Downloaded from Genbank Using Custom Scripts

LABYRINTHULOMYCETES DIVERSITY META-ANALYSIS

Jingwen Pan

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF

THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES

(Botany)

THE UNIVERSITY OF BRITISH COLUMBIA

(Vancouver)

January 2016

Abstract Labyrinthulomycetes are a group of ubiquitous stramenopiles that inhabit a wide range of habitats and play important ecological roles as nutrient recyclers and sometimes disease causing agents. Even though they have had a long history of being studied, their diversity has not yet been fully explored. The lack of a comprehensive reference database with up-to-date phylogeny also hinders any pursuits in understanding the ecological distribution of this group. This study was designed with the purpose of constructing a curated reference database and a phylogenetic tree based on existing 18S rDNA data, and then using this database to uncover any hidden diversity and novelty among Labyrinthulomycetes and provide a reference guidance for future identification. Using the newly-created reference database, I also analyzed high-throughput environmental sequencing data from two databases. My results reveal extensive diversity within the Labyrinthulomycetes, and recover many previously unknown environmental sequences, greatly expanding our knowledge of the ecological distribution of this group. The high- throughput environmental sequencing data analysis also shows some of the newly identified environmental clades to be particularly abundant in the ocean. The phylogenetic framework I have provided in this study, together with the metadata I have compiled, will serve as a useful tool for future ecological and evolutionary studies of this widespread lineage.

ii Preface This thesis is original and unpublished. It is based on work designed by J. del Campo and I. I conducted all the analysis and J. del Campo provided supervisory guidance. I prepared all figures and wrote the manuscript, and J. del Campo and P.J. Keeling revised it.

iii Table of Contents

Abstract ...... ii Preface ...... iii Table of Contents ...... iv List of Tables ...... v List of Figures ...... vi Acknowledgements ...... vii Chapter 1: Introduction ...... 1 1.1 Labyrinthulomycetes Introduction ...... 1 1.1.1 Labyrinthulomycetes Classification ...... 1 1.1.2 Labyrinthulomycetes Morphology and Physiology ...... 3 1.1.3 Labyrinthulomycetes Ecology and Lifestyle ...... 3 1.1.4 Summary ...... 5 1.2 High-throughput Environmental Sequences Studies ...... 5 1.3 Goals ...... 7 Chapter 2: Materials and Methods ...... 9 2.1 Reference Phylogenetic Tree Construction ...... 9 2.2 Reference Database Annotation ...... 10 2.3 Labyrinthulomycetes V9 Reads Database ...... 11 2.4 Abundance and Richness Distribution Patterns ...... 12 Chapter 3: Results ...... 13 3.1 Phylogeny of Labyrinthulomycetes ...... 13 3.2 Examining Abundance and Richness using GenBank Sanger Sequences ...... 14 3.2.1 Host Association ...... 15 3.3 Abundance and Richness using V9 Reads ...... 16 Chapter 4: Discussion ...... 19 4.1 Phylogeny and Classification ...... 19 4.2 Environmental Distribution of Labyrinthulomycetes ...... 22 Chapter 5: Conclusion and Future Directions ...... 24 References ...... 34

iv List of Tables Table 1: List of previously described environmental clades (Collado-Mercado et al. 2010) and the new clades they now belong to according to the present phylogenetic study .... 30 Table 2: Total abundance and richness for the three Labyrinthulomycetes databases ...... 32

v List of Figures Figure 1: Diversity of Labyrinthulomycetes inferred from a maximum likelihood (RAxML) phylogenetic tree constructed using 18S rDNA sequences...... 26 Figure 2: Diversity of Labyrinthulomycetes (full version) ...... 27 Figure 3: Abundance and richness for major groups of Labyrinthulomycetes ...... 31 Figure 4: Heatmap showing the abundance distribution of major groups of Labyrinthulomycetes according to different environmental parameters ...... 33

vi Acknowledgements I would like to thank many people who made this thesis possible. First, I would like to express my sincere gratitude to my supervisor Patrick Keeling for his support, guidance and patience on my study. I also thank the members of my committee, Brian Leander, James Berger and Rosie

Redfield for their advice. I am also grateful to all members from the Keeling lab, the Leander lab and the Fast lab for their help and inspiration along the way, and for creating such a wonderful working environment. I am also thankful to faculty and staff of UBC, especially Veronica

Oxtoby, for general help.

I own particular thanks to Javier del Campo, whose expertise and guidance have helped me in all the time of research and in writing of this thesis, and for being patient with me during stressful moments.

Finally, I wish to thank my parents and friends for their love and continuous support throughout my study and my life. They have walked through every joyful and difficult moment with me.

Special thanks to Fen Liu and Cuiyu Lin for their daily amusement and advice on Excel from the later.

vii Chapter 1: Introduction 1.1 Labyrinthulomycetes Introduction Labyrinthulomycetes are a group of unicellular eukaryotic microorganisms commonly found in marine environments (Raghukumar 2002). The slime-like appearance of most of its members has resulted in previous misplacement of this group within Fungi, Oomycetes (Stramenopiles) and amoebae. Observations of mitochondria with tubular cristae and their heterokont, biflagellate zoospore production, together with support from 18S rRNA gene phylogenies eventually led to the placement of this group within the Stramenopiles, together with the photosynthetic

Ochrophytes (diatom, brown algae, etc) and other non-photosynthetic groups, like the plant pathogenic Oomycetes (Cavalier-Smith et al. 1994; Tsui et al. 2009). Within the stramenopiles, the Labyrinthulomycetes is a monophyletic group and is characterized by having a cell wall made of scales containing proteins and sulphated polysaccharides, as well as the production of an ectoplasmic network (EN), which is a membrane-bound, branched network secreted through a unique organelle called bothrosome (sagenogenetosome).

1.1.1 Labyrinthulomycetes Classification Since the first description of Labyrinthula in 1867 by Cienkowski, the classification of

Labyrinthulomycetes has undergone several changes and rearrangements. During the late 20th century, Labyrinthulomycetes were divided into two groups by Olive (1975) and Porter (1989), with labyrinthulids having only one genus, Labyrinthula, and thraustochytrids having seven genera, Thraustochytrium, Japonochytrium, Schizochytrium, Althornia, Ulkenia, Aplanochytrium and Labyrinthuloides. However, several genera of thraustochytrids were later proven not to be monophyletic, and some species of these genera were subsequently moved into the labyrinthulids by Honda and collaborators (Honda et al. 1999). These include members of Thraustochytrium,

1 Schizochytrium, Aplanochytrium and Labyrinthuloides. In the early 21st century,

Labyrinthulomycetes were split into three groups, with aplanochytrids and labyrinthulids each having one genus, Aplanochytrium and Labyrinthula respectively, while thraustochytrids contained the rest of the genera (Leander and Porter 2001; Leander et al. 2004). In addition, some species of Labyrinthuloides were transferred to the genus Aplanochytrium. Two genera of thraustochytrids, Schizochytrium sensu lato and Ulkenia sensu lato, were subsequently rearranged into seven genera based on combined studies on their morphology, life cycle, biochemistry and phylogeny (Yokoyama and Honda 2007; Yokoyama et al. 2007):

Schizochytrium, Oblongichytrium, Aurantiochytrium, Ulkenia, Botryochytrium, Parietichytrium and Sicyoidochytrium. While aplanochytrids and labyrinthulids usually group together forming a monophyletic Labyrinthulida (Tsui et al. 2009; Beakes et al. 2014), the monophyly of thraustochytrids remains debatable, with Oblongichytrium often seen as sister to Labyrinthulida

(Yokoyama et al. 2007; Anderson and Cavalier-Smith 2012; Gomaa et al. 2013; Takahashi et al.

2014) or sister to both Labyrinthulida and other thraustochytrids (Yokoyama and Honda 2007;

Collado-Mercado et al. 2010). A new genus, Amphifila (Amphifilidae, Thraustochytrida) has been erected to accompany the rearrangement of Diplophrys marina into Amphifila marina

(Anderson and Cavalier-Smith 2012). The original genus Diplophrys with its remaining species is now placed under Diplophryidae (Amphitremida, Labyrinthulomycetes), together with

Amphitrema and Archerella. The later two have traditionally been placed within Cercozoa and

Foraminifera, and are now included in Labyrinthulomycetes under family Amphitremidae

(Amphitremida) due to recent 18S rRNA gene studies (Gomaa et al. 2013; Takahashi et al. 2014) that have them placed sister to Thraustochytrida and Labyrinthulida with strong support.

