Comparative cytogenetics between the species Passiflora edulis and Passiflora cacaoensis

Passiflora edulis Sims is the most economically important species of the genus Passiflora. A new species was described recently, Passiflora cacaoensis Bernacci & Souza, which displayed morphologic characteristics very similar to P. edulis. Due to the need for delimitation of the two species, karyomorphological and banding analyses were carried out. Both species have 2n = 18, with the same karyotype formula 16 m + 2sm. There was variation between the species regarding the location of satellites and the width of chromosome pairs 2, 4 and 8. C banding revealed the presence of constitutive heterochromatin in the centromeric and telomeric regions of all chromosomes in both species. However, only in P. cacaoensis did chromosomes 3 and 9 have a large quantity of heterochromatin. Fluorochrome banding revealed CMA⁺ bands only in the satellites, but no DAPI⁺ bands. Fluorescence in situ hybridisation (FISH) showed that in P. cacaoensis the rDNA 5S probe is located in a single site in the subterminal position of the long arm of chromosome 5. However, for the rDNA 45S probe, two sites were detected in terminal positions of the long arms of chromosome 7, with a bigger and stronger signal, and of chromosome 9. According to the asymmetry index and the quantity of heterochromatin, P. cacaoensis is a more basal species than P. edulis. The cytogenetic data indicate that P. cacaoensis is closely related to P. edulis, but is a different species.     

Sequence-based identification of species belonging to the genus Debaryomyces

In the present study we assessed the identification by sequence analysis of the 15 species belonging to the genus Debaryomyces. We found that the following species can be identified both quickly and correctly by direct sequence comparison of the ribosomal 5.8S-ITS region: D. carsonii, D. etchelsii, D. maramus, D. melissophilus, D. occidentalis and D. yamadae. In contrast, the species D. castellii, D. coudertii, D. hansenii, D. nepalensis, D. polymorphus, D. pseudopolymorphus, D. robertsiae, D. udenii and D. vanrijiae showed high sequence similarity in ribosomal regions with one or several species. In these cases, sequence comparison of the ACT1 gene is proposed to ensure unequivocal strain designation.     

Electrical DNA-chip-based identification of different species of the genus Kitasatospora

The identification of different Kitasatospora strains has been shown with a DNA-chip based on an electrical readout scheme. The 16S-23S rDNA internal transcribed spacer region of these Actinomycetes was used for identification. Two different capture probes per strain were immobilized on the chip. The capture probes were spotted on a DNA-chip with electrode structures for an electrical DNA detection. A biotinylated PCR product of the 16S-23S rDNA region was incubated on the chips and bound to its complementary capture sequences. Followed by a gold nanoparticle or enzyme labeling and a deposition of silver, the binding of the PCR product was detected by an increase of the measured conductivity on the chip. To show the applicability of this detection system, four strains of Kitasatospora were chosen for an identification using the DNA-chip with electrical detection. Each strain was clearly identified using the system. Concentrations of the polymerase chain reaction (PCR) products within the range of 1 ng/ml to 1 μg/ml were detected and identified. These tests are the first application of this novel electrical detection scheme for the identification and classification of microorganisms. The presented results show that the DNA-chip with electrical detection can be used for a robust and cost-efficient DNA analysis.     

Karyology and cytotaxonomy of the genus Passiflora L. (Passifloraceae)

 The chromosomes of 31 species of Passiflora, distributed throughout the subgenera Astrophea, Calopathanthus, Distephana, Dysosmia, Passiflora, Plectostemma and Tacsonia were analysed. Three different karyotypes were observed: 2n = 12, 24, 36; 2n = 18, 72 and 2n = 20. The karyotype of these species was almost always constituted of metacentric and submetacentric chromosomes with variable karyotype symmetry. In the group with x = 6, represented by the subgenus Plectostemma, six diploid species with 2n = 12, one tetraploid with 2n = 24 (P. suberosa) and an intraspecific polyploid with 2n = 12, 36 (P. misera) were analysed. P. pentagona (subgenus Astrophea) may also be included in this karyological group since it presents 2n = 24 and may be of polyploid origin, with x = 6. The interphase nuclei in this group were areticulate, except those of P. morifolia and P. pentagona with semi-reticulate characteristics. Two small terminal heterochromatic blocks, positive for chromomycin A₃, were identified in the largest chromosome pair of P. capsularis and P. rubra, species very closely related, while P. tricuspis displayed four chromosomes with proximal blocks. In the group with x = 9, represented mainly by subgenus Passiflora, 20 species with 2n = 18 and one with 2n = 72 were studied. They presented chromosomes larger than those species with x = 6 and interphase nuclei of semi-reticulate type, except for P. mixta with areticulate nuclei. Four terminal CMA⁺ blocks were observed in P. edulis, six blocks in P. caerulea and P. racemosa, while five blocks were observed in the single P. amethystina plant analysed. P. foetida (subgenus Dysosmia), the only species with 2n = 20, exhibited six chromosomes with CMA⁺ blocks and interphase nuclei of the areticulate type. The meiotic analysis of representatives of the three groups (P. foetida, P. suberosa, P. cincinnata and P. racemosa) always presented regular pairing and regular chromosome segregation, except in P. jilekii where a tetravalent was observed. The analysis of the chromosome variation within the genus and the family suggests that the base number of Passiflora may be x₁ = 6 or x₁ = 12, whereas x₂ = 9 is only an important secondary base number. Received April 11, 2000 Accepted October 5, 2000     

