Candidula coudensis, that show an exceptionally narrow distributional range. The genetic survey presented here shows the existence of five main mitochondrial lineages in Vale da Couda that do not cluster together suggesting independent evolutionary histories. Our results also indicate a departure from the expectation that species with restricted distributions have low genetic variability. The putative past and contemporary models of geographic distribution of Vale da Couda lineages are compatible with a scenario of species co-existence in more southern locations during the last glacial maximum (LGM) followed by a post-LGM northern dispersal tracking the species optimal thermal, humidity and soil physical conditions.The Iberian Peninsula has an extensive record of species displaying strong genetic structure as a result of their survival in isolated pockets throughout the Pleistocene ice ages. We used mitochondrial and nuclear sequence data to analyze phylogeographic patterns in endemic land snails from a valley of central Portugal , putatively assigned to Phylogeography combines evidence from both population genetics and phylogenetics, to understand the evolutionary processes that shape geographic population structure . These eIdentifying the drivers of geographic distribution patterns is also essential to understand the population dynamics in space and time. Species distribution modeling (SDM) allows one to examine the relationship between the identified presence records of a species, lineage or related species, with the environmental characteristics of these locations. From the inferred relationship it is possible to estimate the response, function and contribution of environmental variables , and preLand snails are good models for evolutionary studies, since phylogeographic patterns are often preserved due to their limited dispersal capabilities and specific habitat requirements . Also, sCandidula present in Europe, from eastern Canary Islands to the Balkans and northwards to Scotland and southernmost Sweden are represented by 24 putative species. Portugal has 8 endemic species  from a total of 12   . NeverthCandidula coudensis  is an endemic species described recently with a highly restricted geographic distribution in Vale da Couda, Leiria, Portugal. A broad-scale sampling of this region (ca. 100 km2) revealed that C. coudensis could only be found within a small area of ca. 13.5 km2 (13.5 km2 .C. coudensis: (1) Vale da Couda individuals may form a monophyletic clade, indicative of a single population on a restricted area in the absence of major phylogeographic breaks  and (2) Vale da Couda individuals are expected to show reduced levels of haplotype and nucleotide diversities, consistent with an isolated population on a limited geographical area.The species can be found in open rocky limestone substrata, olive tree grounds, areas of natural vegetation, in roadside areas or even in stone-walls in nearby houses . The extUsing a combination of DNA sequences  and geo-referenced field records of the species we sought to address the above hypotheses by revealing the genetic diversity and geographic structure of contemporary Vale da Couda individuals, and reconstructing its demographic history. Using Iberian environmental data relative to past and current conditions retrieved from public repositories, we inferred locations of the putative refugia during the LGM and provided estimates of relative environmental suitability of Vale da Couda individuals that can assist future fieldwork.Suitable habitat of Vale da Couda individuals consists mainly of boulders and stonewalls, and those were the preferred sites for sampling. Density was very variable, between 0.1 and one individuals per square meter on suitable habitat. The total sampling area at each of the four sites comprised a few square meters, and up to six individuals were collected within a few meters of each other. Samples for genetic assessment were lumped into four sites, which were GPS referenced.Sampling in Vale da Couda resulted in 73 individuals collected from four different sites . We receDNA was extracted from the samples using a CTAB protocol . Univershttp://msr.cs.nthu.edu.tw), which separates the information from the chromatogram into a major and minor sequence, corresponding to each allele, while comparing the sequence information with a given reference sequence  (h)   and haplπ) (h)  diversitπ) (h) . To evalfixation  and Destntiation  statistintiation . The varntiation .Phylogeographic relationships among haplotypes of COI and ITS1 alleles were represented using the Median Joining Network method  implemenCandidula spp. individuals were collected in different locations .To place the Vale da Couda samples in a broader phylogenetic context and to ascertain the non-monophyly of the individuals from Vale da Couda (given the very distant haplogroups found—see Results section below), putative ocations  to ascerriterion  implemenriterion , we seleriterion . The robhttp://cmip-pcmdi.llnl.gov/cmip5/): CCSM, CNRM, IPSL and MIROC3.2.The study area was the Iberian Peninsula. Bioclimatic variables for current conditions were retrieved from WorldClim dataset  in 30 ar2 cells. As the distribution of this recently discovered species is restricted  based on the COI data set yielded the topology depicted in C. olisippensis. The other clade included haplogroup D and Haplogroup E from Vale da Couda that showed an unresolved phylogenetic position. These specimens grouped with C. setubalensis from Arrábida , and the annual precipitation (BIO12) as the three most relevant variables defining the niche for Vale da Couda lineages . ENFA maC. coudensis. Results presented are surprising because they reflect higher genetic variability than expected in species with restricted geographic distributions, low dispersal potential and large estimated population sizes. Despite our results add to a growing list of taxa showing limited distribution and high genetic diversity . Nevertheless, the following assumptions generated from the large population size premise must be considered:The maintenance of diversity in rare species can be explained by the existence of a large effective population size . The 100da Couda  may repr (1)Candidula from Vale da Couda, our network High mutation rates. Possible explanation for the observed diversity could be the occurrence of high mutation rates, as reported in other groups of land snails . High mu (3)Candidula living individuals in Vale da Couda are present in a highly restricted geographic area (13.5 km2), we would expect that most of the individuals would share a common ancestry and generate groups of rather closely related haplotypes given their putative low dispersal abilities. However, our haplotype network showed a large number of gaps within each haplogroup, not supporting the shared ancestry hypothesis;Common ancestry in a restricted area. Because  (4)Existence of cryptic species. Finally, if each haplogroup represents a cryptic species, we would expect smaller population sizes with more related individuals within each group and fewer gaps within haplogroups. This expectation is not met by our results considering the large number of gaps separating haplotypes of the same haplogroup .circa 20 k years). The LGM model shows a wider area that extends to the south with higher probability of occurrence compared with the present-day distribution of the individuals from Vale da Couda  currently occupying these southern locations. The differences between the paleo-model and the contemporary model are somewhat unexpected considering that most of the northern hemisphere terrestrial organisms have contracted their geographic distributions to the south during harsher glacial climate conditions, and have expanded their distribution by re-colonizing former northern territories after deglaciation , as suggested by LGM hindcast. A postglacial change of climatic conditions towards lower precipitation in the Lisbon area may have caused Vale da Couda lineages contraction to its actual distribution using the suitable Mesozoic calcareous rock as a corridor. Although we have addressed some common hindcasting uncertainties by using different GCMs and a clamping mask hindcast approach, we could not resolve the lack of accurate lithological data for emerged coastal land in the LGM. More appropriate habitats not depicted in our models could have existed in regions near the coast.C. setubalensis and C. arrabidensis occur in sympatry, as well as C. olisippensis and C. coudensis  cannot be fully explained with present data. LGM hindcasts revealed the existence of putative glacial refugia south of the current distribution of the lineages of Vale da Couda. These findings have implications for the understanding of the genetic characteristics of rare and endemic species. From a conservation perspective, Vale da Couda lineages do not seem to be endangered, with high genetic diversity within and between lineages maintained by putative large effective population sizes.The genetic survey presented here revealed the existence of four main mitochondrial lineages in Vale da Couda (previously attributed to a single species) with independent evolutionary histories and exhibiting extremely narrow geographic ranges. These results do not corroborate previous morphological studies that considered the existence of a single species, 10.7717/peerj.3069/supp-1Table S1Candidula.Sample location and summary statistics for the genus Click here for additional data file.10.7717/peerj.3069/supp-2Table S2Bioclimatic variables for current conditions retrieved from WorldClim dataset .Click here for additional data file.10.7717/peerj.3069/supp-3Table S3Click here for additional data file.10.7717/peerj.3069/supp-4Figure S1Estimates of net evolutionary divergence between haplogroups  and within lineages , based on Tamura-Nei distances.Click here for additional data file.


