Calibrachoa heterophylla (Solanaceae), an endemic and vulnerable wild petunia species from the South Atlantic Coastal Plain (SACP).The glacial and interglacial cycles that characterized the Quaternary greatly affected the distribution and genetic diversity of plants. In the Neotropics, few phylogeographic studies have focused on coastal species outside of the Atlantic Rainforest. Climatic and sea level changes during the Quaternary played an important role in the evolutionary history of many organisms found in coastal regions. To contribute to a better understanding of plant evolution in this environment in Southern South America, we focused on C. heterophylla originated inland and subsequently colonized the SACP; the data show that the inland haplogroup is more ancient than the coastal one and that the inland was not affected by sea level changes in the Quaternary. The major diversification of C. heterophylla that occurred after 0.4 Myr was linked to sea level oscillations in the Quaternary, and any diversification that occurred before this time was obscured by marine transgressions that occurred before the coastal sand barrier’s formation. Results of the Bayesian skyline plot showed a recent population expansion detected in C. heterophylla seems to be related to an increase in temperature and humidity that occurred at the beginning of the Holocene.We assessed DNA sequences from two cpDNA intergenic spacers and analyzed them using a phylogeographic approach. The present phylogeographic study reveals the influence of complex geologic and climatic events on patterns of genetic diversification. The results indicate that The geographic clades have been formed when the coastal plain was deeply dissected by paleochannels and these correlate very well with the distributional limits of the clades. The four major sea transgressions formed a series of four sand barriers parallel to the coast that progressively increased the availability of coastal areas after the regressions and that may have promoted the geographic structuring of genetic diversity observed today. The recent population expansion for the entire species may be linked with the event of marine regression after the most recent sea transgression at ~5 kya. The glacial and interglacial cycles that characterized the Pleistocene had major effects on the distribution and genetic diversity of plant species in the Northern Hemisphere caused by the advance and retreat of large ice sheets at higher latitudes ,2. In SoAfter the Last Glacial Maximum (LGM) and at the beginning of the Holocene, approximately 11,700 years ago , the cliSeveral studies -8,15,16 2, from 28° north to 34° south. It is a flat area of lowlands occupied mostly by large systems of coastal lakes. It extends NE-SW for approximately 600 Km and has an average width of 60–70 Km in the south central portion and 15–20 Km in the northern portion  Wijsman (Solanaceae) as a model to study phylogeographic patterns in the SACP and thus better understand plant evolution in coastal environments in South America. Calibrachoa heterophylla is an endemic and vulnerable wild petunia species with a distribution range predominantly in the SACP, where it inhabits dunes and sandy grasslands. In addition to being found in the coastal region, this species also inhabits inland regions of the same longitude, including sandy soils that are distant (more than 500 Km) from the South Atlantic Coast. The ancestors of wild petunias have an Andean-Pampean origin, with a recent colonization of the present geographical range coupled with a fast and recent morphological radiation [C. heterophylla make it an excellent model for understanding the Quaternary evolutionary processes of plants in the South Atlantic Coast.In the present work, we chose adiation  and it wadiation  that mucGenetic markers can shed light on phylogeographical history of organisms . ChloropC. heterophylla. Specifically we asked the following questions: (i) Did C. heterophylla colonize the SACP from inland populations growing in ancient soils that were not affected by sea level changes, or did the species arise in the SACP and then were brought or migrated inland later? (ii) Did the environmental and sea level changes during the Quaternary result in allopatric processes that isolated and shaped the genetic structure of populations? (iii) Did Holocene climate changes result in changes of population size in C. heterophylla? To answer these questions, we assessed the DNA sequences of two cpDNA intergenic spacers (trnH-psbA and trnS-trnG) that have been successfully employed in population studies of wild petunias [The main aim of this work was to investigate how past climatic and geological changes in the SAPC could have affected the evolutionary history of petunias ,31. To rtrnH-psbA and trnS-trnG were 456 bp and 784 bp, respectively. Overall, 32 sites were informative, 16 in each region, resulting in a value of 0.41 ± 0.22 for nucleotide diversity (π%) and a value of 0.879 ± 0.010 for haplotype diversity (h). Moreover, 27 different haplotypes were identified from concatenated spacers . Four groups were inferred: a group in the extreme south of the SACP (G1), a group in the central SACP (G2), a group in north of the SACP (G3) and a final group comprising inland populations (G4). Populations in each group are listed in Table The degree of genetic structure of the populations was estimated by AMOVA, which indicated that 85.1% of the variation was among populations and 14.9% was within populations. SAMOVA analysis was used to infer the best grouping of populations on the basis of molecular variation. The configuration of four groups was considered the best by presenting the highest value of Φal SACP G, a groupA Mantel test showed a significant and moderate correlation between genetic and geographical distances between the populations . Spatial autocorrelation analysis with Alleles in Space (AIS), for the whole species, agrees with the above result, but showed a direct correlation between genetic and geographic distances up to approximately 230 km, after that the genetic divergence is maximum and shows no further increase (data not shown). Since the maximum distances between individuals within the groups are around the above value (~300 km), this suggests that, as expected, this isolation by distance pattern occurs mainly within groups. This is supported by the similar results of the spatial autocorrelation analyses for each of the two clusters with large sample sizes (G2 and G3).The haplotype network Figure  resultedC. heterophylla cpDNA haplotypes and the outgroup was 1.13 (0.53-2.26) Myr ago. The most ancient divergence within C. heterophylla was between the South Coast haplogroup and the others at approximately 1 Myr ago. The Central Coast haplogroup separated from the North Coast and Mainland haplogroups approximately 0.89 (0.37-1.68) Myr ago, and the latter two diverged approximately 0.85 (0.34-1.59) Myr ago  for the four haplogroups were 0.23 (0.08-0.49) (North Coast), 0.38 (0.16-0.97) , 0.40 (0.18-1.08) (South Coast) and 0.77 (0.28-1.45) (Mainland) . The Bayesian Skyline Plot for the species  was similar to that reported for Petunia axillaris (h = 0.831), P. altiplana (h = 0.801), P. bonjardinensis (h = 0.822) and P. guarapuavensis (h = 0.747), the species that showed the highest haplotype diversity in Petunia for the same plastidial intergenic spacers. However, the nucleotide diversity for C. heterophylla was higher than that of Petunia spp. e.g., [Calibrachoa has been studied so far, and thus, no comparisons within the genus can be made. The intergenic spacer trnS-trnG has proved to be an effective molecular marker for population studies in Calibrachoa, showing almost equivalent variability to trnH-psbA, in contrast to what was reported for Solanum (Solanaceae) [Haplotype diversity in a h = 0.89 was sims h = 0.8, P. altianaceae) .C. heterophylla populations were genetically structured. This was confirmed by the results of AMOVA, in which most of the observed genetic diversity was found among populations. Similar results were also observed in Petunia exserta and P. axillaris[Petunia species [Petunia[Calibrachoa species  and mechanisms of long distance seed dispersal are absent in petunia species [Comparison of the haplotype network, phylogeny and geographical distribution of the haplotypes showed that axillaris and in s species  using th [Petunia and Cali species , which p species , Europe  species ,19,38, A species , and Nor species .C. heterophylla, two main paleochannels (Jacuí and Jaguarão) correlate very well with the distributional limits of the three haplogroups  diversification [After transgression/regression events, newly formed coastal habitats with the absence of competition provide ecological opportunities for species. These events allow the occurrence of evolutionary processes similar to those occurring on islands and/or mountaintops : rapid dfication .A similar pattern of population expansion was observed in each of our different analyses , suggesting a general demographic pattern for the whole species. However, although all neutrality tests were negative (an indication of population expansion), most were not significant. This absence of significance may be explained by being conservative and not very powerful to detect older population size changes  comparedC. heterophylla was absent in Uruguay and in some Brazilian locations. This may be because of local extinctions that could have occurred naturally, but more likely, it is because of an anthropic influence, as the entire coastal area has undergone severe human-induced changes in recent decades, such as progressive urbanization, human occupation, development of cattle ranching and agriculture [C. heterophylla, specific strategies must be implemented. We propose that populations with higher genetic diversity belonging to each of the four haplogroups must be conservation priorities. Special attention should be given to the South Coast haplogroup represented in only one population (P1) and to P6 and P7 populations, the only ones with haplotypes of distinct haplogroups. The SACP deserves special attention mainly because it has been shaped by the confluence of taxa originating in tropical and temperate regions. These transitional regions are extremely important for the processes of diversification and speciation. The preservation of species that occur in these areas can be used as a model for biological responses to environmental or climatic changes [Despite historical herbarium records, iculture . To pres changes .C. heterophylla reveals the influence of complex geological and climatic events on patterns of diversification and distribution in this wild petunia species. The fact that the inland populations are grouped in a haplogroup found to be older than those from the coast and that the inland was not noticeably affected by Quaternary sea level changes suggest that C. heterophylla originated inland and subsequently colonized the SACP. The major diversification of C. heterophylla that occurred less than 0.4 Myr ago is likely to be related to Quaternary sea level oscillations, and earlier variability may have been lost as a consequence of marine transgressions that occurred before the formation of barriers I-IV. Furthermore, paleochannels may have been important for structuring the three haplogroups found. A recent population expansion detected by the BSP seems to be related to an increase in temperature and humidity in the SACP that occurred in the beginning of the Holocene.The present phylogeographic study of C. heterophylla is representative of multiple taxa or if it is simply an isolated case. If the same or similar patterns were found in other endemic plants, it would suggest that a common mechanism was responsible for driving diversification in this area.The results highlight the substantial contribution of studies of species growing outside of the forests to our knowledge of past vegetation and climate dynamics in the Neotropics. However, more phylogeographic studies