Hybridization and speciation.

Hybridization has many and varied impacts on the process of speciation. Hybridization may slow or reverse differentiation by allowing gene flow and recombination. It may accelerate speciation via adaptive introgression or cause near-instantaneous speciation by allopolyploidization. It may have multiple effects at different stages and in different spatial contexts within a single speciation event. We offer a perspective on the context and evolutionary significance of hybridization during speciation, highlighting issues of current interest and debate. In secondary contact zones, it is uncertain if barriers to gene flow will be strengthened or broken down due to recombination and gene flow. Theory and empirical evidence suggest the latter is more likely, except within and around strongly selected genomic regions. Hybridization may contribute to speciation through the formation of new hybrid taxa, whereas introgression of a few loci may promote adaptive divergence and so facilitate speciation. Gene regulatory networks, epigenetic effects and the evolution of selfish genetic material in the genome suggest that the Dobzhansky-Muller model of hybrid incompatibilities requires a broader interpretation. Finally, although the incidence of reinforcement remains uncertain, this and other interactions in areas of sympatry may have knock-on effects on speciation both within and outside regions of hybridization.

A linkage map for the hybridising toads Bombina bombina and B. variegata (Anura: Discoglossidae).

Stable hybrid zones in which ecologically divergent taxa give rise to a range of recombinants are natural laboratories in which the genetic basis of adaptation and reproductive isolation can be unraveled. One such hybrid zone is formed by the fire-bellied toads Bombina bombina and B. variegata (Anura: Discoglossidae). Adaptations to permanent and ephemeral breeding habitats, respectively, have shaped numerous phenotypic differences between the taxa. All of these are, in principle, candidates for a genetic dissection via QTL mapping. We present here a linkage map of 28 codominant and 10 dominant markers in the Bombina genome. In an F2 cross, markers that were mainly microsatellites, SSCPs or allozymes were mapped to 20 linkage groups. Among the 40 isolated CA microsatellites, we noted a preponderance of compound and frequently interleaved CA-TA repeats as well as a striking polarity at the 5' end of the repeats.

Nuclear markers, mitochondrial DNA and male secondary sexual traits variation in a newt hybrid zone (Triturus vulgaris x T. montandoni).

The smooth and the Montandon's newts (Triturus vulgaris and T. montandoni) are genetically similar sister species with highly divergent male secondary sexual traits involved in complex courtship behaviour. Their parapatric ranges overlap at moderate elevations in the Carpathian Mountains where they hybridize readily. Here we present a detailed study of genetic and morphological variation in populations from the area of sympatry. Analysis of variation at seven nuclear markers, mtDNA and male sexual secondary traits was complemented with an ecological survey of breeding sites characteristics. Extensive hybridization was revealed with back-cross individuals similar to either parental species predominating among hybrids. The hybrid zone exhibited a mosaic pattern: the genetic composition of the populations was correlated only weakly with their geographical position. No association with habitat type was found. Departures from Hardy-Weinberg proportions, significant linkage disequilibria and bimodal distribution of genotypes suggest strongly that assortative mating is an important factor shaping the genetic composition of hybrid populations. The pattern of cytonuclear disequilibria did not indicate much asymmetry in interspecific matings. Changes in the frequency of nuclear markers were highly concordant, whereas mtDNA showed much wider bidirectional introgression with 14% excess of T. montandoni haplotype. We argue that the mosaic structure of the newt hybrid zone results mainly from stochastic processes related to extinction and recolonization. Microgeographical differences in mtDNA introgression are explained by historical range shifts. Since morphologically intermediate males were underrepresented when compared to hybrid males identified by genetic markers, sexual selection acting against the morphological intermediates is implied. We discuss the implications of these findings in the context of reinforcement of prezygotic isolation in newts.

Inheritance of allozyme loci in Bombina: second linkage group established.

