Diversity of Halimeda (Bryopsidales, Chlorophyta) in New ... · Submitted Manuscript 2 20 Abstract 21 22 Halimeda is a genus of calcified and segmented green macroalgae in the order

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Diversity of Halimeda (Bryopsidales, Chlorophyta) in New

Caledonia: a combined morphological and molecular study.

Journal: Journal of Phycology

Manuscript ID: JPY-11-199-ART.R2

Manuscript Type: Regular Article

Date Submitted by the Author: 22-Mar-2012

Complete List of Authors: Dijoux, Laury; IRD, Coreus Verbruggen, Heroen; Ghent University, Phycology Research Group and Center for Molecular Phylogenetics and Evolution; Mattio, Lydiane; University of Cape Town, Botany department & Marine Research Institute Duong, Nathalie; IRD, Coreus

Payri, Claude; IRD, Coreus

Keywords: Biodiversity, Halimeda, New Caledonia, tufA, rbcL, coral reefs, taxonomic revision

Journal of Phycology

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Diversity of Halimeda (Bryopsidales, Chlorophyta) in New Caledonia: a combined 1

morphological and molecular study 1 2

Laury Dijoux ² 3

Institut de Recherche pour le Développement, U227, BPA5, 98848 Nouméa, New Caledonia 4

[email protected] 5

Tél. : (687) 26 07 32 6

Fax : (687) 26 43 26 7

Heroen Verbruggen 8

Phycology Research Group and Center for Molecular Phylogenetics and Evolution, Ghent 9

University, Krijgslaan 281 S8 (WE11), B-9000 Ghent, Belgium 10

Lydiane Mattio 11

Botany department & Marine Research Institute – University of Cape Town 7701 12

Rondebosch, South Africa. 13

Nathalie Duong 14


Claude Payri 16


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Condensed running title: Halimeda diversity in New Caledonia19

Page 2 of 59 Journal of Phycology

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Abstract 20

21

Halimeda is a genus of calcified and segmented green macroalgae in the order Bryopsidales. 22

In New Caledonia, the genus is abundant and represents an important part of the reef flora. 23

Previous studies recorded 19 species identified using morphological criteria. The aim of the 24

present work was to reassess the genus' diversity in New Caledonia using morpho-anatomical 25

examinations and molecular analyses of the plastid tufA and rbcL genes. Our results suggest 26

the occurrence of 22 species. Three of these are reported for the first time from New 27

Caledonia: H. kanaloana, H. xishaensis, and an entity resembling H. stuposa. DNA analyses 28

revealed that the species H. fragilis exhibits cryptic or pseudocryptic diversity in New 29

Caledonia. We also show less conclusive evidence for cryptic species within H. taenicola. 30

31

Keywords: biodiversity, Halimeda, New Caledonia, tufA, rbcL, coral reefs, taxonomic 32

revision. 33

Abbreviation: tufA, elongation factor TU; rbcL, RUBISCO large subunit 34

35

Introduction 36

The genus Halimeda Lamouroux (Bryopsidales, Chlorophyta) is a well-studied group of 37

calcareous green macroalgae. It occurs worldwide in tropical and subtropical marine regions 38

but also in the Mediterranean Sea (Hillis-Colinvaux 1980, Kooistra et al. 2002). It is a 39

conspicuous component of coral reef ecosystems for its biomass and species richness, and 40

occurs from shallow habitats down to 150 m deep on the reef slope (Hillis-Colinvaux 1985, 41

Littler et al. 1986). Halimeda species are composed of calcareous segments connected to each 42

other by non-calcified medullar siphons at the nodal joint. The internal structure of segments 43

is composed of a network of medullar siphons ramifying into a cortex and terminating in 44

inflated peripheral utricles. The outermost utricles adhere to each other forming a continuous 45

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membrane that envelops the internal space of the segment in which calcium carbonate 46

precipitates as aragonite (Hillis-Colinvaux 1980).They are important primary producers, 47

providing food and habitat to several coastal species of invertebrates and parrotfishes (Hillis-48

Colinvaux 1980, Hay et al. 1994). Halimeda is regarded as an important structural component 49

contributing to most of the biogenic carbonate of reefs from the Cenozoic to the present 50

(Payri, 1988, 1995; Taylor et al., 2009). The life history of Halimeda includes periodical 51

sexual reproduction during which the entire cytoplasm turns into reproductive cells and the 52

thallus dies, this is called the holocarpic reproduction (Meinesz, 1980). The calcified 53

segments then break down and crumble, contributing to sand formation. They also reproduce 54

clonally by elongating subterranean rhizoids and fragmentation (Walters et al. 2002). Species 55

display a diversity of forms and various habits colonizing sandy and rocky habitats. Some 56

species feature high intra-specific morphological variability, like H. heteromorpha N’Yeurt 57

which displays trilobed to reniform segment forms and has either a small bulbous attachment 58

structure anchoring it in sand or a small cushion of filaments fixing it to rock. Because of such 59

high levels polymorphism, identification of Halimeda species can be difficult. 60

After its description by Lamouroux (1812), the genus was subdivided in four species groups 61

based on external morphological criteria (Tunae, Pseudo-opuntia, Opuntia and Rhipsalis) by 62

J. G. Agardh (1887:80-89). The analyses of nodal structure and utricles initiated by Askenasy 63

(1888) and expanded by Barton (1901) and Hillis-Colinvaux (1959, 1980), led to the 64

recognition of five lineages: Rhipsalis (eight species), Opuntia (seven species), Halimeda (11 65

species), Micronesicae (three species) and Crypticae (one species). In the molecular 66

phylogenetic studies of Kooistra et al. (2002) and Verbruggen and Kooistra (2004), the 67

classification was further revised, including the reinstatement of section Pseudo-opuntia and 68

the merger of Crypticae with Micronesicae. Additionally, Halimeda melanesica Valet, which 69

was classified in Micronesicae by Hillis-Colinvaux (1980), was transferred to Rhipsalis. 70

Kooistra et al. (2002) further identified the presence of cryptic diversity in the genus. During 71


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the last decade, Verbruggen and co-workers carried out multi-gene phylogenetic studies to 72

further improve our understanding of species delimitation and evolutionary relationships in 73

Halimeda (Verbruggen et al., 2004, 2005a, b, 2006, 2007, 2009a, b). While the section-level 74

lineages have been well-characterized, many taxonomical ambiguities remain at the species 75

level. The number of formally recognized species is 44 (Guiry and Guiry, 2011), although 76

molecular tools indicate at least 52 evolutionarily significant units, some of which are 77

probably cryptic or pseudo-cryptic new species in need of description (Verbruggen et al. 78

2009b). 79

In New Caledonia, Halimeda is common to very abundant in many coralgal reef ecosystems. 80

Most of the sandy or muddy areas of lagoons and back reefs are colonized by species with 81

bulbous holdfasts that form extensive meadows that are sometimes mixed with seagrasses or 82

Caulerpa. These meadows have an important ecological function since they represent one of 83

the three major lagoon habitats structured by macrophytes together with seagrass and 84

Sargassum beds (Garrigue, 1995). Various species form thick sprawling mats or large 85

draperies on hard substrata of reef flats, coral pinnacles and the fore-reef slope. Halimeda 86

contributes significantly to the reef biogenic carbonate and represents one of the dominant 87

bioclastic constituents of lagoon sediments (Chevillon, 1996). The genus’ importance as a 88

structural component of Cenozoic reefs makes it a good indicator in sedimentary archives 89

and, along with coralline red algae, it is used to reconstruct paleoenvironment and climatic 90

histories (Massieux, 1976; Payri & Cabioch, 2004). When Valet (1966, 1968) published his 91

taxonomical work on Chlorophyta from New Caledonia he identified eight species including 92

the new species Halimeda melanesica Valet. Since then, several ecological and floristic 93

studies have been conducted (Garrigue and Tsuda, 1988; Garrigue, 1995) and during the past 94

decade, important new collections have been made in a wide variety of locations around New 95

Caledonia. The numerous specimens collected were identified to currently recognized taxa 96

based mostly on morphological and anatomical analyses. The most comprehensive inventory 97


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to date lists 19 species (Payri 2007). However, the material available has been insufficiently 98

studied in many instances and the diversity may have been underestimated. Furthermore, very 99

little molecular systematic work has been done to verify species identification, and only 100

twelve sequences belonging to five species have been generated from New Caledonian 101

material (Kooistra et al., 2002, Verbruggen et al., 2005a, c , 2009a). 102

Considering the importance of Halimeda in the actual coralgal system and paleoecology of 103

New Caledonia it is clear that the available collections deserve to be completely re-evaluated. 104

Hence, in this paper we aim to revise the species diversity of Halimeda in New Caledonia by 105

characterizing and comparing the morphology and anatomy of New Caledonian morphotypes 106

and evaluating variation in the plastid DNA markers tufA and rbcL. Our strategy consists of 107

defining morphotypes in the collections based on morpho-anatomical analyses and 108

subsequently sequencing the tufA DNA barcodes of a majority of the specimens to evaluate 109

species boundaries, infer the validity of morphotypes and assess the diversity of the genus in 110

the study area. 111

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Material and Methods 113

114

Site and specimens collections 115

New Caledonia is located in the Southwest Pacific Ocean, between 18° and 23° S and 164° 116

and 167° E, about 1,500 km east of Australia (Fig. 1). It is composed of a main island, named 117

“Grande Terre” and several groups of smaller islands, coral reefs and lagoons. The New 118

Caledonian lagoon represents a total surface area of 24,000 km², which makes it one of the 119

biggest in the world (Andréfouët et al., 2004, 2009). It is surrounded by a continuous 1,600 120

km long barrier reef, located from 10 to 70 km away from the Grande Terre coast, and 121

expanding from the Entrecasteaux reefs to the northwest and the Isle of Pines to the south-122


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east. The isolated Chesterfield-Bellona reefs are situated towards the west, half way towards 123

the Australian coast, and the Loyalty Islands are located to the east of the main island. 124

A total of 155 New Caledonian samples pressed as herbarium vouchers and kept in the IRD-125

NOU phycological collection at IRD (Institut de Recherche pour le Développement) in 126

Noumea were considered for morphological analyses. They were collected mainly by SCUBA 127

from the intertidal to 60 m depth. All specimens were morphologically characterized and a 128

selection of them was used for DNA analyses from ethanol or silica gel preserved thallus 129

pieces. 130

The New Caledonian sampling sites included the southwest lagoon of Grande Terre (casual 131

collections between 2004 and 2010), Isle of Pines (BIODIP, November 2005), the Loyalty 132

Islands (BSM-Loyauté, March-April, 2005), La Côte Oubliée (CORALCAL1, March 2007); 133

the Chesterfield –Bellona-Bampton area (CORALCAL2, July 2008), and Le Grand Lagon 134

Nord (CORALCAL3, February 2009). 135

Additional collections from the Maldives (Baa Atoll, 2008), French Polynesia (Moorea, 2007, 136

Biocode Moorea Project –MBP) as well as samples lodged in the Ghent University algal 137

herbarium from Jamaica, Costa Rica, Philippines, Kenya, Indonesia, Maldives, Palau, 138

Australia, Tanzania, Nicaragua, Hawaii, French Polynesia, Thailand, Fiji, Micronesia, Wallis, 139

Chagos and Colombia were included in the analyses. 140

141

Morphology and anatomy 142

The morphological features of the samples were recorded following protocols described by 143

Hillis-Colinvaux (1980) and Verbruggen et al. (2005a, b). We registered both macroscopic 144

criteria such as holdfast type, shape of the thallus or segment form as well as anatomical 145

characters of the nodes and the utricles, which were observed with stereo and light 146

microscopy after dissection and decalcification using HCl 10%. Utricles from non-calcified 147


