What data determine whether a plant taxon is distinct enough to merit legal protection? A case study of Sedum integrifolium (Crassulaceae).

Measures of molecular and morphological genetic variation are often used to set conservation priorities and design management strategies for plant taxa. Evaluated together they can give insights into a taxon's evolutionary status that neither data type alone can achieve. We investigated the distinctness and variability of Sedum integrifolium ssp. leedyi, a federally and state-listed taxon, from its conspecific relatives using 33 random amplified polymorphic DNA (RAPD) markers (253 plants) and 37 morphological characters from 1308 common-garden-grown plants. We included S. integrifolium ssp. leedyi (four populations), its conspecific relatives (11 populations), and S. rosea and S. rhodanthum populations in our study. The morphological and molecular data correspond in showing that S. integrifolium ssp. leedyi populations are highly distinct. However, the data sets differ in their estimates of the relatedness of some S. integrifolium ssp. leedyi populations and in the percentage variation detected due to differences among them (25 and 9-13% for the molecular and morphological data, respectively) suggesting little gene flow among populations and some differentiation, possibly from selective pressures. Given our data, we recommend that S. integrifolium ssp. leedyi merits protection under the U.S. Endangered Species Act and that its populations be managed as distinct units.

Low levels of genetic variation within and high levels of genetic differentiation among populations of species of Abies from southern Mexico and Guatemala.

Populations of Abies in southern Mexico and Guatemala (A. flinckii, A. guatemalensis, A. hickeli, and A. religiosa) have a patchy distribution. This pattern is particularly clear in A. guatemalensis. Genetic diversity within populations, measured by average heterozygosity at 16 isozyme loci, is lower than the range reported for most conifers (mean H(o) ranging from 0.069 in A. guatemalensis to 0.113 in A. flinckii), while differentiation among populations is higher than that observed in most conifer species studied (θ = F(st) ranging from 0.073 in A. hickeli to 0.271 in A. flinckii). Estimated levels of gene flow are low (ranging from 0.672 in A. flinckii to 3.17 in A. hickeli). Populations in most cases had an excess of homozygosity over that expected under Hardy-Weinberg equilibrium, suggesting some inbreeding (F(is) ranging from 0.074 in A. flinckii to 0.235 in A. guatemalensis). A significant relationship between gene flow and geographic distance was observed in A. religiosa, but not in the other three taxa studied. The patterns of genetic variation appear to have been influenced by the distributions and histories of these species. Paleoclimatic evidence suggests that the ranges of these species retreated upwards during the Pleistocene glaciation and became fragmented during the warming period that followed. The populations could have passed through genetic bottlenecks that reduced genetic variation and led to interpopulation differentiation.

Genetic diversity of the endangered endemic Agave victoriae-reginae (Agavaceae) in the Chihuahuan Desert.

Long-lived perennials are a species-rich, ecologically important component of the North American deserts, yet we know little about their genetic structure, information important for their conservation. Agave victoriae-reginae is an endemic of the Chihuahuan Desert of northern Mexico that is endangered by collection for the ornamental trade. We examined levels and patterns of variation at ten polymorphic allozyme loci in ten populations representing the range of the species. Levels of genetic variation (mean H(e)= 0.335) and differentiation (mean F(ST) = 0.236) were high. Phenetic clustering suggested the existence of at least three distinct groups of populations. If this pattern of variation is representative of other long-lived desert perennials, it may explain the species richness of this group and will pose a real challenge to gene conservation efforts.

Evidence that the phytochrome gene family in black cottonwood has one PHYA locus and two PHYB loci but lacks members of the PHYC/F and PHYE subfamilies.

The phytochrome photoreceptors play important roles in the photoperiodic control of vegetative bud set, growth cessation, dormancy induction, and cold-hardiness in trees. Interestingly, ecotypic differences in photoperiodic responses are observed in many temperate-zone tree species. Northern and southern ecotypes of black cottonwood (Populus trichocarpa Torr. & Gray), for example, exhibit marked differences in the timing of short-day-induced bud set and growth cessation, and these responses are controlled by phytochrome. Therefore, as a first step toward determining the molecular genetic basis of photoperiodic ecotypes in trees, we characterized the phytochrome gene (PHY) family in black cottonwood. We recovered fragments of one PHYA and two PHYB using PCR-based cloning and by screening a genomic library. Results from Southern analyses confirmed that black cottonwood has one PHYA locus and two PHYB loci, which we arbitrarily designated PHYB1 and PHYB2. Phylogenetic analyses which included PHY from black cottonwood, Arabidopsis thaliana and tomato (Solanum lycopersicum) suggest that the PHYB/D duplications in these species occurred independently. When Southern blots were probed with PHYC, PHYE, and PHYE heterologous probes, the strongest bands that we detected were those of black cottonwood PHYA and/or PHYB. These results suggest that black cottonwood lacks members of the PHYC/F and PHYE subfamilies. Although black cottonwood could contain additional PHY that are distantly related to known angiosperm PHY, our results imply that the PHY family of black cottonwood is less complex than that of other well-characterized dicot species such as Arabidopsis and tomato. Based on Southern analyses of five black cottonwood genotypes representing three photoperiodic ecotypes, substantial polymorphism was detected for at least one of the PHYB loci but not for the PHYA locus. The novel character of the PHY family in black cottonwood, as well as the differences in polymorphism we observed between the PHYA and PHYB subfamilies, indicates that a number of fundamental macro- and microevolutionary questions remain to be answered about the PHY family in dicots.

