PSEUDO-NITZSCHIA PUNGENS AND P. MULTISERIES (BACILLARIOPHYCEAE): NOMENCLATURAL HISTORY,MORPHOLOGY, AND DISTRIBUTION1

Pseudo-nitzschia pungens f: multiseries is raised in rank from form to species based on morphological, physiological, and genetic features. Distinctive details of the valve face striae, the valve mantle, and the girdle of P. pungens and P. multiseries are outlined. The nomenclatural history and the distribution of the two species and their relationship to other species of the genus are discussed.     

MOLECULAR DELINEATION OF SPECIES AND SPECIES RELATIONSHIPS IN THE RED ALGAL AGAROPHYTES GRACILARIOPSIS AND GRACILARIA (GRACILARIALES)1

Delineation of species in the economically important agarophyte genera Gracilaria and Gracilariopsis has proven extremely difficult using available morphological characteristics. In this study, we examine the usefulness of two transcribed spacers for molecular systematic studies of these genera. The polymerase chain reaction was used to amplify the internal transcribed spacers (ITSs) and the intervening 5.8S ribosomal DNA of the nuclear ribosomal repeat region. In addition, a plastid spacer region and flanking regions of coding genes were amplified from the RUBISCO operon. Both regions were sequenced for individuals and populations of Gracilariopsis lemaneiformis (Bory) Dawson, Acleto, et Foldvik to determine the usefulness of these spacers in delimiting populations. These studies reveal that there is as much variation among individuals of a population as there is between individuals of geographically separate populations. In addition, the ITS spacer regions were compared between different species of Gracilariopsis and Gracilaria. The nuclear ITS spacer region is conserved at a species level in both genera and provides phylogenetically informative characters that can be used to examine species interrelationships among relatively closely related taxa. However, because of the difficulties of aligning this entire region among species from the two genera, the ITS region is not useful for examining intergenera relationships. ITS interspecies sequence comparisons indicate that Gracilariopsis lemaneiformis from California is significantly different from G. lemaneiformis from China and that a species of Gracilariopsis from Peru is more closely related to G. lemaneiformis from North Carolina than it is to the other Gracilariopsis species examined. In addition, these studies indicate that Gracilaria chilensis Bird, McLachlan, et Oliveira from New Zealand and Gracilaria tenuistipitata Chang et Xia from southeast Asia are as closely related as are Gracilaria verrucosa (Hudson) Papenfuss, G. pacifica Abbott, and Gracilaria robusta Kylin. Phylogenetic analysis of aligned plastid spacer sequences from Gracilaria and Gracilariopsis taxa provide similar conclusions about species relationships.     

新科Rhizoplacopsidaceae中的新属及新种Rhizoplacopsis weichingii

本文描述了位于新科盾叶科 Rhizoplacopsidaceae(Umbilicariales,Lecanoromycetes,Ascomycota)中的新属盾叶属 Rhizoplacopsis 和新种蔚青盾叶 Rhizoplacopsis weichingii。蔚青盾叶取名于著名中国真菌学家王云章教授之字“蔚青”,作为庆祝教授百岁生日之纪念。该新种在外形上与 Rhizoplaca 属地衣极为相似,但位于地衣体上的子囊盘却为网衣型。此外,它的子囊顶器结构非常接近于 Umbilicaria-type。基于分子数据,对 Rhizoplacopsis weichingii 及其它相关地衣进行的系统发育分析支持成立新属盾叶属 Rhizoplacopsis 和新科盾叶科 Rhizoplacopsidaceae。     

NUCLEAR RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER REGIONS (ITS1 AND ITS2) DEFINE DISCRETE BIOGEOGRAPHIC GROUPS IN CLADOPHORA ALBIDA (CHLOROPHYTA)1

Nucleotide sequences of the nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2), the 5.8S, and short stretches of the adjacent 18S and 26S coding regions were determined in isolates from four disjunct Cladophora albida (Huds.) Kütz. populations (NE-America, W-Europe, Japan, and W-Australia). The two Pacific isolates share nearly identical ITS sequences as do the two Atlantic isolates. In contrast, interoceanic comparisons exhibit a 21% sequence difference. Variation within ITS regions is useful for identification of population groups on a regional or oceanic scale. However, both spacers are characterized by numerous repeat motifs as well as point mutations, which result in alignment problems at the interspecific level within Cladophora.     

