Evolutionary transitions among dioecy,androdioecy and hermaphroditism in limnadiid clam shrimp (Branchiopoda: Spinicaudata)

Examinations of breeding system transitions have primarily concentrated on the transition from hermaphroditism to dioecy, likely because of the preponderance of this transition within flowering plants. Fewer studies have considered the reverse transition: dioecy to hermaphroditism. A fruitful approach to studying this latter transition can be sought by studying clades in which transitions between dioecy and hermaphroditism have occurred multiple times. Freshwater crustaceans in the family Limnadiidae comprise dioecious, hermaphroditic and androdioecious (males + hermaphrodites) species, and thus this family represents an excellent model system for the assessment of the evolutionary transitions between these related breeding systems. Herein we report a phylogenetic assessment of breeding system transitions within the family using a total evidence comparative approach. We find that dioecy is the ancestral breeding system for the Limnadiidae and that a minimum of two independent transitions from dioecy to hermaphroditism occurred within this family, leading to (1) a Holarctic, all‐hermaphrodite species, Limnadia lenticularis and (2) mixtures of hermaphrodites and males in the genus Eulimnadia. Both hermaphroditic derivatives are essentially females with only a small amount of energy allocated to male function. Within Eulimnadia, we find several all‐hermaphrodite populations/species that have been independently derived at least twice from androdioecious progenitors within this genus. We discuss two adaptive (based on the notion of ‘reproductive assurance’) and one nonadaptive explanations for the derivation of all‐hermaphroditism from androdioecy. We propose that L. lenticularis likely represents an all‐hermaphrodite species that was derived from an androdioecious ancestor, much like the all‐hermaphrodite populations derived from androdioecy currently observed within the Eulimnadia. Finally, we note that the proposed hypotheses for the dioecy to hermaphroditism transition are unable to explain the derivation of a fully functional, outcrossing hermaphroditic species from a dioecious progenitor.     

Molecular diversity of bacterial endosymbionts associated with dagger nematodes of the genus Xiphinema (Nematoda: Longidoridae) reveals a high degree of phylogenetic congruence with their host

Bacterial endosymbionts have been detected in some groups of plant‐parasitic nematodes, but few cases have been reported compared to other groups in the phylum Nematoda, such as animal‐parasitic or free‐living nematodes. This study was performed on a wide variety of plant‐parasitic nematode families and species from different host plants and nematode populations. A total of 124 nematode populations (previously identified morphologically and molecularly) were screened for the presence of potential bacterial endosymbionts using the partial 16S rRNA gene and fluorescence in situ hybridization (FISH) and confocal microscopy. Potential bacterial endosymbionts were only detected in nematode species belonging to the genus Xiphinema and specifically in the X. americanum group. Fifty‐seven partial 16S rRNA sequences were obtained from bacterial endosymbionts in this study. One group of sequences was closely related to the genus ‘Candidatus Xiphinematobacter’ (19 bacterial endosymbiont sequences were associated with seven nematode host species, including two that have already been described and three unknown bacterial endosymbionts). The second bacterial endosymbiont group (38 bacterial endosymbiont sequences associated with six nematode species) was related to the family Burkholderiaceae, which includes fungal and soil–plant bacterial endosymbionts. These endosymbionts were reported for the first time in the phylum Nematoda. Our findings suggest that there is a highly specific symbiotic relationship between nematode host and bacterial endosymbionts. Overall, these results were corroborated by a phylogeny of nematode host and bacterial endosymbionts that suggested that there was a high degree of phylogenetic congruence and long‐term evolutionary persistence between hosts and endosymbionts.     

