Phytoplankton growth after a century of dormancy illuminates past resilience to catastrophic darkness

Abstract

Photosynthesis evolved in the oceans more than 3 billion years ago and has persisted throughout all major extinction events in Earth's history. The most recent of such events is linked to an abrupt collapse of primary production due to darkness following the Chicxulub asteroid impact 65.5 million years ago. Coastal phytoplankton groups (particularly dinoflagellates and diatoms) appear to have been resilient to this biotic crisis, but the reason for their high survival rates is still unknown. Here we show that the growth performance of dinoflagellate cells germinated from resting stages is unaffected by up to a century of dormancy. Our results clearly indicate that phytoplankton resting stages can endure periods of darkness far exceeding those estimated for the Cretaceous-Paleogene extinction and may effectively aid the rapid resurgence of primary production in coastal areas after events of prolonged photosynthesis shut-down.

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Received 9 Dec 2010 | Accepted 13 Apr 2011 | Published 17 May 2011 DOI: 10.1038/ncomms1314

Soa Ribeiro1,2,3, Terje Berge1, Nina Lundholm4, Thorbjrn J. Andersen5, Ftima Abrantes2 & Marianne Ellegaard1

Photosynthesis evolved in the oceans more than 3 billion years ago and has persisted throughout all major extinction events in Earths history. The most recent of such events is linked to an abrupt collapse of primary production due to darkness following the Chicxulub asteroid impact 65.5 million years ago. Coastal phytoplankton groups (particularly dinoagellates and diatoms) appear to have been resilient to this biotic crisis, but the reason for their high survival rates is still unknown. Here we show that the growth performance of dinoagellate cells germinated from resting stages is unaffected by up to a century of dormancy. Our results clearly indicate that phytoplankton resting stages can endure periods of darkness far exceeding those estimated for the Cretaceous-Paleogene extinction and may effectively aid the rapid resurgence of primary production in coastal areas after events of prolonged photosynthesis shut-down.

Phytoplankton growth after a century of dormancy illuminates past resilience to catastrophic darkness

1 Marine Biological Section, Department of Biology, Faculty of Science, University of Copenhagen, ster Farimagsgade 2D, 1353 CPH-K, Denmark. 2 Unidade de Geologia Marinha, LNEG, Estrada da Portela, Zambujal, Apartado 7586, 2720-866 Amadora, Portugal. 3 Centro de Oceanograa, Faculdade de Cincias, Universidade de Lisboa, Campo Grande 1749-016 Lisbon, Portugal. 4 Natural History Museum of Denmark, University of Copenhagen, Slvgade 83 Opg S, 1307 CPH-K, Denmark. 5 Department of Geography and Geology, University of Copenhagen, ster Voldgade 10, 1350 CPH-K, Denmark. Correspondence and requests for materials should be addressed to S.R. (email: [email protected]).

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a b

Photosynthesis in the oceans is responsible for about half of the global carbon xation. This life-sustaining process has been resilient to episodes of catastrophic environmental change

across geological times. Understanding the means by which marine primary producers have survived major crises in the past might help predict their resilience to future changes.

The Cretaceous-Paleogene (K-Pg) mass extinction is the most recent and well-studied biotic crisis of the Phanerozoic (last ca. 500 million years), and its evolutionary consequences extend to modern biota1. Strong evidence indicates that the impact of an ~10-km-wide asteroid on the Earths seaoor 65.5 million years ago ejected large amounts of material into the atmosphere, blocking sunlight to levels below photosynthesis compensation, and contributing to a global collapse of terrestrial and marine food webs2,3. The impact is expected to have resulted in massive earthquakes, widespread tsunamis, acid rain, ozone destruction and wildres4,5, but the collapse of primary productivity is generally accepted to have been a major cause of extinction6. Model calculations suggest an atmospheric blackout lasting at least 69 months7,8, followed by a 50% drop in solar transmission for about a decade8. Moreover, severe cooling is expected to have altered ocean stratication and circulation for several decades, resulting in highly unstable conditions8.

