Botrytis cinerea represents a model organism because of its degrading potential and the huge economic losses caused by its infection. The present review provides an overview of the recent primary physical, chemical, and biological control treatments adopted against the development of B. cinerea in table grapes to extend shelf life. These treatments preserve product quality and safety. This article also focuses on the compliance of different approaches with organic and sustainable production processes. Tailored approaches include those that rely on controlled atmosphere and the application of edible coating and packaging, as well as microbial-based activities. These strategies, applied alone or in combination, are among the most promising solutions in order to prolong table grape quality during cold storage. In general, the innovative design of applications dealing with hurdle technologies holds great promise for future improvements.Consumers highly appreciate table grapes for their pleasant sensory attributes and as good sources of nutritional and functional compounds. This explains the rising market and global interest in this product. Along with other fruits and vegetables, table grapes are considerably perishable post-harvest due to the growth of undesired microorganisms. Among the microbial spoilers,  Vitis vinifera L.) are essential not only for wine production but also for fresh consumption. Table grapes are highly appreciated by consumers, primarily because of their sensory attributes, but also because of their vitamins and bioactive compounds  [Viticulture is one of the major forms of fruit crop cultivation worldwide, and its global diffusion contributes considerably to human nutrition. The fruit has a non-climacteric character with a quite low rate of physiological activity. Grapes (vonoids) . More thvonoids) . AccordiBotrytis cinerea is the main biological cause of post-harvest problems since it is accountable for grey mould formation [Magnaporthe oryzae [Among other factors, fungal decay represents the principal factor responsible for post-harvest deterioration in table grapes . Botrytiormation . Indeed,e oryzae . Fungal Moreover, the infection can occur during storage, marketing, and even after customer purchase. In the vineyard, high relative air humidity and low environmental temperatures reduce the host’s defences. This environment favours the rapid spread of contamination from a single berry to the whole bunch ,7. DurinAmong the economic and social trends, attention to sustainable viticulture and organic production represents a field of high interest, as evidenced by the rising number of cultivated hectares worldwide .Nowadays, this kind of table grape cultivation is still increasing in diffusion and economic importance . The proB. cinerea in order to improve the management of post-harvest decay in table grapes and to prevent quality losses [B. cinerea decay in table grapes, including the primary physical, chemical, and biological approaches.In recent years, different strategies have been proposed to control y losses ,17,18. TB. cinerea survives at low temperatures, and any variation of temperature can promote water condensation, thus favouring fungal growth and sporulation [Physical technologies mainly include modification of several parameters such as temperature, absolute and relative gas pressure, UV irradiation, and sonication. Table grapes for fresh consumption often need a long period of storage for commercial purposes such as export and ready-to-eat. They are usually stored in chambers with strictly controlled temperature and humidity. To this aim, cold storage (~0 °C) is the primary method to avoid post-harvest infections without affecting the main physicochemical features of the product . Howeverrulation . In geneB. cinerea contamination.Surface sanitation is the main strategy implemented to control microbial contamination of fruits and it can be achieved by using different methods. Among these, dipping in hot water (about 50 °C) is an interesting option to prolong the shelf-life of fruits and vegetables ,34. Trea2 at a fixed distance of 25 cm [2), for two illumination periods of 1 min with a specific distance of 60 cm and then maintained at 20 °C for 24 h, followed by cold storage [Ultraviolet irradiation (UV) (wavelengths between 10 to 400 nanometers (nm)) and sonication by ultrasound are non-thermal treatments considered simple, reliable, and eco-friendly emerging technologies for lengthening the shelf life of fresh fruits during storage. Ultraviolet irradiation C  treatment induced a general stimulation of the phenylpropanoid pathway, associated with plant defence mechanisms, leading to an increased resistance to the diseases in artificially inoculated berries . UV-C irof 25 cm . In a re storage  demonstrB. cinerea until one week, showing a decay rate of 2% after ten days of storage at 25 °C [Few studies are reported on the use of high hydrostatic pressure and electrolyzed oxidizing water (EOW), especially on table grapes. Romanazzi et al.  investigat 25 °C . Interesat 25 °C .2)/Carbon dioxide (CO2)) aims to minimize the metabolic activity and oxidative phenomena, thus reducing the physiological decay caused by aerobic microorganisms  [2/low O2 concentrations [3 [2 concentrations >10% reportedly promote off-flavor development, rachis and berries’ browning [3) at 0.3 μL/L was assessed as the minimum concentration to significantly inhibit decay development, in artificially contaminated berries, up to seven weeks in cold storage [2-fumigated grapes [2, both in terms of sensory quality preservation and decay control. Considering that the use of massive doses of gas in a single pre-storage application can be defined as a sanitation procedure, we refer the discussion to chemical methods following section.The modification of absolute and relative gas pressure, in association with low temperatures during storage, is an important strategy to enhance the shelf life of fruits and vegetables . The maicinerea) ,39. In ttrations ,42,43, ations [3 ,42,43. H storage ,44. Reced grapes . Passived grapes . Cefola d grapes  reported2) remains the main method that is used to control the microbial spoilage of post-harvest fruit commodities. The employment of SO2 provides long term storage due to its antioxidant, antibacterial, antifungal and anti-browning properties [2 in berry peels can result in quality deterioration, such as bleached berries, production of off-flavour, or hairline disorder [2 as a pesticide, with maximum tolerance in final products of 10 ppm, and, more generally, sulphur dioxide residuals on table grapes are internationally regulated, including in the European Union [2 in the restraint of B. cinerea in table grapes  (92%), ammonium bicarbonate (NH4HCO3) (91%), sodium silicate (Na2SiO3) (89%), sodium bicarbonate (NaHCO3) (76%) and sodium carbonate (Na2CO3) (74%)  [4 could cause small black spots on the grape surface [2 generating-pads in polyethylene bags [Other chemicals are widely used as dipping solutions to sanitize fruit surfaces. The treatment of grapes by immersion or spraying with solutions of different generally recognized as safe (GRAS) salts at 1% reduced the percentage of spoiled fruit. This was the case with iron sulphate (FeSOharvest) . However surface . Disinfe surface . Neverthene bags . In thisene bags . Furtherene bags .Recently, Gorrasi et al.  demonstr2) is a gaseous disinfectant admitted in the sanitization of uncut and unpeeled fruits and vegetables. In a recent study, Chen et al. [2 during storage. The Food and Drug Administration (FDA) has approved ClO2, given that these treatments might leave chlorite residues on food products at non-hazardous concentrations [2O) is another gas tested to control post-harvest decay in fruit crops. In vitro tests did not show inhibition against grey mould; however, in vivo experiments in table grapes fumigated for 6 h showed a significant reduction in decay development during six days of cold storage [2O was indirectly able to inhibit grey mould by increasing the host′s disease resistance [In addition to ethanol vapours, other gas types have been used as fumigation treatment for the sanitization of bunches. With this scope, chlorine dioxide  is another species of interest, commonly isolated from grapevines/musts [Starm. bacillaris strains, recently isolated from these wines, denoted a significative antifungal activity, probably addressable to the release of volatile organic compounds (VOCs) [C. pyralidae Y1117 and P. kluyveri Y1125, mediated by VOC release in a closed environment, able to inhibit fungal growth for five weeks of storage [Regarding the non- surface ,121. In  surface . The add surface . Moreove surface ,123. Staes/musts ,125 and es/musts ,127. Thrs (VOCs) . The pros (VOCs)  observedWickerhamomyces anomalus BS91, M. pulcherrima MPR3, and Aureobasidium pullulans PI1 were isolated from spontaneous olive fermentation and pomegranate, minimally processed. In detail, M. pulcherrima strain showed the best antifungal activity (disease incidence (DI) = 6.7%, disease severity (DS) = 2.7%), followed by W. anomalus BS91 and A. pullulans PI1, and all of these yeasts were capable of VOC production [W. anomalus seemed to be connected to a killer phenotype [W. anomalus and A. pullulans [M. pulcherrima was probably associated with iron depletion [M. fructicola strain’s biocontrol applications for viticultural applications have been reported [In vivo studies demonstrated that grey mould can be efficiently controlled by various microbial antagonists isolated from a large variety of vegetal matrices. oduction . In parthenotype . Enzyme ullulans , whereasepletion . In the reported .Issatchenkia terricola yeasts isolated from ‘Thompson Seedless’ grapes’ surface have shown the ability to reduce decay caused by B. cinerea up to 80% compared to the untreated control [Candida membranifasciens Kh69 and Meyerozyma guilliermondii Ka21 and Kh59, while bacteria were Bacillus spp. Kh26 and Ralstonia spp. N1. All tested microbes were able to increase B. cinerea inhibition from 23.8% to 54.7%. Among these, the highest level was found for Ralstonia spp. N1(54.7%), while Bacillus spp. Kh26 and M. guilliermondii Ka21 and Kh59 showed inhibition below 50% [Epiphytic  control . In anot control . Yeasts elow 50% .Paenibacillus pasadenensis R16, isolated from grapevine cultivar ‘Barbera’, has shown a reduction in disease incidence of grey mould by 27.5% [Bacillus spp. are reported to have antimicrobial activity against several plant phytopathogens [B. subtilis QST713  and B. amyloliquefaciens FZB24 , are now available and effective against grey mould on grapes. Recently, Chen et al. [Bacillus strains, isolated from various ecological niches, to control decay development in table grapes and other fruit crops. The most vigorous antifungal activity was recorded in B. subtilis Z-14 [Still on the prokaryotic side, a bacterial strain, by 27.5% . It was by 27.5% . A largeathogens ,131,132.n et al.  demonstrlis Z-14 . VOC proRecently, there have been intense investigations conducted in the field of natural antimicrobials and their effectiveness. Many biological compounds have been tested for the bio-control of table grape spoilages. These compounds include classes of chemicals/matrices such as vegetal extracts, essential oils, and defence inducers .Cupressaceae. In a recent study [Among the vegetal compounds, volatiles generated from cellulose soaked with garlic hydro-alcoholic extract and its derived sulfur compounds have shown anti-grey mould activity in packaged table grapes both at 4 and 25 °C, during the 14 days of experimental trials . Howevernt study , no decant study .B. cinerea in artificially inoculated trials during nine days of shelf-life [Essential oils (EOs) from many plants, such as thymus and lemongrass, have revealed great potential in post-harvest disease control . In addielf-life . HoweverB. cinerea in artificially infected table grapes [Another research field involves the use of vegetal hormones, plant activators, and inner signalling molecules. These molecules act through a complex signalling network under the control of salicylic acid, ethylene, jasmonic acid, and phenylpropanoid pathways, which leads to the increase of specific secondary metabolites . Methyl jasmonate is a volatile compound that mediates stress responses in plants and has shown to promote fungal resistance in various fruit crops. Recently, it was found to be effective in lessening the development of e grapes . In thise grapes . The dise grapes .B. cinerea. After six days of incubation at 22 °C, the treatment with a solution at 20 mg/mL FA was found to be effective by reducing decay development [Fulvic acids (FA) are the soluble fraction of natural organic matter and are used in agriculture as a plant growth promoter and to control several plant diseases. Xu et al.  assayed elopment . The autelopment .Among secondary metabolites, phytoalexins are synthesized by the plants as broad-spectrum inhibitors. Stilbenoids, including pterostilbene and piceatannol, are phytoalexins commonly found in vine leaves and wine . “Mare’sSalvia fruticosa Mill. extract [Mentha piperita or M. villosa essential oil [Edible coatings made with natural polymers like chitosan or alginate can act as a cover material able to wrap the berry. Thus, these formulations can extend the shelf-life of fruit crops and maintain quality reducing water losses ,146. Chi extract  and Menttial oil  was repotial oil . HoweverPost-harvest fungal decay of fruits and vegetables is responsible for huge levels of economic loss and account consistently for large quantities of agro-food waste ,149,150.B. cinerea incidence in table grapes, adopting hurdles technology [As in other fields of food technology, an integrated management program (combining two or more different solutions) could be useful to minimize post-harvest losses caused by undesired fungal development ,154,155.chnology ,27,31,51chnology . Moreovechnology ,162,163 chnology ,165: prochnology ,169,170.


