Mongabay.com Open Access Journal - Tropical Conservation Science Vol.7 (4):677-689, 2014

Research Article

Phylogenetic position of the most endangered Chilean bird: the Masafuera Rayadito (Aphrastura masafuerae; Furnariidae)

Javier Gonzalez1 1Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24–25, 14476 Potsdam, Germany. Corresponding author: Dr. Javier Gonzalez Email: [email protected]

Abstract Masafuera Rayadito (Aphrastura masafuerae; Furnariidae) is a Critically Endangered species endemic to Alejandro Selkirk Island (Juan Fernández Archipelago, Chile). Categorized as probably extinct in 1980, later estimates, ranging from 140 (in 2002) to 500 individuals (in 2006–2007), showed a fluctuating population size of the species. The grazing of goats and cattle has increased habitat loss for the species. Other threats are lack of nesting sites, introduced species such as feral cats and rats (Rattus rattus and R. norvegicus), and increased populations of natural predators like the Masafuera Hawk. In order to increase the availability of nesting sites, 81 nest boxes were installed in different habitats in 2006, with evidence of use during subsequent breeding seasons. Despite conservation concerns, however, no genetic studies are yet available for this furnariid. This study reports for the first time the levels of genetic divergence of the species, based on nucleotide sequences of the mitochondrial DNA (cytochrome oxidase subunit 1 gene; COI). Aphrastura masafuerae is closely related to a widespread species of furnariid distributed mainly in Chile on the mainland, the Thorn-tailed Rayadito (A. spinicauda). The Masafuera Rayadito diverged from its mainland sister species probably during the Pleistocene 0.57 ± 0.19 Myr ago. Consistent with mitochondrial and nuclear molecular clocks, the estimated time of the split between A. masafuerae and A. spinicauda is in perfect agreement with the geological origin of the Juan Fernández Archipelago, which is of volcanic origin. In order to assess genetic variability within the population of this fragile bird, further studies using a multi-locus genetic approach at the population level are necessary.

Keywords: Furnariidae, Juan Fernandez Archipelago, mitochondrial DNA, molecular clock, Threatened species

Received: 29 July 2014; Accepted 16 October 2014; Published: 15 December 2014

Copyright: © Javier Gonzalez. This is an open access paper. We use the Creative Commons Attribution 4.0 license http://creativecommons.org/licenses/by/3.0/us/. The license permits any user to download, print out, extract, archive, and distribute the article, so long as appropriate credit is given to the authors and source of the work. The license ensures that the published article will be as widely available as possible and that your article can be included in any scientific archive. Open Access authors retain the copyrights of their papers. Open access is a property of individual works, not necessarily journals or publishers.

Cite this paper as: Gonzalez, J. 2014. Phylogenetic position of the most endangered Chilean bird: the Masafuera Rayadito (Aphrastura masafuerae; Furnariidae). Tropical Conservation Science Vol.7 (4): 677-689. Available online: www.tropicalconservationscience.org

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Introduction Archaeological data show high rates of extinction on the Pacific islands [1]. However, molecular phylogenetic studies reveal radiations of several songbirds that might have started on islands, increasing avian diversity on nearby continental regions [2]. The high endemism found on islands [3] correlates often with high rates of extinction because of inbreeding and human impact [4]. Thus, island ecosystems become biodiversity hotspots and at the same time have a high conservation priority.

Usually found in dense vegetation cover [5], the Masafuera Rayadito (Aphrastura masafuerae; Furnariidae) is a small, buff-brownish bird characterized by reddish spine-like tail feathers and a light superciliary stripe ([6] and references therein). This species is endemic to Alejandro Selkirk Island (Juan Fernández Archipelago, Chile) and is declared a Critically Endangered species by the International Union for Conservation of Nature (IUCN) [7]. In 1980, Vaurie [8] thought that the species was probably extinct, and population size estimates of the early 1990s have been as low as 140 individuals [9]. Subsequent conservative estimates, during the post-breeding season of 2006 and 2007 [10], suggested a larger population size of 500 individuals, indicating a fluctuating population size of the species during recent decades. The grazing of goats and cattle has increased habitat loss for the species. The lack of nesting sites, introduced species such as feral cats and rats (Rattus rattus and R. norvegicus) and increased populations of natural predators like the Masafuera Hawk (Buteo polyosoma exsul) are threatening this bird as well [6,10].

