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第 5回 国立環境研究所琵琶湖分室セミナー
「琵琶湖固有カワニナ属の多様化の歴史」
日時：2017年 9月 20日（水）15：00 -16：00
セミナー講師：三浦 収（高知大学）
日本最大の湖である琵琶湖には、亜種や変種も含めると約 60種の固有種がいることが知られて
いる。その中で最も種数が多いのは淡水棲巻貝のカワニナ属であり、実に 15種もの固有種が報告
されている。琵琶湖以外の地域に分布する日本産カワニナ属は 3種しかいないことを考慮すると、
琵琶湖におけるカワニナ属の多様化が如何に例外的な規模であるかを理解できる。それでは、これ
らの琵琶湖のカワニナ属は、いつの時代にどのようにして多様化したのであろうか？
本発表では、化石記録と DNA解析の結果を基に、琵琶湖固有カワニナ属の進化の歴史について
議論する。
LETTER
doi:10.1002/evl3.92
Recent lake expansion triggered theadaptive radiation of freshwater snailsin the ancient Lake BiwaOsamu Miura,1,2 Misako Urabe,3 Tomohiro Nishimura,1,4 Katsuki Nakai,5 and Satoshi Chiba6
1Faculty of Agriculture and Marine Science, Kochi University, 200 Monobe, Nankoku, Kochi 783-8502, Japan2E-mail: [email protected]
3Department of Ecosystem Studies, School of Environmental Science, The University of Shiga Prefecture, 2500
Hassaka-cho, Hikone, Shiga 522-8533, Japan4Current address: Cawthron Institute, 98 Halifax Street East, Nelson 7010, New Zealand5Lake Biwa Museum, 1091 Oroshimo, Kusatsu, Shiga 525-0001, Japan6Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, Kawauchi 41, Aoba-ku,
Sendai 980-0862, Japan
Received July 3, 2018
Accepted November 9, 2018
Lake expansion that leads to the formation of new habitats has potential to drive intralacustrine diversification. The ancient
Lake Biwa in central Japan has historically experienced substantial changes in the lake size, and it provides a useful system for
evaluating the role of lake-size fluctuations in the diversification of endemic fauna. Here, we used genome-wide DNA analyses and
reconstructed the diversification history of the endemic freshwater snails belonging to the subgenus Biwamelania with respect
to the geological history of Lake Biwa. We found that two genetically distinct snail lineages independently colonized Lake Biwa
and they concurrently and rapidly radiated into 15 extant Biwamelania species. A combination of paleontological evidence and
molecular dating technique demonstrated that the radiation of Biwamelania was tightly linked to the latest enlargement of the
lake about 0.4 million years ago and suggested that increased ecological opportunity associated with the lake expansion drove the
rapid adaptive radiation. We propose that the Biwamelania snails in Lake Biwa offer a promising new system for understanding
the association between the geological history of the lake and rapid intralacustrine diversification.
KEY WORDS: ancient lakes, adaptive radiation, lake-size changes, fossils, genome DNA analysis.
Impact summaryAncient lakes often harbor highly diverse faunas with
many endemics and contributed to advance our under-
standing of intralacustrine diversification. More than
hundreds of cichlid species were rapidly evolved in the
Great East African Lakes and there is also a great di-
versity of amphipods that evolved in the Siberian Lake
Baikal. Here, we introduce a new system for further de-
veloping the model of endemic diversification in ancient
lakes. The Japanese ancient Lake Biwa harbors a suite
of endemic freshwater snails in the subgenus Biwame-
lania. Using a combination of genome DNA analyses
and paleontological evidence, we found that these Bi-
wamelania species rapidly radiated with exploiting a
variety of habitats in response to the latest expansion of
the lake about 0.4 million years ago. The clear geology
and abundant fossils in Lake Biwa can provide a frame-
work for determining the evolutionary factors that have
facilitated rapid species diversifications within ancient
lakes.
4 3C© 2018 The Authors Evolution Letters published by Wiley Periodicals, Inc. on behalf of Society for the Study of Evolution (SSE)and European Society for Evolutionary Biology (ESEB).This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the originalwork is properly cited.Evolution Letters 3-1: 43–54
O. MIURA ET AL.
Lake-size changes are one of the major drivers of extinction
and speciation in lake fauna (Sturmbauer and Meyer 1992;
Sturmbauer 1998; Kornfield and Smith 2000; Sturmbauer et al.
2001; Sturmbauer et al. 2011). A shallowing and reduction of
lake area will elevate the extinction rate because of intense com-
petition in the shrinking environments. In contrast, a deepening
and enlargement of lake will promote population expansion
and increase the speciation rate because of colonization to
new habitats, reduced competition, and population subdivisions
(Sturmbauer 1998). For example, the cichlids in the Great East
African Lakes have shown that intralacustrine radiations occur
upon lake refilling after severe drought (Owen et al. 1990; Sturm-
bauer and Meyer 1992; Sturmbauer 1998; Kornfield and Smith
2000; Sturmbauer et al. 2001; Genner et al. 2010; Sturmbauer
et al. 2011). Severe drought could have extinguished many cichlid
species while survived cichlids could have encountered several
empty niches upon the lake expansion, which are considered
to partly facilitate the rapid adaptive radiation in the African
cichlids (Sturmbauer et al. 2011).
The cichlids in the Great East African Lakes have caused a
major development in models for adaptive radiation (Albertson
et al. 1999; Sturmbauer et al. 2001; Kocher 2004; Seehausen 2006;
Sturmbauer et al. 2011). While these species flocks exhibit intrala-
custrine radiations and will certainly help in obtaining more infor-
mation on evolutionary dynamics of species diversification, the in-
tralacustrine radiations of endemic invertebrates such as mollusks
may also have a great potential in contributing to our knowledge
on species diversification (von Rintelen et al. 2004; Albrecht et al.
2006; Albrecht et al. 2008; Glaubrecht 2008; Schultheiß et al.
2009; Van Bocxlaer and Hunt 2013; Van Bocxlaer 2017). Because
mollusks are less mobile, they are likely to become geographically
isolated to a higher degree. Further, because of their calcareous
shells, mollusks have a high fossilization potential. These behav-
ioral and structural characters of mollusks enable us to effectively
access to the information on both recent and past species
histories.
The subgenus Biwamelania (Mollusca: Caenogastropoda:
Semisulcospiridae) is composed of 15 extant species and 10 fossil
species (Matsuoka 1987; Nishino and Watanabe 2000; Matsuoka
and Miura 2018) and it is the most diverse endemic group in Lake
Biwa (Watanabe and Nishino 1995; Nishino and Watanabe 2000;
Kihira et al. 2003). Lake Biwa is an ancient lake located in central
Honshu in Japan (Fig. 1A). The lake has experienced substantial
lake-size changes throughout its history (Satoguchi 2012) (see
Fig. 1B). First, the relatively shallow and small lake was formed
south of the current Lake Biwa about four million years ago, and
the lake depth and area substantially increased between 3.2 and
2.6 million years ago. After this deep and large lake period, the
lake eventually moved north and almost dried up (but remained
as swamps) until 1.8 million years ago. The lake basin moved
to the location near the current Lake Biwa and was refilled about
1.2 million years ago. The enlargement of Lake Biwa to its present
volume was estimated to have occurred approximately 0.4 million
years ago (Satoguchi 2012). These lake-size changes would have
repeatedly affected lacustrine habitat conditions and should have
influenced the distribution and diversity of the endemic snails
(Matsuoka 1987).
