35b Calosi et al 2013 Phil Tran B Adaptation and acclimatization to ocean acidification in marine...

Electronic Supplementary Material for ‘Adaptation and acclimatisation to 1

ocean acidification in marine ectotherms: an in situ transplant experiment with 2

polychaetes at a shallow CO2 vent system’. 3

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Piero Calosi1*, Samuel P.S. Rastrick1, Chiara Lombardi2, Heidi J. de Guzman3, Laura 6

Davidson4, Marlene Jahnke4, Adriana Giangrande5, Jörg D. Hardege4, Anja 7

Schulze3, John I. Spicer1, Maria-Cristina Gambi6 8

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1Marine Biology and Ecology Research Centre, School of Marine Science and Engineering, Plymouth University, 11

Drake Circus, Plymouth, PL4 8AA, UK. 12

2Marine Ecology Laboratory, Marine Environment and Sustainable Development Unit ENEA, P.O. Box 224, La 13

Spezia, Italy. 14

3Department of Marine Biology, Texas A & M University at Galveston, P. O. Box 1675, Galveston, TX 77554, 15

USA. 16

4Chemical Ecology Group, School of Biological, Biomedical & Environmental Sciences, The University of Hull, 17

Hull, HU6 7RX, UK. 18

5Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, University of Salento, Lecce, Italy. 19

6Stazione Zoologica Anton Dohrn, Laboratory of Functional and Evolutionary Ecology, Villa Comunale 80121, 20

Napoli, Italy. 21

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RUNNING TITLE: Adaptation & acclimatisation to CO2 vent 24

Corresponding author: *Email: [email protected] 25

mailto:[email protected]%20%20ABSTRACT

APPENDIX 1. Sequence generation and phylogenetic analysis for Amphiglena mediterranea and Platynereis dumerilii 26

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Table A1.1: Details of collections and number of specimens of Platynereis dumerilii and Amphiglena mediterranea successfully processed for 28

cytochrome c oxidase subunit I (COI) and the Internal Transcribed Spacer (ITS) sequencing. Genbank Accession numbers for individual 29

specimens are listed in table A 2.3. N/A (north acidified area), S/A (south acidified area). 30

Collecting location

Lat./Long.

Acidified (A)/ Control (C)

Collection

date

Distance

from vents

P. dumerilii

A. mediterranea

n

COI

ITS

n

COI

ITS

Castello N3

40°43’55.00’’N 13°57’48.82’’E

A

8/7/2009

N/A

-

0

0

7

0

1

Castello S3 40°43’51.18’’N 13°57’47.45’’E

A 7/7/2010 S/A 28 12 2 13 5 5

S. Anna, Ischia 40°43’35.76’’N 13°57’36.95’’E

C 11/21/2011 600 m 28 14 3 19 5 11

S. Pietro, Ischia 40°44’47.59’’N 13°56’39.86’’E

C 11/17/2011 4 km 27 17 2 11 8 5

Nisida, Gulf of Naples 40°46’32.60’’N 14°9’45.52’’E

C 11/14/2011 15 km 29 15 3 18 5 12

S. Caterina, Ionian Sea 40°7’50.86’’N 17°59’39.11’’E

C 11/15/2011 > 600 km 11 9 3 7 6 1

Forio, Ischia 40°44’25.08’’N 13°51’41.54’’E

C 5/20/2012 8 km 13 7 0 0 0 0

Bristol Channel

51° 12' 50.48"N 3° 7' 28.84"W

C

28/7/2011

Atlantic

15

15

4

0

0

0

31

Table A1.2: Forward (F) and Reverse (R) primers used for amplification of the two markers 32