ECOLOGICAL DIFFERENTIATION BETWEEN SYMPATRIC PSEUDOCRYPTIC SPECIES IN THE ESTUARINE BENTHIC DIATOM NAVICULA PHYLLEPTA (BACILLARIOPHYCEAE)1

The occurrence of cryptic and pseudocryptic species, often living in sympatry, is widespread among microalgae. This phenomenon raises important questions about niche partitioning between these closely related species. To date, however, few studies have addressed the ecological mechanisms underlying sympatry in cryptic and pseudocryptic species. As a result, we have only a limited understanding of the factors that govern their distribution along environmental gradients. Here, we used the ribosomal internal transcribed spacer (ITS), 18S rRNA gene, and the RUBISCO LSU (rbcL) chloroplast gene sequence data together with cell wall morphology to show that estuarine populations of the widespread and common benthic diatom Navicula phyllepta Kütz. consist of pseudocryptic species. Growth rate measurements in function of salinity showed that N. phyllepta strains assigned to the different species differed in their tolerance to low salinities (<5 practical salinity units, psu), which was reflected by their different (but widely overlapping) distribution in the Westerschelde estuary (the Netherlands). Multiple regression analyses of the factors determining the abundance of the different species in field samples revealed that, in addition to salinity, sediment type and ammonium concentrations were probably equally important. Our results show that N. phyllepta sensu lato comprises different species with specialized ecophysiological characteristics rather than generalists with a broad adaptability to different environmental conditions.     

子囊菌的一个新目Umbilicariales J.C.Wei & Q.M.Zhou

本文基于核糖体SSU rDNA序列对石耳科Umbilicariaceae的系统地位进行了研究.将所获得的石耳科地衣中6个种的SSU rDNA序列与GenBank中其它地衣型及非地衣型真菌的相关序列进行比对用于系统发育研究.结果表明长期以来系统地位不够明确而暂时被置于茶渍目Lecanorales的石耳科不能被包括在茶渍目中,分子数据支持成立石耳目Umbilicariales.基于分子数据并结合形态学和解剖学特征描述了新目Umbilicariales J.C. Wei ＆ Q.M. Zhou（Lecanoromycetes,Ascomycota）.     

Phylogenetic inferences in Antennaria (Asteraceae: Gnaphalieae: Cassiniinae) based on sequences from Nuclear Ribosomal DNA internal transcribed spacers (ITS)

The phylogenetic relationships among sexually reproducing species of Antennaria (Asteraceae) are poorly understood. An earlier cladistic analysis based on morphology did not fully resolve the phylogeny of these taxa and therefore a different approach using molecular data was explored. The internal transcribed spacer regions (ITS-1 and ITS-2) of nuclear ribosomal DNA were sequenced for 30 species of Antennaria and one species from each of the outgroup genera Anaphalis, Ewartia, Leontopodium, and Pseudognaphalium. The ITS-1 sequence in Antennaria ranged from 253 to 260 base pairs (bp) in length, and the proportion of nucleotide differences between pairs of species of Antennaria ranged from 1 to 14%. For ITS-2, the divergence between pairs of species of Antennaria ranged from 0 to 8%. ITS-2 is shorter than ITS-1, ranging from 213 to 219 bp. Phylogenetic analysis indicates that, relative to the outgroups included, Antennaria is a well-supported monophyletic group. Based on the genera surveyed, Leontopodium appears to be the sister genus of Antennaria. The general topology of the molecular trees agrees with that based on previous morphological analyses and indicates that Antennaria is composed of six clades of equal rank, corresponding to the traditionally recognized informal groups, the Geyeriae, Argenteae, Arcuatae, Dimorphae, Pulcherrimae, and Catipes. Sequence and morphological data indicate that the Alpinae and Dioicae are unnatural, polyphyletic units that should be abandoned and redefined as the monophyletic Catipes group. Phylogenetic analysis of ITS sequences also suggests the dissociation of A. stenophylla from the Dimorphae, where it is traditionally placed, and its affiliation with the Argenteae, as well as the placement of A. arcuata in its own group.     