Coffea canephora), collecting missions were conducted to explore its current native distribution in Uganda over a broad climatic range. Wild material from seven forests could thus be collected. We used 19 microsatellite (SSR) markers to assess genetic diversity and structure of this material as well as material from two ex-situ collections and a feral population. The Ugandan C. canephora diversity was then positioned relative to the species’ global diversity structure. Twenty-two climatic variables were used to explore variations in climatic zones across the sampled forests. Overall, Uganda’s native C. canephora diversity differs from other known genetic groups of this species. In northwestern (NW) Uganda, four distinct genetic clusters were distinguished being from Zoka, Budongo, Itwara and Kibale forests A large southern-central (SC) cluster included Malabigambo, Mabira, and Kalangala forest accessions, as well as feral and cultivated accessions, suggesting similarity in genetic origin and strong gene flow between wild and cultivated compartments. We also confirmed the introduction of Congolese varieties into the SC region where most Robusta coffee production takes place. Identified populations occurred in divergent environmental conditions and 12 environmental variables significantly explained 16.3% of the total allelic variation across populations. The substantial genetic variation within and between Ugandan populations with different climatic envelopes might contain adaptive diversity to cope with climate change. The accessions that we collected have substantially enriched the diversity hosted in the Ugandan collections and thus contribute to ex situ conservation of this vital genetic resource. However, there is an urgent need to develop strategies to enhance complementary in-situ conservation of Coffea canephora in native forests in northwestern Uganda.Wild genetic resources and their ability to adapt to environmental change are critically important in light of the projected climate change, while constituting the foundation of agricultural sustainability. To address the expected negative effects of climate change on Robusta coffee trees ( Coffea species, i.e. Coffea arabica and C. canephora [C. canephora exports and the whole coffee sector provides a livelihood for about 8 million people [C. canephora production is thus of major national and global importance, particularly for smallholder farmers [Coffee is a major global commodity and the total value of its industry was estimated to surpass US$200 billion in 2017 [n people . The sus farmers . Unfortu farmers ,6. A glo farmers .C. arabica and C. canephora [The ability of crops to adapt to environmental challenges, such as the effects of climate change, depends on the extent of genetic variation within the crop. The resilience potential of all crop production systems is anchored by the intraspecific trait diversity that has evolved in the species’ natural habitat . There hanephora ,15. The Coffea canephora is a diploid (2n = 2x = 22), self-incompatible [C. canephora natural distribution range in Africa stretches from Guinea in the west to Uganda in the east and Angola in the south [Coffea species, C. canephora has the widest distribution and climatic range e.g rainfall levels differ by 3-fold across the species’ distribution range. Development of molecular markers such as SSR (single sequence repeat), RFLP (restriction fragment length polymorphism) and RAPD (random amplification of polymorphic DNA) markers, or more recently SNPs, derived from DNA sequencing has enabled researchers to further understand the phylogenetic relationships between different Coffea species [C. canephora populations [C. canephora diversity has been greatly facilitated by the availability of a large repository of simple sequence repeat (SSR) based microsatellite markers, which provides efficiency and high-resolution in genetic analyses [C. canephora is linked to the geographical location [C. canephora diversity was pooled within two different genetic groups, namely: (i) a Guinean group including wild populations from Côte d’Ivoire, and (ii) a Congolese group consisting of accessions from the Central African Republic and Cameroon [C. canephora genetic diversity into five genetic groups . Geographically, genetic group A comprised wild populations from Congo and Cameroon, group B from eastern-central Africa, group C from western-central Africa, Cameroon and northeastern Congo, group E from Congo and southern Cameroon, while group D consisted of wild populations from Côte d’Ivoire and Guinea. Musoli et al. [C. canephora genetic diversity with eight distinct genetic groups, including the Ugandan one (group O), thus identifying two new genetic groups, i.e. R (comprising samples from southern Democratic Republic of the Congo) and G (comprising samples from Angola), whereas the differentiation between groups E and R was weaker  determine the level of genetic diversity, population structure and genetic relationships between wild, feral  and cultivated C. canephora genotypes, (ii) position Ugandan C. canephora diversity relative to the species’ global diversity structure, and (iii) identify the relationship between the genetic structure of Ugandan C. canephora wild populations with their climatic profiles.Uganda lies within a dry geographical area of the ribed by . Wild Ugished by , who repC. canephora populations. The hierarchical sampling strategy described by [C. canephora distribution in these three distinct climatic zones. Wild samples were collected in 2015 and geo-referenced from seven natural forests: Zoka, Budongo, Itwara, Kibale, Mabira, Malabigambo and Lutoboka central forest reserve on Kalangala main Island  in Kituza and the Plant Genetic Resources Centre (PGRC) in Entebbe. In addition, individuals described as feral  were collected from Kalangala main Island, Bunyama and Bugala islands of Kalangala district in Lake Victoria. The feral samples were collected from six different sub-sites separated by 10 km and located at least 0.5–2 km from the edges of the Islands. The Kalangala study site thus included both wild and feral populations. The cultivated set of individuals was represented by a total of 52 samples collected from the two germplasm field collections of the Ugandan National Agricultural Research Organization (NARO): 32 samples from the National Coffee Research Institute Kituza and 20 samples from the National Agricultural Research Laboratories at Kawanda. The cultivated genotypes were selected on the basis of their historical and passport data with the aim of representing the total range of traditional and commercially cultivated C. canephora diversity, including the two predominant forms found in Uganda: Erecta, or upright forms, and Nganda, or spreading forms [C. canephora accessions within the overall species diversity by comparing them with a representative set of wild accessions from the C. canephora diversity groups, as previously defined with SSR markers or the SNP array [Based on annual precipitation records, Uganda was broadly categorized in five distinct climatic zones –34, nameribed by  was appln Island . These fng forms  and the NP array ,23 (S2 TC. canephora genotypes. The multiplex PCRs were carried out using the QIAGEN Type-it® Microsatellite PCR kit (Ref 206246), in a 25-μL reaction mixture containing the “Master Mix”, 50 ng template DNA, 0.2 μM of each reverse and fluorescence-labeled primers. The PCR amplification was performed using the following cycling conditions: initial denaturation at 95°C for 5 min, followed by 28 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 1.5 mim, and extension at 72°C for 30 s, followed by a final extension at 60°C for 30 min. Subsequently, ABI 3500 XL sequencer  was used to analyze the PCR products, and their sizes (bp) were determined using GeneMapper® version 6 . The PCR amplification conditions and all information on the markers are also given in MoccaDB, an integrative database for functional, comparative and diversity studies on the Rubiaceae family (http://moccadb.ird.fr/ [http://coffee-genome.org/ [Silica-dried leaves from the 269 study samples were ground in liquid nitrogen with a mortar and pestle. DNA was extracted using the DNeasy® Plant Maxi kit (QIAGEN), with few adjustments to tailor it for dry leaf material. Extractions were performed by adding double amounts of both AP1 and P3 buffers. Nuclear genetic variation was assessed at 19 SSR loci  using ma.ird.fr/ ,36) and ome.org/ ).o and He) and inbreeding coefficient  were estimated for each sampled locality using GenAlEx software version 6.5 [st, [Within-population indices of genetic diversity such as the number of alleles (Na), number of effective alleles (Ne), number of private alleles , observed and expected heterozygosity .The genetic structure of the diversity was analyzed using the Bayesian clustering method of the STRUCTURE version 2.3.4 software package. This model-based clustering method uses a Bayesian approach to detect underlying genetic (sub)-populations within a group of individuals that are genotyped with multiple markers . FurtherTo investigate the genetic relationships between accessions, either at the species level  at 30 arc-second resolution [AI = aridity index, MAP = mean annual precipitation, MAE = mean annual potential evapotranspiration.andPET = potential evapotranspiration, RA = mean monthly extraterrestrial radiation, Tmean = mean monthly temperature, TD = mean monthly temperature range.Twenty-two environmental variables were retrieved for each georeferenced wild sample and used as predictors to characterize the climatic characteristics of each study site. Nineteen out of the 22 environmental variables were taken from a global climate database , and the three others were: elevation, aridity index (AI) and potential evapotranspiration (PET: a measure of the ability of the atmosphere to remove water through evapotranspiration) . These vsolution . Elevatisolution . AI valusolution . PET valsolution . The fort-tests with no assumption of equal variances to determine if the mean differences between specific pairs of locations were statistically significant. Moreover, Pearson correlation coefficients were calculated to test the relationship between all of the studied environmental variables. All tests were deemed significant at p ≤ 0.05 and the analysis was performed with R software version 3.5.0 [Since the locations had unequal sample sizes, we performed Welch’s one-way tests to assess whether there were significant differences in the means of the selected environmental variables across locations. We subsequently performed pairwise on 3.5.0 .p ≤ 0.01.A principal component analysis (PCA) was performed on the 22 above-mentioned environmental variables to describe the variation in environmental