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\begin{document}

\title{Environmental DNA unveils deep phylogeographic structure of a freshwater
fish}



\author[1]{Tetsu Yatsuyanagi}%
\author[1]{Takashi Kanbe}%
\author[1]{Kazuya Fujii}%
\author[1]{Shouko Inoue}%
\author[1]{Hitoshi Araki}%
\affil[1]{Hokkaido University}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Phylogeography bears an important part in ecology and evolution.
However, current phylogeographic studies are largely constrained by
limited numbers of individual samples. Using the newly developed
environmental DNA (eDNA) assay for phylogeographic analyses, this study
provides detailed information regarding the history of Siberian stone
loach \emph{Barbatula toni}, a primary freshwater fish across the whole
range of Hokkaido, Japan. Based on an eDNA metabarcoding on 293 river
water samples, we detected eDNA from \emph{B. toni} in 189 rivers. Among
the \emph{B. toni} eDNA-positive sample set, 51 samples were chosen to
implement the \emph{Barbatula}-specific eDNA assay on a fine scale with
the goal of determining the phylogeographic pattern. As a result, two
regionally restricted, genetically distinct lineages of the species were
revealed. According to a molecular clock analysis, they have been
genetically isolated for at least 1.5 million years, suggesting their
ancient origin and colonization of Hokkaido, presumably in the glacial
periods. These results demonstrate how freshwater fishes can alter their
distributions over evolutionary timescales and how eDNA assay can deepen
our understanding of phylogeography.%
\end{abstract}%



\sloppy


Title:

Environmental DNA unveils deep phylogeographic structure of a freshwater
fish

\textbf{Authors:} Tetsu Yatsuyanagi\textsuperscript{1}, Takashi
Kanbe\textsuperscript{2}, Kazuya Fujii\textsuperscript{1,3}, Shouko
Inoue\textsuperscript{2}, Hitoshi Araki\textsuperscript{2}*

\textbf{Affiliations:}

\textsuperscript{1}Graduate School of Agriculture, Hokkaido University;
Sapporo, 060-8589, Japan.\textsuperscript{2}Research Faculty of
Agriculture, Hokkaido University; Sapporo, 060-8589,
Japan.\textsuperscript{3}Fukuda Hydrologic Center; Sapporo, 001-0024,
Japan. *Corresponding author: Hitoshi Araki Email:
\emph{arakih@agr.hokudai.ac.jp}



\textbf{Abstract:}

Phylogeography bears an important part in ecology and evolution.
However, current phylogeographic studies are largely constrained by
limited numbers of individual samples. Using the newly developed
environmental DNA (eDNA) assay for phylogeographic analyses, this study
provides detailed information regarding the history of Siberian stone
loach \emph{Barbatula toni,} a primary freshwater fish across the whole
range of Hokkaido, Japan. Based on an eDNA metabarcoding on 293 river
water samples, we detected eDNA from \emph{B. toni} in 189 rivers. Among
the \emph{B. toni} eDNA-positive sample set, 51 samples were chosen to
implement the \emph{Barbatula} -specific eDNA assay on a fine scale with
the goal of determining the phylogeographic pattern. As a result, two
regionally restricted, genetically distinct lineages of the species were
revealed. According to a molecular clock analysis, they have been
genetically isolated for at least 1.5 million years, suggesting their
ancient origin and colonization of Hokkaido, presumably in the glacial
periods. These results demonstrate how freshwater fishes can alter their
distributions over evolutionary timescales and how eDNA assay can deepen
our understanding of phylogeography.

\textbf{Short running title}

eDNA-based phylogeography of loach species in Japan

\textbf{Competing interests}

We declare that we have no competing interests.

\textbf{Keywords}

phylogeography, environmental DNA, freshwater fish, haplotypes



\textbf{Main Text:}

Phylogeography relates the formation of species distributions,
ecological shifts, and evolutionary processes to geology (\emph{1} ).
For example, one phylogeographic study has linked multiple glacial
refugia in Europe to genetic diversity in plants (\emph{2} ). Other
phylogeographic studies have revealed the common African origins of
humans and multiple origins of domesticated livestock (\emph{3}
--\emph{5} ). In addition, environmental DNA (eDNA) analysis of sediment
samples has unveiled a multimillion year old ecosystem (e.g., from the
Kap K\selectlanguage{ngerman}øbenhavn Formation in Greenland) (\emph{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 (\emph{7}
--\emph{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 (\emph{10} --\emph{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 \emph{Barbatula toni}
, in Hokkaido (the northernmost large island in Japan). Goto et al.
proposed that \emph{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 (\emph{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
(\emph{14} , \emph{15} ). \emph{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 (\emph{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 \emph{Sabanejewia} ) expanded
rapidly and became genetically homogenized during Pleistocene
glaciations (\emph{17} ) and Mesoamerican fishes expanded and diverged
from their South American origins via historical drainage connectivity
(\emph{18} ). Thus, the intraspecific phylogeographic pattern of
\emph{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 (\emph{11} ,
\emph{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 \emph{b} (cyt-\emph{b} ) gene
of \emph{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 \emph{B. toni} .

