Flow cytometric and cytogenetic analyses of Iberian Peninsula Festuca spp.

Festuca L. has an important diversification centre in the Iberian Peninsula. We used chromosome counting, fluorescence (FISH) and genomic in situ hybridization (GISH), and DNA flow cytometry (FCM) to clarify the taxonomic position of several taxa, to search for phylogenetic relationships and to assess the extent and pattern of genome variation in fescues. The chromosome number of Festuca duriotagana var. barbata is determined for the first time and new ploidy level estimations are given for F. rothmaleri and F. summilusitana. In the latter species, besides the reported decaploid level, dodecaploidy was found in some populations, which points to the existence of an unrecognized taxon. Moreover, these differences were confirmed by FCM and a high positive correlation was found with the type of substrate where F. summilusitana was growing. For each section, a decrease of genome size with increase of polyploidy was observed. In general, in situ hybridization techniques failed to reveal phylogenetic relationships among the selected species. In FISH, a variation in the number of rDNA sites was observed in some species. GISH results indicate that F. henriquesii is not a progenitor of the studied polyploid species.     

A new species of Haplophyllum A. Juss. (Rutaceae) from the Iberian Peninsula: evidence from morphological, karyological and molecular analyses

BACKGROUND AND AIMS: The discovery of a new species, Haplophyllum bastetanum F.B. Navarro, V.N. Suárez-Santiago & Blanca sp. nov., in the south-east of Spain has prompted the comparative study of species of the Iberian Peninsula, and others related, through morphological, cytogenetic, molecular, distributional and ecological characterization. METHODS: The morphological study involved a quantitative analysis of the species present in the Iberian Peninsula and a comparative analysis of the morphological characteristics between H. bastetanum and other related species. Mitotic analyses were made with root meristems taken from germinating seeds. Phylogenetic analyses of the internal transcribed spacer sequences of nuclear ribosomal DNA were performed using neighbour-joining (NJ) and maximum-parsimony methods. KEY RESULTS: Haplophyllum bastetanum is a diploid species (2n = 18) distinguished primarily for its non-trifoliate glabrous leaves, lanceolate sepals, dark-green petals with a dorsal band of hairs, and a highly hairy ovary with round-apex locules. The other two Iberian species (H. linifolium and H. rosmarinifolium) are tetraploid (2n = 36) and have yellow petals. Both phylogenetic methods generated a well-supported clade grouping H. linifolium with H. rosmarinifolium. In the NJ tree, the H. linifolium-H. rosmarinifolium clade is a sister group to H. bastetanum, while in the parsimony analysis this occurred only when the gaps were coded as a fifth base and the characters were reweighted according to the rescaled consistency index. This latter group is supported by the sequence divergence among taxa. CONCLUSIONS: The phylogenies established from DNA sequences together with morphological and cytogenetic analyses support the separation of H. bastetanum as a new species. The results suggest that the change in the number of chromosomes may be the key mechanism of speciation of the genus Haplophyllum in the Iberian Peninsula. An evolutionary scheme for them is propounded.     

Multivariate and cladistic analyses of the purple-flowered species ofErysimum (Cruciferae) from the Iberian Peninsula

A taxonomically difficult purple-flowered group within the genusErysimum, restricted to the Iberian Peninsula, is analyzed by multivariate and cladistic analyses. 51 specimens have been scored for 14 characters. Both principal components and discriminant analyses provide support to the recognition of the five species considered by the author, namelyE. linifolium, E. lagascae, E. baeticum, E. popovii, andE. cazorlense. Cladistic analysis, using 7 characters resulted in a single most parsimonious cladogram containing no homoplasies. The pattern of morphologic divergence follows a clear NW.-SE. trend, which is congruent with the topology of the cladogram. This trend significantly affects growth-form as well as fruit characters, both providing the main grounds for species recognition. The different behavior and significance of several characters in both kinds of analysis is discussed. The co-occurrence of morphologically similar individuals differing in the flower color is discussed, too. Possible explanations for this phenomenon involve hybridization in a wide sense or, alternatively, rejecting the assumption of monophyly for the group.     

