Evolution, epigenetic inheritance, development: a diplo/tetraploid model for Anura evolution

This review deals with innovative concepts of evolution in vertebrates, such as epigenetic mechanisms and transgenerational inheritance. Evolutionary models based on data of fossil records, cytogenetics and molecular genetics are indicated. The 2R-model of vertebrate evolution is focalized as well as the epigenetic mechanisms of gene regulation and variability of polyploid anurans. It is known that science evolves by routes that are sometimes impelled by puzzling questions. The cytogenetic data here reported for Anurans brought some perplexing considerations involving fundamental concepts of neo-Darwinism regarding slow/fast evolution, ploidy, epigenetics, and transgenerational inheritance. Indeed, a growing body of evidence reveals that besides gene mutations, diversity may also be produced by epigenetic mutations of regulatory segments of DNA. Yet, an intriguing point to be explained is whether these types of mutations can promote evolution via transgenerational inheritance.

Whole-genome duplication and hemoglobin differentiation traits between allopatric populations of Brazilian Odontophrynus americanus species complex (Amphibia, Anura).

Two allopatric populations of Brazilian diploid and tetraploid Odontophrynus americanus species complex, both from São Paulo state, had their blood hemoglobin biochemically analyzed. In addition, these specimens were cytogenetically characterized. Biochemical characterization of hemoglobin expression showed a distinct banding pattern between the allopatric specimens. Besides this, two distinct phenotypes, not linked to ploidy, sex, or age, were observed in adult animals of both populations. Phenotype A exhibits dark-colored body with small papillae, ogival-shaped jaw with reduced interpupillary distance and shorter hind limbs. Phenotype B shows yellowish-colored body with larger papillae, arch-shaped jaw with broader interpupillary distance and longer hind limbs. Intermediate phenotypes were also found. Considering the geographical isolation of both populations, differences in chromosomal secondary constrictions and distinct hemoglobins banding patterns, these data indicate that 2n and 4n populations represent cryptic species in the O. americanus species complex. The observed phenotypic diversity can be interpreted as population genetic variability. Eventually future data may indicate a probable beginning of speciation in these Brazilian frogs. Such inter- and intrapopulational differentiation/speciation process indicates that O. americanus species complex taxonomy deserves further evaluation by genomics and metabarcoding communities, also considering the pattern of hemoglobin expression, in South American frogs.

Whole-genome duplication and hemoglobin differentiation traits between allopatric populations of Brazilian Odontophrynus americanus species complex (Amphibia, Anura)

Two allopatric populations of Brazilian diploid and tetraploid Odontophrynus americanus species complex, both from São Paulo state, had their blood hemoglobin biochemically analyzed. In addition, these specimens were cytogenetically characterized. Biochemical characterization of hemoglobin expression showed a distinct banding pattern between the allopatric specimens. Besides this, two distinct phenotypes, not linked to ploidy, sex, or age, were observed in adult animals of both populations. Phenotype A exhibits dark-colored body with small papillae, ogival-shaped jaw with reduced interpupillary distance and shorter hind limbs. Phenotype B shows yellowish-colored body with larger papillae, arch-shaped jaw with broader interpupillary distance and longer hind limbs. Intermediate phenotypes were also found. Considering the geographical isolation of both populations, differences in chromosomal secondary constrictions and distinct hemoglobins banding patterns, these data indicate that 2n and 4n populations represent cryptic species in the O. americanus species complex. The observed phenotypic diversity can be interpreted as population genetic variability. Eventually future data may indicate a probable beginning of speciation in these Brazilian frogs. Such inter- and intrapopulational differentiation/speciation process indicates that O. americanus species complex taxonomy deserves further evaluation by genomics and metabarcoding communities, also considering the pattern of hemoglobin expression, in South American frogs.

