Likely geographic origins derived from analyses of DNA microsatellites were used as priors for Bayesian analyses in which birds were assigned to a breeding-ground origin using their δ2Hf values. As with most applications of Bayesian methods, our approach greatly improved the results . Area of origin decreased by 3 to 5-fold on average, but ranged up to a 10-fold improvement. We recommend this approach in future studies of migratory connectivity and suggest that our methodology could be applied more broadly to the study of dispersal, sources of productivity of migratory populations, and a range of evolutionary phenomena.An enduring problem in avian ecology and conservation is linking breeding and wintering grounds of migratory species. As migratory species and populations vary in the degree to which individuals from distinct breeding locales mix on stop-over sites and wintering grounds, establishing migratory connectivity informs our understanding of population demography and species management. We present a new Bayesian approach for inferring breeding grounds of wintering birds of unknown origins in North America. We incorporate prior information from analysis of genetic markers into geographic origin assignment based upon stable-hydrogen isotope analysis of feathers (δ Migratory species and populations vary in the degree to which individuals from distinct breeding locales mix on different stop-over sites and wintering grounds. Therefore, to understand population demographics, local adaptation, and causes of diversification, the spatial characteristics (i.e. which populations mix on breeding and wintering grounds) and strength  have provided the most direct empirical estimates of movements of migrants throughout the world a priori. For example, inherited phenotypic characteristics that show diagnostic geographic variation have also proven useful for distinguishing among breeding populations of some species on the wintering grounds Intrinsic markers representative of the region of origin require only an initial capture, but to be of use, population-level differences in the marker must exist, and have been quantified 2H) in rainfall show strong and predictable spatial gradients. Hydrogen in precipitation is integrated into the tissues of an organism, and so consumer tissues ultimately reflect the H isotope composition of the hydrosphere driving the local foodweb. A number of assumptions are inherent in the application of H isotope measurements for the study of geographic connectivity Analysis of stable-hydrogen isotopes in feathers is one of the most commonly applied intrinsic marker techniques in studies of migration Genetic markers are also commonly used to infer origins of migratory species. Phylogeographic patterns of variation in birds tend to be longitudinal due to glaciation Genetic and stable isotope markers have been used side-by-side in several studies of migratory connectivity to date, 2Hf) to increase accuracy of assignment of birds moving through stopover sites. However, banding data can be limited for some species despite intensive mark and recapture schemes There are two major challenges in applying Bayesian approaches to assigning geographic origins to samples based upon stable-isotope analysis of feathers: choosing suitable prior probabilities (hereafter priors) and integrating these into Bayesian analysis. Various sources of information can be used to refine the assigned origins of unknown source birds based on isotopic data, and several methods have been applied 2Hf isoscapes generally reflect broad latitudinal clines with little longitudinal variation Lanius ludovicianus) derived from spatial analysis of nuclear DNA microsatellites as priors for Bayesian assignment based on δ2Hf. We use data from the Loggerhead Shrike to illustrate our novel approach. Results suggest that while both markers are capable of assigning individuals to their likely origin, our Bayesian approach was far superior to assessments based upon either genetic or δ2Hf alone.Given that genetic structuring is often longitudinal in northern temperate species in North America, with greater genetic distinction on east-west over north-south axes Mus mustellus) in a protective wire ‘hardware-cloth’ cage. No mouse was used as bait in a trap for more than an hour at a time. While in the hardware-cloth cage, the mouse was provided with a slice of apple for nourishment. In between deployment, the live trap was placed out of the sun and covered with a breathable towel to reduce the stress level of the mouse. In between use, mice were housed in a mouse cage approximately 35 cm(l)×25 cm(w)×30 cm(h), with access to food and water ad libitum. To date, no mouse has been injured by a shrike or in handling.Adult shrikes were captured using a modified Potter trap Loggerhead Shrikes were sampled at 44 North American breeding locales across their geographic range during the breeding season (2004–2008) . Each biAphelocoma coerulescensTotal genomic DNA was extracted from a ∼1 cm clipping of the distal tip of a plucked tail feather, using a QIAGEN  DNEasy Extraction Kit as per Coxon et al. 2Hf) data were obtained from 556 of the shrikes sampled (2Hf were obtained using continuous-flow isotope-ratio mass spectrometry (CF-IRMS) following the ‘comparative equilibration’ technique described by Wassenaar and Hobson 2Hf isoscape for SY and ASY individuals independently using the δ2Hf values of the feathers represented by our best sample of putatively known-origin birds. Methods used to derive our isoscapes are detailed in The distal tip of P1 feather was clipped for use in stable isotope analysis; P1 is a feather that is almost exclusively grown on the breeding grounds in this species  sampled . We adde2Hf data (n = 515), we removed a subset of birds  to assess the impact of the Bayesian approach on the study of connectivity  encompassed within the probable area of origin using both 2∶1 and 3∶1 odds ratios, with and without applying genetic priors. Assignment of the validation samples using deuterium feather values was undertaken using the ‘raster’ package 2Hf values, with and without priors, and with microsatellite data is provided in To compare the probability densities modeled to describe potential origins using our Bayesian approach versus assignments based on δAll sampling completed during this study complied with the current laws of the countries in which it was performed. Queen’s University’s Animal Care Committee approved the protocols used in sampling animals in this study (Protocol number Lougheed-2008-059-Or).2Hf isoscape versus genetic admixture coefficients alone with 71% of individuals assigned ‘correctly’ using microsatellite markers alone, 73% assigned correctly using the δ2Hf alone with 2∶1 odds ratio and 81% assigned correctly using the δ2Hf alone with 3∶1 odds ratio  was weak and Q genetic admixture coefficients were relatively low, the Bayesian methodology resulted in a much-reduced geographic area of probable origin  an individual ASY bird’s putative origin and a 5-fold decrease for individual SY birds . The mage origin .2Hf or genetic data alone. The level of improvement depended on the interplay between the local steepness of the isotope isocline and the strength of genetic admixture coefficient Q.We describe a Bayesian approach for assigning birds of unknown provenance to a breeding ground origin using both genetic and isotope data and validate our results using putatively ‘known source’ birds from across the species range in North America. We found dramatic improvement in the spatial resolution of the assigned area of origin using the Bayesian methodology with two types of intrinsic markers over models using δ2Hf data alone for almost all validation samples and for all sample areas    also corresponded to the level of improved resolution, in particular in areas characterized by a weak isocline. For example, where the isocline was shallow and the Q admixture coefficients were low, as in our southeastern Texas sample locale (Q = 0.53 to 0.62), there was only a moderate decrease in the spatial extent of the likely geographic origin in the model using priors versus that using the δ2Hf data alone, but in areas where the isocline was relatively shallow but Q values were stronger, such as in our Louisiana (Q = 0.71 to 0.77) and northern Texas sample areas (Q = 0.74 to 0.83), the improvement in resolution was greater (The greatest improvement in resolving likely areas of origin using our Bayesian models occurred in areas with a steep isocline and high to 0.98) , but theto 0.52) . However greater .Our results from North Carolina and Nebraska , both ch2Hf isoscape alone, this in part flows from real biological phenomena. Specifically, for philopatric birds, genetic diagnoses of origin will be concordant with those based on acquired markers, such as feather stable isotope values. However, assignments based on genetic and stable isotope data will be discordant for individuals that have dispersed beyond the boundaries of the spatial resolution for the isoscape. Thus, methodological improvements would presumably have little impact on rates of assignment. However, previously developed isoscapes While the proportion of individuals assigned to their putative origin (i.e. sample area) remained essentially unchanged using the Bayesian model versus that based on a δDispersal plays a fundamental role in determining range size and expansion We have presumed that the capture location of each individual during the breeding season was within its natal area, in the case of SY birds, or, in the case of ASY birds, its previous breeding area. Obviously, this will not be true for all samples as some first year breeders and older birds will disperse varying distances between breeding seasons The inclusion of short-distance dispersers when parameterizing our model would likely neither result in discordance between genetic and stable isotope data nor affect probability of assignment, unless isotopic or genetic spatial structure was pronounced. However, long-distance dispersal movements among breeding populations, specifically breeding season dispersal rather than migratory movements, would affect both model parameterization and assignment of individuals of unknown origin (e.g. migratory or wintering). While we did not specifically test for these effects, we suggest that the inclusion of isotopic data obtained from long-distance dispersers when parameterizing our model, regardless of where within the continental range they originated, would affect the resolution of our isocline, with a negative correlation between the number of long-distance dispersers and the strength of the isocline. Thus, a weakened isocline and increased variance in deuterium values would increase the putative area of origin and/or diminish the accuracy of assignment in assignments of unknown individuals to their geographic origins. Long-distance immigrants will be evident as isotopic outliers relative to others sampled within a locale. Deuterium values do vary locally and thus, while more stringent removal of such outliers would allow for finer-scale designation of origin for assigned individuals, it would also undoubtedly diminish the accuracy of results.Long-distance dispersers also complicate analysis of genetic markers. STRUCTURE does not require a priori designation of sample populations, allowing immigrants to be assigned to their ‘correct’  genetic cluster, rather than to that of the genetic cluster representing the area in which they were sampled. The inclusion of immigrants diminishes resolution of the spatial genetic structuring. In population genetic terms, the number and natal origin (i.e. genetic heritage) of F1 hybrids – those individuals that result from successful dispersal and subsequent reproduction – will have a direct impact on the genetic structuring of the species. Dispersal can increase the extent of hybrid zones and result in loss of genetic structuring altogether. From the perspective of assignment of unknown individuals, the probability of assignment will be positively related to the strength of genetic structuring, the spatial resolution of genetic structure, and their own level of admixture.2Hp with δ2Hf have shown general agreement across several avian species Methods for assignment of individuals to their putative breeding population origin have improved markedly since the isotopic method was first proposed for birds Table S1List of samples included in this study. Given are the geographic coordinates for the range over which individuals were sampled in each area, number of samples for which nuclear microsatellite and deuterium data were available and number of samples used to validate methodology from each sample locale.(DOCX)Click here for additional data file.Table S2Assignment of individuals in the validation sample set to origin using various models. Shown is the number of individuals assigned to correct breeding ground origin based on nuclear microsatellite (Msat) data alone, and feather δ2H values using 2∶1 and 3∶1 odds ratios without priors.(DOCX)Click here for additional data file.Table S3Probable area of origin for each individual in the validation sample set using various models. Shown is the number of raster cells (20 km2 blocks) assigned as being the probable area of origin for each individual in each sample locale using δ2Hf data alone and using Bayesian methodology with nuclear microsatellite data as priors for deuterium data with 2∶1 and 3∶1 odds. Numbers in bold indicate individuals whose capture coordinates fell within the probable area of origin. Numbers in bold italics indicate individuals whose capture location fell within 200 km of the probable area of origin. Numbers in italics indicate individuals whose capture location fell within 400 km of the probable area of origin. Admixture coefficient corresponds to the genetic cluster with which the individual most closely assigned.(DOCX)Click here for additional data file.Table S4Statistical comparison of expected versus observed assignment rates in the two Bayesian models. Shown are the average number and cumulative percentage of individuals assigned in the probable area of origin or to within a buffer zone of varying size around the probable area of origin using δ2Hf data with 2∶1 and 3∶1 odds without priors. Bolded values signify tests where the number of correct assignments was greater than expected. χ2 tests performed with one degree of freedom.(DOCX)Click here for additional data file.Text S1(DOC)Click here for additional data file.


