Present-day and future climate pathways affecting Alexandrium blooms in Puget Sound,WA, USA

This study uses a mechanistic modeling approach to evaluate the effects of various climate pathways on the proliferative phase of the toxin-producing dinoflagellate Alexandrium in Puget Sound, WA, USA. Experimentally derived Alexandrium growth responses to temperature and salinity are combined with simulations of the regional climate and Salish Sea hydrology to investigate future changes in the timing, duration, and extent of blooms. Coarse-grid (100–200 km) global climate model ensemble simulations of the SRES A1B emissions scenario were regionally downscaled to a 12-km grid using the Weather Research and Forecasting model for the period 1969–2069. These results were used to: (1) analyze the future potential changes and variability of coastal upwelling winds, and (2) provide forcing fields to a Regional Ocean Model System used to simulate the circulation of the Salish Sea, including Puget Sound, and the coastal ocean. By comparing circa-1990 and circa-2050 climate scenarios for the environmental conditions that promote Alexandrium blooms, we disentangle the effects of three climate pathways: (1) increased local atmospheric heating, (2) changing riverflow magnitude and timing, and (3) changing ocean inputs associated with changes in upwelling-favorable winds. Future warmer sea surface temperatures in Puget Sound from increased local atmospheric heating increase the maximum growth rates that can be attained by Alexandrium during the bloom season as well as the number of days with conditions that are favorable for bloom development. This could lead to 30 more days a year with bloom-favorable conditions by 2050. In contrast, changes in surface salinity arising from changes in the timing of riverflow have a negligible effect on Alexandrium growth rates, and the behavior of the coastal inputs in the simulations suggests that changes in local upwelling will not have major effects on sea surface temperature or salinity or Alexandrium growth rates in Puget Sound.     

Alexandrium and Scrippsiella cyst viability and cytoplasmic fullness in a 60-cm sediment core from Sequim Bay,WA

Many marine protists produce a benthic resting stage during their life history. This non-motile cyst stage can either germinate near the sediment surface to provide the inoculum for subsequent blooms or, be buried by sediment deposits over time and entrained into the sedimentary record. Buried cysts can be resuspended into the water column by mixing events (e.g., storms) or other disturbances (e.g., dredging). It is not clear how long cysts can survive while buried in the sediments and still be capable of germinating given favorable conditions. Here, the germination success of cysts produced by the potentially toxic dinoflagellate genus Alexandrium and the non-toxic dinoflagellate genus Scrippsiella is reported from a 60-cm sediment core collected in Sequim Bay, WA, in December 2011. Cysts of Alexandrium spp. and Scrippsiella spp. were isolated from 2-cm sections of the core, placed in individual wells of a 96-well plate with growth medium, imaged, incubated at favorable conditions and monitored for germination. An image analysis program, DinoCyst, was used to quantitatively measure the amount of granular storage products, presumed energy stores, inside the cytoplasm to test the hypothesis that older cysts located deeper in the sediment core will have fewer energy stores available and will be less likely to germinate. An index of the area of the cytoplasm occupied with granular storage products relative to cyst size, termed ‘cytoplasmic fullness’, and age, based on ²¹⁰Pb dating of surrounding sediments, was compared with germination success or failure. This research indicates that cysts of Alexandrium spp. and Scrippsiella spp. can remain viable in sediments for 60 years or longer, show little visual evidence of cytoplasmic deterioration over this timescale (as measured by cytoplasmic fullness), and that germination success is statistically similar for cysts isolated from 0–60 cm deep in the sediment core. These results suggest that a cyst's cytoplasmic fullness is not indicative of viability and that cysts located as deep as 60 cm in the sediments are as likely to germinate as surface cysts given favorable conditions.     