子囊菌的一个新目Umbilicariales J.C. Wei & Q.M. Zhou

本文基于核糖体SSU rDNA序列对石耳科Umbilicariaceae的系统地位进行了研究.将所获得的石耳科地衣中6个种的SSU rDNA序列与GenBank中其它地衣型及非地衣型真菌的相关序列进行比对用于系统发育研究.结果表明长期以来系统地位不够明确而暂时被置于茶渍目Lecanorales的石耳科不能被包括在茶渍目中,分子数据支持成立石耳目Umbilicariales.基于分子数据并结合形态学和解剖学特征描述了新目Umbilicariales J.C. Wei & Q.M. Zhou(Lecanoromycetes,Ascomycota).     

RESTRICTION FRAGMENT LENGTH POLYMORPHISM OF RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER AND 5.8S REGIONS IN JAPANESE ALEXANDRIUM SPECIES (DINOPHYCEAE)1

The 5.8S ribosomal RNA (rDNA) gene and flanking internal transcribed spacers (ITS1 and ITS2)from 9 isolates of Alexandrium catenella (Whedon and Kofoid) Taylor, 11 isolates of A. tamarense (Lebour) Taylor, and single isolates of A. affine (Inoue et Fukuyo) Balech, A. insuetum Balech, and A. pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov. from various locations in Japan were amplified using the polymerase chain reaction (PCR) and subjected to restriction fragment-length polymorphism (RFLP) analysis. PCR products from all strains were approximately 610 bp, inclusive of a limited region of the 18S and 28S rRNA coding regions. RFLP analysis using four restriction enzymes revealed six distinct classes of rDNA (“ITS types”). Restriction patterns of A. catenella were uniform at the intra-specific level and clearly distinguishable from those of A. tamarense. The patterns associated with A. tamarense (“tamarense group”) were also uniform except for one strain, WKS-1. Some restriction fragments from WKS-1 were in common with those of A. catenella or A. tamarense, whereas some were distinct from all Alexandrium species tested. Alexandrium affine, A. insuetum, and A. pseudogonyaulax carry unique ITS types. The ITSs of the “tamarense group” exhibit sequence heterogeneity. In contrast, the ITSs of all other isolates (including WKS-1) appear homogeneous. RFLP analysis of the 5.8S rDNA and flanking ITSs regions from Alexandrium species reveals useful taxonomic and genetic markers at the species and/or population levels.     

Does high intraspecific variability of two genome fragments indicate a recent speciation process of Paramecium dodecaurelia (P. aurelia species complex,Ciliophora, Protozoa)?

Species of the Paramecium aurelia complex show different levels of intraspecific polymorphism, with P. dodecaurelia revealing a high level of intraspecific variation. Paramecium dodecaurelia strains originating from distant localities in the Palaearctic, North America (USA), and Oceania (Hawaii) were studied in terms of intraspecific differentiation and the degree of speciation. Sequences of genes encoding the ITS1–5.8S-ITS2–5’ end of LSU rDNA (1063–1097 bp) and cytochrome c oxidase subunit I mtDNA (638–644 bp) were obtained from 33 strains of P. dodecaurelia, other P. aurelia species, and another species of the genus Paramecium, with Tetrahymena sp. used as an outgroup. In phylograms, the majority of P. dodecaurelia strains from the Palaearctic appear in one cluster, while strains from Japan, Hawaii and the USA are grouped in another cluster, together with some strains from Italy and representatives of the P. aurelia species complex. Our results tend to support the hypothesis that P. dodecaurelia seems to be a polyphyletic species with several haplotypes similar to or even shared with other members of the P. aurelia species complex. However, it is still an open question whether the revealed intraspecific differences within P. dodecaurelia are the result of ongoing speciation, or perhaps they just indicate genetic differentiation within a species that has a wide geographic distribution.     

