruz; La Pajcha, Ca 37km S Samaipata

JF424428 JF424429 JF424430 JF424431 JF424432 JF424433 JF424434 JF424435 JF424436 JF424437 JF424438 JF424439 JF424440 JF424441 JF424442 JF424443 JF424444 JF424445 JF424446 JF424447

JF424500 JF424501 JF424502 JF424503 JF424504 JF424505 JF424506 — — JF424507 — JF424508 JF424509 JF424510 JF424511 JF424512 — JF424513 JF424514 JF424515

JF424546 — — — — — — — — — JF424547 JF424548 JF424549 — — — JF424550 JF424551 JF424552

JF424586 — — — — — — — — — — JF424587 JF424588 JF424589 — — — JF424590 JF424591 JF424592

LSUMNS-1273 FMNH-433261 FMNH-389772 KUNHM-749 KUNHM-2060

Bolivia: La Paz Peru: Cuzco; La Esperanza, 39 km (road) NE Paucartambo Brazil: Rondonia; Cachoeira Nazare, W bank Rio Jiparana Peru: Madre de Dios Mexico: Campeche

AY959855* — JF424448 JF424449 JF424450

AY959828* — JF424516 JF424517 JF424518

— JF424553 JF424554 JF424555 JF424556

— JF424593 JF424594 JF424595 JF424596

*Sequences from Weckstein 2005. AMNH, American Museum of Natural History; ANSP, Academy of Natural Sciences; COP, Coleccioń Ornitolo´gica Phelps, Venezuela; EBRG, Museo Estacioń Biolo´gica Rancho Grande, Venezuela; FMNH, Field Museum of Natural History; IAvH, Instituto Alexander von Humboldt, Colombia; KUNHM, University of Kansas Natural History Museum; LSUMNS, Louisiana State University Museum of Natural Science; MZFC, Museo de Zoologıá, Facultad de Ciencias, Universidad Nacional Autońoma de Me´xico; ULA, Universidad de los Andes, Venezuela; USNM, National Museum of Natural History, Smithsonian Institution. *Sequences from Weckstein 2005.

Phylogenetic analyses Phylogenetic trees were obtained using maximum parsimony, maximum likelihood (ML), and Bayesian analyses. Prior to conducting model-based analyses, we used Modeltest 3.7 (Posada & Crandall 1998, 2001) to determine the most appropriate model of DNA evolution, using the Akaike information criterion; this procedure was applied to each gene, each data partition (first, second, and third codon positions in mitochondrial genes), and combined datasets (see below). Maximum likelihood trees were estimated using the Genetic Algorithm for Rapid Likelihood Inference (GARLI 0.951; Zwickl 2006), which estimates tree topology, branch lengths, and model parameters that maximize the )ln likelihood, in a simultaneous approach. Analyses were conducted specifying the model ‘family’ obtained by Modeltest, but allowing estimation of parameter values directly from the data. In all analyses, 10 independent runs were completed to assure consistency of likelihood scores among runs. Bootstrap support was assessed via 1000 pseudo replicates, under the same settings used in tree searches. Model parameter val-

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ues estimated in GARLI were used to obtain matrices of ND2 pairwise ML-corrected distances in PAUP v.4.0a109 (Swofford 2009). Because sequencing of A. huallagae resulted in a small fragment [498 base pairs (bp)], we estimated distance values between A. huallagae and all other species based on an ML tree inferred from a pruned matrix of 498 bp. Comparisons of individual-gene ML trees and their non-parametric bootstrap support were used as measures of phylogenetic congruence among genes (Bull et al. 1993; de Queiroz et al. 1995; Wiens 1998); e.g. whenever conflicting topologies were well supported, we considered the potential for gene incongruence. We explored other potential sources of incongruence among genes, such as nucleotide bias among taxa (using a Chi-squared test of homogeneity in PAUP*) and evolutionary rate heterogeneity (using a likelihood-ratio test comparing the likelihood scores of the ML trees with and without enforcing the molecular clock). Final analyses were performed over mitochondrial (ND2 + cytb), nuclear (bfb7 + TGFb2.5F), and combined (mitochondrial + nuclear) datasets, and trees were estimated using ML (with the same settings used in



