Supplemen a y ma e ial associa ed wi h
Op imizing DNA ex ac ion p o ocols o he die analysis o a baleen
whale (Eubalaena aus alis)
Aashi Pa ikh1, Richa d O’Ro ke2, Emma L. Ca oll2, Els Ve meulen3, Robe Ha cou 1,
An hony Cha i on1
1School o Na u al Sciences, Macqua ie Uni e si y, Walluma agal Campus, Macqua ie Pa k
NSW 2109 Aus alia
2School o Biological Sciences, Waipapa Tauma a Rau – Uni e si y o Auckland, Auckland
1142 New Zealand
3Mammal Resea ch Ins i u e Whale Uni , Facul y o Na u al and Ag icul u al Sciences,
Uni e si y o P e o ia, Ha ield 0028 Sou h A ica
Table S1. Lis o SRW samples used in his s udy and associa ed me ada a.
Sample
Da e collec ed
Loca ion
S o age
SA-1
24/10/2006
Sou h A ica – S Helena Bay
E hanol
SA-2
Unknown
Sou h A ica – S Helena Bay
E hanol
SA-3
10/12/1992
Sou h A ica – S Helena Bay
E hanol
NZ-1
15/8/2017
New Zealand – Auckland Islands
F ozen
NZ-2
8/8/2017
New Zealand – Auckland Islands
F ozen
Table S2. PCR condi ions o 18S DNA and C us 16S m DNA.
Amplicon
PCR Mix u e (20 uL)
PCR Condi ions
PCR S age
Tempe a u e
Time
PCR
cycles
18S
2x Ampli aq Gold 360 Mas e mix
Ini ial
dena u a ion
95°C
8
min
P ime -F (0.1mM)
Dena u a ion
95°C
20 s
x 43
P ime -R (0.1mM)
Annealing
58°C
20 s
PNA clamp (0.5mM)*
Elonga ion
72°C
30 s
DNA (2uL)
Final elonga ion
72°C
8
min
C us 16S
2x Ampli aq Gold 360 Mas e mix
Ini ial
dena u a ion
95°C
10
min
P ime -F (0.2mM)
Dena u a ion
95°C
30 s
x 45
P ime -R (0.2mM)
Annealing
51°C
30 s
DNA (2uL)
Elonga ion
72°C
45 s
Final elonga ion
72°C
10
min
*Whale blocking PNA clamp: CGACCGTCTTCTCAGC-Lys
Table S3. Classi iac ion o a ge and non- a ge o ganisms cap u ed by 18S DNA and
C us 16S m DNA.
Ta ge
Non-Ta ge
18S DNA
Kingdom
Me azoa ( ee- loa ing ma ine
zooplank on wi hin his
kingdom)
Amoebozoa, C yp ophyceae,
Discoba, Me amonada,
Opis hokon a, Rhodophy a,
Sa and Vi idiplan ae
Class
Me azoa: Polychae a,
Hexanauplia, Malacos aca,
Os acoda, Thecos aca,
Sagi oidea, Ascidiacea,
Cnida ia, Hyd ozoa,
Scyphozoa, C enopho a,
Lopho ochozoa, Bi al ia,
Gas opoda, P o os omia,
Eu o a o ia
Me azoa:
Palaeacan hocephala,
Cli ella a, A achnida,
Collembola, Insec a, Ces oda
and T ema oda
C us 16S
m DNA
Class
Me azoa: Malacos aca,
Thecos aca
Me azoa: A achnida,
Collembola, Eu o a o ia,
Gymnolaema a, Hyd ozoa,
Insec a, Mammalia
Table S4. Summa y o a ge zOTU ichness cap u ed by each ex ac ion p o ocol o he 18S
DNA da ase .
Ex ac ion
p o ocol
Mean
zOTU
ichness
SD
Ph1P.1B
3.8
3.25
Ph1P.2B
3.67
2.28
Ph1S.1B
3.04
2.67
Ph1S.2B
3.27
3.11
Ph24P.1B
4.16
3.75
Ph24P.2B
4.16
3.7
Ph24S.1B
2.58
3.53
Ph24S.2B
3.24
2.81
Soil.1B
4.6
3.8
Soil.2B
4.73
4.37
S ool.1B
4.69
2.79
S ool.2B
3.51
2.65
Table S5. Pos hoc pai wise compa ison o di e ences in a ge zOTU ichness de i ed om
di e en p o ocols om he 18S DNA da ase .
