Eyprepocnemis plorans plorans harbour supernumerary chromosomes in all natural populations hitherto analyzed from the Circum-Mediterranean region, with the single exception of the headwaters of the Iberian Segura River and several of its tributaries. To ascertain the causes of this distribution pattern, we analyze here the genetic structure of five natural populations collected in this zone (two +B and three -B), by means of ISSR markers. We found significant population structure, with two kinds of populations coinciding with +B and -B ones, separated by strong barriers to gene flow. This gives strong support to the hypothesis that the non-B populations precede B origin, and that B-carrying individuals from coastal zones have been able to colonize upstream areas, until geographical barriers  impeded their advance.Parasitic supernumerary (B) chromosomes show high capability to spread across populations. But the existence of abrupt discontinuities in their distribution demands an explanation. The grasshopper  Gene flow is a main determinant of population structure because different populations may evolve as a unit if gene flow is high but they may accumulate differences when gene flow is low. The amount of gene flow impeding population differentiation depends on its balance with other forces, mainly genetic drift and selection . HoweverEyprepocnemis plorans subsp. plorans lives in humid places next to the coast and in the vicinity of river shores and adjacent irrigated cultivation areas, in a Circum Mediterranean distribution. Farming activities have no doubt served as means for this grasshopper dispersion in the last few thousand years. In natural populations from an irrigated area close to the Segura River at Murcia (Spain), downstream to the populations analysed here, it has been reported that this species displays a single generation per year, from July to March, being abundant in all types of cultivations and plant associations, due to its polyphagous character, a low exigent diet and high dispersal ability, allowing it to find new appropriate biotic conditions [E. plorans an excellent colonizer, since a single gravid female may easily found a new population. Bearing also in mind that egg productivity increases significantly with the number of matings [The grasshopper nditions . In addinditions . This hi matings , the hig matings  has reacThis grasshopper harbours a complex system of B chromosomes in terms of both variation and geographical range, since more than 50 different B-variants have been described in the Iberian Peninsula , 7 and tIn the Iberian Peninsula, B-lacking populations have only been found in the headwaters of several rivers belonging to the Segura river basin, including the Segura River itself and several of its tributaries  . Since iE. p. plorans, reaching almost the entire geographical range of this subspecies, has been facilitated by the existence of a transmissional advantage (drive) during female meiosis [E. plorans [The widespread presence of B chromosomes in  meiosis , a prope meiosis . Gene fl plorans  thus hel plorans .In this paper we test the hypothesis that B chromosomes are absent from the headwaters area of the Segura River basin (Iberian Peninsula) because geographical barriers have impeded their advance from coastal populations . For thi(II), i.e. an analogous parameter to Nei's Gst, was 0.080, thus being very close to the Bayesian estimate (Gst-B = 0.067), and 95% confidence intervals indicated that they are significantly higher than zero (Nem) estimated from this Gst-B value (3.48) indicated the existence of substantial gene flow among populations.B chromosomes were found in two populations  but not in the remaining three  . The sixhan zero , thus suST = 0.15544) and between populations within groups (FSC = 0.09292), but that found between B-carrying and B-lacking groups (FCT = 0.06892) was only marginally significant . Classifying the populations by B chromosome presence or absence, we found significant molecular variance between populations  , with inFst  or using Dice's dissimilarity index . In order to investigate possible causes of absence of IBD despite the significant population genetic differentiation found, we used Barrier v 2.2 to detect possible barriers to gene flow. It detected a very strong barrier between the populations carrying B chromosomes  and the B-lacking ones , with another medium-strength barrier between these latter populations (No IBD was found either using Pairwise-ulations . This su24 chromosome around its place of origin (i.e. the Torrox population in the Spanish province of Málaga) [1 chromosome from the coast to upstream populations in the Segura River basin has been interrupted by strong barriers to gene flow thus explaining the absence of B chromosomes in headwaters' populations.We present here an analysis of B chromosome distribution across a broad area, including estimations of gene flow between populations, which has revealed the existence of strong genetic barriers that coincide with geographical accidents impeding natural migration . The most similar study is our analysis of the spread of the B Málaga)  but, in 1) is predominant in populations from the Iberian Peninsula, Balearic Islands, Morocco, Tunisia and Sicily, suggesting the recent spread of this B chromosome through Western Mediterranean areas [1 is the most widespread variant in the Iberian Peninsula, on which basis it is considered the original B variant [Fst values found between the B-carrying and B-lacking populations analyzed here. This is reinforced by our repeated witnessing of B chromosome invasions in this species, and by the absence of B chromosomes in the headwater areas of the Segura River basin. In the Torrox  population, the B24 chromosome completed its replacement of the B2 variant (from which it was presumably derived) in only a few years [1 chromosome arrived first to the south-west area of the island, and then it spread towards the north, with B chromosome prevalence increasing one order of magnitude in the Pollença population in only ten years [Our present results support the hypothesis by Cabrero et al.  suggestian areas . Bearing variant , we concew years . In the en years . Recentlet al. [E. plorans. Our present results, however, have failed to show IBD for the ISSR markers in inland populations, and genetic differentiation among the analysed populations was low (Fst = 0.08). In the aphid Schlechtendalia chinensis, Ren et al. [Fst = 0.239) but no IBD, and suggested that this might be due to genetic drift or the low spatial scale of the studied populations. Likewise, the moth Hyles tithymali showed scarce genetic differentiation at several of the Canary Islands presumably due to fast and recent colonization [Fst values found in E. plorans.In a study of B chromosome frequency along the Segura River, from coastal populations up to Calasparra, Perfectti et al.  observedn et al.  observednization , a fact E. plorans grasshoppers. Most frequently, rivers pass through narrow canyons with arid soils surrounding them, with absence of irrigated fields and hence of herbaceous vegetation for food. For instance, in the Segura River, B chromosomes are present up to the Cenajo reservoir . In the Northern tributary (Mundo River), however, B chromosomes have reached the Liétor locality (600 m AMSL) , suggestAlthough the strong barriers to gene flow found in the Segura River basin explains the absence of B chromosomes at localities upstream the barrier, we cannot rule out the possibility that B chromosome presence is impeded, at some degree, by natural selection due to a low tolerance for B chromosomes at high altitude environments. A conceivable pathway could be associated with the cell-cycle lengthening effect that B chromosomes have in other species  causing E. plorans were collected at five natural populations in the Segura river basin, on the East of the Iberian Peninsula , following manufacturer's recommendations. DNA quantification was performed in a TBS-380 minifluorometer (Turner Biosystems) using Picogreen dye .E. plorans. The analysis of ISSR allele frequencies was performed by Bayesian methods using the Hickory v1.1 software [f = 0 model because E. plorans is a polygynandric organism [ISSRs markers were amplified by polymerase chain reaction (PCR) using the primers shown in software , under torganism   with the program Arlequin v3.5  with 10,Population structure was also analysed with the Structure 2.3.1 software , which uFst values were calculated with the AFLP-surv software [Fst values between each pair of populations, with the matrix of geographical distances between populations  method.(DOC)Click here for additional data file.S1 TableThe two populations carrying B chromosomes are indicated by an asterisk.(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 TableThe two populations carrying B chromosomes are indicated by an asterisk.(DOC)Click here for additional data file.S4 TableThe two populations carrying B chromosomes are indicated by an asterisk.(DOC)Click here for additional data file.S5 TableThe two populations carrying B chromosomes are indicated by an asterisk.(DOC)Click here for additional data file.S6 TableThe two populations carrying B chromosomes are indicated by an asterisk. SE = Standard error.(DOC)Click here for additional data file.S7 TableThe two populations carrying B chromosomes are indicated by an asterisk.(DOC)Click here for additional data file.S8 Table(XLS)Click here for additional data file.


IA1 and IA2 genes in 12 golden pheasant populations across the Chinese mainland to characterize their genetic variation levels, to understand the effects of historical positive selection and recombination in shaping class I diversity, and to investigate the genetic structure of wild golden pheasant populations.The major histocompatibility complex (MHC) genes are vital partners in the acquired immune processes of vertebrates. MHC diversity may be directly associated with population resistance to infectious pathogens. Here, we screened for polymorphisms in exons 2 and 3 of the IA1 alleles in exon 2 (IA1-E2), 11 IA1-E3 alleles, 27 IA2-E2 alleles, and 28 IA2-E3 alleles. The non-synonymous substitution rate was significantly greater than the synonymous substitution rate at sequences in the IA2 gene encoding putative peptide-binding sites but not in the IA1 gene; we also found more positively selected sites in IA2 than in IA1. Frequent recombination events resulted in at least 9 recombinant IA2 alleles, in accordance with the intermingling pattern of the phylogenetic tree. Although some IA alleles are widely shared among studied populations, large variation occurs in the number of IA alleles across these populations. Allele frequency analysis across 2 IA loci showed low levels of genetic differentiation among populations on small geographic scales; however, significant genetic differentiation was observed between pheasants from the northern and southern regions of the Yangtze River. Both STRUCTURE analysis and F-statistic (FST) value comparison classified those populations into 2 major groups: the northern region of the Yangtze River (NYR) and the southern region of the Yangtze River (SYR).Among 339 individual pheasants, we identified 14 IA2 than IA1 indicate that IA2 has undergone much stronger positive-selection pressure during evolution. Moreover, the recombination events detected between the genes and the intermingled phylogenetic pattern indicate that interlocus recombination accounts for much of the allelic variation in IA2. Analysis of the population differentiation implied that homogenous balancing selection plays an important part in maintaining an even distribution of MHC variations. The natural barrier of the Yangtze River and heterogeneous balancing selection might help shape the NYR-SYR genetic structure in golden pheasants.More extensive polymorphisms in The online version of this article (doi:10.1186/s12862-016-0609-0) contains supplementary material, which is available to authorized users. The major histocompatibility complex (MHC) is a primary factor in initiating immune defenses, and it is composed of glycoproteins that are specialized to present foreign antigens to T lymphocytes . The MHCApart from their prominent role in immune responses, much attention has been paid to MHC genes because of their extensive polymorphism. In recent years, the patterns of genetic variability in MHC class I genes have been intensively investigated across different vertebrate taxa, including mammals , 5, birdGallus gallus, Phasianidae, Galliformes), the first representative of the “minimal essential” MHC, is highly condensed and dramatically simpler than the mammalian MHC, containing only 2 classical class I genes, BF1 and BF2 ), and the other 5 were located near the southern foothills of the QDM [SQDM]), whereas 4 populations were distributed within the SYR  are expressed. To comprehensively characterize variation in the highly variable regions (exons 2 and 3) in IA1 and IA2, we developed locus-specific screening methods based on knowledge of the MHC genomic structure in the golden pheasant .


