Zoroastrianism is one of the oldest extant religions in the world, originating in Persia (present-day Iran) during the second millennium BCE. Historical records indicate that migrants from Persia brought Zoroastrianism to India, but there is debate over the timing of these migrations. Here we present genome-wide autosomal, Y chromosome, and mitochondrial DNA data from Iranian and Indian Zoroastrians and neighboring modern-day Indian and Iranian populations and conduct a comprehensive genome-wide genetic analysis in these groups. Using powerful haplotype-based techniques, we find that Zoroastrians in Iran and India have increased genetic homogeneity relative to other sampled groups in their respective countries, consistent with their current practices of endogamy. Despite this, we infer that Indian Zoroastrians (Parsis) intermixed with local groups sometime after their arrival in India, dating this mixture to 690–1390 CE and providing strong evidence that Iranian Zoroastrian ancestry was maintained primarily through the male line. By making use of the rich information in DNA from ancient human remains, we also highlight admixture in the ancestors of Iranian Zoroastrians dated to 570 BCE–746 CE, older than admixture seen in any other sampled Iranian group, consistent with a long-standing isolation of Zoroastrians from outside groups. Finally, we report results, and challenges, from a genome-wide scan to identify genomic regions showing signatures of positive selection in present-day Zoroastrians that might correlate to the prevalence of particular diseases among these communities. Since there is no context or documentation for the life of Zarathustra, his very existence is a matter for debate. Although some scholars have proposed that he lived in the 6Qisseh-ye Sanjan, a legendary account of the journey by sea from Iran and settlement in India.According to Parsi  tradition, a group of Zoroastrians set sail from Iran to escape persecution by the Muslim majority. They landed on the coast of Gujarat (India) where they were permitted to stay and practice their religion. The date of the arrival has been the cause of speculation and varies between 785 CEZoroastrian communities today (2011 census) are concentrated in India , Southern Pakistan , and Iran—mainly in Tehran, Yazd, and Kerman—. In the last 200 years Zoroastrians, both Parsi and Irani, have formed diaspora communities in North America , Canada , Britain , Australasia , and the Middle East . Zoroastrianism is a non-proselytizing religion, with a hereditary male priesthood of uncertain origins.Genetic data provide a means of examining the biological relationships of different populations and testing claims of common ancestry. Previous studies of Iranian Zoroastrians have suggested that they are genetically differentiated from their neighboring populations. For example, Farjadian et al.Less is known about the genetic landscape and the origins of Zoroastrianism in India, despite Parsis representing more than 80% of present-day Zoroastrians worldwide.1.Which demographic processes  have contributed most to present-day genetic differences between Zoroastrian and non-Zoroastrian groups sampled from the same country?2.Is there evidence of increased genetic homogeneity in Zoroastrian groups relative to non-Zoroastrian groups?3.Is there genetic evidence for admixture events in Zoroastrian groups? Does this support historical records tracing the origin of Indian Zoroastrians (a.k.a. Parsis) to migrants from Persia?4.Is there evidence that any inferred admixture events were sex biased?5.Is there genetic evidence for the patrilineal descent of Parsi priests from a small number of founding individuals?6.Using a genome-wide scan, can we reliably detect genomic signatures of positive selection in the Zoroastrian populations that may relate to the prevalence of diseases or other phenotypic traits in the community?Prompted by these observations, we explored the genetic legacy of Zoroastrianism in more detail by generating genome-wide autosomal and Y/mtDNA genotype data for Iranian and Indian Zoroastrian individuals and comparing them to other publicly available genetic data. With this study we aimed to address the following questions.th century immigrants; the Zoroastrian Association was formed in 1861, at which time there were around 50 Zoroastrians living in the UK.Buccal swabs were collected from a total of 526 men from India, Iran, the United Arab Emirates, and the United Kingdom see . Individ71 of these individuals , all belonging to the lay  population, were genotyped using the Affymetrix Human Origins array. This array targets 627,421 SNPs with well-documented ascertainment, although we note that our inference techniques use haplotype information that has been shown to be less affected by ascertainment bias.The above dataset was merged with modern populations in the Human Origins dataset of Lazaridis et al.,We performed PCA on all the South Asian and West European populations included in the merge using PLINK 1.9 after linkage disequilibrum (LD) pruning using–indep-pairwise 50 5 0.5.We jointly phased the autosomal chromosomes for all individuals in the merge using SHAPEITWe classified our 2,545 modern individuals into 230 groups, with the majority of these groups based on population labels.Based on this tree, exploration of genetic similarity among individuals (see below), and visual inspection of haplotype sharing patterns among our 207 clusters, we classified these clusters into genetically homogeneous groups, choosing a level of the tree where there were 50 final total clusters, among which Iranian Zoroastrians and Parsis each formed their own distinct groups. At this level of the tree, we note that the 10,000-year-old Neolithic Iranian WC1 clustered with other modern Iranians, but nonetheless we re-classified WC1 as its own cluster, so that we ended up with 51 final total clusters we use throughout this paper see .1, …, nK} individuals, respectively, the set of donors is fixed to contain nk − 1 individuals from each of the K groups. This is to account for the fact that individuals cannot be painted using themselves as a donor, so that individuals within each of these K donor groups can only ever be painted using nk − 1 individuals from their own group label. We refer to this second painting protocol where K = 230 as the “all donors painting” throughout. Note that a primary difference between this painting and the fineSTRUCTURE painting described above is that we now use group labels, based in part on clustering results, which are required for our leave-one-out approach. When using haplotype information for this painting, we initially estimated the mutation/emission and switch rate parameters as described above, giving estimated Mut and Ne of 0.000704 and 223.5674, respectively.We then painted all 230 modern and 8 ancient samples using all 230 modern groups as donors, following the “leave-one-out” approach, as described by Hellenthal et al.,We also performed a slightly different version of this painting where Iranian and Indian populations were excluded as donors, using the leave-one-out approach described above for all 216 remaining groups, a third painting protocol with K = 216 that we refer to throughout as the “non-Indian/Iranian donors painting.” We did this to mitigate genetic effects due to recent isolation in our Indian and Iranian groups when inferring the best surrogates to sources of admixture in our groups. Mut and Ne parameters  were re-estimated for this new scenario as described above. To help determine whether admixture from outside groups or independent drift effects due to genetic isolation are driving genetic differences among sampled groups within Iran and Indiak ϵ  as inferred by CHROMOPAINTER. We also inferred pairwise TVD among individuals assigned to the same group as defined by our final 51 fineSTRUCTURE-inferred clusters, results of which are discussed in the previous section.We quantified differences in the painting profiles between all Iranian and Indian groups by applying the metric total variation distance (TVD) as described in Leslie et al.i is the number of SNPs in chromosome i ϵ , L the total number of SNPs across all the 22 chromosomes, and XY was also calculated for the Iranian and Indian groups using the all donors painting.Independent drift effects in groups X and Y since their split can generate genetic differences between them without requiring any outside introgression since this split. To elucidate whether inferred genetic differences are more attributable to ancestry from outside sources, we followed the approach in van Dorp et al.ST for these groups was also calculated based on independent SNPs using PLINK 1.9, which implements the method introduced by Weir and Cockerham.The weighted FFor a comparison of techniques, we used the following three distinct approaches to quantify the relative amounts of genetic diversity within groups.We performed a fourth analysis using CHROMOPAINTER that is analogous to that in van Dorp et al.2 > 0.2) in a sliding window of 250 SNPs.We also inferred within-group genetic diversity across all pairwise combinations of the same 13 individuals within each of the above genetic clusters using the IBD coefficient PI_HAT implemented in PLINK v.1.9, on a dataset where SNPs were first pruned to remove those in high linkage disequilibrium  statisticWe performed 100 permutation tests to establish the empirical distributions of XP-EHH values across the genome for both the Indian and Iranian populations, both to determine significance thresholds and to assess the reliability of our selection inference. For each permutation, we randomly partitioned our Zoroastrians and non-Zoroastrians into two different groups, and then calculated XP-EHH comparing these two groups. The threshold values at significance level of 0.01% (quantiles 0.0001 and 0.9999) from the empirical distribution combining all 100 permutations were used to determine the significance of the XP-EHH test. These values were −4.46 and 4.46 for the Iran and −4.37 and 4.37 for India.In order to explore sex-biased admixture and to evaluate claims of patrilineal inheritance among the Parsi priests, all the 526 samples collected for this study were typed for Y chromosome (490 successful samples) and mitochondrial DNA (518 successful sequencing) see . Y chromThe mitochondrial DNA hyper variable segment 1 (HVS-1) was sequenced as described by Thomas et al.h, and its standard error were calculated using the formula given by Neiz test. Populations were compared using FST based on haplotype or haplogroup frequencies, estimated from analysis of molecular variance (AMOVA) ØST values,ST values was based on 10,000 permutations of the data and 10,000 Markov steps were used in the exact test. Patterns of genetic differentiation were visualized using principal coordinates (PCO) analysis performed on a similarity matrix calculated as 1 − FST, based on Yhg and iMhg frequencies. Values along the main diagonal of the similarity matrix, representing the similarity of each population sample to itself, were calculated from the estimated genetic distance between two copies of the same population sample , where n is the sample size).Unbiased genetic diversity, Y chromosome and mtDNA admixture proportions were estimated using the likelihood-based method LEA,The coalescence