Systematics of Thraupis (Aves, Passeriformes) reveals an extensive hybrid zone between T. episcopus (Blue-gray Tanager) and T. sayaca (Sayaca Tanager).

The Neotropical avian genus Thraupis (Passeriformes, Thraupidae) currently comprises seven species that are widespread and abundant throughout their ranges. However, no phylogenetic hypothesis with comprehensive intraspecific sampling is available for the group and, therefore, currently accepted species limits remain untested. We obtained sequence data for two mitochondrial (ND2, cyt-b) and three non-coding nuclear (TGFB2, MUSK, and βF5) markers from 118 vouchered museum specimens. We conducted population structure and coalescent-based species-tree analyses using a molecular clock calibration. We integrated these results with morphometric and coloration analyses of 1,003 museum specimens to assess species limits within Thraupis. Our results confirm that Thraupis is a monophyletic group and support its origin in the late Miocene and subsequent diversification during the Pleistocene. However, we found conflicts with previous phylogenies. We recovered Thraupis glaucocolpa to be sister to all other species in the genus, and T. cyanoptera to the remaining five species. Our phylogenetic trees and population structure analyses uncovered phylogeographic structure within Thraupis episcopus that is congruent with geographic patterns of phenotypic variation and distributions of some named taxa. The first genetic and phenotypic cluster in T. episcopus occurs east of the Andes and is diagnosed by the white patch on the lesser and median wing coverts, whereas the second group has a blue patch on the wing and distributes to the west of Colombia's eastern Andes. Finally, we present evidence of hybridization and ongoing gene flow between several taxa at different taxonomic levels and discuss its taxonomic implications.

Introduction

The genus Thraupis (Aves, Thraupidae) is widely distributed across the Neotropics from northern Mexico to the Pampas of Argentina. All species are common and easily observed within their distributions, which include open areas, grasslands, scrub vegetation, crops, settlements, second-growth forests, and even urban areas of densely populated cities. Currently, Thraupis sensu stricto comprises seven species [1, 2]: T. glaucocolpa (Glaucous Tanager), T. cyanoptera (Azure-shouldered Tanager), T. abbas (Yellow-winged Tanager), T. ornata (Golden-chevroned Tanager), T. palmarum (Palm Tanager), T. sayaca (Sayaca Tanager), and T. episcopus (Blue-gray Tanager). Specimens of Thraupis are numerous in natural history museums, permitting a proper evaluation of inter- and intra-specific geographic variation of coloration. Moreover, across the lowlands of South and Central America at least one species of this genus occurs, making it an excellent model for investigating evolutionary and biogeographic questions [3], such as the effect of the Andes and other geographic barriers on speciation and color evolution [e.g., 4–8]. However, an objective and testable phylogenetic hypothesis and a robust definition of species limits are fundamental to address some of these questions.

The taxonomic history of Thraupis has been dynamic and convoluted with subspecies shifting between species, species between genera, and taxa changing from subspecies to species level and vice versa [9, 10]. Recent changes included the movement of two species into resurrected genera–Rauenia bonariensis (Blue-and-yellow Tanager) and Sporothraupis cyanocephala (Blue-capped Tanager)–because molecular phylogenetic evidence showed that they are not closely-related to the remainder species of Thraupis [2, 11–13]. Current phylogenetic evidence suggest that Thraupis is a monophyletic group that is closely related to the genera Chalcothraupis, Poecilostreptus, Stilpnia, Tangara, and Ixothraupis. Nonetheless, the latest available phylogenetic hypothesis of the genus lacks statistical support at some inner nodes, yielding unresolved positions for some species, such as T. cyanoptera [14]. Moreover, T. glaucocolpa, a species often considered as sister to T. sayaca due to coloration similarities, remains unsampled [10, 14].

Most taxonomic problems within Thraupis are concentrated within the group formed by T. episcopus and T. sayaca, hereafter referred to as the episcopus-sayaca complex. This complex contains a total of 17 subspecies (T. episcopus episcopus, T. e. cana, T. e. caesitia, T. e. cumatilis, T. e. nesophila, T. e. ehrenreichi, T. e. berlepschi, T. e. quaesita, T. e. leucoptera, T. e. mediana, T. e. coelestis, T. e. caerulea, T. e. major, T. e. urubambae; T. sayaca sayaca, T. s. boliviana and T. s. obscura) classified within the two species [15]. Moreover, due to its plumage similarity, T. glaucocolpa has historically been considered allied to the episcopus-sayaca complex. After its description, the placement of T. glaucocolpa has oscillated between species and subspecies, albeit always assumed to be closely related to T. sayaca [2, 10, 13, 14, 16]. Hilty [17] suggested that it is best treated as a superspecies with T. episcopus and T. sayaca, but its overlapping distribution with T. episcopus makes this relationship unlikely [2].

The Blue-gray Tanager (T. episcopus) is the species with the largest number of named subspecies, which are recognized based on slight variations of the wing-patch coloration, and blue tones on the chest and back. However, several subspecies have poorly defined geographic limits and are almost unidentifiable. Two major geographic groups are distinguishable within T. episcopus [see 18] based on the coloration of the lesser and median wing coverts (hereafter “wing-patch”), which ranges from white to ultramarine, with some populations showing sky blue and flax flower blue wing-patches [19]. All individuals with white wing-patch are found east of the Andes, whereas most taxa with blue wing-patch are west of the eastern Colombian Andes–including the Cauca and Magdalena valleys, and northern Colombia. However, there are some individuals with intermediate phenotypes in the eastern Andes of Colombia with mixed blue and white wing-patches. Thus, population structure and taxonomic limits within the polytypic T. episcopus have yet to be thoroughly assessed [17, 18]. Several hybridization events have been reported between congeners: namely T. episcopus x T. palmarum, T. episcopus x T. ornata, and T. episcopus x T. sayaca [20]. In areas where T. sayaca and T. episcopus overlap, such as northwestern Bolivia and southeastern Peru [21], the correct identification of either species is extremely challenging because they tend to be phenotypically like one another, more so than in other parts of their ranges. Whether these instances of introgressive hybridization are widespread in those areas of parapatry or it is occasional, remains to be assessed.

Hybridization and introgression are important factors that affect evolution, diversification, and speciation processes. They can lead to different outcomes such as boostering speciation by reinforcement [22], merging taxa, generating new reticulate lineages [23, 24] or even transferring advantageous alleles from one lineage to another [25]. However, the underlying mechanisms leading to either outcome remain partially unclear. Hybridization often occurs in nature between closely-related species, and it has been widely reported in various bird families within songbirds (Passeriformes) [26-32]. Currently, one of the reasons that can lead to hybridization is human-induced habitat transformation [33, 34], which in the episcopus-sayaca complex might be relevant. Both species are largely parapatric and meet along the ecotone between Amazonia and the drier habitats in the Cerrado and Caatinga. Because Amazonian limits are receding due to deforestation, the limits and interactions between species may have been changing.

