Hybridisation processes in sympatric populations of pines Pinus sylvestris L., P. mugo Turra and P. uliginosa Neumann

Natural hybridisation was postulated between the closely related pine species Pinus sylvestris and the P. mugo complex, however no clear evidence on propagation of mature hybrids in nature has been documented so far. To test the hybridisation hypothesis we applied chloroplast DNA (cpDNA) markers and isozymes in the analyses of 300 individuals representing the variety of morphological forms in the sympatric populations of P. sylvestris, P. mugo and P. uliginosa at the peat bog complex in the Sudety Mts., Poland. Additionally, the haplotypes of paternally inherited cpDNA of 149 open pollinated progeny derived from seeds were compared to the haplotypes of parental trees to access the intensity and direction of contemporary hybridisation. The morphologically highly variable polycormic (multi-stemmed) hybrids between P. mugo and P. uliginosa were identified. The second group of hybrids was found among the monocormic (single-stemmed) P. sylvestris-like individuals carrying the cpDNA from P. mugo complex. Hybrids of P. sylvestris as a pollen donor and P. mugo or P. uliginosa as a mother were not found, either in the group of examined trees, or among the open pollinated progeny. The results indicate that numerous hybrids can exist in the sympatric population of the species studied and that gene flow can successfully proceed from P. mugo complex to P. sylvestris. Hybridisation and ecological selection seems to play a significant role in diversification and evolution of the investigated species.     

A critical evaluation of reproductive barriers between closely related species using DNA markers - a case study in Pinus

Former controlled crosses between twelve Pinus montana var. rostrata (Pinus mugo complex) and eight P. sylvestris clones revealed that only two P. sylvestris had efficiently fertilised P. montana. Two species-diagnostic chloroplast DNA markers were applied to verify the species purity of the parental clones. All maternal P. montana were unambiguously confirmed to belong to the P. mugo complex at both chloroplast DNA marker loci. Six P. sylvestris clones carried the `sylvestris' haplotypes. However, the same two P. sylvestris clones that had efficiently fertilised P. montana displayed the chloroplast haplotypes diagnostic to the P. mugo complex. The patterns of highly polymorphic cpDNA microsatellite markers in parents and offspring ruled out contamination by foreign pollen. We concluded that the two clones successful in the crosses represent fertile hybrids between the two species with P. mugo as the pollen donor. Consequently, DNA markers are proposed for verifying or falsifying the success of artificial fertilisation in general. The existence of crossing barriers between the two Pinus species, meaningful to the postulated natural hybridisation and the evolution of their populations in sympatric stands, was indicated and is newly discussed.     

Allozyme investigations on the genetic differentiation between closely related pines —Pinus sylvestris, P. mugo, P. uncinata, andP. uliginosa (Pinaceae)

In eight natural European populations of four closely related taxa of pines (Pinus sylvestris, P. mugo, P. uncinata, andP. uliginosa) starch-gel electrophoreses revealed altogether 58 alleles at 15 loci from nine enzyme systems. With Nei's genetic distance (D) the largest mean genetic distance (D = 0.171) was found betweenP. sylvestris andP. mugo, a distance corresponding to that between other closely related pine species. Mean genetic distances between the other taxa were less than half that value and characteristic for subspecies or varieties rather than for species. On the basis of our results we suggest that both,P. uliginosa andP. uncinata, could be the result of the ancient hybridization betweenP. sylvestris andP. mugo.     

Intra- and interspecific genetic differentiation in closely related pines fromPinus subsectionSylvestres (Pinaceae) in the former Soviet Union