Conservation efforts have concentrated on nesting and feeding behavior, breeding population size, censuses, critical habitat, and other ecological features of this furnariid [5,6,10]. In 2006, for example, 81 nest boxes were installed in order to increase the availability of nesting sites. Evidence of use of these nesting boxes by the Masafuera Rayadito was found in only seven boxes located in the southern part of the island [10]. Despite conservation concerns, genetic studies are available only for the sister species (Aphrastura spinicauda) and close relatives [11-13]. Nowadays, wildlife conservation programs nearly always integrate molecular techniques to explore the genetic makeup of species of concern. Considering that the Masafuera Rayadito is threatened with extinction, genetic data are needed for future conservation programs. Using nucleotide sequences of the mitochondrial DNA (cytochrome oxidase subunit 1 gene; COI), this study reports for the first time the levels of genetic divergence of this endangered species from its mainland sister species, the Thorn-tailed Rayadito.

Methods Sampling Two individuals were captured with mist-nets on Alejandro Selkirk (33°45’ S, 80°45’ W, see Fig. 1), the westernmost island of the Juan Fernández Archipelago. This steep volcanic island is located 181 km west of Robinson Crusoe Island and 835 km off the Chilean coast. See Castilla [14] for detailed information about climate and other geographical characteristics of the island. The birds were captured on the summit region of the island (Los Inocentes peak; 1,200 m above sea level) where the species normally reproduces (Fig. 2). The vegetation of the area consists of dominant fern Lophosoria quadripinnata (95–100% cover), tree- ferns (Dicksonia externa and Blechnum cycadifolium) and the tree Drimys confertifolia. See Hahn et al. [6] for detailed information regarding the nesting habitat of the species. Blood samples were obtained by puncturing the brachial vein and stored on FTA cards (Whatman, Germany). Immediately after blood sampling, the birds were released in the same place where they ware captured. The capture of the two individuals in Alejandro Selkirk Island was authorized by CONAF (Corporación Nacional Forestal, Archipiélago de Juan Fernández National Park).

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80º45´ W 78º50´ W Alejandro Selkirk Robinson Crusoe Island Island

Pacific Ocean 34º45´ S 33º39´ S

Santa Clara 4 km Island 5 km

Fig. 1. Map of the study area. Alejandro Selkirk Island, Archipiélago de Juan Fernández National Park, Chile

DNA isolation, PCR amplification and sequencing of COI The DNA was extracted from FTA cards following the manufacturer’s instructions. Briefly, the FTA card sample discs (1.2 mm) were placed in the PCR amplification tubes and washed two times with 200 µl of FTA purification reagent. After removal from the purification reagent, the discs were washed twice with 200 µl of TE buffer. FTA discs were dried at room temperature for 1 hr.

The primers BirdF1 and COIbirdR2 [15] were used to amplify about 700 base pairs (bp) of the mitochondrial cytochrome oxidase subunit 1 gene (COI). The PCR reaction mix (25 µL) contained the following components: 2 µL of DNA template (60 ng of DNA), 2.5 µL AmpliTaq® 360 Buffer (10✕), 2 µL of 25 mM MgCl2, 2 µL of 10 mM solution of dNTP and 0.125 µL AmpliTaq® 360 DNA Polymerase (Applied Biosystems, Germany).

PCR cycles were performed as follows: 94°C for 5 min, 33 cycles of 94°C for 1 min, 50°C for 40 s, 72°C for 40 s, and a final extension at 72°C for 5 min. PCR products were visualized on 1.4% agarose gels. Sequencing of both strands was conducted using an ABI 3730XL Capillary Sequencer (Applied Biosystems, Germany) with the BigDye® Terminator Cycle Sequencing Kit version 3.1 by GATC Biotech AG (Konstanz, Germany).

The sequences generated in this study have been deposited in GenBank under accession numbers JQ739454 and JQ739455.

Data analysis COI sequences were retrieved from the GenBank for two families of the Furnarioidea, i.e. Furnariidae and Dendrocolaptidae [13]. Sequences were aligned with BIOEDIT v. 7.0.9.0 [16], and phylogenetic trees were reconstructed using maximum likelihood (ML) in PAUP* v. 4.0b10a [17], and Bayesian inference (BI) in MRBAYES v. 3.1.2 [18]. We explored the model of sequence evolution that fits the data best with JMODELTEST v. 0.1.1 [19] and MRMODELTEST v. 2.3 [20]. ML heuristic searches were performed with closest stepwise sequence additions, tree-bisection-reconnection, branch-swapping (TBR), ‘multrees’ option and the best model found with JMODELTEST. In the ML analyses, the robustness of each node was

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Mongabay.com Open Access Journal - Tropical Conservation Science Vol.7 (4):677-689, 2014 assessed by 1,000 bootstrap replicates. For BI analyses, two independent runs of 10,000,000 generations each were performed along with four Markov chains. Trees were sampled every 500 generations and the first 4,000 samples were discarded as ‘burn-in’. Uncorrected genetic distances (p-distance) were calculated with MEGA v. 5 [21]. Phylogenetic trees were rooted with five representatives of the tapaculos (Rhinocryptidae) and the Elegant Crescentchest (Melanopareia elegans; see [13,22,23]).

