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Phylogenetic Relationships and Divergence Times among Mustelids (Mammalia: Carnivora) Based on Nucleotide Sequences of the Nuclear Interphotoreceptor Retinoid Binding Protein and Mitochondrial Cytochrome b Genes

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Phylogenetic relationships among 20 species-group taxa of Mustelidae, representing Mustelinae (Mustela, Martes, Gulo), Lutrinae (Enhydra), and Melinae (Meles), were examined using nucleotide sequences of the nuclear interphotoreceptor retinoid binding protein (IRBP) and mitochondrial cytochrome b genes. Neighbor-joining and maximum-parsimony phylogenetic analyses on these genes separately and combined were conducted. While IRBP performed better than cytochrome b in recovering more-inclusive clades, cytochrome b demonstrated more resolving power in recovering less-inclusive clades. Strong support was found for a close affinity of Enhydra with Mustela to the exclusion of Martes and Gulo (causing Mustelinae to be paraphyletic); the most-basal position of Mustela vison within Mustela, followed by Mustela erminea; an association of Mustela lutreola, Mustela itatsi, Mustela sibirica, and the subgenus Putorius (including Mustela putorius and Mustela eversmanii), to the exclusion of Mustela nivalis and Mustela altaica; and a basal position of Mustela itatsi to a clade containing Mustela sibirica and Putorius. Whereas cytochrome b strongly supported Mustela lutreola as the sister species to Putorius, IRBP strongly supported its basal placement to the Mustela itatsi–Mustela sibirica–Putorius clade. The low level of sequence divergence in cytochrome b between Mustela lutreola and Putorius is therefore a result of interspecific mitochondrial introgression between these taxa, rather than a recent origin of Mustela lutreola in a close relationship to Putorius. Time estimates inferred from IRBP and cytochrome b for mustelid divergence events are mostly in agreement with the fossil record.
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2003 Zoological Society of JapanZOOLOGICAL SCIENCE
20
: 243–264 (2003)
Phylogenetic Relationships and Divergence Times among
Mustelids (Mammalia: Carnivora) Based on Nucleotide Sequences
of the Nuclear Interphotoreceptor Retinoid Binding Protein
and Mitochondrial Cytochrome
b
Genes
Jun J. Sato
1
, Tetsuji Hosoda
2
, Mieczys
´
law Wolsan
3
, Kimiyuki Tsuchiya
4
,
Masahiko Yamamoto
5
, and Hitoshi Suzuki
1
*
1
Laboratory of Ecology and Genetics, Graduate School of Environmental Earth Science, Hokkaido University,
Kita-ku, Sapporo 060-0810, Japan
2
Gobo Shoko High School, 43-1 Komatsubara, Yukawa, Gobo 644-0012, Japan
3
Institute of Paleobiology, Polish Academy of Sciences, Twarda 51/55, 00-818 Warszawa, Poland
4
Fuculty of Agriculture, Tokyo University of Agriculture, Atsugi 243-0034, Japan
5
Yokohama City Zoo, Asahi-ku, Yokohama 241-0001, Japan
ABSTRACT
—Phylogenetic relationships among 20 species-group taxa of Mustelidae, representing Mus-
telinae (
Mustela
,
Martes
,
Gulo
), Lutrinae (
Enhydra
), and Melinae (
Meles
), were examined using nucleotide
sequences of the nuclear interphotoreceptor retinoid binding protein (IRBP) and mitochondrial cytochrome
b
genes. Neighbor-joining and maximum-parsimony phylogenetic analyses on these genes separately and
combined were conducted. While IRBP performed better than cytochrome
b
in recovering more-inclusive
clades, cytochrome
b
demonstrated more resolving power in recovering less-inclusive clades. Strong sup-
port was found for a close affinity of
Enhydra
with
Mustela
to the exclusion of
Martes
and
Gulo
(causing
Mustelinae to be paraphyletic); the most-basal position of
Mustela vison
within
Mustela
, followed by
Mus-
tela erminea
; an association of
Mustela lutreola
,
Mustela itatsi
,
Mustela sibirica
,
and the subgenus
Putorius
(including
Mustela putorius
and
Mustela eversmanii
), to the exclusion of
Mustela nivalis
and
Mustela alta-
ica
; and a basal position of
Mustela itatsi
to a clade containing
Mustela sibirica
and
Putorius
. Whereas
cytochrome
b
strongly supported
Mustela lutreola
as the sister species to
Putorius
, IRBP strongly sup-
ported its basal placement to the
Mustela itatsi
-
Mustela sibirica
-
Putorius
clade. The low level of sequence
divergence in cytochrome
b
between
Mustela lutreola
and
Putorius
is therefore a result of interspecific
mitochondrial introgression between these taxa, rather than a recent origin of
Mustela lutreola
in a close
relationship to
Putorius
. Time estimates inferred from IRBP and cytochrome
b
for mustelid divergence
events are mostly in agreement with the fossil record.
Key words:
molecular phylogeny, Mustelidae, cytochrome
b
gene, nuclear IRBP gene, divergence time
INTRODUCTION
Mustelidae is the largest and most-diverse family
among carnivoran mammals (order Carnivora). There are
66 (including
Mustela itatsi
) mustelid species extant today.
They are usually classified in 25 genera and six subfamilies
(e.g., Wozencraft, 1993). These subfamilies are Mustelinae
(weasels, martens, and their allies), Lutrinae (otters), Meli-
nae (badgers), Mellivorinae (honey badger), Taxidiinae
(American badger), and Mephitinae (skunks). Mustelids are
widely distributed geographically and occur throughout Eur-
asia, Africa, and America (introduced into New Zealand).
They have adapted to very varied climatic and biotic condi-
tions and are found in habitats that range from the arctic tun-
dra to tropical rainforest and from deserts to inland
waterways and even the open sea (e.g., Macdonald, 1985;
Nowak, 1991). In many ecosystems of the Northern Hemi-
sphere, mustelids are the most-common predatory mam-
mals.
The increasing interest in mustelids and their phylogeny
is therefore not surprising. There is an extensive bibliogra-
phy related to phylogenetic relationships within this family,
including studies based on either morphological grounds
*
Corresponding author: Tel. +81-11-706-2279;
FAX. +81-11-706-2279.
E-mail: htsuzuki@ees.hokudai.ac.jp
J. J. Sato
et al
.244
(neontology: e.g., Wozencraft, 1989; Bryant
et al.
, 1993;
Wyss and Flynn, 1993; paleontology: e.g., Wolsan, 1993a,
1999; Baskin, 1998; Ginsburg and Morales, 2000) or genetic
grounds (karyology: e.g., Graphodatsky
et al
., 1976; Coutu-
rier and Dutrillaux, 1986; Obara, 1991; serum immunology:
e.g., Seal
et al
., 1970; Belyaev
et al
., 1984; Taranin
et al
.,
1991; protein electrophoresis: e.g., Hartl
et al
., 1988;
O’Brien
et al
., 1989; Dragoo
et al
., 1993; DNA-DNA hybrid-
ization: e.g., Árnason and Widegren, 1986; Lushnikova
et
al
., 1989; Wayne
et al
., 1989; amino-acid sequencing:
Hashimoto
et al
., 1993; Stanhope
et al
., 1993; nucleotide
sequencing: see the next paragraph for references), or inte-
grating morphological and genetic data (Vrana
et al
., 1994;
Dragoo and Honeycutt, 1997; Bininda-Emonds
et al
., 1999).
Despite this considerable accumulation of information, the
mustelid phylogeny remains an issue of uncertainty and
controversy. Although the fossil record is the only source of
direct evidence of past organismal history and uniquely con-
tributes to phylogeny reconstruction by dating divergence
times and providing access to denser taxon sampling than
is otherwise possible (Smith and Littlewood, 1994; Smith,
1998; and references therein), paleontological data have
largely been ignored by students of mustelid phylogeny.
Recent attention in phylogenetic relationships among
mustelids has focused on nucleotide-sequence data (Dra-
goo
et al
., 1993; Hosoda
et al
., 1993, 1997, 1999, 2000;
Masuda and Yoshida, 1994a, b; Vrana
et al
., 1994; Ledje
and Árnason, 1996a, b; Carr and Hicks, 1997; Dragoo and
Honeycutt, 1997; Koepfli and Wayne, 1998, 2001; Davison
et al
., 1999, 2000a, 2001; Demboski
et al
., 1999; Emerson
et al
., 1999; Kurose
et al
., 1999a, b, 2000, 2001; Cassens
et al
., 2000; Flynn
et al
., 2000). Most of these studies come
from analysis of variation in nucleotide sequences of mito-
chondrial genes. An additional, relatively untapped, source
of phylogenetic information is the coding sequences of sin-
gle-copy nuclear loci. Single-copy nuclear genes are an
ideal source of phylogenetic information because homolo-
gous single-copy genes from different species are in all like-
lihood orthologous—that is, derived from a single common
ancestral gene through a series of speciation events—and
therefore suitable for the cladistic examination of relation-
ships among species lineages (e.g., Stanhope
et al
., 1992,
1996).
The gene encoding interphotoreceptor retinoid binding
protein (IRBP) is a single-copy nuclear gene (Bridges
et al
.,
1986; Stanhope
et al
., 1992, 1996; Springer
et al
., 1997).
This gene is expressed in eyes of vertebrates (Bridges
et
al
., 1986), where it functions in the transfer of retinoids dur-
ing light- and dark-phase adaptation (Fong
et al
., 1990; Pep-
perberg
et al
., 1993). Variation in nucleotide sequences of
this gene has proved useful for elucidating higher-level rela-
tionships among mammals (Stanhope
et al
., 1992, 1996;
Springer
et al
., 1997, 1999, 2001; Jansa and Voss, 2000).
Recently, this gene has also been employed in studies of
lower-level mammalian relationships (Jansa and Voss,
2000; Serizawa
et al
., 2000; Suzuki
et al
., 2000).
This paper is the first report on nucleotide sequences of
IRBP in Mustelidae. Here we present results on phyloge-
netic relationships among 20 species-group taxa of this fam-
ily, inferred from nucleotide sequences of IRBP and the
mitochondrial cytochrome
b
gene, the latter obtained from
DNA databases. We also examine differences in perfor-
mance between the two genes in recovering clades at
different taxonomic levels. A new, well-documented fossil-
based time estimate for the mustelid-procyonid divergence
is used to calibrate the molecular clocks of the mustelid
IRBP and cytochrome
b
genes. By means of this calibration
we estimate dates for divergence events within Mustelidae.
MATERIALS AND METHODS
Sampling
A fragment of the first exon of IRBP, 1188 base pairs (bp) in
length (sites 337–1530 in the human reference sequence [Fong
et
al
., 1990], except for sites 1318–1323 that are absent from all mus-
telids), was sequenced from 20 individuals representing 19 species-
group taxa, five genera, and three subfamilies of Mustelidae and
one species of Procyonidae (Table 1). The complete nucleotide
sequences of cytochrome
b
(1140 bp) from 19 species-group taxa
of Mustelidae and the procyonid, as well as nucleotide sequences
of IRBP and cytochrome
b
from two species of the carnivoran sub-
order Feliformia and the order Rodentia (Table 1), were obtained
from the DDBJ/EMBL/GenBank International Nucleotide Sequence
Database.
A 3-bp fragment of IRBP (sites 1311–1313 in the human refer-
ence sequence [Fong
et al
., 1990]) was absent from all examined
representatives of
Mustela
. This fragment was therefore excluded
from phylogenetic analyses, which finally included 1185 bp of IRBP
and 1140 bp of cytochrome
b
, each for 19 species-group taxa of
Mustelidae and an outgroup species. The composite nucleotide-
sequence data consisted of a total of 2325 bp (1185 bp from IRBP
and 1140 bp from cytochrome
b
) for each of 18 species-group taxa
of Mustelidae (for which the data on the two genes were available)
and an outgroup species.
As the outgroup in all phylogenetic analyses, the procyonid
Procyon lotor
was used on the basis of the proposed sister-group
relationship between Mustelidae and Procyonidae, supported by
evidence from both genetics and morphology (Bininda-Emonds
et
al
., 1999; Flynn
et al
., 2000; and references therein).
DNA isolation, amplification, and sequencing
DNA was extracted from tissues preserved in ethanol by the
conventional phenol-chloroform method. The amplification was per-
formed via nested polymerase chain reactions (PCRs), using an
automated thermal cycler (model PJ 2000, TAKARA). Each first
PCR mix contained 10 mM Tris (pH 8.3); 50 mM KCl; 0.01% gela-
tin; 0.1% Triton X-100; 2.5 mM MgCl
2
; 0.2 mM dNTP mix; 0.05
µ
M
of each primer (1 pmol of each primer per reaction); 0.5 units of
Amplitaq DNA polymerase (ABI, Applied Biosystems); and 0.1–0.5
µ
g of template total genomic DNA in a total volume of 20
µ
l. Ther-
mal cycling parameters of the first PCR were as follows: denatur-
ation at 94
°
C for 1 min, annealing and extension at 70
°
C for 3 min
each (Stanhope
et al
., 1992). A 1-
µ
l aliquot of each reaction mixture
after the first PCR was used as a template for the second PCR in
a 20-
µ
l reaction mixture with the same reagents except for the
concentration of MgCl
2
(which was 1.875 mM) and the primer pairs.