In the ocean, extinction rates between the major groups of primary producers were strikingly dierent, as evidenced by the fossil record9. Oceanic calcifying plankton (mainly coccolithophores) suered the most drastic diversity loss ever, for example (ref. 10), resulting in a carbonate production crash that disrupted organic matter uxes to the deep ocean for a period of up to 34 million years11. Much less catastrophic extinction rates were inferred from the K-Pg fossil record of coastal primary producers such as diatoms12 and dinoagellates1315. In fact, life resurgence in coastal areas appears to have been relatively rapid, possibly occurring in less than a century16.

Many modern phytoplankton species are able to combine photo-synthesis with uptake of particulate food by phagocytosis, a nutritional strategy termed mixotrophy. Although mixotrophy might have been a survival advantage to some species17, it cannot solely explain the dierential extinction. Phagotrophic mixotrophy is unknown in diatoms and some mixotrophic dinoagellates have been reported to encyst when exposed to sudden darkness18. The end-Cretaceous asteroid impact is predicted to have released excessive CO2 into the atmosphere and caused nitric- and sulphuric-acid rain4,5,8. Ocean

acidication might have negatively aected biocalcifying organisms and has been proposed as an important cause for the near-extermination of calcareous nannoplankton, although culturing studies have shown that coccolitophores are able to maintain intracellular calcication in calcite-undersaturated waters, and can grow at low pH values19. A few calcareous nannoplankton survivors of the K-Pg event have recently been identied and, notably, these include species with cyst-like morphologies adapted to neritic environments20.

Resting stages are also common among the phytoplankton groups least aected by the K-Pg extinction (diatoms and dinoagellates). It has, therefore, been hypothesized that this life-history trait may have provided an escape from extinction21,22.

In order for phytoplankton resting stages to have conferred a survival advantage at the K-Pg, these would have to remain viable for as long as the darkness and environmental instability lasted (up to several decades) and, more importantly, maintain the ability to resume vegetative growth at high rates aer prolonged dormancy. Phytoplankton resting stages oen result from sexual reproduction and are classically considered life-history traits to survive short-term and seasonal unfavourable conditions. They provide a seeding stock for new populations, aid dispersal, and are an import source of genetic novelty23. A few studies have reported survival of dinoagel-late and diatom resting stages from months to years ex situ24,25 and

up to several decades when naturally preserved (buried in marine

sediments)2628, but the eect of long-term dormancy on the growth performance of revived cells is unknown. Dormant stages have a highly reduced metabolism relative to actively growing vegetative cells (a 60-fold reduction was reported for the dinoagellate Scrippsiella trochoidea)29. Respiratory substrates (storage products) are used throughout the dormancy period, and resting stages also depend upon these endogenous energy reserves to germinate and become photosynthetically active29. Energy losses associated with long-term dormancy are, thus, expected to aect the growth performance of germinated cells.

We isolated phytoplankton resting stages from sediment cores retrieved from a low-oxygen sill fjord, and found these to be viable for up to 87 12 years. We cultured strains of the dinoagellate Pentapharsodinium dalei germinated from cysts isolated from discrete sediment core layers (Fig. 1). The growth performance of the tested strains was not aected by nearly a century of dormancy. As this species belongs to a lineage well-established by the Cretaceous, and presumably little aected by the K-Pg crisis30, we argue that resting stages could eectively have contributed to the survival of phytoplankton groups possessing this life-history trait. Our ndings indicate that resting stages in coastal sediments may play a signicant role for the recovery of primary production aer events of global photosynthesis disruption.

ResultsSediment core chronology. The agedepth model displays a linear relationship between core depth and age (Fig. 2a). Dry bulk density did not change markedly downcore (Fig. 2b), indicating relatively uniform sediment texture and compaction through time, which provides support for the reliability of the age model. The activity of unsupported 210Pb decreased exponentially with depth (Fig. 2c), and was detectable down to a sediment core depth of ~45 cm. The 137Cs prole showed a distinct peak at a core depth of ~10 cm that corresponds to the Chernobyl disaster of 1986 (Fig. 2d). The radionuclide proles indicate that the core represents an undisturbed archive of sediments deposited through time, as supported by the laminations visible in the X-ray image (Fig. 2e).

