doi: 10.1111/j.1420-9101.2005.00975.x
No evidence for the ‘Meselson effect’ in parthenogenetic oribatid mites (Oribatida, Acari) ¨ N,à R. A. NORTON,§ I. SCHAEFER,* K. DOMES,* M. HEETHOFF, K. SCHNEIDER,* I. SCHO S. SCHEU* & M. MARAUN* *Technische Universita¨t Darmstadt, Institut fu¨r Zoologie, Darmstadt, Germany Universita¨t Tu¨bingen, Zoologisches Institut, Abteilung fu¨r Evolutionsbiologie der Invertebraten, Tu¨bingen, Germany àRoyal Belgian Institute of Natural Sciences, Freshwater Biology, Brussels, Belgium §State University of New York, College of Environmental Science and Forestry, 1 Forestry Drive, NY, USA
Keywords:
Abstract
ancient asexuals; ef-1a; hsp82; oribatid mites; parthenogenesis.
It has been hypothesized that in ancient apomictic, nonrecombining lineages the two alleles of a single copy gene will become highly divergent as a result of the independent accumulation of mutations (Meselson effect). We used a partial sequence of the elongation factor-1a (ef-1a) and the heat shock protein 82 (hsp82) genes to test this hypothesis for putative ancient parthenogenetic oribatid mite lineages. In addition, we tested if the hsp82 gene is fully transcribed by sequencing the cDNA and we also tested if there is evidence for recombination and gene conversion in sexual and parthenogenetic oribatid mite species. The average maximum intra-specific divergence in the ef-1a was 2.7% in three parthenogenetic species and 8.6% in three sexual species; the average maximum intra-individual genetic divergence was 0.9% in the parthenogenetic and 6.0% in the sexual species. In the hsp82 gene the average maximum intra-individual genetic divergence in the sexual species Steganacarus magnus and in the parthenogenetic species Platynothrus peltifer was 1.1% and 1.2%, respectively. None of the differences were statistically significant. The cDNA data indicated that the hsp82 sequence is transcribed and intron-free. Likelihood permutation tests indicate that ef-1a has undergone recombination in all three studied sexual species and gene conversion in two of the sexual species, but neither process has occurred in any of the parthenogenetic species. No evidence for recombination or gene conversion was found for sexual or parthenogenetic oribatid mite species in the hsp 82 gene. There appears to be no Meselson effect in parthenogenetic oribatid mite species. Presumably, their low genetic divergence is due to automixis, other homogenizing mechanisms or strong selection to keep both the ef-1a and the hsp82 gene functioning.
Introduction Putative ancient parthenogenetic taxa, such as bdelloid rotifers, darwinulid ostracods and some clusters within oribatid mites (Mark Welch & Meselson, 2000; Scho¨n & Martens, 2003; Maraun et al., 2004), are challenging theories of evolutionary biology. They are species rich Correspondence: Mark Maraun, Technische Universita¨t Darmstadt, Institut fu¨r Zoologie, Schnittspahnstrasse 3, 64287 Darmstadt, Germany. Tel.: +49-6151-165219; fax: +49-6151-166111; e-mail:
[email protected]
184
(darwinulid ostracods: 26 species; bdelloid rotifers: 363 species; oribatid mites: between 54 and 156 species in different clusters) and may have radiated while being parthenogenetic. These taxa have been termed ‘evolutionary scandals’ (Maynard Smith, 1978) because theoretical considerations suggest that long-term advantages of sexual reproduction should eventually overcome the short-term advantages of parthenogenesis. Consequently, parthenogenetic lineages are doomed to extinction in the long term. Understanding how species managed to persist over long periods of time without sex and recombination will contribute significantly to our
J. EVOL. BIOL. 19 (2006) 184–193 ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
No evidence for the ‘Meselson effect’
understanding of the prevalence of sex in the living world. No single evolutionary theory is able to explain why sex is the prevalent reproductive mode in eukaryotes (Bell, 1982; Kondrashov, 1988,1993; Roughgarden, 1991; Browne, 1992; Lynch et al., 1993; Crow, 1994; West et al., 1999; Butlin, 2002). From the selfish DNA point of view parthenogenesis is much easier to explain than sexual reproduction, yet only a minority – about 1% – of the known eukaryotic species reproduce via parthenogenesis (White, 1978). It is still not clear if the main function of sex is the homogenisation of genomes (Roughgarden, 1991; Otto & Nuismer, 2004) or the production of different genotypes that allow adaptation to changing environments (Ghiselin, 1974; Maynard Smith, 1978; Hamilton, 1980; Perlman et al., 2003). Ancient asexual taxa provide the opportunity to test the hypothesis that sexual reproduction evolved not to produce genetic variation in the short term but to homogenize genomes over long periods of time. Contradicting the latter hypothesis, ancient asexual taxa have more genetic variation than sexual taxa of similar age (Barraclough & Herniou, 2003; Barraclough et al., 2003). A precondition for understanding the survival of ancient asexuals is to know (a) whether these taxa really are ancient and (b) whether they are apomicts or automicts. Of the three putative ancient asexual animal taxa, bdelloid rotifers are apomicts (Mark Welch & Meselson, 2000; Birky, 2004). In asexual, nonmarine ostracods, only apomixis has been reported so far (Butlin et al., 1998) although the Darwinulidae have not yet been investigated extensively. The exceptionally low genetic divergence of ITS1 in the ancient asexual species of the family, Darwinula stevensoni, could be attributed to gene