The second PCR was performed under the following conditions:
denaturation at 96
°C for 30 sec, annealing at 50°C for 30 sec,
extension at 60°C for 30 sec. In the first PCR, a 1.3-kb fragment of
IRBP was amplified using primers +IRBP217 and –IRBP1531
Molecular Phylogeny of Mustelidae 245
(Stanhope et al., 1992). In the second PCR, three segments were
amplified against the product of the first PCR, using the three
primer sets: R +IRBP335 and U –IRBP734 (Serizawa et al., 2000);
R +IRBP724 (5'-CAGGAAACAGCTATGACCCCTGCACGTGGAC-
ACCATCT-3') and U –IRBP1145 (5'-TGTAAAACGACGGCCAGT-
GCGGTCCACCAGCGTGTAGT-3'); and R +IRBP1110 and U
–IRBP1530 (Serizawa et al., 2000). Numbers in the primer names
designate the position of the 3' end of the primer in the human ref-
erence sequence (Fong et al., 1990). The prefixes “+” and “–” refer
to the reading and complementary strands, respectively.
The sequencing of the product of the second PCR was carried
out according to the manufacturer’s instructions, using either a Dye
Primer or Big Dye Primer Cycle Sequencing Kit (ABI) and run on
an ABI 373A or ABI 310 automated sequencer.
Phylogenetic analyses
Phylogenetic analyses were performed by two methods,
neighbor-joining (NJ; Saitou and Nei, 1987) and maximum parsi-
mony (MP; Swofford and Olsen, 1990), both implemented by using
PAUP* version 4.0b10 (Swofford, 1998). The NJ analyses
employed matrices of genetic distances generated by using the
Kimura two-parameter method (Kimura, 1980). The MP analyses
were conducted using 100 heuristic tree-bisection reconnection
searches in which the input order of taxa was randomized. All NJ
analyses and the MP analyses of IRBP sequences were based on
equally weighted nucleotide substitutions. For MP analyses of cyto-
chrome b sequences, the following character weightings were used:
(1) equally weighted nucleotide substitutions (MP
TiTv
), and (2) trans-
versions only at third positions of codons and all nucleotide substi-
Table 1. Taxon and gene sampling, with DDBJ/EMBL/GenBank International Nucleotide Sequence Database accession numbers. For new
accessions (this paper), localities of the vouchers are provided.
Taxon
Accession no.; locality or reference
IRBP Cytochrome b
Carnivora
Caniformia
Mustelidae
Mustelinae
Gulo gulo (Wolverine) AB082962; Sakhalin, Russia AB051245; Hosoda et al., 2000
Martes americana (American Marten) AB082963; Maine, USA AB051234; Hosoda et al., 2000
Martes flavigula (Yellow-Throated Marten) AB082964; Primorye, Russia AB051235; Hosoda et al., 2000
Martes foina (Beech or Stone Marten) AB082965; Thuringia, Germany AB051236; Hosoda et al., 2000
Martes martes (Pine Marten) AB082966; Moscow, Russia AB051237; Hosoda et al., 2000
Martes melampus (Japanese Marten) AB082967; Wakayama, Honshu, Japan AB051238; Hosoda et al., 2000
Martes zibellina (Sable) AB012360; Kurose et al., 1999a
Mustela altaica (Mountain Weasel) AB082968; Altai region, Russia AB051239; Hosoda et al., 2000
Mustela erminea (Ermine or Stoat) AB082969; Hokkaido, Japan AB051240; Hosoda et al., 2000
Mustela eversmanii (Steppe Polecat) AB082970; Chita region, Russia AB026102; Kurose et al., 2000
Mustela itatsi (Japanese Weasel) AB082971; Aomori, Honshu, Japan AB026104; Kurose et al., 2000
Mustela lutreola (European Mink) AB082972; Novosibirsk, Russia AB026105; Kurose et al., 2000
Mustela nivalis (Weasel) AB082973; Aomori, Honshu, Japan AB051241; Hosoda et al., 2000
Mustela putorius furo (Domestic Ferret) AB082974; experimental animal AB026103; Kurose et al., 2000
Mustela putorius putorius (European Polecat) AB082975; Moscow, Russia AB026107; Kurose et al., 2000
Mustela sibirica (Siberian Weasel) AB082976; Wakayama, Honshu, Japan AB051242; Hosoda et al., 2000
Mustela vison (American Mink) AB082977; Hokkaido, Japan* AF057129; Koepfli and Wayne, 1999
Lutrinae
Enhydra lutris (Sea Otter) AB082978; Alaska, USA AB051244; Hosoda et al., 2000
Melinae
Meles meles anakuma (Japanese Badger) AB082980; Miyazaki, Kyushu, Japan
Meles meles meles (European Badger) AB082979; Thuringia, Germany X94922; Ledje and Arnason, 1996a
Procyonidae
Procyon lotor (Raccoon) AB082981; Miyazaki, Kyushu, Japan* X94930; Ledje and Arnason, 1996a
Feliformia
Felis catus (Domestic Cat) Z11811; Stanhope et al., 1992 U20753; Lopez et al., 1996
Rodentia
Apodemus speciosus
(Large Japanese Field Mouse)
AB032856; Serizawa et al., 2000 AB032849; Serizawa et al., 2000
* An introduced population.
J. J. Sato et al.246
tutions at first and second positions (MP
–3Ti
). The weighting
decreasing the transition bias was employed to minimize the effects
of saturation.
For either gene, a χ
2
-test of homogeneity was implemented by
using PAUP* 4.0b10 to test the assumption of base-compositional
homogeneity. Prior to combining the two genes into single analy-
ses, the incongruence length difference test (also termed the parti-
tion homogeneity test; Mickevich and Farris, 1981; Farris et al.,
1995) was performed to test significance of incongruence between
the two data sets.
Bootstrap proportions (BS; Felsenstein, 1985) were obtained
by generating 1000 heuristic replicates with PAUP* 4.0b10, each
consisting of 100 heuristic tree-bisection reconnection searches in
which the input order of taxa was randomized. The decay index (DI;
also known as the Bremer support, branch support, clade stability,
length difference; Bremer, 1988, 1994) was calculated using
TreeRot version 2b (Sorenson, 1999).
Estimation of divergence time
Divergence times were estimated assuming the constancy of
the rate of molecular change over time (molecular-clock hypothesis;
Zuckerkandl and Pauling, 1965). To test this hypothesis, the
two-cluster test (Takezaki et al., 1995) was performed.
Gene-specific rates of nucleotide substitution were calibrated
using our fossil-based time estimate for the mustelid-procyonid split
(inferred from the first stratigraphic appearances of these families)
and the average Kimura two-parameter genetic distance between
Procyon lotor and the mustelid species-group taxa sequenced.
Divergence times between mustelid species-group taxa were esti-
mated by means of the gene-specific rate of nucleotide substitution
and the Kimura two-parameter genetic distance between these
taxa. Divergence times between mustelid clades were estimated by
using the rate of nucleotide substitution and the average Kimura
two-parameter genetic distance between the sequenced species-
group taxa contained in these clades. For the cytochrome b gene,
transversion distances only were used to avoid saturation problems
caused by transitions.
RESULTS
Nucleotide variation
IRBP
The studied individuals of Martes americana and Meles
meles meles showed heterozygosity (G-C, silent substitu-
tion) at sites 1209 and 1434, respectively (sites according to
the human reference sequence [Fong et al., 1990]). Each of
the 10 individuals of Mustela shows a single indel of 3-bp
deletion, which corresponds to sites 1311–1313 in the
human reference sequence (Fong et al., 1990). Excepting
the three sites, there are 158 (13.3%) variable sites among
the remaining 1185 sites from the mustelids examined,
including 31 (19.6%), 18 (11.4%), and 109 (69.0%) sites at
first, second, and third codon positions, respectively. Of
these variable sites, 70 (44.3%) are phylogenetically infor-
mative, including nine (12.9%), five (7.1%), and 56 (80.0%)
sites at first, second, and third codon positions, respectively.
The mean frequencies of the four bases are as follows: A,
17.7%; C, 31.9%; G, 32.0%; and T, 18.4%. The null hypoth-
esis of homogeneity in base composition across the mus-
telid taxa was not rejected by the χ
2
-test (P>0.05).
Nucleotide substitutions and Kimura two-parameter
genetic distances among the 1185 bp from the mustelids
studied range from, respectively, one and 0.08% between
the two subspecies of Mustela putorius to, respectively, 51
and 4.45% between Mustela itatsi and Meles meles meles
(Table 2). The value of the genetic distance between the two
subspecies of Meles meles is 0.51%. Among the nine spe-
cies of Mustela, the genetic distances range from 0.17% (M.
putorius furo vs. M. sibirica) to 2.49% (M. itatsi vs. M. vison),
but the maximum value is only 1.89% (M. erminea vs. M.
itatsi) when Mustela vison is excluded from comparison.
Among martens, the distances range from 0.42 to 0.76% (M.
melampus vs. M. foina and M. martes, respectively) within
the subgenus Martes, and to 1.71% (M. flavigula vs. any of
M. martes, M. melampus, and M. americana) within the
genus. The minimum genetic distance between species of
different genera is 0.85% (Martes foina vs. Gulo gulo), and
the minimum distance between species of different subfam-
ilies is 2.50% (Gulo gulo vs. Meles meles anakuma).
Cytochrome b
There was no length variation in nucleotide sequences
of the cytochrome b gene from the mustelids studied. Of the
1140 bp sequenced, 454 (39.8%) sites are variable, includ-
ing 95 (20.9%), 27 (5.9%), and 332 (73.1%) sites at first,
second, and third codon positions, respectively. Of the vari-
able sites, 336 (74.0%) are phylogenetically informative,
including 66 (19.6%), 16 (4.8%), and 254 (75.6%) sites at
first, second, and third codon positions, respectively. The
mean frequencies of the four bases are as follows: A,
29.0%; C, 29.9%; G, 13.5%; and T, 27.5%. The null hypoth-
esis of homogeneity in base composition across the mus-
telid taxa was not rejected by the χ
2
-test (P>0.05).
Nucleotide substitutions and Kimura two-parameter
genetic distances among the 1140 bp from the mustelids
examined range from, respectively, three and 0.26%
between Mustela putorius furo and Mustela eversmanii to,
respectively, 197 and 20.28% between Mustela nivalis and
Meles meles (Table 3). The value of the genetic distance
between the two subspecies of Mustela putorius is 0.62%.
Among the nine species of Mustela, the genetic distances
range from 0.26% (M. putorius furo vs. M. eversmanii) to
15.27% (M. nivalis vs. M. vison); however, when Mustela
vison is excluded, the maximum distance is only 11.03% (M.
itatsi vs. M. altaica). Among species of martens, the dis-
tances range from 2.70% (M. martes vs. M. melampus) to
9.91% (M. foina vs. M. zibellina) within the subgenus Mar-
tes, and to 15.23% (M. flavigula vs. M. americana) within the
genus. The minimum genetic distance between species of
different genera is 12.46% (Mustela erminea vs. Martes
melampus), and the minimum distance between species of
different subfamilies is 13.37% (Mustela erminea vs. Enhy-
dra lutris).
Phylogenetic inference
IRBP
A tree that resulted from NJ analysis of the IRBP nucle-
otide sequences (Fig. 1A) and the strict consensus of the 16
Molecular Phylogeny of Mustelidae 247
shortest trees that resulted from MP analysis of these
sequences (Fig. 1B) consistently indicate that (1) Enhydra
and Mustela are more closely related to each other than
either is to Martes or Gulo, causing the subfamily Mustelinae
to be paraphyletic (NJ BS; MP BS/DI = 100; 100/8); (2) Gulo
and Martes are phylogenetically closer to each other than
Table 2. Numbers of base-pair differences (above diagonal) and Kimura two-parameter percentage genetic distances (below diagonal)
among partial nucleotide sequences of IRBP (1185 bp) from mustelids and Procyon lotor.
Taxon 1234567891011121314151617181920
1 Gulo gulo 12171011113937414439343940393437293163
2 Martes americana 1.02 20 6 8 7 43 42 47 50 46 40 45 46 45 41 39 31 31 68
3 Martes flavigula 1.45 1.71 19 20 20 43 43 47 48 46 42 45 46 45 38 42 37 39 70
4 Martes foina 0.85 0.51 1.62 8 5 42 42 46 49 45 39 44 45 44 37 37 31 31 66
5 Martes martes 0.94 0.68 1.71 0.68 9 41 41 45 48 44 38 43 44 43 38 40 32 34 67
6 Martes melampus 0.94 0.59 1.71 0.42 0.76 42 42 46 49 45 39 44 45 44 39 39 31 31 67
7 Mustela altaica 3.38 3.73 3.73 3.65 3.56 3.64 17 13 16 13 7 11 12 11 24 39 47 50 86
8 Mustela erminea 3.21 3.65 3.73 3.65 3.56 3.65 1.45 21 22 21 16 19 20 19 24 36 43 45 86
9 Mustela eversmanii 3.56 4.09 4.09 4.00 3.91 4.00 1.11 1.80 9 13 11 4 5 4 26 41 47 50 91
10 Mustela itatsi 3.83 4.37 4.18 4.28 4.19 4.27 1.37 1.89 0.77 16 14 7 8 7 29 42 48 51 91
11 Mustela lutreola 3.38 4.00 4.00 3.91 3.82 3.91 1.11 1.80 1.11 1.37 10 11 12 11 25 40 47 50 87
12 Mustela nivalis 2.94 3.47 3.64 3.38 3.29 3.37 0.59 1.37 0.94 1.20 0.85 9 10 9 21 35 44 47 82
13 Mustela putorius furo 3.38 3.91 3.91 3.82 3.73 3.82 0.94 1.62 0.34 0.59 0.94 0.76 1 2 24 39 45 48 89
14 Mustela putorius putorius 3.47 4.00 4.00 3.91 3.82 3.91 1.02 1.71 0.42 0.68 1.02 0.85 0.08 3 25 40 46 49 90
15 Mustela sibirica 3.38 3.91 3.91 3.82 3.73 3.82 0.94 1.62 0.34 0.59 0.94 0.77 0.17 0.25 24 39 45 48 89
16 Mustela vison 2.94 3.56 3.29 3.20 3.29 3.38 2.06 2.06 2.23 2.49 2.14 1.80 2.06 2.14 2.06 36 39 43 82
17 Enhydra lutris 3.21 3.38 3.65 3.20 3.47 3.38 3.38 3.11 3.55 3.64 3.46 3.02 3.37 3.46 3.37 3.11 47 47 88
18 Meles meles anakuma 2.50 2.67 3.20 2.67 2.76 2.67 4.09 3.74 4.09 4.18 4.09 3.82 3.91 4.00 3.91 3.38 4.10 6 71
19 Meles meles meles 2.68 2.67 3.38 2.67 2.94 2.67 4.37 3.92 4.36 4.45 4.36 4.09 4.18 4.27 4.18 3.73 4.10 0.51 72
20 Procyon lotor 5.57 6.03 6.22 5.84 5.94 5.93 7.72 7.73 8.20 8.20 7.81 7.34 8.00 8.10 8.01 7.35 7.94 6.31 6.40
Table 3. Numbers of base-pair differences (above diagonal) and Kimura two-parameter percentage genetic distances (below diagonal)
among the complete nucleotide sequences of cytochrome b (1140 bp) from mustelids and Procyon lotor
.
