Mallotus villosus) is a commercially exploited, key forage-fish species found in the boreal waters of the North Pacific and North Atlantic Oceans. We examined the population structure of capelin throughout their range in the Canadian northwest Atlantic Ocean using genetic-based methods. Capelin collected at ten beach and five demersal spawning locations over the period 2002 through 2008  were genotyped using six polymorphic microsatellite loci. Temporally distinct samples were identified at three beach spawning locations: Chance Cove, Little Lawn and Straitsview, Newfoundland. Four capelin stocks are assumed for fisheries management in the northwest Atlantic Ocean based on meristics, morphometrics, tag returns, and seasonal distribution patterns. Our results suggested groupings that were somewhat different than the assumed structure, and indicate at least seven genetically defined populations arising from two ancestral populations. The spatial mosaic of capelin from each of the two basal cluster groups explains much of the observed geographic variability amongst neighbouring samples. The genetic-defined populations were resolved at Jost’s Dest ≥ 0.01 and were composed of fish collected 1) in the Gulf of St. Lawrence, 2) along the south and east coasts of Newfoundland, 3) along coastal northern Newfoundland and southern Labrador, 4) along coastal northern Labrador, 5) near the Saguenay River, and at two nearshore demersal spawning sites, 6) one at Grebes Nest off Bellevue Beach on the east coast of Newfoundland, and 7) one off the coast of Labrador at Domino Run. Moreover, the offshore demersal spawners on the Scotian Shelf and Southeast Shoal appeared to be related to the inshore demersal spawners at Grebes Nest and in Domino Run and to beach spawners from the Gulf of St. Lawrence.Capelin ( Mallotus villosus Müller) is an energy-rich, small forage-fish for a suite of fish, seabird and marine mammal species throughout their circumpolar range, and their extensive feeding migrations serve to transfer energy from one location and time to another [Arctogadus glacialis) and sandlance (Ammodytes sp.) fulfilling a similar role, though they are less abundant and not as widespread.Capelin ( another –5. As a Capelin spawn on the bottom where their eggs adhere to the sediments and most capelin in the northwest Atlantic Ocean (NWA) spawn in the intertidal zone of gravel beaches, though some spawn on the sea bed at depths of 4 to 50 m; notably on the Southeast Shoal located on the Grand Bank  and in sCapelin in the NWA are considered to be a population complex representing one of four phylogenetic clades , 13 thatCircumpolar basin-scale analyses of microsatellite DNA data have shown that capelin collected in the Newfoundland and Labrador region are genetically distinct from those collected off west Greenland, in the NEA, the Barents Sea and in the eastern and western Pacific Ocean , 17. TheManagement of the capelin fisheries in the NWA assumes a four-stock complex  based, iAll capelin samples for this field study were collected by the Canadian Department of Fisheries and Oceans, the regulatory authority for this species. Capelin are not a protected species and no specific permissions were required. Whole fish were sacrificed by exposing them to freezing temperatures (-20°C or -70°C) or killing them with a scalpel or sharp knife; most fish had a short life expectancy at the time of sampling due to mortality associated with spawning.A total of 3,433 individual capelin tissue samples were collected across several well-known beach and demersal spawning locations in the NWA at or near spawning time , Table 1In most cases, individuals were collected using castnets and dipnets on beaches when the fish were actively spawning. In two exceptions, mature pre-spawning fish were collected using a capelin trap near Straitsview in 2006 and a tuck seine off Twillingate in 2008; each adjacent to a spawning beach. Samples collected from demersal spawning sites relied on a midwater trawl on the Southeast Shoal, a bottom trawl at Domino Run, and a modified crab-pot with a castnet-type closure that was lowered onto spawning aggregations at the Gull Rock and Grebes Nest locations. Immature capelin from the Scotian Shelf were collected during the annual Canadian Department of Fisheries and Oceans summer bottom-trawl survey.Muscle tissue located below the dorsal fin was removed at the time of collection and preserved in 95% ethanol, or entire individuals were frozen and similar tissue was later excised and preserved as above. Each fish was measured for total length (± 1 mm), sex identified, and otoliths were collected for later age determination (± 1 year).For DNA extraction, ~25 mg of the ethanol-preserved tissue was re-hydrated in one mL of DNase free water for 30 to 60 minutes. The water was removed and the DNA was extracted using a DNeasy 96 Blood and Tissue kit  following manufacturer instructions, without the optional RNase treatment. Purified DNA was collected from two elutions of 100 μL from the column and stored separately. The eluted DNA was quantified using the QuantIT PicoGreen assay  according to manufacturer instructions and fluorescence was measured using a FLUOStar OPTIMA  fluorescent-plate reader. All samples were normalized to ten ng/μL using ten mM Tris pH 8.0 prior to PCR (polymerase chain reaction) amplification.Mvi2, Mvi3, Mvi5, Mvi9, Mvi10 and Mvi16 selected from the twelve isolated by Gordos et al. [Mvi1 and Mvi31 could not be resolved on the platform we used; 2) preliminary analyses of 149 samples collected in 2002 indicated that the Mvi14 and Mvi33 loci did not amplify reliably resulting in too many missing data; and 3) the Mvi12 and Mvi22 loci showed extreme deviations from Hardy-Weinberg equilibrium (HWE). Of the six loci we used, only the Mvi5 and Mvi10 loci showed allele sizes that followed a predominantly tetranucleotide pattern; the Mvi3 locus had alleles at two- and four-bp (base-pair) intervals, and the Mvi2, Mvi9 and Mvi16 loci contained interrupted repeats with alleles at one-, two- and four-bp intervals.Each DNA sample was amplified at six microsatellite loci: s et al. . The rem2, 0.2 mM dNTP; 0.25 μM each primer and 0.5U Taq (MBI Fermentas). The PCR cycles consisted of three minutes at 94°C for one cycle; three cycles of one minute each at 94°C, annealing temperature (AT) for 30 seconds, 45 seconds at 72°C; then X cycles of 30 seconds at 94°C, AT for 30 seconds and 45 seconds at 72°C; followed by one cycle of ten minutes at 72°C and a holding temperature of 10°C. Annealing temperatures (AT) and cycles (X) for each locus respectively were: Mvi2 53°C, 28 cycles; Mvi3 54°C, 28 cycles; Mvi5 56°C, 28 cycles; Mvi9 62°C, 30 cycles; Mvi10 55°C, 30 cycles and Mvi16 62°C, 35 cycles.The PCR reactions were performed in a ten μL volume containing approximately 25 ng DNA, 1X KCl PCR buffer   Nonidet P40), 1.5 mM MgClMvi9, Mvi10 and Mvi16 loci were labelled in HEX and the Mvi2, Mvi3 and Mvi5 loci were labelled in FAM, and the loci were combined based on similar allele ranges [Mvi9 and Mvi2; Mvi5 and Mvi10 and Mvi3 and Mvi16. For either detection platform two μL of the sample in loading dye was run on a gel. Relative allele sizes were manually assigned (scored) to each allele against a ladder of known-size fragments and control samples were run on each gel (inter- and intra-gel controls). Further details of the quality control steps that we applied to our data are presented as Supplementary Material  gel containing seven M urea  using 1X TBE buffer (Sigma Aldrich) . The fragments were detected using a FMBIOII or FMBIOIII  as used in Gordos et al.  for these ranges  to allowMaterial .P) using α = 0.05 with subsequent Bonferroni corrections for the number of tests [Complete genotypes across all six microsatellite loci were obtained from 3,433 individuals among 45 collections and 15 locations , Fig 1 rof tests . We addiof tests –30 in orGenepop version 4.0.10 [NA), the number of alleles (RS), the number of private alleles (PS) for a standard sample size of 38 individuals, observed (HO) and expected number of heterozygotes (HE), and Weir and Cockerham’s [FIS) using Genetix version 4.05.2 [Adze version 1.0 [Adze; allele frequencies for each locus were produced with Genetix. Weir and Cockerham’s [F-statistics (θ) were also re-calculated to examine pair-wise genetic differentiation and to test the null hypothesis of panmixia. Samples outside the upper 95% confidence interval for θ were considered to be highly differentiated.We conducted preliminary analyses on the 45 samples to assess linkage disequilibrium (LD) and to determine whether temporal samples from each location could be pooled. Details of those analyses are presented as Supplementary Material  Table. Fn 4.0.10  was usedn 4.0.10 . Comparakerham’s  inbreedin 4.05.2  and Adzesion 1.0 . The numkerham’s  F-statisGeneclass version 2.0 [The Præbel et al. study of genetic variation among NEA capelin  used asssion 2.0  to evalusion 2.0 ; the detDest [Dest parameter is based on allele identities and estimates allelic differentiation by partitioning heterozygosity within and among population components. This improves the estimates of population genetic divergence by not confounding within-group heterozygosity with divergence [Dest is not rooted in F-statistic-based metrics and performs consistently under different modelled levels of haplotype diversity and genetic distance between populations [FST or GST that may underestimate genetic divergence [Dest and F-statistics weight the common alleles more