of SACP plant species are needed to determine if the phylogeographic history of trnG-trnS and psbA-trnH combined are successfully informative to draw the phylogeographic pattern in Calibrachoa species [Petunia[Even single locus phylogenies may not describe exactly the species’ evolutionary history, the cpDNA intergenic spacers  species ,33 and r [Petunia.C. heterophylla were intensively sampled over three years across the species’ known distribution, from Laguna (Santa Catarina Brazilian State) to Cape Polonio (Uruguay), including the adjacent northern and southern areas. Despite the existence of herbarium records, the species was not found in Uruguay or in some Brazilian locations. Furthermore, three populations were found out of the SACP in sandy soils along the riverbanks of the inland that were more than 500 Km distant from the South Atlantic Coast  and nuclear (ITS and Leafy intron 2) loci were tested in representative samples, but no significant variation was found. PCR amplification and sequencing were conducted as described in other petunia species [trnH-psbA, and JQ082455-JQ082470, trnS-trnG).The escribed ,59, resp species . SequencFor all collection localities, no specific permits were required because the land is not privately owned or protected and the field studies did not involve endangered or protected species. This work was conducted under MP 2.186-16 of the Brazilian Federal Government.The sequences were aligned manually using GeneDoc . Becauseh), nucleotide diversity (π) [ST (pairwise differences). Mantel test between genetic and geographical distances and Spatial autocorrelation analyses  were performed with the program Alleles in Space 1.0 (AIS [Standard diversity indices including haplotype diversity (sity (π) , and anasity (π) ) were obsity (π) . The AMO1.0 (AIS ). Close CT).Historical barriers to gene flow were identified using Spatial Analysis of Molecular Variation in SAMOVA 1.0  with 1,0http://www.fluxus-engineering.com). Sequences of Calibrachoa paranensis (Dusén) Wijsman, C. serrulata (L. B. Sm. & Downs) Stehmann & Semir, and C. elegans (Miers) Stehmann & Semir that belong to the same subgenus of C. heterophylla[The evolutionary relationships between haplotypes were estimated using the Median-Joining method (ϵ = 0 ) implemeerophylla were als8 chains were conducted, sampling every 1,000 generations. The settings used were the Yule tree prior, the HKY substitution model with four gamma categories and the strict molecular clock. The substitution rate used was 2.8 × 10-9 (± 5.4 × 10-11) substitutions per site per year, or 0.56% per million years. This rate was chosen because it was previously estimated for Petunia for the same loci studied here [http://beast.bio.ed.ac.uk/Tracer) was used to check for convergence of the Markov chains and adequate effective sample sizes (>200) after the first 2 × 107 chains were deleted as “burn-in”. A maximum clade credibility tree was obtained using TreeAnnotator, part of the BEAST software package. Statistical support was determined by assessing the Bayesian posterior probabilities.Phylogeny and divergence times among haplotypes were estimated using a Bayesian approach with BEAST 1.6.1 . Two runied here  and it iied here ; 0.26–0.ied here ). The beied here  using thied here . Tracer Calibrachoa heterophylla was investigated using several methods, such as Tajima’s D[Fs[C. heterophylla dataset. The settings for the substitution model and substitution rate were the same as those used for the phylogenetic analysis.The demographic history of ajima’s D and Fu’sma’s D[Fs neutralima’s D[Fs; implemetrnH-psbA, and JQ082455-JQ082470, trnS-trnG) and in Dryad repository: http://dx.doi.org/10.5061/dryad.564ms. The concatenated alignment is available in http://dx.doi.org/10.5061/dryad.10v90.Sequences are deposited in GenBank (accession numbers: JQ072006-JQ072022, The authors declare that they have no competing interests.LBF and GM planned, designed, and led the project; GM did the laboratory work; GM, APLL, JNF and SLB ran the analyses; GM and JNF collected the plant material; GM, SLB and LBF wrote most of the text. All authors have contributed in the preparation of the study and commented on and approved the final manuscript.Collectively the group is interested in investigating evolutionary processes, plant speciation, conservation genetics, phylogeographic patterns and molecular systematics.Calibrachoa heterophylla.Haplotypes identified in the cpDNA of Click here for fileInformation about outgroup used in the phylogenetic analysis.Click here for file


Oreochromis mossambicus, a widely used freshwater aquaculture species, is especially imperiled by this phenomenon since it is recognized by the IUCN as an endangered taxon due to genetic admixture with O. niloticus an invasive congeneric species. The Lower Limpopo and the intermittent Changane River (Mozambique) drain large wetlands of potentially great importance for conservation of O. mossambicus, but their populations have remained unstudied until today. Therefore we aimed (1) to estimate the autochthonous diversity and population structure among genetically pure O. mossambicus populations to provide a baseline for the conservation genetics of this endangered species, (2) to quantify and describe genetic variation of the invasive populations and investigate the most likely factors influencing their spread, (3) to identify O. mossambicus populations unaffected by hybridization. Bayesian assignment tests based on 423 AFLP loci and the distribution of 36 species-specific mitochondrial haplotypes both indicate a low frequency of invasive and hybrid genotypes throughout the system, but nevertheless reveal evidence for limited expansion of two alien species (O. niloticus and O. andersonii) and their hybrids in the Lower Limpopo. O. mossambicus populations with no traces of hybridization are identified. They exhibit a significant genetic structure. This contrasts with previously published estimates and provides rather promising auspices for the conservation of O. mossambicus. Especially, parts of the Upper Changane drainage and surrounding wetlands are identified as refugial zones for O. mossambicus populations. They should therefore receive high conservation priority and could represent valuable candidates for the development of aquaculture strains based on local genetic resources.Hybridization between invasive and native species accounts among the major and pernicious threats to biodiversity. The Mozambique tilapia  Estimates of the frequency, composition and expansion dynamics of hybrids are therefore essential data for assessing the future of a hybrid system and to propose management policies.The increasing human influence on earth ecosystems may cause major alterations of patterns of genetic exchange between populations and species OreochromisOreochromis within Africa were reported to induce several cases of interspecific hybridization leading to severe threats for the genetic integrity of native local species .The Mozambique tilapia, O. niloticus and its hybridization with native O. mossambicusO. niloticus was probably introduced in the Upper Limpopo system (Zimbabwe) in the early 1990s from a population previously established in Lake Kariba. Several dams in the Upper Limpopo system were stocked with O. niloticus, in some cases with several tens of thousands of specimens et al. O. niloticus inoculation occurred as the consequence of major floods in the year 2000 leading to the escape of O. niloticus specimens from a fish farm located in the Lower Limpopo River The Limpopo River system provided the most extensive evidence for the spread of O. niloticus is not the first alien Oreochromis species having spread in the Limpopo System. In 1973, Oreochromis andersonii specimens originating from the Okavango River are known to have been released in the Upper Limpopo System (Bostwana) O. andersonii haplotypes recovered in the Upper Limpopo It is noteworthy that Oreochromis populations of the lower reaches of the Limpopo although this zone is of crucial concern for the conservation of O. mossambicus because it potentially shelters genetically pure populations. Moreover, there are no fine-grained genetic data describing the autochthonous structure and diversity of pure O. mossambicus populations at a local scale.All previous genetic studies mainly concerned populations sampled in the Upper Limpopo close to well-known zones of introductions O. mossambicus populations of high conservation value.The Changane drainage, located in the Gaza Province, Mozambique, is the main tributary of the Lower Limpopo and an intermittent dry land river. It represents the largest and least disturbed wetland of the Limpopo system Oreochromis sp. in the Changane-Lower Limpopo O. mossambicus metapopulation as a model system, and to characterize the spread of alien and hybrid genomes across geographical and ecological barriers. We genotyped 376 specimens from 12 populations for 423 nuclear AFLP markers and sequenced a subset of 176 specimens for a fast evolving mitochondrial DNA locus in order (1) to estimate the autochthonous diversity and population structure among genetically pure O. mossambicus populations with the aim to provide a first baseline for the conservation genetics of this endangered species, (2) to quantify and describe genetic variation of the invasive populations, and investigate the main factors likely to influence their spread and genetic introgression with the native O. mossambicus, (3) to identify O. mossambicus populations not affected by hybridization.The general objective of the present study is to assess the invasion of alien Oreochromis species in the Limpopo-Changane system  except in the Limpopo River where O. niloticus haplotypes are dominant (61%). O. andersonii haplotypes were recovered in the Limpopo and in the lower and middle reaches of the Changane River and are absent elsewhere. Six distinct haplotypes were recovered in the O. andersonii sample  while only a single haplotype was found for O. niloticus despite the highest number of sampled mtDNA specimens for this species . This haplotype differs from the three O. niloticus haplotypes previously recovered in the Limpopo . AMOVA indicated a significant differentiation between lacustrine and riverine O. mossambicus populations  and a significant population divergence between localities . The O. mossambicus haplotype network  dominate the samples from the upper and middle reaches of the Changane River and the lakes. Unambiguous evidence for admixed nuclear genotypes was only identified at Chibuto and Chigubo albeit at low frequencies . The newhybrids analysis indicates a very similar pattern as the one obtained with structure  which is likely to be an O. niloticus-dominated hybrid genotype. To summarize, localities likely hosting allochthonous species and hybrid individuals are located in the Limpopo  and in the lower and middle reaches of the Changane sub-drainage . Not surprisingly the highest values of genetic diversity were found in these four samples  using criteria . The ref cluster . Individtructure  and conf samples . A seconO. mossambicus nuclear genotype with an O. niloticus mitochondrial haplotype. All three occur in the Limpopo  and in the Lower Changane . structure results obtained with K = 3 indicate that individuals bearing an O. andersonii haplotype do not tend to be more likely assigned to the third –hypothetically O. andersonii– cluster than specimens with an O. mossambicus haplotype .Individual comparisons of AFLP based assignments and mtDNA  reveal tO. mossambicus was evaluated including the eight localities showing no trace of alien genotypes. Pairwise FST calculations (F = ST0.0000). Of the lakes investigated, Macosse is also not significantly differentiated from the rest of the Changane drainage . The three other lacustrine localities generally exhibit strong levels of differentiation with all or part of the system .Population genetic structure within ulations  reveal ne system . Especiastructure analyses, with and