Comparative analysis of the sperm ultrastructure of three species of Phyllomedusa (Anura, Hylidae)

We describe the sperm ultrastructure of three species of frogs in the genus Phyllomedusa . According to micrographs, total size of the spermatozoon of Phyllomedusa hypochondrialis is significantly smaller than those of Phyllomedusa bicolor and Phyllomedusa tarsius . The acrosome complex consists of two conical structures covering the nucleus, the acrosome vesicle and the subacrosomal cone. The subacrosomal cone of P. bicolor and P. tarsius is less electron-dense and appears more granular in transverse section than in P. hypochondrialis . In P. bicolor and P. tarsius , the nuclear space is reduced and the subacrosomal cone fills most of the space between the acrosome vesicle and nucleus. The anterior region of the nucleus in the spermatozoa of P. bicolor and P. tarsius ends abruptly, while in P . hypochondrialis it is sharp-ended. In P. bicolor and P. tarsius , the axial fibre is much larger than in P . hypochondrialis . The sperm ultrastructure of Phyllomedusa appears conservative at the intrageneric level. Future studies on the sperm ultrastructure of hylids can provide new insights on the systematics of the group and a larger database for a cladistic analysis.     

In situ chromosomal localization of rDNA sites in "Safed Musli" Chlorophytum ker-gawl and their physical measurement by fiber FISH

Fluorescence In Situ Hybridization (FISH) technique has been applied on somatic chromosomes and extended DNA fibers in the medicinally important species of Chlorophytum to elucidate physical localization and measurement of the rDNA sites using two rRNA multigene families homologous to 45S and 5S rDNA. The two species of Chlorophytum, namely C. borivillianum and C. comosum, both with 2n = 28, reveal diversity for copy number and localization of rDNA sites. C. borivillianum is comprised of five 45S-rDNA sites:one each in the secondary constriction region of chromosomes 7, 8, 9; one in the subtelomeric region of the short arm of chromosome 2 and the telomeric region of the short arm of chromosome 12; and one 5S-rDNA site in the subtelomeric region of the long arm of chromosome 1. In C. comosum, there are three 45S-rDNA sites (one each in the short arm of chromosomes 12, 13, and 14) and two 5S-rDNA sites (in the secondary constriction regions of chromosomes 2 and 13). Fiber FISH analysis conducted on extended DNA fibers revealed variation in the size of continuous tandem strings for the two r-DNA families. Taking the standard value of native B DNA equivalent to 3.27 kb for 1 mum, it was estimated that the physical size of continuous DNA strings is of the order of approximately 90 kb, 180 kb, and 300 kb for 45S-rDNA and of the order of 60 kb, 150 kb for 5S-rDNA in C. comosum, grossly in correspondence to their respective physical sizes at metaphase.     

Karyosystematic study of Potentilla L. subgen. Potentilla (Rosaceae) in the Iberian Peninsula

Chromosome numbers in 80 populations belonging to 18 species of Potentilla L. subgen. Potentilla from the Iberian Peninsula and two of P. maura, a North African endemic taxon, have been counted. The basic number of chromosomes is always x = 7 and these chromosomes are small (between 1 and 2 μm). For three species, the number of chromosomes is reported for the first time and, for another six, this number has been established in Iberian representatives. Moreover, new ploidy levels have been obtained for P. hispanica and P. crantzii with regard to their entire distribution area, and in P. cinerea and P. neumanniana for the Iberian Peninsula. Some taxonomic, phylogenetic and phytogeographic comments are made for several species or groups of species from the West Mediterranean region. In 13 species only one ploidy level has been found, but six species have several ploidy levels. Seven ploidy levels occur in the investigated taxa. The frequency of each ploidy level represented within Iberian Potentilla is analysed and the data are compared with those available for taxa from the rest of the distribution area of the genus.     

Unusual primitive heteromorphic ZZ/ZW sex chromosomes in Proceratophrys boiei (Anura, Cycloramphidae, Alsodinae), with description of C-Band interpopulational polymorphism

