Allozyme variation in Mexican species and classification of Datura (Solanaceae)

 Fifty natural Datura populations, belonging to eleven species (D. ceratocaula, D. discolor, D. inoxia, D. kymatocarpa, D. lanosa, D. metel, D. pruinosa, D. quercifolia, D. reburra, D. stramonium, D. wrightii) from Mexico and adjacent USA, were investigated using starch gel electrophoresis. A total of 64 alleles were scored at 17 loci (DIA1, DIA2, GOT1, GOT2, G6PDH, IDH, MDH1, MDH2, MDH3, ME, PGD1, PGD2, PGM1, PGM2, PHI, SAD, SOD). The heterozygosity among the species ranged from 0.166 (D. ceratocaula) to 0.276 (D. wrightii). Most genetic diversity was found within populations (average H_s=0.242), while values between populations are relatively low (average Dst=0.066, Gst=0.171). The analysis of the genetic distance suggested new taxonomic relationships among the species. Rather than supporting the conventional infrageneric classification with three sections, the results revealed that the herbaceous members of the genus Datura form four groups. One group included four of the eight species of the section Dutra and was more similar to the section Ceratocaulis than it was to the other group that contained the remaining taxa of Dutra. Received February 13, 2001 Accepted December 25, 2001     

The arctic-alpine polyploids Cerastium alpinum and C. nigrescens (Caryophyllaceae) in a sympatric situation: breakdown of species integrity?

 The high polyploids Cerastium alpinum (8x) and C. nigrescens (12x) were investigated in a mixed population in central Norway to evaluate whether hybridisation has resulted in continuous variation in morphology and genetic markers, a hypothesis previously proposed to account for the extensive taxonomic confusion in this species group. Isozyme, fertility, and morphological (37 characters) variation were examined among 347, 265, and 237 plants, respectively. A PCO analysis based on 23 quantitative morphological characters identified two main groups, corresponding to C. alpinum and C. nigrescens. The groups were also clearly separated in isozyme markers, several qualitative morphological characters, and chromosome numbers. Only 20 plants (8.4%) were more or less intermediate in the PCO analysis. These plants had a parental or hybrid isozyme multilocus phenotype and typically few well-developed anthers, low pollen stainability, and no seed set. Several plants within the C. nigrescens group also had reduced pollen stainability. These results indicate that later-generation hybrids and/or backcrosses towards C. nigrescens are formed. Thus, interspecific, interploidal gene flow probably occurs, but at rates that are insufficient to break down species integrity. Received July 5, 2000 Accepted August 2, 2001     

Allozyme variation in Carex sect. Digitatae – Evidence of introgression, genetic distinctiveness and evolution of taxa

 A subset of populations of the European taxa of Carex sect. Digitatae, including Carex digitata, C. pallens, C. ornithopoda, C. pediformis ssp. rhizodes and C. humilis have been investigated for allozyme variation. The (presumably) distantly related C. pendula was used as a reference taxon. Carex digitata, C. pallens and C. ornithopoda on the one hand, and C. humilis and C. pediformis on the other, shared the majority of the alleles. Cluster analyses based on genetic distances grouped populations of C. digitata and C. ornithopoda in a mixed subcluster whereas all populations of C. pallens formed a subcluster distinct from the digitata/ornithopoda cluster. This in spite of the fact that C. ornithopoda is morphologically clearly distinct from C. digitata whereas C. pallens is barely distinguishable from the latter. Carex pediformis and C. humilis appeared not more genetically similar to C. digitata than to the reference taxon. It is concluded that 1) C. digitata, C. pallens and C. ornithopoda are closely related and most probably forms a monophyletic group, 2) C. pallens is a genetically distinct species, 3) C. pediformis and C. humilis are not closely related to the rest of C. sect. Digitatae, 4) C. pediformis and C. humilis are relatively closely related, 5) introgression do occur between the investigated species but not to such an extent that it can explain major geographic patterns of variation in C. digitata. Received December 21, 2001; accepted November 14, 2002 Published online: March 24, 2003     

Microsatellite polymorphism and genetic differentiation in three Norwegian populations of Elymus alaskanus (Poaceae)