2 1.1.2 Labyrinthulomycetes Morphology and Physiology Labyrinthulida are characterized by gliding mobility using the ectoplasmic network (EN). Unlike the spindle shaped, colonial Labyrinthula (labyrinthulids) that have their cells enrobed by the

EN, cells of Aplanochytrium are often solitary and are not embedded within an EN (Leander et al. 2004; Tsui et al. 2009). In addition to biflagellate zoospores, Aplanochytrium also produce non-flagellated “crawling spores” that glide using the network (Leander et al. 2004).

Thraustochytrida cells are spherical, unicellular or colonial. They are immobile and the EN is only used to increase surface area for enzyme secretion and nutrient absorption. Amoeboid cell stages have been observed in some genera including Ulkenia, Sicyoidochytrium, Parietichytrium and Botryochytrium. Althornia are free floating and do not have a bothrosome or an ectoplasmic network (Alderman and Jones 1971; Moss 1985; Bower 1987). Amphifila differ from other thraustochytrids in having pseudostomes instead of true bothrosomes, and ectoplasmic elements in the form of pseudopodia (Anderson and Cavalier-Smith 2012; Gomaa et al. 2013). Several refractive granules can often be seen in the cytoplasm of Amphifila under light microscopy. Cells of Amphitremida also possess pseudostomes and pseudopodia. Similar to Amphifila, Diplophrys also bear refractive granules. Amphitrema and Archerella both harbor photosynthetic zoochlorellae endosymbionts and are thus mixotrophic. Biflagellate zoospore production has not been observed in either Amphitremida and Amphifila (Gomaa et al. 2013).

1.1.3 Labyrinthulomycetes Ecology and Lifestyle Labyrinthulomycetes are ubiquitous and can be found in a diverse range of habitats, including both freshwater and marine, from epipelagic surface to deep-sea column (Raghukumar 2002).

They have also been isolated from various kinds of substrates (Raghukumar and Damare 2011), including but not limit to algae, mangrove leaves, seagrass, coral mucus, and mollusks. Some of

3 these associations are parasitic, like the thraustochytrid quahog parasite X of hard-shell clam

Mercenaria mercenaria (Ragan et al. 2000), the wasting disease of eel grass Zostera marina caused by Labyrinthula sp. (Muehlstein et al. 1991), or the turf grass parasite Labyrinthula terrestris (Bigelow et al. 2005). However, Labyrinthulomycetes can also be seen on apparently healthy organisms, suggesting their relationships may be commensal or even mutualistic.

Kramarsky-Winter et al. (2006) showed that the white coating aggregate on the coral mucus of

Fungia granulosa contained several different members of Labyrinthulomycetes. Excised polyp tissues with these inhabitants remained viable and were able to settle and grow. Aplanochytrium minuta has also been isolated from both healthy and detrital brown alga Sargassum cinereum

(Sathe-Pathak et al. 1993). The isolation was done after surface sterilization of the alga frond, suggesting the association was endobiotic. Labyrinthulomycetes have also been proposed to be involved in nutrient recycling. Most Labyrinthulomycetes are saprotrophic feeders through an osmotrophic or phagotrophic mode of nutrient uptake. In fact, they are often seen to be associated with detritus like fallen mangrove leaves, decomposing algae, and fecal pellets of marine invertebrates (Raghukumar and Raghukumar 1999; Tsui et al. 2009).

Evaluation of Labyrinthulomycetes biomass using the acriflavine direct detection method

(AfDD), based on fluorochrome staining of the sulphated polysaccharide containing cell walls, has shown this group to be much more abundant in the ocean than previously suspected

(Raghukumar and Schaumann 1993). Their biomass can sometimes even be equivalent to that of bacteria during phytoplankton decay (Raghukumar et al. 2001). Members of thraustochytrids, especially Aurantiochytrium, are known for high-level production of omega-3 polyunsaturated fatty acids (PUFA), including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).

4 PUFAs are considered to be not only ecologically important for marine animals (Gladyshev et al.

2009), but also commercially valuable as a dietary supplement for human health because they suppress cardiovascular diseases and support brain development in newborns (Simopoulos 1991;

Ruxton et al. 2004). There has also been an increased interest in Aurantiochytrium as a potential candidate for biodiesel production and for squalene synthesis, a natural antioxidant popular in the cosmetic industry (Lee Chang et al. 2012).

1.1.4 Summary Even though some aspects of the Labyrinthulomycetes have been studied in depth, their environmental diversity has not yet been fully explored. Collado-Mercado et al. (2010) and Ueda et al. (2015) both attempted to uncover the hidden diversity of this group. However, they both focussed exclusively on marine samples, and therefore neglected the potential diversity from freshwater and soil environments, especially in the Amphifilidae and Amphitremida. Therefore, in this study I would like to do an exhaustive exploration and analysis of the diversity and novelty of the Labyrinthulomycetes using publically available data.

1.2 High-throughput Environmental Sequences Studies Ecological studies of microbial life often utilize DNA metabarcoding methods that use high- throughput sequencing (HTS) to access the community composition of study sites. The HTS approach is advantageous in the tremendous amount of data it can generate at a fraction of cost and time as compared to traditional Sanger sequencing method (Caporaso et al. 2012; Pawlowski et al. 2014). DNA metabarcoding often relies on amplification of the hypervariable regions

(HVRs) of the SSU rDNA using specifically designed primers that can target a wide range of taxa (Stoeck et al. 2010). The most commonly used regions include the V3 and V6 regions of the

5 16S rDNA for prokaryotes (Huse et al. 2008), the internal transcribed spacer (ITS) regions for fungi (Vobis et al. 2004), and the V4 and V9 regions of the 18S rDNA for eukaryotic microbes

(Amaral-Zettler et al. 2009; Stoeck et al. 2010). These sequence data can representatively sample a community, but interpreting them correctly relies heavily on the reference database to which the environmental samples are compared to make a taxonomic assignment. Some of the existing curated databases include the PR2 database (Guillou et al. 2012), the Greengenes database

(DeSantis et al. 2006) and the SILVA database (Quast et al. 2012). The taxonomical classification for Labyrinthulomycetes in these databases is often not up to date, with most of the sequences only being assigned as labyrinthulids or thraustochytrids. Considering the wide distribution of Labyrinthulomycetes in different environments and their importance, it is useful to create a curated reference database of sequences from this lineage that can be used for future studies. The curated reference data can also be used in identification of high-throughput environmental sequences (HTES) generated from metabarcoding as mentioned above. I will apply the newly generated reference data to the identification of Labyrinthulomycetes in two existing V9 HTES databases, the VAMPS database (Huse et al. 2008) and the Tara Ocean database (de Vargas et al. 2015), both of which also have environmental metadata available.

VAMPS (Visualization and Analysis of Microbial Population Structures) is a website designed to allow users to upload their HTES data for taxonomical annotation and downstream analysis like abundance and richness, by providing an interactive graphical user interface (Sogin et al.

2006; Huse et al. 2008). HTES data uploaded onto VAMPS are also accessible for other users.

For HTES data, VAMPS currently utilizes reference databases constructed from SILVA database

(Quast et al. 2013) for various HVRs. The HVR reference databases are generated by in-silico

6 excision of the corresponding section of the full-length sequences from SILVA. Detailed methodology can be viewed on the VAMPS website (http://vamps.mbl.edu). Among the different HVRs, the V9 database is optimized for eukaryotes. HTES raw data uploaded to the server are first de-replicated, keeping only unique sequences for each dataset. Taxonomical annotation of these unique sequences are done through GAST (Global Alignment for Sequence

Taxonomy) (Sogin et al. 2006) against the HVR reference databases.

