First constitutive heterochromatin characterization and Karyotype of white stork Ciconia ciconia (Aves: Ciconiidae) / Primeira caracterização constitutiva de heterocromatina e cariótipo de cegonhabranca Ciconia ciconia (Aves: Ciconiidae)

The karyotype and constitutive heterochromatin pattern of the white stork Ciconia ciconia samples obtained from Manzala lake, Dimiaat, Egypt was described. Somatic cells of Ciconia ciconia samples have diploid number 2n= 68 chromosomes. Out of 68 chromosomes, 11 pairs including sex chromosomes were macrochromosomes and the remaining pairs were microchromosomes. Of the 11 macrochromosome pairs, no.1, 2, 4 and 5 were submetacentric and pairs no. 6, 7 and 8 were described as metacentric. In addition, the autosome pair no.3 was subtelocentric, while autosome pair no.9 was acrocentric. Also, the sex chromosome Z represents the fourth one in size and it was classified as submetacentric while, W chromosome appeared as medium size and was acrocentric. Furthermore, C-banding pattern (constitutive heterochromatin) revealed variation in their sizes and occurrence between macrochromosomes. Pairs no. 7 and 8 of autosomes exhibited unusual distribution of heterochromatin, where they appeared as entirely heterochromatic. This may be related to the origin of sex chromosomes Z and W. However, there is no sufficient evidence illustrate the appearance of entirely heterochromatic autosomes. Therefore, there is no available cytogenetic literature that describes the C-banding and karyotype of Ciconia Ciconia, so the results herein are important and may assist in cytogenetic study and evolutionary pattern of Ciconiiformes.

O cariótipo e o padrão constitutivo de heterocromatina das amostras de cegonha-branca Ciconia ciconia obtidas no lago Manzala, Dimiaat, Egito, foram descritos. As células somáticas de amostras de Ciconia ciconia possuem número diploide 2n = 68 cromossomos. Dos 68 cromossomos, 11 pares incluindo cromossomos sexuais eram macrocromossomos e os pares restantes eram microcromossomos. Dos 11 pares de macrocromossomos, os nos 1, 2, 4 e 5 eram submetacêntricos, e os pares nos 6, 7 e 8 foram descritos como metacêntricos. Além disso, o par de autossomos no 3 era subtelocêntrico, enquanto o par de autossomos no 9 era acrocêntrico. Além disso, o cromossomo sexual Z representa o quarto em tamanho e foi classificado como submetacêntrico, enquanto o cromossomo W apareceu como de tamanho médio e acrocêntrico. Além disso, o padrão de bandamento C (heterocromatina constitutiva) revelou variação em seus tamanhos e ocorrência entre macrocromossomos. Pares nºs 7 e 8 dos autossomos exibiram distribuição incomum de heterocromatina, onde apareceram como totalmente heterocromáticos. Isso pode estar relacionado à origem dos cromossomos sexuais Z e W. No entanto, não há evidências suficientes para ilustrar o aparecimento de autossomos totalmente heterocromáticos. Portanto, não há literatura citogenética disponível que descreva o bandamento C e o cariótipo de Ciconia ciconia, portanto os resultados aqui apresentados são importantes e podem auxiliar no estudo citogenético e no padrão evolutivo de Ciconiiformes.

The use of microsatellite markers in Neotropical studies of wild birds: a literature review

ABSTRACT Despite extensive habitat fragmentation, the Neotropical region possesses 30% of the world´s bird species. Microsatellites have remained one of the most popular genetic markers and have been used in ecological and conservation studies since the 1990's. We conducted a literature review comparing the number of papers published from January 1990 to July 2015 that used microsatellite markers for studies of wild birds in the Neotropical region, USA and some European countries. We assigned the articles to three categories of studies: population genetics, animal behavior/kinship analysis and the development of species-specific bird microsatellite markers. We also compared the studies in the Neotropics that used heterologous versus species-specific markers and provide a list of heterologous markers of utility in multiple birds. Despite the rich bird fauna in the Neotropics, the number of articles published represents only 5.6% of that published by the USA and selected European countries. Within the Neotropical region, Brazil possessed 60.5% of the total papers published, with the remaining 39.5% shared between five countries. We conclude that the lack of specialized laboratories and resources still represents a limit to microsatellite-based genetic studies of birds within the Neotropical region. To overcome these limitations, we suggest the use of heterologous microsatellite markers as a cost-effective and time-effective tool to assist ecological studies of wild birds.