heavily than the rare alleles [Dest was calculated with the online program Smogd version 1.2.5 (http://www.ngcrawford.com/django/jost) [Dest we used the program SPADE (updated 2009) [Dest for each locus after removing all alleles with an average frequency across samples of less than 0.05. For Mvi9 and Mvi16, no average allele frequencies met this criterion and so 0.01 was used as the threshold. These levels have been used to define rare allele thresholds for examining the effect of rare alleles on estimates of effective population size [Fixation indices systematically underestimate genetic differentiation when evaluated with highly polymorphic markers . We usedDest  as a meavergence . Dest iulations . It is avergence , 41, 42. alleles , 40, 43.go/jost)  separateed 2009)  which alion size .Dest pair-wise distance matrix was imported into Primer version 6.1.5 . Samples within this matrix were visualized on a non-metric (no units) multidimensional scaling plot (nMDS) based on Euclidean distances, such that the ranked differences among samples were preserved. The degree of correspondence between the distances among points implied by the nMDS scatterplot and the Jost Dest matrix input was evaluated using the Kruskal stress formula 1 implemented in Primer. A complete linkage hierarchical cluster analysis was performed on this matrix. In complete linkage hierarchical clustering, the two clusters with the smallest pair-wise distance are merged in each step. This method was chosen as it is sensitive to outliers.Jost’s Barrier version 2.2 [Dest excluding temporally differentiated samples from the same location) to generate a connectivity network of genetic distances based on Delaunay triangulation and Voronoï tessellation. Automatically generated virtual points prevented long edges forming in the Delaunay triangles. Monmonier’s Maximum Difference algorithm [Dest to represent genetic differentiation among capelin from the 15 locations. We ran the analyses excluding the three temporally distinct samples  and by replacing CC, SV and LL samples with the temporally distinct samples to assess the stability of the results. Barrier robustness was evaluated by examining “consensus barriers”, that is the number of loci supporting the individual sections of the barriers constructed from single locus matrices of Dest analyzed as above in the multiple matrix mode of Barrier. We sequentially considered both the consensus support for each barrier and the genetic distance between sample pairs at those barriers. We concluded our assessment  when a barrier was supported by fewer than four of the six loci (67%) in any segment over its length and (or) when average Dest over all segments forming the barrier between adjacent samples connected through the oceanic features, was less than 0.01.The nMDS ordination considers the genetic distance between all possible pairs of samples and highlights those that are most differentiated from the others in Euclidean space. The expression of population differentiation in geographic space and the location of “barriers”, or areas of reduced gene flow among neighbouring samples, were also assessed using sion 2.2 . Geograplgorithm  was thenDest) and geographic  distances among the 15 locations  with the web-service program Isolation By Distance (Ibdws) [Ibdws was used to calculate the slope and intercept of the IbD relation using reduced major axis regression [Isolation by distance (IbD) describes the tendency of individuals to mate with others that are nearby rather than with individuals from some distant population and if present can cause genetic diversity to be clinal over the sample area. Mantel tests  were use (Ibdws) . Ibdws wgression .Structure (version 2.3.4) [We further investigated the genetic population structure of capelin using the Bayesian clustering program in n 2.3.4) ; the detTissue samples used for microsatellite DNA loci assessment were collected from fish ranging in age from one to six years  representing the 1996 through 2006 year-classes, with the majority (99%) in the 1999 through 2006 year-class range. The total length of individuals ranged between 99 and 191 mm and averaged 158.03 mm . There were marginal differences in average and median lengths among the 15 site locations with the exception of the Scotian Shelf (SS) samples that consisted of immature fish of age-one and-two that averaged 135.29 mm and had the greatest variation (1.86 S.E.) in total length . AlthougMvi2, Mvi9 and Mvi16 loci had 137, 153 and 158 alleles respectively, while the Mvi3, Mvi5 and Mvi10 loci were less variable with 36, 25 and 42 alleles respectively. This complexity in allelic variation reflects the one, two and four base pair (bp) variation associated with loci Mvi2, Mvi9, Mvi16 and to a lesser degree Mvi3 and our large sample sizes which enabled the detection of rare alleles. In all of these loci the most common alleles differed in size by four bp consistent with the gain or loss of one or more repeat unit and varied amongst samples. However one and two bp variations were spread throughout the allelic size range, suggestive of interrupted repeats in these loci.The microsatellite loci varied widely in the observed number of alleles and illustrated the high level of variability typically associated with marine fish species. The Our preliminary genetic analyses  indicateRS), was similar among samples, with no sample particularly allelic-rich or-poor (PS) were similar among samples within loci and over all loci. Observed heterozygosity was high and ranged from 0.880 in the Aguanus River (GSL) sample to 0.927 in the St. Lewis, Labrador (SLL) sample. Re-evaluation of the HWE proportions in the 18 samples identified three of the 108 combinations of samples and loci to be significant, after correcting for multiple tests (21 were significant prior to correction). All three were associated with a large FIS  outside the 95% C.I. for the statistic .Allelic richness per locus, by standard sample  did not exhibit a significant effect on θ as determined by jack-knifing across samples at each locus . Therefore, we did not consider it necessary to exclude them from subsequent analyses. Locus Mvi9 had the most influence on the value of multilocus θ  and ranged from < 0 to 0.0045 . Samplesilocus θ .P ≤ 0.05 and 91 significant after correcting for multiple tests. In producing those results the MC appeared to mix well with small standard error and the number of switches always greater than 10,000. Fifty-three of the 153 pair-wise combinations of samples produced estimates of multilocus θ greater than the upper limit of the 95% C.I. indicating that some sample pairs were very highly differentiated  alleles on the value of Dest in our data was minimal  among the 15 sample locations are illustrated in Dest across segments ≥ 0.01 as average genetic distance dropped to 0.006 in the next iterations for barriers 6 to 10 , identified the area with the highest genetic differentiation to occur between capelin collected at the demersal spawning site Grebes Nest (BB65) and those from the two nearby spawning beaches at Bellevue Beach (BB) and Chance Cove (CC) and the demersal spawning site at Gull Rock (BB61)  . This bak (BB61) . Barrierk (BB61) . Replacik (BB61) , and theDest measure of genetic distance . However, this correlation was driven by a single datum (Saguenay River/Unity Bay pair). Removing that datum and removing the Saguenay River sample both produced insignificant results.There was a significant correlation between geographic distance and Jost’s Structure program recovered mean values of Ln P(D) through different K values from 1–15  from those in south and eastern Newfoundland-Grand Bank . Despite low levels of differentiation, we were able to detect structuring with Bayesian analyses which supported two basal groups corresponding to samples which formed clusters based on Jost’s Dest. Given that the Bayesian analyses fit a model of genetic data based upon assumptions in populations mating at random, and Jost’s Dest is based on allele identities, the mutual reinforcement of the genetic patterns that each analysis provides increases our confidence in our conclusions. At least some of the spatial variation revealed by the Barrier analyses can be explained by the sample mosaic pattern produced through interspersion of fish with ancestors from the two basal groups.The patterns that emerged from our analyses suggest subtle population structure within this phylogenetic clade, as opposed to a single panmictic population. This is evidenced by pair-wise tests of significance of the null hypothesis of panmixia that revealed genetic differentiation between demersal and beach spawning capelin, even when samples from the two spawning groups were in close proximity at two nearshore demersal spawning locations. Spatial genetic variation from the Temporal variation in allele frequencies within locations occurred, and our results highlight three strongly differentiated temporal samples from three of the sample locations. This could be due to generational effects produced by the short life span of capelin, occasional incursion of capelin of a different basal population, or other unknown factors.Dest. Our values of multilocus θ were less than that observed amongst capelin samples using comparable statistics in the NEA where FST ranged from -0.001 to 0.027. However, the degree of correct self-assignment  to the same basal unit with high probability, and by pairwise sample differentiation based on θ and Jost’s Dest, as well as the nMDS and complete linkage clustering