We performed cytogenetic analyses on specimens from three population samples of Proceratophrys boiei from southeastern and northeastern Brazil. We stained chromosomes of mitotic and meiotic cells with Giemsa, C-banding and Ag-NOR methods. All specimens of P. boiei presented a karyotype with a full chromosome complement of 2n=22, metacentric and submetacentric. We observed the secondary constriction within the short arm of pair 8, which was in the same position of the nucleolus organizer region (NOR). NOR heteromorphism was observed within two specimens from the municipality of Mata de S?o Jo?o (northeastern Bahia State). The C-banding evidenced an unusual heterochromatic pattern in the genome of P. boiei. In the southern most population samples (S?o Paulo State), we observed large blocks of heterochromatin in the centromeric regions of all chromosomes, whereas the northernmost samples (Bahia State) presented a small amount of constitutive heterochromatin. We suppose that this geographic variation in heterochromatin quantities could be due to heterochromatinization of some chromosome regions in the genome of the S?o Paulo samples. Furthermore, females from S?o Paulo presented, within chromosome pair 1 from C-banded karyotypes, one homologous chromosome almost heterochromatic, whereas males had heterochromatin restricted to the centromeric region. This unusual heterochromatic arrangement led us to assume that P. boiei owns a ZZ/ZW type of sexual determination system. This finding is very important, as this is the first record of ZZ/ZW sex chromosomes within Cycloramphidae. We believe that the cytogenetic differences found between southeastern and northeastern Brazilian population samples of P. boiei strongly supports the existence of a species complex under the name P. boiei, and the requirement of taxonomic and systematic reviews by morphological, bioacoustical, molecular, and cytogenetic data could define this taxonomic issue in the future.     

边陲黄耆和乌拉特黄耆的细胞学研究及其分类学订正

边陲黄耆和乌拉特黄耆间断分布于蒙新荒漠的西侧和东侧,二者在外部形态上接近,是一对易混淆的种。对这两个种的核型研究表明,边陲黄耆的核型公式为２ｎ＝１６＝１０ｍ（２ＳＡＴ）＋６ｓｔ,其中第１、２、３号染色体较长,其余染色体较短,核型呈现出明显的二型性,在第４号染色体（ｍ型）长臂上有明显的随体,而乌拉特黄耆的核型公式为２ｎ＝１６＝６ｍ＋６ｓｍ＋４ｓｔ（２ＳＡＴ）,其中除第１号染色体较长外,其余染色体相互     

Cytogenetic evidence for a complex of species within the taxon Anopheles maculatus (Diptera: Culicidae)

Two largely independent studies of chromosomes from natural populations of Anopheles maculatus provide evidence for several genetic species within the taxon. (1) Polytene chromosome variation shows four different rearrangements of arm 2 and three rearrangements of the X chromosome. There is strong evidence for three species. Two allopatric populations represent either dramatic geographic variation for two independent inversion systems within one of the genetic species, or represent two additional species. Their species status remains unresolved by this work. (2) Heterochromatic variation occurs in both X and Y chromosomes as revealed by Giemsa-banding of mitotic chromosomes from larval brains. The distribution and association of these various sex chromosomes give further evidence of a species complex. A preliminary correlation of these two kinds of chromosomal variation is given.     

The blackburni/murchisona species complex in Australian Pseudotetracha (Coleoptera: Carabidae: Cicindelinae: Megacephalini): evaluating molecular and karyological evidence

Our phylogenetic analysis of three endemic species of the Australian tiger beetle genus Pseudotetracha (Fleutiaux, 1864) from South Australia used sequences of two fragments of the mitochondrial genes 16S rRNA and cytochrome oxidase III. A matrix for each gene and two combined matrices were constructed. We compared these three riparian species, together with data from nine taxa of this genus available in GenBank, using parsimony and Bayesian methods. These molecular results are in agreement with the phylogenetic hypothesis for the blackburni/murchisona species complex previously proposed based on morphology, whereas other recent molecular analyses have questioned the existence of this species complex. In all of our analyses, samples of P. blackburni divided into two statistically supported clades, one of which is more closely related to P. mendacia and P. pulchra than to the other P. blackburni clade. This suggests the existence of a cryptic new species. Additionally, we analysed chromosomes of the second metaphase cells of members of the two clades. The observations showed different karyotypes as blackburni‐1 has two types of second meiotic metaphase cells with 11 and 12 chromosomes, whereas in blackburni‐2, all cells have 12 chromosomes, adding evidence for the putative existence of two species.     

Novel dermaseptins from Phyllomedusa hypochondrialis (Amphibia)

Six new antimicrobial peptides structurally related to the dermaseptin family have been isolated from the skin secretion of the amphibian Phyllomedusa hypochondrialis. The primary structures of these molecules named as DShypo 01, 02, 03, 04, 06, and 07 were determined by de novo MS/MS experiments, Edman degradation, and cDNA sequencing. The fifth peptide was found to be precisely the same DS 01 from Phyllomedusa oreades previously described by our group. The majority of the peptides purified from the crude skin secretion could be directly localized and mapped onto a freshly dissected dorsal skin fragment using mass spectrometry-imaging techniques. Comparisons between peptides and commercial drugs on their antibacterial and anti-Leishmania amazonensis efficiencies, associated with peptide lytic effects on mammalian blood cells and surface plasmon resonance interaction studies on immobilized DMPC vesicles, were also performed.     

