Conserving a threatened North American walnut: a chromosome-scale reference genome for butternut (Juglans cinerea).

With the advent of affordable and more accurate third-generation sequencing technologies, and the associated bioinformatic tools, it is now possible to sequence, assemble, and annotate more species of conservation concern than ever before. Juglans cinerea, commonly known as butternut or white walnut, is a member of the walnut family, native to the Eastern United States and Southeastern Canada. The species is currently listed as Endangered on the IUCN Red List due to decline from an invasive fungus known as Ophiognomonia clavigignenti-juglandacearum (Oc-j) that causes butternut canker. Oc-j creates visible sores on the trunks of the tree which essentially starves and slowly kills the tree. Natural resistance to this pathogen is rare. Conserving butternut is of utmost priority due to its critical ecosystem role and cultural significance. As part of an integrated undergraduate and graduate student training program in biodiversity and conservation genomics, the first reference genome for Juglans cinerea is described here. This chromosome-scale 539 Mb assembly was generated from over 100 × coverage of Oxford Nanopore long reads and scaffolded with the Juglans mandshurica genome. Scaffolding with a closely related species oriented and ordered the sequences in a manner more representative of the structure of the genome without altering the sequence. Comparisons with sequenced Juglandaceae revealed high levels of synteny and further supported J. cinerea's recent phylogenetic placement. Comparative assessment of gene family evolution revealed a significant number of contracting families, including several associated with biotic stress response.

Pan-genome and transcriptome analyses provide insights into genomic variation and differential gene expression profiles related to disease resistance and fatty acid biosynthesis in eastern black walnut (Juglans nigra).

Walnut (Juglans) species are used as nut crops worldwide. Eastern black walnut (EBW, Juglans nigra), a diploid, horticultural important woody species is native to much of eastern North America. Although it is highly valued for its wood and nut, there are few resources for understanding EBW genetics. Here, we present a high-quality genome assembly of J. nigra based on Illumina, Pacbio, and Hi-C technologies. The genome size was 540.8 Mb, with a scaffold N50 size of 35.1 Mb, and 99.0% of the assembly was anchored to 16 chromosomes. Using this genome as a reference, the resequencing of 74 accessions revealed the effective population size of J. nigra declined during the glacial maximum. A single whole-genome duplication event was identified in the J. nigra genome. Large syntenic blocks among J. nigra, Juglans regia, and Juglans microcarpa predominated, but inversions of more than 600 kb were identified. By comparing the EBW genome with those of J. regia and J. microcarpa, we detected InDel sizes of 34.9 Mb in J. regia and 18.3 Mb in J. microcarpa, respectively. Transcriptomic analysis of differentially expressed genes identified five presumed NBS-LRR (NUCLEOTIDE BINDING SITE-LEUCINE-RICH REPEAT) genes were upregulated during the development of walnut husks and shells compared to developing embryos. We also identified candidate genes with essential roles in seed oil synthesis, including FAD (FATTY ACID DESATURASE) and OLE (OLEOSIN). Our work advances the understanding of fatty acid bioaccumulation and disease resistance in nut crops, and also provides an essential resource for conducting genomics-enabled breeding in walnut.

Domestication and selection footprints in Persian walnuts (Juglans regia).

Walnut (Juglans) species are economically important hardwood trees cultivated worldwide for both edible nuts and high-quality wood. Broad-scale assessments of species diversity, evolutionary history, and domestication are needed to improve walnut breeding. In this study, we sequenced 309 walnut accessions from around the world, including 55 Juglans relatives, 98 wild Persian walnuts (J. regia), 70 J. regia landraces, and 86 J. regia cultivars. The phylogenetic tree indicated that J. regia samples (section Dioscaryon) were monophyletic within Juglans. The core areas of genetic diversity of J. regia germplasm were southwestern China and southern Asia near the Qinghai-Tibet Plateau and the Himalayas, and the uplift of the Himalayas was speculated to be the main factor leading to the current population dynamics of Persian walnut. The pattern of genomic variation in terms of nucleotide diversity, linkage disequilibrium, single nucleotide polymorphisms, and insertions/deletions revealed the domestication and selection footprints in Persian walnut. Selective sweep analysis, GWAS, and expression analysis further identified two transcription factors, JrbHLH and JrMYB6, that influence the thickness of the nut diaphragm as loci under selection during domestication. Our results elucidate the domestication and selection footprints in Persian walnuts and provide a valuable resource for the genomics-assisted breeding of this important crop.