Segregation and linkage relationship of nine allozyme loci, which are fixed for alternative alleles in the European fire-bellied toads, Bombinal bombina and B. variegata, were studied using artificial F1 hybrids to obtain backcross and F2 progeny. Alleles coding for electromorphs at nine loci (Ldh-1, Mdh-1, Idh-1, Ck, Ak, Gpi, Aat-1, Np, and G6pd) showed Mendelian ratios. Two of the loci, Ak and G6pd, were found to be closely linked (2 cM apart); the other loci assorted independently.

Mitochondrial DNA variation in the hybridizing fire-bellied toads, Bombina bombina and B. variegata.

Using five restriction enzymes, geographical variation of mitochondrial DNA (mtDNA) in Bombina bombina and B. variegata was studied in samples from 20 locations. Each restriction enzyme produced a species-specific fragment pattern. B. bombina haplotypes A and B were closely related to each other. In contrast, haplotypes A and B of B. variegata formed two distinct lineages. A very distinctive haplotype (C) was found in the Carpathian Mountains, whereas two other haplotypes, D and E (differing by a single AvaI site), were present in western Europe and the Balkans, respectively. Populations polymorphic for haplotypes D and E occurred in the central Balkans where the haplotypes could replace each other clinally. mtDNA sequence divergence between B. bombina and B. variegata was estimated as 6.0-8.1% and 4.7-5.2% between type C and types D/E of B. variegata. The latter divergence is contrary to allozyme and morphological data that place the western and Carpathian B. v. variegata together (Nei's D = 0.07) and separate them from the Balkan subspecies B. v. scabra (Nei's D = 0.18). Broad interspecific correlation among morphology, allozymes and mtDNA types in European fire-bellied toads argues that, despite continuous hybridization (interrupted perhaps during Pleistocene glacial maxima), little or no mtDNA introgression between the species has occurred outside the narrow hybrid zones that separate these parapatric species.

HABITAT PREFERENCE IN THE BOMBINA HYBRID ZONE IN CROATIA.

This paper demonstrates the effect of habitat heterogeneity and a habitat preference on the genetic structure of a hybrid zone between the toads Bombina bombina and B. variegata (Anura: Discoglossidae); 1613 toads from 85 sites across a transect near Pescenica, Croatia, were scored for five unlinked diagnostic allozyme markers. These were found to be largely concordant. Aside from minor systematic deviations, there was little variance in allele frequency among loci within sites. Yet the allele frequencies did not follow a smooth cline, but formed a mosaic in the center, such that neighboring sites could differ markedly in their enzyme score. A detailed ecological survey revealed a correlation between this pattern and habitat. In keeping with the typical breeding sites of the parental taxa, B. bombina-like hybrids were found more often in ponds, whereas B. variegata-like hybrids were more common in puddles. In addition, there was significant heterozygote deficit (FIS ) and strong linkage disequilibrium (R), both of which were stronger on the B. bombina side of the transect, and stronger in puddles than ponds. Mark-recapture data showed: (1) that the animals disperse beyond the scale of the habitat pattern; (2) frequent turn-over of individuals within sites; and (3) nonrandom movement between two sites of different habitat type. We conclude that an active habitat preference must contribute to the observed association between marker alleles and habitat. As a consequence, there is incomplete mixing of the two gene pools, which could explain the high level of FIS and R. The asymmetry in these parameters may reflect asymmetry in the preference or in the distribution of habitats across the zone. We discuss the implications of habitat preference for the dynamics of hybrid zones.

The insect cytochrome oxidase I gene: evolutionary patterns and conserved primers for phylogenetic studies.