Ficus carica L.) is a gynodioecious species with two sexual forms: male trees (caprifigs) with male and female flowers and female trees that produce only female flowers that will result in the edible fig syconium. In this study the genetic diversity of 20 Tunisian accessions of caprifig is analyzed using SSR markers previously developed for this crop.The common fig (The results revealed that the 13 pairs of primers used amplified a total of 37 alleles in the accessions studied. The number of alleles per locus ranged from two to six, with a mean value of 2.85 alleles per locus. Observed and expected heterozygosities showed mean values of 0.33 and 0.29 respectively. UPGMA cluster analysis and Principal Component Analysis grouped the caprifig accessions analyzed in three groups.The results obtained show a low genetic diversity in the Tunisian accessions of caprifig studied and, in spite of analyzing samples from different geographic regions, no clear groupings based on geographical origin are observed suggesting widespread exchange of caprifig plant material through vegetative propagation among different areas in Tunisia. Ficus carica L.; 2n = 2x = 26) [Ficus L. contains about 750 species of woody trees, epiphytes and shrubs, mainly of tropical and subtropical distribution, divided into six subgenera [The common fig 2x = 26)  belongs x = 26  bubgenera  that shaubgenera –6 domestFicus carica L. is a gynodioecious species with two sexual forms: male trees (caprifigs) that produce syconia with separate male and female flowers and female trees that produce syconia with only female flowers that will develop into edible seeded figs if pollinated. Since only male trees produce pollen the common fig is functionally a dioecious species. Three types of female figs are cultivated [Ficus is dependent on the coevolution of Ficus species with pollinating wasps of the family Agaonidae [Blastophaga psenes L. The caprifigs produce fruits in three crop cycles during each growing season, each harboring the larvae, pupae and, temporarily, the adults of the pollinating Blastophaga [Blastophaga psenes in cold mountainous areas and the unavailability of mature caprifig “profichi” when female figs are receptive.ltivated : the comgaonidae . In the stophaga : “proficIn Tunisia, as in other Mediterranean countries , fig hasVarious studies have reported the use of morphological traits –19 and iThe main objective of this study was to characterize and evaluate the genetic diversity of 20 Tunisian caprifig accessions using SSR markers in order to develop strategies to preserve the endangered genetic resources of this species.This study was carried out on 20 Tunisian caprifig accessions originated in different geographic regions and with different phenotypic traits Table . All the−1 with modified TE buffer  and used for PCR amplification.Genomic DNA was extracted from lyophilized young leaves following a CTAB-based method optimized previously for fig . After pTM 2000XL capillary DNA analysis system .A set of 13 SSR loci , previously developed in fig  were useDifferent parameters of genetic diversity were estimated: number of alleles per locus (A), allelic frequencies, observed heterozygosity (Ho), expected heterozygosity (He), Wright’s fixation index and probability of identity (PI). Those parameters were only computed using the SSRs that produced amplification of a single locus . The computations were performed with the program POPGENE 1.32  and IDENThe 13 SSR loci produced successful and repeatable amplification fragments in all the 20 caprifig accessions analyzed, resulting in a total of 37 alleles ranging from two  to six (LMFC30) alleles per locus (Table obs > Hexp) was observed for the LMFC15, LMFC31, LMFC18, LMFC27 loci whereas a deficit of heterozygosity (Hobs < Hexp) was observed in LMFC24 and MFC2 loci (Table Observed heterozygosity ranged from 0.05 (LMFC32 and LMFC21) to 0.80 (LMFC31) with a mean of 0.33  between the cophenetic coefficient and the similarity matrix was chosen. The UPGMA dendrogram obtained showed three main groups among the genotypes analyzed  includes 7 accessions , the second (B) contains 10 accessions  and the third (C) includes 3 accessions . Several homonymies were detected in the genotypes analyzed since genotypes having the same name in the same or different locations are genetically different. This includes all the different samples analyzed of Magouli (2 samples), Bouharrag (3 samples), Bithri (2 samples) and Dhokkar (6 samples). Among all the nodes obtained only three groups have bootstrap values higher than 50 %. These groups include the three undistinguishable accessions  (bootstrap of 95 %), those three accessions and Bouharrag3 (bootstrap of 71 %) and the accessions Limi and Sawoudi (bootstrap of 62 %).Among all the possible UPGMA dendrograms generated, that with the highest cophenetic correlation (zed Fig.  with thrPrincipal Component Analysis shows that the three first principal components explain 50.50 % of the total variability. The contributions of PCA1, PCA2 and PCA3 were 21.33, 19.26 and 9.92 % respectively. Fig. Some of the main groups obtained in the UPGMA and PCA studies are correlated with the geographic origin of the genotypes. Thus the group C in Figs. The present study provides the first molecular database of Tunisian caprifig accessions using SSR markers. The set of SSR markers used indicates that the genetic diversity between accessions studied is overall narrow and that no clear relationship is found between geographical origin and genetic composition suggesting exchange of caprifig genetic material among different regions. Additional studies with caprifigs from other countries should be performed in order to have a clear picture on overall caprifig genetic diversity and optimize collaborative caprifig genetic resource management and conservation.


Marmota himalayana, which plays an essential role in the maintenance, transmission, and prevalence of plague. To achieve a more clear insight into the differentiation of M. himalayana, complete cytochrome b (cyt b) gene and 11 microsatellite loci were analyzed for a total of 423 individuals from 43 localities in the northeast of the QTP. Phylogenetic analyses with maximum likelihood and Bayesian inference methods showed that all derived haplotypes diverged into two primary well-supported monophyletic lineages, I and II, which corresponded to the referential sequences of two recognized subspecies, M. h. himalayana and M. h. robusta, respectively. The divergence between the two lineages was estimated to be at about 1.03 million years ago, nearly synchronously with the divergence between M. baibacina and M. kastschenkoi and much earlier than that between M. vancouverensis and M. caligata. Genetic structure analyses based on the microsatellite dataset detected significant admixture between the two lineages in the mixed region, which verified the intraspecies level of the differentiation between the two lineages. Our results for the first time demonstrated the coexistence of M. h. himalayana and M. h. robusta, and also, determined the distribution range of the two subspecies in the northeast of QTP. We provided fundamental information for more effective plague control in the QTP.The primary host of plague in the Qinghai-Tibet Plateau (QTP), China, is  The massive pandemics of the plague in Eurasia resulted in the death of an estimated 75 to 200 million people and caused significant economic, cultural, and population changes worldwide . RMarmota minor as the oldest marmot fossil at 10.3 mya . Following the protocol of Steppan et al. (2011) , ar (2003) .himalayana and robusta lineages occurred at about 1.03 mya . It happened at almost the same time when the divergence between M. baibacina and M. kastschenkoi  took place, and it was much earlier than the divergence between M. vancouverensis and M. caligata . After that, the two lineages both split into two branches again, respectively at 0.34 mya (95% HPD: 0.22–0.48 mya) and 0.42 mya (95% HPD: 0.26–0.62 mya), with the latter almost simultaneously with the divergence between M. vancouverensis and M. caligata.The BEAST analysis  indicateHo) and expected heterozygosity (He) (averaged across loci), ranged from 0.52 and 0.54 to 0.89 and 0.80  to 14  with a total of 124 observed alleles and an average number of 11.3 . The aveand 0.80 . Only onhimalayana lineage) and the B-I and B-II groups (the mixed range of two lineages), while inside the mixed range, no genetic differences could be observed between the B-I and B-II groups at all, demonstrating the strong gene flow and little reproductive isolation between the two lineages in the mixed range. The meaning of the Fst values confirmed our results. It was estimated as 0.0677 for group A vs. group B-I and 0.0765 for group A vs. group B-II in contrast to a smaller Fst value for group B-I vs. group B-II (0.0067).In total, 23 populations with 259 individuals were allocated to region A, i.e. group A, while the rest 20 populations with 164 individuals were assigned to region B. In region B, 60 samples from 14 populations were divided into group B-I while the remaining 104 samples from 20 populations into group B-II. According to Structure Harvester, the optimal value for K was estimated to be 2, and then 3 and 4 . The barb sequences of all 423 specimens from the northeast of QTP were clustered into two basal well-supported and monophyletic lineages, lineage I and lineage II. The lineages corresponded to the referential sequences representing M. h. himalayana and M. h. robusta, respectively  listed in Steppan et al. (1999) [M. vancouverensis and M. caligata (1.2%). Taking the subspecies taxonomic status of the himalayana and robusta lineages we are going to illustrate here into consideration, this result seems unexpected but it is consistent with the different divergence histories. As revealed in the tMRCA analysis in BEAST  and much earlier than that between M. vancouverensis and M. caligata . M. vancouverensis is a special marmot species living exclusively on an island, the Vancouver Island [M. caligata more recently, which can be explained by the isolation of that island from the mainland after the sea level rise at the end of the last glaciation, little chance for second contact since the isolation enabled them to evolve into different species [M. himalayana, the situation was just the opposite due to frequent and great second contacts as discovered here in the mixed range, leaving the two diverged lineages merely developing into subspecies in M. himalayana. Back to our topic, a certain distribution pattern of haplotypes in the two lineages emerged in the studied range. Only haplotypes in the himalayana lineage were detected in all 259 individuals in a pretty large area in the northeast (region A). However, all haplotypes in the robusta lineage and a few ones in