Climate‐associated genetic variation in Fagus sylvatica and potential responses to climate change in the French Alps

Local adaptation patterns have been found in many plants and animals, highlighting the genetic heterogeneity of species along their range of distribution. In the next decades, global warming is predicted to induce a change in the selective pressures that drive this adaptive variation, forcing a reshuffling of the underlying adaptive allele distributions. For species with low dispersion capacity and long generation time such as trees, the rapidity of the change could impede the migration of beneficial alleles and lower their capacity to track the changing environment. Identifying the main selective pressures driving the adaptive genetic variation is thus necessary when investigating species capacity to respond to global warming. In this study, we investigate the adaptive landscape of Fagus sylvatica along a gradient of populations in the French Alps. Using a double‐digest restriction‐site‐associated DNA (ddRAD) sequencing approach, we identified 7,000 SNPs from 570 individuals across 36 different sites. A redundancy analysis (RDA)‐derived method allowed us to identify several SNPs that were strongly associated with climatic gradients; moreover, we defined the primary selective gradients along the natural populations of F. sylvatica in the Alps. Strong effects of elevation and humidity, which contrast north‐western and south‐eastern site, were found and were believed to be important drivers of genetic adaptation. Finally, simulations of future genetic landscapes that used these findings allowed identifying populations at risk for F. sylvatica in the Alps, which could be helpful for future management plans.     

Assessment of species limits in African ‘brown buntings’ (Emberiza,Passeriformes) based on mitochondrial and nuclear sequence data

We estimated a phylogeny for 10 taxa currently placed in four polytypic species that collectively encompass the African ‘brown buntings’: Cape Bunting Emberiza capensis, Cinnamon‐breasted Bunting Emberiza tahapisi, Lark‐like Bunting Emberiza impetuani and House Bunting Emberiza striolata. We made use of the mitochondrial cytochrome b gene and the nuclear introns 6–7 of ornithine decarboxylase (ODC), and intron 2 of myoglobin. There was substantial cytochrome b sequence divergence between taxa currently treated as conspecific: sahari vs. striolata (2.6–3.1% (uncorrected‐p); 3.0–3.6% (HKY + I)), and goslingi vs. tahapisi (4.4–4.7% (uncorrected‐p); 5.4–5.9% (HKY + I)). The degree of divergence of the nuclear loci among taxa was limited, and these loci lacked reciprocal monophyly, most likely as a consequence of incomplete lineage sorting. A single representative of the taxon septemstriata, generally treated as a member of the dark‐throated tahapisi group, here appears to be genetically consistent with the grey‐throated goslingi, and may be of hybrid origin. All other taxa allocated to E. striolata and E. tahapisi make up four reciprocally monophyletic groups consistent with sahari, striolata, tahapisi and goslingi, respectively. The extent of genetic evidence suggests that these taxa have been evolving as separate evolutionary lineages for a long time. This is further manifested in several morphological and vocal characteristics described previously, and we propose that these divergent taxa be treated as separate species: Cinnamon‐breasted Bunting Emberiza tahapisi, Gosling's Bunting Emberiza goslingi, Striolated Bunting Emberiza striolata and House Bunting Emberiza sahari. We do not propose any taxonomic changes regarding Emberiza impetuani or Emberiza capensis.     

Recent phylogeographic structure in a widespread ‘weedy’ Neotropical tree species,Cordia alliodora (Boraginaceae)

