Leo P. Bruederle

Allozyme evidence for genetic autopolyploidy and high genetic diversity in tetraploid cranberry, Vaccinium oxycoccos (Ericaceae).

Polyploidy has been important in the evolution of angiosperms and may significantly affect population genetic diversity and structure. Nineteen isoenzyme loci were studied in diploid and tetraploid populations of Vaccinium oxycoccos (Ericaceae), and the results are compared with data previously reported for the related V. macrocarpon. Diploid V. oxycoccos and V. macrocarpon were readily discriminated based on their allozymic variation. No evidence for fixed heterozygosity was found in tetraploid V. oxycoccos. In contrast, all polymorphic loci exhibited both balanced and unbalanced heterozygotes, with some individuals exhibiting a pattern consistent with the presence of three alleles. These results support an autopolyploid origin for tetraploid V. oxycoccos. However, tetraploid V. oxycoccos possessed a suite of alleles not found in diploid V. oxycoccos; half of these alleles were shared with V. macrocarpon. This suggests that autotetraploid V. oxycoccos may have undergone hybridization with V. macrocarpon or that the autotetraploid retained the genetic variation present in an ancestral diploid species. Following theoretical expectations, proportion of polymorphic loci, mean number of alleles, and observed heterozygosity were significantly higher for the autotetraploid than for the diploid. Mean inbreeding (F(IS)) was similar for diploid and tetraploid V. oxycoccos. The latter exhibited population differentiation (F(ST)) exceeding both diploid species.

Allozyme Variation in Populations of the Carex crinita Complex (Cyperaceae)

The Carex crinita Lam. complex (Cryptocarpae) comprises eight validly described taxa occurring in eastern North America. Modern taxonomic treatments of the complex differ consid- erably recognizing from three to seven taxa of varying rank. For this study, 36 populations rep- resenting the range of morphological variation in the complex were examined utilizing starch gel electrophoresis and allozyme analysis. Data were collected for 18 putative genetic loci. Although 13 of these loci were variable across the species complex, only 15 populations were polymorphic at one or more loci. The number of alleles per locus averaged 1.06, whereas the average percentage of polymorphic loci was 5.26. The majority of genetic variation is distributed among taxa as evi- denced by the values obtained for Neis gene diversity statistic; a GST of 0.658 indicates that these populations are highly differentiated. Averaged by taxon, Neis unbiased genetic identity ranged from 0.726 for pairwise comparisons of populations of C. gynandra and C. mitchelliana to 0.866 for C. gynandra and C. crinita var. brevicrinis. These data support the recognition of four taxa comprising this species complex. Additionally, statistically significant deviations from Hardy-Weinberg ex- pected heterozygosity and high positive values for Wrights fixation index suggest high levels of inbreeding. These data contrast with morphological evidence suggesting outcrossing through wind dispersal of pollen.

Genetic Differentiation of Carex flava and Carex viridula in West Europe (Cyperaceae)