The Tara Ocean project (2009-2013) is a global expedition sampling plankton at various depths in a wide range of marine ecosystems around the world. Detailed experimental design for it can be found in Pesant et al. (2015). Briefly, plankton samples were collected from pre-determined depths (surface water, deep chlorophyll maximum, or the mesopelagic zone), size filtered and cryopreserved. PCR amplification of the total DNA extract using V9 specific primers was then followed by Illumina Hiseq sequencing. After initial quality filtering of the raw data, they were then clustered into Swarms (Mahé et al. 2014) and taxonomically assigned using a customized reference database called V9_PR2 database (de Vargas et al. 2015).

Because data obtained from both datasets, either unique sequences from VAMPS or Swarms from Tara Ocean, are sequences that have been clustered, the term ‘unique sequence’ will be used in this manuscript when referring to these clustering representatives, and the original data from the sequencing machine will be referred to collectively as ‘raw data’ or ‘raw reads’.

1.3 Goals The goal of this study was to 1) explore the diversity and novelty among Labyrinthulomycetes based on existing 18S rDNA data, 2) construct a curated database and a reference phylogenetic

7 tree, and 3) use my curated database to develop high-throughput environmental sequencing data analyses on the Labyrinthulomycetes.

8 Chapter 2: Materials and Methods 2.1 Reference Phylogenetic Tree Construction All GenBank 18S rDNA sequences taxonomically identified as Labyrinthulomycetes were retrieved using the corresponding taxid (35131). Mitochondrial sequences and complete genomes were excluded, as were sequences shorter than 500bp. The remaining sequences were clustered at 97% identity using USEARCH v7.0.1090 (Edgar 2010). In order to build the tree, 44 other stramenopiles, a Planomonas sp. and a Sabulodinium sp. sequences were used as outgroups

(Massana et al. 2014). All sequences were aligned and trimmed using MAFFT (Katoh and

Standley 2013) with default settings and trimAl (Capella-Gutierrez et al. 2009) respectively. A maximum likelihood phylogenetic tree was constructed with RAxML 8.1.3 (Stamatakis 2014) using the rapid hill climbing algorithm and GTRCATI evolutionary model. Sequences were then assessed for whether they truly belong to Labyrinthulomycetes based on the tree topology, and taxonomic literature (Leander and Porter 2001; Leander et al. 2004; Yokoyama and Honda 2007;

Yokoyama et al. 2007; Gomaa et al. 2013; Liu et al. 2013). The remaining sequences were then used to retrieve more sequences from GenBank using blastn (Camacho et al. 2009) (E- value=10e-5) as previously described (del Campo and Massana 2011; del Campo and Ruiz-Trillo

2013). Only the first 100 new sequences were kept for each query. After removing duplicated sequences, the new ones were added to my dataset. This new dataset was then be used to construct a phylogeny, as described above, and new sequences belonging to

Labyrinthulomycetes were blasted against nt. This cycle was repeated until no more new sequences that cluster with Labyrinthulomycetes were retrieved from GenBank. Sequences were then checked for chimeras using both the built-in function of Qiime v1.9.1

(identify_chimeric_seqs.py) (Caporaso et al. 2010b) against the SILVA database (v119,

9 97%identity) and USEARCH (uchime_denovo). Chimeric sequences were also manually examined.

After chimera cleanup, the final phylogenetic tree was built using RAxML with the settings mentioned above. Statistical support for the consensus tree was calculated using non-parametric bootstrapping with 1,000 replicates. Support from Bayesian posterior probability was examined with MrBayes v3.2.2 (Ronquist and Huelsenbeck 2003; Altekar et al. 2004) using the

GTR+Gamma model. The analysis was performed using 64 MCMC chains with a sampling frequency of every 1,000th generation. A consensus tree was generated after discarding the first

50% of the total generations as “burn-in”.

2.2 Reference Database Annotation Sequences were first identified for annotation from previously published works (Leander and

Porter 2001; Leander et al. 2004; Yokoyama and Honda 2007; Yokoyama et al. 2007; Collado-

Mercado et al. 2010; Gomaa et al. 2013; Liu et al. 2013), excluding environmental clades. I tried to adopt the established taxonomy as my classification method as far as it was supported by my tree. New names based on the most representative cultured strain were added for those undescribed clusters when the bootstrap support for the clade was 70% or higher indicating a moderately well supported clade. If the group contained only environmental sequences, the group was then named THR”0” for the groups belonging to the Thraustochytriidae and LAB”0” for the groups that could be only classified as Labyrinthulomycetes, where “0” is a number.

Environmental singletons (OTUs represented by a single sequence) were unannotated. The annotation for different groups and for individual sequences was then deposited into the database. The classification for the monophyletic, non-environmental groups followed the most

10 updated taxonomy available (Gomaa et al. 2013; Beakes et al. 2014). Metadata for the sequences in my dataset was downloaded from GenBank using custom scripts. For sequences still missing environmental data, their information was then collected manually from the literature.

2.3 Labyrinthulomycetes V9 Reads Database Unique sequences annotated as Labyrinthulomycetes or more generically as Stramenopiles were retrieved from the two V9 databases (Huse et al. 2014; de Vargas et al. 2015). The fasta file containing all the reads was sorted by length using USEARCH and clustered into OTUs with

97% similarity using Qiime with default setting (UCLUST). OTUs were then aligned with the reference alignment using PyNAST (Caporaso et al. 2010a) embedded in Qiime (align_seqs.py).

The reference alignment was the same alignment that was used to generate the reference phylogenetic tree in the previous section. OTUs that failed to align were discarded. The PyNAST alignment output was merged with the reference alignment and filtered for gap positions using

Qiime (filter_alignment.py) with gap filtering threshold set to 0.99 and entropy threshold set to

0.0001. Identification of Labyrinthulomycetes reads was done using a maximum likelihood phylogenetic approach by mapping the OTUs onto the Labyrinthulomycetes reference tree using the Evolutionary Placement Algorithm (EPA) of RAxML (Berger et al. 2011). The use of

RAxML-EPA for identification of short reads and its accuracy has been assessed by several studies (Matsen et al. 2010; Dunthorn et al. 2014; Parfrey et al. 2014; Chesters et al. 2015;

Filipski et al. 2015). OTUs that were not placed within the Labyrinthulomycetes were removed, together with their 97% clustered unique sequences. Trees using the remaining sequences were built consecutively until no more reads were placed outside the Labyrinthulomycetes. OTUs and their clustered unique sequences were then annotated according to their placement. OTUs that

11 were not placed with any previously defined groups were assigned a new name following the rule mentioned in the previous section.

2.4 Abundance and Richness Distribution Patterns For abundance and richness analyses, comparisons between different groups were done at the order level, except for the order Thraustochytrida where the family level was used. Abundance represents the number of sequences found in each group, while richness calculates the number of different OTUs, sequence clusters that are less than 97% similarity to each other. For data from

VAMPS and Tara Ocean, abundance was calculated based on raw data through custom scripts that link the OTU table with their previous clustering frequency tables (available online for each database). The abundance of each group under each environmental category was then calculated using an Excel pivot table and a heatmap was generated to better illustrate the distribution patterns. For example, to calculate the abundance distribution of different Labyrinthulomycetes groups in the freshwater environment, the total number of raw reads from different sample sets was summarized using a pivot table for each group. Groups with no freshwater reads were denoted 0. A two-color scale heatmap was then generated across different groups based on their relative percentage over a grand total for each environmental category. Richness was calculated by summarizing the number of different OTUs that occurred in each group. Because the V9 dataset is a collection of data from different projects that sometimes utilize different sampling protocols, comparison between the distributions of any given group across different environmental parameters is not meaningful.

