Genomic evidence for homoploid hybrid speciation between ancestors of two different genera.

Homoploid hybrid speciation (HHS) has been increasingly recognized as occurring widely during species diversification of both plants and animals. However, previous studies on HHS have mostly focused on closely-related species while it has been rarely reported or tested between ancestors of different genera. Here, we explore the likely HHS origin of Carpinus sect. Distegocarpus between sect. Carpinus and Ostrya in the family Betulaceae. We generate a chromosome-level reference genome for C. viminea of sect. Carpinus and re-sequence genomes of 44 individuals from the genera Carpinus and Ostrya. Our integrated analyses of all genomic data suggest that sect. Distegocarpus, which has three species, likely originates through HHS during the early divergence between Carpinus and Ostrya. Our study highlights the likelihood of an HHS event between ancestors of the extant genera during their initial divergences, which may have led to reticulate phylogenies at higher taxonomic levels.

Comprehensive Transcriptome Analysis of Rare Carpinus putoensis Plants under NO2 stress.

We evaluated a transcriptome using high-throughput Illumina HiSeq sequencing and related it to the morphology, leaf anatomy, and physiological parameters of Carpinus putoensis putoensis under NO2 stress. The molecular mechanism of the C. putoensis NO2 stress response was evaluated using sequencing data. NO2 stress adversely affected the morphology, leaf anatomy, and total peroxidase (POD) activity. Through RNA-seq analysis, we used NCBI to compare the transcripts with nine databases and obtained their functional annotations. We annotated up to 2255 million clean Illumina paired-end RNA-seq reads, and 250,200 unigene sequences were assembled based on the resulting transcriptome data. More than 89% of the C. putoensis transcripts were functionally annotated. Under NO2 stress, 1119 genes were upregulated and 1240 were downregulated. According to the KEGG pathway and GO analyses, photosynthesis, chloroplasts, plastids, and the stimulus response are related to NO2 stress. Additionally, NO2 stress changed the expression of POD families, and the HPL2, HPL1, and POD genes exhibited high expression. The transcriptome analysis of C. putoensis leaves under NO2 stress supplies a reference for studying the molecular mechanism of C. putoensis resistance to NO2 stress. The given transcriptome data represent a valuable resource for studies on plant genes, which will contribute towards genome annotations during future genome projects.

Hybrid speciation via inheritance of alternate alleles of parental isolating genes.

It is increasingly realized that homoploid hybrid speciation (HHS), which involves no change in chromosome number, is an important mechanism of speciation. HHS will likely increase in frequency as ecological and geographical barriers between species are continuing to be disrupted by human activities. HHS requires the establishment of reproductive isolation between a hybrid and its parents, but the underlying genes and genetic mechanisms remain largely unknown. In this study, we reveal by integrated approaches that reproductive isolation originates in one homoploid hybrid plant species through the inheritance of alternate alleles at genes that determine parental premating isolation. The parent species of this hybrid species are reproductively isolated by differences in flowering time and survivorship on soils containing high concentrations of iron. We found that the hybrid species inherits alleles of parental isolating major genes related to flowering time from one parent and alleles of major genes related to iron tolerance from the other parent. In this way, it became reproductively isolated from one parent by the difference in flowering time and from the other by habitat adaptation (iron tolerance). These findings and further modeling results suggest that HHS may occur relatively easily via the inheritance of alternate parental premating isolating genes and barriers.

Chloroplast genomes of seven species of Coryloideae (Betulaceae): structures and comparative analysis.

Coryloideae is a subfamily in the family Betulaceae consisting of four extant genera: Carpinus, Corylus, Ostrya, and Ostryopsis. We sequenced the plastomes of six species of Corylus and one species of Ostryopsis for comparative and phylogenetic analyses. The plastomes are 159-160 kb long and possess typical quadripartite cp architecture. The plastomes show moderate divergence and conserved arrangement. Five mutational hotspots were identified by comparing the plastomes of seven species of Coryloideae: trnG-atpA, trnF-ndhJ, accD-psaI, ndhF-ccsA, and ycf1. We assembled the most complete phylogenomic tree for the family Betulaceae using 68 plastomes. Our cp genomic sequence phylogenetic analyses placed Carpinus, Ostrya, and Ostryopsis in a clade together and left Corylus in a separate clade. Within the genus Corylus, these analyses indicate the existence of five subclades reflecting the phylogeographical relationships among the species. The data offer significant genetic information for the identification of species of the Coryloideae, taxonomic and phylogenetic studies, and molecular breeding.