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apical segments and basal segments were excluded from the analyses as recommended by 148

Verbruggen et al. (2005d). 149

Specimens were subsequently grouped into morphotypes (groups of similar morphology) 150

according to morphological analyses of the criteria listed above. All the morphological and 151

anatomical characters were evaluated simultaneously for all the specimens to place them into 152

their corresponding lineages, according to Verbruggen et al. (2004). Within each lineage, the 153

specimens were separated into different morphotypes looking the morphological and 154

anatomical discontinuities in the arrangement and size ranges of the segments, medullar 155

filaments, utricles and nodal fusions. Finally, the morphotype boundaries often, but not 156

always correspond to species delimitation. Subsequent DNA analyses were used to confirm or 157

reject species placement within these groups. 158

159

DNA extraction and PCR 160

A total of 141 samples preserved in ethanol, ten fresh samples and 60 dried samples from the 161

herbarium were processed. Samples preserved in ethanol were air dried for 12 hours prior to 162

DNA extraction. Few segments or part of one segment (approximately 0.5 cm long) were 163

crushed in liquid nitrogen and total DNA was extracted using the DNeasy Plant minikit 164

(Qiagen) according to the manufacturer’s instructions. PCR was carried out in a total volume 165

of 25 µL containing 2 µL of DNA extract, 0.2 µM of forward and reverse primers (Table S1), 166

0.2 mM of each dNTP, 2.5 µL of reaction buffer 10X (including 1.5 mM of MgCl2) and 1.25 167

U of Taq polymerase (Jump Start Red Taq, Sigma). PCR parameters included an initial 168

denaturation phase at 94°C for 2 min, 40 (tufA) or 35 (rbcL) cycles of 1 min at 94°C, 1 min at 169

annealing temperature (see Table S1), 1 min at 72°C and a final extension of 5 min at 72°C. 170

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DNA sequencing 172


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The majority of samples was sequenced in both directions with the PCR primers by Macrogen 173

(Macrogen Inc., Seoul, Korea) using the BigDye TM terminator method. The other samples 174

were purified on column MinElute (Qiagen) and sequenced with an ABI Prism BigDye 175

Terminator v.3.1 cycle sequencing kit (Applied Biosystems) at the Plateforme du Vivant 176

(IRD, Nouméa, New Caledonia). The 20 µL reaction volume contained 0.8 µL of ready 177

reaction premix, 4 µL of BigDye sequencing buffer, 4 µM of each primer and 10 ng of 178

purified DNA. The sequencing reaction included a denaturation phase at 96°C for 1 min, 60 179

cycles of 15 sec at 98°C, 10 sec at 50°C and 4 min at 60°C. The sequencing products were 180

purified on Cephadex G50. Automated sequencing was performed on an ABI Prism 310 181

Genetic Analyzer (Applied Biosystems). 182

183

Sequence alignment and phylogenetic analyses 184

Sequences were aligned manually using the BioEdit sequence alignment editor (Hall 1999). A 185

total of 124 tufA and rbcL sequences of Halimeda were downloaded from GenBank 186

(Hanyuda et al., 2000, Verbruggen et al., 2005 a, b, c, d, 2006, 2007 and 2009b, Lam et al., 187

2006, Curtis et al., 2008,Cocquyt et al., 2009) and added to our sequence alignments. 188

Our phylogenetic analyses consisted of two parts. First, a global phylogeny of the genus was 189

generated using a concatenated dataset of rbcL and tufA. The purpose of this analysis was to 190

show the major groupings in the genus and their relationships. Second, we performed analyses 191

of only tufA sequences for the five major groupings observed in the first step. These analyses 192

were performed to yield information about species delimitation and relationships within the 193

major groupings of the genus. 194

For the first analysis, tufA and rbcL were concatenated when both were available for a 195

sample. Phylogenies were inferred with neighbour joining (NJ), maximum parsimony (MP), 196

and maximum likelihood (ML) in MEGA4 (NJ, MP; Tamura et al. 2007) or PHYML (ML; 197

Guindon and Gascuel 2003). For ML analyses, the model of nucleotide substitution was 198


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estimated using Findmodel (available at 199

http://www.hiv.lanl.gov/content/sequence/findmodel/findmodel.html). Support for nodes was 200

assessed by the bootstrap method (Felsenstein 1985) using 1,000 replicates for NJ and MP 201

analyses and 100 replicates for ML analyses. Nodes were considered as weak below 70%, 202

moderately supported between 71 and 84%, and strongly supported above 85%. A sequence 203

of Avrainvillea rawsonii was used as outgroup for the first analysis (Verbruggen et al. 2006). 204

For the lineage specific analyses of tufA, a sequence from a related lineage was used as 205

outgroup. The resulting lineage-specific haplotype trees were used to delimit species with a 206

method previously shown to yield accurate species boundaries in integrated morphological-207

molecular studies of Halimeda (e.g. Verbruggen et al 2005a,b). The method consists of 208

identifying well-supported clades that are preceded by a relatively long branch and have 209

comparably low sequence diversity within. Alignments and trees are available through 210

Treebase (http://www.treebase.org). 211

212

Results 213

214

Morphology 215

Morphological investigation of the 187 New Caledonian samples led to the recognition of 20 216

morphotypes (Table 1) into which each individual from New Caledonia could be 217

unambiguously assigned. These morphotypes, numbered A-T matched the descriptions of 218

respectively (A) H. cylindracea, (B) H. borneensis, (C) H. macroloba, (D) H. fragilis, (E) H. 219

micronesica, (F) H. lacunalis, (G) H. discoidea, (H) H. macrophysa, (I) H. magnidisca, (J) H. 220

taenicola, (K) H. gracilis, (L) H. minima, (M) H. opuntia, (N) H. distorta, (O) H. 221

heteromorpha, (P) H. kanaloana, (Q) H. xishaensis, (R) H. velasquezii, (S) H. gigas, and (T) 222

H. melanesica (Table S2). Morphological characters of the morphotypes are summarized in 223

Table 1. 224


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Morphotypes A, B, C, P could easily be distinguished from the others by the presence of 225

pores at the nodal fusion and a well-developed bulbous holdfast for anchorage in sandy 226

substrata.They could be distinguished from one another by their segment form. Morphotype A 227

showed cylindrical segments, simple or trilobed while morphotype B presented flat trilobed 228

segments and morphotype C had large reniform segments. Morphotype P presented basal 229

cylindrical segments similar to morphotype A but its segments were flat and trilobed. 230

Morphotype T also presented pores at the nodal fusion but lacked well-developed holdfasts. 231

Morphotypes D and E were distinguished by the absence of nodal fusion and by the 232

separation of the utricles after decalcification. Morphotype D had a basal segment with many 233

daughter segments in all directions (at 360° all around) whereas morphotype E showed a basal 234

“handlike” segment with numerous branching segments in its upper part (oriented about 235

180°). Both had a single holdfast. 236

Morphotypes F, G, H, I, J, Q and S featured erect thalli with a single holdfast and complete 237

nodal fusion of two or three siphons. Morphotype F could be distinguished by rounded, oval 238

or cuneate segments and utricles with a diameter less than 50 µm in surface view. 239

Morphotype G showed high polymorphism but constant and distinctive anatomical criteria 240

with inflated secondary utricles generally exceeding 100 µm in diameter. Morphotype H has a 241

thallus smaller than morphotype G and utricles visible to the bare eye and featuring a 242

characteristic honeycomb aspect before decalcification. Morphotype I had basal segments 243

forming a long and thin stipe with a well-developed bulbous holdfast. Morphotype J could be 244

distinguished from the other morphotypes by thick cuneate, cylindrical or oval segments and a 245

single rhizoidal holdfast; the anatomical studies pointed out an inflated tertiary layer of 246

utricles with a width over 110 µm. 247

Morphotype Q was very similar to morphotype G for the external morphology but 248

occasionally featured distinctive undulate segments. It had very large peripheral utricles and 249


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relatively small secondary utricles compared to morphotype G (peripheral utricle length: 80 to 250

160 µm, width: 60 to 93 µm; secondary utricles length: 100 to 200 µm, width: 60 to 135 µm). 251

Morphotype S presented flat and glossy segments, most of which were reniform or oval, to 31 252

mm long and 42 mm broad. While the external morphology is reminiscent of morphotype G, 253

it could be easily distinguished by its internal anatomy because it possessed larger peripheral 254

utricles (peripheral utricle length: 92 to 187 µm, width: 130 to 240 µm). 255

Morphotype K presented a complete nodal fusion of pairs of siphons and could be 256

distinguished by a spreading, sprawling or bushy thallus, multiple attachment points, 257

numerous ramifications and long and thin branches with trilobed and cylindrical segments. 258

Morphotypes L, M, N and R shown identical nodal structures with partial fusion of two or 259

three siphons (rarely more) for a short distance. Morphotype L had an erect or pendent 260

curtain-like thallus with a single attachment to the substrata, branching in one plane except 261

sometimes at first segments. Morphotype R was similar to morphotype L but in surface view 262

its peripheral utricles were narrower than those of morphotype L (14 to 16 µm for R Vs 14 to 263

40 µm for L). Morphotype M was characterized by a bushy and compact thallus with multiple 264

attachment points to the substrata, small reniform segments heavily calcified sometimes 265

ribbed and brittle. Morphotype N possessed a sprawling thallus with prostrate and erect 266

portions, with multiple fixation points and reniform or trilobed segments of variable size and 267

organized in perpendicular planes. It can be distinguished from morphotype M by bigger and 268

twisted segments. 269

Morphotype O had a partial nodal fusion forming a single unit with very rare pores (small and 270

not clearly visible) and 3-4 utricle layers, a polymorphous thallus, a basal part sometimes 271

forming a pseudo-stalk, holdfast with matted rhizoids adapted to hard substrata. Segments 272

were small, mostly trilobed but occasionally reniform. 273

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DNA analyses 275


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A total of 223 tufA and 52 partial rbcL sequences, including data downloaded from GenBank 276

(109 and 15 respectively), were aligned. New sequences were submitted to GenBank; 277

accessions are listed in Table S2. The tufA alignment was 857 bp, including gaps. The partial 278

rbcL sequences (first 689 bp) were obtained with the primer pair rbcL1-rbcL3 (Table S1). 279

Other primer pairs did not provide consistent results and were thus not analyzed further. NJ, 280

MP, and ML analyses produced similar tree topologies for both markers analyzed either 281

separately or in combination. A summary of the tree resulting from the ML analysis applied to 282

the concatenated (tufA + rbcL) sequence alignment is shown in Fig.2. While the backbone of 283

the tree did not receive acceptable support, five lineages representing the five sections of 284

Verbruggen and Kooistra (2004) were consistently recovered: Rhipsalis (lineage L1), 285

Micronesicae (L2), Halimeda (L3), Pseudo-opuntia (L4) and Opuntia (L5). The tufA gene, 286

for which the biggest dataset was available (see Table S2), was analyzed separately for each 287

section to get a more detailed picture of species boundaries within these lineages. 288

289

Lineage 1 (Rhipsalis) 290

Morphotypes A, B, C, O, P and T grouped within section Rhipsalis with GenBank sequences 291

available for H. heteromorpha, H. macroloba, H. kanaloana, H. incrassata, H. melanesica, H. 292

borneensis, H. cylindracea, H. monile and H. simulans (Fig. 3). The section was subdivided 293

into five moderately to strongly supported subclades (73% ≤ BP ≤100 %) denoted (i) to (v). 294

The sequences of H. borneensis grouped together (iv) but with two distinctive subgroups from 295

