Diatom communities in marine protected areas in the coral reefs in Lombok, Indonesia, using aqueous and sedimentary environmental DNA (eDNA)

. Diatom has a specific response to particular ecological conditions. They live both planktonic and benthic, which is usually the diversity that has only been documented by microscopic. The information about the diversity of Diatoms is critical as a baseline study for biomonitoring. Thus, in this study, we applied environmental DNA through a next-generation sequencing method to analyze planktonic and benthic communities of Diatom in marine protected areas in Lombok Waters, Indonesia. The result shows that benthic Diatom has more richness than planktonic but lacks significance between spatial. In our study, we detected 25 species of Diatom in West and East Lombok. Diatom from sediment samples has more richness than water, with four species only found in the Water samples and 18 species in the sediment samples. Three species were found both in the water and sediment samples. The highest abundance in the Stramenopiles clades is Diatom. Relative abundance of diatom about 12% to 23.91% from total micro eukaryotic or about 85.71%tol 86.14% from total Stramenopiles clades for East Lombok and West Lombok, respectively. Limitations of the genetic reference database, especially for tropical causes, restricted the determination of genetically identified sequence types at both genus and species levels.


Introduction
The general approach to assessing spatial variation in species composition is a fundamental feature of sustainable resource management.In conservation, measures of compositional and abundance of species for evaluating diversity and similarity are essential.That information is used to study the homogenizing or diversifying effects of human activities, spatial variability, or natural disturbances [1].Information about the diversity of diatom species in the water and sediment is valuable.It allows the monitoring of ecosystem conditions, which is called biomonitoring.It is a crucial component for sustainable management in the aquatic ecosystem [2].In the ocean, assessment of diversity challenges scientists because most of them have limited size ranges and are difficult to identify.Recently, molecular methods have been used to detect organisms in the water and sediment, and it is an alternative method [3] to morphological identification, which is limited in resolution size [4].
Diatom are classified as microeukaryote communities found in almost all environmental conditions and play an important role in marine ecosystems in the cycles in the bacterial loop and food web as primary producers, predators, and parasites [5,6,7].Diatoms are also found in the benthic substrates [8], floating debris, and on macrophytes.Environmental conditions are affected by phytoplankton community composition; depth gradient from the water column to sediment in the marine have different environmental conditions that influence microeukaryotes community composition [7,9,10,11,12] and diatom composition.It is very challenging for scientists to reveal the relationship between environmental conditions and microbial community composition, which are determined by the biogeographical patterns [7,13,14,15].
On the other hand, identification of diatom until level species is challenging, for species identification of diatom by morphology is relatively complicated and requires expertise.Molecular identification is an alternative method to reducing human bias [16,17].Environmental DNA (eDNA) is a molecular identification method that can analyze biodiversity in the ecosystem as an alternative and effective method for biomonitoring.The eDNA is a complex mixture of genomic DNA from marine organisms found in the environment samples: water, sediment, etc. [18,19].It allows the detection of many kinds of organisms at the same time, depending on the target gene and its primer.
This study used primer 1389F and 1510R [20] to detect novel microbial eukaryotic organisms using V9 hypervariable regions of small-subunit (SSU) rRNA genes, the target 18s rRNA gene.The primer is used to detect eukaryotic communities, which the diatom is part of.The studies about identifying microbial eukaryote communities, mainly diatom, are limited.On the other side, diatom is important for biomonitoring water quality and bioassessment for the ecosystem [21,22].Thus, the research aims to describe Diatoms from water and sediment in marine protected areas in Lombok using environmental DNA (eDNA).
For the water sample, we collected from surface water.We collected the sediment sample from the bottom or ocean floor at about 15 m depth and mixed it with the water at the bottom for the sediment sample that the diving collected.About 4 liters of water samples in each location were collected into bottle samples and filtered using a peristaltic pump and through some step filtration.First, the water samples were filtered by an 80 µm pore size sieve to remove grain, large metazoan, and debris; filtration by 14 µas m pore size and, the last filtration used a paper filter with 0.4 µm pore size.Lastly, the samples were transported to the Biodiversity Laboratory at IPB University for DNA extraction, PCR, and electrophoresis.
Thermal cycling for DNA amplification consisted of pre-denaturation at 94 0 C for three minutes, denaturation at 90 0 C for 45 seconds, and annealing at 60 0 C for 30 minutes, with a final 5-minute extension at 72 0 C. PCR products were sent to the University of Rhode Island for sequencing.The sequencing process used next-generation sequencing (NGS), the Illumina Miseq platform.

