A FORENSIC AND PHYLOGENETIC SURVEY OF CAULERPA SPECIES (CAULERPALES,CHLOROPHYTA) FROM THE FLORIDA COAST,LOCAL AQUARIUM SHOPS,AND E‐COMMERCE: ESTABLISHING A PROACTIVE BASELINE FOR EARLY DETECTION1

Baseline genotypes were established for 256 individuals of Caulerpa collected from 27 field locations in Florida (including the Keys), the Bahamas, US Virgin Islands, and Honduras, nearly doubling the number of available GenBank sequences. On the basis of sequences from the nuclear rDNA‐ITS 1+2 and the chloroplast tufA regions, the phylogeny of Caulerpa was reassessed and the presence of invasive strains was determined. Surveys in central Florida and southern California of >100 saltwater aquarium shops and 90 internet sites revealed that >50% sold Caulerpa. Of the 14 Caulerpa species encountered, Caulerpa racemosa was the most common, followed by Caulerpa sertularioides, Caulerpa prolifera, Caulerpa mexicana, and Caulerpa serrulata. None of the >180 field‐collected individuals (representing 13 species) was the invasive strain of Caulerpa taxifolia or C. racemosa. With one exception (a sample of C. racemosa from a shop in southern California belonged to the invasive Clade III strain), no invasive strains were found in saltwater aquarium stores in Florida or on any of the internet sites. Although these results are encouraging, we recommend a ban on the sale of all Caulerpa species (including “live rock”) because: morphological identification of Caulerpa species is unreliable (>12% misidentification rate) and invasive strains can only be identified by their aligned DNA sequences, and because the potential capacity for invasive behavior in other Caulerpa species is far from clear. The addition of the Florida region to the genetic data base for Caulerpa provides a valuable proactive resource for invasion biologists as well as researchers interested in the evolution and speciation of Caulerpa.     

ANALYSIS OF A PLASTID MULTIGENE DATA SET AND THE PHYLOGENETIC POSITION OF THE MARINE MACROALGA CAULERPA FILIFORMIS (CHLOROPHYTA)1

Molecular phylogenetic relationships within the Chlorophyta have relied heavily on rRNA data. These data have revolutionized our insight in green algal evolution, yet some class relationships have never been well resolved. A commonly used class within the Chlorophyta is the Ulvophyceae, although there is not much support for its monophyly. The relationships among the Ulvophyceae, Trebouxiophyceae, and Chlorophyceae are also contentious. In recent years, chloroplast genome data have shown their utility in resolving relationships between the main green algal clades, but such studies have never included marine macroalgae. We provide partial chloroplast genome data (～30,000 bp, 23 genes) of the ulvophycean macroalga Caulerpa filiformis (Suhr) K. Herig. We show gene order conservation for some gene combinations and rearrangements in other regions compared to closely related taxa. Our data also revealed a pseudogene (ycf62) in Caulerpa species. Our phylogenetic results, based on analyses of a 23‐gene alignment, suggest that neither Ulvophyceae nor Trebouxiophyceae are monophyletic, with Caulerpa being more closely related to the trebouxiophyte Chlorella than to Oltmannsiellopsis and Pseudendoclonium.     

MOLECULAR PHYLOGENY OF THE GENUS CAULERPA (CAULERPALES,CHLOROPHYTA) INFERRED FROM CHLOROPLAST tufA GENE1

The genus Caulerpa consists of about 75 species of tropical to subtropical siphonous green algae. To better understand the evolutionary history of the genus, a molecular phylogeny was inferred from chloroplast tufA sequences of 23 taxa. A sequence of Caulerpella ambigua was included as a potential outgroup. Results reveal that the latter taxon is, indeed, sister to all ingroup sequences. Caulerpa itself consists of a series of relatively ancient and species‐poor lineages and a relatively modern and rapidly diversifying clade, containing most of the diversity. The molecular phylogeny conflicts with the intrageneric sectional classification based on morphological characters and an evolutionary scheme based on chloroplast ultrastructure. High bootstrap values support monophyly of C. mexicana, C. sertularioides, C. taxifolia, C. webbiana, and C. prolifera, whereas most other Caulerpa species show para‐ or polyphyly.     

