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The Heteroduplex mobility assay (HMA) has proven to be a robust tool for the detection of genetic variation. Here, we describe a simple and rapid application of the HMA by microfluidic capillary electrophoresis, for phylogenetics and population genetic analyses (pgHMA). We show how commonly applied techniques in phylogenetics and population genetics have equivalents with pgHMA: phylogenetic reconstruction with bootstrapping, skyline plots, and mismatch distribution analysis. We assess the performance and accuracy of pgHMA by comparing the results obtained against those obtained using standard methods of analyses applied to sequencing data. The resulting comparisons demonstrate that: (1) there is a significant linear relationship (R = 0.992) between heteroduplex mobility and genetic distance; (2) phylogenetic trees obtained by HMA and nucleotide sequences present nearly identical topologies; (3) clades with high pgHMA parametric bootstrap support also have high bootstrap support on nucleotide phylogenies; (4) skyline plots estimated from the UPGMA trees of HMA and Bayesian trees of nucleotide data reveal similar trends, especially for the median trend estimate of effective population size; and (5) optimized mismatch distributions of HMA are closely fitted to the mismatch distributions of nucleotide sequences. In summary, pgHMA is an easily-applied method for approximating phylogenetic diversity and population trends. KEYWORDS: bootstrap, heteroduplex mobility assay, mismatch distribution, phylogenetics, skyline plot

To understand the ecosystem dynamics that underpin the year-round presence of a large generalist consumer, the Bryde’s whale (Balaenoptera edeni brydei), we use a DNA metabarcoding approach and systematic zooplankton surveys to investigate seasonal and regional changes in zooplankton communities and if whale diet reflects such changes. Twenty-four zooplankton community samples were collected from three regions throughout the Hauraki Gulf, New Zealand, over two temperature regimes (warm and cool seasons), as well as 20 samples of opportunistically collected Bryde’s whale scat. Multi-locus DNA barcode libraries were constructed from 18S and COI gene fragments, representing a trade-off between identification and resolution of metazoan taxa. Zooplankton community OTU occurrence and relative read abundance showed regional and seasonal differences based on permutational analyses of variance in both DNA barcodes, with significant changes in biodiversity indices linked to season in COI only. In contrast, we did not find evidence that Bryde’s whale diet shows seasonal or regional trends, but instead indicated clear prey preferences for krill-like crustaceans, copepods, salps and ray-finned fishes independent of prey availability. The year-round presence of Bryde’s whales in the Hauraki Gulf is likely associated with the patterns of distribution and abundance of these key prey items.

A current strategy for obtaining haplotype information from several individuals involves short-read sequencing of pooled amplicons, where fragments from each individual is identified by a unique DNA barcode. In this paper, we report a new method to recover the phylogeny of haplotypes from short-read sequences obtained using pooled amplicons from a mixture of individuals, without barcoding. The method, AFPhyloMix, accepts an alignment of the mixture of reads against a reference sequence, obtains the single-nucleotide-polymorphisms (SNP) patterns along the alignment, and constructs the phylogenetic tree according to the SNP patterns. AFPhyloMix adopts a Bayesian model of inference to estimates the phylogeny of the haplotypes and their relative frequencies, given that the number of haplotypes is known. In our simulations, AFPhyloMix achieved at least 80% accuracy at recovering the phylogenies and frequencies of the constituent haplotypes, for mixtures with up to 15 haplotypes. AFPhyloMix also worked well on a real data set of kangaroo mitochondrial DNA sequences.

Cultures in humans and other species are maintained through interactions among conspecifics. Declines in population density could be exacerbated by culture loss, thereby linking culture to conservation. We combined historical recordings, citizen science and breeding data to assess the impact of severe population decline on song culture, song complexity and individual fitness in critically endangered regent honeyeaters ( Anthochaera phrygia ). Song production in the remaining wild males varied dramatically, with 27% singing songs that differed from the regional cultural norm. Twelve per cent of males, occurring in areas of particularly low population density, completely failed to sing any species-specific songs and instead sang other species' songs. Atypical song production was associated with reduced individual fitness, as males singing atypical songs were less likely to pair or nest than males that sang the regional cultural norm. Songs of captive-bred birds differed from those of all wild birds. The complexity of regent honeyeater songs has also declined over recent decades. We therefore provide rare evidence that a severe decline in population density is associated with the loss of vocal culture in a wild animal, with concomitant fitness costs for remaining individuals. The loss of culture may be a precursor to extinction in declining populations that learn selected behaviours from conspecifics, and therefore provides a useful conservation indicator.

