Extensive hybridization between two Andean warbler species with little divergence in mtDNA

A thesis submitted to the Department of Biological Sciences of the Universidad de los Andes in partial fulfillment of the requirements for the degre of Master of Biological Sciences

By Laura N. Céspedes Arias January 2018

Advisor: Daniel Cadena, PhD Full Professor, Universidad de los Andes

Co-advisor: Andrés M. Cuervo, PhD Scientific Director of Biological Collections, Instituto de Investigación de Recursos Biológicos Alexander von Humboldt

1 ABSTRACT: Studying processes occurring after closely related taxa achieve secondary contact, such as hybridization, is important to have a comprehensive understanding on how species are formed and maintained over time. However, studying these processes has been largely overlooked in studies of avian speciation in the Andes, which have largely focused on understanding the role of topographic and ecological barriers promoting divergence in allopatry. In this study I characterized a hybrid zone between two closely related Andean birds, Myioborus ornatus and Myioborus melanocephalus, using genetic, coloration, and song data. Geographic ranges of these species abut around the Colombia-Ecuador border and multiple specimens from the region exhibit intermediate phenotypes suggesting hybridization, but descriptions of patterns of variation were heretofore lacking. I conducted fieldwork to collect specimens and obtain recordings of vocalizations across a ca. 400 km transect around the Colombia-Ecuador border and in localities away from the putative hybrid zone. I described variation in head color pattern, ventral coloration and tail pattern using specimens. To describe acoustic variation, I measured spectral and temporal parameters from song recorded in the field and archived in sound libraries. To describe genetic variation, I sequenced the mitochondrial gene ND2. I extended genetic sampling to encompass all main regions where these species occur, from Venezuela to Bolivia, to describe phylogeographic structure. The hybrid zone is characterized by low genetic structure and is ~200 km wide based on head coloration. Intermediate plumage phenotypes are the most common across the hybrid zone, and parental forms do not overlap geographically, suggesting extensive hybridization. Acoustic divergence is subtle and song duration is the only song parameter that varies across the sampling transect. Genetic variation coincides with at least one topographic break along the Andes but geographic variation in plumage was not always associated with genetic structure within the M. ornatus-M. melanocephalus complex. Specimens from the hybrid zone and Ecuadorian populations of M. melanocephalus belong to the same genetic group as M. ornatus, and there is low genetic structure within this group characterized by high diversity in plumage phenotypes. Patterns of variation in plumage suggest that selection against hybrids is not strong, a hypothesis that should be tested in forthcoming studies using genomic data.

2 INTRODUCTION When populations become geographically isolated, genetic and phenotypic differences tend to accumulate, potentially resulting in divergence in traits relevant for reproductive isolation (Coyne and Orr 2004, Price 2008). If formerly isolated populations come into contact and reproductive barriers are incomplete, then hybridization will occur (Harrison 1993), with potentially different outcomes (Abbott et al. 2013). If isolation is incomplete and strong selection against hybrids does not exist, then incipient reproductive barriers can break down leading to the collapse of differentiated populations into one (Taylor et al. 2006); alternatively, such barriers may strengthen via reinforcement (Liou and Price 1994, Ortiz-Barrientos et al. 2009, Hudson and Price 2014). Hybridization can be a transient phenomenon resulting in either of the two alternative scenarios above, or it may persist over extended periods of time (Harrison 1986) if there is a balance between gene flow between parental populations and selection against hybrids (Slatkin 1973, Barton 1979, Barton and Hewitt 1985) or if selection favors hybrids in the area where they occur (Moore 1977, Wang et al. 1997, Good et al. 2000). Studying hybrid zones is therefore especially illuminating for understanding how species barriers are formed and maintained over time (Harrison 1993, Harrison and Larson 2016).

The tropical Andes are a biodiversity hotspot for multiple taxa (Brummitt and Lughadha 2003) and, notably, for birds (Stotz et al. 1996, Hawkins et al. 2007, Fjeldså et al. 2012). The rich Andean avifauna is characterized by pervasive geographic variation in plumage (Remsen 1984a, Graves 1988) and strong genetic structure within species, with phenotypic and genetic variation often coinciding with physical barriers (Bonaccorso 2009, Chaves et al. 2011, Gutiérrez-Pinto et al. 2012, Valderrama et al. 2014). Therefore, avian diversification in the Andes is thought to have predominantly resulted from allopatric speciation (García-Moreno and Fjeldså 2000, Weir 2009, Caro et al. 2013). Because Andean birds tend to have linear and elevationally restricted distributions, their ranges can be readily fragmented by gaps of unsuitable habitat (e.g. dry valleys for cloud forest species) or become disjunct due to local extinctions, leading to differentiation among isolated populations (Graves 1985, 1988). However, allopatric distributions

3 caused by topographic or climatic barriers or by local extinctions are often not stable over time. For example, elevational movements of vegetation belts due to climatic fluctuations (Haffer 1974, Hooghiemstra and Van der Hammen 2004, Cárdenas et al. 2011) have likely promoted cycles of fragmentation and reconnection of suitable habitat for Andean species, potentially leading to secondary contact between diverging populations (Vuilleumier 1969, Graves 1982). Although we have a growing understanding of how isolation across barriers contributes to phenotypic and genetic differentiation in the montane Neotropics, we know little about processes occurring when geographic barriers are overcome and closely related lineages achieve secondary contact. This is an prominent gap of current knowledge of Andean diversification because understanding processes acting upon secondary contact is key to have a comprehensive understanding of the historical build-up of the rich avifauna of this region (Cadena 2007, Dubay and Witt 2014, Chattopadhyay et al. 2017, Winger 2017).

Because birds employ vocal and plumage signals for species recognition (e.g., Lanyon 1963, Uy et al. 2009a, McEntee 2014), the study of such traits is key to understand how isolating barriers are established during avian speciation (Price 2008). In the context of allopatric differentiation, evidence suggests that genetic divergence -a surrogate for time spent in isolation- strongly predicts differentiation in plumage coloration and song (Winger and Bates 2015). A few recent studies focusing on Andean taxa have provided insights on the mechanisms driving divergence in plumage coloration in allopatry, suggesting an important role for social or sexual selection (Parra 2010, Cadena et al. 2011, Winger and Bates 2015). However, information on patterns of variation in plumage color and in song in taxa that hybridize may provide additional, complementary insights about the role of such signals for communication and reproductive isolation (Stein and Uy 2006, Greig and Webster 2013, Baldassarre et al. 2014). Therefore, studying variation in communication-related traits in taxa that have come upon secondary contact along the Andes can contribute to the ongoing body of work on avian speciation, which has largely focused on describing geographic variation, mostly in plumage (Remsen 1984a, b; Graves 1985), and more recently but less

4 frequently on understanding microevolutionary mechanisms underlying its origin (Parra 2010, Cadena et al. 2011, Winger and Bates 2015).

The 12 species in the genus Myioborus (Aves: Parulidae) inhabit montane areas across the New World (Curson et al. 1994). Ten of the species occur at high elevations and most are allopatrically distributed in mountain ranges separated by lowland gaps (Pérez-Emán 2005). Outstanding exceptions are the Spectacled Redstart (Myioborus melanocephalus) and the Golden-fronted Redstart (Myioborus ornatus), which have abutting ranges along the Andes in southern Colombia and northern Ecuador (Del Hoyo et al. 2010). Previous studies with limited sampling suggest recent divergence (Lovette et al. 2010) and lack of reciprocal monophyly in mitochondrial DNA (mtDNA) between these two species (Pérez-Emán 2005). The M. ornatus/melanocephalus complex, like most highland Myioborus, is characterized by overall similarity in body plumage but marked and complicated geographic variation in head color patterns (Pérez-Emán 2005, Mendoza-Santacruz 2012). Numerous records of individuals with intermediate plumage from the region where ranges abut (Zimmer 1949, Curson et al. 1994, Robbins et al. 1994, Cresswell et al. 1999, Ridgely and Greenfield 2001, McCarthy 2006, Mendoza- Santacruz 2012) suggests the existence of a hybrid zone, but detailed descriptions of patterns of variation in the area are heretofore lacking.

In this study I collected song recordings and museum specimens in multiple localities in Colombia and Ecuador to characterize the putative Myioborus hybrid zone using genetic (mtDNA), plumage coloration, and song data. Specifically, I sought to answer the following questions: (1) What is the extent of hybridization between these taxa? (i.e. what is the frequency of hybrids, what is the location and width of hybrid zone); and (2) How do color, song and mtDNA vary across the M. ornatus- melanocephalus hybrid zone? I also described phylogeographic relationships in the M. ornatus-melanocephalus complex to provide a broader understanding of their evolutionary history based on exhaustive geographic sampling covering the full extent of the distribution of both species.

5 METHODS Study species Myioborus melanocephalus comprises five subspecies distributed along humid slopes of the Andes from southern Colombia to Bolivia (Curson et al. 1994). This species is characterized by a mostly black face and yellow spectacles, but subspecies differ in the amount of black on the submoustachial area and in the presence or absence of a rufous crown (Zimmer 1949, Curson et al. 1994). The southern subspecies M. melanocephalus malaris, M. melanocephalus melanocephalus and M. melanocephalus bolivianus all lack the rufous crown, and replace each other geographically from south of the Marañón River Valley to central Bolivia (Figure 1A). The northern subspecies M. melanocephalus griseonuchus and M. melanocephalus ruficoronatus have a rufous crown, and occur in northern Peru (Piura and Cajamarca) and Ecuador to southern Colombia, respectively (Figure 1A). Myioborus ornatus occurs in all three cordilleras of the Andes of Colombia north to the Táchira Depression in western Venezuela (Curson et al. 1994). A yellow forehead and whitish ear patch characterizes M. ornatus, which comprises two subspecies: M. ornatus chrysops, from the Western and Central cordilleras (Figure 1A), and M. ornatus ornatus, from the Eastern cordillera (Figure 1A); these taxa differ in their facial plumage pattern, with ear coverts and lores being yellow in chrysops and white in ornatus (Salvin 1837, Curson et al. 1994).

I conducted field work to collect songs and specimens of Myioborus warblers in 21 localities along a ~400 km transect around the Colombian-Ecuadorian border (Figure 1B, Table 1) between July 2016 and March 2017. I defined field sites based on the location of previous reports of Myioborus with intermediate plumage (Zimmer 1949, Curson et al. 1994, Robbins et al. 1994, Cresswell et al. 1999, Ridgely and Greenfield 2001, McCarthy 2006, Mendoza-Santacruz 2012). Collecting sites were located in the eastern slope of the Andes, except for the three sites in the central inter-Andean valley of Ecuador (Figure 1B; localities 2, 5 and 6). I also sampled putatively pure allopatric populations of M. o. chrysops in Antioquia (northwestern Colombia) and M. m. ruficoronatus in Tungurahua (south-central Ecuador; Figure 1B). Sampling in the

6 southern extreme of the range of M. m. ruficoronatus was not possible due to logistic restrictions. I also obtained plumage and genetic data from existing specimens at the ornithological collection of Instituto Alexander von Humboldt (IAvH), which allowed me to add one sampling locality (Table 1). To measure distances among localities for geographic cline construction, I fitted a linear regression between latitude and longitude for each of my sampling localities. I then established the position of each sampling locality along this transect using perpendicular lines, and calculated the distance between the southernmost locality (Parroquia El Triunfo, Tungurahua, Ecuador) and each of the points along the transect (Table 1).

Collection of vouchered sound recordings and museum specimens In each locality I collected museum specimens (total n=109) to obtain genetic and plumage data. In most localities I collected at least four specimens (Table 1; mean =5.5 range= 4-8 specimens) except from La Sofía Road, which was excluded from cline construction because only one specimen was collected. Colombian specimens and tissue samples are deposited in the Museo de Historia Natural ANDES at Universidad de los Andes (Bogotá, Colombia), and Ecuadorian specimens in the Museo Ecuatoriano de Ciencias Naturales (Quito, Ecuador). Duplicate tissue samples are deposited in the tissue collection of the Instituto Alexander von Humboldt (IAvH-CT 21121-21330)

Vocalizations of more than half (54%) of the collected individuals were recorded prior to collection, and I also obtained recordings of several individuals not collected. The majority of recordings corresponded to vocalizations in response to playback of songs of M. ornatus, M. melanocephalus, and of individuals from the putative hybrid zone. Playback stimuli elicited seemingly territorial responses, which usually included singing and often approaches to the speaker. Individuals were usually found in pairs or less often in groups or as solitary individuals, and responses to playback often included calls and songs from multiple individuals. Recordings are archived in the Macaulay Library (https://www.macaulaylibrary.org/, Supplementary Table 2).

7 Genetic sampling (ND2 mitochondrial gene) As a first approach to describe patterns of genetic variation across the geographic range of M. ornatus and M. melanocephalus and at their putative hybrid zone I sequenced the NADH dehydrogenase subunit 2 (ND2) mitochondrial gene for specimens collected in this study and available from museum collections (total n=141 for tissue samples, n= 10 for toepad samples; Supplementary Table 1). I extracted genomic DNA from tissue samples using a modified phenol-chloroform protocol (Sambrook 1987). For toepad samples, I extracted DNA using a QIAamp® extraction kit (Qiagen) in a lab where no bird DNA is processed and under a UV-hood.

