Sexual Generation Research Articles

Modelling suggests Wolbachia-induced cytoplasmic incompatibility in oak gall wasps with cyclical parthenogenesis.

Oak gall wasps typically exhibit a life cycle with one sexual and one asexual generation each year. These wasps can carry various endosymbionts, one of which is the maternally inherited bacterium Wolbachia that can induce several reproductive manipulations on its host. Cytoplasmic incompatibility (CI) has been described as the most prominent of these manipulations. CI leads to embryonic mortality in the hosts' offspring when infected males mate with either uninfected females or with females that harbour different Wolbachia strains. It has been hypothesized that Wolbachia can induce CI in oak gall wasps. To address this hypothesis, we derived a mathematical model to investigate the spread of a bacterial infection in naive populations and to determine the plausibility of CI occurrence. To validate our model, we used published data from Wolbachia-infected Belonocnema kinseyi populations in two approaches. Our first approach uses measurements of infection frequencies and maternal transmission in the sexual generation. For the second approach, we extended the model to compare predictions to estimates of mtDNA-haplotypes, which, like Wolbachia, are maternally inherited, and can therefore be associated with the infection. Both approaches indicate that CI is present in these populations. Our model can be generalized to investigate the occurrence of CI not only for oak gall wasps but also for other species.

Comparative transcriptome reprogramming in oak galls containing asexual or sexual generations of gall wasps.

Oak gall wasps have evolved strategies to manipulate the developmental pathways of their host to induce gall formation. This provides shelter and nutrients for the developing larva. Galls are entirely host tissue; however, the initiation, development, and physical appearance are controlled by the inducer. The underlying molecular mechanisms of gall formation, by which one or a small number of cells are reprogrammed and commit to a novel developmental path, are poorly understood. In this study, we sought a deeper insight into the molecular underpinnings of this process. Oak gall wasps have two generations each year, one sexual, and one asexual. Galls formed by these two generations exhibit a markedly different appearance. We sequenced transcriptomes of both the asexual and sexual generations of Neuroterus quercusbaccarum and Neuroterus numismalis. We then deployed Nanopore sequencing to generate long-read sequences to test the hypothesis that gall wasps introduce DNA insertions to determine gall development. We detected potential genome rearrangements but did not uncover any non-host DNA insertions. Transcriptome analysis revealed that transcriptomes of the sexual generations of distinct species of wasp are more similar than inter-generational comparisons from the same species of wasp. Our results highlight the intricate interplay between the host leaves and gall development, suggesting that season and requirements of the gall structure play a larger role than species in controlling gall development and structure.

Helminth Fauna of Bats of the Rostov Region: Review. 2. Trematoda

Introduction. The class of Trematoda includes two subclasses — Aspidogastrea and Digenea. Representatives of the former do not parasitize in bats. The definitive hosts for digenetic trematodes (Digenea) are vertebrates, including bats, in which the sexual generation (maritae) parasitizes. Gastropods or bivalves are the intermediate hosts. Supplementary hosts for the trematodes of bats are larvae and imagos of flying insects. Since in most cases the development of parthenites and cercariae of trematodes is related to water environment, the supplementary hosts are the groups of insects whose larvae live in the water. The list of bat species living in the Rostov region has been compiled in the previous research. In the present article the authors investigate the trematode fauna parasitizing in bats of the region under study. The aim of this review is to analyse the current state of knowledge on the species composition of the helminth parasitizing in chiropterans of the Rostov region.Materials and Methods. The research materials have been obtained from the libraries and open access resources: PubMed (pubmed.ncbi.nlm.nih.gov), CyberLeninka (cyberleninka.ru), Google Scholar (scholar.google.com), BHL (www.biodiversitylibrary.org), JSTOR (www.jstor.org). Some data has been provided by the colleagues.Results. 42 trematode species from 10 genera, 5 families and 1 order can parasitize in 15 bat species living in the Rostov region. The Lecithodendriidae family (24 species) and the genus Paralecithodendrium (9 species) are represented by a large number of species. Of the listed trematodes, 33 species used to be recorded on the territory of Russia.Discussion and Conclusion. The results of the conducted systematic review have revealed that the largest number of trematode species parasitizes in two bat species living in the Rostov region: the common noctule (27 species) and the serotine bat (24 species). The smallest number of species (4 per each) is found in the giant noctule and the soprano pipistrelle bat. Two species (Plagiorchis elegans and Plagiorchis vespertilionis) have been found in the companion cats and dogs includingly, wheras a case of the latter parazitising in humans has also been recorded. The presence of bats in the urban environment enables the full life cycle of these trematodes. There exists possibility of the companion animals and people to get infected through incidental ingestion of the infested insects.

