The horse is a mammalian species showing a high variation among maternal lineages but a limited variability in the paternal inheritance. The female phylogenetic history is commonly investigated by analyzing the maternally transmitted mitochondrial DNA (mtDNA), while the male perspective is provided by the paternally inherited portion of the Y chromosome (NRY). Here we explored the variation of both non-recombining genetic systems in three horse breeds present in Sardinia: Giara, Sarcidano, and Sardinian Anglo-Arab. The analysis of 34 stallions revealed three differentially distributed NRY types: (i) the first and most ancestral one is typical of Sarcidano; (ii) the second is well represented in Giara and seems to derive from Neapolitan/Oriental stallions; (iii) the third confirms the Thoroughbred influence in the Sardinian Anglo-Arab breed. By extending the analysis to 178 mtDNAs, we observed a common maternal origin for Giara and Sarcidano. Contrarily, the outlier behavior of the Sardinian Anglo-Arab is due to its higher mitochondrial variability, testifying for multiple maternal lineages in its current population. Our preliminary findings highlight the importance of a parallel molecular screening of NRYs and mtDNAs to reconstruct both paternal and maternal phylogenetic histories and to fully evaluate the extent of autochthonous genetic resources in the island.Sardinia, an island located to the west of Italy in the Mediterranean Sea, boasts three native horse breeds: Giara, Sarcidano, and Sardinian Anglo-Arab. Here, we have investigated for the first time three loci of the non-recombining region of the Y chromosome (NRY) in 34 stallions from these breeds and performed a phylogenetic analysis of the maternal relationships among 178 previously published mitochondrial control regions. We found that the current NRY diversity of Sardinian horse breeds is linked to three haplotypes (HT), all identified within Sarcidano. Each breed showed a typical HT: HT1  was the most represented in Sarcidano, HT2  in Giara, and HT3 (Thoroughbred wave) in Sardinian Anglo-Arab. The specificity of each haplotype suggests the influence of independent breeding strategies and the effect of genetic drift in each Sardinian population. The female counterpart, extended to 178 horses, showed a low genetic variability and a common maternal origin for Giara and Sarcidano. The higher variability of the Sardinian Anglo-Arab indicates multiple mare lineages in its current population. Further genetic analyses will be crucial to understand the paternal history of male horses, preserve the endangered mares’ and stallions’ lineages, and improve the enhancement of autochthonous genetic resources on this island. Among them, there are three horse breeds native to this island that are classified “at risk” by FAO  , is one of the 15 indigenous horse “breeds of limited distribution” in Italy recognized by the AIA, the Italian breeders’ association. The origin of these animals is shrouded in mystery. Even if it is considered an ancient native breed, some scholars claim that they were introduced from the Middle East. In 1540, Sigismondo Arquer was the first to state that there were these wild horses in Sardinia [http://www.aia.it) as “of limited distribution”, with 115 animals  . With the Giara breed, their local status is critical. They are both bred in semi-feral conditions and recorded in the Anagraphic Register (http://www.anagrafeequidi.it).The Sarcidano Horse is a rare semi-feral breed originated from the plateau of Sarcidano in the Laconi municipality (province of Oristano). It is also recognized by the AIA  and male (Y chromosome) ancestors ,10,11,12On the other hand, the female counterpart of modern horses usually manifests a high genetic variability. In particular, the mitochondrial DNA (mtDNA) control regions of Sardinian horse breeds were already analyzed together with other Italian breeds, highlighting a genetic structure for these breeds, who live in geographically isolated contexts ,14. PrevThe aim of this study was to investigate and reconstruct the paternal and maternal phylogenetic histories of three Sardinian horse breeds   by screeThe analyses were conducted on a dataset of Sardinian horse DNAs previously collected and reported also in Cardinali et al., 2016  and GionFor the Sardinian Anglo-Arab, genealogical information recorded in the Studbook was considered in order to select maternally unrelated samples belonging to dam lines with at least 100 descendants and deriving from all important and widespread sire lines. For the Giara and Sarcidano breeds, living in semi-feral conditions, sampling was performed during the late summer when the drought forces herds to reach the few available springs located within confined areas. These are equipped with corridors or small capture paddocks for the identification of subjects through a transponder, or any health treatments and biological sampling. In these peculiar circumstances, individuals were randomly sampled taking care to select specimens belonging to different groups in order to reduce the probability of sampling the genealogically closest subjects. Groups mainly consist of: solitary subjects (without herd); herds with a dominant stallion, about 8–10 mares and young foals; herds of only sexually mature young males.Our complete dataset resulted in a total of 125 DNA samples  including 34 stallions.Three polymorphic sites of the NRY  were analyzed for the 34 stallions belonging to the Sardinian breeds  as reported in Wallner and colleagues  Table S.Equus caballus haplotype HT1 Y chromosome locus YE3 (JX646942.1), locus YE17 (JX646950.1), and locus YXX (JX647030.1) for the haplotype annotation, and their evolutionary relationships were evaluated through a median-joining tree built using Network software v.10.0 [Referring to Wallner et al., 2013 , we seleN = 39), Sarcidano , and Sardinian Anglo-Arab  (N = 30) and Sarcidano (N = 23) sequences available from GenBank [To provide a more comprehensive overview of the autochthonous horse breeds in Sardinia, the DnaSP 5.1 software  was used N = 56) . Then, w GenBank ,16 , from the nucleotide position (np) 10,592 to np 11,330 for the locus YE3, from nps 1240 to 1410 for YE17, and from nps 25,342 to 25,480 for YXX, showed three different haplotypes  [The Sarcidano is the only breed showing all three haplotypes. On the other hand, each breed is characterized by a most represented HT: HT1 in Sarcidano, HT2 in Giara, and HT3 in Sardinian Anglo-Arab .The overall alignment of 610 base pairs, from np 15,491 to np 16,100, of the 125 control-region sequences from Cardinali et al., 2016  and GionWe observed similar values for Giara and Sarcidano in terms of haplotype diversity  and number of haplotypes , and higher values for Sardinian Anglo-Arab .By analyzing a shorter mtDNA control region , in order to add another 53 published sequences from Giara and Sarcidano, we observed a total of 47 haplotypes, of which nine were shared between two or more Sardinian breeds while the other 38 were present in only one breed: 11 for Giara, 10 for Sarcidano, and 17 in Sardinian Anglo-Arab .All haplotypes were classified in haplogroups, except for three sequences  from Morelli et al., 2014 [Even if whole-genome approaches are now opening up new clues on the livestock genetic complexity and admixture, the two uniparental systems are still widely used to solve questions about breed origins and demographic processes. The traceability and characterization of female and male livestock lineages also offer a unique opportunity to conserve genetic resources and to promote and defend local breeds ,20,21. TThe current Y-chromosomal diversity of Sardinian horse breeds is linked to three haplotypes (HT1-3) that have been previously identified, at relatively high frequencies, across a broad range of horse breeds . The ancThe presence of only six haplotypes in European horses and the low microsatellite variability ,23,24,25The unexpected high value of the overall haplotype diversity in Sardinian breeds is mostly driven by the Sardinian Anglo-Arab that presents 31 of the 38 haplotypes identified in our dataset . The lowTo include all available data, for a total of 178 horses, we performed the network analysis after sequence trimming to make the mtDNA dataset homogeneous. The analysis revealed a total of 47 haplotypes, with 38 unique mtDNA control-region sequences . Among tThe haplogroup classification of all Sardinian sequences showed a total of eight horse maternal lineages out of the 17 previously reported  with oneThe Y-chromosome HT2 and the high number of maternally inherited haplotypes identified in the Sardinian Anglo-Arab breed suggest a sex-bias in the breeding program, where probably stallions from a unique breed (Thoroughbred) have been crossed with mares of different origins, a common breeding procedure used to improve local breeds with Thoroughbred thus creating half-breed individuals. The high mtDNA variability found in the Sardinian Anglo-Arab could be explained by the ancient migratory events that in the past have reached Sardinia and brought mares of different origins  .The semi-feral Sarcidano breed was probably less impacted by these breeding programs and conserved the highest NRY variability and strong traces of the ancestral haplotype HT1. Likewise, on the maternal side, it shows the four most common mtDNA haplogroups in current Italian breeds .In Giara the high incidence of the mtDNA haplogroup G together with the results obtained from the NRY analysis confirms the Middle Eastern origin of this breed and its subsequent isolation in the island. Contemporary Giara horses are probably descendants of the ancient Phoenicia, in agreement with Gratani  and manySardinia, located to the west of Italy in the Mediterranean Sea, is an island characterized by many autochthonous species. Among them, there are three horse breeds native to this island and classified “at risk”, which were the objective of the present study. The peculiar Y-chromosomal distribution together with the high frequencies of certain mtDNA haplogroups highlights the genetic characteristics of these endangered local breeds.Each breed showed a distinctive NRY haplotype: the ancestral one (HT1), most represented in Sarcidano; the HT2, marker of the Neapolitan/Oriental wave, above all found in Giara, and HT3, legacy of the Thoroughbred wave, prominent in the Sardinian Anglo-Arab. As for the female counterpart, the haplotype sharing between Giara and Sarcidano supports inferences of common maternal origins and the high incidence of the four most frequent lineages in present-day Italian horse breeds  suggests female inputs from the peninsula. However, Giara shows strong genetic relationships to ancient Eastern Mediterranean sources, corroborating the hypothesis of a common past migration traced back to Phoenicians: both humans and horses could have reached the island from the Eastern Mediterranean area by sea in the first millennium BCE.The low variability in Giara and Sarcidano confirms the marginal impact of crossbreeding on the indigenous mitochondrial gene pools and the lack of recent gene flow from outside into Sardinia. On the other hand, the high haplotype variability reported for Sardinian Anglo-Arab indicates the presence of multiple mare lineages in the current population.This study suggests that despite their endangered status, these Sardinian horse breeds preserve significant reservoirs of genetic diversity. Distinctive variants and/or lineages have been identified when evaluating the two uniparental genetic markers. Therefore, targeted conservation efforts that take into account both female and male lines of descent are required to safeguard the extant genetic variability and to establish improvements in the selection programs.