I prepared PCR mixes of 25 µl containing 1 µl of DNA, 1.0X of Taq buffer, 3.0mM of MgCl2, 0.2mM of each dNTP, 0.48µM of each primer L5216 and H6313 (Sorenson et al. 1999), and 1U of Taq DNA polymerase recombinant (Invitrogen). I used the following amplification conditions: 94°C for 5min of initial denaturation, 35 cycles of 94°C for 45s for denaturation, 20 cycles of touch down -0.5°C from 62°C to 52°C followed by 15 cycles to 52°C for 45s for annealing, and 72°C for 1min for extension, and a final extension of 72°C for 7 min. To amplify the complete ND2 gene from toe-pad samples, I used two separate PCR reactions, both combining an external and internal primer (L5758 and H1056U, L5216 and H5766; Sorenson et al. 1999, Arbeláez-Cortés et al. 2014). PCR mixes of 25 µl consisted on 1.5 of DNA, 1.0X of Taq buffer, 3.0mM of MgCl2, 0.2mM of each dNTP, 0.48 µM of each primer, 0.066 µM of BSA and 1U of Taq DNA polymerase recombinant (Invitrogen). I used the following amplification conditions: 94°C for 5min of initial denaturation; 35 cycles of 94°C for 45s for denaturation, 51.9°C for 45s for annealing, 72°C for 45s for extension, and a final extension of 72°C for 7 min.

I assembled and aligned consensus sequences using Geneious v 9.0 (Kearse et al. 2012). To assess relationships among populations across the distribution of M. ornatus and M. melanocephalus I compiled a larger ND2 dataset by adding 74 sequences either from previous studies (Pérez-Emán 2005, Cuervo 2013) or generated by M.A. Castro, J. Gómez and E. Bonaccorso for this project. These sequences extended sampling to cover

8 all taxa and main regions where the study species occur in Colombia, Ecuador, Peru, Bolivia and Venezuela (Figure 1, Supplementary Table 1). Sequences were assigned to subspecies ideally through examination of specimens or based on geography (Zimmer 1949). A specimen was included in the group ‘putative hybrids’ irrespective of its plumage phenotype if it was collected from a locality in the area where intermediates occur (Figure 1). Therefore, for genetic structure analyses, the term putative hybrids corresponds to a geographic group and not necessarily to a group of indiviudals with intermediate plumage phenotype. Sample sizes per group were: M. melanocephalus bolivianus (n=8), M. melanocephalus melanocephalus (n=9), M. melanocephalus malaris (n=4), M. m. griseonuchus (n=12), M. melanocephalus ruficoronatus (n=18), M. ornatus ornatus (n=29), M. ornatus chrysops (n=15), and putative hybrids (n=127). For most samples I was able to obtain high-quality sequences for at least 1026 bp; some sequences (20), were shorter than 1026 bp and were excluded from some analyses (see below).

Phylogeography and genetic structure in the M. ornatus/melanocephalus complex. To examine relationships among populations across the distribution of M. ornatus and M. melanocephalus, I inferred ND2 gene trees with maximum-likelihood and Bayesian methods using unique haplotypes identified using DnaSP v6. (Librado and Rozas 2009) and Myioborus albifrons as outgroup. All sequences (222; Supplementary Table 1) were incorporated in this analysis, including those with extensive missing data (28-256bp). The maximum-likelihood analysis was conducted using RaxML v.8 (Stamatakis 2014) with a GTR+Γ model of substitution with 25 rate categories and 1000 bootstrap replicates. The Bayesian analysis was done using MrBayes 3.2.2 (Ronquist and Huelsenbeck 2003) with four MCMC chains of 50 million generations sampling every 1,000 generations and discarding the first 50% as burn-in. For both analyses I implemented a partition scheme by codon as suggested by PartitionFinder 2 (Lanfear et al. 2016). Convergence was assessed using Tracer 1.6 through examination of trace plots and effective sample sizes (Rambaut et al. 2014). All analyses were run through the CIPRES platform (www.phylo.org).

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To further describe relationships among ND2 haplotypes, I built a a median- joining network (Bandelt et al. 1999) in PopArt (Leigh and Bryant 2015). This analysis and subsequent population genetic analyses were based on an alignment of 202 sequences with 1026 unambiguous positions. To test for population structure among subspecies and putative hybrids I calculated FST using functions implemented in in the R package ‘hierfstat’ (Goudet 2005) assessing significance with 2,000 permutations. I also calculated Nei genetic distances between these groups in ‘adegenet’ for R (Jambart 2008).

Plumage coloration I described variation in plumage across the hybrid zone by analyzing multiple traits that are described to differ between M. ornatus chrysops and M. melanocephalus ruficoronatus (Fjeldså and Krabbe 1990, Curson et al. 1994, Mendoza-Santacruz 2012). Traits analyzed were extent of rufous crown, extent of black in forehead, extent of whitish ear patch, extent of black in sides of the face, extent of white on tail feathers and underpart coloration. I included juveniles with adult plumage (identified by the presence of bursa of Fabricius and incomplete ossification) in analyses, but excluding them did not affect results. I analyzed data for both males and females together because there is no sexual dichromatism and sample sizes for females were too small to conduct separate analyses.

Head color pattern. To characterize head coloration, I established categories based on the presence/absence and extent of colored plumage patches. I then standardized these scores and obtained an overall plumage hybrid index by averaging them (Rohwer and Wood 1998, Shriver et al. 2005, Toews et al. 2011), with 1 representing a typical M. melanocephalus ruficoronatus and 0 representing a typical M. ornatus chrysops. Scoring categories (Table 2) were established by a preliminary inspection of the series collected in this study and specimens deposited in other museums (Instituto de Ciencias Naturales, Field Museum of Natural History, Instituto Alexander von Humboldt, Universidad de Nariño). Two observers (myself and A.M Cuervo) independently scored all specimens and scores were averaged for analyses.

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Underpart coloration. Coloration of the underparts has been described as bright yellow in M. melanocephalus ruficoronatus (Fjeldså and Krabbe 1990) and golden- yellow (Curson et al. 1994) or warm yellow (Fjeldså and Krabbe 1990) in M. ornatus chrysops. To quantify variation in color hue in the throat and chest I used reflectance spectrometry. Reflectance measurements were taken using an Ocean Optics USB4000 spectrophotometer and a DH-2000 deuterium halogen light source coupled with an optic fiber QP400-2-UV-VIS with a 400 um diameter. The spectrometer was calibrated using a white standard. Each color patch was measured three times per individual and then the reflectance spectra were averaged using functions implemented in ‘pavo’ for R (Maia et al. 2013). I then obtained values of chroma for the spectral range corresponding to red (S1.red; 605-700 nm; Montgomerie 2006), which is useful to distinguish orange and ochraeaceus hues from a typical yellow. Few specimens were excluded from this measurement due to heavy molt.

Tail color pattern- According to previous descriptions, M. ornatus and M. melanocephalus are characterized by exhibiting mostly white coloration in the two most external rectrices (R5, R6; Fjeldså and Krabbe 1990), but M. m. ruficoronatus appears to have more extensive white in the third rectrix (R4; Curson et al. 1994). To describe variation in tail coloration I measured the maximum extent of white in the three outer rectrices with a dial caliper (following Pérez-Emán et al. 2010) and standardized by the total length of these three feathers. I excluded individuals with active molt or visibly worn or incomplete rectrices.

Song structure I described vocal variation based on quantitative measurements of vocal traits extracted from one example song per individual. I obtained 125 sound recordings in the field from localities in the hybrid zone transect and putatively pure allopatric populations (Table 1). When the original recording included more than one complete song bout, I chose the focal bout for measurements using a random number generator. To complement

11 geographic sampling, I also analized recordings from Macaulay Library (https://www.macaulaylibrary.org/), xenocanto (www.xeno-canto.org/) and personal archives of other recordists for M. melanocephalus ruficoronatus (n=4) and M. ornatus chrysops (n=5), for a total of 134 songs (Supplementary Table 2). Geographic position of all recording sites along the study area were calculated with reference to the southernmost locality as explained above. Including recordings from sound archives, the southernmost locality was in the Zamora-Chinchipe Province in Ecuador (Supplementary Table 2).

Spectrograms were generated using Luscinia (frame length= 5 ms, time step= 1 ms, spectrograph points= 240, 80% frame overlap; Lachlan 2007). I measured notes within songs manually (brush size= 10) due to variation in the quality of the recordings and to exclude notes belonging to vocalizations of other non-focal individuals, which were common in recordings. As a first approach to describe geographic variation in song, I summarized the following temporal and spectral variables for each song, most of which have been studied in other avian hybrid zones (Haavie et al. 2004, Greig and Webster 2013): song duration, mean peak frequency, minimum peak frequency, maximum peak frequency, mean frequency bandwidth per note, song frequency bandwidth, and mean note slope (frequency bandwidth/note duration for each note).

Geographic cline analyses and distribution of phenotypes I fitted traits showing a clear sigmoidal pattern of geographic variation to various equilibrium cline models (Szymura and Barton 1986, Gay et al. 2008) implemented in the R package ‘HZAR’ (Derryberry et al. 2014). Specifically, I ran the three following models: (1) a model that only estimates center and width of the cline (Model I; c , w and varH), (2) a model that also estimates variance and mean values for both tails of the cline (Model II; c, w, varH, muL, muR, varL and varR ), and (3) a model that estimates additional parameters describing the shape of the tails (Model III; c, w, muL, muR, varH , varL, varR, tauL and tauR). I ran three chains of 10 million iterations per model discarding the first 50% as burn-in, and assessed convergence using trace plots. Because runs for models II

12 and III did not converge for some parameters in initial runs, I performed additional runs modifying the tune value as suggested by Derryberry et al (2014). All runs were performed randomizing starting parameters. I chose the best-fit cline model for each trait based on AICc values.

I also described the frequency distribution of plumage phenotypes (i.e., hybrid index scores) because this can provide information on the type of selection acting on hybrid zones (Gay et al. 2008). For example, a bimodal distribution, where both pure forms are common in the center, is expected under disruptive selection and assortative mating; alternatively, a unimodal distribution of phenotypes in the center is expected if intermediates are most common as a result of extensive hybridization (Gay et al. 2008). I described the phenotypic of the hybrid index by only considering localities within the estimated width around the center estimated with ‘HZAR’.

RESULTS Phylogeography and genetic structure. Genetic structure of the M. ornatus/melanocephalus complex along the Andes was partially related to geographic discontinuities. The Bayesian gene tree (Figure 2A) recovered (1) a well-supported southern clade including all but one of the specimens from south of the Marañón River Valley located in northern Peru (corresponding to subspecies M. m. malaris, M. m. melanocephalus and M. m. bolivianus), and (2) a clade with high nodal support including all M. m. griseonuchus and southern samples of M. m. ruficoronatus (i.e. from Loja, Zamora-Chinchipe, Morona-Santiago, Ecuador) and few specimens of putative hybrids (Figure 2A). This clade also included the M. m. malaris sample that did not cluster with the southern clade (Figure 2, haplotype 72). However, these two groups supported in the Bayesian tree (Figure 2A, groups 1 and 2) had low bootstrap support in the maximum-likelihood gene tree (bootstrap values 67 and 56 respectively, tree not shown). The remaining specimens, including M. ornatus, M. o. chrysops, M. m. ruficoronatus, and most putative hybrids, did not form a clade in the Bayesian nor the maximum-likelihood tree.

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Population genetic analyses and haplotype network construction were based on an alignment of 202 sequences with 92 segregating sites and representing 78 different haplotypes (Hd= 0.899, Π=0.00403). The haplotype network showed two genetic clusters consistent with well-supported groups in the Bayesian gene tree (Figure 2). The network further revealed a third genetic cluster including the northern specimens corresponding to M. o. ornatus, M. o. chrysops and M. m. ruficoronatus as well as most putative hybrids (Figure 2B, hereafter M. ornatus + M. m. ruficoronatus group), which also shared the most common haplotype. The remaining putative hybrid specimens were grouped with M. m. griseonuchus and some specimens of M. m. ruficoronatus as in the Bayesian gene tree (Figure 2, group 2). The three genetic clusters were separated from each other by less than five mutational steps in all cases (Figure 2B). The M. ornatus + M. m. ruficoronatus genetic cluster was characterized by star-like shape with a common central haplotype (Figure 2B).

Both FST and Nei genetic distances evidenced the low genetic differentiation within the M. ornatus + M. m. ruficoronatus group (Table 3). FST values were lower than 0.1 and non-significant for all pairwise comparisons (Table 3). Putative hybrids, as a geographic group, were not differentiated from M. m. ruficoronatus nor from either subspecies of M. ornatus (Table 3). Conversely, I found relatively high values of FST for all but two of the pairwise comparisons involving the southern subspecies (M. m. griseonuchus, M. m. malaris, M. m. melanocephalus, M. m. bolivianus; Table 3), although sample sizes for these groups were small, especially in M. m. malaris. I was not able to identify haplotypes more commonly found in M. m. ruficoronatus or in M. o. chrysops to allow building a haplotype frequency cline across the sampling transect; the frequency of the most common haplotype, which was shared by these taxa and putative hybrids, did not show a pattern of clinal variation across the hybrid zone.

