The extinct Sicilian wolf shows a complex history of isolation and admixture with ancient dogs.

The Sicilian wolf remained isolated in Sicily from the end of the Pleistocene until its extermination in the 1930s-1960s. Given its long-term isolation on the island and distinctive morphology, the genetic origin of the Sicilian wolf remains debated. We sequenced four nuclear genomes and five mitogenomes from the seven existing museum specimens to investigate the Sicilian wolf ancestry, relationships with extant and extinct wolves and dogs, and diversity. Our results show that the Sicilian wolf is most closely related to the Italian wolf but carries ancestry from a lineage related to European Eneolithic and Bronze Age dogs. The average nucleotide diversity of the Sicilian wolf was half of the Italian wolf, with 37-50% of its genome contained in runs of homozygosity. Overall, we show that, by the time it went extinct, the Sicilian wolf had high inbreeding and low-genetic diversity, consistent with a population in an insular environment.

Pan-European phylogeography of the European roe deer (Capreolus capreolus).

To provide the most comprehensive picture of species phylogeny and phylogeography of European roe deer (Capreolus capreolus), we analyzed mtDNA control region (610 bp) of 1469 samples of roe deer from Central and Eastern Europe and included into the analyses additional 1541 mtDNA sequences from GenBank from other regions of the continent. We detected two mtDNA lineages of the species: European and Siberian (an introgression of C. pygargus mtDNA into C. capreolus). The Siberian lineage was most frequent in the eastern part of the continent and declined toward Central Europe. The European lineage contained three clades (Central, Eastern, and Western) composed of several haplogroups, many of which were separated in space. The Western clade appeared to have a discontinuous range from Portugal to Russia. Most of the haplogroups in the Central and the Eastern clades were under expansion during the Weichselian glacial period before the Last Glacial Maximum (LGM), while the expansion time of the Western clade overlapped with the Eemian interglacial. The high genetic diversity of extant roe deer is the result of their survival during the LGM probably in a large, contiguous range spanning from the Iberian Peninsula to the Caucasus Mts and in two northern refugia.

Purifying selection shaping the evolution of the Toll-like receptor 2 TIR domain in brown hares (Lepus europaeus) from Europe and the Middle East.

Toll-like receptors (TLRs) are transmembrane proteins of the innate immune system, composed of the ectodomain involved in pathogen recognition and the intracellular Toll/interleukin-1 receptor (TIR) domain important for downstream signal transduction. Here, we analyze the genetic variability of TIR nucleotide and amino-acid sequences of the TLR2 gene in 243 brown hares from Europe and the Middle East and tested for the presence of selection signals and spatial structuring. TLR2 TIR domain sequences were PCR amplified and sequenced, while genotyping was performed by phasing. Genetic diversity indices were calculated in DnaSP and Arlequin, while presence of selection signals was tested using MEGA and the Datamonkey web server. The presence of spatial patterns in TIR sequence distribution was tested by spatial Principal Component Analysis (sPCA) in adegenet. A total of 13 haplotypes were revealed with haplotype diversity of 0.424, and nucleotide diversity (π) of 0.00138. Two spatial clusters were revealed: "Anatolia/Middle East" and "Europe". In Anatolia the two most prevalent amino-acid variants, A and B (the latter being the most ancestral) were maintained at similar frequencies; but in Europe a shift in genotype frequencies was observed as well as a higher number of nonsynonymous substitutions giving rise to novel amino-acid protein variants originating from the evolutionarily younger protein variant. Molecular diversity (haplotype and nucleotide diversity) indices were significantly higher in the "Anatolia/Middle East" cluster. A signal of purifying selection was detected acting on the TIR sequences.

Positive selection and precipitation effects on the mitochondrial NADH dehydrogenase subunit 6 gene in brown hares (Lepus europaeus) under a phylogeographic perspective.

