Rogalski, MA, Leavitt, PR, Skelly, DK. 2017. Daphniid zooplankton assemblage shifts in response to eutrophication and metal contamination during the Anthropocene. doi: 10.1098/rspb.2017.0865

Electronic supplementary material 1. Study lake chemical, physical, and watershed characteristics

Text S1. All study lakes were formed by glacial activity during the last glacial maximum approximately 12,000 years ago. Black Pond, the low-nutrient reference site, is moderate in size and depth (30 ha, 7 m maximum depth), with a watershed that has remained 80-90% forested over the past century (Table S1) [1, 2]. Alexander Lake is the largest and deepest lake (87 ha, 16m deep), with a small watershed relative to its size (308 ha). Over the past eighty years, approximately 80% of the watershed has remained forested, with developed cover shifting from low- to high-density residential development, in addition to the establishment of an industrial park in the 1970s and an electric power plant in 2002 [1, 2]. Roseland Lake has a moderate surface area (36 ha), shallow depth (6.1 m) and extensive watershed area (7,873 ha). Roughly 30% of this watershed has consisted of agricultural and urban cover over at least the past eighty years [1-4]. Cedar Pond is the smallest (9 ha) and shallowest lake (5.2 m) with the most heavily developed watershed: 79% of the 114 ha watershed is developed today. A basalt trap rock quarry was built upstream of Cedar in 1914 and continues operation today; remaining developed cover has shifted from primarily agricultural to suburban residential and urban over the past century [1,2,5]. The selected lakes are all relatively small and shallow, typical of the lakes in Connecticut (Table S2).

References:1) Frink CR, Norvell WA. 1984 Chemical and Physical Properties of Connecticut Lakes,

New Haven, CT, USA: The Connecticut Agricultural and Experiment Station.2) University of Connecticut Libraries Map and Geographic Information Center - MAGIC.

2012 Neighborhood Change in Connecticut, 1934 to Present. Accessed October 24, 2014. http://magic.lib.uconn.edu/otl/dualcontrol_aerialchange.html.

3) Connecticut Department of Environmental Protection. 1978 The causes of algae growth in Roseland Lake, Woodstock, CT, p. 18, Hartford, CT, USA: CT DEP.

4) Eastern Connecticut Conservation District. 2009 Muddy Brook and Little River water quality improvement plan, p. 90, Norwich, CT, USA: Eastern Connecticut Conservation District.

5) Connecticut Department of Environmental Protection. 2005 A total maximum daily load analysis for Cedar Pond in North Branford, Connecticut, p. 30, Hartford, CT, USA: CT DEP.

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Table S1. Lake characteristics and historic limnological data. Zmax = maximum depth (m); SA= Surface Area (hectares); WA = Watershed Area (hectares); TPs/b = Total Phosphorus (TP) surface (1 m depth)/ bottom (1 m from bottom) (µg/L). Secchi = Secchi disc transparency (m).

Lake Zmax SA WA Sampling Date TPs/b SecchiBlack 7 30 186 August 1980b

July 1993c

July 2013e

Sept 2013e

15/2344/-- 9/3210/31

3 4.2 3.63.4

Alexander 16 77 308 Summer 1937-39a

July/Aug 1974b

Aug 1989c

July 2005d

July 2013e

Sept 2013e

11/-- 7/1523/5122/349/688/122

8 8.1 5.8 5.8 5.44.75

Roseland 6.1 39 7873 Summer 1937-39a

July/Aug 1974b

June 1993c

July 2013e

Sept 2013e

13/--38/12096/-- 44/25438/354

2.5 2.8 0.9 1.11.25

Cedar 5.2 9 114 July/Aug 1974b

Aug 2005d

July 2013e

Sept 2013e

38/12024/3523/3429/100

0.9 2.3 22.1

Data sources: a) Deevey ES, Jr. 1940 Limnological studies in Connecticut. V. A contribution to regional

limnology Am. J. Sci. 238, 717-741. (doi:10.2475/ajs.238.10.717)b) Frink CR, Norvell WA. 1984 Chemical and Physical Properties of Connecticut Lakes, p.

