DNA barcodes reveal striking arthropod diversity and unveil seasonal patterns of variation in the southern Atlantic Forest.

The Atlantic Forest harbors 7% of global biodiversity and possesses high levels of endemism, but many of its component taxa remain unstudied. Due to the importance of tropical forests and the urgency to protect them, there is a compelling need to address this knowledge gap. To provide more information on its arthropod fauna, a Malaise trap was deployed for 12 months in a semi-degraded area of the southern Upper Paraná ecoregion of the Atlantic Forest. All specimens were DNA barcoded and the Barcode Index Number (BIN) system was employed to assign each specimen to a species proxy. DNA barcodes were obtained from 75,500 arthropods that included representatives of 8,651 BINs. Nearly 81% of these BINs were first records, highlighting the high rates of endemism and lack of study of arthropods from the Atlantic Forest. Diptera was the most abundant order, followed by Hemiptera, Lepidoptera and Hymenoptera. Diptera was also the most species-rich order, followed by Hymenoptera, Lepidoptera, and Coleoptera, a result consistent with studies in other biogeographic regions. Insects were most abundant in winter and most diverse in autumn and winter. This pattern, however, was caused mainly by the dynamics of dipteran diversity as other orders differed in their seasonal variation. The BIN composition of the insect community varied sharply through the year and also differed between the two consecutive summers included in the sampling period. The study of the 38 commonest BINs showed that seasonal patterns of abundance were not order-specific. Temperature had the strongest impact on seasonal abundance variation. Our results highlight the striking and understudied arthropod diversity of the highly fragmented Atlantic Forest, the predominance of dipterans, and the fact that abundance and richness in this insect community peak in the coolest months. Standardized studies like this generate fast and reliable biodiversity inventories and unveil ecological patterns, thus providing valuable information for conservation programs.

Concerted variation in melanogenesis genes underlies emergent patterning of plumage in capuchino seedeaters.

Coloration traits are central to animal communication; they often govern mate choice, promote reproductive isolation and catalyse speciation. Specific genetic changes can cause variation in coloration, yet far less is known about how overall coloration patterns-which involve combinations of multiple colour patches across the body-can arise and are genomically controlled. We performed genome-wide association analyses to link genomic changes to variation in melanin (eumelanin and pheomelanin) concentration in feathers from different body parts in the capuchino seedeaters, an avian radiation with diverse colour patterns despite remarkably low genetic differentiation across species. Cross-species colour variation in each plumage patch is associated with unique combinations of variants at a few genomic regions, which include mostly non-coding (presumably regulatory) areas close to known pigmentation genes. Genotype-phenotype associations can vary depending on patch colour and are stronger for eumelanin pigmentation, suggesting eumelanin production is tightly regulated. Although some genes are involved in colour variation in multiple patches, in some cases, the SNPs associated with colour changes in different patches segregate spatially. These results suggest that coloration patterning in capuchinos is generated by the modular combination of variants that regulate multiple melanogenesis genes, a mechanism that may have promoted this rapid radiation.

Genetic variation in neotropical butterflies is associated with sampling scale, species distributions, and historical forest dynamics.

Previous studies of butterfly diversification in the Neotropics have focused on Amazonia and the tropical Andes, while southern regions of the continent have received little attention. To address the gap in knowledge about the Lepidoptera of temperate South America, we analysed over 3000 specimens representing nearly 500 species from Argentina for a segment of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Representing 42% of the country's butterfly fauna, collections targeted species from the Atlantic and Andean forests, and biodiversity hotspots that were previously connected but are now isolated. We assessed COI effectiveness for species discrimination and identification and how its performance was affected by geographic distances and taxon coverage. COI data also allowed to study patterns of genetic variation across Argentina, particularly between populations in the Atlantic and Andean forests. Our results show that COI discriminates species well, but that identification success is reduced on average by ~20% as spatial and taxonomic coverage rises. We also found that levels of genetic variation are associated with species' spatial distribution type, a pattern which might reflect differences in their dispersal and colonization abilities. In particular, intraspecific distance between populations in the Atlantic and Andean forests was significantly higher in species with disjunct distributions than in those with a continuous range. All splits between lineages in these forests dated to the Pleistocene, but divergence dates varied considerably, suggesting that historical connections between the Atlantic and Andean forests have differentially affected their shared butterfly fauna. Our study supports the fact that large-scale assessments of mitochondrial DNA variation are a powerful tool for evolutionary studies.