Whole genome based insights into the phylogeny and evolution of the Juglandaceae.

BACKGROUND: The walnut family (Juglandaceae) contains commercially important woody trees commonly called walnut, wingnut, pecan and hickory. Phylogenetic relationships and diversification within the Juglandaceae are classic and hot scientific topics that have been elucidated by recent fossil, morphological, molecular, and (paleo) environmental data. Further resolution of relationships among and within genera is still needed and can be achieved by analysis of the variation of chloroplast, mtDNA, and nuclear genomes. RESULTS: We reconstructed the backbone phylogenetic relationships of Juglandaceae using organelle and nuclear genome data from 27 species. The divergence time of Juglandaceae was estimated to be 78.7 Mya. The major lineages diversified in warm and dry habitats during the mid-Paleocene and early Eocene. The plastid, mitochondrial, and nuclear phylogenetic analyses all revealed three subfamilies, i.e., Juglandoideae, Engelhardioideae, Rhoipteleoideae. Five genera of Juglandoideae were strongly supported. Juglandaceae were estimated to have originated during the late Cretaceous, while Juglandoideae were estimated to have originated during the Paleocene, with evidence for rapid diversification events during several glacial and geological periods. The phylogenetic analyses of organelle sequences and nuclear genome yielded highly supported incongruence positions for J. cinerea, J. hopeiensis, and Platycarya strobilacea. Winged fruit were the ancestral condition in the Juglandoideae, but adaptation to novel dispersal and regeneration regimes after the Cretaceous-Paleogene boundary led to the independent evolution of zoochory among several genera of the Juglandaceae. CONCLUSIONS: A fully resolved, strongly supported, time-calibrated phylogenetic tree of Juglandaceae can provide an important framework for studying classification, diversification, biogeography, and comparative genomics of plant lineages. Our addition of new, annotated whole chloroplast genomic sequences and identification of their variability informs the study of their evolution in walnuts (Juglandaceae).

Improved de novo chromosome-level genome assembly of the vulnerable walnut tree Juglans mandshurica reveals gene family evolution and possible genome basis of resistance to lesion nematode.

Manchurian walnut (Juglans mandshurica Maxim.) is a synonym of J. cathayensis, a diploid, vulnerable, temperate deciduous tree valued for its wood and nut. It is also valued as a rootstock for Juglans regia because of its reported tolerance of lesion nematode. Reference genomes are available for several Juglans species, our goal was to produce a de novo, chromosome-level assembly of the J. mandshurica genome. Here, we reported an improved assembly of J. mandshurica with a contig N50 size of 6.49 Mb and a scaffold N50 size of 36.1 Mb. The total genome size was 548 Mb encoding 29,032 protein coding genes which were annotated. The collinearity analysis showed that J. mandshurica and J. regia originated from a common ancestor, with both species undergoing two WGD events. A genomic comparison showed that J. mandshurica was missing 1657 genes found in J. regia, and J. mandshurica includes 2827 genes not found in of the J. regia genome. The J. mandshurica contained 1440 unique paralogues that were highly enriched for flavonoid biosynthesis, phenylpropanoid biosynthesis, and plant-pathogen interaction. Four gene families related to disease resistance notable contraction (rapidly evolving; LEA, WAK, PPR, and PR) in J. mandshurica compared to eight species. JmaPR10 and JmaPR8 contained three orthologous gene pairs with J. regia that were highly expressed in root bark. JmaPR10 is a strong candidate gene for lesion nematodes resistance in J. mandshurica. The J. mandshurica genome should be a useful resource for study of the evolution, breeding, and genetic variation in walnuts (Juglans).

The de novo genome assembly of Tapiscia sinensis and the transcriptomic and developmental bases of androdioecy.

