The reproductive biology of the genusBaldellia (Alismataceae)

The reproductive biology of the genusBaldellia was studied under cultivation and in nature.B. ranunculoides subsp.repens is self-incompatible whereasB. ranunculoides subsp.ranunculoides andB. alpestris are self-compatible. Bud-pollination occurs in theB. alpestris. Self-incompatibility correlates with the production of fewer, larger, more showy flowers, higher pollen production (but reduced pollen grain size), fewer carpels and smaller seeds. Resource allocation to sexual reproduction is inversely correlated with the degree of vegetative growth.B. ranunculoides subsp.repens produces more ramets than subsp.ranunculoides, which reproduces mainly by seeds.B. alpestris is intermediate between the two since it is self-compatible, produces fewer, larger seed than subsp.ranunculoides and is also able to propagate by vegetative means.     

Global conservation status assessment of the threatened aquatic plant genus Baldellia (Alismataceae): challenges and limitations

In this study, the aquatic monocot Baldellia (Alismataceae) is used as a model for evaluating the general hindrances and shortfalls in the global conservation status assessment of a threatened taxon. Our study clearly shows that Linnean shortfalls (uncertainty in the number of species and taxonomy) and the Wallacean shortfall (fragmentary knowledge regarding distribution) form the basis for all other hindrances. We demonstrate that even in Europe, which has traditionally been very well investigated, between 60 and 75% of regions or countries possess no detailed distribution maps and/or data banks for Baldellia spp. Furthermore, between 50 and 60% of regions do not have any published red list category. Thus, only general conclusions concerning the global conservation status of the three Baldellia taxa are possible—a global assessment of conservation status for B. ranunculoides subsp. repens is nearly impossible. Baldellia ranunculoides s.str. shows a strong decline in practically all regions of its natural range, and thus it is probably the most threatened species in the genus. Baldellia alpestris is the least threatened species in the genus, even though it is a narrow endemic. Our case study clearly shows the need for reinforced coordination of research and conservation activities as well as an urgent need for data accessibility regarding taxonomic, chorological and conservation studies of endangered species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Morpho-environmental characterization of the genus Baldellia Parl. (Alismataceae) in the Iberian Peninsula,Balearic islands and North Morocco

Abstract

The genus Baldellia Parl. has always been a complex taxon. Three species and three subspecies have been proposed for the Iberian Peninsula. The morphological variation of this genus in the Iberian Peninsula, Balearic islands and northern Morocco, and its thermo-pluviometric and altitudinal correlation are the focus of this contribution. Twenty-eight morphological variables were examined and subjected to a multivariate statistical analysis. The three morphotypes observed (Form 1, which includes B. ranunculoides subsp. repens and B. alpestris; Form 2, which includes B. ranunculoides and Form 3 – for the new combination B. ranunculoides subsp. ranunculoides var. tangerina (Pau) J. Rocha, A. Crespí, M. García-Barriuso, R. Almeida, J. Honrado, comb. nova proposed here for the first time) seem to represent different reproductive strategies. Morphological variables related with the presence or absence of stolons and the architecture of the inflorescence; the size and number of fruits are the most discriminating variables. Form 1 represents the asexual morphotype; Forms 2 and 3 correspond to morphotypes for which sexual reproduction is preferential. The environmental approach revealed that the asexual form (Form 1) grows in temperate, in more humid conditions, and within a broad altitudinal range. In contrast, the two sexual forms are more common in warmer and drier conditions, and occur over a narrower interval of altitudes.     

Eutrophication and endangered aquatic plants: an experimental study on <Emphasis Type="Italic">Baldellia</Emphasis> <Emphasis Type="Italic">ranunculoides</Emphasis> (L.) Parl. (Alismataceae)