individual-gene trees), Bayesian, and maximum parsimony analyses. Mitochondrial and nuclear analyses were conducted upon all sequences available, whereas combined analyses were performed on a pruned dataset that included only samples sequenced for all four genes (Table 1). Prior to Bayesian analyses, we performed preliminary short runs (2 · 106 generations) in MrBayes 3.1 (Ronquist & Huelsenbeck 2003) to choose among alternative data partitions, using Bayes factors (BF; Kass & Raftery 1995). Five partition schemes were tested considering mitochondrial and nuclear datasets separately, as follows: (i) two mitochondrial partitions (ND2, cytb); (ii) three mitochondrial partitions by codon (first, second, and third codon positions); (iii) six mitochondrial partitions by gene and codon; (iv) one nuclear partition (Bfib7 and TGF together); and (v) two nuclear partitions (Bfib7 and TGF). The harmonic mean of the log-likelihood values sampled from the stationary phase of the Bayesian runs was used as an estimator of model marginal likelihood (Newton & Raftery 1994). Choosing among models was achieved by calculation of the test statistic 2 ln (BF21), where BF21 is the ratio of marginal likelihoods of competing models; results of these calculations were interpreted following Kass & Raftery (1995). Preliminary analyses were performed assigning each partition its best-fit model of DNA evolution, with all parameters unlinked between partitions (except topology and branch lengths), running four Markov chains (temperature = 0.20) and sampling trees every 1000 generations. Final Bayesian trees were estimated using the best partition schemes identified using BF. Analyses consisted of two independent runs of 10 · 106 generations with the same settings used during preliminary runs. Stationarity was assessed by plotting )lnL per generation in Tracer 1.3 (Rambaut & Drummond 2004) and plotting posterior probabilities of clades as a function of number of generations in AWTY (Wilgenbusch et al. 2004). From the 10 000 resulting trees, the first 2500 were discarded as ‘burn in’; the remaining 7500 were combined to calculate posterior probabilities in a 50% majority-rule consensus tree. Parsimony analyses were conducted in PAUP v.4.0a109 (Swofford 2009), with characters unordered and equally weighted; ambiguities in nuclear genes were coded using degeneracy IUPAC codes and treated as polymorphisms. Heuristic searches for the best tree were conducted running 10 000 stepwise random additions (TBR branch-swapping), and bootstrap support was estimated via heuristic searches with 1000 bootstrap pseudo replicates (Felsenstein 1985), each consisting of 100 stepwise random additions. Hypothesis testing Once the Bayesian tree of the combined dataset was obtained, alternative tree topologies were evaluated based

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on their Bayesian posterior probabilities (Huelsenbeck & Rannala 2004). In practice, this approach consists of taking the post-burn-in trees from the posterior probability distribution, and filtering all trees compatible with each alternative topology (in PAUP). In this case, the percentage of trees retained indicates the posterior probability that the hypothesis is correct [conditional on the model, data, prior probabilities, and convergence of the Markov chain Monte Carlo (MCMC); Huelsenbeck & Rannala 2004]. Alternative hypotheses included topologies compatible with Haffer’s species groups and topologies under the assumption of monophyly of A. derbianus (see Results).