Here, we integrate molecular and morphological data to assess the current molecular hypothesis of the genus Thraupis. The integration of genotypic, and phenotypic information have led to major changes in systematic and taxonomic classifications, furthering the knowledge about the taxonomic limits across many bird groups [35-41]. Even in the genomic era, the integration of phylogenies based on few loci with morphological and bioacoustics analyses have identified evolutionary lineages with few external morphological differences and proposed novel biogeographical hypotheses [42-47]. Here, we infer a species-level phylogeny of the genus Thraupis, evaluate taxonomic boundaries within the episcopus-sayaca complex, and discuss the implications of interspecific introgressive hybridization for the taxonomy and evolution of Thraupis.

Materials and methods

Ethics statement

All newly collected specimens were obtained under regulations of the Brazilian National Council for the Control of Animal Experimentation (Conselho Nacional de Controle de Experimentação Animal–CONCEA; protocol (#001/2016) and Sistema de Autorização e Informação em Biodiversidade, SISBIO (#10013–4).

Genetic sampling

We sampled a total of 113 vouchered specimens of Thraupis housed at eleven scientific collections (S1A Table in S1 File). We included all species in the genus, with emphasis on the episcopus-sayaca complex (15 of 17 subspecies: Fig 1). The distribution of T. e. ehrenreichi is limited to Huitanaã, on the Purús River [15, 48]. However, specimens analyzed from Acre, Brazil, match the original description of the subspecies and we consider these samples to represent ehrenreichi. Additionally, because the type locality of T. e. major is from Junín, Peru and we had no samples from that locality, we consider that subspecies unsampled. Likewise, we did not have access to samples of T. sayaca boliviana. Finally, five samples were included as outgroups (Paroaria baeri, Tangara chilensis, Tangara mexicana, Ixothraupis punctata, and Stilpnia cayana) for a total of 118 samples. Outgroups were selected based on previously published phylogenies [7, 13].

Fig 1

Location and haplotype networks of 112 genetic samples of Thraupis.

(A) Taxonomic identification and geographic distribution of the genetic samples of Thraupis included in this study. Green and lilac areas show the distribution of T. episcopus and T. sayaca, respectively. Haplotype network of (B) the mitochondrial markers ND2 and cyt-b, (C) TGFB2, (D) MUSK, and (E) BF5 from the tissues referenced in Fig 1A. Red circles in the mitochondrial haplotype network highlight haplotype clusters and were named after the taxa with nomenclatural priority in the cluster but do not represent a single subspecies (i.e., the episcopus group includes several subspecies). Maps in this figure were made using the free software Qgis v.3.10.7 and free data layers from DIVA-GIS (https://www.diva-gis.org/) and distribution polygons from IUCN red list webpage [53, 54]. Tanager illustrations made by Fernando Ayerbe-Quiñones.

We extracted total genomic DNA using the Genomic DNA Mini Kit (Invitrogen) and performed PCR amplification of the autosomal nuclear introns beta-fibrinogen intron 5 (BF5) and transforming Growth Factor Beta 2 intron 5 (TGFB2), the Z-linked muscle receptor tyrosine kinase (MUSK), and the mitochondrial markers cytochrome b (cyt-b) and NADH dehydrogenase 2 (ND2). Primers and protocols are summarized in S1B Table in S1 File. We assembled sequences using references from GenBank [49]. We used Geneious v.9.1.8 [50] to clean low-quality fragments, look for ambiguities, and align sequences using the MUSCLE algorithm with a maximum number of seven iterations. We phased nuclear sequences with heterozygous sites using Seqphase [51, 52]. We deposited sequences in GenBank with accession numbers ON552715 –ON552831, ON552832 –ON552949, OP289858 –OP289974, OP289975 –OP290081 and OP290082–290198. Of the 118 individuals, 15 did not produce sequence data for all five markers (i.e., BF5, TGFB2, MUSK, cyt-b, ND2). The total concatenated alignment length was 3,723 base pairs (bp) and most of the sequences yielded high quality reads for over 95% of the sequence length.

Gene tree and population structure analyses

We built gene trees for each locus using RAxML v.8 [55] in the Cipres Science Gateway site v.3.2 [56]. For the mitochondrial locus (concatenated ND2 and cyt-b) we used Partition Finder2 v.2.1.1 [57] to select the best partition scheme. For all loci, we applied the nucleotide substitution model GTR + Γ and a rapid-bootstrap analysis using 999 replicates to assess nodal support. Also, for each locus, we constructed a median-joining haplotype network using the software PopART [58]. Informed by gene-tree and haplotype network analyses, we used the software STRUCTURE v. 2.3.3 [59] on the concatenated sequences (5 markers: 3,723 bp) of 95 samples (including phased nuclear sequences) within the episcopus-sayaca complex. We ran analyses by varying the number of putative populations (k) from 2 to 6 and running 10 iterations per each k value. We ran each Markov Chain Monte Carlo (MCMC) for a total of 50,000 generations, with a burn-in of 2,500. For each value of k, we selected the outcome with the highest likelihood and applied the method of Evanno et al. to assess the number of ancestral populations (best supported K) [60].

Species tree analysis

To infer a species phylogeny, we conducted a Bayesian coalescent species tree analysis calibrated with a molecular clock using *BEAST [61, 62] on Cipres [56]. Taxa were defined a priori guided by results of network, gene-tree, and population structure analyses. Therefore, we used traditionally recognized species-level taxa as currently defined [2, 13], except for T. episcopus, which was separated into three groups. Also, to avoid violations of the assumption of no migration between species in the multispecies coalescent model [63, 64], we removed those individuals with hybrid ancestries in population structure analyses and intermediate genetic profiles (Table 1). Input data were prepared using BEAUti [65] within BEAST 2 v.2.4.6 [66]. Based on results from PartitionFinder2 [57], we chose GTR + Γ + I as the best substitution model for all loci except for ND2, which used the GTR + Γ substitution model. We ran three independent analyses with unlinked substitution models within partitions. For the mitochondrial locus, we used a strict clock with a clock rate of 0.0105 [67]. For the other three loci we used a log-normal relaxed clock, with the clock rate estimated from the data. We used the Yule model for tree shape and 200 million generations, sampling every 10,000 generations, and a burn-in of 25%. We combined log files with LogCombiner from BEAST2 v.2.4.6 [65] and used Tracer v.1.6.0 [68] to check that all ESS values were higher than 200. We also used LogCombiner to fuse the three species tree files and combine all the posterior probability into a single maximum clade credibility tree. We ran TreeAnnotator v.1.10 implemented in BEAST2 with a burn-in of 25% and a posterior probability limit of 0.5. We visualized tree figures with FigTree v.1.4.3 [69].