Taxonomy and Phylogeny of the Aphid Genus Nippolachnus Matsumura, 1917, with Synonymy of the Mysterious Neonippolachnus Shinji, 1924 (Hemiptera: Aphididae: Lachninae).

Nippolachnus Matsumura, 1917 is a small aphid genus from the tribe Tuberolachnini (Hemiptera: Lachninae) occurring in Southeast Asia. Species from this genus are quite characteristic and stand out among lachnids for their morphology and ecological associations. We have performed a revision and phylogenetic analyses to elucidate the relationships within Nippolachnus and other representatives of Tuberolachnini. Here, the taxonomy of the genus is revised based on morphological data to include seven species, three of them newly described: Nippolachnus chakrabartiisp. nov. from India, Nippolachnus sinensissp. nov. from China, and Nippolachnus malayaensissp. nov. from Indonesia. Nippolachnus appear to be non monophyletic genus and a new genus, Indolachnusgen. nov., is described to accommodate Nippolachnus himalayensis (van der Goot, 1917) as Indolachnus himalayensis (van der Goot, 1917) comb. nov. The new genus is a sister group to the remaining Nippolachnus species, which created a monophyletic clade. Neonippolachnus Shinji, 1924 syn. nov. is recognised as a synonym of Nippolachnus, and Neonippolachnus betulae Shinji, 1924 syn. nov. as a synonym of Nippolachnus micromeli Shinji, 1924. For the first time, a scanning electron microscopy study of the sexual generation of N. piri Matsumura, 1917 has been performed. Apterous and alate viviparous females of N. bengalensis Basu and Hille Ris Lambers, 1968, N. piri, and N. micromeli, and alate viviparous females of N. xitianmushanus Zhang and Zhong, 1982 are re-described and illustrated, as well as apterous and alate viviparous females of I. himalayensiscomb. nov. Hitherto unknown morphs of N. micromeli, N. piri, and N. xitianmushanus are described. A lectotype and paralectotypes of N. xitianmushanus are designated herein. Notes on distribution and host plants are given, and keys to apterous and alate viviparous females of the genera Nippolachnus and Indolachnus are also provided.

Life cycle closure of Cerroneuroterus minutulus (Giraud, 1859) with C. aggregatus (Wachtl, 1880) (Hymenoptera: Cynipidae: Cynipini): experimental, taxonomic and molecular approaches.

The life cycle of Cerroneuroterus minutulus (Giraud, 1859), a species previously known only from its asexual generation, is closed. Our study demonstrates that C. minutulus exhibits the heterogony typical of Cynipini species, with alternating sexual and asexual generations. The identity of the sexual generation is demonstrated by laboratory experiments and confirmed by molecular data, with Cerroneuroterus aggregatus (Wachtl, 1880) determined to be the sexual generation, and junior synonym, of Cerroneuroterus minutulus (Giraud, 1859). Illustrations and diagnoses for adults and galls, observations on biology and distribution information are provided as is a detailed redescription of the sexual generation of C. minutulus.

RNA-seq analysis of parasitism by Intoshia linei (Orthonectida) reveals protein effectors of defence, communication, feeding and growth.