conditions across sites. Furthermore, a redundancy analysis (RDA) was conducted to investigate the amount of genetic variation that could be explained by the environmental conditions at the study sites . Then foC. canephora native populations was examined at the 19 SSR loci and in the 191 C. canephora accessions collected from wild populations . In contrast, the population from Mabira had the highest allelic richness (Rs = 4.75) and heterozygosity (He = 0.62) among the wild populations. Notably, populations from Zoka and Budongo, with a relatively high allelic richness  and expected heterozygosity (He = 0.58 for both), appeared to be relatively unique among the study populations. They had the highest average number of private alleles across all loci  compared to Itwara, Kibale or Malabigambo with all less than 0.1.The genetic diversity in Ugandan ulations . An averC. canephora wild genotypes were generally classified into clades according to their Structure clusters, with a clear separation between individuals from the four NW forests and those from SC forests. Furthermore, individuals from the four different NW forests  clustered according with their “forest” counterparts. Few intermixed clusters of individuals from Budongo and Zoka were observed, thus revealing probable gene flow and admixture between adjacent Budongo and Zoka populations. Accessions from SC forests  were spread within a large clade, suggesting population intermixing within the region.Clustering analysis findings for the whole set of native populations clearly revealed that the native diversity broadly differentiated a large group with the accessions from the SC forests  and four forests in northwestern (NW) Uganda  . In the st) values  to 0.18 (differences between Zoka and Itwara), while those for the SC forests, ranged from 0.02 to 0.05. The Fst values indicating the differentiation between each of the four NW forests and other forests, ranged from 0.08 to 0.22. The genetic diversity in Ugandan C. canephora native populations is thus structured into five main genetic groups: Zoka, Budongo, Itwara and Kibale populations in northwestern Uganda and the SC group in southwestern Uganda.This observed genetic structure was supported by the population divergence (F) values . These ve = 0.63). However, they presented no or few private alleles  forests Figs  and S3. C. canephora genotypes were analyzed together with individuals from seven other diversity groups , suggesting their close genetic relationship together with their geographic proximity in northern region of Uganda (C. canephora diversity were evident amidst other genetic groups (When Ugandan ribed by ), they cf Uganda . In addic groups .C. canephora populations were distributed in forests with significantly varying environmental conditions, as exemplified by five environmental variables including the two independent annual bioclimatic variables  -BIO1 and annual precipitation (mm) -BIO12) , which was 4.7°C higher than that of the Itwara the population with the lowest BIO1. Similar BIO1 patterns and elevation values were observed across locations since both were correlated (p< 0.05) (Uganda’s native  -BIO12) . The C. p< 0.05) . For preAnnual precipitation differed significantly across locations, except between Itwara-Malabigambo and Zoka-Kibale . PotentiWhen all climatic variables were analyzed simultaneously through a principal component analysis (PCA), the populations in Zoka, Budongo, Kalangala, Mabira and Malabigambo occurred in distinct climatic envelopes, while the climatic envelopes in Itwara tended to overlap those of Kibale . In thisp<0.001) (i.e. the mean of monthly (max temperature—min temperature) (BIO2) and PET, while being positively correlated with precipitation-related variables, e.g. precipitation of the driest quarter (BIO17), precipitation in the driest month (BIO14) and precipitation of the wettest month (BIO13). The second axis (RDA2) explained 3.33% of the total genetic diversity and was mainly positively correlated with three temperature-related variables, i.e. mean temperature of the wettest quarter (BIO8), mean temperature of the coldest quarter (BIO11), annual mean temperature (BIO1), and negatively correlated with precipitation in the warmest quarter (BIO18). RDA2 differentiated the genetic diversity of populations occurring in warmer zones (Zoka and Budongo) from others. Interestingly, the RDA projection structured individuals according to their forest of origin for northwestern (NW) populations , but individuals from southern-central (SC) populations  all overlapped in the two first axis spaces. However, the low proportion of constrained variance, i.e. 16.3% of the total genetic diversity, suggested that there are also other factors affecting genetic diversity and structure of C. canephora in the region.Comparisons between pairwise individual genetic distances and their geographical distances showed a significant but weak isolation by distance effect (R = 0.347 with p<0.001) . Meanwhip<0.001) . The firC. canephora populations was not present elsewhere in the C. canephora distribution range. The studied material constituted a well-differentiated genetic group, as previously suggested by [Our results showed that the genetic diversity found within Ugandan wild ested by  who studC. canephora populations in these forests compared to that previously described by [Forest populations from the SC cluster had higher allelic richness than those from the NW forests, except Zoka forest. They however constituted a large intermixed genetic group, which could possibly be attributed to a high level of gene flow between nearby forest and plantations. The relatively low allelic diversity that we found for the western Ugandan material might be due to restricted gene-flow between nearby populations isolation by fragmentation, genetic drift and bottleneck caused by anthropogenic disturbances in these forests, or selective mortality under local stress event. This factors could potentially also explain the significant genetic divergence noted for Itwara and Kibale populations and seed dispersal (via primates or bats) of the species [C. canephora plants from SC forests for cultivation use, led to the spread of seeds and/or cuttings over large distances within the C. canephora cultivation areas of southern-central Uganda [Most cultivated germplasm of Uganda seem to have originated from the southern-central forests  see . There imaterial ,35,52. T species ,18 mightl Uganda  that somC. canephora populations is well-distributed in both wild and cultivated accessions.The relative contributions of the aforementioned factors, i.e. (i) use of wild material by farmers, (ii) use of introduced material from abroad, and (iii) natural gene flow between populations are hard to assess. However, when we compare the genetic diversity indices between the SC wild accessions (He = 0.62) with wild accessions from different regions of Africa pooled together (He = 0.61 according to ), they aC. canephora offspring or accessions introgressed with cultivated material. Indeed, local feral individuals, formerly cultivated material returned to the wild, were related to the individuals from the SC group. We found a much greater genetic similarity between the wild and cultivated accessions than Musoli et al. [We expected that wild samples from Kalangala would show a substantial level of originality due to the genetic material collected from the Lutooboka central forest reserve . Howeveri et al. . This miC. canephora populations occurred across a broad environmental range, except for Kibale and Itwara whose climatic envelops tended to overlap  and high productivity. In this study, all six cultivars were shown to be genetically similar to SC wild populations. Our results showed that the genetic diversity in NW forests was distinct from that of the cultivated material and that NW populations occurred in comparatively contrasted climatic zones, thus highlighting a new source of genetic diversity. This unexplored genetic diversity could be utilized in coffee breeding programs to improve the resilience of cultivated material to various adverse effects of climate change, e.g. drought, temperature, pests and diseases.Ugandan C. canephora diversity and genetic structure and the location of the populations in distinct environmental habitats, there is need to devise an efficient complementary conservation strategy that allows for the in-situ and ex-situ conservation of Ugandan coffee genetic resources. As shown earlier, some populations like those of Itwara and Kibale are small spatially isolated populations which could thus be more susceptible to ecological or genetic disturbance [in-situ and ex-situ conservation of these populations is especially important. Conservation strategies should promote the prevention of deforestation and associated habitat destruction in these areas. For ex-situ conservation, our study enriched the diversity conserved by the National Agricultural Research Organization (NARO) at the National Coffee Resources Institute (NaCORI) in Kituza and the Plant Genetic Resources Centre (PGRC) in Entebbe. Our study material was safety duplicated to enhance the ex-situ conservation of these resources and provide material for other related studies geared towards understanding the potential offered by Ugandan coffee genetic diversity.In the light of the substantial level of Ugandan turbance . In our C. canephora populations thrived in contrasting environmental conditions and that the genetic structuring of wild populations was divided into five main environmentally and geographically bounded groups, i.e. the four northwestern forests  and the southern-central group, mainly comprising wild populations from Malabigambo, Mabira and Kalangala, and the cultivated collection material. We also demonstrated that the distinction between populations was partly correlated with the climatic differences in their habitats, suggesting that these populations might present an adaptive potential useful for breeding climate change resilient material. Most importantly, our study showed that the cultivated and current elite genotypes predominantly belonged to the same genetic group (SC group), while material from the NW group  contained genetic material that has not yet been utilized but which could be very useful for coffee improvement in the current climate change setting. Ex-situ conservation strategies must be developed very quickly and the material already collected in the national collections should be evaluated in more detail, in particular for its physiological capacities to cope with the adverse effects of climate change, e.g. drought, higher temperatures or disease pressure [In conclusion, our study revealed that Ugandan pressure .S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.