\textbf{DNA sequencing}

We developed the enhanced eDNA assay to detect intraspecific genetic
diversity of \emph{B. toni} by introducing unique molecular tagging
systems (see Supplementary Materials). Utilizing stringent ASV methods,
this assay minimizes the risk of false positives caused by index hopping
and sequencing errors (\emph{19} , \emph{20} ). To select a river that
hosts \emph{B. toni}across Hokkaido for this study, we analyzed eDNA
samples collected from 293 rivers. In this step, we prepared five
dual-indexed libraries using the fish-universal MiFish primer set
(\emph{21} ) and performed eDNA metabarcoding on a MiSeq sequencing
platform (Illumina, San Diego, CA, USA). Throughout the five MiSeq runs,
the eDNA of \emph{B. toni} was detected in 189 out of 293 river samples
(64.5\%; fig S1). Among these valid samples, we selected 51 river
samples covering the coastline of Hokkaido to implement the
\emph{Barbatula} -specific eDNA assay on a fine scale with the goal of
determining the phylogeographic pattern of\emph{B. toni} (table S1).
Using newly-developed PCR primers amplifying 266 bp of the mitochondrial
DNA cyt-\emph{b} gene of \emph{B. toni} , we prepared a dual-indexed
library comprising four replicates of the 51 river samples, four field
blanks, and eight PCR blanks. After trimming the adapters and primers
from the result of a single MiSeq sequencing run, 9,193,270 reads were
obtained. After read merging, quality filtering, denoising, and chimera
removal, 5,981,225 reads and 65 haplotypes of \emph{B. toni}were finally
obtained. Furthermore, no eDNA was detected in the field and PCR blanks.
A conservative decision was then made to accept 50 out of 65 haplotypes
based on two criteria: 1) detection in all four sequencing replicates
and 2) eDNA concentration \textgreater{} 1.0 copy per liter. The first
criterion eliminated 15 haplotypes (one haplotype with 3 out of 4
detections, two with 2 out of 4 detections, and twelve with 1 out of 4
detections) as potential erroneous haplotypes with low detection
frequencies (\emph{22} ). The eDNA concentrations of all remaining
haplotypes exceeded the second criterion (8.3 copies/L at minimum).

To reliably infer the phylogenetic relationship among the \emph{B.
toni}in Hokkaido and Sakhalin (Russia), we obtained tissue-derived DNA
sequences through Sanger sequencing (Materials and Methods-2). Using the
primer set Le-L4 and Le-H4 (\emph{23} ), we amplified 1,055 bp of the
cyt-\emph{b} DNA sequences of five individual tissue samples collected
from four river systems: Shiribeshi--Toshibetsu River, Koetoi River,
Tokoro River (Nos. 46, 7, and 17 in table S1), and a river in Sakhalin.

\textbf{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) (\emph{R}
\textsuperscript{2} = 0.04, \emph{p} = 0.18; figs. S2, S3, and S4).

The phylogenetic analysis using Sanger-sequenced cyt-\emph{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 \emph{B. toni} and a closely-related species \emph{B.
nuda}collected from the continent (northern China and Amur Basin) were
positioned at the ancestral outgroup, the \emph{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 (\emph{23} --\emph{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 (\emph{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.

\textbf{Spatial demography and population expansion}

To examine the process of regional demography and the potential
occurrence of genetic bottlenecks, we performed a mismatch distribution
analysis using the spatial expansion model in Arlequin ver. 3.5.2.2
(\emph{27} , \emph{28} ). Within each group except Group 1, the models
did not reject the expected spatial expansion scenario with multimodal
distributions of pairwise haplotypic differences (fig. S6 and table S3).
In Group 1, the mismatch distribution exhibited a dominant frequency
peak at one pairwise difference, indicating that the populations in this
group had experienced a genetic bottleneck. The \selectlanguage{greek}τ \selectlanguage{english}value, indicating the
relative time span since a population spatially expanded through a
region, was high in Group 2 (Clade-A) and also in Group 4 (Clade-B).