Gasterosteus aculeatus) populations, little attention has been paid towards the temporal stability of allele frequency distributions, and whether there are consistent differences in effective size (eN) of local populations. We investigated temporal stability of genetic variability and differentiation in 15 microsatellite loci within and among eight collection sites of varying habitat type, surveyed twice over a six-year time period. In addition, eNs were estimated with the expectation that they would be lowest in isolated ponds, intermediate in larger lakes and largest in open marine sites. In spite of the marked differences in genetic variability and differentiation among the study sites, the temporal differences in allele frequencies, as well as measures of genetic diversity and differentiation, were negligible. Accordingly, the eN estimates were temporally stable, but tended to be lower in ponds than in lake or marine habitats. Hence, we conclude that allele frequencies in putatively neutral markers in three-spined sticklebacks seem to be temporally stable – at least over periods of few generations – across a wide range of habitat types differing markedly in levels of genetic variability, effective population size and gene flow.Temporal variation in allele frequencies, whether caused by deterministic or stochastic forces, can inform us about interesting demographic and evolutionary phenomena occurring in wild populations. In spite of the continued surge of interest in the genetics of three-spined stickleback ( The study of evolution is ultimately about the study of changes in allele frequencies within populations over time. Allele frequencies in a given locus can change either due to deterministic (e.g. selection) or stochastic  reasons . In popuDuring the past decade, an increasing number of studies have investigated temporal changes in allele frequencies and population genetic parameters in the wild, both over long  and Ne; reviews in: )Most population genetic studies employ allochronic approaches to assess shifts in allele frequencies, with the underlying assumption of temporal genetic stability. Our results demonstrating short-term stability of genetic parameters in multiple three-spined stickleback populations across a range of habitat types validate this assumption for this species. This finding may not be surprising considering the relatively short time period—roughly two to three stickleback generations—between sampling events. The probability of detecting significant allele frequency differences when samples are collected few years apart may be low (e.g. ). MoreovNe; [Ne between the pond and other populations was too small to generate noticeable allele frequency shifts over the short time span encompassed in our study . TherefNe; ,78. In aNe; ,35,36 foudy (cf. ). For exproblems ,84.Demonstrating temporal stability in the patterns and degree of population structure is a powerful way of confirming the reliability of observed spatial genetic patterns. For example, Tessier and Bernatchez  sampled Although differential allele frequency changes are expected in populations subject to different levels of gene flow , the smaST) among the marine sites SIK and KOT, as well as between KOT and TVA, were significant in this but not in a previous study [ST-estimates across the two studies is high .Finally, we note that the results of this study in respect to the degree and patterns of genetic differentiation among Baltic and North Sea populations are largely in agreement with earlier studies conducted across these sites ,32,36,94Ne estimates were most precise when small. This was most often seen with all methods in the pond populations, and with the single-sample sibship analysis method in all populations. However, there is likely little probability of sampling sibs in our scheme, since sibs are unlikely to be present among breeders [Ne [Ne estimates in the sea and lake populations obtained with most other methods included infinity, which is likely to be a reflection of weak genetic signal relative to sampling noise: the precision of Ne estimates declines as Ne increases [Although the assumptions associated with estimating effective population size are likely violated in many studies , both tebreeders , at leasers [Ne , and henncreases . HoweverNe of the sea populations were lower than expected, as those of many marine fishes are at least an order of magnitude higher . ,98. Ne er . Likne sites . We trieoo e.g. ,100,101),101Ne ostimates , but corstimates ) which aNe estimates for sea and lake populations of three-spined stickleback populations are puzzling given that census population sizes (Nc), even in small ponds, can be in the range of thousands , and thds e.g. ,71). Hen. HenNe ulations . Althougulations ,22, we aize (cf. ). It is Ne-values, the sea and lake populations appear to have substantially higher (and likely underestimated) Nes than the pond populations: for most estimators, both the lower and upper confidence intervals, as well as the point estimates, were higher for sea and lake populations than for pond populations. In spite of the uncertainties involved with Ne estimates, the temporal changes in allele frequencies in all habitat types were small over the six year study interval, and hence, from the point of view estimation of genetic population structure, negligible.As pointed out by Palstra and Ruzzante , the posNe estimates were most precise. Future studies utilizing more widely separated temporal cohort-based samples could be informative in gaining better estimates of effective size of contemporary populations .S1 Table(XLSX)Click here for additional data file.


A polymorphic inversion that lies on chromosome 17q21 comprises two major haplotype families (H1 and H2) that not only differ in orientation but also in copy-number. Although the processes driving the spread of the inversion-associated lineage (H2) in humans remain unclear, a selective advantage has been proposed for one of its subtypes. Here, we genotyped a large panel of individuals from previously overlooked populations using a custom array with a unique panel of H2-specific single nucleotide polymorphisms and found a patchy distribution of H2 haplotypes in Africa, with North Africans displaying a higher frequency of inverted subtypes, when compared with Sub-Saharan groups. Interestingly, North African H2s were found to be closer to “non-African” chromosomes further supporting that these populations may have diverged more recently from groups outside Africa. Our results uncovered higher diversity within the H2 family than previously described, weakening the hypothesis of a strong selective sweep on all inverted chromosomes and suggesting a rather complex evolutionary history at this locus. In contrast to early predictions from classical cytogenetic studies, a particular subtype of balanced rearrangement—chromosomal inversion—has been recently recognized as a common source of genomic variation . A strikKANSL1 gene, and were initially thought to be restricted to European populations , the “derived” haplotypes , enriched in duplicated copies, are found at much higher frequencies in present-day non-African populations . As a coNevertheless, the processes driving the spread of the duplication-specific haplotypes in the human lineage remain unclear, as the observed patterns could also be the result of the complex demographic history of populations, without the need of invoking selection .In this study, we propose to further explore the evolutionary history of the 17q21 region by focusing on the inversion-associated haplotype family . By extending sampling efforts to previously overlooked populations, we characterized the distribution of both H2 subtypes—H2′ and H2D—in a wide panel of individuals, targeting specific geographical groups, to better understand the evolutionary processes that have shaped the 17q21 rearrangement. Moreover, the diversity patterns within the H2 lineage were re-analyzed using a unique panel of H2-specific single nucleotide polymorphisms (SNPs), derived from publicly available resequencing data and explicitly selected for this purpose. Finally, departures of nucleotide variability patterns from neutral expectations were tested using simulated data together with full-sequence information for both major haplotype families .-inv and attempts to clarify important aspects of the evolution of this genomic region.Overall, this work provides a finer picture of the distribution and the genetic diversity of the 17q21n = 137) were retrieved from a recently published database (n = 100). Publicly available genotypes from a diverse population panel were obtained from the Stanford Human Genome Diversity Project (n = 373). Algerian (n = 52) and Portuguese (n = 49) population SNP data were generated at LC laboratory at IGC and at AA laboratory at IPATIMUP, respectively.Genotype data from several North African and European samples  from distinct populations—CEU, FIN, GBR, TSI, IBS, GWD, YRI, MSL, ESN, LWK, BEB, GIH, ITU, PJL, and STU—were retrieved from the 1000 Genomes Project ftp (Phase III release).Also, genotype data of individuals (High coverage resequencing data of two H2 homozygous from Eastern Africa (NA21599)  and Centhttp://www.ccr.coriell.org/, last accessed November 26, 2015).Lymphoblast cell lines from five Hapmap individuals were purchased from Coriell Cell Repository . Given that no incongruence was found between the FISH-determined orientation and the haplotype sequence . Similarly, the duplication status of the H2-chromosomes was assessed mainly using two duplication-markers reported in supplementary table S2, Supplementary Material online). Nevertheless, whenever resequenced data were available , the presence/absence of the duplication was further confirmed using the HMMCopy software .sequence , a set osequence  were usesoftware  . Haplotype frequencies were then estimated for each geographically defined group. Note that we do not wish to give too strong a meaning to the geographical groupings above and, instead, see them as practical units devised to achieve sufficient sample sizes for the estimation of population summary statistics.Once the orientation and duplication statuses were confirmed, each individual was grouped according to geographical origin, and six major continental groups were thus defined, Sub-Saharan Africa, North Africa, Middle East, South Asia, Central Europe, and South Europe  an H2 homozygous from Eastern Africa (NA21599)  and 2) asupplementary table S3, Supplementary Material online), using the GATK-UG . The remaining SNPs (n = 136) were then genotyped in all H2 carriers, for which DNA was available, using SEQUENOM platform . Moreover, 17 inversion- and duplication-tagging SNPs were added to the assay to check for genotype consistency. The quality of the obtained genotypes was then verified