Wednesday Jun 12 2002 12:43 PM phya 41_502 Mp_12 Allen Press • DTPro System GALLEY 12 File # 02tq

Phycologia (2002) Volume 41 (5), 000±000 Published 1 September 2002

Molecular phylogenies of (, ) reveal nonmonophyly for , and

WIEBE H.C.F. KOOISTRA*² Smithsonian Tropical Research Institute, Box 2072, Balboa, Republic of Panama

W.H.C.F. KOOISTRA. 2002. Molecular phylogenies of Udoteaceae (Bryopsidales, Chlorophyta) reveal nonmonophyly for Udotea, Penicillus and Chlorodesmis. Phycologia 41: 000±000.

Generic delineation within and among the udoteacean genera Udotea, Penicillus, and Chlorodesmis has remained provisional because of con¯icting evidence from different sets of traits, especially between reproductive structures and vegetative morphology. The ribosomal DNA phylogeny presented here contradicts the classical generic concepts within the Udoteaceae by revealing traditional genera to be polyphyletic. Based on this study, it was reported that the brush of Penicillus is essentially an uncorticated Udotea blade in which siphons have lost adherence. In fact, this Penicillus mor- phology arises twice independently. Similarly, the simple uniaxial thallus of Chlorodesmis has evolved through secondary reduction or neoteny. Siphon anatomy of vegetative thalli does not delineate natural groups perfectly. However, the shape of reproductive structures and the size of macrogametes appear to circumscribe natural taxa well. Overall sequence diver- gence within Udotea and its allied species in Chlorodesmis, Penicillus and Rhipocephalus is low and indicates relatively young and rapidly diversifying lineages.

INTRODUCTION phons. In several others, the blade siphons bear laterals con- sisting of short protuberances or more elaborate appendages The Udoteaceae (Bryopsidales, Chlorophyta) comprise a similar to those in the cortex of the stipe of most udoteacean strictly marine family of siphonous macroalgae with a pre- genera (Gepp & Gepp 1911; Littler & Littler 1990). However, dominantly tropical distribution. The morphology of the ma- monophyly for Udotea is challenged by the presence, in some ture thalli ranges from simple siphonous ®laments to intricate species, of corticated blades or by segments resembling those multiaxial structures, in which siphon anatomy depends on the seen in certain other genera of Udoteaceae, e.g. Niz- location in the thallus and the life-history stage (Gepp & Gepp zamuddin and J.V. Lamouroux, and by the pres- 1911; Hillis-Colinvaux 1980; Meinesz 1980; Littler & Littler ence, in others, of uncorticated blades or loose brushes that 1990, 1999). Despite the complexity, even the most elabo- point to an af®nity with, e.g. Rhipocephalus KuÈtzing, Rhipi- rately rami®ed system of siphons consists of a single multi- dosiphon Montagne, A. Gepp & E. Gepp, Tyde- nucleate cell, through which nuclei, cytoplasm and organelles mania Weber van Bosse and Penicillus Lamarck (Gepp & can be freely transported. In many Udoteaceae, the intersi- Gepp 1911; Hillis-Colinvaux 1984; Vroom et al. 1998). phonal spaces become calci®ed in photosynthetic parts of the Information on sexual reproduction and life-history strate- thalli (Borowitzka & Larkum 1977), rendering them less pal- gies is available for some Udoteacea but sketchy for others atable (Hay et al. 1994). Reproduction (Kamura 1966; Mei- and not available at all for most species (Meinesz 1980; Hillis- nesz 1980) and protoplasm reallocation (Littler & Littler Colinvaux 1984; Clifton 1997; Vroom et al. 1998; Clifton & 1999) lead to a complete loss of protoplasm in part, if not all, Clifton 1999). Nonetheless, con¯ict abounds between vege- of the , resulting in empty `ghost thalli'. Their calci®ed tative and generative traits. Generally, multiaxial adult thalli remains become part of the substratum (Drew 1993; Freile et develop from uniaxial juvenile tufts composed of rhizoids and al. 1995) and can fossilize with their siphonal ®ngerprints in- dichotomously branching siphons (Meinesz 1980). Several tact. Comparable fossil casts reveal that siphonous algae have species of Chlorodesmis Harvey & Bailey are probably mis- grown in abundance from at least the Ordovician period on- identi®ed juvenile phases of other taxa. For instance, Pseu- wards (Johnson 1961; Wray 1977; Poncet 1989). dochlorodesmis furcellata (Zanardini) Bùrgesen is actually the Udoteaceae have been classi®ed predominantly on the basis juvenile of Mediterranean H. tuna (Ellis & Solander) J.V. La- of vegetative traits of mature thalli (Vroom et al. 1998). For mouroux (Meinesz 1980). Other Chlorodesmis spp. reproduce instance, species of the morphologically diverse and species- sexually and must therefore be mature, though relationships rich genus Udotea J.V. Lamouroux are described as possessing are challenged because gametangia differ conspicuously corticated, calci®ed stipes and funnel- or fan-shaped blades among species. For example, C. baculifera (J. Agardh) Duck- (Littler & Littler 1990). In many species of Udotea, the blades er possesses compound gametangia similar to those of the consist only of a single or several layers of dichotomously morphologically elaborate genus Halimeda, whereas the gam- branching siphons that adhere laterally to neighbouring si- etangia of C. fastigiata (C. Agardh) Ducker (previously known as C. comosa Harvey & Bailey) appear like those of * Corresponding author ([email protected]). uncorticated Udotea spp. (Ducker 1965, 1967; Hillis-Colin- ² Present address: Stazione Zoologica, Villa Comunale, 80121 Na- vaux 1980; Meinesz 1980). ples, Italy. Several authors have attempted to ®nd order in this system- Wednesday Jun 12 2002 12:43 PM phya 41_502 Mp_13 Allen Press • DTPro System GALLEY 13 File # 02tq

Kooistra: Phylogeny of Udoteaceae

Table 1. Specimens and their sample sites.1

GenBank Species Specimen Sample site (ocean) numbers racemosa (ForsskaÊl) J. Agardh 98-197 Portobelo, Panama (A) AF479702 C. sertularioides M. Howe 98-190 Porvenir, Panama (A) AF479703 Udoteaceae Chlorodesmis caespitosa J. Agardh 99-023 Taboga Island, Panama (IP) AF416399 C. caespitosa 99-088 Taboga Island, Panama (IP) AF416400 C. fastigiata (C. Agardh) Ducker 98-132 Lizard Island, Great Barrier Reef (IP) AF416396 Flabellia petiolata (Turra) Nizzamuddin NA Naples, Italy (A) AF416389 F. petiolata F2 Piran, Slovenia (A) AF416390 Halimeda borneensis W.R. Taylor 99-077 Philippines 534 (IP) AF416387 H. cylindracea Decaisne WHS460 Townsville, Great Barrier Reef (IP) AF316388 Lamarck 98-181 Porvenir, Panama (A) AF416404 P. capitatus 99-159 Porvenir, Panama (A) AF416405 P. dumetosus J.V. Lamouroux 98-180 Porvenir, Panama (A) AF416406 P. dumetosus 99-157 Porvenir, Panama (A) AF416407 P. nodulosus Blainville 98-130 Lizard Island, Great Barrier Reef (IP) AF416398 P. pyriformis A. Gepp & E. Gepp 98-154 Bermuda (A) AF416408 P. pyriformis 98-182 Porvenir, Panama (A) AF416409 P. pyriformis 99-158 Portobelo, Panama (A) AF416410 Rhipiliopsis reticulata (C. van den Hoek) Farghali & Denizot 99-125 Galeta, Panama (A) AF416386 Rhipocephalus phoenix (Ellis & Solander) KuÈtzing 98-184 Porvenir, Panama (A) AF416402 Udotea abbottiorum D. Littler & Littler 98-200 Portobelo, Panama (A) AF416401 U. argentea Zanardini 99-103 Guam (IP) AF416394 U. cyathiformis Decaisne BW01416 Cay Zapatilla, Panama (A) AF416403 U. doteyi D. Littler & Littler 00-001 Roatan, Honduras (A) AF416393 U. ¯abellum (Ellis & Solander) M. Howe 98-196 Portobelo, Panama (A) AF416392 U. looensis D. Littler & Littler 00-002 Spanish Harbor, Florida (A) AF416397 U. norrisii D. Littler & Littler 98-160 Bermuda (A) AF416391 U. orientalis A. Gepp & E. Gepp 98-202 Skottsberg, South Africa (IP) AF416395

