Polygalacturonase, Pectate Lyase and Pectin Methylesterase Activity in Pathogenic Strains of Phytophthora capsici Incubated under Different Conditions

Pectolytic enzymes are found mainly in fungi and bacteria. The most widely occurring enzymes are polygalacturonase (PGs), pectin methylesterase (PMEs) and pectate lyase (PLs) produced during the infection process and during culturing. The secretion of these enzymes results in the disorganization of the plant cell walls, which is responsible for the pathogenicity of the pathogens. These enzymes degrade the pectin of plants causing maceration of plant tissues and the enzyme activity increases under favourable environmental conditions. We have found that Phytophthora capsici , a pathogenic oomycete, produces levels of these three enzymes equal to those produced by soft-rotting Erwinia chrysanthemi . The activity of PGs, PLs and PMEs was investigated at the optimum temperature, pH and ionic strength in highly pathogenic P. capsici strains cultivated in two kinds of liquid medium containing either crude pepper extracts plus pectin or pectin as the carbon source. Virulence tests and enzymes activity showed that there was a high correlation between the enzyme activity and the pathogenicity of P. capsici . The effects of different carbon sources on the enzyme activity showed that pepper extract plus pectin was the best source for the carbon source.     

Mating Preferences of the Gynogenetic Amazon Molly Differ Between Populations Sympatric with Different Host Species

Gynogenetic species rely on sperm from heterospecifics for reproduction but do not receive genetic benefits from mating because none of the paternal genome is incorporated into offspring. The gynogenetic Amazon molly (Poecilia formosa) is a species of hybrid origins that are sympatric with one of the two parent species that provide sperm for reproduction, P. latipinna or P. mexicana. Amazons should not prefer to mate with one species over the other because sperm from both species will trigger embryogenesis, but mating preferences may be present in Amazons through other mechanisms. Amazons may prefer the more familiar species (males found in sympatry), or Amazons may prefer males with the greatest lateral projection area (LPA), a preference that is present in the parent species and may be retained within the Amazon hybrid genome. We tested association preferences of two populations of Amazons sympatric with either P. mexicana or P. latipinna. We first performed live trials to test whether Amazons preferred one host species over the other and found that neither population of Amazons showed a preference. We then tested whether Amazons preferred sympatric male (familiar) host or the male with the greatest lateral projection area (LPA) using four animated male models that varied in host species and manipulation of LPA. We found Amazons from a population sympatric with P. latipinna showed no variation in their association preference across the different models. In contrast, Amazons from a population sympatric with P. mexicana (naturally small LPA) spent more time associating with the male models that had smaller LPA, which is more familiar to this population of Amazons. We suggest that Amazons may have population differences in mating preferences, where Amazons sympatric with P. latipinna may not show mating preference for host species, but Amazons sympatric with P. mexicana may show preferences for more familiar‐shaped males.     

Changes in the Distribution of Pectin Methylesterase across Leaf Abscission Zones of Phaseolus vulgaris

Determinations of pectin methylesterase in abscission zonesand surrounding parts of Phaseolus leaves have shown a relativelyhigh activity in young tissues, which decreases as the leafgrows older. The greatest fall occurs in the pulvinus. In non-senescent leaves, the enzyme activity is higher in pulvinithan elsewhere and there is a gradient of activity across theline of abscission from the pulvinus to the petiole or stem.During senescence this gradient falls, and is generally reversedat abscission. Data suggest that the advent of abscission maybe linked to the steepness of this gradient. If abscission of leaves or isolated abscission zones is acceleratedby appropriate treatments there is a more rapid decrease inpectin methylesterase activity than in the controls, and a morerapid fall in the gradient across the line of abscission. Ifabscission is retarded the enzyme activity is sustained andthe gradient is maintained or increased. The results are discussed in relation to the changes that mayoccur in the pectic constituents of cell walls during senescenceand abscission, and in relation to some of the known effectsof auxins on pectin metabolism.     

Changes in the Endosperm Cell Walls of Two Datura Species before Radicle Protrusion

The possibility of an association between changes in cell walls of the micropylar portion of the endosperm and the induction of germination was explored in seeds of Datura ferox and Datura stramonium. The structure of the inner surface of the endosperm was studied by scanning electron microscopy and the composition of cell wall polysaccharides analyzed by gas chromatography and gas chromatography-mass spectrometry. Both scanning electron microscope images and chemical analysis showed changes in the micropylar portion of the endosperm in induced seeds before radicle protrusion. The inner surface of the endosperm appeared eroded, and in some areas, wall material seemed to be missing. The content of the main component of the cell wall polysaccharides, containing predominantly 4-linked mannose, decreased well before the emergence of the radicle through the endosperm. We propose that the degradation of a mannan type polysaccharide is an important factor in the reduction in mechanical strength of the endosperm, thus facilitating germination.     

