Multiannual patterns of genetic structure and mating type ratios highlight the complex bloom dynamics of a marine planktonic diatom.

Understanding the genetic structure of populations and the processes responsible for its spatial and temporal dynamics is vital for assessing species' adaptability and survival in changing environments. We investigate the genetic fingerprinting of blooming populations of the marine diatom Pseudo-nitzschia multistriata in the Gulf of Naples (Mediterranean Sea) from 2008 to 2020. Strains were genotyped using microsatellite fingerprinting and natural samples were also analysed with Microsatellite Pool-seq Barcoding based on Illumina sequencing of microsatellite loci. Both approaches revealed a clonal expansion event in 2013 and a more stable genetic structure during 2017-2020 compared to previous years. The identification of a mating type (MT) determination gene allowed to assign MT to strains isolated over the years. MTs were generally at equilibrium with two notable exceptions, including the clonal bloom of 2013. The populations exhibited linkage equilibrium in most blooms, indicating that sexual reproduction leads to genetic homogenization. Our findings show that P. multistriata blooms exhibit a dynamic genetic and demographic composition over time, most probably determined by deeper-layer cell inocula. Occasional clonal expansions and MT imbalances can potentially affect the persistence and ecological success of planktonic diatoms.

Phenological segregation suggests speciation by time in the planktonic diatom Pseudo-nitzschia allochrona sp. nov.

The processes leading to the emergence of new species are poorly understood in marine plankton, where weak physical barriers and homogeneous environmental conditions limit spatial and ecological segregation. Here, we combine molecular and ecological information from a long-term time series and propose Pseudo-nitzschia allochrona, a new cryptic planktonic diatom, as a possible case of speciation by temporal segregation. The new species differs in several genetic markers (18S, 28S and ITS rDNA fragments and rbcL) from its closest relatives, which are morphologically very similar or identical, and is reproductively isolated from its sibling species P. arenysensis. Data from a long-term plankton time series show P. allochrona invariably occurring in summer-autumn in the Gulf of Naples, where its closely related species P. arenysensis, P. delicatissima, and P. dolorosa are instead found in winter-spring. Temperature and nutrients are the main factors associated with the occurrence of P. allochrona, which could have evolved in sympatry by switching its phenology and occupying a new ecological niche. This case of possible speciation by time shows the relevance of combining ecological time series with molecular information to shed light on the eco-evolutionary dynamics of marine microorganisms.

New alleles in the mating type determination region of West Atlantic strains of Pseudo-nitzschia multistriata.

The cosmopolitan, species-rich diatom genus Pseudo-nitzschia represents a good system for the study of speciation, evolution and diversity. Understanding elements linked to population dynamics and life cycle regulation for these species is of particular importance in view of their ability to produce the toxin domoic acid and cause harmful blooms. Pseudo-nitzschia multistriata, one of the toxic species that represents a model for the study of life cycle related questions, is the only diatom for which a sex determination mechanism has been described. Populations in the Gulf of Naples (Mediterranean Sea), can share four different allelic variants (A, M, B, N) of the mating type determination region, and one of them (A) is responsible for the determination of the mating type + (MT+), defined by the MT+ restricted expression of the gene MRP3. Here, we analysed the sex determination genomic region in three new strains isolated from the Gulf of Mexico and compared it to the alleles previously described in the Mediterranean strains. We first show that these geographically distant strains of P. multistriata belong to different populations but can interbreed. Next, we show that the two populations share an overall similar structure of the genomic locus although differences can be seen in the polymorphic regions upstream of MRP3. In strain P4-C1, we amplified and sequenced an allele (M) identical to one of those previously characterized in the Mediterranean strains. In the other two strains, P4-C2 and P4-C5, we identified three new alleles, which we named A2, B2 and N2. P4-C2 and P4-C5 are heterozygous and share the common allele A2 linked to the monoallelic expression of the MT+ specific sex determining gene MRP3. Our results expand information on the global distribution of P. multistriata and on the level of conservation of the sex determination region in different populations. The definition of the extent of intra- and inter-specific conservation of this region would be a relevant addition to our understanding of Pseudo-nitzschia diversity and evolution.

Clonal expansion behind a marine diatom bloom.

