Growth and Development of Rhizophora spp. Seedlings on Different Substrates and Insertion Level in the Wouri Estuary Mangrove (Douala , Cameroon)

Mangroves are threatened with extinction worldwide. Many mangrove reforestation projects have been developed, but very few have achieved their restoration objectives. With the ambitions to contribute for rehabilitation of mangrove ecosystems, the aim of this study was to assess the effects of substrate composition and level of insertion on the growth and development of Rhizophora seedlings in nurseries. The experiment was conducted in a semi-lighted nursery set up in situ. Rhizophora propagules were reared on three types of mangrove substrates (type 1: 75% of mud and 25% of sand; type 2: 50% of mud and 50% of sand, and type 3: 25% of mud and 75% of sand) with different levels of insertion (at ¼; ½ and ¾). Seedling heights and diameters were recorded, as well as the time of appearance of the first leaves and the variation of number of leaves. The greatest average height (40.2 ± 7.48 cm) and the highest mean diameter (1.2 ± 0.01 cm) were obtained for the substrate composed of 75% mud and 25% sand. The best growth and development of seedlings were recorded on the substrate type 1 with an insertion at ¼. Seedling production at ¼ insertion of substrate type 1 appeared the most effective combination. The production of Rhizophora seedlings on the substrate combining 25% Sand and 75% Mud with an insertion level at 1/4 seems to be the best method for the complete restoration and rehabilitation of the mangrove ecosystem of the Wouri estuary.


INTRODUCTION
Numerous projects to restore severely degraded mangroves are being implemented around the world [Djamaluddin et al., 2019;Leal and Spalding, 2022].These projects involve the reforestation of native species and the study of various techniques related to habitat recovery, such as propagule maintenance, seedling production, assessment of the optimum planting and seedling transplanting period, planting procedures, crop and plantation monitoring, assessment of environmental factors, in particular light, temperature and edaphic parameters [Ellison et al., 2020;Silva & Maia, 2019].
In Africa, some projects to restore damaged mangroves have been reported in several countries, including Benin, Cameroon, Côte d'Ivoire, Kenya, Mozambique, Nigeria, Senegal, and Tanzania [Bocquet, 2018;Egnankou, 2009 Since some years now, mangroves in Cameroon have been the subject of several studies as: descriptive studies on biological and socioeconomic potential [Nfotabong-Atheull et al., 2013]; highlight their ecological role in protecting the coast and synthesising approaches, results, challenges and constraints from many other studies [Ajonina et al., 2016].A number of reforestation projects have been launched to restore mangroves in Cameroon, including CAMERR Project Restoration of mangrove ecosystems.Started on 2020, the project aims to restore 1000 hectares of mangroves and the objective is to support the restoration and sustainable management of the mangrove, the protection of biodiversity and the strengthening of local resilience around the coastline of Douala city to thus contribute to the resilience of ecosystems and residents of the mangrove periphery.
A decade before the above project, funded by the International Tropical Timber Organisation (ITTO), the Participatory Rehabilitation and Management of Mangroves and Watersheds in the Coastal Zone of the Douala-Edea National Park, abbreviated to the "Douala-Edea Mangroves Project", was submitted by the Government of Cameroon, initiated and implemented by the Cameroon Ecology (Cam-Eco) carried out between 2010-2013.
Despite a long tradition of mangrove restoration, it is still difficult to ensure the success of restitution.However, the natural development conditions are optimal, and seeds are abundant and available in all seasons [Rovai et al.,  Most projects involve planting Rhizophora spp.propagules in nurseries, but few of these projects have detailed monitoring and followup plans, and in most cases little documentation or recommendations for modifications to the original planting plan exist [Lee et al., 2019;Kodikara et al., 2017].
With the ambitions to contribute for restoration of mangrove ecosystems through reforestation, a preview work was carried out to evaluate the impact of light and conservation period of propagules on seedling growth of Rhizophora spp.[Boubakary et al., 2019].This paper reports the follow of preview work and the aim is to examine the effect of substrate composition and the level of insertion on the growth and development of seedlings of the genus Rhizophora.
The Douala 1 st site was chosen for reasons of equipment safety and the Douala 3 rd sites because the lush vegetation and the robustness of the trees that suggest a favorable environment for mangroves ecosystem health and the abundance of propagules.Propagules were collected under various trees.The climate of the region belongs to the equatorial domain of a particular type or Cameroonian.It is characterized by a long rainy season (March -November) with abundant rainfall (4000 mm per year) and a short dry season (December -February), high and stable temperatures (26.7 °C), and high humidity throughout the year with a maximum always close to 100% [Din et al., 2008]

