Reproductive Compatibility of European Populations of Two Species of Ulnaria (Bacillariophyta)

Clones of two morphotypes corresponding to two species of pennate araphid diatoms, Ulnaria ulna and U. acus, were isolated from samples taken in Europe. Clonal cultures entered heterothallic sexual reproduction in combinations appropriate to their mating types. Following the concept of biological species, we tested reproductive compatibility between all representatives of geographically distant populations. Isolated clones proved to be reproductively compatible within the limits of each of the species; the progeny (generation F1) were viable and fertile and freely entered into backcrossing. No cases of interspecies hybridization were found. The absence of intraspecific reproductive isolation between distant populations in these two species suggests that the populations of both U. ulna and U. acus are conspecific.

Genus Ulnaria (Kützing) Compère was described for a group of the freshwater diatom species, which had been earlier assigned to a large and heterogenic genus Synedra Ehrenberg [1]. There are 44 species of genus Ulnaria known today [2]; the type species is U. ulna (Nitzsch) P. Compère. Benthic diatom algae U. ulna was isolated by a German natural scientist C.L. Nitzsch in 1817 as Bacillaria ulna; benthic silt deposits of Wittenberg moat (Germany) is the type locality [3]. The species was considered within genera Navicula Bory, Frustulia Rabenhorst, Exilaria Greville, Fragilaria Lyngbye, and Synedra Ehrenberg [4]. Genus Ulnaria (Kütz.) Compère was designated in 2001 with U. ulna becoming the type species. The species occurs in the fresh and brackish water in Europe, Southwest and Southeast Asia, North and South Americas, Australia, and New Zealand [2].
It is often the case, however, that new species are described on the sole basis of minor differences in morphological traits or even a single trait. Separation of such morphological species takes into account neither a fairly wide range of variation of the morphological traits [9] nor the polytypic nature of species, which leads to the difficulties arising in species delimitation. This brings to the center stage a comprehensive approach to the species description, which will comprise both the morphological and molecular-genetic criteria as well as studies on ability or inability of species to cross.
Wide distribution of U. ulna and U. acus undoubtedly raises a question as to whether the species are actually cosmopolitan or that one and the same name may hide a whole assemblage of species. Extensive group of diatom algae comprises, on the one hand, the cosmopolitan widely distributed species [10] and, on the other hand, the species complexes comprising the cryptic and pseudocryptic species [11]. Gaining insight into the problem of boundaries between species and the extent of divergence of the populations occurring in geographically distant water bodies involves application of morphological and molecular methods as well as methods of reproductive biology based on a biological species concept [12]. Studying the mechanisms and the extent of reproductive isolation in combination with methods of the molecular-genetic analysis allows for delimitation of the cryptic species. MOSCOW  Sexual reproduction and the breeding system of U. ulna and U. acus were studied and described [13][14][15], which allows these species to be used in the study of reproductive barriers between populations. The present work is concerned with a matter of the presence or absence of reproductive isolation between the clonal cultures obtained from the European water bodies.
MATERIALS AND METHODS Cells belonging to morphotype Ulnaria ulna were isolated from samples collected over 2009-2013 in the water bodies of the European part of Eurasian continent, namely, a small river in Cardiff (United Kingdom), a canal in Ghent (Belgium), the Sarthe River (La Sarthe, Le Mans, France), the Dnieper River (Kyiv, Ukraine), a mountain brook in Crimea, and the Moscow River in Zvenigorod (Russia). Clones of U. acus were isolated from samples collected in the Erdre (L'Erdre, Nantes, France) and Lake Ritsa (Abkhazia) at different times from 2015 to 2020. The distance between the populations was measured using Google Maps (https://www.google.ru/maps/). Samples were collected by scraping the fouling from rocks and thalli of macrophytes.
Cells were isolated for the clonal culture using the micropipette technique [16] and purified by subculturing multiple times to the fresh medium. The cultures were maintained in the medium similar in the composition to Dm (with the increased concentration of sodium silicate and B-group vitamins) [17]. The culturing was done in the 5-9-cm in diameter and 0.9-1.4-cm in height Petri dishes with the media fill in the volume from 8 to 45 mL corresponding to the dish size. Exponential growth phase was supported by a weekly subculturing into fresh medium. Monocultures were checked through at a regular basis under the microscope for the purpose of detecting the cases of homothallic reproduction. The dishes were held in an isolated room in broad daylight by a north-facing window. A Nib-100 microscope (China) was used for monitoring and manipulations and a Biolar PI microscope (PZO, Poland) was used for imaging. The latter was aligned using the differential interference contrast and illumination technique according to [18]. Photographs were taken with a Canon PowerShort A640 digital camera. Cell dimensions were determined with an ocular scale calibrated by an object micrometer. Morphology of the diatom valves was examined using a scanning electron microscope (SEM); electron microimages were obtained from the Research Equipment Sharing Center at the Papanin Institute for Inland Waters, Russian Academy of Sciences, on a JEOL JSM-6510LV scanning electron microscope (Jeol, Japan). Terminology is used in accordance with dictionaries [19,20].

