Experimental interspecific hybridization in Daphnia

Hybridization is a common phenomenon in Daphnia (Cladocera; Anomopoda); interspecific hybrids have been found between several species and hybrids are found in many European lakes. Although much information on the morphology, ecology and genetics of hybrids is available, little is known about the level of reproductive isolation among species or about the relative fitness of hybrids and parental species. In order to facilitate studies on differentiation and speciation processes and comparative experimental studies on hybrids and recombinant genotypes, we present the first successful laboratory crossing experiments of two different Daphnia species, D. galeata and D. cucullata. Males and sexual females from two D. galeata and two D. cucullata clones were reciprocally crossed, juveniles hatched from resting eggs and reared until maturity. Hatching and juvenile survival rates of hybrids were relatively low (12.1% and 24%, respectively). D. galeata and D. cucullata clones vary in their level of successful interspecific matings and in the number of subsequent offspring. In general, hybrid crosses between D. cucullata females and D. galeata males were more successful than reciprocal crosses.     

Daphnia Galeata × D. Longispina Hybrids in Western Norway

We describe the occurrence of D. galeata×longispina hybrids in two lakes of western Norway. Parental species and interspecific hybrids were characterised by both nuclear and mitochondrial molecular markers. In one of the populations, hybrids were shown to dominate the population over several years. A few individuals in both populations were probably not F1 hybrids, but possibly backcrosses or F2 hybrids. Most (possibly all) F1 hybrids were of D. galeata maternal origin. In addition, interspecific hybrids could be identified based on morphological characters, which were intermediate between the parental species. Interspecific hybridisation between these two species is remarkable, since they are distantly related.     

Nuclear DNA content of Vitis species,cultivars, and other genera of the Vitaceae

The nuclear DNA content was analyzed in Vitis species, hybrid cultivars, and genera of the Vitaceae using flow cytometry. Significant variation was found among Vitis species, hybrids, and other genera of the Vitaceae (Ampelopsis and Parthenocissus). DNA content was estimated to range from 0.98 to 1.05 pg/2C within V. labrusca (ns) and 0.86 to 1.00 pg/2C within V. vinifera (ns). Genotypes from Vitis and Parthenocissus were similar in nuclear DNA content (approximately 1.00 pg/2C) whereas they differed significantly from Ampelopsis (1.39 pg/2C). No correlation between DNA content and the center of origin of genotypes of the Vitaceae was noted. Based on the present study, the Vitis genome size is 475 Mbp, 96% of which is non-coding. Knowledge of DNA content is useful in order to understand the complexity of the Vitis genome and to establish a relationship between the genetic and physical map for map-based cloning.     

RAPD characterisation of two neotropical hybrid legumes

An investigation of randomly amplified polymorphic DNA (RAPD) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) marker distribution was made for two well-characterised hybrids and their parents,Leucaena leucocephala andL. esculenta andParkinsonia aculeata andCercidium praecox. Three chloroplast DNA (cpDNA) markers identified the maternal parent of eachL. leucocephala ×L. esculenta hybrid. Fifteen species-diagnostic RAPD markers (invariant in one taxon and absent from the other) were always present in theLeucaena hybrid and assumed to be of nuclear origin, whilst three RAPD markers showed expression patterns identical to the cpDNA markers and were assumed to be of organellar origin. No RAPD or PCR-RFLP taxon-diagnostic markers were discovered for eitherP. aculeata orC. praecox. However, 21 RAPD markers were species-specific (polymorphic within one taxon but absent from the other) and Southern analysis indicated that none of the markers were of organellar origin. Only 67% additivity of markers specific toP. aculeata andC. praecox was demonstrated in the hybrids between these two species, whilst inLeucaena 97% additivity was demonstrated. Differences between the two hybridising situations were related to the behaviour of the molecular markers and the biology of the species.     

