Metagenomic characterization of unicellular eukaryotes in the urban Thessaloniki Bay

George Tsipas

SCHOOL OF ECONOMICS, BUSINESS ADMINISTRATION & LEGAL STUDIES

A thesis submitted for the degree of Master of Science (MSc) in Bioeconomy Law, Regulation and Management

May, 2019 Thessaloniki – Greece George Tsipas ’’Metagenomic characterization of unicellular eukaryotes in the urban Thessaloniki Bay’’

Student Name: George Tsipas

SID: 268186037282 Supervisor: Prof. Dr. Savvas Genitsaris

I hereby declare that the work submitted is mine and that where I have made use of another’s work, I have attributed the source(s) according to the Regulations set in the Student’s Handbook.

May, 2019 Thessaloniki - Greece

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George Tsipas ’’Metagenomic characterization of unicellular eukaryotes in the urban Thessaloniki Bay’’

1. Abstract

The present research investigates through metagenomics sequencing the unicellular protistan communities in Thermaikos Gulf. This research analyzes the diversity, composition and abundance in this marine environment. Water samples were collected monthly from April 2017 to February 2018 in the port of Thessaloniki (Harbor site, 40o 37’ 55 N, 22o 56’ 09 E). The extraction of DNA was completed as well as the sequencing was performed, before the downstream read processing and the taxonomic classification that was assigned using PR2 database. A total of 1248 Operational Taxonomic Units (OTUs) were detected but only 700 unicellular eukaryotes were analyzed, excluding unclassified OTUs, Metazoa and Streptophyta. In this research-based study the most abundant and diverse taxonomic groups were Dinoflagellata and Protalveolata. Specifically, the most abundant groups of all samples are Dinoflagellata with 190 OTUs (27.70%), Protalveolata with 139 OTUs (20.26%) Ochrophyta with 73 OTUs (10.64%), Cercozoa with 67 OTUs (9.77%) and Ciliophora with 64 OTUs (9.33%). The most diverse taxonomic groups are Dinoflagellata with 109508 reads, Protalveolata with 21977 reads and Ochrophyta with 11338 reads. At parallel, some of the dominant OTUs are Akashiwo with 47673 reads Noctiluca with 18025 reads and Syndiniales Group I with 14723 reads. The most abundant samples occurred to be the samples of 9/5/17 with 208 OTUs, 13/12/17 with 202 OTUs, 23/8/17 with 201 OTUs and 26/7/17 with 196 OTUs, while the least abundant are the samples of 15/11/17 with 93 OTUs, 18/10/17 with 101 OTUs, 6/2/18 with 109 OTUs and 28/6/17 with 124 OTUs. The highest number of OTUs from the Closest Relatives (Table 6) are Ptychodiscus noctiluca, Protodinium simplex, Biecheleriopsis adriatica, Protoperidinium pellucidum, Gyrodinium cf. gutrula and Micromonas pusilla. This study will provide data for future metagenomics studies and will contribute for a better understanding about the unicellular protistan communities in Thermaikos Gulf.

Keywords: Metagenomics sequencing, Thermaikos Gulf, Protist, Diversity, Abundance

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George Tsipas ’’Metagenomic characterization of unicellular eukaryotes in the urban Thessaloniki Bay’’

2. Acknowledgments

First of all, I want to thank my supervising professor Dr. Savvas Genitsaris for his precious guidance, the confidence and appreciation he has shown towards me. Dr. Savvas Genitsaris gave me a huge opportunity to deal with a particularly interesting subject and I thank him for his essential scientific assistance and for his extremely useful critical remarks on this process. In reality, I could not have achieved this current success without the valuable guidelines of Dr. Savvas Genitsaris who was always next to me and ready to answer and guide me in all of my questions or hesitations. My appreciation and my thanks to all the professors who educated me in the interdisciplinary courses of this master’s program from the International Hellenic University of Thessaloniki, since they gave me the appropriate motives and the necessary knowledge to reach this level on my dissertation. A big thanks also to Dr. Madesis Panagiotis who offered us hospitality in the Centre for Research and Technology Hellas – CERTH, Thermi, Thessaloniki, in order to perform the extraction of DNA from the samples. I must thank Dr. Zhaohe Luo, Haifeng Gu and his colleagues for granting me with their research on the Morphology, ultrastructure, and phylogeny of Protodinium simplex and Biecheleriopsis cf. adriatica (Dinophyceae) from the China Sea, which have been a helpful research for this study. I would also like to thank my colleague Tania Tatsika for her support and Vaso Ftaka for their hospitality and encouragement that they have shown towards me. Last but not least, I have to thank my family because without them it would be a very difficult task or even impossible to acquire this degree and conduct this research based dissertation. I thank them for standing by me all these years and for the patience that they have shown. George Tsipas 31/May/2019

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George Tsipas ’’Metagenomic characterization of unicellular eukaryotes in the urban Thessaloniki Bay’’

3. Preface

The present study with title ‘’Metagenomic characterization of unicellular eukaryotes in the urban Thessaloniki Bay’’ was conducted from April 2017 until February 2018, within the framework of the Interdisciplinary Master of Science (MSc) Bioeconomy Law, Regulation and Management offered by the International Hellenic University of Thessaloniki. This study was carried out under the supervision of Dr. Savvas Genitsaris, Academic Assistant at International Hellenic University, School of Economics, Business Administration & Legal studies. Nowadays the technological achievements in genetics and metagenomics are evolving rapidly as the world moves further to a better understanding of the global ecosystems. This research study has led to the generation of data sequencing, which allowed to develop a better understanding for the diversity and composition of the unicellular protistan communities in Thermaikos Gulf. Also, contributes to a better exploration of the unicellular protists that are responsible for many local problems such as red tides. A speculation is that metagenomic technologies are and will be a strong analyzer for the ecosystems, thus the study of Thermaikos Gulf will produce an up to date library and genetic data about unicellular protistan communities of the area.

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4. Contents

1. ABSTRACT ...... 3

2. ACKNOWLEDGMENTS ...... 4

3. PREFACE ...... 5

4. CONTENTS ...... 6

4.1 LIST OF FIGURES ...... 7

4.2 LIST OF TABLES ...... 8

5. INTRODUCTION ...... 9

6. MATERIALS AND METHODS ...... 14

6.1 SAMPLE COLLECTION ...... 14

6.2 THE METHODOLOGY OF DNA EXTRACTION ...... 16 6.2.1 Read Processing ...... 16

7. RESULTS ...... 18

7.1 METAGENOMIC ANALYSIS ...... 18

7.2 PROTISTAN COMMUNITY STRUCTURE ...... 20 7.2.1 Diversity of the protistan community ...... 20 7.2.2 Composition of the protistan community ...... 27 7.2.3 Relative abundance of the protistan community...... 30

7.3 DOMINANT OTUS ...... 32

8. DISCUSSION ...... 35

9. CONCLUSIONS ...... 43

10. BIBLIOGRAPHY ...... 45

APPENDIX ...... 59

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George Tsipas ’’Metagenomic characterization of unicellular eukaryotes in the urban Thessaloniki Bay’’

4.1 List of Figures

Figure 1: Sampling site in Thermaikos Gulf, Harbor site (Esri)...... 14 Figure 2: Appearance of newly recorded OTUs per sample (Appendix Table 1)...... 20 Figure 3: Total OTUs per sample (Table 5)...... 21 Figure 4: Simpson 1-D Diversity index of OTUs in samples (Table 5)...... 21 Figure 5: Dominance-D of OTUs in samples (Table 5)...... 22 Figure 6: Shannon H index of OTUs in samples (Table 5)...... 23 Figure 7: Evenness e^H/S of OTUs in samples (Table 5)...... 24 Figure 8: Berger-Parker dominance of OTUs in samples (Table 5)...... 25 Figure 9: Chao1 index of OTUs in samples (see also Table 5)...... 26 Figure 10: Total relative numbers of OTUs in the taxonomic groups, overall in the entire study (Appendix Table 2)...... 28 Figure 11: Composition of the taxonomic groups per sample, measured in OTUs ...... 29 (Appendix Table 2)...... 29 Figure 12: Composition of the taxonomic groups in samples, measured in OTUs and expressed as a percentage (Appendix Table 2)...... 29 Figure 13: Relative abundance of taxonomic groups overall in the entire study, measured in reads of OTUs (Appendix Table 3)...... 30 Figure 14: Relative abundance of taxonomic groups in the samples measured in reads of OTUs and expressed as a percentage (Appendix Table 3)...... 31 Figure 15: The most dominant OTUs found in all samples, measured in reads of OTUs . 32 (Appendix Table 4)...... 32