DEFINING TAXON BOUNDARIES IN MEMBERS OF THE MORPHOLOGICALLY AND GENETICALLY PLASTIC GENES CAULERPA (CAULERPALES,CHLOROPHYTA)1

Numerous attempts to capture the morphological variability of the genus Caulerpa have resulted in an unstable classification of numerous varieties and formae. In the present study we attempted to test taxon boundaries by investigating morphological and genetic variation within and between seven taxa of Caulerpa, supposedly belonging to four species, sampled at different sites in a Philippine reef system. Using both field and culture observations, we described the relation between the variability of a set of morphological characters and ecological parameters, such as wave exposure, light intensity, and substrate type. Statistical analyses showed that the limits between two (out of three) ecads of the C. racemosa (Forsskål) J. Agardh complex were obscured by the presence of morphological plasticity. Other studied taxa of Caulerpa (i.e. C. cupressoides [Vahl] C. Agardh, C. serrulata [Forsskål] J. Agardh, and two formae of C. sertularioides [S. Gmelin] Howe) could be grouped based on morphology despite the presence of morphological plasticity. Our results indicated a strong association between light intensity and several quantitative morphological variables. Genetic diversity of these taxa was assessed by partial sequencing chloroplast rbcL and tufA genes and the ycf10‐chlB chloroplast spacer. In all phylogenetic analyses, C. serrulata, C. cupressoides, C. sertularioides, and the three ecads of C. racemosa emerged as distinct genetic units. Despite the presence of morphological plasticity and morphological convergence, a subset of morphological characters traditionally used in taxonomic delimitation still had sufficient discriminative power to recognize the terminal phylogenetic clades.     

ORGANIZATION OF THE CHLOROPLAST GENOME OF THE FRESHWATER CENTRIC DIATOM CYCLOTELLA MENEGHINIANA1

We constructed a complete physical map and a partial gene map of the chloroplast genome of Cyclotella meneghiniana Kützing clone 1020-1a (Bacillariophyceae). The 128-kb circular molecule contains a 17-kb inverted repeat, which divides the genome into single copy regions of65 kb and 29 kb. This is the largest genome and inverted repeat found in any diatom examined to date. In addition to the 16S and 23S ribosomal RNA genes, the inverted repeat contains both the ndhD gene (as yet unexamined in other diatoms) and the psbA gene (located similarly in one of two other examined diatoms). The Cyclotella chloroplast genome exists as two equimolar populations of inversion isomers that differ in the relative orientation of their single copy sequences. This inversion heterogeneity presumably results from intramolecular recombination within the inverted repeat. For the first time, we map the ndhD, psaC, rpofi, rpoCl, and rpoC2 genes to the chloroplast genome of a chlorophyll c-containing alga. While the Cyclotella chloroplast genome retains some prokaryotic and land plant gene clusters and operons, it contains a highly rearranged gene order in the large and small single copy regions compared to all other examined diatom, algal, and land plant chloroplast genomes.     

Physical map of chloroplast DNA in sugi,Cryptomeria japonica

Summary To investigate the evolution of conifer species, we constructed a physical map of the chloroplast DNA of sugi, Cryptomeria japonica, with four restriction endonucleases, PstI, SalI, SacI and XhoI. The chloroplast genome of C. japonica was found to be a circular molecule with a total size of approximately 133 kb. This molecule lacked an inverted repeat. Twenty genes were localized on the physical map of C. japonica cpDNA by Southern hybridization. The chloroplast genome structure of C. japonica showed considerable rearrangements of the standard genome type found in vascular plants and differed markedly from that of tobacco. The difference was explicable by one deletion and five inversions. The chloroplast genome of C. japonica differed too from that of the genus Pinus which also lacks one of the inverted repeats. The results indicate that the conifer group originated monophyletically from an ancient lineage, and diverged independently after loss of an inverted repeat structure.     

PHYLOGENETIC RELATIONSHIPS OF CAULERPA (CHLOROPHYTA) BASED ON COMPARATIVE CHLOROPLAST ULTRASTRUCTURE1,2

The ultrastructure of chloroplasts from 28 of the 73 species of Caulerpa Lamouroux (Chlorophyta, Caulerpales) has been studied to aid in interpreting phylogenetic relationships among the 12 recognized sections. Variations of systematic value include pyrenoid occurrence and fine structure, thylakoid architecture and amount of photosynthate storage. Comparisons of field and culture specimens indicate these characters are consistent. Chloroplast thylakoids are grouped into bands, with the distribution of bands differing among species. In the most common arrangement, bands are evenly distributed throughout the chloroplast. A few species show lateral displacement of bands whereas others have a majority of bands arranged at one end of the chloroplast. Starch is stored cither as one or two large grains (> 1 μm diam.) or numerous small grains (< 0.5 μm diam.). Electron-transparent regions are common in other species in which chloroplasts rarely store starch. Simple, embedded pyrenoids are present in several species of section Sedoideae. An opaque region occurs in chloroplasts of C. elongata which may represent an intermediate stage in the evolutionary loss of the pyrenoid. It is suggested that the chloroplast of Caulerpa evolved, from a large, complex, pyrenoid-containing organelle housing both photosynthetic and amylogenic functions, to a small, structurally simpler one, specialized for photosynthesis alone. A phylogeny of the 12 sections of Caulerpa is constructed, based on chloroplast evolution which agrees with an earlier morphology-based hypothesis on the origin and evolution of Caulerpa.     

