High variability and non-neutral evolution of the mammalian avpr1a gene

  • Sabine Fink1,

    Affiliated with

    • Laurent Excoffier1 and

      Affiliated with

      • Gerald Heckel1Email author

        Affiliated with

        BMC Evolutionary Biology20077:176

        DOI: 10.1186/1471-2148-7-176

        Received: 16 April 2007

        Accepted: 27 September 2007

        Published: 27 September 2007

        Abstract

        Background

        The arginine-vasopressin 1a receptor has been identified as a key determinant for social behaviour in Microtus voles, humans and other mammals. Nevertheless, the genetic bases of complex phenotypic traits like differences in social and mating behaviour among species and individuals remain largely unknown. Contrary to previous studies focusing on differences in the promotor region of the gene, we investigate here the level of functional variation in the coding region (exon 1) of this locus.

        Results

        We detected high sequence diversity between higher mammalian taxa as well as between species of the genus Microtus. This includes length variation and radical amino acid changes, as well as the presence of distinct protein variants within individuals. Additionally, negative selection prevails on most parts of the first exon of the arginine-vasopressin receptor 1a (avpr1a) gene but it contains regions with higher rates of change that harbour positively selected sites. Synonymous and non-synonymous substitution rates in the avpr1a gene are not exceptional compared to other genes, but they exceed those found in related hormone receptors with similar functions.

        Discussion

        These results stress the importance of considering variation in the coding sequence of avpr1a in regards to associations with life history traits (e.g. social behaviour, mating system, habitat requirements) of voles, other mammals and humans in particular.

        Background

        The genetic bases of complex phenotypic traits like differences in social and mating behaviour among species and individuals remain largely unknown [1]. Most such traits are probably under polygenic control and the contribution of each gene to the phenotype is often very difficult to assess [2]. Even for genes with large effects, it is highly challenging to identify the causes of particular phenotypic differences because genetic variation is rarely restricted to dichotomous polymorphism in a gene [e.g. [36]]. Genetic variation at a locus is not only shaped by locus- or site-specific selective processes but also by the evolutionary history of the particular species or population.

        One of the best examples of a single gene with large effects involved in very specific phenotypic and behavioural differences is the arginine-vasopressin receptor 1a (avpr1a). This gene has been proposed to play a key role in controlling variation in mammalian social behaviour [711], and it has been particularly well-studied for its role in the formation of mating systems in rodents from the genus Microtus [1215]. Phenotypic differences between species in arginine-vasopressin 1a receptor (V1aR) distribution in the brain and contrasting social behaviour were largely attributed to the presence of a repetitive expansion in the regulatory region upstream of the gene [1215]. The transfer of the entire avpr1a gene region including the repetitive expansion or the coding region from a monogamous vole to non-monogamous voles and other rodents resulted in modified V1aR distributions and changes in social behaviour [12]. Additionally, monogamous voles showed increased affiliative behaviour (measured as time spent in contact with other voles, see in [14]) after injection of the arginine-vasopressin (AVP) hormone in the brain, while non-monogamous voles displayed unchanged social behaviour [14]. However, AVP has two main roles: it controls higher cognitive functions such as memory and learning in the brain, and it acts peripherally by facilitating water absorption in the kidney and by contracting smooth muscle cells from blood vessels [16]. The impact of hormones on behavioural variation may therefore also depend on environmental conditions [see [17]]. A recent study showed further that neither social nor genetic monogamy are strictly associated with the presence of the repetitive expansion in the regulatory region of avpr1a in voles and other mammals [18].

        In contrast to polymorphism in the regulatory region of avpr1a, variation in the coding part is assumed to be low and functionally negligible [14, 19, 20]. Rodent avpr1a patterns have been proposed as mammalian model systems for the study of the role of hormone receptors in the formation of complex social interactions, including human social disorders [21]. Studies of human avpr1a have mainly focused on variation in the non-coding upstream region of the gene, and associations have been reported with autism [2224], eating behaviour [25], self perception [11] and even creative dance performance [26]. Single nucleotide polymorphisms in the human avpr1a gene have been detected [2224, 27], but it is unknown if they affect the encoded protein. Previous studies of the Microtine avpr1a have not explicitly studied levels and patterns of variation in the coding region at the inter- or intra-specific level. Its potential influence on social behaviour and interactions in voles and other mammals remains therefore totally unknown.

        We use here an evolutionary approach to investigate variation in parts of the coding region of the mammalian avpr1a gene. We analyse patterns of nucleotide and amino acid (AA) polymorphism in the Microtus genus represented by 24 species from three continents (Europe, North America, Asia), and compare it to the avpr1a diversity found in various higher mammalian taxa. Furthermore, we examine rate variation among the functionally important regions – the ligand binding site or the G-protein binding domain [16] – and other parts of the V1aR, and we test for the role of selection in shaping variability in the avpr1a gene.

        Results

        Microtine avpr1a diversity

        The analysis of a large fragment (792 bp) of the first exon (total 970 bp) of the avpr1a gene in the genus Microtus revealed unexpectedly high levels of variation with an overall nucleotide diversity of 0.0161. The sequencing of individuals from 24 species revealed 12 heterozygous individuals, while the two individuals obtained from GenBank were apparently homozygous for avpr1a. After cloning of heterozygous PCR-products, a total of 36 different sequences were detected among the 48 chromosomes, which showed overall 103 variable positions (13%). Despite this large amount of diversity, one chromosome sequence (E01, see Table 1) was identical between two individuals of different species (M. tatricus, M. oeconomus).
        Table 1

        Origin of rodent samples and avpr1a sequences

        Microtus species

        continent

        country

        locality

        chromosome label

        accession number

        tatricus

        Europe

        Slovakia

        High Tatra Mountains

        E01

        EU176005

        agrestis

        Europe

        Finland

        Lapua

        E02

        EU175968

            

        E03

        EU175969

        arvalis

        Europe

        Switzerland

        Belp

        E04

        EU175970

            

        E05

        EU175971

        rossiaemeridionalis

        Europe

        Macedonia

        Gradsko

        E06

        EU175972

            

        E07

        EU175973

        multiplex

        Europe

        Switzerland

        Ticino

        E08

        EU175974

        nivalis

        Europe

        Spain

        Avila

        E09

        EU175975

        felteni

        Europe

        Greece

        Thessalia

        E10

        EU175976

        thomasi

        Europe

        Greece

        Nomos Arkadia

        E11

        EU175977

        cabrerae

        Europe

        Portugal

        Cauda

        E12

        EU175978

        schelkovnikovi

        Europe

        Azerbaijan

        Talysh

        E13

        EU175979

            

        E14

        EU175980

        socialis

        Europe

        Azerbaijan

        Stepanakert

        E15

        EU175981

            

        E16

        EU175982

        oeconomus

        North-America

        Canada

        Yukon

        E01

        EU176006

        ochrogaster

        Gene bank sequence

          

        NA01

        AF069304

        ochrogaster

        North-America

        USA

        Kansas

        NA01

        EU175983

        montanus

        Gene bank sequence

          

        NA02

        AF070010

        montanus

        North-America

        USA

        Missoula

        NA03

        EU175984

        pinetorum

        North-America

        USA

        Calloway

        NA04

        EU175985

            

        NA05

        EU175986

        californicus

        North-America

        USA

        Stanislaus

        NA06

        EU175987

            

        NA07

        EU175988

        chrotorrhinus

        North-America

        USA

        Minnesota

        NA08

        EU175989

            

        NA09

        EU175990

        richardsoni

        North-America

        USA

        Minnesota

        NA10

        EU175991

        longicaudus

        North-America

        USA

        Sierra County

        NA11

        EU175992

            

        NA12

        EU175993

        abbreviatus

        North-America

        Alaska

        Hall Island

        NA13

        EU175994

        oregoni

        North-America

        USA

        Oregon

        NA14

        EU175995

            

        NA15

        EU175996

        townsendii

        North-America

        USA

        Oregon

        NA16

        EU175997

            

        NA17

        EU175998

        montebelli

        Asia

        Japan

        Tottori lity

        A01

        EU175999

            

        A02

        EU176000

        kikuchii

        Asia

        Taiwan

        Tao-Yuan

        A03

        EU176001

        other rodents:

             

        Arvicola terrestris

        Europe

        Switzerland

        Bern

        A. terrestris

        EU176002

        Apodemus sylvaticus

        Europe

        Switzerland

        Bern

        A. sylvaticus

        EU176003

        Clethrionomys glareolus

        Europe

        Germany

        Waldbeck

        C. glareolus

        EU176004

        Sequences of heterozygous individuals were resolved by cloning and sequencing of PCR-products (see text). Chromosomes are labelled according to continent of origin of the samples (E = Europe, NA = North America, A = Asia). GenBank accession numbers are given for avpr1a nucleotide sequences.