Plumage coloration across the hybrid zone. I found a wide variety of plumage phenotypes including different combinations of color traits typical of M. o. chrysops and

14 of M. m. ruficoronatus (Figure 3). For example, some individuals had an extensive rufous crown as in M. m. ruficoronatus, but in direct contact with a yellow forehead typical of M. o. chrysops (Figure 3A). Other specimens had also a hint of black in the forehead and below the eye (Figure 3A). The amount of black on the forehead and sides of the face varied substantially among individuals when present (Figure 3A and 3C). Some individuals showed either a complete or partial rufous crown like in M. m. ruficoronatus but also showed a clear whitish ear patch, like in M. o. chrysops. Also, a few specimens had a typical M. o. chrysops forehead and crown but lacked completely the whitish auricular characteristic of this taxon. Although variation in the rufous crown did not appear to be continuous, two intermediate states were found in between the extremes of being fully present or absent: few rufous feathers visible or a fully formed crown but with a clearly smaller area than typical for M. m. ruficoronatus (Figure 3B). Specimens of M. o. chrysops distant from the hybrid zone often had rufous coloration on the base of black feathers fn the crown but this was not noticeable without a detailed examination. I also found a clear intermediate state for the white ear patch, with some specimens having 1-2 whitish feathers per side, whereas typical M. o. chrysops had more than ten and typical M. m. ruficoronatus had no feathers with such coloration (Figure 3C).

Individuals with intermediate plumage phenotype were common along the sampling transect. In localities 13-15 (Hybrid index=0.27-0.75; see Figure 1B) no “pure” individuals were observed. Pure plumage forms (M. o. chrysops and M. melanocephalus ruficoronatus) were never observed nor collected together in the same locality. Head coloration as measured by the hybrid index showed a clear pattern of clinal variation across the transect (Figure 4A). According to variation described by the hybrid index, this hybrid zone is 212 km wide (95% confidence interval: 188 km- 246 km) and is centered at 305 km (95% confidence interval: 297 km -316 km) along the sampling transect, around localities 13-16 (Figure 1). Width and center values were estimated based on the model that fitted the mean and variance of tails (model II), which had the lowest AICc value (model I= -147.84, model II= -182.73, model III= -174.03). The distribution of the hybrid index in the hybrid zone (localities 8–19) was clearly unimodal (Figure 4B).

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Underparts coloration did not show a clear pattern of geographic variation across the hybrid zone (Figure 5B and 5C). Although statistically significant, the linear relationship between chroma on the chest and distance along the study transect was weak (p-value=0.02, R-squared=0.05, m<0.001, df=103 for the chest; p-value=0.81, R- squared<0.001, m<0.001, for the throat). Underpart coloration thus did not clearly vary with respect to its location along the hybrid zone; instead, it varied patchily in space (Figure 5E). For example, individuals at Romerillo (Figure 5E, locality 14) all had underparts with a more orange tinge compared to most specimens from elsewhere. Several specimens from the southernmost localities in the sampling transect (El Triunfo, locality 1; Pasochoa, locality 2) exhibited a marked contrast between a yellow abdomen with an almost orange chest and throat which was lacking in other areas (Figure 5E).

The amount of white in the tail did not exhibit a clear pattern of geographic variation along the sampling transect (Figure 5A). Although I noticed considerable variation among individuals, with some specimens having the third rectrix almost entirely white and others having it entirely black, no overt differences existed between the extremes of the transect (Figure 5D) and there was clinal nor linear variation with distance (p-value=0.11, R-squared=0.03, m<0.001, df= 81 for linear regression).

Acoustic variation Temporal, but not spectral parameters of songs, varied geographically along the distribution ranges of M. o. chrysops and M. m. ruficoronatus. Specifically, song duration tended to be longer towards the south (Zamora-Chinchipe, Ecuador) and shorter in northern populations of M. o. chrysops (Antioquia, Colombia), yet variation was not clinal (Figure 6; p-value<0.001, R-squared=0.32, m=-0.0008, for linear regression after log- transformation). The range of song duration in the southern extreme (Zamora- Chinchipe) was 11.8-19 s whereas in the north (Antioquia) songs were never longer than 5.2 s, with intermediate values across the sampling transect, although sample sizes were relatively small especially in the southern extreme (n=3). Parameters related to

16 frequency did not vary geographically as evidenced by non-significant linear regressions between distance and maximum peak frequency (p-value=0.33, R-squared=0.007), minimimum peak frequency (p-value=0.64, R-squared=0.002), frequency bandwidth (p- value=0.7, R-squared=0.001), mean frequency bandwidth (p-value=0.89, R- squared<0.001), or mean note slope (p-value=0.26, R-squared=0.009).

DISCUSSION The understanding of the origin of Andean bird diversity has been mostly focused on assessing the role of geographic and ecological barriers promoting allopatric differentiation, whereas the study of other processes such as those that occur when formerly isolated populations come into contact (i.e. hybridization and introgression; Dubay and Witt 2014, Winger 2017) has been notably limited. In addition to describing how genetic structure and geographic variation in plumage in the M. ornatus/melanocephalus complex is related to topographical barriers along the Andes, here I explored processes that may act upon secondary contact by describing a hybrid zone between M. o. chrysops and M. m. melanocephalus using mtDNA, plumage and song data. Genetic structure in the M. ornatus/melanocephalus complex partially coincided with physical barriers (e.g. the Marañón River Valley), suporting a role for geographic barriers driving differentiation in these cloud forest warblers. However, geographic variation in plumage was not always associated with genetic structure in mtDNA, which may reflect rapid diversification of head coloration patterns and incomplete lineage sorting, or the influence of gene flow and introgression. I found a 200 km cline in head coloration, but not in other plumage traits or in parameters describing vocalizations, along a continuous cloud forest belt in the area where the ranges of M. o. chrysops and M. m. ruficoronatus abut. The existence of a broad hybrid zone where individuals with intermediate plumage are common suggest that hybridization between these taxa is extensive.

My comprehensive geographic sampling revealed that mtDNA genetic structure in the M. ornatus/melanocephalus complex does not clearly coincide with current species

17 and subspecies limits, but is associated with at least one break in the distribution of cloud forests along the Andes. Specifically, based on mtDNA sequences M. melanocephalus is paraphyletic because most individuals of M. m. ruficoronatus from Ecuador are more closely related to both subspecies of M. ornatus than to southern populations of M. melanocephalus, a result consistent with previous work with more limited sampling (Pérez-Emán 2005). I found evidence of mtDNA differentiation across the Marañón River Valley, a dry area which dissects the distribution of M. melanocephalus and coincides with the transition between rufous- and black-crowned forms (Zimmer 1949, Curson et al. 1994). This arid valley has shown to be important for differentiation in many cloud forest birds (Bates and Zink 1994, Chaves et al. 2011, Gutiérrez-Pinto et al. 2012, Winger and Bates 2015), and likely acts as a strong barrier in the present for Myioborus taxa restricted to humid high-elevation forests and scrub (Curson et al. 1994). An intriguing exception is one specimen from the area south of the Marañón where M. m. malaris occurs (Amazonas, Peru), which groups with individuals from north of the Marañón and seems intermediate between rufous- and black-crowned forms (i.e. it exhibits a few rufous feathers on a mostly black crown; MSB-32521). This specimen likely represents an example of trans-Marañón dispersal and introgression. Another geographic feature that might have acted as an isolating barrier is the dry Zamora River Valley in southern Ecuador (Krabbe 2008, Bonaccorso 2009), which may account for the existence of two different mtDNA groups in M. m. ruficoronatus: most specimens grouped with M. ornatus but few of them, all from southern Ecuador, grouped with M. m. griseonuchus.

In contrast to patterns of genetic structure associated with geographic barriers in the south (i.e. divergence across the Marañón in northern Peru and possibly across the Zamora in southern Ecuador), all analyses suggest that from central Ecuador to Venezuela, a region where a wide variety of plumage forms occur, there is low genetic structure in the M. melanocephalus/ornatus complex. Accordingly, I did not find any evidence of considerable genetic structure across the hybrid zone, a pattern contrasting with numerous avian hybrid zones characterized by sharp clines of haplotype or allele

18 frequencies in mtDNA markers (Irwin et al. 2009a, Carling and Zukerberg 2011, Miller et al. 2014; but see Morales-Rozo et al. 2017). This lack of ND2 divergence may reflect rapid and recent diversification in the M. ornatus/melanocephalus complex in the northern Andes resulting in incomplete lineage sorting, a scenario one would expect especially if effective population sizes are large (Maddison and Knowles 2006). An alternative explanation is mitochondrial introgression, which may lead to discordant patterns of variation in mtDNA relative to nuclear DNA and plumage (Weckstein et al. 2001, Irwin et al. 2009b, Brelsford et al. 2011) and is plausible given phenotypic evidence of hybridization (see below). To explicitly evaluate whether incomplete lineage sorting or mtDNA introgression account for the observed patterns, information on nuclear loci would be necessary (Toews and Brelsford 2012). However, I suggest that the existence of differentiated groups in the south and no genetic structure in the north together with the star-shaped distribution of haplotypes from Ecuador to Venezuela is consistent with a history of recent colonization of the north related to population expansions (Cuervo 2013), a scenario that would lend support to the hypothesis that incomplete lineage sorting accounts for the observed lack of differentiation in mtDNA. Additional information supporting this hypothesis is that M. o. ornatus, a phenotypically distinct form occurring in a different cordillera distant from the hybrid zone, is part of the same ND2 group as all M. o. chrysops, most M. m. ruficoronatus, and most putative hybrids. Phylogeographic studies in co-distributed species have revealed substantial genetic structure associated with the valleys that separate Colombian cordilleras (Cadena and Klicka 2007, Gutiérrez-Pinto et al. 2012, Valderrama et al. 2014), suggesting that these lowland gaps represent effective barriers for cloud forest species and that the lack of divergence in ND2 might be a result of recent colonization (Winger and Bates 2015). Haplotype sharing among all these taxa suggests that differences in coloration patterns have evolved rapidly (Ödeen and Bjorklund 2003; Mila et al. 2007a, b; Campagna et al. 2012, Harris et al. 2017).

A novel aspect of this study, relative to previous work on diversification of Andean birds is that I not only made inferences about about the role of geographic

19 barriers promote differentiation, but I also provided a detailed description of a hybrid zone. Studying processes ocurring after secondary contact has been largely neglected in studies of speciation of birds in the Andes despite its importance for achieving a comprehensive understanding of the origin and maintenance of the rich Andean avifauna. Phenotypic data suggest that M. o. chrysops and M. m. ruficoronatus indeed intergrade in a hybrid zone, which based on head coloration, is centered near the Ecuador-Colombian border. I next turn to describe how my analysis of this hybrid zone contributes to understanding historical biogeography and diversification of Neotropical montane birds.

The geographic area where the Myioborus hybrid zone is centered (Nariño Department; Figure 1A, localities 13-16) has no clear topographic or climatic breaks influencing the continuity of the cloud forest belt (Graham et al. 2010). Diversification within the M. ornatus/melanocephalus complex is estimated to have occurred during the Pleistocene (Pérez-Emán 2005, Cuervo 2013), a period characterized by elevational shifts of vegetation belts (Haffer 1974, Hooghiemstra and Van der Hammen 2004, Cárdenas et al. 2011) leading to changing degrees of fragmentation of cloud forest along the Andes (Ramírez-Barahona and Eguiarte 2013). Therefore, I hypothesize that the connectivity of cloud forest in this area may have become interrupted, promoting allopatric divergence (Graves 1985, 1988), and then it was reestablished allowing secondary contact between forms differentiated in plumage. Pallinological evidence suggest that the area where the Myioborus hybrid zone is centered (i.e. around Laguna de la Cocha, locality 14) has experienced major changes in the connectivity of vegetation belts at least since the Last Glacial Maximum (Flantua et al. 2014). Remarkably, this area also coincides with a concentration of phylogeographic breaks in other cloud forest birds (e.g. Myiothlypis coronata, Hellmayrea gularis, Synallaxis unirufa; Cuervo 2013). This is consistent with the hypothesis that cloud forest connectivity could have been lost promoting divergence across multiple Andean taxa, which can be further tested in a comparative phylogeography framework with dense sampling along this area (Moritz et al. 2009).

20 Although I have been implying that the Myioborus hybrid zone formed by secondary contact, an alternative explanation for the observed cline in head coloration is primary intergradation (Endler 1977); these alternative scenarios are difficult to distinguish based on current patterns of variation (Price 2008, Harrison and Larson 2016). I argue that the most plausible scenario is secondary contact because primary intergradation is expected to occur along environmental gradients with contrasting selective pressures in the extremes, which are not clearly present along the cloud forest band that M. ornatus and M. melanocephalus inhabit (except perhaps for variation in temperature seasonality; Graves 1991), and the hybrid zone does not coincide with an environmental transition (Graham et al. 2010). Moreover, it is unlikely that constrasting environmental conditions lead to differences in specific plumage patches (e.g. head coloration patterns) through divergent natural selection (Cadena et al. 2011).

Regardless of the causes of the origin of the Myioborus hybrid zone, the question arises of whether there is some form of selection against hybridization mantaining the integrity of M. o. chrysops and M. m. ruficoronatus. If interbreeding is unimpeded, then the Myioborus hybrid zone might represent a transient stage leading to the homogeneization of these plumage forms. Alternatively, if there is considerable selection against hybrids, the hybrid zone is expected to be stable and relatively narrow (i.e. a tension zone; Barton and Hewitt 1985) and hybridization and selection may even lead to the completion of reproductive isolation via reinforcement (Liou and Price 1994, Ortiz-Barrientos et al. 2009). My observations in the field and specimens collected across the hybrid zone suggest that pure plumage forms (M. o. chrysops and M. m. ruficoronatus) do not overlap and that intermediate individuals are common across the hybrid zone, with ~100% of the individuals being intermediate in the center. This, together with the existence of a high diversity of intermediate plumage phenotypes, including multiple combinations of M. o. chrysops and M. m. ruficoronatus species facial traits, strongly suggests that many individuals are advanced-generation hybrids or product of backcrosses (Price 2008). This pattern is consistent with hybrids being viable and fertile, which is expected when divergence between hybridizing taxa is recent (Price

21 and Bouvier 2002). Furthermore, I found that the Myioborus hybrid zone is unimodal (parental forms are the least common near the center; Jiggins and Mallet 2000, Gay et al. 2008), which is suggestive of extensive hybridization and little disruptive selection (Gay et al. 2008). However, even unimodal hybrid zones could be characterized by selection against hybrids (Szymura and Barton 1986, Jiggins and Mallet 2000) or by assortative mating acting in the extremes (Smadja et al. 2004), scenarios that could be at play in the Myioborus hybrid zone but cannot be evaluated based solely on patterns of variation in plumage color.