Recent systematic treatments evaluating variation in the Carex flava species complex have divided the West European representatives into two species, C. flava and C. viridula. These treatments contrast with previous reports that have recognized as many as nine species based upon morphological variation. To assess the systematic relationship of C. flava and C. viridula, allozyme analyses were conducted on 34 West European populations distributed from the Alps to the Arctic Circle. Data were obtained from electrophoresis of soluble enzymatic proteins extracted from leaf tissue. Fifteen of the 20 presumptive loci examined for this study were found to be polymorphic; variation was distributed between the two species and within 25 populations. These data support recognition of C. flava as a species distinct from C. viridula at a genetic identity of 0.69. The two species are discriminated by diagnostic alleles at two loci and highly disparate allele frequencies at an additional five loci. There is little genetic differentiation among populations of C. flava in West Europe. Genetic identities exceeding 0.95, which were obtained from infraspecific pairwise comparisons of C. flava, do not support recognition of subspecies. In contrast, C. viridula was highly differentiated with a GST of 0.806 strongly supporting recognition of subspecies. The Carex flava L. species complex is one of the most complicated, yet well-studied groups of sedges in Europe. It is included within sect. Ceratocystis Dumort., which comprises seven species worldwide (Crins and Ball 1988). The literature on the C. flava complex contains numerous reports on its morphology, e.g., Crins and Ball (1989a); regional distribution, e.g., Havlkkova (1983); ecology, e.g., Schmid (1984a, 1984b); nomenclature, e.g., Schmid (1983); floral biology and flowering phenology, e.g., Vonk (1979); karyotype and crossability, e.g., Schmid (1982); taxonomy, e.g., Crins and Ball (1989b); and evolution, e.g., Crins and Ball (1988). Schmid (1982, 1983, 1984a, 1984b), in a series of publications considering the European representatives of this group, reported on the ecology, systematics, and evolution of the Carex flava complex. Schmids treatment divided the complex into two species, C. flava and C. viridula Michaux, recognizing five subspecies comprising C. viridula. Crins and Ball (1989b) also recognized two species in the European flora, but acknowledged less subspecific variation meriting taxonomic status in C. viridula. Starch gel electrophoresis and allozyme analysis are powerful tools for elucidating systematic relationships in Carex (Bruederle and Fairbrothers 1986; Ford and Ball 1989; Waterway 1988; Whitkus 1985). Allozyme data have been useful in discriminating morphologically cryptic taxa, e.g., C. crinita var. brevicrinis Fern. (Bruederle and Fairbrothers 1986), as well as confirming the taxonomic status of taxa, e.g., C. mitchelliana M. A. Curtis (Bruederle et al. 1989). We report here on detected allozyme variation within the C. flava species complex in West Europe. The purpose of this publication is twofold: 1) to present electrophoretic data assessing variation within the complex and discuss the systematic relationship of the two widely recognized species C. flava and C. viridula; and 2) to consider the distribution of infraspecific variation within each of these two species and briefly evaluate subspecific relationships. MATERIALS AND METHODS Plants were collected at 23 sites distributed from Switzerland and Austria to northern Norway (fig. 1; table 1). Whereas 20 to 50 individuals were collected from large populations, a complete sample of all observed individuals was obtained from populations comprising fewer than 20 individuals. The caespitose habit of the species facilitated recognition and collection of individuals for the study. Plants were cultivated and maintained in a common garden or glass

Genetic variation in natural populations of the large cranberry, Vaccinium macrocarpon Ait. (Ericaceae)'

1816, the large cranberry has become a regionally important small fruit crop in the United States. In order to assess genetic variation in naturally occurring populations of the large cranberry, sampling was conducted at ten sites distributed from Wisconsin east to Massachusetts, and south to Delaware. Starch gel electrophoresis and substrate-specific staining techniques were used to resolve 23 putative Mendelian loci. Population data collected as individual genotypes were analyzed, generating measures of population structure and genetic diversity. All populations were found to exhibit low levels of genetic variation, e.g., expected heterozygosity (Hexp 0.00 + 0.002-0.057 + 0.030). Relative to other plant species, total genetic diversity was extremely low (H, 0.048) with the majority of all genetic variation due to differences among individuals within populations. This genetic homogeneity was corroborated by Neis (1978) genetic identity which ranged from 0.977 to one. Recent events in the evolutionary history of this taxon are proposed to have played an important role in determining population genetic structure in this species.

Genetic diversity and endemism in North American Carex section Ceratocystis (Cyperaceae).

