Gene and species trees reveal mitochondrial and nuclear discordance in the Drosophila cardini group (Diptera: Drosophilidae)

Abstract. The Drosophila cardini group includes 15 species, which are subdivided into the D. cardini and D. dunni subgroups. Although many phylogenetic hypotheses have been proposed for this group during the last five decades (based on patterns of reproductive isolation, morphology of male genitalia, chromosomal inversions, isozyme variation, or molecular sequence data), these are mostly discordant with each other. We aimed to clarify some of the evolutionary patterns related to the origin of this incongruence, while also attempting to provide a better-supported phylogenetic hypothesis for the D. cardini subgroup. For this purpose, sequences from three mitochondrial and three nuclear loci were gathered for at least eight species, and both individual gene trees and joint species tree estimates were evaluated. Although there was concordance among gene trees within each of the nuclear and mitochondrial sets, considerable incongruence was revealed in the comparisons between these two data sets. The branching position of D. neocardini was the main source of incongruence, and species trees reconstructed using different approaches with and without this species were particularly incongruent. In addition to providing a better approximation of the evolutionary history of the D. cardini group, this study suggests that incomplete lineage sorting or introgression may be biasing previous species tree estimates. More generally, the results also suggest that the use of supermatrix methods can lead to an overestimation of support for the inferred relationships, and highlight the potential effects of different taxon sampling strategies in phylogenetic reconstruction.     

Cytogenetics of the South American DROSOPHILA MULLERI Complex: The MARTENSIS Cluster. More Interspecific Sharing of Inversions

Three Drosophila mulleri complex species have been found to be endemic to the cactus regions of northern South America. The three species are morphologically distinct, and no hybridization between species has been found in either the laboratory or nature. An analysis of their salivary gland chromosomes revealed that they evolved from a single cytological population, subspecies F of the ancestor of the D. mulleri complex, and comprise a cluster of sister species that are homozygous for inversions not found elsewhere. Within the martensis cluster are also found several inversions that are unique for these species, but have segregated out among the three species. Thus 2f² is found in martensis and starmeri, but not in uniseta, while 2t⁶, 3v and 3w are found in starmeri and uniseta, but not in martensis. This is analogous to the situation found within the remainder of the mulleri complex species, all the members of which share homozygosity for six other inversions. An interpretation of the cytological evolution of the martensis cluster is proposed.     

The actin loci in the genus Drosophila: establishment of chromosomal homologies among distantly related species by in situ hybridization

We have mapped by in situ hybridization the actin genes in selected, distantly related Drosophila species, using the 5C actin gene of D. melanogaster as a probe. In all species six dispersed actin loci were observed, probably corresponding to six genes, and they were similarly distributed among the chromosomes. In conjunction with previously available genetic and cytogenetic evidence, this consistent pattern of actin gene distribution reinforces the hypothesis that the chromosomal elements have maintained their essential identities throughout Drosophila evolution, and permits identification of these elements in very diverse species. Conservation of the actin loci also offers fixed points for the analysis of chromosomal inversions and other rearrangements.     

The LTR retrotransposon micropia in the cardini group of Drosophila (Diptera: Drosophilidae): a possible case of horizontal transfer

The presence of the micropia retroelement from the Ty1-copia family of LTR retroelements was investigated in three species of the Drosophila cardini group. Southern blot analysis suggested the existence of at least four micropia copies in the genomes of D. cardinoides, D. neocardini and D. polymorpha populations. The high sequence similarity between dhMiF2 and Dm11 clones (micropia retroelements isolated from D. hydei and D. melanogaster, respectively) with micropia sequences amplified from D. cardini group genome supports the hypothesis that this retroelement plays an active role in horizontal transfer events between D. hydei and the D. cardini group. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