A physical map of the sorghum chloroplast genome

Summary The chloroplast genome of the IS1112C cytoplasm of sorghum was mapped by the construction of a Bam-HI library in pUC8, and hybridization with BamHI, SalI, and PstI digests of chloroplast DNA (ctDNA) of sorghum and maize. The molecules are extensively colinear, with only one of 13 SalI fragments differing slightly from maize. Seven of 70 restriction sites differed in the two species. A total molecular size of ca. 138 kb was estimated for sorghum. The inverted repeat was not conserved between sorghum and maize, as revealed by a slightly larger BamHI 16S rDNA fragment in sorghum. Homology of a sequence adjacent to the bcl gene and one end of the inverted repeat was detected. These homologies were also observed in maize, and suggest that the ctDNA genomes of sorghum and maize share small reiterations of sequences of the inverted repeat.USDA-ARS     

Chloroplast genome organisation in sugar beet and maize

Summary The XhoI and SmaI restriction map of the chloroplast genome from the fertile cytoplasm of sugar beet has been constructed from overlapping cosmid clones. The genome was found to be typical of that of a dicotyledonous species, being 147.3 kb in size and having an inverted repeat. RbcL for the large subunit of ribulose-1,5-bisphosphate carboxylase, psbA for the 32 kD protein of the photosystem II reaction centre, and the 16S ribosomal RNA were located using heterologous probes. In both sugar beet and maize the inverted repeats recombine giving two isomeric forms of the genome.     

THE PHYLOGENY OF CAULERPA BASED ON RDNA INTERNAL TRANSCRIBED SPACER SEQUENCES

Phylogenetic hypotheses for the pantropical marine green algal genus, Caulerpa , were inferred based on analyses of nuclear-encoded rDNA internal transcribed spacer (ITS) sequences. Results of these analyses were used to assess the correspondence between rDNA phylogeny and traditional sectional taxonomy, to identify synapomorphic morphological characters (including assimilator morphology and chloroplast ultrastructure), and to examine marine biogeographic hypotheses for the genus. Ribosomal DNA ITS sequences were aligned for thirty-three species and intraspecific taxa of Caulerpa. Results indicate limited correspondence between phylogeny and sectional taxonomy for the genus, (e.g., the sections Filicoideae and Sedoideae were not monophyletic). In contrast, chloroplast morphology could be mapped to the tree topology with limited homoplasy. Pantropical isolates of the filicoidean species, Caulerpa sertularioides and Caulerpa mexicana each formed monophyletic groups. Caulerpa reyesii was included as a derived taxon within the Caulerpa taxifolia clade, suggesting that these species were conspecific and affirmed the lack of correspondence between phylogeny and assimilator morphology. Isolates and various intraspecific taxa of Caulerpa racemosa did not form a monophyletic group. Instead, these taxa formed a heterogeneous assemblage with other sedoidean and filicoidean taxa. Within the C. sertularioides clade, Caribbean and Atlantic isolates formed a basal paraphyletic group, whereas eastern and western Pacific isolates formed a more derived monophyletic group. Therefore, these results are not consistent with an Indo-West Pacific origin of this species.     

Gene rearrangements in Chlamydomonas chloroplast DNAs are accounted for by inversions and by the expansion/contraction of the inverted repeat

To gain insight into the mutational events responsible for the extensive variation of chloroplast DNA (cpDNA) within the green algal genus Chlamydomonas, we have investigated the chloroplast gene organization of Chlamydomonas pitschmannii, a close relative of the interfertile species C. eugametos and C. moewusii whose cpDNAs have been well characterized. At 187 kb, the circular cpDNA of C. pitschmannii is the smallest Chlamydomonas cpDNA yet reported; it is 56 and 105 kb smaller than those of its C. eugametos and C. moewusii counterparts, respectively. Despite this substantial size difference, the arrangement of 77 genes on the C. pitschmannii cpDNA displays only three noticeable differences from the organization of the corresponding genes on the collinear C. eugametos and C. moewusii cpDNAs. These changes in gene order are accounted for by the expansion/contraction of the inverted repeat and one or two inversions in a single-copy region. In land plant cpDNAs, these kinds of events are also responsible for gene rearrangements. The large size difference between the C. pitschmannii and C. eugametos/C. moewusii cpDNAs is mainly attributed to multiple events of deletions/additions as opposed to the usually observed expansion/contraction of the inverted repeat in land plant cpDNAs. We also found that the mitochondrial genome of C. pitschmannii is a circular DNA molecule of 16.5 kb which is 5.5 and 7.5 kb smaller than its C. moewusii and C. eugametos counterparts, respectively.     

