Structure, organization and nucleotide diversity of the mitochondrial
control region and cytochrome b of southern water vole
(Arvicola sapidus)
ALEJANDRO CENTENO-CUADROS1 & JOSÉ A. GODOY2
1
Department of Conservation Biology, and 2Department of Integrative Ecology, Estación Biológica de Doñana, CSIC.
C/ Américo Vespucio s/n. Isla de La Cartuja, 41092 Sevilla, Spain
(Received 27 October 2009; revised 5 February 2010; accepted 5 February 2010)
Abstract
The southern water vole (Arvicola sapidus Miller, 1908) is an endangered rodent whose conservation guidelines should
preserve the current genetic variability. We analyze the structure and organization of the mitochondrial control region (CR) in
A. sapidus. The CR of this species is characterized by a low guanine-cytosine content, the absence of any repetitive motif within
the two hypervariable regions, and the presence of the two extended termination-associated sequences and conserved
sequence blocks. Nucleotide diversity comparisons between A. sapidus and the European water vole (Arvicola terrestris)
revealed differences in the distribution of genetic variation. Furthermore, we provide primers for the amplification of short and
highly polymorphic fragments of CR and cytochrome b especially designed for degraded materials. These markers offer
molecular tools to assist in the establishment of future conservation and management guidelines, and will also facilitate studies
at different spatial and evolutionary scales of this species.
Keywords: Conserved sequence blocks, cytochrome b, D-loop, extended termination-associated sequences, Muridae, Rodentia
Introduction
The vertebrate mitochondrial DNA (mtDNA) is a
closed, circular, and maternally inherited molecule
widely used in molecular ecology and phylogeographic
studies (Ballard and Whitlock 2004). Among the 37
genes encoded within mtDNA, the control region
(CR) and cytochrome b (Cytb) have been most
frequently used at intraspecific and interspecific levels.
The CR is a non-coding mitochondrial fragment, the
length of which varies depending on the number of
repeated motifs within the region. The CR of
mammals is flanked by tRNAPro and tRNAPhe and is
divided into two hypervariable domains separated by a
central conserved region—but see, e.g. Matson and
Baker (2001) for two hypervariable regions within
the second domain in Clethrionomys. The latter
is characterized by three conserved sequence boxes
(CSBs) involved in regulatory signals for the processing of the RNAs that prime replication. The two
hyper variable regions are mainly composed of
extended termination- associated sequences (ETAS),
the most rapidly evolving portion of the mitochondrial
genome (Saccone et al. 1993), which are presumably
related to the regulation of replication and transcription of mtDNA. Hypervariable fragments within the
CR usually meet the polymorphism required to
address issues related to population genetic structure
or phylogeography. However, the heterogeneous
distribution of sequence variation along the region,
and the possible occurrence of homopolymers tracts
and tandem repeats and heteroplasmy may hamper
the direct sequencing of polymerase chain reaction
(PCR) products. It is thus necessary to envisage
a thorough characterization of the structure of this
Correspondence: A. Centeno-Cuadros, Estació n Bioló gica de Doñ ana, CSIC, C/Amé rico Vespucio, s/n C.P., Sevilla 41092, Spain.
Tel: 34 954466700. Fax: 34 954621125. E-mail: acenteno@ebd.csic.es
region within every newly targeted taxon previous to
its routine use in molecular ecology and evolutionary
studies.
The two species of the genus Arvicola (Mammalia,
Rodentia, and Cricetidae) are distributed across
Eurasia. The European water vole (Arvicola terrestris)
ranges throughout eastern Asia up to the northern
regions of Iberia and has been the subject of several
molecular ecology studies (Stewart et al. 1998, 1999;
Berthier et al. 2005; Piertney et al. 2005; Oliver and
Piertney 2006). On the other hand, the southern water
vole (Arvicola sapidus) is distributed through Iberia
and France, and nowadays is categorized as Vulnerable by the IUCN (Rigaux et al. 2008) by habitat
fragmentation, contamination of water bodies, and the
introduction of the American mink (Mustela vison);
molecular studies are scarce and mainly focused on its
karyotype (Diaz de la Guardia and Pretel 1978, 1979;
Megı́as-Nogales et al.) and has been only recently
studied at the intraspecific (phylogeography) and
interspecific levels (evolutionary history of Arvicola)
(Centeno-Cuadros et al. 2009a,b). Conservation
biologists and wildlife managers need molecular
tools to go into the knowledge of historical and
contemporary processes in depth and therefore
help them to develop proper conservation programs
of the species. To date, there are no detailed studies on
CR structure and variability in water voles, partially
because of homopolymer sequences that hamper the
sequencing of the entire region (Piertney et al. 2005;
S. Piertney, personal communication).
In the present study, we characterize the organization and variability of the entire mitochondrial CR
sequence in A. sapidus by designing a set of
primers that circumvent sequencing problems. In the
second step, we provide primers that amplify short
and highly polymorphic fragments of CR and Cytb to
study intraspecific variation from degraded materials
and apply these primers on non-invasive samples of
Southern and European water vole species.