Cytogenetic analysis of some Brazilian marsupials (Didelphidae: Marsupialia)

Three species of marsupials from the Amazon region (Marmosa cinerea, Caluromys lanatus, and Didelphis marsupialis) and two from the region of S?o Paulo (Didelphis marsupialis and Didelphis albiventris) were studied. The G-banding pattern of the species with 2n = 14 (M. cinerea and C. lanatus) was very similar, as well as the pattern of G-bands in the species with 22 chromosomes (Didelphis). All of the autosomes of M. cinerea and D. albiventris have centromeric C-bands and the Y chromosome is totally C-band positive. The long arm of the M. cinerea X chromosome is completely C-band positive except for a negative band close to the centromeric region. In D. albiventris the long arm of the X chromosome is C-band positive except for a negative band close to the telomeric region. In M. cinerea the silver-stained nucleolar organizer regions (Ag-NORs) are found in the acrocentric chromosomes, being located in the telomeric region of one pair and in the centromeric region of the other pair. Caluromys lanatus has centromeric Ag-NORs in one acrocentric and in one submetacentric chromosome pairs. Didelphis marsupialis has three chromosome pairs with telomeric Ag-NORs. In D. albiventris the Ag-NORs are terminal and located in both arms of one pair and in the long arm of two pairs of chromosomes.     

Amphibian skin secretomics: application of parallel quadrupole time-of-flight mass spectrometry and peptide precursor cDNA cloning to rapidly characterize the skin secretory peptidome of Phyllomedusa hypochondrialis azurea: discovery of a novel peptide family, the hyposins

This study reports the variety of peptides present in the skin secretory peptidome of Phyllomedusa hypochondrialis azurea. Peptide structures, along with post-translational modifications, were elucidated by QTOF MS/MS analysis, cDNA sequencing, or a combination of both. Twenty-two peptides, including 19 novel structures, were identified from six different structural classes, including tryptophyllins, dermorphins, and a novel group of peptides termed hyposins. The study demonstrates the power of this combined approach to mine the rich peptidome compliment of the amphibian defensive skin secretome.     

Chromatin differentiation between Theobroma cacao L. and T. grandiflorum Schum

A comparative analysis of mitotic chromosomes of Theobroma cacao (cacao) and T. grandiflorum (cupuaçu) was performed aiming to identify cytological differences between the two most important species of this genus. Both species have symmetric karyotypes, with 2n = 20 metacentric chromosomes ranging in size from 2.00 to 1.19 μm (cacao) and from 2.21 to 1.15 μm (cupuaçu). The interphase nuclei of both species were of the arreticulate type, displaying up to 20 chromocentres, which were more regularly shaped in cacao than in cupuaçu. Prophase chromosomes of both species were more condensed in the proximal region, sometimes including the whole short arm. Both species exhibited only one pair of terminal heterochromatic bands, positively stained with chromomycin A ₃ , which co-localized with the single 45S rDNA site. Each karyotype displayed a single 5S rDNA site in the proximal region of another chromosome pair. Heterochromatic bands were also observed on the centromeric/pericentromeric regions of all 20 chromosomes of cacao after C-banding followed by Giemsa or DAPI staining, whereas in cupuaçu they were never detected. These data suggest that the chromosomes of both species have been largely conserved and their pericentromeric chromatin is the only citologically differentiated region.     

Genetic Evidence for the Allodiploid Origin of the Moss Species Polytrichum longisetum

Abstract: Fixed heterozygous banding patterns observed for 4 allozyme and 12 microsatellite loci, in combination with a chromosome number of 14, show that Polytrichum longisetum is an allodiploid species. Comparison of these banding patterns with those of related Polytrichaceae species suggest that Polytrichum formosum, or an ancestor taxon of this species, is one of the haploid progenitors of P. longisetum. The second progenitor species of P. longisetum could not be designated in this study as it was not among the examined possible progenitor species. DNA sequence data for two microsatellite loci, however, suggest that the second progenitor species should possibly be more closely related to P. formosum than any of the other haploid possible progenitor species examined in this study. As the current systematic literature does not mention the existence of such a species, this could indicate that the second progenitor species is already extinct.     

Chromosomal evidence for sibling species of the malaria vector Anopheles cruzii.