French Polynesia and New Caledonia. 296

Sequences of each morphotype grouped together and our identifications of these morphotypes 297

matched with the species names on the downloaded GenBank sequences. One exception is 298

specimen (IRD 5311, see arrow on Fig. 3), which we assigned to morphotype A (H. 299

cylindracea) but formed a lineage clearly separated from other species. While the sequence 300

grouped with GenBank sequences of H. monile, H. simulans and H. incrassata, the genetic 301


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distinctness of this sequence suggests that it represents a separate species. A closer 302

morphological analysis indicated similarities with H. stuposa. 303

304

Lineage 2 (Micronesicae) 305

Sequences available for morphotypes D and E grouped within section Micronesicae with 306

GenBank sequences for H. micronesica, H. fragilis, H. cryptica and H. pygmaea (Fig.4). 307

Morphotype E from the Maldives strongly grouped (BP>99%, Fig. 4) with H. micronesica. 308

Specimens initially ascribed to morphotype D (H. fragilis) appeared to be polyphyletic and 309

grouped in three well-supported subgroups (BP>99%, Fig. 4). One specimen from the 310

Chesterfield grouped with sequences for H. fragilis from Guam and the Northern Mariana 311

Islands (noted H. fragilis 1), whereas the three specimens from the Maldives formed a distinct 312

clade with other sequences for H. fragilis from Philippines, Tanzania and Maldives (noted H. 313

fragilis 2). Two other specimens formed a distinct lineage (H. fragilis 3) sister to H. pygmaea 314

(Fig. 4). 315

316

Lineage 3 (Halimeda) 317

Morphotypes G, I, Q, H, J, F and S grouped within section Halimeda, which included 318

GenBank sequences for H. discoidea, H. gigas, H. magnidisca, H. xishaensis, H. cuneata, H. 319

macrophysa, H. taenicola, H. lacunalis, H. tuna, H. hummii and H. scabra (Fig. 5). This 320

lineage was subdivided into ten moderately to strongly supported clades (78% < BP < 100 %, 321

Fig 45). Overall, the morphotype assignments of our samples were consistent with the 322

identified GenBank sequences with which the samples clustered. However, small deviations 323

did occur. Morphotype G (H. discoidea) is polyphyletic, with widely divergent clades 324

containing Indo-Pacific and Atlantic/Caribbean specimens (see also Kooistra et al 2002, 325

Verbruggen et al. 2005b, Verbruggen et al 2009b). Specimens in morphotype J (H. taenicola) 326

formed a moderately to well supported clade (82% ≤ BP ≤ 90%) but was subdivided into two 327


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divergent subgroups, one with individuals from French Polynesia and the Maldives, and the 328

second with specimens from Chesterfield. 329

330

Lineage 4 (Pseudo-opuntia) 331

Only sequences available for morphotype K (H. gracilis) grouped within the Pseudo-opuntia 332

lineage with GenBank sequences available for H. gracilis and H. lacrimosa (Fig. 6). 333

Morphotype K sequences formed a moderately supported clade (72 % ≤ BP ≤ 81%, Fig. 6) 334

with H. gracilis. As can be seen in Fig. 6, H. gracilis is a complex consisting of at least four 335

genetically distinct species (see also Verbruggen et al., 2009b). Our specimens from New 336

Caledonia and Maldives all fell in clade "H. gracilis 1", which is the clade also containing 337

material from Sri Lanka, the type locality. Interestingly, the H. gracilis 1 lineage exhibits 338

quite divergent subclades, hinting towards the possibility that additional cryptic diversity is 339

present. 340

341

Lineage 5 (Opuntia) 342

Sequences available for morphotypes L, M, N and R grouped within the Opuntia lineage with 343

GenBank sequences for H. renschii, H. minima, H. distorta , H. opuntia, H. velasquezii, H. 344

copiosa and H. goreauii (Fig. 7). As anticipated on morphological grounds, morphotype M 345

grouped with sequences of H. opuntia (75% ≤ BP ≤ 97%, Fig. 7). H. distorta appeared 346

polyphyletic with two clades noted H. distorta 1 and H. distorta 2 weakly to strongly 347

supported (BP = 94% and BP = 76% respectively). Sequences obtained for morphotype N 348

grouped within the subgroup H. distorta 2. Morphotype R grouped strongly with H. 349

velasquezii sequences (BP = 93%, Fig 6). Sequences for H. minima and H. renschii formed a 350

large clade (BP >89 %, Fig 6) including a subgroup for H. renschii (BP =78%). 351

Morphotype L was found in the H. minima / H. renschii group within two subclades: 352

sequences from the Maldives grouped moderately with H. minima 1 from Australia and 353


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Tanzania (BP = 72%) and sequences from New Caledonia grouped strongly with H. minima 4 354

from the Philippines and Fiji. Three unidentified samples from Northern Mariana Islands and 355

Fiji (H. no ID) were strongly grouped together (BP = 100%) and distinctive from other 356

species. 357

358

359

Discussion 360

Our primary aim was to characterize the species diversity of the genus Halimeda in New 361

Caledonia using a combination of molecular tools and morphological investigations. Besides 362

addressing this goal, our results have implications for our knowledge of the taxonomy and 363

global diversity of the genus and warrant reflecting upon the suitability of conventional 364

morphological identification criteria and methods for delimiting species in the genus. 365

366

Approaches towards species delimitation 367

Species delimitation is one of the principal goals of systematic biology. Classically, species 368

were delimited based on morphological differences between perceived entities. Since the 369

early 1990's, DNA sequencing has made it increasingly clear that species boundaries based on 370

perceived morphological differences have not always reflected biological reality. They have 371

shown that morphologically defined entities can encompass various genetic species (cryptic 372

diversity) and that DNA clusters at the species level can be morphologically diverse (e.g., 373

Zuccarello and West 2003; Leliaert et al., 2009). As a consequence of the morphological 374

plasticity of algae and the fact that morphological species delimitation can be deceptive, DNA 375

sequences are very useful complements to morphological data in defining species boundaries. 376

In Halimeda, various approaches have been taken towards species delimitation. All work 377

predating the DNA sequencing era has used external morphological and anatomical features 378

to define species boundaries. More recently, Verbruggen et al. (2005a, 2006) started by 379


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defining species boundaries based on sets of DNA barcodes and subsequently looked for 380

morphological characters coinciding with the DNA-based species boundaries. In this paper, 381

we tried an intermediate approach. We defined morphotypes based on analyses of our external 382

morphological and anatomical datasets and subsequently sequenced the samples and verified 383

whether the morphotypes corresponded to DNA clusters. While this was true in most cases, 384

we were unable to capture the full species diversity in our morphotypes – some of them 385

showed cryptic diversity at the DNA level. 386

The recognition of species-level clades in this study followed previous work on Halimeda, in 387

which species limits are defined manually by inspecting haplotype trees to identify well-388

supported clades that are preceded by a relatively long branch and have comparably low 389

sequence diversity within. We acknowledge that this method is to some degree subjective, in 390

that different workers may disagree on what constitutes a "well-supported clade preceded by a 391

relatively long branch and comparably low sequence diversity within". This is where the 392

taxonomist's judgment comes in, which is based on the joint interpretation of the haplotype 393

tree and the morphological information. In recent years, more objective methods have been 394

developed to pinpoint species boundaries in DNA sequence datasets (Pons et al. 2006, 395

Monaghan et al. 2009, Leliaert et al. 2009). These methods, and the general mixed Yule-396

coalescence approach in particular, correspond well to the method we have used here. It relies 397

on differences between slower above-species tree branching (speciation model) and faster 398

within-species tree branching (haplotype coalescence), which is what is more intuitively 399

assessed with the method we use. 400

Over the years, several DNA markers have been used for questions near the species level in 401

Halimeda. The early work by Kooistra used 18S nrDNA sequences (Hillis et al., 1998, 402

Kooistra et al., 1999), which turned out to be too conservative for fine-grained work and were 403

replaced with the ITS region (Kooistra et al.,2002, Verbruggen et al., 2005a, b, 2006). 404

Slightly later, the plastid marker UCP7 (Provan et al., 2004), comprising partial rps19 and 405


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rps3 sequences and the spacer between them, was introduced for species delimitation in 406

sections Rhipsalis and Halimeda (Verbruggen et al. 2005a, b, 2006, 2009b). Recently, the 407

plastid tufA gene was proposed as the DNA barcode of choice for the Ulvophyceae (Saunders 408

and Kucera 2010). This marker had already been used for phylogenetic purposes in the genus 409

Halimeda (Verbruggen et al., 2005) and for species delimitation in two of its sections 410

(Pseudo-opuntia and Opuntia: Verbruggen et al., 2009b). In the present study, the use of the 411

tufA barcode is extended to all sections of the genus. It clearly follows from our results that 412

the gene has potential as a barcoding marker as it shows enough resolution to clearly 413

distinguish between species-level clades, shows the presence of cryptic diversity within 414

morphotypes (e.g. H. fragilis), and indicates the presence of new species and/or species not 415

currently present in the DNA databases (e.g. H. cf. stuposa, H. no ID). 416

417

Morphological identification criteria 418

Among the nine morphological characters analyzed in this study, many appeared to show 419

overlap between species. For example, it can be difficult to discriminate between H. 420

discoidea, and H. xishaensis using only thallus and segment form. On the other 421

handHowever, the same two characters allow perfect discrimination between other species 422

(e.g. H. cylindracea vs. H. macroloba). Bulbous holdfasts were found to occur in sections 423

Rhipsalis (L1) (except for H. melanesica) as well as Halimeda (L3), which was interpreted by 424

Verbruggen and Kooistra (2004) to be a response to a specific adaptation to growth in sandy 425

or muddy substrata. Verbruggen et al. (2005d) noted that some deviant segments should not 426

be included in morphological analyses because they can mislead species assignment. 427

Following their conclusion that basal, apical and non-calcified segments may not be 428

representative of the average morphology of a species, these segment types were excluded 429

from the present study. It is clear that external morphology may be used for a primary 430

classification of individuals into morphological groups but this enables species identification 431


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only in rare cases. Anatomical characters appear more reliable to identify species. Features 432

like the nodal fusion type and the size and form of utricles show more variation among 433

species (Verbruggen et al., 2005a), and therefore represent the main characters for 434

morphological identification. However, these features show overlap between species, too. A 435

combination of external and anatomical features obviously extends the range of distinctions 436

that can be made and is the preferred strategy for morphological discrimination between 437

species. Even though a well-contemplated combination of external morphological and 438

anatomical characters can provide a powerful source of information for the identification of a 439

majority of Halimeda species, it is certainly possible that they will not permit distinction 440

between the most similar of cryptic species pairs. 441

In the present study, which defined morphotypes prior to DNA analyses, morphological and 442

anatomical identifications were consistent with molecular results in a majority of cases, 443

except for morphotypes A, D and L, which were present in multiple genetic clusters. A hybrid 444

identification approach based on morpho-anatomical features for more distinctive species and 445

DNA tools for the cryptic species complexes may thus be reliable, although an inherent risk 446

of such an approach is that it may leave cryptic diversity within morphologically distinctive 447

taxa undetected. 448

449

Taxonomical implications 450

As mentioned above, there was generally a clear distinction between sequence clusters at the 451

species level and 14 out of 20 morphotypes grouped unambiguously with sequences 452

previously identified to the same species: morphotype B = H. borneensis, C = H. macroloba, 453

E = H. micronesica, F = H. lacunalis, H = H. macrophysa, I = H. magnidisca, J = H. 454

taenicola, M = H. opuntia, O = H. heteromorpha, P = H. kanaloana, Q = H. xishaensis, R = 455