without the locprior option, do not detect differentiation between the O. mossambicus populations unaffected by introgression. The most likely number of clusters is K = 2 (with locprior) and K = 4 or 6 (without locprior)  at K = 2 to 6 revealing no apparent patterns of clustering for both models (ocprior) , with noh models .O. mossambicus populations persist. This situation fits with scenario #2 defined by Allendorf et al.Oreochromis sp. introduction events, distance from the sites of introduction and local ecological conditions.Our results show that interspecific hybridization in the Limpopo system leads to occasional introgressions, except in the upper reaches of the Changane River and investigated surrounding lakes where many pure O. andersonii and O. niloticus. The first one involved O. andersonii, a species released in 1973 approximately 600 km upstream to the Changane River in the Upper Limpopo drainage O. andersonii haplotypes expansion has likely progressed downstream in the Limpopo River and then again upstream up to the middle reaches of the Changane River (Chigubo). The second mtDNA dispersal event has involved O. niloticus mtDNA introgression into the O. mossambicus gene pool and is evidenced by few individuals in the Limpopo as well as in lower Changane. Hybridization events leading to cytonuclear discordance are not rare in related cichlid species O. mossambicus experienced two recent successive events of partial (and possibly ongoing) mtDNA introgression. O. mossambicus x O. niloticus successive backcrosses involving female O. niloticus are reported from experimental conditions O. andersonii to O. mossambicus suggests some similarities in the processes of expansion and introgression for both alien species. Unbalanced sex-ratios of interspecific hybrid progeny, a well-documented pattern in tilapias  O. niloticus was established O. andersonii compared to O. niloticus, which would be in accordance with the relative remoteness of the native ranges of the two alien species (see e.g. O. andersonii only while at least two independent introductions occurred for O. niloticusO. niloticus haplotypes suggests that this species may not have benefitted from admixture of its two low diversity sources (Upper and Lower Limpopo sources). Thus, the much higher genetic diversity of O. andersonii –as estimated from mtDNA– may have favored its broader expansion in the drainage relatively to O. niloticus.Second, a much higher mtDNA diversity was observed in see e.g. . HoweverO. andersonii is phylogenetically closer to O. mossambicus than to O. niloticusO. mossambicus are expected to be fewer with O. andersonii than with O. niloticus leading to weaker intrinsic barriers to introgression and more likely spread of genetic components of the first species. In summary, time since introduction, patterns of genetic diversity and genetic incompatibilities between alien species and O. mossambicus could explain the broader expansion of O. andersonii haplotypes in the system. Time since introduction can be considered as a baseline explanation, but differential genetic introgression of O. andersonii was potentially accelerated by the other two factors (i.e. phylogenetic distance to O. mossambicus and genetic diversity).Third and last, Oreochromis suggests that rapid replacement and even local extinction of the native or resident species can occur. For example, introduction of O. niloticus in the previously established Oreochromis macrochir population in a Madagascan lake has led to the complete replacement of O. macrochir with O. niloticus after only ten years O. mossambicus by O. niloticus in less than ten years. In a second dam, Weyl O. niloticus in less than one year, but with no evidence for the total replacement of O. mossambicus over this short time scale. Less sudden or only a partial genetic replacement can also occur, as exemplified by the partial  introgression of introduced Oreochromis leucosticus mtDNA into a native O. niloticus gene pool of the Lake Baringo, Kenya O. niloticus introduction in the early 1990′s et al.O. niloticus or hybrids and 14% of O. mossambicus (N = 257). Using a similar allozyme based approach for fish sampled in 2002 and 2006, van der Bank and Deacon N = 63) and 35% in 2006 (N = 103) suggesting that the frequency of preserved O. mossambicus genotypes can drastically vary with time in a riverine system.A survey of recent invasion-mediated hybridization in O. niloticus expansion and O. mossambicus local extinction in the Lower Limpopo are not as dramatic as previously reported for invaded lakes O. mossambicus specimens remain dominant in the most isolated and remote parts of the system (here the upstream section and the surrounding lakes). Noteworthy, our population genetic data agree with a recent qualitative ecological study of O. niloticus invasion risk in the Limpopo O. niloticus invasion. Thus, temporarily isolated and still unintrogressed headwater O. mossambicus populations could act as refugia preventing the total replacement by alien species and hybrids of the native Changane populations.Our results also show that hybrid frequency and invasion patterns strongly vary spatially suggesting a pattern of progressive and only localized replacement. Furthermore, O. mossambicus populations. Given the low prevalence of hybridization, competition for food between O. niloticus and locally preadapted O. mossambicus should also be considered as a factor depriving fitness of O. niloticus, as a trophic niche overlap of the two species was documented in the Limpopo O. mossambicus. Diachronic genetic surveys before and after major flood events  and the broad amount of data now available on the Oreochromis genome could provide the opportunity for future investigation of both the genomic location and function of putatively non-neutrally introgressing alleles in the biological invasion context. This could be achieved by SNPs genotyping and mapping using a next generation sequencing approach (e.g. RAD sequencing) A mosaic of extreme environmental conditions brought together is found along the Changane River system, from highly saline or brackish swamps in the lower and middle reaches to eutrophic freshwater swamps in the head river sections . The heterogeneous pattern of admixed genotypes distribution in the Lower and Middle Changane River may partly result from highly variable ecological conditions possibly acting as barriers to the expansion of potentially less resistant alien or admixed genotypes. Extreme habitats characterized by long periods of extreme eutrophic conditions, such as swamps in the river bed, are expected to challenge the establishment and spread of alien species that would be less favored under hypoxia and low water temperature nts e.g.  could asnts e.g.  performents e.g.  would alO. mossambicus populations recovered in the Changane system exhibit a substantial amount of genetic diversity contrasting with the depleted genetic diversity reported for O. mossambicus populations that were exported worldwide during the 20th century O. mossambicus restocking in critically invaded areas (e.g. the Upper and mainstream Limpopo) as well as a diverse autochtonous genetic resource for the development of new local aquaculture strains The preserved O. mossambicus gene pool, a significant differentiation between riverine and lacustrine habitats for both nuclear and mitochondrial markers indicates that the O. mossambicus populations included in this study could represent at least two distinct conservation units related to their geographical distribution and/or ecological versatility. The null FST values found between the four sampling sites of the headwater region despite the current strong isolation of each swamp has to be considered with regards of the recent history water flow variation. A major flood occurred in 2000, which had connected all swamps hydrologically and hereby allowed homogenization of genetic variation. Sampled localities were disconnected two or three years thereafter as a result of increased drought. Riverine populations therefore have remained permanently isolated over the last five or six years. This time lapse was probably too short to lead to detectable genetic differentiation between these localities. Floods have already been evidenced as a radical homogenizing factor erasing isolation by distance patterns in riverine cichlids Although the clustering approach indicates the relative homogeneity of the preserved vs. riverine differentiation is further supported by the highest Fst found among comparisons involving three out of four isolated lacustrine sites  vs. the four riverine headwater sites . Macosse Lake, which represents the largest permanent body of water included in this study, exhibits no significant genetic differentiation from the rest of the system. The presence of several shared and frequent haplotypes between lakes and the river could suggest that lacustrine populations result from multiple colonization events. Overall, geographic isolation over geological time scales (i.e. since the last major Pleistocene sea level fluctuation or extreme floods events connecting lakes to rivers), possibly combined with an increased drift effect in populations from small water bodies could have induced the within-drainage differentiation pattern. Interestingly, a recent study of native O. niloticus populations also reports significant values of local-scale genetic differentiation associated to the levels of geographic connectivity between populations et al.O. mossambicus native range indicated genetic differentiation among drainages. Focusing on a narrower geographical scale with an intensive sampling, the present study provides a finer picture of the O. mossambicus local genetic structure, indicating that the naturally fragmented O. mossambicus habitat induced subtle genetic within-drainage differentiation. These are potential conservation units to be managed locally .The lacustrine O. mossambicus in the face of introductions of allochthonous Oreochromis species in the Limpopo drainage. We provide evidence for only a limited expansion of alien species and their hybrids in the Lower Limpopo despite multiple introductions. This indicates that the spread of hybrids in the system is rather slow probably due to geographical and ecological barriers. The peculiarity of the Limpopo hybrid system is that the endangered O. mossambicus underwent two successive waves of interspecific genetic introgression from introduced species. Currently, these two invasive species exhibit remarkably different levels of genetic variation that potentially correlate with their respective invasive abilities. This result encourages further investigations on the role of propagule pressure and genetic diversity in the success of biological invasions involving genetic introgression of locally adapted native species. In addition, we identified two refugial zones that should henceforth receive high priority for the conservation of O. mossambicus: the head of the Changane drainage and the lakes surrounding the Lower Changane River. Considering that the four investigated lakes are exclusively populated by native O. mossambicus strongly suggests that the surrounding wetland system around the Changane  three river pools within the Changane  and, finally, three pools within the endorheic headwater system . Tinwarina and Chigubo are permanent water bodies respectively characterized by brackish  and saline waters  due to local soil conditions . The last site (Maficuiane) is an ephemeral pool in the river bed situated about 30 km down the river's origin. The three sampling sites chosen within the headwater endorheic system are all also ephemeral. Zinhane and Lipasse are permanent pools with highly alternating water levels in response to drought; both are located on the Nhambandzule River, which runs from the north into the central wetlands of the Banhine National Park (BNP). Linlangalinwe represents a pool located on the Chefu River, the channel connecting the BNP and the Changane main channel during