For the molecular dating, an uncorrelated lognormal (UCLN) model of molecular evolutionary rate heterogeneity was also used in the computer program BEAST v. 1.6.2 [24,25]. The analysis was conducted using the model found by JMODELTEST v. 0.1.1 [19] that fits the data best. The universal avian clock of 2.1% sequence divergence per million years (0.0105 substitutions/site/lineage/million years) was employed in these analyses [26]. Two independent runs of 20,000,000 generations each were performed with sampling once every 1,000 trees. The number of generations required to reach convergence was assessed by TRACER v. 1.5 (http://beast.bio.ed.ac.uk/Tracer).

Fig. 2. View from “Los Inocentes” peak, Alejandro Selkirk Island, Archipiélago de Juan Fernández National Park, Chile. The vegetation cover consists mainly of fern Lophosoria quadripinnata and the tree Drimys confertifolia (left). The Masafuera Rayadito (Aphrastura masafuerae) caught by a mist-net for blood sampling (right)

Results The alignment of the protein-encoding gene COI consisted of 694 bp. No internal stop codons or frame shifts were found in these sequences that translated entirely by using the chicken mitochondrial code. In both individuals of the Masafuera Rayadito, variation was found to be at only one site (0.14%) and corresponds to a synonymous transition (T/C) at the third position of the codon (GCT/GCC) that encodes the amino acid adenine.

Figure 3 shows the phylogeny based on COI nucleotide sequences. Maximum likelihood and Bayesian inference analyses recovered congruent topologies (see Appendices 1 and 2). Topological incongruence was due only to nodes supported by low bootstrap or posterior probability values. The phylogenetic reconstruction reveals a polytomy and a basal position of Aphrastura among the Furnariidae, and one major separate clade for the woodcreepers (Dendrocolaptidae). Aphrastura masafuerae is closely related

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Mongabay.com Open Access Journal - Tropical Conservation Science Vol.7 (4):677-689, 2014 to A. spinicauda on the mainland. Both species clustered together with high bootstrap support (see Fig. 3).

The uncorrected genetic distance (p-distance) between Aphrastura masafuerae and A. spinicauda was 1.2 ± 0.4%. Assuming an avian clock of roughly 2.1%/Myr for the mitochondrial DNA [26] these taxa might have recently diverged 0.57 ± 0.19 Myr ago. The Bayesian inference molecular clock analysis shows a similar divergence time estimate of 0.71 Myr with a 95% confidence interval of 1.18–0.34 Myr (see Appendix 3).

Fig. 3. Majority-rule consensus tree derived from Bayesian inference (BI) analysis and based on 694 bp of the cytochrome oxidase subunit 1 gene (COI). BI posterior probabilities (≥0.90) are indicated above the branches. The model selected for the BI analysis consisted of GTR + gamma distribution shape parameter (G) = 1.42 + proportion of invariable sites (I) = 0.60. In parentheses are the numbers of species represented by the terminal triangles. See Supplementary material for GenBank accession numbers and complete detailed phylogenies

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Discussion Phylogenetic relationships The topology obtained in this study conforms to accepted knowledge about the infraorder Furnariides based on nuclear and mitochondrial DNA [13,27-29]. Regarding Aphrastura, the phylogenetic position of the genus has adopted different phylogenetic positions within the Furnariidae depending on the marker and taxa analyzed. Considering nuclear genes, nucleotide sequences from introns do not provide strong support for the relationship of this genus with any other furnariid species in particular, for instance, Aphrastura clusters with Coryphistera or Leptasthenura [30]. However, these analyses included a reduced number of species [30]. Although the phylogenetic relationships of Aphrastura and other furnariids are only partially clear, a larger data set (about 4,000 bp and additional taxa) based on recombination activating genes supports a basal position for the genus among the Synallaxinae with high/medium bootstrap support [13].

As in this study, the evidence recovered from another mitochondrial gene, cytochrome b, supports an unresolved polytomy at the base of the Furnariidae but a closer relationship of Aphrastura to Synallaxinae and the tribe Furnariini [30]. Combined data sets including sequences of nuclear introns and mitochondrial genes also support a basal position of this lineage among Synallaxinae [12,30].

Based on a molecular clock for the myoblogin intron 2 in passerines [29], the genus Aphrastura probably diverged from other furnariids at the Lower Miocene [12]. In this study, Bayesian molecular clock analyses show also similar ages (19–18 Myr) for the split between the Synallaxinae and Furnariinae (see Appendix 3).