Natural and mass selection during domestication and cultivation favored particular traits of interest in barley. In the present study, population structure, and genetic relationships among 144 accessions of barley landraces and breeding materials from various countries were studied using a set of 77 and 72 EST-SSR and gSSR markers, respectively distributed on seven chromosomes of barley. In total, 262 and 429 alleles were amplified in 77 EST-SSRs and 72 gSSR loci, respectively. Out of which, 185 private/group-specific alleles were identified in the landraces compared with 14 in "cultivar and advanced breeding lines", indicating the possibility to introgress favorite alleles from landraces into breeding materials. Comparative analysis of genetic variation among breeding materials, Iranian landraces, and exotic landraces revealed higher genetic diversity in Iranian landraces compared with others. A total of 37, 15, and 14 private/group-specific alleles were identified in Iranian landraces, exotic landraces, and breeding materials, respectively. The most likely groups for 144 barley genotypes were three as inferred using model- and distance-based clustering as well as principal coordinate analysis which assigned the landraces and breeding materials into separate groups. The distribution of alleles was found to be correlated with population structure, domestication history and eco-geographical factors. The high allelic richness in the studied set of barley genotype provides insights into the available diversity and allows the construction of core groups based on maximizing allelic diversity for use in barley breeding programs. Hordeum vulgare L. subsp. spontaneum in the Near East about 10,000 years ago3. Due to the wide adaptation of barley to various environmental conditions, its germplasm pool with high genetic diversity has the potential to breed for adaptation to different environmental conditions. However, genetic diversity in modern barley cultivars has been declined following intense selection in modern plant breeding programs. Therefore, characterization and the use of barley germplasm arrays such as landraces are essential for identifying candidate genes for important traits6.Barley is one of the oldest known domesticated crops with a large genome and a high number of varieties and accessions worldwide. Archaeological and molecular studies indicate that it was domesticated from two-row wild barley 8. Due to heterogeneous nature of the landraces, huge genetic diversity is structured at the field and farmer levels (between and within populations). Morphological and agronomic traits, as well as molecular tools, have been utilized to assess components of the genetic diversity in landraces10.Domestication and human selection in subsistence agriculture over a long period resulted in the development and evolution of landraces with high adaptation to the local natural environments. In such systems, natural selection, seed exchange, and human migration generated a huge genetic variation which has been maintained by farmers11. Replacement of highly adapted landraces by modern varieties performing under optimal conditions but failing under harsh environments12 resulted in the disappearance of most landraces of crop plants from practical farming13. Although in many countries, landraces were completely replaced by modern varieties starting from a long time back, in some marginal areas, to take advantage of the specific adaptations of landraces to the agro-ecosystem, their cultivation still continues14. The landraces of self-pollinated species such as barley harboring the chromosomal regions consisted of blocks of genes with a low frequency of recombination which may confer a specific adaptation to specific environments15. This shows that the landraces are ideal association panels to identify genes controlling adaptive traits in crop species16. Therefore, for efficient utilization of barley landraces in breeding programs and to develop the strategies for their optimal conservation, it is necessary systematically to evaluate molecular diversity encompassed in the landraces collections17. Among the DNA markers, SSR markers have been extensively use to analyze population structure and genetic diversity in barley germplasm, due to the high polymorphism, reproducibility, co-dominant and multi-allelic nature when compared to most of the marker systems22.Modern plant breeding activities in the recent century resulted in the development of a large number of elite varieties with a higher yield, better quality, and resistance to stresses, but the narrow genetic backgroundThe present study was carried out to (i) characterize the allelic diversity, and population structure in a collection of 119 barley landraces from Iran and across the world including two-row and six-row types with different growth habits and compared with those of barley cultivars and advanced breeding lines and (ii) compare the efficiency of genomic and EST- SSR markers in assessing genetic relationships and population structure.Genetic relationships in a set of 144 barley genotypes were investigated using 72 gSSR and 77 EST-SSR loci distributed at seven barley chromosomes. The interval spanning between markers used varied from 5.80 cM in 4H to 8.10 cM in 6H with an average of 6.93 cM. The number of alleles/locus varied from 3.69 (5H) to 6.04 (4H) and PIC ranged from 0.44 (2H) to 0.62 (4H)  origins and breeding history, (ii) growth habits and (iii) the number of ear rows. Genetic diversity parameters for these groups are presented in Table The mean values of Na, Ne, I and uHe estimated based on EST-SSR data were slightly higher in winter growth habit  than spring growth habit , but significantly higher than facultative genotypes  . A total of 14, 3 and zero EST-SSR private alleles were detected in winter, spring and facultative growth habits, respectively. Higher diversity was observed among the genotypes with various growth habits using gSSR markers compared with EST-SRRs. The mean values of Na, Ne, I, uHe and Npa in winter growth habit genotypes were 5.64, 3.85, 1.32, 0.65, and 44, respectively, compared with 5.25, 3.10, 1.12, 0.55 and 17, respectively, in spring growth habit and 3.39, 2.36, 0.86, 0.48 and zero, respectively, in facultative genotypes.Considering ear row number, mean Na, Ne, I, uHe and Npa of EST-SSRs in two-row genotypes were 3.21, 1.94, 0.72, 0.411 and 8, compared with 3.29, 2.22, 0.84, 048 and 14, respectively in six-row genotypes. The mean of these parameters for gSSRs were 5.37, 3.06, 1.10, 0.54 and 13, in two-row barley and 5.58, 3.77, 1.31, 0.65 and 41, respectively in six-row barley. The analysis revealed slightly higher genetic diversity in six-row genotypes compared with two-row genotypes, but the difference was only significant for Npa. However, allelic diversity calculated based on gSSR data was higher in both groups compared with EST-SSRs.In the model-based clustering using SSR, EST-SSR, and combined data sets, the log-likelihood value [LnP(D)] increased continuously as K changed from 1 to 10, but inflection was evident when K increased from 2 to 3  compared with spring genotypes  and facultative . Genetic diversity based on ear row type revealed no major differences in the number of different alleles per locus and the effective number of alleles found in the two-row genotypes ,  compared with the six-row genotypes , . But the genetic diversity as revealed by Shannon's index and unbiased expected heterozygosity was higher in the six-row genotypes  and  compared with the two-row genotypes  and . The six-row genotypes had also a higher number of private alleles  compared with the two-row Genotypes .23. Besides, to achieve future production under changing environments, greater genetic diversity than that is present in current elite germplasms will be needed24. Fortunately, an extensive reservoir of biodiversity has been stored in genebanks as seeds of historical breeding materials, locally adapted landraces, and crop wild relatives which could be used to enrich cultivated gene pools. Although the ease of mobilization of favorable alleles into breeding materials is inversely related to the degree of adaptation, advances in genomics and molecular breeding technologies can accelerate the use of exotic germplasm for crop improvement25. However, access to novel allelic combinations available in the genebank collections requires thoughtful and renewed genotypic and phenotypic characterization of the materials6.Knowledge of population structure and genetic relationships among the genotypes is a prerequisite for plant breeding programs as well as for emerging genome-wide association studies (GWAS) as an alternate approach for QTL detection in comparison to linkage map-based QTL analysis28.In the present study, a panel of 144 barley genotypes (80 two-rowed and 64 six-rowed) including 69 landraces from various regions of Iran, and 50 landraces from other countries mainly from China and Egypt along with 9 advanced breeding lines and 16 commercial cultivars were genotyped using 149 SSR markers. This set of SSR markers amplified 691 alleles in the genotype under study and could provide a reasonable genotypic tool as a result of relatively high allele number, informativeness, and genomic coverage. The mean number of alleles/locus (4.64), gene diversity (0.55) and PIC (0.50) indicate a relatively high level of genetic variation in our study compared with some previous studies. An average of 3.20, 4.32, and 8.10 alleles/locus and the mean PIC of 0.38, 0.52, and 0.60 were reported in the analysis of diverse germplasms including wild and cultivated barley using SSR markers29. The presence of 17 SSR loci with PIC value of 0.80 or a higher and high number of alleles/locus could provide unique fingerprints for the studied barley genotypes and used for rapid analysis of genetic diversity and population structure in barley germplasm collections. A total of 691 polymorphic alleles detected in our study were enough to assess genetic relationships among barley landraces and cultivars. It was concluded that the presence of 350–550 alleles is enough for objective assessment of the genetic relationship between wheat accessions and it could be applicable for barley accessions as well31.The number of alleles/locus and PIC values have been suggested as a criterion to assess the level of genetic diversity in germplasm collections28. In our study, significant differences were observed between landraces and improved genotypes in number of alleles, number of effective alleles, number of private alleles, and gene diversity. On the other hand, the number of private alleles was 185 for the landraces while it was only 14 for cultivars and advanced breeding lines. It shows that modern breeding programs reduced the level of genetic variation in breeding materials. However, in our study, the number of landraces (119) was higher than that of cultivars and advanced breeding lines (25), but low level of genetic variation in the modern cultivars and breeding materials indicates the need of introducing new alleles from sources such as landraces and wild relatives to tackle climate change through plant breeding and better use of plant genetic resources. Compared with exotic landraces and "cultivars and advanced breeding lines", Iranian landraces had a higher level of gene diversity as revealed by Na, Ne, He, and number of private alleles. Significant differentiation assessed by FST (P = 0.001) was observed among the three groups. The number of private alleles in Iranian landraces  was also higher compared to exotic landraces  and “cultivars and advanced breeding lines” . Although Iranian landraces contained slightly more individuals compared with exotic landraces and “cultivars