Orthonectida is a group of multicellular endoparasites of a wide range of marine invertebrates. Their parasitic stage is a multinuclear shapeless plasmodium infiltrating host tissues. The development of the following worm-like sexual generation takes place within the cytoplasm of the plasmodium. The existence of the plasmodial stage and the development of a sexual stage within the plasmodium are unique features to Bilateria. However, the molecular mechanisms that maintain this peculiar organism, and hence enable parasitism in orthonectids, are unknown. Here, we present the first-ever RNA-seq analysis of the plasmodium, aimed at the identification and characterization of the plasmodium-specific protein-coding genes and corresponding hypothetical proteins that distinguish the parasitic plasmodium stage from the sexual stage of the orthonectid Intoshia linei Giard, 1877,parasite of nemertean Lineus ruber Müller, 1774.We discovered 119 plasmodium-specific proteins, 82 of which have inferred functions based on known domains. Thirty-five of the detected proteins are orphans, at least part of which may reflect the unique evolutionary adaptations of orthonectids to parasitism. Some of the identified proteins are known effector molecules of other endoparasites suggesting convergence. Our data indicate that the plasmodium-specific proteins might be involved in the plasmodium defense against the host, host-parasite communication, feeding and nutrient uptake, growth within the host, and support of the sexual stage development. These molecular processes in orthonectids have not been described before, and the particular protein effectors remained unknown until now.

Phylloxera and Aphids Show Distinct Features of Genome Evolution Despite Similar Reproductive Modes.

Genomes of aphids (family Aphididae) show several unusual evolutionary patterns. In particular, within the XO sex determination system of aphids, the X chromosome exhibits a lower rate of interchromosomal rearrangements, fewer highly expressed genes, and faster evolution at nonsynonymous sites compared with the autosomes. In contrast, other hemipteran lineages have similar rates of interchromosomal rearrangement for autosomes and X chromosomes. One possible explanation for these differences is the aphid's life cycle of cyclical parthenogenesis, where multiple asexual generations alternate with 1 sexual generation. If true, we should see similar features in the genomes of Phylloxeridae, an outgroup of aphids which also undergoes cyclical parthenogenesis. To investigate this, we generated a chromosome-level assembly for the grape phylloxera, an agriculturally important species of Phylloxeridae, and identified its single X chromosome. We then performed synteny analysis using the phylloxerid genome and 30 high-quality genomes of aphids and other hemipteran species. Unexpectedly, we found that the phylloxera does not share aphids' patterns of chromosome evolution. By estimating interchromosomal rearrangement rates on an absolute time scale, we found that rates are elevated for aphid autosomes compared with their X chromosomes, but this pattern does not extend to the phylloxera branch. Potentially, the conservation of X chromosome gene content is due to selection on XO males that appear in the sexual generation. We also examined gene duplication patterns across Hemiptera and uncovered horizontal gene transfer events contributing to phylloxera evolution.

Contrasting Evolutionary Patterns Between Sexual and Asexual Lineages in a Genomic Region Linked to Reproductive Mode Variation in the pea aphid.

Although asexual lineages evolved from sexual lineages in many different taxa, the genetics of sex loss remains poorly understood. We addressed this issue in the pea aphid Acyrthosiphon pisum, whose natural populations encompass lineages performing cyclical parthenogenesis (CP) and producing one sexual generation per year, as well as obligate parthenogenetic (OP) lineages that can no longer produce sexual females but can still produce males. An SNP-based, whole-genome scan of CP and OP populations sequenced in pools (103 individuals from 6 populations) revealed that an X-linked region is associated with the variation in reproductive mode. This 840-kb region is highly divergent between CP and OP populations (FST = 34.9%), with >2,000 SNPs or short Indels showing a high degree of association with the phenotypic trait. In OP populations specifically, this region also shows reduced diversity and Tajima's D, consistent with the OP phenotype being a derived trait in aphids. Interestingly, the low genetic differentiation between CP and OP populations at the rest of the genome (FST = 2.5%) suggests gene flow between them. Males from OP lineages thus likely transmit their op allele to new genomic backgrounds. These genetic exchanges, combined with the selection of the OP and CP reproductive modes under different climates, probably contribute to the long-term persistence of the cp and op alleles.

