THE SIGNIFICANCE OF SARGASSUM ON SINGAPORES REEFS

ilicifolium from seven sites, in the islands south of Singapore, from August 2012 to December 2012.. Data between August 2010 and August 2011 were from three sites, Pulau Hantu H2, Pula

SARGASSUM ON SINGAPORE'S REEFS

LOW KIM YEW JEFFREY

M.Sc National University of Singapore

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE

2015

DECLARATION

I hereby declare that this thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources

of information which have been used in the thesis

This thesis has also not been submitted for any degree in any

university previously

LOW KIM YEW JEFFREY

7 Jan 2015

Prof Chou, for taking me under your wing all those years ago, and your guidance throughout my years as a researcher;

The people from the Reef Ecology and the Experimental Marine Ecology Laboratories: many thanks for your companionship, discussions (heated, frivolous and otherwise), and help on numerous occasions A special shout out to Li Jinghan, for her invaluable work on the epifaunal studies;

To the team from the Marine Ecology Research Group, Nanyang Technological University, past and present: Dr Peter Steinberg (head-honcho); Dr Bryan Wilson (DNA sequencing and analysis); Dr David Feary (fish communities); Dr Adriana Verges (herbivory); Dr Alexandra Campbell (epifaunal studies); Dr Andrew Bauman (coral settlement); Dr James Guest and Rick Leong (coral-algae interaction studies);

To National Parks Board (NParks), for supporting me in my studies;

To my colleagues at NParks, who helped take the weight of the workload during the times of my absence; special shout-out to Rachel for her help with the maps;

My many dive buddies, especially Chay Hoon, Debby, Ben, Vincent, Kee Seng Thanks for taking the time to accompany me in my field work!

Last but not least, I would like to thank my parents for their continuous support of me and my aspirations They are much loved

1.5 Sargassum species as a foundation species 8

CHAPTER 2 S TATUS OF S ARGASSUM IN S INGAPORE : I DENTIFICATION AND

CHAPTER 3 D ISTRIBUTION AND ANNUAL VARIATION OF S ARGASSUM IN THE

4.2 Materials and methods 80

6.5 Protection of marine habitats in Singapore 143

SUMMARY

This thesis presents the first quantitative studies on the

macroalgae Sargassum, which has long been known to comprise a

major component on Singapore's coral reefs, but was never fully studied It comprises chapters that reviewed its classification, quantified its distribution and seasonal variation, and investigated the effects of herbivory within Singapore's reef system

Forty-one valid species were documented from both the Singapore Herbarium (at the Singapore Botanic Gardens) and the Herbarium, Lee Kong Chian Natural History Museum Morphological and DNA analyses of fresh specimens, however, confirmed the

presence of only five species in the field, namely, S ilicifolium, S

granuliferum , S aquifolium and S swartzii, which are subtidal, while the fifth species, S polycystum, was intertidal The occurrence and

distribution of the other 36 species are currently unknown

Sargassum was found to be distributed throughout the islands south of Singapore, with long term reef monitoring data documenting

its occurrence for over two and a half decades Sargassum was

persistent on the reefs (the basal portions are perennial), with higher densities on the reef flat than the reef crest The densities on the reef crest ranged between 15 ± 11.9 m-2 and 30.2 ± 9.4 m-2, and on the reef flat, between 57.4 ± 20.5 m-2 and 93.8 ± 28.4 m-2 All species showed

cyclical growth and die-back, which coincided with the Northeast Monsoon (between August and March) and the Southwest Monsoon (between April and July) respectively Peak average length and

biomass for S ilicifolium were 219 ± 35 cm and 18.5 kg/m2 wet weight

(WW), while for S aquifolium were 88 ± 14 cm and 465.6 g/m2 WW

The biomass for S ilicifolium was 5-40 times that reported elsewhere

Not enough data could be collected for the other two subtidal species

Herbivory pressure was measured in terms of bite rates using underwater video, and was many magnitudes lower than reported elsewhere, at just 0.24 bites/h Consumption rates using macroalgal bioassays were also low, estimated at 0.125 cm/h An indirect measure

of herbivory, using bite marks on leaves, showed marginally higher fish bites at the outer reefs This result was supported by fish surveys which showed non-signifcant differences between sites for herbivorous fish, which were more abundant in the outer islands The macro-

herivores on local reefs comprised two fish genera, Scarus and

Siganus , and the sea urchin, Diadema setosum Both their abundances

were low, with maximum densities of 0.203 individuals/m2 and 0.205 individuals/m2 respectively

Similarly, herbivory by epifauna was low, despite showing similar community structure with studies elsewhere Only about 30% of the fauna observed were herbivores, and occurred in abundances of

than reported for substantial effects on macroalgal cover to be observed No spatial variation could be seen in epifaunal communities

on Sargassum, nor on Hypnea and Bryopsis which were used for

comparative studies There were, however, distinct differences in the

epifaunal communities between Hypnea and Bryopsis, with the assemblage from Sargassum overlapping both

LIST OF TABLES

Table 2.1 The current classification of Sargassum, based on Trono (1997), Mattio

and Payri (2010) and Guiry and Guiry (2014)

Table 2.2 Morphotypes collected from seven sites in the southern islands of

Singapore (total 72 samples from seven morphotypes and one out-group)

Distinguishing characters for Sargassum derived from the morphological study

(H2: Pulau Hantu; TPT: Terumbu Pempang Tengah; S4: Pulau Semakau; J1: Pulau Jong; PSD1: Sisters’ Islands; SJn: St John’s north; and Tk: Pulau Tekukor)

Table 2.3 Sargassum species documented from Singapore Species with specimens

lodged at the Singapore Herbarium and the Herbarium, Lee Kong Chian Natural History Museum are in bold typeface, and marked *1 and *2 respectively Validity

of the species names checked against AlgaeBase (Guiry and Guiry, 2014) unless otherwise stated All locality sources also include Pham et al (2011) and Low and Chou (2013) unless otherwise indicated

Table 2.4 Locations from which Sargassum had been collected, compiled from

herbarium records from Singapore Herbarium and the Herbarium, Lee Kong Chian Natural History Museum Species in bold were observed in field in this study Online catalogues were not used, because the accuracy of the lists was in doubt

Table 2.5 Habitats from which Sargassum had been collected, compiled from

herbarium records of Singapore Herbarium and the Herbarium, Lee Kong Chian Natural History Museum Species in bold were observed in field in this study Online catalogues were not used, because the accuracy of the lists was in doubt