The germination characteristics of Alexandrium minutum (Dinophyceae), a toxic dinoflagellate from the Fal estuary (UK)

The germination characteristics of Alexandrium minutum cysts from the Fal estuary were studied at different conditions of temperature (4–24 °C) and salinity (15–35‰) and in the dark and low light intensity (2 μmol^?2 s^?1). Sediment sub-samples were directly cultured and processed at the end of the experiment for counts of non-germinated cysts. A decrease in the number of cysts was interpreted as germination that was calculated by comparison of the number of cysts over time with that of initial counts. The 50% germination time (time at which 50% of the total initial number of cysts had germinated) was calculated for each condition. A. minutum did not germinate in the dark but it germinated under all other conditions studied. Highest germination occurred at salinities of 30 psu and 35 psu and temperatures from 8 °C to 24 °C (germination rate—expressed as the inverse of the 50% germination time: 1.1–1.2). Lowest germination occurred at 15 psu and 4 °C and 24 °C (germination rate: 3.9–3.8). However, little variation in germination rates occurred across the conditions studied. As these conditions represent those likely in the estuary it is probable that A. minutum cysts on the surface of the sediments represent a constant source of cells to the water column and sediment disturbance (revealing buried cysts) could rapidly inoculate the water column with vegetative cells. This data was used to develop a model for Alexandrium germination from coastal sediments.     

An Alexandrium Spp. Cyst Record from Sequim Bay,Washington State,USA, and its Relation to Past Climate Variability1

Since the 1970s, Puget Sound, Washington State, USA, has experienced an increase in detections of paralytic shellfish toxins (PSTs) in shellfish due to blooms of the harmful dinoflagellate Alexandrium. Natural patterns of climate variability, such as the Pacific Decadal Oscillation (PDO), and changes in local environmental factors, such as sea surface temperature (SST) and air temperature, have been linked to the observed increase in PSTs. However, the lack of observations of PSTs in shellfish prior to the 1950s has inhibited statistical assessments of longer‐term trends in climate and environmental conditions on Alexandrium blooms. After a bloom, Alexandrium cells can enter a dormant cyst stage, which settles on the seafloor and then becomes entrained into the sedimentary record. In this study, we created a record of Alexandrium spp. cysts from a sediment core obtained from Sequim Bay, Puget Sound. Cyst abundances ranged from 0 to 400 cysts · cm^?3 and were detected down‐core to a depth of 100 cm, indicating that Alexandrium has been present in Sequim Bay since at least the late 1800s. The cyst record allowed us to statistically examine relationships with available environmental parameters over the past century. Local air temperature and sea surface temperature were positively and significantly correlated with cyst abundances from the late 1800s to 2005; no significant relationship was found between PDO and cyst abundances. This finding suggests that local environmental variations more strongly influence Alexandrium population dynamics in Puget Sound when compared to large‐scale changes.     

Role of resting cysts in Chilean Alexandrium catenella dinoflagellate blooms revisited

The detection of sparse Alexandrium catenella-resting cysts in sediments of southern Chilean fjords has cast doubts on their importance in the recurrence of massive toxic dinoflagellate blooms in the region. The role of resting cysts and the existence of different regional Chilean populations was studied by culturing and genetic approaches to define: (1) cyst production; (2) dormancy period; (3) excystment success; (4) offspring viability and (5) strain mating compatibility. This study newly revealed a short cyst dormancy (minimum 69 days), the role of key abiotic factors (in decreasing order salinity, irradiance, temperature and nutrients) controlling cyst germination (max. 60%) and germling growth rates (up to 0.36–0.52 div. day⁻¹). Amplified fragment length polymorphism (AFLP) characterization showed significant differences in genetic distances (GD) among A. catenella populations that were primarily determined by the geographical origin of isolates and most likely driven by oceanographic dispersal barriers. A complex heterothallic mating system pointed to variable reproductive compatibility (RCs) among Chilean strains that was high among northern (Los Lagos/North Aysén) and southern populations (Magallanes), but limited among the genetically differentiated central (South Aysén) populations. Field cyst surveys after a massive 2009 bloom event revealed the existence of exceptional high cyst densities in particular areas of the fjords (max. 14.627 cysts cm⁻³), which contrast with low cyst concentrations (<221.3 cysts cm⁻³) detected by previous oceanographic campaigns. In conclusion, the present study suggests that A. catenella resting cysts play a more important role in the success of this species in Chilean fjords than previously thought. Results from in vitro experiments suggest that pelagic–benthic processes can maintain year-round low vegetative cell concentrations in the water column, but also can explain the detection of high cysts aggregations after the 2009-bloom event. Regional drivers that lead to massive outbreaks, however, are still unknown but potential scenarios are discussed.     