186, New Haven, Connecticut: The Connecticut Agricultural and Experiment Station.c) Canavan RW, Siver PA. 1995 Connecticut Lakes: A Study of the Chemical and Physical

Properties of Fifty-six Connecticut Lakes. p. 299, New London, Connecticut: Connecticut College Arboretum.

d) CT Agricultural and Experiment Station, unpublished data: http://www.ct.gov/caes/cwp/view.asp?a=2799&q=377004&caesNav=|

e) Rogalski, previously unpublished data

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Table S2. Summary statistics describing the 83 lakes included in at least one of the three historic surveys conducted in Connecticut since the 1930s.

1st quartile

Median Mean 3rd quartile

Min. Max.

Maximum depth (m) 4.9 8.2 10.2 13.8 1.1 32.9Surface area (hectares) 28.2 47.8 112.6 109.5 6.0 2195.0Watershed: Surface Area 5.7 11.2 88.9 25.7 1.8 3068.9

Data sources:1) Deevey ES, Jr. 1940 Limnological studies in Connecticut. V. A contribution to regional

limnology Am. J. Sci. 238, 717-741. (doi:10.2475/ajs.238.10.717)2) Frink CR, Norvell WA. 1984 Chemical and Physical Properties of Connecticut Lakes,

New Haven, CT, USA: The Connecticut Agricultural and Experiment Station.3) Canavan RW, Siver PA. 1994. Chemical and physical properties of Connecticut lakes,

with emphasis on regional geology. Lake Reserv. Manage. 10,175-188. (doi:10.1080/07438149409354189).

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Text S2. Account of historical fish stocking in the study lakes.

Predation pressure is known to be a strong selective force on zooplankton community structure [1,2], and we attempted to eliminate vertebrate zooplanktivory from landlocked alewives (Alosa pseudoharengus), an anadromous taxon which is widely stocked in this region [3]. However, like most lakes in Connecticut, all four study lakes have been managed for fisheries to some extent over the past century. Historical records show that fish introduction (mainly yellow perch (Perca flavescens), pickerel (Esox sp.), and smallmouth bass (Micropterus dolomieu)) began at least by the early 1900s, far before significant daphniid species shifts over the past few decades [4]. Furthermore, comparison of historic and modern fish surveys show that piscivorous and planktivorous species composition has not changed dramatically in any of the study lakes during the past fifty years [5,6, Cedar Lake Association, unpublished data]. Finally, we note that the fish community composition in the oligotrophic reference lake, Black Pond, is similar to those in the other three lakes [6, Cedar Lake Association, unpublished data]. Taken together, these observations strongly suggest that zooplankivory by fish is unlikely to be the driving force behind daphniid assemblage homogenization.

References:1) Brooks JL, Dodson SI. 1965 Predation, body size, and composition of plankton. Science

150, 28–35. (doi:10.1126/science.150.3692.28)2) Gliwicz ZM. 2003 Between hazards of starvation and risk of predation: the ecology of

offshore animals, Excellence in ecology volume 12. Oldendorf/Luhe: International Ecology Institute.

3) Post DM, Palkovacs EP, Schielke EG, Dodson SI. 2008 Intraspecific variation in a predator affects community structure and cascading trophic interactions. Ecology 89, 2019–2032. (doi:10.1890/07-1216.1)

4) Mollan WK, Manross FN, Pease CH, Atwood JW, Sanford LC, Arnold PC, McMullen W, McLaughlin LH, Crampton JM, Bartle MK. 1920 Thirteenth biennial report of the State Board of Fisheries and Game for the years 1919-1920. p. 71, Hartford, CT, USA: State of Connecticut Office of the Board of Fisheries and Game.

5) Connecticut State Board of Fisheries and Game. 1959 A Fishery Survey of the Lakes and Ponds of Connecticut. p. 395, Hartford, CT, USA: Connecticut State Board of Fisheries and Game.

6) Jacobs RP, O’Donnel EB. 2002 A Fisheries Guide to Lakes and Ponds of Connecticut. p. 354, Hartford, CT, USA: Connecticut Department of Environmental Protection.

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Electronic supplementary material 2. Additional details on sediment analyses.

