INTRODUCTION
⌅Although outcrossing is the most prevalent mating system in angiosperms, a third of flowering plants exhibit mixed mating systems in which reproduction occurs both by self-fertilization and outcrossing (Goodwillie & al. 2005Goodwillie C., Kalisz S. & Eckert C.G. 2005. The evolutionary enigma of mixed mating systems in plants: occurrence, theoretical explanations, and empirical evidence. Annual Review of Ecology, Evolution and Systematics 36: 47-79.). In such cases, the genetic self-incompatibility system (SI) that is the main barrier to selfing in flowering plants (Igić & al. 2008Igić B., Lande R. & Kohn J.R. 2008. Loss of self-incompatibility and its evolutionary consequences. International Journal of Plant Sciences 169: 93-104.) is eventually broken. In Asteraceae, while most species are self-incompatible (63%), a significant proportion are partially (10%) or totally (27%) self-compatible, pointing to a breakdown of their SI (Ferrer & Good-Avila 2007Ferrer M. & Good-Avila S. 2007. Macrophylogenetic analyses of the gain and loss of self-incompatibility in the Asteraceae. New Phytologist 173: 401-14.). Moreover, a complete and irreversible transition from outcrossing to selfing is a prevailing pattern in angiosperm diversification (Stebbins 1974Stebbins G.L. 1974. Flowering plants. Evolution above the species level. Belknap Press, Cambridge. ; Igić & al. 2008Igić B., Lande R. & Kohn J.R. 2008. Loss of self-incompatibility and its evolutionary consequences. International Journal of Plant Sciences 169: 93-104.; Goldberg & al. 2010Goldberg E.E., Kohn J.R., Lande R., Robertson K.A., Smith S.A. & Igić B. 2010. Species selection maintains self-incompatibility. Science 330: 493-495.). However, the maintenance of selfing requires being advantageous enough to counterbalance the effects of inbreeding depression (Charlesworth & Charlesworth 1987Charlesworth D. & Charlesworth B. 1987. Inbreeding depression and its evolutionary consequences. Annual Review of Ecology, Evolution and Systematics 18: 237-268.; Byers & Waller 1999Byers D.L. & Waller D.M. 1999. Do plant populations purge their genetic load? Effects of population size and mating history on inbreeding depression. Annual Review of Ecology and Systematics 30: 479-513.). This situation may occur in populations with severe pollen limitation due to low effective population size and/or scarce pollinator presence (Busch & Schoen 2008Busch J.W. & Schoen D.J. 2008. The evolution of self-incompatibility when mates are limiting. Trends in Plant Science 13: 128-136.; Good-Avila & al. 2008Good-Avila S.V., Mena-Alí J.I. & Stephenson A.G. 2008. Evolutionary consequences of variations in self-fertility in self-incompatible species. In Franklin-Tong E. (ed.), Self-incompatibility in flowering plants-evolution, diversity, and mechanisms. Springer, Berlin.), in which selection for reproductive assurance is a prevalent factor (Busch & Delph 2012Busch J.W. & Delph L.F. 2012. The relative importance of reproductive assurance and automatic selection as hypotheses for the evolution of self-fertilization. Annals of Botany 109: 553-562.). This could be especially relevant in annual plants whose reproductive success is limited to one flowering season (Shivanna 2014Shivanna K.R. 2014. Reproductive assurance through autogamy in some annual weed species. Proceedings of the National Academy of Sciences, India Section B: Biological Sciences 84: 681-687., 2015Shivanna K.R. 2015. Reproductive assurance through autogamous self-pollination across diverse sexual and breeding systems. Current Science 109: 1255-1263.).