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4.2 List of Tables

Table 1: Sample numbering and dates of collection...... 15 Table 2: Samples and their corresponding number of reads...... 18 Table 3: The recovered reads per sample before the chimera search...... 18 Table 4: Total OTUs of unicellular protistan Eukaryotes, Unclassified OTUs, Metazoa and Streptophyta...... 19 Table 5: Estimation of OTUs and indexes...... 26 Table 6: Closest Relatives of OTUs while demonstrating their Trophic Group, Taxonomic Affiliation, Isolation Source and their Appearance in the Samples...... 33

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5. Introduction

The development of new uses of marine living resources and the processing of raw materials by biotechnological methods is called "blue biotechnology". This term refers to the exploration and exploitation of various marine organisms for the purpose of developing high value products. The Organization for Economic Co-operation and Development (OECD, 2004) refers to biotechnology as the implementation of technology and science in living organisms, and also to their products and parts which are intended for modifying living or non-living organisms and materials for the purpose of goods, services and knowledge. The marine environment is particularly important for the European Union because, about 50% of its territory is on the high seas and most Member States have coastlines, approximately 50% of its citizens live about 50 km from the coast and 3.5 million inhabitants of the EU are directly involved in maritime activities (EC, 2017). According to ‘’The 2018 Annual Economic Report on EU Blue Economy’’ (2018), the "blue" economy already covers approximately 5.4 million jobs and a gross added value of almost € 500 billion a year. The European Union is investing in "blue" economy and Greece has an important opportunity to use these investments. However, the ecosystems and biodiversity have been downgraded to a point in which they are no longer able to provide their valuable services to their fullest. The contribution of microbial communities can help alter the downgrading process. Almost all forms of life on planet Earth support its whole existence in microbial communities. Humanity has profited immensely from the study of microbes since they were discovered in the 17th century. However, most of the benefits have been discovered and exploited from studying only a few of the millions of microbial species presented in the environment. The microbes are by far the most abundant and diverse organisms on the planet and are responsible for the conservation of the atmospheric and chemical conditions, required for the survival of all larger organisms (Pearce et al., 2009).

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Protists are defined as eukaryotic organisms with a colonial, unicellular, filamentous or parenchymatous organization that lack vegetative tissue differentiation, except for reproduction (Adl et al., 2005). It counts over 40000 morphological species that are scarcely related and do not show common ways of movement, nourishment or reproduction, while some of them are using photosynthesis like plants and other protists are heterotrophic, such as animals (Cavalier-Smith, 2017). Microscopic eukaryotes are proved to be diverse and abundant while are filling critical ecological roles around the globe, yet there is a recognized gap in understanding their biodiversity (Bik et al., 2011). Adl et al. (2007) denote that the discovery ratio of new species from environmental samples remains high and that a large number of geographic regions have not been sampled or have been inefficiently sampled. The main discussions in the scientific communities are i) the diversity of microbes, ii) their geographical distribution, and iii) the hypothesis that they can be categorized as cosmopolitan and can habit in any geographical area, while scientists have a limited understanding of microbial distribution (Weisse, 2008). The organisms with <1mm size show a tendency to have a cosmopolitan distribution, according to Fenchel & Finlay (2004). Following the great ecological crisis of 1980-1990, modern science has recognized and identified most of the misplaced human interventions in the ecosystem and has proposed several solutions. Metagenomics is a new, revolutionary scientific approach that aims to understand the world of microbial communities. This new field of metagenomics, where the possibilities are offered by genomic analysis, is applied to whole microbial communities, bypassing the need to isolate and cultivate individual species (Chen & Pachter, 2005). Metagenomics involves many different innovative techniques and approaches, while others will most likely be developed, as this field grows new methods. The first step in many metagenomics studies is the same: researchers collect a sample from a specific environment (such as soil, seawater etc.) and mass-export DNA from all sampled microbes, as well as, researchers have been extracting proteins or RNA increasingly often (Vieites et al., 2009).

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Traditional microbiological studies have focused on examining individual species as isolated and cultivable units. However, the vast majority of microbial species has never been successfully isolated as viable specimens for analysis, apparently because their survival depends upon specific microenvironments and niches that have not been, or cannot be, simulated experimentally (Ritz, 2007). Baldauf (2003) stated that molecular techniques reflect one of the most advantageous tools for uncovering the “missing” part of this diversity, and unseal the complicated communities of marine protists, and their associations with biotic and abiotic features. Metagenomics sequencing has proved to be a powerful approach for investigating the diversity and functional relationships in marine ecosystems and can generate a total community (Bik, 2014; Genitsaris et al., 2015). Unquestionably the fast development of metagenomics applications has set the basis for better biodiversity analysis, yet the diversity of these applications adds difficulties to the experimental design of the researches (Klindworth et al., 2012). A major detailed review on sequencing technologies was conducted by Loman et al. (2012). Next generation sequencing (NGS) technologies such as, Illumina's MiSeq which is a major platform for amplicon sequencing in microbial ecology studies, are powerful tools to analyze and characterize the diversity of microbial communities (Wen et al., 2017; Zhou et al., 2015). In recent years, Thermaikos Gulf has been exposed to the consequences of environmental degradation due to reckless human activity. One of the most important environmental issues of Thermaikos Gulf is eutrophication. Eutrophication is the excessive development of autotrophic organisms, mainly phytoplankton and macrophytes, in a surface aquatic body and is the result of the natural or artificial enrichment of its nutrient waters (Khan et al., 2005). The previous definition for eutrophication is by Nixon (1995) who dealt with the phenomenon of the eutrophication in the ocean and coastal marine systems and proposes as a definition that the "eutrophication is the increase of enrichment rate at organic material of a marine ecosystem". Eutrophication is an environmental issue that has the effect of reducing the dissolved oxygen in the deeper layers of water, creating toxins and causing many

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George Tsipas ’’Metagenomic characterization of unicellular eukaryotes in the urban Thessaloniki Bay’’

casualties to fishes (Escalera et al., 2007). Eutrophication in coastal marine systems can 1) Change the composition of phytoplankton community, 2) Increase the phytoplankton biomass, 3) Decrease the populations of fish and shellfish, 4) Reduce the transparency, 5) Cause the losses of habitats and 6) Reduce the biodiversity and the oxygen concentration in deeper water layers (National Research Council, 2000). Thermaikos Gulf is a NATURA 2000 network area which occupies extended coastlines (about 50% of coastal waters) and includes areas of specific ecological value which are also included in lists of protection and of special management of the marine environment. These areas concern the delta of rivers, lagoons and protected habitats (Directive 92/43, Directive 79/409, Nature Network - NATURA 2000), also includes marine parts of the protection zones of Aliakmonas - Loudias - Axios - Gallikos, Kytros and Kalochori lagoon. Τhe ‘’Management Body of Thermaikos Gulf ‘’, (former Axios delta Management Authority), contains many of the areas of Natura network 2000, as well as the sea area which includes the Thessaloniki Bay under the codes GR1250004 and GR1220012 (Ministry of Productive Reconstruction, Environment and Energy, 2015). Collins et al. (2005) claim that the sedimentary evolution by the four rivers (Gallikos, Aliakmon, Loudias, and Axios) and the anthropogenic impact, have presented significant growth, from 1959 to 2000, in Thermaikos Gulf. Freshwater supplies that flow from the rivers along the western coasts are concentrating lighter water, as a result these densitometry differences contribute to the penetration of the waters in the Aegean Sea and in Thermaikos Gulf, thus affecting the speed of renewal of the water masses (Hyder et al., 2002). Furthermore, another study about Thermaikos Gulf by Savvidis et al. (2011) notes that a Harmful Algal Bloom (HAB), causing red tides, occurred in northeast coasts of Thermaikos Gulf, it then spread to northwest and west coasts approximately in 10 days. Pavlidou et al. (2015) found that the highest nitrate rate, of all Greece’s coastal areas, was found in Thermaikos Gulf while they noticed an increase in the abundance of harmful Dinoflagellate species. Nitrate ions are part of the nitrogen cycle, and are found in inorganic fertilizers and in wastewaters acting as an oxidizing agent, who can cause many

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consequences to the human health and can remain in the layers of the seas for decades (World Health Organization, 2003). Human activity has altered the nitrate flow, resulting in the escalation of eutrophication which causes Harmful Algal Blooms (HABs) and red tides (Paerl & Tucker, 1995; Hayakawa et al., 2015). Thermaikos Gulf has restricted water mass circulation and riverine flows, therefore HABs are thriving there, but also the agriculture and aquaculture establishments are related to these phenomena, such as the species Dinophysis acuminata which is related to the intoxication of the mussels (Varkitzi et al., 2018). Harmful Algal Blooms (HABs) appear naturally, yet the human actions aggravate the ecosystem by enriching their nutrients, polluting and modifying the hydrology, and also the invasion of alien species has a certain link to the HABs (Jewett et al., 2008). Caruana & Amzil (2018) categorized the harmful algal species to toxic and non-toxic species, the toxic species can produce toxins with the potential to harm humans and the non-toxic species can reach high levels of toxins by composing higher biomass levels. The composition of the microbial communities, the occurrence of HABs and the dominance of the taxonomic groups contribute in the assessment of the eutrophication merit (EC, 2005). The main objective of this research-based dissertation is to investigate the diversity, composition and abundance of the unicellular eukaryotes in the urban Thessaloniki Bay, via high-throughput metagenomics sequencing. The study of the marine environment is considered to be very important in order to produce data not only for metagenomics approaches but also to other scientific fields such as renewable resources, blue biotechnology and for the management and protection of marine environments. This environmental investigation through modern sequencing technologies with bioinformatics and ecological tools can aims to answers produce solid answers to ‘who, what, when, where, why and how’ of the marine protistan communities (Knight et al. 2012).

