Supplemental Figures:
Supplementary Figure 1. Teleost Na+, K+ ATPase α1 (ATP1A1) gene tree reconstructed using Bayesian inference
(BI) with MrBayes version 3.2.1 (Ronquist et al. 2012) and the GTR+I+Γ model of nucleotide evolution. Blue
branches lead to paralogs up-regulated in freshwater and green branches lead to paralogs up-regulated in seawater.
Supplementary Figure 2. Teleost Na+, K+ ATPase α1 (ATP1A1) gene tree reconstructed using the maximum
likelihood (ML) method with RAxML software version 7.2.7 (Stamatakis 2006) and the GTR+Γ model of
nucleotide evolution. Blue branches lead to paralogs up-regulated in freshwater and green branches lead to
paralogs up-regulated in seawater.
1
Supplementary Table 1. Sequences obtained from Genbank/Ensembl. Sequences were used to design primers
(Supplementary Table 2) to amplify larger portions of the coding regions (Supplementary Table 3). Those that
were directly used in our phylogenetic analysis are noted.
Species
Genbank Accession Probable length Used in
References
number or
identity
(bp)
Phylogenetic
Ensembl ID
analysis?
(name in
alignment)
α1a
α1b-ii
α1b-i
α1b-ii
α1c
α1c-i
α1c-i
1562
3410
1704
1683
1834
1458
<1000
Yes (salmo1b)
-
Gharbi et al. 2005
Leong et al. 2010
Gharbi et al. 2005
Gharbi et al. 2005
Leong et al. 2010
Gharbi et al. 2005
Leong et al. 2010
α1c-ii
α1c-ii
1442
<1000
-
Gharbi et al. 2005
Leong et al. 2010
Oncorhynchus mykiss
Oncorhynchus mykiss
Oncorhynchus mykiss
Oncorhynchus mykiss
Oncorhynchus mykiss
Oncorhynchus masou
Oncorhynchus masou
Coregonus clupeaformis
Coregonus clupeaformis
Coregonus clupeaformis
Coregonus clupeaformis
Coregonus clupeaformis
Coregonus migratorius
AY692142.1
BT058747.1
AY692143.1
AY692144.1
BT072358.1
AY692145.1
GE782721.1
GE779075.1
GE770966.1
GO059849.1
GE783843.1
GO059850.1
GE787361.1
GE794722.1
DW007288.1
GE783309.1
GO053878.1
AY692146.1
GO061624.1
GO058676.1
GO061624.1
GO058676.1
GE778166.1
GE782376.1
GO058677.1
GO061119.1
EG889338.1
GO061118.1
GO055341.1
NM_001124461.1
NM_001124460.1
NM_001124459.1
NM_001124458.1
NM_001124630.1
AB573640.1
AB573639.1
CB484547.1
CB483540.1
CX349535.1
CB483543.1
EV367023.1
GR918019.1
α1a
α1b
α1c
α2
α3
α1a
α1b
α1
α1
α1
α1
α1
α1
3250
3450
3436
3882
3035
3239
3387
586
576
511
442
138
391
Yes (mykiss1a)
Yes (mykiss1b)
Yes (mykiss1c)
Yes (mykissa2)
Yes (mykissa3)
Yes (masu1a)
Yes (masu1b)
-
Thymallus thymallus
Esox lucius
Esox lucius
Plecoglossus altivelis
Plecoglossus altivelis
Plecoglossus altivelis
FF840948.1
GH250173.1
GH250174.1
FN658835.1
EY510425.1
EY510253.1
α1
α1
α1
849
776
635
287
516
516
-
Richards et al. 2003
Richards et al. 2003
Richards et al. 2003
Richards et al. 2003
Richards et al. 2003
Ura et al. Unpublished
Ura et al. Unpublished
Rise et al. 2004
Rise et al. 2004
Rise et al. 2004
Rise et al. 2004
Koop et al. 2008
Bychenko et al. 2010,