Juan Fernández Archipelago colonization The remote Pacific Island Alejandro Selkirk is located 181 km west of Robinson Crusoe and 835 km west of South America. Thus, for dispersing birds like the Thorn-tailed Rayadito [11], the Juan Fernández Archipelago represents a biogeographically remote group of islands isolated by a major but traversable oceanic barrier. The spatial separation of this archipelago has turned out to be a hotspot of avian endemism [31].

The level of genetic divergence between Aphrastura masafuerae and A. spinicuada supports a recent colonization of the Juan Fernández Archipelago by the Masafuera Rayadito in the South Pacific Ocean, approximately 0.65 Myr ago. These time estimates do not predate the origin of the Juan Fernández Archipelago and are in perfect agreement with the geological age based on potassium-argon dating [32]. The three main islands of the Juan Fernández Archipelago are not older than 5.8 ± 2.1 Myr, and Alejandro Selkirk Island is the youngest one with 2.44–1.01 Myr [32]. Previous genetic studies performed on other Juan Fernández passerines recovered similar time divergences. For instance, using mitochondrial genetic markers like the cytochrome b gene, colonization time estimates of 700,000 and 300,000 years have been suggested for the Juan Fernandez Firecrown (Sephanoides fernandensis; [33]) and the Juan Fernandez Tit- Tyrant (Anairetes fernandezianus; [34]), respectively.

The Masafuera Rayadito might have colonized Alejandro Selkirk Island after the emergence of the island during the Pleistocene. However, it remains unclear whether this avian species arrived at Alejandro Selkirk by a direct colonization from the mainland or from neighboring islands like Masatierra or Santa Clara, which are 3.79–4.23 and 5.8 Myr old, respectively. One plausible scenario for this cross-oceanic dispersal is an east-west colonization followed by later extinction events on older eastern islands. Modes of

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Mongabay.com Open Access Journal - Tropical Conservation Science Vol.7 (4):677-689, 2014 speciation such as dispersals from Masatierra to Masafuera and back-dispersal events have been proposed for several species of plants [32,35]. Thus, a step-stone model of colonization for the Juan Fernández Islands by birds, as in other archipelagos (e.g. the Canary Islands; [36]), cannot be discarded.

Implications for conservation Genetic data may provide critical information about population structure [37], genetic variability [38], taxonomic status [39] and conservation management units [40]. Estimation of the phylogenetic distance among species of a community is essential for determining phylogenetic diversity and the functioning of ecosystems [41]. The geographical isolation of the Juan Fernández Archipelago has resulted in the mere morphological description of several endemic species of birds, and only a few genetics studies conducted on Robinson Crusoe Island are available so far [33,34]. For the first time, the new genetic data obtained in the present study provide insights about the level of divergence between the endangered Masafuera Rayadito thriving on Alejandro Selkirk and the widespread Thorn-tailed Rayadito on the mainland.

With approximately 130 species, the Synallaxinae (spinetails and allies) contribute nearly half of the diversity found within the Furnariidae ([42]), and both species Aphrastura masafuerae and A. spinicauda are the sole representative of a probable basal lineage in the phylogeny of this speciose group of Neotropical birds ([12,13]).

Despite the existing hesitation to include phylogenetic diversity in conservation planning ([43]; but see [44]) and in order to keep evolutionary history to the highest level possible, many conservation studies incorporate phylogenetic diversity analyses in order to maximize the number of clades rather than the number of species conserved, giving a high relative weight to species which are taxonomically distinct [45]. Lineages containing few or no sister taxa, which is the case of Aphrastura, may contribute greatly to phylogenetic diversity ([46]). With relatively long-branch lengths leading to the two extant species of Aphrastura in all phylogenetic reconstructions (this study), this genus does contribute importantly to the phylogenetic diversity within the Furnariidae. Thus, coupled with its small population size and restricted distribution on a remote island, the phylogenetic position of the Masafuera Rayadito is an additional biodiversity component supporting the high conservation priority of the species.

Population size fluctuations (e.g. bottlenecks) may be caused by glaciations, climate change, or anthropogenic impact determining the demographic history of the species ([47]). Based on the level of genetic divergence estimated in this study, the Masafuera Rayadito has probably survived in isolation on Alejandro Selkirk Island during the last 600,000 years. We do not know how much the environment of this species has changed during the last hundred thousand years, but human impact has been documented on Alejandro Selkirk during recent decades ([6,10]). Census estimates indicate that the population of this endangered species has probably been fluctuating during this time (for instance, see [9] and [10]).