of samples based on Dest. Capelin have been recruiting to the Scotian Shelf in recent years [Our genetic population structure results show general agreement with the stock structure in Divisions 4RST but extends the stock influence to include those fish on the Scotian Shelf (SS) and St. Lewis, Labrador (SLL) near the northern entrance to the Strait of Belle Isle. This result is strongly supported by the Bayesian clustering method implemented in nt years , 15 whernt years . This puK > 2 , extending at least as far north as Twillingate (TW) in 3K. Tag-return data indicate that pre-spawning aggregations of capelin on the east coast of Newfoundland represent a mixture of capelin distributed across several coastal embayments along the south and east coast of Newfoundland [Genetic data from this study indicate four population complexes in the area of Divisions 2J and 3KL . The anaoundland .Dest shows relatively high genetic differentiation (Dest ≥ 0.01) between the Southeast Shoal and two of the south and east coast of Newfoundland samples  from within that genetic population. We also observed a relatively low level of genetic differentiation between the Southeast Shoal and south and east coast of Newfoundland samples based on θ that was statistically significant but not outside the 95% confidence interval of θ. Lower genetic differentiation could be due to a number of factors, including large population size, recent separation or incursion, occasional homogenization, etc. Immature capelin originating from southern and eastern Newfoundland beaches and from demersal spawning grounds on the Southeast Shoal co-habit the northern Grand Bank [Dest when visualized in the nMDS and complete linkage clustering analyses. Pairwise analyses of differentiation in θ support are consistent with the above in that no differences were found between SES and those samples associated with the genetic population centered in the Gulf of St. Lawrence . The spatial mosaic pattern of basal populations would distinguish the Southeast Shoal capelin spatially from the beach spawners collected in coastal Newfoundland, consistent with results based on meristic and morphometric data [The demersal capelin spawning on the Southeast Shoal in NAFO Div. 3NO (SES) has been differentiated previously from beach spawners collected in coastal Newfoundland based on meristic and morphometric data , 22 and and Bank . When maand Bank , which cric data , 22. Theric data , 22.Dest. The separation of these two samples in the spatial analysis is explained by the St. Lewis (SLL) sample, with its affinities to the Gulf of St. Lawrence, situated between the two. Further north, capelin from the demersal spawning location at Domino Run, Labrador (DRL) were genetically differentiated from beach spawning capelin samples collected to the north  and south (RBB) creating two populations in this area; a result consistent with the spatial organization of the two basal populations identified in the Bayesian analyses. However this was one of the locations for which we were unable to analyze temporal variability and so this uniqueness  may not be representative of the genetic diversity of these fish if our collection in that year was atypical. The capelin collected over three years from Unity Bay have relatively high levels of genetic differentiation from neighbouring samples to the south which are apparent in five of the six loci in barrier-iii in barrier-v of the Barrier analyses. Further, the three temporal replicates from this location were undifferentiated. It is possible that capelin from UB may be more related to capelin found further north and west as they are adjacent to the region associated with the Arctic phylogenetic clade [vice versa. It is unlikely that Unity Bay capelin are mixing with those from west Greenland as the latter have been genetically differentiated from NWA capelin [To the north, a third population is formed by the Red Bay Beach  and Straitsview  beach spawning samples which group together in both Bayesian clustering and nMDS based on Jost’s ic clade . Capelin capelin , 13, 17.Colbeck et al.  also detAn intriguing result of our analyses is that capelin spawning on one of the nearshore demersal spawning sites demonstrated relatively high genetic differentiation from capelin spawning on neighbouring beaches, although they are geographically separated by less than five km; i.e., Grebes Nest (BB65) with Bellevue Beach (BB) and with Chance Cove (CC). The genetic differentiation of BB65 was very well supported by four, five and six of the six loci in each of its three barrier segments in the first barrier. The nearby Gull Rock (BB61) demersal spawning site is 2.5 km from Bellevue Beach (BB) and was not differentiated from the same neighbouring spawning beaches and was differentiated from BB65 in the barrier analyses, Bayesian clustering and in pair-wise population differentiation. We may speculate if capelin are unable to spawn on Bellevue Beach because of warming water temperatures , these fOur other demersal spawning locations were far offshore  or not in the vicinity of sampled spawning beaches (DRL) and so we were unable to directly compare those locations to nearby beaches. We note that collectively, demersally spawning fish were genetically distinct from beach spawners in pair-wise tests of differentiation, but those test results were not as compelling as the direct comparison of fish in the vicinity of Bellevue Beach. Præbel et al.  were unaClupea harengus [The results of our study provide genetically-based evidence that capelin in the NWA comprise several populations that is consistent, to varying extents, with other studies using different data and metrics. Some are composed of multiple beach and demersal spawning locations such as the Gulf of St. Lawrence and the Scotian Shelf, and the south and east coasts of Newfoundland with capelin spawning at Gull Rock, Newfoundland. Others consist of one beach  or one demersal  spawning location. Our interpretation, especially for those sites that were sampled in a single year , is tempered by our inability to test for temporal variability at all locations for all analyses due to our asymmetrical sampling of years and locations, which we did find at three sampling locations . Nevertheless, the identification of genetic differentiation among capelin collections in the northwest Atlantic Ocean, likely arising from two ancestral populations, adds to the growing literature demonstrating spatial genetic heterogeneity in ecologically similar marine fish species such as herring, harengus , 65–67.S1 FigLn P(D)), admixture coefficient (α) and model fit (r)  for each K value.Mean log likelihood of the data ((TIF)Click here for additional data file.S2 FigLocprior model. K is the number of hypothetical clusters each represented by a different colour. The best alignment of the results of individual computer runs for each K is presented. Proportional genetic assignment for each capelin is represented by a vertical bar and shown under simulations for K = 2, 3, 4, 5, 6, 7, 10 and 12. The plots are organized by grouping individuals by sample location.Bar plots displaying the genetic clustering relationships of capelin from 15 sample locations ((TIF)Click here for additional data file.S1 TableHE), observed heterozygosity (HO) and the inbreeding coefficient (FIS) for each of 45 capelin samples at each of 6 loci and overall loci. Codes for samples are found in P ≤ 0.05). * Indicates significant deviation from HWE after correcting for multiple tests (P ≤ 0.0002).Nei’s unbiased expected heterozygosity ((DOCX)Click here for additional data file.S2 TableAllelic sizes (xxx base pairs) at six microsatellite loci in capelin collected at 15 locations (sample codes in (DOCX)Click here for additional data file.S3 TableMvi) across 18 samples (see N) and associated genetic diversity summary statistics that include the number of alleles observed (NA) for each locus, allelic richness (RS) for each locus, the number of private alleles (PS) based on a minimum sample size of 38 individuals, expected heterozygosity (HE), observed heterozygosity (HO) and the inbreeding coefficient (FIS). SD = standard deviation.Details of capelin at 6 loci ((DOCX)Click here for additional data file.S4 TableFIS expresses the variation between individuals within populations; FIT expresses the variation within individuals; FST θ expresses the variation among populations. Jost’s Dest is estimated for each locus (no jack-knife) and overall (Chao’s harmonic mean). 95% confidence intervals were constructed through 1,000 bootstraps for each locus.Re-sampled indices using a jack-knife on the six loci and 18 samples to produce variance estimates and to evaluate the influence of the selection of loci and/or samples on the mean values of parameters. (DOCX)Click here for additional data file.S5 TableDest for six microsatellite loci applied to 18 capelin samples. Values calculated using all allele frequencies are compared with those calculated after removing rare alleles . Confidence intervals were generated through 500 permutations of the data.(DOCX)Click here for additional data file.S6 TableN replicates  for the log likelihood of the data (Ln P(D)), the degree of admixture (α) and model fit (r) from Markov chain Monte Carlo (MCMC) data collection chains of 500,000 steps each, run under 1 to 15 assumed populations (K).Summary statistics of (DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S3 Text(DOCX)Click here for additional data file.S4 Text(DOCX)Click here for additional data file.


