Ancient Asexuals Research Articles

Ancient asexuality: No scandals found with novel data.

Ancient asexuality: No scandals found with novel data.

Description of a new genus and two new species of Darwinulidae (Crustacea, Ostracoda), from Christmas Island (Indian Ocean) with some considerations on the morphological evolution of ancient asexuals

Darwinulidae is believed to be one of the few metazoan taxa in which fully asexual reproduction might have persisted for millions of years. Although rare males in a single darwinulid species have recently been found, they may be non-functional atavisms. The representatives of this family are characterized by a slow evolutionary rate, resulting in a conservative morphology in the different lineages over long time frames and across wide geographic ranges. Differences between species and genera, although often based on small details of valve morphology and chaetotaxy, are nevertheless well-recognizable. Five Recent genera (Darwinula, Alicenula, Vestalenula, Penthesilenula and Microdarwinula) and about 35 living species, including also those left in open nomenclature, are included in this family. Previous phylogenetic analyses using both morphological characters and molecular data confirmed that the five genera are good phyletic units. Here, we report on the results of a study on darwinulid ostracods from Christmas Island (Indian Ocean). The taxonomic investigation led to the description of a new genus and two new species, namely Isabenula humphreysi gen. nov. sp. nov. and Vestalenula sp. E, this latter being left in open nomenclature, because only one individual was available. A new phylogeny of the family, using 30 morphological characters of 23 described species, is here presented. A key to the genera of Recent Darwinulidae, based on the morphology of valves and soft parts in adult females, is also provided. The discovery of Isabenula gen. nov., which shares intermediate morphological features with the genera Vestalenula and Penthesilenula, opens new opportunities to critically reconsider previous hypotheses on tempo and mode of evolution in the family Darwinulidae and on the phylogenetic relationships between its representatives.

Population Genomic Analyses Suggest a Hybrid Origin, Cryptic Sexuality, and Decay of Genes Regulating Seed Development for the Putatively Strictly Asexual Kingdonia uniflora (Circaeasteraceae, Ranunculales).

Asexual lineages are perceived to be short-lived on evolutionary timescales. Hence, reports for exceptional cases of putative 'ancient asexuals' usually raise questions about the persistence of such species. So far, there have been few studies to solve the mystery in plants. The monotypic Kingdonia dating to the early Eocene, contains only K. uniflora that has no known definitive evidence for sexual reproduction nor records for having congeneric sexual species, raising the possibility that the species has persisted under strict asexuality for a long period of time. Here, we analyze whole genome polymorphism and divergence in K. uniflora. Our results show that K. uniflora is characterized by high allelic heterozygosity and elevated πN/πS ratio, in line with theoretical expectations under asexual evolution. Allele frequency spectrum analysis reveals the origin of asexuality in K. uniflora occurred prior to lineage differentiation of the species. Although divergence within K. uniflora individuals exceeds that between populations, the topologies of the two haplotype trees, however, fail to match each other, indicating long-term asexuality is unlikely to account for the high allele divergence and K. uniflora may have a recent hybrid origin. Phi-test shows a statistical probability of recombination for the conflicting phylogenetic signals revealed by the split network, suggesting K. uniflora engages in undetected sexual reproduction. Detection of elevated genetic differentiation and premature stop codons (in some populations) in genes regulating seed development indicates mutational degradation of sexuality-specific genes in K. uniflora. This study unfolds the origin and persistence mechanism of a plant lineage that has been known to reproduce asexually and presents the genomic consequences of lack of sexuality.

Evidence from automixis with inverted meiosis for the maintenance of sex by loss of complementation.

The adaptive value of sexual reproduction is still debated. A short-term disadvantage of asexual reproduction is loss of heterozygosity, which leads to the unmasking of recessive deleterious mutations. The cost of this loss of complementation is predicted to be higher than the twofold cost of meiosis for most types of asexual reproduction. Automixis with terminal fusion of sister nuclei is especially vulnerable to the effect of loss of complementation. It is found, however, in some taxa including oribatid mites, the most prominent group of ancient asexuals. Here, I show that automixis with terminal fusion is stable if it is associated with inverted meiosis and that this appears to be the case in nature, notably in oribatid mites. The existence of automixis with terminal fusion, and its co-occurrence with inverted meiosis, therefore, is consistent with the hypothesis that loss of complementation is important in the evolution of sexual reproduction.