The southwestern part of Hokkaido, in which Group 1 is distributed with
low genetic variation, had unique geological characteristics. For
instance, the Ishikari Lowland (Fig. 3b) was submerged in seawater
during the Mindel--Riss (0.18--0.23 Mya) and Riss--W\selectlanguage{ngerman}ürm (0.07--0.13 Mya)
Interglacial periods (\emph{14} ). Moreover, results of the MiFish
metabarcoding (fig. S1) also suggested the existence of many rivers in
the west of the Ishikari Lowland where \emph{B. toni} could not be
detected using eDNA. This finding is consistent with the conventional
view that the Ishikari Lowland prevented the southward dispersal of
primary freshwater fishes from Siberia (\emph{13} ). In addition, the
Shikotsu--Toya volcanic field have many active volcanoes, where large
caldera-forming eruptions have occurred since approx. 0.10 Mya
(\emph{15} ). Repeated seawater transgressions and large eruptions in
this region have probably isolated the \emph{B. toni} populations in
southwestern Hokkaido, causing their genetic bottleneck. Several
previous studies, mainly on terrestrial animals, have reported similar
phylogeographies in this region. For example, the phylogeographic
vicariance of the brown bear \emph{Ursus arctos} , red fox \emph{Vulpes
vulpes} , and ezo salamander \emph{Hynobius retardatus} in the Ishikari
Lowland (\emph{29} --\emph{31} ) and low genetic variability among the
southwestern populations of the dark red-backed vole \emph{Myodes
rex}(\emph{32} ) support the hypothesis that geological events affected
the historical expansion and demography of terrestrial and freshwater
organisms in southwestern Hokkaido.

Several rivers in our study were found to co-host populations of
different lineages. In the Tokoro River system (No. 17 in Fig. 3b),
phylogenetically distant genetic groups have seemingly and uniquely
experienced secondary contact. The presence of genetically distinct
individuals in this river was further confirmed through Sanger
sequencing of DNA from captured individual fish samples (fig. S5).
Examination of the evolutionary mechanism of this coexistence, such as
reproductive isolation of the lineages, might reveal cryptic diversity
in \emph{B. toni} . Co-occurrences of phylogenetically distant groups
were also found in other rivers, but whether such coexistence results
from natural distribution or artificial translocations cannot be
discerned. Although \emph{B. toni} is not a target for aquaculture,
human transmission through Hokkaido is possible through mixing with
other fish resources such as salmon.

\textbf{Comparative phylogeographic inference}

The eDNA extracted from water scoops revealed the large-scale
phylogeographic structure of \emph{B. toni} . Two distinct lineages were
found in the northern and southern regions of Hokkaido, which were not
separated by obvious geographic barriers. The observed pattern differed
from that of other aquatic species reported in Hokkaido. Besides\emph{B.
toni} , two other species of primary freshwater fishes are also known to
have colonized in Hokkaido from Sakhalin across the Soya Strait: the
pond minnow \emph{Rhynchocypris percnurus sachalinensis} and
eight-barbel loach \emph{Lefua nikkonis} (\emph{13} , \emph{33} ). The
lineages of \emph{R. p. sachalinensis} are clearly differentiated on the
east and west sides of the Hidaka Mountains at the center of Hokkaido
(\emph{34} ). The Japanese crayfish \emph{Cambaroides
japonicus}(\emph{35} ) also exhibits the similar phylogeographic
pattern. The earliest-branched lineage of \emph{L. nikkonis} (belongs to
the Nemacheilidae family, like \emph{B. toni} ) is distributed along the
Sea of Okhotsk (northeastern Hokkaido), while the late-branched lineage
has wide-range distribution without either north--south or east--west
divided structure (\emph{33} ). No prior study on aquatic organisms has
demonstrated clear north--south phylogeographic vicariance in Hokkaido
without current geographic barriers.

Based on the estimated divergence times, we concluded that \emph{B.
toni} immigrated multiple times to Hokkaido via land bridges during the
ice age. The north--south phylogeographic structure of this species
possibly arose from the long time lag (over one million years) between
the independent colonization of Hokkaido by the ancestral populations of
the two clades. The phylogeography of this species is a potentially
important indicator of the historical footprints in regions of Hokkaido
and the past connection between Hokkaido and Sakhalin. Through future
studies on this species in Sakhalin and other Far East regions, we could
understand the distribution expansion of \emph{B. toni} and other
species from the continent to the islands. Our findings highlight
that\emph{B. toni} , which has been overlooked as a common species, is a
valuable source of biogeographic information on past geology and
environments.