Factors leading to endemism, and the evolutionary implications of endemism, can be explored by studying closely related taxa with variously restricted distributions. Such a model is provided by Carex section Ceratocystis (Cyperaceae); Carex cryptolepis, Carex sp. nov., and C. lutea are North American endemics with broad, restricted, and highly restricted distributions, respectively. The prediction that levels of genetic diversity are a consequence of distribution size was tested within a phylogenetic context using population level genetic variation at 18 allozyme loci. In contrast to expectations, mean proportion of loci polymorphic, number of alleles per polymorphic locus, and expected heterozygosity were significantly greater in C. lutea than either C. cryptolepis or Carex sp. nov. Although the possibility of a shift in breeding system, past introgression, or progenitor-derivative relationships could explain the relatively high levels of variation observed in C. lutea, these were dismissed on the basis of allozyme and nuclear ribosomal sequence data. We conclude that C. lutea maintains levels of genetic diversity typical of caespitose carices despite its narrow endemism and that the low levels of genetic variation in C. cryptolepis and Carex sp. nov. are likely the result of population fluctuations during Pleistocene glacial-interglacial cycles.

Genetic Differentiation of Diploid Blueberry, Vaccinium sect. Cyanococcus (Ericaceae)

The most dissected taxonomic treatment of Vaccinium section Cyanococcus, generated by Camp in 1945, recognized nine diploid (2n = 24) taxa. More recently, Vander Kloet recognized six taxa nesting the two hillside blueberries and the three highbush blueberries, respectively. Whereas previous taxonomic treatments have been based primarily upon morphological data, field studies, chromosome counts, and crossability, the present study employs allozyme data to assess genetic relationships of representative diploid blueberry populations. Starch gel electrophoresis was conducted on population samples obtained from 25 sites identified in an extensive field survey of this endemic North American section. Data were collected at 11 polymorphic loci previously observed to have high taxonomic information content. Data were analyzed to obtain similarity, as well as standard measures of population genetic structure. Five genetic lineages were discriminated; these were supported by highly disparate allelic frequencies. Conspecific populations were undif- ferentiated, with distribution of genetic variation similar to that described for other predominantly outcrossing species. In conjunction with patterns of morphological variation, this study supports the recognition of seven species: two highbush species, V. corymbosum and V. elliottii, and five lowbush species, V. boreale, V. darrowii, V. myrtilloides, V. pallidum, and V. tenellum.

Carex viridistellata sp. nov. (Cyperaceae), a New Cryptic Species from Prairie Fens of the Eastern United States

Abstract Divergence between evolutionary lineages is not always marked by the development of obvious species-specific characters, whether morphological, physiological, or ecological. Consequentially, extant biodiversity can easily be overlooked. These cryptic species are often not recognized until genetic data are in hand, as is the case for the novel taxon we describe here. Carex viridistellata in Carex section Ceratocystis is an endemic species restricted to calcareous wetlands of Michigan, Ohio, and Indiana, where it has previously been collected as Carex cryptolepis. Crosses between Carex viridistellata and Carex cryptolepis produce sterile F1 hybrids, and the two species are differentiated by a number of subtle morphological characters, as well as aspects of their respective ecologies. Phylogenetic analyses of nrDNA strongly indicate monophyly of Carex viridistellata and its sister species relationship with the North Carolina narrow endemic Carex lutea. Both species constitute a distinct lineage within a generally poorly resolved section Ceratocystis. This work highlights the broad importance of cryptic taxa, with implications for fields ranging from population genetics to conservation and restoration.

Specimens at the Center: An Informatics Workflow and Toolkit for Specimen-Level Analysis of Public DNA Database Data