Anthropogenic enrichment of aquatic ecosystems is an increasingly common phenomenon, resulting from human population growth and the intensification of industrial and agricultural developments. Eutrophication has been considered to be one of the main causes of the decline of many aquatic plants. These observations, however, have rarely been supported by data or explored in detail using an experimental approach. Our experimental study demonstrates, for the first time, a strong direct negative influence of eutrophication on the performance of the endangered aquatic plant Baldellia ranunculoides s.str. (L.) Parl. (Alismataceae). Both morphological and reproductive traits were significantly affected by this phenomenon. Plants growing on eutrophic substrates were much smaller, possessed fewer and smaller leaves, and their biomass was on average half that of plants growing on a mesotrophic substrate. Additionally, plants growing on eutrophic substrates produced fewer inflorescences and flowers, and they had a smaller number of achenes per capitulum. Consequently, they produced only 15% of the seeds produced by plants growing in non-eutrophic conditions. Thus, the chance of long-term subsistence of B. ranunculoides populations in strongly eutrophic habitats will be significantly reduced, and at the same time, the success of any new establishment of viable B. ranunculoides populations in such habitats will be highly restricted. Conservation efforts for B. ranunculoides, either of existing populations or in newly selected re-introduction sites, should start, therefore, with an assessment of the trophic level of those sites. On the other hand, due to its sensitivity to eutrophication, the presence of viable populations of B. ranunculoides can be used as an indicator of valuable aquatic habitats for oligotrophy-dependent organisms.     

Water table and species identity outweigh carbon and nitrogen availability in a softwater plant community

Performance of aquatic macrophytes is driven by many environmental factors, and a major challenge is to understand how aquatic macrophyte communities are structured in various environments. In softwater lakes in Western Europe, hydrological state (submersed/emersed), carbon dioxide and ammonium levels and species interactions are considered as driving forces in structuring amphibious plant communities. In this study we aimed at evaluating the relative importance of these factors for four species in a competitive neighbourhood. Softwater lake habitat was simulated during one growing season in laboratory conditions, mimicking water level fluctuation, photoperiod and temperature. Artificial communities consisted of small populations of four softwater macrophyte species: Luronium natans, Baldellia ranunculoides ssp. repens, Eleocharis multicaulis and Hydrocotyle vulgaris. These communities were subjected to two levels of carbon dioxide and ammonium. Additionally, monocultures of Baldellia and Eleocharis were grown at a higher nutrient level combination in order to measure their competitive response in a community. Time (hydrological state) and species identity turned out to be the only consistently significant factors determining community composition. Plant performance was clearly species-dependent, while carbon dioxide and ammonium did not have major effects. The competitive response was significant in both Eleocharis and Baldellia. Competition intensity was highest in the emersed state. Carbon dioxide had a supplementary effect on the within-species performance in Luronium, Baldellia and Eleocharis, with high carbon dioxide level mainly resulting in more flowers and more stolons. Community outcomes and competitive responses in aquatic macrophytes appear difficult to predict, because of mixed life strategies and morphological and functional plasticity. We conclude that hydrological state was the only important environmental factor. The identity of the species that were present—implying species interactions—largely determined community outcome.     

Karyological variation in the group ofMyosotis alpestris (boraginacea)

A total of 6 population samples ofMyosotis stenophylla Knaf, a rare species showing great ecological disjunction in its distribution, were examined to clarify the present status of its karyological variation. In order to elucidate relationships between lowland tetraploid populations ofM. stenophylla and diploid and tetraploid montane populations ofM. alpestris F.W. Schmidt, four population samples ofM. alpestris were also examined. The karyotypes of all populations ofM. alpestris s.l. studied were highly asymmetrical and heterogeneous, being composed of metacentric, submetacentric, subtelocentric and satellited acrocentric chromosomes. The karyotype formula for haploid chromosome set was established: n=x=12=6m+2sm+3st+1t^SAT. Multivariate analysis based on chromosome length and shape showed significant differences between diploid and tetraploid forms ofM. alpestris s.l. Four numerical parameters, used to characterize the karyotype ofM. stenophylla, revealed significant differences between populations on serpentine and on non-serpentine substrates. In addition, the noticeable affinity of the karyotype of non-serpentine populations to that ofM. alpestris tetraploids has been shown by means of discriminant analysis. These data suggest that the unique features of serpentine play an important role in the origin of karyotypic differentiation within populations ofM. stenophylla.     

Dynamics of polyploid formation and establishment in the allotetraploid rock fern Asplenium majoricum

Background and Aims

Successful establishment of newly formed polyploid species depends on several interlinked genetic and ecological factors. These include genetic diversity within and among individuals, chromosome behaviour and fertility, novel phenotypes resulting from novel genomic make-up and expression, intercytotypic and interspecific competition, and adaptation to distinct habitats. The allotetraploid rock fern Asplenium majoricum is known from one small population in Valencia, Spain, and several larger populations on the Balearic island of Majorca. In Valencia, it occurs sympatrically with its diploid parents, A. fontanum subsp. fontanum and A. petrarchae subsp. bivalens, and their diploid hybrid A. × protomajoricum. This highly unusual situation allowed the study of polyploid genetic diversity and its relationship to the formation and establishment of nascent polyploid lineages.

