Intraspecific Structure and Reproductive Relationships betweenElymus mutabilisand E. transbaicalensis(Poaceae) in Southern Siberia from the Viewpoint of Taxonomical Genetics

The relationships between StH-genomic species Elymus mutabilis (Drob.) Tzvel. and E. transbaicalensis (Nevski) Tzvel. were examined using biosystematic and biochemical (SDS electrophoresis of endosperm proteins) methods. The results showed the following. (1) There is interspecific morphological discreteness that permits assigning most individuals from the wild to a definite species. However, self-fertile intermediate individuals of introgressive origin were found. (2) Both taxa exhibited marked specificity of electrophoretic characteristics of endosperm proteins. (3) The species form two separate recombination gene pools (RGPs). Thus, E. mutabilisand E. transbaicalensis should be regarded as two distinct species. Nevertheless, there is some genetic exchange and unidirectional gene flow between the RGPs, especially in secondary (successive) habitats where mixed populations are observed. Two RGPs form the single introgressive gene pool (IGP), which includes other StH-genomic species of Northern Eurasia.     

Comparison of gene flow among species that occur within the same geographic locations versus gene flow among populations within species reveals introgression among several Elymus species

One of the challenges in evolutionary biology is to understand the evolution of speciation with incomplete reproductive isolation as many taxa have continued gene flow both during and after speciation. Comparison of population structure between sympatric and allopatric populations can reveal specific introgression and determine if introgression occurs in a unidirectional or bidirectional manner. Simple sequence repeat markers were used to characterize sympatric and allopatric population structure of plant species, Elymus alaskanus (Scribn. and Merr.) Löve, E. caninus L., E. fibrosus (Schrenk) Tzvel., and E. mutabilis (Drobov) Tzvelev. Our results showed that genetic diversity (H_E) at species level is E. caninus (0.5355) > E. alaskanus (0.4511) > E. fibrosus (0.3924) > E. mutabilis (0.3764), suggesting that E. caninus and E. alaskanus are more variable than E. fibrosus and E. mutabilis. Gene flow between species that occurs within the same geographic locations versus gene flow between populations within species was compared to provide evidence of introgression. Our results indicated that gene flow between species that occur within the same geographic location is higher than that between populations within species, suggesting that gene flow resulting from introgressive hybridization might have occurred among the sympatric populations of these species, and may play an important role in partitioning of genetic diversity among and within populations. The migration rate from E. fibrosus to E. mutabilis is highest (0.2631) among the four species studied. Asymmetrical rates of gene flow among four species were also observed. The findings highlight the complex evolution of these four Elymus species.     

Modeling of introgression between Elymus caninus and E. fibrosus (Poaceae) and their registration using one-dimensional SDS electrophoresis

Artificial sexual hybrids between Elymus caninus (L.) L. and E. fibrosus (Schrenk) Tzvel. were experimentally examined in generations F₁–F₅. The possibility of genetic introgression between these species was shown. Morphologically, the hybrid plants can be assigned to either of the parental species or to variety E. caninus var. muticus (Holmb.) Karlsson. Some traits (spike density, leaf blade width, leaf blade pubescence, awns of lemmas) exhibited considerable variation. Polypeptide spectra of endosperm proteins were characterized in the initial parental biotypes and the hybrid progeny, using a gel-buffer SDS electrophoresis system. It was suggested that successful interspecies hybridization requires backcrosses or normalizing crosses. The possibility of sexual genetic exchange enables to utilize the gene pools of the two species to transfer the required traits in selection forage forms.     

Modeling of introgression between Elymus caninus and E. fibrosus (Poaceae) and their registration using one-dimensional SDS electrophoresis

Artificial sexual hybrids between Elymus caninus (L.) L. and E. fibrosus (Schrebk) Tzvel. were experimentally examined in generations F1-F5. The possibility of genetic introgression between these species was shown. Morphologically, the hybrid plants can be assigned to either of the parental species or to variety E. caninus var. muticus (Holmb.) Karlsson. Some traits (spike density, leaf blade width, leaf blade pubescence, awns of lemmas) exhibited considerable variation. Polypeptide spectra of endosperm proteins were characterized in the initial parental biotypes and the hybrid progeny, using a gel-buffer SDS electrophoresis system. It was suggested that successful interspecies hybridization requires backcrosses or normalizing crosses. The possibility of sexual genetic exchange enables to utilize the gene pools of the two species to transfer the required traits in selection forage forms.     