1 Atlantic; IP, Indo-Paci®c. atic tangle (Lamouroux 1812; Ernst 1904; Gepp & Gepp (Caulerpaceae, Bryopsidales) as the close outgroup and those 1911; Feldmann 1946, 1954; Bold & Wynne 1978; Meinesz of dasycladalean species as a more distant outgroup. 1980; Silva 1982; Hillis-Colinvaux 1984; Floyd & O'Kelly 1989; Hoek et al. 1995). Vroom et al. (1998) used cladistics (Hennig 1965) to demonstrate that morphological, anatomical MATERIAL AND METHODS and life-history traits could not resolve evolutionary relation- ships satisfactorily among the type species of most bryopsi- Specimens and their sample sites are listed in Table 1. Species dalean genera. Nevertheless, their results suggested monophy- were identi®ed using the methods of Gepp & Gepp (1911), ly for Udoteaceae, as well as a general trend from an ancestral Taylor (1950, 1960), Ducker (1967), Hillis-Colinvaux (1980) simple thallus morphology to derived, more complex struc- and Littler & Littler (1990, 2000). Detailed illustrations of tures. A similar study among western Atlantic species of Udo- phenotypes and morphological descriptions are available in tea (Littler & Littler 1999) also found a general trend towards these references. Freshly collected thalli were preserved in a more complex, derived blade morphology, with uncorticated 95% v/v ethanol or silica gel desiccant and kept refrigerated blades being ancestral. in the dark until used for morphological analysis and DNA A molecular phylogenetic approach may provide an alter- extraction. Morphological characters and their states were col- native way to a better understanding of udoteacean relation- lected from the specimens included in the molecular study to ships. Several authors have inferred phylogenies at various permit direct comparison and to avoid bias caused by phe- taxonomic levels within the Bryopsidales by using plastid or notypic differences between the type specimens and the spec- nuclear genetic markers (Pillmann et al. 1997; Jousson et al. imens included in this study. The characters and their states 1998; FamaÁ et al. 2000; Hanyuda et al. 2000; Woolcott et al. examined in this study formed a subset of those listed by 2000; Durand et al. 2002). Molecular phylogenies of Hali- Taylor (1960) and Littler & Littler (1990). Character states for meda inferred from nuclear ribosomal DNA markers (Hillis reproductive structures and macrogametes were obtained from et al. 1998; Kooistra et al. 2002) showed remarkable concor- Meinesz (1980) and references therein, and from Clifton & dance with taxonomic groups delimited by phenotypic traits. Clifton (1999). Here, I infer phylogenies from a large part of the nuclear- DNA was extracted from c. 0.5±1 cm2 of clean thallus that encoded ribosomal DNA (nrDNA), encompassing several var- had been air-dried and placed in TE buffer (pH 8.0) before iable regions, to explore relationships among and within the powdering in liquid nitrogen. The frozen powder was added udoteacean genera Chlorodesmis, Rhipocephalus, Penicillus to 0.5 ml preheated (60ЊC) extraction buffer (100 mM Tris± and Udotea. To root the trees, I use sequences of Caulerpa HCl, pH 8.0; 50 mM ethylenediaminetetraacetic acid, pH 8.0; Wednesday Jun 12 2002 12:43 PM phya 41_502 Mp_14 Allen Press • DTPro System GALLEY 14 File # 02tq

Phycologia, Vol. 41 (5), 2002

Table 2. Oligonucleotide primers used for ampli®cation and sequencing.1

Code F/R 5Ј±3Ј Location Use Source TW32 F GCAAGTCTGGTGCCAGCATCT TW3 A, S this study TW52 F AACTTGAAGGAATTGACGGAAG TW5 A, S this study TW62 R GCATCACAGATCTGTTATTGCCTC TW6 A, S this study TW72 F GAGGCAATAACAGATCTGTGATGC TW7 S this study H1R R TCTACGTGCGAAGGTTCAGAG 3Ј end of 18S DNA A, S this study H1F F CTCTGAACCTTCGCACGTAGA 3Ј end of 18S rDNA A, S this study ITS22 R GCTACGTCCTTCATCGACGC ITS2 A, S this study ITS32 F GCGTCGATGAAGGACGTAGC ITS3 S this study ITS43 R TCCTCCGCTTATTGATATGC ITS4 A, S White et al. (1990) WKR R TTTCCTCGCCGACGACATCGC 5Ј end of 28S rDNA A, S this study 26AB R GGTCCGTGTTTCAAGACGGG 500 bp into 28S rDNA A, S F.W. Zechman, personal communication

1 A, used in ampli®cation; S, used for sequencing. 2 Primers modi®ed from White et al. (1990). 3 This primer was used in this study, but a perfectly ®tting one reads TCCTCCGCTTAATGATATGC.