H. velasquezii, S = H. gigas and T= H. melanesica. Clearly, these species can be incorporated 456

unambiguously in the list of New Caledonian Halimeda species. 457


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Conversely, for 6 out of the 20 morphotypes, the situation was more complex due to cryptic 458

or pseudocryptic diversity. We distinguish between two different scenarios. The first is that 459

within New Caledonia, specimens from one morphotype belong to different cryptic entities. 460

This is the case for morphotypes A = H. cylindracea (2 entities), and D = H. fragilis (2 461

entities). The second scenario is that the morphospecies in question is known to contain 462

cryptic diversity, but the New Caledonian samples belong to only one of the constituent 463

cryptic entities. This is the case for morphotypes G = H. discoidea (2 entities), K = H. gracilis 464

(4 entities), L = H. minima (4 entities), and N = H. distorta (2 entities). We will now discuss 465

each of these six problematic cases and their taxonomic consequences in more detail. 466

467

Morphotype A, which was thought to correspond to H. cylindracea, appeared polyphyletic in 468

our phylogenetic analysis. As could be expected the majority of the samples of this 469

morphotype matched Genbank sequences belonging to H. cylindracea. However, a single 470

sequence attributed to morphotype A (IRD 5311) formed a separate lineage related to H. 471

incrassata, H. simulans and H. monile in subgroup (iii) (Fig. 3). A closer morphological 472

analysis demonstrated similarities of this specimen to H. stuposa. First and considering the 473

identification key to Halimeda species of section Rhipsalis (Verbruggen et al., 2005a), some 474

details can help us to differentiate the specimen from H. cylindracea: it has a basal zone 475

(basal segments up to the holdfast and underneath the first ramifications) of 1.6 cm long, 476

segments thicker than H. cylindracea (in average, 1.5 to 2 mm vs less than 1.5 mm for H. 477

cylindracea) and its segments are not cylindrical throughout the thallus but most of them were 478

trilobed. Secondly, it possessed segments always less than 12.5 mm wide (most in the 2-6 mm 479

range), and its peripheral utricles averaged 35.8 µm in width and 58.8 µm in length, while the 480

width of its secondary utricles equaled 28 µm in width with supranodal siphons shorter than 481

300 µm (208 µm). In fact, following Verbruggen et al. (2005a), our specimen presented all 482

the criteria to be identified as H. stuposa, but since no DNA sequence data are available for 483


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confirmed samples of H. stuposa for verification, we converged on "H. cf. stuposa" as the 484

identification for this clade. 485

486

Morphotype D (Lineage 2), identified as H. fragilis, showed clear evidence for cryptic 487

diversity. The New Caledonian samples were only present in two of these (H. fragilis 1 and 488

H. fragilis 3) but our samples from the Maldives, Tanzania and the Philippines formed a third 489

cluster (H. fragilis 2). Closer examination did not reveal obvious morphological differences 490

between the clusters. 491

The morphospecies H. discoidea (lineage 3) appeared polyphyletic with two distinct and 492

unrelated clusters. The first (H. discoidea 1) contained all sequences from Indo-Pacific 493

material, including all our New Caledonian samples belonging to morphotype G. The second 494

cluster comprised all sequences from Atlantic specimens (H. discoidea 2). The genetic 495

distinctness of Atlantic and Indo-Pacific specimens belonging to the morphospecies has been 496

discussed in detail in the literature (Kooistra et al., 1999, Verbruggen et al. 2005b, 497

Verbruggen et al. 2009b). 498

The polyphyly of H. gracilis initially noted by Kooistra et al. (2002) has later been confirmed 499

with better taxon sampling (Verbruggen et al., 2009b, this study). It is now clear that this 500

morphospecies is a complex of four species (Fig. 6), two from the Indo-Pacific region (H. 501

gracilis ip 1 & ip 2) and two from the Atlantic Ocean (H. gracilis flo & car 2). Our specimens 502

from New Caledonia and the Maldives (morphotype K) all fell within H. gracilis ip 1, which 503

includes the specimen from the type locality and can safely be considered as the true H. 504

gracilis. The other groups, which are not the focus of this paper as they do not occur in New 505

Caledonia, should be subjected to more intensive morphological investigation in an attempt to 506

identify diagnostic characters. 507

Our molecular data resolved Halimeda minima into four clusters, one of which (H. minima 4) 508

is present in New Caledonia. The polyphyly of this species was initially noted by Kooistra et 509


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al. (2002), who distinguished three distinct groups, while Verbruggen et al. (2009b) also 510

recognized the fourth cluster noted here. The new data presented here shows that while two of 511

the four clusters are clearly distinct (H. minima 2 and H. minima 4), sequences of H. minima 512

1, H. minima 3 and H. renschii are very closely related and do not always form clear-cut 513

clusters, indicating that the taxonomy of the species H. minima and H. renschii needs to be 514

reconsidered. The principal morphological criterion used to separate H. minima from H. 515

renschii is that the holdfast region of the latter consists of a region at least 1 cm high in which 516

the basal segments are covered in rhizoids and sand particles of several segments, whereas the 517

former has a simple holdfast at the basal segment (Hillis-Colinvaux, 1980). However, it is 518

known that holdfasts can show within-species variation depending on habitat (Verbruggen et 519

al. 2006). As the H. renschii – minima 1 – minima 3 complex does not appear to be present in 520

New Caledonia, we will not treat this any further, but this group needs a more critical 521

evaluation. 522

Our analyses resolved H. distorta in two groups (cf. Verbruggen et al., 2009b). While the 523

cluster noted H. distorta 1 appears to be restricted to the Pacific, H. distorta 2 grouped 524

specimens from the wider Indo-Pacific area, including our specimens from New Caledonia 525

(morphotype N). Kooistra and Verbruggen (2005) merged H. hederacea with H. distorta 526

based on the lack of separation of these taxa in ITS nrDNA sequences, and it is not clear how 527

the two clusters we find within H. distorta relate to these older names. Additional work is 528

clearly needed to sort out the taxonomy of this complex. 529

We wish to make a final taxonomic note about H. taenicola. Even though this species is 530

recovered as monophyletic in our study, it is composed of two distinct clusters one of which 531

has samples only from the Chesterfield reef area. We have not been able to find marked 532

anatomical or morphological differences between these two subgroups, although our 533

specimens from the Maldives did look bushier than specimens from Chesterfield. However 534

this character may not be valuable to discriminate the two subgroups because the analysis is 535


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based on few specimens and the general shape of the thallus may be strongly influenced by 536

the environment. 537

538

Diversity of Halimeda in New Caledonia 539

This study identified 22 species of Halimeda in New Caledonia, representing all five sections 540

of the genus. 541

Eighteen species were confirmed using a combination of DNA analyses and morphological 542

investigation, i.e. H. borneensis, H. cylindracea, H. discoidea, H. distorta, H. fragilis 1, H. 543

fragilis 3, H. gracilis, H. heteromorpha, H. kanaloana, H. lacunalis, H. macroloba, H. 544

macrophysa, H. magnidisca, H. minima, H.melanesica, H. opuntia, H. taenicola and H. 545

velasquezii. Some morpho-species were shown to harbor cryptic diversity. In one case, two 546

cryptic species were present in New Caledonia (H. fragilis 1 and H. fragilis 3). In four other 547

cases, the species in question was shown to contain several cryptic species but only one of 548

these cryptic species was present in New Caledonia. This was the case for H. discoidea (only 549

the Indo-Pacific entity in New Caledonia), H. gracilis (only entity #1 in New Caledonia), H. 550

minima (only entity #4 in New Caledonia) and H. distorta (only entity #2 in New Caledonia). 551

Four Three other species were identified based on morphological criteria only: H. 552

micronesica, H. xishaensis and H. gigas. Regrettably, we were unable to generate DNA data 553

for these species. However, because these species represent clear-cut morphotypes that have 554

been shown to form distinct DNA clusters (Verbruggen et al., 2005b, 2005c; this study) with 555

no reports of cryptic diversity, a high degree of confidence can be attached to these records. 556

We also identified a DNA cluster (H. cf stuposa) that forms a distinct lineage in molecular 557

analyses and thus represents a proper species. While the specimen in question bears 558

resemblance to H. stuposa, this identification is not certain as the morphology does not match 559

exactly and no sequences of typical H. stuposa samples are available. 560


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The report of the species H. kanaloana for New Caledonia represents a major extension of the 561

known distribution range of this species, as this species was previously known only from 562

Hawaii and possibly Japan (Ryu Kyu Islands). The New Caledonian specimens had 563

previously been identified as H. incrassata (Garrigue 1985), which is morphologically close 564

to H. kanaloana in various aspects, but can be distinguished on the basis of six features 565

including the dimensions of subperipheral and peripheral utricles, and the distance between 566

the base of the nodal fusions and the first siphon ramification above the node (Verbruggen et 567

al., 2006). They also differ in their distribution: H. incrassata is restricted to the Atlantic 568

Ocean while H. kanaloana is a Pacific Ocean species (Verbruggen et al., 2006, this study). 569

Similarly, the recognition of H. xishaensis in New Caledonia marks a major extension of the 570

known range of this species, because, to our knowledge, it was previously only recorded from 571

the Xi Sha or Paracel Islands in the South China Sea. Finally, we obtained here the first 572

sequence of H. melanesica from its type locality (Lifou, New Caledonia), which will facilitate 573

accurate molecular identification of specimens of this species from other parts of the world. 574

575

Acknowledgments 576

We acknowledge the IRD divers team and boat pilots for their contribution to this work 577

through kind and careful assistance. Thanks also to all collectors who have contributed to the 578

study: G. Lasne (BIOCENOSE), E. Fontan, O. Cornubert and D. Ponton. Thanks to C. 579

Fauvelot for providing samples from French Polynesia and Antoine de N’Yeurt for help with 580

identification of specimens collected in Moorea. Thanks to Coppejans E., Dargent O., 581

Tyberghein L., Leliaert F., Diaz R., Galanza C., Huisman J., De Clerck O., Schils T., Payo 582

D.A. and Pauly K. for collecting Ghent samples. We sincerely thank C. Majorel (Plateforme 583

du vivant) and S. D'hondt (Ghent University) for assisting with the molecular work. HV is a 584

postdoctoral fellow of the Research Foundation – Flanders. LD is a PhD fellow of the 585

Province Sud- New Caledonia. 586


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587

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Agardh, J. G. 1887. Till algernes systematik. Nya bidrag. (Femte afdelningen.). Acta 589

Universitatis Lundensis. 23(2): 1-174, 5 plates. 590

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Andréfouët, S., Torres-Pulliza, D., Dosdane, M. (collab.), Kranenburg, C. (collab.), Murch, B. 592

(collab.), Muller-Karger, F. E. (collab.), Robinson, J. A. (collab.) 2004. Atlas des récifs 593

coralliens de Nouvelle Calédonie. Nouméa (NCL) ; Nouméa : IRD ; IFRECOR, 26 p. + 22 p. 594

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Andréfouët, S., Cabioch, G., Flamand, B., Pelletier, B. 2009. A reappraisal of the diversity of 596

geomorphological and genetic processes of New Caledonian coral reefs: a synthesis from 597

optical remote sensing, coring and acoustic multibeam observations. Coral Reefs. 28:691-707. 598

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Moebius, O. Nordstedt bearbeitet. In: Forschungsreise S.M.S. "Gazelle". Theil 4: Bot. 601