Megachile sculpturalis (Hymenoptera: Megachilidae), native to East-Asia, was first recognized in Southeast-France in 2008, and has since spread throughout much of Europe. The spread is very fast, and colonization may result from multiple fronts.Invasive species are increasingly driving biodiversity decline, and knowledge of colonization dynamics, including both drivers and dispersal modes, are important to prevent future invasions. The bee species To track the history of this invasion, codominant markers were genotyped using Illumina sequencing and the invasion history and degree of connectivity between populations across the European invasion axis were investigated. Distinctive genetic clusters were detected with east–west differentiations in Middle-Europe.We hypothesize that the observed cluster formation resulted from multiple, independent introductions of the species to the European continent. This study draws a first picture of an early invasion stage of this wild bee and forms a foundation for further investigations, including studies of the species in their native Asian range and in the invaded range in North America. Afranthidium (Immanthidium) repetitum, and, relatedly, wood-nesting bees make up larger proportions of the fauna in more remote islands due to higher dispersal abilities [Invasion occurs when a species is accidentally or intentionally introduced to and establishes within such a new environment. But invasive pollinators, like invasive bee species, often deviate from the negative perspective of invaders, especially when it comes to pollinators of crops. Bees are generally considered beneficial for the pollination services they provide, but they may have surprising negative impacts on local ecosystems where they do not naturally occur –4. Negatbilities , 10. In bilities .Given inbilities , it is cMegachile  sculpturalis Smith, 1853, one of hundreds of species amongst Megachile [Megachile sculpturalis is a protandric wild bee with an active season from June to mid-September in Europe [Megachile sculpturalis is a cavity-nesting wild bee using preexisting holes in wood. The species naturally occurs in East-Asia , but was first established outside its native range in the eastern USA around 1994, and is now considered as invasive species [Styphnolobium japonicum and Ligustrum sp. in Europe. Both plants are native to Asia and often used as ornamental plants in human habitats [M. sculpturalis on the local bee populations [egachile , is the n Europe –17. Malen Europe . Megachi species , 20, 21.habitats , 17, 22.habitats , 24. A rulations .Fig. 1SeVespa velutina first recognized in 2005 also in South-France, and Megachile disjunctiformis found in northern Italy in 2011 [Megachile sculpturalis most likely found its way to Europe as a stowaway on ships while nesting in dead wood [M. sculpturalis colonized many European countries remarkably fast. Its range expansion from France and Italy continued to Spain, Switzerland, Germany, Austria, Hungary, Slovenia, Croatia, Serbia, Bosnia Herzegovina reaching now to the Crimean Peninsula [M. sculpturalis probably colonized most countries by stepwise dispersal. Additionally, accidental transport in wood could explain the large distances-over hundreds of kilometers-between isolated reports eastward [In Europe, this species was first recognized in a small city near Marseille  in 2008 . There a in 2011 –29. Megaead wood , 30. Folead wood . In the eninsula , 30–37. eninsula , Gyöngyöeninsula , and Beleninsula . This sueastward .M. sculpturalis had been tracked relatively well, because of the conspicuous size of the species (for an European bee) and its typical association with anthropogenic habitats [The invasion of habitats . Thus, whabitats –41. Popuhabitats –45.To conduct such analyses, allele frequencies from selected neutral marker loci are compared to assess gene flow, which is a crucial parameter studying population dynamics and invasion biology , 46, 47.M. sculpturalis across Europe. The degree of connectivity between widespread populations is explored to detect signs of one or multiple colonizing events in Central Europe. The present study sets the baseline for further population genetic analyses, including specimens not only from Europe but also from its native range in Asia and North America. It should serve as a model for a deeper understanding of the interdependencies of successful colonization dynamics and mechanisms of dispersals, such that we may better predict and control the spread of invasive species in the future.Here, we present and validate a set of SSR loci as markers suitable for a genotyping by amplicon sequencing approach. We use these markers for a first investigation of genetic variations of M. sculpturalis populations, 48 SSR markers were designed and tested on 81 samples of European M. sculpturalis specimens. Out of all 48 markers, two (FB12 and FB47) were excluded due to an unusually high number of alleles as a possible consequence of duplication or scoring errors. The marker set included 20 pentanucleotide (two were excluded) and another 28 tetranucleotide motifs. Primer information is summarized in Table To study the genetic variability of European The partial Illumina MiSeq run resulted in 3,043,426 paired reads for the library analyzed with an average of around 41,000 reads per sample.o. When females (n = 42) were separated from the males, we saw a similar picture as for all samples: the Ho was lower than He in all groups and for females overall, but not for the group VIE , in contrast to low levels in samples from the group CH = 0.191 and SFR = 0.021. The remaining sample group FR had 0.298. This pattern is also reflected by the level of genetic distance between the groups  was low between samples from the group SFR, FR and CH, in comparison to values between samples from VIE. Multiple approaches generated similar patterns of this differentiation, e.g. GD and sHua between populations Table .Table 3PPCoA provided a visualization of the genetic distance patterns between individuals of the four sample groups. Samples from the groups SFR, FR and CH formed one discrete cluster. Samples from Vienna were separated from the remaining samples by relatively high distances Fig. . No geneOverall, pairwise FST values between populations were high between Vienna and the other groups 0.33–0.36), suggesting different populations, and low between the groups from France and Switzerland , introduced bees have the potential to impact the indigenous biodiversity by altering pollination networks. Invasive pollinators can have effects on the native flora by modifying the pollen transfer patterns, which affects the seed set of indigenous plants . In addiis group . TherefoM. sculpturalis, only long-distance transportation can explain its invasion [M. sculpturalis found its way to North America and Europe on ships following maritime trading routes [Species introduction events begin at their origins, where efficient vectors for single or multiple colonization events are required. Humans most often act as vectors and transport species via commerce and trading networks , 61. Witinvasion , 67. As invasion . More lig routes , 30. As g routes , 68. Aftg routes , 69. Humg routes , 70, 71.M. sculpturalis was first observed in Europe. Marseille constitutes an important maritime trading point in southern France. Ships for maritime trading were also most likely the vector delivering the species unintentionally to European islands, e.g. the most recent finding on Mallorca  [M. disjunctiformis was found near an international trading hub (Interporto) near Bologna, Italy [M. sculpturalis in sharing the same natural biogeography, East-Asia, and it is even a member of the same subgenus . This supports the assumption that invaders are not random samples of the global species pool [M. disjunctiformis based on the method described is currently in progress.This theoretical framework is reflected in the location where Spain; ) . Similara, Italy . This seies pool , 73, 74.M. sculpturalis was already recognized in 2008 [Introduced species often require a demographic lag phase immediately following the initial colonization event, prior to full establishment and subsequent range expansion . Althoug in 2008 , for mancsd locus that is heterozygous in females and homozygous in males in what is described as single-locus complementary sex determination [csd locus, which acts as a coping strategy circumventing the extinction vortex [M. sculpturalis can successfully invade new areas [By implementing SSR-GBAS, fine-scale ecological questions can be answered , e.g. thmination . In smalmination , 80. Hown vortex . Besidesew areas , 82–84. Anthidium manicatum) [A polyandrous mating system is another possible explanation for the observed invasion success despite severe founder effects. Even moderate polyandry, with more than two mates, can maintain genetic diversity and enable populations to overcome founder effects , 86. Butnicatum) , and a fM. sculpturalis in Europe [M. disjunctiformis in Italy, the potential of frequent recurrent introduction of this species should be high. This might be one factor for its high invasive potential [The striking differences detected between Vienna and all other regions represents our most unexpected finding. The genetic structure of both females and all samples, as well as the principal coordinate analysis, differentiated these two groups clearly: a western sample group  and a second, eastern group from Vienna. Vienna contains the lowest number of effective alleles and polymorphic loci. The observed heterozygosity of the sample group VIE exceeded expected heterozygosity. However, specimens from Vienna also had the highest number of private alleles. Given these findings, it seems highly unlikely that the population in Vienna spread from the 2008 origin in France. Instead, we hypothesize that they were transported independently, meaning that Europe was invaded multiple times by this species. This would explain both the discordance between eastern and western genotypes and the remarkably fast and wide-spread distribution of n Europe . In addin Europe –90. Althotential .Multiple introductions can facilitate successful establishment via an increased number of individuals, a broader spatial range and increased genetic diversity , 91, 92.Microsatellite loci enabled the first insights on the demographic dynamics of this introduced wild bee. By better quantifying population genetic structuring, indirect measurements of gene flow revealed striking differences between populations within Europe. As the SSR-GBAS was applied in a relatively early invasion stage before the expected admixture between European populations