time of clusters of Y chromosomes belonging to the same UEP-defined haplogroup was estimated by finding the average square difference (ASD) between the inferred ancestral haplotype  and all observed chromosomes.Most of the Iranian Zoroastrians see . InteresWe clustered some of our sampled individuals, including all Indians and Iranians, into 51 genetically homogeneous groups that exhibited good correlation between genetic similarity and population label , and theThe composition of the remaining genetically homogeneous clusters can be found in STST < 0.04. However, similar to Jewish groups from these regions, the Onge from the isolated Andaman Islands, and the Indian Kharia, an indigenous tribal ethnic group that has been isolated from other groups,ST and from 0.544 to 0.551 for TVD, with each distance measure larger than the maximum such measure between any two non-Zoroastrian, non-Jewish Iranian groups  . Therefore, in both Iran and India, these results indicate a high degree of genetic distance between the Zoroastrians in these countries relative to most other sampled individuals from their respective countries.Among our sampled individuals from Armenia, India, Iran, and Pakistan, we measured genetic distance between pairs of groups using two different techniques: (1) the commonly used, allele frequency-based measure Fctively) . SimilarST between them due to a lack of recent intermixing.XY;Relatively recent isolation may enhance the observed genetic differences between Zoroastrians and other groups, e.g., reflected in how individuals in each Zoroastrian group are inferred to share more recent ancestors with individuals of the same population label than with individuals from other populations under our all donors painting . TherefoXY measure, Iranian Zoroastrians showed a much-reduced genetic distance to other Iranian groups  and (2), we detected admixture in the Parsis dated to 27 (range: 17–38) and 32 (19–44) generations ago, respectively, in each case between one predominantly Indian-like source and one predominantly Iranian-like source. This large contribution from an Iranian-like source (∼64%–76%) is not seen in any of our other 7 Indian clusters, though we detect admixture in each of these 7 groups from wide-ranging sources related to modern day individuals from Bangladesh, Cambodia, Europe, Pakistan, or of Jewish heritage . For IraWe also ran TreeMix in order to infer a bifurcating tree relating five sampled groups: our two Zoroastrian groups, one other Indian group (Indian_C), one other Iranian group (Iranian_A), and Yoruba as an outgroup . This TR59ST values indicate that through the male line the lay Parsis have a closer relationship to the lay Iranian Zoroastrians, but through the female line they have a closer relationship to the non-Zoroastrians from India  and inferred mtDNA haplogroups (iMhg) showed that the Parsi priests sample has the lowest gene diversity values of all population samples studied for both Y and mtDNA , and S10om India . HoweverUsing the likelihood-based estimation of admixture (LEA) method of Chikhi et al.∗(xR1a1a) or Yhg-L (as defined in ∗(xR1a1a) and Yhg-L chromosomes to be 37 generations (95% CI 19 to 61 generations) and 31 generations (95% CI 18 to 46 generations), respectively. Assuming a generation time of 28 years, this translates to 1,036 years  and 868 years , respectively. Noting that these two coalescence date estimates are not significantly different (only 63% of simulated dates for Yhg-P∗(xR1a1a) are older than those for Yhg-L), we re-estimated the coalescence date assuming that both lineages originated at the same time by finding the mean ASD from the respective modal haplotypes for both clusters. This gave a combined within haplogroup coalescence date estimate of 923 years . When uncertainty in the mutation rate estimate is taken into account, the 95% CI widens to 501 to 1,782 years.Y/mtDNA data defined 8 Y chromosome haplogroups and 182 total Y chromosome haplotypes when using biallelic and STR loci  and S10 We calculated XP-EHH values for Iranian Zoroastrians and Parsis using other Iranians and Indians as reference populations . Tables SLC39A10 (Solute Carrier Family 39 Member 10 [MIM: 608733]) with an important role in humoral immunityCALB2 , which plays a major role in the cerebellar physiology.In the case of the Iranian Zoroastrians, most of the regions with the strongest signals of selection  are located in intergenic or intronic regions. Among these, some of the most significant SNPs , associated with neurological disorders like early epilepsy (MIM: 616211),WFDC (acidic protein WAP four-disulfide core domain containing) family and other genes like SPINT4 , SNX21 (sorting nexin family member 21), or TNNC2  , between a source genetically similar to modern Indian groups and a second source best represented genetically by a ∼9,500-year-old Neolithic farmer from Iran . This IrThat our approach inferred the Neolithic Iranian sample WC1 to be a better surrogate for the Iranian admixing source in the Parsis than any modern Iranian groups (including Iranian Zoroastrians)  likely rkohanim patrilineal priesthood, who claim descent from Moses’ brother Aaron and display low Y chromosome diversity, with most Y chromosome STR haplotypes either belonging to or being only a small number of mutation steps away from a modal haplotype.Patterns of mtDNA and NRY variation also provide clear evidence of contrasting maternal and paternal ancestry in Parsis, consistent with previous studies that suggest that migration of the ancestors of the present-day Parsi population from Iran to India was largely sexually asymmetrical.In Iranian Zoroastrians, we inferred a relatively old admixture event between sources best represented genetically by the Neolithic Iranian WC1 and modern-day Cypriots occurring around 70 CE (range: 570 BCE–750 CE). While we infer admixture in each of the three other non-Jewish Iranian groups , this adNotably, when using only modern groups as surrogates and excluding WC1, GLOBETROTTER was not able to detect this older admixture event . In thisOur analyses suggest that present-day Iranian Zoroastrians ceased intermixing with other groups shortly after the Arab conquest of Persia during 633–654 CE.Genetic isolation and endogamous practices can be associated with higher frequencies of disease prevalence. For example, there are reports claiming a high recurrence of diseases such as diabetesIn summary, we explore the genetic landscape and structure of Indian and Iranian Zoroastrians and provide genome-wide genetic evidence that the Parsis descend from an admixture event between ancestral groups consisting predominantly of males with Iranian-related ancestry and females with Indian-related ancestry. We date this event in ancestral Parsis to around 1030 CE, in agreement with historical records. We also provide evidence of a much older admixture event in Iranian Zoroastrians dated to around 74 CE with an unknown historical explanation but overlapping the period where Zoroastrianism acted as state religion in the region. We also demonstrate that Zoroastrians in both countries are genetically homogeneous populations differentiated from other population living locally, likely in part due to religious rules that discourage intermixing with non-Zoroastrians. Further work is required to help understand whether the genetic differences attributable to this isolation correlate with observed differences in disease phenotypes between these communities and other local groups.


Chilina dombeiana, in the Biobío River, one of the largest rivers in Chile. This species has a low potential for dispersal given the absence of a free-swimming larval stage  and given that adults have a low capacity for mobility. We hypothesized that: 1. Females would mate with different males (polyandry) resulting in intrabrood multiple paternity, 2. Individuals from closer sites would be more related than individuals from distant sites, and 3. Male parental contributions would be unevenly distributed within broods. Individuals from three different sites were sampled along the river: upper, mid, and river mouth. In the laboratory, hatching juveniles from a total of 15 broods were collected for paternity analyses. We used microsatellite markers and the programs GERUD and COLONY to determine whether multiple paternity exists and to estimate the contribution of different males to the brood. We found that multiple paternity was very common at all of the sites analyzed with as many as 8 males fertilizing a single female and a mean of 4.2 fathers per brood estimated by COLONY. Sire contribution was skewed to particular males in several broods. In addition, overall relatedness among broods for the three sites ranged from 0.17 to 0.45 with evidence of many half-siblings. Relatedness differed among the three sites. Particularly in upstream sites or in anthropogenically disturbed populations, the high levels of multiple paternity observed in C. dombeiana may be an efficient strategy to avoid inbreeding and prevent the loss of genetic diversity within populations.Studying the mating system of obligate aquatic organisms that inhabit river ecosystems is important for understanding its evolution as well as the role of biological and environmental factors in modulating population dynamics and species distributional patterns. Here, we studied the reproductive strategy of the Chilean endemic freshwater snail,  Polyandry, the act of a female mating with multiple males, is one of the most common mating systems observed in nature and has been reported in many taxonomic groups ,5,6,7,8.The main difficulty in evaluating the effect of polyandry on fitness, especially in males, is obtaining reliable estimates of reproductive success. Various pre- and post-fertilization mechanisms occur that may affect paternal contributions, such as cryptic female choice, sperm viability, sperm competition, sperm-ovum compatibility, and sibling cannibalism ,21. FortLampsilis cardium, [Villosa iris, [Hyriopsis cumingii, [Potamopyrgus antipodarum [For invertebrate species, most of our knowledge of mating systems and reproductive behavior comes from genetic studies of reproductive success in terrestrial and marine systems; far less is known for the freshwater realm . In contcardium, ; Villosasa iris, ; Hyriopsumingii, ). One ofipodarum .Chilina dombeiana  is native to Chile and belongs to an ancient family of freshwater gastropods, Chilinidae Dall 1870 . This family has a single genus, Chilina Gray 1828, and is found only in South America [C. dombeiana exhibits simultaneous hermaphroditism and although there is no evidence, this species like other gastropods from the order Hygrophila, could be capable of self-fertilization and/or biparentality through cross-fertilization [C. dombeiana is oviparous with internal fertilization and direct development . The eggs are encapsulated and embedded in gelatinous zig-zag-like string egg masses that are usually attached to rocky substrates [C. dombeiana is widely distributed in Chilean river ecosystems between 35° and 41° S and has high population densities. It is common to observe aggregations of individuals on or near patchily distributed rocky platforms and boulders (pers. obs.). Furthermore, adults have been observed to aggregate during reproductive periods and when laying of egg masses occurs (pers. obs.). In spite of their high abundance and wide distribution, there is scarce information on the biology and ecology of this organism and even less is known of the mating system and reproductive strategy of C. dombeiana. America . C. domblization ,31,32. Cbstrates . C. dombC. dombeiana populations inhabiting the Biobío River, the third largest river basin in Chile [C. dombeiana in three sites along the Biobío River. We expect to find: 1. multiple paternity in this species, 2. higher relatedness between individuals of closer sites than between individuals of distant sites, and 3. uneven male parental contribution within broods.Here we studied in Chile . The Bioin Chile . The phyC. dombeiana individuals was conducted in accordance with the biosafety and bioethics guidelines of the National Commission for Scientific and Technological Research (CONICYT) of Chile. No conservation or management policies are considered for C. dombeiana and the study was not conducted in protected areas, thus no specific permission was required for sampling individuals.Handling of C. dombeiana individuals from three different sites  along the Biobío River, Biobío Region, Chile. The three sites included the upper Biobío , the mid Biobío near to the locality of Negrete , and close to the mouth of the Biobío River . After collection, specimens were individually stored in 95% ethanol in plastic tubes until later genetic analyses were conducted.During December 2015, we collected by hand a total of 60 adult C. dombeiana adults, five from each site, with shell lengths ranging from 15 to 22 mm were transported alive in a cooler filled with freshwater to the Faculty of Science, Universidad Católica de la Santísima Concepción. In the laboratory, adults were placed in individual plastic boxes (200 ml) filled with freshwater collected from the sample sites. A small rock was added to each box to serve as substrate to stimulate egg mass laying. The boxes were constantly aerated with an air pump, and a 12 h light/dark photoperiod and constant temperature of 18°C were maintained during the entire experimental period. Additionally, the water inside the boxes was changed every ten days. After approximately one week, adult individuals  laid egg masses with lengths ranging from 30 to 70 mm. From each egg mass, 21 pre-hatching juvenile individuals  were randomly chosen for paternity analysis. The females (mothers) and 315 juvenile offspring were placed in individual tubes and stored in 95% ethanol.For parentage analysis, a total of 15 3 of muscle tissue was taken from the foot. DNA extraction was performed using the Thermo Scientific Direct PCR kit following the manufacturer’s instructions. The template genomic DNA was diluted to 10ng/μl. Genotyping of microsatellites was conducted using primers fluorescently labeled with 6FAM, VIC, PET or NED. Developed by Genetic Marker Services, eleven variable microsatellite loci were successfully amplified in Chilina dombeiana . Amplification was conducted in a thermal cycler (Verity) with the following cycling parameters: 35 to 40 cycles for 60 s at 95°C, 60 s at 55°C and 30 s at 72°C. An initial denaturing step  was included and the last cycle was followed by a 10 min extension at 72°C. The PCR products were separated on an ABI3730 XL automated DNA sequencer with the LIZ-500 size standard (Applied Biosystems); this was conducted in the Genomic Facility Center of University of Guelph (Canada). The allele sizes for each female and for the offspring were analyzed using the program GeneMarker v2.2 .DNA extraction of the juveniles was conducted using the whole animal, meanwhile for adult individuals about 3 mmreported ) and werFor each site the assumptions of Hardy–Weinberg and linkage equilibrium were tested in GENEPOP 4.1 , and sigBefore paternity analyses, the probability of exclusion (PE), which considers the probability to discard as a true parent an unrelated individual, was estimated over all loci using GERUD 2.0 . PE was C. dombeiana. Six to eleven microsatellite loci were used in the analysis; in some cases loci with missing data were discarded. COLONY is another computer program that uses mathematical algorithms to determine the number of sires when paternal genotypes remain unknown. While GERUD estimates the minimum number of sires, COLONY estimates the most likely number of sires. Thus, COLONY was used to estimate the maximum number of sires per brood using a maximum-likelihood method to assign parentage and sibship groups; from this, the genotypes of the unsampled parents were also reconstructed [The analysis of paternity was conducted using two different softwares GERUD 2.0  and COLOstructed . Three rIn order to investigate relatedness of offspring within families and whether or not individuals from families within a site were more related compared to individuals from other families and sites, we computed and compared pairwise relatedness estimator values (r) using COANCESTRY 1.0.1.5 . PairwisTo determine if the reproductive contribution of different fathers in the multiple-sired broods differed significantly from the expectation of equality (evidence of significant reproductive skew), we calculated sire evenness (E). This metric was estimated using an index of sire diversity D, based on the Shannon-Wiener index (following ). Sire dS equals the number of genetic sires in a brood and pi equals the proportion of offspring sired by the ith sire. Once D was obtained, evenness E could be calculated by utilizing an additional index, Dmax. This index is a hypothetical diversity score where Dmax = ln S; thus, this value is the maximum possible sire diversity if offspring had been distributed evenly given the number of genetic sires. Whether or not a female has reached maximum diversity within the clutch is limited to a set number of sires (evenness). This is calculated by :E=−∑i=2 tests with the null hypothesis of equal reproductive contribution were made in R 2.13.1 [To examine whether paternal contributions deviated significantly from equality in each brood, goodness of fit χR 2.13.1 .C. dombeiana is a simultaneous hermaphrodite we explored the potential existence of self-fertilization. The classical approach of measuring heterozygote deficiencies (FIS) has been used widely to estimate inbreeding, but this method is associated with typing artefacts, null alleles, and allele drop-out among other common problems that have been discussed in the literature [Given that terature . As suchterature . Selfingterature  and softterature .IS) were determined for each site; FIS values ranged from 0.063 to 0.092 , 0.337 (±0.069), 0.328 (±0.062) for sites UBB, MBB, and MoBB, respectively . OverallChilina dombeiana. The present study provides the first insights into the occurrence and frequency of multiple paternity in this species. Multiple paternity was detected in all 15 families analyzed, using two different programs , originBulinus truncatus, has revealed that this gastropod mainly self-fertilizes [Biomphalaria glabrata primarily outcrosses [Physa acuta can self-fertilize or outcross, but outcrossing is the main mating system in populations of this species [C. dombeiana were not significantly different than 0 in the three sites, and this was seen across the two different methods tested. This suggests that self-fertilization in this species is either very rare or may not occur.Freshwater snails are simultaneous hermaphrodites and most species preferentially self-fertilize or outcross ,56,22,57rtilizes . In cont species ,60. In tBiomphalaria obstructa [Bulinus africanus [B. cernicus [Physa heterostropha pomilia [Ancylus fluviatilis [Lymnaea peregra [Viviparus ater mate with different males and may store sperm [Potamopyrgus antipodarum, multiple paternity ranged from two to four sires as detected using GERUD and between four and seven sires when estimates were made using COLONY. Additionally for P. antipodarum, sires contributed evenly within the broods [Multiple paternity has been frequently observed in marine gastropod mollusks, but to date there are few studies documenting this in freshwater systems . Meanwhile, 20% of the broods had unequal male contributions when estimates were made using COLONY. Inter-mating period and mating order [In the present study, paternity analyses revealed sperm storage and high levels of multiple paternity within broods of ng order , sperm sng order , and theng order  could beC. dombeiana may provide benefits to this species in terms of ensuring fertilization and increasing mean offspring genetic diversity, as has been suggested for other invertebrates [Cornu aspersum has been shown to aid fertilization of eggs [Potamopyrgus antipodarum has been thought to increase genetic diversity within broods [Caridina ensifera blue [Mating with multiple individuals can be a costly behavior , howevertebrates ,74. For  of eggs , and muln broods . In addiera blue . Furtherera blue ,77.C. dombeiana, impacting the abundance of this species and causing severe local bottlenecks or occasional local extinctions. Furthermore, the potential effects of anthropogenic stress could be more critical given the absence of dispersal larvae and the sedentary behavior of C. dombeiana adults. As such, this species could be particularly vulnerable to contamination, habitat fragmentation, and habitat loss [C. dombeiana could help to ensure fertilization success, could increase genetic diversity, and could reduce the adverse effects of inbreeding. For example, in the marine snail Littorina saxatilis it has been suggested that high multiple paternity could potentially increase effective population size when populations experience strong bottlenecks [The Biobío River is exposed to high levels of anthropogenic pressure in the way of hydroelectric dams, industrial waste, industrial and agricultural water consumption, irrigation runoff, sand extraction, and recreational use . Such petat loss ,34,79. Stlenecks .C. dombeiana shows high level of multiple paternity suggesting that populations of this species may be highly genetically diverse; thus, this species may have the evolutionary potential necessary to respond to changes in environmental conditions and natural and anthropogenic perturbations. Different aspects of the ecology and biology of C. dombeiana are still unknown; hence additional studies, such as those measuring spatial demography, mating behavior, and anatomical reproductive features are required to explain the paternity patterns observed for this species.In summary, the