The Evolution of Hermaphroditism from Dioecy in Crustaceans: Selfing Hermaphroditism Described in a Fourth Spinicaudatan Genus

The evolution of reproductive systems has intrigued evolutionary biologists for well over a century. Recent empirical and theoretical work has elucidated the evolution of dioecy (separate males and females) from hermaphroditism in many plant species. The reverse transition, evolving hermaphroditism from dioecy, has occurred many times in animals, and yet is poorly studied relative to its reverse analog in plants. Crustaceans in the sub-order Spinicaudata have evolved hermaphroditism from dioecy three separate times, in some cases forming all-hermaphroditic species and in others forming androdioecious (males + hermaphrodites) species. Herein we report evidence of hermaphroditism in a fourth spinicaudatan genus: the newly described Calalimnadia. We present sex ratio and anatomical evidence that Calalimnadia mahei comprises selfing hermaphrodites, with no males being found in over 10,000 offspring reared. We combine these reproductive results with those of other Spinicaudata to estimate the evolution of hermaphroditism in this crustacean sub-order. We use these genetic data combined with anatomical evidence to suggest that C. mahei represents a fourth, independent derivation of hermaphroditism from dioecy in these reproductively labile crustaceans.     

Sex determination: ways to evolve a hermaphrodite

Most species of the nematode genus Caenorhabditis reproduce through males and females; C. elegans and C. briggsae, however, produce self-fertile hermaphrodites instead of females. These transitions to hermaphroditism evolved convergently through distinct modifications of germline sex determination mechanisms.     

The evolution of mating systems in a group of brooding sea anemones (Epiactis)

Summary

The sea anemone genus Epiactis provides an unusually good opportunity to study the evolution of brooding and mating systems. The four Epiactis species on the Pacific coast of North America all brood their offspring up to the juvenile stage, but each has a different combination of internal vs. external brooding and gonochory vs. simultaneous or gynodioecious hermaphroditism. Two of the four species (E. prolifera and E. lisbethae) were indistinguishable with allozymes (20 loci), but could be differentiated using multilocus DNA fingerprinting. Phylogenetic analyses of the allozyme data by distance and parsimony methods using three outgroups suggest that the four nominal Epiactis species are polyphyletic, with the two internal brooders evolving independently of the two external brooders. This topology does not allow inferences about the evolutionary order of hermaphroditism, dioecy and gynodioecy. Separate sexes and obligate outcrossing are of ten believed to be ancestral, with hermaphroditism and the potential for self-fertilization being favored in taxa where restricted dispersal promotes inbreeding. Previous studies of population genetic structure in these Epiactis species is consistent with this hypothesis, as even the cross-fertile species were highly inbred.     

Computational and phylogenetic validation of nematode horizontal gene transfer

Sequencing of expressed genes has shown that nematodes, particularly the plant-parasitic nematodes, have genes purportedly acquired from other kingdoms by horizontal gene transfer. The prevailing orthodoxy is that such transfer has been a driving force in the evolution of niche specificity, and a recent paper in BMC Evolutionary Biology that presents a detailed phylogenetic analysis of cellulase genes in the free-living nematode Pristionchus pacificus at the species, genus and family levels substantiates this hypothesis.     

Molecular phylogeny of the genus Asparagus (Asparagaceae) explains interspecific crossability between the garden asparagus (A. officinalis) and other Asparagus species

The genus Asparagus comprises approximately 200 species, some of which are commercially cultivated, such as the garden asparagus (A. officinalis). Many Asparagus species, including A. officinalis, are dioecious and have been grouped into a subgenus distinct from that of hermaphroditic species. Although many interspecific crossings have been attempted to introduce useful traits into A. officinalis, only some of the dioecious species were found to be cross-compatible with A. officinalis. Here, molecular phylogenetic analyses were conducted to determine whether interspecific crossability is proportional to the genetic distance between the crossing pairs and to further clarify the evolutionary history of the Asparagus genus. A clade with all cross-compatible species and no cross-incompatible species was recovered in the phylogenetic tree based on analyses of non-coding cpDNA regions. In addition, a sex-linked marker developed for A. officinalis amplified a male-specific region in all cross-compatible species. The phylogenetic analyses also provided some insights about the evolutionary history of Asparagus; for example, by indicating that the genus had its origin in southern Africa, subsequently spreading throughout the old world through intensive speciation and dispersal. The results also suggest that dioecious species were derived from a single evolutionary transition from hermaphroditism in Asparagus. These findings not only contribute towards the understanding of the evolutionary history of the genus but may also facilitate future interspecific hybridization programs involving Asparagus species.     