Cross-species amplification of microsatellite markers in Mycteria leucocephala Pennant 1769: Molted feathers as successful DNA source.

DNA from molted feathers is being increasingly used for genetic studies on birds. However, the DNA obtained from such non-invasive sources is often not of enough quantity and quality for isolation of new microsatellite markers. The present study examined the potential of shed feathers of near threatened Painted Stork as a source of its DNA for cross-species amplification of microsatellites. Thirty-one shed feathers of varying conditions (‘good’ and ‘deteriorated’) and sizes (‘large’, ‘intermediate’ and ‘small’) collected in a north Indian population were used to isolate DNA by a standard isopropanol method and 11 microsatellite markers already developed in the Wood Stork were screened for amplification. Nine plucked feathers from two dead Painted Storks were also used to compare the DNA yield and amplification success. The DNA yield of feathers varied significantly in relation to the calamus size and condition. Among molted feathers, ‘good’ and ‘large’ samples provided more DNA than ‘deteriorated’ and ‘small’ ones, respectively. ‘Large’ plucked feathers yielded more DNA than ‘large’ molted feathers. DNA was almost degraded in all the samples and ratio of absorbance at 260/280 nm varied from 1.0 to 1.8, indicating impurity in many samples. Independent of DNA yields, all microsatellites were cross-amplified in all kinds of feathers, with >80% success in different feather categories. It is concluded that the shed feathers can be successfully used to isolate DNA in the Painted Stork and for cross-species amplification of microsatellites.

Polymorphic heterologous microsatellite loci for population genetics studies of the white-faced ibis Plegadis chihi (Vieillot, 1817) (Pelecaniformes, Threskiornithidae)

We screened 44 heterologous microsatellites isolated in species of the families Threskiornithidae, Ciconiidae and Ardeidae for their use in a migratory waterbird, the white-faced ibis Plegadis chihi (Vieillot, 1817) (Threskiornithidae). Of the screened loci, 57 percent amplified successfully and 24 percent were polymorphic. In two breeding colonies from southern Brazil (N = 131) we detected 32 alleles (2-10 alleles/ locus). Average He over all loci and colonies was 0.55, and the combined probability of excluding false parents, 98 percent. There was no departure from HWE in any loci or population. Eru6 and Eru4 loci were in non-random association in the Alvorada colony, and NnNF5 and Eru5 in both populations. AMOVA analysis indicated that most of the genetic diversity was contained within populations. Structure analysis suggested a single population, and F ST value showed weak genetic structuring (F ST = 0.009, p = 0.05). The two populations are apparently connected through gene-flow. The panel of six microsatellites optimized here was sufficiently informative for characterizing the genetic diversity and structure in these natural populations of the white-faced ibis. The information generated could be useful in future studies of genetic diversity, relatedness and the mating system in Plegadis chihi and related species.

Maintenance of syntenic groups between Cathartidae and Gallus gallus indicates symplesiomorphic karyotypes in new world vultures

Similarities between New World and Old World vultures have been interpreted to reflect a close relationship and to suggest the inclusion of both in Accipitridae (Falconiformes). However, deeper analyses indicated that the placement of the New World vultures (cathartids) in this Order is uncertain. Chromosome analysis has shown that cathartids retained a karyotype similar to the putative avian ancestor. In order to verify the occurrence of intrachromosomal rearrangements in cathartids, we hybridized whole chromosome probes of two species (Gallus gallus and Leucopternis albicollis) onto metaphases of Cathartes aura. The results showed that not only were the syntenic groups conserved between Gallus and C. aura, but probably also the general gene order, suggesting that New World vultures share chromosomal symplesiomorphies with most bird lineages.