Data collection
A total of 360 Rhizophora propagules were harvested, sorted, and stored in situ for seven days.Simultaneously, a space was prepared, a nursery was built, and substrates were prepared during the low tide.Two batches of 180 healthy propagules measuring 25-40 cm in height and 1.1-1.2cm in diameter were used in experiments 1 and 2.

Experience 1: Substrate preparation and seedlings raise
The substrates used were mangrove mud and fine sand extracted in situ [Costa et al., 2016].The use process involved quantitative mix of mangrove sand and mud from three combinations: • a substrate mix of 75% sand and 25% mud; • a substrate mix of 50% sand and 50% mud; • a substrate mix of 25% sand and 75% mud.
Each type of compound substrate was then placed in 60 plastic bags, each 25 cm wide and 35 cm high, and arranged in batches in a semiilluminated nursery.

Experience 2: Impact of insertion level
By referring to the optimal condition of propagules storage, the optimum light condition for seedlings in the nursery (semi-lighted) and on the best mineralogical substrate composition three (03) trials were used to determine the best level of insertion of propagules in the substrate [Boubakary et al., 2019].
A substrate mix composed of 25% sand and 75% mud was introduced into 180 bags (25 cm wide and 35 cm long each).The substrate-filled bags were arranged in three batches of 60.The propagules were inserted into the substrate at specific depths of 1/4, 1/2 and 3/4 of their height, respectively, within each group of pots.The arrangement of the seedlings in the nursery was the same as in the previous experiment.
The nursery site was located in the estuary, along a channel that was neither too high nor too low, ensuring that the plants were regularly submerged during high tide [Fontalvo-Herazo et al., 2011].The dominant species was Rhizophora racemosa Meyer (Fig. 2).Biological growth parameters were recorded, including the appearance of the seedlings, the time taken for the first leaf to appear, the variation in the number of leaves with time.The height of seedlings using a tape measure, and both the base and mid-height diameters of the seedlings using a caliper.In addition, predator activity was also observed.These parameters were recorded weekly for a period of two months.

Data analysis
The "r commander" package of R software version 4.1.3was used to perform descriptive statistic of data collected.The Student's parametric test was used to compare the average of heights and diameters among seedlings.Excel software 2013 was used for graphical representations.

Height growth of seedlings
Student's parametric test showed a significant difference between the mean heights of Rhizophora seedlings produced on the three types of substrates tested (P = 0.064).The mean heights of the seedlings were unevenly distributed over the three types of substrates as a function of time.The greatest average height (40.2 ± 7.48 cm) was obtained for the substrate composed of 75% mud and 25% sand and the lowest (38.76 ± 5.55 cm) for the substrate composed of 25% mud and 75% sand (Figure 3).
Seedling growth is linked to substrate type.The relationship between growth in height, growth in diameter and substrate type is strong for all the combinations tested.For the substrate of 75% mud and 25% sand, the correlation coefficient (R²) is 0.9406 and the linear equation is y = 2.6513x + 29.417.For the substrate composed of 50% mud and 50% sand, the correlation coefficient (R²) is 0.9631 and the linear equation is y = 1.9838x + 31.704.For the substrate of 25% mud and 75% sand, the correlation coefficient (R²) is 0.9653 and the linear equation is y = 1.993x + 30.795.The correlation coefficients are close to 1 in all three types, showing the strong link between seedling height and substrate type (Figure 4).
Analysis of variance showed that substrate type had a significant influence on height growth, diametric growth and leaf development in Rhizophora spp.seedlings (Table 1).