Whole-genome duplication and hemoglobin differentiation traits between allopatric populations of Brazilian Odontophrynus americanus species complex (Amphibia, Anura)

Two allopatric populations of Brazilian diploid and tetraploid Odontophrynus americanus species complex, both from São Paulo state, had their blood hemoglobin biochemically analyzed. In addition, these specimens were cytogenetically characterized. Biochemical characterization of hemoglobin expression showed a distinct banding pattern between the allopatric specimens. Besides this, two distinct phenotypes, not linked to ploidy, sex, or age, were observed in adult animals of both populations. Phenotype A exhibits dark-colored body with small papillae, ogival-shaped jaw with reduced interpupillary distance and shorter hind limbs. Phenotype B shows yellowish-colored body with larger papillae, arch-shaped jaw with broader interpupillary distance and longer hind limbs. Intermediate phenotypes were also found. Considering the geographical isolation of both populations, differences in chromosomal secondary constrictions and distinct hemoglobins banding patterns, these data indicate that 2n and 4n populations represent cryptic species in the O. americanus species complex. The observed phenotypic diversity can be interpreted as population genetic variability. Eventually future data may indicate a probable beginning of speciation in these Brazilian frogs. Such inter- and intrapopulational differentiation/speciation process indicates that O. americanus species complex taxonomy deserves further evaluation by genomics and metabarcoding communities, also considering the pattern of hemoglobin expression, in South American frogs.

Pore-linked filaments in anura spermatocyte nuclei.

Pore-linked filaments were visualized in spreads of anuran spermatocyte nuclei using transmission electron microscope. We used Odontophrynus diplo and tetraploid species having the tetraploid frogs reduced metabolic activities. The filaments with 20-40 nm width are connected to a ring component of the nuclear pore complex with 90-120 nm and extend up to 1 microm (or more) into the nucleus. The filaments are curved and connect single or neighboring pores. The intranuclear filaments are associated with chromatin fibers and related to RNP particles of 20-25 nm and spheroidal structures of 0.5 microm, with variations. The aggregates of several neighboring pores with the filaments are more commonly observed in 4n nuclei. We concluded that the intranuclear filaments may correspond to the fibrillar network described in Xenopus oocyte nucleus being probably related to RNA transport. The molecular basis of this RNA remains elusive. Nevertheless, the morphological aspects of the spheroidal structures indicate they could correspond to nucleolar chromatin or to nucleolus-derived structures. We also speculate whether the complex aggregates of neighboring pores with intranuclear filaments may correspond to pore clustering previously described in these tetraploid animals using freeze-etching experiments.

Pore-linked filaments in anura spermatocyte nuclei

Pore-linked filaments were visualized in spreads of anuran spermatocyte nuclei using transmission electron microscope. We used Odontophrynus diplo and tetraploid species having the tetraploid frogs reduced metabolic activities. The filaments with 20-40 nm width are connected to a ring component of the nuclear pore complex with 90-120 nm and extend up to 1æm (or more) into the nucleus. The filaments are curved and connect single or neighboring pores. The intranuclear filaments are associated with chromatin fibers and related to RNP particles of 20-25 nm and spheroidal structures of 0.5æm, with variations. The aggregates of several neighboring pores with the filaments are more commonly observed in 4n nuclei. We concluded that the intranuclear filaments may correspond to the fibrillar network described in Xenopus oocyte nucleus being probably related to RNA transport. The molecular basis of this RNA remains elusive. Nevertheless, the morphological aspects of the spheroidal structures indicate they could correspond to nucleolar chromatin or to nucleolus-derived structures. We also speculate whether the complex aggregates of neighboring pores with intranuclear filaments may correspond to pore clustering previously described in these tetraploid animals using freeze-etching experiments.