The fossil records have demonstrated that Biwamelania
species experienced extinction and speciation events associated
with the lake-size changes (Fig. 1C). The oldest Biwamelania
species, Semisulcospira (Biwamelania) praemultigranosa,
first appeared at Paleo-lake Tokai about 3.9 million years
ago and it colonized Lake Biwa about 3.6 million years ago
(Matsuoka, 1987, 2001); however, it became extinct during the
deepening event of the lake about 3.2 million years ago. Semisul-
cospira (Biwamelania) reticulataformis MS and Semisulcospira
(Biwamelania) nojirina MS subsequently emerged in the deep
and large lake, but they had been disappeared before or during
shallowing event of the lake about 2.6 million years ago. The
shallow swamps between 2.6 and 1.8 million years ago harbored
Semisulcospira (Biwamelania) gamoensis MS and Semisul-
cospira (Biwamelania) tagaensis MS, and these species were
replaced by six Biwamelania species when the lake was refilled
about 1.2 million years ago. Of the six species, five species
were extirpated before the latest expansion of the lake about 0.4
million years ago (Matsuoka 1987; Matsuoka and Miura 2018).
Fifteen Biwamelania species are present in the current Lake
Biwa, but most of them were not found from fossil records, except
for Semisulcospira (Biwamelania) habei (Matsuoka 1987). This
suggests that the most of extant Biwamelania species may have
rapidly radiated after the latest expansion of the lake (Nishino
and Watanabe 2000). However, this scenario should be carefully
tested because the fossil record is generally less complete at lower
taxonomic levels (Benton 1995). Although it has become possi-
ble to reconstruct the evolutionary history of a focal group on the
basis of DNA variations in the extant species, previous molecular
studies have demonstrated that the molecular phylogenies of the
genus Semisulcospira on the basis of a single or few genes did
not accurately reflect their evolutionary relationship because of
the retention of ancestral polymorphisms or introgression (Lee
et al. 2007; Miura et al. 2013; Kohler 2016). A previous study
performed using several allozyme loci also failed to resolve their
phylogenetic relationships because of insufficient variations in
these loci (Kamiya et al. 2011). Only a method that assays vari-
ations in a large number of unlinked loci is likely to minimize
the risk of inaccurate inference of phylogenetic relationships
(Albertson et al. 1999; Carstens and Knowles 2007). In this study,
we used a genome-wide DNA analyses based on a double digest
restriction site associated DNA library sequencing technique to
infer the robust phylogenetic relationship of Biwamelania and
4 4 EVOLUTION LETTERS FEBRUARY 2019
ADAPTIVE RADIATION IN THE ANCIENT LAKE BIWA
4.0 3.0 2.0 1.0 0 MYA
Shallow
Deep
A
B
C
10km(a)
(b)(c)
(d)(e)
(f)Japan
300 km
lake condition unknown
Rel
ativ
e de
pth
S. (B.) praemultigranosaS. (B.) reticulataformis MSS. (B.) nojirina MSS. (B.) gamoensis MSS. (B.) tagaensis MSS. (B.) spinuliferaS. (B.) pusilla
S. (B.) nakamurai
S. (B.) habei
S. (B.) kokubuensis
S. (B.) pseudomultigranosa
(a)
(b)(c)
(d) (e)
(f)
Figure 1. Histories of Lake Biwa and the Biwamelania snails. The geographical locations and area size of the past and current Lake Biwa
(A). The history of Lake Biwa can be divided into six stages with respect to geographical location (a–f). The relative depth of the past and
current Lake Biwa (B). The relative depth was inferred on the basis of geological information published by Kawabe (1994) and Satoguchi
(2012). Alphabets represent the stage of the lake shown in (A). Fossils of Biwamelania species and their known time ranges (C). The ages
of the fossils were referred from Matsuoka (1987) and were further corrected on the basis of a recent stratigraphic study by Satoguchi
and Nagahashi (2012).
related riverine species. We tested the hypothesis of recent radia-
tion of the Biwamelania snails associated with the latest expansion
of the lake and we further evaluated what ecological factors facil-
itated the radiation of the Biwamelania snails. Finally, we high-
lighted how this system can serve as a useful system to provide in-
sight into what processes drive adaptive radiation in ancient lakes.
MethodsSAMPLE COLLECTIONS
Fifty-four individuals of 14 endemic species in Lake Biwa were
obtained from 15 sites in Lake Biwa and its drainage (see Table S1
for details, see also Fig. S1 for their shell morphology). We used
snorkeling and SCUBA to collect the samples from deeper habi-
tats and obtained Semisulcospira (Biwamelania) multigranosa
and one specimen of Semisulcospira (Biwamelania) reticulata
from a fisherman who dredged near Okishima Island in the lake.
We could not find Semisulcospira (Biwamelania) ourense de-
spite our sampling efforts. Semisulcospira (Biwamelania) ourense
is an extremely rare species, and only a handful of individuals
were recorded after its description (Watanabe and Nishino 1995;
Kihira et al. 2003). Most of the Biwamelania species were col-
lected from their type localities, although the type localities
of Semisulcospira (Biwamelania) decipiens, Semisulcospira (Bi-
wamelania) niponica, and Semisulcospira (Biwamelania) multi-
granosa are recorded just as “Lake Biwa”. In addition to en-
demic species in Lake Biwa, 42 individuals of the riverine species,
Semisulcospira libertina, Semisulcospira reiniana, and Semisul-
cospira kurodai were collected from 29, four, and one sites, re-
spectively. We identified these species based on adult shell fea-
tures, and number and shape of embryos following the procedures
of Davis (1969) and Watanabe and Nishino (1995). Snails were
either fixed in 95% ethanol, stored at –30°C or both for molecular
analyses. We isolated genome DNA using a modified CTAB pro-
cedure described by Miura et al. (2012). Some of these specimens
were used in the former mtDNA study (Miura et al. 2013).
EVOLUTION LETTERS FEBRUARY 2019 4 5
O. MIURA ET AL.
GENOME DNA SEQUENCING AND IDENTIFICATION
OF RAD LOCI
We used 104 genome DNA samples (Table S1) for a double digest
restriction site associated DNA library (ddRAD) sequencing
technique, as described by Peterson et al. (2012) with a slight
modification. Briefly, the extracted DNA from each individual
was further purified using a nucleospin gDNA clean-up kit
(Macherey-Nagel) with addition of RNase A. Approximately
30 ng of DNA were digested using two restriction enzymes
(EcoRI and MspI). P1 and P2 adapters from Peterson et al.
(2012) were ligated to the DNA fragments of each individual.
The ligated samples were multiplexed and purified using a
nucleospin gDNA clean-up kit. An E-gel size select agarose gel
(Invitrogen, CA) was used to collect 300–350 bp DNA fragments.
We amplified the DNA fragments in eight single PCR reactions.
The PCR products were combined and cleaned using E-gel size
select agarose gel and the nucleospin gDNA clean-up kit. The
constructed DNA library was sent to Genome Quebec Innovation
Center and sequenced using Illumina Hiseq 2000 single-end
sequencing, yielding maximum read lengths of 100 bp.
Raw sequence reads were processed using pyRAD 3.0.66
(Eaton 2014). Sequences were de-multiplexed using their
sample-specific barcode without allowing any mismatches. The
restriction site and barcode were removed from each sequence.
A nucleotide base with a FASTQ quality score less than 20
was replaced with N. Sequences having more than 5% Ns were
discarded. Sequences within each sample were clustered using
VSEARCH (https://github.com/torognes/vsearch) with an 85%
similarity threshold, following the pyRAD SE ddRAD tutorial.
Within-sample clusters with fewer than 10 sequences were
excluded to ensure accurate base calls. Consensus sequences
were created based on the clusters with consideration of the error
rate and heterozygosity. Consensus sequences from all samples
were clustered using the same similarity threshold that was
applied in the within-sample clustering. The resulting across-
sample clusters were aligned with MUSCLE (Edgar 2004).
Any clusters having more than 5% shared polymorphic sites
were discarded, because a shared heterozygous site across many
samples likely represents clustering of paralogs (Hohenlohe et al.
2011). Clusters shared among fewer than 50 individuals were
excluded, and the remaining clusters were treated as ddRAD loci
(Table S1).