Transcriptional activity and epigenetic regulation of transposable elements in the symbiotic fungus Rhizophagus irregularis.

Arbuscular mycorrhizal (AM) fungi form mutualistic relationships with most land plant species. AM fungi have long been considered as ancient asexuals. Long-term clonal evolution would be remarkable for a eukaryotic lineage and suggests the importance of alternative mechanisms to promote genetic variability facilitating adaptation. Here, we assessed the potential of transposable elements for generating such genomic diversity. The dynamic expression of TEs during Rhizophagus irregularis spore development suggests ongoing TE activity. We find Mutator-like elements located near genes belonging to highly expanded gene families. Whole-genome epigenomic profiling of R. irregularis provides direct evidence of DNA methylation and small RNA production occurring at TE loci. Our results support a model in which TE activity shapes the genome, while DNA methylation and small RNA–mediated silencing keep their overproliferation in check. We propose that a well-controlled TE activity directly contributes to genome evolution in AM fungi.

A Survey of Transposon Landscapes in the Putative Ancient Asexual Ostracod Darwinula stevensoni

How asexual reproduction shapes transposable element (TE) content and diversity in eukaryotic genomes remains debated. We performed an initial survey of TE load and diversity in the putative ancient asexual ostracod Darwinula stevensoni. We examined long contiguous stretches of DNA in clones from a genomic fosmid library, totaling about 2.5 Mb, and supplemented these data with results on TE abundance and diversity from an Illumina draft genome. In contrast to other TE studies in putatively ancient asexuals, which revealed relatively low TE content, we found that at least 19% of the fosmid dataset and 26% of the genome assembly corresponded to known transposons. We observed a high diversity of transposon families, including LINE, gypsy, PLE, mariner/Tc, hAT, CMC, Sola2, Ginger, Merlin, Harbinger, MITEs and helitrons, with the prevalence of DNA transposons. The predominantly low levels of sequence diversity indicate that many TEs are or have recently been active. In the fosmid data, no correlation was found between telomeric repeats and non-LTR retrotransposons, which are present near telomeres in other taxa. Most TEs in the fosmid data were located outside of introns and almost none were found in exons. We also report an N-terminal Myb/SANT-like DNA-binding domain in site-specific R4/Dong non-LTR retrotransposons. Although initial results on transposable loads need to be verified with high quality draft genomes, this study provides important first insights into TE dynamics in putative ancient asexual ostracods.

Evaluation of the ontogeny and sexual dimorphism in a new species of Middle Triassic Darwinulocopina (Crustacea, Ostracoda) from Argentina

AbstractDarwinulocopina is an interesting group of ostracods that includes some of the first invaders of freshwater habitats during the late Palaeozoic. The Permian–Triassic extinction greatly reduced their diversity, culminating in a single extant family. The darwinulids are regarded as ‘ancient asexuals’ given that a parthenogenetic mode of reproduction is assumed for all post‐Triassic members of the group. However, the high diversity achieved during the late Palaeozoic is often associated with sexual reproduction. Here we studied a monospecific assemblage of ostracods from the Middle Triassic of the Cuyo Basin, Province of Mendoza, Argentina, recognizing a new species of Darwinulocopina,Prasuchonella?huarpesp. nov. We discuss the traditional length/height and length/width graphical method for identifying ontogeny/sexual dimorphism in fossil ostracod assemblages. A geometric morphometric analysis was performed on both lateral and dorsal views of almost 170 carapaces, to evaluate the ontogeny and sexual dimorphism of the species. The best results were obtained from the analysis in dorsal view, discriminating four ontogenetic stages. This revealed a main ontogenetic trend related to the development of the brooding chamber. Although subtle in difference, female carapaces are wider not only at the brooding chamber, but also along the whole length, compared with the presumptive males. We provide full systematic descriptions and discussions, attempting to unify descriptive criteria for recent and fossil darwinulocopine carapaces, and suggest closeness to species previously described from the upper Permian of Russia. As a result, we recommend a review of Mesozoic records of Darwinulocopina, particularly those from the Triassic.

Lineage-Specific Genes and Cryptic Sex: Parallels and Differences between Arbuscular Mycorrhizal Fungi and Fungal Pathogens.

