An hypothesis and evidence concerning the genetic components controlling tumor formation in Nicotiana

Summary The evidence discussed in this paper demonstrates unuqual genetic contribution of N. debneyi-tabacum and N. longiflora to the development of tumors in hybrids between them. Tumor formation depends upon the presence of a specific longiflora chromosome fragment in an otherwise debneyi-tabacum background and consequently is transmitted as a dominant trait. Tumor expression remains relatively constant among those segregants which carry the complete complements of N. debneyi-tabacum or N. tabacum along with the longiflora chromosome, but tumors fail to develop on plants with a few debneyi chromosomes on a diploid longiflora background. These results suggest that gene(s) on a single longiflora chromosome fragment are sufficient, whereas from N. debneyi or N. tabacum a large number of genes distributed over many chromosomes are required for tumor formation. An hypothesis concerning genetic components controlling tumor initiation (I) and expression (ee) is proposed, supported by these observations, and by previous studies both genetic and physiological, on another tumorous hybrid between N. glauca and N. langsdorffii. (I) and (ee), representing unequal contributions from two evolutionarily diverse species, must both be present in the hybrid for tumors to develop. Evidence is presented to indicate that N. longiflora and N. langsdorffii, belonging to the section Alatae, represent species carrying (I) and that N. debneyi, N. tabacum and N. glauca, belonging to different sections of the genus Nicotiana, are (ee) carriers. It is predicted that genetic analyses will reveal that the genes for tumor initiation (I) will be carried invariably by species of the section Alatae, or the so-called plus group of Näf, and genes modifying expression (ee) by species from other sections but belonging to the so-called minus group. Specific characterization of (I) and (ee) in biochemical terms is under investigation.     

Geographical variation in postzygotic isolation and its genetic basis within and between two Mimulus species

The aim of this study is to investigate the evolution of intrinsic postzygotic isolation within and between populations of Mimulus guttatus and Mimulus nasutus. We made 17 intraspecific and interspecific crosses, across a wide geographical scale. We examined the seed germination success and pollen fertility of reciprocal F₁ and F₂ hybrids and their pure-species parents, and used biometrical genetic tests to distinguish among alternative models of inheritance. Hybrid seed inviability was sporadic in both interspecific and intraspecific crosses. For several crosses, Dobzhansky–Muller incompatibilities involving nuclear genes were implicated, while two interspecific crosses revealed evidence of cytonuclear interactions. Reduced hybrid pollen fertility was found to be greatly influenced by Dobzhansky–Muller incompatibilities in five out of six intraspecific crosses and nine out of 11 interspecific crosses. Cytonuclear incompatibilities reduced hybrid fitness in only one intraspecific and one interspecific cross. This study suggests that intrinsic postzygotic isolation is common in hybrids between these Mimulus species, yet the particular hybrid incompatibilities responsible for effecting this isolation differ among the populations tested. Hence, we conclude that they evolve and spread only at the local scale.     

Response of Nicotiana mesophyll protoplasts of normal and tumorous origin to indoleacetic Acid in vitro

Enzymatically isolated mesophyll protoplasts of the two normal, nontumor-forming parent species Nicotiana glauca and N. langsdorffii and two of their tumor-prone interspecific hybrids were maintained in a 0.5 m mannitol solution supplemented with various concentrations of auxin (indoleacetic acid) and the growth inhibitor abscisic acid. The bursting response of protoplasts in medium containing indoleacetic acid in physiological concentrations showed that protoplasts from the tumorous hybrids tolerate auxin in up to 30 times higher concentrations than protoplasts from parent plants. The “survival” of all protoplast preparations in comparable abscisic acid containing media was significantly greater than that in the indoleacetic acid supplemented solutions. Protoplasts in vitro respond with bursting only after the external indoleacetic acid concentrations reach levels comparable to those of endogenous auxins present in these cells. The data are discussed in conjunction with previous observations on uptake and maintenance of indoleacetic acid levels in tumorous Nicotiana tissues.     

