Local selection modifies phenotypic divergence among Rana temporaria populations in the presence of gene flow

In ectotherms, variation in life history traits among populations is common and suggests local adaptation. However, geographic variation itself is not a proof for local adaptation, as genetic drift and gene flow may also shape patterns of quantitative variation. We studied local and regional variation in means and phenotypic plasticity of larval life history traits in the common frog Rana temporaria using six populations from central Sweden, breeding in either open‐canopy or partially closed‐canopy ponds. To separate local adaptation from genetic drift, we compared differentiation in quantitative genetic traits (Q_ST) obtained from a common garden experiment with differentiation in presumably neutral microsatellite markers (F_ST). We found that R. temporaria populations differ in means and plasticities of life history traits in different temperatures at local, and in F_ST at regional scale. Comparisons of differentiation in quantitative traits and in molecular markers suggested that natural selection was responsible for the divergence in growth and development rates as well as in temperature‐induced plasticity, indicating local adaptation. However, at low temperature, the role of genetic drift could not be separated from selection. Phenotypes were correlated with forest canopy closure, but not with geographical or genetic distance. These results indicate that local adaptation can evolve in the presence of ongoing gene flow among the populations, and that natural selection is strong in this system.     

Establishment and maintenance of adaptive genetic divergence under migration, selection, and drift

There is a long tradition in population genetics of exploring the maintenance of variation under migration-selection balance using deterministic models that assume infinite population size. With finite population size, stochastic dynamics can greatly reduce the potential for the maintenance of polymorphism, but this has yet to be explored in detail. Here, classical two-patch models are extended to predict: (1) the probability of a locally beneficial mutation rising in frequency in the patch where it is favored and (2) the critical threshold migration rate above which the maintenance of polymorphism is much less likely. Individual-based simulations show that these approximations provide accurate predictions across a wide range of parameter space.     

Genetic structure of mountain lion (Puma concolor) populations in California

Analysis of 12 microsatellite loci from431 mountain lions (Puma concolor)revealed distinct genetic subdivision that wasassociated with geographic barriers andisolation by distance in California. Levels ofgenetic variation differed among geographicregions, and mountain lions that inhabitedcoastal areas exhibited less heterozygositythan those sampled inland. The San FranciscoBay and Sacramento-San Joaquin River Delta, theCentral Valley, and the Los Angeles Basinappeared to be substantial barriers to geneflow, and allele frequencies of populationsseparated by those features differedsubstantially. A partial barrier to gene flowappeared to exist along the crest of the SierraNevada. Estimated gene flow was high amongmountain lions inhabiting the Modoc Plateau,the western Sierra Nevada, and northern sectionof the eastern Sierra Nevada. SouthernCalifornia mountain lion populations mayfunction as a metapopulation; however, humandevelopments threaten to eliminate habitat andmovement corridors. While north-south geneflow along the western Sierra Nevada wasestimated to be very high, projected loss andfragmentation of foothill habitat may reducegene flow and subdivide populations. Preservation of existing movement corridorsamong regions could prevent population declinesand loss of genetic variation. This studyshows that mountain lion management andconservation efforts should be individualizedaccording to region and incorporatelandscape-level considerations to protecthabitat connectivity.     

Population genetic structure and migration patterns of Dendrothrips minowai (Thysanoptera: Thripidae) in Guizhou,China

Dendrothrips minowai Priesner (Thysanoptera: Thripidae) is one of the most destructive insect pests on tea plants. Although outbreaks of this pest occur annually in South China, especially in Guizhou Province, little is known about its population genetics, such as genetic diversity and gene flow. To investigate its population genetic structure and migration routes in Guizhou Province, we analyzed 24 D. minowai populations across Guizhou using six microsatellite loci. We detected the moderate genetic diversity and the population genetic structure of this thrip species. Neighbor‐joining (NJ) phylogenetic tree and STRUCTURE analyses recognized two clusters within the studied populations. No correlation between genetic and geographical distances (r = 0.0139, P = 0.5830) was detected and more than 89% of the variation occurred among samples within populations. Gene flow analysis revealed high migration rates (74.0 – 894.1) among D. minowai populations. Overall, the trend of asymmetrical gene flow was from northeast to southwest. Our analyses demonstrated that D. minowai derived or originated from multiple sites and could be eventually divided into two groups in Guizhou.     

