Classical and molecular cytogenetics of Atherinella brasiliensis (Teleostei, Atheriniformes) from South coast of Brazil

Cytogenetic studies were performed on specimens of Atherinella brasiliensis from Laranjeiras Bay (Paraná State, Brazil). All specimens had a diploid number of 48 chromosomes, with a karyotype constituted by 4m+14sm+18st+12a and fundamental number of 84. The C-positive heterochromatin was distributed over the nucleolar organizer regions (NORs) in the centromeric regions and on short arms of metacentric and submetacentric chromosomes. Most of this heterochromatin was AT-rich, except in the NORs, which were rich in GC, as detected by double staining with chromomycin A₃/4'-6-diamin-2-phenylindole. Single NORs were located at terminal positions of a submetacentric pair, as confirmed by fluorescent in situ hybridization with 18S rDNA probes. Both techniques showed a size heteromorphism between the homologous chromosomes. The 5S rDNA clusters were located in terminal positions on two chromosomal pairs and also displayed a size heteromorphism. Despite the conserved diploid number, the data on the karyotype microstructure help characterize the cytogenetic profile of this group.     

Frequency and direction of hybridization in sympatric populations of Pinus taeda and P. echinata (Pinaceae)

Two naturally occurring, sympatric, northern Georgia populations of Pinus taeda L. (loblolly pine) and P. echinata Mill. (shortleaf pine) were examined with respect to genetic diversity within populations and the frequency, spatial distribution, and morphology of putative hybrids. Shortleaf pine predominated at the "road" site while loblolly pine predominated at the "granite outcrop" site. Hybrid individuals were identified by their IDH allozyme genotype, the only such locus known to be fixed for different alleles in the two species. All allozymatically detectable hybrids (34 at the road site and two at the granite outcrop site) were juveniles that were distributed in open, sunny patches. A similar pattern of recruitment was seen for juveniles of the parental species. Hybrids were spatially distant from mature shortleaf pine, suggesting that shortleaf pine was not the seed parent. Discriminant analysis on needle characteristics indicated that loblolly pine was easily distinguished from shortleaf pine and the hybrids, but that shortleaf pine and the hybrids were barely distinguishable from each other. A diagnostic cpDNA restriction site marker indicated that shortleaf pine sired all the hybrids at both sites. No evidence of later generation hybridization was found.     

Improving vigour assessment of pine (Pinus nigra Arnold) seedlings before their use in reforestation

ABSTRACT

We investigated whether changes in the root system of pine seedlings induced by stress (lifting of bare-root seedlings from the nursery bed irrespective of dormancy; prolonged storage of bare-root seedlings in a cold room) could provide a measure of plant vigour. Physiological parameters, such as growth potential and root electrolyte leakage, and morphological parameters, such as root length and number of root tips, were calculated. Computerised image analysis was used to measure root growth, overall and based on root-diameter class (0–0.5 mm, 0.5–1.0 mm and 1.0–1.5 mm). The efficiency of vigour assessment was evaluated by correlating the data for each parameter with percentage seedling survival. Root growth potential was more efficient than root electrolyte leakage, but both parameters were affected by seedling age. Total root length was a more efficient indicator of plant vigour than root tip number, particularly when referred to roots of the same diameter class. A comparative analysis of physiological and morphological parameters referred to the root systems improves their relative effciency.     

Comparative cytogenetics among populations of Astyanax altiparanae (Characiformes, Characidae, Incertae sedis)

Cytogenetic data are presented for Astyanax altiparanae populations from three Brazilian hydrographic systems. The chromosomal data obtained in A. altiparanae support the hypothesis of diploid number conservation. However, small differences in the karyotype formula and number of nucleolar organizer regions were observed in these populations. The apparent karyotypical similarity among the studied populations strongly suggests a close relationship among them with some chromosomal divergences due to gene flow restriction.     