took place, we were able to find tremendous genetic patterns. This east–west differentiation as well as fine-scale genetic patterns are best explained by multiple colonization events in Middle Europe. Such multiple colonization events can partly explain its invasion success in Europe. Further, it is a possible explanation for its adaptation potential to its new environment. To confidently determine the number of colonization events, future studies with larger sampling from all invaded regions of Europe will be necessary. By implementing genetic landscape modelling, we are planning to estimate the probability of migration between geographic separated populations and to test the degree of connectivity more explicitly. Furthermore, specimens from its native origin and North America will be included to produce a synthetic view of its global invasion patterns and potential. By including North American samples for genetic structure analyses, we will be able to test if European individuals are bridgehead populations, those from a previously-invaded region rather than the native range, as are often found in other hymenopterans . If the Specimens for the present study were gathered from commercially-circulated trap nests (in France and Switzerland) and an international initiated citizen science project aiming to investigate its distribution in Switzerland, Liechtenstein and Austria . SamplesMarker development was conducted according to Tibihika  and CurtIn total, 83 samples were available for DNA isolation. To extract DNA from larval samples, approximately 1 mm of tissue was used. Tissue samples from adult bees were taken by cutting off the third leg. Bee legs were ground with 5 Zirkonium beads  per tube in a mill at maximum force (700 rpm) for 20 min. Isolation was done using the DNA tissue Kit by Macherey–Nagel GmbH & Co KG, Germany with according to the manufacturers protocol, with few modifications as follows: In each tube, 180 μL Pre-lysis buffer T1 and 10 μL Proteinase K (10 mg/mL) was added to the ground tissue, vortexed, and incubated at 56 °C overnight on a Mixing Block MB-102 . Next, 10 μL RNAse (10 mg/mL) was added and the mixture incubated at 37 °C for 10 min, and subsequently at 70 °C for 10 min with an additional 180 μL Lysis buffer B3. After lysis, the samples were centrifuged with for one minute each at 1000, 2000, 4000, 8000 rpm in a Eppendorf 5430 R centrifuge . The last step was carried out at 11,000 rpm for 7 min. After centrifugation, 360 μL supernatant was mixed with 180 μL Ethanol abs. and loaded onto an EconoSpinTM columns with silica membranes  and centrifuged at 8000 rpm for 1 min. The membrane was washed with 600 μL of 80% Ethanol at 12,000 rpm for 1 min and dried afterwards via centrifugation for 2 min and at 12,000 rpm after discarding the flow through. DNA was eluted at 12,000 rpm for 1 min with 50 μL with 10 Tris (pH of 8) preheated to 65 °C and incubated at least 3 min. The resulting DNA solution was used as the template for amplification.2O and 1 μL DNA were mixed. PCR run with the following program: 95 °C for fifteen minutes, 95 °C denaturation temperature for 30 s, annealing at 55 °C for one minute and elongation at 72 °C for one minute, repeating for 30 cycles. For the second PCR, 1.5 μL from each PCR product from the four multiplex reactions were pooled per sample and cleaned by mixing it with 4.3 μL AMPure XP magnetic beads . Washing steps as well as the second PCR elongating the PCR strands with unique indexes for sequencing, pooling the amplicons and purification followed Curto et al. DNA amplification was carried out by a two-step PCR approach with four sets of primer mixes Table , which we), observed heterozygosity (Ho), and number of alleles (Na), Mean Shannon Values over loci (sHua) using Log Base = 2, number of migrants, AMOVA and G-statistics were calculated with GenAlex. We also calculated Joest’s estimate of differentiation with GenAlex, as for highly polymorphic loci the Fst value depends on the allele number per locus. Pairwise Fst [Data analysis was done using scripts described previously and available at github.com/ mcurto/SSR‐GBS‐pipeline . Sequencwise Fst  was testwise Fst . Demograwise Fst . Geneticwise Fst  and prinwise Fst . For a se > Heq) [e > Heq were counted and tested if regions showed signs of an increase in heterozygosity above zero by calculating sign tests and carried out one-tailed Wilcoxon tests, as in Neophytou et al. (2019). Tests were carried out in the program BOTTLENECK under the infinite allele model (IAM) [Finally, we tested for genetic bottlenecks with the hypothesis of bottlenecked populations showing a higher than expected heterozygosity based on Hardy–Weinberg Equilibrium (HWI) than under the mutation-drift equilibrium (He > Heq) , 115. Nuel (IAM) .Additional file 1: Fig. S1. Box plot of the proportion of heterozygous loci found in females from the sample groups CH (Switzerland), SRF (South-France), VIE (Vienna) and males collected in Vienna , whereas VIE males 2–4 were found to be diploid in several loci.Additional file 1: Table S1. Detailed information of the samples included in the present study. Adult specimens were assigned as males = m, or females = f. As protandric species, females emerge later than males and are positioned at the cavity end: L1 = first larvae positioned at the cavity back, L2 = second larvae positioned nest to L1; Ln = larvae were counted and labeled according to their nest position.


Peromyscus californicus) and the polygynandrous deer mouse (P. maniculatus) and brush mouse (P. boylii). By sampling populations of these species from multiple localities in California, we were able to conduct replicated analyses of the relationship between mating system and immunogenetic variation. Across the four localities sampled, diversity at MHC Class I and II genes was consistently higher in the two polygynandrous species. We found no evidence that sampling location  contributed to observed differences in MHC variation among populations or species. Collectively, our data indicate that immunogenetic variation in Peromyscine mice is associated with reproductive behavior, rather than geographic locality or habitat type. The consistently greater variability detected in the polygynandrous species examined suggests that balancing selection imposed by behaviorally-mediated pathogen exposure is important in maintaining variation at MHC genes in these animals.The number of reproductive partners per individual varies markedly across animal mating systems. This variation may be an important determinant of patterns of immunogenetic diversity, particularly at Major Histocompatibility Complex (MHC) Class I and II loci. To compare immunogenetic variation in taxa with markedly different mating systems, we used RNAseq-generated data to quantify genotypic diversity in three species of Peromyscine rodents: the monogamous California mouse ( Mating systems are complex behavioral phenotypes, the genomic implications of which are generally poorly understood ,2. AmongDespite clear expectations regarding the effects of mating systems on immunogenetic variability, few studies have examined these relationships in natural populations of vertebrates but see ,12). Tho. Tho12])Peromyscus mice that occur in California. The California mouse  occupies oak savannah and chaparral habitats from the San Francisco Bay to northern Baja California [P. boylii) and the deer mouse (P. maniculatus) are both polygynandrous, with individual males and females mating with multiple partners [To explore how differences in mating system shape variation at multiple MHC loci, we compared levels and patterns of immunogenetic diversity in three species of lifornia ,15. Behalifornia ,17, meanpartners ,19. BecaP. californicus is associated with reduced variability and less intense selection at MHC loci compared to sympatric populations of the polygynandrous P. boylii or P. maniculatus. Because the samples analyzed had been obtained from four geographically and ecologically distinct locations in coastal and inland California, we also sought to determine if differences among the study species varied with sampling locality, as expected if local differences in pathogen communities influence selection patterns on MHC loci [To capitalize on this comparative system, we used transcriptomic sequencing to quantify variation at multiple MHC genes in these species. Specifically, we sought to test the prediction that monogamy in MHC loci . CollectPeromyscus that occur in California. P. maniculatus is a small-bodied (10–24 g), ecologically generalized species that occurs in a wide range of habitats in California, including deciduous woodlands, deserts, coastal scrub, chaparral, and grasslands [P. boylii is larger-bodied (22–36 g) and more ecologically restricted, occurring only in chaparral and scrub forest [P. californicus, the largest of the study species (32–54 g), is also found in chaparral and scrub forest habitats from the San Francisco area to northern Baja California [P. maniculatus and P. boylii are polygynandrous, meaning that each individual mates with multiple partners of the opposite sex [P. californicus, is both socially and genetically monogamous [P. californicus and P. maniculatus are more closely related than P. maniculatus and P. boylii [We examined patterns of immunogenetic diversity in three species of asslands ,22. P. bb forest ,22. P. clifornia ,22. P. msite sex ,19. In cnogamous , indicat. boylii  and thusP. californicus (www.cnrfc.noaa.gov); inclusion of populations from both extremes of the range of P. californicus encompassed habitat variation associated with this marked difference in rainfall. Similarly, because habitats differ substantially on the western versus the eastern sides of the coastal mountains in California , at each end of our north-south sampling transect we selected one coastal and one inland population for analysis, resulting in a total of four sampling localities. At both coastal locations, P. californicus co-occurs with P. maniculatus; at both inland locations, P. californicus co-occurs with P. boylii. Thus, this sampling scheme allowed us to compare genetic diversity in the monogamous species to that in two different polygynandrous species, with each interspecific comparison encompassing two distinct habitat types. This sampling scheme was not intended to provide a detailed analysis of the potential role of environmental differences on MHC variability in the study species but, rather, to assess the generality of relationships between MHC variation and mating system across differences in the conditions in which the study species occur.Mice were sampled at four localities, two in the northern and two