Hybrid Lineages Research Articles

A novel 5-enolpyruvoylshikimate-3-phosphate (EPSP) synthase transgene for glyphosate resistance stimulates growth and fecundity in weedy rice (Oryza sativa) without herbicide.

Understanding evolutionary interactions among crops and weeds can facilitate effective weed management. For example, gene flow from crops to their wild or weedy relatives can lead to rapid evolution in recipient populations. In rice (Oryza sativa), transgenic herbicide resistance is expected to spread to conspecific weedy rice (Oryza sativa f. spontanea) via hybridization.Here, we studied fitness effects of transgenic over-expression of a native 5-enolpyruvoylshikimate-3-phosphate synthase (epsps) gene developed to confer glyphosate resistance in rice. Controlling for genetic background, we examined physiological traits and field performance of crop–weed hybrid lineages that segregated for the presence or absence of this novel epsps transgene.Surprisingly, we found that transgenic F2 crop–weed hybrids produced 48–125% more seeds per plant than nontransgenic controls in monoculture- and mixed-planting designs without glyphosate application. Transgenic plants also had greater EPSPS protein levels, tryptophan concentrations, photosynthetic rates, and per cent seed germination compared with nontransgenic controls.Our findings suggest that over-expression of a native rice epsps gene can lead to fitness advantages, even without exposure to glyphosate. We hypothesize that over-expressed epsps may be useful to breeders and, if deployed, could result in fitness benefits in weedy relatives following transgene introgression.

Genomic and environmental selection patterns in two distinct lettuce crop–wild hybrid crosses

Genomic selection patterns and hybrid performance influence the chance that crop (trans)genes can spread to wild relatives. We measured fitness(-related) traits in two different field environments employing two different crop–wild crosses of lettuce. We performed quantitative trait loci (QTL) analyses and estimated the fitness distribution of early- and late-generation hybrids. We detected consistent results across field sites and crosses for a fitness QTL at linkage group 7, where a selective advantage was conferred by the wild allele. Two fitness QTL were detected on linkage group 5 and 6, which were unique to one of the crop–wild crosses. Average hybrid fitness was lower than the fitness of the wild parent, but several hybrid lineages outperformed the wild parent, especially in a novel habitat for the wild type. In early-generation hybrids, this may partly be due to heterosis effects, whereas in late-generation hybrids transgressive segregation played a major role. The study of genomic selection patterns can identify crop genomic regions under negative selection across multiple environments and cultivar–wild crosses that might be applicable in transgene mitigation strategies. At the same time, results were cultivar-specific, so that a case-by-case environmental risk assessment is still necessary, decreasing its general applicability.

Pollinator‐mediated selection on floral morphology: evidence for transgressive evolution in a derived hybrid lineage

Hybridization between closely related lineages is a mechanism that might promote substantive changes in phenotypic traits of descendants, resulting in transgressive evolution. Interbreeding between divergent but morphologically similar lineages can produce exceptional phenotypes, but the potential for transgressive variation to facilitate long-term trait changes in derived hybrid lineages has received little attention. We compare pollinator-mediated selection on transgressive floral traits in both early-generation and derived hybrid lineages of the Piriqueta cistoides ssp. caroliniana complex. The bowl-shaped flowers of morphotypes in this complex have similar gross morphologies and attract a common suite of small insect pollinators. However, they are defined by significant differences in characters that generate pollinator interest and visitation, including floral area and petal separation. In common garden experiments, patterns of pollen deposition in early-generation recombinant hybrids indicate that Piriqueta's pollinators favour flowers with greater area and reduced petal separation. Changes in floral morphology in derived hybrid lineages are consistent with predictions from selection gradients, but the magnitude of change is limited relative to the range of transgressive variation. These results suggest that hybridization provides variation for evolution of divergent floral traits. However, the potential for extreme transgressive variants to contribute to phenotypic shifts may be limited due to reduced heritability, evolutionary constraints or fitness trade-offs.

Review Genomic in situ hybridization in plants

Genomic in situ hybridization (GISH), which is a modification of fluorescent in situ hybridization, has been widely used in the study of plants. It has become one of the most important techniques for molecular cytogenetics. GISH is a technique that allows distinguishing the genomes in a cell. With this technique, it is possible to differentiate the genomes in a hybrid; consequently, this tool has been applied to the study of hybrid lineages, genetic improvement programs, and studies of the evolution of polyploids. Moreover, GISH can be applied to the analysis of the meiotic behavior in hybrids and polyploids, providing information concerning the relationship between species. This review presents the wide application of this technique in plants.

