MOLECULAR PHYLOGENY of the GENUS CAULERPA (CAULERPALES, CHLOROPHYTA) INFERRED from CHLOROPLAST Tufa GENE1
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J. Phycol. 38, 1040–1050 (2002) MOLECULAR PHYLOGENY OF THE GENUS CAULERPA (CAULERPALES, CHLOROPHYTA) INFERRED FROM CHLOROPLAST tufA GENE1 Patrizia Famà2 Laboratoire de Systématique Moléculaire, Département de Zoologie et Biologie Animale, Université de Genève, 1224 Chêne-Bougeries, Switzerland Brian Wysor Department of Biology, University of Louisiana at Lafayette, Lafayette, Louisiana 70504-451, U.S.A. Wiebe H. C. F. Kooistra Marine Botany, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy and Giuseppe C. Zuccarello Marine Biological Association of the United Kingdom, The Laboratory Citadel Hill Plymouth, PL1 2PB U.K. The genus Caulerpa consists of about 75 species cal 1986). However, these very grazer deterrents make of tropical to subtropical siphonous green algae. To bet- these plants an ideal substratum for a suite of cryptic ter understand the evolutionary history of the genus, a meiofauna. Many of these organisms feed on Caulerpa molecular phylogeny was inferred from chloroplast tufA despite the toxins (Hay et al. 1994). sequences of 23 taxa. A sequence of Caulerpella Caulerpa belongs to the Bryopsidophyceae (Van den ambigua was included as a potential outgroup. Re- Hoek et al. 1995), a class of algae with a coenocytic sults reveal that the latter taxon is, indeed, sister to thallus organization. Each thallus is essentially a single all ingroup sequences. Caulerpa itself consists of a cell that develops into an elaborate system of branching series of relatively ancient and species-poor lineages siphons. Caulerpa is defined by the presence of trabecu- and a relatively modern and rapidly diversifying clade, lae: inwardly projecting cylindrical extensions of cell containing most of the diversity. The molecular phylog- wall material passing through the central lumen of eny conflicts with the intrageneric sectional classifica- the siphons (Lamouroux 1809, Bold and Wynne 1985). tion based on morphological characters and an evolu- Thalli are composed of a prostrate rhizome (stolon), tionary scheme based on chloroplast ultrastructure. branched anchoring rhizoids, and upright branches (as- High bootstrap values support monophyly of C. mexi- similators) that bear distinctive branchlets and are used cana, C. sertularioides, C. taxifolia, C. webbiana, and C. in species identification. These units, called metameres prolifera, whereas most other Caulerpa species show (White 1979), can potentially regenerate new ramets para- or polyphyly. after a frond or stipe is cut. Gametogenesis involves Key index words: Caulerpa; chloroplast DNA; phylog- migration of cytoplasm into unspecialized gametangia eny; systematics; tufA where it is transformed into anisogamous gametes (Goldstein and Morrall 1970, Enomoto and Ohba Abbreviation: tufA, elongation factor TU 1987). Just before dawn, micro- and macrogametes are shed in the water column in species-specific brief release intervals (Clifton 1997, Clifton and Clifton 1999). The Bryopsidalean genus Caulerpa comprises a Caulerpa includes about 75 species worldwide group of conspicuous algae distributed in a range of (Weber-van Bosse 1898, Calvert et al. 1976, Price et al. habitats throughout the tropical and subtropical ma- 1998). Many taxa form discrete well-delimited units rine realm (Dawson 1966, Hay et al. 1985, Meinesz with relatively little morphological variability. Yet some and Boudouresque 1996). Recently, the genus at- taxonomically perceived species exhibit rampant mor- tracted considerable research interest because species phological plasticity and ill-defined taxonomic bound- expanded their ranges into more temperate environ- aries. Variability in growth forms and in the photosyn- ments (Meinesz and Hesse 1991, Piazzi et al. 1994, thetic performance of Caulerpa species seem to be Dalton 2000, Kaiser 2000). Most species are well de- related to substrate, light intensity, and water motion fended against large grazers by a suite of toxic com- (Gacia et al. 1996, Collado-Vides and Robledo 1999). pounds (de Paula and de Oliveira 1982, Paul and Feni- Sectional division among taxa (Agardh 1872, Weber- van Bosse 1898) is predominantly supported by differ- ences in assimilator morphology. These assimilators, 1Received 17 December 2001. Accepted 2 June 2002. however, can be highly plastic and seem under strong 2Author for correspondence: e-mail [email protected]. control of the environment (Gilbert 1941, Calvert 1976, 1040 PHYLOGENY OF CAULERPA 1041 Ohba et al. 1992). Therefore, species boundaries, spe- Algal specific forward and reverse primers for the tufA gene cies relationships, and sectional divisions are ques- were designed based on a sequence alignment of 14 algal taxa deposited