Neutral Spores—An Easier Way to Cultivate Pyropia leucosticta (Thuret) Neefus & J.Brodie, 2011 (Bangiales, Rhodophyta) along the Romanian Black Sea Shore

: Macroalgal cultures are currently of great interest worldwide. Although along the Romanian Black Sea coast, there is no tradition in this sense, currently, a major interest in developing this direction exists, taking into consideration that the overexploitation of wild seaweed resources involves the disturbance of the marine environment, with implications that can sometimes be irreversible. Porphyra and Pyropia species (also known as nori algae) are considered the world’s most valuable mariculture algal products due to their high nutritional value and multiple active principles, considered unique representatives of marine algae. Considering these aspects, attention was focused on the only nori species from the Romanian Black Sea coast, namely, Pyropia leucosticta . The species was cultivated in controlled laboratory conditions for 5.5 months by manipulating its reproductive elements. The aim was to establish a simple, low-cost method by avoiding the complicated con-chocelis phase of the species; therefore, reproduction and further species development were achieved by collecting the released neutral spores and isolating them in culture vessels, ensuring, at the same time, proper nutrient conditions, using sterile enriched seawater and mimicking ambient conditions. At the end of the experiment, specimens of almost 80 − 90 mm were obtained, making this method suitable for obtaining ex situ P. leucosticta blades on the Romanian Black Sea shore.


