Gabriele Procaccini

North Atlantic phylogeography and large-scale population differentiation of the seagrass Zostera marina L.

As the most widespread seagrass in temperate waters of the Northern Hemisphere, Zostera marina provides a unique opportunity to investigate the extent to which the historical legacy of the last glacial maximum (LGM18 000–10 000 years bp) is detectable in modern population genetic structure. We used sequences from the nuclear rDNA–internal transcribed spacer (ITS) and chloroplast matK‐intron, and nine microsatellite loci to survey 49 populations (> 2000 individuals) from throughout the species’ range. Minimal sequence variation between Pacific and Atlantic populations combined with biogeographical groupings derived from the microsatellite data, suggest that the trans‐Arctic connection is currently open. The east Pacific and west Atlantic are more connected than either is to the east Atlantic. Allelic richness was almost two‐fold higher in the Pacific. Populations from putative Atlantic refugia now represent the southern edges of the distribution and are not genetically diverse. Unexpectedly, the highest allelic diversity was observed in the North Sea–Wadden Sea–southwest Baltic region. Except for the Mediterranean and Black Seas, significant isolation‐by‐distance was found from ~150 to 5000 km. A transition from weak to strong isolation‐by‐distance occurred at ~150 km among northern European populations suggesting this scale as the natural limit for dispersal within the metapopulation. Links between historical and contemporary processes are discussed in terms of the projected effects of climate change on coastal marine plants. The identification of a high genetic diversity hotspot in Northern Europe provides a basis for restoration decisions.

Intraspecific diversity in Scrippsiella trochoidea (Dinopbyceae): evidence for cryptic species

Abstract Scrippsiella trochoidea is a widely distributed neritic dinoflagellate that produces calcareous resting cysts. We assessed the level of intraspecific diversity at the molecular, morphological and physiological levels among 15 strains identified as S. trochoidea and isolated from the Gulf of Naples (Italy, Mediterranean Sea), and an additional isolate from the Faeroe Islands. We investigated the morphology of motile cells and cysts, mating modality, encystment success, and growth rates at different light irradiances. The ribosomal DNA internal transcribed spacer (ITS) region was sequenced to infer phylogenetic relationships among the S. trochoidea strains and closely related species. The molecular analysis revealed a well-supported lineage comprising strains with a Scrippsiella plate pattern. Within this clade, a number of distinct ITS haplotypes were recorded but the relationships among them were only partially resolved. The 16 S. trochoidea isolates grouped into five single-strain clades and three multi-strain clades. The grouping of haplotypes in a series of distinct clades suggests the existence of cryptic species within what has previously been considered a single species, based on the morphological features of the motile cells and cysts. Some of the ITS haplotypes were distinguishable visually, based on minor morphological features of the motile cells and cysts, but in two cases morphologically almost identical strains fell into different clades. Our results showed that the majority of the strains are homothallic; only S. trochoidea v. aciculifera from the Faeroe Islands is heterothallic. Cyst production rates were notable for their diversity, even among strains grouping with the same ITS haplotype, as were growth rates at different light irradiances. Based on phylogenetic results, two new combinations are proposed: S. operosa (Deflandre) Montresor comb. nov. and S. infula (Deflandre) Montresor comb. nov.

The genome of the seagrass Zostera marina reveals angiosperm adaptation to the sea

Seagrasses colonized the sea on at least three independent occasions to form the basis of one of the most productive and widespread coastal ecosystems on the planet. Here we report the genome of Zostera marina (L.), the first, to our knowledge, marine angiosperm to be fully sequenced. This reveals unique insights into the genomic losses and gains involved in achieving the structural and physiological adaptations required for its marine lifestyle, arguably the most severe habitat shift ever accomplished by flowering plants. Key angiosperm innovations that were lost include the entire repertoire of stomatal genes, genes involved in the synthesis of terpenoids and ethylene signalling, and genes for ultraviolet protection and phytochromes for far-red sensing. Seagrasses have also regained functions enabling them to adjust to full salinity. Their cell walls contain all of the polysaccharides typical of land plants, but also contain polyanionic, low-methylated pectins and sulfated galactans, a feature shared with the cell walls of all macroalgae and that is important for ion homoeostasis, nutrient uptake and O2/CO2 exchange through leaf epidermal cells. The Z. marina genome resource will markedly advance a wide range of functional ecological studies from adaptation of marine ecosystems under climate warming, to unravelling the mechanisms of osmoregulation under high salinities that may further inform our understanding of the evolution of salt tolerance in crop plants.