        After translation into AA, the 36 DNA sequence types coded for 24 different proteins. Individuals from three species remained heterozygous at the AA level (in M. arvalis, M. rossiaemeridionalis and M. oregoni). Two protein types were shared among several species (M. longicaudus, M. socialis and M. tatricus, M. oeconomus, M. multiplex, respectively). The highest number of AA variants was found in the ligand binding domain of the V1aR with up to four different AAs per position (Figure 1). The number of AA changes was significantly different between ligand binding domain, G-protein binding domain and transmembrane regions (χ2 = 13.95, df = 2, p < 0.001). AA changes occurred at eight positions (24%) in the ligand-binding N-terminus of the protein, at six positions (18%) in the G-protein binding domain, and at 20 positions (58%) between these functionally important regions (Figure 2). Significantly more AA changes were present in the N-terminus (χ2 = 13.92, df = 1, p < 0.001) than in the first five transmembrane regions or the G-protein binding domain (χ2 = 13.92, df = 1, p < 0.001). The G-protein binding domain did not show significantly more AA substitutions than the transmembrane regions (χ2 = 0.06, df = 1, p > 0.5). The individuals with two V1aR types differed at the intra-individual level either in the ligand binding domain (M. oregoni, position 26) or in the G-protein binding domain (M. rossiaemeridionalis, position 255; M. arvalis, position 262).
        http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-7-176/MediaObjects/12862_2007_Article_467_Fig1_HTML.jpg
        Figure 1

        Synonymous and non-synonymous changes in the avpr1a gene. A: Schematic overview of the structure of V1a receptor adapted from a model of Mus musculus [95]. The functionally important receptor regions (ligand binding and G-protein binding domains) are shown in red, while six out of seven transmembrane regions are displayed in black (label TM1-TM6). B: Non-synonymous (grey bars) and synonymous (black lines) substitutions in Eutherian mammals and the marsupial Monodelphis domestica (one DNA sequence per species, see text). Highest numbers of non-synonymous substitutions are present in the ligand binding and the G-protein binding domains, while synonymous substitutions are scattered along the whole gene. C: Non-synonymous (grey bars) and synonymous (black lines) changes for 24 species of the Microtus genus (one sequence per species, see text). High numbers of AA variants are found in the ligand binding domain only, while the G-protein binding domain is relatively conserved. Similar to the pattern in higher mammalian taxa, synonymous substitutions are equally frequent along the exon.

        http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-7-176/MediaObjects/12862_2007_Article_467_Fig2_HTML.jpg
        Figure 2

        Structural model of the V1a receptor with amino acid substitutions in the genus Microtus. AA substitutions are spread over the whole protein, but largest numbers of changes are found in the functionally important ligand binding domain. Position of changes and type of changes are marked as: black circle = radical change; white circle = conservative change; grey circle = conservative and radical changes at the same position; white square = deletion; black square = insertion; black triangle = radical change and deletion at the same position. Changes between protein types within an individual occur in the functionally important regions (ligand and G-protein binding domains) and are marked as red diamond for a radical change, and as orange diamond for a conservative change.

        Genetic variation within the Microtus genus included the deletion of two AAs in the ligand binding domain in one species (M. agrestis) and an insertion of two AA in the first transmembrane region in three species (M. agrestis, M. montebelli, M. kikuchii). AA insertions segregate together with an AA change at position 42 for a group of related species (Figure 3). These protein alterations were apparently subsequently lost in one species of this cluster (M. oeconomus, see Figure 3). The two closely related sister species M. arvalis and M. rossiaemeridionalis [28] share two AA changes (58, 85), while otherwise protein types did not obviously segregate with phylogenetic relationships as inferred from the mitochondrial cytochrome b gene.
        http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-7-176/MediaObjects/12862_2007_Article_467_Fig3_HTML.jpg
        Figure 3

        Amino acid alterations of the avpr1a gene plotted onto a mitochondrial cytochrome b phylogeny of the genus Microtus. Positions and types of changes are labelled as in Figure 2. Bootstrap values > 50 (10'000 replicates) of the maximum likelihood method are shown on the branches. AA alterations in Microtus segregate generally independently of the phylogenetic background except for the closely related sister species M. arvalis and M. rossiaemeridionalis which show two identical changes at the same positions (58, 85). Additionally, a two AA long insertion together with an alteration at position 42 appear in the cluster of M. agrestis together with M. montebelli and M. kikuchii, where the changes seem to have been subsequently lost in M. oeconomus.

        23 AA substitutions involved radical (at least one change between physico-chemical classes considering polarity, charge and volume; see in [29]) and 10 conservative (all three categories reveal the same physico-chemical characteristics for the interchangeable AAs) changes. Ten of the radical changes were found in the ligand binding and the G-protein binding domains (see Figure 2).

        Different selection tests detected mostly negative selection on the Microtine avpr1a gene and some ambiguous evidence for positive selection on particular parts. HyPhy detected no signal of positive selection and several sites under negative selection between the functionally important regions (codon positions 50, 67, 68, 82, 170, 186, p < 0.05). PAML results suggested equal substitution rates among Microtus lineages (M0 vs M3: 2Δ l = 6.1684, 4 df, p > 0.05, see Table 2), and no statistical support for positive selection in any part of the gene (M1 vs M2: 2Δ l = 0, 2 df, p > 0.5; M7 vs M8: 2Δ l = 0, 2 df, p > 0.5). Nevertheless, some codon positions had an ω exceeding 1 in the analysis of models for positive selection (M2 and M8: positions 18 and 26; M8 only: position 30), and these sites lie in the ligand binding domain at the N-terminus of the gene. These codons were positively selected against a background of strong purifying selection acting on 96% of the sites, which is in agreement with the results of HyPhy.
        Table 2

        Results of avpr1a selection tests performed with the software PAML

        model

        parameters

        likelihood l

        positively selected sites

        A: Microtus

           

        M0, one ratio

         

        -1970.4456

        Not allowed

        M1, neutral

        p 0 = 0.9600, ω0 = 0.0602

        -1967.7956

        Not allowed

         

        p 1 = 0.0400, ω1 = 1

          

        M2, selection

        p 0 = 0.9600, ω0 = 0.0602

        -1967.7956

        18,26

         

        p 1 = 0.0199, ω1 = 1

          
         

        p 2 = 0.0200, ω2 = 1

          

        M3, discrete

        p 0 = 0.1311, ω0 = 0.0406

        -1967.3614

         
         

        p 1 = 0.7474, ω1 = 0.0407

          
         

        p 2 = 0.1215, ω2 = 0.4609

          

        M7, beta

        p = 0.29056, q = 2.75313

        -1967.4361

        Not allowed

        M8, beta and ω

        p 0 = 1.0000, p = 0.29056, q = 2.75313

        -1967.4361

        18,26,30

         

        p 1 = 0.0000, ω = 1.0000

          

        B: Mammals

           

        M0, one ratio

         

        -3628.0022

        Not allowed

        M1, neutral

        p 0 = 0.8433, ω0 = 0.0452

        -3567.8186

        Not allowed

         

        p 1 = 0.1568, ω1 = 1

          