\textbf{Conclusion}

With eDNA assay, this study revealed the fine-grained and deep
phylogeographic structure of a primary freshwater fish, \emph{B. toni} ,
over its large distribution area. Combining the eDNA assay with
conventional approaches and a molecular clock analysis, novel
phylogeographic events originating from the glacial period over one
million years ago were uncovered. Conventional surveys in
phylogeographic studies are prone to sampling biases due to the
difficulty of directly collecting many individuals from different sites.
The eDNA-based method provides an unbiased understanding of population
genetic structures over a wide geographic range. When combined with a
sufficient number of appropriate capture-based analyses, eDNA-based
analyses can considerably reduce the effort and time required for
acquiring high-resolution phylogeographic information. In fact,
approximately 300 river samples were collected for this study, covering
the entire Hokkaido region with high geographic density in 32 days. One
of the advantages of this approach is that the same eDNA samples can be
reused for detecting many other species in the same river systems,
facilitating comparative phylogeographic studies. At present, extensive
eDNA projects are being executed on national and global scales. For
example, the All Nippon eDNA Monitoring Network includes more than 800
monitoring sites across the Japanese archipelago
(\emph{https://db.anemone.bio/}), whereas the eDNA expedition project of
UNESCO World Heritage marine sites covers more than 15 countries
(\emph{https://www.unesco.org/en/edna-expeditions}). The integration of
broad-scale eDNA biomonitoring with phylogeographic analyses has the
potential to largely advance our understanding of global biodiversity.
Our findings demonstrate the huge advantages of the eDNA technique as an
innovative population genetic method that can rapidly and extensively
detect biodiversity patterns.

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\textbf{Acknowledgments:}

We thank Hokkaido Aquaculture Promotion Corporation, Michio Fukushima
and Hiroki Mizumoto for providing a part of the water samples, Fumito
Tashiro for providing a tissue sample, Ryukoku University Center for
Biodiversity Science for analyzing a part of the eDNA metabarcoding
samples, and Kazusa DNA Research Institute for DNA extraction of a part
of the eDNA samples. We also thank Akira Goto and the members in Animal
Ecology lab in Faculty of Agriculture, Hokkaido University for many
suggestions in the development of this study. We used the DNA Sequencing
Facility of Research Faculty of Agriculture, Hokkaido University.

\textbf{Funding:}

Environment Research and Technology Development Fund of the
Environmental Restoration and Conservation Agency Provided by the
Ministry of Environment of Japan grant no. JPMEERF20204004 (HA)

JSPS KAKENHI grant no. 20H03005 (HA)

JSPS KAKENHI grant no. 23H00329 (HA)

Grant-in-Aid for JSPS Fellow grant no. JP21J12688 (TY)

\textbf{Author contributions:}Conceptualization: TY, TK, HA Methodology:
TY, TK, SI Investigation: TY, KF, HA Visualization: TY Funding
acquisition: TY, HA Project administration: TY, HA Supervision: HA
Writing -- original draft: TY, TK, KF, SI, HA Writing -- review \&
editing: TY, TK, KF, SI, HA\textbf{Data availability:}

Raw sequence reads are deposited in DDBJ Sequence Read Archive (DRA).
For the MiFish sequence data, accession numbers are DRR440535--DRR440728
and DRR488036--DRR488183. For \emph{Barbatula toni} 's
cyt-\emph{b}sequence data, accession numbers are DRR490086--DRR490140.
Full details of the methods, results, supporting tables and figures are
available in Supplementary Information.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Fig. 1.} \textbf{Bayesian phylogenetic relationships among
\emph{Barbatula toni} constructed using 221 bp of the cytochrome
\emph{b} haplotypes} of eDNA. The Sakhalin specimen (top branch) and
reference sequences are shown with their accession numbers in GenBank.
The numbers on the nodes are Bayesian posterior probabilities.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Fig. 2.} \textbf{Minimum-spanning network of eDNA haplotypes
of\emph{Barbatula toni}:} The colors of the circles correspond to the
phylogenetic groups in Fig. 1. Each crossed line on the network
represents one mutation between the connected haplotypes. The black dots
represent missing haplotypes. The size of each circle is related to its
haplotype frequency.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Fig. 3.} \textbf{Maps and distributions of eDNA-based
phylogenetic groups of\emph{Barbatula toni}:} \textbf{(a)} map of the
Japanese archipelago and Sakhalin; \textbf{(b)} distributions of
eDNA-based phylogenetic groups of \emph{B. toni} in Hokkaido. The
different colors are described in the captions of Figs. 1 and 2 and
proportions in the two-color circles indicate the haplotype frequencies
in each phylogenetic group. The numbers are the IDs of the rivers from
which the groups were extracted.

\selectlanguage{english}
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