Tapiscia sinensis (Tapisciaceae) possesses an unusual androdioecious breeding system that has attracted considerable interest from evolutionary biologists. Key aspects of T. sinensis biology, including its biogeography, genomics, and sex-linked genes, are unknown. Here, we report the first de novo assembly of the genome of T. sinensis. The genome size was 410 Mb, with 22,251 predicted genes. Based on whole-genome resequencing of 55 trees from 10 locations, an analysis of population genetic structure indicated that T. sinensis has fragmented into five lineages, with low intrapopulation genetic diversity and little gene flow among populations. By comparing whole-genome scans of male versus hermaphroditic pools, we identified 303 candidate sex-linked genes, 79 of which (25.9%) were located on scaffold 25. A 24-kb region was absent in hermaphroditic individuals, and five genes in that region, TsF-box4, TsF-box10, TsF-box13, TsSUT1, and TsSUT4, showed expression differences between mature male and hermaphroditic flowers. The results of this study shed light on the breeding system evolution and conservation genetics of the Tapisciaceae.

Biocultural diversity of common walnut (Juglans regia L.) and sweet chestnut (Castanea sativa Mill.) across Eurasia.

A biocultural diversity approach integrates plant biology and germplasm dispersal processes with human cultural diversity. An increasing number of studies have identified cultural factors and ethnolinguistic barriers as the main drivers of the genetic diversity in crop plants. Little is known about how anthropogenic processes have affected the evolution of tree crops over the entire time scale of their interaction with humans. In Asia and the Mediterranean, common walnut (Juglans regia L.) and sweet chestnut (Castanea sativa Mill.) have been economically and culturally important crops for millennia; there, in ancient times, they were invested with symbolic and religious significance. In this study, we detected a partial geographic congruence between the ethno-linguistic repartition of human communities, the distribution of major cognitive sets of word-related terms, and the inferred genetic clusters of common walnut and sweet chestnut populations across Eurasia. Our data indicated that isolation by distance processes, landscape heterogeneity and cultural boundaries might have promoted simultaneously human language diversification and walnut/chestnut differentiation across the same geographic macro-regions. Hotspots of common walnut and sweet chestnut genetic diversity were associated with areas of linguistic enrichment in the Himalayas, Trans-Caucasus, and Pyrenees Mountains, where common walnuts and sweet chestnuts had sustained ties to human culture since the Early Bronze Age. Our multidisciplinary approach supported the indirect and direct role of humans in shaping walnut and chestnut diversity across Eurasia from the EBA (e.g., Persian Empire and Greek-Roman colonization) until the first evidence of active selection and clonal propagation by grafting of both species. Our findings highlighted the benefit of an efficient integration of the relevant cultural factors in the classical genome (G) × environmental (E) model and the urgency of a systematic application of the biocultural diversity concept in the reconstruction of the evolutionary history of tree species.

Phylogenomics Reveals an Ancient Hybrid Origin of the Persian Walnut.

Persian walnut (Juglans regia) is cultivated worldwide for its high-quality wood and nuts, but its origin has remained mysterious because in phylogenies it occupies an unresolved position between American black walnuts and Asian butternuts. Equally unclear is the origin of the only American butternut, J. cinerea. We resequenced the whole genome of 80 individuals from 19 of the 22 species of Juglans and assembled the genome of its relatives Pterocarya stenoptera and Platycarya strobilacea. Using phylogenetic-network analysis of single-copy nuclear genes, genome-wide site pattern probabilities, and Approximate Bayesian Computation, we discovered that J. regia (and its landrace J. sigillata) arose as a hybrid between the American and the Asian lineages and that J. cinerea resulted from massive introgression from an immigrating Asian butternut into the genome of an American black walnut. Approximate Bayesian Computation modeling placed the hybrid origin in the late Pliocene, ∼3.45 My, with both parental lineages since having gone extinct in Europe.

Microsatellite Borders and Micro-sequence Conservation in Juglans.