using Plink software (n = 79) were found to be monomorphic, these variants were removed for the remaining of the study as they more likely represent false positives or singletons . Thus, a final list of 74 SNPs were kept for subsequent analysis (genotype rate: 0.998).Afterwards, the two variant sets were merged and a total of 203 previously unidentified SNPs were found to be polymorphic in at least one individual from the list. Due to the highly repetitive architecture of the 17q21 region, the SNP set was further pruned to avoid variants located within known segmental duplications . Phased haplotypes were finally sorted according to the three possible statuses  and grouped by continental origin. Nucleotide diversity estimates were measured for each haplotype using VCFdivstat (v1.0). Genetic differentiation estimates were assessed between all continental pairs using adegenet R package  the full set of H2 homozygous samples available (N = 32), and (**) a subset of randomly selected H1 homozygous samples (N = 150) . To assess the statistical significance of Tajima’s D, genetic data were then simulated under neutrality using invertFREGENE software . The inversion will then be transmitted to subsequent generations until a target frequency is reached. Here, the target frequency was set to 0.337—highest known frequency of the 17q21-inv. Afterwards, Tajima’s D scores were estimated from the simulated data to generate null distributions for both inverted and noninverted chromosomal types.Given that our SNP panel was primarily designed to specifically capture H2 intrahaplotypic variation, and considering the heavy effects of SNP ascertainment bias in standard genetic methods of analysis, full sequence information from the 1000 Genomes Project was next used to estimate summary statistics of population genetic variation for each major haplotype family—H1 and H2. Using the ANGSD software , nucleotsoftware  for a toB and C, supplementary material, Supplementary Material online), the orientation and duplication statuses at 17q21 were assessed using genotype data from 2,120 individuals belonging to several present-day populations (fig. 1D), with cumulative frequencies varying between 0.7% in Sub-Saharan African populations and 33.7% in Southern Europeans. Contrasting with previous estimates  could be reliable inferred from previously described single nucleotide markers   to further refine the diversity patterns within the 17q21 inversion-associated haplotypes. Our results uncovered appreciable levels of haplotype diversity for both inverted subtypes . These rFst estimates between geographical groups within each structural subtype. The results are illustrated in Fst < 0.20), Sub-Saharan African H2′ chromosomes are substantially different from the ones in the remaining geographical groups. Interestingly, the highest levels of genetic differentiation were observed between Sub-Saharan and North African/Middle Eastern populations, where Fst scores exceeded 0.5.Afterwards, genetic differentiation was assessed through pairwise It is important to note that the analyses performed so far mainly rely on allele frequency information to determine whether the two inversion-associated subhaplotypes display distinct patterns of diversity and differentiation between present-day meta-population groups. Although these measures would not be affected by minor subhaplotypic phasing errors , the same would not apply when considering phylogenetic approaches, where possible errors in the phasing step would likely result in inaccurate inference of evolutionary relationships between chromosomes.To circumvent this limitation, we performed a phylogenetic analysis including only heterokaryotype individuals . This approach allows excluding from the analyses incorrectly phased chromosomes, which could be confounded with genetic recombinants. The phylogenetic tree obtained with the 17q21 haplotypes derived from heterokaryotypes is thus shown in As expected, all chromosomes belonging to the H1 lineage cluster together in a single branch, regardless of the continental origin, clearly separated from the H2 haplotypes. Interestingly, the first split corresponds to Sub-Saharan African H2′ copies whereas the second split incorporates the remaining H2′ chromosomes and all H2D copies. This suggests that the H2D evolved from an H2′ copy (or set of copies). Moreover, although there is not enough phylogenetic information to be more precise at this stage, the lack of population stratification between (or within) subtypes suggests that recombination events between inverted copies have likely occurred throughout the evolution of the 17q21 region.D tends to be negative throughout the inversion region.As stated above, another important issue regarding the 17q21 locus is whether selection has played a major role in its evolutionary history. Although a variety of statistical tests have been devised to detect selection from SNP genotype data , one impD.We simulated neutral inversions in population genetic data to generate theoretical null distributions for both chromosomal types  and founinv by extending previous efforts to uncharacterized populations, in order to better understand the processes that have shaped the genetic and architectural diversity of this genomic region. In agreement with recent surveys  the demographic model simulated is fairly simplistic, and 2) selection cannot be robustly detected using information from a single summary statistic. Also, the fact that the observed data fall tangentially outside the neutral distribution for both orientations suggests that demography may have a stronger contribution than selection to the observed departure. KANSL1 produced a similar phenotype in both backgrounds that could be under selection in modern humans and thus, contrary to previous suggestions , with the H1 haplotype showing much higher levels of polymorphism than H2 chromosomes. Moreover, both chromosomal types displayed a negative trend for Tajima’s gestions , selectiIn conclusion, while increasing the known distribution of the inversion associated haplotypes to previously uncharacterized geographical regions, our work showed that the 17q21 H2-family is genetically more diverse and differentiated than initially appreciated . MoreoveSupplementary material, figures S1–S3, and tables S1–S4 are available at Genome Biology and Evolution online .


Simulium tani was inferred from mitochondria-encoded sequences of cytochrome c oxidase subunits I (COI) and II (COII) along an elevational gradient in Cameron Highlands, Malaysia. A statistical parsimony network of 71 individuals revealed 71 haplotypes in the COI gene and 43 haplotypes in the COII gene; the concatenated sequences of the COI and COII genes revealed 71 haplotypes. High levels of genetic diversity but low levels of genetic differentiation were observed among populations of S. tani at five elevations. The degree of genetic diversity, however, was not in accordance with an altitudinal gradient, and a Mantel test indicated that elevation did not have a limiting effect on gene flow. No ancestral haplotype of S. tani was found among the populations. Pupae with unique structural characters at the highest elevation showed a tendency to form their own haplotype cluster, as revealed by the COII gene. Tajima’s D, Fu’s Fs, and mismatch distribution tests revealed population expansion of S. tani in Cameron Highlands. A strong correlation was found between nucleotide diversity and the levels of dissolved oxygen in the streams where S. tani was collected.The population genetic structure of  A wealth of biological diversity is associated with the broad range of abiotic and biotic factors typically inherent in altitudinal variation Black flies are ubiquitous inhabitants of streams and rivers over a wide range of elevations throughout the world. They are hypothesized to have originated in cool, mountainous areas Simulium tani Takaoka & Davies, with 11 known cytoforms S. tani from different geographical regions of Thailand S. tani is in Malaysia.The most cytogenetically diverse black fly in the Oriental Region is S. tani was found at different frequencies at each elevation. Simulium tani was predominantly found at 0–300 m, 601–900 m and 1201–1500 m, but less prevalent at 301–600 m and 901–1200 m . Pupae of S. tani at the highest elevation had unique structural characters (nearly smooth frons and slender gill filaments), whereas those at lower elevations had the typical condition (moderately tuberculate frons and thicker gill filaments). The pupal characters of the high-elevation population were associated with unique cytogenetic features, suggesting some genetic differentiation, although only a single cytoform (‘K’) is recognized in Cameron Highlands In Cameron Highlands, Malaysia, a monthly survey of black flies at five different elevations (0–1500 m above sea level) showed that S. tani and evaluate the degree of genetic differentiation along an elevational gradient, based on mitochondria-encoded COI and COII genes. Selected environmental factors associated with the population genetic structure of S. tani also were investigated. This fine-scale population genetic study complements the broad-scale study by Pramual et al. S. tani.Given this morphological and chromosomal variation, the current study aimed to determine the intraspecific genetic diversity of No national permissions were required for this study, which did not involve endangered or protected species. No specific permissions were required to access the study sites; the collections were made on public lands.S. tani were found every month at Elevations A, C and E; in February–April, June, July, October, and December at Elevation B; and only in February and December at Elevation D. Pupae were removed by hand from in-stream vegetation, placed in moistened 8-ml clear plastic tubes, and held in a cooler to allow emergence of adults, which then were identified using taxonomic keys Black fly pupae were sampled monthly in 2012 from five different elevations (A–E) in Cameron Highlands, Malaysia: A = 0–300 m, B = 301–600 m, C = 601–900 m, D = 901–1200 m, and E = 1201–1500 m . Pupae oThe following stream characteristics were measured at each of the five collection sites during each of the 12 monthly visits: temperature, width, depth, current velocity, conductivity, dissolved oxygen, and pH .S. tani was isolated successfully from all reared adult specimens subjected to analysis (n = 71), using the i-genomic CTB DNA Extraction Mini Kit . All extraction steps were performed according to the manufacturer’s instructions.DNA of S. tani from different elevations in Cameron Highlands, 71 individuals were subjected to mitochondrial COI and COII amplifications. For comparison, amplifications of the nuclear encoded 18S ribosomal RNA (18S) and the 28S ribosomal RNA (28S) genes of S. tani were