Figure S1. Decay of excess (xs) 210Pb to levels supported by 226Ra decay in the sediments of the four study lakes. 226Ra concentrations are based on measures of 214Pb. Age estimates were calculated using the constant rate of supply method (CRS) [1]. Error bars on 226Ra and excess 210Pb levels are one sigma counting uncertainties, and error bars on sediment age estimates are age calculations based on radioisotope counting uncertainties. In cases where error bars are not present they do not extend beyond the points drawn in the figures. Further details on estimation of the sediment ages for these lakes are found in Rogalski [2].

References:1. Appleby, PG, Oldfield, F. 1983 The assessment of 210Pb data from sites with varying

sediment accumulation rates. Hydrobiologia 103, 29–35. 2. Rogalski, MA. 2015 Tainted resurrection: Metal pollution is linked with reduced hatching

and high juvenile mortality in Daphnia egg banks. Ecology 96, 1166–1173. (doi:10.1890/14-1663.1.sm)

Text S3: Detailed methods for sediment fossil pigment analyses

Lipid-soluble pigments were extracted from the bulk sediments by soaking freeze-dried sediments in a mixture of acetone: methanol: water (80:15:5, by volume) for 24 h in darkness and under an inert N2 atmosphere at 4°C. Pigment concentrations were quantified by reversed-phase high performance liquid chromatography (RP-HPLC). Specifically, carotenoid, chlorophyll (Chl), and pigment-derivative concentrations were quantified using an Aligent 1100 HPLC system following the reversed-phase procedure of Leavitt and Hodgson [1]. The Agilent 1100 system was equipped with a C-18 column (5-μm particle size; 10-cm length), and an Agilent model 1100 photodiode array spectrophotometer (435-nm detection wavelength). An

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internal reference standard (3.2 mg L-1) of Sudan II (Sigma Chemical Corp., St. Louis, MO) was injected in each sample.

Pigments isolated from sediments were compared to those from unialgal cultures and authentic standards obtained from US Environmental Protection Agency and other suppliers [1]. Pigment identity was based mainly on spectral characteristics and chromatographic mobility of pigments from all sources. Pigment analysis was restricted to taxonomically-diagnostic carotenoids characteristic of the following algal groups: diatoms, chrysophytes and some dinoflagellates (fucoxanthin), mainly diatoms (diatoxanthin), cryptophytes (alloxanthin), chlorophytes (pheophytin b), chlorophytes and cyanobacteria (lutein-zeaxanthin), filamentous or colonial cyanobacteria (myxoxanthophyll), Nostocales cyanobacteria (canthaxanthin), all cyanobacteria (echinenone), okenone (purple sulfur bacteria), and the major a, b, and c-phorbins (chlorophyll (Chl) and Chl derivatives), although not all compounds were recorded in every sample. Pigment concentrations were expressed as nmol pigment g-1 total C, with total carbon (TC) derived from elemental analyses without correction for inorganic C (normally minimal).

References1) Leavitt P, Hodgson D. 2001 Sedimentary pigments. In Tracking Environmental Change

using Lake Sediments. Volume 3: Terrestrial, Algal and Siliceous Indicators (eds J Smol, H Birks, W Last), pp. 295–325. Dordrecht, The Netherlands: Kluwer Academic Publications.

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Electronic supplementary material 3. Details on traits used to distinguish ephippia produced by different daphniid taxa found in the four study lakes.

Text S4: There were six morphological types of daphniid ephippia found in the four study lakes. After hatching, culturing, and identifying individuals from dozens of ephippia from each lake, the following characteristics were used to identify the species that produced ephippia that did not hatch or that were empty of resting eggs. Images included are cropped but not resized.

Ceriodaphnia ephippia had a single egg chamber and were much smaller in size than Daphnia ephippia. Ceriodaphnia individuals rarely hatched, thus Ceriodaphnia were identified to the genus level.

Daphnia ephippia had two egg chambers. Daphnia pulicaria and Daphnia catawba produced much larger ephippia than the other three species. Both of these species had relatively thick, noticable spinescence along the dorsal edge of the ephippium (greater than any of the other species). Of these two, D. pulicaria ephippia were distinguishable by their elongated shape (with one end being narrower).

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Ceriodaphnia ephippium photographed at 100x power.

Ephippia produced by D. catawba (left) and D. pulicaria (right) in Alexander Lake. Photo is taken at 100 x power

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Similarly long and thick spinescence along the dorsal edge of a D. pulicaria ephippium from Cedar Pond. (400 x power). Note this is not a feature used to distinguish between D. catawba and D. pulicaria.