Insect mitochondrial cytochrome oxidase I (COI) genes are used as a model to examine the within-gene heterogeneity of evolutionary rate and its implications for evolutionary analyses. The complete sequence (1537 bp) of the meadow grasshopper (Chorthippus parallelus) COI gene has been determined, and compared with eight other insect COI genes at both the DNA and amino acid sequence levels. This reveals that different regions evolve at different rates, and the patterns of sequence variability seems associated with functional constraints on the protein. The COOH-terminal was found to be significantly more variable than internal loops (I), external loops (E), transmembrane helices (M) or the NH2 terminal. The central region of COI (M5-M8) has lower levels of sequence variability, which is related to several important functional domains in this region. Highly conserved primers which amplify regions of different variabilities have been designed to cover the entire insect COI gene. These primers have been shown to amplify COI in a wide range of species, representing all the major insect groups; some even in an arachnid. Implications of the observed evolutionary pattern for phylogenetic analysis are discussed, with particular regard to the choice of regions of suitable variability for specific phylogenetic projects.

The sequence and structure of the meadow grasshopper (Chorthippus parallelus) mitochondrial srRNA, ND2, COI, COII ATPase8 and 9 tRNA genes.

The nucleotide sequence of the mitochondrial ND2, COI, COII, ATPase8, srRNA and nine tRNA genes have been sequenced from two individual of the meadow grasshopper Chorthippus parallelus. Comparisons are made to other insects for which the same regions are completely sequenced. Percentage A + T is found to be relatively low in C. parallelus though consistent with that of the other Orthopteran, Locusta migratoria. The relative number of substitutions observed in the different protein-coding genes was analysed between pairs of insect species sharing different levels of relatedness. A clear change in this rate was observed between the within-genus and between-genera comparisons. This change is interpreted in terms of the functional constraints acting on these four different genes. The patterns seem to result from an early saturation of COI and COII genes with synonymous substitutions, and a tolerance of ND2 and ATPase8 function to high levels of amino acid replacements. This analysis highlights a need for further sequence studies and comparisons between taxa of different levels of divergence in order to understand the patterns of mtDNA evolution on which many evolutionary investigations are based.

Inheritance of allozyme loci in Bombina: one linkage group established.

Two hybridizing European species of fire-bellied toads, Bombina bombina and B. variegata, have alternate electromorphs fixed at a number of allozyme loci. Segregation of alleles at seven allozyme loci (Ldh-1, Mdh-1, Ak, Ck, Gpi, Np, and Est-beta) was studied in a backross progeny of an F1 interspecific hybrid male and a B. bombina female. Mendelian inheritance of allozyme forms at all seven loci was ascertained. Except for two loci, Gpi and Est-beta, which were found to be tightly linked (1 cM apart), other loci showed independent segregation.

Evolution and structural conservation of the control region of insect mitochondrial DNA.

The control regions of mitochondrial DNA of two insects, Schistocerca gregaria and Chorthippus parallelus, have been isolated and sequenced. Their sizes are 752 bp and 1,512 bp, respectively, with the presence of a tandem repeat in C. parallelus. (The sequences of the two repeats are highly conserved, having a homology of 97.5%.) Comparison of their nucleotide sequences revealed the presence of several conserved sequence blocks dispersed through the whole control region, showing a different evolutionary pattern of this region in these insects as compared to that in Drosophila. A highly conserved secondary structure, located in the 3' region near the small rRNA gene, has been identified. Sequences immediately flanking this hairpin structure rather than the sequences of this structure themselves are conserved between S. gregaria/C. parallelus and Drosophila, having a sequence consensus of "TATA" at 5' and "GAA(A)T" at 3'. The motif "G(A)nT" is also present in the 3' flanking sequences of mammalian, amphibian, and fish mitochondrial L-strand replication origins and a potential plant mitochondrial second-strand-replication origin, indicating its universal conservation and functional importance related to replication origins. The stem-and-loop structure in S. gregaria/C. parallelus appears to be closely related to that found in Drosophila despite occupying a different position, and may be potentially associated with a second-strand-replication origin. This in turn suggests that such a secondary structure might be widely conserved across invertebrates while their location in the control region may be variable. We have looked for such a conserved structure in the control regions of two other insects, G. firmus and A. mellifera, whose DNA sequences have been published, and their possible presence is discussed. Mitochondrial control regions characterized to date in five different insect taxa (Drosophila, G. firmus, A. mellifera, S. gregaria, and C. parallelus) may be classed into two distinct groups having different evolutionary patterns. It is observed that tandem repetition of regions containing a probable replication origin occurred in some species from disjunct lineages in both groups, which would be the result of convergent evolution. We also discuss the possibility of a mechanism of "parahomologous recombination by unequal crossing-over" in mitochondria, which can explain the generation of such tandemly repeated sequences (especially the first critical repetition) in the control region of mtDNA, and also their convergent evolution in disjunct biological lineages during evolution.