RESULTS AND DISCUSSION
Cells corresponding to morphotype of U. ulna were isolated from six European populations. The farthest populations (Cardiff, United Kingdom, and Moscow River, Zvenigorod) are located at a distance of approximately 2650 km; the closest (Le Mans, France and Ghent, Belgium) are at 420 km. In natural populations, cells of U. ulna grew either as individual cells or in groups of cells attached to a substrate with a common mucilage pad, while their occurrence in plankton was less frequent. In cultures, rapidly growing clones formed colonies at the bottom of Petri dishes providing the conditions were appropriate.
Cells of U. ulna are rod-shaped; valves are linear, slightly tapered, and commonly feature intercalary bands. There is one rimoportula situated on the sternum at an angle to the apical axis at each end of the valve. On the external face of the frustule, the rimoportula have an ellipsoid opening. There are usually two rimoportulae or there may be one. The axial area is narrow and linear (Fig. 1). The central area is rectangular and may contain poorly visible marks of striae or be lacking them. Striae density is five to nine in 10 μm. No significant morphological differences were identified between the populations [9].
We measured apical length of cells in the populations at the time of sampling ( Table 1). None of the populations featured cells with a size close to the maximum species-specific [14]; all the populations had cells whose sizes permitted the engagement in sexual reproduction.
The clones isolated from all the studied populations proved to be capable of heterothallic reproduction in their groups as well as interpopulation crossing. Overall, 32 clones were involved in sexual reproduction; part of them are shown in Table 2. The studied clones crossed freely (Fig. 2) in combinations corresponding to their mating types; characteristic patterns coincided with the ones described earlier for U. ulna [14]. Morphologically and behaviorally distinct male and female gametes in Ulnaria point to two sexes rather than two types of mating. Clones from the European populations produced viable, fertile offspring, which again successfully mated with each other and the parent clones, thus suggesting a total absence of reproductive isolation between these populations.
Cell of U. acus were detected in samples collected at a distance of approximately 3280 km from each other (Lake Ritsa, Abkhazia and Nantes, France). In both populations, morphology of the vegetative cells corresponds to U. acus; they attach to a substrate with one end with the mucilage or by forming small colonies. A fairly large number of cells stay unattached to the substrate suspended in the medium. The cells are elongated with narrow lance-shaped valve. Similar to U. ulna, the valve ends feature one rimoportula each situated on the sternum at an angle to the apical axis. Outside the frustule, the rimoportulae have an ellipsoid opening. The axial area is narrow, sometimes irregular; the central area is rectangular (Fig. 3). There are 9-13 striae in 10 μm.
All the studied U. acus clones proved to be capable of heterothallic reproduction with intra-and interpopulation mating in the opposite-sex clone mixes (Table 3) according to their sexual type [15]. Thus, no apparent prezygotic barriers were detected between clones in either population.
A series of the following basic morphological criteria is commonly employed to describe new species from the genus Ulnaria: length and width of a cell valve, density of striae arrangement, occurrence and the number of rimoportulae, valve shape, and shape of the axial and central areas. In that regard, species-specific apical size of cells often appears underestimated because the biology of the species and current phase of the cell life cycle are overlooked, whereas it is import-    ant to keep in mind that the cell size may increase severalfold as a result of sexual reproduction [14,15]. The reports are available on a large intraspecific variability of traits, such as striae density, cell width, and density of areolae for many diatoms, e.g., Nitzschia rectilonga Takano [21] and Tabularia fasciculata (C.A. Agardh) Williams & Round [22]. Notwithstanding, it has been noted for species T. fasciculata, which is closely related to Ulnaria [23], that the major differences in morphological criteria will not necessarily constitute grounds for species delimitation since there were no reproductive barriers between the two morphological groups [22]. This is also the case with European populations of U. ulna, which freely mated with each other despite a number of morphological distinctions.
Shape of the colony is among the criteria describing the Ulnaria spp. [8]; observations over the clonal cultures of U. acus, however, demonstrate that behavior of the cells varies depending on the stage of the life cycle. At a prereproductive stage of the life cycle, when cells feature a greater length, they largely occur in the medium in a suspended state; only a small fraction of cells settle down to the bottom. As cells reach approximately half of the maximum species-specific size and are ready to mate, the majority of cells meet at the bottom while forming dense colonies containing from a few to dozens of cells. In mixed cultures, colonies frequently happened to have cells of different clones, in the majority of which, in particular, sexual reproduction was observed (Fig. 4).
Concerning the genetic boundary between species, note that even the great genetic distance between the populations does not preclude their complete reproductive compatibility, such as in T. fasciculata [22] and Pseudo-nitzschia pungens (Grunow ex P.T. Cleve) Hasle [10]. Cases of interspecific hybridization have been established in P. pseudodelicatissima (Hasle) Hasle and P. arenysensis S. Quijano-Scheggia, E. Garcés & N. Lundholm in Quijano-Scheggia et al. [24]. As the authors noted, however, it was successful in only 10% of all clones with the known type of mating, while only one of 60 isolated postinitial cells was able to divide and form a viable clone. In the case with the studied Ulnaria species, all crossings among the reproductively compatible clones proved to be successful, while the isolated progeny from interpopulation crossing was viable and fertile. No cases of interspecific hybridization were detected in mixed cultures of U. ulna and U. acus.
Thus, a wide distribution of Ulnaria ulna and U. acus was demonstrated for the European part of the Eurasian continent. Based on the biological concept, a lack of intraspecific reproductive isolation between the distant populations in these two species suggests a conspecific nature of populations of both U. ulna and U. acus.

FUNDING
The study was conducted as a part of the state assignment to T.I. Vyazemsky Karadag Scientific Station-