The annual herb Anacyclus linearilobus Boiss. & Reut. is a narrow endemism occurring in coastal dune ecosystems in Algeria. This species was described in the surroundings of Oran by Boissier & Reuter (1852)Boissier P.E. & Reuter G.F. 1852. Pugillus Plantarum Novarum Africae Borealis Hispaniaeque Australis. Ferd. Ramboz et socii, Geneva. and only few other populations are known so far (Humphries 1979Humphries C.J. 1979. A revision of the genus Anacyclus L. (Compositae: Anthemideae). Bulletin of the British Museum (Natural History) Botany 7: 83-142.). The genus Anacyclus was recently circumscribed to eight species distributed in western Mediterranean (Vitales & al. 2018Vitales D., Nieto Feliner G., Vallès J., Garnatje T., Firat M. & Álvarez I. 2018. A new circumscription of the Mediterranean Anacyclus (Anthemideae, Asteraceae) based on plastid and nuclear DNA markers. Phytotaxa 349: 1-17.; Álvarez 2019Álvarez I. 2019. Anacyclus L. In Benedí C., Buira A., Rico E., Crespo M.B., Quintanar A., Aedo C. (eds.), Flora iberica vol. 16(3), Compositae (partim). Real Jardín Botánico-CSIC, Madrid.). Morphologically, A. linearilobus mainly differs from other Anacyclus species in the degree of basal leaves division, which in A. linearilobus is 1-2 pinnatisect whereas in the remaining species is tri-pinnatisect (Humphries 1979Humphries C.J. 1979. A revision of the genus Anacyclus L. (Compositae: Anthemideae). Bulletin of the British Museum (Natural History) Botany 7: 83-142.; Vitales & al. 2018Vitales D., Nieto Feliner G., Vallès J., Garnatje T., Firat M. & Álvarez I. 2018. A new circumscription of the Mediterranean Anacyclus (Anthemideae, Asteraceae) based on plastid and nuclear DNA markers. Phytotaxa 349: 1-17.). All species in this genus have a relatively wide distribution, except A. linearilobus that is restricted to the Algerian coast, and A. maroccanus Ball that is an endemism of the Morocco plateau (Humphries 1979Humphries C.J. 1979. A revision of the genus Anacyclus L. (Compositae: Anthemideae). Bulletin of the British Museum (Natural History) Botany 7: 83-142.; Vitales & al. 2018Vitales D., Nieto Feliner G., Vallès J., Garnatje T., Firat M. & Álvarez I. 2018. A new circumscription of the Mediterranean Anacyclus (Anthemideae, Asteraceae) based on plastid and nuclear DNA markers. Phytotaxa 349: 1-17.). Additionally, A. linearilobus is the only species in this genus growing in no anthropic habitats.
Anacyclus linearilobus was considered in the past closely related to A. clavatus (Desf.) Pers. and A. homogamos (Maire) Humphries (Humphries 1979Humphries C.J. 1979. A revision of the genus Anacyclus L. (Compositae: Anthemideae). Bulletin of the British Museum (Natural History) Botany 7: 83-142.), however recent phylogenetic analyses indicate A. valentinus L. as its sister species, in a clade including A. clavatus and A. radiatus Loisel. (Oberprieler 2004Oberprieler C. 2004. On the taxonomic status and the phylogenetic relationships of some unispecific Mediterranean genera of Compositae-Anthemideae I. Brocchia, Endopappus and Heliocauta. Willdenowia 34: 39-57.; Vitales & al. 2018Vitales D., Nieto Feliner G., Vallès J., Garnatje T., Firat M. & Álvarez I. 2018. A new circumscription of the Mediterranean Anacyclus (Anthemideae, Asteraceae) based on plastid and nuclear DNA markers. Phytotaxa 349: 1-17.). However, A. linearilobus and A. valentinus are morphologically quite different (i.e., while A. linearilobus show radiate capitula, A. valentinus present rayless ones). All species in Anacyclus are diploid (2n = 18; Humphries 1981Humphries C.J. 1981. Cytogenetic and cladistic studies in Anacyclus (Compositae: Anthemideae). Nordic Journal of Botany 1: 83-96.; Rosato & al. 2017Rosato M., Álvarez I., Nieto Feliner G. & Rosselló J.A. 2017. High and uneven levels of 45S rDNA site-number variation across wild populations of a diploid plant genus (Anacyclus, Asteraceae). PLoS One 12: e0187131.), although significant differences in genome size between them were documented (Agudo & al. 2019Agudo A.B., Torices R., Loureiro J., Castro S., Castro M. & Álvarez I. 2019. Genome size variation in a hybridizing diploid species complex in Anacyclus (Asteraceae: Anthemideae). International Journal of Plant Sciences 180: 374-385.; Vitales & al. 2020Vitales D., Álvarez I., Garcia S., Hidalgo O., Nieto Feliner G., Pellicer J., Vallès J. & Garnatje T. 2020. Genome size variation at constant chromosome number is not correlated with repetitive DNA dynamism in Anacyclus (Asteraceae). Annals of Botany 125: 611-623.). It is remarkable that the two extremes in genome size within the genus are represented by these two sister species, with sizes of 8.22 Gbp/2C in A. valentinus and 13.14 Gbp/2C in A. linearilobus (Vitales & al. 2020Vitales D., Álvarez I., Garcia S., Hidalgo O., Nieto Feliner G., Pellicer J., Vallès J. & Garnatje T. 2020. Genome size variation at constant chromosome number is not correlated with repetitive DNA dynamism in Anacyclus (Asteraceae). Annals of Botany 125: 611-623.). Finally, it is important to consider the possible hybrid origin of A. valentinus suggested by Álvarez & al. (2020)Álvarez I., Agudo A.B., Herrero A. & Torices R. 2020. The Mendelian inheritance of gynomonoecy: insights from Anacyclus hybridizing species. American Journal of Botany 107: 116-125., since it could also involve its sister species, A. linearilobus. In hybrids, allelic incompatibilities (Bateson 1909Bateson W. 1909. Heredity and variation in modern lights. In Seward A.C. (ed.), Darwin and modern science. Cambridge University Press, Cambridge.; Dobzhansky 1936Dobzhansky T. 1936. Studies on hybrid sterility. II. Localization of sterility factors in Drosophila pseudoobscura hybrids. Genetics 21: 113-135.; Muller 1942Muller H.J. 1942. Isolating mechanisms, evolution, and temperature. Biology Symposium 6: 71-125.) and lethal nuclear-cytoplasmic interactions (Levin 2003Levin D.A. 2003. The cytoplasmic factor in plant speciation. Systematic Botany 28: 5-11.) may cause low seed sets and high variation in fertility.
