Josep A. Rosselló

Nuclear ribosomal DNA (nrDNA) concerted evolution in natural and artificial hybrids of Armeria (Plumbaginaceae)

Nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) sequences from artificial hybrids and backcrosses between Armeria villosa ssp. longiaristata and A. colorata were studied to assess the possible effects of concerted evolution in natural hybrids. F1 artificial hybrids show the expected pattern of additive polymorphisms for five of the six variable sites as estimated from direct sequences. However, homogenization of polymorphism is already observed in the F2, and is biased towards A. colorata except for one site. In backcrosses, an expected tendency towards homogenization of polymorphic sites in the direction of the recurrent parent is observed for five sites, although this does not necessarily imply concerted evolution. Conversely, the sixth site appears to elude such a mechanism and thus provides additional support for the occurrence of biased concerted evolution. Our findings are relevant to interpreting phylogeographic patterns involving gene flow and are also consistent with the hypothesis of a hybrid origin of A. villosa ssp. carratracensis.

Phylogenetic Analysis Informed by Geological History Supports Multiple, Sequential Invasions of the Mediterranean Basin by the Angiosperm Family Araceae

Despite the remarkable species richness of the Mediterranean flora and its well-known geological history, few studies have investigated its temporal and spatial origins. Most importantly, the relative contribution of geological processes and long-distance dispersal to the composition of contemporary Mediterranean biotas remains largely unknown. We used phylogenetic analyses of sequences from six chloroplast DNA markers, Bayesian dating methods, and ancestral area reconstructions, in combination with paleogeographic, paleoclimatic, and ecological evidence, to elucidate the time frame and biogeographic events associated with the diversification of Araceae in the Mediterranean Basin. We focused on the origin of four species, Ambrosina bassii, Biarum dispar, Helicodiceros muscivorus, Arum pictum, subendemic or endemic to Corsica, Sardinia, and the Balearic Archipelago. The results support two main invasions of the Mediterranean Basin by the Araceae, one from an area connecting North America and Eurasia in the Late Cretaceous and one from the Anatolian microplate in western Asia during the Late Eocene, thus confirming the proposed heterogeneous origins of the Mediterranean flora. The subendemic Ambrosina bassii and Biarum dispar likely diverged sympatrically from their widespread Mediterranean sister clades in the Early-Middle Eocene and Early-Middle Miocene, respectively. Combined evidence corroborates a relictual origin for the endemic Helicodiceros muscivorus and Arum pictum, the former apparently representing the first documented case of vicariance driven by the initial splitting of the Hercynian belt in the Early Oligocene. A recurrent theme emerging from our analyses is that land connections and interruptions, caused by repeated cycles of marine transgressions-regressions between the Tethys and Paratethys, favored geodispersalist expansion of biotic ranges from western Asia into the western Mediterranean Basin and subsequent allopatric speciation at different points in time from the Late Eocene to the Late Oligocene.

A Phylogeographic Split in Buxus balearica (Buxaceae) as Evidenced by Nuclear Ribosomal Markers: When ITS Paralogues Are Welcome