PHYLOGENETIC ANALYSES AND DEMOGRAPHIC
INFERENCES
The ddRAD sequences were concatenated into a single sequence
alignment by an output function in pyRAD. Phylogenetic anal-
ysis was conducted by maximum likelihood (ML) algorism,
using RAxML 8.0.20 (Stamatakis 2014) with general time re-
versible and gamma model. Node robustness was assessed using
bootstrapping and 100 replicates. Other semisulcospirid species,
Semisulcospira extensa, Parajuga sp., Juga silicula, and Juga pli-
cifera were selected as outgroups, based on Lee et al. (2007),
Strong and Kohler (2009), and Kohler (2016). As concatenation
of genome DNA sequence data can be problematic because of spu-
riously high bootstrap supports for incorrect partitions (Gadagkar
et al. 2005), we also inferred species trees based on multispecies
coalescent model. Gene tree for each locus was estimated by
RAxML with general time reversible and gamma model using the
MAGNET pipeline (http://github.com/justincbagley/MAGNET).
We then used ASTRAL III (Mirarab and Warnow 2015), which
estimates a species tree that agrees with the largest number of
quartet trees within a set of unrooted gene trees.
We estimated divergence time of the Biwamelania species
using BPP 3.3 (Yang 2015). This analysis assumes no recombina-
tion among loci, neutral clock-like evolution with JC69 mutation
model, and no migration among species. Because the Bayesian
analyses using the multispecies coalescent are computationally
expensive, we used the subset of individuals and ddRAD loci
shared among all those individuals to reduce the size of the
dataset. We used species tree topology estimated by ASTRAL
III and estimated relative divergence time (τ) and population size
(θ) at each node. The prior for τ was Gamma (2, 250) and θ is
Gamma (2, 1000). We converted τ value to actual time (t) us-
ing fossil calibration. The oldest fossil Biwamelania species, S.
(B.) praemultigranosa, is recorded from the Ueno and Iga For-
mation of the Kobiwako Group and Kameyama Formation of
the Tokai Group (3.9–3.2 million years ago) (Matsuoka, 1987,
2001). Semisulcospira (Biwamelania) praemultigranosa has an
elongated conical shell outline and a small number (two or three)
of basal cords on the body whorl, which correspond to diagnostic
characters of the subgenus Biwamelania (Matsuoka 1985). This
fossil species is the prospective earliest stem lineage of Biwame-
lania. Therefore, the age of the node representing the split of
the basal group of Biwamelania [S. (B.) habei group] from the
other Japanese Semisulcospira was set for 3.9 million years ago,
which represents the first appearance of the subgenus Biwamela-
nia (Matsuoka 2001) (see also Fig. 1). We used TRACER v. 1.6
and FIGTREE v. 1.4.2 (Drummond and Rambaut 2007) to check
for convergence and to visualize the results. We also estimated
net diversification rate of the Biwamelania and related riverine
species using BAMM 2.5.0 (Rabosky 2014), based on the tree
obtained by the BPP analysis. MCMC simulations in BAMM
were run for 100 million steps, sampling parameters every 10,000
steps, and the MCMC results were analyzed within the R package
BAMMtools (Rabosky et al. 2014).
The demographic histories of the Biwamelania species were
reconstructed using Extended Bayesian Skyline Plot (EBSP) anal-
ysis (Heled and Drummond 2008), implemented in BEAST2
(Bouckaert et al. 2014). For each Biwamelania species, we
4 6 EVOLUTION LETTERS FEBRUARY 2019
ADAPTIVE RADIATION IN THE ANCIENT LAKE BIWA
randomly selected 200 loci that are shared by all individuals within
the species. We used the same evolutionary model (JC69) as in the
above BPP analysis and used the mutation rate estimated by the
BPP analysis. The analyses were run for 1.5 billion generations,
sampled every 50,000 steps and first 10–20% of samples were dis-
carded as burnin. We used TRACER v. 1.6 to check convergence,
and up to three independent runs were combined if the runs were
not converged. We reduced the number of loci to 100 for Semisul-
cospira (Biwamelania) nakasekoae and Semisulcospira (Biwame-
lania) takeshimensis because their parameters did not converge in
a reasonable computation time. The estimated relative population
size was plotted against time for each Biwamelania species using
R v3.3 (https://www.r-project.org).
ECOLOGICAL ASSESSMENTS
To evaluate habitat usage patterns of the Biwamelania species,
we classified substrate types of the sampling locations into the
six categories: mud, sandy mud, sand, sandy gravel, pebble, and
rock. Sandy mud is a mixture of mud and sand, and sandy gravel
is a mixture of sand and gravel. We roughly identified the sub-
strate types by eye at the field based on dominant particle size.
We also recorded the depth range at the sampling locations. We
then integrated our dataset and the habitat information of each Bi-
wamelania species reported in the study of Watanabe and Nishino
(1995), which contain about a hundred sampling points in Lake
Biwa. Note that the numbers of the sampling locations for some
species are limited as these species are distributed in restricted
locations (see Fig. S2). We used chi-square test to evaluate dif-
ference in habitat types among Biwamelania species. We used
mid-range as a representative depth at each sampling location
and evaluated the difference in habitat depth among Biwamela-
nia species. We compared the distribution of Biwamelania species
along the lake depth using a general linear model. These statistical
tests were performed using JMP V. 9.0 (SAS Institute).
ResultsWe obtained 0.4 to 15.3 million reads for each individual af-
ter de-multiplexing the Illumina Hiseq raw dataset (Table S1).
The average number of clusters greater than nine sequences was
15,271.1; the average coverage achieved per individual per loci
was 55.7 (Table S1). There were 6,285 loci with a total align-
ment length of 597,943 bp and the dataset had 31% of missing
data.
The ML tree based on the concatenated ddRAD sequences
is shown in Fig. 2A. There were five genetically well-supported
clades detected in Semisulcospira spp. All of these clades were
supported by the highest bootstrap value (100%). Semisulcospira
libertina L1 was distributed at the east side of Japan while
S. libertina L2 was distributed at the west side of Japan. Semisul-
cospira reiniana was included in S. libertina L2. Semisulcospira
libertina L3 was exclusively observed at the sites in Korea. The
clear monophyly and a high level of divergence among the clades
L1–3 suggest that these clades represent distinct biological
species. However, we do not assign scientific names to these
clades because the taxonomic revision is not the goal of this study.
There were two largely separated clades in the Lake Biwa endemic
species (Fig. 2A). One clade at basal position included Semisul-
cospira (Biwamelania) habei, Semisulcospira (Biwamelania)
dilatata, Semisulcospira (Biwamelania) rugosa, Semisulcospira
(Biwamelania) fuscata, Semisulcospira (Biwamelania) niponica,
and Semisulcospira (Biwamelania) reticulata; this clade also
included non-endemic species, Semisulcospira kurodai. The
other clade included Semisulcospira (Biwamelania) decipiens,
Semisulcospira (Biwamelania) nakasekoae, Semisulcospira (Bi-
wamelania) fluvialis, Semisulcospira (Biwamelania) arenicola,
Semisulcospira (Biwamelania) takeshimensis, Semisulcospira
(Biwamelania) shiraishiensis, Semisulcospira (Biwamelania)
multigranosa, and Semisulcospira (Biwamelania) morii. We
followed the reports by Kamiya et al. (2011) by calling the former
clade as the S. (B.) habei group and the later clade as the S. (B.) de-
cipiens group. The species tree based on multispecies coalescent
models was similar to that of the concatenated ML tree (Fig. 2B).
However, the species tree showed that the phylogenetic positions
of the species within the S. (B.) decipiens groups were often un-
certain. The ddRAD loci shared among all selected individuals (in
total of 403 loci) were used for the divergence time estimates. The
estimated divergence times for major clades and the Lake Biwa
endemics are shown in Fig. 3A. The estimated mutation rate of Bi-
wamelania was 1.06 ×10−9 substitution per year, which is compa-
rable to the mutation rate in other organisms in diverse taxonomic
group (Lynch 2010). The BAMM analysis demonstrated that the
net diversification rate was increased after the latest enlargement
of Lake Biwa (Fig. 3B). The EBSP analyses further exhibited that
the most of Biwamelania species have experienced population
expansions during and after the latest enlargement of the lake
(Fig. 4).
