Short-Term Toxicity Effects of Prymnesium Parvum on Zooplankton Community Composition; Aquatic Sciences; Witt Et Al.; University of Oklahoma; [email protected]

Short-Term Toxicity Effects of Prymnesium Parvum on Zooplankton Community Composition; Aquatic Sciences; Witt Et Al.; University of Oklahoma; Dhambright@Ou.Edu

Aquatic Sciences (2019) 81:55 https://doi.org/10.1007/s00027-019-0651-2 Aquatic Sciences

RESEARCH ARTICLE

Short‑term toxicity efects of Prymnesium parvum on zooplankton community composition

Brenda A. Witt1,2 · Jessica E. Beyer1 · Thayer C. Hallidayschult1 · K. David Hambright1

Received: 13 November 2018 / Accepted: 29 June 2019 © Springer Nature Switzerland AG 2019

Abstract Harmful algal blooms (HABs) can disrupt aquatic communities through a variety of mechanisms, especially through toxin production. Herbivorous and omnivorous zooplankton may be particularly susceptible to HAB toxins, due to their close trophic relationship to algae as grazers. In this study, the acute toxigenic efects of the haptophyte Prymnesium parvum on a zooplankton community were investigated under laboratory conditions. Total zooplankton abundances decreased during 48-h exposure, although species responses to P. parvum densities varied. Changes in community composition were driven by declines in Daphnia mendotae and Keratella spp. abundances, which resulted in an average shift in copepod abundance from 47.1 to 72.4%, and rotifer abundance from 35.0 to 7.1%. Total cladocerans were relatively unchanged in relative abundance (11.1–10.4%), though the dominant cladoceran shifted from Daphnia mendotae (61.3% of cladocerans) to Bosmina longirostris (81.5% of cladocerans). Daphnia mendotae and Keratella spp. are known to be non-selective or generalist feeders and were likely harmed through a combination of ingestion of and contact by P. parvum. Proportional increases in copepod and Bosmina abundances in the presence of P. parvum likely refect selective or discriminate feeding abilities in these taxa. This study corroborates previous feld studies showing that P. parvum can negatively afect zooplankton, refecting species-specifc diferences in zooplankton-P. parvum interactions. Such changes can alter zooplankton community composition, leading to substantial food-web consequences and potential long-term ecosystem-level impacts in lakes that experience blooms.

Keywords Acute toxicity · Harmful algae · Zooplankton community · Zooplankton feeding behavior

Introduction afected community (Lampert 1987; DeMott and Mueller- Navarra 1997; Granéli and Johansson 2003a, b). Addition- Harmful algal blooms (HABs) dominate pelagic phytoplank- ally, HABs directly and deleteriously afect a wide range ton worldwide (Huisman et al. 2005; Granéli and Turner of aquatic organisms, including fsh, mussels, zooplankton, 2006; Hudnell 2008; Shumway et al. 2018). A variety of and other microbial species (James and De La Cruz 1989; mechanisms have been hypothesized to explain HAB domi- Guo et al. 1996; Landsberg 2002; Shumway et al. 2003; nance: toxicity, low nutritional value for grazers, increased Glibert et al. 2005), thereby changing the entire structure of competitive abilities following environmental change, and an aquatic food web (Sunda et al. 2006). subsequent ability to produce strong bottom-up efects in the The toxigenic, haptophyte alga Prymnesium parvum Carter 1937 forms HABs in fresh waters, presumably Electronic supplementary material The online version of this having acclimated from its marine origins (Edvardsen article (https://doi.org/10.1007/s00027-019-0651-2) contains and Paasche 1998). Since the frst known appearance of supplementary material, which is available to authorized users. Prymnesium in the U.S. in the Pecos River, TX in 1985, it has spread to 23 other states (Roelke et al. 2016). A * K. David Hambright [email protected] suite of cytolytic, hemolytic, and ichthyotoxic toxins are produced by Prymnesium, evidenced by massive fsh kills 1 Plankton Ecology and Limnology Laboratory, Department observed during blooms (Johansson and Granéli 1999; of Biology, University of Oklahoma, Norman, OK 73019, Legrand et al. 2001; Granéli and Johansson 2003b; Baker USA et al. 2007; Roelke et al. 2007). While the mechanism of 2 Present Address: Redlands Community College, El Reno, toxin delivery is hypothesized to be the release of toxins OK 73036, USA