Germination fluctuation of toxic Alexandrium fundyense and A. pacificum cysts and the relationship with bloom occurrences in Kesennuma Bay,Japan

While cyst germination may be an important factor for the initiation of harmful/toxic blooms, assessments of the fluctuation in phytoplankton cyst germination, from bottom sediments to water columns, are rare in situ due to lack of technology that can detect germinated cells in natural bottom sediments. This study introduces a simple mesocosm method, modeled after previous in situ methods, to measure the germination of plankton resting stage cells. Using this method, seasonal changes in germination fluxes of toxic dinoflagellates resting cysts, specifically Alexandrium fundyense (A. tamarense species complex Group I) and A. pacificum (A. tamarense species complex Group IV), were investigated at a fixed station in Kesennuma Bay, northeast Japan, from April 2014 to April 2015. This investigation was conducted in addition to the typical samplings of seawater and bottom sediments to detect the dinoflagellates vegetative cells and resting cysts. Bloom occurrences of A. fundyense were observed June 2014 and February 2015 with maximum cell densities reaching 3.6 × 10⁶ cells m⁻² and 1.4 × 10⁷ cells m⁻², respectively. The maximum germination fluxes of A. fundyense cysts occurred in April 2014 and December 2014 and were 9.3 × 10³ cells m⁻² day⁻¹ and 1.4 × 10⁴ cells m⁻² day⁻¹, respectively. For A. pacificum, the highest cell density was 7.3 × 10⁷ cells m⁻² during the month of August, and the maximum germination fluxes occurred in July and August, reaching 5.8 × 10² cells m⁻² day⁻¹. Thus, this study revealed the seasonal dynamics of A. fundyense and A. pacificum cyst germination and their bloom occurrences in the water column. Blooms occurred one to two months after peak germination, which strongly suggests that both the formation of the initial population by cyst germination and its continuous growth in the water column most likely contributed to toxic bloom occurrences of A. fundyense and A. pacificum in the bay.     

Wind and temperature controls on Alexandrium blooms (2000–2007) in Thau lagoon (Western Mediterranean)

Since 1998, blooms of Alexandrium catenella/tamarense in the lagoon of Thau developed regularly each autumn, reaching a maximum of several millions cells per liter in 2004. By contrast, spring blooms occurred only twice (in 2000 and 2007). During these periods, sea surface temperatures (SST) and the wind patterns appear to impact the bloom occurrences much more than the apparent limiting resources such as inorganic nutrients. The analysis of SST and wind from April to June and September to November (from 2000 to 2007) indicates first that there has to be an initial wind stress in order to resuspend the cysts buried in the sediment. Blooms then occur after a period of weak winds (<4 m s⁻¹) and of stable SST close to 20 °C (±2 °C). Those conditions appear to be most favorable for germination of Alexandrium cysts and its ensuing vegetative growth. This period of stability (a few days to a few weeks) allows the development of the inoculum from the cyst's germination, its cohesion because of reduced hydrodynamics, and development of vegetative cells that are sensitive to agitation. Strong winds during 1–2 day periods can interrupt the bloom dynamics by dispersing (advection due to southeasterly winds) and/or eliminating (turbulence due to northwesterly winds) the vegetative cells. In the spring, under the same conditions of optimal SST, strong wind episodes dominate and those, as well as biological factors very likely lead to a lower occurrence of blooms relative to the fall situation.     

Which species,Alexandrium catenella (Group I) or A. pacificum (Group IV), is really responsible for past paralytic shellfish poisoning outbreaks in Jinhae-Masan Bay,Korea?