        M2, selection

        p 0 = 0.8433, ω0 = 0.0452

        -3567.8186

         
         

        p 1 = 0.1356, ω1 = 1

          
         

        p 2 = 0.0212, ω2 = 1

          

        M3, discrete

        p 0 = 0.5084, ω0 = 0.0000

        -3538.7336

         
         

        p 1 = 0.3169, ω1 = 0.1063

          
         

        p 2 = 0.1747, ω2 = 0.4661

          

        M7, beta

        p = 0.23059, q = 1.71469

        -3540.0533

        Not allowed

        M8, beta and ω

        p 0 = 1.0000, p = 0.23059, q = 1.71466

        -3540.0535

        25,36

         

        p 1 = 0.0000, ω = 2.02300

          

        Branch specific models:

           

        MA, foreground branch = M. montanus

         

        -3567.8186

        none

        MA, foreground branch = A. terrestris

         

        -3567.8187

        none

        MA, foreground branch = C. glareolus

         

        -3567.8186

        none

        MA, foreground branch = A. sylvaticus

         

        -3567.8186

        none

        MA, foreground branch = M. musculus

         

        -3567.3630

        none

        MA, foreground branch = R. norvegicus

         

        -3567.8186

        none

        MA, foreground branch = O. aries

         

        -3563.2582

        191,228,231,243,247,260

        MA, foreground branch = B. taurus

         

        -3567.3824

        243

        MA, foreground branch = C. familiaris

         

        -3567.8186

        191

        MA, foreground branch = M. mulatta

         

        -3567.8186

        none

        MA, foreground branch = P. troglodytes

         

        -3567.5837

        247

        MA, foreground branch = H. sapiens

         

        -3567.5242

        259,274

        MA, foreground branch = M. domestica

         

        -3567.7818

        none

        Maximum likelihood methods were applied to compare models allowing for positive or negative selection (M2, M3, M8 and MA) with models without selection (M0, M1, M7). Parameters are represented as p 0 = proportion of sites where ω < 1 (ω0), p 1 = proportion of sites where ω = 1 (ω1), and for models with selection p 2 = proportion of sites where ω > 1 (ω2). For models M7 and M8, p and q represent parameters of the beta distribution. The log likelihood l of each model is given as well as the position of positively selected codons (where ω > 1). A : Overall selection tests in Microtus species (M1 vs. M2, M7 vs. M8) and ratio heterogeneity (M0 vs. M3). B : Overall selection tests of avpr1a and tests for positive selection along specific mammal branches (M1 vs. MA).

        Mammalian avpr1a diversity

        To contrast Microtine avpr1a diversity to variability in other mammals, we sequenced the corresponding fragment of the first exon in different rodent taxa (Arvicola terrestris, Clethrionomys glareolus, Apodemus sylvaticus, see Table 1) and supplemented it with published nucleotide sequences of several Eutherian mammals, as well as a marsupial sequence (Monodelphis domestica) as outgroup. Nucleotide sequence analyses revealed high nucleotide diversity (0.1488) and a high proportion of variable positions (41.7%; 36.38% without marsupial). A phylogenetic tree based on nucleotide sequences using ML and NJ reconstruction methods revealed the same topology (Figure 4), with e.g. rodents and primates forming highly supported clades.
        http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-7-176/MediaObjects/12862_2007_Article_467_Fig4_HTML.jpg
        Figure 4

        Maximum likelihood tree inferred from the nucleotide sequences of exon 1 of the arginine-vasopressin 1a receptor gene for various mammalian taxa. Bootstrap values > 50 are shown for the maximum likelihood method above branches and for neighbour-joining below branches. Positively selected sites (ω > 1) are shown in black circles. Note that most of these sites are found in the G-protein binding domain (231–274). Only two positively selected sites (191; 228) were detected outside this domain in two species (O. aries and C. familiaris).

        The high diversity found at the nucleotide level resulted in high AA diversity after translation with all species showing unique AA sequence types. Most changes occurred in the two functionally important regions of the V1aR: the ligand binding domain and the G-protein binding domain (Figure 1). The latter region included many AA deletions and insertions, resulting in length variation among mammals. Except for a 3 AA long deletion in several rodents (M. montanus, A. terrestris, C. glareolus), the other insertions and deletions occurred in single species only.

        Sliding window analyses of dN/dS ratios along the gene showed a strong signal of positive selection in the ligand binding domain (dN/dS = 2.163), while the dN and dS values in the region around the G-protein binding domain are equal due to relatively more synonymous variation (Figures 1; 5). The transmembrane regions show comparatively few non-synonymous mutations (Figure 1) which leads to small dN/dS ratios (Figure 5).
        http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-7-176/MediaObjects/12862_2007_Article_467_Fig5_HTML.jpg
        Figure 5

        Sliding window analysis of the ratio of non-synonymous substitutions (dN) over synonymous substitutions (dS) along the avpr1a gene of mammals compared to the marsupial Monodelphis domestica (see text). The ratio is drawn over the midpoint window position (window size 30, step size 10) from nucleotide position 50 to 800 from the start codon (due to primer selection). dN/dS exceeds 1 in the ligand binding domain, which indicates positive selection in this region. A second peak of dN/dS close to 1 is found around 750 bp corresponding to the G-protein binding domain of the AVP 1a receptor.

        Despite the evidence for positive selection on the ligand binding domain, further tests rather suggested generally negative selection on avpr1a. PAML detected significant rate variation among the lineages (M0 vs M3: 2Δ l = 178.537, df = 4 p < 0.05), where 88% of all sites were under strong purifying selection, while 12% showed relaxed purifying selection acting on these sites (Table 2). PAML revealed no evidence for positive selection overall (M1 vs M2: 2Δ l = 0, df = 2 p > 0.05; M7 vs M8:2Δ l = 0.0004, df = 2 p > 0.05; Table 2). HyPhy detected five negatively selected sites in functionally important regions and 24 in-between (codon positions 33, 41, 47, 48, 52, 69, 71, 77, 87, 107, 119, 120, 125, 136, 138, 146, 152, 159, 178, 184, 198, 216, 223, 227, 230, 250, 251, 254, 279).

        Considering the phylogenetic background of the species provided further evidence for non-neutral evolution of the avpr1a gene. For the mammalian branches, evolutionary models allowing for selection (MA) were not significantly better than models not incorporating selection (M1; see likelihood values Table 2). Codons with dN/dS ratios exceeding 1 were detected mainly in the G-protein binding domain (231–274), with only two species showing positively selected sites outside (O. aries, C. familiaris; positions 191, 228; see Figure 4).

        Despite high variability among mammals in general and within Microtus, substitution rates of avpr1a are not exceptionally high relative to other nuclear genes in the comparison of mouse, rat and Microtus (Figure 6). Many genes investigated to date show much higher non-synonymous rates than avpr1a and synonymous rates rank this receptor gene only slightly higher. However, it is worth noting that substitution rates of avpr1a are higher than for other hormone receptors with related function like oxytocin, corticothropin or estrogen.
        http://static-content.springer.com/image/art%3A10.1186%2F1471-2148-7-176/MediaObjects/12862_2007_Article_467_Fig6_HTML.jpg
        Figure 6

        Comparison of synonymous and non-synonymous substitutions per site for orthologous nuclear genes in Microtus, mouse and rat. Genes are ranked according to non-synonymous substitutions (black bars) per site. Synonymous substitutions per site are shown as white bars.

        Discussion

        Our analyses of the avpr1a gene, shown to have high behavioural impact in the genus Microtus as well as in other mammals [12, 30], revealed high nucleotide and protein diversity. Variation within Microtus involved many radical physico-chemical amino acid substitutions and deletions, which were located at functionally important regions of the V1aR. The pattern indicates positive selection on few codons in the ligand binding domain and possibly in the G-protein binding domain, but purifying selection on the majority of the gene.

