INTRODUCTIONTop

Seagrasses are declining worldwide due to human and natural causes (Waycott et al. 2009Waycott M., Duarte C.M., Carruthers T.J., et al. 2009. Accelerating loss of seagrasses across the globe threatens coastal ecosystems. Proc. Natl. Acad. Sci. USA 106: 12377-12381.), leading to loss of their important ecosystem functions (Hemminga and Duarte 2000Hemminga M.A., Duarte C.M. 2000. Seagrass ecology. Cambridge University Press. ). Disturbance and stress are well known environmental factors affecting successful plant establishment (Craine 2009Craine J.M. 2009. Resource strategies of wild plants. Princeton University Press.). Catastrophic events in particular can change seagrass cover drastically (den Hartog 1987Den Hartog C. 1987. “Wasting disease” and other dynamic phenomena in Zostera beds. Aquat. Bot. 27: 3-14, Williams 1988Williams S.L. 1988. Disturbance and recovery of a deep-water Caribbean seagrass bed. Mar. Ecol. Prog. Ser. 42: 63-71., Larkum and den Hartog 1989Larkum A.W.D., Den Hartog C. 1989. Evolution and biogeography of seagrasses. Biology of seagrasses. In: Larkum A.W., McComb A.J., et al. (eds), Biology of seagrasses: a treatise on the biology of seagrasses with special reference to the Australian region. Elsevier, Amsterdam, pp. 112-156.) and may trigger energy allocation into sexual reproduction (e.g. Reusch 2006Reusch T.B. 2006. Does disturbance enhance genotypic diversity in clonal organisms? A field test in the marine angiosperm Zostera marina. Mol. Ecol. 15: 277-286., Alexandre et al. 2005Alexandre A., Santos R., Serrão E. 2005. Effects of clam harvesting on sexual reproduction of the seagrass Zostera noltii. Mar. Ecol. Prog. Ser. 298: 115-122., Hammerstrom et al. 2006Hammerstrom K.K., Kenworthy W.J., Fonseca M.S., et al. 2006. Seed bank, biomass, and productivity of Halophila decipiens, a deep water seagrass on the west Florida continental shelf. Aquat. Bot. 84: 110-120.). Although clonal propagation is the major mode of space occupation for many seagrasses (Marbà and Duarte 1998Marbà N., Duarte C.M. 1998. Rhizome elongation and seagrass clonal growth. Mar. Ecol. Prog. Ser. 174: 269-280.), under some environmental conditions sexual reproduction can be very important in population persistence and in particular for recovery from disturbances (Greve et al. 2005Greve T.M., Krause-Jensen D., Rasmussen M.B., et al. 2005. Means of rapid eelgrass (Zostera marina L.) recolonisation in former dieback areas. Aquat. Bot. 82: 143-156., Bell et al. 2008Bell S.S., Fonseca M.S., Kenworthy W.J. 2008. Dynamics of a subtropical seagrass landscape: links between disturbance and mobile seed banks. J. Landsc. Ecol. 23: 67-74., Park et al. 2011Park S.R., Kim Y.K., Kim J.H., et al. 2011. Rapid recovery of the intertidal seagrass Zostera japonica following intense Manila clam (Ruditapes philippinarum) harvesting activity in Korea. J. Exp. Mar. Biol. Ecol. 407: 275-283.).

