Impaginato 345 Adv. Hort. Sci., 2023 37(4): 345­352 DOI: 10.36253/ahsc­13616 Reproductive biology of Sphaeralcea species with ornamental interest A. Gutiérrez 1, 2 (*), P. Monzón 2, S. Micheletto 1, P. Marinangeli 1, 3 1 Center for Renewable Natural Resources of the Semi‐Arid Region (CER‐ ZOS) CONICET ‐ UNS, 8000 B Blanca, Argentina. 2 Department of Biology, Biochemistry and Pharmacy, National University of the South, 8000 B Blanca, Argentina. 3 Department of Agronomy, National University of the South, 8000 B Blanca, Argentina. Key words: Combining ability, Malvaceae, mating system, pollen viability, stigma receptivity. Abstract: The genus Sphaeralcea belongs to the Malvaceae family and has native species from South America. Their attractive morphological characteris­ tics with ornamental value have not yet been explored. The objective of this work was to know the viability of pollen, stigma receptivity, type of pollination and combining ability of four Sphaeralcea species (S. australis, S. bonariensis, S. crispa and S. mendocina), with the aim to develop new ornamental varieties. Fructification, fertility, seed germination and survival seedlings on intraspecific and reciprocal interspecific offspring were assessed. The highest values of stig­ ma receptivity and pollen viability were obtained at 2:00 PM for the four species. S. mendocina also showed high values of pollen viability at 4:00 PM .The species proved to be self­incompatible and allogamous, with different degrees of reproductive compatibility. The interspecific crosses of S. mendocina and the intraspecific of S. crispa did not produce descendants. The crosses between S. australis and S. bonariensis as maternal parent presented the best combining ability with good fruit production, seed germination and survival. This research provides useful information for the formulation and implementa­ tion of breeding strategies, to improve pollination efficiency, and to breed new Sphaeralcea varieties with ornamental potential. 1. Introduction The Malvaceae family is worldwide distributed in regions with temper­ ate and warm climate. In South America, are represented by 63 genus and 533 species of herbs, shrubs and trees, from these 315 are native, 202 endemic and 16 exotics (Zuloaga et al., 2019). Some species are eco­ nomically important, like various Gossypium species, including cotton. Others have medicinal properties (Martínez and Barboza, 2010) and orna­ mental interest (Krapovickas, 2003; Gutiérrez et al., 2021). Some genera of Malvaceae with ornamental potential are Pavonia, Lecanophora, (*) Corresponding author: aguti@criba.edu.ar Citation: GUTIERREZ A., MONZÓN P., MICHELETTO S., MARINANGELI P., 2023 ­ Reproductive biology of Sphaeralcea species with ornamental interest. ­ Adv. Hort. Sci., 37(4): 345­352. Copyright: © 2023 Gutierrez A., Monzón P., Micheletto S., Marinangeli P. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 29 August 2022 Accepted for publication 30 August 2023 AHS Advances in Horticultural Science https://doi.org/10.36253/ahsc-13616 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2023 37(4): 345­352 346 Modiolastrum, Rynchosida and Sphaeralcea (Ponce et al., 2006; Torres et al., 2008; Masini and Rovere, 2015; Gutierrez et al., 2021). In Argentina, the Sphaerlacea genus have native and herbaceous species with attractive characteristics for ornamental cultivation, such as S. australis, S. crispa, S. mendoci‐ na and S. bonariensis (Sriladda et al., 2012; Gutierrez et al., 2021). They are adapted to semi­arid condi­ tions, and have tolerance to water stress, high and low temperatures and high insolation, which make them good candidates for breeding programs in urban ecosystems adapted to extreme weather con­ ditions and for sustainable landscaping. Moreover, the use of native germplasm in breeding programs contributes to the conservation of biodiversity (Masini and Rovere, 2015). In general, native plants make more efficient use of environmental factors such as water and other climate factors, as well as edaphic and biological variables, which result in a lower maintenance demand and in a good perfor­ mance under the restrictive local conditions. Knowledge