The polygalacturonase-inhibiting protein PGIP2 of Phaseolus vulgaris has evolved a mixed mode of inhibition of endopolygalacturonase PG1 of Botrytis cinerea

Botrytis cinerea is a phytopathogenic fungus that causes gray mold in >1,000 plant species. During infection, it secretes several endopolygalacturonases (PGs) to degrade cell wall pectin, and among them, BcPG1 is constitutively expressed and is an important virulence factor. To counteract the action of PGs, plants express polygalacturonase-inhibiting proteins (PGIPs) that have been shown to inhibit a variety of PGs with different inhibition kinetics, both competitive and noncompetitive. The PG-PGIP interaction promotes the accumulation of oligogalacturonides, fragments of the plant cell wall that are general elicitors of plant defense responses. Here, we characterize the enzymatic activity of BcPG1 and investigate its interaction with PGIP isoform 2 from Phaseolus vulgaris (PvPGIP2) by means of inhibition assays, homology modeling, and molecular docking simulations. Our results indicate a mixed mode of inhibition. This is compatible with a model for the interaction where PvPGIP2 binds the N-terminal portion of BcPG1, partially covering its active site and decreasing the enzyme affinity for the substrate. The structural framework provided by the docking model is confirmed by site-directed mutagenesis of the residues that distinguish PvPGIP2 from the isoform PvPGIP1. The finding that PvPGIP2 inhibits BcPG1 with a mixed-type kinetics further indicates the versatility of PGIPs to evolve different recognition specificities.     

A polygalacturonase-inhibiting protein from grapevine reduces the symptoms of the endopolygalacturonase BcPG2 from Botrytis cinerea in Nicotiana benthamiana leaves without any evidence for in vitro interaction

Six endopolygalacturonases from Botrytis cinerea (BcPG1 to BcPG6) as well as mutated forms of BcPG1 and BcPG2 were expressed transiently in leaves of Nicotiana benthamiana using agroinfiltration. Expression of BcPG1, BcPG2, BcPG4, BcPG5, and mutant BcPG1-D203A caused symptoms, whereas BcPG3, BcPG6, and mutant BcPG2-D192A caused no symptoms. Expression of BcPG2 caused the most severe symptoms, including wilting and necrosis. BcPG2 previously has been shown to be essential for B. cinerea virulence. The in vivo effect of this enzyme and the inhibition by a polygalacturonase-inhibiting protein (PGIP) was examined by coexpressing Bcpg2 and the Vvpgipl gene from Vitis vinifera in N. benthamiana. Coinfiltration resulted in a substantial reduction of the symptoms inflicted by the activity of BcPG2 in planta, as evidenced by quantifying the variable chlorophyll fluorescence yield. In vitro, however, no interaction between pure VvPGIP1 and pure BcPG2 was detected. Specifically, VvPGIP1 neither inhibited BcPG2 activity nor altered the degradation profile of polygalacturonic acid by BcPG2. Furthermore, using surface plasmon resonance technology, no physical interaction between VvPGIP1 and BcPG2 was detected in vitro. The data suggest that the in planta environment provided a context to support the interaction between BcPG2 and VvPGIP1, leading to a reduction in symptom development, whereas neither of the in vitro assays detected any interaction between these proteins.     

Antisense expression of the Arabidopsis thaliana AtPGIP1 gene reduces polygalacturonase-inhibiting protein accumulation and enhances susceptibility to Botrytis cinerea

Polygalacturonases (PGs) hydrolyze the homogalacturonan of plant cell-wall pectin and are important virulence factors of several phytopathogenic fungi. In response to abiotic and biotic stress, plants accumulate PG-inhibiting proteins (PGIPs) that reduce the activity of fungal PGs. In Arabidopsis thaliana, PGIPs with comparable activity against BcPG1, an important pathogenicity factor of the necrotrophic fungus Botrytis cinerea, are encoded by two genes, AtPGIP1 and AtPGIP2. Both genes are induced by fungal infection through different signaling pathways. We show here that transgenic Arabidopsis plants expressing an antisense AtPGIP1 gene have reduced AtPGIP1 inhibitory activity and are more susceptible to B. cinerea infection. These results indicate that PGIP contributes to basal resistance to this pathogen and strongly support the vision that this protein plays a role in Arabidopsis innate immunity.     