Unpublished
Koop et al. 2008
Leong et al. 2010
Leong et al. 2010
Lu et al. 2010
Yazawa et al. 2007, Unpublished
Yazawa et al. 2007, Unpublished
Salmo salar
Salmo salar
Salmo salar
Salmo salar
Salmo salar
Salmo salar
Salmo salar
Salmo salar
Salmo salar
α1
α1
2
Plecoglossus altivelis
Plecoglossus altivelis
Plecoglossus altivelis
Osmerus mordax
Osmerus mordax
Osmerus mordax
Oreochromis niloticus
Oreochromis niloticus
EY511140.1
EY511139.1
EY510426.1
EL549256.1
EL517903.1
EL521745.1
ENSONIT00000015703
ENSONIT00000015672
Oreochromis niloticus
Oreochromis niloticus
Oreochromis niloticus
ENSONIT00000015628
ENSONIT00000015603
ENSONIT00000015580
Anabas testudineus
Anabas testudineus
Anabas testudineus
Takifugu rubripes
Takifugu rubripes
Tetraodon nigroviridis
Tetraodon nigroviridis
Gasterosteus aculeatus
Gasterosteus aculeatus
JN180940
JN180941
JN180942
ENSTRUT00000032672
ENSTRUT00000033934
ENSTNIT00000009334
ENSTNIT00000009181
ENSGACT00000018945
ENSGACT00000018961
α1
α1
α1
α1
α1
α1
1a
Possible
1ba
1a
1a
Possible
1aa
α1aa
α1ba
α1ca
α1
α1
α1
α1
α1
α1
613
577
613
243
477
550
3075
3072
Yes (tilapia1)
Yes (tilapia2)
Yazawa et al. 2007, Unpublished
Yazawa et al. 2007, Unpublished
Yazawa et al. 2007, Unpublished
von Schalburg et al. 2008
von Schalburg et al. 2008
von Schalburg et al. 2008
Di Palma et al. Unpublished
Di Palma et al. Unpublished
3072
3072
3069
Yes (tilapia3)
Yes (tilapia4)
Yes (tilapia5)
Di Palma et al. Unpublished
Di Palma et al. Unpublished
Di Palma et al. Unpublished
3069
3069
3048
3126
3108
3090
3069
3078
3087
Yes (anabas1a)
Yes (anabas1b)
Yes (anabas1c)
Yes (fugu1)
Yes (fugu2)
Yes (tetraod1)
Yes (tetraod2)
Yes (stickle2)
Yes (stickle1)
Ip et al. 2012
Ip et al. 2012
Ip et al. 2012
Aparicio et al. 2002
Aparicio et al. 2002
Jaillon et al. 2004
Jaillon et al. 2004
Jones et al. 2012
Jones et al. 2012
Naming of Oreochromis niloticus and Anabas testudineus α1 paralogs is based upon Tipsmark et al.’s (2011) and
Ip et al.’s (2012) designations.
a
Supplementary Table 2. Primers used to amplify Na+, K+ ATPase α1 (ATP1A1) paralogs.
Primer
Direction
F1
F2
F3
R5
R4
R3
R2
R1
F
F
F
R
R
R
R
R
Primer position
relative to O.
mykiss α1b
Primer sequence (5’ to 3’)
Designed from
GGG CTT GKA AAG GGG AAA GAT G
CCC CHG AGT GGR TSA AGT TCT G
CCACGGTGGAGAGCTGAAGGAC
GRG GAG GGT CAA TCA TGG ACA T
GTC CTT CAG CTC TCC ACC GTG
GGC RAY RTC CAT YCC AGG RCA GTA
CTTGAGKGGGTMCATTCTGA
GRGTTYCGTCGCAKGATGTA
O. mykiss α1a and α1b
O. mykiss α1a and α1b, α1c, α2, α3
O. mykiss α1a and α1b
O. mykiss α1a and α1b, α1c, α2, α3
O. mykiss α1a and α1b
O. mykiss α1a and α1b, α1c, α2, α3
O. mykiss α1a and α1b
O. mykiss α1a and α1b
start codon
4-25
272-293
2010-2031
1780-1801
2011-2031
2923-2946
2948-2967
3034-3053
Supplementary Table 3. Na+, K+ ATPase α1 (ATP1A1) sequence information collected in this study.