of the plants reproductive biology is essential for classical breeding programs, because it allows better orientation and planning of the crosses. In the case of the species under study, their repro­ ductive biology is unknown. This knowledge is essen­ tial to be used in pollinations, and to increase the chance of successful fertilization. Some of these aspects are pollen viability, stigma receptivity, polli­ nation type and combining ability since these depend on successful reproduction. Pollen viability and stig­ ma receptivity are aspects that plays an important role in successful hybridizations (Figueiredo et al., 2020). Pollen viability is a measure of male fertility (Liu et al., 2021), viable pollen is critical to the process of reproduction and pollen longevity can be affected by temperature and relative humidity (Ren et al., 2019). Stigma receptivity is the ability to receive the pollen, therefore it directly affects the plant life cycle, allowing the pollen to adhere, hydrate and germinate (Shivanna and Sawhney, 1997). A detailed knowledge of these features will determine the best moment for pollination, to enable successful controlled pollination in breeding programs. Stigma receptivity is related to the activity of enzymes such as peroxidase, esterase and dehy­ drogenase (Galen and Plowright, 1987). Receptive stigmas have high enzyme activity, which can occur in different phases of flower development. The obser­ vation of the activity of these enzymes can be used to characterize stigma receptivity (Zhang et al., 2021). For the pollination process to occur, the trans­ fer of pollen to the stigma must happen during the period in which the stigma is receptive, otherwise, pollen cannot adhere and germinate. The aim of this study was to evaluate the repro­ ductive biology of four native species of the genus Sphaeralcea, determining pollen viability, timing of stigma receptivity, pollination type and combining ability. The hypothesis we followed was that it is pos­ sible to determine aspects of the reproductive biolo­ gy of the native germplasm of Sphaeralcea, with the ultimate objective to develop new ornamental vari­ eties. 2. Materials and Methods Collection area The Pampas region is an extensive plain located to the east of Argentina between 31° and 39° south lati­ tude. Aliaga et al. (2017) characterized the Pampas general climate, considering rainfall, air temperature, humidity, and wind speed as well as the altitude and the alternation between dry and wet events in the area. Based on these elements, they categorized the Southwest of Buenos Aires and the Southeast of La Pampa as semi­arid region. Sphaeralceae seeds were collected between December 2020 and March 2022 in different sites of this semi­arid region (Fig. 1). The sites were characterized by the occurrence of long periods of drought and isolated floods together with windy periods which affect severely the water avail­ ability (Aliaga et al., 2017). Fig. 1 ­ Geographic distribution and collection sites of studied populations of Sphaeralcea australis, S. crispa, S. men‐ docina and S. bonariensis in central Argentina, South America. For labels see the table. Gutiérrez et al. ‐ Reproductive biology of Sphaeralcea species 347 Plant material and experimental design The plant material was collected in the indicated area (Fig. 1) as seeds and preserved at 4°C dry with silica gel. The seeds were germinated after pre­ger­ minative treatments to break dormancy (Gutierrez et al., 2019). After scarification, the seeds were placed in Petri dishes on filter paper moistened with distiller water in a germination chamber at 20°C (ISTA, 2019) with a 12 h photoperiod previously used in other Malvaceae (Erickson et al., 2016; Leperlier et al., 2020). Five plants of each Sphaeralcea species were grown in pots with commercial substrate (GROWMIX MultiPro®) in the greenhouse of the Center of Renewable Natural Resources from the Semi­Arid Region (CERZOS, CONICET ­ UNS), under controlled temperature (18­28°C), irrigation and relative humid­ ity (55­85%). During the day, when the flowers were in anthe­ sis, pollen viability and stigma receptivity were stud­ ied at 8:00 h, 