Glucosylation activity and complex formation of two classes of reversibly glycosylated polypeptides

Reversibly glycosylated polypeptides (RGPs) have been implicated in polysaccharide biosynthesis. In plants, these proteins may function, for example, in cell wall synthesis and/or in synthesis of starch. We have isolated wheat (Triticum aestivum) and rice (Oryza sativa) Rgp cDNA clones to study the function of RGPs. Sequence comparisons showed the existence of two classes of RGP proteins, designated RGP1 and RGP2. Glucosylation activity of RGP1 and RGP2 from wheat and rice was studied. After separate expression of Rgp1 and Rgp2 in Escherichia coli or yeast (Saccharomyces cerevisiae), only RGP1 showed self-glucosylation. In Superose 12 fractions from wheat endosperm extract, a polypeptide with a molecular mass of about 40 kD is glucosylated by UDP-glucose. Transgenic tobacco (Nicotiana tabacum) plants, overexpressing either wheat Rgp1 or Rgp2, were generated. Subsequent glucosylation assays revealed that in RGP1-containing tobacco extracts as well as in RGP2-containing tobacco extracts UDP-glucose is incorporated, indicating that an RGP2-containing complex is active. Gel filtration experiments with wheat endosperm extracts and extracts from transgenic tobacco plants, overexpressing either wheat Rgp1 or Rgp2, showed the presence of RGP1 and RGP2 in high-molecular mass complexes. Yeast two-hybrid studies indicated that RGP1 and RGP2 form homo- and heterodimers. Screening of a cDNA library using the yeast two-hybrid system and purification of the complex by an antibody affinity column did not reveal the presence of other proteins in the RGP complexes. Taken together, these results suggest the presence of active RGP1 and RGP2 homo- and heteromultimers in wheat endosperm.     

The interconversion of UDP-arabinopyranose and UDP-arabinofuranose is indispensable for plant development in Arabidopsis

L-Ara, an important constituent of plant cell walls, is found predominantly in the furanose rather than in the thermodynamically more stable pyranose form. Nucleotide sugar mutases have been demonstrated to interconvert UDP-Larabinopyranose (UDP-Arap) and UDP-L-arabinofuranose (UDP-Araf) in rice (Oryza sativa). These enzymes belong to a small gene family encoding the previously named Reversibly Glycosylated Proteins (RGPs). RGPs are plant-specific cytosolic proteins that tend to associate with the endomembrane system. In Arabidopsis thaliana, the RGP protein family consists of five closely related members. We characterized all five RGPs regarding their expression pattern and subcellular localizations in transgenic Arabidopsis plants. Enzymatic activity assays of recombinant proteins expressed in Escherichia coli identified three of the Arabidopsis RGP protein family members as UDP-L-Ara mutases that catalyze the formation of UDP-Araf from UDP-Arap. Coimmunoprecipitation and subsequent liquid chromatography-electrospray ionization-tandem mass spectrometry analysis revealed a distinct interaction network between RGPs in different Arabidopsis organs. Examination of cell wall polysaccharide preparations from RGP1 and RGP2 knockout mutants showed a significant reduction in total L-Ara content (12–31%) compared with wild-type plants. Concomitant downregulation of RGP1 and RGP2 expression results in plants almost completely deficient in cell wall–derived L-Ara and exhibiting severe developmental defects.     