Population structure of sexually reproducing carp gudgeons: does a metapopulation offer refuge from sexual parasitism?

Australian carp gudgeons (Hypseleotris spp.) of the Murray–Darling basin are a species complex including sexually reproducing taxa and unisexual hybrid lineages that reproduce via hybridogenesis. Unisexual fish require gametes of a sexual species to propagate themselves and can be regarded as ‘sexual parasites’ capable of driving closed populations to extinction. Metapopulation dynamics have been proposed as a mechanism that could facilitate coexistence between a sexual parasite and its ‘host’. This study evaluates whether patterns of spatial genetic variation are compatible with metapopulation dynamics for a sexually reproducing member of the carp gudgeon complex (Hypseleotris sp. HA), in the Granite Creeks system of central Victoria. Genetic differentiation of fish among all study sites was accommodated by a model of migration-drift equilibrium using decomposed pairwise regression analysis. Given that the population was divided into discrete patches in the form of refugial waterholes during the time of this study, we infer that spatially constrained source–sink metapopulation dynamics may be responsible for producing this pattern. It is therefore possible that metapopulation dynamics contribute to coexistence in the Granite Creeks carp gudgeon hybridogenetic system, and further analysis is required to determine the relative importance of environmental versus demographic factors towards patch extinction.

Non-homogeneous combination of two porous genomes induces complex body shape trajectories in cyprinid hybrids

IntroductionHybridization is a common phenomenon in fish and is considered to be a major source of diversification. Deciphering the remoulding of genomic regions and phenotypes in zones where hybrid specimens occur is of particular interest to elucidate the emergence of evolutionary novelties. This approach is particularly challenging because the first step of hybridization seems to be the most important stage in the emergence of hybrid lineages. However, the signal can be significantly altered after only a few generations.ResultsWe studied 41 microsatellites and partial cytochrome b gene sequences in 970 specimens belonging to two fish species (Chondrostoma nasus and Parachondrostoma toxostoma) in allopatric/parapatric zones, hybrids between them in a natural sympatric zone: the Ardèche basin. We showed that the genomic architecture in hybrids presented pattern heterogeneity of selection for the different loci. Indeed, the upstream part of the river (Rosières and Labeaume) presented an overdominant fitness of heterozygotes (12.20%) corresponding to a genomic compatibility, and underselection was observed for 4.88%-7.32% of the loci tested indicating a genomic incompatibility. Moreover the upstream station (Rosières) presented a positive selection of invasive C. nasus homozygotes (17.07% to 21.95%) indicating that hybridization may increase the fitness of admixed individuals.We showed that hybrid morphology (body shape based on 21 landmarks) correlated with genomic dilution indicating a species fingerprint. However, we demonstrated that the hybrid morphology was not a linear modification between the two parental species but a trade-off between several correlated traits.ConclusionsHybrid specimens present a mosaic of genomic combination, showing regions with genomic compatibility and others with genomic incompatibility between the two species. Positive selection (invasive advantage ranging from 9.76% to 21.95% of the loci) was evidenced in the upstream part of the Ardèche indicating that environmental selection makes a substantial contribution. Although the presence of a dam is known to impose heterogeneous hybrid zones between these two species, we demonstrated in this study that a natural environment can also generate a hybrid zone with a large number (and diversity) of hybrids. The combination of the two genomes in the hybrids results in complex ontogenetic trajectories (with different morphological traits evolving at different rates) that correspond to novel developmental pathways.