Chloroplast DNA supports a hypothesis of glacial refugia over postglacial recolonization in disjunct populations of black pine (Pinus nigra) in western Europe

European black pine (Pinus nigra Arn.) is a widely distributed Mediterranean conifer. To test the hypothesis that fragmented populations in western Europe survived in situ during the last glacial rather than having been re-colonized in the postglacial period, genetic variation was assessed using a suite of 10 chloroplast DNA microsatellites. Among 311 individuals analysed, 235 haplotypes were detected revealing high levels of chloroplast haplotype diversity in most populations. Bayesian analysis using a model of linked loci, with no prior assumption of population structure, assigned individuals to 10 clusters that corresponded well with the six predefined sampling regions, while an analysis carried out at the population level and assuming unlinked loci, recovered the original six sampling regions. This regional structure was supported by a biogeographical analysis that detected five barriers, with the two most significant separating Alps from Corsica and southern Italy, and southern Spain from the Pyrenees. No signals of demographic expansion were detected, and comparisons of R(ST) with pR(ST) suggested that a stepwise mutational model was important in regional differentiation, but not in population-within-region differentiation. These tests support long-term persistence of the species within the six regions. The temporal depth estimate, assuming a high mutation rate in coalescent modelling, placed the deepest split between the Alps and the other regions at about 150 000 years ago, and the most recent split of Pyrenees from southern France at about 30 000 years ago. Taken together, the data suggest that chloroplast DNA is structured in black pine and disjunct populations in western Europe are likely to have been present during the Last Glacial Maximum.     

Chemical composition and antimicrobial activity of the essential oil of endemic Dalmatian black pine (Pinus nigra ssp. dalmatica)

The chemical composition and the antimicrobial activity of the essential oil isolated from the needles of endemic Dalmatian black pine (Pinus nigra ssp. dalmatica) from Croatia were investigated. The chemical composition of the essential oil was determined by GC and GC/MS analyses, and the main compounds identified were α-pinene, β-pinene, germacrene D, and β-caryophyllene. Disc-diffusion and broth-microdilution assays were used for the in vitro antimicrobial screening. The Dalmatian black pine essential oil exhibited a great potential of antibacterial activity against Gram-positive bacteria (MIC=0.03-0.50% (v/v)) and a less pronounced activity against Gram-negative bacteria (MIC=0.12-3.2% (v/v)). The volatile compounds also inhibited the growth of all fungi tested, including yeast.     

Genomic relationships within Boronia (Rutaceae) as revealed by karyotype analysis and RAPD molecular markers

 The genus Boronia Sm. section Boronia series Boronia contains species with n=7 (B. megastigma), n=7 or 8 (B. heterophylla), n=8 (B. molloyae) and n=9 (B. purdieana), representing ideal species with which to examine comparative chromosome morphology. Between species there were few chromosomes with similar morphology, indicating numerous genome re-organisations. Karyotypes between and within species of Boronia could be distinguished and inheritance of some chromosomes was observed. Species and hybrids with 2n = 14 or 15 had at least one large chromosome. Chromosome morphology indicated a closer relationship between B. heterophylla and B. molloyae and between B. purdieana and B. megastigma than between these two groups. Whole genomic DNA was extracted from 9 genotypes of Boronia. RAPD bands were analysed and pairwise distance matrices between genotypes were computed. Dendrograms were generated and analysed using unweighted pair-group method with arithmetic average cluster analysis. Dendograms supported cytological results, indicating B. heterophylla and B. molloyae are closely related and clearly distinct from B. megastigma and B. purdieana. The evolution of boronias is discussed. Received February 16, 2001; accepted March 21, 2002 Published online: October 14, 2002 Address of the authors: G. Yan, F. Shan, J. A. Plummer (e-mail: jplummer@cyllene.uwa.edu.au), Plant Sciences, Faculty of Natural and Agricultural Science, The University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009, Australia.     

