J . Zool., Lond. (1 992) 228, 445-453
Albumin evolution and phylogenetic relationships among Greek rodents of the
families Arvicolidae and Muridae
N . P. NIKOLETOPOULOS,
B. P. CHONDROPOULOS
A N D S . E. FRAGUEDAKIS-TSOLIS
Section of Animal Biology, Department of Biology, University of Patra, 260 01 Patra, Greece
(Accepted 30 September 1991)
(With 1 figure in the text)
The phylogenetic relationships of seven rodent species Microtus atticus, M . thomasi, M . epiroticus
(family Arvicolidae) and Mus domesticus, Raiius norvegicus, Apodemus J7avicollis and A .
mystacinus (family Muridae) have been studied. In order to define these relationships we study the
albumin evolution using the micro-complement fixation test (MC’F). No phylogenetic (immunological) distance between M . atticus and M . thomasi was found, a fact which confirms from the
biochemical point of view the opinion that the former taxon is a synonym of the latter one. A
molecular time scale relating MC’F immunological distances and geological time was established
based on the assumption of a rate of 100 amino acid substitutions per 16-20 million years. The
time ofdivergence between M . epiroticus and M . thomasiwas estimated to be 0.5-0.6 million years
ago (Pleistocene). Such a recent divergence corroborates the opinion based on morphological and
protein electrophoretic criteria according to which Terricola (formerly Pitymys) must be
considered as a subgenus of the genus Microtus and not as a distinct genus Piiyrnys, as previously
had been accepted. Apodemusflavicollis and A . mystacinus were separated about 0.65-0.8 million
years ago (Pleistocene). The Rattus norvegicus lineage was separated 10-12.5 million years ago
(end of Miocene), shortly before the Mus and Apodemus divergence. Our data indicate that the
common ancestor of Arvicolidae and Muridae lived 20-25 million years ago (early Miocene). All
these results are in agreement with paleontological and some recent DNA-DNA hybridization
and electrophoretic data.
Contents
Page
. . . . . . . . . . . . . . . . . . .
Introduction
Materials and methods . . . . . . . . . . . . . . .
Animals . . . . . . . . . . . . . . . . . . . . .
. . . .
Albumin isolation and antisera preparation
M C F experiments and evolutionary tree construction
Results and discussion . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . .
445
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446
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451
Introduction
The discrimination of the phylogenetic relationships revealed within the rodent families
Arvicolidae and Muridae have been based on morphological, karyological and biochemical data
(Brownell, 1983; Sarich, 1985; Gill et al., 1987; Modi, 1987; Catzeflis et al., 1987, 1989; Chaline &
Graf, 1988). Moreover, such studies concerning the classification and evolution within and
between the families of Arvicolidae and Muridae in Greece are already known (Ondrias, 1966;
Fraguedakis-Tsolis & Ondrias, 1977;Niethammer & Krapp, 1978, 1982; Fraguedakis-Tsolis et al.,
445
0 1992 The Zoological Society of London
446
N . P. NIKOLETOPOULOS ET A L
1983; Bonhomme e t al., 1984; Fraguedakis-Tsolis & Chondropoulos, 1986; Fraguedakis-Tsolis et
al., 1986; Giagia e t al., 1987). However, many questions regarding the taxonomic status of these
taxa still remain to be answered.
It is generally accepted that molecular data offer a much more regular pattern of evolutionary
change compared to that given by morphological and physiological characters.
As is already known, the immunological studies of serum albumin using the MC’F method in
mammalian and non-mammalian taxa reveal the degree of amino acid differentiation between the
compared proteins. This differentiation is broadly considered as a reliable estimator of the
divergence time of various evolutionary lineages (Sarich, 1969 a, b; Maxson &Wilson, 1974,1975;
Wilson, Carlson & White, 1977; Maxson, 1981). Thus, the albumin system studied by the MC’F
method seems valuable for phylogenetic studies, because this technique provides information on
both the cladistic and chronistic aspects of phylogeny.