Population genetic structure of Picea engelmannii, P. glauca and their previously unrecognized hybrids in the central Rocky Mountains

Areas of geographic overlap between potentially hybridizing species provide the opportunity to study interspecific gene flow and reproductive barriers. Here we identified hybrids between Picea engelmannii and P. glauca by their genetic composition at 17 microsatellite markers, and determined the broad-scale geographic distribution of hybrids in the central Rocky Mountains of North America, a geographic region where hybrids and isolation between species had not previously been studied. Parameter estimates from admixture models revealed considerable variation in ancestry within and among collection sites, suggesting that within this area of geographic overlap, the interaction of the two species varies extensively. The results document a previously unrecognized patchy distribution of hybrids between P. engelmannii and P. glauca, including locations where hybrids were not known or expected to exist. Further, the ancestry of many hybrids was consistent with multiple generations of hybridization, with probable directional backcrossing to P. engelmannii, suggesting a relatively porous species boundary. The identification and characterization of hybridization between these spruce in this region raises the question of what factors maintain barriers to gene flow in these long-lived forest trees. The current research lays the groundwork for future study of the ecological and evolutionary contexts of their hybridization, as well as of differential introgression and permeability of species boundaries.     

Restoration of shooty morphology of a nontumorous mutant of Nicotiana glauca x N. langsdorffii by cytokinin and the isopentenyltransferase gene

The shooty morphology of a nontumorous amphidiploid mutant of Nicotiana glauca Grah. x N. langsdorffii Weinm. was restored by cytokinins, whether exogenously applied or endogenously produced by transformation of the mutant with a transfer DNA (T-DNA) cytokinin-biosynthesis gene (isopentenyltransferase; ipt). Auxins alone did not confer this effect. Similar transformation was not achieved for the parental species. In the case of transformation with the ipt gene, selection of the transformed tissues was based on its hormone-independent growth in the presence of the antibiotic kanamycin. Transformed tissues exhibited a shooty morphology, indistinguishable from that of wildtype genetic tumors N. glauca x N. langsdorffii. This altered phenotype was caused by the presence and constitutive expression of the ipt gene. The insertion and expression of this gene in transformed tissues was confirmed by using the polymerase chain reaction (PCR) technique as well as conventional molecular hybridization analysis. Expression of the ipt gene led to an elevated level of cytokinin in the transformed mutant tissues. This evidence supports the notion that genetic tumors are caused, at least in part, by elevated levels of cytokinin in interspecific hybrids.     

Behavioural mechanisms of sexual isolation involving multiple modalities and their inheritance

Speciation research dissects the genetics and evolution of reproductive barriers between parental species. Hybrids are the “gatekeepers” of gene flow, so it is also important to understand the behavioural mechanisms and genetics of any potential isolation from their parental species. We tested the role of multiple behavioural barriers in reproductive isolation among closely related field crickets and their hybrids (Teleogryllus oceanicus and Teleogryllus commodus). These species hybridize in the laboratory, but the behaviour of hybrids is unusual and there is little evidence for gene flow in the wild. We found that heterospecific pairs exhibited reduced rates of courtship behaviour due to discrimination by both sexes, and that this behavioural isolation was symmetrical. However, hybrids were not sexually selected against and exhibited high rates of courtship behaviour even though hybrid females are sterile. Using reciprocal hybrid crosses, we characterized patterns of interspecific divergence and inheritance in key sexual traits that might underlie the mating patterns we found: calling song, courtship song and cuticular hydrocarbons (CHCs). Song traits exhibited both sex linkage and transgressive segregation, whereas CHCs exhibited only the latter. Calculations of the strength of isolation exerted by these sexual traits suggest that close‐range signals are as important as long‐distance signals in contributing to interspecific sexual isolation. The surprisingly weak mating barriers observed between hybrids and parental species highlight the need to examine reproductive isolating mechanisms and their genetic bases across different potential stages of introgressive hybridization.     

Cytogenetics of modern sugar canes

Modern sugar cane varieties are derived from interspecific crosses involving as many as four species. Because a chromosome increase accompanies certain crosses and backcrosses, modern varieties have very high aneuploid chromosome numbers and complicated genetics. Despite this complexity, the chromosome behavior of some modern varieties approaches that of allopolyploids. In achieving homozygosity, therefore, such varieties should respond to inbreeding almost like diploids. The meiotic chromosome behavior of F₁ hybrids and modern varieties indicates little or no genetic exchange between chromosomes ofSaccharum officinarum andS. spontaneum. Irradiation may break linkages between desirable and undesirableS. spontaneum genes not ordinarily broken by crossing-over between the chromosomes of the two species. The quick success of nobilizingS. spontaneum (recurrently back-crossing to “noble canes”) depends on a peculiar increase of the chromosomes ofS. officinarum. Experience with nobilizingS. spontaneum should not make breeders impatient when they turn to interspecific crosses unaccompanied by chromosome increases.     