        High genetic variation in the avpr1a gene

        Genetic variability in the coding region of the avpr1a gene appears much higher and evolutionarily much more important than previously suggested [30, 31]. DNA sequences of just two M. ochrogaster and M. montanus individuals were taken as evidence that the Microtine avpr1a gene was highly conserved [14, 20, 31]. However, our analyses reveal not only high levels of genetic variation in the coding region between mammalian species, but also within the genus Microtus. We detected up to 23 polymorphic positions in the first exon of the gene within a single Microtus individual compared to other closely related species, whereas studies of human avpr1a revealed a few synonymous and non-synonymous SNP in humans [22, 27]. Population data from at least one Microtus species will be necessary to allow more detailed comparisons with human variation. However, the apparent difference between voles and humans may be explained in part by the longer evolutionary history of Microtus voles of at least several hundred thousand years with about three generations per year [28] and generally elevated mutation rates in rodents compared to primates [32, 33]

        Many AA replacements in the Microtine avpr1a gene involved radical physico-chemical changes, and several vole species showed deletions and insertions of AAs in this hormone receptor gene. Such length-variation in coding DNA within or among closely related species is remarkable, because it is usually restricted to non-coding DNA, where it may influence in particular cases the expression of genes but is generally functionally and selectively neutral [3437]. Additionally, we detected considerable length variation in the G-protein binding region between different mammalian species. The diversity found among mammals might influence signal transduction, since already single AA changes can lead to differences in receptor activation in this region [16].

        Amino acid positions identified as crucial for either ligand binding or G-protein activation in humans, mouse and rat are mainly conserved among Microtus species as well as among other mammals. A highly conserved triplet (Asp148-Arg149-Tyr150) with a role in signal transduction in many G-protein coupled receptors was conserved across all individuals analysed [38]. Additionally, a glycosylation site (Asn27) with a crucial role in protein folding or stabilization [39] remained conserved among all mammals and voles. Glu185, supposed to be involved in agonist and peptide as well as non-peptide antagonist binding to the V1aR [16], was highly conserved among mammals except for one Microtus individual (M. richardsoni), which showed a mutation to His185. This alteration together with an additional mutation at a glycosylation site (Asn198->Thr198) could lead to dysfunctions [39]. It is unclear if this would apply to voles since analyses of the specific roles of these sites in Microtus are lacking.

        The observed high level of diversity and the detection of indels are unlikely to be due to gene duplications of avpr1a or the occurrence of pseudo-genes. avpr1a is a single copy gene in humans [40] and in rat [41], and a duplication has been found only in M. ochrogaster [14]. We cannot exclude that some of the detected variation stems from the presence of very recently duplicated sequences in some species. However, contrary to the truncated and clearly divergent version of avpr1a in M. ochrogaster, we found no indication for non-functionality in any of the sequences, such as reading frame shifts due to insertions or deletions of single nucleotides or the presence of premature stop codons [42]. This suggests that at least the majority of avpr1a gene variants is functional.

        The potential functional relevance of variation in Microtine avpr1a is further emphasized by the detection of alleles coding for different protein types within the same individuals. It is worth noting that this involved again the ligand and G-protein binding domains. However, it is currently unclear if heterozygous individuals express different protein variants in the same tissue or if there is tissue specific expression [see [15]]. Gene expression studies are needed to investigate this further, since some receptor functions can as well be substituted by other receptors for hormones which are involved in very similar pathways [30, 43]. This distinction would be of pharmaceutical importance for human health [see [44, 45]]. Given the high variability in protein types, Microtus voles could serve as ideal models to study the processing and the expression of avpr1a gene products and their functional consequences in homo- and heterozygous individuals since they can be bred in the laboratory [21].

        We further hypothesize that variation in the coding sequence of avpr1a might be related to life history traits (e.g. mating system, habitat), given the peripheral role of the V1aR in water retention in the kidney [16], and the social relevance when expressed in the brain [710]. Although kidney inefficiency was suggested as a major reason for the restriction of some Microtus species to moist habitat [46], we could not detect any connection between receptor type and a given habitat. Species occupying dry habitats (e.g. M. multiplex, M. arvalis, M. lusitanicus, M. pinetorum, M. nivalis, [4750]) or wet habitats (e.g. M. agrestis, M. rossiaemeridionalis, M. tatricus and M. oeconomus [5153]) showed no specific V1aR types or AA change corresponding to habitat requirements, nor were they phylogenetically closely related (Figure 3). Additionally, there was no general association of AA variation in Microtus avpr1a with social or genetic mating system. The socially monogamous (defined by observational studies, see in [54]) species M. ochrogaster, M. multiplex and M. pinetorum [51, 52, 55] shared neither a protein type nor a particular AA change. Similarly, neither showed the socially non-monogamous species such as M. montanus, M. californicus and M. richardsoni [51, 53, 56] nor the genetically non- monogamous species (M. arvalis, M. agrestis and M. ochrogaster, [18, 5759]) identical AA alterations or protein types among each other that could potentially be associated with behavioural patterns. It is obvious that the high level of variation in the genus Microtus makes it very difficult to detect any direct associations of protein types or AA changes with basic life history traits. Since inter- and intra-specific variation in mating behaviour and habitat usage exist (e.g. for M. ochrogaster see [17]), studies on protein variation within species are needed to investigate locally adapted receptor types and their correlation to life history traits.

        Evidence for non-neutral evolution

        Statistical tests for selection indicated mostly purifying selection on the transmembrane partition of V1aR but also a minor role for positive selection in shaping avpr1a diversity in mammals. The sliding window analysis detected positive selection mainly in the ligand binding domain and an increased number of synonymous and non-synonymus changes in the G-protein binding domain. Branch-specific tests across mammals detected positively selected sites mainly in the G-protein binding domain. It is unclear why selection tests failed to detect a deviation from neutrality overall, but the background of purifying selection might be too high in comparison to the positive selection acting on particular domains to allow the signal to be picked up. Additionally, positive selection evidenced by ω > 1 may be difficult to detect as selection could also be acting on synonymous sites [6062].

        The impact of positive selection on avpr1a diversity is less evident within the Microtus genus than in evolutionarily less related mammalian taxa. Overall selection tests remained mostly inconclusive, which may be caused by a lack of power due to the high rate of speciation and the short divergence times between Microtus species [28]. Interestingly, the number of AA variants within Microtus was still significantly higher in the ligand and G-protein binding domains than in the transmembrane regions. This pattern might reflect relaxed selective constraints at the N-terminus and at the G-protein binding domain, and stronger evolutionary constraints on the transmembrane region due to structural limitations because of the embedding in the lipid layer. Alternatively, we suggest that this high diversity in functionally important domains of the gene is compatible with balancing selection maintaining high allele diversity in selected regions [63, 64]. However, we shall need detailed studies at the population level to asses the potential impact of this type of selection on avpr1a diversity in Microtus further [65].

        Evolution of avpr1a variability in comparison to other genes

        The speed of evolutionary change in avpr1a is difficult to assess because of the lack of data on nuclear mammalian genes with similar taxonomic and geographic scope. The only directly comparable data set from a nuclear gene covering several Microtus species comes from the p53 tumor-suppressor gene [66]. Variation in this gene is much lower than in avpr1a with only a few silent mutations in coding regions [66]. Additionally, nucleotide diversity in the fast-evolving nuclear genes IRBP and RAG1 within the mouse genus is lower than in Microtine avpr1a if the longer divergence time in Mus is taken into account (5 to 6 mya for Mus, see in [67] vs. 1.2 to 2 mya for Microtus, see in [28]). Variation in avpr1a appears here even higher after translation of the nucleotide sequences into AAs, because variation in Mus is reduced to 9% variable positions in IRBP and 4% in RAG1 [67] whereas 11% variable positions in the AA sequences remain in the vole avpr1a gene.