conversion (Scho¨n & Martens, 2003) whereas homogenising mechanisms such as highly efficient DNA repair (Scho¨n & Martens, 1998) could keep other genomic regions intact (Scho¨n & Martens, 2003). In contrast, there is evidence for automixis in parthenogenetic taxa of oribatid mites (e.g. Platynothrus peltifer) (Taberly, 1987a,b; Wrensch et al., 1994; Butlin et al., 1998; Gorelick, 2003). Definitions of sexual and parthenogenetic reproduction are sometimes contradicting, which is why we provide our definition below. We use the term sex for the fusion of meiotically and independently produced gametes of two individuals. Parthenogenetic reproduction as used here relates specifically to thelytoky, the production of females from unfertilised eggs. Further, we distinguish between apomictic and automictic thelytoky. Apomixis describes a process in which oocytes are produced by mitotic cell division in the germ line. Daughters inherit a complete unrecombined maternal genome, which results in increased heterozygosity at any given locus over time as mutations accumulate. Automixis refers to the reconstitution of the diploid state from meiotically reduced oocytes within one organism. Several mechanisms for
185
reconstituting the numbers of chromosomes in the presumptive egg are known, each either resulting in increased heterozygosity or homozygosity among the offspring. Doubling the chromosomes before meiosis and pairing of homologous chromosomes increases homozygosity as does post-meiotic fusion of meiotically produced ova (eggs with polar bodies), if nonhomologous chromosomes pair, crossing over can generate genetically diverse offspring. The degree of heterozygosity at a given locus depends on whether the first or the second meiotic division is suppressed. The same holds for the fusion of the first (central fusion) or the second (terminal fusion) polar body with the oocyte. In general, central fusion automixis results in heterozygosity (except in cross-over regions) whereas terminal fusion automixis increases homozygosity (except in cross-over regions) (Maynard Smith, 1978; Bell, 1982; Suomalainen et al., 1987). The patterns of these hypothesized mechanisms can be overwritten by nonmeiotic homogenising processes, such as gene conversion, mitotic recombination, or DNA repair. One way to test for long-term absence of recombination is the so-called ‘Meselson effect’, as proposed by Birky (1996) and Mark Welch & Meselson (2000). The Meselson effect assumes that the two allelic copies at a given locus (in a diploid) become divergent over time as a result of the independent accumulation of mutations. Over millions of years of evolution the intra-individual allelic divergence of ancient apomicts should be higher than that of sexual species. However, the effect should not exist in ancient automicts, in which homogenising mechanisms such as recombination, gene conversion (Butlin, 2000), or DNA repair (Scho¨n & Martens, 2000) should lower allelic diversity (Gorelick, 2003; Gandolfi et al., 2003). Therefore, the power of testing the Meselson effect is asymmetrical. Its presence indicates long-term lack of recombination but its absence does not distinguish sexual reproduction from any other sort of homogenising mechanism (Butlin, 2000). So far, the Meselson effect has been found in bdelloid rotifers (Mark Welch & Meselson, 2000) but not in D. stevensoni (Scho¨n & Martens, 2003) or any other putative ancient parthenogenetic group (Birky, 2004). Even the presence of the Meselson effect does not prove long-term absence of recombination; Ceplitis (2003) showed that the effect is also compatible with low rates of sexual reproduction. In contrast to darwinulid ostracods and bdelloid rotifers, the various putative ancient asexual taxa of oribatid mites have so far hardly been studied in an evolutionary context with molecular techniques. These mites are decomposer animals that reach high densities in virtually all soils of the world, ranging from a few hundreds in agricultural sites up to 500 000 m)2 in northern boreal forests (Maraun & Scheu, 2000). Fossils are known from Devonian sediments indicating an age of at least 380 My (Shear et al., 1984, Norton et al., 1988). There is increasing evidence that several species-rich clusters of oribatid
J. EVOL. BIOL. 19 (2006) 184–193 ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
186
I. SCHAEFER ET AL.
mites represent ancient asexual lineages that radiated while being parthenogenetic (Norton & Palmer, 1991; Norton et al., 1993; Maraun et al., 2003,2004). This study tests for the presence of the Meselson effect in parthenogenetic species of oribatid mites. The intraindividual genetic divergence in parthenogenetic taxa is compared with that of sexually reproducing oribatid mite species. We used the elongation factor-1a (ef-1a) and the heat shock protein 82 (hsp82) genes; both are conserved and most likely single copy genes (Klompen, 2000; Mark Welch & Meselson, 2000). The hsp82 gene has been used for the analysis of the Meselson effect in bdelloid rotifers (Mark Welch & Meselson, 2000) and darwinulid ostracods (Scho¨n & Martens, 2003) facilitating the comparison of results from the different groups of ancient asexuals. The ef-1a gene has been successfully used for phylogenetic studies (e.g. Klompen, 2000). Additionally, the functionality of hsp82 was verified in the current study by sequencing its cDNA. We also applied likelihood permutation tests (McVean et al., 2002) to test for recombination and gene conversion in ef-1a and hsp82 from both sexual and parthenogenetic taxa.
