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Phylogeography relates the formation of species distributions,
ecological shifts, and evolutionary processes to geology (1 ). For
example, one phylogeographic study has linked multiple glacial refugia
in Europe to genetic diversity in plants (2 ). Other
phylogeographic studies have revealed the common African origins of
humans and multiple origins of domesticated livestock
(3 –5 ). In addition, environmental DNA (eDNA) analysis of
sediment samples has unveiled a multimillion year old ecosystem (e.g.,
from the Kap København Formation in Greenland) (6 ). Nevertheless,
the current phylogeographic approaches are largely limited by less
number of samples. To comprehensively understand the ecology and
evolution of a target species, hundreds or thousands of individual
tissue samples are required within a large-scale geographic scheme.
Small or unequal sample sizes leads to errors in statistical models,
while low spatial coverage of the sampling points obscures
phylogeographic patterns (7 –9 ).
Advancements in bioinformatic processing methods, especially amplicon
sequence variant (ASV) methods, can distinguish biological sequence
variants from erroneous ones. These technologies have substantially
improved eDNA-based population genetic studies while negating the need
for sampling target species in the wild (10 –12 ). Because
this technique minimizes the effort of sample collection (for instance,
fish eDNA can be detected from water scooped from water bodies), it can
innovatively reveal the fine-scale phylogeographic structure of a
species without sampling biases.
In this study, we conducted an eDNA-based phylogeographic study of a
primary freshwater fish, the Siberian stone loach Barbatula toni ,
in Hokkaido (the northernmost large island in Japan). Goto et al.
proposed that B. toni colonized rivers in Hokkaido from Sakhalin,
the northern continental Far East, via the Soya Strait, where land
bridges were periodically formed during the ice age (13 ).
Terrestrial and freshwater faunae in Hokkaido are particularly unique to
the Japanese archipelago as they are segregated by a biogeographic
boundary known as the Blakiston Line. In addition, the organisms on this
island have been subjected to numerous large-scale volcanic and glacial
events, leaving behind geological evidences, such as sedimentary layers
(14 , 15 ). B. toni is among the most common primary
freshwater fishes inhabiting the rivers in Hokkaido. Members of this
primary freshwater fish species depend exclusively on their inhabited
river system throughout their life history, often resulting in strong
congruence between their geographic placements and phylogenetic breaks
in their populations (16 ). Biologists have examined the history
of colonization, expansion, and diversification of primary freshwater
fishes in relation to geological and/or climatic changes. For example,
European freshwater fishes (genus Sabanejewia ) expanded rapidly
and became genetically homogenized during Pleistocene glaciations
(17 ) and Mesoamerican fishes expanded and diverged from their
South American origins via historical drainage connectivity (18 ).
Thus, the intraspecific phylogeographic pattern of B. toni might
provide insights into the paleogeological and climatic events that have
shaped the distribution and diversity of living organisms on the
Hokkaido island. Although eDNA analyses have captured large-scale
intraspecific genetic diversity (11 , 12 ), no studies have
addressed the novel histories of freshwater fishes over evolutionary
timescales using eDNA till date.
Applying the eDNA technique, we detected the intraspecific genetic
variations in the mitochondrial cytochrome b (cyt-b ) gene
of B. toni and assessed their phylogeographic pattern throughout
Hokkaido. After revealing the phylogeographic structures, we conducted
molecular analyses of tissue-derived DNA to evaluate the potential
scenarios of population expansion and vicariance of B. toni .