Research Article

Evidence for the Evolution of Bdelloid Rotifers Without Sexual Reproduction or Genetic Exchange

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Science  19 May 2000:
Vol. 288, Issue 5469, pp. 1211-1215
DOI: 10.1126/science.288.5469.1211

Abstract

The Class Bdelloidea of the Phylum Rotifera is the largest metazoan taxon in which males, hermaphrodites, and meiosis are unknown. We conducted a molecular genetic test of this indication that bdelloid rotifers may have evolved without sexual reproduction or genetic exchange. The test is based on the expectation that after millions of years without these processes, genomes will no longer contain pairs of closely similar haplotypes and instead will contain highly divergent descendants of formerly allelic nucleotide sequences. We find that genomes of individual bdelloid rotifers, representing four different species, appear to lack pairs of closely similar sequences and contain representatives of two ancient lineages that began to diverge before the bdelloid radiation many millions of years ago when sexual reproduction and genetic exchange may have ceased.

Few species of animals or plants reproduce only asexually—and those that do seldom make up an entire genus, let alone a taxon of higher rank (1–3). These observations have been taken to mean that the loss of sexual reproduction is a dead end in evolution, leading to early extinction. Against this generalization, the entire Class Bdelloidea of the Phylum Rotifera stands out as an apparently radical exception (4), an “evolutionary scandal” (5).

The Rotifera, a protostome phylum, includes four monophyletic groups: Class Bdelloidea, Class Monogononta, Class Seisonida, and the Acanthocephala (6–10). Seisonids and acanthocephalans reproduce sexually and monogononts reproduce both sexually and asexually, but only asexual reproduction is known in bdelloids (11).

The Class Bdelloidea, comprising four families, 18 genera, and some 360 described species (12–14), is by far the largest metazoan taxon in which no evidence of sexual reproduction has been found (2, 15, 16). Eggs are produced from oocytes in well-differentiated ovaries by two mitotic divisions with no chromosome synapsis and no reduction in chromosome number, each oocyte giving rise to one egg and two polar bodies (17, 18). Bdelloid rotifers have existed for at least 35 to 40 million years, the age of the oldest amber in which they have been identified (19).

Bdelloids are found in fresh water and moist terrestrial habitats worldwide and are easily recognized by their characteristic ciliated head structure. Individuals range from 0.1 to 1 mm in length and have about 1000 nuclei, with muscles, ganglia, tactile and photosensitive sensory organs, feeding and swimming structures, digestive and secretory organs, and gonads. The genomic DNA content in the species in which it has been measured is about 1000 megabase pairs (20,21). Figure 1 shows the four species used in this study, representing three of the four bdelloid families.

Figure 1

Adult bdelloid rotifers of four species. Clockwise from upper left: Philodina roseola(Philodinidae) (eating algae), Macrotrachela quadricornifera (Philodinidae) (the large oval is a mature egg), Adineta vaga (Adinetidae), and Habrotrocha constricta (Habrotrochidae). Scale bar, 100 μm.

The failure to observe males, hermaphrodites, and meiosis throughout the class, although remarkable, does not exclude rare or unrecognized forms of sexual reproduction or some other mode of genetic exchange. Demonstration that bdelloid rotifers engage in sexual reproduction would put to rest the principal apparent exception to the prevailing view that genetic exchange is essential for evolutionary success. Conversely, demonstration that bdelloids evolved without sexual reproduction would challenge this central tenet of current evolutionary theory and would provide a system to test hypotheses for why sexual reproduction is so nearly universal and why other asexual species appear to suffer early extinction (1, 2, 22, 23).

Here, we report a test of the possibility that bdelloid rotifers evolved without sexual reproduction or genetic exchange, based on the analysis of nucleotide sequences in individual genomes of diverse bdelloid species.

Experimental approach. In sexually reproducing species, recombination and segregation allow random genetic drift to drive selectively neutral alleles toward fixation or extinction, limiting the divergence between allelic sequences caused by recurring mutation (24). Reported species averages for synonymous site diversity (corresponding to average synonymous site heterozygosity if mating is random) in a wide variety of invertebrate and vertebrate species range from 0.1 to 4% (25–29).