Occurrence of genetic tumours in Triticum interspecies hybrids

Summary F₁ interspecific hybrids involving nine tetraploid Triticum species were studied. Some developed leaf tumours at the seedling stage. Tumorous hybrids were restricted to crosses involving either T. timopheevi or T. araraticum as one parent. The hybrids from the rest of the crosses, including those of T. timopheevi × T. araraticum, were non-tumorous. Genetically divergent and non-integrated parental species appeared to be inducing spontaneous tumour formation in their hybrids.     

Tumor formation and morphogenesis on different Nicotiana sp and hybrids induced by Agrobacterium tumefaciens T-DNA mutants

A series of experiments are presented that have been performed to observe the interactions between Agrobacterium tumefaciens strains mutated in the T-DNA genes involved in indoleacetic acid and cytokinin biosynthesis and several Nicotiana species and hybrids. Infections were induced on leaf cuttings of Nicotiana debneyi, N. knightiana, N. clevelandii, N. bigelovii var bigelovii, N. bigelovii var quadrivalvis, N. glauca, N. langsdorffii, the amphidiploid tumorous hybrid N. glauca × N. langsdorffii, and a nontumorous mutant of it. The effect of deletions of the Ti plasmid varied according to plant genotype. Insertion mutants in iaaM and iaaH suppressed tumor formation in N. langsdorffii, reduced it in N. bigeloviivar quadrivalvis, had no effect in N. glauca and the two amphidiploid hybrids, and promoted tumorigenesis when compared to the wild-type Agrobacterium strain B6S3 in N. bigelovii N. debneyi, and N. knightiana. The same mutations induced shoot formation in N. glauca, increased it in N. debneyi, and suppressed root formation in N. knightiana. On the other hand, an insertion mutation of the isopentenyl transferase gene (ipt-) had no effect in N. bigelovii var quadrivalvis, N. debneyi, the tumorous hybrid, suppressed tumor formation in N. langsdorffii, and inhibited it in N. glauca, the nontumorous hybrid, N. bigelovii var bigelovii, and N. knightiana. Insertion in ipt suppressed shoot formation in the nontumorous hybrid and inhibited it in the nontumorous amphidiploid and N. debneyi, while promoting root formation in N. glauca and N. debneyi. The suggestion of the existence of specific hormone equilibria necessary for the shift to each morphogenetic pattern was supported by experiments with exogenous hormone treatments of three genotypes (N. glauca, N. langsdorffii, and the nontumorous N. glauca × N. langsdorffii).     

Seven of eight species in Nicotiana section Suaveolentes have common factors leading to hybrid lethality in crosses with Nicotiana tabacum

Background and Aims

Reproductive isolation is a mechanism that separates species, and is classified into two types: prezygotic and postzygotic. Inviability of hybrids, or hybrid lethality, is a type of postzygotic isolation and is observed in some plant species, including Nicotiana species. Previous work has shown that the Q chromosome, which belongs to the S subgenome of N. tabacum, encodes one or more genes leading to hybrid lethality in some crosses.

Methods

Interspecific crosses of eight wild species were conducted in section Suaveolentes (which consists of species restricted to Australasia and Africa) with the cultivated species Nicotiana tabacum. Hybrid seedlings were cultivated at 28, 34 or 36 °C, and PCR and chromosome analysis were performed.

Results and Conclusions

Seven of eight wild species produced inviable hybrids after crossing. Hybrid lethality, which was observed in all crosses at 28 °C, was Type II lethality, with the characteristic symptoms of browning of hypocotyl and roots; lethality was suppressed at elevated temperatures (34 or 36 °C). Furthermore, one or more genes on the Q chromosome of N. tabacum were absolutely responsible for hybrid lethality, suggesting that many species of section Suaveolentes share the same factor that triggers hybrid lethality by interaction with the genes on the Q chromosome. Exceptionally, only one wild species, N. fragrans, produced 100 % viable hybrids after crossing with N. tabacum, suggesting that N. fragrans has no factor triggering hybrid lethality.     