the himalayana lineage, specific to this region  or shared with region A , were found in the southwest (region B)  . UndeniaM. h. himalayana ‘apparently differs in being grayer, without the more ochraceous tints of M. h. robusta, and in having the black of the forehead less extensive’ as described by Howell (1929) [M. h. himalayana was found in our study, and the sampling there was sufficient to strut a compelling assessment. Though an exclusive breeding range for M. h. robusta was not found in the studied area; it is believed to be located somewhere else in the whole species’ range. Third, the two units were proved as not reproductively isolated. The analyses on the genetic structure revealed strong genetic differentiation accompanied by obvious admixture between the unique breeding range for the himalayana lineage and the mixed range of two lineages  were significantly larger than that between group B-I and group B-II (0.0067). These results serve as a sufficient demonstration for the strong gene flow between the two lineages in the mixed range and the absence of reproductive isolation between them. Last, although no defining features based on genetic or developmental facts were verified here, sufficient time has elapsed for some “heritable geographic variation in phenotype” to be built since the lineages split at about 1.03 mya, provided that the story is not too intricate. So, although a reciprocal monophyly is considered not necessary for subspecies differentiation and cannot act as a proper excuse to reject any subspecies, it does be an important positive signal indicating the occurrence of subspecies.The conception and diagnosable criteria of subspecies as a taxonomic unit have gone through a long and impassioned debate –46. Pattl (1929) . Second,lineages . FurtherM. h. himalayana and M. h. robusta has been in use for years. Although no studies especially aimed at their differentiation are available, detailed descriptions of their distribution in China could be found in many taxonomic checklists, two opinions out of which will be discussed here. According to Wang (2002) [M. h. robusta in northwestern Yunnan, western Sichuan and Qinghai (except Qaidam Basin). However, in Smith and Xie (2009) [M. h. himalayana lives in a larger range than Smith and Xie (2009) [M. h. himalayana outside Qaidam Basin.The subspecies rank of g (2002) , the nome (2009) , the fore (2009)  expectede (2009)  about boY. Pestis, which can act as important determinants for the role and contribution of a particular host population in the maintenance and transmission of plague, are recognized to depend on host species and even genetic differences among individuals and populations [Peromyscus maniculatus) and California voles  were already reported, and the resistance in M. californicus was revealed to be genetic [M. himalayana is a question of future studies, which may provide constructive knowledge for the prevention and control of plague. Whatever, it is proved by our study that the genetic differentiation in the Himalaya marmot population in QTP is discrete and heterogeneous, which can serve as reliable supporting information for more precise investigations on plague epizootiology in future. Meanwhile, a unique breeding range for M. h. himalayana and a mixed range where both subspecies are distributed and interbreed fairly frequently are located in the northeast of QTP. If the subspecies contribute to the plague differently, corresponding prevention and control strategies should be developed for different ranges.Resistance and susceptibility to ulations –49. Vari genetic , 51. TheM. himalayana and can act as a guide to the government’s marmot-killing actions to control the marmot population and conserve its biological diversity simultaneously.On the other hand, due to the key role of the Himalayan marmot in the maintenance, transmission, and prevalence of plague , the govM. h. himalayana and M. h. robusta, was confirmed in different aspects, i.e., the well supported reciprocal monophyly, quite a long period of divergence (1.03 mya) and large genetic distance exceeding that between almost any other subspecies pair and even some species pair in the same genus, a unique breeding range for M. h. himalayana and clear signals for admixture between them. Provided all these proofs, we proved that the subspecies differentiation of Himalayan marmot and a unique breeding range for M. h. himalayana, together with a second contact range where both subspecies are distributed and interbreed, took place in the northeast of QTP.As discussed above, the taxonomic rank of two subspecies, S1 File(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


This initiative would generate a number of centers, both economic and political, which would facilitate greater interaction. To explore how information flows are merged and the specific opportunities that may be offered, Chinese cities along “the Belt and Road” are selected for a case study. Furthermore, urban networks in cyberspace have been characterized by their infrastructure orientation, which implies that there is a relative dearth of studies focusing on the investigation of urban hierarchies by capturing information flows between Chinese cities along “the Belt and Road”. This paper employs Baidu, the main web search engine in China, to examine urban hierarchies. The results show that urban networks become more balanced, shifting from a polycentric to a homogenized pattern. Furthermore, cities in networks tend to have both a hierarchical system and a spatial concentration primarily in regions such as Beijing-Tianjin-Hebei, Yangtze River Delta and the Pearl River Delta region. Urban hierarchy based on web search activity does not follow the existing hierarchical system based on geospatial and economic development in all cases. Moreover, urban networks, under the framework of “the Belt and Road”, show several significant corridors and more opportunities for more cities, particularly western cities. Furthermore, factors that may influence web search activity are explored. The results show that web search activity is significantly influenced by the economic gap, geographical proximity and administrative rank of the city. The centrality of these cities has been strengthened; as a result of both policy and attention, they have become popular cities.The initiative of jointly building the “Silk Road Economic Belt and the 21st Century Maritime Silk Road” (hereafter referred to as “the Belt and Road”) was proposed by Chinese President Xi Jinping in 2013. The initiative aims to establish a new pattern of trade and an infrastructure network that connects Asia, Europe and Africa . Since tA city’s position determines its benefit from the initiative. More specifically, a city’s power arises from the forms of interaction and exchange that occur through complex networks . Among tIn this paper, we have three central aims. The first aim introduces the methodological framework and argument that information flow is as an appropriate database for examining the increasing importance of knowledge opportunities for cities. Second, the informational ranking of Chinese cities along “the Belt and Road” is derived. The paper uses the Baidu search volume in a comparative analysis of an informational approach, and Chinese cities along “the Belt and Road” are selected for a case study to determine how these cities are merged in cyberspace and the specific opportunities that are offered in the context of “the Belt and Road”. Third, the research field is extended by examining the factors that affect information flow.The remainder of this paper consists of four main sections. The first section discusses the theoretical rationale for an analysis of urban networks among Chinese cities along “the Belt and Road”. The second section briefly describes the selected cities, calculation method and hypotheses for the potential influencing factors. The results are presented in the third section. The fourth section discusses the main findings and potential drawbacks of the data outlined in this section. Following this section, an overview of potential avenues for further research is presented.The next section discusses the theoretical rationale for an analysis of urban networks among Chinese cities along “the Belt and Road”. We subsequently discuss the debate regarding “the Belt and Road” to provide context and outline its remit. Following that, we acknowledge that exploring urban hierarchies among Chinese cities along “the Belt and Road” is essential to advance our understanding of center cities and their dynamic connections. Then, we argue the conceptualization of hierarchies and networks. We acknowledge that a city’s network of information flows presents an amalgam of hierarchical and networked characteristics. Given this understanding, we subsequently introduce and discuss in detail the feasibility of using the Baidu index as a source of information flows.Cities may be connected at different scales. Empirical studies tend to analyze the spatial organization by examining functional linkages at different scales, including global –13, regi“The Belt and Road” initiative is a “spatial fix”  expectedIn Castells’s seminal work, he argues a network society encompasses a “global network” of cities rather than a few urban cores at the top of the hierarchy . Urban rThe use of inter-city connection data has become an increasingly popular way to examine urban hierarchies . This paIn the present “information age”, the quickening and deepening connections among cities, which are considered characteristic of the contemporary globalizing world, call for novel ways of understanding the relative importance of cities . The rapIt is a truism of the information age that information is everywhere, however, with respect to information relevant to scholars, the identification and utilization of “good” data are relatively new developments . MoreoveTherefore, informational infrastructures and associated material flows between cities must be investigated, as well as the evolving cyberspaces of cities in relation to digital information . The conth China Internet Development Report", by the end of 2016, the number of Chinese internet users reached 731 million [http://news.xinhuanet.com/fortune/2017-02/24/c_129495316.htm), Baidu has become the most widely used web search engine in China.The internet may be considered a window into a city. Web search activity has become an important way for individuals to learn about a city, and it is regarded as the informational link , 47. Acc million . Because million , Baidu ibaidu.com using a combination of the city name and the search content. For example, the combination of “Beijing” and “weather” had the largest search volume when individuals nationwide searched for Beijing. The most common combination for the selected cities was