Arbuscular mycorrhizal fungi (AMF) live as obligate root symbionts on almost all land plants. They have long been regarded as ancient asexuals that have propagated clonally for millions of years. However, genomic studies in Rhizophagus irregularis and other AMF revealed many features indicative of sex. Surprisingly, comparative genomics of conspecific isolates of R. irregularis revealed an unexpected interstrain diversity, suggesting that AMF carry a high number of lineage-specific (LS) genes. Intriguingly, cryptic sex and LS genomic regions have previously been reported in a number of fungal pathogens of plants and humans. Here, we discuss these genomic similarities and highlight their potential relevance for AMF adaptation to the environment and for symbiotic functioning.

Single nucleus sequencing reveals evidence of inter-nucleus recombination in arbuscular mycorrhizal fungi.

Eukaryotes thought to have evolved clonally for millions of years are referred to as ancient asexuals. The oldest group among these are the arbuscular mycorrhizal fungi (AMF), which are plant symbionts harboring hundreds of nuclei within one continuous cytoplasm. Some AMF strains (dikaryons) harbor two co-existing nucleotypes but there is no direct evidence that such nuclei recombine in this life-stage, as is expected for sexual fungi. Here, we show that AMF nuclei with distinct genotypes can undergo recombination. Inter-nuclear genetic exchange varies in frequency among strains, and despite recombination all nuclear genomes have an average similarity of at least 99.8%. The present study demonstrates that AMF can generate genetic diversity via meiotic-like processes in the absence of observable mating. The AMF dikaryotic life-stage is a primary source of nuclear variability in these organisms, highlighting its potential for strain enhancement of these symbionts.

Cross-Contamination Explains “Inter and Intraspecific Horizontal Genetic Transfers” between Asexual Bdelloid Rotifers

A few metazoan lineages are thought to have persisted for millions of years without sexual reproduction. If so, they would offer important clues to the evolutionary paradox of sex itself [1, 2]. Most "ancient asexuals" are subject to ongoing doubt because extant populations continue to invest in males [3-9]. However, males are famously unknown in bdelloid rotifers, a class of microscopic invertebrates comprising hundreds of species [10-12]. Bdelloid genomes have acquired an unusually high proportion of genes from non-metazoans via horizontal transfer [13-17]. This well-substantiated finding has invited speculation [13] that homologous horizontal transfer between bdelloid individuals also may occur, perhaps even "replacing" sex [14]. In 2016, Current Biology published an article claiming to supply evidence for this idea. Debortoli etal. [18] sampled rotifers from natural populations and sequenced one mitochondrial and four nuclear loci. Species assignments were incongruent among loci for several samples, which was interpreted as evidence of "interspecific horizontal genetic transfers." Here, we use sequencing chromatograms supplied by the authors to demonstrate that samples treated as individuals actually contained two or morehighly divergent mitochondrial and ribosomal sequences, revealing cross-contamination with DNA from multiple animals of different species. Other chromatograms indicate contamination with DNA from conspecific animals, explaining genetic and genomic evidence for "intraspecific horizontal exchanges" reported in the same study. Given the clear evidence of contamination, the data and findings of Debortoli etal. [18] provide no reliable support for their conclusions that DNA is transferred horizontally between or within bdelloid species.

General environmental heterogeneity as the explanation of sexuality? Comparative study shows that ancient asexual taxa are associated with both biotically and abiotically homogeneous environments.

Ecological theories of sexual reproduction assume that sexuality is advantageous in certain conditions, for example, in biotically or abiotically more heterogeneous environments. Such theories thus could be tested by comparative studies. However, the published results of these studies are rather unconvincing. Here, we present the results of a new comparative study based exclusively on the ancient asexual clades. The association with biotically or abiotically homogeneous environments in these asexual clades was compared with the same association in their sister, or closely related, sexual clades. Using the conservative definition of ancient asexuals (i.e., age >1 million years), we found eight pairs of taxa of sexual and asexual species, six differing in the heterogeneity of their inhabited environment on the basis of available data. The difference between the environmental type associated with the sexual and asexual species was then compared in an exact binomial test. The results showed that the majority of ancient asexual clades tend to be associated with biotically, abiotically, or both biotically and abiotically more homogeneous environments than their sexual controls. In the exploratory part of the study, we found that the ancient asexuals often have durable resting stages, enabling life in subjectively homogeneous environments, live in the absence of intense biotic interactions, and are very often sedentary, inhabiting benthos, and soil. The consequences of these findings for the ecological theories of sexual reproduction are discussed.