100 mM NaCl; 1% w/v sodium dodecyl sulfate; 1% w/v poly- hLRT parameters obtained. Weighted (Goloboff, K ϭ 2) max- vinylpyrrolidone-40 and 0.5% v/v 2-mercaptoethanol) and imum parsimony (MP) trees were generated using the full was incubated for 10 min at 60Њ C. The mixture was then heuristics, and the tree bisection±reconnection±branch swap- shaken gently with an equal volume of chloroform±isoamyl ping mode. Gaps were omitted in pairwise comparisons. Boot- alcohol [CIA; 24/1 (v/v)] and centrifuged (16,000 ϫ g) for 5 strap values were obtained using MP because this algorithm min. The aqueous layer was collected, re-extracted with CIA consumed less time than ML and because MP and ML recov- and centrifuged as described previously. The DNA in the ered identical tree topologies. Alternative topologies were aqueous phase was precipitated using 0.5 volumes of 8 M evaluated using MacClade 3.07 (Maddison & Maddison ammonium acetate (pH 8.0) and 2 volumes of 100% ethanol, 1997). To root trees, dasycladalean SSU sequences from Olsen stored at Ϫ20Њ C for up to 1 h and then centrifuged for 15 et al. (1994) were chosen as an outgroup, based on the ®nd- min as described previously. Pellets were washed in 70% (v/ ings of Zechman et al. (1999). v) ethanol, air-dried and resuspended in 10 ␮l TAE buffer (pH 8.0). DNA was gel-puri®ed in low melting agarose (TAE); bands were excised under weak UV-radiation and subsequent- RESULTS ly incubated with Gelase following the manufacturers' instruc- tions (Epicentre Technologies Corporation, Madison, Wiscon- The SSU region of all udoteacean and caulerpacean speci- sin). Use of gel-puri®ed DNA greatly improved the success mens included in this study contained an insert (c. 100 bp) of the polymerase chain reaction (PCR). at the same location as the one described for Halimeda by Nuclear ribosomal DNA was PCR-ampli®ed and sequenced Hillis et al. (1998). The inserts of the udoteacean taxa shared for phylogenetic analysis. The target region (c. 2200 bp) ex- a common secondary structure (not shown) and elevated tended from c. position 500 in the small subunit (SSU) rDNA, CG%. The ITS-1 region was very short in all genera, being through an insert in the SSU (c. position 1440 in SSU; Hillis longest in Caulerpa (118 bp), shortest in Rhipiliopsis (54 bp) et al. 1998), the two internal transcribed spacers (ITS-1 and and intermediate in all other Udoteaceae included in this ITS-2) and the intervening 5.8S region, to c. position 500 in study (65±70 bp). The ITS-2 region was also relatively short, the large subunit (LSU). PCR ampli®cation of target regions being c. 330 bp in Caulerpa and 172±182 bp in Udoteaceae. in three overlapping products (TW3±HIR, TW5±ITS4 and The ITS-2 and partial LSU regions of U. looensis could not HIF±26AB), sequencing and alignment of sequences were as be obtained; positions of these regions were marked `N' in described by Kooistra et al. (1999). The ®nal alignment is the alignment. available upon request. The primers used are listed in Table The insert and ITS regions of the two Caulerpa specimens 2. The PCR-product TW5±ITS4 was obtained to verify the could not be aligned with those of the remaining specimens. common origin of the other two sequences, which did not However, to determine relationships between Udoteaceae and overlap. GenBank accession numbers are listed in Table 1. Caulerpaceae and to root Udoteaceae properly an initial align- Multiple specimens per species were included or, if only one ment was generated using the partial SSU sequences of all specimen was available, sequences were generated from dif- specimens included in this study and the corresponding da- ferent DNA extractions to verify the results. sycladalean SSU sequences from Olsen et al. (1994). Posi- Phylogenetic signal among parsimony-informative sites was tions involved in ambivalent alignment were removed before

assessed by comparing the measure of skewedness (g1-value; phylogenetic analysis, if they would affect tree topology. The PAUP*) with the empirical threshold values in Hillis & Huel- 431 parsimony-informative sites in the resulting alignment senbeck (1992). To determine which model of sequence evo- (length ϭ 1258 positions) contained signi®cant phylogenetic

lution best ®ts the data, hierarchical likelihood ratio tests signal [g1 ϭϪ0.821; threshold value g1 ϭ 0.09 (P ϭ 0.01) (hLRTs) were performed using Modeltest Version 3.0 (Posada for 25 taxa and 250 parsimony-informative sites]. Phyloge- & Crandall 1998). Phylogenies were inferred from these se- netic analysis resulted in three equally most parsimonious quences using PAUP* (version 4.0b8, Swofford 2001). Max- trees. In the abridged cladogram (Fig. 1) redrawn from these imum likelihood (ML) analysis was constrained with the trees and with Dasycladales as outgroup, Caulerpa branched Wednesday Jun 12 2002 12:43 PM phya 41_502 Mp_15 Allen Press • DTPro System GALLEY 15 File # 02tq

Kooistra: Phylogeny of Udoteaceae

The tree obtained was topologically identical to a weighted single MP tree. Moreover, MP trees generated from the SSU alone (Fig. 1), from the SSU and insert, from the ITS regions and from the LSU separately (trees not shown) did not con- tradict the topology of the ML tree, indicating coherence among these regions.

DISCUSSION

The nrDNA sequences contain the proper amount of varia- tion to resolve udoteacean relationships at both the intra- and the intergeneric level. In fact, the inferred tree topologies shown in Figs 1 and 2 resolve relationships more precisely than those inferred from nonmolecular traits reported by Lit- tler & Littler (1990) and Vroom et al. (1998). The results indicate that currently used generic delineations of Udotea, Penicillus, Rhipocephalus and Chlorodesmis do not de®ne natural groups.

Sequence comparisons

The identical location of the SSU insert in all tested represen- tatives within the Udoteaceae and Caulerpaceae (Hillis et al. 1998; Kooistra et al. 1999, 2002; this study) indicates its pres- Fig. 1. Abridged cladogram redrawn from three weighted (Goloboff, ence in the last common ancestor of these sister classes within K ϭ 2) equally most parsimonious trees inferred from 431 parsimony- informative positions in the alignment of the partial SSU rDNA se- the Bryopsidales (see Vroom et al. 1998) and suggests the quences (see Materials and Methods) listed in Table 1 and the corre- existence of a novel conserved region in the SSU. sponding regions of all dasycladalean SSU sequences in Olsen et al. Short ITS regions as detected here seem to be con®ned to (1994). An insert in the udoteacean SSU sequences and positions con- Udoteaceae; caulerpacean ITS sequences are considerably taining bases that could not be aligned unambiguously were removed longer (Pillmann et al. 1997; Jousson et al. 1998; FamaÁ et al. from this alignment. Bootstrap values have been generated using 1000 replicates. The three most parsimonious trees were each 987 steps long 2000; Durand et al. 2002; Kooistra et al. 2002; this study). (CI ϭ 0.720, RC ϭ 0.638). Intraindividual ITS sequence heterogeneity appears less con- spicuous in Udoteaceae than in certain Caulerpa spp. (FamaÁ et al. 2000) but this is perhaps to be expected because Cau- off ®rst. Then there followed a clade with Rhipiliopsis and lerpa possesses longer ITS sequences and more positions and Halimeda, which was sister of a clade containing Flabellia, can therefore contribute towards intraindividual heterogeneity. Udotea, Chlorodesmis, Penicillus and Rhipocephalus. All Differences could also result from artefacts of the sequencing clades obtained proper support (Fig. 1). protocols used. In a heterogeneous batch of ITS sequences After exclusion of Caulerpa and dasycladalean outgroups, uncommon types can be identi®ed through cloning, as done complete sequences of the remaining taxa were aligned with- by FamaÁ et al. (2000), whereas they show up as 'background out trouble. Nonetheless, gaps had to be introduced, though noise' in a direct sequencing approach. mainly in the insert and ITS regions. The 529 parsimony- The fact that SSU and ITS sequences both contribute to the informative positions in the alignment contained signi®cant same phylogeny is quite unusual because these sequences are phylogenetic signal (g1 ϭϪ0.807; threshold value as men- expected to evolve at widely different rates. In general, ITS tioned previously). Substitution probability varied among re- sequences become dif®cult to align as soon as the much slow- gions, being highest in ITS-1, ITS-2 and the insert and lowest er SSU becomes phylogenetically useful (Bakker et al. 1994, in the SSU core and 5.8S regions (MacClade). Modeltest re- 1995), although some ITS-2 core positions can retain phylo- sults con®rmed the deviation from random base substitution genetic signal well above the generic level (Hershkovitz & (see Fig. 2). The best substitution model to explain apparent Lewis 1996; Mai & Coleman 1997). However, in Udoteaceae, complexity was a general time reversible model; G was slight- rates of SSU evolution are strongly elevated in comparison ly over-represented. Moreover, C↔T changes occurred c. 1.7 with those in other (Zechman et al. 1990, 1999). times as frequently as A↔G changes. In contrast, if the ITS regions are short, sequence evolution The resulting ML tree recovered Rhipiliopsis and Halimeda can be expected to be slow because of a higher proportion of as sister taxa. Because these two genera were recovered in a functional core positions (Mai & Coleman 1997) than in the sister clade to the remaining Udoteaceae shown in Fig. 1, they much longer ITSs found in other green algae (e.g. Kooistra et were used to root the tree and then pruned away from it. The al. 1993; Bakker et al. 1995). Therefore, rates of SSU and resulting tree topology (Fig. 2) identi®ed Flabellia as sister to ITS sequences are expected to be much closer to one another a clade containing a paraphyletic Udotea, with its allied gen- in Bryopsidales than in other green algae. Despite the differ- era Rhipocephalus, Penicillus and Chlorodesmis nested inside ing substitution rates and modes, no discordance in phyloge- it. Penicillus and Chlorodesmis were also nonmonophyletic. netic history could be detected between the SSU and ITS re- Wednesday Jun 12 2002 12:43 PM phya 41_502 Mp_16 Allen Press • DTPro System GALLEY 16 File # 02tq