The habitat usage pattern was significantly different among
Biwamelania species within each group (Chi-square test, df =25, n = 134, X2 = 82.0, P < 0.01 for the S. (B.) habei group; df =35, n = 120, X2 = 64.5, P < 0.01 for the S. (B.) decipiens group,
see Fig. 5A). There were also differences in habitat depth among
Biwamelania species within each group (GLM, F5, 128 = 5.16,
P < 0.01 for the S. (B.) habei group; F7, 112 = 2.79, P = 0.01 for
the S. (B.) decipiens group, see Fig. 5B). While the majority of
Biwamelania species inhabited at the shallow coastal habitats, S.
(B.) reticulata, S. (B.) takeshimensis, S. (B.) shiraishiensis, and
S. (B.) morii were often found at the habitat about 4 m in depth
and sometimes found at the locations deeper than 10 m.
EVOLUTION LETTERS FEBRUARY 2019 4 7
O. MIURA ET AL.
S. libertina L2
S. libertina L1
S. libertina L3
S. (B.) arenicola
S. (B.) decipiens
S. (B.) nakasekoaeS. (B.) fluvialis S. (B.) shiraishiensisS. (B.) takeshimensisS. (B.) moriiS. (B.) multigranosa
S. (B.) decipiens group
S. (B.) dilatata
S. (B.) habeiS. (B.) rugosaS. (B.) fuscata
S. (B.) niponica
S. (B.) reticulataS. (B.) kurodai
S. (B.) habei group
S. extensaParajuga sp.
Juga plicifera
S. (B.) arenicolaS. (B.) decipiensS. (B.) nakasekoaeS. (B.) fluvialis
S. (B.) shiraishiensisS. (B.) takeshimensisS. (B.) moriiS. (B.) multigranosa
S. libertina L2 S. libertina L1
S. libertina L3S. (B.) dilatataS. (B.) habeiS. (B.) rugosa
S. (B.) fuscataS. (B.) niponica
S. (B.) reticulataS. (B.) kurodai
S. extensaParajuga sp.
Juga silicula
100
100
100
100
100
95
72
58
100
100
100
84
100
100
76
100
97
47
S. (B.) decipiens group
S. (B.) habei group
A
B
Juga silicula
Juga plicifera100
81
93
100
100
100
100
100100
84
100
10058
100
90
100
100
100
100
100
100
100
100
100
100
100
98
100
100
100
100
100
100
100
100
100
0.005
***
0.20
*
Figure 2. Maximum likelihood tree based on the concatenated ddRAD loci (A) and species tree based on the multispecies coalescent
model (B). Asterisks at the terminal node in the concatenated ML tree indicate S. reiniana. Numbers near major nodes are the support
values. The scale bar represents the mean number of nucleotide substitutions per site for the ML tree, and coalescent units for the species
tree.
DiscussionMolecular phylogeny based on the ddRAD sequencing technique
showed that there are two largely distinct groups in the subgenus
Biwamelania (Fig. 2A, B). The S. (B.) habei group is basal and
the oldest clade of Semisulcospira in Japan. The other clade, S.
(B.) decipiens group, is a sister group of clade L2 of the common
riverine species, S. libertina, which is a polyphyletic species
composed of geographically and genetically well-isolated clades
4 8 EVOLUTION LETTERS FEBRUARY 2019
ADAPTIVE RADIATION IN THE ANCIENT LAKE BIWA
S. libertina L1
S. (B.) fluvialisS. (B.) nakasekoae
S. (B.) arenicola
S. (B.) moriiS. (B.) decipiens
S. (B.) shiraishiensis
S. (B.) takeshimensisS. (B.) multigranosa
S. libertina L2
S. libertina L3
S. (B.) habeiS. (B.) dilatata
S. (B.) rugosa
S. (B.) niponicaS. (B.) fuscata
S. (B.) reticulataS. kurodai
4.0 3.0 2.0 1.0 0 MYA
Lake expansion
Shallow
Deep
lake condition unknown
Rel
ativ
e de
pth
(a)
(b)(c)
(d) (e)
(f)
10km(a)
(b)(c)
(d)(e)
(f)
4 3 2 1 0
net d
iver
sific
atio
n ra
te
-2
-1
0
1
2
3
4
MYA
Lake expansion
A
B
Figure 3. Phylogenetic evidence for the recent radiation of the subgenus Biwamelania (A). Divergence times were estimated for the
Biwamelania species and related riverine species, under the multispecies coalescent model. Horizontal bars represent the upper and
lower interval bounds for 95% of the highest posterior densities (HPDs). The geographical locations and relative depth of the past and
current Lake Biwa are also shown (see details in Fig. 1). Plot of net diversification rate through time based on BAMM analysis (B). Shaded
areas denote 90% Bayesian credibility intervals.
(L1–L3). These two endemic species groups and three riverine
clades should be taxonomically revised in future study.
Molecular phylogenies provide a framework for studying
endemic radiations. Our tree rejected the hypothesis that the
Biwamelania species flock originated from a single colonization
event (Fig. 2A, B). The tree topology is most plausibly interpreted
by assuming two independent colonizations of Biwamelania in
Lake Biwa. The first colonization by the S. (B.) habei group
occurred at the initial stage of Lake Biwa, and the second colo-
nization by the S. (B.) decipiens group occurred about 1.90 million
years ago (1.28–2.49 million years ago: 95% HPD; Fig. 3A) when
Lake Biwa was a group of shallow swamps (Satoguchi 2012).
Multiple colonizations have often been reported in other endemic
radiations in ancient lakes. For example, the cichlids in Lake
Malawi were colonized in the lake at least two times (Joyce et al.
2011). Further, Tylomelania snails in ancient lakes in Sulawesi
colonized four times from surrounding rivers (von Rintelen et al.
2004). These colonization events often facilitated diversifications
of lineages in ancient lakes, while not all colonizations resulted
in diversification scenario (e.g., Peart et al. 2014). Matsuoka and
Miura (2018) carefully inspected the adult and embryonic shells
of five fossil species from the Pleistocene Katata Formation
(1.2–0.4 million years ago) and found that S. (B.) nakamurai,
S. (B.) pseudomultigranosa, S. (B.) kokubuensis, and S. (B.)
pusilla have shell characters similar to the extant species in the
S. (B.) decipiens group while the adult and embryonic shell
of S. (B.) spinulifera resembles to those of the extant species
in the S. (B.) habei group. This morphological observation is
consistent with the result of molecular dating, which shows that
two groups have coexisted in the lake since 1.9 million years
ago (Fig. 3A). On the other hand, S. (B.) praemultigranosa, S.
(B.) reticulataformis MS, S. (B.) nojirina MS, S. (B.) gamoensis
MS, and S. (B.) tagaensis MS existed in the lake before the
colonization of the S. (B.) decipiens group (Fig. 1C), suggesting
EVOLUTION LETTERS FEBRUARY 2019 4 9
O. MIURA ET AL.
Time (MYA)00.20.40.6
Rel
ativ
e po
pula
tion
size
S. (B.) habei groupS. (B.) decipiens group
S. (B.) dilatata
0
1
2
3
4
S. (B.) arenicola
0
1
2
3
4
S. (B.) reticulata
0
5
10
15S. (B.) rugosa
0
2
4
6
8
10
S. (B.) decipiens
0
1
2
3
4
S. (B.) niponica
0
1
2
3
4
S. (B.) kurodai
0
0.5
1
1.5
2.5
2
S. (B.) nakasekoae
0
2
4
1
3
5S. (B.) fluvialis
0
1
2
3
4
S. (B.) fuscata
0
1
2
3
4
0.81.0
S. (B.) morii
0
1
2
3
4
S. (B.) habei
0
2
4
1
3
5
00.20.40.60.8
S. (B.) multigranosa
0
1
2
3
1.000.20.40.60.8
S. (B.) shiraishiensis
0
0.2
0.4
0.6
0.8
1.000.20.40.6
S. (B.) takeshimensis
0
2
4
6
0.81.0
Figure 4. The results of Extended Bayesian Skyline Plots for the Biwamelania snails. The dotted line represents the median value for the
relative population size, and the grey area indicates the 95% upper and lower credible limits. The bars at 0.4 million years ago indicate
the time for the latest expansion of Lake Biwa.
these five old fossil species are the ancestral species in the S. (B.)
habei group. Detailed morphological examination of these fossil
species will provide an opportunity to evaluate the validity of this
hypothesis.