A new species of Muscari subgenus Leopoldia from the Iberian Peninsula

The biological analysis of several populations confirms the existence in the Iberian Peninsula of two species of Muscari subgenus Leopoldia: M. comosum and M. matritensis (the latter described as new). Karyologically, they differ in at least a translocation rearrangement, in the presence of heterochromatin, which is lacking in M. matritensis, and in some chromosomal characters. At the genic level, the distance between M. comosum and M. matritensis is high (0.378). Morphologically, they show some differences in floral structure which are correlated with different reproductive systems, allogamy in M. comosum and autogamy in M. matritensis. The possible evolutionary relationships between these taxa is discussed.     

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.     

Behaviour of pollinator insects within inflorescences of Scrophularia species from Iberian Peninsula

Several hypotheses have been proposed to explain the pattern of pollinator visits to vertical inflorescences. These range from a response to a pattern of resources to merely instinctive behaviour. In dichogamous plants, such behaviour has been associated with promoting outcrossing and avoiding geitonogamy. We here analyse behaviour of the principal pollinator groups in five protogynous species of Scrophularia with different flower sizes (S. sambucifolia, S. grandiflora, S. lyrata, S. scorodonia and S. canina), and the distribution of sexual phases along the inflorescences. The results in all cases show that pollinators follow a pattern of ascending visits accompanied by movements between flowers of the same whorl (horizontal movements). The relative frequency of these horizontal movements depends on the flower size, with a higher frequency in species with large flowers. In vertical movements of the three more common pollinator groups to several plant species (bumblebees, wasps and small bees), the behaviour was essentially independent of flower size, with bumblebees having the highest ratio of ascents to descents. Behaviour of the pollinators, together with the absence of a definite pattern of distribution of the sexual phases along the inflorescence, implies that geitonogamy is not avoided in any of the Scrophularia species studied.     

The non-omphalinoid species of Arrhenia in the Iberian Peninsula

A taxonomic study of the species with nutant, pleurotoid and cyphelloid basidiomata of the genus Arrhenia in the Iberian Peninsula is presented. This study is based on the examination of recent specimens collected in the field and from dried herbarium collections. Five species and one variety of this genus are recognized on the basis of morphological and ecological features: Arrhenia acerosa var. acerosa, A. acerosa var. tenella, A. auriscalpium, A. lobata, A. retiruga and A. spathulata. Arrhenia auriscalpium is new to Spain, where it is not restricted to alpine zones of the Eurosiberian region and is more common in the Mediterranean region than previously reported. In contrast, A. acerosa var. tenella currently is known from the Eurosiberian subalpine belt and represents the first report to the Iberian Peninsula, and A. retiruga is known only from the mesomediterranean to supramediterranean belts of the Mediterranean region. Arrhenia acerosa var. acerosa, A. lobata and A. spathulata are widespread in both the Eurosiberian and the Mediterranean regions. Non-Iberian materials of A. acerosa var. tenella and A. spathulata also were studied for comparison. Cyphella cochlearis var. subsessilis, Dictyolus lagunae, Leptoglossum muscigenum var. azonum and Pleurotellus acerosus var. tenellus are lectotypified. Cyphella cochlearis var. subssesilis, Dictyolus lagunae and Leptoglossum muscigenum var. azonum are synonymized with Arrhenia spathulata, and Cyphella cochlearis var. auriformis is placed in synonymy with A. auriscalpium. These taxa are illustrated, described and discussed, based on Iberian material with emphasis on features of the basidioma, pigment of the pileipellis, presence or absence of clamps and shape and size of the basidiospores. A diagnostic key also is given.     