major flood periods.All necessary permits for sampling were obtained from the University Edouardo Mondlane - Faculty of Science (Maputo). A fishing permit was acquired from the Banhine National Park Administration (#0002/2009). At each sampling site local authorities and communities were first contacted and sampling activities always took place with their agreement. The field studies did not involve protected species. Specimens were collected along the Changane drainage and in the Limpopo between 2006 and 2009. A total of 12 sites were sampled, and included the different reaches of the Changane River, surrounding lakes and the Lower Limpopo ; Table 1O. niloticus specimens from a the Bouaké strain  were included in the analysis as O. niloticus reference samples. The Bouaké cultured strain was spread in several regions of Africa O. niloticus strain does not affect the detection of hybrids. First, within the Oreochromis radiation O. niloticus is a phylogenetically clearly distinct from O. mossambicus (and O. andersonii) O. niloticus populations) should have no influence on the following Bayesian approaches and, therefore, can be neglected for the detection of O. niloticus hybrids within the Limpopo-Changane system. Second, the AFLP nuclear genetic diversity  falls within the estimated Mean ± 1SD range of the among O. mossambicus populations variation in diversity (He = 0.0446 ± 0.0071). This supports the high polymorphism of the Bouaké strain hosted in Montpellier . Fragments were separated on an ABI 3130 Genetic Analyzer  with an internal size standard (ROX 500 XL). The quality of each run was visually inspected. Fragments were scored for each primer combination between 80 and 500 bps using GeneMapper® v.4.0 with a fluorescence detection threshold of 50 units.We followed a version of the Vos Genotyping error rate was estimated using the ratio of mismatches to the total number of replicated markers Oreochromis control region haplotypes already revealed a strong species level taxonomic clustering under parsimony in a previous study haplotype and haploNet of the ‘pegas’ v0.4-2 package nuc.div function of ‘pegas’amova function of the ‘pegas’ package MtDNA sequences were aligned using the ClustalW algorithm Fst between years within each sampling site in aflp-survFst values were detected  and samples of each locality from different years were therefore pooled.Since the dataset includes localities sampled over several years , a preliminary analysis was performed to test for significant chronological differentiation in computing aflp-surv was used to estimate expected nuclear genetic diversity (He) per sampling locality using the same procedure. Two different approaches were selected to identify hybrids or pure individuals. The Bayesian clustering method implemented in structure 2.3 K) ranging from 1 to 13 using a 100000 steps burn-in period followed by 400000 MCMC repetitions. We did not use the clustering model considering sample group information (the locprior option) K was determined from the log probability of data given K using the ΔK criterion Oreochromis species where recovered in the Limpopo drainage from mtDNA structure for K = 3 in order to detect a geography-related structure or a correlation with O. andersonii haplotypes for the individual posterior probability of assignment to the third cluster. Results from structure were compared to those of newhybrids software O. mossambicus, O. niloticus, first generation hybrids (F1), second generation hybrids (F2) and backcrosses with each parental species . This program was run with uniform priors and with 100000 steps burn-in period followed by 300000 iterations to estimate individual posterior probabilities.O. mossambicus and O. niloticus are phylogenetically distinct species, an O. niloticus component could hide a consistent amount of genetic variation potentially including a finer scale sub-clustering pattern not detected within the non-niloticus cluster. We therefore used a subset of the original dataset from which the individuals previously assigned by structure to the O. niloticus cluster (P>5%) were removed , and ran structure as before.As O. mossambicus localities likely unaffected by other species, using a second subset of the original dataset including only populations for which no clues for hybridization were detected in the prior analyses . Genetic differentiation was measured by calculating pairwise Fst from allelic frequencies computed with the Bayesian method aflp-survFst was determined based on 10000 permutations with Bonferroni corrections for multiple tests . As for mtDNA (see above), we performed an AMOVA on the AFLP dataset. A structure analysis was finally performed using a similar procedure as for the full dataset (see above). Here, following the recommendations of Hubisz et al. locprior) and the original (no locprior) clustering models. structure analyses were carried out at the Bioportal server of the University of Oslo  structureQ plots correspond to the runs with the most positive log probability of the data for a given K.We then focused on population structure within Figure S1STRUCTURE analysis of the full AFLP dataset. Averaged log probability of the data Ln P(X|K) (upper panel) and the value of the ΔK criteria (lower panel) computed according to Evanno et al. (2005) for each number of cluster K. The ΔK plot clearly supports the presence of two clusters in the data.(TIF)Click here for additional data file.Figure S2STRUCTURE analysis of the AFLP dataset comprising only individuals with no detected O. niloticus component.A. Averaged log probability of the data Ln P(X|K) (upper panel) and the value of the ΔK criteria (lower panel) computed according to Evanno et al. (2005) for each number of cluster K. B. STRUCTURE barplots for K = 2 to 7 showing assignment values (Q) of individuals with no O. niloticus component.(PDF)Click here for additional data file.Figure S3STRUCTURE analysis of the AFLP dataset only comprising O. mossambicus individuals from the eight localities preserved from genetic introgression. A. Averaged log probability of the data Ln P(X|K) (upper panels) and the value of the ΔK criteria (lower panels) computed according to Evanno et al. (2005) for each number of cluster K, with (left) and without (right) the LOCPRIOR option. B. STRUCTURE barplots for K = 2 to 6 showing the assignment values (Q) of O. mossambicus individuals from the eight localities preserved from genetic introgressions, with (left) and without (right) the LOCPRIOR option.(PDF)Click here for additional data file.Figure S4Haplotype genealogy of the genus Oreochromis based on a 385 bp fragment of the mitochondrial control region. The size of the circles representing each haplotype is proportional to log.(PDF)Click here for additional data file.Table S1Abundance and repartition of haplotypes in the Changane-Lower Limpopo System per locality.(PDF)Click here for additional data file.Table S2Number of AFLP loci per primer combination.(PDF)Click here for additional data file.Table S3Control region sequences from GenBank used in this study. Sequences marked with * were included in the haplotypes networks of the (PDF)Click here for additional data file.


The population of Argentina is the result of the intermixing between several groups, including Indigenous American, European and African populations. Despite the commonly held idea that the population of Argentina is of mostly European origin, multiple studies have shown that this process of admixture had an impact in the entire Argentine population. In the present study we characterized the distribution of Indigenous American, European and African ancestry among individuals from different regions of Argentina and evaluated the level of discrepancy between self-reported grandparental origin and genetic ancestry estimates. A set of 99 autosomal ancestry informative markers (AIMs) was genotyped in a sample of 441 Argentine individuals to estimate genetic ancestry. We used non-parametric tests to evaluate statistical significance. The average ancestry for the Argentine sample overall was 65% European (95%CI: 63–68%), 31% Indigenous American (28–33%) and 4% African (3–4%). We observed statistically significant differences in European ancestry across Argentine regions  as well as between the capital and immediate suburbs of Buenos Aires city compared to more distant suburbs . European ancestry among individuals that declared all grandparents born in Europe was 91% (95%CI: 88–94%) compared to 54% (95%CI: 51–57%) among those with no European grandparents (p<0.001). Our results demonstrate the range of variation in genetic ancestry among Argentine individuals from different regions in the country, highlighting the importance of taking this variation into account in genetic association and admixture mapping studies in this population. The current population of Argentina is the result of generations of intermixing between various groups, including Indigenous Americans who originally resided in this part of South America, Spanish conquistadores and Africans brought as slaves starting in the early and late 1500s respectively, and a large European immigrant population that arrived between 1870 and 1950 In spite of this rich history of immigration and admixture, most of the Argentine population self-identifies as of European-descent, with only 1% of the total population self-identifying as descendants of an indigenous group . In contrast to this perception, it has been reported that a considerable proportion of the Argentine population has at least one Indigenous American ancestor In this study we investigated the distribution of genetic ancestry in four regions of Argentina in a relatively large number of individuals (n = 441), taking into account information on the origin of each individual's grandparents. The latter allowed us to evaluate the level of concordance between grandparental origin and genetic ancestry estimates. We also compared the distribution of individual ancestry in Buenos Aires city (N = 168), the largest urban area in Argentina, to those of two other large urban areas in Latin America: Mexico City (N = 502) and San Juan de Puerto Rico (N = 133) to contextualize the observed level of variation in individual ancestry proportions of Buenos Aires with those observed in other major Latin American cities.All participants provided written informed consent. The study was approved by the Human Research Protection Program, Committee of Human Research of the University of California, San Francisco, the Ethics Committee of the Hospital Italiano of Buenos Aires and the Ponce School of Medicine & Health Sciences Institutional Review Board. The Argentine Ministry of Health approved the study and the shipment of samples from Argentina to UCSF for analysis.Argentine men and women were randomly identified between the years 2000 and 2010 from blood donor banks within major hospitals in different regions of the country and invited to participate. All individuals were asked to donate a sample of peripheral blood, and were asked to provide information about the region/country of birth of all grandparents . TherefoData on genetic ancestry from Mexico City was obtained from 502 healthy Mexican women enrolled in a breast cancer case-control study A set of 106 single nucleotide polymorphisms (SNPs) that can discriminate Indigenous American, African, and European ancestry was used to estimate the proportion of genetic ancestry in individuals from Argentina, Mexico City and San Juan de Puerto Rico. Simulation studies have shown that 100 ancestry informative markers (AIMs) with allele frequency differences similar to the ones we used here are required to achieve a correlation higher than >0.9 with true ancestry Six of the 106 AIMs were excluded from the analysis because they had a call rate lower than 90%. Even though AIMs are expected to violate Hardy-Weinberg equilibrium more than other markers, we excluded an additional SNP due to its deviation from expected frequencies under equilibrium (p<0.0005). The final