Walnuts (Juglans spp.) are economically important nut and timber species with a worldwide distribution. Using the published Persian walnut genome as a reference for the assembly of short reads from six Juglans species and several interspecific hybrids, we identified simple sequence repeats in 12 Juglans nuclear and organellar genomes. The genome-wide distribution and polymorphisms of nuclear and organellar microsatellites (SSRs) for most Juglans genomes have not been previously studied. We compared the frequency of nuclear SSR motifs and their lengths across Juglans, and identified section-specific chloroplast SSR motifs. Primer pairs were designed for more than 60,000 SSR-containing sequences based on alignment against assembled scaffold sequences. Of the >60,000 loci, 39,000 were validated by e-PCR using unique primer pairs. We identified primers containing 100% sequence identity in multiple species. Across species, sequence identity in the SSR-flanking regions was generally low. Although SSRs are common and highly dispersed in the genome, their flanking sequences are conserved at about 90 to 95% identity within Juglans and within species. In a few rare cases, flanking sequences are identical across species of Juglans. This comprehensive report of nuclear and organellar SSRs in Juglans and the generation of validated SSR primers will be a useful resource for future genetic analyses, walnut breeding programs, high-level taxonomic evaluations, and genomic studies in Juglandaceae.

Population genetic structure and adaptive differentiation of iron walnut Juglans regia subsp. sigillata in southwestern China.

Southwestern (SW) China is an area of active tectonism and erosion, yielding a dynamic, deeply eroded landscape that influences the genetic structure of the resident populations of plants and animals. Iron walnut (Juglans regia subsp. sigillata) is a deciduous tree species endemic to this region of China and cultivated there for its edible nuts. We sampled 36 iron walnut populations from locations throughout the species' range in SW China and genotyped a total of 765 individuals at five chloroplast DNA regions and 22 nuclear microsatellite loci. Species distribution models were produced to predict the evolution and historical biogeography of iron walnut and to estimate the impacts of climate oscillations and orographic environments on the species' demography. Our results indicated that J. regia subsp. sigillata had relatively low genetic diversity, high interpopulation genetic differentiation, and asymmetric interpopulation gene flow. Based on DIYABC analysis, we identified two lineages of J. sigillata in southwestern China. The lineages (subpopulations) diverge during the last glacial period (~1.34 Ma). Southwestern China was a glacial refuge during the last glacial period, but increasingly colder and arid climates might have fostered the fragmentation of J. regia subsp. sigillata within this refugium. Finally, we found that recent habitat fragmentation has led to a reduction in population connectivity and increased genetic differentiation by genetic drift in isolated populations. Our results support a conclusion that geological and climatic factors since the Miocene triggered the differentiation, evolutionary origin, and range shifts of J. sigillata in the studied region.

Pooled whole-genome sequencing of interspecific chestnut (Castanea) hybrids reveals loci associated with differences in caching behavior of fox squirrels (Sciurus niger L.).

Dispersal of seeds by scatter-hoarding rodents is common among tropical and temperate tree species, including chestnuts in the genus Castanea. Backcrossed (BC) interspecific hybrid chestnuts exhibit wide variation in seed traits: as the parent species (Castanea dentata and C. mollissima) have distinct seed phenotypes and tend to be handled differently by seed dispersers, phenotypic variation in BC trees is likely due to inheritance of genes that have undergone divergent evolution in the parent species. To identify candidate genomic regions for interspecific differences in seed dispersal, we used tagged seeds to measure average dispersal distance for seeds of third-generation BC chestnuts and sequenced pooled whole genomes of mother trees with contrasting seed dispersal: high caching rate/long distance; low caching rate/short distance; no caching. Candidate regions affecting seed dispersal were identified as loci with more C. mollissima alleles in the high caching rate/ long-distance pool than expected by chance and observed in the other two pools. Functional annotations of candidate regions included predicted lipid metabolism, dormancy regulation, seed development, and carbohydrate metabolism genes. The results support the hypothesis that perception of seed dormancy is a predominant factor in squirrel caching decisions, and also indicate profitable directions for future work on the evolutionary genomics of trees and coevolved seed dispersers.

Pollen flow and paternity in an isolated and non-isolated black walnut (Juglans nigra L.) timber seed orchard.

