Low interspecific pollen transfer between invasive aquatic <Emphasis Type="Italic">Ludwigia grandiflora</Emphasis> and native co-flowering plants

Showy invasive alien plants are often integrated in the diet of generalist pollinators and because of the lack of co-evolvement with the native plant community, a high amount of interspecific pollen transfer (IPT) can be expected. We investigated pollinator switching and magnitude plus distance of IPT between the alien aquatic Ludwigia grandiflora and the native Lythrum salicaria in both directions in uninvaded and invaded sites with a different relative abundance of L. grandiflora (% cover of the alien plant: no cover; low cover: <5%; high cover: 50–75%). A field experiment was conducted to include both pollinator interspecific movements and tracking of IPT, using fluorescent dye as a pollen analogue. Despite a substantial overlap in pollinators between L. grandiflora and the native L. salicaria, less than 10% of the observed flights were interspecific. Similar results were found in dye transfer patterns. The proportions of stigmas with conspecific dye were always higher than the proportions of stigmas with heterospecific dye for L grandiflora and L. salicaria. There were no differences in conspecific dye loads for L. salicaria between uninvaded and invaded sites. Conspecific pollen loss (native CPL) and heterospecific pollen deposition (alien HPD) were in general low and species-specific. The distance of HPD ranged respectively from 1.7 to 39 m and from 0.3 to 54.8 m in the low cover and high cover sites while CPL ranged respectively from 6.40 to 68.02 m and from 0.60 to 40.18 m in the low cover and high cover sites. We can conclude that, in this system, CPL and HPD will play a minor role in pollinator-mediated interaction. Furthermore, interspecific competition for pollinators will cover a larger distance than just neighboring individuals. Our results suggest the necessity to consider the combined effect of insect visitation, pollen deposition, relative alien abundance, distance and seed set when investigating pollinator-mediated interactions of invasive plants.     

Effective range of reproductive interference exerted by an alien dandelion, <Emphasis Type="Italic">Taraxacum officinale</Emphasis>, on a native congener

Reproductive interference (RI), defined as the fitness cost of interspecific sexual interactions, such as interspecific pollen transfer (IPT) in plants, is ecologically important. Theoretically, RI could result in competitive exclusion, as it operates in a frequency-dependent manner. Additionally, IPT may have a greater range than resource competition, although information about the range of IPT is lacking. In the present study, we measured the range of IPT exerted by Taraxacum officinale (an alien species) on a native dandelion, T. japonicum. We used two approaches. In one, we analyzed the RI effect on a native seed set at three spatial scales. In the second, we tracked IPT from alien to native flower heads using fluorescent pigments as markers. We estimated that pollination distances were in the order of several meters. These distances exceeded the mean distance from each native plant to the nearest alien. As hypothesized, the effect of RI reached farther than neighboring individuals. These data indicate the spatial range from which alien dandelions should be removed to allow the conservation of natives.     

Mating Behavior Differences and the Cost of Mating in <Emphasis Type="Italic">Allonemobius fasciatus</Emphasis> and <Emphasis Type="Italic">A. socius</Emphasis>

Close range prezygotic barriers are assumed to be present between sister taxa who have overlapping distributions. Here we report the results of studies designed to test the existence of prezygotic barriers between two closely related species, A. fasciatus and A. socius. We finely dissected the courtship and mating rituals and performed Monte Carlo analysis on lengths of time and number of occurrences of particular events in the courtship mating sequence. These detailed investigations of the courtship and mating behavior of conspecific and heterospecific pairs demonstrate that behavioral isolation is non-existent. We also measure the adult lifespan and number of progeny produced from singly and multiply mated males and females in conspecific and heterospecific trials. We found that cost of a heterospecific mating is asymmetric between the sexes with males paying a higher cost.     

Floral morph composition and pollen limitation in the seed set of <Emphasis Type="Italic">Nymphoides indica</Emphasis> populations

We investigated the floral morph composition and seed set of the populations in a heterostylous aquatic plant, Nymphoides indica O. Kuntze (Menyanthaceae). The bias of floral morph ratio in the populations was negatively correlated with the seed set. In the populations with biased floral morph ratio, the pollen on the stigma of the common morphs were dominated by pollen of the same morph, resulting in reduced seed set. The addition of pollen of the opposite morph on the stigmas of the common morph resulted in an increase of fruit and seed set rates. The intermorph pollination rate and the fruit and seed set rates of Nymphoides indica were highest when two floral morphs were close in the populations. Various insect species including Lepidoptera, Diptera and Hymenoptera visited the flowers. However, geitonogamous pollination was common because of the short flying distance of the pollinators. These results indicate that the seed set of Nymphoides indica is limited by the shortage of compatible pollens and that the success of intermorph pollination is influenced by spatial distribution of the two floral morphs. As Nymphoides indica is endangered, conservation of the populations with high fecundity is proposed by us.     