analysis included a total of 99 AIMs. We genotyped 558 individuals from Argentina, 502 from Mexico and 141 from Puerto Rico. We found complete concordance among 10 genotyped duplicates. We excluded individuals with a genotype call rate of <70% . The final analysis included 441 samples from Argentina, 502 from Mexico and 133 from Puerto Rico. The data used in the present study is available from the authors upon request.As part of a different ongoing study, fifty-four out of the 441 individuals were also genotyped with an Affymetrix 250 K StyI array . We excluded SNPs with more than 5% of missing data and a hardy-Weinberg equilibrium p<0.00005. Since the model in ADMIXTURE Individual genetic ancestry was estimated using a maximum likelihood (ML) approach Multidimensional Scaling with pairwise allele sharing distances as implemented in the program PLINK The difference in mean European/Indigenous American ancestry between the different Argentine regions was tested using the two-sample Kolmogorov-Smirnov test for equality of distribution functions. The significance of the difference in mean Indigenous American/European ancestry between the five categories defined by the presence of 0, 1, 2, 3, or 4 grandparents that reside in a particular region of Argentina was evaluated with the Kruskal-Wallis test. Non-parametric approaches were selected because the distribution of genetic ancestry deviated from normality . Both analyses were conducted with the program STATA 11 The distribution of genetic ancestry among the 441 Argentine individuals included in our study varied from 0 to 100% Indigenous American, 0 to 100% European, and 0 to 35% African ancestry . The avest urban belt to individuals from the 2nd urban belt (p = 0.01) . When 2nd urban belt residents were removed from the analysis, there were no significant differences in European ancestry between the two hospitals.Individuals from Buenos Aires city were ascertained from two large hospitals, one private (n = 79) and one public (n = 89). Individuals from the private hospital had more European ancestry  compared to individuals ascertained from the public hospital , p = 0.028. To investigate the potential cause for this heterogeneity within the city of Buenos Aires, we determined the association between place of residence and genetic ancestry. Individuals recruited in these hospitals were residents of either the city of Buenos Aires proper, or the immediate surrounding urban areas  . MoreoveWe collected information about the region/country of birth of each individual's grandparents and we compared the mean estimated proportion of European, African and Indigenous American genetic ancestry between individuals who had 0 to 4 grandparents having been born in a particular region of Argentina, in any other Latin American country or in Europe . As expeIn an effort to validate the ancestry estimates obtained with our set of AIMs, we next compared individual genetic ancestry estimates obtained with the set of 99 AIMs to those obtained with a set of 118,192 SNPs in a group of 54 individuals within our Buenos Aires sample. The correlation coefficients for the European and Indigenous American estimates were 0.90 and 0.93 respectively . Africanst and 2nd dimensions of a multidimensional scaling analysis  within the same space defined by the 1analysis . WhereasWe investigated the individual genetic ancestry proportions among individuals from four regions in Argentina and demonstrated their variation across and within regions, with the NWA region having the most striking difference in European and Indigenous American ancestry proportions compared to all other regions. Moreover, we found that within Buenos Aires City there were modest but statistically significant differences in genetic ancestry across different urban regions. In this respect, our results add to the previously published descriptions of genetic ancestry distribution in Argentina by providing more extensive sampling and genetic ancestry estimates that are based on a relatively large set of AIMs.The genetic diversity of various Latin American populations has been previously described et al.et alet al.There have been previous studies that focused on the genetic admixture characteristics of the Argentine population. Our group has previously investigated the proportion of African ancestry in individuals from Buenos Aires City et alet al ((24 AIMs), and differences in the ascertainment of individuals in each study. The study by Corach et al included 246 male donors from paternity testing clinics.A more recent study by Corach et alIn our study, we found the most significant differences in genetic ancestry proportions among individuals from NWA when compared to individuals from all other regions investigated. Currently, the Argentine northwest has approximately 5,000,000 inhabitants, who represent about 1/8 of the total country population. When the Spanish conquistadores arrived in the early 1500s, this region had the largest population size of the Argentine territory, with an estimated population of 200,000 In the present study we were able to compare the distribution of genetic ancestry of the two northern regions of the country (NWA and NEA). We observed that individuals from NEA had a greater proportion of European ancestry compared to individuals from NWA. However, we also observed a smaller degree of variation of the Indigenous American and European components in NEA compared to NWA (NEA Standard Deviation (SD) = 0.13 and NWA SD = 0.24; variance ratio test p = 0.0014). This can be interpreted as the result of more widespread admixture in NEA. The historical data seems to support this assertion. Specifically, although the Spanish authorities tried to restrict inter-ethnic marriages and the use of indigenous languages across all regions of their colonies, the NEA represented a marginal area where this control was not very effective th and 17th century, and the earlier Spanish conquistadores nd urban belt, where we observed significantly higher Indigenous American ancestry The Buenos Aires Metropolitan Area (Buenos Aires City and surrounding urban areas) is the third most populated metropolis in Latin America, after Mexico City and Sao Paulo. In Buenos Aires City two historical events had a strong influence in the genetic composition of its inhabitants. First, the arrival of a large number of European immigrants, mostly from Italy and Spain, between 1870 and 1950 who intermixed with the smaller local population Buenos Aires City. This was a population that had already resulted from admixture of several generations of original Indigenous Americans, Africans brought as slaves during the Spanish conquest in the 16We validated our ancestry estimates with two approaches. One, by comparing ancestry estimates obtained with our panel of 99 AIMs to those obtained with 118,192 SNPs in a subset of individuals. Overall, we observed that the individual ancestry estimates that we obtained using information from 99 AIMs were strongly correlated with those obtained with genome wide data for the major ancestral components (European and Indigenous American). However, this was not the case for African ancestry, which shows a correlation coefficient of about 0.12. This low level of correlation is likely the result of an overestimation of the African component, as estimated by the 99 AIMs panel. Since genetic ancestry estimates have statistical variance, when the proportion of ancestry is close to zero the estimates of ancestry tend to be biased towards higher numbers . Therefore, care should be taken in interpreting ancestry estimates when the overall proportion of that ancestral group is low (<5%).Another way of validating our results was to compare the obtained genealogical information to the estimated proportions of genetic ancestry, and to investigate how informative one would be of the other. We observed that the average estimated European ancestry among individuals with at least one European grandparent was higher than that of individuals with no European born grandparents (80% versus 54%). Therefore, our data showed that the number of grandparents born in Europe is highly correlated with the proportion of European ancestry as measured by genetic markers. Interestingly, our data indicated that the largest change in average genetic ancestry when considering the number of grandparents born in Europe was between 0 and 1 grandparent. This is probably reflecting the effect of assortative mating Our study contrasted the similarities and differences in the ancestral composition of three Latin American cities with very different demographic histories: Mexico City, with a strong Indigenous American component, Buenos Aires City, with a strong European component, and San Juan de Puerto Rico, with a strong European as well as a relatively important African component. Despite the differences in the average genetic ancestry proportions between the three cities, our results show that the distributions of individual ancestry estimates have a similar degree of dispersion.One limitation of the present study is that we ascertained individuals through a limited network of blood donor centers at hospitals and clinics, instead of using a population-based approach. Therefore, we cannot generalize the ancestry proportions obtained from each group of individuals to those in the region of origin of each group. A larger network of hospitals and clinics for our ascertainment, or a general population-based approach  would have given us a more precise picture of the distribution of ancestry proportions at the regional level. However, in spite of this limitation we were able to achieve our aims of describing the level of heterogeneity within the country as well as testing the reliability of genetic ancestry estimates using AIMs when comparing to grandparental origin.In summary, our results suggest, in concordance with previous studies, that genetic epidemiological research in Latin America should take genetic ancestry into account, preferably by directly estimating it using AIMs or comparable genetic markers (e.g. GWAS data). Studies that are unable to obtain information about genetic ancestry should, at the minimum, take into consideration not only the countries and the regions of origin of all participating individuals, but also the cities from where individuals come from. As we report here, demographic variations at the local level could also affect admixture patterns, and thus confound associations. Self-reported information about grandparents' origins may be useful surrogates, especially in regions with recent immigration patterns. However this information has to be taken with extreme caution given the potential overestimation of European ancestry by self-report.Figure S1Map of Federal District and first, second and third belt.(TIF)Click here for additional data file.Figure S2st and 2nd Multidimensional components of samples from Buenos Aires metropolitan area and ancestral individuals (black triangles).1 Samples from the Capital are represented in green, from the 1st urban belt in pink, and from the 2nd urban in orange.(TIF)Click here for additional data file.Figure S3st and 2nd Multidimensional components of samples from the cities of Buenos Aires, Mexico and San Juan, including European, African and Indigenous American ancestrals (black triangles).1 Samples from Buenos Aires are in gold, samples from Mexico are in light blue and samples from Puerto Rico are in purple.(TIF)Click here for additional data file.Table S1Individual information on the number of grandparents born in Argentina (by region), other South American country or Europe.(XLS)Click here for additional data file.Table S2List of ancestry informative markers (AIMs) with chromosomal position and allele frequency in European, African and Indigenous American samples.(XLSX)Click here for additional data file.Table S3Correlation coefficients between genome wide ancestry estimates and 99 ancestry informative markers (AIMs) estimates.(DOC)Click here for additional data file.