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6. Materials and methods

6.1 Sample collection

The sampling site is located at Thermaikos Gulf in Thessaloniki, Greece near the Port of Thessaloniki (40o 37’ 55 N, 22o 56’ 09 E, picture 1). Twelve samples (Table 1) were collected from the urban site at Thessaloniki Bay (Harbor site), for DNA extraction and metagenomics sequencing. In Figure 1 are depicted, the North-West coast with the many cultivated fields, while in the North-East bay appears the large expansion of the city.

Figure 1: Sampling site in Thermaikos Gulf, Harbor site (Esri).

Samples were collected from April 2017 to February 2018, during this period, every month an additional sample was obtained from the Harbor site. These samples Page 14 of 63

George Tsipas ’’Metagenomic characterization of unicellular eukaryotes in the urban Thessaloniki Bay’’

were collected at a maximum depth of 4m. A sample of 50 mL was filtered through 0.2 μm cellulose acetate filters (Sartorius) and was kept in -20oC until DNA extraction and metagenomics sequencing. The temperature of the sea was measured in situ with the YSI Pro 1030 (YSI Inc. Ohio, USA). The extraction of DNA was completed using the Macherey – Nagel NucleoSpin®, Genomic DNA Isolation Kit and carried out for the twelve (12) marine samples. The samples were analyzed in the Centre for Research and Technology Hellas – CERTH, Thermi, Thessaloniki on June 2018.

Table 1: Sample numbering and dates of collection. Sample Sample Name Date № 1 8 12/4/2017 № 2 15 9/5/2017 № 3 22 7/6/2017 № 4 28 28/6/2017 № 5 35 26/7/2017 № 6 41 23/8/2017 № 7 48 20/9/2017 № 8 55 18/10/2017 № 9 62 15/11/2017 № 10 69 13/12/2017 № 11 76 10/1/2018 № 12 83 6/2/2018

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6.2 The Methodology of DNA extraction

The extracted DNA was subjected to PCR using specific primers targeting the V4 hyper variable region of the 18S rRNA gene (E572F = CYGCGGTAATTCCAGCTC; E1009R = AYGGTATCTRATCRTCTTYG). These primers have been found to successfully amplify approximately 440 bp in the V4 hypervariable region of all the major high-level eukaryotic taxonomic groups (Genitsaris et al., 2016). Next, the PCR products were purified and the amplicon samples were sequenced on Illumina MiSeq using 300+300 bp paired-end chemistry, which allows the overlap and the stitching together of paired amplicon reads into one full-length read of higher quality (http://cgeb-imr.ca/protocols.html). The PCR amplification step and the sequencing were performed at the Integrated Microbiome Resource (IMR) at Dalhousie University, Halifax Canada.

6.2.1 Read Processing

The produced reads were subjected to downstream processing using mothur v.1.34.0 software (Schloss et al., 2009), following the proposed standard operating procedure (Schloss et al., 2011). Briefly, forward and reverse reads were joined, and contigs below 200 bp, with >8 bp homopolymers and ambiguous base calls were removed from further analysis. The remaining reads were dereplicated to the unique sequences and aligned independently against the SILVA 132 database, containing 163916 eukaryotic SSU rRNA gene sequences (Quast et al., 2013). Then, the reads suspected for being chimeras were removed using the UCHIME software (Edgar, 2010). The remaining reads were clustered into Operational Taxonomic Units (OTUs) at 97 % similarity level. In order to obtain a rigorous dataset, OTUs with a single read in the entire dataset were removed from the analysis, as they were suspected of being erroneous sequences (e.g. Kunin et al., 2010; Richards et al., 2015; Genitsaris et al., 2016). The resulting dataset was normalized to 14258 reads, while the samples with lower total number of reads were kept, as were in the dataset. We chose this course of action as the best compromise in order to retrieve the majority of the biodiversity detected by sequencing, but also to include all samples in

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the analysis, even those with lower number of reads. Taxonomic classification was assigned using BLASTN searches against the “Protist Ribosomal Reference database” (PR2 database), with curated protistan taxonomy (Guillou et al., 2013). The reads belonging to OTUs affiliated to Metazoan and Streptophyta sequences, were removed from the dataset. The protistan community structure was estimated using Alpha diversity estimators, such as Simpson’s, Dominance D, Shannon H, Evenness, Berger-Parker, Chao1 diversity indexes and OTUs-S which were analyzed and calculated with the PAST 3. Software (Hammer, Harper & Rayan, 2001).

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7. Results

7.1 Metagenomic analysis

Following the standard operating procedure, specifically MiSeq SOP protocol (https://www.mothur.org/wiki/MiSeq_SOP) with 97% sequence similarity (Schloss et al., 2011), a total of 502130 reads was produced (Table 2).

Table 2: Samples and their corresponding number of reads. Date Sample Number of reads 12/4/2017 8 55142 9/5/2017 15 39278 7/6/2017 22 39772 28/6/2017 28 37302 26/7/2017 35 35499 23/8/2017 41 22377 20/9/2017 48 35758 18/10/2017 55 63378 15/11/2017 62 39540 13/12/2017 69 57387 10/1/2018 76 50233 6/2/2018 83 26464 Total 502130

After continuing the procedure and removing the ambiguous bases and anything shorter than 470 bp, the number of sequences amounted to 228669, before the chimera search, with the unique sequences being at 148756 which is the final number of sequences that was screened. The lowest sequences are observed in the sample of 23/8/2017, while the highest are observed in the sample of 18/10/2017.

Table 3: The recovered reads per sample before the chimera search. Samples Recovered reads S8 12/4/17 22918 S15 9/5/17 18069 S22 7/6/17 17256 S28 28/6/17 17997

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S35 26/7/17 15860 S41 23/8/17 10589 S48 20/9/17 17142 S55 18/10/17 30278 S62 15/11/17 18486 S69 13/12/17 28564 S76 10/1/18 20042 S83 6/2/18 11468 Total 228669

A total of 1248 OTUs were detected, and after the removal of Unclassified OTUs, Metazoa and Streptophyta, a total of 700 unicellular Eukaryotes were produced, as observed in Table 4.

Table 4: Total OTUs of unicellular protistan Eukaryotes, Unclassified OTUs, Metazoa and Streptophyta.

Eukaryota 700

Unclassified OTUs 435 Metazoa 104 Streptophyta 9 Total 1248

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7.2 Protistan Community Structure

7.2.1 Diversity of the protistan community

All the living organisms on Earth are part of one huge interdependent mosaic. Biodiversity is the expression of this mosaic where the variety of life has been created over billions of years of evolution. In Figure 2 are presented in chronological order the amount of the newly recorded OTUs per sample, adding each time the new OTUs to the already counted, from the previous sample/samples.

Appearance of new OTUs per sample

800 700 661 687 700 570 593 600 523 542 455 500 388 400 341 292 300 200 150 100 0

Figure 2: Appearance of newly recorded OTUs per sample (Appendix Table 1).

In Figure 3, are recorded the maximum OTUs in the sample of 9/5/2017 with 208 OTUs and the minimum with 94 OTUs in the sample of 15/11/2017, also the average OTUs are 150 OTUs.

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Total OTUs per sample 250 208 196 201 202 200 153 150 137 150 124 129 109 101 93

OTUs 100

50

0

Figure 3: Total OTUs per sample (Table 5).

Simpson 1-D Diversity index, measures the diversity of the studied habitat while taking into account the abundance of each OTUs. The higher diversity values in samples according to Simpson’s 1-D index (Figure 4) are the samples of 26/7/17 with 0.9132 and 9/5/17 with 0.9003 indices, while the lowest values of diversity are the Samples of 18/10/17 and 12/4/17 with 0.3136 and 0.5955 indices, respectively.

Figure 4: Simpson 1-D Diversity index of OTUs in samples (Table 5). Page 21 of 63

George Tsipas ’’Metagenomic characterization of unicellular eukaryotes in the urban Thessaloniki Bay’’

The most diverse samples, according to Dominance-D (Figure 5) are the samples of 26/7/17, 9/5/17 and 23/8/17, while the most dominated sample is that of 18/10/17. The most dominated samples are that of 18/10/17 with 0.6864 and 12/4/17 with 0.4045 indices, while the two less dominated samples are the samples of 9/5/17 and 26/7/17 with 0.09969 and 0.08676 indices, respectively.

