Evolution, 58(11), 2004, pp. 2486–2497
RATES OF MOLECULAR EVOLUTION IN NUCLEAR GENES OF EAST
MEDITERRANEAN SCORPIONS
BENJAMIN GANTENBEIN1,2
1 School
AND
PETER D. KEIGHTLEY1
of Biological Sciences, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, United Kingdom
Abstract. Scorpions of the genus Mesobuthus represent a useful terrestrial model system for studying molecular
evolution. They are distributed on several Aegean islands and the adjacent mainland, they are believed to have low
rates of dispersal, and evolutionary divergence dates of taxa are available based on biogeographic events that separated
islands from each other and the mainland. Here, we present data on polymorphism and synonymous (Ks) and nonsynonymous (Ka) substitution rates for nine nuclear protein-coding genes of two east Mediterranean scorpion species,
Mesobuthus gibbosus and M. cyprius (Buthidae). Levels of polymorphism tend to be lower in populations from islands
(mean nucleotide diversity ␲ ⫽ 0.0071 ⫾ 0.0028) than in mainland populations (mean ␲ ⫽ 0.0201 ⫾ 0.0085). By
using linear regression of genetic divergence versus isolation time, we estimate Ks to be 3.17 ⫾ 1.54 per (site ⫻ 109
years), and Ka to be 0.39 ⫾ 0.94 per (site ⫻ 109 years). These estimates for both Ks and Ka are considerably lower
than for many other invertebrates, such as Drosophila, and may be attributed to scorpions’ mammal-like generation
times (⬃2 years) and low metabolic rates. Phylogenetic analysis using maximum likelihood revealed a phylogeny that
is congruent with that expected based on biogeographic events and in which divergences at synonymous sites are
proportional to the dates that the taxa are believed to have split. Tests of equality of branch lengths for the Cyprus
and Crete lineages revealed that Ks-estimates are about the same in both lineages, as expected from the biogeographic
events that separated the islands, but Ka was increased in the Cyprus lineage compared to the Cretan lineage.
Key words.
East Mediterranean, local molecular clock, Mesobuthus, nuclear protein-coding DNA, scorpions.
Received July 26, 2004.
The molecular clock hypothesis postulates rate constancy
of molecular evolution (Margoliash 1963; Zuckerkandl and
Pauling 1965; Ohta and Kimura 1971; Gillespie 1989; Li
1997) and has stimulated much interest. If macromolecules
(proteins or DNA sequences) evolve at constant rates, they
can be used to date divergence times between taxa (Hasegawa
et al. 1985, 1987; Kishino et al. 2001). Although the existence
of a universal molecular clock has always been controversial,
several studies have attempted to calibrate local molecular
clocks by focusing on particular groups of organisms, such
as vertebrates (Yoder and Yang 2000). Such local clocks
appear to tick surprisingly linearly through time (Knowlton
et al. 1993; Beerli et al. 1996; Knowlton and Weight 1998).
Calibrating a molecular clock requires information from
reliable external sources, preferably well-dated fossils or biogeographic events (Hillis et al. 1996). Such evidence has been
used to date divergence events in vertebrates (Kumar and
Hedges 1998; Hedges and Kumar 2003), but has been much
less used in invertebrates. Together with the lack of fossil
invertebrates, this has resulted in a lack of information on
rates of molecular evolution for invertebrates, taxa that make
up 98% of worldwide biodiversity (Brusca 2000). Recently,
deep evolutionary splits among arthropods have been dated
using a molecular clock based on nuclear gene sequence data
(Peterson et al. 2004; Pisani et al. 2004). However, the date
of the deuterostome-arthropod split and relative evolutionary
rates of evolution among the different arthopod groups remain controversial. Here, we present new genetic data on
nine nuclear protein-coding genes of two East Mediterranean
scorpion species of the genus Mesobuthus Vachon 1950 for
which well-dated divergence times are available. Due to the
2 Present address: AO Research Institute, Clavadelerstrasse, CH7270 Davos Platz, Switzerland; E-mail: [email protected].
Accepted August 3, 2004.
joining of the Stait of Gibraltar, it is believed that the Mediterranean Basin dried out 5.6 million years ago (known as
the Mediterranean Salinity Crisis, MSC). As a result, many
Mediterranean islands were connected to the mainland via
land bridges over a period of 100,000 years. About 5.33
million years ago, the reopening of the Strait of Gibraltar led
to the refilling of Mediterranean over a period of only about
100 years (Krijgsman et al. 1999; Duggen et al. 2003). Scorpions of the Mediterranean basin therefore offer an excellent
opportunity to study evolutionary rates in invertebrates. First,
it can be assumed that scorpion populations inhabited the
Mediterranean islands during the MSC and exchanged genes
during the establishment of land bridges. Second, it is likely
that the populations subsequently remained isolated, as scorpions show extremely low dispersal rates (annually 1–30 m;
Polis et al. 1985), and genetic data on allozymes and nuclear
and mitochondrial DNA (mtDNA) sequences suggest a strong
population structure with high FST-estimates between subpopulations and low dispersal rates (Yamashita and Polis
1995; Gantenbein et al. 2001; Gantenbein 2004).
The scorpion genus Mesobuthus has its main species diversity in central Asia (Fet et al. 2000) and contains many
species of considerable toxicity to humans (Keegan 1998).
The genus has recently been the subject of molecular studies
on between-species phylogeny and within-species phylogeography (Gantenbein et al. 2003). Molecular and morphological phylogenetic analysis has revealed that populations
of the island of Cyprus represent a divergent lineage; therefore, these have been assigned a species rank (i.e., Mesobuthus cyprius Gantenbein and Kropf 2000; Gantenbein et
al. 2000). A recent phylogeographic study using DNA sequence data from a fragment of the mitochondrial large ribosomal subunit (16S; Gantenbein and Largiadèr 2002) on
the Aegean sister species Mesobuthus gibbosus (Brullé 1832),
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䉷 2004 The Society for the Study of Evolution. All rights reserved.