Morphometric and molecular differentiation between quetzal subspecies of Pharomachrus mocinno (Trogoniformes: Trogonidae)

The resplendent Quetzal (Pharomachrus mocinno) is an endemic Mesoamerican bird species of conservation concern. Within this species, the subspecies P. m. costaricensis and P. m. mocinno, have been recognized by apparent morphometric differences; however, presently there is no sufficient data for confirmation. We analyzed eight morphometric attributes of the body from 41 quetzals: body length, tarsus and cord wing, as well as the length, wide and depth of the bill, body weight; and in the case of the males, the length of the long upper-tail cover feathers. We used multivariate analyses to discriminate morphometric differences between subspecies and contrasted each morphometric attribute between and within subspecies with paired non-parametric Wilcoxon test. In order to review the intraspecific taxonomic status of this bird, we added phylogenetic analysis, and genetic divergence and differentiation based on nucleotide variations in four sequences of mtDNA. The nucleotide variation was estimated in control region, subunit NDH6, and tRNA Glu and tRNA Phe in 26 quetzals from eight localities distributed in five countries. We estimated the genetic divergence and differentiation between subspecies according to a mutation-drift equilibrium model. We obtained the best mutation nucleotide model following the procedure implemented in model test program. We constructed the phylogenetic relationships between subspecies by maximum parsimony and maximum likelihood using PAUP, as well as with Bayesian statistics. The multivariate analyses showed two different morphometric groups, and individuals clustered according to the subspecies that they belong. The paired comparisons between subspecies showed strong differences in most of the attributes analyzed. Along the four mtDNA sequences, we identified 32 nucleotide positions that have a particular nucleotide according to the quetzals subspecies. The genetic divergence and the differentiation was strong and markedly showed two groups within P. mocinno that corresponded to the quetzals subspecies. The model selected for our data was TVM+G. The three phylogenetic methods here used recovered two clear monophyletic clades corresponding to each subspecies, and evidenced a significant and true partition of P. mocinno species into two different genetic, morphometric and ecologic groups. Additionally, according to our calculations, the gene flow between subspecies is interrupted at least from three million years ago. Thus we propose that P. mocinno be divided in two independent species: P. mocinno (Northern species, from Mexico to Nicaragua) and in P. costaricensis (Southern species, Costa Rica and Panama). This new taxonomic classification of the quetzal subspecies allows us to get well conservation achievements because the evaluation about the kind and magnitude of the threats could be more precise. Rev. Biol. Trop. 58 (1): 357-371. Epub 2010 March 01.

El Quetzal (Pharomachrus mocinno) es un ave endémica mesoamericana de interés en conservación. Dentro de esta especie, se reconocen a las subespecies P. m. costaricensis y P. m. mocinno por aparentes diferencias morfométricas, sin embargo, hasta el momento no hay datos suficientes que las confirmen. En este estudio, analizamos ocho rasgos morfométricos de 41 quetzales: la longitud del cuerpo, del tarso y de la cuerda alar, así como la longitud, el ancho y la profundidad del pico, el peso corporal, y en el caso de los machos, la longitud de las plumas cobertoras supracaudales. Usamos análisis multivariados para discriminar diferencias morfométricas entre las subespecies. Comparamos cada rasgo morfométrico dentro y entre las subespecies a partir de comparaciones pareadas con el análisis no-paramétrico de Wilcoxon. Realizamos análisis filogenéticos, y de diferenciación y divergencia genéticas fundamentados en las variaciones nucleotídicas de cuatro secuencias de ADNm con la finalidad de revisar el estatus taxonómico de esta ave. La variación nucleotídica fue estimada en la región control, la subunidad NDH6 y los tRNA Glu y tRNA Phe en 26 quetzales de ocho localidades de cinco países. Estimamos la divergencia y la diferenciación genética entre subespecies con base en el modelo de equilibrio mutación-deriva. Obtuvimos el mejor modelo de mutación nucleotídica siguiendo el procedimiento implementado en el programa Model test. Construimos las relaciones filogenéticas entre las subespecies con máxima parsimonia y máxima verosimilitud usando PAUP, así con estadística Bayesiana. Los análisis multivariados discriminaron dos grupos morfométricos, y los individuos se agruparon de acuerdo con la subespecie a la que pertenecen. Las comparaciones pareadas entre las subespecies mostraron fuertes diferencias en la mayoría de los rasgos analizados. En las cuatro secuencias de ADNmt identificamos 32 posiciones nucleotídicas que tienen un nucleótido particular de acuerdo con la subespecie de quetzal. La divergencia genética y la diferenciación fueron marcadas y mostraron dos grupos dentro de P. mocinno que correspondieron a las subespecies de quetzales. El modelo seleccionado para nuestros datos fue el TVM+G. Los tres métodos filogenéticos usados recuperaron dos clados monofiléticos robustos correspondiendo a cada una de las subespecies. Consideramos que nuestros resultados muestran una significativa y real división de P. mocinno en dos grupos genéticos, morfométricos y ecológicos. Además de acuerdo con nuestras estimaciones, el flujo génico está interrumpido entre las subespecies desde al menos hace tres millones de años. Por ello, proponemos que P. mocinno sea dividido en dos especies independientes: P. mocinno (especie norteña, desde México hasta Nicaragua) y P. costaricensis (especie sureña, Costa Rica y Panamá). Esta nueva clasificación de las subespecies de quetzal permitirá mejores logros en su conservación, dado que la evaluación de la clase y magnitud de las amenazas serán más precisas.

