Interspecific Pollination Research Articles

Pollinator‐mediated isolation in sympatric milkweeds (Asclepias): do floral morphology and insect behavior influence species boundaries?

Summary •We explored whether mechanical or ethological differences provide pollinator-mediated floral isolation capable of reinforcing existing species barriers among sympatric Asclepias with divergent floral morphologies: A. incarnata, A. verticiallata and A. syriaca. •In a common garden, we quantified pollinator visitation and flight patterns, differences in corporal attachment of pollinia to insects, and the potential outcome of putative floral barriers for interspecific pollination and fruit set. •We detected significant variation in the importance, constancy, and behavior of major pollinators on sympatric asclepiads, including Bombus, Xylocopa and large sphecid wasps. Pollinia attach differentially to the arolium on insect legs for A. syriaca, but to the tarsal hairs in other asclepiads. Fruit-set was lower in mixed than unispecific patches of Asclepias. •We detected mechanical isolation between A. syriaca and its congeners and a tendency toward wasp pollination in A. verticillata. All three species appear to show some specialization for long-tongued hymenoptera and lepidopterans. Pre–mating barriers provide a potentially effective means of reducing interspecific pollination, but more study is needed in species visited by generalists.

Development of self‐incompatible Brassica napus: (I) introgression of S‐alleles from Brassica oleracea through interspecific hybridization

AbstractCultivars in Brassica napus var. oleifera, a self‐pollinating, self‐compatible species, have traditionally been developed as open‐pollinated lines or populations. Significant yield gains in this species have been realized through the exploitation of heterosis. Commercial hybrid production has been possible as a result of the development of a number of pollination control systems. Self‐incompatibility was transferred from B. oleracea var. italica to B. napus var. oleifera through interspecific hybridization. The response to interspecific pollination, as measured by pod elongation and initial stages of ovule development, was genotype dependent, and two highly responsive B. napus genotypes were identified. Embryo rescue was used to produce the interspecific hybrids. Isoelectric focusing of stigma proteins was used to identify S‐alleles in the interspecific hybrids to facilitate backcrossing. Segregation of the S‐locus through a series of back‐crosses to B. napus was complicated by aneuploidy; however, the S‐locus was found to segregate as a single gene. Usefulness of B. oleracea as a source of S‐alleles for pollination control in B. napus is discussed.

Controlled and open pollination between Pinus brutia (Ten.) and Pinus halepensis (Mill.) in Israel and hybrid performance

The present study examined the occurrence of open and controlled pollination between Pinus brutia (Ten.) and Pinus halepensis (Mill.) under the environmental conditions of the East Mediterranean climate. Controlled interspecific pollination succeeded only with P. brutia as the maternal parent and P. halepensis as the paternal parent. Open interspecific pollination also occurred under Israeli conditions, due to some overlapping between the flowering periods of P. brutia and P. halepensis. Interspecific pollination was characterized by a low percentage of filled seeds and a low germination percentage compared to seeds from intraspecific pollination of each of the parents. The growth rate of the hybrids in the nursery was inferior to or the same as either of the parents. The susceptibility of the hybrids to Matsucoccus josephi (Bodenheimer et Harpaz) varied between different clones, but was intermediate between P. brutia (low susceptibility) and P. halepensis (high susceptibility). Backcrossing 1 seedlings w...

Production of hybrids between Cajanus acutifolius and C. cajan

There are many wild species of pigeonpea which are endemic to Australia. These wild species are cross incompatible with cultivated species of Indian origin. Cajanus acutifolius is one such species which does not easily cross with cultivated pigeonpea. Interspecific pollinations lead to hybrid seeds which were semi-shrivelled. Very few seeds germinated to give rise to F1 plants. Backcrossing the hybrid plants to C. cajan, the male parent, gave rise to aborting seeds which did not germinate in vivo hence BC1 plants are obtained after saving the aborting embryos in vitro. BC1 plants showed normal meiotic pairing, but had low pollen fertility. The reasons for embryo abortion and low pollen fertility in spite of normal meiosis could be due to the effect of wild species cytoplasm.