performed using a subset of 20 representative individuals. Preliminary data revealed no polymorphic sites in the 18S or 28S sequences of S. tani from different elevations, whereas COI and COII sequences were more valuable in resolving intraspecific relationships. Therefore, meaningful comparisons were made by using COI and COII sequences as the molecular markers.To resolve the phylogeographical and phylogenetic relationships of Amplifications of the COI, COII, 18S, and 28S genes were performed in a final volume of 50 µL containing 0.5–1.0 µg genomic DNA, 25 µL of ExPrime Taq Master Mix , and 10 pmol of each forward and reverse primer. Details of the polymerase chain reaction (PCR) primers are summarized in The amplified fragments were electrophoresed on 2% agarose gels pre-stained with SYBR Safe  The PCR products were purified with MEGAquick-spin PCR & Agarose Gel DNA Extraction System . Purified PCR products were sent to a commercial company for DNA sequencing in both directions. Samples were sequenced using BigDyeH Terminator 3.1 Sequencing Kit and analyzed using an ABI PRISM 377 Genetic Analyzer .S. tani were deposited in the NCBI GenBank. The COII and 18S sequences of S. tani were generated for the first time in this study, filling a void in the GenBank database.Sequencing data were analyzed and edited using ChromasPro 1.7.6  and BioEdit 7.0.9.0 S. tani were analyzed using a median-joining algorithm The genetic diversity or haplotype networks of S. tani in both COI and COII genes, uncorrected (p) pairwise genetic distances were calculated using PAUP 4.0B10 To access the genetic divergence of ST), and gene flow (Nm) values were obtained from these tests. The levels of genetic differentiation can be categorized as STF >0.25 (great differentiation), 0.15 to 0.25 (moderate differentiation), and STF <0.05 (negligible differentiation) 2 statistic of Ramos-Onsins and Rozas http://www.uni-graz.at/zoowww/mismatchcalc/. The expansion time was estimated based on the assumption of 12 generations a year for tropical black flies To determine the level of genetic differentiation among populations, gene flow and genetic differentiation tests were performed with the program DnaSP 5.0 STF) and elevation difference (m) were examined using Mantel tests The associations between genetic distance  and the population with structurally unique pupae at Elevation E (black) showed a slight tendency to form different clusters  than did COII (0.00–1.17%) or COI+COII (0.13–1.32%) for genetic distance , 4.S. tani was based on nucleotide diversity. The COI, COII, and COI+COII sequences produced consistent results, with the highest level of nucleotide diversity at Elevation B and the least diversity at Elevation D. Generally, the levels of nucleotide diversity in descending order were as follows: Elevation B (301–600 m)>Elevation E (1201–1500 m)>Elevation A (0–300 m)>Elevation C (601–900 m)>Elevation D (901–1200 m).In total data estimates, the COI gene revealed higher haplotype diversity (1.000) and nucleotide diversity (0.0089) than did the COII gene, which had 0.9602 for haplotype diversity and 0.0045 for nucleotide diversity . The COISTF <0.05, indicating low differentiation. Moderate differentiation (STF >0.15), evidenced by the COI+COII genes, was found between Elevations A and D. Overall, however, no significant differentiation was observed among populations (P>0.05). High levels of gene flow occurred among populations, as evidenced by the high value of Nm 8.38 in the COI+COII genes  and R2 statistic (P<0.05) from mismatch distribution tests (P<0.05) , 370,000 (COII) and 270,000 (COI+COII) years ago.The median-joining network of COI, COII, and COI+COII revealed “star-like” networks, suggesting population expansion. The unimodal mistmatch distributions, low values of the Raggedness index  , furtherSTF) and elevation (m), suggesting that isolation by distance did not have a limiting effect on gene flow. Spearman rank-order correlation revealed that the nucleotide diversity (COI and COI+COII genes) of S. tani was positively correlated with the levels of dissolved oxygen in the stream water  (P>0.05) was found for the other six stream variables .A Mantel test revealed no significant association between genetic distance (= 0.037) . No signS. tani in Cameron Highlands, with 71 unique haplotypes among 71 examined individuals that did not share the same ancestral haplotype. The high level of genetic variation in S. tani was corroborated by the high degree of haplotype diversity (1.0000) and nucleotide diversity (0.0058–0.0099) and by the uncorrected “p” distance matrix (0.12–1.81%), based on the COI gene. The wide-scale study by Pramual et al. S. tani in Thailand, with 121 unique haplotypes from 147 individuals and haplotype diversity and nucleotide diversity of 0.8570–1.0000 and 0.0030–0.0155, respectively. Based on haplotype frequencies, the genetic diversity of S. tani in the present study was greater than that previously described in Thailand for S. tani (by 1.21 fold), S. feuerborni Edwards (2.03 fold), and S. siamense Takaoka & Suzuki (3.54 fold) S. tani occurring in the mid Pleistocene agreed with the previous findings in Thailand S. tani has older expansion  than Malaysian S. tani .The median-joining network demonstrated a high level of genetic diversity for populations of Simulium gravelyi Puri in India revealed greater genetic diversity at high elevations (>1500 m) than at low elevations (<300 m) A fine-scale study of S. tani in Cameron Highlands cannot be excluded. Over the years, pristine montane forests in Cameron Highlands have been cleared for intensive agricultural activities Moreover, the role of anthropogenic disturbances in structuring the current genetic variability in S. tani in Cameron Highlands. The monthly sampling data indicated low numbers of S. tani at Elevations B and D but high numbers at Elevations A (low elevation), C (mid-elevation) and E (high elevation). Additional sampling at higher elevations (>1600 m) throughout the year did not reveal S. tani (unpublished data). The relatively small samples from Elevations B and D in 2012 and 2013 suggested that these two sites might support temporary, rather than self-sustaining, populations. Although the sample sizes for Elevations B and D were not ideal for measuring the haplotype frequencies in total data estimates, the haplotype diversity and nucleotide diversity revealed by the COI and COII data were not influenced by the total number of individuals along the elevational gradient. In addition, a data set excluding the specimens from Elevations B and D was evaluated; the results were in concordance with the five-elevation data set, indicating low genetic differentiation and high gene flow in S. tani (results not shown).A population model for mosquitoes S. tani in Cameron Highlands. Isolation by distance did not have a limiting effect on gene flow; no significant correlation was found between genetic distance and elevation. Hence, elevation is unlikely to be a physical barrier to gene flow for S. tani. The weakly formed haplotype cluster (COII gene) for the population of S. tani with distinct pupal features reflects the similarly weak, but distinct, cytogenetic differentiation previously reported S. tani in Cameron Highlands. The molecular results, thus, support the conclusion from cytogenetic data that a single taxonomic entity (Cytoform ‘K’) exists in Cameron Highlands S. tani across its range; previous studies S. tani only within the elevational range (0–1500 m) examined in Cameron Highlands. In Thailand, where high levels of genetic differentiation at both chromosomal and molecular scales have been found across regions, the lowlands and Gulf of Thailand have been suggested as physical barriers to gene flow Previous studies have revealed the importance of elevation as a barrier to gene flow for black flies S. tani across an elevational gradient. Congruent results from cytogenetic, morphological, and molecular analyses indicating slight differences in the high-elevation population of S. tani suggest that applying all three analytical approaches provide a powerful means of unfolding the biodiversity hidden at multiple scales in black flies.In conclusion, the present study–the first molecular phylogeographical study of Malaysian black flies–revealed high levels of genetic diversity but low levels of genetic differentiation in


Apis mellifera carnica) are considered as an indigenous subspecies in Hungary adapted to most of the ecological and climatic conditions in this area. However, during the last decades Hungarian beekeepers have recognized morphological signs of the Italian honey bee (Apis mellifera ligustica). As the natural distribution of the honey bee subspecies can be affected by the importation of honey bee queens or by natural gene flow, we aimed at determining the genetic structure and characteristics of the local honey bee population using molecular markers. All together, 48 Hungarian and 84 foreign  pupae and/or workers were used for mitochondrial DNA analysis. Additionally, 53 sequences corresponding to 10 subspecies and the Buckfast hybrid were downloaded from GenBank. For the nuclear analysis, 236 Hungarian and 106 foreign honey bees were genotyped using nine microsatellites. Heterozygosity values, population‐specific alleles, FST values, principal coordinate analysis, assignment tests, structure analysis, and dendrograms were calculated. Haplotype and nucleotide diversity values showed moderate values. We found that one haplotype (H9) was dominant in Hungary. The presence of the black honey bee (Apis mellifera mellifera) was negligible, but a few individuals resembling other subspecies were identified. We proved that the Hungarian honey bee population is nearly homogeneous but also demonstrated introgression from the foreign subspecies. Both mitochondrial DNA and microsatellite analyses corroborated the observations of the beekeepers. Molecular analyses suggested that Carniolan honey bee in Hungary is slightly affected by Italian and black honey bee introgression. Genetic differences were detected between Polish and Hungarian Carniolan honey bee populations, suggesting the existence of at least two different gene pools within A. m. carnica.Carniolan honey bees ( Honey bees provide an important pollination services in commercial crops and in many natural habitats worldwide  was first determined based on morphometric parameters , is native to Hungary, Slovenia, and some regions of the former Yugoslavia, Romania, Bulgaria, and southern Austria (Ruttner The honey bees of lineage (C) are variable in behavior and color and in addition adapted to various climatic zones from Mediterranean climate to colder mountains of the Balkans and Central Europe Ruttner . The Car Ruttner . Lately, Ruttner . Ruttner Ruttner  describe Ruttner  through The adaptation of