Growing Degree-Day Measurement of Cyst Germination Rates in the Toxic Dinoflagellate Alexandrium catenella.

Blooms of many dinoflagellates, including several harmful algal bloom (HAB) species, are seeded and revived through the germination of benthic resting cysts. Temperature is a key determinant of cysts' germination rate, and temperature-germination rate relationships are therefore fundamental to understanding species' germling cell production, cyst bed persistence, and resilience to climate warming. This study measured germination by cysts of the HAB dinoflagellate Alexandrium catenella using a growing degree-day (DD) approach that accounts for the time and intensity of warming above a critical temperature. Time courses of germination at different temperatures were fit to lognormal cumulative distribution functions for the estimation of the median days to germination. As temperature increased, germination times decreased hyperbolically. DD scaling collapsed variability in germination times between temperatures after cysts were oxygenated. A parallel experiment demonstrated stable temperature-rate relationships in cysts collected during different phases of seasonal temperature cycles in situ over three years. DD scaling of the results from prior A. catenella germination studies showed consistent differences between populations across a wide range of temperatures and suggests selective pressure for different germination rates. The DD model provides an elegant approach to quantify and compare the temperature dependency of germination among populations, between species, and in response to changing environmental conditions. IMPORTANCE Germination by benthic life history stages is the first step of bloom initiation in many, diverse phytoplankton species. This study outlines a growing degree-day (DD) approach for comparing the temperature dependence of germination rates measured in different populations. Germination by cysts of Alexandrium catenella, a harmful algal bloom dinoflagellate that causes paralytic shellfish poisoning, is shown to require a defined amount of warming, measured in DD after cysts are aerated. Scaling by DD, the time integral of temperature difference from a critical threshold, enabled direct comparison of rates measured at different temperatures and in different studies.

Evidence for massive and recurrent toxic blooms of Alexandrium catenella in the Alaskan Arctic.

Among the organisms that spread into and flourish in Arctic waters with rising temperatures and sea ice loss are toxic algae, a group of harmful algal bloom species that produce potent biotoxins. Alexandrium catenella, a cyst-forming dinoflagellate that causes paralytic shellfish poisoning worldwide, has been a significant threat to human health in southeastern Alaska for centuries. It is known to be transported into Arctic regions in waters transiting northward through the Bering Strait, yet there is little recognition of this organism as a human health concern north of the Strait. Here, we describe an exceptionally large A. catenella benthic cyst bed and hydrographic conditions across the Chukchi Sea that support germination and development of recurrent, locally originating and self-seeding blooms. Two prominent cyst accumulation zones result from deposition promoted by weak circulation. Cyst concentrations are among the highest reported globally for this species, and the cyst bed is at least 6× larger in area than any other. These extraordinary accumulations are attributed to repeated inputs from advected southern blooms and to localized cyst formation and deposition. Over the past two decades, warming has likely increased the magnitude of the germination flux twofold and advanced the timing of cell inoculation into the euphotic zone by 20 d. Conditions are also now favorable for bloom development in surface waters. The region is poised to support annually recurrent A. catenella blooms that are massive in scale, posing a significant and worrisome threat to public and ecosystem health in Alaskan Arctic communities where economies are subsistence based.

Morphological and phylogenetic data do not support the split of Alexandrium into four genera.

A recently published study analyzed the phylogenetic relationship between the genera Centrodinium and Alexandrium, confirming an earlier publication showing the genus Alexandrium as paraphyletic. This most recent manuscript retained the genus Alexandrium, introduced a new genus Episemicolon, resurrected two genera, Gessnerium and Protogonyaulax, and stated that: "The polyphyly [sic] of Alexandrium is solved with the split into four genera". However, these reintroduced taxa were not based on monophyletic groups. Therefore this work, if accepted, would result in replacing a single paraphyletic taxon with several non-monophyletic ones. The morphological data presented for genus characterization also do not convincingly support taxa delimitations. The combination of weak molecular phylogenetics and the lack of diagnostic traits (i.e., autapomorphies) render the applicability of the concept of limited use. The proposal to split the genus Alexandrium on the basis of our current knowledge is rejected herein. The aim here is not to present an alternative analysis and revision, but to maintain Alexandrium. A better constructed and more phylogenetically accurate revision can and should wait until more complete evidence becomes available and there is a strong reason to revise the genus Alexandrium. The reasons are explained in detail by a review of the available molecular and morphological data for species of the genera Alexandrium and Centrodinium. In addition, cyst morphology and chemotaxonomy are discussed, and the need for integrative taxonomy is highlighted.

Cyst-forming dinoflagellates in a warming climate.