Many fish species utilise nursery habitats to enhance survival and growth through vulnerable juvenile life stages. Nursery habitats, however, are often threatened by a variety of stressors, and it is likely that some nursery habitats or locations are more degraded than others. If degraded nursery habitats impact juvenile fish growth or other aspects of fish physiology, a tool to detect this would have great utility in prioritising habitat conservation efforts. We assessed the ability of metabolic profiling to discriminate hatchery reared snapper (Chrysophrys auratus) that had differing food availability and growth rates. Metabolomic profiles of snapper from the different sample groups (i.e. different combinations of fast or slow growth rate and high and low food availability) had high levels of discrimination. This difference was largely driven by an unknown metabolite, which we suspect is a dipeptide proline compound. It is unclear if this unknown metabolite was specific to the results of the present study, or may have more generic value as a bio-indicator of nursery habitat quality. The next step is to analyse the metabolomic profiles of juvenile snapper from a gradient of locations with low to high nursery habitat quality and assess the ability of metabolomic methods to discriminate individual fish from across this more realistic setting.

Background In short-read DNA sequencing experiments, the read coverage is a key parameter to successfully assemble the reads and reconstruct the sequence of the input DNA. When coverage is very low, the original sequence reconstruction from the reads can be difficult because of the occurrence of uncovered gaps. Reference guided assembly can then improve these assemblies. However, when the available reference is phylogenetically distant from the sequencing reads, the mapping rate of the reads can be extremely low. Some recent improvements in read mapping approaches aim at modifying the reference according to the reads dynamically. Such approaches can significantly improve the alignment rate of the reads onto distant references but the processing of insertions and deletions remains challenging. Results Here, we introduce a new algorithm to update the reference sequence according to previously aligned reads. Substitutions, insertions and deletions are performed in the reference sequence dynamically. We evaluate this approach to assemble a western-grey kangaroo mitochondrial amplicon. Our results show that more reads can be aligned and that this method produces assemblies of length comparable to the truth while limiting error rate when classic approaches fail to recover the correct length. Finally, we discuss how the core algorithm of this method could be improved and combined with other approaches to analyse larger genomic sequences. Conclusions We introduced an algorithm to perform dynamic alignment of reads on a distant reference. We showed that such approach can improve the reconstruction of an amplicon compared to classically used bioinformatic pipelines. Although not portable to genomic scale in the current form, we suggested several improvements to be investigated to make this method more flexible and allow dynamic alignment to be used for large genome assemblies.

Increasing sound in the ocean from human activity potentially threatens marine animals that use sound to communicate, detect prey, avoid predators and function within their ecosystem. The detection and classification of sound produced by marine animals, such as whales and fish, is an important component in noise mitigation strategies, while also providing valuable insights into their ecology. Traditionally, visual surveys are conducted to assess how these animals utilize a specific area, often underestimating the number of individuals as they don’t spend much time at the surface. Long-term passive acoustic monitoring efforts have become more prevalent to monitor such animals. The large datasets collected can be impractical to manually process, necessitating the development of automated detection methods, which often produce mixed results owing to the broad frequency range and variable duration of many biological sounds. Here we describe a novel approach for automated detection of underwater biophonic sounds employing hidden Markov models (HMM). Acoustic data was collected at a single listening station in Hauraki Gulf, from October 2014 to April 2016. HMM detection models were developed for Bryde’s whales (Balaenoptera edeni) that were used as a model organism because they are notoriously hard to study with traditional visual surveys and produce a characteristic call. Bryde’s whale calls also directly overlap the sounds of anthropogenic activity, in particular the sound of vessels transiting to the busiest port in New Zealand; therefore monitoring whale calls is of utmost importance when confronting increasing sound in the ocean. Vocalizations were detected with a sensitivity of 77% and false positive rate of 23%. Bryde’s whale vocalizations were detected on 11% of all recordings. Overall, there were significantly more detections during summer (n = 1716) than winter (n = 447), and significantly more during the day (n = 1991) compared to night (n = 1264). This study shows the feasibility of using HMMs on long-term acoustic datasets. The method has the potential to be used for a wide range of soniferous animals who, like the Bryde’s whale, also produce unique sounds. The detection method would be particularly useful for mitigation and management strategies of species that are difficult to detect using traditional visual methods.