Rimicaris exoculata was studied using microsatellite markers to infer connectivity along the 7100-Km range encompassing the sampled sites. Astonishingly, no genetic differentiation was found between individuals from the different geographic origins, supporting a scenario of widespread large-scale dispersal despite the habitat distance and fragmentation. We hypothesize that delayed metamorphosis associated to temperature differences or even active directed migration dependent on physical and/or chemical stimuli could explain these results and warrant further studies on adaptation and dispersal mechanisms.Dispersal plays a fundamental role in the evolution and persistence of species, and especially for species inhabiting extreme, ephemeral and highly fragmented habitats as hydrothermal vents. The Mid-Atlantic Ridge endemic shrimp species  Hydrothermal vents are extreme habitats that are distributed worldwide in association with volcanic and tectonic events. The fluids emitted at vent sites are geo-thermally heated up to several hundred degrees Celsius and enriched with minerals, metals and reduced chemicals, yet, they support highly populated communities of organisms that are specifically associated to these ecosystems. Most of the vent-restricted species have developed specific adaptations to extreme conditions and close relationships with chemoautotrophic microorganisms that are the food web’s primary producers in the absence of photosynthesis.Hydrothermal vent species fully represent the paradigm of larval dispersal. The high level of endemism observed among The remoteness of hydrothermal vents renders the assessment of migration difficult, and calls for indirect population genetic methods to infer rates and patterns of connectivity in the depth of the ocean. However, vent habitats scattered along a ridge, due to their instability and high extinction rates, might be considered as metapopulation(s), where the migration-drift equilibrium might never be reached Bathymodiolus mussels Phylogeographic studies to date suggest the existence of barriers to dispersal along oceanic ridges, the degree of realized dispersal and the strength of the barrier vary to a certain extent according to life history traits, such as egg and larvae characteristics Rimicaris exoculata, which is often present in high-density swarms  around high-temperature sulphide chimneys at deeper hydrothermal vent sites R. exoculata, corresponding to the adult and juvenile stages R. exoculata is thought to originate mainly from specialized chemosynthetic epibionts present in their gill chamber and gut  using higher resolution genetic markers and at a larger biogeographical scale than applied in any previous MAR studies in any species, and to determine whether the low genetic differentiation found among matrilines Samples were collected from five locations corresponding to different hydrothermal vent fields  along the Mid-Atlantic Ridge . For twoTaq buffer, 200 µM of dNTP’s, 0.3 µM of each primer and 0.5 U of HotStart Taq polymerase (Qiagen). PCR amplifications were conducted on a Perkin-Elmer Gene Amp System 7200  with the following program: 15 min. at 95°C; 30 cycles composed of 30s at the annealing temperature Microsatellite loci were amplified from the 687 individuals sampled, by PCR with 11 fluorescently-labelled forward primers (described in HE) and observed (HO) proportion of heterozygotes, and the inbreeding coefficient (FIS) were estimated using the software GENETIX, 4.05 richA), since the observed number of alleles in a sample is highly dependent on sample size.The mean number of alleles per locus , the expected (F estimator of genetic structure θ F-statistics being greater than zero was determined by permutation (10 000 replicates) using GENETIX, 4.05 The et al.To investigate the number of distinct genetic populations represented by our sampling, we used the software STRUCTURE version 2.1 FST. As searching strategy we used 10 short chains (1000 trees sampled) and three long chains (10 000 trees sampled). For each chain the first 100 000 steps were used as a burn-in and adaptive heating was used to ensure an independent, comprehensive search of the parameter space. In order to directly compare some of the results obtained in this study using microsatellite markers, and the previous results obtained with a mitochondrial marker To assess asymmetrical gene flow between the different hydrothermal vents, we used the software package MIGRATE version 3.2.16 To assess the possible site of origin of the individuals from Ashadzé and South MAR, assignment/exclusion analyses were performed using GENECLASS2 HE) were higher than estimates based on the number of alleles and sample size (HEq). During a bottleneck, allele number decreases faster than heterozygosity, resulting in a transient apparent heterozygosity excess, indicative of a recent bottleneck event HEq were calculated under the single-step mutation model (SMM) and the two-phase model (TPM), allowing for i) 10% and 2) 4% of multi-step mutations. The latter TPM model was chosen after assessing the mutation model of the microsatellite markers using a likelihood approach implemented by MISAT p) to involve changes greater than a single repeat. Model fit was then statistically tested by the differences in likelihoods. For each locus, Markov chains were run for 100,000 generations for SMM and TPM models, and p in the TPM was allowed to vary between 0.0001 and 0.5. Likelihood ratio tests were used as described in 2 distribution (d.f. = 1).To test for a reduction in effective population size linked to bottleneck or founder events, the Wilcoxon sign-rank test was applied to infer if expected heterozygosities estimated from allele frequencies (Ne) of R. exoculata we generated Bayesian skyline plots using mitochondrial data east v.1.5.4 Ne through time using Markov chain Monte Carlo (MCMC) sampling. The reconstructed Bayesian skyline plots (BSP) were obtained using the HKY model as selected by the AIC criterion implemented in Modeltestracer v.1.5 NeT), the median and the corresponding credibility intervals.To characterize past changes in the effective population size  standardized for comparison across a minimum common sample size of 5 individuals , ranged from 4.52 (Rainbow 2005) to 5.35 (SouthMAR). No loci showed evidence of linkage disequilibrium. Unbiased heterozygosity (HE) varied between 0.66 (Rainbow 2005) and 0.71 (Logatchev 2007) and the observed heterozygosity (OH) varied between 0.58 (Rainbow 2005) and 0.68 (Logatchev 2007) for the three sampled sites  and years  were repeated without the 4 possibly problematic loci resulting in the same trends as obtained with all 11 loci  to 52 (Rim26) over all locations . The meand years . Four lo 11 loci . Using t 11 loci : Rainbow 11 loci .OH (OH) varied from 0.58 (2005) to 0.66 (2007) across years , the Logatchev vent did not show significant differences in genetic diversity between years, neither in HO  nor meanHO . For thess years  and meanss years .FST estimates between vent locations (Pairwise ocations  were notocations  demonstrEstimates of gene flow obtained with MIGRATE software ranged from 2.59 (Logatchev to Rainbow) to 3.27 (TAG to Rainbow) with no apparent asymmetrical migration between the three hydrothermal vent sites . In agreThe estimation of the mutation model of 6 microsatellite marker using MISAT revealed that 3 microsatellites followed a SMM model and 3 others a TPM with multi-step mutations with a rate ranging from 3.45% to 8.35% . For theHE) was lower than estimates based on the number of alleles and sample size (HEq). The Wilcoxon sign-rank results revealed significant heterozygosity deficiency for all sites and years sampled , as the expected heterozygosity estimated from allele frequencies  and higher variability in gene diversity (0.17–0.82) were detected using microsatellite markers, on the basis of a much smaller sample size Branchipolynoe seepensisA lack of power due to low genetic variation can also be discarded as an alternative hypothesis, as high genetic diversity was detected within all studied vents. Both the mean number of alleles per locus (3.55–17.91) and gene diversity (0.66–0.71) are within the values found for other hydrothermal vent organisms e.g. . HoweverR. exoculata, yet exhibiting distinct larval development in relation to vertical migration in the water column.A scenario of current persistent large-scale effective dispersal (gene flow) is indicated by the lack of spatial genetic differentiation estimated from the non-significant pairwise differentiation, the high gene flow estimates and the inference of one single population in the MAR. The different demographic analyses based on either microsatellites or DNA markers support a recent expansion, possibly associated to a common (re)colonization event, about 250 Kyr ago  at these demes and/or at the scale of the metapopulation, therefore rendering the effect of genetic drift negligible over the temporal scales considered in this study. Two scenarios may explain the large scale and temporal panmixia, i) present day large-scale effective dispersal and/or ii) an historical event of extinction followed by (re)colonization of all vents from a common source, where insufficient time elapsed since the (re)colonization event, compared to the likely large effective population size, rendering the effects of genetic drift negligible Rimicaris exoculata along the MAR. This large-scale dispersal was not shown to exhibit directionality or source-sink relationships between the studied sites. This is in agreement with the few studies available on circulation along the MAR, the ridge being constrained by very high walls that flank its axial valleys Together with the spatial and demographic analyses, the trends in temporal variability also revealed no genetic differentiation among samples separated by one to a maximum of eleven years for two vent sites (Logatchev and Rainbow). These results may indicate large effective population sizes , expected to reduce dispersal ability, has been reported to have high gene flow along part of the East Pacific Rise Besides showing homogeneity over shorter distances, these species from the EPR exhibit different life history traits at the egg and larval stage, which in different ways might potentially increase dispersal ability. These include free-swimming planktotrophic larvae (in the mussel species Rimicaris exoculata larvae go through a long planktotrophic phase suspected to partly take place in the photic zone due to the photosynthesis-derived lipids R. exoculata to be unaffected by seafloor barriers, but our discovery of large scale effective dispersal across the immensity of oceanic masses still allowing successful recruitment at their rare and fragmented habitat seriously questions the hypothesis of purely passive dispersal.Three-dimensional trajectories may be aided by mesoscale eddies which can affect the seafloor at great depths and potentially transport vent-associated organisms for hundreds of kilometers R. exoculata adults have been shown to possess a dorsal photoreceptor that is highly sensitive to black body radiation associated with high-temperature vents 2S This study challenges the classical view of passive large scale dispersal subject to haphazard trajectories of currents in different layers of the water column. In the three dimensional ocean, the probability of an individual reaching a suitable habitat after being diluted in an incommensurable volume of water seems infinitesimal and calls for the existence of non-passive, dispersal strategy mechanisms such as those delaying metamorphosis or actively guiding larvae or adults towards suitable habitat. Recent studies showed that planktonic larval duration is strongly influenced by water temperature for many fish and invertebrate taxa