Camelus emerged on the North American continent. While the genetic status of the two domestic species has long been established, the wild two-humped camel has only recently been recognized as a separate species, Camelus ferus, based on molecular genetic data. The demographic history established from genome drafts of Old World camels shows the independent development of the three species over the last 100,000 years with severe bottlenecks occurring during the last glacial period and in the recent past. Ongoing studies involve the immune system, relevant production traits, and the global population structure and domestication of Old World camels. Based on the now available whole genome drafts, specific metabolic pathways have been described shedding new light on the camels’ ability to adapt to desert environments. These new data will also be at the origin for genome-wide association studies to link economically relevant phenotypes to genotypes and to conserve the diverse genetic resources in Old World camelids.Old World camels have come into the focus as sustainable livestock species, unique in their morphological and physiological characteristics and capable of providing vital products even under extreme environmental conditions. The evolutionary history of dromedary and Bactrian camels traces back to the middle Eocene , when the ancestors of  Without Old World camelids (Camelini), human survival and prosperity in the marginal agro-ecological zones of African, Arabian, and Asian (semi-) deserts would have been challenging. Since ancient times, camels were exploited as beast of burden, for riding, and as milk and meat source, and today, gain tremendous importance as sustainable livestock species with very specific features . Camels facilitated the trading and cultural dialog between three continents by connecting the Arabian Peninsula with the Sahara and the Levant to the Far East and Asia, with northern Arabia at the crossroads.Deep historical events like climate change or glacial periods as well as more recent anthropogenic interactions triggered demographic processes in Old World camelids, shaping the patterns of genetic diversity in modern populations. By examining this modern genetic diversity and its global distribution, it is possible to gain insight into past demographic events, which reflect the history of Old Word camelids. This review will give an overview about the evolutionary history of Old World camels and the molecular genetic studies performed over the past years, and summarize the standard molecular genetic methods that underlie these studies. In addition, recent advances in next-generation sequencing (NGS) technologies and their careful implementation in current and future camel research will be discussed.Depending on the objectives and size of the project, the choice of the marker is critical. The assessment of genetic diversity and population structure might be important if local or regional groups/breeds are investigated, while large-scale phylogenetic studies will be more interested in the global connectivity and demographic history of populations. Association studies between phenotypic traits and underlying genotypes will be necessary if marker-assisted breeding programs are anticipated or if adaptation to a specific environment is addressed.Molecular markers were previously applied in the camelid family for parentage control  or restriction length polymorphisms (RFLPs) studied in the mitochondrial DNA (mtDNA) reflecting the maternal inheritance  in the nuclear DNA following the co-dominant inheritance  due to related individuals; (2) screening of population structure using Bayesian methods ; (5) phylogenetic and phylogeographic analyses constructing phylogenetic trees   evaluation of the raw data including confirming single haplotypes, testing for allelic dropout and possible null alleles, and checking for possible relatedness in the sample set to avoid a bias in the genetic diversity estimates, i.e., underestimation of heterozygosity and overestimation of the inbreeding coefficient  when the first ancestors of the camelid family were found in North America. After splitting into New World (Lamini) and Old World (Camelini) camels, the latter migrated via the Bering land bridge to the eastern hemisphere (the Old World). The earliest camel remains in Asia date back to 5 mya , C. ferus (n = 9), and C. bactrianus (n = 7)) mapped to the wild camel reference genome  of 18,000 around 350,000 ya, with a sharp reduction of up to 70 % until the last glacial period . During the LGP, the Ne continued declining until it increased again in the Bactrian camel species and reached a plateau in the dromedaries after the end of the last glacial maximum . Similar patterns of demographic changes in large mammals based on climatic conditions during this period have been described  genetic profile of the global camel populations. A panmictic  dromedary population ranging from North and East Africa to Pakistan and Australia was found Charruau . At the The phylogeographic analysis of modern domestic Bactrian camels from Mongolia, Russia, Kazakhstan, and China revealed that mitochondrial haplotypes are shared between animals and countries Charruau . MoreoveAnthropogenic hybridization between domestic animals and their wild relatives has occurred since the early days of animal breeding. In Old World camels, we find two directions, (i) hybridization between Bactrian camels and dromedaries, which facilitates strong and robust individuals; and (ii) introgression (gene flow from one pool into the other over more generations) from domestic Bactrian camels into wild two-humped camels, and survival of the wild species classified as “critically endangered” by the International Union for Conservation of Nature (IUCN).C. bactrianus) and dromedaries (C. dromedarius) was associated with the transportation of goods along multiple routes of the Silk Road. This practice intended to produce animals with the robustness of the Bactrian camel, the endurance of dromedary, and the ability to tolerate sharply contrasting climatic conditions  and Kez-nar  followed by F1-backcrossing with either dromedary for increased milk productivity or Bactrian camel for wool and cold resistance (Faye and Konuspayeva F2 hybrids (F1 × F1) in Old World camels are usually not favored because of a difficult character and weak progeny performance  has been applied in other domestic species for a long time (Goddard and Hayes Ne and linkage disequilibrium in the population), termed the reference population, which have been phenotyped (measured for certain traits) and SNP genotyped. Breeding values for selection candidates solely based on their genotypes can then be calculated with a prediction equation. The candidates are ranked on these estimated breeding values and the best ones are selected to breed the next generation. The advantage of genomic selection is that based on their genomic prediction, individuals can be selected accurately much earlier in life, which results in a reduction of the generation interval and an increase in the rate of genetic gain  as a basis for genomic selection need to go along with a thorough and standardized recording of production traits, pedigree, and progeny performance testing. Potential genomic selection projects in camels need to be preceded and complemented by traditional breeding programs based on pedigrees and phenotypes.In Old World camels, the challenge for future sustainable breeding programs will be to balance the intensification of production traits with the preservation of genetic diversity and local adaptation. The development of high-density SNP chips or potential whole-genome sequencing projects similar to the 1000 bulls genome project  and genetic diversity, while improving vitally important production traits in the amazing and precious Old World camels.The level of genetic variation and as such the ability of a population to evolve and respond to artificial and natural selection is positively associated with the effective population size Charruau . Thought


Megabalanus azoricus , is a Macaronesian endemic whose obscure taxonomy and the unknown relationships among forms inhabiting isolated Northern Atlantic oceanic islands is investigated by means of molecular analysis herein. Mitochondrial data from the 16S rRNA and COX1 genes support its current species status, tropical ancestry, and the taxonomic homogeneity throughout its distribution range. In contrast, at the intraspecific level and based on control region sequences, we detected an overall low level of genetic diversity and three divergent lineages. The haplogroups α and γ were sampled in the Azores, Madeira, Canary, and Cabo Verde archipelagos; whereas haplogroup β was absent from Cabo Verde. Consequently, population analysis suggested a differentiation of the Cabo Verde population with respect to the genetically homogenous northern archipelagos generated by current oceanographic barriers. Furthermore, haplogroup α, β, and γ demographic expansions occurred during the interglacial periods MIS5 (130 Kya - thousands years ago -), MIS3 (60 Kya), and MIS7 (240 Kya), respectively. The evolutionary origin of these lineages is related to its survival in the stable southern refugia and its demographic expansion dynamics are associated with the glacial-interglacial cycles. This phylogeographic pattern suggests the occurrence of genetic discontinuity informative to the delimitation of an informally defined biogeographic entity, Macaronesia, and its generation by processes that delineate genetic diversity of marine taxa in this area.The Azorean barnacle,  Megabalanus [Megabalanus azoricus  [Balanus, which contained the larger forms of Balani, as a variety of Balanus tintinnabulum (Linnaeus). Hoek [Megabalanus, which currently includes 23 species [Megabalanus tintinnabulum [M. azoricus when located in the Azores and as M. tintinnabulum when found in the other Macaronesian archipelagos [M. azoricus from the Azores [Megabalanus species, including M. coccopoma, M. rosa, and M. volcano, whose current cosmopolitan distributions are associated with fouling in ocean-crossing vessels [The Azorean barnacle belongs to the taxonomically problematic genus abalanus . In the y, 1916) . It was y, 1916)  subdiviss). Hoek  proposeds). Hoek  used it ipelagos . Althouge Azores , their t vessels .Megabalanus azoricus, common, native endemic form in the Azores islands, has been reported from different collections [Megabalanus specimens to the M. tintinnabulum taxa. A re-examination of these specimens assigned them to the M. azoricus taxa [M. azoricus in the Canaries, tracing archeological remains of the species back to 800 years ago. Although present throughout the archipelago, it is