Paralytic shellfish poisoning (PSP) caused the deaths of four people in coastal area of Korea, mainly Jinhae-Masan Bay and adjacent areas, in April 1986 and in 1996. The PSP outbreaks were caused by the consumption of mussels, Mytilus edulis. The organism that caused PSP was identified, from morphological data only, as Alexandrium tamarense which is recently renamed as A. catenella, however recent studies have shown that the morphological diagnostic characteristics used to identify Alexandrium species have uncertainties and molecular tools and other criteria should be considered as well. The organism that caused past PSP outbreaks and incidents in Korea therefore need to be carefully reconsidered. The aim of this study was to re-evaluate the species really responsible for past outbreaks of PSP in Jinhae-Masan Bay, Korea. The temporal production and fluxes of the resting cysts of Alexandrium species were investigated for one year (from March 2011 to February 2012) using a sediment trap, and the morphology and phylogeny of vegetative cells germinated from the resting cysts were analysed. The production of Alexandrium species peaked in August and November, when temporal discrepancies were found in the water temperature (22.4 and 22.7 °C in August, 19.1 and 19.6 °C in November) and salinity (29.5 and 26.1 psu in August, 30.5 and 31.8 psu in November). The morphological data revealed that Alexandrium species germinated from resting cysts collected in August have a ventral pore on the 1′ plate, whereas the 1′ plate in Alexandrium species germinated from resting cysts collected in November lacks a ventral pore. Molecular phylogenetic data for the vegetative cells from the germination experiments allowed the August and November peaks to be assigned to Alexandrium catenella (Group I) and A. pacificum (Group IV), respectively. This indicates that the production of resting cysts of A. catenella can be enhanced by relatively high water temperature. This result is not consistent with those of previous studies that A. catenella responsible for PSP outbreaks was found at relatively low water temperature. In addition, large subunit ribosomal sequences data revealed that A. pacificum isolates from Korea were closely related to those from Australia, Japan and New Zealand where the PSP toxicity of shellfish and blooms occurred in the 1990s, indicating that the introduction of toxic dinoflagellates were related to ballast water from bulk-cargo shipping. Based on these results, we concluded that past PSP outbreaks in Jinhae-Masan Bay of Korea could have been caused by A. pacificum rather than by A. catenella.     

Germination response and viability of an endangered tropical conifer Widdringtonia whytei seeds to temperature and light

The tropical conifer Widdringtonia whytei Rendle is an endangered species endemic to Mulanje Mountain in Malawi. A study was conducted for the first time under controlled conditions in order to assess the effects of temperature and light on germination and viability of W. whytei seeds. Seeds incubated at a constant temperature of 20 °C attained the highest cumulative germination percentage (100%) followed by 87% germination under fluctuating temperatures of 15 °C night/25 °C day. No seed germination occurred at temperatures below 15 °C. Seeds that failed to germinate at temperatures below 15 °C showed the highest (> 90%) viability compared to the seeds incubated at 25 °C (60%). Across temperature regimes, germination was significantly higher under light (44.7%) than dark (35.6%) conditions. It is concluded that temperature is one of the critical factors for germination of W. whytei seed. The ability of W. whytei seeds to germinate both in light and darkness implies that the species would unlikely form a persistent soil seed bank, an attribute which is common in species that survive in habitats frequently disturbed by fires.     

Seed storage and germination in Kumara plicatilis,a tree aloe endemic to mountain fynbos in the Boland,south-western Cape,South Africa