and advanced lines” (69 vs 50 and 25), this was consistent with the higher genetic variation in the Iranian landraces as revealed by number of alleles, number of effective alleles, gene diversity and Shannon’s index 35; Bmag382 36; Bmag518 37, chlorophyll content38, septoria speckled leaf blotch39, plump grain and 1000 grain weight40); Bmag740 (adult spot blotch and net type net blotch39 and days to maturity41); Bmag751 (carbon isotope discrimination and grain yield41); EBmac541 (dehydration gene for drought tolerance42); GBM1482 ; GBMS141 (Endosperm hardness40); GMS89 (grain yield26); HVM40  and HVM74 45. In our studies, with K = 2, two- and six-rowed genotypes were assigned into distinct sub-populations. With an optimal number of clusters K = 4, except cluster III, two- and six-rowed genotypes were grouped separately. Cluster I, II, and IV consisted of 89%, 88% and 74% six-row, two-row, and two-row accessions, respectively. Cluster III is structured by 45% two-row and 55% six-row accessions. Our observations showed that Iranian barley landraces are different in comparison to the exotic landraces and mostly grouped separately. This could be due to the different evolutionary and domestication history of barley from Iran offers a plausible explanation for the observed differences33.We employed various statistical approaches to ensure the reliability of the inferences made regarding population structure in the collection. Although the results of model-based clustering were further ascertained by the results from the distance-based cluster analysis Figs. , 2, 3 an32.To further explore the genetic diversity and relationships among the clusters and within the clusters, allelic pattern and various diversity statistics were assessed for each of the STRUCTURE inferred clusters Table . The cluIn the present study, we utilized microsatellite markers to assess the effect of breeding history, origin, and growth type on genetic diversity and population structure of barley genotypes. Assigning of Iranian landraces, exotic landraces and "varieties and advanced breeding lines" into separate groups based on both gSSR and EST-SSt data could be due to their different evolutionary, domestication, and breeding history. The higher genetic diversity in Iranian landraces revealed by high allelic diversity and private alleles number could be due to the fact that a second domestication event may have occurred in barley, possibly in Central Asia at the eastern edge of the Iranian Plateau. The allelic diversity presented in the studied collection, especially in Iranian landraces was found to be correlated with population structure, domestication, and eco-geographical factors. The high allelic richness in the studied set of barley genotype as revealed by genetic and statistical analyses provides insights into the available diversity and allows construction of core groups based on maximizing allelic diversity for use in Iranian barley breeding programs. The Iranian landrace panel comprised lines that were well distributed across all eco-geographical regions with different climatic regimes which could be used for introgression of favorite alleles into breeding lines.A set of 144 barley genotypes (80 two-rowed and 64 six-rowed) including 69 landraces from various regions of Iran, 23 from China, 12 from Egypt and 15 from the USA, England, India, Pakistan and Algeria along with 9 advanced breeding lines and 16 commercial varieties was used. Out of 144 genotypes, 61, 69, and 14 were spring, winter, and facultative growth habits, respectively. The majority of six-row and two-row barley genotype were winter types   agarose gel electrophoresis and spectrophotometer were used to examine the quality and quantity of the DNA samples, respectively. Each genotype was analyzed using a set 77 and 72 polymorphic EST-SSR and gSSR markers, respectively distributed on seven chromosomes of barley. Bin locations of the markers were adapted from Grain Genes Map Data Report: Barley, Steptoe × Morex, SSR (https://wheat.pw.usda.gov/cgi-bin/graingenes/report). The PCR reaction mixture was prepared in a final volume of 10 μL containing 40 ng template DNA, 1 × PCR buffer, 0.025 units Taq DNA polymerase , 0.2 mmol dNTPs, and 0.8 pmol forward and reverse primers. Amplifications were performed under the following conditions: 4 min at 94 °C, followed by 35 cycles at 94 °C for 1 min, primer annealing at 58–68 °C for 1 min depending on primers and 72 °C for 1 min, and a final extension at 72 °C for 7 min. A Gel-Scan 3000 electrophoresis system  based on 4% ultra-thin (0.2 mm) non-denature polyacrylamide gel stained by ethidium bromide was used to visualize PCR products.Genomic DNA was extracted from bulked fresh leaf samples of each genotype using the CTAB method49 and GenAlEx 6.5 software50. Polymorphism Information Content (PIC) values were determined as 51. Gene diversity was calculated as 52. Shannon's information index was estimated as pi and pj are the frequencies of the ith and jth alleles of a given locus, respectively.Number of allele (Na), number of effective allele (Ne), major allele frequency (MAF), Shannon's index (I), gene diversity (He) and polymorphic information content (PIC) were determined for all the analyzed markers across the total population as well as landraces and "varieties and advanced breeding lines" using PowerMarker version 3.2553 was performed by running 100,000 Markov chain Monte Carlo (MCMC) iterations after a burn-in of 100,000 replicates with 5 independent runs per K ranging from 1 to 10, using the admixture model with correlated allele frequencies, which estimates fractions of individual genomes that belong to different ancestry groups. The optimal number of cluster (K) was determined by calculating the mean posterior probability for each K value, LnP(D), which is based on the estimated maximum log-likelihood values53. The ΔK values  was also calculated as suggested by54 using STRUCTURE-HARVESTER, version 0.6.9455.Population structure in a collection of 144 barley genotypes including Iranian and exotic landraces, advanced breeding lines, and cultivars was assessed by two statistical methods. The model-based clustering implemented in STRUCTURE 2.3.427. Finally, the unrooted neighbor-joining (N-J) clustering algorithm under the Reynold 1983 distance coefficient was applied using the software PowerMarker 3.2526 to investigate the relationship of barley accessions.We used principal coordinate analysis (PCoA) for genetic differentiation between two- and six-row genotypes, among spring, winter, and facultative genotypes as well as Iranian landraces, exotics landraces, and “varieties and advanced breeding lines. The PCoA was performed on GenAIEx 6.501 software27 was used the partition of the observed molecular variation among and within the three clusters inferred using STRUCTURE and NJ cluster analyses corresponding to Iranian landraces, exotic landraces and "cultivars and advance breeding lines". The genetic parameters including the number of a different allele with a frequency ≥ 0.05, the number of effective alleles, Shannon's index (I), and unbiased expected heterozygosity (uHe = (2N/(2N − 1)) × He) were estimated for each cluster, where N is group size.A hierarchical analysis of molecular variance (AMOVA) implemented in GenAlex 6.501


Ae. aegypti, the main vector of dengue disease, among 10 collection sites including major ports and inland cities using variations at 11 microsatellite loci. Discriminant analysis of principal components (DAPC) and k-means clustering estimated eight genetic clusters. Analysis of Molecular Variance (AMOVA) estimated equal variances among cities and among collections in Colombo, Sri Lanka. Significant evidence, although weak, was detected for isolation by distance. Analysis of gene flow rates and directions using MIGRATE-n indicated that populations throughout the island served as a source of immigrants for Colombo with abundant gene flow among major commercial cities in Sri Lanka, which appear to receive migrant mosquitoes from throughout Sri Lanka. The observed patterns probably arise through human movement of Ae. aegypti during commerce from throughout Sri Lanka into Colombo increasing the risk of spread. The patterns uncovered in this study are significant for global health as Sri Lanka is situated along a key international shipping route.In Sri Lanka, dengue is the most serious arboviral disease. Recent increases in dengue cases suggest a higher infection rate and spread of the disease to new areas. The present study explores gene flow patterns of  Aedes aegypti, which has been on the rise over the past 50 years with a 30-fold increase in global incidence [Aedes aegypti is an invasive mosquito species originated in Africa and has colonized tropical and subtropical regions of the world during the last 400–500 years with the help of human movement and trade [Viruses transmitted by mosquitoes are a primary health concern driven by multiple factors leading to the loss of human life due to the increased incidence of mosquito-borne diseases globally . Dengue ncidence ,2. AedesSri Lanka is a tropical island situated in the Indian Ocean. The island is situated along a key international shipping route between the Malacca Straits and the Suez Canal. Approximately 36,000 ships, including 4500 oil tankers, use the route annually [In Sri Lanka, DF is considered a serious arboviral disease with progressively larger epidemics over regular periods of time . AlthougAe. aegypti and infected humans from Southeast Asia to the Middle East and Africa. A recent study assessing the genetic relationships of the Sri Lankan population of Ae. aegypti relative to other global collections revealed Sri Lankan mosquitoes cluster with other Asian collections [While DF is a significant public health threat in Sri Lanka, these patterns would have little significance to global health if not for the fact that Sri Lanka is situated along a key international shipping route. Thus, Sri Lanka may be playing a major role in the trafficking of DENVs, its vector lections . Among Alections  have alslections .Ae. aegypti within Sri Lanka gives an understanding regarding the population movements, the flow of resistant genes and new epidemics that could occur.Knowledge of dispersal of a species gives insight into the gene flow levels and the adaptability of that particular species to a new area. Thus, understanding the genetic structure of Aedes aegypti were also collected in three inland sites. Jaffna  and single collections from Jaffna, Galle, Hambanthota, Puttalum, Trincomalee and Kandy . Colombo. Jaffna  is the c. Jaffna . Puttala. Jaffna  is an inAe. aegypti in Sri Lanka. The main aim of the present study was to analyze the genetic structure and gene flow of the populations of Ae. aegypti collected from four major ports cities and three inland study sites in Sri Lanka.The current study analyzed the gene flow patterns and the population genetic structure of Ae. aegypti were collected from 2013 to 2015 from seven districts . The polymerase chain reaction (PCR) was carried out in an Eppendorf® Thermo-cycler in 25 µL reaction volumes. Amplification conditions consisted of initial denaturation at 95 °C for 15 min followed by 35 cycles of 94 °C for 30 s, 55 °C for 90 s and 72 °C for 90 s and a final extension step at 72 °C for 1 min. PCR products were sent to Macrogen Inc. Korea for fragment analysis. The results obtained were genotyped using GeneMapper™ Software 5 with GeneScan™ 1200 LIZ™ dye Size Standard . All loci were polymorphic.Eleven previously published nuclear microsatellite loci AT1, AG7, AC1, AG5, AG4, AC2, AG2, AC5 , B07, H0o and He), allelic richness and the Garza–Williamson (GW) Index [The observed and expected heterozygosities, (HW) Index  