Materials and methods We sequenced between one and eleven clones of a fragment of the ef-1a gene (573 bp) of three sexual (Eupelops plicatus, Achipteria coleoptrata, Steganacarus magnus) and three parthenogenetic species of oribatid mites (Nanhermannia coronata, Nothrus silvestris, P. peltifer). We also sequenced two clones from one individual of the parthenogenetic oribatid species Hypochthonius rufulus (Enarthronota) as outgroup for the phylogenetic analysis (for details of the sample locations see Table S1). Additionally, we sequenced between eight and 17 clones of a fragment of the hsp82 gene (525–537 bp) from the sexual species S. magnus and the parthenogenetic species P. peltifer (genomic DNA and cDNA). We also sequenced one clone of the parthenogenetic species Tectocepheus minor (Oribatida) to root the tree. Mites were collected from litter in different forests in Germany (for details of the sample locations see Table S2). Sample preparation, PCR and sequencing Single animals were placed in an Eppendorf tube, frozen in liquid nitrogen and crushed against the tube wall with a steel rod. Genomic DNA was obtained using Qiagen DNeasy kit for animal tissues following the manufacturer’s protocol for animal tissues (Qiagen, Germany). The ef-1a fragment was amplified using the forward primer 40.71 F (5¢-TCN TTY AAR TAY GCN TGG GGT-3¢) and the reverse primer 52.RC (5¢-CCD ATY TTR TAN ACR TCY TG-3¢; Klompen, 2000). Amplifications of the ef-1a fragment were performed in 50 lL volumes containing 2 lL of each primer (100 pM lL)1), 15 lL DNA and 25 lL of HotStarTaq Mastermix (2.5 units Hot-
StarTaq Polymerase, 200 lM of each dNTP and 15 mM MgCl2 buffer solution; Qiagen, Germany). Amplification conditions were: an initial denaturation step at 95 C for 15 min, followed by 35 cycles of 94 C (30 s) denaturation, 50 C (70 s) annealing and 72 C (90 s) extension and terminated with 10 min at 72 C as final extension. The hsp82 gene fragment of 525–537 bp length was amplified using the forward primer hsp1.2 (5¢-TGC TCT AGA GCA CAR TTY GGT GTN GGT TTY TA-3¢) and the reverse primer hsp8.x (5¢-ACG TTC TAG ART GRT CYT CCC ART CRT TNG T-3¢) (Scho¨n & Martens, 2003). PCR compositions for hsp82 were identical to ef-1a but cycling conditions differed: an initial denaturation step at 95 C for 15 min was followed by nine cycles of 95 C (50 s) denaturation, 50 C (50 s) annealing and 72 C (2 min) extension. Then 34 cycles of 95 C (50 s), 55 C (50 s) and 72 C (2 min) followed. The PCR was again concluded with a final extension of 10 min at 72 C. PCR products were purified on 2% agarose gels; bands were excised from gels, recovered and purified using QIAquick Gel Extraction Kit (Qiagen, Germany). All PCR products were cloned using the Perfectly BluntTM Cloning Kit (Novagen, Germany) and transformed into E. coli Nova Blue SinglesTM competent cells (Novagen, Germany) by heat shock using the manufacturer’s protocol. The plasmids were purified using QIAprep Spin Miniprep System (Qiagen, Germany). The DNA was sequenced by Scientific Research and Development GmbH, Oberursel (Germany), using an ABI sequencer (Applied Biosystems, USA). mRNA isolation, cDNA synthesis and sequencing Total RNA was extracted from single and half individuals of S. magnus and P. peltifer. Animals were placed in reaction tubes, frozen in liquid nitrogen and disrupted using a steel rod. The solution was homogenized using a syringe with a small needle. The procedure followed the protocol for isolation of total RNA from animal tissue as given in the RNeasy Mini Handbook (Qiagen, Germany). Complementary DNA (cDNA) was synthesized using the Qiagen One Step RT-PCR Kit (Qiagen, Germany). Amplification conditions were: 30 min at 50 C for reverse transcription of the RNA template to obtain cDNA, followed by PCR for hsp82 (see above). Cloning was done as described above and the cDNA was sequenced from Scientific Research and Development GmbH, Oberursel (Germany). All ef-1a and hsp82 sequences were deposited in GenBank (see Tables S1 and S2 for accession numbers). Alignment and phylogenetic analysis DNA and cDNA sequences were aligned with the multiple alignment algorithms in the program Clustal X (Thompson et al., 1994,1997) using 10 and 0.1 as parameters for gap opening and gap extension penalty,
J. EVOL. BIOL. 19 (2006) 184–193 ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
No evidence for the ‘Meselson effect’
respectively. The alignment for ef-1a was unambiguous because all sequences had the same length. All sequences except three could be translated into amino acids. Those DNA sequences contained stop-codons; which may be due to PCR errors, or, alternatively, they may be pseudogenes. The sequences were included in the phylogenetic tree and clearly marked with asterisks but were not used for the analysis of the genetic distances within and between the oribatid mite species. The alignment of the amino acids and the nucleic acids for the hsp82 gene was also unambiguous since only a few gaps occurred and these were always 3 bp long, indicating the loss of single amino acids. No stop-codons were found in the hsp82 fragment when the sequences were translated (data not shown). All alignments are available from the corresponding author on request. Phylogenetic trees were constructed using neighbourjoining (NJ), maximum parsimony (MP) and likelihoodbased (ML) algorithms in PAUP* (Version 4b10; Swofford, 1999) treating gaps as missing data. Tree length and statistical indices are given for informative sites only (Posada & Crandall, 1998). Our settings for the ef-1a data corresponded to the TrNef model (Tamura and Nei, equal base frequencies) with gamma correction (a ¼ 0.4667; Yang, 1996) and with substitution parameters A–C, A–T, C–G, G–T ¼ 1.0; A–G ¼ 2.9637; C–T ¼ 3.1584. For the hsp82 data our settings corresponded to the TrN model (Tamura and Nei; base frequencies: A ¼ 0.3788; C ¼ 0.1772; G ¼ 0.2743; T ¼ 0.1696) with gamma correction (a ¼ 0.8411) and with substitution parameters A–C, A–T, C–G, G–T ¼ 1.0; A–G ¼ 3.3943 and C–T ¼ 6.979. Reliability of the nodes was ascertained from 1000 bootstrap replicates. MP trees were constructed with heuristic search of 100 random additions, and the tree bisection-reconnection (TBR) branch-swapping algorithm with the option of collapsing zero branch length. A strict consensus tree was constructed for both ef-1a and the hsp82 sequence data. Sequence divergence in sexual and parthenogenetic taxa The average maximum intra-specific and intra-individual molecular divergence (¼distances based on the evolutionary model obtained by MODELTEST; Posada & Crandall, 1998) in the ef-1a of the three sexual species was compared with those of the three parthenogenetic species by two separate one-way analyses of variance (A N O V A ) with the fixed factor ‘reproductive mode’ using SAS 8e (SAS Insitute Inc., Cary, USA). McVean test for recombination and gene conversion Likelihood permutation tests using the program LDhat Version 2.0 (http://www.stats.ox.ac.uk/mcvean/ LDhat/) for recombination and gene conversion (Mc-
187
Vean et al., 2002) were conducted with the two-allele model, no frequency cut-off of rare alleles, Watterson’s theta estimates (hW) of population-scaled mutation rates per site and 10 000 permutations as parameters. Likelihood permutation tests were run with 101 grid points and different values of 4Ner until the value with ML as suggested by the program were obtained. Nonparametric permutation tests on measures of linkage disequilibrium and physical distance are calculated to provide values of PLKmax for recombination and PLKmax for gene conversion, which are new statistical parameters of this particular likelihood permutation test (McVean et al., 2002).