        Our comparison of all currently available homologous nuclear genes for the mouse-rat-vole trio showed for the avpr1a gene a relatively high synonymous substitution rate but a comparatively low non-synonymous substitution rate (Figure 6). It is unclear to which extent this comparison is somewhat biased by a generally stronger interest and more published sequences of genes with high mutation rates (e. g. MHC, BRCA [68, 69]). It is nevertheless noteworthy that this comparison revealed higher nucleotide and protein diversity in avpr1a than in other related hormone receptors with similar functions (e.g. oxytocin, see in [16, 20, 70]; serotonin, see in [71, 72]; corticothropin, see in [73, 74]).

        Conclusion

        Our analyses show that genetic diversity in the avpr1a gene is much higher than previously claimed, and that part of this variation might be functionally relevant. We provide evidence for extensive variation in avpr1a at all taxonomic levels of mammals, with many changes in functionally important regions. We suggest that positive selection acting on these operative domains helps to maintain variation despite the presence of overall purifying selection. The role of balancing selection, particularly within the genus Microtus, should nevertheless deserve further investigation at the intra-specific level. The effects of genetic variation in avpr1a on phenotypic traits like mating systems, social behaviour or habitat requirements in Microtus and other mammals are far from being characterized. As this study shows, it seems particularly important to characterize abundant genotypic and phenotypic variation thoroughly before establishing general causal links between genotypes and phenotypes.

        Methods

        Samples

        The V1aR is encoded by two exons: exon1 (~970 bp) and exon2 (~290 bp). We sequenced part (792 bp) of the first exon of the avpr1a gene since this fragment covers the two functionally important regions (ligand and G-protein binding domains) of the receptor. Sequences were analysed for 24 Microtus species which cover the entire Palearctic range of the genus (Europe, North America, Asia; Table 1). Tissue samples were obtained by live trapping with Longworth small mammal traps (Penlon Ltd), or from ecologists studying the species. Genomic DNA was extracted using a standard phenol-chloroform protocol [75] or Magnetic beads (MagneSil™ BLUE, Promega). We used two sequences from GenBank from M. ochrogaster and M. montanus (Accession numbers AF069304 and AF070010) to confirm locus identification.

        Moreover, we sequenced three rodent taxa (Arvicola terrestris, Apodemus sylvaticus and Clethrionomys glareolus, see Table 1) and retrieved additional mammalian avpr1a sequence information from GenBank [76] and Ensembl [77] to compare sequence diversity and substitution rates for the avpr1a gene in mammals. Accession numbers in GenBank are: BC024149 for Mus musculus, NM_053019 for Rattus norvegicus, L41502 for Ovis aries, U19906 for Homo sapiens; Accession numbers in Ensembl:ENSCAFG00000000339 for Canis familiaris ENSBTAG00000007175 for Bos taurus, ENSMMUG00000000549 for Macaca mulatta, ENSPTRG00000005167 for Pan troglodytes, and ENSMODG00000014334 for Monodelphis domestica.

        DNA sequencing

        We amplified avpr1a sequences in a reaction volume of 25 μl in a GeneAmp® PCR System 9700 (Applied Biosystems) using Quiagen Taq polymerase. We used two primer pairs for amplification and sequencing reactions: V1aR-5'exon-ProtF 5'-GAGCTTAGGACAGGCTTTCTCG-3' and V1aR-5'exon-ProtR 5'-CGATCACGAAGGTCATCTTCAC-3', Mus-Mic-exon1f 5'-CCGACAGCATGAGTTTCC-3' together with Mus-Mic-exon1r 5'-CCACATCTGGACGATGAAGA-3'. The PCR amplification profile included an initial denaturation step at 92°C for 2 min, followed by 40 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 90 sec. A final extension step of 72°C for 10 min was performed. Amplified fragments were controlled for size on a 1.5% agarose gel by comparing them with a 100 base pair (bp) ladder (Invitrogen). After cleaning with GenElute™ PCR clean-up kit (Sigma) and dissolving products in 50 μl bi-distilled water, the sequencing reaction was carried out in a 10 μl reaction volume. Terminator Ready Reaction Mix 'Big Dye' Version 3.1 from Applied Biosystems was used. Both strands were sequenced using the following PCR conditions: An initial step of denaturation at 96°C for 10 sec, followed by 30 cycles of denaturation at 96°C for 10 sec, annealing at 55°C for 10 sec, and extension at 72°C for 4 min 30 sec. The products were cleaned using a DyeEx 96 spin kit (Quiagen), and were separated and detected on an ABI Prism 3100 Genetic Analyser from Applied Biosystems.

        Cloning and sequencing of PCR products

        PCR products of individuals showing heterozygous sites in direct sequencing were cloned using the Qiagen PCR Cloning Kit. Purified PCR products were quantified in a Spectrophotometer (Gene Quant pro RNA/DNA Calculator, Biochrom) and approximately 65 ng of the product were ligated into pDrive Cloning Vector (Qiagen) in 10 μl reactions. Reactions were incubated for 45 min at 4°C before heat shock transformation into QIAGEN EZ Competent Cells. An additional incubation step of 45 min at 37°C with shaking was done before plating to allow recombinant growth. Cells were plated onto Kanamycin-IPTG-X-Gal agar and cultured for 17 h at 37°C. Ten positive clones per individual were randomly selected and further grown in LB broth for 17 h at 37°C with shaking. Plasmid miniprep columns (QIAprep® Spin Miniprep Kit, Qiagen) were used to purify each clone before sequencing with both M13 universal 5'-GTAAAACGACGGCCAGT-3'and M13 reverse 5'-CAGGAAACAGCTATGAC-3' primers. Sequencing conditions were as follows: An initial step of denaturation at 90°C for 50 sec, followed by 25 cycles of denaturation at 90°C for 10 sec, annealing at 50°C for 10 sec, and extension at 60°C for 4 min. After a final cleaning step with a DyeEx 96 spin kit (Quiagen), the sequences were run on an ABI Prism 3100 Genetic Analyser from Applied Biosystems.

        Statistical analyses

        Sequences were aligned using the Clustal W algorithm [78] implemented in the program BioEdit 5.0.9 [79], and were revised manually. Shared sequence types were detected using the program Arlequin 3.1 [80]. Phylogenetic relationships among sequenced chromosomes were reconstructed by obtaining neighbour-joining (NJ) [81] and maximum likelihood (ML) trees rooted with Monodelphis domestica for the mammalian taxa and rooted with Arvicola terrestris for the Microtus genus with 10,000 bootstrap replicates in Mega 3 [82] and Paup 4.0 b [83]. For the ML analysis, Modeltest 3.06 [84] implemented in Paup 4.0 b [83] was used to estimate the most suitable model of DNA substitution, by performing hierarchical likelihood ratio tests to compare 52 different models and by applying the Akaike Information Criterion [85]. For the Microtus genus, the best substitution model was the transversion model with gamma distribution (TVM+G) with the following parameters: Substitution rate matrix: A↔C 2.7903; A↔G and C↔T 9.3807; A↔T 1.1820; C↔G 0.9720; G↔T 1.0000; and gamma distribution shape parameter 0.1986. The base frequencies were estimated as: A: 0.1801, C: 0.2951, G: 0.2963, T: 0.2285.

        For the mammalian phylogeny, the best substitution model was the general time reversible model with invariable sites and gamma distribution (GTR+I+G) [86, 87]. The following parameters for the model were estimated: Substitution rate matrix: A↔C 1.7329; A↔G 5.3823; A↔T 0.6124; C↔G 1.4182; C↔T 3.8055; G↔T 1.0000; proportion of invariable sites 0.4474 and gamma distribution shape parameter 3.0860. The base frequencies were estimated as: A: 0.1566, C: 0.3344, G: 0.3228, T: 0.1862.

        The nucleotide sequences were translated into AA sequences in Mega 3 using the universal code. The positions of the AA changes were determined using the structural model of the arginine-vasopressin 1a receptor of Mus musculus [16]. To determine whether changes are equally distributed across the model, we applied Chi-Square tests for the different structural regions (ligand binding domain, transmembrane regions and G-protein binding domain). AA changes were classified as radical or conservative by comparing physicochemical properties of AAs such as charge, polarity and volume following Zhang [29].