Flow cytometric and cytogenetic analyses in human spontaneous abortions

Cytogenetic and flow cytometric analyses were performed on 38 human spontaneous abortions in an attempt to obtain information on karyotype abnormalities and to compare the two approaches of analysis. In 19 cases, it was not possible to perform cytogenetic analysis because too long a time had passed between surgical sampling and cell culture, and in vitro culture failed. Of the 19 cases analyzed, 10/19 showed a normal karyotype and 5/19 showed a single trisomy (2/5 trisomies involved chromosome 16, 1/5 trisomy involved chromosome 18, 1/5 trisomy involved chromosome 20, and 1/5 was Klinefelter syndrome). Of the remaining 4/19 cases, 2/19 showed a polyploid condition (1 tetraploidy and 1 triploidy), 1/19 a double trisomy (chromosomes 13 and 21), and 1/19 a pentasomy of the sex chromosomes (49,XXXXY). Flow cytometric analysis was performed on all abortive samples. The samples were subdivided, when possible, into two portions conventionally named amniotic and chorionic, using the amniotic membrane as an anatomical reference. Maternal blood lymphocytes were used as a diploid standard for each sample. In the 19 cases not analyzed by the cytogenetic approach, flow cytometric analysis showed 9 diploid and 10 aneuploid DNA distributions. In the remaining 19 cases, analyzed with both approaches, the comparison of DNA estimations using cytogenetic and flow cytometric analyses showed good agreement. In the cases with karyotype abnormalities, flow cytometric measurement provided evidence of an alteration of DNA content with respect to the diploid standard. Flow cytometric analysis showed a diploid distribution, whereas cytogenetic analysis revealed chromosomal abnormalities in only 4/19 cases. These discordant results could be related to mosaic conditions or maternal cell contamination. Moreover, cytogenetic and flow cytometric analyses were performed on 2 amniotic cell cultures, and concordant results were obtained. The results obtained suggest that a combination of these techniques is beneficial in attempts to obtain information about DNA content alterations, even when cultures fail, and in screening studies of human abortions.     

Flow cytometric comparison of haemocytes from three species of bivalve molluscs

Haemocyte subpopulations from three bivalve species (the clams Ruditapes philippinarum and Mercenaria mercenaria and the oyster, Crassostrea virginica) were characterised using light-scatter flow cytometry and a standard set of methods. Two parameter (forward and side scatter) plots for the three species were very similar and resembled plots for mammalian white blood cells. Two haemocyte groups (granulocytes and agranulocytes) were found in both the haemolymph and the extrapallial fluid of the clams while those two groups and an additional third group were found in the haemolymph of the oyster. All subpopulations were sorted on to glass slides, identified, photographed, and measured microscopically. Sorting of the bivalve granulocyte and agranulocyte groups indicated varying degrees of heterogeneity within each population in terms of either size or granularity, or both. However, subsorting of selected regions within the major groupings produced highly pure haemocyte populations. The comparison showed both similarities and differences among species. For instance, a distinct subpopulation of small granulocytes was present only in oysters and a subpopulation of spindle-shaped haemocytes, only in M. mercenaria. The haemocyte subpopulations delineated by light-scatter flow cytometry underscore questions about cell lineages, but the instrument also offers a powerful technique for answering them.     

Scilla merinoi (Liliaceae), a new species from Galicia, northwestern Iberian Peninsula

A new species, Scilla merinoi , from Galicia is described and illustrated. The similarities between the new taxon and two related species, S. odorata and S. verna , are discussed. Together with a morphological comparison of the three species, scanning electron micrographs of seeds and a map of the new species' known distribution are presented.     

Contributions to the karyological study of the genus Ranunculus L. subgenus Batrachium (DC.) A Gray from the Iberian Peninsula

DIOSDADO. J. C, PASTOR, J. E. & VALDÉS, B. 1993. Contributions to the karyological study of the genus Ranunculus L. subgenus Batrachium (DC.) A. Gray from the Iberian Peninsula. The somatic and gametic chromosome numbers and detailed chromosome morphology are presented for eight taxa of aquatic ranunculi from the Iberian Peninsula. Within the subgenus Batrachium diploid and tetraploid levels have been observed (2 n = 16, 32) the commoner of which is the diploid. From the karyological data relationships have been established between the subgenus Batrachium and subgenus Ranunculus.     