Daphnia mendotae produced moderate sized ephippia. They were distinguishable by having egg chambers in a slanted position. In addition, the dorsal edges of the ephippia were very smooth when viewed at 400x

Daphnia ambigua and Daphnia parvula produced smaller ephippia. The key reliable feature that differentiated D. ambigua and D. parvula ephippia is the nature of the spinescence along the dorsal edge of the ephippium. D.

ambigua ephippia had small, coarse spinescence along the dorsal edge. D. parvula had either very thin, fine spinescence or no observable spinescence. The shape of D. ambigua and D. parvula ephippia was not a very useful distinguishing trait, however, D. ambigua did tend to be deeper in height. In addition, D. ambigua ephippia sometimes had dark pigmentation around the egg chambers.

9 D. parvula ephippium from Cedar Pond, showing slightly shallower depth relative to width compared with D. ambigua. (100x power)

Dorsal edge of the same D. mendotae ephippium pictured to the left, from Cedar Pond (400x power). Note the lack of spinescence.

D. mendotae ephippium from Cedar Pond. Note the slanted placement of the egg chambers. (100 x power)

Two D. ambigua ephippia from Roseland Lake, showing dark pigmentation that is sometimes present. (100x power)

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Very sparse, thin spinescence on dorsal edge of a D. parvula ephippium from Cedar Pond (400 x power).

Spinescence on a D. ambigua ephippium from Cedar Pond. (400x power)

Short, stout spinescence on D. ambigua ephippium from Black Pond (400 x power).

Electronic supplementary material 4. Additional results from the principal component analyses conducted on eutrophication and metals data, as well as daphniid assemblages, and species richness rarefaction.

Table S3. Results from principal component analyses (PCA) of eutrophication and metals data for all time periods available from each of the four study lakes. The third principal component had eigenvalues of 0.576-0.071 and is not presented. Concentrations of six heavy metals (Cd, Cr, Cu, Hg, Pb, and Zn) were included in the metals PCAs, with the exception of Cedar Pond, where Cu was excluded. Variables included in the PCA of eutrophication included C: N; concentrations of β-carotene, okenone (if present), the sum of taxonomically diagnostic pigments (total pigments); and the percent of total pigments produced by cyanobacteria taxa.

Black Alexander Cedar RoselandPC1 PC2 PC1 PC2 PC1 PC2 PC1 PC2

EutrophicationEigenvalue 1.924 1.108 3.498 0.900 4.492 0.292 3.152 0.463Prop. explained 0.481 0.277 0.700 0.180 0.898 0.058 0.788 0.116Loadings C: N 0.411 -1.302 -1.118 -0.129 -1.492 0.217 -1.434 0.014 β-carotene 1.063 -0.538 0.887 0.354 1.397 0.522 1.305 0.752 Okenone N/A N/A 1.155 0.106 1.445 0.266 N/A N/A Total pigments 1.208 0.403 1.150 -0.040 1.481 -0.024 1.505 0.014 Prop. Cyano 1.199 0.518 0.506 -1.057 1.407 -0.536 1.303 -0.754

MetalsEigenvalue 5.817 0.101 4.564 1.012 4.134 0.619 4.144 1.128Prop. explained 0.970 0.017 0.761 0.169 0.827 0.124 0.691 0.189Loadings Cr -1.174 -0.083 -1.038 -0.052 1.029 -0.940 0.469 -1.072 Cu -1.178 -0.770 -1.053 -0.411 N/A N/A 0.964 0.667 Zn -1.158 -0.147 -1.058 0.227 1.283 0.543 1.258 0.164 Cd -1.182 -0.108 -1.083 -0.331 1.375 0.103 1.238 0.233 Pb -1.177 0.142 -0.551 0.985 1.301 0.134 1.144 -0.339 Hg -1.156 0.276 -1.055 0.059 1.331 -0.035 1.093 -0.227

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Figure S2. Sediment profiles for chromium, copper, cadmium, lead, zinc and mercury (Cd, Cu, Cd, Pb, Zn, Hg) for each lake. Note differences in scale among metals and difference in units for Hg. Each row represents data from one of four lakes. PC1 values show first principal component scores from analysis of the six metals, with the exception of Cedar Pond, where the PCA excluded copper. Red data points indicate sediment metal concentrations that exceed probable effect concentrations (sediment toxicity is likely for aquatic organisms). Variation in absolute metal concentrations among lakes may reflect differences in lake basin shape, watershed characteristics (e.g., size and land cover), sedimentation rate, pollution source (e.g. atmospheric deposition vs. surface runoff), and natural variation (e.g., mineralogy). For the purposes of this study, temporal variation in metals within lakes is of much greater interest than differences in absolute concentrations across lakes.