Tetraena mongolica (Zygophyllaceae), an endangered endemic species in western Inner Mongolia, China. For endemic species with a limited geographical range and declining populations, historical patterns of demography and hierarchical genetic structure are important for determining population structure, and also provide information for developing effective and sustainable management plans. In this study, we assess genetic variation, population structure, and phylogeography of T. mongolica from eight populations. Furthermore, we evaluate the conservation and management units to provide the information for conservation.atpB-rbcL noncoding spacer region of the chloroplast DNA were used to reconstruct the phylogeography of T. mongolica. A total of 880 bp was sequenced from eight extant populations throughout the whole range of its distribution. At the cpDNA locus, high levels of genetic differentiation among populations and low levels of genetic variation within populations were detected, indicating that most seed dispersal was restricted within populations.Sequence variation and spatial apportionment of the T. mongolica. For setting a conservation management plan, each population of T. mongolica should be recognized as a conservation unit.Demographic fluctuations, which led to random losses of genetic polymorphisms from populations, due to frequent flooding of the Yellow River and human disturbance were indicated by the analysis of BEAST skyline plot. Nested clade analysis revealed that restricted gene flow with isolation by distance plus occasional long distance dispersal is the main evolutionary factor affecting the phylogeography and population structure of  Tetraena mongolica Maxim, a monotypic genus of the Zygophyllaceae, is endemic to the western part of Inner Mongolia around the basin of the Yellow River [T. mongolica, up to 0.5 m in height, flower from mid-May till early June, and set fruits in July. The species is restrictedly distributed in the western Gobi, the largest desert in Asia characterized by extreme low annual rainfall [T. mongolica with a fully developed root system is well adapted and becomes locally dominant. T. mongolica plays an important ecological role as windbreak for stabilizing river bank [Genetic variation within and among natural populations is crucial for the long-term survival of a species. An accurate estimate of the level and distribution of genetic diversity of threatened species provides fundamental information in designing conservation programs ,2. Tetraow River , and is ow River . Plants rainfall , where Tver bank . Human'sT. mongolica. As the high rate of ovule abortion after anthesis [T. mongolica was quite low (1.3 - 2.8%) in the natural populations [T. mongolica [Previous studies have been focusing on the biological characters causing the population decline of anthesis , the see 1.3 - 2.% in the ulations . Previouongolica .Understanding levels and spatial partitioning of genetic polymorphisms in an endangered species provides sufficient information for conservation practices. This kind of researches has become increasingly popular in the recent years, with the development of analytical methods to take phylogenetic distinctiveness into account when setting conservation priorities ,12. DuriT. mongolica from eight populations throughout the entire distribution range. Several aims are pursued: 1) to examine the levels of genetic variation within and between populations, 2) to reconstruct phylogeographical patterns and examine the extent of genetic differentiation among populations, and 3) to identify the conservation and management units based on genetic evidence, to provide the information for the development of effective and efficient conservation practices for this species.For endemic species with a limited geographical range and declining populations, historical patterns of demography and hierarchical genetic structure are important for determining population structure, and also provide information for developing effective and sustainable management plans . In thisatpB and rbcL genes of the chloroplast DNA. Identical sequences were obtained from five clones derived from the same amplification reaction, indicating no PCR artifacts caused by Taq polymerase or sequencing errors. The atpB-rbcL intergenic region of cpDNA in T. mongolica varied from 872 to 880 base pairs (bp) in length. The cpDNA sequences were aligned with a consensus length of 881 bp, of which 46 sites (5.2%) were variable.No within-individual variation was detected in the non-coding spacer between HQ142910-HQ142986) were identified from 77 individuals of T. mongolica, with an estimated haplotype diversity of h = 0.962 ± 0.009 (Table θ = 0.00447 ± 0.0003) and within populations, ranging from θ = 0.00099 (TST) to 0.00405 (HN).The chloroplast spacer is A/T rich with an average content of 73.6%, which is consistent with the nucleotide composition of most noncoding spacers and pseudogenes because of low functional constraints . In totaT. mongolica.A neighbor-joining tree obtained using MEGA recovered eight cpDNA clades Figure . Most ofA nested clade analysis was accomplished by linking nucleotide haplotypes in a hierarchical manner based on mutational changes. After linking the haplotypes into a clade, closely related clades were linked further to form a higher-level group; via such hierarchical linking, a nested network was drawn Figure . In totaNm of 0.04-0.71 and FST of 0.38-0.90 indicated high levels of genetic differentiation between all populations, with three exceptional pairs of HN-TST, HN-XD and XD-TST .A nested contingency analysis detected significant geographical associations within several clades  and the whole cladogram. The phylogeographical inferences are listed in Figure Dc and Dn for each clade representing contemporary distributions of haplotypes were used to interpret historical and contemporary gene flow processes  [Hygrophila pogoncalyx (θ = 0.00343) [θ = 0.01018 for the cpDNA trnD-trnT spacer of Cunninghamia konishii [θ = 0.01268 for the cpDNA atpB-rbcL spacer of Cycas taitungensis [T. mongolica may be ascribed to its extremely small effective population size associated with the low seed set in wild (1.3 - 2.8%) [In this study, we investigated the phylogeographical pattern and population structure of endangered  0.0022) , and Hyg0.00343) , but is konishii , and θ =ungensis . The two - 2.8%) .T. mongolica populations [T. mongolica experienced relatively little disturbance due to low accessibilities [Recent habitat loss has reduced the number and size of ulations . Small pulations . Interesbilities ,10.T. mongolica based on ISSR data [T. mongolica is primarily pollinated by insects [Our previous study revealed medium levels of genetic differentiation among populations of SSR data . In cont insects . Gene fl insects , likely  insects , whereas insects . In thisT. mongolica has long been used as firewood  needs to be defined. Various criteria for ESUs have been suggested, including reciprocal monophyly [T. mongolica, given high levels of genetic differentiation and reciprocal monophyly between most populations and lower genetic variation within populations, each population should be treated as different evolutionarily significant units for conservation , 5 μl dNTP mix (8 mM), 10 pmole of each primer, and 4 U of Taq polymerase . The reaction was programmed on an MJ Thermal Cycler with first cycle of denaturation at 95°C for 2 min, then 30 cycles of denaturation at 94°C for 45 sec, annealing at 48°C for 1 min, and extension at 72°C for 1 min 30 sec, followed by 72°C extension for 10 min and 4°C for storage.Leaves were powdered in liquid nitrogen and stored in a -70°C freezer. Genomic DNAs were extracted from the powdered tissue following a CTAB procedure and gel-quantified . The int primers . Each 50atpB-rbcL fragment, both strands were cycle-sequenced using the Taq Dye Deoxy Terminator Cycle Sequencing Kit . Products of the cycle sequencing reactions were run on an ABI 377XL automated sequencer (Applied Biosystem). Cloned PCR products were sequenced using universal T7 forward (5'- TAATACGACTCACTATACGGG-3') and SP6 reverse (5'-TATTTAGGTGACACTATAG-3') primers located on pGEM-T easy vector termination sites. Five clones for each individual were randomly chosen and sequenced.All PCR products were purified from an agarose gel using the PCR product purification kit  and cloned into a pGEM-T easy cloning vector (Promega). For each cpDNA The chloroplast DNA sequences were aligned with the program Clustal X 1.81 . Indels The nested clade analysis was accomplished by linking nucleotide haplotypes in a hierarchical manner based on mutational changes. The linking rules start at the tips of the cladogram and move one mutational step into the interior, uniting all haplotypes connected by this procedure into a '1-step clade'. Following pruning off the initial 1-step clades from the tips, the procedure is repeated on the more interior portions of the haplotype tree until all haplotypes have been placed into 1-step clades. The next level of nesting uses the 1-step clades as units, rather than individual haplotypes. The linking rules are the same; however, '2-step clades' are now formed. The nesting procedure is repeated until a nesting level is reached such that the next higher nesting level would result in only a single category spanning the entire original haplotype network; via such hierarchical linking, a nested network was drawn.h) [θ) [Nm, the number of female migrants per generation between populations. Nm was estimated using the expression FST = 1/(1 + 2 Nm), where N is the female effective population size and m is the female migration rate [Levels of inter- and intra-population genetic diversity based on cpDNA were quantified by indices of haplotype diversity (h)  and estih) [θ)  using Dnh) [θ) . Patternion rate .Nm values against geographical distance [Nm and distance was determined by a regression of F-test over distances using SPSS program version 6.0 [Dc, a measure of the geographical spread of a clade, and 2) the nested clade distance, Dn, a measure of the geographical distribution of a clade relative to other clades in the same higher level nesting category. These measures of geographical distribution were used to infer historical processes following the methods of Templeton et al. [A model of "isolation by distance" was assessed by plotting pairwise distance . The corsion 6.0 . Geograpsion 6.0 . Two stan et al. . The nesn et al. . The debn et al. -66 have T. mongolica, we analyzed the cpDNA noncoding spacer data with BEAST v1.4 [atpB-rbcL spacer of 3 × 10-9 substitutions per site per year was used [To estimate the population demographic trends for AST v1.4 . BayesiaAST v1.4 . In the was used ,70. The TYC designed and conceived this study. XJG collected the samples and analyzed the data. WKW, ZHL, CCH, KHH and WHH collected the genetic data. WKW and TYC analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.