Therefore, it is important to monitor the genetic diversity in commercial germplasms as breeding practices, registration and marketing of new cultivars can lead to a significant reduction of the genetic diversity. Reduced genetic variation may lead to crop vulnerabilities, food insecurity and limited genetic gains following selection. The aim of this study was to evaluate the genetic vulnerability risk by examining the relationship between the commercial Brazilian maize germplasms and the Nested Association Mapping (NAM) Parents. For this purpose, we used the commercial hybrids with the largest market share in Brazil and the NAM parents. The hybrids were genotyped for 768 single nucleotide polymorphisms (SNPs), using the Illumina Goldengate® platform. The NAM parent genomic data, comprising 1,536 SNPs for each line, were obtained from the Panzea data bank. The population structure, genetic diversity and the correlation between allele frequencies were analyzed. Based on the estimated effective population size and genetic variability, it was found that there is a low risk of genetic vulnerability in the commercial Brazilian maize germplasms. However, the genetic diversity is lower than those found in the NAM parents. Furthermore, the Brazilian germplasms presented no close relations with most NAM parents, except B73. This indicates that B73, or its heterotic group , contributed to the development of the commercial Brazilian germplasms.A few breeding companies dominate the maize (Zea mays L.) hybrid market in Brazil: Monsanto The maize crop has drastically changed with the adoption of hybrid cultivars, in 1933 in the United States, 1944 in Brazil . AccordiExserohilum turcicum) epidemic in 1970, which elicited concern regarding the genetic diversity among American breeder hybrids [According to Romay et al. , the lim®, 35% of the market-share, followed by Dupont Pioneer® (30%), Dow Agrosciences® (15%), Syngenta® (10%), Helix Sementes® (4%), EMBRAPA (3%) and other companies (3%). According to Reif et al. [Currently, the maize crop in Brazil makes use of improved seeds in 14 million hectares , in addiyngenta® %, Helix Genetic diversity is mainly estimated through genotypic information. Due to the large increase in characterized markers and automated sequencing platforms available, the use of single nucleotide polymorphisms (SNPs) has been increasing. These markers are biallelic and have good coverage across the maize genome , which eThus, aiming to provide genetic resources for the study of quantitative traits and diversity in maize, McMullen et al.  developeConsidering all of the factors above, the objectives of this study were to estimate the risk of genetic vulnerability in the Brazilian commercial maize germplasms and the genetic kinship of these germplasms with the NAM parents.® was represented by the Dekalb® brand, while Dow Agrosciences® and Agromen Tecnologia® were self-represented. Similarly, Helix Sementes® was represented by the brands Biomatrix® and Santa Helena Sementes®. These hybrids (Population one) display high adaptability, and the majority of them are transgenic organisms  and the Mo17 inbred line [Aiming to learn the main sources of the current Brazilian germplasm, it was included along with previously described hybrids, the NAM parental genotypes , with an array of 768 SNPs [Samples of leaf tissue from each hybrid were sent to the DuPont Pioneer768 SNPs . The SNP768 SNPs  to make ® platform, and the data are available at www.panzea.org. Using the NAM genotype data, only the SNPs shared with the Brazilian commercial germplasms were analyzed.The NAM parental lines were genotyped using an array of 1,536 SNP markers on the Illumina GoldenGateThe SNP genotypic data were converted into numeric digits  by means of the Scrime package  of the RFor each SNP marker, estimates were generated among the Brazilian commercial hybrid population and for the combined population (hybrids and NAM parents) by means of PowerMarker 3.25 software :Dlpli is the frequency of the i-th allele of the l-th SNP.Gene Diversity—Ql is the sum of heterozygous loci at the l-th SNP, Pl is the sum of homozygous loci for the i-th allele at the l-th SNP, and Rl is the sum of homozygous loci for the alternative, lower frequency i-th allele at the l-th SNP.Minor Allele Frequency—MAFHlQl is the sum of heterozygous loci at the l-th SNP, Pl is the sum of homozygous loci for the ith- allele at the l-th SNP, and Rl is the sum of homozygous loci for the i-th, lower frequency allele at the l-th SNP.Heterozygosity—pli and plj are the frequency of the i-th and j-th alleles, respectively, at the l-th SNP.Polymorphism Information Content—PICxij and yij are the frequencies of the i-th allele at the l-th SNP on X and Y populations, respectively. r is the number of loci studied.From the allelic frequencies we estimated the genetic distances as described by Nei et al. :DA=1r∑jSubsequently, a dendrogram was generated using a bootstrap method , using the unweighted pair group method average (UPGMA). These bootstrap analyses were performed using the PowerMarker 3.25 software, and the node consistency dendrogram was generated using the MEGA 5.1 software .Ne) of the Brazilian commercial hybrids was obtained using the following equation:Ne estimate of the hybrid progenitors was obtained using:Fst is the withinpopulation- inbreeding coefficient of the commercial germplasm. The latter was estimated as described by Resende et al. [G is estimated by:W is the incidence matrix to fix effects of alleles at bialellic markers, and pi and qi are allelic frequencies of biallelic markers at the i-th loci. So Fst was obtained by the mean of the diagonal of the G matrix less one.The estimates of effective population size  [The identification of private alleles of each company was performed with the Convert 1.31 software . Privateene.org) .To quantify the kinship between the germplasms of the market share companies, Pearson correlation analysis was performed between the companies using the allele frequencies of the SNPs.K) ranging from 1 to 10, with 10 repetitions for each K. For each run, the Markov Chain Monte Carlo (MCMC) number was one million, with burn-in of the first 500 thousand MCMC.Aiming to identify the population structure of the elite Brazilian germplasms of maize breeding companies (Population one) and their relationship with NAM parents (Population two), two population structure analyses were performed using the Structure 2.3.4 Software . In the ΔK [L(K) is the average of the natural logarithm of the probability of the data at each step of the MCMC minus half the variance for K populations as estimated by the Structure Harvester app [To estimate the number of ancestral populations that best fit the structure of the genotypes, we used the criterion ΔK :ΔK=m[|Lster app .Qik), where Qik is the estimated proportion of the genome of the i-th genotype derived from the k-th ancestral population [Qik a bar chart of each genotype was built representing the relationship of the i-th genotype with the k-th ancestral population. Genotypes were considered to be from the same group or population when Qk exceeded 60%. Genotypes were considered to belong to the mixed population if they did not fit in any population, as previously described [Given the results of the population structure, the contribution of each hybrid to the population could be determined according to their average value of association probability , respectively. In the combined population, comprising commercial hybrids and the NAM parents , 340 comThe germplasm of the companies have low estimates of gene diversity (GD), polymorphism information content (PIC), heterozygosity (H) and MAF . The onewww.gramene.org/).Only three of the companies have private alleles, Biomatrix, DuPont Pioneer and Syngenta, with 1, 3, and 11 private alleles, respectively . Again, Fst) of the 20 hybrids was 0.08, which corresponds to an effective population size (Ne) of 18.54 individuals. In contrast, the Ne estimate of the commercial hybrid parents was 127.22 individuals.The within-population inbreeding coefficient  and the Santa Helena Sementes hybrid (SHS 5560). The second population comprised 3 Syngenta hybrids . The third population comprised the remaining Dow Agrosciences hybrids  and the Agromen Tecnologia hybrid (30A37PW). The fourth population comprised the Biomatrix hybrids , the last Syngenta hybrid (Fórmula TL), 1 DuPont Pioneer hybrid (30F53H) and 1 Dekalb hybrid (DKB 177 PRO). The fifth population comprised the remaining hybrids from Dekalb . Note that of the 5 structured populations, 2 were exclusive to Syngenta and Dekalb.The dendrogram obtained by bootstrap analysis  separateThe optimal population structure to combine the populations was estimated at K = 2 . The NAMThe dendrogram method separated the genotypes into 7 groups . Of thesThe estimates of GD and PIC were low for the companies Agromen Tecnologia and Santa Helena Sementes, due to the low number of hybrids considered for each of these companies. However, the estimates of MAF and H were informative, so there is no predominance of heterozygous loci in these hybrids.Through the estimates of GD and PIC the genetic diversity of the germplasms and the kinship between hybrids can be inferred. The greater the estimate of these indices, the greater the genetic diversity in the company germplasms and, hence, the lower the kinship between the hybrids of these companies . In thisSyngenta was notably the company with the largest number of private alleles. According to Guo et al. , privateFst between them. According to Sebbenn and Seoane [Ne, or greater similarity to the actual studied population, occurs when there is negative or close to zero genetic correlation estimates between the genotypes. In this context, the commercial American maize germplasms of the 1980s [Ne of 55.9 individuals, corresponding to a Fst of 0.84. This estimate indicates higher genetic vulnerability of these germplasms than that of the current Brazilian commercial germplasms.The results allow us to infer that the current composition of Brazilian commercial hybrids offers little risk of genetic vulnerability, considering the estimates of genetic variability and d Seoane , a highehe 1980s , showed However, it is important to emphasize that there are other factors related to genetic vulnerability that were not considered in this study , suggesting that the low vulnerability found here should be carefully considered.www.dowagro.com/br/agromen/nossa/brasil.htm, accessed January 28, 2015). Despite the high similarity between Dow Agrosciences with Biomatrix and Santa Helena Sementes, there is no known knowledge of any shared germplasms. Nevertheless, it is known that the Helix Sementes owns Biomatrix and Santa Helena Sementes. Santa Helena Sementes was acquired by Helix Sementes in 2011 and its seed stocks, germplasm bank and the commercial structure were transferred to Biomatrix . Although these companies are in the same group, they do not have as high of a correlation with each other (0.55) as they do with Dow Agrosciences. This is likely due to the short time between the exchange of material and subsequent development and launch of the evaluated cultivars.Through the estimates of allelic frequency correlation, it was observed that there are relationships between the germplasms of the different companies. Of these, the germplasm of Dow Agrosciences has the greatest similarity to other companies. The correlation between Dow Agrosciences and Agromen Tecnologia may be explained by the fact that the latter is affiliated of the former, although they have independent management and production structures. However, there is the possibility of sharing technologies and joint access to the unified germplasm bank . On the other hand, the DKB 177 PRO Dekalb hybrid was the most distinct from the hybrids from the other companies.The population structure not only indicated similarity between Dow Agrosciences and Agromen Tecnologia but also clearly indicated the most similar hybrids between them (30A37PW and 2A550PW). This confirms the free access of Agromen Tecnologia to the Dow Agrosciences germplasm bank Click here for additional data file.