12 Chapter 3: Results 3.1 Phylogeny of Labyrinthulomycetes In total, 1,181 18S rDNA sequences were retrieved from GenBank after our phylogenetic analysis. These sequences were used to build our reference database together with their environmental metadata. The final phylogeny for these sequences was constructed with 332

OTU97 representatives of the 1,181 sequences. Two versions of the tree are shown to better assist our discussion: a summarized version (Figure 1A) with all the sequences belonging to the same phylogenetic group collapsed together and a full version showing each individual sequence

(Figure 2). Virtually all of the previously defined genera of Labyrinthulomycetes were recovered with strong support (>70/0.7). While most genera of Thraustochytriidae form a monophyletic group with support of 88/1, this does not include Oblongichytrium, which instead groups as sister to both Labyrinthulida and Thraustochytrida (Figure 1A). Considering the uncertain placement of Oblongichytrium among different studies, and to avoid forming non-monophyletic group, I proposed a separate, new order Oblongichytrida for this genus. However, because the proposition is based solely on the phylogenetic position of its 18S rDNA, this proposal requires more evidence to validate the new order. Within Thraustochytriidae, species of

Thraustochytrium group at various locations, often interspersed with other genera, suggesting that this genus is polyphyletic. The phylogenetic tree also revealed 20 new environmental clades, most of which have over 70% bootstrap support and Bayesian posterior probability of 1 (Figure

1A). Among these new clades, 16 of them do not belong to any of the previously defined major groups. Previously identified uncultivated groups uTh1, uLa1 to uLa7 from Collado-Mercado et al. (2010) can also be identified from the tree, but because each group now contains more sequences, new corresponding labels THR1, LAB1 to LAB7 are given to avoid confusion (Table

1). The former clades uTh2 and uLa8 are, according to our phylogeny (Figure 2), well within

13 Aurantiochytrium (70/1) and Labyrinthulida ANT10_3 (-/0.85), suggesting these are not unique groups. The ancestral branch of LAB1, LAB6 and LAB8 is 50/0.99 supported and are placed into supergroup LAB1/6/8.

I was able to retrieve environmental metadata for 924 sequences (out of 1,181) through a combination of GenBank data retrieval and manual literature search. The 257 sequences that have no metadata available are all cultured strains.

3.2 Examining Abundance and Richness using GenBank Sanger Sequences Abundance and richness analyses were conducted by comparing different phylogenetic groups using a variety of parameters. The total abundance distribution between cultured and environmental sequences, as illustrated by the upper bars in Figure 3, reveals that over half of the sequences in most of the groups are environmental, except for Thraustochytriidae, which contains mostly sequences from cultured species. 18S rDNA sequences of Amphifilidae are highly variable as illustrated by the over two-fold differences in abundance versus richness.

Based on the metadata information collected, Thraustochytriidae and Labyrinthulida are often isolated from marine environments. Mangrove forests, which are saltwater ecosystems found between terrestrial and marine environments, are also a common habitat for

Labyrinthulomycetes. This is especially true for Aurantiochytrium, where 11% of the sequences in the database were collected from mangroves. In contrast, Amphifilidae, a distinct member of

Thraustochytrida, are found primarily in freshwater and soil samples, with only three sequences from the marine environment. Similarly, Amphitremida also contain many freshwater sequences.

The environmental clade AMP1 is the main marine representative of this lineage. Two sequences belonging to the Am. wrightianum group were also collected from marine environments. All the

14 environmental clades recovered in this study were from marine samples, including LAB7,

LAB14 and LAB15, which were common according to the total abundance analysis (Figure 3).

3.2.1 Host Association

A total of 208 Labyrinthulomycetes sequences (69 OTU97), belonging to various phylogenetic groups, were isolated directly from biological substrates and they are denoted as being “host- associated” in this study (Figure 1B). Over half of the Labyrinthulida sequences are host- associated, of which two-third form associations with plants, mainly Labyrinthula with seagrass.

A second common association is between Aplanochytrium and coral mucus. Within

Aplanochytrium, sequences from the subclade containing OTU97 representatives FJ389839,

FJ389848, FJ389840 and FJ389872 are all associated with the massive coral Favia sp. and all come from the same study (Siboni et al. 2010). Another subclade of OTU97, containing

AF348521, AF348517, AF348518 and AF348516, is also host-associated entirely, but with a more diverse range of hosts, including coral, seagrass and algae. In addition to Labyrinthulida, coral association can also be seen in Thraustochytriidae and Oblongichytrida. The T. striatum group also contains a large number of sequences associated to Favia sp. (Siboni et al. 2010).

Other groups of Thraustochytriidae that contain coral-associated sequences include

Sicyoidochytrium, Thraustochytriidae HK10 and Ulkenia. Roughly 4% of Thraustochytriidae sequences are isolated from other invertebrates. These include the clam parasite quahog parasite

QPX (Quahog parasite group), the abalone parasite Labyrinthuloides haliotidis (L. haliotidis group) and all the sequences in T. caudivorum, which belong to flatworm parasitic species.

15 3.3 Abundance and Richness using V9 Reads The curation of the 18S rRNA V9 region reads using a phylogenetic approach led to an increase in the specificity of classification when comparing to their original taxonomic assignation. 2,210 unique sequences from 11 different studies were retrieved from VAMPS. 1,030 of those unique sequences were discarded based on the RAxML-EPA assignment. Among these discarded unique sequences, 862 were previously annotated as Stramenopiles or Stramenopiles environmental samples, and 168 were previously annotated as Stramenopiles-Labyrinthulida-

Oomycetes by the reference V9 SILVA database. 1,180 unique sequences were placed within

Labyrinthulomycetes, of which 1,170 were assigned to a more specific classification than their original annotation. In most cases these were previously identified only as stramenopiles environmental samples. 474 unique sequences were selected from the Tara Ocean database. Of the 76 discarded unique sequences, 51 were previously identified as Labyrinthulomycetes, in the

RAxML-EPA tree fell outside of this group. 398 unique sequences were placed within

Labyrinthulomycetes and the classification of 295 was improved. Overall, these HTES studies greatly increased the amount of Labyrinthulomycetes data available to study abundance and richness. In total, the final Labyrinthulomycetes V9 dataset contains 520 OTUs representing a total of 760,593 raw reads from VAMPS and Tara Ocean (Table 2). For the Tara Ocean database alone, the improved annotation by our study has resulted in an over two-fold increase in the abundance of Labyrinthulomycetes present in the collected samples (compared with Database

W6 in de Vargas et al. 2015).

In addition, 19 environmental clades were identified from the V9 dataset, seven of which were found to branch within previously defined lineages. Five of these belong to Thraustochytrida, one to Amphitremida and one to Labyrinthulida. The remainder were not found to belong to any

16 previously identified lineages, and their phylogenetic positions within Labyrinthulomycetes can be seen in the dendrogram in Table 2 as well as in Figure 4. Taxa with formally described members, such as Thraustochytriidae and Labyrinthulida, are generally better-studied, and also have more data in GenBank, but large-scale HTES studies help in revealing the abundance and richness for novel taxa, some of which surpass that of the better-studied groups. Analysis on the

Tara Ocean database shows that LAB14, an environmental clade identified for the first time in this study, to be the most abundant of all subgroups, accounting for over 50% of the raw data.

LAB7 and LAB15 also rank third and fourth in abundance, suggesting that they too are ecologically significant but under-studied. In the case of VAMPS (Table 2) most of the raw data belongs to Oblongichytrida and Labyrinthulida (67% and 25% respectively), and many of the environmental clades are absent from this dataset.

Based on the environmental information available for both the VAMPS and Tara Ocean databases, the abundance distribution was also compared across the different phylogenetic groups using different environmental parameters. Among the 760,593 raw reads, 423 are from freshwater environment and 760,170 are from marine; 99% of the raw data is derived from marine samples. Oblongichytrida is the dominant group in freshwaters while LAB14 is the most abundant in marine data (Figure 4A). Labyrinthulida are common in both freshwater and marine.

As the marine samples dominate the databases, I further analyzed marine metadata for depth

(Figure 4B) and temperature (Figure 4C). Over 98% of Labyrinthulomycetes are recovered from the photic zone. Among all the lineages compared, Labyrinthulida is the only one common in all three regions, even though it is not the most abundant (Figure 4B). The most representative

Labyrinthulomycetes subgroup in the photic zone is LAB14, whereas Oblongichytrida is the

17 dominant taxon in the aphotic zone and the sediment. Although LAB14 continues to be the dominant taxon across all temperature ranges in the ocean (Figure 4C), its dominance is reduced with increasing temperature, and other taxa increase in abundance, such as Labyrinthulida,

LAB7 and LAB15. While Oblongichytrida is generally the minority under most temperature range, its abundance increases under 5-10°C.

