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.

A change of expression in the conserved signaling gene MKK7 is associated with a selective sweep in the western house mouse Mus musculus domesticus.

Changes in gene expression are known to occur between closely related species, but it is not yet clear how many of these are due to random fixation of allelic variants or due to adaptive events. In a microarray survey between subspecies of the Mus musculus complex, we identified the mitogen-activated protein-kinase-kinase MKK7 as a candidate for change in gene expression. Quantitative PCR experiments with multiple individuals from each subspecies confirmed a specific and significant up-regulation in the testis of M. m. domesticus. Northern blot analysis shows that this is due to a new transcript that is not found in other tissues, nor in M. m. musculus. A cis-trans test via allele specific expression analysis of the MKK7 gene in F1 hybrids between domesticus and musculus shows that the expression change is mainly caused by a mutation located in cis. Nucleotide diversity was found to be significantly reduced in a window of at least 20 kb around the MKK7 locus in domesticus, indicative of a selective sweep. Because the MKK7 gene is involved in modulating a kinase signalling cascade in a stress response pathway, it seems a plausible target for adaptive differences between subspecies, although the functional role of the new testis-specific transcripts will need to be further studied.

When invaders meet locally adapted types: rapid moulding of hybrid zones between sculpins (Cottus, Pisces) in the Rhine system.

Hybrid zones are commonly studied to dissect the processes that drive divergence among lineages, which have incomplete barriers of reproduction. Most hybrid zones have existed for an extended time making inferences on the initial mode of formation difficult. It is a priori unclear how fast a nascent hybrid zone would form as a response to endogenous and exogenous factors. We have studied several hybrid zones between two lineages of sculpins (Cottus spp.), which emerged due to a recent range expansion of one of the lineages along the river Rhine in the early 1990s. Applying a dense sampling across two contact areas and using a highly informative set of 45 microsatellite markers we found pronounced genetic structure. Steep genetic clines suggest that strong selective forces have shaped the respective hybrid zones from the beginning. We find that the zones are coupled to ecological transitions from small streams to larger rivers. The width of these zones is much smaller than estimates of annual individual dispersal distances, as estimated outside of the hybrid zones. The pattern is apparently not strongly affected by pre- or postzygotic reproductive isolation because numerous backcross hybrids occur within the zones. This suggests that strong natural selection acts against immigrant genotypes. The study exemplifies how local adaptation can play a key role in preventing admixture in dependence of the ecological context.

A genetic map of Cottus gobio (Pisces, Teleostei) based on microsatellites can be linked to the physical map of Tetraodon nigroviridis.

To initiate QTL studies in the nonmodel fish Cottus gobio we constructed a genetic map based on 171 microsatellite markers. The mapping panel consisted of F1 intercrosses between two divergent Cottus lineages from the River Rhine System. Basic local alignment search tool (BLAST) searches with the flanking sequences of the microsatellite markers yielded a significant (e < 10(-5)) hit with the Tetraodon nigroviridis genomic sequence for 45% of the Cottus loci. Remarkably, most of these hits were due to short highly conserved noncoding stretches. These have an average length of 40 bp and are on average 92% conserved. Comparison of the map locations between the two genomes revealed extensive conserved synteny, suggesting that the Tetraodon genomic sequence will serve as an excellent genomic reference for at least the Acanthopterygii, which include evolutionarily interesting fish groups such as guppies (Poecilia), cichlids (Tilapia) or Xiphophorus (Platy). The apparent high density of short conserved noncoding stretches in these fish genomes will highly facilitate the identification of genes that have been identified in QTL mapping strategies of evolutionary relevant traits.

Homologues of c-hairy1 (her9) and lunatic fringe in zebrafish are expressed in the developing central nervous system, but not in the presomitic mesoderm.