Introduction
Seaweeds are a diverse group of organisms that are as important to our nearshore coastal marine world as land plants are to our terrestrial world. Seaweeds were the evolutionary precursors to land plants, and like land plants, they are critical primary producers, forming living links between the inorganic and organic worlds by using photosynthesis to convert CO 2 and nutrients into living biomass. These primary producers support other marine life by producing oxygen, contributing to marine food webs, and providing structures and habitats for fishes and invertebrates. Seaweeds are also an important resource for humans. Historically, coastal peoples have relied on seaweeds for food, minerals, medicine, insulation, fertilizer, and fodder. Pyropia leucosticta (formerly Porphyra leucosticta) belongs to the red algal order Bangiales. The red seaweed Porphyra and other closely related genera such as Pyropia, known collectively by the Japanese name nori, are of particular interest for their use in aquaculture and are the most economically valuable maricultured seaweeds in the world [1]. These bladed Bangiales are highly valuable and extensively farmed in Southeast Asia, gaining popularity and acceptance as a sustainable and locally grown food ingredient, as they are high in health-beneficial substances, such as antioxidants, minerals, vitamins, and proteins. The cultivation of Atlantic Porphyra species still faces several difficulties due to their complex heteromorphic life cycle, which is currently not fully understood.
More than 40% of the dry weight of Porphyra is made up of porphyran, which is evaluated to have nutritional and health benefits [9] and is considered to have a potential status as an antiviral agent [10]. Several antioxidant molecules have been identified in the genus Porphyra: histidine-related compounds, chlorophyll analogs, mycosporine-like amino acids, sulfated polysaccharides, and oligosaccharides. In particular, it has been suggested that the accumulation of UV-absorbing mycosporine-like amino acids, such as porphyra-344, provides photoprotection to P. leucosticta, and thus, these compounds may function as biological antioxidants. This screening emphasized the great antioxidant potential of P. leucosticta [11]. P. leucosticta could be utilized as a sea vegetable, a source of pigments (namely, R-phycoerythrin, which is used as a fluorescent tag), a protein substitute for fish meal, and countless other applications [5].
An ideal candidate for aquaculture would have several attributes, including a fast growth rate, a high capacity for nutrient accumulation, high protein content, extended seasonality, and a life history that allows for easy propagation. In addition, it should be native to the locality where it will be grown [12]. These aspects are fully covered by P. leucosticta, hence its selection for laboratory cultivation along the Romanian Black Sea coast. The seasonal fluctuations in environmental conditions on temperate intertidal rocky shores result in the necessity of organisms to withstand wide temperature, irradiance, and nutrient ranges. One adaptation of marine macrophytes to ensure survival in these highly dynamic and extreme habitats is a complex life cycle, with alterations in preferred conditions depending on the life history stage. Changing environmental conditions can be important triggers of life history events, such as initiating reproduction, and the temperature and photoperiod have been found to be especially important factors in the life history of bladed Bangiales [4]. Before starting a large-scale culture, it is important to know the influence of environmental factors on Porphyra/Pyropia species, as these are natural triggers of the reproductive process, for a more faithful simulation under laboratory-controlled conditions. It is also necessary to know the onset of the reproductive process in the natural environment in order to accurately determine the period when aquaculturists can collect wild fertile nori specimens to start the cultivation process.