Although phenotypic evidence suggest that hybridization is ongoing and hybrids are viable, a more direct evaluation of selection against hybrids should involve the comparison of the estimated cline width of ~200 km for head coloration with cline width expected under neutral diffusion (Barton and Hewitt 1985, Barton and Gale 1993). Specifically, the neutral cline should be wider than observed clines if some form of selection against hybrids is acting (Toews et al. 2011, Baldassarre et al. 2014, Seneviratne et al. 2016). Although there are estimates of generation time (two years; Pérez-Emán et al. 2010) and natal dispersal distances (935 m for females and 485 m for males; Mumme 2015) for a closely related Myioborus, we lack information about a plausible time since secondary contact and this precludes an assessment of cline width expected under neutral diffusion. However, compared to other hybrid zones in Parulidae with similar characteristics (i.e. excluding mosaic hybrid zones; Vallender et al. 2007) the Myioborus hybrid zone is relatively wide (Oporornis tolmei x O. philadelpia: 130 km, Irwin et al. 2009a; Setophaga towsendi x S. virens: 40-87 km, Toews et al. 2011; Setophaga coronata audoboni x S. c. coronata: 132 km, Brelsford and Irwin 2009; Setophaga occidentalis x S. towsendi: 100-125 km, Rohwer and Wood 1998, Rohwer et al. 2001). This difference in width between the Myioborus hybrid zone and other hybrid zones involving New World warblers appears especially remarkable because Myioborus are tropical resident birds, predicted to disperse shorter distances than migratory species like all of the above (Paradis et al. 1998). In sum, the data suggest that either selection against hybrids is weak or that the Myioborus hybrid zone is relatively old. Using multi-locus nuclear data

22 will be fundamental to properly test for selection against hybrids, for example by calculating levels linkage disequilibria across the hybrid zone, which are predicted to be low if selection against hybrids is weak and high if there is substantial reproductive isolation between hybridizing taxa (Brelsford and Irwin 2009, Taylor et al. 2012).

Although head coloration is the most striking difference between M. o. chrysops and M. m. ruficoronatus, I sought to describe variation in additional plumage traits and song parameters to potentially gain insights into their relative role for communication and reproductive isolation. In contrast to head color patterns, which showed a clear cline, body coloration did not vary along my sampling trasect. Instead, my data suggested that variation in yellow underpart coloration varies more among individuals within sites or among populations not necessarily separated by long distances than across geography, possibly due to subtle differences in access to dietary carotenoids (Hill 1993, Mcgraw et al. 2001, Hill et al. 2002). Similarly, I found no clear differentiation between the extremes of the sampling transect in the amount of white on tail feathers. Although body coloration has been described as different in M. o. chrysops and M. m. ruficoronatus (Fjeldså and Krabbe 1990, Curson et al. 1994), my data indicate that differentatiation in plumage is restricted to head coloration pattern. The clade of high- elevation Myioborus, which comprises ten species including M. ornatus and M. melanocephalus, is characterized by an overall similarity in body coloration and striking variation in head color pattern (Pérez-Emán 2005). Plumage patches in the head are often involved in intraspecific communication and mate choice (Andersson et al. 1998, Rémy et al. 2010) and are exhibited during displays (Price and Pavelka 1996), suggesting that such traits might be under strong sexual selection and are therefore likely to evolve rapidly (West-Eberhard 1983). Additionally, differences in head color patches might be controlled by few genetic regions with large phenotypic effects (Uy et al. 2009b, Poelstra et al. 2014, Campagna et al. 2016, Toews et al. 2016), which would facilitate rapid divergence when populations become isolated.

23 New world warblers, such as Myioborus, rely importantly on acoustic cues for intraspecific communication (Curson et al. 1994) and similarity in song predicts the incidence of hybridization between species of the family (Willis et al. 2014), which makes the study of such signals relevant relevant when describing hybrid zones. Using song recordings obtained across the Myioborus hybrid zone I found an increase in song duration towards the south, where songs in pure populations of M. o. chrysops tend to be shorther than those in M. m. ruficoronatus, a pattern previously mentioned in passing with no quantitative analysis (van Perlo 2015). However, I found that this acoustic variation is not clinal. Given that my results suggest that at least some song attributes (e.g. song duration) vary geographically and there is high variation within sites, a sampling design including fewer and more spaced localities but with a higher number of recordings would provide more robust insights into acoustic variation across this hybrid zone. Moreover, having a greater number of recordings in allopatric populations of both taxa would allow one to identify song attributes that best describe acoustic variation across the Myioborus hybrid zone (Kenyon et al. 2011) and characterize vocal repertoires in these warblers, which likely include several song types (Lemon et al. 1987, Byers 1995). Additionally, I acknowledge playback may have an effect on the recorded songs (e.g. increase vocal amplitude, Brumm and Todt 2004; matching song types and strophe length; McGregor et al. 1992), and this could have introduced additional variance in song parameters, therefore obscuring more subtle patterns of variation. Regardless, except for differences in song duration, acoustic divergence between M. o. chrysops and M. m. ruficoronatus appears to be relatively low and anecdotal playback experiments suggest that individuals respond strongly to heterospecific songs (L. Céspedes, personal observation), which suggest that acoustic cues might not be importantly involved in assortative mating in this hybrid zone. This hypothesis could be explicitly tested by a more detailed description of acoustic variation, which may reveal more notorious differences in song (e.g. in syntax), and by conducting systematic playback experiments.

24 Concluding remarks and next steps. M. ornatus and M. melanocephalus are two widespread Andean birds characterized by low genetic divergence in mtDNA and outstanding geographic variation in color patterns. Phenotypic evidence suggests that these species hybridize extensively where their ranges abut, along more than 200 km where individuals with intermediate plumage are the most common. Future studies should focus on evaluating whether there is considerable selection against hybrids acting in the Myioborus hybrid zone, mantaining the integrity of M. m. ruficoronatus and M. o. chrysops. Obtaining genomic data would allow one to explicitly assess selection against hybrids (e.g. by measuring linkage disequilibrium; Brelsford and Irwin 2009, Taylor et al. 2012) and to identify regions of limited introgression (Poelstra et al. 2014, Toews et al. 2016, Walsh et al. 2016), potentially associated with incipient reproductive isolation. Further, behavioral experiments could advance our knowledge of the relative role of different communication signals (e.g. song, specific head color patches) for recognition and mate choice. More generally, conducting behavioral studies is important to evaluate the relevance of geographically variable communication signals (i.e. head coloration in M. ornatus/melanocephalus), common among Andean birds (Remsen 1984a, Graves 1988), for recognition and reproductive isolation when taxa achieve secondary contact. This study represents the starting point for future research on the dynamics of the Myioborus hybrid zone, which will further our understanding of how species are formed and maintained over time in the highly diverse Andean mountains.

ACKNOWLEDGEMENTS First of all, I want to thank my advisor Daniel Cadena for his guidance and encouragement during the development of this project and for being an extraordinary mentor since the beginning of my research career. I also want to thank my co-advisor Andrés Cuervo for his support and guidance in every stage of this process, and for sharing with me his passion for birds and his vast knowledge of the Neotropical

25 avifauna. Thank you Daniel and Andrés for being both critic and supportive when I needed it.

This project had the financial support of the following organizations: Universidad de los Andes (Proyecto Semilla), Society of Systematic Biologists (Graduate Student Research Award), Association of Field Ornithologists (Bergstrom Award), Neotropical Ornithological Society (Francois Vuillemier Fund) and National Geographic Society (Young Explorers Grant). I am very grateful to the following people for their generous contribution to the projects’ crowd funding campaign on www.experiment.com: Irby Lovette, Liam Revell, Maria C. Arias, Jairo Céspedes, John Pollinger, Mary McElaney, Andrea Ordoñez, Nancy O’Keefe, Sean McElaney, Greg Hesch, Mathew Miller, Virginia Renate Wesselingh, Mary Hesch, Jonatha Caspian, Paulo Pulgarín, Catalina Palacios, Nick Mason, Natalie Wright, Natalia Gutierrez-Pinto, Max Wilson, Valentina Gómez-Bahamón, Christopher Witt, Laura Forero, Colby Harrell, Christina Tran, Victor Hugo Quiroz-Herrera, Nicholas Nemeth, Alfredo Barrera, Eric Damon Walters and three anonimous donors. I am also very thankful to all the ones that helped get the message out.

I am tremendously grateful to many people and institutions that made fieldwork possible and welcomed us in their properties and communities. In Ecuador: Anita and José Iglesias (El Triunfo, Tungurahua), Yanayacu Biological Station, Termas de Papallacta, Hacienda Zuleta and Guandera Reserve and Biological Station. In Colombia: Cuayal family (Ipiales), Laura Florez (Córdoba), Laos family (Puerres), Nelly Figueroa (Funes), local community at Vereda Romerillo and Reserva La Sombra de un Árbol (Laguna de la Cocha), Popayan-Zambrano family (Yacuanquer), Álvaro Cárdenas and Angélica Medina (San Francisco), Leonardo Molina (La Cruz), Muñoz family and Secretaria de Agricultura (San Pablo), Ángela Montoya (Popayan) and Lucila Arias (Inzá). I am very thankful to many people who contributed their time and skills to complete fieldwork: Alejandro Mendoza-Santacruz, Paola Montoya, Sean McElaney, Paulo Pulgarín, Fanny González, Eduardo Obando, Maria A. Meneses and David Ocampo. Universidad Tecnológica de

26 Indoamérica and Instituto Alexander von Humboldt provided important logistical support regarding collecting and importation-exportation permits.

ND2 sequences from key localities were available thanks to collaboration with Elisa Bonaccorso (Universidad San Francisco de Quito), Jorge Pérez-Emán (Universidad Central de Venezuela), Maria Alejandra Castro (Universidad Central de Venezuela), and Christopher Witt (University of New Mexico). Tissue and toepad samples were kindly provided by curators and collection managers at the following institutions: Kansas University Museum (KU), Lousiaina State University Museum of Natural Science (LSUMNS), National Museum of Natural History of the Smithsonian Institution (USNM), Academy of Natural Sciences of Drexel University (ASNP), Pontificia Universidad Católica de Ecuador (PUCE), Instituto Alexander Von Humboldt (IAvH), Instituto de Ciencias Naturales (ICN) and Universidad de Nariño (UN). I am thankful to all collectors and museum staff that made these samples available for this and many other studies. I also thank Ben Marks, John Bates (FMNH), Gary Stiles (ICN), Sergio Córdoba, Socorro Sierra (IAvH) and John Jairo Calderón (UN) for allowing me to inspect specimens under their care, and Nathan Rice (ANSP) and Christopher Milensky (USNM) for kindly providing pictures of Myioborus specimens.

For insigthful conversations and tremendous support during the development of this project I thank all members of the Laboratorio de Biología Evolutiva de Vertebrados. I am grateful to Catalina Palacios and Jorge Avendaño for guidance with laboratory procedures and to Robert Lachlan for advice on the use of Luscinia. Interpretation of results benefited greatly from conversations with Santiago Herrera, Elkin Tenorio and David Toews. I am especially thankful to Alejandro Mendoza-Santacruz who contributed greatly with logistics and fieldwork and shared with me critical data on distribution and plumage variation of Myioborus, and to Elisa Bonaccorso who made fieldwork in Ecuador a successful and extraordinary experience.

27 Lastly I want to thank my parents, Maria C. and Jairo, my brothers and friends (many of them already named above) because their love and support was critical to overcome the many challenges that this research project represented.

PERMIT STATEMENT Specimens from Colombia were collected as part of the project “Patrones de divergencia fenotipica y genotipica en aves neotropicales” under a countrywide collection permit granted to Universidad de los Andes by the Colombian government (Permiso Marco de Recolección, Resolución 1177, Octuber 9th 2014). Specimens from Ecuador were collected under the “Contrato de Acceso a Recursos Genéticos: Aplicando la genética molecular como herramienta para la investigación de la diversidad, ecología y conservación de las especies del Ecuador” (MAE-DNB-CM-2015-0017) suscribed between the Ministerio del Ambiente del Ecuador and Universidad Tecnológica de Indoamerica. Tissue samples duplicates were exported from Ecuador to Colombia under the permit Nº38-2017-EXP-CM-2015-DNB/MA issued by the Ministerio del Ambiente del Ecuador.

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40

Figure 1. Study sites encompassing the distribution of M. ornatus and M. melanocephalus. In both maps, gray shades represent elevation, with medium gray corresponding to the elevation band inhabited by the study species (2000-3500 m) A) Localities for which I obtaied mtDNA sequences encompassing all currently recognized subspecies in the complex and putative hybrids, as defined for the population genetic analyses B) Sampling localities across the putative hybrid zone for which I obtained mtDNA, color and song data. Localities captured by the inset were classified as putative hybrids in population genetic analyses. The numbers correspond to localities in Table 1. Illustrations by Maria Paula Bustamante.