were estW) Index . This soW) Index . The allW) Index . k-means clustering. The lowest Bayesian Information Criterion (BIC) was used to infer the ideal value of K. The first two principal components of the DAPC were plotted to evaluate the relationship among clusters. A cross-validation function  was used to confirm the correct number of principal components (PC) to be retained.The population structure was analyzed by a principal component analysis (PCA) and discriminant analysis of principal components (DAPC) using adegenet 2.1.1  for R vePopulations, which have experienced a recent reduction of their effective population size, exhibit a correlative reduction in allele numbers and heterozygosities at polymorphic loci. Data were analyzed for evidence of a bottleneck using the program (BOTTLENECK version 1.2.02) . To deteem values estimated in the first and second replicates to test for the consistency of estimates from the two replicate runs. The same was done with θ. The immigration parameter M (not m) among populations and θ within populations were estimated using maximum likelihood in Migrate-n 3.5.1 [Migrate-n was used to estimate two parameters: the effective migration rate (4NeM) and θ = 4Neµ. Two replicate maximum likelihood runs were made in MIGRATE-n 3.5.1 . As reco-n 3.5.1 . The effe) exceeded the observed heterozygosity (Ho) in eight of the 10 loci ) and the natural log (geographic distance) using a Mantel test   when excluding the Colombo populations that were close to one another (ARLEQUIN was used to test the correlation between the linear genetic distance (Ftel test . A posit 0.0001) A. This r another B.k-means clustering show that Clusters 1, 3, 6 and 8 contained individuals from many (8–10) collection sites. In contrast, Clusters 2 and 4 contained primarily mosquitoes from Trincomalee while Cluster 7 consisted almost exclusively of Puttalum individuals. Cluster 5 consists primarily of individuals from Galle, Hambanthota and Kandy , as were values of θ  . Mixed gAedes aegypti have a close association with humans and are very well adapted to anthropophilic environments. Passive migration of Ae. aegypti to new geographical areas via human activities is a frequently observed scenario. Aedes aegypti have been reported in Sri Lanka since the 1930s [he 1930s  and thisAe. aegypti populations within Sri Lanka to investigate gene flow and the population genetic structure. Our results indicate that the populations throughout Sri Lanka are less genetically differentiated with the exception of the Kandy population. PCA and DAPC analysis revealed low genetic structure among the populations studied. The effective migration rate between other cities and Kandy is limited with the exception of moderate gene flow directed toward Colombo, the main commercial capital of Sri Lanka. Although continuous tourist and business activities take place between Kandy and many cities, Kandy reveals an isolated population. Kandy is geographically isolated due to location in the central mountain range in the country. The low temperatures in the mountains may also have played a role in the life cycle of the passively transported mosquito eggs [The present study is the first to analyze multiple ito eggs ,27.Aedes aegypti mosquitoes have a limited active flight range but can move between 10 and 800 m around their larval habitats [Ae. aegypti mosquitoes in the form of eggs and larvae via tire trade [Aedes mosquitoes [Ae. aegypti populations were connected with human-aided passive migration of the mosquito. habitats ,29. It hhabitats ,35,36,37re trade . This stre trade . Severalsquitoes . Weak issquitoes . A studyOur analysis suggests high levels of gene flow into the city of Colombo. Colombo is the main commercial city in Sri Lanka with the main harbor and port situated in the city. Many people move to the city from urban areas daily for their employments. It was also of interest to note that, apart from Colombo, gene flow is also directed toward Galle, which is also another commercial city and a regional port within Sri Lanka with the southern express highway connecting the city of Colombo directly with Galle promoting daily transportation. Similarly, a high level of gene flow is also directed toward Jaffna. With the end of the civil war in 2002, Jaffna has become one of the major tourist attractions. Jaffna and Colombo are connected through direct bus and railway transportation systems. Tourists and commercial supplies are been transported to and from the city of Jaffna via Trincomalee or Puttalum, through which major bus and train routes exist. Major resettlement programs that are being initiated in Jaffna require commercial cargo to be transported to Jaffna. Thus, human-aided passive migration resulting from the high level of commercial traffic between these cities, likely explains the patterns observed in the present study. This conclusion is also strengthened by the weak isolation by distance signal which is likely driven by human-aided movements. Ae. aegypti dispersal is restricted by the availability of oviposition sites [The lack of migration between other populations suggests that populations within individual cities may be initiated by very few individuals. It has also been reported that on sites  and the on sites . It has on sites . The limAe. aegypti populations in Sri Lanka have been exposed to adulticides and larvicides frequently as dengue eradication campaigns rely on insecticides and removal of breeding sites to lower the disease spread. Populations have therefore experienced intense selection by insecticides that probably resulted in periodic population bottlenecks. A similar scenario has been discussed in Phnom Penh, Cambodia, where treatment of discarded containers with temephos affected the population differentiation [Our analysis with the Garza–Williamson indices suggests genetic drift caused by founder effect. The lowest average Garza Williamson values for the Colombo J and Hambanthota populations suggest recent bottlenecks. Vector control through source reduction and insecticide treatment of breeding sites may also create periodic population bottlenecks leading to genetic drift. ntiation . Aedes aegypti females tend to distribute their eggs in multiple oviposition sites, thus a single container may contain a mixture of several female oviposition events, consequently decreasing genetic differentiation [k-means clustering also revealed an exclusive cluster consisting of Puttalum individuals. This small-scale clustering and the genetic differentiation may be due to the clustering of oviposition sites and hosts. It has been reported that Ae. aegypti tends to cluster with a house or a unit of houses [The levels of genetic differentiation detected in the current study are low with only one population (Kandy) being genetically differentiated. In comparison, other microsatellite-based studies carried out in the region have recorded much higher levels of genetic differentiation within the study sites ,33. Althntiation . The Kanf houses . HoweverAe. aegypti in the present study revealed genetically similar populations with high levels of gene flow. The gene flow was directed toward the major commercial cities. High level of gene flow associated with passive dispersal, aided by human activities and transportation, represents a major threat of dispersal of insecticide-resistant genes to susceptible populations [The geneflow rates and the population genetic structure of ulations ,45,46,47Ae. aegypti toward the cities of Colombo, Galle and Jaffna via transportation routes. Passive migration of the mosquito to distant areas far from its own flight range may introduce insecticide resistance genes and new pathogens to the more susceptible populations. This could have a serious impact on control programs and the possible threat of the emergence of a disease epidemic. As for future research on the dispersal patterns of the mosquito, examination of Ae.aegypti populations from locations in direct trade with Colombo may be informative. Further studies on transportation routes and control strategies could provide better understanding of the population dynamics and the potential spread of Ae. aegypti in Sri Lanka thus enabling more informed strategies to control the spread of dengue and other mosquito-borne diseases.Our results indicate a passive migration of the


However, there are concerns about the hypothesized low diversity among the few K. grandifoliola germplasm sources introduced in the country. Using eight microsatellite markers, we evaluated the genetic diversity and divergence among 53 superior trees selected from three provenances of K. grandifoliola located in the state of Para. These populations are among the oldest plantations (>15 years) in Brazil and, therefore, the country's main seed sources. The average number of alleles per locus was 5.9, expected heterozygosity was moderate (^=0.56) and lower than the high observed heterozygosity (HO=0.74). Therefore, the intrapopulation fixation index was negative (f=-0.31) indicating the possibility that selection of superior trees might have favored heterozygous plants with heterosis. No genetic structure was observed between provenances. The genetic diversity observed within selected trees, with an effective population size (Ne) of 30.4, is comparable to that of natural populations of African and Brazilian mahoganies. Therefore, our results contradict the idea that the genetic diversity of K. grandifoliola introduced in Brazil is low and show that our germplasm can be exploited for breeding purposes.Given its high-valued wood, the African mahogany  Although they are not as fast growing as Eucalyptus and Pinus species, African mahoganies exhibit excellent wood quality, with similar physicochemical properties to the Brazilian mahogany (Swietenia macrophylla)  of mother trees  stand out because of their co-dominance, multiallelism, and high level of detectable polymorphism. As such, estimates of genetic differences can be obtained even between related individuals. The information obtained from these markers allows estimation of different genetic parameters, such as level of heterozygosity or diversity, as well as the degree of genetic structure and differentiation between different groups of individuals. This information is very useful in monitoring and managing the genetic variability available in natural ecosystems, germplasm banks, or genetic improvement programs .K. grandifoliola that are, currently, the main seed sources for K. grandifoliola plantations in Brazil. Our hypotheses were: 1) the Brazilian germplasm of K. grandifoliola has low genetic diversity; 2) populations introduced from Africa (Ivory Coast and Tanzanian provenances) have higher genetic diversity than progenies of the first five K. grandifoliola trees introduced in the country; 3) populations introduced from different regions of Africa will present genetic structure. For the analyses of genetic diversity, we successfully transferred K. senegalensis microsatellites . This farm contains more than 1500 trees (~13 years old in 2012) originating from two African provenances: Ivory Coast and Tanzania. The third provenance was located at the farm owned by Hiroshi Okajima (03º04’13.6”S and 47º28’58.9”W), whose trees (~19 years old in 2012) originated from seeds collected from the first five K. grandifoliola trees introduced in Brazil. These five trees were planted in 1976 at Embrapa Amazônia Oriental . Fiftythree superior quality trees were selected, 12 from the Mr Norton’s Ivory Coast provenance, 21 from Mr. Norton’s Tanzania, and 20 from Mr. Okajima’s. These trees were cloned and are being evaluated in clonal tests conducted in different regions of the country.Selection was performed in three provenances located