Seedling diameter growth
Student's parametric test showed a significant difference between the mean seedling diameters according to the substrate types (P= 0.064).The mean diameters of the seedlings were unequal on the three types of substrates tested.The highest mean diameter (1.2 ± 0.01 cm) was recorded on the substrate of 75% mud and 25% sand and the lowest mean diameter (1.18 ± 0.02 cm) on the substrate of 25% mud and 75% sand (Figure 5).The growth in diameter of the seedlings is linked to the type of substrate.For the substrate of 75% mud and 25% sand, the correlation coefficient (R²) is 0.9449 and the linear equation is y = 0.0067x + 1.1789.For the substrate composed of 50% mud and 50% sand, the correlation coefficient (R²) 0.6998 and the linear equation is y = 0.0052x + 1.1682.On the substrate composed of 25% mud and 75% sand, the correlation coefficient (R²) 0.8963 and the linear equation is y = 0.0073x + 1.1507.In all, the analysis shows that diametric growth depends on the type of substrate on which the seedlings develop.The correlation is strongest for the substrate of 75%   mud and 25% sand, with an R² value of 0.9449, which is closest to 1 (Fig. 6).

Leaf development according to substrate type
The appearance of leaves in the seedlings was uneven across the different substrates and observation time.In all batches of propagules that were harvested, collected and transplanted without prior preservation, no propagule showed a leaf after two weeks on the three substrate combinations.However, the appearance of leaves was recorded on all batches from the third week onwards, with a higher rate (40%) for the substrate of 75% mud and 25% sand and a lower rate (20%) for the substrate of 25% mud and 75% sand.In the fourth week, 53% of the seedlings for the substrate of 25% mud and 75% sand had 4 leaves, 61.66% of the seedlings on the substrate of 50% mud and 50% sand had 4 leaves and 55% of the seedlings for the substrate of 25% mud and 75% sand bore 4 leaves.In the fifth week, 93.33% of the seedlings on the 25% mud and 75% sand substrate had at least 4 leaves, 90% of the seedlings for the 50% mud and 50% sand substrate had at least 4 leaves and 98.33% of the seedlings on the 75% mud and 25% sand substrate also had at least 4 leaves.By the sixth week, all seedlings were bearing at least 4 leaves.By the seventh week, 11% of seedlings on the 25% mud and 75% sand substrate had 6 leaves, 5% on the 50% mud and 50% sand substrate and 20% on the 75% mud and 25% sand substrate.By the eighth week, 66.66% of seedlings for the 25% mud and 75% sand substrate had 6 leaves, 70% on the 50% mud and 50% sand substrate and 91.66% for the 75% mud and 25% sand substrate (Table 2).Among the three types of substrates analysed, the substrate of 75% mud and 25% sand was more favourable to leaf development.

Relationship between diameter and height growth of seedlings and substrate type
For the three substrates tested, diametric growth is linked to the seedling growth in height.The correlation coefficient R² = 0.9709867 is stronger on the substrate of 75% Mud and 25% Sand and is closest to 1.The characteristics of the substrate therefore determine the life of the mangrove community (Table 3).

Effect of insertion level in the substrate on the growth and development of Rhizophora spp. seedlings
Analysis of variance shows that the level of insertion of the propagules in the substrate has a very significant influence on the growth in height of the seedlings; the same observation is pertinent for growth in diameter and leaf development (Table 4).

Mortality
The mortality rate of seedlings over eight weeks was observed as follows: • 6.66% in the group of propagules with ¼ of their height inserted in the substrate; • 15% in the group of propagules with ½ of their height inserted in the substrate; • 33.33% in the group of propagules with ¾ of their height inserted into the substrate.

Height variation of seedling according to propagule insertion level
The growth of the seedlings was influenced by the depth of insertion of propagules in the substrate.In eight weeks, the seedlings showed different heights depending on the propagule's insertion level in the substrate.The highest mean height (44.43 ± 6.59 cm) was recorded in the batch of propagules inserted at ¼ into the substrate; the lowest mean height (20.19 ± 8.30 cm) in the batch of propagules inserted at ¾ into the substrate.For the batch of propagules inserted at ½ the average height recorded over eight weeks is (35.58 ± 6.70) (Figure 7).Plantlet height growth is related to the insertion level of propagules into the substrate.Propagules inserted at ¼ grow faster (Figure 8A).The linear equation for growth is y = 2.532x + 34.639 and the correlation coefficient R² = 0.9455.Propagules inserted at ½ grow normally, the linear equation for growth is: y = 2.3467x + 26.196 and the correlation coefficient is R 2 = 0.9173.Propagules inserted at ¾ on the other hand, grow slowly.The linear equation for growth is y = 1.1945x + 8.8853 and the correlation coefficient R² = 0.9633 (Figure 8C).In all cases, growth in height is strongly linked to the level of propagules in the substrate with correlation coefficients close to 1.