weather. The large search content also includes Yaohao, subway, university, and coach when people nationwide searched for Beijing. When individuals searched for Shanghai, the predominant search content included Disney, university, subway and traffic. For the famous tourist city Sanya, the largest search content included photograph, tenement, wedding veil and Haikou. Most of these search contents imply strong connections among cities. For example, when individuals in Beijing search for weather, subway and other transportation routes into Shanghai, they tend to have the following two reasons: (1) they would go to Shanghai in the following days, or (2) they have family members or friends in Shanghai. In this case, some aimless search volume that is also included is assumed to be temporal. The previously described search content listed showed a strong desire for connections  behind these web search activities.To understand the type of web search activity among cities, six cities from the study area are selected, and the nationwide search contents for these cities are sorted . The larWhen analyzing the Baidu web search, most of the search content may be traced back to the connection among cities. Specifically, the flow is expressed as information flow, however, in essence, it is an integrated flow including people, material and information. Interestingly, Baidu web search activity provides the possibility for a city-based analysis based on users’ geo-tagged places, which typically correspond to their location. Baidu is a web search engine for data sharing based on the massive behaviors of internet users. The Baidu index may provide researchers with the scale of a keyword, its evolution over time, and the distribution of internet users who search for these words. For example, when a user in Shanghai searches for a combination of content that includes the key word “Beijing”, the search volume referred to as the Baidu index would be recorded. Key words such as “Beijing road” are not included. Specifically, when users in Beijing are searching for combinational contents including the key word “Shanghai”, subtitled Disney, or university, these web search data are subsequently stored in the Baidu index. Therefore, the connection between Shanghai and Beijing, which is bidirectional, may be calculated based on the web search activity of users.In contrast to infrastructure-orientated approaches, in which information flows are measured, the Baidu index approach deals with virtual flows. The Baidu index is operated standardly based on its web search data that may present not only information infrastructures but also their associated material flows between cities, which indicates an overview of urban networks among the cities.Against this background, the exploration of urban hierarchy among Chinese cities along “the Belt and Road” has two meanings. First, this work presents a way to capture connections and identities of places using associated material flows in China, which has the largest number of web search engine users worldwide. Second, this study mapping urban networks among Chinese cities along “the Belt and Road” also has substantial realistic significance, particularly for understanding the center city along “the Belt and Road”.Chinese cities along “the Belt and Road” have been introduced as a trial to examine how information flows may be used as a framework to empirically study the contemporary global political economy. On March 28, 2015, the National Development and Reform Commission, Ministry of Foreign Affairs, and Ministry of Commerce jointly issued the “Vision and Actions on Jointly Building Silk Road Economic Belt and 21st-Century Maritime Silk Road”. In the Chinese cities along “the Belt and Road” were selected for a case study to investigate how these cities are merged in cyberspace and the specific opportunities that are offered in the context of “the Belt and Road”. The results of this study indicated which of the 36 cities are nearly in intensive collaboration and which cities are removed from this type of collaboration. This research may help the Chinese government provide relatively preferential policies to these node cities in the future.https://index.baidu.com/, and the web search volumes may be represented by the Baidu index between two cities, an inter-city informational connection matrix may be constructed with data from the Baidu search engine. There are 1260 links among the 36 cities each year, thus, three matrices spanning the years 2011, 2014, and 2016 are built. All collected data comply with the terms of service for the Baidu website.Given that web search activity produced between two cities may be explored via The data process in this paper includes four steps.①ij represents the web search volume of city i, in connection with city j; Standardizing the connectional data among 36 cities. The specific steps are as follows:②i represents the city’s external connection index which reflects the city’s external contact strength in the entire network system; Based on the first step, the second step is to calculate the city’s external connection index. The external connection index reflects the urban hierarchy in the network system.③ij represents the connection degree between cities i and j, The third step is to calculate the connection degree between cities based on the first step. The indicator characterizes the informational contacting degree among cities in urban networks. The calculation is as follows.④i represents the connection degree of city i, Zij represents the connection degree between cities i and j, and Zii represents the connection degree between cities i and i.The fourth step is to calculate the connection degree of each city for characterizing each city’s connection to other cities within the urban network system. The specific calculation is as follows.Euclidean distances between 36 cities are calculated, and a spatial distance matrix is established. The research considers the significant changes that have occurred in inter-city transportation distances over the previous 20 years as well as the vast territory of China. This paper examines only straight-line distances; in the 36×36 matrix, the minimum distance is 74 km from Xiamen to Quanzhou, and the maximum distance is 3504 km from Urumqi to Sanya.Factors such as city size (in demographic or economic terms), policies, physical distance and borders may affect the spatial interactions between cities . From th(1)Assumption 1: cities with high levels of economic development tend to have high web search activity.The presence of information cyberspace often depends more on informational infrastructure . In 1977(2)Assumption 2: web search activity tends to occur between cities with large economic gaps.ij as the economic gap to show the extent of the economic gap between two cities. In this paper, GDP per capita is chosen to minimize the impact of the population and GDP. The economic gap between two cities is calculated asi represents the GDP per capita of city i, and Gj represents the GDP per capita of city j. A smaller Aij indicates a larger economic gap between cities i and j whereas a larger Aij indicates a smaller the economic gap between cities i and j.We define the variable AThis article assumes that web search activity tends to occur between cities with a large economic gap.(3)Assumption 3: high administrative rank cities tend to have more opportunities to gain high web search activity.The influence of local government is mainly reflected in the intervention of the regional economic pattern, which is referred to as the administrative region economy . High ad(4)Assumption 4: web search activity is affected by geographical proximity.Urban network theory suggests that links among cities are not equivalent. Moreover, a rule of distance attenuation exists . TherefoCombined with the city location, informational connection matrices of 2011, 2014 and 2016 were used to investigate the spatial characteristics of the network evolution . OverallUrban networks have gradually become more balanced. Among all cities, informational connections among 18 cities account for 68% of the total, and the information connection of Beijing, Guangzhou, Shenzhen, and Shanghai in 2011 accounts for 19.7% of the total. Specifically, we identify 22,843 links among four cities  in 2011, 47,643 in 2014, and 51,034 in 2016, which account for 19.7%, 18.9%, and 18.5%, respectively, of the total connections among the selected cities. Furthermore, a polycentric pattern is explicitly present in 2011, and a polycentric structure is maintained in Beijing and Shanghai in 2014, whereas a homogenized structure develops prior to 2016.The network has undergone continuous adjustment, in which web search activity experienced a process of weakening between certain cities and strengthening between other cities. Compared with 2011, links among Hohhot, Lhasa and other cities significantly weakened in 2014. In contrast, links among Beijing, Shanghai, Guangzhou and other cities have significantly strengthened, which led to the central role of these cities. During 2014 and 2016, the links among Beijing, Kunming and other cities significantly weakened, resulting in a relatively homogeneous structure across the entire network in 2016. Although the links among cities have been reduced (or enhanced) in the previous two years, the links in 2016 have increased to varying degrees compared with those in 2011 .The web search process between cities is bidirectional, indicating that the web search activity between cities includes two aspects. For example, web search activities of city i on other cities and other cities on city i are both included. In this paper, two definitions are provided as follows: the sending Baidu index is the total web search volume of city i for other cities, and the receiving Baidu index is the total web search volume of other cities for city i.Using the method of natural breaks, an apparent pyramidal hierarchy is present as indicated in The map of the receiving Baidu index is presented in In contrast, the sending and receiving Baidu indexes for the selected three years are largely correlated. That is, a city with a larger sending Baidu index tends to have a larger receiving Baidu index. Thus, a city that searches more for other cities is reciprocally searched for by other cities.Some findings arise if the context of “the Belt and Road” is considered. “The Belt and Road” cities in China have significant centrality. In essence, the connection degree shows a substantial difference among cities .As China’s political, economic and cultural center, Beijing has the strongest network connectivity with 0.074, which is higher than that of the secondary city of Xiamen. In addition, cities such as Shenzhen, Hangzhou, Chengdu, Sanya, Shanghai, Xi’an, Wuhan and Chongqing have similar and strong network