Post-pollination hybridization barriers in <Emphasis Type="Italic">Nicotiana</Emphasis> section <Emphasis Type="Italic">Alatae</Emphasis>

Nicotiana section Alatae contains eight species with variable flower sizes and morphologies. Section members readily hybridize in the glasshouse, but no hybrids have been observed in natural sympatric and parapatric populations. To investigate interspecific crossing relationships with respect to mechanisms preventing hybridization, all members of section Alatae were intercrossed in a complete diallel. We found positive correlation between the pistil length of the pollen donor and interspecific seed set relative to the conspecific cross. Pollen tube growth rate and pollen donor pistil length were positively correlated as well. Furthermore, pollen from short-pistil members of section Alatae could only grow a maximum distance proportional to, but greater than, their own pistil lengths. Our results show that pollen tube growth capacity (i.e., rate and distance), provides a hybridization barrier in long-pistil species × short-pistil species crosses. We also found another hybridization barrier not specifically related to pollen tube growth capacity in short-pistil species × long-pistil species. Taken together, these barriers can generally be described by a ‘pistil-length mismatch’ rule; in section Alatae, pollen has the most success fertilizing ovules from species with pistil lengths similar to their own. This rule could contribute to hybridization barriers in Section Alatae because the species display dramatically different pistil lengths. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sympatric reinforcement of reproductive barriers between <Emphasis Type="Italic">Neotinea tridentata</Emphasis> and <Emphasis Type="Italic">N. ustulata</Emphasis> (Orchidaceae)

Reinforcement is the process by which selection favors traits that decrease mating between two incipient species in response to costly mating or the production of maladapted hybrids, causing the evolution of greater reproductive isolation between emerging species. I have studied a pair of orchids, Neotinea tridentata and N. ustulata, to examine the level of postmating pre- and post-zygotic isolating mechanisms that maintain these species, and the degree to which the boundary may still be permeable to gene flow. In this study, I performed pollen tube growth rate experiments and I investigated pre- and post-zygotic barriers by performing hand pollination experiments in order to evaluate fruit set, embryonate seed set and seed germination rates by intra- and interspecific crosses. Fruit set, the percentage of embryonate seeds and germinability of interspecific crosses were reduced compared to intraspecific pollinations, showing significant differences between sympatric and allopatric populations. While in allopatric populations the post-pollination isolation index ranged between 0.40 and 0.11, in sympatric populations orchid pairs showed total isolation due to post-pollination prezygotic barriers, guaranteed at the level of pollen–stigma interactions. Indeed, in sympatric populations, pollen tubes reached the ovary after 24 h in only 8 out of 45 plants; in the remaining cases, the pollen tubes did not enter the ovary, and thus no fruit set occurred. This pair of orchids is characterized by postmating pre-zygotic reproductive isolation in sympatric populations that prevents the formation of hybrids. This mechanism of speciation, starting in allopatry and triggering the reinforcement mechanisms of reproductive isolation in secondary sympatry, is the most likely explanation for the pattern of evolutionary transitions found in this pair of orchids.     

Males,but not females,contribute to sexual isolation between two sympatric species of <Emphasis Type="Italic">Gambusia</Emphasis>

Reproductive isolation restricts genetic exchange between species. Various pre- and post-mating barriers, such as behavior, physiology and gametic incompatibility, have been shown to evolve in sympatry. In certain scenarios, isolation can be asymmetrical, where species differentially prefer conspecifics. We examined sexual isolation via conspecific mate preference between Gambusia affinis and G. geiseri in both sexes. To investigate male contribution to sexual isolation, we compared the number of mating attempts (gonopodial thrusts) directed at either a conspecific or a heterospecific female, in both species. We also examined sperm priming and expenditure in males in the presence of conspecific or heterospecific females. We then measured female preference for either a conspecific or heterospecific male, in both species. We found that males of both species preferred to mate with conspecific females, but showed no difference in sperm production or expenditure between conspecific and heterospecific females. Females of both species did not prefer conspecific over heterospecific males. Our results suggest that sexual isolation might be mediated by male mate choice in this system and not female choice, suggesting that there is asymmetrical reproductive isolation between the sexes in G. affinis and G. geiseri, but symmetrical species isolation.     