Aristeus antennatus and Aristaeomorpha foliacea, was compared through multiple genetic markers. These marine shrimp species are of high commercial importance, and are exploited in the Mediterranean Sea (MED) and in Mozambique Channel (MOZ) where they occur in partial sympatry. Aristeus antennatus (N = 50) from Western and Eastern Mediterranean , Atlantic Ocean (AO) and MOZ, and Aristaeomorpha foliacea (N = 40) from WM, EM, MOZ North-Western Australia (AUS) were analyzed with two nuclear genes (PEPCK and NaK) and one mitochondrial (COI) gene. Within the study area differences were found between the two species in their phylogeographical patterns, suggesting distinct responses to environmental changes. Monophyly of Aristeus antennatus was found across its distributional range. This pattern contrasted by a deep evolutionary split within Aristaeomorpha foliacea where genetic diversity followed geography distinguishing MED-MOZ and AUS. We propose that the AUS lineage of A. foliacea warrants consideration as a distinct species, with consequent implications in systematics and resource management.Phylogeographical studies can reveal hidden patterns in the evolutionary history of species. Comparative analyses of closely related species can further help disentangle the relative contributions of processes responsible for such patterns. In this work, the phylogeography of two aristeid species,  Aristeus antennatus  and Aristaeomorpha foliacea  are the two most economically valuable species of the Aristeidae family. They occur in sympatry in a large part of their geographical and bathymetrical distribution. Aristeus antennatus can be found between 80 Aristaeomorpha foliacea is distributed between 120 and 1000 m depth Aristeus antennatus is distributed in the Mediterranean Sea and adjacent Atlantic Ocean, from Portugal to Cape Verde Islands Aristaeomorpha foliacea is found within the same geographical range of Aristeus antennatus, but in the Indian Ocean its distribution is much wider, reaching North-Western Australia coastal waters Aristeus antennatusAristaeomorpha foliaceaAristeid shrimps  are a group of commercially important species within the superfamily Penaeoidea (Rafinesque 1815), which are also known as red shrimps because of their body coloration Aristeus virilis) Aristaeomorpha foliacea) Aristeus antennatus and Aristaeomorpha foliacea were consistent in showing a general picture of relative genetic homogeneity within the Mediterranean Sea Aristeus antennatus detected significant genetic differentiation among Western Mediterranean, Eastern Mediterranean, Atlantic Ocean and Mozambique Channel Aristaeomorpha foliacea detected three mtDNA monophyletic lineages corresponding to Mediterranean Sea, Mozambique Channel and North-Western Australia Despite the high economic value of both species, little is known about their systematics, or their biology, ecology and population genetics outside the Mediterranean Sea. Information on phylogenetic relationships within Aristeidae and with other Penaeoidea families is very scarce. Only two surveys included aristeid species as a part of reviews on the phylogeny of Decapoda , we include nuclear molecular markers in this study: phosphoenolpyruvate carboxykinase (PEPCK) and sodium–potassium ATPase α -subunit (NaK). These markers have proven satisfactory to resolve phylogenies of crustacea and insecta [10 and references therein].Given their close taxonomic relationship, partial sympatry and large similarities in adult ecology and reproductive biology Aristeus antennatus and Aristaeomorpha foliacea to contrast how these two species have been influenced by present or historical factors. Such comparisons can provide sound information on the evolutionary history of species, as well as help in the identification of evolutionary isolated areas and unveil hidden cryptic species A. antennatus and A. foliacea.Our objective was to compare the phylogeographical patterns of Aristeus antennatus and 40 Aristaeomorpha foliacea individuals were selected from previous works based on mtDNA Aristeus antennatusA. antennatus between the Ionian Sea and Aegean Sea are genetically similar based on the fact that A. foliacea were genetically similar between these two Seas Aristaeomorpha foliaceaAristeus virilis were also included in the analysis  for comparative purposes. Available GenBank sequences for the three studied genes were found only for Penaeus monodon  and Solenocera crassicornis , which belongs to the same superfamily (Penaeoidea), and were used as outgroup species in phylogenetic analyses.Sub-samples of 50 5′-CGGAGGATCAATCATCGACA-3′, for Aristeus spp.), NaK-rAf . Amplifications for PEPCK and NaK were carried out in a reaction mix containing 1–3 µl of template DNA (25 ng µl−1), 1X PCR reaction buffer, 3 mM MgCl2, 200 µM dNTPs, 200 nM of each primer, and 0.03 U of DNA polymerase  in a 30 µl final volume. The PCR profile for both nuclear genes was as follows: 3 min at 94°C for initial denaturation, followed by 35 cycles of 30 s at 94°C, 30 s at 57°C and 55°C annealing temperatures for PEPCK and for NaK, respectively, 90 s at 72°C with a final extension for 10 min at 72°C. Non-template controls were run in all PCRs to ascertain that no cross contamination took place. PCR products were verified on 1% agarose gel with ethidium bromide (0.5 mg/ml). Sequences were cleaned for sequencing by treating with exonuclease I and shrimp alkaline phosphatase Laboratori d’Ictiologia Genètica, Universitat de Girona, Spain.DNA extraction of ethanol-preserved samples, polymerase chain reaction (PCR) and sequencing of COI followed the procedures outlined in Fernández et al. Aristaeomorpha foliacea . Final edition and concatenation of the three genes were performed with BioEdit v7.0.4.1 Nucleotide sequences were aligned and edited in SeqScape v2.5 (Applied Biosystems) employing as reference the partial regions of COI, PEPCK and NaK genes from 2) statistic, with Bonferroni correction Genetic variability estimates were calculated on each gene, separately. Haplotype and nucleotide diversity were calculated for COI gene with DnaSP v5, and expected heterozygosity was obtained for PEPCK and NaK genes with the online version of Genepop v4.0.10 i = 0.563, and base frequency A = 0.261, C = 0.255, G = 0.215, T = 0.269.A partition homogeneity test i = 0.576, and equal base frequencies; successively, a Bayesian phylogenetic tree was constructed with MrBayes v3.1.2 6 generations. The chain was sampled every 100 generations to obtain 20 000 sampled trees. The first 5 000 trees (25%) were discarded as the burn-in phase. A final consensus tree with branch length and clade credibility (posterior probability) was generated with the 75% remaining samples.Maximum likelihood (ML) Aristeus antennatus and Aristaeomorpha foliacea datasets. Tamura-Nei genetic distances i = 0.69 for COI, TN i = 0.636 and α = 0.466 for NaK, and K2P i = 0.926 for PEPCK. Standard errors were obtained after 10 000 replicates. Correction between groups for all genes was calculated (DA) Analysis of molecular variance (AMOVA) Aristeus antennatus, 42 individuals were successfully sequenced for PEPCK (536 bp) and 27 for NaK (498 bp) gene, providing five and 26 different genotypes, respectively  haplotypes plus one PEPCK and two NaK different genotypes. Sequences were deposited in GenBank (P = 0.194) allowing their combination for successive analyses. The final concatenated dataset consisted of 55 sequences  and the two outgroup sequences that presented 236 parsimony informative sites from a total of 1 548 bp.From a total of 50 ectively . From a  GenBank . The parh >0.378), with the highest values for Aristeus antennatus and Aristaeomorpha foliacea in MOZ and AUS, respectively  than in Aristeus antennatus (HO = 0.43)  and AUS (Af-ph20), and some alleles of those genotypes  were private