Table 1

Specimens with gene tree conflicts between mitochondrial and nuclear markers.

Names in bold indicate specimens that clustered within a clade that differed from plumage-based assignments from museum identifications.

CATALOGUE NUMBER	PLUMAGE-BASED ID	MUSK-Z CLADE	TGFB2 CLADE	ND2 –cyt-b CLADE
MZUSP 90269	T. sayaca	T. sayaca	T. episcopus	T. sayaca
MZUSP 98621	T. sayaca	T. episcopus	T. sayaca	T. episcopus
MZUSP (UFG4362)	T. sayaca	T. sayaca	T. episcopus	T. sayaca
KU 115635	T. episcopus	T. episcopus	T. episcopus	T. sayaca
MZUSP 101479	T. episcopus	T. episcopus	T. episcopus	T. sayaca
LSUMZ 9554	T. episcopus	T. episcopus	T. episcopus	T. sayaca
MSB 27433	T. episcopus	T. episcopus	T. episcopus	T. sayaca
MSB 36846	T. episcopus	T. episcopus	T. episcopus	T. sayaca
MPEG T11969	T. episcopus	T. episcopus	T. episcopus	T. sayaca
MZUSP 107246	T. episcopus	T. episcopus	T. episcopus	T. sayaca

Morphometrics

We measured a total of 1,003 specimens housed at six museum collections (S1C Table in S1 File) covering the geographic distribution of the complex [15]. Using a digital caliper, we measured total culmen (TC), culmen from nares to tip (CN), tarsus length (TS), wing chord (Wing), and tail length (Tail) following standard methods [70, 71]. When available, we obtained body weight from specimen labels. When coordinates were not specified, we used gazetteers to georeference specimens [72-75]. Because weight was the variable with the most missing data, we used a linear regression model between weight and the other measurements from specimens with full information to fill missing weight data. We excluded T. glaucocolpa from morphometric comparisons because it is not closely related to the episcopus-sayaca complex (see results). Moreover, comparisons were made grouping specimens into the resulting categories of the molecular analyses: one for T. sayaca and two within T. episcopus. To assess the variation in multivariate morphometric space, we performed a Principal Component Analysis (PCA), and to assess whether mean morphometric values among groups were significantly different, we conducted a Multivariate Analysis of Variance (MANOVA), with and without missing data replacement. Finally, we conducted a Linear Discriminant Analysis (LDA) to evaluate whether groups were morphometrically diagnosable.

Coloration

We photographed 353 specimens that represent a subset of those used in morphometric analyses (S1D Table in S1 File), covering most of the geographic distribution and the color variation observed in the episcopus-sayaca complex. Also, we had access to photographs of type specimens, most of which were not taken within the following standardized conditions and were only used for visual comparisons (folder C available at figshare.com - S1 folder).

We used a Nikon D800 camera with a lens AF-S NIKKOR 28-300mm f/3.5–5.6G ED VR, a black background, and a grey standard. We standardized the angle of view by using a tripod and placing specimens 35 cm from the lens. We used a shutter speed of 1/250, F 10.0, ISO 200, built-in flash -2.0, and white balance for flash +3.0 B6. Using Photoshop V. 19.0 and a gray card in each picture, we standardized white balance across pictures, making pictures comparable even with different light conditions across different collections. We extracted the HTML color code from the crown, chest, mantle, distal edging of the primary feathers, and the wing patch. We used the HTML code to color the georeferenced points of each specimen. Thus, we ended with a color distribution map for each of the four body parts. Lastly, we quantified the amount of white on the greater coverts with a categorical scale from 0 (no white) to 5 (the maximum amount of white found on T. episcopus) (S1 Fig).

Results

Phylogenetic relationships within Thraupis

Nuclear gene trees were poorly resolved but showed novel patterns (S2 Fig). For instance, in the TGFB2 and MUSK gene trees, most individuals clustered with conspecifics based on the morphological identification of voucher museum specimens. However, three individuals morphologically identified as T. sayaca clustered with individuals identified as T. episcopus (one bird in MUSK and two in TGFB2; S3 and S4 Figs). Unlike nuclear gene trees, the mitochondrial gene tree (ND2 and cyt-b) contained high bootstrap support for most relationships and clustered most samples with conspecifics (S5 Fig). Nevertheless, a total of eight individuals, seven T. episcopus and one T. sayaca were recovered within the other species (Table 1). Additionally, the mitochondrial gene tree recovered T. glaucocolpa as the sister species of all other members in the genus, and the remaining Thraupis species form three clades with unresolved relationships among them. The first clade includes Thraupis cyanoptera only. The second clade comprises Thraupis abbas, T. ornata and T. palmarum, whereas the third clade is formed by T. sayaca and T. episcopus (S5 Fig). Moreover, mitochondrial markers revealed a geographic pattern of genetic structure within T. episcopus, with samples clustering into three groups (Fig 1B and S5 Fig), hereafter referred to as the “episcopus,” “cana” and “quaesita” groups. The quaesita group was recovered as the sister of the other two. The group with the most subspecies and wider distribution is episcopus, which included samples from Amazonia, Llanos, and Trinidad and Tobago. Its distribution coincides with the nominate subspecies episcopus and the subspecies coelestis, leucoptera, major, nesophila, mediana, urubambae, berlepschi, and caerulea. The cana group includes populations from the Magdalena and Cauca valleys and the adjacent Andean slopes, northern Colombia, Central America, and nearby islands, and coincides with the subspecies cana, cumatilis, and caesitia. The last group, quaesita, contains only individuals of the subspecies quaesita, which is found in the Pacific lowlands of western Colombia, Ecuador, and northwestern Peru.

Population structure and species tree analyses recovered the same three genetic groups within T. episcopus but with cana and quaesita as sister taxa (Fig 2A and 2B) recognizing as many as four populations in the episcopus-sayaca complex. However, the Evanno et al.’s method suggests that the maximum number of individual populations is three (best supported K = 3) [60], merging cana and quaesita groups into a single cluster, which are the most similar groups based on plumage. Similarly, the species tree analysis also recovered cana and quaesita as sister lineages.

Fig 2

Phylogenetic relationships within Thraupis and geographic distribution of the genetic structure within the episcopus-sayaca complex.

(A) Phylogenetic relationships of Thraupis. Names in brackets represent subspecies groups, with the name corresponding to the taxon with nomenclatural priority within each genetic cluster (i.e., a single group can include several subspecies). Thickness of the branches is associated with the posterior probability value on the corresponding node. (B) STRUCTURE results of the episcopus-sayaca complex, specimens in group 2 represents the putative hybrids (Table 1), best supported K = 3. (C) Geographic distribution of the episcopus-sayaca complex. Colored circles represent different lineages in the species tree and groups in the STRUCTURE plots. Orange triangles represent genotypically inconsistent specimens (Table 1), presumed to be hybrids between episcopus and sayaca. Color continuum from withe to black represent changes in elevation (withe = 0 m, black ≥ 2500 m). Maps in this figure were made using the free software Qgis v.3.10.7, free data layers from DIVA-GIS (https://www.diva-gis.org/). Tanager illustrations by Fernando Ayerbe-Quiñones.