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 2004Hughes A.R., Stachowicz, J.J. 2004. Genetic diversity enhances the resistance of a seagrass ecosystem to disturbance. Proc. Natl. Acad. Sci. 101: 8998-9002., Massa et al. 2013Massa S.I., Paulino C.M., Serrão E.A., et al. 2013. Entangled effects of allelic and clonal (genotypic) richness in the resistance and resilience of experimental populations of the seagrass Zostera noltii to diatom invasion. BMC Ecol. 13: 39., Evans et al. 2017Evans S.M., Vergés A., Poore A.G. 2017. Genotypic diversity and short-term response to shading stress in a threatened seagrass: Does low diversity mean low resilience? Front. Plant. Sci. 8: 1417.). 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. 2005Reusch T.B., Ehlers A., Hämmerli A., et al. 2005. Ecosystem recovery after climatic extremes enhanced by genotypic diversity. Proc. Natl. Acad. Sci. USA 102: 2826-2831., Qin et al. 2014Qin L.Z., Li W.T., Zhang X.M., et al. 2014. Sexual reproduction and seed dispersal pattern of annual and perennial Zostera marina in a heterogeneous habitat. Wetl. Ecol. Manag. 22: 671-682.). 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 1970Den Hartog C. 1970. The sea-grasses of the world. North-Holland Pub. Co., Amsterdam.), forming perennial and annual populations (Bos et al. 2007Bos A.R., Bouma T.J., de Kort G.L., et al. 2007. Ecosystem engineering by annual intertidal seagrass beds: sediment accretion and modification. Estuar. Coast. Shelf. 74: 344-348.). 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 1978Keddy C.J., Patriquin D.G. 1978. An annual form of eelgrass in Nova Scotia. Aquat. Bot. 5: 163-170., Santamarı́a-Gallegos et al. 2000Santamaría-Gallegos N.A., Sánchez-Lizaso J.L., Félix-Pico E.F. 2000. Phenology and growth cycle of annual subtidal eelgrass in a subtropical locality. Aquat. Bot. 66: 329-339., Jarvis et al. 2014Jarvis J.C., Moore K.A., Kenworthy W.J. 2014. Persistence of Zostera marina L. (eelgrass) seeds in the sediment seed bank. J. Exp. Mar. Biol. Ecol. 459: 126-136.). 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 1994Olesen B., Sand-Jensen K. 1994. Patch dynamics of eelgrass Zostera marina. Mar. Ecol. Prog. Ser. 106: 147-156., Olesen 1999Olesen B. 1999. Reproduction in Danish eelgrass (Zostera marina L.) stands: size-dependence and biomass partitioning. Aquat. Bot. 65: 209-219., Kim et al. 2008Kim T.H., Rark S.R., Kim Y.K., et al. 2008. Growth dynamics and carbon incorporation of the seagrass Zostera marina L. in Jindong Bay and Gamak Bay on the Southern Coast of Korea. Algae 23: 241-250.). Despite these general predictions, high sexual allocation has been observed in some perennial populations (van Lent and Verschuure 1994van Lent F., Verschuure J.M. 1994. Intraspecific variability of Zostera marina L. (eelgrass) in the estuaries and lagoons of the southwestern Netherlands. I. Population dynamics. Aquat. Bot. 48: 31-58., Meling-López and Ibarra-Obando 1999Meling-López A.E., Ibarra-Obando S.E. 1999. Annual life cycles of two Zostera marina L. populations in the Gulf of California: contrasts in seasonality and reproductive effort. Aquat. Bot. 65: 59-69., Olesen 1999Olesen B. 1999. Reproduction in Danish eelgrass (Zostera marina L.) stands: size-dependence and biomass partitioning. Aquat. Bot. 65: 209-219.). Such unpredicted behaviour in persistent populations may be linked to disturbance events (Cabaço and Santos 2012Cabaço S., Santos R. 2012. Seagrass reproductive effort as an ecological indicator of disturbance. Ecol. Indic. 23: 116-122., Qin et al. 2014Qin L.Z., Li W.T., Zhang X.M., et al. 2014. Sexual reproduction and seed dispersal pattern of annual and perennial Zostera marina in a heterogeneous habitat. Wetl. Ecol. Manag. 22: 671-682.) 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.

METHODSTop

Location

The studied meadow is located at the tip of the Troia Peninsula (Ponta do Adoxe) at the mouth of the Sado estuary, Portugal (38°29.525’N 08°54.507’W, Fig. 1). The area is a sand point characterized by very strong tidal currents. The perennial Z. marina bed is located at the beach, starting at 1.5 m depth and extending to 3 m depth.

