Seagrasses are declining worldwide due to human and natural causes (Waycott et al. 2009), leading to loss of their important ecosystem functions (Hemminga and Duarte 2000). Disturbance and stress are well known environmental factors affecting successful plant establishment (Craine 2009). Catastrophic events in particular can change seagrass cover drastically (den Hartog 1987, Williams 1988, Larkum and den Hartog 1989) and may trigger energy allocation into sexual reproduction (e.g. Reusch 2006, Alexandre et al. 2005, Hammerstrom et al. 2006). Although clonal propagation is the major mode of space occupation for many seagrasses (Marbà and Duarte 1998), under some environmental conditions sexual reproduction can be very important in population persistence and in particular for recovery from disturbances (Greve et al. 2005, Bell et al. 2008, Park et al. 2011).

Several studies have focused on the relationship between genotypic diversity and resilience and resistance to perturbations and extreme stress events in seagrasses (e.g. Hughes and Stachowicz 2004, Massa et al. 2013, Evans et al. 2017). These studies report that higher genotypic diversity can have several positive effects on the response to perturbations, including increased resistance to loss and faster recovery. A better understanding of how seagrass meadows recover from extreme events and of the relative role of sexual versus clonal propagation (i.e. genotypic diversity) may thus be an important concern in conservation and management decisions (Reusch et al. 2005, Qin et al. 2014). However, most of the evidence is based on short-term experiments or at most a year of recovery. Very little is known about the relationship between genotypic diversity and disturbances in the long term, analysed at scales encompassing several years. Therefore, this paper aims to focus on understanding the relationship between sexual versus clonal propagation and disturbance over a multi-year time scale.

Zostera marina (L.), a monoecious seagrass species, is the most widely distributed seagrass species in the northern hemisphere (den Hartog 1970), forming perennial and annual populations (Bos et al. 2007). Annual populations are characterized by high sexual allocation, where all vegetative shoots turn into flowering shoots, creating seed banks which can germinate when conditions are favourable (Keddy and Patriquin 1978, Santamarı́a-Gallegos et al. 2000, Jarvis et al. 2014). The genotypic (i.e. clonal) diversity of annual Z. marina meadows is therefore expected to be very high. Perennial populations are characterized by lower sexual allocation, and therefore lower genotypic diversity, with clonal growth being the favoured strategy for area occupation (Olesen and Sand-Jensen 1994, Olesen 1999, Kim et al. 2008). Despite these general predictions, high sexual allocation has been observed in some perennial populations (van Lent and Verschuure 1994, Meling-López and Ibarra-Obando 1999, Olesen 1999). Such unpredicted behaviour in persistent populations may be linked to disturbance events (Cabaço and Santos 2012, Qin et al. 2014) such as storms.

The aim of this study was to assess how the importance of sexual and clonal reproduction varies before, during and after recovery from natural storm disturbance in a natural Zostera marina population followed over a five-year period. We proposed to test two alternative hypotheses that resulted in contrasting genotypic diversity in the recovered meadow; we hypothesized that, in the long term, seagrass recovery may lead to either 1) clone dominance due to vegetative propagation being more effective than sexual reproduction, resulting in low genotypic diversity; or 2) high genotypic diversity due to recurrent seedling recruitment.

Our results show that both sexual and clonal reproduction of Z. marina played an important role, but contributed differentially, in different stages of recovery of a meadow from a severe natural disturbance event that had caused its disappearance. At an early stage, the seed bank played a crucial role in the meadow recovery, shown by seedling recruitment. It has been hypothesized (Orth et al. 2006) that the natural recover of Z. marina banks is likely to be possible due to small remnant stands. We made a considerable effort to cover the total former area covered by the seagrass meadow after its decay and found no evidence of remaining shoots. Therefore, our results demonstrate the important role of the seed bank in recovering a population in which all plants were lost.

On the long term, clonal growth increased shoot density, increasing the vegetated area. This perennial meadow behaved as an annual meadow during extreme climate conditions, demonstrating the species’s capacity to recover from large scale losses, via seedling recruitment, as similarly observed in other locations (Jarvis and Moore 2010, Kim et al. 2014, Qin et al. 2016). Most of all, it shows that the contribution of sexual and clonal propagation is a population trait that is very variable (Rafajlović et al. 2017), and this plasticity plays a key role in population persistence despite catastrophic disturbance events.