Aim Although hundreds of tree species have broad geographic ranges in the Neotropics, little is known about how such widespread species attained disjunct distributions around mountain, ocean and xeric barriers. Here, we examine the phylogeographic structure of a widespread and economically important tree, Cordia alliodora, to: (1) test the roles of vicariance and dispersal in establishing major range disjunctions, (2) determine which geographic regions and/or habitats contain the highest levels of genetic diversity, and (3) infer the geographic origin of the species. Location Twenty‐five countries in Central and South America, and the West Indies. Methods Chloroplast simple sequence repeats (cpSSR; eight loci) were assayed in 67 populations (240 individuals) sampled from the full geographic range of C. alliodora. Chloroplast (trnH–psbA) and nuclear (internal transcribed spacer, ITS) DNA sequences were sampled from a geographically representative subset. Genetic structure was determined with samova , structure and haplotype networks. Analysis of molecular variance (AMOVA) and rarefaction analyses were used to compare regional haplotype diversity and differentiation. Results Although the ITS region was polymorphic it revealed limited phylogeographic structure, and trnH–psbA was monomorphic. However, structure analysis of cpSSR variation recovered three broad demes spanning Central America (Deme 1), the Greater Antilles and the Chocó (Deme 2), and the Lesser Antilles and cis‐Andean South America (Deme 3). samova showed two predominant demes (Deme 1 + 2 and Deme 3). The greatest haplotype diversity was detected east of the Andes, while significantly more genetic variation was partitioned among trans‐Andean populations. Populations experiencing high precipitation seasonality (dry ecotype) had greater levels of genetic variation. Main conclusions Cordia alliodora displayed weak cis‐ and trans‐Andean phylogeographic structure based on DNA sequence data, indicative of historical dispersal around this barrier and genetic exchange across its broad range. The cpSSR data revealed phylogeographic structure corresponding to three biogeographic zones. Patterns of genetic diversity are indicative of an origin in the seasonally dry habitats of South America. Therefore, C. alliodora fits the disperser hypothesis for widespread Neotropical species. Dispersal is evident in the West Indies and the northern Andean cordilleras. The dry ecotype harbours genetic variation that is likely to represent the source for the establishment of populations under future warmer and drier climatic scenarios.     

High‐Throughput DNA sequencing of ancient wood

Reconstructing the colonization and demographic dynamics that gave rise to extant forests is essential to forecasts of forest responses to environmental changes. Classical approaches to map how population of trees changed through space and time largely rely on pollen distribution patterns, with only a limited number of studies exploiting DNA molecules preserved in wooden tree archaeological and subfossil remains. Here, we advance such analyses by applying high‐throughput (HTS) DNA sequencing to wood archaeological and subfossil material for the first time, using a comprehensive sample of 167 European white oak waterlogged remains spanning a large temporal (from 550 to 9,800 years) and geographical range across Europe. The successful characterization of the endogenous DNA and exogenous microbial DNA of 140 (~83%) samples helped the identification of environmental conditions favouring long‐term DNA preservation in wood remains, and started to unveil the first trends in the DNA decay process in wood material. Additionally, the maternally inherited chloroplast haplotypes of 21 samples from three periods of forest human‐induced use (Neolithic, Bronze Age and Middle Ages) were found to be consistent with those of modern populations growing in the same geographic areas. Our work paves the way for further studies aiming at using ancient DNA preserved in wood to reconstruct the micro‐evolutionary response of trees to climate change and human forest management.     

Forest phenology and a warmer climate – growing season extension in relation to climatic provenance

Predicting forest responses to warming climates relies on assumptions about niche and temperature sensitivity that remain largely untested. Observational studies have related current and historical temperatures to phenological shifts, but experimental evidence is sparse, particularly for autumn responses. A 4 year field experiment exposed four deciduous forest species from contrasting climates (Liquidambar styraciflua, Quercus rubra, Populus grandidentata, and Betula alleghaniensis) to air temperatures 2 and 4 °C above ambient controls, using temperature‐controlled open top chambers. Impacts of year‐round warming on bud burst (BB), senescence, and abscission were evaluated in relation to thermal provenance. Leaves emerged earlier in all species by an average of 4–9 days at +2 °C and 6–14 days at +4 °C. Magnitude of advance varied with species and year, but was larger for the first 2 °C increment than for the second. Effect of warming increased with early BB, favoring Liquidambar, but even BB of northern species advanced, despite temperatures exceeding those of the realized niche. Treatment differences in BB were inadequately explained by temperature sums alone. In autumn, chlorophyll was retained an average of 4 and 7 days longer in +2 and +4 °C treatments, respectively, and abscission delayed by 8 and 13 days. Growing seasons in the warmer atmospheres averaged 5–18 days (E2) and 6–28 days (E4) longer, according to species, with the least impact in Quercus. Results are compared with a 16 years record of canopy onset and offset in a nearby upland deciduous forest, where BB showed similar responsiveness to spring temperatures (2–4 days °C^?1). Offset dates in the stand tracked August–September temperatures, except when late summer drought caused premature senescence. The common garden‐like experiment provides evidence that warming alone extends the growing season, at both ends, even if stand‐level impacts may be complicated by variation in other environmental factors.     