Methods

Genetic variation for isozyme and chloroplast DNA markers was determined for A. majoricum and A. × protomajoricum sampled thoroughly from known sites in Majorca and Valencia. Results were compared with variation determined previously for the diploid parent taxa.

Key Results

A highly dynamic system with recurring diploid hybrid and allotetraploid formation was discovered. High diversity in the small Valencian A. majoricum population indicates multiple de novo origins from diverse parental genotypes, but most of these lineages become extinct without becoming established. The populations on Majorca most probably represent colonization(s) from Valencia rather than an in situ origin. Low genetic diversity suggests that this colonization may have occurred only once.

Conclusions

There is a striking contrast in success of establishment of the Majorcan and Valencian populations of A. majoricum. Chance founding of populations in a habitat where neither A. fontanum subsp. fontanum nor A. petrarchae subsp. bivalens occurs appears to have been a key factor enabling the establishment of A. majoricum on Majorca. Successful establishment of this polyploid is probably dependent on geographic isolation from diploid progenitor competition.     

Flavonoids in the species of Cyrtomium (Dryopteridaceae) and related genera

Flavonoids in 19 Cyrtomium, three Cyrtogonellum and two Phanerophlebia taxa were surveyed. Major flavonoids were flavonol O-glycosides based on kaempferol, quercetin, and sometimes myricetin, and C-glycosylflavones, such as isovitexin, vitexin, isoorientin, orientin and their O-glycosides. The C-methylflavanones, farrerol and cyrtominetin, and their 7-O-glucosides were isolated from Cyrtomium devexiscapulae and Cyrtomium laetevirens. Flavanones have been reported from Cyrtomium falcatum sensu lato. Though C. falcatum sensu lato is divided into four taxa, i.e. C. falcatum subsp. falcatum, C. falcatum subsp. australe, C. falcatum subsp. littorale, and C. devexiscapulae, the occurrence of the flavanones was restricted to C. devexiscapulae, and they did not occur in C. falcatum sensu stricto.     

Ecological differentiation and cladogenesis of Baldellia (L.) Parl. (Alismataceae)

The Iberian Peninsula is home to a large number of endemic species that often show strong genetic subdivisions which indicate possible isolation of populations in the past. Numerous phylogeographic and phylogenetic studies on Iberian flora and fauna have revealed this territory to be the origin of genetic differentiation during the Pleistocene Ice Age. To better understand the influence of Plio-Pleistocene climatic oscillations on the cladogenesis and population structuring of refugial taxa restricted to freshwaters of the Iberian Peninsula, we studied the evolutionary history of the small European genus Baldellia, both from a phylogenetic/phylogeographic perspective and regarding its ecological characteristics. Molecular analyses were based on four plastid regions and nuclear ITS2 ribosomal spacer from individuals belonging to geographically isolated Baldellia populations, while the relationships between climate and Baldellia species distribution was investigated by redundancy analysis applied to the climatic data with the occurrence of Baldellia species as external variables. The absence of a clear phylogenetic signal most likely reflects a common and recent cladogenesis for Baldellia. In contrast, the more rapidly evolving plastid sequences indicate a recent phylogeographic history of refugial repartition and ecological segregation probably occurring during the last glaciation, a scenario well supported by marked differences in species?? ecological preferences and climate analysis.     

Soil salinity as a selection pressure is a key determinant for the evolution of salt tolerance in Blue Panicgrass (Panicum antidotale Retz.)