Passiflora tacanensis, a new species of Passiflora subgenus Decaloba supersection Cieca from Mexico

Passiflora tacanensis, a new species from southern Mexico, is described and illustrated. It was discovered during the course of a taxonomic revision ofPassiflora subgenusDecaloba supersectionCieca and is assigned to that group of apetalous passion flowers.Passiflora tacanensis is known only from one fruiting herbarium specimen that was collected in southern Mexico on Volcán Tacaná in a high mesophytic forest. It is distinguished from its closest relatives,P. coriacea, P. sexocellata, P. mcvaughiana, andP. xiikzodz, by its foliose stipules, leaves that lack laminar nectaries but possess petiolar nectaries, and wide seeds with a chalazal beak that is not well developed.     

Genetic identification of three species of the genus Clarias using allozyme and mitochondrial DNA markers

Genetic variability in three clariid species, Clarias batrachus, C. gariepinus and C. macrocephalus, were investigated by allozyme electrophoresis and mitochondrial DNA (ND 5/6) RFLP markers. The catfish species C. batrachus and C. macrocephalus are native to the Indian subcontinent and Southeast Asia, respectively, while the African catfish C. gariepinus was introduced to Asian countries for culture. Sixteen gene loci from 12 enzyme systems were analyzed. Fixed allelic differences were evident between pairs of species at least at four loci. In mtDNA RFLP analysis, eight composite haplotypes were observed and each species was characterized by a set of haplotypes. The UPGMA phylogenetic tree revealed three distinct clusters: C. batrachus; C. gariepinus (India and Thailand); and C. macrocephalus.     

DNA barcoding and microsatellites help species delimitation and hybrid identification in endangered galaxiid fishes

The conservation of data deficient species is often hampered by inaccurate species delimitation. The galaxiid fishes Aplochiton zebra and Aplochiton taeniatus are endemic to Patagonia (and for A. zebra the Falkland Islands), where they are threatened by invasive salmonids. Conservation of Aplochiton is complicated because species identification is hampered by the presence of resident as well as migratory ecotypes that may confound morphological discrimination. We used DNA barcoding (COI, cytochrome b) and a new developed set of microsatellite markers to investigate the relationships between A. zebra and A. taeniatus and to assess their distributions and relative abundances in Chilean Patagonia and the Falkland Islands. Results from both DNA markers were 100% congruent and revealed that phenotypic misidentification was widespread, size-dependent, and highly asymmetric. While all the genetically classified A. zebra were correctly identified as such, 74% of A. taeniatus were incorrectly identified as A. zebra, the former species being more widespread than previously thought. Our results reveal, for the first time, the presence in sympatry of both species, not only in Chilean Patagonia, but also in the Falkland Islands, where A. taeniatus had not been previously described. We also found evidence of asymmetric hybridisation between female A. taeniatus and male A. zebra in areas where invasive salmonids have become widespread. Given the potential consequences that species misidentification and hybridisation can have for the conservation of these endangered species, we advocate the use of molecular markers in order to reduce epistemic uncertainty.     

Identification of the pattern of heterochromatin distribution in Passiflora species with C-banding

We tested four C-banding protocols to obtain heterochromatic bands in the passion fruit species Passiflora edulis and P. cacaoensis (Passifloraceae). Three of these protocols had been previously described. The three published protocols were not adequate to obtain C-bands in these species. An adapted protocol demonstrated heterochromatin distribution in metaphasic chromosomes of species of Passiflora for the first time. The differentiated coloration for C-bands was obtained with immersion of the slides in 99% ethanol, 45% acetic acid (additional step), 0.2 N hydrochloric acid, hydroxide of barium, 45% acetic acid, and 2X standard saline citrate at four different temperatures. The C-bands were observed in the satellites and in the telomere and centromere regions of all chromosomes, both in P. edulis and in P. cacaoensis.     