Evolution of seedling diameters according to insertion level propagule
As with height growth, the diametric growth of seedlings is unevenly distributed as a according to time and insertion level of propagules in the substrate.Deeper the propagule is slower it grows.The largest average diameter (1.13 ± 0.06 cm) was obtained in the batch of propagules inserted at ¼.The lowest average diameter (0.77 ± 0.01 cm), is obtained in the batch of propagules inserted at ¾.For the batch of propagules inserted at ½ in the substrate, the average diameter obtained in eight weeks is (0.95 ± 0.02 cm) (Figure 9).
The diametric growth of seedlings is related to the level of insertion of the propagule in the substrate.For propagules inserted at ¼ in the substrate, the correlation coefficient R² = 0.6157 and the linear equation y = 0.0098x + 1.0851; growth was rapid for the first four weeks, then a slowdown is observed for three weeks and an acceleration from the seventh week onwards (Figure 10A), whereas in propagules inserted at ½ in the substrate, diametric growth was rapid for the first three weeks and slowed for a fortnight before continuing.The linear equation is y = 0.0092x + 0.9189 and the correlation coefficient (R² = 0.9298) indicates that the relationship is very strong (Figure 10B).For the propagules inserted at ¾ in the substrate, diametric growth is rapid for the first two weeks then slows from the fifth week before resuming at the seventh week.The linear equation is y = 0.0053x + 0.7487 and the correlation coefficient (R² = 0.8533) indicates that the relationship is strong (Figure 10C).

Relationship between diameter growth and height growth of seedlings at different insertion levels
Diametric and height growth of seedlings are linked to the insertion levels of propagules into the substrate.The strongest correlation was observed for propagules inserted at ½ into the substrate, followed by propagules inserted at ¾ and the lowest for propagules inserted at ¼ (Table 5).

Collar height
Average collar heights were constant throughout the period during which the seedlings were observed in the nursery.No changes were observed (Figure 11).

Leaf development according to insertion level of propagules
The appearance of leaves was recorded from the third week in the three different batches of seedlings.The leaf appearance rate was 40%, 31.66% and 20% respectively for propagules inserted at ¼, ½ and ¾.At the fifth week, seedlings in all batches had at least 2 leaves and 4 leaves at the sixth and seventh week.At the eighth week, 61.66% of the seedlings transplanted at ¼ had 6 leaves, 6.66% of the seedlings inserted at ½ in the substrate had 6 leaves and none of the seedlings in the batches of seedlings inserted at ¾ had 6 leaves (Table 6).Inserting seedlings at ¼ in the substrate is more favourable for leaf development.

Development of branches in seedlings
Branching development in seedlings was influenced by the different depths of propagules insertion in the substrate.The highest percentage of branching in seedlings (62.5%) was obtained after eight weeks of observation for propagules at ¼ in the substrate.For propagules inserted at ½ the percentage of branching was 58.82% and the lowest percentage (2.5%) was recorded for propagules inserted at ¾ (Figure 12).The production of Rhizophora seedlings by insertion of propagules at ¼ is more favorable to branching development.