Figure 5: Dominance-D of OTUs in samples (Table 5).

The combination of the number of OTUs and their relative abundance, determines the diversity of OTUs and one of the most suggested indexes is Shannon H. Figure 6, is measuring the entropy from Shannon H index and results in the most diverse samples which are the samples of 9/5/17 with 3.35, 23/8/17 with 3.24 and 26/7/17 with 3.48 indices. At parallel the least diverse samples are the samples of 18/10/17 with 1.02 and 15/11/17 with 1.85 indices.

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Figure 6: Shannon H index of OTUs in samples (Table 5).

Figure 7 estimates how even the communities of the samples are, resulting in the most even samples being that of 9/5/17 with 0.1255, 26/7/17 with 0.1583 and 23/8/17 with 0.1212 indices. The least even samples are the samples of 18/10/17 with 0.02703, 13/12/17 with 0.03323 and 12/4/17 with 0.0446 indices.

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Figure 7: Evenness e^H/S of OTUs in samples (Table 5).

The Berger-Parker index (Figure 8) also agrees with the Dominance-D (Figure 5) that the most diverse samples are the samples of 26/7/17, 9/5/17 and 23/8/17 and that the most dominated, by other OTUs, is the sample of 18/10/17. Berger-Parker (Figure 8) presents that the most diverse samples are the samples of 26/7/17 with 0.2478, 9/5/17 with 0.25 and 23/8/17 0.3251 values, while the most dominated, by other OTUs, are the samples of 18/10/17 with 0.8268, 12/4/17 with 0.6268 and 20/9/17 with 0.6106 values. Figure 8, also points out two more samples dominated by other OTUs, (dominated relative to the number of OTUs) which are the samples of 12/4/17 and 20/9/17.

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Figure 8: Berger-Parker dominance of OTUs in samples (Table 5).

By Chao1 index (Figure 9) occurs that the most diverse samples are those of 9/5/17 with 227.2, 23/8/17 with 211.3, 13/12/17 with 208.2 and 26/7/17 with 205.1 values. On the contrary, the least diverse samples are those of 15/11/17 with 94.41, 18/10/17 with 103 and 6/2/18 with 112.3 indices.

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Figure 9: Chao1 index of OTUs in samples (see also Table 5).

In Table 5, are presented the OTUs-S, Simpson 1-D, Dominance D, Shannon H, Evenness, Berger-Parker and Chao1 indexes of OTUs in samples. Within the table are marked the highest and the lowest values calculated.

Table 5: Estimation of OTUs, Simpson’s, Dominance D, Shannon H, Evenness, Berger-Parker, Chao1 indexes of OTUs in samples, while marking the highest and lowest indices.

OTUs Simpson Dominance Shannon Evenness- Berger- Samples -S 1-D D H e^H/S Parker Chao1

S8 12/4/17 150 0.5955 0.4045 1.901 0.0446 0.6268 150.5

S15 9/5/17 227 0.9003 0.09969 3.349 0.1255 0.25 227.2

S22 7/6/17 146 0.6633 0.3367 2.231 0.06376 0.5709 146.1

S28 28/6/17 130 0.6316 0.3684 1.889 0.05086 0.5869 130.6

S35 26/7/17 205 0.9132 0.08676 3.48 0.1583 0.2478 205.1 Page 26 of 63

George Tsipas ’’Metagenomic characterization of unicellular eukaryotes in the urban Thessaloniki Bay’’

S41 23/8/17 211 0.8485 0.1515 3.242 0.1212 0.3251 211.3

S48 20/9/17 138 0.6185 0.3815 2.133 0.06116 0.6106 138

S55 18/10/17 103 0.3136 0.6864 1.024 0.02703 0.8268 103

S62 15/11/17 94 0.7457 0.2543 1.85 0.06768 0.4049 94.41

S69 13/12/17 208 0.6881 0.3119 1.933 0.03323 0.4407 208.2

S76 10/1/18 159 0.7952 0.2048 2.568 0.08202 0.4263 159.2

S83 6/2/18 112 0.7783 0.2217 2.299 0.08896 0.4236 112.3

7.2.2 Composition of the protistan community

In Figure 10 the OTUs that were calculated and found to be <1% were integrated in the groups of Other Protists, Hacrobia and Opisthokonta. The produced taxonomic groups were 15 with Dinoflagellata, Protalveolata, and Ochrophyta, overcoming the 50% of the total number of OTUs, having the 58.60% of OTUs. Specifically, the most abundant taxonomic groups of all samples are Dinoflagellata with 190 OTUs (27.70%), Protalveolata with 139 OTUs (20.26%), Ochrophyta with 73 OTUs (10.64%), Cercozoa with 67 OTUs (9.77%), Ciliophora with 64 OTUs (9.33%), Chlorophyta with 38 OTUs (5.54%) and Prymnesiales with 34 OTUs (4.96%), with the rest of the taxonomic groups, Fungi, Cryptophyceae, Retaria, Marine Stramenopiles (MAST), Charophyta, Other Opisthokonta, Other Protists, Other Hacrobia being less than 2%.

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Total relative numbers of OTUs in the taxonomic groups Dinoflagellata Other Protists. 1.17% Protalveolata

Other Opisthokonta. Ochrophyta 1.17% Charophyta. Other Hacrobia. Cercozoa MAST. 1.46% 1.31% 1.75% Ciliophora Retaria. 1.60% Dinoflagellata. 27.70% Cryptophyceae. 1.60% Chlorophyta

Fungi. 1.75% Prymnesiales Fungi Prymnesiales. 4.96% Cryptophyceae Chlorophyta. 5.54% Retaria Ciliophora. 9.33% MAST

Charophyta

Cercozoa. 9.77% Other Opisthokonta Other Protists Ochrophyta. 10.64% Protalveolata. 20.26% Other Hacrobia

Figure 10: Total relative numbers of OTUs in the taxonomic groups, overall in the entire study (Appendix Table 2).

The abundance of taxonomic groups in the samples (Figures 11 & 12) demonstrates the most abundant samples, greater than 200 OTUs and those are the samples of 9/5/17 with 208 OTUs, 13/12/17 with 202 OTUs and 23/8/17 with 201 OTUs. The least abundant samples, lesser than 130 OTUs, are the samples of 15/11/17 with 93 OTUs, 18/10/17 with 101 OTUs, 28/6/17 with 124 OTUs and 20/9/17 with 129 OTUs. The OTUs that were calculated and found to be <1% were integrated in the groups of Other Protists and Opisthokonta.

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Composition of the taxonomic groups in samples Cercozoa Chlorophyta S83 6/2/18 Ciliophora S76 10/1/18 Cryptophyceae

S69 13/12/17 Dinoflagellata

S62 15/11/17 Fungi

S55 18/10/17 Haptophyta MAST S48 20/9/17 Ochrophyta S41 23/8/17 Samples Other Opisthokonta S35 26/7/17 Protalveolata S28 28/6/17 Prymnesiales S22 7/6/17 Retaria S15 9/5/17 Picozoa S8 12/4/17 Labyrinthulomycetes 0 50 100 150 200 250 Other Protists

Figure 11: Composition of the taxonomic groups per sample, measured in OTUs (Appendix Table 2).

Composition of the taxonomic groups in samples (%) Cercozoa Chlorophyta S83 6/2/18 Ciliophora S76 10/1/18 Cryptophyceae S69 13/12/17 Dinoflagellata S62 15/11/17 Fungi S55 18/10/17 Haptophyta MAST S48 20/9/17 Ochrophyta S41 23/8/17 Samples Other Opisthokonta S35 26/7/17 Protalveolata S28 28/6/17 Prymnesiales S22 7/6/17 Retaria S15 9/5/17 Picozoa S8 12/4/17 Labyrinthulomycetes 0% 20% 40% 60% 80% 100% Other Protists

Figure 12: Composition of the taxonomic groups in samples, measured in OTUs and expressed as a percentage (Appendix Table 2). Page 29 of 63

George Tsipas ’’Metagenomic characterization of unicellular eukaryotes in the urban Thessaloniki Bay’’

7.2.3 Relative abundance of the protistan community

Biodiversity in practice tends to be measured based on the abundance of species, while it is a good substitute as it acts as a "unifier" on the multiple faces and variations of biodiversity. Abundance is usually measurable in practice and there is a significant amount of information, about relative abundance and patterns of species. The classification of OTUs with 97% similarity has produced 145485 reads. The most diverse taxonomic groups, presented in Figure 13, is Dinoflagellata with 109508 reads, the next highest taxonomic groups are Protalveolata with 21977 reads, Ochrophyta with 11338 reads, and Ciliophora with 7803 reads. The OTUs that were calculated and found to be <1% were integrated in the groups of Other Protists and Opisthokonta.