Geographic patterns of song variation are common in passerines and can develop as a consequence of the mechanisms of song acquisition and dispersal. In particular, the timing of dispersal relative to the sensory learning phase and the time of song crystallization is important. For example, when the sensory phase continues after dispersal or when males learn new songs every breeding season, i.e. open-ended learner, neighborhoods can develop where males share their songs. In this study, we utilize a unique, comprehensive dispersal and song recording dataset to investigate the existence and development of micro-geographic song variation in a wild passerine population. Machine learning song analysis methods allow us to overcome perceptual bias in the classification of the songs of New Zealand hihi (Notiomystis cincta). We show that males share more song elements of their repertoire with their neighbors than with more distant males or with males from the same natal area, implying that repertoire is acquired post-dispersal. Finally, we suggest that high levels of male competition have driven the development of post-dispersal vocal learning behavior in this species.

Passive acoustic monitoring (PAM) coupled with automated species identification is a promising tool for species monitoring and conservation worldwide. However, high false positive rates are still an important limitation and a crucial factor for acceptance of these techniques in wildlife surveys. Here we present the Assemblage of Focal Species Recognizers - AFSR, a novel approach for decreasing false positives and increasing models’ precision in multispecies contexts. AFSR focusses on decreasing false positives by excluding unreliable sound file segments that are prone to misidentification. We used MatlabHTK, a hidden Markov models interface for bioacoustics analyses, for illustrating AFSR technique by comparing two approaches, 1) a multispecies recognizer where all species are identified simultaneously, and 2) an assemblage of focal species recognizers (AFSR), where several recognizers that each prioritise a single focal species are then summarised into a single output, according to a set of rules designed to exclude unreliable segments. Both approaches (the multispecies recognizer and AFSR) used the same sound files training dataset, but different processing workflow. We applied these recognisers to PAM recordings from a remote island colony with five seabird species and compared their outputs with manual species identifications. False positive rates and precision improved for all the five species when using AFSR, achieving remarkable 0% false positives and 100% precision for three of five seabird species, and < 6% false positive rates, and >90% precision for the other two species. AFSR’ output was also used to generate daily calling activity patterns for each species. Instead of attempting to withdraw useful information from every fragment in a sound recording, AFSR prioritises more trustworthy information from sections with better quality data. AFSR can be applied to automated species identification from multispecies PAM recordings worldwide.

Summary 1.Passive bioacoustic recording devices are now widely available and able to continuously record remotely located sites for extended periods, offering great potential for wildlife monitoring and management. Analysis of the huge datasets generated, in particular for specific biotic sound recognition, remains a critical bottleneck for widespread adoption of these technologies as current methods are labour intensive. 2.Several methods borrowed from speech processing frameworks, such as hidden Markov models, have been successful in analysing bioacoustic data but the software implementations can be expensive and difficult to use for non-specialists involved in wildlife conservation. To remedy this, we present a software interface to a popular speech recognition system making it possible for non-experts to implement hidden Markov models for bioacoustic signal processing. Octave/Matlab functions are used to simplify the set up and the definition of a bioacoustic signal recogniser as well as the analysis of the results. 3.We present the different functions as a workflow. To demonstrate how the package can be used we give the results of an analysis of a bioacoustic monitoring dataset to detect the nocturnal presence and behaviour of a cryptic seabird species, the common diving petrel Pelecanoides urinatrix urinatrix, from Northern New Zealand. 4.We show that the package matlabHTK can be used efficiently to reconstruct the daily patterns of colony activity in the common diving petrel. This article is protected by copyright. All rights reserved.

Background Pooling techniques, where multiple sub-samples are mixed in a single sample, are widely used to take full advantage of high-throughput DNA sequencing. Recently, Ranjard et al. (PLoS ONE 13:0195090, 2018) proposed a pooling strategy without the use of barcodes. Three sub-samples were mixed in different known proportions (i.e. 62.5%, 25% and 12.5%), and a method was developed to use these proportions to reconstruct the three haplotypes effectively. Results HaploJuice provides an alternative haplotype reconstruction algorithm for Ranjard et al.’s pooling strategy. HaploJuice significantly increases the accuracy by first identifying the empirical proportions of the three mixed sub-samples and then assembling the haplotypes using a dynamic programming approach. HaploJuice was evaluated against five different assembly algorithms, Hmmfreq (Ranjard et al., PLoS ONE 13:0195090, 2018), ShoRAH (Zagordi et al., BMC Bioinformatics 12:119, 2011), SAVAGE (Baaijens et al., Genome Res 27:835-848, 2017), PredictHaplo (Prabhakaran et al., IEEE/ACM Trans Comput Biol Bioinform 11:182-91, 2014) and QuRe (Prosperi and Salemi, Bioinformatics 28:132-3, 2012). Using simulated and real data sets, HaploJuice reconstructed the true sequences with the highest coverage and the lowest error rate. Conclusion HaploJuice provides high accuracy in haplotype reconstruction, making Ranjard et al.’s pooling strategy more efficient, feasible, and applicable, with the benefit of reducing the sequencing cost.