A number of genes that are involved in somitogenesis in vertebrates are cyclically expressed in the presomitic mesoderm. These include homologues of the Drosophila genes fringe and hairy. We have analysed here two genes that belong to these classes in the zebrafish, namely the apparent orthologues of lunatic fringe (l-fng) and of c-hairy1 (called her9). However, unlike the respective mouse and chicken genes, they are not expressed cyclically in the presomitic mesoderm. Instead, both genes are mainly expressed in the central nervous system. her9 is predominantly expressed in the fore- and midbrain, and transiently in the hindbrain. Thus, the previously identified and only very distantly related her1 gene of zebrafish has more similarities to the expression of the c-hairy1 gene than its apparent orthologue her9, indicating that sequence similarity and similarity of function are not necessarily linked in this case. l-fng expression is found in alternating pre-rhombomeres, comparable to the equivalent mouse gene expression and in the anterior compartments of the mature somites, which was also shown for the chicken l-fng gene. The latter expression indicates that it might be involved in boundary definition and cell fate decision processes, rather than in pre-patterning of the somites. Interestingly, a similar role has previously been inferred for the grasshopper homologue of l-fng. This suggests that the function of l-fng in boundary definition of the somites might be ancestral, while its recruitment to the pre-patterning process of the somites might be a derived feature in higher vertebrates.

Mitochondrial protein phylogeny joins myriapods with chelicerates.

The animal phylum Arthropoda is very useful for the study of body plan evolution given its abundance of morphologically diverse species and our profound understanding of Drosophila development. However, there is a lack of consistently resolved phylogenetic relationships between the four extant arthropod subphyla, Hexapoda, Myriapoda, Chelicerata and Crustacea. Recent molecular studies have strongly supported a sister group relationship between Hexapoda and Crustacea, but have not resolved the phylogenetic position of Chelicerata and Myriapoda. Here we sequence the mitochondrial genome of the centipede species Lithobius forficatus and investigate its phylogenetic information content. Molecular phylogenetic analysis of conserved regions from the arthropod mitochondrial proteome yields highly resolved and congruent trees. We also find that a sister group relationship between Myriapoda and Chelicerata is strongly supported. We propose a model to explain the apparently parallel evolution of similar head morphologies in insects and myriapods.

Neurogenesis in the spider Cupiennius salei.

To uncover similarities and differences in neurogenesis in arthropod groups, we have studied the ventral neuroectoderm of the spider Cupiennius salei (Chelicerata, Aranea, Ctenidae). We found that invaginating cell groups arose sequentially, at stereotyped positions in each hemisegment and in separate waves, comparable with the generation of neuroblasts in Drosophila. However, we found no evidence for proliferating stem cells that would be comparable with the neuroblasts. Instead, the whole group of invaginating cells was directly recruited to the nervous system. The invagination process is comparable with Drosophila, with the cells attaining a bottle-shaped form with the nuclei moving inwards, while actin-rich cell processes remain initially connected to the surface of the epithelium. This general pattern is also found in another spider, Pholcus phalangioides, and appears thus to be conserved at least among the Araneae. We have identified two basic helix-loop-helix encoding genes -- CsASH1 and CsASH2 -- that share sequence similarities with proneural genes from other species. Functional analysis of the genes by double-stranded RNA interference revealed that CsASH1 was required for the formation of the invagination sites and the process of invagination itself, whereas CsASH2 seemed to be required for the differentiation of the cells into neurones. Our results suggest that the basic processes of neurogenesis, as well as proneural gene function is conserved among arthropods, apart of the lack of neuroblast-like stem cells in spiders.

Genetic and ecological divergence of a monophyletic cichlid species pair under fully sympatric conditions in Lake Ejagham, Cameroon.

Although there is mounting evidence that speciation can occur under sympatric conditions, unambiguous examples from nature are rare and it is almost always possible to propose alternative allopatric or parapatric scenarios. To identify an unequivocal case of sympatric speciation it is, therefore, necessary to analyse natural settings where recent monophyletic species flocks have evolved within a small and confined spatial range. We have studied such a case with a cichlid species flock that comprises five Tilapia forms endemic to a tiny lake (Lake Ejagham with a surface area of approximately 0.49 km2) in Western Cameroon. Analysis of mitochondrial D-Loop sequences shows that the flock is very young (approximately 10(4) years) and has originated from an adjacent riverine founder population. We have focused our study on a particular pair of forms within the lake that currently appears to be in the process of speciation. This pair is characterized by an unique breeding colouration and specific morphological aspects, which can serve as synapomorphic characters to prove monophyly. It has differentiated into a large inshore and a small pelagic form, apparently as a response to differential utilization of food resources. Still, breeding and brood care occurs in overlapping areas, both in time and space. Analysis of nuclear gene flow on the basis of microsatellite polymorphisms shows a highly restricted gene flow between the forms, suggesting reproductive isolation between them. This reproductive isolation is apparently achieved by size assortative mating, although occasional mixed pairs can be observed. Our findings are congruent with recent theoretical models for sympatric speciation, which show that differential ecological adaptations in combination with assortative mating could easily lead to speciation in sympatry.

