The studied breeds showed a medium level of genetic differentiation; the mean number of alleles per locus ranged from 3.4 to 6.6, and the effective number of alleles from 2.1 to 3.5. The mean degree of heterozygosity varied from 49% to 69% and from 47% to 68% for HO and HE, respectively. The population inbreeding coefficient (FIS) indicated an absence of inbreeding in the studied breeds. The average polymorphism information content (PIC) values for most of the breeds were higher than 0.5. The cumulative power of discrimination (PD) for all the markers in all breeds reached high values (close to 1.0), while the probability of identity (PID) was low, ranging between 10−11 and 10−19. The cumulative exclusion probability when the genotypes of one (PE1) and both parents (PE2) are known and showed that the parentage can be confirmed with a probability of 94.92% to 99.95% and 99.78% to 99.9999%, respectively.There is growing concern that extreme breed standardization contributes to a reduction of the effective population size and high levels of inbreeding, resulting in the loss of genetic diversity in many breeds. This study examined genetic diversity among eight popular dog breeds in Poland and evaluated the effectiveness of a 21-microsatellite (STR) panel recommended by the International Society for Animal Genetics (ISAG) for parent verification. The following breeds were characterized: German Shepherd, Maltese, Irish Wolfhound, Yorkshire Terrier, Biewer Yorkshire Terrier, Golden Retriever, Labrador Retriever, and French Bulldog. STRUCTURE analysis showed breed distinctiveness among all the dog breeds under study. Reynold’s distance ranged between θ Canis familiaris) is one of the first animals domesticated by man and has accompanied humans in their day-to-day life for thousands of years. Historically, dogs were used as working animals to herd livestock, hunt, and guard the home, and now they are also treated as companion animals [The dog  markers are a well-known effective and powerful tool widely used to investigate the genetic structure and diversity of dog breeds ,11,12,13n = 260), Maltese , Irish Wolfhound , Biewer Yorkshire Terrier , Yorkshire Terrier , Golden Retriever , Labrador Retriever , and French Bulldog . For all breeds, sampling was a selection of at least 70 unrelated animals  from different kennels, except for the GR breed for which 48 samples were collected.The study was conducted based on the results of analysis of microsatellite polymorphism performed as part of canine pedigree testing at the National Research Institute of Animal Production in 2018–2020. A total of 903 samples were used from eight breeds, namely, German Shepherd , using the following thermal profile: 5 min of initial DNA denaturation at 98 °C, followed by 30 cycles of denaturation at 98 °C for 15 s, annealing at 58 °C for 75 s, elongation of starters at 72 °C for 30 s, and final elongation of starters at 72 °C for 5 min. Analysis of the obtained PCR products was performed using an ABI 3130xl capillary sequencer . The amplified DNA fragments were subjected to electrophoresis in 7% denaturing POP-7 polyacrylamide gel in the presence of a standard length of 500 Liz and a reference sample. The results of the electrophoretic separation were analyzed automatically using the GeneMapper® Software 4.0 .This analysis uses the core panel of STR markers recommended by ISAG for individual identification and parentage analysis and a gender identification marker. The following microsatellite markers were used: AHTk211, CXX279, REN169O18, INU055, REN54P11, INRA21, AHT137, REN169D01, AHTh260, AHTk253, INU005, INU030, FH2848, AHT121, FH2054, REN162C04, AHTh171, REN247M23, AHTH130, REN105L03, REN64E19, and Amel locus. DNA was extracted from swabs and blood using the Sherlock AX Kit , following the manufacturer’s protocol. The extracts were quantified with a NanoDrop 2000 spectrophotometer . The STR loci were amplified using Phusion U Hot Start DNA Polymerase , and the PCR reaction was performed on Veritiw [u, Yu are allele frequencies from the first and second populations.Population structure was analyzed using STRUCTURE software version 2.3.3  considerw .(1)θw=∑O, expected heterozygosity—HE, and inbreeding coefficient—FIS, were calculated according to Nei and Roychoudhury [Based on this genetic distance, an unweighted pair-group method with averages (UPGMA) dendrograms were constructed to illustrate the similarities between the populations. Observed heterozygosity—Hhoudhury , and Wrihoudhury . The Harhoudhury . The genPolymorphic information content—PIC ,jk is the allele frequency j,k for i-locus; CPD is the cumulative power of discrimination.Power of discrimination—PD ,(2)PD=1ID [i p,j is allele frequencies j,i; CPID is the cumulative probability of identity.Probability of identity—PID ,(4)PID=1 and PE2) and the cumulative probability of parentage exclusion (CPE) [jk is allele frequency j,k for i-locus; CPE1, CPE2 are cumulative probabilities of identity.Probability of parentage exclusion for each locus, when the genotypes of one and both parents are known (PEon (CPE) ,(6)PE1=The statistical analysis was performed using the IMGSTAT software, ver. 2.10.1 (2009), which supports the laboratory of the National Research Institute of Animal Production.A total of 185 alleles were detected at 21 STR loci across all breeds. The total number of alleles per locus ranged between 6 for AHTk211 and 12 for AHTk137 and REN169018.The genetic population structure of each breed was determined based on the admixture level for each individual dog using the correlated allele frequencies model implemented within the STRUCTURE software. The results of Delta K indicated that the optimal number of genetic clusters representing most similar individuals for breeds was at K = 8 . The avew = 0.634), and the smallest between BYT and YT (θw = 0.260). The UPGMA dendrogram revealed that the YT and BYT were grouped together, and the Irish Wolfhound breed was farthest away from others (Reynold’s (1983) genetic distance between eight breeds is summarized in m others .The greatest number of alleles was noted for the breeds BYT  and M , and the smallest for IW . The mean number of alleles per locus for breeds ranged from 3.4 for IW to 6.6 for BYT, whereas the effective number from 2.1 for WI to 3.5 for M and YT .Out of the 185 alleles found within these breeds, 20 were breed specific . The greE = 0.677), YT (Ho = 0.662 and HE = 0.698), and BYT (Ho = 0.661 and HE = 0.658). Among the eight dog breeds considered, the lowest values of heterozygosity were found for IW (Ho = 0.491 and HE = 0.474). The population inbreeding coefficient (FIS) ranged from −0.049 (GR) to 0.053 (YT). The average p-value of HWE for each breed was higher than 0.05  and two parental genotypes available (PE2). The probability of exclusion for one parent available (PE1) ranged between 0.005 (REN169DO1 in IW) and 0.467 (CXX279 in YT) and when two parents were available (PE2) between 0.047 (REN169DO1 in IW) and 0.642 (REN169DO1 in YT) across different markers and breeds. The cumulative exclusion probability for PE1 and PE2 varied from 0.949242 (IW) to 0.999495 (YT) and from 0.997768 (IW) to 0.999999 (YT), respectively . Both breeds showed genetic closeness to Maltese (θw < 0.4), which is considered, alongside the Black and Tan Terrier and Skye Terrier breeds, as the ancestor of the Yorkshire Terrier. Similar genetic proximity (θw = 0.41) was observed between Maltese and French Bulldog. The genetic distance between the other breeds was θw > 0.50. The differences between GR and LR, which belong to the same group of retrievers and flushing dogs (pointer type), may be due to their different origins. Golden Retriever is a breed developed in the 19th century in Great Britain, while Labrador Retriever dates back from the 18th century and comes from Newfoundland. Labrador Retriever shows similar genetic distance (θw = 0.55) with German Shepherd. Phylogenetically the most distant breed is Irish Wolfhound which originated in Ireland and was used to hunt and protect against wolves. The breed became extinct as wolf numbers decreased and post-19th century it was presumably recreated by dog fanciers [The two clusters formed by BYT and YT are suggestive of genetic sub-structuring that resulted from using animals belonging to divergent selection lines. The Biewer breed was founded in Germany in 1984 as a result of the selection for white hair genes to produce tricolor dogs. The Biewer Yorkshire Terriers were ultimately recognized as a distinct breed in 1989 by the Allgemeiner Club der Hundefreunde Deutschland (ACH). The Biewer Terrier became the first breed proven to be distinct and unique using genetic testing . This brfanciers .O to be 0.742. In the same study, higher values were also observed for the Labrador Retriever breed (HO = 0.675) than in our study. A degree of heterozygosity below 50% was observed only for IW. This breed was characterized by the lowest total number of identified alleles. Furthermore, only two alleles each were determined in as many as four loci, among which 131 bp (AHTh130) and 216 bp alleles (REN169D01) had a frequency of 0.912 and 0.945, respectively. The mean effective number of alleles per locus was only 2.1. Limited genetic variation may be the result of the origin of this breed from a common founder population involving four founder lines [O = 0.502 for 33 STR, and when a 21-STR panel was used, HO = 0.483, and this value was similar to that obtained in our study (HO = 0.491) [O and HE values were similar to one another. The inbreeding coefficient for five breeds was negative, but the mean FIS had low negative values (from −0.004 to −0.049), which suggests no inbreeding in these breeds. The lack of observed genetic bottlenecking in any breed, despite a breeding system, may be due to multiple pedigree lines used. The lack of inbreeding was also reported in Bracco Italiano (FIS = 0.061), Tatra Shepherd (FIS = −0.005), six livestock guard dog breeds , and in Polish Hunting Dog (FIS = −0.01) [The highest within-breed diversity was characteristic of the M, BYT, and YT breeds, in which the largest number of alleles was identified. In Maltese, 4 out of 135 identified alleles were specific for this breed, but their frequency was low (less than 3%). In contrast, a 128 bp allele at the CXX279 locus, common only to BYT and YT, was present in both breeds with a frequency of more than 16%. For these breeds, the highest degree of heterozygosity (over 65%) was also noted. For Yorkshire Terrier, Ho = 0.662, which is similar to the studies in the UK , where H= 0.491) . In the = −0.01) ,13,36.ID genetic parameters. As reported by Botstein et al. [ID shows the probability with which two unrelated, randomly selected individuals in the population will have the same genotype. It is accepted that CPID values ranging between 10−3 and 10−4 are sufficiently low for the identification of individuals in natural animal populations [ID value estimated only for 15 STR markers in canine amounted to 10−8 [ID ranged between 10−11 for IW and 10−19 for M and YT. For all breeds, the obtained low CPID values should be sufficient to distinguish individual dogs. The usefulness of the investigated panel of markers for parentage verification was determined by calculating the probability of exclusion (PE). For a previously used commercial panel of 10 STR, CPE was 0.994 [1 above 0.99 and CPE2 above 0.999, except for GS (CPE1 = 0.986) and IW breeds . The values higher than CPE1 of 0.9999 and CPE2 of 0.99999 were obtained for YT, M, and BYT. The highest CPE2 = 0.999999 was observed for YT.The usefulness of the studied markers for individual identification of dogs was evaluated based on standard PIC, PD, and Pn et al. , markersn et al. ,38,39,40n et al. . The PD n et al. . The proulations , whereas to 10−8 . In our as 0.994 . The useas 0.994 . For all1 (0.949), and PE2 (0.998) with the highest PID (6.71 × 10−11) for the Irish Wolfhound breed, illustrating the lower effectiveness of the STRs panel. In contrast, Yorkshire Terrier and Maltese breeds had the highest PIC (0.655 and 0.640), PE1 (0.9995 and 0.9994), and PE2 (0.999999 and 0.9999987) with the lowest PID (2.34 × 10−19 and 4.47 × 10−19). The results suggest the popular breeds in Poland have sufficient diversity relative to other populations that have been studied. The research here provides baseline data for monitoring and managing the breeds.Analysis of the microsatellite DNA markers provides valuable information about canine diversity, and a 21-STR panel is an effective tool for individual identification and parentage testing. Our study showed the lowest PIC (0.414), PE


Then we consider the role of STAT3 and the parallels between STA3/STAT5 in cardioprotection by conditioning, giving emphasis to the human heart and confounders.Ischemia–reperfusion injury (IRI) is one of the biggest challenges for cardiovascular researchers given the huge death toll caused by myocardial ischemic disease. Cardioprotective conditioning strategies, namely pre- and post-conditioning maneuvers, represent the most important strategies for stimulating pro-survival pathways essential to preserve cardiac health. Conditioning maneuvers have proved to be fundamental for the knowledge of the molecular basis of both IRI and cardioprotection. Among this evidence, the importance of signal transducer and activator of transcription 3 (STAT3) emerged. STAT3 is not only a transcription factor but also exhibits non-genomic pro-survival functions preserving mitochondrial function from IRI. Indeed, STAT3 is emerging as an influencer of mitochondrial function to explain the cardioprotection phenomena. Studying cardioprotection, STAT3 proved to be crucial as an element of the survivor activating factor enhancement (SAFE) pathway, which converges on mitochondria and influences their function by cross-talking with other cardioprotective pathways. Clearly there are still some functional properties of STAT3 to be discovered. Therefore, in this review, we highlight the evidence that places STAT3 as a promoter of the metabolic network. In particular, we focus on the possible interactions of STAT3 with processes aimed at maintaining mitochondrial functions, including the regulation of the electron transport chain, the production of reactive oxygen species, the homeostasis of Ca Global health is constantly threatened by cardiovascular diseases (CVDs) and every year 31% of worldwide deaths are caused by fatal CVDs. According to the World Health Organization, CVDs caused 17.9 million deaths in 2016. Among these, 85% are attributed to ischemic heart disease or stroke .Over the past twenty to thirty years, mortality from ischemic heart disease has significantly decreased. However, currently available therapies improve symptoms and slow pathological heart remodeling but fail to further reduce CVD mortality . A complCoronary reperfusion is currently the “gold standard” for saving the lives of patients with acute myocardial infarction (MI). Reperfusion is mandatory to avoid fatal damage to the ischemic myocardium. However, the reperfusion itself contributes to exacerbating the damage and the extent of the infarct size (IS). In this regard, cardiovascular research over the years has investigated how to limit ischemia–reperfusion injury (IRI), leading to the development of cardioprotective maneuvers, which allowed researchers to analyze and understand the molecular aspects of IRI .Already in 1972 Braunwald and collaborators suggested that the infarct area was not only a function of the duration of ischemia, the area at risk and collateral flow, but could also be reduced with cardioprotective interventions . HoweverThe cardioprotective pathways are not yet fully understood, but in the early 2000s, Yellon's group , 182 propharmacological conditioning) .Although, STAT3 is involved as a transcription factor inducing the upregulation of antioxidant, antiapoptotic and pro-angiogenic genes in SWOP , 251, it2+ homeostasis, the mitochondria quality control and the inhibition of mPTPs opening. We also provide a critical evaluation of pathophysiological roles of STAT3 and the parallels between STA3/STAT5 in the context of cardiac IRI. Finally, we discuss the role of STAT3 in the cardioprotection by ischemic and pharmacological/hypothermic conditioning and the effects of common risk factors and confounders on STAT3-mediated cardioprotection in cells, in animal models and in the human heart.Therefore, in this review we first briefly describe the structure of STAT3 and the principal STAT3 post-translational modifications. Then we focus on the role of STAT3 within mitochondria highlighting the evidence that places STAT3 as a promoter of the metabolic network. In particular, we focus on the possible interactions of STAT3 with the ETC, the production of ROS, the Castat genes, their regulation and the subcellular STAT3 localization. For a more detailed description the readers are kindly redirected to recent reviews , opening the horizon towards the understanding of the pro-survival functions of this protein.2+ and ROS homeostasis and preventing mPTPs opening, contributes to the recovery of mitochondrial function following IR episode, which is a fundamental prerequisite in the mechanism of efficient cardiac conditioning.In cardioprotection scenario, STAT3, by regulating mitochondrial CaAlthough the most recent data suggest that optimal cardioprotection can be reached with the combination of additive or synergistic multitarget therapies , understHere we have discussed some recent studies that highlight the key role of STAT3 in cardioprotection, which, together with other regulatory  and pathways, plays a central role also in humans and in experimental models relevant for translation from bench research to clinic .We hope that the results reported in this review will encourage more and more researchers to deepen their studies and clarify the remaining doubts about STAT3 pro-survival role in the cardiovascular field. In particular, the function exerted on the ETC to maintain its efficiency and prevent ROS formation represents an excellent starting point to justify the interest in understanding the mechanisms underlying this role of STAT3 within mitochondria.