for regions as well (Genetic variability of PEPCK was higher in nly four . This ou = 0.43) . Each sp as well .Aristeus antennatus than in Aristaeomorpha foliacea, as revealed by the higher number of alleles detected in the former species (38 vs. 6) , than in Aristeus antennatus (HO≥0.75)  . HoweverO≥0.75) . Almost be drawn . InsteadAristeus and Aristaeomorpha genera were identified. Within Aristeus, Aristeus virilis and Aristeus antennatus clustered into exclusively monophyletic lineages. Within Aristeus antennatus no clear associations between geographical distribution and sequences obtained was detected. Within Aristaeomorpha, two major phylogroups and geographical association of genetic diversity were detected. One group corresponding to AUS and the second including MED and MOZ, where MED appears monophyletic   supported the existence of genetic differentiation at the regional level (Aristeus antennatus (D = 0.0033±0.0008) than that found in Aristaeomorpha foliacea (D = 0.0094±0.0015)  was about the half of genetic distance between Aristeus species (DA = 0.0492±0.0062)  . Conversal level . The ave±0.0015) ; net gen±0.0062) . Deep diDA = 0.0301±0.0064 to DA = 0.1051±0.0171) than for PEPCK (DA = 0.0056±0.0022 to DA = 0.0238±0.0044). These values fall within the range previously reported by Ma et al. D = 0.01–0.099, PEPCK D = 0.002–0.036). Very low genetic distance values were obtained between MED and MOZ regions of Aristaeomorpha foliacea, which were then pooled together to estimate the genetic distance between MED-MOZ and AUS regions . The values of mitochondrial genetic distance between Aristeidae species (DA = 0.1143–0.1949) were of the same order of magnitude of those values with outgroup species (Aristaeomorpha foliacea regions MED-AUS (DA = 0.0690±0.0117) and MOZ-AUS (DA = 0.0698±0.0117) are equivalent to the 61% genetic distance between true congeneric species ; however, the genetic distance between MED and MOZ Aristaeomorpha foliacea geographical regions is equivalent to the 8% (DA = 0.0090±0.0040)  .Aristeus antennatus, Aristeus virilis and Aristaeomorpha foliacea by a large number of mutational steps and connected the three species in a circle , whilst Aristaeomorpha foliacea presented three subnetworks, each corresponding to one of the regions considered. The subnetwork corresponding to MOZ connected with i) MED subnetwork through four MSs, ii) AUS subnetwork through 32 MSs, and iii) Aristeus antennatus through 89 MSs. The subnetwork corresponding to AUS connects with Aristeus virilis through 89 MSs.The median-joining network of haplotypes clearly separated a circle . AristeuGenealogical concordance is expected to be found among closely related taxa, particularly if they share the same habitat and are co-distributed Aristeus antennatus showed concordant patterns across genes. Previous mitochondrial genetic analysis detected significant genetic differences between the Mediterranean Sea (MED), the Atlantic Ocean (AO) and the Mozambique Channel (MOZ) A. antennatus is a monophyletic lineage. This outcome is corroborated by i) the homogeneity in the distribution of nuclear alleles frequencies between regions (ii) the combined analyses of mitochondrial and nuclear genes carried out in the present work   loci. Previous mtDNA genetic analysis detected three highly differentiated lineages that were geographically characterized: MED, MOZ and North-Western Australia (AUS) ST = 0.874, P<0.001), confirming the existence of a clearly differentiated AUS lineage  = 0.079) . Convers lineage . The mit lineage  was not  lineage , which p lineage . However lineage . IncomplAristeus antennatus and Aristaeomorpha foliacea co-occur (MED and MOZ) prompted the following hypotheses: i) a vicariant event separated the MED and MOZ populations of these species, where ii) evolutionary forces and/or ecological processes had a lesser effect on A. antennatus than on A. foliacea, shaping intraspecific phylogenies differently. These hypotheses are discussed below.Discordant patterns detected within the regions in which Aristaeomorpha foliacea has been estimated at ca. 0.5 Mya (Aristeus antennatus and Aristaeomorpha foliacea within its area of influence (between Cape Verde and South Africa) where currently there is no knowledge of their presence Based on COI genetic distances and using 0.83–1.2% evolutionary rate for COI gene  was almost the half of that detected between true congeneric species  from the Mediterranean Sea and Eastern Atlantic, and A. rostridentata (Spence Bate 1888) from the Indo-Pacific A. rostridentata from Fiji Islands (Pacific Ocean) with i) individuals of A. foliacea from the Mediterranean Sea and Atlantic coast of Morocco and ii) individuals of A. rostridentata from the Indian Seas . Finding no single constant morphological difference between A. rostridentata holotype and the individuals of A. foliacea from the Mediterranean Sea, Calman A. rostridentata holotype, from Pacific Ocean, and the individuals from the Indian Seas. This distinction led him to consider A. rostridentata from Fiji Islands as a synonym of A. foliacea and to recognize individuals from the Indian Seas as representatives of a distinct species, for which he proposed the name Aristaeomorpha woodmasoni, after Wood-Mason, Alcock and Kemp, who had already pointed out some of these morphological differences Before 1920’s there were two recognized species in the genus A. foliacea from MED-MOZ and AUS  was almost the half of that detected between true congeneric species (A. foliacea i) showed strong support from multilocus genealogical concordance, ii) inhabits in a recognized distinct biogeographical province  expresses substantial levels of multilocus divergence since the split from a parental lineage, we suggest that A. foliacea in NorthWestern Australia should be considered a distinct species. We are confident that if the holotype of A. rostridentata from the Fiji Islands, or any paratype, can ever be genetically analyzed, this will result genetically like the AUS lineage of A. foliacea. Thus, following the rules of the International Commission on Zoological Nomenclature, we propose to resuscitate Aristaeomorpha rostridentata for the Australian lineage. Furthermore, within MED-MOZ lineage Aristaeomorpha foliacea expressed monophyletic mitochondrial clusters, with significant divergence in the distribution of allele frequencies , nucleotide diversity (π), standard deviation (S.D.). ALB: Alborán Sea, WM: Western Mediterranean, EM: Eastern Mediterranean, AO: Atlantic Ocean, MOZ: Mozambique Channel, AUS: North-Western Australia.(DOC)Click here for additional data file.Table S2GenBank accession numbers for each individual analyzed. COI haplotype number (ch), PEPCK (ph) and NaK (nh) genotype number and allele number. Empty rows correspond to individuals with unsuccessful amplification. The individuals used in phylogenetic analyses are in bold. Location codes are as in (DOC)Click here for additional data file.Table S3Matrix of Tamura-Nei genetic distance measures for concatenated data (1 548 bp) for species and lineages detected in.(DOC)Click here for additional data file.Table S4Matrix of genetic distances between all lineages for nuclear genes. Below diagonal, genetic distances for NaK following Tamura-Nei, and genetic distances for PEPCK are given above diagonal following K2P.(DOC)Click here for additional data file.Table S5Matrix of Tamura-Nei genetic distances calculated for COI dataset (514 bp) between all lineages  and estimated times (Mya) since divergence , using 0.83–1.2% evolutionary rate .(DOC)Click here for additional data file.Table S6Allele-frequencies homogeneity test for nuclear loci (NaK and PEPCK) between regions for both species. Contingency chi-squared value (χ2), degrees of freedom (df), p-value (P).(DOC)Click here for additional data file.