Results Sequence attributes Mitochondrial gene sequences, including those for A. huallagae, showed no patterns suggesting amplification of nuclear pseudogenes (Sorenson & Quinn 1998), i.e. they lacked unexpected stop codons and had patterns of nucleotide substitution and base composition characteristic of protein-coding mtDNA. Although sequence length for mitochondrial genes varied among samples, we obtained an average of 1000 bp for ND2 (range = 754–1041 bp) and 1002 bp for cytb (range = 844–1014 bp). Using ancient DNA techniques, we obtained 498 bp of ND2 sequence data from a single toepad sample of A. huallagae. Overall, nuclear-gene sequence lengths were homogeneous, with most samples having 587 bp for TGFb2.5 and 628 bp for bfb7. Nuclear alignments were unambiguous, showing few autapomorphic (1–2 bp) indels for both genes. Also, two synapomorphic indels (2 bp) were observed for TGFb2.5, the first uniting all Aulacorhynchus and the second uniting all Aulacorhynchus except the A. prasinus complex. Substitution parameters for all genes are summarized in Table 2. Nucleotide composition bias across lineages was nonsignificant for all genes (P = 0.999); clock-like behaviour was rejected for both mitochondrial genes (P = 0.038 for ND2 and P = 0.0072 for cytb), but not for the nuclear genes (P = 0.999). GenBank accession numbers are provided in Table 1 and all data matrices are available in TreeBase (http://purl.org/phylo/treebase/phy lows/study/TB2:S11275). Phylogenetic analyses Maximum likelihood trees of individual loci produced similar topologies, with one exception (see Discussion), and thus were combined in further analyses. Comparison of Bayes factors produced negligible differences between models (2 ln [BF21] 85%). Also, individually or together, nuclear genes showed a sister relationship between the A. prasinus complex and a



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Fig. 2 Bayesian 50% majority rule consensus tree estimated from combined analysis of ND2 and cytb. Bayesian posterior probabilities, maximum likelihood, and maximum parsimony bootstrap values are indicated whenever nodes were recovered with less than 1.00 Bayesian posterior probability or 100% bootstrap support.

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Hypothesis testing Final tree topologies obtained by means of maximum parsimony, maximum likelihood, and Bayesian analysis were inconsistent with the monophyly of Haffer’s A. haematopygus superspecies and in conflict with the current taxonomy of A. derbianus. From the Bayesian perspective, trees showing the monophyly of A. derbianus showed a probability of 0.0017, and no tree from the posterior probability distribution showed a topology consistent with Haffer’s A. haematopygus superspecies, implying that the Bayesian posterior probability of this topology is close to zero. These results indicate that the posterior probabilities of such groups being real are extremely low (given the model, data, prior probabilities, and convergence of the MCMC; Huelsenbeck & Rannala 2004).

Discussion

Fig. 3 Bayesian 50% majority rule consensus tree estimated from the nuclear introns TGFb2.5, and bfb7. Bayesian posterior probabilities, maximum likelihood, and maximum parsimony bootstrap values are indicated whenever nodes were recovered with less than 1.00 Bayesian posterior probability or 100% bootstrap support.

clade containing all other Aulacorhynchus (68 ML bootstrap for TGFb2.5), the node uniting A. derbianus + A. sulcatus (62 ML bootstrap for TGFb2.5), monophyly of A. coeruleicinctis (54 ML bootstrap for bfb7), monophyly of A. derbianus [Andes] (65 ML bootstrap for TGFb2.5), and monophyly of A. derbianus [Pantepui] (64 ML bootstrap for TGFb2.5; 61 ML bootstrap for bfb7). Combining the two nuclear genes, the same relationships were recovered in all phylogenetic reconstructions with higher nodal support (Fig. 3). Combined analyses of mitochondrial and nuclear genes recovered the same major nodes shown by the mitochondrial trees; in addition, the ML and Bayesian trees showed the sister relationship between the A. prasinus complex and all other Aulacorhynchus. To summarize, this key sister relationship was obtained in individual analyses of cytb, bfb7, and TGFb2.5, and was fully consistent with one synapomorphic indel in the alignment of TGFb2.5. 344