Filamentos ligados a poros foram visualizados em núcleos de espermatócitos de anuros através da técnica de espalhamento para microscopia eletrônica de transmissão. Os animais usados pertencem ao gênero Odontophrynus com espécies cripticas diplo e tetraplóides naturais, tendo os tetraplóides atividade metabólica reduzida. Os filamentos com 20-40 nm de largura são ligados a um anel componente do complexo poro nuclear de 90-120 nm e estendem-se até 1 æm (ou mais) para dentro do núcleo. Os filamentos são curvos e ligam poros simples ou poros vizinhos. Os filamentos intranucleares são associados a fibras de cromatina e relacionados a partículas de RNP de 20-25 nm e a estruturas esféricas de 0.5æm, com variações. Os agregados de poros vizinhos com os filamentos longos são mais freqüentemente observados em núcleos 4n. Concluímos que os filamentos intranucleares podem corresponder aos emaranhados fibrilares descritos em núcleos de oócitos de Xenopus e possivelmente relacion ados ao transporte de RNA. A base molecular desse RNA não é conhecida. Contudo, os aspectos morfológicos das estruturas esféricas parecem indicar que elas podem corresponder à cromatina nucleolar ou a estruturas derivadas do nucléolo. Também, especulamos se os agregados complexos de poros vizinhos com os filamentos intranucleares podem corresponder aos aglomerados de poros previamente descritos nesses animais tetraplóides através da técnica "freeze-etching".

Evolution by polyploidy and gene regulation in Anura.

The evolution of the metazoa has been characterized by gene redundancy, generated by polyploidy, tandem duplication and retrotransposition. Polyploidy can be detected by looking for duplicated chromosomes or segments of orthologous chromosomes in post-polyploid animals. It has been proposed that the evolutionary role of polyploidy is to provide extra-copies of genes, whose subsequent alteration leads to new functions, increased biological complexity, and, ultimately, speciation. We review the theory of evolution by genome duplication, basing our arguments on findings from autopolyploid anurans and fish, undergoing post-polyploidy diploidization. We conclude that: 1) the high genetic variability of autotetraploid anurans is a result of tetrasomic expression, based on studies of isozymes and other proteins. 2) Epigenetic mechanisms mediate the reduced expression or silencing of redundant copies of genes in the regulation of gene expression of these tetraploids. This conclusion is based on data concerning ribosomal and hemoglobin gene activity. 3) Duplication of the genome may have occurred more than once in the phylogeny of the anurans, as exemplified by 4n and 8n Leptodactylidae species.

Evolution by polyploidy and gene regulation in Anura

The evolution of the metazoa has been characterized by gene redundancy, generated by polyploidy, tandem duplication and retrotransposition. Polyploidy can be detected by looking for duplicated chromosomes or segments of orthologous chromosomes in post-polyploid animals. It has been proposed that the evolutionary role of polyploidy is to provide extra-copies of genes, whose subsequent alteration leads to new functions, increased biological complexity, and, ultimately, speciation. We review the theory of evolution by genome duplication, basing our arguments on findings from autopolyploid anurans and fish, undergoing post-polyploidy diploidization. We conclude that: 1) the high genetic variability of autotetraploid anurans is a result of tetrasomic expression, based on studies of isozymes and other proteins. 2) Epigenetic mechanisms mediate the reduced expression or silencing of redundant copies of genes in the regulation of gene expression of these tetraploids. This conclusion is based on data concerning ribosomal and hemoglobin gene activity. 3) Duplication of the genome may have occurred more than once in the phylogeny of the anurans, as exemplified by 4n and 8n Leptodactylidae species.

Somatic pairing, endomitosis and chromosome aberrations in snakes (Viperidae and Colubridae)