Salvia sclarea L.), a perfume, medicinal and aromatic plant, is used for sclareol production and ornamental purposes. Despite its wide use in the field of cosmetics, the phenotypic and genetic diversity of wild and cultivated clary sages remains to be explored. We characterized the genetic and phenotypic variation of a collection of six wild S. sclarea populations from Croatia, sampled along an altitudinal gradient, and, of populations of three S. sclarea cultivars. We showed low level of genetic diversity for the two S. sclarea traditional cultivars used for essential oil production and for ornamental purposes, respectively. In contrast, a recent cultivar resulting from new breeding methods, which involve hybridizations among several genotypes rather than traditional recurrent selection and self-crosses over time, showed high genetic diversity. We also observed a marked phenotypic differentiation for the ornamental clary sage compared with other cultivated and wild clary sages. Instead, the two cultivars used for essential oil production, a traditional and a recent one, respectively, were not phenotypically differentiated from the wild Croatian populations. Our results also featured some wild populations with high sclareol content and early-flowering phenotypes as good candidates for future breeding programs. This study opens up perspectives for basic research aiming at understanding the impact of breeding methods on clary sage evolution, and highlights interesting avenues for clary breeding programs.A road-map of the genetic and phenotypic diversities in both crops and their wild related species can help identifying valuable genetic resources for further crop breeding. The clary sage ( A road-map of the genetic and phenotypic diversities in both crops and their wild related species can help identifying valuable genetic resources for further crop breeding. Over the past century, agricultural practices caused a dramatic erosion of biodiversity in major crop plants . The conDespite the increasing use of medicinal aromatherapy, genetic and phenotypic variation among wild and cultivated perfume, aromatic and medicinal plants (PAMP) are still little explored . PlaSalvia sclarea L., is a biennial or short-lived perennial with a native distribution in Northern Mediterranean, but has a vast geographic distribution towards Central Asia and the Middle East [i.e., oil production or ornamental purposes, and result from traditional or recent breeding methods. Traditional clary sage breeding methods consist of successive cycles of selection and inbreed crosses among individuals from the same population sharing close candidate phenotypes. For oil production, candidate phenotypes show high sclareol and linalool content and, when possible, early flowering. Indeed, the most valued compounds of clary sage is sclareol, a diterpene extracted from clary sage straw, used as starting material for the synthesis of Ambroxide®, characterized by a musky odor and scent fixative ability, and linalool, a monoterpene and its acetylated derivative linalyl acetate [e.g., the “Milly” cultivar. Clary sage used for oil production has pink to purple flowers, a phenotype which stands in stark contrast with cultivars used for ornamental purposes which shows white flowers, e.g., the “Vatican White” cultivar. More recently, the raising global demand for sclareol in countries where clary sage is cultivated, including Hungary, Bulgaria, Moldavia, Romania and France, has encouraged new breeding initiatives. The recent clary sage breeding approach involves recurrent selection with successive cycles of three steps: intermating between best-performing populations, evaluation of traits of interest, and selection of best-performing individuals. This new breeding method that involves the mixing of several best performing populations led to the creation of a clary sage variety with high sclareol yield, named “Scalia”. However, the “Scalia” variety displays a late-flowering phenotype, an unwanted phenotype. Further recurrent selection cycles led to the development of the “Toscalia” variety, which is characterized by a high sclareol yield without a late-flowering phenotype. While the Toscalia has an attractive phenotype for breeders, it is still not flowering early enough. Breeders are therefore still seeking for candidate populations with early-flowering, and if possible higher sclareol and linalool contents. Wild clary sage populations represent a potential source of untapped allelic variation. However, phenotypic variation for flowering time, sclareol and linalyl acetate contents of S. sclarea cultivars used for different purposes, as well as of wild clary sage populations, are still lacking. The genetic relationships among cultivated and wild clary sages, and their respective genetic diversity, also remained unknown.The clary sage, dle East , 11, anddle East , and it  acetate –16. Earl acetate . TherebyWe investigated genetic and phenotypic variation among populations of three clary sage cultivars  and wild diploid Croatian clary sage populations . The wilS. sclarea individuals were collected at six sites in Croatia , Blato , Zaostrog , Kljenak , Ravca , Banja . We chose Croatia because wild clary sage is widespread in this country and there is no cultivation of clary sages there. We therefore avoided the potential sampling of feral populations. Seeds were stored at 4°C and 40% humidity until sowing. We also obtained seeds of cultivated S. sclarea from the “Vatican White”, “Toscalia” and “Milly” cultivars. Note that under the “cultivar” umbrella fall cultivated individuals resulting for several crosses between individuals sharing close phenotypes for sclareol and linalool contents, flowering time and flower color. As explained in the introduction, the Milly cultivar is traditionally used for perfume and aromatherapy purposes, whereas the Vatican White population is propagated and commercialized for ornamental purposes. The Toscalia variety is originated from recent breeding for commercial purpose for essential oil. Toscalia and Milly seeds were kindly donated by the French National Conservatory of Perfume, Medicinal, Aromatic and Industrial Plants (CNPMAI) and the French Interprofessional Institute of Perfume, Medicinal, Aromatic and Industrial Plants (ITEIPMAI), respectively. There, the Milly cultivar is maintained as a population of clary sage that are recurrently interbred. Salvia sclarea ‘Vatican white’ seeds were purchased from Jelitto (Germany). Croatian wild seeds were collected in 2017 but are not considered a strictly protected species according to the Croatian Ordinance and therefore do not fall under the Nagoya protocol. Note that the Vatican white has white flowers whereas ‘Milly’, ‘Toscalia’ and wild S. sclarea studied have different shades of purple-pink.During summer 2016, seeds from 60 wild  Croatia . These sS. sclarea, and each S. sclarea cultivar, three to four individuals were grown in growth chamber  for six weeks. DNA was extracted from leaves using the Qiagen DNeasy 96 Plant Kit following manufacturer recommendations. Genomic fragments from DXS2, CMK and ITS 1 loci were amplified by PCR using primers listed in For each sampled site for the wild Tajima’s D test [Tajima’s D test was non-significant, we assumed the sequences were evolving neutrally. However, Tajima’s D can be significant in case of bottleneck or expansion, regardless selection. We therefore further tested for neutrality of the sequences with the McDonald and Kreitman test (MKT) [Salvia outgroup species was available on NCBI for a given marker. The resulting neutrally-evolving sequences were then concatenated. Because of the occurrence for each sequenced marker of heterozygous sites encoded with the IUPAC code, haploid sequences were phased for each individual with the PHASE algorithm implemented the DnaSP software [We checked the neutrality of the markers to be used for population genetic analyses with s D test . When Tast (MKT) . The MKTsoftware . HaploidWatterson’s θ [Tajima’s D [S. salvia and each cultivar . We investigated genetic relationships among wild and cultivated samples with a splitstree and a principal component analysis (PCA). For the splitstree, we used the NeighborNet distance transformation and equal angle splits transformation implemented in SplitsTree v4 [http://www.R-project.org).We computed rson’s θ  and Tajijima’s D  with thejima’s D  for eachsTree v4 . For thesTree v4  and adegsTree v4  R packagi.e., Makarska-coast, Ravca-mountain and Banja-plateau, rd, 2017, and then grown in a growth chamber  for four months. On November 7th, 2017, 93 seedlings (between 15 and 23 per site or cultivar) were transplanted in the field in Chemillé  to allow the plants overwintering. Plants were placed every 0.4 m x 0.7 m according to a randomized layout. Flowering time was defined as the date of opening of the first flower and was recorded between May 31st and June 29th, 2018, until all plants have flowered. For calyx sampling, inflorescences were cut on July 19th, 2018 at the beginning of the afternoon. Mature calyces (harboring green achenes) were cut from all over the inflorescence, immediately placed in dry ice and stored at -80°C at the end of the day until further analysis.Seeds from three wild clary sage sites, representing the three macro-environments  may have led to natural variation in phenotypes. In order to study the influence of the use of the cultivated sages and the wild status of each plant, we fitted a generalized linear model as follows:μ is the overall mean of the phenotypic trait , ‘wild_site_or_cult_use’ is the fixed effect of the status of each plant  and e is the residual. We ran a general linear model, with a residual term assumed to be normally distributed. Statistical significance of the difference between status was tested with a pairwise Kruskal-Wallis rank test.In the presence of a domestication syndrome, we expect differences in phenotypes of cultivated accessions compared to wild accessions. We can also