We assessed population structure and the spatio-temporal pattern of diversification in the Glossy Antshrike Sakesphorus luctuosus (Aves, Thamnophilidae) to understand the processes shaping the evolutionary history of Amazonian floodplains and address unresolved taxonomic controversies surrounding its species limits. By targeting ultraconserved elements (UCEs) from 32 specimens of S. luctuosus, we identified independent lineages and estimated their differentiation, divergence times and migration rates. We also estimated current and past demographic histories for each recovered lineage. We found evidence confirming that S. luctuosus consists of a single species, comprising at least four populations, with some highly admixed individuals and overall similar levels of migration between populations. We confirmed the differentiation of the Araguaia River basin population (S. l. araguayae), and gathered circumstantial evidence indicating that the taxon S. hagmanni may represent a highly introgressed population between 3 distinct phylogroups of S. luctuosus. Divergence time estimates between populations seem to be recent, occurring during the last 183 kya. Signs of population expansions were detected for populations attributed to subspecies S. l. luctuosus, but the S. l. araguayae population had probably maintained its effective size through time. Our results support that S. luctuosus has had a complex population history, resulting from a high dependence on southeastern “clear-water” habitats and their availability through time. Spatial and demographic expansions towards the western “white water” flooded forests might still be ongoing. Our study reinforces the view that isolation due to absence of suitable habitat has been an important driver of population differentiation within Amazonian flooded forests, but also that differences between várzeas (“white water” floodplains, mostly in southwestern Amazonia) and igapós (“clear- water” floodplains, especially located in the east) should be further explored as powerful drivers of micro-evolution.

Genomic DNA was extracted from tissues of 32 specimens of Sakesphorus luctuosus from across its known range and a S. canadensis specimen. We used DNeasy Blood & Tissue kit (Qiagen) for DNA extraction, and Qubit® 2.0 Fluorometer (Life Technologies) to assess quantity and quality of the extracted DNA. Sequence capture and sequencing of Ultra Conserved Elements (hereafter UCEs) were performed according to Faircloth et al. (2012) by RAPiD Genomics (Gainesville, FL, USA). More than 2,300 UCEs and 97 exons were targeted (Harvey, Aleixo, Ribas, & Brumfield, 2017; Zucker et al., 2016).

We followed the PHYLUCE pipeline (Faircloth, 2016, 2017) to first remove adapters, barcodes and low quality sequence regions using Illumiprocessor 2.0.7 (Faircloth, 2013), with the trimming tool Trimmomatic 0.32.1 (Bolger, Lohse, & Usadel, 2014); and to assemble trimmed reads using Trinity (Grabherr et al., 2011). These analyses were performed using default parameters. Contigs were blasted against the probe set of exons and UCEs using phyluce_match_contigs_to_probes. The annotated set was divided by locus, and each was aligned with MAFFT using the scripts phyluce_assembly_get_match_counts, phyluce_assembly_get_fastas_from_match_counts, and phyluce_align_seqcap_align. A complete matrix comprising only loci without missing data was obtained following the UCE Phylogenomics tutorial from PHYLUCE pipeline (Faircloth, 2016).

A complete DNA sequence matrix, comprising loci without missing data, from 32 specimens of Sakesphorus luctuosus and 1 specimen of Sakesphorus canadensis (sample ID: A16764) is here deposited (Silva, Ribas & Aleixo, Ecology and Evolution, doi: 10.1002/ece3.7951). This dataset was obtained after sequence capture and sequencing of more than 2,300 Ultra Conserved Elements and 97 exons. The PHYLUCE pipeline was used to remove adapters, barcodes and low quality sequence regions using Illumiprocessor 2.0.7, with the trimming tool Trimmomatic 0.32.1; and to assemble trimmed reads using Trinity. These analyses were performed using default parameters. Contigs were blasted against the probe set of exons and UCEs using phyluce_match_contigs_to_probes. The annotated set was divided by locus, and each was aligned with MAFFT using the scripts phyluce_assembly_get_match_counts, phyluce_assembly_get_fastas_from_match_counts, and phyluce_align_seqcap_align.