Negligible nuclear introgression despite complete mitochondrial capture between two species of chipmunks

The idea that species boundaries can be semipermeable to gene flow is now widely accepted but the evolutionary importance of introgressive hybridization remains unclear. Here we examine the genomic contribution of gene flow between two hybridizing chipmunk species, Tamias ruficaudus and T. amoenus. Previous studies have shown that ancient hybridization has resulted in complete fixation of introgressed T. ruficaudus mitochondrial DNA (mtDNA) in some populations of T. amoenus, but the extent of nuclear introgression is not known. We used targeted capture to sequence over 10,500 gene regions from multiple individuals of both species. We found that most of the nuclear genome is sorted between these species and that overall genealogical patterns do not show evidence for introgression. Our analysis rules out all but very minor levels of interspecific gene flow, indicating that introgressive hybridization has had little impact on the overall genetic composition of these species outside of the mitochondrial genome. Given that much of the evidence for introgression in animals has come from mtDNA, our results underscore that unraveling the importance introgressive hybridization during animal speciation will require a genome‐wide perspective that is still absent for many species.     

Genetic entities and mating system in hermaphroditic Fucus spiralis and its close dioecious relative F. vesiculosus (Fucaceae, Phaeophyceae)

To date, molecular markers have not settled the question of the specific status of the closely related, but phylogenetically unresolved, brown seaweeds, hermaphroditic Fucus spiralis and dioecious Fucus vesiculosus, nor their propensity for natural hybridization. To test the degree of species integrity and to assess effect of the mating system on the population genetic structure, 288 individuals coming from parapatric (discontinuous) and sympatric (contiguous) spatial configurations at two sites were genotyped with five microsatellite loci. Using a Bayesian admixture analysis, our results show that F. spiralis and F. vesiculosus comprise clearly distinct genetic entities (clusters) generally characterized by cosexual and unisexual individuals, respectively. Genetic diversity within each entity suggests that F. spiralis reproduces primarily through selfing while F. vesiculosus is characterized by an endogamous breeding regime. Nevertheless, aberrant sexual phenotypes were observed in each cluster, no diagnostic alleles were revealed and 10% of study individuals were intermediate between the two genetic entities. This pattern can be explained by recent divergence of two taxa with retention of ancestral polymorphism or asymmetrical, introgressive hybridization. However, given (i) coincident monomorphism at three loci in spiralis clusters and (ii) that significantly more intermediates were observed in sympatric stations than in parapatric stations, we argue that interspecific gene flow has occurred after divergence of the two taxa. Finally, we show that whether recently separated or recently introgressive, the divergent breeding systems probably contribute to species integrity in these two taxa.     

History repeated: recent and historical mitochondrial introgression between the current darter Etheostoma uniporum and rainbow darter Etheostoma caeruleum (Teleostei: Percidae)

Incongruence between recognized taxonomy and phylogenetic relationships between two species from a diverse clade (Percidae: Etheostomatinae) of stream fishes was found in a mitochondrial (mt) DNA gene tree. Two darters in subgenus Oligocephalus , Etheostoma uniporum current darter and Etheostoma caeruleum rainbow darter were sampled throughout their sympatric distribution in the Ozark Highlands of the central United States. Sequences from cytochrome (cyt) b and the first intron of the nuclear marker S7 were analysed separately using maximum parsimony and Bayesian methods. Cyt b recovered both species as polyphyletic; E. uniporum haplotypes were interspersed within E. caeruleum . However, both species were monophyletic and non-sister taxa based on S7. The cyt b gene tree pattern is caused by introgressive hybridization resulting in the mtDNA replacement of E. uniporum haplotypes by those of E. caeruleum . Some E. uniporum haplotypes are shared with geographically proximate E. caeruleum , and this is consistent with recent hybridization, while other E. uniporum haplotypes indicate historical sorting of introgressed lineages. The mechanism of introgression is likely asymmetric sneaking behaviour by male E. uniporum , a mating tactic observed in related species. MtDNA replacement may have occurred in E. uniporum due to drift fixation in a historically small female effective population. Additional evidence for darter hybridization will likely be discovered in future molecular genetic surveys of the nearly 200 species in eastern North America.     

Morphological, cytogenetic, and molecular evidence for introgressive hybridization in birch.