MOLECULAR CLOCK OF MEDITERRANEAN SCORPIONS
a species with a vicariant distribution across the Greek Balkans, Anatolia, and numerous Aegean islands, revealed an
excellent linear correlation between time and geological separation time. An independent estimate for the evolutionary
rate of the orthologous mtDNA fragment was also obtained
from a clock calibration of Buthus occitanus populations from
both sides of the Strait of Gibraltar (Gantenbein and Largiadèr 2003). However, little is known about evolutionary
rate of nuclear genes in scorpions and in arthropods in general. With few exceptions, studies of arthropods outside Drosophila have dealt with mitochondrial genes or nuclear ribosomal sequences (Giribet et al. 2001; Gaunt and Miles
2002). In scorpions, genetic research of nuclear DNA has
focused on toxin genes, and comparative phylogenetic studies
are very scarce (Loret and Hammock 2001; Froy and Gurevitz
2003).
In this paper we estimate rates of molecular evolution for
East Mediterranean scorpions in nuclear protein-coding
genes, using nine loci that have recently been isolated from
a cDNA library of this species (our data, in progress). We
calibrate the clock using a linear regression approach and
phylogenetic methods.
MATERIALS
AND
METHODS
Sampling Strategy
Scorpions of the two buthid species M. cyprius Gantenbein
and Kropf 2000 and M. gibbosus (Brullé 1832) were collected
between 1996–2002 (exact collection dates are available on
request). The distribution of the two species, the sampling
localities, and the sample sizes are shown in Figure 1 and
Table 1 (Kinzelbach 1975). Generally specimens were collected from a circular area of about 300 m2.
For the purpose of rooting the phylogeny, we included data
from two congeneric species M. eupeus (C. L. Koch 1839)
and M. caucasicus (Nordmann 1840) (Table 1), which are
sister clades to the ingroup (Fet et al. 2003; Gantenbein et
al. 2003).
DNA Analysis
DNA was extracted from muscle tissue (pedipalp or legs)
using either chloroform/phenol ethanol precipitation (Sambrook et al. 1989) or by using the Gentra (Ashby de la Zouch,
Leicestershire, U.K.) Puregene DNA isolation kit. Genomic
DNA was eluted in 50 ␮l of 10 mM Tris-HCl/1 mM EDTA
(pH 8.0). Polymerase chain reaction (PCR) primers were designed from two tissue-specific (body and tail) expressed
sequence tag (EST) libraries of an adult M. gibbosus specimen
(B. Gantenbein, M. Thomson, A. Rosie, J. Parkinson, I. Gantenbein, and M. Blaxter, unpubl. data). From a PCR screen
of 52 randomly chosen genes, only nine could be amplified.
The amplification failures might be due to the presence of
long introns. Table 2 indicates likely gene identities derived
from BLAST E-values and sequence similarity of BLASTX
matches against the nucleotide database, the primer sequences, and cross-links to Flybase reference numbers. To amplify
these genes, a general PCR cycling profile was used; initial
denaturation 95⬚C for 5 min followed by 35 cycles of 25 sec
at 95⬚C, 20 sec at 50⬚C, and 90 sec at 72⬚C. PCR reactions
2487
were carried out in 20-␮l volumes using Qiagen (Crawley,
West Sussex, U.K.) Taq polymerase with 2 ␮l of genomic
DNA (about 50–100 ng) in a MJ-Research (Rayne/Brayntrace, Essex, U.K.) PTC-100 thermocycler. Products were inspected on 1.5% agarose gels for multiple bands and were
then purified using a PEG8000 precipitation protocol (Andolfatto et al. 2003). Gel purification (1-to-1 Biology, Qbiogene, Cambridge, U.K.) and subsequent subcloning of the
PCR product was applied in the case of loci 1 and 3, where
sequencing failed due to difficult templates containing repeats, and for the amplification of the outgroups. For cloning
of PCR products, amplicons were cloned into the plasmid
vector pCR2.1 using the TOPO Cloning Kit (Invitrogen
Corp., Paisley, U.K.). At least four and up to 10 positive
clones were chosen per transformation. M13 universal primers were used to screen colonies by PCR. PCR primers were
again removed by polyethylene glycol (PEG 8000) precipitation, and purified templates were sequenced on both strands
using M13for and M13rev primers and DYEnamic ET Dye
Terminator Kit (Amersham Biosciences, Little Chalfont,
Buckinghamshire, U.K.). PCR products were sequenced using the forward PCR primer (Table 2). Sequencing reactions
were precipitated with ethanol and sodium acetate and run
on an ABI377XL sequencer or on an ABI3730 capillary sequencer (Applied Biosystems, Foster City, CA). All sequences were checked manually for sequencing errors. Consensus
sequences of subcloning (loci 1 and 3) were assembled using
the Seqman (Dnastar, Inc., Madison, WI) package. All sequences were deposited in the EMBL nucleotide sequence
database (http://www.ebi.ac.uk) under the following accession numbers; loci 01–03: AJ605341–AJ605513; loci 04–06:
AJ605780–AJ605953; loci 07–09: AJ606701–AJ606874.
Analyses of Multilocus DNA Sequences
A total of 58 sequences were aligned using the multiple
alignment algorithm of ClustalX (Thompson et al. 1997).
Alignment of protein-coding sequences was unambiguous
due to the presence of open reading frames. We calculated
the number of polymorphic sites, GC-content, and nucleotide
diversity (␲) within exons and intronic regions (Nei and Li
1979) from the polymorphism data within each major phylogenetic clade.
Calibration of a Molecular Clock
We calculated estimates of pairwise Ks and Ka, DNA substitution rates using the method of Nei and Gojobori (1986),
which corrects for multiple hits assuming the model of Jukes
and Cantor (1969). Ks and Ka were calculated using DNASP
4.0 (Rozas and Rozas 1999). We then calculated the mean
of Ks and Ka (and their standard errors) between major clades
as identified from phylogenetic analysis (see below) and plotted these estimates against geological separation time. We
corrected the mean of Ks and Ka between clades for ancestral
polymorphism using the following formula:
¯ net ⫽ K
¯ AB ⫺ (K
¯A ⫹ K
¯ B)/2,
K
(1)
where K̄net is the net evolutionary rate, K̄AB is the mean evolutionary rate between clades A and B, and K̄A and K̄B are
2488
B. GANTENBEIN AND P. D. KEIGHTLEY
FIG. 1. (A) Sampling sites of the two buthid scorpion species Mesobuthus cyprius Gantenbein and Kropf 2000 and M. gibbosus (Brullé
1932) in the east Mediterranean area. Abbreviations of sampling sites are given in Table 1, numbers refer to the number of localities
per major biogeographic region. (B) Enlargement of Aegean region. Arrows indicate isolation times based on geological evidence (see
Material and Methods section).
the mean evolutionary rates within clade A and B, respectively (Nei and Li 1979).
