Evolutionary Discontinuity of the Carabine Ground Beetles

Abstract

The Carabine ground beetles are mostly hind wing-less and cannot fly, so that there is more chance of diversification by
geographic isolation compared with winged insects. The relationships between morphological diversification and phylogeny of
the ground beetles of the world have been inferred mainly by comparisons of mitochondrial ND5 gene sequences. Based on dating
by a mitochondrial DNA ``clock,'' it has been deduced that an explosive radiation of the major carabine groups took place
50–40 MYA. This was followed by occasional radiations on various scales, sometimes accompanied by parallel morphological changes.
There are also a good number of examples showing that the fundamental morphology has remained unchanged for a long time among
geographically isolated populations within the same species. Thus, carabid evolution would have proceeded discontinuously,
with phases of rapid morphological change alternating with silent phases.

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Evolutionary Discontinuity of the Carabine Ground Beetles
Zhi-Hui Su,1Yu ˆki Imura,2Syozo Osawa1
1JT Biohistory Research Hall, Takatsuki, Osaka 569-1125, Japan
2Department of Gynecology, To ˆkyu ˆ General Hospital, Oˆta-ku, Tokyo 145-0062, Japan
Received: 26 October 2000 / Accepted: 16 February 2001
Abstract.
hind wing-less and cannot fly, so that there is more
chance of diversification by geographic isolation com-
pared with winged insects. The relationships between
morphological diversification and phylogeny of the
ground beetles of the world have been inferred mainly by
comparisons of mitochondrial ND5 gene sequences.
Based on dating by a mitochondrial DNA “clock,” it has
been deduced that an explosive radiation of the major
carabine groups took place 50–40 MYA. This was fol-
lowed by occasional radiations on various scales, some-
times accompanied by parallel morphological changes.
There are also a good number of examples showing that
the fundamental morphology has remained unchanged
for a long time among geographically isolated popula-
tions within the same species. Thus, carabid evolution
would have proceeded discontinuously, with phases of
rapid morphological change alternating with silent
phases.
The Carabine ground beetles are mostly
Key words:
tree — Ground beetles — Discontinuous evolution
Mitochondrial ND5 gene — Phylogenetic
Introduction
The subfamily Carabinae (Coleoptera, Carabidae) con-
sists of two tribes, the Cychrini and the Carabini. The
Carabini are further classified into two subtribes, Cara-
bina and Calosomina. The Carabina ground beetles are
hind wing-less and cannot fly, and they consist of about
800 species (Brezina 1999), mostly inhabiting the North-
ern Hemisphere. They are divided into nine divisions
(Imura et al. 1998a) and 94 (sub)genera (Imura and Mi-
zusawa 1996). We report the pattern of evolution of the
carabids based mainly on comparisons of the mitochon-
drial NADH dehydrogenase subunit 5 (ND5) gene se-
quences from about 2000 specimens consisting of repre-
sentative species and subspecies covering most of the
known genera (Table A1). The sequences of nuclear 28S
rDNA and the trehalase gene were also used for some
groups to determine their consistency with the ND5 data.
Materials and Methods
Sampling of the carabine beetles, DNA extraction, PCR amplification,
and sequencing were carried out as described (Su et al. 1996a, 1998).
An approximately 1.1-kb fragment of the mitochondrial genome con-
taining 1069 bp of the ND5 gene was amplified using a primer set
(forward, 5?-CCTGTTTCTGCTTTAGTTCA-3?; reverse, 5?-
GTCATACTCTAAATATAAGCTA-3?) (Su et al. 1996a), and the
ND5 region was used for construction of the tree. For amplication of
nuclear 28S rDNA, an 834- to 879-bp fragment comprising the variable
domains D1 and D2 of the 28S rRNA gene was amplified using a
primer set designed from the Drosophila 28S rDNA sequence (forward,
5?-GACTACCCCCTGAATTTAAGCAT-3? at position 3313; reverse,
5?-GACTCCTTGGTCCGTGTTTCAAG-3? at position 4053) (Tautz et
al. 1988; Kim et al. 2000).