connectivities, with connection degrees between 0.03 and 0.05. Cities such as Guangzhou, Qingdao, Nanchang, Tianjin, Dalian, Ningbo, Lanzhou, Changsha, Haikou, Harbin, Shenyang, Zhengzhou and Nanning have connection degrees between 0.02 and 0.03. Furthermore, cities such as Quanzhou, Hefei, Fuzhou and Kunming have lower connectivity degrees between 0.01 and 0.02. In addition, cities such as Zhanjiang and Yantai have the lowest connection degrees in the network.The connection degree of cities has substantially improved from 2011, 2014 to 2016. For example, the connection degree of Beijing was only 0.0019 in 2011, however, it increased to 0.07 in 2014, which is more than three times the value in 2011. The connection degree of cities in 2014 showed some changes, in which the connection degree represents an abnormal value compared with the values in 2011 and 2016, the apex of which was Kunming with an increment of more than 8 times. A further examination of the Baidu index showed that Kunming significantly increased because of the violent incidents that occurred there in early March 2014, which explained the sudden increase in web search activity. However, the connection degree returned to a relatively normal state in 2016.As shown in the Although discussions of urban hierarchy, in which “senior” cities have power over and control those below, may be unfashionable or unhelpful, it makeA shared opportunity for the 36 cities has been shown from the increase of national and regional centers since the implementation of “the Belt and Road” initiative. First, Beijing, Shanghai and Shenzhen are always in the list of national centers in 2016, and cities such as Chongqing, Xiamen, and Chengdu rank significantly as national centers. Second, the number of regional centers has increased since 2014. More regional sub-centers such as Hefei, Nanning and Nanchang have transformed into regional centers in 2016. Moreover, cities such as Guangzhou, Chongqing, and Qingdao are assumed to be regional centers. This phenomenon also verifies that the urban hierarchy based on web search activity is not exactly the same as the urban hierarchy based on physical and infrastructure links, 59. UrbThe connection degree is consistent with the external connection degree of cities. As shown in The connection degree under web search activity is not influenced by its own economic development. As shown in These results show that there is no obvious correlation between urban network connectivity and economic development. However, what about the correlation between the connection degree and economic gap? Will the web search activity tend to occur between cities with similar economic development or cities with a large economic gap?ij, which presents the economic gap between cities, and the ordinate is the web search activity. The results show that web search activity tended to occur between cities with a large economic gap in 2011, 2014, and 2016. Thus, when the Aij value is lower, both the economic gap and web search activity are also lower. With an increase in Aij, the economic gap between cities decreases, which causes a reduction in web search activity.The results indicate that web search activity tends to occur between cities with a large economic gap. As shown in Figs ij≈0, the economic gap between cities is large; the value of the web search activity was 860 in 2011, 1700 in 2014 and 1965 in 2016.The web search activity under the same economic gap has been increasing yearly. When A2 + 1942.3x + 16,565. This is a quadratic equation with a negative slope and a relatively high fitting degree of 0.7161 (R2 = 0. 7161). The results show that within a certain distance range, the increment of distance leads to an increment of web search activity between cities. However, this increasing process is quite limited and begins to decrease beyond a certain range of distance. The result indicates that the distribution of city nodes is related to distance. Specifically, nodes closer to the city are associated with greater inter-city web search activity, whereas nodes farther away are associated with less web search activity. From the fitting function and its correlation coefficient (R2) for each year, the impact of geographical proximity on inter-city web search activity has the same trend. For the years of 2011, 2014 and 2016, the correlation coefficients (R2) are 0.7123, 0.7103 and 0.7158, respectively, which also indicates a strong correlation between distance and web search activity.To explore the relationship between geographical proximity and web search activity and the change that occurs in this relationship, a fitting analysis of inter-city web search activity at different distances is undertaken . Each fiIn this paper, the largest web search activity among cities occurs within the distance of 1000–1500 km, accumulated to 34,937 among selected years, whereas the least amount of inter-city web search activity occurs within a distance of 3,300–3,500 km, accumulated to 3,443 among selected years.As previously discussed, the large search volumes between cities occur in the range of 1000–1500 km; 187 total links among cities occur in this distance. Among these links, 178 links based on at least one provincial capital city occurred, accounting for 95%. Moreover, 115 links between provincial capitals occur, accounting for 62%. Based on these data, it is tentatively speculated that web search activity is significantly influenced by administrative rank. This impact is estimated as follows.Among the 36 cities selected, 26 cities are provincial capitals, and 10 cities are non-capital cities. Therefore, 650 links between provincial capital cities and 260 links based on at least one provincial capital city are supposed to occur. Accordingly, 36 links based on at least one provincial capital city and 4 links between non-capital cities are expected. However, the results show that large search volumes tend to occur among provincial capital cities . In the “The Belt and Road” is expected to facilitate interactions among numerous city centers. This exploratory study would contribute to the literature on “the Belt and Road” by providing a systematic assessment of the urban hierarchies for Chinese cities along “the Belt and Road”. Furthermore, with the rapid development of internet technology and increase in Chinese internet user scale, the internet has become an important window to show inter-city links in China. The study introduces the Baidu database as a comparative analysis of information flows in China, which is an effective supplement to the existing research. Specifically, the Baidu index represents an under-utilized database of information that has been characterized as geo-tagged and massive. The proposed research is specifically concerned with how information flows are merged and the specific opportunities that are offered for Chinese cities along “the Belt and Road”. Therefore, research on cities in this area would not only be helpful to guide the long-term development of “the Belt and Road” but would also make economic centers more attractive by providing productivity benefits.Overall, the results show that urban networks have undergone continuous adjustment, in which web search activity experienced a process of weakening between certain cities but strengthening between other cities. Some cities, especially western cities in China, have shared more opportunity since the initiative of “the Belt and Road”. Our research also confirm that there are significant urban hierarchies under informational flows among cities along “the Belt and Road”. Our research also examine some factors that could influence the urban network based on web search activity. (1)The results show that it is influenced more by the economic gap between cities than by the economic development of its own city. In other words, cities with a large economic gap tend to be more attractive for high web search activity. This finding can be explained by the traditional theory of flow space that cities with large economic gaps tend to have large potential energy differences. As a result, more elements flow among cities, and cities become more attractive. (2)The web search activity is affected by geographical proximity, which is consistent with the "distance decay". Nodes closer to the city are associated with greater inter-city web search activity, whereas nodes farther away are associated with less web search activity. Thus, the development of information technology does not cause the demise of geospatial relationships. Why is web search activity affected by geographical proximity? Traditional geographical proximity plays an important role in searching for other cities by network users. The reason is that although information dissemination on the internet is no longer affected by the friction of distance cost, the searching behavior for information is remain limited by real life. These limitations cause a relationship increase to occur in neighboring cities. (3)The web search activity is a selection process for administrative rank. Regression models indicate that conditioning on administrative characteristics is positively associated positively with a high degree of informational flows in China. The distance among provincial cities is always larger than 600 km. For this reason, web search activity increases consistent with a distance increase in the range of 0 to 600 km. This finding is consistent with the previous research results that urban networks of information flows/exchange present as an altogether messier and more dynamic amalgam of hierarchical characteristic .This paper discusses the web search activity, connection degree, urban hierarchy and its influencing factors, which is a new attempt to investigate not only “the Belt and Road” but also urban networks under the influence of information flows. The results also demonstrate the effectiveness of using web search activity to study urban network systems. Our analysis suffers from several limitations, which indicate potential directions for future research. First, web search activity is vulnerable to emergencies; for example, we cannot ignore the fact that in some years, cities that are affected by emergencies, such as the violent incidents in Kunming had a higher web search activity and a greater connection degree in network. Second, web search activity is influenced not only by the economy, distance, and administrative rank of a city but also factors such as infrastructure construction and industrial division. Third, a proportion of the web search volume without a semantic connection cannot be excluded. Therefore, it is necessary to introduce multivariate assessments.