The mean nucleotide substitutions per (site ⫻ 10⫺9 year)
was estimated by linear regression including an intercept in
the model for each gene and for a concatenated supergene.
A test for equality of slopes was performed as described in
Sokal and Rohlf (1995), based on deviations from the mean
slope and an analysis of variance. Intronic data of each gene
were treated as noncoding DNA in DNASP 4.0, and estimates
of Ks were regressed against divergence time for each gene
separately and for a concatenated superintron. Evolutionary
rates for intronic data (obtained as slopes of regressions) were
then tested pairwise for significant deviations from the slope
(i.e., rate) of the codon data using the F-test statistics from
Sokal and Rohlf (1995).
Separation times for Mesobuthus populations are based on
the dating of the MSC and on tectonic movements of the
Hellenic arc and are as follows: Cyprus (CY) versus mainland
(B, P, CA, WA, SA) and, CY versus Crete (CR): 5.33 million
years; Karpathos (KA) versus West Anatolia (WA): 3 million
years; KA versus Rhodes (RH): 3 million years; and RH
versus WA: 1.8 million years) (Hsü et al. 1977; Beerli et al.
1996; Krijgsman et al. 1999; Gantenbein and Largiadèr 2002;
Duggen et al. 2003). The divergence date of 5.33 million
years is based on the assumption of isolation of populations
since the refilling of the Mediterranean Basin. The other two
estimates come from tectonic movements of the Hellenic arc,
which includes the islands of Crete, Karpathos, and Rhodes.
The island of Karpathos’ connection to the mainland was
broken at some time during the middle or late Pliocene (3
million year ago; Kuss 1975). Rhodes itself was connected
to the Anatolian mainland until the late Pliocene or early
Pleistocene (1.8 million years ago; Meulenkamp et al. 1972).
Greek islands such as Samos, Kos, and Lesvos, which are
surrounded by shallow waters (less than 120 m deep) were
most likely connected to the Anatolian mainland during the
last Pleistocene ice age (25,000 years ago). The sea level
during that time was 120 m lower than the present sea level
(Fairbanks 1989).
Tree reconstruction of DNA sequences was carried out to
resolve the relationships between island and adjacent mainland populations and to estimate evolutionary rates from
branch lengths. For phylogenetic analyses all ambiguities
(132 bp) and gaps (369 bp) were stripped out, as recom-
2489
MOLECULAR CLOCK OF MEDITERRANEAN SCORPIONS
TABLE 1.
Biogeographic
region
Sampling sites and sample sizes.
Locality,
country1
Abbreviation
N
Mesobuthus cyprius Gantenbein and Kropf 2000
Cyprus
Tepebasi (TR)
Kantara (TR)
McyCY1a-d
McyCY2a-b
4
2
A. Scholl
A. Scholl
Mesobuthus gibbosus (Brullé, 1832)
Greek Balkan
Parga (GR)
Visitsa (GR)
Mt. Olympus (GR)
Central Anatolia
Avanos (TR)
Hacibectas (TR)
Crete
Vai (GR)
Zakros (GR)
Agios Ioanis (GR)
Kares (GR)
Vraskes (GR)
Camares (GR)
Omalos Plateau (GR)
Nida Plateau (GR)
locality NA (GR)
Fourni Island
Fourni island (GR)
Gavdos
Gavdos (GR)
Ikaria
Ikaria (GR)
Karpathos
Pigadia (GR)
Amoopi (GR)
Kos
locality NA (GR)
Lesvos
locality NA (GR)
Megisti
Megisti (GR)
Peloponnesus
Mathia (GR)
Rhodes
Lindos (GR)
Kolympia (GR)
Monolithos (GR)
South Anatolia
Selale (TR)
Aspendos (TR)
Samos
Chora (GR)
Kusneika (GR)
West Anatolia
Yerkesik (TR)
Mugla (TR)
Ula (TR)
MgiB1a
MgiB2a
MgiB3a
MgiCA1a-b
MgiCA2a-b
MgiCR1a
MgiCR2a-e
MgiCR3a
MgiCR4a
MgiCR5a
MgiCR6a
MgiCR7a
MgiCR8a
MgiCR9a
MgiFO1a
MgiGA1a
MgiIK1a
MgiKA1a-b
MgiKA2a-b
MgiKO1a
MgiLE1a-b
MgiME1a
MgiP1a-d
MgiRH1a-b
MgiRH2a-b
MgiRH3a-b
MgiSA1a-b
MgiSA2a
MgiSAM1a
MgiSAM2a
MgiWA1a-b
MgiWA2a-b
MgiWA3a-b
1
1
1
2
2
1
5
1
1
1
1
1
1
1
1
1
1
2
2
1
2
1
4
2
2
2
2
1
1
1
1
1
2
V. Fet
V. Fet
V. Fet
A. Scholl
A. Scholl
I. Stathi
I. Stathi
C. Kropf
M. Braunwalder
M. Braunwalder
B. Lüscher
I. Stathi
I. Stathi
E. Aistleitner
I. Stathi
I. Stathi
I. Stathi
A. Scholl
A. Scholl
I. Stathi
I. Stathi
I. Stathi
B. and I. Gantenbein
A. Scholl
A. Scholl
A. Scholl
A. Scholl
A. Scholl
A. Scholl
A. Scholl
A. Scholl
A. Scholl
A. Scholl
Outgroup
Mesobuthus eupeus
Central Anatolia
Cemilköy (TR)
Meu
1
A. Scholl
Mesobuthus caucasicus
Central Asia
Jarkurgan (UZ)
Mca
1
A. Gromov, V. Fet
1
Collector
Country codes: GR, Greece; TR, Turkey; UZ, Uzbekistan.
mended by Swofford et al. (1996), leaving 3355 bp of concatenated DNA data for all taxa. We followed the total-evidence approach by concatenating DNA sequences into a supergene, including introns (Huelsenbeck et al. 1996). For
phylogenetic tree search using maximum likelihood (ML) we
used the general time reversible model with gamma correction (GTR ⫹ ⌫) as a DNA substitution model for the supergene. This model was suggested by comparing hierarchical
likelihood-ratio tests (log likelihood ratios between models
are available on request). Rate heterogeneity among sites was
assumed to follow distribution (the shape parameter ␣ was
estimated using ML) with four categories, each parameterized
by its mean (Yang 1996). The tree space was explored by
10 heuristic tree searches without enforcing a molecular clock
and by randomizing the order of the sequence input in PAUP*
4.0b10 (Swofford 1998).
