A. chinensis populations using simple sequence repeat (SSR) molecular markers. The potential suitable distribution of tetraploid A. chinensis estimated under the current climate and predicted for the future climate was generated with ecological niche modeling (ENM). The results indicated that the polyploid populations of A.chinensis have high levels of genetic diversity and that there are distinct eastern and western genetic clusters. The population structure of A. chinensis can be explained by an isolation-by-distance model. The results also revealed that potentially suitable areas of tetraploids will likely be gradually lost and the habitat will likely be increasingly fragmented in the future. This study provides an extensive overview of tetraploid A. chinensis across its distribution range, contributing to a better understanding of its germplasm resources. These results can also provide the scientific basis for the protection and sustainable utilization of kiwifruit wild resources.Understanding genetic diversity and structure in natural populations and their suitable habitat response to environmental changes is critical for the protection and utilization of germplasm resources. We evaluated the genetic diversity and structure of 24  Actinidia species . Previous studies have shown that Actinidia has experienced at least eight interspecific hybridization events in the process of evolution [Actinidia chinensis is the species with the highest domestication level and the greatest economic benefit among the species of Actinidia [Kiwifruit is a perennial, dioecious economic fruit that is native to China. It is one of the best examples of the successful domestication and commercialization of crops in the early 20th century . Kiwifructinidia .A. chinensis has been increasing, providing us with a better understanding of this fruit crop [A. chinensis can produce fertile offspring, which is often accompanied by excellent traits for crop improvement [A. chinensis are scarce, and the origin of tetraploid A. chinensis remains unclear [A. chinensis in limited regions [A. chinensis have been reported.In recent years, the number of studies on the genetic diversity of uit crop ,11. Crosrovement ,9. The mrovement . Tetraplrovement . Among t unclear . In prev regions  or focus regions . NeitherA. chinensis dominated by tetraploid individuals based on 40 microsatellite markers. In addition, 52 distribution points of tetraploid A. chinensis were used for niche simulation. The objectives of this study were to (1) evaluate the genetic diversity of the tetraploid component of A. chinensis; (2) describe the tetraploid population structure; (3) predict the potential suitable distribution for tetraploid A. chinensis in both current and future climate; and (4) provide breeding and conservation strategies for kiwifruit germplasm.Simple sequence repeat (SSR) markers, which are based on genome sequences, are easy to use, relatively low in cost, and have high polymorphism and extensive genetic information. It is the most widely used type of DNA molecular marker to characterize genetic germplasms, with many alleles at each locus . PreviouA. chinensis populations. The average number of alleles per locus was 18.9, of which, the minimum number of alleles detected at each locus was 9 (at UDK96-009 locus) and the maximum number was 28 (at UDK96-034 locus). The polymorphism information content (PIC) varied from 0.213 (UDK96-028) to 0.924 (UDK96-019), with an average of 0.808 (Ne) ranged from 3.037 in ZG to 6.437 in ZY, averaging 5.124 alleles per population. The inbreeding coefficients (Fis) were all greater than zero, ranging from 0.080 (LA) to 0.389 (DN), with an average of 0.024 (He) as estimated using GENODIVE ranged from 0.670 (NL) to 0.855 (DN), whereas the He estimated using POLYGENE ranged from 0.636 (ZY) to 0.795 (DN). However, the value of observed heterozygosity (Ho) calculated using POLYGENE ranged from 0.681 (NL) to 0.808 (LA), and the same trend has been observed in GENODIVE. The observed gene heterozygosity was lower than the expected gene heterozygosity. On average, the genetic diversity of the tetraploid population was higher than that of the diploid population, although it was different in some populations such as LA and PN.  among populations , indicating the presence of an isolation-by-distance effect  was to see if there was any genetic variation across populations as well as within populations. According to our results , the AMOK = 2 was the best value when LnP(K) was found to increase and ΔK was maximized , and the results were generally consistent with those of STRUCTURE , indicating that all models performed well in predicting the suitable habitat under all climate scenarios. Under the current climatic scenarios, the potentially suitable area was a good representation of the actual distribution of tetraploid ectively .A. chinensis decreased substantially under eight different future climate scenarios. The tetraploids’ highly suitable habitats were predicted to decrease by up to about 95.3% under the 2081–2100, SSP5_8.5 scenario, the highest level of the greenhouse gas emission scenarios. Additionally, the moderately and hardly suitable distribution of tetraploid A. chinensis showed the same decreasing trend. Additionally, under the 2081–2100, SSP5_8.5 scenario, the reduction rate of tetraploids’ moderately and hardly suitable habitats was found to be the highest, with a total of 50.8%  than diploid varieties, which has been observed in many orchards.In general, the level of genetic diversity possessed by a species reflects its prepared evolutionary potential. Therefore, species with low levels of genetic diversity are more likely to become extinct . During A. chinensis individuals formed two genetic groups in both principal coordinate analysis (PCoA) and Bayesian model-based clustering (FST) and geographical distance   . The dif studies .A. chinensis [A. chinensis is a desirable food source for frugivory animals. Additionally, the seeds of A. chinensis can germinate readily upon maturation and are potentially capable of establishing a new population. Thus, high levels of gene flow among A. chinensis population are expected.In the present study, of the total genetic variation partitioned, 9.01% was attributed to the differences among populations, and 90.99% to the differences among individuals within populations, in agreement with the findings of previous studies on hinensis ,14. The A. chinensis. Although previous studies [A. chinensis is generally considered to be an autopolyploid, and diploid A. chinensis was one of its ancestors [Galax urceolata [G. pentaphyllum [A. chinensis arose once or multiple times will require other data such as high-throughput DNA sequence methods.Polyploidy is widely distributed in plants, and polyploidization is regarded as a major force driving plant evolution and speciation ,24,25. P studies ,31 have ncestors ,33. All rceolata  and G. paphyllum . More deA. chinensis is listed in the list of the national key protected wild plants. From a conservation perspective, genetic diversity estimates can be used in making decisions about the management of extant populations of endangered species. In the present study, the high level of genetic diversity maintained within wild populations of A. chinensis is encouraging. However, the result of the ENM showed significant decreases in the area of the potential distribution of tetraploid A. chinensis under various future climate scenarios. This suggests that climate change will shrink the potential suitable habitat of tetraploid A. chinensis under future different emission scenarios. This mainly results from changes in the distribution of temperature and precipitation, which directly affects the boundaries and trends of plant growth [A. chinensis will tend to migrate to high elevations, and its habitat will be more fragmented. Under these scenarios, there will be more pressure on the conservation and management of tetraploid A. chinensis resources in the future.t growth . With clA. chinensis populations suggests their great potential for kiwifruit breeding. For example, the yellow flesh cultivars “Jintao” is a tetraploid, which is a Chinese selection from wild resources and is widely planted in Europe, South America, and China. Moreover, two genetic clusters were revealed in the A. chinensis populations, suggesting that intraspecies crosses using the individuals from each of these clusters would be useful in cultivar development of kiwifruit.The high genetic diversity observed in tetraploid A. chinensis individuals from the National Actinidia Germplasm Repository of China, which were collected from 24 wild populations  method . The polGenotyping microsatellite data corresponding to polyploids can be problematic because of the difficulties in assigning the correct allele dosage for each locus and individual ,41,42. IFST) in this study could not be calculated using traditional analytics software [A. chinensis samples and the dosage effect of polyploid alleles. Therefore, we used POLYSAT 1.5-0 [The standard population genetic diversity statistics , effective number of alleles (Ne), and observed (Ho) and expected (He) heterozygosity and inbreeding coefficient (Fis), all of which were calculated with GENODIVE. As software developed specifically for the analysis of polyploid genetic data, POLYGENE v1.2 can take into account both polyploid genotypic ambiguities and double reduction [Ho) and expected (He) heterozygosity, polymorphic information content (PIC), and Shannon diversity index (I) were also estimated for each population and locus in POLYGENE v1.2. Differentiation among A. chinensis populations was assessed with GST [A. chinensis populations, analysis of molecular variance (AMOVA) [FST was adopted as the measure of genetic distance [Genetic diversity was evaluated through the following descriptive statistics: the number of alleles (eduction  and can with GST . In addi (AMOVA)  was impldistance . Principdistance . Previoudistance .K was inferred with the online program STRUCTURE HARVESTER [DA genetic distance was established for A. chinensis populations with POPTREE v.2 [To reveal the number of clusters, a Bayesian analysis under an admixture model with correlated allele frequencies was performed with the program STRUCTURE 2.3.4 ,54. The ARVESTER ,56. The ARVESTER  was usedARVESTER  and ArcMTREE v.2 .A. chinensis with Maxent v.3.4.0 [A. chinensis was represented by 52 data points extracted from previous studies [http://www.worldclim.org/, accessed on 1 December 2021) [Ecological niche modeling (ENM) was used to predict suitable current and future distribution ranges of tetraploid  v.3.4.0 ,61. The  studies . In addi studies . These per 2021) . The futer 2021) . In thiser 2021) . For furA. chinensis. It also compared the genetic diversity and structure between two cytotypes within the A. chinensis. In addition, changes in potentially suitable regions for tetraploid A. chinensis were modeled with ENM. Based on the results of our analyses, considerable levels of genetic diversity exist among tetraploids in A. chinensis, and its potential suitable area will likely be reduced in the future. These results can serve as basic information by providing options to breeders to develop, through selection and breeding, new and more productive varieties that are adapted to changing environments. In addition, this will also provide a reference basis for the protection of wild tetraploid A. chinensis resources.This study revealed the genetic diversity and structure of 19 tetraploid populations in