more abundant in the eastern islands, closest to the African coast [M. azoricus has been cited for the Cabo Verde Islands, were it is also consumed as a seafood, there is no detailed study of this population [lections . Historilections . Topotyplections  and theilections . Darwin lections  assignedcus taxa . Gonzálecus taxa  reportedan coast . Althougpulation , 14.M. azoricus means that morphological characters have been exclusively used for species diagnosis to date. The wall plates, terga, and scuta have been well described [M. volcano (NC_006293). Homologous data has been used in similar biological and biogeographic studies to explore taxonomic and phylogeographic issues. Consequently, the highly complex taxonomic ranks of barnacles have been phylogenetically evaluated [Heteralepas japonica, located in Philippine waters, shows a distinctive clade divergent from the Japanese and Taiwanese populations and is now a new species, Heteralepas cantelli [Tretaclita squamosa complex in Asia [Pollicipes pollicipes detected a distinctive, monophyletic population in Cabo Verde [Chthamalus malayensis, along the Indo-West Pacific [Semibalanus balanoides was investigated by analyzing control region variation [Tetraclita rubescens, focusing on migration load and current climate change effects, have also been investigated [The absence of genetic data for escribed  and faciescribed . Howeverescribed . Availabvaluated –19. Howecantelli . Similar in Asia , 22. Anabo Verde , which wbo Verde . Phyloge Pacific . The effariation . The mecstigated .A prerequisite for Macaronesian endemism is the existence of a well-defined biographic entity supporting it. Throughout the last 60 My (million years) the Macaronesian region has been evolving geologically, and at present is comprised of diverse seamounts and the Azores, Madeira, Canaries, and Cabo Verde archipelagos . HoweverM. azoricus species are digonic hermaphroditism in sessile adults, development of planktotrophic larvae, dependence on exposed rocky shores in the intertidal habitat, and insular distribution restricted to the northeastern Atlantic oceanic islands [Some crucial features of the  islands . These M islands . They em islands , 36, 37.M. azoricus has been placed on the OSPAR 2010 list of threatened and/or declining species and habitats [The high level of direct exploitation as a food source has been pinpointed as the cause of declining populations in the Azores archipelago , 11, a shabitats . In thishabitats .M. azoricus allow us to evaluate hypotheses on their taxonomic and endemic status throughout their Macaronesian geographic range. Subsequently, inferences on gene flow and historical demography facilitate the definition of a specific phylogeographic pattern consistent with the hypothesis of a putative Macaronesian biogeographic marine entity, and with the local hydrography and paleoclimatology processes.The present work includes the characterization of diverse conserved codifying and non-codifying hypervariable mitochondrial sequences to allow both taxonomic and population structure analysis. Thus, the phylogenetic and descriptive analysis of the intraspecific genetic diversity in Megabalanus azoricus  samples  were collected from each of the Macaronesian archipelagos, including the Azores, Madeira (including Savage), Canaries, and Cabo Verde Islands  was also collected in the Canaries. A muscle tissue section was excised to obtain a 30 mg tissue sample for total DNA isolation with the E.Z.N.A.® Tissue mollusk kit . DNA was spectrophotometrically quantified with SmartSpec Plus  and its integrity was then evaluated by 1.5% agarose gel electrophoresis. The BANGEMAC Network (http://bangen-pct.org/) maintains a collection of the analyzed tissues and DNA samples.Four  Islands , Table 1M. azoricus [The complete individuals and the external and internal views of the scuta and terga plates were observed to verify their congruence with the morphological descriptions available for azoricus , 8.The intraspecific analysis was carried out with the complete mitochondrial control region sequences flanked by the 12S rRNA and tRNA-Ile genes. Moreover, the partial mitochondrial 16S rRNA and COX1 gene sequences were obtained in a subset of the sample for the interspecific comparative analysis of sequences in phylogenetic reconstruction and DNA barcoding. The near complete mitochondrial 16S rRNA was amplified with Cirr-16S-5F (5′-CAACCTGGCTYACGCCGGTCT-3′) and Cirr-Val-3R (5′-GCTGTTTAAGCATTTCATTTACACTGAAAAGG-3′) primers. The fragment in the COX1 mitochondrial gene was amplified using the LCO1490 and HCO2198 primers . Finally2, 200 μM each dNTP, 0.13 μM each primer, 0.15 units of AmpliTaq 360 (Applied Biosystems), and 10–50 ng of total DNA. However, amplification within the COX1 gene was performed with 2.5mM MgCl2. The consensus PCR profile was 94°C for 3 min, followed by 36 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 60s, then 72°C for 7 min.Amplifications were performed in a 15 μL reaction volume using GeneAmp 9700 thermocyclers . PCR reactions comprised 1X AmpliTaq 360 buffer (Applied Biosystems), 3.5mM MgClThe PCR products were digested with Exo-SAP-It , to remove primers and to deactivate unused dNTPs, and sequenced in both directions using the BigDye 3.1 sequencing kit (Applied Biosystems). The extension products were purified with DyeEx columns  and separated and detected in a 3730xl Genetic Analyzer (Applied Biosystems). Following chromatogram revision and trimming with Sequence Scanner software (Applied Biosystems), the sequences were aligned with ClustalX  implemenMegabalanus taxa based on the available homologous 16S rRNA partial gene data was performed to corroborate the phylogenetic singularity of the sampled M. azoricus and define their phylogenetic relationships in a limited but informative data set. Additionally, our dataset (N = 54) was compared with the available Megabalanus COX1 sequences through BLAST (http://blast.ncbi.nlm.nih.gov/) and BOLD [The external morphology of the specimens was verified for compliance with previous descriptions of the Azorean barnacle , 2, 8. Aand BOLD  to find and BOLD . The HKYand BOLD  was seleand BOLD  and implh), and Tajima’s D in populations and observed haplogroups.Genetic diversity estimations for the control region and COX1 sequences were obtained in Arlequin v. 3.5  and DnaSτ) and mutational parameters θ0 and θ1, before and after the expansion event, were also obtained in Arlequin by parametric bootstrapping [The demographic expansion hypothesis within this species was tested with alternative tests based on different criteria. First, using the distribution of the observed number of differences between sequence pairs obtained with Arlequin v3.5 , the so trapping .Fs statistic shows large negative values when estimated from a population undergoing demographic expansion [R2 test is superior in detecting population growth [We also performed the Tajima’s  and the xpansion . For sman growth .FST among the four sampled populations and their significance were computed in Arlequin v.3.5 [Ne) through time was obtained with BEAST (version 1.7.5) [The pairwise in v.3.5 . The sigin v.3.5 . The popin v.3.5 . A Bayesn 1.7.5)  under thn 1.7.5)  with sitRelationships among the sampled haplotypes, their relative frequencies, and geographic locations were graphically described by means of a median-joining network  in NetwoMegabalanus taxonomy include Megabalanus azoricus at the species rank and define their external diagnostic characters [M. azoricus [Major revisions of the complex aracters , 2. The M. azoricus (Mazo CAN03) and M. tulipiformis (Mtul 01) specimens, both sampled from the Canary Islands, were 1143 bp and 1192 bp length. The ribosomal alignment consisted of 11 sequences, 484 characters long and comprising 8 sequences from the available partial mitochondrial 16S rRNA gene from diverse Megabalanus species (GenBank/EMBL/DDBJ accession numbers: KM575947 and KM575948). This limited phylogenetic inference allowed us to verify the genetic distinctiveness and taxonomic congruence of the sample analyzed, belonging to a species without any available phylogenetically relevant molecular information at present. The Megabalanus species (97%) formed a monophyletic group that included Austromegabalanus psittacus and M. tulipiformis in a divergent and basal position. M. azoricus belonged to a clade that includes its closest relatives M. spinosus, and basally, M. occator. The sister clade, which comprises the rest of the species for which 16S rRNA data is available, assembled M. tintinnabulum in a basal position, but with low support (46%), with respect to the derived clade containing M. stultus and the closely related M. volcano and M. californicus  with respect to M. zebra in the BOLD repository and to M. rosa (JX503004), M. coccopoma, and M. ajax records in the Genbank database.Moreover, the publicly available COX1 barcode data from alternative N = 54, length = 658 bp), was 0.0036 (S.D. = 0.0013), which is in the range of other intraspecific barnacle values: 0.006–0.011 in Pollicipes species [Balanus glandula [The intraspecific variability, calculated from the COX1 partition data set  collected along the Macaronesian range, at sites located in the Azores, Madeira, Savage, Canary, and Cabo Verde archipelagos  was 0.9649, being 133 haplotypes with a nucleotide diversity (π) = 0.01813. The population values suggest lower values in the northern Azores population  compared to those of the southern Cabo Verde population   and π  slope values were significant according to the latitudinal regression analysis . These COX1 sequences were deposited in GenBank/EMBL/DDBJ under the accession numbers KM575949—KM575966. The mean intraspecific p-distances among COX1 and control region (CR) haplotypes were 0.004 and 0.016, respectively.The population genetic structure of ipelagos , Fig 1. 