41 Figure 2. Genetic structure in the M. ornatus-M. melanocephalus complex inferred from ND2 sequences. A) Bayesian 50% majority-rule tree for unique haplotypes, showing strong support for two clades: one including all specimens from M. m. bolivianus, M. m. melanocephalus and most of M. m. malaris and other including all M. m. griseonuchus, M. m. ruficoronatus, one M. m. malaris and some putative hybrids. Tip labels correspond to unique haplotype numbering associated with specimens in Supplementary Table 1. The outgroup (M. albifrons) is not shown for clarity B) MJ haplotype network revealing an additional genetic cluster to those identiefied in the gene tree, which includes specimens of M. o. ornatus and M. o. chrysops and most M. m. ruficoronatus and putative hybrids.

42

Figure 3. A sample of the diversity of plumage phenotypes present across the hybrid zone. A) Head color patterns, from top to bottom: typical phenotype of M. ornatus chrysops; intermediate phenotype, showing a solid yellow face and forehead and an extensive rufous crown; intermediate with only a hint of black in the rear of the yellow forehead; intermediate with moderate amount of black between surrounding the rufous crown, and typical M. melanocephalus ruficoronatus B) Variation in crown coloration, ranging from non-visible rufous feathers (top) to a full rufous crown (bottom) C) Variation in the presence and extent of the whitish ear patch, and width of the black malar line, ranging from specimens showing a whitish ear patch and no black malar line, to specimens with no ear patch and a solid black malar line. Photo credits: Paulo Pulgarín (A), David Ocampo (A)

43

Figure 4. A) Plumage cline describing the Myioborus hybrid zone. The hybrid index was calculated taking into account only facial plumage patches, where 1 (one) represents a typical M. melanocephalus ruficoronatus and 0 (zero) a typical M. ornatus chrysops. Gray dots represent individual score values, crosses are mean values per locality and the vertical line is the estimated center. B) Unimodal distribution of plumage phenotypes as measured by the hybrid index (localities 8-19).

44

Figure 5. Body plumage coloration do not have clear geographic variation A) Variation in the amount of white in tail feathers (white index) across the sampling transect B,C) Variation in red chroma (605-700 nm) of the throat and chest D) Specimens showing variation in the amount of white in the tail. Arrows are pointing to R4 in all specimens showing that is almost completely white in the one in the middle (from Vereda Romerillo, locality 14) and almost all black in the ones of the extremes that correspond to allopatric populations (La Montañita, locality 22; El Triunfo, locality 1) E) Specimens showing variation in underpart coloration. Two in the left (from San José de Córdoba, locality 13; Yanayacu Biological Station, locality 3) showing a bright yellow, and the two on the right (from Romerillo, locality 14) showing an orange tinge. Specimen in the middle (from El Tiunfo, locality 1) is characterized by a throat and chest with an orange hue, contrasting with a bright yellow abdomen.

45

Figure 6. Song duration length, but not variables related to frequency, vary along the distribution of M. o. chrysops and M. m. ruficoronatus. Gray dots correspond to each recording measured. Only statistically significant regression line are shown.

46 Table 1. Sampling localities across the hybrid zone and putative allopatric populations (1, 21 and 22). Numbers correspond to those in Figure 1. Sampling sizes are shown for number of specimens collected in this study, specimens with plumage data included in cline analyses and number of high quality recordings included in acoustic analyses. Coordinates for locality 21 (Neira and Aguadas) corresponded to the average of coordinates for specimens collected in the Caldas Department (Colombia) and collected prior to this study.

Distance in Locality Country Department/Province n (specimens) n (color) n (recordings) transect (km) Latitude Longitude 5 5 9 1 Parroquia El Triunfo Ecuador Tungurahua 0 -1.3161 -78.4028 2 Refugio de Vida Silvestre Pasochoa Ecuador Pichincha 5 5 8 83 -0.4250 -78.5168 3 Yanayacu Biological Station Ecuador Napo 5 5 5 92 -0.6398 -77.9129 4 Termas de Papallacta Ecuador Napo 5 4 7 108 -0.3578 -78.1508 5 Hacienda Zuleta Ecuador Imbabura 6 6 6 168 0.1984 -78.0716 6 Bosque Nueva America Ecuador Imbabura 5 4 7 178 0.2597 -77.9824 7 La Sofia Road Ecuador Sucumbios 1 0 0 215 0.4389 -77.5871 8 Guandera Reserve and Biological Station Ecuador Carchi 4 4 6 225 0.5887 -77.7043 9 Vereda El Mirador Colombia Nariño 5 5 7 242 0.7363 -77.6523 10 Vereda La Cumbre Colombia Nariño 5 4 6 258 0.8149 -77.4799 5 5 8 11 Vereda La Esperanza Colombia Nariño 264 0.8666 -77.4600 12 Corregimiento Chapal Colombia Nariño 6 4 5 270 0.9234 -51.6357 Vereda San José de Córdoba and Bosque El 5 5 5 13 Zanjón Colombia Nariño 297 1.1545 -77.3886 14 Vereda Romerillo at Laguna de la Cocha Colombia Nariño 8 10 7 300 1.0807 -77.1687 15 Vereda San Felipe Colombia Nariño 5 5 6 305 1.1980 -77.3149 16 Alto de Daza Colombia Nariño 7 5 6 315 1.2674 -77.2538 17 Vereda La Siberia and Minchoy Colombia Putumayo 5 5 7 324 1.1613 -76.8472 18 Vereda La Palma Colombia Nariño 6 6 5 358 1.5429 -76.9232 19 Cerro de la Campana and Vereda Ramal Alto Colombia Nariño 5 5 6 375 1.7148 -76.9335 20 Vereda Riosucio Colombia Cauca 5 5 7 485 2.4641 -76.2099 21 Aguadas and Neira* Colombia Caldas 0 4 0 816 5.3704 -75.4049 22 Finca La Montañita Colombia Antioquia 6 6 4 914 6.4558 -75.6012

47 Table 2. Scoring categories established to calculate the hybrid score. Higher scores correspond to typical M. melanocephalus ruficoronatus traits and lower to M. ornatus chrysops.

Rufous crown Description 0 No visible rufous crown. Hint of rufous on the base of black feathers might be present. 1 Few visible rufous feathers 2 Small crown, almost like a band (3-5 mm) 3 Full rufous crown (> 6 mm) Black in forehead a 0 No black on front of rufous crown. Rufous is in direct contact with yellow forehead. 1 Few spots of black between rufous and yellow on forehead. Extensive yellow on forehead 2 Narrow black band in front of rufous crown or transition between rufous and yellow heavily spotted with black. 3 Moderate amount of black on the front with a narrow band of yellow feathers that separates black from the culmen. 4 Wide black band on front of the rufous crown. Black is in direct contact with culmen, but few dispersed yellow feathers might be present. Black stripes under eye 0 Sides of the face are completely yellow 1 Barely visible black stripe under eye. Often incomplete being only present near the beak. 2 Clear black stripe under the eye, always continuous from the beak but narrow. 3 Wide and complete black stripe (>1.5 mm) under eye and wider near the beak. Whitish ear patch 0 White auricular composed by whitish yellow feathers insert next to tympanum continuously (usually >10) 1 White auricular composed by 3-6 whitish feathers insert next to tympanum. 2 1-2 whitish feathers insert next to tympanum. When only one feather present, may be found only on one side. 3 No whitish feathers insert next to the tympanum. a The score in this category when no rufous crown was present was 0. Therefore, these traits are not independent by definition so I calculated a hybrid index treating both as one and two separate traits.

48 Table 3. Genetic differentiation between groups in the the M. melanocephalus- M. ornatus complex. Numbers above the diagonal correspond to the FST values, that are in bold if p-value<0.05. Below the diagonal, mean Nei-genetic distances between defined groups.

M.m. bolivianus M.o. chrysops M. m. griseonuchus Putative hybrids M. m. malaris M. m. melanocephalus M. o. ornatus M. m. ruficoronatus M.m. bolivianus (n=7) 0.5092 0.5390 0.1599 0.1716 0.1479 0.4102 0.2957 M.o. chrysops (n=15) 0.1145 0.5182 0.0079 0.3758 0.5035 0.0657 0.0885 M. m. griseonuchus (n=9) 0.1460 0.0882 0.1545 0.4156 0.5403 0.4191 0.2122 Putative hybrids (n=116) 0.1131 0.0021 0.0822 0.0735 0.1529 0.0242 0.0323 M. m. malaris (n=4) 0.0351 0.0809 0.0989 0.0774 0.0787 0.2613 0.1659 M. m. melanocephalus (n=7) 0.0252 0.1083 0.1418 0.1063 0.0138 0.4073 0.2910 M.o. ornatus (n=28) 0.1112 0.0059 0.0869 0.0044 0.0797 0.1077 0.0863 M. m. ruficoronatus (n=16) 0.1094 0.0140 0.0539 0.0102 0.0716 0.1052 0.0137

49

Supplementary Table 1. Information of specimens associated with ND2 sequences. Haplotypes correspond to numbering in Figure 2A.

Catalog/collector Sample number Group Haplotype Locality Museum type1 1 LSUMZ-B1257 M. m. bolivianus 88 Bolivia: depto. La Paz, ca 1 km S Chuspipata LSUMNS Tissue 2 MSB34428 M. m. bolivianus 76 Peru: depto. Cuzco, Abra Bella Vista MSB Tissue 3 MSB33222 M. m. bolivianus 80 Peru: depto. Cuzco, Abra Bella Vista MSB Tissue 4 MSB33529 M. m. bolivianus 81 Peru: depto. Cuzco, Chile MSB Tissue 5 KU21181 M. m. bolivianus 90 Peru: depto. Puno, below Sina KU Tissue 6 KU21196 M. m. bolivianus 91 Peru: depto. Puno, below Sina KU Tissue 7 KU21240 M. m. bolivianus 90 Peru: depto. Puno, below Sina KU Tissue 8 KU21175 M. m. bolivianus 87 Peru: depto. Puno, below Sina KU Tissue 9 LSUMZ-B8036 M. m. melanocephalus 82 Peru: depto. Pasco, Playa Pampa, 8 km NW Cushi on trail to Chaglia LSUMNS Tissue 10 LSUMZ-B822 M. m. melanocephalus 86 Peru: depto. San Martín LSUMNS Tissue 11 LSUMZ-B823 M. m. melanocephalus 84 Peru: depto. San Martín, Puerta del Monte, ca 30 km NE Los Alicios LSUMNS Tissue 12 MSB 31628 M. m. melanocephalus 92 Peru: depto. Húanuco, near Carpish Tunnel MSB Tissue 13 KU 14793 M. m. melanocephalus 76 Peru: depto. Junin, along Rio Satipo KU Tissue 14 KU 14762 M. m. melanocephalus 75 Peru: depto. Junin, along Rio Satipo KU Tissue 15 KU 16876 M. m. melanocephalus 77 Peru: depto. Ayacucho, Tutumbaro KU Tissue 16 KU 17043 M. m. melanocephalus 79 Peru: depto. Ayacucho, 2 Km S Cano KU Tissue 17 KU 17042 M. m. melanocephalus 89 Peru: depto. Ayacucho, 2 Km S Cano KU Tissue 18 MSB32101 M. m. malaris 85 Peru: depto. Amazonas, 4.5 km N Tullanya MSB Tissue 19 MSB32317 M. m. malaris 83 Peru: depto. Amazonas, 4.5 km N Tullanya MSB Tissue 20 MSB32177 M. m. malaris 78 Peru: depto. Amazonas, 4.5 km N Tullanya MSB Tissue 21 MSB32521 M. m. malaris 72 Peru: depto. Amazonas, 4.5 km N Tullanya MSB Tissue 22 LSUMZ-B32703 M. m. griseonuchus 73 Peru: depto. Cajamarca, Quebrada las Palmas, ca 13 km WSW Chantali LSUMNS Tissue 23 LSUMZ-B31701 M. m. griseonuchus 71 Peru: depto. Cajamarca LSUMNS Tissue 24 LSUMZ-B31773 M. m. griseonuchus 71 Peru: depto. Cajamarca LSUMNS Tissue 25 LSUMZ-B31795 M. m. griseonuchus 71 Peru: depto. Cajamarca LSUMNS Tissue 26 LSUMZ-B31947 M. m. griseonuchus 66 Peru: depto. Cajamarca LSUMNS Tissue 27 LSUMZ-B32519 M. m. griseonuchus 71 Peru: depto. Cajamarca LSUMNS Tissue 28 LSUMZ-B32702 M. m. griseonuchus 68 Peru: depto. Cajamarca LSUMNS Tissue 29 LSUMZ-B33832 M. m. griseonuchus 66 Peru: depto. Cajamarca LSUMNS Tissue 30 MSB 41931 M. m. griseonuchus 64 Peru: depto. Cajamarca, ca. 2.8 km NW Agua Azul MSB Tissue 31 MSB 42121 M. m. griseonuchus 64 Peru: depto. Cajamarca, ca. 2.8 km NW Agua Azul MSB Tissue 32 MSB 41958 M. m. griseonuchus 69 Peru: depto. Cajamarca, ca. 2.8 km NW Agua Azul MSB Tissue 33 MSB 42040 M. m. griseonuchus 66 Peru: depto. Cajamarca, ca. 2.8 km NW Agua Azul MSB Tissue