Seed storage under appropriate conditions is a relatively inexpensive means of safeguarding plant genetic material for ex situ conservation. Post-storage germination trials are used to determine the viability of stored seeds, and hence the efficacy of the particular storage treatment. Kumara plicatilis (= Aloe plicatilis) is a tree aloe endemic to mountain fynbos in the Boland, south-western Cape. The viability and germination behaviour of K. plicatilis seeds were assessed for seeds stored for four and nine months at − 80 °C, 4 °C, 25 °C and under ambient conditions in a laboratory. Seeds were germinated under controlled conditions and germination rates and percentages determined. Ungerminated seeds were tested for viability using tetrazolium salt. Seed viability was not significantly reduced during storage. Seeds stored at − 80 °C for four and nine months exhibited the fastest germination rate overall (both 5.9 ± 0.3 weeks, mean ± S.E.), and slowest was for seeds stored under ambient conditions for four and nine months (both 7.8 ± 0.4 weeks). All seed lots showed similar percentage germination after four months of storage (78.0–90.4%). The highest percentage germination overall was for seeds stored at − 80 °C for four months (90.4%) and the lowest was for seeds kept at 4 °C and − 80 °C for nine months (39.2 and 39.6%, respectively). Respective percentage viability for ungerminated seeds in these two treatments was 82% and 87%, respectively, indicating the induction of secondary dormancy. Induced dormancy triggered by protracted cold temperatures may be an adaptation that enables seeds to survive prolonged extreme conditions that are unfavourable for germination. Further research on the long-term storage of aloe seeds would be beneficial for developing long-term seed storage and germination testing protocols for ex situ conservation.     

Resting cysts,and effects of temperature and salinity on the growth of vegetative cells of the potentially harmful species Alexandrium insuetum Balech (Dinophyceae)

The potentially harmful species Alexandrium insuetum established by the incubation of resting cysts isolated from sediment trap samples collected at Jinhae-Masan Bay, Korea was characterized by morphological and phylogenetic analysis. The effects of temperature and salinity on the growth of A. insuetum were also investigated. The resting cysts are characterized by a spherical shape, a small size (20–25 μm) and the presence of either three or four red accumulation bodies. The similarity of morphological features of the resting cysts to those of other species of the minutum group (consisting of Alexandrium minutum and A. tamutum) indicates that the morphological features of resting cysts might improve the accuracy of the grouping of Alexandrium species. A. insuetum germinated from the resting cysts is morphologically consistent with vegetative cells reported from Korean and Japanese coastal areas, and has an partial large subunit (LSU) rDNA sequence identical to that from Japanese strains. The growth of A. insuetum was observed between salinity 20 and 35, with increasing temperature; however at 25 °C, A. insuetum could grow even at the salinity of 15. The highest growth rate (0.60 d⁻¹) was observed at 25 °C and the salinity of 25, which is higher than the previously reported growth rate of A. tamarense, which is responsible for outbreaks of paralytic shellfish poisoining and blooms in Jinhae-Masan Bay. These results suggest that the proliferation of A. insuetum in Jinhae-Masan Bay is likely to be highest during the summer.     

Ecophysiological requirements on seed germination of a Mediterranean perennial grass (Stipa tenacissima L.) under controlled temperatures and water stress

Stipa tenacissima L. (alpha grass) steppes are one of the most representative ecosystems in arid Mediterranean ecosystems. On the one hand these steppes, which are perpetually exposed to climate and strong anthropogenic pressure, have undergone severe degradation. On the other hand, the ability of S. tenacissima to regenerate naturally is significantly reduced. In this study the germination response and seedling emergence of S. tenacissima are examined in relation to the main environmental factors (water stress and temperature) under laboratory-controlled conditions. The main aim of this paper was to investigate the influence of temperature over a temperature range (10 °C–30 °C) and water stress induced by the solutions of polyethylene glycol (PEG)-6000 (0 to − 1.6 MPa) for a period of 30 days, on the germination behavior of S. tenacissima seeds. The results showed that temperatures between 10° and 20 °C seem to be favorable for the germination of this species, with optimum temperatures among accessions found in 20 °C. When seeds were water-stressed, germination severely decreased at − 0.8 MPa, indicating that the accession resistance limits to the water stress, and was completely inhibited at − 1.6 MPa. Consequently, the final germination percentage (FGP) decreased and the mean time germination (MTG) increased. Based on the empirical data of the germination rate, we estimated that the parameters of the thermal time and hydrotime models showed different values in all accessions which proves the difference between accession adaptive capacities.     