10:00 h, 12:00 h in the morning and at 2:00 h, 4:00 h, and 6:00 h in the afternoon. The same experimental designs were used to eval­ uate pollen viability and stigma receptivity. Four species (treatments), five plants per species (replica­ tions) and three flowers per plant, were used to eval­ uate a total of 60 anthers and 60 stigmas in a com­ pletely randomized experiment. Stigma receptivity The Osborn method was used to evaluate the stig­ ma receptivity, based on the reaction of the peroxi­ dase enzyme. The stigma is classified as receptive when placing a drop of hydrogen peroxide at 40% on the flowers stigmas a bubble production is observed (Osborn et al., 1988). The reaction of stigma receptiv­ ity was examined with a stereomicroscope. Pollen viability The estimation of viable pollen was carried out with the Alexander technique (Alexander, 1980). The grain dyed in an intense violet color was taken as viable and the one that was colored green was taken as non­viable. Pollen grain counts were performed in an optical microscope of four random fields per preparation to estimate the percentage of pollen via­ bility (%) = [(Number of viable pollen grains/Number of total pollen grains) x 100]. Mating system The experimental trial to test the reproductive system in Sphaeralcea genus was based on four experiments and is seen in Table 1. In the green­ house, the plants of experiments 2, 3, and 4 were iso­ lated with a fine mesh in a cage that excludes poten­ tial pollinators. The plants of experiment 1 were used as control and located outside the cage. For experiment 1, five plants of each species (S. australis, S. crispa, S. mendocina and S. bonariensis) were used and three flowers of each plant were marked the day before anthesis. The flowers were allowed to develop normally without any manipula­ tion, as control. The developed fruits were properly identified and covered until harvest. For experiment 2, five plants of each species were used, and three flowers of each plant were marked the day before anthesis. The fruits were properly identified and covered until fully developed and har­ vest. For experiment 3, five plants of each species were used, and three flowers of each plant were marked the day before anthesis. The flowers were allowed to develop normally and without any type of manipula­ tion to evaluate natural self­pollination. The devel­ oped fruit were properly identified and covered until harvest. For experiment 4, inter­specific reciprocal and intra­specific crossing were performed between S. australis, S. crispa, S. mendocina and S. bonariensis Table 1 ­ Experimental management on Sphaeralcea australis, S. crispa, S. mendocina and S. bonariensis. Normal seed set “+” and great­ ly reduced or zero seed set “­” (modified from Simpson, 2019) Experimental management Seed production 1. Flowers left to develop normally, as control. + Fertile − Infertile 2. Isolated flowers, then self­pollinated by hand. + Self­fertile − Not self­fertile 3. Flowering plants in caged, then left freely. + Self­pollinating − Not self­pollinating 4. Isolated flowers, then emasculated and outcrossed. + Outcrossing − Not outcrossing Adv. Hort. Sci., 2023 37(4): 345­352 348 (Table 2). Five plants of each species were used, and three flowers for each plant were marked on the day before anthesis. The flower buds were emasculated, the anthers were removed prior to pollen release and reciprocal outcrossing were made once a day during the flowering period. Other flowers were used as male parent. After pollination, the flowers were properly identified and covered until fruit harvest. The fruiting and fertility results of the four experi­ ments were assessed by counting the seed set in relation to the number of pollinated flowers [Fructification (%) = (number of fruits produced / number of pollinated flowers) x 100] and assessment the full seed in relation to the total seed [Fertility (%) = (number of full seeds/total number of seeds (full + empty)) x 100]. A classification range based on fructi­ fication and fertility percentage was used, therefore 0 to 35% was considered low, 36 to 65% intermedi­ ate and 66 to 100% high. Combining ability The full seeds