Table 2.6a Characters for Sargassum morphotypes in Singapore waters (seeAjisaka,

Noro and Yoshida, 1995; Phang, Noro and Yoshida, 1995; Tseng and Lu, 1995; Noiraksar, Ajisaka & Ogawa, 2007; Noiraksar and Ajisaka, 2008; Mattio & Payri, 2009)

Table 2.6b Characters for Sargassum morphotypes in Singapore waters (seeAjisaka,

Noro and Yoshida, 1995; Phang, Noro and Yoshida, 1995; Tseng and Lu, 1995; Noiraksar, Ajisaka & Ogawa, 2007; Noiraksar and Ajisaka, 2008; Mattio & Payri, 2009)

Table 3.1 A list of the sampling sites and what types of surveys were carried out Table 3.2 Qualitative descriptions of the reefs, observed during swim-through

surveys Si = S ilicifolium; Sa = S aquifolium; Ss = S swartzi; Sg = S

granuliferum ; Sp = S polycystum

Table 3.3 Results for Two-Way Crossed Analysis using ANOSIM in PRIMER 6 on

growth data of S ilicifolium from seven sites, in the islands south of Singapore,

from August 2012 to December 2012 Figures indicate Global R statistics, those in parenthesis are significance levels Cells in bold show significant differences (a) Pairwise test between sites, (b) Pairwise tests between sampling times, (c) Month- on-month comparisons between years

Table 3.4 Global R statistics and their significance levels from a Two-Way Crossed

Analysis using ANOSIM in PRIMER 6 on growth data of S aquifolium (4 sites), S

swartzii (4 sites) and S granuliferum (2 sites), in the islands south of Singapore,

from August 2012 to December 2012

the islands south of Singapore between 2010 and 2012 (Legend: "DICT" =

Dictyoca; "CCA" = crustose coralline algae; "GREEN" = all green algae; "RED" =

all other red algae; "SARG" = Sargassum)

Table 3.6 Growth cycles for three species of Sargassum Peak length data for S

ilicifolium / cristaefolium was extracted from Trono and Luisma, 1990;

Aterweberhan et al., 2005, 2006, 2008; and this study S cristaefolium is a

synonym of S ilicifolium Data for S siliquosum from Ang (1985) and Low and Chou (2013) were also included Peak length data for S aquifolium / binderi was extracted from Wong and Phang (2004) and Yeong and Wong (2013) S binderi is

a synonym for S aquifolium MCR = Cape Rachado, Malaysia; MPD = Port

Dickson, Malaysia; PB = Bolinao, Philippines; RS = Sheik Said Island, Red Sea; S

= Singapore

Table 4.1 Fish survey sites and sampling times between 1987 and 2012

Table 4.2 Time and locations for the mobile invertebrate surveys under the Reef Friends programme

Table 4.3 ANOSIM results for bite marks at seven sites in the islands south of Singapore, between Decemebr 2011 and March 2013 Global R between sites: 0.403; p<0.001; Global R between samples: 0.325, p<0.001

Table 5.1 Overview of algal species collected over sampling period

Presence/absence (+/-) of algal species found in sampling sites and months is

indicated Sargassum was collect throughout the sampling period

Table 5.2 Monthly average of environmental parameters for the period July to December 2012, except for the month of September due to missing data

Table 5.3 Pair-wise comparisons of environmental data between months R values and significance level of sample statistic (in parentheses), derived from ANOSIM

on environmental data (temperature, pH, dissolved oxygen and Secchi disc readings) from seven sites for the months of July (7), August (8), October (10), November (11) and December (12) of 2012 September was omitted due to missing data ANOSIM between months: Global R = 0.847, p=0.001

Environmental parameters did not varay among sites for each month (Global R = 0.172, p=1)

-Table 5.4 Percentage contribution and cumulative contribution of all 15 epifaunal taxa observed across all samples (n=779) Potential mesograzers indicated with (*) Densities were calculated as number of individuals per 100g wet weight of algae Note: Only 1 crab (Brachyura) was only encountered in one sample of

Sargassum, so its effective contribution to the analysis was zero!

Table 5.5 Mean and standard deviation of epifauna density on Sargassum, Hypnea and Bryopsis collected from the islands south of Singapore, between July and

December 2012 Densities were calculated as individuals per 100g wet weight of algae

Table 5.6 Pair-wise comparisons between months for Sargassum, Hypnea and

Bryopsis R values (with sample statistic values in parenthesis) were calculated using ANOSIM in PRIMER 6

Table 5.7 Pair-wise comparison between Sargassum, Hypnea and Bryopsis R

values (with sample statistic values in parenthesis) were calculated using ANOSIM

in PRIMER 6 Global R: 0.367, p=0.001

Table 5.8 Densities for two of the most abundant taxa, Amphipoda and Isopoda, per 100g wet weight of algae

LIST OF FIGURES

Figure 1.1 Location of Singapore and the islands south of Singapore

Figure 2.1 Sites surveyed for Sargassum in the islands south of Singapore throughs were conducted at all sites to assess the occurrence of Sargassum

Swim-Collection of specimens for herbarium, morphological and DNA analysis were carried out only at Terumbu Pempang Tengah (TPT); Pulau Hantu (H2); Pulau Semakau (S4); Pulau Jong (J1); Pulau Tekukor (Tk1); Sisters’ Islands (PSD1); St John’s north (SJn); and Raffles Lighthouse (R2)

Figure 2.2 (a) PCR of genomic DNA extracted from putative Sargassum species

using primers for the nuclear its2 region; (b) Genomic DNA extracted from putative

Sargassum species'

Figure 2.3 The NJ tree of Sargassum collected from Singapore Hormophysa

cuneformis was collected as an out-group Seven clusters can be seen,

corresponding to: S swartzii (Ss), S aquifolium (Sa), S polycystum (Sp), S

granuliferum (Sg) and S ilicifolium (Si, Si1 and Si2)

Figure 2.3 The NJ tree of Sargassum collected from Singapore Hormophysa

cuneformis was collected as an out-group Seven clusters can be seen,

corresponding to: S swartzii (Ss), S aquifolium (Sa), S polycystum (Sp), S

granuliferum (Sg) and S ilicifolium (Si, Si1 and Si2)

Figure 2.4 Phylogenetic tree of S ilicifolium (derived from supplementary material of Chan et al [in review]), along with sequences derived from GenBank (S

polycystum , S aquifolium, S decurrens and Turbinaria ornata (outgroup)