in GenBank. The forward and reverse primers an- tionable. neal at position 210 (tufAF 5Ј-TGAAACAGAAMAWCGTCATT Ultrastructural traits and DNA sequence differences ATGC-3Ј) and 1062 (tufAR 5Ј-CCTTCNCGAATMGCRAAW have been applied to resolve phylogenetic relationships CGC-3Ј), respectively, of the Codium fragile (Suringar) Hariot among taxa within Caulerpa. A phylogeny of 28 Caulerpa tufA gene sequence (GenBank accession number U09427). species, based on chloroplast ultrastructure (Calvert et Double-stranded DNAs were amplified using PCR following two protocols. al. 1976), reflected an evolutionary trend from puta- In the first PCR procedure, reactions were performed in a tively ancestral, large, pyrenoid-containing chloro- total volume of 50 L consisting of 5 mM MgCl2, 0.3 mM each plasts to small chloroplasts lacking pyrenoids. More primer, 0.2 mM each dNTP, 0.5 units of Taq DNA polymerase recently, molecular studies using allozymes (Benzie et (Roche Diagnostics, Rotkreuz, Switzerland), and 1.0 L of 10ϫ dilution of template DNA. The reactions were exposed to the al. 1997), chloroplast DNA RFLP (Satoh et al. 1992, following PCR profile: 40 cycles of denaturation (94Њ C for 1 Lehman and Manhart 1997), and nuclear rDNA or chlo- min), primer annealing (52Њ C for 1 min), and extension (72Њ C roplast DNA sequences (Pillman et al. 1997, Jousson et for 2 min). A 5-min final extension cycle at 72Њ C followed the al. 1998, 2000, Olsen et al. 1998, Famà et al. 2000, 40th cycle to ensure the completion of all novel strands. In the Hanyuda et al. 2000) showed high intraspecific or even second protocol, a PCR master mix of 13 L was prepared con- sisting of 2.5 mM MgCl2, 0.5 mm each primer, 0.2 mM each intraindividual differences in chloroplast DNA size and dNTP, 1.0 M Betaine, 0.5 units of Taq DNA polymerase (PE Ap- nuclear rDNA polymorphism. Such patterns hamper de- plied Biosystems, Foster City, CA, U.S.A.), and 0.5–1.0 L of 1ϫ termination of evolutionary relationships in this genus. or 100ϫ dilution of template DNA. This procedure involved an The chloroplast gene tufA encodes for elongation initial denaturation at 94Њ C for 3 min, followed by 40 cycles of denaturation (94Њ C for 1 min), primer annealing (45Њ C for 1 factor TU, a molecule that mediates the entry of an min), and extension (72Њ C for 2 min) followed by a final exten- amino-acyl-tRNA into the acceptor site of a ribosome sion step at 72Њ C for 4 min. In instances in which a very small during elongation of the nascent polypeptide chain in amount of PCR product was obtained, the faint band was ex- protein synthesis (Lewin 1997). This gene is encoded cised from a low melting point agarose gel and used as tem- by the chloroplast genome of photosynthetic algae plate in a subsequent amplification with the same primers and PCR conditions described above. but is nuclear encoded in some Charophyceae and in Double-stranded PCR products were cleaned using the land plants (Baldauf et al. 1990, Bonny and Stutz High Pure PCR Product Purification Kit (Roche Diagnostics) 1993). The tufA gene is a good candidate for phyloge- or excised from a low melting point agarose gel and digested netic studies above the species level because of its con- using the GELase™ Agarose Gel-Digesting Preparation (Epi- centre Technologies, Madison, WI, U.S.A.) before sequencing. served nature across a wide range of organisms. Until The double-stranded PCR products were used as templates in recently, however, tufA sequences have been used cycle sequencing reactions. Sequencing primers were the same only to address phylogenetic questions at suprage- as those used for amplification. PCR products were sequenced neric levels (Ludwig et al. 1990, Delwiche et al. 1995, using the Big Dye Terminator Cycle Sequencing Ready Reac- Baldauf et al. 1996). tion Kit (Applied Biosystems, Rotkreuz, Switzerland) on an ABI-3100 or an ABI-377 DNA automated sequencer (Applied In this study, we inferred a phylogeny from partial Biosystems), following manufacturer’s instructions. chloroplast tufA sequences among 23 described taxa Molecular data analysis. Sequences were aligned manually using and a taxon morphologically divergent from all de- the Genetic Data Environment software, version 2.2 (Larsen et al. scribed Caulerpa species to test the usefulness of this 1993). The complete alignment is submitted under EMBL acces- sion number ALIGNϪ000315. Phylogenetic signal among parsi- gene in resolving phylogenetic relationships at the ge- mony-informative sites was assessed by comparing the measure nus level. A sequence of a putative close outgroup, Caul- of skewedness (g1-value, PAUP* version 4.0b6, Swofford 2000) erpella ambigua (Prud’homme van Reine and Lokhorst with empirical threshold values in Hillis and Huelsenbeck 1992), was also examined to root the obtained phylog- (1992). To determine