Apis mellifera, queens are essential for reproduction and colony functioning. Therefore, any negative effect of these agricultural chemicals on the mating success of queens may have serious consequences for the fitness of the entire colony. Queens were exposed to the common neonicotinoid pesticides thiamethoxam and clothianidin during their developmental stage. After mating, their spermathecae were dissected to count the number of stored spermatozoa. Furthermore, their worker offspring were genotyped with DNA microsatellites to determine the number of matings and the genotypic composition of the colony. Colonies providing the male mating partners were also inferred. Both neonicotinoid and control queens mated with drones originating from the same drone source colonies, and stored similar number of spermatozoa. However, queens reared in colonies exposed to both neonicotinoids experienced fewer matings. This resulted in a reduction of the genetic diversity in their colonies . As decreased genetic diversity among worker bees is known to negatively affect colony vitality, neonicotinoids may have a cryptic effect on colony health by reducing the mating frequency of queens.Neonicotinoid insecticides can cause a variety of adverse sub-lethal effects in bees. In social species such as the honeybee,  Pollinating insects provide important ecosystem and economic services by foraging on wild plants and agricultural crops . Recent Neonicotinoids are neurotoxic insecticides that are ubiquitously employed in agriculture for pest control. The widespread use of such neurotoxic insecticides results in residual accumulation of low concentrations in the environment ,5. ActinGiven the various effects of neonicotinoids on honeybees, it is possible that queens may be particularly susceptible to neonicotinoids during the demands of mating . HoneybeExtreme polyandry of the honeybee queen results in lowering average relatedness among workers within a colony, which translates into greater genetic diversity. The ability of a queen to mate with multiple drones is paramount to her own the fitness, as well as the fitness of the colony. Extreme polyandry may benefit the colony for various reasons , includiPoor queen quality has been frequently observed by beekeepers, and is considered to be a major driver of overwintering colony mortality . RecentlGiven that factors affecting queen mating can affect colony productivity –40, and Our study did not involve endangered or protected species.A. m. carnica colonies reported by Williams et al. [We used the same six s et al. . Three ts et al. . For this et al. . Each read libitum for 36 days to ensure that young nurse workers exposed to the experimental treatments during their entire development period were available for queen rearing. Three control colonies received patties free of neonicotinoids and three treatment colonies received patties spiked with 4 ppb thiamethoxam and 1 ppb clothianidin . This is within the concentration ranges found in pollen of treated crops [Colonies were fitted with hive entrance pollen traps to limit external pollen foraging, and fed daily 100 g honey/pollen (3:1) patties ed crops ,56. Henced crops ,58. Thered crops ,49,59.Queens were collected four weeks post initial oviposition. Spermathecae were removed and placed in Kiev buffer . The numWe genotyped 20–24 worker offspring per mated queen that emerged seven weeks post oviposition initiation.Taq-polymerase and HPLC water to a final volume of 10 μl. The temperature profile for the PCR was as follows: 5 min denaturation at 95°C, 35 cycles of 30 sec each for denaturation (95°C), annealing Tm   follows :me=1∑qir using [me = Observed Effective Mating and qi the proportion of offspring sired by the ith male.We calculated the Observed Relatedness r using :r=0.25+me) and average intracolonial genetic relatedness (r) both reflect intracolonial genetic diversity. However, in the highly polyandrous honeybee, r does not measure intracolonial genetic diversity well as it quickly approaches the limit of r = 0.25 when the number of matings increase.Effective number of matings , which is the probability of obtaining two identical genotypes in two different individuals by chance, we employed [qi are the allele frequencies at the first locus, ri are the allele frequencies at the second locus, and zi are the allele frequencies at the last locus.To determine the non-detection error  or not. To estimate the number of Drone Source Colony that remained undetected because of finite sample size (“non-sampling error”) , we fittThe proportion of neonicotinoid and control queens that mated with drones originating from each Drone Source Colony was determined using Fisher’s exact test.NDE < 0.007 (2.18 drones), thus providing confidence in our data set (6 ± 1.68) was not significantly different from controls  and control  colonies. The non-detection error for not discriminating between the genotypes of two siring drones because they share the same genotype by chance was data set . Sperm CSuccessful mating of the honeybee queen is paramount to colony health and fitness . Our resMany possible mechanisms can non-exclusively explain the reduced number of queen matings caused by exposure to neonicotinoids, including behavioural, physiological, or anatomical impairment of queens . AlthougOur analyses represent a snap-shot of the intracolonial worker patriline distribution in time. Although the frequency of various subfamilies may vary over time , the totIntracolony genetic diversity generated by polyandry is an important fitness parameter that contributes to enhanced colony survival and disease resistance . AlthougOur data suggest that combined exposure to the neonicotinoids thiamethoxam and clothianidin can have a negative long term effect on colony health by reducing intracolonial genetic diversity resulting from few matings. The data highlight an important sublethal effect of neonicotinoids for eusocial species relying on one or few primary reproductives .S1 TableTweenty to 24 worker offspring  per queen were genotyped using five closely linked microsatellite loci . Alleles were scored as fragment lengths in base pairs. Colony source refers to the colony from which queens were reared. Treatments are noted “P” when the queens were exposed to neonicotinoids (thiamethoxam and clothianidin) during developmental stage, or “C” for controls.(DOCX)Click here for additional data file.


Lymnaea stagnalis. We estimated family-level variation and covariation in life history  and constitutive immune defence traits  in snails experimentally exposed to typical (15 °C) and heat wave (25 °C) temperatures. We also assessed variation in the reaction norms of these traits between the treatments.On-going global climate change poses a serious threat for natural populations unless they are able to evolutionarily adapt to changing environmental conditions . A prerequisite for evolutionary change is within-population heritable genetic variation in traits subject to selection. In relation to climate change, mainly phenological traits as well as heat and desiccation resistance have been examined for such variation. Therefore, it is important to investigate adaptive potential under climate change conditions across a broader range of traits. This is especially true for life-history traits and defences against natural enemies (e.g. parasites) since they influence organisms’ fitness both directly and through species interactions. We examined the adaptive potential of fitness-related traits and their responses to heat waves in a population of a freshwater snail, We found that at the heat wave temperature, snails were larger and reproduced more, while their immune defence was reduced. Snails showed high family-level variation in all examined traits within both temperature treatments. The only negative genetic correlation  appeared at the high temperature. However, we found no family-level variation in the responses of most examined traits to the experimental heat wave . Only the reduction of PO-like activity when exposed to the high temperature showed family-level variation, suggesting that the cost of heat waves may be lower for some families and could evolve under selection.Our results suggest that there is genetic potential for adaptation within both thermal environments and that trait evolution may not be strongly affected by trade-offs between them. However, rare differences in thermal reaction norms across families indicate limited evolutionary potential in the responses of snails to changing temperatures during extreme weather events.The online version of this article (doi:10.1186/s12862-017-0988-x) contains supplementary material, which is available to authorized users. Natural populations of numerous species are currently threatened by anthropogenic environmental changes such as habitat loss, chemical pollution, and invasive species. One of the factors with wide ecological effects is global climate change –4. ClimaA necessary prerequisite for evolutionary change by means of natural selection is within-population heritable genetic variation in the traits subject to selection. Typically, fitness-related traits are considered to show heritable variation and thus evolutionary potential in nature . This isLymnaea stagnalis. We chose to focus on heat waves since their increasing frequency and severity has been suggested to have an even greater impact on natural populations than the gradual increase in average temperatures )Drosophila, especially species with narrow geographical distribution show very little genetic variation in desiccation and cold resistance )L. stagnalis snails responded to an experimental heat wave by increasing reproduction and size while reducing expression of immune defence traits. Most of these traits showed high family-level variation at both control and heat wave temperatures. However, responses to the high temperature were similar in most traits across different families . Only PO-like activity showed family-level variation in reaction norms between temperatures, which suggests that the cost snails need to pay for increased reproduction and size shows genetic variation and thus potential for adaptive evolution. The only genetic trade-off was found at the high temperature. Thus, our results suggest abundant adaptive potential in the examined traits, but only limited genetic variation in their thermal reaction norms.L. stagnalis inhabits lakes and ponds throughout the Holarctic region. When exposed to high temperatures it is known to initially increase reproduction and growth [L. stagnalis is an important host for various parasites, including several highly virulent trematode species [The hermaphroditic snail d growth . After 1d growth , 50. Thi species , 52 that species , 54. Thu species , 50 