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Phylogeny and systematics of Chiroxiphia and Antilophia manakins (Aves, Pipridae)
Molecular Phylogenetics and Evolution 127 (2018) 706–711
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Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev
Phylogeny and systematics of Chiroxiphia and Antilophia manakins (Aves, Pipridae)
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Sofia Marques Silvaa, , Carlos Eduardo Agneb, Alexandre Aleixoa, Sandro L. Bonattob a
Museu Paraense Emílio Goeldi, Department of Zoology, Av. Perimetral, 1901, 66077-530 Belém, PA, Brazil Laboratório de Biologia Genômica e Molecular, Escola de Ciências, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6681, 90619-900 Porto Alegre, RS, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Amazonia Biodiversity Neotropics Paraphyly Speciation Taxonomy
Chiroxiphia and Antilophia manakins are recognized as closely related genera. Nonetheless, Chiroxiphia has been recovered as paraphyletic in some studies with limited taxonomic coverage. This genus currently comprises five species, although this arrangement is still unsettled. Chiroxiphia pareola is the most widespread species, with four recognized subspecies, but their taxonomic status are also uncertain. Finally, the phylogenetic relationships amongst the majority of Chiroxiphia and Antilophia taxa are unknown. Here, we use multilocus DNA sequences from multiple individuals of all currently accepted species and subspecies of both genera to infer their phylogenetic relationships and its implications on their classification. Our results suggest Chiroxiphia, as currently defined, is a paraphyletic group, since C. boliviana is more closely related to Antilophia than to the remaining Chiroxiphia taxa. Within C. pareola, our results support that C. p. regina and C. p. napensis should be treated as independent species. We found three divergent clades in C. p. pareola likely corresponding to distinct subspecies: one in which the isolated and endemic Tobago Island C. p. atlantica individuals are grouped with C. p. pareola from the north bank of the lower Amazon River; and two sister clades comprising individuals distributed south of the Amazon river, and those from the Atlantic Forest.
1. Introduction The closely related genera Chiroxiphia and Antilophia (Aves: Pipridae) (McKay et al., 2010) currently comprise five and two species, respectively (Fig. 1A) (Snow, 2004). Most of these species inhabit lowland humid forests, while A. galeata and C. boliviana are found respectively in gallery forests in Cerrado (a dry biome within Brazil) and montane forests in the Yungas (Snow and de Juana, 2017a, 2017b). As most manakins, females of both genera are olive, but more greenish in Chiroxiphia, and brownish in Antilophia (Snow, 2004); and males present exuberant plumage colors, with bright crowns, and long rectrices (tail feathers) in some species (Snow, 2017a, 2017b, 2017c, 2017d; Snow et al., 2017; Snow and de Juana, 2017b, 2017a). Besides differences in the plumage colors, the division of Chiroxiphia and Antilophia is based on a single morphological character (different shapes of the syrinx cartilaginous A1–B1 bridge) (Prum, 1992). However, the validity of this taxonomic arrangement is not settled yet. To some, Chiroxiphia and Antilophia might correspond to a species complex (Snow, 2004). Moreover, occasional hybridization between C. caudata and A. galeata has been recorded (Pacheco and Parrini, 1995; Rezende et al., 2013). Most phylogenies including species of these genera were ⁎
Corresponding author. E-mail address: sofi[email protected] (S.M. Silva).
https://doi.org/10.1016/j.ympev.2018.06.016 Received 22 February 2018; Received in revised form 22 May 2018; Accepted 6 June 2018
Available online 12 June 2018 1055-7903/ © 2018 Elsevier Inc. All rights reserved.
interested in solving relationships among superior taxonomic ranks, and so only used up to three Chiroxiphia/Antilophia species (Mayr, 2014; McKay et al., 2010; Ohlson et al., 2013; Tello et al., 2009). In one of those molecular phylogenies, C. caudata grouped with A. galeata instead of with C. boliviana (Tello et al., 2009), highlighting the doubts on Chiroxiphia and Antilophia relationships and taxonomy. Among Chiroxiphia species, C. pareola has the most controversial taxonomy, and the widest distribution (Fig. 1A) (Snow, 2017b). Current taxonomy recognizes four subspecies in this species: C. p. pareola, C. p. napensis, C. p. regina, and C. p. atlantica. However, Miller (1908) has considered all these taxa as full-species, and several authors have treated them as a species complex (Kirwan et al., 2011; Miller, 1908). Moreover, C. boliviana was included as a fifth C. pareola subspecies until recently, although morphological, ecological, vocal and displaying differences supported it as a distinct species (Snow and de Juana, 2017b). The status of A. galeata and A. bokermanni as two distinct species has also been questioned. Nonetheless, these seem to represent a monophyletic group made of two recently diverged species (Luna et al., 2017; Rêgo et al., 2010). Here we use multilocus molecular data of multiple individuals
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Fig. 1. A. Geographic distribution of Chiroxiphia and Antilophia species (IUCN, 2017) and C. pareola subspecies. Main Amazon Basin rivers are also depicted. B. Maximum likelihood and Bayesian inference of Chiroxiphia and Antilophia based on mitochondrial and nuclear genes. Node numbers indicate bootstrap support (> 90%) and Bayesian posterior probability (> 0.90) for both analyses with full and subsampled datasets, respectively (see text for details). C. Chronogram estimated for Chiroxiphia and Antilophia species and lineages derived from a species tree Bayesian analysis. Node numbers indicate time estimates in million years, with confidence intervals in brackets. Branch numbers correspond to the posterior probability (> 0.50). Asterisks (*) represent BPP posterior probabilities > 0.90 under the three models tested. Chiroxiphia pareola pareola is divided in N – northern, AF – Atlantic Forest, and S – southern. Light green: C. linearis; dark green: C. lanceolata; dark brown (arrow): C. p. atlantica; brown: C. p. pareola; dark red: C. p. napensis; dark grey: C. p. regina; light grey: C. caudata; dark blue: C. boliviana; light blue: A. bokermanni; and blue: A. galeata. B Numbers represent the localities sampled as described in Table 1, as follows: C. p. regina 1–6; C. p. pareola 7–16; C. p. napensis 18; C. p. atlantica 19–21; C. caudata 22–23; C. boliviana 24–25; C. linearis 26–27; C. lanceolata 28–29; A. bokermanni 30–31; and A. galeata 32. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
representing all species and subspecies of Chiroxiphia and Antilophia to (1) estimate the phylogenetic relationships amongst species; (2) test the monophyly of Chiroxiphia and Antilophia; and (3) infer species limits within the C. pareola complex, and other species with controversial taxonomic statuses.
Terminator Cycle Sequencing Standard Version 3.1 kit in an automated sequencer MegaBACE 1000. Sequences were visually inspected and edited using Geneious™ (Kearse et al., 2012). Sequences were codon aligned using MUSCLE (Edgar, 2004) implemented in Geneious, and no insertion/deletion or stop codons were found.
2. Material and methods
2.2. Phylogenetic reconstruction
2.1. Sampling and genetic protocols
Different approaches were used to infer the phylogenetic relationships amongst Chiroxiphia and Antilophia individuals. Bayesian phylogenetic inferences (BI) were obtained using: (1) the full multilocus dataset; (2) mtDNA sequences only; (3) a reduced multilocus dataset with no missing data, given that some individuals had a relatively large proportion of missing sites (e.g. A. bokermanni; Table 1). Bayesian phylogenetic inferences were obtained using BEAST 1.6.2 (Drummond and Rambaut, 2007) with a partition for each gene. The substitution model for the mtDNA genes was HKY + four gamma categories, while for the nDNA genes the selected model was HKY (Hasegawa et al., 1985; Yang, 1994). We used a molecular strict clock with a substitution rate for the mitochondrial genes following a normal distribution with mean of 0.01105/site/year and a standard deviation of 0.0034 (Weir and Schluter, 2008). The nuclear genes substitution rates were set to be estimated based on the mitochondrial rates. The Yule process of speciation was set as tree prior (Drummond and Rambaut, 2007). Two MCMC independent runs were performed, with 5 × 107 generations, sampling each 5000 generations. Tracer 1.6 (Drummond and Rambaut, 2007) was used to check for convergence and stability of log likelihoods. The maximum clade credibility tree was obtained using TreeAnnotator 1.6.2 (Drummond and Rambaut, 2007) after the first 10% samples were discarded as burn-in.