Artificial pollination of black walnut (Juglans nigra L.) is not practical and timber breeders have historically utilized only open-pollinated half-sib families. An alternate approach called "breeding without breeding," consists of genotyping open-pollinated progeny using DNA markers to identify paternal parents and then constructing full-sib families. In 2014, we used 12 SSR markers to genotype 884 open-pollinated half-sib progeny harvested from two clonal orchards containing 206 trees, comprised of 52 elite timber selections. Seed was harvested in 2011 from each of two ramets of 23 clones, one upwind and one downwind, based on prevailing wind direction from the west-southwest. One orchard was isolated from wild black walnut and composed of forward selections while the other orchard was adjacent to a natural forest containing mature black walnut composed of backward selections. Isolation significantly increased within-orchard pollination (85%) of the progeny from the isolated orchard compared to 42% from the non-isolated orchard. Neither prevailing wind direction nor seed tree position in the orchard affected paternity patterns or wild pollen contamination. Genetic diversity indices revealed that progeny from both orchards were in Hardy-Weinberg equilibrium with very little inbreeding and no selfing. A significant level of inbreeding was present among the forward selected parents, but not the first generation (backward selected) parents. Some orchard clones failed to sire any progeny while other clones pollinated upwards of 20% of progeny.

The Phytogeographic History of Common Walnut in China.

Common walnut (Juglans regia L.) is an economically important hardwood tree species cultivated worldwide for its high quality wood and edible nuts. It is generally accepted that after the last glaciation J. regia survived and grew in almost completely isolated stands in Asia, and that ancient humans dispersed walnuts across Asia and into new habitats via trade and cultural expansion. The history of common walnut in China is a matter of debate, however. We estimated the genetic diversity and spatial genetic structure of 31 walnut populations sampled across its Chinese range using 22 microsatellite markers (13 neutral and 9 non-neutral). Using historical data and population genetic analysis, including approximate Bayesian analysis (ABC), we reconstructed the demographic history of J. regia in China. The genetic data indicated the likely presence of J. regia in glacial refugia in the Xinjiang province (Northwest China), Northeastern China (Beijing, Shandong, and Changbai Mountains), Central China (Qinling and Baishan Mountains and Xi'an), and Southwestern China (Tibet, Yunnan, Guizhou, and Sichuan provinces). Based on DIY-ABC analysis, we identified three ancient lineages of J. regia in China. Two lineages (subpopulation A and subpopulation B+C) diverged about 2.79 Mya, while Southwestern China, and Qinling and Baishan Mountains lineages diverged during the Quaternary glaciations (about 1.13 Mya). Remnants of these once-distinct genetic clusters of J. regia may warrant ecological management if they are to be retained as in situ resources. A population size expansion in Northeastern China was detected in the last five centuries. The present distribution of walnut in China resulted from the combined effects of expansion/contraction from multiple refugia after the Last Glacial Maximum and later human exploitation.

Signatures of Selection in the Genomes of Chinese Chestnut (Castanea mollissima Blume): The Roots of Nut Tree Domestication.

Chestnuts (Castanea) are major nut crops in East Asia and southern Europe, and are unique among temperate nut crops in that the harvested seeds are starchy rather than oily. Chestnut species have been cultivated for three millennia or more in China, so it is likely that artificial selection has affected the genome of orchard-grown chestnuts. The genetics of Chinese chestnut (Castanea mollissima Blume) domestication are also of interest to breeders of hybrid American chestnut, especially if the low-growing, branching habit of Chinese chestnut, an impediment to American chestnut restoration, is partly the result of artificial selection. We resequenced genomes of wild and orchard-derived Chinese chestnuts and identified selective sweeps based on pooled whole-genome SNP datasets. We present candidate gene loci for chestnut domestication and discuss the potential phenotypic effects of candidate loci, some of which may be useful genes for chestnut improvement in Asia and North America. Selective sweeps included predicted genes potentially related to flower phenology and development, fruit maturation, and secondary metabolism, and included some genes homologous to domestication candidates in other woody plants.

Genomic Variation Among and Within Six Juglans Species.