Bipolar distribution of the cyst-forming dinoflagellate Polarella glacialis

Morphological investigations of motile cells and cysts of a small dinoflagellate (strain CCMP 2088) isolated from Canadian Arctic waters were carried out under both light and scanning electron microscopy. This species strongly resembled Polarella glacialis (strain CCMP 1383), which up to now was known only from Antarctic sea ice. The photosynthetic pigment composition of strain CCMP 2088 is typical of dinoflagellates, with peridinin as a major accessory pigment. Phylogenetic relationships between the two strains and other dinoflagellate species were inferred from SSU nrDNA using Neighbour Joining and weighted parsimony analyses. Our results showed that strain CCMP 2088 and P. glacialis (strain CCMP 1383) grouped in the same clade (Suessiales clade), showing high similarity values (0.99%). Morphological and molecular data support the assignment of the Arctic strain to P. glacialis. The free-living Gymnodinium simplex and the two P. glacialis strains have a basal position in the Suessiales clade, as compared to Symbiodinium spp.

Toxic Pseudo-nitzschia multistriata (Bacillariophyceae) from the Gulf of Naples: morphology, toxin analysis and phylogenetic relationships with other Pseudo-nitzschia species

The genus Pseudo-nitzschia includes several species capable of producing domoic acid, the causative agent of Amnesic Shellfish Poisoning. Some of these species have been recorded frequently in the Gulf of Naples. For one of the species, P. multistriata, which has been recurrently found in our sampling area since 1995, this is the first report for European waters. Here we provide further details on the fine structure of this species. Pseudo-nitzschia multistriata was the only one found to produce domoic acid among all the Pseudo-nitzschia species from the Gulf of Naples, and this finding raises the number of potentially toxic species in this genus to nine. Phylogenetic relationships among several Pseudo-nitzschia species were assessed using the hypervariable domains (D1–D3) of the large subunit (LSU) rDNA. The match between the phylogeny obtained and important taxonomic characters used in this genus are discussed. Results show that P. multistriata clusters with wider species lacking a central larger interspace in the raphe. Close genetic relationships were determined between P. fraudulenta and P. subfraudulenta, and between P. pungens and P. multiseries. Genetic differences among these pairs of species are comparable to those among isolates of P. pseudodelicatissima from the Gulf of Naples, indicating high intraspecific genetic diversity of Pseudo-nitzschia species in the relatively conserved LSU region. This could explain the problematic results obtained when testing a match between species-specific Pseudo-nitzschia LSU probes and our sequences.

Population genetics of dwarf eelgrass Zostera noltii throughout its biogeographic range

The marine angiosperm Zostera noltii (dwarf eelgrass), an important facilitator species and food source for invertebrates and waterfowl, predominantly inhabits intertidal habitats along eastern Atlantic shores from Mauritania to southern Norway/Kattegat Sea and throughout the Mediterranean, Black and Azov seas. We used 9 microsatellite loci to characterize population struc- ture at a variety of spatial scales among 33 populations from 11 localities throughout the entire biogeographic range. Isolation by distance analysis suggested a panmictic genetic neighborhood of 100 to 150 km. At the global scale, a neighbor-joining tree based on Reynolds distances revealed strongly-supported groups corresponding to northern Europe, Mauritania and the Black/Azov Sea; separate Mediterranean and Atlantic-Iberian groups were poorly supported. Clones (genets with multiple ramets) were present in most populations but were generally small (ca. <3 m 2 ). Exceptions were found in Mauritania (ca. 29 m in length), the Azov Sea (ca. 40 m in length) and the Black Sea (ca. 50 m in length). Although genetic diversity and allelic richness generally decreased from Mauri- tania to Denmark, the putative post-glacial recolonization route, both were unexpectedly high among populations from the German Wadden Sea.

Phylogeography of the invasive seaweed Asparagopsis (Bonnemaisoniales, Rhodophyta) reveals cryptic diversity.

The rhodophyte seaweed Asparagopsis armata Harvey is distributed in the northern and southern temperate zones, and its congener Asparagopsis taxiformis (Delile) Trevisan abounds throughout the tropics and subtropics. Here, we determine intraspecific phylogeographic patterns to compare potential causes of the disjunctions in the distributions of both species. We obtained specimens throughout their ranges and inferred phylogenies from the hypervariable domains D1‐D3 of the nuclear rDNA LSU, the plastid spacer between the large and small subunits of RuBisCo and the mitochondrial cox 2–3 intergenic spacer. The cox spacer acquired base changes the fastest and the RuBisCo spacer the slowest. Median‐joining networks inferred from the sequences revealed the absence of phylogeographic structure in the introduced range of A. armata, corroborating the species’ reported recent introduction. A. taxiformis consisted of three nuclear, three plastid and four mitochondrial genetically distinct, lineages (1–4). Mitochondrial lineage 3 is found in the western Atlantic, the Canary Islands and the eastern Mediterranean. Mitochondrial lineages 1, 2, and 4 occur in the Indo‐Pacific, but one of them (lineage 2) is also found in the central Mediterranean and southern Portugal. Phylogeographic results suggest separation of Atlantic and Indo‐Pacific lineages resulted from the emergence of the Isthmus of Panama, as well as from dispersal events postdating the closure event, such as the invasion of the Mediterranean Sea by mitochondrial lineages 2 and 3. Molecular clock estimates using the Panama closure event as a calibration for the split of lineages 3 and 4 suggest that A. taxiformis diverged into two main cryptic species (1 + 2 and 3 + 4) about 3.2–5.5 million years ago (Ma), and that the separation of the mitochondrial lineages 1 and 2 occurred 1–2.3 Ma.