The species tree analysis recovered T. glaucocolpa as sister to all other congeners, as the mitochondrial gene tree, and T. cyanoptera as sister to the remaining species in the genus. Also, the species tree suggested that Thraupis emerged between 5 and 5.5 million years ago at the Miocene–Pliocene boundary. Finally, all current lineages diversified between the Pliocene and the Pleistocene.

Following results of the population structure and the species tree analyses, we considered T. sayaca as a single group and divided T. episcopus into two groups for morphometric analyses: episcopus–east of the Andes–and cana–west of the eastern Andes. The principal component analysis did not show isolation between any of the groups (S6 Fig). However, we found that mean morphometric values are significantly different, with or without replacing missing data (P<0.001 in both cases). Similarly, linear discriminant analyses showed that all groups clustered together with only 6.1% of individuals identifiable with a 0.9 posterior probability or higher and 19.2% of the individuals identified with a posterior probability of 0.5 or lower. Most specimens clustered toward center of morphometric space (Fig 3).

Fig 3

Linear Discriminant Analysis (LDA) of morphometric traits within the episcopus-sayaca species complex.

Coefficients of linear discriminants for LDA1 are: -6.557557*Weight + 7.451838*CN + 1.987242*CT + 3.975692*TS + 16.869735*Wing + 2.702621*Tail. Coefficients of linear discriminants for LDA2 are: 14.460641*Weight + 1.987242*CN—4.708751*CT + 15.982253*TS—11.342798*Wing—1.458385*Tail. Individuals marked with gray dots were identified with a posterior probability of 0.9 or higher and represent 6.1% of the birds. Individuals marked with black dots were identified with a posterior probability of 0.5 or lower and represent 19.2% of the birds.

The plumage analysis of the episcopus-sayaca species complex recovered three geographic patterns. Within T. episcopus, wing-patch color varies geographically. Birds west of the Andes have a blue wing-patch, whereas populations east of the Andes have a white wing-patch, except for populations from the Llanos in Colombia and Venezuela, and Trinidad and Tobago, which have white, blue, and intermediate wing-patches (Figs 4 and 5). Birds from Trinidad and Tobago have intermediate wing-patches but were more morphologically homogeneous than individuals from continental regions. Moreover, putative genetic intermediates (Table 1) between T. episcopus and T. sayaca also have intermediate wing-patch coloration. Within the episcopus group, the turquoise coloration of the chest and the crown is more iridescent and brighter in western Amazonia and becomes duller away from this region (S7 and S8 Figs), followed by the decrease in the amount of white on the greater wing coverts (S9 Fig). We did not detect any clear geographic pattern in the coloration of the back and primary feathers (S10 and S11 Figs).

Fig 4

Geographic distribution of wing-patch color in the episcopus-sayaca species complex.

Each symbol represents a photographed specimen of T. episcopus (circles), T. sayaca (squares), or intermediate specimens (triangles). Symbol colors denote the actual color of the wing patch, as extracted from photography with the HTML code. Pink crosses represent the type locality of each subspecies. Color scale from black to withe represent changes in elevation (black = 0 m, white ≥ 3000 m). Maps in this figure were made using the free software Qgis v.3.10.7 and free data layers from DIVA-GIS (https://www.diva-gis.org/).

Fig 5

Morphological variation in the episcopus-sayaca complex and a presumed T. episcopus x T. abbas hybrid.

(A) typical specimen of T. episcopus from the western side of the Andes. (B—D) intermediate individuals between western and eastern populations of T. episcopus in Colombia and Venezuela Llanos, showing the color variation of intermediate individuals. (E) Representative bird from eastern of the Andes, although this is the most morphologically variable group. (F—G) specimens of T. episcopus x T. sayaca morphological and molecular hybrids, with intermediate colors in the wing patch. (H). The typical phenotype of T. sayaca, and (I) presumed T. episcopus x T. abbas hybrid next to (J) a typical T. abbas individual.

Discussion

A phylogenetic hypothesis for the genus Thraupis: A story of pervasive hybridization?

Our phylogenetic hypothesis uncovers key aspects of the relationships within Thraupis and confirms some previous phylogenetic assessments [7, 13, 14]. We corroborate that, as currently defined, Thraupis is monophyletic and we provide new insights into the phylogenetic position of previously unsampled taxa. We found that T. glaucocolpa is not sister or closely-related to T. sayaca, as previously thought [14], but rather it is the sister species to all species in the genus. This finding suggests that the plumage similarity between T. glaucocolpa and T. sayaca, which was interpreted as evidence of their close relationship, may reflect either an ancestral plumage pattern retained in both lineages or parallel evolution of plumage coloration.

Although the phylogenetic position of T. glaucocolpa and T. cyanoptera was well supported, we recovered T. abbas with a low posterior probability in the species tree. Possible explanations for this low resolution include incomplete lineage sorting or recent introgression [64] with other Thraupis species. Evidence for hibridization comes from the TGFB2 haplotype network, which nests T. abbas within T. episcopus at this locus (Fig 1C). Moreover, this possibility is further supported by a putative hybrid specimen T. episcopus x T. abbas (Fig 5I), housed at the Museum of Comparative Zoology (MCZ 163160), and collected at Comayagüela, Honduras, where the only two co-ccurring species are T. episcopus and T. abbas. This specimen is entirely purple with the head and back coloration of T. abbas (Fig 5J) but shows only a slight contrast between its head and body and does not have black marks on the lores, as in T. episcopus. Introgression can lead to low resolution across the phylogeny complicating the phylogenetic placement of some taxa. Genomic analyses will provide further insights into the phylogeny of the genus and will inform the extent of past on on-going hybridization between congeners [e.g., 76].

The critical role of hybridization in the evolutionary history of Thraupis is also evident between parapatric populations belonging to sister species. We found that T. episcopus and T. sayaca diverged ca. .5 million years ago and currently hybridize over a broad region of central South America, along the ecotone between Amazonia and the South American Dry Diagonal, where they are parapatric (S12 Fig). There are several specimens showing intermediate traits between the two species (Fig 5F and 5G) that also were confirmed as hybrids by gene trees and population structure analyses (Table 1). Of the ten hybrids reported here, two were collected in central Amazonia in Pará, Brazil (S12 Fig), far from the range of T. sayaca. It is possible that anthropogenic disturbances, such as deforestation, in the Amazon basin created similar environmental conditions to those found across the native range of T. sayaca, and generated opportunities for its dispersal into the distribution of T. episcopus. These hybrids were collected in 2009 in an area bisected by the Trans-Amazonian Highway (Rodovia Transamazônica), whose construction started in the early 1970’s and is a powerful driver of deforestation in central Amazonia [77]. Vegetation transformation from primary forest to open and secondary edge vegetation may have favored the dispersal of T. sayaca into Amazonia. Further analyses based on genomic data will be critical to attain a comprehensive understanding of the history of hybridization along ecotones.