Genomic analysis in Juglans (walnuts) is expected to transform the breeding and agricultural production of both nuts and lumber. To that end, we report here the determination of reference sequences for six additional relatives of Juglans regia: Juglans sigillata (also from section Dioscaryon), Juglans nigra, Juglans microcarpa, Juglans hindsii (from section Rhysocaryon), Juglans cathayensis (from section Cardiocaryon), and the closely related Pterocarya stenoptera While these are 'draft' genomes, ranging in size between 640Mbp and 990Mbp, their contiguities and accuracies can support powerful annotations of genomic variation that are often the foundation of new avenues of research and breeding. We annotated nucleotide divergence and synteny by creating complete pairwise alignments of each reference genome to the remaining six. In addition, we have re-sequenced a sample of accessions from four Juglans species (including regia). The variation discovered in these surveys comprises a critical resource for experimentation and breeding, as well as a solid complementary annotation. To demonstrate the potential of these resources the structural and sequence variation in and around the polyphenol oxidase loci, PPO1 and PPO2 were investigated. As reported for other seed crops variation in this gene is implicated in the domestication of walnuts. The apparently Juglandaceae specific PPO1 duplicate shows accelerated divergence and an excess of amino acid replacement on the lineage leading to accessions of the domesticated nut crop species, Juglans regia and sigillata.

Population genetics, phylogenomics and hybrid speciation of Juglans in China determined from whole chloroplast genomes, transcriptomes, and genotyping-by-sequencing (GBS).

Genomic data are a powerful tool for elucidating the processes involved in the evolution and divergence of species. The speciation and phylogenetic relationships among Chinese Juglans remain unclear. Here, we used results from phylogenomic and population genetic analyses, transcriptomics, Genotyping-By-Sequencing (GBS), and whole chloroplast genomes (Cp genome) data to infer processes of lineage formation among the five native Chinese species of the walnut genus (Juglans, Juglandaceae), a widespread, economically important group. We found that the processes of isolation generated diversity during glaciations, but that the recent range expansion of J. regia, probably from multiple refugia, led to hybrid formation both within and between sections of the genus. In southern China, human dispersal of J. regia brought it into contact with J. sigillata, which we determined to be an ecotype of J. regia that is now maintained as a landrace. In northern China, walnut hybridized with a distinct lineage of J. mandshurica to form J. hopeiensis, a controversial taxon (considered threatened) that our data indicate is a horticultural variety. Comparisons among whole chloroplast genomes and nuclear transcriptome analyses provided conflicting evidence for the timing of the divergence of Chinese Juglans taxa. J. cathayensis and J. mandshurica are poorly differentiated based our genomic data. Reconstruction of Juglans evolutionary history indicate that episodes of climatic variation over the past 4.5 to 33.80 million years, associated with glacial advances and retreats and population isolation, have shaped Chinese walnut demography and evolution, even in the presence of gene flow and introgression.

Localized gene expression changes during adventitious root formation in black walnut (Juglans nigra L.).

Cutting propagation plays a large role in the forestry and horticulture industries where superior genotypes need to be clonally multiplied. Integral to this process is the ability of cuttings to form adventitious roots. Recalcitrance to adventitious root development is a serious hurdle for many woody plant propagation systems including black walnut (Juglans nigra L.), an economically valuable species. The inability of black walnut to reliably form adventitious roots limits propagation of superior genotypes. Adventitious roots originate from different locations, and root induction is controlled by many environmental and endogenous factors. At the molecular level, however, the regulation of adventitious root formation is still poorly understood. In order to elucidate the transcriptional changes during adventitious root development in black walnut, we used quantitative real-time polymerase chain reaction to measure the expression of nine key genes regulating root formation in other species. Using our previously developed spatially explicit timeline of adventitious root development in black walnut softwood cuttings, we optimized a laser capture microdissection protocol to isolate RNA from cortical, phloem fiber and phloem parenchyma cells throughout adventitious root formation. Laser capture microdissection permitted high-resolution, site-specific analysis of gene expression that differentiated between participatory and non-participatory root progenitor cells. Results indicated mRNA abundance was altered in all nine rooting-related genes in response to auxin treatment in both juvenile and mature cuttings. SCARECROW LIKE-1 (SCL) had the greatest change in expression in juvenile rooting-competent cells at days 16 and 18, with a 24- and 23-fold increase relative to day 0, respectively. Tissues not linked to root organogenesis had little change in SCL expression at similar time points. AUXIN RESPONSE FACTOR (ARF)6 and ARF8 as well as SHORTROOT expression also increased 2- to 4-fold in rooting-competent tissue. The greatest transcript abundance in rooting-competent cuttings was restricted to root progenitor cells, while recalcitrant cuttings had a diffuse mRNA signal among tissue types.