Relative abundance of taxonomic groups Dinoflagellata Protalveolata Fungi. MAST. Labyrinthulomycetes. Ciliophora 0.10% 0.10% 0.01% Cercozoa. 1.20% Chlorophyta Other Opisthokonta. Retaria. 1.09% Haptophyta. 0.10% Prymnesiales 0.58% Other Protists. 0.02% Ochrophyta Cryptophyceae. 1.60% Cryptophyceae Ochrophyta. 6.82% Picozoa. 0.04% Haptophyta Prymnesiales. 2.68% Cercozoa Chlorophyta. 1.86% Fungi MAST Ciliophora. 4.69% Other Opisthokonta Protalveolata. 13.22% Retaria Labyrinthulomycetes Other Protists Dinoflagellata. 65.89% Picozoa

Figure 13: Relative abundance of taxonomic groups overall in the entire study, measured in reads of OTUs (Appendix Table 3).

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Figure 14 presents the richest samples, which are the samples of 13/12/17 with a total of 25675 reads, 12/4/17 with a total of 21697 reads, 28/6/17 with 16926 reads, 10/1/18 with 16429 reads, 9/5/17 with 16409 reads, 15/11/17 with 13317 reads. The least rich samples are the samples of 23/8/17 with 4266 reads, 20/9/17 with 8819 reads 18/10/17 with 10214 reads, 26/7/17 with 10358 reads, 6/2/18 with 10513 reads and 7/6/17 with 11583 reads.

Picozoa Relative abundance of the taxonomic groups in Samples (%) Other Protists 100% Labyrinthulomycetes 90% Retaria 80% Other Opisthokonta 70% MAST

60% Fungi Cercozoa 50% Haptophyta 40% Cryptophyceae 30% Ochrophyta 20% Prymnesiales

10% Chlorophyta Ciliophora 0% Protalveolata Dinoflagellata

Figure 14: Relative abundance of taxonomic groups in the samples measured in reads of OTUs and expressed as a percentage (Appendix Table 3).

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7.3 Dominant OTUs

In this chapter are presented the most dominant OTUs, existing in all samples. The most dominant OTUs, above the 10% of the dominance rate, are Akashiwo with 47673 reads (41.49%), Noctiluca with 18025 reads (15.69%) and Syndiniales Group I with 14723 reads (12.81%). Below of the 10% of the dominance rate, but above the 4% of the reads are Gonyaulax with 10450 reads (9.09%) and Chaetoceros with 5273 reads (4.59%).

Dominant OTUs

Tintinnopsis Protodinium Skeletonema Teleaulax Gymnodinium Arthracanthida Strombidium Islandinium Thalassiosira Amoebophrya Chaetoceros Gonyaulax Syndiniales Group I Noctiluca Akashiwo

0 10000 20000 30000 40000 50000 60000

Figure 15: The most dominant OTUs found in all samples, measured in reads of OTUs (Appendix Table 4).

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In Table 6 are recorded the OTUs that have presence in all samples or in most samples and are having the highest number of OTUs. The Closest relatives are detected through the GenBank® (Clark et al., 2015) using the BLAST (Altschul et al., 1990) program, which is a sequence similarity search tool. Also, many Unclassified OTUs were blasted through the GenBank® and are presented in the Appendix (Table 5).

Table 6: Closest Relatives of OTUs while demonstrating their Trophic Group, Taxonomic Affiliation, Isolation Source and their Appearance in the Samples. Taxonomic Closest Relative (100%) Isolation Appearance OTUs Trophic Group Affiliation [Accession Number] Source in Samples Microplankton Ptychodiscus noctiluca (100%) Sea water Otu0001 grazers Dinoflagellata [KU640194.1] All Protodinium simplex (100%) BH41_57 Otu0052 Autotrophs Protalveolata [KY980102.1] All Microplankton Biecheleriopsis adriatica (100%) Coastal waters Otu0025 grazers Dinoflagellata [LC068843.1] All Uncultured haptophyte (100%) Clone Otu0065 Autotrophs Coccolithales [JX680341.1 ] All Marine biome, Uncultured marine picoeukaryote fjord, coastal Otu0027 Autotrophs Protalveolata (100%) [FR874830.1] water All Microplankton Protoperidinium pellucidum (99%) Coastal waters Otu0041 grazers Dinoflagellata [AY443022.1] All Microplankton Gyrodinium cf. gutrula (100%) Clone; Otu0028 grazers Dinoflagellata [FN669511.1] All Subtropical Microplankton Uncultured eukaryote clone coastal Otu0034 grazers Dinoflagellata 10D10P08 (99%), [KU743847.1 ] ecosystem All Uncultured eukaryote clone Clone Otu0081 Heterotrophic Kathablepharidae SGUH1328 (99%) [KJ762973.1] All Uncultured eukaryote clone All except Clone Otu0059 Autotrophs Leucocryptos SGYP1492 (100%) [KJ763642.1 ] S8 Uncultured eukaryote clone All except Marine water Otu0120 Autotrophs Prymnesiales SHBA568 (99%) [HQ869236.1 ] S8 Adenoides eludens (99%) All except BH46_94 Otu0014 Autotrophs Protalveolata [KY980212.1 ] S28 Uncultured haptophyte clone All except Seawater Otu0029 Autotrophs Prymnesiales 897st16-1 (100%) [KF620969.1 ] S55

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Uncultured marine Geminigeraceae clone Marine RA071004C.032 (100%) plankton1 All except Otu0023 Autotrophs Teleaulax [FJ431465.1] S28 Haptolina fragaria (99%) All except Otu0018 Autotrophs Prymnesiales [AM491013.2 ] Clone; S62 Micromonas pusilla (99%) Mediterranean All except Otu0074 Autotrophs Chlorophyta [KF501033.1 ] Sea S55 Nanoplankton Sinistrostrombidium cupiformum China: All except Otu0063 grazers Ciliophora (100%) [JX310366.1 ] Qingdao S28 Teleaulax gracilis (100%) Estuarine- All except Otu0105 Autotrophs Teleaulax [JQ966995.1 ] coastal water S28 Uncultured eukaryote clone All except Marine water Otu0095 Parasites Cercozoa SHAX767 (99%) [HQ868539.1 ] S35 Uncultured marine eukaryote clone UEPACDp4 (99%) Coastal water All except Otu0043 Autotrophs Protalveolata [AY129046.1 ] S83 Thalassiosira pseudonana (99%) United All except Otu0013 Autotrophs Ochrophyta [AJ535169.1 ] Kingdom S8, S48 Uncultured eukaryote clone All except Clone Otu0101 Autotrophs Leucocryptos SGUH1318 (100%) [KJ762967.1 ] S62, S8

1 Marine plankton sorted by flow cytometry (PE-eukaryotes) from Roscoff Astan Station, English Channel, 5 m, 2007. Page 34 of 63

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8. Discussion

Many studies have been conducted for the investigation of marine and coastal environments through metagenomic approaches. Hofrichter (2002) in a systematic summary about protists described approximately 4400 species in the Mediterranean Sea. Some of the lists created for the Mediterranean Sea about marine protists are from Velasquez and Cruzado (1995), Velasquez (1997) who recorded 736 species and 96 genera for Diatoms, and Gómez (2003) who recorded 673 Dinoflagellata species and 104 genera. A study by Genitsaris et al. (2016) in the English Channel, conducted with NGS methods, found 1174 OTUs divided in 9 supergroups with the richest supergroups being Alveolata and Stramenopiles. In our study the richest supergroup is Alveolata with Dinoflagellata and Protalveolata being the most rich and abundant groups. These huge numbers of diversity and abundance of Marine Alveolates (MALVs) indicate interactions with many different hosts e.g. Dinoflagellates (Skovgaard et al., 2005, Massana & Pedrós- Alió, 2008, Christaki et al., 2015). Many of these interactions with MALVs and their hosts are categorized as symbtiotic relationships (Bråte et al., 2012). Another NGS study in six distant European marine sites, about pelagic and benthic protists, by Massana et al. (2015) found that the most abundant and dominant groups were MALVs and were dominant over picoplankton. They also found four micro/mesoplankton protist groups to be most dominant (Acantharia, Dinophyceae, Diatomea and Ciliophora). In the present study Ciliophora are represented by 9.33% of relative abundance and 4.69% of diversity, while Acantharia are represented by only 11 OTUs and Diatomea (Stramenopiles) only with 47 OTUs, from the total of 700 eukaryotic OTUs. Moreover, many studies have been conducted for the Mediterranean Sea based also on NGS approaches. One of them is by Ghai et al. (2012), describing and comparing the Mediterranean coastal lagoons, Mar Menor (hypersaline) and Albufera de Valencia (freshwater), Spain, using high throughput metagenomic sequencing with three metagenomic datasets. Even if this study is mainly focused on prokaryotes, many