honeybees to their local environment has not been well studied . Five haplotypes were initially described within the C lineage: C1 in A. m. ligustica, C2a in A. m. carnica, C2b in A. m. caucasica  analysis has become a widely used approach in studying the genetic diversity among populations because of its conserved gene content, high level of nucleotide substitutions, and maternal inheritance. The most widely used marker was the intergenic region between the cytochrome oxidase I and II (cox1–cox2) genes in A. m. mellifera populations from Norway, Sweden, Denmark, England, Scotland, and Ireland were checked for introgression  populations in Polish Augustów Forest derived from nonnative bees.Microsatellites are biparentally inherited markers and give useful information about population events such as introgression and hybridization through mating between foreign drones and local queens  at 16 different locations. Additional populations located in Italy (A. m. ligustica), Liberia (A. m. adansonii), Spain (A. m. iberiensis), Poland (A. m. mellifera/carnica), and the Buckfast line from Hungary were used for comparison  from Hungary.One honey bee worker pupa per colony was used for mtDNA analysis  (N = 240). All used samples are presented in Tables Three honey bee pupae per colony were used for microsatellite analysis from Hungary N = 40. All uA. m. ligustica complete mitochondrial genome (Acc. no.: L06178.1) , 3 mmol/L MgCl2 (Applied Biosystem), 10× reaction buffer , 0.75 U Taq polymerase (Applied Biosystem), and 20 ng/μL of template. The amplification cycle consisted of an initial denaturation step of 10 min at 95°C, followed by 35 cycles of 15 sec at 95°C, 30 sec at 63°C, and 30 sec at 73°C, followed by a final extension step of 25 min at 73°C. PCR products were purified using a Clean‐Up DNA fragment purification kit  and sequenced by the Eurofins MWG Operon Company .The cox1 intergenic region was PCR‐amplified with the newly designed (due to fail in PCR amplification with the commonly used primers) forward (5′‐CTGATATAGCATTCCCCCGAATA‐3′) and reverse (5′‐AGAATTGGATCTCCACGTCCTA‐3′) primers. These primers were designed from 2056 to 2401 nucleotide position detected in the Apis cerana as an out‐group (Acc. no.: DQ020237.1) .Each sequence obtained was manually checked and aligned with the published sequences for comparison using ClustalX program , 4.3 mmol/L MgCl2 , 5× reaction buffer , 0.8 U Taq polymerase , and 20 ng/μL of extracted DNA. The amplification cycle consisted of an initial denaturation step of 2 min at 94°C, followed by 35 cycles of 30 sec at 94°C, 30 sec at 55°C (A (B) 24, A43), 56°C , 57°C (A35), 58°C , 60°C (A113), and 30 sec at 72°C, followed by a final extension step of 10 min at 72°C.Nine polymorphic microsatellite loci A7, A113, A107, A28, A88, A14, A35, A(B)24  (Nei Alleles were subsequently scored using PeakScanner version 1.0 software (Applied Biosystem). Population genetic parameters were calculated with GenAlEx 6.4  Nei  were cal Ds) Nei .Principal coordinate analysis (PCoA) and assignment test  iterations. Ten runs for each K value (2 ≤ K ≤ 10) were used, and the number of populations was reasoned from the value of ∆K as described in Evanno et al. . In 11 cases, the nucleotide exchanges were transitions, while in three cases, transversions , and in one case either a transition (G/A) or a transversion (G/C) took place in position 2169.The sequence dataset sized 345 base pairs with 329 conserved and 15 variable positions from 180 Seventeen different haplotypes H1–17) were detected (GenBank accession numbers: under submission). Seven haplotypes have been characterized in the Hungarian population . The H9 is at high frequency in central European localities and is increasing in frequency toward the south. The ratio of H9 at Bialowieza (77.5%), Siedlce (80%), and Krakow (60%) line is relatively high, but from Augustów Forest (10%) to Wroclaw (20%) line is very low in Poland. The H8, H11, and H12 in Hungary; H4, H13, and H14 in Spain; and H5, H6, and H7 in Poland were detected at first. H15 was found only in Liberia  was also found in the black honey bee  were detected in Hungary, three  in Poland, and other three in Spain . As expected, the Spanish and Liberian (H15) haplotypes were well differentiated from the Hungarian haplotypes , thus varied between 0.634  and 0.846 (Liberia). Honey bee populations from Hungary deviated significantly from the Hardy–Weinberg equilibrium (P < 0.05). The Fis values of the ten groups varied between −0.104  and −0.460 , thus reflecting a heterozygote excess within all the populations with negative or close to zero values.Overall parameters of the ten investigated populations and the Fst genetic distance among individual samples. The results of principal coordinate analysis showed African (AF) and Spanish (SP) populations were well separated, while the Hungarian population (HU) appeared to be separated into two. On the other hand, Polish A. m. mellifera populations (Augustów Forest and Wroclaw) clustered separately from A. m. carnica subspecies from Krakow, Bialowieza, and Siedlce  was performed to investigate population patterns based on the lce Fig. .K = 2). This is supported by the A. m. iberica (Spain) and A. m. mellifera (Augustów) subspecies belong to the common M lineage. When the model assumed four populations (K = 4), the clustering revealed Buckfast and Italian populations separated from the other stock. The clustering together with the aforementioned two populations on the phylogenetic tree is also visible. Finally, K = 6 alignment the A. m. adansonii (Liberia) subspecies separation was seen by the A. m. iberica and A. m. mellifera (Poland‐Augustów) subspecies. The results showed the best K value after Evano correction (K = 2) (not shown).Cluster analysis of the honey bee populations Fig.  identifiFst values and the Nei's corrected standard genetic divergence (Ds) among the ten groups. Multilocus Fst values varied between −0.001  and 0.291 (Hungary and Spain). Not surprisingly, the Liberian (0.216) and Spanish (0.291) populations were significantly distinct from the Hungarian population. Honey bees from Poland, which lie in the natural distribution range of A. m. mellifera, showed differentiated admixture of Carniolan bees. According to the examined microsatellite markers, the Polish Augustów Forest (0.167) and Wroclaw (0.136) populations showed strong divergence values from the Hungarian populations, stronger than the Bialowieza (0.084), Krakow (0.029), and Siedlce (0.045) populations. A genetic barrier has been identified among these populations, which is validated by the results of the PCoA at the population level  (2.5%), and the black bee from Poland (A. m. mellifera) (2.2%). We confirmed that the Liberian individuals belong to the A. m. adansonii subspecies.Assignment tests to determine the breed of origin of individuals were performed. Only one individual from Wroclaw could be assigned to the es Table . Based oIn Central Europe, the haplotype 9 was the most frequent haplotype and its frequency decreased to the north. With the help of mtDNA and nine polymorph microsatellite markers, we proved that the Hungarian honey bee populations are nearly homogeneous. We identified heterozygosity in the domestic Carniolan Pannonian bees; therefore, inbreeding is not typical. In conclusion, the apicultural practices in the Hungarian honey bee colonies were appropriate for the conservation of indigenous honey bees. The results of this research provide new knowledge about genetic variability and useful information for conservation proposes by developing and supporting breeding programs. As it is well known, the health of honey bee colonies cannot be understood without considering the genetic diversity, and the locally adapted bees survived better than introduced bees.A. m. ligustica). Migratory beekeeping might explain weak introgression, very high excess of heterozygotes, and other rare haplo‐ and genotypes detected in the Hungarian population. In spite of this relative homogeneity, populations have developed one ecotype that showed relatively high heterozygosity and can survive in our climates, all features that are indicative of high evolutionary potential for local adaptation.The Hungarian population has been isolated geographically by the Carpathians Mountains northward, probably giving rise to the endemic honey bee population during the last glaciation .Microsatellite analysis is a suitable diagnostic method for confirming the origin of subspecies . The population structure and genetic diversity of native Carniolan subspecies in Slovakia (He = 0.705)  in Hungary were also at a similar level as the African Guinean (A. m. adansonii) population (Ho = 0.861)  and wild bees (Apoidae)   population. Our data comparison differs in the results of western European populations because the number of heterozygosity of microsatellite alleles was reduced  than Dall'Olio et al.  for the z et al.  could acFst values and Nei's corrected standard genetic divergence values (Ds) revealed the strong differentiation among Liberian, Spanish, and Hungarian populations. This suggested that the geographic distance was an impediment to the gene flow among colonies. The Hungarian Carniolan honey bee population showed slight distance values from the black honey bee and Italian subspecies. Dall'Olio et al. , although a small‐scale gene flow was observed. It was established that the presence of the black honey bee in Hungary was negligible. De la Rúa et al. (A. m. carnica populations still exist in Croatia, Serbia, and Slovenia. Because of the small‐scale indigenous gene affect, the Hungarian populations also reckon among these countries.The assignment test also revealed the presence of non‐a et al.  mentioneA. m. carnica and A. m. caucasica and widely propagated them in Poland. The native honey bee populations, such as A. m. mellifera, has been replaced by A. m. carnica in several regions of Central Europe, which may be ascribed to insufficient mating control (Kotthoff et al. Based on the results, the Polish population was considerably heterogeneous. Oleksa et al.  confirmeWe suggest using instrumental insemination with sufficiently examined sperm donors throughout Hungary, which is important in preventing introgression and hybridization. Furthermore, it is important to assess the prevalence of the H9 from neighboring regions. We hope that our results may provide additional important novel evidences for the conservation of the native Carniolan honey bee populations in Central and eastern Europe. Data about mitochondrial and microsatellite DNA polymorphism in native Hungarian honey bees were reported here for the first time, and the two molecular tools showed near‐concordant result.None declared.Figure S1. Neighbour‐joining tree using Nei genetic distance.Click here for additional data file.


