Spatial and Seasonal Variabilities of Picoeukaryote Communities
in a Subtropical Eutrophic Coastal Ecosystem
Based on Analysis of 18S rDNA Sequences
CHEUNG, Man Kit
A Thesis Submitted in Partial Fulfillment
of the Requirements for the Degree of
Master of Philosophy
in
Biology
© The Chinese University of Hong Kong
Sept 2007
The Chinese University of Hong Kong holds the copyright of this thesis. Any person(s) intending to use a part or whole of the materials in the thesis in a proposed publication must seek copyright release from the Dean of the Graduate School. 1
/M/
2 Sff M jij
>g}VNsLIBRARr SYSTEM Thesis/Assessment Committee
Professor KWAN, Hoi Shan (Chair) Professor WONQ Chong Kim (Thesis Supervisor) Professor CHU, Ka Hou (Committee Member) Professor QIAN, Pei Yuan (External Examiner) Abstract
Picoeukaryotes are eukaryotes smaller than 2-3 |im in diameter. They occur in photic zones worldwide and play fundamental roles in marine ecosystems. Lacking distinctive external features, picoeukaryotes are difficult to be identified by conventional methods such as electron microscopy. Recent studies based on cloning and sequencing of small subunits (SSU) of ribosomal RNA (rRNA) genes directly from environmental samples have revealed high diversity of picoeukaryotes and identified many novel lineages. While numerous studies have been carried out in various ecosystems and geographical regions, information on subtropical coastal waters of the western Pacific is not available. In addition, information on the relationships between picoeukaryotic diversity and environmental factors are crucial for understanding ecosystem functioning. While several studies have been initiated to work on the effects of various environmental variables such as pH and temperature, data relating marine picoeukaryotic diversity and trophic status is still lacking.
In this study, the diversity of picoeukaryotes in coastal waters of the western
Pacific is characterized for the first time. In addition, spatial and seasonal variations in picoeukaryotic community composition in Tolo Harbour, a semi-enclosed eutrophic bay, and Mirs Bay, an oligoinesotrophic bay strongly influenced by water circulations, were studied. Eight 18S rRNA gene clone libraries were constructed using coastal seawater samples collected at the two study sites in January, April, July and October 2006. About a hundred clones per library were assessed by restriction fragment length polymorphism (RFLP) using the restriction enzyme Hae\\\. Clones showing the same RFLP patterns were grouped into distinct operational taxonomic units (OTUs). Clone representative from each OTU was partially sequenced.
I Examination of 733 picoeukaryotic clones revealed 186 different RFLP patterns, representing 186 OTUs. At least 17 higher-level taxonomic groups of picoeukaryotes were observed. Three additional higher-level groups were potentially novel.
Alveolates group II, ciliates and stramenopiles comprised 37%, 17% and 11 %, respectively of the picoeukaryote assemblages and represented the most dominant groups in both Tolo Harbour and Mirs Bay. These observations highlighted the importance of parasitism in aquatic ecosystems and the presence of potentially pico-sized ciliates. Members from Dinophyceae, Prasinophyceae and Cercozoa also showed significant contributions.
Spatial variations in composition and diversity of picoeukaryotes were recognized. Non-photosynthetic members were common in both study sites irrespective of trophic status. However, a decrease in the proportion of photosynthetic members was generally observed in eutrophic Tolo Harbour libraries.
A hump-shaped pattern between primary productivity and diversity was suggested for marine picoeukaryotes for the first time. Seasonal variations in picoeukaryote composition were more pronounced in the oligomesotrophic Mirs Bay than in the eutrophic Tolo Harbour. Diversity of picoeukaryotes seemed to be affected by water temperature, but other biotic and/or abiotic factors may also pay a role.
This study provides the first piece of information on the diversity of picoeukaryotes in the western Pacific, allowing a better understanding of the true dimensions of picoeukaryotic diversity. In addition, data on spatial and seasonal variations of marine picoeukaryotes with different degrees of eutrophication is provided here, creating basis for formulating hypotheses on ecosystem functioning.
II 摘要
微微型浮游真核生物是直徑少於2 - 3微米的真核生物。他們出現在全世
界的透光層及在海洋生態系統中扮演著重要的角色。由於它們欠缺獨特的外在
表徵，使用傳統的方法（例如電子顯微鏡）很難把它們辨認出來。最近一些應
用在環境樣本上的核糖體小亞基基因克隆與序列分析硏究’展現了這些生物的
豐富多樣性，並且在當中分辨出很多新的演化系群。雖然很多相關的硏究已應
用於不同的生態系統及地理位置上，但到目前爲止’我們仍然沒有位於亞熱帶
西太平洋海岸的這些生物的資料。另一方面’知道這些生物的多樣性及環境因素
之間的關係，有助我們理解生態系統的工能運作。雖然一些硏究已著手於不同
的環境變數（例如酸驗値及水溫）對這些生物多樣性的影響，但我們仍不清楚
有關營養位階對海洋中的微微型浮游真核生物多樣性的影響。
在這個硏究’我們首次描續了西太平洋海岸微微型浮游真核生物的多樣
性。另外，我們亦分別於富營養的吐露港及貧至中度營養的大鵬灣’硏究了這
些生物的空間及季節性變化。我們在二零零六年的一月，四月，七月及十月，
分別在兩個實驗站採集了海水樣本’並建立了八個18 S核糖體基因克隆文
庫。我們在每個文庫當中抽取了約一百個殖株’並使用限制晦Haelll對它們作
出了限制晦片段長度多型性分析。我們把那些展示出相同限制晦片段長度多型
性形態的殖株歸納作同一個分類運算單位’繼而從每一個分類運算單位當中選
取了殖株代表以作部份序列分析。
我們考察了7 3 3個微微型浮游真核生物殖株，並發現了18 6個不同的
限制腺片段長度多型性形態，分別代表著18 6個不同的分類運算單位。我們
從中發現了最少17個高階分類群’而另外三個高階分類群更可能是新發現
的°第二組囊泡蟲，纖毛蟲及不等鞭毛生物分別佔微微型浮游真核生物群總數
III 的3 7 %，17 %及1 1%，是在兩個實驗站中最豐富的群組。這展示了寄生
性在水域生態系統中的重要性，亦建議了微微型纖毛蟲的存在。另外’橫裂甲
藻綱，綠色鞭毛藻綱及絲足蟲類的成員也佔著相當的比重。
另一方面，我們發現了微微型浮游真核生物成份及多樣性的空間變化。非
光合成員在兩個不同營養位階的實驗站也很普遍，而光合成員佔有的比例在富
營養的吐露港特別少。我們首次發現初級生產及海洋微微型浮游真核生物多樣
性之間可能呈現著峰形形態。相比起富營養的吐露港，這些生物成份的季節變
化在貧至中度營養的大鵬灣中比較比顯。微微型浮游真核生物的多樣性似乎受
著水溫影響，但其他生物的或非生物的因素也許亦有影響。
這個硏究首次提供了西太平洋微微型浮游真核生物多樣性的資料，令我們
對這些生物的真正多樣性有了更佳的了解。另外’這個硏究提供了海洋微微型
浮游真核生物的季節變化，以及對不同營養富度的空間變化’爲生態系統的工
能運作假設的制定建立了基礎。
IV Acknowledgements
First of all, I would like to thank my supervisor Prof. C. K. Wong for his guidance throughout the two years of my M.Phil study in The Chinese University of Hong
Kong. I would also like to thank Prof. K. H. Chu who taught me basic knowledge of molecular biology that allowed me to finish my M.Phil project smoothly. In addition,
I would like to thank my thesis committee members for their valuable opinions that leaded to apparent improvements of my project.
1 want to thank Mr. Y. H. Yung who helped me to collect samples during field trips. I would also like to thank Mr. C. P. Li and Mr. K. C. Cheung for their helpful technical assistance provided. I want to thank all my labmates in Prof. Wong and Prof. Chu's labs who together created a great atmosphere for pursuing scientific knowledge.
1 want to thank my parents who gave born to me. Last but not least, special thanks should be given to my girlfriend who gave me plenty of supports when I got exhausted in cases of experimental failure.
V Table of Contents
Page
Abstract (English) i
Abstract (Chinese) m
Acknowledgements V
Table of contents VI
List of figures IX
List of tables XI
List of Appendices XII
Chapter 1. General introduction l
1.1. Picoeukaryotes 1
1.2. Conventional characterization techniques 1
1.3. Cloning and sequencing approach 3
1.3.1. Applications in prokaryotic plankton 3
1.3.2. Applications in eukaryotic picoplankton 3
1.4. Variations in diversity with environmental factors 5
1.5. Study site 6
1.6. Objectives 8
Chapter 2. Materials and methods 9
2.1. Study site 9
2.2. Sample collection 9
2.3. DNA extraction and 18S rRNA gene amplification \ \
VI 2.4. Clone library construction and screening 12
2.5. Sequencing and phylogenetic analysis 13
2.6. Statistical analyses 14
Chapter 3. Results 15
3.1. Hydrological parameters of study site 15
3.2. Clone libraries 15
3.3. Higher-level taxonomic distribution 21
3.4. Phylogenetic affiliations of OTUs 22
3.4.1 Alveolates 35
3.4.2 Stramenopiles 36
3.4.3 Rhizaria 36
3.4.4 Other lineages 37
3.4.5 Novel higher-level groups 38
3.5. Diversity estimates of picoeukaryotes 39
Chapter 4. Discussion 42
4.1. Picoeukaryotic diversity 42
4.1.1 Overall diversity 42
4.1.2 Diversity of individual taxonomic groups 44
4.1.2.1 Most represented lineages 44
4.1.2.2 Other photosynthetic lineages 52
4.1.2.3 Other non-photosynthetic lineages 55
4.1.2.4 Novel higher-level lineages 56
VII 4.2. Spatial and seasonal variations of picoeukaryotes 58
4.2.1 Spatial variations 59
4.2.1.1 Compositional variations 60
4.2.1.2 Variations in diversity 61
4.2.2 Seasonal variations 65
4.2.2.1 Compositional variations 65
4.2.2.2 Variations in diversity 66
4.3. Methodological aspects 67
4.3.1 Sample collection 67
4.3.2 PGR amplification 68
4.3.3 Cloning and RFLP screening 69
4.3.4 Statistical estimates 71
4.3.5 Future directions 71
Chapter 5. General conclusion 73
References 81
Vlll List of figures
Figure Page
Fig. 1. 10
Map showing the sampling locations of the current study.
Fig. 2. 17
Gel photo showing 16 restriction fragment length polymorphism (RFLP) patterns from library THOl.
Fig. 3. 19
Rank abundance curve for the pooled dataset of 733 picoeukaryote clones, representing 186 OTUs.
Fig. 4. 20
Histogram of GenBank BLAST similarities of sequences obtained in this study.
Fig. 5. 23
Relative abundance of the six most represented picoeukaryote groups in the eight clone libraries.
Fig. 6. 24
Relative abundance of the photosynthetic and non-photosynthetic groups in the eight clone libraries.
IX Fig. 7. 25
Maximum-likelihood (ML) phylogenetic tree of 18S rDNA sequences from all major picoeukaryotic groups observed in the current study.
Fig. 8. 27
ML phylogenetic tree of alveolate 18S rDNA sequences.
Fig. 9. 31
ML phylogenetic tree of stramenopile 18S rDNA sequences.
Fig. 10. 33
ML phylogenetic tree of 18S rDNA sequences of rhizaria.
Fig. 11. 34
ML phylogenetic tree of 18S rDNA metazoan sequences.
Fig. 12. 41
Rarefaction curves for the eight clone libraries.
Fig. 13. 64
Graphs showing the relationship between diversity indices and chlorophyll a concentration.
X List of tables
Table Page
Table 1. 16
Collection information, hydrological parameters and phytoplankton cell counts of water samples collected in this study.
Table 2. 18
Picoeukaryote OTU distribution of higher-level taxonomic groups
Table 3. 40
Picoeukaryote diversity estimates for different combinations of the eight clone
libraries.
Table 4. 48
Detailed number of OTU(s) of different taxonomic groups with % similarity values
equals to or higher than a certain threshold.
XI List of appendices
Appendix Page
Appendix 丨. 74
Equations for the statistics used in the present study.
Appendix 2. 76
List of all the OTUs recovered in the current study.
XII Chapter 1. General introduction
1.1 Picoeukaryotes
Picoeukaryotes, eukaryotes smaller than 2-3 |im in diameter, occur in photic zones of the world's oceans at concentrations ranging from 10^ to 10'^ cells ml"'
(Sherr & Sherr 2000). Photosynthetic picoeukaryotes are important primary producers in the world's oceans. In the North Atlantic, picoeukaryotes account for up to 79% of the total primary production (Li 1994). On the other hand, heterotrophic picoeukaryotes, mainly flagellates, are important grazers of prokaryotes (Sherr & Sherr 2000). Compared with their prokaryotic counterparts, picoeukaryotes are highly diverse, comprising members from nearly every algal phylum (Stockner & Antia 1986). Despite their ecological importance, the diversity of picoeukaryotes is poorly known. To date, only about 40 species belonging to nine algal classes of photosynthetic picoeukaryotes have been described (Vaulot et al.
2004). Information on the true dimensions of eukaryotic diversity is believed to be essential to fully understand the ecological complexity of marine microbial food webs (Lovejoy et al. 2006).
1.2 Conventional characterization techniques
Many experimental techniques have been used to characterize marine plankton, but each individual technique has its own limitations. Cells from Prasinophyceae genera such as Bathycoccus and Ostreococcus are too small to be identified by optical microscopy (Zhu et al. 2005). Indeed, even larger cells are difficult to be identified to the class level by conventional optical microscopy (Murphy & Haugen
1 1985) due to the lack of distinctive morphological features (Thomsen 1986).
Epifluoresence microscopy or flow cytometry have been used to estimate the total abundance of eukaryotic picoplankton (Campbell et al. 1994), but these techniques do not allow discrimination of taxa (Simon et al. 1994). Scanning and transmission electron microscopies generally allow taxonomic assignment of picoplankton to classes (Andersen et al. 1996), but most coccoid cells do not have enough ultrastructural features for identification at lower taxonomic levels (Potter et al.
1997). Microscopic methods are usually time consuming and are affected by heavy cell losses during sample preparation. Monoclonal antibodies allow detection of certain species or ecotypes (Anderson et al. 1999), but they are expensive to produce and cannot be applied to higher taxonomic levels due to their high specificity.
Organisms belonging to different algal classes contain different diagnostic chlorophyll and carotenoid marker pigments that can be identified and quantified by high performance liquid chromatography (HPLC) (Jeffrey et al. 1999). While
HPLC pigment analysis is very useful for characterizing new isolates, it cannot resolve diversity at lower taxonomic levels. Interpretation of the complex pigment patterns of environmental samples is difficult because most pigments are found in more than one algal class and some pigments are not present in every member of the same class. Cultivation is probably the best way to characterize a natural organism, but there is no guarantee that organisms grown in culture are representative of populations in the natural community (Guillou et al. 1999b). At best, many of the conventional characterization techniques have limited phylogenetic capacity and are cumbersome or time-consuming (Moon-van der Staay et al. 2000).
2 1.3 Cloning and sequencing approach
1.3.1 Applications in prokaryotic plankton
Molecular techniques based on cloning and sequencing of genes directly from natural samples provide a new and powerful way to analyse the diversity of plankton, particularly picoplankton (Guillou et al. 2004). Genetic libraries of small subunit ribosomal RNA (SSU rRNA) genes constructed from environmental samples have proven very valuable in the study of the taxonomic composition of marine prokaryotic plankton. In several early studies (Giovannoni et al. 1990, Delong
1992)，marine prokaryotic assemblages have been found to compose largely of novel bacterial and archaeal lineages without any known cultured representatives. More studies on the diversity of prokaryotes have been conducted in recent years (e.g.
Fuhrman et al. 1993; Giovannoni et al. 1995). Two general conclusions can be drawn from these studies. First, many sequences obtained from natural samples do not correspond to those obtained from cultured representatives (Giovannoni et al.