Specialist <Emphasis Type="Italic">Osmia</Emphasis> bees forage indiscriminately among hybridizing <Emphasis Type="Italic">Balsamorhiza</Emphasis> floral hosts

Pollinators, even floral generalists (=polyleges), typically specialize during individual foraging bouts, infrequently switching between floral hosts. Such transient floral constancy restricts pollen flow, and thereby gene flow, to conspecific flowers in mixed plant communities. Where incipient flowering species meet, however, weak cross-fertility and often similar floral traits can yield mixed reproductive outcomes among pollinator-dependent species. In these cases, floral constancy by polyleges sometimes serves as an ethological mating barrier. More often, their foraging infidelities instead facilitate host introgression and hybridization. Many other bee species are oligolectic (taxonomic specialists for pollen). Oligoleges could be more discriminating connoisseurs than polyleges when foraging among their limited set of related floral hosts. If true, greater foraging constancy might ensue, contributing to positive assortative mating and disruptive selection, thereby facilitating speciation among their interfertile floral hosts. To test this Connoisseur Hypothesis, nesting females of two species of oligolectic Osmia bees were presented with randomized mixed arrays of flowers of two sympatric species of their pollen host, Balsamorhiza, a genus known for hybridization. In a closely spaced grid, the females of both species preferred the larger flowered B. macrophylla, evidence for discrimination. However, both species’ females showed no floral constancy whatsoever during their individual foraging bouts, switching randomly between species proportional to their floral preference. In a wider spaced array in which the bouquets reflected natural plant spacing, foraging oligolectic bees often transferred pollen surrogates (fluorescent powders) both between conspecific flowers (geitonogamy and xenogamy) and between the two Balsamorhiza species. The Connoisseur Hypothesis was therefore rejected. Foraging infidelity by these oligolectic Osmia bees will contribute to introgression and hybridization where interfertile species of Balsamorhiza meet and flower together. A literature review reveals that other plant genera whose species hybridize also attract numerous oligolectic bees, providing independent opportunities to test the generality of this conclusion.     

Interspecific social interactions and behavioral responses of <Emphasis Type="Italic">Apodemus agrarius</Emphasis> and <Emphasis Type="Italic">Apodemus flavicollis</Emphasis> to conspecific and heterospecific odors

The social interactions between Apodemus agrarius and A. flavicollis, and their behavioral responses to conspecific and heterospecific odors, were studied in male–male and female–female interspecific dyadic encounters, and an attraction–avoidance test was used in order to clarify the behavioral mechanisms which control their relationships in wild populations. The experiments were carried out at the beginning and at the end of the breeding season—in spring and in autumn. In spring the aggressiveness was higher than in autumn. Males of both species showed attraction to conspecific odors from the opposite sex, while the females were indifferent. In autumn both males and females displayed attraction to conspecific odors from the same sex. However, mice of both species showed avoidance to heterospecific odors from the same and the opposite sex in spring, and indifference to heterospecific odors from the same and the opposite sex in autumn. Based on these findings, it could be assumed that the patterns of social interactions and responses to conspecific and heterospecific odors undergo seasonal changes in their life cycle. Probably the avoidance response to heterospecific odors could serve as a spacing mechanism to avoid aggressive encounters between A. agrarius and A. flavicollis in syntopic habitats during the breeding period.     

