Population structure and seed set of heterostylous species Pulmonaria obscura (Boraginaceae)

Self-incompatibility in plants is the mechanism preventing autogamy and promoting xenogamy. The vast majority of heteromorphic species are heterostylous. Populations of distylous species include two types of plants, S-morph (with “thrum” flowers) and L-morph (with “pin” flowers). Since the first work on this special reproductive feature, many aspects of heterostyly have been studied: its distribution in flowering plants, flower morphology, inheritance patterns, the ratio of S-morphs and L-morphs in populations, seed sets of plants with different types of flowers, etc. Despite the long history of studying heterostyly, many of these features remain unclear or unexplored for many heterostylous species. In the genus Pulmonaria, distyly was first described and studied in detail in the species Pulmonaria officinalis, while Pulmonaria obscura Dumort (Unspotted Lungwort) has thus far not been studied regarding distyly. The latter is a perennial herbaceous polycarpic plant with a short rhizome. Fragmentariness and contradictory data on the ratio of S- and L-morphs in the plant populations led us to identify the structure of Pulmonaria obscura populations in Moscow Oblast and compare the seed sets of plants with different floral types. The ratio of S- vs. L-morphs and the seed set ofP. obscura were studied in Moscow Oblast from 2020 to 2022. The frequencies of plants with thrum and pin flowers were studied in three populations. In all populations, the number of generative S- and L-morphs was counted on 1-m-wide transects. In each population, the number of studied plants was at least 100. The ratio of S- vs. L-morphs was investigated over three years in all three populations. The study of seed set in P. obscura was carried out according to the generally accepted methods. The number of ovules and seeds per flower and plant and the percentage ratio between these indicators were estimated. The number of flowers/fruits in the inflorescence/infructescence, and the number of seeds in the fruit were counted on 60 plants (10 plants with different types of flowers from three populations). The crossing system in P. obscura was carried out by hand pollination applying the following kinds: 1) legitimate pollination, 2) illegitimate pollination, 3) autogamy. Ten S- and L-morphs were used in each case when counting seeds. All obtained data were processed by the methods of variation statistics. Analysis of the variability of morphological parameters was performed using descriptive statistics (minimum and maximum of the indicator), and range diagrams (mean value ± error of the means and standard deviation). A comparison of the obtained values of medians of traits in different populations was carried out using the standard Mann-Whitney U test. The ordination of samples from populations was performed using the method of principal components (PCA). The ratio of plants with pin and thrum flowers in three populations of P. obscura did not deviate significantly from 1: 1 and remained stable without noticeable fluctuations during the three years of observations (see Tables 1 and 2). Pin and thrum flowers of P. obscura and plants with different types of flowers, do not differ significantly (see Fig. 1). Such features of seed sets persisted during all three years of observations. The number of ovules and seeds per flower and per generative shoot is slightly higher in thrum flowers, but these differences are not statistically significant. The average number of seeds was 1.6 pcs. in pin flowers and 1.8 pcs. in thrum ones (see Fig. 1). Both types of flowers are characterized by an average value of the seeds/ovules ratio (from 32.7 to 55.4%), which did not depend on the year of observation. The formation of 1 or 2 seeds per fruit is most common in both floral types (see Fig. 1). As a result, such kinds of coenobia account for 71.6-87.5% of fruits. Coenobia without seeds or, on the contrary, with 4 ones are extremely rare. The frequency of fruits from both floral types does not statistically differ if 0, 1, 2 or 4 seeds are set in them. Thrum flowers have more fruits with 3 seeds than pin ones: 21.1 and 5.8%, respectively (see Fig. 1 and Table 3). The arrangement of L- and S-morphs from different populations in the first two principal components (PCA) (see Fig. 2) did not reveal differences between them and between different plants of the three populations. The first principal component (PC1 = 33.2% of variance) is closely related positively to the number of flowers, number of ovules and seeds per plant and negatively to the number of fruits that do not set seeds. The second principal component (PC2 = 27.9% of variance) correlates positively with the seeds/ovules ratio and the number of fruits with 3 seeds, and negatively with the number of fruits with 1 seed. The largest number of seeds and fruits was set in L- and S-morphs as a result of legitimate pollination (see Tables 3 and 4). In this type of pollination, the most common kind is fruits with 4 seeds. As a result of illegitimate pollination and autophily, few seeds are set and a small number of fruits are formed in Land S-morphs (see Tables 3 and 4). In these kinds of crossings, two types of fruits are formed: some do not contain seeds, and the second sets contain 1 seed. The number of ovules and seeds in pin and thrum flowers of P. obscura, and S- and L-morphs, do not differ significantly, which contributes to maintaining an equal ratio of morphs in populations. The number of ovules and seeds per flower and plant in P. obscura did not differ statistically, which contributed to maintaining an equilibrium ratio of forms in the studied populations. For this study, Pulmonaria obscura appears to be characterized by partial self-compatibility: few fruits and seeds are set in the case of illegitimate pollination and autophily. The article contains 5 Tables, 44 References. The Authors declare no conflict of interest.

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