 Variation at seven microsatellite loci was investigated in three local E. alaskanus populations from Norway and microsatellite variation was compared with allozyme variation. The percentage of polymorphic loci was 81%, the mean number of alleles per polymorphic locus was 5.7 and expected heterozygosity was 0.37. An F-statistic analysis revealed an overall 48% deficit of heterozygotes over Hardy-Weinberg expectations. Gene diversity is mainly explained by the within population component. The averaged between population differentiation coefficient, F _st , over 7 loci is only 0.13, which accounts for only 13% of the whole diversity and was contrary to allozyme analysis. The mean genetic distance between populations was 0.12. However, a χ² -test showed that allele frequencies were different (p < 0.05) among the populations at 5 of the 7 loci. In comparison with the genetic variation detected by allozymes, microsatellite loci showed higher levels of genetic variation. Microsatellite analysis revealed that population H10576 possesses the lowest genetic variation among the tested three populations, which concur with allozyme analysis. The dendrogram generated by microsatellites agreed very well with allozymic data. Our results suggest that natural selection may be an important factor in shaping the genetic diversity in these three local E. alaskanus populations. Possible explanations for deficit heterozygosity and incongruence between microsatellites and allozymes are discussed. Received November 6, 2001; accepted April 24, 2002 Published online: November 14, 2002 Addresses of the authors: Genlou Sun (e-mail: Genlou.sun@STMARYS.CA), Biology Department, Saint Mary's University, Halifax. Nova Scotia, B3H 3C3, Canada. B. Salomon, R. von Bothmer, Department of Crop Science, The Swedish University of Agricultural Sciences, P.O. Box 44, SE-230 53, Alnarp, Sweden.     

Infraspecific genetic variation in Biscutella laevigata (Brassicaceae): new focus on Irene Manton's hypothesis

 Genetic variation in 42 populations throughout the range of Biscutella laevigata L. (Brassicaceae), a morphologically variable central European species, has been investigated by enzyme electrophoresis with three loci (Amy1, Amy2, and Gpi2). Genetic identities and the Fitch-Margoliash tree suggest differentiation into four regional groups: 1) a northwestern diploid group (northern France and northern Germany), 2) a northeastern diploid group (southern Germany, Upper Austria, northern Lower Austria, Poland, and Romania), 3) a central diploid group in southern Lower Austria corresponding to subspecies austriaca, and 4) a southern tetraploid group in Alpine areas of France, Germany, Switzerland, Austria, Italy, and Slovenia corresponding to subspecies laevigata. Geographically isolated diploid relic populations that are genetically depauperate are found in the NW and NE diploid groups. On the other hand, the diploid relic subspecies austriaca from the NE Prealps and Alps is highly variable. Subspecies laevigata appears to be a genetical autotetraploid with multiple origins involving several diploid progenitors (the NW diploids, subspecies austriaca and B. prealpina). Received April 6, 2001; accepted March 6, 2002 Published online: October 14, 2002 Addresses of the authors: Karin Tremetsberger (e-mail: k.tremetsberger@gmx.net), Christiane K?nig, Rosabelle Samuel, Tod F. Stuessy, Department of Higher Plant Systematics and Evolution, Institute of Botany, University of Vienna, Rennweg 14, A-1030 Vienna, Austria. Wilhelm Pinsker, Institute of Medical Biology, University of Vienna, Waehringerstra?e 10, A-1090 Vienna, Austria.     

Comparative analysis of genetic diversity in the mangrove species Avicennia marina (Forsk.) Vierh. (Avicenniaceae) detected by AFLPs and SSRs