CHROMOSOMAL REPATTERNING AND LINKAGE GROUP CONSERVATION IN MOSQUITO KARYOTYPIC EVOLUTION

Chromosome number and morphology in mosquitoes is remarkably uniform: virtually all mosquitoes have a diploid chromosome number of six (2N = 6), and their chromosomes are invariably metacentric or submetacentric. Numerical changes obviously have not been important in mosquito chromosomal evolution, and because of the morphological similarity of their chromosomes, it appears that structural changes have played little or no role in mosquito karyotypic evolution. The goal of the present study was to identify the types and relative numbers of chromosomal changes in mosquito evolution and to extend the comparison where possible to the higher diptera. To do this, we compared the enzyme linkage maps of six species of Aedes to each other and to enzyme maps of seven other mosquito species and to Drosophila melanogaster. Our results indicate that Aedes chromosomes have been modified by inversions, most which were paracentric, and by translocations, most which were Robertsonian. Intrageneric comparison of Aedes enzyme maps also revealed groups of linked enzyme loci whose integrity has been maintained throughout Aedes evolution (conserved linkages/syntenies). Intergeneric comparisons of Aedes enzyme maps with those of species in the genera Culex, Anopheles, and Toxorhynchites disclosed conserved associations of enzyme loci between mosquito genera. These findings lead us to postulate that the ancestral mosquito karyotype consisted of six chromosomal elements which, other than being combined in different ways in various mosquito groups, have remained essentially intact during mosquito evolution. Furthermore, the identification of groups of linked enzyme loci common to mosquitoes and to D. melanogaster indicates that linkage group conservation may characterize the karyotypic evolution of all dipteran insects.     

The <Emphasis Type="Italic">Drosophila bipectinata</Emphasis> species complex: phylogenetic relationship among different members based on chromosomal variations

Making interspecific hybridizations, where possible remains an unparalleled option for studying the intricacies of speciation. In the Drosophila bipectinata species complex comprising of four species, namely D. bipectinata, D. parabipectinata, D. malerkotliana and D. pseudoananassae, interspecific hybrids can be obtained in the laboratory, thus bequeathing an ideal opportunity for studying speciation and phylogeny. With the view of investigating the degree of divergence between each species pair, we planned to study the polytene chromosomes of the F ₁ hybrids, as it would mirror the level of compatibility between the genomes of the parental species. Two sets of crosses were made, one involving homozygous strains of all four species from India and the other including homozygous strains from different places across the globe. Polytene chromosomes of F ₁ larvae from both sets of crosses had similar configurations. In F ₁ larvae from crosses involving D. bipectinata, D. parabipectinata and D. malerkotliana, complex configurations (depicting overlapping inversions) could be detected in different arms. However, they were fairly synapsed, indicating that the differences are only at the level of gene arrangements. The polytene chromosomes of larvae obtained by crossing D. pseudoananassae with the other three species were very thin with gross asynapsis in all the arms, demonstrating that the genome of D. pseudoananassae is widely diverged from rest of the species. The overlapping inversions (reflected in complex configuration), are inferred in the light of earlier chromosomal studies performed in this complex.     

Cytogenetic studies of Chironomus circumdatus from India (Diptera: Chironomidae)

Chironomus circumdatus is one of the most common and wide-spread species in India. Cytogenetic studies pertaining to the mitotic and polytene chromosomes, nucleolar organizer regions, C-banding and naturally occurring chromosomal polymorphism have been carried out for the first time in this Indian species. Altogether seven inversions comprising six paracentric and one pericentric are detected in the Indian populations. The distribution of inversions in relation to different environmental conditions is discussed.     

Oogenesis and chromosomal heterozygosity in the thelytokous midge,Lundstroemia parthenogenetica (Diptera,Chironomidae)

The thelytokous triploid midge Lundstroemia parthenogenetica (Diptera: Chironomidae:Chironominae), 3n = 9, is always found to be heterozygous for two inversions and a deletion. Parthenogenesis is maintained by an apomiotic restitutional oogenesis as found in the subfamily Orthocladiinae. This mechanism is compared with other groups exhibiting a restitutional or disordered first division. Possible modes of evolution of the apomictic restitutional system, origin of triploidy in apomictic forms, and chromosomal polymorphisms are discussed.     

INVERSION LENGTH AND BREAKPOINT DISTRIBUTION IN THE DROSOPHILA BUZZATII SPECIES COMPLEX: IS INVERSION LENGTH A SELECTED TRAIT?