Eunicella cavolini, along with insights into the demographic history of this species. We sampled populations in four areas of the Mediterranean Sea: continental France, Algeria, Turkey, and the Balearic and Corsica islands. Along French coasts, three sites were sampled at two depths (20 and 40 m). We demonstrated a high genetic structure in this species , and most pairwise differentiation tests were significant. We did not detect any difference between depths at the same site. Clustering analyses revealed four differentiated groups corresponding to the main geographical areas. The levels of allelic richness and heterozygosity were significantly different between regions, with highest diversity in Algeria and lowest levels in Turkey. The highest levels of private allelic richness were observed in Algeria followed by Turkey. Such contrasted patterns of genetic diversity were not observed in other Mediterranean octocorals and could be the result of different evolutionary histories. We also provide new empirical evidence of contrasting results between tests and model‐based studies of demographic history. Our results have important consequences for the management of this species.Understanding the factors explaining the observed patterns of genetic diversity is an important question in evolutionary biology. We provide the first data on the genetic structure of a Mediterranean octocoral, the yellow gorgonian  E. cavoE. cavolini in different parts of the Mediterranean Sea. First, we will describe the genetic structure of this species at different spatial scales. We include a comparison between depths to test the differentiation along an environmental gradient. We will then test whether populations from different geographical areas present the same levels of diversity and similar demographic histories. We will study past demographic events with tests of mutation–drift equilibrium and with estimates of current and past effective sizes. These results will be useful for the management of this ecologically important species  and southwestern Mediterranean (Algeria), Aegean Sea, and Marmara Sea  following the manufacturer's instructions or a salting‐out procedure  and unbiased expected heterozygosities (Hexp). Linkage disequilibrium was tested among all pairs of loci with a permutation test (n = 1000). GENETIX was used to compute single and multilocus FIS on the basis of the estimator of Weir and Cockerham  on one population: We used either the same distributions for Nanc and Ncurr, or distributions indicating either reduction or expansion of populations. As this led to similar results indicating population decline (data not shown), we focused for the main analyses on an approach without a priori, with the same distributions for Nanc and Ncurr. Considering computation time, we analyzed separately a subset of two samples per area: KIA/DDC (pooled) and SPI for Algeria; MJS and RIS for France; SIV and AYV for Turkey; and MEN and REV for the islands group. The two Algerian samples KIA and DDC were pooled according to a nonsignificant differentiation comparison (see results). For continental France, the two retained samples corresponded to two depths and sites, and gave different results with BOTTLENECK. We also analyzed each region separately by grouping the corresponding population samples. In order to evaluate the impact of mutation model on the obtained results, we analyzed the results for the seven loci separately in the Algerian region. As genetic structure can impact the results of MSVAR analysis, an analysis at the deme level inside the French region was launched using two population samples in that region. We also pooled two individuals from the 12 population samples  from the Marseille area, as an approximation to the method proposed by Chikhi, Sousa, Luisi, Goossens, and Beaumont  and scaled parameters  over the four replicated data sets. All outputs were analyzed with the R CODA package  and the logarithm of geographical distances (shortest distance by sea) between populations  in IBDWS 3.16  at the Mediterranean scale and within French region only (thanks to the number of samples in this region) was tested through the correlation between Rousset, . The corFST and RST estimators with ARLEQUIN v.3.5  was performed with The relationships between populations were further investigated using principal coordinate analysis (PCoA) with GenAlEx 6.5  criterion  . Observed and unbiased expected heterozygosities ranged from 0.31 for SIV to 0.62 for KIA, and from 0.34 for SIV to 0.71 for DDC, respectively  . When considering the scaled parameters, the current value of θ = 4Neμ was again highest in Algeria, intermediate in France and islands, and lowest in Turkey. The θ estimate was ten times higher in Algeria than in Turkey . The inference of demographic decline was coherent along all our loci in the Algerian area, all indicating signatures of population declines .The analysis of departure from mutation–drift equilibrium using BOTTLENECK indicated no significant heterozygosity excess, expected following a bottleneck, on the basis of one‐tailed Wilcoxon test Table . On the 3.3FST values ranged from 0  to 0.36  with an overall FST = 0.13. All populations were significantly differentiated except KIA and DDC (Algeria) that are separated by a distance of 721 m, and in Marseille for samples from the same site but different depths . A sample from Marseille (POU) with 25% missing data showed also five nonsignificant differentiation tests  after FDR correction. Pairwise FST values corrected for null alleles showed similar values of differentiation between samples . The smallest geographical distance for which significant genetic differentiation was observed was 763 m, with FST = 0.02 for KIA vs SPI.Pairwise FCT = 0.19 and FCT = 0.04 with FST‐like and RST‐like analyses, respectively; Table FST but not significant with RST . A significant positive correlation was evident between genetic distances and the logarithm of the geographical distances, indicating a pattern of IBD at the Mediterranean scale  and within the French region . At the global scale, the IBD pattern seemed to be separated in two parts with a lower slope at short distance compared to a much higher slope at higher distances.The AMOVA indicated significant differences between geographical groups of samples, both by separating the two islands Table  and by gThe principal coordinate analysis (PcoA) confirmed the distinctiveness of the different geographical areas: Axis 1 separated Algeria from other samples, while axis 2 separated Turkey from other ones Figure . Island 3.4K = 2 separated samples from Algeria and Turkey in cluster 1 and samples from France, Menorca, and Corsica in cluster 2 . At K = 3, four replicates over twelve grouped Algerian and Turkish samples and were not retained here. For K = 4, samples from islands Menorca and Corsica were assigned to a new group, while other samples were clustered as above but with France partly admixed with the islands cluster  criterion indicated K = 5 as the best clustering solution . With K = 5, Algerian samples were in cluster 1, Menorca and Corsica in cluster 2, Turkey in cluster 3 and all French samples subdivided between clusters 4 and 5 but with high admixture between these two putative clusters.The first round of STRUCTURE with 2 Figure . For K =r Figure . The DelK = 3 was the best solution followed by K = 6 , but there was no clear genetic structuring . A STRUCTURE analysis on Turkish samples alone indicated a clear separation of both populations at K = 2 . The STRUCTURE analysis on Menorca and Corsica samples indicated a distinction between these two islands but with quite high levels of admixture .For the second STRUCTURE analysis on French samples, K = 15 and 21 but without a single clear informative value . We present here the results obtained with K = 15 clusters, and other analyses around this value gave similar results. Higher K values did not bring more information on the general structure at the Mediterranean scale. The analysis confirmed the main groupings evidenced with STRUCTURE but with the additional separation between the two Turkish samples from Marmara Sea and Aegean Sea in clusters 11 and 14, respectively  was minimal between y Figure . Samplesy Figure . High pe4We have demonstrated (1) strong genetic structure between samples from different regions in the Mediterranean, (2) we did not observe any significant differentiation between depths for a given site in France, and (3) we have shown significant differences in the levels of genetic diversity between regions with the highest values in southwestern Mediterranean (Algeria) and the lowest in the eastern part (Turkey), which could be correlated to different evolutionary histories or levels of effective sizes.4.1FST values indicated that the highest differentiation was observed between eastern (Turkish) and western populations and differentiation was higher when comparing western samples with Marmara Sea (mean FST = 0.3) than with Aegean Sea (mean FST = 0.22). Such deep genetic break between eastern and western Mediterranean populations has been reported in various species such as fish, molluscs, or the seagrass Posidonia oceanica , or to intrinsic different patterns of genetic structure. Here again, the distribution range of E. cavolini could partly explain these differences between E. singularis and E. cavolini: There is a notable absence of E. cavolini along Mediterranean Spanish coasts. This could have promoted a North–South coastline connectivity for E. singularis which is more frequent there  displayed a genetic distinction between Balearic Islands and Corsica  and Corsica islands were identified as a separate cluster without substructure . The Fe et al. . The geoFST value (0.24) and to DAPC. This was not visible with the global STRUCTURE analysis probably because of the reduced number of Turkish samples analyzed. Indeed unbalanced sampling affects such clustering method . It will thus be necessary to study more loci to test for possible genetics–environment associations.No significant differentiation was evidenced between samples from different depths within the same sites near Marseille as observed in a preliminary study  or with an important private diversity  should be a priority. This is particularly important when considering the current pressures on this species. In Turkey, some relatively dense E. cavolini populations, restricted to few areas in the Marmara Sea, are under various threats such as fisheries (Topçu & Öztürk, The yellow gorgonian 10.5061/dryad.8vj25.All genotypes are deposited on Dryad. doi: Click here for additional data file. Click here for additional data file. Click here for additional data file.