P o ocols
Es ima e
S d E o
z alue
P (>|z|)
Ph1P.2B-Ph1P.1B
-0.13333
0.4486
-0.297
1
Ph1S.1B-Ph1P.1B
-0.75556
0.4486
-1.684
0.8758
Ph1S.2B-Ph1P.1B
-0.53333
0.4486
-1.189
0.9899
Ph24P.1B-Ph1P.1B
0.35556
0.4486
0.793
0.9997
Ph24P.2B-Ph1P.1B
0.35556
0.4486
0.793
0.9997
Ph24S.1B-Ph1P.1B
-1.22222
0.4486
-2.725
0.2146
Ph24S.2B-Ph1P.1B
-0.55556
0.4486
-1.238
0.9859
Soil.1B-Ph1P.1B
0.8
0.4486
1.783
0.8278
Soil.2B-Ph1P.1B
0.93333
0.4486
2.081
0.6369
S ool.1B-Ph1P.1B
0.88889
0.4486
1.981
0.7065
S ool.2B-Ph1P.1B
-0.28889
0.4486
-0.644
1
Ph1S.1B-Ph1P.2B
-0.62222
0.4486
-1.387
0.9662
Ph1S.2B-Ph1P.2B
-0.4
0.4486
-0.892
0.9992
Ph24P.1B-Ph1P.2B
0.48889
0.4486
1.09
0.9952
Ph24P.2B-Ph1P.2B
0.48889
0.4486
1.09
0.9952
Ph24S.1B-Ph1P.2B
-1.08889
0.4486
-2.427
0.3883
Ph24S.2B-Ph1P.2B
-0.42222
0.4486
-0.941
0.9987
Soil.1B-Ph1P.2B
0.93333
0.4486
2.081
0.6368
Soil.2B-Ph1P.2B
1.06667
0.4486
2.378
0.4217
S ool.1B-Ph1P.2B
1.02222
0.4486
2.279
0.4923
S ool.2B-Ph1P.2B
-0.15556
0.4486
-0.347
1
Ph1S.2B-Ph1S.1B
0.22222
0.4486
0.495
1
Ph24P.1B-Ph1S.1B
1.11111
0.4486
2.477
0.355
Ph24P.2B-Ph1S.1B
1.11111
0.4486
2.477
0.3546
Ph24S.1B-Ph1S.1B
-0.46667
0.4486
-1.04
0.9968
Ph24S.2B-Ph1S.1B
0.2
0.4486
0.446
1
Soil.1B-Ph1S.1B
1.55556
0.4486
3.468
0.0258
*
Soil.2B-Ph1S.1B
1.68889
0.4486
3.765
<0.01
**
S ool.1B-Ph1S.1B
1.64444
0.4486
3.666
0.0134
*
S ool.2B-Ph1S.1B
0.46667
0.4486
1.04
0.9968
Ph24P.1B-Ph1S.2B
0.88889
0.4486
1.981
0.7056
Ph24P.2B-Ph1S.2B
0.88889
0.4486
1.981
0.707
Ph24S.1B-Ph1S.2B
-0.68889
0.4486
-1.536
0.9309
Ph24S.2B-Ph1S.2B
-0.02222
0.4486
-0.05
1
Soil.1B-Ph1S.2B
1.33333
0.4486
2.972
0.1163
Soil.2B-Ph1S.2B
1.46667
0.4486
3.269
0.0499
*
S ool.1B-Ph1S.2B
1.42222
0.4486
3.17
0.0671
.
S ool.2B-Ph1S.2B
0.24444
0.4486
0.545
1
Ph24P.2B-
Ph24P.1B
0
0.4486
0
1
Ph24S.1B-
Ph24P.1B
-1.57778
0.4486
-3.517
0.0221
*
Ph24S.2B-
Ph24P.1B
-0.91111
0.4486
-2.031
0.6727
Soil.1B-Ph24P.1B
0.44444
0.4486
0.991
0.9979
Soil.2B-Ph24P.1B
0.57778
0.4486
1.288
0.9808
S ool.1B-Ph24P.1B
0.53333
0.4486
1.189
0.9899
S ool.2B-Ph24P.1B
-0.64444
0.4486
-1.437
0.9563
Ph24S.1B-
Ph24P.2B
-1.57778
0.4486
-3.517
0.0223
*
Ph24S.2B-
Ph24P.2B
-0.91111
0.4486
-2.031
0.6724
Soil.1B-Ph24P.2B
0.44444
0.4486
0.991
0.9979
Soil.2B-Ph24P.2B
0.57778
0.4486
1.288
0.9807
S ool.1B-Ph24P.2B
0.53333
0.4486
1.189
0.9899
S ool.2B-Ph24P.2B
-0.64444
0.4486
-1.437
0.9563
Ph24S.2B-
Ph24S.1B
0.66667
0.4486
1.486
0.9446
Soil.1B-Ph24S.1B
2.02222
0.4486
4.508
<0.01
***
Soil.2B-Ph24S.1B
2.15556
0.4486
4.805
<0.01
***
S ool.1B-Ph24S.1B
2.11111
0.4486
4.706
<0.01
***
S ool.2B-Ph24S.1B
0.93333
0.4486
2.081
0.6366
Soil.1B-Ph24S.2B
1.35556
0.4486
3.022
0.1025
Soil.2B-Ph24S.2B
1.48889
0.4486
3.319
0.0426
*
S ool.1B-Ph24S.2B
1.44444
0.4486
3.22
0.0578
.
S ool.2B-Ph24S.2B
0.26667
0.4486
0.594
1
Soil.2B-Soil.1B
0.13333
0.4486
0.297
1
S ool.1B-Soil.1B
0.08889
0.4486
0.198
1
S ool.2B-Soil.1B
-1.08889
0.4486
-2.427
0.3871
S ool.1B-Soil.2B
-0.04444
0.4486
-0.099
1
S ool.2B-Soil.2B
-1.22222
0.4486
-2.725
0.2141
S ool.2B-S ool.1B
-1.17778
0.4486
-2.625
0.265
Table S6. Summa y o a ge zOTU ichness cap u ed by each ex ac ion p o ocol o he
C us 16S m DNA da ase .
Ex ac ion
p o ocol
Mean
zOTU
ichness
SD
Ph1P.1B
8.31
5
Ph1P.2B
6.31
5.22
Ph1S.1B
5.33
3.05
Ph1S.2B
7.6
6.06
Ph24P.1B
7.58
6.02
Ph24P.2B
7.6
5
Ph24S.1B
3.82
3.68
Ph24S.2B
5.73
2.67
Soil.1B
7.18
4.3
Soil.2B
6.62
7.15
S ool.1B
4.07
4.23
S ool.2B
4.98
3.35
Table S7. Pos hoc pai wise compa ison o di e ences in a ge zOTU ichness de i ed om
di e en p o ocols om he C us 16S m DNA da ase .