调控拟南芥花瓣脱落的果胶甲酯酶基因表达和功能分析

在基因序列和表达信息分析的基础上,重点研究初选出的两个拟南芥果胶甲酯酶基因At8g47550和At4g02330的表达和功能。半定量RT-PCR和转基因植株检测表明,At2g47550主要在花粉粒中大量表达,其次在微管组织中有明显表达。然而,At4g02330在拟南芥花和荚果整个发育时期都有不同程度的表达。主要在花的离层组织、柱头、微管组织和花粉粒中表达。结果说明,At2g47550可能参与花粉粒和花粉管的发育,而At4g02330则可能通过参与细胞壁中果胶代谢,从而达到调控细胞分离和花瓣脱落的目的。     

Analysis of the Pectin Methylesterase Activity in a Segregating Population of a Tobacco Transgenic Plant using Callus Tissue

With the aim to develop a callus-based tool to study the activity of transgenes, we induced callus from a segregating population of a tobacco transgenic plant expressing a single copy of a transgene designed to overexpress a pectin methylesterase isoenzyme (pmeu1). Pectin methylesterase (PME) activity in the whole plant showed 4-fold induction whereas in callus tissue we found up to 20-fold induction. Furthermore, determination of PME activity of 38 segregating transgenic calluses showed the presence of four groups: calluses with activity like control (calluses-c), calluses with 5-fold induction (calluses-5); calluses with 10-fold induction (calluses-10) and calluses with 20-fold induction (calluses-20) of PME activity. Based on the expected Mendelian segregation of azygous:hemizygous:homozygous, we placed calluses-c, calluses-10 and calluses-20 as azygous, hemizygous and homozygous, respectively. This is a behavior that can be explained by the gene-dosage phenomena. Chi-square statistics was used to test whether the calluses-5 are azygous (²= 4.24) or either homozygous or heterozygous (²= 0.034). The Chi-square critical value is ² _{(=0.05, 1 df)}= 3.84 which allowed to conclude that calluses-5 are either homozygous or heterozygous. Next, we tested whether calluses-5 are either hemizygous (²= 6.36) or homozygous (²= 0.314). The critical value of ² _{(=0.05, 2 df)}= 5.99 was highly significant for concluding that calluses-5 are homozygous for the transgene. This is a behavior that can be explained by the co-silencing phenomena. It is concluded that the use of callus tissue can allowed to find changes in expression of transgenes that are difficult to observe in the whole plant.     

The Pectin Methylesterase Gene Complement of Phytophthora sojae: Structural and Functional Analyses,and the Evolutionary Relationships with Its Oomycete Homologs

Phytophthora sojae is an oomycete pathogen that causes the disease known as root and stem rot in soybean plants, frequently leading to massive economic damage. Additionally, P. sojae is increasingly being utilized as a model for phytopathogenic oomycete research. Despite the economic and scientific importance of P. sojae, the mechanism by which it penetrates the host roots is not yet fully understood. It has been found that oomycetes are not capable of penetrating the cell wall solely through mechanical force, suggesting that alternative factors facilitate breakdown of the host cell wall. Pectin methylesterases have been suggested to be important for Phytophthora pathogenicity, but no data exist on their role in the P. sojae infection process. We have scanned the newly revised version of the annotated P. sojae genome for the presence of putative pectin methylesterases genes and conducted a sequence analysis of all gene models found. We also searched for potential regulatory motifs in the promoter region of the proposed P. sojae models, and investigated the gene expression levels throughout the early course of infection on soybean plants. We found that P. sojae contains a large repertoire of pectin methylesterase-coding genes and that most of these genes display similar motifs in the promoter region, indicating the possibility of a shared regulatory mechanism. Phylogenetic analyses confirmed the evolutionary relatedness of the pectin methylesterase-coding genes within and across Phytophthora spp. In addition, the gene duplication events that led to the emergence of this gene family appear to have occurred prior to many speciation events in the genus Phytophthora. Our results also indicate that the highest levels of expression occurred in the first 24 hours post inoculation, with expression falling after this time. Our study provides evidence that pectin methylesterases may be important for the early action of the P. sojae infection process.     