State‐of‐the‐art CRISPR/Cas9 Technology for Genome Editing in Trypanosomatids

CRISPR/Cas9 technology has revolutionized biology. This prokaryotic defense system against foreign DNA has been repurposed for genome editing in a broad range of cell tissues and organisms. Trypanosomatids are flagellated protozoa belonging to the order Kinetoplastida. Some of its most representative members cause important human diseases affecting millions of people worldwide, such as Chagas disease, sleeping sickness and different forms of leishmaniases. Trypanosomatid infections represent an enormous burden for public health and there are no effective treatments for most of the diseases they cause. Since the emergence of the CRISPR/Cas9 technology, the genetic manipulation of these parasites has notably improved. As a consequence, genome editing is now playing a key role in the functional study of proteins, in the characterization of metabolic pathways, in the validation of alternative targets for antiparasitic interventions, and in the study of parasite biology and pathogenesis. In this work we review the different strategies that have been used to adapt the CRISPR/Cas9 system to Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp., as well as the research progress achieved using these approaches. Thereby, we will present the state‐of‐the‐art molecular tools available for genome editing in trypanosomatids to finally point out the future perspectives in the field.     

Interaction of MC1R and PMEL alleles on solid coat colors in Highland cattle

Six solid colors occur in Highland cattle: black, dun, silver dun and red, yellow, and white. These six coat colors are explained by a non‐epistatic interaction of the genotypes at the MC1R and PMEL genes. A three base pair deletion in the PMEL gene leading to the deletion of a leucine from the signal peptide is observed in dilute‐colored Highland cattle (c.50_52delTTC, p.Leu18del). The mutant PMEL allele acts in a semi‐dominant manner. Dun Galloway cattle also have one copy of the deletion allele, and silver dun Galloway cattle have two copies. The presence of two adjacent leucine residues at the site of this deletion is highly conserved in human, horse, mouse and chicken as well as in cattle with undiluted coat colors. Highland and Galloway cattle thus exhibit a similar dose‐dependent dilution effect based on the number of PMEL :c.50_51delTTC alleles, as Charolais cattle with PMEL :c.64G>A alleles. The PMEL :c.64G>A allele was not found in Highland or Galloway cattle.     

Diversification and asymmetrical gene flow across time and space: lineage sorting and hybridization in polytypic barking frogs

Young species complexes that are widespread across ecologically disparate regions offer important insights into the process of speciation because of their relevance to how local adaptation and gene flow influence diversification. We used mitochondrial DNA and up to 28 152 genomewide single nucleotide polymorphisms from polytypic barking frogs (Craugastor augusti complex) to infer phylogenetic relationships and test for the signature of introgressive hybridization among diverging lineages. Our phylogenetic reconstructions suggest (i) a rapid Pliocene–Pleistocene radiation that produced at least nine distinct lineages and (ii) that geographic features of the arid Central Mexican Plateau contributed to two independent northward expansions. Despite clear lineage differentiation (many private alleles and high between‐lineage F_ST scores), D‐statistic tests, which differentiate introgression from ancestral polymorphism, allowed us to identify two putative instances of reticulate gene flow. Partitioned D‐statistics provided evidence that these events occurred in the same direction between clades but at different points in time. After correcting for geographic distance, we found that lineages involved in hybrid gene flow interactions had higher levels of genetic variation than independently evolving lineages. These findings suggest that the nature of hybrid compatibility can be conserved overlong periods of evolutionary time and that hybridization between diverging lineages may contribute to standing levels of genetic variation.     