Studies of genetic diversity are essential in understanding the extent of differentiation between breeds, and in designing successful diversity conservation strategies. The objective of this study was to evaluate the level of genetic diversity within and between North American Brown Swiss , Jersey  and Holstein  cattle, using genotyped bulls. GENEPOP and FSTAT software were used to evaluate the level of genetic diversity within each breed and between each pair of the three breeds based on genome-wide SNP markers .P < 0.01), which could be a result of selection, genetic drift and inbreeding within each breed. Hardy-Weinberg test also confirmed significant heterozygote deficit in each breed over several loci. Moreover, results from population differentiation tests showed that the majority of loci have alleles or genotypes drawn from different distributions in each breed. Average gene diversity, expressed in terms of observed heterozygosity, over all loci in BS, JE and HO was 0.27, 0.26 and 0.31, respectively. The proportion of genetic diversity due to allele frequency differences among breeds (Fst) indicated that the combination of BS and HO in an ideally amalgamated population had higher genetic diversity than the other pairs of breeds.Hardy-Weinberg equilibrium (HWE) exact test within breeds showed a significant deviation from equilibrium within each population (Results suggest that the three bull populations have substantially different gene pools. BS and HO show the largest gene differentiation and jointly the highest total expected gene diversity compared to when JE is considered. If the loss of genetic diversity within breeds worsens in the future, the use of crossbreeding might be an option to recover genetic diversity, especially for the breeds with small population size. The importance of genetic diversity in livestock is directly related to the need for genetic improvement of economically important traits as well as to facilitate rapid adaptation to potential changes in breeding goals . EstimatHardy-Weinberg Equilibrium (HWE) states that in a large random mating population with no selection, mutation, or migration, the allele frequencies and the genotype frequencies are constant from generation to generation, and, hence, a simple relationship between the allele frequencies and the genotype frequencies exists . The theis, Fit and Fst) are the most widely used parameters for studying the genetic differentiation of populations. These indices have been originally defined in terms of the correlations of two uniting gametes [it is the correlation between uniting gametes that generate an individual relative to the gametes of the total population. Fis is the average over all subpopulations of the correlation between uniting gametes that generate an individual relative to the gametes of their own subpopulation. Fst is the correlation between random gametes within subpopulations, relative to gametes of the total population. For example, in this study, an ideally amalgamated population of Brown Swiss and Jersey bulls would have each breed as a subpopulation. Furthermore, the relationship between fixation indices and measures of identity by decent have been illustrated in previous studies [Tests for departures from Hardy-Weinberg proportions are often used to check on random mating in populations, and the deviations from the expected frequency of homozygotes are used to estimate inbreeding coefficients . The sam gametes -14. Acco studies ,16.st is equal to 0 when the same allele is fixed in all populations [st and Gst, respectively) have been extensively used to describe the level of genetic diversity [Fixation indices can also be formulated entirely in terms of the allelic and genotypic frequencies in the population ,17,18. Iulations . Alleliculations . In viewiversity ,17.The objective of this study was to assess the status of genetic diversity within and between BS, JE and HO breeds, using bulls genotyped with a dense SNP marker map through detailed analyses carried out via GENEPOP and FSTAT software.Genome-wide SNP data for the three breeds were received from the Animal Improvement Programs Laboratory, USDA  in November 2009. The data consisted of 900, 2,922 and 3,535 Brown Swiss (BS), Jersey (JE) and Holstein (HO) bulls, respectively, all genotyped with the Illumina BovineSNP50K BeadChip  as part of the North American collaboration in genomic prediction in dairy cattle . Genotypo), and the expected heterozygosity (He) under HWE. The significance of breed differences was tested using the exact test of population differentiation in GENEPOP software based on allele frequencies.Estimates of genetic diversity and statistical analyses were performed using the software GENEPOP, version 4.0 . The exast coefficient proposed by Wright [Genetic differentiation between breeds was also estimated using the Fy Wright  and compst was estimated by a “weighted” analysis of variance [st is:The software FSTAT  was usedvariance . The mosp2 the sample variance of allele frequencies over populations [st can therefore be described as the amount of allele frequency variance in a sample relative to the maximum possible variance. Fst can also be defined as follows [Where: δulations . Fst can follows :it is the correlation between uniting gametes that generate an individual, relative to the gametes of the total population; Fis is the average, over all subpopulations, of the correlation between uniting gametes that generate an individual relative to those of their own subpopulation.Where: Ft) in the total population was also defined regardless of structure of the population, in terms of total population gene frequency (qt) [The amount of heterozygosis (Yncy (qt) :m) was estimated, assuming the n-island model of population structure, on the basis of the relationship:Indirect estimates of gene flow were implemented in FSTAT  accordinst and Gst) or independent (Dst’ and Gst’) of the number of subpopulations [st is the average gene diversity between subpopulations. The gene diversity in the total population is equivalent to the sum of gene diversities within each subpopulation. Coefficient of gene differentiation (Gst) was computed as the ratio of Dst to the total population diversity.Furthermore, FSTAT was used to calculate inter-population gene differences and coefficients of gene differentiation that are either dependent (Dulations . Dst is P < 0.01). Moreover, results of the exact test for HWE showed lower observed heterozygosity (Ho) than expected heterozygosity (He) in each breed . Jersey showed higher percentage of loci with fixed alleles followed by BS and HO (Table The exact test for Hardy-Weinberg Equilibrium (HWE) within breeds showed a significant deviation in each breed P < 0.0. Moreoveo, are shown in Figure o than JE and BS across all chromosomes. BS and JE had similar overall Ho, however, depending on the chromosome, one or another of the two breeds had higher Ho. Higher Ho for HO than BS and JE and similar overall Ho for BS and JE is consistent with the effective population sizes of this three breeds, which is higher for HO and lower and similar for BS and JE [o in HO has reduced from 0.361, when 4 generations were traced back in the pedigree, down to 0.3534, when one generation was traced back.Average gene diversity over all loci, per chromosome, in BS, JE and HO, expressed in terms of HS and JE  Moreoverst . This may indicate the fact that there has not been new outbred genetic material introduced to the bull population over the last four generations, except the use of commonly used sires of good genetic merit over generations. The relatedness between individuals within breed in BS vs. HO populations relative to the total population was higher (0.28) than that in BS vs. JE (0.23) and JE vs. HO (0.22), which also implies that BS and HO gene pools are more differentiated compared to the other pairs of breeds.Population genetic differentiation of BS, JE and HO, as measured by Ft Figure  showed trs Table . Howeverst values. In addition, indirect estimates of gene flow indicated the presence of higher effective number of migrants (Nm) between populations of JE and HO followed by BS and JE, while BS and HO populations showed the least Nm. This might be one explanation for the higher values of population differentiation measures, in particular higher Fst between BS and HO.A summary of allelic richness, average fixation indices, frequency of private alleles per breed pair are presented in Table The exact-test for population differentiation of each breed pair across all loci showed highly significant differences among breeds regarding the distributions from which the alleles and genotypes were drawn from. Accordingly, the majority of loci have alleles or genotypes drawn from different distributions in the three breeds. However, there are some loci with alleles or genotypes drawn from the same distribution in all the breeds. For example, loci with alleles drawn from the same distribution in BS, JE and HO are shown for Chromosome 14 and 18 Figure . This imst = 0.03) and independent (Dst’ = 0.07) on the number of subpopulations, and also the highest expected total heterozygosity (Yt = 0.33) compared with the ideally amalgamated populations of BS vs. JE, or JE vs. HO. Similarly, the highest Gst and Gst’  were also observed in an ideally amalgamated population of BS vs. HO. Therefore, the measure of inter-population gene differences also revealed that BS vs. HO population had the highest genetic differences compared to the other two pairs of breeds (BS vs. JE and JE vs. HO). Overall, the results indicated higher genetic diversity in an ideally amalgamated population of BS vs. HO.The amalgamated BS vs. HO population showed the highest average inter-population gene differentiation both dependent (DP < 0. 01). The populations also showed several loci with a significant heterozygote deficit (P < 0.01) but no loci with significant heterozygote excess, which implies the application of genetic selection and inevitably the role of random genetic drift and inbreeding in each breed. Generally, the results have showed that there are some loci (from 3.1 to 7.8%) with alleles drawn from the same distribution in all the populations. This may suggest the fact that, over time and through forces like selection and random genetic drift, the allele frequencies have been largely changed in the breeds, where very little of the original genomes are preserved.The recent decline in diversity is sufficiently rapid that loss of diversity should be of concern to animal breeders . SeveralEach breed showed considerable difference between the observed and expected number of heterozygous individuals across loci. However, in the ideally amalgamated pairs of the populations, the difference between the observed and expected number of heterozygous individuals appears to be smaller suggesting that crossbreeding could be carefully considered for increasing diversity in the future if needed. In livestock species, heterozygote deficiencies can be interpreted as the consequence of many factors, such as selection, population subdivision, or inbreeding .st[st among the last four generations in HO was below 0.1, suggesting that there has been no significant introduction of more outbreed gene pool into the HO population over the last four generations. These results imply measures of population differentiation based on Fst have been described as reliable. For example, pair-wise Fst values were significantly correlated between bi-allelic loci and microsatellite datasets in Atlantic salmon, and similar result was found with regard to the overall heterozygosity [Populations are said to be undifferentiated if Fst. In thiszygosity .st results were reliable and most consistent with Reynold’s distances, Nei’s minimum distance measures and eight other genetic distance measures for ordering populations, which are widely used and well-established measures of genetic differentiation [st indicated that BS vs. HO population has higher genetic diversity than BS vs. JE and JE vs. HO ideally amalgamated populations. The average estimates, based on microsatellites, of Fst in 20 Northern European cattle breeds was 0.11 ± 0.01 [The highest proportion of total genetic variation attributed to between breed differentiation was observed in BS vs. HO. The proportion of between breed genetic variation observed in this study was comparable to the average between breed variation (7.03%) reported in nine populations of Argentinean Creole cattle populations . Studiesntiation . In this1 ± 0.01 , which iTo summarize, Brown Swiss, Jersey and Holstein bull populations have substantially different gene pools. An interesting result was the heterozygote deficit observed in each of the populations in this study. In livestock species, heterozygote deficiencies can be explained by several factors, such as selection, population subdivision, drift and inbreeding. Each breed showed a considerable difference between the observed and expected number of heterozygous individuals across loci. However, in the ideally amalgamated pairs of the populations, the difference between the observed and expected number of heterozygote individuals across loci appears to be smaller, suggesting that crossbreeding could be carefully considered for increasing diversity if needed in the future. At the present level of genetic diversity, crossbreeding is not a necessity, however if loss of genetic diversity within each breed worsens in the future, crossing can be considered as an option to increase total genetic diversity within breeds.st) compared to the other pairs (BS vs. JE and JE vs. HO). If BS and HO were to be amalgamated, higher total expected gene diversity would be obtained as compared to the other pairs of breeds (BS vs. JE and JE vs. HO). If the loss of genetic diversity within breeds worsens in the future, the use of crossbreeding might be an option to recover genetic diversity, especially for the breeds with small population size.The results suggested that the within population genetic diversity accounts for a higher proportion of the total genetic diversity in ideally amalgamated populations than the diversity between populations. The results of private alleles frequencies in this study indicated that each breed might contain unique genes or gene combinations that are absent in another breed. The study demonstrates that even with a much smaller population size, BS showed similar gene diversity to the Jersey breed, while Holstein showed higher gene diversity than both breeds in agreement with their reported effective population sizes. BS and HO seem to have higher population differentiation (Fis is the average over all subpopulations of the correlation between uniting gametes relative to those of their own subpopulation. Fst is the correlation between random gametes within subpopulations relative to gametes of the total population, which is a measure of subpopulations differentiation. Fit is the correlation between uniting gametes that generated the individual relative to gametes of the total population; subscripts is, st and it stand for individual relative to subpopulation, subpopulation relative to total population, and individual relative to total population, respectively.FBS = Brown Swiss; JE = Jersey; HO = Holstein; HWE = Hardy-Weinberg Equilibrium.MGM conducted the analyses and wrote the manuscript. FSS supervised the study and critically reviewed the manuscript. All authors read and approved the final manuscript.The authors declare that they have no competing interests.