The historical biogeography of Pteroglossus aracaris (Aves, Piciformes, Ramphastidae) based on Bayesian analysis of mitochondrial DNA sequences

Most Neotropical birds, including Pteroglossus aracaris, do not have an adequate fossil record to be used as time constraints in molecular dating. Hence, the evolutionary timeframe of the avian biota can only be inferred using alternative time constraints. We applied a Bayesian relaxed clock approach to propose an alternative interpretation for the historical biogeography of Pteroglossus based on mitochondrial DNA sequences, using different combinations of outgroups and time constraints obtained from outgroup fossils, vicariant barriers and molecular time estimates. The results indicated that outgroup choice has little effect on the Bayesian posterior distribution of divergence times within Pteroglossus, that geological and molecular time constraints seem equally suitable to estimate the Bayesian posterior distribution of divergence times for Pteroglossus, and that the fossil record alone overestimates divergence times within the fossil-lacking ingroup. The Bayesian estimates of divergence times suggest that the radiation of Pteroglossus occurred from the Late Miocene to the Pliocene (three times older than estimated by the ôstandardõ mitochondrial rate of 2% sequence divergence per million years), likely triggered by Andean uplift, multiple episodes of marine transgressions in South America, and formation of present-day river basins. The time estimates are in agreement with other Neotropical taxa with similar geographic distributions.

Genetic diversity and evidence of recent demographic expansion in waterbird populations from the Brazilian Pantanal

The present study determined nuclear and mitochondrial (mtDNA) levels of genetic variability and phylogeographic patterns in breeding populations of Roseate Spoonbill (N = 57), Wood Stork (N = 89), and Jabiru Stork (N = 30), sampled in the Brazilian Pantanal. These species were selected since they are bioindicators of wetlands’ health and are threatened in other parts of their distribution. As they are in close association with this ecosystem, they are appropriate for studying the effects of Pleistocene climatic changes on their demographic patterns. Levels of nuclear genetic diversity in Pantanal populations were not significantly different from those of other populations throughout the American continent, where they are considered threatened or of special concern. Reduced levels of mtDNA genetic diversity were observed in the Central American population of Jabiru Stork in comparison to the Pantanal population. Recent demographic expansion in the Pantanal was markedly evidenced by unimodal patterns of mismatch distribution and Fu’s Fs neutrality test in these three species. We hypothesize that the average time of population expansion (between 30,843 and 14,233 years before present) is associated to responses of these birds’ populations to paleoclimatic changes in these wetlands during the last glaciation period. We recommend special conservation efforts with the Jabiru Stork populations, a genetic monitoring program based on mtDNA, and an ecological characterization of these waterbirds species throughout their distribution range.