in the southern portions of the range of fornicus . The twoAll trapping of the study populations was completed between February and April 2016. At each sampling locality, animals were captured using Sherman live-traps baited with rolled oats and containing a small ball of synthetic batting that the animals used as nesting material. A total of 180 traps were set per locality, with traps placed in pairs at 10 m intervals to create a grid measuring 150 m x 60 m and containing 90 trap stations (pairs of traps). At each sampling locality, traps were opened at 1600 hrs and closed 0300 hrs for 20 consecutive nights. All animals captured were identified to species using standard pelage and body size characters .At each sampling locality, we collected a subset of seven adult males and seven adult females per species  for use in transcriptomic analyses. These animals were euthanized via overdose with Isoflurane followed by cervical dislocation. Immediately postmortem, a sample of liver tissue (0.5 cm x 0.5 cm) was collected from each individual. Liver samples were placed in 1.5 mL of RNAlater for 24 hrs at 4° C, after which samples were frozen in liquid nitrogen. In addition, a sample of ear pinna was collected from each animal for use in mitochondrial sequence confirmation of species identity. Pinna samples (2 mm x 5 mm) were stored in 95% ethanol at ambient temperature until analysis. At the end of each field trip, samples were transported to the Museum of Vertebrate Zoology on the UC Berkeley campus; once on campus, the samples were stored in a -80°C freezer until analysis.All field work involving live mice was approved by the Animal Care and Use Committee at the University of California, Berkeley, and was consistent with the Guidelines for the Use of Wild Mammals in Research published by the American Society of Mammalogists .cytochrome b (cyt-b) locus. Genomic DNA was isolated from ear pinna samples using a salt extraction protocol [cyt-b locus was performed using primers MVZ 05 and MVZ 16 [2O, 2.75μL of 10x buffer (with MgCl2), 2.2μL of MgCl2 (50mM), 2μL of Betaine (5M), 1μL of BSA (10mg/ml) (Bovine Serum Albumin), 0.44μL of dNTPs (10mM), 0.44μL of each of the primers (10μM) (forward and reverse), 0.15μL of Taq polymerase  (New England Bio Labs), and 1μL of the DNA template. Amplification conditions consisted of an initial denaturation at 95°C for 4:00 min and 35 cycles of the following: denaturation at 95°C for 0:35 min, annealing at 49°C for 0:40 min, and extension at 72°C for 0:50 min. Our cycle sequencing mix consisted of 9μL reactions containing the following: 5.94μL of ddH2O, 1.43μL of the forward primer (MVZ 05), 1.98μL of the 5x Big Dye Buffer, 0.5μL of Big Dye, and 1μL of the PCR template. Sequences were edited and aligned using Geneious 7.1.7 [Because the study species can be difficult to distinguish based solely on external pelage characteristics, for the subset of animals collected for transcriptomic analyses, we confirmed species assignments made in the field using sequences from the mitochondrial protocol . PCR ampd MVZ 16 . Our PCRus 7.1.7 , after w® 24 equipped with ceramic beads , after which RNA was extracted from the homogenized sample using the UltraClean® Tissue and Cells RNA isolation kit . The concentration of each extract was determined using a NanoDrop spectrophotometer (ThermoFisher); typically, extractions yielded ~ 200 ng/μL of RNA. The quality of each extract was assessed using RNA 6000 Pico BioAnalyzer chips (Agilent); the mean RNA integrity number (RIN) score for our samples was 8 (range = 7.1 to 8.9). RNA extractions were then further purified using the DNase Max® kit (MO BIO) or—for samples requiring additional concentration—RNeasy spin columns (Qiagen). Finally, samples were diluted to a standard concentration of 80 ng/μL in 25 μL of RNAase-free water  for library preparation.To quantify transcriptomic patterns of genetic diversity, we first isolated RNA from a subset of the liver samples collected during this study , resulting in extracts from 64 individuals. For each sample, a 15 mg portion of liver was homogenized using a PowerLyzer® platform KAPA Stranded mRNA-Seq Kit (KAPA Biosystems), with the manufacturer’s protocol modified to accommodate half reactions. For each sample, a total of 2μg of high-quality RNA (RIN > 7) was suspended in 25μL of RNase-free water. Samples were fragmented at 94°C for 4 min in order to achieve a typical library insert size of 250bp. We made two modifications to the manufacturer’s protocol. First, to minimize artifacts resulting from over-amplification and to maximize the number of unique fragments amplified, we divided the cleaned, adaptor ligated cDNA samples  in to two amplification reactions. Each of these sub-reactions was amplified for 10 cycles, after which the reactions were pooled to produce a single final library per individual. Second, the final, uniquely barcoded libraries were cleaned using low ratio SeraMag beads (Sigma-Aldrich) instead of KAPA pure beads.cDNA libraries were prepared using the IlluminaInitially, the size and concentration of each library were assessed visually by running samples on a 1% agarose gel that was then stained with ethidium bromide. More precise measures of library sizes were obtained using a BioAnalyzer DNA 1000 chip; mean library size (without primer sequences) was 264 (±13bp). More precise measures of library concentrations were obtained using a Qubit fluorometer (ThermoFisher) high sensitivity assay. Libraries measuring < 10ng/μL were further concentrated using a 2x Solid Phase Reversible Immobilization (SPRI) bead clean up procedure. A 10μL aliquot of each final library was submitted to the Vincent J. Coates Genomics Sequencing Laboratory at the University of California, Berkeley for sequencing. The 64 samples submitted were pooled equimolarly and then distributed across four lanes of Illumina HiSeq4000, 100PE sequencing.https://github.com/dstreett/Super-Deduper). Finally, we identified reads resulting from bacterial contamination by aligning all sequences in our dataset to the Escherichia coli genome, after which contaminant reads were removed using Bowtie2 [Raw sequence data were cleaned following the protocols of Singhal (2013)  and Bi e Bowtie2 .P. maniculatus available from the NCBI RefSeq database (GCF_000500345.1_Pman_1.0_cds_from_genomic.fna). Such CDS references typically contain multiple transcripts per gene; because we were interested in genic level patterns of diversity and selection, we used custom Biopython scripts [P. maniculatus that contained only the single longest transcript variant for each gene in the assembly.To examine genetic diversity and patterns of selection on protein coding genes, we created species-specific coding sequence (CDS) reference assemblies for each of our study taxa. We began by downloading the existing CDS reference assembly for  scripts  to creatP. californicus and P. boylii. We selected one male and one female per species per study population and then concatenated the reads for all conspecifics. We indexed the single-copy P. maniculatus CDS reference using the LAST aligner lastdb command, after which we used the LAST aligner fastq-interleave module to merge the concatenated sequences for P. californicus and P. boylii and to align the resulting reads for each species to the indexed single-copy CDS reference for P. maniculatus. Each aligned sequence was converted to BAM format using SAMtools [samtools sort and indexed using samtools index. These alignments were then converted to FASTA format CDS references for each species using the samtools mpileup and seqtk seq functions (https://github.com/lh3/seqtk); the resulting reference sequences were evaluated for completeness by comparing them to the single-copy P. maniculatus CDS reference. Missing data for P. californicus and P. boylii were replaced with the corresponding portion of the P. maniculatus reference. Sites denoted by IUPAC ambiguity codes were replaced by randomly selecting one of the possible nucleotides present at that location (e.g. inserting a C or a T for a nucleotide denoted as Y).To increase the efficiency and accuracy of read mapping for the other study species and to reduce mapping bias caused by use of a taxonomically distinct reference, we constructed species-specific CDS references for SAMtools , after whttp://www.popgen.dk/angsd/index.php/ANGSD) [−06.We used SNP markers to quantify genetic variation in each study species. To account for potential errors during base calling, alignment, or assembly, whenever possible we used genotype likelihoods instead of fixed genotypes to quantify genetic variability. Genotype likelihoods were calculated using the empirical Bayesian framework in ANGSD (p/ANGSD) . This prTo examine relationships between mating behavior and immunogenetic diversity, we identified SNPs in our data set that were associated with MHC Class I and II loci based on gene nomenclature. We extracted genes that matched known naming systems for MHC . Similarly, we identified genes coding for MHC transactivator proteins and regulatory factors . Collectπ = 4Neμ = θ. To test for possible deviations from this expectation, we used ANGSD to calculate Tajima's D [For all MHC loci as well as for all neutral loci in our data set, overall genetic variation was estimated in ANGSD as both the average number of pairwise differences, π , and thejima's D  for eachjima's D . Taken tjima's D .DQα and DRβ genes. Positive selection is only expected to operate within the peptide-binding regions (PBR) of Class II loci [DQβ) and thus tests for selection were not performed on this gene.To test the prediction that polygynandry is associated with enhanced selection on MHC loci, we quantified the apparent strength of selection on these genes in each of our study species. To begin, we reconstructed full sequences for our loci and used the KaKs_calculator  to test  II loci ; we werecodeml package in PAML [DQα and exon 2 of DRβ in each of our study species as well as to identify individual codons that appeared to be subject to positive selection. For these analyses, we first downloaded available exon 2 sequences for P. californicus MHC-DQα (Genbank JN703316) and P. maniculatus MHC-DRβ (NCBI AF516929.1). We then generated a consensus sequence for each locus and species (N = 6 sequences) in Clustal Omega [2ΔlnL = 2(lnL1- lnL0)) was compared to a X2 distribution with one degree of freedom.We used the branch-site model of diversifying selection as implemented in the  in PAML ,45 to teal Omega . We examST for all pairwise combinations of conspecifics from different populations. Within species, we used this procedure to estimate FST for neutral loci in our data set as well