Genetic diversity is what selection acts on, thus shaping the adaptive potential of populations. We studied micro-evolutionary patterns of the key planktonic diatom Pseudo-nitzschia multistriata at a long-term sampling site over 2 consecutive years by genotyping isolates with 22 microsatellite markers. We show that both sex and vegetative growth interplay in shaping intraspecific diversity. We document a brief but massive demographic and clonal expansion driven by strains of the same mating type. The analysis of an extended data set (6 years) indicates that the genetic fingerprint of P. multistriata changed over time with a nonlinear pattern, with intermittent periods of weak and strong diversification related to the temporary predominance of clonal expansions over sexual recombination. These dynamics, rarely documented for phytoplankton, contribute to the understanding of bloom formation and of the mechanisms that drive microevolution in diatoms.

Pseudo-nitzschia arctica sp. nov., a new cold-water cryptic Pseudo-nitzschia species within the P. pseudodelicatissima complex.

A new nontoxic Pseudo-nitzschia species belonging to the P. pseudodelicatissima complex, P. arctica, was isolated from different areas of the Arctic. The erection of P. arctica is mainly supported by molecular data, since the species shares identical ultrastructure with another species in the complex, P. fryxelliana, and represents a new case of crypticity within the genus. Despite their morphological similarity, the two species are not closely related in phylogenies based on LSU, ITS and rbcL. Interestingly, P. arctica is phylogenetically most closely related to P. granii and P. subcurvata, from which the species is, however, morphologically different. P. granii and P. subcurvata lack the central larger interspace which is one of the defining features of the P. pseudodelicatissima complex. The close genetic relationship between P. arctica and the two species P. granii and P. subcurvata is demonstrated by analysis of the secondary structure of ITS2 which revealed no compensatory base changes, two hemi-compensatory base changes, and two deletions in P. arctica with respect to the other two species. These findings emphasize that rates of morphological differentiation, molecular evolution and speciation are often incongruent for Pseudo-nitzschia species, resulting in a restricted phylogenetic value for taxonomic characters used to discriminate species. The description of a new cryptic species, widely distributed in the Arctic and potentially representing an endemic component of the Arctic diatom flora, reinforces the idea of the existence of noncosmopolitan Pseudo-nitzschia species and highlights the need for combined morphological and molecular analyses to assess the distributional patterns of phytoplankton species.

Distribution, occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis.

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n=256) from European waters, collected 2009-2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2×2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.

Interspecific plastidial recombination in the diatom genus Pseudo-nitzschia.

Plastids are usually uni-parentally inherited and genetic recombination between these organelles is seldom observed. The genus Pseudo-nitzschia, a globally relevant marine diatom, features bi-parental plastid inheritance in the course of sexual reproduction. This observation inspired the recombination detection we pursued in this paper over a ~1,400-nucleotide-long region of the plastidial rbcL, a marker used in both molecular taxonomy and phylogenetic studies in diatoms. Among all the rbcL-sequences available in web-databases for Pseudo-nitzschia, 42 haplotypes were identified and grouped in five clusters by Bayesian phylogeny. Signs of hybridization were evident in four of five clusters, at both intra- and interspecific levels, suggesting that, in diatoms, (i) plastidial recombination is not absent and (ii) hybridization can play a role in speciation of Pseudo-nitzschia spp.

Specificity of LSU rRNA-targeted oligonucleotide probes for Pseudo-nitzschia species tested through dot-blot hybridisation.

In the scope of the development of a microarray PhyloChip for the detection of toxic phytoplankton species, we designed a large series of probes specific against targets in the nuclear large subunit (LSU) rRNA of a range of Pseudo-nitzschia species and spotted these onto the microarray. Hybridisation with rRNA extracted from monoclonal cultures and from plankton samples revealed many cross-reactions. In the present work, we tested the functionality and specificity of 23 of these probes designed against ten of the species, using a dot-blot procedure. In this case, probe specificity is tested against the target region in PCR products of the LSU rRNA gene marker region blotted on nitrocellulose filters. Each filter was incubated with a species-specific oligoprobe. Eleven of the tested probes showed specific responses, identifying seven Pseudo-nitzschia species. The other probes showed non-specific responses or did not respond at all. Results of dot-blot hybridisations are more specific than those obtained with the microarray approach and the possible reasons for this are discussed.