Chloroplast DNA rearrangements are more frequent when a large inverted repeat sequence is lost

We examined the arrangement of sequences common to seven angiosperm chloroplast genomes. The chloroplast DNAs of spinach, petunia and cucumber are essentially colinear. They share with the corn chloroplast genome a large inversion of approximately 50 kb relative to the genomes of three legumes--mung bean, pea and broad bean. There is one additional rearrangement, a second, smaller inversion within the 50 kb inversion, which is specific to the corn genome. These two changes are the only detectable rearrangements that have occurred during the evolution of the species examined (corn, spinach, petunia, cucumber and mung bean) whose chloroplast genomes contain a large inverted repeat sequence of 22-25 kb. In contrast, we find extensive sequence rearrangements in comparing the pea and broad bean genomes, both of which have deleted one entire segment of the inverted repeat, and also in comparing each of these to the mung bean genome. Thus there is a relatively stable arrangement of sequences in those genomes with the inverted repeat and a much more dynamic arrangement in those that have lost it. We discuss several explanations for this correlation, including the possibility that the inverted repeat may play a direct role in maintaining a conserved arrangement of chloroplast DNA sequences.     

Chloroplast DNA variation among five species ofRanunculaceae: Structure,sequence divergence,and phylogenetic relationships

A restriction site map of the chloroplast genome ofCaltha palustris L. (Ranunculaceae) has been constructed for 13 restriction endonucleases using filter hybridization with cloned tobacco chloroplast DNA fragments. A size of 153.8 kb has been estimated for theCaltha chloroplast genome. Forty-six chloroplast genes and four open reading frames have been mapped using small tobacco chloroplast gene probes. Chloroplast DNA sequence divergence has been estimated for all pairs of five species ofRanunculaceae, Caltha palustris, Ranunculus bulbosus, R. fascicularis, R. recurvatus, andTrollius ledebourii, and ranges between 0.2% and 9.6% for the total genome. Divergence values are much higher in the small and large single copy regions than in the inverted repeat. Phylogenetic relationships between the five species have been hypothesized using chloroplast DNA restriction site mapping. One hundred and six informative restriction site mutations have been detected using eleven restriction endonucleases. Cladistic analyses of the restriction site mutations have been performed using Wagner and Dollo parsimony algorithms, and confidence intervals have been calculated for the resulting monophyletic groups using bootstrapping. It is demonstrated that restriction site comparisons are applicable to theRanunculaceae on intergeneric level, with the exception of groups having extensive genomic rearrangements. Moreover, sequence divergence is low enough at the interspecific level to allow phylogenetic analyses within genera such asRanunculus.     

AN ULTRASTRUCTURAL STUDY OF THE GIANT GREEN ALGAL COENOCYTE, CAULERPA PROLIFERA

The general fine structure of the giant coenocyte Caulerpa prolifera (Forsskål) Lamouroux is presented. The cytoplasm forms a parietal layer throughout the plant without any regular membranous separations within organs or between organs. The vacuome is similar in structure. There are distinct patterns of organelle distribution in the highly polar cytoplasm of rhizoids and blades. The organelles are compared to those of other members of the division Chlorophyta and are typical. Amyloplast structure is compared to that of chloroplasts. A possible developmental sequence from chloroplast buds through an unusual circular body is suggested.     

Physical and gene mapping of chloroplast DNA from Atriplex triangularis and Cucumis sativa.

A rapid and simple method for constructing restriction maps of large DNAs (100-200 kb) is presented. The utility of this method is illustrated by mapping the Sal I, Sac I, and Hpa I sites of the 152 kb Atriplex triangularis chloroplast genome, and the Sal I and Pvu II sites of the 155 kb Cucumis sativa chloroplast genome. These two chloroplast DNAs are very similar in organization; both feature the near-universal chloroplast DNA inverted repeat sequence of 22-25 kb. The positions of four different genes have been localized on these chloroplast DNAs. In both genomes the 16S and 23S ribosomal RNAs are encoded by duplicate genes situated at one end of the inverted repeat, while genes for the large subunit of ribulose-1,5-bisphosphate carboxylase and a 32 kilodalton photosystem II polypeptide are separated by 55 kb of DNA within the large single copy region. The physical and genetic organization of these DNAs is compared to that of spinach chloroplast DNA.     

