Reduced pollen viability and achene development in Solidago × niederederi Khek from Poland

The phenomenon of plant hybridization between alien species and their native congeners that support biotic homogenization significantly reflects global changes in native floras. This process is a major threat to genetic integrity of indigenous plant species [1] and leads to an increase in the number of new taxa which should be characterized as alien plants [2]. The spontaneous occurrence of alien-native hybrids can be very helpful in circumscribing the invasiveness of alien plant species involved in such hybridization. Unfortunately, hybridogenous specimens are often overlooked during the field studies because of the morphological resemblance to their parental species. In Europe, Solidago ×niederederi Khek, a natural hybrid between the alien S. canadensis L. and the native S. virgaurea L. [3–5] has rarely been recorded in Austria, Denmark, Sweden, Norway, the United Kingdom, Germany, and in Poland [3–13]. Its occurrence is undoubtedly an indirect result of naturalization and invasion of North American S. canadensis [14,15]. It is usually intermediate in the shape of inflorescence, the size of the capitulum, and the shape and venation of its leaf [4,5]. It has either restricted fertility, able to produce only a few well-developed achenes [4], or is completely sterile [6]. However, this plant seems to be a long-lived perennial, which forms characteristic clumps of stems like those of S. canadensis. There is a need for further research on its vegetative propagation as a way to become established [5]. Solidago ×niederederi grows in open disturbed areas (e.g., abandoned arable fields, quarries, clay pits, railway and river embankments), usually together with the parental species [4–12]. The aim of this work is to confirm the hybrid nature of specimens of S. ×niederederi collected from a newly discovered locality in Poland [5] using pollen grain and achene characters. We hypothesized that male meiosis in a hybrid between two Solidago species that are not closely related, becomes disturbed due to the presence of non-homologous genomes. This leads to significantly decreased pollen viability and seed production as compared to its parental species S. canadensis and S. virgaurea.


Introduction
The phenomenon of plant hybridization between alien species and their native congeners that support biotic homogenization significantly reflects global changes in native floras.This process is a major threat to genetic integrity of indigenous plant species [1] and leads to an increase in the number of new taxa which should be characterized as alien plants [2].The spontaneous occurrence of alien-native hybrids can be very helpful in circumscribing the invasiveness of alien plant species involved in such hybridization.Unfortunately, hybridogenous specimens are often overlooked during the field studies because of the morphological resemblance to their parental species.
The aim of this work is to confirm the hybrid nature of specimens of S. ×niederederi collected from a newly discovered locality in Poland [5] using pollen grain and achene characters.We hypothesized that male meiosis in a hybrid between two Solidago species that are not closely related, becomes disturbed due to the presence of non-homologous genomes.This leads to significantly decreased pollen viability and seed production as compared to its parental species S. canadensis and S. virgaurea.

Origin of plant material
Plants in blooming and fruit development of S. virgaurea and S. ×niederederi were collected in September 2011 and identified by Dr. Artur Pliszko in a newly found locality in Mieruniszki village (54°10'43" N, 22°32'04" E) in the Zachodniosuwalskie Lakeland in NE Poland [5].The distance between the hybrid plants and parental species did not exceed two meters.Individuals of S. canadensis were collected in the same season in Balice (50°05'10.3"N, 19°47'01.9"E)near Kraków (southern Poland) and identified by Dr. Maria Kościńska-Pająk.All analyzed Solidago taxa formed compact belowground rhizome systems developing many genets.For each taxon, pollen grains and achenes were isolated from dry inflorescences of three plants (probably genets).

Estimation of pollen stainability (viability)
For acetocarmine and Alexander tests, dry pollen grains from 32 flowers of each parental species and from 40 flowers of hybrid S. ×niederederi were shed on slides with drops of 1% acetocarmine or Alexander's dye [16].In the acetocarmine test cytoplasm stains red in viable pollen and remains transparent in nonviable pollen, while in the Alexander test viable pollen grains appear red and nonviable pollen grains stain green.Stamens of each flower isolated from inflorescence were divided into two equal parts.Pollen from each part was stained with acetocarmine or with Alexander.

Pollen grain diameter
Diameter of 500 pollen grains from 32 flowers of each parental species and from 40 flowers of hybrid S. ×niederederi was measured under Eclipse E400 optical microscope (Nikon) using NIS Elements (ver.4.0.)program.Measurements of each grain were performed including exine along polar and equatorial axis.

Achene size
One hundred achenes of each taxon were used to estimate disturbances in achene development.Length of achenes without pappus was determined on 50 normally developed achenes of both parental species and on 6 well developed achenes of hybrid S. ×niederederi.Measurements were carried out under Opta-Tech SK series stereoscopic microscope using OptaView (ver.7.3.1.7.) program.

Scanning electron microscopy (SEM)
Dried pollen grains shed directly from inflorescences were dusted onto stubs with SPI carbon-conductive double-sided adhesive discs, sputtered with gold (SPI SUPPLIES ionsputtering system), and observed with a scanning electron microscope (HITACHI S-4700).

Statistics/data analysis
Comparison of achene size, pollen size, pollen viability between taxa, and both viability tests were conducted using Kruskal-Wallis ANOVA with Wilcoxon signed rank tests with Bonferroni correction post hoc.Statistical analyses were performed in R program (ver.3.1.1.).

Results and discussion
Interspecific hybrids, especially between species that are not closely related, usually have disturbed generative reproduction due to abnormalities in male (microsporogenesis) and female (macrosporogenesis) meiosis.Such abnormalities result from the presence of non-homologous chromosomes in the hybrid genome as was observed in several interspecific Viola hybrids [17][18][19][20].
Pollen grains of all examined taxa observed in SEM were isopolar, 3-aperturate with circular equatorial outline and echinate ornamentation.Solidago canadensis grains had sunken apertures, while those of S. virgaurea had colporate apertures.Hybrid pollen grains seemed to have rather sunken apertures [27].
In the inflorescences of S. canadensis and S. virgaurea the frequency of normally developed achenes was very high reaching 90% and 89%, respectively but they differed in size.Achenes of S. canadensis reached 1.52 mm, in S. virgaurea 3.14 mm (Tab.3, Fig. 2).Achene development in hybrids was dramatically reduced, in that only 6 achenes of 100 analyzed were normal, while the rest were empty and deformed (Fig. 2d).The length of 6 measured achenes ranged from 1.11 mm to 2.47 mm (Tab.3).

Conclusion
Pollen grain characters (pollen stainability, pollen diameter) and achene development confirmed the hybrid origin of specimens from the newly found locality in Poland and based on morphological characters they could be recognized as S. ×niederederi, a natural hybrid between S. canadensis and S. virgaurea.
Further studies on hybrids from other localities in Poland should be analyzed using similar characters, but also its hybrid origin should be confirmed using molecular markers.

Tab. 1
Pollen viability (stainability) of Solidago taxa by two staining tests.Values bearing different letters along one column differ significantly and indicate differences among taxa at P = 0.05.P values are given for tests' comparisons.
Pollen size of Solidago taxa.Achene size of Solidago taxa.
Values bearing different letters differ significantly and indicate differences among taxa at P = 0.05.Values bearing different letters differ significantly and indicate differences among taxa at P = 0.05.