Comments on the taxonomy of T. episcopus and T. sayaca

Based on the unified species concept proposed by de Queiroz [78], which posits that species are “separately evolving metapolulation lineages” that can be delimited by several not obligatory or exclusive species criterion (i.e., reproductive isolation, phenotypically distinguishable, ecologically distinct to mention some), and the results presented herein, we suggest maintaining the species rank for T. episcopus and T. sayaca. Excluding hybrids, adults of both species are morphologically distinct and easily diagnosed by the body and wing-patch coloration (Fig 5). Moreover, these taxa were recovered as two well-supported groups in our phylogenetic and population structure analyses (Fig 2). They tend to occur in different ecosystems and hybrids seem to be restricted to the ecotone between Amazon and the South American Dry Diagonal. At the subspecies level, we suggest taxonomic changes, especially within T. episcopus. First, we recognize two major groups within T. episcopus, one east of the Andes with almost all individuals exhibiting a white wing patch, and a second group west of the Andes with a blue wing patch. We suggest these groups be maintained as a single species given the pervasive hybridization in the llanos of Colombia and Venezuela, as indicated by the common occurrence of intermediate birds showing the full spectrum of wing-patch colors, from white to blue, including violet and lilac, away from populations of the “parental” subspecies. Finally, both our phylogenetic and population structure analyses support the treatment of both wing-patch groups as a single species.

The group east of the Andes comprises a total of ten subspecies; eight of them–T. e. episcopus [9], T. e. coelestis [79], T. e. leucoptera [80], T. e. major [81], T. e. ehrenreichi [82], T. e. caerulea [83], T. e. mediana and T. e. urubambae [84]–have a white wing patch; and two–T. e. nesophila [85, 86] and T. e. berlepschi [87]–have a violet and ultramarine wing patch. We did not find a single discrete character that separates the white wing patch taxa from the nominal form. Instead, we found a clinal color variation (S7–S11 and S13 Figs, and folder A available at figshare.com - S1 folder) of the characters used in the original descriptions, namely the amount of white in the wing, the color of the wing patch, head, chest, and back. The rump color was a common character used in the description of the subspecies. However, it is commonly hidden under the wings of museum specimens, and it was difficult to observe in most specimens. Birds near the Napo region, in western Amazonia, are bluer and with some iridescent feathers on chest and head. They have a white wing patch, the maximum category of white in the greater wing coverts (S9 Fig), a turquoise head that contrasts with the back, a blue iridescent chest, and completely blue wings and tail. Specimens away from this region become duller and with less white on the wing patch and greater coverts (see supplemental material). Furthermore, the hypothesis that different subspecies are intermediate along a continuous cline comes from the original descriptions. Zimmer wrote about T. episcopus mediana: “This form is admittedly intermediate between episcopus and coelestis, but it is relatively consistent over a very extensive range and deserves recognition as a distinct form” and also reported: “There is more tendency toward integration between mediana and episcopus than between mediana and coelestis where the distinction is more abrupt” [84]. Here, we confirm that most subspecies in T. episcopus are points along a continuous cline that perhaps seemed discrete to past authors because of sampling gaps. Thus, because there are no discrete characters that permit the diagnosis of geographic populations from others, we suggest synonymizing all subspecies with white wing-patch into T. e. episcopus.

The taxa with violet and ultramarine colors on the wing patch, T. e. nesophila and T. e. berlepschi, are a special case. Within the continental specimens, it is possible to find birds with all intermediate colors of wing-patch between white and blue across the llanos of Colombia and Venezuela. These birds are classified as T. episcopus nesophila. However, because they are intermediate between two groups and its diagnostic character, the violet-blue wing-patch [85], is not stable, we suggest the continental group should not represent a separate taxon. On the other hand, most specimens from the islands of Trinidad and Tobago have a consistent morphology with almost completely ultramarine wing-patch and rump, and blue iridescent feathers on the chest, distinct from similar specimens from the mainland. This form should be considered a valid subspecies due to its discrete morphology and insular distribution. Since its original description in 1880 [85], the type locality of T. episcopus nesophila is assumed to be Trinidad [10], and so, it is the senior synonym of T. e. berlepschi. It is important to highlight that some specimens from Trinidad may exhibit violet colors on the wing-patch and specimens from the mainland may have similar colorations on the rump, chest, and wing-patch as the island group.

The second group is located west of the Andes and has a prevalent blue wing-patch, being formed by T. episcopus cana [88], T. episcopus quaesita [89], T. episcopus cumatilis [90] and T. episcopus caesitia [91]. Excluding T. e. caesitia–not included in our analyses–we did not find any consistent character that distinguished these subspecies from T. e. cana (folder B available at figshare.com - S1 folder). However, because we found that phylogenetic and population structure analyses recovered T. e. quaesita as a distinct group (Fig 1B), we suggest maintaining this taxon until more comprehensive sampling across the Chocó rainforest is available. Thus, T. e. cana is the senior synonym of T. e. cumatilis, and we suggest maintaining T. e. quaesita and T. e. caesitia, given our incomplete sampling.

Finally, within T. sayaca [9], we did not analyze many specimens of the subspecies T. s. obscura [92] and T. s. boliviana [93]. However, the type specimen of T. s. boliviana was analyzed from high quality photos (Supplemental Material). This specimen was collected near the Bolivia–Peru border–a region where we confirmed four hybrids–and shows a wing patch with almost identical coloration of one of the hybrids we sequenced (Fig 5G). Thus, we consider T. s. boliviana to be intermediate between T. episcopus and T. sayaca, and, therefore, not a valid taxon.

Here, we showed that Thraupis is a genus comprised of seven species and originated at least 5 million years ago near the Miocene–Pliocene boundary. We found evidence that suggests that hybridization has been an important factor in Thraupis evolution, especially between closely related species such as T. episcopus and T. sayaca, and current anthropogenic activity as deforestation can be important factors that modify current hybridization dynamics. Based on our morphological and genetic data, we conclude that T. episcopus and T. sayaca should be treated as a separated species with a maximum of five subspecies. Finally, we want to emphasize the importance of scientific collections on this study and the importance of studying common and abundant species.

Alternative language abstract.

Abstract in Spanish.

(PDF)

Click here for additional data file.