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eukaryotes are identified. The most abundant species in Mar Menor was the Alexandrium genus, which includes autotrophic and heterotrophic species. Alexandrium, which have presence in our samples, is known for producing Harmful Algal Blooms (HABs) and many toxins which can cause negative effects to humans, while it raises effects on many marine organisms, such as fishes and birds (Anderson et al., 2012). A study from Saridou et al. (2009) who studied Saronic Gulf and stated that, taking into account the abundances of the organisms examined, the internal Saronic Gulf showed concentrations familiar to other similar areas in Greece, such as Pagasitikos, Thermaikos and Maliakos gulfs. The latest NGS study on Thermaikos Gulf is by Stefanidou et al. (2018), who compared unicellular eukaryotic plankton communities from the Baltic and the Mediterranean Seas. Stefanidou et al. (2018), analyzed a total of 839 OTUs and categorized them in 8 most abundant taxonomic groups (Dinophyceae, Bacillariophyceae, Haptophyceae, Chlorophyceae, Ciliophora, Fungi, Cercozoa and MAST) from the Mediterranean Sea· all these 8 groups exist in our study. Stefanidou et al. (2018) recorded that most abundant taxonomic groups occur from one sample are Fungi (38%), Dinophyceae (10%) and Haptophyceae (5%), while in the present study the Fungi and Haptophyta were recorded to be less than 2%, from all twelve samples’ relative abundances, while Dinophyceae has 165 (23.57%) OTUs from the 700 OTUs. Protalveolata and Dinoflagellata are having the highest abundance values in all samples. Dinoflagellates include autotrophs, mixotrophs grazers and parasites. Dinoflagellates are commonly known for their toxic and Harmful Algal Blooms (HABs) species, which are causing red tides (Hallegraeff et al., 2003), and often lead to fish and other animals’ casualties, while being a source of various types of human illnesses caused by their toxic substances (Landsberg, 2002). Nikolaidis et al. (2005) are also noting that Thermaikos Gulf is influenced by the human activities and the sewage system resulting in Harmful Algal Blooms (HABs) and discoloration of the waters with huge numbers of Dinoflagellates. An important finding from previous studies (Mouratidou et al., 2004; Koukaras & Nikolaidis, 2004) is that the first human intoxication happened in Thermaikos

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Gulf on January 2000, by the Dinoflagellate species Dinophysis cf. acuminate, which are present in our samples as Dinophysis genus. The most abundant and diverse groups from the taxonomic group of Alveolata (MALV) are Dinoflagellates, specifically class Dinophyceae, subclass Gymnodiniphycidae and Noctilucales order. Alveolates are usually characterized as intracellular symbionts or parasites, when occurring in MASTs (Harada et al., 2007). Dinophyceae are characterized mainly by species of Diatom grazers (Grattepanche et al., 2011a, b). Based on the chromalveolate hypothesis Heterokonta, Haptophyta, Cryptophyta and Alveolata, which are present in this research, have obtained their red-algal plastids in a single secondary endosymbiotic incident (Cavalier-Smith, 1999). Ochrophyta are consisted mainly of autotrophic Heterokonts (Cavalier-Smith & Chao, 2006). Heterokonts or Stramenopiles belong to one of the most diverse clades of protists and include important algae (e.g. diatoms, brown seaweeds) and also, include MAST groups and parasitic species (Derelle et al., 2016). Cercozoans which are represented with 9.77% of the total abundance and 1.2% of the total diversity are proposed to maintain mostly parasitic behavior (Schnepf & Kühn, 2000). Ciliophora or Ciliates are represented in this study with 9.33 indices of relative abundance and 4.69% of diversity and are characterized as a key factor of aquatic ecosystems who act as a predator of Protozoa and Bacteria while providing nutrients at higher trophic levels (Dopheide et al., 2009). The Fungi kingdom is known to be involved in the degradation of organic matter (Raghukumar, 2004) however, in this study the Fungi kingdom is only represented with 0.1% of the total diversity and 1.75% of the total abundance. The results of these twelve samples with 700 unicellular Eukaryotes showed the great diversity and abundance that exists in Thermaikos Gulf. Importance must be given in the Closest relatives (Table 6) and more specifically in species Ptychodiscus noctiluca Stein 1883, Otu0001, [KU640194.1], for which Gómez et al., (2016) noted that Ptychodiscus noctiluca has never appeared to reach high abundance values in aquatic ecosystems and is recorded only incidental. Also, they are known to have a huge range of appearances

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from tropic to arctic waters. In our study it is presented with the genus Noctiluca amounting to 18025 reads. One major species of the Noctiluca genus is Noctiluca scintillans and is considered to be a Harmful Algal Bloom species (HAB) because during its blooming it produces high concentrations of ammonium, which may be toxic to fish and it has also been associated with mortality events of many marine invertebrate species (Horner, 2002; Smithsonian, 2012; Escalera et al., 2007). In the present study, was found only the genus Noctiluca and its Closest relative (Table 6), but it seemed appropriate to record it, due to the reason that Ignatiades & Gotsis-Skretas, (2010) have recorded Noctiluca scintillans species in its blooming period, as it caused red tides, in Thermaikos Gulf. In addition, a study by Petala et al., (2018) found the species Noctiluca scintillans in all of their samples and also the species Gonyaulax cf. fragilis and Chaetoceros spp, which are present in the samples of this study. Furthermore, Petala et al., (2018) are concluding that the incidental presence of Dinoflagellates, the often blooms and the high-level of phytoplankton biomass, suggest a lesser than good quality of the waters. The most dominant genus in our samples is Akashiwo with 47673 reads. Akashiwo sanguinea is an easily identifiable and prominent species, which has been known to form huge noticeable blooms (Horner et al., 1997). Akashiwo sanguinea is also known for red tide phenomena which are produced by its blooming (Hargraves, 2011). The second most dominant genus is Noctiluca with 28025 reads and the third is Syndiniales Group I with 14723 reads. The eukaryotic lineage Dinophyceae (Alveolata) includes Syndiniales, which are a parasitic order and are important marine producers while it has been confirmed to be one of the most diverse and well represented taxonomical group in environmental studies (Guillou et al., 2008). In a study by Cleary & Durbin (2016) on the Antarctic marine ecosystem, while the researchers expected to find low abundances of parasite sequences, yet over the half of their sequences (52%) were parasites of the Syndiniales group I and II. Also, a genus of Syndiniales group is Amoebophrya with 3155 reads in our study, for which Miller et al. (2011) have stated that this genus is is a syndinian parasite that destroys Harmful Bloom (HAB) forming algae, by developing within the host cytoplasm, or inside the host nucleus,

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depending on the particular Dinoflagellate species being parasitized (Kim et al., 2008). Approximately, all references are resulting that the genus Amoebophrya are thought to have an important impact on host population dynamics and it has been proposed for many years to be a biological key factor for controlling Harmful Algal Blooms (HABs) (Park et al., 2007). Another important observation concerns the Closest relatives which are Otu0052, [KY980102.1], Protodinium simplex Lohmann. 1908 and Otu0025, [LC068843.1], Biecheleriopsis adriatica Moestrup, Lindberg & Daugbjerg 2009. Luo et al. (2015) in their phylogeny, morphology and genetic research are resulting that these two OTUs have huge similarity levels, even greater than the similarities among each species. A species that could be a tool for the eutrophication problem may be Otu0041, [AY443022.1], Protoperidinium pellucidum Bergh. 1881 which is proposed by Buskey (1997) and Gribble et al. (2007), as one of the most important microplankton grazers with very high levels of ingestion on other diatoms. Another species of particular interest is Otu0074, [KF501033.1], Micromonas pusilla (Butcher) Manton & Parke. 1960, which is considered to be an important element of the Mediterranean Sea. It is the only species in the genus Micromonas and it belongs to the group of Prasinophyceae (Not et al., 2004). The group of Prasinophyceae, according to Swofford (2002), is the most primitive and ancient group of the green lineage which has led to the evolution of the terrestrial, higher plants. Besides the above, the genus Micromonas is one of the most abundant genus existing in the marine environments. McKie-Krisberg & Sanders (2014) are pointing out the ability of Micromonas to consume very rapidly other prokaryotes and they are suggesting that Micromonas will dominate in the primary productivity. Another dominant genus is Gonyaulax with 10450 reads, for which Pitcher et al. (2019), analyzed a large Dinoflagellate bloom in Walker Bay (South Africa, January 2017), which affected many abalone farming areas and caused the death of many millions of animals. In this study researchers found that the Dinoflagellate Gonyaulax spinifera was linked to the red tide in Walker Bay, thus the genus Gonyaulax is maybe linked to the red