Specificity of lipoxygenase pathways supports species delineation in the marine diatom genus Pseudo-nitzschia.

Oxylipins are low-molecular weight secondary metabolites derived from the incorporation of oxygen into the carbon chains of polyunsaturated fatty acids (PUFAs). Oxylipins are produced in many prokaryotic and eukaryotic lineages where they are involved in a broad spectrum of actions spanning from stress and defense responses, regulation of growth and development, signaling, and innate immunity. We explored the diversity in oxylipin patterns in the marine planktonic diatom Pseudo-nitzschia. This genus includes several species only distinguishable with the aid of molecular markers. Oxylipin profiles of cultured strains were obtained by reverse phase column on a liquid chromatograph equipped with UV photodiode detector and q-ToF mass spectrometer. Lipoxygenase compounds were mapped on phylogenies of the genus Pseudo-nitzschia inferred from the nuclear encoded hyper-variable region of the LSU rDNA and the plastid encoded rbcL. Results showed that the genus Pseudo-nitzschia exhibits a rich and varied lipoxygenase metabolism of eicosapentaenoic acid (EPA), with a high level of specificity for oxylipin markers that generally corroborated the genotypic delineation, even among genetically closely related cryptic species. These results suggest that oxylipin profiles constitute additional identification tools for Pseudo-nitzschia species providing a functional support to species delineation obtained with molecular markers and morphological traits. The exploration of the diversity, patterns and plasticity of oxylipin production across diatom species and genera will also provide insights on the ecological functions of these secondary metabolites and on the selective pressures driving their diversification.

Strengths and weaknesses of microarray approaches to detect Pseudo-nitzschia species in the field.

The planktonic diatom genus Pseudo-nitzschia contains several genetically closely related species. Some of these can produce domoic acid, a potent neurotoxin. Thus, monitoring programs are needed to screen for the presence of these toxic species. Unfortunately, many are impossible to distinguish using light microscopy. Therefore, we assessed the applicability of microarray technology for detection of toxic and non-toxic Pseudo-nitzschia species in the Gulf of Naples (Mediterranean Sea). Here, 11 species have been detected, of which at least 5 are potentially toxic. A total of 49 genus- and species-specific DNA probes were designed in silico against the nuclear LSU and SSU rRNA of 19 species, and spotted on the microarray. The microarray was tested against total RNA of monoclonal cultures of eight species. Only three of the probes designed to be species-specific were indeed so within the limits of our experimental design. To assess the effectiveness of the microarray in detecting Pseudo-nitzschia species in environmental samples, we hybridized total RNA extracted from 11 seasonal plankton samples against microarray slides and compared the observed pattern with plankton counts in light microscopy and with expected hybridization patterns obtained with monoclonal cultures of the observed species. Presence of species in field samples generally resulted in signal patterns on the microarray as observed with RNA extracted from cultures of these species, but many a-specific signals appeared as well. Possible reasons for the numerous cross reactions are discussed. Calibration curves for Pseudo-nitzschia multistriata showed linear relationship between signal strength and cell number.

Recent and ancient signature of balancing selection around the S-locus in Arabidopsis halleri and A. lyrata.

Balancing selection can maintain different alleles over long evolutionary times. Beyond this direct effect on the molecular targets of selection, balancing selection is also expected to increase neutral polymorphism in linked genome regions, in inverse proportion to their genetic map distances from the selected sites. The genes controlling plant self-incompatibility are subject to one of the strongest forms of balancing selection, and they show clear signatures of balancing selection. The genome region containing those genes (the S-locus) is generally described as nonrecombining, and the physical size of the region with low recombination has recently been established in a few species. However, the size of the region showing the indirect footprints of selection due to linkage to the S-locus is only roughly known. Here, we improved estimates of this region by surveying synonymous polymorphism and estimating recombination rates at 12 flanking region loci at known physical distances from the S-locus region boundary, in two closely related self-incompatible plants Arabidopsis halleri and A. lyrata. In addition to studying more loci than previous studies and using known physical distances, we simulated an explicit demographic scenario for the divergence between the two species, to evaluate the extent of the genomic region whose diversity departs significantly from neutral expectations. At the closest flanking loci, we detected signatures of both recent and ancient indirect effects of selection on the S-locus flanking genes, finding ancestral polymorphisms shared by both species, as well as an excess of derived mutations private to either species. However, these effects are detected only in a physically small region, suggesting that recombination in the flanking regions is sufficient to quickly break up linkage disequilibrium with the S-locus. Our approach may be useful for distinguishing cases of ancient versus recently evolved balancing selection in other systems.