Fine-scale environmental variation contributes to introgression in a three-species spruce hybrid complex

Hybridization is common for many forest trees, where weak barriers to reproduction obscure species boundaries. We characterized the genomic structure of Picea populations comprising three species spanning two well-known contact zones, the Picea sitchensis?×?Picea glauca and the P. engelmannii?×?P. glauca hybrid zones, using a set of 71 candidate-gene single nucleotide polymorphisms. The genetic structure of populations suggests a complex genomic architecture shaped by interspecific gene flow and strong environmental selection, with increased genetic diversity in hybrids. The presence of admixture among all three species suggests that three-way hybrids with mixed ancestry occur where species ranges overlap in transitional environments. Significant clinal variation and associations with climatic variables (including continentality, temperature, and precipitation) differ between hybrid zones, indicating that individual species and their hybrids are adapted to distinct environmental niches. Allele–environmental association analysis revealed that most of the candidate genes with evidence of selection were unique to either the Sitka?×?white or the Engelmann?×?white hybrid zones, with few shared between these zones. Management of these widespread and diverse gene pools will be best served through development of climate-based seed transfer, with recommended seed sources informed by a combination of genetic and climatic information for future climates.     

Plant genetic differences influence herbivore community structure: evidence from a hybrid willow system

To determine the influence of plant genetic variation on community structure of insect herbivores, we examined the abundances of 14 herbivore species among six genetic classes of willow: Salix eriocephala, S. sericea, their F₁ and F₂ interspecific hybrids, and backcross hybrids to each parental species. We placed 1-year-old plants, grown from seeds generated from controlled crosses, in a common garden. During the growing season, we censused gall-inducing flies and sawflies, leaf-mining insects, and leaf-folding Lepidoptera to determine the community structure of herbivorous insects on the six genetic classes. Our results provided convincing evidence that the community structure of insect herbivores in this hybrid willow system was shaped by genetic differences among the parental species and the hybrid genetic classes. Using MANOVA, we detected significant differences among genetic classes for both absolute and relative abundance of herbivores. Using canonical discriminant analysis, we found that centroid locations describing community structure of the insect herbivores differed for each genetic class. Moreover, the centroids for the four hybrid classes were located well outside of the range between the centroids for the parental species, suggesting that more than additive genetic effects of the two parental species influenced community formation on hybrid classes. Line-cross analysis suggested that plant genetic factors responsible for structuring the herbivore community involved epistatic effects, as well as additive and dominance effects. We discuss the ramifications of these results in regard to the structure of insect herbivore communities on plants and the implications of our findings for the evolution of interspecific interactions.     

Transport and Accumulation of IAA-14C in Tumour-forming Nicotiana Hybrids

The polar transport of indol-3yl-acetic acid (IAA-2-¹⁴C) instem explants and decapitated shoots of tumour-prone Nicotianahybrids (2n, 3n, and 4n) was compared with that in the normal,non-tumorous parent species N. glauca and N. langsdorffii. Thetotal uptake of the auxin from donor blocks was greatest inthe hybrids and N. glauca. The velocity of the basipetal movementof IAA-¹⁴C was the same in all species tested, i.e. 8 mm/h.The transport capacity for the hormone, however, was decreasedin the three tumour-prone hybrids. Gas chromatography showedthat between 70 and 90 per cent of the transported auxin waspresent in the form of IAA, between 10 and 30 per cent in theform of indol-3yl-aldehyde (IAld). The basipetal transport exceeded the acropetal transport inyoung (third) intemodes of all plants studied, whereas in olderstem segments (tenth intenodes) the reverse was found. The polarity of auxin transport was less well expressed in thetumorous hybrids. Blocking the active transport by pre-treatment of stem cuttingswith 2,4-dinitrophenol (2,4-DNP) caused a drastic reductionin the polar IAA-¹⁴C movement; in all plants tested the auxintransport was reduced to the same low level. The accumulation of auxin at the base of cuttings was higherin N. glauca and the 2n hybrid than in N. langsdorffii, i.e.about seven times higher after 1-h and three times higher after12-h transport experiments. The release of ¹⁴C from the cuttinginto an agar receiver block, however, was markedly reduced inthe 2n hybrid, whereas in N. glauca the labelled substancesmoved more freely into the receiver blocks. Differences in the capacity for the accumulation and the releaseof IAA-¹⁴C in hybrid and N. glauca stem tissues were studiedusing decapitated greenhouse plants wounded by incision abovethe fourth internode. Accumulation of the auxin occurred onlyabove the wound-cut in hybrid plants. This observation is consistentwith the view that tumour formation on hybrid stems occurs atsites of wounding. Our data suggest an elevated auxin levelto be present during tumour initiation at these sites. These results on polar transport and accumulation of IAA-¹⁴Cin tumorous Nicotiana plants together with our previous dataon various endogenous auxins suggest that the induction of neoplasticgrowth in tobacco plants is correlated with increased auxinlevels and an accumulation of the hormone at sites of wounding.     