18 Chapter 4: Discussion 4.1 Phylogeny and Classification The current study is an attempt to clarify and update the phylogeny of Labyrinthulomycetes through analysis of all available 18S rDNA sequences from both cultured and environmental samples (GenBank last searched in June 2015). Alongside the construction of a curated reference tree and database, I have also discovered putative novel phylogenetic groups, and showed some of these to be very abundant in the ocean. Overall, the topology of my phylogenetic tree is in agreement with several previous studies for the placement of most groups, like the sister placement of Labyrinthula and Aplanochytrium into Labyrinthulida, and the basal branching of

Amphifilidae to Thraustochytriidae in the order Thraustochytrida. My analyses also agree on the placement of Amphitremida deep within Labyrinthulomycetes and the placement of Diplophrys sister to Amphitrema and Archerella (Gomaa et al. 2013; Takahashi et al. 2014). The placement of Oblongichytrium outside of Thraustochytrida in this study is not very surprising considering other studies have also shown similar results, even when different methods were used to obtain the phylogenetic tree (Yokoyama and Honda 2007; Yokoyama et al. 2007; Collado-Mercado et al. 2010; Anderson and Cavalier-Smith 2012; Gomaa et al. 2013; Takahashi et al. 2014; Ueda et al. 2015). Similar to these studies, members of the genus Thraustochytrium are also found scattered throughout Thraustochytriidae, suggesting this to be a polyphyletic genus that should be revised in the future.

Even though the clade comprising Oblongichytrium does not have strong bootstrap support and only moderate Bayesian posterior probability (0.87), this group contains many annotated sequences. The same happens with the genus Aplanochytrium, where the support from both bootstrap analysis and Bayesian posterior probability are low, but the group is identified based

19 on the annotated sequences it contained. At 97% clustering, sequences from cultured strains including A. kerguelense, A. stocchinoi and A. minuta are all clustered under KJ761355. These sequences are often used in other studies, without clustering, to generating phylogenetic trees

(Leander et al. 2004; Collado-Mercado et al. 2010; Takahashi et al. 2014). Because my study used more OTUs97 for Aplanochytrium, I cannot find any other comparable studies to fully verify the phylogenetic structure of this group in my tree.

As with any other phylogenetic analysis, the variations in the tree of Labyrinthulomycetes between this study and other studies may be a result of using different alignment and phylogenetic tree construction methods, as well as the larger number of sequences included in this study. The phylogenetic tree presented by Collado-Mercado et al. (2010) is the closest to mine in terms of method (both used RAxML) and the number of sequences used (over 300).

Even then, the 97% clustering step performed prior to tree construction in my study means that many sequences present in their tree are absent from mine, for example the KJ761355 case mentioned above. While it remains debatable as to what taxonomical level 97% OTU clustering represents for different lineages, it may be one reason for the differences in tree topology.

Two genera of the Thraustochytriidae, Althornia and Japonochytrium, were not included in this study due to lack of publicly available sequences. The genus Althornia was named after isolation of Althornia crouchii Jones & Alderman (1971) from diseased oyster shells. According to

Alderman and Jones, it is ‘a monocentric, biflagellate phycomycete with free-floating globose sporangia with a thick laminate wall’ (Alderman and Jones 1971). It was placed in the

Thraustochytriales based on a morphological study by Alderman et al. (Alderman et al. 1974).

20 Since then, very little work has been done on this genus and to date no 18S rRNA gene sequence has been published. Japonochytrium was originally described by Kobayashi and Ookubo in 1953 for the species Japonochytrium marinum. Later, Harrison and Jones described the morphology and ultrastructure of a species that closely resembled Japonochytrium marinum, denoted

Japonochytrium sp. (Harrison and Jones 1974). The cultured strain ATCC28207 (sequence

AB022104) was labeled as Japonochytrium marinum by Tsui et al. (2009). However, this strain has previously been revised to Ulkenia sp. by Yokoyama et al. in 2007. AB022104 was clustered with AB022116 at 97%, and phylogenetic analyses also confirmed its placement within Ulkenia

(97/1) (Figure 2). It remains uncertain whether Japonochytrium is a real genus or misidentified

Ulkenia. If future 18S phylogenetic studies can confirm the placement of these two genera within

Labyrinthulomycetes, it is then very possible that sequences belonging to them have already been included in my data.

In total, 39 new environmental clades have been identified in my study. While most new clades branch well within Labyrinthulomycetes, the basal branching position of some of them in the majority of my analyses (e.g., LAB15 and LAB16), suggest that some of these lineages may represent sister lineages to the Labyrinthulomycetes sensu stricto; morphological observations would be required to tell if they match the descriptions applied to Labyrinthulomycetes or not.

Additionally, 97% clustering of the hypervariable V9 data may have resulted in more novel clades than there would be if full-length sequences were used. Future morphological and physiological studies will help in confirming the identity of these new clades in general.

Nevertheless, the discovery of the new environmental clades, together with the fact that most of them are placed outside of Thraustochytrida, Amphitremida and Labyrinthulida, shows the

21 limitation of traditional culture-dependent approaches in uncovering the diversity of

Labyrinthulomycetes and the benefit of large-scale environmental samplings.

4.2 Environmental Distribution of Labyrinthulomycetes Several studies have been carried out to investigate Labyrinthulomycetes viability and metabolic activity in deep-sea columns under cold temperature and high water pressure. Using a combination of the AfDD staining technique and culturing methods, Raghukumar et al. (2001) were able to detect presence of thraustochytrids from water samples collected from the Arabian

Sea up to 2000m in depth. In another study, Raghukumar and Raghukumar (1999) demonstrated that during a seven-day incubation thraustochytrids cultures were able to grow and maintain protease production and enzyme activity under 10°C and 10MPa. Riemann and Schaumann

(1993) have also reported, using both AfDD staining and Nomarski microscopy, dense populations of thraustochytrids-like protists in a fast ice core drilled close to the southern shelf ice margin of the Weddell Sea. However, considering the time when these experiments were conducted, it remained unclear as to which group of thraustochytrids (including

Oblongichytrium) they were referring to. Both of the reference and V9 databases contain sequences collected from deep-sea column and many of them belong to either Oblongichytrium or Labyrinthulida. According to the V9 database, Oblongichytrium is the most abundant taxon in the deep-sea (>2,000m), followed by Labyrinthula. Deep-sea environments are usually associated with low temperature, and this may explain the increased dominance of

Oblongichytrium at 5-10°C, as shown in Figure 4C. Other lineages were either not detected or occurred in very low numbers in these conditions. However, it should be noted that all deep-sea data are acquired from VAMPS, and the projects included in this database do not have sample sizes as large as that of Tara Oceans. With improved species-identification tools, it would be

22 useful to re-visit the deep-sea column to see if a clearer idea as to which subgroups of

Labyrinthulomycetes live there, as their role in this ecosystem and how they survive there are potentially interesting.

Based on the environmental metadata I have collected, Labyrinthulomycetes have also been frequently isolated from oxygen minimum zones (OMZs) and anoxic environments. However, they were not analyzed further in this study due to lack of solid evidence for being truly anaerobic. Cathrine and Raghukumar (2009) reported successful isolation and cultivation of species of thraustochytrids from oxygen-limited environments, suggesting some members of

Labyrinthulomycetes might be able to survive in anoxic habitats, perhaps by some facultative anaerobic metabolism.

Although the potential ecological roles of Labyrinthulomycetes to their associated hosts have been discussed in detail by several studies (e.g., Raghukumar 2002; Raghukumar and Damare

2011), the evolutionary origin of these associations among different taxa, as well as any shared morphological or physiological characteristics, have rarely been discussed. My study shows that host-association has evolved independently in many Labyrinthulomycetes lineages, and that the same group of taxa can be found from many, often very different, hosts. For example

Labyrinthula sp. have been isolated from the surface of seagrass and from the cytoplasm of single-celled amoebozoan protists (Dyková et al. 2008). It also remains unclear as to what defence mechanisms different Labyrinthulomycetes use to protect themselves against different host immune responses.