50 34 LSUMZ-B6243 M. m. ruficoronatus 36 Ecuador: prov. Morona-Santiago, W slope Cordillera del Cutucu LSUMNS Tissue 35 LSUMZ-B6244 M. m. ruficoronatus 37 Ecuador: prov. Morona-Santiago, W slope Cordillera del Cutucu LSUMNS Tissue 36 LSUMZ-B6256 M. m. ruficoronatus 37 Ecuador: prov. Morona-Santiago LSUMNS Tissue 37 ASNP19603 M. m. ruficoronatus 65 Ecuador: prov. Zamora-Chinchipe ANSP Tissue 38 ASNP19541 M. m. ruficoronatus 65 Ecuador: prov. Zamora-Chinchipe ANSP Tissue 39 SEN638 M. m. ruficoronatus 74 Ecuador: prov. Loja, parroquia San Sebastián PUCE Tissue 40 SEN445 M. m. ruficoronatus 65 Ecuador: prov. Morona-Santiago, Parque Nacional Sangay PUCE Tissue 41 SEN910 M. m. ruficoronatus 70 Ecuador: prov. Loja, Bosque Protector Colambo Yacuri PUCE Tissue 42 SEN466 M. m. ruficoronatus 29 Ecuador: prov. Morona-Santiago, Parque Nacional Sangay PUCE Tissue 43 SEN842 M. m. ruficoronatus 12 Ecuador: prov. Tungurahua, parroquia Sucre PUCE Tissue 44 SEN450 M. m. ruficoronatus 66 Ecuador: prov. Morona-Santiago, Parque Nacional Sangay PUCE Tissue 45 SEN806 M. m. ruficoronatus 20 Ecuador: prov. Tungurahua, parroquia Sucre, Parque Nacional Llanganates PUCE Tissue 46 SEN723 M. m. ruficoronatus 44 Ecuador: prov. Pastaza, Reserva Privada Ankaku PUCE Tissue 47 LNCA48 M. m. ruficoronatus 59 Ecuador: prov. Tungurahua, cantón Patate, parroquia El Triunfo IAvH Tissue 48 LNCA49 M. m. ruficoronatus 15 Ecuador: prov. Tungurahua, cantón Patate, parroquia El Triunfo IAvH Tissue 49 LNCA50 M. m. ruficoronatus 51 Ecuador: prov. Tungurahua, cantón Patate, parroquia El Triunfo IAvH Tissue 50 LNCA51 M. m. ruficoronatus 31 Ecuador: prov. Tungurahua, cantón Patate, parroquia El Triunfo IAvH Tissue 51 LNCA52 M. m. ruficoronatus 15 Ecuador: prov. Tungurahua, cantón Patate, parroquia El Triunfo IAvH Tissue 52 ASNP18884 Putative hybrids 10 Ecuador: prov. Carchi ASNP Tissue 53 ASNP18837 Putative hybrids 35 Ecuador: prov. Carchi ASNP Tissue 54 ASNP18798 Putative hybrids 46 Ecuador: prov. Carchi ASNP Tissue 55 ASNP18839 Putative hybrids 59 Ecuador: prov. Carchi ASNP Tissue 56 ASNP18844 Putative hybrids 53 Ecuador: prov. Carchi ASNP Tissue 57 ASNP3878 Putative hybrids 45 Ecuador: prov. Carchi, Impuerán ASNP Tissue 58 USNM608538 Putative hybrids 19 Ecuador: prov. Sucumbios USNM Tissue 59 USNM608540 Putative hybrids 19 Ecuador: prov. Sucumbios USNM Tissue 60 USNM608535 Putative hybrids 51 Ecuador: prov. Sucumbios USNM Tissue 61 USNM608536 Putative hybrids 55 Ecuador: prov. Sucumbios USNM Tissue 62 USNM608537 Putative hybrids 56 Ecuador: prov. Sucumbios USNM Tissue 63 SEN427 Putative hybrids 63 Ecuador: prov. Imbabura, Cordillera de Toisán PUCE Tissue 64 CARS257 Putative hybrids 44 Ecuador: prov. Imbabura, La Rinconada - Zuleta PUCE Tissue 65 AMC1374 Putative hybrids 5 Colombia: depto. Putumayo, San Francisco ANDES Tissue 66 AMC1375 Putative hybrids 39 Colombia: depto. Putumayo, San Francisco ANDES Tissue 67 AMC1376 Putative hybrids 20 Colombia: depto. Putumayo, San Francisco ANDES Tissue 68 AMC1377 Putative hybrids 61 Colombia: depto. Nariño, Alto de Daza ANDES Tissue 69 AMC1378 Putative hybrids 49 Colombia: depto. Nariño, Alto de Daza ANDES Tissue 70 AMC1379 Putative hybrids 20 Colombia: depto. Nariño, Alto de Daza ANDES Tissue 71 AMC1380 Putative hybrids 20 Colombia: depto. Nariño, Alto de Daza ANDES Tissue 72 AMC1381 Putative hybrids 20 Colombia: depto. Nariño, Pasto, vereda San Felipe ANDES Tissue 73 AMC1382 Putative hybrids 60 Colombia: depto. Nariño, Pasto, vereda San Felipe ANDES Tissue

51 74 AMC1383 Putative hybrids 18 Colombia: depto. Nariño, Pasto, vereda San Felipe ANDES Tissue 75 AMC1384 Putative hybrids 60 Colombia: depto. Nariño, Yacuanquer ANDES Tissue 76 AMC1385 Putative hybrids 4 Colombia: depto. Nariño, Pasto, vereda Romerillo ANDES Tissue 77 AMC1386 Putative hybrids 51 Colombia: depto. Nariño, Pasto, vereda Romerillo ANDES Tissue 78 AMC1387 Putative hybrids 39 Colombia: depto. Nariño, Pasto, vereda Romerillo ANDES Tissue 79 AMC1388 Putative hybrids 20 Colombia: depto. Nariño, Pasto, vereda Romerillo ANDES Tissue 80 AMC1389 Putative hybrids 42 Colombia: depto. Nariño, Pasto, vereda Romerillo ANDES Tissue 81 AMC1390 Putative hybrids 23 Colombia: depto. Nariño, Pasto, vereda Romerillo ANDES Tissue 82 AMC1391 Putative hybrids 23 Colombia: depto. Nariño, Pasto, vereda Romerillo ANDES Tissue 83 AMC1392 Putative hybrids 51 Colombia: depto. Nariño, Pasto, vereda Romerillo ANDES Tissue 84 AMC1393 Putative hybrids 20 Colombia: depto. Nariño, Puerres ANDES Tissue 85 AMC1394 Putative hybrids 45 Colombia: depto. Nariño, Puerres ANDES Tissue 86 AMC1395 Putative hybrids 20 Colombia: depto. Nariño, Puerres ANDES Tissue 87 AMC1396 Putative hybrids 58 Colombia: depto. Nariño, Puerres ANDES Tissue 88 AMC1397 Putative hybrids 3 Colombia: depto. Nariño, Córdoba ANDES Tissue 89 AMC1398 Putative hybrids 20 Colombia: depto. Nariño, Córdoba ANDES Tissue 90 AMC1399 Putative hybrids 51 Colombia: depto. Nariño, Córdoba ANDES Tissue 91 AMC1400 Putative hybrids 20 Colombia: depto. Nariño, Córdoba ANDES Tissue 92 AMC1401 Putative hybrids 51 Colombia: depto. Nariño, Córdoba ANDES Tissue 93 AMC1402 Putative hybrids 59 Colombia: depto. Nariño, Ipiales ANDES Tissue 94 AMC1403 Putative hybrids 20 Colombia: depto. Nariño, Ipiales ANDES Tissue 95 AMC1404 Putative hybrids 20 Colombia: depto. Nariño, Ipiales ANDES Tissue 96 AMC1405 Putative hybrids 13 Colombia: depto. Nariño, Ipiales ANDES Tissue 97 AMC1406 Putative hybrids 51 Colombia: depto. Nariño, Ipiales ANDES Tissue 98 AMC1408 Putative hybrids 48 Colombia: depto. Nariño, Pasto, vereda San Felipe ANDES Tissue 99 AMC1409 Putative hybrids 20 Colombia: depto. Nariño, Pasto, vereda San Felipe ANDES Tissue 100 AMC1410 Putative hybrids 20 Colombia: depto. Nariño, Alto de Daza ANDES Tissue 101 AMC1411 Putative hybrids 61 Colombia: depto. Nariño, Alto de Daza ANDES Tissue 102 AMC1412 Putative hybrids 38 Colombia: depto. Nariño, Alto de Daza ANDES Tissue 103 AMC1413 Putative hybrids 59 Colombia: depto. Nariño, Yacuanquer ANDES Tissue 104 AMC1414 Putative hybrids 59 Colombia: depto. Nariño, Yacuanquer ANDES Tissue 105 AMC1415 Putative hybrids 59 Colombia: depto. Nariño, Yacuanquer ANDES Tissue 106 AMC1416 Putative hybrids 18 Colombia: depto. Nariño, Yacuanquer ANDES Tissue 107 AMC1417 Putative hybrids 20 Colombia: depto. Nariño, Funes ANDES Tissue 108 AMC1418 Putative hybrids 20 Colombia: depto. Nariño, Funes ANDES Tissue 109 AMC1419 Putative hybrids 67 Colombia: depto. Nariño, Funes ANDES Tissue 110 AMC1420 Putative hybrids 20 Colombia: depto. Nariño, Funes ANDES Tissue 111 AMC1421 Putative hybrids 20 Colombia: depto. Nariño, Funes ANDES Tissue 112 AMC1422 Putative hybrids 38 Colombia: depto. Nariño, La Cruz ANDES Tissue 113 AMC1423 Putative hybrids 59 Colombia: depto. Nariño, La Cruz ANDES Tissue

52 114 AMC1424 Putative hybrids 20 Colombia: depto. Nariño, La Cruz ANDES Tissue 115 AMC1425 Putative hybrids 20 Colombia: depto. Nariño, La Cruz ANDES Tissue 116 AMC1426 Putative hybrids 16 Colombia: depto. Nariño, La Cruz ANDES Tissue 117 AMC1427 Putative hybrids 16 Colombia: depto. Nariño, La Cruz ANDES Tissue 118 AMC1428 Putative hybrids 51 Colombia: depto. Nariño, San Pablo ANDES Tissue 119 AMC1429 Putative hybrids 20 Colombia: depto. Nariño, San Pablo ANDES Tissue 120 LNCA15 Putative hybrids 51 Colombia: depto. Putumayo, San Francisco ANDES Tissue 121 PAM65 Putative hybrids 20 Colombia: depto. Putumayo, San Francisco ANDES Tissue 122 PAM66 Putative hybrids 45 Colombia: depto. Nariño, Puerres ANDES Tissue 123 LNCA16 Putative hybrids 47 Colombia: depto. Nariño, Funes ANDES Tissue 124 LNCA17 Putative hybrids 49 Ecuador: prov. Carchi, Guandera Reserve and Biological Station IAvH Tissue 125 LNCA18 Putative hybrids 20 Ecuador: prov. Carchi, Guandera Reserve and Biological Station IAvH Tissue 126 LNCA19 Putative hybrids 44 Ecuador: prov. Carchi, Guandera Reserve and Biological Station IAvH Tissue 127 LNCA20 Putative hybrids 7 Ecuador: prov. Carchi, Guandera Reserve and Biological Station IAvH Tissue 128 LNCA21 Putative hybrids 49 Ecuador: prov. Sucumbios, La Sofia-La Bonita road IAvH Tissue 129 LNCA22 Putative hybrids 20 Ecuador: prov. Napo, Yanayacu Biological Station IAvH Tissue 130 LNCA23 Putative hybrids 14 Ecuador: prov. Napo, Yanayacu Biological Station IAvH Tissue 131 LNCA24 Putative hybrids 25 Ecuador: prov. Napo, Yanayacu Biological Station IAvH Tissue 132 LNCA25 Putative hybrids 20 Ecuador: prov. Napo, Yanayacu Biological Station IAvH Tissue 133 LNCA26 Putative hybrids 31 Ecuador: prov. Napo, Yanayacu Biological Station IAvH Tissue 134 LNCA27 Putative hybrids 20 Ecuador: prov. Napo, Termas de Papallacta IAvH Tissue 135 LNCA28 Putative hybrids 20 Ecuador: prov. Napo, Termas de Papallacta IAvH Tissue 136 LNCA29 Putative hybrids 20 Ecuador: prov. Napo, Termas de Papallacta IAvH Tissue 137 LNCA30 Putative hybrids 20 Ecuador: prov. Napo, Termas de Papallacta IAvH Tissue 138 LNCA31 Putative hybrids 20 Ecuador: prov. Napo, Termas de Papallacta IAvH Tissue 139 LNCA32 Putative hybrids 20 Ecuador: prov. Imabura, Hacienda Zuleta IAvH Tissue 140 LNCA33 Putative hybrids 59 Ecuador: prov. Imabura, Hacienda Zuleta IAvH Tissue 141 LNCA34 Putative hybrids 20 Ecuador: prov. Imabura, Hacienda Zuleta IAvH Tissue 142 LNCA35 Putative hybrids 20 Ecuador: prov. Imabura, Hacienda Zuleta IAvH Tissue 143 LNCA36 Putative hybrids 63 Ecuador: prov. Imabura, Hacienda Zuleta IAvH Tissue 144 LNCA37 Putative hybrids 17 Ecuador: prov. Imabura, Hacienda Zuleta IAvH Tissue 145 LNCA38 Putative hybrids 20 Ecuador: prov. Imbabura, Bosque de Nueva América IAvH Tissue 146 LNCA39 Putative hybrids 17 Ecuador: prov. Imbabura, Bosque de Nueva América IAvH Tissue 147 LNCA40 Putative hybrids 20 Ecuador: prov. Imbabura, Bosque de Nueva América IAvH Tissue 148 LNCA41 Putative hybrids 20 Ecuador: prov. Imbabura, Bosque de Nueva América IAvH Tissue 149 LNCA42 Putative hybrids 59 Ecuador: prov. Imbabura, Bosque de Nueva América IAvH Tissue 150 LNCA43 Putative hybrids 60 Ecuador: prov. Pichincha, Refugio de Vida Silvestre Pasochoa IAvH Tissue 151 LNCA44 Putative hybrids 30 Ecuador: prov. Pichincha, Refugio de Vida Silvestre Pasochoa IAvH Tissue 152 LNCA45 Putative hybrids 20 Ecuador: prov. Pichincha, Refugio de Vida Silvestre Pasochoa IAvH Tissue 153 LNCA46 Putative hybrids 63 Ecuador: prov. Pichincha, Refugio de Vida Silvestre Pasochoa IAvH Tissue