In the present work a study based on the immunological study of serum albumin evolution was
made to elucidate the phylogenetic problems between and within the rodent families Arvicolidae
and Muridae represented here by three and four species, respectively. Analogous investigations on
rodents are also known (Ellis & Maxson, 1979, 1980; Fuller et al., 1984; Sarich, 1985).
Materials and methods
Animals
A total of 74 individuals of the species Microtus (Terricola) atticus, Microtus (Terricola) thomasi, Microtus
(Microtus) epiroticus belonging to the family Arvicolidae, and Mus domesticus, Rattus norvegicus, Apodemus
flavicollis, Apodemus mystacinus of the family Muridae, were live-trapped at 3 localities of the Greek
mainland, Veria ( M . epiroticus), Itea ( M . thomasi) and Patra (all other taxa), during the period 1985-1988.
The above systematic nomenclature was based on the recent taxonomic revision of the European groundvoles of the genus Microtus according to which 2 distinct subgenera are proposed, Microtus and Terricola
(Brunet-Lecomte, 1988, 1990; Chaline, Brunet-Lecomte & Graf, 1988). The species included in the latter
subgenus was formerly classified to the subgenus Pitymys that is now considered to occur only in North
America.
Albumin isolation and antisera preparation
Blood was obtained by cardiac puncture of the anaesthetized animals and albumin was isolated by using
Sephadex G- 150 and DEAE-cellulose columns.
Antisera were prepared in New Zealand white rabbits against the purified albumins of the following
species, Microtus ( T . ) thomasi, Microtus ( T . ) atticus, Mus domesticus, Rattus norvegicus and Apodemus
Javicollis, according to the procedure described by Prager & Wilson (1971). Immunization of 3 rabbits
against each albumin was carried out. Such a procedure helps the reduction of errors in irnmunological
distance estimates, resulting from variation in specificity of antisera produced by different rabbits. In each
case, the resulting antisera against a particular rodent albumin were titrated using the MC’F procedure and
pooled in an inverse proportion to their titres (Prager & Wilson, 1971).
MC‘F experiments and evolutionary tree construction
Albumin/anti-albumin cross-reactions were measured by quantitative micro-complement fixation tests
(MC’F) according to the method of Champion et al. (1974). Cross-reactions data are given in immunological
PHYLOGENETIC RELATIONSHIPS I N RODENTS
447
distance units (ID), which are thought to be a measure of amino acid sequence differences between the
albumins of the species being compared. It is known that for albumin one unit of immunological distance is
roughly equivalent to one amino acid difference (Maxson & Wilson, 1974).
An evolutionary tree based on units of immunologicaldistance was constructed, using the method of Fitch
& Margoliash (1967). This method is evaluated as the most reasonable to use for constructing phylogenetic
trees from distance matrices when immunological data are available (Prager & Wilson, 1978). For the
construction of this tree the average immunologicaldistances obtained in reciprocal tests were used. For the
species M . ( M . ) epiroticus and A . mystacinus a correction of the unidirectional immunologicaldistances was
made, using Beverley & Wilson’s (1982) method of unidirectional attachment.
Results and discussion
The average titre and slope of the five rodent antisera used in this work were about 4,200 and
332, respectively (Table I). The values of these two parameters are not mentioned in other MC’F
studies concerning rodent albumin phylogeny; but our values lie within the limits given in the
literature for other mammals, reptiles and amphibians for which a variety of values (especially for
the titre) is known (Wallace, King & Wilson, 1973; Champion et al., 1974; Maxson & Szymura,
1979; Chen, Mao & Ling, 1980; Maxson, 1981; Collier & O’Brien, 1985). In Table I1 the results of
the MC’F tests made for seven species of rodent against five antisera are given. It must be noted
here that Beverley & Wilson (1982) have suggested that, in the cases of reciprocal MC’F tests, the
reliability of immunological data for estimating the amino acid sequence divergence between
proteins is proportional to the percentage standard deviation from reciprocity. In our
experiments, the standard deviation value was found to be 6.0, which is within the range reported
for similar works (Ellis & Maxson, 1980; Beverley & Wilson, 1982; Fuller, Lee & Maxson, 1984).