Phycologia, Vol. 41 (5), 2002 gions, which is to be expected because these sequences are Gepp 1911; Littler & Littler 1990), which is not the nearest linked. Therefore, all these nrDNA regions were combined in sister to these two Penicillus spp. a single phylogenetic analysis. Calci®cation may have been gained in the last common ancestor of Udoteaceae and then lost independently in at least Gene trees and species trees Chlorodesmis, Flabellia and Rhipiliopsis. Alternatively, cal- ci®cation may have appeared independently in Halimeda and Good resolution of gene trees does not guarantee that the trees in the Udotea-clade and may have been lost in Chlorodesmis. will accurately re¯ect taxon genealogy (Nichols 2001). Poten- Hillis-Colinvaux (1984) proposed monophyly for both calci- tial causes of such mismatch include paralogy, lineage sorting ®ed and uncalci®ed Udoteaceae. Results by Vroom et al. and lateral gene transfer between distantly related species (1998) and the tree topologies shown of Figs 1 and 2 reject (Doyle 1992; De Queiroz 1993; Wendel et al. 1995). The such a division. Although calci®cation has appeared and has nrDNA sequences used in the present study are members of been lost several times, lack of calci®cation characterizes in- a multicopy gene family, in which more than one haplotype dividual genera such as Caulerpa, Rhipiliopsis and the mono- can occur. Concerted evolution (Arnheim 1983), which is sup- typic Flabellia. The last was even separated from Udotea for posed to homogenize such variation, may accidentally ®x hap- this very reason (Gepp & Gepp 1911; Nizzamuddin 1987). lotypes so as not to re¯ect genealogy. Notably, certain Cau- Loss of traits seems to have been common during the evo- lerpa spp. tolerate remarkable amounts of intraindividual lution of the Udoteaceae. Such secondary loss is demonstrated nrDNA ITS variation (Pillmann et al. 1997; FamaÁ et al. 2000), to an extreme in Chlorodesmis. Although some members of affecting intra- and interspeci®c phylogenetic reconstruction. this morphologically simple genus may represent ancestral Durand et al. (2002) actually uncovered signs of hybridization forms within the Udoteaceae, the two species included in this in their study of C. racemosa (ForsskaÊl) J. Agardh, in the form study are highly derived. Their phenotypic simplicity results of con¯icting topologies between trees inferred from different from secondary loss of complexity: the multiaxial thallus or- nrDNA regions. However, gross distortion of the tree topology ganization, differentiation into blade and stipe, cortication and shown in Figs 1 and 2 is not expected because only a few calci®cation have all gone. The nearest neighbours of C. caes- ambiguities were encountered along the obtained sequences pitosa are Penicillus spp. Apparently C. caespitosa has lost and trees inferred from the different gene regions corroborated the stipe and calci®cation of cap siphons, whereas adherence one another. Nevertheless, my results ought to be tested by of the cap siphons was lost earlier. In C. fastigiata, however, comparison with trees inferred, e.g. from plastid sequences. stipe, calci®cation and adherence of blade siphons must have become lost along the internode leading to this species be- Phylogeny, morphology and life history cause its nearest relative, U. orientalis, still possesses these character states. Comparison between the morphological features and the mo- Recovery of uncalci®ed Chlorodesmis with calci®ed taxa lecular trees shows that patterns of change in morphological as the nearest and next nearest neighbours suggests secondary characters are not fully congruent with the molecular tree to- loss of calci®cation. However, this is not necessarily correct. pology. As an example, loss of lateral adherence among blade Chlorodesmis spp. may simply represent juvenile stages of siphons ± thus giving rise to a Penicillus-shape ± has occurred other udoteacean species not included in Fig. 2, just as Pseu- at least twice independently because Penicillus is not mono- dochlorodesmis furcellata (Zanardini) Bùrgesen and Espera phyletic (Fig. 2). mediterranea Decaisne proved to be juveniles of Mediterra- Siphonal appendages (see illustrations in Taylor 1960; Lit- nean H. tuna (Ellis & Solander) J.V. Lamouroux and Penicil- tler & Littler 1990) are encountered in all specimens included lus capitatus, respectively (Meinesz 1980). But if Chlorodes- in this study except Chlorodesmis. In Halimeda, these ap- mis constitutes the mature thallus, then secondary loss may pendages form the cortical utricles, whereas in Caulerpa they have happened in two different ways. First, these simple thalli form the fronds and rhizoids along the stolons. Comparison may result from loss of stipe and calci®cation in Penicillus, with Figs 1 and 2 reveals that acquisition of appendages pre- thus permitting cap siphons to sprawl unimpeded over the dates the emergence of the Caulerpaceae and Udoteaceae. In substratum. Indeed, Penicillus spp. are recovered as the near- Halimeda, Flabellia, Udotea, Penicillus and Rhipocephalus, est and next nearest neighbours of C. caespitosa. Second, the appendages ramify profusely to form cortical layers. Equal Chlorodesmis spp. may represent cases of neoteny, in which cortication of blade and stipe in stipitate species must be an- the juvenile stage becomes fertile (Sonder 1871; Ducker 1965, cestral because, in F. petiolata, U. dotyi, U. norrisii and U. 1967). The results presented here do not allow these hypoth- ¯abellum, the siphonal appendages in the blade are similar to eses to be tested but they could be examined in future molec- those in the stipe. The siphonal appendages have become re- ular research. If particular Chlorodesmis spp. evolved through duced to small stumps in U. argentea and to spine-like struc- neoteny, the genes needed for reproduction could be expected tures near the base of the blade in U. looensis. Siphonal ap- to remain functional both in this species and in its phenotyp- pendages are absent from the blade in the remaining Udotea ically more elaborate neighbour. In contrast, any genes needed spp. included in this study and in Penicillus and Rhipoce- for the development of the mature thallus might be expected phalus. to become pseudogenes or to be lost altogether, whereas they The stipes are generally corticated throughout. Most of the must remain functional in non-Chlorodesmis relatives. siphonal appendages in the stipes of Udoteaceae possess blunt, Features associated with the udoteacean life history appear rounded tips, whereas those in the stipes of P. dumetosus and to be more congruent with the molecular phylogenies than is its nearest neighbour, P. pyriformis, have pointed tips. This vegetative morphology. Gametangial shape and macrogamete same character state is also observed in U. looensis (Gepp & size seem to delimit natural groups among taxa included in Wednesday Jun 12 2002 12:43 PM phya 41_502 Mp_17 Allen Press • DTPro System GALLEY 17 File # 02tq