Demographically idiosyncratic responses to climate change and rapid Pleistocene diversification of the walnut genus Juglans (Juglandaceae) revealed by whole-genome sequences.

Whether species demography and diversification are driven primarily by extrinsic environmental changes such as climatic oscillations in the Quaternary or by intrinsic biological interactions like coevolution between antagonists is a matter of active debate. In fact, their relative importance can be assessed by tracking past population fluctuations over considerable time periods. We applied the pairwise sequentially Markovian coalescent approach on the genomes of 11 temperate Juglans species to estimate trajectories of changes in effective population size (Ne ) and used a Bayesian-coalescent based approach that simultaneously considers multiple genomes (G-PhoCS) to estimate divergence times between lineages. Ne curves of all study species converged 1.0 million yr ago, probably reflecting the time when the walnut genus last shared a common ancestor. This estimate was confirmed by the G-PhoCS estimates of divergence times. But all species did not react similarly to the dramatic climatic oscillations following early Pleistocene cooling, so the timing and amplitude of changes in Ne differed among species and even among conspecific lineages. The population histories of temperate walnut species were not driven by extrinsic environmental changes alone, and a key role was probably played by species-specific factors such as coevolutionary interactions with specialized pathogens.

Comparative Genomics of Pathogenic and Nonpathogenic Beetle-Vectored Fungi in the Genus Geosmithia.

Geosmithia morbida is an emerging fungal pathogen which serves as a model for examining the evolutionary processes behind pathogenicity because it is one of two known pathogens within a genus of mostly saprophytic, beetle-associated, fungi. This pathogen causes thousand cankers disease in black walnut trees and is vectored into the host via the walnut twig beetle. Geosmithia morbida was first detected in western United States and currently threatens the timber industry concentrated in eastern United States. We sequenced the genomes of G. morbida in a previous study and two nonpathogenic Geosmithia species in this work and compared these species to other fungal pathogens and nonpathogens to identify genes under positive selection in G. morbida that may be associated with pathogenicity. Geosmithia morbida possesses one of the smallest genomes among the fungal species observed in this study, and one of the smallest fungal pathogen genomes to date. The enzymatic profile in this pathogen is very similar to its nonpathogenic relatives. Our findings indicate that genome reduction or retention of a smaller genome may be an important adaptative force during the evolution of a specialized lifestyle in fungal species that occupy a specificniche, such as beetle vectored tree pathogens. We also present potential genes under selection in G. morbida that could be important for adaptation to a pathogenic lifestyle.

Rethinking the history of common walnut (Juglans regia L.) in Europe: Its origins and human interactions.

Common walnut (Juglans regia L) is an economically important species cultivated worldwide for its high-quality wood and nuts. It is generally accepted that after the last glaciation J. regia survived and grew in almost completely isolated stands in Asia, and that ancient humans dispersed walnuts across Asia and into new habitats via trade and cultural expansion. The history of walnut in Europe is a matter of debate, however. In this study, we estimated the genetic diversity and structure of 91 Eurasian walnut populations using 14 neutral microsatellites. By integrating fossil pollen, cultural, and historical data with population genetics, and approximate Bayesian analysis, we reconstructed the demographic history of walnut and its routes of dispersal across Europe. The genetic data confirmed the presence of walnut in glacial refugia in the Balkans and western Europe. We conclude that human-mediated admixture between Anatolian and Balkan walnut germplasm started in the Early Bronze Age, and between western Europe and the Balkans in eastern Europe during the Roman Empire. A population size expansion and subsequent decline in northeastern and western Europe was detected in the last five centuries. The actual distribution of walnut in Europe resulted from the combined effects of expansion/contraction from multiple refugia after the Last Glacial Maximum and its human exploitation over the last 5,000 years.