(Engler, A. Eds), Berlin, pp. 1-58. 602

603

Barton, E. S. 1901. The genus Halimeda. Siboga-Expeditie Monographe LX of: Uitkomsten 604

op Zoologisch, Botanisch, Oceanographisch en Geologisch Gebied verzameld in 605

Nederlandsch Oost-Indie 1899-1900 60: 1-32, 2 figs, 4 pls. 606

Chevillon, C. 1996. Skeletal composition of modern lagoon sediments in New Caledonia: 607

coral, a minor constituent. Coral Reefs. 15:199-207. 608

Cocquyt, E., Verbruggen, H., Leliaert, F., Zechman, F. W., Sabbe, K. & De Clerck, O. 2009. 609

Gain and loss of elongation factor genes in green algae. BMC Evol. Biol. 9:39. 610


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Curtis, N., Dawes, C. & Pierce, S. 2008. Phylogenetic analysis of the large subunit Rubisco 611

gene supports the exclusion of Avrainvillea and Cladocephalus from the Udoteaceae 612

(Bryopsidales, Chlorophyta). J. Phycol. 44:761-67. 613

Dong, M. L., Tseng, C. K. 1980. Studies on the Chlorophyceae from Xi Sha Islands, China. 614

Studia Marina Sinica 17:1-10. 615

Famà, P., Wysor, B., Kooistra, W. H. C. F. & Zuccarello, G. C. 2002. Molecular phylogeny of 616

the genus Caulerpa (Caulerpales, Chlorophyta) inferred from chloroplast tufA gene. J. 617

Phycol. 38:1040-50. 618

Felsenstein, J. 1985. Confidence limits on phylogenies: An approach using the Bootstrap. 619

Evolution 39:783-91. 620

Garrigue, C., & Tsuda, R. O. Y. T. 1988. Catalog of Marine Benthic Algae from New 621

Caledonia. Micronesica 21:53-70. 622

Garrigue, C. 1995. Macrophyte associations on the soft bottoms of the south-west lagoon of 623

New Caledonia: Description, structure and biomass. Bot. Mar. 38:481–92. 624

Guindon, S. & Gascuel, O. 2003. A simple, fast, and accurate algorithm to estimate large 625

phylogenies by maximum likelihood. Syst. Biol. 52:696-704. 626

Guiry, M. D. & Guiry, G. M. 2011. AlgaeBase. World-wide electronic publication, National 627

University of Ireland, Galway. http://www.algaebase.org; searched on 23 August 2011. 628

Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis 629

program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41:95-98. 630

Hanyuda, T., Arai, S. & Ueda, K. 2000. Variability in the rbcL introns of Caulerpalean algae 631

(Chlorophyta, Ulvophyceae). J. Plant Res. 113:403-413. 632


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Use the "Insert Citation" button to add citations to this document. 633

Zuccarello, G.C. & West, J.A., 2003. Multiple cryptic species: molecular diversity and 634

reproductive isolation in the Bostrychia radicans/ B. moritziana complex (Rhodomelaceae, 635

Rhodophyta) with focus on North American isolates. J. Phycol., 39(5):948-959. 636

637

638

639

Figure legends: 640

641

642

Figure 1. Geographic position of New Caledonia in the South West Pacific (A) and map of the 643

New Caledonian islands and reefs (B) 644

645

Figure 2. Overall structure of the ML phylogenetic tree obtained for the genus Halimeda 646

based on concatened partial tufA + rbcL sequences. Bootstrap proportion values (only 647

BP>70%) are indicated for ML/NJ/MP analyses. Triangles represents sequences grouping per 648

clade. Outgroup: Avrainvillea lacerata (FJ432651) 649

650

Figure 3. NJ analysis for section Rhipsalis (L1) based on tufA sequence alignment. Bootstrap 651

values (BP>70%) are indicated for NJ/MP/ML analyses. Outgroup: Halimeda micronesica. 652

Localities are noted as follows, FP: French Polynesia, Phi: Philippines, NC: New Caledonia, 653

Tanz: Tanzania, H: Hawaii, Ye: Yemen, Pan: Panama, Jam: Jamaica, Bah: Bahamas and Car: 654

Caribbean. Asterisks indicate type localities and the arrow points to an unexpected grouping 655

and boxes highlight New Caledonian samples 656

657


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Figure 4. NJ analysis of section Micronesicae (L2) based on tufA sequence alignment. 658

Bootstrap values (BP>70%) are indicated for NJ/MP/ML analyses. Outgroup: Halimeda 659

distorta. Localities are noted as follows, Mal: Maldives; Phi: Philippines; Tanz: Tanzania; 660

NC: Grande terre, New Caledonia; Chest-NC: Chesterfield, New Caledonia; Jam: Jamaica; 661

NM: North Marianna; Aust: Australia; Wa: Wallis and Cha: Chagos. Asterisks indicate type 662

localities, arrows point at unexpected groupings and boxes highlight New Caledonian 663

samples. 664

665

Figure 5. MP analysis of section Halimeda (L3) based on tufA sequence alignment. Bootstrap 666

values (BP>70%) are indicated for MP/ NJ/ML analyses. Outgroup: Halimeda incrassata. 667

Localities are noted as followed, Nic : Nicaragua; FP: French Polynesia; Mal: Maldives; 668

Chest-NC: Chesterfield; New Caledonia; NC: New Caledonia; Ye: Yemen; SrL: Sri-Lanka; 669

Aust W: Western Australia; Sth Af: South Africa; Tanz: Tanzania; Bre: Brasil; It: Italia; Can: 670

Canaries; Jam: Jamaica; Pan: Panama; Mex: Mexico; Bah: Bahamas; Thai: Thailand; Phi : 671

Philippines; CR: Costa Rica. Asterisks indicate type localities and boxes highlight New 672

Caledonian samples. 673

674

Figure 6. NJ analysis of section Pseudo-opuntia (L4) based on tufA sequence alignments. 675

Bootstrap values (BP>70%) are indicated for NJ/MP/ML analyses. Outgroup: Halimeda 676

macrophysa. Localities are noted as follows, Mal: Maldives; SrL: Sri Lanka; Ha: Hawaii; 677

Chest-NC: Chesterfield, New Caledonia; PF: French Polynesia; Aust W: West Australia; Phi: 678

Philippines; Sth Af: South Africa;Tanz: Tanzania; Flo: Florida; Jam: Jamaica; Bah: Bahamas. 679

Asterisks indicate type localities and the box highlight New Caledonian sample. 680

681

Figure 7. ML analysis of section Opuntia (L5) based on tufA sequence alignments. Bootstrap 682

values (BP>70%) are indicated for ML/ NJ/MP analyses. Outgroup: Halimeda micronesica. 683


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Localities are noted as followed, FP: French Polynesia, Aust: Australia, Phi: Philippines, 684

Tanz: Tanzania, C. Isl: Cook Islands, Mal: Maldives, Soc: Socotra, NC: New Caledonia, Wa: 685

Wallis, Micro: Micronesia, Pal: Palau, NMI: North Marianna Island, Jam: Jamaica, Pan: 686

Panama. Asterisks indicate type localities and boxes highlight New Caledonian samples. 687

. 688

689


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690


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Table 1. Summary of the morphology for morphotypes recognized in this study. Abbreviations: L = length, W = width. Digits following these

abbreviations indicate: 1 = primary utricle, 2 = secondary utricle.

Holdfast

type

General

shape

Basal region Segments of top

half of thallus

Nodal

siphons

Number

of

utricles

layers

Cortical utricles

dimensions

Utricles after

decalcification

Utricles

form in

surface

view

Number of

specimens

studied

Identification

A bulbous erect pseudo-stipe of

barrel-shaped

segments with a

single

ramification

cylindrical or

trilobed; L: 1-9

mm; W: 1-8 mm

single

unit with

pores

3-5 L1 = 20-80 µm;

W1 = 10-36

µm; L2 = 10-70

µm; W2 = 10-

44 µm

detached rounded or

polygonal

7 H.

cylindracea

B bulbous erect well developed,

broad fan-shaped

segment bearing

multiple daughter

segments

flat, trilobed to

discoid; L: 4-11

mm; W: 4-14 mm

single

unit with

pores

3 or

more

L1 = 40-80 µm;

W1 = 20-48

µm; L2 = 28-88

µm; W2 = 24-

48 µm

attached polygonal 10 H. borneensis



31

C bulbous erect broad pseudo-

stipe with a

single

ramification

flat, discoid to

reniform; L: 8-20

mm; W: 14-30

mm

single

unit with

pores

3-4 L1 = 56-120

µm; W1 = 34-

60µm; L2 = 60-

93 µm; W2 =

34-54 µm

attached rounded or

polygonal

10 H. macroloba

D single

tight

rhizoidal

mat

compact,

bushy

funnel-shaped

basal segment

bearing many

daughter

segments

flat, reniform; L:

3-8 mm; W: 5-13

mm

siphons

go

through

node

unfused

2-3 L1 = 40-88 µm;

W1 = 20-40

µm; L2 = 14-50

µm; W2 = 12-

32 µm

detached rounded 6 H. fragilis

E single

tight

rhizoidal

mat

bushy; dense

branching

near base

fan-shaped basal

segment bearing

many daughter

segments

flat, reniform to

wedge-shaped and

trilobed; L: 3-8

mm; W: 5-14 mm

siphons

go

through

node

unfused

2-3 L1 = 20-88 µm;

W1 = 16-44

µm; L2 = 12-

100 µm; W2 =

8-40 µm

detached but

remaining in

groups

rounded 7 H.

micronesica



32

F single

tight

rhizoidal

mat

erect broad, barrel-

shaped segments

flat, rounded-oval,

some cuneate; L:

3-9 mm; W: 3-12

mm

complete

fusion of

2-3

siphons

3 L1 = 48-120

µm; W1 = 10-

48 µm; L2 =

20-88 µm; W2

= 20-32 µm

attached polygonal 6 H. lacunalis

G single

tight

rhizoidal

mat or

bulbous

erect, very

polymorphic

variable, often

broad and small

basal segment

flat, highly

polymorphic

(discoid, oval,

reniform, rarely

cylindrical to

wedge-shaped); L:

up to 29 mm; W:

up to 33 mm

complete

fusion of

2-3

siphons

2, rarely

3

L1 = 49-160

µm; W1 = 16-

48 µm; L2 =

80-240 µm; W2

= 72-176 µm

attached polygonal 11 H. discoidea



33

H single

tight

rhizoidal

mat

small, erect single small

basal segment

flat, discoid to

reniform; L:

15mm; W: 24 mm

complete

fusion of

2-3

siphons

2 L1 = 80-120

µm; W1 = 40

µm; L2 = 96-

160 µm; W2 =

56-80 µm

attached polygonal 2 H.

macrophysa

I bulbous erect basal segments

often long and

thin, stalk-like

flat, broad,

discoid; L: 22-25

mm; W: up to 22-

26 mm

complete

fusion of

2-3

siphons

2 L1 = 100-150

µm; W1 = 30-

65 µm

attached polygonal 1 H. magnidisca

J single

tight

rhizoidal

mat

erect basal segments

often broad and

square, forming a

stipe

thick flattened,

broad cuneate to

oval; L: 2-16 mm;

W: 2-19 mm

complete

fusion of

2-3

siphons

3 L1 = 40-88 µm;

W1 = 24-48

µm; L2 = 68-

184 µm; W2 =

28-100µm

attached polygonal 10 H. taenicola



34

K multiple

dispersed

rhizoidal

holdfasts

sprawling,

sparse

ramifications

segments

somewhat

flattened with 4

or more daughter

segments

flat, trilobed,

broad ovoid to

narrow cuneate; L:

2-6 mm; W: 1-6

mm

complete

fusion of

2 (rarely

3) siphons

2 L1 = 40-88 µm;

W1 = 20-32

µm; L2 = 60-

300 µm; W2 =

24-60 µm

attached polygonal,

sometimes

rounded

7 H. gracilis

L single

tight

rhizoidal

mat

erect or

pendent,

curtain like;

branching in

single plane

basal segment

trilobed,

generally bearing

3 daughter

segments

flat, reniform or

oval; L: 3-4 mm;

W: 4-7 mm

short

fusion of

2 or

rarely 3

siphons

1-2 L1 = 20-40 µm;

W1 = 14-40

µm; L2 = 24-96

µm; W2 = 12-

48 µm

attached polygonal 7 H. minima

M multiple

dispersed

rhizoidal

holdfasts

sprawling,

forming

compact,

bushy mats

basal segment

could not be

identified

flat, reniform,

sometimes ribbed;

L: 3-7 mm; W: 4-8

mm

short

fusion of

2 or

rarely 3

siphons

1-3 L1 = 12-16 µm;

W1: 15-19 µm;

L2 = 12-37 µm;

W2 = 9-12 µm

attached polygonal,

sometimes

rounded

3 H.opuntia



35

N multiple

dispersed

rhizoidal

holdfasts

sprawling

mats

basal segment

could not be

identified

flat, reniform to

trilobed, often

keeled; L = 4-8

mm; W = 6-20

mm

short

fusion of

2-5

siphons

3 L1 = 30-53 µm;

W1 = 21-37

µm; L2 = 19-

109 µm; W2 =

16-31 µm

attached polygonal 8 H. dist

O single

tight

rhizoidal

mat

erect, bushy small and broad

segments,

sometimes

forming stipe

flat, trilobed to

reniform; L = 3-8

mm; W = 1-10

mm

single

unit or

groups

without

pores

2-3 L1 = 31-94 µm;

W1 = 22-47

µm; L2 = 47-

125 µm; W2 =

25-40 µm

attached polygonal 4 H.

heteromorpha

P bulbous erect barrel-shaped

segments

forming stipe

flat, broad trilobed

to flabellate; L:

12mm; W: 15-20

mm

single

unit with

pores

3 L1 = 67-113

µm; W1= 47-73

µm; L2 = 47-87

µm; W2 = 33-

66 µm

attached polygonal 2 H. kanaloana



36

Q single

tight

rhizoidal

mat

erect subcylindrical

basal segment

flat but often

undulate, discoid

to reniform; L: 4-

20 mm; W: 5-32

mm

complete

fusion of

2-3

siphons

1-2 L1 = 80-160

µm; W1 = 60-

93 µm; L2 =

100-200 µm;

W2 = 60-135

µm

attached polygonal 7 H. xishaensis

R single

tight

rhizoidal

mat

erect or

pendent

subterete or

compressed basal

segment

flat, broad oval to

reniform; L: 2-7

mm; W: 3-10 mm

short

fusion of

2-3

siphons

2 L1 = 24-30 µm;

W1 = 14-16

µm; L2 = 16-60

µm; W2 = 16-

20 µm

adhering

slightly or

separated

rounded or

polygonal

2 H. velasquezii

S single

tight

rhizoidal

mat

erect or

pendent

single small

basal segment

flat, discoid to

reniform; L: 25-31

mm; W: 40-42

mm

complete

fusion of

2-3

siphons

2, rarely

3

L1 = 130-240

µm; W1 = 96-

150 µm; L2 =

117 µm; W2 =

attached polygonal 2 H. gigas



37

128 µm

T single

tight

rhizoidal

mat

erect multiple

segments larger

and broader than

the upper ones

flat, cuneate,

trilobed;

L: 3-5 mm ; W: 4-

5 mm

single

unit with

small

pores

3 L1 = 40-70 µm;

W1 = 40-56

µm; L2 = 44-92

µm; W2=32-

52µm

attached polygonal 3 H. melanesica


Submitted M

anuscript

Geographic position of New Caledonia in the South West Pacific (A) and map of the New Caledonian islands and reefs (B)

297x420mm (300 x 300 DPI)


Submitted M

anuscript

Overall structure of the ML phylogenetic tree obtained for the genus Halimeda based on concatened partial tufA + rbcL sequences. Bootstrap proportion values (only BP>70%) are indicated for ML/NJ/MP analyses.

Triangles represents sequences grouping per clade. Outgroup: Avrainvillea lacerata (FJ432651)

63x47mm (300 x 300 DPI)


Submitted M

anuscript

NJ analysis for section Rhipsalis (L1) based on tufA sequence alignment. Bootstrap values (BP>70%) are indicated for NJ/MP/ML analyses. Outgroup: Halimeda micronesica. Localities are noted as follows, FP:

French Polynesia, Phi: Philippines, NC: New Caledonia, Tanz: Tanzania, H: Hawaii, Ye: Yemen, Pan: Panama, Jam: Jamaica, Bah: Bahamas and Car: Caribbean. Asterisks indicate type localities and the arrow points to

an unexpected grouping and boxes highlight New Caledonian samples 215x176mm (300 x 300 DPI)


Submitted M

anuscript

NJ analysis of section Micronesicae (L2) based on tufA sequence alignment. Bootstrap values (BP>70%) are indicated for NJ/MP/ML analyses. Outgroup: Halimeda distorta. Localities are noted as follows, Mal:

Maldives; Phi: Philippines; Tanz: Tanzania; NC: Grande terre, New Caledonia; Chest-NC: Chesterfield, New Caledonia; Jam: Jamaica; NM: North Marianna; Aust: Australia; Wa: Wallis and Cha: Chagos. Asterisks

indicate type localities, arrows point at unexpected groupings and boxes highlight New Caledonian samples. 204x151mm (300 x 300 DPI)


Submitted M

anuscript

MP analysis of section Halimeda (L3) based on tufA sequence alignment. Bootstrap values (BP>70%) are indicated for MP/ NJ/ML analyses. Outgroup: Halimeda incrassata. Localities are noted as followed, Nic : Nicaragua; FP: French Polynesia; Mal: Maldives; Chest-NC: Chesterfield; New Caledonia; NC: New

Caledonia; Ye: Yemen; SrL: Sri-Lanka; Aust W: Western Australia; Sth Af: South Africa; Tanz: Tanzania; Bre: Brasil; It: Italia; Can: Canaries; Jam: Jamaica; Pan: Panama; Mex: Mexico; Bah: Bahamas; Thai: Thailand; Phi : Philippines; CR: Costa Rica. Asterisks indicate type localities and boxes highlight New

Caledonian samples. 125x289mm (300 x 300 DPI)


Submitted M

anuscript

NJ analysis of section Pseudo-opuntia (L4) based on tufA sequence alignments. Bootstrap values (BP>70%) are indicated for NJ/MP/ML analyses. Outgroup: Halimeda macrophysa. Localities are noted as follows, Mal: Maldives; SrL: Sri Lanka; Ha: Hawaii; Chest-NC: Chesterfield, New Caledonia; PF: French Polynesia; Aust W: West Australia; Phi: Philippines; Sth Af: South Africa;Tanz: Tanzania; Flo: Florida; Jam: Jamaica; Bah:

Bahamas. Asterisks indicate type localities and the box highlight New Caledonian sample.

189x130mm (300 x 300 DPI)


Submitted M

anuscript

ML analysis of section Opuntia (L5) based on tufA sequence alignments. Bootstrap values (BP>70%) are indicated for ML/ NJ/MP analyses. Outgroup: Halimeda micronesica. Localities are noted as followed, FP:

French Polynesia, Aust: Australia, Phi: Philippines, Tanz: Tanzania, C. Isl: Cook Islands, Mal: Maldives, Soc:

Socotra, NC: New Caledonia, Wa: Wallis, Micro: Micronesia, Pal: Palau, NMI: North Marianna Island, Jam: Jamaica, Pan: Panama. Asterisks indicate type localities and boxes highlight New Caledonian samples.

.

194x289mm (300 x 300 DPI)



Table S1. Oligonucleotide primers used for amplification and sequencing with detail of sequences, annealing temperature and previous

publication.

Primer name Sequence T (°C) source

tufA F : 5’ - TGAAACAGAAMAWCGTCATTATGC-3’

R : 5’ -CCTTCNCGAATMGCRAAWCGC-3’

52 Fama et al. 2002

RbcL binding site

rbcL1 : bp 7–26

rbcL2 : bp 6–29

rbcL3 : bp 689–667

rbcL 4 :bp 905–886

rbcL5 : bp 427–449

rbcL6 : bp 1396–1372

F : 5’-CCAMAAACWGAAACWAAAGC-3’

F : 5’-TCCAAAAACTGAAACTAAAGCAGG-3’

R : 5’-GCTTGWGMTTTRTARATWGCTTC-3’

R : 5’-TCAATAACCGCATGCATTGC-3’

F : 5’-GCTTATGCWAAAACATTYCAAGG-3’

R : 5’-AATTTCTTTCCAAACTTCACAAGC-3’

49

57

49

49

56

56

Hanyuda et al. (2000)




Lam et al.,(2006)

Lam et al. (2006)

UCP7 F : 5’-ATWTCDGCDCCATTWAGDCKVCC-3’

R : 5’-ATGGTWGGWCAWAAATTDGGTGAGTTT-3’

56 Provan et al. (2004)


Submitted M

anuscript

Table S2: List of Halimeda specimens and sequences used in this study.* represented samples

collected by G. Lasne, Biocenose. These are from biological points of environmental survey

of Vale New Caledonia, © Biocénose SARL.

Species

Specimen

number Geographical location GenBank Source

TufA rbcL

H. borneensis

W.R. Taylor

HV183b Tahiti, French

Polynesia (CP) AM049955

Verbruggen

et al 2006

FJ624514

Verbruggen

et al 2009b

CP08 1081

Moorea, French

Polynesia (CP) JN644647 this study

CP08 1157

Moorea, French


CP08 886

Moorea, French


CP08 908

Moorea, French


IRD 5309

Grande Terre, New

Caledonia (WP) JN644651 JN644729 this study

IRD 5310

Grande Terre, New

Caledonia (WP) JN644730 this study

H. copiosa

Goreau &

E.A. Graham

H.0330 Discovery Bay,

Jamaica (CAR) EF667065

Verbruggen

et al.2007

FJ624508

Verbruggen

et al 2009b

HV460

Saint Ann, Saint Ann's

Bay, Lee Reef, Jamaica

(CAR) FJ624647

Verbruggen

et al. 2009b

H. cryptica

Colinvaux &

E.A. Graham H.0237

Discovery Bay,

Monitor Reef, Jamaica

(CAR) EF667056

Verbruggen

et al 2007b

HV958

Saint Ann, Priory, The

Wall, Jamaica (CAR)

HV

unpublished

HV483 St. Ann Parish, Priory,

Jamaica (CAR) EF667057

Verbruggen

et al 2007

FJ624496

Verbruggen

et al 2009b

H. cuneata

Hering H.0500 Brazil (WA) AY826358

Verbruggen

et al 2005

HEC 15286

Dickwella, Sri Lanka

(CI) FJ624537

Verbruggen

et al. 2009b

SOC252 Socotra, Yemen (WI) AY826356

Verbruggen

et al 2005

KZN2K4-21 South Africa (WI) AY826353

Verbruggen

et al 2005

KZNb2352 South Africa (WI) AY826354

Verbruggen

et al 2005

WA182 Western Australia, AY826355 Verbruggen


Submitted M

anuscript

Australia (EI) et al 2005

JH12

Western Australia,

Australia (EI) FJ624538

Verbruggen

et al. 2009b

H.0550

Western Australia,

Rottnest Island,


Verbruggen

et al 2009b

H.0502

Espirito Santo State,

Manguinhos, Praia,

Brazil (WA) FJ624518

Verbruggen

et al 2009b

KZN-b 2263

KwaZulu-Natal,

Sodwana, Jesser Point,

South Africa (WI) FJ624533

Verbruggen

et al 2009b

SOC 348 Socotra, Yemen (WI) FJ624532

Verbruggen

et al 2009b

HV560

Cebu, Daanbantayan,

Malapascua,

Philippines (WP) FJ624531

Verbruggen

et al 2009b

HV823

Luzon, Sorsogon,

Bulusan, Dancalan,


Verbruggen

et al 2009b

H.0195-2

KwaZulu-Natal,

Scottsburgh, South

Africa (WI) FJ624527

Verbruggen

et al 2009b

H.