P o ocols
Es ima e
S d E o
z alue
P (>|z|)
Ph1P.2B-Ph1P.1B
-2
0.99471
-2.011
0.6869
Ph1S.1B-Ph1P.1B
-2.97778
0.99471
-2.994
0.1109
Ph1S.2B-Ph1P.1B
-0.71111
0.99471
-0.715
0.9999
Ph24P.1B-
Ph1P.1B
-0.73333
0.99471
-0.737
0.9999
Ph24P.2B-
Ph1P.1B
-0.71111
0.99471
-0.715
0.9999
Ph24S.1B-
Ph1P.1B
-4.48889
0.99471
-4.513
<0.01
***
Ph24S.2B-
Ph1P.1B
-2.57778
0.99471
-2.591
0.2845
Soil.1B-Ph1P.1B
-1.13333
0.99471
-1.139
0.9929
Soil.2B-Ph1P.1B
-1.68889
0.99471
-1.698
0.8698
S ool.1B-Ph1P.1B
-4.24444
0.99471
-4.267
<0.01
**
S ool.2B-Ph1P.1B
-3.33333
0.99471
-3.351
0.038
*
Ph1S.1B-Ph1P.2B
-0.97778
0.99471
-0.983
0.9981
Ph1S.2B-Ph1P.2B
1.28889
0.99471
1.296
0.9799
Ph24P.1B-
Ph1P.2B
1.26667
0.99471
1.273
0.9825
Ph24P.2B-
Ph1P.2B
1.28889
0.99471
1.296
0.9798
Ph24S.1B-
Ph1P.2B
-2.48889
0.99471
-2.502
0.3382
Ph24S.2B-
Ph1P.2B
-0.57778
0.99471
-0.581
1
Soil.1B-Ph1P.2B
0.86667
0.99471
0.871
0.9994
Soil.2B-Ph1P.2B
0.31111
0.99471
0.313
1
S ool.1B-Ph1P.2B
-2.24444
0.99471
-2.256
0.5074
S ool.2B-Ph1P.2B
-1.33333
0.99471
-1.34
0.9738
Ph1S.2B-Ph1S.1B
2.26667
0.99471
2.279
0.4912
Ph24P.1B-
Ph1S.1B
2.24444
0.99471
2.256
0.5081
Ph24P.2B-
Ph1S.1B
2.26667
0.99471
2.279
0.4915
Ph24S.1B-
Ph1S.1B
-1.51111
0.99471
-1.519
0.9357
Ph24S.2B-
Ph1S.1B
0.4
0.99471
0.402
1
Soil.1B-Ph1S.1B
1.84444
0.99471
1.854
0.7875
Soil.2B-Ph1S.1B
1.28889
0.99471
1.296
0.9798
S ool.1B-Ph1S.1B
-1.26667
0.99471
-1.273
0.9825
S ool.2B-Ph1S.1B
-0.35556
0.99471
-0.357
1
Ph24P.1B-
Ph1S.2B
-0.02222
0.99471
-0.022
1
Ph24P.2B-
Ph1S.2B
0
0.99471
0
1
Ph24S.1B-
Ph1S.2B
-3.77778
0.99471
-3.798
<0.01
**
Ph24S.2B-
Ph1S.2B
-1.86667
0.99471
-1.877
0.7742
Soil.1B-Ph1S.2B
-0.42222
0.99471
-0.424
1
Soil.2B-Ph1S.2B
-0.97778
0.99471
-0.983
0.9981
S ool.1B-Ph1S.2B
-3.53333
0.99471
-3.552
0.02
*
S ool.2B-Ph1S.2B
-2.62222
0.99471
-2.636
0.259
Ph24P.2B-
Ph24P.1B
0.02222
0.99471
0.022
1
Ph24S.1B-
Ph24P.1B
-3.75556
0.99471
-3.776
<0.01
**
Ph24S.2B-
Ph24P.1B
-1.84444
0.99471
-1.854
0.7875
Soil.1B-Ph24P.1B
-0.4
0.99471
-0.402
1
Soil.2B-Ph24P.1B
-0.95556
0.99471
-0.961
0.9984
S ool.1B-
Ph24P.1B
-3.51111
0.99471
-3.53
0.0215
*
S ool.2B-
Ph24P.1B
-2.6
0.99471
-2.614
0.2719
Ph24S.1B-
Ph24P.2B
-3.77778
0.99471
-3.798
<0.01
**
Ph24S.2B-
Ph24P.2B
-1.86667
0.99471
-1.877
0.7743
Soil.1B-Ph24P.2B
-0.42222
0.99471
-0.424
1
Soil.2B-Ph24P.2B
-0.97778
0.99471
-0.983
0.9981
S ool.1B-
Ph24P.2B
-3.53333
0.99471
-3.552
0.0202
*
S ool.2B-
Ph24P.2B
-2.62222
0.99471
-2.636
0.2589
Ph24S.2B-
Ph24S.1B
1.91111
0.99471
1.921
0.7458
Soil.1B-Ph24S.1B
3.35556
0.99471
3.373
0.0366
*
Soil.2B-Ph24S.1B
2.8
0.99471
2.815
0.1732
S ool.1B-
Ph24S.1B
0.24444
0.99471
0.246
1
S ool.2B-
Ph24S.1B
1.15556
0.99471
1.162
0.9917
Soil.1B-Ph24S.2B
1.44444
0.99471
1.452
0.9529
Soil.2B-Ph24S.2B
0.88889
0.99471
0.894
0.9992
S ool.1B-
Ph24S.2B
-1.66667
0.99471
-1.676
0.8797
S ool.2B-
Ph24S.2B
-0.75556
0.99471
-0.76
0.9998
Soil.2B-Soil.1B
-0.55556
0.99471
-0.559
1
S ool.1B-Soil.1B
-3.11111
0.99471
-3.128
0.0762
.
S ool.2B-Soil.1B
-2.2
0.99471
-2.212
0.5408
S ool.1B-Soil.2B
-2.55556
0.99471
-2.569
0.2972
S ool.2B-Soil.2B
-1.64444
0.99471
-1.653
0.889
S ool.2B-S ool.1B
0.91111
0.99471
0.916
0.999
Table S8. Pos hoc pai wise compa ison o signi ican di e ences in Shannon di e si y o
a ge species de i ed om di e en p o ocols om he 18S DNA da ase .
P o ocols
Es ima e
S d. E o
Z- alue
P (>|z|)
S ool.1B-Ph1S.2B
0.3891827
0.0788487
4.936
<0.01
***
S ool.2B-Ph1S.2B
0.2919968
0.0788487
3.703
0.0117
*
S ool.1B-Ph24S.1B
0.3765767
0.0788487
4.776
<0.01
***
S ool.2B-Ph24S.1B
0.2793909
0.0788487
3.543
0.0205
*
S ool.1B-Ph24S.2B
0.3221138
0.0788487
4.085
<0.01
**
Figu e S1. Compa ison o Shannon di e si y o a ge species cap u ed by he di e en
ex ac ion p o ocols in he 18S DNA da ase .