Necrotizing activity of five Botrytis cinerea endopolygalacturonases produced in Pichia pastoris

Five Botrytis cinerea endopolygalacturonase enzymes (BcPGs) were individually expressed in Pichia pastoris, purified to homogeneity and biochemically characterized. While the pH optima of the five enzymes were similar (approximately pH 4.5) the maximum activity of individual enzymes differed significantly. For hydrolysis of polygalacturonic acid (PGA), the V(max,app) ranged from 10 to 900 U mg(-1), while the K(m,app) ranged from 0.16 to 0.6 mg ml(-1). Although all BcPGs are true endopolygalacturonases, they apparently have different modes of action. PGA hydrolysis by BcPG1, BcPG2 and BcPG4 leads to the transient accumulation of oligomers with DP < 7, whereas PGA hydrolysis by BcPG3 and BcPG6 leads to the immediate accumulation of monomers and dimers. The necrotizing activity (NA) of all BcPGs was tested separately in tomato, broad bean and Arabidopsis thaliana. They showed different NAs on these plants. BcPG1 and BcPG2 possessed the strongest NA as tissue collapse was observed within 10 min after infiltration of broad bean leaves. The amino acid (aa) D192A substitution in the active site of BcPG2 not only abolished enzyme activity but also the NA, indicating that the NA is dependent on enzyme activity. Furthermore, deletion of the Bcpg2 gene in B. cinerea resulted in a strong reduction in virulence on tomato and broad bean. Primary lesion formation was delayed by approximately 24 h and the lesion expansion rate was reduced by 50-85%. These data indicate that BcPG2 is an important virulence factor for B. cinerea.     

Evolutionary analysis of endopolygalacturonase-encoding genes of Botrytis cinerea

Sequence analysis of five of the six endopolygalacturonase-encoding genes ( Bcpg1 , Bcpg2 , Bcpg3 , Bcpg4 , Bcpg5 ) from 32 strains of Botrytis cinerea showed marked gene to gene differences in the amount of among-strains diversity. Bcpg4 was almost invariable in all strains; Bcpg3 and Bcpg5 showed a moderate variability, similar to that of non-pathogenicity-associated genes examined in other studies. Conversely, Bcpg1 and Bcpg2 were highly variable and were shown to be under positive selection based on the McDonald–Kreitman test and likelihood ratio test. The evolution of the five endopolygalacturonase genes is explained by their different ecophysiological role. Diversification and balancing selection, as detected in Bcpg1 and Bcpg2 , can be used by the pathogen to escape recognition by the host and delay plant reaction in the early phases of infection. The analysis of the polymorphisms and the location of the sites with high probability of being positively selected highlighted the relevance of variability of the BcPG1 and BcPG2 proteins at their C-terminal end. By contrast, the absence of variability in Bcpg4 suggests that the efficiency of the product of this gene is critical for B. cinerea growth in late phases of infection or during intraspecific competition, thus markedly affecting strain fitness.     

The BOS loci of Arabidopsis are required for resistance to Botrytis cinerea infection

Three Botrytis-susceptible mutants bos2, bos3, and bos4 which define independent and novel genetic loci required for Arabidopsis resistance to Botrytis cinerea were isolated. The bos2 mutant is susceptible to B. cinerea but retains wild-type levels of resistance to other pathogens tested, indicative of a defect in a response pathway more specific to B. cinerea. The bos3 and bos4 mutants also show increased susceptibility to Alternaria brassicicola, another necrotrophic pathogen, suggesting a broader role for these loci in resistance. bos4 shows the broadest range of effects on resistance, being more susceptible to avirulent strain of Pseudomonas syringae pv. tomato. Interestingly, bos3 is more resistant than wild-type plants to virulent strains of the biotrophic pathogen Peronospora parasitica and the bacterial pathogen P. syringae pv. tomato. The Pathogenesis Related gene 1 (PR-1), a molecular marker of the salicylic acid (SA)-dependent resistance pathway, shows a wild-type pattern of expression in bos2, while in bos3 this gene was expressed at elevated levels, both constitutively and in response to pathogen challenge. In bos4 plants, PR-1 expression was reduced compared with wild type in response to B. cinerea and SA. In bos3, the mutant most susceptible to B. cinerea and with the highest expression of PR-1, removal of SA resulted in reduced PR-1 expression but no change to the B. cinerea response. Expression of the plant defensin gene PDF1-2 was generally lower in bos mutants compared with wild-type plants, with a particularly strong reduction in bos3. Production of the phytoalexin camalexin is another well-characterized plant defense response. The bos2 and bos4 mutants accumulate reduced levels of camalexin whereas bos3 accumulates significantly higher levels of camalexin than wild-type plants in response to B. cinerea. The BOS2, BOS3, and BOS4 loci may affect camalexin levels and responsiveness to ethylene and jasmonate. The three new mutants appear to mediate disease responses through mechanisms independent of the previously described BOS1 gene. Based on the differences in the phenotypes of the bos mutants, it appears that they affect different points in defense response pathways.     