can 3 generation) originating from a pond in Zürich, Switzerland . In this region, the summer water temperature in ponds typically remains low (< 16 °C), although it depends on the hydrology of the ponds . During heat waves, water temperature can, however, rapidly increase to 20–30 °C and remain high for over 2 weeks . To start the stock population, we collected 45 adult snails from the pond. Since L. stagnalis prefers outcrossing [The snails we used for breeding experimental snails came from a laboratory stock population .Spirulina until they reached approximately 5 mm shell length. After that, we fed the snails with spinach and lettuce ad libitum. We changed water in the containers once a week. When the snails from all families were mature (shell length > 20 mm), we randomly chose 40 snails per family and placed them individually in 0.2 L perforated plastic cups sunk into a water bath (15 ± 1 °C) as above. Since pathogen resistance in snails may show age-related patterns [L. stagnalis is a simultaneous hermaphrodite [We haphazardly collected 15 adult snails (shell length > 20 mm) from the stock population to produce experimental snails. We maintained these snails individually in 0.2 L perforated plastic cups that were sunk into a water bath (15 ± 1 °C) connected to a biological filter. We fed the snails with fresh lettuce ad libitum and maintained them until each of them had produced two egg clutches (> 30 eggs per clutch). We used these clutches to obtain family lines  for the experiment and transferred each clutch individually into plastic containers with 6 L of aged tap water (15 ± 1 °C). After the juveniles hatched, we fed them with patterns , it is iphrodite  and can phrodite , snails Using maternal sibships, our goal was to estimate whether the responses of snails to high temperatures vary across different genetic backgrounds (i.e. families), and how much of the phenotypic variation in the examined traits is expressed at the family level. It is important to note, however, that variation among maternal sibships may not directly reflect the amount of additive genetic variation in the examined traits. This is because variation among families could also be due to other genetic factors such as dominance variance, and/or due to non-genetic maternal effects (reviewed in ). It is We maintained the snails as described above and randomly exposed them to two different temperature treatments (15 ± 1 °C and 25 ± 1 °C). We chose 25 °C as a high (i.e. heat wave) temperature as it lies above the thermal optimum of snails  and occuTo examine whether possible family-level variation in traits studied could be due to differences in neutral genetic variation caused, for example, by inbreeding, we assessed heterozygosity at microsatellite loci in snail families, as described in Additional file To estimate the reproductive output of snails, we collected all the egg clutches oviposited by the snails during the experiment and photographed them from approximately 10 cm above with a Fujifilm FinePix F30 digital camera . From each picture, we measured the two-dimensional area of the clutch that contained eggs using ImageJ software . Then, we measured the area containing 10 eggs in each clutch and calculated an estimate of the total number of eggs in the clutch. We summed up the number of eggs in the clutches oviposited by each snail to get a measure of its reproductive output during the experiment.L. stagnalis.We measured haemocyte concentration, PO-like activity, and antibacterial activity of snail haemolymph to examine variation in snail immune function. In invertebrates, haemocytes, through phagocytosis, constitute the main part of the cellular immune response . FurtherEscherichia coli cells, and measured the decrease in OD of the solution followed by a reaction in which antibacterial enzymes lyse E. coli cells.We took the haemolymph samples and measured the immune parameters as described in . BrieflyN = 76; PO-like activity: N = 67; antibacterial activity: N = 64) to estimate repeatability (R) of the measurements. Repeatability describes the proportion of variance in a character occurring among, rather than within, individuals. We calculated it from variance components derived from an analysis of variance (ANOVA) where snail individual was used as a factor [R = 0.985, F76, 77 = 33.630, P < 0.001; PO-like activity: R = 0.985, F67, 68 = 33.001, P < 0.001; antibacterial activity: R = 0.880, F64, 65 = 3.487, P < 0.001).We measured all immune parameters twice from a subsample of snails  software. To examine the effect of temperature on the probability of snails reproducing during the experiment, we used a generalized linear model (GLM) with the reproductive status of snails  as a binomial response variable. In the analysis, we used temperature as a fixed factor. Since there was no variation in some families and blocks  we could not include family and block as factors in the model. After that, we analysed the variation in the number of oviposited eggs using only those snails that reproduced using an ANOVA. In the model, we used temperature as a fixed and family as a random factor. Additionally, we included the main effect of block (random factor) to reduce possible noise in the data that could arise from examining the snails on different days. To meet the assumptions of ANOVA, we ln transformed the number of produced eggs.To analyse the variation in snail size and immune parameters  we used similar ANOVAs as above. To meet the assumptions of ANOVA, we ln transformed haemocyte concentration and PO-like activity. When statistically significant temperature-by-family interactions were observed in the above analyses, we analysed the variation in response variables separately for different temperatures. We did this to examine whether family-level variation was observed under both environmental conditions. Snails that did not reproduce were excluded from the analyses of snail size and immune parameters. This was done because reproduction can be traded off with these traits  and the In addition to the above ANOVAs, we examined the variation in responses of snails to temperature treatments by analysing the data using the approach in . We formt-values to assess their statistical significance as the quotient of each genetic correlation and its standard error.We calculated genetic correlations among traits separately in both temperature treatments using components of covariance and variance estimated, based on sums of cross-products and sums of squares in the analyses of covariance and variance, respectively . We incl


Ochotona curzoniae). Totally, 1,352 individual samples were collected, and 10 microsatellite loci were analyzed. Results revealed that plateau pika possessed high genetic diversity and inbreeding coefficient in a fine‐scale population. Dispersal distance is short and restricted in about 20 m. An effective sex‐biased dispersal strategy is employed by plateau pika: males disperse in breeding period for mating while females do it after reproduction for offspring and resource. Inbreeding avoiding was shown as the common driving force of dispersal, together with the other two factors, environment and resource. In addition, natal dispersal is female biased. More detailed genetic analyzes are needed to confirm the role of inbreeding avoidance and resource competition as ultimate cause of dispersal patterns in plateau pika.Dispersal is an important aspect in organism's life history which could influence the rate and outcome of evolution of organism. Plateau pika is the keystone species in community of grasslands in Tibetan Plateau. In this study, we combine genetic and field data to character the population genetic pattern and dispersal dynamics in plateau pika ( They mainly distribute within and around the Tibetan Plateau. They are highly social burrow dwellers with seasonally breeding , Tibetan Autonomous Prefecture of Golog, Qinghai Province, People's Republic of China, where the average altitude is about 3,846 m , observed heterozygosity (Ho), expected heterozygosity (He), allelic richness (AR), gene diversity (GD), polymorphism information content (PIC), and Hardy–Weinberg equilibrium  repetitions and a 100,000 burn‐in period. The most likely K was identified by web‐based tool STRUCTURE HARVESTER v0.6.8 , mean/variance of assignment index (mAIc/vAIc), and the average relatedness of individuals within a population relative to the whole sample  were analyzed for both sexes using 10,000 permutations in the menu of biased dispersal.Assignment tests were executed to evaluate the sex‐bias dispersal of population via the program FSATAT version 2.9.3.2 Goudet, . Genetic2.8r by drawing with replacement within the set of relevant pairwise comparisons for a given distance class. Second, the standard spatial autocorrelation analysis of sexes was performed in separate populations, and then the nonparametric heterogeneity tests were computed  were computed. In our study, the distance class is 10 m (first distance class is 0–10 m). The first distance class was determined based on the family zone of plateau pika from previous study and our observation of field  ranges from 0.11 to 0.21 among 5 years , but it is in contrary with the observation in the field. Considering the result of observation, the clusters are defined 22 (K = 22).We identified genetic clusters following the procedure described by Evanno et al. . In inde3.3FST and r), assignment index (mAIc and vAIc), or inbreeding index , as indicated by significantly lower spatial autocorrelation coefficients of males in May to July, while the result is opposite in August. The result indicates that male dispersed while female was more philopatric in mating period from May to July, but the situation reversed in the end of breeding in August Figures  and 4.For immaturity group, both sexes displayed higher spatial autocorrelation coefficients in May and June, while male was with much higher than female in July and August Figures  and 4. I44.1Genetic diversity is an important aspect of population dynamics which directly relates with evolutionary potential and inbreeding  is considered as a great limiting factor for performance of alpine biota. So in this analysis, dispersal time has been in full consideration. The timing of dispersal could provide interesting clues to ultimate pattern. Natal dispersal was thought of a fundamental parameter in life history, and population biology , the dispersal is also related to breeding avoiding, that is reducing the risk of father–daughter mating (Lawson Handley & Perrin, The dispersal strategy of plateau is effective, but there are still some defects. Firstly, it is cannot completely avoid inbreeding Table . This maNone declared. Click here for additional data file. Click here for additional data file.