The Short antennae gene of Tribolium is required for limb development and encodes the orthologue of the Drosophila Distal-less protein.

Insects bear a stereotyped set of limbs, or ventral body appendages. In the highly derived dipteran Drosophila melanogaster, the homeodomain transcription factor encoded by the Distal-less (Dll) gene plays a major role in establishing distal limb structures. We have isolated the Dll orthologue (TcDll) from the beetle Tribolium castaneum, which, unlike Drosophila, develops well-formed limbs during embryogenesis. TcDll is initially expressed at the sites of limb primordia formation in the young embryo and subsequently in the distal region of developing legs, antennae and mouthparts except the mandibles. Mutations in the Short antennae (Sa) gene of Tribolium delete distal limb structures, closely resembling the Dll phenotype in Drosophila. TcDll expression is severely reduced or absent in strong Sa alleles. Genetic mapping and molecular analysis of Sa alleles also support the conclusion that TcDll corresponds to the Sa gene. Our data indicate functional conservation of the Dll gene in evolutionarily distant insect species. Implications for evolutionary changes in limb development are discussed.

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 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.

Phylogeography of the bullhead Cottus gobio (Pisces: Teleostei: Cottidae) suggests a pre-pleistocene origin of the major central European populations.

The bullhead Cottus gobio is a small, bottom-dwelling fish consisting of populations that have not been subject to transplantations or artificial stocking. It is therefore an ideal model species for studying the colonization history of central European freshwater systems, in particular with respect to the possible influences of the Pleistocene glaciation cycles. We sampled Cottus populations across most of its distribution range, with a special emphasis on southern Germany where the major European drainage systems are in closest contact. Mitochondrial D-loop sequencing of more than 400 specimens and phylogenetic network analysis allowed us to draw a detailed picture of the colonization of Europe by C. gobio. Moreover, the molecular distances between the haplotypes enabled us to infer an approximate time frame for the origin of the various populations. The founder population of C. gobio stems apparently from the Paratethys and invaded Europe in the Pliocene. From there, the first colonization into central Europe occurred via the ancient lower Danube, with a separate colonization of the eastern European territories. During the late Pliocene, one of the central European populations must have reached the North Sea in a second step after which it then started to colonize the Atlantic drainages via coastal lines. Accordingly, we found very distinct populations in the upper and lower Rhine, which can be explained by the fact that the lower Rhine was disconnected from the upper Rhine until approximately 1 million years ago (Ma). More closely related, but still distinct, populations were found in the Elbe, the Main and the upper Danube, all presumably of Pleistocene origin. Intriguingly, they have largely maintained their population identity, despite the strong disturbance caused by the glaciation cycles in these areas. On the other hand, a mixing of populations during postglacial recolonization could be detected in the lower Rhine and its tributaries. However, the general pattern that emerges from our analysis suggests that the glaciation cycles did not have a major impact on the general population structure of C. gobio in central Europe.

Conserved and divergent aspects of terminal patterning in the beetle Tribolium castaneum.

To infer similarities and differences in terminal pattern formation in insects, we analyzed several of the key genes of this process in the beetle Tribolium castaneum. We cloned two genes of the terminal pattern cascade, namely tailless (tll) and forkhead (fkh), from Tribolium and studied their expression patterns. In addition, we analyzed the pattern of MAP kinase activation at blastoderm stage as a possible signature for torso-dependent signaling. Further, we analyzed the late expression of the previously cloned Tribolium caudal (Tc-cad) gene. Finally, we used the upstream region of Tc-tll to drive a reporter gene construct in Drosophila. We find that this construct is activated at the terminal regions in Drosophila, suggesting that the torso-dependent pathway is conserved between the species. We show that most of the expression patterns of the genes studied here are similar in Drosophila and Tribolium, suggesting conserved functions. There is, however, one exception, namely the early function of Tc-tll at the posterior pole. In Drosophila, the posterior tll expression is involved in the direct regulation of the target genes of the terminal pathway. In Tribolium, posterior Tc-tll expression occurs only for a short time and ceases before the target genes known from Drosophila are activated. Thus, we infer that Tc-tll does not function as a direct regulator of segmentation genes at the posterior end. It is more likely to be involved in the early specification of a group of "terminal" cells, which begin to differentiate only at a later stage of embryogenesis, when much of the abdominal segmentation process is complete. Thus, there appears to have been a major shift in tll function during the evolutionary transition from short germ to long germ embryogenesis.