Alexandrium pacificum Litaker sp. nov (Group IV): Resting cyst distribution and toxin profile of vegetative cells in Bizerte Lagoon (Tunisia,Southern Mediterranean Sea)

A high spatial resolution sampling of Alexandrium pacificum cysts, along with sediment characteristics (% H₂O, % organic matter (OM), granulometry), vegetative cell abundance and environmental factors were investigated at 123 study stations in Bizerte Lagoon (Tunisia). Morphological examination and ribotyping of cells obtained from a culture called ABZ1 obtained from a cyst isolated in lagoon sediment confirmed that the species was A. pacificum. The toxin profile from the ABZ1 culture harvested during exponential growth phase was simple and composed of the N-sulfocarbamoyl toxins C1 (9.82 pg toxin cell⁻¹), the GTX6 (3.26 pg toxin cell⁻¹) and the carbamoyl toxin Neo-STX (0.38 pg toxin cell⁻¹). The latter represented only 2.8% of the total toxins in this strain.High abundance of A. pacificum cysts correlated with enhanced percentages of water and organic matter in the sediment. In addition, sediment fractions of less than 63 μm were examined as a favorable potential seedbed for initiation of future blooms and outbreaks of A. pacificum in the lagoon. A significant difference in the cyst distribution pattern was recorded among the lagoon's different zones, with the higher cyst abundance occurring in the inner waters. Also, no correlation due to the specific hydrodynamics of the lagoon was observed in the spatial distribution of A. pacificum cysts and vegetative cells.     

Algicidal and growth-inhibiting bacteria associated with seagrass and macroalgae beds in Puget Sound,WA, USA

The algicidal and growth-inhibiting bacteria associated with seagrasses and macroalgae were characterized during the summer of 2012 and 2013 throughout Puget Sound, WA, USA. In 2012, Heterosigma akashiwo-killing bacteria were observed in concentrations of 2.8 × 10⁶ CFU g⁻¹ wet in the outer organic layer (biofilm) on the common eelgrass (Zostera marina) in north Padilla Bay. Bacteria that inhibited the growth of Alexandrium tamarense were detected within the biofilm formed on the eelgrass canopy at Dumas Bay and North Bay at densities of ∼10⁸ CFU g⁻¹ wet weight. Additionally, up to 4100 CFU mL⁻¹ of algicidal and growth-inhibiting bacteria affecting both A. tamarense and H. akashiwo were detected in seawater adjacent to seven different eelgrass beds. In 2013, H. akashiwo-killing bacteria were found on Z. marina and Ulva lactuca with the highest densities of ∼10⁸ CFU g⁻¹ wet weight at Shallow Bay, Sucia Island. Bacteria that inhibited the growth of H. akashiwo and A. tamarense were also detected on Z. marina and Z. japonica at central Padilla Bay. Heterosigma akashiwo cysts were detected at a concentration of 3400 cysts g⁻¹ wet weight in the sediment from Westcott Bay (northern San Juan Island), a location where eelgrass disappeared in 2002. These findings provide new insights on the ecology of algicidal and growth-inhibiting bacteria, and suggest that seagrass and macroalgae provide an environment that may influence the abundance of harmful algae in this region. This work highlights the importance of protection and restoration of native seagrasses and macroalgae in nearshore environments, in particular those regions where shellfish restoration initiatives are in place to satisfy a growing demand for seafood.     

Effects of seed maturation time and dry storage on light and temperature requirements during germination in invasive Prosopis juliflora

The effects of time of seed maturation and dry seed storage and of light and temperature requirements during seed incubation on final germination percentage and germination rate were assessed for the invasive shrub Prosopis juliflora (Sw.) D.C., grown under desert environmental conditions of the United Arab Emirates (UAE). Seeds were collected from Fujira on the northern coast of the UAE at different times during the growing seasons (autumn, winter and spring) and were germinated immediately and after 8 months of dry storage under room temperature (20±3 °C). Seeds were germinated at three temperatures (15, 25 and 40 °C) in both continuous light and darkness. The results showed significant effects for time of seed collection, seed storage, light and temperature of seed incubation and many of their interactions on both germination percentage and rate. Fresh seeds matured during autumn and winter germinated significantly greater at 40 °C and in light than at lower temperatures and in dark. Storage significantly increased germination percentage and rate; the increase was greater for seeds matured during winter than for seeds matured during spring. This indicates that dormancy breakage was greater in seeds of winter than seeds of spring. The need for high temperature to achieve greater germination was significantly reduced after seed storage, especially for seeds matured in autumn and winter.     