Adaptive Significance and Long-Term Survival of Asexual Lineages

Important questions remain about the long-term survival and adaptive significance of eukaryotic asexual lineages. Numerous papers dealing with sex advantages still continued to compare parthenogenetic populations versus sexual populations arguing that sex demonstrates a better fitness. Because asexual lineages do not possess any recombination mechanisms favoring rapid changes in the face of severe environmental conditions, they should be considered as an evolutionary dead-end. Nevertheless, reviewing literature dealing with asexual reproduction, it is possible to draw three stimulating conclusions. (1) Asexual reproduction in eukaryotes considerably differs from prokaryotes which experience recombination but neither meiosis nor syngamy. Recombination and meiosis would be a driving force for sexual reproduction. Eukaryotes should therefore be considered as a continuum of sexual organisms that are more or less capable (and sometimes incapable) of sexual reproduction. (2) Rather than revealing ancestral eukaryotic forms, most known lineages of asexual eukaryotes have lost sex due to a genomic conflict affecting their sexual capacity. Thus, it could be argued that hybridization is a major cause of their asexuality. Asexuality may have evolved as a reproductive mechanism reducing conflict within organisms. (3) It could be proposed that, rather than being generalists, parthenogenetic hybrid lineages could be favored when exploiting peculiar restricted ecological niches, following the “frozen niche variation” model. Although hybrid events may result in sex loss, probably caused by genomic conflict, asexual hybrids could display new original adaptive traits, and the rapid colonization of environments through clonal reproduction could favor their long-term survival, leading to evolutionary changes and hybrid speciation. Examination of the evolutionary history of asexual lineages reveals that evolutionary processes act through transitional stages in which even very small temporary benefits may be enough to counter the expected selective disadvantages.

Molecular diagnosis to discriminate pathogen and apathogen species of the hybrid Verticillium longisporum on the oilseed crop Brassica napus.

The cruciferous fungal pathogen Verticillium longisporum represents an allodiploid hybrid with long spores and almost double the amount of nuclear DNA compared to other Verticillium species. V. longisporum evolved at least three times by hybridization. In Europe, virulent A1xD1 and avirulent A1xD3 hybrids were isolated from the oilseed crop Brassica napus. Parental A1 or D1 species are yet unknown whereas the D3 lineage represents Verticillium dahliae. Eleven V. longisporum isolates from Europe or California corresponding to hybrids A1xD1 or A1xD3 were compared. A single characteristic type of nuclear ribosomal DNA could be assigned to each hybrid lineage. The two avirulent A1xD3 isolates carried exclusively D3 ribosomal DNA (rDNA) which corresponds to V. dahliae. The rDNA of all nine A1xD1 isolates is identical but distinct from D3 and presumably originates from A1. Both hybrid lineages carry two distinct isogene pairs of four conserved regulatory genes corresponding to either A1 or D1/D3. D1 and D3 paralogues differ in several single nucleotide polymorphisms. Southern hybridization patterns confirmed differences between the A1 and D1/D3 isogenes and resulted in similar patterns for D1 and D3. Distinct signatures of the Verticillium transcription activator (VTA)2 regulatory isogene pair allow identification of V. longisporum hybrids by a single polymerase chain reaction and the separation from haploid species as V. dahliae or Verticillium albo-atrum. The combination between VTA2 signature and rDNA type identification represents an attractive diagnostic tool to discriminate allodiploid from haploid Verticillia and to distinguish between A1xD1 and A1xD3 hybrids which differ in their virulence towards B. napus.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-012-4530-1) contains supplementary material, which is available to authorized users.

Hybridization betweenPyrola grandifloraandPyrola minorin West Greenland: a tension zone maintained by clonality

Interspecific hybridization may lead to novel hybrid lineages and transfer of genetic material, possibly enabling species to expand into new habitats. Alternatively, hybrids may be inviable or sterile, constraining movement of parental species and genes into each others range. Pyrola minor (L.) and Pyrola grandiflora (Rad.) hybridize on Disko Island in Greenland. A recent study found most hybrids to be F1plants produced by P. minor mothers. Here, we test if later generation hybrids are indeed absent in a larger sample, using morphological and molecular markers. We further test if unidirectional hybridization is due to reproductive barriers. Intermediate plants were all F1hybrids from P. minor mother plants. Our analyses indicate that hybrids originated from few hybridization events and have been maintained by clonal reproduction since then. Barriers to pollen flow between the species result from a combination of few pollinators, different flower morphology and phenology, high proportions of abnormal pollen in hybrids, and reduced pollen tube growth of heterospecific and hybrid pollen. In general, pollen from P. grandiflora grew better than pollen from P. minor, which may explain the unidirectional hybridization. Hybridization between P. grandiflora and P. minor therefore seems to operate like a tension zone in which F1hybrids are maintained by clonal propagation.