The viability of interspecific crosses between all Anacyclus annual species has already been documented (Humphries 1981Humphries C.J. 1981. Cytogenetic and cladistic studies in Anacyclus (Compositae: Anthemideae). Nordic Journal of Botany 1: 83-96.), but the breeding system has only been assessed in A. clavatus, A. homogamos, and A. valentinus (Álvarez & al. 2020Álvarez I., Agudo A.B., Herrero A. & Torices R. 2020. The Mendelian inheritance of gynomonoecy: insights from Anacyclus hybridizing species. American Journal of Botany 107: 116-125.). In all these cases, the species are considered self-incompatible and inter-fertile although reproductively isolated by postzygotic barriers.
Our goal here was to assess the breeding system in two populations of Anacyclus linearilobus and compare it with other annual species in the genus including its sister species, A. valentinus. We consider that this study is relevant not only for conservation purposes of A. linearilobus, but also for designing future research on the ecological and evolutionary singularity of this species.
MATERIAL AND METHODS
⌅Plant material
⌅Ripened capitula of Anacyclus linearilobus were collected in 2016 from two natural populations (Fig. 1; Table 1). The Bousfer population occupied a wide area along Bomo and Corales beaches where it presented a discontinued distribution. Therefore, three subareas were sampled in this case: two in Bomo beach (BMO and MOB), and one in Corales beach (COR). Our access to La Macta population (MAC) was restricted to a small sampling area. During the same collection trip two other localities were visited, although without success: the surroundings of Oran that was cited by Humphries (1979)Humphries C.J. 1979. A revision of the genus Anacyclus L. (Compositae: Anthemideae). Bulletin of the British Museum (Natural History) Botany 7: 83-142., and “Les Andalouses” near El-Ançor, represented by several herbarium sheets. For seed germination, 10-30 achenes from each individual were sown on wet desiccant paper into Petri dishes during 90 days under a 16h light/8h dark regime and 10-27ºC in the Research Greenhouse of the Real Jardín Botánico (CSIC) in Madrid. Once germinated, seedlings were grown individually in a mix of COMPO SANA® Universal Potting Soil (COMPO GbmH, München, Germany) and siliceous sand (3:1) until the first 4 to 6 leaves developed. We selected fifteen grown plants from Bousfer (i.e., five from each subarea), and five from La Macta as pollen receptors, and 8-20 grown plants from each population as pollen donors.
| Population | Area or subarea code | Number of individuals collected | Origin and voucher information |
|---|---|---|---|
| Bousfer | BMO | 3 | Algeria: Bousfer, Bomo beach, 35º45’0.32’’N, 0º49’50.52’’W, 4 m, 23 May 2016, Álvarez 2339 |
| MOB | 1 | Algeria: Bousfer, Bomo beach, 35º44’52.08’’N, 0º49’56.6’’W, 19 m, 23 May 2016, Álvarez 2340 | |
| COR | 1 | Algeria: Bousfer, Corales beach, 35º45’37”N, 0º49’18”W, 33 m, Jun. 2016, Kaid-Harche s/n | |
| La Macta | MAC | 3 | Algeria: La Macta, 35º47’25.26’’N, 0º9’24.8’’W, 2 m, 24 May 2016, Álvarez 2345 |
Breeding system and experimental crosses
⌅To estimate the breeding system in Anacyclus linearilobus we followed a protocol similar to that previously performed in other Anacyclus species (Álvarez & al. 2020Álvarez I., Agudo A.B., Herrero A. & Torices R. 2020. The Mendelian inheritance of gynomonoecy: insights from Anacyclus hybridizing species. American Journal of Botany 107: 116-125.). Four different experiments (one per inflorescence -capitulum-) were performed on each individual designated as pollen receptor: (1) no pollen addition to test autogamy; (2) pollen addition of the same individual to test self-compatibility; (3) pollen addition of individuals from the same population than the pollen receptor to test intra-population outcrossing; and (4) pollen addition of individuals from a different population to test inter-population outcrossing. All experiments were carried out in 2017 at the Research Greenhouse of the Real Jardín Botánico (CSIC). All manipulated capitula were bagged before anthesis until fruits were collected. Bags were only open for pollen addition. In experiment 1 no additional manipulation was done once the capitula were bagged. In experiment 2, pollen from different capitula of the same individual was added to produce pollen saturation. In experiments 3 and 4, a mix of pollen from different individuals was used in each case to ensure viability, avoiding individual effects, and to favour pollen saturation. In Asteraceae it is difficult to emasculate without damaging the gynoecium due to the joint development of the sexual organs. Furthermore, in