For example, the results of a phenological survey of Porphyra umbilicalis indicate that the season has the most influence on reproduction in this alga. Plants are reproductive from fall through early spring, when ample nutrients and cold temperatures support neutral spore production. During the summer months, depleted nutrients and high temperatures cause plants to die back and become mostly vegetative. P. umbilicalis is highly exposed to incoming wave action, something that appears to benefit reproduction [13]. This is also the case for P. leucosticta, which prefers shallow exposed coastal areas, with an optimal reproductive period during winter. P. leucosticta has a characteristic appearance during the reproductive period, with clearly differentiated thallus edges with an alternation of darker and lighter areas, indicating the so-called "neutral spores-rich margins" [13]. Specimens with this characteristic appearance can be found along the Romanian coast, usually between January and early April (depending on the water temperature). Royer (2011) demonstrated an interesting fact: the storage of reproductively mature P. umbilicalis blades at −20 • C only showed a significant effect on the number of sporelings that germinated but did not divide. The small number of sporelings affected by this difference from controls suggested that it is not a negative indicator for the storage of frozen seed stock for aquaculture. This promising result indicates that short periods of freezing may have little effect on spore viability when reproductively mature P. umbilicalis blades are frozen for storage [13]. This aspect should also be verified for other nori species. For the same species, P. umbilicalis, Gavrielidis and Neefus (2016) established that maximum growth rates (>9% day −1 ) were observed when blades were grown at 10 to 15 • C with at least 12 h of light in the day and ≥110 µmol photons m −2 s −1 . At the same time, the authors demonstrated that growing P. umbilicalis under low light (≤60 µmol photons m −2 s −1 ) and day-neutral/long-day conditions will result in higher pigment content and higher protein content, making the blades more suitable for either an aquaculture feed substitute or a human food product [12].
Regarding the light influence, it has been shown that many red algae can be described as subtidal algae, areas where blue and green light prevails, so the specific photopigment of red algae allows efficient absorption. In addition, several red algal species' growth rates and photosynthesis depend on the light quality during the culture period and on the pigment composition under these conditions. In conclusion, light characteristics (spectral quality, quantity, and duration) have a profound influence on plant and seaweed metabolism and development. Wu (2016) performed some experiments on Pyropia haitanensis and showed that fluorescent tubes, along with blue and green light, were more efficient in promoting algal growth than thalli grown under red lighting. Similarly, some studies have shown that blue and green light could play an advantageous role in red algal growth and development [14].
These are extremely important aspects for establishing a large-scale cultivation protocol, and all of the conditions stated above must be tested and applied particularly to P. leucosticta from the Romanian Black Sea coast, since each species has its own ecological valences. The present study aims to highlight the importance of asexual reproduction for the red alga P. leucosticta collected from the Romanian Black Sea coast in obtaining new specimens under laboratory-controlled conditions by manipulating its reproductive stages. At the same time, a short review of the main existing studies addressing this topic was carried out, and the results of this study are presented in the frame of the most important results obtained by other researchers. Along the Romanian coast of the Black Sea, there is no tradition regarding either the ex situ or in situ cultivation of macroalgae for subsequent capitalization. In this regard, this study established the basic conditions for cultivating P. leucosticta under laboratory-controlled conditions, wishing to support future Romanian seaweed growers; since most studies regarding the culture of Porphyra/Pyropia species refer to the sexual reproduction of the species (the conchocelis phase) and fewer are related to asexual reproduction, particularly along the Romanian Black Sea coast, these differentiated aspects have not been addressed. In this study, we tested a simple methodology that is easier to apply in comparison with the complicated conchocelis stage method and allowed new specimens to be obtained under controlled conditions without high costs and complicated infrastructure. In a previous study [15], intermediate results obtained after only 3 months of P. leucosticta laboratory culture were presented in a general manner, so the current study aims to present the final results while also providing additional information and the main conclusions of the experiment and highlighting future research directions.

