Paper S Title: the Regime of Climate-Related Disturbance and Nutrient Enrichment Modulate

Paper’s title: The regime of climate-related disturbance and nutrient enrichment modulate macroalgal invasions in rockpools

Journal’s name: Biological Invasions

Authors: Iacopo Bertocci*, J. Domínguez Godino, C. Freitas, M. Incera, R. Araújo, A. Bio, F. Arenas, I. Sousa-Pinto, P. A. Reis, R. Domínguez

*Corresponding author. Affiliation: CIIMAR/CIMAR, Centro Interdisciplinar de Investigação Marinha e Ambiental, Rua dos Bragas, 289, 4050-123, Porto, Portugal. E-mail:

Appendix 3 – Effectiveness of the experimental treatment of nutrient enrichment

Water samples for nutrient analysis were collected at the beginning and the end (first and last two weeks, respectively) of each of three bi-monthly periods the fertilizer was maintained in the field over the duration of the experiment: February-March 2012, March-April 2013 and June-July 2013. Data on the concentration of each nutrient from two water replicates collected in eight natural and eight enriched rock pools was analysed, separately for each sampling occasion, with two-way analysis of variance (ANOVA) including the following factors: Condition (fixed, two levels: natural vs. enriched) and Pool (random, 8 levels, nested in Condition), with two replicates. The assumption of homogeneity of variances was checked with Cochran’s C test and data were log(x+1) transformed when necessary. When this was not possible, untransformed data were analysed and tests considered robust if not significant (at p < 0.05) or significant at p < 0.01, to compensate for the increased probability of Type I error. When the test for the factor ‘Pool’ was not-significant at p > 0.25, it was pooled from the linear model and the F value for ‘Condition’ was calculated over the pooled MS, providing a more powerful test (Winer et al. 1991; Underwood 1997).

Results indicated that the concentration of nitrate and phosphate were significantly larger in enriched than in natural pools at both the beginning and the end of the periods of experimental enrichment February-March 2012 and June-July 2013 (Tab. 1 A, C; Fig. 1 and Fig. 3), while only a slight not-significant trend in the same direction was detected in the period March-April 2013 (Tab. 1 B; Fig. 2). Noting that the mean concentration of both nutrients recorded in unmanipulated pools was clearly larger during this period than in the other two, such exception could be explained with upwelling events that, normally, are particularly intense at the study area in April (Lemos and Pires 2004). This interpretation would be further supported by the almost doubling of the concentration of both nitrate and phosphate at the end compared to the start of the period March-April 2013 (Fig. 2). The naturally increased availability of nutrients might have made the experimental enrichment virtually irrelevant in March-April 2013. Although the same treatment-buffering effect might have occurred also in the corresponding period of the previous year, present findings collectively made us confident about the ability of the experimental treatment to effectively increase the average concentration of nutrients in rock pools over the whole period of the study.

References

Lemos RT, Pires, HO (2004) The upwelling regime off the west Portuguese coast, 1941–2000. Int J Climatol 24:511-524

Underwood AJ (1997) Experiments in Ecology: their logical design and interpretation using analysis of variance. Cambridge University Press, Cambridge, UK

Winer BJ, Brown DR, Michelis KM (1991) Statistical principles in experimental design. Mc Graw-Hill, New York.

Table 1. ANOVA testing for differences in the concentration of nutrients in ‘natural’ and ‘enriched’ rock pools at the beginning and the end (first and last two weeks, respectively) of each of three bimonthly periods the fertilizing treatment was maintained in the field over the duration of the experiment. * p < 0.05, ** p < 0.01, *** p < 0.001.

(A) FEBRUARY-MARCH 2012 Nitrate Phosphate

Source of start end start end

variation df MS F MS F MS F MS F

Condition = C 1 22.61 8.1 ** 104.62 6.0 ** 1.12 9.1 ** 0.41 6.5 **

Pool(C) 14 2.87 9.3 *** 17.35 8.2 *** 0.12 1.8 0.06 6.7 ***

Residual 16 0.26 2.11 0.07 0.01

Cochran’s test C = 0.603, p < 0.01 C = 0.814, p < 0.01 C = 0.791, p < 0.01 C = 0.466

Transformation None None None Ln(x+1)

(B) MARCH-APRIL 2013 Nitrate Phosphate

Source of start end start end

variation df MS F MS F MS F MS F

Condition = C 1 0.45 1.4 9.04 0.6 0.35 4.6 *a 0.02 0.8

Pool(C) 14 0.32 4.4 ** 41.54 9.9 *** 0.11 0.03 1.5

Residual 16 0.07 4.19 0.04 0.02

Pooledb 30 0.08

Cochran’s test C = 0.395 C = 0.262 C = 0.791, p < 0.01 C = 0.217

Transformation Ln(x+1) None None None

Source of start end start end

variation df MS F MS F MS F MS F

Condition = C 1 0.27 7.1 ** 11.58 4.4 *a 0.34 6.0 **a 0.21 7.7 **a

Pool(C) 14 0.04 1.3 5.24 0.11 0.05

Residual 16 0.03 0.30 0.01 0.01

Pooledb 30 2.61

Cochran’s test C = 0.355 C = 0.413 C = 0.680, p < 0.01 C = 0.177

Transformation Ln(x+1) None None None

a Tested against the pooled MS

b Pooled term = Pool(C) + Residual

Figure 1. Concentration (mean + SE, n = 16) of nutrients in natural and treated rockpools at the start (first two weeks) and the end (last two weeks) of the bimonthly period February-March 2012 of experimental enrichment.

Figure 2. Concentration (mean + SE, n = 16) of nutrients in natural and treated rockpools at the start (first two weeks) and the end (last two weeks) of the bimonthly period March-April 2013 of experimental enrichment.

Figure 2. Concentration (mean + SE, n = 16) of nutrients in natural and treated rockpools at the start (first two weeks) and the end (last two weeks) of the bimonthly period June-July 2013 of experimental enrichment.