Dynamic Formation of Asexual Diploid and Polyploid Lineages: Multilocus Analysis of Cobitis Reveals the Mechanisms Maintaining the Diversity of Clones

Given the hybrid genomic constitutions and increased ploidy of many asexual animals, the identification of processes governing the origin and maintenance of clonal diversity provides useful information about the evolutionary consequences of interspecific hybridization, asexuality and polyploidy. In order to understand the processes driving observed diversity of biotypes and clones in the Cobitis taenia hybrid complex, we performed fine-scale genetic analysis of Central European hybrid zone between two sexual species using microsatellite genotyping and mtDNA sequencing. We found that the hybrid zone is populated by an assemblage of clonally (gynogenetically) reproducing di-, tri- and tetraploid hybrid lineages and that successful clones, which are able of spatial expansion, recruit from two ploidy levels, i.e. diploid and triploid. We further compared the distribution of observed estimates of clonal ages to theoretical distributions simulated under various assumptions and showed that new clones are most likely continuously recruited from ancestral populations. This suggests that the clonal diversity is maintained by dynamic equilibrium between origination and extinction of clonal lineages. On the other hand, an interclonal selection is implied by nonrandom spatial distribution of individual clones with respect to the coexisting sexual species. Importantly, there was no evidence for sexually reproducing hybrids or clonally reproducing non-hybrid forms. Together with previous successful laboratory synthesis of clonal Cobitis hybrids, our data thus provide the most compelling evidence that 1) the origin of asexuality is causally linked to interspecific hybridization; 2) successful establishment of clones is not restricted to one specific ploidy level and 3) the initiation of clonality and polyploidy may be dynamic and continuous in asexual complexes.

Genetic diversity, conservation, and utilization of Theobroma cacao L.: genetic resources in the Dominican Republic

Cacao (Theobroma cacao L.) is a significant agricultural commodity in the Dominican Republic, which ranks 11th in the world for cacao exports. To estimate genetic diversity, determine genetic identity, and identify any labeling errors, 14 SSR markers were employed to fingerprint 955 trees among cacao germplasm accessions and local farmer selections (LFS). Comparisons of homonymous plants across plots revealed a significant misidentification rate estimated to be 40.9 % for germplasm accessions and 17.4 % for LFS. The 14 SSRs amplified a total of 117 alleles with a mean allelic richness of 8.36 alleles per locus and average polymorphism information content (PIC) value of 0.67 for the germplasm collection. Similar levels of variation were detected among the LFS where a total of 113 alleles were amplified with a mean of 8.07 alleles per locus and PIC of 0.57. The observed heterozygosity (Hobs) was 0.67 for the germplasm collection and 0.60 for LFS. Based on population structure analysis 43.9 % of the germplasm accessions and 72.1 % of the LFS are predominantly of the Amelonado ancestry. Among these Amelonado, 51.7 % for the germplasm collection and 50.6 % for LFS corresponded to Trinitario hybrid lineage. Criollo ancestry was found in 7.6 and 9.5 % of the germplasm accessions and LFS, respectively. The Contamana, Nacional, and Iquitos backgrounds were also observed in both populations, but the Curaray background was only detected in the germplasm accessions. No Purus or Guiana ancestry was found in either of the populations. Overall, significant genetic diversity, which could be exploited in the Dominican Republic breeding and selection programs, was identified among the germplasm accessions and LFS.

Identification of putative hybrid and natural lineages in genus Potamogeton revealed by chloroplast and nuclear DNA markers

Identification of putative hybrid and natural lineages in genus Potamogeton revealed by chloroplast and nuclear DNA markers

Hybrid vigor in the biological control agent, Longitarsus jacobaeae

Hybridization is an important evolutionary mechanism that can increase the fitness and adaptive potential of populations. A growing body of evidence supports its importance as a key factor contributing to rapid evolution in invasive species, but the effects of hybridization have rarely been assessed in intentionally introduced biological control agents. We investigated hybrids between a Swiss and an Italian population of the beetle, Longitarsus jacobaeae, a biological control agent of Jacobaea vulgaris, by reciprocally crossing individuals in the laboratory. Phenological traits of F1 and F2 hybrid lineages showed intermediate values relative to their parental populations, with some maternal influence. Fitness of the F2 generation, measured as lifetime fecundity, was higher than that of the Italian parent in one of the lineages and higher than that of either parent in the other hybrid lineage. The increased fecundity of hybrids may benefit tansy ragwort biological control by increasing the establishment success and facilitating a more rapid population buildup in the early generations. Even though the long-term consequences of hybridization in this and other systems are hard to predict, intentional hybridization may be a useful tool in biological control strategies as it would promote similar microevolutionary processes operating in numerous targeted invasive species.