A dual role for integrin‐linked kinase and β1‐integrin in modulating cardiac aging

Cardiac performance decreases with age, which is a major risk factor for cardiovascular disease and mortality in the aging human population, but the molecular mechanisms underlying cardiac aging are still poorly understood. Investigating the role of integrin‐linked kinase (ilk) and β1‐integrin (myospheroid, mys) in Drosophila, which colocalize near cardiomyocyte contacts and Z‐bands, we find that reduced ilk or mys function prevents the typical changes of cardiac aging seen in wildtype, such as arrhythmias. In particular, the characteristic increase in cardiac arrhythmias with age is prevented in ilk and mys heterozygous flies with nearly identical genetic background, and they live longer, in line with previous findings in Caenorhabditis elegans for ilk and in Drosophila for mys. Consistent with these findings, we observed elevated β1‐integrin protein levels in old compared with young wild‐type flies, and cardiac‐specific overexpression of mys in young flies causes aging‐like heart dysfunction. Moreover, moderate cardiac‐specific knockdown of integrin‐linked kinase (ILK)/integrin pathway‐associated genes also prevented the decline in cardiac performance with age. In contrast, strong cardiac knockdown of ilk or ILK‐associated genes can severely compromise cardiac integrity, including cardiomyocyte adhesion and overall heart function. These data suggest that ilk/mys function is necessary for establishing and maintaining normal heart structure and function, and appropriate fine‐tuning of this pathway can retard the age‐dependent decline in cardiac performance and extend lifespan. Thus, ILK/integrin‐associated signaling emerges as an important and conserved genetic mechanism in longevity, and as a new means to improve age‐dependent cardiac performance, in addition to its vital role in maintaining cardiac integrity.     

Efficacy of Acibenzolar‐S‐Methyl and β‐Aminobutyric Acid for Control of Downy Mildew in Greenhouse Grown Basil and Peroxidase Activity in Response to Treatment with these Compounds

Downy mildew, caused by the oomycete pathogen Peronospora belbahrii, is a devastating foliar disease of basil in the United States and worldwide. Currently there are very few chemistries or organic choices registered to control this disease. In this study, two systemic acquired resistance (SAR) inducers, acibenzolar‐S‐methyl (ASM) and β‐aminobutyric acid (BABA), were evaluated for their in vitro effects on the pathogen, for their potential to control basil downy mildew in greenhouses, and for changes in peroxidase activity in basil plants treated with these two SAR inducers. No significant inhibition of sporangial germination was detected in water agar amended with ASM at concentrations lower than 100 mg/l or with BABA at concentrations lower than 500 mg/l. Efficacy of ASM and BABA in greenhouses varied depending on the rate, method and timing of application. The area under the disease progress curve (AUDPC) of disease severity was significantly reduced compared to the non‐treated control when ASM was sprayed (in all experiments) or drenched (in one out of two experiments) pre‐, or pre‐ + post‐inoculation at rates of 25–400 mg/l. Three weekly post‐inoculation sprays of ASM at the rate of 50 mg/l reduced AUDPC by 93.0 and 47.2% when started 3 and 7 days after inoculation (DAI), respectively. The AUDPC of disease severity was also significantly reduced when BABA was sprayed pre‐ + post‐inoculation at rates of 125–500 mg/l. According to the prediction using a log‐logistic function, 50% maximum disease protection was achieved at a concentration of 27.5 mg/l of ASM. Basil plants treated with these two SAR inducers and challenged with the pathogen showed significantly higher peroxidase activity than the non‐treated control at 8 DAI. Temporally, the highest activity of peroxidase was detected at 8 DAI, decreased at 15 DAI and waned further at 23 DAI.     

Identification and Pathogenicity of Lasiodiplodia Species from Eucalyptus urophylla × grandis,Polyscias balfouriana and Bougainvillea spectabilis in Southern China

Species of Lasiodiplodia are important pathogens of a wide variety of plants covering a wide geographical distribution. These fungi can be associated with different symptoms such as stem cankers, shoot blights, fruit rots, dieback and gummosis. Diseases caused by Lasiodiplodia were surveyed on Eucalyptus urophylla × grandis, Polyscias balfouriana and Bougainvillea spectabilis in a nursery in southern China. Based on morphology characteristics and phylogenetic analyses of ITS rDNA sequences and translation elongation factor 1‐alpha (TEF‐1α) gene regions, four species of Lasiodiplodia were identified. Lasiodiplodia theobromae was identified from E. urophylla × grandis, P. balfouriana and B. spectabilis. L. hormozganensis, L. iraniensis and L. pseudotheobromae were identified from B. spectabilis. To our knowledge, with the exception of L. theobromae on E. urophylla × grandis, this study represents the first report of these fungi on the host plants. Pathogenicity tests showed that all Lasiodiplodia spp. obtained in this study are virulent to E. urophylla × grandis and B. spectabilis, and L. theobromae was virulent to P. balfouriana.     