Kooistra: Phylogeny of Udoteaceae

Fig. 2. Maximum likelihood phylogram inferred from nuclear rDNA sequences comprising the SSU from c. base pair 500 onwards, an insert in this region, ITS-1, 5.8S rDNA, ITS-2 and the ®rst c. 500 base pairs of the LSU rDNA. Maximum likelihood analysis has been constrained with results from Modeltest. Substitution model: R[A↔C] ϭ 0.9316, R[A↔G] ϭ 2.0478, R[A↔T] ϭ 1.2213, R[C↔G] ϭ 0.4870, R[C↔T] ϭ 3.4396 relative to R[G↔T] (ϭ 1.0000); base frequencies: A ϭ 0.2092, C ϭ 0.2666, G ϭ 0.3118, T ϭ 0.2124; proportion of invariable sites ϭ 0.5185; unequal distribution of variable sites with gamma distribution shape parameter ϭ 0.6588. Bootstrap values have been generated using weighted (Goloboff, K ϭ 2) maximum parsimony (1000 replicates) because this algorithm produced a tree topology identical to that of the ML tree. this study, though their states are still unknown for some of Penicillus, Rhipocephalus, U. abbottiorum and U. cyathifor- these species. Compound gametangia are found in Halimeda mis possess large macrogametes with numerous ¯agella (Mei- (see illustrations in Meinesz 1980) and differ from the di- nesz 1980; Clifton & Clifton 1999). Most Halimeda spp. also chotomously branched or unbranched gametangia in Flabellia have small macrogametes, but the lineage with sand-adapted [as U. petiolata: illustrated by Gepp & Gepp (1911) and Mei- species possesses in addition somewhat enlarged macroga- nesz (1980)], C. fastigiata (Ducker 1965), Penicillus, Rhipo- metes with many ¯agella (Clifton & Clifton 1999). Within the cephalus and Udotea. Relatively small bi¯agellate macroga- con®nes of Udotea, Penicillus and Rhipocephalus, the pos- metes are present in both Flabellia and U. ¯abellum, whereas session of large macrogametes is probably a derived character. Wednesday Jun 12 2002 12:43 PM phya 41_502 Mp_18 Allen Press • DTPro System GALLEY 18 File # 02tq

Phycologia, Vol. 41 (5), 2002

Most phycological taxonomic systems have been founded on Alternatively, because the anatomy of the reproductive struc- such reproductive features and on ultrastructural traits of the tures and the characteristics of the macrogametes seem to de- gametes (Hoek et al. 1995). The Udoteaceae, and possibly lineate clades and grades in Udoteaceae, these traits could be Bryopsidales as a whole, appear to be no exception to this used to construct a more natural . Even Chlorodes- generalization. mis spp. may be classi®ed properly once their reproductive Phylogenies inferred from nonmolecular traits in Udotea- features are known. However, taxonomic revision should ceae, though noisy, reveal structure (Littler & Littler 1990; await the addition of sequence data and life-history informa- Vroom et al. 1998) that is more or less in agreement with tion from many more udoteacean taxa. what is shown in Figs 1 and 2. A cladogram inferred from nonmolecular data for bryopsidalean genera (Vroom et al. Phylogeny and gamete release intervals 1998, ®g. 1) has Caulerpa branching off ®rst, followed by Flabellia and then by U. ¯abellum, Penicillus and Halimeda. Udoteacean species shed their gametes just before dawn but, According to the present study, only Halimeda is misplaced. in contrast to corals (Veron 1995) and related organisms, they Moreover, the ®rst tree in my study (Fig. 1) separates the do this at particular times, which differ among species (Clifton Caulerpaceae from the Udoteaceae, as proposed by Silva & Clifton 1999). Clifton & Clifton hypothesized that closely (1982). Results of a similar cladistic approach in western At- lantic Udotea by Littler & Littler (1990) also agree with the related species avoid each other's gamete release intervals to molecular phylogenies presented here. Unfortunately, their diminish the risk of hybridization. Correlation of the release tree is rooted incorrectly with an ingroup taxon, R. phoenix. times given by Clifton (1997) and Clifton & Clifton (1999) If Flabellia and Halimeda had been chosen as the outgroup, and the trees presented here reveal that two closely related the branching order would have been similar to that shown in udoteacean species have overlapping periods of release at the Fig. 2. Inclusion of Penicillus or Rhipocephalus or both would sites where the authors made their observations; R. phoenix then render Udotea paraphyletic. Moreover, fully corticated sheds gametes 23.4 Ϯ 1.9 min before sunrise and U. cyathi- Udotea blades would have been identi®ed as ancestral and formis 25.5 Ϯ 0.5 min before sunrise, thus challenging the uncorticated ones (or brushes) as derived. Cortication of Clifton hypothesis. Explanations for the overlap might be that blades and stipes, as in U. ¯abellum and its relatives and Fla- the species do not normally occur close together or do not bellia, may seem more advanced, yet this state is in fact the shed their gametes during the same mornings. Alternatively, undifferentiated one: stipe and blade are both corticated alike. these taxonomically perceived species may merely represent extreme forms of the same species along an extended plastic- Phylogeny and generic concepts ity range. Such cases have led to over-taxonomization in Hal- imeda (Kooistra et al. 2002). A third possibility is that there According to this study, the genera Chlorodesmis, Penicillus are prezygotic barriers. In this case there would be no selec- and Udotea are not monophyletic and consist of species tion for temporal separation of release intervals in these close- lumped together, based on super®cial morphological similar- ly related species. But then it remains dif®cult to explain why ity. Nonmonophyletic species and genera are not uncommon this incompatibility evolved only in these closely related spe- among green algae (e.g. Kooistra et al. 1993; Bakker et al. cies, whereas more distantly related ones shed their gametes 1994, 1995), both in morphologically simple taxa and in taxa at different moments. where simple features or processes generate intricate thalli. Indeed, drastic differences in appearance among the genera Sequence variation and tempo of morphological change Udotea, Penicillus and Rhipocephalus generally result from only minor anatomical modi®cation. Recovery of Penicillus The uncorticated Udotea spp. and their allies show rapid clad- and Rhipocephalus within a paraphyletic Udotea is not so sur- ogenesis among closely related species, accompanied by con- prising once it is realized that the brush of Penicillus is little siderable phenotypic changes. This natural group must have more than an uncorticated Udotea blade in which the siphons have lost lateral adherence. Similarly, R. phoenix is essentially originated relatively recently because their nrDNA ITS se- a Udotea sp. composed of a whorl of blades instead of a single quences remain alignable. Moreover, SSU distances remain blade. small among these taxa, despite the highly elevated substitu- Moreover, Rhipocephalus, Penicillus and Udotea are not tion rate of this marker in Bryopsidales and Dasycladales very well differentiated from each other. For instance, the (Zechman et al. 1999). In contrast, Flabellia and the clade boundary between Penicillus and Udotea is vague because U. containing those Udotea spp. with completely corticated ®brosa Littler & Littler can be interpreted as having a brush- blades represent more ancient, species-poor lineages. Species like blade or a blade-like brush, depending on one's point of in these clades appear similar, suggesting that phenotypic view. Blade siphons of this species adhere only over part of change proceeds more slowly here than in the species-rich their length (see illustrations in Littler & Littler 1990, 2000). clade. Several taxa still have to be added to the udoteacean The boundary between Rhipocephalus and Penicillus is also phylogeny before a more complete insight can be gained into fuzzy because R. oblongus (Decaisne) KuÈtzing (see illustra- the distribution of taxa among the various lineages. However, tions in Littler & Littler 2000) appears to be a Rhipocephalus general concordance between the cladograms recovered by in which the blade siphons have lost adherence. Littler & Littler (1990) ± if they are regarded as unrooted ± One way to delineate natural genera would be to place all and the molecular phylogeny shown in Fig. 2 suggests that species currently in Udotea, as well as its morphologically most Udotea spp. belong to the species-rich, more rapidly distinct allies Penicillus and Rhipocephalus, in a single genus. diversifying clade. Wednesday Jun 12 2002 12:43 PM phya 41_502 Mp_19 Allen Press • DTPro System GALLEY 19 File # 02tq