DISCUSSION
Rhizophora spp.seedlings on different type of substrates and insertion level showed significant difference.Jumawan et al. [2015] showed that the soil's quality affects largely the diversity of mangroves.The ability of soils to retain C, water and nutrient ions are strongly influenced by the soil texture [Havlin et al., 2014].Basically, soil texture is determined through the particle size percentage of clay, silt and sand [Ashman and Puri, 2002].Mangrove plants may grow in different types of soil reason why Jorquia [2022] recommends more essential that planters consider the type of substrate in planting mangroves.
In this work, the seedlings of Rhizophora spp.showed good growth and development on all the types of substrates tested in nursery, with the best results on the substrate composed of 75% Mud and 25% Sand.The combination of 75% Mud and 25% Sand forms an excellent substrate.The results obtained are similar to those of Costa et al. [2016] who examined the growth and survival of Rhizophora spp., Avicennia and Laguncularia seedlings on three different types of substrates in nursery on the coast of Brazil.Dewiyanti et al.
[2023] obtained the different results in the station where the substrate was composed of: 84.6 % Sand, 10.2 % Dust and 5.1 % Clay.The research was carried out at mangrove rehabilitation.The highest value of stem height and stem diameter measured was at station 1, where the average value of stem height reached 1.22 cm per week and stem diameter was 0.25 mm per week.The lowest values for measuring stem height, and stem diameter were at stations 3 with a stem height value of 0.57 cm per week, and stem diameter 0.12 mm per week.Bocquet [2018] investigated on the significant impact of muddy substrate on seedling growth and development.According to these authors, the physical and chemical characteristics of muddy substrates allow the retention of moisture and nutrients required by seedlings.The soil pores are different for clay, silt and sand.For clay, the soil pores are the smallest when compared to silt and sand.Hence, this type of soil has a higher ability to retain water and nutrients [Ashman and Puri, 2002].
In mangrove forest, the nutrients are supplied by the litter fall from trees and suspended materials from surface runoff above ground, fauna activities and microorganisms' decomposition mechanism [Cannicci et al., 2008].The litter fall or surface materials were utilized by macro invertebrates such as Scylla serrata and that is broken down into smaller sizes.Then, the process was taken over by microorganisms until all the materials deposit into the soil permanently.
The substrate factor also has a significant effect on the number of leaves, depending on the different substrates tested.There was a significant effect of composite substrates on the parameters measured.The number of leaves at the end of  Dewiyanti et al. [2023] from the station 1, 2, and 3 were 21, 25, and 5 strands.In all study stations, the average addition of leave was one strands, Delvian et al. [2019], reported that at twelve weeks of observation, the highest number of leaves was 7 strands.Parameters of water physics, water chemistry, and substrate are environmental factors that affect mangrove growth [Wantasen, 2014].Nursinar et al. [2023] showed that the number of Rhizophora apiculata leaves that were sown every month increased and the number of leaves increased by one leaf every month.
However, it is important to remember that Rhizophora spp.propagules are able to develop and grow independently of the substrates tested because according to Windusari et al. [2014], mangroves develop in mud clay (silt) mixed with organic matter, but they are also found in areas with peat or substrate with a high sand content.An adequate insertion height of propagules in substrate of precise composition is essential for the rapid growth and development of seedlings of the genus Rhizophora.The production of seedlings in a nursery by insertion at ¼ in the substrate would seem to be a better approach.These results are in line with those of Poveda and Guiraud [2014] who proved during reforestation that, propagules should be planted neither deep nor too much on the surface and oppose those of Nakouzi et al. [2018] in the Practical guide to the production and planting of mangrove species in Benin (Cotonou) who found that propagules should be sunk 1 / 3 straight into the mud for best results.The difference in results could be linked to the edaphic condition of the experimental environment in which this work was carried out, opposite to the edaphic condition of the natural environment.
Mortality of species of the genus Rhizophora observed in mangroves is due to several factors, including intra and interspecific competition, diseases, herbivores, predation and the natural senescence of trees.It should be noted that all stages of development are affected, including propagules, young trees and adult trees.However, mangroves in the early stages of development experience higher mortality, and small seedlings are more at risk because of competition with larger trees for light and/or nutrients.
Mortality of mangrove seedlings is influenced by several factors, including intra and interspecific competition, diseases, herbivores, predation and the natural senescence of trees propagules, young trees and adult trees and level of insertion into the substrate; according to Jimenez and Lugo [1984].The observations of these authors are similar to those made in the context of this work for batch of seedlings resulting from the insertion of propagules at ¾ in the substrate.The high mortality rate observed in the range of propagules inserted deep into the substrate reflects the poor adaptability of the seedlings at this level of insertion [Poveda and Guiraud, 2014].In the group of seedlings inserted slightly into the substrate the mortality rate was low, reflecting the favourable condition for the survival of seedlings of the genus Rhizophora.
The viability of reforestation depends on duration and ecological conditions.Plant growth and development parameters are not constant over time.In the work carried out in the mangroves of the Wouri estuary, the growth in diameter and height of the seedlings, the development of primary and secondary branches and stilt roots were faster for the batch of seedlings resulting from the insertion of propagules at ¼ in the substrate and very slow for the batch of propagules inserted deep in the substrate.The observed variability could be explained by the individual or combined effects of environmental parameters such as propagule retention time, the effect of light, the effect of substrate composition, and the level of insertion of propagules into the substrate, the salinity of the environment [Jorquia, 2022].The results obtained as part of the work carried out in the mangroves of the Wouri estuary are similar with those of Bocquet [2018] obtained at the end of the ecological assessment of reforested mangroves in the Saloum delta in Senegal.