桉树杂交种对桉树枝瘿姬小蜂的抗性变异分析

植物种间杂交是一种普遍自然现象,杂交往往造成植物表型及生理变异,从而改变杂种抗虫性。与亲本种相比,杂种抗虫性可能增强或减弱,也有可能处于与亲本相似水平。初生、次生代谢物的质变与量变是引起杂种抗虫性变异的重要原因。近年来,桉树杂交育种已在世界范围内广泛应用并取得了显著成效,桉树杂交种间抗虫性表现参差不齐,因此,桉树是研究杂交种抗虫性变异机制的理想材料。以2个桉树杂交种巨细桉DH201-2、巨尾桉G9及桉树重要害虫桉树枝瘿姬小蜂为研究对象,比较了2个杂交种与其纯亲本种[(巨桉×细叶桉),(巨桉×尾叶桉)]间的抗虫性差异;同时,综合比较了品系间叶片性状(叶片厚度、含水率、比叶面积)、初生化合物(C、N、可溶性糖、可溶性蛋白)及次生化合物(总酚、单宁)差异,以研究桉树杂交种抗虫性变异的理化机制。结果表明:DH201-2感染桉树枝瘿姬小蜂的虫瘿数目显著高于其双亲本种,而G9上虫瘿数目显著低于其双亲本种。DH201-2与G9的叶片厚度与巨桉相近,而显著薄于另一亲本种。DH201-2叶片含水率显著高于细叶桉、与巨桉相近;G9叶片含水率则显著低于其双亲本种。相似的是,DH201-2和G9的比叶面积均显著高于其双亲本种。初生化合物方面,DH201-2叶片可溶性糖和可溶性蛋白含量均显著高于其亲本种,N含量则仅高于细叶桉;而G9叶片可溶性蛋白含量虽高于其双亲本种,可溶性糖含量则无显著差异,N含量显著低于其双亲本种。次生化合物方面,DH201-2叶片总酚和单宁含量显著低于其双亲本种,而G9则显著高于其双亲本种。因此,与其亲本种相比,DH201-2感虫性增加,而G9抗虫性增加;与桉树枝瘿姬小蜂发育相关的营养指标(如含水率、可溶性糖、N含量)及次生防御物质(如总酚、缩合单宁)在桉树杂交种组织内的含量差异影响了桉树杂交种对桉树枝瘿姬小蜂的抗性。在全球推行桉树杂交育种且桉树害虫数量逐年增加的大背景下,应加强对桉树杂交种抗虫性机制研究,为选育高抗品系及桉树产业可持续发展提供理论指导。     

Chloroplast DNA variation inCoreopsis nuecensoides andC. nuecensis (Asteraceae), a presumed progenitor-derivative species pair

Coreopsis nuecensoides andC. nuecensis are narrowly distributed endemics of southeastern Texas. While they overlap in range, they differ in chromosome number, and F₁ hybrids exhibit strong sterility barriers. Previous morphological, cytogenetic, and allozyme studies suggested thatC. nuecensoides andC. nuecensis are very closely related members of a progenitor-derivative species pair. The two species differ substantially in terms of their leaf flavonoid chemistry, but the flavonoid data do not address the exact nature of the relationship between them. We have further examined the relationship between the species by analyzing genetic diversity within both species using chloroplast DNA (cpDNA) restriction site data. Sixteen restriction site changes were used to define thirteen distinct cpDNA haplotypes. The pattern of relationships among haplotypes provides some support for the presumed ancestor-descendent relationship, but other interpretations are possible. Only one cpDNA haplotype was shared by the two species; of the remaining twelve, seven were unique to one species and five to the other. This result is consistent with results of flavonoid studies, in which each species exhibited flavonoid compounds not seen in the other, but differs from the allozyme results, in which the vast majority of alleles were shared by both species and only the presumed progenitor exhibited unique alleles. Taken together, the data support a very close relationship between the species, but are equivocal with regard to a progenitor-derivative relationship.     

Genetic variation and hybridization in SwedishSchoenus (Cyperaceae)