Kooistra: Phylogeny of Udoteaceae

Sequence variation and evolutionary age to hide and as a food source because migration would be prohibitively risky (Hay et al. 1994). These grazers are gen- The age of the last common ancestor of all extant Udoteaceae erally so small that, instead of consuming the thallus whole- cannot be estimated accurately from fossil data. Of the Udo- sale, they puncture and devour the contents of individual utri- teaceae, only Halimeda possesses a fossil record (Elliott 1965; cles. Older Halimeda segments often show curved rows or Bucur 1994) but its ®rst appearance is dif®cult to determine patches of empty peripheral utricles (my observations). Dur- because of taxonomic uncertainties concerning pre-Cretaceous ing daylight, plastids are indirectly protected against this cryp- Halimeda fossils and the relationship between Halimeda and tofauna because small organisms venturing upon the thallus possible ancestor genera (see references in Hillis 2000). More- are highly susceptible to predation by a multitude of small over, possible gaps in the Halimeda fossil record (Hillis 2000) ®sh (ibid.). could lead to serious underestimates of the ®rst appearance Yet, the activity of these small herbivores does not explain (Springer 1995). Phylogeographical patterns may offer an al- why heavily calci®ed, completely corticated blades are ances- ternative tool for dating dichotomies in the tree topology. tral and uncorticated blades are derived. Notably, the latter are Kooistra et al. (2002) suggest a Mid- to Late Miocene age found in species-rich clades. Loss of blade cortication in Udo- (15±10 MaBP) for the last common ancestor of the extant tea and its allies could be an adaptation against nocturnal sea Halimeda diversity from patterns of vicariance in their mo- urchin grazing. In ecorticate Udotea spp., as well as in Pen- lecular phylogeny; ®ve Halimeda lineages separated almost icillus and Rhipocephalus, plastids migrate from the blade or perfectly in Atlantic and Indo-Paci®c sister clades, possibly whorl all the way into the densely calci®ed stipe. There they resulting from the closure of the pan-tropical connection are relatively protected against sea urchins. If blade or brush through the emergence of the Isthmus of Panama. Since that siphons are devoured, only polysaccharide material time, the tropical parts of the Indo-Paci®c and Atlantic have is lost; more precious nuclei and plastids are readily deployed been strictly separate. Comparable data are not yet available in a replacement (see also Littler & Littler 1999). for Udotea and its allies but may be recoverable. Indeed, many Atlantic groups of Udotea spp. (Littler & Littler 1990) have morphologically similar counterparts in the Indo-Paci®c CONCLUSIONS (Gepp & Gepp 1911). The molecular phylogeny presented here suggests polyphyly Phylogeny and adaptation to grazing for several currently perceived genera within Udoteaceae. Tra- Tropical marine benthic algae experience high grazing pres- ditionally, taxa were grouped into genera based on the super- sure (Hay et al. 1983; Hay & Taylor 1985; Lewis 1985; Lewis ®cial appearance of the vegetative thallus. However, drastic & Wainwright 1985). The apparent success of the Udoteaceae changes in thallus form result mainly from secondary losses depends on adaptations that check this herbivore onslaught and minor, often homoplasious alterations among anatomical (Hay et al. 1988, 1994; Duffy & Hay 1990; Hay 1997). Ara- traits. The anatomy of reproductive structures and the char- gonite casts, in combination with acidic, toxic secondary com- acteristics of the macrogametes may delimit natural groups pounds (Paul & Fenical 1986; Hay et al. 1994) and nocturnal because their states correlate with molecular clades. Perfectly growth (Hay et al. 1988), are proven defences against various alignable ITS sequences and little variation among SSU se- types of large grazers. When calci®ed bryopsidalean thalli are quences across Udoteaceae indicate a comparatively young crushed, as would happen in the stomach of a parrot®sh, for extant diversity with rapidly diversifying lineages. example, the intersiphonal aragonite, in combination with the intrasiphonal acids, will produce carbon dioxide gas, a pro- foundly unpleasant experience for a ®sh in the water column ACKNOWLEDGEMENTS (Hay et al. 1994). In Bryopsidales with multiaxial thalli, organelles commute Financial support was received from the A.W. Mellon Foun- through the siphonal system between the outer parts, where dation through the Smithsonian Tropical Research Institute. I they photosynthesize throughout the day, and the inner re- am grateful L. Hillis for introducing me to Halimeda speci- gions, where they retreat during darkness (Drew & Abel mens and for additional ®nancial support. I also thank K. Ar- 1990). Especially at night, the outer cortex must be a danger- ano, P. Barnes, R. Collin, W. Freshwater, L. Hillis, L. Kirken- ous place. Otherwise, there would be no reason for the mass dale, A. N'Yeurt, G. Procaccini, F. Rindi, R. Tsuda, M. Way- retreat. The behaviour cannot have evolved as a defence cott and B. Wysor for their help with sample collections. Two against large herbivores because grazing ®sh are inactive at anonymous reviewers and B. Wysor are thanked for their con- night and nocturnal sea urchins, like ®sh, devour large pieces structive comments on an earlier version of the manuscript. of the blade, including the medullary region. Instead, this commuting must have evolved primarily as a defence against small, possibly nocturnal cryptic meiofauna that are able to REFERENCES puncture individual siphons. Indeed, various small species have become adapted to eating nothing else beside bryopsi- ARNHEIM N. 1983. Concerted evolution of multigene families. In: Evo- dalean algae (Hay et al. 1994). The thalli form an ideal sub- lution of genes and proteins (Ed. by M. Nei & M. Koehn), pp. 38± 61. Sinauer Associates, Sunderland, Massachusetts. strate for such organisms because the risk of large grazers BAKKER F. T. , O LSEN J.L., STAM W.T. & VAN DEN HOEK C. 1994. The accidentally consuming them together with their habitat re- Cladophora complex: new views based on 18S rDNA gene se- mains small for reasons explained previously. However, these quences. Molecular Phylogeny and Evolution 3: 365±382. small species generally have to use the thalli both as a place BAKKER F. T. , O LSEN J.L. & STAM W.T. 1995. Evolution of nuclear Wednesday Jun 12 2002 12:43 PM phya 41_502 Mp_20 Allen Press • DTPro System GALLEY 20 File # 02tq