To assess the role of selection pressure in plant adaptation to saline environment, a hydroponic experiment was conducted on six Panicum antidotale Retz. populations collected from a wide range of habitats with varying selection pressure in the form of soil salinity. The soil electrical conductivity of six different habitats ranged from 3.39 to 19.23 dS m⁻¹ and pH from 5.86 to 7.65. Plants of all populations collected from varying habitats were established in pots containing normal soil and allowed to grow for 6 months. Newly grown tillers from each plant were separated and 10 of them each formed a composite sample for a particular population. They were then transplanted in plastic containers each containing 10 l of half strength Hoagland's nutrient solution alone or with 150 mol m⁻³ NaCl. After 42 days growth in salt treatment, the populations collected form highly saline habitats proved to be more salt-tolerant compared with those from mild or non-saline habitats in terms of growth performance. The populations adapted to high salinity showed less decrease in leaf K⁺/Na⁺ and Ca²⁺/Na⁺ ratios under salinity stress. Moreover, under stress the salt-tolerant populations showed less reduction in photosynthetic capacity than the salt-sensitive populations. In addition, hyper-accumulation of organic solutes such as glycinebetaine and proline and thereby higher osmotic adjustment seemed to be associated with the higher degree of adaptability of the salt-tolerant populations to salt stress. From the data presented, it is plausible to conclude that selection pressure (soil salinity) must have been one of the important determinants bringing about the evolution of salt-tolerance trait in Blue Panic grass.     

Comparative karyotype analysis of populations in the Alstroemeria presliana Herbert (Alstroemeriaceae) complex in Chile

Alstroemeria L., one of the most diverse genera of the Chilean flora and of high floricultural value, is represented by 35 species, most of them distributed between 28–38° S in the Mediterranean zone of Central Chile. There are 24 complex-forming taxa, of which 18 have conservation problems (8 are considered “endangered” and 10 as “vulnerable”). One of these complexes is Alstroemeria presliana Herb. with two subspecies: subsp. presliana and subsp. australis Bayer. Alstroemeria presliana grows in Chile and Argentina: subsp. presliana is distributed from Reserva Nacional Siete Tazas (35°27′ S, Region of Maule) to Antuco, (37°25′ S, Region of Bío-Bío), and is also found in Neuquén, Argentina; subsp. australis is endemic to the Cordillera of Nahuelbuta. A comparative karyotype study was carried out among six populations of A. presliana subsp. presliana and five populations of A. presliana subsp. australis. The eleven populations presented an asymmetric karyotype, with 2n = 2× = 16 chromosomes but with different karyotype formulae. A. presliana subsp. presliana shows the haploid formula 2m + 2m-sat + 1sm-sat + 1st-sat + 1t + 1 t-sat, and A. preslianasubsp. australis presents a formula 1m + 2m-sat + 1sm + 2t + 2t-sat chromosomes. The architecture of the karyotype between the subspecies is very different. The scatter plot among CV_CL vs. M_CA shows different groupings between populations of the two subspecies. According to the results obtained it is possible to consider raising Alstroemeria presliana subsp. australis at species level.     

A taxonomical revision ofThymus sect.Teucrioides (Lamiaceae)

TheThymus teucrioides Boiss. & Spruner aggregate is revised and the following new taxa, all from the alpine zone in the Greek mountains, are described:Th. leucospermus Hartvig from the calcareous mountains of Pindhos and Mt Parnassos in Sterea Ellas,Th. rechingeri Hartvig with the subsp.macrocalyx Hartvig from calcareous mountains in Sterea Ellas and N Peloponnissos, andTh. teucrioides subsp.alpinus Hartvig from the serpentine areas of N Pindhos. In the variableTh. teucrioides s. str. many characters have turned out to be markedly geographically correlated and many local populations can be distinguished by a particular combination of characters.Dedicated to Prof.K. H. Rechinger on the occasion of his 80th birthday.     

Cuticular hydrocarbons corroborate the distinction between lowland and highland Natal fruit fly (Tephritidae,Ceratitis rosa) populations

The cuticular hydrocarbons (CHs) and morphology of two Ceratitis rosa Karsch (Diptera: Tephritidae) populations, putatively belonging to two cryptic taxa, were analysed. The chemical profiles were characterised by two-dimensional gas chromatography with mass spectrometric detection. CHs of Ceratitis rosa that originated from the lowlands and highlands of Kenya comprised of n-alkanes, monomethylalkanes, dimethylalkanes and unsaturated hydrocarbons in the range of the carbon backbone from C₁₄ to C₃₇. Hydrocarbons containing C₂₉, C₃₁, C₃₃ and C₃₅ carbon atoms predominated in these two populations. 2-Methyltriacontane was the predominant compound in both populations. Quantitative differences in the distribution of hydrocarbons of different chain lengths, mainly the C₂₂, C₃₂, C₃₃ and C₃₄ compounds of these two populations, were observed despite indistinct qualitative differences in these hydrocarbons. Morphological analyses of male legs confirmed that the flies belong to different morphotypes of Ceratitis rosa previously labelled as R1 and R2 for lowland and highland populations, respectively. A statistical analysis of the CH compositions of the putative R1 and R2 species showed distinct interspecific identities, with several CHs specific for each of the lowland and highland populations. This study supports a hypothesis that the taxon Ceratitis rosa consists of at least two biological species.     