A chromosome-level reference genome of the hornbeam, Carpinus fangiana.

Betulaceae, the birch family, comprises six living genera and over 160 species, many of which are economically valuable. To deepen our knowledge of Betulaceae species, we have sequenced the genome of a hornbeam, Carpinus fangiana, which belongs to the most species-rich genus of the Betulaceae subfamily Coryloideae. Based on over 75 Gb (~200x) of high-quality next-generation sequencing data, we assembled a 386.19 Mb C. fangiana genome with contig N50 and scaffold N50 sizes of 35.32 kb and 1.91 Mb, respectively. Furthermore, 357.84 Mb of the genome was anchored to eight chromosomes using over 50 Gb (~130x) Hi-C sequencing data. Transcriptomes representing six tissues were sequenced to facilitate gene annotation, and over 5.50 Gb high-quality data were generated for each tissue. The structural annotation identified a total of 27,381 protein-coding genes in the assembled genome, of which 94.36% were functionally annotated. Additionally, 4,440 non-coding genes were predicted.

Is Betula carpatica genetically distinctive? A morphometric, cytometric and molecular study of birches in the Bohemian Massif with a focus on Carpathian birch.

Until recently, Czech taxonomists often treated Betula carpatica as a distinct species. Several morphological traits for distinguishing B. carpatica from B. pubescens or other birches are described in literature; however, it has been proven impossible to reliably identify B. carpatica in the field. With the use of morphological and molecular approaches, we intended to assess the position of B. carpatica in the context of other birch taxa reported from the Bohemian Massif and to find more reliable morphological traits for their identification. In our dataset, we distinguished the following birch taxa referred to in the recent Czech literature: B. pendula, B. pubescens, B. carpatica, B. oycoviensis, B. nana, B. petraea and B. ×seideliana. We complemented them with triploids and several diploid and tetraploid "working units" into which we included intermediate individuals that in terms of morphology did not unambiguously match any of the abovementioned birch taxa. Holoploid genome size was measured to determine the ploidy level. To identify genetic relationships between selected taxa and "working units", microsatellite analyses were performed. Model-based STRUCTURE analysis together with principal coordinates analysis (PCoA) based on genetic distances was performed to identify the similarities in multilocus genotype data between groups distinguished in the dataset. The applied analyses were not able clearly to distinguish any group among tetraploid individuals. In this light, it was of no use to search for any more reliable morphological traits of B. carpatica and also B. petraea. Among diploids, B. nana was always distinguished, in contrast to B. oycoviensis, which was not genetically recognized despite being usually morphologically distinct. Based on our results and a literature review, we suggest that B. carpatica and also the closely similar B. petraea should not be considered separate species. A similar conclusion seems relevant also for B. oycoviensis; however, further verification is desirable in this case.

Interspecific delimitation and relationships among four Ostrya species based on plastomes.

BACKGROUND: The genus Ostrya (Betulaceae) contains eight species and four of them are distributed in China. However, studies based on limited informative sites of several chloroplast markers failed to resolve interspecific delimitation and relationships among the four Chinese species. In this study, we aimed to use the whole chloroplast genomes to address these two issues. RESULTS: We assembled and annotated 33 complete chloroplast genomes (plastomes) of the four Chinese species, representing 17 populations across most of their geographical distributions. Each species contained samples of several individuals that cover most of geographic distributions of the species. All plastomes are highly conserved in genome structure and gene order, with a total length of 158-159 kb and 122 genes. Phylogenetic analyses of whole plastomes, non-coding regions and protein-coding genes produced almost the same topological relationships. In contrast to the well-delimitated species boundary inferred from the nuclear ITS sequence variations, three of the four species are non-monophyletic in the plastome trees, which is consistent with previous studies based on a few chloroplast markers. CONCLUSIONS: The high incongruence between the ITS and plastome trees may suggest the widespread occurrences of hybrid introgression and incomplete lineage sorting during the divergence of these species. In addition, the plastomes with more informative sites compared with a few chloroplast markers still failed to resolve the phylogenetic relationships of the four species, and further studies involving population genomic data may be needed to better understand their evolutionary histories.

Host plant phylogeny and geographic distance strongly structure Betulaceae-associated ectomycorrhizal fungal communities in Chinese secondary forest ecosystems.