Phycologia, Vol. 41 (5), 2002

rDNA ITS sequences in the Cladophora albida/cericea clade (Chlo- patterns in herbivory on a Caribbean fringing reef: the effects on rophyta). Journal of Molecular Evolution 40: 640±651. plant distribution. Oecologia 58: 299±308. BOLD H.C. & WYNNE M.J. 1978. Introduction to the algae: structure HAY M.E., PAUL V.J., Lewis S.M., Tucker J. & Trindell R.N. 1988. and reproduction. Prentice-Hall, Englewood Cliffs, New Jersey. Can tropical seaweeds reduce herbivory by growing at night? Diel 720 pp. patterns of growth, nitrogen content, herbivory, and chemical versus BOROWITZKA M.A. & LARKUM A.D.W. 1977. Calci®cation in the green morphological defenses. Oecologia 75: 233±245. alga Halimeda. V. An ultrastructure study of the thallus develop- HAY M.E., KAPPEL Q.E. & FENICAL W. 1994. Synergism in plant de- ment. Journal of Phycology 13: 6±16. fenses against herbivores: interactions of chemistry, calci®cation BUCUR I.I. 1994. Lower Cretaceous and Gymnocodi- and plant quality. Ecology 75: 1714±1726. aceae from southern Carpatians and Apuseni Mountains (Romania) HENNIG W. 1965. Phylogenetic systematics. Annual Review of Ento- and the systematic position of the Gymnocodiaceae. BeitraÈge an mology 10: 97±116. der Paleontologie 19: 13±37. HERSHKOVITZ M.A. & LEWIS L.A. 1996. Deep-level diagnostic value CLIFTON K.E. 1997. Mass spawning by green algae on coral reefs. of the rDNA-ITS region. Molecular Biology and Evolution 13: Science 275: 1116±1118. 1276±1295. CLIFTON K.E. & CLIFTON L.M. 1999. The phenology of sexual repro- HILLIS D.M. & HUELSENBECK J.P. 1992. Signal, noise, and reliability duction by green algae (Bryopsidales) on Caribbean coral reefs. in molecular phylogenetic analyses. Journal of Heredity 83: 189± Journal of Phycology 35: 24±34. 195. DE QUEIROZ A. 1993. For consensus (sometimes). Systematic Biology HILLIS L.W. 2000. Phylogeny of Halimeda (Bryopsidales): linking pa- 42: 368±372. leontological, morphological and molecular data. Acta Palaeonto- DOYLE J.J. 1992. Gene trees and species trees: molecular systematics logica Romaniae, Cluj-Napoca 2: 183±189. as one-character taxonomy. Systematic Botany. 17: 144±163. HILLIS L.W., ENGMAN J.A. & KOOISTRA W.H.C.F. 1998. Morphological DREW E.A. 1993. Halimeda biomass, growth rates and sediment gen- and molecular phylogenies of Halimeda (Chlorophyta, Bryopsida- eration on reefs in the central Great Barrier Reef province. Coral les) identify three evolutionary lineages. Journal of Phycology 34: Reefs 2: 101±110. 669±681. DREW E.A. & ABEL K.M. 1990. Studies on Halimeda. III. A daily HILLIS-COLINVAUX L. 1980. Ecology and taxonomy of Halimeda: pri- cycle of chloroplast migration within the segments. Botanica Ma- mary producer of coral reefs. Advances in Marine Biology 17: 1± rina 33: 31±45. 327. DUCKER S.C. 1965. The structure and reproduction of the green algae HILLIS-COLINVAUX L. 1984. Systematics of the Siphonales. In: System- Chlorodesmis bulbosa. Phycologia 4: 149±162. atics of the green algae (Ed. by D.E.G. Irvine & D. John), pp. 271± DUCKER S.C. 1967. The genus Chlorodesmis (Chlorophyta) in the 296. Academic Press, London. Indo-Paci®c region. Nova Hedwigia 13: 145±182. HOEK C. VAN DEN,MANN D.G. & JAHNS H.M. 1995. Algae. An intro- DUFFY J.E. & HAY M.E. 1990. Seaweed adaptations to herbivory. duction to phycology. Cambridge University Press, Cambridge. 623 Bioscience 40: 368±375. pp. DURAND C., MANUEL M., BOUDOURESQUE C.F., MEINESZ A., VERLAQUE JOHNSON J.H. 1961. Limestone-building algae and algal limestones. M. & LEPARQO Y. 2002. Molecular data suggest a hybrid origin for Johnson Publishing Company, Boulder, Colorado. 297 pp. the invasive (Caulerpales, Chlorophyta) in the JOUSSON O., PAWLOWSKI J., ZANINETTI L., MEINESZ A. & BOUDOUR- Mediterranean Sea. Journal of Evolutionary Biology 15: 122±133. ESQUE C.F. 1998. Molecular evidence for the aquarium origin of the ELLIOTT G.F. 1965. The interrelationships of some Cretaceous Codi- green alga introduced to the Mediterranean Sea. aceae (calcareous algae). Palaeontology 8: 199±203. Marine Ecology Progress Series 172: 275±280. ERNST A. 1904. BeitraÈge zur Kenntnis der Codiaceen. Beihefte zum KAMURA S. 1966. On the sexual reproduction of two species of Hal- Botanischen Zentralblatt 13: 115±148. imeda (Chlorophyta). Bulletin of Arts and Science, University of the FAMAÁ P. , O LSEN J.L., STAM W.T. & P ROCACCINI G. 2000. High levels Ryukyus, Mathematics and Natural Sciences 9: 302±313. of intra- and inter-individual polymorphism in the rDNA ITS1 of KOOISTRA W.H.C.F., OLSEN J.L., STAM W.T. & H OEK C. VAN DEN. 1993. Caulerpa racemosa (Chlorophyta). European Journal of Phycology Problems related to species sampling in phylogenetic studies: an 35: 349±356. example of non-monophyly in Cladophoropsis and Struvea (Si- FELDMANN J. 1946. Sur l'heÂteÂroplastie de certaines Siphonales et leur phonocladales, Chlorophyta). Phycologia 32: 419±428. classi®cation. Comptes Rendus Hebdomadaires des SeÂances de KOOISTRA W.H.C.F., CALDERO N M. & HILLIS L.W. 1999. Development l'AcadeÂmie des Sciences, Paris 222: 752±753. of the extant diversity in Halimeda is linked to vicariant events. FELDMANN J. 1954. Sur la classi®cation des ChlorophyceÂes siphoneÂes. Hydrobiologia 398/399: 39±45. VIII CongreÁs International de Botanique. Rapports et Communi- KOOISTRA W.H.C.F., COPPEJANS E.G.G. & PAYRI C. 2002. Molecular cations 17: 96±98. systematics, historical ecology and phylogeography of Halimeda FLOYD G.L. &. O'KELLY C.J. 1989. Phylum Chlorophyta. Class Ul- (Bryopsidales). Molecular Phylogenetics and Evolution. In press. vophyceae. In: Handbook of the Protoctista (Ed. by L. Margulis, LAMOUROUX J.V.F. 1812. Extrait d'un meÂmoire sur la classi®cation des t J.O. Corliss, M. Melkonian & D.J. Chapman), pp. 617±635. Jones polypes coralligeÁnes non entieÁrement pierreux. Nouveau Bulletin & Bartlett, Boston. Scienti®que de la SocieÂte Philomatique 3: 181±188. FREILE D., MILLIMAN J.D. & HILLIS L. 1995. Leeward bank margin LEWIS S.M. 1985. Herbivory on coral reefs: algal susceptibility to Halimeda meadows and draperies and their sedimentary importance herbivorous ®shes. Oecologia 65: 370±375. on the western Great Bahama Bank slope. Coral Reefs 14: 27±33. LEWIS S.M. & WAINWRIGHT P.C. 1985. Herbivorous abundance and GEPP A. & GEPP E.S. 1911. The of the Siboga expedition grazing intensity on a Caribbean coral reef. Journal of Experimental including a monograph of Flabellarieae and Udoteae. Siboga-Ex- Marine Biology and Ecology 87: 215±228. peditie, Monographie 62: 1±150. LITTLER D.S. & LITTLER M.M. 1990. Systematics of Udotea species HANYUDA T. , A RAI S. & UEDA K. 2000. Variability in the rbcL introns (Bryopsidales, Chlorophyta) in the tropical western Atlantic. Phy- of Caulerpalean algae (Chlorophyta, ). Journal of cologia 29: 206±252. Plant Research 113: 403±413. LITTLER D.S. & LITTLER M.M. 1999. Blade abandonment/proliferation: HAY M.E. 1997. Calci®ed seaweeds on coral reefs: complex defenses, a novel mechanism for rapid epiphyte control in marine macro- trophic relationships, and value as habitats. Proceedings of the phytes. Ecology 80: 1736±1746. Eighth International Coral Reef Symposium 1: 713±718. LITTLER D.S. & LITTLER M.M. 2000. Caribbean reef : an iden- HAY M.E. & TAYLOR P.R. 1985. Competition between herbivorous ti®cation guide to the reef plants of the Caribbean, Bahamas, Flor- ®shes and urchins on Caribbean reefs. Oecologia 65: 591±598. ida and the Gulf of Mexico. Offshore Graphics, Washington, D.C. HAY M.E., COLBURN T. & D OWNING D. 1983. Spatial and temporal 542 pp. Wednesday Jun 12 2002 12:43 PM phya 41_502 Mp_21 Allen Press • DTPro System GALLEY 21 File # 02tq