The meadow had been monitored and sampled for population genetic analysis before its disappearance in 2008 (Diekmann and Serrão 2012Diekmann O.E., Serrao E.A. 2012. Range-edge genetic diversity: locally poor extant southern patches maintain a regionally diverse hotspot in the seagrass Zostera marina. Mol. Ecol. 21: 1647-1657.). In March 2010, after intense winter storms, we observed that all 1.12 ha were lost (Cunha et al. 2013Cunha A.H., Assis J.F., Serrão E.A. 2013. Seagrasses in Portugal: a most endangered marine habitat. Aquat. Bot. 104: 193-203.). Z. marina recovery was monitored during the spring and summer of 2010. By 2013, recovery of the meadow was observed (continuous seagrass cover of 0.57 ha) and it was resampled for genetics.

Field work

The total area of the meadow was estimated in the spring of 2009 using GPS in tracking mode while following its contour from the boat on a day of good visibility. A comparison between the GPS tracking data and Google Earth images from the same year confirmed that the areas were the same. In 2007, 2013 and 2015, the area was estimated using Google Earth images.

Before the disturbance, shoot density had been recorded in 2009. Immediately after the winter disturbance, we recorded seedling and shoot densities in April, June and September 2010. The meadow was resampled in July 2013 (three years after the disturbance), when it was visually estimated to have attained 100% of the cover it had had before its disappearance. SCUBA divers monitored along two 50 m transects perpendicular to the shore. Every 2 m along the transects, the divers systematically counted seedling and shoot density using a 25×25 cm quadrat at the interception point, and measured leaf lengths of the longest leaf within each shoot using a ruler, in a total of 25 quadrats per transect.

We performed ANOVA and, when necessary, Tukey tests (R Core Team 2017R Core Development Team. 2017. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.r-project.org/) to test mean differences before and after disturbance for the following variables: seedling density (seedling m^–2), shoot density (shoots m^–2) and leaf length (cm).

Genetics

Plant leaves were collected haphazardly by SCUBA diving, maintaining a distance of 1.5 m between sampled shoots. The pre-disturbance samples were collected in May 2008 (n=83) and the post-recovery samples were collected in May 2013 (n=31). The number was not equal between pre- and post-disturbance because the sampling was opportunistic, with SCUBA divers collecting shoots haphazardly and only counting them when they arrived at the boat. Samples were conserved in silica gel desiccant.

Plant DNA was extracted following the CTAB method (Doyle and Doyle 1988Doyle J.J., Doyle J.L. 1988. Natural interspecific hybridization in eastern North American Claytonia. Am. J. Bot. 75: 1238-1246.). After extraction, two chloroform⁄isoamyl alcohol (24:1) extractions and one ethanol (100%) precipitation were used to purify the DNA. Samples were genotyped for eight microsatellite loci (Stam et al. 1999Stam T.B.R., Wytze T., Olsen J.L. 1999. Microsatellite loci in eelgrass Zostera marina reveal marked polymorphism within and among populations. Mol. Ecol. 8: 317-321., Reusch 2000Reusch T.B.H., Stam W.T., Olsen J.L. 2000. A microsatellite-based estimation of clonal diversity and population subdivision in Zostera marina, a marine flowering plant. Mol. Ecol. 9: 127-140.) and followed the protocols of Reusch et al. (2000)Reusch T.B.H., Stam W.T., Olsen J.L. 2000. A microsatellite-based estimation of clonal diversity and population subdivision in Zostera marina, a marine flowering plant. Mol. Ecol. 9: 127-140. for multiplexing. Fluorescently labelled PCR fragments were analysed on an ABI PRISM 3130 Genetic Analyser (Applied Biosystems) at CCMAR. The STRAND software (http://www.vgl.ucdavis.edu/informatics/strand.php) was used to score raw allele sizes. The R package MsatAllele (Alberto 2009Alberto F. 2009. MsatAllele_1.0: an R package to visualize the binning of microsatellite alleles. J. Hered. 100: 394-397.) was used to bin the allele sizes, and ambiguities were manually reviewed.