Sequence alignments were carried out using the multiple-alignment
program CLUSTAL W, version 1.5 (Thompson et al. 1994). There
were no insertions/deletions in the aligned ND5 gene sequences
throughout the Carabina species examined. A few insertions/deletions
were found in some 28S rDNA sequences. The G + C content was
The sequences reported in this paper have been deposited in the DDBJ/
EMBL/GenBank nucleotide sequence database under the accession
numbers listed in Table A1 (Appendix).
Correspondence to: Zhi-Hui Su; email: su.zhihui@ims.brh.co.jp
J Mol Evol (2001) 53:517–529
DOI: 10.1007/s002390010242
© Springer-Verlag New York Inc. 2001

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nearly constant (21 ± 1%) for the ND5 gene and 58 ± 0.8% for 28S
rDNA in the Leptocarabus and Rhigocarabus species analyzed. Phy-
logenetic trees were constructed by the neighbor-joining (NJ) method
(Saitou and Nei 1987) and UPGMA (unweighted pair-group method
with arithmetic mean), with the evolutionary distance (D) computed by
Kimura’s (1980) two-parameter method. To obtain the reliability of the
inferred phylogenetic tree, the bootstrap test based on 500–1000 re-
samplings (Felsenstein 1985) was applied. All procedures of the phy-
logenetic analysis were performed by the DNA sequence package
SINCA, version 3.0 (Fujitsu System Engineering, Japan).
Results and Discussion
Chronology
Since a lack of chronology greatly restricts the interpre-
tations of the phylogenetic data, the base substitution rate
of the ND5 gene was calculated using several geographi-
cal data as follows. (1) Two races of the carabid Eulep-
tocarabus porrecticollis kansaiensis in the Kinki district,
Japan, are separated by the Yodogawa River–Biwako
Lake line which formed 3 MYA (million years ago) (Yo-
koyama 1973; Takemura 1985), and Kimura’s evolution-
ary distance (D) between these two races was calculated
to be 0.0076 ± 0.0011. Thus, these two races would have
started to diversify upon establishment of the river, ca. 3
MYA. (2) The paleomagnetic evidence points out that
the ancient Japan area split into the northeast arc and
southwest arc from the eastern periphery of the Eurasian
continent about 15 MYA (Otofuji et al. 1991, 1994).
Since the carabid Damaster blaptoides is endemic to
Japan, the proto-Damaster which inhabited the ancient
Japan area of the continent would have started to diver-
sify upon separation of Japan from the continent ca. 15
MYA. Diversification of Damaster began with the sepa-
ration of the northeastern (E) lineage and the southwest-
ern (W) lineage. The D value between E and W is 0.042
± 0.0029 (Su et al. 1998). (3) The distribution ranges of
two races of both Phricocarabus glabratus and To-
mocarabus convexus are clearly separated by the Alps,
which were formed about 20 MYA. Then the two races
of each species would have been isolated ∼20 MYA. The
D values between them are 0.065 ± 0.0012 and 0.051,
respectively. The relationship between chronology and
evolutionary distance shows that an accumulation of the
base substitution increases almost linearly with time
(Fig. 1), suggesting the near-constancy of the base sub-
stitution rate probably throughout the carabids. This en-
ables us to use the UPGMA for construction of a phy-
logenetic tree using the ND5 gene sequences. From Fig.
1, 0.01 D unit corresponds to 3.6 MYR (million years)
for the ND5 gene.
Radiation
Figure 2 shows an ND5 phylogenetic tree of the repre-
sentative Carabina species. The branching order of vari-
ous lineages is obscure because of short branch lengths
with low bootstrap values. Unresolved branching orders
near the root of the tree would not be the result of satu-
ration of the base substitutions, because actual substitu-
tion percentages are linearly correlated with Kimura’s
(1980) evolutionary distance (D), the value corrected for
multiple substitutions. The linearity is also the case for
separately plotted transitions (Ts) and transversions (Tv).
One plausible explanation would be that the major Cara-
bina groups explosively radiated within a short time, re-
sulting in considerable morphological diversification
among the descendant species. The radiation was calcu-
lated to have taken place 50–40 MYA.