We generated sequences from all Chiroxiphia species and subspecies, and A. bokermanni (Fig. 1A; Table 1). Antilophia galeata was represented by one specimen already sequenced for the same molecular markers we have used (Table 1) (McKay et al., 2010; Tello et al., 2009). Since Luna et al. (2017) already showed with strong support that Antilophia is monophyletic with two recently diverged species; our sampling effort was focused on Chiroxiphia. As outgroup we used sequences of representatives of the genera Ilicura, Masius, Corapipo, Xenopipo, Manacus and Dixiphia (Table 1) (McKay et al., 2010; Tello et al., 2009). Total DNA was extracted from muscle or feathers, using DNeasy blood & tissue kit (QIAGEN). Two mitochondrial DNA fragments (mtDNA: cytochrome oxidase I, COI and NADH dehydrogenase 2, ND2), and two recombination-activating gene fragments (nDNA: RAG1 and RAG2) were analyzed. COI was amplified following Ivanova et al. (2007), with a M13 tail to aid sequencing procedure, and ND2 following Sorenson et al. (1999), using L5216 and H6313 primers. Amplifications of RAG1 were obtained using primers 17–20, 19–22, 21–24, 23–2b, and for RAG2, primers 1–16 (Barker et al., 2004; Groth and Barrowclough, 1999), following Tello et al. (2009). Products were purified with ExoSAP-IT™ (GE Healthcare), and sequenced with Big Dye 707
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Table 1 List of taxa sampled, and respective museum voucher numbers, locality, GenBank accession numbers, and references in brackets, except for the sequences obtained from this study. Sample number corresponds to localities in Fig. 1. Sample
Taxon
Voucher
Locality
ND2
COI
RAG1
RAG2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Chiroxiphia pareola regina Chiroxiphia pareola regina Chiroxiphia pareola regina Chiroxiphia pareola regina Chiroxiphia pareola regina* Chiroxiphia pareola regina Chiroxiphia pareola pareola Chiroxiphia pareola pareola* Chiroxiphia pareola pareola* Chiroxiphia pareola pareola Chiroxiphia pareola pareola Chiroxiphia pareola pareola* Chiroxiphia pareola pareola Chiroxiphia pareola pareola* Chiroxiphia pareola pareola* Chiroxiphia pareola pareola* Chiroxiphia pareola pareola* Chiroxiphia pareola napensis Chiroxiphia pareola atlantica Chiroxiphia pareola atlantica Chiroxiphia pareola atlantica Chiroxiphia caudata* Chiroxiphia caudata* Chiroxiphia boliviana* Chiroxiphia boliviana Chiroxiphia linearis* Chiroxiphia linearis* Chiroxiphia lanceolata* Chiroxiphia lanceolata Antilophia bokermanni Antilophia bokermanni Antilophia galeata* Manacus manacus Dixiphia pipra Xenopipo sp. Ilicura militaris Masius sp. Corapipo sp.
MPEG 59653 INPA 238 MPEG 62360 MPEG 68986 MPEG 71228 MPEG 60257 INPA 1768 FMNH 391547 FMNH 391546 FMNH 391545 FMNH 427143 MPEG 70494 MZUSP 84531 MPEG 70561 MPEG 61598 FMNH 391549 MZUSP 83685 LSUMZ 5470 FMNH 394508 FMNH 394504 FMNH 394505 MCP 1482 MZUSP 80752 FMNH 433671 LSUMZ 22643 FMNH 434063 FMNH 434067 LSUMZ 26896 LSUMZ 26922 ABO 1B ABO 2 LSUMZ B13809
Brazil, Amazonas, Novo Airão Brazil, Amazonas, RDS Amanã Brazil, Amazonas, Coari Brazil, Pará, Jacareacanga Brazil, Rondônia, Machadinho d'Oeste Brazil, Amazonas, Jutaí Brazil, Roraima, Boa Vista Brazil, Amapá Brazil, Amapá Brazil, Amapá, Tartarugalzinho Brazil, Pernambuco, Mata do Estado Brazil, Alagoas, Ibateguara Brazil, Sergipe, Capela Brazil, Pará, Curuçá Brazil, Mato Grosso, Querência Brazil, Pará, Melgaço Brazil, Pará, Porto de Moz Peru, San Martín Trinidad and Tobago, Tobago Trinidad and Tobago, Tobago Trinidad and Tobago, Tobago Brazil, Rio Grande do Sul Brazil, São Paulo, Cotia Peru, Cusco, Paucartambo Bolívia, La Paz El Salvador, Ahuachapan El Salvador, Sonsonate Panama, Panama Panama, Panama Brazil, Ceará Brazil, Ceará Bolivia, Santa Cruz Department
MH157498 MH157499 MH157500 MH157501 MH157502 MH157503 MH157504 MH157505 MH157506 MH157507 MH157508 MH157509 MH157510 MH157511 MH157512 MH157513 MH157514 MH157515 MH157516 MH157517 MH157518 MH157519 MH157520 MH157521 MH157522 MH157523 MH157524 MH157525 MH157526
MH157528 MH157529 MH157530 MH157531 MH157532 MH157533 MH157534 MH157535 MH157536 MH157537 MH157538 MH157539 MH157540 MH157541 MH157542 MH157543 MH157544 MH157545 MH157546 MH157547 MH157548 MH157549 MH157550 MH157551 MH157552 MH157553 MH157554 MH157555 MH157556
GU985490(1) DQ363987(2) EF501952(4) GU985517(5) AY136621(6) GU985505(1) GU985495(1)
EF111037(1) FJ027769(3) EF111031(1) EF111047(5) JN801754(7) EF111035(1) EF111038(1)
MH157558 MH157559 MH157560 MH157561 MH157562 MH157563 MH157564 MH157565 MH157566 MH157567 MH157568 MH157569 MH157570 MH157571 MH157572 MH157573 MH157574 MH157575 MH157576 MH157577 MH157578 MH157579 MH157580 MH157581 MH157582 MH157583 MH157584 MH157585 MH157586 MH157587 MH157588 FJ501780(1) FJ501664(1) FJ501625(1) FJ501766(1) FJ501645(1) FJ501666(1) FJ501621(1)
MH157590 MH157591 MH157592 MH157593 MH157594 MH157595 MH157596 MH157597 MH157598 MH157599 MH157600 MH157601 MH157602 MH157603 MH157604 MH157605 MH157606 MH157607 MH157608 MH157609 MH157610 MH157611 MH157612 MH157613 MH157614 MH157615 MH157616 MH157617 MH157618 MH157619 MH157620 FJ501780(1) FJ501844(1) FJ501805(1) FJ501946(1) FJ501825(1) FJ501846(1) FJ501801(1)
(1) Tello et al. (2009), (2) Barber et al. (2007), (3) Kerr et al. (2009), (4) Rheindt et al. (2008), (5) McKay et al. (2010), (6) Marini et al. (2002), (7) Tavares et al. (2011). * Samples used in the phylogenetic analysis with less missing data (Fig. S1).