23 Chapter 5: Conclusion and Future Directions My phylogenetic study of Labyrinthulomycetes based on existing data has revealed this group to be extremely diverse and ubiquitous. The 39 new environmental clades I have identified in my study indicates this group to be more diverse than previously expected, and that culture- dependent approach alone is not adequate to describe its full diversity. While some of these newly identified environmental clades are abundant in the ocean, little is known regarding their morphology and physiology, and these merit further study. This is particularly obvious in the case of LAB14, which can be found abundantly across all temperature ranges in the photic zone of the marine environment. Based on the metadata I collected, Labyrinthulomycetes can be found in both freshwater and marine environments. The large numbers of sequences originating from anoxic and deep-sea environments strongly suggest that they are capable of living under extreme conditions. Their associations with different kinds of hosts and evidence that suggest host-association may include endobiotic associations are fascinating, and raise new questions about the ecology, physiology and evolution of the Labyrinthulomycetes.

The curated reference database and phylogenetic tree I have created are also an up-to-date resource that will provide a more accurate annotation for future studies, as illustrated by my analysis on the two V9 databases. However, the lack of information from the morphological aspects for many lineages obstructs my effort in sorting out the taxonomic ranking of several groups. To further improve the reference database, 18S rRNA gene data for Althornia will need to be added in the near future, and problems with the polyphyletic genus Thraustochytrium will also need to be resolved with additional data on the morphology, physiology and biochemistry of the various species currently included within this genus. In addition, the authenticity of

24 Japonochytrium should be examined and clarified to avoid additional confusion and prevent future faulty taxonomy.

25 A -/0.97 THR1 T. kinnei group -/0.98 8 4/1 L. haliotidis group -/1 THR*2 -/0.87 91/1 Thraustochytriidae SEK690 THR3 94/1 Schizochytrium Thraustochytriidae Parietichytrium 96/1 Botryochytrium T. aureum group Thraustochytriidae HK10 98/1 T. striatum* group Thraustochytrida -/1 97/1 Ulkenia * 70/1 * Aurantiochytrium Thraustochytriidae SEK706 * 98/1 Thraustochytriidae KB8 -/0.98 -/0.77 FN690479 Sicyoidochytrium 88/1 T. caudivorum* Quahog* parasite group -/0.98 Thraustochytriidae SEK704 * Amphifilidae group -/0.99 75/1 Am. wrightianum group * 97/1 -/0.8 Ar. flavum group * Amphitremida Diplophrys AMP1 92/1 LAB1 99/1 LAB8 * LAB1/6/8 -/0.99 LAB6 78/1 LAB11 98/1 LAB5 98/1 Labyrinthula -/0.98 EF659869 * FJ389881 Aplanochytrium Labyrinthulida -/0.85 Labyrinthulida* ANT10_3 90/1 Labyrinthulida G41 * AY916573 99/1 LAB4 -/0.92 LAB9 -/1 LAB3 LAB10 -/0.87 -/0.98 Oblongichytrium Oblongichytrida 95/1 LAB2 * B -/0.79 90/1 LAB13 Thraustochytriidae 353 -/0.9 LAB7 98/1 LAB14 -/0.94 Amphifilidae 69 -/1 77/1 LAB12 91/1 LAB15 -/0.74 Amphitremida 28 95/1 LAB16 LAB1/6/8 24 -/0.99 Other Stramenopiles -/0.74 Labyrinthulida 245 92/1 Outgroups Oblongichytrida 68

92/1 0% 20% 40% 60% 80% 100% Free living Other invertebrates Algae Coral Plants

0.1 Figure 1. A) Diversity of Labyrinthulomycetes inferred from a maximum likelihood (RAxML) phylogenetic tree constructed using 18S rDNA sequences. Numbers at nodes represent bootstrap support/Bayesian posterior probability. Only values >70% or 0.7 are shown. Nodes with support values of 100/1 are highlighted as black dot. Groups containing host-associated sequences are indicated by . Genus abbreviation is as follows: T in T. kinnei group, T. aureum group, T. striatum group, and T. caudivorum means Thraustochytrium. L in L. haliotidis group means Labyrinthuloides. Amphitrema for Am. wrightianum group, Archerella for Ar. flavum group. B) Host-associated sequence abundance in Labyrinthulomycetes major groups. The total number of sequences, with metadata available, is indicated at the end of the bar for each group.

26 9 5 JN418985 7 8 JF791020 AY256273 HQ393981 AY256317 THR1 100 GU823956 DQ455713 8 0 HQ869652 JF275536 FJ800587

7 8 JN675247 9 6 JN675253 9 4 JN675251 9 6 JN675270 JN675273 T. kinnei group 100 L34668 8 9 KF718865 DQ459553

8 5 AB073307 9 8 AB810951 L. haliotidis group 8 4 HQ228982 U21338 JN547303 100 FJ800594 THR2

9 1 AB973545 Thraustochytriidae SEK690 100 FN690668 FJ800589 THR3

9 8 JN675266 HQ228969 AB073309 AB290578 Schizochytrium AB542115 100 AB052556 9 4 AB073306 AY705753 Labyrinthulomycetes 100 AB073304 100 AB810971 Parietichytrium 7 6 AB810953 100 AB073305

100 DQ023614 AB810984 Botryochytrium Stramenopiles JX993841 9 6 8 1 100 JX993844 100 JX993840 T. aureum group 9 5 JX993839 100 AB022110 HQ228972 FJ389858 8 1 FJ821500 Thraustochytriidae HK10 100 FJ389815 FJ800627

7 4 FJ389802 FJ389799 KM402873 AF265337 7 5 FJ389760

8 5 FJ389765 100 FJ389808 T. striatum group T

FJ389875 h 100 9 8 FJ389768 r aus 100 FJ389776 FJ389851 AB810969 t o 100

HQ116831 c

HQ228968 h 9 6 JX993842 ytriidae 8 5 AB022116 HQ228981 8 2 HQ228979 9 7 L34054 Ulkenia FJ389788 100 FJ389790

9 8 DQ834736 8 8 DQ525180 DQ834737 DQ834732 JF266572 9 8 EU871043 DQ834733 DQ834738 JN675250 DQ023610 DQ023611 AB073303 AB362211

GU295222 T

JX847368 h

8 3 JX863672 r aus 7 8 FJ004948 EU728656 FJ821479 t

FJ560900 o

JX993843 Aurantiochytrium c

KM023693 h

AB810978 ytrida KJ938302 100 DQ834734

7 3 DQ834735 DQ525181 9 1 JN675256 JN675262 100 JN675245 JN675255 8 5 AB073308 FJ821482 7 5 JN675275 9 3 KM374695 100 AF265338 DQ374149 FJ821497 DQ367051 9 6 AB810966 9 9 AB973555 100 9 9 AB973557

9 1 JF972654 EF100273 7 0 100 EF100298 AB973558 AF257314 9 7 KM196563 AB973559 Thraustochytriidae SEK706 JX457319 9 8 FJ800613 Thraustochytriidae KB8 FN690479 8 8 AB183654 Sicyoidochytrium 100 EF114353 T. caudivorum

7 8 AF474172 9 8 AB973562 Quahog parasite group 100 AB022113 AF261664 AB810959 100 FN690483 Thraustochytriidae SEK704 LN581594 LN581766 9 8 LN575027 LN586640 LN582041 Figure 2. Diversity of LabyrinthulomycetesDQ 10460(full0 version). Numbers at nodes indicate bootstrap 8 9 9 2 DQ104585 DQ104602 support. Only those >70% are shown. LN580137 L

LN587488 a

AB970288 b

AB970183 yri 3AB572131 9 KC454889 AB627973 n

AB970424 thulo LN585946 9 8 LN577577 AB695490 GU919334 27 AB721051 Amphi lidae group m KC911795 AY919801 y 7 6 AY821979 c JQ692229 e t

AB856528 es AY919687 8 5 AY919701 100 AY919755 9 3 8 8 AY919705 GQ330584 8 8 AB721042 GU922746

FJ410599 S 7 7 AB769304 t 9 5 100 AF265331

AY046668 r

AY082983 amenopiles 100 GU918930 100 AB622307 DQ104601 FJ592426

GU949602 A 100 Am. wrightianum group

7 2 JQ244217 mphit 7 5 GU923337 GQ330589 EF586082 Ar. !avum group 9 7 KC245096 r

AB856527 emida 8 1 Diplophrys 100 100 GU923285 AF304465

100 GU823043 GU824170 AMP1 FJ800640 9 8 GU823297 100 LAB1

GU218917 L 9 2 100 AB275111 AB1/6/8 FJ800605 GU823135 100 AB505496 100 LAB8 9 9 AB505497 AF290070 8 5 DQ310278 EF100295 LAB6 100 GU823597 AB505562 AB191425 FJ800611 7 8 HQ867171 LAB11 AY381210 9 8 FJ800624 LAB5 AB290454 100 DQ073056 AB290456 FJ389849 7 4 AY835688 AY821970 GQ499192