Thirty-seven natural populations of four closely related species ofPinus subsect.Sylvestres, P. mugo, P. funebris, P. pallasiana, andP. sylvestris, occurring in the former Soviet Union were investigated by starch-gel electrophoresis. In the populations assayed 127 allelic variants at 25 loci were revealed.Nei's distance coefficient (Dn) was used to estimate the level of genetic differentiation amongP. sylvestris races and among closely related species. A dendrogram constructed using Dn values shows that of the fiveP. sylvestris races analyzed only the geographically isolated var.hamata exhibited sufficient differences at theDia-2 locus (a mean Dn value relative to the other four races is 0.025) to recognize it as a distinct taxon. The remaining races, sylvestris, cretacea, lapponica, and sibirica, have a similar gene pool (Dn values are not greater than 0.010), and they should be regarded as a single taxon,P. sylvestris var.sylvestris. Interspecific comparisons revealed thatP. sylvestris andP. mugo have the closest genetic affinities to each other withNei's genetic distance of 0.108. The dendrogram demonstrates thatP. funebris is closer toP. sylvestris andP. mugo thanP. pallasiana. The available paleontologic data allowed us to conclude thatNei's (1975) time scale estimate for the time of divergence of the taxa was more accurate thanNei's (1971) time scale estimate.     

Abortive embryogenesis in hybrid swarm populations of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> L. and <Emphasis Type="Italic">Pinus muga</Emphasis> Turra

Comparative study on fertilization process in Pinus sylvestris, Pinus mugo and in their putative hybrid swarm individuals was done involving pre-zygotic and post-zygotic stages. The amount of surviving ovules from open pollination reflecting the mode of interaction between pollen grains and nucellar tissue of an ovule averaged at 8.1 of sound ovules per conelet in Pinus sylvestris, 7.3 ovules in the hybrid swarm population and at 4.9 ovules in Pinus mugo. A strong correlation was observed between the number of surviving ovules and the proportion of germinating seeds in the compared species and hybrids. Normal course of embryogenesis in Pinus sylvestris and Pinus mugo contrasted with increased frequency of disturbances observed in the hybrid swarm individuals. The differential survival rates of the ovules and deviations from typical pattern of embryogenesis are discussed from the standpoint of cross-ability relationship between Pinus sylvestris and Pinus mugo.     

Population structure,gene diversity,and differentiation in natural populations of Cedar pines (Pinus subsect.Cembrae,Pinaceae) in the USSR

Twelve natural populations of four cedar pine species,Pinus sibirica, P. cembra, P. pumila, andP. koraiensis, occurring in the Soviet Union were investigated by starch-gel electrophoresis. Frequencies of 55 alleles at 19 loci were determined. Interpopulation genetic diversity inP. sibirica andP. pumila was only 2–4 per cent of the total genetic diversity. Nei's distance coefficient (Dn) was used to estimate the level of genetic differentiation among conspecific populations and among species. Dn values among populations ranged from 0.006 to 0.038. A dendrogram constructed using Dn values divided cedar pines species into 2 clusters:sibirica-cembra (Dn = 0.030) andpumila-koraiensis (Dn = 0.143). Nei's distance between these clusters was 0.232. On the basis of the data obtained it was possible to draw the following conclusion:P. sibirica, P. pumila, andP. koraiensis are distinct species, whileP. cembra should apparently be regarded as geographicalP. sibirica race.     

Genetic relationships among Mexican white pines (Pinus, Pinaceae) based on RAPD markers

Genetic relationships among Mexican white pines have not been completely resolved by DNA sequencing analyses. The use of random amplified polymorphic DNA (RAPD) markers for the study of interspecific relationships has been questioned because of the possible lack of homology of co-migrating bands between species. However, several RAPD based studies on pines have provided sufficient information to discriminate between closely related taxa. Genetic relationships among four species of Mexican white pines (Pinus ayacahuite, Pinus strobiformis, Pinus lambertiana and Pinus chiapensis) were estimated based on RAPD markers. Sixty-nine primers generated 247 bands in pooled DNA samples from ten populations. In addition, four selected primers generated 27 bands in 176 individual DNA samples. Unweighted Pair Group Method with Arithmetic Average (UPGMA) dendrograms based on Jaccard similarity indices were constructed. The results suggest that the closest pine species analyzed were P. ayacahuite and P. strobiformis, followed by P. lambertiana. The most genetically distant species was P. chiapensis. Cluster analyses did not support P. strobiformis as a distinct species from P. ayacahuite.     