Many phytoplankton species, including many harmful algal bloom (HAB) species, survive long periods between blooms through formation of benthic resting stages. Because they are crucial to the persistence of these species and the initiation of new blooms, the physiology of benthic stages must be considered to accurately predict responses to climate warming and associated environmental changes. The benthic stages of dinoflagellates, called resting cysts, germinate in response to the combination of favorable temperature, oxygen-availability, and release from dormancy. The latter is a mechanism that prevents germination even when oxygen and temperature conditions are favorable. Here, evidence of temperature-mediated control of dormancy duration from the dinoflagellates Alexandrium catenella and Pyrodinium bahamense-two HAB species that cause paralytic shellfish poisoning (PSP)-is reviewed and presented alongside new evidence of complementary, temperature-based control of cyst quiescence (the state in which cysts germinate on exposure to favorable conditions). Interaction of the two temperature-based mechanisms with climate is explored through a simple model parameterized using results from recent experiments with A. catenella. Simulations demonstrate the importance of seasonal temperature cycles for the synchronization of cysts' release from dormancy and are consistent with biogeography-based inferences that A. catenella is more tolerant of warming in habitats that experience a larger range of seasonal temperature variation (i.e., have higher temperature seasonality). Temperature seasonality is much greater in shallow, long-residence time habitats than in deep, open-water ones. As warming shifts species' ranges, cyst beds may persist longer in more seasonally variable, shallow inshore habitats than in deep offshore ones, promoting HABs that are more localized and commence earlier each year. Recent field investigations of A. catenella also point to the importance of new cyst formation as a factor triggering bloom termination through mass sexual induction. In areas where temperature seasonality restricts the flux of new swimming cells (germlings) to narrow temporal windows, warming is unlikely to promote longer and more intense HAB impacts-even when water column conditions would otherwise promote prolonged bloom development. Many species likely have a strong drive to sexually differentiate and produce new cysts once concentrations reach levels that are conducive to new cyst formation. This phenomenon can impose a limit to bloom intensification and suggests an important role for cyst bed quiescence in determining the duration of HAB risk periods.

Quantitative Response of Alexandrium catenella Cyst Dormancy to Cold Exposure.

Many dinoflagellate cysts experience dormancy, a reversible state that prevents germination during unfavorable periods. Several of these species also cause harmful algal blooms (HABs), so a quantitative understanding of dormancy cycling is desired for better prediction and mitigation of bloom impacts. This study examines the effect of cold exposure on the duration of dormancy in Alexandrium catenella, a HAB dinoflagellate that causes paralytic shellfish poisoning (PSP). Mature, dormant cysts from Nauset Marsh (Cape Cod, MA USA) were stored at low but above freezing temperatures for up to six months. Dormancy status was then determined at regular intervals using a germination assay. Dormancy timing was variable among temperatures and was shorter in colder treatments, but the differences collapse when temperature and duration of storage are scaled by chilling-units (CU), a common horticultural predictor of plant and insect development in response to weather. Cysts within Nauset meet a well-defined chilling requirement by late January, after which they are poised to germinate with the onset of favorable conditions in spring. Cysts thus modulate their dormancy cycles in response to their temperature history, enhancing the potential for new blooms and improving this species' adaptability to both unseasonable weather and new habitats/climate regimes.

Bloom termination of the toxic dinoflagellate Alexandrium catenella: Vertical migration behavior, sediment infiltration, and benthic cyst yield.

New resting cyst production is crucial for the survival of many microbial eukaryotes including phytoplankton that cause harmful algal blooms. Production in situ has previously been estimated through sediment trap deployments, but here was instead assessed through estimation of the total number of planktonic cells and new resting cysts produced by a localized, inshore bloom of Alexandrium catenella, a dinoflagellate that is a globally important cause of paralytic shellfish poisoning. Our approach utilizes high frequency, automated water monitoring, weekly observation of new cyst production, and pre- and post-bloom spatial surveys of total resting cyst abundance. Through this approach, new cyst recruitment within the study area was shown to account for at least 10.9% ± 2.6% (SE) of the bloom's decline, ∼ 5× greater than reported from comparable, sediment trap based studies. The observed distribution and timing of new cyst recruitment indicate that: (1) planozygotes, the immediate precursor to cysts in the life cycle, migrate nearer to the water surface than other planktonic stages and (2) encystment occurs after planozygote settlement on bottom sediments. Near surface localization by planozygotes explains the ephemerality of red surface water discoloration by A. catenella blooms, and also enhances the dispersal of new cysts. Following settlement, bioturbation and perhaps active swimming promote sediment infiltration by planozygotes, reducing the extent of cyst redistribution between blooms. The concerted nature of bloom sexual induction, especially in the context of an observed upper limit to A. catenella bloom intensities and heightened susceptibility of planozygotes to the parasite Amoebophrya, is also discussed.

Insights into the loss factors of phytoplankton blooms: The role of cell mortality in the decline of two inshore Alexandrium blooms.