in two farms with the oldest K. senegalensis seedlings, obtained from a local nursery, were also used. These genotypes served as an external group in the analyses of genetic structure to compare with K. grandifoliola trees.For this study, leaf samples were collected from selected trees. In the field, they were immediately packed in ice and then stored in a freezer (-80 ºC). Samples from a control group consisting of 12 The Doyle and K. grandifoliola. However, for the congener K. senegalensis, two studies were found that describe 13 , reaction buffer (1x), BSA (2.5 mg/mL), and dNTP (2.5 mM). PCR thermocycling included an initial denaturation at 95 ºC for 7 min, followed by 35 cycles with denaturation at 94 ºC for 30 s, annealing for 30 s at 50–55 ºC depending on the primer pair, and extension at 72 ºC for 1 min. After thermocycling, a final extension step was performed at 72 ºC for 5 min. Locus amplification was conducted in a T100 Thermal Cycler (Bio-Rad).A literature search revealed the nonexistence of SSR primers developed for PCR products visualized in 3% agarose gel were obtained for 21 of the 24 SSR loci assessed in this screening. For 19 of the 21 SSR loci, primers were labeled with either 6-FAM, HEX or NED-Replacement fluorescent dyes  for the genotyping of this study.The 19 pairs of SSR primers labeled with fluorescent dye were submitted to further screening via capillary electrophoresis, using the DNA of six individuals. Based on this screening, eight easily genotyped polymorphic loci  were selCapillary electrophoresis was performed in an ABI-3100 genetic analyzer (Applied Biosystems). The electropherograms were analyzed for genotyping in the GeneMapper 3.5 program (Applied Biosystems). The genotypes were organized in a spreadsheet, with microsatellite loci on the lines and individuals (selected trees) in the columns.For population genetics and statistical analyses, the Norton population was subdivided into two groups: one with 12 trees from the Ivory Coast and another with 21 trees from Tanzania. Thus, analyses were carried out considering the Okajima, Norton Tanzania (Tz), and Norton Ivory Coast (IC) provenances as subpopulations, totaling 53 selected individuals.https://phylogeny.uconn.edu/software/) and https://www2.unil.ch/popgen/softwares/fstat.htm). The GDA program estimates the number of alleles per polymorphic loci (Ap), observed (HO) and expected (HE) heterozygosity, and the intrapopulation fixation index (f). The FSTAT program was used to estimate the allelic richness (Ar) of the subpopulations. Effective population size was estimated with NeEstimator v2  ((PE) (https://homepage.uni-graz.at/de/kristina.sefc/).Since the markers were originally developed for ity (PI)  and of pI) ((PE)  in the sF, theta (θ) and f, corresponding to Wright’s total fixation (FIT), interpopulation (Fst), and intrapopulation IS) was assessed with the GDA program using the The possibility of genetic structure among samples was further investigated with a principal component analysis (PCA) to depict the genetic distance among samples. The PCA was performed in R with package ape (K (number of sub-populations or clusters) varying from one (no genetic structure) to eight, with 30 iterations for each K. The best K value was assessed using the method developed by The genetic structure was also assessed by the Structure 2.3.4 program, which uses a Bayesian approach  was 5.9, ranging between 3 (locus ssrKs16) and 16 alleles (locus Ks040). Average heterozygosity was 0.738, with locus ssrKs7 exhibiting the highest observed heterozygosity (Ho= 0.979) and Ks022 the lowest (oH= 0.442). Expected heterozygosity e)(H was lower than observed (OH), with a mean of 0.563, varying from 0.365 for locus Ks22 to 0.835 for Ks040. Given the greater heterozygote frequency than expected by Hardy-Weinberg equilibrium, the estimated intrapopulation fixation index (f) was negative and equal to -0.314  and identity (PI). The value of PE varied from 0.180 (Ks022) to 0.663 (Ks040) for individual loci and was 0.970 considering all the loci. PI, considering all the loci, was low (7.612 x 10-6), indicating that these markers are also useful for genotyping K. grandifoliola.Since the SSR markers were originally developed for oH = 0.746, and the Norton_Tz subpopulation 0.710, close to the values found in the Okajima provenance (0.758).Genetic diversity analyses for the population of Norton trees subdivided into two provenances showed little difference between them . Norton_EH also varied little between the Norton_IC E(H = 0.550) and Norton_Tz E(H = 0.553) provenances. These values are also close to that of the Okajima subpopulation, which exhibited EH = 0.586. Estimated allelic richness (Ar), calculated based on 11 individuals per subpopulation, was greater for the Okajima (Ar = 3.946) and Norton_Tz (Ar = 3.896) subpopulations compared to the Norton_IC (Ar = 2.958) subpopulation. These results contradict the hypothesis of low variability between K. grandifoliola populations introduced in Brazil.Average (Ne) was 30.4 for all 53 samples, with 95% confidence interval ranging from 18.3 to 58.2. Considering each subpopulation individually, the Okajima population had Ne of 11.5, Norton_Tz of 14.2 and Norton_IC of 22.8. However, the confidence interval for the estimates of Ne for each subpopulation were high, making these estimates unreliable.Estimate of linkage disequilibrium-based contemporary effective population size f) ranged from -0.377 (Norton_IC subpopulation) to -0.296 (Norton_Tz subpopulation), with an average of -0.324. The total fixation estimate (F) was also high and negative, with an average of -0.311, influenced by the high negative f value.The estimate of interpopulation fixation index (θ = 0.008) was not statistically significant, with a 95% confidence interval varying from -0.007 to 0.028. This result indicates little differentiation or genetic structure between the subpopulations. The average intrapopulation fixation index . Therefore, there is no apparent sub-population clusters among K. grandifoliola individuals. As such, no apparent genetic difference was observed between the three provenances   of the Roger’s genetic distance between the samples . The PCArton_Tz) .K. grandifoliola selected trees. The K = 5. However, the highest value obtained was very low (ΔK < 8) . Under the hypothesis that K=5, analysis of the bar graph  with the allocation of 53 individuals in the five populations, indicated that all the individuals had uniform distributions of allocation probability. This result provides, therefore, strong evidence for the absence of structure between the genotypes. A more detailed analysis of the estimated average and standard deviation of the likelihood of the different models (K ranging from 1 to 8) indicates that the most likely model is the one with K = 1 (absence of structure). This model exhibited the highest likelihood and lowest standard deviation among the 30 repetitions assessed . It is important to underscore that the The results obtained from the Structure program also suggested the nonexistence of genetic structure between the 53 K. grandifoliola populations introduced in Brazil. The population of 53 K. grandifoliola individuals exhibits relatively high genetic diversity E(H = 0.563 and OH = 0.738). Curiously, the 20 genotypes selected from the Okajima provenance, which originated from only five parental trees, had the highest levels of genetic diversity E(H = 0.586 and OH = 0.758). This result indicates that the five parental trees that originated the Okajima subpopulation were likely genetically diverse.Our results contradict the hypothesis of low genetic diversity in the (K. grandifoliola) in Brazil. It is important to underscore that the populations of the present study (Okajima and Norton) are among the only ones in the country at reproductive age. As such, these farms are the major sources of seeds and account for nearly all the K. grandifoliola seedlings currently produced in Brazil. This relatively high genetic diversity can be exploited for breeding purposes. In addition, it justifies plantations with higher density  to allow selection of the best trees during thinning. The stems of many K. grandifoliola genotypes have tortuosity problems  adult trees from the Amazon forest found average Ho and EH values of 0.73 and 0.84, respectively . By sampling 192 Cabralea canjerana (Meliaceae) trees in seven fragments of the Atlantic Forest in the state of Minas Gerais, OH and EH of 0.70 and 0.73, using six microsatellite loci.High diversity can be observed, primarily when the results of this investigation are compared with studies of genetic diversity in natural populations of economically important tropical wood species. Studies on phylogenetically close tree species, belonging to the family S. macrophylla) and that the diversity values observed are close to those obtained in the selected trees of K. grandifoliola from this study O(H = 0.738 and EH = 0.563). This demonstrates that, although the selected trees originate from provenances with a restricted genetic base, their genetic diversity is comparable to that observed in natural populations of species belonging to the family Meliaceae.It is important to highlight that these studies were conducted with natural populations of Brazilian mahogany  than that obtained in natural populations of phylogenetically close species.To date, no genetic studies have been carried out in populations of The estimate of heterozygosity, together with the number of alleles per locus and allelic richness, indicate that all three provenances  display genetic diversity with similar magnitudes. The magnitude of diversity demonstrates that these populations can be exploited for breeding purposes.K. grandifoliola  was lower than estimates reported in research involving K. senegalensis, which ranged from 8.5 to 10.8 alleles per locus  and found 6.6 alleles per locus. A larger number of alleles results in greater capacity to generate new genotype combinations, thereby broadening the genetic base for both conservation and breeding purposes  obtaining the lowest number of alleles. The Norton_IC obtained lower Ap (3.000) and Ar (2.958) values than those observed in the Okajima subpopulation (Ap = 4.625 and Ar = 3.946). On the other hand, the Norton_Tz population had values closer (Ap = 4.375 and Ar = 3.896) to those obtained for the Okajima population.When the Okajima, Norton_IC and Norton_Tz provenances were compared, they exhibited approximately the same values for K. grandifoliola introduced in the country and there are no reports of adult populations, in the proximity, that could pollinate these parental trees.These results were surprising since they contradict the initial hypothesis that the provenances collected from Africa (Norton Ivory Coast and Tanzânia) would display greater diversity than that of the Okajima. The Okajima provenance originated from only five parental trees located at Embrapa Amazônia Oriental. These five trees were the first f indicates that heterozygote frequency is higher than expected by the Hardy-Weinberg equilibrium. Inbreeding depression is especially prevalent in adult populations of perennial species .The intrapopulation fixation index is an important measure in population genetics studies, since it may indicate the presence of endogamy and other forces that cause deviations in the expected equilibrium between homozygotes and heterozygotes . Negative values, closer to zero (f = -0.038), were also reported in another investigation with K. senegalensis . Similar findings were recorded in several natural populations of S. macrophylla, with average f values of 0.015  provenances. The partition of genetic variability between and within populations  and Tanzania (Tz), both