The fossil records and the geology of the lake suggested that
lake-size change is a major factor for extinction and speciation
(Fig. 1C). The latest extinction event in Lake Biwa occurred about
0.4 million years ago, when the lake basin was substantially en-
larged and deepened as a result of a fault-block movement called
Rokko Movements (Matsuoka 1987). Six Biwamelania species
were present in the fossil record before this extinction event
(Matsuoka 1987; Matsuoka and Miura 2018). However, all but
one species were extirpated, and S. (B.) habei is the only species
that appears both in the past and current lake (Fig. 1C). Therefore,
the fossil evidence suggests that more than 10 species of extant
Biwamelania snails rapidly radiated following the latest expan-
sion of the lake (Nishino and Watanabe 2000). If this fossil-based
inference is correct, we should expect to find a shallow, bushy
phylogeny among Biwamelania species. Consistent with this
expectation, we found that the phylogenetic relationships within
the S. (B.) habei and S. (S.) decipiens groups were characterized
by short branches (Fig. 2). The molecular dating analysis demon-
strated that the divergence event of the S. (B.) habei group began
about 0.35 million years ago and the S. (S.) decipiens group began
to diverge about 0.17 million years ago (Fig. 3A), demonstrating
that the radiation events in Biwamelania occurred following the
latest increase in lake size. The net diversification rate also in-
creased after the enlargement of the lake (Fig. 3B), further sup-
porting the recent radiation of the Biwamelania snails.
The demographic reconstruction of the Biwamelania species
demonstrated that the population of the most of Biwamelania snail
expanded after the latest enlargement of the lake about 0.4 million
years ago (Fig. 4). Similar patterns were reported in several fish
populations in Lake Biwa. The demographic inferences based on
mtDNA variations showed that 22 fish species or clades in Lake
Biwa expanded their populations after the latest enlargement of
the lake (Tabata et al. 2016). These demographic patterns sug-
gest that the latest enlargement of the lake provided new stable
5 0 EVOLUTION LETTERS FEBRUARY 2019
ADAPTIVE RADIATION IN THE ANCIENT LAKE BIWA
S. (B.) morii
S. (B.) multigranosa
S. (B.) shiraishiensis
S. (B.) takeshimensis
S. (B.) nakasekoae
S. (B.) fluvialis
S. (B.) arenicola
S. (B.) decipiens
S. (B.) reticulata
S. (B.) dilatata
S. (B.) rugosa
S. (B.) fuscata
S. (B.) niponica
S. (B.) habei
0 0.2 0.4 0.6 0.8 1
Mud
Sandy mud
Sand
Sandy gravel
Pebble
Rock
10
8
6
4
2
0
Dep
th (m
)
S. ( B
.) m
orii
S. ( B
.) m
ultig
rano
sa
S. ( B
.) sh
irais
hien
sis
S. ( B
.) ta
kesh
imen
sis
S. ( B
.) na
kase
koae
S. ( B
.) flu
vial
is
S. ( B
.) ar
enic
ola
S. ( B
.) de
cipi
ens
S. ( B
.) re
ticul
ata
S. ( B
.) di
lata
ta
S. ( B
.) ru
gosa
S. ( B
.) fu
scat
a
S. ( B
.) ni
poni
ca
S. ( B
.) ha
bei
Frequency
A
B
No. sites=58
No. sites=41
No. sites=4
No. sites=4
No. sites=4
No. sites=23
No. sites=8
No. sites=3
No. sites=1
No. sites=3
No. sites=2
No. sites=18
No. sites=2
S. (
B.)
deci
pien
s gr
oup
S. (
B.)
habe
i gro
up
S. (B.) decipiens groupS. (B.) habei group
No. sites=83
Figure 5. Ecological divergence in the Biwamelania snails. (A) The habitat types occupied by the Biwamelania snails. (B) The average
habitat depth of the Biwamelania snails. Error bars indicate ± S.E.
habitats for faunas in Lake Biwa and resulted in the concurrent
demographic increases across the diverse faunas.
New ecological niches should become available with the
expansion of Lake Biwa. Adaptive radiation can take place when
founders enter a new environment with a number of discrete
ecological niches (Gavrilets and Losos 2009), and thus, the
exploitation and specialization to new habitats is likely to be an
important process in rapid diversification of the Biwamelania
snails. Consistent with this idea, the Biwamelania species radiated
from two ancestral species after the latest lake expansion now
use a variety of habitats in Lake Biwa. In the S. (B.) habei group,
S. (B.) niponica, and S. (B.) dilatata were often found on rocky
habitats, while S. (B.) reticulata was found on muddy-sandy
bottoms (Fig. 5A). Similarly, in the S. (B.) decipiens group, S. (B.)
EVOLUTION LETTERS FEBRUARY 2019 5 1
O. MIURA ET AL.
takeshimensis, S. (B.) shiraishiensis, and S. (B.) morii were
observed on rocky habitats, while S. (B.) arenicola and S. (B.)
multigranosa were found on muddy-sandy bottoms (Fig. 5A).
Further, some Biwamelania snails also extend their habitat
to offshore. While most of the Biwamelania species prefer
coastal habitats, S. (B.) reticulata, S. (B.) takeshimensis, S.
(B.) shiraishiensis, and S. (B.) morii are mainly distributed in
deeper habitats (Fig. 5B). These habitat usage patterns can be
independently evolved between two groups. The exploitation of
new habitats could result in the reduction of resource competition
and are suggestive of a significant role for ecological factors in
rapid diversification in Biwamelania snails.
The studies on species radiation in the Great East African
Lakes have shown that cichlids inhabit the littoral zone were
allopatrically differentiated at a small geographical scale during
the expansion of the lake, owing to extreme territoriality and lack
of dispersal opportunities during any life stage (Sturmbauer et al.
2011). Indeed, many cichlids are narrow endemics present only
in a single stretch of continuous habitat (Ribbink et al. 1983)
and exhibited low level of gene flow among local populations
(Genner et al. 2010). This pattern was also reported in a shallow
water catfish in Lake Tanganyika (Peart et al. 2018). Similar to
the case of the cichlids and catfish in African ancient lakes, the
geographic distribution of nine Biwamelania species is confined
to small regions in the lake (Watanabe and Nishino 1995, see
Fig. S2). For instance, S. (B.) fuscata, S. (B.) ourense, S. (B.)
dilatata, and S. (B.) rugosa have been observed in only a single or
few locations in the lake. Three species, S. (B.) takeshimensis, S.
(B.) shiraishiensis, and S. (B.) morii, inhabit only isolated islets
in the lake (Fig. S2). Further, two species, S. (B.) fluvialis and S.
(B) nakasekoae are distributed at only a part in Uji River, which
is a major outlet of Lake Biwa (Fig. S2). Biwamelania species
are ovoviviparous snails with no planktonic stages, and thereby,
the different coasts within the lake may have been sufficient to
isolate their populations. These distribution patterns suggest that,
in addition to ecological factors, spatial factors can also play an
important role in the radiation of the Biwamelania snails in the
lake.
