Ecological Archives E096-177-A1

Lloyd S. Peck, Michael A. S. Thorne, Joseph I. Hoffman, Simon A. Morley, and Melody S. Clark. 2015. Variability among individuals is generated at the gene expression level. Ecology 96:17141724. http://dx.doi.org/10.1890/14-0726.1

Appendix A. Primer combinations for AFLP analysis; Variance inflation factors for L. elliptica and N. concinna metrics; STRUCTURE programme output for Bayesian analyses; gene expression frequency distributions; and raw data figures for oxygen consumption, activity, gene expression, shell dimensions and mass measures of L. elliptica and N. concinna.

Table A1. Primer combinations used for AFLP genotyping and numbers of AFLP loci generated in N. concinna and L. elliptica (n = 48 and 45 individuals respectively).

TaqI primer (5'-3')

EcoRI primer (5'-3')

N. concinna

L. elliptica

 

 

No. loci

No. polymorphic loci

% polymorphic loci

No. loci

No. polymorphic loci

% polymorphic loci

GATGAGTCCTGACCGA–CTG

GACTGCGTACCAATTC–AGC

32

25

78.1

15

11

73.3

GATGAGTCCTGACCGA–CGA

GACTGCGTACCAATTC–AGC

27

18

66.7

14

11

78.6

GATGAGTCCTGACCGA–CAG

GACTGCGTACCAATTC–AGC

8

5

62.5

16

11

68.8

GATGAGTCCTGACCGA–CAC

GACTGCGTACCAATTC–AGC

27

20

74.1

14

13

92.9

GATGAGTCCTGACCGA–CCA

GACTGCGTACCAATTC–AAC

34

27

79.4

GATGAGTCCTGACCGA–CCA

GACTGCGTACCAATTC–ACA

33

30

90.9

24

17

70.8

GATGAGTCCTGACCGA–CAC

GACTGCGTACCAATTC–ATG

41

35

85.4

 

GATGAGTCCTGACCGA–CTG

GACTGCGTACCAATTC–ATG

9

7

77.8

Total

 

202

160

79.2

92

70

76.1

 

Table A2. Variance Inflation Factors showing the level of colinearity in the measures taken for Laternula elliptica. There are two separate sets as those organisms which were tested for HSP expression were different from the set tested for upper lethal temperature. Type refers to metric type (gene expression (G); physiological (P); morphological (M)). )). The largest measures were removed in subsequent analyses until all remaining values were less than 10.

Measure (for animals in HSP expression trials)

VIF

VIF excluding Tissue dry mass

Type of measure

ManHSP70A

11.23

7.69

G

ManHSP70B

12.16

7.42

G

SiphHSP70A

  2.45

2.11

G

SiphHSP70B

  2.39

2.07

G

Whole animal O2 Consumption

  2.46

1.99

P

Burial duration

  1.61

1.52

P

Age

  2.13

1.87

M

Animal

  3.13

2.80

M

Tissue dry mass

75.77

 

M

Tissue AFDM

59.55

3.87

M

Shell dry mass

  4.86

4.47

M

Shell length

 2.48

2.36

M

Measure ( for animals used in UTL trials)

 

 

 

While animal O2 Consumption

 1.63

1.61

P

Burial duration

 1.50

1.75

P

UTL

 1.29

1.22

P

Tissue dry mass

289.90

 

M

Tissue AFDM

253.01

4.51

M

Shell dry mass

6.27

5.27

M

Shell length

1.45

1.43

M

 

Table A3. Variance Inflation Factors showing the level of colinearity in the measures taken for Nacella concinna. There are two separate sets as those organisms which were tested for HSP expression were different from the set tested for upper lethal temperature. Type refers to metric type (gene expression (G); physiological (P); morphological (M)). )). The largest measures were removed in subsequent analyses until all remaining values were less than 10.

Measure (for animals used in HSP expression trials)

VIF

Values after removal of Tissue dry mass

Values after removal of shell length

Type

HSP70B

1.41

1.41

1.37

G

Whole animal O2 Consumption

2.71

2.06

2.06

P

Tenacity

2.63

2.45

2.26

P

Shell length

  12.91

11.77

 

M

Shell width

7.90

7.87

5.61

M

Shell height

3.10

2.86

2.68

M

Tissue dry mass

201.70

 

 

 

Tissue AFDM

141.69

10.75

8.73

M

Shell dry mass

5.82

  5.39

5.31

M

Measure (for animals used in UTL expression trials)

 

 

 

 

Whole animal O2 Consumption

1.79

1.74

1.64

P

Tenacity

2.15

2.12

2.10

P

UTL

3.13

2.56

1.81

P

Shell length

17.21

11.83

4.78

M

Shell width

13.15

12.75

 

M

Shell height

2.74

2.47

2.38

M

Foot area

3.91

3.70

3.69

M

Tissue dry mass

212.92

 

 

M

Tissue AFDM

179.24

7.33

7.32

M

Shell dry mass

8.28

6.08

4.73

M

 

FigA1

Fig. A1. Results of Bayesian analyses of population structure, implemented in the program STRUCTURE (Pritchard et al. 2000).  For each species, the estimated log probability of the data, denoted Ln P(D), was calculated for each value of K, the hypothesised number of distinct genetic clusters, allowing estimation of the most likely number of populations (see Methods for details).  Mean ± SE Ln P(D) values based on five replicates for each value of K are presented for (a) N. concinna and (b) L. elliptica.  For both species, Ln P(D) peaked at K = 1, indicating support for a single population (i.e. a lack of cryptic population substructure).


 

FigA2

Fig. A2. Respiration rate (oxygen consumed, µmol O2 g-1 AFDM h-1 vs tissue AFDM, g) of A. Laternula elliptica, and B. Nacella concinna. Each data point represents the value for an individual.


 

FigA3

Fig. A3. Activity plots, assessed as burial duration (sec) vs tissue AFDM (g) for Laternula elliptica and tenacity (sec) vs tissue AFDM (g) for Nacella concinna. Each data point represents the value for an individual.


 

FigA4

Fig. A4. Gene expression plots for Laternula elliptica. Data presented are HSP70A and 70B expression for mantle (upper) and siphon (lower). Each data point represents the value for an individual.


 

FigA5

Fig. A5. Gene expression plot for Nacella concinna. Data presented are HSP70A and 70B expression for foot tissue. Each data point represents the value for an individual.


 

FigA6

Fig. A6. Upper Temperature Limit (UTL) values plotted vs tissue AFDM for A. Laternula elliptica, and B. Nacella concinna. Each data point represents the value for an individual and indicates the temperature where that individual became unresponsive to external stimuli.


 

FigA7

Fig. A7. Shell dimension and shell mass values plotted vs tissue AFDM for A. Laternula elliptica (length, mm), B. Nacella concinna (length, breadth, height, mm). Each data point represents the value for an individual, C. Laternula elliptica (shell dry mass, g) and D. Nacella concinna (shell dry mass, g). Each data point represents the value for an individual.


 

FigA8

Fig. A8. Age, as number of growth rings on the shell plotted vs tissue AFDM for Laternula elliptica. Each data point represents the value for an individual.


 

FigA9

Fig. A9. Frequency distributions for expression levels of 305 genes shared between all 24 L. elliptica investigated obtained by tagged 454 sequence analysis. The Hsp70 value obtained from QPCR of 0.66, represented by the red line, was close to the mean of the distribution (0.62).


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