Singapore's contribution is labelled with pre-fix "SI" and indicated with arrows Other samples (not indicated here) were collected from Indonesia, Malaysia, Vietnam, Philippines, Cambodia, Thailand, Taipei and Australia by the papers’ co- authors

Figure 3.1 Sites surveyed for Sargassum in the islands south of Singapore (refer to

Table 3.1 for type of surveys carried out) Starred: Swim-throughs to assess the

occurrence of Sargassum; H2, S4, R2, J1 and PSD1: line-intercept transects and

growth and density studies; TPT and TPL: only growth and density studies

Figure 3.2 Mean and standard deviation of S ilicifolium density at the reef flat and

crest, from Nov 2011 to Dec 2012 ANOSIM tests showed significant differences between crest and flat (Global R=0.702, p<0.001) There were no significant differences among sites over time (Global R=0.035, p>0.122)

Figure 3.3 Mean and standard deviation of S aquifolium density at the reef flat and

crest, between Nov 2011 and Dec 2012 ANOSIM tests showed significant

differences between crest and flat (Global R=0.524, p<0.001) There were no significant differences among sites over time (Global R=0.005, p>0.331)

Figure 3.4 Mean and standard deviation of S ilicifolium lengths, from August 2010 to

December 2012 Data between August 2010 and August 2011 were from three sites, Pulau Hantu (H2), Pulau Semakau (S4) and Pulau Jong (J1), and data from September 2011 to December 2012 included four additional sites, Terumbu Pempang Laut (PL), T.P Tengah (PT), Pulau Satumu (R2) and Sisters’ Islands (SD) Significant differences in lengths between sampling months indicated by ( ) and significant differences in heights between growth year indicated by ( ) Refer also to ANOSIM results in Table 3.4 Receptacles were observed for almost all

Figure 3.5 Mean and standard deviation of Sargssum aquifolium lengths, from four

sites (Jong, Terumbu Pempang Laut, T.P Tengah and Sisters’) in the islands south of Singapore, between September 2011 and December 2012

Figure 3.6 Mean and standard deviation of Sargssum granuliferum lengths, from two

sites (Hantu and Semakau) in the islands south of Singapore, between September

2011 and December 2012

Figure 3.7 Mean and standard deviation of Sarsraum swartzii lengths, from three

sites (Jong, Terumbu Pempang Laut and T.P Tengah) in the islands south of Singapore, between August 2010 and Dec 2012

Figure 3.8 Length-wet weight correlation plot for S ilicifolium Data was collected

from five sites in the islands south of Singapore in November 2011

Figure 3.9 Length-wet weight correlation plot for S aquifolium Data was collected

from four sites in the islands south of Singapore in November 2011

Figure 3.10 Mean and standard deviation of S ilicifolium biomass, estimated from

length, length-wet weight correlation and density studies Data was collected in the islands south of Singapore between November 2011 and December 2012

Figure 3.11 Mean and standard deviation of S aquifolium biomass, estimated from

length, length-wet weight correlation and density studies Data was collected is the islands south of Singapore between November 2011 and December 2012

Figure 3.12 Data from shallow transects in the reefs south of Singapore, collected between 1988 and 2012 (a) Multi-dimentional scaling (MDS) plot of benthic

community, showing trajectory of community change over time; (b) mean percent cover of hard corals and macroalage Bleaching events are indicated

Figure 3.13 Data from deep transects in the reefs south of Singapore, collected between 1988 and 2012 (a) Multi-dimentional scaling (MDS) plot of benthic

community, showing trajectory of community change over time; (b) mean percent cover of hard corals and macroalage Bleaching events are indicated

Figure 4.1 Map of Singapore, showing survey sites for reef fish in the islands south

of Singapore ( ) indicates sites where indirect herbivory (bites) counts were carried out Refer to Table 4.1 for times when fish surveys were carried out

Figure 4.2 Categories of bite marks on S ilicifolium blades: intact blades; blades with

fish bite marks; blades with gastropod bite marks; and bite marks of other

invertebrates

Figure 4.3 Mean and standard deviation of Scarus and Siganus abundance in the

reefs south of Singapore, from fish visual census surveys conducted between 1987 and 2012

Figure 4.4 Multi-dimensional scaling (MDS) plot of Siganus and Scarus in the reefs

south of Singapore, at the deep transects from data collected between 1987 and

2012 (a) Siganus abundance, shallow; (b) Scarus abundance, shallow; (c)

Siganus abundance, deep; (d) Scarus abundance, deep

Figure 4.5 Multidimenional scaling (MDS) plot of the bite marks data, from seven sites in the islands south of Singapore, between December 2011 and March 2013 Cluster is indicative of site differences as shown in Table 4.2

Figure 4.6 Incidence of bites on S ilicifolium blades at seven sites in the islands

south of Singapore, between December 2011 and March 2013 (a) Fish, (b)

Gastropods; (c) Other invertebrates (amphipods and urchins), and (d) Intact blades

Figure 4.7 Bite rates on macroalgae (MA) and epilithic algal matrix (EAM) from video assays conducted in Singapore Colour code: lime green = herbivores; orange = omnivores (algae, zooplankton, zoobenthos); dark maroon = carnivores

Figure 4.8 Mean and standard deviation of sea urchin (Diadema setosum) density at

shallow and deep transects at nine sites in the islands south of Singapore from

2004 to 2012

Figure 4.9 Multi-dimension Scaling plots showing distribution of sea urchins

(Diadema setosum) among nine sites in the islands south of Singapore, from 2004

to 2012 Bubble plot scales indicate number of individuals per transect (a)

Between shallow and deep transects; (b) Distribution among years, shallow and deep; (c) Among shallow sites; and (d) Among years, shallow sites only

Figure 4.10 Mean and standard deviation of reef fish biomass in Singapore,

estimated from fish surveys carried out between 2011 and 2012 (Feary,

unpublished data) Top left and right shows data collected in from eight sites in the islands south of Singapore; bottom left and right shows data collected from

Tioman, Malaysia

Figure 5.1 Map of the islands south of Singapore showing the locations of the seven sampling sites (starred) Pulau = Island, Terumbu = patch reef

Figure 5.2 Multi-dimensional scaling (MDS) plot of environmental data (temperature,

pH, dissolved oxygen and Secchi disc readings) from seven sites for the months of July (7), August (8), October (10), November (11) and December (12) of 2012 September was omitted due to missing data ANOSIM results between months shown in Table 5.3