Previous studies in hares and jackrabbits have indicated that positive selection has shaped the genetic diversity of mitochondrial genes involved in oxidative phosphorylation, which may affect cellular energy production and cause regional adaptation to different environmental (climatic) pressures. In the present study, we sequenced the NADH dehydrogenase subunit 6 (MT-ND6) gene of 267 brown hares (L. europaeus) from Europe and Asia Minor and tested for positive selection and adaptations acting on amino acid sequences (protein variants). Molecular diversity indices and spatial clustering were assessed by DnaSP, Network, and Geneland, while the presence of selection signals was tested by codeml in PAML, and by using the Datamonkey Adaptive Evolution web server. The SPSS software was used to run multinomial regression models to test for possible effects of climate parameters on the currently obtained protein variants. Fifty-eight haplotypes were revealed with a haplotype diversity of 0.817, coding for 17 different protein variants. The MT-ND6 phylogeographic pattern as determined by the nucleotide sequences followed the earlier found model based on the neutrally evolving D-loop sequences, and reflected the earlier found phylogeographic Late Pleistocene scenario. Based on several selection tests, only one codon position consistently proved to be under positive selection. It did occur exclusively in the evolutionarily younger hares from Europe and it gave rise to several protein variants from the southeastern and south-central Balkans. The occurrence of several of those variants was significantly favored under certain precipitation conditions, as proved by our multinomial regression models. Possibly, the great altitudinal variation in the Balkans may have lead to bigger changes in precipitation across that region and this may have imposed an evolutionarily novel selective pressure on the protein variants and could have led to regional adaptation.

Maternal genomic variability of the wild boar (Sus scrofa) reveals the uniqueness of East-Caucasian and Central Italian populations.

The phylogeography of the European wild boar was mainly determined by postglacial recolonization patterns from Mediterranean refugia after the last ice age. Here we present the first analysis of SNP polymorphism within the complete mtDNA genome of West Russian (n = 8), European (n = 64), and North African (n = 5) wild boar. Our analyses provided evidence of unique lineages in the East-Caucasian (Dagestan) region and in Central Italy. A phylogenetic analysis revealed that these lineages are basal to the other European mtDNA sequences. We also show close connection between the Western Siberian and Eastern European populations. Also, the North African samples were clustered with the Iberian population. Phylogenetic trees and migration modeling revealed a high proximity of Dagestan sequences to those of Central Italy and suggested possible gene flow between Western Asia and Southern Europe which was not directly related to Northern and Central European lineages. Our results support the presence of old maternal lineages in two Southern glacial refugia (i.e., Caucasus and the Italian peninsula), as a legacy of an ancient wave of colonization of Southern Europe from an Eastern origin.

Genetic differentiation and asymmetric gene flow among Carpathian brown bear (Ursus arctos) populations-Implications for conservation of transboundary populations.

The abundance and distribution of large carnivores in Europe have been historically reduced. Their recovery requires multilevel coordination, especially regarding transboundary populations. Here, we apply nuclear and mitochondrial genetic markers to test for admixture level and its impact on population genetic structure of contemporary brown bears (Ursus arctos) from the Eastern, Southern, and Western Carpathians. Carpathian Mountains (Europe). Nearly 400 noninvasive brown bear DNA samples from the Western (Poland) and Eastern Carpathians (Bieszczady Mountains in Poland, Slovakia, Ukraine) were collected. Together with DNA isolates from Slovakia and Romania, they were analyzed using the set of eight microsatellite loci and two mtDNA regions (control region and cytochrome b). A set of 113 individuals with complete genotypes was used to investigate genetic differentiation across national boundaries, genetic structuring within and between populations, and movement between populations. Transboundary brown bear subpopulations (Slovakia and Poland) did not show significant internal genetic structure, and thus were treated as cohesive units. All brown bears from the Western Carpathians carried mitochondrial haplotypes from the Eastern lineage, while the Western lineage prevailed in the brown bears from the Bieszczady Mountains. Despite similar levels of microsatellite variability, we documented significant differentiation among the studied populations for nuclear markers and mtDNA. We also detected male-biased and asymmetrical movement into the Bieszczady Mountains population from the Western Carpathians. Our findings suggest initial colonization of the Western Carpathians by brown bears possessing mtDNA from the Eastern lineage. Genetic structuring among populations at microsatellite loci could be a result of human-mediated alterations. Detected asymmetric gene flow suggests ongoing expansion from more abundant populations into the Bieszczady Mountains and thus supports a metapopulation model. The knowledge concerning this complex pattern can be implemented in a joint Carpathian brown bear management plan that should allow population mixing by dispersing males.

Genetic variation in Tertiary relics: The case of eastern-Mediterranean Abies (Pinaceae).