Genetic variation dynamics of small and isolated populations like that of Masafuera Rayadito are explained mainly by two phenomena: genetic drift, which may lead to loss of adaptive alleles or the fixation of deleterious alleles; and inbreeding, which may increase homozygosity within the population ([48]). As a consequence of these phenomena, the viability of populations may be affected by a fitness reduction or inbreeding depression. How did the founder population of the Masafuera Rayadito manage to overcome inbreeding depression or survive probably successive bottlenecks on a remote island like Alejandro Selkirk during the last hundred thousand years? The species has probably been able to maintain a certain level of genetic variability. For instance, the differences found on nucleotide sequences obtained from two individuals (this study) indicate some degree of intrapopulation genetic variability, enabling the

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Mongabay.com Open Access Journal - Tropical Conservation Science Vol.7 (4):677-689, 2014 persistence and survival of the lineage. However, these data are not enough to determine the genetic makeup of the species, since they are based on only two individuals and one genetic marker. In order to understand the time and mode of evolution of the Masafuera Rayadito as part of an insular endemic avifauna inhabiting the remote Juan Fernández Archipelago, further studies using a multilocus genetic approach at the species and population level are necessary.

Acknowledgements I am grateful to Javiera Meza (Alejandro Selkirk, Archipiélago de Juan Fernández National Park, Corporación Nacional Forestal, CONAF), and Horacio Merlet and Charif Tala from the Servicio Agrícola Ganadero (SAG) for allowing the collection of biological samples. I thank the biologists Hernán Díaz, who helped me with the logistics, Gastón Correa and Ramon Schiller (CONAF), who advised and supported me in the field. I thank Christina Renk (Whatman GmbH), who kindly provided the FTA cards. The Bundesministerium für Bildung und Forschung (BMBF-DLR: CHL 01/016; many thanks to Dr. Hans-Ulrich Peter, Friedrich-Schiller-Universität Jena) funded the fieldwork and laboratory costs partially. I thank also an anonymous referee who kindly provided valuable comments and advice to improve this manuscript.

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Synallaxis albescens FJ028349 100 Synallaxis azarae FJ028355 Synallaxis frontalis EU233026 Synallaxis spixi FJ028362 96 Synallaxis ruficapilla JN801997 Synallaxis rutilans JN801998 Synallaxis scutata JN802002 Certhiaxis cinnamomeus FJ027330 80 Spartonoica maluroides FJ028294 Schoeniophylax phryganophilus FJ028248 96 Asthenes patagonica FJ027188 Asthenes steinbachi FJ027200 e 99 a Asthenes anthoides FJ027173 n

80 i Asthenes modesta FJ027184 88 87 x

Asthenes pyrrholeuca FJ027191 a

l

100 Asthenes baeri FJ027176 l Asthenes dorbignyi FJ027181 a 86 n

Cranioleuca pyrrhophia FJ027453 y

Pseudoseisura gutturalis FJ028167 S 97 Phacellodomus ruber FJ027987 F Phacellodomus maculipectus FJ027986 100 Phacellodomus striaticollis FJ027997 94 Phacellodomus rufifrons FJ027995 u Phacellodomus striaticeps FJ027996 92 92 Anumbius annumbi FJ027133 r Coryphistera alaudina FJ027437 88 98 Leptasthenura aegithaloides FJ027721 100 Leptasthenura fuliginiceps FJ027731 n Leptasthenura platensis FJ027734 Sylviorthorhynchus desmursii FJ028346 a

Cinclodes atacamensis FJ027378 i 94

Cinclodes patagonicus FJ027389 n

85 i Cinclodes fuscus FJ027383 i r

r

Upucerthia dumetaria FJ028530 a 95 Furnarius rufus FJ027595 n r i 88 Phleocryptes melanops FJ028019

u

Lochmias nematura JN801778 F Philydor lichtensteini FJ028015 i 91 Philydor ruficaudatum JN801912 Anabacerthia amaurotis JN801485 i n d Heliobletus contaminatus JN801710 i 82 r