Upg-2: a urinary pepsinogen variant located on chromosome 1 of the mouse.

Acrylamide gel electrophoresis of mouse urine distinguishes two groups of pepsinogens, the fast-migrating group A and the slow-migrating group B. Inbred mouse strains fall into two categories with respect to group B pepsinogens, one expressing a single-banded and the other a double-banded phenotype. This variation is controlled by a single locus, urinary pepsinogen-2 or Upg-2, which has at least two alleles, Upg-2s and Upg-2d. Typing of recombinant inbred strains suggests that the location of the Upg-2 locus is on chromosome 1, in the vicinity of loci coding for other peptidases, namely, Pep-2 and Rnr, and of the Acf-1 locus.

Mouse mitochondrial superoxide dismutase locus is on chromosome 17.

The hamster X mouse hybridoma cell line GCL28 carries only one copy of mouse chromosome 17 but expresses H-2 antigens controlled by the major histocompatibility complex of the mouse. The cell line and clones derived from it were subjected to treatment with H-2 specific antisera and complement and a series of H-2 antigen-negative clones was produced. Typing of the clones for the mouse enzyme glyoxalase 1, which is encoded by an H-2-linked gene, revealed that the loss of H-2 antigen expression was accompanied by the loss of chromosomes 17 in these clones. This suggestion was verified by karyotype analysis of selected clones. Typing of the clones and subclones for the mouse mitochondrial superoxide dismutase (SOD-2) indicated complete concordance between loss of chromosome 17 and loss of SOD-2 activity. This finding suggests that the locus controlling the expression of SOD-2 is located on chromosome 17. Since a similar locus in the human is linked to HLA, the human major histocompatibility complex, extensive homology must exist between the mouse and human MHC-bearing chromosomes.

Quantitative immunological studies of the albumins of several species of fire bellied toads, genus Bombina.

1. Rabbit antisera against purified serum albumin of Bombina bombina were used to study relationships between B. bombina, B. variegata, and B. orientalis. 2. Quantitative micro-complement fixation tests indicated the albumins of B. bombina from central Poland and Bulgaria were indistinguishable. The albumins from several populations of B. variegata differed very slightly from that of B. bombina. The albumin of B. orientalis was quite distinct from that of B. bombina. 3. Using albumin as a molecular clock, we estimated B. bombina and B. variegata diverged within the last million years, whereas the B. orientalis lineage diverged roughly 10-12 mil yr ago.

Inheritance and linkage analysis of five enzyme loci in interspecific hybrids of toadlets, genus Bombina.

Progeny produced from Bombina bombina, B. variegata, and field-collected interspecific hybrids have been analyzed for the inheritance of five enzyme loci, which are fixed for alternate alleles in the parental species. Lactate dehydrogenase (Ldh-1), NAD-dependent malate dehydrogenase (Mdh-1), creatine kinase (Ck), adenylate kinase (Ak), and glucosephosphate isomerase (Gpi) are all inherited in a Mendelian manner as codominant alleles at nuclear loci. Both parental alleles are equally functional in artificial F1 hybrids (female B. bombina x male B. variegata) at each of the loci studied. No linkage between any pair of loci was observed. Discovery of this inherited biochemical variation combined with a technique for assaying individual genotypes without killing the animals makes feasible studies of hybrid population structure heretofore impossible.