Taxon 1234567891011121314151617181920
1 Gulo gulo 152 142 149 142 143 147 160 149 168 163 167 162 169 164 177 175 168 185 216
2 Martes americana 15.25 152 103 58 53 63 147 143 150 162 154 152 150 148 151 172 170 180 218
3 Martes flavigula 14.12 15.23 152 140 135 147 166 152 169 163 169 176 170 167 173 181 175 181 208
4 Martes foina 14.86 9.90 15.22 94 89 103 147 149 167 168 167 156 168 163 168 180 164 181 209
5 Martes martes 14.09 5.35 13.87 8.95 30 31 143 134 146 156 150 155 147 142 155 179 167 178 216
6 Martes melampus 14.19 4.86 13.29 8.43 2.70 45 137 129 139 154 143 142 140 135 146 174 160 177 219
7 Martes zibellina 14.68 5.84 14.69 9.91 2.79 4.11 147 139 149 160 153 154 150 145 162 176 175 184 220
8 Mustela altaica 15.98 14.46 16.70 14.47 14.01 13.33 14.47 80 87 114 93 82 88 87 92 146 166 195 198
9 Mustela erminea 14.72 14.02 15.05 14.71 13.02 12.46 13.58 7.49 94 91 91 89 95 94 91 128 137 180 200
10 Mustela eversmanii 16.97 14.83 17.05 16.86 14.38 13.58 14.73 8.20 8.95 64 9 92 3 4 41 140 154 182 199
11 Mustela itatsi 16.35 16.20 16.31 16.95 15.53 15.26 15.98 11.03 8.62 5.94 59 95 65 63 60 136 153 176 202
12 Mustela lutreola 16.85 15.29 17.05 16.86 14.83 14.03 15.19 8.82 8.63 0.80 5.45 91 12 13 40 141 152 184 201
13 Mustela nivalis 16.20 15.04 17.83 15.49 15.39 13.90 15.28 7.68 8.41 8.72 9.02 8.62 93 92 95 153 161 197 209
14 Mustela putorius furo 17.07 14.82 17.15 16.97 14.48 13.68 14.83 8.30 9.04 0.26 6.04 1.06 8.82 7 42 141 155 183 200
15 Mustela putorius putorius 16.50 14.62 16.82 16.40 13.94 13.14 14.28 8.20 8.94 0.35 5.84 1.15 8.72 0.62 45 139 153 178 197
16 Mustela sibirica 18.03 14.93 17.50 16.95 15.39 14.35 16.22 8.72 8.63 3.73 5.54 3.63 9.04 3.82 4.10 141 161 176 208
17 Mustela vison 17.86 17.41 18.52 18.39 18.26 17.63 17.90 14.48 12.48 13.85 13.37 13.96 15.27 13.95 13.72 13.94 162 184 215
18 Enhydra lutris 17.01 17.19 17.87 16.49 16.83 16.02 17.81 16.71 13.37 15.32 15.18 15.09 16.09 15.42 15.19 16.11 16.29 191 216
19 Meles meles meles 19.01 18.34 18.44 18.50 18.12 17.96 18.84 20.02 18.21 18.51 17.78 18.76 20.28 18.62 18.04 17.76 18.74 19.61 226
20 Procyon lotor 22.55 22.78 21.41 21.63 22.55 22.89 23.05 20.18 20.44 20.31 20.71 20.56 21.54 20.42 20.08 21.44 22.32 22.39 23.89
J. J. Sato et al.248
Fig. 1. Phylogenetic relationships among mustelids, based on partial nucleotide sequences of IRBP (1185 bp). A, tree resulted from neigh-
bor-joining analysis. The horizontal length of each branch is proportional to the number of nucleotide substitutions per site. Numbers at
branches are percentage bootstrap values in support of adjacent nodes. B, strict consensus of the 16 shortest trees (length, 218 steps; consis-
tency index, 0.83; retention index, 0.88) resulted from maximum-parsimony analysis. Numbers above branches are percentage bootstrap val-
ues in support of adjacent nodes; numbers below branches are the decay indices.
Molecular Phylogeny of Mustelidae 249
either is to any of the remaining genera under study (70; 66/
2); (3) the subgenus Martes (M. martes, M. melampus, M.
americana, M. foina) is monophyletic (96; 91/1); (4) Mustela
is monophyletic (98; 98/4), with Mustela vison (95; 93/3) and
Mustela erminea (96; 92/3) as successively more closely
related to a clade containing the remaining studied species
of the genus; (5) within the last clade, large-sized species
(M. lutreola, M. itatsi, M. sibirica, M. eversmanii, M. putorius)
and small-sized species (M. nivalis, M. altaica) are con-
tained in separate subclades (75;72/1 and 54; 63/1; respec-
tively); (6) within the subclade of large-sized species, Mus-
tela lutreola is basal to the remaining species (98; 96/3); and
(7) Mustela putorius furo is more closely related to Mustela
putorius putorius than to Mustela eversmanii (64; 64/1).
The phylogenetic position of Melinae is uncertain. While
NJ analysis provided weak evidence (BS = 48) of a basal
position of Meles with respect to a clade encompassing
Enhydra, Mustela, Martes, and Gulo, MP analysis supported
a trichotomy among Meles, the Enhydra-Mustela clade, and
the Martes-Gulo clade. There is also inconsistent evidence
for phylogenetic relationships among Martes subgenera and
Gulo. Whereas NJ analysis placed Gulo in a sister-group
relationship with the subgenus Martes, causing the genus
Martes to be paraphyletic (BS = 66), MP analysis supported
a trichotomy among Gulo and the subgenera Martes and
Charronia (Martes flavigula). Interrelationships among spe-
cies of the subgenus Martes are also equivocal. Although
NJ analysis linked Martes foina with Martes melampus (BS
= 60), recognizing Martes americana and Martes martes as
successively more distant outgroups to this clade, MP anal-
ysis supported a polytomy among the four species. Finally,
the placement of Mustela itatsi is equivocal and there is no
support for the monophyly of the subgenus Putorius (Mus-
tela putorius, Mustela eversmanii). While NJ analysis nested
Mustela itatsi as basal to a trichotomy of Mustela sibirica,
Mustela eversmanii, and Mustela putorius (BS = 83), MP
analysis supported a tetrachotomy among the four species.
Cytochrome b
A tree that resulted from NJ analysis of the nucleotide
sequences of the cytochrome b gene (Fig. 2A) and the strict
consensus of the two shortest trees that resulted from
MP
TiTv
analysis of these sequences (Fig. 2B), as well as the
strict consensus of the 12 shortest trees that resulted from
the MP
–3Ti
analysis (Fig. 2C), all uniformly indicate that (1)
Mustelinae is paraphyletic relative to Enhydra; (2) Gulo and
Martes are more closely related to each other than either is
to any of the remaining genera under study (NJ BS; MP
TiTv
BS/DI; MP
–3Ti
BS/DI = 75; 57/4; 70/2); (3) the subgenus
Martes (M. martes, M. zibellina, M. melampus, M. ameri-
cana, M. foina) is monophyletic (100; 100/15; 92/5), with
Martes foina as basal to the remaining species (100; 100/15;
72/2), and Martes martes and Martes zibellina as sister spe-
cies (92; 92/9; 79/2); (4) the genus Mustela is monophyletic
(72; 56/3; 88/5), with Mustela vison (100; 96/10; 97/6) and
Mustela erminea (48; 69/2; 59/1) as successively more
closely related to a clade comprising the sister subclades
recognized by the IRBP analyses, one including large-sized
species (100; 99/14; 97/6) and the other including small-
sized species (77; 75/3; 53/1); and (5) the subgenus Puto-
rius is monophyletic (86; 94/4; 60/1).
There is conflicting evidence for placement of Melinae.
Although maximum-parsimony analyses consistently recog-
nized Meles as basal to a clade containing Enhydra, Mus-
tela, Martes, and Gulo (MP
TiTv
BS/DI = 56/10; MP
–3Ti
BS/DI
= 88/7), NJ analysis linked this genus with the Martes-Gulo
clade, causing Mustelinae to be paraphyletic (BS = 48).
Both NJ and MP
–3Ti
analyses nested Enhydra as the sister
taxon to Mustela (NJ BS = 63; MP
–3Ti
BS/DI = 70/1), but
MP
TiTv
analysis placed Enhydra in the sister-group position
to the Martes-Gulo clade, albeit with very weak support (BS/
DI = 21/1). Interrelationships among Martes subgenera and
Gulo are also equivocal. While NJ (BS = 73) and MP
TiTv
(BS/DI = 55/5) analyses recognized Gulo as the sister taxon
to the subgenus Charronia, causing the genus Martes to be
paraphyletic, MP
–3Ti
analysis supported a trichotomy among
Gulo and the subgenera Charronia and Martes. Within the
last subgenus, there is discrepant evidence for placement of
Martes melampus and Martes americana. Although NJ and
MP
TiTv
analyses nested Martes melampus and Martes
americana as successively more closely related to the Mar-
tes martes-Martes zibellina clade (NJ BS = 94 and 92,
respectively; MP
TiTv
BS/DI = 87/5 and 92/9, respectively),
MP
–3Ti
analysis supported a trichotomy among this clade,
Martes melampus, and Martes americana. Within the clade
of large-sized Mustela, NJ and MP
TiTv
analyses were con-
sistent in placing Mustela itatsi, Mustela sibirica, and Mustela
lutreola as successively more closely related to the subgenus
Putorius (NJ BS = 95, 100, and 86, respectively; MP
TiTv
BS/
DI = 74/2, 100/9, and 94/4, respectively), but MP
–3Ti
analysis
supported a tetrachotomy among this subgenus and the three
species. Within Putorius, in turn, NJ analysis united Mustela
putorius furo with Mustela eversmanii to the exclusion of Mus-
tela putorius putorius (BS = 70), but maximum-parsimony
analyses supported a trichotomy among these taxa.
Combined IRBP and cytochrome b
The incongruence length difference test did not reject
the null hypothesis of homogeneity in phylogenetic signal
between the IRBP and cytochrome b nucleotide sequences,
enabling them to be combined into a single analysis. The NJ
analysis of the concatenated nucleotide sequences of the
two genes resulted in a tree presented in Fig. 3A. The
MP
TiTv
analysis of these sequences yielded a single shortest
tree (Fig. 3B), and the MP
–3Ti
analysis revealed six shortest
trees which produced a strict consensus tree shown in Fig.
3C. A number of phylogenetic observations can be made
from these trees. First, a basal position of Meles in relation
to the remaining mustelids used in this study is weakly sup-
ported by NJ analysis (BS = 47), moderately supported by
MP
TiTv
analysis (BS/DI = 78/7), and strongly supported by
MP
–3Ti
analysis (BS/DI = 99/11). Second, there is strong
J. J. Sato et al.250
Fig. 2. Phylogenetic relationships among mustelids, based on the complete nucleotide sequences of cytochrome b (1140 bp). A, tree
resulted from neighbor-joining analysis. The horizontal length of each branch is proportional to the number of nucleotide substitutions per site.
Numbers at branches are percentage bootstrap values in support of adjacent nodes. B, strict consensus of the two shortest trees (length, 1155
steps; consistency index, 0.50; retention index, 0.57) resulted from maximum-parsimony analysis with all nucleotide substitutions included.
Numbers above branches are percentage bootstrap values in support of adjacent nodes; numbers below branches are the decay indices. C,
strict consensus of the 12 shortest trees (length, 413 steps; consistency index, 0.59; retention index, 0.68) resulted from maximum-parsimony
analysis with third-position transitions excluded. Numbers above branches are percentage bootstrap values in support of adjacent nodes; num-
bers below branches are the decay indices.