1995). Second, most sequences obtained from open sea samples fall into a few distinct yet diverse phylogenetic groups (Fuhrman et al. 1993). Indeed, an amazing prokaryotic diversity has been found in many different environments, including some of the most inhospitable regions on our planet (Pace 1997, Rothschild & Mancinelli
2001).
1.3.2 Applications in eukaryotic picoplankton
Despite the transcendence of the cloning and sequencing approach for prokaryotic microbiology, the technique has rarely been applied to studies of
3 microbial eukaryotes. The first SSU rRNA-based surveys of small eukaryotic
plankton were dated back to 2001. Using universal eukaryotic primers, Moon-van
der Staay et al. (2001) identified a high diversity of lineages including
photosynthetic classes such as Prasinophyceae, Haptophyceae and Pelagophyceae,
typical heterotrophic groups such as choanoflagellates and Acantharea, and groups
with phototrophic, heterotrophic and mixotrophic representatives such as
dinoflagellates. Also retrieved were sequences not clearly affiliated to any known
organisms. Di'ez et al. (2001) and Lopez-Garci'a et al. (2001) also observed large
picoeukaryotic diversities in the Mediterranean Sea, the North Atlantic, Antarctic
seas and the Antarctic Polar Front. The results of these studies reveal a hidden
diversity of tiny protists in the ocean.
More recently, similar studies on picoeukaryote diversity have been performed in other open oceans (Lovejoy et al. 2006, Not et al. 2007a) and in aquatic ecosystems such as deep-sea hydrothermal vents (Lopez-Garci'a et al. 2003), anoxic environments (Stoeck et al. 2003), coastal waters (Massana et al. 2004a, Romari &
Vaulot 2004，Medlin et al. 2006, Worden 2006) and freshwater lakes and ponds
(Lefranc et al. 2005, Richards et al. 2005, Slapeta et al. 2005). All these studies have revealed an unexpectedly high diversity of picoeukaryotes. While some of the new phylotypes obtained can be assigned to novel species in already known genera, families or orders, others represent novel lineages within already known eukaryotic groups such as alveolates and stramenopiles. More importantly, some of these new phylotypes do not seem to be related to any known lineages and may represent novel high-level taxonomic groups (Stoeck et al. 2003).
Among these studies, only four were conducted at coastal areas (Massana et al.
4 2004a, Romari & Vaulot 2004, Medlin et al. 2006, Worden 2006). Coastal ecosystems deserve particular attention because they are exposed to terrestrial influences and may harbour picoeukaryotic assemblages which are different from those of the open sea. Coastal areas are also prone to large temporal fluctuations induced by episodic events such as freshwater run off, sediment deposition and algal blooms. Picoeukaryotes are known to be ecologically important in coastal regions
(Worden et al. 2004). Among the four studies conducted on coastal picoeukaryotic diversity, three were performed along the western Atlantic coast (Massana et al.
2004a, Romari & Vaulot 2004，Medlin et al. 2006) and one was performed in the eastern Pacific coast (Worden 2006). Information on picoeukaryote communities in the subtropical western Pacific coast is lacking. It is clear that a comprehensive description of the diversity of marine picoeukaryotes requires the investigation of more habitats. And, it is worth investigating whether the western Pacific coast embeds a similarly high picoeukaryotic diversity as in other coastal sites.
1.4 Variations in diversity with environmental factors
In addition to determining picoeukaryotic diversity, studies of their variations in relation to environmental factors are crucial to understanding ecosystem functioning.
Scientists have just begun to extend their focus from measurements of protist diversity to studies of the relationships between protist diversity and environmental variables. Countway et al. (2005) reported that incubation of natural seawater samples at ambient light and temperature resulted in relatively minor changes in the overall protistan diversity but substantial changes in the dominance of particular phylotypes. Behnke et al. (2006) recorded a higher richness of microeukaryotes from the upper H2S boundary than in the H2S-free oxic-anoxic upper layer and the
5 highly sulfidic bottom layer in a supersulfide anoxic Qord (Framvaren) in Norway.
Brown & Wolfe (2006) reported a general decline in protistan diversity with
decreasing pH or increasing temperature. Lepere et al. (2006) showed that the
dynamics of small eukaryotes could be regulated by both top-down (e.g. zooplankton
grazing) and bottom-up (e.g. nutrient concentrations and bacterial abundance) factors.
Lefevre et al. (2007) observed a similarly high diversity of picoeukaryotes between
two contrasting zones in the Lake Pavin, irrespective of oxygen levels. Marine
euphotic zones have been shown to harbour more diverse assemblages of protists
(Countway et al. 2007) and picoeukaryotes (Not et al. 2007a) than the deep sea.
Variations in species diversity in relation to eutrophication have been intensively
studied for marine macroalgae (Worm et al. 1999), marine microalgae (Sundback &
Snoeijs 1991, Hillebrand & Sommer 2000) and freshwater microalgae (Marcus 1980，
Miller et al. 1992) using traditional approaches, including field experiments.
Lefranc et al. (2005) initiated the first molecular study to investigate the relationships
between the diversity of freshwater small eukaryotes and lake trophic status using
cloning and sequencing techniques. A hump-shaped progression of freshwater
small eukaryote diversity along an eutrophication gradient was reported. No
analogous studies are available for marine picoeukaryotes. Thus, it is worth testing
whether the hump-shape relationship is also valid between marine picoeukaryotic
diversity and different trophic status.
1.5 Study site
Hong Kong is located at the northern part of the South China Sea, a large water mass belonging to the western Pacific Ocean. Tolo Harbour and Mirs Bay are
6 located in the northeastern corner of Hong Kong. Tolo Harbour is a semi-enclosed
bay with an average depth of about 10 m and a maximum depth of 22 m. Its
semi-enclosed topography impedes tidal flushing. Studies conducted in the 1970s
revealed that Tolo Harbour was organically polluted (Wear et al. 1984). Indeed,
Tolo Harbour accounted for about 40% of all harmful algal blooms reported in Hong
Kong during the last 30 years (HKEPD 2003). High chlorophyll a levels indicate
that Tolo Harbour is highly eutrophic. Tolo harbour is connected to Mirs Bay via a
narrow channel. Compared to Tolo Harbour, Mirs Bay is relatively unpolluted and
is continuously influenced by water circulations from the South China Sea (HKEPD
2003).
Microscopy-based studies have identified diatoms and dinoflagellates as the
dominant components of the marine phytoplankton in Tolo Harbour (Lam & Ho
1989). More recently, Yung et al. (1997) have reported that unidentified small
flagellates are also common in Tolo Harbour. Pigment analyses confirmed that
most water samples from Tolo Harbour also contained prymnesiophytes,
cryptophytes and chlorophytes (Wong & Wong 2004). Long-term data published
by the HKEPD (2003) suggested that algal blooms in Hong Kong are caused by a
diverse array of causative organisms. While massive growth of diatoms and dinoflagellates have caused most of the algal blooms, at least 14% of the algal blooms recorded between 1980 and 2002 are believed to be caused by small algal species which have not been properly identified (HKEPD, 2003). No information on the taxonomic composition and diversity of eukaryotic picoplankton in coastal seas around Hong Kong is available. Investigation of seasonal and spatial patterns in diversity and compositions in eukaryotic picoplankton communities was conducted in Tolo Harbour and Mirs Bay because of the availability of long-term
7 data on phytoplankton composition and physio-chemical variables such as temperature and salinity (HKEPD 2003).
1.6 Objectives
The objectives of the current study are: (1) to study the diversity of picoeukaryotes in coastal waters of the western Pacific for the first time and more specifically, to test the hypothesis that 'a highly diverse picoeukaryote assemblage is also present in subtropical coastal waters of the western Pacific', (2) to study the spatial variations of picoeukaryotes in coastal waters with different trophic status and to test the hypothesis on the hump-shaped relationship between marine picoeukaryotic diversity and chlorophyll a concentration, and (3) to investigate the seasonal variations in picoeukaryote communities in coastal waters of Hong Kong.
8 Chapter 2. Materials and methods
2.1 Study site
The study was conducted at two coastal stations in Hong Kong waters, a
subtropical marine ecosystem in the western Pacific Ocean: the eutrophic inner Tolo
Harbour (TH; 22�26，N，丨 14�13’E an) d the oligomesotrophic Mirs Bay (MB; 22�30,N，
114°21，E) (Fig. 1). Tolo Harbour is a semi-enclosed bay with a long history of
eutrophication. Mirs Bay is a relatively unpolluted water body which is strongly
influenced by water currents from the South China Sea. Mirs Bay also contains two
marine parks and most of the coral beds in Hong Kong. Based on data from the
Hong Kong Environmental Protection Department (HKEPD) from 2002 to 2005
(http://www.eDd.gov.hk/). mean concentrations of chlorophyll a (Chi a), total
phosphorus and nitrate nitrogen were 11.89 jag 1'', 32.71 |ig 1"' and 10.23 jug 1"',
respectively for Tolo Harbour, and 1.94 ^ig I"', 6.25 ^g 1"' and 6.50 jug I"',
respectively for Mirs Bay.
2.2 Sample collection
Surface seawater samples (2.5 I) were collected in January, April, July and
October, 2006. Immediately after collection, the water samples were filtered
through a 200 ^im mesh sieve to remove most of the mesozooplankton and large
particles. Filtered samples were returned to the laboratory in polycarbonate carboys
within 2 h. Water temperatures, salinities and dissolved oxygen (DO) levels were
measured on board using a Hydro Lab sensor. Chi a concentrations were determined fluorometrically. In brief, 10 ml water samples were filtered through
9 •丨丨 / T MB China / • /kr、， K 一'、，一 ,, o _)� J ^ Hong Kong / _^一’：>—‘ r产'�� '1 \
，\ Ir-^ : _
South China Sea \ , / , ^ 广、从’欢.；^、
Figure 1. Map showing the sampling locations of the current study. TH and MB represent Tolo Harbour and Mirs Bay, respectively.
10 0.45 |Lim Millipore filters (Whatman) and extracted overnight in 5 ml 90% acetone
(analytical grade) at 4°C in dark. Fluorescence of the acetone extracts before and
after acidification (10% HCI) was measured with a Turner Designs 10-AU
fluorometer and the amounts of chlorophyll a were calculated using the method of
Yentsch and Menzel (1963). Diatom and dinoflagellate concentrations were
determined under inverted microscope by the Utermohl technique (1958). In brief,
100 ml water samples were preserved in Lugol's iodine solution and then
concentrated to 10 ml by settling for a week in glass cylinders. One ml of the
concentrated samples were transferred to a counting chamber and phytoplankton
were counted under a Leica DMIL inverted microscope. Two liters of water was
prefiltered through a 3 网 pore size Nuclepore membrane (Whatman) and the
microbial biomass was then collected onto a GF/F filter (Whatman). A gentle
vacuum (<20 cmHg) created by a hand pump was used to facilitate the filtration
processes. The filter was then transferred into a 15 ml centrifuge tube containing
DNA lysis buffer (0.75 M sucrose, 40 mM EDTA, 50 mM Tris-HCl, pH 8),
immediately frozen in liquid nitrogen and stored at -80°C until DNA extraction.
2.3 DNA extraction and 18S rRNA gene amplification
DNA was extracted using a cetyltrimethylaininonium bromide (CTAB) extraction
procedure (Doyle and Doyle 1990). Briefly, filter paper containing sample was
thawed and ground in liquid nitrogen. The powder was then transferred to a 50 ml
Falcon tube containing 3% (w/v) CTAB and incubated at 60°C for 30 min in a water
bath. Nucleic acids were extracted once with an equal volume of chloroform-
isoamyl alcohol (24:1). After centrifugation for 10 min at 12,000 rpm, the aqueous
phase was transferred to a clean centrifuge tube and 2/3 volume of cold isopropanol
26 was added to precipitate the nucleic acids at room temperature overnight. Nucleic
acids were recovered by centrifugation, washed once with 70% ethanol and
re-suspended in milli-Q water. Extracts were purified with the Geneclean II Kit
(BIO 101) and stored at -20% until use.
The 18S rRNA gene was amplified by PCR using the eukaryotic primers
Euk328f(5'-ACCTGGTTGATCCTGCCAG-3') and Euk329r
(5 -TG ATCCTTC YGC AGGTTC AC-3'), complementary to regions of conserved
sequences proximal to the 5’ and 3' termini of the 18S rRNA gene (Moon-van der
Staay et al. 2000). Each PCR mixture (50 )li1) contained 2 ii\ ofDNA template, 5 ^il
of lOx PCR buffer (50 mM KCI; 10 mM Tris-HCl; 1.5 inM MgCb), 200 )nM of
dNTP, 0.2 iiM of each primer and 2.5 U Tag polymerase (Promega). The PCR
thermal regime consisted of an initial denaturation of 3 min at 94°C, followed by 32
cycles of 45 sec at 94°C, 30 sec at 58°C, 2 min at 72°C，and a final cycle of 10 min at
72°C. PCR products of the expected size (ca. 1,800 bp) were purified with the
Qiaquick gel purification kit according to manufacturer's instructions (QIAGEN).
2.4 Clone library construction and screening
The purified PCR products were cloned using the TA cloning kit (TaKaRa
Biotechnology) following the manufacturer's recommendations. Putative positive clones were picked and transferred to a multiwell plate with LB medium and 5% glycerol, and stored at -80°C. About one hundred putative positive clones from each library were randomly selected, and the presence of the 18S rRNA gene insert was checked by PCR amplification with the same primers. Five microliters of PCR product were digested for 2 h at 37°C with 2.5 U of restriction enzyme HaelU
12 (Promega). Restriction fragments were separated by electrophoresis at 90 V for 50
min in a 2.5% agarose gel. A 50-bp DNA ladder (Invitrogen) was included in each
gel to aid the Restriction Fragment Length Polymorphism (RFLP) pattern
comparison. Clones showing the same RFLP pattern (DNA fragments of equal size)
were grouped together and considered to belong to the same operational taxonoinic
unit (OTU).
2.5 Sequencing and phylogenetic analysis
At least one clone of each RFLP type were purified and then partially sequenced
using the internal primer ELik528f(5' - GCGGTAATTCCAGCTCCAA -3') (Elwood
et al. 1985) and ABI PRISM® BigDye™ Terminators V 3.1 Kit (Applied Biosystems)
on an ABI PRISM® 3700 DNA Analyzer (Applied Biosystems). This primer binds
upstream of the most variable V4 region of the 18S rRNA gene, which is sufficient
for phylogenetic identification of eukaryotes at roughly the genus level. In order to
determine the phylogenetic affiliation, all sequences were subjected to a BLAST
search (Oct-15-2006 database) using the National Center for Biotechnology
Information web server (http://w\vvv.ncbi.nlin.nih.gov/). Potential chimerical
artefacts were checked using the CHECK_CHIMERA program at the Ribosomal
Database Project II (http://35.8.164.52/cgis/chimera.cgi?su=SSlJ). Nucleotide
sequences from the current study were deposited in GenBank under accession
numbers EF538969 - EF539172.
High quality non-metazoan sequences that passed the chimera checking process, together with their closest GenBank relatives, were first aligned using the ClustalW multiple alignment tool integrated in MEGA 3.1 (Kumar et al. 2004) and the
13 alignments were then manually improved. Ambiguously aligned regions were
identified and removed by Gblocks v.0.91b (Castresana 2000) (minimum block
length = 5; allowed gap positions = with half). Maximum-likelihood (ML)
phylogenetic trees were constructed using PHYML v.2.4.4 (Guindon and Gascuel
2003)，with the general time-reversible (GTR) nucleotide substitution model.
Parameters for gamma distribution (G) and invariable sites (I) were estimated by the program. Statistical confidence on the inferred phylogenetic relationships was assessed by 100 ML bootstrap replicates. The inferred trees were displayed using
MEGA.
2.6 Statistical analyses
Rarefaction curves were plotted using the program Analytic Rarefaction 1.3
(http://vv\vvv.Liga.edu/~strata/software/). Library coverage values, nonparametric species richness estimators Chaol and ACE, the Shannon diversity index H and the
Simpson's diversity index D'' were calculated using the software SPADE
(http://chao.stat.nthu.edu.tw/$oftwareCE.html). Equations for the statistics are listed in Appendix 1.