Table S9. Pos hoc pai wise compa ison o signi ican di e ences in Shannon di e si y o
a ge species de i ed om di e en p o ocols om he C us 16S m DNA da ase .
P o ocols
Es ima e
S d. E o
Z- alue
P (>|z|)
Soil.1B-Ph1P.2B
0.41254
0.122292
3.373
0.036
*
Soil.1B-Ph24P.1B
0.430272
0.122292
3.518
0.0226
*
Soil.1B-Ph24P.2B
0.51634
0.122292
4.222
<0.01
**
S ool.1B-Soil.1B
-0.515106
0.122292
-4.212
<0.01
**
S ool.2B-Soil.1B
-0.437476
0.122292
-3.577
0.0181
*
Figu e S2. Compa ison o Shannon di e si y o a ge species cap u ed by he di e en
ex ac ion p o ocols in he C us 16S m DNA da ase .
Table S10. Pos hoc pai wise compa ison o signi ican di e ences in a ge species
abundance composi ion de i ed om di e en p o ocols om he 18S DNA da ase .
Obse ed s a is ic F ee S epdown Adjus ed P-Value
Ph24S.1B s S ool.1B 119.757 0.005 **
Ph24S.1B s Soil.2B 104.095 0.010 **
S ool.1B s Soil.2B 102.376 0.010 **
S ool.1B s Soil.1B 99.982 0.010 **
Ph1P.2B s Ph24S.1B 97.004 0.010 **
Ph1P.2B s Soil.2B 92.456 0.015 *
Ph1P.2B s S ool.1B 92.327 0.015 *
S ool.2B s Soil.2B 90.866 0.015 *
Ph24S.1B s Soil.1B 89.635 0.015 *
Ph1P.1B s S ool.1B 88.172 0.015 *
Ph1P.2B s Soil.1B 87.412 0.015 *
S ool.2B s Soil.1B 86.460 0.015 *
Ph1P.1B s Soil.2B 86.076 0.015 *
Ph1P.1B s Ph24S.1B 85.243 0.015 *
Ph1S.1B s S ool.1B 85.224 0.015 *
Ph24S.2B s S ool.1B 80.202 0.020 *
Ph1P.1B s Soil.1B 77.748 0.030 *
Ph24P.1B s S ool.1B 74.187 0.035 *
Ph1P.2B s Ph24P.1B 73.204 0.035 *
Ph24P.2B s Ph24S.1B 73.140 0.035 *
Ph24P.2B s S ool.2B 72.983 0.035 *
Ph24P.2B s S ool.1B 72.282 0.035 *
Ph1P.1B s S ool.2B 70.222 0.045 *
Ph24S.2B-Ph1S.2B
0.28889
1.1778
0.245
1
Soil.1B-Ph1S.2B
5.15556
1.1778
4.377
<0.01
***
Soil.2B-Ph1S.2B
6.15556
1.1778
5.226
<0.01
***
S ool.1B-Ph1S.2B
5.93333
1.1778
5.038
<0.01
***
S ool.2B-Ph1S.2B
2.95556
1.1778
2.509
0.3331
Ph24P.2B-
Ph24P.1B
-1.8
1.1778
-1.528
0.9332
Ph24S.1B-
Ph24P.1B
-4.51111
1.1778
-3.83
<0.01
**
Ph24S.2B-
Ph24P.1B
-3.42222
1.1778
-2.906
0.1383
Soil.1B-Ph24P.1B
1.44444
1.1778
1.226
0.987
Soil.2B-Ph24P.1B
2.44444
1.1778
2.075
0.6406
S ool.1B-Ph24P.1B
2.22222
1.1778
1.887
0.7678
S ool.2B-Ph24P.1B
-0.75556
1.1778
-0.641
1
Ph24S.1B-
Ph24P.2B
-2.71111
1.1778
-2.302
0.4753
Ph24S.2B-
Ph24P.2B
-1.62222
1.1778
-1.377
0.9679
Soil.1B-Ph24P.2B
3.24444
1.1778
2.755
0.2004
Soil.2B-Ph24P.2B
4.24444
1.1778
3.604
0.0163
*
S ool.1B-Ph24P.2B
4.02222
1.1778
3.415
0.0314
*
S ool.2B-Ph24P.2B
1.04444
1.1778
0.887
0.9992
Ph24S.2B-
Ph24S.1B
1.08889
1.1778
0.925
0.9989
Soil.1B-Ph24S.1B
5.95556
1.1778
5.056
<0.01
***
Soil.2B-Ph24S.1B
6.95556
1.1778
5.906
<0.01
***
S ool.1B-Ph24S.1B
6.73333
1.1778
5.717
<0.01
***
S ool.2B-Ph24S.1B
3.75556
1.1778
3.189
0.0641
.
Soil.1B-Ph24S.2B
4.86667
1.1778
4.132
<0.01
**
Soil.2B-Ph24S.2B
5.86667
1.1778
4.981
<0.01
***
S ool.1B-Ph24S.2B
5.64444
1.1778
4.792
<0.01
***
S ool.2B-Ph24S.2B
2.66667
1.1778
2.264
0.5025
Soil.2B-Soil.1B
1
1.1778
0.849
0.9995
S ool.1B-Soil.1B
0.77778
1.1778
0.66
1
S ool.2B-Soil.1B
-2.2
1.1778
-1.868
0.779
S ool.1B-Soil.2B
-0.22222
1.1778
-0.189
1
S ool.2B-Soil.2B
-3.2
1.1778
-2.717
0.2184
S ool.2B-S ool.1B
-2.97778
1.1778
-2.528
0.3197
Table S16. Pos hoc pai wise compa ison o di e ences in Shannon di e si y de i ed om
di e en p o ocols om he aw 18S DNA da ase .