Avicennia marina is an important mangrove species with a wide geographical and climatic distribution which suggests that large amounts of genetic diversity are available for conservation and breeding programs. In this study we compare the informativeness of AFLPs and SSRs for assessing genetic diversity within and among individuals, populations and subspecies of A. marina in Australia. Our comparison utilized three SSR loci and three AFLP primer sets that were known to be polymorphic, and could be run in a single analysis on a capillary electrophoresis system, using different- colored fluorescent dyes. A total of 120 individuals representing six populations and three subspecies were sampled. At the locus level, SSRs were considerably more variable than AFLPs, with a total of 52 alleles and an average heterozygosity of 0.78. Average heterozygosity for AFLPs was 0.193, but all of the 918 bands scored were polymorphic. Thus, AFLPs were considerably more efficient at revealing polymorphic loci than SSRs despite lower average heterozygosities. SSRs detected more genetic differentiation between populations (19 vs 9%) and subspecies (35 vs 11%) than AFLPs. Principal co-ordinate analysis revealed congruent patterns of genetic relationships at the individual, population and subspecific levels for both data sets. Mantel testing confirmed congruence between AFLP and SSR genetic distances among, but not within, population comparisons, indicating that the markers were segregating independently but that evolutionary groups (populations and subspecies) were similar. Three genetic criteria of importance for defining priorities for ex situ collections or in situ conservation programs (number of alleles, number of locally common alleles and number of private alleles) were correlated between the AFLP and SSR data sets. The congruence between AFLP and SSR data sets suggest that either method, or a combination, is applicable to expanded genetic studies of mangroves. The codominant nature of SSRs makes them ideal for further population-based investigations, such as mating-system analyses, for which the dominant AFLP markers are less well suited. AFLPs may be particularly useful for monitoring propagation programs and identifying duplicates within collections, since a single PCR assay can reveal many loci at once. Received: 3 October 2000 / Accepted: 19 February 2001     

Classification of rice germplasm: III. High-resolution fingerprinting of cytoplasmic genetic male-sterile (CMS) lines with AFLP

 The cytoplasmic genetic male-sterile (CMS) lines developed at the International Rice Research Institute are valuable in producing tropical rice hybrids. Efficient use of CMS lines in hybrid rice production will depend on their level of genetic diversity. Aside from morphological characterization, molecular analysis based on DNA markers can provide information on the genetic diversity of the germplasm. The Amplified Fragment Length Polymorphism (AFLP) technique was used to fingerprint 71 CMS lines and four rice cultivars, ‘IR64’, ‘Azucena’, ‘IR74’, and ‘FR13A’. Eleven primer pair combinations specific to the enzymes PstI and MseI were used to generate 530 AFLP markers, 176 of which were polymorphic. Each CMS line revealed a distinct fingerprint. The AFLP marker-based dendrogram depicted genetic variation among the CMS lines. The CMS lines developed in japonica background grouped with ‘Azucena’, a japonica cultivar. None of the CMS lines clustered with ‘FR13A’, a flood-tolerant traditional indica variety. ‘IR64’ was found to be distinct from the other indica CMS lines and clustered with lines developed in its background. The grouping of CMS lines into a few groups is useful for breeders in selecting genetically diverse CMS lines for hybrid rice production and in avoiding test crossing every CMS line empirically. This study demonstrated that AFLP is a powerful and reliable tool in determining the genetic relationships and in producing distinct fingerprints of rice cultivars. Received: 20 December 1996 / Accepted: 9 October 1997     

Pollen morphology and infra-generic evolutionary relationships in some Chinese species of Pedicularis (Scrophulariaceae)

 The pollen grains of 32 Chinese species of Pedicularis representing 13 groups within the genus were investigated using light microscopy and scanning electron microscopy. The genus is eurypalynous, the apertures may be tricolpate, trisyncolpate, or bisyncolpate and five types of surface ornamentation (microscabrate, microrugulate, retipilate, microreticulate and microfoveolate) were observed. Comparison of the different aperture types using light microscopy allows three pollen types to be distinguished. Examination of exine ornamentation with scanning electron microscopy enabled each pollen type to be divided into two or three sub-types (giving a total of seven sub-types). Pollen morphology within the genus is discussed in relation to infrageneric relationships, evolutionary and pollination biology. There is little correlation with the existing infrageneric taxonomy (some taxonomic groups have more than one pollen type, while the same pollen type may be found in several different species-groups), but greater correlation with the corolla morphology. Received January 10, 2002; accepted March 5, 2002 Published online: March 10, 2003     

Chromosome and breeding system evolution of the genus Mercurialis (Euphorbiaceae): implications of ITS molecular phylogeny