Mechanisms involved in the production of differently colored feathers in the structurally colored swallow tanager (Tersina viridis; Aves: Thraupidae).

Non-iridescent, structural coloration in birds originates from the feather's internal nanostructure (the spongy matrix) but melanin pigments and the barb's cortex can affect the resulting color. Here, we explore how this nanostructure is combined with other elements in differently colored plumage patches within a bird. We investigated the association between light reflectance and the morphology of feathers from the back and belly plumage patches of male swallow tanagers (Tersina viridis), which look greenish-blue and white, respectively. Both plumage patches have a reflectance peak around 550 nm but the reflectance spectrum is much less saturated in the belly. The barbs of both types of feathers have similar spongy matrices at their tips, rendering their reflectance spectra alike. However, the color of the belly feather barbs changes from light green at their tips to white closer to the rachis. These barbs lack pigments and their morphology changes considerably throughout. Toward the rachis, the barb is almost hollow, with a reduced area occupied by spongy matrix, and has a flattened shape. By contrast, the blue back feathers' barbs have melanin underneath the spongy matrix resulting in a much more saturated coloration. The color of these barbs is also even along the barbs' length. Our results suggest that the color differences between the white and greenish-blue plumage are mostly due to the differential deposition of melanin and a reduction of the spongy matrix near the rachis of the belly feather barbs and not a result of changes in the characteristics of the spongy matrix.

Rapid speciation via the evolution of pre-mating isolation in the Iberá Seedeater.

Behavioral isolation can catalyze speciation and permit the slow accumulation of additional reproductive barriers between co-occurring organisms. We illustrate how this process occurs by examining the genomic and behavioral bases of pre-mating isolation between two bird species (Sporophila hypoxantha and the recently discovered S. iberaensis) that belong to the southern capuchino seedeaters, a recent, rapid radiation characterized by variation in male plumage coloration and song. Although these two species co-occur without obvious ecological barriers to reproduction, we document behaviors indicating species recognition by song and plumage traits and strong assortative mating associated with genomic regions underlying male plumage patterning. Plumage differentiation likely originated through the reassembly of standing genetic variation, indicating how novel sexual signals may quickly arise and maintain species boundaries.

Evolution between forest macrorefugia is linked to discordance between genetic and morphological variation in Neotropical passerines.

The central Andean rainforests and the Atlantic Forest are two similar biomes that are fully isolated by xerophytic and open-vegetation regions (the Chaco and Cerrado, respectively). Even though there is evidence suggesting that these rainforests have been connected in the past, their dynamics of connection, the geographic areas that bridged these regions, and the biological processes that have promoted diversification between them remain to be studied. In this research, we used three passerine species (Poecilotriccus plumbeiceps, Phylloscartes ventralis and Cacicus chrysopterus) as models to address whether the Andean and the Atlantic forests have acted as a refugia system (macrorefugia), and to evaluate biogeographic hypotheses of diversification and connection between them. In order to achieve these goals, we performed traditional phylogeographic analyses and compared alternative biogeographic scenarios by using Approximate Bayesian Computation. Additionally, we performed morphological analyses to evaluate phenotypic divergence between these regions. Our findings support that both rainforest regions acted as refugia, but that the impact of their isolation was stronger on the genetic than on the morphologic characters. Our results provided evidence that both geographic isolation as well as ecological factors have modeled the external traits of forest organisms in the region. Regarding the connection routes between the Andes and the Atlantic Forest, the genetic data rejected the hypothesis of a Chaco connection in the tested species, providing evidence for a connection through the Cerrado or through the transition between the Cerrado and Chaco, in a process that could have started as early as the Late Miocene.