Schoenus ferrugineus andS. nigricans have restricted distributions in Sweden and are almost exclusively confined to calcareous fen habitats. AtS. nigricans sites,S. ferrugineus is usually also present, and hybrids are frequently found. In this report, I used allozymes to estimate the amount of gene flow between the two species, and to compare the partitioning of genetic diversity in each of them. Thirteen loci were analysed at eight different enzyme systems. Seven loci were variable between or within the species. The two species had completely different alleles at two of the seven variable loci, whereas there was overlap at five loci. In all, 22 different alleles were found. Six of these alleles were confined toS. nigricans, and five alleles were confined toS. ferrugineus. Nei's genetic identity was 0.55.—InS. ferrugineus, three loci (23%) were polymorphic, and the average number of alleles per polymorphic locus was 2.0 (each polymorphic locus had two alleles). InS. nigricans, three loci (23%) were polymorphic, and the average number of alleles per polymorphic locus was 2.3.—The proportion of genetic diversity due to variation among sites (G _ST) was fairly similar in the two species, mean over loci = 0.12 inS. ferrugineus and 0.15 inS. nigricans. However, the proportion of genetic diversity due to variation among individuals within sites (G _IS) differed markedly between the two species, mean over loci = 0.54 inS. ferrugineus and 0.17 inS. nigricans. Accordingly, there was a much higher individual heterozygosity inS. nigricans than inS. ferrugineus. — Most hybrids were interpreted as F₁ hybrids. However, a small proportion, 0.5–1.6 %, were F_n hybrids or back-crosses.—On the Swedish mainland, all former occurrences ofS. nigricans are extinct, but viable hybrids are still present at a few sites in southernmost Sweden.     

Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion

Summary With the idea to develop a selection system for asymmetric somatic hybrids between oilseed rape (Brassica napus) and black mustard (B. nigra), the marker gene hygromycin resistance was introduced in this last species by protoplast transformation with the disarmed Agrobacterium tumefaciens strain C58 pGV 3850 HPT. The B. nigra lines used for transformation had been previously selected for resistance to two important rape pathogens (Phoma lingam, Plasmodiophora brassicae). Asymmetric somatic hybrids were obtained through fusion of X-ray irradiated (mitotically inactivated) B. nigra protoplasts from transformed lines as donor with intact protoplasts of B. napus, using the hygromycin resistance as selection marker for fusion products. The somatic hybrids hitherto obtained expressed both hygromycin phosphotransferase and nopaline synthase genes. Previous experience with other plant species had demonstrated that besides the T-DNA, other genes of the donor genome can be co-transferred. In this way, the produced hybrids constitute a valuable material for studying the possibility to transfer agronomically relevant characters — in our case, diseases resistances — through asymmetric protoplast fusion.     

Interspecific hybridization between the cultivated potato Solanum tuberosum subspecies tuberosum L. and the wild species S. circaeifolium subsp. circaeifolium Bitter exhibiting resistance to Phytophthora infestans (Mont.) de Bary and Globodera pallida (Stone) Behrens

Summary Crossability between the diploid species S. circaeifolium subsp. circaeifolium (crc) and other diploid species, primarily diploid S. tuberosum subsp. tuberosum (tbr-2x), was studied. Forty-seven hybrids were obtained from crosses between crc as female parent and tbr-2x and some other species from series Tuberosa as male parents. Of these hybrids 17% were diploids; the other 83% were triploids, probably carrying two genomes of crc. Female fertility was sufficient to obtain offspring from backcrosses with the cultivated parent. Pollen stainability of the f₁ varied, and micro-pollen as well as unreduced pollen occurred. During meiosis of the diploids and triploids a rather high proportion of univalents was found, and in the triploids on average two or three trivalents per cell were found. All hybrids were resistant to Globodera pallida pathotypes 2 and 3, and 75% of the tested genotypes were highly resistant to Phytophthora infestans. Solanidine, tomatidine, tomatidenol, and demissidine glycosides were found in tubers of the hybrids. Comparisons with somatic hybrids between crc and tbr-2x are made. It is concluded that crc is a valuable Solanum species that can and should be included in potato breeding programs.     

Behavioral sterility of hybrid males in acoustically communicating grasshoppers (Acrididae,Gomphocerinae)

The effectiveness of hybridization barriers determines whether two species remain reproductively isolated when their populations come into contact. We investigated acoustic mating signals and associated leg movements responsible for song creation of hybrids between the grasshopper species Chorthippus biguttulus and C. brunneus to study whether and how songs of male hybrids contribute to reproductive isolation between these sympatrically occurring species. Songs of F1, F2, and backcross hybrids were intermediate between those of both parental species in terms phrase number and duration. In contrast, species-specific syllable structure within phrases was largely lost in hybrids and was produced, if at all, in an irregular and imperfect manner. These divergences in inheritance of different song parameters are likely the result of incompatibility of neuronal networks that control stridulatory leg movements in hybrids. It is highly probable that songs of hybrid males are unattractive to females of either parental species because they are intermediate in terms of phrase duration and lack a clear syllable structure. Males of various hybrid types (F1, F2, and backcrosses) are behaviorally sterile because their songs fail to attract mates.     