The genotype data were analysed to estimate the number of sampled genets (G) for comparison with the number of sampled ramets (N= sample size). The probability that identical multilocus genotypes (MLG) were produced by sexual recombination (Psex) rather than being clones was estimated using the GenClone software (Arnaud-Haond and Belkhir 2007Arnaud-Haond S., Belkhir K. 2007. GENCLONE: a computer program to analyse genotypic data, test for clonality and describe spatial clonal organization. Mol. Ecol. Resour. 7: 15-17. ). Genotypic diversity, the proportion of genets per population, was estimated following Dorken and Eckert (2001)Dorken M.E., Eckert C.G. 2001. Severely reduced sexual reproduction in northern populations of a clonal plant, Decodon verticillatus (Lythraceae). J. Ecol. 89: 339-350.: R = (G-1) ⁄ (N–1). We also analysed samples from before and after disturbance together in GenClone to infer whether we could find the same clones in both populations from different years.

Population genetic parameters were calculated after the removal of clone replicates for pre-disturbance and post-disturbance samples. The observed and expected heterozygosity (H_O; H_E) and the inbreeding coefficient (G_IS) were estimated using the GenoDive software (Meirmans and van Tienderen 2004Meirmans P.G., Van Tienderen P.H. 2004. GENOTYPE and GENODIVE: two programs for the analysis of genetic diversity of asexual organisms. Mol. Ecol. Notes 4: 792-794.) and G_IS were tested for significant difference from zero using a permutation test (Goudet 1995Goudet J. 1995. FSTAT (version 1.2): a computer program to calculate F-statistics. J. Hered. 86: 485-486.) to detect departure from Hardy-Weinberg equilibrium. Pairwise population differentiation (F_ST) was estimated and significance (difference from zero) was tested by permutations (AMOVA, Excoffier 1992Excoffier L., Smouse P.E., Quattro J.M. 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131: 479-491., Michalakis and Excoffier 1996Michalakis Y., Excoffier L. 1996. A generic estimation of population subdivision using distances between alleles with special reference for microsatellite loci. Genetics 142: 1061-1064.) using the same software (Meirmans and van Tienderen 2004Meirmans P.G., Van Tienderen P.H. 2004. GENOTYPE and GENODIVE: two programs for the analysis of genetic diversity of asexual organisms. Mol. Ecol. Notes 4: 792-794.).

Using the allele frequencies in the two samples (pre- and post-disturbance), we calculated the likelihood of an individual genotype being found in each of the samples (Paetkau et al. 1995Paetkau D., Calvert W., Stirling I., et al. 1995. Microsatellite analysis of population structure in Canadian polar bears. Mol. Ecol. 4: 347-354.), allowing us to assign the sample from which each post-recovery individual is most likely to come. Individual population assignment was inferred in Genodive (Meirmans and van Tienderen 2004Meirmans P.G., Van Tienderen P.H. 2004. GENOTYPE and GENODIVE: two programs for the analysis of genetic diversity of asexual organisms. Mol. Ecol. Notes 4: 792-794.) based on a Monte Carlo test with an alpha of 0.002 on 100 replicated datasets and 11400 resampled individuals.

RESULTSTop

Before and after meadow comparison

The seagrass meadow increased in area from 0.79 ha in 2007 to 1.12 ha in 2009. After disturbance in the winter of 2009/2010, seagrass cover was completely absent. Meadow recovery occurred throughout the next five years; in 2013 it had 0.57 ha and in 2015 it reached 1.47 ha.