morphology and behavior, were added to BPP results to assign the most valid taxonomic status to the lineages identified as independent. To establish which groups would be tested, we have considered the current taxonomy (Snow, 2004), and the known distribution of the lineages (Fig. 1A) (Snow, 2004). For instance, despite northern C. p. pareola and C. p. atlantica being recovered as paraphyletic in our analyses (Fig. 1B), we tested their validity given that C. p. atlantica is restricted, and isolated within Tobago island, and no gene flow is likely to currently occur between these subspecies (Fig. 1A) (Snow, 2017b). Similarly, A. bokermanni was paraphyletic in our analyses, but this might be due to their recent split from A. galeata (Luna et al., 2017). Therefore, we tested the delimitation of 12 clades: A. galeata, A. bokermanni, C. boliviana, C. caudata, C. p. napensis, C. p. regina, C. p. atlantica, northern Amazonian C. p. pareola, southern Amazonian C. p. pareola, Atlantic Forest C. p. pareola, C. lanceolata and C. linearis. The joint species delimitation and species tree analysis was run after several initial runs to optimize the convergence of the following runs. Convergence of initial tests was assessed using Tracer (Drummond and Rambaut, 2007; Yang, 2015). We chose the rjMCMC (reversible-jump Markov Chain Monte Carlo) analysis, algorithm 0 and ε = 2, for 500,000 generations (sampling interval of five), and a burn-in of 50,000 generations. We tested different scenarios, considering relatively large and small ancestral population sizes, θ ∼ G(1,10) and θ ∼ G(2,2000), respectively, and shallow and deep divergence times, τ ∼ G(2,2000) and τ ∼ G(1,10), respectively. The different inheritance patterns of our
A maximum likelihood (ML) tree based on the full dataset (all individuals) was obtained using RAxML (Stamatakis, 2014), implemented in the CIPRES Science Gateway (Miller et al., 2010). Four partitions were used corresponding to each gene fragment. Other parameters were maintained as the default settings. A majority rule consensus tree was obtained after 200 bootstrap replications. Finally, a species tree with divergence times was estimated, using *BEAST algorithm and the same parameters as explained for the BI tree (Heled and Drummond, 2011). Specimens were assigned to twelve lineages (see next section, and results), which corresponded to known species and subspecies, except for C. p. pareola, which was split into three separate lineages (northern Amazonian, southern Amazonian, and Atlantic Forest) in accordance with our results. 2.3. Lineage delimitation Lineage delimitation within Chiroxiphia and Antilophia was validated using BPP 3.2 (Yang, 2015). BPP (Bayesian Phylogenetics and Phylogeography) uses a multispecies coalescent model, under distinct evolutionary scenarios, to account for gene tree-species tree conflicts, while solving phylogenetic relationships, and describing evolutionary independence between lineages (Yang, 2015). Nonetheless, BPP should be used critically, as it is a robust method to validate phylogeographic structure, but not species limits (Sukumaran and Knowles, 2017). Thus, independent evidences retrieved from the literature, such as 708
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genes (Luna et al., 2017; Rêgo et al., 2010). However, as plumage coloration, song and geographic location clearly differentiate these species (Snow et al., 2017), we agree that the taxonomic and conservation status of A. bokermanni should be maintained, at least until more powerful multilocus datasets are available (BirdLife International, 2016; Luna et al., 2017; Snow et al., 2017). Our results clearly support C. boliviana as a fully independent species (Snow and de Juana, 2017b), well-differentiated from C. pareola, whith which it was sometimes lumped as a subspecies (e.g. Snow, 2004). Furthermore, C. boliviana has a distinct and extreme ecological niche within Pipridae, occurring in cold and drier areas, in the Yungas of the southern Andes (Anciães and Townsend, 2009; Snow and de Juana, 2017b). Although occasionally, C. linearis and C. lanceolata, the only representatives of Chiroxiphia occurring north of the Isthmus of Panama, were considered conspecific with C. pareola (e.g. Snow, 2004). However, their clade diverged from C. pareola around 4 Mya, clearly not supporting that past classification. The sister species relationship between C. linearis and C. lanceolata is corroborated by their vocal and morphological proximity (Haffer, 1987; Miller, 1908; Snow, 2017d, 2017c). Yet, their divergence around 1.5 Mya is consistent with their status as full species, as accepted by most researchers (del Hoyo et al., 2017). The C. p. regina and C. p. napensis clade diverged from the C. p. pareola + C. p. atlantica by about 2.3 Mya, and although their songs and displays are unknown, morphologically they are quite dissimilar between themselves, and from C. p. pareola + C. p. atlantica, mainly by the crown color and size (Snow, 2017b). Furthermore, C. p. regina and C. p. napensis have diverged from each other for roughly the same amount of time (i.e. 1.4 My) as C. linearis and C. lanceolata. Thus, we consider there is enough genetic and morphological support to consider C. regina and C. napensis as separate species (Miller, 1908; Ridgway, 1907). At the intra-specific level, the majority of authors do not recognize C. regina alicei as a valid subspecies, due to great morphological similarity (Kirwan et al., 2011; Snow, 1979). Our results support this arrangement, since all C. regina individuals (specimens 1 and 2 are from the supposed area of occurrence of C. r. alicei) are also genetically very similar. Our results show that individuals of the Tobago endemic C. p. atlantica group with individuals of C. p. pareola from the Guiana shield, north of the Amazon River. Similarly, BPP analyses did not recover them as statistically significant distinct lineages. These subspecies are morphologically very similar, although C. p. atlantica is bigger and present some small differences in the plumage (Snow, 2017b, 1971). Chiroxiphia pareola atlantica is an isolated population that may have originated as recently as the separation of Tobago from Trinidad and the continent, following the sea rise at the end of the last glacial age (Snow, 1985). Similar events were reported for other birds (Höglund and Shorey, 2004). Given its isolated distribution and the presence of some diagnostic morphological characters, we recommend that C. p. atlantica continue to be treated as a distinct subspecies under the polytypic C. pareola. However, the situation with C. p. pareola is more complex. Some BPP models and some phylogenetic analyses failed to recover strong statistical support for the separation of C. p. pareola clades. In addition, their diversification likely occurred more recently than 1 Mya. Nonetheless, the absence of rainforest within central Brazil, and the presence of the Amazon river might represent ecological or pure vicariant barriers for these lineages (Ribas et al., 2012; Thom and Aleixo, 2015). This suggests that three taxa, at least at the subspecies level, may exist in C. p. pareola, one comprising the nominal northern lineage, another within south of the Amazon River, and a third within the Atlantic Forest. However, given the general lack of information on phenotypic and behavioral differences between these groups, we would not propose any new taxonomic arrangement here, but we strongly emphasize the importance of further multi-character studies on them.