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Figure S3. Palaeolimnological records of historic eutrophication for the lakes. Note differences in units among indices and scale among pigments. Total pigments include combined concentrations of taxonomically diagnostic pigments: fucoxanthin, alloxanthin, diatoxanthin, leutein-zeaxanthin, myxoxanthin, canthaxanthin, and echinenone. Percent cyanobacteria represents the proportion of primarily cyanobacteria pigments (myxoxanthin, canthaxanthin, and echinenone) to total pigments. PC1 represents first principal component scores for PCA of the following variables: C: N, β-carotene, okenone, total pigments, and % cyanobacteria. Changes in % cyanobacterial pigments reflect changes in the relative abundance of cyanobacteria over time in a given lake, but do not necessarily reflect the exact percentage they made up in the water column. Owing to differences in lability of individual pigments and properties of the lake basin, % cyanobacteria, and indeed overall abundance of algal pigments, cannot be accurately compared among lakes.

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Table S4. Results of PCA of Hellinger transformed daphniid species densities based on resting egg banks in the sediments of the four study lakes. Species with the heaviest loadings for each principal component axis are emphasised with bold text. Note that changes in D. pulicaria are reflected strongly in PC3 only. For this reason, figure 3 in the main text displays a biplot of PC1 vs. PC3, in order to show patterns related to all six of the species.

PC1 PC2 PC3 PC4 PC5 PC6Eigenvalue 2.281 1.545 1.227 0.670 0.204 0.072Prop. explained 0.380 0.258 0.205 0.112 0.034 0.012PC axis loadings Ceriodaphnia 1.781 -0.787 0.424 -2.583 -1.598 0.472 D. ambigua 2.174 -0.319 -0.149 1.742 0.408 2.317 D. catawba -1.249 2.415 -0.213 -0.577 -1.081 2.115 D. mendotae -1.286 -1.981 -1.230 1.000 -2.253 0.499 D. parvula -1.518 -1.698 1.397 -0.949 1.556 1.720 D. pulicaria 0.051 -0.236 -3.115 -1.216 1.424 0.383

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Figure S4. PCA biplots showing temporal shifts in daphniid assemblage composition in each lake, comparing PC1 and 2 (panel A) and PC2 and 3 (panel B). The PCA is based on Hellinger transformed species densities estimated from sediment diapausing egg banks. A

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plot of PC1 vs PC3 is available in the main text (figure 4). Species scores are represented by abbreviated species names: Ceriodaphnia = CER, D. ambigua = AMB, D. catawba = CAT, D. mendotae = MEN, D. parvula = PAR, D. pulicaria = PUL. Site scores are labelled with the approximate age of the sediment for that time period. Lines are drawn to show the temporal changes in daphniid composition within each lake. Alexander=blue, Cedar=gold, Roseland=red, Black=black.

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Figure S5. Rarefied estimates of species richness at each time period, based on resampling the lowest count of all of the lakes and time periods (42 ephippia). Error bars show 1 SE.

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Electronic supplementary material 5. Modern daphniid composition and eutrophication history in Connecticut lakes

Text S5. In choosing the four lakes for this palaeolimnological study we visited a total of 14 lakes that have been surveyed at least twice in historic surveys conducted over the past several decades (Deevey 1940; Frink and Norvel 1984; Canavan and Siver 1995; CT Agricultural and Experiment Station, unpublished data). We sampled the daphniid zooplankton assemblages in these candidate lakes in the summer of 2011. We collected an integrated sample from the water column in the deepest basin of each lake using a 12 cm diameter conical net (80 um mesh). The daphniid species composition for each of these lake samples, as well as nutrient and transparency data from historic surveys, is found in table S5. The location of these lakes is shown in figure S4.