A test of the riverine barrier hypothesis in the largest subtropical river basin in the Neotropics.

The riverine barrier hypothesis proposes that large rivers represent geographical barriers to gene flow for terrestrial organisms, leading to population differentiation and ultimately allopatric speciation. Here we assess for the first time if the subtropical Paraná-Paraguay River system in the Del Plata basin, second in size among South American drainages, acts as a barrier to gene flow for birds. We analysed the degree of mitochondrial and nuclear genomic differentiation in seven species with known subspecies divided by the Paraná-Paraguay River axis. Only one species showed genetic differentiation concordant with the current river channel, but another five species have an east/west genetic split broadly coincident with the Paraná River's dynamic palaeochannel, suggesting this fluvial axis has had a past role in shaping present-day genetic structure. Moreover, dating analyses show that these splits have been asynchronous, with species responding differently to the riverine barrier. Comparisons informed by the geological history of the Paraná River and its influence on the ecological and climatic differences among ecoregions in the study area further bolster the finding that responses to this geographical barrier have been species-specific.

Contrasting evolutionary histories in Neotropical birds: Divergence across an environmental barrier in South America.

Avian diversity in the Neotropics has been traditionally attributed to the effect of vicariant forces promoting speciation in allopatry. Recent studies have shown that phylogeographical patterns shared among codistributed species cannot be explained by a single vicariant event, as species responses to a common barrier depend on the biological attributes of each taxon. The open vegetation corridor (OVC) isolates Amazonia and the Andean forests from the Atlantic Forest, creating a notorious pattern of avian taxa that are disjunctly codistributed in these forests. Here, we studied and compared the evolutionary histories of Ramphotrigon megacephalum and Pipraeidea melanonota, two passerines with allopatric populations east and west of the OVC that represent different subspecies. These species differ in their biological attributes: R. megacephalum is a sedentary, forest specialist mostly confined to bamboo understorey, whereas P. melanonota is a seasonal migrant and generalist species that ranges in a variety of closed and semi-open environments. We performed genetic and genomic analyses, complemented with the study of coloration and behavioural differentiation, to assess population divergence across the OVC. We found that the evolutionary histories of both R. megacephalum and P. melanonota have been shaped by this environmental barrier. However, these species responded in different and asynchronous manners to the establishment of the OVC and to past connections between the currently isolated South American forests, which can be mostly explained by their distinct ecologies and dispersal abilities. Our results support the fact that the biological attributes of species can make their evolutionary histories idiosyncratic.

Phylogeographic variation within the Buff-browed Foliage-gleaner (Aves: Furnariidae: Syndactyla rufosuperciliata) supports an Andean-Atlantic forests connection via the Cerrado.

We evaluated whether the Andean and the Atlantic forests acted as refugia during the Quaternary, and tested biogeographic hypotheses about the regions involved in the connectivity between those biomes (through the Chaco or the Cerrado). To achieve these goals we selected the Buff-browed Foliage-gleaner Syndactyla rufosuperciliata (Aves, Furnariidae) as a study system, a taxon distributed between the Andean and Atlantic forest. We first explored the historical connectivity between regions through niche modeling. We subsequently used DNA sequences (n = 71 individuals) and genomic analyses (ddRADseq, n = 33 individuals) to evaluate population genetic structure and gene flow within this species. Finally, we performed population model selection using Approximate Bayesian Computation. Our findings indicate that the Andean and the Atlantic forests acted as refugia, and that the populations of the focal species from both regions contacted through the Cerrado region, thus suggesting that the historical dynamics of Andean and Atlantic forests are important for the evolution of forest birds in the region. The results are in agreement with studies of other organisms and may indicate a more general pattern of connectivity among biomes in the Neotropics. Finally, we recommend recognizing both the Andean and the Altantic forests lineages of S. rufosuperciliata as independent species.