O presente estudo determinou níveis de variabilidade nuclear e mitocondrial (DNAmit) e padrões filogeográficos de populações reprodutivas de colhereiro (N = 57), cabeça-seca (N = 89) e tuiuiú (N = 30) do Pantanal brasileiro. Estas espécies foram selecionadas porque são bioindicadoras da integridade das áreas alagáveis e se encontram ameaçadas em outras partes da sua área de distribuição. Devido à sua estreita associação com esse tipo de ecossistema, seu estudo permite verificar os efeitos das mudanças climáticas do Pleistoceno nos seus padrões demográficos. Níveis de diversidade genética nuclear nas populações pantaneiras não diferiram significativamente dos encontrados em outras populações ao longo do continente americano onde são consideradas ameaçadas ou como de especial interesse para conservação. Níveis reduzidos de variabilidade genética DNAmit foram observados nas populações de tuiuiú da América Central, quando comparadas às populações do Pantanal. A expansão demográfica recente das três espécies no Pantanal ficou marcadamente evidenciada pelos padrões unimodais da distribuição das diferenças pareadas e pelo teste de neutralidade de Fs de Fu. Hipotetizamos que o tempo médio de expansão populacional (entre 30.843 e 14.233 anos antes do presente) está associado as respostas destas populações de aves às mudanças climáticas ocorridas nas áreas alagáveis durante o último período de glaciação. Recomendamos, esforços especiais na conservação das populações do tuiuiú, um programa de monitoramento genético baseado no DNAmit e a caracterização ecológica dessas espécies de aves aquáticas ao longo de suas áreas de distribuição.

Genetic structure in Brazilian breeding colonies of the Roseate Spoonbill (Platalea ajaja, Aves: Threskiornithidae)

Roseate Spoonbills (Platalea ajaja, Linnaeus) are wading birds present in two of the most important Brazilian wetlands: the Pantanal wetlands and Rio Grande do Sul marshes. Natural populations of these species have not been previously studied with variable nuclear molecular markers. In order to support decision making regarding the management and conservation of these populations, we estimated and characterized the distribution of genetic variability among five Brazilian breeding colonies. The average observed heterozygosity in Brazilian Roseate Spoonbill populations (Ho = 0.575) did not differ significantly from the value determined in a U.S. wild-caught sample of 15 individuals, using data generated by the same set of microsatellite loci. Considering that the U.S. population underwent a recent reduction in size, we discuss this result supposing that the U.S. population was not genetically affected or that both populations had suffered a bottleneck. Global F(ST) indicated the lack of genetic differentiation among colonies, indicating the occurrence of past and/or present gene flow among them. Analysis of molecular variance revealed that most of the genetic variation is distributed within the colonies. Results are explained by a recent origin of colonies or by high levels of gene flow. Management decisions should take into consideration the fact that, even in the presence of high genetic exchange, ecological adaptations to different environments are important for species survival.

The karyotype of the critically endangered Lear's macaw, Anodorhynchus leari Bonaparte 1856 (Aves, Psittaciformes)

We used conventional chromosomal staining to describe the karyotype of the critically endangered Lear's macaw, Anodorhynchus leari Bonaparte 1856. A diploid number of 2n = 70 and a karyotype similar to that of its congener Anodorhynchus hyacinthinus suggests that chromosomal rearrangements were not the main evolutionary mechanism in the genus.

Demographic history of wood stork (Mycteria americana) Brazilian Pantanal colonies revealed by mitochondrial DNA analysis

We used mitochondrial DNA (mtDNA) sequences to investigate the demographic history of the wood stork (Mycteria americana) populations in the Brazilian Pantanal. Sequences of 390/460 bp fragment of the mtDNA control region were analyzed in 62 wood stork specimens from 8 colonies using neutrality tests, phylogeographic, and coalescent analyses. Population expansion was supported by the significantly negative values of Tajima's (D = -2.071) and Fu's (Fs = -14.544) statistics and the unimodal pattern of mismatch distribution. Nested clade analyses indicated a historic range expansion event and recurrent gene flow that was restricted by isolation by distance as explanations for the haplotype distribution among the sampled colonies. High genetic diversity and the strictly unidirectional gene flow pattern emphasized the conservation importance of preserving the southern Pantanal colonies. Coalescence analyses suggested that northern and southern colonies diverged approximately 6,250 years before the present (YBP), and that their most recent common ancestor was approximately 18,900 YBP. Our results suggest that the contemporary wood stork Pantanal population originated from a more geographically limited founder population. Potential source populations may have occurred in the southern Pantanal or ancestry may reside in populations inhabiting the Brazilian central plateau or areas closer to the equatorial region.