as for the MHC genes examined. As described above, neutral loci excluded both MHC genes and outlier SNPs identified using ‘BayeScan' [P. californicus and P. boylii, and population was the highest level for P. maniculatus. To execute AMOVAs, we used the R packages ‘adegenet’ [P. californicus and P. boylii; data from P. maniculatus were excluded from these analyses because samples for this species were only available from two localities  on SNPsdegenet’ ,50, ‘popdegenet’ , ‘pegas’degenet’  and ‘apedegenet’ . Signifidegenet’ . Finallydegenet’  and geogdegenet’ . Signifidegenet’ . Mantel calities . Finallycyt-b sequences confirmed that all individuals identified in the field as P. californicus (n = 32) and P. boylii (n = 16) were correctly assigned to these species. Of the 16 animals identified in the field as P. maniculatus, two males and two females from the northern, coastal BCR population were identified genetically as P. boylii. As a result, the final sample size for each species was 32 for P. californicus, 20 for P. boylii, and 12 for P. maniculatus; the final data set for the BCR population included data from all three study species , indicating that the error rate for reading nucleotides was < 0.1%. Average coverage for the 64 individuals sequenced was 42% (± 7%).P. californicus, 247,602 for P. maniculatus, and 53,022 for P. boylii. Of these, the number associated with MHC genes was 988 for P. californicus, 1,847 for P. maniculatus and 1,712 for P. boylii. The number of distinct MHC loci represented per study species was 28, 29 and 33, respectively; overall, 89% of the MHC genes represented in the SNP data set were shared across all three species.A total of 125,720 SNPs were identified for Between-species comparisons of genetic diversity were restricted to genes detected in all three species; this restriction was applied to analyses of MHC loci as well as analyses of all SNPs examined . At the At the population level, measures of genetic diversity also tended to be greater for the polygynandrous study species. For the two localities at which P. maniculatus and P. californicus co-occurred , populatDQα and DQβ genes, values of π were greater in P. boylii and P. maniculatus than in P. californicus . Coefficients of relatedness calculated using only data from MHC loci were also low (mean r = 0.02 ± 0.07). Similarly, pairwise coefficients of relatedness for P. maniculatus tended to be low, with mean values of 0.01 ± 0.07  and 0.10 ± 0.25 (MHC loci). Pairwise estimates of relatedness for P. boylii were comparable, with mean values of 0.02 ± 0.05 , and 0.04 ± 0.10 (MHC loci). Thus, overall, conspecifics sampled at the same locality were not closely related to one another.Coefficients of relatedness calculated based on data from all SNPs revealed that in D for MHC loci approximated normality  and thus we expected ~ 95% of locus-specific values of D to fall between –2 and 2. The average (± SD) value of Tajima’s D in the BCR population of P. californicus was weakly negative (-0.05 ± 0.96) but was weakly positive in the remaining three populations of this species ; no loci displayed significant departures from neutral expectations . In P. maniculatus, values of Tajima’s D were also weakly negative in the BCR population (-0.36 ± 0.80) but weakly positive in the TPSNR population (0.04 ± 0.59), with no departures from neutral expectations . In contrast, in P. boylii, average values of D were weakly positive in all populations , with several genes displaying apparent departures from neutrality at EOR and HNHR (D > 2 or D < -2) (Ciita) locus, significantly positive values of Tajima’s D were obtained for MHC genes  . With thHC genes .P. californicus, an average of 64% (± 7%) of the SNPs examined (N = 988) differed significantly from Hardy-Weinberg expectations. This value was 85% (± 5%)  for P. maniculatus and 76%  for P. boylii. When we examined the specific MHC genes that contained these SNPs, we found that ~ 70% of these loci were shared among data sets for all three of the study species. In contrast, two of these genes were detected only in P. californicus , one was detected only in P. maniculatus , and two were detected only in P. boylii . Thus, the vast majority of SNPs that were characterized by significant departures from neutrality were present in the monogamous as well as the polygynandrous study species.Multiple SNPs associated with MHC genes displayed evidence of departures from Hardy-Weinberg expectations. Across populations of DQα, DRβ, and DQβ), the Class II DQα locus appeared to be under purifying selection in all three species (p < 0.001 Fishers exact test). In contrast, rates of synonymous substitutions at the Class II DRβ and DQβ genes were greater for P. maniculatus and P. boylii than for P. californicus, with evidence for purifying selection detected only for P. californicus . Branch-site tests for the peptide-binding regions (exon 2) of the DQα and DRβ genes revealed no evidence of positive selection in any of the study species .In general, the MHC genes that displayed significant evidence of selection appeared to be subject to purifying selection . Of the ST based on neutral loci as well as those based only on MHC genes indicated that genetic dissimilarity tended to increase with the geographic distance between populations  was substantial, although not statistically significant  and geographic distance (km) for both MHC loci  and neutral loci . Co-inertia analyses revealed that variability in the MHC and neutral data sets were highly correlated .For each study species, results of AMOVAs for neutral loci as well as those for MHC loci revealed that the majority of genetic variation was contained within populations. Consistent with our estimates of FP. maniculatus and P. boylii displaying greater diversity at MHC genes than the monogamous P. californicus. Despite these differences in diversity, we found no evidence for enhanced selection at MHC loci in either of the polygynandrous species relative to the monogamous species. Further, we found no evidence to suggest that variation in habitat conditions contributed to patterns of immunogenetic variation among populations or species. Because our findings are based on analyses of more than a single population per species and represent animals sampled from diverse habitats and geographic regions, these data imply that mating systems play an important role in shaping patterns of immunogenetic diversity in these mammals.Differences in immunogenetic diversity among our study species were consistent with predictions based on the mating systems of these animals, with the polygynandrous Although differences in mating systems provide a logical explanation for the interspecific differences in immunogenetic diversity reported here, other factors may have contributed to this outcome, notably differences in the underlying genetic structures of the study species as well as local differences in the communities of pathogens to which the study animals were exposed. Within some populations, overall genetic diversity was greater for the two polygynandrous species and this tendency may have influenced variability at the subset of immunogenetic loci examined. This possibility is particularly relevant for animals sampled at EOR and TPSNR, where estimates of genetic diversity for all SNP loci were also greater for the two polygynandrous species than for the monogamous species . Our anaST) among populations of conspecifics tended to increase with geographic distance in each species. For neutral loci, although populations of P. maniculatus and P. boylii were typically less genetically distinct than those of P. californicus, estimates of FST for all species fell within values typically interpreted as evidence of moderate differentiation, providing no suggestion of pronounced interspecific variation in the spatial structure of genetic diversity. Consistent with this, the results of AMOVAs indicated that within each study species, population differentiation increased with geographic distance, a pattern that was confirmed using Mantel tests. Finally, co-inertia analyses revealed that variability at MHC and neutral loci were highly correlated in each species, providing no indication that genetic structure differed between these two categories of loci. Collectively, these results suggest that in our study animals it is geographic distance–not habitat or location–that underlies genetic differentiation at both MHC loci and neutral loci. Although this outcome appears to be robust, it is possible that our sampling scheme, in particular our sampling of just one population per geographic region  precluded detection of fine scale variation differences in genetic structure among the study populations that contribute to the differences in MHC variability reported here. Future studies that include additional populations of each species from a greater variety of habitats should further elucidate the roles of distance and environment in contributing to the patterns of immunogenetic variability reported here.Interspecific differences in the spatial distribution of genetic variation may arise due to demographic differences that affect processes such as gene flow and genetic drift –59. For At the same time, because MHC class I and II genes are thought to be subject to selection imposed by pathogens ,4, variaP. californicus. This decrease in spatial differentiation for immune loci may reflect the effects of balancing selection on MHC genes [P. maniculatus reported a similar pattern of reduced genetic differentiation at MHC relative to neutral genes [P. boylii, these analyses failed to detect similar signatures of selection in P. californicus or P. maniculatus and, overall, evidence for selection on MHC genes was inconsistent. For example, while Ka/Ks ratio tests for selection revealed that MHC loci were typically subject to purifying selection , our anispidus: ) and woovaticus: ) also favaticus: ,68.P. californicus versus P. maniculatus and P. boylii is consistent with reduced pathogen exposure due to the limited number of reproductive partners per individual in the monogamous species [P. californicus [Hypogeomys antimena: [Canis lupus: [Spheniscus magellanicus: [Although the mechanisms by which individual-level decisions (mate choice) and population-level dynamics (pathogen-mediated selection) are expected to affect variation at MHC genes are generally understood, interactions at the interface between individuals and populations have not been as well-characterized. Mating systems provide a logical target for exploring this intersection, as they reflect both individual reproductive decisions and population level patterns of exposure to selective