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tides caused in Thermaikos Gulf. Another abundant genus is considered to be Chaetoceros with 5273 reads, which includes approximately 400 species (Rines & Theriot, 2003). Johansen et al. (1990) mentioned that many of them are found in inland waters and have very high growth rates. They also develop high lipid contents, which make them significant primary producers in saline inland waters and they are considered as a candidate for synthetic fuel production (Stoermer et al., 2003). Additionally, Thalassiosira with 2763 reads includes species which are acting as spring bloomers and primary producers in aquatic environments and considered to be an important component of phytoplankton populations while most of its species are considered as cosmopolitans (Park et al., 2016). Complete genome sequencing has now been reported for the Thalassiosira pseudonana diatoms with its’ 34 million–base pair draft nuclear genome and 129 thousand–base pair plastid adding the 44 thousand–base pair mitochondrial genomes, which revealed 24 diploid nuclear chromosomes (Armbrust et al., 2004). The analysis of Montsant et al. (2007) on Thalassiosira pseudonana reveals an unusual mixture of elements that have never previously been found together in the same organism, demonstrating the uncommon phylogenetic history of diatoms. Also, there is strong evidence that a small RNA expression exists in the transcriptome of Thalassiosira pseudonana and these small RNAs are closer to plant RNAs (Norden- Krichmar et al., 2011). In the present study the genus Islandinium corresponds to 1944 reads. Islandinium species were characteristic species of polar and subpolar environments of modern and cold paleo environments of the Quaternary. A new study has revealed a novel species named Islandinium brevispinosum, which is a new organic-walled Dinoflagellate cyst that is locally common and probably of heterotrophic affinity (Pospelova & Head, 2002). The species Islandinium minutum and Islandinium? cezare are generally characterizing the cold polar and subpolar waters of the high northern and southern latitudes and exist in waters that seldom exceed 7°C (Head et al., 2001; Kunz-Pirrung, 1998; Kunz-Pirrung, 1999; Rochon et al., 1999; de Vernal et al., 1999). Pospelova & Head (2002) are noting that Islandinium brevispinosum commonly occurs in shallow nutrient-rich estuarine

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waters that are generally characterized by mean August temperatures ranging from 23°C to 25° C and salinities from 27 to 31 psu. The Dinoflagellate genus Gymnodinium with 1687 reads, includes genera of Alexandrium and Pyrodinium, which are responsible for the production of Paralytic Shellfish Toxins (PST) in the aquatic environment and are potent neurotoxic alkaloids (Reis Costa et al., 2018). In a research study from Montoya et al. (2006) it is suggested that in the autumn of 2003, during a red tide in the coastal waters of Mar del Plata, Argentina, the red tide was consisted mainly of the chain-forming Dinoflagellate Gymnodinium catenatum. The status of the genus Gymnodinium remains uncertain while nowadays more and more species are explored and identified (Thessen et al., 2012). A particular order that should be mentioned are Tintinnids which are also known as lorica-bearing Ciliates because the lorica acts as a shell which protects their protoplasts (Balkıs & Koray, 2014), while the lorica is the basis for identification and classification. Tintinnids are key component energy-transporters which carry energy within the microbial food web and the classic food chain in the marine planktonic ecosystems (Pierce & Turner, 1992), but are presented only by 1049 reads, which are the lowest reads in the present study. However, these species are easily identified based on their morphological features (Santiago & Lagman, 2018) which made them perfectly capable to play the role of bio-indicators of different water masses (Kim et al., 2012), as well as to other research interests such as diversity, distribution and changes of micro zooplankton communities (Dolan & Gallegos, 2001). Last but not least, Tintinnids, have a fossil record of the loricae, which is very easily fossilized, thus they are a promising opportunity to meet and track Ciliate evolution over the years (Dolan et al., 2012). Even if Cryptophytes have less than 2% of the total abundance, they are major ecological phototrophic components in aquatic environments, causing red tides in many countries (Yeong Du Yoo et al., 2017). Kim et al. (2015) support that the genus Teleaulax has donated its plastid to Dinoflagellates Dinophysis caudate, Amylax triacantha but firstly to the Ciliate Mesodinium rubrum through the trophic web. More importantly these organisms have preserved this acquired plastid, causing Harmful Algal Blooms (HABs) in

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aquatic environments. Jewett et al. (2008) linked the presence of the Harmful Algal Blooms (HABs) with the invasion of alien species. Skeletonema species with 1572 reads, especially in coastal and marine environments, often form dense blooms and are common phytoplankters (Castillo et al., 1995; Huang et al., 2007; Kooistra et al., 2008). They are a major autotrophic key component of the aquatic ecosystems acting as primary producers which maneuver carbon, oxygen, minerals, vitamin B, temperature, pH, food web flow (Boyce et al., 2010). Family Kareniaceae has only 83 reads and two species (Karlodinium and Takayama). This family causes many losses of invertebrates such as oysters (Nézan et al., 2014), as well as fish deaths in many countries (Jeong et al., 2016). On the other hand, six new species were characterized and all of them are containing fucoxanthin or its derivatives, as the main carotenoid pigments (Salas, 2008). A recent study (Zhang et al., 2015) suggests that fucoxanthin is a special carotenoid and has many bioactivities such as preventing and treating lifestyle-related diseases, as obesity, diabetes, cancer, cardiovascular disease, and other chronic diseases. In this study, from the Closest relatives of Table 5 in the Appendix, there was recorded the potentially harmful Otu0056, [MG877164.1] Hydroides elegans Haswell, 1883. Nedved & Hadfield (2009) characterized this species as a problematic fouling organism that can cause damage in ships and habits in warm water all around the globe. The frequent changes in environmental conditions in the interior Thermaikos gulf lead to variations of different parameters, allowing for different protist organisms to grow in a given time period.

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9. Conclusions

To conclude, this study suggests that the protistan communities of Thessaloniki Bay can be diverse and abundant. Many of the protists that were analyzed are pathogenic or potentially harmful for invertebrates and vertebrates. This research provided evidence about the major taxonomic groups who dominate in DNA quantities and are Dinoflagellata and Protalveolata in Thermaikos Gulf. Specifically the most important conclusions are that: • The final number of sequences that was screened was 148756. The lowest sequences are observed in the sample of 23/8/2017, while the highest are observed in the sample of 18/10/2017. • The maximum OTUs are found in the sample of 9/5/2017 and the minimum in the sample of 15/11/2017 (Figure 3). • The highest diversity values are in the samples of 26/7/17 and 9/5/17, while the lowest are in the samples of 18/10/17 and 12/4/17 (Simpson 1-D Diversity index, Figure 4). • The most diverse samples, according to Dominance-D (Figure 5) are the samples of 26/7/17, 9/5/17 and 23/8/17, while the most dominated sample is that of 18/10/17. • The most even protistan communities are in the samples of 9/5/17, 26/7/17 and 23/8/17 (Evenness e^H/S, Figure 7). • The most diverse samples according to Chao1 index (Figure 9) are those of 9/5/17, 23/8/17, 13/12/17 and 26/7/17. On the other hand, the least diverse samples are those of 15/11/17, 18/10/17 and 6/2/18. • The produced taxonomic groups were 15 with Dinoflagellata, Protalveolata, and Ochrophyta, overcoming 50% of the total number of OTUs (Figure 10). • The most abundant samples are the samples of 9/5/17, 13/12/17 and 23/8/17 (Figures 11 & 12). • The most diverse taxonomic group is Dinoflagellata and the next highest taxonomic group is Protalveolata (Figure 13). Page 43 of 63

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• The richest samples are the samples of 13/12/17, 12/4/17, 28/6/17, 10/1/18, 9/5/17 and 15/11/17. The least rich samples are the samples of 23/8/17, 20/9/17, 18/10/17, 26/7/17, 6/2/18 and 7/6/17 (Figure 14). • The most dominant OTUs are Akashiwo, Noctiluca and Syndiniales Group I (Figure 15).

• The highest number of OTUs from the Closest relatives (Table 6) are Ptychodiscus noctiluca, Protodinium simplex, Biecheleriopsis adriatica, Protoperidinium pellucidum, Gyrodinium cf. gutrula, Adenoides eludens, Micromonas pusilla, Thalassiosira pseudonana and Teleaulax gracilis The estimation and identification of the protistan communities in Thermaikos Gulf improved the understanding of the whole protistan communities including the interactions between biotic and abiotic elements. Even if, more scientific research must be developed in this ecosystem, this research is a contribution for current and future metagenomics studies while the produced data will help understand the functions of this sensitive ecosystem. This metagenomics analysis suggests that the protistan communities are characterized as diverse with the new OTUs appearing across the samples. Thermaikos Gulf is affected a lot by the protists that assist eutrophication and red tides phenomena. On the contrary, many protists are found to have positive effects on the ecosystem, having abilities of symbiosis and helping maintain the equilibrium of their ecosystem. This study also demonstrates the need for integrated environmental marine assessment. However, in a marine ecosystem with continuous anthropogenic impacts it is difficult to determine the environmental conditions that favor the development of phytoplankton and its distribution. This study can constitute the starting point for monitoring programs which can investigate further the interactions between biotic and abiotic components. The marine environment is a valuable heritage that must be protected, preserved and, if possible, to restore itself by maintaining and ensuring the diversity and dynamics of the oceans and seas, which must be clean, healthy and productive.