Monitoring the physiological activity of plants by means of EPR spectroscopy. Mn(II) signals in Pinus nigra Arnold

Differently aged needles from a Pinus nigra Arnold tree growing in a typical urban area have been examined by means of electron paramagnetic resonance (EPR) spectroscopy, and the variation of the observed six-line Mn (II) signal was monitored for 1 year. An inverse trend has been established between the photosynthetic efficiency of the plant and the measured content of Mn (II). The possibility of using EPR spectroscopy in studying ageing and in assessing stress situations in plants is considered. Received: 23 April 1999 / Accepted: 13 January 2000     

The Influence of the urban environment on the composition of terpenes in the needles of Black Pine (Pinus nigra Arnold)

 The composition of terpenoid substances in needles of Pinus nigra Arnold with respect to air pollution is discussed. Trees grown in the urban environment of the city of Nitra, which is contaminated by immissions, and trees grown in the relatively clean environment of the Mlyňany Arboretum SAS were investigated. The urban environment caused a change in the species composition and the content of terpenes. In the city of Nitra needles of P. nigra contained a smaller number of terpenoid substances (12 terpenes) in comparison with the Arboretum (15 terpenes). At Nitra P. nigra contained four terpenes with a relative portion above 1% and at the Mlyňany Arboretum P. nigra needles contained six terpenes above 1%. At the polluted site citral, eugenol, and bisabolol are completely missing in the terpene pattern of needles and myrcene, carvone and alfa-humulene were notably lower. The decrease of the alfa-pinene content and the increase of the beta-pinene content in needles from Nitra significantly lowered the ratio of alfa-pinene to beta-pinene from 2.80 to 0.86. Received: 27 October 1995 / Accepted: 29 March 1996     

Genetic diversity and phylogenetic relationships among five endemic Pinus taxa (Pinaceae) of China as revealed by SRAP markers

The genetic diversity and phylogenetic relationships among five endemic Pinus taxa of China (Pinus tabulaeformis, P. tabulaeformis var. mukdensis, P. tabulaeformis f. shekanensis, Pinus massoniana and Pinus henryi) were studied by SRAP markers. Using 10 SRAP primer pairs, 247 bands were generated. The percent of polymorphic bands (94.8%), Nei's genetic diversity (0.2134), and Shannon's information index (0.3426) revealed a high level of genetic diversity at the genus-level. At the taxon level, P. tabulaeformis f. shekanensis and P. henryi showed a higher genetic diversity than the others. The coefficient of genetic differentiation among taxa (0.3332) indicated a higher level of genetic diversity within taxon, rather than among taxa. An estimate of gene flow among taxa was 1.0004 and implied a certain amount of gene exchange among taxa. The results of neighbor-joining cluster analysis and principal co-ordinate analysis revealed that P. tabulaeformis, P. tabulaeformis var. mukdensis and P. tabulaeformis f. shekanensis were conspecific, which was in agreement with the traditional classification. Phylogenetic relationships analysis also indicated that P. henryi might be a distinct species closely related to P. tabulaeformis.     

Variation in the nuclear ribosomal DNA internal transcribed spacer (ITS) region of Pinus rzedowskii revealed by PCR-RFLP

 In the genus Pinus the internal transcribed spacers (ITS1 and ITS2) and the 5.8s region of the nuclear ribosomal DNA are approximately 3000 bp in length. ITS1 is considerably longer than ITS2 and partial sequences of ITS1 indicate that this region is evolving rapidly and exhibits intraspecific variation. The ITS2 and 5.8s regions are relatively conserved. We surveyed restriction fragment length variability of PCR-amplified fragments (PCR-RFLP) of the ITS region in four populations (86 individuals) of Pinus rzedowskii, a pine endemic to western Michoacán, Mexico. Five of the restriction endonucleases assayed revealed variation, with a total of 13 variants, most of which were length mutations of 300–900 bp. A moderate degree of population differentiation was detected. The average diversity (Shannon’s index) of ITS fragment size patterns was 1.19, with 34% of the variation due to differences among populations and 66% due to differences among individuals within populations. The same individuals were assayed for nine polymorphic isozymes, which gave diversity measures similar to those of each restriction endonuclease. Received: 25 August 1997 / Accepted: 19 September 1997     