Adaptive population differentiation in phenology across a latitudinal gradient in European aspen (Populus tremula, L.): a comparison of neutral markers, candidate genes and phenotypic traits

A correct timing of growth cessation and dormancy induction represents a critical ecological and evolutionary trade-off between survival and growth in most forest trees (Rehfeldt et al. 1999; Horvath et al. 2003; Howe et al. 2003). We have studied the deciduous tree European Aspen (Populus tremula) across a latitudinal gradient and compared genetic differentiation in phenology traits with molecular markers. Trees from 12 different areas covering 10 latitudinal degrees were cloned and planted in two common gardens. Several phenology traits showed strong genetic differentiation and clinal variation across the latitudinal gradient, with Q(ST) values generally exceeding 0.5. This is in stark contrast to genetic differentiation at several classes of genetic markers (18 neutral SSRs, 7 SSRs located close to phenology candidate genes and 50 SNPs from five phenology candidate genes) that all showed F(ST) values around 0.015. We thus find strong evidence for adaptive divergence in phenology traits across the latitudinal gradient. However, the strong population structure seen at the quantitative traits is not reflected in underlying candidate genes. This result fit theoretical expectations that suggest that genetic differentiation at candidate loci is better described by F(ST) at neutral loci rather than by Q(ST) at the quantitative traits themselves.     

Evolution of adaptive diversity and genetic connectivity in Arctic charr (Salvelinus alpinus) in Iceland

The ecological theory of adaptive radiation predicts that the evolution of phenotypic diversity within species is generated by divergent natural selection arising from different environments and competition between species. Genetic connectivity among populations is likely also to have an important role in both the origin and maintenance of adaptive genetic diversity. Our goal was to evaluate the potential roles of genetic connectivity and natural selection in the maintenance of adaptive phenotypic differences among morphs of Arctic charr, Salvelinus alpinus, in Iceland. At a large spatial scale, we tested the predictive power of geographic structure and phenotypic variation for patterns of neutral genetic variation among populations throughout Iceland. At a smaller scale, we evaluated the genetic differentiation between two morphs in Lake Thingvallavatn relative to historically explicit, coalescent-based null models of the evolutionary history of these lineages. At the large spatial scale, populations are highly differentiated, but weakly structured, both geographically and with respect to patterns of phenotypic variation. At the intralacustrine scale, we observe modest genetic differentiation between two morphs, but this level of differentiation is nonetheless consistent with strong reproductive isolation throughout the Holocene. Rather than a result of the homogenizing effect of gene flow in a system at migration-drift equilibrium, the modest level of genetic differentiation could equally be a result of slow neutral divergence by drift in large populations. We conclude that contemporary and recent patterns of restricted gene flow have been highly conducive to the evolution and maintenance of adaptive genetic variation in Icelandic Arctic charr.     

华木莲居群遗传结构与保护单元

华木莲(Sinomanglietia glauca)仅分布于江西宜春和湖南永顺, 是我国一级重点保护植物。前人采用RAPD、ISSR以及叶绿体SSCP(single-stranded conformation polymorphism)标记对华木莲进行了居群遗传学研究, 但未包括后发现的湖南居群或未检出居群内遗传变异。为了全面检测华木莲遗传多样性及其空间分布格局, 并据此确定保护单元, 本研究采用细胞核微卫星标记对华木莲所有4个居群共77个个体进行了居群遗传学分析。结果表明, 华木莲具有较低的遗传多样性(平均等位基因数A = 2.604, 平均期望杂合度H_E = 0.423)和较高的遗传分化(F_ST = 0.425)。STRUCTURE和主成分分析(Principal Coordinated Analysis, PCA)将4个居群首先分为江西、湖南两组, 江西的2个居群实际上是同一个自然繁育居群, 而湖南的2个居群则为2个分化明显的自然繁育居群。研究还发现湖南居群存在明显的杂合子过剩现象, 可能是小居群内随机因素造成的。研究结果表明华木莲可能在近期历史上遭受过强烈的瓶颈效应, 导致种群缩小、遗传多样性丧失和居群分化加剧, 需要加强对其进化潜力的保护。在制定保护措施时, 需要考虑其较高的遗传分化水平, 根据遗传结构可以将其划分为3个保护单元, 即湖南居群和江西居群分别为2个进化显著单元, 湖南居群进一步划分为2个管理单元(分别为朗溪乡云盘村和小溪乡鲁家村居群)。     