Several other factors may facilitate radiation in the Biwame-
lania species. Variation in the radula morphology of S. (B.)
decipiens, S. (B.) multigranosa, and S. (B.) reticulata has been ob-
served (Watanabe 1970; Prozorova and Rasshepkina 2006). The
radula morphology often reflects the trophic system, such as food
resource usage pattern (Hawkins et al. 1989). It suggests that the
trophic specialization may also, in part, contribute to the diversifi-
cation in Biwamelania, as it observed in the radiation of Tylomela-
nia snails at ancient lakes in Sulawesi (von Rintelen et al. 2004). In
addition, the Biwamelania species exhibited a high level of kary-
otype variations among species (Burch 1968; Kobayashi 1986).
Reproduction between species with different karyotypes can yield
hybrids that are heterozygous for chromosomal rearrangements,
and these hybrids typically have reduced fertility because of error
during the first meiotic division (Forejt 1996). Therefore, these
karyotype variations may also account for the evolution of rapid
reproductive isolation among the Biwamelania species.
We demonstrated that two distinct lineages of the Biwamela-
nia snails were concurrently radiated during the latest expansion
of Lake Biwa. Our results exhibited the potential of the Biwamela-
nia snails in Lake Biwa to serve as a useful system for determining
the evolutionary factors in speciation and adaptive radiation.
Further development of evolutionary models for Biwamelania
snails with ecological, paleontological, and karyotypic perspec-
tives should provide insight into the relative importance of each
evolutionary factor on radiation in Biwamelania snails.
AUTHOR CONTRIBUTIONSO.M., U.M., and S.C. designed research, O.M., U.M., T.N., and K.N.performed the field and laboratory works, O.M. took the lead in analyzingthe dataset and writing the manuscript with major contributions from S.C.The all authors reviewed, edited, and commented on the manuscript.
ACKNOWLEDGEMENTSWe thank K. Matsuoka for sharing the information on the fossils ofSemisulcospira and valuable discussion. We also thank M. Hayashi, Y.Sasaki, N. Takahashi, K. Kimura, E. Noguchi, and A. Takami for field as-sistance in Japan and T. Kotsuka and T. Katano for collecting the samplesin Korea. D. O Foighil, T. Lee, E. Strong, K. Van Norman, E. Johannes,D. Campbell, L. Prozorova, T. Hirano, and T. Saito kindly shared the out-group samples. We are indebted to R. Kaustuv for invaluable discussionand for providing helpful comments and edits on the earlier draft. Wethank the contribution of the high throughput sequencing platform of theMcGill University and Genome Quebec Innovation Center. Computationswere partially performed on the NIG supercomputer at ROIS National In-stitute of Genetics. Support was provided by the Program to DisseminateTenure Tracking System of the MEXT. This work was facilitated by JSPSKAKENHI Grant Number 16K18606, 16K07522, and 18H01330.
CONFLICT OF INTEREST
We have no competing interests.
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Supporting InformationAdditional supporting information may be found online in the Supporting Information section at the end of the article.
Figure S1. Shells of representatives of the genus Biwamelania used in this study.Figure S2. Geographical distribution of nine Biwamelania species with limited distribution ranges.Table S1. The sampling localities of Semisulcospira spp. and the summary of RAD loci analyzed in this study.
5 4 EVOLUTION LETTERS FEBRUARY 2019
Supporting figures for
Recent lake expansion triggered the adaptive radiation of freshwater snails in the
ancient Lake Biwa
Osamu Miura, Misako Urabe, Tomohiro Nishimura, Katsuki Nakai, Satoshi Chiba
This supplement contains:
Figure S1
Figure S2
Figure S1. Shells of representatives of the genus Biwamelania used in this study.
Specimens a-u shows the species in the S. (B.) habei group. a-c: S. (B.) habei
(KUZ-Z1702, KUZ-Z1733, Z1734), d-f: S. (B.) niponica (KUZ-Z1689, Z1720, Z1726),
g-i: S. (B.) fuscata (KUZ-Z1711－1713), j-l: S. (B.) dilatata (KUZ-Z1687, Z1695,
Z1696), m-o: S. (B.) rugosa (KUZ-Z1717－1719), p-r: S. (B.) reticulata (KUZ-Z1684,
Z1705, Z1706), s-u: S. (B.) kurodai (KUZ-Z1739－1741). Specimens v-as shows the
species in the species in the S. (B.) decipiens group. v-x: S. (B.) decipiens (KUZ-Z1701,
Z1716, Z1724), y-aa: S. (B.) arenicola (KUZ-Z1685, Z1693, Z1694), ab-ad: S. (B.)
fluvialis (KUZ-Z1729－1731), ae-ag: S. (B.) nakasekoae (KUZ-Z1735－37), ah-aj: S.
(B.) takeshimensis (KUZ-Z1690－1692), ak-am: S. (B.) shiraishiensis (KUZ-Z1721－
1723), an-ap: S. (B.) multigranosa (KUZ-Z1681－ 1683), aq-as: S. (B.) morii
(KUZ-Z1698－1700). The bar at the right bottom indicates 1 cm.
Figure S2. Geographical distribution of nine Biwamelania species with limited
distribution ranges. The other five species (S. (B.) habei, S. (B.) niponica, S. (B.)
decipiens, S. (B.) arenicola, and S. (B.) multigranosa) have broad distributions in Lake
Biwa and are not shown on the map.
Table S1. The sampling localities of Semisulcospira spp. and the summary of RAD loci analyzed in this study. *; type locality of the species.