Successive radiations after the explosive radiation of
the Carabina are seen in most of the Carabina divisions,
which are best represented by the division Procrustimor-
phi. Shortly after the Carabina radiation, the Procrusti-
morphi lineage again radiated into six geographically
linked groups 30–20 MYA (to be published). Among
them, the Chinese group radiated into at least six clus-
ters, having resulted in most remarkable morphological
diversification among the descendants (Fig. 3). Another
example of radiation is recognized in various Tomocara-
Fig. 1.
ogy. a Split of two populations of Euleptocarabus porrecticollis kan-
saiensis by the Biwako Lake–Yodogawa River line, which was formed
3 MYA. b Split of the E and W lineages of Damaster blaptoides,
presumably by cleavage of ancient Japan into NE and SW arcs ca. 15
MYA. c, d Split of two populations of Phricocarabus glabratus (c) and
those of Tomocarabus convexus (d) by the Alps, which was completed
ca. 20 MYA. For the localities where the specimens of Euleptocarabus
porrecticollis, Damaster blaptoides, Phricocarabus glabratus, and To-
mocarabus convexus were collected, see Kim et al. (1999a), Su et al.
(1998), and Fig. 6, respectively.
Relationship between evolutionary distance (D) and chronol-
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Fig. 2.
the Carabina. The taxonomic divisions are indicated in the tree. Boot-
strap values (%) based on 500 resamplings are shown at each branching
point throughout all the trees in this paper. Two Cychrus species (tribe
Cychrini) were taken as the outgroup. Taxonomic divisions roughly
coincide with the lineages on the ND5 tree, except that the Latitarsi are
heterogeneous and separated into at least three lineages (to be pub-
UPGMA tree of the ND5 gene for representative species of lished). The yellow shadowing indicates the radiation of the major
groups. The UPGMA and the NJ method yielded trees with essentially
the same topology throughout this paper as exemplified in Fig. 4.
Photographs of the specimens are shown below the tree, and the num-
bers correspond to those following the scientific names in the tree
throughout the figures. Scale bar: 20 mm.
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Fig. 3.
for representative species of the Chinese
group in the division Procrustimorphi. The
specimens from China are marked by
asterisks. The blue shadings indicate
radiations which occurred twice in this
group. Photographs of 16 species are
shown below the tree. Scale bar: 20 mm.
UPGMA tree of the ND5 gene
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bus species (division Latitarsi) which radiated ca. 25
MYA (see Fig. 6).
Explosive radiation is known to have occurred during
evolution, well-known examples being the Cambrian ex-
plosion of various animal phyla and the extensive diver-
sification of cichlids in Lake Victoria and its satellite
lakes in Africa (see Stanley 1981).
Discontinuous Evolution
As described above, radiation includes the multiple oc-
currence of various lineages within a short time, presum-
ably accompanied by morphological diversification as
deduced from the descendant species. An apparently dis-
continuous but singular emergence of a morphologically
different species is occasionally recognized in various
carabine groups. Some examples are described below.
Euleptocarabus porrecticollis (division Lepidospinulati)
is strictly endemic to Japan, while another species in the
same division, Limnocarabus clathratus, is distributed in
the northern parts of the Eurasian continent and Japan.
The much closer phylogenetic relatedness of the Japa-
nese L. clathratus with E. porrecticollis compared with
that between the Japanese and the continental L. clath-
ratus suggests that E. porrecticollis branched off from
the Japanese L. clathratus, accompanied by a rapid mor-
phological differentiation long after the separation of the
two L. clathratus races (Kim et al. 1999a) (Fig. 2). Ap-
otomopterus sauteri (division Spinulati) radiated into
three lineages ca. 20 MYA. A. hupeensis, which is
clearly distinguishable from A. sauteri, emerged from
one of the A. sauteri stems (Fig. 2). Hemicarabus tuber-
culosus (division Crenolimbi) is widely distributed
throughout northeastern Asia including Japan, Korea,
and Primorskii. Two other Hemicarabus species, H. ni-
tens and H. macleayi, inhabit Europe and East Asia, re-
spectively. These three species, which are clearly distin-
guishable morphologically from one another, are very
close in their ND5 gene sequences (Fig. 2), suggesting
their recent (rapid) morphological differentiations. This
Fig. 4.
outgroup. Photographs of 11 species including Acathaicus, Cathaicus, and Eupachys are shown below the trees.
Phylogenetic positions of Acathaicus, Cathaicus, and Eupachys: UPGMA (left) and NJ (right) trees. Megodontus kolbei was taken as an
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