Sexual reproduction in the cork oak ( Quercus suber L). II. Crossing intra- and interspecific barriers

Intraspecific barriers promote outcrossing while interspecific mechanisms may contribute to the isolation of species; both control the exchange of genes between plants. In this paper we establish that post-pollination mechanisms promote outcross and act at different temporal and spatial levels to control seed set and quality in Quercus species. Controlled pollinations were performed to investigate intraspecific crossing barriers in Q. suber and pollen-pistil interactions following interspecific pollinations with some simpatric Quercus species. Cytological data of intraspecific crosses in Q. suber and Q. ilex have shown different kinetics on pollen tube growth after self and outcross pollination, but pollen tubes were able to reach the base of the styles in both species and seed set was successful. Although some pre-zygotic interaction is occurring at the style, the most important interaction takes place at ovary. The cross between Q. ilex and Q. suber is possible only in one direction, revealing a case of unilateral incongruity. We show that the lack of seed set observed in the cross Q. suber×Q. ilex is due to the inability of pollen tubes to penetrate the transmitting tissue after germination. With Q. suber mainly as female parent, pollinations with other simpatric Quercus species have shown different levels of constraint on pollen tube progression at stigma and style levels. Eventual hybridisation between Quercus species will depend on the compatibility of pollen-pistil interactions, on the competitive ability of pollen genotypes, and, most important, on the overlapping of geographical, phenological and ecological factors. Differences in seed set and seed allocation was evident in all crosses, and was particularly outstanding in interspecific and in self intraspecific crosses, determining the ultimate level of seed production and quality in Quercus species.

Natural hybridization between Vanilla claviculata (W.Wright) Sw. and V. barbellata Rchb.f. (Orchidaceae): genetic, morphological, and pollination experimental data

Vanilla claviculata (W.Wright) Sw. and V. barbellata Rchb.f. grow sympatrically in the southwestern part of Puerto Rico. At three localities in the range of the two species, individuals with flowers that appeared different from previously known species were found. To test whether these were hybrids between V. claviculata and V. barbellata, 11 morphological floral characters were recorded at seven populations and allozyme profiles of the three taxa compared. Principal component analysis on the morphological characters gave three distinct groups with minor overlaps. The first component axis reflects variation in overall size of the flowers, while the second component reflects variation in the size of the distal aperture of the labellum. Allozyme data revealed that the putative hybrids have a significant surplus of heterozygotes (FIS=−0.65) as expected in a F1generation. Both data sets support the hypothesis that the individuals are hybrids, which is further augmented by interspecific pollination experiments between the putative parental species. This is the first case of natural hybridization reported in the genus Vanilla.

Natural hybridization between Vanilla claviculata (W.Wright) Sw. and V. barbellata Rchb.f. (Orchidaceae): genetic, morphological, and pollination experimental data

Vanilla claviculata (W.Wright) Sw. and V. barbellata Rchb.f. grow sympatrically in the southwestern part of Puerto Rico. At three localities in the range of the two species, individuals with flowers that appeared different from previously known species were found. To test whether these were hybrids between V. claviculata and V. barbellata, 11 morphological floral characters were recorded at seven populations and allozyme profiles of the three taxa compared. Principal component analysis on the morphological characters gave three distinct groups with minor overlaps. The first component axis reflects variation in overall size of the flowers, while the second component reflects variation in the size of the distal aperture of the labellum. Allozyme data revealed that the putative hybrids have a significant surplus of heterozygotes (FIS=−0.65) as expected in a F1generation. Both data sets support the hypothesis that the individuals are hybrids, which is further augmented by interspecific pollination experiments between the putative parental species. This is the first case of natural hybridization reported in the genus Vanilla.

Sistema reprodutivo de Oxypetalum appendiculatum Mart. e Oxypetalum banksii Roem. & Schult. subsp. banksii (Asclepiadaceae)

Estudos sobre o sistema reprodutivo de O. appendiculatum e O. banksii subsp. banksii foram realizados em Viçosa, MG, sudeste do Brasil. Ambas são autocompatíveis e a viabilidade das sementes, resultantes de autopolinizações e polinizações cruzadas, é alta. A baixa taxa de produção de frutos em flores de O. banksii subsp. banksii expostas aos visitantes (polinizações abertas) deve estar relacionada à falta de polinização (fator extrínseco). Em O. appendiculatum, a baixa taxa de produção de frutos deve estar relacionada a fatores intrínsecos, regulados pela planta. Polinizações interespecíficas resultaram em sementes viáveis e os híbridos F1 apresentaram 64% de flores férteis; esses híbridos não foram encontrados na natureza. Autopolinizações em flores de F1 resultaram em sementes também viáveis.