The region of Transoxiana underwent an early agricultural-demographic transition leading to the earliest proto-urban centers in Central Asia. The agronomic details of this cultural shift are still poorly studied, especially regarding the role that long-generation perennials, such as grapes, played in the cultivation system. In this paper, we present directly dated remains of grape pips from the early urban centers of Sapalli and Djarkutan, in south Uzbekistan. We also present linear morphometric data, which illustrate a considerable range of variation under cultivation that we divide into four distinct morphotypes according to pip shape. While some of the pips in these two assemblages morphologically fall within the range of wild forms, others more closely resemble modern domesticated populations. Most of the specimens measure along a gradient between the two poles, showing a mixed combination of domesticated and wild features. We also point out that the seeds recovered from the Djarkutan temple were, on average, larger and contained more affinity towards domesticated forms than those from domestic contexts. The potential preference of morphotypes seems to suggest that there were recognized different varieties that local cultivators might aware and possibly propagating asexually. Stretching back to Possehl’s2 Middle Asian Interaction Spheres, it has been archaeologically clear that the early populations of southern Central Asia were intertwined into long-distance trade networks, which, among other cultural attributes, fostered an early globalization process in the agricultural arena5. Across the river ways and oases of southern Central Asia, a complex agricultural system gradually developed from 7000 to 5000 BP8. By the early 5000 BP, large-scale irrigated agriculture projects were fostering early urbanization and the establishment of an elite class10. Increased localized human density and regional demographic growth presumably further increased the demand for crop diversity and appears to have promoted the emergence and development of arboriculture, which would, hypothetically, have been tied to increased sedentism, greater stability in land tenure, and more reliable political oversight of irrigation systems. Recently Fuller and Stevens11 resurrected Childe’s13 concepts, suggesting that there was a direct link within Eurasian cultural development between the rise of arboriculture and the expansion of urbanism. The earliest evidence for the cultivation of grapes (Vitis vinifera), and possibly apples  and plums (Prunus cf. insititia) in Central Asia comes from this time period and region14. The cultivation of long-generation perennials requires different cultivation techniques from those used for annual crops, including grafting and cloning, and, in these arid regions, reliable irrigation systems16. The grape seeds that we present in this manuscript represent some of the best evidence for the beginnings of perennial cultivation in this understudied ancient agricultural center.The Greater Khorasan Civilization18. Archaeologists working in this region have emphasized the magnitude of exchange networks and globalization processes of this part of the world during this period21. The unique cultural repertoire that came to represent these clusterings of cities has been variably referred to in archaeological literature as the Oxus Civilization22, Bactria-Margiana Archaeological Complex (BMAC)23 and Greater Khorasan Civilization1. In many aspects, these peoples reached a demographic zenith around four millennia ago, when agricultural investment and craft production was heavily intensified18.The burgeoning urban centers across the oases of the southern Kara Kum and river valleys of the Kopet Dag grew in scale and population size starting around 5500–5000 BPVitis vinifera ssp. sylvestris populations in parallel across the broad area of southeastern Europe and southwest Asia24. The domesticated grape gradually spread into Europe and East Asia and became one of the most important woody perennial crops in the world , and relatively drought, heat, and frost (winter not spring frosts) tolerant. Once peoples across Eurasia mastered viticulture, the transition into other forms of arboriculture, notably of asexually propagated rosaceous species, was a logical next step, and the grape may have paved the way for the domestication of the apple and plum.It is well accepted that the domesticated grape evolved from wild rld Fig. . Several27; Although, it remains unclear when humans began recognizing distinct forms and locking them in place as genets through cloning28. The main characteristics related to domestication do not preserve archaeologically. While aDNA and population genetics of historical landraces has made headway in answering many of these questions over the past decade31, directly dated evidence for trait evolution is still needed. Some studies have attempted to link historical and archaeological lineages genetically33, but most archaeobotanical plant remains are carbonized and therefore not suitable for genetic analysis. The use of image analysis techniques for multivariate comparisons and classification of modern and archaeological samples has been increasing in prominence 40, dates (Phoenix dactylifera)41, and olives (Olea europaea)42. More recently, the integration of 3D seed scanning techniques, machine-learning software, and genetic tests are further proving the effectiveness of the morphometric approaches43. Despite the integration of new measurement methods, the effectiveness and reliability of linear morphometrics for the study of variation in grape seeds has been illustrated through repeated studies in the Mediterranean.The most significant evolutionary changes under cultivation within the grape include an increase in the mass of the pericarp and the size of the inflorescence, greater sugar content, and more regular yeildsIn this study, we examine two populations that represent some of the earliest cultivated grape assemblages in southern Central Asia, originating from the early urban sites of Sapalli n = 21) and a temple at Djarkutan (n = 103)  is correlated with the position of the chalaza .Building on the growing number of studies of grape pip morphometrics, we seek to add a new perspective, as most previous studies were conducted in the Mediterranean. While the scholars conducting many of these other studies attempted to link modern or historical landraces to archaeobotanical remains, we approach this practice cautiously. However, we did add an assemblage of modern wild and cultivar grape pipsThe UPGMA cladogram Fig.  shows 4 p value > 0.75), which further confirms that class 2 has obvious characteristic traits of modern domesticated forms.Subsequent LDA results also confirmed the conclusion of a strong discrimination between all 4 classes. The model shows that 93.60% of the grape seeds have been correctly assigned, consistent with the results of UPGMA cluster analysis, proving the reliability of the UPGMA classification. However, according to the confusion matrix and classification results obtained by LDA, some specimens may be overlapping between classes 2 and 1 and class 4 and 3. The potential for overlap further attests to the similarity between these paired groups, which is also consistent with our statistical results in L, B, LS, and PCH. Also, taking the grape morphological groups found in archeological sites as a reference, using the DA method to classify modern cultivated grapes, we show that all of the modern local cultivated grapes that we collected are classified into class 2 (Vitis vinifera L. ssp. sylvestris), is dioecious and produces small, black berries, which naturally grows across the Mediterranean, east to the western slopes of the Himalayas, but the domesticated grape (V. vinifera L. ssp. sativa), is characterized by larger berries and hermaphroditism27. The earliest evidence of grape processing and utilization by humans can be traced back to 8000 BP in West Asia and the Caucasus, and scholars have argued that people were foraging and even fermenting grapes for many millennia prior to their cultivation49. Current research suggests that wild grapes produce roundish pips with short stalks, while domesticated seed varieties are more elongated with longer stalks. These differences have been acknowledged for a long time and widely used in archaeobotanical literature to discriminate wild and domesticated pips in archeological grape seeds50. Although, it is also well recognized that there is considerable overlap in these variables in early cultivated forms and all early cultivated forms are morphologically classified as wild and many domesticated populations contain individual seeds that would overlap with wild forms. Ultimately, discussions of morphometrics in grapes are only meaningful on a population level. Meanwhile, carbonization process also can changes the seed form, some researcher even questioned the reliability of distinguishing wild or cultivated varieties by measuring charred seeds28. However, according to recent experimental research, the deformation of the charring process does not greatly affect the result of morphological identification52. These realizations, combined with the traits that we just discussed, seems to suggest that there is variation in the archaeological specimens that we present here, with some having more wild affinities and others representing more derived forms.The domestication and dispersal of grapes remains a heavily debated topic. The wild grape 26. There has been a considerable focus on the spread of crops across Eurasia and archaeobotanical inquiry over the past decade56, and, despite need for more early archaeobotanical assemblages from this region, it seems likely that the lack of earlier evidence is an indication of the fact that the rise of the Oxus city states marked the advent of viticulture in Central Asia. Grape cultivation had already made its way into the Indus region by 5000 BP, but does not really become prominent until the rise of the Indus Civilization around 4500 BP57.In Transoxiana, wild grape relatives may have been present across western Turkmenistan and the Caspian Sea region in the past, but as of yet no grape seeds have been recovered from early archaeological village sites, such as JeitunThe PCA and LDA results show that the grape seed morphology within cultivated populations dating to roughly 4000 BP in Central Asia can be effectively divided into 4 class. Among them, class 2 has the strongest affinity towards domesticated characteristics, but the quantity of seeds in this grouping is relatively low, accounting for only 15.2% of all seeds. Compared with the intermediate class 1 and the strongly domesticated class 2, the most marked characteristics of class 3 and 4 are shorter seed lengths and stalks, and the lengths of seeds in class 4 are shorter than those in class 3 and the breath is larger, features which are generally considered as indicators of wild grapes, accounting for 40% of all grape seeds in the study.37. Morphological studies of modern cultivate grape variation in Europe have shown that the elongation gradient of grape seed length and stalk length might be represented as a marked continuum of the domestication syndrome. Again, this leaves open the question of whether different forms were being selected and preferred, notably whether class 2 might have represented a more recently introduced and more derived form or an example of further localized parallel evolution of domestication traits. It also leaves open the question of whether class 1 represents a more ancestral form, possibly remaining in its ancient form due to continued asexual propagation. Also, we must admit that the pips deformation process during carbonization might affect our judgment on these issues.Most of the grape seeds found at Djarkutan and Sapalli belong to classes 1 and 3, accounting for 80% of the overall assemblage. Among them, class 1, which accounts for 45%, has the most similar morphology to