Vol.:(0123456789)1 3 55 Page 2 of 8 B. A. Witt et al. into the surrounding water (Tillmann 1998; Skovgaard Methods and Hansen 2003; Tillmann 2003; Blossom et al. 2014), evolutionary considerations and controlled experimenta- Study site and zooplankton collection tion reveal that cell-to-cell contact may be necessary to produce toxicity to fsh (Lewis 1986; Jonsson et al. 2009; Lake Texoma has a history of Prymnesium blooms that Remmel and Hambright 2012). typically have been isolated to the western regions of the At the front line of interaction with harmful algae (HA), lake where salinity is higher due to infow from the Red such as Prymnesium, are zooplankton—the nexus of pri- River. Intermittent Prymnesium blooms have occurred mary production and higher-level consumers, such as fsh. in Lake Texoma since 2001. The environmental condi- Much is known about the physiological responses of indi- tions conducive to blooms of Prymnesium have been well vidual zooplankton exposed to HA (Colin and Dam 2002; studied. While lab studies and lake surveys have revealed Remmel et al. 2011). Feeding experiments have confrmed a wide range of salinity tolerances for P. parvum (Roe- that non-selective suspension feeders like Daphnia and lke et al. 2007; Hambright et al. 2014; Rashel and Patiño Brachionus are highly vulnerable to P. parvum (Barreiro 2017), a salinity of 1.67 psu and cooler water temperatures et al. 2005; Remmel et al. 2011), and a long-term monitor- are generally required for bloom formation in Lake Tex- ing study of a recurrent bloom of P. parvum led Hambright oma (Hambright et al. 2015), the salinity tolerance perhaps et al. (2010) to hypothesize that species’ feeding guilds refecting Prymnesium’s origins in coastal marine systems. may explain observed species-specific responses. For Blooms in Texoma have tended to be restricted to coves example, copepods and some cladocerans (e.g., Bosmina) where salinity elevations are common during relatively dry are more selective than other zooplankters (DeMott 1982, winters (Hambright et al. 2010, 2015). Large and devastat- 1995; Hambright et al. 2007b), and this feeding behav- ing fsh kills have resulted from these blooms, though fsh ior may mediate responses to HA, as has been shown for community recovery has been swift, likely due to the small cyanobacterial blooms (Sommer et al. 2001; Hambright scale of blooms and subsequent fsh immigration from et al. 2007b). Other studies, including short-term mes- unafected areas of the lake (Zamor et al. 2014). The fate ocosm manipulations, have also indicated differential of afected zooplankton communities is less well studied. vulnerabilities of herbivorous zooplankton to P. parvum Long-term monitoring of Prymnesium cell densities (Errera et al. 2008; Schwierzke et al. 2010). Addition- across the lake, both littoral and pelagic, has provided a ally, cladoceran and rotifer abundances declined before clear picture of the seasonal dynamics of blooms (Ham- copepod abundances during a P. parvum bloom in Lake bright et al. 2010) and ofered the opportunity to study Koronia, further supporting diferential responses among zooplankton communities based on prior exposure. zooplankton taxa (Michaloudi et al. 2009). But despite Because adaptation or acclimation may lead to toxin all the knowledge of the efects of HA on individual zoo- resistance due to long-term exposure, and could confound plankters and zooplankton taxa, little is known about the interpretation of the immediate efects on a zooplankton immediate efects of HABs on zooplankton communities. community during a bloom, this study used a community The aim of this study was to separate the direct acute that has not experienced high bloom densities, yet has the toxicity impacts of Prymnesium on zooplankton commu- potential to encounter a Prymnesium bloom in the future nity structure in Lake Texoma (OK-TX, USA) from the (Hambright et al. 2014). Zooplankton were collected longer-term indirect impacts, such as zooplankton com- from the Soldier Creek Inlet (33.927829, − 96.682097, munity interactions and shifts in algal food quality. An see Fig. 1 in Hambright et al. 2010), an area of the lake environmentally controlled, two-day microcosm study for which Prymnesium cell densities to date have never with a semi-natural zooplankton assemblage and varying exceeded 3000 cells mL −1, thus reducing the chances of Prymnesium cell densities was used to test for diferen- exposure-mitigated toxin resistance. tial survivorship within a zooplankton community. Rem- Zooplankton were collected on 9 May 2014 using verti- mel et al. (2011) hypothesized that zooplankton feeding cal tows of 63-µm- and 350-μm-mesh Wisconsin plankton behavior might provide an explanation of the community nets. In addition, water was collected at ~ 1-m depth using shifts observed in southern US reservoir during P. parvum a Van Dorn sampler and fltered through 20-μm-mesh blooms (Hambright et al. 2010), as feeding mechanisms Nitex netting to remove all zooplankton and large colonial and selectivity are likely to mediate exposure to toxins. algae, while still providing a suitable algal community to The prediction was that, when exposed to increasing con- serve as food for the zooplankton. This procedure provided centrations of P. parvum, non-selective grazers would drop a semi-natural environment inclusive of the grazeable out of the community frst and selective feeders would assemblage of phytoplankton [but not containing Prym- dominate. nesium; checked microscopically with a detection limit of