Environmental filtering and dispersal limitation are two of the primary drivers of community assembly in ecosystems, but their effects on ectomycorrhizal (EM) fungal communities associated with wide ranges of Betulaceae taxa at a large scale are poorly documented. In this study, we examined EM fungal communities associated with 23 species from four genera (Alnus, Betula, Carpinus and Corylus) of Betulaceae in Chinese secondary forest ecosystems, using Illumina MiSeq sequencing of the ITS2 region. Effects of host plant phylogeny, soil, climate and geographic distance on EM fungal community were explored. In total, we distinguished 1738 EM fungal operational taxonomic units (OTUs) at a 97% sequence similarity level. The EM fungal communities of Alnus had significantly lower OTU richness than those associated with the other three plant genera. The EM fungal OTU richness was significantly affected by geographic distance, host plant phylogeny, soil and climate. The EM fungal community composition was significantly influenced by host plant phylogeny (12.1% of variation explained in EM fungal community), geographic distance (7.7%), soil (4.6%) and climate (1.1%). This finding highlights that environmental filtering linked to host plant phylogeny and dispersal limitation strongly influence EM fungal communities associated with Betulaceae plants in Chinese secondary forest ecosystems.

Genomic effects of population collapse in a critically endangered ironwood tree Ostrya rehderiana.

Increased human activity and climate change are driving numerous tree species to endangered status, and in the worst cases extinction. Here we examine the genomic signatures of the critically endangered ironwood tree Ostrya rehderiana and its widespread congener O. chinensis. Both species have similar demographic histories prior to the Last Glacial Maximum (LGM); however, the effective population size of O. rehderiana continued to decrease through the last 10,000 years, whereas O. chinensis recovered to Pre-LGM numbers. O. rehderiana accumulated more deleterious mutations, but purged more severely deleterious recessive variations than in O. chinensis. This purging and the gradually reduced inbreeding depression together may have mitigated extinction and contributed to the possible future survival of the outcrossing O. rehderiana. Our findings provide critical insights into the evolutionary history of population collapse and the potential for future recovery of the endangered trees.

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.

The complete chloroplast genome of Ostrya rehderiana.

The complete chloroplast sequence of Ostrya rehderiana is 159 347 bp in length, containing 85 protein-coding genes, 8 ribosomal RNA genes, and 31 transfer RNA genes. The circular genome exhibits a typical chloroplast genome structure comprising a large single copy region of 88 552 bp, a small single copy region of 18 941 bp and a pair of inverted repeats of 25 927 bp. The overall GC content of the chloroplast genome is 36.5%. Phylogenetic analysis of O. rehderiana sequence together with 12 complete chloroplast genomes revealed a basal placement of O. rehderiana within the Fagales species.

Diploid hybrid origin of Ostryopsis intermedia (Betulaceae) in the Qinghai-Tibet Plateau triggered by Quaternary climate change.

Despite the well-known effects that Quaternary climate oscillations had on shaping intraspecific diversity, their role in driving homoploid hybrid speciation is less clear. Here, we examine their importance in the putative homoploid hybrid origin and evolution of Ostryopsis intermedia, a diploid species occurring in the Qinghai-Tibet Plateau (QTP), a biodiversity hotspot. We investigated interspecific relationships between this species and its only other congeners, O. davidiana and O. nobilis, based on four sets of nuclear and chloroplast population genetic data and tested alternative speciation hypotheses. All nuclear data distinguished the three species clearly and supported a close relationship between O. intermedia and the disjunctly distributed O. davidiana. Chloroplast DNA sequence variation identified two tentative lineages, which distinguished O. intermedia from O. davidiana; however, both were present in O. nobilis. Admixture analyses of genetic polymorphisms at 20 SSR loci and sequence variation at 11 nuclear loci and approximate Bayesian computation (ABC) tests supported the hypothesis that O. intermedia originated by homoploid hybrid speciation from O. davidiana and O. nobilis. We further estimated that O. davidiana and O. nobilis diverged 6-11 Ma, while O. intermedia originated 0.5-1.2 Ma when O. davidiana is believed to have migrated southward, contacted and hybridized with O. nobilis possibly during the largest Quaternary glaciation that occurred in this region. Our findings highlight the importance of Quaternary climate change in the QTP in causing hybrid speciation in this important biodiversity hotspot.

Environmental and genetic control of insect abundance and herbivory along a forest elevational gradient.