of the intra­ and interspecific cross­ es of the previous experiments were subjected to mechanical scarification because native Sphaeralcea species present physical dormancy in its seeds (Gutierrez et al., 2019). Scarified seeds were then germinated in a culture chamber. The seeds that ger­ minated were sown in seedling trays with commer­ cial GROWMIX MultiPro® substrate and cultivated in the greenhouse under controlled light (shading net 50% of light extinction), temperature (18­28°C) and humidity (55­85%) conditions (early growth stage). The seedlings that developed three to four true leaves were transplanted into 7x7x9 cm pots with a substrate composed of 50% sandy soil, 35% peat, 10% perlite and 5% compost (advanced growth stage). Survival of germinated seeds and seedlings from intraspecific offspring (siblings) and reciprocal interspecific offspring (hybrids) were evaluated to quantify combining ability at each stage of develop­ ment (early growth stage and advanced growth stage). A classification range was used for the per­ centages of germination and seedling survival as low (0 to 35%), intermediate (36 to 65%) and high (66 to 100%). 3. Results Stigma receptivity The four species of Sphaeralcea had different behaviors in terms of stigma receptivity, reaching dif­ ferent maximum percentages and at different times of the day. S. bonariensis showed high values at 8:00 am and sustained over time until 2:00 PM when it was 100%. S. australis and S. crispa had similar behaviors with two high peaks of receptivity, at 8:00 am and the maximum at 2:00 PM (99% S. australis and 93% S. crispa). S. mendocina during the morning hours showed a different behavior from the rest, with very low stigma receptivity values with an exponential growth between 12:00 to 2:00 PM where it reached the highest percentage of receptivity (92%). After 2:00 PM, when all the species had their maximum peaks of stigma receptivity, the values began to decrease in different ways. For S. australis and S. bonariensis the decrease was marked, reaching val­ ues of 0% at 6:00 PM. For S. crispa and S. mendocina it was gradual until 6:00 PM, when receptivity was null (Fig. 2). Pollen viability The four species of Sphaeralcea obtained high percentages of pollen viability, although these values varied throughout the day and between species. The highest values were recorded at 2:00 PM for S. aus‐ tralis (99%), S. bonariensis (99%) and S. crispa (98%), with no statistically significant differences between Table 2 ­ Combinations of interspecific reciprocal and intraspe­ cific crosses that originated the hybrid and sibling off­ spring, respectively Female parent (♀) x Male parent (♂) Interspecific reciprocal crosses S. australis S. crispa S. australis S. mendocina S. australis S. bonariensis S. bonariensis S. crispa S. bonariensis S. australis S. bonariensis S. mendocina S. crispa S. australis S. crispa S. mendocina S. crispa S. bonariensis S. mendocina S. crispa S. mendocina S. australis S. mendocina S. bonariensis Intraspecific crosses S. australis S. australis S. bonariensis S. bonariensis S. crispa S. crispa S. mendocina S. mendocina Gutiérrez et al. ‐ Reproductive biology of Sphaeralcea species 349 them, and at 4:00 PM for S. mendocina (99%). At 12:00 PM the percentages were also high for all species and the lowest values were recorded at 6:00 PM for all species (Fig. 3). Mating system All combinations, inter and intraspecific crosses, managed to form fruits (Table 3) except S. mendocina x S. bonariensis. In the case of self­pollinations, fruits were not observed. The species used as female parent produced dif­ ferences in the percentage of fruit production. The values were low when S. crispa was used as female (7 to 33%), intermediate when it was S. mendocina (47 to 53%) and high (67 to 100%) with S. australis and S. bonariensis. In the intraspecific crosses, the same pattern was repeated, showing low fruiting percent­ ages for S. crispa (13%), intermediate in S. mendocina (40%) and high in S. australis (67%) and S. bonariensis (87%). Regarding fertility, most of the interspecific cross­ Table 3 ­ Number of pollinated flowers (NPF), number of fruits produced (NFP), fructification percentage (FP), number of full seeds (NFS), number of empty seeds (NES) and seed fertility percentage (SFP) for intraspecific and interspecific reciprocal crosses between S. australis (Sa), S. bonariensis (Sb), S. crispa (Sc), and S. mendocina (Sm) Fig. 2 ­ Stigma receptivity percentage in Sphaeralcea australis (Sa), S. bonariensis (Sb), S. crispa (Sc) and S. mendocina (Sm) flowers in function of time of the day. Fig. 3 ­ Pollen viability percentage in Sphaeralcea australis (Sa), S. bonariensis (Sb), S. crispa (Sc) and S. mendocina (Sm) flowers at different times of the day. Means with differ­ ent letters indicate significant differences with the Fisher's LSD Test, p<0.05. Each vertical bar represents mean ± standard error. (♀ x ♂) NPF NFP FP (%) NFS NES SFP (%) Interspecific reciprocal crosses SaxSb 15 15 100 277 43 83.1 SaxSc 15 15 100 188 43 78.3 SaxSm 15 10 66.7 90 68 55.7 SbxSm 15 8 53.3 18 115 4.5 SbxSc 15 15 100 367 43 89.5 SbxSa 15 10 66.7 179 14 90.2 ScxSb 15 2 13.3 14 0 78.6 ScxSm 15 1 6.7 1 9 10 ScxSa 15 5 33.3 68 6 90.5 SmxSc 15 7 46.7 30 8 78.9 SmxSa 15 7 46.7 58 12 79.7 Intraspecific crosses SaxSa 15 10 66.7 121 74 62.1 SbxSxb 15 13 86.7 283 17 94.3 ScxSc 15 2 13.3 21 9 70 SmxSm 15 6 40 63 8 86.7 350 Adv. Hort. Sci., 2023 37(4): 345­352 es yielded high percentages of full seed production with values between 78 to 91%. However, in the cross of S. australis x S. mendocina it was intermedi­ ate (56%) and it was low for S. bonariensis x S. men‐ docina (4%) and S. crispa x S. mendocina (10%). For intraspecific crosses, fertility was high for S. bonar‐ iensis (94%), S. mendocina (89%) and S. crispa (70%) and intermediate for S. australis. Combining ability The germination percentage after mechanical scarification was high for all crosses except for S. aus‐ tralis x S. australis, which showed intermediate val­ ues (Table 4). Seedling survival decreased throughout development in all descendants, with high and inter­ mediate values predominating in the first growth stage (plant tray) and the majority being low in the advanced stage of development (pot). In the case of the hybrid, the offspring from S. australis x S. bonar‐ iensis showed the highest values of final survival (56%). In the intraspecific crosses, the highest values of descendant survival were from S. australis x S. aus‐ tralis (66%). The crosses that failed to develop live seedlings were S. bonariensis x S. 4. Discussion and Conclusions The methods used by Osborn et al. (1988) and Alexander (1980) to evaluate stigma receptivity and pollen viability, respectively, were effective to achieve successful crosses in the genus Sphaeralcea. Stigma receptivity is a highly variable trait among species of the plant kingdom. There are species such as Carica papaya L. where the flowers are receptive before the floral opening and until the closing (Parés et al., 2002), others such as Passiflora edulis are receptive during anthesis until flowers closed (Ángel Coca et al. , 2011). Our results for the genus Sphaeralcea showed that S. australis, S. crispa and S. bonariensis had high stigma receptivity at flowers opening, except for S. mendocina, which obtained positive results after they opened, and the bubbling was null before flower closure (6:00 PM) for all species. Ambient heat can serve to attract insects to an open flower through volatilization of floral scent during anthesis, and also helps to maintain a period of maximum stigma receptivity (Consiglio and Bourne, 2001). In our results, stigma receptivity was high between 12:00 PM and 2:00 PM which are coinci­ Table 4 ­ Number of full seeds (NFS), germination percentage (GP), number of seedlings in early growth stage (NSEGS), survival percen­ tage of early growth stage (SPEGS), number of seedlings in advanced growth stage (NSAGS) and survival percentage of advan­ ced growth stage (SPAGS) for intraspecific and interspecific reciprocal crosses between S. australis (Sa), S. bonariensis (Sb), S. crispa (Sc), and S. mendocina (Sm) (♀ x ♂) NFS GP (%) NSEGS SPEGS (%) NSAGS SPAGS (%) Interspecific reciprocal crosses SaxSb 277 88.8 181 73.6 102 56.4 SaxSc 188 83.5 139 88.5 12 8.6 SaxSm 90 100 90 100 35 38.9 SbxSm 