Quantitative real-time PCR detection of Pseudomonas oleovorans subsp. lubricantis using TaqMan-MGB assay in contaminated metalworking fluids

Metalworking fluids (MWFs) are highly prone to microbial contamination, which leads to their degradation and biofouling. Pseudomonas oleovorans subsp. lubricantis, a newly described subspecies, was found to be important to MWF fouling. However, the actual distribution of P. oleovorans subsp. lubricantis in MWF is difficult to study using standard culturing techniques. To overcome this, a study was conducted to design a specific quantitative real-time PCR (qPCR) assay using TaqMan^®MGB (minor groove binding) probe for its identification and estimated quantification in contaminated MWFs. The gyrB housekeeping gene sequence was selected for designing a TaqMan^® MGB primer-probe pair using the Allele ID^® 5.0 probe design software for the assay. Whole-cell qPCR was performed with MWF spiked directly with P. oleovorans subsp. lubricantis (eliminating DNA extractions using commercial kit); the primer-probe pair’s sensitivity was 10¹ colony forming units (CFU) ml⁻¹. The assay provided no amplification with other closely related Pseudomonas species found in MWFs indicating its specificity. It was successful in identifying and enumerating P. oleovorans subsp. lubricantis from several used MWFs having between 10⁴ and 10⁶ CFU ml⁻¹. The designed TaqMan^® MGB probe thus can be successfully used for the subspecies-specific identification of P. oleovorans subsp. lubricantis and facilitates the study of its impact on MWFs.     

Ribosomal protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for phylogenety-based subspecies resolution of Bifidobacterium longum

The taxonomic positions of the subspecies of Bifidobacterium longum (B. longum subsp. longum, subsp. infantis, and subsp. suis) have been controversial. A current proposal is that the former two species “B. infantis” and “B. suis” be unified with B. longum and all three reclassified as three subspecies. To test this proposal, ribosomal protein profiling as observed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied to the classification of 17 strains of B. longum, including three subspecies. Among 41 different kinds of ribosomal proteins selected as biomarkers whose masses were calculated from their amino acid sequences, 31-41 ribosomal proteins were observed in sample strains with the same masses as the references. The high matching rate indicates high conservation of ribosomal proteins within the sample strains, and therefore strongly supports the unification of the former species. However, the masses of some ribosomal proteins varied within species. The phylogenetic tree constructed from the profiles of ribosomal proteins matched the references, showing a clear cluster of the subsp. longum and the subsp. infantis strains. This result supports the proposal to reclassify B. longum into subsp. longum and subsp. infantis. The subsp. suis strains formed an individual sub-cluster within the infantis cluster. However, their ribosomal proteins have both characters of longum and infantis types. This result suggests that the taxonomic position of the subsp. suis should be reconsidered.     

Are carbon and nitrogen exchange between fungi and the orchid Goodyera repens affected by irradiance?

Background and Aims The green orchid Goodyera repens has been shown to transfer carbon to its mycorrhizal partner, and this flux may therefore be affected by light availability. This study aimed to test whether the C and N exchange between plant and fungus is dependent on light availability, and in addition addressed the question of whether flowering and/or fruiting individuals of G. repens compensate for changes in leaf chlorophyll concentration with changes in C and N flows from fungus to plant.Methods The natural abundances of stable isotopes of plant C and N were used to infer changes in fluxes between orchid and fungus across natural gradients of irradiance at five sites. Mycorrhizal fungi in the roots of G. repens were identified by molecular analyses. Chlorophyll concentrations in the leaves of the orchid and of reference plants were measured directly in the field.Key Results Leaf δ¹³C values of G. repens responded to changes in light availability in a similar manner to autotrophic reference plants, and different mycorrhizal fungal associations also did not affect the isotope abundance patterns of the orchid. Flowering/fruiting individuals had lower leaf total N and chlorophyll concentrations, which is most probably explained by N investments to form flowers, seeds and shoot.Conclusions The results indicate that mycorrhizal physiology is relatively fixed in G. repens, and changes in the amount and direction of C flow between plant and fungus were not observed to depend on light availability. The orchid may instead react to low-light sites through increased clonal growth. The orchid does not compensate for low leaf total N and chlorophyll concentrations by using a ¹³C- and ¹⁵N-enriched fungal source.     