In April 2010, the spring after disturbance, the number of seedlings was significantly higher than in any of the subsequent monitoring times (June 2010, September 2010 and July 2013) (Tukey HSD P<0.05 between all comparisons; Fig. 2). There were no differences in seedling density between June 2010, September 2010 and July 2013 (Tukey HSD P>0.05 between all comparisons). Seedling density was maximum in April 2010 (10.2 seedlings m^–2 ±2.71 SE), decreasing to 0.07 seedlings m^–2 (±0.04 SE) in June 2010 and no seedlings were detected in September 2010 and July 2013 (Fig. 2).

Shoot density also varied over time, with an opposite trend relative to that of seedling density. It changed from 288.48 shoots m^–2 (±19.30 SE) in July 2009 (before the disturbance), to total loss of shoots in February 2010. In that same year, in April we only observed seedlings, none of which had initiated clonal growth. By June 2010, shoot density was 12.62 (±3.91 SE) and by September it had nearly duplicated to 22.19 (±4.05 SE) shoots m^–2. Finally, the population reached densities similar to the pre-disturbance values in July 2013 (314.80 shoots m^–2 ±9.35 SE), representing 100% plant cover (Fig. 3).

There were significant shoot density differences between all dates (Tukey HSD P<0.05) with the exception of June and September 2010 (Tukey HSD P=0.73). Leaf length also increased significantly from April 2010 to July 2013, as shown by significant differences between all dates during the monitoring period (Tukey HSD P<0.05) (Fig. 4).

Population genetic structure

Different clones were identified in the samples collected before and after disturbance. The genotypic diversity estimated decreased from R=0.89 in 2008 to R=0.60 in 2013 (Table 1). Based on allelic frequencies, the individual population assignment test tagged only a single individual from the post-disturbance population as belonging to the pre-disturbance population.

Expected and observed heterozygosity were similar in both samples (permutation test, P=1) (Table 1). Although the inbreeding coefficient was similar between populations (permutation test, P=1) (Table 1) the G_IS of the pre-disturbance population revealed heterozygote deficiency (P=0.001), whereas the post-disturbance showed no deviation from Hardy-Weinberg equilibrium (P=0.134). The pairwise population differentiation fixation index, F_ST, between samples was 0.091 and not significantly different from zero (permutation test P=0.001).

DISCUSSIONTop

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. 2006Orth R.J., Luckenbach M.L., Marion S.R., et al. 2006. Seagrass recovery in the Delmarva coastal bays, USA. Aquat. Bot. 84: 26-36. ) 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 2010Jarvis J.C., Moore K.A. 2010. The role of seedlings and seed bank viability in the recovery of Chesapeake Bay, USA, Zostera marina populations following a large-scale decline. Hydrobiologia 649: 55-68., Kim et al. 2014Kim S.H., Kim J.H., Park S.R., et al. 2014. Annual and perennial life history strategies of Zostera marina populations under different light regimes. Mar. Ecol. Prog. Ser. 509: 1-13., Qin et al. 2016Qin L.Z., Li W.T., Zhang X., et al. 2016. Recovery of the eelgrass Zostera marina following intense Manila clam Ruditapes philippinarum harvesting disturbance in China: The role and fate of seedlings. Aquat. Bot. 130: 27-36.). Most of all, it shows that the contribution of sexual and clonal propagation is a population trait that is very variable (Rafajlović et al. 2017Rafajlović M., Kleinhans D., Gulliksson C., et al. 2017. Neutral processes forming large clones during colonization of new areas. J. Evol. Biol. 30: 1544-1560.), 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 2012Diekmann O.E., Serrao E.A. 2012. Range-edge genetic diversity: locally poor extant southern patches maintain a regionally diverse hotspot in the seagrass Zostera marina. Mol. Ecol. 21: 1647-1657.). 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 2012Diekmann O.E., Serrao E.A. 2012. Range-edge genetic diversity: locally poor extant southern patches maintain a regionally diverse hotspot in the seagrass Zostera marina. Mol. Ecol. 21: 1647-1657.).