The eastern-Mediterranean Abies taxa, which include both widely distributed species and taxa with minuscule ranges, represent a good model to study the impacts of range size and fragmentation on the levels of genetic diversity and differentiation. To assess the patterns of genetic diversity and phylogenetic relationships among eastern-Mediterranean Abies taxa, genetic variation was assessed by eight nuclear microsatellite loci in 52 populations of Abies taxa with a focus on those distributed in Turkey and the Caucasus. Both at the population and the taxon level, the subspecies or regional populations of Abies nordmanniana s.l. exhibited generally higher allelic richness, private allelic richness, and expected heterozygosity compared with Abies cilicica s.l. Results of both the structure analysis and distance-based approaches showed a strong differentiation of the two A. cilicica subspecies from the rest as well as from each other, whereas the subspecies of A. nordmanniana were distinct but less differentiated. ABC simulations were run for a set of scenarios of phylogeny and past demographic changes. For A. ×olcayana, the simulation gave a poor support for the hypothesis of being a taxon resulting from a past hybridization, the same is true for Abies equi-trojani: both they represent evolutionary branches of Abies bornmuelleriana.

Survival and divergence in a small group: The extraordinary genomic history of the endangered Apennine brown bear stragglers.

About 100 km east of Rome, in the central Apennine Mountains, a critically endangered population of ∼50 brown bears live in complete isolation. Mating outside this population is prevented by several 100 km of bear-free territories. We exploited this natural experiment to better understand the gene and genomic consequences of surviving at extremely small population size. We found that brown bear populations in Europe lost connectivity since Neolithic times, when farming communities expanded and forest burning was used for land clearance. In central Italy, this resulted in a 40-fold population decline. The overall genomic impact of this decline included the complete loss of variation in the mitochondrial genome and along long stretches of the nuclear genome. Several private and deleterious amino acid changes were fixed by random drift; predicted effects include energy deficit, muscle weakness, anomalies in cranial and skeletal development, and reduced aggressiveness. Despite this extreme loss of diversity, Apennine bear genomes show nonrandom peaks of high variation, possibly maintained by balancing selection, at genomic regions significantly enriched for genes associated with immune and olfactory systems. Challenging the paradigm of increased extinction risk in small populations, we suggest that random fixation of deleterious alleles (i) can be an important driver of divergence in isolation, (ii) can be tolerated when balancing selection prevents random loss of variation at important genes, and (iii) is followed by or results directly in favorable behavioral changes.

Unique postglacial evolution of the hornbeam (Carpinus betulus L.) in the Carpathians and the Balkan Peninsula revealed by chloroplast DNA.

The Balkan Peninsula is one of the largest and most important European glacial refugia. However, the evolutionary history and phylogeographic pattern of temperate tree species that survived in the Balkans glacial refugia and their contribution to the genetic structure of the current population in the Carpathian Mountains remains poorly understood. Using polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP), and extensive population sampling, we explored the phylogeographic pattern of Carpinus betulus in both the Balkan Peninsula and the Carpathian region. We aimed to determine the locations of potential glacial refugia, in order to delineate post-glacial colonization routes, and also to test if northern cryptic refugia had persisted during the Last Glacial Maximum (LGM). Our results provide strong support for the existence of multiple refugia similar to the 'refugia-within-refugia' scenario, which would suggest that Carpinus betulus has experienced a complex evolutionary history. In agreement with pollen data, our findings corroborate with previous hypotheses suggesting that hornbeam has a distinct postglacial evolution in the southeast of Europe (Carpathians and Balkan Peninsula) compared with that in Western Europe. Three postglacial re-colonization routes, from three distinct effective glacial refugia: (1) the Dinaric Alps; (2) the Pirin and Rhodope Mountains and (3) the Strandzha Mountains, were detected within the Balkan Peninsula. The pattern of cpDNA haplotypes distribution across the Ukrainian Carpathians revealed a "suture zone", which is a consequence of contact due to postglacial re-colonization between hornbeam populations originating from all three effective glacial refugia. The peculiar haplotype geographical structure found in C. betulus and the identified multiple glacial refugia in the Balkans may have direct consequences in the management and conservation of hornbeam forest genetic resources.

Origin and genetic differentiation of pink-flowered Sorbus hybrids in the Western Carpathians.