Materials and methods
We ear punched 47 individuals of A. sapidus trapped
(and released live) in the Natural Region of Doñ ana
(southwestern Spain, 378100 N, 68230 W), which were
used to describe nucleotide variation in the CR. We
also obtained fresh tissue samples from distant
populations in Spain (GenBank accession numbers
FJ895499, FJ895500, FJ895507, and FJ895577; see
Table I) in order to obtain a representative sampling of
haplotypes and nucleotide polymorphism. We also
used 22 non-invasive samples (see below) of A. terrestris
scherman from the north of Spain in order to test the
cross-amplification of CR and Cytb primers designed in
this study. Fresh tissue DNA was extracted with a
“salting-out” protocol (Mü llenbach et al. 1989). We also
used DNA extracted from non-invasive samples—
mainly bones and skins obtained from pellets of owl
(Tyto alba) and eagle owl (Bubo bubo)—and from
museum specimens (housed at the scientific collections
of the Biological Station of Doñ ana) to test the suggested
primers targeting short and polymorphic fragments of
CR and Cytb (see Results and discussion). We found
homopolymer tracts within domain II that hampered
sequencing reactions in CR of A. sapidus. We solved this
problem by using primers F15708 and R92 (Piertney
et al. 2005), primer 50 -TCCCCACCATCAGCACCCAAAGC-30 designed by Stacy et al. (1997) (hereafter
F15374) and four specifically designed internal primers
yielding partially overlapping fragments (F15816, 50 ATGTTTTATCGTCCATACGTTCC-30 ; F15872,
50 -AATCAGCCCATGCCTAACAT-30 ; R15946, 50 TAGCCGTCAAGGCATGAAG-30 ; RCRasa 50 -AAAAACAACTCAAAATTCCAAAA-30 ). Primer3 (Rozen
and Skaletsky 2000) was used to optimize the locations
of these primers with the default user-set primer design
parameters, targeting conserved sequence overall
haplotypes and restricting the overall size of the
amplified fragment to less than 250 bp. Cytb was
amplified and sequenced using primers H15288 and
L14115 (Martin et al. 2000). PCR amplifications were
performed as follows: 948C for 5 min, 40 cycles at 928C
for 30 s, 628C (CR) or 608C (Cytb) for 30 s, and 728C for
30 s, finishing with 728C for 5 min. Five microliters of
mitochondrial PCR products were purified with 2 ml
ExoSAP-IT enzyme (USB Corp. Cleveland, OH,
USA). Sequencing reactions were performed using the
BigDye Terminator Cycle Sequencing Kit v.1.1
(Applied Biosystems, Inc. Foster City, CA, USA)
following the manufacturer’s instructions, and the same
primers were used for the amplifications. Sequencing
products were run on an Applied Biosystems 3130 £ l
Genetic Analyzer. Forward and reverse sequences were
edited and aligned using Sequencher 4.6 (Gene Codes
Corp. Ann Arbor, MI, USA). We described the
distribution of nucleotide diversity along CR using
a sliding window of 100 sites (step size of 25 sites)
as implemented in the software DnaSP 4.50.3
(Rozas et al. 2003).
Results and discussion
Herein we report the first analysis of the structure of
the entire CR of a representative of the genus Arvicola
(A. sapidus) (Figure 1; GenBank accession number
FJ502319). By using four partially overlapping
fragments, we were able to obtain complete sequences
from 47 southern water voles. We identified a central
region that includes two CSBs. While CSB3 was
completely conserved in A. sapidus, CSB2 differed by
two indels with respect to corresponding sequences in
Mus musculus mitochondrial genome. We could not
allocate Arvicola CSB1, indicating large divergences
between M. musculus and A. sapidus sequences.
However, the degree of conservation and functionality
Table I. Geographic distribution, type of sample, and GenBank accession numbers for all CR (204 bp) and Cytb (208 bp) haplotypes
sequenced for A. sapidus from Spain (SP), Portugal (POR), and France (FR).