Sampling
P. leucosticta blades are thin and smooth, with different shades of red and undulating margins. It is a stenothermic species reported to live at shallow depths on hard substrates or as an epiphytic species. It does not develop significant biomass in the natural environment and can only be sampled between November and April, when the water temperature is still low [16], since P. leucosticta is unable to withstand higher temperatures and concomitant desiccation [2]. The algal donor material was collected from the shallow area, up to 1 m depth, from a natural hard substrate at the beginning of February during the full reproductive period. The freshly harvested algal material was stored in Ziploc bags without water and transported as quickly as possible to the laboratory in a refrigerated box to prevent its deterioration [1].

Reproductive Tissue
In the laboratory, samples were washed in sterilized seawater to remove epibionts and epiphytic algae. Afterwards, the algal material was carefully analyzed in order to identify the reproductive tissue. For this, a light microscope (Zeiss Axio Imager A1) with ×60 magnification was used. It was identified on the edges of the thallus, from which a portion of 1 × 1 mm was excised, taking care to minimize the amount of non-fertile tissue, as it is an additional contamination source for the future culture. Sori with neutral spores can be seen as brownish-red bands around the margins of the thallus [17]. Excised pieces from the upper portion of mature blades were kept for 24 h in Petri dishes, which were wrapped in sterile moist cheesecloth at 4 • C without light in order to create stress conditions, which will stimulate spore release. On the second day, the reproductive tissue was introduced into sterile Petri dishes in sterile seawater at a temperature of 15 • C under low light in order to release reproductive spores [1]. To measure the light intensity, a portable lux meter was used. To support the release of reproductive spores, cool white fluorescent tubes were used under low-light conditions (approx. 800 lux) for about 2 h. Spore release occurred quickly, after approximately 2 h, since the algal material was placed under optimal light (approx. 800 lux) and temperature conditions (max. 15 • C). Macroscopically, the tissue appeared highly disintegrated, a sign of spore release. Neutral spores released from wild reproductive tissues were collected under a Stereo microscope (OLYMPUS SZX10) with an autoclaved Pasteur pipette and transferred into sterile plastic Petri dishes. For this study, spores were transferred into 10 Petri dishes. A small-scale experiment was preferred so that the biological material could easily be monitored and handled. Neutral spores were released evenly across the entire surface, since they lack flagella and will not move. To facilitate spores' fixation and easily monitor their further evolution under the microscope, sterilized glass microscope slides were attached to Petri dishes. For this early stage's development, a light intensity of approx. 3300 lux (cool white fluorescent tubes) and a water temperature of 15 • C were used.