The positioning of macrochromosomes of Bothrops jararaca and Bothrops insularis (Viperidae) was studied in undistorted radial metaphases of uncultured cells (spermatogonia and oogonia) not subjected to spindle inhibitors. Colchicinized metaphases from uncultured (spleen and intestine) and cultured tissues (blood) were also analyzed. We report two antagonic non-random chromosome arrangements in untreated premeiotic cells: the parallel configuration with homologue chromosomes associated side by side in the metaphase plate and the antiparallel configuration having homologue chromosomes with antipolar distribution in the metaphase ring. The antiparallel aspect also appeared in colchicinized cells. The spatial chromosome arrangement in both configurations is groupal size-dependent and maintained through meiosis. We also describe, in untreated gonia cells, endomitosis followed by reductional mitosis which restores the diploid number. In B. jararaca males we observed that some gonad regions present changes in the meiotic mechanism. In this case, endoreduplicated cells segregate the diplochromosomes to opposite poles forming directly endoreduplicated second metaphases of meiosis with the suppression of first meiosis. By a successive division, these cells form nuclei with one set of chromosomes. Chromosome doubling in oogonia is known in hybrid species and in parthenogenetic salamanders and lizards. This species also presented chromosome rearrangements leading to aneuploidies in mitosis and meiosis. It is suggested that somatic pairing, endomitosis, meiotic alterations, and chromosomal aberrations can be correlated processes. Similar aspects of nuclei configurations, endomitosis and reductional mitosis were found in other Viperidae and Colubridae species

Somatic pairing, endomitosis and chromosome aberrations in snakes (Viperidae and Colubridae).

The positioning of macrochromosomes of Bothrops jararaca and Bothrops insularis (Viperidae) was studied in undistorted radial metaphases of uncultured cells (spermatogonia and oogonia) not subjected to spindle inhibitors. Colchicinized metaphases from uncultured (spleen and intestine) and cultured tissues (blood) were also analyzed. We report two antagonic non-random chromosome arrangements in untreated premeiotic cells: the parallel configuration with homologue chromosomes associated side by side in the metaphase plate and the antiparallel configuration having homologue chromosomes with antipolar distribution in the metaphase ring. The antiparallel aspect also appeared in colchicinized cells. The spatial chromosome arrangement in both configurations is groupal size-dependent and maintained through meiosis. We also describe, in untreated gonia cells, endomitosis followed by reductional mitosis which restores the diploid number. In B. jararaca males we observed that some gonad regions present changes in the meiotic mechanism. In this case, endoreduplicated cells segregate the diplochromosomes to opposite poles forming directly endoreduplicated second metaphases of meiosis with the suppression of first meiosis. By a successive division, these cells form nuclei with one set of chromosomes. Chromosome doubling in oogonia is known in hybrid species and in parthenogenetic salamanders and lizards. This species also presented chromosome rearrangements leading to aneuploidies in mitosis and meiosis. It is suggested that somatic pairing, endomitosis, meiotic alterations, and chromosomal aberrations can be correlated processes. Similar aspects of nuclei configurations, endomitosis and reductional mitosis were found in other Viperidae and Colubridae species.

Chromosome structure in man and Amphibia-Anura, restriction enzymes

Cromossomos metafásicos fixados de humano e de anuros poliplóides (Odontophynus amricanus, 4n = 44) foram tratados com enzimas de restriçäo MspI, Hpa II, EcoRI, Bgl II, Dra I e Hae III, seguidos pela coloraçäo Giemsa. Msp I (C CGG) e Hae III (GG CC) produziram bandas similares a G nos cromossomos humanos, indicando que as bandas säo causadas pela remoçäo de classes específicas de DNA ricas em CG. Contudo, padröes comparáveis de banda G foram também obtidos nos cromossomos humanos com Eco RI (G AATTC), Bgl II (A GATCT) e Dra I (TTT AAA), cujos sítios de digestäo näo revelam abundância particular em CG. Nos anuros 4n, Msp I removeu DNA cromossômico do braço longo de dois a três homólogos do grupo I (lq), da constricçäo secundária do grupo 11 bem como as bandas centroméricas, teloméricas e intercalares. Essas duas näo foram removidas pela Hpa II (C CGG), a qual näo corta DNA metilado. Eco RI causou bandas 2q e 3q. Os resultados sugerem que: 1. a açäo dessas enzimas depende da acessibilidade da enzima a regiöes cromossômicas com compactaçöes diferentes do DNA; 2. a obtençäo difícil de bandas intersticiais em Anura pode resultar da menor quantidade de seqüências ricas em CG provavelmente arranjadas em cachos menores, ou da maior condensaçäo cromatínica nos cromômeros