Sequences from the ribosomal nuclear internal transcribed spacers (ITS) have been widely used to infer evolutionary hypotheses across a broad range of living organisms. Intraspecific sequence variation is assumed to be absent or negliable in most species, but few detailed studies have been conducted to assess the apportionment of ITS sequence variation within and between plant populations. Buxus balearica was chosen as a model species to assess the levels of infraspecific and intragenomic ITS variation in rare and endangered species occurring in disjunct populations around the Mediterranean basin. Intragenomic polymorphic sites were detected for western and eastern accessions of B. balearica and in two accessions of the sister species B. sempervirens. Overall, 19 different ribotypes were found in B. balearica after sequencing 48 clones, whereas 15 ribotypes were detected in 19 clones of B. sempervirens. The integrity and secondary structure stability of the ribosomal sequences suggest that they are not pseudogenes. The high number of ribotypes recovered through cloning suggested that some sequences could be chimeric or generated in vivo by partial homogenization through gene conversion or unequal crossing-over. Average sequence divergence among B. balearica clones was 0.768%, and the most divergent sequences differed by 1.62%. Available evidence does not suggest that B. balearica paralogues have been obtained from other extant Buxus species through interspecific hybridization. The presence of several ribosomal sequences in box implies that the molecular forces driving the concerted evolution of this multigene family are not fully operational in this genus. Phylogenetic analyses of cloned ITS sequences from B. balearica displayed very poor resolution and only two clades received moderate bootstrap support. Despite the marked intragenomic sequence divergence found, ribosomal data suggest a clear phylogeographic split in B. balearica between western and eastern accessions. The distinct, nonchimeric sequences that are postulated as being present in each biogeographic group suggest that box populations from Anatolia (eastern Mediterranean) are relict.

Relationships of the Woody Medicago Species (Section Dendrotelis) Assessed by Molecular Cytogenetic Analyses

BACKGROUND AND AIMS The organization of rDNA genes in the woody medic species from the agronomically important Medicago section Dendrotelis was analysed to gain insight into their taxonomic relationships, to assess the levels of infraspecific variation concerning ribosomal loci in a restricted and fragmented insular species (M. citrina) and to assess the nature of its polyploidy. METHODS Fluorescence in situ hybridization (FISH) was used for physical mapping of 5S and 45S ribosomal DNA genes in the three species of section Dendrotelis (M. arborea, M. citrina, M. strasseri) and the related M. marina from section Medicago. Genomic in situ hybridization (GISH) was used to assess the genomic relationships of the polyploid M. citrina with the putatively related species from section Dendrotelis. KEY RESULTS The diploid (2n = 16) M. marina has a single 45S and two 5S rDNA loci, a pattern usually detected in previous studies of Medicago diploid species. However, polyploid species from section Dendrotelis depart from expectations. The tetraploid species (2n = 32) M. arborea and M. strasseri have one 45S rDNA locus and two 5S rDNA loci, whereas in the hexaploid (2n = 48) M. citrina four 45S rDNA and five 5S rDNA loci have been detected. No single chromosome of M. citrina was uniformly labelled after using genomic probes from M. arborea and M. strasseri. Instead, cross-hybridization signals in M. citrina were restricted to terminal chromosome arms and NOR regions. CONCLUSIONS FISH results support the close taxonomic interrelationship between M. arborea and M. strasseri. In these tetraploid species, NOR loci have experienced a diploidization event through physical loss of sequences, a cytogenetic feature so far not reported in other species of the genus. The high number of rDNA loci and GISH results support the specific status for the hexaploid M. citrina, and it is suggested that this species is not an autopolyploid derivative of M. arborea or M. strasseri. Further, molecular cytogenetic data do not suggest the hypothesis that M. arborea and M. strasseri were involved in the origin of M. citrina. FISH mapping can be used as an efficient tool to determine the genomic contribution of M. citrina in somatic hybrids with other medic species.

Genetic variability in a narrow endemic snapdragon (Antirrhinum subbaeticum, Scrophulariaceae) using RAPD markers

Antirrhinum subbaeticum is an endangered species inhabiting fragmented limestone cliffs. In the last 3 years, a drastic population decline has been observed in three of four known populations and the estimated number of surviving individuals is now close to 400. A RAPD study was conducted to evaluate the levels of genetic variation present in this species to improve conservation guidelines. Thity-nine polymorphic products identified 66.1% of the samples by unique RAPD multilocus profiles. A cluster analysis grouped the samples into two broad groups corresponding to northern or southern provenances. AMOVA analysis showed that only 17.7% of the genetic diversity was partitioned within populations. These results are in contrast to data available for other Antirrhinum species. This genetic structure could be explained by the predominant selfing behaviour exhibited by A. subbaeticum as opposed to the allogamy of other congeners. Genetic diversity within populations does not seem to be strongly related to population size and historical factors could be responsible for the very low levels of genetic diversity found in one population. Given the low genetic diversity within populations, it is suggested that an extensive sampling of individuals be made for recovering appropriate levels of the gene pool for ex situpreservation. However, translocation of individuals to the genetically weakened Bogarra population from other sources is not recommended.