expect phenotypic differences between the cultivated clary sages depending on their uses , and between wild D and MKT showed that the ITS, and the two genes involved in the diterpene production pathway, DXS2 and CMK, evolved neutrally  flowered between May 31P = 0.046, P<0.0001, Overall, we observed significant differences in flowering time, sclareol and linalyl acetate among wild clary sages, clary sages cultivated for ornamental purposes and for oil production . For wilWe provided a first insight into crop and wild genetic and phenotypic variation of a perfume, medicinal and aromatic plant, the clary sage. Our results showed a lower level of genetic diversity for the traditional clary sage cultivars, whatever their uses, compared with the Croatian wild populations. In contrast, the recent Toscalia cultivar, resulting from new breeding methods, showed a high genetic variation, not significantly different from the Croatian wild populations. Phenotypically, a strong divergence is observed for the ornamental clary sage compared with the two cultivars used for essential oil production. Interestingly, the cultivars used for essential oil production were not significantly differentiated from the Croatian wild populations, whatever the area/altitude of origin of the wild population. We also did not observe phenotypic variation among wild Croatian clary sage populations. Our results finally pinpoint certain Croatian wild populations with interesting sclareol and linalyl acetate contents for future clary sage breeding.S. sclarea quality though raising the capacity of essential oil accumulation from the same population. Such a new breeding method has resulted in the recent Toscalia cultivar, which, not surprisingly showed high level of genetic diversity, equivalent to the one found in the wild Croatian populations studied here.We did find a lower genetic diversity for the traditional cultivars used for essential oil production (Milly) and for ornamental purposes (Vatican White). Traditional clary sage breeding methods consist of successive cycles of selection and inbreed crosses among individuals from the same population sharing close candidate phenotypes, either for sclareol content and/or early-flowering for oil production, or flower color for ornamental purposes. The recurrent selection and bottlenecks implied by traditional breeding methods have likely played a role in the loss of genetic diversity observed for the two cultivars. In contrast, the recent breeding strategy consists of hybridizations of different genotypes to diversify the genetic basis for The lower diversity of traditional clary sage cultivars may reflect a bottleneck during domestication but also a post-domestication erosion . Furthere.g., Milly or Toscalia), or of S. sclarea wild populations, the latters displaying darker shades of pink and purple. In other plant species, the pink or purple color of flowers is often due to the presence of specialized metabolites belonging to the flavonoid family [Artemisia annua, the amount of artemisinin found in leaves is tightly correlated to glandular trichome density [We showed a strong phenotypic divergence for the ornamental clary sage cultivar, with an earlier flowering and much lower sclareol and linalyl acetate contents than the wild Croatian clary sage populations and the cultivar used for essential oil production. Indeed, Vatican White plants flowered 10 days earlier and produced 20% less sclareol and 30% less linalyl acetate than the cultivar used for oil production. In addition, Vatican White cultivar has light flowers that stand in stark contrast with the flowers of other cultivars used for oil production . Finally, planted in a common environment, the six wild populations sampled across an environmental gradient did not show significant differences in metabolite content. This result suggests no genetic difference for metabolic contents among wild populations. Such a lack of variation can be explained by the narrow geographic area sampled in this study.Salvia officinalis [In conclusion, while plants from the wild Croatian populations can be used for breeding programs as a whole regarding their metabolic content, the Ravca population can be used specifically for its early flowering. Additional common gardens including several wild and cultivated clary sage populations in different sites across an altitudinal gradient are now required to guide future clary sage breeding programs. Specific environmental conditions may induce variation yield and chemical composition in metabolite contents, as previously shown in a related species, icinalis .Clary sage produces sclareol, a terpenoid widely used in perfume industry for the hemisynthesis of ambroxide, a valued perfume component with remarkable fixative properties. In response to the development of ambroxide use in functional perfumery, the global demand for sclareol currently raises, prompting efforts aiming at increasing sclareol supply. Breeding programs aims at increasing the yield of sclareol production from clary sage through targeted genetic improvement. The purpose of our study was to enhance knowledge on phenotypic and genetic variation of wild and cultivated clary sages to provide a first basis for clary sages breeding programs. A larger number of markers and samples of wild and cultivated sages  across the clary sage distribution is however now required to better understand the clary sage evolution, and provide further directions for breeding programs.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


The European Robin is a small passerine bird associated with woodlands of Eurasia and North Africa. Despite being relatively widespread and common, little is known of the species’ breeding biology and genetic diversity. We used Next Generation Sequencing (NGS) to develop and characterize microsatellite markers for the European Robin, designing three multiplex panels to amplify 14 microsatellite loci. The level of polymorphism and its value for assessing parentage and genetic structure was estimated based on 119 individuals, including seven full families and 69 unrelated individuals form Poland’s Białowieża Primaeval Forest and an additional location in Portugal. All markers appeared to be highly variable. Analysis at the family level confirmed a Mendelian manner of inheritance in the investigated loci. Genetic data also revealed evidence for extra-pair paternity in one family. The set of markers that we developed are proven to be valuable for analysis of the breeding biology and population genetics of the European Robin. Erithacus rubecula, a small (c.19 g) passerine bird, is a common and widespread species of wooded habitats in Europe, parts of western Asia and North Africa1. Although it is a familiar and moderately well-known species in Western Europe, there is little information about the species’ breeding biology across much of its range, and studies of its population genetics are mostly lacking9. This absence of genetic studies is probably related to the European Robin’s elusiveness during the breeding season, when nests are notoriously difficult to locate1.The European Robin 4. However, these studies may not be representative of the European Robin’s ecology in more natural forests elsewhere in its range. This study bias and difficultly of observing breeding European Robins means that there is no information characterising the species’ typical patterns of behaviour and breeding biology in primaeval forest habitats, to which it was originally adapted10.Most of the available fragmentary data on the breeding biology of European Robins in Britain comes from highly transformed environments, such as gardens and secondary woodlands12. Despite this basic knowledge there is no information on genetic relatedness or extra-pair paternity within or between broods and their parents.The European Robin is considered to be socially monogamous and highly territorial in the breeding season, when pairs may have up to three consecutive breeding attempts between spring and early summer. Nests are typically located in recesses or cavities on standing or fallen trees within several meters of the ground, in banks or walls, or under overhanging vegetation on the ground itself; nests generally contain 4–5 eggs that are incubated for around 15 days before hatching altricial nestlings that leave after a further 15 days, becoming independent around 10 days later17. These techniques have revealed that extra-partner mating is a much more frequent phenomenon among birds than was previously thought18, and extra-pair nestlings occur frequently even among socially monogamous species14. Such studies are important in advancing the understanding of female mate choice and reproductive fitness20. The benefits to females of adopting the extra-pair mating strategy are still debated, and there is no certain evidence that the advantages of such breeding activity outweigh the costs. Conversely, males obviously benefit from this behaviour through the increased reproductive success while engaging in the care of nestlings17.Molecular methods have made an invaluable contribution to the study of the breeding biology and mating systems of birds21. This study revealed that birds from Gran Canaria island should be considered as a distinct subspecies that is genetically separated from birds on continental Europe and the western Canary Islands. In contrast, analysis of population from the Azores indicated a lack of neutral genetic differences between group of birds from the archipelago and the group from mainland Portugal, suggesting a recent colonization of these islands22. Another resident population, from southern Italy, was investigated using cross-amplified microsatellites23, which suggested significant genetic differentiation between populations with long- and short-distance migratory behaviour.Only three studies have applied molecular techniques to describe population genetics of the European Robin. The genetic structure of a resident population from the Canary