Evolutionary biology: evidence for sympatric speciation?

Sympatric speciation is difficult to demonstrate in nature and remains a hotly debated issue. Barluenga et al. present a case of putative sympatric speciation for two cichlid species in the Nicaraguan crater lake Apoyo, but they overlook or reinterpret some key published information on the system. Although sympatric speciation is possible in theory, we show here that, when this information is taken into account, the results of Barluenga et al. do not provide conclusive evidence for sympatric speciation: this is because the null hypothesis of multiple invasion with introgression cannot be rejected.

Social structure of pilot whales revealed by analytical DNA profiling.

Long-finned pilot whales swim in large, extremely cohesive social groups known as pods. Molecular typing revealed that pod members form a single extended family. Mature males neither disperse from nor mate within their natal pods, a situation unusual for mammals. Such behavior could be explained in terms of inclusive fitness benefits gained by adult males helping the large number of female relatives with which they swim.

Evolution of transcriptional regulation.

Major advances have been made in understanding the evolution of transcriptional regulation using microevolutionary and macroevolutionary experimental approaches. The roles of stabilising selection and compensatory changes in an enhancer region have been elucidated in Drosophila. The molecular dynamics of regulatory alleles have been studied in plants. Evidence is accumulating for the involvement of regulatory evolution in morphological changes between closely related species, as well as in major changes of body plans.

Simple sequences.

Simple sequences (or microsatellites) are stretches of monotonous repetitions of short (1-5bp) nucleotide motifs that are distributed across the whole genome in eukaryotes. They are probably generated by slippage during replication and their primary mutation rate seems to be controlled predominantly by the efficiency of the mismatch repair system. Although most mutations in simple sequence loci appear to be neutral, some mutations in particular stretches have been implicated as having a role in human genetic diseases.

Chromosomal homogeneity of Drosophila ribosomal DNA arrays suggests intrachromosomal exchanges drive concerted evolution.

BACKGROUND: The individual copies of tandemly repeated genes, such as ribosomal DNA (rDNA), evolve coordinately within a species. This phenomenon has been called concerted evolution, and is thought to be caused by sequence-homogenizing mechanisms, such as gene conversion or unequal crossing-over between individual copies of the gene family. As these processes would act between the arrays on homologous and non-homologous chromosomes, the whole family of repeats would be expected to undergo homogenization in a given interbreeding population. RESULTS: In order to study the homogenization process, we have examined polymorphisms within the internal transcribed spacer (ITS) of the rDNA in populations of Drosophila melanogaster at the sequence level, by DNA sequencing and temperature-gradient gel electrophoresis. Among 84 ITS clones sequenced from five different wild-type strains, we found three polymorphic sites that are apparently in the process of homogenization. However, these three sites, as well as combinations of them, occurred at different frequencies in the different strains. Moreover, temperature-gradient gel electrophoresis analysis of an ITS fragment including these three sites shows that single chromosomes from locally interbreeding populations can harbor rDNA arrays that are largely homogenized for different sequence variants. CONCLUSIONS: The presence of chromosomal arrays that are homogeneous for different variants in interbreeding populations of Drosophila melanogaster indicates that there is little recombination between the chromosomes while new mutations are being homogenized along the individual arrays. The most likely explanation for this finding is that intrachromosomal recombination events occur at much higher rates than recombination between homologous chromosomes. Thus, the first step of the homogenization process would occur mainly within chromosomal lines. Such behavior of tandem repeat arrays suggests a simple explanation of how selection can act on a multigene family, namely by acting on whole chromosomally confined repeat arrays rather than on individual repeat units.

A genetic uncertainty problem.

The existence of genes that, when knocked out, result in no obvious phenotype has puzzled biologists for many years. The phenomenon is often ascribed to redundancy in regulatory networks, caused by duplicated genes. However, a recent systematic analysis of data from the yeast genome projects does not support a link between gene duplications and redundancies. An alternative explanation suggests that genes might also evolve by very weak selection, which would mean that their true function cannot be studied in normal laboratory experiments. This problem is comparable to Heisenberg's uncertainty relationship in physics. It is possible to formulate an analogous relationship for biology, which, at its extreme, predicts that the understanding of the full function of a gene might require experiments on an evolutionary scale, involving the entire effective population size of a given species.

Evolution of segmentation genes in insects.

Systematic genetic analysis of the segmentation process in Drosophila has established a paradigm for the molecular control of the formation of metameric segments. However, it has been suggested that some of the mechanisms involved in this process in Drosophila are uniquely adapted to the syncytial mode of embryogenesis in such higher dipterans. A particularly contentious problem is the role of early segmentation genes in short-germ insects, in which development proceeds by sequential addition of segments in a cellular environment. However, analysis of the expression of presumptive homologs of segmentation genes in holometabolous short-germ insects suggests that they do indeed have a role in segmentation and that the Drosophila paradigm may be more widely applicable than is usually assumed. Most interestingly, these results suggest that the molecular mechanisms of pattern formation in noncellular and in cellular environments may not be as radically different as it is often thought to be.