Sexual reproduction in someScrophularia species (Scrophulariaceae) from the Iberian Peninsula and the Balearic Islands

The reproductive biology of someScrophularia species in the Iberian Peninsula and Balearic Islands has been studied. Levels of fruit-set in bagged and unbagged inflorescences, the production of seeds/capsule, the effectiveness of seed-set following cross-pollination in the genus and the pollen/ovule ratios have been analyzed.Some observations on floral and reproductive biology in some species ofScrophularia from the Iberian Peninsula and Balearic Islands. II. For first part seeOrtega Olivencia & Devesa 1993.     

Flow cytometric DNA analyses of epithelial dysplasia of the esophagus

OBJECTIVE: To investigate, with flow cytometry, DNA aneuploidy as a marker of early carcinogenesis in dysplastic esophageal lesions. STUDY DESIGN: DNA content of exfoliated cells from 789 cases of esophageal dysplasia (including mild dysplasia, 195 cases; moderate dysplasia, 383 cases; and severe dysplasia, 211 cases) was determined with a FACS 420 flow cytometer. RESULTS: Cellular DNA content was closely related to the severity of dysplasia. The carcinogenesis rate in patients with dysplasia showed that DNA aneuploidy was significantly higher than in patients showing DNA diploidy. CONCLUSION: DNA aneuploidy in dysplastic lesions is a very important early signal of carcinogenesis. Patients with dysplastic lesions showing DNA aneuploidy should be treated and closely followed.     

Weight–length relationships of some fish species of the Iberian Peninsula

This study reports weight–length relationships (WLR) for Salmo trutta (Linnaeus, 1758), Phoxinus phoxinus (Linnaeus, 1758), Chondrostoma Chondrostoma miegii (Steindachner, 1866), Barbus graellsii (Steindachner, 1866), Barbatula barbatula (Linnaeus, 1758) and Gobio gobio (Linnaeus, 1758). Specimens were collected by electrofishing during summer between 1996 and 1998. The b values in the WLR W = aL^b varied between 2.97 and 3.42. To the best knowledge of the authors, this study presents the first reference on WLRs for four of the species in Spanish waters.     

Floral rewards in someScrophularia species (Scrophulariaceae) from the Iberian Peninsula and the Balearic Islands

The genusScrophularia in the Iberian Peninsula and Balearic Islands comprises two sections,Scrophularia andCaninaG. Don. Analyses were carried out on flower production, flower duration and their sexual phases, pollen and nectar production together with observations on their pollinators. Nectar production is correlated with corolla size and pollen production with anther size. The taxa of sect.Scrophularia show greater nectar and pollen production than those of sect.Canina. Also, those of the first section produce more ovules per ovary than those of the second group, production being correlated with the ovary size.Some observations on floral and reproductive biology in some species ofScrophularia from the Iberian Peninsula and the Balearic Islands. I.     

