Giuseppe C. Zuccarello

DNA-based species delimitation in algae

Given the problems of species delimitation in algae using morphology or sexual compatibility, molecular data are becoming the standard for delimiting species and testing their traditional boundaries. The idea that species are separately evolving metapopulation lineages, along with theoretical progress in phylogenetic and population genetic analyses, has led to the development of new methods of species delimitation. We review these recent developments in DNA-based species delimitation methods, and discuss how they have changed and continue to change our understanding of algal species boundaries. Although single-locus approaches have proven effective for a first rapid and large-scale assessment of species diversity, species delimitation based on single gene trees falls short due to gene tree–species tree incongruence, caused by confounding processes like incomplete lineage sorting, trans-species polymorphism, hybridization and introgression. Data from unlinked loci and multi-species coalescent methods, which combine principles from phylogenetics and population genetics, may now be able to account for these complicating factors. Several of these methods also provide statistical support regarding species boundaries, which is important because speciation is a process and therefore uncertainty about precise species boundaries is inevitable in recently diverged lineages.

A mitochondrial marker for red algal intraspecific relationships

Intraspecific studies of red algae have relied on nuclear or plastid markers rather than mitochondrial data to address questions of systematics, biogeography or population genetics. In this study, primers were developed that spanned the noncoding intergenic region between the mitochondrial cytochrome oxidase subunit 2 and cytochrome oxidase subunit 3 genes. These primers were demonstrated to be successful on a variety of red algae in different orders: Gracilariales, Bonnemaisoniales and Ceramiales (families: Delesseriaceae, Ceramiaceae and Rhodomelaceae). Amplification products were between 450 and 320 bp in length, with variation in length shown among geographically distant isolates within a species. The region was variable within a single species, as shown for Bostrychia moritziana and B. radicans, and within populations of Caloglossa leprieurii. In the latter species, four mitochondrial haplotypes were observed in isolates from a single locality in Woolooware Bay, New South Wales, Australia. Analysis of hybrids between different mitochondrial haplotypes of B. moritziana revealed that the mitochondria are maternally inherited in this species. This is the first report of a mitochondrial marker that is variable within red algal populations and may lead to a better understanding of the population ecology of these important marine organisms.

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.

LOW MOLECULAR WEIGHT CARBOHYDRATE PATTERNS IN THE BANGIOPHYCEAE (RHODOPHYTA)

The qualitative and quantitative occurrence of low molecular weight carbohydrates (LMWCs) in the Bangiophyceae is surveyed. Members of the orders Erythropeltidales (Sahlingia and Erythrotrichia), Compsopogonales (Compsopogon), and Bangiales (Bangia and Porphyra) all contain floridoside and, in some cases, D‐ and L‐isofloridoside, making the group chemotaxonomically the most homogeneous; L‐isofloridoside occurred only in the Bangiales. The Porphyridiales showed great variation in carbohydrate types: Chroodactylon contained only sorbitol, Porphyridium contained only floridoside, Dixoniella and Rhodella species exhibited only mannitol, and Rhodosorus showed digeneaside and sorbitol, whereas Stylonema contained floridoside, D‐isofloridoside, digeneaside, and sorbitol. The extensive variation in LMWC composition within and between the different orders suggests that the enzyme systems for each might have developed repeatedly in different Bangiophyceae and Florideophyceae. Nonetheless, a phylogenetic tree based on small‐subunit ribosomal RNA supports the LMWC patterns in the Bangiophyceae.