Genetic structure of hybrid zones between Pinus pumila and P. parviflora var. pentaphylla (Pinaceae) revealed by molecular hybrid index analysis

Pinus species have three differently inherited genomes: paternal chloroplast, maternal mitochondrial, and biparental nuclear. Our previous study on the hybrid zones between alpine Pinus pumila and montane to subalpine P. parviflora var. pentaphylla demonstrated contrasting patterns of introgression of two cytoplasmic genomes, i.e., the paternal cpDNA flowed from P. parviflora var. pentaphylla to P. pumila, and the maternal mtDNA flowed in the reverse direction. In the present study, we developed codominant nuclear DNA markers diagnostic or mostly diagnostic for each parental species by single-strand conformation polymorphism (SSCP) of polymerase chain reaction (PCR) products, using expressed sequence tag (EST) primers of Pinus taeda. To describe the introgressive patterns of the nuclear genes, the molecular hybrid index (MHI) showing the overall proportion of alleles inferred to be derived from P. pumila was determined for each plant collected in hybrid zones on Mt. Asahidake and Mt. Higashiazuma, Japan. At Mt. Asahidake, the MHI values changed clinally according to the altitudes at which the plants were collected. However, at Mt. Higashiazuma, there was a gap in the MHI values between the plants above and below the Abies and Tsuga forest zone (alt. 1800-1900 m). This suggested that the zone plays a role in creating an effective barrier to gene flow in the hybrid zone.     

Diversity and genetic differentiation among populations of Indian and Kenyan tea (Camellia sinensis (L.) O. Kuntze) revealed by AFLP markers

AFLP markers were successfully employed to detect diversity and genetic differentiation among Indian and Kenyan populations of tea (Camellia sinensis (L.) O. Kuntze). Shannon's index of diversity was used to partition the total phenotypic variation into between and within population components. On average, most of the diversity was detected within populations, with 79% of the variation being within and 21% being between populations of Indian and Kenyan tea. A dendrogram constructed on the basis of band sharing distinctly separated the three populations of tea into China type (sinensis), Assam type (assamica) and Cambod type (assamica ssp. lasiocalyx) in a manner consistent with the present taxonomy of tea, the known pedigree of some of the genotypes and their geographical origin. Principal coordinate (PCO) analysis grouped Assam genotypes both from India and Kenya supporting the suggestion that the Kenyan clones have been derived from collections made in this region. The China types were more dispersed on the PCO plot which is a reflection of wider genetic variation. As would be expected, clones collected from the same region exhibited less overall genetic variation. AFLP analysis discriminated all of the tested genotypes from India and Kenya, even those which cannot be distinguished on the basis of morphological and phenotypic traits. Received: 2 May 1996 / Accepted: 14 June 1996     

Analysis of volatiles from black pine (Pinus nigra): significance of wounding and egg deposition by a herbivorous sawfly

The composition of headspace volatiles of black pine (Pinus nigra) was analysed by coupled gas chromatography-mass spectrometry (GC-MS). It has been shown in a previous study that egg deposition of the sawfly Diprion pini on P. sylvestris induces a quantitative change of the pine volatile blend . Chrysonotomyia ruforum, an egg parasitoid of D. pini, is known to be attracted by volatiles from egg-carrying P. sylvestris, but not by odour from egg-laden P. nigra. Therefore, the present study focused on the question whether also P. nigra as another host plant of this sawfly responds to egg deposition by change of its volatile blend. The headspace of untreated, egg-carrying, and artificially wounded P. nigra twigs were compared. The artificial damage inflicted to the twigs mimicked the damage by the sawfly female prior to egg deposition. Thirty five mainly terpenoid compounds that were identified in more than 50% of the egg-carrying P. nigra twigs could also be detected in the headspace of untreated and artificially wounded twigs. Quantitative differences of the blends of volatiles from differently treated P. nigra twigs were compared by multivariate data analyses. PLS-DA (projection to latent structures-discriminant analysis) revealed that blends of volatiles from differently treated P. nigra differed significantly. When comparing volatiles from egg-carrying and artificially wounded P. nigra with respective P. sylvestris samples, qualitative and quantitative differences were detected. The differences in volatile composition of P. nigra and P. sylvestris are discussed with special respect to the egg parasitoid's response to odours of egg-carrying pine twigs.     