Co-occurrence Patterns in a Subtropical Ant Community Revealed by Complementary Sampling Methodologies.

Ants are abundant and ecologically dominant insects in most terrestrial communities. In subtropical ecosystems, there is a high turnover of species from the canopy to the top layers of the soil. Additionally, ant communities are often influenced by inter-specific competition. Collectively, these two processes (abiotic filtering and competition) make ants ideal for studies of community structure. We examined composition, co-occurrence, and species interactions in a sub-tropical forest ant community to examine how ground-foraging ant species partition microhabitats. We used four methods: pitfall traps, litter samples, surface baits, and subterranean baits. Surface baiting was employed at three different time periods to examine how foraging activity and species interactions at baits varied with time of day and temperature. Each method sampled a particular assemblage of the 97 total ant species. Pitfall traps shared ~50% of species with surface baits and litter samples. Subterranean baits had the fewest total species but included some uncommonly sampled ants. The majority of interactions between species at baits were neutral, but a few agonistic interactions were also observed when bait occupancy was highest. Species co-occurrence patterns suggest that this ant community may not be heavily influenced by interspecific competition. Our results reinforce the advantages of applying complementary sampling techniques to examine ant community structure, and suggest that competition and dominance is best considered in the context of resource type, foraging strategy and time of sampling. Finally, we discuss the lack of two conspicuous Neotropical groups in our samples, leaf-cutting ant and army ants.

Forest corridors between the central Andes and the southern Atlantic Forest enabled dispersal and peripatric diversification without niche divergence in a passerine.

The central Andean rainforests and the Atlantic Forest are separated by the Chaco and the Cerrado domains. Despite this isolation, diverse evidence suggests that these rainforests have been connected in the past. However, little is known about the timing and geographic positions of these connections, as well as their effects on diversification of species. In this study, we used the Black-goggled Tanager (Trichothraupis melanops, Thraupidae) as a model to study whether the Andean and the Atlantic forests have acted as a refugia system, and to evaluate biogeographic hypotheses of diversification and connection between these rainforests. We compared alternative biogeographic scenarios by using Approximate Bayesian Computation (ABC), modeled range shifts across time, and assessed niche divergence between regions. The results indicated that the major phylogeographic gap within T. melanops is located between these rainforests. The ABC analysis supported peripatric diversification, with initial dispersal from the Atlantic Forest to the Andes during the Mid-Pleistocene. Also, the results supported an Andean-Atlantic forests connection through the current Cerrado-Chaco transition, linking the southern Atlantic Forest with the central Andes. Our findings, taken together with other studies, support that the connection between these biomes has been recurrent, and that has occurred mostly through the Cerrado and/or the Cerrado-Chaco transition. The data also support that the connection dynamic has played an important role in the biological diversification, by promoting peripatric divergence in some forest taxa restricted to both biomes.

Barcoding the butterflies of southern South America: Species delimitation efficacy, cryptic diversity and geographic patterns of divergence.

Because the tropical regions of America harbor the highest concentration of butterfly species, its fauna has attracted considerable attention. Much less is known about the butterflies of southern South America, particularly Argentina, where over 1,200 species occur. To advance understanding of this fauna, we assembled a DNA barcode reference library for 417 butterfly species of Argentina, focusing on the Atlantic Forest, a biodiversity hotspot. We tested the efficacy of this library for specimen identification, used it to assess the frequency of cryptic species, and examined geographic patterns of genetic variation, making this study the first large-scale genetic assessment of the butterflies of southern South America. The average sequence divergence to the nearest neighbor (i.e. minimum interspecific distance) was 6.91%, ten times larger than the mean distance to the furthest conspecific (0.69%), with a clear barcode gap present in all but four of the species represented by two or more specimens. As a consequence, the DNA barcode library was extremely effective in the discrimination of these species, allowing a correct identification in more than 95% of the cases. Singletons (i.e. species represented by a single sequence) were also distinguishable in the gene trees since they all had unique DNA barcodes, divergent from those of the closest non-conspecific. The clustering algorithms implemented recognized from 416 to 444 barcode clusters, suggesting that the actual diversity of butterflies in Argentina is 3%-9% higher than currently recognized. Furthermore, our survey added three new records of butterflies for the country (Eurema agave, Mithras hannelore, Melanis hillapana). In summary, this study not only supported the utility of DNA barcoding for the identification of the butterfly species of Argentina, but also highlighted several cases of both deep intraspecific and shallow interspecific divergence that should be studied in more detail.

