An analysis of genetic variation in Astragalus cremnophylax var. cremnophylax, a critically endangered plant, using AFLP markers

A recently developed molecular technique (amplified fragment length polymorphisms, AFLP) was used for characterizing genetic heterogeneity within and among populations of a critically endangered species of plant, Astragalus cremnophylax var. cremnophylax. Using AFLP, up to 50 polymorphic genetic markers per AFLP-PCR amplification were generated, and a total of 220 variable markers overall. This information was used first to assess genetic diversity within each of the three known populations of Astragalus cremnophylax var. cremnophylax from Grand Canyon National Park in Arizona, USA: North Rim (NR; n= 970), South Rim Site 1 (SR1; n= 500), and South Rim Site 2 (SR2; n= 2). Diversity in the form of average heterozygosity (H) and the proportion of polymorphic genes (P) was greatest in the NR population ((H) = 0.13 and (P) = 0.38) and least in the SR2 population ((H) = 0.02 and (P) = 0.04). Diversity was also quite low for the SR1 population ((H) = 0.04 and (P) = 0.10). In addition, substantial genetic differentiation among populations was indicated by both phenetic (AMOVA) and genetic analyses (overall corrected F_ST= 0.41). This finding was corroborated by the results of several multivariate analyses which utilized the genetic data, including a UPGMA cluster analysis and a principal coordinate analysis which revealed the existence of discrete groups corresponding to the populations. Population structure was further revealed within the NR population which was known to consist of four spatially separated groups of plants. Several recommendations for the future management of the species are discussed.     

Population variation and phylogeny in the endangered <Emphasis Type="BoldItalic">Chamaesyce skottsbergii</Emphasis> (Euphorbiaceae) based on RAPD and ITS analyses

Chamaesyce skottsbergii var. skottsbergii is federally listed as an endangered taxon, and is found in small and isolated populations restricted to calcareous soils in dry shrubland habitats on the Hawaiian islands of Oahu and Molokai. Concern over the genetic relationship among these disjunct populations arose as a result of threats to the habitat of the Oahu population. The populations were examined using random amplified polymorphic DNA (RAPD) markers and sequence analysis of the internal transcribed spacer (ITS) region of the rDNA cistron. Chamaesyce skottsbergii var. vaccinioides, a closely related variety found in several small populations on Molokai, was used for baseline comparison of the genetic divergence among populations. RAPD analysis demonstrated that variation within and among populations is the highest for any Hawaiian species examined. Polymorphism was greater than 95% within populations and was 99.4% at the species level. Similarly, measures of genetic similarity indicate that differentiation among these populations is higher than is known for some species. Both RAPD and ITS sequence analysis indicate that populations of C. skottsbergii var. skottsbergii on Oahu and Molokai are genetically distinct, and the extent of this genetic differentiation supports the recognition of these populations as distinct varieties. The Molokai population is in fact much more closely related to var. vaccinioides than to var. skottsbergii on Oahu, and thus should be recognized by the previously used variety name, C. skottsbergii var. audens. Further conservation measures for each of the varieties are addressed.     

The taxonomic status of Astragalus mokiacensis (Fabaceae), a species endemic to the southwestern United States

Astragalus mokiacensis has been a problematic species since it was first described in 1877. Every major revision delimited this taxon differently. A principal components analysis of morphological data from herbarium specimens was used to determine the affinities between type specimens and extant populations ofA. mokiacensis. The taxon recently recognized asA. lentiginosus var.trumbullensis is morphologically similar to the lectotype ofAstragalus mokiacensis. Astragalus lentiginosus var.trumbullensis is herein recognized as a lowelevation minor variant and considered a synonym ofA. mokiacensis. A taxonomic key and complete synonymy are included.     

Phylogenetic Relationships between Asplenium bourgaei (Boiss.) Milde and A. jahandiezii (Litard.) Rouy Inferred from Morphological Characters and rbcL Sequences

Abstract: Micromorphological analysis reveals that A. jahandiezii, which occurs as a local endemic taxon in the Grand Canyon du Verdon in southern France, shows substantial differences to A. bourgaei, from the eastern Mediterranean region, in the surface structure of perispores, sporangial indehiscence and the size of stomatal subsidiary cells. Nucleotide sequences and genetic distances (0.9 % nucleotide substitutions) of the rbc L gene support the view that both taxa are related but distinct sibling species. Both species form a monophyletic group together with Asplenium fontanum and A. majoricum.     