C and AgAs bands of the octaploed untanha frog Ceratophyrs dorsat (C. aurita) (8n = 104, Amphibia, Anura)

O presente trabalho analisa a forma craniana de três gêneros de roedores caviíneos. A análise estatística multivariada mostrou que a semelhança de forma craniana é provavelmente devida à convergência com o tamanho nas trajetórias ontogenéticas, estima através da análise de regressäo de escores individuais derivados da análise dos componentes principais dos caracteres morfométricos cranianos

Extrachromosomal circular DNA-containing structures in lymphocytes of normal and x-fragile individuals: electron microscopy

Por microscopia eletrônica, evidenciou-se seqüências de ADN ricas em CG, ativas na transcriçäo de ARN em "spreads" de linfócitos in-vitro, de indivíduos normais e com fra-X. Esta classe de DNA ocorre em dois estados dentro dos núcleos. Apresenta-se como círculos extracromossômicos ou em associaçäo com alças cromatínicas "multiforked". Assumiu-se que os círculos säo formados pelo anelamento de segmentos excisos, neosintetizados nas alças cromatínicas complexas. As configuraçöes encontradas levaram à discussäo sobre a ocorrência eventual da amplificaçäo gênica ou de elementos similares a transposons em células humanas somáticas

Active genes visualized in pachytene and lymphocyte nuclei from normal and affected individuals: Ag-As-staining, light and electron microscopy and high-resolution autoradiography

Aglomerados de genes nucleolares transcritos foram evidenciados por microscopia óptica e eletrônica como grânulos de 0.1 a 1.0 micron m, corados com prata e ácido fosfotungstico alcoólico (PTA), em espermatócitos e linfócitos humanos. Os grânulos encontrados nos núcleos paquitênicos acham-se aglomerados numa regiäo nuclear distinta, contendo bivalentes autossômicos associados e o par sexual XY. Um grânulo corado pela prata foi encontrado estreitamente relacionado com o bivalente sexual XY. Esta específica regiäo nuclear foi designada "corpo cromatínico" (c cr), neste trabalho. Os grânulos, encontrados nos nucléolos de linfócitos, correspondem a aglomerados de sítios ativos em transcriçäo, tendo fibrilas enroladas de ribonucleoproteínas (RNP), as quais incorporam uridina [3H], em experimentos de marcaçäo de pulso. Os genes ativos foram encontrados nos componentes fibrilares do nucléolo e seus níveis de transcriçäo, estimados pela quantidade de grânulos corados, variaram em indíviduos normais e afetados e durante o tratamento com ácido fólico

Circular Chromatin complexes in human lymphocytes: high-resolution autoradiography

Fibras de cromatina ativas em transcriçäo foram observadas em cromossomos humanos. Esses cromossomos mostram configuraçöes do tipo "loops/scaffold". As fibras ativas têm nucleossomos alterados e apresentam aspectos "multi-forked" os quais levam à formaçäo de anéis. Os transcritos de Ribonucleoproteína (RNP) aparecem como emaranhados de 0,1 micronm ou múltiplos dispostos em série ao longo de fibra. Sugere-se que os complexos circulares de cromatina pertencem ao genoma humano. A possibilidade de que os anéis provêm de procariotos é também discutida

Aspects of active chromatin in chromosomes displaying loops/scaffold configuration: high-resolution autoradiography

Cromossomos profásicos apresentando a configuraçäo "loops/scaffold" foram obtidos em espalhamentos de células do sangue periférico humano de indivíduos normais e afetados. Observou-se uma distinta classe de alças condensadas formando um corpo cromatínico de diâmetro variável. Essas alças säo intensamente dobradas, têm nucleossomos alterados, apresentam fibrilas laterais e fragmentam-se formando anéis. Estas fibrilas incorporam 5 3H uridina, como demonstrado por autoradiografia de alta resoluçäo. As alteraçöes dos nucleossomos foram explicadas pela remoçäo de histonas sendo os anéis indicativos de seqüências repetitivas do DNA