Background and Aims: Diversity of the genus Sorbus has been affected by interspecific hybridizations. Pink-flowered hybrid species have been insufficiently studied so far. They comprise bigenomic hybrid species derived from crosses S. aria s.l. × S. chamaemespilus and trigenomic ones, where S. aucuparia was involved as well. The main objective of the present study was to reconstruct their hybrid origins as well as to assess genetic distinction among several morphologically recognized hybrid species. Methods: Samples from putative maternal species and eight pink-flowered and two white-flowered hybrid species were collected in the Western Carpathians and the Sudetes. In total, 370 specimens were analysed. Six chloroplast microsatellites were used to infer parentage, whereas nuclear amplified fragment length polymorphism (AFLP) markers were employed for the identification of clones and patterns of genetic variation. Ploidy levels were estimated by flow cytometry on a subset of 140 individuals. Key Results: Genetic data supported their hybrid origins proposed based on flower and leaf morphology, and chloroplast DNA (cpDNA) revealed recurrent origins ( S. caeruleomontana , S. haljamovae ), even from bidirectional hybridization events ( S. zuzanae ). All bigenomic and trigenomic hybrid species (except triploid S. zuzanae ) were found to be tetraploid. In addition to polyploidy, low genetic variation and the presence of clones within and among populations were observed, suggesting predominantly apomictic reproduction of the hybrid species. Most of the described hybrid species appeared also genetically distinct. Conclusions: The data suggest that multiple hybridization events in the Western Carpathian Sorbus have led to the formation of separate, partially reproductively isolated genetic lineages, which may or may not be discriminated morphologically. Even bidirectional hybridization can produce individuals classified to the same taxon based on phenotype. For some hybrid taxa, hybridization pathways were proposed based on their genetic proximity to parental species and differences in genome sizes.

Wolf population genetics in Europe: a systematic review, meta-analysis and suggestions for conservation and management.

The grey wolf (Canis lupus) is an iconic large carnivore that has increasingly been recognized as an apex predator with intrinsic value and a keystone species. However, wolves have also long represented a primary source of human-carnivore conflict, which has led to long-term persecution of wolves, resulting in a significant decrease in their numbers, genetic diversity and gene flow between populations. For more effective protection and management of wolf populations in Europe, robust scientific evidence is crucial. This review serves as an analytical summary of the main findings from wolf population genetic studies in Europe, covering major studies from the 'pre-genomic era' and the first insights of the 'genomics era'. We analyse, summarize and discuss findings derived from analyses of three compartments of the mammalian genome with different inheritance modes: maternal (mitochondrial DNA), paternal (Y chromosome) and biparental [autosomal microsatellites and single nucleotide polymorphisms (SNPs)]. To describe large-scale trends and patterns of genetic variation in European wolf populations, we conducted a meta-analysis based on the results of previous microsatellite studies and also included new data, covering all 19 European countries for which wolf genetic information is available: Norway, Sweden, Finland, Estonia, Latvia, Lithuania, Poland, Czech Republic, Slovakia, Germany, Belarus, Russia, Italy, Croatia, Bulgaria, Bosnia and Herzegovina, Greece, Spain and Portugal. We compared different indices of genetic diversity in wolf populations and found a significant spatial trend in heterozygosity across Europe from south-west (lowest genetic diversity) to north-east (highest). The range of spatial autocorrelation calculated on the basis of three characteristics of genetic diversity was 650-850 km, suggesting that the genetic diversity of a given wolf population can be influenced by populations up to 850 km away. As an important outcome of this synthesis, we discuss the most pressing issues threatening wolf populations in Europe, highlight important gaps in current knowledge, suggest solutions to overcome these limitations, and provide recommendations for science-based wolf conservation and management at regional and Europe-wide scales.

Adapting through glacial cycles: insights from a long-lived tree (Taxus baccata).

Despite the large body of research devoted to understanding the role of Quaternary glacial cycles in the genetic divergence of European trees, the differential contribution of geographic isolation and/or environmental adaptation in creating population genetic divergence remains unexplored. In this study, we used a long-lived tree (Taxus baccata) as a model species to investigate the impact of Quaternary climatic changes on genetic diversity via neutral (isolation-by-distance) and selective (isolation-by-adaptation) processes. We applied approximate Bayesian computation to genetic data to infer its demographic history, and combined this information with past and present climatic data to assess the role of environment and geography in the observed patterns of genetic structure. We found evidence that yew colonized Europe from the East, and that European samples diverged into two groups (Western, Eastern) at the beginning of the Quaternary glaciations, c. 2.2 Myr before present. Apart from the expected effects of geographical isolation during glacials, we discovered a significant role of environmental adaptation during interglacials at the origin of genetic divergence between both groups. This process may be common in other organisms, providing new research lines to explore the effect of Quaternary climatic factors on present-day patterns of genetic diversity.