Inferring the ancestral sexuality and reproductive condition in sponges (Porifera)

Considerable diversity abounds among sponges with respect to reproductive and developmental biology. Their ancestral sexual mode (gonochorism vs. hermaphroditism) and reproductive condition (oviparity vs. viviparity) however remain unclear, and these traits appear to have undergone correlated evolution in the phylum. To infer ancestral traits and investigate this putative correlation, we used DNA sequence data from two loci (18S ribosomal RNA and cytochrome c oxidase subunit I) to explore the phylogenetic relationships of 62 sponges whose reproductive traits have been previously documented. Although the inferred tree topologies, using the limited data available, favoured paraphyly of sponges, we also investigated ancestral character‐state reconstruction on a phylogeny with constrained sponge monophyly. Both parsimony‐ and likelihood‐based ancestral state reconstructions indicate that viviparity (brooding) was the likely reproductive mode of the ancestral sponge. Hermaphroditism is favoured over gonochorism as the sexual condition of the sponge ancestor under parsimony, but the reconstruction is ambiguous under likelihood, rendering the ancestry of sexuality unresolved in our study. These results are insensitive to the constraint of sponge monophyly when tracing the reproductive characters using parsimony methods. However, the maximum likelihood analysis of the monophyletic hypothetical tree rendered gonochorism as ancestral for the phylum. A test of trait correlation unambiguously favours the concerted evolution of sexuality and reproductive mode in sponges (hermaphroditism/viviparity, gonochorism/oviparity). Although testing ecological hypotheses for the pattern of sponge reproduction is beyond the scope of our analyses, we postulate that certain physiological constrains might be key causes for the correlation of reproductive characters.     

Phylogenetic relationships within phylum Rotifera: orders and genus Notholca

We investigated evolutionary relationships among orders in phylum Rotifera and among species in genus Notholca (Rotifera) by computing parsimonious cladograms. All of the most-parsimonious cladograms generated for the ordinal level confirm the view that class Monogononta, superclass Eurotatoria, and phylum Rotifera are monophyletic. Species within the genus Notholca were separated into six groups (clades), but some species have been defined based on highly variable characters not reliably studied using cladistics. Therefore, phenetic studies are warranted, especially for species possessing caudal processes.     

Extremely high molecular diversity within the East Asian nematode Caenorhabditis sp. 5

Most relatives of the self-fertilizing hermaphroditic nematode model organism Caenorhabditis elegans reproduce via obligate outbreeding between males and females, which also represents the ancestral mode of reproduction within the genus. However, little is known about the scope of genetic diversity and differentiation within such gonochoristic species, especially those found outside of temperate Europe and North America. It is critical to understand the evolutionary processes operating in these species to provide a framework for deciphering the evolution of hermaphroditism and a baseline for the application of outcrossing Caenorhabditis to problems in evolutionary genetics. Here, we investigate for the first time molecular sequence variation for Caenorhabditis sp. 5, a species found commonly in eastern Asia. We identify enormous levels of standing genetic variation that approach the levels observed in the marine broadcast-spawning sea squirt, Ciona savignyi. Although we document significant isolation by distance, we demonstrate that the high polymorphism within C. sp. 5 is not because of strong differentiation among populations or to the presence of cryptic species. These findings illustrate that molecular population genetic approaches to studying obligately outbreeding species of Caenorhabditis will prove powerful in identifying and characterizing functionally and evolutionarily important features of the genome.     