the case of Anacyclus, the small size of the hermaphroditic flowers (~ 5 mm) and their high number per capitulum (150-200), make emasculation unfeasible. In this case, we consider outcrossing valid when there is a significant difference between self-fertilization and outcrosses within the same individual. To give time to match phenology and to get the highest number of mature capitula available by pollen donors, the first two capitula formed in each plant selected as pollen receptor were used for experiments 1 and 2. In most Asteraceae, the mature pollen is released and pushed out of the floret as the style elongates, being exposed at the tip of the style (Leins & Erbar 2006Leins P. & Erbar C. 2006. Secondary pollen presentation syndromes of the Asterales-a phylogenetic perspective. Botanische Jahrbücher fur Systematik, Pflanzengeschichte und Pflanzengeographie 127: 83-103.). We collected pollen from style tips with tweezers daily and once a day (between 9-11 am) and blended in 1.5 mL Eppendorf Tubes® (Eppendorf, Hamburg, Germany) for immediate application. Pollen addition was performed using a paintbrush for each capitulum, until all flowers in the capitulum were ripened (usually 2-3 weeks, depending on the capitulum size). Ripened capitula were collected at least 4 weeks after the pollen addition was finished in each case. Since in the Asteraceae each flower may produce only one seed, the breeding system was estimated by the seed set (i.e., number of ripened seeds per capitulum/total number of flowers per capitulum) of each individual designated as pollen receptor.
Statistical analyses
⌅The probability of setting a viable seed was analysed by fitting generalised linear mixed models (GLMMs), via restricted maximum likelihood (Patterson & Thompson 1971Patterson H.D. & Thompson R. 1971. Recovery of inter-block information when block sizes are unequal. Biometrika 58: 545-554.) using the ‘lme4’ package for R (Bates & al. 2015Bates D., Mächler M., Bolker B.M. & Walker S. 2015. Fitting Linear Mixed-Effects Models using lme4. Journal of Statistical Software 67: 1-48.). We followed recommendations in Zuur & al. (2010)Zuur A.F., Leno E.N. & Elphick C.S. 2010. A protocol for data exploration to avoid common statistical problems. Methods in Ecology and Evolution 1: 3-14. to ensure that our data met the assumptions of linear modelling, plotting residuals against fitted values, and against each explanatory variable before fitting any model. We modelled the probability of producing viable seeds using a binomial distribution with a logit link function. This model included the type of pollen addition (i.e., experiments enumerated above) as a fixed factor, and the pollen receptor (plant) and the population where it belongs (site) as random factors. We also fitted the same models for each site separately, removing the site as a random factor. All P-values of post-hoc comparisons were corrected using Holm’s adjustment. The versatility of GLMMs makes them a better choice than linear models for modelling the variables that we considered and discerning between fixed and random factors in the model.
To assess the level and variation of self-incompatibility among these populations, we estimated the index of self-incompatibility for each individual following Lloyd (1992)Lloyd D.G. 1992. Self- and cross-fertilization in plants II. The selection of self-fertilization. International Journal of Plant Sciences 153: 370-380.: ISI = 1 - relative selfed success / relative outcrossed success. For this equation, we used the seed set rate resulting from the selfing experiment as the relative selfed success and that from the intra-population experiment as the outcrossed success. In the Bousfer population that includes three subareas (BMO, MOB and COR), the intra-population experiment considered was that of the corresponding subarea in each case. According to Raduski & al. (2012)Raduski A.R., Haney E.B., Igić B. 2012. The expression of self-incompatibility in angiosperms is bimodal. Evolution 66: 1275-1283., self-incompatible individuals score ≥ 0.8; partial self-incompatible plants show values between 0.2 and 0.8, and self-compatible ones ≤ 0.2. We used analyses of variance (ANOVAs) to test for the significant differences of ISI calculated for each population of each species, using both variables (species and populations) as fixed factors. Seed set rates for A. clavatus, A. homogamos, and A. valentinus were those previously obtained in Álvarez & al. (2020)Álvarez I., Agudo A.B., Herrero A. & Torices R. 2020. The Mendelian inheritance of gynomonoecy: insights from Anacyclus hybridizing species. American Journal of Botany 107: 116-125..