A Chromosome study of four species and a hybrid of the killifish genus Fundulus (Cyprinodontidae)

The chromosomes of four killifish species (F. diaphanus, F. heteroclitus, F. majalis and F. parvipinnis) and a hybrid (F. diaphanus × F. heteroclitus) were studied. The karyotype studies were based on observations from both squash and air-dry preparations, the latter of which were prepared from in vitro primary cell cultures from ovaries. The consistent presence of one to several pairs of species-specific marker chromosomes, and the differential expression of acrocentric short arms in karyotypes of different species were useful in determining interspecific relationships. These characters did not show any noticeable variation interand intra-individually, and retained their original forms even in the hybrid complement which was the composite of chromosome complements of both parental species. Male heterogamety was confirmed in F. diaphanus and F. parvipinnis in which the X chromosomes resemble each other being submetacentric and having an identifiable secondary constriction. The occurrence of the cytological heterogamety in fishes is briefly discussed.     

Co-occurrence of Valine/Isoleucine-derived and cyclopentenoid cyanogens in a Passiflora species

The valine/isoleucine-derived cyanogenic glycosides linamarin and lotaustralin have been isolated together with the cyclopentenoid cyanogen passibiflorin from Passiflora lutea. This is only the second report of the production of cyanogenic glycosides from more than one biosynthetic pathway in individuals of a single species.     

Passiflora linda,a new species from southern Ecuador

A new species ofPassiflora subgenusTacsonia from the province of Zamora-Chinchipe, Ecuador, is described. The species is illustrated and its affinities to other species within the subgenus are discussed.     

Flow cytometric analysis of genome size variation in some Passiflora species

Nuclear genome size variation was studied in eight taxa of Passiflora. Nuclear DNA content was estimated by flow cytometry of nuclei stained by propidium iodide. 2C DNA content ranged from 3.16-5.36 pg for diploids and 1.83 pg for tetraploid. Differences in nuclear genome size were observed among Passiflora species (pg): P. suberosa 1.83, P. edulis f. edulis 3.16, P. edulis f. flavicarpa (Brazil) 3.19, P. edulis f. flavicarpa (Mexico) 3.21, P. mucronata 3.40, Passiflora edmundoi 3.43, P. laurifolia 3.88, P. giberti 3.92, P. quadrangularis 5.36, the largest value being up to 192% greater than the smallest. The means of 2C DNA content were compared by the Tukey test, and the differences in genome size permitted the recognition of five taxa groups. The result was the same for the means 2C genome size (Mbp) values. The genetic parameters were studied with their respective estimators, phenotypic variance (sigma2F), genotypic variability (PhiG), and the genotypic determination index (H2). The genotypic determination index presented high magnitude estimates (greater than 99%) emphasizing the reliability of the results and demonstrating the efficiency of determining the DNA content in the species using only one leaf per plant. Passiflora species show great phenotypic variability and have different geographic distribution that might implicate in genetic diversity.     

Studies in Brazilian Passifloraceae III. A new species of Passiflora

A new species ofPassiflora from Minas Gerais, Brazil, is described and namedP. saccoi. This species belongs to subgenusPassiflora seriesLobatae harms because of its lobed leaves, foliaceous, semi-ovate to semi-oblong stipules that are attached on one side above base and, hence, often appear sub-reniform, and foliaceous, verticillate bracts borne near the apex of the peduncle.     

Skin peptide and cDNA profiling of Australian anurans: genus and species identification and evolutionary trends

Host defense peptides of 35 species of Australian frogs from the hylids Cyclorana and Litoria, and the myobatrachids Crinia, Limnodynastes and Uperoleia have been identified. The biological activities of the majority of these peptides have been determined and include hormones, neuropeptides, opioids, immunomodulators, membrane active peptides [including antimicrobial, anticancer, antiviral (enveloped viruses like HIV and Herpes) and antifungal peptides], neuronal nitric oxide synthase inhibitors, pheromones and individual peptides with other specific activities. The host defense peptide skin profile can be diagnostic at both the species and higher taxonomic levels; for example, species of Crinia, Litoria and Uperoleia each produce quite different types of peptides. Species of Cyclorana and Limnodynastes are more difficult to characterize by skin peptides alone: species of both genera produce similar peptides with no apparent activity. The skin peptide profiles of frogs from the genera Crinia, Litoria and Uperoleia may be used together with morphological and cognate methods, to differentiate between sub-species and even different population clusters of the same species. Nucleotide sequencing of cDNAs of precursors (pre-pro peptides) of bioactive peptides from the skin glands of various species of the genus Litoria show that the majority of these peptides originated from a single ancestor gene before the break away of Australia from Gondwana. The exceptions are the caerulein neuropeptides {e.g. caerulein [pEQDY(SO₃H)TGWMDF(NH₂)]} which have a different origin to that of other Litoria peptides. Disulfide containing peptides from skin glands of species of Crinia show a different evolutionary route to peptides from species of Litoria.