Can Extensive Reticulation and Concerted Evolution Result in a Cladistically Structured Molecular Data Set

Hierarchy is the main criterion for informativeness in a data set, even if no explicit reference to evolution as a causal process is provided. Sequence data (nuclear ribosomal DNA ITS) from Armeria (Plumbaginaceae) contains a certain amount of hierarchical structure as suggested by data decisiveness and distribution of tree lengths. However, ancillary evidence suggests that extensive gene flow and biased concerted evolution in these multicopy regions have significantly shaped the ITS data set. This argument is discussed using parsimony analysis of four data sets, constructed by combining wild sequences with those from different generations of artificial hybrids (wild + F1, F2, and backcrosses; wild + backcrosses; wild + F1; wild + F2). Compared to the F1 hybrids, F2 show a certain degree of homogenization in polymorphic sites. This effect reduces topological disruption caused by F1 and is considered to be illustrative of how extensive gene flow and biased concerted evolution may have modeled the wild ITS data. The possibility that hierarchy has arisen as a result of—or despite a significant contribution from—those two such potentially perturbing forces raises the question of what kind of signal are we recovering from this molecular data set.

New national and regional bryophyte records, 36

ub lis he d by M an ey P ub lis hi ng ( c) B rit is h B ry ol og ic al S oc ie ty Bryological Notes New national and regional bryophyte records, 36 L T Ellis, V A Bakalin, E Baisheva, H Bednarek-Ochyra, R Ochyra, E A Borovichev, S S Choi, B-Y Sun, P Erzberger, V E Fedosov, R Garilleti, B Albertos, P Gorski, P Hajkova, N G Hodgetts, M Ignatov, A Koczur, L E Kurbatova, M Lebouvier, A Mežaka, J Miravet, P Pawlikowski, R D Porley, J A Rossello, M S Sabovljevic, J Pantovic, A Sabovljevic, W Schroder, S Ştefănuţ, G M Suarez, M Schiavone, O T Yayintas, J Vaňa The Natural History Museum, London, UK, Botanical Garden-Institute, Vladivostok, Russia, Institute of Biology and Soil Science, Vladivostok, Russia, Institute of Biology of Ufa, Scientific Centre of Russian Academy of Sciences, Ufa, Russia, Institute of Botany, Poland, Polar-Alpine Botanical Garden-Institute, Kirovsk, Russia, Chonbuk National University, Jeonju, Korea, Berlin, Germany, M.V. Lomonosov Moscow State University, Russia, Universidad de Valencia, Burjassot, Spain, Poznan University of Life Sciences, Poland, Masaryk University, Brno, Czech Republic, Institute of Botany, Brno, Czech Republic, Earlish, Isle of Skye, UK, Main Botanical Garden, Moscow, Russia, Institute of Nature Conservation, Krakow, Poland, Komarov Botanical Institute, Russia, Universite de Rennes 1, France, University of Latvia, Riga, Latvia, Marimurtra Botanical Garden, Catalonia, Spain, University of Warsaw, Poland, Cerca dos Pomares, Portugal, Universidad de Valencia, Spain, University of Belgrade, Serbia, Ludwigsstadt, Germany, Institute of Biology of Romanian Academy, Bucharest, Romania, Facultad de Ciencias Naturales e I.M.L., Tucuman, Argentina, Canakkale Onsekiz Mart University, Biga, Canakkale, Turkey, Charles University, Praha, Czech Republic