The average reciprocal values of albumin immunological distance were used to construct the
phylogenetic tree shown in Fig. 1. The goodness of fit of this tree to the input data can be
statistically evaluated by either the Fitch & Margoliash (1967) percentage standard deviation (s) or
the Prager & Wilson (1976) percentage error (F). In the case of our data, the above-mentioned
values are s = 7.4 and F = 5.6, respectively. These values are comparable to those given in other
phylogenetic studies using albumin MC’F data. Actually, Prager & Wilson (1978), Ellis & Maxson
(1980), Fuller et al. (1984) and Collier & O’Brien (1985), have reported percentage standard
deviation values ranging from 1.4 to 21.8 and percentage error values from 1.1 to 16-5.
The taxonomic status of M . ( T . )atticus (Miller, 1910)and M . ( T . )thomasi (Barett & Hamilton,
1903) is controversial. Thus, Kratochvil(l97 l), using morphological criteria, considered them as
two distinct species; Stamatopoulos (1 985) reached the same conclusion using morphological and
TABLEI
Micro-complementJixationtitre and slope valuesfor
each antiserum pool prepared against the serum
albumin of the species referred
Species
Titre
Slope
Apodemus flauicollis
Rattus noruegicus
Mus domesticus
Microtus (Terricola) atticus
M . (Terricola) thomasi
3,550
1,600
4,200
5,120
6,500
355
280
380
335
312
N . P. NIKOLETOPOULOS ET A L .
448
TABLEI1
Albumin immunological distances (ID units) among the studied species of the rodent families
Muridae and Arvicolidae
Immunological distance (antiserum against)
Species
A.f.
R.n.
M.d.
M. (T.)a.
M. (T.) t.
Apodemus flavicollis
Rattus norvegicus
Mus domesticus
Microtus (Terricola) atticus
Microtus (Terricola) thomasi
Microtus (Microtus) epiroticus
Apodemus mystacinus
0
61
61
57
102
102
100
4
0
63
65
116
138
156
0
118
136
152
0
0
3
117
61
0
118
132
132
134
65
118
119
60
0
3
118
A.f: Apodemusflavicollis, R.n: Rattus norvegicus, M.d: Mus domesticus, M. (T.) a: Microtus
(Terricola) atticus, M. (T.) t: Micrvtus (Terricola) thomasi
ethological data. In contrast, Niethammer (1974), ZivkoviC, Petrov & Rimsa (1975), Petrov &
ZivkoviC (1977, 1979) and Corbet (1978) suggested that these two taxa represent the same species.
On the basis of the data presented in this study (Table II), the opinion that the M . ( T . ) thomasi and
M . ( T . ) atticus are not biochemically separated species is strongly supported. Thus, the M . ( T . )
atticus could be considered a synonym of M . ( T . ) thomasi. In corroboration of this view is the fact
that, at least in the laboratory, no detectable reproductive isolation exists between these two forms
20
2 Apodemus mystacinus
c
38
L
Microtus (Tefficola)
thomasi
92
k! Microtus (Micfotus)
epiroticus
MYA
I
I
I
1
30
20
10
0
I
Oligocene
I
I
Miocene
Pliocene
I
Pleistocene
FIG.1. Phylogenetic tree ofthe seven studied taxa of rodents constructed on the basis oftheir albumin relationships. The
numbers on the branches are the amounts of albumin change (expressed in immunological distance units) estimated to have
occurred along each branch. This tree was rooted at midlength of the deepest branch joining the clades Muridae and
Arvicolidae.
PHYLOGENETIC RELATIONSHIPS I N RODENTS
449
(Giagia & Stamatopoulos, unpubl. data), although they exhibit some karyotypic differences
(Giagia, 1985).