Intraspecific morphological and genetic differentiation in <Emphasis Type="Italic">Scrophularia grayana</Emphasis> (Scrophulariaceae)

Scrophularia grayana, which is distributed throughout northern Japan and Sakhalin, and its locally endemic variety var. grayanoides, have been examined morphometrically and genetically. Principal-component analysis using a total of 26 morphological characteristics revealed that these taxa are morphologically differentiated, but that the difference is not distinct. These two taxa have the same number of chromosomes in the somatic cells, 2n = 94, suggesting that ploidal level difference is not relevant to their divergence. The distributions of the taxa are adjoining in the north of Japanese mainland Honshu. Nevertheless, principal-coordinate analysis using putative 112 ISSR loci indicated they are genetically very distinct. Many taxon-specific alleles were found, and many of the alleles were fixed in each taxon. This genetic information suggests that a relatively long time has passed since the taxa became differentiated and that gene flow has rarely occurred between them, although morphological similarity has been maintained, probably because of natural selective forces. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Ecology of two geographically restricted Astragalus species (Fabaceae), A. bibullatus and A. tennesseensis, of the eastern United States

Astragalus bibullatus is endemic to limestone glades in the Central Basin of Tennessee, and except for one population in Illinois,A. tennesseensis is endemic to limestone glades in the Central Basin and in the Moulton Valley of Alabama. However,A. tennesseensis has been extirpated from four counties in Illinois, from its only known site in Indiana, from one county in Tennessee, and from one county in Alabama.Astragalus bibullatus is closely related to the geographically wide-spread Great Plains taxonA. crassicarpus var.crassicarpus (sectionSarcocarpi), whereasA tennesseensis is the only taxon in sectionTennesseensis. Both species are shallow-rooted, hemicryptophyte perennials without vegetative reproduction, have no effective means of seed dispersal, form long-lived seed banks, have similar life cycle phenologies, are intolerant of heavy shade, and have moderate amounts of genetic diversity. Much. additional information is available on the autecology ofA. tennesseensis. Its primary habitat is the transition zone between open glades and glade woods, where physical environmental factors are intermediate between those of the adjacent zones. Seedling-juvenile survival is low. Plants flower first in their second to fifth year and only a few times before dying, are self-incompatible, respond to drought by shedding leaves and by accumulating large amounts of proline, and compete poorly. Populations exhibit high fluctuations in number of individuals and have high turnover rates. A greenhouse study ofA bibullatus andA crassicarpus var.crassicarpus did not identify any difference in responses of these species to light or soil moisture that could account for the great differences in geographic ranges. Thus, historical factors were also considered to explain the narrow endemism ofA. bibullatus. Finally, we present previously unpublished data on the effect of light level and of competition with the cedar glade dominantSporobolus vaginiflorus in several watering regimes on growth ofA. tennesseensis. The role of these factors in restriction of this species to its transition zone microhabitat is discussed.     

Genetic variation in populations of <Emphasis Type="Italic">Allothrombium pulvinum</Emphasis> (Acari: Trombidiidae) from Northern Iran revealed by mitochondrial <Emphasis Type="Italic">cox</Emphasis>I and nuclear rDNA <Emphasis Type="Italic">ITS</Emphasis>2 sequences

Allothrombium pulvinum Ewing is a common natural enemy of aphids and some other arthropods. So far, there are no studies that have addressed genetic variation of this predatory mite. We investigated genetic variation of A. pulvinum across its whole known range in Iran. A 410 bp portion of the mitochondrial cytochrome c oxidase subunit I gene (coxI) and 797–802 bp portion of the internal transcribed spacer 2 of rDNA (ITS2) were sequenced for 55 individuals from 11 populations, resulting in 12 and 26 haplotypes, respectively. In the coxI region, haplotype and nucleotide diversities varied among populations from 0.00 to 0.90 and from 0.0000 to 0.0110, respectively. In the ITS2 region they varied from 0.20 to 0.91 and from 0.0006 to 0.0023, respectively. For both gene regions the highest haplotype and nucleotide diversities were detected in population Mahmoud Abad from northern Iran. Statistically significant population differentiation (F _ST) was detected in most pair-wise population comparisons. The results of population differentiation for both gene regions were generally congruent indicating that A. pulvinum from Iran consists of genetically different populations. This suggests that A. pulvinum comprises at least two geographically distinct populations or even more than one species. This study is an initial step towards understanding genetic variation of A. pulvinum, a taxon for which little molecular information is available. More intensive sampling and analysis of additional DNA regions are necessary for more detailed classification of this taxon.     