Exploring the boundary between pollination syndromes: bats and hummingbirds as pollinators of<Emphasis Type="Italic"> Burmeistera cyclostigmata</Emphasis> and<Emphasis Type="Italic"> B. tenuiflora</Emphasis> (Campanulaceae)

In this study I documented the degree of specialization in the pollination systems of Burmeistera cyclostigmata and B. tenuiflora (Campanulaceae) to explore the potential role of floral isolation in the diversification of the genus. I asked which floral characteristics are important in specializing on either bat or hummingbird pollination, and whether overlap between these floral syndromes can exist. I examined nocturnal and diurnal pollen deposition, pollinator visitation rates, and single visit effectiveness and related them to intra- and interspecific variation in Burmeistera floral characteristics at Monteverde, Costa Rica. Bats and hummingbirds visited both Burmeistera species, and bats pollinated both species. Owing to differences in floral morphology, however, hummingbirds effectively pollinated only B. tenuiflora. The generalized pollination system of B. tenuiflora demonstrates that there can be overlap in the boundary between ornithophily and chiropterophily, and that nectar production and timing of anthesis do not serve as barriers between these syndromes. The high intraspecific variation in floral color from green to red or purple did not correlate with either nocturnal or diurnal pollen deposition. Degree of flower accessibility did affect pollination; nocturnal pollen deposition significantly decreased as flowers become more obstructed. In Burmeistera, floral morphology and accessibility appear to be the most important floral characteristics for specialization at the boundary between ornithophily and chiropterophily.     

Influence of reproductive traits on pollination success in two <Emphasis Type="Italic">Daphne</Emphasis> species (Thymelaeaceae)

Taxonomically related species can differ in a number of reproductive traits, which may translate into a differential mating system and pollination success. Here we compare two hermaphroditic insect-pollinated Daphne species (D. rodriguezii and D. gnidium) which differ in distribution (island endemic vs. mediterranean) and floral traits (long- vs. short-tube corolla). We investigated their mating system and pollen limitation by means of hand-pollination experiments and quantified the diversity and abundance of flower visitors by direct observations. Plant size and five reproductive traits (flower production, proportion of viable anthers, pollen production, flower tube length and tepal area) were studied to assess how they contribute to reproductive success, measured as proportion of pollen grains germinated per stigma and fruit set. Selfing was very low and pollen limitation existed in both species, though was higher in D. rodriguezii probably due to the scarcity of flower visitors. The low fruit set in both species suggests that most of the pollen grains found on stigmas are self-pollen. Pollinators appeared to favour some floral traits (specifically, flower tube length or tepal area) in both species, although flower crop in D. rodriguezii was the only reproductive trait influencing fruit set. In both species, the highest variability in reproductive traits and pollination success was within individuals. Our findings suggest that despite both species showed similar mating system, dependency on outcrossing pollen and selection of floral traits, pollen limitation was higher in D. rodriguezii, probably as a higher proportion of self-pollen arrives to its stigmas.     

Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.     

Behavioral Sequence Leading to Sexual Isolation Between <Emphasis Type="Italic">Drosophila ananassae</Emphasis> and <Emphasis Type="Italic">D. pallidosa</Emphasis>

Drosophila ananassae and D. pallidosa are closely related, sympatric species that lack postmating isolation. Sexual isolation has been considered important in maintaining them as independent species. To clarify the behavioral processes leading to sexual isolation, we analyzed behavioral sequences and examined the effect of courtship song on mating success and on behaviors of both sexes by surgically removing male wings (song generators), female aristae (song receivers), or female wings (means of fluttering). We found that heterospecific courtship songs evoked female wing fluttering, whereas conspecific courtship song did not. Furthermore, female wing fluttering made courting males discontinue courtship. These findings suggest that strong sexual isolation is achieved through the following behavioral sequence: heterospecific song→female wing fluttering→courtship discontinuation.     

Hybridization rate and genotypic diversity of apomictic hybrids between native (<Emphasis Type="Italic">Taraxacum japonicum</Emphasis>) and introduced (<Emphasis Type="Italic">T. officinale</Emphasis>) dandelions in western Japan

Hybridization between the introduced and native plants may enhance invasiveness, especially in asexually reproducing species. Hybrid apomictic dandelions between native (Taraxacum platycarpum and T. japonicum) and exotic (T. officinale) species are distributed widely throughout Japan. To estimate the origin(s) and dispersal of the hybrids, we investigated the hybridization rate and genotypic diversity in mixed populations of T. japonicum, T. officinale and their hybrids at two green parks in western Japan. Among the plants identified as exotics from flower morphology, 86–96% were hybrids by genetic analysis. Genetic data with simple sequence repeat markers revealed a high clonal diversity of the hybrid both within and between populations, indicating multiple origins. A hybrid seed was found from among the 1891 seeds collected from T. japonicum in the parks, indicating ongoing hybridization in the field. T. officinale and hybrids were genetically differentiated between the two parks independent of the ploidy level; the allele frequency of T. officinale and tri- and tetraploid hybrids were similar within each park but different between the two parks. This suggests that the origins of hybrids were similar within the park but different between the parks. Overall, our results suggest that hybridization, including backcross, is an ongoing process, and that genetically diverse hybrids with various origins have been spreading in western Japan, probably because hybridization enhanced invasiveness at native habitat.     