Androdioecy and hermaphroditism in five species of clam shrimps (Crustacea: Branchiopoda: Spinicaudata) from India and Thailand

Crustaceans in the order Spinicaudata display a broad range of reproductive strategies, ranging from pure hermaphroditism to pure dioecy (separate males and females), and intermediate combinations. One particularly interesting genus of these “clam shrimps” is Eulimnadia. Based on offspring sex ratios, it has been suggested that all members of the genus are androdioecious: populations consist of mixtures of males and hermaphrodites. However, only two of the ~40 species in this genus have been examined histologically to confirm the presence of ovotestes in the purported hermaphrodites of this group. Here, we report both sex ratio and histological evidence showing that populations of five additional Eulimnadia species from India and Thailand are indeed mixes of males and hermaphrodites (four species) or hermaphrodite only (one species). Sex ratios of adults and offspring from isolated hermaphrodites are in accordance with those previously reported for 15 Eulimnadia species, and histological assays of four of the five species show the presence of both testicular and ovarian tissue in these hermaphrodites. As has been previously reported, the testicular tissue in members of these Eulimnadia spp. is located in a small section at the distal end of the gonad. In addition, the sperm produced in these hermaphrodites forms distinct plaques of compacted chromatin. Overall, these data are consistent with a single origin of hermaphroditism in Eulimnadia, and support the notion that all members of the genus are either androdioecious or all‐hermaphroditic.     

A molecular phylogenetic analysis of reproductive trait evolution in the soft coral genus Alcyonium

The soft coral genus Alcyonium is among the most reproductively diverse invertebrate taxa known: The genus includes species that vary both in mode of reproduction (including broadcast spawners, internal brooders, and external brooders) and sexual expression (gonochores, hermaphrodites, and a unisexual parthenogen). Such diversity offers a unique opportunity to examine associations between reproductive and morphological traits in a phylogenetic context. We used an approximately 900-bp sequence of the nuclear ribosomal gene complex spanning the internal transcribed spacer (ITS) regions to construct a molecular phylogeny for 14 European and North American species of Alcyonium onto which we mapped the known distribution of reproductive and morphological traits. The phylogeny suggests that hermaphroditism or parthenogenesis has evolved independently at least twice in this genus, and always in internally brooding species. Broadcast spawning and external brooding only occur in species with large colony size, whereas all species with small colony size brood their larvae internally. Internal brooding and small size appear to be ancestral in this genus; if this is the case, an association between broadcast spawning and large colony size has evolved independently in at least two clades. This tendency of small adults to brood their larvae while large adults broadcast spawn them into the plankton has been observed in a variety of solitary invertebrate taxa, but to date has not been documented in any other colonial invertebrates. Moreoever, it has been suggested that organisms with a colonial growth form should not experience the allometric constraints on brood space that have been proposed to explain the association between adult size and mode of reproduction in solitary organisms. Unlike many other colonial groups, however, module (polyp) size is strongly correlated with colony size in Alcyonium, and constraints on brooding may be imposed by module, rather than colony, allometry. The very close genetic relationship (< 1% sequence divergence) and shared polymorphisms among A. digitatum (a large, gonochoric broadcast spawner), A. siderium, and A. sp. A (intermediate-sized and small hermaphroditic, internal brooders) suggest that evolutionary transitions between broadcast spawning and brooding and between gonochorism and hermaphroditism can occur easily and rapidly in this group.     

Understanding plant reproductive diversity

Flowering plants display spectacular floral diversity and a bewildering array of reproductive adaptations that promote mating, particularly outbreeding. A striking feature of this diversity is that related species often differ in pollination and mating systems, and intraspecific variation in sexual traits is not unusual, especially among herbaceous plants. This variation provides opportunities for evolutionary biologists to link micro-evolutionary processes to the macro-evolutionary patterns that are evident within lineages. Here, I provide some personal reflections on recent progress in our understanding of the ecology and evolution of plant reproductive diversity. I begin with a brief historical sketch of the major developments in this field and then focus on three of the most significant evolutionary transitions in the reproductive biology of flowering plants: the pathway from outcrossing to predominant self-fertilization, the origin of separate sexes (females and males) from hermaphroditism and the shift from animal pollination to wind pollination. For each evolutionary transition, I consider what we have discovered and some of the problems that still remain unsolved. I conclude by discussing how new approaches might influence future research in plant reproductive biology.     