        To test for a link between V1aR types and phylogenetic relationships between Microtus, we checked for branch specific AA changes of avpr1a on a mitochondrial cytochrome b gene phylogeny [see in [28]] with sequences obtained from GenBank (accession numbers: AF119280, AF159400, AF163890 –AF163891, AF163893, AF163896, AF163900 –AF163901, AF163903–AF163906, AF187230, AY167210, AY220028, AY220770, AY513788, AY513798, AY513816, AY513819, AY513829, AY513837, AY513840, AY513845). To contrast the synonymous and non-synonymous diversity found in the avpr1a gene to other nuclear genes, we performed an exhaustive GenBank search for all annotated gene sequences available for Microtus species (up to december 20th, 2006). The resulting 31 sequences were aligned with homologous genes from Mus musculus and Rattus norvegicus and synonymous and non-synonymous substitution rates for each gene were computed with Mega 3.

        Tests for selective neutrality

        We tested for regions under positive selection along the mammalian avpr1a by estimating the ratio ω of non-synonymous changes (dN) over synonymous changes (dS) per site. We used a sliding window approach with a window size of 30 and a step size of 10 with the program DnaSP 4.10 to compare mammalian species against the marsupial Monodelphis domestica.

        To further test for the impact of selection on particular sites in avpr1a, we used a maximum likelihood approach with the single likelihood ancestor counting (SLAC) method implemented in HyPhy which makes no assumption about rate variation between lineages [8890]. Further statistical tests for selection involved the computation of lineage-specific ratios of ω using codon-based maximum likelihood methods implemented in the program "codeml" from the PAML package [91]. As a basis for these analyses, we used a phylogenetic tree tested for consistent topology between ML and NJ as well as with data from 3rd codon positions only [see [92]].

        We used likelihood ratio tests in PAML to compare different neutral (MO, M1, M7) and selection (M2, M8) models of DNA sequence evolution of avpr1a. In all these tests, two times the log-likelihood difference (2Δ l) between models is compared to a χ2 distribution with the number of degrees of freedom (dF) equal to the difference in the number of parameters between the models [93]. We tested for rate heterogeneity among lineages by comparing the one ratio model M0 against the discrete model M3 where different rates are allowed [93]. This test is mainly used to check for rate variation of ω, but it can also be used to detect positive selection [94]. Additionally, the neutral model M1 with two ratio classes of ω (< 1 and 1) was compared to the selection model M2 which allows for an additional class where ω > 1 [93]. A similar comparison was carried out between a neutral model assuming a beta distribution of ω (M7), and a model with similar characteristics but allowing for positively selected sites (M8) [93]. We performed branch specific tests to examine whether avpr1a evolves differently in the higher mammalian taxa by comparing the neutral model M1 with model MA which allows for positively selected sites on a pre-selected branch [91, 94].

        Declarations

        Acknowledgements

        We thank I. Dupanloup for helpful discussions and S. Tellenbach for technical assistance. We are grateful to the following people and institutions for providing access to samples: Museum of Vertebrate Zoology of the University of California, A. Bannikova, S. Braaker, R. Burri, Bündner Naturmuseum, F. Catzeflis, C. Conroy, B. Cushing, T. Derting, M. Jaarola, T. Maddalena, N. Martinkova, J.-P. Müller, M. Pfunder, R. Pita, J. Suchomel, J. Robovsky, J. Runge, L. Vinciguerra, P. Vogel. The Swiss National Science Foundation partly financed this study (project no. 112072).

        Authors’ Affiliations

        (1)
        Computational and Molecular Population Genetics (CMPG), Zoological Institute, University of Bern