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Phylogeny of Aulacorhynchus Combined mitochondrial + nuclear analyses provided a strongly supported hypothesis of relationships for the Aulacorhynchus toucanets. Monophyly of the genus was expected based on morphological similarity and previous mitochondrial DNA analyses (Puebla-Olivares et al. 2008), as well as from myological characters uniting the group (Prum 1988). Also, analysis of multiple samples per species corroborated the monophyly of the A. prasinus complex and placed this lineage as sister to all other Aulacorhynchus. Our results do not support the monophyly of the A. haematopygus superspecies (sensu Haffer 1974), given that A. haematopygus is more closely related to A. sulcatus + A. derbianus than to A. coeruleicinctis + A. huallagae (Fig. 4). The most unexpected results are the paraphyly of A. derbianus and the sister relationship between A. derbianus [Andes] and A. sulcatus. This relationship is puzzling given the disjunct distributions of these two taxa across the northern Andes: A. sulcatus in the northern ranges and Andes of Venezuela and the eastern mountains of Colombia, and A. derbianus [Andes] from northern Ecuador south to Bolivia (Fig. 1). Alternative biogeographic scenarios may imply either long-distance dispersal (highly improbable) or extinction of an ancestral species across the Colombian Andes. A third possibility, an undiscovered population in the Andes of Colombia (discussed in Haffer [1974]) is improbable in light of the conspicuousness of Aulacorhynchus toucanets. More detailed analyses of the biogeography of Aulacorhynchus will be necessary to support any of these possibilities. Taxonomy of Aulacorhynchus In recognizing species, we adhere to the evolutionary species concept, first proposed by Simpson (1961) and



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Fig. 4 Bayesian 50% majority rule consensus tree estimated from the combined, pruned analysis (ND2, cytb, TGF 2.5, and bfb7). Bayesian posterior probabilities, maximum likelihood, and maximum parsimony bootstrap values are indicated whenever nodes were recovered with less than 1.00 Bayesian posterior probability or 100% bootstrap support.

modified by Wiley (1978). This concept incorporates important theoretical factors such as lineage independence, identity, and evolutionary tendencies, and provides a flexible framework for delimitating species when reproductive isolation is difficult to test (e.g. among allopatric populations). Evidence supporting the validity of species may come from different sources (morphology, genetics, behaviour), and no trait alone may be considered a biological property that the species must have to be recognized as such (de Queiroz 2005). Our molecular phylogeny in combination with previous information on geographic distributions and characters of morphology and behaviour, allow us to address the following issues: (i) the taxonomic status of A. sulcatus calorhynchus, (ii) the species-level status of A. huallagae, and (iii) the paraphyly of A. derbianus and the species-level status of A. derbianus [Pantepui].

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Analyses of samples across most of the range of A. sulcatus show that haplotypes of A. s. calorhynchus form a monophyletic group nested well within A. sulcatus. Although monophyly of A. s. calorhynchus may suggest incipient geographic isolation, lack of vocal differentiation, and potential hybridization with other populations (Schwartz 1972) suggest that specific status is not warranted. Regardless of our limited sampling, phylogenetic analyses show A. huallagae as sister to A. coeruleicinctis. Furthermore, sequence divergence between these species is in the order of 0–0.003 substitutions per site. When considering the same mitochondrial fragment, this distance is lower that recorded among samples of A. coeruleicinctis (0–0.005). Although molecular divergence may suggest that A. huallagae could be considered no more than a geographic isolate of A. coeruleicinctis, molecular divergence alone is not sufficient for assessing taxonomic status (de Queiroz 2005).