P o ocols
Es ima e
S d E o
z alue
P (>|z|)
Ph1P.2B-Ph1P.1B
0.0104359
0.0926887
0.113
1
Ph1S.1B-Ph1P.1B
0.2779648
0.0926887
2.999
0.1096
Ph1S.2B-Ph1P.1B
0.1382541
0.0926887
1.492
0.9431
Ph24P.1B-Ph1P.1B
-
0.2363032
0.0926887
-2.549
0.3091
Ph24P.2B-Ph1P.1B
-
0.2632176
0.0926887
-2.84
0.1627
Ph24S.1B-Ph1P.1B
0.0810135
0.0926887
0.874
0.9993
Ph24S.2B-Ph1P.1B
-
0.1262406
0.0926887
-1.362
0.9707
Soil.1B-Ph1P.1B
-
0.2181565
0.0926887
-2.354
0.4382
Soil.2B-Ph1P.1B
-0.21896
0.0926887
-2.362
0.4331
S ool.1B-Ph1P.1B
0.0768936
0.0926887
0.83
0.9996
S ool.2B-Ph1P.1B
-
0.0630501
0.0926887
-0.68
0.9999
Ph1S.1B-Ph1P.2B
0.2675289
0.0926887
2.886
0.1463
Ph1S.2B-Ph1P.2B
0.1278182
0.0926887
1.379
0.9676
Ph24P.1B-Ph1P.2B
-
0.2467391
0.0926887
-2.662
0.2458
Ph24P.2B-Ph1P.2B
-
0.2736535
0.0926887
-2.952
0.1235
Ph24S.1B-Ph1P.2B
0.0705776
0.0926887
0.761
0.9998
Ph24S.2B-Ph1P.2B
-
0.1366765
0.0926887
-1.475
0.9477
Soil.1B-Ph1P.2B
-
0.2285924
0.0926887
-2.466
0.3611
Soil.2B-Ph1P.2B
-
0.2293959
0.0926887
-2.475
0.3557
S ool.1B-Ph1P.2B
0.0664577
0.0926887
0.717
0.9999
S ool.2B-Ph1P.2B
-0.073486
0.0926887
-0.793
0.9997
Ph1S.2B-Ph1S.1B
-
0.1397108
0.0926887
-1.507
0.9391
Ph24P.1B-Ph1S.1B
-0.514268
0.0926887
-5.548
<0.01
***
Ph24P.2B-Ph1S.1B
-
0.5411825
0.0926887
-5.839
<0.01
***
Ph24S.1B-Ph1S.1B
-
0.1969513
0.0926887
-2.125
0.6044
Ph24S.2B-Ph1S.1B
-
0.4042054
0.0926887
-4.361
<0.01
***
Soil.1B-Ph1S.1B
-
0.4961214
0.0926887
-5.353
<0.01
***
Soil.2B-Ph1S.1B
-
0.4969248
0.0926887
-5.361
<0.01
***
S ool.1B-Ph1S.1B
-
0.2010713
0.0926887
-2.169
0.5723
S ool.2B-Ph1S.1B
-
0.3410149
0.0926887
-3.679
0.0126
*
Ph24P.1B-Ph1S.2B
-
0.3745572
0.0926887
-4.041
<0.01
**
Ph24P.2B-Ph1S.2B
-
0.4014717
0.0926887
-4.331
<0.01
**
Ph24S.1B-Ph1S.2B
-
0.0572406
0.0926887
-0.618
1
Ph24S.2B-Ph1S.2B
-
0.2644946
0.0926887
-2.854
0.1581
Soil.1B-Ph1S.2B
-
0.3564106
0.0926887
-3.845
<0.01
**
Soil.2B-Ph1S.2B
-
0.3572141
0.0926887
-3.854
<0.01
**
S ool.1B-Ph1S.2B
-
0.0613605
0.0926887
-0.662
1
S ool.2B-Ph1S.2B
-
0.2013042
0.0926887
-2.172
0.5706
Ph24P.2B-
Ph24P.1B
-
0.0269144
0.0926887
-0.29
1
Ph24S.1B-
Ph24P.1B
0.3173167
0.0926887
3.423
0.0304
*
Ph24S.2B-
Ph24P.1B
0.1100626
0.0926887
1.187
0.99
Soil.1B-Ph24P.1B
0.0181467
0.0926887
0.196
1
Soil.2B-Ph24P.1B
0.0173432
0.0926887
0.187
1
S ool.1B-Ph24P.1B
0.3131968
0.0926887
3.379
0.0352
*
S ool.2B-Ph24P.1B
0.1732531
0.0926887
1.869
0.7785
Ph24S.1B-
Ph24P.2B
0.3442311
0.0926887
3.714
0.011
*
Ph24S.2B-
Ph24P.2B
0.136977
0.0926887
1.478
0.9469
Soil.1B-Ph24P.2B
0.0450611
0.0926887
0.486
1
Soil.2B-Ph24P.2B
0.0442576
0.0926887
0.477
1
S ool.1B-Ph24P.2B
0.3401112
0.0926887
3.669
0.0128
*
S ool.2B-Ph24P.2B
0.2001675
0.0926887
2.16
0.5791
Ph24S.2B-
Ph24S.1B
-
0.2072541
0.0926887
-2.236
0.5238
Soil.1B-Ph24S.1B
-0.29917
0.0926887
-3.228
0.0571
.
Soil.2B-Ph24S.1B
-
0.2999735
0.0926887
-3.236
0.0552
.
S ool.1B-Ph24S.1B
-
0.0041199
0.0926887
-0.044
1
S ool.2B-Ph24S.1B
-
0.1440636
0.0926887
-1.554
0.9251
Soil.1B-Ph24S.2B
-
0.0919159
0.0926887
-0.992
0.9979
Soil.2B-Ph24S.2B
-
0.0927194
0.0926887
-1
0.9977
S ool.1B-Ph24S.2B
0.2031342
0.0926887
2.192
0.5552
S ool.2B-Ph24S.2B
0.0631905
0.0926887
0.682
0.9999
Soil.2B-Soil.1B
-
0.0008035
0.0926887
-0.009
1
S ool.1B-Soil.1B
0.2950501
0.0926887
3.183
0.0648
.
S ool.2B-Soil.1B
0.1551064
0.0926887
1.673
0.8805
S ool.1B-Soil.2B
0.2958536
0.0926887
3.192
0.0638
.