Phylogeography of the Bostrychia calliptera–B. pinnata complex (Rhodomelaceae, Rhodophyta) and divergence rates based on nuclear, mitochondrial and plastid DNA markers

The species Bostrychia calliptera and B. pinnata, separated by cortication of main axis, were analysed by sequencing regions from the three genomes (nuclear: large-subunit ribosomal RNA; mitochondrial: cox2–3 spacer; and plastid: Rubisco spacer) to determine the phylogeographic relationships among pantropical isolates. All genetic regions produced congruent phylogenetic relationships, with three major lineages being revealed within this species complex. One lineage comprised isolates from the Americas, another comprised isolates from the Indo-Pacific and a third contained isolates from both the Indo-Pacific and the Americas. Analyses testing for molecular clock-like rates of base substitutions show that certain lineages within the species complex evolve in a clock-like manner. Phylogenies do not support separation of B. calliptera and B. pinnata based on cortication of the main axis, and samples found in close proximity can belong to different highly divergent lineages. Molecular clock calibrations of divergence times within this complex, based on the vicari ant event of the last closure of the Isthmus of Panama, reveal that the estimated divergence rates in the different genetic elements are slower than most published rates. Furthermore, divergence times indicate that this species complex is very old, having remained morphologically identical at least since the Oligocene (23–40 Ma); it may possibly have been associated with mangrove vegetation since the establishment and worldwide spread of mangroves in the Tertiary. Biogeographical patterns are complex and may reflect ancient Tethyan distributions. Molecular analyses may be the only way that the biogeography and evolution of morphologically static and ancient red algal species can be resolved.

Molecular phylogeny of the genus Tribonema (Xanthophyceae) using rbcL gene sequence data: monophyly of morphologically simple algal species

G.C. Zuccarello and G.M. Lokhorst. 2005. Molecular phylogeny of the genus Tribonema (Xanthophyceae) using rbcL gene sequence data: monophyly of morphologically simple algal species. Phycologia 44: 384–392. The genus Tribonema (Xanthophyceae) is a common component of many freshwater ecosystems. It is distinguished by its H-shaped bipartite walls and filamentous habit. Species identification has relied on few morphological characters, especially filament size and plastid numbers. We approached the question of interspecific relationships within the genus by sequencing the plastid-encoded rbcL gene of 10 species of Tribonema. We sequenced multiple samples of seven of these species from throughout Europe. Our data suggest that T. hormidioides (Vischer) Lokhorst is closely related to the genus Xanthonema from which it has recently been removed, and should be returned to that genus. Most other samples of Tribonema belong to a single clade, which includes Bumilleriopsis filiformis Vischer. None of the repeatedly sampled species of Tribonema, except T. microchloron Ettl (two samples), are monophyletic. Trends in character divergence within clades were seen with a clade containing mostly species with wide filaments and another with mostly thin filaments. The question of gene trees vs organismal phylogeny is discussed. Our data suggest that presently identified morpho-species of Tribonema may not be useful as evolutionary units or in biodiversity assessment of freshwater ecosystems.

Low molecular weight carbohydrates of the Bangiophycidae (Rhodophyta)

In the order Porphyridiales there are three clades based on molecular evidence. These show parallels with the low molecular weight carbohydrate (LMWCs) in different genera. Clade Porphyridiales 1 includes Dixoniella, Glaucosphaera, Rhodella, and one undescribed genus (3987) that all contain mannitol. Clade Porphyridiales 2 comprises taxa of the Stylonematales Rhodosorus and Stylonema species and contains digeneaside and sorbitol, whereas Chroodactylon has only sorbitol. In clade Porphyridiales 3 Flintiella, Porphyridium, and the undescribed genus (3797) all possess only floridoside. In the Erythropeltidales Rhodochaete contains floridoside and digeneaside, Erythrotrichia species contain only floridoside, Sahlingia subintegra has floridoside and traces of D‐floridoside, and Smithora has L‐isofloridoside plus floridoside. In the Compsopogonales Boldia and Compsopogon have only floridoside. Within these genera as presently circumscribed, the LMWCs appear to be a reliable character to supplement the usual cytological characters.