Extensive morphological variation of tetraploid birch (Betula pubescens) in Iceland is believed to be due to gene flow from diploid dwarf birch (B. nana) by means of introgressive hybridization. A combined morphological and cytogenetic approach was used to investigate this phenomenon in two geographically separated populations of natural birch woodland in Iceland. The results not only confirmed introgressive hybridization in birch, but also revealed bidirectional gene flow between the two species via triploid interspecific hybrids. The populations showed continuous morphological variation connecting the species, but karyotypically they consisted of only three types of plants: diploids, triploids, and tetraploids. No aneuploids were found. Some of the tetraploid plants had B. pubescens morphology as expected, but most of them had intermediate characters. Most of the diploid plants were B. nana, but some were intermediates and a few had B. pubescens morphology. The triploid plants were either intermediates or they resembled one of the two species. Similar introgressive variation was observed among the diploid and triploid progeny of open-pollinated B. nana in a garden. Birch samples including field plants and artificial hybrids were further examined using a molecular method based on genomic Southern hybridization. The experiments verified introgression at the DNA level.     

Alteration of the proximal bond energy in the unliganded form of the homodimeric myoglobin from Nassa mutabilis. Kinetic and spectroscopic evidence.

CO binding kinetics to the homodimeric myoglobin (Mb) from Nassa mutabilis has been investigated between pH 1.9 and 7.0. Protonation of the proximal imidazole at low pH (less than or equal to 3.0) and the consequent cleavage of the HisF8NE2-Fe proximal bond brings about a approximately 20-fold increase of the second-order rate constant for CO binding. This process displays a pKa = 4.0 +/- 0.2, significantly higher than that observed in all other deoxygenated hemoproteins investigated up to now. Such a feature underlies a decreased energy for the HisF8NE2-Fe proximal bond in the unliganded form and it also appears supported by resonance Raman spectroscopy in the low frequency region of the Fe(II) deoxygenated hemoprotein. Further, the pH-rate profile of N. mutabilis Mb, like that of the homodimeric hemoglobin (Hb) from Scapharca inaequivalvis (Coletta, M., Boffi, A., Ascenzi, P., Brunori, M. and Chiancone, E. (1990) J. Biol. Chem. 265, 4828-4830), can be described only by assuming a concerted proton-linked transition with n = 1.8 +/- 0.1. Such a characteristic suggests, also on the basis of the amino acid sequence homology between N. mutabilis Mb and S. inaequivalvis Hb in the region forming the subunit interface, that the interaction mechanism is similar for the two homodimeric proteins, and drastically different Hb in the region forming the subunit interface, that the interaction mechanism is similar for the two homodimeric proteins, and drastically different from that operative in other hemoproteins.     

Introgressive hybridization between the Atlantic and Pacific herring (<Emphasis Type="Italic">Clupea harengus</Emphasis> and <Emphasis Type="Italic">Clupea pallasii</Emphasis>) in the White Sea,Barents and Kara Seas evidenced by microsatellites

In the North of Europe two sister species of herring—the Atlantic Clupea harengus and Pacific Clupea pallasii have post-glacially come into secondary contact. Although the breeding areas of the two species are thought to be separate, previous genetic studies based on allozyme and mitochondrial DNA data have supported the existence of introgression from the Atlantic herring to the Pacific herring. In this study, we employed microsatellite markers to explore the extent of introgressive hybridization between these fishes. Bayesian methods for assigning individuals to populations and identifying admixture proportions were applied to the genetic data based on ten microsatellites. Results indicated that 18 out of 464 Pacific herring (3.88%) were likely admixed. Some of the hybrid individuals contained genotypes suggesting that introgressive hybridization is an ongoing process. Despite some admixture, the Atlantic and Pacific herring gene pools remain sharply distinct (the average proportions of membership to “Atlantic” and “Pacific” clusters were Q_A = 0.998 and Q_P = 0.974, respectively), suggesting that hybridization was not frequent. The discovery of introgression among herring species opens new questions about the mechanisms underlying the observed pattern of genetic population differentiation in the North European Pacific herring.     