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Enhalus acoroides across the Indo-Malay Archipelago. Heterozygosities were consistently high, and significant isolation-by-distance, consistent with restricted gene flow, was observed. Both a neighbour joining tree based on DA distance and Bayesian clustering revealed three major clusters of E. acoroides. Our results indicate that phylogeographic patterns of E. acoroides have possibly been influenced by glaciation and deglaciation during the Pleistocene. Recent physical oceanography such as the South Java Current and the Seasonally Reversing Current may also play a role in shaping the genetic patterns of E. acoroides.The Indo-Malay Archipelago is regarded as a barrier that separates organisms of the Indian and Pacific Oceans. Previous studies of marine biota from this region have found a variety of biogeographic barriers, seemingly dependent on taxon and methodology. Several hypotheses, such as emergence of the Sunda Shelf and recent physical oceanography, have been proposed to account for the genetic structuring of marine organisms in this region. Here, we used six microsatellite loci to infer genetic diversity, population differentiation and phylogeographic patterns of  Ceriops , separating the Indian and Pacific Oceans . AlthougCeriops  and the norrhiza . Similarnorrhiza , the marlbicilla , and ther planci . Despiter planci .Haptosquilla pulchella have revealed a sharp genetic break across the Java Sea, which divides the population into north (Pacific) and south (Indian) , these islands coalesced to form a large land mass known as the Sunda shelf . Despite(Indian) . Other sa crocea  and frig affinis , showed ps tagal  and the a ovalis , revealeRhizophora mucronata Lam. in the Malay Peninsula and Sumatra  of this species is also possible due to strong currents  following the manufacturer’s protocol. Eight microsatellite loci  developed by ®, Foster City, CA, USA) (Silica gel-dried leaves (5 cm in length) from each shoot were ground using a mortar and pestle. Genomic DNA was extracted using DNeasy plant mini kit  .2O, 5 µl of 2× PCR Master Mix, 1 µl of 0.2 µM primer mix and 1 µl template DNA. PCR cycling was carried out for 5 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 1.5 min at 57 °C and 30 s at 72 °C with an extension of 30 min at 60 °C in the final cycle. (ii)three loci  were amplified in a 20 µl total reaction containing 7.8 µlddH2O, 2 µl of 1× PCR gold buffer, 2 µl MgCl2 (2.5 mM), 2 µl dNTPs (200 µm), 1.5 µl of each 0.75 µm primer, 1U taq polymerase  and 3 µl DNA template. PCR cycling was carried out for 15 min at 95 °C, followed by 32 cycles of 30 s at 94 °C, 1.5 min at 58 °C and 60 s at 72 °C with an extension of 30 min at 60 °C in the final cycle. All PCRs were performed on a 2720 Thermal Cycler . GeneScan™ 500 LIZ®  was used as an internal lane standard and PCR products were sent to UC Berkeley DNA Sequencing Facility, USA, where they were run on an ABI 3130 Xl automated DNA sequencer . Individual genotypes were scored using Geneious ver. 7.0.6 (Polymerase Chain Reaction (PCR) was performed into two ways. (i) five loci  were amplified using the Qiagen multiplex PCR Kit in a 10 µl total reaction containing 3 µl ddHr. 7.0.6 .HO) and expected heterozygosities (HE) were calculated using Genalex ver. 6.5 , observed heterozygosities  among localities were imported from Arlequin. A geographic distance matrix was generated using the PATH tool implemented in Google Earth (Google Earth Plus for Windows) that calculates the shortest distance by sea. In the third matrix used to run the partial Mantel test, populations belonging to the same clustering group were coded with 0, and populations belonging to different clustering groups with 1. Both Mantel and partial mantel tests were performed using Isolation-by-distance Web Service (IBDWS) version 3.23 . To control for the potential existence of hierarchical population structure , we alsoion 3.23  with 10,Two loci (Eaco_052 and Eaco_050) were discarded after initial examination because of quality control issues. Eaco_052 had only a single allele in all populations (monomorphic), while Eaco_050 was not successfully amplified in most samples. Thus, only six loci  were used for further analysis . All samHE) from 0.458 to 0.605, respectively. The TD population had the highest HE value (0.605), while the ANS showed the lowest HE (0.458).A total of 89 alleles were detected across these six microsatellite loci, ranging from one allele at the locus Eaco_019 in BM, KJ, BK, and ANS populations to 13 alleles at locus Eaco_054 in the BM population. The mean number of alleles per locus ranged from 1.57 to 9.57, and the mean number of alleles per population ranged from 4.33 to 7.00 . The BM P < 0.05) prior to a multiple test correction with a false discovery rate. After correction for multiple tests, only two loci significantly deviated from Hardy-Weinberg equilibrium (Eaco_001 and Eaco_054).Deviation from HWE is evidence that simple genetic models may not be appropriate . Hardy-WFST tests by including or excluding locus Eaco_054 (in which three out of seven locations were deviated from HWE after FDR correction); removing Eaco_054 did not change the results of pairwise comparisons, therefore all loci were included in further analysis. Individual genotypes for these loci are reported in Microchecker detected homozygote excess at locus Eaco_054 in TD and ANS and at locus Eaco_001 in TD and NK, likely a result of null alleles. Null alleles may also explain the significant deviation from Hardy-Weinberg expectations at these loci (Eaco_054 and Eaco_001), although inbreeding due to population substructure (Wahlund effect) and natural selection are also possible. Null alleles were potentially implicated in 16 out of 42 locus-population combinations, with an estimated frequency of <0.2 . Null alFST values ranged from 0.127 to 0.359. FST were significant between all pairs of samples with P < 0.001 (FST = 0.359), while the smallest pairwise difference was found between TD and PR and between BM and BK (FST = 0.127).Pairwise  < 0.001 . The larDA distance identified three major clusters   . Isolati = 0.003 . Due to HO, HE, and the number of alleles), genetic differentiation and phylogeographic patterns of E. acoroides in the Indo-Malay Archipelago. Despite the limited number of loci (six microsatellites) used in this study, high observed heterozygosity (HO) is found in all populations. Our results are consistent with the general patterns of limited dispersal and restricted gene flow at various geographical scales suggested by previous studies , while the northernmost population (ANS) possessed lower observed heterozygosity. Sample collection at BK and PR was conducted in undisturbed area where the seagrasses growth in a dense and continues bed . Seagrasses in undisturbed areas tend to be more genetically diverse . AlthougE. acoroides in Lembongan  and Waigeo (Papua), found that observed heterozygosities were 0.436–0.582, respectively . Observed heterozygosities of E. acoroides varied from 0.100 to 0.567 in China  showed a significant correlation between genetic differentiation and geographic distance, even when the most distant and differentiated population (NK) is excluded. These results indicate that there is low gene flow between populations, possibly due to limited dispersal potential of E. acoroides. For example, E. acoroides is limited over a small spatial scale, usually less than 10 km. The low gene flow discovered in the present study and from previous studies . Differentiation of the western Indonesian (Indian population) is common in other organisms and is believed to be largely a result of Pleistocene vicariance    respectiE. acoroides between NK and the other sites.The geographical partitioning of genetic groups observed in the present study may be a result of various events in the past, but the effect of recent physical oceanography cannot be ignored . The impHippocampus  across the Java, Natuna, and South China Seas . These lFinally, although we found significant isolation-by-distance overall, two sites (KJ and ANS) showed high genetic similarity despite being geographically distant from one another. This genetic pattern may also have been influenced by sea level changes during the Pleistocene. When the sea level was at 30 m Below Present Level (BPL), land bridges connecting Sumatra and Borneo via Bangka Belitung seem to have been lost . Thus, bSeagrass beds provide important habitat for a wide variety of organisms in marine ecosystems . HoweverHere we show there is limited connectivity and significant genetic differentiation between all sampling sites. This study also revealed there are three different cluster based on genetic distance and Bayesian clustering. Therefore, we recommend resource managers treat populations across our sampling region as three Management Units (MUs) to maintain the unique genetic characteristics of each region to support seagrass conservation goals. Any restoration activities conducted using local stocks (the same structure cluster) would be beneficial because their genetic diversities have been assessed. Population with higher genetic diversity increases the fitness and reproductive success of the transplants . Geneticoutbreeding depression). Although, this has not been documented or recorded in seagrasses so far, this event has been found in wild populations of scarlet gilia (Ipomopsis aggregata)  .E. acoroides, as evidenced by significant genetic differentiation among sites, and significant isolation-by-distance. Strong genetic differences were observed between the Indian Ocean population (NK) and all six others. These differences probably represent Pleistocene vicariance between Indian and Pacific populations, consistent with similar patterns in other marine flora and fauna. Seasonally variable currents and extreme storm events may play a role in long-distance dispersal of E. acoroides because the phylogeographic pattern connecting the two peripheral locations that are outliers from the IBD pattern match these alternating currents instead. Our data will help to guide management and restoration efforts by confirming high heterozygosity among most local populations, and highlighting boundaries between which translocations are most desirable. In conclusion, when planning a restoration project, genetically guided criteria should be used in the selection of donor material. Therefore, management plans for seagrass in Indonesia should add such genetic evaluation of seagrasses to conservation and restoration planning.Despite the loss of seagrass habitat, these plants manage to maintain good genetic diversity and heterozygosity. Genetic diversity analysis revealed high levels of heterozygosity in all sampling sites. Our results also indicate low gene flow among sites of 10.7717/peerj.4315/supp-1Figure S1Click here for additional data file.10.7717/peerj.4315/supp-2Figure S2Click here for additional data file.10.7717/peerj.4315/supp-3Data S1Click here for additional data file.