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy to use and widely utilized by laboratories analyzing the adipogenic potential of mesenchymal cells. However, they are not specific to changes in gene transcription. We have developed a gene-specific differentiation assay to analyze when a mesenchymal cell has switched its fate to an adipogenic lineage. Immuno-labelling against fatty acid binding protein-4 (FABP4), a lineage-specific marker of adipogenic differentiation, enabled visualization and quantification of differentiated cells. The ability to quantify adipogenic differentiation potential of mesenchymal cells in a 96 well microplate format has promising implications for a number of applications. Hundreds of clinical trials involve the use of adult mesenchymal stromal cells and it is currently difficult to correlate therapeutic outcomes within and especially between such clinical trials. This simple high-throughput FABP4 assay provides a quantitative assay for assessing the differentiation potential of patient-derived cells and is a robust tool for comparing different isolation and expansion methods. This is particularly important given the increasing recognition of the heterogeneity of the cells being administered to patients in mesenchymal cell products. The assay also has potential utility in high throughput drug screening, particularly in obesity and pre-diabetes research.

Human expert analyses are commonly used in bioacoustic studies and can potentially limit the reproducibility of these results. In this paper, a machine learning method is presented to statistically classify avian vocalizations. Automated approaches were applied to isolate bird songs from long field recordings, assess song similarities, and classify songs into distinct variants. Because no positive controls were available to assess the true classification of variants, multiple replicates of automatic classification of song variants were analyzed to investigate clustering uncertainty. The automatic classifications were more similar to the expert classifications than expected by chance. Application of these methods demonstrated the presence of discrete song variants in an island population of the New Zealand hihi (Notiomystis cincta). The geographic patterns of song variation were then revealed by integrating over classification replicates. Because this automated approach considers variation in song variant classification, it reduces potential human bias and facilitates the reproducibility of the results.

Within breath-hold diving endotherms, procellariiform seabirds present an intriguing anomaly as they regularly dive to depths not predicted by allometric models. How this is achieved is not known as even basic measures of physiological diving capacity have not been undertaken in this group. To remedy this we combined time depth recorder (TDR) measurements of dive behaviour with haematology and oxygen store estimates for 3 procellariiform species (common diving petrels Pelecanoides urinatrix urinatrix; grey-faced petrels Pterodroma macro ptera gouldi; and sooty shearwaters Puffinus griseus) during their incubation phase. Among species, we found distinct differences in dive depth (average and maximal), dive duration and dives h �1 , with sooty shearwaters diving deeper and for longer than grey-faced petrels and common diving petrels. Conversely, common diving petrels dove much more frequently, albeit to shallow depths, whereas grey-faced petrels rarely dived whatsoever. Such differences in dive behaviour were reflected in haematological parameters, with sooty shearwaters having higher red blood cell counts and haematocrit (Hct) values compared to common diving and grey-faced petrels; whereas common diving petrels had significantly lower Hct but possessed higher haemoglobin concentrations per cell and greater respiratory oxygen stores than both sooty shearwaters and grey-faced petrels. Such results provide the first insights into the physiological traits underpinning procellariiform dive behaviour, and confirm the trend for deep-diving seabirds to have proportionally lower blood and respiratory oxygen stores than shallow divers.

Competition between organisms influences the processes governing the colonization of new habitats. As a consequence, species or populations arriving first at a suitable location may prevent secondary colonization. Although adaptation to environmental variables (e.g., temperature, altitude, etc.) is essential, the presence or absence of certain species at a particular location often depends on whether or not competing species co-occur. For example, competition is thought to play an important role in structuring mammalian communities assembly. It can also explain spatial patterns of low genetic diversity following rapid colonization events or the "progression rule" displayed by phylogenies of species found on archipelagos. Despite the potential of competition to maintain populations in isolation, past quantitative analyses have largely ignored it because of the difficulty in designing adequate methods for assessing its impact. We present here a new model that integrates competition and dispersal into a Bayesian phylogeographic framework. Extensive simulations and analysis of real data show that our approach clearly outperforms the traditional Mantel test for detecting correlation between genetic and geographic distances. But most importantly, we demonstrate that competition can be detected with high sensitivity and specificity from the phylogenetic analysis of genetic variation in space. (Competition; dispersal; phylogeography.)