agents such as socially transmitted pathogens. Our finding that patterns of immunogenetic diversity differed with mating system underscores the potential for such analyses to generate new insights into the processes mediating individual- versus population-level impacts on genetic variability. For example, the lower variability at MHC genes reported here for  species . At the  species . Under tfornicus , the Malntimena: ), grey ws lupus: ), and Malanicus: ). ComparPeromyscus typically examined allelic or nucleotide level variation at one to a few MHC genes [DQα locus in P. californicus and P. maniculatus from the Big Creek Reserve (BCR), one of the sampling sites examined here. Their analyses revealed greater allelic and nucleotide level variation, as well as greater evidence for selection, in the polygynandrous P. maniculatus. Bacterial diversity in vaginal swabs collected from female mice was also greater for P. maniculatus than for P. californicus [DQα locus in the polygynandrous P. maniculatus. While our results regarding overall patterns of MHC diversity are consistent with this conclusion, we did not find evidence for enhanced selection at the DQα gene nor any of the other individual MHC loci examined. Potential explanations for this disparity include differences in sample size, in the relative ability of allele sequences versus SNPs to detect selection, and in the environmental factors imposing selective pressures on these genes [Peromyscus, mating behavior is associated with patterns of immunogenetic diversity. In particular, locus-specific studies have revealed greater MHC diversity [Previous analyses of MHC diversity in HC genes ,75,76. Pfornicus , leadingse genes . Howeveriversity  and geneiversity  in polygP. polionotus–an independently evolved example of monogamy within the genus Peromyscus [Although our analyses suggest that mating systems are important contributors to patterns of immunogenetic variability, the effects of behavior on genetic diversity are embedded within a complex network of interactions involving elements of behavior, ecology, demography, and immunology. As a result, while patterns of mating behavior–in particular the number of reproductive partners per individual–appear to be important, there are clearly other factors that need to be considered to understand patterns of immunogenetic diversity in natural populations of animals. As a first step, it would be valuable to assess the generality of the relationships reported here. Thus, future studies should include romyscus ; if compS1 FigEstimates of per-site nucleotide diversity (π) for (A) SNPs associated with MHC loci and (B) all SNPs identified in each study species. Data for each population of conspecifics were analyzed separately; these data are summarized in (TIF)Click here for additional data file.S1 TableShaded cells denote genes (N = 27) used in species-level estimates of genetic diversity; bolded cells denote genes (N = 13) associated with population-level estimates of diversity See .(DOCX)Click here for additional data file.


Northwestern Italy is a wine region of the world with the highest of reputations, where top quality wines of remarkable economic value are produced from traditional, long-cultivated varieties. Kinship analyses were performed using 32 microsatellite loci and more than 10 K single-nucleotide polymorphism markers on 227 traditional grapes mostly from Northwestern Italy—including those that have been neglected or are threatened. This was done to better understand the genetic grapevine origins and history of this reputable wine producing area, thus enhancing its cultural value and the marketing appeal of its wines. The work revealed a complex network of genetic relationships among varieties, with little contribution of genotypes from other areas. It revealed the major role played by a few ancient grape varieties as parents of numerous offspring, including some that are endangered today. The ancestry of many cultivars is proposed. Among these are Dolcetto, Barbera and Riesling italico. Through the inference of parent–offspring and sibling relations, marker profiles of ungenotyped putative parents were reconstructed, suggesting kinship relations and a possible parentage for Nebbiolo, one of the most ancient wine grapes worldwide. Historic and geographic implications from the resulting kinships are discussed. Current production is based on cultivars selected or created for fresh consumption, and on a wide range of varieties for wine production, both vegetatively propagated. The natural diversity in this species was most likely the key to its great spread over time throughout the Mediterranean and Continental Europe, where modern wine industry expanded, further broadening from the Old to the New World1.Grapevines are one of the most economically valuable agricultural crops in the world, accounting for a total area of 7,450,000 ha and a grape production of 75,500,000 t  the likely geographic origin of ancient grape varieties5, (2) their descendants and relatives10, and (3) their history and migrations over time. Forgotten grapes, currently of little importance, were discovered and in some cases provided missing links that reveal the history of more relevant progenies13.Both the geographic and genetic origins of ancient Factors and events that contribute to the high variation among today’s cultivars (and wines) have long been a matter of speculation, not only in regard to the economic relevance of grapevine as a crop, but also in regard to their indubitable cultural heritage. This speculation has not only contributed to scientific advancement in grapevine genetics, but also to a better understanding about the origin of cultivated grapes, complementing the high interest on variety history for wine marketing.18, and relation between currently cultivated varieties and wild Vitis vinifera subsp. sylvestris genetic pool23. In turn, this provides insight into domestication processes and helps us trace the evolution and dissemination of viticulture and wine culture.When applied on a large scale, genotyping offers information about cultivated grapevine genetic clusters24, Simple Sequence Repeats (SSRs) or microsatellites have been favoured molecular markers for varietal identification along with parentage analyses for several decades28. Due to their stability, high reproducibility and polymorphism combined with co-dominant Mendelian inheritance, nuclear-SSRs (nSSRs) were widely applied to grapevines. They were also used for building reference databases .Until the development of Single Nucleotide Polymorphisms (SNPs) for grapevine typingVitis spp.31. Their use is increasing, taking advantage of automated processing, high repeatability, and the richness of information they provide, depending on the number of SNPs scored33.More recently, several SNP sets have been developed for 34. Grapevine chloroplasts are maternally inherited. Chlorotype identification within V. vinifera germplasm has allowed the definition of the maternal lineage of cultivars, along with the investigation on their relations to the sylvestris subspecies and their geographic distribution35. Therefore, despite the low genetic diversity, grape chlorotypes can give insight to the origin of ancient cultivars and their dissemination patterns26.The analysis of chloroplast DNA has been used in phylogenetic studiesvinifera genetic diversity, both in wild and cultivated forms. Eco-geographic factors , together with human history  have, in all likelihood, contributed to the large number of grapevine cultivars found on the Italian peninsula.Among grape growing countries, Italy is one of the richest in https://www.oiv.int/en/statistiques/?year=2016&countryCode=ITA). However, according to the Vitis International Variety Catalogue (VIVC)  the number of traditional vinifera wine varieties designated to be of Italian origin are 1,038, compared to 348 and 552 for Spain and France, respectively. The Italian varietal assortment for wine production is wide and complex, with more than 500 wines under Protected Denominations of Origin (PDO) and Protected Geographical Indications (PGI) currently recognized. This amounts to a third of European appellation wines.Italy is the third largest wine grape growing country after Spain and France, with 710,000 ha under vines  is one of the most renowned wine areas of the world where premium wines from the centuries-old cultivar Nebbiolo such as Barolo, Barbaresco, Ghemme and Gattinara are produced. The region is also the likely home of Barbera and Dolcetto, respectively the major and the third regional black grapes that contribute to fine PDO wines. Moreover, Piedmont harbors White muscat , a fragrant grape that goes into the popular sparkling wine Asti, as well as several other aromatic varieties. There are 47 local varieties permitted for cultivation on the current 45,000 ha under vine of the region. They all arise from natural hybridization and selection rather than breeding. Twenty other wine grapes grown in the region have been introduced from abroad. Nearly 200, however, are the grape varieties of presently very scarce cultural importance, neglected, or even severely threatened of extinction. They have been recovered and collected over the last four decades, mostly in ancient vineyards located in today’s marginal wine areas and are all gathered in a central regional ex situ collection located in Grinzane Cavour (south western Piedmont). This ancient, local grape germplasm is a potential key to a reconstructed genetic history of today’s most popular cultivars from Piedmont.With this in mind we have focused on traditional grape varieties cultivated today or in the past in northwestern Italy. Our research had three main objectives: (1) to assess the correct identification of traditional varieties, using DNA fingerprinting, together with ampelographic resources and historical reports; (2) to investigate the genealogy and geographic origin of traditional grapes , with the final aim of (3) revealing local wine history and culture.The work relies on the inheritance of both nuclear and chloroplast SSR markers along with SNPs. An original approach was applied for inferring un-genotyped individuals and this played a key role in the resulting reconstructed pedigree.https://catalogoviti.politicheagricole.it/catalogo.php), as indicated in Supplementary Table All the 186 unique genotypes from north-western Italy included in this study  were specified as “presumed”. For the remaining 12 genotypes no name could be found or assigned. Therefore, they were indicated as “unknown white” or “unknown black” according to their berry colour.37 led us to identify this unknown accession as the historical