345

A. prasinus

A. prasinus

A. cognatus A. caeruleogularis

Eastern Panama Costa Rica and western Panama

Goldman’s Blue-throated Toucanet Blue-throated Toucanet

A. prasinus

A. prasinus

A. prasinus

A. prasinus

Eastern Mexico to Nicaragua

Emerald Toucanet

A. wagleri A. cyanolaemus A. albivitta

A. prasinus A. prasinus A. prasinus


A. wagleri A. albivitta A. albivitta

Wagler’s Toucanet White-throated Toucanet

A. lautus A. atrogularis A. dimidiatus



A. A. A. A.

A. caeruleocinctus (sic)

A. coerulei-cinctis (sic)

A. coeruleicinctis

A. coeruleicinctis

n⁄a

A. huallagae

A. huallagae

A. huallagae

A. haematopygius (sic)

A. haematopygus

A. haematopygus

A. haematopygus

A. derbianus

A. derbianus

A. derbianus

A. derbianus

A. whitelyanus (sic)

A. derbianus

A. derbianus

A. whitelianus

Southwestern Mexico Eastern Andes of Colombia to Sierra de Perija´ and Andes of Venezuela Central Andes of Colombia Sierra Santa Marta, Colombia Andes in Ecuador and Peru, and foothills in Peru, Bolivia, and westernmost Mato Grosso. Eastern Andes from central Peru to southeast Bolivia Eastern Andes of central Peru´ (very restricted) Western Venezuela (Sierra de Perija´), Andes of Colombia, and western Andes of Ecuador Eastern Andes from central Bolivia to northern Ecuador Highlands of the Guianan Shield (Venezuela to Surinam)

A. sulcatus A. calorhynchus

A. sulcatus A. calorhynchus

A. sulcatus A. sulcatus

A. sulcatus A. sulcatus

Northeastern Colombia to eastern Venezuela

Groove-billed Toucanet


griseigularis lautus* atrogularis atrogularis

Distribution

Common name, based on Cory (1919) or Meyer de Shauensee (1970)a

Subspecies included sensu Dickinson (2003) and Clements (2007)

A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A. A.

Plumbeus-throated Toucanet Santa Marta Toucanet Black-throated Toucanet

A. coeruleicinctis

Blue-banded Toucanet Yellow-browed Toucanet

prasinus cognatus p. caeruleogularis p. maxillaris p. prasinus p. virescens p. warneri p. chiapensis p. volcanius p. stenorhabdus p. wagleri p. cyanolaemus p. albivitta p. phaeolaeumus? p. griseigularis p. lautus p. atrogularis p. dimidiatus

a

A. huallagae

Crimson-rumped Toucanet

A. h. haematopygus A. h. sexnotatus

Earl of Derby’s Toucanet

A. d. derbianus A. d. nigrirostris** A. derbianus duidae A. d. osgoodi A. d. whitelianus A. s. sulcatus A. s. erythrognathus A. s. calorhynchus

Whitely’s Toucanet


A. caeruleogularis

d

Peters (1948)

*Not included in Puebla-Olivares et al. (2008) or this study. **Only in Dickinson (2003).

Puebla-Olivares et al. (2008) and this contribution

d

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Cory (1919)

Short & Horne (2002), Dickinson (2003), Clements (2007), Remsen et al. (2010)