Nutrient Medium
There are several different types of media, but all provide a mixture of essential macronutrients (nitrogen, phosphorus, calcium, potassium, sodium, chloride, etc.) and trace elements (iron, manganese, zinc, molybdenum, copper, cobalt, zinc, etc.), metal chelators (EDTA), vitamins (cobalamin, thiamine, and biotin), and hydrogen-ion and metalion buffers (TRIS and EDTA). It is very important to provide the culture with biologically available iron. Iron forms very strong complexes in seawater that make it biologically unavailable. In this regard, the iron must be mixed with EDTA prior to adding it to the stock solution. EDTA is a manmade "chelator" that loosely binds to iron molecules, making it available to juvenile blades. In this regard, a slightly modified Von Stosch growth medium was used (Table 1), since it has been proven that red seaweeds have greater success in Von Stosch compared with other nutrient media (e.g., Provasoli) [1]. Stock solutions were made and stored in the refrigerator, from which fresh culture medium was prepared as needed. The stock solutions (only the salts) were sterilized in an autoclave (steam sterilizer Raypa AES75) for 20 min at 121 • C; the vitamins were sterilized by filtration (filter of 0.20 µm, Ø 25 mm). A volume of 1 mL of stock solution was used per 1 L of seawater, which was filtered and sterilized by autoclaving in advance (at 121 • C for 40 min). Seawater must be changed regularly during the culture period to replenish nutrients for growing plants. When the specimens were kept in Petri dishes (in early stages, during the first 1-2 months, depending on the degree of development of new blades), the water was exchanged every two weeks in order to not create additional disturbances and to ensure spore attachment. Later, when the specimens were transferred to Erlenmeyer flasks, the water was exchanged weekly to prevent nutrient deficiency. The culture medium was changed by pouring out the old medium and quickly filling it with a fresh one.

Diatom Control
In general, macroalgal cultures have a common problem-the presence of diatoms and/or other microorganisms in culture vessels, which limits the development of future seedlings. In this experiment, diatoms were eliminated by adding 0.07 mL of a saturated solution of germanium dioxide (GeO 2 ) to each Petri dish once per week and after a water change. For 500 mL Erlenmeyer flasks, 1 mL of GeO 2 was used in each flask, while for 1000 mL Erlenmeyer flasks, 2 mL of GeO 2 was used. Germanium dioxide inhibits silica uptake, which is necessary for cell wall formation. It keeps diatoms from being able to reproduce and effectively eliminates diatoms in the culture [1]. Red algae apparently have a high tolerance level for GeO 2 compared to other macroalgae [8].

Ensuring Sterile Conditions
To successfully maintain effective and robust cultures, preventing contamination and maintaining a stable hatchery environment are key [18]. Although it is difficult to maintain an axenic environment to grow macroalgae, special attention was paid to the workspaces (disinfected every time before handling the specimens) and glassware used (sterilized every time before use). In this sense, all glassware was sterilized in advance in a CALORIS EC 100 thermoregulating oven, with a measuring range of 50-240 • C (at 180 • C for 1 h). Moreover, to prevent any problems, it is recommended to have a cupboard full of clean autoclaved glassware (flasks, beakers, and stirrers) with the openings closed to ensure that the insides are kept as sterile as possible. A selection of glass and plastic pipettes, tubing, microscope slides, coverslips, gloves, cotton wool, autoclave tape, bottle brushes, cleaning cloths, laboratory rolls, Petri dishes (varying sizes), tin foil, scalpels/blades, and scissors should be kept well stocked and accessible [18]. In addition, seawater used during the entire experiment was filtered and sterilized in an autoclave since microscopic and juvenile phases are very sensitive to contamination. It is well known that the most effective method for sterilizing seawater is autoclaving, which ensures sterility through a treatment that applies extreme heat and pressure to seawater [18].

Handling Young Blades
After 1.5-2 months, young blades were carefully dislodged with tweezers from the Petri dishes and moved into 500 mL and later into 1000 mL Erlenmeyer flasks. This is a mandatory condition, since the blades compete with each other for nutrients, light, and space, so bigger vessels should be provided for the specimens to grow faster. The culture dishes were kept at 15 • C ± 2 • C under a day-neutral photoperiod (12:12 L:D) using cool white fluorescent tubes mounted approx. 50 cm above the Erlenmeyer flasks and connected to an interval timer to control the photoperiod. Further development of P. leucosticta was performed under a light intensity of 3300-3500 lux, considering that a higher light intensity is required for the foliose phase compared to the conchocelis phase, which is well adapted to low light intensities and a water temperature of 15 • C ± 2 • C. These were considered to be specific optimal conditions for the development of P. leucosticta collected from the Romanian Black Sea coast under laboratory-controlled conditions. Continuous aeration was applied after the blades were transferred to Erlenmeyer flasks. The goal is to create a circulation pattern within the flasks in order to prevent any dead zones [1]. In the first month, daily photographic documentation of the growth and development of the blades was performed with the help of a light microscope (Zeiss Axio Imager A1) with an attached CANON camera. After one month, all blades were easy to visualize macroscopically.