Classification of the amount of white on greater wing coverts in Thraupis episcopus.

Illustration showing the difference of amount of white. Dash line denotes the main rachis of the greater coverts and the black area the amount of white on the feather. Values of 0 represent no white and 5 the maximum amount of white.

(PNG)

Click here for additional data file.

Maximum-likelihood gene tree of the nuclear marker BF5.

Raw tree files at figshare.com–S2 folder.

(PNG)

Click here for additional data file.

Maximum-likelihood gene tree of the nuclear marker MUSK.

Raw tree files at figshare.com–S2 folder.

(PNG)

Click here for additional data file.

Maximum-likelihood gene tree of the nuclear marker TGFB2.

figshare.com–S2 folder.

(PNG)

Click here for additional data file.

Maximum-likelihood gene tree of the concatenate mitochondrial markers ND2 and cyt-b.

(PNG)

Click here for additional data file.

Principal component analyses of morphmetic traits within Thraupis episcopus.

(A) Loadings of the PCA. (B) PCA of the three main groups recovered in genetic analyses. The names episcopus, cana and sayaca were given based on nomenclatural priority in each group. All comprise more than one named subspecies (see main S1 Text).

(PNG)

Click here for additional data file.

Geographic distribution of chest color variation in the episcopus-sayaca species complex.

Each symbol represents a photographed specimen of T. episcopus (circles), T. sayaca (squares) or intermediate specimens (triangles). Symbol colors represent the actual color of the chest, as extracted from photography with the HTML code. Pink crosses denote the type locality of each subspecies. Raw pictures available at figshare.com—S1 folder.

(PNG)

Click here for additional data file.

Geographic distribution of crown color variation in the episcopus-sayaca species complex.

Each symbol represents a photographed specimen of T. episcopus (circles), T. sayaca (squares) or intermediate specimens (triangles). Colors of the symbols reflects the actual color of the crown, as extracted from photography with the HTML code. Pink crosses mark the type locality of each subspecies. Raw pictures available at figshare.com—S1 folder.

(PNG)

Click here for additional data file.

Geographic distribution of amount of white on the wing greater coverts in the Thraupis episcopus.

Each white circle represents a photographed specimen of T. episcopus. The amount of white was estimated using S1 Fig. The size of the with circles represent the amount of white on the wing greater coverts. Individuals with category 0 or no white are not on the map. Raw pictures available at figshare.com—S1 folder.

(TIF)

Click here for additional data file.

Geographic distribution of back color in the episcopus-sayaca species complex.

Each symbol represents a photographed specimen of T. episcopus (circles), T. sayaca (squares) or intermediate specimens (triangles). Colors of the symbols reflects the actual color of the back, as extracted from photography with the HTML code. Pink crosses mark the type locality of each subspecies. Raw pictures available at figshare.com—S1 folder.

(PNG)

Click here for additional data file.

Geographic distribution of primaries color in the episcopus-sayaca species complex.

Each symbol represents a photographed specimen of T. episcopus (circles), T. sayaca (squares) or intermediate specimens (triangles). Colors of the symbols reflects the actual color of the primaries, as extracted from photography with the HTML code. Pink crosses mark the type locality of each subspecies. Raw pictures available at figshare.com—S1 folder.

(PNG)

Click here for additional data file.

Geographic distribution of molecular hybrids and deforestation path connecting to inner Amazon hybrids.

Pink triangles represent the locality of hybrid specimens in Table 1. Green and Purple represents the geographic distribution of T. episcopus and T. sayaca respectively [53, 54]. Black arrows indicate deforestation path that connect the Cerrado grasslands with the hybrid molecular specimens on the middle of the Amazon. Most of the highlighted deforestation path belongs to the Trans-Amazonian Highway.

(PNG)

Click here for additional data file.

Geographic distribution of Thraupis episcous subspecies and coloration examples of collected individuals (See pictures in S1 folder).

Pink crosses mark the type locality of each subspecies. Blue circles with numbers are link with the pictures on S1.

(PNG)

Click here for additional data file.

A Sequenced specimen information; B Protocol and details of Polymerase Chain Reactions; C Morphometric data; D Photographed specimen information.

(XLSX)

Click here for additional data file.

16 Feb 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Tzen-Yuh Chiang

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following financial disclosure:

“DC

2015/22981-9 and 2017/03900-3

FAPESP – Fundação de Amparo à Pesquisa do Estado de São Paulo

https://fapesp.br/en

No

DC

No grant number

The Frank M. Chapman Memorial Fund of the American Museum of Natural History

https://www.amnh.org/research/vertebrate-zoology/ornithology/grants

No”

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide

4. We note that Figure 1, 2 and 4 in your submission contain map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure 1, 2 and 4 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The MS presents unique and novel data on the phylogenomics and the evaluation of the species in Thraupis genus. It also provides new insights in the distribution of the species and their boundaries with morphometric evaluations. Study was well design and the results were presented in detail. So I conclude that this MS could highly contribute to the current knowledge in the era of Thraupis.

Reviewer #2: In this study the authors investigated the systematics of Thraupis and the hybridization of Thraupis episcopus. The manuscript is very interesting and well-written. The study falls into the scope of Plos one, thus it could be published. Before this can be done, a minor revision should be made as specified below.

- I suggest changing the title. The authors found evidence of hybridization between T. episcopus and T. sayaca, hence, “Systematics of Thraupis reveals hybridization of Thraupis episcopus (Blue-gray Tanager) at multiple scales” is not appropriated. It should be hybridization in the genus Thraupis, or between T. episcopus and T. sayaca.

- I suggest to the authors to explore more the impact of the hybridization between the Thraupis species. In the introduction they que cite other examples of hybridization among Passeriformes.

- Can you add in the discussion the divergence time between T. episcopus and T. sayaca?

Reviewer #3: This manuscript investigated the species boundaries of neotropical tanagers with plumage, mitochondrial and nuclear genetic analyses of hundreds of individuals across south America. This is a valuable study and I find the results very interesting with nice figures (though the resolution should be higher). However, the clarity of the writing can be largely improved and streamlined around the main taxonomic adjustment that T. episcopus and T. sayaca should be one species with at most five subspecies. Figures should really guide readers to inspect the plumage, mtDNA, and nuclear genetic evidence to reach this conclusion.

Major concerns:

First, he evidence that T. episcopus and T. sayaca should be a single species should be clearly presented with plumage, mtDNA and nuclear genetic evidence. The mtDNA evidence is almost clear, but authors should highlight the admixture between the species by labeling individuals sampled from T. episcopus and T. sayaca. The nuclear genetic evidence is unclear to me, please highlight this evidence on the phylogenetic tree.

Second, I am not convinced that there are at most five subspecies within this species complex. It depends on what K is the best fit to the data, and there are also possibly unsampled subdivisions.