346 Table 4 Taxa in Aulacorhynchus according to a set of major revisions of the taxonomy and this contribution


This argument is valid, given that genetic distance varies strongly from lineage to lineage because of the different phenomena involved in the divergences at the population level (Whitlock 2003). Therefore, we embrace current taxonomy in recognition of A. huallagae as an independent evolutionary lineage and a valid species, based on its morphological uniqueness, its apparent (not significantly supported) reciprocal monophyly with respect to A. coeruleicinctis, and its restricted and disjunct geographic range. Nevertheless, more data are needed to evaluate the species-level status of A. huallagae. Our well-supported conclusion of paraphyly of A. derbianus indicates that this ‘species’ is not a natural group. Moreover, evidence for substantial independent molecular evolution for A. sulcatus, A. derbianus [Andes], and A. derbianus [Pantepui] at the mitochondrial level (Fig. 2), as well as monophyly of A. derbianus [Andes] and A. derbianus [Pantepui] at the nuclear level (Fig. 3), suggests that these well-differentiated lineages are, in fact, different evolutionary species (sensu Wiley 1978). Patterns of geographic and ecological isolation reinforce this idea, given that the ranges of these three lineages are effectively isolated in the montane forests of north Venezuela and eastern Colombia (A. sulcatus), the Andes from northern Ecuador south to Bolivia (A. derbianus [Andes]), and the Guianan Shield (A. derbianus [Pantepui]). Morphological differences among these lineages are relatively subtle, but each is diagnosable based on different combinations of plumage characters. Populations of A. derbianus [Pantepui] have a washed blue collar that resembles the wider and more defined blue collar of A. haematopygus, A. coeruleicinctis, and A. huallagae; this character is not present in either A. sulcatus or A. derbianus [Andes]. On the other hand, populations of A. derbianus [Andes] possess a prominent blue nuchal patch that is not present in any other Aulacorhynchus. Body mass differences, although overlapping in range, are noteworthy; whereas A. derbianus [Andes] weighs 141– 262 g, and A. sulcatus weighs 150–200 g, A. derbianus [Pantepui] weighs 117–160 g (Short & Horne 2002). Also, qualitative analyses based on song spectrograms show that vocalizations of A. derbianus [Andes] and A. sulcatus differ in both frequency and pattern (Schwartz 1972). Thus, based on the molecular, morphological, and acoustic evidence available, we propose that the name A. derbianus be restricted to populations of the Andean form (A. d. derbianus from central Bolivia to northern Ecuador), and that the Pantepuian populations (A. d. whitelianus, A. d. duidae, and A. d. osgoodi) be treated as a separate species. Given taxonomic priority, A. whitelianus Salvin and Godman (1882) is the appropriate name for the Pantepuian species.

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Finally, our results also contribute to the growing body of evidence supporting the existence of several independent lineages within A. prasinus (NavarroSiguënza et al. 2001; Puebla-Olivares et al. 2008). Data showing that samples of the three Mesoamerican populations analyzed herein (A. p. prasinus, A. p. wagleri, and A. p. caeruleogularis) form a monophyletic group (Fig. 2), provide further evidence for reproductive isolation between Mesoamerican and South American (A. p. albivitta) lineages. A comparative synthesis of the taxonomic history of Aulacorhynchus, including proposals by Puebla-Olivares et al. (2008) and this study, is presented in Table 4. Puebla-Olivares et al. (2008) and this contribution have identified several independently evolving lineages that, we argue, deserve species-level status. Since appropriate recognition of diversity is tightly linked to conservation biology, it is important that these taxonomic proposals are adopted, if appropriately justified. Only in this way, the conservation status of these independent entities may be reassessed.

Acknowledgements We are deeply indebted to all the collectors that made possible this work, and to the following individuals and institutions that granted tissue samples under their care: J. Cracraft and P. Sweet (AMNH); N. Rice (ANSP); M. Lentino (COP); S. Hackett and D. E. Willard (FMNH); J. D. Palacio and C. Villafane (IAcH); R. Brumfield and J. V. Remsen (LSUMNS); and J. Dean (USNM). Many other people contributed significantly to this work, as follows: Jorge Pe´rez-Emań and M. Lentino collected samples of A. prasinus and A. calorhynchus in Sierra de Perija´, N. Malaver granted institutional support for obtaining collection permits for Venezuela (Permit 01-03-03-2306), A. Bonaccorso and C. Rengifo provided valuable logistic support during fieldwork in Venezuela, R. Fleischer extracted DNA from A. huallagae, and N. Rice and M. B. Robbins corroborated identifications of A. derbianus subspecies. Funding for developing diverse stages of this study was obtained from a NSF Dissertation Improvement Grant (DEB-0508910), the AMNH Frank Chapman Memorial Fund, the KU Natural History Museum Panorama Society, Secretarıá Nacional de Ciencia y Tecnologıá of Ecuador (SENACYT: PIC-08-470), and SEMARNATCONACyT Sectorial Funds of Mexico (C01-0265). Special thanks to Instituto Nacional de Parques (INPARQUES), for kindly allowing use of its great infrastructure across Venezuela.

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