 The internal transcribed spacers (ITS1 and ITS2) of nuclear ribosomal DNA were amplified and sequenced from 19 samples representing all species of the genus Mercurialis and two outgroup species, Ricinus communis and Acalypha hispida. The length of ITS1 in the ingroups ranged from 223 to 246 bp and ITS2 from 210 to 218 bp. Sequence divergence between pairs of species ranged from 1.15% to 25.88% among the ingroup species in the combined data of ITS1 and ITS2. Heuristic phylogenetic analyses using Fitch parsimony on the combined data of ITS1 and ITS2 with gaps treated as missing generated 45 equally parsimonious trees. The strict consensus tree was principally concordant with morphological classification. Within the genus, the ITS sequences recognised two main infrageneric clades: the M. perennis complex including three Eurasian stoloniferous species (M.␣leiocarpa, M. ovata and M. perennis) and the western Mediterranean group including eight both annual and perennial species. Of the western Mediterranean clade, the annual and perennial species grouped respectively into two different groups, and the annual life form is revealed as a synapomorphic character derived from perennial, whereas in the Eurasian clade ITS phylogeny suggested M. leiocarpa as basal clade sister to M.␣perennis and M. ovata. ITS phylogeny failed to resolve the relationships among the different cytotypes of M. ovata and M. perennis. ITS phylogeny also suggested rapid karyotypic evolution for the genus. The karyotypic divergence among the perennial species of western Mediterranean region did not corroborate the nucleotide sequence divergence among the species. Optimisation of chromosome numbers onto the ITS phylogeny suggested x=8 to be the ancestral basic chromosome number of the genus. ITS phylogeny confirmed that the androdioecy of M. ambigua is derived from dioecy. The nucleotide heterozygosity and additivity in ITS sequences clearly confirm the interspecific hybridisation in the genus Mercurialis. Received December 22, 2001; accepted May 21, 2002?Published online: November 14, 2002 Address of the authors: Martin Kr?henbühl, Yong-Ming Yuan (correspondence) and Philippe Küpfer, Institut de Botanique, Laboratoire de botanique évolutive, Université de Neuchatel, Emile-Argand 11, CH-2007 Neuchatel, Suisse. (e-mail: yong-ming.yuan@unine.ch)     

A phylogenetic analysis of Primulaceae s.l. based on internal transcribed spacer (ITS) DNA sequence data

 Phylogenetic relationships in Primulaceae were investigated by analysis of nuclear rDNA ITS sequences. Thirty-four species of Primulaceae, two of Myrsinaceae and four outgroup taxa were analyzed. In accordance to the results of recently published papers on the phylogeny of Primulaceae we found the family to be paraphyletic and resolved the positions of some genera. Our results show (a) the rather basal position of Centunculus within Lysimachieae, the genus thus being rather distantly related to Anagallis, (b) the close relationship between Lysimachia sect. Lerouxia, Anagallis, Asterolinon, and Pelletiera, (c) the well-supported monophyly of a group consisting of the four genera Hottonia, Omphalogramma, Bryocarpum, and Soldanella, and (d) the affinity of Stimpsonia to the Myrsinaceae-Lysimachieae-Ardisiandra clade. The ITS sequence data do not provide sufficient information to resolve basal relationships within the Primulaceae s.l. There is evidence against the monophyly of the large genera Primula, Androsace, and Lysimachia. In contrast to the phylogenetic reconstructions based on plastid gene sequences, Cyclamen does not appear as a member of the Myrsinaceae-Lysimachieae clade, but its position remains unclear. Revised July 10, 2002; accepted November 21, 2002 Published online: March 20, 2003     

Nuclear DNA contents of all species of Helleborus (Ranunculaceae) discriminate between species and sectional divisions

 Genome size (C-values) and pollen viability staining were applied as new criteria to investigate the species of the genus Helleborus Linnaeus (Ranunculaceae). All species have the same chromosome number (2n=32). However, the nuclear DNA content, as measured by flow cytometry with propidium iodide, could be demonstrated to range between 19 pg to 35.7 pg. The different genome sizes of the species coincided to a large extent with earlier determined section boundaries based on morphology. Flow cytometry can be a convenient method to discriminate between some species. Received April 17, 2001 Accepted May 7, 2001     

Delineation and phylogeny of Centaurea sect. Acrocentron based on DNA sequences: a restoration of the genus Crocodylium and indirect evidence of introgression