S ool.2B-Soil.2B
0.1559099
0.0926887
1.682
0.8766
S ool.2B-S ool.1B
-
0.1399437
0.0926887
-1.51
0.9383
Table S17. Summa y o zOTU ichness cap u ed by each ex ac ion p o ocol o he aw
C us 16S m DNA da ase .
Ex ac ion
p o ocol
Mean
zOTU
ichness
SD
Ph1P.1B
10.5
3.41
Ph1P.2B
7.71
4.66
Ph1S.1B
8.18
2.94
Ph1S.2B
8.58
3.91
Ph24P.1B
9.18
3.46
Ph24P.2B
9.98
3.15
Ph24S.1B
6.36
2.9
Ph24S.2B
8.64
2.18
Soil.1B
8.44
3.64
Soil.2B
8.56
4.7
S ool.1B
4.51
3.74
S ool.2B
7.02
3.08
Table S18. Pos hoc pai wise compa ison o di e ences in zOTU ichness de i ed om
di e en p o ocols om he aw C us 16S m DNA da ase .
P o ocols
Es ima e
S d E o
z alue
P (>|z|)
Ph1P.2B-Ph1P.1B
-2.75556
0.72663
-3.792
<0.01
**
Ph1S.1B-Ph1P.1B
-2.28889
0.72663
-3.15
0.0703
.
Ph1S.2B-Ph1P.1B
-1.88889
0.72663
-2.6
0.2793
Ph24P.1B-Ph1P.1B
-1.28889
0.72663
-1.774
0.8323
Ph24P.2B-Ph1P.1B
-0.48889
0.72663
-0.673
0.9999
Ph24S.1B-Ph1P.1B
-4.11111
0.72663
-5.658
<0.01
***
Ph24S.2B-Ph1P.1B
-1.82222
0.72663
-2.508
0.3337
Soil.1B-Ph1P.1B
-2.02222
0.72663
-2.783
0.1857
Soil.2B-Ph1P.1B
-1.91111
0.72663
-2.63
0.2627
S ool.1B-Ph1P.1B
-5.95556
0.72663
-8.196
<0.01
***
S ool.2B-Ph1P.1B
-3.44444
0.72663
-4.74
<0.01
***
Ph1S.1B-Ph1P.2B
0.46667
0.72663
0.642
1
Ph1S.2B-Ph1P.2B
0.86667
0.72663
1.193
0.9897
Ph24P.1B-Ph1P.2B
1.46667
0.72663
2.018
0.6809
Ph24P.2B-Ph1P.2B
2.26667
0.72663
3.119
0.0769
.
Ph24S.1B-Ph1P.2B
-1.35556
0.72663
-1.866
0.781
Ph24S.2B-Ph1P.2B
0.93333
0.72663
1.284
0.9813
Soil.1B-Ph1P.2B
0.73333
0.72663
1.009
0.9975
Soil.2B-Ph1P.2B
0.84444
0.72663
1.162
0.9916
S ool.1B-Ph1P.2B
-3.2
0.72663
-4.404
<0.01
***
S ool.2B-Ph1P.2B
-0.68889
0.72663
-0.948
0.9986
Ph1S.2B-Ph1S.1B
0.4
0.72663
0.55
1
Ph24P.1B-Ph1S.1B
1
0.72663
1.376
0.9681
Ph24P.2B-Ph1S.1B
1.8
0.72663
2.477
0.3544
Ph24S.1B-Ph1S.1B
-1.82222
0.72663
-2.508
0.3345
Ph24S.2B-Ph1S.1B
0.46667
0.72663
0.642
1
Soil.1B-Ph1S.1B
0.26667
0.72663
0.367
1
Soil.2B-Ph1S.1B
0.37778
0.72663
0.52
1
S ool.1B-Ph1S.1B
-3.66667
0.72663
-5.046
<0.01
***
S ool.2B-Ph1S.1B
-1.15556
0.72663
-1.59
0.913
Ph24P.1B-Ph1S.2B
0.6
0.72663
0.826
0.9996
Ph24P.2B-Ph1S.2B
1.4
0.72663
1.927
0.7428
Ph24S.1B-Ph1S.2B
-2.22222
0.72663
-3.058
0.0926
.
Ph24S.2B-Ph1S.2B
0.06667
0.72663
0.092
1
Soil.1B-Ph1S.2B
-0.13333
0.72663
-0.183
1
Soil.2B-Ph1S.2B
-0.02222
0.72663
-0.031
1
S ool.1B-Ph1S.2B
-4.06667
0.72663
-5.597
<0.01
***
S ool.2B-Ph1S.2B
-1.55556
0.72663
-2.141
0.5932
Ph24P.2B-
Ph24P.1B
0.8
0.72663
1.101
0.9948
Ph24S.1B-
Ph24P.1B
-2.82222
0.72663
-3.884
<0.01
**
Ph24S.2B-
Ph24P.1B
-0.53333
0.72663
-0.734
0.9999
Soil.1B-Ph24P.1B
-0.73333
0.72663
-1.009
0.9975
Soil.2B-Ph24P.1B
-0.62222
0.72663
-0.856
0.9995
S ool.1B-Ph24P.1B
-4.66667
0.72663
-6.422
<0.01
***
S ool.2B-Ph24P.1B
-2.15556
0.72663
-2.967
0.1184
Ph24S.1B-
Ph24P.2B
-3.62222
0.72663
-4.985
<0.01
***
Ph24S.2B-
Ph24P.2B
-1.33333
0.72663
-1.835
0.7989
Soil.1B-Ph24P.2B
-1.53333
0.72663
-2.11
0.6165
Soil.2B-Ph24P.2B
-1.42222
0.72663
-1.957
0.7231
S ool.1B-Ph24P.2B
-5.46667
0.72663
-7.523
<0.01
***
S ool.2B-Ph24P.2B
-2.95556
0.72663
-4.067
<0.01
**
Ph24S.2B-
Ph24S.1B
2.28889
0.72663
3.15
0.071
.