Speciation with gene flow and the genetics of habitat transitions

Whether speciation can advance to completion in the face of initially high levels of gene flow is a very controversial topic in evolutionary biology. Extensive gene exchange is generally considered to homogenize populations and counteract divergence. Moreover, the role of introgressive hybridization in evolution remains largely unexplored in animals, particularly in freshwater zooplankton in which allopatric speciation is considered to be the norm. Our work investigates the genetic structure of two young ecological species: the pond species, Daphnia pulex and the lake species, Daphnia pulicaria. Phylogenetic and population genetics analyses were conducted on mitochondrial NADH dehydrogenase 5 (ND5) gene, the nuclear Lactate dehydrogenase (Ldh) gene and 21 nuclear microsatellite markers in 416 individuals from habitats with various degrees of permanence. The strong and consistent phylogenetic discordance between nuclear and mitochondrial markers suggests a complex evolutionary history of multiple independent habitat transition events that involved hybridization and introgression between lake and pond Daphnia. On the other hand, the low level of contemporary gene flow between adjacent populations indicates the presence of effective habitat isolating barriers. The Daphnia system provides strong evidence for a divergence-with-gene flow speciation model that involves multiple habitat transition events.     

Interspecific hybridizations among species of theElymus semicostatus andElymus tibeticus groups (Poaceae)

Interspecific hybridizations were made between species of theE. semicostatus group, viz.,E. semicostatus (Nees exSteud.)Meld.,E. validus (Meld.)B. Salomon,E. abolinii (Drob.)Tzvel., andE. fedtschenkoi Tzvel., and species of theE. tibeticus group, viz.,E. pendulinus (Nevski)Tzvel.,E. tibeticus (Meld.)Singh,E. shandongensis B. Salomon, andE. gmelinii (Ledeb.)Tzvel., as well as among species within theE. tibeticus group. All species are tetraploid (2n = 4x = 28) and possess SY genomes. Meiotic pairing data from 24 hybrids involving 17 interspecific combinations are presented. The average number of chiasmata per cell ranged from 17.91 to 26.20 in hybrids within theE. tibeticus group, compared with 7.26 to 22.04 in hybrids between the two species groups. Despite the extensive collection of cytological data, there was no definite evidence for confirming or disproving the separate existence of the two groups.     

Genetic analysis of anthocyanin of the anthers and culm pigmentation in common wheat

Anthocyanin pigmentation of various organs develops during plant ontogeny in response to adverse and damaging abiotic and biotic stressors (environmental factors). Using the monosome method, the genes responsible for anther and culm anthocyanin pigmentation (Pan1 and Pc2, respectively) were localized to 7D chromosome in introgressive lines from crosses between common wheat Triticum aestivum L. and the species Triticum timopheevii Zhuk. Genetic analysis of ten common wheat genotypes using testers carrying genes Pan1, Pn1, and Pn2 showed that these genotypes contained Pan1 and Pn2 genes. Visual examination of plants from 70 and 76 varieties of respectively winter and spring common wheat revealed anthocyanin pigmentation of anthers and culms in 36 varieties. Pan1 and Pn2 genes were presumably introduced into common wheat from Aegilops tauschii (Fig.) Tzvel., a donor of the D genome.     

Mechanical barriers to introgressive hybridization revealed by mitochondrial introgression patterns in Ohomopterus ground beetle assemblages

To reveal the role of diverged body size and genital morphology in reproductive isolation among closely related species, we examined patterns of, and factors limiting, introgressive hybridization between sympatric Ohomopterus ground beetles in central Japan using mitochondrial NADH dehydrogenase subunit 5 (ND5) gene sequences. We sampled 17 local assemblages that consisted of two to five species and estimated levels of interspecific gene flow using the genetic distance, D(A), and maximum-likelihood estimates of gene flow. Sharing of haplotypes or haplotype lineages was detected between six of seven species that occurred in the study areas, indicating mitochondrial introgression. The intensity and direction of mitochondrial gene flow were variable among species pairs. To determine the factors affecting introgression patterns, we tested the relationships between interspecific D(A) and five independent variables: difference in body size, difference in genital size, phylogenetic relatedness (nuclear gene sequence divergence), habitat difference, and species richness of the assemblage. Body and genital size differences contributed significantly to preventing gene flow. Thus, mechanical isolation mechanisms reduce the chance of introgressive hybridization between closely related species. Our results highlight the role of morphological divergence in speciation and assemblage formation processes through mechanical isolation.     