Expression patterns of hairy, even-skipped, and runt in the spider Cupiennius salei imply that these genes were segmentation genes in a basal arthropod.

There is an ongoing discussion on whether segmentation in different phyla has a common origin sharing a common genetic program. However, before comparing segmentation between phyla, it is necessary to identify the ancestral condition within each phylum. Even within the arthropods it is not clear which parts of the genetic network leading to segmentation are conserved in all groups. In this paper, we analyze the expression of three segmentation genes of the pair-rule class in the spider Cupiennius salei. Spiders are representatives of the Chelicerata, a monophyletic basic arthropod group. We find that in spider embryos, the orthologues for the Drosophila primary pair-rule genes hairy, even-skipped, and runt are expressed in stripes in the growth zone, where the segments are forming, suggesting a role for these genes in chelicerate segmentation. These data imply that the involvement of hairy, even-skipped, and runt in arthropod segmentation is an ancestral character for arthropods and is not restricted to a particular group of insects.

Mitochondrial sequence analysis of Salamandra taxa suggests old splits of major lineages and postglacial recolonizations of central Europe from distinct source populations of Salamandra salamandra.

Representatives of the genus Salamandra occur in Europe, Northern Africa and the Near East. Many local variants are known but species and subspecies status of these is still a matter of dispute. We have analysed samples from locations covering the whole expansion range of Salamandra by sequence analysis of mitochondrial D-loop regions. In addition, we have calibrated the rate of divergence of the D-loop on the basis of geologically dated splits of the closely related genus Euproctus. Phylogenetic analysis of the sequences suggests that six major monophyletic groups exist (S. salamandra, S. algira, S. infraimmaculata, S. corsica, S. atra and S. lanzai) which have split between 5 and 13 million years ago (Ma). We find that each of the Salamandra species occupies a distinct geographical area, with the exception of S. salamandra. This species occurs all over Europe from Spain to Greece, suggesting that it was the only species that has recolonized Central Europe after the last glaciation. The occurrence of specific east and west European haplotypes, as well as allozyme alleles in the S. salamandra populations suggests that this recolonization has started from at least two source populations, possibly originating in the Iberian peninsula and the Balkans. Two subpopulations of S. salamandra were found that are genetically very distinct from the other populations. One lives in northern Spain (S. s. bernardezi) and one in southern Italy (S. s. gigliolii). Surprisingly, the mitochondrial lineages of these subpopulations group closer together than the remainder S. salamandra lineages. We suggest that these populations are remnants of a large homogeneous population that had colonized Central Europe in a previous interglacial period, approximately 500 000 years ago. Animals from these populations were apparently not successful in later recolonizations. Still, they have maintained their separate genetic identity in their areas, although they are not separated by geographical barriers from very closely related neighbouring populations.

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.

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.

A comparison of homologous developmental genes from Drosophila and Tribolium reveals major differences in length and trinucleotide repeat content.

The flour beetle Tribolium castaneum has become an important model organism for comparative studies of insect development. Many developmentally important genes have now been cloned from both Tribolium and Drosophila and their expression characteristics were studied. We analyze here the complete coding sequences of 17 homologous gene pairs from D. melanogaster and T. castaneum, most of which encode transcription factors. We find that the Tribolium genes are on average 30% shorter than their Drosophila homologues. This appears to be due largely to the almost-complete absence of trinucleotide repeats in the coding sequences of Tribolium as well as the generally lower degree of internal repetitiveness. Clusters of polar and other amino acids such as glutamine, proline, and serine, which are often considered to be important for transcriptional activation domains in Drosophila, are almost completely absent in Tribolium. Codon usage is generally less biased in Tribolium, although we find a similar tendency for the preference of G- or C-ending codons and a higher bias in conserved subregions of the proteins as in Drosophila. Most of the aminoacid substitutions in the DNA-binding domains of the transcription factors occur at residues that do not make a specific contact to DNA, suggesting that the recognition sequences are likely to be conserved between the two species.