Population history of European mountain pines Pinus mugo and Pinus uncinata revealed by mitochondrial DNA markers

The dwarf mountain pine (Pinus mugo) and the Pyrenean pine (P. uncinata) constitute a pair of closely related coniferous taxa of poorly resolved evolutionary history and affinity, which inhabit numerous stands scattered over subalpine environments of European mountain ranges. The aim of the study was to investigate their phylogeography and mutual relationships, shedding new light on their taxonomy and the past of the alpine flora. Previous evolutionary reconstructions of the mountain pines relied mainly on bi‐parentally or paternally inherited markers that quickly homogenize between populations, showing rather shallow and recent differentiation of gene pools. Therefore, to contrast these pictures, we analyzed diversity and differentiation within a large set of new mitochondrial loci, inherited in maternal line and distributed by seeds at short geographical distances. Samples of the taxa were taken from 27 natural populations representing their range‐wide distributions—17 populations of P. mugo and 10 of P. uncinata. All markers appeared polymorphic, providing a total of 31 multilocus haplotypes. Two of the loci proved to be species‐diagnostic and nearly fixed between analyzed samples. Distribution of mitotypes indicate that allopatric populations of the taxa constitute separate mitochondrial haplogroups, and the two mountain pines have independent evolutionary history. However, introgression of P. mugo mitotypes by P. uncinata specimens revealed in the species contact zone in Western Alps shows that their speciation is not fully completed.     

Contribution to the taxonomy of Pinus uncinata (Pinaceae) based on cone characters

Eight natural populations of Pinus uncinata Ramond from the Spanish Pyrenees, five of P. mugo Turra from the Carpathians, the Sudethians and Abruzzian Apennines, three of P. sylvestris L. from the Central-European Lowland and the Iberian Peninsula and one of P. uliginosa Neumann from SW Poland have been compared biometrically in 16 cone characters. P. uncinata appears the most distant from all other taxa, differing by having the largest and most asymmetric cones and the thickest apophyses with the most prominent umbo. In cone characters, P. mugo is more similar to P. sylvestris than to P. uncinata. Nevertheless, P. mugo and P. sylvestris remain distant from each other in a number of morphological characters examined. P. uliginosa has cones intermediate between the other three taxa and is moreover very variable. The locality of P. uliginosa in the Silesian Lowland may be of a relict character and conserve genes from P. uncinata.     

Breeding systems and hybridization inPetrorhagia sect.Kohlrauschia (Caryophyllaceae)

In the diploid speciesP. prolifera, the evolution of autogamy has resulted in the presence of distinct selfing and outcrossing races. The change in breeding system toward autogamy is associated with a reduction in the size of floral features and a lower pollen production. The outbreeding populations have only been found in Greece, while the selfing populations have colonized much of Europe. The two races appear to be reproductively isolated from one another. —Petrorhagia sect.Kohlrauschia contains four species.P. glumacea is outbreeding and shows unilateral incompatibility with the small-flowered race ofP. prolifera and interfertility with the large-flowered race; althoughP. glumacea is sympatric with the large-flowered race, they do not appear to hybridize in the wild. Amongst the allopatric species, internal breeding barriers are also found. These may take the form of hybrid sterility or seed incompatibility.P. velutina is autogamous and reproductively isolated from all the other species of the section regardless of whether they are sympatric or allopatric. It is suggested that the evolution of autogamy has been an important factor in the spread ofP. velutina, the tetraploidP. nanteuilii, and the small-flowered race ofP. prolifera in Europe.     

Taxonomic differences among closely related pines Pinus sylvestris, P. mugo, P. uncinata, P. rotundata and P. uliginosa as revealed in needle sclerenchyma cells

The purpose of the present study was to test the taxonomic value of sclerenchyma in distinguishing Pinus sylvestris and P. mugo, P. uncinata, P. rotundata and P. uliginosa, all representing the subsection Sylvestres within the genus Pinus. Thirty-six samples were gathered in natural populations. Every sample was represented with 30 individuals, every individual with 10 brachyblasts. Three types of sclerenchymatic cells surrounding the resin canals and four between vascular bundles were distinguished. Relations among samples and taxa were verified using discriminant analysis and clustering based on Euclidean distances. The types of sclerenchymatic cells surrounding the resin canals and located between the vascular bundles differentiate the compared taxa when used as average frequencies but are extremely variable and do not allow the classification of every individual. The study demonstrated that the type of sclerenchymatic cells surrounding the resin canals and between the vascular bundles in needles could have an important taxonomic value in distinguishing the taxa of two-needle pines of the subsection Sylvestres in Europe at the population level. The distinguishing of individuals was difficult because of very high variation of sclerenchyma characters.     