GENETIC DIVERSITY AND INTROGRESSION IN TWO CULTIVATED SPECIES (PORPHYRA YEZOENSIS AND PORPHYRA TENERA) AND CLOSELY RELATED WILD SPECIES OF PORPHYRA (BANGIALES,RHODOPHYTA)1

We investigated the genetic variations of the samples that were tentatively identified as two cultivated Porphyra species (Porphyra yezoensis Ueda and Porphyra tenera Kjellm.) from various natural populations in Japan using molecular analyses of plastid and nuclear DNA. From PCR‐RFLP analyses using nuclear internal transcribed spacer (ITS) rDNA and plastid RUBISCO spacer regions and phylogenetic analyses using plastid rbcL and nuclear ITS‐1 rDNA sequences, our samples from natural populations of P. yezoensis and P. tenera showed remarkably higher genetic variations than found in strains that are currently used for cultivation. In addition, it is inferred that our samples contain four wild Porphyra species, and that three of the four species, containing Porphyra kinositae, are closely related to cultivated Porphyra species. Furthermore, our PCR‐RFLP and molecular phylogenetic analyses using both the nuclear and plastid DNA demonstrated the occurrence of plastid introgression from P. yezoensis to P. tenera and suggested the possibility of plastid introgression from cultivated P. yezoensis to wild P. yezoensis. These results imply the importance of collecting and establishing more strains of cultivated Porphyra species and related wild species from natural populations as genetic resources for further improvement of cultivated Porphyra strains.     

MASS SEXUAL REPRODUCTION IN THE TOXIGENIC DIATOMS PSEUDO‐NITZSCHIA AUSTRALIS AND P. PUNGENS (BACILLARIOPHYCEAE) ON THE WASHINGTON COAST,USA1

Sexual reproduction is documented for the first time in field populations of the pennate diatoms Pseudo‐nitzschia australis Freng. and P. pungens (Grunow ex Cleve) Hasle (var. cingulata Villac and hybrids between var. cingulata and var. pungens). A bloom dominated by these species began on June 26, 2006, along Kalaloch Beach, Washington, USA, coincident with a drop in the Si(OH)₄:NO₃ ratio to below two. Multimodal size distributions were detected for both species, and synchronous auxosporulation occurred within the smallest size class during a 3‐week window. Auxospores and initial cells created a new class of large cells, and cells in the intermediate size classes increased in abundance during auxosporulation. Mating cells of both species were attached to colonies of surf‐zone diatoms. Paired gametangia, gametes, zygotes, auxospores, and large initial cells were found. Auxosporulation began first for P. pungens (June 30), apparently once a critical, high cell concentration was reached, followed by P. australis (July 5), when the total Pseudo‐nitzschia cell concentration reached 929,000 cells · L^?1. Low frequencies of auxosporulation occurred throughout the bloom but increased 4‐fold for P. australis and 3‐fold for P. pungens when macronutrients were reduced to low levels on July 11. A 2‐year life cycle was estimated for P. australis and 3 years for P. pungens, both with annual auxosporulation. Domoic acid (DA) in razor clams reached a maximum of 38 μg DA · g^?1 on July 18. A significant relationship existed between the percent of cells within the new size range and DA concentrations in razor clams on the same beach.     

MORPHOLOGICAL,TOXICOLOGICAL, AND GENETIC DIFFERENCES AMONG PSEUDO‐NITZSCHIA (BACILLARIOPHYCEAE) SPECIES IN INLAND EMBAYMENTS AND OUTER COASTAL WATERS OF WASHINGTON STATE,USA1

Plankton samples from three inland embayments and several outer coastal sites of Washington State were collected from 1997 through 1999 and were examined for the presence of diatoms of the genus Pseudo‐nitzschia and levels of the toxin, domoic acid (DA). Seven species were observed, including Pseudo‐nitzschia pungens (Grunow ex Cleve) Hasle, P. multiseries (Hasle) Hasle, P. australis Frenguelli, P. fraudulenta (Cleve) Hasle, P. cf. heimii Manguim, P. pseudodelicatissima (Hasle) Hasle, and P. delicatissima (Cleve) Heiden. The coastal Pseudo‐nitzschia species assemblages differed significantly from those observed within embayments. The dominant species observed at coastal sites were P. pseudodelicatissima and P. cf. heimii. Pseudo‐nitzschia assemblages found in embayments included one or more of the following species: P. pungens, P. multiseries, P. australis, P. pseudodelicatissima, and P. fraudulenta. The nuclear large subunit rRNA gene was sequenced for six of the seven species identified. This sequence revealed that P. multiseries, P. pungens, P. australis, and P. heimii were genetically similar to those found in California, whereas P. delicatissima and P. pseudodelicatissima were distinct from the California isolates. Although the concentrations of DA in razor clams along Washington State coasts have exceeded regulatory limits several times since 1991, levels of DA in shellfish from Washington State embayments have not yet exceeded regulatory limits. The widespread presence of toxin‐producing Pseudo‐nitzschia species suggests, however, that toxic blooms are likely to occur within embayments in the future. In conjunction with the monitoring of environmental conditions conducive to toxic bloom formation, the development of species‐specific probes for rapid and accurate detection of potentially toxic Pseudo‐nitzschia species in this region would enable the forecasting of a toxic event before DA accumulates in shellfish, thereby reducing the impacts to coastal communities.     