The experimental life cycle closure of Andricus truncicolus (Giraud, 1859) (Hymenoptera: Cynipidae: Cynipini) and taxonomic description of its sexual generation.

The life cycle of Andricus truncicolus (Giraud, 1869), a species previously known only from its asexual generation, is closed. Laboratory rearing and field sampling have revealed the sexual generation of this species, which develops in a hypertrophic degeneration of the terminal or lateral shoot buds appearing as a muddled agglomerate of leaves on Quercus, section Cerris oaks. We describe for the first time the newly discovered sexual generation of A. truncicolus and provide illustrations and diagnosis for adults and galls of both generations, further observations on biology and information on the species distribution.

Secondary Rhinaria Contribute to Major Sexual Dimorphism of Antennae in the Aphid Semiaphis heraclei (Takahashi)

Sexual generation is an important generation in the life cycle of host-alternating aphids, and its population size determines the intensity of the peak in the next spring. Although male trapping techniques based on olfactory stimuli have been successfully established in the field, the biological basis of olfactory perception in males is unclear. In this study, we compared the morphology of antennae and the types, sizes, numbers, and distribution of sensilla between males and sexual females in the host-alternating aphid Semiaphis heraclei (Hemiptera: Aphididae). We found that flagellum length differentiation contributed to the majority of the sexual dimorphism of antennae. Most sensillum types or subtypes, including trichoid sensilla subtype I, campaniform sensilla, and primary rhinaria subtypes I and II, were enlarged in males. In addition, males bore more trichoid sensilla subtype I than sexual females. In particular, secondary rhinaria were present in males only and could not be detected in sexual females. These results revealed the structural basis of male olfactory perception. Our findings provide insight into the mechanism underlying chemical communication between sexual aphids and could thus be useful for pest control.

Biodiversity of rolB/C-like Natural Transgene in the Genus Vaccinium L. and Its Application for Phylogenetic Studies.

A variety of plant species found in nature contain agrobacterial T-DNAs in their genomes which they transmit in a series of sexual generations. Such T-DNAs are called cellular T-DNAs (cT-DNAs). cT-DNAs have been discovered in dozens of plant genera, and are suggested to be used in phylogenetic studies, since they are well-defined and unrelated to other plant sequences. Their integration into a particular chromosomal site indicates a founder event and a clear start of a new clade. cT-DNA inserts do not disseminate in the genome after insertion. They can be large and old enough to generate a range of variants, thereby allowing the construction of detailed trees. Unusual cT-DNAs (containing the rolB/C-like gene) were found in our previous study in the genome data of two Vaccinium L. species. Here, we present a deeper study of these sequences in Vaccinium L. Molecular-genetic and bioinformatics methods were applied for sequencing, assembly, and analysis of the rolB/C-like gene. The rolB/C-like gene was discovered in 26 new Vaccinium species and Agapetes serpens (Wight) Sleumer. Most samples were found to contain full-size genes. It allowed us to develop approaches for the phasing of cT-DNA alleles and reconstruct a Vaccinium phylogenetic relationship. Intra- and interspecific polymorphism found in cT-DNA makes it possible to use it for phylogenetic and phylogeographic studies of the Vaccinium genus.

Molecular genetic and bioinformatic approaches for the allele reconstruction of the <i>rolB/C</i>-like gene in representatives of the genus <i>Vaccinium</i> L.