class 2, with some derived characteristics. The cross-validation results and confidence ellipses between classes 1 and 2 in the LDA also show some overlap. All of these observations are consistent with previous research in Europe that found a large number of archaeological grape specimens that fall along a continuum between domesticated and wild groups from southern and western Europe58. Due to limited water availability, summer agriculture mainly relies on irrigation in these regions59. Fruit cultivation outside the foothills, notably in the arid desert, requires a significant investment in labor and time, arguably indicating the presence of local-scale Wittfoglian-style hydraulic city states and some form of centralized authority by this time. Although, small-scale garden-style grape cultivation is possible if farmers were familiar with the necessary soil conditions, drainage requirements, and need for water inputs in the summer60. Grape vines are also a delayed return crop, and when asexually reproduced they tend to require a minimum of four years before the first harvest is procured61. At the same time, early urbanization of southern Central Asia, economic development, and population increases would all have led to an increased demand for fruit, wine, and cash crops.In Central Asia, archaeological sites are disproportionately more prominent in areas where there is current human activity, notably in the piedmont oases, as the arid climate of the deserts and steppe makes agricultural pursuits more challenging63 Historical sources attest to the cultural significance of wine across the Mediterranean and from most cultures of the ancient Silk Road64. Historically, grapes and wine making have been an important part of the Central Asian cultural repertoire, art historical depictions of grape vines are prominent across medieval sites and wine is readily attested from Sogdian and later sources65. The earliest archaeological evidence for wine production comes from the Lake Urmia basin in northwestern Iran and from roughly the same time period in the mountains of Georgia , all of which is presumed to have relied on truly wild populations of grapes66. Clearer evidence for wine production, likely on a larger scale, dates to ca. 5000 BP in Anatolia62 and northern Greece68. Other research suggests that the earliest domesticated grapes were present in the Caucasus by around 6000 BP69 and the first cultivated morphotypes introduced to that region were still on a trajectory towards a more modern-looking domesticated form, some of these morphologically look more derived and others looking more plesiomorphic.Archaeological and historical evidence attests to the economic and ritual significance of wine across Eurasia. Wine-related artifacts are readily found in tombs and ceremonial contexts, and are generally considered to have been used as mortuary offerings, during feasting events, and as expressions of conspicuous consumption in the past71. Large-scale wine production is also attested in the earliest Chinese accounts of peoples in Central Asia 72 and the earliest Classical sources that discuss cultural attributes of peoples to the north 73. The continuing importance of viticulture across southern Central Asia is attested by the discovery of an ancient winery at Kerkidon with two large vats, each having the capacity to hold 400 L, dating to 2200–1700 BP74. The best evidence that grape cultivators were selecting specific varieties and preferentially propagating favorable morphotypes comes from the ostraca at Nisa (2300–1600 BP.), which contain written references to different types of wine, classified by color and age, and noting if it had turned to vinegar75. The grape pips that we present in this paper are temporally on par with the oldest ever recovered in southern Central Asia. Ancient grape seeds have been previously reported at Anau South in Turkmenistan 76, and both Gonur Depe and Djarkutan 77. Artifacts that archaeologists ascribe to pottery wine vessels have also been found at early sites, such as Sarazm and Sapalli, but they require further investigation78.The deeper cultural legacy of prehistoric viticulture in southern Central Asia requires further investigation. Grape pips have been recovered 3500–3000 BP site across the Tien Shan and into Xinjiang, attesting to widespread cultivation of grapes in ecologically amicable regions by that time period79, possibly representing ritual offerings or consumption. By further comparing the grapes found in the temple with the grapes found in the domestic contexts, we note that those recovered from the temple are significantly larger . The raw projection was converted into a tomographic slice in .raw format by using IVPP225kVCT_Recon software, 2048 * 2048 pixels consisted each tomogram. The raw tomogram was imported into VGstudio 2.2 software and saved as a .Tiff file, then imported into Mimics 19.0 software to separate the pedestal and sample to obtain the 3D structure. Separation was performed by multiple modification and thresholding function tools in Mimics 19.0. Related measurements were also performed using Mimics 19.0.A total of 125 grape seed remains were selected with complete shape, less deformation and less clear alteration during carbonization for further analyze. The grape seeds were subjected to 225 kV three-dimensional (3D) fossil micro-CT (225-3D-μCT) scanning 50, we selected 4 linear measurements, which are regarded as the most efficient to discriminate between wild and domesticated grapes and are easily observable, including total length (L), length of stalk (LS), position of the chalaza (PCH) and total width (B)  was carry out by log-shape ratio to obtain grape seed shape variation. Cluster analysis was also performed using the unweighted pair-group method with arithmetic means (UPGMA) on the PCA centroid coordinates to explain the highest levels of morphological variation (PC1 and PC2). At the same time, linear discriminant analysis (LDA) was performed using log-shape ratios to test the validity of the subgroups identified by PCA and UPGMA cluster result. All statistical analyses were performed using XLSTAT 2019 .According to previous research(B) Fig. a. Also rSupplementary Information 1.Supplementary Information 2.Supplementary Information 3.


Resource-based cities (RBCs) face serious environmental pollution, and there are efforts to try to overcome those challenges by transforming industrial structure through investing in new technologies. Based on the panel data of 114 prefecture-level resource-based cities in China, this paper uses the difference-in-differences (DID) method to identify the effects of the “Broadband China” pilot policy on the carbon emission efficiency of resource-based cities. The results show that the “Broadband China” pilot policy has a significant effect on the improvement of carbon emission efficiency of resource-based cities, and the results are still valid after parallel trend test, PSM-DID estimation and placebo test. This study also finds that there are differences in the carbon emission efficiency of different locations and types of resource-based cities. In addition, the results of the mechanism analysis show that the “Broadband China” pilot policy can promote the improvement of carbon emission efficiency by promoting the upgrading of the industrial structure, the accumulation of human capital and the improvement of the level of urban innovation of resource-based cities. The findings provide a reference for China’s resource-based cities to develop the Broadband infrastructure, realize industrial upgrading, accumulate human capital and improve urban innovation level, and promote low-carbon transformation and improve carbon emission efficiency. With the rapid development of urbanization and industrialization, the control of carbon emissions has long been an important issue faced by all mankind ,2. ChinaSpecifically, resource-based cities, as special cities, have gradually developed into having an important economic status in China by relying on their own rich mineral resources development and operation. However, in recent years, as the global resource market has entered a recession, coupled with the gradual depletion of resources, the overall economic development of resource-based cities in China has been slow, and some cities have even experienced a decline. Among them, as a typical old industrial base and resource-based city agglomeration in China, the economic development situation in the three northeastern provinces is still unsatisfactory and the brain drain is serious, despite a series of special policies introduced by the state to revitalize the northeast. In addition, many resource-based cities still make articles around resource-based industries in the process of transformation, such as coal resource-based cities around coal vigorously develop coal power, coal chemical industry, coal machinery and equipment manufacturing industries, which has achieved certain economic benefits in the short term. However, in the long term, the transformation mode of extending the traditional industrial chain and relying on high energy consumption industries such as mineral resources mining and related energy industries to develop the economy will still lead to high carbon emissions in resource-based cities, and the transformation of resource-based cities to develop the economy in the context of carbon neutrality also faces the pressure of controlling carbon emissions. Therefore, a reasonable assessment of the carbon emission efficiency of resource-based cities is conducive to their low-carbon economic transformation and the achievement of China’s “dual carbon” goal.The rise of the Internet has increased opportunities to transform economic development and promote sustainable development, and to a certain extent, it can alleviate environmental pressure . As a keIn this context, it is particularly important to objectively and accurately assess the effect of broadband infrastructure construction on economic and social development. The “Broadband China” strategy has elevated broadband facilities to national strategic public infrastructure for the first time, which helps regions to build and improve broadband infrastructure. Therefore, taking the “Broadband China” pilot policy as the entry point to study the construction of broadband infrastructure and its relationship with carbon emission efficiency of resource-based cities is of great theoretical and practical significance to explore new paths and new models for low-carbon economic transformation and high-quality development.Regarding carbon emission efficiency, domestic and foreign scholars have conducted extensive research, mainly focusing on the analysis of carbon emission efficiency measurement. Tone ,7, by co2 emission characteristics of 126 resource-based cities in China, and analyzed the CO2 emission trends and emission reduction potential of resource-based cities in the future. Sun et al. [In recent years, resource-based cities have gradually become the object of carbon emission research in academia, and the studies on carbon emission in resource-based cities are mainly divided into two categories. One category is to study all resource-based cities as research objects, such as Feng et al.  based onn et al.  selectedn et al.  take modn et al.  used eigThe role of information and communication technologies (ICT), especially broadband, in the development of individuals and socio-economics has been widely discussed ,24,25. SAt present, as the network information infrastructure and carbon emissions have gradually attracted the attention of various countries, the relationship between the two has also become a hot research topic in academia. According to the existing theoretical research ,35,36, tIt can be seen that the existing