RESULTS
⌅Seed germination rate, flowering, and pollen production
⌅In average, the rate of germination in individuals from Bousfer population (70.8%, n = 30) was within the range found in other Anacyclus species (54-91% in A. clavatus, 66-94% in A. homogamos, and 56-85% in A. valentinus; Torices & al. 2013Torices R., Agudo A. & Álvarez I. 2013. Not only size matters: achene morphology affects time of seedling emergence in three heterocarpic species of Anacyclus (Anthemideae, Asteraceae). Anales del Jardín Botánico de Madrid 70: 48-55.), and it was slightly lower in La Macta population (43%, n = 30). However, a detailed analysis by pollen receptor indicated a high variation in seed germination rate at the individual level, ranging from 26.7% to 86.7% in Bousfer, and from 10% to 90% in La Macta.
Generally, cultivated plants produced the first capitulum around 30-40 days after transplantation. Individuals COR 1 and BMO 1 from Bousfer, and MAC 8 from La Macta showed difficulties in pollen releasing, and therefore their pollen availability was lower.
Breeding system
⌅In line with results obtained for other Anacyclus species (Álvarez & al. 2020Álvarez I., Agudo A.B., Herrero A. & Torices R. 2020. The Mendelian inheritance of gynomonoecy: insights from Anacyclus hybridizing species. American Journal of Botany 107: 116-125.), most A. linearilobus individuals showed seed set values < 0.1 for self-compatibility treatments (Table 2). However, in the two studied populations, discordant results with this pattern were observed in two individuals from Bousfer, and in four out of five individuals from La Macta.
Seed set for outcrossing, including both intra- and inter-population treatments, was ≥ 0.4, except for six individuals from Bousfer (Table 2). Variation in seed set under outcrossing pollination treatments was already observed in A. valentinus (Álvarez & al. 2020Álvarez I., Agudo A.B., Herrero A. & Torices R. 2020. The Mendelian inheritance of gynomonoecy: insights from Anacyclus hybridizing species. American Journal of Botany 107: 116-125.), ranging between 0.351-0.981. On the contrary, seed set in the outcrossing treatments were higher and with lower variation in A. clavatus (0.831-0.939) and in A. homogamos (0.789-0.978). Additionally, in A. linearilobus, the variation was observed also at individual level in several cases. For example, the individual COR 1 showed seed sets < 0.4 and ~ 0.9 after different intra-population treatments (Table 2).
| Popula- tion | ID | auto | self | intra-population outcrosses | inter-population outcrosses | ISI | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| BMO | COR | MOB | MAC | - | - | |||||
| Bousfer | BMO 7 | 0.000 [172 ] | 0.014 [138 ] | 0.603 [136 ] | - | 0.692 [120 ] | 0.647 [119 ] | - | - | 0.976 |
| BMO 1 | 0.076 [225 ] | 0.056 [195 ] | 0.481 [206 ] | - | - | - | - | - | 0.883 | |
| BMO 6 | 0.190 [163 ] | 0.054 [205 ] | 0.828 [169 ] | 0.413 [80 ] | 0.000 [117 ] | 0.727 [110 ] | - | - | 0.935 | |
| BMO 2 | 0.000 [223 ] | 0.017 [120 ] | 0.871 [209 ] | 0.708 [137 ] | 0.628 [94 ] | 0.608 [102 ] | - | - | 0.981 | |
| BMO 3 | 0.000 [126 ] | 0.016 [125 ] | 0.906 [139 ] | 0.923 [156 ] | 0.739 [165 ] | 0.796 [103 ] | - | - | 0.982 | |
| COR 1 | 0.021 [146 ] | 0.012 [161 ] | 0.907 [97 ] | 0.034 [146 ] | 0.006 [158 ] | 0.344 [128 ] | - | - | 0.637 | |
| COR 2 | 0.079 [202 ] | 0.229 [144 ] | 0.193 [109 ] | 0.688 [157 ] | 0.444 [81 ] | 0.650 [143 ] | - | - | 0.667 | |
| COR 6 | 0.519 [154 ] | 0.669 [130 ] | - | - | - | 0.629 [97 ] | - | - | - | |