Simple DNA sequences of Drosophila virilis isolated by screening with RNA.

We isolated clones from different types of genomic Drosophila virilis libraries by screening with mRNA labelled in vitro. The DNA regions of the clones showing cross-hybridization with the RNA were sequenced. All of them contained different types of simple sequences, which most probably were solely responsible for the hybridization. We show that simple sequences and their transcription are not a phenomenon that is restricted to Drosophila. Simple sequences can be detected in all eucaryotes and at least three types (poly[d(G--T)] X poly[d(C--A)], poly[d(G--A)] X poly-[d(C--T)] and poly(dA) X poly(dT] are also widely transcribed.

Conservation of major nuclease S1-sensitive sites in the non-conserved spacer region of ribosomal DNA in Drosophila species.

We have analysed nuclease S1-sensitive sites in cloned ribosomal DNA repeats from Drosophila melanogaster, D. hydei and D. virilis. All species contain major S1-sensitive sites in the spacer near the region of transcription termination, albeit with somewhat different positions and sensitivities. The same sites are also sensitive to the single-strand specificity of Bal31 nuclease at neutral pH. Additional major sites exist at each end of the intervening sequence within the 28 S gene of non-transcribed intervening-sequence-positive ribosomal DNA units of D. hydei. Only minor sites, however, were detected in the Pol I promoter regions. This is in contrast to Pol II transcribed genes, where S1 hypersensitivity becomes apparent at the 5' ends during gene expression. We have sequenced and mapped the S1 sites in the D. hydei spacer. They consist mainly of alternating A and T nucleotides that could form small cruciform structures. Cross-hybridization at low stringencies between the relevant S1-sensitive spacer regions of the three species indicates that the sites lie within very divergent sequences. We discuss the potential functional significance of S1 sites in rDNA spacers and intervening sequences, and the manner in which they might be maintained during rDNA sequence divergence.

Evolutionary divergence of promoters and spacers in the rDNA family of four Drosophila species. Implications for molecular coevolution in multigene families.

The organization and sequence of the rDNA multigene family of four Drosophila species (melanogaster, orena, virilis and hydei) have been compared in order to understand the quality and quantity of the differences which are involved with interspecific divergence of promoters and the polymerase I complexes (molecular coevolution). Each species has an intergenic spacer (IGS) made up of subrepeats which contain duplications of the promoter. Major structural and point-mutational differences exist, most of which have been spread by unequal crossingover through the family and species. Structural differences involve the types, lengths and copy-number of the IGS subrepeats, and the lengths and position of "unique" regions between blocks of repeats. The 240 base-pair repeat array shared by D. melanogaster and D. orena has been replaced by a 220 base-pair repeat, and the 95 and 330 base-pair arrays are absent altogether in D. virilis and D. hydei. The length of the "unique" region between the 240/220 base-pair arrays and the start of transcription varies, with the unusual situation of the last of the 220 repeats ending at the external transcribed spacer (ETS) boundary in D. virilis. Other structural differences involve regions of high cryptic simplicity arising from slippage in D. virilis and D. hydei IGSs. Sequence analysis of IGS and the ETSs indicates that the rDNA is not uniformly divergent throughout its length. Apart from the genes, there are regions of relatively high conservation covering the promoter regions and at some but not all potential RNA processing sites. The conserved promoter regions are more extensive within each pair of species D. melanogaster versus D. orena and D. virilis versus D. hydei, in keeping with their phylogenetic distances. Slippage-like mechanisms are involved with large numbers of deletions/insertions that make up the ETS differences between the species. Patterns of shared mutations between IGS subrepeats indicate stages of transition during rDNA differentiation by continual homogenization. The simultaneous operation of different turnover mechanisms, at different periodicities and rates, generates a complex picture of reorganization, some of which would influence the process of molecular coevolution in the family.

Polymorphism and locus-specific effects on polymorphism at microsatellite loci in natural Drosophila melanogaster populations.