molecular markers were considered. Each analysis was run at least twice to confirm consistency of results (Yang, 2015). Some lineages had low sampling sizes (n = 1), but BPP was shown to perform well even for poorly sampled taxa (Yang and Rannala, 2017). 3. Results Final alignments comprised 1583 base pairs (bp) for mtDNA, and 2981 bp for nDNA, totalizing 688 variable and 501 parsimony informative sites. All new sequences were deposited in GenBank (Table 1). Maximum likelihood (ML) and Bayesian (BI) phylogenetic inferences gave essentially identical results, including the BI with mtDNA only and the analysis with fewer terminal taxa (reduced dataset), with only a few topological differences within some minor clades (Fig. 1B; Fig. S1). Most major clades presented the highest support in all phylogenies. Chiroxiphia and Antilophia formed a highly supported monophyletic group. However, Chiroxiphia was not recovered as monophyletic, since C. boliviana and Antilophia, although very divergent, grouped as sister taxa, with high support. All Chiroxiphia species and two C. pareola subspecies were recovered as highly supported and divergent clades. The sequences of the three Antilophia individuals analyzed are extremely similar, and they grouped in an unresolved polytomy. According to the BPP analyses, C. boliviana, C. caudata, C. p. napensis, C. p. regina, C. lanceolata, and C. linearis represent statistically well supported independent evolutionary lineages under all models tested (PP ≥ 0.96; Fig. 1C; Table S1). Southern C. p. pareola, and Atlantic Forest C. p. pareola had lower statistical support under some coalescence scenarios (PP ≥ 0.84). Antilophia galeata and A. bokermanni might represent independent lineages (PP varied between 0.58 and 0.81, among the different models tested), and were less likely to represent a single lineage (0.19 ≤ PP ≤ 0.42). Finally, under some coalescence models, C. p. atlantica and northern C. p. pareola were more likely to represent one than two independent lineages, albeit with no statistical support (0.21 ≤ PP ≤ 0.79). The species tree with divergence time estimated the split between Antilophia + C. boliviana and all the other Chiroxiphia species as the oldest one for the ingroup (∼4.6 Mya) (Fig. 1C). The divergence between C. p. napensis and C. p. regina was very similar to the divergence between C. linearis and C. lanceolata (∼1.4 Mya). The split between the northern C. p. pareola + C. p. atlantica and southern Amazon + Atlantic Forest C. p. pareola was very recent (∼0.69 Mya), and the split between A. bokermanni and A. galeata was even more recent (∼0.43 Mya). 4. Discussion A previous molecular study on Tyrannides (Tello et al., 2009) also recovered Chiroxiphia as paraphyletic (C. caudata was closer to A. galeata than to C. boliviana), but the authors were not confident with this result given its relatively low statistical support. In our much more comprehensive dataset, Chiroxiphia paraphyly is due to C. boliviana being more closely related to Antilophia, although the split between these taxa is very old (∼3 Mya), and our multilocus species tree did not recovered their sister relationship with high statistical support (Fig. 1C). Interestingly, C. boliviana was considered morphologically an “intermediate evolutionary step” for Chiroxiphia towards Antilophia (Miller, 1908). Nonetheless, C. boliviana is morphologically, ecologically, and behaviorally dissimilar from both Antilophia and the other Chiroxiphia species (Snow et al., 2017; Snow and de Juana, 2017b, 2017a). Furthermore, the phylogenetic position of C. caudata is still not fully resolved (see below), and, although unlikely, it could group with the Antilophia + C. boliviana clade or even as the sister clade to all remaining Chiroxiphia and Antilophia species. We confirm that A. galeata and A. bokermanni are very recently diverged species that have not yet reached reciprocal monophyly for most 709
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Finally, the phylogenetic position of C. caudata remains somewhat uncertain. Although in all our phylogenies it was placed as the sister clade to all Chiroxiphia except the C. boliviana + Antilophia clade, the support values for this position varied. However, we confirmed that this species corresponds to an isolated lineage endemic to the Atlantic Forest, and that it diverged from its sister clade around 4.4 Mya. Our comprehensive molecular analyses with multiple individuals per species, from all Chiroxiphia and Antilophia species and subspecies, and nuclear and mtDNA sequences sets the ground to nomenclatural and taxonomic changes within Chiroxiphia that better suits their relationship and diversity. To solve the recovered paraphyly of Chiroxiphia as found here (se also Tello et al., 2009), two main taxonomic changes are possible, Antilophia could be synonymized with Chiroxiphia (the name with priority) or C. boliviana could be transferred to Antilophia. Although we favor the former solution, here we would not recommend any taxonomic change at this moment, as ongoing subgenomic studies might better resolve the few remaining phylogenetic ambiguities recovered in our trees, such as the position of C. caudata, and propose more stable nomenclature changes for these manakins.
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(Eds.), Handbook of the Birds of the World Alive. Barcelona, p. 57082. Snow, D., 2017d. Long-tailed Manakin (Chiroxiphia linearis). In: del Hoyo, J., Elliott, A., Sargatal, J., Christie, D.A., de Juana, E. (Eds.), Handbook of the Birds of the World Alive. Barcelona, p. 57081. Snow, D., 2004. Family Pipridae (Manakins). In: del Hoyo, J., Elliot, A., Christie, D.A. (Eds.), Handbook of the Birds of the World. Barcelona, pp. 110–169. Snow, D., de Juana, E., 2017a. Helmeted Manakin (Antilophia galeata). In: del Hoyo, J., Elliott, A., Sargatal, J., Christie, D.A., de Juana, E. (Eds.), Handbook of the Birds of the World Alive. Barcelona, p. 57079. Snow, D., de Juana, E., 2017b. Yungas Manakin (Chiroxiphia boliviana). In: del Hoyo, J., Elliott, A., Sargatal, J., Christie, D.A., de Juana, E. (Eds.), Handbook of the Birds of the World Alive. Barcelona, p. 57084. Snow, D., de Juana, E., Bonan, A., Sharpe, C.J., 2017. Araripe Manakin (Antilophia bokermanni). 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Acknowledgments We thank the curators and curators' assistants for allowing access to the biological samples under their care: Mario Cohn-Haft and Camila Ribas (INPA), Péricles Sena Rêgo (UFPA – Campus Bragança), Luís Fábio Silveira (MZUSP), Carla Fontana (MCP – PUCRS), Robb T. Brumfield and Donna L. Dittman (LSUMNS), and John Bates and David Willard (FMNH); Alex Lees, Guy Kirwan, Fernando Pacheco, and Marina Anciães for their valuable comments on early versions of this manuscript; and Paola Pulido-Santacruz for laboratory assistance. We also thank the anonymous reviewers whose comments improved this manuscript. The data generated by this research was mostly funded by CNPq and FAPERGS grants to S.L.B. C.E.A. was supported by a scholarship by CAPES, and S.M.S. was supported by a PDJ fellowship from CNPq (150657/2017-0). A.A. is supported by a CNPq research productivity fellowship (# 306843/2016-1). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.ympev.2018.06.016. References Anciães, M., Townsend, P.A., 2009. Ecological niches and their evolution among Neotropical manakins (Aves: Pipridae). J. Avian Biol. 40, 591–604. http://dx.doi. org/10.1111/j.1600-048X.2009.04597.x. Barber, B.R., Rice, N.H., Klicka, J., 2007. Systematics and evolution in the Tityrinae (Passeriformes: Tyrannoidea). Auk 124, 1317–1329. http://dx.doi.org/10.1642/ 0004-8038(2007) 124[1317:SAEITT]2.0.CO;2. Barker, F.K., Cibois, A., Schikler, P., Feinstein, J., Cracraft, J., 2004. Phylogeny and diversification of the largest avian radiation. Proc. Natl. Acad. Sci. U. S. A. 101, 11040–11045. http://dx.doi.org/10.1073/pnas.0401892101. BirdLife International, 2016. Antilophia bokermanni. IUCN Red List Threat. Species. (accessed 2.22.17). del Hoyo, J., Elliott, A., Sargatal, J., Christie, D.A., de Juana, E. (Eds.), 2017. Handbook of the Birds of the World Alive. Lynx Edicions, Barcelona. Drummond, A.J., Rambaut, A., 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7, 214. http://dx.doi.org/10.1186/1471-2148-7-214. Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. http://dx.doi.org/10.1093/nar/ gkh340. Groth, J.G., Barrowclough, G.F., 1999. Basal divergences in birds and the phylogenetic utility of the nuclear RAG-1 gene. Mol. Phylogenet. Evol. 12, 115–123. http://dx.doi. org/10.1006/mpev.1998.0603. Haffer, J., 1987. Biogeography of Neotropical birds. In: Whitmore, T.C., Prance, G.T. (Eds. ), Biogeography and Quaternary History of Tropical America. Oxford, pp. 105–150. Hasegawa, M., Kishino, H., Yano, T., 1985. Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol. 22, 160–174. http://dx.doi.org/10. 1007/BF02101694. Heled, J., Drummond, A.J., 2011. Calibrated tree priors for relaxed phylogenetics and divergence time estimation. Syst. Biol. syr087. http://dx.doi.org/10.1093/sysbio/
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Tello, J.G., Moyle, R.G., Marchese, D.J., Cracraft, J., 2009. Phylogeny and phylogenetic classification of the tyrant flycatchers, cotingas, manakins, and their allies (Aves: Tyrannides). Cladistics 25, 429–467. http://dx.doi.org/10.1111/j.1096-0031.2009. 00254.x. Thom, G., Aleixo, A., 2015. Cryptic speciation in the white-shouldered antshrike (Thamnophilus aethiops, Aves-Thamnophilidae): the tale of a transcontinental radiation across rivers in lowland Amazonia and the northeastern Atlantic Forest. Mol. Phylogenet. Evol. 82, 95–110. http://dx.doi.org/10.1016/j.ympev.2014.09.023. Weir, J.T., Schluter, D., 2008. Calibrating the avian molecular clock. Mol. Ecol. 17, 2321–2328. http://dx.doi.org/10.1111/j.1365-294X.2008.03742.x. Yang, Z., 2015. The BPP program for species tree estimation and species delimitation. Curr. Zool. 61, 854–865. http://dx.doi.org/10.1093/czoolo/61.5.854. Yang, Z., 1994. Maximum likelihood phylogenetic estimation from DNA sequences with variable rates over sites: approximate methods. J. Mol. Evol. 39, 306–314. http://dx. doi.org/10.1007/BF00160154. Yang, Z., Rannala, B., 2017. Bayesian species identification under the multispecies coalescent provides significant improvements to DNA barcoding analyses. Mol. Ecol. 26, 3028–3036. http://dx.doi.org/10.1111/mec.14093.