53 154 LNCA47 Putative hybrids 60 Ecuador: prov. Pichincha, Refugio de Vida Silvestre Pasochoa IAvH Tissue 155 IAvH-CT-7276 Putative hybrids 20 Colombia: depto. Huila, San Agustín IAvH Tissue 156 IAvH-CT-17378 Putative hybrids 63 Colombia: depto. Nariño, Pasto, Laguna de la Cocha IAvH Tissue 157 IAvH-CT-18945 Putative hybrids 67 Colombia: depto. Nariño, Pasto IAvH Tissue 158 IAvH-CT-18947 Putative hybrids 30 Colombia: depto. Nariño IAvH Tissue 159 IAvH-CT-18953 Putative hybrids 20 Colombia: depto. Nariño, Puerres, Monopamba IAvH Tissue 160 IAvH-CT-18964 Putative hybrids 20 Colombia: depto. Nariño, Gualmatán IAvH Tissue 161 LNCA55 Putative hybrids 20 Colombia: depto. Cauca, Inzá ANDES Tissue 162 LNCA56 Putative hybrids 20 Colombia: depto. Cauca, Inzá ANDES Tissue 163 LNCA57 Putative hybrids 20 Colombia: depto. Cauca, Inzá ANDES Tissue 164 LNCA58 Putative hybrids 20 Colombia: depto. Cauca, Inzá ANDES Tissue 165 LNCA59 Putative hybrids 40 Colombia: depto. Cauca, Inzá ANDES Tissue 166 LNCA60 Putative hybrids 20 Colombia: depto. Nariño, San Pablo ANDES Tissue 167 LNCA61 Putative hybrids 44 Colombia: depto. Nariño, San Pablo ANDES Tissue 168 LNCA62 Putative hybrids 6 Colombia: depto. Nariño, San Pablo ANDES Tissue 169 UN 1048 Putative hybrids 18 Colombia: depto. Nariño, Azufral UN Toepad 170 UN627 Putative hybrids 20 Colombia: depto. Nariño, Pasto, Laguna de la Cocha UN Toepad 171 UN1047 Putative hybrids 26 Colombia: depto. Nariño, Azufral UN Toepad 172 UN139 Putative hybrids 57 Colombia: depto. Nariño, Cumbal UN Toepad 173 UN562 Putative hybrids 67 Colombia: depto. Nariño, Pasto, Laguna de la Cocha UN Toepad 174 UN192 Putative hybrids 52 Colombia: depto. Nariño, Pasto, Laguna de la Cocha UN Toepad 175 UN620 Putative hybrids 20 Colombia: depto. Nariño, Pasto, Laguna de la Cocha UN Toepad 176 UN1049 Putative hybrids 45 Colombia: depto. Nariño, Pasto, Laguna de la Cocha UN Toepad 177 UN518 Putative hybrids 20 Colombia: depto. Nariño, Pasto, Laguna de la Cocha UN Toepad 178 UN623 Putative hybrids 18 Colombia: depto. Nariño, Pasto, Laguna de la Cocha UN Toepad 179 IAvH-CT-1711 M. o. chrysops 63 Colombia: depto. Antioquia IAvH Tissue 180 ICN34345 M. o. chrysops 13 Colombia: depto. Antioquia, Jardin, Finca La Sierra ICN Tissue 181 ICN34479 M. o. chrysops 51 Colombia: depto. Antioquia, Jardin, Finca La Sierra ICN Tissue 182 AMC1430 M. o. chrysops 51 Colombia: depto. Antioquia, San Pedro de los Milagros ANDES Tissue 183 AMC1431 M. o. chrysops 20 Colombia: depto. Antioquia, San Pedro de los Milagros ANDES Tissue 184 AMC1432 M. o. chrysops 54 Colombia: depto. Antioquia, San Pedro de los Milagros ANDES Tissue 185 AMC1433 M. o. chrysops 51 Colombia: depto. Antioquia, San Pedro de los Milagros ANDES Tissue 186 AMC1434 M. o. chrysops 51 Colombia: depto. Antioquia, San Pedro de los Milagros ANDES Tissue 187 AMC1435 M. o. chrysops 11 Colombia: depto. Antioquia, San Pedro de los Milagros ANDES Tissue 188 IAvH-CT-1830 M. o. chrysops 62 Colombia: depto. Caldas, Neira IAvH Tissue 189 IAvH-CT-4610 M. o. chrysops 20 Colombia: depto. Antioquia, Caldas IAvH Tissue 190 IAvH-CT-14967 M. o. chrysops 20 Colombia: depto. Caldas, Aguadas IAvH Tissue 191 IAvH-CT-14928 M. o. chrysops 2 Colombia: depto. Caldas, Aguadas IAvH Tissue 192 KC305 M. o. chrysops 20 Colombia: depto. Antioquia, Jardin ICN Tissue 193 KC311 M. o. chrysops 20 Colombia: depto. Antioquia, Jardin ICN Tissue

54 194 IAvH-CT-1118 M. o. ornatus 41 Colombia: depto. Boyacá, SFF Iguaque IAvH Tissue 195 IAvH-CT-1120 M. o. ornatus 21 Colombia: depto. Boyacá, SFF Iguaque IAvH Tissue 196 IAvH-CT-1719 M. o. ornatus 27 Colombia: depto. Norte de Santander, Cucutilla IAvH Tissue 197 IAvH-CT31 M. o. ornatus 32 Colombia: depto. Norte de Santander, Orocue IAvH Tissue 198 JP228 M. o. ornatus 28 Venezuela: Estado Táchira, PN Tama COP Tissue 199 JP229 M. o. ornatus 20 Venezuela: Estado Táchira, PN Tama COP Tissue 200 JP232 M. o. ornatus 33 Venezuela: Estado Táchira, PN Tama COP Tissue 201 JP233 M. o. ornatus 8 Venezuela: Estado Táchira, PN Tama COP Tissue 202 ICN3603 M. o. ornatus 20 Colombia: depto. Norte de Santander, Calvario IAvH Tissue 203 IAvH-A-14832 M. o. ornatus 22 Colombia: depto. Norte de Santander, Orocue IAvH Tissue 204 AMC1371 M. o. ornatus 20 Colombia: depto. Cundinamarca, Guasca, Rincón del Oso ANDES Tissue 205 AMC1372 M. o. ornatus 20 Colombia: depto. Cundinamarca, Guasca, Rincón del Oso ANDES Tissue 206 AMC1373 M. o. ornatus 20 Colombia: depto. Cundinamarca, Guasca, Rincón del Oso ANDES Tissue 207 LNCA12 M. o. ornatus 20 Colombia: depto. Cundinamarca, Subachoque, La Pradera ANDES Tissue 208 LNCA6 M. o. ornatus 20 Colombia: depto. Cundinamarca, Subachoque, La Pradera ANDES Tissue 209 PAM60 M. o. ornatus 23 Colombia: depto. Cundinamarca, Subachoque, La Pradera ANDES Tissue 210 PAM62 M. o. ornatus 20 Colombia: depto. Cundinamarca, Subachoque, La Pradera ANDES Tissue 211 IAvH-CT-53 M. o. ornatus 1 Colombia: depto. Norte de Santander IAvH Tissue 212 IAvH-CT-1124 M. o. ornatus 43 Colombia: depto. Boyacá, Villa de leyva IAvH Tissue 213 IAvH-CT-1129 M. o. ornatus 42 Colombia: depto. Boyacá, Villa de leyva IAvH Tissue 214 IAvH-CT-7913 M. o. ornatus 42 Colombia: depto. Boyacá, Villa de leyva IAvH Tissue 215 IAvH-CT-1661 M. o. ornatus 33 Colombia: depto. Norte de Santander, Cucutilla IAvH Tissue 216 IAvH-CT-2524 M. o. ornatus 20 Colombia: depto. Meta, San Juanito IAvH Tissue 217 IAvH-CT-2566 M. o. ornatus 20 Colombia: depto. Cundinamarca, Guasca IAvH Tissue 218 IAvH-CT-4159 M. o. ornatus 24 Colombia: depto. Boyacá, Villa de leyva IAvH Tissue 219 IAvH-CT-4178 M. o. ornatus 34 Colombia: depto. Boyacá, Villa de leyva IAvH Tissue 220 IAvH-CT-6822 M. o. ornatus 50 Colombia: depto. Cundinamarca, Bojacá IAvH Tissue 221 IAvH-CT-6961 M. o. ornatus 21 Colombia: depto. Boyacá, Villa de leyva IAvH Tissue 222 IAvH-CT-9642 M. o. ornatus 9 Colombia: depto. Meta IAvH Tissue 1 Toepad samples were taken from study skins collected between 2005 and 2008.

55 Supplementary Table 2. Sound recordings used for acoustic measurements, organized from southernmost to northernmost localities. ML: Macaulay Library, XC: xeno-canto.

Catalog number Source Locality Recordist Latitude Longitude 1 XC8016 xeno-canto Ecuador: prov. Zamora-Chinchipe, Reserva Tapichalaca Nick Athanas -4.496 -79.132 2 - Personal archive Ecuador: prov. Zamora-Chinchipe, Reserva Tapichalaca Manuel Sánchez -4.496 -79.132 3 XC155429 xeno-canto Ecuador: prov. Loja, Lagunas de Manú Leonardo Ordoñez -3.574 -79.427 4 ML48357671 This study Ecuador: prov. Tungurahua, cantón Patate, parroquia El Triunfo Laura Céspedes -1.316 -78.403 5 ML48358061 This study Ecuador: prov. Tungurahua, cantón Patate, parroquia El Triunfo Laura Céspedes -1.316 -78.403 6 ML48358631 This study Ecuador: prov. Tungurahua, cantón Patate, parroquia El Triunfo Laura Céspedes -1.316 -78.403 7 ML48359161 This study Ecuador: prov. Tungurahua, cantón Patate, parroquia El Triunfo Laura Céspedes -1.316 -78.403 8 ML48359721 This study Ecuador: prov. Tungurahua, cantón Patate, parroquia El Triunfo Laura Céspedes -1.316 -78.403 9 ML48360591 This study Ecuador: prov. Tungurahua, cantón Patate, parroquia El Triunfo Laura Céspedes -1.316 -78.403 10 ML48361381 This study Ecuador: prov. Tungurahua, cantón Patate, parroquia El Triunfo Laura Céspedes -1.316 -78.403 11 ML48363001 This study Ecuador: prov. Tungurahua, cantón Patate, parroquia El Triunfo Laura Céspedes -1.316 -78.403 12 ML48363671 This study Ecuador: prov. Tungurahua, cantón Patate, parroquia El Triunfo Laura Céspedes -1.316 -78.403 13 XC242857 xeno-canto Ecuador: prov. Napo, Salcedo-Tena road Niels Krabbe -0.994 -78.288 14 ML46744121 This study Ecuador: prov. Napo, Yanayacu Biological Station Laura Céspedes -0.640 -77.913 15 - This study Ecuador: prov. Napo, Yanayacu Biological Station Paulo Pulgarín -0.640 -77.913 16 - This study Ecuador: prov. Napo, Yanayacu Biological Station Paulo Pulgarín -0.640 -77.913 17 - This study Ecuador: prov. Napo, Yanayacu Biological Station Paulo Pulgarín -0.640 -77.913 18 - This study Ecuador: prov. Napo, Yanayacu Biological Station Paulo Pulgarín -0.640 -77.913 19 ML83147 Macaulay Library Ecuador: prov. Pichincha, Refugio de Vida Silvestre Pasochoa Theodore A. Parker -0.433 -78.483 20 ML48390161 This study Ecuador: prov. Pichincha, Refugio de Vida Silvestre Pasochoa Laura Céspedes -0.425 -78.517 21 ML48392891 This study Ecuador: prov. Pichincha, Refugio de Vida Silvestre Pasochoa Laura Céspedes -0.425 -78.517 22 ML48393181 This study Ecuador: prov. Pichincha, Refugio de Vida Silvestre Pasochoa Laura Céspedes -0.425 -78.517 23 ML48393831 This study Ecuador: prov. Pichincha, Refugio de Vida Silvestre Pasochoa Laura Céspedes -0.425 -78.517 24 ML48394441 This study Ecuador: prov. Pichincha, Refugio de Vida Silvestre Pasochoa Laura Céspedes -0.425 -78.517 25 ML48394681 This study Ecuador: prov. Pichincha, Refugio de Vida Silvestre Pasochoa Laura Céspedes -0.425 -78.517 26 ML48394901 This study Ecuador: prov. Pichincha, Refugio de Vida Silvestre Pasochoa Laura Céspedes -0.425 -78.517 27 ML48395121 This study Ecuador: prov. Pichincha, Refugio de Vida Silvestre Pasochoa Laura Céspedes -0.425 -78.517 28 ML46745091 This study Ecuador: prov. Napo, Termas de Papallacta Laura Céspedes -0.358 -78.151 29 ML46745591 This study Ecuador: prov. Napo, Termas de Papallacta Laura Céspedes -0.358 -78.151 30 ML46745731 This study Ecuador: prov. Napo, Termas de Papallacta Laura Céspedes -0.358 -78.151 31 ML46746451 This study Ecuador: prov. Napo, Termas de Papallacta Laura Céspedes -0.358 -78.151 32 ML46746971 This study Ecuador: prov. Napo, Termas de Papallacta Laura Céspedes -0.358 -78.151

