Localization of 18S ribosomal genes in suckermouth armoured catfishes Loricariidae (Teleostei, Siluriformes) with discussion on the Ag-NOR evolution

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Nucleolus organizer regions in Physalaemus cuvieri (Anura, Leptodactylidae), with evidence of a unique case of Ag-NOR variability

We studied ten specimens of Physalaemus cuvieri collected at different localities in Brazil using conventional staining and banding techniques. All specimens had 2n = 22. There were karyotypic variants: distinct patterns in the number and chromosome localization of Ag-NORs as well as in the corresponding secondary constrictions. Preliminary C-banding patterns obtained for specimens From two localities are also suggestive of karyotypic differentiation in P. cuvieri.

Cytogenetic analysis of three sea catfish species (Teleostei, Siluriformes, Ariidae) with the first report of Ag-NOR in this fish family

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cytogenetic characterization of Metynnis maculatus (Teleostei; Characiformes): the description in Serrasalminae of a small B chromosome bearing inactive NOR-like sequences

Mitotic chromosomes of Metynnis maculatus (KNER 1860) (Teleostei, Characiformes), a fish species that occurs in the Amazon and Parana-Paraguay river basins, were analyzed for the first time by Giemsa and Ag-NOR staining, C-banding and fluorescence in situ hybridization (FISH) with 18S and 5S rDNA sequences. The basic chromosome number of the species is 2n=62 (32M+22SM+4ST+4A) and, in addition to the 62 regular chromosomes, one small acrocentric supernumerary B chromosome was found in part of the specimens analyzed. Four active NORs were present, and constitutive heterochromatin blocks were found in the pericentromeric region of several chromosomes. A heterochromatic block was also present in the interstitial portion of the submetacentric NOR-bearing pair and the B chromosome was entirely heterochromatic. FISH using an 18S rDNA probe confirmed the results obtained with AgNO(3) staining, and an additional signal was also present on the B chromosomes. 5S rDNA sequences mapped only to the largest acrocentric pair. This is the first description of supernumerary B chromosomes in Serrasalminae, and this karyotype characterization may be useful in further studies about chromosome evolution in this fish group.

Chromosomal differentiation of Hyla nana and Hyla sanborni (Anura, Hylidae) with a description of NOR polymorphism in H-nana

Specimens of Hyla nana and Hyla sanborni from a syntopic population were studied cytogenetically. These species are morphologically very similar and are frequently misidentified, confused with each other. Both species had a diploid chromosome number, 2n = 30. However, the karyotypes of H. nana and H. sanborni differed considerably from each other in the number of submetacentric and telocentric chromosomes. The two species also differed in their primary NOR-bearing chromosomes (metacentric pair 13 in H. nana and telocentric pair 12 in H. sanborni). Additional nucleolus organizer regions (NORs) were detected by Ag-NOR staining and FISH in chromosome pairs 1, 5, 6, 12, and 14 in seven specimens of H. nana. Thus, a total of six patterns of NOR were identified. These differences in karyotype and in NOR location allowed the unambiguous identification of syntopic individuals of the two species. However, the chromosomal morphology of both species differed from that reported for populations from other geographic regions, suggesting that a systematic reevaluation of this group of Hyla may be necessary.

Paracentric inversion involving a NOR-bearing chromosome of rainbow trout (Oncorhyncus mykiss): Electron microscopy studies of the synaptonemal complex

Cytogenetic studies conduced on a sample of 19 specimens of rainbow trout (Oncorhyncus mykiss) showed that 6 presented two chromosomes with a single subterminal AgNOR on the short arms and 13 presented one chromosome with a single subterminal Ag-NOR and a second chromosome with two small subterminal Ag-NORs. Synaptonemal complex studies showed the presence of a pairing failure involving the two lateral elements (LEs) of the NOR-bearing chromosomes only in those specimens with two different NOR-bearing chromosomes, suggesting that the chromosomes with two Ag-NORs originated from a paracentric inversion involving a terminal segment of the original NOR-bearing chromosome. A comparative analysis between three normal and three heterozygous fishes suggest that in the latter group the LEs of the NOR-bearing chromosomes initiate the synaptic process in a more delayed manner, the synapsis develops more quickly, and the nucleolus stays associated with chromosomes for a longer time.

Morphometric study by image analysis of Ag-stained nucleoli in thyroids bearing proliferating lesions

The Ag-NOR staining technique and image analysis were used to evaluate morphological parameters (area, perimeter and axis ratio) in nucleoli from normal thyroids and from thyroids bearing proliferating lesions (carcinomas, adenomas and hyperplasias). Regions with normal appearance located close to adenomatous and carcinomatous regions, in the thyroid of every patient, were also analyzed for comparison with the respective pathological regions and with normal thyroids. Statistical analysis of data for the nucleolar area and perimeter allowed the separation of adenomas and carcinomas from hyperplasias and normal tissue but not the two components in each of these two groups. However, if we look at the numbers, a sequence of increasing nucleolar mean areas in the order: normal, hyperplasia, adenoma and carcinoma may be observed, indicating the sequence of increasing rRNA requirements in these different kinds of cells. The axis ratio that denotes the nucleolar shape (round or oblong) did not show significant differences among tissues, suggesting that shape is not important in the characterization of these pathologies. Differences in nucleolar areas and perimeter between normal and affected regions from each patient were statistically significant for adenomas and carcinomas. When these normal regions were compared with the normal thyroids, significant differences were not obtained in the three evaluated parameters. The observations and their importance for histopathological diagnosis are discussed.