The Lancet in 1971, states that the availability of good medical care tends to vary inversely with the need of the population served.Tudor Hart’s inverse care law, set out in an article in Worldwide, our cities and towns are a growing example of the inverse care law. The inequalities in health-seeking and health outcomes among city sub-populations are well documented.,,In many parts of the world, cities open up opportunities for making a living, however basic, for people from rural areas. Cities can also offer an escape from the constraints of patriarchy-, gender-, caste- and race-based hierarchies, and this can have positive ramifications for health.What aggravates the predicament of poor migrants to cities is that they are often regarded as illegitimate non-citizens by the state, even though their contributions to building and running our cities are indispensable.A state that is actively hostile to its most vulnerable urban residents uses many strategies to perpetuate exclusion. One of these is simply making certain urban populations invisible. Across cities in Africa, Asia and Latin America, street children and homeless adults have for generations lived precarious, sometimes violent, and unprotected lives. In a literal sense, they are the most visible of all urban populations, as they have no walls or roof to hide their every move from observers. Yet people of privilege as well as the state treat them as though they do not exist, ignoring any obligations to pursue positive policies for their housing, protection, food and nutrition, health care and education.Instead, these groups must resort to precarious and exploitative relationships with private intermediaries for shelter, employment and other basic needs.A second, often overlapping, strand of public policy – not just in the industrializing but also the industrialized world – is to treat as illegal the self-help efforts of the urban poor in cities. This includes their efforts towards housing  or employment (such as street-vending or the work of undocumented migrants). This adds a layer of social stigma and prejudice to the existing precariousness of these people, and reinforces state neglect, as has been seen with Roma populations across Europe.,This process of illegalization sometimes becomes more vicious when the efforts of the poor to survive and cope are treated as criminal acts. In cities across the globe, working-class areas with high concentrations of ethnic minorities are treated as unsafe and potentially criminal by the state as well as by better-off citizens.In this scenario, impoverished migrants to cities must cope with life under conditions that are precarious by the very design of public policy and law. They are not legally entitled to the housing, sanitation, employment and security that would enable them to have better health.Exclusion in urban areas is associated with unique and pernicious risks. From the Philippines to South Africa, Brazil to India, slum-dwellers face a greater risk of violence, particularly gender-based, than the general population.,Increasingly, however, examples are emerging of the state acting to reverse exclusion, by becoming the guarantor and provider of land rights, housing, water, livelihoods, education and health services.These examples are rare: the norm remains one in which poor populations in cities continue to be treated as interlopers by the state, always facing the risk of removal and relocation. Tudor Hart argued that “this inverse care law operates more completely where medical care is most exposed to market forces, and less so where such exposure is reduced.”


Eothenomys melanogaster) by integrating geological and ecological factors. We analysed the genetic structure and divergence time of E. melanogaster based on fast-evolving mitochondrial and nuclear markers using Bayesian trees and coalescent species tree approaches. Historical scenarios of distribution range and demography were reconstructed based on spatial interpolations of genetic diversity and distance, extended Bayesian skyline plots, phylogeographic diffusion analysis, and ecological niche modelling (ENM) during different periods. We also assessed the relationships between geographical distance/ecological vicariance and genetic distance .South China encompasses complex and diverse landforms, giving rise to high biological diversity and endemism from the Hengduan Mountains to Taiwan Island. Many species are widely distributed across South China with similar disjunct distribution patterns. To explore the causes of these disjunct distribution patterns and their genetic consequences, we investigated the endemic species Père David’s Chinese Vole  and the mountains around the Sichuan Basin, respectively—that have mostly developed since the Pleistocene. IBD played an important role in early divergence, and geological events  and IBE further reinforced genetic differentiation. ENM shows the importance of suitable habitats and elevations.E. melanogaster is the high dependence on middle-high-altitude habitat in the current period. Mountains in the occurence range have served as “sky islands” for E. melanogaster and hindered gene flow. Pleistocene climatic cycles facilitated genetic admixture in cold periods and genetic diversification in warm periods for inland clades. During cold periods, these cycles led to multiple colonization events between the mainland and Taiwan and erased genetic differentiation.Our results suggest that the primary cause of the disjunct distribution in The online version of this article (10.1186/s12862-018-1168-3) contains supplementary material, which is available to authorized users. South China encompasses complex and diverse landforms. The high-altitude Hengduan Mountains (HM) and Yunnan-Guizhou Plateau (YGP) are in the west, and the terrain gradually descends to hilly areas in the east. The HM and adjacent areas are characterized by a series of parallel alpine ridges with dramatic ecological stratification and environmental heterogeneity , 2. ThisThe intraspecific diversification of montane species of South China is related to the uplift of the QTP since the Pliocene and the topographical complexity of the hilly areas , 12. PreEothenomys is a typical endemic genus of the South China mountainous regions  and theim.org/) . To reduls 1.4.4  by examils 1.4.4 . The sete x mutation rate, μ) and effective migration rates  between sample sites were calculated using MIGRATE 3.6.11 .Fig.FigE. meP < 0.001. The ENM shows the importance of suitable habitats and elevations , whereas its suitable habitat was lower in the north and expanded southward during the LGM .In the current ENM, the area under the ROC (Receiver operating characteristic) curve  was close to one (0.997 ± 0.002), indicating better than random prediction , and the binomial probability was r = 0.4363, P = 0.0001).Gene flow among the three clades showed similar patterns for all six genes: the SW and SE clades produced migrants entering the C clade, and the reverse gene flows from the C clade to the other two clades were much lower  that led to expansion in the glacial period and contraction in the interglacial period. This result is similar to results found for some previously studied species in this region [Although the main divergence occurred as a result of IBD and geological events, Pleistocene climatic changes were profoundly imprinted in the genetic structure, as was clearly supported by the ENM results Fig. . Unlike ulengeri , and Musmusculus , E. melas region . The midE. melanogaster shifted to lower elevations because of the expansion of habitat in low-altitude areas such as the Sichuan Basin (elevation range from 200 to 750 m) and Middle-Lower Yangtze Plain and degradation of habitat in high-altitude areas such as large mountains in the northwest of Sichuan Basin, Wuyi Mountains and the Central Mountains of Taiwan . The mean elevation shifts of E. melanogaster-applicable habitat between the LGM and current conditions intuitively reflected the tendency that facilitated genetic exchange. In warm periods, high-altitude localities acted as “sky islands” for E. melanogaster and resulted in genetic divergence [During the ice ages, populations of Populations from different sites in the SE clade showed a paraphyletic structure. According to the results of the phylogeographic diffusion analysis, the whole SE clade population originated and expanded from the Wuyi Mountains area Fig. . Taiwan E. melanogaster lives between 700 m and 3,000 m above sea level, we suspect that large mountain ridges  can act not only as corridors for this species but also as barriers. Therefore, a stable environment led to low diversification, and the dual effects of mountains shaped distant phylogenetic relationships within clades.Two long-distant branches with weak geographic association are found within the SW clade Fig. . This paHaplotype diversity (h) and nucleotide diversity (Pi) can reveal the general demographic history of populations . The C cAmong the three clades, the SW clade shows the lowest overall genetic diversity and genetic divergence Table ; howeverE. melanogaster throughout the Quaternary and to obtain insights into the processes that shaped this species’ disjunct distribution pattern. The genetic analysis revealed three deeply divergent clades, Southeast, Southwest and Central, which derived from the Wuyi Mountains, the YGP and the mountains around the Sichuan Basin, respectively—mostly since the Pleistocene. IBD played important roles in early divergence, and geological events  and IBE further reinforced genetic differentiation. The primary cause of the disjunct distribution in E. melanogaster was the preference for middle-high-altitude habitats in the current period. High-altitude localities acted as “sky islands” for E. melanogaster and blocked genetic exchange among populations. Pleistocene climatic cycles had various impacts on the diversification of different clades. Genetic admixture in cold periods and genetic diversification in warm periods were facilitated in inland clades but led to multiple colonization events between the mainland and Taiwan and erased genetic differentiation during cold periods.In this study, comprehensive phylogeographic analyses were performed to infer the evolutionary history of Additional file 1:Table S1. Sample information and the GenBank accession numbers. Sampling information and the GenBank accession numbers of all sequences of Eothenomys melanogaster used in the study. Table S2. Outgroups of phylogenetic analysis. Outgroups used for fossil calibration and phylogenetic analyses of E. melanogaster. Table S3. Gene flow of three maternal clades. Gene flow of three maternal clades of E. melanogaster estimated by MIGRATE. C represents the Central Clade, SE represents the Southeast Clade and SW represents the Southwest Clade. (DOCX 32 kb)Additional file 2:Figure S1. Bayesian phylogenetic trees of nuclear loci and species tree. Bayesian phylogenetic trees of nuclear loci and species tree estimated by BP&P and *BEAST for E. melanogaster. Red indicates individuals belonging to the Central clade; blue indicates individuals belonging to the Southwest clade; and yellow indicates individuals belonging to the Southeast clade. Values beside branches indicate Bayesian posterior probability. (TIFF 24617 kb)Additional file 3:Figure S2. Phylogenetic trees of concatenated cytb and nDNA genes. Phylogenetic trees of concatenated cytb and nDNA genes for E. melanogaster. Values beside branches indicate posterior probability and bootstrap. (TIFF 28922 kb)