Resting stage viability and germination. We were able to revive resting cysts of P. dalei naturally preserved in the fjord sediments down to a core depth of 34 cm, corresponding to year 1,922 12 (Fig. 2a). Sediments deeper than 34 cm (1,922 12) were barren in viable resting cysts. Germination experiments targeted this species, but we encountered resting stages of 28 other dinoagellate cyst taxa with visible cell contents. Viable diatoms were found in fjord sediments down to core depths corresponding to ca. 40 years of age, the noncalcifying haptophyte Isochrysis sp. was found alive aer incubation

Figure 1 | Resting cyst and vegetative cell of the model species P. dalei. (a) Resting cyst isolated from coastal sediments. Scanning electron microscopy micrograph. Average resting cyst diameter = 25 m (n = 15).

(b) Vegetative motile stage after cyst germinations. Scanning electron microscopy. Cell length = 19 m (n = 820). Scale bars in (a) and (b) = 5 m.

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a

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Figure 2 | Sediment core chronology and growth of revived cells. (a) Agedepth model for the sediment record from Kolj Fjord, resulting from a combined constant rate of supply and constant initial concentration model. Error bars are modelled standard errors. (b) Dry bulk density prole, showing relatively constant values down-core, which indicate no major changes in sediment structure or compaction through time. (c) Prole of unsupported 210Pb,

revealing an exponential decrease with core depth. (d) Prole of 137Cs, with a distinct peak at ~10 cm, corresponding to the 1986 Chernobyl disaster. (e) X-ray radiograph of K1, revealing undisturbed laminated sediments. Arrows to the right of the X-ray image indicate the sediment layers from which

P. dalei cysts were germinated for the growth experiment. (f, g) Cumulative cell concentration as a function of incubation time for the 18 P. dalei strains germinated from sediment core layers dated to 2006 (f), 1,960 5 (g), and 1,922 12 (h). Errors in the dates correspond to the modelled standard errors given in (a). Different points in fh represent mean values of three replicates ( standard error).

of sediments dated to ca. 25 years, and lamentous cyanobacteria germinated from sediments ca. 10 years old.

From a total of ~450 isolated cysts, 193 Petapharsodinium dalei laboratory cultures (hereaer refereed to as strains) were established by single-cell isolation of vegetative stages germinated from individual cysts (Fig. 1). All germinations took place within the rst two weeks of incubation.

Growth experiment. Strains selected for the growth experiment were revived from three discrete sediment core layers: the surface layer, dated to 2006; an intermediate layer at 21 cm depth (1,960 5); and the layer at 34 cm depth (1,922 12) (arrows in Fig. 2e). Germination frequency varied between 28% for the recent layer, 61% for the intermediate and 5% for the oldest layer, and more than 50 cysts were isolated from each layer. We randomly picked 18 strains for the growth experiment, comprising 6 strains revived from each of the three discrete layers.

Vegetative cell concentrations increased exponentially as a function of time for all the tested strains (Fig. 2fh). Balanced growth was successfully achieved, as division rates between successive samplings were nearly constant throughout the exponential growth phase. Cell division rates diered signicantly among the P. dalei strains and varied between 0.17 0.07 and 0.60 0.07 day 1 (Linear

Model (LM), F = 4.48, d.f. = 17, 225, P < 2.210 16) (Table 1, Fig. 2fh). The strain with the highest cell division rate originated from the recent layer, and the slowest from the oldest layer (Table 1). Production rates, calculated as the product of division rate and cell size, also varied signicantly between the strains (LM, F = 264.5, d.f. = 17, 11,325, P < 2.210 16; Table 1). The lowest and highest production rates (521 31 and 2,279 36 m3 day 1, respectively) corresponded to strains germinated from the recent layer (Table 1). Strain represented an important source of variation for the growth variables studied. When taking into account the random variation due to strain, no signicant dierences were found in cell division rates, cell size, and production rates between the three age depths tested (Linear Mixed Eects Model (LMEM), cell division rate 2 = 0.45, d.f. = 2, P = 0.80; cell size 2 = 5.63, d.f. = 2, P = 0.06; production rate 2 = 1.94, d.f. = 2, P = 0.37; Fig. 3). Hence, growth performance was independent of cyst dormancy period.