Population genetic consequences of feeding habits in some forest lepidoptera

By surveying variation at allozyme loci in several phytophagous lepidopteran species (Geometridae), we have tested two hypotheses about the relationship of genetic variation to environmental heterogeneity: (1) that allozyme polymorphisms may exist because of associations between genotypes and "niches" (different host plants, in this instance), and (2) that the overall genetic variation of a species is correlated with environmental heterogeneity (or breadth of the species' overall ecological niche).—Genetic differentiation among samples of oligophagous or polyphagous species taken from different host species was observed in one of three species, at only one of seven polymorphic loci. The data thus provide no evidence for pronounced genetic substructuring, or "host race" formation in these sexually reproducing species, although host plant-genotype associations in a parthenogenetic moth give evidence of the potential for diversifying selection.—In a comparison of allozyme variation in polyphagous ("generalized") and oligophagous ("specialized") species, heterozygosity appeared to be higher in specialized species, at all polymorphic loci but one. It is possible that this unexpected result arises from a functional relation between breadth of diet and genetic variation.     

Complex genetics control natural variation in Arabidopsis thaliana resistance to Botrytis cinerea

The genetic architecture of plant defense against microbial pathogens may be influenced by pathogen lifestyle. While plant interactions with biotrophic pathogens are frequently controlled by the action of large-effect resistance genes that follow classic Mendelian inheritance, our study suggests that plant defense against the necrotrophic pathogen Botrytis cinerea is primarily quantitative and genetically complex. Few studies of quantitative resistance to necrotrophic pathogens have used large plant mapping populations to dissect the genetic structure of resistance. Using a large structured mapping population of Arabidopsis thaliana, we identified quantitative trait loci influencing plant response to B. cinerea, measured as expansion of necrotic lesions on leaves and accumulation of the antimicrobial compound camalexin. Testing multiple B. cinerea isolates, we identified 23 separate QTL in this population, ranging in isolate-specificity from being identified with a single isolate to controlling resistance against all isolates tested. We identified a set of QTL controlling accumulation of camalexin in response to pathogen infection that largely colocalized with lesion QTL. The identified resistance QTL appear to function in epistatic networks involving three or more loci. Detection of multilocus connections suggests that natural variation in specific signaling or response networks may control A. thaliana-B. cinerea interaction in this population.     

Herbivore host-associated genetic differentiation depends on the scale of plant genetic variation examined

Herbivore adaptation to plant genetic variation can lead to reproductive isolation and the formation of host-associated lineages (host-associated differentiation, or HAD). Plant genetic variation exists along a scale, ranging from variation among individual plant genotypes to variation among plant species. Along this scale, herbivores may adapt and diverge at any level, yet few studies have examined whether herbivore differentiation exhibits scaling with respect to host variation (e.g., from genotypes to species). Determining at which level(s) herbivore differentiation occurs can provide insight into the importance of plant genetic variation on herbivore evolution. Previous studies have found strong genetic differentiation in the eriophyid mite, Aceria parapopuli, between hybrid Populus hosts and parental Populus species, but minimal neutral-locus differentiation among individual trees of the same species. We tested whether genetic differentiation in A. parapopuli scales with genetic variation in its Populus hosts. Using mite ITS1 sequence data collected among host species and among host populations, two key patterns emerged. (1) We found strong differentiation of A. parapopuli among Populus species, supporting the hypothesis that plant species differences drive reproductive isolation and HAD. (2) We did not find evidence of host-driven genetic differentiation in mites at the level of plant populations, suggesting that this level of plant variation is insufficiently strong to drive differentiation at a neutral locus. In combination with previous studies, we found that HAD occurs at the higher levels of plant genetic variation, but not at lower levels, and conclude that HAD depends on the scale of plant genetic variation examined.     

