Phylogenetic relationship and geographic structure
A Bayesian phylogenetic analysis classified the eDNA haplotypes into two
distinct lineages (Fig. 1; Clade-A and Clade-B), each containing three
genetic groups. The network analysis also confirmed their phylogenetic
relationships (Fig. 2). Groups 1 and 6 revealed a large genetic
divergence between the two distinct lineages, with the dominant
haplotypes BTW-00 and BTO-00 occupying the centers of each group.
Additionally, the haplotypes in other groups were connected in a
star-like manner to Groups 1 and 6. Moreover, the distributions of these
phylogenetic groups were regionally restricted in Hokkaido (Fig. 3).
Clade-A and Clade-B were separately distributed in southern and northern
Hokkaido, respectively. While the
eDNA concentrations largely varied among rivers, the detected haplotype
richness was not significantly correlated with eDNA concentration
(copy/L) (R 2 = 0.04, p = 0.18; figs. S2,
S3, and S4).
The phylogenetic analysis using Sanger-sequenced cyt-b sequences
(868 bp) supported the phylogenetic relationship constructed using the
eDNA-based analysis (fig. S5). In the haplotype derived from
tissue-derived DNA of the Sakhalin specimen, a 221-bp sequence of the
region targeted by the eDNA assay was identical to BTO-00 of Group-6,
the major haplotype found in northern Hokkaido. While the reference
sequences of B. toni and a closely-related species B. nudacollected from the continent (northern China and Amur Basin) were
positioned at the ancestral outgroup, the B. toni haplotype
obtained from the Sakhalin specimen was included within the northern
lineage (Clade-B) in Hokkaido.
A molecular clock analysis
(2.5%–2.8%/Myr
(23 –25 )) informed that the two clades diverged in the
early Pleistocene (1.59–1.78 Mya, table S2). During the early
Pleistocene, the glacial and interglacial periods repeated in cycles and
land bridges emerged at the Soya Strait due to sea-level drops during
glaciation (26 ). Therefore, it is suggested that the ancestors of
Clade-A first colonized Hokkaido from Sakhalin during a low-sea-level
period in the early-Pleistocene glaciation. During an interglacial
period, they were long-term isolated by the opening of the Soya Strait.
In contrast, Clade-B includes the northern Hokkaido and Sakhalin
populations, which diverged circa 0.16–0.18 Mya. This estimate
indicates that the ancestors of Clade-B dispersed southward to Hokkaido
from Sakhalin via land bridges formed around that period.