Tumbleweed (Salsola, section Kali) species and speciation in California

Tumbleweeds (Salsola species, section Kali) are road side and rangeland pest plants throughout the 48 contiguous states in the US. Three described tumbleweed species and two undescribed Salsola taxa occur in California. The known species are Russian thistle, Salsola tragus, introduced from Eurasia in the 1800s, Russian barbwire thistle, S. paulsenii, which grows in the desert regions of California, and is also native to Eurasia, and the recently identified S. kali subspecies austroafricana, possibly native to South Africa. Our goals were to investigate karyology, genome size, and molecular genetic affinities of the described species and the other taxa within their ranges in California using recently developed microsatellite loci, dominant nuclear DNA markers (RAPD and ISSR), and DNA sequence data. Chromosome counts and genome size assessments made with flow cytometry were compared. These analyses indicated that one undescribed taxon is a new allopolyploid hybrid between S. tragus and S. kali subspecies austroafricana, and the other undescribed taxon appears to be a complex hybrid involving all three described species. The invasion potentials for the hybrid taxa are unknown. Tumbleweeds are the focus of biological controls efforts but the identification of suitable agents for the hybrid taxa may be problematic because of the large amount of genetic variability encompassed within this evolving Salsola complex.     

Implications of molecular systematic analyses on the conservation of rare and threatened taxa: Contrasting examples from Malvaceae

Systematic research provides essential evidence for setting conservation priorities for rare and endangered taxa. Phylogenetic analyses can identify cryptic, genetically distinct lineages as well as actively interbreeding, and hence, non-distinctive lineages earlier perceived as separate taxa. A major aim of this study was to identify genetically distinct, rare lineages within two Malvaceae sister-genera, Sidalcea and Eremalche. The focus was two taxon-pairs each consisting of one rare and one more common taxon. The results demonstrate that even within two closely related genera, with a large number of rare taxa, molecular phylogenetic analyses can reveal contrasting degrees of evolutionary divergence and thus contrasting conservation implications for threatened taxa. Contrary to expectations, the substitution rate in the nuclear ribosomal transcribed spacers for annual Eremalche did not correspond to the faster evolutionary rate of annuals – compared to perennials – detected earlier within Sidalcea. Branch lengths in the (annual) Eremalche clade were shorter than those of annual members of Sidalcea. The phylogenetic analyses showed that the rare and endangered S. keckii and E. kernensis each are most closely related to a common species that has been regarded as insufficiently distinct to warrant separate taxonomic status. An additional aim of the study was to test the utility of the Phylogenetic Diversity (PD) measure to formalize the procedure of prioritizing conservation efforts. The measure demonstrated S. keckii (but not E. kernensis) to be genetically distinct from its closest relative and a good candidate for conservation. The PD measure was earlier used for assessing conservation priorities for areas, but proved useful to more objectively suggest conservation priorities among threatened taxa. Because this measure is calculated directly from the data, it retains more character information and gives a better representation of genetic diversity than other measures relying on tree topologies.     

Revised treatment of <Emphasis Type="Italic">Memecylon</Emphasis> sect. <Emphasis Type="Italic">Afzeliana</Emphasis> (<Emphasis Type="Italic">Melastomataceae: Olisbeoideae</Emphasis>), including three new species from Cameroon