We have studied the natural variation at microsatellite loci in two African and five non-African populations of Drosophila melanogaster. Ten dinucleotide simple sequence loci were cloned from chromosomally mapped P1 clones and typed for single individuals from isofemale lines of the respective populations. We find that the African populations harbor the largest degree of diversity, while the non-African populations show a lower diversity. This supports previous results that D. melanogaster originated in Africa and spread across the rest of the world in historic times. Using genetic distance measures, we find also a distinct population subdivision between the non-African populations. Most interestingly, we find for some loci in some populations a strongly reduced variability, which cannot be explained by bottleneck effects. Employing a conservative test based on the variance in repeat number, we find that at least one locus in one population deviates significantly from the expectations of mutation-drift equilibrium. We suggest that this may be due to a recent selective sweep in this chromosomal region that may have been caused by a linked locus that was involved in local adaptation of the population.

Differential regulation of target genes by different alleles of the segmentation gene hunchback in Drosophila.

hunchback (hb) is a key regulatory gene in the early segmentation gene hierarchy of Drosophila. It codes for a transcription factor of the Cys2-His2 zinc finger type and shows two separate zinc finger domains in its coding region. hb forms a morphogenetic gradient in the middle of the embryo that is required for setting the spatial boundaries of several target genes. We have analyzed the molecular lesions found in the different hb alleles and have studied the differential effects of these alleles on a number of such target genes. We find that in mutants in which the HB protein lacks a functional second finger domain, the regulation of the target genes Krüppel (Kr) and knirps (kni) is differentially affected. While this domain is required for the correct regulation of Kr, it is not necessary for the repression of kni. Furthermore, mutations affecting this domain lead to a decreased protein stability. The integration of the expression pattern of target genes was found to be distorted in a second class of mutants between the two finger domains which lead to gain of function or neomorphic phenotypes. The effects of these mutations were studied in detail and it was found that they fall into two classes, the first one interfering with the function of the maternal hb product, the second leading to a delayed segmentation. The function of the latter class appears to be linked to the secondary expression of hb in the parasegment 4 (PS4) stripe at blastoderm stage.

Large number of replacement polymorphisms in rapidly evolving genes of Drosophila. Implications for genome-wide surveys of DNA polymorphism.

We present a survey of nucleotide polymorphism of three novel, rapidly evolving genes in populations of Drosophila melanogaster and D. simulans. Levels of silent polymorphism are comparable to other loci, but the number of replacement polymorphisms is higher than that in most other genes surveyed in D. melanogaster and D. simulans. Tests of neutrality fail to reject neutral evolution with one exception. This concerns a gene located in a region of high recombination rate in D. simulans and in a region of low recombination rate in D. melanogaster, due to an inversion. In the latter case it shows a very low number of polymorphisms, presumably due to selective sweeps in the region. Patterns of nucleotide polymorphism suggest that most substitutions are neutral or nearly neutral and that weak (positive and purifying) selection plays a significant role in the evolution of these genes. At all three loci, purifying selection of slightly deleterious replacement mutations appears to be more efficient in D. simulans than in D. melanogaster, presumably due to different effective population sizes. Our analysis suggests that current knowledge about genome-wide patterns of nucleotide polymorphism is far from complete with respect to the types and range of nucleotide substitutions and that further analysis of differences between local populations will be required to understand the forces more completely. We note that rapidly diverging and nearly neutrally evolving genes cannot be expected only in the genome of Drosophila, but are likely to occur in large numbers also in other organisms and that their function and evolution are little understood so far.

Zebrafish zic1 expression in brain and somites is affected by BMP and hedgehog signalling.

Here we report the expression of the zebrafish zic1 gene, also known as opl, a homologue to other vertebrate Zic genes and the Drosophila odd-paired gene. zic1 expression starts during epiboly stages in lateral parts of the neural plate and eventually comes to lie in dorsal regions of the developing brain following the morphogenetic movements of neural tube formation. To address the question whether BMP2 signalling affects the extent of zic1 expression, we analysed swirl and chordino mutant embryos. Expanded Zic1 expression in swirl and reduced expression in chordino as well as in bmp2 injected embryos suggest that BMP2 and its antagonists define the extent of zic1 expression in the neural plate. By searching for factors responsible for the dorsal restriction of Zic1 expression, we found zic1 expression is eliminated in sonic hedgehog (shh) injected embryos. The most rostral expression however is not affected by Shh suggesting that Shh plays a different role in dorso-ventral patterning of the future telencephalon. During somitogenesis zic1 is expressed in the dorsal most part of the developing somites. Here zic1 marks cells that are distinct from the main adaxial somite portion, the future myomere. zic1 expression in the somites is expanded in swirl but reduced in shh injected embryos, suggesting these factors have opposing activity in dorsoventral patterning of the somites. Later, a growing mass of zic1 expressing cells occurs in a dorsal mesenchyme that eventually invades the dorsal fin fold, suggesting a somitic contribution to the dorsal fin mesenchyme.

Elimination of EVE protein by CALI in the short germ band insect Tribolium suggests a conserved pair-rule function for even skipped.