The quartile variation coefficient (QVC), a measure of variability, varied between the three layers (see height of the boxes in Fig. 3ac). The recent layer was the most diverse in terms of cell division rates (QVC = 31%), and the deepest layer was the least diverse (QVC = 3%), while the intermediate layer had a coefficient of 23% (Fig. 3a). The quartile variation coefficient of production rate was 35% for the recent layer, 22% for the intermediate and 12% for the

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a

0.8

Table 1 | Summary statistics of growth performance for the Pentapharsodinium dalei strains tested.

Cyst dormancy period (years)

Strain Division rate (day 1)

Production rate (m3 day 1)

3 1 0.32 (0.06) 2,121 (228) 678 (33)

2 0.28 (0.06) 3,140 (229) 888 (33)3 0.20 (0.06) 2,813(229) 553 (33)4 0.22 (0.06) 2,390 (229) 521 (31)5 0.60 (0.07) 3,760 (229) 2,279 (36) 6 0.42 (0.06) 2,786 (229) 1,163 (33) 49 5 7 0.41 (0.06) 2,322 (199) 943 (26)

8 0.45 (0.06) 2,717 (210) 1,231 (28) 9 0.36 (0.06) 2,080 (228) 742 (29) 10 0.28 (0.06) 2,767 (231) 788 (33) 11 0.46 (0.06) 3,613 (229) 1,688 (33) 12 0.23 (0.06) 4,482 (229) 1,049 (31) 87 12 13 0.36 (0.04) 1,750 (162) 628 (20)

14 0.37 (0.05) 2,423 (209) 907 (26) 15 0.35 (0.06) 2,411 (229) 836 (28) 16 0.35 (0.06) 2,213 (229) 771 (33) 17 0.17 (0.07) 3,826 (229) 659 (36) 18 0.37 (0.07) 2,077 (229) 764 (36)

Mean cell division rate, cell size, and production rate values are given with standard errors (in brackets) as obtained from linear models. The growth experiment was conducted using three replicates per strain.

Cell size (m3)

0.6

Cell division rate (d1 )

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3 495 8712

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3,000

oldest layer (Fig. 3c). This indicates an important drop in P. dalei growth performance variability aer 49 5 years of dormancy.

Discussion

By germinating resting stages of the cyst-forming dinoagellate P. dalei deposited in fjord sediments, we were able to show that the dormancy period had no reducing eect on the growth performance of revived cells. The sediment record used here was retrieved from a sill fjord experiencing recurrent anoxic conditions and virtually no bioturbation31. A lack of sediment disturbance was evident from the X-ray image, showing discrete laminations, and was also supported by the activity of 210Pb and 137Cs, providing a robust age control. In addition, any vertical contamination by smearing between layers was avoided by discarding the outer portion of each sediment layer while slicing the cores. We can therefore assume that the cysts isolated from the sediments represent, indeed, resting stages deposited in bottom sediments of the fjord over a time span from 2006 (the year of core collection) to 1,922 12. As P. dalei cysts were germinated in early 2009, we have tested growth performance aer up to 87 12 years of cyst dormancy.

The variation in growth parameters of P. dalei was comparable between the recent group and the group of strains revived from sediments dated to 1,960 5, but notably lower for the oldest layer (1,922 12). This drop in variability aer ca. 50 years of dormancy suggests a link between the ability to optimally utilize respiratory substrates, and the ability to resume vegetative growth at high rates. This is reected by the low germination success of the oldest layer (5%). Although a higher percentage of cysts with cell contents were no longer viable in the deepest sediment layer, those able to germinate had the same potential to actively grow in the water-column as cells germinated from recently formed cysts. For P. dalei cysts to have remained viable for nearly a century, an extremely low respiration rate and/or extensive reserves would have been needed. Carbohydrate reserves are more suitable as storage than lipids in anoxic conditions, but previous studies with dinoagellate cysts have shown that this storage product would quickly be depleted at a constant rate of degradation29. Therefore, either respiration rates decrease with cyst age or other respiratory substrates (lipids or proteins) are recruited aer depletion of carbohydrates29.