Pollinators of Oxypetalum (Asclepiadaceae) in Southeastern Brazil

Floral visitors of seven species of Oxypetalum were registered in Viçosa, State of Minas Gerais. O. appendiculatum, O. banksii subsp, banksii, O. alpinumn var. alpinum and O. pachyglossum are pollinated by wasps, being Polybia ignobilis (Vespidae) a pollinator of these four species. It seems that P ignobilis promotes interspecific pollinations mainly between O. alpinum var. alpinum and O. pachyglossum, two species with very similar floral morphology. O. jacobinae, O. mexiae and O. subriparium are pollinated by bees. Wasps and bees carry one, two, three or several pollinaria in the mouthparts. O. mexiae, an endemic species in Viçosa, seems to present reproductive limitations, since its flowers are seldom visited.

Reproductive systems and crossing potential in three species ofBulbophyllum (Orchidaceae) occurring in Brazilian ?campo rupestre? vegetation

Experimental self-, cross- and interspecific pollinations were carried out inBulbophyllum weddellii, B. involutum andB. ipanemense (Orchidaceae). The last two species are closely related, butB. weddellii is somewhat more distant. The three species are self-compatible, with pollination by a vector necessary for fruit formation. In all crossing types, high rates of fruit abortion and formation of seeds without embryos were observed, particularly in interspecific crosses that involvedB. weddellii. Crosses between the more closely relatedB. involutum andB. ipanemense produced fruit formation rates and seed viability similar to those obtained in intraspecific crosses. These results agree with current hypotheses that suggest that interspecific crossing rates reflect phylogenetic proximity. Examination of pollen tube growth and aborted fruits suggests that a series of factors may be involved in the high abortion rate for fruits and large proportion of seeds without embryos. The low interspecific fertility noted forB. weddellii andB. involutum is important in the maintenance of isolation between these species that are sympatric, flower in the same period and share the same pollinators.

Pre-zygotic breeding barriers between the wild diploid potato species Solanum maglia and S. kurtzianum from Argentina

Solanum maglia Schldl. is a wild tuber-bearing species from the Quebrada de Alvarado (Mendoza, Argentina) and the Valparaiso area (Chile). Only diploid cytotypes are known from Argentina, while diploids and triploids are found in Chile. Five diploid accessions from Argentina did not produce seeds upon intercrossing. Thus, the basis of this behaviour and the crossability relations of this species with S. kurtzianum, Bitt. et Wittm., another diploid species that grows in its vicinity were investigated. After checking pollen viability, intraspecific and interspecific pollinations were carried out following an incomplete diallel mating design. Five emasculated flowers were pollinated per each one of 135 different combinations of genotypes (crosses); three flowers were fixed in FAA and analyzed under fluorescent microscopy, whereas the remaining two were left on the plant for fruit and seed set. Pollen tube growth was arrested in the first third of the style and no seeds were obtained in intraspecific S. maglia crosses. In interspecific crosses and according to their direction, pollen tubes either grew normally to the base of the style or were arrested in the last third, producing no fruits or fruits with a variable number of seeds in each. It is concluded that the five S. maglia accesions are, possibly, one clone and, thus, the gametophytic auto-incompatibility system prevents seed formation upon intercrossing. On the other hand, the incompatibility reaction observed in the interspecific crosses might be controlled by one or more genes independent of the S-locus.

Temporal variation in pollinarium size after its removal in species of Bulbophyllum: A different mechanism preventing self-pollination in Orchidaceae

InBulbophyllum involutum andB. ipanemense (Orchidaceae), two closely related species, shortly after removal the pollinarium has a diameter of approximately twice that of the entrance of the stigmatic cavity, requiring a mean time of 105 to 135 minutes to shrink in width and allow pollination. Because the pollinators of these species remain for some minutes in the same flower after removing the pollinia this mechanism, previously unknown inOrchidaceae, may be very important in preventing self-pollination. This mechanism does not occur inB. weddellii, and the pollinator does not remain in the flower after removing the pollinia. The smaller diameter of the stigmatic cavity inB. involutum reduces by 50% the chances of interspecific pollination withB. weddellii, and interspecific crossing is strictly unidirectional. This is important in maintaining isolation between these sympatric species, which share the same pollinators and have synchronized flowering.

Pollen-stigma capture is not species discriminant within the Brassicaceae family

The capture of pollen on the stigma surface is the first stage of interactions following the initial contact of the male gametophyte with the female tissues. In order to analyse pollen adhesion, we used a biomechanical assay based on Archimedes flotation of pollinated pistils to measure the forces necessary to detach pollen populations from stigmas. A considerable homogeneity of early pollen-stigma adhesion in intra- and interspecific situations among the Brassicaceae was observed. On the contrary, pollination of Brassica oleracea stigmas by pollen from species of more distantly related families showed a significantly lower level of pollen adhesion. Crosses involving interspecific pollinations among the Brassicaceae are not blocked at the stage of pollen capture but rather at later stages of pollination such as hydration, germination or penetration of the pollen tube into the style. This control is not totally stringent, confirming the potential risk of escape of transgenes from cultivated Brassicaceae crops to their allied adventices through interspecific pollination.