Auditory experience, gene regulation and auditory memories in songbirds

Long-lasting auditory memories of the songs a songbird hears are essential for key aspects of its natural life, including song learning, and territorial and reproductive behaviors. Exposure of songbirds to the songs of other birds of the same species induces marked expression of genes encoding transcription regulators in brain regions that subserve auditory processing. A combination of neuroanatomical, electrophysiological and molecular approaches has been used to investigate the functional significance of this induction response. The emerging theme is that the genomic events triggered by song may represent a regulatory cascade that leads to long-term modification of the physiological properties of song-responsive neurons. It is postulated that such a cascade constitutes a possible basis for the establishment of long-term song auditory memories. Intriguing similarities to more "complex" brains in both structure and function suggest that at least some of the principles uncovered in the songbird brain may be of general applicability.

DNA fingerprinting in the rare black-fronted piping guan Pipile jacutinga (Cracidae, Aves)

Brazilian Cracidae are threatened by heavy environmental degradation and hunting. The Black-fronted piping-guan (Pipile jacutinga) used to inhabit the Atlantic coastal highland forests. Now it occurs in limited forest areas where it is rarely seen. Interative management, including captive breeding, might be an important action for its survival. We present data on DNA fingerprinting using Jeffreys' human minisatlelite probes 33.6 and 33.15. Our results show that this technique is useful for estimating the genetic variability of natural populations and may help to maintain the genetic variability of captive bred individuals of this species. A linkage analysis of the fingerprint profiles in a family with 7 chicks was performed (to estimate the number of independently segregating loci detected in this species) and at least 16 highly polymorphic independent loci were identified for each probe.

Cytogenetic analysis of thrush (Turdus, muscicapidae) species

Foram estudados citogeneticamente 4 espécies de Turdus do Estado de Säo Paulo, a T. leucomela (Tle), T. rufiventris (Tru), T. amaurochalinus (Tam), T. albicollis (Tal) e uma da América do Norte a T. migratorius (Tmi). Foram utilizadas técnicas convencionais para obtençäo de metáfases de células de medula óssea, para biometria e classificaçäo dos cromossomos. Verificou-se a existência de 2 grupos cariotípicos, denominados tipo I e II. No primeiro estäo incluídas as 3 espécies brasileiras (Tam, Tru e Tal) e as espécies norte americana (Tmi) e no segundo grupo a espécie Tle cuja diferença principal diz respeito ao macroautossomo telocêntrico de número 6 no tipo I e submetacêntrico (e de número 4 pela classificaçäo proposta) no tipo II. Quanto aos demais macroautossomos, estes säo semelhantes em morfologia e porte em todas as espécies analisadas. Observou-se 2n= ñ 78 nas espécies Tal e Tru verificando-se nas demais 2n = ñ 80. O par sexual constitui-se de Z metacêntrico, o 4§ em porte na Tmi, Tam, Tal e Tle e o 3§ na Tru, o W, submetacêntrico e o 8§ na Tmi, Tam, Tru e Tle e 7§, quanto ao tamanho, na Tal. Comparando os resultados com espécies de Turdus européias e analisadas por Ryttman (1984) verificou-se que o cariótipo da T. pilaris enquadra-se no Tipo I e o da T. merula no tipo II. Dados de literatura indicam ser mais frequente o tipo II para o gênero Turdus

Karyotype of teh little blue macaw Cyanopsitta spixii (psittaciformes, aves)

A Ararinha azul (Cyanopsitta) considerada extinta na natureza foi analisada por método citogenético com objetivo de sexagem e estudos evolutivos. A espécie, com 2n = ñ 70 cromossomos, NF = 88, apresentou heteromorfismo de cromossomos sexuais o que permitiu a sexagem de quatro aves mantidas em cativeiro dentro do programa de cooperaçäo com o "Comitê da Arara Spixii