Length and position of breakpoints are characteristics of inversions that can be precisely determined on the polytene chromosomes of Drosophila species, and they provide crucial information about the processes that govern the origin and evolution of inversions. Eighty-six paracentric inversions described in the Drosophila buzzatii species complex and 18 inversions induced by introgressive hybridization in D. buzzatii were analyzed. In contrast to previous studies, inversion length and breakpoint distribution have been considered simultaneously. We conclude that: (1) inversion length is a selected trait; rare inversions are predominantly small while evolutionarily successful inversions, polymorphic and fixed, are predominantly intermediate in length; a nearly continuous variation in length, from small to medium sized, is found between less and more successful inversions; (2) there exists a significant negative correlation between length and number of polymorphic inversions per species which explains 39% of the inversion length variance; (3) natural selection on inversion length seems the main factor determining the relative position of breakpoints along the chromosomes; (4) the distribution of breakpoints according to their band location is non-random, with chromosomal segments that accumulate up to eight breakpoints.     

Chromosomal polymorphism in two species of Hypancistrus (Siluriformes: Loricariidae): an integrative approach for understanding their biodiversity

Structural chromosome changes are widely described in different vertebrate groups and generate genetic, phenotypic and behavioral diversity. During the evolution of loricariids, several rearrangements (fissions, fusions, inversions) seem to have occurred. Hypancistrus, tribe Ancistrini, are highly demanded for fishkeeping around the world. In this tribe, the diploid chromosome number 2n = 52 is considered a synapomorphy, and paracentric-type inversions appear to be involved in the chromosomal evolution of the tribe. The present study investigated the karyotypes of H. zebra and H. cf. debilittera using cytogenetic, classical and molecular tools, as well as DNA barcoding. Data reveal that, although diploid number in both species corroborates the proposed synapomorphy for the tribe, there is a complex karyotype dynamics, reflected in the intense chromosomal polymorphism, resulting from rearrangements involving ribosomal regions (5S and 18S rDNA), which are suggested to be paracentric inversions. Besides, DNA barcode confirms reciprocal monophyletism between the species, validating the existence of two species, only. This scenario, coupled with genomic instability caused by exogenous sequences such as Rex-3 retrotransposons and the species’ sedentary lifestyle, which helps the fast polymorphism fixation, may reflect different phenotypic color patterns in natural populations, as observed in H. cf. debilittera.     

Cytogenetic Mapping of Enzyme Loci on Chromosomes J and U of DROSOPHILA SUBOBSCURA

Enzyme loci located on chromosome J and U were mapped cytologically by means of a Y translocation technique. A linkage map of the two chromosomes was established in a parallel experiment and the recombination frequency in different regions of the chromosomes determined. A comparison of the cytogenetic localization of the enzyme genes in D. subobscura and D. melanogaster indicates that many paracentric inversions must have taken place in the course of divergent evolution. However, no displacements of genes from one element to another due to pericentric inversions, reciprocal translocations or transposing elements can be observed. In spite of the large number of structural rearrangements that have occurred in the phylogeny of the genus Drosophila, gross similarities of banding pattern in homologous regions of the chromosomes of the two species become apparent.     

Comparative cytogenetic analysis of four species of <Emphasis Type="Italic">Dendropsophus</Emphasis> (Hylinae) from the Brazilian Atlantic forest

We conducted a cytogenetic study of four hyline frog species (Dendropsophus elegans, D. microps, D. minutus and D. werneri) from southern Brazil. All species had 2n = 30 chromosomes, with interspecific and intraspecific variation in the numbers of metacentric, submetacentric, subtelocentric and telocentric chromosomes. C-banding and fluorochrome staining revealed conservative GC-rich heterochromatin localized in the pericentromeric regions of all species. The location of the nucleolus organizer regions, as confirmed by fluorescent in situ hybridization, differed between species. Telomeric probes detected sites that were restricted to the terminal regions of all chromosomes and no interstitial or centromeric signals were observed. Our study corroborates the generic synapomorphy of 2n = 30 chromosomes for Dendropsophus and adds data that may become useful for future taxonomic revisions and a broader understanding of chromosomal evolution among hylids.     

Chromosomal number and evolution in two Brazilian species of Callithrix

The Karyotypes of the Brazilian marmosets C. jacchus and C. penicillata are very similar. Both have 2n = 46. C. jacchus has one more metacentric pair and C. penicillata has one more submetacentric pair, whilc the sex chromosomes show distinet morphological differences.On the basis of the chomosome complement of these species some considerations about the process of chromosomal evolution are presented.A short comparative study of these and other species of the same genus shows that basic mechanisms such as centric fusion and pericentric inversions can explain the observed morphologic chromosomal variations.     