Involvement of SLR1 genes in pollen adhesion to the stigmatic surface in Brassicaceae

The S-locus-related gene SLR1 is highly conserved and highly expressed in several species of the Brassicaceae family. Its function has not been determined, although several features would suggest a fundamental role in pollination. A second related gene (SLR2) is conserved and expressed in a subset of Brassica genotypes. We analysed the stigmatic expression of SLR1 and SLR2 genes among 11 different plants from various species or genera of the Brassicaceae and examined the extent of the pollen-stigma interaction during intraspecific, interspecific and intergeneric pollinations between them. Appropriate statistical tests on these variables (pollen adhesion, germination, penetration into the stigma, style and ovary, and SLR gene expression) showed that expression of SLR1 (but not SLR2) may be a factor in pollen-stigma adhesion. This hypothesis was supported by the observation of reduced pollen-stigma adhesion in transgenic B. napus plants modified for SLR1 expression.

Intra- and Inter-specific Pollination of Santalum spicatum and S. album

The flower morphology, receptivity and sexual compatibility between genotypes and species were determined in Western Australian sandalwood (Santalum spicatum) and Indian sandalwood (S. album). The results showed that the stigma of both species became receptive at anthesis and reached a peak at 3 or 4 days after anthesis. Pollen tubes took 2 days to grow to the ovary when pollinated at anthesis, and 1 day when pollinated 2 or 3 days after anthesis. The egg apparatus matured at least 2 days after pollination and varied between genotypes. Fertilisation occurred 2 or 3 days following cross pollination. Although 10–40% of ovules were fertilised following intra-specific crosses of both species, the average initial fruit set was much lower: 4% in S. spicatum and 19% in S. album. Most immature fruit (75–80%) abscised following intra-specific pollination. The number of pollen tubes that grew in styles after self-and inter-specific pollination was lower than that for intra-specific pollination. Following self and inter-specific pollination, growth of pollen tubes was arrested in the style, ovary and around the embryo sac; a few penetrated the embryo sac. Initial fruit set was low and developing fruit abscised prematurely. The results indicated that pre- and post-fertilisation mechanisms control self-incompatibility and inter-specific incompatibility between the sandalwood species.

Pollination genetics of hybridization in sympatric populations of asclepias exaltata and a. syriaca (asclepiadaceae)

Enzyme electrophoresis of pollinia and seeds was used to identify pollinia on pollinators, interspecific pollinations, and hybrid seed production in sympatric populations of Asclepias exaltata and A. syriaca. The frequency of mixed pollinia loads on pollinators was low. Only 4.1% (N= 169 pollinia) and 8% (N= 244 pollinia) of the pollinia collected from pollinators of A. syriaca and A. exaltata, respectively, were identified as pollinia from the other species. Natural levels of interspecific pollination, 3.6% (N = 166 pollinia) on A. syriaca and 3.1% (N = 228 pollinia) on A. exaltata, were typically lower than the number of foreign pollinia carried by pollinators. Hybrid seeds were identified in only two of 208 A. syriaca fruits and one of 178 A. exaltata fruits. Hybrid seeds were largely underdeveloped in the single A. exaltata fruit. High fruit set (34.7%) and near normal seed set following hand‐pollination of A. syriaca with A. exaltata pollinia suggest that hybridization is more likely between these taxa when A. syriaca is the maternal parent. Our findings indicate that the likelihood of hybridization between A. exaltata and A. syriaca is remote. Nevertheless, introgressive hybridization has been documented in these species, which suggests that hybridization remains a strong evolutionary force even for species that infrequently interbreed.