Bioinformatics analysis
Raw data (fastq files) as sequencing results were analyzed using bioinformatics.We used Cutadapt 1.15 [27,28] to trim the primers and remove the adapter and index sequences from \raw reads; Cutadapt [27,28] was also used for demultiplexing and single-end and pairedend reads [26] PCR-amplified metagenome data then denoised by Dada2 [27,28].It allows for reduced errors, dereplicating sequences, removing low-quality reads, filtering chimeras, and merging forward and reversed reads [27,28].Truncation parameters in this study were decided using visual assessment from read quality plots where truncation was performed when read quality dropped off [28].In addition, we used MAFFT for the sequence alignment or perform de novo multiple sequence alignment [29].MAFFT is a Multiple sequence alignment program that is important in biological sequences, a similarity-based method.Moreover, to construct or build a phylogenetic tree FastTree [30].All that process for bioinformatics analysis of raw data in QIIME 2 version 2019.10 [31].This study used the SILVA 132 database 18S rRNA as a reference database for taxonomic assignment.The process for bioinformatic analysis using the command line in the conda and QIIME2 environment.On the other side, some feature ID that represent OTU were not detected until species.Thus, we collaborate between SILVA and BLASTn in GenBank (NCBI) for identification to a lower taxonomic level.We separated feature ID into unassigned sequences or those only assigned to phylum, family, and genus level, then blast it using BLASTn to identify until species level.In addition, we also used a two-way ANOVA test to analyze the difference of assemblage diatom along spatial (Management areas) and environment (water and sediment) based on frequency of sequence.