Results The aligned ef-1a sequences were 573 bp and those of the hsp82 gene were 525 or 537 bp long (see Tables S1 and S2). Of the 573 aligned sites of ef-1a 283 were constant, 48 were variable but parsimony-uninformative and 242 characters were parsimony-informative. Of the 537 aligned sites of the hsp82 gene 360 were constant, 72 were variable but parsimony-uninformative and 105 were parsimony-informative. Phylogeny and Meselson effect For ef-1a (Fig. 1) the bootstrap replications of the NJ analysis clearly support the separation of the species but rarely separated individuals within the sexual or parthenogenetic species. The topologies of the MP and the ML tree for ef-1a were very similar to the NJ tree and are therefore not shown (both trees are available from the corresponding author on request). All species investigated were separated in the NJ tree. For hsp82 (Fig. 2) the bootstrapping supports the separation of the two species P. peltifer and S. magnus but within these two species few splits were supported by bootstrap values higher than 50. The MP and ML trees were nearly identical to the NJ tree (both trees are available from the corresponding author on request). Intra-specific and intra-individual molecular variation was generally low in each of the species. However, the average maximum intra-specific genetic divergence of ef-1a in the three sexual species (8.6%) was larger than that of the three parthenogenetic species (2.7%). Additionally, the average maximum intraindividual genetic divergence of the sexual species (6.0%) was larger than in the parthenogenetic species (0.9%). However, neither difference was statistically significant (A N O V A : F1,4 ¼ 3.28, P ¼ 0.14 and F1,4 ¼ 3.81, P ¼ 0.12, respectively). The average maximum intra-individual genetic divergence of the hsp82 gene was 1.1% in the sexual species S. magnus and 1.2% in the parthenogenetic species P. peltifer. Results of the likelihood permutation tests indicate that recombination occurs in ef-1a of all three sexual species (Table S3). In addition, there is also evidence for
J. EVOL. BIOL. 19 (2006) 184–193 ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
188
I. SCHAEFER ET AL.
100
E. plicatus KW1 b E. plicatus KW1 d E. plicatus KW1 h E. plicatus KW1 a E. plicatusKW1 e E. plicatus KW2 a E. plicatus KW1 c E. plicatus KW1 g 95 E. plicatus KW2 e 68 E. plicatus KW2 g E. plicatus KW1 i E. plicatus KW2 d 100 E. plicatus KW2 b E. plicatus KW2 c E. plicatus KW2 f E. plicatus KW1 f * E. plicatus W1 a 97 E. plicatus W1 f plicatus W1 c 99 100 E. E. plicatus W1 i E. plicatus W1 g E. plicatus W1 d 100 E. plicatus W1 h E. plicatus W1 b E. plicatus W1 e 100 A. coleoptrata KW1 a A. coleoptrata KW1 c A. coleoptrata KW1 d A. coleoptrata KW1 f A. coleoptrata KW1 e A. coleoptrata KW1 h 100 A. coleoptrata KW1 g A. coleoptrata KW1 b A. coleoptrata KW2 a A. coleoptrata KW2 b N. silvestris S4 14 N. silvestris S4 19 N. silvestris S4 17 N. silvestris S4 22 N. silvestris S4 23 N. silvestris KW3 3 N. silvestris KW3 2 N. silvestris KW4 1 N. silvestris S4 21 N. silvestris KW4 2 N. silvestris S4 18 100 N. silvestris S4 16 N. silvestris KW3 1 N. silvestris KW3 4 N. silvestris S4 15 N. coronata S2 d N. coronata S2 b N. coronata S1 a N. coronata S3 a N. coronata S1 g N. coronata S2 c N. coronata S2 h N. coronata S1 i N. coronata S2 i N. coronata S2 f N. coronata S3 b N. coronata S1 c N. coronata S1 b N. coronata S1 l N. coronata S2 g N. coronata S2 a N. coronata S1 d N. coronata S1 k N. coronata S1 e N. coronata S1 f N. coronata S2 e N. coronata S1 h N. coronata S2 k
Eupelops plicatus (sexual)
Achipteria coleoptrata (sexual)
Nothrus silvestris (parthenogenetic )
78
Nanhermannia coronata (parthenogenetic )
100 100 66
100
P. peltifer KW1 c P. peltifer KW1 d P. peltifer KW1 b P. peltifer KW1 a P. peltifer KW3 P. peltifer KW2 a P. peltifer KW2 c P. peltifer KW2 b
100 H. rufulus S1 a H. rufulus S1 b
Hypochthonius rufulus
Platynothrus peltifer (parthenogenetic )
S. magnus KW3 S. magnus KW1 a S. magnus KW1 f S. magnus KW2 c 60 S. magnus KW2 b 76 S. magnus KW1 d * S. magnus KW1 c * S. magnus KW1 b S, magnus KW1 e S, magnus KW1 g S. magnus KW2 a
Steganacarus magnus (sexual)
0.1
Fig. 1. Neighbour joining tree of several clones of three parthenogenetic species and three sexual species of oribatid mites and the parthenogenetic out-group species H. rufulus. The tree was constructed using the 573 bp alignment of the partial sequence of the elongation factor 1a gene. Numbers at the nodes represent supporting percentages of 1000 bootstrap replicates (only values above 50% are reported). For abbreviations see Table S1.