Species Locality Coordinate Group Sample ID Voucher Raw read mean depth (depth >= 10) No. RAD lociS. libertina Yurihonjo, Akita, Japan 39° 19' N, 140° 00'E L1 AKN1 KUZ-Z065 694118 36.8 3761S. libertina Kurobe, Toyama, Japan 36° 50' N, 137° 29'E L1 TYK1 KUZ-Z066 1289141 52.6 4494S. libertina Hitachi, Ibaraki, Japan 36° 40' N, 140° 42'E L1 IBJ1 KUZ-Z067 804935 38.0 3850S. libertina Fujioka, Gunma, Japan 36° 14' N, 139° 03'E L1 GMF5 KUZ-Z068 835905 42.0 3714S. libertina Matsusaka, Mie, Japan 34° 33' N, 136° 36'E L1 MEM2 KUZ-Z071 1458146 54.4 4629S. libertina Yabu, Hyogo, Japan 35° 24' N, 134° 46'E L1 HYK5 KUZ-Z073 851399 39.4 3989S. libertina Ohtsu, Shiga, Japan 35° 00' N, 135° 51' E L1 SGO2 KUZ-Z099 1311005 51.3 4533S. libertina Yonago, Tottori, Japan 35° 26' N, 133° 24'E L2 TTY2 KUZ-Z0382 887943 39.3 4072S. libertina Hongo, Hiroshima, Japan 34° 25' N, 132° 57'E L2 HSH1 KUZ-Z0383 1318494 46.3 4969S. libertina Hongo, Hiroshima, Japan 34° 25' N, 132° 57'E L2 HSH4 KUZ-Z0385 1095087 44.9 4785S. libertina Masuda, Shimane, Japan 34° 45' N, 131° 53'E L2 SHM1 KUZ-Z0391 985855 41.5 4618S. libertina Ichikikushikino, Kagoshima, Japan 31° 44' N, 130° 17'E L2 KGY6 KUZ-Z0437 1113599 44.0 4693S. libertina Wakayama, Wakayama, Japan 34° 15' N, 135° 09'E L2 WAW1 KUZ-Z074 1387979 47.4 5037S. libertina Yonago, Tottori, Japan 35° 26' N, 133° 24'E L2 TTY4 KUZ-Z075 866699 40.4 3941S. libertina Mannou, Kagawa, Japan 34° 09' N, 133° 51'E L2 KWM1 KUZ-Z076 953937 41.5 4704S. libertina Niihama, Ehime, Japan 33° 54' N, 133° 16'E L2 EHN1 KUZ-Z077 1142554 47.0 4844S. libertina Ohta, Shimane, Japan 35° 10' N, 132° 26'E L2 SHO1 KUZ-Z078 815608 38.8 4194S. libertina Masuda, Shimane, Japan 34° 45' N, 131° 53'E L2 SHM5 KUZ-Z079 1042862 45.1 4353S. libertina Misumi, Yamaguchi, Japan 34° 20' N, 131° 18'E L2 YGM1 KUZ-Z080 1312581 51.9 4862S. libertina Misumi, Yamaguchi, Japan 34° 20' N, 131° 18'E L2 YGM2 KUZ-Z081 1397172 54.8 4822S. libertina Chikushino, Fukuoka, Japan 33° 29' N, 130° 30'E L2 FOT4 KUZ-Z082 1018099 41.1 4735S. libertina Kunisaki, Oita, Japan 33° 28' N, 131° 43'E L2 OIK1 KUZ-Z083 661899 31.0 4310S. libertina Kunisaki, Oita, Japan 33° 28' N, 131° 43'E L2 OIK2 KUZ-Z084 689917 31.3 4320S. libertina Usuki, Oita, Japan 33° 06' N, 131° 47'E L2 OIU2 KUZ-Z085 759680 35.2 4316S. libertina Uto, Kumamoto, Japan 32° 38' N, 130° 32'E L2 KMU8 KUZ-Z086 1086573 44.1 4721S. libertina Izumi, Kagoshima, Japan 32° 06' N, 130° 20'E L2 KGI1 KUZ-Z087 1385276 46.8 4839S. libertina Ichikikushikino, Kagoshima, Japan 31° 44' N, 130° 17'E L2 KGY4 KUZ-Z088 1137077 44.4 4719S. libertina Kokubu, Kagoshima, Japan 31° 42' N, 130° 45'E L2 KGK1 KUZ-Z089 683257 31.8 4218S. libertina Miyazaki, Miyazaki, Japan 31° 57' N, 131° 26'E L2 MZM3 KUZ-Z090 1499413 55.2 4859S. libertina Nakatane, Kagoshima, Japan 30° 32' N, 131° 00'E L2 KTN1 KUZ-Z091 710737 38.6 3891S. libertina Motobu, Okinawa, Japan 26° 37' N, 127° 53'E L2 ONM1 KUZ-Z092 982389 46.9 4317S. reiniana Sugaura, Nagahama, Shiga, Japan 35° 27' N, 136° 08' E L2 SGC1 KUZ-Z1704 2031170 67.8 5229S. reiniana Ohtsu, Shiga, Japan 35° 00' N, 135° 51' E L2 OC1 KUZ-Z1727 1103929 48.5 4740S. reiniana Nango, Ohtsu, Shiga, Japan 34° 56' N, 135° 54' E L2 NC1 KUZ-Z1732 1307568 52.5 4879S. reiniana Uji, Kyoto, Japan 34° 54' N, 135° 47' E L2 UC2 KUZ-Z1738 799307 38.4 4263S. libertina Chuncheon, Gangwon, Korea 37° 57' N, 127° 41'E L3 S1 KUZ-Z0463 1236724 49.8 4128S. libertina Seongju, Gyeongsangbuk, Korea 35° 55' N, 128° 09'E L3 I3 KUZ-Z0480 1044115 46.0 4035S. libertina Damyang, Jeollanam, Korea 35° 22' N, 126° 58'E L3 D1 KUZ-Z0484 975420 45.9 4056S. libertina Damyang, Jeollanam, Korea 35° 18' N, 126° 56'E L3 Y3 KUZ-Z0493 1035102 44.5 4163
S . (B .) arenicola Hassaka, Hikone, Shiga, Japan 35° 15' N, 136° 12' E S . (B .) decipiens group IH1 KUZ-Z1685 1106084 54.1 4959S . (B .) arenicola Hassaka, Hikone, Shiga, Japan 35° 15' N, 136° 12' E S . (B .) decipiens group IH2 KUZ-Z1686 881375 47.9 4661S . (B .) arenicola Iso, Maibara, Shiga, Japan 35° 18' N, 136° 15' E S . (B .) decipiens group ISH1 KUZ-Z1693 1252900 56.4 5164S . (B. ) arenicola Iso, Maibara, Shiga, Japan 35° 18' N, 136° 15' E S . (B .) decipiens group ISH2 KUZ-Z1694 1651048 69.8 5295S . (B. ) decipiens Sugaura, Nagahama, Shiga, Japan 35° 27' N, 136° 08' E S . (B .) decipiens group SGT1 KUZ-Z1701 1669336 67.0 5351S . (B .) decipiens Ohura, Nagahama, Shiga, Japan 35° 29' N, 136° 07' E S . (B .) decipiens group OUT1 KUZ-Z1707 1332287 57.3 5161S . (B .) decipiens Nihonmatsu, Nagahama, Shiga, Japan 35° 27' N, 136° 06' E S . (B .) decipiens group NT1 KUZ-Z1710 1593228 66.6 5246S . (B .) decipiens Kitafunaki, Takashima, Shiga, Japan 35° 19' N, 136° 04' E S . (B .) decipiens group KT1 KUZ-Z1716 734974 42.0 4392S . (B .) decipiens Shirahige, Takashima, Shiga, Japan 35° 16' N, 136° 01' E S . (B .) decipiens group ST2 KUZ-Z1724 1576387 66.6 5301S . (B .) decipiens Ohtsu, Shiga, Japan 35° 00' N, 135° 51' E S . (B .) decipiens group OT1 KUZ-Z1725 1428699 60.5 5218S . (B .) decipiens Nango, Ohtsu, Shiga, Japan 34° 56' N, 135° 54' E S . (B .) decipiens group NGT1 KUZ-Z1728 1067739 51.9 4946S . (B .) fluvialis Nango, Ohtsu, Shiga, Japan* 34° 56' N, 135° 54' E S . (B .) decipiens group NN3 KUZ-Z1729 957018 49.3 4740S . (B .) fluvialis Nango, Ohtsu, Shiga, Japan* 34° 56' N, 135° 54' E S . (B .) decipiens group NN4 KUZ-Z1730 888513 46.7 4674S . (B .) fluvialis Nango, Ohtsu, Shiga, Japan* 34° 56' N, 135° 54' E S . (B .) decipiens group NN5 KUZ-Z1731 428059 31.2 3279S . (B .) morii Chikubu-shima, Nagahama, Shiga, Japan* 35° 25' N, 136° 08' E S . (B .) decipiens group TKM1 KUZ-Z1698 1501107 63.5 5224S . (B .) morii Chikubu-shima, Nagahama, Shiga, Japan* 35° 25' N, 136° 08' E S . (B .) decipiens group TKM2 KUZ-Z1699 1447370 62.8 5188S . (B .) morii Chikubu-shima, Nagahama, Shiga, Japan* 35° 25' N, 136° 08' E S . (B .) decipiens group TKM3 KUZ-Z1700 1456753 63.9 5188