Islands, which migrate short distances between breeding and wintering grounds, was characterized using polymorphism of mitochondrial DNA26. Also, diversification of the species may lead to mutations in sequences of flanking regions of some alleles, preventing the primer annealing during PCR28. This, in turn, may result in a high frequency of ‘null alleles’. Moreover, the success rate of amplification frequently decreases in proportion with the genetic distance between species29.The use of cross-amplified microsatellites in genetic studies is known to have several potential disadvantages, including particular markers being less polymorphic in target species than in the species of originHowever, to date, there appears to have been no species-specific microsatellite markers described for the European Robin, including for the Central European population that represents the nominative and highly-migratory subspecies. Furthermore, this knowledge gap compounds the lack of data, supported by genetic information, for the diversity, ecology and biology of the European Robin’s populations in natural forest ecosystems across Europe. Such studies from primary forest conditions are informative and highly valuable as a benchmark for understanding a species’ biology in modified ecosystems and more outlying populations in the species’ range.30. Such data provides an important reference system for further studies of evolutionary and ecological processes in this forest, in addition to those occurring in managed forests under strong anthropogenic pressure.In this study we provide the first genetic data for European Robins from the last significant expanse of European temperate primaeval forest, the Białowieża Forest in north-eastern Poland, and from a second novel location on the western margin of the species’ European range, in Portugal. Information from the Białowieża Forest provides a unique opportunity to extend our knowledge of the European Robin’s biology in the natural, unmodified forest that still retains an almost complete suite of flora and faunaThe main aim of the study is to describe the species-specific microsatellite markers for the European Robin, as a resource for population genetics and breeding biology studies. We then apply these molecular markers to investigate the paternity of multiple broods and individuals of European Robins from the Białowieża Forest, and additional samples from individuals resident approximately 2730 km away in Portugal, to estimate genetic diversity of the species across this geographical span.This study was carried out in compliance with the ARRIVE guidelines. All methods were performed in accordance with the relevant guidelines and regulations. The methods used for animal care and handling were approved by 2nd Ethical Committee for Animal Experimentation at Warsaw University of Life Sciences, Poland  and Portuguese Institute for Conservation, Nature and Forests .10. Genetic material was collect on three sample plots (about 30 ha each) at representative locations in the forest.Samples of genetic material from European Robins were collected in two widely separated areas of Europe, in order to isolate microsatellite markers and investigate relatedness. In north-east Poland, birds were sampled between 2018 and 2019 in the Strict Reserve of Białowieża National Park   were caught using mist-nets (fine sheets of netting suspended between poles) and a recording of songs and calls as a lure. Further samples of a single chick (8–10 days old) from each of 28 nests were also collected. These combined samples enabled genetic analysis for 47 presumably unrelated birds. Additionally, we collected samples from seven ‘full’ families  to verify Mendelian inheritance of the isolated microsatellites. These seven ‘full families’ included 14 of the 19 adult birds reported above. The material for genetic analysis was collected during standard capture and marking  procedures for adult birds, and during nest inspections for nestlings, and involved plucking a single tail feather from each individual. Samples were stored in Eppendorf tubes filled with 96% pure ethyl alcohol.In Portugal, where the breeding population of European Robins is resident all year round31, 24 samples of adult birds were collected from between April and June 2020 at five locations: (A) Fradelos ; (B) Azurara ; (C) Arvore ; (D) Larça ; and (E) Tapada de Mafra  , 95 individuals from the Białowieża forest were sampled for genetic analysis. Adult birds  and a dense and diverse understory. These four locations are integrated in a landscape mainly composed by small forest patches, intertwined with agriculture and urbanized areas. Finally, Tapada de Mafra is a fenced property of more than 800 ha, managed for eco-tourism and big-game hunting with a mosaic of open areas and diverse forests dominated by Quercus spp. The birds were captured with mist nests placed along dirt tracks or with spring traps baited with mealworms (Tenebrio molitor). A blood sample was collected from each bird by puncturing the brachial vein near the joint between the humerus and ulna with a sterile needle (27G), following standard methods32. Approximately 75 µl of blood was collected with a capillary tube and then transferred to a 2 ml tube filled with 98% ethanol, which was stored at 4 °C.Fradelos, Azurara and Arvore consist of mixed plantations of 24, or blood clots within the rachis of fully grown feathers33. Hence, approximately 0.5 cm of the calamus was extracted and cut into smaller pieces using a sterile scalpel on sterile Petri dish. Cut parts of the calamus were then incubated with a Proteinase K overnight at 56 °C.For the feather samples, the main source of DNA is cells attached to the base of the calamus (quill) when feathers are in the growth phaseFor samples from Portugal, blood clots were removed from the alcohol and put into the buffer LT (EURx Polska) and incubated with a Proteinase K overnight at 56 °C. We then used a Tissue DNA Purification Kit (EURx Polska) to extract DNA from the calamus and blood following standard protocols. Each extract was analysed using Nanodrop (Thermo Fisher Scientific) to check the purity and concentration of DNA. The extracted DNA was stored at -20 °C.An amount of 7.4 µg of genomic DNA consisting of four equally (20 µl) mixed DNA samples was used for a 2 kb library construction. DNA was fragmented, using Covaris System according to protocol, into sizes between 1.5 and 3 kb, applying the following modifications: intensity was set to 0.2, and time of sonication to 500 s. Fragmented DNA was purified using AMPure PB Beads applying 0.6 × volume ratio.ExoIII and ExoVII enzymes. To separate the enzymatic reactions, DNA was purified with 0.6 × volume ratio of AMPure PB Beads. Size distribution of DNA fragments and its integrity was monitored on 0.5% agarose gels during and after completing the procedure of library preparation. The final library was double purified with 0.6 × volume ratio of AMPure PB Beads. Binding Calculator v.2.3.1.1 (PacBio) was applied for protocol generation in order to prepare the library for sequencing applying the DNA/Polymerase Binding Kit P6 V2 and MagBeads reagents. Sequencing was performed on a PacBio RS II sequencer running three SMRT Cells in the 360-min data collection mode.Subsequently, 750 ng of shared and concentrated DNA was used for library preparation with SMRTbell Template Prep Kit 1.0, applying the online Pacific Biosciences protocol (PacBio). This procedure consisted of: 1. DNA damage and ends repair, 2. blunt ligation of hairpin adaptors at both ends of the DNA fragments, and 3. removal of failed ligation products with 34 program in order to identify microsatellite sequences and design primers. A threshold of at least five repetitions for tri- and tetra-nucleotide repeats, excluding mononucleotide repeats, was set in this analysis. Primer design parameters including the msatcommander option “combine loci” were as follows: length of the primers 18–22 bp, annealing temperature (Tm) 58 °C–62 °C, GC content 30%–70%, and amplicon product size 80–400 bp with minimum of 20–30 bp DNA sequence flanking from both sides of the tandem repeat of the microsatellite.Data generated through the PacBio sequencing platform was analysed to obtain Reads of Insert (ROI) according to the RS_ReadsofInsert.1 protocol applying default settings. The ROI reads were further analysed with the msatcommander v.1.08Selection of microsatellite markers was performed in several steps. Firstly, based on information obtained for the set of microsatellite sequences, identified in the genome of the European Robin from the above analyses, loci with the highest number of tandem repeats were selected . In the second step, loci were filtered based on criteria calculated in silico: (1) maximal PCR efficiency, (2) minimal penalty score, (3) no dimer formation, (4) a maximal difference in melting temperature of 2 °C between a primer pair, and (5) sequence length of the microsatellite ranging from 100 to 350 bp. This gave 40 selected microsatellite loci, which were then divided based on product size into three groups: (1) short < 200 bp, (2) medium 200–300 bp, and (3) long > 300 bp. The reason for this division was optimization of designed multiplex PCR.35 and Austin et al. (2011)36. Briefly, the forward primers were tailed by a 5′-CACGACGTTGTAAAACGAC sequence tag, which was identical to four fluorescent labelled dyes: 6-Fam, Hex, Tamra and Rox. The PCR reaction mix contained: 1 µl of DNA, 7.5 µl of 2 × Dream Taq Hot Start Green PCR Master Mix (Thermo Fisher Scientific), 0.02 M tagged forward locus specific primer, 0.2 M locus specific reverse primer and 0.2 M fluorescent labelled universal forward primers. The reaction was performed under the following conditions: initial denaturation for 15 min at 95 °C, 35 cycles of 30 s at 95 °C, 50 s at 57 °C, 45 s at 72 °C and final elongation of 5 min at 72 °C. Microsatellite loci were amplified separately and visualised on 1.5% agarose gel. The genotyping analyses was