Abundance and distribution of toxic Alexandrium tamarense resting cysts in the sediments of the Chukchi Sea and the eastern Bering Sea

Abundance and distribution of the toxic dinoflagellate Alexandrium tamarense species complex resting cyst were investigated in the eastern Bering Sea and the Chukchi Sea for the first time. Sediment samples (top 0–3 cm depth) were collected from the continental shelf of the eastern Bering Sea (17 stations) and the Chukchi Sea (13 stations) together with a long core sample (top 0–21 cm depth) from one station in the Chukchi Sea during 2009–2012. The cysts were enumerated using the primuline staining method. Species identification of the cysts was carried out with multiplex PCR assay and the plate morphology of vegetative cells germinated from cysts in the both areas. Alexandrium cysts were widely detected in the both areas, ranging from not detected (<1 cysts cm⁻³) to 835 cysts cm⁻³ wet sediment in the eastern Bering Sea and from not detected (<1 cysts cm⁻³) to 10,600 cysts cm⁻³ in the Chukchi Sea, and all isolated cysts were genetically and morphologically identified as the North American clade A. tamarense. Their cysts were mainly distributed in the shallow continental shelf where the water depth was less than 100 m in both areas. The cysts were detected from the deep layer (18–21 cm depth of sediment core) of the long core sample. The present study confirmed the abundant existence of A. tamarense with wide range of distribution in these areas. This fact suggests that A. tamarense vegetative cells have appeared in the water column in the both areas. Furthermore, these abundant cyst depositions indicate that this species originally distributed in the Arctic and subarctic regions and well adapted to the environments in the marginal ice zone.     

Temperature requirements for dormancy break and seed germination vary greatly among 14 wetland Carex species

We evaluated dormancy loss in seeds of 14 Carex species (C. atherodes, C. brevior, C. comosa, C. cristatella, C. cryptolepis, C. granularis, C. hystericina, C. lacustris, C. pellita, C. scoparia, C. stipata, C. stricta, C. utriculata, C. vulpinoidea) under growing season and stratification conditions and determined the temperature requirements for germination. Seeds were germinated for 1 year at a diel temperature regime (5/1 °C, 14/1 °C, 22/8 °C, or 27/15 °C) or a seasonal regime (seeds moved among the four diel regimes to mimic seasonal temperatures). All species had conditionally dormant seeds at maturity. The optimal temperature for germination of most species was 27/15 °C. The 14 species were grouped by their seed viability, dormancy, and germination with a Seed Regeneration Index (SRI; range 0–1) using the results of this study and a previously published paper on stratification effects on Carex seed dormancy and germination. The eight species that had an SRI value >0.5 (C. brevior, C. comosa, C. cristatella, C. cryptolepis, C. hystericina, C. scoparia, C. stipata, C. vulpinoidea) had high seed viability (>60%) and required little to no stratification to germinate readily over a broad range of temperatures. The six species with an SRI value <0.5 (C. atherodes, C. granularis, C. lacustris, C. pellita, C. stricta, C. utriculata) generally had low seed viability (<50% and often <1%) and required stratification or particular temperatures (35/30 °C or 5/1 °C for C. stricta; 35/30 °C for C. utriculata; 27/15 °C for C. atherodes, C. lacustris, C. pellita; 5/1 °C for C. granularis) for germination ≥50%. These six species will require more attention from restoration practitioners to ensure that there are sufficient viable seeds to meet revegetation goals, that dormancy break is achieved, and that seeds are sown when temperatures are optimal for germination. The different seed germination syndromes that we found for these Carex species likely contribute to variable seed bank formation and emergence patterns, and species coexistence.     