Soil.1B-Ph24S.1B
2.08889
0.72663
2.875
0.1493
Soil.2B-Ph24S.1B
2.2
0.72663
3.028
0.1009
S ool.1B-Ph24S.1B
-1.84444
0.72663
-2.538
0.3155
S ool.2B-Ph24S.1B
0.66667
0.72663
0.917
0.999
Soil.1B-Ph24S.2B
-0.2
0.72663
-0.275
1
Soil.2B-Ph24S.2B
-0.08889
0.72663
-0.122
1
S ool.1B-Ph24S.2B
-4.13333
0.72663
-5.688
<0.01
***
S ool.2B-Ph24S.2B
-1.62222
0.72663
-2.233
0.5256
Soil.2B-Soil.1B
0.11111
0.72663
0.153
1
S ool.1B-Soil.1B
-3.93333
0.72663
-5.413
<0.01
***
S ool.2B-Soil.1B
-1.42222
0.72663
-1.957
0.7224
S ool.1B-Soil.2B
-4.04444
0.72663
-5.566
<0.01
***
S ool.2B-Soil.2B
-1.53333
0.72663
-2.11
0.6153
S ool.2B-S ool.1B
2.51111
0.72663
3.456
0.0269
*
Table S19. Pos hoc pai wise compa ison o di e ences in Shannon di e si y de i ed om
di e en p o ocols om he aw C us 16S m DNA da ase .
P o ocols
Es ima e
S d E o
z alue
P (>|z|)
Ph1P.2B-Ph1P.1B
-
0.1513174
0.107553
-1.407
0.962
Ph1S.1B-Ph1P.1B
0.1113204
0.107553
1.035
0.997
Ph1S.2B-Ph1P.1B
0.0909475
0.107553
0.846
1
Ph24P.1B-Ph1P.1B
0.0898855
0.107553
0.836
1
Ph24P.2B-Ph1P.1B
0.010241
0.107553
0.095
1
Ph24S.1B-Ph1P.1B
0.1200061
0.107553
1.116
0.994
Ph24S.2B-Ph1P.1B
0.1108741
0.107553
1.031
0.997
Soil.1B-Ph1P.1B
0.1522184
0.107553
1.415
0.961
Soil.2B-Ph1P.1B
-0.048796
0.107553
-0.454
1
S ool.1B-Ph1P.1B
-
0.1529458
0.107553
-1.422
0.959
S ool.2B-Ph1P.1B
0.048904
0.107553
0.455
1
Ph1S.1B-Ph1P.2B
0.2626378
0.107553
2.442
0.377
Ph1S.2B-Ph1P.2B
0.242265
0.107553
2.253
0.511
Ph24P.1B-Ph1P.2B
0.241203
0.107553
2.243
0.518
Ph24P.2B-Ph1P.2B
0.1615584
0.107553
1.502
0.94
Ph24S.1B-Ph1P.2B
0.2713235
0.107553
2.523
0.326
Ph24S.2B-Ph1P.2B
0.2621916
0.107553
2.438
0.381
Soil.1B-Ph1P.2B
0.3035358
0.107553
2.822
0.17
Soil.2B-Ph1P.2B
0.1025215
0.107553
0.953
0.999
S ool.1B-Ph1P.2B
-
0.0016283
0.107553
-0.015
1
S ool.2B-Ph1P.2B
0.2002215
0.107553
1.862
0.782
Ph1S.2B-Ph1S.1B
-
0.0203729
0.107553
-0.189
1
Ph24P.1B-Ph1S.1B
-
0.0214349
0.107553
-0.199
1
Ph24P.2B-Ph1S.1B
-
0.1010794
0.107553
-0.94
0.999
Ph24S.1B-Ph1S.1B
0.0086857
0.107553
0.081
1
Ph24S.2B-Ph1S.1B
-
0.0004463
0.107553
-0.004
1
Soil.1B-Ph1S.1B
0.040898
0.107553
0.38
1
Soil.2B-Ph1S.1B
-
0.1601164
0.107553
-1.489
0.944
S ool.1B-Ph1S.1B
-
0.2642662
0.107553
-2.457
0.367
S ool.2B-Ph1S.1B
-
0.0624164
0.107553
-0.58
1
Ph24P.1B-Ph1S.2B
-0.001062
0.107553
-0.01
1
Ph24P.2B-Ph1S.2B
-
0.0807065
0.107553
-0.75
1
Ph24S.1B-Ph1S.2B
0.0290585
0.107553
0.27
1
Ph24S.2B-Ph1S.2B
0.0199266
0.107553
0.185
1
Soil.1B-Ph1S.2B
0.0612708
0.107553
0.57
1
Soil.2B-Ph1S.2B
-
0.1397435
0.107553
-1.299
0.979
S ool.1B-Ph1S.2B
-
0.2438933
0.107553
-2.268
0.5
S ool.2B-Ph1S.2B
-
0.0420435
0.107553
-0.391
1
Ph24P.2B-
Ph24P.1B
-
0.0796445
0.107553
-0.741
1
Ph24S.1B-
Ph24P.1B
0.0301206
0.107553
0.28
1
Ph24S.2B-
Ph24P.1B
0.0209886
0.107553
0.195
1
Soil.1B-Ph24P.1B
0.0623328
0.107553
0.58
1
Soil.2B-Ph24P.1B
-
0.1386815
0.107553
-1.289
0.981
S ool.1B-Ph24P.1B
-
0.2428313
0.107553
-2.258
0.508
S ool.2B-Ph24P.1B
-
0.0409815
0.107553
-0.381
1
Ph24S.1B-
Ph24P.2B
0.1097651
0.107553
1.021
0.997
Ph24S.2B-
Ph24P.2B
0.1006331
0.107553
0.936
0.999
Soil.1B-Ph24P.2B
0.1419774
0.107553
1.32
0.977
Soil.2B-Ph24P.2B
-0.059037
0.107553
-0.549
1
S ool.1B-Ph24P.2B
-
0.1631868
0.107553
-1.517
0.936
S ool.2B-Ph24P.2B
0.038663
0.107553
0.359
1
Ph24S.2B-
Ph24S.1B
-0.009132
0.107553
-0.085
1
Soil.1B-Ph24S.1B
0.0322123
0.107553
0.3
1
Soil.2B-Ph24S.1B
-0.168802
0.107553
-1.569
0.92
S ool.1B-Ph24S.1B
-
0.2729518
0.107553
-2.538
0.316
S ool.2B-Ph24S.1B
-
0.0711021
0.107553
-0.661
1
Soil.1B-Ph24S.2B
0.0413442
0.107553
0.384
1
Soil.2B-Ph24S.2B
-
0.1596701
0.107553
-1.485
0.945
S ool.1B-Ph24S.2B
-
0.2638199
0.107553
-2.453
0.37
S ool.2B-Ph24S.2B
-
0.0619701
0.107553
-0.576
1
Soil.2B-Soil.1B
-
0.2010143
0.107553
-1.869
0.779
S ool.1B-Soil.1B
-
0.3051641
0.107553
-2.837
0.164
S ool.2B-Soil.1B
-
0.1033143
0.107553
-0.961
0.998
S ool.1B-Soil.2B
-
0.1041498
0.107553
-0.968
0.998
S ool.2B-Soil.2B
0.0977
0.107553
0.908
0.999
S ool.2B-S ool.1B
0.2018498
0.107553
1.877
0.774
Taxonomic composi ion
Fo bo h 18S DNA and C us 16S m DNA he e was a signi ican e ec o ex ac ion
p o ocol on composi ion based on bo h abundance and p esence-absence (P < 0.001 in all
cases). Pai wise p o ocol compa isons may be ound below a e he igu e.