Garra barreimiae from the Al Hoota Cave and nearby Hoti Pit in Northern Oman. Surface morphotypes of this cyprinid species are common throughout the distribution area. Very rarely individuals with intermediate phenotypes can be found. In the present study, potential gene flow between cave and surface populations was tested and population structure within five sampling sites was assessed. Overall, 213 individuals were genotyped at 18 microsatellite loci. We found that the cave populations have lower genetic diversity and are clearly isolated from the surface populations, which seem to be sporadically in contact with each other. The results indicate a recent genetic bottleneck in the cave populations. Thus, it can be assumed that during climatic changes the connection between cave and surface water bodies was disjoined, leaving a subpopulation trapped inside. Nevertheless, occasional gene flow between the morphotypes is detectable, but hybridisation seems only possible in cave habitat with permanent connection to surface water. Individuals from surface sites bearing intermediate phenotypes but cave genotypes imply that phenotypic plasticity might play a role in the development of the phenotype.Cave-dwelling taxa often share the same phenotypic modifications like absence of eyes and pigmentation. These “troglomorphic characters” are expressed in the populations of  The best studied organism concerning this matter is the characid Astyanax mexicanus 10. Besides Astyanax, the Afro-Asian cyprinid genus Garra, which comprises over 100 freshwater fish species can be considered as an exemplary system. It includes several cave-dwelling species or populations within species exhibiting varying degrees of troglomorphism. A mental disc (modified lower lip used to adhere to the substrate) is characteristic to most species of the strictly continental genus which is supposed to have originated in the early Miocene around 23 Myr in Southwestern China13. Typical troglomorphic characters as complete lack of pigmentation and complete anophthalmy are displayed in the Iranian cave barb Garra typhlops 14 and in the Iraqui blind barb Garra widdowsoni 15, both species being cave-dwelling without any surface occurrences known. This is also the case for the Somalian cave-dwelling species Phreatichthys andruzzii Vinciguerra, 1924, for which Colli and colleagues16 suggested a taxonomic update due to the position of P. andruzzii within the genus Garra in their phylogenetic tree. This species, which according to Calderoni and colleagues17 evolved in complete darkness for at least 2.8 million years, exhibits also pronounced troglomorphic traits18.Troglomorphic cave fish always share a similar phenotype exhibiting reduction or absence of eyes and pigmentation. These “typical” cave characters developed independently among taxa and can vary strongly in their expression, often even within species or populationsGarra barreimiae FOWLER & STEINITZ, 1956, which, like A. mexicanus comprises both, surface and cave dwelling populations. Garra barreimiae is endemic to the southeastern Arabian Peninsula. It is quite common in the United Arab Emirates and northern Oman along the Hajar Mountains where it inhabits all kinds of water puddles, streams and even artificial water drainage systems  used by the locals to water their gardens.In the centre of the present study is G. barreimiae in the Al Hoota Cave near the Jebel Akhdar in northern Oman. Due to the morphological similarities, it was assigned to the species G. barreimiae by Banister19. Yet, the individuals of this population exhibit a typical troglomorphic phenotype such as no pigmentation and lack of externally visible eyes, while scales are present. Besides the more obvious differences, Banister also described the troglomorphic specimens to exhibit smaller optic lobes and reduced squamation. In the closely located cave Hoti Pit20, we discovered another population of G. barreimiae which also displayed the cave morphotype.In 1980 Dunsire and Gallagher discovered a subterranean population of 21 were the first to conduct molecular genetic analyses in this species. They sequenced a part of the mitochondrial (mt) control region (CR) in individuals from both the Al Hoota Cave population as well as from several surface sampling sites. Their results showed that all individuals collected in the cave belong to the same haplogroup. However, this haplogroup also contained individuals from nearby surface localities. Most of the latter individuals exhibited an intermediate phenotype suggesting that the cave and surface populations might be still in contact or have been until recently. The genetic distances between individuals from Al Hoota Cave and individuals from nearby surface localities are quite small implying a recent origin of the cave population. Yet, the question remained whether a nuclear marker would confirm the close phylogenetic relationships between surface and cave populations as suggested by the mt CR. Within the genus Garra, G. barreimiae represents the rare case where both morphotypes (troglomorphic/surface) are still existing within one species. The aim of the present study was to better understand this system in which both forms co-exist. More specifically, we asked, if there is recent gene flow between those populations and whether it does occur equally in both directions? Are the individuals exhibiting an intermediate phenotype the result of recent hybridization between cave and surface individuals or a result of phenotypic plasticity? What is the genetic structure and diversity of cave and surface populations of G. barreimiae? Do the nuclear markers confirm the presumed bottleneck events as suggested by mt data?Kruckenhauser and colleaguesG. barreimiae by a 454 sequencing approach22 and analysed them in Al Hoota Cave as well as several surface populations in Northern Oman. Moreover, morphological measurements of the eye size were taken to assess the amount of reduction. The results were compared with those derived from mt CR sequences.To address those questions we utilized 18 of the 19 microsatellite loci designed for 20. These floods occur occasionally every few years or sometimes more than once a year. Between such events there is almost no running water in the cave, but there are four extensive lakes and some smaller ones. These water bodies hold only troglomorphic fish20. Between the lakes there are several vertical drops that seem to be an insurmountable barrier for fish (from lower to higher lakes). From the passage morphology and transported cave sediment it can be estimated that during floods flow velocities in some parts reach more than 10 m/s and discharge rises up to few hundred litres per second. The average temperature in the cave is 25 °C and humidity between 40–70% up to 100% after floods when the water in the lakes is high and sumps stop air ventilation23. Except for floods, the wadis up- and downstream of the cave are dry. The lower entrance of the cave was developed as a show cave. Geographic coordinates: longitude 57.3572; latitude 23.0844.The Al Hoota Cave  is a 5 km long spacious cave southeast of the Jebel Shams mountain peak (2980 m above sea level (asl)) that developed in cretaceous limestone. The cave forms an underground flood system: during flood events water from the above lying Wadi Hoti sinks into the upper entrance of the cave (990 m asl) and re-emerges 262 m deeper at the lower entrance into the Wadi Falahi3/s. The entrance is a 12 m deep pit and in the lake at its bottom no fish were found. The known end of the 380 m long cave is formed by another lake harbouring only troglomorphic fish. The part of the cave where the passage ceiling reaches into the water and forms a sump, has not been explored yet and the reappearance of the water is unknown. As there are no signs of backflooding at the terminal sump it can be concluded that the cave behind the sump is spacious. Geographic coordinates: longitude 57.3726; latitude 23.1015.Hoti Pit (HP) is a pitfall in Wadi Hota 400 m upstream and some 20 m higher than the upper entrance to Al Hoota Cave. During floods Hoti Pit takes some water from the Wadi Hoti but according to sediments and morphology only few mGhubrat Tanuf (GT) is a cave located in the Wadi Falahi at 670 m asl and approx. 1 km downstream of the lower Al Hoota Cave entrance. A small stream emerges from the cave entrance that feeds a pool in the Wadi . The rather narrow cave is an almost horizontal water table cave with a length of 690 m. Estimated discharge values reach from less than 1 l/s to few l/s. During floods, water from the Wadi enters the cave for some 100 m as indicated by wood and garbage transported into the cave. In the upstream end the cave splits up into two separate streams which both emerge from sumps. The upper stream is interrupted by a 9 m high cascade where no fish were found in the upstream part. The lower one is only marginally higher than the entrance, and surface fish can be found throughout this part. In this cave, mostly individuals displaying the surface morphotype occur, but also few individuals exhibiting typical cave or intermediate phenotypes were found. Geographic coordinates: longitude 57.3681; latitude 23.0718.The sampling site Wadi Falahi (WF) is a surface water body about 200 m underneath the lower Al Hoota Cave entry which represents the beginning of the Wadi Falahi, a riverbed which reaches ~4.5 km downstream comprising also the sampling site Ghubrat Tanuf/Ain al Msalla. Most of the time, the wadi lies dry, but single puddles may remain for several weeks or even months after rains. Fish were collected at a rather shallow water body located about 680 m asl which appears to be drying out regularly during summer. At this sampling site, usually the surface form occurs but also few individuals exhibiting intermediate phenotype were found. Geographic coordinates: longitude 57.3539; latitude 23.0804.G. barreimiae from Misfat al Abriyeen had the surface phenotype and all specimens analysed were collected from this aflaj. Geographic coordinates: longitude 57.3159; latitude 23.1481.Misfat al Abriyeen  is a small mountain village located at an impressive wadi at 1000 m asl about 7 km northwest of Al Hoota Cave. At the end of (0.7 km behind) the village a spring emerges from big boulders in the wadi and is tapped into an artificial water drainage system (aflaj). All Tissue samples (finclips) were taken from a total of 213 individuals collected at five sampling sites located at the south side of Central Hajar Mountain chains in northern Oman, some 25 km northwest of the town Nizwa. These specimens were captured via fishing net and immediately anesthetized with clove oil and stored in 80% ethanol. Two of the sampling sites are mere cave locations, two are typical surface sites and one location consists of subterranean water bodies connected to surface water bodies , only the troglomorphic form was found. The water body of Ghubrat Tanuf cave is constantly connected to the surface water body of Ain Al Msalla and harbours mostly fish bearing a typical surface phenotype, but also few individuals with intermediate or troglomorphic phenotype. A similar phenotypic composition also applies for the surface water body Wadi Falahi, whereas in Misfat only fish with the surface phenotype were found.G. barreimiae22. One microsatellite marker published in ref. We extracted genomic DNA from tissue (finclips preserved in 80% ethanol) following the QIAGEN DNeasy Blood & Tissue extraction kit protocol. All samples were profiled with 19 microsatellite loci by using primers that were specifically designed for 24, and aligned with the program MEGA version 6.0625 together with the 122 sequences retrieved from Kruckenhauser et al.21. The final alignment was 399 bp long and comprised a total of 218 sequences from nine sampling sites. The haplotype network was constructed using the software PopART version 1.7 (available at http://popart.otago.ac.nz) and the Median-joining (MJ) algorithm proposed by Bandelt, Forster and Röhl26.In 96 selected samples, we amplified a section of the mt CR gene via PCR reaction  Mg2+, 0.25 µl of each primer , 13.9 µl a.d.). The following primers were used: Thr1+ 5′-GCATCGGTCTTGTAATCCGA-3′, and CR4- 5′-TTGGGCGTCGGCGGTGAGAG-3′; designed by ref. R version 3.1.327. After checking our data set for completeness and missing values (0.57% mean missing data within the whole data set), we calculated general information like the number of alleles, the diversity index and evenness per locus with the R-package poppr version 2.1.028. Furthermore, we calculated the expected (He) and observed (Ho) heterozygosity per locus in each population with the R-package adegenet version 2.0.029 and the mean He and Ho over all loci per population with the R-package diveRsity version 1.9.8930. In addition, we tested for genotypic linkage disequilibrium with the software Genepop version 4.331. Deviations from the Hardy-Weinberg equilibrium (HWE) were estimated using both the global test and exact test in Genepop executing 10000 Markov chain Monte Carlo (MCMC) runs for 100 batches, each with 5000 iterations. The software Micro-Checker v. 2.2.332 was used to check for the existence of microsatellite null alleles in the data set. Although there was only one population, i.e., Hoti Pit, with no signal of microsatellite null alleles at any locus, the tests showed that microsatellite null alleles at a low frequency might be present in populations Ghubrat Tanuf  and Wadi Falahi , whereas in the populations Al Hoota and Misfat potential microsatellite null alleles are only present at one