performed using ABI 3500XL Genetic Analyzer (Applied Biosystems) and the PCR product sizes were read using GENEMAPPER v.4.1 software (Applied Biosystems). Based on the results obtained, 17 microsatellite loci were selected for further analysis and design of multiplex panels. These loci were characterized by a lack of amplification artefacts, a high number of alleles and no more than two peaks per locus on chromatograms.Finally, for further testing, we selected 11 short loci, 14 medium loci and 15 long loci. These loci were initially tested on 17 unrelated individuals following the method of Schuelke (2000)The multiplex PCR reaction mix contained: 1 µl of DNA extract (containing 47–283 ng of DNA in the case of young birds) or 3 µl of DNA extract (containing 1–78 ng of DNA in the case of adult birds), 7.5 µl of PCR Master Mix (QIAGEN), 0.6 µl of the primer mix  and 4.65 µl or 2.65 µl water for PCR depending on the amount of DNA extract used. This time forward primers were fluorescently labelled on their 5′ end with one of the following dyes: 6-FAM, TAMRA, HEX and ROX (Sigma-Aldrich). The amplification conditions were as follows: initial denaturation for 15 min at 95 °C, 40 cycles of 30 s at 94 °C, 90 s at 57 °C, 90 s at 72 °C and final elongation for 10 min at 72 °C.Analysis of the amplification of the microsatellite loci in different combinations of multiplex sets was verified using ABI 3500XL Genetic Analyzer. As a size standard we used GeneScan 600 LIZ (Applied Biosystems). The genotyping was performed using a mixture of formamide with LIZ-600 and PCR product, which was denatured for 5 min. at 95 °C. Genotypes were then identified using GENEMAPPER v.4.1.E) and observed heterozygosity (HO) using GenAlEx version 6.537 and Hardy–Weinberg equilibrium (HWE), and also Genepop on the Web version 439. Additionally, the fixation index (FIS), allelic richness and FST were calculated using FSTAT version 2.9.340.All statistical analyses were performed on genotypes of 69 presumably unrelated individuals . For each locus we assessed the number of alleles (A), unbiased expected heterozygosity , F(null) , NE-1P (average non-exclusion probability for the first parent), NE-2P (average non-exclusion probability for the second parent), NE-PP (average non-exclusion probability for a candidate parent pair), NE-I  and NE-SI (average non-exclusion probability for identity of two siblings). Probability of identity (PI) and probability of identity for siblings (PISibs) were estimated using GenAlEx version 6.537.Parameters of usefulness of the selected microsatellites in paternity verification and population genetics studies were also estimated, using Cervus 3.0RSII sequencing of the SMRT library generated 4,330 Mb of raw data covering 192,298 reads for the three SMRT Cells analysed jointly. The average length of inserts in these runs equalled 1,959, 1,873 and 2,997 bp, respectively . The final output length of reads reached 3,800 Mb. Of these sequence sets, 65,162 reads of insert (ROI) were acquired and accumulated with 87,123,089 bp. The average ROI length equalled 1,337 bp and the average number of passes exceeded 16.The final sequence list consisted of 62,872 DNA sequences that were subjected to microsatellite loci identification. Altogether 1932 microsatellite loci covering tri- or tetra-nucleotide repeats in at least five repetitions were identified. For the a priori chosen parameters of microsatellite selection for amplification testing, primer pairs for 961 loci were successfully designed. From this set, 18 tri-nucleotide and 22 tetra-nucleotide markers (named ER1-ER40), with the number of repeats ranging between 37 and 10, were further chosen for amplification and polymorphism description (Supplementary Table Agarose gel electrophoresis indicated that 36 out of 40 tested primer pairs (90%) were successfully amplified. The amplification was evaluated as successful if the length of the band was similar to the size of alleles indicated by mstacommander. Genotyping using ABI 3500XL Genetic Analyzer (Applied Biosystems) showed that 23 primer pairs amplified microsatellite fragments, meaning that the peaks on chromatograms were sharp and the score of the peaks was similar to the expected product size. Finally, 17 primer pairs were selected for designing multiplex PCR. In the case of three primer pairs, amplification failed in multiplex reaction, despite applying several combinations. Hence, we finally designed three multiplex panels  to 26 (locus ER31).O = 0.913;) and ER31 (HE = 0.935). The lowest value of heterozygosity was observed in locus ER24   and significantly lower than the expected heterozygosity . The estimated frequency of null alleles [F(null)] was very low, reaching 10% only in two cases , although FIS was low and non-significant after Bonferroni correction . Similarly, in the group of samples from Portugal the observed heterozygosity was lower than expected , with an associated low value of FIS (FIS = 0.07). The mean number of alleles was higher in Poland (mean A = 13.64) than in Portugal (mean A = 10.71), although allelic richness was similar in both regions .Considering the two separate locations, analysis of the 45 individuals from the population from Poland also indicated significant deviation from Hardy–Weinberg Equilibrium due to heterozygote deficiency  but this was statistically significant (P < 0.05).Genetic differentiation, assessed as FSibs = 0.28) and the highest were in loci ER24  and ER32 . The respective values for the combination of 14 loci were also very low  indicated extra-pair paternity in only one case Table .Table 3M44. It is a method of choice in cases of non-model organisms with no available genomic data, as well as allowing the obtaining of long reads (here ca. 2 kb) that cover the whole microsatellite sequence together with a large flanking region. This method, coupled with a universal primer labelling system35, was proven to be highly cost effective and efficient when compared to the traditional approaches of microsatellite description45, or testing via individual fluorescent labelling of all of the tested primers46.The PacBio RSII sequencing enabled microsatellite markers for European Robins to be discovered in a sufficient number, and the restricted selection using narrow criteria enhanced the possibility of choosing high quality markers for population analysis. This method is often chosen for de novo microsatellite characterization, and is recognised as being reliable and efficient46.In our study, we selected 14 microsatellite loci amplified in three multiplex reactions, which are useful for further analysis of the breeding biology and population genetics of the European Robin. The criteria of loci selection concentrated on tri- and tetra-nucleotide microsatellites, as they were reported to be less biased with amplification errors, especially stuttering, although they were less polymorphic and frequent than di-nucleotides. However, such an approach as ours results in far fewer problems in reading, and also later coding of alleles23. Indeed, we showed that the identified microsatellites amplified well in the two populations that were separated by a large distance across Europe (c. 2730 km). Comparison of birds from these two populations showed that resident birds from Portugal have slightly lower genetic diversity  than the migratory population from Poland.The results of the analysis of nearly 70 unrelated birds from Poland and Portugal showed a high level of polymorphisms of the identified microsatellites, indicated by the values of allelic diversity and heterozygosity. Such a set of markers should allow adequate investigation of genetic diversity and population structure in the European Robin, complementing and advancing on the work of previous studiesRegulus satrapa)47.Higher genetic diversity of migratory populations compared to resident ones has also been confirmed in North American Golden‑crowned Kinglets 1.We showed that genetic differentiation was low between the two populations, yet it was statistically significant. This supports previous studies that showed significant differentiation even within a limited geographical area, such as approximately 100 kmIn the analysis of the genotypes of the seven full families of European Robins from Białowieża, we found strong evidence of extra-pair paternity in one nest. Although the sample size was low in this initial study , the result suggests a potentially low frequency of EPP in the study population . However, this observation should be verified in a more extensive study, preferably by sampling a large number of representative families and populations across the species’ range.Phylloscopus sibilatrix and Willow Warblers P. trochilus, which vary between zero and c. 50% of nests54. These rates are also similar to many other bird species55. Determining rates of EPP can be important for understanding strategies of breeding biology, settlement patterns and mate selection58.The rate of EPP that we found for European Robins was within the range of other European forest songbirds, such as the Wood Warbler 4, but has nevertheless been poorly studied across much of its range until the present time. We encourage further studies of the genetic diversity of European Robins across its broad geographical range, which may be valuable in understanding any differences between migratory and sedentary populations. Analysis of genetic diversity may also assist in the understanding of any differences between populations inhabiting the varying climatic spectrum in the European Robin’s range  and the wide variety of modified habitats compared to the primeval forest of Białowieża. Comparisons in the genetic variability between other species across this range of enviornments and behaviours would also be valuable.In summary, our results are the first to provide the essential basis for similar ecological and genetic studies of European Robins, which is an iconic and culturally significant species in parts of EuropeSupplementary Table S1.