泉水唇鱼的核型和带型研究

该文用上皮细胞培养方法对金沙江水系的泉水唇鱼(Semilabeo p rochilus)染色体进行了研究. 其二倍体数2n=50, 核型组成为16m+20sm+14st, NF=86. 阳性C-带位置有几种类型:(1)着丝粒区; (2)短臂异染色质化; (3)插入异染色质带. Ag-NOR、有7条, 出现在Nos. 10-13染色体短臂. 根据以上所述的特征, 推测泉水唇鱼可能是较为特化的种类。

寇氏隐甲藻(Cryp the codinium cohnii 涡鞭毛虫类)核仁的分裂

对涡鞭毛虫寇氏隐甲藻（Cryp thecodinium cohnii）细胞周期中的核仁进行了电镜连续超薄切片三维重组观察，并进行了Ag-NOR银染的光镜和电镜观察。在整个细胞周期中，寇氏隐甲藻的核仁内都有由核仁染色体发出的染色质索，又由染色质索发出细胞染色质丝进入核仁纤维区中。细胞进入分裂期后，核仁内的染色质索逐渐变粗。在核仁拉长前，核仁内大部分染色质索凝集成为“核仁内染色体”。Ag-NOR染色结果表明，有少量的rDNA也收回到核仁内染色体上，但绝大部分rDNA始终分散地分布在核仁内。核仁染色体象其它染色体一样，也是以一端结合在贯穿细胞核的细胞质管道的壁上，看来也会以同样的方式移到两极。核仁染色体向二极移动，而与核仁染色体相连的rDNA的绝大部分又始终分散地分布在核仁纤维区中，这显然是甲藻核仁拉长并分裂为二的根本原因。核仁内始终存在分散的rDNA也是它们的核仁不瓦解的根本原因。我们观察到，寇氏隐甲藻的染色体不仅连接于细胞质管道壁上，而且还与管道中的微管形成结构上的联系，从而可以认为它们的染色体移动也有微管参与。这点修正了kubai与Ris(1969)的报导。本文根据涡鞭毛虫（甲藻）的核仁在核仁分裂过程中只是拉长和分裂，而并不瓦解和重建的根本原因，探讨了典型有丝分裂过程中核仁的瓦解与重建的起源问题。

灵长类的减数分裂细胞遗传学研究

I.四种猕猴属(Macaca)动物精母细胞联会复合体的比较研究 本工作采用去污剂微铺展－－硝酸银染色技术比较研究了熊猴、平顶猴、藏酋猴、恒河猴及其亚种毛耳猴的精母细胞联会复合体（SC）核型和SC的结构、形态及其在减数分裂过程的行为。结果表明：1.这几种动物的SC核型及SC的发育过程基本一致。粗线期SC的相对长度和臂比与体细胞染色体的相对长度和臂比具有较好的吻合性。SC的形成开始于线期，成熟于粗线期，解体于双线期。2.在减数分裂前期，性染色体轴表现强烈的嗜银性，配对明显落后于常染色体。根据性染色体的形态和行为，可分为五种类型。此外，本文还对XY染色体的同源性和侧轴加粗等现象进行了讨论。II.食蟹猴（M.fascicularis）和熊猴（M.assamensis）杂种（F1）的细胞遗传学研究 本工作采用染色体显带、组织学观察以及低渗铺张－－硝酸银染色等方法较为详细地研究了食蟹猴和熊猴种间杂种（F1）体细胞染色体的G带、C带、Ag-NOR、精母细胞联会复合体的结构、形态和行为以及精子发生。结果表明，①杂种亲本的染色体级具有高度的同源性；②杂种的精子发生过程完全正常；③食蟹猴和熊猴的种间生殖屏障可能主要是生态隔离。此外，本文还对食蟹猴和熊猴染色体高度同源的原因、二者的分类地位以及杂种细胞中Ag-NOR的多态性等问题进行了分析和讨论。

Comparative Cytogenetics and Molecular Phylogeography in the Group Astyanax altiparanae - Astyanax aff. bimaculatus (Teleostei, Characidae)