cylindracea

Decaisne

SOC364 Socotra, Yemen (WI)

AM049956

Verbruggen

et al 2006

FJ624510

Verbruggen

et al 2009b

IRD 5325

Grande Terre, New


IRD 5326

Grande Terre, New


H. discoidea

1 Decaisne IRD 5349*

Grande Terre, New


IRD 5320

Chesterfield, New


CP08 413

Moorea, French


CP08 454

Moorea, French


IRD 5303

Grande Terre, New


IRD 5305

Grande Terre, New


IRD 5306

Grande Terre, New


IRD 5307

Grande Terre, New


G.678

Bahía Culebra: Bahía

Virador, Costa Rica

(EP)

HV

unpublished


Submitted M

anuscript

G.677

Bahía Culebra: Punta

Arenita, Costa Rica

(EP)

HV

unpublished

G.672

Bajo Tres Hermanas,

Costa Rica (EP)

HV

unpublished

ODC1745

Basco, Philippines

(WP)

HV

unpublished

ODC1525

Diani Beach, Kenya

(WI)

HV

unpublished

G.673

Isla Ballena, Costa

Rica (EP)

HV

unpublished

G.195

Kepulauan Seribu, Air,

Indonesia (WP)

HV

unpublished

G.679

León: Playa El

Tránsito, Nicaragua

(EP)

HV

unpublished

G.687

León: Playa Hermosa,

Nicaragua (EP)

HV

unpublished

HV585

Logon Bay,

Malapascua Island,

Philippines (WP)

HV

unpublished

G.686

Managua: Masachapa,

Nicaragua (EP)

HV

unpublished

G.510

Molokai, Penguin

Bank, Hawaii, USA

(CP)

HV

unpublished

G.683

Nicaragua: El Hueco,

Nicaragua (EP)

HV

unpublished

OMII118 Oman (WI) AY826359

Verbruggen

et al 2005

HV58

Paea,Tahiti, French

Polynesia (CP)

HV

unpublished

G.684

Rivas: Guacalito,

Nicaragua (EP)

HV

unpublished

G.681

Rivas: La Peña Rota,

Nicaragua (EP)

HV

unpublished

G.676

San Josecito, Costa

Rica (EP)

HV

unpublished

SOC299 Socotra Yemen (WI) AY826360

Verbruggen

et al 2006

G.644 Thailand (WP)

HV

unpublished

H. discoidea

2 Decaisne H.0207

Gran Canaria, Spain

(EA) AY826361

Verbruggen

et al 2005

HV 495 Jamaica (CAR)

HV

unpublished

HV396 Jamaica (CAR) AY826362

Verbruggen

et al 2005

H.discoidea

Decaisne DML61724

Barrier Reef, Belize

(CAR) FJ624519

Verbruggen

et al 2009b


Submitted M

anuscript

HV431

Saint Ann, Priory Bay,

Jamaica (CAR) FJ624520

Verbruggen

et al 2009b

AB038488

Hanyuda et

al 2000

H. distorta 1

(Yamada)

Hillis-

Colinvaux

WLS422.02

Wallis Island, Wallis

and Futuna (WP) FJ624644

Verbruggen

et al. 2009b

LL0378 Fiji (WP)

HV

unpublished

HV767

Luzon: Sorsogon,

Bulusan, Dapdap,


Verbruggen

et al. 2009b

HV275

Rangiroa Atoll, Tiputa,

French Polynesia (CP) FJ624709 FJ624505

Verbruggen

et al. 2009b

HV263

Rangiroa, Avatoru,

French Polynesia (CP) FJ624643

Verbruggen

et al. 2009b

H. distorta 2

(Yamada)

Hillis-

Colinvaux

H.0507

Cebu, Mactan Island,

Lapu Lapu, Philippines

(WP) FJ624653

Verbruggen

et al. 2009b

TZ0118

Kunduchi, Tanzania

(WI) FJ624658

Verbruggen

et al. 2009b

HV600

Malapascua,


Verbruggen

et al. 2009b

HV572

Malapascua, Shark

Point, Philippines

(WP) FJ624649

Verbruggen

et al. 2009b

HV1817 Maldives (CI)

HV

unpublished

W0135 Micronesia (WP)

HV

unpublished

W0165 Micronesia (WP)

HV

unpublished

HV127

Moorea, Entre deux

baies, French Polynesia

(CP) FJ624655

Verbruggen

et al. 2009b

W0028 Palau (WP)

HV

unpublished

W0069 Palau (WP)

HV

unpublished

HV199

Tahiti, Faaa, French

Polynesia (CP) FJ624656

Verbruggen

et al. 2009b

H.0287

Tahiti, French


Verbruggen

et al. 2009b

HV188

Tahiti, Punaauia,


Verbruggen

et al. 2009b

WLS391.02


and Futuna (WP)

HV

unpublished

CP08 450

Moorea, French


CP08 910

Moorea, French



Submitted M

anuscript

IRD 4926 Maldives (CI) JN644664 this study


IRD 5350*

Grande Terre, New



H.0280 New Caledonia (WP) FJ624652

Verbruggen

et al. 2009b

H.0475

Queensland, Australie

(WP) FJ624648

H. fragilis 1

W.R. Taylor

H.0125 Bile Bay, Guam (WP)

EF667058

Verbruggen

et al 2007

FJ624498

Verbruggen

et al 2009b

G.014

Tinian, Northern

Mariana Islands (WP) FJ624640

Verbruggen

et al. 2009b

IRD 5318

Chesterfield, New


H. fragilis 2

W.R. Taylor HV 1816 Maldives (CI)

HV

unpublished

HV1828 Maldives (CI)

HV

unpublished

TZ0233

Mtwara, Mnazi Bay,

Ruvula, Tanzania (WI) FJ624642

Verbruggen

et al. 2009b

HV766

Palawan, Busuanga,

Tangat, Philippines

(WP) FJ624641

Verbruggen

et al. 2009b

ODC1772 Philippines (WP)

HV

unpublished




H. fragilis 3

W.R. Taylor IRD 5319

Chesterfield, New

Caledonia JN644672 JN644739 this study

IRD 5300

Grande Terre, New


H. gigas

W.R. Taylor HV1820

Dhaaparuh,Thiladhun

mathi, Maldives (CI)

HV

unpublished

HEC12910 Guam (WP) AY826364

Verbruggen

et al 2005

G.752

James Price

Point,Western

Australia, Australia

(EI)

HV

unpublished

HV769

Luzon, Sorsogon,

Bulusan, Dapdap,

Philippines (WP)

HV

unpublished

TZ0323

Mikindani, Tanzania

(WI) FJ624539

Verbruggen

et al. 2009b



Submitted M

anuscript

H. goreauii

W.R. Taylor H.0258

Galeta Island, Panama

(CAR) FJ624659 FJ624509

Verbruggen

et al. 2009b

HV360


Bay, Drax Hall,

Jamaica (CAR) FJ624661

Verbruggen

et al. 2009b

H. gracilis ip

1 Harvey ex

J.Agardh

HV317 Avatoru, Rangiroa,

French Polynesia (CP) AM049965

Verbruggen

et al 2006

FJ624492

Verbruggen

et al 2009b

ODC 894

Oahu, Lanikai, Hawai,

USA (CP) FJ624723

Verbruggen

et al. 2009b

HV312

Rangiroa, Avatoru,


Verbruggen

et al. 2009b

HEC 15289

Weligama, Surfers

Point, Sri Lanka (CI) FJ624724

Verbruggen

et al. 2009b

IRD 5312

Chesterfield, New







H. gracilis ip

2 Harvey ex

J.Agardh

KZN-b 2280

KwaZulu-Natal,

Sodwana, Wright,

Canyon, South Africa

(WI) FJ624726

Verbruggen

et al. 2009b

HV824

Luzon, Sorsogon,

Bulusan, Dancalan,

Philippines (WP) FJ624727 FJ624493

Verbruggen

et al. 2009b

TZ0001

Mbudya Island,

Tanzanie (WI) FJ624729

Verbruggen

et al. 2009b

H.0861

Western Australia,


Verbruggen

et al. 2009b

H. gracilis flo

Harvey ex

J.Agardh DML59047

Palm Beach, Florida

(CAR) FJ624731 FJ624491

Verbruggen

et al. 2009b

H. gracilis

car2 Harvey

ex J.Agardh HV959

Saint Ann, Priory, The

Wall, Jamaica (CAR) FJ624721

Verbruggen

et al. 2009b

H.gracilis

Harvey ex

J.Agardh HV461


Bay, Lee Reef, Jamaica

(CAR) FJ624494

Verbruggen

et al 2009b

H.

heteromorph

a N'Yeurt

PH 197

Cebu, Mactan Island,

Lapu Lapu City,

Marigondon,Philippine

s (WP) FJ624535

Verbruggen

et al. 2009b

HV146 Moorea, French

Polynesia (CP) AM049957

Verbruggen

et al 2005

FJ624511

Verbruggen

et al 2009b


Submitted M

anuscript

CP08 518

Moorea, French


IRD 5330*

Grande Terre, New


IRD 5346

Moorea, French


H. hummii

D.L.

Ballantine

H.0253

San Blas, Panama

(CAR) AM049988

Verbruggen

et al. 2005

H.0232

Portobelo, Isla Mamey,

Panama (EP) FJ624522

Verbruggen

et al 2009b

H. incrassata

(J.Ellis) J.V.