Figure 5.3 Multi-dimensional scaling (MDS) plot of the epifauna from the tip, middle

and base of Sargassum Stress value is an indication of fit of data on a

two-dimensional plane; any stress value below 0.2 is considered a good fit (Clarke and Warwick, 2001) ANOSIM between parts, Global R: 0.12, p=0.001

Figure 5.4 MDS plot of epifauna in Sargassum, Bryopsis and Hypnea between July

and December, averaged over all sites at each sampling time Arrows added to show shift in epifaunal communities over sampling period (brown arrows:

Sargassum ; red arrows: Hypnea; green arrows: Bryopsis) Stress value is an

indication of fit of data on a two-dimensional plane; any stress value below 0.2 is considered a good fit (Clarke and Warwick, 2001)

Figure 5.5 MDS plot of epifauna on Sargassum, Hypnea and Bryopsis at seven sites

in the islands south of Singapore, July to December 2012 Stress is an indication of fit of data on a two-dimensional plane; any value below 0.2 is considered a good fit (Clarke and Warwick, 2001) The overlay shows correlation of environmental

parameters (Pearson’s correlation > 0.2) to the epifaunal data (a) Sargassum; (b)

Hypnea ; (c) Bryopsis

Figure 5.6 MDS plot showing the epifaunal composition in Sargassum, Bryopsis and

Hypnea between July and December Stress value is an indication of fit of data on

a two-dimensional plane; any stress value below 0.2 is considered a good fit (Clarke and Warwick, 2001)

Figure 5.7 MDS plots of epifauna on Sargassum, Bryopsis and Hypnea between July

CHAPTER 1 INTRODUCTION

1.1 Brief profile of Singapore

Singapore is located south of the West Malaysian Peninsula at 1.3667° N, 103.7500° E It is an island city-state with 5.4 million people

on 716 km2 of real estate (Anon., 2014a) It territorial waters are roughly equivalent to its land area, at approximately 600 km2, containing 72 islands (Figure 1.1; Chou, 2006)

Figure 1.1 Location of Singapore and the islands south of Singapore

Singapore has been heavily developed over the last five decades, resulting in the loss not only of its terrestrial flora and fauna, but also most of its coastline (Chia et al., 1988; Hilton and Manning,

1995) Similarly, many islands were also reclaimed in the late 1970s, some to increase land for industrial capacity (Jurong Island and Pulau Bukom, for petro-chemicals), while others for recreation (Pulau Hantu, Pulau Sakijang Bandera [St John's Island], Pulau Tembakul [Kusu Island]) In the 1980s a series of reclamation works were carried out to expand Pulau Blakang Mati (Sentosa), also for recreation In the late 1990s, Pulau Semakau and Pulau Sakeng were reclaimed as an offshore landfill for incinerated material (Ng, 2009) It is estimated that Singapore has lost up to 60% of its coral reefs owing to these developments (Chou, 1995; 2008)

Singapore's first coastal marine "protected" areas came into existence in the 1990s as well After the Singapore Green Plan (SGP) was launched in 1992 (Anon., 2014b), marine interest groups rallied together to produce an addendum, which they dubbed the "Singapore Blue Plan", that outlined marine areas (primarily coral reefs areas) that should be conserved The blue plan was adopted by the SGP committee, and four "nature areas" were zoned in the islands south of Singapore that were afforded a basic, administrative-level type of protection Ten years on, several milestones marked a shift in the mindset of policy makers: two new "Nature Reserves" were announced, Labrador Nature Reserve (coastal rocky shore) and Sungei Buloh Wetland Reserve (mangroves) (Anon., 2001), resulting in

a total of four legally protected areas Also, in the revised version of the

and Pulau Subar Laut (The Sisters’, or Sisters' Islands) were

"upgraded" in status, and captured in the country's premier planning document, the Singapore Master Plan Most recently, it was announced that these islands (and the western reefs of two other islands) are part of a marine park (Chua, 2014) It must be noted here that this "marine park" status is not the same as the international definition of the term, but is very well advanced for a nation that is striving for economic prosperity while balancing the needs of conservation

1.2 Coral reef studies in Singapore

Hard corals have been the focus of much of the ecology research locally, with two major projects standing out: the ASEAN-USAID Coastal Resources Management Project (CRMP), and the ASEAN-Australia Living Coastal Resources project (LCR), which ran from 1985 to 1989 and 1986 to 1994 respectively The CRMP focussed

on management-related issues, for example, on restocking of fish in the Singapore River (Lee and Low, 1991), soft-bottom benthic communities (Khoo, 1991), artificial reefs (Chou, 1991; Chou, 1994; Chua and Chou, 1994; Low and Chou, 1999) and coastal land-use (Chia et al., 1988; Chia, 1992) The LCR focussed on biodiversity and habitat monitoring, eventually producing monitoring techniques for coral reef monitoring that are still in use in the region today (Dartnall

and Jones, 1986; English et al., 1994; 1997) These two projects set the tone for coral reef research in Singapore for two decades after their completion

Note-worthy outcomes from the work during the lifespan of these two projects include three symposia, numerous workshops and joint-study trips, and for Singapore, a team of over 20 researchers and graduates (Chou, 1991b) Locally, enough capacity was built up to implement a local Reef Survey and Conservation Project (RSCP), which contributed to the development of the "Blue Plan" mentioned earlier The Blue Plan advocated the conservation of marine biodiversity and protection of coral reef areas in the islands south of Singapore This effort was jointly organised by the Singapore Institute

of Biology, Republic of Singapore Yacht Club and the Singapore Underwater Federation, and trained over 150 volunteer scuba divers to survey over 65 coral reef sites (Chou, 1991c; Chua and Chou, 1992) Two decades later, the coral monitoring programme continues as a volunteer-run programme called Reef Friends, and is funded and supported by the National Parks Board (Low and Yaakub, in prep) The Blue Plan itself was refreshed and resubmitted to government agencies

in 2009, resulting in two outcomes, the implementation of the Comprehensive Marine Biodiversity Survey, and the announcement of Singapore’s first marine park

1.3 Importance of algae

While algae are a major component of a coral reef habitat, not many studies have been carried out on them, at least locally We do know that they act both as primary producers as well as indicators of nutrient input into the system (Graham & Wilcox, 2000) Their uses are many (Trono, 1997), primarily as a source of raw material for the production of agars, carrageenans and alginates (collectively called phycocolloids), but also as a vegetable and flavouring in foods, as well

as ingredients in traditional Chinese medicine It is also used as fertiliser for nutrient poor soils, and is mixed in the feed of farm animals for their salt