Interspecific hybridization betweenDaphnia hyalina,D. galeata,andD. cucullata and seasonal abundances of these species and their hybrids

The three cladoceran speciesDaphnia hyalina, D. galeata, andD. cucullata frequently coexist in the lakes of northern Germany. Although there are some problems in distinguishing them morphologically, they are easily determined by gelelectrophoresis: each species carries a different allele at the glutamate oxaloacetate transaminase (GOT) locus. Animals morphologically intermediate between two species are heterozygous for the alleles carried by the species they resemble. This pattern is in agreement with the findings at other loci, where also diagnostic alleles exist. These findings are most easily explained by interspecific hybridization between the three species. No evidence is found for backcrosses involving hybrids ofD. cucullata, whereas some backcrosses betweenD. hyalina, D. galeata, and their hybrids are found in some lakes. In four lakes the seasonal abundances of the three species and their hybrids are determined.     

Cytogenetic and genomic relationships ofThinopyrum elongatum with twoPsathyrostachys species and withLeymus secalinus (Poaceae)

Intergeneric hybridizations were made betweenT. elongatum, and twoPsathyrostachys and fiveLeymus species. The seed set obtained onT. elongatum ×Leymus hybrids ranged from 5.65% to 20.00%, depending onLeymus species. The seed set obtained onT. elongatum ×Psathyrostachys hybrids ranged from 16.07% to 19.70%. Meiotic pairing at metaphase-I in JN diploid hybrids ofT. elongatum ×Psathyrostachys species revealed a very low level homology between the basic J and N genomes, and further demonstrated that the two genomes are quite diverged. Chromosome pairing in theT. elongatum ×Leymus secalinus hybrid averaged 15.19 univalents + 2.62 rod bivalents + 0.26 ring bivalents + 0.02 trivalents, suggesting that the partial J^e chromosomes ofT. elongatum has homology withLeymus secalinus genomes.L. secalinus might have 3–4 chromosomes originating from J^e genome.     

Spontaneous hybridization between a male-sterile oilseed rape and two weeds

Spontaneous interspecific hybrids were produced under natural conditions (pollination by wind and bees) between a male-sterile cybrid Brassica napus (AACC, 2n = 38) and two weeds Brassica adpressa (AdAd, 2n = 14) and Raphanus raphanistrum (RrRr, 2n = 18). After characterization by chromosome counts and isozyme analyses, we observed 512 and 3 734 inter-specific seeds per m² for the B. napus-B. adpressa and B. napus-R. raphanistrum trials respectively. Most of the hybrids studied had the expected triploid structure (ACX). In order to quantify the frequency of allosyndesis between the genomes involved in the hybrids, their meiotic behavior was compared to a haploid of B. napus (AC). For the B. napus-B. adpressa hybrids, we concluded that probably no allosyndesis occurred between the two parental genomes, and that genetic factors regulating homoeologous chromosome pairing were carried by the B. adpressa genome. For the B. napus-R. raphanistrum hybrids, high chromosome pairing and the presence of multivalents (in 9.16% of the pollen mother cells) indicate that recombination is possible between chromosomes of different genomes. Pollen fertility of the hybrids ranged from 0 to 30%. Blackleg inoculation tests were performed on the three parental species and on the interspecific hybrids. BC₁ production with the weeds and with rapeseed was attempted. Results are discussed in regard to the risk assessment of transgenic rapeseed cultivation, F₁ hybrid rapeseed variety production, and rapeseed improvement.     

Novel class of rDNA repeat units in somatic hybrids between Nicotiana and Atropa

Summary Behavior of ribosomal RNA genes in the process of somatic hybridization was analyzed using hybrids Nicotiana tabacum + Atropa belladonna. Blothybridization of parental species DNAs to ³²P-rDNA specific probes revealed two classes of ribosomal repeats in both tobacco and nightshade; their length was 11.2 kb, 10.4 kb (tobacco) and 9.4 kb, 10.2 kb (night-shade). For analysis of hybrids, labelled ³²P rDNA specific probes were hybridized to DNA of parental species and somatic hybrids digested with restriction endonucleases EcoR1, EcoRV and BamH1. A new class of ribosomal DNA repeat, absent in parental species, was found in hybrid line NtAb-1. Possible mechanisms of appearence of a new rDNA class in the process of somatic cell fusion are discussed.     