Fungal surface remodelling visualized by atomic force microscopy

Most fungal growth is localized to the tips of hyphae, however, early stages of spore germination and the growth of certain morphological mutant strains exhibit non-polarized expansion. We used atomic force microscopy (AFM) to document changes in Aspergillus nidulans wall surfaces during non-polarized growth: spore germination, and growth in a strain containing the hypA1 temperature sensitive morphogenesis defect. We compared wall surface structures of both wild-type and mutant A. nidulans following growth at 28 ° and 42 °C, the latter being the restrictive temperature for hypA1. There was no appreciable difference in surface ultrastructure between wild-type and hypA1 spores, or hyphal walls grown at 28 °C. When dry mature A. nidulans conidia were wetted they lost their hydrophobin coat, indicating an intermediate stage between dormancy and swelling. The surface structure of hypA1 germlings grown at 42 °C was less organized than wild-type hyphae grown under the same conditions, and had a larger range of subunit sizes. AFM images of hyphal wall surface changes following a shift in growth temperature from restrictive (42 °C) to permissive (28 °C), showed a gradient of sizes for wall surface features similar to the trend observed for wild-type cells at branch points. Changes associated with the hyphal wall structure for A. nidulans hypA1 offer insight into the events associated with fungal germination, and wall remodelling.     

Some dynamics of spread and infection by aeciospores of Puccinia punctiformis,a biological control pathogen of Cirsium arvense

Systemic disease of Cirsium arvense caused by Puccinia punctiformis depends on teliospores, from telia that are formed from uredinia, on C. arvense leaves. Uredinia result from infection of the leaves by aeciospores which are one main source of dispersal of the fungus. However, factors governing aeciospore spread, germination, infection, and conversion to uredinia and telia have not been extensively investigated. In this study, effective spread of aeciospores from a source area in a field was fitted to an exponential decline model with a predicted maximum distance of spread of 30 m from the source area to observed uredinia on one leaf of one C. arvense shoot. However, the greatest number of shoots bearing leaves with uredinia/telia was observed within 12 m of the source area, and there were no such shoots observed beyond 17 m from the source area. Aeciospore germination under laboratory conditions was low, with a maximum of about 10%. Temperatures between 18 °C and 25 °C were most favorable for germination with maximum germination at 22 °C. Temperature and dew point data collected from the Frederick, MD airport indicated that optimum temperatures for aeciospore germination occurred in late spring from about May 18 to June 20. Dew conditions during this period were favorable for aeciospore germination. A total of 122 lower leaves, 2 per shoot, on 61 C. arvense shoots were individually inoculated in a dew tent in a greenhouse by painting suspensions of aeciospores onto the leaves. Of these inoculated leaves, 47 produced uredinia within an average of 21.2 ± 6.9 days after inoculation. Uredinia were also produced, in the absence of dew, on 17 non-inoculated leaves of 12 shoots. These leaves were up to 4 leaves above leaves on the same shoots that had been individually and separately inoculated. Results of PCR tests for the presence of the fungus in non-inoculated leaves that were not bearing uredinia, showed that 44 leaves above inoculated leaves on 27 shoots were positive for the presence of the fungus. These leaves were up to 5 leaves above inoculated leaves on the same shoot. Uredinia production and positive PCR results on leaves above inoculated leaves on the same shoot indicated that aeciospore infection was weakly systemic. In other tests in which all leaves of plants were spray-inoculated with aeciospores, uredinia were produced by 10 days after inoculation and converted to telia and sole production of teliospores in about 63 days after inoculation. Successful systemic aeciospore infections in late spring would be expected to result in uredinia production in excess of a 1:1 ratio of aeciospore infections to uredinia and ultimately telia production in late summer. In this manner, systemic aeciospore infections would promote increased density of telia that lead to systemic infections of roots in the fall.