The Regulatory Properties of Rubisco Activase Differ among Species and Affect Photosynthetic Induction during Light Transitions

Rubisco’s catalytic chaperone, Rubisco activase (Rca), uses the energy from ATP hydrolysis to restore catalytic competence to Rubisco. In Arabidopsis (Arabidopsis thaliana), inhibition of Rca activity by ADP is fine tuned by redox regulation of the α-isoform. To elucidate the mechanism for Rca regulation in species containing only the redox-insensitive β-isoform, the response of activity to ADP was characterized for different Rca forms. When assayed in leaf extracts, Rubisco activation was significantly inhibited by physiological ratios of ADP to ATP in species containing both α-Rca and β-Rca (Arabidopsis and camelina [Camelina sativa]) or just the β-Rca (tobacco [Nicotiana tabacum]). However, Rca activity was insensitive to ADP inhibition in an Arabidopsis transformant, rwt43, which expresses only Arabidopsis β-Rca, although not in a transformant of Arabidopsis that expresses a tobacco-like β-Rca. ATP hydrolysis by recombinant Arabidopsis β-Rca was much less sensitive to inhibition by ADP than recombinant tobacco β-Rca. Mutation of 17 amino acids in the tobacco β-Rca to the corresponding Arabidopsis residues reduced ADP sensitivity. In planta, Rubisco deactivated at low irradiance except in the Arabidopsis rwt43 transformant containing an ADP-insensitive Rca. Induction of CO₂ assimilation after transition from low to high irradiance was much more rapid in the rwt43 transformant compared with plants containing ADP-sensitive Rca forms. The faster rate of photosynthetic induction and a greater enhancement of growth under a fluctuating light regime by the rwt43 transformant compared with wild-type Arabidopsis suggests that manipulation of Rca regulation might provide a strategy for enhancing photosynthetic performance in certain variable light environments.The activity of Rubisco, the enzyme that catalyzes CO₂ assimilation in photosynthesis, is regulated by Rubisco activase (Rca), a specific catalytic chaperone (Spreitzer and Salvucci, 2002; Portis, 2003). Like other AAA+ ATPases (Snider et al., 2008), Rca uses the energy from ATP hydrolysis to remodel the conformation of its target protein, Rubisco. The conformational changes induced by Rca restore catalytic competence to Rubisco active sites that have been inactivated by the unproductive binding of sugar phosphates, including the substrate ribulose 1,5-bisphosphate (RuBP; Wang and Portis, 1992). Because of the requirement for ATP hydrolysis and the inhibition of activity by ADP (Robinson and Portis, 1988, 1989), Rca adjusts the rate of CO₂ fixation to the rates of electron transport activity via changes in the activation state of Rubisco (Salvucci et al., 1985). As a result of this coordinate regulation, the light response of Rubisco activation closely resembles the light response of CO₂ assimilation, and the levels of RuBP under steady-state conditions are relatively constant over a wide range of irradiance levels (Perchorowicz et al., 1981; Dietz and Heber, 1984).Many plant species express two isoforms of Rca, α and β, that are both active in ATP hydrolysis and Rubisco activation (Shen et al., 1991; Salvucci et al., 2003). In some plant species, these isoforms are the products of an alternative splicing event that generates two polypeptides, which are identical except for a 20- to 30-amino acid extension at the C terminus of the longer α-isoform (Werneke et al., 1989). In cotton (Gossypium hirsutum), soybean (Glycine max), and presumably other plant species, separate genes encode the two isoforms of Rca (Salvucci et al., 2003; Yin et al., 2010). In these species, the amino acid sequences of the overlapping regions of the α- and β-polypeptides are very similar, and the C-terminal extension of the longer α-isoform is similar to the extension produced by alternative splicing (Supplemental Fig. S1).Our current understanding of the role of the two Rca isoforms is based primarily on investigations with Arabidopsis (Arabidopsis thaliana; Zhang and Portis, 1999; Zhang et al., 2001; Wang and Portis, 2006). The C-terminal extension of the α-Rca contains two redox-regulated Cys residues that are modulated by thioredoxin f (Zhang and Portis, 1999). When these residues are oxidized to a disulfide, the affinity for ATP decreases and enzyme activity is more sensitive to inhibition by ADP. Physiological ratios of ADP to ATP significantly inhibit the activity of the Arabidopsis α-Rca when in the oxidized state, but inhibition is much less when this isoform has been reduced by thioredoxin. In contrast, the shorter Arabidopsis β-Rca is not redox regulated and is less sensitive to inhibition by ADP (Zhang and Portis, 1999). Mixing experiments with recombinant Rca have shown that the properties of α-Rca are conferred to the heterooligomer, providing a mechanism for redox regulating the Rca holoenzyme (Zhang et al., 2001). In this way, Rca is similar to the chloroplastic glyceraldehyde 3-P dehydrogenase (GAPDH), which also has both redox-regulated (GAP-B) and non-redox-regulated (GAP-A) forms that differ by a C-terminal extension (Baalmann et al., 1996). Like Rca (Zhang and Portis, 1999; Zhang et al., 2001), redox regulation of two Cys residues in the extension exerts master control over the mixed GAPDH oligomer.Some plant species, including members of the Solanaceae family, as well as maize (Zea mays) and green algae, express only the shorter β-Rca (Salvucci et al., 1987). The β-Rca in these species is not responsive to redox regulation, even though the activation state of Rubisco in these plants is modulated by irradiance (Salvucci and Anderson, 1987) and seems to be associated with the redox status of the chloroplast (Ruuska et al., 2000). With GAPDH, all higher plants appear to have both chloroplastic isoforms, but the non-redox-sensitive form, Gap-A, can be regulated indirectly by thioredoxin through the binding of the small chloroplast protein CP12 (Trost et al., 2006). By analogy, a similar association with CP12 or a CP12-like protein could provide a means of conferring redox sensitivity to β-Rca in species that have only this Rca isoform. However, no association of Rca was observed when the native CP12 complex and other high-molecular-mass species were isolated from tobacco (Nicotiana tabacum) chloroplasts (Carmo-Silva et al., 2011b).In this study, the regulation of β-Rca activity was examined both in vivo and in vitro in plant species that contain only one (i.e. β-) or both (α- and β-) Rca isoforms. The response of enzyme activity to physiological ratios of ADP to ATP was measured for the native Rca in leaf extracts, as well as for recombinant Arabidopsis and tobacco enzymes, to determine the sensitivity of β-Rca to ADP in different species. In addition, experiments were conducted with transgenic Arabidopsis plants containing variants of β-Rca to determine the link between Rca regulation and photosynthetic induction. The results suggest a new strategy for enhancing photosynthetic performance under variable light environments based on altering the regulatory properties of Rca to increase the rate of photosynthetic induction.     