References:1) Deevey ES, Jr. 1940 Limnological studies in Connecticut. V. A contribution to regional

limnology Am. J. Sci. 238, 717-741. (doi:10.2475/ajs.238.10.717)2) Frink CR, Norvell WA. 1984 Chemical and Physical Properties of Connecticut Lakes, p.

186, New Haven, Connecticut: The Connecticut Agricultural and Experiment Station.3) Canavan RW, Siver PA. 1995 Connecticut Lakes: A Study of the Chemical and Physical

Properties of Fifty-six Connecticut Lakes. p. 299, New London, Connecticut: Connecticut College Arboretum.

4) CT Agricultural and Experiment Station, unpublished data; http://www.ct.gov/caes/cwp/view.asp?a=2799&q=377004&caesNav=|

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Table S5. Lake properties, historic nutrient data, and daphniid species composition of 14 candidate study lakes sampled in the summer of 2011. Z max = maximum depth (meters); SA= Surface Area (hectares); TPs/b = Total Phosphorus (TP) surface (1 m depth)/ bottom (1 m from bottom) (µg/L). Secchi = Secchi disc transparency (m). Nutrient and transparency data for Roseland lake in 2000s is from 2013, collected by Rogalski. Daphnia assemblage composition and densities are based on sampling the water column on a single date in summer 2011.

Lake SA Z max Historic data (Secchi, TPs/b) Daphniid species composition(density/L lake water)

1930s 1970s 1990s 2000s

1) Alexander 87.0 15.5 8, 11 8.1, 7/16 5.8, 29/-- 5.8, 22/34 D. mendotae (2.83/L); D. pulicaria (1.84/L)2) Bantam 383.2 7.9 2.3, 18 1.8, 35/70 1.7, 42/50 1.4, 54/59 D. retrocurva (0.37/L); D. mendotae (0.20/L); D.

ambigua (0.03/L)3) Bashan 110.5 14.3 8.2, 17 5.5,5/15 6, 12/23 4.2, 8/1 D. catawba (1.47/L)4) Beseck 46.9 7.9 1.5, 31 2.8, 34/165 2.5, 32/46 2.5, 21/60 D. mendotae (6.37/L)5) Black 29.7 7.0 N/A 3, 15/23 4.2, 25 3.8, 1/32 D. catawba (7.02/L)6) Cedar 8.7 5.2 N/A 0.9, 71/98 N/A 2.3, 24/35 D. mendotae (2.21/L); D. parvula (10.81/L)7) Hayward 70.4 10.7 5.4, 11 3.3, 15/16 3.6, 22/25 2.2, 13/15 D. pulicaria (0.61/L); D. retrocurva (0.99/L); D.

ambigua (0.05/L)8) Mohawk 6.6 7.9 N/A N/A 4.2, 27/29 7.1, 9/27 D. catawba (0.98/L); D. ambigua (0.28/L)9) Norwich 12.2 10.1 N/A 3.2, 12/15 2.5, 18/27 1.7, 3/15 D. catawba (0.02/L)10) Roseland 38.9 5.8 2.5, 13 2.75,

38/1140.9, 96/-- 1.1, 44/254 D. parvula (13.97/L)

11) Tyler 75.7 7.0 N/A 3.8, 19/16 3, 42/62 2.1, 24/25 D. mendotae (5.32/L); D. catawba (0.17/L)12) West Hill 105.6 19.2 8.1, 11 7, 7/16 7.6, 12/33 10.2, 5/5 D. mendotae (4.8/L); D. pulicaria (2.63/L)13) West Side 17.0 9.8 N/A 4, 11/38 3.5, 38/74 5.1, 15/326 D. catawba (0.94/L); D. mendotae (0.12/L)14) Wyassup 37.1 8.5 N/A 4.3, 10/11 3.7, 24/-- 2.2, 20/52 no Daphnia present

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Figure S6. Geographic location of 14 candidate study lakes. “+” indicates the presence of D. mendotae and “*” indicates the presence of D. pulicaria in the surveyed lakes in summer of 2011. D. mendotae and D. pulicaria presence in Cedar Pond and Roseland Lake includes presence in surface sediments. Lake numbers reference labels in table S5.

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