For the calibration of a local molecular clock within the
previously revealed phylogenetic tree, we constrained particular branch lengths to have equal evolutionary rates (Yoder
and Yang 2000). For this purpose we analyzed the concatenated intron-exon data (3856 bp) using the HYPHY package
(Kosakovsky-Pond and Muse 2002) and defined 15 partitions
in total, one partition per coding gene (nine in total) and one
partition for each intronic data per gene (six in total, Table
3). For coding partitions we applied the MG94 ⫻ HKY85
DNA substitution model (Hasegawa et al. 1985; Muse and
Gaut 1994) and for noncoding sequences we chose the
HKY85 model. To reduce the parameter space, we decided
to use majority-rule consensus sequences (one sequence per
major phylogeographic clade). This resulted in 14 ingroup
sequences and two outgroup sequences in the data matrix.
We then maximized the ML function and tested whether constrained topologies, that is, local molecular clocks (null hypothesis), differ significantly from the more parameter-rich
Lysozyme precursor C (Lys-C)
9
TAACAGTTGTTATCATTGATAAATTGG
CG1385
BU092283/BU092304/
CB334110
BU092099/BU092259/
BU092303/CB333841/
CB333849/CB334134/
CB334149
Defensin 4kD
8
CG9111
CG8137
BU092239
Serinproteinase inhibitor Spn2
7
CG3315
BU092065
Thioredoxin1 (Trxr-1)
6
ATGGCTGGATTAGGAAGACGTCTTGTC
TAACTTGACCATTTACCTAGAATGTCAC
CATGTACTGACGCTGGCATTGCC
ATTGGCGGGATATTACTTGTG
TGAACAGTTAGCTAAGGC
TTAACCCATTGATTAACTTCAT
GCCATGAAAGCCGTTGCTATTCT
ACGACACAAATACAGGTGA
ATGGCTTTCAAGTTTTCATTTTTCG
Chaperonin10-Heat shock protein
5
CG11267
Serin-type endopepdidase
4
TTATTGTATCCCTATTAGAATCGCAGTTTAAGG
CG10527
BU092199/
BU092224/CB334031
BU092102/BU092213/
BU092333/CB333837/
CB333846/CB334064/
CB334077/BU091908
BU091831
Methyl transferase
3
Locus
Number
CG7532
CG11221
BU091820
Protein kinase
2
GAGTGTCATGCCAATAGATTACAG
ATCCACACATCTTCTAAAACGGTTAATTC
TCTGATGTATGGCAGATGGCAATG
CGAACTCAAGATCCACTCCTGTACTCG
TGGGTTCCAGCTCGCAGCGGTAACG
AACTTCGTAGTCGGAATACGAATGTTCTC
AGTTCTTATTGGTGTTCTTCTTTTGG
CG9896
BU092066/BU092133
Unknown protein
1
Drosophila
EST Accession
numbers
Primer
sequences (5⬘ -3⬘ )
B. GANTENBEIN AND P. D. KEIGHTLEY
TABLE 2.
Gene identification and primers for amplification of nine nuclear protein-encoding genes.
2490
model, that is, assuming no clock in any of the branches
(Huelsenbeck and Hillis 1996). In particular, we were interested to know whether the branch length of the Cyprus lineage equals the branch of the Crete lineage, since both of
these islands were expected to have diverged from each other
5.33 million years ago. As a second constraint, we tested
whether the branch of the island of Karpathos lineage equals
3/5.33 times the branch length of the Cyprus or the Crete
lineage. The third constraint concerned the branch length of
the Rhodes lineage to the ancestral node connecting the island
with the Anatolian mainland, which was set to 1.8/5.33 times
the Cyprus lineage. We then tested these constraint models
against the alternative parameter-rich model using the ␹2approximated likelihood ratio statistics.
Hybridization on the Island of Rhodes
Molecular clock calibrations may be affected by recurrent
gene flow (hybridization) (Fleischer et al. 1998; Schierup and
Hein 2000; Posada and Crandall 2002). A previous allozyme
study of M. gibbosus found evidence for recent artificial transplantation from the Greek mainland to the island of Rhodes,
where they apparently hybridize with the autochthonous population (Gantenbein and Largiadèr 2002). In this study genotype classes were assigned based on a three-locus hybrid
index ranging from 0 to 6. A hybrid index of 0 was assigned
to a genotype homozygous for Greek mainland alleles, and
an index of 6 was assigned to a genotype homozygous for
Anatolian mainland samples. Here, we exclusively sequenced
specimens (n ⫽ 6) that were assigned a hybrid index of 6.
Thus, disturbance of the clock calibration and phylogenetic
tree reconstruction should be minimized.
RESULTS
Genetic Variation within Major Clades
All nine genes analyzed showed some level of polymorphism (Table 3). Locus 8 (defensin), was the most variable
of the nine genes (␲ ⫽ 0.0704 ⫾ 0.007) due to a high number
of polymorphic sites (S) for a relatively short gene fragment
(78 bp of 147 bp ⫽ 0.53%). The least polymorphic gene (26
polymorphic sites of 248 bp ⫽ 0.10%) is locus 6 (Trxr-1).
Estimates of ␲ and S were generally lower within the population samples of major islands than within mainland samples. For Cyprus samples, estimates of ␲ range from 0 to
0.1149 (gene 8, defensin), and similar low estimates are observed for the island of Crete, ranging from 0.0042 to 0.0533.
The genetic variability within the two populations sampled
from the relatively small island of Karpathos, was negligible
in all nine genes compared to Cyprus, Crete, and Rhodes.
Sequence variability on populations of Rhodes, however, was
in the same range as Cyprus and Crete.