RESOURCE-ASSOCIATED POPULATION SUBDIVISION IN A SYMBIOTIC CORAL-REEF SHRIMP

The importance of sympatric speciation remains controversial. An empirical observation frequently offered in its support is the occurrence of sister taxa living in sympatry but using different resources. To examine the possibility of sympatric differentiation in producing such cases, I measured genetic, behavioral, and demographic differentiation between populations of the tropical sponge-dwelling shrimp Synalpheus brooksi occupying two alternate host species on three reefs in Caribbean Panama. This species belongs to an apparently monophyletic group of ≥ 30 species of mostly obligate, host-specific sponge-dwellers, many of which occur in sympatry. Demographic data demonstrated the potential for disruptive selection imposed by the two host species: shrimp demes from the sponge Agelas clathrodes were consistently denser, poorer in mature females, more heavily parasitized by branchial bopyrid isopods, and less parasitized by thoracic isopods, than conspecific shrimp from the sponge Spheciospongia vesparium. Laboratory assays demonstrated divergence in host preference: shrimp on all three reefs tended to choose their native sponge species more often than did conspecific shrimp from the other host. Because S. brooksi mates within the host, this habitat selection should foster assortative mating by host species. A hierarchical survey of protein-electrophoretic variation also supported host-mediated divergence, revealing the following: (1) shrimp from the two hosts are conspecific, as evidenced by absence of fixed allelic differences at any of nine allozyme loci scored; (2) strong genetic subdivision among populations of this philopatric shrimp on reefs separated by 1–3 km; and (3) significant host-associated genetic differentiation within two of the three reefs. Finally, intersexual aggression (a proxy for mating incompatibility) between shrimp from different host species was significantly elevated on the one reef where host-associated genetic differences were strongest, demonstrating concordance between genetic and behavioral estimates of divergence. Adjacent reefs appear to be semi-independent sites of host-associated differentiation, as evidenced by differences in the degree of host-associated behavioral and genetic differentiation, and in the specific loci involved, on different reefs. In philopatric organisms with highly subdivided populations, such as S. brooksi, resource-associated differentiation can occur independently in different populations, thus providing multiple “experiments” in differentiation and resulting in a mosaic pattern of polymorphism as reflected by neutral genetic markers. Several freshwater fishes, an amphipod, and a snail similarly show independent but remarkably convergent patterns of resource-associated divergence in different conspecific populations, often in the absence of obvious spatial barriers. In each case, substantial differentiation has occurred in the face of continuing gene flow.     

Population subdivision and gene flow among wild orangutans

Genetic variability among populations of orangutans from Borneo and Sumatra was assessed using seven SSR loci. Most SSR loci were highly polymorphic and their allele frequencies exhibited substantial variation across subpopulations. While significant genetic subdivision was observed among the island populations, genetic distance did not increase with geographic distance and sufficient gene flow persists to prevent marked genetic subdivision. Since it is unlikely that the Bornean Orangutans dispersed naturally among locations separated by such formidable geographic barriers, human assistance might already have altered their genetic structure. Our data suggests that there may be at least two subspecific clades of orangutans within Borneo while Central Kalimantan animals may have become more genetically related to animals in Sumatra due to human intervention.     