DNA marker based identification of spontaneous hybrids between Pinus mugo and P. sylvestris at the Lithuanian sea‐side

The objective was to identify spontaneous hybrids between P. mugo and P. sylvestris using organelle DNA markers in sympatric zones at the sea‐side spit of Kursiu Nerija in western Lithuania. A field inventory was carried out over the entire Lithuanian part of the spit and 203 individuals morphologically intermediate between P. sylvestris and P. mugo were tested for their male parent with chloroplast DNA PCR‐RFLP markers and for their female parent with mitochondrial DNA PCR markers. Unfortunately, the mitochondrial DNA Nad7 marker failed to identify the female parent species in our study. However, the chloroplast DNA PCR‐RFLP marker revealed that out of 203 tested individuals only 23 had a paternity different than that indicated by morphology. Of these, 13 individuals were morphologically identified as P. sylvestris but possessed cpDNA of P. mugo (putative hybrids with P. sylvestris (female) ×P. mugo (male parent), and 10 individuals morphologically identified as P. mugo possessed cpDNA of P. sylvestris and may be hybrids with P. mugo (female) ×P. sylvestris (male parent). The remainder of the 177 individuals identified in the field inventory were were considered as pure species. In conclusion, our study indicates ongoing spontaneous hybridization between P. mugo and P. syvestris in Kursiu Nerija. Human impact via seed transfer on altered hybridization rates as well as evolutionary consequences are discussed.     

Field-based artificial crossings indicate partial compatibility of reciprocal crosses between <Emphasis Type="Italic">Pinus sylvestris</Emphasis> and <Emphasis Type="Italic">Pinus mugo</Emphasis> and unexpected chloroplast DNA inheritance

Crossability relationships between Scots pine (Pinus sylvestris L.) and mountain dwarf pine (Pinus mugo Turra) was studied, using artificial pollination approach. Partial compatibility of the reciprocal crossings of these species was proved experimentally, validating the idea of a spontaneous formation of their hybrid swarms under natural conditions. The hybrids were validated using organellar DNA markers and nuclear DNA microsatellites. Based on the percentage of filled seeds, the interspecific crossings were less efficient than the intraspecific cross-pollinations of P. sylvestris and P. mugo individuals. Both species were found to intercross readily with individuals of their putative hybrid swarm, P. mugo exhibiting a higher hybridological affinity towards putatively hybrid individuals than P. sylvestris. Validation of the hybrids confirmed the paternal inheritance of chloroplast DNA (cpDNA) in the combination P. sylvestris × P. mugo only. Surprisingly, in the reciprocal crossing P. mugo × P. sylvestris, maternal inheritance of cpDNA was revealed. Obtained results offer a new insight into the direction and intensity of gene flow within the hybrid swarms of Scots pine and mountain dwarf pine.     

Genome size and base composition of five<Emphasis Type="Italic"> Pinus</Emphasis> species from the Balkan region

The 2C DNA content and base composition of five Pinus (2n=24) species and two Pinus subspecies from the Balkan region have been estimated by flow cytometry. P. heldreichii (five populations) and P. peuce (one population) were assessed for the first time, as also were subspecies of P. nigra (three populations—two of subspecies nigra and one of subspecies dalmatica) along with P. sylvestris, and P. mugo from the same region. The 2C DNA values of these Pinus ranged from 42.5 pg to 54.9 pg (41.7–53.8×10⁹ bp), and the base composition was quite stable (about 39.5% GC). Significant differences were observed between two subspecies of P. nigra and even between two populations of subsp. nigra. The two other species (P. sylvestris and P. mugo) had 2C values of 42.5 pg and 42.8 pg, respectively, while that of P. peuce was 54.9 pg. These genome sizes are in accordance with published values except for P. sylvestris, which was 20% below estimates made by other authors.Communicated by M. Beckert     