research has laid an important foundation for understanding the relationship between information infrastructure construction and carbon emission efficiency. However, the analytical framework, research objects and research methods for studying the relationship between information infrastructure construction and carbon emission efficiency still have certain limitations and need to be further deepened. First, in terms of analytical framework, the existing research mainly focuses on the carbon emission reduction effect of Internet infrastructure development, and few scholars have conducted in-depth studies on its influence mechanism. Second, in terms of research objects, the existing research mainly studies carbon emissions from the macro perspective of countries, regions or industries, and rarely evaluates the influence of information/digital infrastructure on carbon emission efficiency from the perspective of cities  with obvious high carbon emissions characteristics. Third, in terms of research methods, most of the existing research on the development of information infrastructure uses a single indicator for measurement, and the analysis process rarely involves the discussion of endogeneity. Therefore, based on the exogenous policy influence of “Broadband China”, this paper examines the effects of information infrastructure development on carbon emission efficiency and its influence mechanism from the city level, in an attempt to supplement the existing literature. The marginal contributions of this paper are mainly reflected in: First, this paper uses the DID method to evaluate the effects of “Broadband China” pilot policy on the carbon emission efficiency of resource-based cities from the city level. To a large extent, the endogeneity problems faced by research at the micro-level are avoided. At the same time, in order to further test whether there is an endogeneity problem in the regression results, this paper conducts a series of robustness tests to ensure the reliability of the empirical results. Second, this paper further examines the difference in the influence of “Broadband China” pilot policy on the carbon emission efficiency of different locations and different types of resource-based cities. Third, based on the mediation effect model, this paper analyzes the mechanism by which “Broadband China” pilot policy promotes the upgrading of industrial structure, the accumulation of human capital, and the improvement of urban innovation levels of resource-based cities, thereby promoting carbon emission efficiency.With the vigorous development of the information industry, broadband, as a carrier of information, plays a fundamental role in economic development that cannot be ignored ,50. At pThe “Broadband China” strategy puts forward phased development goals, which include: first, to achieve full speed-up development by the end of 2013. Focus on strengthening the construction of fiber optic network and 3G network, increasing the access rate of broadband network, improving and enhancing the user’s Internet experience; second, from 2014 to 2015, vigorously promote the popularization. By 2015, the basic realization of urban fiber to the building into the home, rural broadband into the countryside into the village, fixed broadband household penetration rate of 50%, 3G and its long-term evolution technology (3G/LTE) user penetration rate of 32.5%, the proportion of administrative villages with broadband reached 95%, schools, libraries, hospitals and other public welfare institutions to basically achieve broadband access. At the same time, we will strengthen the research and development of new technologies in key areas such as optical communications, broadband wireless communications, next-generation Internet, next-generation radio and TV networks, and cloud computing, and achieve original innovation results in some key areas. Third, from 2016 to 2020, to achieve optimization and upgrading. Focus on promoting broadband network optimization and technology evolution and upgrading, broadband network service quality, application level and broadband industry support capacity to reach the world advanced level. In addition, the “Broadband China” pilot policy as an important “Broadband China” strategic special action to implement the “Broadband China” strategy as the core, the construction of “Broadband China” demonstration cities as a precursor to the implementation of the two mandatory national standards of fiber to the home as a grasp to promote the cities and counties in urban areas with full coverage of fiber optic network, and constantly expand the number of Internet broadband access ports, 4G base stations, fiber transformation of old neighborhoods, in order to solidly promote the construction of broadband cities. To this end, in 2014, 2015 and 2016, around 119 “Broadband China” demonstration cities were selected in three batches after comprehensive consideration of regional distribution, geographical conditions, and existing network facilities . After bAt present, informatization and digitalization have gradually become a breakthrough topic in China’s economic and environmental governance . BroadbaHypothesis 1 (H1).“Broadband China” pilot policy plays a positive role in promoting carbon emission efficiency of resource-based cities in China.Resource-based cities have obvious stage characteristics in the development process. Different resource-based cities face different political backgrounds, geographical characteristics and humanistic thoughts , and theHypothesis 2 (H2).The influence of “Broadband China” pilot policy on carbon emission efficiency of different locations and types of resource-based cities is heterogeneous.(1) “Broadband China” pilot policy affects the carbon emission efficiency of resource-based cities by promoting the upgrading of industrial structure.The promotion effect of the “Broadband China” pilot policy to accelerate industrial structure upgrading is mainly reflected in the development of new industries and transformation of traditional industries. On the one hand, the “Broadband China” pilot policy promotes the continuous improvement of Internet-based network infrastructure construction and the emergence of new economic growth modes, such as new industries, new business modes and new models, which will help the development of a new generation of information technology and give better play to the role of data as a factor of production, drive industrial upgrading and promote the optimization of industrial structure. For example, in the field of services, intelligent transformation of services will be realized to promote the formation of a new industrial production and service system, which is conducive to optimizing the industrial structure of resource-based cities and realizing the transformation of traditional industries with high energy consumption, high carbon emissions and high pollution to new green industries with low energy consumption, low carbon emissions and low pollution, thus solving the contradiction between industrial development and environmental protection  and prom(2) “Broadband China” pilot policy affects the carbon emission efficiency of resource-based cities by promoting the accumulation of human capital.The effects of the implementation of the “Broadband China” pilot policy on the accumulation of human capital is mainly reflected in the rich information resources brought by the continuous improvement of information infrastructure. With the implementation of the “Broadband China” strategy, it has promoted the continuous improvement of Internet-based network infrastructure construction and accelerated the progress of urban information technology, which is conducive to the formation of various data resource platforms, better collection, storage, development and utilization of various resources and data, and has a positive effect on innovative organizations ,67, and (3) “Broadband China” pilot policy affects the carbon emission efficiency of resource-based cities by promoting the improvement of innovation level.The implementation of the “Broadband China” strategy can effectively improve the innovation level of resource-based cities. On the one hand, broadband infrastructure construction promotes the development of the Internet, which has the ability to accelerate the cross-regional integration of capital, talent, technology and other innovation factors. This will lead to knowledge spillover effects and encourage the proliferation of technological innovation systems characterized by manufacturing, learning, research and use. On the other hand, with the improvement of broadband infrastructure, enterprises’ broadband network capabilities and network-based process re-engineering and business innovation capabilities are gradually increasing, which drives technological advances in manufacturing, energy conservation, and environmental protection, and will help enterprises to enhance their technological innovation capabilities in research and development (R&D) and production sectors . TechnolBased on the above analysis, hypothesis 3 is proposed:Hypothesis 3 (H3).“Broadband China” pilot policy can improve the carbon emission efficiency of resource-based cities in China by promoting the upgrading of industrial structure, accumulation of human capital, and the improvement of innovation level.This paper takes 116 prefecture-level resource-based cities among the 262 resource-based cities identified in the National Sustainable Development Plan for Resource-based Cities (2013–2020) issued by the State Council in 2013 as the research objects. In view of the completeness and availability of the data, this paper excludes Bijie and Jinchang from the urban sample, and uses the mean method to supplement the missing data, and finally selects the annual data of 114 resource-based cities from 2004 to 2018 as the research sample, with a total of 1710 observations. The data are mainly from the “China Statistical Yearbook”, “China City Statistical Yearbook”, “Urban Construction Statistical Yearbook” from 2005 to 2019, and statistical bulletins of national economic and social development of prefecture-level cities. Some missing data are filled using the mean method.i and t represent city and time, respectively. itCEE is the core explained variable of this paper, representing the carbon emission efficiency of the ith city in the t year. broadbandit represents whether city i is determined as the “Broadband China” demonstration city in the t year. If a city is determined as the “Broadband China” demonstration city in the current year and subsequent years, let broadbandit = 1; if the city is not identified as a “Broadband China” demonstration city, then broadbandit = 0. itControl represents the other control variables affecting the carbon emission efficiency of resource-based cities with time and individual changes, tγ represents the time-fixed effect, iη represents the fixed effect of individual cities, and itε is the random error term.This paper takes the construction of the “Broadband China” pilot policy as a quasi-natural experiment and uses the DID method to test the influence of the “Broadband China” pilot policy on the carbon emission efficiency of resource-based cities. Since the “Broadband China” demonstration cities are determined in batches, this paper draws on the research of Beck et al.  to constitMid represents the intermediary variable, which is industrial structure upgrade (industrs), human capital (humancs) and innovation level (innova), respectively, and the meanings of other variables are the same as model (1). The mediation effect of the model test is divided into three steps: The first step is to estimate the coefficient α1 of the model (1), and test the total effect of the “Broadband China” demonstration cities construction