Integrated signaling network involving calcium, nitric oxide, and active oxygen species but not mitogen-activated protein kinases in BcPG1-elicited grapevine defenses

We have already reported the identification of the endopolygalacturonase 1 (BcPG1) from Botrytis cinerea as a potent elicitor of defense responses in grapevine, independently of its enzymatic activity. The aim of the present study is the analysis of the signaling pathways triggered by BcPG1 in grapevine cells. Our data indicate that BcPG1 induces a Ca2+ entry from the apoplasm, which triggers a phosphorylation-dependent nitric oxide (NO) production via an enzyme probably related to a NO synthase. Then NO is involved in (i) cytosolic calcium homeostasis, by activating Ca2+ release from internal stores and regulating Ca2+ fluxes across the plasma membrane, (ii) plasma membrane potential variation, (iii) the activation of active oxygen species (AOS) production, and (iv) defense gene expression, including phenylalanine ammonia lyase and stilbene synthase, which encode enzymes responsible for phytoalexin biosynthesis. Interestingly enough, mitogen-activated protein kinase (MAPK) activation is independent of this regulation pathway that closely connects Ca2+, NO, and AOS.     

PATTERNS OF GENETIC ARCHITECTURE FOR LIFE-HISTORY TRAITS AND MOLECULAR MARKERS IN A SUBDIVIDED SPECIES

Abstract Understanding the utility and limitations of molecular markers for predicting the evolutionary potential of natural populations is important for both evolutionary and conservation genetics. To address this issue, the distribution of genetic variation for quantitative traits and molecular markers is estimated within and among 14 permanent lake populations of Daphnia pulicaria representing two regional groups from Oregon. Estimates of population subdivision for molecular and quantitative traits are concordant, with Q _ST generally exceeding G _ST. There is no evidence that microsatellites loci are less informative about subdivision for quantitative traits than are allozyme loci. Character-specific comparison of Q _ST and G _ST support divergent selection pressures among populations for the majority of life-history traits in both coast and mountain regions. The level of within-population variation for molecular markers is uninformative as to the genetic variation maintained for quantitative traits. In D. pulicaria , regional differences in the frequency of sex may contribute to variation in the maintenance of expressed within-population quantitative-genetic variation without substantially impacting diversity at the genic level. These data are compared to an identical dataset for 17 populations of the temporary-pond species, D. pulex .     

Demic structure and its relation with the distribution of an adaptive trait in Danish flea beetles

The flea beetle Phyllotreta nemorum is an oligophagous species using crucifers as host plants. In Denmark two populations have been found which use Barbarea vulgaris ssp. arcuata (G-type) as a host plant, whereas this plant is unsuitable for the survival of the majority of P. nemorum. In the locations in which these two populations occur, alternative host plants are also present. The plants occur in patches, some of which contain a mixture of host plants. In this study of allozyme variation, genetic differentiation between P. nemorum using different host plants in patches in the two different localities was studied hierarchically to assess substructuring of the populations. Evidence was found for low, but significant, amounts of genetic differentiation between (sub)populations using spatially separated plant patches at a distance of approximately 100 m to 1 km (theta = 0.009) and between localities approximately 44 km apart (theta = 0.026), and there was an association between genetic differentiation and geographical distance. No genetic differentiation was found between beetles from different host plants with overlapping local distributions. No evidence was thus found for sympatric host race formation. The geographical distribution of genes enabling P. nemorum to use B. vulgaris as a host plant (100% 'resistant' beetles in samples from B. vulgaris, but much fewer on patches containing only alternative host plants) contrasts with the relatively low amount of genetic differentiation at the neutral allozyme loci. This distribution of 'resistant' beetles (to B. vulgaris defence) is likely to be influenced by local differences in selection and asymmetric gene flow.     

Evolution of drought tolerance and defense: dependence of tradeoffs on mechanism, environment and defense switching