In contrast, segregation can no longer occur in a lineage that has abandoned sexual reproduction and in which reproduction is only mitotic and is without nonsister exchange. After millions of years under these conditions, descendants of formerly allelic sequences within individual genomes, if not lost by deletion, gene conversion, or nondisjunction, will be highly divergent. Suppose, for example, that sex and genetic exchange were abandoned in a diploid ancestor of modern bdelloids 80 million years ago and that the average rate of neutral nucleotide substitution was 5 × 10 9 per site per year. In that case, individual genomes will no longer be made up of closely similar haplotypes. Instead, ignoring any contribution from preexisting heterozygosity and correcting for multiple substitutions at the same site, individual bdelloid genomes will contain descendants of formerly allelic sequences differing at about 50% of neutral sites (29).

Sequence divergence in genomes of bdelloid and nonbdelloid rotifers was investigated in four genes: hsp82 (82-kD heat shock protein), tbp (TATA-box binding protein),rpol3I (RNA polymerase III large subunit), andtpi (triosephosphate isomerase). BLAST searches of all invertebrate sequences in available databases, including the complete genome sequences of Caenorhabditis elegans and Drosophila melanogaster, revealed no species in which any of these genes is accompanied by a paralog that arose since the origin of metazoans.

The experimental procedure was intended to isolate and sequence every copy of a given gene present in the genome of a single individual of each species. DNA was extracted from a population recently propagated from a single individual or, in the case of some of the nonbdelloid species, prepared from a small natural isolate or a single animal. For each gene, 24 to 38 amplicons from two to four polymerase chain reaction (PCR) amplifications were cloned in plasmids and sequenced in both directions (30). As a measure of neutral difference in coding regions, we used the uncorrected percentage difference at fourfold degenerate sites, here designated D4, a quantity that is slower to saturate and less sensitive to transition-transversion bias than are differences at twofold and threefold degenerate sites (31). For the genes examined, 11 to 18% of all coding sites are fourfold degenerate.

Sequence divergence of hsp82. We examinedhsp82 in four bdelloid species representing the three major families within the class and, for comparison, seven species of sexually reproducing rotifers (30). The region examined was the 843- to 888-bp segment corresponding to D. melanogastercodons 13 to 302, except in Macrotrachela quadricornifera and Brachionus plicatilis RUS, for which the regions examined were the 518- and 498-bp segments, respectively, corresponding to D. melanogaster codons 152 to 320.

Genomes of each of the seven sexually reproducing rotifers were found to contain either two nearly identical hsp82sequences, as depicted for B. plicatilis RUS in Fig. 2A, or only a single sequence (Table 1). The highest D4 in this group of nonbdelloid rotifer genomes was 2.4% (average 1%). A similar average D4 (0.5%) may be calculated for the corresponding region ofhsp82 in a natural population of Drosophila pseudoobscura (28).

Figure 2

Sequences of the two copies ofhsp82 found in (A) the monogonont rotiferB. plicatilis RUS and (B) the bdelloid rotifer M. quadricornifera. For each species, the copies are aligned codon by codon with the consensus beneath; identities are indicated as carets () and differences are shaded in red. Gaps are inserted in the B. plicatilis sequences to maintain register with the M. quadricorniferasequences.

Table 1

Divergence of hsp82 copies in sexually reproducing rotifers. Percent difference (%) is indicated above the total number of differences found (no.) in each species at all nucleotide sites (Total), synonymous sites (Syn.), and fourfold degenerate sites (D4). Average sequence differences for theD. pseudoobscura region corresponding to D. melanogaster codon positions 1 to 270 (the longest length available) are calculated for 11 isolates reported in (28).

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Findings for bdelloid rotifers were very different. Each of the four bdelloid genomes examined contains two or more highly divergent copies of hsp82. No copies were found that are as closely similar to each other as those in nonbdelloid rotifers (Tables 1 and2). Two copies were found inM. quadricornifera, differing at fourfold degenerate sites by 54% (Fig. 2B). Three copies were found inAdineta vaga and Habrotrocha constricta and four in Philodina roseola. The highest values of D4 betweenhsp82 copies in each of these three bdelloid genomes are 30, 26, and 49%, respectively, and the corresponding lowest values are 6.0, 6.6, and 3.5%. All three copies of hsp82 in A. vaga contain a 57- to 58-bp intron, and the divergence between intron copies parallels D4 for adjacent exons.