Results
Spore germination, blade growth, and further development were followed for 5.5 months, from February to mid-July. After handling the reproductive material (Figure 1a), as described in the "Materials and Methods" section (Section 2.2), the evolution of juvenile blades was carefully monitored. The released reproductive cells were initially round (Figure 1b-d). The evolution of newly formed blades obtained exclusively under laboratory-controlled conditions started after only one week, when germinated spores (with a germ tube and cell division taking place- Figure 1e-g) were noticed in Petri dishes. After 2 weeks, neutralspore-germinated thalli with single-row cell division were observed (Figure 1h).
tions. Continuous aeration was applied after the blades were transferred to Erlenmeyer flasks. The goal is to create a circulation pattern within the flasks in order to prevent any dead zones [1]. In the first month, daily photographic documentation of the growth and development of the blades was performed with the help of a light microscope (Zeiss Axio Imager A1) with an attached CANON camera. After one month, all blades were easy to visualize macroscopically.

Results
Spore germination, blade growth, and further development were followed for 5.5 months, from February to mid-July. After handling the reproductive material (Figure 1a), as described in the "Materials and Methods" section (Section 2.2), the evolution of juvenile blades was carefully monitored. The released reproductive cells were initially round (Figure 1b-d). The evolution of newly formed blades obtained exclusively under laboratorycontrolled conditions started after only one week, when germinated spores (with a germ tube and cell division taking place- Figure 1e-g) were noticed in Petri dishes. After 2 weeks, neutral-spore-germinated thalli with single-row cell division were observed (Figure 1h).  Cellular division on multiple planes started immediately after this process (Figure 2a-d). After 4 weeks of controlled culture, the macroscopic phase began (Figure 3a,b). Although with reduced dimensions of up to approx. 5 mm, young foliose blades started to become macroscopically visible (Figure 3a,b). At these reduced dimensions, they were still growing while attached to microscopic slides or the bottom of Petri dishes (Figure 3c,d). As they grew, some of the specimens detached, because the microscope slides, having no asperities, do not ensure good adhesion, and it was necessary to transfer them to Erlenmeyer containers. The delay in transferring them to larger containers suitable for their new dimensions caused the twisting of the newly formed specimens and a drastic reduction in the growth rate. started to become macroscopically visible (Figure 3a,b). At these reduced dimensions, they were still growing while attached to microscopic slides or the bottom of Petri dishes (Figure 3c,d). As they grew, some of the specimens detached, because the microscope slides, having no asperities, do not ensure good adhesion, and it was necessary to transfer them to Erlenmeyer containers. The delay in transferring them to larger containers suitable for their new dimensions caused the twisting of the newly formed specimens and a drastic reduction in the growth rate. After approximately 6 weeks, a maximum of 30 neutral-spore-germinated thalli were counted in one of the Petri dishes (the largest was approximately 10 mm). After about 4 weeks, some specimens presented some rhizoids, indicating that holdfasts had developed (Figure 4a-c). Although with reduced dimensions of up to approx. 5 mm, young foliose blades started to become macroscopically visible (Figure 3a,b). At these reduced dimensions, they were still growing while attached to microscopic slides or the bottom of Petri dishes (Figure 3c,d). As they grew, some of the specimens detached, because the microscope slides, having no asperities, do not ensure good adhesion, and it was necessary to transfer them to Erlenmeyer containers. The delay in transferring them to larger containers suitable for their new dimensions caused the twisting of the newly formed specimens and a drastic reduction in the growth rate.
After approximately 6 weeks, a maximum of 30 neutral-spore-germinated thalli were counted in one of the Petri dishes (the largest was approximately 10 mm). After about 4 weeks, some specimens presented some rhizoids, indicating that holdfasts had developed (Figure 4a-c). A temperature above 15 °C triggered the entry of newly formed blades into an early reproductive period, which led to spore release (Figure 5a,b). The evolution of the spores followed the same pattern as the adult reproductive material, and after 10 days, these new thalli became macroscopically visible (Figure 5c). Although Abdel-Rahman (2015) mentions that neutral-spore production was higher at 20 °C than at other temperatures [19], for P. leucosticta, an increase in temperature was considered to be a stress factor, hence this biological response. A temperature above 15 • C triggered the entry of newly formed blades into an early reproductive period, which led to spore release (Figure 5a,b). The evolution of the spores followed the same pattern as the adult reproductive material, and after 10 days, these new thalli became macroscopically visible (Figure 5c). Although Abdel-Rahman (2015) mentions that neutral-spore production was higher at 20 • C than at other temperatures [19], for P. leucosticta, an increase in temperature was considered to be a stress factor, hence this biological response. A temperature above 15 °C triggered the entry of newly formed blades into an early reproductive period, which led to spore release (Figure 5a,b). The evolution of the spores followed the same pattern as the adult reproductive material, and after 10 days, these new thalli became macroscopically visible (Figure 5c). Although Abdel-Rahman (2015) mentions that neutral-spore production was higher at 20 °C than at other temperatures [19], for P. leucosticta, an increase in temperature was considered to be a stress factor, hence this biological response. At the end of the experiment, the maximum dimensions were 80-90 mm (Figure 6ac). During the release of neutral spores, some filamentous conchocelis were also generated, but at low quantities. During five and a half months of the experiment, an upward trend of specimens' growth was noticed (considering the blade's average length, expressed in mm) ( Figure 7). P. leucosticta maintained positive growth during the culture period for the majority of the thalli developed under experimental conditions. At the end of the experiment, the maximum dimensions were 80-90 mm (Figure 6a-c). During the release of neutral spores, some filamentous conchocelis were also generated, but at low quantities. A temperature above 15 °C triggered the entry of newly formed blades into an early reproductive period, which led to spore release (Figure 5a,b). The evolution of the spores followed the same pattern as the adult reproductive material, and after 10 days, these new thalli became macroscopically visible (Figure 5c). Although Abdel-Rahman (2015) mentions that neutral-spore production was higher at 20 °C than at other temperatures [19], for P. leucosticta, an increase in temperature was considered to be a stress factor, hence this biological response. At the end of the experiment, the maximum dimensions were 80-90 mm (Figure 6ac). During the release of neutral spores, some filamentous conchocelis were also generated, but at low quantities. During five and a half months of the experiment, an upward trend of specimens' growth was noticed (considering the blade's average length, expressed in mm) ( Figure 7). P. leucosticta maintained positive growth during the culture period for the majority of the thalli developed under experimental conditions.

Discussion
Our study demonstrated that the discharge of neutral spores capable of generating new thalli is massive under favorable conditions, namely, if adequate temperature and light regimes are ensured. The optimal development of foliose blades will be achieved