An estimated US$94 million was exported from the Philippines

in 1996 (Trono, 1997) and seaweed farming represented a major increase of income as an alternative to other forms of farming (Gao and McKinley, 1994), with Asia contributing 98% of the world’s production (Holmyard, 2011) Much of the classification work has also revolved around "economic" seaweeds (see Trono, 1997; Abbott [and others], 1985; 1988; 1992a; 1994; 1995; 1997; 1999; 2002; 2004a)

1.4 About Sargassum

Many studies have focussed on Sargassum's engineering potential as a bio-absorbent of heavy metals (Davis et al., 2000;

Fourest and Volesky, 1995; Chen and Yang, 2006; Yang and Chen,

2008; Vijayaraghavan et al., 2009), or its biomedical properties as an anti-tumour therapeutic agent (Itoh et al., 1993) However, macroalgae,

especially the brown, have been recently identified as potential sources

of biofuels Their ability to rapidly gain biomass (Subhadra and Edwards, 2010) means that they have significantly higher production yields per unit area than terrestrial plants (Gao and McKinley, 1994; McHugh, 2003) Their high productivity also makes them ideal potential candidates for CO2 removal (Gao and McKinley, 1994; Lee et al., 2011, Rajkumar et al., 2014) Many challenges still need to be overcome, however, in particular the impact to the environment as seaweed farms expand to accommodate the demand for feedstock (Wei et al., 2013)

Sargassum is one of the most diverse and taxonomically complex algae, which as a genus, is well characterised (Trono, 1997; Mattio and Payri, 2010): it has a thallus that is not filamentous and composed of a fixation holdfast, one to several main axes (also described as “strap-shaped branches”) that ramify into “branches” (or axes) of several orders, and distinct “leaves” (or foliar appendages or fan-shaped blades), vesicles (aerocysts) and receptacles (reproductive organs)

However, it has over 1000 holotypes, syntypes, variants and forma

worldwide, and over 190 species of Sargassum have been reported

regionally (Ang et al., 2008) encompassing tropical and temperate waters, Indian and Pacific Ocean

This complexity stems from the expression of its variable morphology, which can be dependent on not only the environmental factors, but also on the age of the alga and its reproductive state (Kilar

et al., 1992) It expresses this variability either between populations, within the same population, or even within one individual The ranks were originally developed based on differences in the size and shape

of the leaves, vesicles and receptacular morphology, often based on fragmentary evidence, and without taking into account the extreme

phenotypic variability (Mattio and Payri, 2010; Mattio et al 2010) that

these characters exhibit This has led to a proliferation of not only species names, but also the complex, and oftentimes confusing, classification structure (Mattio and Payri, 2010)

Recent attempts to standardise the characters used in describing the species and in simplifying the phylogenetic relationships between species (Phillips, 1995; Trono, 1997; Abbott, 2004b; among others) have been greatly enhanced by the advent of molecular techniques (Mattio and Payri, 2010), and less than 40% of the species

are now recognised as valid (Mattio et al., 2010; Mattio & Payri, 2010)

Many recent workers of this genus agree that re-examining the

classification of this extensive genus is needed, with many now considering the lower ranking classifications superfluous (Wormersley, 1987; Yoshida, 1987), too vague to be useful (Abbott, 1992b), and in many cases, taxonomically invalid (Mattio and Payri, 2009; 2010) The current, accepted classification is presented in Chapter 2 (Table 2.1)

1.5 Sargassum species as a foundation species

The role of foundation species in facilitating entire communities

of organisms through the creation and provision of habitats is widely recognized in many terrestrial and marine ecosystems (Dayton, 1972; Bertness and Callaway, 1994; Bruno and Bertness, 2001; Stachowicz 2001; Ellison et al., 2005) In marine ecosystems, benthic plant communities such as kelp (Carr, 1989; Estes and Duggins, 1995; Graham, 2004), seagrasses (Orth and Heck, 1980; Heck and Crowder, 1991; Reed and Hovel, 2006), and other seaweeds (John et al., 1992; Hay, 1997) contribute to habitat complexity and provide refuge for organisms seeking shelter from biological and physical stress (Dayton, 1972; Stachowicz, 2001) In addition to being primary producers within systems, the biogenic structure formed by dense beds of benthic plants plays a role in sustaining ecosystems, providing shelter for significant biodiversity (Ornellas and Coutinho, 1998) or functioning as crucial

brooding sites for the eggs and juveniles of marine invertebrates (Poore and Steinberg, 1999) Through the provision of such ecosystem services, the presence of foundation species enhances the diversity and abundance of associated organisms (Bracken et al., 2007) within natural systems

Large phaeophycean algae form the cornerstone of community organization of many marine ecosystems (Dayton and Tegner, 1984; Lüning, 1990; Hawkins et al., 1992; Åberg, 1992; Bertness and Leonard, 1997; Jenkins et al., 1999a, b; Petraitis and Latham, 1999;

Witman and Dayton, 2001; Connell 2003) Several species of Fucus and Ascophyllum nodosum in the North Atlantic Ocean act as

ecosystem engineers of rocky intertidal shores, regulating conditions and resources for other species (Lubchenco, 1980; Bertness and Leonard, 1997; Petraitis and Latham, 1999; Leonard, 2000) An investigation of macroalgae as a co-occurring species by Dijkstra et al (2011) revealed that macroalgae enhanced species abundance in a manner surpassing that of the primary foundation species

1.6 Sargassum as an invasive species

On a broader geographical scale, there is also the threat of biological invasion, particularly to other coasts, exemplified by the

spread of S muticum on the Pacific coast of northern America and on

the Atlantic coast of Europe (Critchley et al 1990) The proliferation of

Sargassum has also been linked to environmental changes, particularly

in relation to enhanced siltation and nutrient loads (McCook 1999; Schaffelke 1999), thus making some species potentially good indicators to monitor ecosystem changes, including increases of anthropogenic pressure

1.7 Sargassum studies in Singapore

As already mentioned, the research focus on local coral reefs has largely neglected the algae, although some recent work has provided substantial updates to the biodiversity of algae in general These studies were largely on biofouling algae (Lee et al., 2009), or collections from a limited area (Noiraksar et al., 2012), or basic compilation of existing records (Pham et al., 2011) Other work on