Decoupling of genetic and phenotypic divergence in a headwater landscape

In stream organisms, the landscape affecting intraspecific genetic and phenotypic divergence is comprised of two fundamental components: the stream network and terrestrial matrix. These components are known to differentially influence genetic structure in stream species, but to our knowledge, no study has compared their effects on genetic and phenotypic divergence. We examined how the stream network and terrestrial matrix affect genetic and phenotypic divergence in two stream salamanders, Gyrinophilus porphyriticus and Eurycea bislineata, in the Hubbard Brook Watershed, New Hampshire, USA. On the basis of previous findings and differences in adult terrestriality, we predicted that genetic divergence and phenotypic divergence in body morphology would be correlated in both species, but structured primarily by distance along the stream network in G. porphyriticus, and by overland distance in E. bislineata. Surprisingly, spatial patterns of genetic and phenotypic divergence were not strongly correlated. Genetic divergence, based on amplified DNA fragment length polymorphisms, increased with absolute geographic distance between sites. Phenotypic divergence was unrelated to absolute geographic distance, but related to relative stream vs. overland distances. In G. porphyriticus, phenotypic divergence was low when sites were close by stream distance alone and high when sites were close by overland distance alone. The opposite was true for E. bislineata. These results show that small differences in life history can produce large differences in patterns of intraspecific divergence, and the limitations of landscape genetic data for inferring phenotypic divergence. Our results also underscore the importance of explicitly comparing how terrestrial and aquatic conditions affect spatial patterns of divergence in species with biphasic life cycles.     

Marine landscapes and population genetic structure of herring (Clupea harengus L.) in the Baltic Sea

Numerically small but statistically significant genetic differentiation has been found in many marine fish species despite very large census population sizes and absence of obvious barriers to migrating individuals. Analyses of morphological traits have previously identified local spawning groups of herring (Clupea harengus L.) in the environmentally heterogeneous Baltic Sea, whereas allozyme markers have not revealed differentiation. We analysed variation at nine microsatellite loci in 24 samples of spring-spawning herring collected at 11 spawning locations throughout the Baltic Sea. Significant temporal differentiation was observed at two locations, which we ascribe to sympatrically spawning but genetically divergent 'spawning waves'. Significant differentiation was also present on a geographical scale, though pairwise F(ST) values were generally low, not exceeding 0.027. Partial Mantel tests showed no isolation by geographical distance, but significant associations were observed between genetic differentiation and environmental parameters (salinity and surface temperature) (0.001 < P < or = 0.099), though these outcomes were driven mainly by populations in the southwestern Baltic Sea, which also exhibits the steepest environmental gradients. Application of a novel method for detecting barriers to gene flow by combining geographical coordinates and genetic differentiation allowed us to identify two zones of lowered gene flow. These zones were concordant with the separation of the Baltic Sea into major basins, with environmental gradients and with differences in migration behaviour. We suggest that similar use of landscape genetics approaches may increase the understanding of the biological significance of genetic differentiation in other marine fishes.     

Random amplified polymorphic DNA analysis of southern brown bandicoot (Isoodon obesulus) populations in Western Australia reveals genetic differentiation related to environmental variables

Random amplified polymorphic DNA (RAPD) markers were used to analyse genetic variation within and between populations of Isoodon obesulus in Western Australia. Genetically controlled geographical variation in body size associated with habitat type and rainfall exists in this species, raising the question of whether local conditions may influence gene flow in I. obesulus. The RAPD markers displayed substantial genetic variation, with all animals possessing unique RAPD phenotypes over 39 polymorphic bands produced by three primers. Significant geographical subdivision was apparent (PhiST = 0.208) with southwest locations being divergent from all others, despite there being no physical barriers to gene flow. The pattern of subdivision was unrelated to physical distance between the locations, but was related to both annual rainfall and habitat type. Therefore, the most reasonable explanation for this pattern of subdivision appears to be that gene flow is restricted by selection against migrants between local populations with substantially different habitat type or rainfall. Restriction of gene flow through selection against migrants is rarely investigated, and the results of this study suggest that the importance of this process in the formation of population structure may be underestimated.     