The new meadow originated exclusively from sexual recombination from the previous population and not from seed dispersal from a differentiated population, as shown by the absence of genetic differentiation between the pre- and post-disturbance samples. All other populations in the region are very differentiated and separated by >100 km (Diekmann and Serrão 2012). Closer to this study site there were small patches within a distance viable for seed dispersal but only one survived the storm. This patch was less than 10 m², a very small patch inside the Sado estuary, most likely not containing sufficient plant material to have given rise to this new population. Also, the small surviving patch in the Sado River Estuary was studied in previous works and had very low genotypic richness, leading to a prediction of low sexual allocation (Diekmann and Serrão 2012).

Our study site is located in an extremely dynamic area, mainly due to strong tidal currents and storms during winter months (Paulo et al. 2019 - Supplementary materials). In such environments, high sexual allocation by seagrasses is well documented (van Lent and Verschuure 1994, Meling-López and Ibarra-Obando 1999, Santamaría-Gallegos et al. 2000). The high genotypic diversity found in the pre-disturbance population demonstrates that this meadow allocated resources into sexual reproduction in the years previous to disturbance. As a result, a seed bank allowed the population to recover. The observed reduction in genotypic diversity in 2013 shows that shoot recruitment from sexual propagules, which started as 100% seedlings (R=1), was diluted with clonal growth over time. Nevertheless, the post-disturbance population genetic diversity was still high.

The pre-disturbance population showed heterozygosity deficiency, which may be explained by inbreeding (Diekmann and Serrão 2012), suggesting that in the past, sexual reproduction occurred between closely related individuals or selfing. The post-disturbance population, sampled after three years of recovery, had no heterozygote deficiency, indicating random mating. This change in mating system might hypothetically be a consequence of the different clonal structure of the population. Although male and female flowers in the same inflorescence are not mature in synchrony, those in different flowering shoots from the same clone can reproduce with each other (Reusch 2001). In contrast, in a recent population formed entirely by smaller clones, each clone is not likely to have many more flowering shoots than any other one.

Southern edge populations, such as the one studied here, are particularly important for the persistence and evolution of Z. marina as a species. These populations are small and reproductively isolated from others (Diekmann and Serrão 2012). Although at the worldwide scale, eelgrass conservation status is of least concern, the current trend is for populations to decline (Short el al. 2010). The southern populations in particular are considered highly endangered and many have recently disappeared (Cunha et al. 2013). As an example of the species vulnerability in this region of the world, 5 km northwest from our study area, 10 ha of eelgrass meadow were recently lost and did not recover naturally (Cunha et al. 2014, Paulo et al. 2019). Due to the opportunistic nature of this research, we did not have the opportunity to study sexual and clonal allocation. As future recommendations, we suggest the study of the sexual investment in these populations to better understand the chances of recolonization after localized extinction. Seedbank size should be assessed yearly, together with the meadow flowering rate, information that is vital to understand the chances of survival in case of another mass mortality event. Such knowledge can be used to plan seed-based restoration programmes (Tanner and Parham 2010, Marion and Orth 2010). Because Z. marina seed banks have a transient nature, the window of opportunity for recolonization might be short. Seed banks’ highest germination success is limited to 6 months and falls to less than 5% in 15 months (Jarvis et al. 2014). It is still unknown how long it takes for a seagrass population to undertake another extreme event and still recover. Theory predicts that after colonization is completed, sexual reproduction can be favoured, decreasing clonal dominance (Rafajlović et al. 2017). Our study period was too short to confirm this hypothesis. Therefore, in future research, it is very important to quantify sexual investment (flower and seed density) and seedling success vs. clonal growth to determine long term trade-offs and vulnerability status to seagrass meadows.

This study demonstrated that the relative roles of sexual versus clonal reproduction in partially-clonal organisms, such as seagrasses, vary with the temporal scales of population disturbance and are best understood when analysed on multi-year scales. The study also revealed that sexual reproduction is of extreme importance in population maintenance and resilience in the face of unpredicted future climate regimes.