Intron-Exon Structure of Scorpion Genes
We identified 11 introns in total, based on the comparison
of the genomic sequences with the respective EST-clone (Table 3). The lengths of the introns are given in Table 3 for
each major phylogenetic clade. Splicing sites were generally
of the exon/GT-type in the 5⬘-region and of the AG/exontype in the 3⬘-region. This pattern was also recently found
2491
MOLECULAR CLOCK OF MEDITERRANEAN SCORPIONS
TABLE 3.
Population1
Locus
1
2
3
4
5
6
7
8
9
1-9
B, P,
CY
CR
KA
RH
all
B, P,
CY
CR
KA
RH
all
B, P,
CY
CR
KA
RH
all
B, P,
CY
CR
KA
RH
all
B, P,
CY
CR
KA
RH
all
B, P,
CY
CR
KA
RH
all
B, P,
CY
CR
KA
RH
all
B, P,
CY
CR
KA
RH
all
B, P,
CY
CR
KA
RH
all
CA, WA, SA
CA, WA, SA
CA, WA, SA
CA, WA, SA
CA, WA, SA
CA, WA, SA
CA, WA, SA
CA, WA, SA
CA, WA, SA
Genetic polymorphism within and among populations.
Coding2
Noncoding2
Exons
Polymorphic2
Parsimony
informative2
Nucleotide
diversity
SE
357
607
612
606
609
357
328
332
335
336
336
327
339
360
360
351
360
402
258
261
258
261
261
261
183
186
186
192
186
156
375
378
372
378
375
363
342
342
342
342
339
318
72
72
78
78
90
66
309
312
315
315
312
288
254
250
255
249
252
257
0
0
0
0
0
0
73
66
62
74
64
42
76
74
76
51
51
51
65
66
62
67
68
74
0
0
0
0
0
0
0
0
0
0
0
0
108
112
100
122
116
81
495
520
518
523
517
476
4
4
4
4
4
4
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
4
4
4
4
4
4
29
7
5
5
4
67
3
1
1
0
0
11
23
1
6
2
1
28
4
1
2
0
0
57
26
12
10
0
2
32
0
1
7
0
7
13
26
0
4
0
18
44
57
49
37
2
27
78
123
64
8
34
44
223
11
1
3
1
1
33
2
0
1
0
0
7
13
0
5
0
0
16
3
0
0
0
0
21
15
0
6
0
0
18
1
0
2
0
2
11
15
0
4
0
7
31
42
13
15
0
15
49
99
28
0
2
5
165
0.0012
0.0042
0.0023
0.0044
0.0025
0.0003
0.0018
0.0010
0.0008
0.0000
0.0000
0.0046
0.0090
0.0008
0.0042
0.0024
0.0013
0.0000
0.0040
0.0010
0.0009
0.0000
0.0000
0.0179
0.0236
0.0159
0.0000
0.0000
0.0026
0.0151
0.0010
0.0014
0.0042
0.0000
0.0055
0.0046
0.0180
0.0000
0.0036
0.0000
0.0217
0.0237
0.0812
0.1149
0.0553
0.0050
0.0605
0.0704
0.0415
0.0215
0.0032
0.0214
0.0195
0.0346
⫾0.0018
⫾0.0016
⫾0.0006
⫾0.0018
⫾0.0008
⫾0.0016
⫾0.0005
⫾0.0007
⫾0.0004
⫾0.0000
⫾0.0000
⫾0.0005
⫾0.0000
⫾0.0005
⫾0.0005
⫾0.0013
⫾0.0004
⫾0.0011
⫾0.0000
⫾0.0006
⫾0.0008
⫾0.0000
⫾0.0000
⫾0.0008
⫾0.0062
⫾0.0103
⫾0.0037
⫾0.0000
⫾0.0011
⫾0.0024
⫾0.0003
⫾0.0005
⫾0.0018
⫾0.0000
⫾0.0016
⫾0.0007
⫾0.0024
⫾0.0000
⫾0.0008
⫾0.0000
⫾0.0000
⫾0.0030
⫾0.0158
⫾0.0199
⫾0.0095
⫾0.0017
⫾0.0124
⫾0.0075
⫾0.0046
⫾0.0028
⫾0.0010
⫾0.0044
⫾0.0039
⫾0.0029
1 Populations are B, Balkan; CR, Crete; CY, Cyprus; KA, Karpathos; P, Peloponnesus; CA, Central Anatolia; RH, Rhodes; SA, South Anatolia; WA,
Western Anatolia.
2 All base-pair counts exclude ambiguities and gaps in the alignment.
in the intron-exon structure of sodium and potassium channel
toxins in scorpions (Froy et al. 1999) and is generally observed in eukaryotes (Levin 1994). The splicing site at the
5⬘-end is GTAAG in six of 11 cases. The splicing site at the
3⬘-end was found to be AG in all introns. The TACTAACbox, a conserved gene sequence reported for introns, involved
in the splicing process, is usually located about 30 bp upstream in the 3⬘ region of other eukaryotes. We observed the
following deviations from this box in scorpions: TTATAAC
in intron 1, TGACAAC in intron 2, GATTAAT in intron3,
all in gene 1, TTATAAT in gene 3, and GTATAAT (most
likely) in gene 4. There was no obvious box in the intron of
gene 5.
Mean GC-content of the noncoding region was 18.6% and
GC-content of the coding region was 42.0% (mean of GC2
and GC3 was 40.6% and 31.3%, respectively).
2492
B. GANTENBEIN AND P. D. KEIGHTLEY
TABLE 4. Estimates of net nonsynonymous (Ka) and net synonymous (Ks) substitution rates (substitutions per site) between clades with
known divergence times (million years). Net Ka and Ks are also given for between-mainland comparisons.