Summary  Seven new names at species rank are proposed in Memecylon sect. Afzeliana Jacq.-Fél., a group of forest shrubs and small trees confined to Guineo-Congolian Africa. The group is centred in Cameroon, where 17 of the 20 species occur. A new flower type, the “star-flower” in Memecylon is revealed, and its taxonomic and ecological importance discussed. Three new, locally endemic species from the South West Province of Cameroon are described, mapped and illustrated: M. kupeanum R. D. Stone, Ghogue & Cheek, M. bakossiense R. D. Stone, Ghogue & Cheek, and M. rheophyticum R. D. Stone, Ghogue & Cheek. Two new names, M. accedens R. D. Stone, Ghogue & Cheek and M. hyleastrum R. D. Stone & Ghogue and one new combination, M. mamfeanum (Jacq.-Fél.) R. D. Stone, Ghogue & Cheek are provided at species level for three taxa originally proposed as varieties of M. afzelii G. Don. The taxon M. arcuatomarginatum var. simulans Jacq.-Fél. is also elevated to species status, as M. simulans (Jacq.-Fél.) R. D. Stone & Ghogue. Conservation assessments are provided for all the newly named taxa. A key is provided to the species of Memecylon sect. Afzeliana.     

Isoenzyme diversity in Reynoutria (Polygonaceae) taxa: escape from sterility by hybridization

The genus Reynoutria is represented by four taxa in the Czech Republic – R. japonica var. japonica and compacta, R. sachalinensis and R. × bohemica. Using isoenzyme analysis, we determined the degree of genotype variability in all taxa and compared clones of R. japonica var. japonica from the Czech Republic with those from Great Britain. While the rarely occurring tetraploid variety R. japonica var. compacta possesses low variability, the octoploid female clone of R. japonica var. japonica is genetically uniform in the 93 clones sampled and belongs to the same genotype that is present in the whole Europe. R. japonica var. japonica can be fertilized by the pollen of tetraploid R. sachalinensis and a hexaploid hybrid R. × bohemica is produced. In R. sachalinensis, 16 genotypes were found in the 50 clones sampled. R. × bohemica is genetically the most diverse taxon in the study area, with 33 genotypes recorded among 88 clones sampled.     

Novelties in Begonia (Begoniaceae) from the coastal forests of Brazil

Two new narrow endemic species ofBegonia from the Atlantic coastal forests of Brazil,B. espiritosantensis andB. ibitiocensis, are described and illustrated.Begonia schenkii var.calvescens, an endemic taxon from the states of Santa Catarina and Rio Grande do Sul, is transferred to species rank, and described and illustrated. A distribution map is provided for all three taxa.     

Conservation genetics and taxonomic status of the rare Kentucky lady's slipper: Cypripedium kentuckiense (Orchidaceae)

Cypripedium kentuckiense is a recently described rare orchid found in Arkansas (predominantly) and in eight other states. Much debate has focused on whether this taxon should be recognized as a distinct species or considered to be an extreme manifestation of the variability present in the widespread taxon Cypripedium parviflorum var. pubescens. In this study, 12 isozyme loci were analyzed for 14 populations of C. parviflorum var. pubescens and eight populations of C. kentuckiense. These data were used to examine the genetic similarity of these taxa, assess whether isozyme data support the continued recognition of C. kentuckiense as a distinct species, and assess whether a newly discovered disjunct Virginia population of C. kentuckiense is genetically isolated from other C. kentuckiense populations. The isozyme data revealed that the two taxa are very closely related with a high interspecific genetic identity. However, C. kentuckiense populations contain a subset of the variation present in C. parviflorum var. pubescens, and they have expected levels of heterozygosity that are one-quarter that of C. parviflorum var. pubescens populations. Cypripedium kentuckiense also possesses one widespread unique allele and a unique multilocus genotype. These data suggest that C. kentuckiense should be recognized as a distinct species, possibly of recent origin from C. parviflorum. Lastly, the isozyme data support the hypothesis that gene flow between the Virginia population and other populations of C. kentuckiense has been restricted.     

A rangewide herbarium‐derived dataset indicates high levels of gene flow in black cherry (Prunus serotina)

Isolation by Distance (IBD) is a genetic pattern in which populations geographically closer to one another are more genetically similar to each other than populations which are farther apart. Black cherry (Prunus serotina Ehrh.) (Rosaceae) is a forest tree species widespread in eastern North America, and found sporadically in the southwestern United States, Mexico, and Guatemala. IBD has been studied in relatively few North American plant taxa, and no study has rigorously sampled across the range of such a widespread species. In this study, IBD and overall genetic structure were assessed in eastern black cherry (P. serotina Ehrh. var. serotina), the widespread variety of eastern North America. Eastern North America. Prunus serotina Ehrh. var. serotina (Rosaceae). Dense sampling across the entire range of eastern black cherry was made possible by genotyping 15 microsatellite loci in 439 herbarium samples from all portions of the range. Mantel tests and STRUCTURE analyses were performed to evaluate the hypothesis of IBD and genetic structure. Mantel tests demonstrated significant but weak IBD, while STRUCTURE analyses revealed no clear geographic pattern of genetic groups. The modest geographic/genetic structure across the eastern black cherry range suggests widespread gene flow in this taxon. This is consistent with P. serotina's status as a disturbance‐associated species. Further studies should similarly evaluate IBD in species characteristic of low‐disturbance forests.     

