DOI: 10.5061/dryad.xpnvx0kh7
title: Local adaptation through countergradient selection in northern populations of Skeletonema marinoi

description: Marine microorganisms have the potential to disperse widely with few
 obvious barriers to gene flow. However, among microalgae, several studies
 have demonstrated that species can be highly genetically structured with
 limited gene flow among populations, despite hydrographic connectivity.
 Ecological differentiation and local adaptation have been suggested as
 drivers of such population structure. Here we tested whether multiple
 strains from two genetically distinct Baltic Sea populations of the
 diatom Skeletonema marinoi showed evidence of local
 adaptation to their local environments; the estuarine Bothnian Sea and the
 marine Kattegat Sea. We performed reciprocal transplants of multiple
 strains between culture media based on water from the respective
 environments, and we also allowed competition between strains of estuarine
 and marine origin in both salinities. When grown alone, both marine and
 estuarine strains performed best in the high salinity environment, and
 estuarine strains always grew faster than marine strains. This result
 suggests local adaptation through countergradient selection, i.e. genetic
 effects counteract environmental effects. However, higher growth rate of
 the estuarine strains appears to have a cost in the marine environment and
 when strains were allowed to compete, marine strains performed better than
 estuarine strains in the marine environment. Thus, other traits are likely
 to also affect fitness. We provide evidence that tolerance to pH could be
 involved, and that estuarine strains which are adapted to a more
 fluctuating pH continue growing at higher pH than marine
 strains. 
description: Experiment performed by
 Josefin Sefbom at Gothenburg University. Statistical analyses performed by
 Karin Rengefors. <strong>Material and
 Methods</strong> Skeletonema marinoi<em> strains</em> We used a total of nine strains originating from the Bothnian Sea, northern Baltic Sea, and six strains originating from the Kattegat-Skagerrak area. From here on, estuarine Bothnian Sea strains are referred to as E1-E9, and the marine Kattegat-Skagerrak strains are referred to as M1-M6, for each experiment (Table 1). All strains had previously been genotyped using eight polymorphic microsatellite markers (S.mar1-8) (Almany<em> et al.</em>, 2009). The algal cultures were maintained in native seawater-based silica enriched f/2-medium (Guillard, 1975) with salinities resembling their native environment, 26 PSU (marine) or 7 PSU (estuarine). Experimental cultures were kept in 50 mL-culture flasks (Nunc EasYFlasks™ Nunclon™Δ) at 10°C, the light-dark photo period was 12:12h at a light intensity of 50 μmol photons m<sup>-2</sup> s<sup>-1</sup> (36W Cool Daylight). Light and temperature conditions remained unchanged during all experiments. <em>Experiment 1 - Reciprocal transplant experiments (estuarine vs. marine) </em> To test whether strains were locally adapted to home conditions, we carried out reciprocal transplant experiments whereby maximum growth rate and maximum biomass increase were measured for strains growing in native and in non-native water. Six estuarine strains (E1-E6) and six marine strains (M1-M6) were pre-acclimatised (as described below) to new conditions before maximum growth rate, and biomass increase was measured in the non-native treatment. Growth medium was prepared with natural seawater collected from the Skagerrak (marine water) and northern Baltic proper (estuarine) and double filtered through GF/F glass microfiber filter (pore size 0.7 µm) (Whatman), and then Pall Supor Membrane filter (pore size 0.2 µm) (Pall corporation). Pre-acclimatisation was done in a stepwise manner by transferring estuarine strains from 100% Baltic Sea water to 75-50-25% (adding Skagerrak water) and finally 100% Skagerrak water, every seven days. Marine strains were acclimatised using the same procedure but starting from 100% Skagerrak water and finishing in 100% Baltic Sea water. Pre-acclimatised strains were kept in new conditions for seven days before measuring growth. The total duration of the acclimatisation procedure was four weeks, equivalent to 31 generations (7 generations per step). Generally, at least 6 generations are recommended for acclimatization in microalgal work, while mutations and potential selections are expected to arise after 100 or more generations although no definite threshold has been determined (Zhang<em> et al.</em>, 2021). Maximum growth rates and maximum biomass increase were measured for each strain in triplicates. Growth was monitored daily by transferring 1 mL of each culture to a 48-well plate and measuring chlorophyll fluorescence on a Thermo Scientific Varioskan<sup>®</sup> Flash (microplate reader) with SkanIt<sup>® </sup>Software 2.4.3 (Wavelength: Excitation (nm) 425, Emission (nm) 680).  Maximum growth rate (<em>µ</em><sub>max</sub>) was calculated as <em>µ</em><sub>max</sub> = Ln (F2/ Ln F1)/(t2-t1), where F1 and F2 is the fluorescence at time point t1 and t2. We used a sliding window approach where time points were taken on 3-day intervals (Wood<em> et al.