Here are some minor concerns.

Title: vague, what scales?

Abstract:

(1) most people aren’t familiar with this species complex. Please highlight the part of your results that are inconsistent with the existing taxonomic understanding.

(2) Where is the evidence of population structure within T. episcopus? Did you mean episcopus-sayaca complex? These were already described as different taxonomic units?

Introduction, last sentence, “interspecific gene flow” should be hybridization.

Result

Please report best supported K.

Discussion

- “T. glaucocolpa is not related…” Here the “related” is vague. This shoud be “T. glaucocolpa is not sister species to”?

-

Fig.1 B-E, please label sayaca in the haplotype network. Since the purple cluster contains the green individuals, shouldn’t this be evidence against sayaca and episcopus being distinct species?

very hard to see cyan points on the map, consider either enlarge the dots, or change a color.

Fig.2 font problem, can’t see the text clearly. B, please use consistent color scheme as Fig. 1

Fig. 4, It’s a good idea to plot plumage color the map. The pink crosses are similar to the circles and squares in size, readers could be confused about the colors. Please increase the resolution.

Please try to combine Fig. 4 and Fig. 5, or should type specimens on Fig. 4.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Alparslan YILDIRIM

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

3 Jun 2022

Louisiana State University

119 Foster Hall, Baton Rouge, LA 70803

April 2, 2021

Dear editor and reviewers,

Thank you for your time and comments reviewing this manuscript. We try to improve the manuscript as much as we could following your suggestions. Please follow the individual answers to each comment:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at [links].

Thank you for drawing our attention to this point. Indeed, there were several things out of format. The format is now per PLoS One standards.

2. Thank you for stating the following financial disclosure:

“DC

2015/22981-9 and 2017/03900-3

FAPESP – Fundação de Amparo à Pesquisa do Estado de São Paulo

https://fapesp.br/en

No

DC

No grant numbers

The Frank M. Chapman Memorial Fund of the American Museum of Natural History

https://www.amnh.org/research/vertebrate-zoology/ornithology/grants

No”

Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

No funder had any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. I add this statement in a new Financial Disclosure Statement. Additionally, we add some information form in the Financial Disclosure Statement.

3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide

Yes, I understand that you will hold my manuscript until I submit the sequences. Once the manuscript is accepted, I will upload the sequences to GenBank so accession number can be included in the manuscript.

4. We note that Figure 1, 2 and 4 in your submission contain map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

All images were produced by the authors, I included the following information on the figures with maps:

All maps in the paper were made using the free software Qgis v.3.10.7 and freedata from DIVA-GIS (https://www.diva-gis.org/).

Reviewer #1: The MS presents unique and novel daqta on the phylogenomics and the evaluation of the species in Thraupis genus. It also provides new insights in the distribution of the species and their boundaries with morphometric evaluations. Study was well design and the results were presented in detail. So I conclude that this MS could highly contribute to the current knowledge in the era of Thraupis.

Thank you.

Reviewer #2: In this study the authors investigated the systematics of Thraupis and the hybridization of Thraupis episcopus. The manuscript is very interesting and well-written. The study falls into the scope of Plos one, thus it could be published. Before this can be done, a minor revision should be made as specified below.

- I suggest changing the title. The authors found evidence of hybridization between T. episcopus and T. sayaca, hence, “Systematics of Thraupis reveals hybridization of Thraupis episcopus (Blue-gray Tanager) at multiple scales” is not appropriated. It should be hybridization in the genus Thraupis, or between T. episcopus and T. sayaca.

We accept the suggestion. We changed the title to “Systematics of Thraupis (Aves, Passeriformes) reveals an extensive hybrid zone between T. episcopus (Blue-gray Tanager) and T. sayaca (Sayaca Tanager)”

- I suggest to the authors to explore more the impact of the hybridization between the Thraupis species. In the introduction they que cite other examples of hybridization among Passeriformes.

Purposedly, we avoided talking deeply about hybridization because we are already working on a second paper where address this issue in further detail using genomic data (RADseq). Nonetheless, we decided to include a paragraph almost at the end of the introduction to mention some more information about hybridization in line 125.

Hybridization and introgression are important factors that affect evolution, diversification, and speciation processes. They can lead to different outcomes such as boostering speciation by reinforcement [22], merging taxa, generating new reticulate lineages [23,24] or even transferring advantageous alleles from one lineage to another [25]. However, the underlying mechanisms leading to either outcome remain partially unclear. Hybridization often occurs in nature between closely-related species, and it has been widely reported in various bird families within songbirds (Passeriformes) [26-31]. Currently, one of the reasons that can lead to hybridization is human-induced habitat transformation [32], which in the episcopus-sayaca complex might be relevant. Both species are largely parapatric and meet along the ecotone between Amazonia and the drier habitats in the Cerrado and Caatinga. Because Amazon limits are moving backward due to deforestation, the limits and interactions between species may have been changing.

Furthermore, we developed part of the discussion around the putative role of hybridization in driving the observed patterns.

- Can you add in the discussion the divergence time between T. episcopus and T. sayaca?

We added the information in the sentence that starts in line 542:

We found that T. episcopus and T. sayaca diverged ca.1.5 million years ago and currently hybridize over a broad region of central South America, along the ecotone between Amazonia and the South American Dry Diagonal, where they are parapatric (S12 Fig).

Reviewer #3: This manuscript investigated the species boundaries of neotropical tanagers with plumage, mitochondrial and nuclear genetic analyses of hundreds of individuals across south America. This is a valuable study and I find the results very interesting with nice figures (though the resolution should be higher). However, the clarity of the writing can be largely improved and streamlined around the main taxonomic adjustment that T. episcopus and T. sayaca should be one species with at most five subspecies. Figures should really guide readers to inspect the plumage, mtDNA, and nuclear genetic evidence to reach this conclusion.

Major concerns:

First, he evidence that T. episcopus and T. sayaca should be a single species should be clearly presented with plumage, mtDNA and nuclear genetic evidence.

We do not propose that T. episcopus and T. sayaca should be a single species. On the contrary, on line 559 we explicitly state that “we suggest maintaining the species rank for T. episcopus and T. sayaca.” It is due to their morphological and ecological differences and the presence of a solid genetic structure. Eventhough there is a hybrid zone between these species in the Amazonia-Cerrado ecotone, it seems to be narrow and species integrities elsewhere are mainatined.

The mtDNA evidence is almost clear, but authors should highlight the admixture between the species by labeling individuals sampled from T. episcopus and T. sayaca. The nuclear genetic evidence is unclear to me, please highlight this evidence on the phylogenetic tree.

Trees in the supplemental material already have all individuals’ labels including museum where the sample is from, catalog number and species (same birds in Table 1). Moreover, individuals with mixed ancestry are already highlighted in magenta. The only tree without highlighted individuals was the BF5 tree (S2 Fig) because its low support across almost all the branches we do not consider it as evidence for the introgression. However, we remade FIG S2 highlighting hybrid individuals.