西双版纳热带雨林中两种生态型蕨类植物的光合特性比较研究

通过比较分布于西双版纳热带雨林林下生境中的附生鸟巢蕨(Neottopteris nidus)和地生网脉铁角蕨(Asplenium finlaysonianum)的光合特征和光合诱导特性, 来研究不同生态型蕨类植物的光斑利用策略。研究结果表明, 2种蕨类植物的最大净光合速率、暗呼吸速率、表观量子效率、光饱和点和光补偿点没有显著差异, 但网脉铁角蕨的最大气孔导度远远高于鸟巢蕨, 表明后者具有更强的光合水分利用效率。在暗处理3小时接着光照(光强为20 mmol .m-2.s-1)30分钟后, 网脉铁角蕨的初始气孔导度显著高于鸟巢蕨。连续照射饱和强光后, 网脉铁角蕨达到最大净光合速率50%(T50%)和90%的时间(T90%)比鸟巢蕨短: 网脉铁角蕨和鸟巢蕨的T50%分别为0.57和5.31分钟, T90%分别为5.85和26.33分钟。诱导过程中, 气孔导度对强光的响应明显滞后于净光合速率。鸟巢蕨达到最大气孔导度的时间明显比网脉铁角蕨慢, 但在光合诱导消失过程中2种蕨类植物的 光合诱导维持能力却没有显著差异。上述结果表明, 与大多数地生林下植物(如网脉铁角蕨)相比, 附生鸟巢蕨的水分保护比碳获得更重要, 但却限制了附生蕨对光斑的利用。     