An analysis of the ovarian polytene chromosomes of Anopheles cruzii from three localities in Southeast Brazil revealed the existence of two genetic entities within this morphologically uniform taxon. These cryptic species differed in the banding patterns of the X chromosome and 3L arm. A pattern of bands that cannot be explained by the fixation of any of the known inversions in chromosome X was revealed and named chromosomal form B to distinguish it from the standard pattern of this X chromosome, form A. Each chromosomal form is characterized by a different set of inversions. The lack of heterozygotes (A/B) for these X chromosome forms in populations where both forms coexist is evidence of absence or limited gene flow between the two groups.     

A consistent nomenclature of antimicrobial peptides isolated from frogs of the subfamily Phyllomedusinae

A growing number of cationic antimicrobial peptides have been isolated from the skin of hylid frogs belonging to the Phyllomedusinae subfamily. The amino acid sequences of these peptides are currently located in several databases under identifiers with no consistent system of nomenclature to describe them. In order to provide a workable terminology for antimicrobial peptides from Phyllomedusid frogs, we have made a systematic effort to collect, analyze, and classify all the Phyllomedusid peptide sequences available in databases. We propose that frogs belonging to the Phyllomedusinae subfamily should be described by the species names set out in Amphibian Species of the World: http://research.amnh.org/herpetology/amphibia/index.php, American Museum of Natural History, New York, USA. Multiple alignments analysis of at least 80 antimicrobial peptides isolated from 12 Phyllomedusinae species were distributed in seven distinct peptide families including dermaseptin, phylloseptin, plasticin, dermatoxin, phylloxin, hyposin and orphan peptides, and will be considered as the name of the headgroup of each family. The parent peptide's name should be followed by the first upper letter of the species for orthologous peptides and publication date determines priority. For example, the abbreviation B for bicolor and H for hypochondrialis. When two species begin with the same letter, two letters in upper case should be used (the first letter followed by the second or the third letter and so on). For example, the abbreviation DI for distincta, DU for duellmani, VA for vaillanti and VN for vanzolinii. Paralogous peptides should bear letter(s) in upper case followed by numbers.     

18-26S rDNA在4种重楼属植物中的定位

为探讨rDNA在重楼属Paris L.中的分布规律,利用荧光原位杂交（FISH）对4种重楼属植物 的18-26S rDNA进行了定位。所有植物均为二倍体,基因组由A、B、C、D和 E5条染色体构成。（1）滇重楼P.polyphylla var.yunnanensis:2n=10=6m+4t,C和D染色体的 短臂上各有1个18-26S rDNA位点;（2）长柱重楼P.forrestii:2n=10=6m+4t,B染色体的长臂 、C和D染色体的短臂上各有1个位点;（3）五指莲P.axialis:2n=10=6m（2sat）+4t（2sat） +1-2B,C和D染色体的短臂上各有1个位点;在有1个B染色体的细胞中,B染色体没有信号点, 而有2个B染色体的细胞中,只有1个B染色体上有信号点,表明B染色体上有基因存在且其分裂 不均等;（4）大理重楼P.daliensis:2n=10=4m+2sm+2st+2t,C染色体的短臂上有1个位点。1 8-26S rDNA位点不仅出现在染色体的次缢痕上,也出现在非次缢痕位点。另外,4个种中C染 色体短臂末端均有18-26S rDNA。     

The karyotype of Nothoscordum arenarium Herter (Gilliesioideae, Alliaceae): A populational and cytomolecular analysis

The genus Nothoscordum Kunth comprises approximately 20 species native to South America. Karyologically, the genus is remarkable for its large chromosomes and Robertsonian translocations. Variation in chromosome number has been recorded in a few polyploid species and it is unknown among diploids. This study presents the chromosome number and morphology of 53 individuals of seven populations of N. arenarium Herter (2n = 10). In addition, karyotype analyses after C-banding, staining with CMA and DAPI, and in situ hybridization with 5S and 45S rDNA probes were performed in six individuals from one population. All individuals exhibited 2n = 10 (6M + 4A), except for one tetraploid (2n = 20, 12M + 8A) and one triploid (2n = 15, 9M + 6A) plant. C-banding revealed the presence of CMA(+) /DAPI (-) heterochromatin in the short arm and in the proximal region of the long arm of all acrocentric chromosomes. The 45S rDNA sites co-localized with the CMA (+) regions of the acrocentrics short arms, while the 5S rDNA probe only hybridized with the subterminal region of a pair of metacentric chromosomes. A change in the pattern of CMA bands and rDNA sites was observed in only one individual bearing a reciprocal translocation involving the long arm of a metacentric and the long arm of an acrocentric chromosome. These data suggest that, despite isolated cases of polyploidy and translocation, the karyotype of N. arenarium is very stable and the karyotypic instability described for other species may be associated with their polyploid condition.     