Population structure in Arabidopsis lyrata: evidence for divergent selection on trichome production

Leaf trichomes may serve several biological functions including protection against herbivores, drought, and UV radiation; and their adaptive value can be expected to vary among environments. The perennial, self-incompatible herb Arabidopsis lyrata is polymorphic for trichome production, and occurs in a glabrous and a trichome-producing form. Controlled crosses indicate that the polymorphism is governed by a single gene, with trichome production being dominant. We examined the hypothesis that trichome production is subject to divergent selection (i.e., directional selection favoring different phenotypes in different populations) by comparing patterns of variation at the locus coding for glabrousness and at eight putatively neutral isozyme loci in Swedish populations of A. lyrata. The genetic diversity (He) and allele number at isozyme loci tended to increase with population size and decreased with latitude of origin, whereas genetic diversity at the locus coding for glabrousness did not vary with population size and increased with latitude of origin. The degree of genetic differentiation at the glabrousness locus was much higher than that at isozyme loci. Genetic identity at isozyme loci was negatively related to geographic distance, suggesting isolation by distance. In contrast, there was no significant correlation between genetic identity at the glabrousness locus and at isozyme loci. The results are consistent with the hypothesis that divergent selection contributes to population differentiation in trichome production in A. lyrata.     

Population structure and genetic variation in the endangered Giant Kangaroo Rat (<Emphasis Type="Italic">Dipodomys ingens</Emphasis>)

Populations of the endangered giant kangaroo rat, Dipodomys ingens (Heteromyidae), have suffered increasing fragmentation and isolation over the recent past, and the distribution of this unique rodent has become restricted to 3% of its historical range. Such changes in population structure can significantly affect effective population size and dispersal, and ultimately increase the risk of extinction for endangered species. To assess the fine-scale population structure, gene flow, and genetic diversity of remnant populations of Dipodomys ingens, we examined variation at six microsatellite DNA loci in 95 animals from six populations. Genetic subdivision was significant for both the northern and southern part of the kangaroo rat’s range although there was considerable gene flow among southern populations. While regional gene diversity was relatively high for this endangered species, hierarchical F-statistics of northern populations in Fresno and San Benito counties suggested non-random mating and genetic drift within subpopulations. We conclude that effective dispersal, and therefore genetic distances between populations, is better predicted by ecological conditions and topography of the environment than linear geographic distance between populations. Our results are consistent with and complimentary to previous findings based on mtDNA variation of giant kangaroo rats. We suggest that management plans for this endangered rodent focus on protection of suitable habitat, maintenance of connectivity, and enhancement of effective dispersal between populations either through suitable dispersal corridors or translocations.     

Linked selection,ancient polymorphism,and ecological adaptation shape the genomic landscape of divergence in Quercus dentata

Multiple evolutionary forces contribute to heterogeneous genomic landscapes; however, disentangling their relative contributions is challenging. We sampled nine populations across the distribution of Quercus dentata, a dominant forest tree in East Asia, and used whole-genome sequencing data to investigate mechanisms underlying divergence. We identified two genetic groups (north and south) that diverged ~1.84 million years ago, consistent with the uplift of the Qinling Mountains during the Pleistocene. The north group experienced a bottleneck during the middle–late Pleistocene and expanded from multiple refugia. The south group experienced a more severe bottleneck and showed high population differentiation, probably due to long-term isolation and habitat fragmentation. We detected genomic islands with elevated relative differentiation (F_ST) scattered across the genome. Among these, 65.9% showed reduced absolute divergence (d_XY) consistent with linked selection, while the remaining (34.1%) showed elevated d_XY suggestive of divergent sorting of ancient polymorphisms. The recombination rate in genomic islands was lower than background, suggesting the importance of genome structure in shaping the genomic landscape. We detected 108 single nucleotide polymorphisms significantly associated with environmental factors, 12 of which clustered in a region of ~500 kb. This region showed multiple signals of positive selection in the north group, including the enrichment of XP-extended haplotype homozygosity scores, an elevated population branch statistic, and an excess of high-frequency derived alleles. In addition, we found that linkage disequilibrium was low and derived haplotypes declined rapidly in this region, indicating selection on standing variation. Our results clarify the evolutionary processes driving genomic divergence in Q. dentata.     