Handbook of the Birds of the World Alive. Barcelona, p. 57080. Snow, D.W., 1985. Affinities and recent history of the avifauna of Trinidad and Tobago. Ornithol. Monogr. 238–246. http://dx.doi.org/10.2307/40168285. Snow, D.W., 1979. Family Pipridae. In: Traylor Jr., M.A. (Ed.), Check-list of Birds of the World, vol. 8, pp. 245–280. Snow, D.W., 1971. Social organization of the blue-backed manakin. Wilson Bull. 35–38. Sorenson, M.D., Ast, J.C., Dimcheff, D.E., Yuri, T., Mindell, D.P., 1999. Primers for a PCRbased approach to mitochondrial genome sequencing in birds and other vertebrates. Mol. Phylogenet. Evol. 12, 105–114. http://dx.doi.org/10.1006/mpev.1998.0602. Stamatakis, A., 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313. http://dx.doi.org/10.1093/ bioinformatics/btu033. Sukumaran, J., Knowles, L.L., 2017. Multispecies coalescent delimits structure, not species. Proc. Natl. Acad. Sci. 114, 1607–1612. http://dx.doi.org/10.1073/pnas. 1607921114. Tavares, E.S., Gonçalves, P., Miyaki, C.Y., Baker, A.J., 2011. DNA barcode detects high genetic structure within Neotropical bird species. PLoS One 6, e28543. http://dx.doi. org/10.1371/journal.pone.0028543.
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Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev
Phylogeny and systematics of Chiroxiphia and Antilophia manakins (Aves, Pipridae)
T
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Sofia Marques Silvaa, , Carlos Eduardo Agneb, Alexandre Aleixoa, Sandro L. Bonattob a
Museu Paraense Emílio Goeldi, Department of Zoology, Av. Perimetral, 1901, 66077-530 Belém, PA, Brazil Laboratório de Biologia Genômica e Molecular, Escola de Ciências, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6681, 90619-900 Porto Alegre, RS, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Amazonia Biodiversity Neotropics Paraphyly Speciation Taxonomy
Chiroxiphia and Antilophia manakins are recognized as closely related genera. Nonetheless, Chiroxiphia has been recovered as paraphyletic in some studies with limited taxonomic coverage. This genus currently comprises five species, although this arrangement is still unsettled. Chiroxiphia pareola is the most widespread species, with four recognized subspecies, but their taxonomic status are also uncertain. Finally, the phylogenetic relationships amongst the majority of Chiroxiphia and Antilophia taxa are unknown. Here, we use multilocus DNA sequences from multiple individuals of all currently accepted species and subspecies of both genera to infer their phylogenetic relationships and its implications on their classification. Our results suggest Chiroxiphia, as currently defined, is a paraphyletic group, since C. boliviana is more closely related to Antilophia than to the remaining Chiroxiphia taxa. Within C. pareola, our results support that C. p. regina and C. p. napensis should be treated as independent species. We found three divergent clades in C. p. pareola likely corresponding to distinct subspecies: one in which the isolated and endemic Tobago Island C. p. atlantica individuals are grouped with C. p. pareola from the north bank of the lower Amazon River; and two sister clades comprising individuals distributed south of the Amazon river, and those from the Atlantic Forest.
1. Introduction The closely related genera Chiroxiphia and Antilophia (Aves: Pipridae) (McKay et al., 2010) currently comprise five and two species, respectively (Fig. 1A) (Snow, 2004). Most of these species inhabit lowland humid forests, while A. galeata and C. boliviana are found respectively in gallery forests in Cerrado (a dry biome within Brazil) and montane forests in the Yungas (Snow and de Juana, 2017a, 2017b). As most manakins, females of both genera are olive, but more greenish in Chiroxiphia, and brownish in Antilophia (Snow, 2004); and males present exuberant plumage colors, with bright crowns, and long rectrices (tail feathers) in some species (Snow, 2017a, 2017b, 2017c, 2017d; Snow et al., 2017; Snow and de Juana, 2017b, 2017a). Besides differences in the plumage colors, the division of Chiroxiphia and Antilophia is based on a single morphological character (different shapes of the syrinx cartilaginous A1–B1 bridge) (Prum, 1992). However, the validity of this taxonomic arrangement is not settled yet. To some, Chiroxiphia and Antilophia might correspond to a species complex (Snow, 2004). Moreover, occasional hybridization between C. caudata and A. galeata has been recorded (Pacheco and Parrini, 1995; Rezende et al., 2013). Most phylogenies including species of these genera were ⁎
Corresponding author. E-mail address: sofi[email protected] (S.M. Silva).
https://doi.org/10.1016/j.ympev.2018.06.016 Received 22 February 2018; Received in revised form 22 May 2018; Accepted 6 June 2018
Available online 12 June 2018 1055-7903/ © 2018 Elsevier Inc. All rights reserved.
interested in solving relationships among superior taxonomic ranks, and so only used up to three Chiroxiphia/Antilophia species (Mayr, 2014; McKay et al., 2010; Ohlson et al., 2013; Tello et al., 2009). In one of those molecular phylogenies, C. caudata grouped with A. galeata instead of with C. boliviana (Tello et al., 2009), highlighting the doubts on Chiroxiphia and Antilophia relationships and taxonomy. Among Chiroxiphia species, C. pareola has the most controversial taxonomy, and the widest distribution (Fig. 1A) (Snow, 2017b). Current taxonomy recognizes four subspecies in this species: C. p. pareola, C. p. napensis, C. p. regina, and C. p. atlantica. However, Miller (1908) has considered all these taxa as full-species, and several authors have treated them as a species complex (Kirwan et al., 2011; Miller, 1908). Moreover, C. boliviana was included as a fifth C. pareola subspecies until recently, although morphological, ecological, vocal and displaying differences supported it as a distinct species (Snow and de Juana, 2017b). The status of A. galeata and A. bokermanni as two distinct species has also been questioned. Nonetheless, these seem to represent a monophyletic group made of two recently diverged species (Luna et al., 2017; Rêgo et al., 2010). Here we use multilocus molecular data of multiple individuals
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Fig. 1. A. Geographic distribution of Chiroxiphia and Antilophia species (IUCN, 2017) and C. pareola subspecies. Main Amazon Basin rivers are also depicted. B. Maximum likelihood and Bayesian inference of Chiroxiphia and Antilophia based on mitochondrial and nuclear genes. Node numbers indicate bootstrap support (> 90%) and Bayesian posterior probability (> 0.90) for both analyses with full and subsampled datasets, respectively (see text for details). C. Chronogram estimated for Chiroxiphia and Antilophia species and lineages derived from a species tree Bayesian analysis. Node numbers indicate time estimates in million years, with confidence intervals in brackets. Branch numbers correspond to the posterior probability (> 0.50). Asterisks (*) represent BPP posterior probabilities > 0.90 under the three models tested. Chiroxiphia pareola pareola is divided in N – northern, AF – Atlantic Forest, and S – southern. Light green: C. linearis; dark green: C. lanceolata; dark brown (arrow): C. p. atlantica; brown: C. p. pareola; dark red: C. p. napensis; dark grey: C. p. regina; light grey: C. caudata; dark blue: C. boliviana; light blue: A. bokermanni; and blue: A. galeata. B Numbers represent the localities sampled as described in Table 1, as follows: C. p. regina 1–6; C. p. pareola 7–16; C. p. napensis 18; C. p. atlantica 19–21; C. caudata 22–23; C. boliviana 24–25; C. linearis 26–27; C. lanceolata 28–29; A. bokermanni 30–31; and A. galeata 32. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
representing all species and subspecies of Chiroxiphia and Antilophia to (1) estimate the phylogenetic relationships amongst species; (2) test the monophyly of Chiroxiphia and Antilophia; and (3) infer species limits within the C. pareola complex, and other species with controversial taxonomic statuses.