Hybrid crosses and the genetic basis of interspecific divergence in lifespan in Pristionchus nematodes

Characterizing the genetic basis of among‐species variation in lifespan is a major goal of evolutionary gerontology research, but the very feature that defines separate species – the inability to interbreed – makes achieving this goal impractical, if not impossible, for most taxa. Pristionchus nematodes provide an intriguing system for tackling this problem, as female lifespan varies among species that can be crossed to form viable (although infertile) hybrids. By conducting reciprocal crosses among three species – two dioecious (long‐lived Pristionchus exspectatus and short‐lived Pristionchus arcanus) and one androdioecious (short‐lived Pristionchus pacificus) – we found that female lifespan was long for all hybrids, consistent with the hypothesis that the relatively short lifespans seen for P. pacificus hermaphrodites and P. arcanus females are caused by independent, recessive alleles that are masked in hybrid genomes. Cross‐direction had a small effect on survivorship for crosses involving P. exspectatus, indicating that nuclear–mitochondrial interactions may also influence Pristionchus longevity. Our findings suggest that long lifespan in P. exspectatus reflects the realization of an ancestral potential for extended longevity in the P. pacificus species complex. This work demonstrates the utility of interspecific hybrids for ageing research and provides a foundation for future work on the genetic architecture of interspecific lifespan variation.     

Vigour-related traits and immunity in hybrids of evolutionary divergent cyprinoid species: advantages of hybrid heterosis?

Hybrid advantage, described as the superiority of hybrids in some traits over their parents and termed the “heterosis effect,” is widely documented in the case of reciprocal crosses of parental species (i.e., hybrids representing the F1 generation). In fish, high survival, fast growth and better health status have been widely documented in F1 hybrids. Nonetheless, the effects of interspecific hybridization on vigour, physiology and immunity-related traits in fish are largely unknown, especially concerning native systems of coexisting parental and hybrid genomes in the same habitat. The present study examined the potential physiological and immune aspects of hybrid heterosis by comparing condition status (measured especially by indexes), haematological profile, glucose concentration and selected parameters of non-specific and specific immunity between the evolutionarily divergent non-congeneric cyprinoid species Abramis brama and Rutilus rutilus and their hybrids representing the F1 generation, all of them caught in nature. Clear differences were documented for vigour-related, physiological and immune parameters between the two divergent species. Hybrids generally tended to express intermediate characters of the measured traits, likely generated by the evolutionary divergence of the hybridizing species; nonetheless, for some traits, hybrids exhibited a character that was more similar to one parental species than to the other. This was interpreted as the heterozygote advantage for F1 hybrids. It is suggested that a maternally inherited genetic background may potentially influence the expression of some branches of non-specific immunity or other aspects related to the fish health status.     

Drosophila Interspecific Hybrids Phenocopy piRNA-Pathway Mutants

The Piwi-interacting RNA (piRNA) pathway defends the germline of animals from the deleterious activity of selfish transposable elements (TEs) through small-RNA mediated silencing. Adaptation to novel invasive TEs is proposed to occur by incorporating their sequences into the piRNA pool that females produce and deposit into their eggs, which then propagates immunity against specific TEs to future generations. In support of this model, the F1 offspring of crosses between strains of the same Drosophila species sometimes suffer from germline derepression of paternally inherited TE families, caused by a failure of the maternal strain to produce the piRNAs necessary for their regulation. However, many protein components of the Drosophila piRNA pathway exhibit signatures of positive selection, suggesting that they also contribute to the evolution of host genome defense. Here we investigate piRNA pathway function and TE regulation in the F1 hybrids of interspecific crosses between D. melanogaster and D. simulans and compare them with intraspecific control crosses of D. melanogaster. We confirm previous reports showing that intraspecific crosses are characterized by derepression of paternally inherited TE families that are rare or absent from the maternal genome and piRNA pool, consistent with the role of maternally deposited piRNAs in shaping TE silencing. In contrast to the intraspecific cross, we discover that interspecific hybrids are characterized by widespread derepression of both maternally and paternally inherited TE families. Furthermore, the pattern of derepression of TE families in interspecific hybrids cannot be attributed to their paucity or absence from the piRNA pool of the maternal species. Rather, we demonstrate that interspecific hybrids closely resemble piRNA effector-protein mutants in both TE misregulation and aberrant piRNA production. We suggest that TE derepression in interspecific hybrids largely reflects adaptive divergence of piRNA pathway genes rather than species-specific differences in TE-derived piRNAs.     