Environmental conditions and plant genotype may influence insect herbivory along elevational gradients. Plant damage would decrease with elevation as temperature declines to suboptimal levels for insects. However, host plants at higher elevations may exhibit traits that either reduce or enhance leaf quality to insects, with uncertain net effects on herbivory. We examined folivory, insect abundance and leaf traits along six replicated elevational ranges in Nothofagus pumilio forests of the northern Patagonian Andes, Argentina. We also conducted a reciprocal transplant experiment between low- and high-elevation sites to test the extent of environmental and plant genetic control on insect abundance and folivory. We found that insect abundance, leaf size and specific leaf area decreased, whereas foliar phosphorous content increased, from low-, through mid- to high-elevation sites. Path analysis indicated that changes in both insect abundance and leaf traits were important in reducing folivory with increasing elevation and decreasing mean temperature. At both planting sites, plants from a low-elevation origin experienced higher damage and supported greater insect loads than plants from a high-elevation origin. The differences in leaf damage between sites were twofold larger than those between plant origins, suggesting that local environment was more important than host genotype in explaining folivory patterns. Different folivore guilds exhibited qualitatively similar responses to elevation. Our results suggest an increase in insect folivory on high-elevation N. pumilio forests under future climate warming scenarios. However, in the short-term, folivory increases might be smaller than expected from insect abundance only because at high elevations herbivores would encounter more resistant tree genotypes.

Transcriptomics of actinorhizal symbioses reveals homologs of the whole common symbiotic signaling cascade.

Comparative transcriptomics of two actinorhizal symbiotic plants, Casuarina glauca and Alnus glutinosa, was used to gain insight into their symbiotic programs triggered following contact with the nitrogen-fixing actinobacterium Frankia. Approximately 14,000 unigenes were recovered in roots and 3-week-old nodules of each of the two species. A transcriptomic array was designed to monitor changes in expression levels between roots and nodules, enabling the identification of up- and down-regulated genes as well as root- and nodule-specific genes. The expression levels of several genes emblematic of symbiosis were confirmed by quantitative polymerase chain reaction. As expected, several genes related to carbon and nitrogen exchange, defense against pathogens, or stress resistance were strongly regulated. Furthermore, homolog genes of the common and nodule-specific signaling pathways known in legumes were identified in the two actinorhizal symbiotic plants. The conservation of the host plant signaling pathway is all the more surprising in light of the lack of canonical nod genes in the genomes of its bacterial symbiont, Frankia. The evolutionary pattern emerging from these studies reinforces the hypothesis of a common genetic ancestor of the Fabid (Eurosid I) nodulating clade with a genetic predisposition for nodulation.

Chloroplast DNA phylogeography of Betula maximowicziana, a long-lived pioneer tree species and noble hardwood in Japan.

Betula maximowicziana is an ecologically and economically important tree species in Japan. In order to examine the phylogeographical pattern of the species in detail, maternally inherited chloroplast (cp) DNA variations of 25 natural populations of Betula maximowicziana and a total of 12 populations of three related species were evaluated by PCR-RFLP analysis. Two main haplotypic groups of B. maximowicziana populations (northern and southern) were detected, with the main boundary passing through the Tohoku region in northeastern Japan; in addition there was high genetic differentiation among the 25 populations studied (GST = 0.950, G'ST =0:977). The phylogeographical pattern exhibited by B. maximowicziana was much more similar to that of alpine plants than to that of beech and oak. Comparison of the patterns of genetic structure obtained from the cpDNA with previously and newly acquired data on bi-parentally inherited nuclear DNA indicates that the nuclear genome was transferred via pollen from the northern haplotypic group to the southern group more frequently than it moved in the opposite direction. Although common haplotypes were detected among B. maximowicziana and the two related species examined, these haplotypes were not shared sympatrically, suggesting very rare hybridization among the species currently occurring in their natural populations.

Effects of sugar beet chitinase IV on root-associated fungal community of transgenic silver birch in a field trial.

Heterogenous chitinases have been introduced in many plant species with the aim to increase the resistance of plants to fungal diseases. We studied the effects of the heterologous expression of sugar beet chitinase IV on the intensity of ectomycorrhizal (ECM) colonization and the structure of fungal communities in the field trial of 15 transgenic and 8 wild-type silver birch (Betula pendula Roth) genotypes. Fungal sequences were separated in denaturing gradient gel electrophoresis and identified by sequencing the ITS1 region to reveal the operational taxonomic units. ECM colonization was less intense in 7 out of 15 transgenic lines than in the corresponding non-transgenic control plants, but the slight decrease in overall ECM colonization in transgenic lines could not be related to sugar beet chitinase IV expression or total endochitinase activity. One transgenic line showing fairly weak sugar beet chitinase IV expression without significantly increased total endochitinase activity differed significantly from the non-transgenic controls in the structure of fungal community. Five sequences belonging to three different fungal genera (Hebeloma, Inocybe, Laccaria) were indicative of wild-type genotypes, and one sequence (Lactarius) indicated one transgenic line. In cluster analysis, the non-transgenic control grouped together with the transgenic lines indicating that genotype was a more important factor determining the structure of fungal communities than the transgenic status of the plants. With the tested birch lines, no clear evidence for the effect of the heterologous expression of sugar beet chitinase IV on ECM colonization or the structure of fungal community was found.