J. EVOL. BIOL. 19 (2006) 184–193 ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
No evidence for the ‘Meselson effect’
189
P. peltifer KW2_d P. peltifer KW2_g P. peltifer KW2_c P. peltifer KW2_b P. peltifer KW3d cDNA P. peltifer KW1a cDNA P. peltifer KW1b cDNA P. peltifer KW3g cDNA P. peltifer KW2_f P. peltifer KW2j P. peltifer KW3h cDNA
79 100
P. peltifer KW1e cDNA P. peltifer KW1_a Platynothrus P. peltifer KW1b peltifer P. peltifer KW1c (parthenogenetic) P. peltifer KW3a cDNA P. peltifer KW3c cDNA P. peltifer KW3f cDNA P. peltifer KW3b cDNA P. peltifer KW1f cDNA P. peltifer KW3e cDNA P. peltifer KW2_h P. peltifer KW2_a P. peltifer KW2_e P. peltifer KW2_i P. peltifer KW1d cDNA P. peltifer 1c cDNA P. peltifer KW1_d S. magnus KW7h cDNA S. magnus KW7i cDNA S. magnus KW7 cDNA S. magnus KW7 cDNA S. magnus KW8_a S. magnus KW8b cDNA S. magnus KW8e cDNA S. magnus KW8_c S. magnus KW7b cDNA S. magnus KW7f cDNA S. magnus KW7d cDNA S. magnus KW7_f 61 Steganacarus S. magnus KW7_a magnus S. magnus KW7_b (sexual) S. magnus KW7_d S. magnus KW7_c S. magnus KW7_e S. magnus KW7c cDNA S. magnus KW7g cDNA S. magnus KW8f cDNA S. magnus KW7e cDNA S. magnus KW7a cDNA S. magnus KW8d cDNA S. magnus KW8c cDNA 100 S. magnus KW8a cDNA S. magnus KW8_b Tectocepheus minor
0.1
Fig. 2. Neighbour joining tree of several clones of the parthenogenetic oribatid mite species P. peltifer, the sexual species S. magnus and the parthenogenetic species T. minor. The tree was constructed using the 537 bp alignment of the partial sequence of the heat shock protein 82 gene. Numbers at the nodes represent supporting percentages of 1000 bootstrap replicates (only values above 50% are reported). For abbreviations see Table S2.
two of the three sexual species (E. plicatus and S. magnus) that gene conversion takes place in ef-1a. None of the parthenogenetic species showed any evidence for
recombination or gene conversion. For hsp 82, likelihood permutation tests provided neither for the sexual species S. magnus nor the parthenogenetic species
J. EVOL. BIOL. 19 (2006) 184–193 ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
190
I. SCHAEFER ET AL.
P. peltifer any evidence for recombination or gene conversion.
Discussion Absence of the Meselson effect Results of this study provide no evidence for the Meselson effect in parthenogenetic oribatid mites. Intra-individual molecular variation in both the ef-1a and the hsp82 genes in parthenogenetic species was very low and similar to that of sexual species. In the phylogenetic tree the DNA sequences of the parthenogenetic (and also of the sexual) species formed distinct clusters suggesting that neither the ef-1a nor the hsp82 alleles evolved independently, i.e. without homogenising mechanisms, over a long period of time. Additionally, the results of the RT-PCR supported the hypothesis that the hsp82 gene is transcribed; indicating that at least one of the two alleles did not deteriorate during evolution. Therefore, the studied parthenogenetic oribatid mite species do not appear to be ancient apomicts, which is consistent with the cytological evidence of Taberly (1987a,b), who found P. peltifer and another parthenogenetic oribatid species to be automictic. Due to the asymmetric power of the Meselson effect (Butlin, 2000) it is difficult to explain its absence in parthenogenetic oribatid mites, but a number of mechanisms that may have contributed to the homogenisation of the genome should be considered. Several mechanisms can be dismissed as unlikely. Rare (spanandric) males are known from many parthenogenetic oribatid mite species, including those we studied (Palmer & Norton, 1991); if these males are functional, then rare sexual reproduction could explain the similarity of intra-individual genetic divergence in sexual and putative parthenogenetic species. However, our results show no evidence for recombination in the parthenogenetic species studied, so spanandric males appear to be non-functional, as was suggested by several previous lines of evidence. For example, spanandric males that have been studied cytologically were functionally incompetent and were ignored by females (Taberly, 1988). Furthermore, Palmer & Norton (1991) found the population sex ratios of 136 species of Desmonomata (including our studied parthenogenetic species) to be either approximately equal (50 : 50) or strongly female biased (about 95% females). Such a bimodal sex ratio distribution also eliminates frequent hybridisation as a possible contributor to the low intra-individual genetic variation. An allozyme study of a number of parthenogenetic Desmonomata (Palmer & Norton, 1992) suggested clonal population structure and examples of fixed heterozygosity despite the presence of rare males, and the population with the most males (6%; Mucronothrus nasalis) had nearly the lowest genotypic diversity. In a general context, spanandric males are found in many
