Salinity and temperature significantly influence seed germination, seedling establishment, and seedling growth of eelgrass Zostera marina L

Abstract

Globally, seagrass beds have been recognized as critical yet declining coastal habitats. To mitigate seagrass losses, seagrass restorations have been conducted in worldwide over the past two decades. Seed utilization is considered to be an important approach in seagrass restoration efforts. In this study, we investigated the effects of salinity and temperature on seed germination, seedling establishment, and seedling growth of eelgrass Zostera marina L. (Swan Lake, northern China). We initially tested the effects of salinity (0, 5, 10, 15, 20, 25, 30, 35, and 40 ppt) and water temperature (5, 10, 15, and 20 °C) on seed germination to identify optimal levels. To identify levels of salinity that could potentially limit survival and growth, and, consequently, the spatial distribution of seedlings in temperate estuaries, we then examined the effect of freshwater and other salinity levels (10, 20, and 30 ppt) on seedling growth and establishment to confirm suitable conditions for seedling development. Finally, we examined the effect of transferring germinated seeds from freshwater or low salinity levels (1, 5, and 15 ppt) to natural seawater (32 ppt) on seedling establishment rate (SER) at 15 °C. In our research, we found that: (1) Mature seeds had a considerably lower moisture content than immature seeds; therefore, moisture content may be a potential indicator of Z. marina seed maturity; (2) Seed germination significantly increased at low salinity (p < 0.001) and high temperature (p < 0.001). Salinity had a much stronger influence on seed germination than temperature. Maximum seed germination (88.67 ± 5.77%) was recorded in freshwater at 15 °C; (3) Freshwater and low salinity levels (< 20 ppt) increased germination but had a strong negative effect on seedling morphology (number of leaves per seedling reduced from 2 to 0, and maximum seedling leaf length reduced from 4.48 to 0 cm) and growth (seedling biomass reduced by 46.15-66.67% and maximum seedling length reduced by 21.16-69.50%). However, Z. marina performed almost equally well at salinities of 20 and 30 ppt. Very few germinated seeds completed leaf differentiation and seedling establishment in freshwater or at low salinity, implying that freshwater and low salinity may potentially limit the distribution of this species in coastal and estuarine waters. Therefore, the optimum salinity for Z. marina seedling establishment and colonization appears to be above 20 ppt in natural beds; (4) Seeds germinated in freshwater or at low salinity levels could be transferred to natural seawater to accomplish seedling establishment and colonization. This may be the optimal method for the adoption of seed utilization in seagrass restoration. We also identified seven stages of seed germination and seedling metamorphosis in order to characterize growth and developmental characteristics. Our results may serve as useful information for Z. marina habitat establishment and restoration programs.

Keywords: Salinity; Seagrass; Seed germination; Seedling establishment; Seedling growth; Temperature; Zostera marina L..

Figure 1. Germination rates (%) of Zostera marina L. seeds subjected to the different salinities and temperatures after four weeks.

Different letters above error bars indicate significant differences at p < 0.05 (one-way analysis of variance) (mean ± standard error (SE), n = 3) (bars represent SE).

Figure 2. Seed germination, length, and weight of Zostera marina L. seedlings subjected to different salinities at 15 °C after three and six weeks.

Different letters indicate significant differences at p < 0.05 (one-way ANOVA) (means ± SE, n = 3).

Figure 3. Morphology of Zostera marina L. seedlings after six weeks (bar represents 1 cm) at 5 °C.

The germinated seeds were incubated in freshwater (A), 10 ppt (B), 20 ppt (C), and 30 ppt (D). (A) Germinated seeds with a cotyledon exceeding 1 cm; (B) Cotyledon exceeds 3 cm; (C) Leaf and adventitious root differentiation with the leaf length exceeding 4 cm; (D) Leaves exceed 5 cm in length.

Figure 4. Seedling establishment rate for Zostera marina L. seeds germinated at reduced salinities and transferred to natural seawater (32 ppt).

Different letters indicate significant differences at p < 0.05 (mean ± standard error).

Figure 5. Metamorphosis of Zostera marina L. seeds and seedlings during and after germination.

(A) Stage 0: a mature seed coated with a dark brown seed coat. (B, C) Stage 1: seed germination. Arrowed lines indicate portions of the sectioned embryo: CB, cotyledonary blade and CS, cotyledonary sheath, respectively; AH, axial hypocotyls and BH, basal hypocotyls, respectively. (D) Stage 2: pre-seedling establishment stage in which germinated seeds have no true leaf or adventitious roots. (E) Stage 3: initial stage of a seedling with first true leaf (L1) differentiation at the base of the cotyledonary blade. (F) Stage 4: seedling with adventitious root (AR) differentiation at the base of the cotyledonary sheath. (G) Stage 5: seedling with second true leaf (L2) differentiation at the base of the cotyledonary blade. (H) Stage 6: intact seedling lacking cotyledonary blade.