Grapes are one of the most economically and culturally important crops worldwide, andthey have been bred for both winemaking and fresh consumption. Here we evaluate patternsof diversity across 33 phenotypes collected over a 17-year period from 580 table and winegrape accessions that belong to one of the world’s largest grape gene banks, thegrape germplasm collection of the United States Department of Agriculture. We find thatphenological events throughout the growing season are correlated, and quantify the markeddifference in size between table and wine grapes. By pairing publicly available historicalphenotype data with genome-wide polymorphism data, we identify large effect locicontrolling traits that have been targeted during domestication and breeding, includinghermaphroditism, lighter skin pigmentation and muscat aroma. Breeding for larger berriesin table grapes was traditionally concentrated in geographic regions where Islampredominates and alcohol was prohibited, whereas wine grapes retained the ancestralsmaller size that is more desirable for winemaking in predominantly Christian regions. Weuncover a novel locus with a suggestive association with berry size that harbors asignature of positive selection for larger berries. Our results suggest that religiousrules concerning alcohol consumption have had a marked impact on patterns of phenomic andgenomic diversity in grapes. Vitis vinifera L.), one of the first domesticated perennials, originatedin the Near East 5000–8000 years ago1 andremain an economically and culturally important crop. In 2015, grapevines covered 7.5million hectares and produced 76 million tons of grapes globally.2 Over the past millennia, human selection for traits of interest,especially those important to fruit production, have shaped the appearance of grapes. Inparticular, selection for hermaphroditic flowers increased grape production, aspropagating both male and female plants was no longer required. While nearly half of allgrapes grown are vinified into wine, 36% are consumed fresh and the rest are dried or usedfor juice.2 Desirable berry traits differdepending on the use of the grapes, and, thus, the different breeding targets for tableand wine grapes have led to differences in berry and bunch size.5There is also evidence of selection for white berry color.5Grapes  in grapes offered a cost-saving of 16–34%.9While grape breeding has resulted in selection for several traits over the pastmillennia, current consumer preference is focused on a small number of elite cultivars. Asa result, most grape cultivars have been grown for centuries—such as ‘PinotNoir’, which has existed for more than a millennium—using vegetativepropagation. These genetically frozen cultivars are highly susceptible to continuallyevolving pathogens.10 Usinggenetic markers, individuals can be tested for a trait at the seed or seedling stage.Thus, MAS offers the greatest potential for traits that are difficult and expensive tophenotype, such as disease resistance, or time-consuming to measure, such as fruit traitsonly visible after several years.8 WildVitis relatives have been previously used for hybrid grapebreeding11 and are a promising source ofresistance loci for introgression through MAS.12For example, V. arizonica was used in the development of Pierce’sdisease-resistant wine grapes,13 whileMuscadinia rotundifolia was used to pyramid resistance from both powdery anddowny mildew into V. vinifera.14Markers have also been identified for many other traits in grape including berrycolor,16flower sex,17 seedlessness18 and muscat aroma.19The ability to save time and money when breeding makes grapes an attractive candidate forMAS.20 Decreasing DNA sequencing costs will continue toaccelerate both marker discovery and the implementation of MAS in grape breeding. Whilesequencing costs have decreased, phenotyping remains a slow and expensiveprocess.21 Fortunately, historical phenotypeinformation already available in gene banks can be linked with genomic information forgenetic mapping of important traits. The ability to leverage historical data from genebanks for genetic mapping has previously been demonstrated in potato,22 barley23 andapple.24 Similarly, in grape, years ofphenotype information may already be available and exploitable for the purposes of geneticmapping. Unfortunately, standardized data formatting and annotation are not yet widelyadopted in grapevine and remain an essential goal.25The discovery of markers for agriculturally important traits has facilitated the use ofMAS in grapes; however, the technique is only worthwhile when the cost of phenotyping ishigher than the cost of discovering new markers and genotyping cultivars.V. vinifera accessions from the United StatesDepartment of Agriculture (USDA) grape germplasm collection. We report several significantgenome-wide association study (GWAS) results, demonstrate the use of signatures ofselection as complementary to GWAS and find that phenotype relationships as well aspatterns of genetic variation have been shaped by human culture and geography.To investigate the history of selection in grape, as well as the future potential of MAS,we evaluated associations between 33 phenotypes and 6114 genome-wide single-nucleotidepolymorphisms (SNPs) in 580 et al.26 Briefly, samples were collected from the USDA grape germplasmcollections in Davis, CA, and Geneva, NY. DNA was extracted using commercial extractionkits. A custom Illumina Vitis9KSNP array  assaying 8898 SNPs wasused to generate genotype data.27 Followingquality filters , 6114 SNPs with<20% missing data in 1817 Vitis samples remained for analysis.26The sample collection and genotype calling for the accessions used in this study werethe result of previous work described in Myles http://www.ars-grin.gov). Only accessions reliably identified as V.vinifera in Myles et al.26were included. Measurements for flower sex were combined across years and samples withdiscordant values for flower sex across years were removed. Additional phenotype dataincluding skin color, berry length, berry width, berry size and cluster density werecollected as part of the present study. Our cluster density measures were merged withmeasurements available from GRIN, and when discrepancies between measurements existed,those values were removed. In some cases, phenotype data were recoded to facilitategenetic mapping. A complete description of the phenotype data, including recodingprocedures, is available in Phenotype data were downloaded from the USDA Germplasm Resources Information Network between all pairwise phenotype comparisons were computedusing R v3.2.0.29 Correlations betweenbinary/binary, quantitative/quantitative and quantitative/binary phenotype pairs weretested using Pearson’s correlation. Correlations between quantitative/ordinal andbinary/ordinal phenotype pairs were tested using Spearman's rank correlationcoefficient. Finally, correlations between ordinal/ordinal phenotype pairs were testedusing Kendall’s rank correlation. To correct for multiple comparisons, aBonferroni correction was applied by multiplying P-values by the number ofpairwise comparisons (528).The correlations  as well as geographic origin as predictors. McFadden’s pseudo-R2 was calculated forlogistic regression using the ‘pscl’ package32 in R v3.0.1. We define the phenotypic variance explained as theR2 of these models, for PC1, PC2 and PCs 3–10.Before assessing population structure, the genotype data were pruned forlinkage-disequilibrium (LD) using PLINK by considering a window of 10 SNPs, removing 1of a pair of SNPs if LD>0.5, and then shifting the window by 3 SNPs and repeating theprocedure. Principal component analysis was performed on the resulting 3196 SNPsgenotyped in 580 accessions using the smartpca program in the EIGENSOFTpackage.28 resulting in 4602 SNPs genotypedin 580 accessions. Missing genotype data were then imputed using LinkImpute33 with optimized values of 7 for parameter kand 23 for l, resulting in an estimated accuracy of 88.8%. Genomic predictionwas performed on all phenotypes using imputed data and the x.val function in the Rpackage PopVar.34 We selected the rrBLUP modeland assessed prediction accuracy with a fivefold (nFold=5) cross-validation procedurerepeated three times (nFold.reps=3) and no further filtering (min.maf=0). All otherdefault parameters were used. The seedlessness phenotype was removed for this analysisbecause of an uneven binary trait distribution, which did not allow forcross-validation. Genomic prediction accuracy was calculated as the correlation(r) between the predicted phenotypes and the observed values.To perform genomic prediction, SNPs with a MAF threshold of <0.01 were removed usingPLINK,35 and anidentity-by-state kinship matrix calculated using the default EMMAX settings. The numberof accessions included for each GWAS varied based on the phenotype, and, thus, a minorallele frequency (MAF) threshold of 0.01 was applied for each GWAS individually,resulting in 4409–4680 SNPs.GWAS were performed using a linear mixed-model method, efficient mixed-modelassociation expedited (EMMAX)36 asin Myles et al.26 SNPs with aMAF>0.05 and <10% missing data were included, resulting in 3397 SNPs. To identifypotential regions of the V. vinfera genome under selection during domesticationand breeding, we calculated the xpEHH statistic across the genome using selscansoftware.37 The xpEHH statistic requiresthe user to divide the samples into two groups to identify regions of the genome withunusually long stretches of low haplotype diversity in one group compared with theother. As this pattern of haplotype diversity is expected in regions subjected topositive selection, the identified regions are considered candidate regions that mayharbor functional variants selected for during domestication or breeding. For berrysize, we compared accessions with large berries  to accessions with small berries . For skin color, we compared dark-skinned accessions to light-skinned accessions . We also compared groups divided based on several binary traits:Muscat versus non-Muscat, dioecious accessions versus hermaphrodites, and table versuswine grapes.Haplotypes were inferred using fastPHASEWe analyzed each of the 33 phenotypes in this study to uncover potential relationshipsbetween phenotypes, as well as confirm the reliability of the data. A correlation matrixbetween all phenotypes is shown in r=0.89,P<1×10−15) and berry size and weight. Thus, berries that arelarge according to one measure, such as length, also tend to be larger in othermeasures, such as width. In addition, all berry size measurements were positivelycorrelated with berry firmness . Both large size and berry firmness aredesirable traits in table grapes,38 and mayhave both been targeted by table grape breeders.We found that all measurements describing berry size were significantly correlated,including berry length and width  and size, indicatingthat larger berries were found in less dense clusters. This observation may have arisenfrom the divergent breeding targets of table and wine grape breeders: less denseclusters of large grapes are preferred in table grapes and smaller more densely packedberries are preferred in wine grapes.38 Asexpected, larger berries also tend to be found on larger clusters.In addition to larger berries being firmer, we found that all berry size measurementswere negatively correlated with cluster density including length and width . Tartrate synthesis stops at veraison,the onset of ripening; therefore, an increase in berry size post veraison dilutes theamount of tartaric acid in the berry.39Similarly, larger berries also tend to have a lower concentration of sugar.40 We found a negative