Sargassum was sparse, despite Sargassum beds constituting a key

coastal habitat and providing significant shelter for biodiversity (Mukai, 1971; Rossier and Kulbicki, 2000; Tanaka and Leite, 2003) The only known quantitative study on algae zonation on a Singapore reef (Chou and Wong, 1984) reported the zonal distribution of 8 species of

macroalgae and 1 seagrass from Pulau Salu While Sargassum was

the dominant species, covering the reef flat from edge of shore to the

components of Turbinaria ornata and five other chlorophytes (green

alga) Other types of reef surveys, using line intercept transects, for example, might provide further evidence for the occurrence of algae on

Singapore’s reefs, but since the survey protocol lumped Sargassum

with other “macroalgae” (or MA, as outlined in English et al., 1997), it may not be always possible to tease out such information

Other studies examined in-situ measurements of its productivity

(although the species was not indicated, it was most likely the

dominant species, S ilicifolium) using an automated respirometer (Tun

et al., 1994) This was carried out as part of a preliminary study of reef

productivity Finally, Ang et al (undated) looked at Sargassum as a

possible bio-indicator of sedimentation, and Yusfiandi (2001) described

a small study to determine if Sargassum could be used to remediate

water quality, but the results of both studies were inconclusive

1.8 Sargassum as a study subject

Sargassum was chosen for this study for several reasons Firstly, Sargassum is known to occur abundantly throughout the year

on Singapore's reefs, in significant density and biomass (although this was not sufficiently quantified) Its perennial nature ensured a steady supply of material and study sites, if needed In fact, Singapore reefs

were dubbed "Sargassum" reefs by Chuang (1977), and combined with

studies that indicate it to be a "foundation species" (Dayton, 1972; Bruno et al., 2003; Bruno et al., 2005), the study of their ecology becomes paramount to understanding the interactions on local reefs Secondly, there was recognition that there were other habitats adjacent

to the coral reefs that were very much understudied This realisation arose from a mini "renaissance" for marine biodiversity and ecology in Singapore, with the infusion of new research laboratories in both the National University of Singapore (NUS) and the Nanyang Techonoligal University (NTU), in the increased focus on marine habitats by the National Parks Board (NParks), and with the setting up of the Technical Committee on Coastal and Marine Environment (Low and Lim, 2012) These efforts were further blostered by the increased activity of marine interest groups, for example, Blue Water Volunteers, Team Seagrass, Hantu Bloggers and Wild Singapore, leading the charge to spur volunteer-driven, "citizen-science" locally Thirdly, there were records

of its occurrence that date back to 1890, and some earlier publications also alluded to its abundance (e.g., Johnson, 1964) This meant that

Sargassum was part of the seascape, and not a recent introduction The collection record was surprisingly sparse, given its apparent density and biomass, although it must be noted that most of the collections of old were from the reef flat areas

1.9 Research objectives

The overall objective of this study is to fill in the gaps on our

knowledge about Sargassum on Singapore reefs In summary, the

studies examined the classification of the local genera through morphological and DNA analyses (Chapter 2); quantifying the extent the species and their seasonality of growth (Chapter 3); and investigating the roles of both macrograzers (fish and urchins, Chapter 4) and mesograzers (epifauna, Chapter 5) in regulating the distribution

of the dominant Sargassum species Data were collected through

methods devised for the study, and also recalled reef monitoring data

collected over the years, providing a retrospective look at Sargassum

on our reefs The final chapter (6) encapsulates the conclusions of the previous studies and discusses future work

CHAPTER 2 STATUS OF SARGASSUM IN SINGAPORE: IDENTIFICATION AND MORPHOLOGY

2.1 Introduction

The genus Sargassum is one of the most diverse and

taxonomically complex algae, with over 1000 holotypes, syntypes, variants and forma worldwide (Mattio and Payri, 2010), and 191 species in the South China Sea region alone (Ang et al., 2008) Part of the complexity stems from the expression of its morphology, which can

be very variable and dependent on not only the environmental factors, but also on the age of the alga and its reproductive state (Kilar et al., 1992) This variability can be expressed either between populations, within the same population, or even within one individual Additionally, the ranks ascribed to this genus are very complex (see Table 1), and were developed based on differences in the size and shape of the leaves, vesicles and receptacular morphology, all of which can exhibit extreme phenotypic variability (Mattio and Payri, 2010) Often the evidence for this classification is fragmentary, thus leading to a proliferation of not only species names, but also a confusing and complex classification structure (Mattio and Payri, 2010) Many researchers now consider the lower ranking classifications superfluous (Wormersley, 1987; Yoshida, 1987), too vague to be useful (Abbott,

2009; 2010) It is estimated that less than 40% of the species names are now considered valid (Mattio and Payri, 2009; 2010; Mattio et al., 2009; 2010)

Table 2.1 The current classification of Sargassum, based on Trono (1997),

Mattio and Payri (2010) and Guiry and Guiry (2014)

Empire: Eukaryota

Malacocarpicae and Acanthocarpicae, as proposed by Mattio and

Payri, 2010)

Recent attempts to standardise the characters used in describing the species and in simplifying the phylogenetic relationships between species (Phillips, 1995; Trono, 1997; Abbott, 2004b; among others) have been greatly enhanced by the advent of molecular techniques (Mattio and Payri, 2010) Many inconsistencies in the morphological

characters selected to classify Sargassum suggest that much of the

older descriptions did not take into account the wide variation in growth

characters that the species in this genus are capable of (Mattio and Payri, 2010; Mattio et al 2010) Almost all recent workers agree that re-examining the classification of this extensive genus is needed

Sargassum is characterised (based on Trono, 1997; Mattio and Payri, 2010) by a thallus that is not filamentous and composed of a fixation holdfast, one to several main axes (also described as “strap-shaped branches”) that ramify into “branches” (or axes) of several orders, and distinct “leaves” (or foliar appendages or fan-shaped blades), vesicles (aerocysts) and receptacles (reproductive organs)

The vesicles are stipitate, borne on stalks of the leaves or receptacles

Documentation of local Sargassum species is surprisingly poor,

with details on only a few species (e.g Teo and Wee, 1983; Wee 1994), or simply a reporting of a species list (Lee et al., 2009) Their distribution locally has only been broadly described as occurring in the reefs of the southern islands of Singapore, which have been described

as being "Sargassum" reefs (Chuang, 1977) Despite constituting a key

coastal habitat, providing significant shelter for biodiversity (Mukai 1971; Rossier and Kulbicki 2000; Tanaka and Leite 2003), no quantitative studies on species distribution or zonation have been made, save a detailed assessment of their zonation for one island (Chou and Wong, 1984) There is little knowledge as to whether these functions are the result of a single species or a mixed group complex It

clarified, as an important first step before effective conservation and management actions can be implemented to maintain ecosystem function Some of the results of this study have been reported in Pham

et al (2011) and Low and Chou (2013)