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–1 0.2 L of zooplankton concentrate. Additions of Prymnesium 300 .L culture to bottles led to average salinity increases of 0.35, ind ( while nutrient additions were negligible. Nevertheless, to control for any potential efects of salinity or nutrient additions, six replicates of both nutrient and salinity controls 200 were established using 0 psu salinity, 16:1 N:P COMBO abundance and 6 psu salinity, no-nutrient COMBO, respectively, in equivalent amounts to those in Prymnesium treatments. As a proxy for initial conditions, an additional six repli- 100 cate bottles were flled with 2.3 L of lake water and 0.20 L of the zooplankton concentrate, harvested immediately by

Total zooplankton fltration (20-µm mesh), and preserved in 70% ethanol-5% Initial Nutrients Salinity 50K 100K 200K glycerol. Experimental bottles were placed on a laboratory Controls Prymnesium bottle roller at 0.5 rotations min−1, with the roller direction –1 (cells mL ) alternated every minute, to create gentle mixing and prevent settling of particles. Incubation was maintained on a 12-h −1 Fig. 1 Box plot showing abundance of zooplankton (ind. L ) for light:12-h dark cycle at a temperature of ~ 23 °C, represent- each treatment. Abundance was signifcantly diferent between con- ing lake conditions at the time of zooplankton collection. trol groups and treatment groups as determined by ANOVA and Tukey post hoc tests (see Table 1). Box plots for each treatment indi- After 48 h, each bottle was fltered through 20-μm mesh cate the median (center horizontal line), frst and third quartiles (top to collect all zooplankton, which were preserved in a 70% and bottom of box), range (end of whiskers), and outliers (circles) ethanol-5% glycerol solution. Identifcation (to lowest taxonomic level possible) and enumeration of adult crustacean zooplankton was conducted using a dissecting microscope 300 cells mL −1 (Zamor et al. 2012)], as well as the ability (50–100× magnifcation). Extremely abundant copepod nau- to control the abundance of zooplankton in the experiment. plii, zebra mussel veligers, and rotifers were subsampled At the time of zooplankton and water collection, salinity (20% of the total sample) and identifed and counted on an was 0.91 psu and temperature was 23.8 °C. inverted microscope at 200× magnifcation.

Zooplankton community composition Statistical analysis

To evaluate the efects of Prymnesium on the zooplankton Diferences in total zooplankton abundance (individuals L−1) assemblage, experiments were conducted in 2.5-L Nalgene between treatments were tested using one-way ANOVA and bottles with the addition of lab-cultured Prymnesium. Batch Tukey post hoc tests following confrmation of normal dis- cultures of Prymnesium (University of Texas Culture Collec- tributions (Shapiro–Wilk test) and homogeneity of variances tion of Algae, UTEX; culture LB 2797; originally isolated (Levene’s test) (R version 3.5.1, R Core Development Team from the Colorado River, TX) were maintained in COMBO 2018). Non-Metric Multidimensional Scaling (NMDS) of medium (Kilham et al. 1998) with a molar N:P of 16:1 Bray–Curtis dissimilarity indices based on taxon abundances (800 μmol L−1 nitrate N: 50 μmol L−1 phosphate P) and was used to visualize the community composition of each salinity of 6 psu (i.e., 6 g L −1 Instant Ocean ®) (Hambright treatment (vegan, 2.5-2, Oksanen et al. 2018). Species that et al. 2014). Approximately 1 month prior to experimen- did not occur in at least 10% of samples were removed to tation, fresh cultures were inoculated and maintained on avoid skewing results by rare taxa. PERMANOVA (999 per- a 12-h light:12-h dark cycle at 20 °C, with constant aera- mutations) was then used to test for signifcant diferences in tion by bubbling with fltered ambient air. Because Prym- community composition between treatments (vegan, 2.5-2) nesium blooms in Lake Texoma associated with fsh kills (Anderson 2001). Post hoc comparisons between treatment have ranged between 25,000 and 200,000 cells mL−1 of pairs were carried out using pairwise PERMANOVAs with Prymnesium, zooplankton community responses to a gra- Holm-Bonferroni adjusted probability values. To determine dient of cell densities: 50,000, 100,000, and 200,000 cells which species were driving any observed community difer- mL−1 were examined. Cultures of Prymnesium were diluted ences, an indicator species analysis (ISA) (Dufrêne and Leg- with 6 psu salinity, 16:1 N:P COMBO to acquire the desired endre 1997) was conducted (labdsv, 1.8-0, Roberts 2016). cell densities for each treatment [verifed using microscopic Lastly, linear regression was used to test if the cell density of counts (Zamor et al. 2012)]. Six replicate bottles were cre- Prymnesium had an efect on zooplankton taxonomic abun- ated for each treatment, containing 2.13 L of fltered lake dances. A Bonferroni correction for multiple comparisons water, 0.17 L of the respective Prymnesium dilution, and was used based on an initial α = 0.05 with 10 comparisons