Evolutionary patterns in the antR-Cor gene in the dwarf dogwood complex (Cornus, Cornaceae)

The evolutionary pattern of the myc-like anthocyanin regulatory gene antR-Cor was examined in the dwarf dogwood species complex (Cornus Subgenus Arctocrania) that contains two diploid species (C. canadensis and C. suecica), their putative hybrids with intermediate phenotypes, and a tetraploid derivative (C. unalaschkensis). Full-length sequences of this gene (∼4 kb) were sequenced and characterized for 47 dwarf dogwood samples representing all taxa categories from 43 sites in the Pacific Northwest. Analysis of nucleotide diversity indicated departures from neutral evolution, due most likely to local population structure. Neighbor-joining and haplotype network analyses show that sequences from the tetraploid and diploid intermediates are much more strongly diverged from C. suecica than from C. canadensis, and that the intermediate phenotypes may represent an ancestral group to C. canadensis rather than interspecific hybrids. Seven amino acid mutations that are potentially linked to myc-like anthocyanin regulatory gene function correlate with petal colors differences that characterize the divergence between two diploid species and the tetraploid species in this complex. The evidence provides a working hypothesis for testing the role of the gene in speciation and its link to the petal coloration. Sequencing and analysis of additional nuclear genes will be necessary to resolve questions about the evolution of the dwarf dogwood complex.     

Genetic differentiation between Coffea liberica var. liberica and C. liberica var. Dewevrei and comparison with C. canephora

Coffea liberica Hiern includes C. liberica var. liberica (LIB) from western Africa and C. liberica var. dewevrei (DEW) from central Africa. This geographical distribution also includes cultivated C. canephora for which a within-species clustering in two interfertile groups is known. In this study, genetic differentiation between LIB and DEW was evaluated on the basis of morphological traits, molecular markers (AFLP) and male fertility of F1 hybrids. The results were also compared with the within-species differentiation in C. canephora. The morphological traits differed significantly but the distributions overlapped. By contrast, LIB and DEW constituted two distinct groups with high genetic differentiation (G_st=0.25) when using AFLP markers. In addition, the pollen viability of F1 hybrids between LIB and DEW was weak (44.2%) and similar to interspecific C. canephora x DEW F1 hybrids or C. canephora x LIB F1 hybrids, indicating that there are marked reproductive barriers between LIB and DEW.     

Phyletic and evolutionary relationships ofBrachyscome lineariloba (Compositae)

A comparison of karyotypes ofBrachyscome breviscapis (2n = 8),B. lineariloba cytodemes E (2n = 10), B (2n = 12) and C (2n = 16) suggests that these species have a homoelogous basic set of four chromosome pairs, two large pairs and two small, and that theB. lineariloba cytodemes E, B and C are related toB. breviscapis by successive additions of small chromosomes. A pronounced asynchrony of chromosome condensation between these large and small chromosomes has been observed. In the artificial hybrids betweenB. dichromosomatica (2n = 4) ×B. breviscapis, and theB. lineariloba cytodemes, theB. dichromosomatica chromosomes are similar in size and condensation behaviour to the small chromosomes ofB. breviscapis and ofB. lineariloba cytodemes E, B and C. Meiotic pairing in these hybrids also demonstrates the strong affinities between these chromosomes. It is suggested thatB. breviscapis may be of amphidiploid origin between a species with two large early condensing chromosome pairs and another,B. dichromosomatica-like species with two small late condensing pairs. It seems most likely that the additional small and late condensing chromosomes inB. lineariloba cytodemes E, B and C are derived from theB. dichromosomatica-like parent, and that each addition increases vigour, fecundity and drought tolerance, allowing these cytodemes to colonize more open and arid environments. Transmission of the univalents in the quasidiploidB. lineariloba cytodeme E was verified as being via the pollen, and not via the embryo sacs.The cytology ofBrachyscome lineariloba (Compositae, Asteroidae), 10.     