Flow cytometric analyses of lectin binding to rat alveolar macrophages

The binding characteristics of ten FITC-labeled plant lectins (Con-A, MPA, BPA, PNA, WGA, SBA, UEA-I, DBA, GS-I, GS-II) to lavaged rat alveolar macrophages were assessed by flow cytometry. The alveolar macrophages (AM) were incubated with varying concentrations of each lectin in a pinocytosis-inhibiting buffer. In addition to measuring lectin-associated green fluorescence, the electronic cell volumes and axial light loss characteristics of the AM were also measured flow cytometrically. These latter parameters were found to be good indicators of cell agglutination caused by some of the lectins, and, in conjunction with green fluorescence measurements, usefully serve to determine optimal or nonagglutinating lectin concentrations for flow cytometric studies. With the exception of UEA-I, all of the lectins examined bound to AM, although a wide range of binding was observed among the lectins. At subagglutinating concentrations, Con A, MPA, BPA, PNA, WGA, SBA, and GS-I bound to the AM with unimodal patterns. Histograms of lectin-associated fluorescence intensity obtained with DBA clearly presented a pattern consistent with a more complex, bimodal distribution of labeled AM, suggesting the presence of at least two subset populations. The low-intensity distribution of AM represented congruent to 70% of the cells, while the more strongly labeled subset represented congruent to 15% of the parent AM population. The remaining balance of the AM was identified as another subpopulation by the failure to detectably bind to the DBA. While GS-II bound to all of the AM, this lectin labeled about 5% of the cells much more intensely than the bulk of the population. Thus, two subset populations of AM could be resolved according to their differing avidities for the GS-II lectin.     

Trans-atlantic distribution of a mangrove oyster species revealed by 16S mtDNA and karyological analyses

Three species of mangrove oysters, Crassostrea rhizophorae, C. brasiliana, and C. gasar, have been described along the Atlantic shores of South America and Africa. Because the distribution of these molluscs is of great biological and commercial interest, their taxonomy and distribution deserve further clarification. Therefore, 15 populations were sampled from both continents. Their 16S mitochondrial polymorphism was studied by sequencing and PCR-RFLP analysis. Two haplotypes were identified. Haplotype a was the only one observed in Africa, but it was also observed in South America together with haplotype b. Because C. gasar is the only mangrove oyster identified on the west coast of Africa, haplotype a was attributed to this species, which has thus been shown to occur in South America. Haplotype b is attributed to C. rhizophorae. The karyotypes of specimens of C. gasar, from Africa and from South America, were very similar, and both species were observed at the same location in Brazil. The occurrence of C. gasar in South America adds a third species-in addition to C. rhizophorae and C. brasiliana-to the list of species present along these coasts. The predominant surface circulation patterns in this part of the Atlantic Ocean favor the hypothesis that C. gasar was transported from Africa to America. Finally, a phylogenetic tree built with seven 16S sequences from Crassostrea and Saccostrea species showed that C. gasar is intermediate between the American Crassostrea species (C. virginica and C. rhizophorae) and the Asian species (C. gigas and C. ariakensis).     

Camelostrongylus mentulatus in domestic goats from the Iberian Peninsula

Two male worms of Camelostrongylus mentulatus were found in one of 84 murciano-granadina goats in Murcia, south-eastern Spain. This is the first report of C. mentulatus in goats in the Iberian peninsula and the possible origin and transmission of this nematode in Spain are discussed.     

Flow cytometric and morphological analyses of Pinus pinaster somatic embryogenesis

An approach combining morphological profiling and flow cytometric analysis was used to assess genetic stability during the several steps of somatic embryogenesis in Pinus pinaster. Embryogenic cell lines of P. pinaster were established from immature zygotic embryos excised from seeds obtained from open-pollinated trees. During the maturation stage, phenotype of somatic embryos was characterized as being either normal or abnormal. Based upon the prevalent morphological traits, different types of abnormal embryos underwent further classification and quantification. Nuclear DNA content of maritime pine using the zygotic embryos was estimated to be 57.04 pg/2C, using propidium iodide flow cytometry. According to the same methodology, no significant differences (P ≤ 0.01) in DNA ploidy were detected among the most frequently observed abnormal phenotypes, embryogenic cell lines, zygotic and normal somatic embryos, and somatic embryogenesis-derived plantlets. Although the differences in DNA ploidy level do not exclude the occurrence of a low level of aneuploidy, the results obtained point to the absence of major changes in ploidy level during the somatic embryogenesis process of this economically important species. Therefore, our primary goal of true-to-typeness was assured at this level.