GenBank accession number
Sample size
Source
Locality
Latitude
Longitude
Huelva (SP)
Huelva (SP)
É vora (POR)
Cá diz (SP)
Sevilla (SP)
Badajoz (SP)
Ávila (SP)
Gerona (SP)
Navarra (SP)
Tarragona (SP)
Navarra (SP)
É vora (POR)
Sevilla (SP)
Haute-Vienne (FR)
Santander (SP)
Haute-Vienne (FR)
Haute-Vienne (FR)
Creuse (FR)
Jaén (SP)
Burgos (SP)
La Rioja (SP)
Pyrénées-Orientales (FR)
Setú bal (POR)
Setú bal (POR)
Auvergne (FR)
Auvergne (FR)
Granada (SP)
Burgos (SP)
36.9876
36.9876
38.7047
36.0616
37.9337
38.8666
40.4271
41.8490
42.7539
40.8541
42.7539
38.7047
37.9337
45.8053
42.9892
45.8053
45.8053
45.8736
37.9108
42.1729
42.3269
42.8332
37.9383
37.9383
45.7344
45.7344
36.9728
42.1729
2 6.4814
2 6.4814
2 7.4000
2 5.6363
2 5.7621
2 6.4274
2 5.3032
2.3902
2 1.0998
2 1.096
2 1.0998
2 7.4000
2 5.7621
0.9337
2 3.9710
0.9337
0.9337
1.6440
2 3.0024
2 3.7077
2 3.0377
2.9191
2 8.7766
2 8.7766
2.6273
2.6273
2 3.4531
2 3.7077
Auvergne (FR)
Huelva (SP)
Setú bal (POR)
Sevilla (SP)
Creuse (FR)
Haute-Vienne (FR)
Granada (SP)
Navarra (SP)
Evora (POR)
Santander (SP)
Sevilla (SP)
Burgos (SP)
Gerona (SP)
Badajoz (SP)
Haute-Vienne (FR)
Jaén (SP)
La Rioja (SP)
Pyrénées-Orientales (FR)
Tarragona (SP)
45.7344
36.9876
37.9383
37.9337
45.8736
45.8053
36.9728
42.7539
38.7047
42.9892
37.9337
42.1729
41.8490
38.8666
45.8053
37.9108
42.3269
42.8332
40.8541
2.6273
2 6.4814
2 8.7766
2 5.7621
1.6440
0.9337
3.4531
2 1.0998
2 7.4000
2 3.9710
2 5.7621
2 3.7077
2.3902
2 6.4274
0.9337
2 3.0024
2 3.0377
2.9191
2 1.0960
CR
FJ895495
FJ895496
FJ895497
FJ895499
FJ895500
FJ895504
FJ895507
FJ895519
1
2
1
1
1
1
1
3
FJ895521
FJ895522
FJ895530
FJ895543
1
1
1
2
FJ895544
FJ895545
FJ895547
FJ895552
FJ895555
FJ895559
FJ895563
FJ895565
FJ895566
FJ895568
FJ895569
FJ895576
FJ895577
1
1
1
1
1
1
1
1
1
1
1
1
1
Fresh tissue
Fresh tissue
Bones
Fresh tissue
Fresh tissue
Bones
Fresh tissue
Fixed tissue
Bones
Bones
Bones
Bones
Bones
Bones
Bones
Bones
Bones
Bones
Bones
Fixed tissue
Bones
Bones
Fresh tissue
Fresh tissue
Fresh tissue
Fresh tissue
Fixed tissue
Fresh tissue
2
3
2
1
1
1
1
2
2
1
1
1
1
1
2
1
1
1
1
Fresh tissue
Fresh tissue
Fresh tissue
Fresh tissue
Bones
Bones
Bones
Bones
Bones
Bones
Bones
Fixed tissue
Fixed tissue
Bones
Bones
Bones
Bones
Bones
Bones
Cytb
FJ895410
FJ895416
FJ895459
FJ895467
FJ895474
FJ895478
FJ895488
of this sequence block has been controversial. For
example, whereas Sbisa et al. (1997) reported the
CSB1 as the least conserved sequence block and
suggested it was the most important in terms of
functionality, studies in the red-backed vole Clethrionomys (Matson and Baker 2001) showed CSB1 as a
sequence block with 40% of variability. The authors
eventually suggested that only a single element of
CSBs and ETAS was involved in mitochondrial
replication and, consequently, any variation within
any additional element should not have any
detrimental effect. Base composition and distances
between CSBs might be variable among species but
are usually conserved at the intraspecific level (Sbisa
et al. 1997). The global nucleotide composition in
Arvicola is AT-rich and follows the common biased
base content found in other organisms—A. sapidus
average percentages: A (32.4%), T (30.7%), C
(23.8%), and G (13.1%) (Saccone et al. 1987;
Zhang and Hewitt 1997). However, A. sapidus showed
slight deviations from the expected A . T . C . G
pattern of mammals in ETAS and CSBs—ETAS: T
Figure 1. Structure and organization of the complete Cytb and mitochondrial CR of A. sapidus. Forward and reverse primers are indicated
with an arrow above their respective position in the mitochondrial genome (a,b) and as underlined sequences (c). Numbers on the upper line
(b) specify the initial position and nucleotide polymorphism (100-bp sliding window, see text) on the consensus sequences of A. sapidus
obtained in this study. Numbers below the lower line (b) show the relative position on M. musculus mtDNA. The graph above the CR
map (b) reveals the distribution of nucleotide diversity along the sequence. An asterisk on a nucleotide position highlights a polymorphic site
among 47 individual sequences. Arrows on (c) delimit conserved blocks in CR sequences. DI, first hypervariable domain; DII, second
hypervariable domain.