1,000

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Production rate (m3 d1 )

1,500

500

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Cyst dormancy period (years)

Figure 3 | Growth performance according to cyst dormancy period. (a) Box plots of vegetative cell division rates (day 1). (b) Cell sizes (m3)

calculated assuming the shape of an elliptical sphere, based on length and width measurements of 60 exponentially growing cells per strain. (c) Production rates (m3 day 1) calculated as the product of division rate and cell size. Lines in bold represent the median, edges of the boxes are quartiles, and whiskers indicate the maximum and minimum values. The height of the boxes indicates interquartile range and provides a measure of variability among the strains. According to maximum likelihood ratio tests of the linear mixed effects model, the mean levels were similar for the three cyst dormancy periods tested.

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The cysts retrieved from Kolj Fjord were naturally preserved in a conned environment experiencing recurrent bottom anoxia31.

Anaerobiosis has been shown to be a crucial factor for resting cyst preservation, as it inhibits early germination without aecting cyst survival32. At the time of the asteroid impact, eustatic sea level was 100200 m higher than today33 and shallow marine environments experiencing oxygen deciency were the rule34. It is, therefore, reasonable to assume that at the end-Cretaceous low-oxygen shallow environments would have provided suitable conditions for resting stage survival.

The cysts isolated from Kolj Fjord sediments were able to germinate quickly aer being placed at 15 C in light and nutrient-replete conditions, regardless of dormancy period. Many studies have shown resting-stage germination to be triggered by an increase in temperature, oxygen and light conditions23. In the natural environment, resting stages, buried deep in the sediment, experience anoxia, darkness and lower temperatures and are unlikely to germinate unless they are brought back to the surface. This might be achieved by the movement of benthic fauna (bioturbation) or by physical processes.

Impact-driven tsunamis and earthquakes are imprinted in K-Pg sediment sequences worldwide and would have had sufficient magnitude to re-suspend resting stages buried deep in shelf sediments across the globe5. These immediate physical processes could have transported resting stages closer to the surface, which would then contribute to the seed pool aer light was restored. Normal wave and current-induced resuspension of ne-grained deposits in suband inter-tidal environments would then provide sufficient changes in the micro-scale environment to trigger resting-stage germination35. Modelling studies indicate that cooling of the surface ocean would result in strong upwelling for over a decade, also leading to sediment re-suspension8.

While screening the Kolj fjord sediments for Pentapharsodinum dalei cysts, we found viable resting stages of diatoms in sediments dated to ca. 40 years. This was not surprising, as several diatom species had previously been reported to germinate aer decades of dormancy27, and Skeletonema marinoi over a period of more than 100 years36. Our observations on resting stages of other primary producers such as cyanobacteria, and non-calcifying haptophytes are relevant to discuss dierential extinction, as these groups are generally not fossilizable.

Our observations indicate that coastal cyanobacteria are also capable of forming resting stages that are viable for at least a decade. Many cyanobacteria produce akinetes, which are spore-like reproductive structures containing reserve material. The viability of akinetes is enhanced by low temperatures, darkness and anaerobic conditions37, and these can survive in lake sediments for up to 64 years38. A high-resolution geochemical and biomarker record from the Fiskeler K-Pg boundary layer clearly shows that whereas there was a marked decrease in chlorophyll-c-containing eukaryotic phytoplankton, cyanobacteria crossed the boundary unscathed16.

We observed that non-calcifying haptophytes can produce resting stages able to survive in coastal sediments for at least 25 years. Isochrysis, Pavlova and Prymnesium species had previously been shown to survive in darkness, but not for periods exceeding 1 year39.

Molecular-clock studies indicate that numerous clades of non-calcifying species with coastal affinities have survived the K-Pg extinction40. In contrast, calcifying (coccolitophorid) and non-calcifying oceanic clades were severely aected10,40. The fact that haptophyte

resting stages have only been found in coastal species might be related to the major divergence occurring at the K-Pg between coastal and oceanic forms.