9 9 AF265332 7 1 AB095092 AB220158 FJ389767 EU431330 Labyrinthula AB246795 100 FR875360 8 6 EF100370 EF100254 GU385668 8 9 GU385599 9 6 GU385638 GU385653 L 9 8 AF265335 a 9 8 100 AF265334 b

FR875331 yri 100 AF265330

GQ499191 n

GQ499190 thulida EF100308 EF659869 FJ389881 AF348521 AF348517 8 2 AF348518 AF348516 AF348520

7 4 FJ389839 FJ389848 Aplanochytrium 7 3 FJ389840 FJ389872 AY046604 AF265333 AY046781 KJ761355 DQ103805 9 4 DQ103777 AB695481 AB695476 7 4 AY916582 AB695479 EF100337 FJ389846 Labyrinthulida ANT10_3 9 0 FJ153754

100 EF100382 9 0 EF100381 EF100392 AB695533 LM653283 JN825662 DQ459554 Labyrinthulida G41 AY916573 GU823652 JF275406 LAB4 7 3 FJ800609 9 9 GU823017 EF100352

100 FJ800607 HM369758 LAB9 FJ800595 LAB3 AB252772 LAB10 AB505563 AY256299 JQ243385 JQ243362 JQ243227

7 9 JQ243386 O JQ243669 blongic 7 3 KP187825

7 6 FN598249 EF100270 Oblongichytrium EF100252 HQ228948 FJ821480 h AB022108 ytrida 100 DQ459550 AF265336 FJ389824 FJ389892 FJ800643 FJ800641 AY870339 DQ459551 AB022111 FJ799794 HQ866358 8 5 HQ870626 HQ866848 FJ800599 LAB2 HQ866052 AB191423 9 5 FJ800623 FJ153715 HQ868118 7 7 LAB13 9 0 HQ868804 GQ863784 AY381171 8 7 HQ869583 LAB7 100 JQ226597

100 GU219191 KF129615

7 7 HQ869768 9 2 HQ869190 HQ869614 7 8 GU823529 LAB14 9 8 GU822961 GU824035 7 5 FJ431786 DQ918581 7 7 JX188369 LAB12

100 KJ762709 100 GU824784 KJ763928 JN832718 LAB15 9 1 GU823939

9 9 GU823099 GU823102

8 7 GU823656 7 5 GU823167 9 6 GU823444 LAB16 9 5 KJ758913 JX188294 Ochrophyta 100 Hyphochytriales U37107_Developayella_elegans 100 Pirsonia 100 Oomycetes 6 7 MAST1 MAST2 100 MAST3 100 MAST6 100 MAST8 9 9 MAST10 MAST20 Outgroups 100 MAST11 100 MAST4 100 MAST7 9 2 100 MAST9 9 9 MAST22 100 Bicosoecida MAST25 9 2 EU349230_Planomonas_micra DQ975473_Sabulodinium_undulatum

0.1 9 5 JN418985 7 8 JF791020 AY256273 HQ393981 AY256317 THR1 100 GU823956 DQ455713 8 0 HQ869652 JF275536 FJ800587

7 8 JN675247 9 6 JN675253 9 4 JN675251 9 6 JN675270 JN675273 T. kinnei group 100 L34668 8 9 KF718865 DQ459553

8 5 AB073307 9 8 AB810951 L. haliotidis group 8 4 HQ228982 U21338 JN547303 100 FJ800594 THR2

9 1 AB973545 Thraustochytriidae SEK690 100 FN690668 FJ800589 THR3

9 8 JN675266 HQ228969 AB073309 AB290578 Schizochytrium AB542115 100 AB052556 9 4 AB073306 AY705753 100 AB073304 100 AB810971 Parietichytrium 7 6 AB810953 100 AB073305

100 DQ023614 AB810984 Botryochytrium JX993841 9 6 8 1 100 JX993844 100 JX993840 T. aureum group 9 5 JX993839 100 AB022110 HQ228972 FJ389858 8 1 FJ821500 Thraustochytriidae HK10 100 FJ389815 FJ800627

7 4 FJ389802 FJ389799 KM402873 AF265337 7 5 FJ389760

8 5 FJ389765 100 FJ389808 T. striatum group T

FJ389875 h 100 9 8 FJ389768 r aus 100 FJ389776 FJ389851 AB810969 t o 100

HQ116831 c

HQ228968 h 9 6 JX993842 ytriidae 8 5 AB022116 HQ228981 8 2 HQ228979 9 7 L34054 Ulkenia FJ389788 100 FJ389790

9 8 DQ834736 8 8 DQ525180 DQ834737 DQ834732 JF266572 9 8 EU871043 DQ834733 DQ834738 JN675250 DQ023610 DQ023611 AB073303 AB362211

GU295222 T

JX847368 h

8 3 JX863672 r aus 7 8 FJ004948 EU728656 FJ821479 t

FJ560900 o

JX993843 Aurantiochytrium c

KM023693 h

AB810978 ytrida KJ938302 100 DQ834734

7 3 DQ834735 DQ525181 9 1 JN675256 JN675262 100 JN675245 JN675255 8 5 AB073308 FJ821482 7 5 JN675275 9 3 KM374695 100 AF265338 DQ374149 FJ821497 DQ367051 9 6 AB810966 9 9 AB973555 100 9 9 AB973557

7 8 AF474172 9 8 AB973562 Quahog parasite group 100 AB022113 AF261664 AB810959 100 FN690483 Thraustochytriidae SEK704 LN581594 LN581766 9 8 LN575027 LN586640 LN582041

8 9DQ104600 9 2 DQ104585 Thraustochytrida DQ104602

LN580137 L

LN587488 a

AB970288 b

AB970183 yri 3AB572131 9 KC454889 AB627973 n

AB970424 thulo LN585946 9 8 LN577577 AB695490 GU919334 AB721051 Amphi lidae group m KC911795 AY919801 y 7 6 AY821979 c JQ692229 e t

AB856528 es AY919687 8 5 AY919701 100 AY919755 9 3 8 8 AY919705 GQ330584 8 8 AB721042 GU922746

FJ410599 S 7 7 AB769304 t 9 5 100 AF265331

AY046668 r

AY082983 amenopiles 100 GU918930 100 AB622307 DQ104601 FJ592426

GU949602 A 100 Am. wrightianum group

7 2 JQ244217 mphit 7 5 GU923337 GQ330589 EF586082 Ar. !avum group 9 7 KC245096 r

AB856527 emida 8 1 Diplophrys 100 100 GU923285 AF304465

100 GU823043 GU824170 AMP1 FJ800640 9 8 GU823297 100 LAB1

9 9 AF265332 7 1 AB095092 AB220158 FJ389767 EU431330 Labyrinthula AB246795 100 FR875360 8 6 EF100370 EF100254 GU385668 8 9 GU385599 9 6 GU385638 GU385653 L 9 8 AF265335 a 9 8 100 AF265334 b

FR875331 yri 100 AF265330

GQ499191 n

GQ499190 thulida EF100308 EF659869 FJ389881 AF348521 AF348517 8 2 AF348518 AF348516 AF348520

100 EF100382 9 0 EF100381 EF100392 AB695533 LM653283 JN825662 DQ459554 Labyrinthulida G41 AY916573 GU823652 JF275406 LAB4 7 3 FJ800609 Figure 2. (continued) 9 9 GU823017 EF100352