Terminator Cycle Sequencing Standard Version 3.1 kit in an automated sequencer MegaBACE 1000. Sequences were visually inspected and edited using Geneious™ (Kearse et al., 2012). Sequences were codon aligned using MUSCLE (Edgar, 2004) implemented in Geneious, and no insertion/deletion or stop codons were found.
2. Material and methods
2.2. Phylogenetic reconstruction
2.1. Sampling and genetic protocols
Different approaches were used to infer the phylogenetic relationships amongst Chiroxiphia and Antilophia individuals. Bayesian phylogenetic inferences (BI) were obtained using: (1) the full multilocus dataset; (2) mtDNA sequences only; (3) a reduced multilocus dataset with no missing data, given that some individuals had a relatively large proportion of missing sites (e.g. A. bokermanni; Table 1). Bayesian phylogenetic inferences were obtained using BEAST 1.6.2 (Drummond and Rambaut, 2007) with a partition for each gene. The substitution model for the mtDNA genes was HKY + four gamma categories, while for the nDNA genes the selected model was HKY (Hasegawa et al., 1985; Yang, 1994). We used a molecular strict clock with a substitution rate for the mitochondrial genes following a normal distribution with mean of 0.01105/site/year and a standard deviation of 0.0034 (Weir and Schluter, 2008). The nuclear genes substitution rates were set to be estimated based on the mitochondrial rates. The Yule process of speciation was set as tree prior (Drummond and Rambaut, 2007). Two MCMC independent runs were performed, with 5 × 107 generations, sampling each 5000 generations. Tracer 1.6 (Drummond and Rambaut, 2007) was used to check for convergence and stability of log likelihoods. The maximum clade credibility tree was obtained using TreeAnnotator 1.6.2 (Drummond and Rambaut, 2007) after the first 10% samples were discarded as burn-in.
We generated sequences from all Chiroxiphia species and subspecies, and A. bokermanni (Fig. 1A; Table 1). Antilophia galeata was represented by one specimen already sequenced for the same molecular markers we have used (Table 1) (McKay et al., 2010; Tello et al., 2009). Since Luna et al. (2017) already showed with strong support that Antilophia is monophyletic with two recently diverged species; our sampling effort was focused on Chiroxiphia. As outgroup we used sequences of representatives of the genera Ilicura, Masius, Corapipo, Xenopipo, Manacus and Dixiphia (Table 1) (McKay et al., 2010; Tello et al., 2009). Total DNA was extracted from muscle or feathers, using DNeasy blood & tissue kit (QIAGEN). Two mitochondrial DNA fragments (mtDNA: cytochrome oxidase I, COI and NADH dehydrogenase 2, ND2), and two recombination-activating gene fragments (nDNA: RAG1 and RAG2) were analyzed. COI was amplified following Ivanova et al. (2007), with a M13 tail to aid sequencing procedure, and ND2 following Sorenson et al. (1999), using L5216 and H6313 primers. Amplifications of RAG1 were obtained using primers 17–20, 19–22, 21–24, 23–2b, and for RAG2, primers 1–16 (Barker et al., 2004; Groth and Barrowclough, 1999), following Tello et al. (2009). Products were purified with ExoSAP-IT™ (GE Healthcare), and sequenced with Big Dye 707
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Table 1 List of taxa sampled, and respective museum voucher numbers, locality, GenBank accession numbers, and references in brackets, except for the sequences obtained from this study. Sample number corresponds to localities in Fig. 1. Sample
Taxon
Voucher
Locality
ND2
COI
RAG1
RAG2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Chiroxiphia pareola regina Chiroxiphia pareola regina Chiroxiphia pareola regina Chiroxiphia pareola regina Chiroxiphia pareola regina* Chiroxiphia pareola regina Chiroxiphia pareola pareola Chiroxiphia pareola pareola* Chiroxiphia pareola pareola* Chiroxiphia pareola pareola Chiroxiphia pareola pareola Chiroxiphia pareola pareola* Chiroxiphia pareola pareola Chiroxiphia pareola pareola* Chiroxiphia pareola pareola* Chiroxiphia pareola pareola* Chiroxiphia pareola pareola* Chiroxiphia pareola napensis Chiroxiphia pareola atlantica Chiroxiphia pareola atlantica Chiroxiphia pareola atlantica Chiroxiphia caudata* Chiroxiphia caudata* Chiroxiphia boliviana* Chiroxiphia boliviana Chiroxiphia linearis* Chiroxiphia linearis* Chiroxiphia lanceolata* Chiroxiphia lanceolata Antilophia bokermanni Antilophia bokermanni Antilophia galeata* Manacus manacus Dixiphia pipra Xenopipo sp. Ilicura militaris Masius sp. Corapipo sp.