56 33 ML46747041 This study Ecuador: prov. Napo, Termas de Papallacta Laura Céspedes -0.358 -78.151 34 ML46747361 This study Ecuador: prov. Napo, Termas de Papallacta Laura Céspedes -0.358 -78.151 35 ML47834081 This study Ecuador: prov. Imabura, Hacienda Zuleta Laura Céspedes 0.198 -78.072 36 ML47834191 This study Ecuador: prov. Imabura, Hacienda Zuleta Laura Céspedes 0.198 -78.072 37 ML47834771 This study Ecuador: prov. Imabura, Hacienda Zuleta Laura Céspedes 0.198 -78.072 38 ML47835911 This study Ecuador: prov. Imabura, Hacienda Zuleta Laura Céspedes 0.198 -78.072 39 ML48320471 This study Ecuador: prov. Imabura, Hacienda Zuleta Laura Céspedes 0.198 -78.072 40 ML48320821 This study Ecuador: prov. Imabura, Hacienda Zuleta Laura Céspedes 0.198 -78.072 41 ML48349351 This study Ecuador: prov. Imbabura, Bosque de Nueva América Laura Céspedes 0.260 -77.982 42 ML48349731 This study Ecuador: prov. Imbabura, Bosque de Nueva América Laura Céspedes 0.260 -77.982 43 ML48350251 This study Ecuador: prov. Imbabura, Bosque de Nueva América Laura Céspedes 0.260 -77.982 44 ML48350561 This study Ecuador: prov. Imbabura, Bosque de Nueva América Laura Céspedes 0.260 -77.982 45 ML48350941 This study Ecuador: prov. Imbabura, Bosque de Nueva América Laura Céspedes 0.260 -77.982 46 ML48351201 This study Ecuador: prov. Imbabura, Bosque de Nueva América Laura Céspedes 0.260 -77.982 47 ML48352071 This study Ecuador: prov. Imbabura, Bosque de Nueva América Laura Céspedes 0.260 -77.982 48 ML48352541 This study Ecuador: prov. Imbabura, Bosque de Nueva América Laura Céspedes 0.260 -77.982 49 ML45664351 This study Ecuador: prov. Carchi, Guandera Reserve and Biological Station Laura Céspedes 0.589 -77.704 50 ML45665191 This study Ecuador: prov. Carchi, Guandera Reserve and Biological Station Laura Céspedes 0.589 -77.704 51 ML46082461 This study Ecuador: prov. Carchi, Guandera Reserve and Biological Station Laura Céspedes 0.589 -77.704 52 ML46083031 This study Ecuador: prov. Carchi, Guandera Reserve and Biological Station Laura Céspedes 0.589 -77.704 53 - This study Ecuador: prov. Carchi, Guandera Reserve and Biological Station Paulo Pulgarín 0.589 -77.704 54 - This study Ecuador: prov. Carchi, Guandera Reserve and Biological Station Paulo Pulgarín 0.589 -77.704 55 ML41832831 This study Colombia: depto. Nariño, Ipiales Laura Céspedes 0.736 -77.652 56 ML41833101 This study Colombia: depto. Nariño, Ipiales Laura Céspedes 0.736 -77.652 57 ML41833671 This study Colombia: depto. Nariño, Ipiales Laura Céspedes 0.736 -77.652 58 ML41833561 This study Colombia: depto. Nariño, Ipiales Laura Céspedes 0.736 -77.652 59 ML41833521 This study Colombia: depto. Nariño, Ipiales Laura Céspedes 0.736 -77.652 60 - This study Colombia: depto. Nariño, Ipiales Laura Céspedes 0.736 -77.652 61 - This study Colombia: depto. Nariño, Ipiales Laura Céspedes 0.736 -77.652 62 ML43107971 This study Colombia: depto. Nariño, Córdoba Laura Céspedes 0.815 -77.480 63 ML43108181 This study Colombia: depto. Nariño, Córdoba Laura Céspedes 0.815 -77.480 64 ML43108611 This study Colombia: depto. Nariño, Córdoba Laura Céspedes 0.815 -77.480 65 ML43109081 This study Colombia: depto. Nariño, Córdoba Laura Céspedes 0.815 -77.480 66 ML43109421 This study Colombia: depto. Nariño, Córdoba Laura Céspedes 0.815 -77.480 67 ML43109661 This study Colombia: depto. Nariño, Córdoba Laura Céspedes 0.815 -77.480 68 ML41831571 This study Colombia: depto. Nariño, Puerres Laura Céspedes 0.867 -77.460

57 69 ML40466281 This study Colombia: depto. Nariño, Puerres Andrés Cuervo 0.867 -77.460 70 ML41831891 This study Colombia: depto. Nariño, Puerres Laura Céspedes 0.867 -77.460 71 ML41831831 This study Colombia: depto. Nariño, Puerres Laura Céspedes 0.867 -77.460 72 ML41831771 This study Colombia: depto. Nariño, Puerres Laura Céspedes 0.867 -77.460 73 ML43106201 This study Colombia: depto. Nariño, Puerres Laura Céspedes 0.867 -77.460 74 ML43106751 This study Colombia: depto. Nariño, Puerres Laura Céspedes 0.867 -77.460 75 ML38070901 This study Colombia: depto. Nariño, Funes Laura Céspedes 0.923 -77.454 76 ML38058841 This study Colombia: depto. Nariño, Funes Laura Céspedes 0.923 -77.454 77 ML36756501 This study Colombia: depto. Nariño, Funes Andrés Cuervo 0.923 -77.454 78 ML43113341 This study Colombia: depto. Nariño, Funes Laura Céspedes 0.923 -77.454 79 ML43114261 This study Colombia: depto. Nariño, Funes Laura Céspedes 0.923 -77.454 80 ML41831081 This study Colombia: depto. Nariño, Pasto, vereda Romerillo Laura Céspedes 1.081 -77.169 81 ML41830931 This study Colombia: depto. Nariño, Pasto, vereda Romerillo Laura Céspedes 1.081 -77.169 82 ML41830871 This study Colombia: depto. Nariño, Pasto, vereda Romerillo Laura Céspedes 1.081 -77.169 83 ML41830851 This study Colombia: depto. Nariño, Pasto, vereda Romerillo Laura Céspedes 1.081 -77.169 84 ML41830781 This study Colombia: depto. Nariño, Pasto, vereda Romerillo Laura Céspedes 1.081 -77.169 85 ML40537031 This study Colombia: depto. Nariño, Pasto, vereda Romerillo Andrés Cuervo 1.081 -77.169 86 ML38024671 This study Colombia: depto. Nariño, Yacuanquer Laura Céspedes 1.155 -77.389 87 ML43140041 This study Colombia: depto. Nariño, Yacuanquer Laura Céspedes 1.155 -77.389 88 ML43142151 This study Colombia: depto. Nariño, Yacuanquer Laura Céspedes 1.155 -77.389 89 ML43142671 This study Colombia: depto. Nariño, Yacuanquer Laura Céspedes 1.155 -77.389 90 ML41829441 This study Colombia: depto. Putumayo, San Francisco Laura Céspedes 1.161 -76.847 91 ML40500961 This study Colombia: depto. Putumayo, San Francisco Andrés Cuervo 1.161 -76.847 92 ML40503341 This study Colombia: depto. Putumayo, San Francisco Andrés Cuervo 1.161 -76.847 93 ML40502621 This study Colombia: depto. Putumayo, San Francisco Andrés Cuervo 1.161 -76.847 94 ML40502381 This study Colombia: depto. Putumayo, San Francisco Andrés Cuervo 1.161 -76.847 95 ML43105541 This study Colombia: depto. Putumayo, San Francisco Laura Céspedes 1.161 -76.847 96 ML43105711 This study Colombia: depto. Putumayo, San Francisco Laura Céspedes 1.161 -76.847 97 ML40484591 This study Colombia: depto. Nariño, Pasto, vereda San Felipe Andrés Cuervo 1.198 -77.315 98 ML40483161 This study Colombia: depto. Nariño, Pasto, vereda San Felipe Andrés Cuervo 1.198 -77.315 99 ML41830451 This study Colombia: depto. Nariño, Pasto, vereda San Felipe Laura Céspedes 1.198 -77.315 100 ML41830561 This study Colombia: depto. Nariño, Pasto, vereda San Felipe Laura Céspedes 1.198 -77.315 101 ML36740451 This study Colombia: depto. Nariño, Pasto, vereda San Felipe Andrés Cuervo 1.198 -77.315 102 ML38019201 This study Colombia: depto. Nariño, Municipo Pasto, Alto de Daza Laura Céspedes 1.267 -77.254 103 ML38019491 This study Colombia: depto. Nariño, Municipo Pasto, Alto de Daza Laura Céspedes 1.267 -77.254 104 ML38020281 This study Colombia: depto. Nariño, Municipo Pasto, Alto de Daza Laura Céspedes 1.267 -77.254

58 105 ML38021051 This study Colombia: depto. Nariño, Municipo Pasto, Alto de Daza Laura Céspedes 1.267 -77.254 106 ML41829961 This study Colombia: depto. Nariño, Municipo Pasto, Alto de Daza Laura Céspedes 1.267 -77.254 107 ML36755081 This study Colombia: depto. Nariño, Municipo Pasto, Alto de Daza Andrés Cuervo 1.267 -77.254 108 ML38085121 This study Colombia: depto. Nariño, La Cruz Laura Céspedes 1.543 -76.923 109 ML38095851 This study Colombia: depto. Nariño, La Cruz Laura Céspedes 1.543 -76.923 110 ML36823331 This study Colombia: depto. Nariño, La Cruz Andrés Cuervo 1.543 -76.923 111 ML36823041 This study Colombia: depto. Nariño, La Cruz Andrés Cuervo 1.543 -76.923 112 ML36803201 This study Colombia: depto. Nariño, La Cruz Andrés Cuervo 1.543 -76.923 113 ML38105701 This study Colombia: depto. Nariño, San Pablo Laura Céspedes 1.715 -76.934 114 ML38105791 This study Colombia: depto. Nariño, San Pablo Laura Céspedes 1.715 -76.934 115 ML36825041-ML36826091 This study Colombia: depto. Nariño, San Pablo Andrés Cuervo 1.715 -76.934 116 ML54029311 This study Colombia: depto. Nariño, San Pablo Laura Céspedes 1.715 -76.934 117 ML54030081 This study Colombia: depto. Nariño, San Pablo Laura Céspedes 1.715 -76.934 118 ML54029711 This study Colombia: depto. Nariño, San Pablo Laura Céspedes 1.715 -76.934 119 ML54021731 This study Colombia: depto. Cauca, Inzá Laura Céspedes 2.464 -76.210 120 ML54021961 This study Colombia: depto. Cauca, Inzá Laura Céspedes 2.464 -76.210 121 ML54023191 This study Colombia: depto. Cauca, Inzá Laura Céspedes 2.464 -76.210 122 ML54023171 This study Colombia: depto. Cauca, Inzá Laura Céspedes 2.464 -76.210 123 ML54023201 This study Colombia: depto. Cauca, Inzá Laura Céspedes 2.464 -76.210 124 ML54023671 This study Colombia: depto. Cauca, Inzá Laura Céspedes 2.464 -76.210 125 ML54024291 This study Colombia: depto. Cauca, Inzá Laura Céspedes 2.464 -76.210 126 ML54025151 This study Colombia: depto. Cauca, Inzá Laura Céspedes 2.464 -76.210 127 XC128054 xeno-canto Colombia: depto. Tolima, Reserva Ibanasca Oscar Marín 4.484 -75.480 128 - Personal archive Colombia: depto. Tolima, Reserva Ibanasca Oscar Laverde 4.591 -75.249 129 XC128596 xeno-canto Colombia: depto. Quindío, Salento, AICA La Patasola Oscar Marín 4.686 -75.547 130 - Personal archive Colombia: depto. Caldas, Reserva Río Blanco Oscar Laverde 5.074 -75.438 131 ML44479681 This study Colombia: depto. Antioquia, San Pedro de los Milagros Andrés Cuervo 6.456 -75.601 132 ML44479691 This study Colombia: depto. Antioquia, San Pedro de los Milagros Andrés Cuervo 6.456 -75.601 133 ML44480091 This study Colombia: depto. Antioquia, San Pedro de los Milagros Andrés Cuervo 6.456 -75.601 134 ML44480421 This study Colombia: depto. Antioquia, San Pedro de los Milagros Andrés Cuervo 6.456 -75.601

59