Our study site is located in an extremely dynamic area, mainly due to strong tidal currents and storms during winter months (Paulo et al. 2019Paulo D., Cunha A.H., Boavida J., et al. 2019. Open coast seagrass restoration. Can we do it? Large scale seagrass transplants. Front. Mar. Sci. 6: 52. - Supplementary materials). In such environments, high sexual allocation by seagrasses is well documented (van Lent and Verschuure 1994van Lent F., Verschuure J.M. 1994. Intraspecific variability of Zostera marina L. (eelgrass) in the estuaries and lagoons of the southwestern Netherlands. I. Population dynamics. Aquat. Bot. 48: 31-58., Meling-López and Ibarra-Obando 1999Meling-López A.E., Ibarra-Obando S.E. 1999. Annual life cycles of two Zostera marina L. populations in the Gulf of California: contrasts in seasonality and reproductive effort. Aquat. Bot. 65: 59-69., Santamaría-Gallegos et al. 2000Santamaría-Gallegos N.A., Sánchez-Lizaso J.L., Félix-Pico E.F. 2000. Phenology and growth cycle of annual subtidal eelgrass in a subtropical locality. Aquat. Bot. 66: 329-339.). 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 2012Diekmann O.E., Serrao E.A. 2012. Range-edge genetic diversity: locally poor extant southern patches maintain a regionally diverse hotspot in the seagrass Zostera marina. Mol. Ecol. 21: 1647-1657.), 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 2001Reusch T.B.H. 2001. Fitness-consequences of geitonogamous selfing in a clonal marine angiosperm (Zostera marina). J. Evol. Biol. 14: 129-138.). 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 2012Diekmann O.E., Serrao E.A. 2012. Range-edge genetic diversity: locally poor extant southern patches maintain a regionally diverse hotspot in the seagrass Zostera marina. Mol. Ecol. 21: 1647-1657.). Although at the worldwide scale, eelgrass conservation status is of least concern, the current trend is for populations to decline (Short el al. 2010Short F.T., Carruthers T.J.R., Waycott M., et al. 2010. Zostera marina. The IUCN red list of threatened species. Version 2014.2.). The southern populations in particular are considered highly endangered and many have recently disappeared (Cunha et al. 2013Cunha A.H., Assis J.F., Serrão E.A. 2013. Seagrasses in Portugal: a most endangered marine habitat. Aquat. Bot. 104: 193-203.). 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. 2014Cunha A.H., Erzini K., Serrão E.A., et al. 2014. Biomares, a LIFE project to restore and manage the biodiversity of Prof. Luiz Saldanha Marine Park. J. Coast. Conserv. 18: 643-655., Paulo et al. 2019Paulo D., Cunha A.H., Boavida J., et al. 2019. Open coast seagrass restoration. Can we do it? Large scale seagrass transplants. Front. Mar. Sci. 6: 52.). 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 2010Tanner C.E., Parham T. 2010. Growing Zostera marina (eelgrass) from seeds in land-based culture systems for use in restoration projects. Rest. Ecol. 18: 527-537., Marion and Orth 2010Marion S.R., Orth R.J. 2010. Innovative techniques for large-scale seagrass restoration using Zostera marina (eelgrass) seeds. Restor. Ecol. 18: 514-526.). 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. 2014Jarvis J.C., Moore K.A., Kenworthy W.J. 2014. Persistence of Zostera marina L. (eelgrass) seeds in the sediment seed bank. J. Exp. Mar. Biol. Ecol. 459: 126-136.). 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. 2017Rafajlović M., Kleinhans D., Gulliksson C., et al. 2017. Neutral processes forming large clones during colonization of new areas. J. Evol. Biol. 30: 1544-1560.). 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.

ACKNOWLEDGEMENTSTop

This work received national funds through FCT - Foundation for Science and Technology through the projects CCMAR/Multi/04326/2013 and Extant and a doctoral fellowship SFRH/BD/81086/2011 to Diogo Paulo. The authors wish to thank Cristina Paulino and Marta Valente for the help in genotyping and Dr Joana Boavida and Sandra Rodrigues during the field work.

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