| COR 7 | 0.000 [215 ] | 0.014 [147 ] | 0.518 [110 ] | 0.801 [136 ] | 0.376 [85 ] | 0.836 [110 ] | - | - | 0.983 | |
| COR 9 | 0.006 [168 ] | 0.023 [130 ] | - | 0.456 [79 ] | - | 0.648 [108 ] | - | - | 0.949 | |
| MOB 3 | 0.000 [182 ] | 0.000 [175 ] | 0.447 [199 ] | 0.565 [184 ] | 0.579 [178 ] | 0.490 [202 ] | - | - | 1.000 | |
| MOB 4 | 0.000 [255 ] | 0.012 [247 ] | 0.521 [177 ] | - | 0.416 [209 ] | 0.523 [107 ] | - | - | 0.971 | |
| MOB 5 | 0.000 [222 ] | 0.000 [158 ] | - | - | 0.248 [113 ] | - | - | - | 1.000 | |
| MOB 6 | 0.000 [141 ] | 0.007 [138 ] | - | - | - | - | - | - | - | |
| MOB 8 | 0.000 [307 ] | 0.004 [226 ] | 0.186 [253 ] | 0.826 [121 ] | 0.895 [275 ] | 0.813 [166 ] | - | - | 0.995 | |
| Popula- tion | ID | auto | self | intra-population outcrosses | inter-population outcrosses | ISI | ||||
| MAC | - | - | BMO | COR | MOB | |||||
| La Macta | MAC 7 | - | 0.231 [212 ] | 0.908 [196 ] | - | - | 0.880 [183 ] | - | 0.560 [100 ] | 0.745 |
| MAC 1 | 0.320 [250 ] | 0.502 [207 ] | 0.528 [233 ] | - | - | 0.602 [226 ] | - | - | 0.048 | |
| MAC 8 | 0.602 [211 ] | 0.682 [173 ] | 0.497 [143 ] | - | - | - | - | - | 0.374 | |
| MAC 17 | 0.013 [235 ] | 0.016 [256 ] | 0.953 [255 ] | - | - | 0.649 [191 ] | 0.824 [140 ] | 0.890 [145 ] | 0.984 | |
| MAC 18 | 0.060 [216 ] | 0.439 [230 ] | 0.775 [218 ] | - | - | 0.832 [125 ] | 0.600 [100 ] | - | 0.434 | |
As in other Anacyclus species studied, in A. linearilobus, the probability of setting a viable seed differed significantly between the outcrossing experiments on the one hand, and the autogamy and selfing ones on the other (Fig. 2). In the same way, the type of pollen addition was a significant explanatory variable at population level (Table 3). In both sites, the analysis of all paired treatments revealed significant differences between them, being the inter- and intra-population outcrosses those producing the highest seed sets (Fig. 2a, b). There were two main differences between sites: inter-population outcrossing led to the highest seed set in Bousfer but not in La Macta, in which they did not significantly differ from the intra-population ones. In addition, in La Macta the probabilities of setting a seed by autogamy and selfing treatments were remarkably higher than in Bousfer (Fig. 2a, b).
| Sites | n | N | d.f. | χ2 | P |
|---|---|---|---|---|---|
| All sites | 15645 | 20 | 3 | 3138.1 | < 0.001 |
| Bousfer | 11370 | 15 | 3 | 1973.0 | < 0.001 |
| La Macta | 4275 | 5 | 3 | 793.8 | < 0.001 |
The self-incompatibility index (ISI) only differed significantly at population level (ANOVA, n = 32, F 6,22 = 3.32, P = 0.018), and not at the species level (ANOVA, n = 32, F 3,22 = 2.10, P = 0.142). In most of the cases, ISI was > 0.8, indicating self-incompatibility (Table 2). The exceptions found included individuals COR 1 and COR 2 that with values of 0.637 and 0.667, respectively were considered partially self-incompatible, as well as individuals MAC 8, MAC 18 and MAC 7 showing values of 0.374, 0.434, and 0.745, respectively. In addition, individual MAC 1 turned out to be self-compatible with ISI = 0.048. In contrast, all individuals of A. clavatus, A. homogamos, and A. valentinus analysed from a previous study (Álvarez & al. 2020Álvarez I., Agudo A.B., Herrero A. & Torices R. 2020. The Mendelian inheritance of gynomonoecy: insights from Anacyclus hybridizing species. American Journal of Botany 107: 116-125.) were self-incompatible showing ISI values = 0.868-1 (N = 16, 5-6 individuals from two populations from each species).