Evidence Supporting the Uptake and Genomic Incorporation of Environmental DNA in the "Ancient Asexual" Bdelloid Rotifer Philodina roseola.

Increasing evidence suggests that bdelloid rotifers regularly undergo horizontal gene transfer, apparently as a surrogate mechanism of genetic exchange in the absence of true sexual reproduction, in part because of their ability to withstand desiccation. We provide empirical support for this latter hypothesis using the bdelloid Philodina roseola, which we demonstrate to readily internalize environmental DNA in contrast to a representative monogonont rotifer (Brachionus rubens), which, like other monogononts, is facultative sexual and cannot withstand desiccation. In addition, environmental DNA that was more similar to the host DNA was retained more often and for a longer period of time. Indirect evidence (increased variance in the reproductive output of the untreated F1 generation) suggests that environmental DNA can be incorporated into the genome during desiccation and is thus heritable. Our observed fitness effects agree with sexual theory and also occurred when the animals were desiccated in groups (thereby acting as DNA donors), but not individually, indicating the mechanism could occur in nature. Thus, although DNA uptake and its genomic incorporation appears proximally related to anhydrobiosis in bdelloids, it might also facilitate accidental genetic exchange with closely related taxa, thereby maintaining higher levels of genetic diversity than is otherwise expected for this group of “ancient asexuals”.

Functional Significance of Meiotic Proteins in the DNA Repair System of Bdelloid Rotifers

Bdelloid rotifers are microinvertebrates that repair DNA double‐strand breaks (DSBs) with an efficiency unparalleled in other eukaryotes. These animals inhabit transient aquatic environments and survive frequent desiccation by entering a state of anhydrobiosis. Their DNA repair system is necessary for repairing the DSBs induced by extreme desiccation. Bdelloids have long been considered ancient asexuals that have survived without males and sex for millions of years. Evidence for canonical meiosis and sexual reproduction in bdelloids is absent, although some form of atypical genetic exchange might occur in some populations. Despite the long‐standing absence of meiosis in bdelloids, their genomes contain four genes that encode proteins (HOP1, SPO11, MSH4, and MSH5) known to be specific to meiosis in other eukaryotes. Since bdelloids lack meiosis yet possess a noteworthy DNA repair system, the objective is to investigate the potential role of meiotic proteins during DNA repair. We used real‐time PCR to quantify meiotic gene expression in the bdelloid Adineta vaga following exposure to 280 Gray of ionizing radiation (which induces DSBs). Genes encoding meiotic proteins were upregulated, relative to non‐irradiated A. vaga. Western blots using custom antibodies for HOP1, SPO11, MSH4, and MSH5 from A. vaga revealed that these proteins are produced in bdelloids. To characterize binding partners of these meiotic proteins during DNA repair, we generated 6×histidine‐tagged copies of HOP1, SPO11, MSH4, and MSH5 from A. vaga. Pull‐down assays using the histidine‐tagged proteins were done to identify the proteins that interact with HOP1, SPO11, MSH4, and MSH5 in irradiated and non‐irradiated bdelloids. These findings will shed light on the functional significance of these meiotic proteins in bdelloids. Overall, this study represents the first genetic and proteomic data to address whether meiotic genes in bdelloids have evolved a novel function in DNA repair, which could justify the maintenance of these genes in an ameiotic lineage. Data supporting a role for “meiotic proteins” in DNA repair would be the first evidence of a non‐meiotic function for these genes and could be used to further our understanding of DNA repair in eukaryotes.Support or Funding InformationThis work was supported by grants from the NCRR (5P20RR016460) & NIGMS (8P20GM103429) from the NIH to AMS, and a Summer Undergraduate Research Fellowship from the Arkansas Department of Higher Education to LED.

Ancient and modern colonization of North America by hemlock woolly adelgid, Adelges tsugae (Hemiptera: Adelgidae), an invasive insect from East Asia.