in terms of diversity level and genetic differentiation. Thus, it is highly likely that the seeds used to plant the first five trees at Embrapa Amazônia Oriental, which supplied Mr. Okajima’s farm, and the seeds bought in Africa that supplied the Norton farm, came from the same population of K. grandifoliola, or from genetically connected populations in Africa. Another important factor to underscore is that Tanzania, a country from which the Norton_Tz seeds originated, does not contain natural populations of K. grandifoliola. Thus, this Tanzania subpopulation must have originated from the same population that gave rise to the other provenances.This low genetic structure among the 53 K. grandifoliola. The information obtained in this study regarding the breeding population of K. grandifoliola is important because it shows that the genetic diversity is not low, contradicting initial worries of a possible lack of genetic variation that could compromise the long-term sustainability of plantations in Brazil. We also demonstrated that the introduced provenances of K. grandifoliola do not seem to have genetic structure. This information is important for the conservation of genetic resources of the species in the country, as well as to guide future genetic improvement in K. grandifoliola. Knowing the genetic variability between and within populations is essential to strengthen genetic improvement and germplasm conservation programs (To the best of our knowledge this is the first report of a genetic study in K. senegalensis were successfully transferred to K. grandifoliola. Second, the level of genetic diversity between K. grandifoliola trees from the Okajima and Norton provenances, which are the primary sources of seeds in Brazil, can be considered at least moderate, contradicting the initial hypothesis of low diversity in the country’s plantations. Third, this genetic diversity is comparable to that observed in natural populations of African and Brazilian mahoganies, enabling exploitation of these K. grandifoliola selected trees in a breeding program. And fourth, there is no genetic structure among selected trees, indicating that the different K. grandifoliola introductions in Brazil may all have originated from the same African sources.The following conclusions could be drawn. First, microssatellite primers designed for


Historic Cities: Issues of Urban Conservation. The book consolidates thinking about, investigations into and actions taken that embarks us on a journey to educate us about and rescue historic cities for the value they represent to us now and into the future. The world is now URBAN and as much as the Development Set still romanticizes rural development at the expense of urban areas, it is cities—all sizes and kinds of cities—that are the future.Jeff Cody and Francesco Siravo have captured the crisis confronting historic cities and issued a call to action in The book is divided into eight thematic parts preceded by an introduction from the editors, and concluded with illustrative images in the shape of visual summaries. These different sections address the changing nature of historic cities, their geographical diversity, the transformation of traditional cities, how to read the ciy. They address the issues of continuity, identifying heritage values of cities, the challenge of sustainable urban conservation as managing historic cities. For each part, the editors have selected a range of key authors from the early 19th century to 2017, from different professional backgrounds, and regions of the world to offer as wide as possible a panorama. The format allows the reader to take in slowly the sequence of chosen selections. The selections track and illuminate thinking that is bringing change to how we consider historic cities and integrate them into the future. The short bursts of input allow the reader to digest what is presented and the cumulative effect brings home a clarity of why and what has to happen now to make historic cities the resource they are.From the outset we are presented, through variegated sources, disciplines and perspectives with clear admonitions to not ignore the multiple values and purposes historic cities hold. Most importantly historic cities reflect and provide backdrop to our humanity if only we would look. The book calls out the human aspects pushed aside in discussions of a new and different value system that threatens historic cities—the market value of land and the restive impacts of societies in change. From the very first page we are told ‘the historic city demands that we understand the profound disconnect that occurred in city building since the industrial revolution … we need to recognize and appreciate the subtle clues that set historic places apart from the look-alike sameness of most present-day urban developments.’ (p. 1)The book points out that the world economy since the industrial revolution has changed the rules of the game. The slow evolution that created cities historically has been taken over by a different set of values and pace of development. Historic cities have become commodities and it is land value and space for new-world toys—cars—that rule and despoils the humanity of places relegated to a footnote by new-world developers and politicians. There is more to cities and neighborhoods than returns on investment.The sense of loss that permeates the book is palpable in the first parts. It calls on those working in the field to balance and move from theory to practice. One of the more poignant expressions of that loss exposes the impacts of the quest for misguided ‘modernity’ and the mass-tourism made possible by prosperity that has made frivolous places once regal and sacred. Salvatore Settis in his selection ‘If Venice Dies’ cuts no slack in his assessment of what has happened in Venice. Seyyed Hossein Nasr forcefully states ‘Euro-centrism also sees its view of the world as the only worldview … Not even Genghis Khan could do what we are doing through “development” … There is nothing more important than to realize that what remains of our historic cities, of our sacred sites and sacred places, is significant not only for national identity or archeological records but also for our own identity as human beings.’ (p. 20–21)This book records the results of the wonton destruction of exceptional places destroyed in the name of Modernism and economic development. The book puts into perspective and helps explain this reviewer’s personal sense of loss, and perhaps that of others who may read it, who have been witness to the destruction of wondrous places and their cultures as a result of the overriding forces of change that Cody and Siravo explain. Examples abound but the two that won’t leave me alone are St. Louis, Missouri where I grew up and studied architecture and Cartagena, Colombia where I was a Peace Corps Volunteer in the 1960’s. In St. Louis, it was to see one of, if not THE best, Victorian housing stock knocked down to be replaced by the then award winning Pruitt-Igoe project. The other is Cartagena, Colombia overtaken by tourism. Pruitt-Igoe in St. Louis was designed for the urban poor that became the most dangerous place to live in the city and was ultimately dynamited in a huge implosion to remove the entire development from existence. It forced the residents, who never made the transition to high-rise living, to disburse and left a gaping wound in the fabric of the city that to this day has never quite healed. The ‘reason’ was the new modernity of Corbu’s Garden City that only masked the racism behind the decisions to rebuild downtown St. Louis.La Heroica, the Heroic, for its defense against Vernon the pirate among others; it is now Cartagena La Turistica, and that is not an improvement. It lost its soul as ‘economic’ pressures drove out the people and destroyed the lively, inclusive central market that really gave the city its flavor to make room for the International Convention Center, surrounded by a determined fence to keep the ‘Cartageneros’ out with a design not unlike the Kunsthaus blobstyle, the Friendly Alien—or not so friendly—in Graz Austria. Settis clearly states, ‘Citizens—and not tourists—are the real life blood of the city.’ (p. 189) Poor Venice! Poor Barcelona! Poor Cartagena!Cartagena, Colombia painfully displays what the selections emphatically lament - the danger of mass-tourism and its impact on what was once one of my favorite places on earth. The 16th century city was the gateway to exporting the gold plundered from the societies the Spanish conquered as well as the gateway of slavery into the Andean region. Once Cartagena The book provides through a diverse, inclusive chorus of authors a new way of thinking about historic cities that changes the static concept of conservation as monuments to a dynamic one that requires the rethinking of historic cities as living resources for future urban growth. Local context is key. Senam Okudzeto, provides an African voice that reminds us that ‘anti-colonialist movements identified Ghanaian urban heritage very closely with social uses of space and on going cultural traditions. Historic structures, including recent ones, are the physical vessels in which social traditions can survive.’ (p. 436–437)Non Arkaraprasertkul and Matthew Williams reflect on the irony of the events in Shanghai, that now, after wonton destruction, ‘regards historic preservation of selected sites, including the traditional alleyways of Shanghai, as essential to the branding of a city with global ambitions.’ (p. 444) The book highlights that the destruction in Shanghai of the alleyway houses, not unlike the destruction of the shop houses in Singapore only to be rebuilt ‘modern,’ is mirrored in the selections on urban renewal of the 1950’s and 60’s in the USA. Gratz with Mintz build on Jane Jacobs’ struggle against car-induced demolitions, slum clearances and urban renewal projects from the 1930s to the 1970s. Cederna offers a glimpse of positive thinking ‘We must fix in our minds on the conviction that integral preservation of the old and creation of the new in cities are complementary tasks … That ancient and modern have distinct material and spiritual prerogatives and are mutually necessary.’ (p. 10)What needs to be made clear is that for all the good that the book points out that historic cities can contribute to the quality of life of the people of a city, it is and will be the political will of those that govern, that foment ‘development’, that provide the guidance a local culture values, and that invests in the maintenance of historic cities that will make the difference. The battle is on and is far from over. The latest assault that misses the point altogether is the reverence for technology and the new systems that would have us believe that they are an improvement in our lives. Shannon Mattern shatters that notion stating clearly ‘The city is not a computer and technologists and political actors speak as if they could reduce urban planning to algorithms’. (p. 575) ‘Smart Cities’ are but a distraction and were they Smart the future of historic cities, as this well conceived book militates for, would be bright.In sum, the book’s length and/or cost might be daunting for some readers, but that it is well worth the effort to take the time to savor the prose, learn from the graphic examples, and determine how to take the lessons implied by the book and apply them to actual challenges. In our post-Covid world, with so many unknown changes to come in historic cities worldwide, this book’s importance and significance is even more critical than it was, before Covid-19 came into our lives, when Cody and Siravo assembled such a stimulating cluster of readings, comments on those readings, and images that complement them.