and original references. 33

Marker, name of primer

Sequence Reference

COI

F: LCO-1490 GGTCAACAAATCATAAAGATATTGG Folmer et al. 1994

R: HCO-2198 TAAACTTCAGGGTGACCAAAAAATCA Folmer et al. 1994

F: LepF1_t1 TGTAAAACGACGGCCAGTATTCAACCAATCATAAAGATATTGG Ivanova et al. 2007

F: VF1_t1 TGTAAAACGACGGCCAGTTCTCAACCAACCACAAAGACATTGG Ivanova et al. 2007

F: VF1d_t1 TGTAAAACGACGGCCAGTTCTCAACCAACCACAARGAYATYGG Ivanova et al. 2007

F: VF1i_t1 TGTAAAACGACGGCCAGTTCTCAACCAACCAIAAIGAIATIGG Ivanova et al. 2007

R: LepRI_t1 CAGGAAACAGCTATGACTAAACTTCTGGATGTCCAAAAAATCA Ivanova et al. 2007

R: VR1d_t1 CAGGAAACAGCTATGACTAGACTTCTGGGTGGCCRAARAAYCA Ivanova et al. 2007

R: VR1_t1 CAGGAAACAGCTATGACTAGACTTCTGGGTGGCCAAAGAATCA Ivanova et al. 2007

R: VR1i_t1 CAGGAAACAGCTATGACTAGACTTCTGGGTGICCIAAIAAICA Ivanova et al. 2007

F: M13F TGTAAAACGACGGCCAGT Ivanova et al. 2007

R: M13R CAGGAAACAGCTATGAC Ivanova et al. 2007

F: AmpF 5'- GGA CAA CCT GGA TCG TTA CTG GGC -3' This study

R: AmpR 5'- AGA CTT CTG GGT GGC CGA AGA -3' This study

ITS

F:ITS18SFPoly GAGGAAGTAAAAGTCGTAACA Nygren et al. 2009

R:ITS5.8SRPoly GTTCAATGTGTCCTGCAATTC Nygren et al. 2009

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Reference 35

Folmer, O., Black, M., Lutz, R. & Vrijenhoek, R. 1994 DNA primers for amplification of 36

mitochondrial cytochrome c oxidase subunit 1 from diverse metazoan invertebrates. Mol. 37

Mar. Biol. Biotechn. 3, 294-299. 38

Ivanova, N. V., Zemlak, T. S., Hanner, R. H. & Hebert, P. D. N. 2007 Universal primer 39

cocktails for fish DNA barcoding. Mol. Ecol. Notes 7, 544-548. 40

Nygren, A., Eklöf, J. & Pleijel, F. 2009 Arctic-boreal sibling species of Paranaitis (Polychaeta, 41

Phyllodocidae). Mar. Biol. Res. 5, 315-327. 42

43

Table A1.3: Voucher numbers/abbreviations for individual specimens, their collection 44 locations and GenBank accession numbers for COI and ITS. 45

Voucher

number/abbreviation

Location

COI

ITS

Amphiglena mediterranea (voucher numbers refer to AS’s database at TAMUG)