Rising atmospheric CO2 reduces sequestration of root-derived soil carbon.

Forests have a key role as carbon sinks, which could potentially mitigate the continuing increase in atmospheric carbon dioxide concentration and associated climate change. We show that carbon dioxide enrichment, although causing short-term growth stimulation in a range of European tree species, also leads to an increase in soil microbial respiration and a marked decline in sequestration of root-derived carbon in the soil. These findings indicate that, should similar processes operate in forest ecosystems, the size of the annual terrestrial carbon sink may be substantially reduced, resulting in a positive feedback on the rate of increase in atmospheric carbon dioxide concentration.

Ecological and genetic effects of cutting in an Alnus trabeculosa Hand.-Mazz. (Betulaceae) population.

In order to assess the ecological and genetic effects of cutting, we compared two portions of Alnus trabeculosa population at Yuda (Iwate Prefecture, Japan): one that has been cut about 30 years ago and one that has remained uncut. These portions were compared in terms of the degree of sprouting, genetic variation and gene distribution using isozyme markers. First, we determined the multilocus genotype (MLG) of all ramets, then sorted them into individuals according to the distribution of the MLGs. The average (+/- SE) of largest distance between ramets in one individual was 2.1 (+/- 0.18) m, which is consistent with the distance (2.0 (+/- 0.20) m) obtained by tracing physical connections between ramets. We found no significant differences in genetic variation between the two portions, but there were significant differences in their degree of sprouting. Furthermore, there were striking differences in gene distribution: the cut portion showed greater clustering of individuals with identical genetic components, which may be due to regeneration in the gaps made by cutting, reflecting the location of the mother trees, and seed and pollen dispersal from them.

Isozyme polymorphisms provide evidence of clinal variation with elevation in Nothofagus pumilio.

Variable physical conditions along elevational gradients strongly influence patterns of genetic differentiation in tree species. Here, the hypothesis is tested that different growth forms of Nothofagus pumilio, which characterizes the subalpine forests in the southern Andes, will display continuous genetic variation with elevation. At each of four elevational strips in three different mountain ranges, fresh leaf tissue was sampled from 30 randomly selected individuals to be analyzed by protein electrophoresis. Allelic frequencies were used to test for heterogeneity across populations and to classify populations into different elevational strips by discriminant analysis. The degree of population divergence was estimated by F(ST). Clinical variation on within-population genetic characteristics was analyzed by linear regressions against elevation. Seven enzyme systems coded for 14 putative isozyme loci, 57% of which were polymorphic in at least one population. Allele frequencies significantly varied with elevation and discriminant analysis separated populations at different elevational strips. Among-population divergence within any mountain range was small, but greater than among different mountain ranges. Overall, low-elevation populations were more variable than high-elevation populations, and regression analyses suggested continuous variation in populations of N. pumilio 100 m apart. Marked stepwise phenological differences on mountain slopes are most probably responsible for the isolation of nearby populations.

Differences in spatial autocorrelation between four sub-populations of Alnus trabeculosa Hand.-Mazz. (Betulaceae).

To investigate the spatial structure of Alnus trabeculosa Hand.-Mazz, we compared three sub-populations at Imaichi in Tochigi Prefecture and one sub-population at Juo in Ibaraki Prefecture, Japan. A total of 269 trees, covering 0.71 ha in total, were mapped and genetically analyzed using nine enzyme systems encoding 13 isozyme loci. There were no significant differences between the four sub-populations in terms of Na,Ne, H(o), H(e )and F(IS). However, according to spatial autocorrelation analysis, the Juo sub-population, which is younger than the others, showed an aggregation of multilocus genotypes, especially within 25 m radii. In contrast with the Juo sub-population, those in Imaichi showed no clear spatial structuring. In the three Imaichi sub-populations, gametic phase disequilibrium is attributable to heterogeneity of genotype frequencies, but in the Juo sub-population it seems to be due to other factors, connected with the composition of the trees in the sub-population and/or its founder population, gene flow and asexual propagation. To conserve as much as possible of the genes or genotypes in restricted areas, conservation of populations that do not show clear family structures, such as those in Imaichi, would be most effective.