Table 2

Divergence (D4) of hsp82 copies in bdelloid rotifers. Each copy is designated by the first letters of the genus and species followed by a number. Divergences between copies within the same genome are shown in boldface. The A. vagaintron is located after D. melanogaster codon position 88; the total divergence between copies Av1 and Av2, Av1 and Av3, and Av2 and Av3 in the intron is 47, 49, and 5.3%, respectively. A 58-bp intron is present after D. melanogaster codon position 241 in Hc1. Neither intron is present in any other rotifer examined, indicating that they appeared late in bdelloid evolution. Nohsp82 introns were found in any nonbdelloid rotifer.

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In agreement with the above results, Southern blots of genomic DNA probed with hsp82 revealed restriction fragments diagnostic for each of the four copies of hsp82 in P. roseola and for each of the three copies in H. constricta and no other fragments (32). As expected, Southern blots of the monogonont B. plicatilis AUS showed only a single fragment, corresponding to the two nearly identical copies of hsp82 in this species.

Relative rate tests show no significant difference between bdelloids and monogononts in hsp82 fourfold degenerate substitution rates, using the acanthocephalan M. moniliformis as an outgroup (32). There is therefore no indication that nucleotide mutation rates in bdelloids differ from those in monogononts.

Sequence divergence of tbp,rpol3I, and tpi. Two divergent copies and no closely similar copies of each of these three genes were found in individual bdelloid genomes. The region of tbp corresponding to D. melanogaster codons 187 to 295 was examined in the bdelloids P. roseola, M. quadricornifera, andH. constricta and in the monogonont B. plicatilisAUS. D4 values for tbp in the bdelloid genomes are 14, 16, and 44%, respectively. Each copy contains two introns of about 60 bp, and the average difference between intron copies is about equal to D4 for the surrounding exons (Table 3). Only a single tbp sequence, without introns, was found inB. plicatilis.

Table 3

Divergence of tbp copies in bdelloid rotifers. Divergences between copies within the same genome are shown in boldface. Values are D4 except for introns, for which total difference is given. Intron 1 is located afterD. melanogaster codon position 271 and intron 2 after the second nucleotide of codon 295, a conserved intron-exon boundary (39).

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The tpi and rpol3I genes were examined only in the bdelloid P. roseola, giving D4 values of 73 and 12%, respectively. Both copies of tpi have three introns of about 60 bp, and the average difference between intron copies is 36% (Table 4).

Table 4

Divergence of rpol3I andtpi copies in P. roseola. Introns are located in both copies of tpi at three conserved positions, numbered after (40). Copy 1 of tpi contains two additional introns, 61 and 59 bp in length, at conserved positions 3 and 9, respectively. No introns were found in rpol3I. For bothtpi and hsp82, the differences in the number of introns between gene copies in individual bdelloid genomes are comparable to those between species with similarly high levels of synonymous sequence difference in surrounding exons (41,42).

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Nucleotide differences between gene copies in coding regions of all four genes examined in bdelloid genomes are mainly at codon third positions and are distributed throughout the sequenced regions, as illustrated in Fig. 2B. No in-frame stop codons are present in any copy, synonymous substitutions consistently outnumber replacement substitutions, and amino acid sequences closely match those of the corresponding genes in other organisms. Evidently, all copies are functional. It may be that nonfunctional copies are deleterious even in the presence of functional copies and are eliminated by selection, consistent with the partial dominance of deleterious recessives observed in heterozygotes of diverse species (33–35). Also, in the absence of segregation, strong heterotic interactions may evolve (1), making the inactivation of gene copies involved in such interactions disadvantageous.

Phylogeny of hsp82 and tbp in bdelloid rotifers. Relationships among the 12 copies ofhsp82 and among the 6 copies of tbp sequenced in bdelloid rotifers are depicted in Fig. 3. All copies of hsp82 belong to one or the other of two ancient lineages, designated a and b, that began to diverge after the separation of bdelloids and monogononts but before the separation of the bdelloid families we examined (Fig. 3A). Representatives of both lineages are present in individual genomes of P. roseola andM. quadricornifera. In addition, apparent duplications and losses of hsp82 occurred occasionally during the bdelloid radiation.