Philydor atricapillus FJ028013 o

Syndactyla rufosuperciliata FJ028364 d a

97 Megaxenops parnaguae JN801795 y

l Automolus ochrolaemus JN801511 i

h e 99 Automolus leucophthalmus FJ027213

84 P Hyloctistes subulatus JN801730 Ancistrops strigilatus JN801489 100 Eremobius phoenicurus FJ027556 90 Upucerthia ruficaudus FJ028542 Pygarrhichas albogularis FJ028180 Aphrastura spinicauda FJ027137 93 Aphrastura spinicauda FJ027138 Aphrastura spinicauda FJ027140 Aphrastura spinicauda FJ027141 100 Aphrastura spinicauda FJ027142 Aphrastura spinicauda FJ027139 Aphrastura masafuerae JQ739454 97 Aphrastura masafuerae JQ739455 Upucerthia certhioides FJ027917 98 Xenops minutus FJ028566 Xenops rutilans JN802101 99 Xiphorhynchus ocellatus JN802111 82 Xiphorhynchus pardalotus JQ176659 85 Xiphorhynchus fuscus FJ028574 Xiphorhynchus elegans JN802104 D 90 94 Campylorhamphus procurvoides JQ174265 100 Campylorhamphus trochilirostris FJ027291 Campylorhamphus pusillus JQ174269 e Campylorhamphus falcularius JN801531 84 Lepidocolaptes falcinellus FJ027720 n 87 Lepidocolaptes angustirostris FJ027713 95 Lepidocolaptes souleyetii JQ175231 Lepidocolaptes albolineatus JQ175228 d Drymornis bridgesii JQ174721 98 Xiphorhynchus flavigaster JN802105 r 92 Xiphorhynchus lachrymosus JN622082 93 Xiphorhynchus guttatus JQ176651 Xiphorhynchus obsoletus JQ176654 o Xiphorhynchus erythropygius JQ176647 Xiphorhynchus picus JN801634 c 82 Xiphocolaptes falcirostris JN802102 Xiphocolaptes major FJ028571 o 100 Xiphocolaptes promeropirhynchus JQ176639 Xiphocolaptes albicollis FJ028569 Xiphocolaptes promeropirhynchus JQ176641 l Nasica longirostris JN801860 Hylexetastes uniformis JN801728 a 85 Dendrocolaptes picumnus JQ174654 88 100 Dendrocolaptes platyrostris FJ027493 97 96 Dendrocolaptes certhia JN801633 p Dendrocolaptes concolor JQ174653 80 Deconychura stictolaema JQ174645 Glyphorynchus spirurus JN801688 t 98 Dendrocincla fuliginosa JN801628 Dendrocincla turdina FJ027490 i Dendrocincla fuliginosa JN622069 94 Dendrocincla homochroa JN622072 Dendrocincla anabatina EU442298 d Dendrocincla fuliginosa JN622068 Dendrocincla merula JN622077 a Dendrocincla tyrannina JN622079 95 97 Deconychura longicauda JN801623 Deconychura longicauda JQ174643 e Sittasomus griseicapillus FJ028293 100 83 Geositta cunicularia FJ027615 To outgroups Geositta punensis FJ027617 Sclerurus rufigularis JN801983 Scleruridae 0.04

Appendix 1. Detailed maximum likelihood (ML) phylogram based on 694 base pairs of the cytochrome oxidase subunit 1 gene (COI). Bootstrap values (≥0.75%; 1,000 replicates) are indicated for each node. The model selected for the ML analysis consisted of GTR + gamma distribution shape parameter (G) = 0.87 + proportion of invariable sites (I) = 0.58. GenBank accession numbers indicated for each species.

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1.00 Synallaxis albescens FJ028349 Synallaxis frontalis EU233026 0.99 Synallaxis azarae FJ028355 Synallaxis spixi FJ028362 1.00 Synallaxis ruficapilla JN801997 Synallaxis scutata JN802002 Synallaxis rutilans JN801998 1.00 Asthenes anthoides FJ027173 Asthenes modesta FJ027184 0.96 Asthenes pyrrholeuca FJ027191 1.00 Asthenes baeri FJ027176

Asthenes dorbignyi FJ027181 e 1.00 a

Asthenes patagonica FJ027188 n Asthenes steinbachi FJ027200 i

0.99 x

Certhiaxis cinnamomeus FJ027330 a

l

Schoeniophylax phryganophilus FJ028248 l 0.93 Cranioleuca pyrrhophia FJ027453 a

n

Pseudoseisura gutturalis FJ028167 y F

Spartonoica maluroides FJ028294 S Phacellodomus maculipectus FJ027986 1.00 Phacellodomus ruber FJ027987 u 1.00 Phacellodomus striaticollis FJ027997 0.99 Phacellodomus rufifrons FJ027995 r Phacellodomus striaticeps FJ027996 0.96 1.00 Anumbius annumbi FJ027133 n Coryphistera alaudina FJ027437 Leptasthenura aegithaloides FJ027721 1.00 0.98 a 1.00 Leptasthenura fuliginiceps FJ027731 Leptasthenura platensis FJ027734 Sylviorthorhynchus desmursii FJ028346 0.92 r Cinclodes atacamensis FJ027378 i 0.98 Cinclodes patagonicus FJ027389 n

i

i i

Cinclodes fuscus FJ027383 r

Upucerthia dumetaria FJ028530 a

1.00 n

Furnarius rufus FJ027595 r i 1.00 Lochmias nematura JN801778 u

Phleocryptes melanops FJ028019 F 1.00 Aphrastura masafuerae JQ739454 d Aphrastura masafuerae JQ739455 Aphrastura spinicauda FJ027137 a 1.00 Aphrastura spinicauda FJ027142 Aphrastura spinicauda FJ027141 e Aphrastura spinicauda FJ027140 Aphrastura spinicauda FJ027139 1.00 Aphrastura spinicauda FJ027138 Philydor lichtensteini FJ028015 0.97 Philydor ruficaudatum JN801912 Anabacerthia amaurotis JN801485 i

n Heliobletus contaminatus JN801710 i

r

Philydor atricapillus FJ028013 o

Megaxenops parnaguae JN801795 d

1.00 y Syndactyla rufosuperciliata FJ028364 l Automolus leucophthalmus FJ027213 i 1.00 h