Cyanoliseus patagonus  across its wide distributional range in Chile and Argentina.While the gene flow in some organisms is strongly affected by physical barriers and geographical distance, other highly mobile species are able to overcome such constraints. In southern South America, the Andes  may constitute a formidable barrier to dispersal. In addition, this region was affected by cycles of intercalating arid/moist periods during the Upper/Late Pleistocene and Holocene. These factors may have been crucial in driving the phylogeographic structure of the vertebrate fauna of the region. Here we test these hypotheses in the burrowing parrot Our data show a Chilean origin for this species, with a single migration event across the Andes during the Upper/Late Pleistocene, which gave rise to all extant Argentinean mitochondrial lineages. Analyses suggest a complex population structure for burrowing parrots in Argentina, which includes a hybrid zone that has remained stable for several thousand years. Within this zone, introgression by expanding haplotypes has resulted in the evolution of an intermediate phenotype. Multivariate regressions show that present day climatic variables have a strong influence on the distribution of genetic heterogeneity, accounting for almost half of the variation in the data.Here we show how huge barriers like the Andes and the regional environmental conditions imposed constraints on the ability of a parrot species to colonise new habitats, affecting the way in which populations diverged and thus, genetic structure. When contact between divergent populations was re-established, a stable hybrid zone was formed, functioning as a channel for genetic exchange between populations. Current molecular genetic methods allow the understanding of the genetic structure underlying different populations of a species with previously unforeseen resolution e.g. -3]. As a. As a3]]However, when contact between divergent populations is re-established, hybrid zones can form with resultant consequences for the evolutionary trajectories of the interbreeding populations [e.g. ]. Two scAlthough Pleistocene climate conditions played an important role in initiating major phylogeographical structuring in today's fauna , very liCyanoliseus patagonus) . This species is distributed in a particularly heterogeneous arid to semi-arid landscape, across an extensive ~1,000,000 km2 range in Chile and Argentina. A previous study , which are based on the WorldClim database [http://www.worldclim.org], version 1.3, at 2.5 minutes resolution . While y Figure , we alsots range , this rets range  also sugOur data show that up to 48% of the genetic variation can be accounted for by present-day climatic variables, considerably greater than the variation accounted for phenotype and ecoregions. This is even more surprising since ecoregions would normally be expected to co-vary with climate. This lack of variation attributable to ecoregion might be explained by the fact that the two major constraints on burrowing parrots habitat suitability, namely availability of water and cliffs, are not included in ecoregion definitions. Even the significant correlation with present-day climate appears counterintuitive since climatic conditions in the past are more likely to have had an influence over population structure. However, if we consider that the majority of the haplotypes within Patagonus1/2 and Andinus most likely evolved after the LGM 14 kya or more recently Figure , and thabloxami are distributed in the IV and VII regions of the country . This a country ,85. The The situation differs considerably in Argentina, where burrowing parrots are officially considered an abundant agricultural pest , despite agricultural damage occurring only locally ,86, withA negative side effect of the recent protection of Patagonian populations is the noticeable increase in commercial value of Andinus populations in the Cuyo region. All available data from this study Tables , 2 &3 anMost alterations in the environment, like climate change, are potential sources of new or intensified directional selection on traits important for the fitness of the species living in it [e.g. ]. EvolutThe authors declare that they have no competing interests.JFM and PQ conceived and designed the study. YM, GS and MS planned the genetic analyses. JFM, PQ, MF and MC carried out the extensive fieldwork. GKM, NK and JFM generated the molecular data. JFM, NK and YM participated in bioinformatic analyses. YM, JFM and PQ were responsible for data analysis and drafted the manuscript. GS and MS reviewed the final draft of the manuscript. All authors read and approved the final manuscript.Table S1. Classification, GenBank accession numbers, and sample locations with detailed climate parameters of 150 burrowing parrots (Cyanoliseus patagonus).Click here for fileTable S2. Haplotype distribution of 150 burrowing parrots (Cyanoliseus patagonus).Click here for fileTable S3. Divergence estimates using fossil calibration with rate smoothing across three gene partitions in comparison with cytochrome b mutation rates.Click here for fileFigure S1. Sample locations (black dots), main places, regions, and ecoregions mentioned in the text, and rivers of Southern South America.Click here for file


Corythoichthys haematopterus, in the northwest Pacific was investigated based on the partial mitochondrial DNA cytochrome b (589 bp) and 16S rRNA (528 bp) region sequences of 108 individuals collected from six sites along the coast of the Japanese archipelago and one site on Mactan Island, the Philippines. A total of 60 and 28 haplotypes were obtained from the cytochrome b and 16S rRNA regions, respectively. Two genetically distinct lineages were detected: lineage A and B, which are separated by mean pairwise genetic distances of 23.3 and 14.1% in the partial cytochrome b and 16S rRNA genes, respectively. Such a huge genetic divergence between lineages, which is comparable to or even higher than the interspecific level, and the difference in their geographical distributions and habitat preferences suggests that they are distinct species, although there is no marked difference in their morphology. Haplotype network and gene and nucleotide diversity statistics indicate that the two lineages have different biogeographic histories: lineage A experienced rapid population expansion after a population bottleneck whereas lineage B has a long evolutionary history in a large stable population. In contrast, the levels of genetic variation among populations are relatively low in both lineages, probably because of frequent gene flow among populations resulting from the dispersal of pelagic larvae by the Kuroshio Current. These results indicate that past climatic events and contemporary oceanographic features have played a major role in establishing the population genetic structure of C. haematopterus.The population genetic structure of the messmate pipefish, The online version of this article (doi:10.1186/2193-1801-2-408) contains supplementary material, which is available to authorized users. Elucidating the population genetic structure of a species can aid conservation decisions, enable us to identify demographically independent populations that should be managed as separate units , even though transportation of larvae may occur Palumbi . In the Syngnathid fishes , in which males provide all parental care to the developing eggs that are glued to the male’s body or brooded in specialized pouch , judging from the timing of juvenile recruitment and the morphology of hatchlings. Adult fish show strong site fidelity and stay within a small home range (often less than 100 m2) for their entire lives  to examine the potential influence of the Kuroshio Current on dispersal and genetic connectivity among C. haematopterus populations. A preliminary study revealed that there is geographical variation in C. haematopterus habitat preferences; the habitat of the main Japan islands population is a steep slope consisting of boulder and bedrock at around 2–10 m depth, whereas that of the Ryukyu Islands population is a sandy bottom partly covered with seagrass meadows in a shallow (less than 1 m depth) reef lagoon (A. Sogabe unpubl data). We therefore examined if such geographical variation equates to genetic differentiation as a consequence of local adaptation.The messmate pipefish c Dawson . For synCorythoichthys haematopterus individuals were collected with a hand net from six sites along the coast of the Japanese archipelago and one site on Mactan Island, the Philippines  consisting of boulder and bedrock in Morode and Bounotsu, whereas they resided in shallow reef lagoons (less than 1 m depth) partly covered with seagrasses  in Bisezaki, Sesoko, Kuroshima, and Mactan. In Kin, however, they occurred on the vertical surface of a wharf constructed on a sandy beach (0–7 m depth). Although four species of Corythoichthys pipefish  are known to inhabit the coast of the Japanese archipelago, they are easily distinguished from each other based on their coloration and snout to postorbital length ratio  was amplified using the primers L14725 and H15926, and L2510 and H3058, respectively , 55°C (45 s), 72°C (2 min), and a final extension at 72°C for 6 min. The PCR products were purified with ExoSAP-IT . DNA sequencing was performed using the BigDye® terminator v3.1 Cycle Sequencing Kit  and an ABI 3100 automated DNA sequencer (Applied Biosystems). The primer used for sequencing the partial 16S RNA was the same as that used for the PCR. However, we designed two internal primers, Coh-F43 and Coh-L80 for sequencing the partial cytochrome b gene .Total DNA was extracted from the fin tissue preserved in 99.5% ethanol using the QuickGene DNA Tissue Kit S , according to the manufacturer’s instructions, and stored at −4°C. A partial sequence of the mitochondrial cytochrome h) and nucleotide diversity (π) statistics were calculated for each sampling site and for each locus using Arlequin 3.5  and geographic distance was examined by a Mantel test  and S. scovelli (AF356068), were used as outgroups for partial cytochrome b-based phylogenetic analysis, and Doryrhamphus dactyliophorus collected in the present study as an outgroup for partial 16S rRNA-based phylogenetic analysis. Cytochrome b and 16S rRNA sequences of other congeneric pipefishes extracted from the GenBank database , obtained from a museum , and collected in the present study  were also included in the analyses. Corythoichthys amplexus, C. flavofasciatus, and C. schultzi were only included in the phylogenetic analysis based on 16S rRNA sequences because of the difficulty in sequencing the cytochrome b region. Preliminary sequence analyses using Mega indicated that the cytochrome b fragment best fits an Tamura-Nei model with gamma shape parameter = 0.24  method (Saitou and Nei n = 11). Fish were measured for standard length (SL) to the nearest 0.1 mm using a slide