Morphological and molecular characterization of somatic hybrid plants between Lycopersicon esculentum and Solanum nigrum

Summary Mesophyll protoplasts of tomato (Lycopersicon esculentum Mill. var. cerasiforme) and of an atrazine-resistant biotype of black nightshade, (Solanum nigrum L.), were fused by using polyethylene glycol/dimethyl sulfoxide (PEG/DMSO) solution and three somatic hybrid plants, each derived from a separate callus, were recovered. A twostep selection system was used: (1) protoplast culture medium (modified 8E) in which only tomato protoplasts formed calluses; and (2) regeneration medium (MS2Z) on which only S. nigrum calluses produced shoots. These selective steps were augmented by early isozyme analysis of putative hybrid shoots still in vitro. Phosphoglucoisomerase (PGI) and glutamate oxaloacetate transaminase (GOT) mapped to five loci on four chromosomes in tomato confirmed the hybrid nature of the nuclei of regenerated shoots. The somatic hybrid plants had simple leaves, and intermediate flower and bud morphology, but anthesis was reduced to 5% due to premature bud abscission and the pollen grains were non-viable. Southern DNA blot hybridization using a pea 45 S ribosomal RNA gene probe reconfirmed the hybrid nature of the nuclear genome of the three plants. A ³²P-labeled probe of Oenothera chloroplast DNA (cpDNA) hybridized to cpDNA restricted with EcoRI or EcoRV indicated the presence of the tomato cpDNA pattern in all three hybrids. Likewise, the plants were all found to be atrazine sensitive. Analysis with two mitochondrial (mt)DNA-specific probes, maize cytochrome oxidase subunit II and PmtSylSa8 from Nicotiana sylvestris, showed that, in addition to typical mitochondrial rearrangements, specific bands of both parents were present or missing in each somatic hybrid plant.Michigan Agricultural Experiment Station Journal Article No. 12433     

Seed quality in hybrid swarm populations of Pinus mugo Turra and P. sylvestris L.

Crop characteristics involving cone size, average number of seeds per cone, and seed germinability have been studied for three hybrid swarm populations of Pinus mugo and P. sylvestris, two pure P. mugo populations and one pure P. sylvestris population, in northern Slovakia. Generally, reproduction capability characteristics were significantly reduced in hybrid swarms in relation to the control populations. Although there were no consistent differences between hybrid swarms and control populations in the average number of seeds per cone or cone size, seed germination rates and full seed production in hybrid swarms were lower than for the control population of P. sylvestris and one of the pure populations of P. mugo. Based on these data a conclusion has been drawn postulating partial rather than full fertility of the hybrid swarm populations of P. mugo and P. sylvestris.     

中国假尾孢属的研究V

本文报导中国假尾孢属的30个种,其中有2个新种:透骨草假尾孢(Pseudocercospora phrymae),槐假尾孢(P. sophorae), 6个新组合:无花果假尾孢(P. fici),爵床假尾孢(P. justiciae),木犀生假尾孢(P. osmanthicola),海桐花假尾孢(P. pittospori),色柱假尾孢(P. polygonorum),花椒假尾孢(P. xanthoxyli)和5个中国新记录。文中对新种和新组合进行了描述并绘图,其他种作了简要描述或讨论。研究的标本保存在中国科学院微生物研究所真菌标本室(HMAS)。     

Somatic hybridization in the gramineae: Pennisetum americanum (L.) K. Schum. (Pearl millet) +Panicum maximum Jacq. (Guinea grass)