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Table 1 Tukey post hoc (I) treatment (J) treatment Mean diference 95% confdence interval p results for ANOVA test of (I–J) signifcance of zooplankton Lower bound Upper bound densities between all treatment groups (α = 0.05). Where Salinity Nutrient − 12.6 − 57.5 32.4 0.92 ‘salinity’ represents the salinity Salinity 50 83.1 40.2 126 < 0.001* adjusted control treatment, and Salinity 100 100 57.3 143 < 0.001* ‘nutrient’ represents the nutrient adjusted control treatment. Salinity 200 127 83.8 170 < 0.001* ‘50’, ‘100’, and ‘200’ represent 50 Nutrient − 95.6 − 141 − 50.6 < 0.001* Prymnesium treatments of 50 100 17.1 − 25.8 60.0 0.77 50,000, 100,000, and 200,000 50 200 43.6 0.71 86.5 0.04* cells/mL, respectively 100 Nutrient − 113 − 158 − 67.7 < 0.001* 200 Nutrient − 139 − 184 − 94.2 < 0.001* 200 100 − 26.5 − 69.4 16.4 0.38

Results denoted by asterisk (*) represent treatments with signifcant diferences in zooplankton abundances

(i.e., α = 0.005). All R scripts used in this study can be found Prymnesium Controls in the on-line supplementary material. treatments 0.2 50K Salinity

Results 2 0.0 Nutrients Identifcation and enumeration of the zooplankton revealed NMDS a community composed of three cladocerans (Daphnia men- –0.2 100K dotae Birge 1918, Bosmina longirostris Müller 1776, and 200K Chydorus sphaericus Müller 1785), two calanoid copepods (Eurytemora afnis Poppe 1880 and Diaptomus sp.), uniden- –0.4 tifed cyclopoid copepods and copepod nauplii, zebra mus- –0.2 0.000.2 .4 sel (Dreissena polymorpha, Pallas 1771) veligers, Keratella NMDS 1 spp. (K. cochlearis Gosse 1851, K. quadrata Müller 1786, K. tecta Gosse 1851), Lecane closterocerca Schmarda 1859, Fig. 2 Non-metric multidimensional scaling (NMDS) analysis Polyarthra sp., Synchaeta sp., and Brachionus angularis depicting diferences in overall zooplankton community composi- Gosse 1851 (Fig. S1). Overall abundances of zooplankton tion between treatments (stress value = 0.19). Polygons represent were signifcantly diferent between the Prymnesium treat- treatment groups with symbols representing individual replicates (blue stars—nutrient controls; blue circles—Salinity controls; yel- ments and the controls after 48 h of exposure to P. parvum low circles—50,000 P. parvum cells mL −1; yellow squares—100,000 −1 −1 (F4,24 = 34.35; p < 0.0001, Table 1). Mean abundances of cells mL ; yellow stars—200,000 cells mL ). Distinct diferences zooplankton ranged between 217 and 229 individuals L−1 between the controls and the treatment are evident and confrmed to in the control treatments compared to 90–134 individuals be signifcant using PERMANOVA (Table 2) (color fgure online) L−1 in the Prymnesium treatments, a reduction in total zooplankton abundances by at least 62% (Fig. 1). Note that one nutrient control bottle was identifed as an outlier using Leys et al.’s (2013) median absolute deviation with a threshold of −1 3, with a total abundance of only 36 individuals L , and was Table 2 PERMANOVA post hoc adjusted p-values comparing com- therefore removed prior to statistical analysis. munity composition diferences between treatments based on Bray– Community composition was significantly different Curtis dissimilarity indices (9999 permutations) between the Prymnesium treatments and controls as visual- Nutrient Salinity 50 100 200 ized through NMDS (stress value = 0.19, Fig. 2) and veri- Nutrient – 1.000 0.010 0.018 0.014 fed by PERMANOVA (p = 0.001; F4,24 = 8.01). There were Salinity 1.000 – 0.024 0.024 0.024 no signifcant diferences in composition between the two 50 0.010 0.024 – 1.00 0.024 controls (Table 2). The composition of the highest Prym- 100 0.018 0.024 1.00 – 0.303 nesium treatment difered signifcantly from all other treat- 200 0.024 0.024 0.024 0.303 – ments except the intermediate Prymnesium treatment. The intermediate Prymnesium treatment resulted in a diferent P-values were adjusted using Holm–Bonferroni correction (α = 0.05)