The genus Astyanax comprises small characin fish of the neotropical region. The so-called `yellow-tailed characins` compose one of the most widely distributed Astyanax groups. A. altiparanae and A. aff. bimaculatus, are evolutionarily closely related and commonly found in several Brazilian hydrographic basins. In the present work, chromosomal data of specimens of A. altiparanae and A. aff. bimaculatus from 4 hydrographic basins in the states of Sao Paulo (Upper Tiete, Paranapanema, Ribeira de Iguape) and Rio de Janeiro (Guapimirim) are shown. All the populations showed 50 chromosomes, with different karyotypic formula. Although only a single Ag-NOR bearing chromosome pair was observed, all populations possess multiple cistrons of 18S rDNA. FISH with the 5S rDNA probe showed single signals at the interstitial position of one metacentric chromosome pair. C-bands are distributed in the terminal and interstitial regions of several chromosomes. However, the As-51 satDNA are frugally located in a few chromosomes of fishes from Upper Tiete, Paranapanema and Guapimirim Rivers, being absent in individuals of A. aff. bimaculatus from Ribeira de Iguape River basin. Beside these 4 populations, molecular phylogeography studies were also performed in individuals from Middle and Lower Tiete River basin and from 2 additional collection sites in the Paranapanema and Ribeira de Iguape River basins. The phylogeographic analysis using 2 mtDNA regions (totalizing 1.314 bp of ND2 and ATPase6/8 genes) of 8 populations of the group of `yellow-tailed characins` from 3 major hydrographic basins showed structuring of populations, suggesting a correlation between chromosomal (nuclear) and molecular (mitochondrial) data. Copyright (C) 2011 S. Karger AG, Basel

Chromosomal studies in four species of genus Chaunus (Bufonidae, Anura): localization of telomeric and ribosomal sequences after fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) using telomeric and ribosomal sequences was performed in four species of toad genus Chaunus: C. ictericus, C. jimi, C. rubescens and C. schneideri. Analyses based on conventional, C-banding and Ag-NOR staining were also carried out. The four species present a 2n = 22 karyotype, composed by metacentric and submetacentric chromosomes, which were indistinguishable either after conventional staining or banding techniques. Constitutive heterochromatin was predominantly located at pericentromeric regions, and telomeric sequences (TTAGGG)(n) were restricted to the end of all chromosomes. Silver staining revealed Ag-NORs located at the short arm of pair 7, and heteromorphism in size of NOR signals was also observed. By contrast, FISH with ribosomal probes clearly demonstrated absence of any heteromorphism in size of rDNA sequences, suggesting that the difference observed after Ag-staining should be attributed to differences in chromosomal condensation and/or gene activity rather than to the number of ribosomal cistrons.

Differential staining and microchromosomal variation in karyotypes of four Brazilian species of Tupinambinae lizards (Squamata : Teiidae)

Kayotypes of four neotropical teiid lizard species (Tupinambinae) were herein studied after conventional as well as silver staining and CBG-banding: Crocodilurus amazonicus (2n = 34), Tupinambis teguixin (2n = 36), Tupinambis merianae and Tupinambis quadrilineatus (2n = 38). The karyological data for T. quadrilineatus as well as those obtained using differential staining for all species were unknown until now. The karyotypes of all species presented 12 macrochromosomes identical in morphology, but differed in the number of microchromosomes: 22 in C. amazonicus, 24 in T. teguixin and 26 in T. quadrilineatus and T. merianae. The Ag-NOR located at the secondary constriction at the distal end of pair 2 is shared by all species, contrasting with the variability observed for this character in species of the related Teiinae. CBG-banding revealed a species-specific pattern in T. quadrilineatus with conspicuous interstitial C-blocks at the proximal region of the long arm of pair 4 and the whole heterochromatic short arm of pair 6. The karyological data reported here corroborates the relationship hypothesis obtained for Tupinambis based on molecular characters. T. teguixin presents the putative ancestral karyotype for the genus with 2n = 36 whereas T. merianae and T. quadrilineatus exhibit 2n = 38, due to an additional pair of microchromosomes.

Chromosome polymorphism in the Brazilian dwarf brocket deer, Mazama nana (Mammalia, Cervidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Inferências carioevolutivas sobre grupos crípticos de peixes marinhos e estuarinos

Cytogenetic studies have been revealing a great diversity not detected, until then, in several families of fishes. Many of these groups, especially those that exhibit great diversity, like Perciformes and Siluriformes, possess species with difficult morphologic characterization, called cryptic species, commonly detected through karyotypic analyses, which reveals outstanding interespecific variations with relationship to the number and its chromosomal structures. Thus, the present work intends to contribute for the cytogenetic knowledge of marine and brackish fish species, because they peculiar life habits and by lack of cytogenetic data of your genetic aspects. Therefore, cytogenetic studies were developed in a species of Apogonidae (Perciformes), two species of sea catfishes of the family Ariidae (Siluriformes) and brackish fish Paurachenipterus galeatus (Siluriformes, Auchenipteridae), through C banding, Ag-NOR, use of base-specific flourochromes (DAPI and CMA3), as well as FISH (Fluorescent in situ hybridization) using ribosomal DNA probes 5S and 18S. The present results contribute to a better understanding of the processes of differentiation patterns and chromosome evolution in these groups. The use of other approaches (the morphology and molecular tools) will allow a larger understanding of the genetic and biological diversity of the Brazilian ichthyofauna.