Sexual system in the tanaidacean Falsapseudes bowmani (Crustacea: Malacostraca: Peracarida)

With more than 40,000 species, Malacostraca is the most diverse crustacean class. Most malacostracans are gonochoristic, but simultaneous hermaphrodites are also known. Tanaidacea is one of two malacostracan orders that includes simultaneously hermaphroditic species; so far, simultaneous hermaphroditism has been confirmed externally and internally in only two tanaidacean species, both in the genus Apseudes (Apseudidae). Here we show, through external and internal morphological observations of fixed specimens, that the apseudid Falsapseudes bowmani is a simultaneous hermaphrodite, making Falsapseudes the second tanaidacean genus in which simultaneous hermaphroditism has been confirmed both externally and internally. In this species, the epistome (a projection on the clypeus) was thick and elongate in large specimens but was thin and spiniform in smaller specimens; the brooding of eggs or embryos was observed only in thin‐epistome individuals, although a pair of ovaries was confirmed in both thick‐ and thin‐epistome individuals. This suggests that individuals with a thick epistome may act as males while also retaining the female reproductive organs.     

The Root-Knot Nematode Producing Galls on Spartina alterniflora Belongs to the Genus Meloidogyne: Rejection of Hypsoperine and Spartonema spp.

Root-knot nematodes are a major group of plant-parasitic nematodes, but their sister group within the Tylenchida remains to be identified. To find the sister group and for any investigation of the evolutionary biology of the genus Meloidogyne, it would be useful to identify the most basal species within Meloidogyninae. Meloidogyne spartinae, a root-knot nematode parasitic on cordgrass (Spartina spp.), constitutes a potentially interesting early diverging (or at least highly divergent) root-knot nematode because it was originally described in a different genus, Hypsoperine (and later Spartonema), due to its unique anatomy and biology (although it was later put in synonymy by some, but not all, taxonomists). We have sequenced the whole 18S rDNA of this species and compared it to other sequences of this region that are available in GenBank for numerous Meloidogyne species. Phylogenetic analysis unambiguously locates the branch corresponding to M. spartinae as a lately diverging species, more closely related to M. maritima, M. duytsi or the M. ardenensis-hapla group. Thus, the distinction of a separate genus (Hypsoperine or Spartonema) for this species is not justified.     

Macroevolutionary synthesis of flowering plant sexual systems

Sexual system is a key determinant of genetic variation and reproductive success, affecting evolution within populations and within clades. Much research in plants has focused on evolutionary transitions away from the most common state of hermaphroditism and toward the rare state of dioecy (separate sexes). Rather than transitions predominantly toward greater sexual differentiation, however, evolution may proceed in the direction of lesser sexual differentiation. We analyzed the macroevolutionary dynamics of sexual system in angiosperm genera that contain both dioecious and nondioecious species. Our phylogenetic analyses encompass a total of 2145 species from 40 genera. Overall, we found little evidence that rates of sexual system transitions are greater in any direction. Counting the number of inferred state changes revealed a mild prevalence of transitions away from hermaphroditism and away from dioecy, toward states of intermediate sexual differentiation. We identify genera in which future studies of sexual system evolution might be especially productive, and we discuss how integrating genetic or population‐level studies of sexual system could improve the power of phylogenetic comparative analyses. Our work adds to the evidence that different selective pressures and constraints act in different groups, helping maintain the variety of sexual systems observed among plants.     