1 3 Page 5 of 8 55 Short‑term toxicity efects of Prymnesium parvum on zooplankton community composition community composition than the controls, but no diference taxon-specifc as predicted based on diferences in feed- relative to either other Prymnesium treatment. The commu- ing behavior, with declines in two indicator taxa, Daph- nity composition of the lowest Prymnesium treatment was nia mendotae and Keratella spp., explaining most of the distinguishable from that of the two control treatments as observed community changes between control and P. par- well as the highest Prymnesium treatment. The composi- vum treatments. tional diferences seen between the controls and Prymnesium Our results lead us to hypothesize that differential treatments were the result of reductions in the abundances responses to Prymnesium among zooplankton were medi- of two specifc taxa. The indicator species analysis (Dufrêne ated by feeding mechanism and apparatus. Generally, Daph- and Legendre 1997) revealed that D. mendotae and Kera- nia mendotae is considered to be a non-selective flter feeder, tella spp. drove the change (Table 3), as both taxa declined and Keratella spp. are generally considered less selective precipitously in Prymnesium treatments (Fig. S1). Regres- than most other rotifers, while the majority of other taxa sion analysis showed that Prymnesium cell density had a (e.g., copepods, Bosmina) are generally considered more signifcant efect on four taxa (Fig. S1). With increasing selective in collecting planktonic food (Boyd 1976; Stark- Prymnesium cell densities, the abundances of D. mendotae, weather 1980; DeMott 1982, 1990, 1995). These diferences Keratella spp., Diaptomus, and zebra mussel veligers sig- in degree of feeding selectivity align with our observed nifcantly declined. efect size of Prymnesium on zooplankton taxa over 48 h. Previous research on efects of Prymnesium also supports this hypothesis. Copepod exposure to Prymnesium patel- Discussion liferum has been shown to result in depressed feeding and minimal mortality, although reductions in fecundity were As the frequency and duration of HABs in freshwater observed (Nejstgaard and Solberg 1996). Additionally, ecosystems increases, so does the possibility of extensive Sopanen et al. (2008) investigated the efect of Prymne- changes to aquatic communities, including zooplankton sium on the copepod E. afnis and, while it was shown that that serve as an important link between algal producers and copepods can succumb to the presence of Prymnesium at planktivorous fsh (Hallegraef 1993). To date, studies have high densities, ingestion was reduced and survival was high, focused on long-term monitoring and long-term mesocosm particularly in mixed feeding suspensions where high-qual- experiments to describe and quantify the impact of P. par- ity food was available. Additional high-quality food may vum on zooplankton communities in afected systems. This explain the higher copepod survival rate seen in our study, study set out to explore direct acute toxicity efects as a as alternative food sources were available from the ambi- means of eliminating, or at least discerning, the confound- ent plankton community included in the microcosms. As ing efects of indirect community interactions within the shown in the regression analysis (Fig. S1), some copepods zooplankton assemblage that may develop over time. Based (i.e., Diaptomus sp.) were signifcantly afected by Prym- on previous monitoring results in a reservoir experiencing nesium cell density, however these efects were not strong recurrent blooms, it was hypothesized that the diferential enough to defne any as indicator species infuencing the efects observed across bloom and non-bloom sites might observed assemblage changes. While our results are most refect diferences in zooplankton feeding behavior and a consistent with the hypothesis of diferential exposure due resultant diference in susceptibilities to P. parvum toxins. to diferences in feeding mechanisms, further experiments Indeed, across the taxonomic spectrum, overall abundances measuring feeding selectivity in the presence of Prymnesium of zooplankton in this experiment were reduced by P. par- are needed to fully elucidate the mechanisms underlying the vum by as much as 60% in the highest P. parvum treatment, observed diferential survival. although reductions were high across all Prymnesium den- Studies on the toxic cyanobacteria Microcystis have sities. Nevertheless, acute toxicity responses were highly shown similar susceptibilities by Daphnia sp. (Hansson et al. 2007; Bednarska et al. 2014), corroborating the idea that feeding strategy plays an important role in governing Table 3 Results of indicator species analysis (Dufrêne and Legendre 1997), which identifes the species driving assemblage changes using changes to zooplankton community structure during a toxic permutation tests algal bloom (Hambright et al. 2007b; Sikora and Dawido- wicz 2014). However, other studies have demonstrated Indicator value p mechanisms that lead to population resiliency, including Keratella spp. 0.460 0.006 both selective feeding and toxin resistance for Daphnia spp. D. mendotae 0.432 0.005 fed Microcystis, further complicating the story as to how and why species responses to HABs are so varied (DeMott Abundances of each taxon are reported in Supplemental Fig. S1. The two species shown represent those responsible for driving the 1990). Additionally, it has been shown that zooplankton observed changes in the zooplankton assemblage composition populations with prior exposure to HABs often have higher