The question of the degree of evolutionary conservation of the pair-rule patterning mechanism known from Drosophila is still contentious. We have employed chromophore-assisted laser inactivation (CALI) to inactivate the function of the pair-rule gene even skipped (eve) in the short germ embryo of the flour beetle Tribolium. We show that it is possible to generate pair-rule type phenocopies with defects in alternating segments. Interestingly, we find the defects in odd numbered segments and not in even numbered ones as in Drosophila. However, this apparent discrepancy can be explained if one takes into account that the primary action of eve is at the level of parasegments and that different cuticular markers are used for defining the segment borders in the two species. In this light, we find that eve appears to be required for the formation of the anterior borders of the same odd numbered parasegments in both species. We conclude that the primary function of eve as a pair rule gene is conserved between the two species.

Regulatory and coding regions of the segmentation gene hunchback are functionally conserved between Drosophila virilis and Drosophila melanogaster.

The segmentation gene hunchback (hb) is involved in setting up the anterior-posterior axis of the Drosophila embryo. It is expressed maternally and zygotically and it plays a key role in integrating the effects of the anterior and posterior maternal systems. The hb gene from D. virilis has previously been cloned and was shown to be well conserved in its coding region, but less so in its upstream region which shows a more patchy pattern of conserved and diverged sequences. This work deals with the functional conservation of hb between the two species. We have mapped two additional regulatory elements for the expression of hb in the early embryo, namely the enhancer for the maternal expression and the enhancer region for the late blastoderm expression. Fragments containing these two elements, the previously identified bicoid dependent element for the early blastoderm expression of hb and the coding region were taken from D. virilis and tested in the D. melanogaster background. We find that all enhancer elements as well as the coding region are functionally conserved between the two species. Comparison of the upstream sequences that include the enhancer region for the late blastoderm expression reveal seven highly conserved blocks. Some of these contain consensus binding sites for transregulatory factors that are likely to control the respective expression domains.

Insect calcium channels. Molecular cloning of an alpha 1-subunit from housefly (Musca domestica) muscle.

The complete amino acid sequence of an invertebrate calcium channel alpha 1-subunit from housefly (Musca domestica) larvae (designated Mdl alpha 1) has been deduced by cDNA cloning and sequence analysis. Mdl alpha 1 shares higher percent sequence identity with 1,4-dihydropyridine (DHP)-sensitive L-type than with DHP-insensitive calcium channels. As shown by whole mount in situ hybridization and immunostaining Mdl alpha 1 is predominantly expressed in the larval body wall musculature.

Intracommunity relationships, dispersal pattern and paternity success in a wild living community of Bonobos (Pan paniscus) determined from DNA analysis of faecal samples.

Differences in social relationships among community members are often explained by differences in genetic relationships. The current techniques of DNA analysis allow explicit testing of such a hypothesis. Here, we have analysed the genetic relationships for a community of wild bonobos (Pan paniscus) using nuclear and mitochondrial DNA markers extracted from faecal samples. Bonobos show an opportunistic and promiscuous mating behaviour, even with mates from outside the community. Nonetheless, we find that most infants were sired by resident males and that two dominant males together attained the highest paternity success. Intriguingly, the latter males are the sons of high-ranking females, suggesting an important influence of mothers on the paternity success of their sons. The molecular data support previous inferences on female dispersal and male philopatry. We find a total of five different mitochondrial haplotypes among 15 adult females, suggesting a frequent migration of females. Moreover, for most adult and subadult males in the group we find a matching mother, while this is not the case for most females, indicating that these leave the community during adolescence. Our study demonstrates that faecal samples can be a useful source for the determination of kinship in a whole community.

Evolutionary analysis of genes involved in early embryonic pattern formation in Drosophila.

Segmentation and homeotic genes have originally been identified and analyzed in Drosophila. Molecular techniques such as low stringency hybridization or PCR now allow to clone homologs of these genes from different organisms. This provides a basis to study the evolution of pattern formation mechanisms between organisms at the gene level, creating a new discipline: molecular comparative embryology. This chapter discusses the practical and conceptual problems arising from this approach.

Notes on the definition and nomenclature of tandemly repetitive DNA sequences.

Tandemly repetitive DNA is a major component of all eukaryotic genomes. This fact has been known for almost 30 years and research on this class of DNA is still being done. Its biology and evolution are therefore now becoming fairly well understood. DNA-fingerprint techniques rely very much on this knowledge. However, the large amount of research on these sequences has inevitably led to a large number of different concepts and theories about their nature. This has also resulted in some confusion as to the nomenclature. The following notes are intended to resolve this confusion somewhat and to give some definitions for the major classes of tandemly repetitive DNA.