INTERSPECIFIC INCOMPATIBILITY IN SOLANUM SPECIES

Pandey , K.K. (Crop Res. Div., D.S. & I.R., Lincoln, Christchurch, New Zealand.) Interspecific incompatibility in Solanum species. Amer. Jour. Bot. 49(8): 874–882. Illus. 1962.—A diallel cross involving 11 self-incompatible and 3 self-compatible species of Solanum was made to study the genetic basis of interspecific incompatibility. Interspecific incompatibility was not limited to crosses in which a self-compatible species was used as the male parent onto a self-incompatible species (unilateral incompatibility). A number of crosses between self-incompatible species were incompatible. In one cross, Q vernei X verrucosum, a self-compatible species was successful as a pollen parent with a self-incompatible species. Unlike other hybrids between self-compatible and self-incompatible species, which are self-incompatible, these F₁ hybrids were self-fertile, and cross-fertile among themselves and with both parents. The self-fertile S. polyadenium was cross-incompatible as a female as well as a male parent with all other species. It is suggested that the unilateral incompatibility is a property of the allele S_C which originated as a consequence of one kind of breakdown of the S_I gene; the S_C allele produces “bare” pollen growth substances which are inactivated in an incompatible style. It is proposed that the failure of the principle of unilateral interspecific incompatibility in solanaceous species may be due to the action of alleles at the second incompatibility locus revealed in certain Mexican species. It is assumed that the South American species are selected intraspecifically only for the action of S alleles but that in certain interspecific crosses and rarely in intraspecific crosses the alleles at the second locus may be expressed, thus interfering with the usual action of S alleles. The F₁ hybrids Q verrucosum (self-fertile) X simplicifolium (self-sterile) were self-incompatible at the tetraploid as well as the diploid level, and their cross-compatibility behavior was consistent with the expected activity of the S_C and S_I alleles of the 2 parents respectively.     

GENETIC EXCHANGE BETWEEN BACILLUS SUBTILIS AND BACILLUS LICHENIFORMIS: VARIABLE HYBRID STABILITY AND THE NATURE OF BACTERIAL SPECIES

Experiments employing both broth and soil cultures demonstrated the capacity for bidirectional genetic exchange between the eubacterial species Bacillus subtilis and Bacillus licheniformis. The process was studied using standard laboratory strains and wild isolates of these species. The genetic exchange in soil occurs spontaneously. The interspecific recombination involved markers for antibiotic resistance and for the use of specific carbon sources (API characters). Hybrids frequently had unstable phenotypes, i.e., lacked a consistent expression of foreign genes over repeated transfer and growth. This instability often involved a “correction” back toward the phenotype of one or the other of the parental species for many differentiating characters; the final phenotype was always that of the more probable or actually known recipient species. This “correction” process is reminiscent of phenomena associated with the instability of artificial fusion protoplasts or noncomplementing diploids of B. subtilis, as well as the merodiploids formed by intergeneric crosses with enteric bacteria. The hybrids observed here must also be diploid, in some manner, because they sequentially express traits of both parental species at rates well above the frequency of mutation. Among the unstable changes in hybrids of the wild strains there was a 3:1 bias in favor of “correction.” The dynamics of the hybridization process in soil are described. It appears that the hybrids are formed most rapidly following outgrowth from spores and during the early growth of parental vegetative cell populations. Later on, the hybrids are much less frequent in the soil cultures, suggesting that they are competitively inferior to the parental species. It is argued that the capacity for recombination found between B. subtilis and B. licheniformis could locally erase their distinctness, even though they possess only about 15% DNA sequence homology. Yet they remain distinct in the wild. The methods and results of these experiments prepare the way for detailed studies of the nature of species and species boundaries throughout the genus Bacillus.