14 Chapter 3. Results
3.1 Hydrological parameters of study site
Water samples collected at two stations in the subtropical coastal waters of Hong
Kong in January, April, July and October 2006 produced a total of eight libraries
(Table 1). Water temperatures at the two stations showed similar seasonal pattern
and an increasing trend was observed from January to October (Table 1). Surface
DO levels varied between 5.52 mg I"' and 9.40 mg I"' and were slightly lower in Tolo
Harbour than in Mirs Bay. Averaged over the study period, Chi a concentrations
varied from 7.25 ^ig p' (2.36-16.21 |Lig p') to Tolo Harbour and 1.78 |ig 1"'
(0.79-2.78 ^g I"') in Mirs Bay. Concentrations of diatoms and dinoflagellates were
generally higher in Tolo Harbour than in Mirs Bay.
3.2 Clone libraries
Examination of 733 picoeukaryotic clones revealed 186 different RFLP patterns
(an example is given in Fig. 2)，representing 186 OTUs (Table 2). Fifteen
additional OTUs of metazoan origin (ca. 7.5% of total OTUs), including 13 from
Copepoda, 2 from Cnidaria, 1 from Ctenophora and 11 potential chimerical
sequences, were detected and excluded from further analyses. The average length of sequences generated was about 650 bp. The shape of the rank abundance curve
(Fig. 3) was of a typical shape for a diverse assemblage (Lunn et al. 2004). About
50% and 75% of sequences obtained in the current study were of> 98% and > 95% identity to GenBank sequences, respectively (Fig. 4).
15 Table 1. Collection information, hydrological parameters and phytoplankton cell
counts of water samples collected in this study.
Station Sampling Library Temp. DO Chi. a Diatoms Dinoflagellates ^ (°C) (mg r') gig 丨）(cells ml-') (cells ml'') Tolo 16/01/06 THOl 19.50 9.25 5.65 507.9 23.1 Harbour 18/04/06 TH04 21.56 6.24 2.36 340.0 8.4 14/07/06 TH07 26.49 5.52 16.21 424.4 7.6 26/10/06 THIO 27.48 5.72 4.79 141.0 136.5 Mirs 16/01/06 MBOl 16.81 9.40 0.79 27.3 9.1 Bay 18/04/06 MB04 20.84 7.14 2.04 262.0 5.2 14/07/06 MB07 27.04 6.16 2.78 122.4 11.8 26/10/06 MBIO 27.35 7.30 1.52 6.2 ^
16 ••bi Figure 2. Gel photo i>li� mim- 16 restriction fragment length polymorphism (RFLP) patterns from library Ti 1 ...
17 Table 2. Picoeukaryotc O TU distribution of higher-level taxonomic groups in pooled
Mirs Bay (MB), pooled Tolo Harbour (TH) and total libraries.
Number ofOTUs (clones) Taxonomic group MB TH Total Alveolates group II 22 (125) 34 (146) 46 (271) Alveolates group I 4 (16) 6 (49) 8 (65) Dinophyceae 12 (40) 7 (14) 16 (53) Ciliophora 23 (62) 20 (65) 34 (127) Stramenopiles 11 (40) 25 (42) 31 (82) Prasinophyceae 9 (28) 7(13) 11 (41) Picobiliphyta 2(7) 4(19) 4(26) Cercozoa 11 (22) 5(9) 14(31) Haptophyceae 2 (4) 3 (3) 4 (7) Cryptophyta 1(1) 2(5) 2(6) Cryptophyte nucleomorph 2 (2) 1 (2) 2 (4) Katablepharidophyta 0 (0) 丨（2) 1 (2) Telonemia 1(1) 1(1) 2(2) Fungi 2 (4) 0 (0) 2 (4) Choanoflagellida 0(0) 1(1) 1(1) Radiozoa 1(1) 2(2) 3(3) Chlorarachniophyte luicljoinorph 1 (1) 0(0) 1(1) Novel Clade I 1(1) 1(1) 2(2) Novel Clade II 1(2) 1(1) 1(3) Novel Clade III 1(1) 1(1) 丨⑵ Total 107 (358) 122(375) 186 (733)
18 45 -1
40 -
35 -
V：‘ 30 - o 0 二- — DMB
1二 20 -i illiL
芸 15 - 4
10 - ： ： E
0 _li J hi If . iP.llii：……_！ i ：；： :llll|llippppijllll|llll|^ 1 10 19 28 37 46 55 64 "3 S2 91 100 109 118 127 136145 154 165 1"2 ISl
Operational Taxonoiiiic Unit (OTU)
Figure 3. Rank abundance curve for the pooled dataset of 733 picoeukaryote clones, representing 186 OTUs.
19 35 n
30 - •
I ill
100 99 98 97 96 95 94 93 92 91 90 BLAST similarity (%)
Figure 4. Histogram of GenBank BLAST similarities of sequences obtained in this study.
20 3.3 Higher-level taxonomic distribution
At least 17 higher-level taxonomic groups of picoeukaryotes were observed in
the current study, with three potential higher-level novel groups (Table 2). Novel
groups were defined here using the following set of criteria: (1) at least three
sequences were involved; (2) sequences were retrieved from at least three
independent environmental libraries; and (3) a high ML bootstrap value > 95% was
recorded.
Combining all the datasets, alveolates group II, with 46 OTUs (ca. 25% of total)
and 271 clones (ca. 37% of total), was the most dominant group. Ciliates and
stramenopiles were also abundant, representing, respectively, about 18% and 17% of
the total OTUs (ca. 17% and 11 % clones). Members from Dinophyceae,
Prasinophyceae and Cercozoa also showed significant contributions, composing 16
OTUs (53 clones), 11 OTUs (41 clones) and 14 OTUs (31 clones), respectively.
Other groups were less pronounced, each contributing fewer than ten OTUs.
Members from the two newly defined phyla, Picobiliphyta (Not et al. 2007b) and
Telonemia (Shalchian-Tabrizi et al. 2006)，were also obtained in this study.
When the datasets were grouped according to sampling stations and thus trophic
status, alveolates group II and stramenopiles harboured more OTUs in the eutrophic
Tolo Harbour libraries even though the total number of contributed clones was
almost comparable between Tolo Harbour and Mirs Bay (Table 2). While alveolates group I，picobiliphytes and cryptophytes were more abundant in Tolo
Harbour, dinoflagellates and prasinophytes showed greater contribution in Mirs Bay.
Cercozoa was both more diverse and abundant in Mirs Bay. The numbers of OTUs
21 and clones for ciliates were similar for both stations. While katablepharidophytes
and choanoflagellates were only observed in Tolo Harbour libraries, clones from
fungi and chlorarachniophyte nucleomorphs could only be retrieved from Mirs Bay
libraries, although these groups were only represented by a few clones. The rank
abundance curve showed a differential spatial distribution of OTUs, with 64 unique
to Mirs Bay libraries and 79 OTUs unique to Tolo Harbour libraries. Only 43
OTUs occurred in both stations (Fig. 3). Seasonally, while all six major taxonomic groups appeared in all eutrophic Tolo Harbour libraries throughout the year, varying only in relative abundance, occurrence in oligomesotrophic Mirs Bay libraries tended to be more sporadic, with all six groups present in April, five in January and July, and only four in the October (Fig. 5). Non-photosynthetic picoeukaryotes dominated all the clone libraries, and the relative contributions of photosynthetic groups were generally higher in the oligomesotrophic Mirs Bay libraries than the eutrophic Tolo
Harbour libraries (Fig. 6).
3.4 Phylogenetic affi 1 iations of OTUs
ML trees were constructed to investigate the phylogenetic positions of sequences obtained in the present study. Most of the higher-level taxonomic groups were supported by high bootstrap values (Fig. 7). Alveolates, stramenopiles and Rhizaria were diverse and independent phylogenetic trees were drawn (Figs. 8, 9 & 10). [A metazoan tree was also included as reference (Fig. 11).]
22 MBOl MB04 MB07 MBIO
THOl TH04 TH07 THIO
• Alveolates group II • Alveola tes group I 園 Dinophyceae 0 Ciliophora _ Stramenopiles E Prasinopliyceae • Others
Figure 5. Relative abundance of the six most represented picoeukaryote groups in the eight clone libraries.
23 • Photosynthetic 因 Non-photosviithetio 111111111 MBOl THOl MB04 TH04 MB07 TH07 MBIO THIO
Figure 6. Relative abundance of the photosynthetic and non-photosynthetic groups in the eight clone libraries.
24 I Leptomyxa reticulata ^ Echlnamoeba exundans 89 •^^•― Stramenopiles 66 r—— A MB04.38 ^�SCM38C2 0 Telonemia 1— Tetonema antarcticum p rRA001219.38 �RA000907.33 -參 MB04.45 Picobiliphyta I pRA000907.18 100 73L0THIO.ll • TH10.69 100 r Leucocryptos marina I lilT Katablepharidophyta ^•TH01.39 I — 100 r Plagioselmis prolonga i— I ^•TH04.10 Cryptophyta 90 Teleaulax acuta p- »lL參 MB04.26 �•TH10.32 ®^J_RA010613.138 I Novel Clade I I P RA010613.131 100 I I I-AMB04.9 S5|AMB10.47 fljL NAMAKO-37 J- TAGIRI-23 Fungi 73" AMB01.14 i— Triparticaicar arcticum Mcromonas pusilla
A MB04.46 AMBOI.S •， L會 thiO.5 n�•MB01.31 —•MB01.38 10_0 65 眷 MB01.22 I MIcromonas pusilla MBIC10095
100 |T TH01.40 Prasinophyceae L- • Ostreococcus sp. 7. NW414.32 I I^ • MB01.30 100 Bathycoccus prasinos 100 MB07.17 *“ Pycnococcus sp. ai !i|•"“•画7.31 [n MBIC10622 7® L_ Prasinoderma coloniale —Rhizaria P Chrysochromulfna scutellum 100 I TTHOI.17 们 |># MB07.32 Haptophyceae J A MB07.26 73IbL010625.10 L TH04.31 30 I Teleaulax amphloxeia I—^―^― rA MB01.32 Cryptophyte nucleomorph WlHeOOIOOS.lOa 1O0JAMB07.44 TH01.7 Novel Clade II LamB10.44 I_ 9n-AMB07.52 100 "LYTH07.19 Novel Clade III LpD6.12 I • MB07.10 L———— —I Chlorarachnlophyte nucleomorph 1001 Lothareila sp. li^—MBBI Alveolates
Figur‘―“M~e 8C. (Legen‘ d on next page)
25 Figure 7. Maximum-likelihood (ML) phylogenetic tree of 18S rDNA sequences from all major picoeukaryotic groups observed in the current study. The tree was based on 79 partial sequences of 447 positions after Gblocks processing. Sequences obtained in the current study were in bold. Symbol-codes to the left of sequence names indicate the sampling location (• = MB, • = TH, •= both). Numbers at nodes are ML bootstrap percentages from 100 replicates. Values < 50% were not shown. The tree was rooted using two Amoebozoa sequences {Leptomyxa reticulate and Echinamoeba exundans). Scale bar = 0.1 substitution per base.
Collapsed clusters (Stramenopiles, Rhizaria and Alveolates) are expanded in the next three figures.
26 I Leptocyfindais danicus
‘ Pseudobodo tremulans I—•TH04.2 I 100 r#• MB01.26 10^NW414.24 I • TH01.13 Prostomatea 90 I I 99 I NCMS0601 „ L~•MB07.38 Oyptocaryon irritans I • TH10.63 ^―^―—H Colpodda 100 I DH147-EKD23 100jSCM28C124 |"WTH10.20 96JMD65.18 [1•鲁 MB01.13 一 ENI42482.00244 ” MB01.12 Strom A • B L ••TH04.37 "-•THCM.IG Strombidium sp. SNB99-2 I — �•THIO.M Lamboi.7 I • MB07.30 731 T TH10.37 ^ P Strombidium sp. SBB99-1 I 121 92r~.TH04.39 I~^ Strom c Ciliophora I— ENI47296.00059 m 80 TH04.42 loot UEPACAIpS pENI42482.00186 MB01.37 Splrotrichea L—-AMB04. 2 Strom D I I 92 ENI40076.00749 lee ®® I AMB01.35 Ly 7^10.24 100pAMB01.21 I L M065.05 一 I~• TH10.68
^ Eutintinnus pectinis
IStrobilidium caudatum ~TTM10.18 1� � 65 I" II~SCM15C2 L 100J~ • MB10.31
L Tintinnopsis dadayi L 100pAMB07.25 I (••MB10.33 65 rAMB10.16 • TH10.33 I •MB10.40 T Parastrombidinofysis shimi Figure 8A. (Legend on page 30)
27 100 I— Toxoplasma gondii I I— I Apicomplexa _ Sarcocystis muris • r Noctiluca scintillans I — •MB10.52 I •MB10.23 100fAMB01.33 I~—1001 LSCM15C23 NDCI .•TH07.15 1 ⑶ CDS. 10
99 I • MB04.34
PyrcxJinium bahamense var. compressum
r I • MB04.15
• Kariodinium micrum jAMB01.6 "L SCM37C24
p Lepidodinium viride
丄『•MB01.27 10 \ Dinoflaqellata IBL001221.15 ^
~• MB04.39
r SCM37C27
‘r • MB01.40
•TH10.6 81 51 Prorocentmrn micans
60 •MB10.29
r Gyrodinium spimle
95 r AP-picoclone12 SHIythio.IO L •TH01.22 "LBL001221.42 100 厂書 MB10.3 I Cemtium longipes 100 r Pof^insus marinus I I I Perkinsozoa Perlcinsus sp. •
97 • MB07.2
96 -81001221.41 I LJr ^ TH10.45 NAGI-1 I iooItagirmo 77 100 I L E214 “ i-A MB07.40 一 rTTH10.57 “ NAGI-3 NAG I 100 I-SCM38C41 98 69 I • TH07.4
I I 65
__lO0rAMBO4.17
L ENVP21819.00079 I- SCM37C30 I NAGI-5 100 TH10.8
Figure 8B. (Legend on page 30)
28 100 厂眷 TH01.1 I L UEPACCp3 I •MB07.15 「 1 • TH07.6 * NAGIM3 e7URA010613.20 loorAMB07.21 I ：~I-OLI1100 9 NAGIM6 100 1~AMBOI.62 I I ：_ NAGII-11 100 UEPACAFpS I • MB10.61 I~T TH07.43 j I • TH10.22 — 「•TH10.46 H NAGIM jy »9 LAP.picoclone8 —•TH04.14 E• TH07.42 I I Amoebophrya sp. ex Karlodinium micrum “ 2 I • MB01.42 r I~ I~• MB04.25 NAGII-2 »8 L Amoebophrya sp. ex Scrippslella sp. “�•MB01.11 p. MB07.42 I 1� �1001 UEPACEp3 NAGII-3 TTH10.12 L Amoebophrya sp. r眷 MB10.46 NAGIM I 00 \ Amoebophrya sp. ex Ceratium tripos 100 jA MB07.29 79 I- AP-picoclone20
•II -•TH10.4 BB01-«3 P •TH10.1•TH10.297 NAG 11 I•~3 J_•TH01.37 NAGIt-5 7a1_ Q2A03
•TH07.6 100 rW TH07.32 ~RA010613.126 2J參 MB07.9 I n-UEPAC41p3 - NAGIMO • TH07.27 sL UEPACAPp3 BL001221.40 I • MB01.3 I~「•MB04.24 NAG1U7 54 •TH10.7 — LDH147-EKD16 I •TH10.49 L 100 r •州似,18 I I NOR26.14r NAGII-6 “I~DH147-EKD6 打「•MB01.19 r^ pA MB10.27 NAGII-8 IOOIRAOI 0613.44 B^TTHIO.I P~OLI11012 ioorTTHOI.8 — I L UEPACBp4 99 I • MB01.16 I NAGIM4 « I AP-picoclone15 I NOR50.19 厂 • TH07.2 - ？71 I •TH07.12 sTl® TH07.26
• TH04.26 ‘0.1 -
Figure 8C. (Legend on next page)
29 Figure 8. ML phylogenetic tree of alveolate 18S rDNA sequences. The tree was based on 162 partial sequences of 505 positions after Gblocks processing. The tree was rooted using two stramenopile sequences {Pseudobodo tremulans and
Leptocylindrus danicus). Other definitions as in figure 7.