Results and discussion
A total of 12 samples have been analyzed from water samples and sediment.Diatoms are organisms found in the waters as debris, sediment, and on the macrophyta.But in this study, we only focus on diatom that lives in the environment samples such as water and sediment.In this study, every representative sequence has been categorized by feature ID, which means not all sequences could be described into species.Some Diatom in West Lombok is similar in East Lombok based on feature ID, which represents a unique sequence and no significant difference between Diatom in West and East Lombok.However, the representative sequence from East Lombok is higher than in West Lombok.Conversely, richness and assemblages between benthic and planktonic diatom are different (p<0.05) in the West and East Diatom.
In addition, planktonic diatom from West and East Lombok is dominated by the genus Chaetoceros.The relative abundance of Chaetoceros in the water was about 28% to 48%, with a sequence frequency of about two to 26 sequences, respectively.For benthic diatom, the relative abundance of Amphora is about 2%, Chaetoceros 7%, Minutocellus 4%, and two taxa below genus level: Bacillariophytina (53%) and Diatomea (32%).Benthic diatom in the open access consists of Chaetoceros (12%), and the other level below genus Bacillariophyceae about 12% to 86% and for benthic diatom in the tourism zone is dominated by genera Navicula about 65% and about 35% is another taxon below genus level (Figure 2).The limitation of the genetic reference database for tropical causes is that some of the sequence only describes until level 5 or 6.It is causing restricted the determination of genetically identified sequence types at both genus and species levels.From the bioinformatic analyses, we got the sequence from the table feature ID, which represents one sequence for each feature ID.Thus, we re-blast the sequence that only reads until ordo or family to get a high level.Furthermore, we found about 25 species of Diatom (Figure 2).It consists of four species only found in the Water samples, 18 species in the sediment samples, and three species found both in the water and sediment samples (Figure 2).Species found in the water samples, namely Uncultured stramenopile, Bacteriastrum hyalinum, Chaetoceros tenuissimus, and Stramenopile sp.Furthermore, species that found in the sediment is Chaetoceros affinis, Chaetoceros cf.vixvisibilis, Chaetoceros diadema, Chaetoceros lorenzianus, Cocconeis cf.molesta, Cylindrotheca closterium, Fistulifera pelliculosa, Amphora coffeiformis, Bacteriastrum mediterraneum, Minutocellus polymorphus, Navicula arenaria, Navicula veneta, Nitzschia amphibia, Nitzschia microcephala, Papiliocellulus elegans, Synedra sp., Tryblionella apiculata, Tursiocola cf.ziemanii.Species of Diatom, which are found both in the water and sediment, are Chaetoceros anastomosans, Chaetoceros diversus, Chaetoceros didymus.
For Coral reefs are usually the most diverse ecosystems, but little is known about the diversity of micro eukaryotic, especially diatoms, in the surrounding water and sediment [32].Microeukaryotic, detected in the surface water until the water column, is categorized as a planktonic.Furthermore, the microeukaryotic, detected from the bottom into the sediment, is classified as a benthic [7].Thus, in this research, we categorized diatom as planktonic and benthic.We also show the number of sequences of Diatom, both planktonic and benthic (Table 1).It shows that the number of representative sequences and frequency of benthic diatom is higher than planktonic diatom.It represents those assemblages and taxa detection in the sediment with much more variation than in the water.The ANOVA test proved that the assemblage of diatom is a significant difference (p<0.05) between water and sediment but lack significance for spatial areas or zone (tourism zone, core zone, open access).It is similar to micro eucaryotic studies [7,11,33,34].The composition and structure of planktonic and benthic are significantly different, and Diatom is dominant from column water into sediment [34,35,36].It differs from other phytoplankton, whose assemblages are almost found in the oxic surface water until the column is about 5 meters [37,38].Furthermore, the difference between diatoms in the water and the sediment is unspecified ecological guild, but in the sediment, commonly found motile guild species and planktic guild were dominant in the water sample [38].
In this study, we found both planktonic and benthic diatom.Planktonic diatom; Uncultured stramenopile, Bacteriastrum hyalinum, Chaetoceros tenuissimus and Stramenopile sp.Genus Bacteriastrum was reported as a marine diatom floating freely [39] and Chaetoceros in the water and sediment.They are usually found in brackish water.In the coastal ecosystem, Chaetoceros is dominant.Chaetoceros is a cosmopolitan group.They live in the water with high salinity, such as in the high land, brackish water, coastal to ocean.In this study, we found some species Chaetoceros such as Chaetoceros affinis, Chaetoceros anastomosans, Chaetoceros cf.vixvisibilis, Chaetoceros diadema, Chaetoceros didymus, Chaetoceros diversus, Chaetoceros lorenzianus, and Chaetoceros tenuissimus from the coral reefs water which is saline water, some of them also found in the studies about diversity phytoplankton in the brackish water world heritage site Indian Sundarbans [40].Diatom which is found in the sediment or benthic, namely; Chaetoceros affinis, Chaetoceros cf.vixvisibilis, Chaetoceros diadema, Chaetoceros lorenzianus, Cocconeis cf.molesta, Navicula arenaria, Navicula veneta, Amphora coffeiformis, Bacteriastrum mediterraneum, Tursiocola cf.ziemanii, Fistulifera pelliculosa, Cylindrotheca closterium, Minutocellus polymorphus, Nitzschia amphibia, Nitzschia microcephala, Papiliocellulus elegans, Synedra sp., Tryblionella apiculate.
In this research, Chaetoceros are found in the water and sediment and have more richness than others.Cocconeis is known as an epiphytic and benthic marine diatom.Also found, Cocconeis cf.molesta was in the sediment sample.Some species from this genus are reported as rare and less known species, with recently found new species Cocconeis; Cocconeis magnoareolata Al-Handal, Riaux-Gob., R.Jahn & A.K.Wulff sp.nov., Cocconeis vrangoensis Al-Handal & RiauxGob.sp.nov.and some species were also identified as newly recorded in Mexico Cocconeis thallasiana [41] and [42].Navicula is a benthic diatom, sometimes found in the water column as a planktonic [43].Amphora is a genera found in marine and freshwater.In this study, we detected Amphora, especially Amphora coffeaeformis.This species is often found in brackish water [10] as a marine benthic diatom.Bacteriastrum is usually found as a pelagic diatom [39], but in this study, we found two kinds of Bacteriastrum: Bacteriastrum hyalinum from water sample and Bacteriastrum mediterraneum from sediment.We also found Tursiocola cf.Ziemanii, from some studies, is also known as a novel species [44].Thus, the information about new species is limited.On the other hand, we know some species und in tropical waters from this research.Fistulifera, wide geographical distribution, and ecological tolerance [45].Cylindrotheca, Nitzschia, Synedra, Tryblionella, and Papiliocellulus were reported as marine benthic diatom [8,46,47,48,49,50].It is similar to this research in that Cylindrotheca Closterium, Nitzschia amphibia, Nitzschia microcephala, Tryblionella apiculate, and Papiliocellulus elegans were found in a sediment sample.
In the structure community of phytoplankton, diatom is the most significant component of all primary production.Diatom contains about 20% to 25% of the phytoplankton community [51,52].It also showed in this study that there is a relative abundance of diatom, about 23%, from all microeukaryotic communities.Further, each species has a different relative abundance.It depends on many factors.Nutrien is one of many factors and plays an important role in changing the composition of marine phytoplankton.The sensitivity of each species to specific nutrients will be affected by blooming one of them.The Chaetoceros also has some phases when the nutrient concentration in the water changes.Some Chaetoceros will be in spora or resting phase when the nutrient is low [52].Some studies found diatom in the water or as a planktonic.Despite this, we found Chaetoceros in the water and sediment in this study.This study analyzed using environmental DNA, which is will detect all organisms where the DNA found.The DNA Chaetoceros detected in the sediment, caused by dying diatom, will fall into the sediment.Furthermore, the study of molecular biogeography of planktonic and benthic diatoms in the Yangtze River [43] said that the difference between diatoms in the water and the sediment is unspecified ecological guild, but in the sediment commonly found motile guild species and planktic guild were dominant in the water sample.