 The Mediterranean group Acrocentron of the genus Centaurea is defined mainly on the basis of pollen type, but also by achene characters and involucral bracts morphology. We have revised the delineation of the group by cladistically comparing the sequences of the ITS spacers of the nuclear ribosomal DNA. Our results confirm that the Acrocentron group is a natural one, with a different delimitation from the commonly accepted. The ITS phylogeny supports that Centaurea sect. Chamaecyanus and sect. Stephanochilus belong to the Acrocentron group and suggests that sect. Chamaecyanus should be merged in sect. Acrocentron as a subsection. Contrary, sect. Aegialophila and sect. Crocodylium form a natural group that cannot be placed in the Acrocentron group and should be considered a different genus. The inclusion of Centaurea crocodylium in Aegialophila makes that the prioritary name for the generic level is Crocodylium; thus, two new nomenclatural combinations are proposed: Crocodylium creticum and Crocodylium pumilum. The groups suggested by the ITS sequences are correlated to the main geographical centers of speciation of Acrocentron. However, support for internal nodes of the tree is extremely poor. The low support within the tree and the absence of correlation between karyology and molecular phylogeny suggest that hybridization has played an important role in the diversification of Acrocentron. Received June 6, 2001; accepted April 2, 2002 Published online: November 7, 2002 Address of the authors: Mònica Font, Teresa Garnatje, Núria Garcia-Jacas, and Alfonso Susanna, Botanic Institute of Barcelona (CSIC-Ajuntament de Barcelona), Av.Muntanyans s.n., E-08038 Barcelona, Spain.     

Self-pollination in Euphrasia willkommii Freyn (Scrophulariaceae), an endemic species from the alpine of the Sierra Nevada (Spain)

 The reproductive ecology of Euphrasia willkommii (Scrophulariaceae), an endemic species from the Mediterranean alpine environments of the SE Spain, has been experimentally studied during two reproductive seasons. The flowers of this plant species were visited by very few insects belonging only to two generalist taxa, thrips and ants. Nevertheless, reproduction is not pollen limited in E. willkommii. Hand-pollinations demonstrated that this plant species is capable of selfing, reproductive success being similar in autogamous and allogamous crosses. Moreover, pollinator-exclusion experiments also showed that, under natural conditions, this plant relies predominantly on selfing, seed production being similar in presence or absence of pollinators. Selfing in E. willkommii is presumably an ecological mechanism to ensure successful reproduction in a harsh environment where pollinator availability is extremely low. Received May 2, 2001 Accepted December 6, 2001     

Genetic variation in Scandinavian Anthericum liliago (Anthericaceae): allopolyploidy, hybridization and immigration history

 We investigated patterns of isozyme variation and the hierarchic structure of genetic diversity in 25 Scandinavian populations of the lilioid herb, Anthericum liliago. Isozyme data suggest that tetraploid A. liliago has an allopolyploid origin and that A. ramosum may be one of its diploid progenitors. Two populations contained known or suspected hybrids between A. liliago and A. ramosum. Isozymes show that one population from S Sweden contains both triploid (hybrid) and tetraploid individuals whereas a putatively hybrid Danish population represents diploid A. liliago. There is an overall northward and eastward decline in allelic richness in the tetraploid populations, with the highest numbers of alleles in Denmark and SW Sweden. This pattern is consistent with a progressive loss of allelic variation during the species' postglacial colonization of Scandinavia. The between-population component of genetic diversity is 4% (compared with 12% in diploid A. ramosum), the between-region diversity component is 7% and 89% of the total diversity is stored within populations. Received March 13, 2002; accepted September 24, 2002 Published online: December 11, 2002     

Genetic variation in Banksia saxicola (Proteaceae), a rare Australian plant with a markedly disjunct distribution

 Banksia saxicola A.S. George (Proteaceae) is a rare Australian endemic, found in only two locations in Victoria that are separated by approximately 500 kms: the Grampians and Wilson's Promontory National Parks. The organisation of genetic variation between and within populations at the two locations was assessed using the Amplified Fragment Length Polymorphism (AFLP) technique. Two populations were sampled in the Grampians National Park and one population was sampled at Wilson's Promontory. The three populations were genetically divergent from each other, in particular the Grampians and Wilson's Promontory populations, and this relates to the ancient geographic isolation of these two regions. The Wilson's Promontory population had lower genetic diversity than either Grampians populations, consistent with its smaller population size. The findings are important for strategies to conserve B. saxicola. Received October 3, 2000 Accepted January 3, 2001     