A. One-third of the tree, containing all ciliate sequences.
B. Another one-third of the tree, containing all dinoflagellate and NAI sequences.
C. Last one-third of the tree, containing all NAGII sequences.
30 lOqT TH07.3 I ieptocyiindfvs danicus I6~7 • TH10.47 Baciliariophyceae 9 fTTH01.20 � 100 1.BL001221.14 L Pseudo-nitzschia miMtiseries toojT THOI.43 L LHe0i02l6.6 Bolldophyceae Phototrophic stramenopiles 一 51 I—^― Bolidomonas paciHca 100 I • TH01.5 I CD8.06 100 丨•MBM.23 I—Chrysophyceae _1L L He000803 4 L t— Phaeoplaca thallosa 64
Paraphysomonas imperforata 12 I~IFV18.3H10 一 pT TH10.43 MAST-1 100lcD8.09 Pirsonia y/emtcosa | PIrsonia r 眷 MB04.36 I MAST-2 100LDHU8.5-EKD53 lOOf FV23-1D5 叫 't TH01.38 MAST-6 ^ l'MEI.24 一 I • MB01.34 p ENVP10203.00002 9»LytH10.39 100 [A MB04.21 121 L C3-E035 Oomycetes UncuHured Pythlum sp.
I L Pseudobodo tremulans Bicosoecida 时 I •MBIO.W r"^ rT TH07.21 — 10O1-ME1-28 r 100 r^ MB01.2S J i BL010625.35 ^•TH04.32 MAST-3 «3 «^BL010320.17 IQJ-AMB04.3 ^ MD0S.32 74 jVTH04.3 UEPACRpS IOOJVTHOI.2 n WD60.8 I ^ 0 TH01.6 H I UEPACCp4 MAST^ « r-V TH07.16 »8'He001005.47 L I • TH10.61 IfA MB07.19 > 1 MAST-11 100LBL000921 26 IOOtTTHOI.24 L r~L UEPACLpS 100 I BL010320.6 MAST-7 rAMB04.18 I '®1|LNW617 17 ®"LyTH07.36 I Labyhnthuloides haliotidis
^ I D107 La byrinthulida 941 • TH01.44
j Noctiluca scintillans
^^ Ceratium longipes
‘0.1 ‘
Figure 9. (Legend on next page)
31 Figure 9. ML phylogenetic tree of stramenopile 18S rDNA sequences. The tree was based on 64 partial sequences of 517 positions after Gblocks processing. The tree was rooted using two dinoflagellate sequences {Noctiluca scintillam and Ceratium longipes). Other definitions as in figure 7.
32 j Pseudobodo tremulans
* Leptocyiindms danicus
71「•MB10.21
••MB07.4 100 NCCI r-^ L NOR46.27
MAMAKOe
I 1(MFu21
「 100「SCM27C7 |J MB07.22
^•TH07.17 ^~•TH04.43 • core Cercozoa J • MB07.6
•， 56 _ TAGIRI-3 ®L 「 L-C1 Cercozoa r I~• TH10.54 NC2 ^ «RL •MB07.23
I • MB07.50
Alias sp. I Thaumatomonadida
* Cryothecomonas aestivalis Cryomonadida • THOl.15 I RD010517.43 ——^―^―^―I 在 Plasmodiophorida 100 I • MB07.8
99 r NOR26.21 I Chlorarachnea
ioorfTmo.26 I—I Polycystinea ^^^ I Pseuckxubus obeliscus ；T >1 ““， ， Radiozoa 99 1— Amphibelone anomala ^―^―——Acantharea
‘0.1 ‘
Figure 10. ML phylogenetic tree of 18S rDNA sequences of Rhizaria. The tree was
based on 28 partial sequences of 513 positions after Gblocks processing. The tree was rooted using two stramenopile sequences {Pseudobodo tremulans and
Leptocylindrus danicus). Other definitions as in figure 7.
33 Rhizophlyctis harden
丨眷 MB04.31 rMD65.36 64 H • p> MB10.2S Copepoda; Cyclopoida p L SCM27C52 • MB10.42 91 pENI42482.00368 100 I— — ••TH10.2 ^ • p SCM38C38 森 Copepoda; Calanoida 「鲁 MB10.39 51 i„ i 88 100 •MB10.32 【•MB10.8 �Junceella aquamata . Cnidaria; Anthozoa 100 •MB04.12 I Garveia sp. •—I • Cnidaria; Hydrozoa lOoL參 TH01.34 • MB04.42 Ctenophora 10 0 Bolinopsis Infundibulum
I 1 0.1
Figure 11 • ML phylogenetic tree of 18S rDNA metazoan sequences. The tree was based on 18 partial sequences of 597 positions after Gblocks processing. The tree was rooted using a fungus sequence {Rhizophlyctis harden). Other definitions as in figure 7.
34 3.4.1 Alveolates
Alveolate sequences were divided into four major groups: alveolates group I
(NAGI), alveolates group II (NAGII), ciliates and dinoflagellates (Fig. 8). Within the most diverse NAGII, members from 13 of 16 previously defined clusters
(Groisillier et al. 2006) were recovered in the current study. Within the
Amoebophrya clade formed by NAGI-1 to NAGI-5, MB07.29 was > 98% similar to
AP-picoclone20 and TH10.4 was 93.7% similar to BBOl-83. TH07.6, TH07.32 and
RAO 10613.126 formed a distinct clade with 100% bootstrap support. Another clade was formed by TH07.2, TH07.12 and TH07.26 with 99% bootstrap support.
Sequence identity between THOl.l and UEPACCp3 and between THOl.8 and
UEPACBp4 was >98%. Both UEPACCp3 and UEPACBp4 were from the eastern
Pacific. THlO.l was 95.7% similar to OLIl 1012 from the equatorial Pacific. The phylogenetic positions of MB07.15，MBO1.42, TH10.49 and TH04.26 were unclear from the current analysis using partial sequences.
All five previously defined alveolates group I (NAGI) lineages (Groisillier et al.
2006) were recovered in the current study. MB04.17 had > 98% identity with
ENVP21819.00079. TH07.4 represented a distant lineage of unclear phylogenetic position. Most dinoflagellate sequences were closely related to known species including Pyrodinium bahamense, Karlodiniuni niicnim, Lepidodiniurn viride,
Prowcentrum micons, Gyrodinium spirale, Ceratium longipes and Noctiluca scintillam. MBO 1.33 and TH07.15 had 99% and 100% identity with SCM15C23 and CDS. 10, respectively, forming a novel dinoflagellate clade, designed as NDC here.
35 A diverse lineage of ciliates including members from Prostomatea, Colpodea and
Spirotrichea was also recovered. Most of the sequences retrieved were closely related to environmental sequences but distantly related to cultured organisms. In addition to the recovery of sequences belonging to previously defined Strombidium clades Strom A and Strom B (Lovejoy et al. 2006), two potential novel Strombidium clades, Strom C and Strom D, with high bootstrap supports were described here.
3.4.2 Stramenopiles
Stramenopiles represented another diverse assemblage in the current study (Fig.
9). Most of the stramenopile sequences obtained (25 out of 31 OTUs) were from heterotrophic lineages, particularly from previously described novel marine stramenopile (MAST) clusters (Massana et al 2004b). Members from seven of the
12 MAST groups were retrieved in the current study. For other heterotrophic members, MB04.21 had 99% identity with oomycete clone C3-E035; TH04.36 was closely related (> 98%) to Pseudobodo trenmlans (Bicosoecida); and THOl.44 was loosely related to D107 and the closest known labyrinthulid relative Labyrinthuloides haliotidis. TH 10.39 was > 97% similar to ENVP 10203.00002. MBOl .34 and
TH 10.51 occupied unclear phylogenetic positions in the current analysis using partial sequences. Photosynthetic stramenopiles formed a monophyletic lineage. Six current stramenopile sequences belonging to three typical algal classes
(Bacillariophyceae, Bolidophyceae and Chrysophyceae) were retrieved in the current study.
3.4.3 Rhizaria
36 Sequences belonging to Cercozoa and Radiozoa were retrieved from the current
study (Fig. 10). Most of the cercozoan sequences belonged to the core Cercozoa
cluster. Except for THOl. 15 which had > 98% identity with Cryothecomonas
aestivalis^ all other core cercozoan sequences were closely related to environmental
sequences. MB07.6, TH 10.54, MB07.23 and IVIB07.50 grouped with TAG丨RI-3
and CI to form a cluster previously referred to as NC2 (Bass & Cavalier-Smith 2004).
MB 10.21 and MB07.4 formed a highly supported group (100% bootstrap) with
NOR46.27 and NAMAKO-6, possibly representing a novel cercozoan clade,
designated here as NCCI. MB07.22 had > 99% identity with SCM27C7 and
TH07.17 was related to TH04.43. These four were grouped together with 82%
bootstrap support. Besides the core cercozoan sequences, MB04.43 was closely
related (> 98%) to NOR26.21, an environmental sequence belonging to
Chlorarachnea, while MB07.8 was loosely related to plasmodiophorid clone
RDO10517.43. For Radiozoa, one sequence closely related to Pseudocubus
obeliscus (Polycystinea) and another to Amphibelone anomala (Acantharea) were
retrieved.
3.4.4 Other lineages
Prasinophyceae was the second diverse photosynthetic assemblage in the current study (Fig. 7). Sequences highly similar (> 98%) to cultured organisms belonging to genera Mcromonas, Ostreococcus, Bathycoccus, Pycnococcus were retrieved, except for MB07.31 which was 95.8% similar to coccoid prasinophyte clone
MBIC10622. Three recorded haptophyte sequences were all closely related to the genus Chrysochromulina (Prymnesiophyceae). Two cryptophyte sequences were retrieved, one closely related to Plagioselmis prolonga and the other to Teleaulax
37 acuta. One katablepharidophyte sequence shared > 99% identity with Leucocryptos
marina was retrieved. Three sequences closely related (>98%) to environmental
relatives belonging to Picobiliphyta, a newly defined algal phylum (Not et al. 2007b),
were also recovered.
Among the other heterotrophic lineages, one sequence belonged to another newly
defined phylum Telonemia (Shalchian-Tabrizi et al. 2006). The sequences
MB 10.47 and MB01.14 appeared to be loosely related to the closest known fungi
relative Triparticalcar arcticum and closely related to environmental clones
NAMAKO-37 and TAGIRI-23, respectively. Two sequences closely related to
cryptophyte nucleomorph and one sequence loosely related to chlorarachniophyte
nucleomorph were also found.
3.4.5 Novel higher-level groups
Three potential novel higher-level clades were discovered in the current study,
and all of them were composed of environmental sequences from at least three
different libraries and supported by 100% bootstrap values (Fig. 7). Novel Clade I
was composed ofTH 10.32, MB04.9 and their closest GenBank relatives
RAO 10613.138 and RA010613.131. Novel Clades II and III were distantly related to other major eukaryotic lineages and could be placed among the cryptophyte and chlorarachniophyte nucleomorph lineages. While Novel Clade II was only composed of sequences obtained from the current study (MB07.44, THOU and
MB 10.44), Novel Clade III included MB07.52 and TH07.19 which were closely related to GenBank sequence PD6.12.
38 3.5 Diversity estimates of picoeukaryotes
Seventy-one to 100 clones were examined for each library, generating 32 to 44
OTUs (Table 3). Although rarefaction curves showed little sign of saturation (Fig.
12), coverage values calculated for most individual libraries were high (ca. 80%),
except for libraries MB 10 and THIO with values of around 70%. The Shannon
diversity indices (H) and the Simpson's diversity indices (D"') of individual libraries
ranged from 3.2 to 4.0 and 10.0 to 25.3, respectively. Species richness estimators
Chaol and ACE ranged from 48 to 98 and 55 to 214，respectively. Nevertheless,
results from both estimators generally agree with each other. However, estimates
from independent libraries were not statistically different from each other (p < 0.05) based on their overlapping 95% CI (Hughes et al. 2001). The number of OTUs, coverage values, H, DChaol and ACE generally increased for sample sets grouped seasonally and spatially.
39 Table 3. Picoeukaryote diversity estimates for different combinations of the eight clone libraries. N is the number of clones; library coverage value is calculated according to Good's equation; H is the Shannon diversity index; D"^ is the Simpson's diversity index; Chaol and ACE are non-parametric species richness estimators.
Sample set N OTUs 二已零只 Chaol (95% CI) ACE (95% CI)
MBOl 100 34 84.0 3.3 11.0 48 (38 - 80) 55 (42 - 90) THOl 96 32 79.2 3.2 10.0 98 (50 - 275) 214 (84- 667) MB04 87 36 78.2 3.6 16.7 61 (44- 115) 80 (50- 169) TH04 99 41 79.8 3.7 19.6 61 (47- 99) 65 (50- 101) MB07 100 41 76.0 3.6 16.4 89 (57- 182) 127 (67- 319) TH07 98 39 78.6 3.5 12.1 61 (46- 102) 99 (57 - 235) MBIO 71 34 71.8 3.6 21.3 84 (48 - 202) 63 (45 - 111) THIO 82 44 67.1 4.0 25.3 84 (59- 151) 135 (76-302) Jan 196 60 84.2 3.8 20.0 108 (79- 181) 140 (92-259) Apr 186 67 84.4 4.1 30.0 93 (77- 132) 101 (83 - 141) Jul 198 65 84.3 3.9 22.7 99 (79- 148) 105 (83 - 154) Oct 153 71 73.2 4.3 37.7 141 (101 -231) 177 (116- 321) MB 358 107 86.6 4.4 44.3 189 (145 -284) 292 (151 - 876) TH 375 122 84.0 4.5 44.9 212 (167-299) 268 (198-403) Total 733 186 89.1 4.8 58.6 289 (243 - 370) 352 (282 - 472)
40 45 - 1 to 1 40 I\fBOl -t:s- THOI 35 - ~1B04 -e- TH04 30 -+- IvIB07 -...r/J -4- TH07 ~ 25 0 -*- tvIBIO <+-c 0 --- THIO o _0 Z 15
la
5
0 0 10 20 30 40 50 60 70 80 90 100 No. of clones eXaJ.nilled
Figure 12. Rarefaction curves for the eight clone libraries. OTUs were defined as clones sharing the same RFLP pattern. A 1 to 1 standard line was included for reference.
41 Chapter 4. Discussion
The discussion section is divided into three main parts: (1) Picoeukaryotic
diversity, (2) spatial and seasonal variations of picoeukaryotes, and (3)
methodological aspects.
4.1 Picoeukaryotic diversity
Picoeukaryotic diversity has been a hot topic of marine research in recent years.
Unfortunately, information is very limited in Pacific coastal waters. Yuan et al.
(2004) conducted a study in the southern South China Sea using un-filtered coastal
water samples collected in the pelagic zone. Worden (2006) initiated the first study
on picoeukaryotic diversity in coastal waters of California. To the best of my
knowledge, no data are available on the diversity of picoeukaryotes in the western
Pacific coastal waters. One of the objectives of this study is to characterize the
diversity of such an ecologically important group of organisms in the poorly studied
Pacific coastal region. Discussion on the diversity issue can be further divided into
two parts: (1) overall diversity, and (2) diversity of individual taxonomic groups.
4.1.1 Overall diversity
In the current study, at least 17 higher-level taxonomic groups of picoeukaryotes
were recognised. This finding is comparable to those observed in other coastal
studies. While 16 higher-level picoeukaryotic groups were recovered at a coastal site of the English Channel (Romari & Vaulot 2004), Massana et al. (2004b) and
Medlin et al. (2006) retrieved 17 and 15 higher-level picoeukaryotic groups from
42 coastal waters at the Mediterranean Sea and the Helgoland coast, respectively.
Major difference between the picoeukaryotic assemblages collected in the current study and those from other coastal studies lies on the relative abundance of dominant groups involved and the presence/absence of some less prominent groups, including
Mesomycetozoa, Perkinsozoa and Apicomplexa, which are not recovered in the current study.