Long-term effective population sizes, temporal stability of genetic composition and potential for local adaptation in anadromous brown trout (Salmo trutta) populations

We examined the long-term temporal (1910s to 1990s) genetic variation at eight microsatellite DNA loci in brown trout (Salmo trutta L) collected from five anadromous populations in Denmark to assess the long-term stability of genetic composition and to estimate effective population sizes (Ne). Contemporary and historical samples consisted of tissue and archived scales, respectively. Pairwise thetaST estimates, a hierarchical analysis of molecular variance (amova) and multidimensional scaling analysis of pairwise genetic distances between samples revealed much closer genetic relationships among temporal samples from the same populations than among samples from different populations. Estimates of Ne, using a likelihood-based implementation of the temporal method, revealed Ne >or= 500 in two of three populations for which we have historical data. A third population in a small (3 km) river showed Ne >or= 300. Assuming a stepping-stone model of gene flow we considered the relative roles of gene flow, random genetic drift and selection to assess the possibilities for local adaptation. The requirements for local adaptation were fulfilled, but only adaptations resulting from strong selection were expected to occur at the level of individual populations. Adaptations resulting from weak selection were more likely to occur on a regional basis, i.e. encompassing several populations. Ne appears to have declined recently in at least one of the studied populations, and the documented recent declines of many other anadromous brown trout populations may affect the persistence of local adaptation.     

Population genetic structure of diploid sexual and polyploid apomictic hawthorns (Crataegus; Rosaceae) in the Pacific Northwest

Polyploidy and gametophytic apomixis are two important and associated processes in plants. Many hawthorn species are polyploids and can reproduce both sexually and apomictically. However, the population genetic structure of these species is poorly understood. Crataegus douglasii is represented exclusively by self-compatible tetraploid pseudogamous apomicts across North America, whereas Crataegus suksdorfii found in the Pacific Northwest is known to include self-incompatible diploid sexuals as well as polyploid apomicts. We compare population structure and genetic variability in these two closely related taxa using microsatellite and chloroplast sequence markers. Using 13 microsatellite loci located on four linkage groups, 251 alleles were detected in 239 individuals sampled from 15 localities. Within-population multilocus genotypic variation and molecular diversity are greatest in diploid sexuals and lowest in triploid apomicts. Apart from the isolation of eastern North American populations of C. douglasii , there is little evidence of isolation by distance in this taxon. Genetic diversity in western populations of C. douglasii suggests that gene flow is frequent, and that colonization and establishment are often successful. In contrast, local populations of C. suksdorfii are more markedly differentiated. Gene flow appears to be limited primarily by distance in diploids and by apomixis and self-compatibility in polyploids. We infer that apomixis and reproductive barriers between cytotypes are factors that reduce the frequency of gene flow among populations, and may ultimately lead to allopatric speciation in C. suksdorfii . Our findings shed light on evolution in woody plants that show heterogeneous ploidy levels and reproductive systems.     

Population genetic structure of Cydia pomonella: a review and case study comparing spatiotemporal variation

Analysis of population genetic structure is a key aspect to understand insect pest population dynamics in agricultural scenarios. Here the role of geography, hosts and time on the population genetic structure of codling moth Cydia pomonella (Linnaeus) (Lep., Tortricidae) populations is described. Temporal variation was examined in two French orchards among each of three adult flights during two successive years. Analyses were conducted using two insecticide resistance markers (variation at the sodium channel gene and enzymatic activity of cytochrome P450 oxidases) and three microsatellite loci. Levels of genetic variation among temporal populations were not significant based on variation in the sodium channel gene and microsatellite loci. However, P450 oxidase activity differed significantly during both flights and years, decreasing during the three flights of the first year and increasing during the second. These results suggest that phytosanitary measures are among the factors shaping the genetic structure of C. pomonella populations over temporal and geographical scales. We discuss the relative importance of natural and passive dispersal related to anthropogenic activities affecting C. pomonella population genetics and highlight population genetic research needs in order to design more efficient pest management practices.     