18 100 9 50 0 SbxSc 367 80.5 186 67.4 70 37.6 SbxSa 179 100 77 43 7 9.1 ScxSb 14 100 13 92.9 3 23.1 ScxSm 1 100 0 0 ­ ScxSa 68 69.1 30 63.8 0 SmxSc 30 71.4 7 35 1 14.3 SmxSa 58 92.6 20 40 0 Intraspecific crosses SaxSa 121 63.6 64 85.3 42 65.6 SbxSxb 283 82.3 120 51.5 47 39.2 ScxSc 21 100 11 55 0 0 SmxSm 63 73.8 42 93.3 6 14.3 Gutiérrez et al. ‐ Reproductive biology of Sphaeralcea species 351 dent with the time of day when the maximum ambi­ ent temperatures were recorded, with an average of 30.4°C (National Meteorological Service, https://www.smn.gob.ar/). In some species high temperatures affect pollen viability (Rao et al., 1992; Radice et al., 2020; Iovane et al., 2022). In Sphaeralcea, there is still no evidence of how environmental factors affect pollen viability, but our results are indirect evidence that pollen would not be affected by high summer temperatures. One possible explanation for these results is that they are native species adapted to local climate con­ ditions and therefore high temperatures do not gen­ erate the thermal stress that affects viability during pollen development or in its mature state. The results of this research indicate that the pollinations that take place between 12:00 PM and 2:00 PM have a greater probability of generating fruits and seeds, since it is when most of the open flowers are recep­ tive, and the viability of pollen is optimal. These species demonstrated to be self­incompati­ ble and allogamous, with different degrees of repro­ ductive compatibility and combining ability between them. The S. mendocina x S. bonariensis cross pro­ duced aborted fruits and were not able to produce offspring. The crosses S. bonariensis x S. mendocina, S. crispa x S. mendocina, S. mendocina x S. australis and S. crispa x S. crispa managed to produce viable seeds that germinated, but with no descendants since the seedlings were not fully develop. These effects are probably the product of reproductive incompatibility between the species since they have different chromosome numbers, S. mendocina is 2n = 30 and the rest of the Sphaeralcea are 2n = 10 (Krapovickas, 1949). It would be interesting to achieve offspring with the germplasm of S. mendoci‐ na since it has very attractive and particular orna­ mental features such as the color of the leaves with shades in the range of gray and pink flowers (Gutiérrez et al., 2021). The null survival of the intraspecific crosses for S. crispa could be due to the rapid loss of vigor of the seeds, since at the time of germination they were smaller plants with a very weak appearance. Except for S. bonariensis x S. mendocina and S. australis x S. mendocina crosses, which were not compatible due to chromosomal differences; the crosses with the best combining ability were those that had S. australis and S. bonariensis as maternal parent with good fruit production. Regarding com­ bining ability, the crosses with the best survival off­ spring were S. australis x S. bonariensis and S. aus‐ tralis x S. australis. Both produced the greatest quan­ tity and quality of descendant plants that prospered over time and had adequate growth and develop­ ment. These novel results will allow us to improve the pollination efficiency and to design a strategic plan for the ornamental improvement of Sphaeralcea genus Our study provides the first data on the reproduc­ tive biology and mating system of Sphaeralceae genus belonging to four native species, which pro­ vides valuable information for the formulation and implementation of new approaches for genetic improvement programs. This facilitates the develop­ ment of new varieties of Sphaeralcea hybrids with ornamental qualities. Acknowledgements Authors thank to National Research Council of Argentina (CONICET) and Department of Agronomy, National University of the South. This work was sup­ ported by the National University of the South (UNS) [grant number PEU 237/19]. References ALEXANDER M., 1980 ­ A versatile stain for pollen, fungi, yeast and bacteria. ­ Stain Technol., 55: 13­18. ALIAGA V., FERRELLI F., PICCOLO C., 2017 ­ Regionalization of climate over the Argentine Pampas. ­ Int. J. 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