1 3 55 Page 6 of 8 B. A. Witt et al. survival than naive populations with no prior exposure (Dam to the development of this project, Caryn Vaughn, Lara Souza, and 2013). Gustafsson et al. (2005) showed Daphnia individu- Dani Glidewell for comments on earlier versions of this manuscript, and Gary Wellborn, Richard Page, and Donna Cobb for assisting with als can develop tolerance to toxins after exposure, and this coordination and facility usage at the University of Oklahoma Bio- resistance can also be passed on to ofspring. By contrast, logical Station. This study was funded, in part, by Grants from the Beyer and Hambright (2017) found that maternal exposure Oklahoma Department of Wildlife Conservation through the Sport Fish to toxigenic Microcystis in rotifers resulted in reduced ftness Restoration Program (Grant F-61-R), the Oklahoma Water Resources Research Institute, and the Ofce of the University of Oklahoma Vice of ofspring as compared to maternal individuals provided President for research to KDH. BAW was funded, in part, by the Uni- high quality algal food sources. In this case, exposure of ear- versity of Oklahoma Biological Station. An earlier version of this paper lier generations to Microcystis decreased ftness, as opposed was submitted in partial fulfllment of the requirements for the Master to the increased ftness observed by Gustafsson et al. (2005). of Science in Biology by BAW. Thus, maternal efects in response to toxigenic algae may Authors’ contribution BAW and KDH conceived and designed the be either benefcial or deleterious; similar experiments test- experiment; BAW, JEB, and TCH conducted the experiment; BAW ing for detoxifcation and maternal efects in response to performed all sample analysis; BAW, JEB, and TCH conducted data Prymnesium are needed to elucidate the relative roles of analysis; BAW, JEB, and KDH wrote the manuscript. these mechanisms in zooplankton resistance to this species Data accessibility of toxigenic algae. Resistance to toxins is thus likely infu- Data will be made available on PANGAEA upon publication. enced by a multitude of factors, including species-specifc responses in detoxifcation, feeding selectivity, maternal efects, and nutrition. Therefore, it is difcult to infer when or why resistance may arise. The community in our study References had limited prior exposure to Prymnesium, although genetic infux from other areas of the reservoir with a history of Anderson MJ (2001) A new method for non-parametric multivariate blooms introduces the possibility that resistance to Prym- analysis of variance. Austral Ecol 26:32–46. https://doi.org/10.1 111/j.1442-9993.2001.01070 nesium toxins may have factored into the results. However, Baker JW, Grover JP, Brooks BW, Urena-Boeck F, Roelke DL, Errera the species-specifc responses corresponding to variation in R, Kiesling RL (2007) Growth and toxicity of Prymnesium par- known feeding strategies leads us to conclude that selec- vum (Haptophyta) as a function of salinity, light, and temperature. tive feeding is a primary mechanism driving the observed J Phycol 43:219–227 Barreiro A et al (2005) Relative importance of the diferent negative changes in species abundances. efects of the toxic haptophyte Prymnesium parvum on Rho- This study provides additional solid evidence that Prym- domonas salina and Brachionus plicatilis. Aquat Microb Ecol nesium blooms can have detrimental and predictable efects 38:259–267 on the structure of zooplankton assemblages, and supports Bednarska A, Pietrzak B, Pijanowska J (2014) Efect of poor manage- ability and low nutritional value of cyanobacteria on Daphnia the hypothesis that less-selective suspension-feeding zoo- magna life history performance. J Plankton Res 36:838–847 plankton may be more susceptible to P. parvum compared Beyer JE, Hambright KD (2017) Maternal effects are no match with selective grazers (Remmel et al. 2011). The short-term for stressful conditions: a test of the maternal match hypoth- impacts seen with non-selective-feeding and other vulner- esis in a common zooplankter. Funct Ecol. https://doi. org/10.1111/1365-2435.12901 able zooplankton species may produce larger-scale impacts Blossom HE, Rasmussen SA, Andersen NG, Larsen TO, Nielsen KF, in the structure of entire zooplankton communities, which Hansen PJ (2014) Prymnesium parvum revisited: relationship may propagate throughout invaded food webs. But caution between allelopathy, ichthyotoxicity, and chemical profles in 5 should be exercised as microcosm studies, such as presented strains. Aquat Toxicol 157:159–166. https://doi.org/10.1016/j. aquatox.2014.10.006 here, only provide a snapshot of short-term acute efects Boyd CM (1976) Selection of particle sizes by flter-feeding copepods: of toxigenic Prymnesium, not a full picture of the complex a plea for reason. Limnol Oceanogr 21:175–180 ecosystem interactions that may enhance or mitigate these Colin SP, Dam HG (2002) Testing for toxic efects of prey on zoo- efects over time. Larger-scale experimentation coupled with plankton using sole versus mixed diets. Limnol Oceanogr 47:1430–1437 long-term monitoring can more accurately demonstrate how Dam HG (2013) Evolutionary adaptation of marine zooplankton zooplankton communities respond to harmful algae (sensu to global change. Annu Rev Mar Sci 5:349–370. https://doi. Hambright et al. 2007a; Errera et al. 2008). Such eforts are org/10.1146/annurev-marine-121211-172229 labor intensive and expensive, but the larger-scale repercus- DeMott WR (1982) Feeding selectivities and relative ingestion rates of Daphnia and Bosmina. Limnol Oceanogr 27:518–527 sions of P. parvum establishment in a new system could DeMott WR (1990) Retention efciency, perceptual bias, and active result in dramatically altered ecosystem function (Sunda choice as mechanisms of food selection by suspension-feeding et al. 2006) and thus warrant continued investigation. zooplankton. In: Hughes RN (ed) Behavioural mechanisms of food selection. 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1 3 Short-term toxicity effects of Prymnesium parvum on zooplankton community composition; Aquatic Sciences; Witt et al.; University of Oklahoma; [email protected]