Hemlock woolly adelgid, Adelges tsugae, is an invasive pest of hemlock trees (Tsuga) in eastern North America. We used 14 microsatellites and mitochondrial COI sequences to assess its worldwide genetic structure and reconstruct its colonization history. The resulting information about its life cycle, biogeography and host specialization could help predict invasion by insect herbivores. We identified eight endemic lineages of hemlock adelgids in central China, western China, Ulleung Island (South Korea), western North America, and two each in Taiwan and Japan, with the Japanese lineages specializing on different Tsuga species. Adelgid life cycles varied at local and continental scales with different sexual, obligately asexual and facultatively asexual lineages. Adelgids in western North America exhibited very high microsatellite heterozygosity, which suggests ancient asexuality. The earliest lineages diverged in Asia during Pleistocene glacial periods, as estimated using approximate Bayesian computation. Colonization of western North America was estimated to have occurred prior to the last glacial period by adelgids directly ancestral to those in southern Japan, perhaps carried by birds. The modern invasion from southern Japan to eastern North America caused an extreme genetic bottleneck with just two closely related clones detected throughout the introduced range. Both colonization events to North America involved host shifts to unrelated hemlock species. These results suggest that genetic diversity, host specialization and host phylogeny are not predictive of adelgid invasion. Monitoring non-native sentinel host trees and focusing on invasion pathways might be more effective methods of preventing invasion than making predictions using species traits or evolutionary history.

Unseen sex in ancient virgin fungi

Unseen sex in ancient virgin fungi

How does the "ancient" asexual Philodina roseola (Rotifera: Bdelloidea) handle potential UVB-induced mutations?

Like other obligate asexuals, bdelloid rotifers are expected to suffer from degradation of their genomes through processes including the accumulation of deleterious mutations. However, sequence-based analyses in this regard remain inconclusive. Instead of looking for historical footprints of mutations in these ancient asexuals, we directly examined the susceptibility and ability to repair point mutations by the bdelloid Philodina roseola by inducing cyclobutane-pyrimidine dimers (CPDs) via exposure to UVB radiation (280-320 nm). For comparison, we performed analogous experiments with the facultative asexual monogonont rotifer Brachionus rubens. Different strategies were found for the two species. Philodina roseola appeared to shield itself from CPD induction through uncharacterized UV-absorbing compounds and, except for the genome reconstruction that occurs after desiccation, was largely unable to repair UVB-induced damage. By contrast, B. rubens was more susceptible to UVB irradiation, but could repair all induced damage in ~2 h. In addition, whereas UV irradiation had a significant negative impact on the reproductive output of P. roseola, and especially so after desiccation, that of B. rubens was unaffected. Although the strategy of P. roseola might suffice under natural conditions where UVB irradiation is less intense, the lack of any immediate CPD repair mechanisms in this species remains perplexing. It remains to be investigated how typical these results are for bdelloids as a group and therefore how reliant these animals are on desiccation-dependent genome repair to correct potential DNA damage given their obligate asexual lifestyle.

Biochemical Diversification through Foreign Gene Expression in Bdelloid Rotifers

Bdelloid rotifers are microinvertebrates with unique characteristics: they have survived tens of millions of years without sexual reproduction; they withstand extreme desiccation by undergoing anhydrobiosis; and they tolerate very high levels of ionizing radiation. Recent evidence suggests that subtelomeric regions of the bdelloid genome contain sequences originating from other organisms by horizontal gene transfer (HGT), of which some are known to be transcribed. However, the extent to which foreign gene expression plays a role in bdelloid physiology is unknown. We address this in the first large scale analysis of the transcriptome of the bdelloid Adineta ricciae: cDNA libraries from hydrated and desiccated bdelloids were subjected to massively parallel sequencing and assembled transcripts compared against the UniProtKB database by blastx to identify their putative products. Of ∼29,000 matched transcripts, ∼10% were inferred from blastx matches to be horizontally acquired, mainly from eubacteria but also from fungi, protists, and algae. After allowing for possible sources of error, the rate of HGT is at least 8%–9%, a level significantly higher than other invertebrates. We verified their foreign nature by phylogenetic analysis and by demonstrating linkage of foreign genes with metazoan genes in the bdelloid genome. Approximately 80% of horizontally acquired genes expressed in bdelloids code for enzymes, and these represent 39% of enzymes in identified pathways. Many enzymes encoded by foreign genes enhance biochemistry in bdelloids compared to other metazoans, for example, by potentiating toxin degradation or generation of antioxidants and key metabolites. They also supplement, and occasionally potentially replace, existing metazoan functions. Bdelloid rotifers therefore express horizontally acquired genes on a scale unprecedented in animals, and foreign genes make a profound contribution to their metabolism. This represents a potential mechanism for ancient asexuals to adapt rapidly to changing environments and thereby persist over long evolutionary time periods in the absence of sex.