correlation between lab brix andberry size , aswell as all other berry size measurements, providing further evidence that sugarconcentration decreases as berry size increases.Titratable acidity, or the concentration of tartaric acid, was negatively correlatedwith berry size measurements including length , leaf date andbloom date , bloom dateand veraison  are allsignificantly correlated. Thus, the timing of a grapevine’s escape from dormancyis predictive of subsequent developmental milestones, like bud break, leaf productionand the onset of veraison. This observation suggests that the genetic control ofgrapevine phenology may be at least partially coordinated by a single regulatorymechanism, rather than independent mechanisms for each developmental event.Several phenological events, describing the timing of development, were assessed byvarious researchers and deposited into the USDA-GRIN database. Despite the noiseexpected from data collection across multiple years by multiple observers, manyphenology traits are correlated with each other. For example, bud burst date and leafdate , width, size and weight whencompared with wine grapes. Smaller berries may be beneficial for winemaking, as smallerberries often have more desirable characteristics for vinification, including highersugar concentration.41 Consistent with previous work, we also found that titratableacidity  andsugar content were significantly higher in wine grapes.42–45In addition to the relationships between phenotypes, we examined differences betweenaccessions based on use (table and wine) and geographic origin  and had significantly less denseclusters  than winegrapes, indicating that selective breeding likely created a divergence in these traitsbetween table and wine grapes.In addition to berry size, cluster density is important for table grapes, as verycompact clusters are often damaged during packing and transport. Broken berries leakjuice, which may spoil the entire cluster. A firm pulp texture that is not easily brokenis therefore essential for table grapes.W=10564.5, P=4.65×10−8) and larger insize including length ,width  and weight. Eastern accessionsalso had less dense ,longer  and heavier clusters when comparedwith the West.We also compared each phenotype for grapes originating in the East, primarily theMiddle East, to those accessions originating in the West, primarily Western Europe. Grapes 6 Conversely, as Christianity doesnot prohibit alcohol consumption and it has been the dominant religion in WesternEurope, grapes from the West have generally been selected for their ability to producehigh-quality wine. Thus, an analysis of the phenotype data alone reveals the stronginfluence of religion on shaping global patterns of grape phenotypic diversity.The similarities between phenotype comparisons based on use and geography are expected,given that most table grapes are from the East and most wine grapes are from the West.In our data set there are 282 accessions with use, geography and phenotype information:30% are Eastern table grapes but only 16% are Western table grapes. In comparison, 51%of the accessions are Western wine grapes and only 4% are Eastern wine grapes . The pauW=1819,P<1×10−15) as well as table and wine grapes and origin  and plotted along PC1 and PC2. The prithe West , it is nPhenotypes that are strongly correlated with population structure are more likely tohave been targeted by selection. Moreover, as population structure is a confoundingeffect in GWAS, phenotypes strongly correlated with population structure can beproblematic to map using association mapping. We therefore examined the degree to whicheach phenotype is correlated with population structure. We found that the proportion ofthe phenotypic variance explained by genetic PCs 1 through 10 ranged from 2 to 43%across phenotypes . Most no46 A single grape cultivar ‘Sultanina’ is a primarysource of seedlessness in table grapes and is a parent of many commercial seedless tablegrape varieties.48 Consistent with these observations, previous work on theaccessions studied here found that Sultanina has 28 first-degree relationships  with other accessions in our dataset.26 The repeated use of ‘Sultanina’ in thebreeding of seedless accessions, and the resulting high degree of relatedness among allseedless accessions, is a likely contributor to the correlation between seedlessness andpopulation structure observed here.In addition to berry traits, the only other phenotype for which the first 10 genotypicPCs explain over 30% of the phenotypic variance is seedlessness (38%). In contrast toberry phenotypes, only a small proportion of the variance in seedlessness is explainedby PC1 (5%). Instead, PCs 3–10 explain 31% of the total variance. Seedlessness isa valued trait in commercially grown table grapes.49 Usingfivefold cross-validation, we calculated prediction accuracies for all phenotypes(r) range from 0.10 for leaf size to 0.76for berry length. We detected the highest prediction accuracies for phenotypesdescribing berry traits including berry length (0.76), size (0.74), shape (0.68), width(0.66), skin color (0.65), weight (0.63) and firmness (0.58). These predictionaccuracies are slightly higher than those previously reported in apple and rice, whichhad a maximum value of 0.55 for harvest season and 0.63 for flowering time,respectively.50 Complex quantitative traits such as those describing berry shapeand size are better targets for improvement through genomic prediction than from singlemarker MAS. A genomics-assisted breeding scheme in which both MAS and GS areincorporated has been proposed in apple and may be a viable option in order to selectfor both monogenic and polygenic traits in grape.51An extension of using PCs to explain phenotypic variance is to perform genomicprediction, which uses all markers to predict phenotypes. Especially for complex traitscontrolled by numerous small effect loci, genomic prediction is emerging as a powerfultool in genomics-assisted breeding.enotypes. Predictet al.,24 genomic prediction accuracy was also highlycorrelated with the proportion of phenotypic variance explained by genetic PCs1–10  believed to harbor thecausal flower sex locus.56 We thereforedemonstrate that, even with only 50 accessions (9% of the sample) carrying the ancestraldioecy phenotype, we successfully map the flower sex locus at relatively high resolutionusing GWAS relative to traditional linkage-mapping approaches.A key transition in grapevine domestication was the switch from dioecy tohermaphroditism: all wild mosome2 . The mos57 However, this region is several Mb from the locushighlighted in 58 It is unclear why suchmismapping occurs with the Vitis9KSNP array data, but unexpected hybridization ofnon-targeted paralogous regions may possibly contribute to these observations.A genome-wide Fst scan comparing dioecious to hermaphroditic accessions also revealedthat the SNP most strongly associated with flower sex had the highest Fst valuegenome-wide, consistent with the effect of selection for hermaphroditism at this locus. If grapMYBA1 gene results in a loss in pigmentationby disrupting anthocyanin biosynthesis.59 Although rare, white-skinned grapes have beenobserved without this associated retrotransposon insertion, suggesting that thisphenotype has arisen via mutation elsewhere in the genome.60 We find SNPs significantly associated with skin color within adiffuse peak on chromosome 2 between 10 and 17 Mb (VvmybA1 gene, the most significantly associated SNP found here is3.6 Mb from the known causal mutation. Our inability to map the known color locuswith precision is consistent with results from rice61 and Arabidopsis62where markers with the strongest association signals were not found directly at knowncausative loci. Moreover, this result is unsurprising given the relatively low markerdensity of the SNP array employed here.Skin color in grapes is largely controlled by a single locus on chromosome 2, where aretrotransposon insertion in the nd 17 Mb . AlthougVitis, while white skin color likely arose after thedomestication of V. vinifera and was subsequently targeted during the breedingof both wine and table grapes.1 We observeextreme Fst values and a significant reduction of haplotype diversity in light-skinnedgrapes that overlap with our observed association signals for skin color (1While the diffuse association signal for grape color spanning nearly 7 Mbindicates that we have poor mapping resolution for this phenotype, it also suggests thepresence of long-range LD potentially caused by selection. Dark skin is the ancestralstate in the genus in color . These oDXS, on chromosome 5.63–65 We scored muscat aroma as abinary trait by simply assigning the 29 accessions carrying the word‘muscat’ in their names to one group, and assigning the remaining 491accessions to another group. The two SNPs exceeding our GWAS significance threshold didnot colocate with the known locus, but were instead located on chromosome 8 and anunanchored contig (26 While GWAS alone would not have allowed us tounequivocally identify the muscat locus without prior knowledge of its location, thepresence of strong signals of positive selection for muscat aroma directly adjacent tothe DXS gene (In addition to flower sex and color, muscat aroma is a phenotype that was likelytargeted by breeders and is inherited in a largely Mendelian manner. Grapes with themuscat aroma are characterized by high concentrations of monoterpenoids whose expressionis largely controlled by a nonsynonymous mutation in a transcription factor,d contig . HoweverDXS gene  providesOur detection of overlapping signals of association and positive selection at the knownloci underlying flower sex, color and muscat aroma suggests that combining GWAS andselective sweep mapping can reveal genomic regions underlying traits targeted by grapebreeders. Our observation of marked differences in berry size between table and winegrapes suggests that large berry size was likely also a target of selection during tablegrape breeding. A GWAS for berry size did not result in any SNPs exceeding oursignificance threshold . We reasGiven the difficulty of mapping berry size using GWAS, we aimed to identify suggestivepeaks that also show evidence of positive selection. Of the peaks identified in We demonstrated that the primary axis of genetic structure differentiates wine fromtable grapes , and thaGene banks are often characterized phenotypically and the data are frequently madepublicly available. An analysis of historical phenotype data collected over a 17-yearperiod from the USDA grape germplasm collection revealed novel insights into patterns ofgrape phenotypic diversity, and enabled high-resolution genetic mapping when paired withgenomic data. LD decays rapidly in grapes and the SNP density in the current study isarguably inadequate for well-powered GWAS. However, we demonstrate that a modest number ofgenetic markers is sufficient to uncover loci targeted during domestication and breedingbecause of the extended LD at these loci caused by positive selection for hermaphroditism,lighter skin pigmentation and muscat aroma. We extend this reasoning to uncover a novellocus associated with berry size that harbors a signature of selection, and suggest thatpatterns of nucleotide diversity at this locus have been shaped by table grape breedersselecting for larger berries predominantly in regions where alcohol consumption has beenprohibited. The present study reveals how religious rules concerning alcohol consumptionhave had a marked impact on patterns of phenotypic and genetic diversity in grapes, thushighlighting the powerful role of human culture in shaping the genomes and phenomes ofagricultural species.