Clade1 comparison
(N)
Ka
SE
Ks
SE
Ka/Ks
Between island-mainland populations
CY(6)-B(3)
5.33
CY(6)-P(4)
5.33
CY(6)-CA(4)
5.33
CY(6)-CR(12)
5.33
CY(6)-SA(3)
5.33
CY(6)-WA(4)
5.33
CR(12)-B(3)
5.33
CR(12)-CA(4)
5.33
CR(12)-P(4)
5.33
CR(12)-SA(3)
5.33
CR(12)-WA(4)
5.33
K(33-36)-WA(4)
3
K(33-36)-RH(6)
3
RH(6)-WA(4)
1.8
0.0156
0.0160
0.0177
0.0168
0.0178
0.0146
0.0053
0.0060
0.0042
0.0068
0.0069
0.0057
0.0056
0.0038
0.0011
0.0015
0.0018
0.0011
0.0011
0.0011
0.0004
0.0007
0.0009
0.0009
0.0012
0.0004
0.0003
0.0011
0.0284
0.0268
0.0261
0.0198
0.0267
0.0266
0.0290
0.0227
0.0265
0.0156
0.0261
0.0170
0.0117
0.0088
0.0011
0.0008
0.0007
0.0007
0.0005
0.0002
0.0009
0.0021
0.0003
0.0003
0.0002
0.0005
0.0005
0.0011
0.551
0.595
0.676
0.848
0.669
0.550
0.184
0.266
0.158
0.434
0.263
0.335
0.477
0.434
Between mainland-mainland populations
B(3)-P(4)
NA
B(3)-CA(4)
NA
B(3)-SA(3)
NA
B(3)-WA(4)
NA
CA(4)-SA(3)
NA
CA(4)-P(4)
NA
CA(4)-WA(4)
NA
P(4)-WA(3)
NA
P(4)-SA(3)
NA
0.0000
0.0031
0.0038
0.0039
0.0088
0.0029
0.0054
0.0042
0.0050
0.0002
0.0019
0.0019
0.0020
0.0033
0.0023
0.0020
0.0015
0.0014
0.0022
0.0123
0.0157
0.0141
0.0186
0.0096
0.0094
0.0151
0.0210
0.0009
0.0011
0.0003
0.0019
0.0005
0.0029
0.0038
0.0035
0.0031
0.000
0.256
0.243
0.279
0.473
0.304
0.568
0.277
0.238
1
Time
Clades are defined in Table 1.
Linear Regression Analysis of Clock Calibration
(Supergene Approach)
Linear Regression-Based Clock Calibration
(Single-Gene Approach)
Means of Ks and Ka estimated for mainland and island
clades and divergence time are given in Table 4. The ratio
of Ka/Ks, a general indicator of adaptive changes, ranges from
0.158 (CR-P comparison) to 0.848 (CY-CR comparison).
Thus, the CY-CR comparison shows more amino acid replacements than all other comparisons. The genetic differentiation among mainland clades (i.e., B, CA, P, SA, WA)
is in some cases also considerable (Table 4) and range up to
0.021 for Ks between P and SA. The differentiation between
CA and SA with 0.0186 for Ks is also remarkable. These two
population groups are separated by a high mountain chain,
which represents a natural geographic dispersal barrier.
Translating these rates into evolutionary time estimates
points to the existence of very old population groups and
that these mainland populations in some cases diverged from
each other for about the same time frame as island populations diverged from mainland populations (5.33 million years
ago). The linear regression of the concatenated supergene is
shown in Figure 2. The regression shows a linear increase
in the synonymous substitution rate (Ks) with expected divergence time (slope ␣ ⫽ 2.28 ⫾ 0.44 substitutions per [site
⫻ 109 years], P ⬍ 0.0001, intercept ␤ ⫽ 2.26 ⫾ 4.80, P ⫽
0.89; Table 5, Fig. 2A). The slope for the introns is slightly
steeper (Fig. 2B), due to the two extreme values at the 5.33
Million years ago calibration point (i.e., the comparisons CYSA and CR-P). The value of the nonsynonymous substitution
rate (Ka) is less clear, since the slope is not significantly
different from zero (␣ ⫽ 1.18 ⫾ 0.60, P ⫽ 0.07, ␤ ⫽ ⫺0.50,
P ⫽ 0.87).
The mean of estimates of Ks and Ka between major clades
of known divergence times were calculated for each gene
separately in the same way as for the concatenated supergene
and plotted against divergence time. Fitting linear regressions
through these plots reveals variation in the regression slope
estimates across the nine genes. The slopes and their F-statistics for the individual genes and R2 for each regression are
summarized in Table 5, computed including an intercept. The
intercepts are not significantly different from zero except for
genes 7 and 9 for Ks and for genes 4, 6, and 8 for Ka. Leastsquare estimates of slopes are not significantly different from
zero in five of the nine genes for Ks and are only significant
for Ka in one case. Analysis of variance among the slopes
gave significant F-ratio statistics (Table 5), supporting the
hypothesis that these genes vary in their selective constraints
or in their mutation rates. The standard errors of the slopes
(Ks and Ka) for the supergene approach are downwardly biased, and thus, are generally smaller than the standard errors
calculated from the mean of slopes (Sokal and Rohlf 1995;
Hillis et al. 1996).
The evolutionary rates for the introns (expressed as Ks) of
the six genes vary between ⫺4.96 and 6.20 (mean 3.45 ⫾
1.18) nucleotide substitutions per (site ⫻ 109 years). This is
similar to the mean Ks at synonymous sites of the proteincoding data.
Phylogenetic Inference
The heuristic ML tree search, calculated without enforcing
a molecular clock, revealed a single tree (Fig. 3) containing
MOLECULAR CLOCK OF MEDITERRANEAN SCORPIONS
2493
solved, but sequences from the same sampling locality tend
to cluster together with high bootstrap support. The four sequences from the island of Karpathos, a geographically wellseparated island from the Anatolian mainland, are also well
separated from neighboring islands. Sequences from the island of Rhodes cluster at a relatively low divergence level
compared to the Cyprus lineage.