Cryptic Species in Putative Ancient Asexual Darwinulids (Crustacea, Ostracoda)

BackgroundFully asexually reproducing taxa lack outcrossing. Hence, the classic Biological Species Concept cannot be applied.Methodology/Principal FindingsWe used DNA sequences from the mitochondrial COI gene and the nuclear ITS2 region to check species boundaries according to the evolutionary genetic (EG) species concept in five morphospecies in the putative ancient asexual ostracod genera, Penthesilenula and Darwinula, from different continents. We applied two methods for detecting cryptic species, namely the K/θ method and the General Mixed Yule Coalescent model (GMYC). We could confirm the existence of species in all five darwinulid morphospecies and additional cryptic diversity in three morphospecies, namely in Penthesilenula brasiliensis, Darwinula stevensoni and in P. aotearoa. The number of cryptic species within one morphospecies varied between seven (P. brasiliensis), five to six (D. stevensoni) and two (P. aotearoa), respectively, depending on the method used. Cryptic species mainly followed continental distributions. We also found evidence for coexistence at the local scale for Brazilian cryptic species of P. brasiliensis and P. aotearoa. Our ITS2 data confirmed that species exist in darwinulids but detected far less EG species, namely two to three cryptic species in P. brasiliensis and no cryptic species at all in the other darwinulid morphospecies.Conclusions/SignificanceOur results clearly demonstrate that both species and cryptic diversity can be recognized in putative ancient asexual ostracods using the EG species concept, and that COI data are more suitable than ITS2 for this purpose. The discovery of up to eight cryptic species within a single morphospecies will significantly increase estimates of biodiversity in this asexual ostracod group. Which factors, other than long-term geographic isolation, are important for speciation processes in these ancient asexuals remains to be investigated.

Molecular Evidence for Ancient Asexuality in Timema Stick Insects

Asexuality is rare in animals in spite of its apparent advantage relative to sexual reproduction, indicating that it must be associated with profound costs [1-9]. One expectation is that reproductive advantages gained by new asexual lineages will be quickly eroded over time [3, 5-7]. Ancient asexual taxa that have evolved and adapted without sex would be "scandalous" exceptions to this rule, but it is often difficult to exclude the possibility that putative asexuals deploy some form of "cryptic" sex, or have abandoned sex more recently than estimated from divergence times to sexual relatives [10]. Here we provide evidence, from high intraspecific divergence of mitochondrial sequence and nuclear allele divergence patterns, that several independently derived Timema stick-insect lineages have persisted without recombination for more than a million generations. Nuclear alleles in the asexual lineages displayed significantly higher intraindividual divergences than in related sexual species. In addition, within two asexuals, nuclear allele phylogenies suggested the presence of two clades, with sequences from the same individual appearing in both clades. These data strongly support ancient asexuality in Timema and validate the genus as an exceptional opportunity to attack the question of how asexual reproduction can be maintained over long periods of evolutionary time.

Secretive ciliates and putative asexuality in microbial eukaryotes

Facultative sexuality is assumed to have occurred in the ancestor of all extant eukaryotes, but the distribution and maintenance of sex among microbial eukaryotes is still under debate. In this paper, we address the purported asexuality in colpodean ciliates as an exemplary lineage. Colpodeans are a primarily terrestrial clade thought to have arisen up to 900 MYA and contain one known derived sexual species. We conclude that the putative asexuality of this lineage is an observational artifact. We suggest that the same might hold for other microbial eukaryotes, and that many are secretively sexual as well. Theoretical work from the distantly related plants and animals suggests that both the evolutionary success of ancient asexuals and the reversal of the loss of sex are highly unlikely, further suggesting that colpodeans are secretively sexual. However, it remains to be seen to what extent sexual theories and predictions derived from macro-organismic lineages apply also to microbial eukaryotes.