Evolution of Vocal Diversity through Morphological Adaptation without Vocal Learning or Complex Neural Control.

The evolution of complex behavior is driven by the interplay of morphological specializations and neuromuscular control mechanisms [1-3], and it is often difficult to tease apart their respective contributions. Avian vocal learning and associated neural adaptations are thought to have played a major role in bird diversification [4-8], whereas functional significance of substantial morphological diversity of the vocal organ remains largely unexplored. Within the most species-rich order, Passeriformes, "tracheophones" are a suboscine group that, unlike their oscine sister taxon, does not exhibit vocal learning [9] and is thought to phonate with tracheal membranes [10, 11] instead of the two independent sources found in other passerines [12-14]. Here we show tracheophones possess three sound sources, two oscine-like labial pairs and the unique tracheal membranes, which collectively represent the largest described number of sound sources for a vocal organ. Birds with experimentally disabled tracheal membranes were still able to phonate. Instead of the main sound source, the tracheal membranes constitute a morphological specialization, which, through interaction with bronchial labia, contributes to different acoustic features such as spectral complexity, amplitude modulation, and enhanced sound amplitude. In contrast, these same features arise in oscines from neuromuscular control of two labial sources [15-17]. These findings are supported by a modeling approach and provide a clear example for how a morphological adaptation of the tracheophone vocal organ can generate specific, complex sound features. Morphological specialization therefore constitutes an alternative path in the evolution of acoustic diversity to that of oscine vocal learning and complex neural control.

Repeated divergent selection on pigmentation genes in a rapid finch radiation.

Instances of recent and rapid speciation are suitable for associating phenotypes with their causal genotypes, especially if gene flow homogenizes areas of the genome that are not under divergent selection. We study a rapid radiation of nine sympatric bird species known as capuchino seedeaters, which are differentiated in sexually selected characters of male plumage and song. We sequenced the genomes of a phenotypically diverse set of species to search for differentiated genomic regions. Capuchinos show differences in a small proportion of their genomes, yet selection has acted independently on the same targets in different members of this radiation. Many divergent regions contain genes involved in the melanogenesis pathway, with the strongest signal originating from putative regulatory regions. Selection has acted on these same genomic regions in different lineages, likely shaping the evolution of cis-regulatory elements, which control how more conserved genes are expressed and thereby generate diversity in classically sexually selected traits.

The niche and phylogeography of a passerine reveal the history of biological diversification between the Andean and the Atlantic forests.