Assessment of genetic variation in Sicilian Helichrysum (Asteraceae) and implication to germplasm conservation

Abstract

Some taxa of Sicilian Helichrysum related to sect. Stoeachadina have an uncertain systematic position; moreover, they are rare and endangered. In order to preserve these taxa in a genebank, a strategy to keep their genetic variation as high as possible is required. The high informative content of AFLP markers was used to assess genetic variations within and between populations and their relationships. The results show that all populations are genetically similar, even though there are two distinct groups: the first is the population referred to H. pendulum, and the second is represented by other populations belonging to several different specific and intraspecific taxa. The H. pendulum population is homogeneous, and characterised by the lowest genetic variation among all the populations considered. The second group shows a greater variation as evidenced by the presence of three subgroups: (i) populations referred to H. panormitanum var. panormitanum and H. panormitanum var. stramineum, (ii) populations referred to H. errerae var. errerae and H. errerae var. messerii, (iii) a population referred to H. hyblaeum, which, despite morphological and chorological differences, is similar to the other populations of the second group from a genetic stand point. Each population of the above-mentioned taxa has local fragment markers whose frequency is useful to determine how many plants should be sampled in order to collect the seeds necessary to preserve the population's genetic diversity in a genebank.     

Inter-simple sequence repeat (ISSR) diversity within Monarda fistulosa var. brevis (Lamiaceae) and divergence between var. brevis and var. fistulosa in West Virginia

Inter-simple sequence repeat (ISSR) banding patterns were used to examine genetic diversity within and among populations ofMonarda fistulosa var.brevis, a rare taxon restricted to several populations in limestone glades and barrens in eastern West Virginia and Virginia. More than 34% of the total ISSR diversity in var.brevis occurred among populations, which is high when compared to the few other rare species that have been examined for ISSR variation. Prior studies demonstrated that var.brevis is morphologically distinct from the more widespread var.fistulosa, and that the differences are maintained when the two varieties are grown together in a uniform environment. The present study utilizing ISSR markers indicated that the two varieties are distinct, though quite similar genetically, and this is concordant with prior investigations documenting their morphological and habitat differences. However, the ISSR results suggest that the two varieties have diverged relatively recently and/or there is a low level of gene flow between them.     

Characterization of microsatellite markers in the endangered Sinojackia xylocarpa (Styracaceae) and cross‐species amplification in closely related taxa

Twenty‐four polymorphic microsatellite loci were isolated and characterized from an AAG‐enriched genomic library of Sinojackia xylocarpa. The average allele number of these microsatellites was 3.3 per locus, ranging from two to seven. The observed and expected heterozygosities at population level were 0.10–0.83 and 0.10–1.00, respectively. In addition, successful cross‐species amplification of this set of microsatellites in three other species of Sinojackia and a closely related taxon, Changiostyrax dolichocarpa, suggested that this set of microsatellite markers should provide a useful tool for genetic and conservation studies of Sinojackia species and other closely related taxa in the Styracaceae.     

Karyology of theScorzonera (Compositae) species from the Iberian Peninsula

A karyological study of 15 taxa ofScorzonera L. from the Iberian Peninsula has been made. The chromosome numbers found inS. hispanica var.pinnatifida, S. baetica, S. reverchonii, S. angustifolia, S. laciniata var.calcitrapifolia and var.subulata (2n = 14) are new. Diploid cytotypes with 2n = 14 and 2n = 12 prevail, andS. hispanica var.crispatula is the only taxon which exhibits autopolyploidy (2n = 14, 28). x = 7 is considered to be the base chromosome number within the genus, with x = 6 being derived from it by translocation. This and detailed karyotype analyses allow to group the Iberian Peninsula species ofScorzonera into three groups.