0.02115) . Moreoveanalysis . As is eNeμ) based on segregating sites exceeded those for π, as is expected for expanding populations. Thus, Tajima’s D estimate was generally negative although without significant values (P > 0.05). Therefore, we could not reject the null model of a neutral genealogy with a constant effective population size, with the exception of the Cabo Verde sample (P = 0.012). For the complete set of populations, D was negative, being −1.78818 (P = 0.005). The Fu’s Fs was significant for all samples whereas the R2 estimators were only significant for the Canary and Cabo Verde samples, with similarly low Fs and R2 values at lower latitudes  includes two highly frequent haplotypes. One of them is located mainly in the Azores and Canary Islands; the other occurs, in identical proportion, in the Madeira and Canary Islands. Additionally, the majority of the other divergent and rare haplotypes have been detected in the Cabo Verde sample. The β haplogroup  is compact, absent from the Cabo Verde, with a most common haplotype (64%), and forms a star conformation with closely related haplotypes. The third haplogroup, γ , consists of a small number of low frequency haplotypes, mainly from the Cabo Verde sample  values for the α and β haplogroups as is expected from an expansion event; whereas a non-significant value was observed for haplogroup γ. The R2 values, being lower values for the α and β haplogroups under the demographic expansion scenario, are congruent with the Tajima’s D and Fu’s F values  disjointed by five mutations. They are equally distributed among the Azores, Madeira, and Canary archipelago samples, whereas in the Cabo Verde sample haplogroup β is absent . The α he sample . This hae sample . The meaF values .FST were only significant (P < 0.001) for the population pairwise comparisons involving the Cabo Verde population with a mean value of 0.366 (S.D. = 0.009). The rest of the populations comparisons were close to 0 and non-significant  was significantly allocated within populations within the defined groups, resulting in a ΦST of 0.35. The percentage variation associated with the separation of the Cabo Verde sample from the other populations had (35.33%) an identical and also significant ΦCT of 0.35  but with a signal of significant growth after 100 Kya. However, this haplogroup has the smallest population size, almost an order of magnitude less than that of haplogroups α and β. The emergence and exponential growth of haplogroup α also occurred around this date (110 Kya). Haplogroup β was dated back to 40 Kya, showing the highest exponential growth to achieve a present population size similar to that of haplogroup α .Euraphia genus) around the Panamanian Isthmus, a divergence rate of 3.1% My-1 (million years) was estimated for the COX1 gene being used for time divergence estimations in Chthamalus and Pollicipes genera [N = 54). From these data, we estimated the relationship between COX1 and CR gene mutation rates to apply an indirect estimation of the mutation rate to the CR sequences. This approach was based on the estimation of the ratio between each gene-specific θ . Because the effective population size (N) is identical among linked loci within the same sample, the ratio between θCOX1 and θCR equals the ratio of mutation rates between loci. The θ estimates were 3.731 and 8.558 for the COX1 and CR data sets, respectively. Consequently, the control region mutation rate (μCR) was 2.294 times the value observed for COX1 gene and, thus, it is estimated as 3.56 x 10-8 per site per year. This estimated μCR value was useful for population time estimation using the t = τ/2u formula, t being the number of generations since demographic expansion, τ the mismatch-distribution parameter value, and u the substitution rate per sequence per generation. Considering a generation time of 0.583 years [In geminate barnacle species  the geographically isolated sample sites, ii) the complex taxonomy, with incomplete descriptions, major revisions, and a number of synonymies , 9; iii)M. azoricus type locality , and their monophyly with relatively low level of intraspecific genetic variability. Comparison of our mitochondrial sequences with the homologous data available for other Megabalanus species, M. tulipiformis, M. occator, M. spinosus, M. stultus, M. californicus, M. tintinnabulum, M. volcano, M. zebra, M. rosa, M. coccopoma, and M. ajax, suggest the distinctiveness and taxonomic singularity of the present taxa. Moreover, among the species not considered above and without available genetic information only M. vesiculosus, with distinctive characters (e.g. spinose ribs), has an Atlantic distribution, although it is apparently extinct from the Brazilian coast. Among the species analyzed in the 16S rRNA data set, M. azoricus was closely related to M. spinosus, which shows a southern distribution across the Guinean Gulf, the Principe, Sao Tomé, and Annobon islands [M. azoricus. The phylogenetically divergent M. tulipiformis, with shared distribution in the East Atlantic although absent in the Azores [M. azoricus [The taxonomic assignation of the samples analyzed was based on morphological characterization , 8, the  islands , suggeste Azores , also shazoricus , 13.Megabalanus data supplements the extensive barnacle phylogeny inferred in other studies [M. azoricus, M tulipiformis, and other Megabalanus species considered here. These phylogenies included independent molecular and fossil calibrations that support the validity of our COX1 calibration. Thus, Austromegabalanus victoriensis [Megabalanus genus, which has been dated to 11.6–23 Mya. The highest Megabalanus interspecific distance, among the available COX1 sequences, was 0.215 when comparing the pairwise M. coccopoma and M. ajax. Using the 1.55%/My mutation rate a minimum speciation time for the origin of the Megabalanus genus can be roughly dated to ~14 Mya, which agrees with barnacle divergence time estimations based on fossil calibration [Despite the limited ribosomal sequence data, both in taxa and sequence length, the present  studies , 19, whioriensis  has beenibration .Ne and abundant gene flow [h) (0.9649) differs from the higher h value estimated for haplogroup γ (0.9921). The maximum values from haplogroup γ agree with the expected estimates for a population characterized by a stable, large and long-term Ne [The haplogroup pattern of ancestral lineages retained after a lineage sorting process falls within the category II in Avise’s classification of phylogeographic patterns. This category is characterized by the presence of highly divergent and broadly sympatric lineages, theoretically occurring in species with a large evolutionary ene flow . Neverthterm Ne . In the h values at the species level  were lower than those estimated for other Cirripedia species. Considering the homologous control region sequence, the stalked barnacle, Pollicipes pollicipes, had a π = 0.0330 and h = 0.9936 [Semibalanus balanoides, π = 0.0298 and h = 0.9967 were estimated from a European sample  [h COX1 values are obtained in other species such as Tetraclita rubescens , which also included the control region [Chthamalus malayensis (π = 0.005–0.017 and h = 0.95–1.00) [M. volcano  and M. coccopoma  species, but were similar to those for M. rosa  [h observed in this insular endemic species [Both π and = 0.9936 . Also in N = 43) . Similar N = 43) . Higher l region  and Chth95–1.00) . Further= 0.819) . Repeate species  when comh and π) observed is a recurrently described pattern, also observed in diverse crustacean species [The negative relationship between latitude and genetic diversity  was similar to the mean D value (−1.087) estimated from Cirripedia COX1 data [D estimation for the species  for haplogroups α and β  when using the non-codifying control region sequence. Although a recurrent negative skewness in the empirical distribution of D statistic values for Cirripedia [M. azoricus, negative, significant D values are not universal, thus haplogroup γ shows a non-significant D value as expected for a long standing stable lineage in agreement with inferences from sequence diversity, mismatch distributions, and dating estimates. Moreover, we could not reject the null hypothesis based on the Fu’s Fs statistic of constant size for the haplogroup γ, whereas that of the R2, was a higher, although significant value (P = 0.026) for this haplogroup  comprising the majority. Xavier et al. [Phorbas fictius, except for the Madeira differentiated population. Second, an isolated southern population in the Cabo Verde Islands, where genetic diversity is high but haplogroup β is absent. Gene flow between these two population sets is low enough to promote the significant genetic isolation observed between northern and southern populations. The northern populations are subject to common oceanographic and paleoclimatic regimes. These archipelagos are under the influence of the Azores and Canary Currents, they have a similar geological origin including historically emerged intermediate seamounts available as stepping-stones during colonization [The r et al.  observednization  and a lonization . Consequnization . As a renization , 24, 76.nization . At the nization . The sounization . Therefonization .In contrast, mesoscale hydrographic structures play a key role in the settlement and recruitment of other crustacean species , 79, parM. azoricus. During maximum glacial episodes the glacial front reached as far south as 40° N [Pleistocene climate changes with the associated sea surface temperature (SST) and sea-level fluctuations are a paradigm in marine biogeography and sustain the current phylogeographic hypothesis. Both variables affect the highly sensitive rocky intertidal habitat, particularly for the sessile as 40° N  and is aas 40° N . Howeveras 40° N .The small populations inhabiting isolated insular habitats and/or glacial refugia fuel population differentiation . The intPollicipes pollicipes, populations date back to the same paleoclimatic period 87–118 Kya, with the most recent common ancestor dated between 200 and 170 Kya [Within the Macaronesian marine region, it is noteworthy that the paleoclimatic records from ODP Site 658, off Mauritania, agree with the abundant paleo-oceanographic data. Both ln (Si/Al) and records of percentage sedimentary opal reflect the deglacial events, allowing an accurate dating of glacial termination II and the start of interglacial period MIS 5 to 130 Kya, whereas termination III and MIS 7 interglacial occurred 240 Kya . The MIS 170 Kya , 64.M. azoricus and the eastern Atlantic tropical M. spinosus. The higher diversity values along a latitudinal gradient for both the α and γ haplogroups suggest that these lineages originated in the stable refuge habitat of the Cabo Verde islands. Additionally, haplogroup α possesses a limited number (2) of highly frequent haplotypes in contrast to a number of private haplotypes from Cabo Verde, as is expected from few founder events or genetic diversity erosion. In contrast, the most recent haplogroup, β (40–61 Kya), is distributed exclusively throughout northern locations  suggesting the persistence of an effective oceanographic barrier to gene flow, as detailed above, from MIS3, selection against Cabo Verde subtropical habitat, and/or a strong contemporary human-mediated northern dispersion. This dispersion is sustained by a high level of marine connectivity through fouling on vessels circulating among the northern archipelagos, which is significantly less in Cabo Verde. This human-mediated vagility is responsible for the cosmopolitan distribution of the congeneric species M. coccopoma, M. rosa, and