Marked genetic divergence revealed by allozymes among populations of the guppy Poecilia reticulata (Poeciliidae), in Trinidad

Populations of the guppy Poecilia reticulata from six locations in N. Trinidad were examined by starch gel electrophoresis to estimate their degree of genetic divergence. Variability at seven enzyme-coding loci demonstrated that populations differed markedly in allele frequencies with some allelic substitution between sites (for 23 loci, Nei's mean genetic identity, Ī ranged from 0.869–1.00; the coefficient of gene differentiation, G _ST= 0.086; and the absolute differentiation between populations, _m= 0.044). There was a good correspondence between degree of physical isolation and extent of genetic differentiation, as exemplified by the partitioning of gene diversity (Nei's gene diversity analysis = 66% between river basins; 32% within river basins; 2% within rivers), though there was considerable variability in the contribution of individual loci. Most populations were in Hardy-Weinberg equilibrium, and any significant deviations were due exclusively to heterozygote deficiencies. The patterns of population differentiation are discussed in relation to probable past geological and historical events, and present-day evolutionary forces. Notable among these are previous continental land connections, periodic immigration from N.E. South America, and post-colonization events including differential predation, sexual selection, apparent restricted vagility and small effective population sizes.     

Genetic diversity and structure of Manila clam (Ruditapes philippinarum) populations from Liaodong peninsula revealed by SSR markers

Manila clam (Ruditapes philippinarum) is an important commercially marine bivalve, and its wild populations have been severely declining in the coast of China during the last decade. In this study, a set of 7 genomic simple sequence repeat (SSR), and 5 expressed sequences tag (EST)-derived SSR markers were analyzed on eight wild R. philippinarum populations to assess the genetic diversity and population differentiation. A total of 114 alleles were detected on 12 loci, and the number of alleles per locus in each population ranged from 2 to 11, and allelic richness per locus varied from 2.00 to 10.88 for each sample. The average of observed and expected heterozygosity ranged from 0.386 to 0.550, and from 0.533 to 0.707, respectively. Pairwise F_ST values indicated that all population pairs had significant genetic differentiation (overall F_ST = 0.242, P < 0.01). Cluster analysis using unweighted pair group method with the arithmetic mean (UPGMA) separated the eight populations into two groups. This study will shed light on the domestication and cultivation on population genetic diversity of R. philippinarum, and also provide the foundation for conservation of R. philippinarum germplasm resources in clam breeding practices.     

Genetic divergence in the Atlantic-Mediterranean Montagu's blenny, Coryphoblennius galerita (Linnaeus 1758) revealed by molecular and morphological characters

Coryphoblennius galerita is a small intertidal fish with a wide distribution and limited dispersal ability, occurring in the northeastern Atlantic and Mediterranean. In this study, we examined Atlantic and Mediterranean populations of C. galerita to assess levels of genetic divergence across populations and to elucidate historical and contemporary factors underlying the distribution of the genetic variability. We analyse three mitochondrial and one nuclear marker and 18 morphological measurements. The combined dataset clearly supports the existence of two groups of C. galerita: one in the Mediterranean and another in the northeastern Atlantic. The latter group is subdivided in two subgroups: Azores and the remaining northeastern Atlantic locations. Divergence between the Atlantic and the Mediterranean can be the result of historical isolation between the populations of the two basins during the Pleistocene glaciations. Present-day barriers such as the Gibraltar Strait or the 'Almeria-Oran jet' are also suggested as responsible for this isolation. Our results show no signs of local extinctions during the Pleistocene glaciations, namely at the Azores, and contrast with the biogeographical pattern that has been observed for Atlantic-Mediterranean warm-water species, in which two groups of populations exist, one including the Mediterranean and the Atlantic coast of western Europe, and another encompassing the western tropical coast of Africa and the Atlantic islands of the Azores, Madeira and Canaries. Species like C. galerita that tolerate cooler waters, may have persisted during the Pleistocene glaciations in moderately affected locations, thus being able to accumulate genetic differences in the more isolated locations such as the Azores and the Mediterranean. This study is one of the first to combine morphological and molecular markers (mitochondrial and nuclear) with variable rates of molecular evolution to the study of the relationships of the Atlantic and Mediterranean populations of a cool-water species.     