Species
Paralog
Used in Phylogenetic
Accession number
analysis? (name in
alignment)
Arctic Char (Salvelinus alpinus)
Yes (char1a)
KJ175154
1a
Arctic Char (Salvelinus alpinus)
Yes (char1b)
KJ175155
1b
Atlantic Salmon (Salmo salar)
Yes (salmo1a)
KJ175156
1a
Atlantic Salmon (Salmo salar)
No (partial sequence)
KJ756510
1b-i
Atlantic Salmon (Salmo salar)
No
(partial
sequence)
KJ756511
1b-ii
Atlantic Salmon (Salmo salar)
Yes (salmo1c)
KJ175157
1c-ii
Arctic Grayling (Thymallus arcticus)
Yes (gray1a)
KJ175158
1a
Arctic Grayling (Thymallus arcticus)
Yes (gray1b)
KJ175159
1b
3
Lake Whitefish (Coregonus clupeaformis)
Lake Whitefish (Coregonus clupeaformis)
Northern Pike (Esox lucius)
Northern Pike (Esox lucius)
Northern Pike (Esox lucius)
Northern Pike (Esox lucius)
Rainbow Smelt (Osmerus mordax)
Rainbow Smelt (Osmerus mordax)
1a
1b
1a-x
1a-y
1b
1c
1-1
1-2
Yes (white1a)
Yes (white 1b)
Yes (pike1ax)
Yes (pike1ay)
Yes (pike1b)
Yes (pike1c)
Yes (smelt1)
Yes (smelt2)
KJ175160
KJ175161
KJ175162
KJ175163
KJ175164
KJ175165
KJ175166
KJ175167
4
Supplementary Table 4. Results of PAML’s lineage-specific models run with 36 (Table 1), 34 and 20 sequences,
to test for the effects of tree topology.
Model
Free-ratio
lnL (np)
36 species:
-27285.32 (139)
κ
36 species:
1.99
34 species:
-24420.21 (131)
34 species:
2.10
20 species:
-13187.78 (75)
20 species:
1.95
ω
 varies among
all lineages
LRT test result (df) & P value
36 species:
732.28 (68)
p < 0.000001
34 species:
712. 41 (64)
p < 0.000001
20 species:
281.12 (36)
p < 0.000001
One-ratio
36 species:
-27651.46 (71)
36 species:
1.97
34 species:
-24776.41 (67)
34 species:
20 species:
-13328.34 (39)
20 species:
1.94
36 species:
 0= 0.097 for all
lineages
34 species:
 0= 0.100 for all
lineages
20 species:
 0= 0.112 for all
lineages
Two-ratios, branch
D, Salmoniform
and Esociform α1a
36 species:
-27608.60 (72)
36 species:
1.97
34 species:
-24734.45 (68)
34 species:
2.09
20 species:
-13290.89 (40)
20 species:
1.95
36 species:
1 = 1.66 for
Branch D
 0=0.093 for all
other lineages
34 species:
1 = 1.645 for
Branch D
 0=0.096 for all
other lineages
36 species:
1.48 (1)
p = 0.22
34 species:
1.46 (1)
p = 0.23
20 species:
0.46 (1)
p = 0.50
20 species:
1 = 1.284 for
Branch D
 0=0.101 for all
other lineages
Two-ratios, branch
D, ω=1
36 species:
-27609.34 (71)
36 species:
1.97
34 species:
-24735.18 (67)
34 species:
2.09
20 species:
-13291.12 (39)
20 species:
1.94
36 species:
 1= 1.00*
 0= 0.093
34 species:
 1= 1.00*
 0= 0.096
20 species:
 1= 1.00*
 0= 0.101
5
Supplementary Table 5. Results of PAML’s Branch-site models run with 36 (Table 2), 34 and 20 sequences, to
test for the effects of tree topology.