Shell Structure and Embryo Development of Pistacia vera L. and P. atlantica Desf. (Anacardiaceae) Following Intra- and Interspecific Pollination

Fruits of Pistacia vera and Pistacia atlantica were sampled up to fruit maturity following intra- and interspecific pollination. Embryo and shell structure were investigated in relation to shell indehiscence and premature splitting of P. vera. The ponticulus of P. atlantica was formed from the funicle only, in contrast to that of P. vera, which developed from both the funicle and the ovary wall. This may be one of the causes of reduced interspecific as compared with intraspecific fertility. Shell indehiscence and premature splitting were more common following interspecific pollination of P. vera. Ovary wall and shell thickness at the suture were lower in P. atlantica than in P. vera, while the suture angle was greater. Following interspecific pollination of P. vera, both ovary wall and shell thickness at the suture were lower and the suture angle was greater than following intraspecific pollination. In the shells of some fruits, more than one groove was observed in areas other than the usual split zone. W...

Interspecific Hybridization between Banksia hookeriana Meisn. and Banksia prionotes Lindl. (Proteaceae)

Interspecific hybridization of Banksia hookeriana and Banksia prionotes was investigated via pollen tube growth, seed set, morphological measurements, and analysis of RAPD polymorphisms. Pollen tube growth to the ovary was observed following selfing and crossing pollination of each species and following interspecific hybridization to B. hookeriana as the female parent, but not in the reciprocal cross. All crosses resulted in seed set, except for self-pollination of B. prionotes and interspecific pollination to B. prionotes as the female parent. Mortality of hybrid seedlings was high. RAPD analysis of hybrid seedlings from two families showed paternal B. prionotes bands in all 11 tested seedlings. Leaf length or width of nine hybrid seedlings that survived to the 10-leaf stage was intermediate between that of intraspecific seedlings of both parents at the same age. A mature natural interspecific hybrid was fertile following controlled selfing and backcrossing to each parental species, as well as following open pollination, but seed set was lower, than for the parental species. It was concluded that hybridization between B. hookeriana and B. prionotes is unilateral, with interspecific seed set of B. hookeriana comparable to that following intraspecific pollination.

Is resource overlap disadvantageous to three sympatric legumes?

Abstract The sympatric legumes Dillwynia hispida, Dillwynia uncinata and Pultenaea densifolia overlap in flowering phenology, floral characters and native bee pollinators. Tests for pollen‐limitation were conducted to determine whether or not the overlap in pollinator use was detrimental to individuals of these species. Pollen limitation was only detected in D. hispida. Pollen loads on bees were examined and it was found that pollen loads from Dillwynia and P. densifolia were spatially segregated on the pollinator's body reducing the likelihood of pollen transfer between these species. The pollen of D. hispida and D. uncinata overlapped on the heads of bees suggesting that interspecific pollinations may be responsible for lowered fruit‐set in D. hispida. However, hand pollinations involving heterospecific pollen and controlled floral visits by bees to the two Dillwynia species revealed that heterospecific pollen loads did not significantly depress fruit‐set in either D. hispida or D. uncinata. Pollinator preferences were examined in detail and it was found that D. uncinata flowers received twice as many bees in a day compared with D. hispida and P. densifolia. Competition through pollinator preference is implicated here as a mechanism of competition. Intraspecific competition is also implicated as D. hispida was pollen limited in the absence of D. uncinata and P. densifolia.

Embryology of Eucalyptus Spathulata and E. platypus (Myrtaceae) Following Selfing, Crossing and Reciprocal Interspecific Pollination

Embryology of Eucalyptus spathulata Hook. and E. platypus Hook. (subgenus Symphyomyrtus, section Bisectaria) was investigated by bright field microscopy to determine the cause of ovule failure at 2 months following selfing, crossing and reciprocal interspecific pollination. Eucalyptus spathulata retained more capsules and produced more seeds following cross- than self-pollination, whereas there was no difference between selfing and crossing for E. platypus. Both species produced seeds following interspecific pollination, but germination was low. Highest ovule penetration by a pollen tube, and ovule growth at 2 months, was observed following crossing of E. spathulata and following interspecific pollination of E. platypus. Most self-pollinated ovules of both species were degenerating and were either unfertilised or had undivided zygotes and free nuclear endosperm. Muticellular pro-embryos were more common in cross-pollinated and interspecific pollinated ovules than following selfing. It was concluded that ovule degeneration of E. spathulata and E. platypus following selfing resulted from low levels of pollen tube penetration and fertilisation, and that in those ovules which were fertilised, the zygote generally failed to divide. Degeneration following cross-pollination of E. spathulata also resulted from failure of the zygote to divide, and in E. platypus additional reasons were low levels of pollen tube penetration and fertilisation, and lack of cellularisation of the endosperm. Ovule degeneration of both species following interspecific pollination included failure of the zygote to divide, but in E. spathulata it also resulted from slower embryo development and reduced cellularisation of the endosperm.