Sclerostylus n. gen. from Panama and Other Neotropical Species of LeptonIchoidea (Dorylaimida)

Leptonchoid species found in soil samples taken in lowland and mid-elevation seasonal forests at four locations in Panama were as follows: Sclerostylus karri n. gen., n. sp., Basirotyleptus saccatus n. sp., B. nemoralis, B. acus, B. penetrans, Doryllium neotropicum n. sp., D. minor, Tyleptus gymnochilus, Gerthus jamesi, Dorylaimoides micoletzkyi, and Tylencholaimellus macrophallus. The discoveries corroborate our earlier suggestion of a Gondwanian origin for Tyleptus and Basirotyleptus and provide data for additional biogeographical study.     

Vergleichende cytotaxonomische Untersuchungen anRanunculus seguieri und der Artengruppe desR. alpestris (Ranunculaceae)

Morphological and cytological investigations as well as crossing experiments were carried out withRanunculus seguieri Vill. and 4 species of theRanunculus alpestris L. group (R. alpestris L.,R. traunfellneri Hoppe,R. bilobus Bertol.,R. crenatus Waldst. & Kit.). ForR. seguieri andR. alpestris, localities and distribution are given in addition to extensive diagnoses. A key to the species includes morphological characteristics and distribution data forR. traunfellneri, R. bilobus, andR. crenatus. New diagnostic characters are described. Crossing experiments betweenR. seguieri and the species of theR. alpestris group were unsuccessful. All 5 species have a chromosome number of 2n = 16, the record forR. bilobus is new. There is no statistically significant difference between the karyotypes ofR. seguieri andR. alpestris s. str. Nevertheless, according to morphological evidence and crossing experiments,R. seguieri is not closely related to theR. alpestris group.

     

Systematic significance of seed coat morphology inSilene L. s. str. (sileneae-caryophyllaceae) from Korea

Seeds of eight species ofSilene s. str. in Korea were examined with a dissecting stereomicroscope and a scanning electron microscope (SEM) to evaluate the systematic significance of seed coat microstructure. The seeds ofSilene s. str. were characterized by triangular or reniform shape, planed or concave dorsal surface in outline, tuberculate or colliculate testa, and sinuated cell margin. Based on the degree of grooves and invagination at dorsal surface, the seeds of the examined taxa ofSilene s. str. fall into two distinct groups: (I) dorsal grooves inconspicuous, surface planed (Silene jenisseensis andS. repens), (II) dorsal grooves conspicuous, surface concave (six remaining taxa). Within each group, seeds of all species may be further distinguished based on shape, size, and testa surface sculpturing. A key to the different species or groups of species is provided, and the systematic significance between/among the taxa is also briefly discussed.     

Hybridizations and genetic relationships among Lindernia species (Scrophulariaceae): L. procumbens and two subspecies of L. dubia

Lindernia procumbens and L. dubia are common annual weeds in flooded rice fields of Japan. Two subspecies of L. dubia, subsp. major and subsp. dubia, are usually recognized in Japan but they are both regarded as synonyms of L. dubia elsewhere. In a cluster analysis based on AFLP, most L. dubia subsp. major formed a separate cluster from L. dubia subsp. dubia although 11% of haplotypes classified using AFLP were not coincident with classification using the shape of leaf bases, which is the commonly used identification trait. Artificial F₁ plants between L. procumbens and L. dubia subsp. major, and between L. procumbens and L. dubia subsp. dubia did not produce seed. Forty percent of capsules produced by F₁ plants from these two subspecies were slimmer and 80% pollen were sterile in slimmer capsules. However, seed number of most F₁ capsules was not different from that of self-fertilized plants, suggesting that there was no complete reproductive isolation between the subspecies. Natural hybridization of these subspecies may have occurred but we are not aware of it because F₁ plants are rare and F₂ plants are indistinguishable from these subspecies.