Aristotelia chilensis (Molina) Stuntz (‘Maqui’) is a widely distributed native species in Chile. Its berry is considered a “super fruit” with an increasing interest in the food industry. This study investigated the phenotypic variation of growth, fruit, and ecophysiological traits of 20 A. chilensis clones originated from six provenances along the latitudinal gradient and established in a common-garden experiment in the Mediterranean zone of central Chile (center part of the species distribution). Differences among provenances were observed for most of the  traits under study, especially between the northern and southernmost provenances . Northern provenances showed higher development of vegetative tissue and fruit yield but lower intrinsic water use efficiency (WUEint) compared with southern ones. Clonal variation within provenances was found significant for the ripening index, WUEint, and fruit number and weight but not significant for traits related to the crown and leaf morphology. A genetic differentiation due to latitudinal cline was not evident in this study, but differences among provenances suggest local adaptation for some traits. The genotypic variation in productive traits must be considered in the outgoing domestication of the species and future selection programs.The domestication of forest species has traditionally relied on productivity issues. However, today there are concerns about the potential responses of natural populations and new cultivars to extreme climatic conditions derived from climate change and how to incorporate this knowledge into the domestication programs.  Forests have an essential role in future food security as they represent a valuable source of vital nutrients and natural antioxidants3. Among fruits, berries have an enormous potential for the food industry as they are a rich source of bioactive compounds such as phenolic acids and anthocyanins4. Nonetheless, there are concerns about how the increasing temperatures and droughts due to climate change might affect the natural populations of important fruit plant species contributing to food security.The global consumption of fruits is increasing due to the higher awareness of their health benefits and nutritional valuesAristotelia chilensis (Molina) Stuntz, commonly named “Maqui”, is in Chile a native species whose range of distribution spans over 15° latitude (c.a. 1700 km) from Mediterranean semi-arid to temperate sub-humid and humid climates6. Its 6-mm red/purple colored berries are considered a “super fruit” because of their great health properties8. Its soluble solids are higher than in other berries such as blueberries (Vaccinium corymbosum), pomegranates (Punica granatum), blackberries (Rubus fruticosus), raspberries (Rubus idaeus), and cranberries (Vaccinium oxycoccos)9. They also have the highest antioxidant content among the fruits currently traded worldwide5. Recent studies have demonstrated its powerful antioxidant, cardioprotective, anti-inflammatory, gastroprotective, antidiabetic, and antibacterial capacity15. Currently, Maqui berries are wildcrafted directly from the natural populations, which contributes to the degradation and loss of genetic diversity16. To sustain the industry's growing demand, domestication studies of the species have focused on genetic selection and adaptation to cultural management techniques. However, there is little knowledge about the phenotypic and genetic variation of fruit and ecophysiological traits of natural maqui populations, information that might guide the future breeding and selection of outstanding genotypes.18. Studies suggest that some species covering large geographical and environmental gradients may develop local adaptation and phenotypic clines in growth and functional traits19, but this ability may change by species20. For instance, some forest species exhibit a higher genetic differentiation in growth and phenological traits than others describing wood anatomy or leaf physiology across their natural distribution19, whereas others exhibit genetic differentiation for physiological traits such as photosynthetic capacity and leaf water status22. Moreover, studies in common garden experiments have reported high provenance differentiation in the fruit and leave morphology of fruit species25, and in the accumulation of bioactive compounds such as polyphenols and anthocyanins in berries species28.One of the most important approaches to understanding the phenotypic variation and adaptation to local conditions is the use of common garden experimentsA. chilensis. In 1-year old clonal plants, Vogel et al.16 found differences in growth, leaf morphology, and fruit set traits among provenances that originated across the latitudinal gradient of the species. In a nursery experiment, Moya et al.29 also reported differences in these traits at the clone level, although this study considered only three commercial varieties. Otherwise, there are only a few studies addressing the genetic diversity and structure of A. chilensis. Cona et al.30 found a high genetic diversity in the species in a latitudinal range of 15 degrees, which following Hamrick31, is expected for a non-domesticated species with anemophily and entomophile pollination. Salgado et al.32 found that most of the genetic variation, evaluated by AFLP and chloroplast microsatellites, was explained by the differences within provenances  than among provenances. These authors point out the existence of little genetic structure and relatively high gene flow between populations. However, it is known that although these types of molecular techniques grasp well the neutral genetic variation, they have a lower performance on the variation of adaptive traits34. Thus, the extent of variation of adaptive traits of the species at the clonal and population level still remains unknown, and little is known in broadly distributed species such as A. chilensis, especially in the southern hemisphere. Regarding that climate conditions vary across the latitudinal gradient of the species distribution, we explored the question of What is the extent of genetic differentiation of fruit and ecophysiological traits between and within provenances of A. chilensis? The present study investigated the phenotypic variation of fruit, ecophysiological traits, and leaf traits of 20 A. chilensis clones from six provenances exhibiting latitudinal-related climate differences in Central Chile, and assessed in a 7-years old common-garden experiment.Some studies suggest a potential genetic variation that may be used in the domestication process of −1, P = 47 mg kg−1, K = 209 mg kg−1, Mn = 42.8 mg kg−1, Zn = 12.3 mg kg−1, Cu = 3.9 mg kg−1, Fe = 51.7 mg kg−1, B = 0.87 mg kg−1, pH = 6.1, and organic matter = 1.1%. Before planting, the soil was plowed to a depth of 40 cm, harrowed, and mounding of 40 cm height. On the planting rows, weeds were controlled by mulching and mechanically and between rows by mowing. A drip irrigation system watered the plants during the growing seasons, from October to March.A common garden experiment was established at the Experimental Station of Universidad de Talca in Panguilemo, Maule Region, Chile . This location is nearly the center of the latitudinal distribution of the species. The site is characterized by a Mediterranean climate, with a mean annual temperature of 14.7 °C and precipitation of 758 mm. The soil is classified as San Rafael soil series , with sandy clay loam texture, moderate drainage, and 50–90 cm deep. Soil nutrient analysis showed a content of N = 8 mg kgA. chilensis clones from eight wild provenances covering an area over 750 km or a latitudinal range of six degrees were arranged in a randomized design with 4–5 replicates. The experimental unit was a single-tree plot with 3.5 m between and 1.2 m within rows. For optimal free pollination, five male clones were located in the central row within the trial , and the number of clones per each provenance. The experiment was performed in accordance with relevant guidelines and regulations.In May 2012, 45 female Asat, μmol m−2 s−1), stomatal conductance  and intrinsic water use efficiency  were measured on one fully expanded leaf in the upper third of the plant, using a portable gas exchange system LI 6800 . Initial chamber conditions were set up close to the ambient conditions during measurements, with a temperature of 20 °C, CO2 concentration of 400 ppm, relative humidity of 50%, and PAR of 1800 µmol m−2 s−1. Measurements were performed between 09.00 and 12.00 h at local time.Leaf-level physiological measurements were taken nine times during the study period  at each plant per clone. Net saturated photosynthesis rate , reddish (beginning of ripening), and black purple (end of ripening). A representative branch of each plant was selected, and the percentage of fruit in each category was visually evaluated. Then, we elaborated a ripening index representing a weighted average at the plant level: Ripening index = (1 × Percentage green fruit + 2 × Percentage of reddish fruit + 3 × Percentage of black-purple fruit) 100–1. The index varies between 1  and 3 .The ripening stage of 16: LA = [(Ll − Lw/2)Lw/2 + п/2(Lw/2)2], where Ll is lamina length and Lw is lamina width. Then, the leaves were oven-dried at 65 °C to calculate the specific leaf area (SLA) as the leaf area to dry weight ratio.At the beginning of summer, 20 leaves per plant were randomly collected in the upper half of the crown for leaf morphological assessments. First, we measured the ratio between the wide of the curved leaf relative to the wide when it is flattened, from now on denominated leaf curve ratio. The same foliage was used to estimate the lamina area (LA) using the formula by Vogel et al.Similarly, at the end of ripening, four twigs were randomly collected at each plant to measure the growth of the previous and current growing season (2017–2018). The foliage and fruit were detached from the twigs and stored in plastic bags for further analysis. The leaves were scanned for leaf area (FA) determination using a bed scanner and the software Image J . The fruit on each twig was counted and weighted in fresh (FFW). Then, it was oven-dried at 65 °C for 48 h for dry mass determination. Additionally, we calculated a productivity index as the FFW to FA ratio. Twigs were also measured in the basal diameter and length using a digital caliper and a metric tape, respectively.35 and Giusti and Wrolstad36, with some modifications as described by González et al.37.Additionally, 60 g of fruit was collected from each plant to assess fruit quality. In a subsample of 20 randomly collected berries, fruit diameter was determined with a digital caliper. The remaining fruit was put in plastic bags and sent to the laboratory for polyphenols and anthocyanins analysis according to Folin-Ciocalteu and differential pH colorimetric methods described by Singleton and Rossi38.From each plant, two photos were taken perpendicular to the planting row with white cloth behind as a high contrast backdrop using a 28–70 mm 1:2.8–4.0 D zoom lens  mounted on a D70 digital SLR . The plant height was measured with a graduated pole. Then, images were processed with the software UrbanCrowns for Windows  to determine the crown volume and density39. A multivariate cluster analysis based on FFW, twigs growth, FA, and WUEint was carried out on the clones using the Ward’s method in the software JMP GENOMICS 7.1. Because of the low replication of the experiment, the statistical significance of the analyses was considered at a probability level of 0.1.First, to assess the interaction between provenances and measured dates within the growing season for the ripening index and leaf-physiological parameters, the following linear mixed model was considered:P = 0.0075, Table P = 0.0850, Table In all studied provenances, the ripening period, from the green to the black-purple stage, lasted approximately one month, although provenances varied in the ripening index by date , likely explained by the greater clonal differences in provenance SanFer  tended to have higher WUEint than northern provenances   to specific edaphoclimatic conditions. However, other studies have shown a good performance of provenances out of their geographic origin, which are signs of maladaptive or non-adaptive genetic changes due to genetic drift or stresses induced by soil resource limitations43. Contrarily to our expectations, the