Molecular Phylogeny of Mustelidae 251
Fig. 3. Phylogenetic relationships among mustelids, based on concatenated nucleotide sequences of IRBP and cytochrome b (2325 bp). A,
tree resulted from neighbor-joining analysis. The horizontal length of each branch is proportional to the number of nucleotide substitutions per
site. Numbers at branches are percentage bootstrap values in support of adjacent nodes. B, single shortest tree (length, 1352 steps; consis-
tency index, 0.56; retention index, 0.60) resulted from maximum-parsimony analysis with all nucleotide substitutions included. Numbers above
branches are percentage bootstrap values in support of adjacent nodes; numbers below branches are the decay indices. C, strict consensus of
the six shortest trees (length, 627 steps; consistency index, 0.67; retention index, 0.73) resulted from maximum-parsimony analysis with cyto-
chrome b third-position transitions excluded. Numbers above branches are percentage bootstrap values in support of adjacent nodes; num-
bers below branches are the decay indices.
J. J. Sato et al.252
support (NJ BS = 99; MP
TiTv
BS/DI = 94/8; MP
–3Ti
BS/DI =
99/9) for a sister-group relationship between Enhydra and
Mustela, causing Mustelinae to be paraphyletic. Third, the
genera Gulo and Martes are moderately to relatively
strongly supported as the closest relatives (NJ BS = 93;
MP
TiTv
BS/DI = 73/4; MP
–3Ti
BS/DI = 81/3), but details of this
relationship are equivocal. While NJ and MP
TiTv
analyses
linked Gulo with Martes flavigula, causing the genus Martes
to be paraphyletic (NJ BS = 74; MP
TiTv
BS/DI = 48/3), MP
–3Ti
analysis supported a trichotomy among Gulo, Martes flav-
igula, and the subgenus Martes. Fourth, the monophyly of
the subgenus Martes is strongly supported (NJ BS = 100;
MP
TiTv
BS/DI = 100/16; MP
–3Ti
BS/DI = 98/9). Fifth, the
most-basal position of Martes foina in the subgenus Martes
is strongly supported by NJ (BS = 100) and MP
TiTv
(BS/DI =
99/14) analyses, but relatively weakly supported by MP
–3Ti
analysis (BS/DI = 73/2). Sixth, interrelationships among
other species of the subgenus Martes are equivocal. While
NJ and MP
TiTv
analyses moderately to strongly supported a
close relationship between Martes martes and Martes
melampus to the exclusion of Martes americana (NJ BS =
97; MP
TiTv
BS/DI = 80/3), MP
–3Ti
analysis provided weak
support (BS/DI = 54/1) for a basal position of Martes melam-
pus with respect to a clade containing Martes martes and
Martes americana. Seventh, the monophyly of Mustela is
strongly supported (NJ BS = 95; MP
TiTv
BS/DI = 93/8; MP
–3Ti
BS/DI = 100/11), with Mustela vison strongly supported (NJ
BS = 100; MP
TiTv
BS/DI = 99/12; MP
–3Ti
BS/DI = 100/10)
and Mustela erminea less strongly supported (NJ BS = 83;
MP
TiTv
BS/DI = 90/5; MP
–3Ti
BS/DI = 87/3) as successively
more closely related to a clade encompassing the remaining
studied species of the genus. Eighth, within the last clade,
there are two separate subclades recognized by the individ-
ual IRBP and cytochrome b analyses: the strongly sup-
ported subclade of large-sized species (NJ BS = 100; MP
TiTv
BS/DI = 100/18; MP
–3Ti
BS/DI = 100/8) and the moderately
supported subclade of small-sized species (NJ BS = 77;
MP
TiTv
BS/DI = 84/4; MP
–3Ti
BS/DI = 76/2). Ninth, within the
subclade of large-sized species, interrelationships among
the subgenus Putorius and other species are equivocal.
While NJ analysis strongly supported Mustela lutreola as the
sister taxon to Putorius (BS = 100), maximum-parsimony
analyses supported this species, albeit relatively weakly
(MP
TiTv
BS/DI = 67/2; MP
–3Ti
BS/DI = 60/1), as the most
basal within the subclade. Moreover, Mustela itatsi was rec-
ognized by NJ (BS = 91) and MP
TiTv
(BS/DI = 99/10) anal-
yses as less closely related to Putorius than is Mustela sibir-
ica, but MP
–3Ti
analysis supported a trichotomy among the
three taxa. Tenth, the monophyly of the subgenus Putorius
is strongly supported by NJ (BS = 99) and MP
TiTv
(BS/DI =
97/5) analyses, but relatively weakly supported by MP
–3Ti
analysis (BS/DI = 65/2). Finally, Mustela putorius furo and
Mustela putorius putorius are relatively weakly supported as
the closest relatives (NJ BS = 50; MP
TiTv
BS/DI = 60/1;
MP
–3Ti
BS/DI = 57/1).
Performance of IRBP versus cytochrome b
Nucleotide sequences of IRBP appear to contain more
resolving power than those of cytochrome b in recovering
more-inclusive mustelid clades. Although our analyses of
IRBP were unable to completely or strongly resolve phylo-
genetic relationships of Meles, they resulted in much greater
support—than analyses of cytochrome b—for the sister-
group status of Enhydra to Mustela, the monophyly of the
genus Mustela, and a basal position of Mustela erminea with
respect to a clade containing Mustela nivalis, Mustela alta-
ica, Mustela lutreola, Mustela itatsi, Mustela sibirica, and the
subgenus Putorius (compare Figs. 1 and 2). On the other
hand, nucleotide sequences of cytochrome b were consid-
erably more efficient than those of IRBP in recovering less-
inclusive clades, such as those within the subgenus Martes
and within the clade of large-sized Mustela. In addition,
analyses employing both transitions and transversions of
cytochrome b (Figs. 2A, B; 3A, B) resulted in much better
resolved relationships among closely related species than
analyses excluding third-position transitions of this gene
(Figs. 2C, 3C). On the other hand, analyses that excluded
cytochrome b third-codon-position transitions generally per-
formed better than analyses using total nucleotide substitu-
tions of this gene in recovering more-inclusive clades.
A comparison of per site substitution rates between
nucleotide sequences of IRBP and cytochrome b (Fig. 4)
indicates that cytochrome b transitions are likely to be satu-
rated among supraspecific taxa. This is in contrast to cyto-
chrome b transversions which appear to accumulate with
time approximately proportionally to IRBP total substitutions
as far back as the caniform-feliform divergence. Neverthe-
less, interordinal comparisons (Carnivora vs. Rodentia) indi-
cate saturation also in cytochrome b transversions (Fig. 4B).
One factor that affects the efficacy of the two genes and
the two types of cytochrome b nucleotide substitutions in
resolving phylogenetic relationships at different taxonomic
levels is apparently the rate of substitution. Although the
higher rates of substitution in cytochrome b and in its tran-
sitions—as compared to the lower rates of substitution in
IRBP and in cytochrome b transversions, respectively—pro-
vide more phylogenetically informative characters over
shorter look-back times, which promotes increase in resolu-
tion at lower taxonomic levels, they also result in saturation
at reduced time spans, which decreases resolution at higher
taxonomic levels.
Dating of divergence events
Calibration of substitution rates
The earliest mustelid known is Plesictis (Wolsan, 1999).
Its mustelid nature is evidenced by an array of cranial and
dental synapomorphies of this family (e.g., Wolsan, 1993a),
including the mustelid suprameatal fossa (sensu Wolsan,
1992, 1993a, b, 1994, 1996, 1999; Wolsan and Lange-
Badré, 1996; not Schmidt-Kittler, 1981). The first strati-
graphic appearance of this genus is in the upper Oligocene
strata of Cournon, France, where remains of Plesictis plesic-
Molecular Phylogeny of Mustelidae 253
tis were found (Laizer and Parieu, 1839, their Mustela
plesictis; Pomel, 1853; Viret, 1929; their Plesictis gene-
toïdes). These remains are referred to a level between the
European Paleogene Mammal Reference Levels MP 28 and
29 (e.g., Hugueney, 1997), which corresponds to an age
between 24.3 and 24.7 Ma (megannum; Schmidt-Kittler et
al., 1997, and references therein).
The earliest procyonid known is Pseudobassaris (Wol-
san, 1993a, 1997a, b, 1998, 1999). The procyonid status of
this genus is indicated by the middle-ear synapomorphies of
this family, the procyonid suprameatal fossa (sensu Wolsan,
1992, 1993a, b, 1994, 1996, 1997a, b, 1998, 1999; Wolsan
and Lange-Badré, 1996; not Schmidt-Kittler, 1981) and the
procyonid epitympanic recess (Wolsan, 1998, 1999).
Pseudobassaris first appears in the late Oligocene of Bel-
garric 1 and Belgarrite 4A, France, from where Pseudobas-
saris riggsi has been recorded (Wolsan and Lange-Badré,
1996, and references therein). The fossil faunal assem-
blages of these localities are assigned to levels intermediate
between the European Paleogene Mammal Reference Lev-
els MP 24 and 25 (e.g., BiochroM’97, 1997) dated at 28.5
and 28.0 Ma, respectively (Schmidt-Kittler et al., 1997, and
references therein). Thus, the earliest record of Procyonidae
is older than that of Mustelidae.
The morphological characteristics of Pseudobassaris
are congruent with the primitive procyonid morphology
inferred from character analysis of the early Miocene to
present-day representatives of this family (Wolsan, 1997a,
b, 1998). The procyonid synapomorphies of Pseudobassaris
are of variable occurrence within this genus (Wolsan and
Lange-Badré, 1996; Wolsan, 1997a, b, 1998). Furthermore,
cranial features synapomorphic for the mustelid-procyonid
clade (lack of the alisphenoid canal; separation of the pos-
terior carotid foramen from the posterior lacerate foramen by
the caudal entotympanic; posterior inflation of the caudal
entotympanic in between the posterior lacerate and stylo-
mastoid foramina [Wolsan, 1993a]) are also variably present
within Pseudobassaris, so that the primitive conditions char-
acteristic of musteloids basal to the mustelid-procyonid
clade also occur (Wolsan and Lange-Badré, 1996; Wolsan,
1997b, 1998). All this indicates that Pseudobassaris is very
close to mustelid-procyonid split. Therefore, taking into
account that Pseudobassaris dates to an age between 28.0
and 28.5 Ma, we assume the latter date as the time of diver-
gence between Mustelidae and Procyonidae.
The constancy of IRBP and cytochrome b evolutionary
rates for mustelids through NJ trees (Figs. 1A, 2A) was
found to be moderate by the two-cluster test. Assuming the
constancy in evolutionary rates of the two genes within Mus-
telidae, the divergence time of 28.5 Ma between Mustelidae
and Procyonidae indicates an approximate rate of 0.0025
nucleotide substitutions per site per million of years for the
mustelid IRBP and an approximate rate of 0.0021 transver-
sions per site per million of years for the mustelid cyto-
chrome b.
Estimates of divergence dates
Total substitutions of IRBP and transversions of cyto-
chrome b suggest that the lineage of Meles diverged from
those leading to Enhydra, Mustela, Martes, and Gulo about
14.5 or 18.1 million years (Myr) ago, respectively, with the
lineages of the two studied subspecies of Meles meles sep-
arating from each other approximately 2 Myr ago (Table 4).
The divergence between a clade containing Enhydra and
Fig. 4. Comparison of per site substitution rates between partial IRBP (1101 bp) and complete cytochrome b (1140 bp) nucleotide sequences
as estimated by using the Kimura two-parameter method. A, number of per site transitions and transversions for IRBP against the number of
per site transitions and transversions for cytochrome b. B, number of per site transitions and transversions for IRBP against the number of per
site transversions for cytochrome b. Open circles are for pairwise comparisons among mustelid species-group taxa. Open triangles are for
pairwise comparisons among species-group taxa of different mustelid genera. Open squares are for pairwise comparisons between species-
group taxa of Mustelidae and Procyon lotor (Procyonidae). Open diamonds are for pairwise comparisons between species-group taxa of Mus-
telidae (Caniformia) and Felis catus (Feliformia). Filled circles are for pairwise comparisons between mustelid species-group taxa (Carnivora)
and Apodemus speciosus (Rodentia). Mustelids used in the comparisons are those listed in Table 1 except Martes zibellina and Meles meles
anakuma.
J. J. Sato et al.254
Mustela and that comprising Gulo and Martes is dated at
14.7–14.8 Ma. The split between the lineages of Enhydra
and Mustela is placed at 13.5–14.1 Ma. The divergence of
the Gulo lineage from those of martens, in turn, is suggested
to be at 3.8–5.8 Ma by IRBP total substitutions, but cyto-
chrome b transversions indicate a substantially older date of
6.5–8.1 Ma. Correspondingly, the origin of the subgenus
Martes is placed at 3.8–6.8 Ma based on IRBP, but an age
of 7.8–8.1 Ma is indicated by cytochrome b. A date of about
430 ka (kiloannum) for the divergence between Martes mar-
tes and Martes zibellina, or their lineages, is inferred from
cytochrome b transversions.
Within the genus Mustela, the lineage of Mustela vison
extends as far back as 8.5–9.9 Ma and that of Mustela
erminea as far back as 3.9–6.7 Ma. The divergence
between the clades of small- and large-sized species dates
to 4.0–4.8 Ma. The split between the lineages of Mustela
nivalis and Mustela altaica is placed at 2.4 Ma by IRBP, but
a considerably older date of 5.6 Ma is suggested by cyto-
chrome b transversions. Mustela itatsi and Mustela sibirica,
or their lineages, appear to diverge from each other within a
time span from 1.7 to 2.4 Ma.
The subgenus Putorius originated about 1.0–1.1 Myr
ago. The time of divergence between Mustela putorius and
Mustela eversmanii is estimated at 1.5 Ma on the basis of
IRBP, but cytochrome b transversions indicate a consider-
ably younger date of approximately 430 ka. Age estimates
for the divergence of the Mustela putorius furo lineage also
substantially differ between the genes. Whereas IRBP dates
this event at about 340 ka, cytochrome b transversions sug-
gest a date of approximately 860 ka.