        References

        1. Robinson GE, Grozinger CM, Whitfield CW: Sociogenomics: Social life in molecular terms. Nature Reviews Genetics 2005, 6: 257–270.View ArticlePubMed
        2. Bucan M, Abel T: The mouse: Genetics meets behaviour. Nature Reviews Genetics 2002, 3: 114–123.View ArticlePubMed
        3. Lank DB, Smith CM, Hanotte O, Burke T, Cooke F: Genetic polymorphism for alternative mating behaviour in lekking male ruff Philomachus pugnax . Nature 1995, 378: 59–62.View Article
        4. Campesan S, Dubrova Y, Hall JC, Kyriacou CP: The nonA gene in Drosophila conveys species-specific behavioural characteristics. Genetics 2001, 158: 1535–1534.PubMed
        5. Shuster SM, Sassaman C: Genetic interaction between male mating strategy and sex ratio in a marine isopod. Nature 1997, 388: 373–377.View Article
        6. Wheeler DA, Charalambos PK, Greenacre ML, Yu Q, Rutila JE, Rosbash M, Hall JC: Molecular transfer of a species-specific behavior from Drosophila simulans to Drosophila melanogaster . Science 1991, 251: 1082–1085.View ArticlePubMed
        7. Francis DD, Young LJ, Meaney MJ, Insel TR: Naturally occurring differences in maternal care are associated with the expression of oxytocin and vasopressin (V1a) receptors: gender differences. Journal of Neuroendocrinology 2002, 14: 349–353.View ArticlePubMed
        8. Young LJ, Winslow JT, Nilsen R, Insel TR: Species differences in V1a receptor gene expression in monogamous and nonmonogamous voles: behavioral consequences. Behavioral Neuroscience 1997, 111: 599–605.View ArticlePubMed
        9. Phelps SM, Young LJ: Extraordinary diverstiy in vasopressin (V1a) receptor distribution among wild prairie voles ( Microtus ochrogaster ): Patterns of variation and covariation. Journal of Comparative Neurology 2003, 466: 564–576.View ArticlePubMed
        10. Keverne EB, Curley JP: Vasopressin, oxytocin and social behaviour. Current Opinion in Neurobiology 2004, 14: 777–783.View ArticlePubMed
        11. Bachner-Melman R, Zohar AH, Bacon-Shnoor N, Elizur Y, Nemanov L, Gritsenko I, Ebstein RP: Link between vasopressin receptor AVPR1A promotor region microsatellites and measures of social behaviour in humans. Journal of Individual Differences 2005, 26: 2–10.View Article
        12. Lim MM, Wang Z, Olazabal DE, Ren X, Terwilliger EF, Young LJ: Enhanced partner preference in a promiscuous species by manipulating the expression of a single gene. Nature 2004, 429: 754–757.View ArticlePubMed
        13. Lim MM, Hammock EAD, Young LJ: The role of vasopressin in the genetic and neural regulation of monogamy. J Neuroendocrinol 2004, 16: 325–332.View ArticlePubMed
        14. Young LJ, Nilsen R, Waymire KG, MacGregor GR, Insel TR: Increased affiliative response to vasopressin in mice expressing the V1a receptor from a monogamous vole. Nature 1999, 400: 766–768.View ArticlePubMed
        15. Hammock EAD, Young LJ: Functional microsatellite polymorphism associated with divergent social structure in vole species. Mol Biol Evol 2004, 21: 1057–1063.View ArticlePubMed
        16. Barberis C, Mouillac B, Durroux T: Structural bases of vasopressin/oxytocin receptor function. Journal of Endocrinology 1998, 156: 223–229.View ArticlePubMed
        17. Cushing BS, Martin JO, Young LJ, Carter CS: The effects of peptides on partner preference formation are predicted by habitat in prairie voles. Hormones and Behavior 2001, 39: 48–58.View ArticlePubMed
        18. Fink S, Excoffier L, Heckel G: Mammalian monogamy is not controlled by a single gene. Proceedings of the National Academy of Science of USA 2006, 103: 10956–10960.View Article
        19. Insel TR, Young LJ: The neurobiology of attachment. Nature Reviews Neuroscience 2001, 2: 129–136.View ArticlePubMed
        20. Nair HP, Young LJ: Vasopressin and pair-bond formation: genes to brain to behavior. Physiology 2006, 21: 146–152.View ArticlePubMed
        21. Hammock EAD, Young LJ: Microsatellite instability generates diversity in brain and sociobehavioral traits. Science 2005, 308: 1630–1634.View ArticlePubMed
        22. Wassink TH, Piven J, Vieland VJ, Pietila J, Goedken RJ, Folstein SE, Sheffield VC: Examination of AVPR1a as an autism susceptibility gene. Molecular Psychiatry 2004, 9: 968–972.View ArticlePubMed
        23. Yirmiya N, Rosenberg C, Levi S, Salomon S, Shulman C, Nemanov L, Dina C, Ebstein RP: Association between the arginine vasopressin 1a receptor (AVPR1a) gene and autism in a family-based study: mediation by socialization skills. Molecular Psychiatry 2006, 11: 488–494.View ArticlePubMed
        24. Kim SJ, Young LJ, Gonen D, Veenstra-VanderWeele J, Courchesne R, Courchesne E, Lord C, Leventhal BL, Cook EH, Insel TR: Transmission disequilibrium testing of arginine vasopressin receptor 1A (AVPR1A) polymorphisms in autism. Molecular Psychiatry 2002, 7: 503–507.View ArticlePubMed
        25. Bachner-Melman R, Zohar AH, Elizur Y, Nemanov L, Gritsenko I, Konis D, Ebstein RP: Association between a vasopressin receptor avpr1a promoter region microsatellite and eating behavior measured by a self-report questionnaire (eating attitudes test) in a family-based study on a nonclinical population. International Journal of Eating Disorders 2004, 36: 451–460.View ArticlePubMed
        26. Bachner-Melman R, Dina C, Zohar AH, Constantini N, Lerer E, Hoch S, Sella S, Nemanov L, Gritsenko I, Lichtenberg P, Granot R, Ebstein RP: AVPR1a and SLC6A4 Gene Polymorphisms Are Associated with Creative Dance Performance. PLoS Genetics 2005, 1: e42.View ArticlePubMed
        27. Saito S, Iida A, Sekine A, Kawauchi S, Higuchi S, Ogawa C, Nakamura Y: Catalog of 178 variations in the Japanese population among eight human genes encoding G protein-cupled receptors (GPCRs). Journal of Human Genetics 2003, 48: 461–468.View ArticlePubMed
        28. Jaarola M, Martinkova N, Gunduz I, Brunhoff C, Zima J, Nadachowski A, Amori G, Bulatova NS, Chondropoulos B, Fraguedakis-Tsolis S, Gonzalez-Esteban J, Lopez-Fuster MJ, Kandaurov AS, Kefelioglu H, da Luz Mathias M, Villate I, Searle JB: Molecular phylogeny of the speciose vole genus Microtus (Arvicolinae, Rodentia) inferred from mitochondrial DNA sequences. Molecular Phylogenetics and Evolution 2004, 33: 647–663.View ArticlePubMed
        29. Zhang J: Rates of conservative and radical nonsynonymous nucleotide substitutions in mammalian nuclear genes. Journal of Molecular Evolution 2000, 50: 56–68.PubMed
        30. Young LJ: Oxytocin and vasopressin receptors and species-typical social behaviors. Hormones and Behavior 1999, 36: 212–221.View ArticlePubMed
        31. Hammock EAD, Young LJ: Variation in the vasopressin V1a receptor promoter and expression: implications for inter- and intraspecific variation in social behaviour. Eur J Neurosci 2002, 16: 399–402.View ArticlePubMed
        32. Catzeflis FM, Sheldon FH, Ahlquist JE, Sibley CG: DNA-DNA hybridization evidence of the rapid rate of muroid rodent DNA evolution. Molecular Biology and Evolution 1987, 4: 242–253.PubMed
        33. Li WH, Ellsworth DL, Krushkal J, Chang BHJ, Hewett-Emmett D: Rates of nucleotide substitution in primates and rodents and the Generation-Time Effect Hypothesis. Molecular Phylogenetics and Evolution 1996, 5: 182–187.View ArticlePubMed
        34. King MC, Wilson AC: Evolution at two levels in humans and chimpanzees. Science 1975, 188: 107–116.View ArticlePubMed
        35. Sinnett D, Beaulieu P, Bélanger H, Lefebvre JF, Langlois S, Théberge MC, Drouin S, Zotti C, Hudson TJ, Labuda D: Detection and characterization of DNA variants in the promoter regions of hundreds of human disease candidate genes. Genomics 2006, 87: 704–710.View ArticlePubMed
        36. Lo HS, Wang Z, Hu Y, Yang HH, Gere S, Buetow KH, Lee MP: Allelic variation in gene expression is common in the human genome. Genome Research 2003, 13: 1855–1862.View ArticlePubMed
        37. Yan H, Yuan W, Velculescu VE, Vogelstein B, Kinzler KW: Allelic variation in human gene expression. Science 2002, 297: 1143.View ArticlePubMed
        38. Hawtin SR: Charged residues of the conserved DRY triplet of the vasopressin V1a receptor provide molecular determinants for cell surface delivery and internalization. Molecular Pharmacology 2005, 68: 1172–1182.View ArticlePubMed
        39. Hawtin SR, Davies ARL, Matthews G, Wheatley M: Identification of the glycosylation sites utilized on the V1a vasopressin receptor and assessment of their role in receptor signalling and expression. Biochemical Journal 2001., 357:
        40. Thibonnier M, Graves MK, Wagner MS, Auzan C, Clauser E, Willard HF: Structure, sequence, expression, and chromosomal localization of the human V1a vasopressin receptor gene. 1996.
        41. Murasawa S, Matsubara H, Kijima K, Maruyama K, Mori Y, Inada M: Structure of the rat V1a vasopressin receptor gene and characterization of its promotor region and complete cDNA sequence of the 3'end. Journal of Biological Chemistry 1995, 270: 20042–20050.View ArticlePubMed
        42. Slade RW, Moritz C, Heideman A, Hale PT: Rapid assessment of single-copy nuclear DNA variation in diverse species. Molecular Ecology 1993, 2: 359–373.View ArticlePubMed