obligate parthenogenetic animals (Lynch, 1984) and, under realistic assumptions of heterozygote superiority or epistatic interactions between loci, rare sexual reproduction in an otherwise asexual population may actually be disadvantageous (Peck & Waxman, 2000). Two possible explanations relate to the age of taxa. One is that very limited differences between alleles might result from a parthenogenetic oribatid species being recently split from a sexual ancestor. This idea is unlikely to have general explanatory power, since some taxa of oribatid mites (e.g. various clades in Desmonomata) are species-rich, yet include no sexual species. A contention that these taxa radiated while being parthenogenetic is much more parsimonious than one in which parthenogenesis evolved frequently and independently in these lineages while all sexual relatives have become extinct is highly unlikely (Norton et al., 1993; Maraun et al., 2004). A second idea is that if oribatid mites, as a group, radiated relatively recently, low intra-individual genetic divergence could still characterize both sexual and parthenogenetic taxa. This also is unlikely since oribatid mites existed at least since the Devonian; about 380 My ago (Shear et al., 1984; Norton et al., 1988), and rich fossil evidence shows that the main radiations are quite old as well (Labandeira et al., 1997). Biogeographic evidence even suggests that one parthenogenetic species (M. nasalis) predates the breakup of Pangea (Hammer & Wallwork, 1979). Estimates of the age of sexual and parthenogenetic oribatid mites using molecular markers that are thought to have clock-like divergence (e.g. COI, 18S rDNA) are needed to better refute this idea. It has been proposed that parthenogenetic taxa might possess efficient mechanisms of DNA repair (Scho¨n & Martens, 1998), which contribute to keeping alleles homogeneous. Together with strong selection pressure this might explain the limited genetic variability of the ef-1a and hsp82 genes. However, neither DNA repair nor strong selection pressure explain why even the third codon position changed little. Other explanations for low allelic divergence might also be relevant here but await confirmation. (1) Those parthenogenetic oribatid mites that have been studied cytologically are automicts. Automixis can homogenize the genome by terminal fusion of (meiotically produced) oocytes with the second polar body. While oogenesis has been well studied in only two parthenogenetic oribatid mite species, our study species P. peltifer is one of them (Taberly, 1987a). We assume that automictic reproduction in our studied parthenogenetic oribatid mites contributed to their low genetic divergence. (2) Gene conversion also is an effective homogenising mechanism that can occur on nonhomologous pairs of chromosomes (Carpenter, 1994). The strongest evidence for gene conversion comes from gene families or multi-copy regions, such as rRNA clusters and the ITS region (Benevolenskaya et al., 1997; Butlin et al., 1998; Fuertes Aguilar et al., 1999), but it probably occurs everywhere
J. EVOL. BIOL. 19 (2006) 184–193 ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
No evidence for the ‘Meselson effect’
in the genome (e.g. Bertran et al., 1997; Haubold et al., 2002). Gene conversion in the ancient asexual ostracod D. stevensoni has been restricted to the ITS1 region only (Scho¨n & Martens, 2003) and might also act in bdelloid rotifers (David Mark Welch, personal communication). In the current study, the likelihood permutation tests provided only evidence for gene conversion in the ef-1a gene of sexually reproducing but not of parthenogenetic oribatid mites. Thus, it seems unlikely that gene conversion, at least in the screened genomic regions, caused the observed, low genetic divergence of parthenogenetic taxa. (3) Mitotic recombination and aneuploidy are other mechanisms that can potentially contribute to the homogenisation of genomes (Birky, 2004). The importance of these mechanisms for limiting intra-individual genetic diversity should be explored in more detail. A puzzling result of this study is that parthenogenetic oribatid mites might rather be ancient automicts instead of apomicts but no evidence for recombination could be found. We therefore assume that the homogenisation of the genome by automixis has been ongoing for long periods of time. Consequently, any effects of recombination have been superimposed and cannot be detected anymore in parthenogenetic oribatid mites. However, this explanation does not solve the enigma why different lineages of parthenogenetic species have similar DNA sequences. Over evolutionary long periods of time, at least some mutations should have accumulated. In conclusion, it seems that the majority of ancient asexual animal taxa might posses some kind of homogenizing mechanisms detaining their genomes from infinitely accumulating mutations (Scho¨n & Martens, 2002). The main mechanisms suggested to date are automixis, gene conversion and highly efficient DNA repair. This imposes that the Meselson effect might not exist at all since it has not been confirmed for any putative ancient asexual taxon (Tramini: Normark, 1999; D. stevensoni: Scho¨n & Martens, 2003; Giardia lamblia: Baruch et al., 1996; oribatid mites: this study), and even the presumed high allelic diversity in bdelloid rotifers may be the result of an ancient hybridisation effect between two taxa (Mark Welch et al., 2004, M. Meselson, personal communication). Then the very interesting question remains why not more taxa are ancient asexuals if the long-term disadvantages of parthenogenetic reproduction can be defeated.
Acknowledgments We thank an anonymous referee and Koen Martens for providing valuable comments on this manuscript. We also thank Michael Laumann and Sonja Migge for commenting on earlier drafts. The first and the second author equally contributed to this work. The study was supported by a European Community Marie Curie Fellowship and by the German Research Foundation (DFG, SPP 1127, Ma 2461/1-2).