100541

San Pietro

KC591782

100542 San Pietro KC591783 KC591899

100543 San Pietro KC591784

100544 San Pietro KC591785 KC591900


100547 San Pietro KC591787 KC591901

100549 San Pietro KC591788 KC591902



100557 Castello S3 KC591790





100563 Castello S3 KC591793 KC591906

100564 Castello S3 KC591794 KC591907


100570 Nisida KC591795 KC591909

100572 Nisida KC591796 KC591910

100573 Nisida KC591797 KC591911

100574 Nisida KC591912

100576 Nisida KC591913

100579 Nisida KC591914

100580 Nisida KC591798

100581 Nisida KC591915

100582 Nisida KC591916

100583 Nisida KC591917

100584 Nisida KC591918

100585 Nisida KC591919

100586 Nisida KC591799 KC591920

100590 S. Anna KC591800 KC591921

100591 S. Anna KC591801 KC591922

100594 S. Anna KC591802 KC591923

100595 S. Anna KC591924

100596 S. Anna KC591803

100597 S. Anna KC591804 KC591925

100598 S. Anna KC591926

100599 S. Anna KC591927

100600 S. Anna KC591928

100601 S. Anna KC591929

100603 S. Anna KC591930

100608 S. Anna KC591931

100612 S. Caterina KC591805 KC591932

100613 S. Caterina KC591806





100627 Castello N3 KC591933

Platynereis dumerilii

S Pietro 1 S. Pietro KC591811




S Pietro 7 S. Pietro KC591815 KC591934


S Pietro 9 S. Pietro KC591817 KC591935










S3 1 Castello S3 KC591827









S3 10 Castello S3 KC591836 KC591936


S3 12 Castello S3 KC591838 KC591937

Nisida 1 Nisida KC591938


















S Anna 3 S. Anna KC591854



S Anna 9 S. Anna KC591857 KC591941


S Anna 11 S. Anna KC591858 KC591943










S Cat 1 S. Caterina KC591868 KC591944



S Cat 4 S. Caterina KC591871






Forio 1 Forio KC591877







Bristol 1 Bristol Channel KC591884











Bristol 12 Bristol Channel KC591895 KC591947




APPENDIX 2. Determination of metabolic rate 46

For the larger species, S. spallanzanii, individuals were placed individually in cylindrical 47

incubation chambers (vol. = 195 mL), which were then placed in the water bath to maintain 48

temperature (T = 24 °C) conditions stable. The sea water in the chamber was also 49

characterised by salinity 38 and mean pH 8.15 for low pCO2 conditions, and mean pH 7.16 50

for high pCO2 conditions. Each incubation chamber was connected, via capillary tubing, to a 51

measurement chamber (vol. 0.25 mL), with sea water circulating between the incubation 52

chamber and the measuring chamber through fine tubing by a micropump (M100S, TCS 53

micro Ltd, Ospringe, UK). Preliminary experiments showed no difference between the pO2 of 54

the sea water in the two chambers when the water was circulating. This method allowed for 55

the circulation of water within the chambers while preventing the formation of pO2 gradients 56

within the respirometric apparatus. For all other (smaller) species, worms were placed 57

directly in the measurement chamber, the volume of which was adjusted (between 0.25 mL 58

and 0.8 mL) depending on the body size of the species: washers were added to the 59

measuring chamber to allow volumetric adjustments. Sea water used in the chambers was 60

taken from the water bath and filtered using 0.22 μm filters. 61

The pO2 of the water in the measurement chambers was determined adapting the method 62

of Rastrick & Whiteley (2011). All individuals were allowed to settle in the experimental 63

apparatus for 1 h, the minimum time required for establishing resting ṀO2. The decline in 64

pO2 within each closed system was determined using an optical oxygen analyser system 65

(OxySense GEN III 5000 series, OxySense, Dallas, TX). The optical oxygen analyser 66

determines water pO2 levels by measuring the fluorescent signal of an oxygen-sensitive dot 67

placed onto the inside surface of the measurement chamber and in contact with the water. 68

Changes in the fluorescent signal occur in proportion to changes in water pO2 and are 69

detected by a fluorimeter connected to an optical probe on the outside surface of the 70

chamber. Each measurement chamber had a recess so that the probe could be aligned 71

parallel to the oxygen-sensitive dot. Oxygen partial pressure measurements were taken 72

sequentially at regular intervals for every measurement chamber for a period of approx. 2 h. 73

For each respirometer the decline in pO2 was linear over the measurement period and never 74

let fall below pO2 = 17 Pa to avoid hypoxia. All measurements were made in low light 75

conditions to minimize any disturbance to the worm. OxySense software (OxySense Inc., 76

Dallas, Texas, USA) converted fluorescent readings into changes in seawater pO2 against 77

time. ṀO2 was calculated as the change in pO2 h-1 from the linear least-squares regression 78

of pO2 (mbar) plotted against time (h). This was multiplied by the solubility coefficient for 79

oxygen, adjusted for salinity and temperature (Harvey 1955), and the volume of water within 80

each respirometer. Whole animal values for ṀO2 were calculated as μlO2 h-1 and corrected 81

for standard conditions for temperature and pressure (STPD), and expressed as log10 μmol 82