Comparisons among populations and individuals to evaluate pollen–pistil interaction as a mechanism of reproductive interference in Taraxacum

Reproductive interference (RI), an interspecific mating interaction that reduces the fitness of at least one of the species involved, can lead to exclusive distributions in closely related species. A hypothesis previously proposed is that RI in plants may occur by ovule usurpation, in which pistils lack interspecific incompatibility and mistakenly accept heterospecific pollen, thereby losing an opportunity for conspecific pollen fertilization. However, few comparative studies have evaluated the consistency of the inferred mechanism within and among individuals and populations. We conducted hand-pollination experiments in six populations of three native Taraxacum species that suffered from different levels of RI from an alien congener, T. officinale, and compared pollen–pistil interactions among populations. We also investigated the interactions for eight individual T. japonicum plants whose response to heterospecific pollen deposition had been previously measured. Our results revealed that pollen tubes often penetrated native ovaries following heterospecific pollination in populations suffering from strong RI, whereas they seldom did in populations suffering from marginal RI. However, the relative frequency of the pollen tube penetration was not significantly related to the strength of alien RI. Not all pistils on an individual plant showed the same pollen receptivity following heterospecific pollination; rather, some accepted and some refused the pollen tubes. The relationship between pollen tube penetration following heterospecific pollination and the strength of the alien RI was also not significant among individuals. Our present results generally support the ovule usurpation hypothesis, but suggest that other factors, such as competition for pollinator services, variation in the effects of heterospecific pollen donors, and condition of the native inflorescences, might also affect the observed RI strength.

     

Phylogenetic Community Context Influences Pollen Delivery to <Emphasis Type="Italic">Allium cernuum</Emphasis>

Few studies have examined how the number and identity of species in the neighbouring community influences the reproductive success of particular focal species. Pollen delivery, an important component of fitness of sexual plants, is a function of not just the floral traits of any particular individual, but of features of the population and community as it depends on pollinator abundance and preferences. Many pollinators in flowering communities will prefer patches with high floral abundance or diversity yet may exhibit lower floral constancy when more flowering species are present. Thus, pollination efficiency could increase or decrease with increased species richness and this will alter the selection pressures placed upon the floral traits (such as floral colour or reward) of any member of a particular community. Moreover, recent studies have indicated that plant-pollinator networks are phylogenetically structured (pollinators visit related plant species more than expected by chance) and this may be an important factor structuring flowering plant communities. Thus, the sheer number of species within a patch may be less important than the number of closely-related species. We investigate whether species richness or phylogenetic proximity of coflowering species influences the amount and proportion of conspecific pollen delivered to nodding onion, Allium cernuum, in fragment patches of Garry Oak meadows in South Western British Columbia, Canada. We find that pollen delivery depended upon the presence of close relatives far more than on species richness or population density, indicating a central role of the community structure on pollination in flowering plant communities. Insofar as pollen delivery relates to the relative number of seeds produced by members of the community, pollination may in turn determine the community structure of the next generation.     

Interspecific hybridization of <Emphasis Type="Italic">Cucumis anguria</Emphasis> and <Emphasis Type="Italic">C. zeyheri via</Emphasis> embryo-rescue

Embryo-rescue was used to facilitate interspecific hybridization of Cucumis anguria L. and C. zeyheri Sond. Embryos were excised from developing fruits at one week intervals for six weeks after hand pollination. Medium containing coconut water was the most suitable for initial germination, and a medium with ascorbic acid was the best for embryo development and plant recovery. Viable plants were obtained from embryos and these plants showed morphological characteristics different from both parents. The analysis of the leucine aminopeptidase (LAP) locus revealed three hybrid types, H1.1, H1.2 and H2.     