caliper. The number of fin rays on the dorsal, pectoral, and caudal fins and the number of body rings  were counted under a microscope.Morphological assessment was conducted in three individuals each from Morode, Kin, Bisezaki, and Kuroshima, and all fish collected in Mactan (b (589 bp) and 16S rRNA regions (528 bp) were determined for a 108 Corythoichthys haematopterus individuals. Alignment of partial cytochrome b sequences detected 136 variable sites without any indels, resulting in 60 haplotypes  and nucleotide diversity (π) ranged from 0.844 to 0.977 and 0.0025 to 0.0930, respectively, for partial the cytochrome b region. For the partial 16S rRNA region gene (h) and nucleotide diversity (π) ranged from 0.538 to 0.895 and 0.0012 to 0.0625, respectively  for both the cytochrome b and 16S rRNA regions . A statistical parsimony haplotype network is shown in Figure b and 16S rRNA regions, respectively; see also Additional files b and 1–13 for 16S rRNA; hereafter lineage A), and the other comprised haplotypes from Kin, Bisezaki, Kuroshima, and Mactan . Within lineage A, the most common haplotype (haplotype 10 for cytochrome b and 8 for 16S rRNA) related by one or two mutational steps to numerous singleton haplotypes and five haplotypes of moderate frequency  for both mitochondrial regions were observed. Within lineage B, however, there were two main haplotypes (haplotype 31 and 45 for cytochrome b and 17 and 18 for 16S rRNA), which were connected by either one or several mutational steps to each other.Most of the population pairwise ns Table . There wb and 16S rRNA gene sequences also revealed two distinct lineages with high bootstrap probabilities  comprised 51 individuals collected in Morode, Bounotsu, and Kin, and lineage B (haplotype 25–60 for cytochrome b and 14–28 for 16S rRNA) was composed of 57 individuals from Kin, Bisezaki, Kuroshima, and Mactan. Mean pairwise genetic distances between two lineages were 23.3 and 14.1% in the partial cytochrome b and 16S rRNA genes, respectively, whereas intra-lineage genetic distance was extremely low for both regions . For the partial 16S rRNA gene, mean pairwise genetic distance between lineages was comparable to or higher than that among congeneric species . The phylogenetic tree based on 16S rRNA revealed that C. schultzi and C. flavofasciatus are sister groups to lineage B and thus C. haematopterus collected in this study was a paraphyletic group. Unexpectedly, the C. intestinalis (sampled in Indonesia) sequence data extracted from GenBank was included in a branch of lineage B, although that of C. haematopterus (sampled in Japan) was included in a branch of lineage A  and nucleotide diversity (π) ranged from 0.844 to 0.933 and 0.0025 to 0.0033, respectively, for the partial cytochrome b region, and from 0.538 to 0.756 and 0.0012 to 0.0018, respectively, for the partial 16S rRNA  and nucleotide (π) diversity ranged from 0.909 (Kuroshima) to 0.973 (Kin) and 0.0064 (Kuroshima) to 0.0109 (Mactan), respectively, for the partial cytochrome b region, and from 0.601 (Kuroshima) to 0.806 (Kin) and 0.0017 (Kuroshima) to 0.0048 (Mactan), respectively, for the partial 16S rRNA .We reanalyzed the genetic divergence and population structure of C. haematopterus morphological characters are shown in Table Corythoichthys haematopterus lineages inhabiting the Japanese archipelago and the Philippines. The distribution of lineage A was restricted to the main islands of Japan and a site in Okinawa Island. However, lineage B has a broad range from Okinawa Island southward, indicating that some biogeographic boundary exists between the two lineages. The genetic divergence between lineages (ca. 14% K2P + Γ distance for 16S rRNA region) is similar to or even higher than at the interspecific level: a phylogenetic tree based on 16S rRNA showed that C. haematopterus does not form a monophyletic group, two congeneric pipefishes, C. flavofasciatus and C. schultzi, were identified as sister groups to lineage B. Assuming a molecular clock for cytochrome b gene, calibrated at 1.4% sequence divergence per million years estimated from a study of seahorses sampled either side of the Isthmus of Panama  indicates that two lineages diverged in 16.6 Ma ago. Consequently, these results strongly suggest that the two C. haematopterus lineages should be treated as two species rather than as two divergent populations within a single species.The present study demonstrates that there are two distinct C. intestinalis, whereas that of C. haematopterus is included in the lineage A clade. This indicates that the C. haematopterus lineage B should be a taxonomically recognized species, C. intestinalis, rather than either an unrecognized sub-species of C. haematopterus or an undescribed cryptic species. Therefore, the present study provides the first record of C. intestinalis in Japan where four species of Corythoichthys pipefishes have been recognized thus far  and south of Luzon Island (14°N) in the Pacific  than previously recognized. Although it is not clear whether C. intestinalis inhabits southern Japan because of a recent range expansion or merely by the misidentification of C. intestinalis as C. haematopterus, our morphological assessment of the two lineages suggests that the latter is a plausible scenario.This view is well supported by the fact that the lineage B sequence data is very similar to that of the congeneric pipefish, r Nakabo . The norCorythoichthys pipefish species, mentioned that C. haematopterus and C. intestinalis are distinguishable based on the number of trunk rings (mode is 17 in the former and 16 in the later) or body marking differences. However, it has also been reported that the number of trunk rings varies among individuals of a single species . Consequently, such huge intraspecific variation in the characters employed for species recognition would lead to incorrect identification. Moreover, the genus Corythoichthys, which comprises at least 23 species, is distributed over a broad Indo-Pacific range includes members of species-complexes that were previously treated as a single widespread taxon , which are reproductively isolated from each other in their natural habitat because of differences in depth and spawning substrate (e.g. seaweed or bryozoans) preferences, can hybridize in artificial environments consisting of steep slopes of complexly stacked concrete structures near a breakwater . In their natural habitat, lineage A inhabited slopes consisting of boulder and bedrock around 2–10 m depth, whereas lineage B dwelled in seagrass meadows in shallow reef lagoons (less than 1 m depth). Clearly the two lineages differ in habitat preferences and are consequently spatially isolated from each other. However, artificial structures, such as wharves and breakwaters, at the Kin site provide both shallow and deep habitats within a small spatial scale, allowing the two lineages to cohabit and breed adjacently. There are ample examples that artificial habitat construction and alteration disrupts reproductive isolation among closely related species that are spatially isolated from each other in the natural environment Arnold . For insC. haematopterus lineages exhibited different population genetic structures. Low nucleotide and high gene diversity (as appeared in the star-shaped haplotype network of cytochrome b) in lineage A suggests that any previous phylogeographical structure in northwest Pacific populations has been erased by more recent range-expansion events  compared with those marine animals (a few days to a week), and enables them to disperse beyond these straits. This may indicate that not only the hydrodynamic characters of ocean currents but also the characters of pelagic larvae  should be taken into account when determining larval dispersal and its effect on population genetic structure.The present study also showed that the levels of genetic variation among populations is low in both p Figure  that divp Figure . The difC. haematopterus in the northwest Pacific by mitochondrial DNA sequences indicates the existence of two genetically differentiated lineages, lineage A and B. We consider these two distinctive species of very similar appearance, C. haematopterus and C. intestinalis. The two lineages are speculated to have different biogeographic histories: rapid population expansion after a bottleneck event in lineage A and a long evolutionary history in a large stable population in lineage B, indicating that past climatic events and contemporary oceanographic features have played major roles in establishing the population genetic structure of C. haematopterus. Although the levels of genetic variation among populations was low in both lineages in the northwest Pacific, additional phylogeographic research with faster evolving genetic markers and intensive sampling covering the entire distributional range is required to elucidate the overall population genetic connectivity and inform conservation strategies for this species.In conclusion, population genetic analysis of Polymorphic nucleotide sites and haplotype frequency of partial mitochondrial DNA cytochromebgene. Polymorphic nucleotide sites and haplotype frequency of partial mitochondrial DNA cytochrome b gene (589 bp) detected in 108 individuals of Corythoichthys haematopterus. The number in parentheses indicates the number of fish collected in Sesoko. (PDF 26 KB)Additional file 1: Polymorphic nucleotide sites and haplotype frequency of partial mitochondrial 16S rRNA gene. Polymorphic nucleotide sites and haplotype frequency of partial mitochondrial 16S rRNA gene (528 bp) detected in 108 individuals of Corythoichthys haematopterus. The number of parentheses indicates the number of fish collected in Sesoko. (PDF 24 KB)Additional file 2:


Salvelinus namaycush) from Great Bear Lake (GBL), NT and one population outside of this lake  at 11 microsatellite loci and the mtDNA control region (d-loop). Overall, population subdivision was low, but significant , and pairwise comparisons indicated that significance was heavily influenced by comparisons between GBL localities and Sandy Lake. Our data indicate that there is no obvious genetic structure among the various basins within GBL  despite the large geographic distances between sampling areas. We found evidence of low levels of contemporary gene flow among arms within GBL, but not between Sandy Lake and GBL. Coalescent analyses suggested that some historical gene flow occurred among arms within GBL and between GBL and Sandy Lake. It appears, therefore, that contemporary  and historical  factors contribute to the lack of neutral genetic structure in GBL. Overall, our results illustrate the importance of history  and contemporary dispersal ecology in shaping genetic population structure of Arctic faunas and provide a better understanding of the evolutionary ecology of long-lived salmonids in pristine, interconnected habitats.Resolving the genetic population structure of species inhabiting pristine, high latitude ecosystems can provide novel insights into the post-glacial, evolutionary processes shaping the distribution of contemporary genetic variation. In this study, we assayed genetic variation in lake trout ( NE), as well as reduced levels of DNA sequence and phylogenetic divergence compared with those from unglaciated areas  has proven useful in a variety of studies for isolating specific evolutionary processes involved in driving contemporary population structure  are one of the most abundant and widely distributed fishes in this system, found at all depths and temperatures (Johnson N = 5) for a mitochondrial DNA analysis of restriction fragment length polymorphism (RFLP) to assess phylogeographic structure and post-glacial dispersal of lake trout throughout its Canadian range  were obtained between 2002 and 2006 from seven locations within GBL. Samples from GBL were collected from all five major arms  following modifications from Power et al. #b. BrieflyA), expected  and observed (HO) heterozygosities and the fixation index (FIS) were calculated in FSTAT  and private allelic richness .For each sampling location, MICROCHECKER  for each locus–population combination and linkage disequilibrium (LD) for each locus–population-pair combination were detected using GENEPOP . As allele number declines more rapidly than heterozygosity during bottlenecks, recently bottlenecked populations should exhibit heterozygosity excess relative to that expected given the observed number of alleles. Both the step-wise mutation (SMM) and the two-phase mutation (TPM) models , we employed the allele size randomization procedure  as implemented in SPAGeDi 1.1 (Hardy and Vekemans ST is significantly larger than that generated through the permutation process (pRST) would suggest that stepwise mutations are important in the current structuring of differentiation  and therefore this estimator was used instead of RST. Global FST, specifically Weir and Cockerham's . Patterns of IBD were tested for all samples combined and then for those including GBL only by examining the correlation between fluvial distance and genetic distance (FST/(1−FST)). The significance of these relationships was tested using Mantel  were performed using Rousset's  frameworg Mantel  tests inK)) given our multi-locus genotype data without any a priori designation of populations. We performed STRUCTURE analyses for two data sets: (1) one consisting of all samples; and (2) one containing only samples from GBL. We employed an admixture model and assumed that allele frequencies were correlated. Given initial evidence of weak population structure in this system (see Results), STRUCTURE analyses were performed using location (LOCPRIOR) information  was identified using 20 replicate runs for each possible value of K (1–10) using a burn-in of 10,000 followed by 10,000 MCMC steps . We report, for the number of clusters, both the posterior probability of the data (ln P[D]) and the post hoc ΔK statistic of Evanno et al.  as those that display genotypic disequilibrium relative to their sampled population  among sampling locations. Similar to the detection of F0 migrants using GENECLASS2, immigrants in BAYESASS are identified as individuals that display genotypic disequilibrium relative to the population from where they were sampled. BAYESASS, however, estimates recent migration within the past one to three generations , inbreeding (f), and migration rate (m)) were all varied until acceptance rates were ∼30%. Once mixing rates were optimized, ten independent runs, with varying random seed numbers, were performed and results are presented as averages of these runs. We first assessed recent migration among arms within GBL and then between GBL and Sandy Lake. The program TRACER  among arms within GBL and between Sandy Lake and GBL was estimated using the maximum-likelihood approach in MIGRATE  and population size  and nucleotide diversity (П) were calculated using ARLEQUIN ver. 3.01  ranged from 0.34 (Smm21) to 0.86 (Sco202 and SnaMSU12) and averaged 0.70 across all loci. Conformation to Hardy–Weinberg equilibrium (HWE) was rejected in 14 of a possible 80 population–locus comparisons (P < 0.05). All of these involved deficits of heterozygotes and deviations from HWE appear to be distributed randomly among loci and sampling locations. Subsequent to adjustments of alpha based on the false discovery rate procedure, eight comparisons involved a significant deviation (P < 0.00979) from HWE. Significant linkage disequilibrium was detected in 28 of 360 tests (P < 0.05), but after employing the false discovery rate procedure, it was detected in only nine comparisons (P < 0.0073), four of which involved the Manitou sample from Keith Arm.The locus SnaMSU6 was identified to contain a null allele at all but one sampling location, and therefore, was removed from all subsequent analyses. Microsatellite variation in lake trout was therefore assayed at 10 loci across 596 individuals from eight sampling locations . OverallP > 0.05).There was no evidence of recent bottlenecks in any of the samples under either of the models of mutation employed . AlthougP < 0.05, P < 0.0127) including all comparisons with Sandy Lake and three that included the McVicar Arm of GBL.G-based exact tests revealed that 21 of 28 pairwise comparisons among samples were significantly differentiated from each other (FST (θ) (and not RST) was considered in further analyses. Overall, low, but significant, genetic differentiation was found  and this was highly inflated by comparisons which included Sandy Lake. Among the GBL only samples, global FST was still significant, but tenfold lower . Among sampling locations, pairwise FST ranged from −0.006 between two locations within Keith Arm of GBL to 0.150 between Sandy Lake and Smith Arm of GBL (FST = 0.0053). Thirteen of these 28 pairwise comparisons were significant (P < 0.05), but only 10 pairwise comparisons remained significant after employing the false discovery rate procedure (P < 0.0127). Pairwise comparisons that included Sandy Lake were always significant and produced the highest FST estimates ) and geographic distance was much less pronounced and was non-significant  were present and this was the case regardless of the test statistic employed  and lnP[D] , q) clearly show the genetic discontinuity between GBL and Sandy Lake while highlighting the genetic homogeneity within the GBL samples. Indeed, when STRUCTURE was performed solely on samples from GBL, the number of inferred genetic groups was one  of one).Similarly, results from Bayesian clustering implemented in STRUCTURE clearly suggested that two genetic populations/clusters  estimated using the likelihood approach in MIGRATE tended to be variable and asymmetrical among arms within GBL  and this haplotype was relatively well represented within each arm of GBL and within Sandy Lake , was found throughout GBL and was represented by one sample in Sandy Lake (Snam02 and Snam03) or two samples (haplotypes Snam04 and Snam05). Polymorphism within each sample was consistent with neutral expectations  and much lower in Sandy Lake . Of the 468 base pairs sequenced, eight variable positions were resolved  resultinndy Lake , Fig. 6.ndy Lake . The othFST values . Genetic homogeneity across our study area was also evidenced by the parsimonious haplotype network (Snam01 and Snam06) were virtually equally represented in all arms of GBL and were also found in Sandy Lake. The remaining four haplotypes, separated by varying numbers of mutations, were quite rare and were only represented in a total of six individuals.Similar to the analysis of microsatellite population structure, analysis of mtDNA sequences revealed no significant population structure among GBL locations as evidenced by the  network . The twoFST ∼ −0.013/0.002) while showing considerable differentiation among samples from GBL and Sandy Lake, which is separated from GBL by distances comparable to those between the various GBL sampling sites .In this study, we assessed population structure of lake trout in one of Canada's largest, relatively undisturbed freshwater ecosystems. Using a combination of molecular markers, this study is one of the first contributions to understanding genetic structure in Arctic populations of this species. Overall, our results suggest a lack of genetic structure among samples of lake trout from GBL  between lake trout from Lake Superior and an out-group population from another nearby lake, Lewis Lake, which harbors populations of lake trout descended from a northern Lake Michigan population . The overall higher levels, and more pronounced structure, among lake trout in this system may be the result of differing sampling strategies and landscape/lake features. Northrup et al. . Lack of intra-lake structure is common in systems such as GBL that appear to be “evolutionarily young”  also revealed relatively little lake-wide variation, although some range-wide geographic structure was evident . The lack of intra-lake differentiation among populations of salmonid fishes, as measured using mtDNA markers, appears to be a common phenomenon . For instance, D'Amelio and Wilson , or combinations of these influences. Historical gene flow and the retention of ancestral polymorphisms have been implicated in the lack of differentiation and obscured contemporary population structure in a variety of other northern and Arctic salmonids )t (Nei and Chakravarti t is the elapsed time (in generations) the populations diverged from a common ancestor. This resulted in an expected global FST within GBL under complete isolation of 0.0066, more than three times as high as our empirical estimate of 0.002. This result is consistent with the idea that gene flow among populations has occurred, and likely continues to occur, since the founding of GBL. In fact the lack of detectable differentiation in this system , it is likely that both contemporary  and historical  factors contribute to this lack of neutral genetic structure. Further physical tagging studies to assess movements within the lake and molecular assessments will be important for distinguishing among these alternative (or complementary) hypotheses more fully.Second, the lack of genetic differentiation among the samples from GBL may be the result of factors such as a strong signal of historical gene flow and the retention of ancestral allelic polymorphism, large historical effective population sizes , which we did not anticipate given the size and complexity of GBL: our study reports some of the lowest values of differentiation among salmonid populations across such a large geographic scale. We also observed genetic differences between Sandy Lake and GBL highlighting significant inter-lake structure; results that are consistent with the only other assessment of lake trout genetic structure among pristine, northern populations (see Northrup et al.