西双版纳热带雨林中两种生态型蕨类植物的光合特性比较研究

通过比较分布于西双版纳热带雨林林下生境中的附生鸟巢蕨（Neottopteris nidus）和地生网脉铁角蕨（Asplenium finlaysonianum）的光合特征和光合诱导特性,来研究不同生态型蕨类植物的光斑利用策略。研究结果表明,2种蕨类植物的最大净光合速率、暗呼吸速率、表观量子效率、光饱和点和光补偿点没有显著差异,但网脉铁角蕨的最大气孔导度远远高于鸟巢蕨,表明后者具有更强的光合水分利用效率。在暗处理3／J＇,时接着光照（光强为20I~mol-m-2,s。‘）30分钟后,网脉铁角蕨的初始气孔导度显著高于鸟巢蕨。连续照射饱和强光后,网脉铁角蕨达到最大净光合速率50％（T50％）和90％的时间（T90%）比鸟巢蕨短：网脉铁角蕨和鸟巢蕨的T50%分别为0．57和5．31分钟,T90％分别为5．85和26．33分钟。诱导过程中,气孔导度对强光的响应明显滞后于净光合速率。鸟巢蕨达到最大气孔导度的时间明显比网脉铁角蕨慢,但在光合诱导消失过程中2种蕨类植物的光合诱导维持能力却没有显著差异。上述结果表明,与大多数地生林下植物（如网脉铁角蕨）相比,附生鸟巢蕨的水分保护比碳获得更重要,但却限制了附生蕨对光斑的利用。     

Patch Connectivity and Genetic Variation in Two Congeneric Grasshopper Species with Different Habitat Preferences

Two congeneric species of grasshopper, Stenobothrus lineatus and S. stigmaticus, are compared in an analysis of genetic structure relative to their observed mobility, and to the spatial structure of their habitat networks. The species differ in their habitat requirements, the latter being rarer and more restricted to isolated patches. We tested for different patch connectivity between the two species in an analysis of genetic variance (based on allozymes) under the assumption that, besides isolation, rarity influences the genetic parameters. Between the species we found no differences in genetic structure as estimated by F_ST; i.e., no isolation effects and no apparent differences between the species in the potential to move between habitat fragments on either a local or regional scale were found. However, the amount of genetic variation in the more widely distributed and less xerothermic S. lineatus was significantly higher than in S. stigmaticus. Some consistency with observed philopatry within patches was found (F_IS > 0), but we consider regular dispersal events of medium and especially long distance to cause the habitat linking. We conclude that the connectivity between occupied patches inferred by genetic analyses can seldom be derived from low observed life-time movements recorded by conventional marking studies. Consequences of applying observed relative to indirect dispersal estimates for the examination of grasshopper metapopulations are discussed.     

Chemical Properties of the Diets of Two Lemur Species in Southwestern Madagascar

Seasonal dietary variations demonstrate the importance of certain plant parts during the year. A parallel analysis of their nutritional constituents provides further information on underlying patterns of consumption of the plant parts and the relative importance of key nutrients. I studied the diets of Lemur catta (ring-tailed lemurs) and Propithecus verreauxi verreauxi (sifakas), for 9 mo over a 13-mo period in the highly seasonal tropical dry forest site of Beza Mahafaly in southwestern Madagascar. I tested dietary plant parts for nutrients—protein, free amino acids, and sugars—and for 2 potential deterrents, phenolics and tannins, using plant extracts prepared in the field. I compared consumption of nutrients and secondary compounds throughout the year and between seasons. Nutrients are balanced throughout the year. The 2 lemur species do not appear nutrient-starved in either season, though actual quantities of nutrients and contributing food parts differ for each species. Lemur catta consumes high levels of sugar throughout the year, whereas Propithecus takes in higher levels of protein. The effects of phenolics and tannins are quantitative, and they appear to deter consumption of plant parts only past a certain threshold. Sifakas consume them in greater quantities than those of ring-tailed lemurs, which appear more sensitive to their effects. Sifakas may have a higher tolerance for secondary plant metabolites, which is consistent with reports for other folivores. The overall stability of nutrients throughout the year indicates no lean period that coincides with the decline in food abundance during the dry season, though actual caloric intake probably decreases.     