Conclusions
In our study, we detected 25 species of Diatom in West and East Lombok.Diatom from sediment samples has more richness than water, with four species only found in the Water samples and 18 species in the sediment samples.Three species were found both in the water and sediment samples.

Fig. 1 .
Fig. 1.Sampling location and horizontally stacked bar chart of Diatom communities from water and sediment in each zone (tourism zone, core zone, open access) and every region; West Lombok (left) and East Lombok (right) based on relative abundance.The bar chart comes from filtering the relative abundance of eukaryotic communities using QIIME2.

Fig. 2 . 1 Sediment
Fig. 2. Comparison diatom community (a) between water and sediment (b) West and East Lombok and (c) zonation is vein diagrams showing presence absence species of diatom which is part of microeukaryotic community that have identified by eDNA.( 1 Sediment sampel, 2 water sampel).
The results of diatom composition in the west and east Lombok in each zona are shown in Figure2.The bar chart (left) shows the relative abundance of diatom in west Lombok water and sediment in each zone (core zone, tourism zone, and open access).That bar chart comes from filtering microeukaryotic composition because of the target primer we used to be for microeukaryotic communities.Diatom is one of many microeukaryotic communities that are part of Straminopiles.The highest abundance in the Stramenopiles clades is Diatom.Relative abundance of diatom about 12% to 23.91% from total microeukaryotic or about 85.71% to 86.14% from total Stramenopiles clades for East Lombok and West Lombok, respectively.

Table 1 .
The number of e-DNA sequences is based on the water and sediment samples taken from each zone in West and East Lombok, including the Core zone, tourism areas, and open access zones.
spatial condition both location (West and East Lombok) and zone (Core zone, tourism zone, and open access) showed that 25 species diatom was detected which is 11 species detected in West Lombok; Chaetoceros affinis, Uncultured stramenopile, Amphora coffeiformis, Chaetoceros cf.vixvisibilis, Chaetoceros lorenzianus, Chaetoceros tenuissimus, Minutocellus polymorphus, Navicula arenaria, Papiliocellulus elegans, Tryblionella apiculate, and Tursiocola cf.ziemanii, and ten species found in East Lombok; Cocconeis cf.molesta, Cylindrotheca closterium, Fistulifera pelliculosa, Bacteriastrum hyalinum, Bacteriastrum mediterraneum, Navicula veneta, Nitzschia amphibia, Nitzschia microcephala, Stramenopile sp., Synedra sp.Four species are found in the West and East Lombok: Chaetoceros didymus, Chaetoceros anastomosans, Chaetoceros diversus, and Chaetoceros diadema.This study compared diatoms from water samples and sediment in west and east Lombok.The highest relative abundance of planktonic diatom in the core zone, tourism zone, and open access in West Lombok is uncultured stramenopile, Chetoceros diadema, and Chaetoceros anastomosans; benthic diatom is Amphora coffeiformis, Tursiocola cf.ziemanii and Minutocellus polymorphus.In east Lombok, the water sample shows that Chaetoceros anastomosans, Chaetoceros diversus, and Stramenopile sp.dominate in the open access, tourism zone, and core zone.Still, for sediment samples, Chaetoceros anastomosans is highest in the tourism zone and open access.Chaetoceros is the most commonly high in marine water, which is eight species of 25 species is Chaetoceros; Chaetoceros affinis, Chaetoceros anastomosans, Chaetoceros cf.vixvisibilis, Chaetoceros diadema, Chaetoceros didymus, Chaetoceros diversus, Chaetoceros lorenzianus, Chaetoceros tenuissimus.The second is Navicula: Navicula arenaria, Navicula veneta, and Nitzchia; Nitzschia amphibia, Nitzschia microcephala.It reveals one of many diatoms in tropical water that have records in Indonesia, especially West and East Lombok, which Chaetoceros dominated during this research.