Strong thermal acclimation of photosynthesis in tropical and temperate wet‐forest tree species: the importance of altered Rubisco content

Understanding of the extent of acclimation of light‐saturated net photosynthesis (A_n) to temperature (T), and associated underlying mechanisms, remains limited. This is a key knowledge gap given the importance of thermal acclimation for plant functioning, both under current and future higher temperatures, limiting the accuracy and realism of Earth system model (ESM) predictions. Given this, we analysed and modelled T‐dependent changes in photosynthetic capacity in 10 wet‐forest tree species: six from temperate forests and four from tropical forests. Temperate and tropical species were each acclimated to three daytime growth temperatures (T_growth): temperate – 15, 20 and 25 °C; tropical – 25, 30 and 35 °C. CO₂ response curves of A_n were used to model maximal rates of RuBP (ribulose‐1,5‐bisphosphate) carboxylation (V_cmax) and electron transport (J_max) at each treatment's respective T_growth and at a common measurement T (25 °C). SDS‐PAGE gels were used to determine abundance of the CO₂‐fixing enzyme, Rubisco. Leaf chlorophyll, nitrogen (N) and mass per unit leaf area (LMA) were also determined. For all species and T_growth, A_n at current atmospheric CO₂ partial pressure was Rubisco‐limited. Across all species, LMA decreased with increasing T_growth. Similarly, area‐based rates of V_cmax at a measurement T of 25 °C (V_cmax²⁵) linearly declined with increasing T_growth, linked to a concomitant decline in total leaf protein per unit leaf area and Rubisco as a percentage of leaf N. The decline in Rubisco constrained V_cmax and A_n for leaves developed at higher T_growth and resulted in poor predictions of photosynthesis by currently widely used models that do not account for T_growth‐mediated changes in Rubisco abundance that underpin the thermal acclimation response of photosynthesis in wet‐forest tree species. A new model is proposed that accounts for the effect of T_growth‐mediated declines in V_cmax²⁵ on A_n, complementing current photosynthetic thermal acclimation models that do not account for T sensitivity of V_cmax²⁵.     

Functional analysis of endo‐1,4‐β‐glucanases in response to Botrytis cinerea and Pseudomonas syringae reveals their involvement in plant–pathogen interactions

Plant cell wall modification is a critical component in stress responses. Endo‐1,4‐β‐glucanases (EGs) take part in cell wall editing processes, e.g. elongation, ripening and abscission. Here we studied the infection response of Solanum lycopersicum and Arabidopsis thaliana with impaired EGs. Transgenic TomCel1 and TomCel2 tomato antisense plants challenged with Pseudomonas syringae showed higher susceptibility, callose priming and increased jasmonic acid pathway marker gene expression. These two EGs could be resistance factors and may act as negative regulators of callose deposition, probably by interfering with the defence‐signalling network. A study of a set of Arabidopsis EG T‐DNA insertion mutants challenged with P. syringae and Botrytis cinerea revealed that the lack of other EGs interferes with infection phenotype, callose deposition, expression of signalling pathway marker genes and hormonal balance. We conclude that a lack of EGs could alter plant response to pathogens by modifying the properties of the cell wall and/or interfering with signalling pathways, contributing to generate the appropriate signalling outcomes. Analysis of microarray data demonstrates that EGs are differentially expressed upon many different plant–pathogen challenges, hormone treatments and many abiotic stresses. We found some Arabidopsis EG mutants with increased tolerance to osmotic and salt stress. Our results show that impairing EGs can alter plant–pathogen interactions and may contribute to appropriate signalling outcomes in many different biotic and abiotic plant stress responses.     