Figu e S7. Rela i e abundance o (a) unique classes de ec ed in he aw 18S DNA da ase
and (b) unique species de ec ed in he aw C us 16S m DNA da ase .
Table S20. Pos hoc pai wise compa ison o signi ican di e ences in species abundance
composi ional da a de i ed om di e en p o ocols om he aw 18S DNA da ase .
P o ocols
Obse ed
s a is ic
Adjus ed P- alue
Soil.1B s S ool.1B
1147.8
0.005
**
Soil.2B s S ool.1B
1098.2
0.005
**
Ph1P.2B s Ph24S.1B
938.4
0.005
**
Soil.1B s S ool.2B
931.9
0.005
**
Ph1P.1B s S ool.1B
930.5
0.005
**
Ph1S.1B s S ool.1B
921.4
0.005
**
Ph1P.2B s S ool.1B
914.2
0.005
**
Ph24S.1B s S ool.1B
911.9
0.005
**
Soil.2B s S ool.2B
907.5
0.005
**
Ph24P.1B s S ool.1B
893.5
0.005
**
Ph1P.1B s Ph24S.1B
847.6
0.005
**
Ph1P.2B s Ph1S.1B
835
0.005
**
Ph24S.1B s Soil.2B
828.3
0.005
**
Ph1S.1B s Soil.1B
810
0.005
**
Ph24P.2B s S ool.1B
809.9
0.005
**
Ph1S.1B s Soil.2B
802.6
0.005
**
Ph24S.1B s Soil.1B
799.3
0.005
**
Ph1P.1B s S ool.2B
781.1
0.005
**
Ph1S.2B s S ool.1B
776.6
0.005
**
Ph24S.2B s Soil.1B
774.2
0.005
**
Ph1P.1B s Ph1S.1B
771.2
0.005
**
Ph1P.2B s S ool.2B
770.4
0.005
**
Ph24S.2B s S ool.1B
769
0.005
**
Ph24S.2B s Soil.2B
766.1
0.005
**
Ph24P.1B s S ool.2B
733.8
0.005
**
Ph1S.1B s Ph24P.1B
720
0.005
**
Ph1P.2B s Ph24S.2B
718.7
0.005
**
Ph24P.1B s
Ph24S.1B
713.5
0.005
**
Ph1S.1B s Ph24P.2B
710.2
0.005
**
Ph24P.2B s
Ph24S.1B
707.4
0.005
**
Ph24P.2B s S ool.2B
688.5
0.005
**
Ph1S.1B s S ool.2B
684.9
0.005
**
Ph1S.2B s Soil.1B
682.8
0.005
**
Ph24S.1B s S ool.2B
676.4
0.005
**
Ph1S.2B s Soil.2B
660.3
0.005
**
Ph1P.1B s Ph24S.2B
654.4
0.005
**
Ph1P.2B s Ph1S.2B
647.5
0.005
**
Ph24P.1B s
Ph24S.2B
618.5
0.005
**
Ph24P.2B s
Ph24S.2B
580.3
0.005
**
Ph1S.2B s Ph24P.1B
574.9
0.005
**
Ph1S.2B s S ool.2B
572.5
0.005
**
Ph24S.2B s S ool.2B
567.7
0.005
**
Ph1P.1B s Ph1S.2B
552.4
0.005
**
Ph1P.2B s Soil.2B
537
0.005
**
Ph1S.2B s Ph24P.2B
512.4
0.005
**
Ph1P.1B s Soil.2B
502.7
0.005
**
Ph1P.2B s Soil.1B
485.9
0.005
**
Ph1P.1B s Soil.1B
473.8
0.005
**
Ph24S.1B s
Ph24S.2B
459.4
0.005
**
Ph24P.2B s Soil.1B
439.9
0.005
**
Ph24P.2B s Soil.2B
433
0.005
**
Ph1S.2B s Ph24S.1B
414.7
0.005
**
Ph1S.1B s Ph24S.1B
401.3
0.005
**
Ph24P.1B s Soil.2B
398.9
0.005
**
Ph24P.1B s Soil.1B
389.2
0.01
**
Ph1P.2B s Ph24P.1B
384.1
0.01
**
Ph1S.1B s Ph1S.2B
370
0.015
*
Ph1S.1B s Ph24S.2B
369.3
0.015
*
Ph1P.1B s Ph24P.1B
339.8
0.03
*
Ph1P.2B s Ph24P.2B
337.9
0.03
*
Ph1S.2B s Ph24S.2B
324.3
0.05
*