DISCUSSION
Nuclear versus mitochondrial genes in phylogenetic
reconstruction
Nuclear genes have been found to have more resolving
power than mitochondrial genes in recovering ordinal and
supraordinal mammalian taxa (Springer et al., 2001). Our
results indicate that the difference in performance between
nuclear and mitochondrial genes, although less well pro-
nounced, can also be seen at lower taxonomic levels. Our
analyses of the nuclear IRBP generally performed better
than analyses of the mitochondrial cytochrome b in recover-
ing more-inclusive mustelid clades. On the other hand, the
cytochrome b gene demonstrated considerably more resolv-
ing power in recovering less-inclusive mustelid clades. For
cytochrome b, analyses employing both transitions and
transversions resulted in much better resolved relationships
among closely related species than analyses excluding
third-position transitions. On the other hand, more-inclusive
mustelid clades were recovered with generally greater effi-
ciency by analyses that excluded cytochrome b third-posi-
tion transitions.
In conclusion, both nuclear and mitochondrial genes
and—within the latter—both transitions and transversions
are more or less complementary to each other in phyloge-
netic information. For this reason, analyses based on con-
catenated nucleotide sequences of the two types of genes,
and using a weighting scheme that includes both all substi-
tutions and transversions only at third codon position for
mitochondrial genes, appear to provide more reliable esti-
mation of phylogenetic relationships below the family level
than analyses based on individual genes alone, and using a
single weighting for mitochondrial genes.
Mustelid phylogeny
Relationships among Melinae, Lutrinae, and Mustelinae
Melinae (excluding Melogale and Mydaus) has recently
been hypothesized to be more closely related to either Mus-
telinae (combined cytochrome b and 12S rRNA: Ledje and
Árnason, 1996b; morphology: Wozencraft, 1989) or Lutrinae
(morphology: Bryant et al., 1993; Baskin, 1998); placed in a
polytomy with the two subfamilies (12S rRNA: Ledje and
Árnason, 1996b; combined genetics and morphology: Bin-
inda-Emonds et al., 1999; morphology: Wyss and Flynn,
1993); proposed as an outgroup to the musteline-lutrine
clade (cytochrome b: Ledje and Árnason, 1996a; Koepfli
and Wayne, 1998; combined cytochrome b, 12S and 16S
rRNA, and morphology: Dragoo and Honeycutt, 1997; α-
and β-hemoglobin: Stanhope et al., 1993); or linked with
some mustelines within the musteline-lutrine clade, thereby
causing Mustelinae to be paraphyletic (cytochrome b:
Masuda and Yoshida, 1994b; 12S rRNA: Emerson et al.,
1999; combined cytochrome b and 12S and 16S rRNA: Dra-
goo and Honeycutt, 1997; morphology: Abramov and Bary-
shnikov, 1995; Baryshnikov and Abramov, 1998). Most our
analyses corroborated the placement of Melinae basal to a
clade encompassing the lutrine Enhydra and the mustelines
Mustela, Gulo, and Martes, with strongest support yielded
by MP
–3Ti
analysis of the combined genes (Fig. 3C). One
analysis only, NJ of cytochrome b alone, found evidence,
albeit very weak, to suggest a close relationship between
Melinae and the Gulo-Martes clade to the exclusion of Mus-
tela and Enhydra (Fig. 2A).
The placement of Lutrinae within Mustelinae, causing
the latter subfamily to be paraphyletic, has recently been
proposed by an array of both molecular and morphology-
based studies (Bryant et al., 1993; Masuda and Yoshida,
1994b; Vrana et al., 1994; Ledje and Árnason, 1996a; Dra-
goo and Honeycutt, 1997; Baryshnikov and Abramov, 1998;
Koepfli and Wayne, 1998; Emerson et al., 1999; Hosoda et
al., 2000). Most of these studies considered the phyloge-
netic position of Enhydra in relation to both Mustela and
Martes or Gulo. The four resultant hypotheses are that (1)
Enhydra is more closely related to Mustela than to Martes
or Gulo (cytochrome b: Ledje and Árnason, 1996a; Koepfli
and Wayne, 1998; combined cytochrome b and 12S and
16S rRNA: Dragoo and Honeycutt, 1997; morphology: Bry-
ant et al., 1993); (2) Enhydra is more closely related to Mar-
tes and Gulo than to Mustela (cytochrome b: Hosoda et al.,
2000); (3) Enhydra and Mustela vison are successively
Molecular Phylogeny of Mustelidae 255
more distant outgroups to a clade encompassing Martes
and the remaining Mustela (cytochrome b: Masuda and
Yoshida, 1994b); and (4) Enhydra is basal to a clade con-
taining Mustela, Martes, and Gulo (combined cytochrome b,
12S and 16S rRNA, and morphology: Dragoo and Honey-
cutt, 1997; morphology: Baryshnikov and Abramov, 1998).
Only one of our analyses (MP
TiTv
of cytochrome b alone)
yielded evidence, albeit very weak, to suggest that Enhydra
is phylogenetically closer to the Gulo-Martes clade than to
Mustela (Fig. 2B). All other analyses corroborated an asso-
ciation of Enhydra and Mustela to the exclusion of Martes
and Gulo, with robust support coming from IRBP alone (Fig.
1) and the combined genes (Fig. 3).
Relationships among Gulo, Martes, and Mustela
Although Bryant et al. (1993) provided morphological
evidence to suggest a basal placement of Martes with
respect to a clade comprising Gulo and Mustela, all other
recent studies of interrelationships among these genera,
based on both morphological and genetic data (Abramov
and Baryshnikov, 1995; Dragoo and Honeycutt, 1997; Bary-
shnikov and Abramov, 1998; Koepfli and Wayne, 1998; Bin-
inda-Emonds et al., 1999; Hosoda et al., 2000), consistently
indicate a close relationship between Gulo and Martes to the
exclusion of Mustela. The latter interrelationship is con-
firmed by all our analyses, with strongest support demon-
strated by the combined genes (Fig. 3).
Relationships within Martes
The genus Martes may be paraphyletic. Of the four
recent studies of interrelationships among Gulo and species
of Martes, Koepfli and Wayne (1998, cytochrome b) and
Bininda-Emonds et al. (1999, combined genetic and mor-
phological evidence) supported the monophyletic status of
the genus Martes, but Baryshnikov and Abramov (1998,
morphology) and Hosoda et al. (2000, cytochrome b) sug-
gested its paraphyly. This paraphyly was due to the hypoth-
esized close relationship of Gulo and either Martes flavigula
and Martes pennanti (Hosoda et al., 2000) or Martes flav-
igula and some other mustelids (Baryshnikov and Abramov,
1998) to the exclusion of the subgenus Martes. While most
our analyses indicated weak to moderate support for the
paraphyletic status of the genus Martes relative to Gulo
(Figs. 1A; 2A, B; 3A, B), the others were unable to com-
pletely resolve phylogenetic relationships of Gulo with
respect to Martes flavigula and the subgenus Martes.
The subgenus Martes has constantly been regarded as
monophyletic (e.g., Anderson, 1970; Bininda-Emonds et al.,
1999; Hosoda et al., 2000), and its monophyly is strongly
supported by all our analyses. Within this subgenus, Martes
foina has generally been recognized as basal to a clade
containing the remaining present-day species (Anderson,
1970; Wolsan, 1987; Carr and Hicks, 1997; Bininda-Emonds
et al., 1999; Hosoda et al., 2000). This basal position is sup-
ported by our analyses of cytochrome b alone and the com-
bined genes, with very strong support coming from the NJ
and MP
TiTv
analyses (Figs. 2A, B; 3A, B). In contrast to
these, our NJ analysis of IRBP alone (Fig. 1A) provided sup-
port, albeit weak, for a sister-group relationship of Martes
foina to Martes melampus, and the MP analysis of this gene
(Fig. 1B) failed to resolve interrelationships within the sub-
genus.
Martes martes and Martes zibellina have mostly been
hypothesized as sister species, with Martes melampus and
Martes americana as successively more distant outgroups
to this clade (cytochrome b: Carr and Hicks, 1997; Hosoda
et al., 1997, 2000; rDNA: Hosoda et al., 1997; morphology:
Wolsan, 1987). Our results largely support this hypothesis.
Our cytochrome b analyses—that is, all our analyses that
included Martes zibellina—supported the sister-group status
of this species to Martes martes (Fig. 2), and most our anal-
yses of cytochrome b alone (Fig. 2A, B) and the combined
genes (Fig. 3A, B) strongly supported Martes melampus and
Martes americana as successively more distant outgroups
to the Martes martes-Martes zibellina clade. Only one of our
analyses (NJ of IRBP alone) yielded support, albeit weak
and only in part, for an alternative hypothesis that links Mar-
tes melampus with Martes americana (cytochrome b: Carr
and Hicks, 1997) and postulates Martes zibellina and Martes
martes as successively more distant outgroups to the Mar-
tes melampus-Martes americana clade (morphology: Bin-
inda-Emonds et al., 1999, based on Anderson’s [1970] phy-
logenetic scenario).
Relationships within Mustela
Although some evidence has been provided to suggest
that the genus Mustela is paraphyletic relative to Martes,
Vormela, some lutrines, or Meles (cytochrome b: Masuda
and Yoshida, 1994b; 12S rRNA: Emerson et al., 1999;
serum immunology: Belyaev et al., 1980, 1984; Taranin et
al., 1991; combined cytochrome b, 12S rRNA, and morphol-
ogy: Vrana et al., 1994; morphology: Baryshnikov and Abra-
mov, 1998), there is prevailing evidence from both genetics
and morphology (e.g., Dragoo and Honeycutt, 1997; Bin-
inda-Emonds et al., 1999; Hosoda et al., 2000) in support of
the monophyletic status for Mustela. Our results confirm the
monophyly of this genus, with strongest support demon-
strated by IRBP alone (Fig. 1) and the combined genes (Fig.
3).
Mustela vison has long been regarded as closely
related to or even conspecific with Mustela lutreola. How-
ever, overwhelming genetic and morphological evidence
have been presented to indicate that the two species are
only distantly related, and that Mustela vison is indeed an
outgroup to a clade comprising all other extant species of
the genus (Volobuev and Ternovsky, 1974; Graphodatsky et
al., 1976; Belyaev et al., 1980, 1984; Youngman, 1982;
Lushnikova et al., 1989; Taranin et al., 1991; Masuda and
Yoshida, 1994b; Koepfli and Wayne, 1998; Bininda-Emonds
et al., 1999; Davison et al., 1999; Emerson et al., 1999;
Abramov, 2000a; Hosoda et al., 2000; Kurose et al., 2000).
This basal placement of Mustela vison is very strongly sup-
J. J. Sato et al.256
ported by all our analyses.
Our analyses also unanimously indicated a basal posi-
tion of Mustela erminea in relation to a clade encompassing
the remaining studied species of the genus except Mustela
vison, with strongest support coming from the analyses of
IRBP alone (Fig. 1). This phylogenetic position of Mustela
erminea has also been suggested by previous studies of
cytochrome b nucleotide sequences (Masuda and Yoshida,
1994b; Davison et al., 1999; Kurose et al., 2000), as well as
by evidence from protein electrophoresis (Hartl et al., 1988),
serum immunology (Taranin et al., 1991), karyology (Gra-
phodatsky et al., 1976; Obara, 1991), and morphology
(Rabeder, 1976). An alternative hypothesis that has recently
received some support (cytochrome b: Hosoda et al., 2000;
protein electrophoresis: Hartl et al., 1988; karyology: Zima
and Král, 1984; combined genetics and morphology: Bin-
inda-Emonds et al., 1999; morphology: Youngman, 1982;
Abramov, 2000a) associates Mustela erminea with Mustela
nivalis and Mustela altaica to the exclusion of Mustela itatsi,
Mustela lutreola, Mustela sibirica, and the subgenus Puto-
rius.
All our analyses supported a sister-group relationship
between clades comprising the small-sized species Mustela
nivalis and Mustela altaica, on the one hand, and the large-
sized species Mustela lutreola, Mustela itatsi, Mustela sibir-
ica, Mustela eversmanii, and Mustela putorius, on the other.
Strongest support for the monophyletic statuses of the two
species groupings come from the concatenated genes (Fig.
3) and NJ and MP
TiTv
analyses of cytochrome b alone (Fig.
2A, B), with the monophyly of the large-sized species being
consistently much stronger supported. That the two species
groupings are contained in separate clades has already
been indicated by both genetic (Volobuev et al., 1975; Gra-
phodatsky et al., 1976; Zima and Král, 1984; Taranin et al.,
1991; Hosoda et al., 1993, 2000; Masuda and Yoshida,
1994a, b; Davison et al., 1999; Kurose et al., 2000) and
morphological (e.g., Abramov, 2000a) data, as well as by
the combined evidence (Bininda-Emonds et al., 1999). The
only recent source to provide evidence in favor of an alter-
native relationship is a morphologically based study of
Youngman (1982). Although his study identified Mustela
nivalis and Mustela altaica as close relatives, it included
their clade within the grouping of the large-sized species.
Mustela itatsi has generally been regarded as conspe-
cific with Mustela sibirica, especially by non-Japanese and
non-Russian authors. However, notable genetic and mor-
phological evidence have been accumulated (Graphodatsky
et al., 1979; Watanabe and Kawamoto, 1984; Watanabe et
al., 1985; Masuda and Yoshida, 1994a, b; Abramov, 2000a,
b; Hosoda et al., 2000; Kurose et al., 2000) in support of the
specific distinctness between the two taxa. Our results pro-
vide further evidence of this distinctness. Excepting three
analyses that failed to resolve relationships between these
species, all other analyses indicated a basal placement of
Mustela itatsi with respect to a clade comprising Mustela
sibirica and the subgenus Putorius, with strongest support
yielded by the MP
TiTv
analysis of the combined genes (Fig.