2.2 Materials and methods

2.2.1 Examination of herbarium records

Specimen records from the Singapore Herbarium at the Singapore Botanic Gardens and from the Herbarium, Lee Kong Chian Natural History Museum, were examined The material described in Teo and Wee (1983), which were presumably the source for Wee (1994), were also examined Information was extracted from AlgaeBase (http://www.algaebase.org) (Guiry and Guiry, 2014), and the online catalogue of the Herbarium of the Bishop Museum (http://ucjeps.berkeley.edu/rlmoe/tioc/ioctoc.html), together with listings and descriptions from Silva et al (1996) and Ajisaka (2002) The information was tabulated and valid names were applied (based on Ajisaka et al., 1999; Ajisaka, 2002; Mattio and Payri, 2009; Mattio and Payri, 2010; Mattio et al., 2009; Mattio et al., 2010; Mattio et al., 2013) Locality information was also noted from the herbarium records These sites were investigated for any physical changes over the years

2.2.2 Sampling sites

Field observations and collections were carried out at a total of

19 sites in the islands south of Singapore (Fig 1.1) Initial surveys comprised swim-throughs at all sites (starred in Fig 1.1) from 2009 to

2010 and collection for the morphological study was also carried out during this time Collection for DNA analysis was done at the seven labelled sites (TPT, H2, S4, R2, J1, PSD1, Tk and SJn) in 2013 All specimens collected for the herbarium were pressed-dried in newspapers before transfer to herbarium quality mounting paper for curation at the Singapore Herbarium

Figure 2.1 Sites surveyed for Sargassum in the islands south of Singapore

Swim-throughs were conducted at all sites to assess the occurrence of

analysis were carried out only at Terumbu Pempang Tengah (TPT); Pulau Hantu (H2); Pulau Semakau (S4); Pulau Jong (J1); Pulau Tekukor (Tk1);

2.2.3 Collection for morphological study

Samples were collected based on differences in the following morphological characters (based on Yoshida, 1983): shape of cross-section of primary axis and/or secondary axis (cylindrical or flattened); shape of leaves (oblong, lanceolate, rounded); margin of leaves (smooth or serrated); leaf dimensions (length and width); shape of vesicles (spherical to elliptical) and presence or absence of crown growths; and vesicle stipule characteristics (winged/simple)

2.2.4 Collection for DNA analysis

Three samples of each morphotype were collected from each sampling site, and preserved in silica gel (based on Chase and Hills, 1991) Branch tips were about 10cm long and were cleaned of epiphytes before preservation Each sample was stored separately in ziplock bags and silica gel was changed several times during the drying process Out-group samples were also collected, comprising six

samples of Hormophysa cuneformis (a mono-specific genus within the Phaeophyceae) The preferred out-group species, Turbinaria ornata,

suggested by Stiger et al (2003) and Phillips et al (2005), occurs only

very rarely in Singapore A total of 66 samples of Sargassum from seven morphotypes, and six samples of the out-group Hormophysa

were collected for analysis (Table 2.2)

Table 2.2 Morphotypes collected from seven sites in the southern islands of Singapore (total 72 samples from seven morphotypes and one out-group)

Distinguishing characters for Sargassum derived from the morphological

study (H2: Pulau Hantu; TPT: Terumbu Pempang Tengah; S4: Pulau Semakau; J1: Pulau Jong; PSD1: Sisters’ Islands; SJn: St John’s north; and Tk: Pulau Tekukor)

1

SJn Tk

S aquifolium (flattened stems;

narrow serrated leaves; oval,

muronate vesicles)

S swartzii (flattened stems;

narrow serrated leaves; globular

vesicles)

S ilicifolium (cylindrical stems;

large serrated leaves; large

heavily muricated stems; leaves

small; small globular vesicles)

3

S granuliferum (cylindrical,

sparsely muricated stems;

medium-sized, serrated leaves;

vesicles small, bunched)

2.2.5 DNA extraction, PCR and sequencing

Genomic DNA was extracted and amplified from samples using a modified protocol of Mattio et al (2008) Briefly, 20 mg of silica gel-dried materials were disrupted together with a 3mm tungsten carbide bead (Qiagen) using a TissueLyser II (Qiagen) for 1 min at 30 Hz and then extracted using a DNeasy Plant Mini Kit (Qiagen), according to

the manufacturer’s instructions DNA was eluted in 50 µL buffer AE

using a GeneClean III kit (MP Biomedicals, UK) PCR reaction mixtures

comprised 12.5 µL Taq Master Mix (Promega, USA), 200 nM of

primers 5.8S-BF (5’-CGATGAAGAACGCAGCGAAATGCGAT-3’) and 25BR-2 (5’-TCCTCCGCTTAGTATATGCTTAA-3’) (Yoshida et al

2000), 200 ng µL−1 non-acetylated BSA, 1 µL (2-10 ng) of genomic DNA, and nuclease-free water to bring the total volume to 25 µL

Reactions were initially denatured for 1 min at 94◦C, followed by 40 cycles of denaturation at 94◦C for 40 s, primer annealing at 55◦C for 30

s and extension at 72◦C for 45 s This was followed by a final extension step of 72◦C for 7 min Amplicons were visualised by agarose gel electrophoresis using ethidium bromide and assessed using the Quantity One 1-D Analysis Software (Bio-Rad, USA)

Sequencing was performed by Geneius Laboratories Ltd., UK; briefly, amplicons were treated with Exonuclease I and Alkaline Phosphatase (Thermo-Scientific, UK), as per the manufacturer’s instructions, prior to sequencing on a 3730xl DNA Analyzer (Applied Biosystems, UK) using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) Sequences were trimmed and aligned using a customised python pipeline; briefly, sequence chromatograms were parsed using the BioPython SeqIO module (Cock et al., 2009) and concatenated and the resulting FastQ files trimmed using Sickle (Joshi and Fass, 2011) Trimmed FastQ files were converted to FastA files using the BioPython SeqI0 module and aligned using MUSCLE (Edgar,