Desmodus rotundus, ranges from South America into northern Mexico in North America. This sanguivorous species of bat feeds primarily on medium to large‐sized mammals and is known to rely on livestock as primary prey. Each year, there are hotspot areas of D. rotundus‐specific rabies virus outbreaks that lead to the deaths of livestock and economic losses. Based on incidental captures in our study area, which is an area of high cattle mortality from D. rotundus transmitted rabies, it appears that D. rotundus are being caught regularly in areas and elevations where they previously were thought to be uncommon. Our goal was to investigate demographic processes and genetic diversity at the north eastern edge of the range of D. rotundus in Mexico. We generated control region sequences (441 bp) and 12‐locus microsatellite genotypes for 602 individuals of D. rotundus. These data were analyzed using network analyses, Bayesian clustering approaches, and standard population genetic statistical analyses. Our results demonstrate panmixia across our sampling area with low genetic diversity, low population differentiation, loss of intermediate frequency alleles at microsatellite loci, and very low mtDNA haplotype diversity with all haplotypes being very closely related. Our study also revealed strong signals of population expansion. These results follow predictions from the leading‐edge model of expanding populations and supports conclusions from another study that climate change may allow this species to find suitable habitat within the U.S. border.The common vampire bat,  As carnivore rabies has come under greater control, rabies in Latin America now is transmitted to humans and livestock principally by common vampire bats  border in the Mexican state of Tamaulipas , unpublished data), which is north of their documented range limit. The fossil record shows that between 30,000 and 5,000 years before present (yBP), D. rotundus ranged within the current borders of the United States  from deposits in present‐day New Mexico, Arizona, California, Texas, Florida, and West Virginia . Recent incidental records of D. rotundus within 50 km of the Mexico/U.S. border and apparent increased captures in areas and elevations where D. rotundus was thought to be uncommon  suggest that this process may be in progress. The potential establishment of populations in the US is of concern given that a rabies epidemic in livestock (vampire‐bat variant) has expanded from the southeast to northwest across the states of San Luis Potosí and Tamaulípas, Mexico since 2001 (SAGARPA). Population genetic analyses can be used to test predictions from the leading‐edge range expansion model and provide an additional test of the potential for D. rotundus to expand its geographic distribution across the Mexico/U.S. border.In another study using samples from this edge D. rotundus at the north eastern edge of the species’ distribution. We evaluate signals of demographic and spatial population expansion and levels of genetic diversity. We assessed demographic events with more rapidly evolving markers than used in previous studies of D. rotundus . In the second sampling effort . DNA from all samples was extracted using a DNeasy Blood and Tissue Kit .Materials for this study came from the combination of two independent sampling efforts Figure . In 20032.2P*  with 1 μmol/L primer, 0.25 μL BigDye v3.1, and 2.275 μL 5× sequencing buffer . Genotypes from 12 microsatellite loci were generated for individual D. rotundus following Piaggio et al. . Sequences were reviewed, edited, and aligned with sequencher v.5.2 . Microsatellite genotypes were converted from genemapper into genepop files for downstream conversion into appropriate input files for various analytical software through gmconvert . The number of haplotypes was assessed, and a haplotype network was calculated using a median‐joining approach , which estimates diversity of alleles while accounting for evenness in the representation of those alleles across the samples and correcting for uneven sample sizes  rather than bats caught on the wing. A test of IBD by nonroost capture localities may pool individuals belonging to different populations and thus be susceptible to bias from the Wahlund (N = 602) in pairwise comparisons. This test uses log‐transformed geographical distances, which are tested for correlation to probability of identity of genes between individuals  were performed: one among roosts and another among individuals Rousset, . arlequi Wahlund  effect, baps v.6.0  distributed among samples, and among females separately, informed by spatial information about capture locality  implemented in Population demographics were evaluated from microsatellite data by calculating the imbalance index β  and 12‐locus genotypes for 602 O and HE were the same two loci that demonstrated significant evidence of null alleles. The Information Index was low to moderate across loci (I = 0.56–2.57) with an average I = 1.37 ± 0.16, suggesting that most loci are characterized by a few very common alleles and more rare alleles. This pattern is evident when graphs of allele frequencies per locus are observed , which we interpret as being not biologically meaningful. No significant genotypic disequilibrium was detected in pairwise comparisons between all loci.Evidence of possible null alleles were detected at two microsatellite loci: Dero_G10F_B03R (frequency = 0.07) and Dero_H02F_C03R (frequency = 0.19). Because the frequency of potential null alleles was low (Dero_G10F_B03R) to moderate (Dero_H02F_C03R), we kept these loci in the analyses as they were not expected to bias our estimates because they occurred at < 0.20 . An analysis of IBD between individuals for the full dataset was highly significant (p < 0.00001). Bayesian clustering analyses in both baps and structure provided the strongest support for a single genetic cluster (K = 1) among all individuals  and then we ran structure , which showed stronger support for a single cluster after removing loci with null alleles.Microsatellite‐based tests of IBD considering genetic and geographic distance between roosts were significant  was not significantly different from zero while Fu's FS  was significant. This is not unexpected, as Tajima's D has been shown to lack power in analyses of population growth  and spatial  expansion could not be rejected. The “starburst” pattern observed in the mtDNA network  in populations from the core of the species’ range. In comparison, Ditchfield  as compared to our 14 segregating sites across a smaller fragment size (=3.2% divergence). Finally, Pinto (D. rotundus from either side of the Andes in Ecuador and sequenced the entire cyt b gene (1140 bp). From 134 individuals that were sampled, there were 22 haplotypes with 147 segregating sites. The samples in these previous studies were collected across the core of the range of D. rotundus (South America into southern Mexico), rather than on the edge of the species’ distribution as in our study. Although we also employ a different mtDNA locus, it is evident that populations of D. rotundus from more southerly core portions of their range have higher genetic diversity than occurs at the northern leading edge of their range.The control region of the mitochondrial DNA genome is known to accumulate mutations more rapidly than cytochrome b Meyer, . With onb Meyer,  and 0.5%tchfield  sequences et al.  sequences et al.  added toy, Pinto  sampled D. rotundus . As areas north of the current distribution of D. rotundus become more suitable for their physiological needs, it is likely that they will colonize those areas and transport their rabies virus variants to new regions and newly susceptible hosts.Fossil evidence demonstrates that A.J.P., A.E.T., G.F.M., A.M‐V., and L.L. conceived the study and/or study design; I.A.O., A.E.T., and A.J.R. collected samples and data; A.J.P., A.L.R., J.W.F., A.E.T., and J.L.N. analyzed the data; and A.J.P. led the writing.None declared. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Galeocerdo cuvier) at seven locations across the Indo-Pacific, and one location in the southern Atlantic. Genetic analyses revealed considerable genetic structuring  between all Indo-Pacific locations and Brazil. By contrast, no significant genetic differences were observed between locations from within the Pacific or Indian Oceans, identifying an apparent large, single Indo-Pacific population. A lack of differentiation between tiger sharks sampled in Hawaii and other Indo-Pacific locations identified herein is in contrast to an earlier global tiger shark nDNA study. The results of our power analysis provide evidence to suggest that the larger sample sizes used here negated any weak population subdivision observed previously. These results further highlight the need for cross-jurisdictional efforts to manage the sustainable exploitation of large migratory sharks like G. cuvier.Population genetic structure using nine polymorphic nuclear microsatellite loci was assessed for the tiger shark ( This finding was in contrast to that identified in the global study by Bernard et al. , which r [et al.  may expld et al. , a few i [et al.  demonstre effect . Moreovee effect ,41). Notre small .G. cuvier across the world [Continued advances in tagging technology that allow for multi-year data collection from mature sharks, and further developments in molecular analyses, is likely to improve our understanding of how partial migration, sex-biased dispersal and reproductive mixing influences population structure and connectivity of tiger sharks within and among global ocean basins. Given the evidence of exploitation-driven declines in he world ,15,43,44