Lamouroux DML30456

British virgin island,

Jost van Dyke Island

(CAR) FJ624534

Verbruggen

et al. 2009b

H.0179

Lee Stocking, Bahamas

(CAR) AM049958

Verbruggen

et al 2005

AY942167

Lam et al

2006

HEC-15752 FJ715719

Cocquyt et

al 2009

Curtis 2 DQ469328

Curtis et al

2008

H. kanaloana

Vroom H.0649

Honolua Bay, Mau’i

Hawai’i , USA (CP) AM049959

Verbruggen

et al 2006

IRD 5323

Grande Terre, New


IRD 5324

Grande Terre, New


H.0650

Maui, Honolua Bay,

Hawaii, USA (CP) FJ624512

Verbruggen

et al 2009b

H. lacrimosa

M.A. Howe H.0308 Bahamas (CAR) EF667064

Verbruggen

et al.2007

DML68301 Bahamas (CAR) FJ624730 FJ624495

Verbruggen

et al. 2009b

H. lacunalis

W.R. Taylor H.0121 Guam (WP) AY826363

Verbruggen

et al. 2005

IRD 5316

Chesterfield, New


IRD 5317

Chesterfield, New


IRD 5302

Grande Terre, New


IRD 5327

Grande Terre, New


H.0743

Western Australia,

Scott Reef, Australia

(EI) FJ624521

Verbruggen

et al 2009

H. macroloba

Decaisne

HV38 Nungwi, Zanzibar,

Tanzania (WI) AM049960

Verbruggen

et al 2006

FJ624513

Verbruggen

et al 2009


Submitted M

anuscript

IRD 5308

Grande Terre, New


IRD 5321

Grande Terre, New


IRD 5322

Grande Terre, New


IRD 5304

Grande Terre, New


H.

macrophysa

Askenasy

HV822 Luzon: Sorsogon:

Bulusan: Dancalan,

Philippines (WP)

HV

unpublished

FJ624526

Verbruggen

et al 2009

H.0271 New Caledonia (WP) AM049989

Verbruggen

et al 2005

H.0024

Queensland: Lizard

Island, Australia (WP)

HV

unpublished

IRD 5328

Grande Terre, New


H.

magnidisca

J.M. Noble H.0814

Heron

Island,Queensland,

Australia (WP)

HV

unpublished

H.0816

Heron

Island,Queensland,

Australia (WP)

HV

unpublished

H.0817

Heron

Island,Queensland,

Australia (WP)

HV

unpublished

H.0819

Heron

Island,Queensland,

Australia (WP)

HV

unpublished

H.0822

Queensland, Australie

(WP) FJ624536 FJ624530

Verbruggen

et al. 2009b

IRD 5329

Grande Terre, New


H.

melanesica

Valet

HV790 Dapdap, Luzon,

Philippines (WP) AM049961

Verbruggen

et al 2006

FJ624515

Verbruggen

et al 2009b

IRD 5810

Lifou, New Caledonia

(NC) JQ432551 this study

H.

micronesica

Yamada

H.0011 Australia

HV

unpublished

H.0025 Australia

HV

unpublished

H.0083 Chagos (CI)

HV

unpublished

H.0084 Chagos (CI)

HV

unpublished

H.0014

Great Barrier Reef,

Australia (WP) EF667060

Verbruggen

et al 2007


Submitted M

anuscript

DML40033

North Astrolabe Reef,

Fiji (WP) EF667061

Verbruggen

et al 2007

WLS184-02


and Futuna (WP) EF667059

Verbruggen

et al 2007

WLS420-02 Wallis Island, Wallis

and Futuna (WP) AM049964

Verbruggen

et al 2006

FJ624499

Verbruggen

et al 2009b


H. minima

(W.R.

Taylor)

Hillis-

Colinvaux

HV148

Moorea, French


Verbruggen

et al. 2009b

PH 526

Mindanao,

Zamboanga, Santa

Cruz Island,


Verbruggen

et al 2009b

IRD 5351*

Grande Terre, New


CP08 415

Moorea, French


CP08 486

Moorea, French


CP08 786

Moorea, French


IRD 5347

Moorea, French






IRD 5298

Grande Terre, New


IRD 5301

Grande Terre, New


IRD 5299

Grande Terre, New


IRD 5297

Grande Terre, New


H. minima 1

(W.R. Taylor)

Hillis-

Colinvaux

H.0380 Bile Bay, Guam (WP) FJ624706

Verbruggen

et al. 2009b

HV1812 Maldives (CI) XX

TZ0002

Mbudya Island,

Tanzania (WI) FJ624666

Verbruggen

et al. 2009b

TZ0005

Mbudya Island,


Verbruggen

et al. 2009b

SOC384 Socotra, Yemen (WI)

EF667067

Verbruggen

et al. 2007

FJ624500

Verbruggen

et al 2009b


Submitted M

anuscript

H.0848

Western Australia,

Rowley Shoals,

Mermaid Reef,


Verbruggen

et al. 2009b

H.0746

Western Australia,

Scott Reef,

Australia(EI) FJ624663

Verbruggen

et al. 2009b

H. minima 2

(W.R.

Taylor)

Hillis-

Colinvaux

H.0382

Apra Harbour, Guam

(WP) FJ624669 FJ624503

Verbruggen

et al. 2009b

H.0381 Guam (WP) FJ624707

Verbruggen

et al. 2009b

G.167 Indonesia (WP)

HV

unpublished

HV765

Palawan, Busuanga,

Tangat, Philippines

(WP) FJ624668

Verbruggen

et al. 2009b

H. minima 3

(W.R.

Taylor)

Hillis-

Colinvaux

HV656

Bohol, Cabilao,


Verbruggen

et al. 2009b

HV693

Bohol, Panglao, Alona

Beach, Philippines

(WP) FJ624682

Verbruggen

et al. 2009b

HV695

Bohol, Panglao, Alona

Beach, Philippines

(WP) FJ624676

Verbruggen

et al. 2009b

H.0395 Cook Islands (CP) FJ624683

Verbruggen

et al. 2009b

HV63

Moorea, Cook Bay,


Verbruggen

et al. 2009b

HV720

Panglao, Philippines

(WP) FJ624675

Verbruggen

et al. 2009b

H.0336

Queensland, Lizard

Island, Australia (WP) FJ624684

Verbruggen

et al. 2009b

H.0332

Queensland, Lizard

Island, Australia (WP) FJ624674

Verbruggen

et al. 2009b

HV163

Tahiti, Arue, French


Verbruggen

et al. 2009b

H.0286

Tahiti, French


Verbruggen

et al. 2009b

HV189

Tahiti, Punaauia,


Verbruggen

et al. 2009b

H. minima.4

(W.R.

Taylor)

Hillis-

Colinvaux

HV791

Luzon: Sorsogon,

Bulusan, Dapdap,

Philippines (WP) FJ624687 FJ624502

Verbruggen

et al. 2009b

H.0990

Vanua Levu,

Cakaulevu reef, Fiji

(WP) FJ624686

Verbruggen

et al. 2009b

H.0991

Vanua Levu,

Cakaulevu reef, Fiji

(WP) FJ624688

Verbruggen

et al. 2009b

H. monile

(J.Ellis &

H.0034 Galeta, Panama (CAR)

AM049962

Verbruggen

et al 2005


Submitted M

anuscript

Solander)

J.V.

Lamouroux

FJ624516

Verbruggen

et al 2009b

Curtis 3

Florida, Tarpon

Springs, USA (CAR) DQ469329

Verbruggen

et al 2009b

H. opuntia

(Linnaeus)

Lamouroux HV591

Cebu, Daanbantayan,

Malapascua, Logon

Bay, Philippines (WP) FJ624690

Verbruggen

et al. 2009b

HV61

Cook Bay, Moorea

French, Polynesia (CP) AM049967

Verbruggen

et al 2005

CP08 414

Moorea, French


CP08 484

Moorea, French


CP08 909

Moorea, French


IRD 5352*

Grande Terre, New


AY942174

Lam et al

2006

AB038489

Hanyuda et

al 2000

H. pygmaea

Verbruggen,

D.S.Littler &

M.M.Littler

DML40143 North Astrolabe Reef,

Fiji (WP) EF667062

Verbruggen

et al 2007

FJ624497

Verbruggen

et al 2009b

H. renschii

Hauck ODC1671 Philippines (WP)

HV

unpublished


HV

unpublished


HV

unpublished

HV7

Zanzibar, Mnemba

Atoll, Tanzania (CP) FJ624691 FJ624501

Verbruggen

et al. 2009b

TZ0468

Zanzibar, Mnemba,


Verbruggen

et al. 2009b

TZ0480

Zanzibar, Mnemba,


Verbruggen

et al. 2009b

H. scabra

M.A.Howe DML68229

Green Turtle Cay,

Bahamas (CAR) FJ624540

Verbruggen

et al. 2009b

DML68311

Bahamas: Green Turtle

Cay (CAR) FJ624523

Verbruggen

et al 2009b

H. simulans

M.A.Howe

HV449 Discovery Bay,

Jamaica (CAR) AM049963

Verbruggen

et al 2005

FJ624517

Verbruggben

et al 2009

H. cf stuposa IRD 5311

Grande Terre, New


H. taenicola

W.R. Taylor HV285-1

Rangiroa, French

Polynesia (CP) AY826365

Verbruggen

et al 2005


Submitted M

anuscript

HV1813

Thiladhunmathi:

Hanimaadhoo,

Maldives (CI)

HV

unpublished

HV285-1 Tiputa, Rangiroa

French Polynesia (CP) AM049966

Verbruggen

et al 2005

FJ624529

Verbruggen

et al 2009b

IRD 5314

Chesterfield, New


IRD 5315

Chesterfield, New


IRD 5313

Chesterfield, New


CP08 411

Moorea, French


CP08 457

Moorea, French


CP08 458

Moorea, French


CP08 709

Moorea, French



IRD 4861 Maldives (CI) JN644723 JN644753 this study

H. tuna

(J.Ellis &

Solander)

J.V.Lamouro

ux

H.0113 Italy (MED) AY826366

Verbruggen

et al 2005

H.0231

Puerto Morelos,

Mexico (CAR) AY826367

Verbruggen

et al 2005

H.0231

Zanzibar: Pongwe,

Tanzania (WI) AY826367

Verbruggen

et al 2005

DML51688

Jamaica: Morant Cays

(CAR) FJ624524

Verbruggen

et al 2009b

AY942177

Verbruggen

et al 2009b

H.

velasquezii

W.R. Taylor HV768

Luzon: Sorsogon,

Bulusan, Dapdap,


Verbruggen

et al. 2009b

HV779

Luzon: Sorsogon,

Bulusan, Dapdap,


Verbruggen

et al. 2009

TZ0066

Mbudya Island,


Verbruggen

et al. 2009b

HEC14003

Mtwara, Mnazi Bay,

Mana Hawanja Island,


Verbruggen

et al. 2009b

TZ0163

Mtwara, Mnazi Bay,

Ruvula, Tanzania (WI) FJ624701

Verbruggen

et al. 2009b

HV746

Palawan, Busuanga,

Cagbatan, Philippines

(WP) FJ624697

Verbruggen

et al. 2009b

G.015 Talofofo Bay, Guam FJ624695 Verbruggen


Submitted M

anuscript

(WP) et al. 2009b

IPAN010

Talofofo, Mana Bay,

Ipan, Guam (WP) FJ624711

Verbruggen

et al. 2009b

TZ0464

Zanzibar, Mnemba,


Verbruggen

et al. 2009b

TZ0547

Zanzibar, Nungwi,


Verbruggen

et al. 2009b

TZ0839

Zanzibar, Paje,


Verbruggen

et al. 2009b

TZ0745

Zanzibar, Pongwe,


Verbruggen

et al. 2009b

IRD 5296

Grande Terre, New


IRD 4935 Maldives (CI) JN644725

HV779

Luzon, Sorsogon,

Bulusan, Dapdap,


Verbruggen

et al 2009b

H. xishaensis

Tseng and

M.L. Dong

H.0122 Guam (WP) AY826357

Verbruggen

et al 2005

ODC1564

Kinondo Reef, Kenya

(WI)

HV

unpublished

TZ0188

Mtwara: Ruvula,

Tanzania (WI)

HV

unpublished

HV731

Palawan: Busuanga:

Uson, Philippines

(WP)

HV

unpublished

H.0389

Providencia, Colombia

(CAR)

HV

unpublished

HV1814

Thiladhunmathi:

Hanimaadhoo,

Maldives (CI)

HV

unpublished

HV33

Zanzibar: Pongwe,

Tanzania (WI)

HV

unpublished


IRD 4867 Maldives (CI) JN644727

this study


H. no.ID.1 G.007

Maug, Northern


Verbruggen

et al. 2009b

H. no.ID.1 G.011

Asuncion, Northern


Verbruggen

et al. 2009b

H. no.ID.1 HVLL0367 Fiji (WP)

HV

unpublished


Documents

Diversity of Halimeda (Bryopsidales, Chlorophyta) in New ... · Submitted Manuscript 2 20 Abstract 21 22 Halimeda is a genus of calcified and segmented green macroalgae in the order