The Atlantic Forest is separated from the Andean tropical forest by dry and open vegetation biomes (Chaco and Cerrado). Despite this isolation, both rainforests share closely related lineages, which suggest a past connection. This connection could have been important for forest taxa evolution. In this study, we used the Saffron-billed Sparrow (Arremon flavirostris) as a model to evaluate whether the Andean and the Atlantic forests act as a refugia system, as well as to test for a history of biogeographic connection between them. In addition, we evaluated the molecular systematic of intraspecific lineages of the studied species. We modeled the current and past distribution of A. flavirostris, performed phylogeographic analyses based on mitochondrial and nuclear genes, and used Approximate Bayesian Computation (ABC) analyses to test for biogeographic scenarios. The major phylogeographic disjunction within A. flavirostris was found between the Andean and the Atlantic forests, with a divergence that occurred during the Mid-Pleistocene. Our paleodistribution models indicated a connection between these forest domains in different periods and through both the Chaco and Cerrado. Additionally, the phylogeographic and ABC analyses supported that the Cerrado was the main route of connection between these rainforests, but without giving decisive evidence against a Chaco connection. Our study with A. flavirostris suggest that the biodiversity of the Andean and of the Atlantic forests could have been impacted (and perhaps enriched?) by cycles of connections through the Cerrado and Chaco. This recurrent cycle of connection between the Andean and the Atlantic Forest could have been important for the evolution of Neotropical forest taxa. In addition, we discussed taxonomic implications of the results and proposed to split the studied taxon into two full species.

The multiple applications of DNA barcodes in avian evolutionary studies.

DNA barcodes of birds are currently available for 41% of known species and for many different geographic areas; therefore, they are a rich data source to answer evolutionary questions. We review studies that have used DNA barcodes to investigate evolutionary processes in birds using diverse approaches. We also review studies that have investigated species in depth where taxonomy and DNA barcodes present inconsistencies. Species that showed low genetic interspecific divergence and lack of reciprocal monophyly either are the result of recent radiation and (or) hybridize, while species with large genetic splits in their COI sequences were determined to be more than one independent evolutionary unit. In addition, we review studies that employed large DNA barcode datasets to study the molecular evolution of mitochondrial genes and the biogeography of islands, continents, and even at a multi-continental scale. These studies showed that DNA barcodes offer high-quality data well beyond their main purpose of serving as a molecular tool for species identification.

Difference between the vocalizations of two sister species of pigeons explained in dynamical terms.

Vocal communication is an unique example, where the nonlinear nature of the periphery can give rise to complex sounds even when driven by simple neural instructions. In this work we studied the case of two close-related bird species, Patagioenas maculosa and Patagioenas picazuro, whose vocalizations differ only in the timbre. The temporal modulation of the fundamental frequency is similar in both cases, differing only in the existence of sidebands around the fundamental frequency in the P. maculosa. We tested the hypothesis that the qualitative difference between these vocalizations lies in the nonlinear nature of the syrinx. In particular, we propose that the roughness of maculosa's vocalizations is due to an asymmetry between the right and left vibratory membranes, whose nonlinear dynamics generate the sound. To test the hypothesis, we generated a biomechanical model for vocal production with an asymmetric parameter Q with which we can control the level of asymmetry between these membranes. Using this model we generated synthetic vocalizations with the principal acoustic features of both species. In addition, we confirmed the anatomical predictions by making post mortem inspection of the syrinxes, showing that the species with tonal song (picazuro) has a more symmetrical pair of membranes compared to maculosa.

Convergent Evolution of Hemoglobin Function in High-Altitude Andean Waterfowl Involves Limited Parallelism at the Molecular Sequence Level.

A fundamental question in evolutionary genetics concerns the extent to which adaptive phenotypic convergence is attributable to convergent or parallel changes at the molecular sequence level. Here we report a comparative analysis of hemoglobin (Hb) function in eight phylogenetically replicated pairs of high- and low-altitude waterfowl taxa to test for convergence in the oxygenation properties of Hb, and to assess the extent to which convergence in biochemical phenotype is attributable to repeated amino acid replacements. Functional experiments on native Hb variants and protein engineering experiments based on site-directed mutagenesis revealed the phenotypic effects of specific amino acid replacements that were responsible for convergent increases in Hb-O2 affinity in multiple high-altitude taxa. In six of the eight taxon pairs, high-altitude taxa evolved derived increases in Hb-O2 affinity that were caused by a combination of unique replacements, parallel replacements (involving identical-by-state variants with independent mutational origins in different lineages), and collateral replacements (involving shared, identical-by-descent variants derived via introgressive hybridization). In genome scans of nucleotide differentiation involving high- and low-altitude populations of three separate species, function-altering amino acid polymorphisms in the globin genes emerged as highly significant outliers, providing independent evidence for adaptive divergence in Hb function. The experimental results demonstrate that convergent changes in protein function can occur through multiple historical paths, and can involve multiple possible mutations. Most cases of convergence in Hb function did not involve parallel substitutions and most parallel substitutions did not affect Hb-O2 affinity, indicating that the repeatability of phenotypic evolution does not require parallelism at the molecular level.