on the carbon emission efficiency of resource-based cities. If α1 is significantly positive, it means that the “Broadband China” demonstration cities construction can significantly improve carbon emission efficiency of Resource-Based Cities. In the second step, the coefficients φ1 and λ2 of model (2) and model (3) are estimated, respectively. If both are significant, this indicates the existence of a mediating effect. On this basis, if λ1 is not significant, it indicates that the mediating variable has played a role of complete mediation; if λ1 is significantly positive and less than α1, it indicates that the mediating variable has played a partial mediating role. Finally, if at least one of the coefficients φ1 and λ2 is not significant, the Sobel test is used to judge whether the mediating effect exists or not.In order to further identify the intermediate mechanism of the “Broadband China” pilot policy affecting the carbon emission efficiency of resource-based cities, this paper draws on the practice of Baron and Kenny  and uses1) Explained variable: carbon emission efficiency (itCEE). The current measurement of carbon emission efficiency is mainly through the construction of carbon emission input-output index system and the use of DEA and SFA models to measure the efficiency [ Core explanatory variable: “Broadband China” pilot policy variable (broadband). This paper adopts the form of policy dummy variable to set. Specifically, in the year when a resource-based city is selected as the “Broadband China” demonstration city and the years after, the value of broadband is 1; otherwise, the value of broadband is 0. The Ministry of Industry and Information Technology (MIIT) and the National Development and Reform Commission (NDRC) designated 119 broadband China demonstration cities in 2014, 2015, and 2016, including 36 resource-based cities. Due to the serious lack of data in some cities, this paper excluded Bijie and Jinchang. Finally, 36 resource-based cities identified as “Broadband China” demonstration cities are included in the research sample, which constitutes the treatment group of the study sample, and the other 78 resource-based cities are the control group.(3) Mediating variables: ① Industrial structure upgrading (industrs). Industrial structure upgrading, as a key way to achieve low-carbon economic development, reflects the process in which the focus of urban economic development and industrial structure gradually shifts from low level to high level. In this paper, the proportion of output value of the tertiary industry in GDP is selected to represent industrial structure upgrading. ② Human capital (humancs). Human capital is an important driving force of urban innovation activities; in particular, senior human capital is conducive to promoting the development of green and low-carbon technology innovation, thus reducing carbon emissions and improving carbon emission efficiency. This paper selects the proportion of students in institutions of higher learning to measure the stock of human capital in cities. ③ The level of innovation (innova). Urban technological innovation is the core driving force to realize low-carbon transformation and promote carbon emission efficiency. Therefore, the proportion of urban invention patents in the total patents is selected in this paper to represent the innovation level of a city.(4) Control variables. This paper mainly controls the following variables in the empirical research process: ① Transportation (traff). As a basic and leading industry in the national economy, the transportation, storage and postal industry is also a major contributor to energy consumption and direct carbon emissions, and is closely related to urban economic development and environmental pollution, its scale plays an important role in the development of low-carbon urban transformation, and the number of employees in the transportation, storage and postal industry can, to a certain extent, reflect the size of the industry, so this paper selects the number of employees in the transportation, storage and postal industry as a representative indicator to measure the level of transportation. ② Employment level (unemprate). The unemployment rate of a city reflects the employment situation of the city, and the change of employment status will affect the change of household consumption embodied carbon emissions, and then affect the carbon emission efficiency. Therefore, this paper refers to existing studies [perra). There is a direct relationship between road infrastructure on traffic CO2 emissions and the increase of population density, and the increase of population density will reduce the level of CO2 emissions per capita, so this paper refers to the existing studies [cityscale). The city scale reflects the level of urbanization development to a certain extent, among which the city population size is directly related to carbon emission, so this paper refers to the existing studies [education). To a certain extent, education investment reflects the importance that the city attaches to education and the degree of human capital demand, and human capital can accelerate the transformation of production factor allocation, improve economic efficiency, promote clean production, and thus have an important impact on carbon emission efficiency. Therefore, this paper selects the proportion of education expenditure to total fiscal expenditure to measure the education investment of the city. ⑥ Gross Domestic Product Growth rate (iGDP). The economic growth of resource-based cities is inextricably linked to the growth of mineral energy consumption and carbon emissions, so the GDP growth rate is chosen to characterize the economic development level of cities in this paper. ⑦ Physical capital level (phycl). Physical capital is an important guarantee to promote scientific and technological innovation and achieve economic transformation, which is conducive to the development and promotion of green and low-carbon technologies and will have a certain impact on carbon emission efficiency, so this paper uses the proportion of fixed asset investment to GDP to characterize the level of physical capital in resource-based cities with reference to the study of Liu et al. [, the value of itbroadbandj− (itjbroadband) is 1; otherwise, it is 0. The meanings of other variables are the same as those of model (1). In particular, itbroadband4 denotes the treatment group that was identified as a “broadband China” demonstration city for 4 years or more. j < 0), the estimated coefficients of all policy dummy variables are not significant. This shows that, before the implementation of the policy, there is no systematic difference in the trend of carbon emission efficiency of resource-based cities in the treatment group and the control group, and the parallel trend assumption is satisfied.Satisfying the assumption of parallel trend is a prerequisite for the use of the DID method. Therefore, to evaluate the implementation effect of “Broadband China” pilot policy, it is necessary to conduct parallel trend test on the explained variable, the carbon emission efficiency of resource-based cities, that is, to test whether there is a significant difference in the trend of carbon emission efficiency of resource-based cities before they are identified as “Broadband China” demonstration cities. Therefore, referring to existing research , this paIn order to further control the influence of the difference between the demonstration cities identified as “Broadband China” and the resource-based cities not identified as “Broadband China” demonstration cities under the “Broadband China” pilot policy on carbon emission efficiency, referring to Rosenbaum and Rubin , this paThe matching and balance test was carried out using the city-level control variables as matching covariates, and the results are shown in In order to exclude the influence of other factors on the carbon emission efficiency of resource-based cities during the implementation of the policy, this paper draws on the practice of Cantoni et al. . There aSpecifically, this paper randomly selects 36 cities from 114 resource-based cities as the treated group, and assumes that these 36 cities are identified as “Broadband China” demonstration cities, and other resource-based cities are the control group. A “pseudo-policy dummy” for a placebo test, and thus an erroneously estimated coefficient. Since the “pseudo-treat group” is randomly generated, the simulated policy dummy variable will not have an influence on the explained variable, and its erroneous estimated coefficient should be close to 0. This paper conducts 500 random samplings, and heter is the proxy variable of the dummy variable of location type (area) and the dummy variable of city type (city); ξ1 is the estimated coefficient of the interaction term between the dummy variable of “Broadband China” pilot policy, the dummy variable of location type and city type, indicating that the heterogeneous influence of the “Broadband China” pilot policy on its carbon emission efficiency due to the differences in the location and type of resource-based cities; itξ, imξ and tmξ are the interaction effects, which are used to control the “Broadband China” pilot policy variables, the interaction terms of the city dummy variables and the heterogeneity dummy variables of the “Broadband China” pilot policy, and the interaction terms of the time dummy variables and the heterogeneity dummy variables of the “Broadband China” pilot policy; the other variables have the same meaning as in model (1).Although the influence of the “Broadband China” pilot policy on the carbon emission efficiency of resource-based cities has been demonstrated above, are there certain differences in the responses of different regions and different types of resource-based cities within the pilot scope to policy shocks? The discussion of this issue is conducive to an in-depth understanding of the conditions for the construction of information network infrastructure to promote the improvement of carbon emission efficiency in resource-based cities. In view of this, this paper further discusses and analyzes the heterogeneity of urban location and urban type. Furthermore, the model (5) is built for heterogeneity regression analysis:Resource-based cities are important energy security bases in China and they play an irreplaceable role in economic development. However, the problem of unbalanced and insufficient development among resource-based cities in different locations is still prominent. Resource-based cities in the eastern region, supported by a series of experimental policies such as reform and opening up, took the lead in developing with convenient transportation and location advantages. Compared with resource-based cities in the central and western regions, factors such as economy, human resources, and scientific and educational resources are superior. In addition, the coastal resource-based cities have a good agricultural foundation, relatively developed industries, and more convenient water and land transportation, while the development of inland cities is relatively backward. Therefore, the differences in location advantages between resource-based cities in the east, central and western regions and the differences between resource-based cities in coastal and inland regions may lead to differences in the policy effects of “Broadband China” demonstration cities construction.area) and assigned the value of cities in the eastern region to be 1 and cities in the central and western regions to be 0. Then, this variable was multiplied by the dual difference item (broadband) of the pilot policy into the regression. The results are shown in column (1) and column (2) of To this end, this paper constructed the location dummy variable ” (referred to as the “Plan”) issued by the State Council in 2013, resource-based cities are classified into four types—growth, mature, declining, and regenerative—according to their resource security capabilities and economic and socially sustainable development capabilities. This paper further divides resource-based cities into three categories—growth period, mature period, and decline period—according to their life cycle characteristics. The resource-based cities in the growth stage are the growth and regeneration resource-based cities listed in the plan, and the resource-based cities in the mature and declining stages are the mature and declining resource-based cities listed in the plan, respectively. Additionally, based on model (5), regression is carried out to analyze the heterogeneity of the influence of the “Broadband China” pilot policy on the carbon emission efficiency of different types of resource-based cities. The regression results are shown in From This paper takes the “Broadband China” pilot policy as a quasi-natural experiment and uses the DID method to investigate the influence of broadband infrastructure on the carbon emission efficiency of resource-based cities and the influence mechanism. The main conclusions are as follows: First, the broadband infrastructure has a significant role in promoting the carbon emission efficiency of resource-based cities. Secondly, the influence of broadband infrastructure on the carbon emission efficiency of resource-based cities has city location heterogeneity and city type heterogeneity. Finally, the “Broadband China” pilot policy can promote the improvement of carbon emission efficiency of resource-based cities by promoting the upgrading of industrial structure, the accumulation of human capital, and the improvement of urban innovation level.First, local governments should actively promote the construction of broadband infrastructure. All localities should seize the opportunity of the “Broadband China” pilot policy to increase investment in broadband network infrastructure development. At the same time, it is necessary, in order to achieve high-quality development of city, to vigorously strengthen the construction of 5G networks, data centers, and the Internet of Things, give full range to the functions of broadband infrastructure, and activate the organic connection between urban innovation subjects, elements, and industries, truly internalize the new generation of information technology as the driving force for urban innovation and low-carbon development. Second, policies should be classified according to different locations and types of resource-based cities. Combined with various factors such as the development level of network infrastructure, technology level, and geographical location in each region, the “Broadband China” strategic action is carried out in a targeted manner to better play the role of broadband infrastructure construction in improving the carbon emission efficiency of resource-based cities. On the one hand, for resource-based cities in different locations, the government should steadily advance the pace of broadband infrastructure in the eastern region and increase the financial and technological support needed for the development of the “Broadband” infrastructure in the central and western regions. On the other hand, for resource-based cities of different city types, the government should focus on supporting resource-based cities in the recession period and guide them to vigorously develop the information technology industry, so as to stimulate their latecomer advantages. Meanwhile, it is necessary for resource-based cities in the growth stage to accelerate the cultivation of innovation talent, and gradually improve the construction of broadband infrastructure. Additionally, for resource-based cities in the mature stage, the steady promotion of the construction of broadband infrastructure should be continued. Third, we should make full use of the influence mechanism of broadband infrastructure construction on the carbon emission efficiency of resource-based cities. Above all, according to the development strategies of different cities, more resource-based cities in the dilemma of industrial transformation should be included in the pilot scope, so that they can rely on the construction of network infrastructure to stimulate the vitality of industrial transformation, and realize the transformation of manufacturing from the traditional raw material manufacturing industry with high pollution and high energy consumption to the high-end equipment manufacturing industry with high intelligent processing degree. Additionally, this will further promote the transformation of the service industry to the high-end industry of the value chain, form a multi-dimensional transformation and upgrading path of the industrial structure, and help reduce city pollution. Furthermore, relevant departments should strengthen the introduction of talents, guiding the inflow of high-quality human capital into high-tech industries on the basis of improving the introduction mechanism of high-tech talents. At the same time, relevant departments also need to pay attention to the secondary training of talents, improve the training and reward mechanism of talents, and give full play to the mechanism of capital accumulation on carbon emission efficiency improvement. Lastly, local governments should actively guide and encourage scientific research institutions and enterprises to use broadband networks for informatization, digitization, and intelligent construction, in particular, increasing support for R&D institutions, enterprises, and scientific research institutes related to cleaner production, pollution prevention and green ecology, and building a comprehensive network innovation research and development platform. Promoting the formation of the green innovation research and development platform is especially necessary to fully stimulate the vitality of city green innovation, improve the city green technology innovation ability, and realize the improvement of carbon emission efficiency of resource-based cities.Combining the above conclusions, the policy implications are as follows: There is still much room for improvement in this study. In this paper, a quasi-natural experiment is constructed through the “Broadband China” pilot policy to assess the effects of broadband infrastructure development on the carbon efficiency of resource-based cities and the mechanisms of effects. Although the findings of this paper are supported by a rigorous analytical approach, more in-depth exploration is needed to determine whether the policy choice can truly cover all aspects of broadband development and whether there are more appropriate policies. In addition, the research object of this paper is limited to resource-based cities with special development characteristics, and different countries, regions and city clusters are at different levels of development, and further research is needed to see whether the findings of this paper can be extended to countries, other types of cities or regions. As the world enters the era of digital economy, the development of digital economy cannot be separated from the development of broadband network, and further evaluation of the effects of broadband infrastructure and other policies related to digital economy is of great significance to the national digital economy and digital transformation. For example, building high-tech smart cities has become one of the important ways to solve urban problems such as environmental pollution, and the effects of smart city construction on carbon emission efficiency can be further analyzed.


Salix famelica and Salix eriocephala collected from 33 sites of origin were analyzed using 26,016 single nucleotide polymorphisms to reveal patterns of population structure and genetic diversity. Analyses by Bayesian methods and principal component analysis detected five main clusters that appeared to be largely shaped by geoclimatic variables including mean annual precipitation and the number of frost-free days. The overall observed (HO) and expected (HE) heterozygosity were 0.126 and 0.179, respectively. An analysis of molecular variance revealed that the highest genetic variation occurred within genotypes (69%), while 8% of the variation existed among clusters and 23% between genotypes within clusters. These findings provide new insights into the extent of genetic variation that exists within native shrub willow species which could be leveraged in pan-Canadian willow breeding programs.Perennial shrub willow are increasingly being promoted in short-rotation coppice systems as biomass feedstocks, for phytoremediation applications, and for the diverse ecosystem services that can accrue. This renewed interest has led to widespread willow cultivation, particularly of non-native varieties. However, Canadian willow species have not been widely adopted and their inherent diversity has not yet been thoroughly investigated. In this study, 324 genotypes of  Salix spp.) occur naturally in mesic areas adjacent to watercourses and wetlands across much of North America1. They were traditionally used in the production of woven baskets and as a source for analgesics3. Willow has been planted extensively as riparian buffers and shelterbelts in agricultural regions, and it has been promoted as a promising biomass feedstock for bioenergy applications4. The carbon dioxide that is released from burning willow wood is largely offset by its fixation during photosynthesis, making willow biomass a nearly carbon–neutral energy source5. In addition, shrub willow responds well to coppicing and is capable of reaching maximum annual growth rates at high planting densities in just a few growing seasons6. However, the non-native species that are routinely deployed can suffer from pests and disease, and may elicit concerns with regards to invasiveness7. As locally adapted genotypes offer unparalleled resistance to climatic and biotic stressors, the development of genetic resources for native willow species could facilitate the development of more resilient and productive commercial willow cultivars adapted for Canadian landscapes8.Shrub willow , Salix famelica  Argus , and Salix prolixa Andersson 11. Previously, these taxa have been circumscribed as subspecies: S. eriocephala ssp. eriocephala , S. eriocephala ssp. famelica , and S. eriocephala ssp. mackenzieana (Hooker) Dorn . However, the most recent taxonomic revision of North American Salix12 recognizes these as three separate species: S. eriocephala sensu stricto, S. famelica, and S. prolixa. Argus et al.12 note that although a small portion of the ranges of these taxa overlap, the distribution and morphology are sufficiently distinct to support separate species designations; however, genetic support for this has been lacking13.Willow are dioecious and obligate outcrossers. There are approximately 450 recognized willow species in the world, falling into six sub-genera, of which over 100 occur in North AmericaVetrix, are the most diverse and currently has an estimated 125 species. Only a small fraction of these is currently employed in short-rotation coppice (SRC) systems, whereby willow productivity is maximized by exploiting the propensity of shrub willow to regrow quickly after cutting the stems near the base. Over the past fifty years, willow breeding efforts in New Zealand, Sweden, the UK, and the USA have resulted in the development and deployment of both intra- and interspecific hybrids with important agronomic traits needed for SRC16. Yields of 4–6 oven dry tonnes (odt) ha−1 y−1 have been reported for hybrids when grown in commercial biomass production systems, while experimental plots have produced as much as 24–30 odt ha−1 y−1 in some