M. volcano [Cabo Verde has well-established refugia and ancestral area features , as sugg volcano  and othe volcano . However volcano .Tectarius striatus [A similar colonization pattern was suggested for another Macaronesian endemic, striatus , 92. HerP. pollicipes and C. montagui are absent from the Azorean intertidal, whereas they are recorded from other Macaronesian islands. Similarly, other intertidal taxa such as mussels, patellid limpets, and large trochids are absent or barely represented, likely because of limited food availability for filter feeders [The level of local endemism is highly variable throughout Macaronesia. Cabo Verde and the Azores have the lowest species richness and endemism ranking for oceanic islands, in terms of native higher plant species . The bio feeders .M. azoricus were obscured by the lack of information regarding the relationships between the Azorean type and specimens from the rest of Macaronesia [Until now endemic character and evolutionary origin hypotheses concerning aronesia . Thus, tsensu lato, including Cabo Verde, spans two classic biogeographic provinces, Lusitanian and West African, with their common boundary located in the Cabo Verde Islands [Ophioblennius atlanticus from the Azores and Cabo Verde cluster in a divergent Lusitanian clade differentiated from the West-African province populations [The Macaronesia  Islands . Around  Islands . Congrue Islands . Howeverulations .sensu lato biogeographic unit or with respect to the Macaronesian sensu stricto, originating from and sustained by paleoclimatology and oceanography, respectively. Any effort in pattern definition is crucial in guaranteeing the conservation of genetic diversity and the sustainability of marine resources, particularly for those species that are heavily exploited, as is the case for M. azoricus.The concept of a Macaronesian biogeographic entity has been challenged because of its flora, for example, the divergent phytogeographic affinities of the Cabo Verde pteriodophytes . Present


Adansonia gregorii. The genus Adansonia is a charismatic tree endemic to Africa, Madagascar, and northwest Australia that has long been valued by humans for its multiple uses. The distribution of genetic variation in baobabs in Africa has been partially attributed to human-mediated dispersal over millennia, but this relationship has never been investigated for the Australian species. We combined genetic and linguistic data to analyse geographic patterns of gene flow and movement of word-forms for A. gregorii in the Aboriginal languages of northwest Australia. Comprehensive assessment of genetic diversity showed weak geographic structure and high gene flow. Of potential dispersal vectors, humans were identified as most likely to have enabled gene flow across biogeographic barriers in northwest Australia. Genetic-linguistic analysis demonstrated congruence of gene flow patterns and directional movement of Aboriginal loanwords for A. gregorii. These findings, along with previous archaeobotanical evidence from the Late Pleistocene and Holocene, suggest that ancient humans significantly influenced the geographic distribution of Adansonia in northwest Australia.This study investigates the role of human agency in the gene flow and geographical distribution of the Australian baobab,  Adansonia, the baobab tree, an iconic genus endemic to Africa, Madagascar, and the Kimberley region of northwest Australia [Adansonia digitata L., has been closely linked to human dispersal and settlement patterns [Adansonia gregorii F. Muell., has been based on the assumption of long-term natural processes [A. gregorii by determining whether the geographic distribution of genetic diversity is explained, in part, by patterns of human migration, as inferred from linguistic analysis.The role of humans in shaping crop genetic diversity has always been considered an integral factor in the evolution of agriculture in various regions of the world –3. Interustralia ,8. Theseustralia –17. Althpatterns ,19. Thispatterns , 16. In rocesses –24 whererocesses ,26. We eA. gregorii separated from other Adansonia species more recently than the break-up of Gondwana, but before the arrival of humans in Australia [A. digitata seeds retain their viability in seawater, making oceanic current dispersal feasible. From this it can be inferred that A. gregorii has been in Australia for longer than humans. There is also the possibility, albeit a less parsimonious explanation, that the species arrived more recently from an unknown population which is now extinct. One hypothesis outlining how A. gregorii may have arrived in Australia with humans has been explored in more detail by Pettigrew [Levels of genetic divergence show that ustralia ,8. Leongustralia  demonstrettigrew .Adansonia gregorii, known in Australia as ‘boab’, is mainly distributed across the Kimberley region of northwest Australia, with a small extension eastward into the Victoria River District in the Northern Territory. The distribution of A. gregorii in the Kimberley extends from the northern coastline to the edge of the Great Sandy Desert and the Tanami Desert [i Desert ,29–32. Ti Desert ,33. The i Desert .Petrogale spp.) and other species suggest an East-West Divide running through Central Kimberley [A. gregorii has demonstrated that there is little genetic structure, with FST values non-significant between most populations [The AMT biome is bounded to the south by arid habitats, which began developing in the Late Cenozoic and contain distinctly different biota ,35. The imberley ,37,39. Dulations . Low geoulations , high diA. gregorii are due to high levels of gene flow and, specifically, that human-mediated seed dispersal has been an important evolutionary factor in the history of this species. Pollination in A. gregorii might occasionally entail birds or bats, but it appears that hawkmoths are the major pollinating agents [A. gregorii pericarp [Petrogale spp.), other wallabies and kangaroos (Macropus spp.), which eat the fruit and disperse the seeds in their scat [P. brachyotis), showed strong genetic structure, suggesting that at least this species has limited dispersal ability across biogeographic barriers in the Kimberley [In this paper we sought to evaluate the latter hypothesis – that the low levels of genetic structure within g agents ,33,41,42g agents –45. Floog agents ,16, but pericarp ,33. Otheeir scat . Howeverimberley ,46.A. gregorii, despite archaeological evidence of its long-term use by Aboriginal groups in the region [A. gregorii [A. gregorii by testing for congruence between the spatial distribution of genetic variation in A. gregorii trees and associated word-forms in the Aboriginal languages of northwest Australia. We demonstrate a high level of spatial overlap between the genetic and linguistic data. Our results indicate that, as previously shown for A. digitata in Africa [A. gregorii in northern Australia, probably by acting as seed dispersal agents over long distances.Anthropogenic agency has not been formally considered as a gene flow vector for e region ,47–50. Te region –3, couldgregorii . Specifin Africa ,51,52, aA. gregorii from different fields of research. The first of these is genetic data from six nuclear microsatellite loci, of 220 A. gregorii individuals [This study makes use of two recently published data sets relating to ividuals   and ten or more non-Pama-Nyungan (nPNy) families [PNy language groups are most widespread across the continent, while the nPNy families (excluding Tasmania) exist primarily in northwest Australia across the Kimberley and western Arnhem Land [PNy language subgroups and nPNy families have been distinguished by the comparative method [The Aboriginal languages of Australia are broadly classified into a number of families, Pama-Nyungan  and two PNy language subgroups (Marrngu and Ngumpin-Yapa)  . Three sA. gregorii populations and language areas was examined by superimposing the genetic data on the phylogenetic tree of the main Kimberley language groups analysed by McGregor and Rumsey [A. gregorii genetic populations and the language areas they occupy would be consistent with the idea that people have moved boabs extensively within language areas, but not so much between language areas.Geographic congruence between d Rumsey , and cond Rumsey  in whichAnalysis of molecular variance (AMOVA ) was useDC method [A partial Mantel test of genetic distance vs. language family area with geographic distance as a covariate  was usedC method , and boaWe used a permutation test, implemented in Mesquite , to testnPNy language families were examined to identify languages with higher and lower numbers of boab word-forms  sub-groups, and the associated language groups. The word-forms are represented in a standardized orthography. A hyphen indicates a morpheme break, e.g., separating a stem from a suffix. The subsequent columns list the reconstructed or loan-source form and the probable original meaning of the reconstructed source form. Section A lists word-forms for the boab tree; Section B lists word-forms for boab tree-parts.Data on boab word-forms and related terminology were drawn from both published and unpublished sources . A partial Mantel test of genetic distance vs. language family area with geographic distance as a covariate was not significant , indicating that the statistical significance of the AMOVA could be due to spatial autocorrelation in both languages and genetic variation. However, it is noteworthy that there seems to be a geographically sharp distinction between the EK and NEC genetic clusters, and this break coincides with the distinction between the Jarragan and Worrorran language families.The association of the five tic tree  and vicetic tree , did notThe mapping of genetic population assignments onto the language tree required 4 steps for the original, unpermuted data whereas the permuted characters required from 5 to 10 steps, with a mean of 8.55. With 999 permutations, this implies a p-value ≤ 0.001, indicating a strongly non-random pattern of association between the language tree and the genetic clusters. Because geography was not included as a covariate, we cannot rule out the possibility that this association is driven by geography rather than a causal relationship between boab genetics and human language variation.A. gregorii is found in the northern coastal areas encompassing the Worrorran language family, followed by those in the Jarragan family  has the forms potkurri and wajarr in the Wunambal language; the former word is restricted to this specific language, while the latter is also used in Wurla (Eastern Worrorran). Wajarr has either been borrowed into the Bunuban family or from it: evidence is equivocal at this