Phylogeography of the cosmopolitan marine parasite Kudoa thyrsites (Myxozoa: Myxosporea)

Kudoa thyrsites (Myxozoa: Multivalvulida) is a cosmopolitan marine parasite of fishes associated with post-mortem tissue degradation. Financial losses incurred as a result of these infections are of concern to commercial fisheries. There is conflicting evidence whether K. thyrsites represents a cryptic species complex. Myxospore morphology is very similar for K. thyrsites across its range, but preliminary genetic analyses show some differences. Kudoa thyrsites and the morphologically similar Kudoa histolytica were examined from hosts in British Columbia, Canada, Oregon, USA, Chile, England, South Africa, Australia, and Japan. We compared myxospore morphology and DNA sequences of heat shock protein 70 and the small subunit, large subunit, and internal transcribed spacer 1 of the ribosomal DNA. There was some morphological variation between regional representatives, inconsistent with genetic analyses. Phylogenetically, major separations correlated to four broad geographic regions: Japan, Australia, eastern Pacific, and eastern Atlantic. Within these regions there was little additional genetic structure. These data are evidence for regional subdivision of K. thyrsites suggesting global transplantation of fishes has yet to homogenize these distinctions. Within regions, parasite gene flow appears to be high between host species, suggesting little host specificity and minimal cryptic speciation. Our data also indicate that K. histolytica is not a valid species, as it was morphologically and genetically indistinguishable from K. thyrsites.     

ANALYSIS OF ALEXANDRIUM (DINOPHYCEAE) SPECIES USING SEQUENCES OF THE 5.8S RIBOSOMAL DNA AND INTERNAL TRANSCRIBED SPACER REGIONS1

The 5.8S ribosomal RNA gene (rDNA) and flanking internal transcribed spacers 1 and 2 (ITS1 and ITS2) from 7 isolates of Alexandrium catenella (Wedon et Kofoid) Taylor, 13 isolates of A. tamarense (Lebour) Balech, 2 isolates of A. affine (Fukuyo et Inoue) Balech, and single isolates of A. fundyense Balech, A. insuetum Balech, and A. pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov. from Japan, Thailand, and the United States were amplified using the polymerase chain reaction (PCR), sequenced, and subjected to phylogenetic analysis. The sequences ranged from 518 to 535 base pairs (bp) exclusive of the 18S and 28S rDNA coding regions. Sequence comparisons revealed seven divergent “ITS types” designated as follows: 1) catenella type, 2) tamarense type, 3) WKS-1 type, 4) Thai type, 5) affine type, 6) insuetum type, and 7) pseudogonyaulax type. Isolates of the tamarense type from various locations in Japan and the United States and of A. fundyense from the United States were closely related to each other and were clearly divergent from isolates of A. tamarense WKS-1 (WKS-I type) or A. tamarense CU-15 (Thai type). These latter two strains carried unique ITS types, although they were not distinguishable from isolates of the tamarense type by morphological criteria. Distance values between isolates of the tamarense type and the WKS-1 or Thai type were quite high (about 0.21 and 0.39, respectively). Seven isolates of A. catenella from Japan (catenella type) clearly diverged from the other ITS types already mentioned. Distance values between isolates of the catenella type were extremely low (<0.01), whereas distance values of ITS between the catenella type and the tamarense, WKS-1, or Thai type were 0.17, 0.18, and 0.40, respectively. Isolates of A. affine, A. insuetum, and A. pseudogonyaulax all carried unique ITS types. The ITSs of the tamarense type exhibited two distinct ITS sets, the “A gene” and the “B gene.” The two sequences occurred in a 1:1 ratio in PCR products. In contrast, the ITSs of all other isolates appeared homogeneous. Sequence comparisons also showed that the variations in the 3′ end of ITS1 (150-177 bp) were low within each ITS type but extremely high between ITS types. The number of different nucleotides among the seven Alexandrium types in this 28-bp region is more than 10. High diversity of this region may facilitate the design of DNA probes specific for each ITS type/species of Alexandrium.     