We describe here the first complete genome assembly of the New Zealand green-lipped mussel, Perna canaliculus, mitochondrion. The assembly was performed de novo from a mix of long nanopore sequencing reads and short sequencing reads. The genome is 16,005 bp long. Comparison to other Mytiloidea mitochondrial genomes indicates important gene rearrangements in this family.

Uncovering the population genetic histories of non-model organisms is increasingly possible through advances in next generation sequencing and DNA sampling of museum specimens. This new information can inform conservation of threatened species, particularly those for which historical and contemporary population data are unavailable or challenging to obtain. The critically endangered, nomadic regent honeyeater Anthochaera phrygia was abundant and widespread throughout south-eastern Australia prior to a rapid population decline and range contraction since the 1970s. A current estimated population of 250-400 individuals is distributed sparsely across 600,000 km2 from northern Victoria to southern Queensland. Using hybridization RAD (hyRAD) techniques, we obtained a SNP dataset from 64 museum specimens (date 1879-1960), 102 'recent' (1989-2012) and 52 'current' (2015-2016) wild birds sampled throughout the historical and contemporary range. We aimed to estimate population genetic structure, genetic diversity and population size of the regent honeyeater prior to its rapid decline. We then assessed the impact of the decline on recent and current population size, structure and genetic diversity. Museum sampling showed population structure in regent honeyeaters was historically low, which remains the case despite a severe fragmentation of the breeding range. Population decline has led to minimal loss of genetic diversity since the 1980's. Capacity to quantify the overall magnitude of both genetic diversity loss and population decline was limited by the poorer quality of genomic data derived from museum specimens. A rapid population decline, coupled with the regent honeyeater's high mobility, means a detectable genomic impact of this decline has not yet manifested. Extinction may occur in this nomadic species before a detectable genomic impact of small population size is realised. We discuss the implications for genetic management of endangered mobile species and enhancing the value of museum specimens in population genomic studies.

International audience; Niche divergence is expected for species that compete for shared resources, including migrants that occupy similar regions during the non-breeding season. Studies of temperate seabirds indicate that both spatial and behavioural segregation can be important mechanisms for reducing competition, but there have been few investigations of resource partitioning by closely related taxa in low productivity, tropical environments. We investigated niche partitioning in 3 gadfly petrel taxa, Pterodroma leucoptera leucoptera (n = 22), P. leucoptera caledonica (n = 7) and P. pycrofti (n = 12), during their non-breeding season in the eastern tropical Pacific Ocean by combining tracking data from geolocator-immersion loggers with remotely sensed environmental data in species distribution models (SDMs), and by comparing feather stable isotope ratios. The 3 taxa showed spatial partitioning: two foraged in the North Equatorial Counter Current and one in the South Equatorial Current. This reflected differences in their realised habitat niches, with significant taxon-specific responses to thermocline depth, sea surface temperature and bathymetry. There were also differences among taxa in activity patterns, and all birds spent a much larger proportion of time in flight at night than during the day, suggesting predominance of nocturnal foraging behaviour. Comparison of stable isotope ratios in feathers suggests that P. l. leucoptera and P. pycrofti mainly consume vertically migrating mesopelagic fishes, whereas the diet of P. l. caledonica also includes some lower trophic levels including crustaceans and squid. Unique insights can be gained from studies of the foraging ecology of tropical pelagic seabirds, in comparison with temperate and polar waters, and are urgently required for understanding and protecting tropical avifauna in key marine habitats.