The problem of the determination of an evolutionary rate in mammals has been thoroughly
discussed but a generally accepted opinion does not exist. Sarich & Wilson (1967 a, b), working
with primates, proposed the use of the serum albumin as a molecular clock, accepting a constant
rate of this protein evolution; such a rate has also been applied to other mammal groups (Sarich,
1969, a, 6) and estimated at 100 amino acid substitutions per about 60 MY. Since then more and
more evidence consistent with this proposal concerning mammalian and non-mammalian taxa has
accumulated (e.g. Sarich, 1970, 1971, 1972; Maxson, Sarich & Wilson, 1975; Chen et al., 1980;Ellis
& Maxson, 1980; Maxson, 1981; Fuller et al., 1984; Lutz & Mayer, 1985; Sarich, 1985; Mayer &
Lutz, 1989). However, the divergence times of various mammalian lineages resulting from studies
using the above-mentioned rate are usually not in congruence with the respective times given by
fossil records, although some notable exceptions are known (e.g. Ellis & Maxson, 1980). This
disagreement has been attributed to the poorness of paleontological data and the methods of
geochronometry used (Jacobs & Pilbeam, 1980), as well as to the fact that many rodent fossils
show combinations of shared derived characters which make obscure the assignation of each
record to a particular lineage. Also, some diagnostic characters of a group may be absent from its
particular members (Brownell, 1983).
Arguments against the constant rate of mammalian genomic evolution, based on DNA
hybridization and nucleotidic sequence studies have arisen, since it has been proved that DNA of
rodents evolved much faster than that of some birds and the larger mammals. More specifically,
Britten (1986) found that the rate of DNA evolution in rodents is approximately five times faster
than in hominoid primates and some birds. Also, Wu & Li (1985), Li, Tanimura & Sharp (1987)
and Li & Tanimura (1987) suggest that the average evolutionary rate of the synonymous sites in
rodents is 4-10 times higher than that in hominoids, 3-6 times higher than that in monkeys and 2-4
times higher than that in artiodactyls. A detailed discussion on the rate differences among various
animal groups is given by Catzeflis et al. (1987). It is very remarkable that the acceptance of such a
rapid evolution in rodent lineages leads to estimations of their splitting times which are usually in
agreement with those revealed by the adequate fossil evidence of this mammalian order (Catzeflis
et al., 1987, 1989).
The above-mentioned make clear that for the amino acid substitutions specific fast rates of
evolution analogous to that used for the nucleotide substitutions have not been proposed so far,
although Sarich (1985) accepted that muroid rodents have a faster rate of amino acid substitution
than Primates and/or Carnivores. In order to overcome this failure in the case of the rodent
albumin, we combined the existing MC'F data for this protein with the respective divergence times
coming from the study of the fossil records to try to determine a reliable albumin evolutionary rate.
We actually use the only available immunological distances given by Sarich & Cronin (1980) and
Fuller et al. (1984) who also summarized the relevant paleontological data at the state of our
knowledge in the seventies and early eighties. The correlation of these two types of data (i.e. the
immunological and paleontological ones) lead us to the conclusion that for the rodent albumin a
rate equal to 100 amino acid substitutions per 16-20 MY should be possible. Although the
available data for such an estimation are limited, this rate seems to be a satisfactory approximation
for the time being, since its application to our immunological results gives splitting times
congruent to those coming from the paleontological evidence (see below).
The phylogenetic tree (Fig. 1) shows that the Microtus ( M . ) epiroticus and M . ( T . ) thomasi
lineages split at the Pleistocene period, about 0.5-0.6 MYA. A comparable time of divergence
450
N. P. NIKOLETOPOULOS ET A L .
between various species of the subgenera Microtus and Pitymys (now Terricola) has also been
derived from paleontological data (Chaline & Graf, 1988). This relatively recent diversification
explains the existence of very close phylogenetic relationships between these two taxa, which is also
corroborated by biochemical evidence (Graf, 1982; Gill et al., 1987). All this information justifies
the taxonomic position of Terricola as a subgenus in the genus Microtus (Niethammer & Krapp,
1982; Chaline & Graf, 1988).