        43. Wang Z, Yu G, Cascio C, Liu Y, Gingrich B, Insel TR: Dopamine D2 receptor-mediated regulation of partner preferences in female prairie voles ( Microtus ochrogaster ): A mechanism for pair bonding? Behavioral Neuroscience 1999, 113: 602–611.View ArticlePubMed
        44. Shearman AM, Cooper JA, Kotwinski PJ, Miller GJ, Humphries SE, Ardlie KG, Jordan B, Irenze K, Lunetta KL, Schuit SCE, Uitterlinden AG, Pols HAP, Demissie S, Cupples LA, Mendelson ME, Levy D, Housman DE: Estrogen receptor alpha gene variation is associated with risk of myocardial infarction in more than seven thousand men from five cohorts. Circulation Research 2006, 98: 590–592.View ArticlePubMed
        45. Vaclavicek A, Hemminki K, Bartram CR, Wagner K, Wappenschmidt B, Meindl A, Schmutzler RK, Klaes R, Untch M, Burwinkel B, Forsti A: Association of prolactin and its receptor gene regions with familial breast cancer. Journal of Clinical Endocrinology and Metabolism 2006, 91: 1513–1519.View ArticlePubMed
        46. Getz LL: A comparison of water balance of the prairie and meadow vole. Ecology 1963, 44: 202–207.View Article
        47. Tamarin RH: Biology of New World Microtus. Shippensburg, Pennsylvania, American Society of Mammologists 1985.
        48. Mitchell-Jones AJ, Amori G, Bogdanowicz W, Krystufek B, Reijnders PJH, Spitzenberger F, Stubbe M, Thissen JBM, Vohralik V, Zima J: The atlas of European mammals. London, T. Poyser, A. D. Poyser 1999.
        49. Niethammer J, Krapp F: Handbuch der Säugetiere Europas. Wiesbaden, Akademische Verlagsgesellschaft 1982, Rodentia II: 649.
        50. Jurdíková N, Žiak D, Kocian L: Habitat requirements of Microtus tatricus : macrohabitat and microhabitat:; Banská Bystrica. (Edited by: Urban P). ŠOP COPK 2000, 41–49.
        51. Shapiro LE, Dewsbury DA: Male dominance, female choice and male copulatory behavior in two species of voles ( Microtus ochrogaster and Microtus montanus ). Behavioral Ecology and Sociobiology 1986, 18: 267–274.View Article
        52. Marfori MA, Parker PG, Gregg TG, Vandenbergh JG, Solomon NG: Using DNA fingerprinting to estimate relatedness within social groups of pine voles. Journal of Mammalogy 1997, 78: 715–724.View Article
        53. Pearson OP: Habits of Microtus californicus revealed by automatic photographic records. Ecological Monographs 1960, 30: 231–445.View Article
        54. Reichard UH, Boesch C: Monogamy: Mating Strategies and Partnerships in Birds, Humans and Other Mammals., Cambridge University Press 2003.
        55. Salvioni M: Home range and social behavior of three species of European Pitymys (Mammalia, Rodentia). Behavioral Ecology and Sociobiology 1988, 22: 203–210.View Article
        56. Ludwig DR: The population biology and life history of the water vole ( Microtus richardsoni ). Calgary, Alberta, PhD thesis, University of Calgary 1981.
        57. Agrell J: Experimental testing of female mating strategies in microtine rodents:; Acapulco, Mexico. 1997, 5.
        58. Agrell J, Wolff JO, Ylonen H: Counter-strategies to infanticide in mammals: costs and consequences. Oikos 1998, 83: 507–571.View Article
        59. Solomon NG, Keane B, Knoch LR, Hogan PJ: Multiple paternity in socially monogamous prairie voles ( Microtus ochrogaster ). Canadian Journal of Zoology 2004, 82: 1667–1671.View Article
        60. Chamary JV, Parmley JL, Hurst LD: Hearing silence: non-neutral evolution at synonymous sites in mammals. Nature Reviews Genetics 2006, 7: 98–108.View ArticlePubMed
        61. Pond SK, Muse SV: Site-to-site variation of synonymous substitution rates. Molecular Biology and Evolution 2005, 22: 2375–2385.View ArticlePubMed
        62. Hurst LD, Pál C: Evidence for purifying selection acting on silent sites in BRCA1. Trends in Genetics 2001, 17: 62–65.View ArticlePubMed
        63. Soussi T, May P: Structural aspects of the p53 protein in relation to gene evolution: a second look. Journal of Molecular Biology 1996, 260: 623–637.View ArticlePubMed
        64. Kreitman M, Hudson RR: Inferring the evolutionary histories of the Adh and Adh-dup loci in Drosophila melanogaster from patterns of polymorphism and divergence. Genetics 1991, 127: 565–582.PubMed
        65. Excoffier L, Heckel G: Computer programs for population genetics data analysis: a survival guide. Nature Reviews Genetics 2006, 7: 745–758.View ArticlePubMed
        66. DeWoody JA: Nucleotide variation in the p53 tumor suppressor gene of voles from Chernobyl, Ukraine. Mutation Research 1999, 439: 25–36.PubMed
        67. Suzuki H, Shimada T, Terashima M, Tsuchiya K, Aplin K: Termporal, spatial, and ecological modes of evolution of Eurasian Mus based on mitochondrial and nuclear gene sequences. Molecular Phylogenetics and Evolution 2004, 33: 626–646.View ArticlePubMed
        68. Nei M: Molecular Evolutionary Genetics. New York, Columbia University Press 1987.
        69. Figueroa F, Günther E, Klein J: MHC polymorphism pre-dating speciation. Nature 1988, 335: 265–271.View ArticlePubMed
        70. Cushing BS, Kramer KM: Mechanisms underlying epigenetic effects of early social experience: The role of neuropeptides and steroids. Neuroscience and Biobehavioral Reviews 2005, 29: 1089–1105.View ArticlePubMed
        71. de Almeida RMM, Ferrari PF, Parmigiani S, Miczek KA: Escalated aggressive behavior: Dopamine, serotonin and GABA. European Journal of Pharmacology 2005, 526: 51–64.View ArticlePubMed
        72. Schiller L, Jähkel M, Oehler J: The influence of sex and social isolation housing on pre- and postsynaptic 5-HT1A receptors. Brain Research 2006, 1103: 76–87.View ArticlePubMed
        73. DeVries AC, Guptaa T, Cardillo S, Cho M, Carter CS: Corticotrophin-releasing factor induces social preferences in male prairie voles. Psychoneuroendocrinology 2002, 27: 705–714.View ArticlePubMed
        74. Lim MM, Nair HP, Young LJ: Species and sex differences in brain distribution of corticotropin-releasing factor receptor subtypes 1 and 2 in monogamous and promiscuous voles species. Journal of Comparative Neurology 2005, 487: 75–92.View ArticlePubMed
        75. Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: A laboratory manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press 1989.
        76. GenBank [http://​www.​ncbi.​nlm.​nih.​gov/​]
        77. Ensembl [http://​www.​ensembl.​org]
        78. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 1997, 25: 4876–4882.View ArticlePubMed
        79. Hall TA: BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 1999, 41: 95–98.
        80. Excoffier L, Laval G, Schneider S: Arlequin ver. 3.0: An integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online 2005, 1: 47–50.PubMed
        81. Saitou N, Nei M: The neighbor-joining method: A new method for reconstructing phylogentic trees. Molecular Biology and Evolution 1987, 4: 406–425.PubMed
        82. Kumar S, Tamura K, Nei M: MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Briefings in Bioinformatics 2004, 5: 150–163.View ArticlePubMed
        83. Swofford DL: PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4 Edition Sunderland, Mass., Sinauer 1999.
        84. Posada D, Crandall KA: MODELTEST: testing the model of DNA substitution. Bioinformatics 1998, 14: 817–818.View ArticlePubMed
        85. Akaike H: A new look at the statistical model identification. IEEE Transactions on Automatic Control 1974, 19: 716–723.View Article
        86. Rodriguez F, Oliver JL, Marin A, Medina JR: The general stochastic model of nucleotide substitution. Journal of Theoretical Biology 1990, 142: 485–501.View ArticlePubMed
        87. Tavaré S: Some probabilistic and statistical problems on the analysis of DNA sequences. Lectures of Mathematical Life Sience 1986, 17: 57–86.
        88. www.datamonkey.org: HyPhy.
        89. Pond SL, Frost SDW: Datamonkey: rapid detection of selective pressure on individual sites of codon alignments. Bioinformatics 2005, 21: 2531–2533.View ArticlePubMed
        90. Pond SL, Frost SDW: Not so different after all: A comparison of methods for detecting amino-acid sites under selection. Molecular Biology and Evolution 2005, 22: 1208–1222.View Article
        91. Yang Z: PAML: a program package for phylogenetic analysis by maximum likelihood. CABIOS 1997, 13: 555–556.PubMed
        92. Suzuki Y, Nei M: False-positive selection identified by ML-based methods: Examples from the Sig1 gene of the diatom Thalassiosira weissflogii and the tax gene of a human T-cell lymphotropic virus. Molecular Biology and Evolution 2004, 21: 914–921.View ArticlePubMed
        93. Yang Z, Nielsen R, Goldman N, Pedersen AMK: Codon-substitution models for heterogeneous selection pressure at amino acid sites. Genetics 2000, 155: 431–449.PubMed
        94. Yang Z, Nielsen R: Codon-substitution models for detecting molecular adaptation at individual sites along specific lineages. Molecular Biology and Evolution 2002, 19: 908–917.PubMed
        95. Kikuchi S, Tanoue A, Goda N, Matsuo N, Tsujimoto G: Structure and sequence of the mouse V1a and V1b vasopressin receptor genes. Japanese Journal of Pharmacology 1999, 81: 388–392.View ArticlePubMed

        Copyright

        © Fink et al. 2007

        This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

        Advertisement