Kooistra: Phylogeny of Udoteaceae

MADDISON W.P.&MADDISON D.R. 1997. MacClade: analysis of phy- (* and other methods), version 4.0b8. Sinauer Associates, Sunder- logeny and character evolution, version 3.07. Sinauer Associates, land, Massachusetts. Sunderland, Massachusetts. 398 pp. TAYLOR W.R. 1950. Plants of Bikini and other northern Marshall MAI J.C. & COLEMAN A.W. 1997. The internal transcribed spacer 2 Islands. University of Michigan Press, Ann Arbor. 227 pp. exhibits a common secondary structure in green algae and ¯owering TAYLOR W.R. 1960. Marine algae of the eastern tropical and sub- plants. Journal of Molecular Evolution 44: 258±271. tropical coasts of the Americas. University of Michigan, Ann Arbor. MEINESZ A. 1980. Connaissances actuelles et contribution aÁ l'eÂtude de 870 pp. la reproduction et du cycle des UdoteÂaceÂes (Caulerpales, Chloro- VERON J.E.N. 1995. Corals in space and time: the biogeography and phytes). Phycologia 19: 110±138. evolution of the Scleractinia. Cornell University Press, New York. NICHOLS R. 2001. Gene trees and species trees are not the same. 321 pp. Trends in Ecology and Evolution 16: 358±364. VROOM P.S., SMITH C.M. & KEELEY S.C. 1998. Cladistics of the NIZZAMUDDIN M. 1987. Observations on the genus Flabellia (Cauler- Bryopsidales: a preliminary analysis. Journal of Phycology 34: pales, Chlorophyta). Nova Hedwigia 44: 175±188. 351±360. OLSEN J.L., STAM W.T., B ERGER S. & MENZEL D. 1994. 18S rDNA WENDEL J.F., SCHNABEL A. & SEELANAN T. 1995. Bidirectional inter- and evolution in the Dasycladales (Chlorophyta): modern living fos- locus concerted evolution following allopolyploid speciation in cot- sils. Journal of Phycology 30: 729±744. ton (Gossypium). Proceedings of the National Academy of Sciences PAUL V.J. & FENICAL W. 1986. Chemical defense in tropical green of the United States of America 92: 280±284. algae, order Caulerpales. Marine Ecology Progress Series 34: 157± WHITE T.J., LEE S. & TAYLOR J. 1990. Ampli®cation and direct se- 169. quencing of fungal ribosomal RNA genes for phylogenetics. In: PILLMANN A., WOOLCOTT G.W., OLSEN J.L., STAM W.T. & K ING R.J. PCR protocols (Ed. by M.A. Innis, D.H. Gel¯and, J.J. Sninsky & 1997. Inter- and intraspeci®c genetic variation in Caulerpa (Chlo- T.J. White), pp. 315±322. Academic Press, New York. rophyta) based on nuclear rDNA ITS sequences. European Journal WOOLCOTT G.W., KNOÈ LLER K. & SING R.J. 2000. Phylogeny of the of Phycology 32: 379±386. (Bryopsidales, Chlorophyta): cladistic analyses of PONCET J. 1989. PreÂsence du genre Halimeda Lamouroux 1812 (algue morphological and molecular data. Phycologia 39: 471±481. verte calcaire) dans le Permien supeÂrieur du Sud Tunesien. Revue WRAY J.L. 1977. Calcareous algae. Elsevier, New York. 186 pp. MicropaleÂontologie 32: 40±44. ZECHMAN F. W. , T HERIOT E.C., ZIMMER E.A. & CHAPMAN R.L. 1990. POSADA D. & CRANDALL K.A. 1998. Modeltest: testing the model of Phylogeny of the Ulvophyceae (Chlorophyta): cladistic analysis of DNA substitution. Bioinformatics 14: 817±818. nuclear-encoded rRNA sequence data. Journal of Phycology 26: SILVA P. 1982. Chlorophyceae. In: Synopsis and classi®cation of living 700±710. organisms, vol. 1. (Ed. by S.P. Parker), pp. 154±156. McGraw-Hill, ZECHMAN F. W. , K OOISTRA W.H.C.F., OLSEN J.L. & STAM W.T. 1999. New York. Current perspectives on the phylogeny of ulvophycean green algae. SONDER O.G. 1871. Die Algen des tropischen Australiens. Abhan- In: Abstracts of the Sixteenth International Botanical Congress, 144 dlungen des Naturwissenschaftlichen Vereins, Hamburg 5: 33±74. pp. St Louis, Missouri. SPRINGER M.S. 1995. Molecular clocks and the incompleteness of the fossil record. Journal of Molecular Evolution 41: 531±538. SWOFFORD D.L. 2001. PAUP* phylogenetic analysis using parsimony Accepted 27 March 2002