不同光照和营养条件下两种不同寿命热带先锋种的对比研究

探讨了热带次生演替早期阶段短命先锋种山黄麻(Trema tomentosa)和长命先锋种中平树(Macaranga denticulata)在两种光照(4%和20%的全光照)和营养梯度(低肥和高肥)下,叶片和细根营养物质含量、非结构性碳水化合物含量、生物量分配模式以及形态结构特征的差异.结果表明:(1)两种先锋种叶片和细根氮、磷含量随着光照和营养的增强显著上升,并且山黄麻叶片和细根氮、磷含量要显著高于中平树.(2)山黄麻各器官淀粉和总非结构性碳水化合物(TNC)浓度在高光下低于中平树,但在低光下高于中平树.(3)山黄麻和中平树的叶片都是可溶性糖的主要存储器官,而淀粉和TNC则主要储存在根中,并且淀粉都是二者TNC库最主要的组成形态和成分.(4)山黄麻拥有更高的根生物量比、细根生物量比、根冠比、比叶面积、叶面积比、比细根长、比细根表面积及更小的细根直径等更强大的资源捕获能力的结构和生物量分配特征.总之,与长命先锋种中平树相比,短命先锋种山黄麻有着更适合快速生长的结构和生物量分配特征、更高的组织营养物质含量、更低的非结构性碳水化合物浓度,因而具有更高的生长速率,能够在次生演替的早期阶段占领统治地位,为以后的次生演替创造条件.     

Differences in the Composition of the Cell Walls of Two Morphologically Different Lines of Suspension-Cultured Catharanthus roseus Cells

Differences in the composition of cell walls of two morphologicallydifferent lines (A and B) of suspension-cultured Catharanthusroseus cells, which have the same origin, were investigated.The cells of strain A are nearly spherical, while those of strainB are cylindrical. In strain A, the amount of cell wall pergram fresh weight of cells increased during the logarithmicphase. In strain B, the amount of cell wall per cell decreasedduring the logarithmic phase. The level of matrix polysaccharides increased markedly duringthe logarithmic phase in strain A. The amount of cellulose incell wall was relatively larger in strain B than in strain A.The following differences in sugar composition between the twostrains were observed: (a) there was an increase in the relativelevels of 4-linked galactose in the EDTA-soluble fraction andof 3-linked glucose in the 5% KOH-soluble fraction during thelogarithmic phase in strain A; (b) there were significantlyhigher levels of arabinose, probably derived from 2,5- and/or3,5-linked arabinan, in the EDTA-soluble fraction and in theextracellular polysaccharides in strain B; (c) there were decreasesin the relative amounts of some kinds of sugar, probably thosederived from xyloglucan, during the stationary phase in strainB. (Received March 31, 1989; Accepted October 12, 1989)     

棉属栽培种与野生种杂交的不亲和性

本文研究了棉属栽培种与野生种杂交的不亲和性,试验材料涉及5个染色体组,包括2个栽培种(陆地棉和中棉)和5个野生种(戴维逊氏棉、瑟伯氏棉、三裂棉、阿拉伯棉和比克氏棉)。以陆地棉作母本,异己花粉管在花柱中生长缓慢,有花粉管胚珠低于10%,陆地棉×戴维逊氏棉杂种胚在子叶期坏死。以中棉作母本,不亲和性主要表现在受精后的胚胎发育过程中。     

Admixture in Humans of Two Divergent Plasmodium knowlesi Populations Associated with Different Macaque Host Species

Human malaria parasite species were originally acquired from other primate hosts and subsequently became endemic, then spread throughout large parts of the world. A major zoonosis is now occurring with Plasmodium knowlesi from macaques in Southeast Asia, with a recent acceleration in numbers of reported cases particularly in Malaysia. To investigate the parasite population genetics, we developed sensitive and species-specific microsatellite genotyping protocols and applied these to analysis of samples from 10 sites covering a range of >1,600 km within which most cases have occurred. Genotypic analyses of 599 P. knowlesi infections (552 in humans and 47 in wild macaques) at 10 highly polymorphic loci provide radical new insights on the emergence. Parasites from sympatric long-tailed macaques (Macaca fascicularis) and pig-tailed macaques (M. nemestrina) were very highly differentiated (FST = 0.22, and K-means clustering confirmed two host-associated subpopulations). Approximately two thirds of human P. knowlesi infections were of the long-tailed macaque type (Cluster 1), and one third were of the pig-tailed-macaque type (Cluster 2), with relative proportions varying across the different sites. Among the samples from humans, there was significant indication of genetic isolation by geographical distance overall and within Cluster 1 alone. Across the different sites, the level of multi-locus linkage disequilibrium correlated with the degree of local admixture of the two different clusters. The widespread occurrence of both types of P. knowlesi in humans enhances the potential for parasite adaptation in this zoonotic system.