Intergeneric (intersubtribe) hybridization between Moricandia arvensis and Brassica A and B genome species by ovary culture

Summary Intergeneric hybrids between Moricandia arvensis (C₃–C₄ intermediate species) and Brassica A and B genome species (B. campestris and B. nigra) were produced via ovary culture. When M. arvensis was used as a female parent, the hybrid embryo yield (0.25–0.45 embryo per pollination) was similar between two genomes, regardless of the male parent. The reciprocal hybrid using B. campestris as a female was also obtained, although yield of embryo was lower (0.02 embryo per pollination). On the other hand, no hybrids were obtained without the in vitro technique. As most hybrid embryos could not develop normal shoots, plants were regenerated by inducing shoots on the cultured hypocotyl. The hybrid nature of the regenerated plant was confirmed morphologically and cytogenetically. A certain amount of bivalents (2.52-2.71) in the hybrids indicated the existence of partial chromosome homology between two genera. The present results indicate that ovary culture is an effective technique for overcoming the crossing barrier between M. arvensis and Brassica cultivated species.     

Crossing experiments of lettuce cultivars and species (Lactuca sect.Lactuca,Compositae)

The degree of relationships withinLactuca sativa and three wild relativesL. serriola, L. saligna, andL. virosa was studied by observing the performance, vigour and fertility of the F 1 hybrids obtained from crosses made in and between the four species. The crosses ofL. saligna ×L. virosa and the reciprocal crosses produced no hybrids.L. saligna andL. virosa are the least related of the four species.L. sativa ×L. serriola and the reciprocal crosses were successful and produced fertile hybrids These two species are genetically very closely related.L. saligna is known to produce, as a female parent, hybrids withL. sativa andL. serriola. Now the reciprocal cross was successful for the first time, so the unability to obtain hybrids in the past was based on the choice of accessions and not caused by unilateral incompatibility.L. virosa ×L. sativa and the reciprocal combination produced hybrids. The combinationL. serriola ×L. virosa produced hybrids with very limited fertility. In contrast to earlier reports (sterile hybrids) one combination of the reciprocal cross too produced hybrids with very limited fertility.—Some of theL. saligna ×L. sativa (and reciprocal) hybrids were found to look strikingly likeL. serriola. This adds evidence for the descent ofL. serriola andL. sativa:L. saligna also made part of the ancestral complex of the cultivated lettuce.     

Host adaptation in the anther smut fungus Ustilago violacea (Microbotryum violaceum): infection success,spore production and alteration of floral traits on two host species and their F1-hybrid

It is often assumed that host specialization is promoted by trade-offs in the performance of parasites on different host species, but experimental evidence for such trade-offs is scant. We studied differences in performance among strains of the anther smut fungus Ustilago violacea from two closely related host plant species, Silene alba and S. dioica, on progeny of (1) the host species from which they originated, (2) the alternative host species, and (3) inter-specific hybrids. Significant intra-specific variation in the pathogen was found for both infection success on a range of host genotypes (virulence) and components of spore production per infected host (aggressiveness) (sensu Burdon 1987). Strains did not have overall higher virulence on conspecifics of their host of origin than on strains from the heterospecific host, but they did have a significantly (c. 3 times) higher spore production per infected male host. This finding suggests that host adaptation may have evolved with respect to aggressiveness rather than virulence. The higher aggressiveness of strains on conspecifics of their host of origin resulted both from higher spore production per infected flower (spores are produced in the anthers), and greater ability to stimulate flower production on infected hosts. The latter indicates the presence of adaptive intraspecific variation in the ability of host manipulation. As transmission of the fungus is mediated by insects that are both pollinators of the host and vectors of the disease, we also assessed the effect of strains on host floral traits. Infection resulted in a reduction of inflorescence height, flower size, and nectar production per flower. Strains did not differ in their effect on nectar production, but infection with strains from S. alba resulted in a stronger reduction of inflorescence height and petal size on both host species. Vectors may therefore in principle discriminate among hosts infected by different strains and affect their efficiency of transmission. Contrary to assumptions of recent hypotheses about the role of host hybrids in the evolution of parasites, hybrids were not generally more susceptible than parental hosts. It is therefore unlikely that the rate of evolution of the pathogen on the parental species is slowed down by selection for specialization on the hybrids.