At first sight, branch lengths of the tree seem to agree very
well with the presumed separation time from geology (Figs. 1,
3). To further investigate this qualitative finding, we tested
whether the branch lengths of the Cyprus and Cretan lineage
could be constrained to equal length and whether the branch
length of the Karpathos lineage (blKA) corresponds to 3/5.33th
the branch length of the Cyprus (blCY) and Createan lineage
(blCR). In addition, the Rhodes lineage (blRH) was constrained
to have diverged 1.8/5.33th of the branch length of the Cyprus
or Crete lineage. The phylogeny of the 16 majority-rule consensus sequences reveals a slightly different tree topology from
the 58 taxa phylogeny, but the major conclusion that the Cyprus,
Cretan, and Karpathos island sequences are well-separated remains (tree not shown). The constraint model (null hypothesis;
blCY ⫽ blCR and blKA ⫽ 3/5.33blCY and blRH ⫽ 1.8/5.33blCY)
is not significantly different from the unconstrained model (alternative hypothesis) if only synonymous rates are considered;
2␦ ⫽ 2(l0 ⫺ l1) ⫽ 2(13,553.80 ⫺ 13,542.60) ⫽ 22.40, df ⫽
56, P ⫽ 0.66. Thus, Ks of each exon partition fits the proposed
molecular clock model fairly well. However, if Ks and Ka are
constrained according to the above branch length ratios, the
difference in log likelihood between the trees is significantly
lower (2␦ ⫽ 2(l0 ⫺ l1) ⫽ 2(10,584.95 ⫺ 10,542.60) ⫽ 84.70,
df ⫽ 52, P ⫽ 0.0028). This is mainly caused from the Cyprus
lineage, which has an increased Ka, as calculated from the
branch length (mean of nine branch lengths ⫽ 7.40 ⫾ 3.88
substitutions per [site ⫻ 109 years]) as compared to the Cretan
lineage (mean 2.70 ⫾ 1.84 substitutions per [site ⫻ 109 years]).
DISCUSSION
Estimates of Evolutionary Rates in Protein-Coding Genes
FIG. 2. (A) Mean estimates of synonymous (Ks) and (C) nonsynonymous (Ka) DNA substitutions per site based on a supergene
(⫽ nine concatenated single genes) plotted against evolutionary
time. For comparison, Ks for concatenated introns (B) plotted
against time is also given. The 95% confidence limits of the mean
are shown.
11 lineages, each supported by rather high bootstrap values.
The outgroup species, Mesobuthus eupeus and M. caucasicus
are well separated by long branches from the ingroup sequences. The most conspicuous ingroup lineage is the Cyprus
lineage, and its branch length is even longer than that of the
Cretan lineage. This finding supports the recent elevation of
the Cyprus lineage to the species level, M. cyprius, based on
morphological and allozyme data (Gantenbein et al. 2000).
The phylogenetic relationships among the mainland samples
(B, P, CA, SA, WA) and the several small Greek island
samples (KO, SAM, IK, FO, LE, ME) are less clearly re-
The range of variation in evolutionary rates among our
sample of nine loci is considerable and could originate from
sampling, different mutation rates, and/or different evolutionary constraints acting on each gene. Li (1997) provides
estimates for evolutionary rates for Drosophila (33 proteins)
and mammals (47 proteins). For Drosophila proteins, the average Ks was 15.6 ⫾ 5.5 (per site ⫻ 109 years) and for mammals 3.51 ⫾ 1.01. Estimates for Ka were 1.91 ⫾ 1.42 in
Drosophila genes and 0.74 ⫾ 0.67 in mammals. Our estimates
for Ks (3.17 ⫾ 1.54 per [site ⫻ 109 years]) and Ka (0.39 ⫾
0.94 per [site ⫻ 109 years]) are therefore comparable to rates
estimated in mammalian genes. However, these datasets differ from each other in the choice of protein sequences, and
our estimates are potentially biased by the fact that primers
were designed from a cDNA library, which tends to overrepresent highly expressed genes.
To our knowledge there is no other study with a similar
DNA dataset from any other invertebrate with a comparably
well-calibrated molecular clock (Zhang and Hewitt 2003).
Since buthid scorpions have a generation time of about 24
months, which is more like mammals than invertebrates, the
2494
B. GANTENBEIN AND P. D. KEIGHTLEY
TABLE 5. Estimates of evolutionary rates for nine single protein-coding genes from linear regression analysis and from a concatenated
supergene. Asterisks mark significant P-values at the 0.05 level.
Locus
Protein-coding
Ks
1
2
3
4
5
6
7
8
9
Mean
F8,108
Ks supergene
Slope
⫾SE
P-value
Intercept
⫾SE
P-value
R2
4.07
1.76
1.35
3.30
2.67
1.73
⫺1.58
14.71
0.49
3.17
1.50
0.77
1.13
1.16
1.66
1.15
1.72
3.89
1.78
1.54
0.02*
0.04*
0.25
0.01*
0.13
0.16
0.37
0.00*
0.79
5.29
0.15
0.34
⫺5.18
⫺6.63
⫺2.69
26.17
⫺13.62
27.05
2.26
7.30
3.76
5.50
5.65
8.09
5.60
8.39
18.98
8.70
4.80
0.48
0.97
0.95
0.38
0.43
0.64
0.01*
0.49
0.01*
0.38
0.30
0.11
0.40
0.18
0.16
0.07
0.54
0.01
2.28
0.44
0.32
2.17
0.89
0.69
2.28
0.51
0.29
⫺0.10
0.44
⫺0.27
5.42
⫺5.40
0.32
0.39
0.55
0.22
0.27
0.36
0.88
0.27
3.35
5.50
1.17
0.94
⫺4.64
⫺1.26
⫺0.72
4.24
1.18
3.47
⫺6.85
77.76
7.38
8.95
2.69
1.09
1.31
1.75
4.28
1.33
16.34
26.84
5.71
8.72
0.11
0.27
0.59
0.03*
0.79
0.02*
0.68
0.01*
0.22
0.59
0.30
0.09
0.01
0.02
0.07
0.18
0.07
0.01
1.18
0.60
⫺0.50
2.92
0.87
0.24
4.17
5.73
4.74
6.20
⫺4.96
3.44
3.22
4.42
0.61
1.30
2.21
2.95
6.51
2.24
1.69
1.30
⫺4.33
⫺14.22
⫺6.05
⫺8.39
74.12
6.18
7.88
⫺7.00
2.97
6.34
10.79
14.42
31.79
10.96
13.52
6.36
0.17
0.04*
0.59
0.57
0.04*
0.58
0.80
0.62
0.28
0.27
0.05
0.16
0.29
0.49
⬍0.0001*
0.00*
Ka
1
2
3
4
5
6
7
8
9
Mean
F8,108
Ka supergene
Noncoding/intronic
Introns gene 1
3
4
5
8
9
Mean
All Introns
F5,72
low estimates could be a consequence of a long generation
time. However, it is also possible that this is a consequence
of differences in metabolic rates, scorpions surely being at
the lower end of the range (Martin and Palumbi 1993; Lighton
et al. 2001); a recent comparative study on metabolic rates
in arachnids and insects revealed that scorpion metabolic
rates are about one-quarter of those of insects, spiders, mites,
and solpugids of the same size (Lighton et al. 2001), and
scorpions are among the animals having the lowest metabolism rates in the animal kingdom. It is plausible that generation time and metabolic rate influence evolutionary rate
synergistically (Martin and Palumbi 1993). Finally, it has
also been shown that body size is positively correlated with
evolutionary rate, at least in mammals (Martin and Palumbi
1993). It is unknown whether such correlations exist in poikilothermic animals or invertebrates in general.