0.87 Hyloctistes subulatus JN801730 P Automolus ochrolaemus JN801511 Ancistrops strigilatus JN801489 1.00 Eremobius phoenicurus FJ027556 Upucerthia ruficaudus FJ028542 Pygarrhichas albogularis FJ028180 Upucerthia certhioides FJ027917 1.00 Xenops minutus FJ028566 Xenops rutilans JN802101 1.00 Xiphorhynchus flavigaster JN802105 Xiphorhynchus lachrymosus JN622082 0.97 Xiphorhynchus guttatus JQ176651 Xiphorhynchus obsoletus JQ176654 0.86 Xiphorhynchus erythropygius JQ176647 D 1.00 Xiphorhynchus ocellatus JN802111 Xiphorhynchus pardalotus JQ176659 e Xiphorhynchus fuscus FJ028574 Xiphorhynchus elegans JN802104 Lepidocolaptes angustirostris FJ027713 n 0.91 Lepidocolaptes falcinellus FJ027720 0.98 Lepidocolaptes souleyetii JQ175231 d Lepidocolaptes albolineatus JQ175228 0.93 Drymornis bridgesii JQ174721 0.98 0.98 Campylorhamphus procurvoides JQ174265 r Campylorhamphus trochilirostris FJ027291 1.00 1.00 Campylorhamphus pusillus JQ174269 o Campylorhamphus falcularius JN801531 Xiphorhynchus picus JN801634 c 0.88 Xiphocolaptes falcirostris JN802102 Xiphocolaptes promeropirhynchus JQ176641 1.00 Xiphocolaptes promeropirhynchus JQ176639 o Xiphocolaptes major FJ028571 Xiphocolaptes albicollis FJ028569 l Hylexetastes uniformis JN801728 1.00 Dendrocolaptes certhia JN801633 0.97 Dendrocolaptes concolor JQ174653 a 1.00 Dendrocolaptes picumnus JQ174654 1.00 Dendrocolaptes platyrostris FJ027493 p Nasica longirostris JN801860 0.99 Dendrocincla fuliginosa JN622069 t Dendrocincla turdina FJ027490 Dendrocincla fuliginosa JN801628 1.00 Dendrocincla homochroa JN622072 i Dendrocincla anabatina EU442298 Dendrocincla fuliginosa JN622068 d Dendrocincla merula JN622077 0.87 Dendrocincla tyrannina JN622079 1.00 Deconychura longicauda JN801623 a Deconychura longicauda JQ174643 Sittasomus griseicapillus FJ028293 e e Deconychura stictolaema JQ174645 a

d

Glyphorynchus spirurus JN801688 i

1.00 r To outgroups Geositta cunicularia FJ027615

Geositta punensis FJ027617 u

Sclerurus rufigularis JN801983 r

0.1 e

l

c

S

Appendix 2. Detailed Bayesian inference (BI) tree based on 694 base pairs of the cytochrome oxidase subunit 1 gene (COI). BI posterior probabilities (≥0.90) are indicated above the branches. The model selected for the BI analysis consisted of GTR + gamma distribution shape parameter (G) = 1.42 + proportion of invariable sites (I) = 0.60. GenBank accession numbers indicated for each species.