Individual inversions or their combinations: which is the main selective target in a natural population of Drosophila subobscura?

It is generally accepted that chromosomal inversions have been key elements in adaptation and speciation processes. In this context, Drosophila subobscura has been, and still is, an excellent model species due to its rich chromosomal polymorphism. In this species, many analyses from natural populations have demonstrated the adaptive potential of individual inversions (and their overlapped combinations, the so‐called arrangements). However, little information is available on the evolutionary role of combinations generated by inversions located in homologous and nonhomologous chromosomes. The aim of this research was to ascertain whether these combinations are also a target for natural selection. For this objective, we have studied the inversion composition of homologous and nonhomologous chromosomes from a D. subobscura sample collected in a well‐studied population, Mount Avala (Serbia). No significant deviation from H‐W expectations was detected, and when comparing particular karyotypic combinations, likelihood ratios close to 1 were obtained. Thus, it seems that for each pair of homologous chromosomes inversions no deviation from randomness was detected. Finally, no linkage disequilibrium was observed between inversions located in different chromosomes of the karyotype. For all these reasons, it can be assumed that, at the cytological level, the individual inversions rather than their combinations in different chromosomes are the main target of selection.     

Molecular Cytogenetic Characterization of Multiple Intrachromosomal Rearrangements in Two Representatives of the Genus Turdus (Turdidae,Passeriformes)

Turdus rufiventris and Turdus albicollis, two songbirds belonging to the family Turdidae (Aves, Passeriformes) were studied by C-banding, 18S rDNA, as well as the use of whole chromosome probes derived from Gallus gallus (GGA) and Leucopternis albicollis (LAL). They showed very similar karyotypes, with 2n = 78 and the same pattern of distribution of heterochromatic blocks and hybridization patterns. However, the analysis of 18/28S rDNA has shown differences in the number of NOR-bearing chromosomes and ribosomal clusters. The hybridization pattern of GGA macrochromosomes was similar to the one found in songbirds studied by Fluorescent in situ hybridization, with fission of GGA 1 and GGA 4 chromosomes. In contrast, LAL chromosome paintings revealed a complex pattern of intrachromosomal rearrangements (paracentric and pericentric inversions) on chromosome 2, which corresponds to GGA1q. The first inversion changed the chromosomal morphology and the second and third inversions changed the order of chromosome segments. Karyotype analysis in Turdus revealed that this genus has derived characteristics in relation to the putative avian ancestral karyotype, highlighting the importance of using new tools for analysis of chromosomal evolution in birds, such as the probes derived from L. albicollis, which make it possible to identify intrachromosomal rearrangements not visible with the use of GGA chromosome painting solely.     

Chromosomal structure of populations of drosophila flavopilosa studied in larvae collected in their natural breeding sites

Summary D. flavopilosa Frey, is a neotropical species of Drosophila living in Chile that, together with 13 other species, has been included by Wheeler, Takada und Brncic (1962) in the flavopilosa species group. Despite the fact that it has not been possible to breed this species in the laboratory, the circumstance that its habitat is well known, has allowed the study of the chromosomal structure of some natural populations of this species, analyzing larvae directly taken to the laboratory from their natural breeding sites: the flowers of the solanacean Cestrum parqui L'Héritier.The present paper describes the mitetic and salivary gland chromosomes of D. flavopilosa, and gives a composite map of the Standard gene arrangement. In central Chile, natural populations of this species are polymorphic with respect to the gene orders in their chromosomes, due to the presence of four independent inversions, all located in one of the six chromosomes (the right arm of the V-chromosome). Quantitative data on the distribution of the inversions shows that there is an altitudinal gradient in the frequencies of two of the four inversions. Heterozygotes for Inversion A, are more frequent at high altitudes than at sea level. On the contrary, heterozygotes for inversion B, are abundant at sea level, but practically disappear at high altitudes. These altitudinal clines have been observed in two valleys near Santiago, Chile, which run from the Andes mountains to the Pacific coast.The work reported in this article has been carried out under Contract AT (30-1) 2465 US Atomic Energy Commission, and partially supported by Grants from the University of Chile, Faculty of Medicine and the Rockefeller Foundation under a joint program.     