Functional diversity of isoamylase oligomers: the ISA1 homo-oligomer is essential for amylopectin biosynthesis in rice endosperm

Rice (Oryza sativa) endosperm has two isoamylase (ISA) oligomers, ISA1 homo-oligomer and ISA1-ISA2 hetero-oligomer. To examine their contribution to starch synthesis, expression of the ISA1 or ISA2 gene was differently regulated in various transgenic plants. Although suppression of ISA2 gene expression caused the endosperm to have only the homo-oligomer, no significant effects were detected on the starch phenotypes. In contrast, ISA2 overexpression led to endosperm having only the hetero-oligomer, and starch synthesis in the endosperm was drastically impaired, both quantitatively and qualitatively, because the starch was devoid of typical starch features, such as thermal and x-ray diffraction properties, and water-soluble highly branched maltodextrins were accumulated. In the ISA2 overexpressed line, about 60% to 70% of the ISA1-ISA2 hetero-oligomer was bound to starch, while the ISA homo- and hetero-oligomers from the wild type were mostly present in the soluble form at the early milking stage of the endosperm. Detailed analysis of the relative amounts of homo- and hetero-oligomers in various lines also led us to the conclusion that the ISA1 homo-oligomer is essential, but not the ISA1-ISA2 oligomer, for starch production in rice endosperm. The relative amounts of ISA1 and ISA2 proteins were shown to determine the ratio of both oligomers and the stoichiometry of both ISAs in the hetero-oligomer. It was noted when compared with the homo-oligomer that all the hetero-oligomers from rice endosperm and leaf and potato (Solanum tuberosum) tuber were much more stable at 40°C. This study provides substantial data on the structural and functional diversity of ISA oligomers between plant tissues and species.     

Speciation via introgressive hybridization in East African cichlids?

Speciation caused by introgressive hybridization occurs frequently in plants but its importance remains controversial in animal evolution. Here we report a case of introgressive hybridization between two ancient and genetically distinct species of Lake Tanganyika cichlids that led to the formation of a new species. Neolamprologus marunguensis contains mtDNA haplotypes from both parental species varying on average by 12.4% in the first section of the control region and by 5.2% in a segment of the cytochrome b gene. All individuals have almost identical DNA sequences in the flanking regions of the single-copy nuclear DNA locus TmoM27, and show a mosaic of alleles derived from both parental lineages in six microsatellite loci. Hence, our finding displays another mode of speciation in cichlid fishes. The increase of genetic and phenotypic diversity due to hybridization may contribute to the uniquely rapid pace of speciation in cichlids.     

Evidence for broadscale introgressive hybridization between two redfish (genus Sebastes) in the North-west Atlantic: a rare marine example

The evolutionary importance of introgressive hybridization has long been recognized by plant evolutionists, and there is now a growing recognition for its potential role in animals as well. Detailed empirical investigations of this evolutionary process, however, are still lacking in many animal groups, particularly in the marine environment. Using integrated microsatellite DNA data (eight loci analysed over 803 individuals representing 17 sampling locations) and multivariate statistical procedures (principal component, factorial correspondence and admixture proportion analyses), we: (i) provide a detailed dissection of the dynamics of introgressive hybridization between Sebastes fasciatus and S. mentella, two economically important redfishes from the North-west Atlantic; and (ii) infer the factors potentially involved in the maintenance of the hybrid zone observed in the gulf of St. Lawrence and south of Newfoundland. This study provided one of the rare examples of extensive introgressive hybridization in the ocean, and highlighted the predominant role of this process in shaping the extent of genetic diversity, interspecific differences and population structuring among redfishes from the North-west Atlantic. The extensive (average rate of introgression = 15%) but geographically circumscribed and asymmetrical pattern of introgressive hybridization, the sympatric persistence of two reproductively isolated introgressed groups, the differential patterns of linkage disequilibrium among samples, and the maintenance of genetic integrity of both species outside the defined zone of introgression despite high potential for gene flow, all implicated selection in promoting and maintaining the observed pattern of introgression.