3B).
The phylogenetic position of Mustela lutreola in relation
to Mustela itatsi, Mustela sibirica, and the species of Puto-
rius continues to be problematic. There is conflicting evi-
dence in support of a close affinity of Mustela lutreola with
Mustela itatsi and Mustela sibirica to the exclusion of Puto-
rius (combined genetics and morphology: Bininda-Emonds
et al., 1999; morphology: Youngman, 1982); a grouping of
Mustela lutreola with species of Putorius to the exclusion of
Mustela itatsi and Mustela sibirica (combined IRBP and
cytochrome b: this paper, NJ BS = 100; cytochrome b: Davi-
son et al., 1999, 2000a; Hosoda et al., 2000; Kurose et al.,
2000; this paper, NJ BS = 100, MP
TiTv
BS/DI = 100/9; D-
loop: Davison et al., 2000a; serum immunology: Taranin et
al., 1991); or a basal placement of Mustela lutreola with
respect to a clade containing Mustela itatsi, Mustela sibirica,
and Putorius (IRBP: this paper, NJ BS = 98, MP BS/DI = 96/
3; combined IRBP and cytochrome b: this paper, MP
TiTv
BS/
DI = 67/2, MP
–3Ti
BS/DI = 60/1). The considerable strengths
of support for alternative hypotheses, revealed by our anal-
yses of individual IRBP and cytochrome b nucleotide
sequences alone, indicate significant conflict in the phyloge-
netic signal between the two genes in resolving Mustela
lutreola relationships. As can be inferred from IRBP and
cytochrome b genetic distances among Mustela lutreola,
Mustela itatsi, Mustela sibirica, and Putorius species-group
taxa (Tables 2, 3), this conflict is related to a drastic differ-
ence between the two genes in relative levels of sequence
divergence between Mustela lutreola and species of Puto-
rius. The low level of sequence divergence in cytochrome b
between Mustela lutreola and Putorius species was inter-
preted by Davison et al. (1999, 2000a, b) as a result of rel-
atively recent speciation of Mustela lutreola in a close rela-
tionship to species of Putorius or the effects of interspecific
introgressive hybridization between Mustela lutreola and
Putorius. Our results point to the mitochondrial introgression
(including cytochrome b), rather than a recent origin of Mus-
tela lutreola.
Although some analyses of mitochondrial nucleotide
sequences (cytochrome b: Davison et al., 1999, 2000a;
Hosoda et al., 2000; D-loop: Davison et al., 2000a) found
evidence in favor of the Putorius paraphyly relative to Mus-
tela lutreola, results of other genetically based studies (cyto-
chrome b: Kurose et al., 2000; karyology: Graphodatsky et
al., 1976), as well as morphological (e.g., Youngman, 1982;
Abramov, 2000a) and combined (Bininda-Emonds et al.,
1999) evidence indicate that this subgenus is monophyletic.
Our analyses of IRBP alone were unable to resolve relation-
ships of Putorius, but all our cytochrome b and combined
analyses uniformly confirmed its monophyly, with strongest
support shown by NJ and MP
TiTv
analyses of the concate-
nated genes (Fig. 3A, B).
Whereas a close relationship of Mustela putorius furo to
Mustela putorius putorius and Mustela eversmanii has prob-
ably never been questioned, the particular placement of
Molecular Phylogeny of Mustelidae 257
Mustela putorius furo within this clade has often been con-
sidered controversial (e.g., Blandford, 1987; Davison et al.,
1999, 2000b) in spite of the fact that compelling evidence
from karyology (Zima and Král, 1984, and references
therein), morphology (Rempe, 1970; Wolsan, 1993c, d;
Kitchener et al., 1999), and developmental biology (Volo-
buev et al., 1974; Ternovsky, 1977), as well as additional
supporting evidence from genetics (combined cytochrome b
and D-loop: Davison et al., 1999; serum immunology:
Belyaev et al., 1980), have been presented to indicate that
Mustela putorius furo and Mustela putorius putorius are
either conspecific or at least phylogenetically closer to each
other than either is to Mustela eversmanii. All our analyses
of IRBP alone and the concatenated genes weakly but con-
sistently supported this interrelationship. Apparently, the
only factual evidence contrary to this placement of Mustela
putorius furo comes from analyses of cytochrome b
sequences (Kurose et al., 2000; this paper). Analyses of
Kurose et al. (2000) resulted in weak support for Mustela
putorius furo as either basal to the Mustela putorius puto-
rius-Mustela eversmanii clade or the sister taxon to Mustela
eversmanii. Our NJ analysis of cytochrome b alone also
weakly supported a pairing of Mustela putorius furo and
Mustela eversmanii (Fig. 2A), but maximum-parsimony anal-
yses revealed insufficient resolving power of this gene to
differentiate among any of the alternative hypotheses of
interrelationships among the three taxa.
Timescale for mustelid phylogeny
Origin of Mustelidae
Our fossil-based time estimate for the origin of Mustel-
idae (mustelid-procyonid split), at 28.5 Ma, is close to Bin-
inda-Emonds et al.’s (1999) estimate of 28.1 Ma derived
from six literature estimates (median = 28.1 Ma, mean =
29.4 Ma). Other recent time estimates of the mustelid emer-
gence suggested older dates ranging from approximately 31
to 45 Ma (O’Brien et al., 1989; Wayne et al., 1989, 1991;
Garland et al., 1993; Flynn, 1996; Byrnes et al., 1998; Koep-
fli and Wayne, 1998; Flynn et al., 2000). Most of these esti-
mates were based on either the erroneously assumed (by
McKenna and Bell, 1997) early Oligocene age for the mus-
telids Plesictis and Plesiogale (which are indeed late Oli-
gocene-early Miocene and early Miocene in age, respec-
tively [e.g., Wolsan, 1993a]) or the alleged mustelid status
of the late Eocene or early Oligocene Mustelavus, Mustelic-
tis (both following Baskin, 1998), or Amphicticeps (following
McKenna and Bell, 1997). However, neither Mustelictis
(contrary to Wolsan, 1992, 1993a; Wolsan and Lange-
Badré, 1996) nor Mustelavus or Amphicticeps have a
suprameatal fossa synapomorphic for Mustelidae. Instead,
they show primitive shallow suprameatal fossae that are
present in musteloids basal to the mustelid-procyonid clade
and also occur outside of Musteloidea (e.g., Wolsan, 1999).
This, in addition to other cranial and dental features (see,
e.g., Schmidt-Kittler, 1981), indicates that Mustelavus, Mus-
telictis, and Amphicticeps are neither mustelids nor mem-
bers of the mustelid-procyonid clade.
Origin of Melinae
The species that have most recently been considered
the earliest known melines are the early Miocene Dehmictis
vorax and Trochictis artenensis (Ginsburg and Morales,
2000). The first stratigraphic occurrences of these species
are within the European Neogene Land Mammal Zone MN
3 (e.g., Ginsburg and Morales, 2000). This biostratigraphic
unit corresponds in age to the interval of 18.0–20.5 Ma
(Steininger, 1999, and references therein). This fossil-based
minimum age for the origin of Melinae is in close agreement
with our estimate of 18.1 Ma inferred from cytochrome b
transversions for the divergence between the lineage of
Meles and a clade encompassing the remaining mustelids
used in this study (Table 4), and it disagrees with our IRBP
estimate of this event at 14.5 Ma. That the latter is an under-
estimate is also suggested by our other results. This esti-
mate is 0.2–0.3 Myr younger than our IRBP and cytochrome
b estimates of the divergence time between the Enhydra-
Mustela and Gulo-Martes clades (Table 4), although these
clades were recognized by most our phylogenetic analyses
as diverged from each other after the emergence of the lin-
eage of Meles (Figs. 1A; 2B, C; 3A–C).
Dates of about 7.9 and 13.7 Ma for the divergence of
Melinae (excluding Melogale and Mydaus), suggested by
Hosoda et al. (1993) and Bininda-Emonds et al. (1999),
respectively, are too young and contradict both the fossil
record and our molecular results.
Divergence between the European and Japanese badger lin-
eages
The earliest known finds referred to Meles meles are of
middle Pleistocene age and have been recorded from both
Europe (e.g., Wolsan, 1993e; Griffiths, 1994; Palombo and
Mussi, 2001) and Japan (Kawamura et al., 1989), as well as
from the mainland Asia (Baryshnikov and Batyrov, 1994).
From deposits older than the late Middle Pleistocene and
extending as far back as the Late Pliocene, an array of other
Meles species have been reported (see, e.g., Wolsan,
2001). The morphological and size characteristics of these
putative extinct species are well within the variability range
observed throughout the current Eurasian distribution of
Meles meles, which justifies the conclusion that the late
Pliocene to present-day badgers are conspecific (Wolsan,
2001). If this is true, then our IRBP estimate of a late
Pliocene age, at 2 Ma, for the split between the lineages of
the European Meles meles meles and the Japanese Meles
meles anakuma is consistent with the widely accepted view
that the two taxa are conspecific. Nevertheless, if Meles
meles is really no older than the Middle Pleistocene, as
traditionally conceived, then our divergence-time estimate
provides evidence of specific distinctness between the Euro-
pean and Japanese badgers, a view that has recently
received some support from morphology (Baryshnikov and
Potapova, 1990; Lüps and Wandeler, 1993; Lynch, 1994;
J. J. Sato et al.258
Stubbe et al., 1998; Abramov, 2001).
Origin of Lutrinae
Four genera have recently been considered to be the
oldest lutrines known. These are Kenyalutra (e.g., Hunt,
1996; McKenna and Bell, 1997; Made, 1999), Potamothe-
rium (Thenius, 1989; Stubbe, 1993), Mionictis (= Lartetictis;
e.g., Willemsen, 1992; Baskin, 1998; Ginsburg, 1999), and
Paralutra (e.g., McKenna and Bell, 1997; Heizmann and
Morlo, 1998; Ginsburg, 1999). However, Kenyalutra is
indeed a viverrid (Morales et al., 2000), Potamotherium is a
musteloid very distantly related to Lutrinae (e.g., Baskin,
1998), and Mionictis is a musteline (Heizmann and Morlo,
1998). Therefore, Paralutra appears to be the earliest known
actual lutrine. The first stratigraphic appearance of this
genus has recently been suggested to be during the Early
Miocene (McKenna and Bell, 1997; Ginsburg, 1999, p. 126),
but the last author on p. 147, Willemsen (1992), and Heiz-
mann and Morlo (1998) indicate that it is middle Miocene in
age and within the European Neogene Land Mammal Zone
MN 7+8. This zone covers a time span from 11.1 to 13.5 Ma
(Steininger, 1999, and references therein).
Our molecular results correspond well with the fossil
record. Our time estimates of the split between the lineages
of Enhydra and Mustela, inferred from IRBP total substitu-
tions (at 13.5 Ma) and cytochrome b transversions (at 14.1
Ma), are close to each other and only slightly older than the
earliest fossil record of Lutrinae. These dates are also in
harmony with our other results, being younger than our esti-
mate of 14.7–14.8 Ma for the divergence between the Enhy-
dra-Mustela and Gulo-Martes clades.
A similar date for the lutrine origin, at approximately 13–
14 Ma, results from Koepfli and Wayne’s (1998) study, and
Bininda-Emonds et al. (1999) place this event between 9.9
and 17.1 Ma. Assuming that Lutrinae is monophyletic, our
estimate of 14.7–14.8 Ma for the split between the Enhydra-
Mustela and Gulo-Martes clades indicates that dates of 15–
23 and 20–25 Ma proposed for the divergence of Lutrinae
by Hosoda et al. (2000) and Wayne et al. (1989), respec-
tively, are overestimates.
Origin of Mustelinae
The earliest mustelines known are the early Miocene
Plesiogale angustifrons and Paragale huerzeleri (e.g.,
Schmidt-Kittler, 1981; Wolsan, 1993a). These species have
been recorded from the older part of the European Neogene
Land Mammal Zone MN 2 (e.g., Hugueney, 1997). This part,
MN 2a, corresponds to a span of time from about 21.5 to
22.5 Ma (Schlunegger et al., 1996). This minimum age for
the origin of Mustelinae, inferred from fossils, is older than
the fossil-based minimum divergence ages for Melinae and
Lutrinae. It is also older than our IRBP and cytochrome b
time estimates of the splits between the lineages leading to
Meles or Enhydra, on the one hand, and to mustelines
examined, on the other (Table 4). This provides further evi-
dence supporting the paraphyletic status of Mustelinae with
respect to Lutrinae, and also indicates that the melines, too,
share a musteline ancestor.
Bininda-Emonds et al.’s (1999) estimate of 11.4 Ma for
the divergence of Mustelinae (which they constrained to be
Table 4. Estimates of divergence times (in millions of years before present) for mustelids, based on a mus-
telid-procyonid split at 28.5 Ma and Kimura two-parameter genetic distances derived from total substitutions
among partial nucleotide sequences of IRBP (1185 bp) and from transversions among the complete nucle-
otide sequences of cytochrome b (1140 bp).