Second, I am not convinced that there are at most five subspecies within this species complex. It depends on what K is the best fit to the data, and there are also possibly unsampled subdivisions.

For the species complex (T. episcopus and T. sayaca), we recognize seven subspecies not five: T. episcopus episcopus, T. episcopus berlepschi, T. episcopus quaesita, T. episcopus cana, T. episcopus caesitia, T. sayaca sayaca and T. sayaca obscura. Thus, it is not clear to us what the comment of the reviewer refers to. However, we suppose that he refers to five subspecies within T. episcopus and the STRUCTURE plot in Fig 2B In that case, the information was not understood correctly.

First, subspecies are discrete morphological geographic units that are not required to be reproductively isolated, otherwise they would be classified as species. Genetic structure and monophyly are not requirements for subspecific treatment, especially when analyzing few molecular markers that are not related with the phenotypically differences [1]. Therefore, we did not base our subspecific classification on molecular data only but rather on diagnosable phenotypic differences among named taxa within obtained genetic clusters.

Second, we believe that results of the STRUCTURE plot (Fig 2B) is misinterpreted. It actually includes all individuals of both T. episcopus and T. sayaca and genetic clusters do not represent subspecies but rather groups of individuals with high levels of shared ancestry. These individuals represent different subspecies i.e., phenotypically diagnosable populations not reproductively isolated among them.

Finally, we are aware that we may have unsampled populations (it is not clear what means by subdivisions) that may alter the levels of resolution of our analyses. However, we would like to highlight that this is the most comprehensive phylogeographic study of the T. episcopus – T. sayaca to date and our sampling is an accurate representation of the distribution of these taxa. Future genomic analyses will provide further insights into the genetic structure of these populations.

Here are some minor concerns.

Title: vague, what scales?

The title has been modified following suggestions by reviewers 2 and 3.

Abstract:

(1) most people aren’t familiar with this species complex. Please highlight the part of your results that are inconsistent with the existing taxonomic understanding.

Thank you for this suggestion. We added the following sentences to the abstract: However, we found conflicts with previous phylogenies. We recovered Thraupis glaucocolpa to be sister to all other species in the genus, and T. cyanoptera to the remaining five species.

(2) Where is the evidence of population structure within T. episcopus? Did you mean episcopus-sayaca complex? These were already described as different taxonomic units?

The structure evidence can be seen in the phylogenetic and haplotype network analyses as well as on maps. Specifically, among the episcopus, cana and quaesita groups (Fig 1B and Fig 2A). These genetic groups are not necessarily taxonomic units formed by only one named taxon but rather represent clusters of several subspecies. We refer to each group using the senior synonym within each group.

To clarifu this point in abstract, we included the following sentence: “Our phylogenetic trees and population structure analyses uncovered phylogeographic structure within Thraupis episcopus that is congruent with geographic patterns of phenotypic variation and distributions of some named taxa.”

Introduction

last sentence, “interspecific gene flow” should be hybridization.

We replaced “interspecific gene flow” for “Introgressive hybridization” in line 123.

Results

Please report best supported K.

We reported the best supported K using the Evanno’s method. However, we made it more explicit in the sentence in line 391: “However, the Evanno et al.'s method suggests that the maximum number of individual populations is three (best supported K=3) [60]”

Discussion

- “T. glaucocolpa is not related…” Here the “related” is vague. This shoud be “T. glaucocolpa is not sister species to”?

We replaced “We found that T. glaucocolpa is not sister or closely-related to T. sayaca” in line 500

-Fig.1 B-E, please label sayaca in the haplotype network. Since the purple cluster contains the green individuals, shouldn’t this be evidence against sayaca and episcopus being distinct species?

We think Fig 1 B-E is clear. The color of the haplotypes is related to the name of the species in Fig 1 A. Purple haplotypes correspond to T. sayaca.

The green dots within the T. sayaca cluster (purple) and the purple dot within T. episcopus correspond to the individuals that are hybrids (exactly same individuals in Table 1). Some of these individuals had intermediate phenotypic characteristics. However, they were classified as T. episcopus because its appearance was closer to it. When mapped, these few birds were located in the contact zone between T. episcopus and T. sayaca in the Amazonia-Cerrado ecotone. A few hybrid individuals in a contact zone are not evidence against sayaca and episcopus being distinct species

very hard to see cyan points on the map, consider either enlarge the dots, or change a color.

Thank you for the comment. We suspect that the reviewer did not have access to the original file. The figure is high resolution and one-page size. All dots are easy to find, even the white ones.

Fig.2 font problem, can’t see the text clearly. B, please use consistent color scheme as Fig. 1

Thank you for the comment. We suspect the reviewer did not have access to the original file. The figure is high resolution and one-page size. We cannot use the same color schemes as in Fig 1. Because we are talking about different taxa. Fig 1 was current species within the genus Thraupis, while Fig 2 are the genetically structured population within Thraupis episcopus.

Fig. 4, It’s a good idea to plot plumage color the map. The pink crosses are similar to the circles and squares in size, readers could be confused about the colors. Please increase the resolution.

Thank you. We suspect the reviewer did not have access to the original file. The figure is high resolution and one-page size.

Please try to combine Fig. 4 and Fig. 5, or should type specimens on Fig. 4.

We tried to combine Fig 4 and 5 in different ways before the first submission. However, the figure looked overcharged and confusing. After trying different alternatives, we decided to include proceed with two separate figures. The geographic distribution of the plumages is better seen on Fig 1 and 2, S13 Fig and pictures in S1 folder.

Sincerely,

Diego Cueva, M.Sc.

Ph.D. Student – Louisiana State University

dacuevac@alumni.usp.br

On behalf of all co-authors

Submitted filename: Response to Reviewers_GAB.docx

Click here for additional data file.

20 Jun 2022

Systematics of Thraupis (Aves, Passeriformes)reveals an extensive hybrid zone between T. episcopus (Blue-gray Tanager) and T. sayaca (Sayaca Tanager)

PONE-D-21-37811R1

Dear Dr. Cueva ,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Tzen-Yuh Chiang

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The quality of the MS is now increased by addressing each comment indicated by reviewers. I think the MS could be published in PLOS One.

Reviewer #2: Dear Authors, I am happy with the new version of your manuscript. It can be accepted for publication now.

Best wishes

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

26 Sep 2022

PONE-D-21-37811R1

Systematics of Thraupis (Aves, Passeriformes) reveals an extensive hybrid zone between T. episcopus (Blue-gray Tanager) and T. sayaca (Sayaca Tanager)

Dear Dr. Cueva:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Tzen-Yuh Chiang

Academic Editor

PLOS ONE