The evolution of Spartina anglica G. E. Hubbard (Gramineae): genetic variation and status of the parental species in Britain

The perennial salt marsh grass Spartina anglica is one of the classic examples of allopolyploid speciation. It originated on the south coast of England at the end of the nineteenth century following chromosome doubling in S. × townsmdii , a hybrid between the native British S. marilima and a species introduced from the United States, S. alterniflora. The nature of the origin of S. anglica is beyond doubt; however, it is not known whether it had a single or multiple origin. In order to address this problem we undertook a survey of the genetic variation in the parental species of S. anglica using isozyme electrophoresis. The results show that S. alterniflora has no detectable variation and that S. maritima has extremely low levels of variation. These results, unfortunately, prevent the question of a single or multiple origin from being answered. Possible reasons for the low levels of variation and its influence on the future of the species are discussed. Another problem concerning the parental species is the rapid decline of S. maritima in Britain. It is often assumed that the major factor in this regression is the invasion of its habitats by S. anglica. We have examined the status of S. marilima throughout its range in Britain and have found that S. anglica rarely co-occurs with S. maritima. We propose that the decline of S. maritima is largely due to the physical erosion of its habitats and that this erosion may produce suitable habitats for colonization by S. anglica.     

Morphology and ecology of two new species of Polypleurum (Podostemaceae) from Kerala, India

 Two new species of the Asiatic genus Polypleurum (Podostemaceae – Podostemoideae), Polypleurum prostratum and Polypleurum disciforme from Kerala, India are described. They differ in the shape of the thalloid plant body from the other species of Polypleurum. A key to the species occurring in India is provided. Though sharing the habitat with some other species of the Podostemaceae, the new species inhabit special niches in the rocky riverbed. – The chemical components of the (hard) water in which they grow differ considerably from what is known from black water rivers of the New World. The results thus widen considerably our knowledge of the ecological needs of Podostemaceae. Received December 10, 2002; accepted January 10, 2003 Published online: March 31, 2003     

The phylogeny of Lens (Leguminosae): new insight from ITS sequence analysis

 Lens includes L. culinaris subsp. culinaris (the cultivated lentil) and several wild species distributed from the Mediterranean region to western Asia. We compared sequence variation in the ITS region among species of Lens in an effort to end persisting uncertainty regarding the phylogeny of the genus. The parsimony analysis revealed a single minimum-length tree with a topology congruent with patterns derived by previous studies of nuclear and chloroplast DNA RFLPs. The basal and highly divergent status of the L. nigricans clade is depicted, and the progenitor-derivative relationship between L. culinaris subsp. orientalis and L. culinaris subsp. culinaris is reaffirmed. Resolution in the tree was improved by combining the ITS data set with a pre-existing set of chloroplast DNA restriction site data obtained from the same group of samples. Received May 8, 2000 Accepted October 26, 2001     

Pollenkitt formation in Ilex paraguariensis A.St.Hil. (Aquifoliaceae)

 We investigated the cellular and organelle transformations during the formation of the pollenkitt in the secretory tapetum of Ilex paraguariensis. After the dissolution of the callose surrounding the young microspores, the elaioplasts of the tapetum produce many globules of saturated and unsaturated lipids (plastoglobules). Further on, oleosomes with unsaturated lipids, synthesized in the endoplasmic reticulum, accumulate in the tapetal cytoplasm. In contrast to other species, the plastoglobule production seems to precede the oleosome synthesis. The tapetum shows signs of cellular maturation in the late vacuolated microspore stage, when the plastoglobules and oleosomes coalesce and form the pollenkitt mass. In mature stages of the tapetum the pollenkitt is released into the loculus. Finally, it is mainly deposited on the exine, according to the entomophilous character of this species. The mode of pollenkitt formation in Ilex para guariensis and its transfer to the pollen surface is slightly dissimilar to other Angiosperms. Received October 24, 2002; accepted December 2, 2002 Published online: March 20, 2003