Transcriptome changes after genome‐wide admixture in invasive sculpins (Cottus)

Models on hybrid speciation assume that hybridization generates increased phenotypic variance that is utilized to invade new adaptive peaks. We test to what extent this prediction can be traced using gene expression data in the fish species Cottus perifretum and Cottus rhenanus as well as a natural hybrid lineage referred to as invasive sculpins. In addition, interspecies crosses were used to explore evolutionary trajectories from initial stages to the hybrid lineage. EST (expressed sequence tag) libraries were sequenced to design an oligonucleotide microarray that was calibrated for probe-specific differences in binding behaviour. Levels of gene expression divergence between species correlate with genetic divergence at neutral markers and, accordingly, invasive sculpins were intermediate between the parental species overall. However, the hybrid lineage is distinguished through unique patterns of gene expression that are enriched for biological functions which represent candidates for the fitness properties of invasive sculpins. We compare F(2) crosses with natural invasive sculpins to show that the variance in gene expression decreases in invasives. Moreover, few of the transgressive patterns of gene expression that distinguish invasives can be directly observed in F(2) crosses. This suggests that the invasive transcriptome was subject to secondary changes after admixture. The result is in line with an evolutionary process that reduces maladaptive variance and optimizes the phenotype of an emerging hybrid lineage.

Hybridization, cryptic diversity, and invasiveness in introduced variable‐leaf watermilfoil

Hybridization may be important in the evolution of invasiveness, but few empirical studies compare introduced hybrid and parental lineages. Invasive ‘variable-leaf watermilfoil’ (Myriophyllum heterophyllum) in the northeastern United States consists of at least three distinct lineages: an interspecific hybrid (M. heterophyllum × Myriophyllum laxum) and two historically allopatric lineages of pure M. heterophyllum. Previous observations suggested that hybrid populations of variable-leaf watermilfoil may be comparatively more ‘invasive’ than pure lineages. However, no quantitative data comparing hybrid and parental lineages have been collected, nor has invasiveness been compared between parental lineages. Here, we demonstrate that these distinct lineages are also ecologically distinct. We find some support for the hypothesis that hybridization has played a role in the evolution of invasiveness: hybrids exhibited higher biomass, individual plant size, and greater branching than at least one parental lineage of M. heterophyllum. However, parental lineages did not differ from the hybrid for some traits, demonstrating that pure parental lineages can also be invasive. In addition, we found no evidence for a role of intraspecific hybridization in the evolution of invasiveness in these lineages of variable-leaf watermilfoil, even where they co-occurred locally. Our study suggests that distinguishing among cryptic lineages will help prioritize rapid response control efforts.

No evidence for niche segregation in a North American Cattail (Typha) species complex.

Interspecific hybridization can lead to a breakdown of species boundaries, and is of particular concern in cases in which one of the parental species is invasive. Cattails (Typha spp.) have increased their abundance in the Great Lakes region of North America over the past 150 years. This increase in the distribution of cattails is associated with hybridization between broad-leaved (Typha latifolia) and narrow-leaved cattails (T. angustifolia). The resulting hybrids occur predominantly as F1s, which are known as T. × glauca, although later-generation hybrids have also been documented. It has been proposed that in sympatric populations, the parental species and hybrids are often spatially segregated according to growth in contrasting water depths, and that this should promote the maintenance of parental species. In this study, we tested the hypothesis that the two species and their hybrids segregate along a water-depth gradient at sites where they are sympatric. We identified the two parental species and their hybrids using molecular genetic markers (SSR), and measured shoot elevations (a proxy for water depth) at 18 sites in Southern Ontario, Canada. We found no evidence for niche segregation among species based on elevation. Our data indicate that all three lineages compete for similar habitat where they co-occur suggesting that there is potential for an overall loss of biodiversity in the species complex, particularly if the hybrid lineage is more vigorous compared to the parental species, as has been suggested by other authors.

Cytogenetic characterization of F1, F2 and backcross hybrids of the Neotropical catfish species Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum (Pimelodidae, Siluriformes)

The cytogenetic characteristics of Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum and their F1, F2 and backcross hybrids were assessed by using chromosome banding techniques. The diploid number of 56 chromosomes was constant in all species and lineages, with a karyotypic formula containing 20 metacentric, 12 submetacentric, 12 subtelocentric and 12 acrocentric chromosomes. Nucleolar organizer regions (NORs) were identified in two subtelocentric chromosomes in the parents and hybrids, with partial nucleolar dominance in F1 and F2 specimens. Heterochromatic blocks were detected in the terminal and centromeric regions of some chromosomes in all individuals. For parental and hybrid lineages, 18S ribosomal clusters corresponding to NORs and 5S ribosomal genes were identified in distinct pairs of chromosomes. The striking conservation in the chromosomal macrostructure of the parental species may account for the fertility of their F1 hybrids. Similarly, the lack of marked alterations in the chromosomal structure of the F1 hybrids could account for the maintenance of these features in post-F1 lineages.