MPEG 59653 INPA 238 MPEG 62360 MPEG 68986 MPEG 71228 MPEG 60257 INPA 1768 FMNH 391547 FMNH 391546 FMNH 391545 FMNH 427143 MPEG 70494 MZUSP 84531 MPEG 70561 MPEG 61598 FMNH 391549 MZUSP 83685 LSUMZ 5470 FMNH 394508 FMNH 394504 FMNH 394505 MCP 1482 MZUSP 80752 FMNH 433671 LSUMZ 22643 FMNH 434063 FMNH 434067 LSUMZ 26896 LSUMZ 26922 ABO 1B ABO 2 LSUMZ B13809
Brazil, Amazonas, Novo Airão Brazil, Amazonas, RDS Amanã Brazil, Amazonas, Coari Brazil, Pará, Jacareacanga Brazil, Rondônia, Machadinho d'Oeste Brazil, Amazonas, Jutaí Brazil, Roraima, Boa Vista Brazil, Amapá Brazil, Amapá Brazil, Amapá, Tartarugalzinho Brazil, Pernambuco, Mata do Estado Brazil, Alagoas, Ibateguara Brazil, Sergipe, Capela Brazil, Pará, Curuçá Brazil, Mato Grosso, Querência Brazil, Pará, Melgaço Brazil, Pará, Porto de Moz Peru, San Martín Trinidad and Tobago, Tobago Trinidad and Tobago, Tobago Trinidad and Tobago, Tobago Brazil, Rio Grande do Sul Brazil, São Paulo, Cotia Peru, Cusco, Paucartambo Bolívia, La Paz El Salvador, Ahuachapan El Salvador, Sonsonate Panama, Panama Panama, Panama Brazil, Ceará Brazil, Ceará Bolivia, Santa Cruz Department
MH157498 MH157499 MH157500 MH157501 MH157502 MH157503 MH157504 MH157505 MH157506 MH157507 MH157508 MH157509 MH157510 MH157511 MH157512 MH157513 MH157514 MH157515 MH157516 MH157517 MH157518 MH157519 MH157520 MH157521 MH157522 MH157523 MH157524 MH157525 MH157526
MH157528 MH157529 MH157530 MH157531 MH157532 MH157533 MH157534 MH157535 MH157536 MH157537 MH157538 MH157539 MH157540 MH157541 MH157542 MH157543 MH157544 MH157545 MH157546 MH157547 MH157548 MH157549 MH157550 MH157551 MH157552 MH157553 MH157554 MH157555 MH157556
GU985490(1) DQ363987(2) EF501952(4) GU985517(5) AY136621(6) GU985505(1) GU985495(1)
EF111037(1) FJ027769(3) EF111031(1) EF111047(5) JN801754(7) EF111035(1) EF111038(1)
MH157558 MH157559 MH157560 MH157561 MH157562 MH157563 MH157564 MH157565 MH157566 MH157567 MH157568 MH157569 MH157570 MH157571 MH157572 MH157573 MH157574 MH157575 MH157576 MH157577 MH157578 MH157579 MH157580 MH157581 MH157582 MH157583 MH157584 MH157585 MH157586 MH157587 MH157588 FJ501780(1) FJ501664(1) FJ501625(1) FJ501766(1) FJ501645(1) FJ501666(1) FJ501621(1)
MH157590 MH157591 MH157592 MH157593 MH157594 MH157595 MH157596 MH157597 MH157598 MH157599 MH157600 MH157601 MH157602 MH157603 MH157604 MH157605 MH157606 MH157607 MH157608 MH157609 MH157610 MH157611 MH157612 MH157613 MH157614 MH157615 MH157616 MH157617 MH157618 MH157619 MH157620 FJ501780(1) FJ501844(1) FJ501805(1) FJ501946(1) FJ501825(1) FJ501846(1) FJ501801(1)
(1) Tello et al. (2009), (2) Barber et al. (2007), (3) Kerr et al. (2009), (4) Rheindt et al. (2008), (5) McKay et al. (2010), (6) Marini et al. (2002), (7) Tavares et al. (2011). * Samples used in the phylogenetic analysis with less missing data (Fig. S1).
morphology and behavior, were added to BPP results to assign the most valid taxonomic status to the lineages identified as independent. To establish which groups would be tested, we have considered the current taxonomy (Snow, 2004), and the known distribution of the lineages (Fig. 1A) (Snow, 2004). For instance, despite northern C. p. pareola and C. p. atlantica being recovered as paraphyletic in our analyses (Fig. 1B), we tested their validity given that C. p. atlantica is restricted, and isolated within Tobago island, and no gene flow is likely to currently occur between these subspecies (Fig. 1A) (Snow, 2017b). Similarly, A. bokermanni was paraphyletic in our analyses, but this might be due to their recent split from A. galeata (Luna et al., 2017). Therefore, we tested the delimitation of 12 clades: A. galeata, A. bokermanni, C. boliviana, C. caudata, C. p. napensis, C. p. regina, C. p. atlantica, northern Amazonian C. p. pareola, southern Amazonian C. p. pareola, Atlantic Forest C. p. pareola, C. lanceolata and C. linearis. The joint species delimitation and species tree analysis was run after several initial runs to optimize the convergence of the following runs. Convergence of initial tests was assessed using Tracer (Drummond and Rambaut, 2007; Yang, 2015). We chose the rjMCMC (reversible-jump Markov Chain Monte Carlo) analysis, algorithm 0 and ε = 2, for 500,000 generations (sampling interval of five), and a burn-in of 50,000 generations. We tested different scenarios, considering relatively large and small ancestral population sizes, θ ∼ G(1,10) and θ ∼ G(2,2000), respectively, and shallow and deep divergence times, τ ∼ G(2,2000) and τ ∼ G(1,10), respectively. The different inheritance patterns of our
A maximum likelihood (ML) tree based on the full dataset (all individuals) was obtained using RAxML (Stamatakis, 2014), implemented in the CIPRES Science Gateway (Miller et al., 2010). Four partitions were used corresponding to each gene fragment. Other parameters were maintained as the default settings. A majority rule consensus tree was obtained after 200 bootstrap replications. Finally, a species tree with divergence times was estimated, using *BEAST algorithm and the same parameters as explained for the BI tree (Heled and Drummond, 2011). Specimens were assigned to twelve lineages (see next section, and results), which corresponded to known species and subspecies, except for C. p. pareola, which was split into three separate lineages (northern Amazonian, southern Amazonian, and Atlantic Forest) in accordance with our results. 2.3. Lineage delimitation Lineage delimitation within Chiroxiphia and Antilophia was validated using BPP 3.2 (Yang, 2015). BPP (Bayesian Phylogenetics and Phylogeography) uses a multispecies coalescent model, under distinct evolutionary scenarios, to account for gene tree-species tree conflicts, while solving phylogenetic relationships, and describing evolutionary independence between lineages (Yang, 2015). Nonetheless, BPP should be used critically, as it is a robust method to validate phylogeographic structure, but not species limits (Sukumaran and Knowles, 2017). Thus, independent evidences retrieved from the literature, such as 708
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genes (Luna et al., 2017; Rêgo et al., 2010). However, as plumage coloration, song and geographic location clearly differentiate these species (Snow et al., 2017), we agree that the taxonomic and conservation status of A. bokermanni should be maintained, at least until more powerful multilocus datasets are available (BirdLife International, 2016; Luna et al., 2017; Snow et al., 2017). Our results clearly support C. boliviana as a fully independent species (Snow and de Juana, 2017b), well-differentiated from C. pareola, whith which it was sometimes lumped as a subspecies (e.g. Snow, 2004). Furthermore, C. boliviana has a distinct and extreme ecological niche within Pipridae, occurring in cold and drier areas, in the Yungas of the southern Andes (Anciães and Townsend, 2009; Snow and de Juana, 2017b). Although occasionally, C. linearis and C. lanceolata, the only representatives of Chiroxiphia occurring north of the Isthmus of Panama, were considered conspecific with C. pareola (e.g. Snow, 2004). However, their clade diverged from C. pareola around 4 Mya, clearly not supporting that past classification. The sister species relationship between C. linearis and C. lanceolata is corroborated by their vocal and morphological proximity (Haffer, 1987; Miller, 1908; Snow, 2017d, 2017c). Yet, their divergence around 1.5 Mya is consistent with their status as full species, as accepted by most researchers (del Hoyo et al., 2017). The C. p. regina and C. p. napensis clade diverged from the C. p. pareola + C. p. atlantica by about 2.3 Mya, and although their songs and displays are unknown, morphologically they are quite dissimilar between themselves, and from C. p. pareola + C. p. atlantica, mainly by the crown color and size (Snow, 2017b). Furthermore, C. p. regina and C. p. napensis have diverged from each other for roughly the same amount of time (i.e. 1.4 My) as C. linearis and C. lanceolata. Thus, we consider there is enough genetic and morphological support to consider C. regina and C. napensis as separate species (Miller, 1908; Ridgway, 1907). At the intra-specific level, the majority of authors do not recognize C. regina alicei as a valid subspecies, due to great morphological similarity (Kirwan et al., 2011; Snow, 1979). Our results support this arrangement, since all C. regina individuals (specimens 1 and 2 are from the supposed area of occurrence of C. r. alicei) are also genetically very similar. Our results show that individuals of the Tobago endemic C. p. atlantica group with individuals of C. p. pareola from the Guiana shield, north of the Amazon River. Similarly, BPP analyses did not recover them as statistically significant distinct lineages. These subspecies are morphologically very similar, although C. p. atlantica is bigger and present some small differences in the plumage (Snow, 2017b, 1971). Chiroxiphia pareola atlantica is an isolated population that may have originated as recently as the separation of Tobago from Trinidad and the continent, following the sea rise at the end of the last glacial age (Snow, 1985). Similar events were reported for other birds (Höglund and Shorey, 2004). Given its isolated distribution and the presence of some diagnostic morphological characters, we recommend that C. p. atlantica continue to be treated as a distinct subspecies under the polytypic C. pareola. However, the situation with C. p. pareola is more complex. Some BPP models and some phylogenetic analyses failed to recover strong statistical support for the separation of C. p. pareola clades. In addition, their diversification likely occurred more recently than 1 Mya. Nonetheless, the absence of rainforest within central Brazil, and the presence of the Amazon river might represent ecological or pure vicariant barriers for these lineages (Ribas et al., 2012; Thom and Aleixo, 2015). This suggests that three taxa, at least at the subspecies level, may exist in C. p. pareola, one comprising the nominal northern lineage, another within south of the Amazon River, and a third within the Atlantic Forest. However, given the general lack of information on phenotypic and behavioral differences between these groups, we would not propose any new taxonomic arrangement here, but we strongly emphasize the importance of further multi-character studies on them.