Abstract Major public DNA databases — NCBI GenBank, the DNA DataBank of Japan (DDBJ), and the European Molecular Biology Laboratory (EMBL) — are invaluable biodiversity libraries. Systematists and other biodiversity scientists commonly mine these databases for sequence data to use in phylogenetic studies, but such studies generally use only the taxonomic identity of the sequenced tissue, not the specimen identity. Thus studies that use DNA supermatrices to construct phylogenetic trees with species at the tips typically do not take advantage of the fact that for many individuals in the public DNA databases, several DNA regions have been sampled; and for many species, two or more individuals have been sampled. Thus these studies typically do not make full use of the multigene datasets in public DNA databases to test species coherence and select optimal sequences to represent a species. In this study, we introduce a set of tools developed in the R programming language to construct individual-based trees from NCBI GenBank data and present a set of trees for the genus Carex (Cyperaceae) constructed using these methods. For the more than 770 species for which we found sequence data, our approach recovered an average of 1.85 gene regions per specimen, up to seven for some specimens, and more than 450 species represented by two or more specimens. Depending on the subset of genes analyzed, we found up to 42% of species monophyletic. We introduce a simple tree statistic—the Taxonomic Disparity Index (TDI)—to assist in curating specimen-level datasets and provide code for selecting maximally informative (or, conversely, minimally misleading) sequences as species exemplars. While tailored to the Carex dataset, the approach and code presented in this paper can readily be generalized to constructing individual-level trees from large amounts of data for any species group.

Evidence for genetic allopolyploidy in Eutrema edwardsii (Brassicaceae): implications for conservation

Eutrema edwardsii R.Br. (Brassicaceae) is an arctic-alpine mustard with a circumpolar distribution. Its closest relative, Eutrema penlandii Rollins, is a federally listed, threatened species that is endemic to the Mosquito Range in the Southern Rocky Mountains of Colorado, USA. As part of a larger project addressing the systematics of this species complex in North America, we conducted chromosome counts, flow cytometry, and allozyme analysis to test the hypothesis that these taxa comprise an autopolyploid complex. Within that context, it should be noted that a chromosome count has not been reported previously for E. penlandii. Results obtained from mitotic counts obtained for two populations of E. penlandii reveal this taxon to be diploid. Diploidy was confirmed using flow cytometry for an additional 15 individuals representing four populations. Previously published chromosome counts for E. edwardsii reveal a polyploid complex of tetraploid, hexaploid, and octaploid populations for which an autopolyploid origin has been presumed. However, allozyme analysis revealed an allopolyploid origin for E. edwardsii, as evidenced from fixed heterozygosity at six loci. Although our data suggest that E. penlandii is a close relative of one of the progenitors of E. edwardsii, the taxonomic identity of the other progenitor(s) cannot be elucidated from these data. The data reported herein support the recognition of E. penlandii as taxonomically distinct, which has implications for conservation, and reveal cryptic variation within E. edwardsii.

Development of nuclear microsatellite loci for Pinus albicaulis Engelm. (Pinaceae), a conifer of conservation concern

Pinus albicaulis (whitebark pine) is a widely-distributed but rapidly declining high elevation western North American tree and a candidate for listing under the U.S. Endangered Species Act. Our objectives were to develop reliable nuclear microsatellite markers that can be used to assess within-population genetic diversity as well as seed and pollen migration dynamics, and to validate markers using two geographically proximal P. albicaulis populations. We identified 1,667 microsatellite-containing sequences from shotgun DNA libraries of P. albicaulis. Primer pairs were designed for 308 unique microsatellite-containing loci, and these were evaluated for PCR amplification success and segregation in a panel of diploid needle tissue. DNA was extracted with an SDS protocol, and primers were screened through gel electrophoresis. Microsatellites were genotyped through fluorescent primer fragment analysis. Ten novel and 13 transferred loci were found to be reproducible in analyses based on 20 foliage samples from each of two locations: Henderson Mountain, Custer Gallatin National Forest, Montana, and Mt. Washburn, Yellowstone National Park, Wyoming (USA). Transferred loci had higher numbers of alleles and expected heterozygosities than novel loci, but also revealed evidence for a higher frequency of null alleles. Eight of the 13 transferred loci deviated significantly from Hardy-Weinberg Equilibrium, and showed large positive FIS values that were likely inflated by null alleles. Mantel’s tests of transferred and novel markers showed no correlation between genetic and geographic distances within or among the two sampled populations. AMOVA suggests that 91% of genetic variability occurs within populations and 9% between the two populations. Studies assessing genetic diversity using these microsatellite loci can help guide future management and restoration activities for P. albicaulis.