Continental-scale analysis reveals deep diversification within the polytypic Red-crowned Ant Tanager (Habia rubica, Cardinalidae).

We explored the phylogeographic patterns of intraspecific diversity in the Red-crowned Ant Tanager (Habia rubica) throughout its continent-wide distribution, in order to understand its evolutionary history and the role of evolutionary drivers that are considered to promote avian diversification in the Neotropics. We sampled 100 individuals of H. rubica from Mexico to Argentina covering the main areas of its disjunct distribution. We inferred phylogenetic relationships through Bayesian and maximum parsimony methodologies based on mitochondrial and nuclear markers, and complemented genetic analyses with the assessment of coloration and behavioral differentiation. We found four deeply divergent phylogroups within H. rubica: two South American lineages and two Mexican and Middle American lineages. The divergence event between the northern and southern phylogroups was dated to c. 5.0 Ma, seemingly related to the final uplift of the Northern Andes. Subsequently, the two South American phylogroups split c. 3.5 Ma possibly due to the development of the open vegetation corridor that currently isolates the Amazonian and Atlantic forests. Diversification throughout Mexico and Middle America, following dispersion across the Isthmus of Panama, was presumably more recent and coincident with Pleistocene climatic fluctuations and habitat fragmentations. The analyses of vocalizations and plumage coloration showed significant differences among main lineages that were consistent with the phylogenetic evidence. Our findings suggest that the evolutionary history of H. rubica has been shaped by an assortment of diversification drivers at different temporal and spatial scales resulting in deeply divergent lineages that we recommend should be treated as different species.

Phylogeography and genetic structure of two Patagonian shag species (Aves: Phalacrocoracidae).

We compared the phylogeographic and genetic structure of two sympatric shag species, Phalacrocorax magellanicus (rock shag) and Phalacrocorax atriceps (imperial shag), from Patagonia (southern South America). We used multilocus genotypes of nuclear DNA (microsatellite loci) from 324 individuals and mitochondrial DNA sequences (ATPase) from 177 individuals, to evaluate hypotheses related to the effect of physical and non-physical barriers on seabird evolution. Despite sharing many ecological traits, the focal species strongly differ in two key aspects: P. magellanicus has a strong tendency to remain at/near their breeding colonies during foraging trips and the non-breeding season, while P. atriceps exhibits the converse pattern. Both species showed similar mtDNA genetic structure, where colonies from the Atlantic Coast, Pacific Coast and Fuegian region were genetically divergent. We also found similarities in the results of Bayesian clustering analysis of microsatellites, with both species having four clusters. However population differentiation (e.g. Fst, Φst) was higher in P. magellanicus compared to P. atriceps, and average membership probabilities of individuals to specific clusters (Q-values) were also higher in the former. Phalacrocorax magellanicus has strong phylogeographic structure, consistent with the impact of Pleistocene glaciations, with diagnostic haplotypes associated with each of the three mentioned regions. The same pattern was not as evident for P. atriceps. Migration rate estimators were higher for P. atriceps than for P. magellanicus; however both species followed an n-island-like model of gene flow, this implies that dispersal occurs across the continental land mass that separates Atlantic and Pacific Oceans. Our results supported the hypothesis that non-physical barriers are important drivers of the genetic and phylogeographic structure in seabirds, and also that physical barriers constitute effective but not absolute impediments to gene flow.