CHLOROPHYLL C PIGMENT PATTERNS IN 18 SPECIES (51 STRAINS) OF THE GENUS PSEUDO-NITZSCHIA (BACILLARIOPHYCEAE)(1).

The pigment composition of 18 species (51 strains) of the pennate diatom Pseudo-nitzschia was examined using HPLC. The carotenoid composition was typical for diatoms, with fucoxanthin (the major xanthophyll), diadinoxanthin, diatoxanthin, and ß,ß-carotene. However, a diverse array of chl c pigments was observed in the studied strains. All Pseudo-nitzschia strains contained chl a and chl c2 , traces of Mg-2,4-divinyl phaeoporphyrin a5 monomethyl ester (MgDVP), and traces of a chl c2 -like pigment originally found in the haptophyte Pavlova gyrans. The distribution of chl c1 and chl c3 was variable among species (present in seven and 14 species, respectively). Based on chl c distribution, three major pigment types were defined: type 1 (chl c1 + c2 , four species: P. australis, P. brasiliana, P. multiseries, and P. seriata), type 2 (chl c1 + c2 + c3 , three species: P. fraudulenta, P. multistriata, and P. pungens), and type 3 (chl c2 + c3 , 11 species: P. arenysensis, P. calliantha, P. cuspidata, P. decipiens, P. delicatissima, P. galaxiae, P. mannii, P. pseudodelicatissima, P. subcurvata, P. cf. subpacifica, and a novel Pseudo-nitzschia species). Type 1 and 2 species also shared the absence of a particular morphological character, the central nodule in the raphe, with the only exception of P. fraudulenta. The implications of such pigment diversity in chemotaxonomy, HAB monitoring, ecology, and phylogeny of Pseudo-nitzschia species are discussed.

Hitch-hiking to a locus under balancing selection: high sequence diversity and low population subdivision at the S-locus genomic region in Arabidopsis halleri.

Hitch-hiking to a site under balancing selection is expected to produce a local increase in nucleotide polymorphism and a decrease in population differentiation compared with the background genomic level, but empirical evidence supporting these predictions is scarce. We surveyed molecular diversity at four genes flanking the region controlling self-incompatibility (the S-locus) in samples from six populations of the herbaceous plant Arabidopsis halleri, and compared their polymorphism with sequences from five control genes unlinked to the S-locus. As a preliminary verification, the S-locus flanking genes were shown to co-segregate with SRK, the gene involved in the self-incompatibility reaction at the pistil level. In agreement with theory, our results demonstrated a significant peak of nucleotide diversity around the S-locus as well as a significant decrease in population genetic structure in the S-locus region compared with both control genes and a set of seven unlinked microsatellite markers. This is consistent with the theoretical expectation that balancing selection is increasing the effective migration rate in subdivided populations. Although only four S-locus flanking genes were investigated, our results suggest that these two signatures of the hitch-hiking effect are localized in a very narrow genomic region.

The rDNA ITS region in the lessepsian marine angiosperm Halophila stipulacea (Forssk.) Aschers. (Hydrocharitaceae): intragenomic variability and putative pseudogenic sequences.

Halophila stipulacea is a dioecious marine angiosperm, widely distributed along the western coasts of the Indian Ocean and the Red Sea. This species is thought to be a Lessepsian immigrant that entered the Mediterranean Sea from the Red Sea after the opening of the Suez Canal (1869). Previous studies have revealed both high phenotypic and genetic variability in Halophila stipulacea populations from the western Mediterranean basin. In order to test the hypothesis of a Lessepsian introduction, we compare genetic polymorphism between putative native (Red Sea) and introduced (Mediterranean) populations through rDNA ITS region (ITS1-5.8S-ITS2) sequence analysis. A high degree of intraindividual variability of ITS sequences was found. Most of the intragenomic polymorphism was due to pseudogenic sequences, present in almost all individuals. Features of ITS functional sequences and pseudogenes are described. Possible causes for the lack of homogenization of ITS paralogues within individuals are discussed.