Apodemus Jlavicollis and A . mystacinus are two species easily distinguished morphologically
(Niethammer, 1978 a, b; Corbet, 1978). The electrophoretic results of Gemmeke (1980) showed
that the genetic distance between A . ,pavicollis and A . mystacinus is slightly greater than that
between A . Jlavicollis and A . sylvaticus which are regarded to be very close relatives. However,
Filippucci, Simson & Nevo (1989) found a high genetic distance between A . Jzavicollis and
A . mystacinus. On the basis of our albumin immunological comparisons, these species diverged
0-65-0.8 MYA (Pleistocene). Such a recent dichotomy is well explained by an obviously lower
number of albumin amino acid substitutions along the Apodemus lineage, as compared to those of
Mus and Rattus (Table 11, Fig. 1). Concerning the phylogenetic relationships of the murid taxa,
Catzeflis et al. (1987), using DNA-DNA hybridization data and taking into consideration the
paleontological evidence (Jacobs, 1977, 1978; Flynn, Jacobs & Lindsay, 1985; Jaeger, Tong &
Denys, 1986), concluded that the Rattus lineage diverged 1-2 MY before the Mus-Apodemus
separation. Our results indicate that Ruttus is phylogenetically slightly more distant (by 1 I D unit)
from both Mus and Apodemus than these two genera are from each other. At first glance, such a
small distance makes the acceptance of two successive dichotomies in the murid tree difficult.
However, the relative rate tests (as defined and used by Sarich, 1969 a, b; Sarich & Wilson, 1973;
Wilson et al., 1977; Beverley & Wilson, 1984), based on the data of Table I1 and using Microtus as
outgroup and the three murids as ingroup taxa, proved that the favoured phylogenetic scheme is
(Mus+ Apodemus) + Rattus. The results of these tests apparently reinforce the two closely spaced
splittings shown in our tree. A quite different scheme of phylogenetic relationships is supported by
Misonne (1969) who, using molar teeth characters, came to the conclusion that Mus and Rattus are
more closely related than Mus and Apodemus.
Regarding the Mus-Rattus separation, paleontological data derived from material found inthe
circummediterranean area give a splitting time of about 10-12 MYA (Ameur, Jaeger & Michaux
1976; Chaline, 1977). More recent fossil data from S. Asia (Pakistan) revealed that the lineages
leading to Mus and Rattus diverged between 8-14 MYA (Jacobs, 1978; Jacobs & Pilbeam, 1980;
Flynn et al., 1985;Jaeger et al., 1986). According to our results, the time of divergence of the Rattus
lineage is 10-12.5 MYA and for Mus and Apodemus is 9.5-12 MYA (late Miocene), which is in
congruence with the above-mentioned paleontological data.
The taxonomic and phylogenetic position of arvicolids ( = microtines) within rodents is still
under investigation. Some authors place this group at a subfamily level (Microtinae) within the
family Cricetidae (Simpson, 1945; Corbet, 1978), while others consider it as a distinct family
Arvicolidae, including all microtines, well separated from Cricetidae (Niethammer & Krapp,
1982). The estimated splitting time of the Muridae-Arvicolidae lineages is controversial. Brownell
(1983), using DNA-DNA hybridization methods, found that the Muridae-Cricetidae divergence
took place 38.5-58.0 MYA. Furthermore, Sarich (19 8 9 , based on immunological criteria, reached
the conclusion that this divergence happened 35-40 MYA. However, considerably smaller dates
are derived from the fossil records. So, Jaeger et al. (1985) give for murine-microtine divergence a
splitting time longer than 20 MY, while Lindsay (1978) accepts as more probable a time of 25-30
MY. Our results give a respective time of 20-25 MY (early Miocene), which is in good accordance
with the paleontological data.
PHYLOGENETIC RELATIONSHIPS I N R O D E N T S
45 1
In conclusion, we could say that this MC’F study of Greek rodent lineages supports earlier
morphological and karyotypic analyses, but adds a new dimension of a time scale to suggested
divergences.
We would like to express our thanks to Dr C. Stamatopoulos who supplied us with some of the animal
material, Dr A. Mintzas for his assistance in the chromatographic isolation of albumins and Prof. Dr S.
Alahiotis for his helpful suggestions on the manuscript drafts. We are also indebted to the anonymous
reviewers for their critical remarks and comments on the manuscripts.
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