studies17. On Prince Edward Island, the willow cultivars S. viminalis (5027) and S. dasyclados (SV1) can yield up to 18–20 odt ha−1 y−1 when planted as riparian buffer strips along watercourses18. Consequently, shrub willow are increasingly garnering attention in SRC systems for their uses in managing wetlands19, intercepting excess nutrient runoff20, remediating industrial soils and wastewater22, and improving the biodiversity of birds and insects23.The largest subgenus, 24. Nonetheless, molecular markers have been used to explore the genetic diversity in willow species, including those in a study of European S. viminalis that formed the foundations for the development of an association mapping population25. As in other tree species with well-developed genetic resources, single nucleotide polymorphisms (SNPs) have become the preferred marker as they are both easily distinguished and abundant across the genome, providing broad coverage of the species’ genetic architecture by capturing both adaptive and neutral diversity. For example, SNP markers were recently used to assess the genetic diversity of S. purpurea from Europe that has naturalized in the northeastern USA26. Genome-wide approaches are advantageous because they can simultaneously discern population structure and facilitate marker development for molecular breeding.Despite the economic and ecological importance of willow, the development of genetic resources has lagged behind other woody species such as poplar and eucalyptusSalix  germplasm collection. The priority has been to amass large genetically diverse base populations from which parents can be selected to develop locally adapted genotypes tailored for specific applications. Historically, progeny selection has been performed both under controlled and field environments for traits such as cold hardiness, salinity tolerance, and disease resistance. Moving forward, the objectives are to expand the breeding pool and select new cultivars with agronomic traits that maintain high biomass yields.At Agriculture and Agri-Food Canada (AAFC), in-house willow resources are being managed to develop biomass feedstocks for bioenergy and environmental applications, which has led to the establishment of the Agriculture Canada 27. Similar success has been achieved at Rothamsted in the UK and in upstate New York in the USA, where commercial cultivars have been established that perform well in those respective climates28. As such, there is every reason to believe that concerted efforts with Canadian shrub willow germplasm will help cultivate a productive short-rotation coppice industry in Canada.Willow breeding programs have already had some success in developing new cultivars optimized for SRC production. For example, the Swedish breeding pipeline has generated several high-yielding willow varieties with improved disease resistance and better frost tolerance by crossing genotypes from across Europe, most notably from central and eastern RussiaSalix collection exhibits vast phenotypic diversity in terms of growth, phenology, physiology, and wood chemistry30, we anticipated that there would be considerable underlying genetic diversity to enable future breeding work. In this study, we investigated the geographic pattern of genetic diversity of S. famelica and S. eriocephala using genotyping-by-sequencing (GBS) to: (1) determine whether geoclimatic variables influenced the population structure, and (2) determine the level of genetic variation within and among populations. By uncovering the extent of diversity within the AgCanSalix collection and the relatedness of genotypes, this work will bolster future efforts to breed willow varieties with superior adaptive traits for a host of Canadian climatic regions. The ultimate aim is to develop multi-functional feedstocks that can simultaneously support a burgeoning bioenergy industry and also deliver environmental benefits.Given that the AgCanS. amygdaloides, S. bebbiana, S. discolor, S. eriocephala, S. famelica, S. interior, and S. petiolaris during the summer of 2012. This collection, known as AgCanSalix, includes willow genotypes from Alberta, Saskatchewan, Manitoba, Ontario, Quebec, New Brunswick, Prince Edward Island, Nova Scotia, and Newfoundland and Labrador , 55° in longitude (57–113 °W), and 796 m in elevation (4–800 m). Sampling was done across a west–east transect to capture the geographic variation that these species occupy. The risk of clonal sampling was mitigated by selecting genotypes that were separated by a minimum of 1 km. The cuttings were collected during the dormancy period, bagged separately, and stored at − 4 °C. In the spring, dormant cuttings were dipped in rooting hormone powder  and induced to root in Spencer-Lemaire rootrainers . Willow plants were grown in a greenhouse located at Indian Head, Saskatchewan  with daytime and nighttime temperatures maintained at 23 and 18 °C, respectively, and a relative humidity of 40%. After two months of growth in the greenhouse, the willow plants were transferred to a shade house and allowed to undergo natural senescence. In late October, the frozen root plugs were individually bagged and stored at − 4 °C until the following spring when a common garden plot was established at Indian Head.For 31 to provide environmental variables associated with the sites of origin, including frost-free days , mean annual precipitation , mean summer temperature , and growing degree days > 5 °C   were obtained from nearby government-run weather stations operated by Environment CanadaC) Table . We defiSalix collection, 324 genotypes were selected . Young leaf tissues from the common garden were collected in Eppendorf tubes, immediately flash-frozen in liquid nitrogen, and stored at − 80 °C.For this study on the genetic diversity of the AgCan32 and quantified using a PicoGreen dsDNA kit . Library generation was based on the 96-plex GBS protocol described by Poland et al.33, employing a double-digest with PstI and MspI as the restriction enzymes. The resulting libraries were then sequenced using an Illumina HiSeq 2000 system .DNA was isolated from frozen leaf tissue using the CTAB method34, using the Salix purpurea v1.0 genome35 as a mapping reference. This resulted in an initial dataset comprising 55,453 putative markers. Quality filtering and missing marker imputation were completed using the synbreed package36 in R version 3.5.137. First, markers were recoded to reflect the number of copies of a reference allele: aa = 0, Aa = 1, and AA = 2. Then, markers with a minor allele frequency (MAF) below 1% and > 50% missing data were removed. Missing markers (16.2%) were imputed using the random method in the codeGeno function, in which the missing values were sampled from the existing marginal allele distribution. This resulted in a final filtered set of 26,016 polymorphic markers for 324 genotypes. Among these markers, the highest number of genotypes with missing data was 162 out of 324 total genotypes. However, most of the markers had far fewer genotypes requiring imputation. The mean number of markers with imputed data was 52 out of 324 genotypes, with a standard deviation of 49.Putative GBS markers were identified following the TASSEL 5.0 GBSv2 Discovery PipelineglPca function in the R package adegenet38. Genotypes were broadly classified as either western or eastern, based on the reported ranges for S. famelica and S. eriocephala discerned by examining plant morphology39.To gain preliminary insights into population structure, a principal component analysis (PCA) was carried out using the 40. The default convergence threshold was selected and runs were performed for a number of clusters ranging from K = 1 to K = 10 using all 324 genotypes. The output was then visualised using Distruct for many K’s as implemented in CLUMPAK41. The optimal value of K was determined using the chooseK.py algorithm, from which K = 4 was identified as the number of model components that maximized marginal likelihood, and K = 5 was the number of model components used to explain structure in data. The R package ggplot242 was then used to plot the average membership across fastSTRUCTURE runs at K = 5.The population structure was assessed using a Bayesian approach for posterior inference implemented in fastSTRUCTURE43 and were plotted onto a geographical map of Canada imported from the R package rnaturalearth44 using R packages ggplot242 and ggspatial45. The species ranges proposed by Argus39 were added manually onto this map using Adobe Illustrator .Pie charts representing admixture results for K = 5 were generated by the R package scatterpiedapc function in the R package adegenet38. Here, the optimal number of clusters, K, was determined for the 324 genotypes sourced from 33 sites of origin using the find.clusters function in the R package adegenet38. K = 5 was inferred by applying the Elbow method, a heuristic commonly used to manually evaluate the number of clusters as the point of inflection on a plot of the Bayesian information criterion (BIC) values against sequential numbers of clusters .For comparison, another evaluation of the population structure was carried out by performing a discriminant analysis of principal components (DAPC) using the HE), observed heterozygosity (HO), Nei’s gene diversity (GD), polymorphism informative content (PIC), and the χ2 statistic for the Hardy–Weinberg equilibrium test and its corresponding P-value were calculated for each cluster using the R package snpReady46. To determine the level of population genetic differentiation, pairwise fixation indexes (FST) for each cluster were calculated based on the method of Weir and Cockerham47, using the R package StAMPP48.To evaluate genetic diversity, the allelic frequency (p and q), MAF, expected heterozygosity  and geographic distance among 33 sites of origin was assessed by performing an isolation-by-distance (IBD) analysis using the R package adegenet49. The significance of the associations was tested based on a Mantel test with 10,000 permutations using the mantel.randtest function. Local density was plotted with two-dimensional kernel density estimations determined using the R package MASS50.The correlation between genetic distance (pairwise F-statistics) using the R package poppr51. The AMOVA partitioned variation among geographic clusters, between genotypes within clusters, and within genotypes.To calculate the extent of hierarchical population structure, an analysis of molecular variance (AMOVA) was performed based on the infinite alleles model  was conducted using the R package Plots were formatted using Adobe Illustrator .S. famelica and S. eriocephala genotypes, 26,016 polymorphic markers were obtained that were distributed evenly across all 19 chromosomes. Using this dataset, we set out to characterize the AgCanSalix collection by examining the population structure with PCA  is depicted in Fig. K = 2 to K = 10 are provided as Fig. Next, we used fastSTRUCTURE to further assess the population structure and evaluate genetic variation within the collection. This analysis revealed the existence of five clusters, based on the optimal model complexity Fig. A. The prK = 5  was performed Fig. A. This a= 5 Fig. . Again tS. famelica and S. eriocephala genotypes , followed closely by West and Central (0.090). This could again be an indication that the Central genotypes represent a transition zone from west to east. Alternatively, the genetic diversity across this region could be underestimated due to the relatively low number of genotypes and sites of origin included from Ontario and southern Quebec. Significant isolation-by-distance was evident across the range, and the correlation between geographic distance and FST was high .The hierarchical AMOVA revealed that 8.2% of the genetic variance existed among the five