DNA BARCODING AND GENETIC DIVERGENCE IN THE GIANT KELP MACROCYSTIS (LAMINARIALES) 1

The brown alga Macrocystis C. Agardh is widely distributed throughout the cold temperate waters of the Northern and Southern hemispheres, forming ecologically diverse and productive kelp forests. The taxonomy of this alga has been under constant discussion. Since the first description, species have been mostly described by holdfast and blade morphology; however, the importance of these taxonomic characters has been questioned. Based on a morphological study, the genus has recently been synonymized into a single species, M. pyrifera (L.) C. Agardh, but additional genetic evidence is still lacking. Using the “DNA‐barcoding” gene (COI), we examined the taxonomy of Macrocystis collected from 19 sites worldwide, covering the distribution of the four ecomorphs (M. “pyrifera,” M. “angustifolia,” M “integrifolia,” and M. “laevis”). Our molecular data strongly support the recognition of a single species; therefore, the genus should contain only one species, M. pyrifera, the oldest name. Results also reveal shared haplotypes in several distant sites around the Southern Hemisphere and very low variability among samples. Additionally, samples of the ecomorphs M. “integrifolia” and M. “pyrifera” from a sympatric population in California had the same haplotype. The revised taxonomy changes questions of Macrocystis distribution from interspecific dispersal and evolutionary questions to intraspecific ecological questions on the maintenance of Macrocystis in certain environments that produce particular morphologies.

A rapid method to score plastid haplotypes in red seaweeds and its use in determining parental inheritance of plastids in the red alga Bostrychia (Ceramiales)

A method for the rapid identification of plastid haplotypes in red algae is presented. This method is based on single-stranded conformation polymorphism (SSCP) to detect variation in DNA sequence of PCR-amplified plastid-encoded ribulose bisphosphate carboxylase/oxygenase large and small subunit intergenic spacer. The method detects variation within a population of the mangrove red alga Caloglossa leprieurii from New South Wales, Australia. Three haplotypes (plastid variants) were easily scored in our samples, and a spatial variation of haplotypes within the intertidal is suggested. This method also reveals maternal inheritance of plastids in crosses between isolates of Bostrychia radicans and in multiple crosses between isolates of B. moritziana. Fluorescence microscopy shows that plastids are contained within the spermatia of these species and that bi- or paternal inheritance of plastids is possible. SSCP is a simple and rapid method for the detection of plastid variation (haplotypes) within populations of red algae that should also be applicable to brown seaweeds.

Diversity within red algal species: variation in world-wide samples of Spyridia filamentosa (Ceramiaceae) and Murrayella periclados (Rhodomelaceae) using DNA markers and breeding studies

Molecular and breeding studies on two pan-tropical marine red algae reveal vastly different levels of genetic variation and reproductive isolation. Sequenced DNA regions from the nuclear, mitochondrial and plastid genomes (partial LSU rRNA, cox2-3 spacer, RuBisCo spacer, respectively) revealed high genetic variation among individuals of Spyridia filamentosa. An rbcL analysis shows that Spyridia is a monophyletic genus distinct from other Ceramiaceae sampled, suggesting that the Ceramiaceae could be paraphyletic. There is complete congruence between all data sets of S. filamentosa, showing a well-supported phylogeographic pattern with samples from the Pacific distinct from Atlantic and Indian Ocean samples. One western Mediterranean sample is associated with Atlantic specimens, while an eastern Mediterranean sample has closer affinities to Pacific samples, possibly indicating a recent cryptic introduction into the eastern Mediterranean. Limited breeding studies imply that these samples are mostly reproductively isolated, whereas a successful cross demonstrated maternal inheritance of organellar DNA. These data indicate that S. filamentosa exists as several cryptic species. Murrayella periclados exhibits low levels of genetic variation and no phylogeographic structure, and almost complete reproductive compatibility between isolates. This suggests that all M. periclados samples share a recent common ancestor that may have dispersed relatively rapidly, or that rates of base pair substitution between these two species vary greatly. Rapid longdistance dispersal of M. periclados is not indicated by what is known of the biology of M. periclados, especially in comparison with S. filamentosa, which appears to be a much better candidate for long-distance dispersal. These data demonstrate that red algal morphospecies are not equivalent units of diversity, with implications for our view of red algal biodiversity and evolution.