Germination aer long-term dormancy appears to be common for resting stages of diatoms, non-calcifying haptophytes and cyanobacteria, indicating that the ability to maintain high-growth performance, as shown here for the dinoagellate P. dalei, is also

taxonomically widespread. We, thus, propose that phytoplankton resting stages accumulated in coastal sediments have aided survival across the K-Pg and played a crucial role for the resurgence of primary production and ecosystem recovery. Phytoplankton resting stage banks in coastal sediments should be incorporated in scenarios aimed at reconstructing or predicting recovery from catastrophes. It should be noted, however, that photosynthesis and biodiversity recovery do not occur at the same rates. Considering that only a fraction of modern phytoplankton groups are able to form resting stages (for example, less than 20% of modern dinoagellate species are known to produce cysts), removing non-cyst formers at the K-Pg likely resulted in a larger diversity loss than that inferred from the fossil record (because non-cyst formers do not fossilize). Although the recovery of primary productivity aer the K-Pg was relatively rapid, the recuperation in biodiversity is expected to have been delayed, due to intrinsic limits to how quickly global biodiversity can recover aer extinction events41.

The benthic realm has recently been shown to have served as an important refuge not only for primary producers but also foraminifera42. Benthicpelagic coupling probably contributed to the resilience and diversication of marine life across other biological crisis in Earth history.

Methods

Sediment-core sampling and dating. Five sediment cores were retrieved at 45 m depth from Kolj Fjord, north of Gothenburg, Sweden (5813N, 1134E) in April 2006. This is a sill fjord with limited oxygen supply, minimum tidal activity and virtually no benthic macrofauna (resulting in nearly no bioturbation)31. The cores were taken with a modied micro-Kullenberg piston corer. All cores were X-rayed while intact and correlated by easily distinguishable features (lamination patterns). The cores were sliced at 1 cm intervals by extrusion from the bottomof the tube. Apart from the rst sample, which had high water content, the outer 510 mm of each slice was scraped o to eliminate potential contamination by smearing between the layers while pushing the core upwards, and the equipment used was thoroughly rinsed between samples. The inner portion of each slice (aer discarding the outer ring) was collected in individually labelled plastic bags and stored in the dark at 4 C. Dating was based on the activity of 210Pb, 226Ra and

137Cs via gamma-spectrometry measured on K4 on a Canberra low-background Germanium well detector. The activity of 210Pb was measured via its gamma-peak at 46.5 keV, 226Ra via the granddaughter 214Pb (peaks at 295 and 352 keV) and 137Cs via its peak at 661 keV. A combined constant rate of supply and constant initial concentration model43 was applied, where the inventory below 45 cm (below the

210Pb detection limit) was calculated on the basis of a regression.

Resting stage isolation and germination. To separate aggregated particles, sediment samples (ca. 5 cm3 of wet sediment) were placed in an ultrasonic bathfor 2 min, and wet-sieved with ltered seawater through a 100 m onto a 25 m metallic-mesh calibrated sieve. The resting stage fraction was separated from the sediments by density centrifugation (sieved aliquot added to a solution of sodium metatungstate with density 1.3 g ml 1 and centrifuged at 1,500 g for 10 min). We registered the taxonomic affinity of all resting stages encountered with cell contents (that is, potentially viable). Single resting cysts of the target species, P. dalei, were transferred with a micropipette into 96-microwell plates containing ~0.25 ml of TL medium44. The plates were placed in a temperature controlled room at 15 C, under an irradiance of ca. 80 mol photons m 2 s 1, and a light:dark cycle of 16 h:8 h. The microwells were examined for motile stages on a regular daily-to-weekly basis. Successful germinations were recorded and individual P. dalei motile cells were transferred to culture asks and kept in TL medium under the same conditions as those for germination. A total of 193 P. dalei strains were established.

Experimental conditions. To test the eect of dormancy time on growth performance, we randomly picked 18 P. dalei strains revived from three discrete sediment layers (K3) dated to recent, 1,960 5 and 1,922 12. The selected strains were subjected to similar laboratory conditions until the start of the experiment. Vegetative cells were acclimated for 12 days at salinity 30 to an irradiance of150 mol photons m 2 s 1, and a light:dark cycle of 16 h: 8 h. Aer the acclimation period, 300 vegetative cells ml 1 were transferred to fresh TL medium (pH 7.5) in closed triplicate sterile polycarbonate asks (70 ml) lled to capacity. The54 replicates were le to grow for 4 additional days before the rst sampling for cell-concentration determination. Volumes of 5 ml were xed with Lugols acid and cell concentrations determined by counting at least 300 cells in Sedgewick Raer chambers. To avoid growth limitation by increased pH due to high photosynthetic production in the closed containers45, we monitored pH at each sampling and assured it did not increase above the levels that aected the growth rate ( < 8.7).