Model
lnL (np)
κ
Site class 1
(all
branches)
Site class 2
(all
branches)
Site class 3
(backgroun
d and
foreground
branches
can vary)
LRT test
result (df)
& P value
BEB posterior probabilities
for specified residues
(Posterior probabilities for
models with 34 and 20
sequences in presented in
brackets)
Model A, branch D
(Salmoniform &
Esociform α1a)
36 species:
-27029.63
(74)
36 species:
2.17
36 species:
p0=0.732;
0=0.051
36 species:
p1=0.100;
1= 1.000*
36 species:
p2a=0.148
B=0.051
F=5.422
36 species:
6.50 (1)
p = 0.011
101 C 0.952 (0.911/0.902)
120 T 0.808 (0.753/0.385)
137 A 0.855 (0.746/0.882)
142 I 0.920 (0.921/0.921)
182 T 0.946 (0.910/0.936)
232 S 0.955 (0.955/0.921)
266 I 0.947 (0.948/0.930)
275 M 0.952 (0.951/0.901)
284 L 0.841 (0.843/0.803)
289 D 0.952 (0.952/0.933)
293 E 0.813 (0.813/0.503)
299 S 0.886 (0.886/0.825)
307 L 0.956 (0.955/0.936)
311 V 0.947 (0.953/0.937)
319 P 0.806 (0.786/0.424)
320 S 0.958 (0.957/0.939)
454 T 0.927 (0.920/0.810)
576 V 0.942 (0.928/0.434)
604 S 0.938 (0.700/0.518)
608 C 0.958 (0.957/0.938)
737 A 0.857 (0.915/0.599)
783 K 0.953 (0.952/0.935)
787 M 0.947 (0.947/0.930)
792 F 0.952 (0.945/0.921)
793 L 0.954 (0.953/0.934)
801 A 0.952 (0.936/0.746)
817 I 0.947 (0.948/0.919)
873 M 0.955 (0.954/0.936)
891 E 0.952 (0.951/0.923)
922 Y 0.840 (0.841/0.795)
929 A 0.898 (0.879/0.434)
941 K 0.953 (0.953/0.935)
953 V 0.935 (0.897/0.638)
960 S 0.969 (0.991/0.677)
Salmoniform/Eso
ciform ancestor
fully freshwater
p2b=0.020
B=1.000*
F=5.422
34 species:
-24237.12
(70)
34 species:
2.28
34 species:
p0= 0.745;
0=0.050
34 species:
p0= 0.099;
0=1.000*
34 species:
p2a=0.138
B=0.050
F=5.450
34 species:
6.8 (1)
p = 0.009
p2b=0.018
B=1.000*
F=5.450
20 species:
-13088.32
(42)
20 species:
2.08
20 species
p0= 0.750;
0=0.044
20 species
p0= 0.102;
0= 1.000*
20 species
p2a=0.131
B=0.044
F=4.121
p2b=0.018
B=1.000*
F=4.121
Model A, branch D,
ω=1 (null)
36 species:
-27032.88
(73)
36 species:
2.17
36 species:
p0=0.454
0=0.051
36 species:
p1=0.062
1= 1.000*
20 species:
4.16 (1)
p = 0.041
36 species:
p2a=0.426
B=0.051
F=1.000*
p2b=0.058
B=1.000*
F=1.000*
34 species:
-24240.52
(69)
34 species:
2.28
34 species:
p0= 0.491;
0=0.050
34 species:
p0=0.066 ;
0=1.000*
34 species:
p2a=0.390
B=0.050
F=1.000*
p2b=0.052
B=1.000*
F=1.000*
6
20 species:
13090.40
(41)
20 species:
2.08
20 species
p0= 0.573;
0=0.044
20 species
p0= 0.079;
0= 1.000*
20 species
p2a=0.305
B=0.044
F=1.000*
p2b=0.042
B=1.000*
F=1.000*
7
Supplementary Table 6. Results of PAML’s Clade models run with 36 (Table 3), 34 and 20 sequences, to test for
the effects of tree topology.