100 FJ800607 HM369758 LAB9 FJ800595 LAB3 AB252772 LAB10 AB505563 AY256299 JQ243385 JQ243362 JQ243227

7 9 JQ243386 O JQ243669 blongic 7 3 KP187825

7 6 FN598249 EF100270 Oblongichytrium EF100252 HQ228948 FJ821480 h 28 ytrida AB022108 100 DQ459550 AF265336 FJ389824 FJ389892 FJ800643 FJ800641 AY870339 DQ459551 AB022111 FJ799794 HQ866358 8 5 HQ870626 HQ866848 FJ800599 LAB2 HQ866052 AB191423 9 5 FJ800623 FJ153715 HQ868118 7 7 LAB13 9 0 HQ868804 GQ863784 AY381171 8 7 HQ869583 LAB7 100 JQ226597

100 GU219191 KF129615

7 7 HQ869768 9 2 HQ869190 HQ869614 7 8 GU823529 LAB14 9 8 GU822961 GU824035 7 5 FJ431786 DQ918581 7 7 JX188369 LAB12

100 KJ762709 100 GU824784 KJ763928 JN832718 LAB15 9 1 GU823939

9 9 GU823099 GU823102

0.1 9 5 JN418985 7 8 JF791020 AY256273 HQ393981 AY256317 THR1 100 GU823956 DQ455713 8 0 HQ869652 JF275536 FJ800587

7 8 JN675247 9 6 JN675253 9 4 JN675251 9 6 JN675270 JN675273 T. kinnei group 100 L34668 8 9 KF718865 DQ459553

8 5 AB073307 9 8 AB810951 L. haliotidis group 8 4 HQ228982 U21338 JN547303 100 FJ800594 THR2

9 1 AB973545 Thraustochytriidae SEK690 100 FN690668 FJ800589 THR3

9 8 JN675266 HQ228969 AB073309 AB290578 Schizochytrium AB542115 100 AB052556 9 4 AB073306 AY705753 100 AB073304 100 AB810971 Parietichytrium 7 6 AB810953 100 AB073305

100 DQ023614 AB810984 Botryochytrium JX993841 9 6 8 1 100 JX993844 100 JX993840 T. aureum group 9 5 JX993839 100 AB022110 HQ228972 FJ389858 8 1 FJ821500 Thraustochytriidae HK10 100 FJ389815 FJ800627

7 4 FJ389802 FJ389799 KM402873 AF265337 7 5 FJ389760

8 5 FJ389765 100 FJ389808 T. striatum group T

FJ389875 h 100 9 8 FJ389768 r aus 100 FJ389776 FJ389851 AB810969 t o 100

HQ116831 c

HQ228968 h 9 6 JX993842 ytriidae 8 5 AB022116 HQ228981 8 2 HQ228979 9 7 L34054 Ulkenia FJ389788 100 FJ389790

9 8 DQ834736 8 8 DQ525180 DQ834737 DQ834732 JF266572 9 8 EU871043 DQ834733 DQ834738 JN675250 DQ023610 DQ023611 AB073303 AB362211

GU295222 T

JX847368 h

8 3 JX863672 r aus 7 8 FJ004948 EU728656 FJ821479 t

FJ560900 o

JX993843 Aurantiochytrium c

KM023693 h

AB810978 ytrida KJ938302 100 DQ834734

7 3 DQ834735 DQ525181 9 1 JN675256 JN675262 100 JN675245 JN675255 8 5 AB073308 FJ821482 7 5 JN675275 9 3 KM374695 100 AF265338 DQ374149 FJ821497 DQ367051 9 6 AB810966 9 9 AB973555 100 9 9 AB973557

7 8 AF474172 9 8 AB973562 Quahog parasite group 100 AB022113 AF261664 AB810959 100 FN690483 Thraustochytriidae SEK704 LN581594 LN581766 9 8 LN575027 LN586640 LN582041

8 9DQ104600 9 2 DQ104585 DQ104602

LN580137 L

LN587488 a

AB970288 b

AB970183 yri 3AB572131 9 KC454889 AB627973 n

AB970424 thulo LN585946 9 8 LN577577 AB695490 GU919334 AB721051 Amphi lidae group m KC911795 AY919801 y 7 6 AY821979 c JQ692229 e t

AB856528 es AY919687 8 5 AY919701 100 AY919755 9 3 8 8 AY919705 GQ330584 8 8 AB721042 GU922746

FJ410599 S 7 7 AB769304 t 9 5 100 AF265331

AY046668 r

AY082983 amenopiles 100 GU918930 100 AB622307 DQ104601 FJ592426

GU949602 A 100 Am. wrightianum group

7 2 JQ244217 mphit 7 5 GU923337 GQ330589 EF586082 Ar. !avum group 9 7 KC245096 r

AB856527 emida 8 1 Diplophrys 100 100 GU923285 AF304465

100 GU823043 GU824170 AMP1 FJ800640 9 8 GU823297 100 LAB1

9 9 AF265332 7 1 AB095092 AB220158 FJ389767 EU431330 Labyrinthula AB246795 100 FR875360 8 6 EF100370 EF100254 GU385668 8 9 GU385599 9 6 GU385638 GU385653 L 9 8 AF265335 a 9 8 100 AF265334 b

FR875331 yri 100 AF265330

GQ499191 n

GQ499190 thulida EF100308 EF659869 FJ389881 AF348521 AF348517 8 2 AF348518 AF348516 AF348520

100 EF100382 9 0 EF100381 EF100392 AB695533 LM653283 JN825662 DQ459554 Labyrinthulida G41 AY916573 GU823652 JF275406 LAB4 7 3 FJ800609 9 9 GU823017 EF100352

100 FJ800607 HM369758 LAB9 FJ800595 LAB3 AB252772 LAB10 AB505563 AY256299 JQ243385 JQ243362 JQ243227

7 9 JQ243386 O JQ243669 blongic 7 3 KP187825

7 6 FN598249 EF100270 Oblongichytrium EF100252 HQ228948 FJ821480 h AB022108 ytrida 100 DQ459550 AF265336 FJ389824 FJ389892 FJ800643 FJ800641 AY870339 DQ459551 AB022111 FJ799794 HQ866358 8 5 HQ870626 HQ866848 FJ800599 LAB2 HQ866052 AB191423 9 5 FJ800623 FJ153715 Labyrinthulomycetes HQ868118 7 7 LAB13 9 0 HQ868804 GQ863784 Stramenopiles AY381171 8 7 HQ869583 LAB7 100 JQ226597

100 GU219191 KF129615

7 7 HQ869768 9 2 HQ869190 HQ869614 7 8 GU823529 LAB14 9 8 GU822961 GU824035 7 5 FJ431786 DQ918581 7 7 JX188369 LAB12

100 KJ762709 100 GU824784 KJ763928 JN832718 LAB15 9 1 GU823939

9 9 GU823099 GU823102

0.1

Figure 2. (continued)

29 Table 1. List of previously described environmental clades (Collado-Mercado et al. 2010) and the new clades they now belong to according to the present phylogenetic study.

Clade ID Comment uTh1 Belongs to THR1 uTh2 Belongs to Aurantiochytrium uLa1 AB219774 is chimera (Labyrinthulid and Coniferophyta), the rest belong to LAB1 uLa2 Belongs to LAB2

uLa3 Belongs to LAB3, all belongs to OTU97 FJ800595 uLa4 Belongs to LAB4 uLa5 Equivalents to LAB5 uLa6 Belongs to LAB6 uLa7 Belongs to LAB7 uLa8 Belongs to Labyrinthulida ANT10_3

30 Figure 3. Abundance and richness for major groups of Labyrinthulomycetes. On the left is a phylogenetic tree showing relationships among different groups (based on Figure 1A). On the right is a stacked bar graph of richness and abundance. For each group, the upper bar represents the total abundance while the lower bar indicates the richness. Upper x-axis: abundance, lower x- axis: richness. Cul Total Abundance: total abundance for cultured sequences, Env Total Abundance: total abundance for environmental sequences.

31 Table 2. Total abundance and richness for the three Labyrinthulomycetes databases. Numbers are calculated from raw data. Phylogenetic relationships among different groups are indicated by the dendrogram on the left.

Figure 4. Heatmap showing the abundance distribution of major groups of Labyrinthulomycetes according to different environmental parameters. A) Freshwater and marine. B) Photic water column, aphotic water column, and sediment. C) Temperature range, in °C. Abbreviations: F: freshwater, M: marine, P: photic zone, A: aphotic zone, S: sediment. For B & C only marine samples were used since they were numerically dominant.

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