While considerable effort has been devoted to understanding the factors regulating the development of phytoplankton blooms, the mechanisms leading to bloom decline and termination have received less attention. Grazing and sedimentation have been invoked as the main routes for the loss of phytoplankton biomass, and more recently, viral lysis, parasitism and programmed cell death (PCD) have been recognized as additional removal factors. Despite the importance of bloom declines to phytoplankton dynamics, the incidence and significance of various loss factors in regulating phytoplankton populations have not been widely characterized in natural blooms. To understand mechanisms controlling bloom decline, we studied two independent, inshore blooms of Alexandrium fundyense, paying special attention to cell mortality as a loss pathway. We observed increases in the number of dead cells with PCD features after the peak of both blooms, demonstrating a role for cell mortality in their terminations. In both blooms, sexual cyst formation appears to have been the dominant process leading to bloom termination, as both blooms were dominated by small-sized gamete cells near their peaks. Cell death and parasitism became more significant as sources of cell loss several days after the onset of bloom decline. Our findings show two distinct phases of bloom decline, characterized by sexual fusion as the initial dominant cell removal processes followed by elimination of remaining cells by cell death and parasitism.

Temperature and residence time controls on an estuarine harmful algal bloom: Modeling hydrodynamics and Alexandrium fundyense in Nauset estuary.

A highly resolved, 3-d model of hydrodynamics and Alexandrium fundyense in an estuarine embayment has been developed to investigate the physical and biological controls on a recurrent harmful algal bloom. Nauset estuary on Cape Cod (MA, USA) consists of three salt ponds connected to the ocean through a shallow marsh and network of tidal channels. The model is evaluated using quantitative skill metrics against observations of physical and biological conditions during three spring blooms. The A. fundyense model is based on prior model applications for the nearby Gulf of Maine, but notable modifications were made to be consistent with the Nauset observations. The dominant factors controlling the A. fundyense bloom in Nauset were the water temperature, which regulates organism growth rates, and the efficient retention of cells due to bathymetric constraints, stratification, and cell behavior (diel vertical migration). Spring-neap variability in exchange altered residence times, but for cell retention to be substantially longer than the cell doubling time required both active vertical migration and stratification that inhibits mixing of cells into the surface layer by wind and tidal currents. Unlike in the Gulf of Maine, the model results were relatively insensitive to cyst distributions or germination rates. Instead, in Nauset, high apparent rates of vegetative cell division by retained populations dictated bloom development. Cyst germination occurred earlier in the year than in the Gulf of Maine, suggesting that Nauset cysts have different controls on germination timing. The model results were relatively insensitive to nutrient concentrations, due to eutrophic conditions in the highly impacted estuary or due to limitations in the spatial and temporal resolution of nutrient sampling. Cell loss rates were inferred to be extremely low during the growth phase of the bloom, but increased rapidly during the final phase due to processes that remain uncertain. The validated model allows a quantitative assessment of the factors that contribute to the development of a recurrent harmful algal bloom and provides a framework for assessing similarly impacted coastal systems.

Outbreeding lethality between toxic Group I and nontoxic Group III Alexandrium tamarense spp. isolates: Predominance of heterotypic encystment and implications for mating interactions and biogeography.

We report the zygotic encystment of geographically dispersed isolates in the dinoflagellate species complex Alexandrium tamarense, in particular, successful mating of toxic Group I and nontoxic Group III isolates. However, hypnozygotes produced in Group I/III co-cultures complete no more than three divisions after germinating. Previous reports have suggested a mate recognition mechanism whereby hypnozygotes produced in co-cultures could arise from either homotypic (inbred) or heterotypic (outbred) gamete pairs. To determine the extent to which each occurs, a nested PCR assay was developed to determine parentage of individual hypnozygotes. The vast majority of hypnozygotes from pairwise Group I/III co-cultures were outbred, so that inviability was a result of hybridization, not inbreeding. These findings support the assertion that complete speciation underlies the phylogenetic structure of the Alexandrium tamarense species complex. Additionally, the ribosomal DNA (rDNA) copy numbers of both hybrid and single ribotype hypnozygotes were reduced substantially from those of haploid motile cells. The destruction of rDNA loci may be crucial for the successful mating of genetically distant conjugants and appears integral to the process of encystment. The inviability of Group I/III hybrids is important for public health because the presence of hybrid cysts may indicate ongoing displacement of a nontoxic population by a toxic one (or vice versa). Hybrid inviability also suggests a bloom control strategy whereby persistent, toxic Group I blooms could be mitigated by introduction of nontoxic Group III cells. The potential for hybridization in nature was investigated by applying the nested PCR assay to hypnozygotes from Belfast Lough, Northern Ireland, a region where Group I and III populations co-occur. Two hybrid cysts were identified in 14 successful assays, demonstrating that Group I and III populations do interbreed in that region. However, an analysis of mating data collected over an 18-year period indicated a leaky pre-mating barrier between ribosomal species (including Groups I and III). Whether the observed selectivity inhibits hybridization in nature is dependent on its mechanism. If the point of selectivity is the induction of gametogenesis, dissimilar ribotypes could interbreed freely, promoting displacement in cases where hybridization is lethal. If instead, selectivity occurs during the adhesion of gamete pairs, it could enable stable coexistence of A. tamarense species. In either case, hybrid inviability may impose a significant obstacle to range expansion. The nested PCR assay developed here is a valuable tool for investigation of interspecies hybridization and its consequences for the global biogeography of these important organisms.