Widespread prevalence but contrasting patterns of intragenomic rRNA polymorphisms in nematodes: Implications for phylogeny,species delimitation and life history inference

Ribosomal RNA genes have long been a favoured locus in phylogenetic and metabarcoding studies. Within a genome, rRNA loci are organized as tandem repeated arrays and the copies are homogenized through the process of concerted evolution. However, some level of rRNA variation (intragenomic polymorphism) is known to persist and be maintained in the genomes of many species. In nematode worms, the extent of rRNA polymorphism (RP) across species and the evolutionary and life history factors that contribute to the maintenance of intragenomic RP is largely unknown. Here, we present an extensive analysis across 30 terrestrial nematode species representing a range of free‐living and parasitic taxa isolated worldwide. Our results indicate that RP is common and widespread, ribosome function appears to be maintained despite mutational changes, and intragenomic variants are stable in the genome and neutrally evolving. However, levels of variation were varied widely across rRNA locus and species, with some taxa observed to lack RP entirely. Higher levels of RP were significantly correlated with shorter generation time and high reproductive rates, and population‐level factors may play a role in the geographic and phylogenetic structuring of rRNA variants observed in genera such as Rotylenchulus and Pratylenchus. Although RP did not dramatically impact the clustering and recovery of taxa in mock metabarcoding analyses, the present study has significant implications for global biodiversity estimates of nematode species derived from environmental rRNA amplicon studies, as well as our understanding of the evolutionary and ecological factors shaping genetic diversity across the nematode Tree of Life.     

Reticulate evolution and sea‐level fluctuations together drove species diversification of slipper orchids (Paphiopedilum) in South‐East Asia

South‐East Asia covers four of the world's biodiversity hotspots, showing high species diversity and endemism. Owing to the successive expansion and contraction of distribution and the fragmentation by geographical barriers, the tropical flora greatly diversified in this region during the Tertiary, but the evolutionary tempo and mode of species diversity remain poorly investigated. Paphiopedilum, the largest genus of slipper orchids comprising nearly 100 species, is mainly distributed in South‐East Asia, providing an ideal system for exploring how plant species diversity was shaped in this region. Here, we investigated the evolutionary history of this genus with eight cpDNA regions and four low‐copy nuclear genes. Discordance between gene trees and network analysis indicates that reticulate evolution occurred in the genus. Ancestral area reconstruction suggests that vicariance and long‐distance dispersal together led to its current distribution. Diversification rate variation was detected and strongly correlated with the species diversity in subg. Paphiopedilum (~80 species). The shift of speciation rate in subg. Paphiopedilum was coincident with sea‐level fluctuations in the late Cenozoic, which could have provided ecological opportunities for speciation and created bridges or barriers for gene flow. Moreover, some other factors (e.g. sympatric distribution, incomplete reproductive barriers and clonal propagation) might also be advantageous for the formation and reproduction of hybrid species. In conclusion, our study suggests that the interplay of reticulate evolution and sea‐level fluctuations has promoted the diversification of the genus Paphiopedilum and sheds light into the evolution of Orchidaceae and the historical processes of plant species diversification in South‐East Asia.     

An endosymbiotic bacterium in a plant-parasitic nematode: Member of a new Wolbachia supergroup

Wolbachia is an endosymbiotic bacterium widely present in arthropods and animal-parasitic nematodes. Despite previous efforts, it has never been identified in plant-parasitic nematodes. Random sequencing of genes expressed by the burrowing nematode Radopholus similis resulted in several sequences with similarity to Wolbachia genes. The presence of a Wolbachia-like endosymbiont in this plant-parasitic nematode was investigated using both morphological and molecular approaches. Transmission electron microscopy, fluorescent immunolocalisation and staining with DAPI confirmed the presence of the endosymbiont within the reproductive tract of female adults.16S rDNA, ftsZ and groEL gene sequences showed that the endosymbiont of R. similis is distantly related to the known Wolbachia supergroups. Finally, based on our initial success in finding sequences of this endosymbiont by screening an expressed sequence tag (EST) dataset, all nematode ESTs were mined for Wolbachia-like sequences. Although the retained sequences belonged to six different nematode species, R. similis was the only plant-parasitic nematode with traces of Wolbachia. Based on our phylogenetic study and the current literature we designate the endosymbiont of R. similis to a new supergroup (supergroup I) rather than considering it as a new species. Although its role remains unknown, the endosymbiont was found in all individuals tested, pointing towards an essential function of the bacteria.