Extraordinary transgressive phenotypes of hybrid tomato are influenced by epigenetics and small silencing RNAs

Hybrid organisms may fail to develop, be sterile or they may be more vigorous than either of the parents. Examples of hybrid vigour or hybrid necrosis in the F1 are often not inherited stably in subsequent generations if they are associated with overdominance. There can also be transgressive phenotypes that are inherited stably in these later generations, but the underlying mechanisms are not well understood. Here we have investigated the possibility that stable transgressive phenotypes in the progeny of crosses between cultivated tomato (Solanum lycopersicum cv. M82) and a wild relative (Solanum pennellii, accession LA716) are associated with micro or small interfering(si) RNAs. We identified loci from which these small(s)RNAs were more abundant in hybrids than in either parent and we show that accumulation of such transgressive sRNAs correlated with suppression of the corresponding target genes. In one instance this effect was associated with hypermethylation of the corresponding genomic DNA. Our results illustrate a potential role of transgressive sRNAs in plant breeding and in natural evolution with wild plants.

Recent, independent and anthropogenic origins of Trypanosoma cruzi hybrids.

The single celled eukaryote Trypanosoma cruzi, a parasite transmitted by numerous species of triatomine bug in the Americas, causes Chagas disease in humans. T. cruzi generally reproduces asexually and appears to have a clonal population structure. However, two of the six major circulating genetic lineages, TcV and TcVI, are TcII-TcIII inter-lineage hybrids that are frequently isolated from humans in regions where chronic Chagas disease is particularly severe. Nevertheless, a prevalent view is that hybridisation events in T. cruzi were evolutionarily ancient and that active recombination is of little epidemiological importance. We analysed genotypes of hybrid and non-hybrid T. cruzi strains for markers representing three distinct evolutionary rates: nuclear GPI sequences (n = 88), mitochondrial COII-ND1 sequences (n = 107) and 28 polymorphic microsatellite loci (n = 35). Using Maximum Likelihood and Bayesian phylogenetic approaches we dated key evolutionary events in the T. cruzi clade including the emergence of hybrid lineages TcV and TcVI, which we estimated to have occurred within the last 60,000 years. We also found evidence for recent genetic exchange between TcIII and TcIV and between TcI and TcIV. These findings show that evolution of novel recombinants remains a potential epidemiological risk. The clearly distinguishable microsatellite genotypes of TcV and TcVI were highly heterozygous and displayed minimal intra-lineage diversity indicative of even earlier origins than sequence-based estimates. Natural hybrid genotypes resembled typical meiotic F1 progeny, however, evidence for mitochondrial introgression, absence of haploid forms and previous experimental crosses indicate that sexual reproduction in T. cruzi may involve alternatives to canonical meiosis. Overall, the data support two independent hybridisation events between TcII and TcIII and a recent, rapid spread of the hybrid progeny in domestic transmission cycles concomitant with, or as a result of, disruption of natural transmission cycles by human activities.

Should hybrids be protected by listing; Betula × sandbergii and Botrychium minganense in Vermont1

Recent consideration of two taxa, a fern (Botrychium minganense Vict.) and a birch (Betula × sandbergii Britton), for addition to the list of protected plants by the Vermont Scientific Advisory Group on Flora revealed the need for an assessment of hybrids in the context of the listing of rare taxa for protection. Though both are hybrids in that they are the product of secondary contact between divergent lineages, the two lie at opposite ends of the hybrid continuum. A consideration of the known array of hybrid biologies in the context of the conservation literature leads to the argument that the fern should be listed, but not the birch. Botrychium minganense is reproductively competent, fully isolated from its progenitors, and evidences a genetic heritage unique relative to them. However, Betula × sandbergii does not have the integrity or genetic uniqueness typical of the rare hybrid lineages that have been listed for protection, either at the state or national level. It lacks a unique gene pool vulnerable to extinction. The two candidates for listing in Vermont present contrasting biologies that together inform a substantive understanding of the issues relating to the conservation biology of plant hybrids.