Model
lnL (np)
κ
Site class 1
(all branches)
Site class 2
(all branches)
Site class 3
(Clade 1,
Clade 2 and
background
branches can
vary)
LRT test
result (df) &
P value
Sites with ω > 1 and a
Bayes Empirical Bayes
Probability of > 0.95
Clade model C,
Salmoniform
and Esociform
α1a (clade 1),
Salmoniform
and Esociform
α1b (clade 2)
36 species:
-26675.955
(76)
36 species:
2.02
36 species:
p0=0.673
0=0.010
36 species:
p1= 0.039
1= 1.000*
36 species:
p2=0.288
Branch 0=0.207
Branch 1=0.493
Branch 2= 0.126
36 species:
88.612 (2)
p < 0.0001
None
34 species:
-23964.802
(72)
34 species:
2.13
34 species:
p0=679;
0=0.011
34 species:
p0=0.042;
0=1.000*
34 species:
p2=0.279
Branch 0=0.204
Branch 1=0.524
Branch 2= 0.121
34 species:
99.912 (2)
p < 0.0001
20 species:
-13017.016
(44)
20 species:
1.98
20 species
p0=0.655;
0=0.004
20 species
p0=0.047;
0=1.000*
20 species:
79. 534 (2)
p < 0.0001
36 species:
-26700.995
(76)
36 species:
2.01
36 species:
p0=0.681
0=0.011
36 species:
p1= 0.036
1= 1.000*
20 species
p2=0.298
Branch 0=0.182
Branch 1=0.482
Branch 2= 0.100
36 species:
p2=0.283
Branch 0=0.237
Branch 1=0.490
Branch 2= 0.097
34 species:
-23992.452
(72)
34 species:
2.13
34 species:
p0=0.683;
0=0.011
34 species:
p0=0.041;
0=1.000
34 species:
p2=0.275
Branch 0=0.236
Branch 1=0.536
Branch 2= 0.088
34 species:
44.612 (2)
p < 0.0001
20 species:
-13037.047
(44)
20 species:
1.97
20 species
p0=0.695;
0=0.010
20 species
p0=0.042;
0=1.000*
20 species:
39.472 (2)
p < 0.0001
36 species:
-26713.791
(76)
36 species:
2.01
36 species:
p0=0.687
0=0.012
36 species:
p1= 0.034
1= 1.000*
20 species
p2=0.263
Branch 0=0.298
Branch 1=0.563
Branch 2= 0.086
36 species:
p2=0.279
Branch 0=0.251
Branch 1=0.290
Branch 2= 0.075
34 species:
-24007.340
(72)
34 species:
2.12
34 species:
p0=0.692;
0=0.012
34 species:
p0=0.037;
0=1.000*
34 species:
p2=0.271
Branch 0=0.257
Branch 1=0.296
Branch 2= 0.065
34 species:
14.836 (2)
p = 0.0006
Clade model C,
Salmoniform
α1a (clade 1),
Salmoniform
α1b (clade 2)
Clade model C,
Salmonid α1a
(clade 1),
Salmonid α1b
(clade 2)
36 species:
38. 532 (2)
p < 0.0001
36 species:
12.940 (2)
p = 0.0015
None
None
8
M2a_rel
20 species:
-13048.164
(44)
20 species:
1.96
20 species
p0=0.738;
0=0.015
20 species
p0=0.30;
0=1.000*
36 species:
-26720.261
(74)
36 species:
2.01
36 species:
p0=0.690
0=0.012
36 species:
p1=0.033
1= 1.000*
20 species
p2=0.232
Branch 0=0.386
Branch 1=0.385
Branch 2= 0.061
36 species:
p2=0.277
2=0.248
34 species:
-24014.758
(70)
34 species:
2.12
34 species:
p0=0.696
0=0.012
34 species:
p1=0.036
1= 1.000*
34 species:
p2=0.268
2=0.255
20 species:
-13056.783
(42)
20 species:
1.97
20 species
p0=0.716;
0=0.012
20 species
p0=0.041;
0=1.000*
20 species
p2=0.243
2=0.318
20 species:
17.238 (2)
p = 0.0002
Supplementary Table 7. Primers used to quantify pike (Esox lucius) Na+, K+ ATPase α1 (ATP1A1) mRNA levels
using quantitative real time qPCR
Gene
Forward primer
Reverse primer
1a -y
CAC AGC AAG CCC TGG TCA
GAA GGG AGC ACC TGA GAG
GAT
1a -x
1b
CCT CGG TGT TGA TGC TACT CT TTT
TTG GCC TCG ACC AGG ATG
TTT CTG CCA TTT CCA GCT GC
TCA CAA TCA AAC TCA AAG
CCT TCT
α1c
CAA AAG TTC AAA GAT CAT GGA
CTC A
GGG CTT GCT GCG GGA CTA G
18S rRNA
GGT ACT TTC TGT GCC TAC CAT GGT
CCG GAA TCG AAC CCT GAT T
9
References:
Aparicio S, Chapman J, Stupka E, et al. (2002) Whole-genome shotgun assembly and analysis of the genome of
Fugu rubripes. Science, 297, 1301-1310.