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Fresh medium replaced the sample volume (5 ml). To eliminate the potential eects of slight spatial dierences in irradiance, the positions of the asks were changed randomly during the course of the experiment.

Cultures used in the study are kept at the Scandinavian Culture Collection for Algae and Protozoa (http://www.sccap.dk).

Calculations and statistics. We determined cell division rates (day 1), cell size (m3) and production rates (m3 day 1) for the 18 tested strains. Cell division rates were calculated for each of the 54 asks (3 replicates per strain) as the logarithm of the change in cell concentration divided by the time elapsed for each successive sampling interval (13 days intervals for up to 14 days of exponential growth). Cumulative cell concentrations were calculated, to account for the dilution rate (as 5 ml of sample was replaced by fresh medium at each sampling). The end of the exponential growth phase was dened as the point where the mean division rates of successively added sampling points changed in comparison to the sampling time interval day 48 (LM, P > 0.05). Cell size was calculated assuming the shape of an elliptical sphere, based on length and width measurements of 60 exponentially growing cells per strain. Production rates were estimated as the product of cell size and division rate.

To test the growth performance of revived P. dalei strains, we used a linear model of the response variables (cell division rate, cell size and production rate) as a function of strain. Accounting for random variation between the strains, we used an LMEM to study the eect of cyst dormancy period on growth performance. Initially, we modelled log-transformed cell concentrations using a random-slope LMEM, including all possible sources of random variation due to the variables: ask (a 54-level factor), time of sampling and strain (an 18-level factor). Random variation due to strain was ~7 times higher than random variation between sampling times and no variation was explained by the random ask eect, indicating low replication error and good reproducibility in our experiment. Therefore, we chose a simplied LMEM including only the response variables (division rate, cell size and production rates) as a function of dormancy period (a 3-level factor) with strain (an 18-level factor) as a random eect. P-values were estimated based on maximum likelihood by comparing the null model including dormancy period (0-model) with the reduced nested model without the xed eect using 2-tests. The mean values (coefficients) for the three groups of strains (representing three distinct cyst dormancy periods) were calculated based on restricted maximum likelihoods. All statistical analyses were performed using R version 2.10.1 (ref. 46). For the LMEM we used the R package lme4 (ref. 46).

The QVC was calculated as a means to assess the growth performance variability for each of the three cyst dormancy periods, according to the formula: QVC = 100((Q3 Q1)/(Q3 + Q1)), where Q1 and Q3 are the rst and third quartile, respectively.

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46. R Development Core Team. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2011) http://www.R-project.org.

Acknowledgments

This study was funded by the Danish Research Council through the project: Changes in Community Structure and Microevolution in Marine Protists. S.R. and T.B. hold doctoral grants from the Portuguese Foundation for Science and Technology (SFRH/ BD/30847/2006) and University of Copenhagen, respectively. We thank T. Gilbert, G. Simpson, T. Fenchel, P. Cermeo and P.J. Hansen for comments and suggestions, P. Frandsen for laboratory assistance, and A.-M. Nyby for the cyst SEM micrograph. The sediment core was kindly X-rayed at Hvidovre Hospital by J. Sgaard, D. Mller and A. Vestergaard.

Author contributions

M.E. developed the research project leading to this study. S.R., N.L., M.E., and T.J.A. collected the sediment cores. T.J.A. dated the samples and constructed the agedepth

model. S.R., N.L. and M.E. performed resting stage isolations and germinations. S.R., T.B., M.E. and N.L. designed the growth experiment. S.R. and T.B. conducted the growth experiment and analysed the data. F.A. contributed with ideas to the study framework. S.R. and T.B. wrote the manuscript. All the authors discussed the results and commented on the manuscript.

Additional information

Competing nancial interests: The authors declare no competing nancial interests.

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How to cite this article: Ribeiro, S. et al. Phytoplankton growth aer a century of dormancy illuminates past resilience to catastrophic darkness. Nat. Commun. 2:311 doi: 10.1038/ncomms1314 (2011).

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