191
References Barraclough, T.G., Birky, C.W. & Burt, A. Jr. 2003. Diversification in sexual and asexual organisms. Evolution 57:2166–2172. Barraclough, T.G., & Herniou, E. 2003. Why do species exist? Insights from sexuals and asexuals. Zoology 106:275–282. Baruch, A.C., Isaac-Renton, J. & Adam, R.D. 1996. The molecular epidemiology of Giardia lamblia: a sequence-based approach. J. Inf. Dis. 174:233–236. Bell, G. 1982. The Masterpiece of Nature. The Evolution and Genetics of Sexuality. University of California Press, California, USA. Benevolenskaya, E.V., Kogan, G.L., Tulin, A.V., Philipp, D. & Gvozdev, V.A. 1997. Segmented gene conversion as a mechanism of correction of 18S rRNA pseudogene located outside of rDNA cluster in D. melanogaster. J. Mol. Evol. 44:646–651. Bertran, E., Rozas, J., Navarro, A. & Barbadilla, A. 1997. The estimation of the number and the length distribution of gene conversion tracts from population sequence data. Genetics 146:89–99. Birky, C.W. 1996. Heterozygosity, heteromorphy, and phylogenetic trees in asexual eukaryotes. Genetics 144:427–437. Birky, C.W. 2004. Bdelloid rotifers revisited. Proc. Natl. Acad. Sci. USA 101:2651–2652. Browne, R.A. 1992. Population genetics and ecology of Artemia: insights into parthenogenetic reproduction. Trends Ecol. Evol. 7:232–237. Butlin, R.K. 2000. Virgin rotifers. Trends Ecol. Evol. 15:389–390. Butlin, R.K. 2002. The costs and benefits of sex: new insights from old asexual lineages. Nature Rev. Genet. 3:311–317. Butlin, R., Scho¨n, I. & Martens, K. 1998. Asexual reproduction in non-marine ostracods. Heredity 81:473–480. Carpenter, A.T.C. 1994. The recombination nodule story: seeing what you are looking at. BioEssays 6:232–236. Ceplitis, A. 2003. Coalescence times and the Meselson effect in asexual eukaryotes. Genet. Res. 82:183–190. Crow, J.F. 1994. Advantages of sexual reproduction. Devel. Gen. 15:205–213. Fuertes Aguilar, J., Rossello, J.A. & Nieto Feliner G. 1999. Nuclear ribosomal DNA (nrDNA) concerted evolution in natural and artificial hybrids of Ameria (Plumbaginacea). Mol. Ecol. 8:1341–1346. Gandolfi, A., Sanders, I.R., Rossi, V. & Menozzi, P. 2003. Evidence for recombination in putative ancient asexuals. Mol. Biol. Evol. 20:754–761. Ghiselin, M.T. 1974. The Economy of Nature and the Evolution of Sex. University of California Press, California, USA. Gorelick, R. 2003. Transposable elements suppress recombination in all meiotic eukaryotes, including automictic ancient asexuals: a reply to Scho¨n and Martens. J. Nat. Hist. 37:903– 909. Hamilton, W.D. 1980. Sex versus non-sex versus parasite. Oikos 35:282–290. Hammer, M. & Wallwork, J.A. 1979. A review of the world distribution of oribatid mites (Acari: Cryptostigmata) in relation to continental drift. Biol. Skr. Dan. Vid. Selsk. 22:1–31. Haubold, B., Kroymann, J., Ratzka, A., Mitchell-Olds, T. & Wiehe, T. 2002. Recombination and gene conversion in an 170-kb genomic region of Arabidopsis thaliana. Genetics 161:1269–1278. Klompen, H. 2000. A preliminary assesment of the utility of elongation factor 1a in elucidating relationships among basal Mesostigmata. Exp. Appl. Acarol. 24:805–820.
J. EVOL. BIOL. 19 (2006) 184–193 ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
192
I. SCHAEFER ET AL.
Kondrashov, A.S. 1988. Deleterious mutations and the evolution of sexual reproduction. Nature 336:435–440. Kondrashov, A.S. 1993. Classification of hypotheses on the advantage of amphimixis. J. Hered. 84:372–387. Labandeira, C.C., Phillips, T.L. & Norton R.A. 1997. Oribatid mites and the decomposition of plant tissues in Paleozoic coalswamp forests. Palaios 12:319–353. Lynch, M. 1984. Destabilizing hybridization, general-purpose genotypes and geographic parthenogenesis. Quart. Rev. Biol. 59:257–290. Lynch, M., Burger, R., Butcher, D. & Gabriel, W. 1993. The mutational meltdown in asexual populations. J. Hered. 84:339–344. Maraun, M. & Scheu, S. 2000. The structure of oribatid mite communities (Acari, Oribatida): patterns, mechanisms and implications for future research. Ecography 23:374–383. Maraun, M., Heethoff, M., Scheu, S., Norton, R.A., Weigmann, G. & Thomas, R.H. 2003. Radiation in sexual and parthenogenetic oribatid mites (Oribatida, Acari) as indicated by genetic divergence of closely related species. Exp. App. Acarol. 29:175–187. Maraun, M., Heethoff, M., Schneider, K., Scheu, S., Weigmann, G., Cianciolo, J., Thomas, R.H. & Norton, R.A. 2004. Molecular phylogeny of oribatid mites (Oribatida, Acari): evidence for multiple radiations of parthenogenetic lineages. Exp. Appl. Acarol. 33:183–201. Mark Welch, D.B. & Meselson, M. 2000. Evidence for the evolution of bdelloid rotifers without sexual reproduction or genetic exchange. Science 288:1211–1215. Mark Welch, D.B., Cummings, M.P., Hillis, D.M. & Meselson, M. 2004. Divergent gene copies in the asexual class Bdelloidea (Rotifera) separated before the bdelloid radiation or within bdelloid families. Proc Natl. Acad. Sci. USA 101:1622–1625. Maynard Smith, J. 1978. The Evolution Of Sex. Cambridge University Press, Cambridge, UK. McVean, G., Awadalla, P. & Fearnhead, P. 2002. A coalescentbased method for detecting recombination from gene sequences. Genetics 160:1231–1241. Normark, B.B. 1999. Evolution in a putatively ancient asexual aphid lineage: recombination and rapid karyotype change. Evolution 53:1458–1469. Norton, R.A. & Palmer, S.C. 1991. The distribution, mechanisms, and evolutionary significance of parthenogenesis in oribatid mites. In: The Acari: Reproduction, Development and Life-history Strategies (R. Schuster & P. W. Murphy, eds), pp. 107–136. Chapman and Hall, London, UK. Norton, R.A., Bonamo, P.M., Grierson, J.D. & Shear, W.A. 1988. Oribatid mite fossils from a terrestrial Devonian deposit near Gilboa, New York. J. Paleont. 62:259–269. Norton, R.A., Kethley, J.B., Johnston, D.E. & OConnor, B.M. 1993. Phylogenetic perspectives on genetic systems and reproductive modes of mites. In: Evolution and Diversity of Sex Ratios (D.L. Wrensch & M.A. Ebbert, eds), pp. 8–99. Chapman and Hall, New York, USA. Otto, S.P. & Nuismer, S.L. 2004. Species interactions and the evolution of sex. Science 304:1018–1020. Palmer, S.C. & Norton, R.A. 1991. Taxonomic, geographic, and seasonal distribution of thelytokous parthenogenesis in Desmonomata (Acari: Oribatida). Exp. Appl. Acarol. 12:67–81. Palmer, S.C. & Norton, R.A. 1992. Genetic diversity in thelytokous oribatid mites (Acari; Acariformes: Oribatida). Biochem. Syst. Ecol. 20:219–231.