Further data are available on evolutionary rates in mitochondrial genes in scorpions. These genes generally evolve
faster than nuclear genes (Lynch and Jarrell 1993; Martin
and Palumbi 1993; Simon et al. 1994), and several mechanisms have been formulated to account for this effect: the
lack of recombination in animal mtDNA (Rokas et al. 2003)
and less efficient DNA repair mechanisms (Martin and Pal-
0.00*
0.04*
0.30
0.80
0.62
0.35
0.13
0.34
0.79
⬍0.0001*
0.07
0.00*
0.00*
0.05*
0.06
0.46
0.15
0.01*
⬍0.0001*
umbi 1993; Avise 1994). For vertebrate mtDNA, the rate of
molecular evolution has been estimated at 2% per Million
years ago (or 1 ⫻ 10⫺8 substitutions per [year ⫻ site] per
lineage; Brown et al. 1979). This often cited mitochondrial
molecular clock for vertebrates has recently been evaluated
using mtDNA clock calibrations of invertebrate species such
as Hawaiian Drosophila (DeSalle et al. 1987; Fleischer et al.
1998), Hawaiian crickets of the genus Laupala (Fleischer et
al. 1998), South American butterflies of the genus Heliconius
(Brower 1994), and in North African-Iberian carabids of the
genus Carabus (Prüser and Mossakowski 1998) and Timarcha
(Gómez-Zurita et al. 2000). These rates range between 4.9
and 7.0 ⫻ 10⫺9 substitutions per (years ⫻ site) per lineage,
and are surprisingly close to rates observed in vertebrates
(Brown et al. 1979). In scorpions, the evolutionary rate of
two mitochondrial genes, the subunit I of cytochrome oxidase, and the large ribosomal subunit (16S rRNA), have been
estimated at ⬃1% divergence per million years for the Aegean species M. gibbosus, which translates to 5.0 substitutions per (site ⫻ 109 year) per lineage (Gantenbein and Largiadèr 2002, 2003; Gantenbein et al. 2003). In scorpions,
synonymous sites in mtDNA genes evolve about two times
faster than in nuclear DNA proteins. However, it has been
MOLECULAR CLOCK OF MEDITERRANEAN SCORPIONS
2495
FIG. 3. Maximum likelihood (ML) tree of east Mediterranean scorpion populations of the genus Mesobuthus based on a concatenated
supergene of exon-intron data of nine nuclear protein genes. The ML-model was a GTR ⫹ G with the following parameters: ␲A ⫽ 0.342,
␲C ⫽ 0.174, ␲G ⫽ 0.207, ␲T ⫽ 0.277; gamma shape parameter ␣ ⫽ 0.785; proportion of invariable sites I ⫽ 0.460; rate parameters, AC ⫽ 0.798, A-G ⫽ 0.887, A-T ⫽ 0.635, G-C ⫽ 0.743, C-T ⫽ 1.743, G-T ⫽ 1.0, respectively. Nodes at branches refer to bootstrap
values ⬎65% inferred from neighbor-joining using 1000 ML-estimated pairwise distance-pseudo replicates as the input matrix. Designations for sequences are given in Table 1. Arrows indicate well-dated branching points of lineages.
shown that the ratios between mean nuclear DNA and mtDNA
differ significantly among animal groups (Caccone et al.
1988).
Biogeographic Conclusions and the Reliability of
Calibration Dates
The inferred phylogeny (Fig. 3) supports the hypothesis
that all of the analyzed specimens are autochthonous, and
there is no obvious case of recent artificial transplantation
having affected the clock calibration. The Cyprus lineage is
well separated from M. gibbosus, and the tree confirms M.
eupeus and M. caucasicus as outgroup species. This tree shape
observed for nuclear genes is also in agreement with a recent
DNA study using combined DNA data of two mtDNA fragments and the PK gene (locus 2 of this study; Fet et al. 2003;
Gantenbein et al. 2003). The clustering among island and
mainland populations of M. gibbosus is in general agreement
with the allozyme tree of Gantenbein and Largiadèr (2002).
The fact that the mainland populations of M. gibbosus are
genetically very distinct (genetic divergence CA-SA, SAWA) supports the hypothesis that the genus Mesobuthus is
2496
B. GANTENBEIN AND P. D. KEIGHTLEY
of central Asian origin, where it is most diverse and where
several species and subspecies have been described. The Aegean island system is particularly well understood geologically (Meulenkamp et al. 1972; Kuss 1975; Le Pichon and
Angelier 1979; Meulenkamp 1985), and the geological isolation dates are believed to be reliable. Although our initial
hypothesis of colonization and subsequent isolation of scorpion populations is a very strong assumption (see Materials
and Methods) the estimated Ks-values appear to follow a
linear molecular clock model (Fig. 2). Thus, other factors,
such as different ancestral population sizes between mainland
and island samples, might not be important influences on
evolutionary rates in scorpions.
ACKNOWLEDGMENTS
We thank I. Gantenbein-Ritter for laboratory assistance. F.
Balloux, A. Eyre-Walker, and D. Gaffney critically commented on an earlier version of the manuscript. BG was supported by a Swiss National Science Foundation IHP grant
(83-EU-065528), the Royal Society (R-36579), and the Basler Stiftung für experimentelle Zoologie (c/o Eidgenössisches
Tropeninstitut). Field trips were supported by the commission
for travelgrants of the Swiss Academy of Natural Science.
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Corresponding Editor: M. Nachman