Daphnia mendotae cyclopoid copepods

Bosmina sp. copepod nauplii ) 1 -

Chydorus sp. Keratella sp.

Zooplankton density (no. L (no. density Zooplankton Eurytemora affinis Diaptomus sp.

other rotifers Dreissena polymorpha (Brachionus, sp., veligers Synchaeta sp., Lecane sp., Polyarthra sp.)

Prymnesium parvum density (cells mL-1)

Supplementary Fig. 1 Results of regression analyses of the effect of Prymnesium density on the abundances of zooplankton taxa. Significant negative relationships (α = 0.005) are based on a Bonferroni correction for multiple comparisons (N = 10) with an initial α = 0.05 Short-term toxicity effects of Prymnesium parvum on zooplankton community composition; Aquatic Sciences; Witt et al.; University of Oklahoma; [email protected]

#R script for all analyses in Witt et al.

#### Load packages #### library(openxlsx) library(car) library(vegan) library(labdsv) library(pairwiseAdonis) # How to install package for pairwise Adonis from github: # library(devtools) # install_github("pmartinezarbizu/pairwiseAdonis/pairwiseAdonis") library(dplyr)

#### Load and clean data #### abundance <- read.csv("Witt et al 2019 Aquatic Sciences Data.csv") names(abundance)

#calculate total abundance (#/L) abundance$totalAbundance <- rowSums(abundance[,3:20])

#### Compare total abundance between treatments #### #first reorder the factors abundance$Treatment <- factor(abundance$Treatment, levels=c("Initial", "0ppt", "6ppt", "50K", "100K", "200K")) quartz()

#now make a boxplot to look at total abundance by treatment with(abundance, boxplot(totalAbundance~Treatment))

#define a subset that does not include initial measurements abundance.noInit <- subset(abundance, Treatment != "Initial")

#ANOVA to test for differences in total abundance (among final measurements) zoop.aov <- aov(totalAbundance ~ Treatment, data = abundance.noInit) # Summary of the analysis summary(zoop.aov) # Do Tukey post hoc TukeyHSD(zoop.aov)