Genetic differentiation of the Western Capercaillie highlights the importance of South-eastern Europe for understanding the species phylogeography.

The Western Capercaillie (Tetrao urogallus L.) is a grouse species of open boreal or high altitude forests of Eurasia. It is endangered throughout most mountain range habitat areas in Europe. Two major genetically identifiable lineages of Western Capercaillie have been described to date: the southern lineage at the species' southernmost range of distribution in Europe, and the boreal lineage. We address the question of genetic differentiation of capercaillie populations from the Rhodope and Rila Mountains in Bulgaria, across the Dinaric Mountains to the Slovenian Alps. The two lineages' contact zone and resulting conservation strategies in this so-far understudied area of distribution have not been previously determined. The results of analysis of mitochondrial DNA control region sequences of 319 samples from the studied populations show that Alpine populations were composed exclusively of boreal lineage; Dinaric populations of both, but predominantly (96%) of boreal lineage; and Rhodope-Rila populations predominantly (>90%) of southern lineage individuals. The Bulgarian mountains were identified as the core area of the southern lineage, and the Dinaric Mountains as the western contact zone between both lineages in the Balkans. Bulgarian populations appeared genetically distinct from Alpine and Dinaric populations and exhibited characteristics of a long-term stationary population, suggesting that they should be considered as a glacial relict and probably a distinct subspecies. Although all of the studied populations suffered a decline in the past, the significantly lower level of genetic diversity when compared with the neighbouring Alpine and Bulgarian populations suggests that the isolated Dinaric capercaillie is particularly vulnerable to continuing population decline. The results are discussed in the context of conservation of the species in the Balkans, its principal threats and legal protection status. Potential conservation strategies should consider the existence of the two lineages and their vulnerable Dinaric contact zone and support the specificities of the populations.

Unexpected presence of Fagus orientalis complex in Italy as inferred from 45,000-year-old DNA pollen samples from Venice lagoon.

BACKGROUND: Phylogeographic analyses on the Western Euroasiatic Fagus taxa (F. orientalis, F. sylvatica, F. taurica and F. moesiaca) is available, however, the subdivision of Fagus spp. is unresolved and there is no consensus on the phylogeny and on the identification (both with morphological than molecular markers) of Fagus Eurasiatic taxa. For the first time molecular analyses of ancient pollen, dated at least 45,000 years ago, were used in combination with the phylogeny analysis on current species, to identify the Fagus spp. present during the Last Interglacial period in Italy. In this work we aim at testing if the trnL-trnF chloroplast DNA (cpDNA) region, that has been previously proved efficient in discriminating different Quercus taxa, can be employed in distinguishing the Fagus species and in identifying the ancient pollen. RESULTS: 86 populations from 4 Western Euroasistic taxa were sampled, and sequenced for the trnL-trnF region to verify the efficiency of this cpDNA region in identifying the Fagus spp.. Furthermore, Fagus crenata (2 populations), Fagus grandifolia (2 populations), Fagus japonica, Fagus hayatae, Quercus species and Castanea species were analysed to better resolve the phylogenetic inference. Our results show that this cpDNA region harbour some informative sites that allow to infer relationships among the species within the Fagaceae family. In particular, few specific and fixed mutations were able to discriminate and identify all the different Fagus species. Considering a short fragment of 176 base pairs within the trnL intron, 2 transversions were found able in distinguishing the F. orientalis complex taxa (F. orientalis, F. taurica and F. moesiaca) from the remaining Fagus spp. (F. sylvatica, F. japonica, F. hayataea, F. crenata and F. grandifolia). This permits to analyse this fragment also in ancient samples, where DNA is usually highly degraded. The sequences data indicate that the DNA recovered from ancient pollen belongs to the F. orientalis complex since it displays the informative sites characteristic of this complex. CONCLUSION: The ancient DNA sequences demonstrate for the first time that, in contrast to current knowledge based on palynological and macrofossil data, the F. orientalis complex was already present during the Tyrrhenian period in what is now the Venice lagoon (Italy). This is a new and important insight considering that nowadays West Europe is not the natural area of Fagus orientalis complex, and up to now nobody has hypothesized the presence during the Last Interglacial period of F. orientalis complex in Italy.