POLARELLA GLACIALIS, GEN. NOV., SP. NOV. (DINOPHYCEAE): SUESSIACEAE ARE STILL ALIVE!

The culture CCMP 1383, obtained from sea‐ice brine collected in McMurdo Sound (Ross Sea, Antarctica), is a small gymnodinioid dinoflagellate. This species is very abundant in the upper land‐fast sea ice, where it can both grow and overwinter as a spiny encysted stage. The motile vegetative stage and the cyst produced in the culture were studied by scanning electron microscopy (SEM) and transmission electron micrscopy (TEM). The amphiesma of the vegetative cells is constituted by thin vesicles that are organized into nine latitudinal series of plates: three in the epitheca, two in the cingulum, and four in the hypotheca. The same tabulation is reflected in the cyst wall by acicular processes arising from the center of paraplates, with the exception of the paracingulum, in which acicular processess are absent. On the basis of the peculiar plate pattern of this dinoflagellate, we establish the new genus Polarella and the new species Polarella glacialis (family Suessiaceae, order Suessiales). This species has a remarkable similarity with fossil Suessiaceae cysts dating back to the Triassic and Jurassic and represents, up to now, the only extant member of the subfamily Suessiaceae. Phylogenetic analysis based on the small‐subunit ribosomal RNA gene confirmed the placement of this species in the order Suessiales and its close relationship with the genus Symbiodinium Freudenthal.

Genetic structure in the Mediterranean seagrass Posidonia oceanica: Disentangling past vicariance events from contemporary patterns of gene flow

The Mediterranean Sea is a two‐basin system, with the boundary zone restricted to the Strait of Sicily and the narrow Strait of Messina. Two main population groups are recognized in the Mediterranean endemic seagrass Posidonia oceanica, corresponding to the Western and the Eastern basins. To address the nature of the East–West cleavage in P. oceanica, the main aims of this study were: (i) to define the genetic structure within the potential contact zone (i.e. the Strait of Sicily) and clarify the extent of gene flow between the two population groups, and (ii) to investigate the role of present water circulation patterns vs. past evolutionary events on the observed genetic pattern. To achieve these goals, we utilized SSR markers and we simulated, with respect to current regime, the possible present‐day dispersal pattern of Posidonia floating fruits using 28‐day numerical Lagrangian trajectories. The results obtained confirm the presence of the two main population groups, without any indices of reproductive isolation, with the break zone located at the level of the Southern tip of Calabria. The populations in the Strait of Sicily showed higher affinity with Western than with Eastern populations. This pattern of genetic structure probably reflects historical avenues of recolonization from relict glacial areas and past vicariance events, but seems to persist as a result of the low connectivity among populations via marine currents, as suggested by our dispersal simulation analysis.

Evolution of the Nitric Oxide Synthase Family in Metazoans

Nitric oxide (NO) is essential to many physiological functions and operates in several signaling pathways. It is not understood how and when the different isoforms of nitric oxide synthase (NOS), the enzyme responsible for NO production, evolved in metazoans. This study investigates the number and structure of metazoan NOS enzymes by genome data mining and direct cloning of Nos genes from the lamprey. In total, 181 NOS proteins are analyzed from 33 invertebrate and 63 vertebrate species. Comparisons among protein and gene structures, combined with phylogenetic and syntenic studies, provide novel insights into how NOS isoforms arose and diverged. Protein domains and gene organization--that is, intron positions and phases--of animal NOS are remarkably conserved across all lineages, even in fast-evolving species. Phylogenetic and syntenic analyses support the view that a proto-NOS isoform was recurrently duplicated in different lineages, acquiring new structural configurations through gains and losses of protein motifs. We propose that in vertebrates a first duplication took place after the agnathan-gnathostome split followed by a paralog loss. A second duplication occurred during early tetrapod evolution, giving rise to the three isoforms--I, II, and III--in current mammals. Overall, NOS family evolution was the result of multiple gene and genome duplication events together with changes in protein architecture.