</em>, 2005). Maximum biomass increase (Δ<sub>BM</sub>) was computed as: Δ<sub>BM</sub>= F<sub>max </sub>- F<sub>min</sub>, where F<sub>max </sub>is the highest fluorescence reading (stationary phase) and F<sub>min </sub>is the starting fluorescence reading. Statistical test of fitness (maximum growth rates and maximum biomass increase) differences was done using a mixed-model two-way ANOVA in IBM SPSS 27 for Mac. “Strain origin” (marine or estuarine) and “medium” (marine or estuarine seawater-based) were defined as fixed factors, where “strains” was denoted as a random factor nested within strain origin. Residuals were tested for normality with the Kolmogorov-Smirnov test. In addition, overall means for each set of strains was calculated, and differences were tested statistically using a t-test in Microsoft Excel 16.54 for Mac. <em>Experiment 2 - Reciprocal transplant experiments with pH monitoring</em> In a second experiment, the same setup was used, with the specific aim to monitor pH, a potential abiotic factor to which populations can adapt. Experimental conditions were identical as above, except that strains E1, E4, and E5 were replaced by strains E7, E8 and E9. In this second experiment, cell counts were estimated by measuring minimum fluorescence values (F0) using a Pulse amplitude-Modulation (PAM) (WALZ®, MAXI version of IMAGING-PAM <em>M-Series</em>, model IMAG-K4). The imaging-PAM measures of chlorophyll <em>a</em> fluorescence were done with a Kappa DX4-285 (MAXI) camera. The parameter settings were as follows: intensity of the light 7, frequency 1, gain 3 and damping 2. The absorptivity of the Red Gain was 40, Red Intensity 4 and NIR Intensity 3. The intensity of the Saturation Pulse was 8. To standardize cell counts, dilutions were made to achieve 100%, 50%, 10%, 5%, 1% and 0.1% of medium concentration in a 48-well plate. The 48-well plate was placed in the dark for 15min, to allow cultures to acclimatize before measuring. Cell density was monitored at the same time daily, 11 days in a row. After the fluorescence/density was measured with the PAM, a subsample (1 mL) from each bottle was fixed with Lugol’s solution in a 48-well plate. The pH of all the 72 bottles was measured at 3-day intervals using the HI 2221 Calibration check pH/ORP Meter of Hanna Instruments (pH electrode HI 1131P, temperature probe HI 7662) (accuracy: ± 0.01 pH and ± 0.2 °C). This was calibrated with buffers of 4.01, 7.01 and 10.01 pH. During calibration, the pH value was automatically calibrated to the corresponding temperature as this was measured too. Statistical analyses were performed as above. Moreover, cell density was plotted against pH to establish the correlation coefficient. <em>Experiment 3 - Competition experiment</em> After acclimatisation, we had two batches of each strain from Experiment I; one batch that had remained in its native water (<em>adapted</em> to estuarine or marine water), and the other in the new (non-native) water (<em>acclimatised</em> to estuarine or marine water). Using a common garden setup, we inoculated an <em>adapted</em> strain together with an <em>acclimatised</em> strain to the more marine Skagerrak water and estuarine Baltic Sea water. In total, there were seven different two-strain combinations of an estuarine and a marine strain: M1 and E3 (P1), M3 and E3 (P2), M3 and E4 (P3), M3 and E5 (P4), M5 and E3 (P5), M5 and E4 (P6), M5 and E5 (P7). The strain composition at the end of the experiment was determined using a microsatellite allele-specific quantitative PCR technique (Sefbom<em> et al.</em>, 2015) (see details below). Strain combinations had been chosen so that the relative abundance of each strain in combination with another could reliably be quantified based on fragment peak-heights in an electropherogram. Each of the seven strain combinations, in the two treatments, was grown in triplicates. Both strains were inoculated at the same time at equal concentrations of 5,000 cells mL<sup>-1</sup> (total starting concentration of 10,000 cells mL<sup>-1</sup>). Growth was monitored daily using fluorescence as described for Experiment I. The experiments were terminated in the early stationary phase (between day 8-10 depending on strain combination). On the final day of the experiments, each replicate was filtered onto 3.0 µm filters (Ø25 mm Versapore<sup>®</sup>-3000, Pall Corporation) and kept at -80°C until DNA extraction. <em>DNA extraction and microsatellite analysis</em> Genomic DNA was extracted using a cetrymethylammonium bromide (CTAB) protocol based on Kooistra<em> et al.</em> (2003). Four microsatellite loci were amplified, S.mar1, S.mar 4, S.mar5 and S.mar6, (Almany<em> et al.</em>, 2009) with PCR conditions as described by Godhe &  Härnström (2010). The products were analysed in an ABI 3730 (Applied Biosystems) and allele sizes were assigned relative to the internal standard GS600LIZ. Binning and peak-height determination were performed using GeneMapper (ABI Prism®GeneMapper™Software Version 3.0). <em>Allele-specific Quantitative PCR (AsQ-PCR)</em> To assess relative abundance of the experimental mixed strain cultures we used an AsQ-PCR method described by Meyer <em>et al.</em> (2006) and optimized for <em>S. marinoi</em> in Sefbom et al (2015). The respective peak-heights from the two strains in the electropherograms are used as a relative quantification measurement. To establish that PCR amplification did not favour one strain over the other, we mixed the seven strain combinations in five known proportions (based on microscopic cell counts), ranging from 10:90 to 90:10 (3 replicates each) and carried out DNA extraction and microsatellite analysis as described above. Four different microsatellite markers were tested (S.mar1, S.mar 4, S.mar5 and S.mar6) in order to find the least biased PCR reaction. Peak-height relative abundances were plotted against known relative abundances to obtain <em>r<sup>2</sup></em>-values. Strain combinations with peak-height ratios representative of the mixed cell ratios (<em>r<sup>2 </sup></em>&gt; 0.9) were considered as having unbiased PCR reactions and reliable for assessing competition experiments. To determine whether the relative proportion of a strain was significantly higher when competing in its native environment we utilized a one-way randomized block ANOVA in IBM SPSS 27. “Growth medium” (marine or estuarine) was used as a fixed factor, and “strain combinations” were designated as random factors.  The proportion of marine strains was used as the response variable (not both, since the proportion of one strain is always one minus the other strain). Residuals were tested for normality.
description: Original strain designations were reassigned new names in the
 manuscript. A README file with new names is included.