Summary Protoplasts from Pennisetum americanum resistant to S-2-amino-ethyl-l-cysteine (AEC) were fused with protoplasts of Panicum maximum utilizing polyethylene glycol-dimethylsulfoxide after inactivation of the Pennisetum protoplasts with 1 mM iodoacetic acid. The iodoacetate treatment prevented division of Pennisetum protoplasts; therefore, only Panicum protoplasts and heterokaryons potentially could give rise to colonies. A second level of selection was imposed by plating 3–4-week-old colonies on AEC medium. Putative somatic hybrid calli were analyzed for alcohol dehydrogenase, 6-phosphogluconate dehydrogenase, aminopeptidase, and shikimate dehydrogenase isozymes. Three somatic hybrid cell lines (lines 2, 3, and 67) were identified which showed two bands of alcohol dehydrogenase activity representing homodimers of P. maximum and P. americanum as well as a novel intermediate band of activity where Panicum-Pennisetum heterodimers would be expected. Aminopeptidase and shikimate dehydrogenase were useful for identifying presumptive hybrid calli but the isozyme patterns were additive-evidence which would not preclude the selection of chimeric callus. A more complex isozyme pattern which varied among the somatic hybrids was observed for 6-phosphogluconate dehydrogenase. In the hybrid calli, the presence of DNA sequences homologous to both P. maximum and P. americanum sequences was confirmed by hybridization of a maize ribosomal DNA probe to XbaI and EcoRI restriction fragments. Growth of hybrid lines on various concentrations of AEC was either similar to the AEC-resistant parent (hybrid line 2) or intermediate between the resistant and sensitive parents (hybrid lines 3, 67).     

Fertile asymmetric somatic hybrids between Lycopersicon esculentum Mill. and Lycopersicon peruvianum var. dentatum Dun.

Summary Thirteen nuclear asymmetric hybrids were regenerated under selective conditions following fusion of chlorophyll-deficient protoplasts from cultivated tomato (Lycopersicon esculentum Mill.) and -(-irradiated protoplasts from the wild species Lycopersicon peruvianum var. dentatum Dun. All hybrid plants were classified as being asymmetric based on morphological traits, chromosome numbers and isozyme patterns. The majority of the hybrids inherited Lycopersicon peruvianum var. dentatum chloroplasts. Mitochondrial DNA analysis revealed mixed mitochondria populations deriving from both parents in some of the hybrids and rearranged mitochondrial DNA in others. The asymmetric hybrids express some morphological traits that are not found in either of the parental species. Fertile F₁ plants were obtained after self-pollination of the asymmetric hybrids in four cases. The results obtained confirm the potential of asymmetric hybridization as a new source of genetic variation, and as a method for transferring of a part of genetic material from donor to recipient, and demonstrate that it is possible to produce fertile somatic hybrids by this technique.     

中甸灯台报春与鹅黄灯台报春的系统演化

中甸灯台报春和鹅黄灯台报春在形态上较为相似,通常被认为是亲缘关系较近的物种。该研究选取核基因ITS序列和叶绿体trn L-F为分子标记,利用贝叶斯的方法构建系统树,在用两种分子标记构建的系统树中,两种报春均以0.81的支持率聚成一个分支,明确了两种报春的姐妹种关系。首先,通过选取两个物种的五个形态学性状,进行主成分分析,聚类结果中两种报春各自的个体分别聚在两块区域,表明两种报春的形态学性状产生了明显的分化,且中甸灯台报春的花部各性状值明显大于鹅黄灯台报春,表明花部综合征和繁育系统间的相关联系。然后,通过用8对SSR分子标记对两种报春进行STRUCTURE聚类分析,结果检测K的最适值为2,此时中甸灯台报春和鹅黄灯台报春的个体各自以不同颜色聚成界限清晰的两块区域,揭示了两种报春在分子水平上也产生了显著而稳定的分化,通过Ima2软件,依据叶绿体联合序列,计算了两种报春的分化时间大约是在更新世,推测这两个姐妹种的物种分化受更新世气候动荡和一些地质历史事件的影响。最后,运用maxent软件分析两个物种的生态位差异,结果表明最干季节降水量、年均降水量和季节性降水量对中甸灯台报春的分布有重要影响,最冷季节平均温度、最干月降水量、年均降水量和季节性降水量对鹅黄灯台报春的分布有重要影响,两个物种间的生态位产生了分化。由此推测,这种生态位的差异是物种分化的结果,同时也是物种间系统演化的重要驱动力。