Plants evolve defenses against herbivores and pathogens in stressful environments; however, plants that evolve tolerances to other environmental stressors may have compromised defenses. Such tradeoffs involving defenses may depend on limited resources or otherwise stressful environments; however, the effect of stressful environments on defense expression might be different for different genotypes (G×E). To test these predictions, we studied genetic variation and co‐variation of drought stress tolerance and defenses at two levels of genetic variation: between and within closely related species. We did this across an experimental drought stress gradient in a growth room for species for which genetic variation in drought tolerance was likely. In apparent contrast to predictions, the species Boechera holboellii (Brassicaceae) from lower and dryer elevations had slower inherent growth rates and correspondingly higher total defensive glucosinolate concentrations than the closely related species B. stricta from higher elevations. Thus, B. holboellii was both drought tolerant and defended; however, optimality theory does predict tradeoffs between defense and growth. Differences between species in the direct effect of water deficiency on glucosinolate production did not obscure the grow‐or‐defend tradeoff. B. holboellii may also have been more resistant to the specialist herbivore Plutella xylostella; a trend that was less clear because it depended on plant development and water deficient conditions. At finer scales of genetic variation, there was significant variation among families and naturally occurring inbred lines of B. stricta in drought tolerance measured as inherent growth, the reaction norm of growth across drought treatments, shoot water potential, and transpiration rates. Evidence for tradeoffs was also found within B. stricta in genetic correlations between resistance and transpiration rates, or glucosinolates and growth rates. No G×E was detected at these finer scales of genetic variation, although sometimes the tradeoff was dependent on drought conditions. Direct effects of drought stress resulted in an apparent plastic switch between resistance and tolerance to damage, which might be a cost avoidance mechanism because tradeoffs never involved tolerance to damage. Thus, when drought tolerance is manifest as slow inherent growth rates, plants may also have relatively high defense levels, especially in stressful environments. Otherwise, defenses may be compromised by drought‐coping mechanisms, although plastic switches to less costly defenses may alleviate constraints in stressful environments.     

Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.     

The D-galacturonic acid catabolic pathway in Botrytis cinerea

D-galacturonic acid is the most abundant component of pectin, one of the major polysaccharide constituents of plant cell walls. Galacturonic acid potentially is an important carbon source for microorganisms living on (decaying) plant material. A catabolic pathway was proposed in filamentous fungi, comprising three enzymatic steps, involving D-galacturonate reductase, L-galactonate dehydratase, and 2-keto-3-deoxy-L-galactonate aldolase. We describe the functional, biochemical and genetic characterization of the entire D-galacturonate-specific catabolic pathway in the plant pathogenic fungus Botrytis cinerea. The B. cinerea genome contains two non-homologous galacturonate reductase genes (Bcgar1 and Bcgar2), a galactonate dehydratase gene (Bclgd1), and a 2-keto-3-deoxy-L-galactonate aldolase gene (Bclga1). Their expression levels were highly induced in cultures containing GalA, pectate, or pectin as the sole carbon source. The four proteins were expressed in Escherichia coli and their enzymatic activity was characterized. Targeted gene replacement of all four genes in B. cinerea, either separately or in combinations, yielded mutants that were affected in growth on D-galacturonic acid, pectate, or pectin as the sole carbon source. In Aspergillus nidulans and A. niger, the first catabolic conversion only involves the Bcgar2 ortholog, while in Hypocrea jecorina, it only involves the Bcgar1 ortholog. In B. cinerea, however, BcGAR1 and BcGAR2 jointly contribute to the first step of the catabolic pathway, albeit to different extent. The virulence of all B. cinerea mutants in the D-galacturonic acid catabolic pathway on tomato leaves, apple fruit and bell peppers was unaltered.     

Behavior and Genetic Variation in Natural Populations

An analysis of allelic variation at genetic loci controllingseveral esterases and hemoglobin, as demonstrated by electrophoresis,indicates that wild populations of the house mouse (Mus musculus)are characterized by fine-scale genetic subdivision, which,through the territorial behavior of family groups (tribes),is achieved even in the absence of physical or ecological barriersto migration. Heterogeneity in allele frequencies among samples from farmsin the same region and from barns on the same farm was demonstrated.Spatial variation in allele frequencies within single barns,involving a clustering of like genotypes, was shown by grid-trapping,thus providing direct evidence of tribal subdivision in continuouslydistributed populations. For two loci, Es-3 and Hbb, an excess of heterozygotes appearedin samples from small populations, while a deficit characterizedsamples from large populations. The evolutionary significance of subdivision and consequentdrift in house mouse populations cannot properly be evaluatedat this time. Although stochastic processes may play the dominantrole in determining, at a given locus, the genotypes of individualsand frequencies of alleles in small populations, geographicpatterns of variation, as studied in Texas, are characterizedby uniformity of allelic frequency in major physiographic orclimatic regions, as would be expected if selection is determiningthe frequencies.