A variety of bioacoustics distance metrics have been used to assess similarities in the vocalizations of different individuals. Here, we provide a detailed analysis of several acoustic similarity indices, some of which have been developed with the specific aim of characterizing the sensory coding of auditory stimuli. We compare different approaches through the analysis of begging calls of several passerine species and specialist brood parasitic cuckoos that putatively evolved to mimic their hosts. The different bioacoustics distances did not provide consistently corre- lated similarity patterns, implying that they are sensitive to different sound features. However, the encoded spectro- gram alignment method was correlated with all other acoustic distance metrics, suggesting that this method

The study of the evolution of sexual differences in behavioral and morphological displays requires analyses of the extent of sexual dimorphism across various sensory modalities. In the seabird family Sulidae, boobies show dramatic sexual dimorphism in their vocalizations, and gannet calls have also been suggested to be dimorphic to human observers. This study aimed to evaluate the presence of sexually dimorphic calls in the Australasian gannet (Morus serrator) through the first comprehensive description of its vocalizations recorded at two localities; Cape Kidnappers, where individuals were banded and sexed from DNA samples, and at the Muriwai gannetry, both on the North Island of New Zealand. Calls were first inspected using basic bioacoustic features to establish a library of call element types for general reference. Extensive multivariate tests, based on a dynamic time warping algorithm, subsequently revealed that no sexual differences could be detected in Australasian gannet calls. The analyses, however, indicated extensive and consistent vocal variation between individuals, particularly so in female gannets, which may serve to signal individual identity to conspecifics. This study generates predictions to identify whether differences in Australasian gannet vocalizations play perceptual and functional roles in the breeding and social biology of this long-lived biparental seabird species.

BackgroundOur understanding of the molecular pathways that underlie melanoma remains incomplete. Although several published microarray studies of clinical melanomas have provided valuable information, we found only limited concordance between these studies. Therefore, we took an in vitro functional genomics approach to understand melanoma molecular pathways.Methodology/principal findingsAffymetrix microarray data were generated from A375 melanoma cells treated in vitro with siRNAs against 45 transcription factors and signaling molecules. Analysis of this data using unsupervised hierarchical clustering and Bayesian gene networks identified proliferation-association RNA clusters, which were co-ordinately expressed across the A375 cells and also across melanomas from patients. The abundance in metastatic melanomas of these cellular proliferation clusters and their putative upstream regulators was significantly associated with patient prognosis. An 8-gene classifier derived from gene network hub genes correctly classified the prognosis of 23/26 metastatic melanoma patients in a cross-validation study. Unlike the RNA clusters associated with cellular proliferation described above, co-ordinately expressed RNA clusters associated with immune response were clearly identified across melanoma tumours from patients but not across the siRNA-treated A375 cells, in which immune responses are not active. Three uncharacterised genes, which the gene networks predicted to be upstream of apoptosis- or cellular proliferation-associated RNAs, were found to significantly alter apoptosis and cell number when over-expressed in vitro.Conclusions/significanceThis analysis identified co-expression of RNAs that encode functionally-related proteins, in particular, proliferation-associated RNA clusters that are linked to melanoma patient prognosis. Our analysis suggests that A375 cells in vitro may be valid models in which to study the gene expression modules that underlie some melanoma biological processes (e.g., proliferation) but not others (e.g., immune response). The gene expression modules identified here, and the RNAs predicted by Bayesian network inference to be upstream of these modules, are potential prognostic biomarkers and drug targets.

Pelagic seabirds are highly mobile, reducing the likelihood of allopatric speciation where disruption of gene flow between populations is caused by physically insurmountable, extrinsic barriers. Spatial segregation during the non-breeding season appears to provide an intrinsic barrier to gene flow among seabird populations that otherwise occupy nearby or overlapping regions during breeding, but how this is achieved remains unclear. Here we show that the two genetically distinct populations of Cook's petrel (Pterodroma cookii) exhibit transequatorial separation of non-breeding ranges at contemporary (ca. 2-3 yrs) and historical (ca. 100 yrs) time scales. Segregation during the non-breeding season per se appears as an unlikely barrier to gene flow. Instead we provide evidence that habitat specialization during the non-breeding season is associated with breeding asynchrony which, in conjunction with philopatry, restricts gene flow. Habitat specialization during breeding and non-breeding likely promotes evolutionary divergence between these two populations via local adaptation.

Evolution of bird vocalizations is subjected to selection pressure related to their functions. Passerine bird songs are also under a neutral model of evolution because of the learning process supporting their transmission; thus they contain signals of individual, population, and species relationships. In order to retrieve this information, large amounts of data need to be processed. From vocalization recordings, songs are extracted and encoded as sequences of syllables before being compared. Encoding songs in such a way can be done either by ear and spectrogram visual analysis or by specific algorithms permitting reproducible studies. Here, a specific automatic method is presented to compute a syllable distance measure allowing an unsupervised classification of song syllables. Results obtained from the encoding of White-crowned Sparrow (Zonotrichia leucophrys pugetensis) songs are compared to human-based analysis.