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Table 1 Summary of the number of haplotypes obtained in each step of the data screening process
and those exactly matching any of the reference sequences (≥96% identity percentage).
Data screening step: 0, non-data screening; 1 , <1 copy/L replaced with 0 copy/L; 2, <1%
in frequency at each site replaced with 0%; 3_1/2, the haplotypes detected at less than half
of the proportion of the most predominant haplotype replaced by 0%; 3_1/3, the
haplotypes detected at less than half of the proportion of the most predominant haplotype
replaced by 0% (see Fig. S3)
Target species
Data
Total detected
Decrease rate
screening
no. of
from step 0
step
Odontobutis obscura
and O.hikimius
No. of reference haplotypes: 5
haplotypes
Number of
haplotypes
100% matching
(%)
with refereces
100%
Recovery rate
Matching rate
of refences
(%)
(%)
Figure of
results
step 0
102
0.0
2.9
60.0
Fig. S5a
step 1
92
9.8
3.3
60.0
Fig. S5b
step 2
54
47.1
5.6
60.0
Fig. S5c
step 3_1/2
19
81.4
15.8
60.0
Fig. 2
step 3_1/3
20
80.4
15.0
60.0
Fig. S5d
Nipponocypris temminckii
step 0
269
0.0
1.1
100.0
Fig. S6a
No. of reference haplotypes: 3
step 1
163
39.4
1.8
100.0
Fig. S6b
step 2
89
66.9
3.4
100.0
Fig. S6c
step 3_1/2
18
93.3
16.7
100.0
Fig. 3
step 3_1/3
18
93.3
16.7
100.0
Fig. S6d
Zacco platypus
step 0
747
0.0
17
2.3
51.5
Fig. S7a
No. of reference haplotypes: 33
step 1
517
30.8
17
3.3
51.5
Fig. S7b
step 2
148
80.2
15
10.1
45.5
Fig. S7c
step 3_1/2
39
94.8
12
30.8
36.4
Fig. 4
step 3_1/3
46
93.8
12
26.1
36.4
Fig. S7d
888
889
890
891
892
893
894
895
36
896
Figuire legends
897
898
899
900
901
902
903
904
Fig.1 (a) Approximately distribution ranges of each target species in Japan and (b) eDNA
sampling locations. Pink circles and blue diamonds indicates samples collected between
2017-2020 and stored in the laboratory and newly collected sample in 2021, respectively.
Photo copyright: O. obscura and O. hikimius for Mr. S. Kunumatsu ; N. temminckii, N.
sieboldii and Z. platypus for ffish.asia (https://ffish.asia, 2022.06.01 downloaded).
905
906
37
907
908
909
910
911
912
913
914
915
916
917
918
919
Fig. 2 Odontobutis obscura and O. hikimius; (a) NJ tree and distribution map based on partial
12S sequence (366 bp) obtained by eDNA analysis and (b) NJ tree based on the deposited
partial 12S sequence (690 bp) in NCBI by Mukai and Nishida (2003) and distribution
map of each group revealed using allozyme analysis by Sakai et al. (1988). Numbers at
internodes of both NJ trees represent bootstrap probability values (≥40 %) for 1,000
replicates. The colours of each group are common in both panels, NJ trees and
distribution maps. IDs in NJ trees: ‘Dhap No.’, haplotype detected by eDNA analysis;
‘FKC No.+ LC7199xx’, ID and accession No. of individuals captured and sequenced in
st. 88 and 89 (Fukuchi River); ‘AB0955xx.’, accession No. of deposited sequence in
NCBI by Mukai and Nishida (2003). Pie chart shows the ratio of detected haplotypes of
each group and the relative total number of haplotypes detected (Table S7a).
38
920
921
922
923
924
925
926
927
928
929
930
931
932
Fig. 3 Nipponocypris temminckii; (a) NJ tree and distribution map based on partial D-loop
sequence (270 bp) obtained by eDNA analysis and (b) ML and Bayesian tree and
distribution map based on partial ND2 sequence (600 bp) provided by Taniguchi et al.
(2020). Numbers at internodes of NJ tree represent bootstrap probability values (≥30 %)
for 1,000 replicates. The colours of each group are common in the panels, trees and
distribution maps. IDs in NJ trees: ‘Thap No.’, haplotype detected by eDNA analysis. Pie
chart shows the ratio of detected haplotypes of each group and the relative total number of
haplotypes detected (Table S7b).
39
933
934
935
936
937
938
939
940
941
942
943
Fig. 4 Nipponocypris sieboldii; NJ tree and distribution map (a) based on partial D-loop sequence
(270 bp) obtained by eDNA analysis and (b) based on partial cytb sequence (715 bp)
obtained by Sanger sequence. Numbers at internodes of both NJ trees represent bootstrap
probability values (≥30 %) for 1,000 replicates. The colours of each group are common in
the panels, NJ trees and distribution maps. IDs in NJ trees: ‘Shap No.’, haplotype detected
by eDNA analysis; ‘LC7185xx’, NCBI accession No. Pie chart shows the ratio of detected
haplotypes of each group and the relative total number of haplotypes detected (Table
S7c).
944
945
946
40
947
948
949
950
951
952
953
954
955
Fig. 5 Zacco platypus; NJ tree and distribution map (a) based on partial D-loop sequence (270 bp)
obtained by eDNA analysis and (b) based on partial cytb sequence (1,004 bp) provided by
Kitanishi et al. (2016). Numbers at internodes of NJ tree in (a) represent bootstrap
probability values (≥30 %) for 1,000 replicates. The colours of each group are common in
the panels, NJ trees and distribution maps. IDs in NJ trees: ‘Zhap No.’, haplotype detected
by eDNA analysis. Pie chart shows the ratio of detected haplotypes of each group and the
relative total number of haplotypes detected (Table S7d).
41
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