New representatives from five of the eight major marine eukaryotic lineages
(Baldauf 2003) were obtained here. Many of the current sequences are highly similar to those retrieved at diverse sites around the world, supporting the hypothesis concerning the global distribution of protists (Fenchel & Finlay 2004). Many current picoeukaryote OTUs are present as singletons which appeared once (80 OTUs) or doubletons which appeared twice (31 OTUs) (Fig. 3), indicating a large number of rare taxa and thus the need of sequencing even a larger number of clones from libraries.
The non-parametric richness estimator Chaol (Chao 1984) is considered to be most suitable for analyzing microbial clone libraries (Hughes et al. 2001). However, it has only been incorporated in picoeukaryotic / protistan diversity studies recently.
Massana et al. (2004b) computed Chaol for surface water samples of a coastal
Mediterranean site as 77-171 in four independent seasonal clone libraries, based on a mean phylotype cutoff level of 98.7%. Countway et al. (2005) computed Chao 1
(without replacement) for a sampling site off North Carolina coast as 282 (229 -381，
95% CI), including non-protistan clones, using a phylotype cutoff of 95%.
Zuendorf et al. (2006) computed Chaol for a sampling site below the chemocline of the anoxic Mariager Fjord in Denmark as 120 and 187, based on 97% and 98.5%
43 cutoff levels, respectively. Lefevre et al. (2007) computed Chaol for four clone libraries in the oxic zone and oxycline of the freshwater Lake Pavin. The values ranged from 13.5-77.2, with OTUs defined according to distinct RFLP patterns. In this study, OTUs are also grouped according to RFLP patterns, and Chaol is computed as 289 (243-370, 95% CI) for the pooled library (Table 3). The high
Chaol value, together with the inclusion of strictly picoeukaryotes and the possibility of underestimation caused by defining OTU by RFLP, suggest that the western
Pacific coast may be one of the most highly diverse aquatic ecosystems ever encountered. This speculation is further supported by the current high H values computed (Table 3) which are comparable to those calculated for the protistan assemblages of the western North Atlantic (Countway et al. 2007).
4.1.2 Diversity of individual taxonomic groups
Taxonomic groups recovered in the current study could be generally classified into: (1) the most represented lineages, (2) other photosynthetic lineages, (3) other non-photosynthetic lineages, and (4) novel higher-level lineages.
4.1.2.1 Most represented lineages
The most represented lineages recovered in the current study include representatives from alveolates, stramenopiles and Rhizaria.
Alveolates
Alveolates are a group of organisms characterized by the presence of
44 membrane-bound flattened vesicles named alveoli (Cavalier-Smith 1993).
Alveolates are composed of four protistan groups (ciliates, dinoflagellates,
apicomplexans and perkinsids). Two additional novel alveolate groups (NAGI &
NAGII) were discovered recently in an 18S rRNA-based environmental diversity
survey of small eukaryotes (< 5 (xm) in the Antarctic polar front (Lopez-Garci'a et al.
2001). The four major groups of alveolates retrieved in this study include novel alveolates groups I and II，ciliates and dinoflagellates.
Novel alveolates groups I and II
Following the discovery of the two novel alveolate groups in the Antarctic Ocean, sequences closely affiliated with the two novel groups are frequently recovered in environmental diversity surveys conducted in various geographical locations and different ecosystems. Groisillier et al. (2006) performed a comprehensive meta-analysis on the genetic diversity and ecological distributions of the two novel groups of alveolates from 62 environmental clone libraries and identified five clusters of NAGI and 16 clusters of NAGII. While NAGII is considered to be abundant in coastal and oceanic ecosystems, NAGI is usually dominant in permanent anoxic waters and hydrothermal ecosystems (Groisillier et al. 2006).
NAGI-1 is the most diverse and dominant NAGI in the current study (Fig. 8B).
Within this group, MB07.2 is retrieved from six of the eight current clone libraries
(Appendix 2). This together with the fact that it was highly similar to BLOO1221.41 retrieved from a coastal site in Blanes Bay, suggest that the cluster may represent members which are common in coastal waters. Interestingly, we obtained one sequence (TH 10.57) belonging to NAGI-3. Previous representatives for NAGI-3
45 have come exclusively from deep marine waters or hydrothermal vent sediments
(Groisillier et al. 2006). This result suggests that the ecological distribution of the cluster might extend to coastal waters.
Some current sequences could not be clearly assigned to previously defined NAG clusters, raising the possibility of additional rare clusters that would need further analysis as the sequence database of the group expands. Specific examples included a moderately supported (79%) NAGII cluster composed of MB07.29,
TH10.4, AP-picoclone 20 and BBOl-83 within the Amoebophrya clade, and two highly supported (> 99%) NAGII clusters composed of sequences retrieved from less than three libraries (TH07.6 + TH07.32 + RA010613.126 and TH07.2 + TH07.12 +
TH07.26) (Fig. 8C). Some of these sequences were highly similar to sequences retrieved from other environmental studies conducted in the Pacific Ocean (i.e.
UEPACCp3, AP-picoclone20, OLI11012 and UEPACBp4), suggesting that they might represent phylotypes restricted to the Pacific Ocean. Also, 39% of the current NAGII sequences and 25% of the current NAG I sequences were <95% similar to GenBank sequences (Table 4), suggesting that the vast diversity of these two novel groups deserve further examination.
The ubiquitous distribution of the two novel alveolates groups in marine ecosystems suggests that these organisms are fundamental components of marine microbial ecosystems in which parasitism may play a key role (Groisillier et al.
2006).
Ciliates
46 Ciliates, free-living marine predators that can be easily distinguished due to their complex morphology, are proposed to be almost comprehensively discovered, with nearly 4000 species identified (Finlay 1998). However, as in other recent molecular environmental surveys (e.g. Romari & Vaulot 2004，Lovejoy et al. 2006)，this study has recovered ciliate phylotypes which are distantly related to described species but closely resemble environmental sequences. Although some of these phylotypes may correspond to described species whose SSU rDNA sequences are not yet available (Baldauf 2003), the majority of them probably represent undetected ciliate diversity. About one-third (32%) of the current ciliate sequences retrieved are
<95% similar to GenBank sequences (Table 4). These findings are in agreement with Foissner (1999) who stated that the number of free-living ciliate species may exceed by at least one order of magnitude of those already described. Therefore, it is too early to establish limits for protistan diversity even for well-studied taxa such as the ciliates.
47 Table 4. Detailed number of OTU(s) of different taxonomic groups with % similarity values equals to or higher than a certain threshold.
%similarity NAGIINAGI D Ci S Pr Pi Ce H Cr CrN K T F Ch R ChN NC Total 100 3 99.5 3 3 5 6 5 25 99 3 2 5 4 7 3 2 32 98.5 4 2 3 3 4 4 2 25 98 3 1 5 2 13 97.5 2 4 1 11 97 2 8 96.5 3 5 96 2 3 95.5 5 3 11 95 1 2 4 94.5 3 3 6 94 2 4 93.5 2 4 6 93 3 2 2 7 92.5 1 2 92 2 3 91.5 2 2 4 91 2 2 90.5 3 4 90 <90 2 7 Total 46 8 16 34 31 11 4 14 4 2 2 2 2 3 4 186 NAGII = Alveolates group II; NAGI = Alveolates group I; D = Dinophyceae; Ci = Ciliophora; S = Stramenopiles; Pr = Prasinophyceae; Pi = Picobiliphyta; Ce = Cercozoa; H = Haptophyceae; Cr = Cryptophyta; CrN = Cryptophyte nuc1eomorph; K = Katablepharidophyta; T = Telonemia; F = Fungi; Ch = Choanoflagellida; R = Radiozoa; ChN = Chlorarachniophyte nucleomorph; NC = Novel c1ades I + 11 + Ill.
48 Members from three groups of ciHates (Prostomatea, Colpodea and Spirotrichea) are recovered in the current study (Fig. 8A). Within Prostomatea, all recovered sequences are loosely related to Cryptocaryon irritans, a fish parasite with low host specificity (Burgess & Mattews 1995). Within Spirotrichea, in addition to the two
Strombidium lineages, Strom A and Strom B, recently discovered by Lovejoy et al.
(2006), two potential novel Strombidium clades, Strom C and Strom D�supported with high bootstrap values were described here. Within the Strom A + B clade,
MBOl.12 and TH 10.34 are retrieved, respectively, in six and seven of the eight current clone libraries (Appendix 2). These results point to the importance of
Strombidium spp. in the ecosystem.
Dinoflagellates
Dinoflagellates include both autotrophic and heterotrophic organisms and are an important component of marine plankton, frequently causing algal blooms in coastal waters. While most current dinoflagellate sequences are closely related to known species, a highly supported novel clade NDCI is defined (Fig. 8B), suggesting the potential presence of novel pico-dinoflagellates.
Stramenopiles
Stramenopiles represent another pronounced lineage recovered in this study.
Stramenopiles form an extremely diverse phylogenetic group including both phototrophs and heterotrophs. Phototrophic stramenopiles forms a deeply branched monophyletic clade in the current study (Fig. 9). This together with the fact that heterotrophic stramenopiles are paraphyletic and occupied a basal position
49 in the phylogenetic tree, validates previous assumption that stramenopiles are primarily heterotrophic and phototrophic groups were resulted from secondary endosymbiosis (Bhattacharya & Medlin 1995). Within the phototrophic lineages, members from the class Bolidophyceae are typically picoplanktonic. However, the presence of Bacillariophyceae sequences highly similar to those of large species such as Pseudo-nitzschia multiseries and Leptocylindrus danicus suggests either cell breakage during filtration or collection of cells via their smallest dimensions.
Nonetheless, they were never dominant members in our study. Chrysophyceae is well recognized as being an important component of freshwater phytoplankton
(Nicholls KH 1995). The retrieval of two current Chrysophyceae sequences highly similar to two other marine environmental sequences suggests that the class may also be prevalent in marine ecosystems.
Novel marine stramenopiles (MAST) constitute the major diversity of stramenopiles in this study (Fig. 9). Massana et al. (2004b) performed a comparative analysis on the genetic diversity and ecological distributions of MAST from 37 marine environmental clone libraries and defined 12 MAST clusters.
Within the seven MAST clusters recovered here, MAST-1, -3, -4 and -7 are large clusters frequently found in coastal systems and open seas, MAST-2 appears to be rare but widespread, and MAST-6 and -11 are minor components of marine picoeukaryotic assemblages (Massana et ai. 2004b). MAST-1 has been found to be more pronounced in open oceans than in coastal seas (Massana et al. 2004b), but
THOl.31 and TH 10.43 retrieved from the current study, together with CD8.09 retrieved from Norwegian Sea coast, indicate the potential importance of the cluster in coastal ecosystems. While MAST-1, -2, -3 and -4 were considered to be heterotrophic flagellates from enrichment cultures (Massana et al. 2002; 2004b;
50 2006)，other MAST clusters were also considered to be heterotrophic flagellates according to their phylogenetic positions among other known heterotrophic groups, such as labyrinthulids and oomycetes (Massana et al. 2002). Heterotrophic flagellates are the primary grazers of prokaryotes and play an important role in nutrient remineralization (Fenchel 1986). In addition to the vast diversity of
MAST groups, a few current sequences affiliate with known heterotrophic stramenopile groups (eg. labyrinthulids, oomycetes and bicosoecids) (Fig, 9) which are more abundant in coastal waters than in open oceans (Massana et al. 2004b).
The vast diversity of heterotrophic stramenopiles recovered here supports the idea that heterotrophic eukaryotes are probably more diverse than their photosynthetic counterparts (Vaulot et al. 2002) and suggests their importance in eutrophic coastal ecosystems, particularly as microbe grazers.
Rhizaria
Rhizaria is the third most prominent lineage recovered in the current study. It is a supergroup comprising the phyla Cercozoa, Radiozoa and Foraminifera
(Nikolaev et al. 2004). Cercozoa is one of the most diverse and species-rich groups of flagellated protists (Bass & Cavalier-Smith 2004). After the erection of the phylum Cercozoa based on molecular data alone (Cavalier-Smith 1998), the composition of Cercozoa has been continuously expanded, and its phylogeny and classification have been periodically updated (Bass & Cavalier-Smith 2004). A recent example is the placement of ebriids within the phylum Cercozoa (Hoppenrath
& Leander 2006). Bass & Cavalier-Smith (2004) conducted a comprehensive phylum-specific environmental 18S rRNA survey of Cercozoa from 41 libraries and revealed at least nine novel cercozoan clades, several possibly at the level of order
51 or above, suggesting that the diversity of this important group of protists could be
much higher than anticipated previously. In our study, additional environmental
representatives from the previously defined NC2 clade were retrieved (Fig. 10). A
totally novel cluster NCC1 was also recovered. These together with the fact that
50% of the current cercozoan sequences are <95% similar to GenBank sequences
(Table 4) suggest that diversity of Cercozoa could be further expanded. Phylotypes
closely related to Cryothecornonas aestivalis, a free-living heterotrophic
nanoflagellate that feeds on small-sized cells, such as diatoms (Drebes et al. 1996)，
are recovered in five of the eight current clone libraries (Appendix 2). Though in
low abundance, this observation suggests that cercozoans may play a role in
controlling the growth of diatoms and thus the occurrence of algal blooms.
Acantharea and Polycystinea are often grouped together (Lopez-Garcia et al. 2002)
to form the phylum Radiozoa (Cavalier-Smith 1993).
4.1.2.2 Other photosynthetic lineages
In addition to the three major eukaryotic lineages, alveolates, stramenopiles and rhizarians discussed above, representatives from other known photosynthetic and non-photosynthetic lineages are also retrieved in the current clone libraries. The following discussion focuses on the photosynthetic lineages which included prasinophyceae, haptophyceae, cryptophytes and picobiliphytes.
Prasinophyceae
Prasinophyceae is the most primitive class of green algae. It includes many picoeukaryotic members (Guillou et al. 2004). Micromonas, Ostreococcus and
52 Bathycoccus of the order Mamiellales are the most commonly recovered genera in genetic libraries (Guillou et al. 2004). Micromonas is the most diverse prasinophyte group in this study (Fig. 7). Indeed, this genus is regarded as a complex group including three independent lineages (Guillou et al. 2004). Unlike the ciliates, most of the current prasinophyte sequences are closely related to cultured species, suggesting that prasinophytes may be the picoeukaryotic group most readily studied by culturing approaches.
Haptophyceae
Haptophyceae is the most important picoeukaryotic group in the open oceans according to pigment analysis (Moon-van der Staay et al. 2000). All haptophyceae sequences retrieved here belong to a single genus Chrysochromulina (Fig. 7)，a diverse group of phagotrophic phytoflagellate (Laybourn-Parry 1992) in which picoplanktonic members are known to exist (e.g. Chfysochromulina leadbeateri)
(Eikrem & Throndsen 1998). The recovery of haptophyceae clones here agrees with previous pigment data (Wong & Wong 2004) and confirms the presence of haptophytes in Hong Kong waters.
Cryptophytes
Cryptophytes are a group of algae that have acquired their chloroplasts by a secondary endosymbiosis between a protozoan and a red alga (Douglas et al. 1991).
To date, only a single Cryptophyte species {Hillea marina) of picoplanktonic size has been described (Butcher 1952), but no sequence for it is available. The two cryptophyte sequences obtained in this study are closely related to Plagioselmis
53 prolonga and Teleaulax acuta (Fig. 7) which belong to genera that have been retrieved in other picoeukaryotic diversity studies (Massana et al. 2004a; 2006;
Romari & Vaulot 2004; Medlin et al. 2006). Again, the recovery of cryptophyte clones here agrees with previous pigment data (Wong & Wong 2004) and confirms the presence of cryptophytes in Hong Kong waters.
Picobiliphytes
Picobiliphytes are a novel picoplanktonic algal group with a phycobiliprotein-containing plastid and possibly represent a secondary endosymbiotic algal group (Not et al. 2007b). Picobiliphyte sequences have been retrieved from a variety of marine systems, including the European coast, the North
Atlantic and the Arctic Ocean (Not et al. 2007b). To the best of my knowledge, this is the first time to recover picobiliphyte sequences in the Pacific coast. All current picobiliphyte sequences fall into one of the three potential clades proposed recently (Not et al. 2007b) and are closely related to environmental sequences collected from a coastal site in the English Channel in France. These results suggest that these phylotypes may be more pronounced in coastal waters (Fig. 7).