# Check for ANOVA assumptions # First, homogeneity of variances leveneTest(totalAbundance ~ Treatment, data = abundance.noInit) # -> no evidence that variance across groups is significantly different based on treatment

#now, normality # Extract the residuals aov_residuals <- residuals(object = zoop.aov) # Run Shapiro-Wilk test shapiro.test(x = aov_residuals) # -> no evidence that residuals are not normally distributed

#### Compare composition between treatments #### #subset data (exclude initials, exclude taxa that aren't in at least 10% (3.5) of samples) #rare taxa: Trichotria, Collotheca, Ascomorpha Short-term toxicity effects of Prymnesium parvum on zooplankton community composition; Aquatic Sciences; Witt et al.; University of Oklahoma; [email protected] abundance.noInit.noRare <- subset(abundance.noInit, select = -c(Trichotria, Collotheca, Ascomorpha, totalAbundance))

#Make NMDS plot set.seed(2) full.nmds <- metaMDS(abundance.noInit.noRare[,3:17], k=2, trymax=100) full.nmds

#make a stress plot stressplot(full.nmds)

#make a quick plot of NMDS results quartz() ordiplot(full.nmds, type="n", display="sites") points(full.nmds, display="sites", type= "p", pch=c(0,1,2,3,4,5)[as.factor(abundance.noInit.noRare$Treatment)]) ordihull(full.nmds,groups=abundance.noInit.noRare$Treatment,draw="polygon",col="grey90",lab el=F) legend(x="bottomright", bty="n",legend=c("0ppt", "100K", "200K", "50K", "6ppt"), pch=c(0,1,2,3,4,5)) # -> very clear separation of controls and treatments

#PERMANOVA full.permanova <- adonis(abundance.noInit.noRare[,3:17]~abundance.noInit.noRare$Treatment) full.permanova

#run post-hoc permanovas (using Holm-Bonferroni correction for multiple comparisons) set.seed(5) pairwise.adonis(abundance.noInit.noRare[,3:17], abundance.noInit.noRare$Treatment, p.adjust.m ='holm') # note that this uses the pairwiseAdonis package which must be installed from github

#run indicator species analysis levels.zoop <- as.numeric(as.factor(abundance.noInit.noRare$Treatment)) true.indic.sp <- indval(abundance.noInit.noRare[,3:17],levels.zoop) summary(true.indic.sp)

#### Test for trends in taxa abundances over Prymnesium density #### # Run regression of each common taxon against prymnesium abundance (set 6ppt and 0ppt equal to zero, first) # start by defining a column that is Prymnesium density # 6ppt = 0 # 0ppt = 0 # 50k = 50000 # 100k = 100000 # 100k = 200000

# Make a new dataframe where weird taxa are dropped (harpacticoid) # Combine Brachionus with 'other rotifers' (Lecane, Polyarthra, Trichotria, Synchaeta, Collotheca, Ascomorpha) abundance.reg <- subset(abundance, Treatment != "Initial") abundance.reg <- subset(abundance.reg, select=-c(totalAbundance,Harpacticoid)) abundance.reg$OtherRotifers <- with(abundance.reg, Brachionus+Lecane+Polyarthra+Trichotria+Synchaeta+Collo theca+Ascomorpha) Short-term toxicity effects of Prymnesium parvum on zooplankton community composition; Aquatic Sciences; Witt et al.; University of Oklahoma; [email protected] abundance.reg <- subset(abundance.reg, select=-c(Brachionus, Lecane, Polyarthra, Trichotria, Synchaeta, Collotheca, Ascomorpha)) abundance.reg$PrymnesiumAbundance <- dplyr::recode(abundance.reg$Treatment, "6ppt" = 0, "0ppt" = 0, "50K"=50000, "100K"=100000, "200K"=200000)

#for each taxon, calculate R2, p-value, for regression against PrymnesiumAbundance, and plot quartz() j <- 1 rvals=NULL pvals=NULL rnames=NULL par(mfrow=c(4,4)) for(i in names(abundance.reg[,3:13])){ tmp <- abundance.reg[[i]] mod <- lm(abundance.reg$PrymnesiumAbundance~tmp) rvals[j] <- summary(mod)$r.squared pvals[j] <- summary(mod)$coefficients[2,4] rnames[j] <- i plot(abundance.reg$PrymnesiumAbundance, tmp, xlab="", ylab=i) j <- j+1 }

#make a dataframe regression.results <- data.frame(rnames, rvals, pvals)

#bonferroni correction - p-value must be < 0.045/10 = 0.005 to be significant regression.results$sig <- regression.results$pvals < 0.0045