Divergence event IRBP Cytochrome b
Meles to Enhydra-Mustela-Gulo-Martes 14.5 18.1
Meles meles meles to Meles meles anakuma 2.0
Enhydra-Mustela to Gulo-Martes 14.8 14.7
Enhydra to Mustela 13.5 14.1
Gulo to Martes flavigula 5.8 6.5
Gulo to subgenus Martes 3.8 8.1
Subgenus Martes to Martes flavigula 6.8 7.8
Martes martes to Martes zibellina 0.43
Mustela vison to other Mustela 8.5 9.9
Mustela erminea to other Mustela except M. vison 6.7 3.9
Mustela nivalis-M. altaica to Mustela itatsi-M. sibirica-M. lutreola-Putorius 4.0 4.8
Mustela nivalis to Mustela altaica 2.4 5.6
Mustela itatsi to Mustela sibirica 2.4 1.7
Putorius to Mustela itatsi 2.7 1.1
Putorius to Mustela sibirica 1.0 1.1
Mustela putorius to Mustela eversmanii 1.5 0.43
Mustela putorius putorius to Mustela putorius furo 0.34 0.86
Molecular Phylogeny of Mustelidae 259
monophyletic) is clearly an underestimate as indicated by
both the fossil record and our molecular results.
Origin of Gulo and the subgenus Martes
The earliest known finds of Gulo are from the Pliocene
deposits of the Adycha River basin and Udunga, Russia
(Sotnikova, 1995; Vangengeim et al., 1998). The fossil local-
ity of Udunga dates to around 3.3 Ma and that of the Adycha
River basin may be about 0.5 Myr older (Vislobokova et al.,
2001).
The early Miocene “Martes” laevidens has recently
been proposed as the oldest known member of the subge-
nus Martes (Anderson, 1994). However, the basicranial
anatomy of “Martes” laevidens (as evidenced by its only pre-
served cranium stored under register number 1937 II 13539
in the Bayerische Staatssammlung für Paläontologie und
historische Geologie, Munich, Germany) clearly indicates
that this species is not congeneric with living martens.
Hence, the earliest known actual representative of the sub-
genus Martes appears to be Martes wenzensis (Anderson,
1970, 1994; Rabeder, 1976). The earliest record of this spe-
cies is from the Pliocene deposits of We
˛˙
ze 1, Poland (Wol-
san, 1989, and references therein), which date to 3.3–4.0
Ma (G
´
lazek et al., 1976; G
´
lazek and Szynkiewicz, 1980).
The fossil-based minimum ages for the origin of Gulo
and the subgenus Martes are in harmony with our molecular
results on the time of divergence between Gulo and martens
and that between the subgenus Martes and other mustel-
ines, which are contained within a span that ranges in age
from 3.8 to 8.1 Ma (Table 4). Date estimates of about 5 and
5–6 Ma for the divergence of the subgenus Martes, derived
from cytochrome b by Carr and Hicks (1997) and Hosoda et
al. (1997), respectively, are within this time span. Slightly
older divergences of the subgenus Martes and the genus
Gulo, at 8.2 Ma, were postulated by Bininda-Emonds et al.
(1999). A substantially older date of 10–14 Ma proposed for
these events by Hosoda et al. (2000) is apparently an over-
estimate.
Origin of Martes martes and Martes zibellina
The earliest undoubted record of Martes martes is from
the last interglacial period, the Eemian (e.g., Wolsan, 1993e;
Anderson, 1994; Kolfschoten, 2000), at around 120 ka
(Sánchez Go
~
ni et al., 2000). In addition, fossils that may
represent this species have also been reported from depos-
its extending as far back as 400 ka (Wolsan, 1993e). The
earliest known finds of Martes zibellina, in turn, are late
Pleistocene in age (e.g., Vereshchagin and Baryshnikov,
1985; Anderson, 1994) and thus are younger than 130 ka
(Kolfschoten and Gibbard, 2000; Sánchez Go
~
ni et al., 2000).
The fossil record of the extinct Martes vetus, which has
been considered ancestral to both Martes martes and Mar-
tes zibellina (e.g., Anderson, 1970, 1994; Wolsan, 1989),
ranges from almost the beginning of the Pleistocene (which
is dated at nearly 1.8 Ma [e.g., Lindsay, 2001]) to about 400
ka, with a possible extension to approximately 300 ka (Wol-
san, 1993e). Thus, the paleontological data are in good
agreement with our estimate of about 430 ka, inferred from
cytochrome b transversions, for the split between the spe-
cies Martes martes and Martes zibellina or their lineages. In
view of the paleontological and molecular evidence pre-
sented here, dates of 1.8 and 2–4 Ma postulated for this
event by Bininda-Emonds et al. (1999) and Hosoda et al.
(2000), respectively, are overestimates.
Origin of Mustela
The earliest undoubted Mustela remains recovered
come from lower Pliocene strata dated at 3.4–4.2 Ma (Morlo
and Kundrát, 2001). This minimum divergence age for Mus-
tela, derived from paleontological data, is close to Hosoda
et al.’s (1993) and O’Brien et al.’s (1989) estimates of this
event at less than 3.9 Ma and over 4 Ma, respectively.
Nonetheless, the fossil record does not contradict our
molecular results, which place the beginning of this genus
between 8.5–9.9 Ma (divergence of Mustela vison) and
13.5–14.1 Ma (Enhydra-Mustela split). Other recent esti-
mates of the Mustela origin at 10.4–11.4 Ma (Bininda-
Emonds et al., 1999), between 8.4–11.6 and 13.7–17.4 Ma
(Kurose et al., 2000), at 12–20 Ma (Wayne et al., 1989), and
between 10–14 and 15–23 Ma (Hosoda et al., 2000) either
closely approach our estimate or suggest a more remote
emergence of the genus.
Origin of polecats
Three extant species are currently recognized as mem-
bers of the subgenus Putorius. These are Mustela putorius
(including Mustela putorius furo), Mustela eversmanii, and
Mustela nigripes (e.g., Wolsan, 1993c; Abramov, 2000a;
Owen et al., 2000). While Mustela nigripes first appears at
the Early-Middle Pleistocene boundary, about 750–850
thousand years (kyr) ago (Owen et al., 2000), the fossil
record of Mustela putorius and Mustela eversmanii com-
mences in the late Middle Pleistocene, about 400 kyr ago
(Wolsan, 1993c-e). These data are in close agreement with
our estimate of 430 ka inferred from cytochrome b transver-
sions for the divergence between Mustela putorius and Mus-
tela eversmanii. This estimate approaches Kurose et al.’s
(2000) and Wolsan’s (1993c) estimates of this divergence at
520 ka and 600–700 ka, respectively. Our IRBP date of 1.5
Ma for this event, as well as dates of 1–3 Ma (Wayne et al.,
1991), about 2–3 Ma (O’Brien et al., 1989), and 2.8 Ma (Bin-
inda-Emonds et al., 1999) are overestimates in view of our
IRBP and cytochrome b dates of 1.0 and 1.1 Ma, respec-
tively, for the split between the lineages of Putorius and
Mustela sibirica and our cytochrome b date of 1.1 Ma for the
split between the lineages of Putorius and Mustela itatsi.
Our results place the origin of the subgenus Putorius at
approximately 1 Ma (Table 4). This date is in harmony with
the fossil-based minimum divergence time (at 750–850 ka)
derived from the earliest stratigraphic occurrences of the
extant species of Putorius. Recent age estimates of the
Putorius divergence included dates of less than 340 ka
J. J. Sato et al.260
(Hosoda et al., 1993), 520 ka to 1.06 Ma (Kurose et al.,
2000), 2.8 Ma (Bininda-Emonds et al., 1999), 2–4 Ma
(Hosoda et al., 2000), and around 2–5 Ma (O’Brien et al.,
1989).
Thus, both our results and the majority of recent time
estimates of the emergence of Putorius contradict the view
that the extinct Mustela stromeri is a member of this subge-
nus. Mustela stromeri has persistently been considered the
earliest known representative of Putorius and a probable
ancestor of both Mustela putorius and Mustela eversmanii
since it was first described by Kormos (1934). The
undoubted records of this poorly known species are from the
Late Pliocene, and its questionable finds have also been
reported from the Early and Middle Pleistocene (e.g., Wol-
san, 1993c, e). The late Pliocene records come from Hun-
garian localities Beremend 5 and Osztramos 7 (Jánossy,
1986), which date to around 3.1–3.2 Ma (Montuire, 1996).
Taking account of this age and in view of our divergence-
time estimates (Table 4), the inclusion of Mustela stromeri
in Putorius causes this subgenus to be paraphyletic with
respect to both Mustela itatsi and Mustela sibirica.
Origin of Mustela putorius furo
Both Kurose et al.’s (2000) date of 1.06 Ma and our
cytochrome b date of 860 ka for the split between the lin-
eages of Mustela putorius furo and Mustela putorius puto-
rius are overestimates in view of our cytochrome b date of
about 430 ka for the separation between Mustela putorius
and Mustela eversmanii. Our IRBP date of 340 ka for the
divergence of the Mustela putorius furo lineage, however, is
in agreement with both our other divergence-time estimates
(Table 4) and the fossil-based minimum age of approxi-
mately 400 ka for the emergence of Mustela putorius (Wol-
san, 1993e).
Domestic Ferret has been recorded since the fourth
century BC when Aristotle mentioned it in his work (e.g.,
Davison et al., 1999). It is generally thought to have been
domesticated somewhere in the Mediterranean region,
although Strabo explicitly indicated its African origin (e.g.,
Davison et al., 1999) and Linnaeus (1758) named this taxon
as being from Africa. The earliest African record of Mustela
putorius is late Pleistocene in age (Aouraghe, 2000) and
thus younger than 130 ka (Kolfschoten and Gibbard, 2000;
Sánchez Go
~
ni et al., 2000). Our IRBP date of 340 ka for the
separation between the lineages of Mustela putorius furo
and Mustela putorius putorius is in harmony with this fossil
evidence from North Africa and supports the view that
Domestic Ferret was domesticated from the African branch
of Mustela putorius, which diverged from the European
branch in the late Middle Pleistocene to immigrate to North
Africa.
ACKNOWLEDGMENTS
We thank Han Sang-Hoon (Korea Wildlife Information and
Research Center), Daniel J. Harrison (Maine University), Mitsuhiro
Hayashida (Yamagata University), Alexei P. Kryukov (Russian
Academy of Sciences), Yoshitaka Obara (Hirosaki University), and
Zhang Ya-Ping (Chinese Academy of Sciences) for their help in col-
lecting samples; Shumpei P. Yasuda (Hokkaido University) for
assistance at laboratory work; and Kurt Heissig (Bayerische
Staatssammlung für Paläontologie und historische Geologie) for
access to specimens of “Martes” laevidens. This study was sup-
ported in part by grants-in-aid for scientific research from the Min-
istry of Education, Science, Sports, and Culture, Japan.
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... To date, works by Volobuev and Ternovsky [16], Volobuev et al. [17], Graphodatsky et al. [18], Graphodatsky et al. [19], and Graphodatsky and Radjabli [20], strongly affiliated with the Siberian Branch of the Russian Academy of Sciences in Novosibirsk (Russia), are the primary sources of information on M. lutreola karyotype. Further research on the genetics of European mink relates primarily to genetic markers [9,21], the phylogenetic relationships of the species [22-26], noninvasive methods of identification [27], assessment of intraspecies genetic diversity [8,14,[28][29][30], and mitochondrial DNA (mtDNA) studies [24,28,31,32]. Worth mentioning are studies concerning molecular ecology on issues relating to European mink and implementing genetic research methods [33][34][35][36]. ...
... Due to its high cytogenetic similarity and proven close phylogenetic relationships [22,24,34,42], the size of the nuclear genome of European mink can be estimated on the basis of the sequenced genome of the ferret (MusPutFur1.0, RefSeq assembly accession: GCF_000215625.1) and M. putorius (polecat_10x_lmp_bionano, GenBank assembly accession: GCA_902207235.1) as being about 2.411-2.474 million bp, the content of GC pairs as about 42%, and the number of genes as about 27,300 [58]. ...
... The phylogenetic analysis based on the nucleotide sequence of the nuclear irbp gene and the mitochondrial cytb gene, performed by Sato et al. [24], proved that M. lutreola belongs in the clade including European polecat, steppe polecat, Siberian weasel, and Japanese weasel ( Figure 1A,B). The close evolutionary relationship between European mink, European polecat, and steppe polecat was also evidenced by the results of a phylogenetic analysis based on the sequences of mitochondrial genes 12S rRNA [31,42,95] and cytb [22,25,31], and the gene encoding the NADH dehydrogenase subunit 2 [31], as well as the nuclear genes (the gene for thyroxine-binding globulin [31], irbp [31,95], the transthyretin-encoding gene [31], and the Mel08 complex repetitive flanking regions [25]). ...
Article
Full-text available
The purpose of this review is to present the current state of knowledge about the genetics of European mink Mustela lutreola L., 1761, which is one of the most endangered mammalian species in the world. This article provides a comprehensive description of the studies undertaken over the last 50 years in terms of cytogenetics, molecular genetics, genomics (including mitogenomics), population genetics of wild populations and captive stocks, phylogenetics, phylogeography, and applied genetics (including identification by genetic methods, molecular ecology, and conservation genetics). An extensive and up-to-date review and critical analysis of the available specialist literature on the topic is provided, with special reference to conservation genetics. Unresolved issues are also described, such as the standard karyotype, systematic position, and whole-genome sequencing, and hotly debated issues are addressed, like the origin of the Southwestern population of the European mink and management approaches of the most distinct populations of the species. Finally, the most urgent directions of future research, based on the research questions arising from completed studies and the implementation of conservation measures to save and restore M. lutreola populations, are outlined. The importance of the popularization of research topics related to European mink genetics among scientists is highlighted.