Long-distance colonization,isolation by distance,and historical demography in a relictual Mexican pinyon pine (Pinus nelsonii Shaw) as revealed by paternally inherited genetic markers (cpSSRs)

Pinus nelsonii is a relictual pinyon pine distributed across a wide altitudinal range in semiarid zones in Mexico near the border between the States of Nuevo León and Tamaulipas. It also occurs in small patches in the State of San Luis Potosí. Pinus nelsonii is classified in the monotypic subsection Nelsoniae, separated from other pinyon pines (subsection Cembroides), because it possesses several distinctive characters including persistent fascicle sheaths, connate needles, and a distinctive wood anatomy. In the present study, chloroplast simple sequence repeats (cpSSRs) were used to estimate genetic variation in most known populations (nine) of P. nelsonii. The genetic variation (HT = 0.73; 27 haplotypes in 256 individuals) is moderate when compared to other pine species. Population differentiation ranged between low and moderate (FST = 0.13 and RST = 0.05), as did the Nei and Goldstein genetic distances between populations. However, this pattern varied depending on whether the infinite alleles or stepwise mutation model was used. In the former case a significant isolation by distance was found, but not in the latter. A significant association between geographical and genetic structure in one clade, through a nested clade analysis, was found, which suggested long-distance colonization between 125000 and 309000 years ago. We found weak evidence for a population expansion. A mismatch distribution suggests that P. nelsonii populations underwent an expansion 4.25 times their size between 59000 and 146000 years ago. On the other hand, the populations' star-like phylogeny and a slight parabolic relationship between coalescence times and lineage number also suggest weak population expansion. Overall, this species appears to have been in demographic stasis for a large proportion of the time detected by the markers used.     

Using chromosomal data in the phylogenetic and molecular dating framework: karyotype evolution and diversification in Nierembergia (Solanaceae) influenced by historical changes in sea level

Karyotype data within a phylogenetic framework and molecular dating were used to examine chromosome evolution in Nierembergia and to infer how geological or climatic processes have influenced in the diversification of this solanaceous genus native to South America and Mexico. Despite the numerous studies comparing karyotype features across species, including the use of molecular phylogenies, to date relatively few studies have used formal comparative methods to elucidate chromosomal evolution, especially to reconstruct the whole ancestral karyotypes. Here, we mapped on the Nierembergia phylogeny one complete set of chromosomal data obtained by conventional staining, AgNOR‐, C‐ and fluorescent chromosome banding, and fluorescent in situ hybridisation. In addition, we used a Bayesian molecular relaxed clock to estimate divergence times between species. Nierembergia showed two major divergent clades: a mountainous species group with symmetrical karyotypes, large chromosomes, only one nucleolar organising region (NOR) and without centromeric heterochromatin, and a lowland species group with asymmetrical karyotypes, small chromosomes, two chromosomes pairs with NORs and centromeric heterochromatin bands. Molecular dating on the DNA phylogeny revealed that both groups diverged during Late Miocene, when Atlantic marine ingressions, called the ‘Paranense Sea’, probably forced the ancestors of these species to find refuge in unflooded areas for about 2 Myr. This split agrees with an increased asymmetry and heterochromatin amount, and decrease in karyotype length and chromosome size. Thus, when the two Nierembergia ancestral lineages were isolated, major divergences occurred in chromosomal evolution, and then each lineage underwent speciation separately, with relatively minor changes in chromosomal characteristics.