DISCUSSION
⌅Our results indicate that Anacyclus linearilobus has a mixed breeding system, in which most individuals are self-incompatible and coexist with other self-compatible and partially self-compatible ones. In contrast, its sister species, A. valentinus, as well as two other annual species in the genus, A. clavatus and A. homogamos, do not show a mixed reproductive system and are all considered self-incompatible (Álvarez & al. 2020Álvarez I., Agudo A.B., Herrero A. & Torices R. 2020. The Mendelian inheritance of gynomonoecy: insights from Anacyclus hybridizing species. American Journal of Botany 107: 116-125.). Therefore, it can be assumed that a breakage of the SI occurred along A. linearilobus evolution, allowing a shift to self-compatibility. The fact that the mating mixed system is present in the two populations studied and that transition to selfing is unidirectional (Igić & al. 2008Igić B., Lande R. & Kohn J.R. 2008. Loss of self-incompatibility and its evolutionary consequences. International Journal of Plant Sciences 169: 93-104.) suggests that this reproductive strategy might be fixed at species level. However, occasionally, mixed mating systems may stabilize. For example, in the annual Hypochaeris salzmanniana DC. (Asteraceae), the proportion of selfing depends on pollinators availability (Arista & al. 2017Arista M., Berjano R., Viruel J., Ortiz M.A., Talavera M., Ortiz P.L. 2017. Uncertain pollination environment promotes the evolution of a stable mixed reproductive system in the self-incompatible Hypochaeris salzmanniana (Asteraceae). Annals of Botany 120: 447-456.), maintaining a mixed mating system over time. The paucity of pollinators might also be related with a partial shift to selfing in two isolated populations of the Madeiran endemism Tolpis succulenta (Aiton) Lowe (Asteraceae) (Crawford & al. 2019Crawford D.J., Moura M., Borges Silva L., Mort M.E., Kerbs B., Schaefer H. & Kelly J.K. 2019. The transition to selfing in Azorean Tolpis (Asteraceae). Plant Systematics and Evolution 305: 305-317.; Kerbs & al. 2020Kerbs B., Crawford D.J., White G., Moura M., Borges Silva L., Schaefer H., Brown K., Mort M.E. & Kelly J.K. 2020. How rapidly do self-compatible populations evolve selfing? Mating system estimation within recently evolved self-compatible populations of Azorean Tolpis succulenta (Asteraceae). Ecology and Evolution 10: 13990-13999.). The shift to self-compatibility may be associated with the floral biology, life cycle, and ecology of the species (Barrett 2014Barrett S.C.H. 2014. Evolution of mating systems: outcrossing versus selfing. In Losos J.B. (ed.), The Princeton Guide to Evolution. Princeton University Press, Princeton.; Arista & al. 2017Arista M., Berjano R., Viruel J., Ortiz M.A., Talavera M., Ortiz P.L. 2017. Uncertain pollination environment promotes the evolution of a stable mixed reproductive system in the self-incompatible Hypochaeris salzmanniana (Asteraceae). Annals of Botany 120: 447-456.; Barrett & Harder 2017Barrett S.C.H. & Harder L.D. 2017. The ecology of mating and its evolutionary consequences in seed plants. Annual Review of Ecology, Evolution and Systematics 48: 135-157.). Interestingly, unlike its sister species and others closely related, A. linearilobus is the only one growing in sand dunes, occupying a different habitat than its congeners. Besides, A. linearilobus individuals grow under shrubs canopy and therefore their occurrence is contingent to the shrub’s presence. While other Anacyclus species that occupy anthropic habitats may form dense continuous populations, in A. linearilobus the density of individuals might be lower with a more scattered distribution. Moreover, the presence and abundance of pollinators in these ecosystems might be heterogeneous and dependent as well on “vegetation islands” within the sand dune matrix. Under this situation pollen availability might be a limiting factor for outcrosses, while the effective population size decreases, driving to a need for a reproductive assurance strategy (Busch & Schoen 2008Busch J.W. & Schoen D.J. 2008. The evolution of self-incompatibility when mates are limiting. Trends in Plant Science 13: 128-136.; Good-Avila & al. 2008Good-Avila S.V., Mena-Alí J.I. & Stephenson A.G. 2008. Evolutionary consequences of variations in self-fertility in self-incompatible species. In Franklin-Tong E. (ed.), Self-incompatibility in flowering plants-evolution, diversity, and mechanisms. Springer, Berlin.), which is essential for species with short life cycles like annuals (Shivanna 2014Shivanna K.R. 2014. Reproductive assurance through autogamy in some annual weed species. Proceedings of the National Academy of Sciences, India Section B: Biological Sciences 84: 681-687.). A similar scenario occurs in oceanic islands, in which selfing and mixed mating systems may be advantageous. In fact, in a study comparing species of Asteraceae, Brassicaceae and Solanaceae from islands and mainland, 66% of island species resulted self-compatible, compared to 41% of mainland ones (Grossenbacher & al. 2017Grossenbacher D.L., Brandvain Y., Auld J.R., Burd M., Cheptou P.-O., Conner J.K., Grant A.G., Hovick S.M., Pannell J.R., Pauw A., Petanidou T., Randle A.M., Rubio de Casas R., Vamosi J., Winn A., Igic B., Busch J.W., Kalisz S. & Goldberg E.E. 2017. Self-compatibility is over-represented on islands. New Phytologist 215: 469-478.).