O2 h-1 STPD. 83

As S. spallanzanii encase themselves in tubes, after measuring ṀO2, worms were removed 84

from their tubes and the ṀO2 of each tube (the tube is typically covered by epibionts) was 85

quantified and subtracted from the original ṀO2 value to give the ṀO2 of the worm without 86

the effect of the organisms associated with the tubes. For all other species, background 87

respiration was taken into account by running blanks, and the average value across a 88

number of blanks was subtracted from the original ṀO2 value. 89

90

Reference 91

Harvey, H.W. 1955 The Chemistry and Fertility of Seawaters. Cambridge, UK: Cambridge 92

University Press. 93

Rastrick, S.P.S. & Whiteley, N.M. 2011 Congeneric amphipods show differing abilities to 94

maintain metabolic rates with latitude. Physiol. Biochem. Zool. 84, 154-165. 95

APPENDIX 3. Mean values ± SE for metabolic rate (measure as oxygen uptake and expressed as log10 μmol O2 h-1 STPD) in the non-calcifying 96

marine worm species (T) ‘tolerant’ and (S) ‘sensitive’ to elevated pCO2/low pH: (C-C) collected from low pCO2/elevated pH conditions outside 97

the vents and exposed to low pCO2 conditions, (C-A) collected from low pCO2/elevated pH conditions and exposed to elevated pCO2 conditions 98

within the vents; (A-A) collected from elevated pCO2/low pH conditions and exposed to elevated pCO2 conditions; (A-C) collected from elevated 99

pCO2/low pH conditions and exposed to low pCO2. 100

101

102

Species C-C C-A A-A A-C

Amphiglena mediterranea (T) 1.226 ± 0.037 1.105 ± 0.103 0.870 ± 0.136 1.035 ± 0.105

Platynereis dumerilii (T) 0.629 ± 0.158 0.641 ± 0.133 0.969 ± 0.081 1.117 ± 0.134

Polyophtalmus pictus (T) 0.910 ± 0.249 1.070 ± 0.097 - -

Syllis prolifera (T) - - 1.211 ± 0.079 1.632 ± 0.056

Lysidice collaris (s) 0.906 ± 0.117 0.293 ± 0.090 - -

Lysidice ninetta (s) 1.189 ± 0.129 0.428 ± 0.158 - -

Sabella spallanzanii (s) -0.082 ± 0.083 0.008 ± 0.069 - -

APPENDIX 4. Mean values ± SE for wet body mass (expressed in mg), and relative number of specimens tested (n), in non-calcifying marine

worm species (T) ‘tolerant’ and (S) ‘sensitive’ to elevated pCO2/low pH: (C-C) collected from low pCO2/elevated pH conditions outside the vents

and exposed to low pCO2 conditions, (C-A) collected from low pCO2/elevated pH conditions and exposed to elevated pCO2 conditions within

the vents; (A-A) collected from elevated pCO2/low pH conditions and exposed to elevated pCO2 conditions; (A-C) collected from elevated

pCO2/low pH conditions and exposed to low pCO2 conditions.

Species C-C n C-A n A-A n A-C n

Amphiglena mediterranea (T) 0.483 ± 0.061 12 0.373 ± 0.045 11 0.392 ± 0.062 12 0.467 ± 0.050 12

Platynereis dumerilii (T) 21.292 ± 4.543 12 4.513 ± 0.068 8 2.850 ± 1.240 12 2.492 ± 0.711 12

Polyophtalmus pictus (T) 7.240 ± 5.526 4 3.933 ± 1.014 3 - -

Syllis prolifera (T) - - 0.825 ± 0.110 12 1.033 ± 0.125 12

Lysidice collaris (S) 10.925 ± 1.979 4 11.029 ± 1.793 7 - -

Lysidice ninetta (S) 7.825 ± 1.901 4 6.400 ± 3.049 4 - -

Sabella spallanzanii (S) 6366.667 ± 1411.658 12 5059.183 ± 398.339 12 - -

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