CONCLUSIONS
The type of substrate use and the insertion level of propagule affect seedling germination and growth of Rhizophora spp.These are therefore important to consider in the ecological restoration of mangroves, which are essential ecosystems for climate regulation, coastal protection and biodiversity.Ideal substrate and insertion depth are two important factors that affect the survival and growth of mangrove seedlings or propagules in restoration projects.The ideal substrate is usually soft, muddy, and rich in organic matter, while the insertion depth is the depth at which the seedling or propagule is planted in the substrate.
Practical limitations related to the ideal substrate and insertion depth may include the availability and accessibility of suitable sites for mangrove restoration, which may be constrained by land use, ownership, and governance issues.Furthermore, the variability and uncertainty of environmental conditions, such as salinity, tidal inundation, and storm events, that may affect the suitability of the substrate and the survival of the seedlings or propagules.It's also important to indicate the trade-offs and synergies between different restoration objectives, such as carbon sequestration, coastal protection, and biodiversity conservation, that may require different substrate types and planting densities.We can also add the costs and benefits of different restoration methods, such as direct planting, natural regeneration, or assisted natural regeneration, that may have different impacts on the substrate and the insertion depth.
. The tides are semi-diurnal with amplitudes ranging from 1.35 to 3 m [Nfotabong-Atheull et al., 2013].The soil of the region is characterised by a hydromorphic horizon that is generally sandymuddy in texture and dark grey in colour [Balzer et al., 1995].

Figure 1 .
Figure 1.Location of the study sites

Figure 2 .
Figure 2. Seedlings in nursery in situ: (a) at low tide; (b) at high tide

Figure 3 .
Figure 3. Variation in seedling heights according to the substrates: S -sand, M -mud

Figure 5 .Figure 6 .
Figure 5. Mean variation of seedling diameters according to substrate type and time: S -sand, M -mud

Figure 7 .Figure 8 .
Figure 7. Variation in mean plantlet height seedlings according to insertion levels

Figure 9 .Figure 10 .
Figure 9. Variation of seedling diameters depending to insertion levels and time

Figure 11 .
Figure 11.Average crown height of seedlings in nurseries
2012; Sarwom et al., 2022].The failures observed in mangrove reforestation are linked with preplanting, planting and post-planting procedures [Lewis et al., 2019; Mukherjee et al., 2015].Among main failures, there are the poor use of ecological knowledge describing the conditions in which mangroves thrive and insufficient knowledge of the physical disturbances linked to the geomorphological processes of mangrove ecosystems [Balke et al., 2013; Elliott et al., 2016; Laurenda et al., 2022; Rovai et al., 2012], lack of involvement of local communities, inappropriate choice of species and plantation sites, and inadequate monitoring of plantations.Some projects fail to meet technical criteria such as substrate composition and the degree to which propagules of Rhizophora spp.are inserted when setting up nurseries.

Table 1 .
Analysis of variance of seedling growth on different types of substrates

Table 2 .
Leaves variation according to time and substrate composition

Table 3 .
Correlation between diameter and height of seedlings on different substrates

Table 4 .
Analysis of variance of the growth of seedlings at different level

Table 5 .
Diameter and height of seedlings at different insertion levels

Table 6 .
Average percentage of seedlings with leaves as a function of time and level of insertion of seedlings in the substrate