locus. However, since none of these loci showed a constant significant deviation in all populations, and further analysis indicated population admixture between Ghubrat Tanuf and Wadi Falahi (see below) we did not exclude these loci from our analyses. We tested the data set also for linkage disequilibrium and the results showed that some locus pairs exhibit evidence of linkage disequilibrium in single populations, but never in all populations.We explored the data mostly by employing the statistical computing software environment R-package pegas version 0.8.233. This function computes Fit, Fst and Fis for each locus in the data set according to the formulae of Weir and Cockerham34. Subsequently, we also calculated the pairwise Fst-values between all populations with the Microsoft Excel Add-In GenAlEx version 6.536. The inbreeding coefficient Fis after Weir and Cockerham over all loci per population was calculated with the program FSTAT37.In order to statistically describe the level of inbreeding or differentiation among and within populations, we calculated the fixation indices as implemented in the R-package diveRsity. Private alleles were estimated with the R-package poppr and the percentage of private alleles per population was calculated based on the actual amount of alleles per population.Allelic richness per locus per population was computed to take the differing sampling sizes of populations into account. This was done by using 1000 re-samples of the smallest sample in the data set (Hoti Pit with N = 17) via the structure38 was used to perform population structure analyses. We employed the admixture model and tested for clusters from K = 1 to K = 7 for the whole data set and K = 1 to K = 3 for the cave subset, and for each assumed K 20 iterations per run were performed to find the correct number of clusters (K). Length of burnin was set to 100000 and the number of MCMC repeats after burnin was 500000. The results were then revised with the online program structure Harvester web version 0.6.9439 to explore which K has the highest probability and ΔK. The next step was to summarize the iterations and to minimize the variance across all twenty iterations of the selected ΔK value with the software CLUMPP version 1.1.240. For high quality visualization of the combined results obtained via CLUMPP, we used the software Distruct version 1.141.The software R-package ade4 version 1.7–242 was performed. By using the scaleGen function in the R-package adegenet, which replaces the missing values with generated mean allele values, it was possible to include all individuals.A principle component analysis using the 43 for real-time estimation of first-generation migrants. We used the L_home likelihood computation and the Bayesian method criterion44 and applied the simulation algorithm suggested by Paetkau et al.45. The calculation was conducted with a number of 10000 simulated individuals and Threshold was set on 0.05.To infer whether gene flow is recent we used the program GeneClass2We measured eye diameter of intermediate individuals which were collected at the sampling sites Ghubrat Tanuf (N = 11) and Wadi Falahi (N = 14). As not all specimens were fully grown adults, we scaled the eye diameter as percentage proportional to the head length (from snout tip to end of operculum) to allow comparison between individuals of different sizes. Subsequently, we also measured eye diameter of surface individuals with external fully developed eyes and pigmentation . The according measurements and photographs are provided in Supplementary Table At first, in order to assess the genetic variation among and between specimens from different sampling sites in general, we calculated the descriptive statistics Table  for the e over all loci was higher in surface populations  compared to the cave populations . In the populations Ghubrat Tanuf and Wadi Falahi He was higher than Ho, whereas both parameters were similar in the other populations. The most private alleles were found in the Al Hoota and Misfat populations . In the populations Ghubrat Tanuf and Wadi Falahi, less private alleles were detected (8.40% and 9.76%), whereas the fewest private alleles were found in the Hoti Pit population (2.44%).The number of alleles  and the proportion of polymorphic loci  were generally higher in surface than in cave populations. Regarding the genetic variability, we corrected these results by taking the variable sampling sizes into account and calculated the allelic richness according to the sample with the fewest individuals . The average allelic number turned out to be slightly lower in the cave populations  than in the surface populations . Furthermore, the calculated mean H46.Deviations from Hardy-Weinberg equilibrium (HWE) for each population and locus showed that the surface populations exhibit more loci with significant deviation (5% level) than the cave populations . The results from the global HW test indicate that the Ghubrat Tanuf, Misfat and Wadi Falahi populations are not in HW equilibrium, whereas the two cave populations Al Hoota and Hoti Pit showed no significant deviation for HW equilibrium. These results remained after repeating the global HW test excluding individuals with an intermediate phenotype (data not shown). The reason for deviations from HWE in the surface sites is most likely due to the presence of microsatellite null alleles. The few loci showing deviations from HWE are the same for which potential microsatellite null alleles were detected. Another reason could be occasional migration from one surface population to another  in Hoti Pit, Al Hoota and Misfat, while the highest values were found again in Ghubrat Tanuf (0.14) and Wadi Falahi (0.21).The inbreeding coefficient Fstructure analysis K = 2 yielded the highest probability, and also the ΔK was highest for K = 2 , we split the data set accordingly into two groups, a cave cluster including the Al Hoota and Hoti Pit individuals and a surface cluster comprising individuals from Ghubrat Tanuf, Misfat and Wadi Falahi.In respect to the result of the structure analyses with a subset of individuals exclusively collected at the two cave sites  to explore whether they are genetically homogenous or if they are differentiated from each other. The analysis resulted in the highest probability of K = 2, with the two clusters correlating with the two sampling sites  and pairwise Fst-values (Nei’s unbiased genetic distance) between all pairs of populations were calculated (heterozygosities are weighted by group sizes). The values range from 0.296 (Misfat and Hoti Pit) to 0.020   display a tripartition roughly dividing the data set in a group comprising most of the Ghubrat Tanuf and Wadi Falahi samples, one cave group containing both Al Hoota and Hoti Pit samples and the third group including all samples from Misfat Fig. . In Fig.19. Hence, we chose a conservative approach and treated them as intermediate phenotypes  on the species’ phenotypic variability. These intermediate phenotypes show the whole palette from normally developed eyes without pigmentation to pigmentation without visible eyes at all  one individual is truly troglomorphic  with a 100% cave genotype, five individuals exhibit an intermediate phenotype  with 50% to 90% share of cave genotypes, and the remaining five individuals were juveniles with unusually small eyes and weak pigmentation and their genotypes exhibit 80% to 90% cave affiliation Table .Table 3LIn Wadi Falahi 14 conspicuous individuals  were collected, five of which were completely troglomorphic without visible eyes and weak or no pigmentation, three were categorized as intermediate due to the presence of microphthalmic eyes and weak pigmentation and six individuals were still juvenile, but exhibited either microphthalmic eyes or no eyes at all and were only very weakly pigmented  from Ghubrat Tanuf and Wadi Falahi  supports the hypothesis that the cave populations probably went through repeated genetic bottlenecks as suggested by Kruckenhauser et al.21. Moreover, it implies that the cave populations originated from a group of surface ancestors holding only a fraction of the genetic diversity of the whole species. Given the low genetic distance between cave and surface populations as reported by Kruckenhauser et al.21 who further suggested that the cave population of G. barreimiae is phylogenetically young, it seems plausible that this population originated at a time, when the climate started to change from wet to arid resulting in dropping water levels48. In southern Arabia the climate changed repeatedly in the Late Pleistocene, with two major wet periods, one of them coinciding with the last interglacial period (125 to 120 ka) and another during the early Holocene (9.7 to 6.2 ka)48. One of these changes probably disrupted the connection between surface and cave water bodies, leaving a subpopulation trapped inside the cave and isolated from surface water bodies, which also became more fragmented. However, it cannot be excluded that the cave population emerged from parallel speciation as found in Astyanax9. Concerning the surface populations, He is higher than Ho in Ghubrat Tanuf and Wadi Falahi, whereas they are nearly similar in the other localities. Since the surface individuals from Ghubrat Tanuf, Wadi Falahi and Misfat all belong to the same cluster in the structure analyses . Since not a single individual from one of the cave sites bears any degree of surface genotype, we conclude that there is no gene flow from the surface populations into the caves. The structure analysis of only the troglomorphic cave individuals revealed a differentiation between the individuals of Al Hoota Cave and Hoti Pit  of individuals from several surface populations and found no geographic pattern within this group. Yet, this differentiation is only weak suggesting occasional gene flow. Although there is no direct connection between these sites during dry periods, there might be a temporal water junction (probably underground) enabling single individuals from Misfat to migrate into the Wadi Falahi when the water level rises during flood events.The differentiation of the Misfat population in the structure analyses . Phenotypic plasticity seems a reasonable explanation considering that the troglomorphic phenotype is of rather recent origin21. Similar observations were conducted with individuals of the Micos cave population of Astyanax, which is considered to be phylogenetically young. Therefore, individuals of this population bear rudimentary eyes that are less reduced compared to phylogenetically old troglodyte populations (e.g. El Abra region) whereas pigmentation is almost as pronounced as in the surface form but increases even more after exposure to light50. Effects of light exposure can also be observed in the Atlantic molly Poecilia mexicana, a species occurring in the Mexican Cueva del Azufre – a system comprising both surface as well as cave habitats. Interestingly, individuals of this species show a gradual decrease of eye-size from the entrance and the front chambers, which receive dim light through numerous skylights, to the innermost chamber of the cave. Interestingly, the differences in eye sizes seem to be related to different amounts of light received in the chambers51. Subsequently, to clarify and quantify the phenotypic variability as a consequence of light exposure during ontogeny in individuals of G. barreimiae, comparative breeding and rearing experiments under controlled conditions are inevitable.However, the situation with specimens bearing an intermediate phenotype from Wadi Falahi is different, because they all have a “pure” cave genotype , in which typical surface forms of G. barreimiae occur frequently and seem to survive well in the cave habitat.The fact that we found not a single hybrid individual at Wadi Falahi leads to the conclusion that hybridisation between cave individuals and surface individuals from Wadi Falahi is either not occurring at all or none of the F1 generation survives and reaches reproductive stage in the surface environment. Probably, hybrid larvae, which are pale and blind or with debility of sight, are at a disadvantage and more prone to predation compared to surface larvae. In addition, the cannibalistic nature of G. barreimiae, we found that gene flow between the two morphotypes is still possible in cave environment but occurs only unidirectional from the cave to surface populations. Hence the integrity of the Al Hoota and Hoti Pit population with their peculiar phenotypes is currently not threatened by admixture with surface morphotypes. However, the restricted local occurrence in only two caves makes them vulnerable to extinction due to environmental impacts. Furthermore, there are indications for phenotypic plasticity, probably due to light exposure, in individuals collected from surface populations with an allele composition typical for cave specimens but exhibiting an intermediate phenotype. The finding of hybrid specimens and cave individuals outside the caves indicate that at least a temporary connection, probably underground, between Hoti Pit cave and surface water bodies must exist. These inferences are supported by results deduced from a combination of mitochondrial, nuclear and morphological data. Furthermore, we found a substructure between the investigated surface populations leading to the conclusion that gene flow between surface water bodies occurs sporadically.Although our results showed significant genetic differentiation between the surface and cave form of Supplementary Figures and TablesSupplementary Table S1Supplementary Table S5