Coccalona from Piedmont. This conclusion arose from several factors, including the extraordinary abundance of leaf hairiness, the attribute to over-crop when grown in deep soils, many morphological features in common with varieties resulting from our data genetically related, and similar places of current recovery and growth in the past. Through SSRs comparison, we discovered this variety was identical to Orsolina from Reggio Emilia province  and to Rohrtraube blaurot included in VIVC (https://www.vivc.de/index.php?r=passport%2Fview&id=10162). It was reported in the first half of the 19th century by Babo and Metzger39 as an ancient variety from south western Germany.The accession Coccalona nera, for example, a key grape in our work, was recovered in southeastern Piedmont as an un-named variety. Some key traits found in historical descriptions of Coccalona nera40.The specification “false” was used to indicate a mislabeled accession. For example, Aleatico (from Paderna) is wrongly named, as the true Aleatico is a different cultivar mainly grown in central and southern Italy. Hence, Aleatico (from Paderna) was designated as “false”. Bottagera, another key grape in our work, is probably in-correctly labeled as well. Unlike the accession we collected under this name, the true historical Bottagera is reported to exhibit large berries and poor qualityV. vinifera genotypes.The sample consisted of: 196 traditional grape varieties from northwestern Italy and from other European regions linked by parent/offspring relationships, 12 cultivars used as references for the pedigree study, along with 19 outgroup 41 was slightly higher than the expected one for most of the loci. Polymorphic Information Content42 was very high, ranging from 17 to 5, as expected especially for the markers with a high number of alleles. The average non-exclusion probabilities of the cumulative nSSR marker set were very high, strengthening parentage analysis outcomes. As for the estimated null allele frequency, it is worth noting the poor performance of VvMD36, with a value slightly higher than the threshold of 0.5 generally suggested for locus exclusion.Population genetics parameters  kinship pairs involving 169 grapevine varieties were detected by using 32 nSSRs and about 10 K SNPs (Supplementary Table One hundred and twelve (112) of the PO related cultivars were grown or collected in Piedmont  were still related at a higher degree. Quagliano, Pelaverga piccolo (the variety behind Verduno wine), Verdea and Favorita  are examples of unrelated varieties.PO relationships were consistently detected by nSSR and SNPs, except for few cases. For the few pairs showing 1 or 2 SSR mismatches (out of the 32 loci), IBD coefficients and SNP Mendelian errors strongly supported parent/offspring relationships. Fifty-six percent of total microsatellite discrepancies were observed at locus VvMD36 (Supplementary Table Forty-four trios (each made up by the two parents and the offspring) were detected by Cervus using SSR data Table . Most pe43. As to the group of flavored grape varieties descending from Moscato bianco, the study revealed the second parent of Moscato nero di Acqui, a fragrant specialty from south east of the region.Some resulting parentages refer to well-known pedigrees, already published and repeatedly confirmed, like Cabernet Sauvignon, Chardonnay, Muscat rouge de Madère. They were used as reference for LOD scores and IBD coefficient evaluation. Others refer to traditional varieties from Piedmont already investigated , often sharing as genitor Malvasia aromatica di Parma27. Our findings support Nebbiolo as true parent, definitely excluding Avanà as Freisa’s second genitor.Our results did not confirm some pedigrees already proposed. Freisa was believed descended from Nebbiolo and Avanà (Hibou noir in France)44, was confirmed here. It arose from two ancient grapes no longer cultivated, now surviving in the regional repository. Lambrusca di Alessandria, a robust dark-coloured variety used for blending, originated from today’s minor or even neglected genitors. Nebbiolo’s descendant Vespolina is an original specialty used in northern Piedmont for varietal wines or in blends with Nebbiolo. Barbera bianca (White Barbera)—not the white somatic variant of Barbera but a distinct seedling—did not reveal any genetic proximity to Barbera. The findings confirmed the parentage of Teinturier ad acino rotondo (Round berry Teinturier), a red-fleshed variety distributed across northern Italy and mentioned by Crespan et al.45 as Tintoria Lloyd. This suggests Piedmont is its cradle due to: (1) its closeness to France where its genitor Teinturier du Cher has been widely spread in the past, and (2) the scattered presence in Piedmont of its second parent Neretto duro , quoted here since XVII century.Most of the proposed trios (28 out of 44) are new insights, involving highly reputed and widely cultivated varieties. Dolcetto, whose pedigree was documented in a preliminary workIn 14 of the proposed trios one of the two parents displayed a chlorotype different from the other, allowing us to define motherhood and fatherhood Table . MotherhEvidence of full-sibling (FS) relationships among the investigated varieties was obtained by nSSRs and SNPs  were around 930 for the whole sample set when inferred by SSR data (data not shown), while more than 20 pairs were linked by full-sibling kinship (Supplementary Table 46). They suggest instead a half-brothers relationship for both pairs, as in the one between Neyret and Petit rouge. As to Nebbiolo’s kingroup, relatedness analyses from SNP and SSR profiles clearly matched, indicating Nebbiolo, Nebbiolo rosé, Pignola and Rossola nera share both parents.The pair Chardonnay–Gamay, used as reference, was correctly confirmed as full-brothers, whilst an uncertain interpretation remains for the debated FS relationship between Syrah and Viognier. The full-brother relationship between Biestro (presumed) and Dolcetto, both offspring of Dolcetto bianco and Moissan according to pedigree outcomes by Cervus Table , was inc43, Moissan, Coccalona nera and Bottagera . Two further varieties, Lambrusca di Alessandria and Nebbiolo, have also an important genetic role as parents, showing 5–8 presumed descendants  and Aleatico . These are, in fact, compatible with a full-siblings relationship , Spergola, Rossoletta and Orsanella. The first three mentioned varieties are typical of north eastern Italy, Ortrugo and Spergola of western Emilia Romagna, while Rossoletta and Orsanella are minor grapes of the central foot Alps. According to this assumed family tree, Teroldego, the popular specialty of Trentino , might also be Nebbiolo’s half-sibling, and in some ways linked to Pinot , and Coccalona nera are unimportant grapes today, but would have been widespread or intensely cultivated in the past.49 who first realized the morphology of its flowers. Later lost from Piedmont, it luckily survived under the name of Malvasia odorosissima  around Parma50, where it has been more recently brought back to cultivation.Malvasia aromatica di Parma seems the closest fit to the Malvasia bianca, shown to be in Piedmont from the XVI Century and definitely not later than early 1900. It was Sannino51 emphasizing the red stem bunch, a feature inherited by its descendants.Moissan was likely identified as the Mauzanetto , described by CroceBottagera  recovered under the name of Bottagera in Valtellina (north Lombardy) had apparently disappeared from Piedmont. As already mentioned, the name Bottagera is questionable, so the history of this genotype is still unknown. However, one would expect it to have been widespread in the past in northern Italy, since it gave rise to a large number of cultivars geographically distributed across a large area from Lombardy to central and western Piedmont.39. It is certainly not uncommon that genotypes of bad grape quality turn out to give rise to premium varieties.Coccalona nera, highly neglected today, because of the revealed synonyms and the large geographic distribution of its offspring or relatives, was likely prevalent across a large area in previous times. Among the relevant PO related to this grape are Barbera and Riesling italico, the latter better known as Welschriesling in Austria and Graševina or Olasz Rizling in the Balkan area. Our findings excluded Barbera and Graševina as putative parents of Coccalona, whose origin seems to lie in the crossing of Bottagera  with a genotype (#1) reconstructed by Colony Fig. . Hence BThe discovery of a grape variety’s geographic origin, especially when referring to traditional renowned wine grapes, is significant for the enhancement and marketing of wines and wine territories. In this regard, the detection of cultivar genetic origin opens the way to speculate on geographic implications. An ancestor’s geographic distribution, especially when retrieved from historic documents, allows one to assume the possible place of birth of descendants. In this work, all the resulting parentages Table  likely oDolcetto, the third most common variety by surface area in Piedmont covering 6,000 ha, resulted the offspring of Moissan and Dolcetto bianco . Historical documentation locates Moissan and its other descendants in the most western part of Piedmont, and in the region of Liguria nearby. Dolcetto bianco would have been grown in a more limited area in the South of Piedmont. Hence the likely cradle for Dolcetto would correspond to a region where the parents’ distribution areas overlap, and this broadly occurs in the present Langhe Fig. . This in52 would seem to indicate a possible origin in the south, as the terms "wälsch" or “welsch” let assume. Similarly, an origin from the southern side of the Alps could be inferred for Coccalona’s progeny Riesling italico, called Welschriesling in Germany and Austria. The absence of its second parent prevent us from establishing if its cradle was central Europe or north western Italy. Here, Riesling italico could be identified with the local ancient “Trebbianino dall’occhio” (“Little Trebbiano with the eye”), term that refers to the much evident pistil scar on the berry. The Coccalona nera PO relationship seems to exclude its cradle from the Balkans, unless Coccalona would have been present there, an un-proved growing place as far as we know.Coccalona nera was supposed to be born from Bottagera and an un-known genotype that, in turn, generated cultivars typical of both sides of the northern Apennines  Fig. . When coLabruscae, the Latin designation of wild or feral grapevines. Some features, like vine endurance and grape content high in colour and tannins, are in fact in common between V. vinifera subsp. sylvestris and cultivated