The female reproductive success in Anacyclus linearilobus was variable both at individual and at population level (Fig. 2a, b). Similar type of variation has been observed at population level in A. valentinus (Fig. 2c, d), but not in other Anacyclus species studied (Fig. 2e-h). This variation in A. valentinus has been attributed to its possible hybrid origin (Álvarez & al. 2020Álvarez I., Agudo A.B., Herrero A. & Torices R. 2020. The Mendelian inheritance of gynomonoecy: insights from Anacyclus hybridizing species. American Journal of Botany 107: 116-125.). As A. linearilobus is the sister species of A. valentinus, we might consider a similar hybrid origin, and therefore, the variation observed in female reproductive success could also have similar causes. Other evidence supporting this hypothesis is the high variation in germination rates in A. linearilobus as well as in A. valentinus (Álvarez & al. 2020Álvarez I., Agudo A.B., Herrero A. & Torices R. 2020. The Mendelian inheritance of gynomonoecy: insights from Anacyclus hybridizing species. American Journal of Botany 107: 116-125.), and the difficulty in pollen release observed in some individuals of A. linearilobus, which is in agreement with the lower fitness expected in hybrids. Genic and allelic incompatibilities between pollen receptors and donors reduce mating availability, giving rise to a reduction in effective population size, which might be increased by inbreeding depression (Angeloni & al. 2011Angeloni F., Ouborg N.J. & Leimu R. 2011. Meta-analysis on the association of population size and life history with inbreeding depression in plants. Biological Conservation 144: 35-43.). In this case, it would be expected that seed set values for intra-population crosses were lower than for the inter-population ones. This was observed in the Bousfer population (Fig. 2a), but not in La Macta (Fig. 2b). As La Macta population has partially lost self-incompatibility, it is not expected that intra-population incompatibility would act as much as in the Bousfer population, resulting in a less variable and higher female reproductive success in La Macta (Fig. 2a, b). Other factors, such as species distribution, and population size and structure, have also relevant effects on reproductive performance. This seems evident in A. valentinus, which shows the highest variation in female reproductive success overall, but in which the seed set values for the intra-population crosses are significantly higher than for the inter-population ones (Fig. 2c, d). In this case, the large size of its populations occurring along roadsides, and its wide distribution in western Mediterranean coasts might have counterbalanced the effects of incompatibilities between individuals from the same population.
The assessment of breeding systems of rare or endemic species may be critical for the development of successful conservation strategies (Pérez & al. 2018Pérez M.E., Meléndez-Ackerman E.J. & Monsegur-Rivera O.A. 2018. Breeding system and pollination of Gesneria pauciflora (Gesneriaceae), a threatened Caribbean species. Flora 242: 8-15.). Since mixed mating systems may reduce species vulnerability (Yates & Ladd 2004Yates C.J. & Ladd P.G. 2004. Breeding system, pollination and demography in the rare granite endemic shrub Verticordia staminosa ssp. staminosa in south-west Western Australia. Austral Ecology 29: 189-200.), the finding that Anacyclus linearilobus shows this strategy as reproductive assurance is hopeful for the viability of its populations. However, the fact that this endemic species may experience high pollen limitation in the communities where it occurs (Alonso & al. 2010Alonso C., Vamosi J.C., Knight T.M., Steets J.A. & Ashman T.L. 2010. Is reproduction of endemic plant species particularly pollen limited in biodiversity hotspots? Oikos 119: 1192-1200.), its scattered distribution contingent to shrub canopy in the habitat it occupies, and its limitation in successful mating, make this endemic species a good candidate to be considered in conservation programmes. In addition, populations of A. linearilobus might be heavily impacted by development of industrial and tourism activities as well as the increase in the urban population in northern Algerian coastline (Snoussi & Aoul 2000Snoussi M. & Aoul E.H.T. 2000. Integrated coastal zone management programme northwest African region case. Ocean & Coastal Management 43: 1033-1045.). It might be possible that both the population of “Les Andalouses” near El-Ançor and that of the surroundings of Oran may have become extinct due to urbanistic development in these areas. Anacyclus linearilobus has a restricted distribution on the Algerian coast, with an extent of occurrence estimated as 4,227 km2 and an area of occupancy estimated as 8-36 km2 (of which only around 7 km2 are suitable habitat). Therefore, we have suggested to the International Union for Conservation of Nature (IUCN) its assessment for the category of EN (Endangered).
In conclusion, in contrast with other species in Anacyclus that are considered self-incompatible, the narrow endemism A. linearilobus showed a mixed mating system in which most individuals were self-incompatible whereas others were partially self-incompatible, and others were self-compatible. Effective population sizes in A. linearilobus are diminished by intrinsic factors such the genic incompatibilities between different individuals giving rise to limitation in mating, but also maybe by environmental factors such as scattered habitat availability. Reproductive assurance under these circumstances may be favoured by the mixed mating system. Our results are also congruent with the hypothesis of a possible hybrid origin for A. linearilobus that showed a variable and relatively low female reproductive success both at population and individual levels. However, further research including genomic and cytogenetic data is required to be conclusive on the origin of this singular evolutionary entity. In the same line, the search for other populations of A. linearilobus along coastal sand dunes in Algeria and their demographic tracking would help in better assessing the state of conservation of this species.