Fruit abortion in the Chihuahuan-Desert endemic cactus <Emphasis Type="Italic">Opuntia microdasys</Emphasis>

Resource and pollen limitation, as well as pollen/ovule incompatibility, have been proposed as causes to explain fruit abortion. To assess whether abortion in Opuntia microdasys was due to resource and/or pollen limitation and could therefore be reversed fruit set and seed set were studied using controlled pollination experiments on 60 plants that had been randomly assigned a combination of watering and fertilization treatments. On the other hand, to test whether fruit abortion was irreversible, due to pollen/ovule incompatibility, we examined the reproductive biology of the species. This included observations on floral phenology, nectar production, flower visitors, numbers of pollen grains and ovules, and self-pollination experiments. Results showed that O. microdasys is a fully self-incompatible species and its floral biology and the activity of the main pollinator allow constant deposition of incompatible pollen onto stigmas, which may contribute to fruit abortion. Reproductive success was limited by nutrients and pollen, but the fruit set increased only by 58%, compared to 47% of the control, after the experimental addition of pollen, nutrients and water. The magnitude of pollen and resource limitation suggests that similar levels of abortion will be present in good as well as in bad years. Selfing as well as incompatibility between ramets from the same clone and between closely related plants seem plausible candidates to explain the large proportion of fruit abortion, and experimental cross pollination between genotypes identified through molecular markers are necessary to fully understand the considerable abortion rate that remains unexplained after pollen and resource addition. Interestingly, the possible reason why the abortion of energetically expensive fruits has not been eliminated by natural selection is that the aborted fruits are propagules able to root and produce new plants with the same genotype of the mother. Abortion would have a dramatic effect on cross-fertilized genotypes because they result in zero fitness, but it would have a positive effect on the fitness of the maternal genotype because a clonal offspring is produced. Evidently, the exact fitness consequences to the maternal plant will depend on the differences in survival and reproduction of these different offspring types.     

A test for conspecific cueing in two sympatric species of pupfish (<Emphasis Type="Italic">Cyprinodon beltrani</Emphasis>, <Emphasis Type="Italic">C. simus</Emphasis>)

In many fishes, individuals prefer to associate with phenotypically similar or conspecific individuals (conspecific cueing). Such phenotypic segregation can be an important evolutionary driver, for example, in intralacustric sympatric speciation processes. I examined conspecific cueing in two species of sympatric pupfish from Laguna Chichancanab in southern Mexico: the little shoaling and highly territorial Cyprinodon beltrani and the highly shoaling but non-territorial C. simus. Females were tested for shoal species preferences in two testing scenarios: (1) a sequential choice test where shoals of four conspecific or four heterospecific (Cyprinodon sp. or Poecilia reticulata) females were presented in succession, and (2) a simultaneous choice test where female shoals of both Cyprindon species were presented concurrently. Overall, higher shoaling in C. simus was corroborated in this study. In the sequential test, no effect of the type of stimulus shoal (con- or heterospecific) on shoaling behavior was detected. In the simultaneous tests, C. beltrani, but not C. simus females showed a preference for the conspecific shoal. It seems possible that C. simus females did not evolve species recognition mechanisms because no other Cyprinodon species in the Laguna Chichancanab shows equally high shoaling, which automatically leads to the formation of single-species (i.e., C. simus-) shoals. C. simus males do not establish long-term territories, but rather spawn within shoals, whereas C. beltrani females approach males in their breeding territories to spawn. I discuss that this behavioral difference still provides a powerful reproductive isolation mechanism even in the absence of conspecific cueing in C. simus.     

Expression of a polygalacturonase enzyme in germinating pollen of <Emphasis Type="Italic">Brassica napus</Emphasis>

Penetration of pollen tubes through stigmatic tissues in Brassica napus L. may involve the release of cell wall modifying enzymes from the pollen tube tip. We examined the expression of a pectin-degrading polygalacturonase (PG) enzyme in unpollinated and early and late pollinated stigmas via immunoblotting and immuno-light microscopy using a PG polyclonal antibody. Immunoblotting analysis indicated that PG enzyme was present at low levels in unpollinated stigmas and at high levels in pollinated stigmas. The level of PG did not detectably increase between early and late pollinated stigmas. Immuno-light microscopy demonstrated that PG enzyme was present in ungerminated pollen grains, stigmatic papillae and in the tip of pollen tubes growing into the papillar wall. This latter evidence suggests that PG enzyme may play an important role in papillar cell wall penetration during pollination although other interpretations of the role of pollen PG should not be discounted. Received: 9 November 2000 / Accepted: 7 December 2000