This is in particular for clone MB04.45, which is retrieved from seven of the eight current clone libraries (Appendix 2). Clone library and FISH data indicate that picobiliphytes are well represented in polar and cold temperate coastal marine ecosystems (Not et al. 2007b). However, two-third of the picobiliphyte clones were recovered in libraries from July and October when water temperature was around 27°C (Table 1 ； Appendix 2). This suggests the cosmopolitan distribution of picobiliphytes in both warm and cold marine waters.
54 4.1.2.3 Other non-photosynthetic lineages
In addition to the three major eukaryotic lineages (alveolates, stramenopiles and rhizarians) discussed in section 4.1.2.1, representatives from other known non-photosynthetic lineages, including katablepharids, Telonemia, Fungi and nucleomorphs are also retrieved in this study. Detailed discussion on each of these lineages is provided below.
Katablepharids
Katablepharids are predatory heterotrophic flagellates closely related to cryptophytes (Okamato & Inouye 2005). Although only partial sequences are employed in the current analysis, katablepharids are found to cluster with cryptophytes with moderate bootstrap support (Fig. 7).
Telonemia
Telonemia is a new protist phylum with affinity to chromist lineages
(Shalchian-Takrizi et al. 2006). Although early microscopic studies have suggested a worldwide distribution of Telonemia-like organisms (Lee & Patterson 1998), this study provides the first record of Telonemia sequences in the Pacific coast.
Fungi
The presence of fungi is considered to be a specific feature of lacustrine ecosystems (Lepere et al. 2006) as these sequences are either absent or rare in
55 marine environments. Two fungal phylotypes retrieved in the current study are
found to be closely related to environmental sequences NAMAKO-37 and
TAGIRI-23 from two anoxic sites in Japan (Fig. 7). They appear to belong to the
lower fungi Chytridiomycota whose phylogenetic positions of taxa are still unstable.
Takishita et al. (2007) hypothesized that these fungal phylotypes may be anaerobic
in nature. However, the retrieval of two closely related fungal phylotypes in the
well-oxygenated waters of Mirs Bay in the current study suggests that these
phylotypes may not be strictly anaerobic. Nevertheless, they may represent
deep-branching novel fungal lineages never detected before using traditional ‘
methods.
»
Nucleomorphs
Two groups of nucleomorph, the cryptophyte nucleomorphs and the
chlorarachniophyte nucleomorphs, were detected (Fig. 7). Nucleomorphs are relict
nuclei and contain a remnant genome produced by the endosymbiosis of an alga and
another eukaryote (Douglas et al. 2001). The nucleomorph endosymbiont has been
subject to unique evolutionary pressures leading to the reduction of their genomes
and the evolution of highly divergent SSU rRNA gene sequences (Douglas et al.
2001). It has been shown that nucleomorph SSU rRNA genes have a higher rate of
nucleotide substitution than their nuclear counterparts, explaining why the
nucleomorph genes usually show long branches in phylogenetic trees (Ishida et al.
1999).
4.1.2.4 Novel higher-level lineages
56 The above three sections (4.1.2.1 -4.1.2.3) have focused on clone representatives from known and previously defined eukaryotic lineages. Three potentially novel higher-level lineages are recovered in this study (Fig. 7). These are discussed in the following paragraphs.
TH10.32 and MB04.9 clustered with RA010613.131 and RA010613.138 from a coastal site of the English Channel in France to form the novel clade I. This group of organisms may be unique in coastal waters as all the four sequences involved were retrieved from coastal libraries. The two English Channel sequences were regarded as "uncultured cryptophytes" in the GenBank entry. However, from the current ML tree (Fig. 7), this group seems to be divergent enough to form an independent higher-level novel clade. Since only partial sequences are available for this group of organisms, its actual phylogenetic position has to be verified in future studies employing full length sequences.
Novel clade II’ composed of three highly similar sequences collected in the current study, occupies a basal position in the eukaryotic tree (Fig. 7). It has been suggested that the number of novel higher-level lineages had been overestimated due to undetected chimeric sequences formed during PGR amplification, long-branch attraction artifacts and incomplete molecular sampling of described eukaryotes
(Berney et al. 2004). However, sequences composing novel clade II are probably not chimeras as they have successfully passed the CHECK—CHIMERA process. In addition, they are highly similar and were retrieved in three independent clone libraries. The possibility of long-branch attraction can also be discarded as the phylogenetic tree was not rooted using prokaryotic sequences which are considered to be problematic in phylogenetic studies of eukaiyotes (Berney et al. 2004).
57 MB07.52 and TH07.19 retrieved here clustered with PD6.12 from the GenBank and formed another highly divergent novel clade III (Fig. 7). PD6.12 is from a polar site in Norwegian Sea. Two additional environmental sequences affiliated closely with this group are RA000609.30 from a coastal site of the English Channel
in France and the Antarctic sequence ANT12.13. These findings suggest that the group is robust and widely distributed.
Baldauf (2003) suggested that most of the eukaryotes can be distributed into eight 'supergroups' and the number of possibly independent, smaller, higher-level lineages is limited. Examples include apusozoans or centroheliozoans
(Cavalier-Smith & Chao 2003). Also, most of the taxa that were traditionally considered early diverging branches of the eukaryotic tree are now regarded as highly derived members of groups belonging to the crown of eukaryotes.
Therefore, the presence of any potentially novel higher-level lineages should be treated carefully and verified by further works.
4.2 Spatial and seasonal variations of picoeukaryotes
Two additional objectives of the current study are: (1) to study the spatial variations of marine coastal picoeukaryotes along a gradient of eutrophication never described before by molecular techniques, and (2) to investigate the seasonal variations of picoeukaryote communities in the western Pacific coast for the first time. Spatial and seasonal changes in both the composition and diversity of picoeukaryotes are discussed below.
58 4.2.1 Spatial variations
Spatial variation refers to changes with respect to space. Changes in species diversity along a eutrophication gradient have been intensively studied. For instance, decreases in marine macroalgal (Worm et al. 1999) and microalgal
(Hillebrand & Sommer 2000) diversity due to eutrophication have been reported.
Nutrient enrichment results in lower freshwater microalgal diversity (Miller et al.
1992), but there are cases of increase in freshwater microalgal diversity following nutrient enrichment, especially in nutrient-poor environments (Pringle 1990).
Indeed, a unimodal relationship between primary productivity and diversity has been proposed as a universal pattern in ecology (Rosenzweig 1992). Recently, Lefranc et al. (2005) performed a study on the relationship between genetic diversity of small eukaryotes in lakes with different trophic status and found that the diversity of small eukaryotes seemed to follow a hump-shaped progression along an eutrophication gradient, with eutrophic lakes having the least diversity and oligomesotrophic lake the most diversity. However, no comparable study has been performed on marine picoeukaryotes.
In the current study, picoeukaryotic clone libraries are constructed for two coastal sites in Hong Kong. Inner Tolo Harbour is eutrohpic, while Mirs Bay is oligomesotrophic (HKEPD 2003). Samples collected at the two sampling sites in different seasons generated eight clone libraries representing waters of widely different nutrient status. This provided the basis for a comprehensive investigation of the relationships between marine picoeukaryotes and trophic status for the first time.
59 4.2.1.1 Compositional variations
The phylogenetic position of a clone implies whether an organism is pigmented or not. Regarding the composition of picoeukaryotes, all current clone libraries are dominated by non-photosynthetic groups (Fig. 6). Similar findings in other coastal areas (Massana et al. 2004b, Romari & Vaulot 2004, Medlin et al. 2006) suggest that a higher proportion of non-photosynthetic picoeukaryotes may be a general characteristic of coastal waters. Also, the relative contribution by photosynthetic groups was generally higher in the Mirs Bay libraries than the Tolo Harbour libraries
(Fig. 6). Reduced importance of photosynthetic groups in libraries for eutrophic waters has also been observed for freshwater small eukaryotes (Lefranc et al. 2005).
These, together with the fact that larger diatoms are more dominant in the eutrophic libraries (Table 1), suggest a division of labour in photosynthesis by large and pico-sized eukaryotes in coastal waters of different trophic status.
The Tolo Harbour and Mirs Bay libraries contained similar total number of contributed clones for the most represented alveolates group II and stramenopiles
(Table 2). This, together with the parasitic nature of alveolates group II and the prevalence of microbe-grazing in stramenopiles, suggest that parasitism and microbe-grazing are important ecological functions in coastal waters irrespective of the nutrient levels. Ciliates also showed a similar contribution at both sites (Table
2) and the trend was consistent throughout the year (Fig. 5). This contradicts with previous observations that ciliates are usually more abundant in eutrophic waters
(Dolan et al. 1999). One possible explanation for this is that the response of pico-sized ciliates to eutrophication is still unknown and may be quite different from that of large ciliates.
60 Alveolates group I, picobiliphytes and cryptophytes were more abundant in the eutrophic Tolo Harbour libraries than in the oligomesotrophic Mirs Bay libraries
(Table 2). The higher abundance of potentially parasitic alveolates group I in the eutrophic libraries suggests that parasitism is important in eutrophic waters. Since the discovery of picobiliphytes, no information is yet available on their distribution in relation to trophic status. The data here provide the first evidence to suggest the dominance of this group in eutrophic waters. Contribution of dinoflagellate clones 4 was higher in the oligomesotrophic Mirs Bay libraries (Table 2). In contrast, larger i dinoflagellates are generally more dominant in eutrophic libraries (Table 1). This provides evidence on the possible presence of pico-sized members in the group that may be specifically adapted to an environment of reduced nutrients by reducing their body size. The higher percentage of prasinophyceae clones in the current oligomesotrophic libraries is consistent with report of higher contribution by this group in oligotrophic open sea libraries (Diez et al. 2001).
The oligomesotrophic Mirs Bay and eutrophic Tolo Harbour libraries harboured, respectively, 64 and 79 unique OTUs whereas only 43 OTUs occurred in both stations (Fig. 3). The low number of shared OTUs and the high number of unique
OTUs suggest that the composition of picoeukaryote communities is influenced by trophic status. Detailed information on the distribution of specific OTUs collected in the current study is provided in the phylogenetic trees (Figs. 7 to 11) and
Appendix 2.
4.2.1.2 Variations in diversity
61 Statistical tools were used to study the relationship between marine picoeukaryotic diversity and trophic status. Briefly, the Shannon diversity indices
(H), the Simpson's diversity indices (D"') and non-parametric species richness estimators Chaol and ACE were computed and compared among clone libraries.
H and D'' are diversity indices commonly used in ecological studies (Morris et al.
2002). Values of both indices range from zero to, theoretically, infinity, and a higher value indicates a higher diversity estimated. While Chaol is generally accepted as an accurate lower bound of species richness (Bohannan & Hughes 2003),
ACE is usually regarded as a point estimate (Chao & Shen 2003-2006). Values computed for these estimators represent numbers of species estimated.
In the study of Lefranc et al. (2005)，only three freshwater clone libraries were constructed and only 39 to 47 clones were analyzed per library. In addition, no statistical test was implemented for hypothesis testing. In contrast, the current study involves a larger number of more comprehensively analyzed clone libraries
(eight clone libraries; 71 to 100 clones analyzed per library), creating a better basis for studying the relationships between marine picoeukaryotic diversity and water trophy.
The eight libraries from the two sampling sites vary in trophic status and provide a series of libraries spanning a wide spectrum of chlorophyll a concentrations.
While low H values (3.2 - 3.5) were recorded in clone libraries of both low (0.79 |ig r') and high (5.65 and 16.21 昭 1"') chlorophyll a concentrations, higher H values
(3.6 - 4.0) were found in clone libraries of intermediate chlorophyll a concentrations
(1.52 to 4.79 |ig 1-1) (Table 3). Similarly, low D ' values (10 - 12.1) were recorded in clone libraries of both low and high chlorophyll a concentrations, and higher D"'
62 values (16.4 - 25.3) were found in clone libraries of intermediate chlorophyll a concentrations (Table 3). These trends (Fig. 13) are similar to the general unimodal pattern proposed by Rosenzweig (1992) and reported for freshwater small eukaryotes (Lefranc et al. 2005). The pattern is believed to be resulted from a compromise between competition and predation at eutrophic environments, and the accessibility of nutrient resources at oligotrophic environments (Lefranc et al. 2005).
Our finding suggests that that this pattern may also be applied to marine picoeukaryotes.
f.
Regarding variations of species richness, the values of Chaol computed are comparable between samples collected in the same month and also between the pooled MB and TH datasets (Table 3). However, the wide range of the confidence intervals, made the detection of potential differences among our clone libraries difficult (Table 3).
While higher numbers of contributing OTUs are observed for alveolates group II ！ and stramenopiles in the eutrophic Tolo Harbour libraries, the numbers of OTUs for ciliates are similar for both sites (Table 2). The result for alveolates group II and stramenopiles implies that the more complex communities in eutrophic waters may require a more diverse picoeukaryotic community to perform the vast ecological functions. For example, an elevated diversity of species- or genus-specific microbe-grazing stramenopiles may be needed to cope with the high diversity of microbes in eutrophic waters. On the other hand, the rather consistent but relatively high OTU contribution of ciliates in both coastal areas suggests the importance of pico-sized ciliates in coastal waters irrespective of the water trophy.
63 4.2 �
4.0 - 广
3.8 - / n: 3.6 - r^
3 Q I L 1 1 I I I 1 1 1 1 1 1 1 1 1 I 1 I
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Chi. a conc. (jxg/1) 30 「 』:M -J 5 -
0 I L- 1 1 I I I 1 1 1 1 1 1 1 1 I I • I 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Chi. a conc. (jig/l) Figure 13. Graphs showing the relationship between diversity indices and chlorophyll a concentration. The upper panel is the results from the Shannon diversity index (H) and the lower panel from the Simpson's diveristy index (1/D).
64 4.2.2 Seasonal variations
Seasonal clone libraries are valuable for formulating and testing hypotheses
(Worden 2006). This is particularly applicable to coastal ecosystems which are
prone to large temporal fluctuations induced by episodic events such as tidal
currents, introduction of freshwater and algal blooms. Indeed, seasonal clone
libraries of picoeukaryotes have been constructed in four studies performed at the
Atlantic coast (Massana et al. 2004a; Romari & Vaulot 2004; Medlin et al. 2006)
and the eastern Pacific coast (Worden 2006). Single Strand Conformational
Polymorphism (SSCP) results showed that picoeukaryote community structure can
change monthly or even within days (Medlin et al. 2006), supporting previous
speculation that plankton are distributed heterogeneously over short time scales
(Scheffer et al. 2003). Also, reappearance of similar SSCP bands on a yearly basis
suggests that there may be certain degree of seasonality in picoeukaryote
communities (Medlin et al. 2006). However, no data is available on seasonal
variations of picoeukaryotes in the western Pacific coast. In this study, four
seasonal picoeukaryotic clone libraries are constructed for each of the two coastal
sampling sites at the subtropical western Pacific for the first time.
4.2.2.1 Compositional variations
While alveolates group II contributes significantly to the picoeukaryote community throughout the whole year, ciliates occur in all clone libraries but were more pronounced in April and October (Fig. 5). The large and substantial contribution by alveolates group II observed here is comparable to those reported in
Roscoff�Helgolan and d California coasts (Romari & Vaulot 2004; Medlin et al.
65 2006; Worden 2006). All these reports suggest that parasitism is of potential
importance in coastal ecosystems. Year-round occurrence of ciliates in the
pico-sized fraction has also been observed in Roscoff and Helgoland coastal stations
(Romari & Vaulot 2004; Medlin et al. 2006)，suggesting that pico-sized ciliates are
particularly dominant in coastal ecosystems. These findings are in contrast to those
observed at Blanes coastal stations where alveolates group I and dinoflagellates
dominated the clone libraries (Massana et al. 2004a). Nonetheless, prasinophytes
(especially the order Mamelliales) figure prominently in picoeukaryotic clone
libraries in this study (Fig. 5) as well as in all other coastal studies (Massana et al.
2004a; Romari & Vaulot 2004; Medlin et al. 2006; Worden 2006).