molecular markers were considered. Each analysis was run at least twice to confirm consistency of results (Yang, 2015). Some lineages had low sampling sizes (n = 1), but BPP was shown to perform well even for poorly sampled taxa (Yang and Rannala, 2017). 3. Results Final alignments comprised 1583 base pairs (bp) for mtDNA, and 2981 bp for nDNA, totalizing 688 variable and 501 parsimony informative sites. All new sequences were deposited in GenBank (Table 1). Maximum likelihood (ML) and Bayesian (BI) phylogenetic inferences gave essentially identical results, including the BI with mtDNA only and the analysis with fewer terminal taxa (reduced dataset), with only a few topological differences within some minor clades (Fig. 1B; Fig. S1). Most major clades presented the highest support in all phylogenies. Chiroxiphia and Antilophia formed a highly supported monophyletic group. However, Chiroxiphia was not recovered as monophyletic, since C. boliviana and Antilophia, although very divergent, grouped as sister taxa, with high support. All Chiroxiphia species and two C. pareola subspecies were recovered as highly supported and divergent clades. The sequences of the three Antilophia individuals analyzed are extremely similar, and they grouped in an unresolved polytomy. According to the BPP analyses, C. boliviana, C. caudata, C. p. napensis, C. p. regina, C. lanceolata, and C. linearis represent statistically well supported independent evolutionary lineages under all models tested (PP ≥ 0.96; Fig. 1C; Table S1). Southern C. p. pareola, and Atlantic Forest C. p. pareola had lower statistical support under some coalescence scenarios (PP ≥ 0.84). Antilophia galeata and A. bokermanni might represent independent lineages (PP varied between 0.58 and 0.81, among the different models tested), and were less likely to represent a single lineage (0.19 ≤ PP ≤ 0.42). Finally, under some coalescence models, C. p. atlantica and northern C. p. pareola were more likely to represent one than two independent lineages, albeit with no statistical support (0.21 ≤ PP ≤ 0.79). The species tree with divergence time estimated the split between Antilophia + C. boliviana and all the other Chiroxiphia species as the oldest one for the ingroup (∼4.6 Mya) (Fig. 1C). The divergence between C. p. napensis and C. p. regina was very similar to the divergence between C. linearis and C. lanceolata (∼1.4 Mya). The split between the northern C. p. pareola + C. p. atlantica and southern Amazon + Atlantic Forest C. p. pareola was very recent (∼0.69 Mya), and the split between A. bokermanni and A. galeata was even more recent (∼0.43 Mya). 4. Discussion A previous molecular study on Tyrannides (Tello et al., 2009) also recovered Chiroxiphia as paraphyletic (C. caudata was closer to A. galeata than to C. boliviana), but the authors were not confident with this result given its relatively low statistical support. In our much more comprehensive dataset, Chiroxiphia paraphyly is due to C. boliviana being more closely related to Antilophia, although the split between these taxa is very old (∼3 Mya), and our multilocus species tree did not recovered their sister relationship with high statistical support (Fig. 1C). Interestingly, C. boliviana was considered morphologically an “intermediate evolutionary step” for Chiroxiphia towards Antilophia (Miller, 1908). Nonetheless, C. boliviana is morphologically, ecologically, and behaviorally dissimilar from both Antilophia and the other Chiroxiphia species (Snow et al., 2017; Snow and de Juana, 2017b, 2017a). Furthermore, the phylogenetic position of C. caudata is still not fully resolved (see below), and, although unlikely, it could group with the Antilophia + C. boliviana clade or even as the sister clade to all remaining Chiroxiphia and Antilophia species. We confirm that A. galeata and A. bokermanni are very recently diverged species that have not yet reached reciprocal monophyly for most 709
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Finally, the phylogenetic position of C. caudata remains somewhat uncertain. Although in all our phylogenies it was placed as the sister clade to all Chiroxiphia except the C. boliviana + Antilophia clade, the support values for this position varied. However, we confirmed that this species corresponds to an isolated lineage endemic to the Atlantic Forest, and that it diverged from its sister clade around 4.4 Mya. Our comprehensive molecular analyses with multiple individuals per species, from all Chiroxiphia and Antilophia species and subspecies, and nuclear and mtDNA sequences sets the ground to nomenclatural and taxonomic changes within Chiroxiphia that better suits their relationship and diversity. To solve the recovered paraphyly of Chiroxiphia as found here (se also Tello et al., 2009), two main taxonomic changes are possible, Antilophia could be synonymized with Chiroxiphia (the name with priority) or C. boliviana could be transferred to Antilophia. Although we favor the former solution, here we would not recommend any taxonomic change at this moment, as ongoing subgenomic studies might better resolve the few remaining phylogenetic ambiguities recovered in our trees, such as the position of C. caudata, and propose more stable nomenclature changes for these manakins.
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Acknowledgments We thank the curators and curators' assistants for allowing access to the biological samples under their care: Mario Cohn-Haft and Camila Ribas (INPA), Péricles Sena Rêgo (UFPA – Campus Bragança), Luís Fábio Silveira (MZUSP), Carla Fontana (MCP – PUCRS), Robb T. Brumfield and Donna L. Dittman (LSUMNS), and John Bates and David Willard (FMNH); Alex Lees, Guy Kirwan, Fernando Pacheco, and Marina Anciães for their valuable comments on early versions of this manuscript; and Paola Pulido-Santacruz for laboratory assistance. We also thank the anonymous reviewers whose comments improved this manuscript. The data generated by this research was mostly funded by CNPq and FAPERGS grants to S.L.B. C.E.A. was supported by a scholarship by CAPES, and S.M.S. was supported by a PDJ fellowship from CNPq (150657/2017-0). A.A. is supported by a CNPq research productivity fellowship (# 306843/2016-1). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.ympev.2018.06.016. References Anciães, M., Townsend, P.A., 2009. Ecological niches and their evolution among Neotropical manakins (Aves: Pipridae). J. Avian Biol. 40, 591–604. http://dx.doi. org/10.1111/j.1600-048X.2009.04597.x. Barber, B.R., Rice, N.H., Klicka, J., 2007. Systematics and evolution in the Tityrinae (Passeriformes: Tyrannoidea). Auk 124, 1317–1329. http://dx.doi.org/10.1642/ 0004-8038(2007) 124[1317:SAEITT]2.0.CO;2. Barker, F.K., Cibois, A., Schikler, P., Feinstein, J., Cracraft, J., 2004. Phylogeny and diversification of the largest avian radiation. Proc. Natl. Acad. Sci. U. S. A. 101, 11040–11045. http://dx.doi.org/10.1073/pnas.0401892101. BirdLife International, 2016. Antilophia bokermanni. IUCN Red List Threat. Species.
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