Habitat fragmentation in forests affects relatedness and spatial genetic structure of a native rodent,Rattus lutreolus

Habitat fragmentation can have a range of negative demographic and genetic impacts on disturbed populations. Dispersal barriers can be created, reducing gene flow and increasing population differentiation and inbreeding in isolated habitat remnants. Aggregated retention is a form of forestry that retains patches of forests as isolated island or connected edge patches, with the aim of ‘lifeboating’ species and processes, retaining structural features and improving connectivity. Swamp rats (Rattus lutreolus) are a cover‐dependent species that are sensitive to habitat removal. We examined the effects of aggregated retention forestry and forestry roads in native wet Eucalyptus forests on swamp rat gene flow and population genetic structure. We characterized neighbourhood size in unlogged forest to provide a natural state for comparison, and examined population structure at a range of spatial scales, which provided context for our findings. Tests of pairwise relatedness indicated significant differentiation between island and edge populations in aggregated retention sites, and across roads in unlogged sites. Spatial autocorrelation suggested a neighbourhood size of 42–55 m and revealed male‐biased dispersal. We found no genetic isolation by geographical distance at larger (>2.3 km) scales and populations were all significantly differentiated. Our results suggest that removal of mature forest creates barriers for swamp rat dispersal. In particular, roads may have long‐term impacts, while harvesting of native forests is likely to create only short‐term dispersal barriers at the local scale, depending on the rate of regeneration.     

Isolation and characterization of polymorphic microsatellite loci in Hemibarbus labeo (Cyprinidae) using PCR‐based isolation of microsatellite arrays (PIMA)

A total of 10 polymorphic microsatellite loci from Hemibarbus labeo were isolated and characterized using an optimized protocol to construct a microsatellite‐enriched genomic library. The analysis of variability was performed in 24 specimens of mainland China. The mean number of alleles across loci was 3.10 ± 1.10 and the level of expected heterozygosity varied from 0.0417 to 0.7482. Frequencies of null alleles of the 10 loci are not significantly greater than zero. No linkage disequilibrium was detected between loci in either population. Five primer pairs cross‐amplify the microsatellites in other species, indicating transportability of the markers within the family Cyprinidae.     

Population structure in Mediterranean islands and risk of genetic invasion in Culexpipiens L. (Diptera: Culicidae)

The mosquito Culex pipiens is subjected to organophosphate treatments in both Corsica and southern Sardinia, but the resistance gene A2-B2, which is currently in a worldwide expansion, has only reached Sardinia. In order to understand this situation, the genetic structure of populations sampled in Sardinia and Corsica was assessed using 15 isozymes. Two loci (HK1 and HK2) were not taken into account because of the possibility of selection. For trie other loci, statistical independence was not rejected for all possible pairs, and no deviation from Hardy-Weinberg expectations was apparent. Low but significant genie differentiation was present between Corsica and Sardinia, as well as between northern and southern Sardinia, despite a large number of effective migrants per generation. These results are discussed in the context of the high probability of extinction/recolonization of breeding sites, the flight migration ability of this mosquito, and the pleiotropic cost of insecticide resistances genes. It is concluded that A2-B2 resistance is unlikely to reach Corsica from southern Sardinia, unless accidental human transportation occurs.     

基于微卫星标记的印度中部Madhya Pradesh地区疟疾强化控制和非强化控制区的斯氏按蚊基因流和种群遗传结构分析

[目的]斯氏按蚊Anopheles stephensi是亚洲东南部城市人体疟疾的主要媒介,印度12％的疟疾病例由其引起.本实验研究了印度中部Madhya Pradesh地区东北部的疟疾强化控制(EMCP)区和非强化控制(非EMCP)区斯氏按蚊的基因流.在EMCP区,由于采用了各种疟疾防控措施因而疟疾病例首先降低,但是很快回升,说明总的疟疾风险维持稳定.[方法]应用7个微卫星位点,对印度中部Madhya Pradesh地区东北部的4个EMCP区和非EMCP区采集的斯氏按蚊进行基因分型,以分析各种群参数.[结果]发现各标记在所有种群中表现出高度的多态性.在两区间未发现很大的遗传多样性.观察到EMCP区的东部种群(FST=0.0485,RST =0.1112)比非EMCP区的北部种群(FST=0.020,RST =0.0145)具有较高的遗传分化,在EMCP区和非EMCP区之间观察到较高的基因流(12.90,6.16,5.06和2.38).RST的灵敏度高于FST,说明分化可能是由于突变而非遗传漂变引起的.[结论]本研究表明,在EMCP区和非EMCP区内以及EMCP区和非EMCP区之间存在很高的基因流.基因流水平高以及抗虫性的发展似乎是EMCP区和非EMCP区疟疾病例发生增加的重要原因.