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10. Bibliography

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Appendix

Table 1: This table presents in chronological order the amount of the newly recorded OTUs per sample, adding each time the new OTUs to the already counted, from the previous sample/samples. (Figure 1).

OTUs New OTUs per Samples presence/absence sample S8 12/4/17 150 150 S15 9/5/17 292 142 S22 7/6/17 341 49 S28 28/6/17 388 47 S35 26/7/17 455 67 S41 23/8/17 523 68 S48 20/9/17 542 19 S55 18/10/17 570 28 S62 15/11/17 593 23 S69 13/12/17 661 68 S76 10/1/18 687 26 S83 6/2/18 700 13

Table 2: Total relative number of OTUs in the taxonomic groups (Figures 10, 11, 12).

Groups Groups Groups Groups Groups Groups Samples Cercozoa Chlorophyta Ciliophora Cryptophyceae Dinoflagellata Fungi S8 12/4/17 8 13 19 3 48 1 S15 9/5/17 11 16 9 6 51 0 S22 7/6/17 12 7 9 6 40 2 S28 28/6/17 19 19 2 4 28 2 S35 26/7/17 16 16 18 6 49 4 S41 23/8/17 18 22 16 7 46 0 S48 20/9/17 8 12 21 6 36 3 S55 18/10/17 11 3 8 5 41 5 S62 15/11/17 6 5 7 3 38 2 S69 13/12/17 15 12 24 7 41 4 S76 10/1/18 17 11 19 9 47 2 S83 6/2/18 7 4 13 5 41 1 Groups Groups

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Samples Haptophyta MAST Ochrophyta Other Opisthokonta Protalveolata Prymnesiales S8 12/4/17 2 5 16 2 22 7 S15 9/5/17 6 7 27 3 47 15 S22 7/6/17 3 3 12 0 30 8 S28 28/6/17 4 0 16 0 16 12 S35 26/7/17 4 5 17 3 35 15 S41 23/8/17 3 2 21 4 45 10 S48 20/9/17 1 0 8 0 24 8 S55 18/10/17 3 1 7 1 12 3 S62 15/11/17 1 1 9 1 17 2 S69 13/12/17 4 3 30 1 47 8 S76 10/1/18 2 3 14 1 23 4 S83 6/2/18 1 0 12 2 17 6 Groups Samples Retaria Picozoa Labyrinthulomycetes Other Protists S8 12/4/17 1 0 0 0 S15 9/5/17 9 1 0 0 S22 7/6/17 3 2 0 0 S28 28/6/17 1 0 1 0 S35 26/7/17 2 1 2 3 S41 23/8/17 2 2 1 2 S48 20/9/17 0 2 0 0 S55 18/10/17 1 0 0 0 S62 15/11/17 0 0 0 1 S69 13/12/17 1 3 1 1 S76 10/1/18 1 0 0 0 S83 6/2/18 0 0 0 0

Table 3: Relative abundance of taxonomic groups from the samples (Figures 13, 14).

S8 S15 S22 S28 S35 S41 Groups/Samples 12/4/17 9/5/17 7/6/17 28/6/17 26/7/17 23/8/17 Dinoflagellata 17832 8736 9349 12319 5043 1915 Protalveolata 2147 2308 916 499 1133 408 Ciliophora 644 259 229 11 381 173 Chlorophyta 443 401 28 552 211 145 Prymnesiales 232 1930 303 615 654 126 Ochrophyta 174 824 70 2406 2249 1220 Cryptophyceae 92 212 139 20 195 130 Haptophyta 76 343 57 110 115 49

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Cercozoa 34 106 51 366 130 62 Fungi 8 0 9 16 19 0 MAST 8 59 7 0 20 6 Other Opisthokonta 4 9 0 0 17 10 Retaria 3 1206 414 10 159 10 Labyrinthulomycetes 0 0 0 2 11 3 Other Protists 0 0 0 0 12 6 Picozoa 0 16 11 0 9 3

S48 S55 S62 S69 S76 S83 Groups/Samples 20/9/17 18/10/17 15/11/17 13/12/17 10/1/18 6/2/18 Dinoflagellata 6128 9318 9982 10013 11778 7095 Protalveolata 619 115 736 12089 624 383 Ciliophora 1180 32 49 985 2264 1596 Chlorophyta 140 12 44 793 266 49 Prymnesiales 112 18 3 78 270 118 Ochrophyta 330 94 2447 424 81 1019 Cryptophyceae 224 34 12 986 565 56 Haptophyta 11 80 4 85 5 32 Cercozoa 48 476 30 110 521 53 Fungi 12 28 6 51 18 2 MAST 0 2 1 42 24 0 Other Opisthokonta 0 2 1 2 12 110 Retaria 0 3 0 0 1 0 Labyrinthulomycetes 0 0 0 0 0 0 Other Protists 0 0 2 8 0 0 Picozoa 15 0 0 9 0 0

Table 4: Dominant OTUs (Figure 4).

OTUs Reads Percentages Akashiwo 47673 41,49% Noctiluca 18025 15,69% Syndiniales Group I 14723 12,81% Gonyaulax 10450 9,09% Chaetoceros 5273 4,59% Amoebophrya 3155 2,75% Thalassiosira 2767 2,41% Islandinium 1944 1,69% Strombidium 1880 1,64% Page 61 of 63

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Arthracanthida 1790 1,56% Gymnodinium 1685 1,47% Teleaulax 1664 1,45% Skeletonema 1572 1,37% Protodinium 1253 1,09% Tintinnopsis 1049 0,91%

Table 5: Unclassified OTUs after the GenBank® Blast (Clark et al. 2015)

OTUs Closest Relative (100%) [Accession Number] Isolation source Uncultured metazoan partial 18S rRNA gene Otu0009 (99%), LN717274.1 Sponge tissue Otu0017 Protodrilus adhaerens(100%), KJ451197.1 Synstellicola paracarens voucher LEGO- Otu0022 POE021(95%), KT030270.1 South Korea Uncultured cercozoan clone DH1321F11 (94%), surface layer sediment Otu0026 KM067448.1 from the East China Sea Uncultured ciliate clone BBL1SF10.66 (100%), Ocean water at 3 m Otu0032 KC488368.1 depth at station Otu0036 Teleaulax amphioxeia(99%), KY980249.1 BH46_160 Uncultured marine eukaryote clone Otu0044 MB07.8(99%), EF539120.1 western Pacific coast Uncultured marine eukaryote clone Otu0048 MB07.8(99%), EF539120.2 western Pacific coast Uncultured Rhizaria clone F.13_4115(99%), surface layer sediment Otu0053 KP685345.1 from the East China Sea Amoebophrya sp. isolate BH46_67 (100%), Otu0054 KY980195.1 BH46_67 Hydroides elegans voucher AM W48217 Otu0056 (100%) MG877164.1 Uncultured eukaryote clone dhot2b7 (100%), Otu0061 EU500001.1 ocean surface waters marine plankton from Roscoff Astan Station, Uncultured marine ciliate clone English Channel, 5 m, Otu0062 RA071004T.013 (99%), FJ431819.1 2007 seawater; sample collection Uncultured marine eukaryote clone depth:chlorophyll Otu0107 MO.011.CM.00343(89%), HM858746.1 maximum (29 m)

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Uncultured syndiniales clone PROSOPE.C9- Otu0108 5m.47(99%), EU792993.1 Mediterranean Sea;2 Otu0109 Chaetoceros sp. Na28A1(97%), MG972364.1 marine Uncultured Dinoflagellate clone 18.f1.3 (84%), Otu0121 KX602133.1 surface sea water Leptocylindrus aporus strain SZN-B651(99%), LTER-MC, Gulf of Otu0123 KC814810.1 Naples Uncultured alveolate clone F.18_129(97%), surface layer sediment Otu0124 KP685290.1 from the East China Sea Otu0127 Uncultured metazoan (99%) LN717273.1 sponge tissue Uncultured eukaryote clone Lake Pontchartrain, 051011_T2S1_W_T_SDP_052 (99%), Transect 2 Station1, Otu0132 FJ349697.1 water, unfractionated

2 Mediterranean Sea; marine picoplankton 3 micrometer from cruise PROSOPE, 1999, Stn 9, Depth 5m Page 63 of 63