Phylogenetic relationships among Lactuca (Asteraceae) species and related genera based on ITS-1 DNA sequences

Internal transcribed spacer (ITS-1) sequences from 97 accessions representing 23 species of Lactuca and related genera were determined and used to evaluate species relationships of Lactuca sensu lato (s.l.). The ITS-1 phylogenies, calculated using PAUP and PHYLIP, correspond better to the classification of Feráková than to other classifications evaluated, although the inclusion of sect. Lactuca subsect. Cyanicae is not supported. Therefore, exclusion of subsect. Cyanicae from Lactuca sensu Feráková is proposed. The amended genus contains the entire gene pool (sensu Harlan and De Wet) of cultivated lettuce (Lactuca sativa). The position of the species in the amended classification corresponds to their position in the lettuce gene pool. In the ITS-1 phylogenies, a clade with L. sativa, L. serriola, L. dregeana, L. altaica, and L. aculeata represents the primary gene pool. L. virosa and L. saligna, branching off closest to this clade, encompass the secondary gene pool. L. virosa is possibly of hybrid origin. The primary and secondary gene pool species are classified in sect. Lactuca subsect. Lactuca. The species L. quercina, L. viminea, L. sibirica, and L. tatarica, branching off next, represent the tertiary gene pool. They are classified in Lactuca sect. Lactucopsis, sect. Phaenixopus, and sect. Mulgedium, respectively. L. perennis and L. tenerrima, classified in sect. Lactuca subsect. Cyanicae, form clades with species from related genera and are not part of the lettuce gene pool.     

ITS sequence data support a single origin for North American Astereae (Asteraceae) and reflect deep geographic divisions in Aster s.l

The Astereae is the largest tribe of Asteraceae in North America. Morphological diversity suggests that the North American assemblage is polyphyletic as 12 endemic genera, as well as lineages of the genus Erigeron and Conyza (Conyzinae), have been hypothesized to represent at least five separate invasions of North America from Africa, Australia, Eurasia, and South America. This hypothesis was tested with a phylogenetic analysis of nucleotide sequence data from the internal transcribed spacers (ITS) of nuclear ribosomal DNA. Sequences for 62 taxa represent seven outgroup taxa and all major Northern and Southern Hemisphere groups of Astereae, including broad taxonomic and geographic sampling of Conyzinae and Aster s.l. (sensu lato). Parsimony analyses indicate that all North American Astereae are members of a strongly supported clade, and that a diverse group of predominantly woody taxa from Africa, Australia, and South America, are basal Astereae. Furthermore, Aster s.l. is deeply polyphyletic as Eurasian taxa, including Aster s.s. (sensu stricto), appear more closely related to Southern Hemisphere taxa than to North American Aster segregates. There is only low to moderate agreement between proposed higher level Astereae relationships based on ITS and those based either on morphology or chloroplast restriction site data.     

The Genus Clusia L.: Molecular Evidence for Independent Evolution of Photosynthetic Flexibility

Abstract: Members of the Clusiaceae genus Clusia (tropical trees and shrubs) belong to the small group of dicotyledonous trees which are able to perform crassulacean acid metabolism (CAM). Most of the species are able to switch between C₃ and CAM modes of photosynthesis and only a few are restricted to either C₃ or CAM. In order to discover possible phylogenetic relationships with regard to the mode of photosynthesis, we investigated 17 species of the genus Clusia, and one species each of the Clusiaceae genera Oedematopus and Hypericum on the basis of internal transcribed spacer (ITS) sequences between the 18S and 26S coding regions of nuclear ribosomal DNA. Little length variation was detected in the ITS region of Clusia species. ITS1 sequences ranged from 255 to 260 bp and ITS2 sequences from 208 to 210 bp. Neighbour-joining and parsimony analyses of these sequences resulted in considerable differences in cluster formation when compared to a classification based on morphological characteristics. The molecular data also give no indication of a group-specific evolution of modes of photosynthesis, i.e., C₃ and CAM. We thus conclude that CAM has evolved independently several times within the genus Clusia.