THE DEVELOPMENT AND POTENTIAL ROLES OF CELL‐WALL TRABECULAE IN CAULERPA MEXICANA (CHLOROPHYTA)1

Siphonous plants represent an alternate scheme to the way most macroscopic plants are constructed. They are single, often large (1–2 m), sometimes morphologically complex, multinucleate (coenocytic) cells where the whole of the cytoplasm is a continuum. Caulerpa mexicana Sond. ex Kütz. is a siphonous tropical marine green alga characterized by four morphologically distinct regions and, as with other members of the genus, by the presence of a dense network of anastomosing cylindrical cell wall in growths called trabeculae. Based on the results of this study, we propose several roles for trabeculae: (i) They are structural components, which likely add some small amount of support in compression but add considerable strength in tension. (ii) As extensions of the cell wall and plasma membrane, they act as diffusion channels from the cell exterior to the interior cytoplasm. It is possible that trabeculae also play a role in determining cell shape through developmental positioning and placement patterns, thus facilitating the diverse shapes found in the morphologically distinct regions of Caulerpa sp.     

Unusual characteristics of Codium fragile chloroplast DNA revealed by physical and gene mapping

Summary A complete physical map of the Codium fragile chloroplast genome was constructed and the locations of a number of chloroplast genes were determined. Several features of this circular genome are unusual. At 89 kb in size, it is the smallest chloroplast genome known. Unlike most chloroplast genomes it lacks any large repeat elements. The 8 kb spacer region between the 16 S and 23 S rRNA genes is the largest such spacer characterized to date in chloroplast DNA. This spacer region is also unusual in that it contains the rps12 gene or at least a portion thereof. Three regions polymorphic for size are present in the Codium chloroplast genome. The psbA and psbC genes map closely to one of these regions, another region is in the spacer between the 16 S and 23 S rRNA genes and the third is very close to or possibly within the 16 S rRNA gene. The gene order in the Codium genome bears no marked resemblance to either the consensus vascular plant order or to that of any green algal or bryophyte genome. Present address: Department of Biology, Texas A&M University, College Station, TX 77843; USA     

COLINEARITY OF CHLOROPLAST GENOMES IN DIVERGENT ECOTYYES OF THE MARINE DIATOM SKELETONEMA COSTATUM (BACILLARIOPHYTA)1

The chloroplast genomes of three isolates of the marine diatom Skeletonema costatum (Grev.) Cleve were mapped and found to be 131 ± 2 kb with inverted repeats (IRs) of approximately 20 kb. In contrast to higher plants, the psbA gene mapped to the IR, and rbcS mapped to the same fragment as the rbcL gene in the large single-copy region. The maps of the three isolates were colinear and revealed as many as 20 site mutations out of a total of 47 sites. The number of site mutations among isolates was consistent with previous data on their genetic diversity and physiology. Comparisons of gene order among our maps and those of three other diatom species showed that closely related genera retained similar gene orders but that more distantly related taxa exhibited extensive rearrangements. We conclude that simple restriction fragment analysis of chloroplast DNA is useful in comparative studies of diatom populations and species but that other analytical methods are more appropriate for phylogenetic studies at higher levels.     

Morphological and molecular clarification of the enigmatic Caulerpa floridana W.R. Taylor (Chlorophyta,Bryopsidales) from the Dry Tortugas,Florida

Morphological and molecular evidence is provided to further document the status of the enigmatic taxon known as Caulerpa floridana W.R. Taylor from White Shoal, Dry Tortugas, Florida. DNA sequencing of three historical herbarium specimens (WRT329, WRT345 and WRT349) housed at the University of Michigan Herbarium (MICH) demonstrated the molecular separation of this species based on the reconstruction of 931 nucleotides of the chloroplast gene tufA. Caulerpa floridana is sister to the western Atlantic endemic C. ashmeadii Harvey and an unknown Caulerpa taxon from the Florida Middle Grounds. Caulerpa floridana most reliably differs from C. ashmeadii by the presence of a sharp, unequivocal apiculus at the tip of each pinnule. A morphological review of southwestern Atlantic records of C. floridana from Brazil excludes these reports as representative of the species.