Colluricincla megarhyncha). Specifically, we test the mechanisms hypothesized to cause geographic and genetic variation . To accomplish this, we surveyed three regions of the mitochondrial genome and a nuclear intron and assessed differences among 23 of the 30 described subspecies from throughout their range. We found support for eight highly divergent lineages within C. megarhyncha. Genetic lineages were found within continuous lowland habitat or on smaller islands, but all individuals within clades were not necessarily structured by predicted biogeographic barriers. There was some evidence of isolation by distance and potential founder-effects. Mitochondrial DNA sequence divergence among lineages was at a level often observed among different species or even genera of birds (5–11%), suggesting lineages within regions have been isolated for long periods of time. When topographical barriers were associated with divergence patterns, the estimated divergence date for the clade coincided with the estimated time of barrier formation. We also found that dispersal distance and range size are positively correlated across lineages. Evidence from this research suggests that different phylogeographic mechanisms concurrently structure lineages of C. megarhyncha and are not mutually exclusive. These lineages are a result of evolutionary forces acting at different temporal and spatial scales concordant with New Guinea's geological history.New Guinea is a biologically diverse island, with a unique geologic history and topography that has likely played a role in the evolution of species. Few island-wide studies, however, have examined the phylogeographic history of lowland species. The objective of this study was to examine patterns of phylogeographic variation of a common and widespread New Guinean bird species ( Phylogeography integrates geographical information and DNA-based genealogies to infer the evolutionary history of modern taxa 2, located north of Australia, from 12°S latitude to the equator. It is home to an unusually diverse biota, supporting over 8.6% of the world's bird species in under 0.75% of the world's land area New Guinea is the world's largest tropical island, roughly 786,000 kmDespite having broad geographical ranges in continuous habitat, many lowland animal species have diversified tremendously. For example, birds are among the best-studied animals in New Guinea, and there are many taxonomic breaks at the species and subspecies level across the island Colluricincla megarhyncha , exhibits a tremendous amount of morphological variation across its range with thirty recognized subspecies C. megarhyncha is a small (35 g mean weight) passerine that feeds primarily on understory arthropods in or near the rainforest interior C. megarhyncha is therefore considered resident and relatively sedentary The focal species for this study, C. megarhyncha. This upper bound is corroborated by evidence from phylogenetic information between a sister taxon, the White-bellied Pitohui (Pitohui incertus) and C. megarhyncha, and suggests coalescent times within C. megarhyncha are 6 Mya or less Several major geological events in New Guinea occurred within the last 6 million years Mya; . We postC. megarhyncha populations throughout New Guinea and surrounding islands.Given the geological evidence , we hypoThis study was carried out in strict accordance with the recommendations in the Guide to the Use of Wild Birds in Research . Blood samples were taken from the brachial (wing) vein and any birds collected were euthanized using recommended techniques and all efforts were made to minimize suffering. Field protocols were approved by the Smithsonian National Zoological Park Institutional Animal Care and Use Committee (IACUC). All specimens were exported legally under Papua New Guinea export permits  and imported under USDA Animal and Plant Inspection Services (APHIS) import permits 42797 (to Robert Fleischer) or 50431 (to John Dumbacher) and declared to US Fish and Wildlife Service using form 3–177.C. megarhyncha , following manufacturer's recommended animal tissue protocol. DNA from museum skins was isolated in a dedicated ancient extraction laboratory in which no modern or post-polymerase chain reaction (PCR) samples are handled. A small toepad fragment was digested overnight at 56°C in lysis buffer , phenol and chloroform extracted, and dialyzed in centrifuge-assisted Centricon® tubes .PCR involved a number of different primer pairs Sequences were aligned and edited with SEQUENCHER 4.7 . PHASE v2.1 was used to resolve nuclear alleles when heterozygous sequences were detected Pitohui incertus) We searched for the best-fit tree with GARLI v0.951 st  for both mtDNA and nDNA in Arlequin v3.11 st uses haplotype frequencies for mtDNA and there were no instances of shared haplotypes among basins, we estimated Fst with the Kimura 2-parameter (K2P) genetic distance, which takes differences among haplotypes into account, as implemented in Arlequin v3.11 An analysis of molecular variance (AMOVA) was performed with mtDNA pairwise estimates of FMolecular clock estimates of divergence were performed to corroborate the geologic timing of hypothesized isolating barriers . We usedxyd−(xd+yd)/2), where xyd is the average number of pairwise differences between basins and xd and yd are the average number of pairwise differences within each basin being compared For the first divergence date estimate, the K2P genetic distance was used because this method takes into account a difference in substitution rates between transitions and transversions while permitting multiple substitutions per site Pitohui incertus). We ran a chain of 20,000,000 generations in BEAST v1.5beta2, logging parameters every 1000 generations. The chain converged by 14,000,000 generations such that effective sample sizes (ESS) for each parameter evaluated in Tracer v1.4.1 were all above 100 and most well above 200 (given a 10% burn-in).For the second divergence date estimate we used Bayesian analysis in BEAST v1.5beta2 to estimate dates of various nodes in the reconstructed phylogeny. BEAST allows input of a mean rate as a prior in the analysis, but allows branches to vary in rate locally across the tree s), transversions (nv), and substitutions (nd) as implemented in Arlequin v3.11 Haplotypes within basins were compared to estimate the number of transitions . Furthermore, there is no difference in the significance of the correlation of distance calculated with a linear or exponential model assumed for the friction grid.Isolation by distance was assessed by testing whether there is a correlation between genetic distances with geographic distances among sampling localities. We calculated geographic distance using Pathmatrix v1.0 st analysis) and effective geographic distances estimates among sites. Significance of the correlation between genetic distance and effective geographic distance were estimated using a Mantel test in Arlequin v3.11 A correlative test for isolation by distance was performed with pairwise K2P genetic distance , a positive correlation between the dispersal distances estimated in PhyloMapper analyses and range size estimated by computing the square root of the potential range area within a basin should be observed. To test for this correlation, the area of each geographic basin was estimated using Google Earth Pro  and was based on putative barriers outlined in The maximum likelihood tree and Bayesian analysis of the mtDNA support five distinct clades on mainland New Guinea and three well supported lineages on surrounding islands . For disThe mtDNA haplotype network illustrates eight major haplotype groups separated by at least 8 base pairs and some as great as 35 base pairs between basins . The nucct = 0.96, p<0.05; 1.8% of the variation explained among sites within basins, Φst = 0.98, p<0.05; 2.4% variation explained within sites Φsc = 0.43, p<0.05). The pairwise analysis of Fst also supports a high degree of genetic differentiation for both mtDNA and nDNA among lowland basins ranging from 0.30–0.96 and 0.10–0.52 respectively and excluding non-significant values  for the nuclear dataset (modern and historic samples), divergence dating was only performed with mtDNA data. Estimates of K2P genetic distances among basins were relatively high and significant. Corrected pairwise genetic distances ranged from 1% to 11% . With ths values were not significant except for BB .We found strong evidence for evolution in dispersal distance with statistical support for the most complex model, which contained six dispersal classes . The perC. megarhyncha. The high level of divergence (5–11% from combined mtDNA loci) observed among C. megarhyncha lineages are similar to the levels separating different genera and even families of birds in North America Our study reveals a remarkable level of genetic divergence within C. megarhyncha, but the genealogical exclusivity of these subspecies, or their interrelationships, had not been examined with genetic data. Data from this study of 23 of those subspecies demonstrates there are eight highly divergent lineages that are geographically distributed in regions corresponding to historically isolated lowland basins or islands, and these do not always correspond to currently recognized subspecies patterns. Furthermore, levels of genetic divergence estimated in this study  among the eight lineages are much higher than those found among congeneric species  and pre- and post-zygotic isolating mechanisms to determine any additional ecological and genetic characteristics that are important in isolating the eight putative lineages found in this study.Thirty subspecies have been described within st estimates, and phylogenetic structure , which implies that they diverged recently, there is substantial gene flow from Australia, or the Australian form is a recent migrant from New Guinea.Another notable vicariance pattern was that observed among the South Papuan Basin and the Trans-Fly Lowlands south of the Central Ranges. In the southern lowlands, ecological divergence in climate and vegetation may have caused a decrease in gene flow for many populations C. megarhyncha. This unsuitable habitat may act as an ecological barrier to dispersal, and further restrict gene flow between the SPB and TF. There are no publications containing data on the timing or historical extent of these savanna-like habitats, so at this time we are not able to test the timing of ecological changes with genetic data.As an alternative to the geographic vicariance model, ecological vicariance may also affect this area between South Papuan Basin and the Trans-Fly Lowlands. Lowlands in this region include dry savanna-like habitats that are unsuitable for Lastly, the Aure Trough was an inland sea that filled around 3–4 Mya, however the dates are disputed st and gene diversity would be higher had we been able to phase all alleles sampled.Nuclear data, while not as strongly structured as mtDNA, still support many of the observed patterns in the mtDNA dataset. Systematic biases in estimates of diversity were created by removing unresolved nuclear alleles from the dataset st pairwise estimates from both the mtDNA and nDNA between CVB and SPP indicate much lower differentiation, and in the case of nDNA are non-significant and Fst is zero. It should be noted, however, that the number of sites sampled within the CVB was low (only two) and further sampling could reveal a different pattern.There are two basins with phylogenetic signatures that are similar to that expected under the founder effect with dispersal across New Guinea. These are (1) CVB and SPP and (2) SPB and BB. First, the topology of the phylogenetic tree indicates that samples from CVB are found within the larger SPP clade. This is a typical pattern observed when a small portion of the haplotype diversity moves into a new area through dispersal, see st values are low or non-significant for both mtDNA and nDNA across the western expanse of the island indicating that there are not many historical barriers to gene flow. Lastly, the Fu Fs test of recent expansion was significant for only BB, but both D and Fs are negative indicating a possible recent expansion.The second group reflecting a FED pattern is SPB and BB basins. The haplotypes sampled from the eastern side (Ekame) of the SPB are found within the diversity of haplotypes sampled within SPB and BB. This node is not significant in the Bayesian analysis (0.82); therefore, support for the FED hypothesis in this geographic region is weak compared to that of SPP and CVB. Molecular clock estimates between SPB and BB are low (0.4–0.7 Mya) and FWithin each major clade, correspondence to geographic basins was evident and birds sampled from multiple sites within a basin had lower molecular diversity and less population genetic structure within compared to among basins . Isolation by distance analysis was significant, but when the intra- versus inter-basin comparison is separated, it becomes apparent that the within basin comparisons are likely driving the significant pattern of isolation by distance. In Colluricincla megarhyncha shows tremendous subspecific variation. Their current classification as polytypic species reflects the complex and confusing patterns of variation in the region; first, the assumption that populations are connected in the lowlands, and second, the historical preference of avian taxonomists to use polytypic species concepts and trinomials in classification. For example, perhaps the most influential compilation of avian taxonomic names in New Guinea was Ernst Mayr's checklist that includes 22 races of C. megarhynchaLike many lowland Papuan species, C. megarhyncha. Our work additionally warns that even lowland taxa with continuous ranges are subject to major taxonomic revision. In many cases, currently-recognized species probably include multiple good biological species and/or phylogenetic species, but additional work will be required to reliably identify functional biological units.For ornithologists and conservationists, it is important to recognize that the current taxonomy of polytypic species should often be considered a place-holder and working hypothesis until more data can be leveraged to test biological and phylogenetic species boundaries. Many polytypic species are likely “oversplit” The data presented here support several potential isolating barriers to gene flow on the island of New Guinea. First, our study has helped identify phylogeographic basins within lowland rainforests. This information is important for identifying and prioritizing conservation areas and is currently underutilized in conservation planning Figure S1Consistent divergence results between nuclear and mitochondrial DNA. Fst data from (TIF)Click here for additional data file.Table S1Location of sites sampled in New Guinea and surrounding islands.(XLS)Click here for additional data file.Table S2Detailed specimen data for all samples used in this study.(XLS)Click here for additional data file.Table S3Phylomapper estimates of evolution in dispersal distance among basins.(XLS)Click here for additional data file.Table S4Molecular diversity estimates and tests of recent population expansion.(XLS)Click here for additional data file.