THE NUCLEAR CYTOLOGY OF BIVALENT AND RING-FORMING RHOEOS AND THEIR HYBRIDS

The structural hybridity of the chromosomes of Rhoeo spathacea (R. discolor) that produces a ring of 12 chromosomes at meiosis is probably maintained by a balanced lethal system active in the zygotic or embryonic stage. Bivalent-forming plants have been identified that belong to the taxon R. spathacea var. concolor. In the clone studied the arm arrangement in R. s. var. concolor is very nearly the same as the arm arrangement in one of the balanced translocation complexes of R. spathacea.     

Cytogenetic analyses of two Curimatidae species (Pisces; Characiformes) from the Paranapanema and Tietê Rivers.

Cytogenetic analyses were performed in two Curimatidae species (Steindachnerina insculpta and Cyphocharax modesta) from the Paranapanema and Tietê Rivers (S?o Paulo State, Brazil), showing a karyotype composed of 54 meta-submetacentric chromosomes in both species. Silver- and chromomycyn-staining and fluorescent in situ hybridization (FISH) using a 18S rDNA probe indicated that the nucleolar organizer regions (NORs) of both species are localized in the terminal region of the long arm of two metacentric chromosomes. Although a single NOR system was evidenced in both analyzed species, S. insculpta and C. modesta presented the nucleolar organizer regions in distinct chromosome pairs, indicating that these cistrons can be considered cytogenetic markers. Variation on the amount and distribution of the constitutive heterochromatin (C-bands) could also be detected between the two species - while S. insculpta presented few heterochromatic blocks, intensely stained C-bands were evidenced in C. modesta specially in the terminal region of the long arm of the NOR-bearing chromosomes. Although most Curimatidae species have been characterized by homogeneous karyotypes, isolated populations could be established under different environmental conditions leading to karyotype micro-structure variations specially related to the NORs localization and C-banding distribution. The obtained data were useful for the cytogenetic characterization and differentiation of S. insculpta and C. modesta and could be used in evolutionary inferences in the Curimatidae group.     

Mapping of18-26S rDNA loci in four species of the genus Paris by fluorescence in situ hybridization（FISH）

18-26S rDNA loci were mapped on chromosomes in four species of Par is,and the num-ber and position of rDNA sites in these species were compared f or analysis of the distribution of the sites. All the plants were diploids,and t he genome consisted of five chromosomes,A,B,C,D and E. （1）P. polyphylla var. yunnanensis,2n=10=6m+4t. Two18-26S rDNA loci were de-tected on the short arms o f C and D chromosomes;（2）P. forrestii,2n=10=6m+4t. One locus was detected on th e long arm of B chromosome,and also two loci on the short arms of C and D chromosomes;（3）P. axialis. 2n=10=6m（2sat）+4t（2sat）+1-2B. Two loci were detected o n the short arms of C and D chromosomes. One locus was detected in the cell with t wo B-chromosomes（B）,but none was detected in that with only one B chromosome, indicating that rRNA gene existed on B chromsome,and an unequal division occurr ed during mitotic cycle of B-chromosomes. （4）P. daliensis,2n=10=4m+2sm+2st+2t. O ne locus was detected on the short arm of D chromo-some. The signals of18-26S rD NA appeared not only in the second constriction but also in the other regions of chromosome. It is noteworthy that one locus was detected in the terminal region o n the short arm of C chromosome in all the four species studied.     

126 Porphyra Birdiae Neefus et Mathieson (Bangiales, Rhodophyta): A New Species from the Northwest Atlantic

Recent studies combining biochemical, molecular, and traditional morphological and ecological traits have shown that some currently recognized species of the red algal genus Porphyra are actually "form species" or "complexes" comprising several morphologically similar but genetically distinct taxa. Conflicting reports of chromosome numbers and differences in DNA sequences reported for Porphyra purpurea (Roth) C. Agardh have raised suspicion that more than one taxon has been confused under this name in the Northwest Atlantic. We have identified one of these cryptic taxa and have recently described it as a new species, Porphyra birdiae . Like P. purpurea (Roth) C. Agardh, it has an ovate to broadly elongate, foliose blade with reproductive areas segregated by a distinct line into male and female sectors. While reproductive specimens have historically been confused with P. purpurea , non-reproductive specimens of P. birdiae have been incorrectly identified as P. umbilicalis Kützing. Although P. birdiae is morphologically similar to both of these species, sequences of SSU (nuclear small subunit rRNA gene) and rbc L (plastid ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene) indicate that it is not closely related to either one. Based on rbc L sequences, P. birdiae is closely related to P. aestivalis Lindstrom et Fredericq, a proposed new species from Alaska.