provenance Talca, which is close to the common-garden experiment , had a moderate fruit production at expenses of a higher water loss   and southernmost provenance Enlagos. The poor performance of provenance Enlagos suggests a local adaptation of this provenance to the temperate oceanic climate where it comes from.Some studies comparing provenances in common garden experiments have shown better performance for local provenance origin than others more distant to the study site E) Fig. . MoreoveA. chilensis has one of the highest relative growth and photosynthetic capacity of the Chilean temperate rainy forest44 and even comparable to the values reported for some of the most productive sclerophyllous species in central Chile45. In this study, the values for the leaf-physiological parameters were in the range reported for the species47, although differences among provenances were found since the end of the ripening stage. All provenances experienced a higher variation Asat within the maturation period, peaked at the end of the ripening period, and remained approximately steady during the rest of the growing period. In other species, this higher variation and increased photosynthetic activity within the ripening period has been related to a higher demand of photosynthates by fruits , which drives physiological processes such as transpiration and gas exchange49. Thus, after berry ripening, this sink decreases the demand for photosynthates in A. chilensis, likely down-regulating the photosynthetic capacity to steady levels as has been reported in apple trees by Kelc et al. [50].Genetic differentiation was found for physiological, morphological, and crown traits. gs and E observed in the local provenance Talca manifested in a significantly lower WUEint compared to southern provenances . Northern provenances came from sites with pronounced water restrictions compared to the southern provenances, so they were expected to be better adapted to the condition at the study site. However, specific adaptation to dry sites such as lower values for crown growth, gas exchange, foliar area, and leaf size were observed in the southernmost provenance Enlagos. The results of the provenance Talca disagree with the theory of specialization51 in which genotypes adapted to unfavorable conditions  may have superior performance in restrictive environments but are unable to take advantage of favorable conditions , leading to low plasticity. Overall, the northernmost provenance SanFer seemed to take advantage of the climatic conditions at the study site maintaining higher productivity and WUEint than the local provenance Talca, suggesting a high potential phenotypic plasticity for those provenances. Unlike the provenance Enlagos, which is likely well-adapted to wetter conditions, decreased physiological and morphological attributes when established in the drier conditions of the study site. This assertion is also supported by the lower leaf curvature ratio of this provenance. In this study, the leaf curvature ratio represents a measure of leaf rolling, which has been reported as a mechanism of avoidance of water loss52.The high 53. The variation in SLA with the latitude is mainly associated with the gradient in light and temperature, but it may also be affected by other factors such as nutrient availability and water stress54. Interestingly, northern provenances marked the differences in SLA among provenances. SLA was the highest for provenances SanFer and Talca and lower for provenance Romer. In A. chilensis, a drier growth environment with more light availability tended to decreased SLA47, a potential local adaptation that was observed only in the provenance Romer. However, in other species, SLA tended to decrease with increasing precipitation at the site provenance origin, but it also showed to increase with water added by irrigation22, which likely applied to the results found for provenances SanFer and Talca.SLA did not increase with the latitude as has been found in other plant species22, while the latter is mainly influenced by climate and nutrients53. Clones taken from a provenance develop under a similar climate, thus there is no climatic differentiation that could drive the genetic diversity on morphological traits. However, some provenances were not well-represented, which needs further research. Despite the low number of genotypes tested, there was a significant variation among clones in Asat, while the clonal performance on gs and WUEint was relatively stable within the growing period. In the end, E-BLUP of clones with higher and lower gs differed 2-fold, while for WUEint 3.7-fold, which offers the possibility to explore the selection of genotypes based on these traits.Within provenances , the genetic differentiation was found significant in gas exchange parameters but not in the crown and leaf-morphological traits. Physiological traits may be more strongly affected by genetic than structural traits55. It has been shown in other fruit tree species that only part of the genes influencing fruit maturation is affected by the environment and explains the varying responses of genotypes to specific environments56. Since the genotypes in our study grew in a common site, the results suggest a high genetic variation within and between provenances for berry ripening that overpasses a latitudinal cline. This high spontaneous genetic variation might explain the species’ success to prosper in different environmental conditions and agrees with the high genetic differentiation observed in genotypes with a common origin of other berries species. For instance, genetic differentiation in berry ripening was found between and within white and red grapevine cultivars in response to temperature58. Nesmith59 found high genotypic differentiation in southern-origin blueberry genotypes in the flowering time and fruit development in response to the chill hours at specific locations. Otherwise, Uleberg et al.28 mentioned potential local adaptations for bilberry clones (Vaccinium myrtillus L.), in which the berry ripening of northern origin genotypes (colder areas) was faster than the southern ones. In A. chilensis the genetic variation on berry ripening highlights the potential for genetic selection within the species to define early and late-ripening varieties.Ripening lasted approximately 1 month, with significant differences in the ripening stage between provenances and clones throughout the dates. However, a clinal pattern was unclear, as the southernmost provenance Enlagos ripened fast even compared with the northernmost provenances, while the two clones from the mid-range of the distribution  consistently had a delay in the ripening. Berry ripening is a complex process affected by soil resources, meso and micro-climate, and genetics60 showed, for another native superfruit (Berberis microphylla G. Forst), that higher radiation tended to explain better the decrease in fruit size and weight than temperature. This might apply to our study, regarding that the gradient for the provenance origin was higher for global radiation than for the temperature. However, for A. chilensis, the decrease in the shortwave radiation  might increase flower production and fruit set in some genotypes29. This partially explained our results because the PAR radiation is similar between the southernmost and northernmost provenance origin, whereas the UV radiation is higher in the southern provenance origin61. Moreover, in our experiment, plants of all provenances received the same amount of water by irrigation, which apparently was not enough for provenance EnLagos (adapted to high precipitation sites). This provenance had the lowest fruit traits  and did not privilege reproductive over vegetative growth. It is known that water irrigation strategies that diminish water usually reduces vegetative growth promoting reproductive growth, i.e., number of fruits63, but this was not the case for EnLagos plants and might be explained by the high plasticity of this provenance, climate differences, or both. Thus, it might be possible that the higher radiation of the common-garden experiment dampened reproductive growth of the EnLagos provenance, particularly clone 528. However, this hypothesis needs further research.Similar to other traits, the fruit size and yield differed mainly between the southernmost (EnLagos) and northern provenances, which suggest some local adaptation for the Enlagos provenance for these traits. There was also less variation in fruit production among clones within this provenance compared with the other provenances. Romero-Román et al.67. Apparently, none of the above-mentioned factors affected the berry quality in the genotypes under study. It is known that an excess or deficit of water influence the acidity of berry crops68, but results depend on the species, the intensity of the water stress applied, and the maturity stage. In apples, bioactive compounds such as anthocyanins tended to decreased with higher temperatures and increased with solar radiation while also differing between accessions69. In the present study, the higher temperatures and radiation experienced by the southern provenances at the experimental site were not likely sufficient to drive changes or local adaptations in the biosynthesis of these secondary compounds. According to Gonzalez-Villagra et al.70, in A. chilensis, severe water stress may increase the leaf anthocyanins and polyphenols content. In grapes, limited irrigation increased the berries anthocyanin concentration, the galloydation of seed, and skin proanthocyanidins, all related to greater wine properties and antioxidant activity71. In our study, the irrigation was provided continuously by a drip-irrigation system, alleviating any potential water stress on plants and the differences among genotypes in secondary compounds. In the case of A. chilensis, the pedoclimatic conditions likely excerpt little influence on anthocyanin content, as has been found in blueberry cultivars by Spinardi et al.65.Although genotypic differences in the fruit productivity were expressed in the common-garden experiment, these were not significant for the anthocyanin and polyphenol content. The accumulation of these secondary compounds depends on the fruit ripening stage, genetic, and environmental factors such as temperature, water stress, irradiation, soils, and altitudeA. chilensis. The domestication of the species might reduce the anthropogenic pressure on natural populations. Our common-garden experiment represents the standard management practices currently implemented in other fruit trees in the Mediterranean zone of Chile (including irrigation) and likely the most productive age. The combined analysis of traits by the clustering analysis suggests the potential genetic variation of the species that can guide the domestication process. Moreover, although three clusters were defined and related to latitude, there are some exceptions . The genetic variation in this study offers the possibility of defining ideotypes for specific sites . Then, further research is needed on multi-site trials to estimate clonal genetic parameters.There is a need to select better genotypes and cultivated under intensive management practices to respond to the future demand for berries of A. chilensis, this study suggests that the species has a great phenotypic variation, as southern provenances (except the southernmost) had a good performance in the climatic conditions of the study site, compared to the northern provenances in some traits. However, the southernmost provenance differed in most of the traits compared with the other provenances, which suggests a local adaptation to wetter sites and with the consequent decrease in performance in drier climates. Regarding the latitudinal gradient in most climatic variables, the variation among provenances was not clearly explained by a cline. Genetic differentiation was higher between than within provenances. Overall, differences among provenances were evident for leaf-physiological and morphological, fruit, and crown traits, whereas at clone level, the differences were expressed only for physiological and fruit traits. The higher phenotypic variation of the provenances understudy suggests a great potential for selection within the species and resilience under the expected changes in the environment due to climate change.Regarding the broad distribution of