Peck, J. R. & Waxman, D. 2000. What’s wrong with a little sex? J. Evol. Biol. 13:63–69. Perlman, S.J., Spicer, G.S., Shoemaker, D.D. & Jaenike, J. 2003. Associations between mycophagous Drosophila and their Howardula nematode parasites: a worldwide phylogenetic shuffle. Mol. Ecol. 12:237–249. Posada, D. & Crandall, K.A. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics 14:817–818. Roughgarden, J. 1991. The evolution of sex. Am. Nat. 138:934– 953. Scho¨n, I. & Martens, K. 1998. Opinion: DNA repair in an ancient asexual: a new solution for an old problem? J. Nat. Hist. 32:943–948. Scho¨n, I. & Martens, K. 2000. Transposable elements and asexual reproduction. Trends Ecol. Evol. 15:287–288. Scho¨n, I. & Martens, K. 2002. Are ancient asexuals less burdened? Selfish DNA, transposons and reproductive mode. J. Nat. Hist. 36:379–390. Scho¨n, I. & Martens, K. 2003. No slave to sex. Proc. R. Soc. Lond. B 270:827–833. Shear, W.A., Bonamo, M., Grierson, J.D., Rolfe, W.D.I., Smith, E.L. & Norton, R.A. 1984. Early land animals in North America: evidence from Devonian age arthropods from Gilboa, New York. Science 224:492–494. Suomalainen, E., Saura, A. & Lokki, J. 1987. Cytology And Evolution In Parthenogenesis. CRC Press, Boca Raton, Florida. Swofford, D. 1999. PAUP*: Phylogenetic Analysis Using Parsimony (And Other Methods). Version 4.0. Sinauer Associates, Sunderland, Massachusetts. Taberly, G. 1987a. Recherches sur la parthe´nogene`se the´lytoque de deux espe`ces d’acariens oribatides: Trhypochthonius tectorum (Berlese) et P. peltifer (Koch). II: E´tude anatomique, histologique et cytologique des femelles parthe´noge´ne´tiques. 1re partie. Acarologia 28:285–293. Taberly, G. 1987b. Recherches sur la parthe´nogene`se the´lytoque de deux espe`ces d’acariens oribatides: Trhypochthonius tectorum (Berlese) et Platynothrus peltifer (Koch). III: E´tude anatomique, histologique et cytologique des femelles parthe´nogene´tiques. 2eme partie. Acarologia 28:389–403. Taberly, G. 1988. Recherches sur la parthe´nogene`se the´lytoque de deux espe`ces d’acariens oribatides: Trhypochthonius tectorum (Berlese) et Platynothrus peltifer (Koch). IV: Observations sur les males ataviques. Acarologia 29:95–107. Thompson, J.D., Higgins, D.G. & Gibson, T.J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic. Acids Res. 22:4673–4680. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic. Acids Res. 24:4876–4882. West, S.A., Lively, C.M. & Read, A.F. 1999. A pluralist approach to sex and recombination. J. Evol. Biol. 12:1003–1012. White, M.D., 1978. Modes of Speciation. W.H. Freeman, San Francisco. Wrensch, D.L., Kethley, J.B. & Norton, R.A. 1994. Cytogenetics of holokinetic chromosomes and inverted meiosis: keys to the evolutionary success of mites, with generalizations on eukaryotes. In: Mites: Ecological and Evolutionary Analyses of Life-history Pattern (M. A. Houck, ed.), pp. 282–343. Chapman and Hall, New York.
J. EVOL. BIOL. 19 (2006) 184–193 ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
No evidence for the ‘Meselson effect’
Yang, Z. 1996. Among-site variation and its impact on phylogenetic analysis. Trends Ecol. Evol. 11:367–371.
Supplementary material The following material is available from http://www. blackwellpublishing.com/products/journals/suppmat/jeb/ jeb975/jeb975sm.htm Table S1 Numbers and names of clones and individuals, DNA type, fragment length, collection locality and GenBank accession numbers of the elongation factor 1a gene fragment of the oribatid mite species analysed in this study.
193
Table S2 Numbers and names of clones and individuals, DNA type, fragment length, collection locality and GenBank accession numbers of the heat shock protein 82 gene fragment of the oribatid mite species analysed in this study. Table S3 Results of the likelihood permutation test for recombination and gene conversion for three sexual species and three parthenogenetic species. Received 25 January 2005; revised 27 April 2005; accepted 17 May 2005
J. EVOL. BIOL. 19 (2006) 184–193 ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