Berthelot C, Brunet F, Chalopin D, et al. (2014) The rainbow trout genome provides novel insights into evolution
after whole-genome duplication in vertebrates. Nature Communications, 5, doi: 10.1038/ncomms4657.
Bychenko OS, Sukhanova LV, Skvortsov TA, et al., (2009) Differences in brain transcriptomes of Baikal
coregonids closely related species. Unpublished.
Di Palma F, Johnson J, Lander ES, and Lindblad-Toh K. The Genome Sequence of Oreochromis niloticus (Nile
Tilapia). Broad Institute Genome Assembly Team. Unpublished.
Gharbi K, Ferguson MM, Danzmann RG (2005) Characterization of Na, K-ATPase genes in Atlantic salmon
(Salmo salar) and comparative genomic organization with rainbow trout (Oncorhynchus mykiss).
Molecular Genetics and Genomics, 273, 474-483.
Ip YK, Loong AM, Kuah JS, et al. (2012) Roles of three branchial Na+-K+-ATPase alpha-subunit isoforms in
freshwater adaptation, seawater acclimation, and active ammonia excretion in Anabas testudineus.
American Journal of Physiology-Regulatory Integrative and Comparative Physiology, 303, R112-R125.
Jaillon O, Aury JM, Brunet F, et al. (2004) Genome duplication in the teleost fish Tetraodon nigroviridis reveals
the early vertebrate proto-karyotype. Nature, 431, 946-957.
Jones FC, Grabherr MG, Chan YF, et al. (2012) The genomic basis of adaptive evolution in threespine
sticklebacks. Nature, 484, 55-61.
Koop BF, von Schalburg KR, Leong J, et al. (2008) A salmonid EST genomic study: genes, duplications,
phylogeny and microarrays. Bmc Genomics, 9.
Leong JS, Jantzen SG, von Schalburg KR, et al. (2010) Salmo salar and Esox lucius full-length cDNA sequences
reveal changes in evolutionary pressures on a post-tetraploidization genome. Bmc Genomics, 11, 17.
Lu XJ, Chen J, Huang ZA, Shi YH, Wang F (2010) Proteomic analysis on the alteration of protein expression in
gills of ayu (Plecoglossus altivelis) associated with salinity change. Comparative Biochemistry and Physiology Part D. 5, 185-189.
Richards JG, Semple JW, Bystriansky JS, Schulte PM (2003) Na+/K+-ATPase alpha-isoform switching in gills of
rainbow trout (Oncorhynchus mykiss) during salinity transfer. Journal of Experimental Biology, 206, 44754486.
Rise ML, von Schalburg KR, Brown GD, et al. (2004) Development and application of a salmonid EST database
and cDNA microarray: Data mining and interspecific hybridization characteristics. Genome Research, 14,
478-490.
Ronquist F, Teslenko M, van der Mark P, et al. (2012) MrBayes 3.2: Efficient bayesian phylogenetic inference and
model choice across a large model space. Systematic Biology, 61, 539-542.
Stamatakis A (2006) RAxML-VI-HPC: Maximum likelihood-based phylogenetic analyses with thousands of taxa
and mixed models. Bioinformatics, 22, 2688-2690.
10
Ura K, Mizuno S, Misaka N, et al., (2009) Characterization of Na+K+-ATPase in masu salmon. Unpublished.
von Schalburg KR, Leong J, Cooper GA, et al. (2008) Rainbow smelt (Osmerus mordax) genomic library and EST
resources. Marine Biotechnology, 10, 487-491.
Yazawa R, Beetz-Sargent M, Davidson WS, Koop BF (2007) EST analysis of Plecoglossus altivelis. Unpublished.
11