Figure 3

Phylogeny of hsp82 andtbp in bdelloid rotifers. (A) hsp82.(B) tbp. The ancient lineages a and b ofhsp82 and of tbp began to diverge at the nodes indicated (*). Both lineages of hsp82 are still present in genomes of P. roseola and M. quadricornifera, as are both lineages of tbp inH. constricta. Other nodes are attributed to duplication events (D) or to separations of taxa (S). Dashed lines indicate lineages that should have originated at species separations but were not found and may have been lost or undetected. Numbers at nodes are percentages of 1000 bootstrapped alignments (when over 50%) of fourfold and threefold degenerate codon positions (and, in the case oftbp, intron positions) supporting each clade by maximum parsimony (above the line) or by neighbor-joining (below the line) using B. plicatilis AUS, a member of the sister class Monogononta, as an outgroup (43). The same tree topologies are found using neighbor-joining of synonymous site differences. The same tbp tree was also obtained with only fourfold and threefold degenerate sites or only introns. In all analyses, fewer than 10% of bootstrapped alignments resulted in trees that lacked an ancient a-b divergence.

As with hsp82, the six copies of tbpidentified in bdelloids belong to one or the other of two ancient lineages, also designated a and b, that began to diverge after the separation of bdelloids and monogononts but before the bdelloid radiation (Fig. 3B). Descendants of both tbp lineages are present in the genome of H. constricta.

The average fourfold degenerate difference between the a and b lineages of tbp (53%, SD 5.5) is not significantly different from that between the a and b lineages of hsp82 (49%, SD 4.2), consistent with the indication from phylogenetic analysis that the a and b lineages of both genes began to diverge during the same interval. For comparison, the average D4 values between bdelloids and the monogonont B. plicatilis AUS for hsp82 andtbp are 75% (SD 6) and 79% (SD 3), respectively.

Discussion. We find that genomes of rotifers of the Class Bdelloidea are strikingly different from genomes of rotifers belonging to the other three classes of the phylum, in which reproduction is known to be obligately or facultatively sexual, and from genomes of sexually reproducing species generally.

First, nearly identical pairs of genes were found in nonbdelloid rotifers, as expected in sexually reproducing diploids, but were not found in bdelloids. Even the most similar copies found in any bdelloid genome are more divergent than the most divergent pair found in any other rotifer (Tables 1 to 4). Although not conclusive, a further suggestion that bdelloid genomes are not composed of allelic pairs of haplotypes comes from the observation that at least 3 of the 13 chromosomes in the karyotype of P. roseola and 2 of the 13 chromosomes in the karyotype of Habrotrocha rosa have no morphological homologs (18, 36).

A formal possibility that could account for a lack of allele pairs in individual genomes is that bdelloids are haploid females of sexually reproducing species that have an evanescent or unrecognized diploid form. The failure to observe male bdelloids and the lack of haploid females in any metazoan life cycle make this possibility remote.

A second remarkable feature of bdelloid genomes is the consistent presence in individual genomes of divergent copies of each of the four genes examined, a condition not encountered in any of the nonbdelloid rotifers (Tables 1 to 4) or in reported sequences of any other invertebrate. Although several all-female lines in diverse animal taxa are known to have arisen as alloploids (1, 2), the consistent finding of highly divergent gene copies in bdelloids cannot be attributed to recent species hybridization, for this would require a multitude of separate hybridizations between highly divergent parents and the disappearance of or failure to recognize the parental sexual species. Moreover, phylogenetic analysis of hsp82 andtbp, the two genes examined in more than one bdelloid species, reveals that each copy of hsp82 and each copy oftbp belongs to one or the other of two lineages that began to diverge before the bdelloid radiation and after the separation of bdelloids from monogononts (Fig. 3). These ancient divergences cannot be attributed to speciation because both hsp82 lineages are still present in individual genomes of P. roseola andM. quadricornifera and both tbp lineages are still present in H. constricta. Such stable association could result, however, if the two lineages descend from ancient duplications or from an ancient polyploid ancestor or if they descend from former allele pairs that neither recombined nor segregated throughout bdelloid evolution. In addition, apparent duplications and losses have occurred occasionally during the bdelloid radiation as may have resulted, for example, from nondisjunction.

Our results exclude the possibility that bdelloid rotifers are ordinary diploids that engage in rare or cryptic sex. It remains possible that, unlike any other metazoan that has been examined, bdelloids are ancient polyploids or diploids with ancient duplications of every gene we studied and that they engage in some elusive form of sexual reproduction but are unusually homozygous. However, consistent with the failure to find males, hermaphrodites, or meiosis, it appears more plausible to interpret our findings as further evidence that the Class Bdelloidea has evolved for tens of millions of years without sexual reproduction or genetic exchange between former alleles.

  • * To whom correspondence should be addressed. E-mail: msm{at}wjh.harvard.edu

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