2004) The neighbour-joining method (using the ClustalW package [Larkin et al., 2007]) was used to construct phylogenetic trees, which

were visualised using Figtree (Rambaut, 2009); Hormophysa

cuneformis was included as an out-group Sequence data for partial its2 genes were submitted to NCBI GenBank

It should be noted here that amplification of seaweed genomic DNA using the published rbcLS (Phillips et al., 2005; Peters and Ramirez, 2001) and cox3 (Kogame et al, 2005) primers was ultimately unsuccessful A number of PCR optimisation steps were attempted but

to no avail and time constraints meant that a full sequence data set could only be obtained for its2 genes

2.3 Results

2.3.1 Updating the Sargassum species list for Singapore

Forty-one valid names (Table 2.3), out of a total of 50 species of

Sargassum, remained after taking into account the synonymies (Mattio and Payri, 2009; 2010; Mattio et al., 2009; 2010) Several species listed Singapore as the type locality (from Silva et al., 1996), and while they are still considered valid names, were not observed in the field AlgaeBase (Guiry and Guiry, 2014) was the best source of compiled

(2002) The list of Sargassum species compiled by Lee et al (2009)

was derived from AlgaeBase, without any assessment of their status Subsequently, Pham et al (2011) and Low and Chou (2013) provided

updates; the list herein is the most updated species list for Sargassum

in Singapore

Many of the species names listed are now known to be synonyms (Mattio and Payri, 2009; Mattio et al., 2010) or misidentifications

(Ajisaka, 2002) For example, S binderi is the synonym for S

aquifolium (Mattio et al., 2009), as S myriocystum is for S polycystum

(Mattio and Payri, 2009) Ajisaka et al (1999) reported several misidentifications by Teo and Wee (1983), presumably based on their

drawings: S duplicatum should be S siliquosum and S

spathulaefolium and S asperifolium should be S polycystum It would

be useful to note that: firstly, none of the material by Teo and Wee

(1983) could be located; and secondly, the Sargassum records of

Pham et al (2011) were supplied by this author

AlgaeBase had yet to update its records for S asperifolium (from

Teo and Wee, 1983); it is still listed as documented from Singapore (see Pham et al., 2011; Low and Chou, 2013) The location record of

Sargassum grevillei J Agardh was also not updated It has however, updated two of the three previously undocumented records from the

Singapore Herbarium, namely S vulgare and S latifolium var

seychellarum It was assumed that the record for the latter species was

a misspelling of S latifolium var seychallense Grunow, since

"seychellarum" does not exist (also see Table 2.3) Guiry and Guiry (2014) also listed a record of S capillare Kutzing from Singapore, citing Pham et al (2011) However, it, nor its synonym, S gracile Greville,

was listed there Similarly, Low and Chou (2013) did not list it as well The online records of both the Bishop Museum and the Singapore Herbarium seem not to be updated, or show only limited information on this subgenus The Herbarium, Lee Kong Chian Natural History Museum, does not have an online catalogue

The local herbaria contained a total of 248 specimens, comprising 11 species, including the three newly reported specimens The Singapore Herbarium collection, amounting to 223 specimens, was largely made up of collections from the late 1800s and early

1900s, and then a handful of "Sargassum sp." from the mid-2000s These were later identified as S polycystum and S aquifolium by this

author The older specimens had limited information on where they were collected from, and were brittle and prone to breakage Some specimens were without holdfasts, and most were of non-fertile individuals The collection at the Herbarium, Lee Kong Chian Natural History Museum’s were more recently (mid- to late-2000s) collected, but was much smaller (only 25 specimens), and contained a few that were labelled as "jetsam" Most of the specimens were labelled as

Sargassum sp., but upon examination, showed to comprise mainly of

Table 2.3 Sargassum species documented from Singapore Species with specimens lodged at the Singapore Herbarium and the Herbarium,

Lee Kong Chian Natural History Museum are in bold typeface, and marked *1 and *2 respectively Validity of the species names checked against AlgaeBase (Guiry and Guiry, 2014) unless otherwise stated All locality sources also include Pham et al (2011) and Low and Chou (2013) unless otherwise indicated

Sargassum assimile

Harvey *1 This species is neither listed in the online catalogue of the Bishop Museum nor mentioned in Ajisaka (2002) Silva et al (1996)

Sargassum baccularia

(Mertens) C Agardh Silva et al (1996) indicated some uncertainty in the locality record

Teo & Wee (1983) reported this species for Singapore, but called it Sargassum asperifolium Hering & G Martens ex J Agardh

(Ajisaka et al., 1999) No physical record of the specimen could be found The record for this species is not updated in Algaebase

Tseng & Lu (1992)

Sargassum baccularia

var subcompressum

Grunow

This species is neither listed in the online catalogue of the Bishop Museum nor mentioned in Ajisaka (2002) Singapore is the

var pergracile Grunow

This specie is neither listed in the online catalogue of the Bishop Museum, nor in Ajisaka (2002) Silva et al (1996) suggested that this species falls within the circumspection of S polycystum Singapore is the only known locality for this species

First documented by Teo & Wee (1983), but voucher specimen could not be located locally Although also reported in Silva et

al (1996), it is neither in the online catalogue of the Bishop Museum, nor mentioned in Ajisaka (2002) Tseng and Lu (1988) describe this as a primarily Chinese species, but also found in Ceylon and Australia

Teo & Wee (1983); Phillips (1995); Silva

var dubiosum Grunow

This species is neither in the online catalogue of the Bishop Museum, nor mentioned in Ajisaka (2002) Silva et al (1996)

Agardh *1

The report of this species from Singapore was based on a specimen in the Singapore Herbarium, and was reported by Pham

et al (2011) from a list provided by this author (see also Low and Chou, 2013) Record is not yet updated in AlgaeBase

Pham et al (2011); Low and Chou (2013)

Table 2.3 … continued

Table 2.3 … continued

The voucher sheet indicates that the name was determined by Setchell (1929), and this species may be a mis-spelling of S

latifolium var seychallense Grunow, as "seychellarum" does not exist! This is the first mention of this specimen (assuming S

latifolium var seychallense is the correct name) from Singapore

The report of this species from Singapore was based on a specimen in the Singapore Herbarium, and was reported by Pham

et al (2011) from a list provided by this author (see also Low and Chou, 2013)

Pham et al (2011); Low and Chou (2013)

This species is neither in the online catalogue of the Bishop Museum, nor mentioned in Ajisaka (2002) Type locality is listed

as “near Singapore”, and the only known record is from Singapore

Silva et al (1996)

Continued on next page … Table 2.3 … continued