Pinus banksiana has at least seven expressed alcohol dehydrogenase genes in two linked groups.

The alcohol dehydrogenase (Adh) gene family is much more complex in Pinus banksiana than in angio-sperms, with at least seven expressed genes organized as two tightly linked clusters. Intron number and position are highly conserved between P. banksiana and angiosperms. Unlike angiosperm Adh genes, numerous duplications, as large as 217 bp, were observed within the noncoding regions of P. banksiana Adh genes and may be a common feature of conifer genes. A high frequency of duplication over a wide range of scales may contribute to the large genome size of conifers.

Comparison of allozyme, RFLP, and RAPD markers for revealing genetic variation within and between trembling aspen and bigtooth aspen.

We examined genetic variation in allozyme loci, nuclear DNA restriction fragment length polymorphisms (RFLPs), and random amplified polymorphic DNAs (RAPDs) in 130 trembling aspen (Populus tremuloides) and 105 bigtooth aspen (P. grandidentata) trees. In trembling aspen 10 out of 13 allozyme loci assayed (77%) were polymorphic (P), with 2.8 alleles per locus (A) and an expected heterozygosity (He) of 0.25. In contrast, bigtooth aspen had a much lower allozyme genetic variability (P=29%; A=1.4; He=0.08). The two species could be distinguished by mutually exclusive alleles at Idh-1, and bigtooth aspen has what appears to be a duplicate 6PG locus not present in trembling aspen. We used 138 random aspen genomic probes to reveal RFLPs in HindIII digests of aspen DNA. The majority of the probes were from sequences of low copy number. RFLP results were consistent with those of the allozyme analyses, with trembling aspen displaying higher genetic variation than bigtooth aspen (P=71%, A=2.7, and He=0.25 for trembling aspen; P=65%, A=1.8, and He=0.13 for bigtooth aspen). The two species could be distinguished by RFLPs revealed by 21 probes (15% of total probes assayed). RAPD patterns in both species were studied using four arbitrary decamer primers that revealed a total of 61 different amplified DNA fragments in trembling aspen and 56 in bigtooth aspen. Assuming a Hardy-Weinberg equilibrium, estimates of P=100%, A=2, and He=0.30 in trembling aspen and P=88%, A=1.9, and He=0.31 in bigtooth aspen were obtained from the RAPD data. Five amplified DNA fragments were species diagnostic. All individuals within both species, except for 2 that likely belong to the same clone, could be distinguished by comparing their RAPD patterns. These results indicate that (1) RFLPs and allozymes reveal comparable patterns of genetic variation in the two species, (2) trembling aspen is more genetically variable than bigtooth aspen at both the allozyme and DNA levels, (3) one can generate more polymorphic and species-specific loci with DNA markers than with allozymes in aspen, and (4) RAPDs provide a very powerful tool for "fingerprinting" aspen individuals.

Constraints on the evolution of plastid introns: the group II intron in the gene encoding tRNA-Val(UAC).

The evolution of the group II intron in the plastid gene encoding tRNA(Val)UAC (trnV) from seven plant taxa was studied by aligning secondary and other structural features. Levels of evolutionary divergence between six angiosperms and a liverwort, Marchantia polymorpha, were compared for the six domains commonly demonstrated for group II introns and were shown to be statistically heterogeneous. Evolutionary rates varied substantially among various domains and other features. Domain II showed the highest evolutionary rate, approaching the synonymous substitution rate reported for cpDNA-encoded genes, while domain VI and the helix and loop region bearing EBS1 evolved at rates similar to those for nonsynonymous substitutions of a number of cpDNA-encoded genes. The minimum free-energy structure of domain I varied among the seven taxa, suggesting that possible protein-RNA or tertiary interactions are important for intron processing.

Evolution of the ribosomal DNA spacers of Drosophila melanogaster: different patterns of variation on X and Y chromosomes.

Length variation of the ribosomal gene spacers of Drosophila melanogaster was studied. Analysis of 47 X chromosomal and 47 Y chromosomal linked rDNA arrays collected from five continents indicates that the arrays on the two chromosomes differ qualitatively. The Y-linked arrays from around the world share little or no similarity for either their overall length or the organization of their spacers. Most of the X-linked arrays do, however, share a major length spacer of 5.1 kb. In addition, those X-linked arrays that have a major 5.1-kb band have similar spacer organization as demonstrated by genomic DNA digestions with several restriction enzymes. These data strongly support the hypothesis that spacer length patterns on only X-linked genes are maintained primarily by natural selection.

Chloroplast DNA polymorphisms in lodgepole and jack pines and their hybrids.

Samples taken from throughout the ranges of distribution of lodgepole pine (Pinus contorta Dougl. ex. Loud.) and jack pine (Pinus banksiana Lamb.) were assayed for Sal I and Sst I chloroplast DNA restriction fragment variation. Although the chloroplast genome is often regarded as highly conserved, at least 2 distinct Sal I and 13 distinct Sst I restriction fragment banding patterns occur in these closely related species. None of the chloroplast DNA restriction fragment banding patterns observed in allopatric lodgepole pine was observed in allopatric populations of jack pine, and vice versa, even though the two species share an extensive zone of sympatry, and gene flow between the species has been reported for nuclear genes. However, several atypical Sst I restriction fragment banding patterns occur only in or near the zone of sympatry. Chloroplasts have been reported to be inherited maternally in the great majority of species studied; however, restriction fragment analyses indicated that chloroplasts are inherited paternally in controlled matings between lodgepole pine (female) and jack pine (male).