Species-specific localization of DNA from pericentromeric heterochromatin on polytene chromosomes in the salivary gland cells and 3D-nuclear organization nurse cells in Drosophila virilis and Drosophila kanekoi (Diptera: Drosophilidae)

Microdissection of the chromocenter of D. virilis salivary gland polytene chromosomes has been carried out and the region-specific DNA library (DvirIII) has been obtained. FISH was used for DvirIII hybridization with salivary gland polytene chromosomes and ovarian nurse cells of D. virilis and D. kanekoi. Localization of DvirIII in the pericentromeric regions of chromosomes and in the telomeric region of chromosome 5 was observed in both species. Moreover, species specificity in the localization of DNA sequences of DvirIII in some chromosomal regions was detected. In order to study the three-dimensional organization of pericentromeric heterochromatin region of polytene chromosomes of ovarian nurse cells of D. virilis and D. kanekoi, 3S FISH DvirIII was performed with nurse cells of these species. As a result, species specificity in the distribution of DvirIII signals in the nuclear space was revealed. Namely, the signal was detected in the local chromocenter at one pole of the nucleus in D. virilis, while the signal from the telomeric region of chromosome 5 was detected on another pole. At the same time, DvirIII signals in D. kanekoi are localized in two separate areas in the nucleus: the first belongs to the pericentromeric region of chromosome 2 and another to pericentromeric regions of the remaining chromosomes.     

Genetic differentiation in theAedes atropalpus complex. II. Chromosomal divergence betweenAe. atropalpus andAe. epactius

Analysis of meiotic chromosomes from hybrids betweenAedes atropalpus andAe. epactius has revealed that the two species are fixed for alternate arrangements of four inversions: a paracentric inversion of chromosome 1, two paracentric inversions of chromosome 2, and a pericentric inversion of chromosome 3. This chromosomal heterozygosity in the interspecific hybrids has resulted in extensive meiolic chromosomal asynapsis. Dicentric bridges, acentric fragments, and chromosomal breakage were also associated with the heterozygous inversions. This disruption of meiosis was sufficient to account for the partial sterility observed in interspecific hybrids. No chromosomal polymorphisms, aberrations, or reduction in fertility was observed in parental strains of intraspecific hybrids of the two species.     

A comparative cytogenetic study of five piranha species (Serrasalmus,Serrasalminae) from the Amazon basin

Cytogenetic studies were conducted on five piranha species belonging to the genus Serrasalmus, subfamily Serrasalminae (Serrasalmus altispinis, S. compressus, S. elongatus, S. manuelli, and S. spilopleura). All the species were collected in the Amazon basin: confluence of Negro and Solimõoes Rivers (CatalãoLake), Solimões River (Marchantaria Island – Camaleão Lake), Uatumã River (Hydroelectric Power Station of Balbina), and Pitinga River (Hydroelectric Power Station of Pitinga). All the five species possess 2n = 60 chromosomes with 5–12 subtelo- and acrocentric chromosomes bearing nucleolar organizer regions. A proximal C-band positive heterochromatin block was evident on the long arms of a medium-sized metacentric chromosome pair in all the analized species, thus making it a cytogenetic marker for the genus. It is hypothesized that 2n = 60 chromosomes represents a derived feature in terms of the chromosomal evolution of piranhas because the basal lineages possess 2n = 62. Both Robertsonian centric fusion and non-Robertsonian rearragements such as pericentric inversions seem implicated in the chromosomal evolution of this group.     

Chromosomal locations of U2 snDNA clusters in <Emphasis Type="Italic">Megaleporinus</Emphasis>, <Emphasis Type="Italic">Leporinus</Emphasis> and <Emphasis Type="Italic">Schizodon</Emphasis> (Characiformes: Anostomidae)

The U small nuclear RNA (U snRNA) genes comprise a multigene family and are required for splicing of pre-mRNA. In this paper, we aimed to study the chromosomal location of the U2 snRNA gene in Megaleporinus, Leporinus and Schizodon species, which constitute interesting models for the study of repetitive DNA and genomic evolution in fish once the group comprises species with and without heteromorphic sex chromosomes. The all six species showed 2n?=?54 chromosomes: Megaleporinus elongatus, Megaleporinus macrocephalus, Leporinus striatus, Leporinus friderici, Schizodon borelli and Schizodon isognathus. The U2 snDNA clusters were evident in only one medium-sized submetracentric pair in all analyzed species and this may represent a condition shared by Anostomidae family.