Discussion
Our study demonstrated that the discharge of neutral spores capable of generating new thalli is massive under favorable conditions, namely, if adequate temperature and light regimes are ensured. The optimal development of foliose blades will be achieved with a constant temperature regime, as fluctuations are extremely harmful; in other words, large temperature changes are unfavorable compared to a constant maximum temperature. In our experiment, when the temperature reached 17 • C, the new specimens formed exclusively in controlled culture entered an early reproductive stage, leading to the massive release of neutral spores, with the formation of new thalli. These were considered stress conditions, and in this case, the tissue that housed the spores disintegrated, while the rest of the blade continued its evolution. However, these thalli appeared depigmented, which can be a problem if the intention is to harvest the biomass for further capitalization. The biological material could have a lower biochemical composition, which is not suitable for the extraction of various compounds.
There are also studies that indicate the growth of P. leucosticta even at temperatures of 20 • C, but the final conclusions stated that temperatures above 15 • C may be suboptimal [5]. Green and Neefus (2015) mentioned that P. leucosticta blades grown under day-neutral (12:12 light-dark) and long-day (16:8 light-dark) conditions had higher growth rates compared with those grown under short-day (8:16 light-dark) conditions. However, blades grown under short-day conditions had higher phycobilin content than blades grown under day-neutral or long-day conditions. The authors mentioned that newly formed blades may effectively dilute the photosynthetic pigment concentration as they expand rapidly. For example, if the intention is to use P. leucosticta as a sea vegetable, production should focus on producing highly pigmented biomass, so the optimal conditions would range from 10 to 15 • C, from 30 to 110 µmol photons m −2 s −1 , and more than 12 h of light per day [5].
Although these biochemical analyses were not performed on the newly formed blades, since the purpose of our study was only to initiate the cultivation process of this species, our data may support this hypothesis, taking into account that the blades appeared depigmented towards the end of the experiment, with a possible cause being a reduced amount of phycoerythrin. Although only standard light conditions (day-neutral) were used, in the future, other light regimes will be tested, particularly short and long days, to see the influence of light on the biochemical composition of P. leucosticta.
Nianci and co-workers (2019) mentioned that large numbers of neutral spores could serve as "seeds" that can be used directly for seedling cultivation [20]. Our study confirms this theory for the species P. leucosticta, collected from the Romanian coast. The experiment was based entirely on the asexual reproduction of the species. Although carried out on a small scale, it led to the development of numerous neutral-spore-germinated thalli and opens new paths for the development of macroalgal cultures along the Romanian coast, a field that has not been intensely explored to date but in which there is a major interest. The method used in this study has been tested worldwide on various species of Porphyra. Compared with the traditional aquaculture mode based on sexual reproduction via shell conchocelis cultivation, this method requires minimal investments in instruments and space for efficient sectioning and is easy for farmers to adopt [20]. Moreover, the whole propagation cycle took only 7 days, which is dramatically faster than conchosporedependent seedling production. Our results support Nianci's (2019) theory (although referring to a different species-Pyropia yezoensis) and suggest that the neutral-spore-based method could be considered as an alternative P. leucosticta cultivation methodology, with important technical support for innovation in Romanian Black Sea macroalgal aquaculture. The main conclusions of all of these studies were that asexually generated blades from neutral spores grow more quickly than those generated from conchospores. Considering that after only 5.5 months, specimens of even 90 mm in length were obtained, we confirm this aspect.
For the large-scale expansion of cultures, it would be interesting to test the introduction of specific ropes into the culture vessels, before the release of neutral spores, as substrates for adhesion. The method has already been tested and provided favorable results in other countries. In this regard, Nianci and co-workers (2019) mentioned that to test the adhesion capability, seedling ropes were incubated in the neutral-spore culture, and they found that 63.5% of the neutral spores could successfully attach to the seedling ropes after 24 h, and 81.3% attached after 48 h [20]. Moreover, Blouin and co-workers (2007) [21] highlighted that nets seeded with asexually derived spores have several advantages. The use of neutral spores, as opposed to conchospores, eliminates the need to complete the sexual life history of Porphyra. This means that the considerable time, expense, and infrastructure necessary to maintain conchocelis cultures can be avoided. Further experiments are also necessary to investigate the importance of the initial spore density for maximal germination.

Conclusions
This study demonstrated the great reproductive capacity of P. leucosticta under laboratory-controlled conditions, and it can be the first step in initiating the aquaculture of P. leucosticta along the Romanian Black Sea shore. The neutral spores, as part of the species' reproductive cycle, proved to be very efficient for obtaining new biological material for future needs. This experiment can be extended to large-scale testing, depending on requirements and interest. However, further research is necessary to optimize the method and to make the transition to a large-scale cultivation approach.