Maria José de Souza

Phylogeography, phylogeny and hybridization in trichechid sirenians: implications for manatee conservation

The three living species of manatees, West Indian (Trichechus manatus), Amazonian (Trichechus inunguis) and West African (Trichechus senegalensis), are distributed across the shallow tropical and subtropical waters of America and the western coast of Africa. We have sequenced the mitochondrial DNA control region in 330 Trichechus to compare their phylogeographic patterns. In T. manatus we observed a marked population structure with the identification of three haplotype clusters showing a distinct spatial distribution. A geographic barrier represented by the continuity of the Lesser Antilles to Trinidad Island, near the mouth of the Orinoco River in Venezuela, appears to have restricted the gene flow historically in T. manatus. However, for T. inunguis we observed a single expanding population cluster, with a high diversity of very closely related haplotypes. A marked geographic population structure is likely present in T. senegalensis with at least two distinct clusters. Phylogenetic analyses with the mtDNA cytochrome b gene suggest a clade of the marine Trichechus species, with T. inunguis as the most basal trichechid. This is in agreement with previous morphological analyses. Mitochondrial DNA, autosomal microsatellites and cytogenetic analyses revealed the presence of hybrids between the T. manatus and T. inunguis species at the mouth of the Amazon River in Brazil, extending to the Guyanas and probably as far as the mouth of the Orinoco River. Future conservation strategies should consider the distinct population structure of manatee species, as well as the historical barriers to gene flow and the likely occurrence of interspecific hybridization.

Karyotype, C-and fluorescence banding pattern, NOR location and FISH study of five Scarabaeidae (Coleoptera) species

Meiotic chromosomes obtained from members of the coleopteran subfamilies Rutelinae and Dynastinae were studied using standard and silver nitrate staining, C-banding, base-specific fluorochromes and fluorescent in situ hybridization (FISH). The study presents detailed karyotipic descripitions of three Rutelinae species (Geniates borelli, Macraspis festiva and Pelidnota pallidipennis), and two Dynastinae species (Lygirus ebenus and Strategus surinamensis hirtus) with special emphasis on the distribution and variability of constitutive heterochromatin and the nucleolar organizer region (NOR). We found that for G. borelli, P. pallidipennis, L. ebenus and S. s hirtus the karyotype was 2n = 20 (9II + Xyp), with G. borelli, P. pallidipennis and L. ebenus showed meta-submetacentric chromosomes which gradually decreased in size. For Macraspis festiva the karyotype was 2n = 18 (8II + Xyp). In L. ebenus we found that the NOR was located on an autosome, but in the other four species it occurred on the sex bivalents. In all five species the constitutive heterochromatin (CH) was predominantly pericentromeric while the X chromosomes were almost completely heterochomatic, although CMA3/DA/DAPI staining showed intra and interspecific variation in the bright fluorescence of the constitutive heterochromatin. The FISH technique showed rDNA sites on the X chromosome of the Rutelinae species.

Cytogenetic mapping of 5S and 18S rRNAs and H3 histone genes in 4 ancient Proscopiidae grasshopper species: contribution to understanding the evolutionary dynamics of multigene families.

This paper reports on the chromosomal location of 18S rRNA, 5S rRNA and H3 histone multigene families in 4 species of a relatively ancient and diversified group of grasshoppers belonging to the family Proscopiidae. The 5S rRNA and H3 histone genes were highly conserved in the number of sites and chromosomal position in the 4th chromosome pair in all species analyzed, whereas the 18S rRNA genes showed slightly more variation because they were present on one or 2 chromosome pairs, depending on the species. The 5S and 18S rRNA gene families occurred in different chromosomes; in contrast, H3 histone and 5S rRNA genes co-localized in the same chromosomal position, with an apparently interspersed organization. Considering that the Proscopiidae family is a relatively ancient group compared with the Acrididae family, the association of the H3 histone and 5S rRNA multigene families can represent a basal condition for grasshoppers, although more research is needed on other representatives of this insect group to confirm this statement. The presence of such an association of 5S rDNA and H3 histone in mussels and arthropods (beetles, grasshoppers and crustaceans) suggests that this linked configuration could represent an ancestral pattern for invertebrates. These results provide new insights into the understanding of the genome organization and the evolution of multigene families in grasshoppers and in insects as a whole.

Possible autosomal origin of macro B chromosomes in two grasshopper species.

The acrocentric macro B chromosomes of Rhammatocerus brasiliensis (Acrididae, Gomphocerinae) and Xyleus discoideus angulatus (Romaleidae, Romaleinae) are highly similar to the X chromosome in each species in terms of morphology, size, and pycnosis. However, the results of FISH experiments using 45S and 5S rDNA probes suggest that in both species the B chromosomes are most likely of autosomal origin. In R. brasiliensis, the B chromosome presented 5S rDNA but not 45S rDNA, in resemblance to the L2, L3, M5 and S11 autosomes, but the X chromosome lacks both rDNA families. In X. d. angulatus, 45S rDNAs is absent from the B chromosome, whereas the X chromosome contains one of the two 45S rDNA clusters in the genome. The occurrence of B chromosomes in all nine R. brasiliensis populations analyzed indicates that they are widely distributed in Northeastern Brazil, and the small amount of interpopulation variation found for B chromosome prevalence suggests the existence of high gene flow, presumably due to the abundance of this grasshopper species on several types of vegetation and its relatively high flight capability.