HT proteins contribute to S‐RNase‐independent pollen rejection in Solanum

Plants have mechanisms to recognize and reject pollen from other species. Although widespread, these mechanisms are less well understood than the self‐incompatibility (SI) mechanisms plants use to reject pollen from close relatives. Previous studies have shown that some interspecific reproductive barriers (IRBs) are related to SI in the Solanaceae. For example, the pistil SI proteins S‐RNase and HT protein function in a pistil‐side IRB that causes rejection of pollen from self‐compatible (SC) red/orange‐fruited species in the tomato clade. However, S‐RNase‐independent IRBs also clearly contribute to rejecting pollen from these species. We investigated S‐RNase‐independent rejection of Solanum lycopersicum pollen by SC Solanum pennellii LA0716, SC. Solanum habrochaites LA0407, and SC Solanum arcanum LA2157, which lack functional S‐RNase expression. We found that all three accessions express HT proteins, which previously had been known to function only in conjunction with S‐RNase, and then used RNAi to test whether they also function in S‐RNase‐independent pollen rejection. Suppressing HT expression in SC S. pennellii LA0716 allows S. lycopersicum pollen tubes to penetrate farther into the pistil in HT suppressed plants, but not to reach the ovary. In contrast, suppressing HT expression in SC. Solanum habrochaites LA0407 and in SC S. arcanum LA2157 allows S. lycopersicum pollen tubes to penetrate to the ovary and produce hybrids that, otherwise, would be difficult to obtain. Thus, HT proteins are implicated in both S‐RNase‐dependent and S‐RNase‐independent pollen rejection. The results support the view that overall compatibility results from multiple pollen–pistil interactions with additive effects.     

Amplicon‐pyrosequencing‐based detection of compositional shifts in bryophyte‐associated fungal communities along an elevation gradient

Although bryophytes are a dominant vegetation component of boreal and alpine ecosystems, little is known about their associated fungal communities. HPLC assays of ergosterol (fungal biomass) and amplicon pyrosequencing of the ITS2 region of rDNA were used to investigate how the fungal communities associated with four bryophyte species changed across an elevational gradient transitioning from conifer forest to the low‐alpine. Fungal biomass and OTU richness associated with the four moss hosts did not vary significantly across the gradient (P > 0.05), and both were more strongly affected by host and tissue type. Despite largely constant levels of fungal biomass, distinct shifts in community composition of fungi associated with Hylocomium, Pleurozium and Polytrichum occurred between the elevation zones of the gradient. This likely is a result of influence on fungal communities by major environmental factors such as temperature, directly or indirectly mediated by, or interacting with, the response of other components of the vegetation (i.e. the dominant trees). Fungal communities associated with Dicranum were an exception, exhibiting spatial autocorrelation between plots, and no significant structuring by elevation. Nevertheless, the detection of distinct fungal assemblages associated with a single host growing in different elevation zones along an elevational gradient is of particular relevance in the light of the ongoing changes in vegetation patterns in boreal and alpine systems due to global climate warming.     

Mutant Alleles at the Brown Locus Encoding Tyrosinase‐Related Protein‐1 (TRP‐1) Affect Proliferation of Mouse Melanocytes in Culture *

Tyrosinase related protein‐1 (TRP‐1) is a melanocyte‐specific gene product involved in eumelanin synthesis. Mutation in the Tyrp1 gene is associated with brown pelage in mouse and oculocutaneous albinism Type 3 in humans (OCA3). It has been demonstrated that TRP‐1 expresses DHICA oxidase activity in the murine system. However, its actual function in the human system is still unclear. The study was designed to determine the effects of mutation at two Typr1 alleles, namely the Tyrp1^b (brown) and Tyrp1^b‐cj (cordovan) compared with wild type Tyrp1^B (black) on melanocyte function and melanin biosynthesis. The most significant finding was that both of the Tyrp1 mutations (i.e. brown expressing a point mutation and cordovan expressing decreased amount of TRP‐1 protein) resulted in attenuation of cell proliferation rates. Neither necrosis nor apoptosis was responsible for the observed decrease in cell proliferation rates of the brown and cordovan melanocytes. Ultrastructural evaluation by electron microscopic analysis revealed that both mutations in Tyrp1 affected melanosome maturation without affecting its structure. These observations demonstrate that mutation in Tyrp1 compromised tyrosinase activity within the organelle. DOPA histochemistry revealed differences in melanosomal stages between black and brown melanocytes but not between black and cordovan melanocytes. There were no significant differences in tyrosine hydroxylase activities of tyrosinase and TRP‐1 in wild type black, brown and cordovan melanocyte cell lysates. We conclude that mutations in Tyrp1 compromise cell proliferation and melanosomal maturation in mouse melanocyte cultures.