Early evolutionary colocalization of the nuclear ribosomal 5S and 45S gene families in seed plants: evidence from the living fossil gymnosperm Ginkgo biloba

In seed plants, the colocalization of the 5S loci within the intergenic spacer (IGS) of the nuclear 45S tandem units is restricted to the phylogenetically derived Asteraceae family. However, fluorescent in situ hybridization (FISH) colocalization of both multigene families has also been observed in other unrelated seed plant lineages. Previous work has identified colocalization of 45S and 5S loci in Ginkgo biloba using FISH, but these observations have not been confirmed recently by sequencing a 1.8 kb IGS. In this work, we report the presence of the 45S–5S linkage in G. biloba, suggesting that in seed plants the molecular events leading to the restructuring of the ribosomal loci are much older than estimated previously. We obtained a 6.0 kb IGS fragment showing structural features of functional sequences, and a single copy of the 5S gene was inserted in the same direction of transcription as the ribosomal RNA genes. We also obtained a 1.8 kb IGS that was a truncate variant of the 6.0 kb IGS lacking the 5S gene. Several lines of evidence strongly suggest that the 1.8 kb variants are pseudogenes that are present exclusively on the satellite chromosomes bearing the 45S–5S genes. The presence of ribosomal IGS pseudogenes best reconciles contradictory results concerning the presence or absence of the 45S–5S linkage in Ginkgo. Our finding that both ribosomal gene families have been unified to a single 45S–5S unit in Ginkgo indicates that an accurate reassessment of the organization of rDNA genes in basal seed plants is necessary.

Intragenomic diversity and phylogenetic systematics of wild rosemaries (Rosmarinus officinalis L. s.l., Lamiaceae) assessed by nuclear ribosomal DNA sequences (ITS)

Nuclear ribosomal sequences (ITS) were used to study species boundaries and to infer phylogenetic patterns in wild rosemaries (Rosmarinus officinalis, R. eriocalyx, R. tomentosus). Intragenomic polymorphisms (overlapping peaks and in some cases unreadable sequences) were found throughout the sequencing electrophoretograms of most Rosmarinus accessions. Sequencing the cloned ITS products from representative individuals resulted in 25 ribotypes differing at 59 variable sites. Average sequence divergence among clones was 1.75%, and the most divergent sequences differed by 3.48%. No single ribotype was shared between any two-paired species. The highest values of intragenomic divergence were similar in R. officinalis (1.63%) and R. eriocalyx (1.14%–2.12%), and contrast with those shown by R. tomentosus (0.97%). Sequence data suggest that most divergent rDNA sequences within individuals belong to paralogous loci that apparently are not pseudogenes. A detailed inspection of direct and cloned sequences does not show evidence that the intragenomic polymorphism found is due to interspecific hybridization. Phylogenetic analyses of cloned sequences suggested that both R. officinalis and R. tomentosus were monophyletic, whereas R. tomentosus clones were nested within a paraphyletic R. eriocalyx.

A SYNOPSIS OF SILENE SUBGENUS PETROCOPTIS (CARYOPHYLLACEAE)

Petrocoptis, a small genus of chasmophytic plants endemic to the Iberian Peninsula, is included as a subgenus within a widely defined Silene. Thus, in Europe, Silene comprises subg. Silene itself, subg. Lychnis, subg. Viscaria, subg. Conoimorpha, and subg. Petrocoptis. It is anticipated that, based on nuclear rDNA sequences, S. subg. Petrocoptis will be found to be monophyletic. The bearded hilum is the only morphological synapomorphy supporting S. subg. Petrocoptis. Four species (seven taxa) are recognised within S. subg. Petrocoptis: S. glaucifolia subsp. glaucifolia and subsp. pseudoviscosa, S. laxipruinosa, S. montserratii subsp. montserratii and subsp. crassifolia, S. pardoi subsp. pardoi and subsp. guarensis. Morphological features of diagnostic value are discussed. A key and maps of the recognised taxa are provided.