Regarding the occurrence of the six most represented picoeukaryotic groups
recovered in the current study, it was found that all six groups appear in all eutrophic
Tolo Harbour libraries throughout the year, varying only in relative abundance. In
contrast, occurrence in oligomesotrophic Mirs Bay libraries tended to be more
sporadic, with all six groups present in April, five in January and July, and only four
in the October (Fig. 5). These observations suggest that seasonal variations in
picoeukaryotic composition may be more pronounced in oligomesotrophic coastal waters than in eutrophic waters. However, it must be noted that the semi-enclosed nature of Tolo Harbour might also reduce seasonal variations of picoeukaryotes due to limited water circulation.
4.2.2.2 Variations in diversity
Statistical methods used to investigate spatial variations were also used to investigate seasonal variations in marine picoeukaryotic diversity. The H and D"'
66 values of the January sample were lower than that of the October sample, implying a
less diverse community in January (Table 3). Water temperature increased from
January to October (Table 1)’ suggesting that higher water temperatures may support
more diverse picoeukaryotic communities. Indeed, a notable positive correlation
between zooplankton species richness and water temperature has been reported in
the Nanji Islands National Nature Reserve in China (Ji et al. 2006). Also, as one of
the numerous examples of the latitudinal diversity gradient phenomenon, terrestrial
algae have been shown to be more diverse in warm tropical regions than in cold
polar regions (Broady 1996). However, it must be noted that while water
temperatures in July were similar to those in October, July had low H and D"' values
compared to October (Table 1 and Table 3). Also, Brown & Wolfe (2006) reported
a general decline in protistan diversity with increasing temperature. These suggest
that additional biotic and/or abiotic factors may influence picoeukaryote
communities, making prediction of community diversity more difficult.
4.3 Methodological aspects
Although the cloning and sequencing approach employed in this study has been widely adopted, some inherent biases and potential problems must be addressed.
This section will discuss problems related to: (1) sample collection, (2) PGR amplification, (3) cloning and RFLP screening, and (4) statistical estimates. Finally, some suggestions for future directions are provided.
4.3.1 Sample collection
Despite using 3 jim pre-filtration, 18S rRNA gene sequences from larger
67 organisms, notably dinoflagellates, ciliates and metazoans are recovered in the
current study. This phenomenon has been reported elsewhere, particularly in
coastal studies (e.g. Romari & Vaulot 2004; Medlin et al. 2006; Worden 2006), and
is believed to be the result of flexible cells that can be forced through the 3 (xm filter
pores, cell breakage during sample collection, sloppy feeding by zooplankton and,
life history stages of small sizes or undescribed picoplanktonic species. The
diversity of metazoan sequences obtained suggests retention of free DNA adhering
to small particles, rather than contamination by one intact animal. In any case,
metazoan sequences are neither common (ca. 7.5% of total OTUs) nor the target of
the current study and therefore have been excluded from further analyses. Future
researches comparing the diversity of the large size fraction may help to resolve the
origin of seemingly large-sized organisms in the pico-size fractions.
4.3.2 PGR amplification
A number of biases and/or problems are associated with gene amplification,
including primer specificity, variable gene copy number and the formation of chimeric sequences.
Regarding primer specificity, although the universal primers used in the current study could recognize most protists, some of them might not be successfully amplified due to one or more mismatches of their primer-binding locations. These included certain members belonging to Chrysophyceae, Chlorarachniophyceae,
Apicomplexa, Ciliophora and primitive eukaryotes such as Metamonada or
Euglenazoa (Moon-van der Staay et al. 2001).
68 Variable gene copy number brings along another bias. Indeed, the copy number
of 18S rRNA gene varies significantly in microalgae. For instance, while
Nannochloropsis has only 1 copy of the gene, some nanoplanktonic dinoflagellates
can have over 1000 copies (Zhu et al. 2005).
Formation of chimeric sequences during PCR amplification may cause problems
in data interpretation. Factors implicated in chimera formation include short
elongation time, long annealing time, and high number of PCR cycles (Qiu et al.
2001). Our amplification protocol has a long elongation time and a short annealing step but includes 32 cycles, which is within the range where increased likelihood of chimera formation has been recorded. Therefore, the identification of several potentially chimeric clones was not unexpected. However, they were not dominant and can be easily identified using the CHECK—CHIMERA program and then discarded from further analyses.
4.3.3 Cloning and RFLP screening
One of the major concerns about the cloning approach is that PCR-based clone libraries rarely become saturated. This was observed even in the analysis of 500 clones from a seawater sample collected in the western North Atlantic (Countway et al. 2005). Therefore, it has been suggested that clone library studies may only reflect the diversity of the major groups involved (Pedros Alio 2006). For our clone libraries, although rarefaction curves show little sign of saturation (Fig. 12), the coverage values calculated are quite high (67-84% for the 71-100 clones analyzed) (Table 3), and the results thus provide a basis for comparison among clone libraries. Indeed, our coverage values are much higher than those obtained from a
69 coastal site of the English Channel (32-59% for 100 clones analyzed per library;
Romari & Vaulot 2004), fall within the upper ranges reported from the oligotrophic
Blanes Bay in the NW Mediterranean (54-76% for 78-111 clones analyzed; Massana et al. 2004) and from contrasting oceanic regions (47-82% for 17-67 clones analyzed;
Diez et al. 2001), and are only slightly lower than those reported in the meromictic
Lake Pavin in France (76-92% for 38-75 clones analyzed; Lefevre et al. 2007).
Therefore, we consider the majority of the picoeukaryotic diversity is correctly sampled in the current study. Theoretically, more OTUs would be retrieved if we had analyzed more clones. However a more exhaustive analysis of the libraries probably would not change the overall picture significantly (Massana et al. 2004a).
The method of screening clones and defining OTU may also be controversial.
While Stoeck et al. (2003) found no to low sequence divergence within a single restriction fragment pattern using a single restriction enzyme {Hae\\\\ Romari &
Vaulot (2004) showed that identical RFLP patterns generated using the same restriction enzyme could correspond to different sequences, resulting in an underestimation of diversity based on RFLP pattern. Using multiple restriction enzymes in the RFLP analysis could improve the resolution and give more accurate estimation of the species diversity (Baldwin et al. 2005). However, the more complex banding pattern and the increased number of small fragments would make the interpretation of results more difficult. Sequence data provide a sharper image of the actual diversity of the community than RFLP patterns. However, in such an approach, it is necessary to group sequences according to their sequence similarity.
Although 98% is a widely adopted microbial standard for genus-level diversity, choosing adequate thresholds is far from trivial because sequence identities vary widely among taxa. For instance, the inter-generic similarities of Pelagophyceae
70 could be higher than the intra-specific similarities of Micromonaspusilla (Romari &
Vaulot 2004). Therefore, in the current study, OTUs are conservatively defined according to RFLP patterns.
4.3.4 Statistical estimates
Many previous 18S rRNA diversity studies have only intensively focused on
identifying phylotypes via the construction of phylogenetic trees, neglecting
quantitative information on protistan diversity (Countway et al. 2007). In the
current study, non-parametric species richness estimators (Chaol and ACE) and
univariate diversity indices (H and D"') are used to provide quantitative statistical
estimates of the diversity of picoeukaryotes under investigation. These estimators
have been applied previously to molecular diversity studies of bacteria (Dunbar et al.
1999; Hughes et al. 2001; Hill et al. 2003). Although these estimators do not
permit comparisons of the specific types of OTUs in different samples, they are
useful for summarizing the overall diversity in an ecosystem resulting from changes
in environmental conditions (Countway et al. 2007). However, it must be noted
that some of the non-parametric species richness estimators tend to underestimate
richness in highly diverse communities (Wang & Lindsay 2005) such as those
studied here.
4.3.5 Future directions
Regarding the inherent biases and limitations of the cloning and sequencing
approach in diversity studies, new methods with fewer biases and higher throughput
could be employed in future works. These may include multiple primer approach
71 (Stoeck et al. 2006) and high-throughput sequencing of short sequence tags (Sogin et al. 2006).
The multiple primer approach (Stoeck et al. 2006) involves application of multiple PGR primer sets during gene amplification. Using three primer sets, the approach tripled the number of protistan species detected in a study performed in the
Cariaco Basin (Stoeck et al. 2006). However, even with this approach, a substantial part of protistan diversity was still shown to be missing.
Sogin et al. (2006) employed a massively parallel tag sequencing strategy to study the diversity of deep-sea bacteria and found an extremely diverse bacterial community. The method involves amplification of a hypervariable gene region with adaptor-modified primers and subsequent high-throughput pyrosequencing steps, recovering thousands of sequences reads. A major concern about this method is the short (ca. 100 bp) nature of the sequence reads generated. However, the limitation is improved in the second generation Genome Sequencer FLX System, which allows generation of sequence reads averaged 200 to 300 bp long. This approach is believed to be applicable for eukaryotes (Sogin et al. 2006).
72 Chapter 5. General conclusion
Picoeukaryotic diversity in the western Pacific coast was explored for the first time. High diversity was recorded with novel alveolates group II, ciliates and stramenopiles being dominant. Three potentially novel higher-level lineages were also recognized. In addition, novel clades were reported for dinoflagellates, ciliates and cercozoans. Geographical ranges of some recently discovered picoeukaryotic lineages were also extended.
Spatial variations in composition and diversity of picoeukaryotes were recognized. Non-photosynthetic members were common in both Tolo Harbour and
Mirs Bay, but a decrease in the proportion of photosynthetic members was generally observed in the eutrophic Tolo Harbour libraries. A hump-shaped pattern between primary productivity and diversity was suggested for marine picoeukaryotes for the first time.
Seasonal variations in picoeukaryote composition were more pronounced in the oligomesotrophic Mirs Bay than the eutrophic Tolo Harbour. High year-round contribution by alveolates group II and ciliates points, respectively, to the importance of parasitism and pico-sized ciliates in coastal ecosystems. Diversity of picoeukaryotes seemed to be affected by water temperature, but other biotic and/or abiotic factors may also pay a role.
73 Appendix 1. Equations for the statistics used in the present study.
1. Coverage value:
C=l-(/i/«)
2. Chaol:
— iD + ZiC/； -1)/2， if/,=0
3. ACE:
《，=D -4- Drare f\ ^ 一 ^abun ^ X J：^ / rare C C rare rme
4. Shannon diversity index (H):
i — f (k(l - /�/…/r Olog[允(1 - /i / n) /»] 一 1 -[1 一叩-/i"o/"r
5. Simpson's diversity index (non-reciprocal form; D):
it=i 打
where
D = number of distinct species discovered in the sample; fj= number of species that are represented j times in the sample, y = 0, 1，…，n\
Dabun = number of distinct species for the "abundant" group;
^ratv number of distinct species for the "rare" group; 74 Cran.' = estimated sample coverage for the "rare" group; yrarv： = estimated coefficient of variation; n = sample size; k = cut-off value which separates species into "abundant" and "rare" groups
75 Appendix 2. List of all the OTUs recovered in the current study. For each OTU, its GenBank closest relative with accession number, the sequence percent similarity, BLAST score, Query/Subject ratio and frequency from each clone library were provided.