Karyotypic analysis, constitutive heterochromatin and NOR distribution in five grasshopper species of the subfamily Leptysminae (Acrididae)

Abstract Cornops aquaticum, C. frenatum frenatum, Stenopola dorsalis, Ste-nacris xanthochlora and Tucayaca parvula were analyzed cytologically by standard staining, C-banding, silver nitrate staining (AgNO3) and base-specific fluorochromes. All species had 23,XO karyotypes in males and 24,XX in females, acrocentric chromosomes and pericentromeric C-bands. In addition, distal bands were revealed in C. aquaticum, C. f. frenatum and S. dorsalis. Triple staining CMA3/DA/DAPI revealed CMA3-positive bands in three biva-lents of C. aquaticum and S. xanthochlora, in four of T. parvula, five of S. dor-salis, and in all chromosomes of C. f. frenatum. Staining with DAPI fluo-rochrome was uniform. The nucleolar organizer regions detected by AgNO3 staining were limited to autosomes in all the species and showed correspondence with GC base pairs distribution detected by sequential staining (AgNO3/CMA3/DA).

Constitutive heterochromatin, NOR location and FISH in the grasshopper Xyleus angulatus (Romaleidae)

Summary C-banding, fluorochrome staining, silver staining and fluorescence in situ hybridization (FISH) were used to characterization of the meiotic chromosomes in the grasshopper Xyleus angulatus. The C-banding pattern showed perincentromeric heterochromatin blocks in all chromosomes, interstitial blocks in the L1, L2 and L3 pairs and distal blocks in medium and small-sized pairs. CMA3/DA staining revealed that these heterochromatic regions were characterized by high content of GC bases pairs. The ribosomal DNA probe (pTa71) showed strong hybridization signal in the pericentromeric regions at bivalents L3, M4 and X chromosome, corresponding to the silver-stained chromosomal positions of active NORs and the heterochromatic area CMA3 + of these chromosomes.

Supernumerary System in Proechimys Iheringi Iheringi (Rodentia, Echimyidae), from the State of São Paulo, Brazil

SUMMARYIn a sample of 13 animals (8 males and 5 females) of the spiny rat Proechimys iheringi theringi from three localities of the State of Sao Paulo (Casa Grande, Ubatuba and Iguape) the diploid numbers were 2n = 61, 62, 63, 64 and 65 chromosomes due to the presence of 1, 2, 3, 4 and 5 minute supernumerary chromosomes, respectively. G- and C-band patterns, localization of the nucleolus organizer regions (NORs) and R-banding obtained after 5-BrdUrd incorporation are presented.

Karyotype, C- and fluorescence banding patterns, NOR location and FISH in the grasshopper Xestotrachelus robustus (Romaleidae)

Abstract Different techniques involving fluorochrome staining, C-banding, NOR location and FISH were used in order to characterize the karyotype and to determine the characteristics of the constitutive heterochromatin in the genome of grasshopper Xestotrachelus robustus. This species presents uniform karyotype in terms of chromosome number (2n=23,XO in males) but differed in the morphology of some chromosomes of the complement. Fluorescence in situ hybridization (FISH) was applied to the location of 45S genes. The results of FISH are compared with those coming from classical cytogenetics (C, AgNO3 and CMA3) banding procedures.

Comparative cytogenetics of three species of Dichotomius (Coleoptera, Scarabaeidae)

Meiotic and mitotic chromosomes of Dichotomius nisus, D. semisquamosus and D. sericeus were analyzed after conventional staining, C-banding and silver nitrate staining. In addition, Dichotomius nisus and D. semisquamosus chromosomes were also analyzed after fluorescent in situ hybridization (FISH) with an rDNA probe. The species analyzed had an asymmetrical karyotype with 2n = 18 and meta-submetacentric chromosomes. The sex determination mechanism was of the Xyp type in D. nisus and D. semisquamosus and of the Xy r type in D. sericeus. C-banding revealed the presence of pericentromeric blocks of constitutive heterochromatin (CH) in all the chromosomes of the three species. After silver staining, the nucleolar organizer regions (NORs) were located in autosomes of D. semisquamosus and D. sericeus and in the sexual bivalent of D. nisus. FISH with an rDNA probe confirmed NORs location in D. semisquamosus and in D. nisus. Our results suggest that chromosome inversions and fusions occurred during the evolution of the group.

Cytogenetic analysis of two Coprophanaeus species (Scarabaeidae) revealing wide constitutive heterochromatin variability and the largest number of 45S rDNA sites among Coleoptera.

The Coleopterans of Scarabaeinae clade presents Coprophanaeus (Megaphanaeus) ensifer and C. (Coprophanaeus) cyanescens (Scarabaeidae) when they are studied cytogenetically by different techniques. The species present symmetric karyotypes, diploid number of 2n=20, and meta-submetacentric chromosomes. C. (M.) ensifer present an XY sex-determining mechanism and C. (C.) cyanescens an XY(p) parachute mechanism. Analysis of constitutive heterochromatin (CH) in the two species revealed the presence of diphasic autosomes, with log arm heterochromatics. Moreover, an additional heterochromatic block in four autosomal bivalents were observed in C. (M.) ensifer. CMA(3)/DA/DAPI fluorochrome staining detected CMA(3) positive heterochromatic blocks restricted to the sex chromosomes in C. (C.) cyanescens, whereas in C. (M.) ensifer CMA(3) positive pericentromeric blocks were present in all autosomes, in the Y chromosome and in the four additional heterochromatic blocks. DAPI staining was neutral in both species. Silver nitrate (AgNO(3)) staining was inefficient for the detection of the nucleolar organizer region (NORs), but showed affinity for the heterochromatic regions. Fluorescence in situ hybridization (FISH) revealed the presence of 45S rDNA sites in the terminal region of the three autosomal bivalents of C. (C.) cyanescens and in seven bivalents and the Y chromosome of C. (M.) ensifer. These results contribute to a better understanding of chromosome evolution in the genus Coprophanaeus, and demonstrate a wide CH variability and the largest number of ribosomal sites among Coleoptera.