S . (B .) multigranosa Okishima, Ohmihachiman, Shiga, Japan - S . (B .) decipiens group OSI1 KUZ-Z1681 1589492 64.9 5220S . (B .) multigranosa Okishima, Ohmihachiman, Shiga, Japan - S . (B .) decipiens group OSI2 KUZ-Z1682 1458900 60.0 5161S . (B .) multigranosa Okishima, Ohmihachiman, Shiga, Japan - S . (B .) decipiens group OSI3 KUZ-Z1683 1471388 62.0 5205S . (B .) nakasekoae Uji, Kyoto, Japan* 34° 54' N, 135° 47' E S . (B .) decipiens group UN4 KUZ-Z1735 881755 47.2 4608S . (B .) nakasekoae Uji, Kyoto, Japan* 34° 54' N, 135° 47' E S . (B .) decipiens group UN5 KUZ-Z1736 1212050 55.2 5053S . (B .) nakasekoae Uji, Kyoto, Japan* 34° 54' N, 135° 47' E S . (B .) decipiens group UN6 KUZ-Z1737 1152101 31.3 758
S . (B .) shiraishiensis Okinoshiraishi, Takashima, Shiga, Japan* 35° 17' N, 136° 07' E S . (B .) decipiens group SRS1 KUZ-Z1721 2051614 78.2 5332S . (B .) shiraishiensis Okinoshiraishi, Takashima, Shiga, Japan* 35° 17' N, 136° 07' E S . (B .) decipiens group SRS2 KUZ-Z1722 2204155 80.4 5355S . (B .) shiraishiensis Okinoshiraishi, Takashima, Shiga, Japan* 35° 17' N, 136° 07' E S . (B .) decipiens group SRS3 KUZ-Z1723 1919684 73.2 5291S . (B .) takeshimensis Takeshima, Hikone, Shiga, Japan* 35° 17' N, 136° 10' E S . (B .) decipiens group TT1 KUZ-Z1690 1887575 70.9 5334S . (B .) takeshimensis Takeshima, Hikone, Shiga, Japan* 35° 17' N, 136° 10' E S . (B .) decipiens group TT2 KUZ-Z1691 1773941 69.1 5310S . (B .) takeshimensis Takeshima, Hikone, Shiga, Japan* 35° 17' N, 136° 10' E S . (B .) decipiens group TT3 KUZ-Z1692 1676480 66.9 5261
S . (B .) dilatata Hassaka, Hikone, Shiga, Japan 35° 15' N, 136° 12' E S . (B .) habei group IF1 KUZ-Z1687 1329717 58.1 4463S . (B .) dilatata Hassaka, Hikone, Shiga, Japan 35° 15' N, 136° 12' E S . (B .) habei group IF2 KUZ-Z1688 1444708 58.9 4535S . (B .) dilatata Iso, Maibara, Shiga, Japan* 35° 18' N, 136° 15' E S . (B .) habei group ISF1 KUZ-Z1695 932927 48.0 4022S . (B .) dilatata Iso, Maibara, Shiga, Japan* 35° 18' N, 136° 15' E S . (B .) habei group ISF2 KUZ-Z1696 1102921 49.5 4285S . (B .) fuscata Nihonmatsu, Nagahama, Shiga, Japan* 35° 27' N, 136° 06' E S . (B .) habei group NK7 KUZ-Z1711 2134729 76.5 4728S . (B .) fuscata Nihonmatsu, Nagahama, Shiga, Japan* 35° 27' N, 136° 06' E S . (B .) habei group NK8 KUZ-Z1712 910536 47.0 3953S . (B .) fuscata Nihonmatsu, Nagahama, Shiga, Japan* 35° 27' N, 136° 06' E S . (B .) habei group NK10 KUZ-Z1713 1031161 49.5 4199S . (B .) habei Sugaura, Nagahama, Shiga, Japan 35° 27' N, 136° 08' E S . (B .) habei group SGH1 KUZ-Z1702 1163890 53.4 4328S . (B .) habei Ohura, Nagahama, Shiga, Japan 35° 29' N, 136° 07' E S . (B .) habei group OUH1 KUZ-Z1708 1084595 51.4 4251S . (B .) habei Ohura, Nagahama, Shiga, Japan 35° 29' N, 136° 07' E S . (B .) habei group OUH2 KUZ-Z1709 1007646 47.4 4204S . (B .) habei Uji, Kyoto, Japan* 34° 54' N, 135° 47' E S . (B .) habei group UH1 KUZ-Z1733 1500221 61.9 4559S . (B .) habei Uji, Kyoto, Japan* 34° 54' N, 135° 47' E S . (B .) habei group UH2 KUZ-Z1734 1256039 56.6 4407
S . (B .) niponica Takeshima, Hikone, Shiga, Japan 35° 17' N, 136° 10' E S . (B .) habei group TY1 KUZ-Z1689 2042060 74.9 4685S . (B .) niponica Chikubu-shima, Nagahama, Shiga, Japan 35° 25' N, 136° 08' E S . (B .) habei group TKY1 KUZ-Z1697 2175451 74.6 4732S . (B .) niponica Sugaura, Nagahama, Shiga, Japan 35° 27' N, 136° 08' E S . (B .) habei group SGY1 KUZ-Z1703 1857808 69.6 4648S . (B .) niponica Nihonmatsu, Nagahama, Shiga, Japan 35° 27' N, 136° 06' E S . (B .) habei group NY3 KUZ-Z1714 1237140 55.2 4389S . (B .) niponica Nihonmatsu, Nagahama, Shiga, Japan 35° 27' N, 136° 06' E S . (B .) habei group NY4 KUZ-Z1715 1471065 58.7 4531S . (B .) niponica Okinoshiraishi, Takashima, Shiga, Japan 35° 17' N, 136° 07' E S . (B .) habei group SRY1 KUZ-Z1720 1748076 67.6 4603S . (B .) niponica Ohtsu, Shiga, Japan 35° 00' N, 135° 51' E S . (B .) habei group OY1 KUZ-Z1726 1851607 69.1 4662S . (B .) reticulata Okishima, Ohmihachiman, Shiga, Japan* - S . (B .) habei group OSK1 KUZ-Z1684 1340699 52.4 4491S . (B .) reticulata Okude, Nagahama, Shiga, Japan 35° 28' N, 136° 07' E S . (B .) habei group OKK2 KUZ-Z1705 1833595 68.1 4643S . (B .) reticulata Okude, Nagahama, Shiga, Japan 35° 28' N, 136° 07' E S . (B .) habei group OKK3 KUZ-Z1706 1546151 62.8 4533S . (B .) rugosa Kitafunaki, Takashima, Shiga, Japan* 35° 19' N, 136° 04' E S . (B .) habei group KTJ1 KUZ-Z1717 2008001 73.2 4731S . (B .) rugosa Kitafunaki, Takashima, Shiga, Japan* 35° 19' N, 136° 04' E S . (B .) habei group KTJ2 KUZ-Z1718 1910226 71.9 4687S . (B .) rugosa Kitafunaki, Takashima, Shiga, Japan* 35° 19' N, 136° 04' E S . (B .) habei group KTJ3 KUZ-Z1719 2082244 76.0 4675
S. kurodai Sasayama, Hyogo, Japan* 35° 04' N, 135° 16' E S . (B .) habei group SK1 KUZ-Z1739 1267022 56.5 4196S. kurodai Sasayama, Hyogo, Japan* 35° 04' N, 135° 16' E S . (B .) habei group SK4 KUZ-Z1740 1684870 68.7 4393S. kurodai Sasayama, Hyogo, Japan* 35° 04' N, 135° 16' E S . (B .) habei group SK5 KUZ-Z1741 1362590 57.8 4283S. extensa South Korea, Chungcheongnam-do - outgroup EX UMMZ300253 5262837 68.8 2740
Parajuga sp. Primorsky Krai, Mikhaylovsky District, Russia 44° 06' N, 132° 01' E outgroup P11108 Pl1108 536916 26.7 894Parajuga sp. Primorsky Krai, Mikhaylovsky District, Russia 44° 06' N, 132° 01' E outgroup P11119 Pl1119 1942372 27.9 2216Juga silicula Castle Rock, Washington, USA 46° 22' N, 122° 56' E outgroup JUGA1 - 15312573 117.8 1046
Juga silicula Castle Rock, Washington, USA 46° 22' N, 122° 56' E outgroup JUGA2 - 14391618 104.5 1040Juga silicula Castle Rock, Washington, USA 46° 22' N, 122° 56' E outgroup JUGA3 - 7368338 82.4 953Juga plicifera Portland, Oregon, USA - outgroup JUGAO1 - 7786246 85.5 962Juga plicifera Portland, Oregon, USA - outgroup JUGAO2 - 10379620 113.9 958