(32.3%), A (31.5%), C (25%), and G (11.3%); CSB:
C (33.3%), A (29.3%), T (25.8%), and G (11.6%).
The distribution of nucleotide diversity along the CR
reveals the allocation of most of the overall polymorphism ( p ¼ 0.0406, nucleotide midpoint position
150, representing 14 out of 19 total polymorphic sites)
at the hypervariable domain I (sliding window
analysis, Figure 1), whereas the maximum peak of
nucleotide diversity in the second hypervariable
domain was four times lower ( p ¼ 0.0103, midpoint
position 725). Our results do not show any evidence
for the existence of a third hypervariable domain
within CR in A. sapidus (as suggested in Clethrionomys;
see Introduction and Matson and Baker 2001). The
distribution of nucleotide diversity in A. sapidus is
biased toward the first hypervariable domain as in
other species of rodents (e.g. Spalax galili; Reyes et al.
2003) and differs from the most commonly observed
patterns where the polymorphism is divided between
both hypervariable regions or even greatly biased
toward the second hypervariable domain (Baker and
Marshall 1997; Matson and Baker 2001; Roques et al.
2004). None of the haplotypes analyzed showed
tandemly repeated sequences. Considering the
observed distribution of polymorphisms and that the
CSBs are flanked by homopolymer tracts (presumably
involved in forming a stable hairpin structure in CR),
we propose to target a 246-bp CR fragment in the first
hypervariable region that contains most of the total
polymorphism in A. sapidus using forward F15468
(5 0 -GCATTAAATTATATTCCCCATGC-3 0 ) and
reverse R15713 (5 0 -TTGTTGGTTTCACGGAGGAT-30 ) primers.
We also characterized the complete 1143-bp coding
region of Cytb from six individuals from different
geographic locations in Spain (GenBank accession
numbers FJ539341 – FJ539346). We found five different haplotypes defined by 10 polymorphic nucleotide
positions (nine synonymous and one non-synonymous
mutations). In order to maximize the polymorphism
amplified in a single short fragment, we suggest
the targeting of a 248-bp fragment using specific
primers F14559 (5 0 -TCCTTTTGAGGGGCTACAGT-30 ) and R14806 (50 -TGGAAGGGAATTTTGTCTGC-30 ).
Furthermore, we applied primers F15468 and
R15713 (CR), and F14559 and R14806 (Cytb) to
non-invasive and museum samples (see Materials and
methods) of A. sapidus (n ¼ 26) (Table I) and A. t.
scherman (n ¼ 22) (Northern Spain) in order to check
the amplification success and usefulness of these two
polymorphic fragments on degraded genetic material.
Nucleotide diversity in Cytb was lower in A. sapidus
( p ¼ 0.00374) than in A. terrestris ( p ¼ 0.00912),
whereas in CR it was higher in A. sapidus
( p ¼ 0.03623) than in A. terrestris ( p ¼ 0.0077).
Comparison of CR polymorphism between species
must be considered cautiously, because European
water voles apparently assemble higher nucleotide
diversity in the second hypervariable domain, a region
that showed scarce variation in A. sapidus.
Following a thorough characterization of the
structure and nucleotide variation in A. sapidus
mitochondrial CR, we propose the first hypervariable
domain in A. sapidus as the most informative
mitochondrial marker for addressing studies at both
intraspecific and interspecific scales. We report
primers for the amplification of highly variable yet
short fragments of both CR and Cytb (246 and 248 bp,
respectively) that are especially useful for highly
degraded genetic material, such as museum specimens
and ancient DNA. Developed primers should prove
useful for studies addressing evolutionar y and
population genetic issues on A. sapidus (and probably
in closely related species) upon which future
conservation and management guidelines should be
based, and provided an efficient and cost-effective tool
for species identification from materials such as feces
or unidentifiable bones obtained in raptor pellets.
Acknowledgments
The authors are especially grateful to J. M. Llanes,
M. Gutiérrez, M. Gonzá lez, F. Alda, J. Romá n,
M. Delibes, the Molecular Ecology Laboratory and
scientific collections housed at the Biological Station
of Doñ ana. They are also indebted to a long list of
biologists who provided samples for this study. The
present work was funded by the Direcció n General de
Investigació n (project BOS2001-2391-C02-01).
A. C.-C. was funded by the Spanish Ministry of
Education and Science.
Declaration of interest: The authors report no
conflicts of interest. The authors alone are responsible
for the content and writing of the paper.
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