clusters identified by the Bayesian analysis Table . GreaterS. famelica and S. eriocephala genotypes.RDA analysis was performed in an effort to tease apart the impact of geoclimatic variables on population structure. The first three constrained axes explained 86.4% of the total variance. RDA1  spanning much of the temperate and boreal zones of Canada. Our results reveal that the AgCanSalix collection falls into a clear pattern of western and eastern genotypes and simultaneously provides genetic evidence to support separate designations for S. famelica and S. eriocephala.To our knowledge, this is the most comprehensive range-wide genetic study of 54, the Canadian genotypes likely arose following northward expansion from glacial refugia. Notably, Allard and Leonard55 described disjunct S. eriocephala populations as far south as Georgia and Alabama in the USA. Following the northward range expansion, there may have been physical and/or climatic barriers that contributed to genetic isolation and divergence. Furthermore, it is likely that willow habitats have been drastically altered by centuries of human colonization and extensive changes to the landscapes across North America56.The contemporary distribution of these taxa has been shaped by both ongoing and historical processes. As the majority of the present-day range of these species was covered by the Laurentide Ice Sheet during the Pleistocene epochSalix collection does not include genotypes from west or north of the Rocky Mountains. Further work in this region is needed to examine whether any additional western cluster(s) may exist or whether these genotypes would group with those from Alberta in the Far West cluster. There is reason to believe that such analyses could capture additional clusters as salicologists have previously described shrub willow west of the Rocky Mountains and in the northwestern boreal region as a separate species, S. prolixa 12. Moreover, in this scenario, the Alberta genotypes in the Far West cluster could perhaps represent a transition between S. prolixa and S. famelica.In this study, willow genotypes sourced from broad natural ranges were examined using Bayesian and principal component analyses to elucidate population structure. Both approaches captured five clusters corresponding to five geographic areas. At present, the AgCanS. famelica and S. eriocephala. In addition, the barrier of the Great Lakes and the Canadian Shield along the longitudinal axis could plausibly limit gene flow between western and eastern populations. However, more genotypes from areas north of the Great Lakes throughout Ontario and southern Quebec are needed to validate this hypothesis. Evident genetic discontinuities along latitudinal and longitudinal transects have previously been observed for other North American tree and shrub species, including Pinus contorta, Salix melanopsis, Populus trichocarpa, and Populus balsamifera60.After the last glacial retreat, range expansion and recolonization patterns may have contributed to the modern-day regional population structure in S. famelica) and eastern  genotypes. Of particular note are the genotypes from Batchawana Bay in Ontario in which significant admixture was observed with genotypes from the West, Central, East, and even Far East clusters. The genotypes from Newfoundland and Labrador are also noteworthy as they appear to be the most divergent, an observation which conforms with decades of research on the genetic isolation of Newfoundland flora61.With the evidence at hand, it appears that germplasm collected from Ontario may represent a zone of transition between western (HO) ranged from 0.125 to 0.162 for the five clusters that were identified by Bayesian analysis. Comparable levels of HO were reported among 45 genotypes of S. eriocephala collected from Ontario . Conversely, 58 genotypes collected from Quebec and New Brunswick exhibited higher heterozygosity . However, it is difficult to directly compare SNP-based studies on genetic diversity, such as ours, with earlier techniques.High recombination rates among outcrossing and sexually propagated plant species generally result in high genetic diversity. Among the genotypes sampled in this study, the observed heterozygosity  for 416 genotypes of S. eriocephala collected from three watersheds in upstate New York, USA. S. silicicola, a willow species endemic to the sand dunes of northern Saskatchewan, also displayed a higher FST = 0.156 in a study of 204 genotypes66.Generally, levels of genetic differentiation are relatively lower among wind-pollinated taxa than for insect-pollinated species. Shrub willow are mainly, but not exclusively, insect-pollinatedFST values in comparison with all other clusters. This observation challenges the results of the population structure analyses which suggested that this cluster is the most divergent. It could be that the smaller sample size in this cluster is the source of the high level of heterozygosity. These contrasting observations bring into question the validity of the Far East cluster, particularly given the vast geographic distances between many of these genotypes. Of the 30 genotypes from Newfoundland, only five (from Stephenville) grouped in the Far East cluster, along with two genotypes from Ontario (Batchawana Bay and Kenora), and one from Quebec (Quebec City). Further analyses of the Far East genotypes and of additional germplasm from Newfoundland are needed to assess the extent of genetic differentiation in this region.The Far East cluster showed the highest levels of heterozygosity and also had the lowest S. famelica and S. eriocephala are highly outcrossing species. Previous molecular genetics studies on S. eriocephala have also reported that, although there exists a high level of allelic diversity, the majority of genetic variation occurs within populations and subpopulations rather than between populations65. Keller et al.60 analysed a collection of Populus balsamifera, a species with a comparably extensive range, and similarly found a low variance (4.4%) among groups. Such patterns of genetic diversity offer both challenges and opportunities for breeding efforts since highly heterozygous outcrossing populations can exhibit diverse phenotypes in crossings, but it can be difficult to tease apart adaptive alleles of interest from the abundant neutral genetic diversity.Analysis of isolation-by-distance revealed that genetic diversity was strongly correlated with the geographic distance between genotypes. However, the AMOVA revealed that the greatest variance occurred within genotypes (68.9%), while variance among the five clusters represented only 8.2%, indicating that S. famelica and S. eriocephala rely on wind for long-distance pollen and seed dispersal67, extensive gene flow could theoretically occur between distant populations. More locally, shrub willow are prone to extensive clonal reproduction since broken twigs and branches frequently root and take hold nearby68.A redundancy analysis that asked whether biogeographic factors can help explain some of the genetic diversity and population structure pointed to precipitation, frost tolerance, and summer temperatures. This is hardly surprising given the vastly different climates that occur across the west–east spatial separation of the clusters. A single willow plant produces thousands of small seeds which lack storage reserves and require suitably mesic sites for establishment. Since both 69 found that the growth of shrub willow in southern Quebec was evidently more limited by precipitation than by summer temperatures. On the other hand, western genotypes which occur in more continental climes may be better adapted to hotter, drier conditions in spring and summer and could be more resistant to frost damage in winter. Western sites of origin had MST values ranging from 11.2 to 15.5 °C and eastern sites ranged from 9.4 to 15.4 °C, while the average number of FFD was 189 for the eastern sites of origin compared to 166 for western sites. It is highly plausible that the west–east differences in climate contributed to the divergence of S. famelica and S. eriocephala. Of course, the willow genotypes examined in this study occur across gradients of climate and geography such that genotypes in the Central cluster may be adapted to intermediate conditions between the western and eastern regions.While western sites of origin in this study receive less annual precipitation compared to eastern sites, they are also exposed to more extreme summer and winter temperatures. Higher precipitation coupled with high soil moisture offer biologically suitable habitats for seed germination and seedling establishment. On average, the sites of origin from the Central, East, and Far East clusters received two-and-a-half times more precipitation annually  compared to West and Far West sites of origin (468 mm). Labrecque and TeodorescuS. eriocephala genotypes consistently performed the best on more productive sites70. However, in a study on the effectiveness of willow in phytoremediation of contaminated soils from industrial sites, it was observed that genetics accounted for the majority of performance differences71. Accordingly, it will be vital to account for the effects of local growing conditions when testing the performance of novel cultivars by conducting breeding trials in common garden experiments.Although geoclimatic variables clearly influenced the diversification of Canadian shrub willow, the enduring question is whether genetics trump environmental conditions with regards to performance in SRC and environmental applications. While the growth and yield of willow clones showed strong genetic-by-environmental interactions in common garden coppicing trials, S. famelica and S. eriocephala. One of the goals in establishing the AgCanSalix collection was to identify traits that influence climate adaptation, particularly with regards to drought, frost, and thermal stresses. Given that these are evidently key drivers for diversity in S. famelica and S. eriocephala, it should be eminently feasible to select for such traits in controlled crosses. Ultimately, the aim is to develop cultivars that are better suited to warmer and drier environments, particularly in the face of rapid global climate change.Opportunities abound to breed advanced willow cultivars by exploiting the expansive genetic diversity that exists across the ranges of S. eriocephala has been promoted for bioenergy production as it is known to produce good yields of high-quality biomass72. Future breeding work with the AgCanSalix collection will seek to prioritize traits for high productivity in SRC. There have already been some successes in identifying genotypes of S. eriocephala for greater biomass yields73 and for phytoremediation applications as well74. However, the breeding work to date has been limited to a relatively small number of genotypes, just a fraction of those available in the AgCanSalix collection.In many ways, the use of Canadian shrub willow in SRC and environmental applications is still in its infancy. Salix collection. For example, genotypes with improved resistance to insect herbivores were readily identified among a collection of S. eriocephala hybrids75. However, the challenge of selecting and maintaining such adaptive traits from highly heterozygous outcrossing populations must not be underestimated76. The next steps will be to identify candidate genotypes for breeding in order to harness the inherent diversity of Canadian shrub willow and advance efforts to develop elite cultivars.This study helps lay the groundwork for future controlled crossing experiments. Locally adapted genotypes that are more resilient to environmental and biotic stresses could be selected by exploiting the allelic diversity of the AgCanSupplementary Information.