The increasing aridity during the Last Glacial Maximum (LGM) has been proposed as a major factor affecting Neotropical species. The character and intensity of this change, however, remains the subject of ongoing debate. This review proposes an approach to test contrasting paleoecological hypotheses by way of their expected demographic and genetic effects on Neotropical cloud forest species. We reviewed 48 paleoecological records encompassing the LGM in the Neotropics. The records show contrasting evidence regarding the changes in precipitation during this period. Some regions remained fairly moist and others had a significantly reduced precipitation. Many paleoecological records within the same region show apparently conflicting evidence on precipitation and forest stability. From these data, we propose and outline two demographic/genetic scenarios for cloud forests species based on opposite precipitation regimes: the dry refugia and the moist forests hypotheses. We searched for studies dealing with the population genetic structure of cloud forest and other montane taxa and compared their results with the proposed models. To date, the few available molecular studies show insufficient genetic evidence on the predominance of glacial aridity in the Neotropics. In order to disentangle the climatic history of the Neotropics, the present study calls for a general multi-disciplinary approach to conduct future phylogeographic studies. Given the contradictory paleoecological information, population genetic data on Neotropical cloud forest species should be used to explicitly test the genetic consequences of competing paleoecological models. The Pleistocene glacial periods have been regarded as major factors influencing the geographical distribution, demographic dynamics, and patterns of genetic diversity of species , using the following search terms combinations: cloud forests* AND phylogeography*. This resulted in only two studies dealing with plant species, both in Mexico and upper Central America. Given the paucity of information, we included other relevant studies for Neotropical montane forest taxa in the discussion.In the last section, we searched for relevant articles dealing with the intra-specific phylogeography of cloud forest taxa on the Web of Knowledge database . Many of these taxa, particularly those from cloud forests, probably inhabited areas of wet microclimates immersed in a matrix of dry conditions  were growing 500–800 m lower than their present range.It has been suggested that the summer precipitation regime was collapsed, resulting in arid conditions extending over much of Mexico, Central America, and the Caribbean  increased in abundance at lower elevations over cooler periods, suggesting stable humidity conditions. However, González et al. , which were subsequently displaced upslope during the Holocene and are today largely absent around Lake Consuelo. Taken together, these records show an expansion of montane taxa into lower elevations during the LGM. This is taken as evidence of prevailing cool and moist conditions in the region.The montane forests around Lake Pacucha (southern Peru) were replaced by grassland vegetation  and the inferred moist conditions have been confirmed by the high percentage of fresh water diatoms and low levels of carbonates in sediment cores from Lake Titicaca, Peru  and others had a significantly reduced precipitation . This is not surprising given the complex nature of the atmospheric-oceanic circulation system over the Neotropics  has been used to describe geographical patterns of genetic diversity. Nevertheless, the use of multiple genetic markers from different genomes would be advantageous to evaluate distinct attributes of the genetic structure and diversity of populations. In all cases, marker resolution would be the limiting factor when studying the demographic history of species. The choice of markers should be done bearing in mind relevant attributes of the molecular dynamics of distinct markers in particular taxa .For the cloud forests, still there are virtually no genetic data for testing the above predictions  in cloud forests of Mexico and upper Central America. Their results appear to support the dry refugia hypothesis and its demographic consequences. The authors found a high genetic differentiation among populations and several common alleles with widespread distributions in distinct regions. In this context, the different common alleles are present in populations located around Toledo's , a species inhabiting cloud forests in Mexico and upper Central America. There was significantly high population differentiation between populations within Toledo's  in Mexico and upper Central America. B. nelumbiifolia is a moist-adapted species living in altitudes from 70 to 1200 m, whereas B. heracleifolia is adapted to more seasonal conditions with an altitude range of 300-1700 m  and nuclear DNA (nDNA) lineages occupying separate mountain ranges. In this respect, the authors claim that populations from distinct mountain ranges were continually isolated during cold periods, including the LGM, as a result of enhanced dryness. The authors performed several demographic tests with inconsistent results, but in general there are no signs of population changes during the last glacial period. In conclusion, Barker et al. , the Atlantic tree rat (Phyllomis pattoni), lizards (Gymnodactylus darwinii), and the three-toed sloth (Bradypus variegatus). In the case of B. variegatus and G. darwinii, the authors observed higher levels of genetic diversity in inferred refugial populations than in populations outside refugia. In addition, these two species showed evidence of population expansions in accordance with the postulated history of refugia. Carnaval and Moritz , pitivers (Bothrops), the lesser wood creeper (Xyphorynchus fuscus), vampire bat (Desmodus rotundus), and frogs . Accordingly, de Mello-Martins  in eastern South America, a species confined to seasonally dry tropical forests (Caetano et al. The direct test of the refugia theory has also been applied for seasonally dry tropical forests (Caetano et al. It is clear that further genetic analyses are needed to establish the validity of the refugia hypothesis for cloud forests species. The inconsistency between studies can be the result of a differential response of species to climate change. The congruence among the inferred glacial dynamics of different species may be dependent on their distribution, ecology, or phylogenetic relationship. However, this inconsistency can also be the result of the difference in the analytical framework of distinct studies.In this respect, the few molecular studies show little refinement in explicitly testing the genetic expectations of the dry refugia model and thus provide ambiguous evidence regarding the effect of widespread aridity on cloud forest taxa. Most of these studies do not incorporate available paleoecological data in their discussions. So far, the most detailed tests of the dry refugia model have relied on the direct comparison of the geographical location of proposed refugia and genetic data. Although valuable, the direct geographical inspection of proposed refugia is by itself inconclusive because these proposals are based on limited and inaccurate paleoecological data. This highlights the difficulty in pinpointing the exact location of former refugia without incorporating sufficient paleoecological data in phylogeographic studies.The above discussion focuses on the genetic outcomes of the last glacial period and in doing so assumes that the genetic imprints of this period have overridden the effects of past glaciations. As observed in these studies, it is highly probable that the genetic divergence of many species pre-dates the last glacial period. However, the demographic events accompanying the LGM would have affected the distribution and frequency of mutations that occurred in the more distant past.The present study is not an exhaustive review of the literature. This review is intended to highlight the need of a general framework for conducting future studies on Neotropical phylogeography . DependiThe climatic history of the Neotropics might be more complicated than stated herein. We are aware that other hypotheses might be more fitting to the data (e.g., microrefugia; Rull Further genetic studies should deal with co-distributed species, in particular those living within ecologically narrow forest communities, such as cloud forests. This might prove useful in uncovering general ecological response patterns of species to climate change, particularly precipitation. There is need of detailed molecular studies of Neotropical cloud forest taxa that include species with distinct ecologies and that represent different lineages. A first requirement would be to undertake broad scale latitudinal and altitudinal surveys and test them against paleoecological data. This can provide evidence of multiple refugial lineages and diversity gradients. If there is evidence of refugia, studies on the genetic structure can be conducted on finer spatial scales to evaluate the location and extent of these forest refugia. On a wider level, comparative research across species from different biomes, such as seasonally dry forests and deserts, is needed to determine the universality of the refugia hypothesis.