Lambrusco or Lambrusca di Alessandria. But this last grape is genetically very far from Lambrusco.The genetic origin of Lambrusca di Alessandria suggests its homeland. It is attested that the traditional area of cultivation of its parents Neretto di Marengo and Crovìn is in south eastern Piedmont and the neighboring region of Liguria respectively. This suggests these areas are the cradle of Lambrusca. Its name, like that of Lambrusco , refers to 53.The inferred sibship clusters of Nebbiolo Fig.  would su54. Spergola-Vernaccia is also PO related to Citronino and Rapallino, two minor grapes attested in Piedmont and in the neighboring Liguria respectively  includes many present-day cultivars from central and eastern France, central Europe and the Balkan peninsula61. In the last region there has been a further contribution as genitors of Coarna alba, Vulpea, and a few others27.Genealogical reconstruction of grape varieties has been the goal of numerous studies, either done on a large scaleExplanations for such pattern frequently repeated in different areas are not completely clear. Some varieties could have had higher chances of generating a larger offspring number because of their ancient and widespread cultivation or because of the transmission of favorable characters (e.g. plantlet vigour).Quite a few of the mentioned European genetic founders, namely Marufo, Hebén, Corna alba, exhibit female flowers. So too does one of the main genitors shown in our work, Malvasia aromatica di Parma. A number of possible reasons can account for these varieties being successful as mothers. Female varieties could be, on average, more ancient (having more chances to generate offspring), since hermaphroditism was one of the traits selected during domestication. Moreover, a female plant’s progeny derives from outcrossing only, and it is expected to be less prone to inbreeding depression. Finally there is also the fascinating, but unproven role, of grape breeders played by viticulturists centuries ago (monks in abbeys?) and the ease of pollinating a female inflorescence, hence the choice to use a female individual as a genitor.62. Minor or even currently neglected grapes were shown to be the founders of present-day renowned cultivars, as also emerged by other studies13. Local germplasm collection and preservation is therefore an essential step for retrace grape and wine history of a region. In Piedmont, the genetic contribution from varieties introduced from abroad seems limited to a few genotypes  the southeast of Italy, where it was found under the local name of Arciprete67 and where several local varieties were revealed as its offspring68. A link with the Greece’s colonization in ancient times of both southern Italy (Magna Graecia) and Marseilles, in Provence, is a fascinating yet un-proven possibility.Some investigated varieties, like Verdea, Pelaverga piccolo, Quagliano and Favorita (syn. Vermentino), were genetically unrelated to the sample set from north western Italy. So, we can assume an outside origin for them. However, it is most likely Favorita/Vermentino was introduced in ancient times, being documented in Piedmont as early as 1658 under the name of Fermentino69, reinforcing the evidence that few generations, if not one, separate many cultivars of today from those of ancient times. This would imply a considerable age for the putative parents of the centuries-old varieties revealed by our study.Recent paleogenomic analyses of grape seeds from French archeological sites indicated direct genetic links between cultivars from Roman period and present varieties26, up to now most reference genetic profiles have consisted of SSR markers which have been widely used over the last 20 years25. Available Vitis genetic databases and literature, as well as internal laboratory databases are, in fact, mostly based on SSR markers. Hence the first steps of kinship analyses, as well as a variety’s true-to-type controls were performed relying on SSRs. Variety trueness to type establishment is crucial for further insights into parentage10. After checking synonyms through SSR markers, in our work varietal identity of the examined genotypes was established based on plant morphology, historical documents, ampelographic descriptions and local surveys. Genotypes were labeled as “unknown” when varietal identity could not be achieved.The combination of SSRs and SNPs in the investigation of grapevine parentages presents an interesting opportunity. Although the recent application of SNPs to grapevine genotyping showed clear advantagesIn our analyses SNP markers strengthened kinship evidence and reinforced outcomes especially when related to second-degree relationships. Direct relationships (duos and trios) were consistently detected by the two marker types  and to its local descendants. It is even more true for the northwestern region of Piedmont. The major genetic influence of Gouais, as an ancestor of a large number of progeny in France and Central Europe, may have played a role in spreading chlorotype C in those areas. Due to the maternal inheritance of chloroplast markers, it was possible to infer the male or the female contribution of genitors in a few parentages Tables . Due to This work has demonstrated that the combination of different markers as nSSRs and SNPs can give reliable results in a parentage study. The probabilistic reconstruction of “missing links”  appears promising in grapevine genealogy studies: the extinction of some key genotypes is highly probable within a long-cultivated gene pool.The recovery of every possible local variety, even the severely neglected or currently nameless, turned to be crucial to reconstructing the greatest number of genetic relationships between cultivars, including those of high relevance today. Therefore, the activities of endangered resources collection and preservation, implemented across grape growing regions, would greatly contribute not only to the conservation of threatened genetic resources, but also to variety parentage studies. The act of uncovering a grape’s historical background  remains of great appeal in promoting wines, especially within the context of developing better engagement marketing oriented to grape variety and wine culture dissemination.The disclosure of genetic relations between the cultivated gene pool examined in this work and the one of other European wine areas, might be a further development of this study, as the reconstructed parentage of Nebbiolo suggests.vinifera cultivars from Piedmont . Here major plant morphological traits were observed that focused on recognizing inherited traits supporting parentage and kinship. Moreover, these observations were addressed to outline leaf and bunch morphology. This proved useful to confirm the identification with historical descriptions and plates.All the examined accessions are grown in an ex-situ germplasm collection located in Grinzane Cavour (Piedmont)  74.DNA was extracted from young green tissues or from woody canes following the procedure described by Thomas et al.Vitis75, and the 3 markers suggested as common reference for grape variety identification within the European Vitis Database (www.eu-vitis.de—“Descriptors/file format—detailed SSR-marker specific information”). The other nSSRs were chosen according to polymorphism, stability and their membership of different linkage groups  markers, enough to identify the eight major chlorotypes present in 30. Genotypes were called using the standard procedure for diploids implemented in GenomeStudio . SNP markers with missingness rate > 1% and minor allele frequency (MAF) < 5% were removed. After SNP curation, missingness of individuals was checked and individuals with more than 1% of missing values were excluded. Data curation resulted in a dataset containing 140 individuals and 11,419 nuclear SNPs—this was used for pedigree reconstruction.Around 2/3 of the samples . Where possible, several accessions for each variety were analyzed, producing a genetic consensus profile.Putative POs at 20 nSSR loci. Grape cultivars sharing at least one allele at 9 nSSR markers were then analyzed at further 11 loci , discarding at this step all the genotypes showing less than 19 common alleles.196 cvs (out of the 186 + 606) resulted putatively linked by PO at steps 1 and 2;12 genotypes (used as references for family relationships) notoriously related from prior publications based on molecular markers and/or from confirmed breeders’ notations: Cabernet franc, Cabernet Sauvignon, Charmont, Chasselas blanc, Garanoir, Heptakilo, Moscato d’Amburgo, Perla di Csaba, Pinot, Sauvignon blanc, Savagnin, Sciaccarello  were assayed on a set of 227 genotypes , full sib (FS) and half sib (HS) relations were simultaneously inferred in order to identify the highest-likelihood pedigree configuration. Colony parameters were set up in order to perform one medium-length run with the full likelihood method, allelic dropout rate 0 and other genotyping error rate 0.0050. Although the occurrence of selfing is rare in grapevine, an inbreeding model has been chosen to account for parental relatedness and population structure. Reconstructed parental genotypes were tested for additional PO relationships.For nSSR markers, parent/offspring relationships and full parentage (family trios) were investigated by the exclusion method (based on counts of Mendelian inconsistencies) and by the likelihood-based method implemented in the software Cervus78, in order to detect PO relationships. Following the PCA-based approach of Morrison79, we removed ancestrally informative markers and checked that our sample genetic structure did not represent a bias for the PLINK estimator. Then, family trios were identified using the exclusion method based on the count of Mendelian errors. Due to high computational demand, Colony sibship clustering was not performed on the whole SNP dataset, rather on a subset of individuals resulting in HS relationships by the SSR analysis, with the main aim of supporting those results. A long run (length = 3) with the full likelihood method, the highest likelihood precision and a genotyping error rate of 0.00015 was performed. Allele frequencies observed in the whole dataset  were provided as known. The exclusion method based on Mendelian errors was used to test all possible pairs of grandparents (from a list of candidate second degree relatives or HS-like) for a certain offspring, in a pedigree including offspring, known parent and two candidate grandparents (putative parents of the un-genotyped parent).As for the SNP dataset, identity by descent (IBD) Cotterman’s coefficients were estimated using PLINK’s Method of MomentsSupplementary file1Supplementary file2