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1.0 Synallaxis albescens FJ028349 1.2 2.8 Synallaxis azarae FJ028355 3.4 Synallaxis frontalis EU233026 Synallaxis spixi FJ028362 4.6 Synallaxis ruficapilla JN801997 5.0 Synallaxis scutata JN802002 6.5 Synallaxis rutilans JN801998 2.8 Asthenes patagonica FJ027188 Asthenes steinbachi FJ027200 7.5 Schoeniophylax phryganophilus FJ028248 Certhiaxis cinnamomeus FJ027330 5.6 Cranioleuca pyrrhophia FJ027453 9.7 Pseudoseisura gutturalis FJ028167 Spartonoica maluroides FJ028294 1.8 5.7 Asthenes modesta FJ027184 Asthenes anthoides FJ027173 11.3 Asthenes pyrrholeuca FJ027191 7.1 0.6 Asthenes dorbignyi FJ027181 Asthenes baeri FJ027176 1.9 2.5 Phacellodomus ruber FJ027987 12.3 4.8 Phacellodomus striaticollis FJ027997 Phacellodomus maculipectus FJ027986 Phacellodomus striaticeps FJ027996 2.5 Phacellodomus rufifrons FJ027995 12.9 9.2 Coryphistera alaudina FJ027437 Anumbius annumbi FJ027133 2.9 4.9 Leptasthenura fuliginiceps FJ027731 8.1 Leptasthenura aegithaloides FJ027721 Leptasthenura platensis FJ027734 Sylviorthorhynchus desmursii FJ028346 2.8 Cinclodes atacamensis FJ027378 3.9 7.4 Cinclodes patagonicus FJ027389 9.4 Cinclodes fuscus FJ027383 Upucerthia dumetaria FJ028530 Furnarius rufus FJ027595 11.0 Lochmias nematura JN801778 8.3 Phleocryptes melanops FJ028019 Upucerthia certhioides FJ027917 Aphrastura spinicauda FJ027140 Aphrastura spinicauda FJ027139 Aphrastura spinicauda FJ027141 14.9 0.2 Aphrastura spinicauda FJ027138 0.7 Aphrastura spinicauda FJ027137 Aphrastura spinicauda FJ027142 Aphrastura masafuerae JQ739455 0.1 Aphrastura masafuerae JQ739454 3.6 9.9 Upucerthia ruficaudus FJ028542 Eremobius phoenicurus FJ027556 Pygarrhichas albogularis FJ028180 3.2 Philydor lichtensteini FJ028015 13.0 17.6 Philydor ruficaudatum JN801912 4.1 Anabacerthia amaurotis JN801485 7.6 Syndactyla rufosuperciliata FJ028364 Heliobletus contaminatus JN801710 8.6 5.7 Philydor atricapillus FJ028013 9.7 Megaxenops parnaguae JN801795 3.6 Hyloctistes subulatus JN801730 7.7 Automolus leucophthalmus FJ027213 Automolus ochrolaemus JN801511 Ancistrops strigilatus JN801489 8.8 Xenops rutilans JN802101 Xenops minutus FJ028566 2.5 Lepidocolaptes falcinellus FJ027720 Lepidocolaptes angustirostris FJ027713 3.42.8 5.6 Lepidocolaptes souleyetii JQ175231 Lepidocolaptes albolineatus JQ175228 Drymornis bridgesii JQ174721 1.0 2.0 Campylorhamphus procurvoides JQ174265 3.3 Campylorhamphus trochilirostris FJ027291 Campylorhamphus pusillus JQ174269 Campylorhamphus falcularius JN801531 3.5 Xiphorhynchus guttatus JQ176651 4.4 Xiphorhynchus obsoletus JQ176654 1.7 Xiphorhynchus lachrymosus JN622082 18.5 5.0 Xiphorhynchus flavigaster JN802105 7.8 Xiphorhynchus erythropygius JQ176647 1.1 Xiphorhynchus ocellatus JN802111 4.0 6.2 Xiphorhynchus pardalotus JQ176659 Xiphorhynchus fuscus FJ028574 4.7 Xiphorhynchus elegans JN802104 9.9 Xiphorhynchus picus JN801634 Xiphocolaptes falcirostris JN802102 1.0 Xiphocolaptes major FJ028571 Xiphocolaptes promeropirhynchus JQ176641 6.4 Xiphocolaptes promeropirhynchus JQ176639 1.7 Xiphocolaptes albicollis FJ028569 8.0 Hylexetastes uniformis JN801728 0.8 Dendrocolaptes concolor JQ174653 5.3 Dendrocolaptes certhia JN801633 0.9 Dendrocolaptes platyrostris FJ027493 19.8 Dendrocolaptes picumnus JQ174654 Glyphorynchus spirurus JN801688 2.1 Dendrocincla fuliginosa JN622068 12.0 Dendrocincla anabatina EU442298 3.2 Dendrocincla homochroa JN622072 Dendrocincla turdina FJ027490 6.2 Dendrocincla fuliginosa JN622069 2.6 Dendrocincla fuliginosa JN801628 12.4 8.4 Dendrocincla merula JN622077 5.1 Dendrocincla tyrannina JN622079 Deconychura longicauda JQ174643 3.9 Deconychura longicauda JN801623 To outgroups 10.2 Sittasomus griseicapillus FJ028293 Deconychura stictolaema JQ174645 Nasica longirostris JN801860 3.4 15.9 Geositta cunicularia FJ027615 3.0 Geositta punensis FJ027617 Sclerurus rufigularis JN801983

Appendix 3. Bayesian inference chronogram (GTR + G + I) inferred from COI in 114 species of the Furnarioidea. Bayesian age estimates are shown for each node. Bars indicate 95% highest posterior density (HPD) intervals. GenBank accession numbers indicated for each species.

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