Similar gender dimorphism in the costs of reproduction across the geographic range of Fraxinus ornus.

BACKGROUND AND AIMS: The reproductive costs for individuals with the female function have been hypothesized to be greater than for those with the male function because the allocation unit per female flower is very high due to the necessity to nurture the embryos until seed dispersal occurs, while the male reproductive allocation per flower is lower because it finishes once pollen is shed. Consequently, males may invest more resources in growth than females. This prediction was tested across a wide geographical range in a tree with a dimorphic breeding system (Fraxinus ornus) consisting of males and hermaphrodites functioning as females. The contrasting ecological conditions found across the geographical range allowed the evaluation of the hypothesis that the reproductive costs of sexual dimorphism varies with environmental stressors. METHODS: By using random-effects meta-analysis, the differences in the reproductive and vegetative investment of male and hermaphrodite trees of F. ornus were analysed in 10 populations from the northern (Slovakia), south-eastern (Greece) and south-western (Spain) limits of its European distribution. The variation in gender-dimorphism with environmental stress was analysed by running a meta-regression between these effect sizes and the two environmental stress indicators: one related to temperature (the frost-free period) and another related to water availability (moisture deficit). KEY RESULTS: Most of the effect sizes showed that males produced more flowers and grew more quickly than hermaphrodites. Gender differences in reproduction and growth were not minimized or maximized under adverse climatic conditions such as short frost-free periods or severe aridity. CONCLUSIONS: The lower costs of reproduction for F. ornus males allow them to grow more quickly than hermaphrodites, although such differences in sex-specific reproductive costs are not magnified under stressful conditions.

A new scenario for the quaternary history of European beech populations: palaeobotanical evidence and genetic consequences.

Here, palaeobotanical and genetic data for common beech (Fagus sylvatica) in Europe are used to evaluate the genetic consequences of long-term survival in refuge areas and postglacial spread. Four large datasets are presented, including over 400 fossil-pollen sites, 80 plant-macrofossil sites, and 450 and 600 modern beech populations for chloroplast and nuclear markers, respectively. The largely complementary palaeobotanical and genetic data indicate that: (i) beech survived the last glacial period in multiple refuge areas; (ii) the central European refugia were separated from the Mediterranean refugia; (iii) the Mediterranean refuges did not contribute to the colonization of central and northern Europe; (iv) some populations expanded considerably during the postglacial period, while others experienced only a limited expansion; (v) the mountain chains were not geographical barriers for beech but rather facilitated its diffusion; and (vi) the modern genetic diversity was shaped over multiple glacial-interglacial cycles. This scenario differs from many recent treatments of tree phylogeography in Europe that largely focus on the last ice age and the postglacial period to interpret genetic structure and argue that the southern peninsulas (Iberian, Italian and Balkan) were the main source areas for trees in central and northern Europe.

Genetic effects of air pollution on forest tree species of the Carpathian Mountains.

The effects of air pollution on the genetic structure of Norway spruce, European silver fir and European beech were studied at four polluted sites in Slovakia, Romania and Czech Republic. In order to reduce potential effects of site heterogeneity on the health condition, pair-wise sampling of pollution-tolerant and sensitive trees was applied. Genotypes of sampled trees were determined at 21 isozyme gene loci of spruce, 18 loci of fir and 15 loci of beech. In comparison with Norway spruce, fewer genetic differences were revealed in beech and almost no differentiation between pollution-tolerant and sensitive trees was observed in fir. In adult stands of Norway spruce, sensitive trees exhibited higher genetic multiplicity and diversity. The decline of pollution-sensitive trees may result thus in a gradual genetic depletion of pollution-exposed populations of Norway spruce through the loss of less frequent alleles with potential adaptive significance to altered stressing regimes in the future. Comparison of the subsets of sensitive and tolerant Norway spruce individuals as determined by presence or absence of discolorations ("spruce yellowing") revealed different heterozygosity at 3 out of 11 polymorphic loci.