... It remains to be discussed whether these Miocene forms should be assigned to Martes (e.g., Jiangzuo et al., 2021), and emerges as an urgent task to perform a comparison with the type species of the genus. While some of these forms have morphological features closely related to Guloninae, others could instead represent stem groups outside of the crown clade Guloninae (Anderson, 1970(Anderson, , 1994Sato et al., 2003;Wang et al., 2012;Samuels and Calvin, 2013;Li et al., 2014;Samuels et al., 2018). For instance, Anderson (1994) proposed Martes laevidens Dehm, 1950, from the lower Miocene (MN3) of Wintershof-West (Germany) as the earliest member of the genus, but such assignation to Martes has been recently discarded (Sato et al., 2003)though it is not fully discussed (as occurred with the remaining European middle Miocene species, Valenciano et al., 2020a). ...
... While some of these forms have morphological features closely related to Guloninae, others could instead represent stem groups outside of the crown clade Guloninae (Anderson, 1970(Anderson, , 1994Sato et al., 2003;Wang et al., 2012;Samuels and Calvin, 2013;Li et al., 2014;Samuels et al., 2018). For instance, Anderson (1994) proposed Martes laevidens Dehm, 1950, from the lower Miocene (MN3) of Wintershof-West (Germany) as the earliest member of the genus, but such assignation to Martes has been recently discarded (Sato et al., 2003)though it is not fully discussed (as occurred with the remaining European middle Miocene species, Valenciano et al., 2020a). Within this complex scenario, it is debatable whether the early late Miocene Martes melibulla Petter, 1963, from the Vallès-Penedès and Madrid basins (Spain) represents the first true Martes (Valenciano, 2017;Valenciano et al., 2020a), or it is instead the late Pliocene Martes wenzensis Stach, 1959 from Wezė 1 (Poland) (Sato et al., 2003). ...
... For instance, Anderson (1994) proposed Martes laevidens Dehm, 1950, from the lower Miocene (MN3) of Wintershof-West (Germany) as the earliest member of the genus, but such assignation to Martes has been recently discarded (Sato et al., 2003)though it is not fully discussed (as occurred with the remaining European middle Miocene species, Valenciano et al., 2020a). Within this complex scenario, it is debatable whether the early late Miocene Martes melibulla Petter, 1963, from the Vallès-Penedès and Madrid basins (Spain) represents the first true Martes (Valenciano, 2017;Valenciano et al., 2020a), or it is instead the late Pliocene Martes wenzensis Stach, 1959 from Wezė 1 (Poland) (Sato et al., 2003). Unfortunately, this systematics issue is far from being fully clarified with the known mustelid material. ...
Article
Small to medium-sized mustelids from the last 18 million years represent a heterogeneous group of carnivorans with a wide-ranging record in the northern hemisphere. They were first referred to the genera Mustela and Martes, but lately ascribed to the latter, and hence considered as the longest-lived genus within Mustelidae. However, a great many of these forms have been based upon fragmentary material and Martes has conformed progressively to a wastebasket nomen for species of uncertain relationships. Here, we describe dentognathic material of a small-sized mustelid from three middle Miocene (MN7 + 8, latest Aragonian) localities of the Iberian Peninsula that constitutes a new genus and species. Aragonictis araid, gen. et sp. nov. represents a distinct taxon if compared with early/middle Miocene forms ascribed to "Martes" spp., especially the similar-sized early Miocene Circamustela? laevidens and the middle Miocene "Martes" caedoti and "Martes" delphinensis. The finding of particular features in A. araid (low p2-3, loss or reduction of the p4 accessory cuspid with its main cuspid centrally located, presence of a sharp, beveled and lingually open m1 talonid, and reduction of M1 lingual platform) indicates affinities with the late Miocene Circamustela in the range of hypercarnivory. Our reassessment of "Martes" indicates possible evidence of cladogenesis for Miocene mustelidae with, at least, two different events being recognized in Europe-the latter during MN7 + 8 to MN9 with presence of Aragonictis and Circamustela. The finding of A. araid further confirms the presence of more densely forested environments than expected in inner Iberia during the latest middle Miocene.
... The domestic ferret (Mustela furo or M. putorius furo), generally thought to be domesticated from M. putorius (see, e.g. Sato et al. 2003), was bred in captivity as early as the fourth century BC and was introduced to many parts of the world (Nowak and Paradiso 1983). ...
... The genetic differentiation was the same as the level of intraspecific variations of other mustelids. M. eversmanii likely diverged from M. putorius approximately 1.5 million years ago based on the nuclear DNA region inter-receptor binding protein (IRBP), though cytochrome b (CytB) transversions indicate a younger date of 430,000 years (Sato et al. 2003). Since these species are occasionally reported to hybridize where they have an overlap in their distribution, the reality of a true species split has been debated (Blandford 1987), and some authors have also considered if M. putorius, M. eversmanii and M. nigripes could be viewed as a single Holarctic species (Anderson 1977;Anderson et al. 1986;O'Brien et al. 1989). ...
Article
Full-text available
European mustelids include the European polecat, Mustela putorius, and the steppe polecat, M. eversmanii. Both occur sympatrically in the Pannonian Basin, where M. eversmanii hungarica represents the westernmost part of the latter species and they allegedly hybridize. We investigated the morphological relationships in sympatric and allopatric populations of these mustelids with representative sampling, taxonomic and geographic coverage. We evaluated inter- and intraspecific patterns of morphological differentiation of 20 cranial measurements and four external traits by distance-based morphometric approaches and multivariate analyses. Our results revealed a considerable heterogeneity in cranial morphology. The two species appeared to be clearly differentiated although sympatric populations were closer to each other and had a slight overlap in the morphometric space. Within M. eversmanii, the subspecies and the nominal taxon only partially overlapped, and M. eversmanii eversmanii was more distant from M. putorius than subspecies hungarica. Although morphometric analyses revealed several intermediate individuals in size in sympatric M. eversmanii and M. putorius populations, only a small fraction of such specimens showed conflict in discrete morphological characterswith the diagnostic discriminant function.We interpret these results as an indication of ongoing hybridisation between sympatric populations, but the low number of hybrids identified suggests limited genetic exchange between the species.
... To clarify the spatiotemporal evolutionary reasons for the extensive ecomorphological diversification of this family, and to address the subfamily classification issue, molecular systematic analyses using a pluralistic phylogenetic method with a supermatrix containing many nucDNA sequences have been conducted (e.g. Koepfli & Wayne, 2003;Sato et al., 2003Sato et al., , 2004Sato et al., , 2006Sato et al., , 2009Fulton & Strobeck, 2006;Koepfli et al., 2008;Wolsan & Sato, 2010;Yu et al., 2011b). used data from 18 genera and 38 species of the family Mustelidae with 8492-bp nucleotide characters from 10 genetic loci, 9 nuclear genes, and 1 mitochondrial gene (Table 2.1). ...
Chapter
Recent advancements in phylogenetic resolution at higher taxonomic levels within the mammalian order Carnivora have been stimulated by the increasing application of nuclear DNA, which is less homoplastic than mitochondrial DNA, and therefore better suited for studying deep‐level (e.g. among genera or older) relationships. Immense progress in sequencing nuclear and mitochondrial DNAs from carnivoran species has resulted in a wealth of data in publicly available DNA databases, allowing an improved understanding of phylogenetic relationships at every taxonomic level using the ‘total evidence’ supermatrix or supertree method. Here, we review recent molecular systematic studies for one of the most enigmatic species, the red panda, Ailurus fulgens , and show that the use of nuclear DNA, Bayesian and maximum likelihood phylogenetic inference, and the supermatrix approach have improved the resolution of the phylogenetic position of this species. Secondly, we show that such methodological improvements have also clarified the evolution of the family Mustelidae (weasels, martens, otters, badgers, and allies). We demonstrate this in light of phylogeny, chronology, and historical biogeography and provide an up‐to‐date subfamily classification of the Mustelidae. Finally, we discuss the implications of molecular systematics to setting and defining conservation priorities on the basis of the EDGE (Evolutionarily Distinct and Globally Endangered) value, and conclude that the supermatrix‐based priority setting is preferable to the supertree‐based one.
... Japanese weasel Japan Y Long regarded as a subspecies of Siberian weasel, M. sibirica, but morphometric and genetic studies strongly support its elevation to species level (Abramov, 2000;Kurose et al., 2000;Sato et al., 2003;Suzuki et al., 2011;Masuda et al., 2012). ...
Chapter
Full-text available
This Appendix provides a detailed (but non-exhaustive) list of the main small carnivoran taxa (n = 72) that have been – to date – the subject of discussions as to whether they should be attributed species or subspecies level.
... The degree of similarity between the complete sequence of the European mink mitogenome and the known mitochondrial genomes of other animals supports previous fin-dings regarding phylogenetic relationships within the class Mammalia L., 1758 and the taxonomic position of M. lutreola [24][25][26][27][28]55,60,[151][152][153]. This similarity reflects the European mink belonging to taxa from successive systematic levels. ...
Article
Full-text available
In this paper, a complete mitochondrial genome of the critically endangered European mink Mustela lutreola L., 1761 is reported. The mitogenome was 16,504 bp in length and encoded the typical 13 protein-coding genes, two ribosomal RNA genes and 22 transfer RNA genes, and harboured a putative control region. The A+T content of the entire genome was 60.06% (A > T > C > G), and the AT-skew and GC-skew were 0.093 and −0.308, respectively. The encoding-strand identity of genes and their order were consistent with a collinear gene order characteristic for vertebrate mitogenomes. The start codons of all protein-coding genes were the typical ATN. In eight cases, they were ended by complete stop codons, while five had incomplete termination codons (TA or T). All tRNAs had a typical cloverleaf secondary structure, except tRNASer(AGC) and tRNALys, which lacked the DHU stem and had reduced DHU loop, respectively. Both rRNAs were capable of folding into complex secondary structures, containing unmatched base pairs. Eighty-one single nucleotide variants (substitutions and indels) were identified. Comparative interspecies analyses confirmed the close phylogenetic relationship of the European mink to the so-called ferret group, clustering the European polecat, the steppe polecat and the black-footed ferret. The obtained results are expected to provide useful molecular data, informing and supporting effective conservation measures to save M. lutreola.
... Le Furet (Mustela putorius furo) est une forme domestiquée du Putois d'Europe, qui aurait divergé de la sous-espèce type voilà 340 000 ans selon Sato et al. (2003). C'est cette forme, dont la présence est attestée en Afrique du Nord par des écrits depuis l'Antiquité, qui a été domestiquée. ...
Technical Report
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Le Putois d’Europe (Mustela putorius) est un petit carnivore de la famille des mustélidés. Ses effectifs ont fortement chuté au cours du XXème siècle, en France comme ailleurs en Europe. Parmi les nombreuses causes identifiées, les principales semblent être les pratiques d’élimination directe et la perte, dégradation et fragmentation de son habitat. Bien que suscitant des inquiétudes, l’état de conservation de l’espèce en France était peu documenté jusqu’à l’enquête de la Société française pour l'étude et la protection des mammifères (SFEPM) de 2017 « Protéger le Putois », qui met en évidence la situation défavorable des populations de putois au niveau national. Ce constat est corroboré par l’État à travers les publications de l’Office français de la biodiversité. La demande d'inscription du Putois d'Europe sur la liste des mammifères protégés en France, portée par la SFEPM, est appuyée par les instances scientifiques (Muséum national d’Histoire naturelle, Conseil national de protection de la nature, Union internationale pour la conservation de la nature). Dans l’optique où l’espèce serait à court terme protégée par la loi française, ce document propose un ensemble de mesures cohérentes et opérationnelles permettant de restaurer durablement les populations de Putois en France tout en assurant un suivi de l’état des populations sur le territoire.
... It was originally identified as Martes (Downs 1951); however, Anderson (1994) and Hughes (2012) have questioned whether many "Martes" taxa prior to the late Miocene were related to the extant genus. Sato et al. (2003) suggested the oldest true Martes is M. wenzensis Frisch, 1775 from the Pliocene of Poland (Wolsan 1989). Anderson (1994) suggested the extant Martes americana Turton, 1806 is a late Pleistocene immigrant to North America. ...
... The 'ineptus' clade observed in the mitochondrial gene trees was retained, whereas the second clade comprised the 'Limpopo' and 'Malawi' groups observed in the mitochondrial gene regions. The IRBP gene is a single-copy gene and is ideal for phylogenetic and evolutionary studies (Stanhope et al. 1996;Sato et al. 2003). Phylogenetic analyses using nuclear gene regions have been known to be compounded by incomplete lineage sorting (Maddison and Knowles 2006). ...
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Results are reported of a phylogenetic analysis of Mustelida. Phylogenetic definitions and diagnoses are provided for Mustelida, Pinnipedia, Ailuridae, Procyonidae and Mustelidae.
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The locality Untermaßfeld yielded a rich invertebrate and vertebrate fauna of an age within the latest Waalian to latest Bavelian interval of the Early Pleistocene. Among numerous remains collected, two specimens representing the family Mustelidae have been recognized. The specimens consist of a partial skull with some incisor, premolar, and molar teeth and a fragment of mandible with molar teeth. Both the specimens are described and referred to the extinct badger Meles hollitzeri Rabeder, 1976 known from the Austrian sites Deutsch-Altenburg 2C1 (type locality) and 4B of Waalian age, as well as from layers 6 and 4b of Treugolnaya Cave in the Caucasus Mountains, representing different ages within the late Early to early Middle Pleistocene interval. — Monographien des Römisch-Germanischen Zentralmuseums Mainz 40(2):659–671.
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