oru. oru~y Taxon Library Clone Clo.ut relative CenBank Accenion" BUSTKore Query/Subject MBOl MB04 MB07 MBlO IHDl mD4 mD7 " rimilarit;y noo AlveoWe~pn MBOl 3 U11CUltured marine ~veolate Group II clone DH147-EKD16 AF29OO71 949 1,210 751ml 25 1 7 11 bno.coph13'a sp . .AF239260 97.6 1,279 730n48 1 6 16 U11CI1ltured rnari%1e picopLmlcton AP-picoclone15 DQ386751 94.8 1,232 754m5 5 2 4 15 19 U11CUltured ~wo~te clone RAOI0613 .44 AY295708 96.1 861 522/543 1 42 bno.coph1]la sp. ex Scrippsi.lla sp. AF472555 919 1,096 733n98 2 1 52 U11CUltured marine eWc:aryote clone UEPACAFp5 AY 129057 96.6 1,319 770n97 8 3 THOl 1 Uncultured marine eWc:aryote clone UEPACCp3 AY 129036 989 1,317 745n53 4 4 1 8 8 U11CUltured marine eWc:aryote clone UEP ACBp4 AY 129045 98.8 1,330 738n47 3 35 U11CUltured eukaryote clone ENI40076.Q0788 AY937888 94.1 678 430/457 2 2 TI bno.boph1]la sp . ex q,mnodinium imtriaJum AF472554 942 1,003 629/668 16 42 U11CUltured marine picopiankton clone HeOOO803 31 AJ965140 963 660 3881403 1 1 MB04 24 Uncultured marine eWc:uvote clone ENVPI0203.00015 00917938 99.4 965 528/531 2 25 lImo~bof'ma Stl . ex Scril'l'si,lla Stl . AF472555 97 .8 1199 680/695 3 TH04 14 bno~bol'hnta Stl . ex Xarlodinium micrum AF472553 95 1194 727n65 5 18 U11CUltured marine eWc:arvote clone NOR26.l4r 00119913 99 .7 1.280 697/699 2 26 U11CUltured marine eWc:arvote clone NOR50.19 DOl19916 90.5 839 589/651 2 MB07 9 U11CUltured marine eWc:aryote clone UEPAC41 tl3 AY129042 99 .6 1400 765n68 1 17 4 21 15 U11CUltured eukarvote clone SCMTIC36 AY664989 932 1031 662nll 1 21 Eukarvote clone OLI11009 AJ402348 982 1323 765n79 4 29 U11CUltured marine picotlLmlcton AP-tlicoclone20 00386756 98.8 1345 747n56 2 42 U11CUltured marine eWc:arvote clone UEP ACEtl3 AY 129038 989 1434 796/805 3 1 TH07 2 U11CUltured marine eukaryote clone NW41431 00119928 92.6 1051 689n44 2 5 U11CUltured ~veo~te clone RAOl061320 -00186535 933 1079 691n41 5 10 6 U11CUltured eukarvote clone SCMTIC21 AY664990 93.8 1101 695n41 2 9 U11CUltured marine eukaryote clone ENVP36162.00243 00918787 949 459 2791294 3 12 U11CUltured eukaryote clone SCM37C21 AY664990 90 .8 987 689n59 5 5 18 U11CUltured marine tlicotlLmlcton clone HeOO0327 39 AJ965089 99 .4 647 355/357 1 26 U11CUlturedmarine eukuvote clone BLoo1221.40 AY426886 90 .6 983 681n52 2 3 27 U11CUlturedmarine eWc:aryote clone UEPACAPtl3 AY129041 99.6 1.301 710m3 3 32 U11CUltured ~veo~te clone RAOl0613.126 DO 186534 95.7 1182 709n41 1 42 bnoIJbol'ma Stl . ex .Karlodinium micrum AF472553 97.4 1.227 702n21 1 1 1 43 Amo/Jb;;;'JU:"a Stl . ex .Karlodinium micrum AF472553 96.5 1123 656/680 1 MBI0 27 U11CUltured~veo~te clone RA010412.70 D0186533 98.2 1,127 646/658 1 30 AmoIJbol'h17la Stl. ex C.ralium m'oos AY208892 99.3 1293 712m7 2 46 Amo.col'h1J1a m. ex C.ralium m·f'OJ AY208892 100 1,260 682/682 1 3 1 51 U11CUltured marine tlicotlLmlcton AP-tlicoclone8 00386744 92.4 1090 720n79 1 TH10 1 Eukaryote clone OLIlI012 AJ402330 95 .7 1,240 749n83 4 4 Uncultured eukarvote clone BBOl 83 AY885027 93.7 798 508/542 8 7 Uncultured marine alveo~te GrouP II clone DH147-EKD16 AF29OO71 95 .7 1,201 718n50 3
76 Appendix 2. (cont' d)
12 hno6bophrya $p. 'Dinophyns' AF239260 96..5 1.219 714n40 2 19 kno6bophrya SJ) . AY175284 93 1149 7421798 2 22 kno"bophrya SJ). ex J:arlodinium micnlm AF472553 922 1,110 734m6 1 25 Uncultured alveoWe clone RA0OO907 2 AY295491 95.6 682 409/428 1 27 Uncultured eukazyote clone Q2A03 m 73006 95.8 1194 715n46 2 46 Uncultured marine picopW1lcton AP-pi.coclone8 00386744 98.4 1179 660/671 1 49 Uncultured marine alveolate GrouP n clone OH147-EKD6 AF290068 91.5 1070 72Dn87 1 Alveolate I70IlP I MB04 17 Uncultured marine eukaryote clone ENVP21819.0oo79 00918313 98.6 1105 6161625 2 MB07 2 Uncultured marine eukaryote clone BLOO1221.41 AY426887 989 1.332 739n47 1 11 10 5 5 2 40 Unculturedeukaryote isolate CAR E214 AY256282 98.1 1.301 736n50 1 TH07 0 Uncultured eukazyote isolate A3 E031 AY046173 89.4 806 579/648 1 4 Uncultured marine alveolate isolate 65 DQ916410 88.1 881 667n57 23 TH10 8 Uncultured eukaryote clone SCM37C30 AY665056 97 1,345 7811805 1 45 Uncultured eukaryote clone TAGIRI-10 AB191418 99.4 1,297 711nl5 1 1 57 Uncultured eukaryote clone SCM38C41 AY664995 99.1 1,367 755n62 1 Alveolata; Ciliophora MBOl 7 StrombidWm sp. AYl43564 99.3 1208 665/670 2 3 12 Uncultured marine eukazyote clone CD8.16 00647517 97.8 1.347 763f78O 2 1 1 3 1 3 13 Uncultured marine eukaryote clone CD8.16 DQ647517 99.6 1273 696/699 1 1 18 Strombidium sp~ AY143564 989 1421 789m8 1 21 Uncultured marine eukaryote clone MD65.05 00119933 97.6 1347 771n90 5 1 1 26 Uncultured marine eukaryote clone NW41424 00120009 99.1 1421 785n92 1 2 1 35 Uncultured eukaryote clone ENI40076.00328 AY937624 95.5 1068 640/670 1 36 Uncultured eukaryote clone ENI42482.00244 AY938119 99.8 948 5151516 1 1 37 Uncultured eukazyote clone ENI4oo76.00328 AY937624 99.1 1201 6611667 1 3 1 THOI 13 Ciliate sp . NCMS060l AM412525 94.8 1138 698n36 4 MB04 2 Uncultured eukazyote clone ENI4oo76.00749 AY937854 99.6 953 5201522 3 7 Uncultured eukazyote clone ENI40076.00328 AY937624 99.1 1,201 661/667 1 TH04 2 Uncultured marine eukazyote clone NW414 24 DQ120009 97.1 1,210 700n21 1 16 Uncultured marine eukazyote clone MD65.18 DQ119937 97.6 1,256 718m6 1 21 Uncultured marine eukazyote clone UEPACAIp5 AYI29053 989 1,290 715m3 7 4 37 Uncultured marine eukazyote clone M065.18 DQ119937 979 1,321 748n64 1 39 Uncultured eukazyote clone ENI47296.00059 AY938245 97.2 1,098 6321650 8 4 42 Uncultured marine eukazyote clone UEPACAIp5 AYI29053 98.4 880 494/502 2 1 2 MB07 25 Uncultured eukazyote clone 03P05C08 Efl 00279 93.8 1,062 675n20 2 30 StrombidWm SI). AY 143564 953 1,166 707n42 1 38 Cryptocaryon iTritam AF351579 93.6 1,129 714n63 1 MB10 16 Strobilidium caudatum AY143573 949 1,110 682n19 1 31 rintinnop~iJ dadaJli AY143562 95..5 1,240 748n83 2 33 Strobilidium caudatum AY143573 94.4 1,092 678n18 1 36 Strombidinop~is $p. AM412524 95.8 688 408/426 2 40 Pal'a~trombidinopn~ ~himi An86648 93.8 989 623/664 1
77 Appendix 2. (cont' d)
THlO 18 Unc:ultured eukaryote clone SCMl5C2 AY665093 94.5 1,229 7611805 1 20 Unc:ulturedeukazyote clone SCM28C124 AY665085 99.5 1,423 7801784 2 24 Uncultuzed eukazyote clone ENI42482.00186 AY938072 98.6 913 508/515 1 33 Strobilidium caudatum AY143573 93 1,075 6921743 7 2 2 34 Strombidium sp. AY 143564 99.5 1,356 743n47 1 6 1 1 4 1 3 37 Strombidium Sl> . AY143565 93.4 1,146 7361788 1 53 Uncultuzedmarine co1podunciJate clone DH147-EKD23 AF290076 88.9 941 7061794 1 1 1 58 El11intinnw p~ctinis AF399170 93.9 1,105 694n39 2 1 A1veolata; Dinophyceae MEal 6 Uncultured eukazyote clone SCM37C24 AY664894 98.7 1,395 777n87 8 4 27 Uncultured marine eukarvote clol\2 BLool221.15 AY426867 99.1 1421 785n92 1 2 33 Uncultuzed eukarvote clone SCM15C23 AY664993 99 1413 7831791 2 1 40 Unculturedeukuyote clol\2 SCM37C27 AY664962 99.4 1432 786n91 1 1 2 TH01 22 Uncultured marine eukaryote clone BLOOl221 .42 AY426888 99.l 1238 6821688 1 MB04 15 Uncultured eukarvote clone SCM28C60 AY664961 95.8 1.255 749n82 3 34 ,FYrodinium baham~7U~ vu. com"r~SJum 150500120 96.1 1199 709n38 1 39 Unculturedeukarrote clone SCM27C15 AY664924 98.5 1297 724n35 1 1 TH07 15 Uncultured marine eukaryote clone CD8.10 DQ647514 100 1,295 70 1n01 2 1 MBI0 3 OIratium longJpeJ DQ388462 99.6 1,291 705n08 2 3 1 2 10 OIratium longip~J DQ388462 99.3 981 5391543 1 23 lhcn·luca Jcintilla7U DQ388461 97.1 1,158 667/687 1 29 P.ntapharJodinium sp . AF274270 96.7 1,240 722n47 3 52 lhcn·luca Jcintilla7U DQ388461 99.6 1,459 7971800 1 5 THI0 6 Uncultuzed eukaryote clone SCM37C27 AY664962 99.8 1,016 5521553 1 10 Uncultuzed marine picoplankton AP-picoclone12 DQ386748 98.5 1,301 7291740 1 ChIorophyta; Prasinophyceae MEal 5 Micromonas pwilla AY425316 99.5 1,430 783n87 4 22 MicromonaJ pusilla AB 183589 99.7 1,454 792n94 3 1 2 30 Uncultured prasinophyte clone NW414.32 DQ055165 99.7 1,437 782n84 2 1 3 31 Mic7omonaJ "willa AB183589 97.5 1.351 7751795 1 38 MicromonaJ pWI"lla AB 183589 98 1,040 589/601 1
TH01 40 OJtr~OCOCCUJ sp . AY425310 99.6 913 498/500 1 1 MB04 46 MicromonaJ pUJilla AY425316 99.7 1,376 749n51 1 MB07 17 I P.YcnococcuJ sp . AB058359 98.4 1,347 755n67 3 1 31 Coccoid prasinophyte MBICI0622 AB058375 95.8 1,186 709n40 4 2 1 TH07 44 Uncultured prasinophyte clone NW41429 DQ055172 99.4 848 465/468 1 I THI0 5 MicromonaJ pwilla AY425316 98.6 1,393 777n88 5 1 2 i Stramenopiles MBOl 25 Uncultured eukaryote clone BLOI0625.35 AY381219 99.7 1,264 688/690 2 1 2 34 Eukaryote marine clol\2 MEl-24 AF363207 93.4 1,116 713n63 1 2 THOl 2 Uncultured marine eukaryote clol\2 IND60.8 DQ234593 99.6 1,447 790n93 1 5 Uncultured marine eukaryote clone CD8 .06 DQ647511 95.3 1,109 671n04 1 6 Uncultuzed marine eukaryote clol\2 UEPACCp4 AY129066 100 1,435 793n93 2 2 3 14 LabJm·nrhuloid~J minuta AF265339 98.l 821 462/471 2
78 Appendix 2. (cont' d)
19 U ncultuzed marine picoplanlcton clone He000803 6 AJ965030 95.1 636 3871407 1 20 Uncultured marine eWcuyote clone BLOOl221.14 AY426866 99.6 983 5361538 7 1 24 Uncultured marine eWcuyote clone UEPACLp5 AY 129067 99.9 1,424 mm4 1 31 Uncultured marine eukazvote clone FV18 3HI0 D0310211 99.4 1208 6631667 3 3 38 Uncultuzedmarine eWcAl'VOte clone lV23 ID5 00310262 99.5 1456 795n99 5 2 43 Uncultuzed marine tllcOtl1anc:ton clone HeOl0218 6 AJ965071 99 1050 5801586 1 44 UncultuzedeWcarvote isolate CAR DI07 AY256317 94.3 1206 7541800 1 MB04 3 Uncultuzed marine eokuyote clone MD6532 D0062487 98.1 1343 757m2 14 18 Uncultured marine eWcarvote clone NW617.17 DOO62467 99.1 1002 552/557 2 21 Uncultured eWcarvote isolate C3 E035 AY046863 99 1391 767n75 2 23 Uncultuzed marine plcop1anc:ton clone HeOOO803 4 AJ965031 98.9 957 5301536 4 36 Uncultuzedmarine str~nopile clone DHl48-5-EKD53 AF290083 98.9 1275 708nl6 1 1 TH04 3 Uncultuzedmarine eWcuyote clone UEPACRp5 AY 129069 99 1,310 724n31 2 32 UncultuzedeuJcaryote clone BLOl032O.17 AY381209 98.7 1,345 749n59 1 36 PJlludobodo ITImrulam AF315604 98.7 989 5501557 1 MB07 19 Uncultured eWcaryote clo~ BLOOO92126 AY381196 99 1,279 708nl5 1 TH07 3 L4ptoq)llindrw danicuJ AJ535175 99.7 1,223 667/669 1 16 Uncultuzed marine plcoplanlcton clone HeOOl005 47 AJ965040 992 904 4981502 1 21 Uncultuzed euJcaryote clone MEl-28 AY116221 98.4 1,199 6751686 1 36 Uncultured eukaryote clone BLOI0320.6 AY381207 98.1 1258 709n23 1 MBI0 48 Uncultured eWcaryote clone BLOl 0320.17 AY381209 933 1092 705n56 1 THI0 39 Uncultured marine eWcaryote clone ENVPl 0203 .00002 D0917930 973 985 5691585 1 43 Uncultuzedmarine eWcarvote clone CD8.09 D0121426 97.7 12n 726n43 1 47 PJlludo-nitzJchia multiJim'IIJ AY221947 98.1 1354 766n81 1 51 Eukarvote marine clone MEl-24 AF363207 89.4 926 681n62 1 Cerc:ozoa THOl 15 QyothllcomonaJ aluti'PaiiJ AF290539 98.7 1,236 703nl2 1 1 1 1 1 MB04 43 Uncultured marine eWcaryote clone NOR2621 DQ314817 98.5 1,299 734n45 1 1 4 TH04 43 Uncultured cerc:=an clone 10-4Fu21 AY360723 91.9 909 599/652 1 MB07 4 Uncultured marine eWcaryote clone NOR4627 DQ314814 98.8 1,360 755n64 1 6 Uncultured eWcaryote clone TAGIRI-3 AB191411 91.5 822 560/612 3 8 Uncultured plasmodiophorid clone RD010517.43 AY295738 902 689 4891542 4 1 22 UncultuzedeWcaryote clone NAMAKO-13 AB252753 99.4 1,122 6151619 1 23 Uncultured eWcarvote clone SCM27C7 AY665094 92.4 1,003 669n24 1 45 Uncultured plasmodiophorid clone RDOI0517 .43 AY295738 89.1 656 4841543 1 50 Uncultured cerc:ozoan clone Cl AY620329 97.8 922 524/536 1 TH07 17 Uncultured cerc:ozoan clone 10-4Fu21 AY360723 92.9 1,003 642/691 2 MBIO 13 Uncultured cerc:=an clone 9-1.7 AY620309 90.6 457 3171350 1 21 UncultuzedeWcaryote clone NAMAKO-6 AB252746 98.5 961 5361544 1 THI0 54 Uncultuzed euJcaryote clone SCM27C7 AY665094 96.8 1,038 606/626 2 Haptophyceae THOl 17 ~JOchromub'na Jcuullum AJ246274 99 1,199 664/671 1 23 Unidentified prym.esiophyte clone OLI16029 AFlO7080 99 715 395/399 1
79 Appendix 2. (cont'd)
I MH07 26 Uncultured marine eulcaryote clone BLOI0625.1 0 AY426921 97.4 1,284 735n55 1 I 32 Uncultmed marine euhzyote clone BLOI0625.10 AY426921 98.6 1,371 765n76 2 1 1 I Cryptophyta MH04 26 r"/,,au/a!( QCUta AF508275 99.6 1,243 6791682 1 1 f TH04 10 Piagios"bnis prolol1ga AF508272 99 1,267 701n08 3 1 Cryptophyta. (NucleOn1Olph) MBOl 32 Uncultmed marine picoplancton clone He001005 108 A.J965235 99.4 976 534/537 1 I TH04 31 r"l"auJa.,; amphiouia AJ421146 99.8 963 5231524 1 2 ; K.t~lepharidophvta THOl 39 UuCOC7J'l?tOS marina AB 193602 99.8 983 5331534 1 1 Telonemia. THOl 26 U ncultmed eulcarvote clone RAOOO907 .3 AY295501 99.3 813 446/449 1 1 MB04 38 Uncultured eulcarvote clone SCM38C20 AY665037 982 1.227 6921705 1 PXobiliphyta. MB04 45 Uncultured phototrophic eulcarvote done RAOO0907 .33 D 222876 992 1.367 753n59 2 1 1 1 5 2 2 I
TH07 20 Uncultured phototrophic eulcaryote done HeOOO803 .72 D 222873 99.3 726 3991402 5 I
! THI0 11 Uncultured phototrophic eulcaryote clone RAOOO907.18 D 222879 99.4 1.301 7121716 1 1 4 I i 59 Uncultmed phototrophic eulcaryote clone RAOO1219.38 D 222878 98.5 1426 797/809 1 Fungi MBOl 14 Uncultured eulcarvote clone T AGIRI-23 AB191431 95.9 998 588/613 2 1 t MBIO 47 Uncultmed eukarvote clone N AMAKO-37 AB252777 99.4 1420 770n75 1 I ChoaJlO~llida THOl 41 Uncultured eulcarvotic: picoplankton clone B360 EF196793 97.8 641 3631371 1 I Radiolaria. MBlO 6 AmJ,hib"IOl1" anoma]a AB178582 98.5 1173 6551665 1 ! THIO 26 PSBUdocubus ob
I 31 Uncultured marine eulcarvote clone MD6536 DQ344788 98.7 1.301 754n64 1 2 12 5 I i I 42 Bolil107JsiJ il1fimdibulum AF293687 99.9 1322 717m8 3 I i MBIO 1 Uncultured eulcarvote clone SCM38C38 AY665127 90.6 318 2211244 25 I 8 Uncultmed copepod isolate MENSA3 AY437861 94.8 1168 716n55 7 ! 15 Uncultured eulcarvote clone ENI40076.00862 AY937955 92.3 274 179/194 3 1 25 Uncultured marine eulcaryote clone NORS021 D0344797 97.4 1149 676/694 11 i 26 Uncultured eulcaryote clone ENI40076.00443 AY937695 96.5 514 3001311 3 32 Uncultured cope pod clone 73 AY491394 95.1 1000 607/638 1 39 Uncultured eulcaryote clone SCM38C38 AY665127 95.5 1144 687m9 37 11 J 42 Uncultured eulcaryote clone SCM27C52 AY665125 96.1 1.255 745n75 1 6 1 1 i 45 Uncultured marine eulcarvote clone ENVP36162.00349 DQ918849 94.1 562 3521374 1 THI0 2 Unculturedeukarvote clone ENI42482.00368 AY938215 99.2 1092 601/606 2 Ii- D0344788 95.5 712 428/448 1 35 - Uncultured marine eulcaryote clone MD65.36
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