stage. Forms of the Jarragan proto-word jumulu have been borrowed into northern Worrorran and into the PNy Ngumpin subgroup. Jarragan also has the term kertewun that has been borrowed further east into the nPNy Mirndi language family.The greatest diversity of word-forms for n family . Each halarrkarti, which is from a language subgroup associated with the desert area south of the main bioregions of A. gregorii distribution. Analyzability of this word into two morphemes  suggests that it comes from the Karajarri (and/or the closely related and neighbouring Mangarla) languages of the PNy Marrngu subgroup on the southwest periphery of the Kimberley and is of relatively recent origin [larr ‘split lengthways’ and the suffix –karti ‘towards; side’ do occur in other languages in the region separately and with somewhat different meanings, their coincidence with Karajarri and Mangarla languages is a strong indication that this is the origin of the new coined term for the boab. As a whole, larrkarti means ‘split side’, which could be an allusion to the hollow trunks of old trees or the dehiscent nature of the boab fruit pod, or more likely to the manner in which the shelled fruit splits into longitudinal segments. The coining of this word may well have come about when the Marrngu speaking people moved from the desert into the west Kimberley areas where they would have encountered these unfamiliar trees in the landscape. This Marrngu word for boab has been borrowed and incorporated into the vocabularies of some of the nPNy Worrorran (Western and Eastern) – Bunuban – Nyulnyulan language families and one Jarragan language alongside other inherited word-forms. Larrkarti has also been borrowed into the PNy Ngumpin language subgroups from desert areas southeast of the Kimberley.Of particular interest is the boab word-form t origin . While bA. gregorii words-forms and population phylogeny. The NC population, which appears to be sister to the remaining populations, has the inherited Worrorran boab word-forms junguri and potkurri, and possibly wajarr. The NWC population has the inherited form junguri. The WK population, which clusters with NWC and is distributed across a number of nPNy language family areas has three words, junguri, wajarr, and larrkarti. The NEC population has wajarr, jungulan (a probable cognate of junguri) and the Jarragan loanword jumulu. The EK population, which occurs across the Jarragan and Mirndi families, has the word-forms jumulu and kertewun. Each pair of genetic clusters shares at least one boab word-form suggesting extensive word exchange.Statistically significant gene flow was recorded across all boab populations in the Kimberley [imberley . Migratiimberley .jumulu and kertewun have diffused further to the east, but not into west Kimberley, whereas the word junguri remains in the northwest and has not diffused into eastern Kimberley. Larrkarti has diffused widely from its inferred source in the southwest into western and central Kimberley languages, but not further into northern Worrorran languages.We compared these gene flow patterns with inferred loanword diffusion patterns . The linLarrkarti and kertewun have diffused West→East, but there are no cases of East→West boab loanword diffusion. Thus, the direction and rates of migration of A. gregorii gene flow show correspondence with the inferred diffusion of boab loanwords.If human-mediated dispersal were responsible for the East ↔West and Coast ↔ Inland dispersal, we might expect to see corresponding loanword diffusion along these axes. Furthermore, since there is evidence of much more west-to-east gene flow than east-to-west gene flow across the inferred East-West divide, we might expect more west-to-east diffusion of loanwords than the reverse. These directional patterns accord well with the diffusion of boab loanwords shown in A. gregorii. The limited morphological and genetic divergence of the Adansonia genus in northwest Australia, despite the presence of biogeographic barriers, can be attributed to high gene flow within A. gregorii [A. gregorii. Loanword movements across all boab populations provide an indication of patterns of interaction between nPNy language families and PNy subgroups that would have influenced the geographic distribution of A. gregorii in the Kimberley region.Our study supports the hypothesis that human-mediated dispersal has played a role in shaping the geographical distribution of gregorii . Given tAdditional evidence of anthropogenic agency in facilitating boab gene flow comes from archaeological excavations at Carpenter’s Gap Shelter located in the Napier ranges of the Kimberley, which falls within the Bunuba language area ,49. LithA. gregorii dispersal were most likely in the extreme northwest Kimberley, potentially overlapping with the inferred NC population area. The range of this source population is likely to have extended beyond the current coastline at the Last Glacial Maximum  when sea levels were over 120 m below present-day, and the northwest continental shelf was exposed to the maximum extent [Based on the above evidence, we postulate that recent boab evolution and geographic distribution have been shaped primarily through ancient human agency. The phylogenetic tree of boab populations and predominant direction of gene flow together imply that the source populations for m extent . The incm extent  and cream extent  in whichA. gregorii, as the current distribution of the species coincides with areas receiving at least 700 mm of annual seasonal rainfall [rainfall . Thereforainfall , it is lA. gregorii populations and possibly created a genetic bottleneck from which current populations would have expanded [Subsequent sea-level rise and restoration of monsoonal activity during the post-glacial period and the Pleistocene-Holocene transition between 17 and 6 ka flooded the Sahul shelf and established the present-day coastlines of northern Australia ,73. The expanded . We propLate Pleistocene records of boab remnants at the Carpenter’s Gap archaeological site ,49 may rA. gregorii gene flow through fruit and seed dispersal, accompanied by diffusion of associated word-forms.The increased mobility of Aboriginal groups in the southern Kimberley during the Late Holocene is likely to have been influenced by greater climate variability in northern Australia. In contrast to the Early Holocene, the Late Holocene (∼1000 BCE – 500 CE) climate in the Australian Monsoon Tropics was marked by periods of increased seasonality and aridity –82. ThesA. gregorii (PNy subgroup) speakers and led to their migration into southern and western Kimberley. The word-form larrkarti was probably coined by Marrngu speakers during this period when they would have encountered the boab tree in the Kimberley landscape. At the same time, the mobility and cultural interactions of these PNy groups with neighbouring nPNy language groups such as Nyikina and Bunuba would have increased. Evidence that the diffusion of the loanword larrkarti is of a relatively recent nature is demonstrated in the way it has been incorporated into other languages, generally in an unchanged form [jumulu for the tree and adopted larrkarti for the fruit pod, perhaps indicating the salience and portability of the edible seed pod in the more recent borrowing. Likewise, the Jaru language (PNy Ngumpin subgroup) has no inherited words for boab and uses both jamula (modified from Gija) and larrkarti for the tree. Other examples of recent boab expansion and loanword diffusion further east can be found in the Ngarinyman language (Ngumpin) in the Northern Territory, where the words jang-nge  and jumulu (borrowed from Gija) are used for the fruit or its edible pith, and the tree respectively.The linguistic data provides additional indication of patterns of migration and social interaction that could have contributed to the spread of gregorii . The perged form . The GijPNy Marrngu term larrkarti and other pre-existing nPNy language words such as wajarr, jumulu, and kertewun moved across the Kimberley in patterns corresponding with multi-directional boab gene flow as shown in our study.The possible climatic influences on human migration and boab loanword movement in the Kimberley echoes some aspects of Bostoen et al.’s  descriptA. gregorii appears similar to the case of A. digitata in Africa, where human agency has been involved in dispersal of the species [A. digitata. He notes that the genetic diversity of baobabs in the ecological zones of West Africa and the diversity of vernacular names for the tree in African languages suggests considerable antiquity as well as significant east-west movement along trade routes and exchanges of associated ideas and terminology. However, despite the diversity of baobab names, he points out that two competing Bantu roots, #mbuyu and #muramba, and variations of these, are found in the Bantu languages of southern and eastern Africa. The Bantu expansion from the tropical forest areas of West Africa is said to have begun from around the middle of the first millennium BC (∼ 2500 BP onwards) and reached southern Africa by about 500 CE [mbuyu and muramba in the Bantu languages of eastern and southern Africa would thus indicate the movement of Bantu into these areas [A. digitata in eastern and southern Africa detected by Leong Pock Tsy et al. [The high gene flow in  species ,51,52. T species  account t 500 CE ,83. Blense areas . The lowy et al.  may likeA. gregorii in Australia is most likely due to high gene flow mediated by human agency, similar to that inferred for A. digitata in continental Africa [Adansonia over many thousands of years in both continents would have contributed to gene flow over long distances and across biogeographical barriers. In contrast, it could be that the divergence of Adansonia into six species in Madagascar was possible in part because of the lack of humans until ca. 2 ka. However, this hypothesis can only be tested by further investigation of the ecological, physiological and biogeographic processes contributing to speciation within the Adansonia clade from Madagascar.Our study demonstrates that the limited intraspecific divergence within l Africa , 51, coml Africa . Human uMusa spp.), taro (Colocasia esculenta) and some yams (Dioscorea spp.) in northern Australia [Livistona palms in Central Australia [Microseris scapigera) in southeastern Australia [This study contributes new evidence for the role of ancient humans in influencing the evolution and distribution of non-domesticated plant species in Australia. Australia has long been viewed as a continent of hunter-gatherers  where prustralia , Livistoustralia , and yamustralia . Our finS1 Dataset(XLS)Click here for additional data file.S2 Dataset(XLS)Click here for additional data file.