Agrobacterium mediated transformation is one of the most studied examples of horizontal gene transfer between pro- and eukaryotes. During this process a part of the Ti-plasmid T-DNA is transferred into the plant cell genome. These sequences could be preserved in the genomes during evolution and inherited in a series of sexual generations. Such plants are described within the genus Vaccinium L. [1]. Our research team is currently analyzing natural transgenes in V. oxycoccos L., V. japonicum Miq., V. conchophyllum Rehder, V. emarginatum Hayata, V. myrtilloides Michx., V. virgatum Ait., V. corymbosum L., V. darrowii Camp, V. smallii A. Gray, V. praestans Lamb., V. ovalifolium Sm., V. myrtillus L., V. uliginosum L., V. vitis-idaea L. Previously, analyzing the natural transgenes in another genus (Camellia L.) [2], we showed the importance of reconstructing the allelic states of transgenes for phylogenetic studies. In this paper, we present a comprehensive approach for studying the allelic state of the rolB/C-like gene in plants of the genus Vaccinium. It combines molecular genetic and bioinformatic research methods. Molecular genetic methods involve Sanger sequencing of a gene sequence in a large number of samples, while for each sample the sequence is presented as a set of polymorphic positions in binary form. Allele resolution occurs based on the description of alleles in homozygotes and a series of subtractions of known alleles in heterozygous samples. The second method involves the analysis of SRA (Sequence Read Archive) sequences available in the databases. SRA is a repository of high-throughput sequencing raw data. Based on our work, we can conclude that both of these approaches make it possible to describe the allelic state of the rolB/C-like gene in representatives of the genus Vaccinium. The work was performed using the equipment of the Resource Center of Saint Petersburg State University Development of Molecular and Cellular Technologies with the support of the Ministry of Science and Higher Education of the Russian Federation in accordance with agreement No. 075-15-2022-322 dated 04/22/2022 on the provision of a grant in the form of a subsidy from the Federal budget of the Russian Federation. The grant was provided as part of the state support for the creation and development of the world-class Scientific Center Agrotechnologies for the Future.

High level of somatic mutations detected in a diploid banana wild relative Musa basjoo.

Plants are thought to lack an early segregating germline and often retain both asexual and sexual reproduction, both of which may allow somatic mutations to enter the gametes or clonal progeny, and thereby impact plant evolution. It is yet unclear how often these somatic mutations occur during plant development and what proportion is transmitted to their sexual or cloned offspring. Asexual "seedless" propagation has contributed greatly to the breeding in many fruit crops, such as citrus, grapes and bananas. Whether plants in these lineages experience substantial somatic mutation accumulation is unknown. To estimate the somatic mutation accumulation and inheritance among a clonal population of plant, here we assess somatic mutation accumulation in Musa basjoo, a diploid banana wild relative, using 30 whole-genome resequenced samples collected from five structures, including leaves, sheaths, panicle, roots and underground rhizome connecting three clonal individuals. We observed 18.5 high proportion de novo somatic mutations on average between each two adjacent clonal suckers, equivalent to ~ 2.48 × 10-8 per site per asexual generation, higher than the per site per sexual generation rates (< 1 × 10-8) reported in Arabidopsis and peach. Interestingly, most of these inter-ramet somatic mutations were shared simultaneously in different tissues of the same individual with a high level of variant allele fractions, suggesting that these somatic mutations arise early in ramet development and that each individual may develop only from a few apical stem cells. These results thus suggest substantial mutation accumulation in a wild relative of banana. Our work reveals the significance of somatic mutation in Musa basjoo genetics variations and contribute to the trait improvement breeding of bananas and other asexual clonal crops.

On the Mechanistic Basis of Killer Meiotic Drive in Fungi.

Spore killers are specific genetic elements in fungi that kill sexual spores that do not contain them. A range of studies in the last few years have provided the long-awaited first insights into the molecular mechanistic aspects of spore killing in different fungal models, including both yeast-forming and filamentous Ascomycota. Here we describe these recent advances, focusing on the wtf system in the fission yeast Schizosaccharomyces pombe; the Sk spore killers of Neurospora species; and two spore-killer systems in Podospora anserina, Spok and [Het-s]. The spore killers appear thus far mechanistically unrelated. They can involve large genomic rearrangements but most often rely on the action of just a single gene. Data gathered so far show that the protein domains involved in the killing and resistance processes differ among the systems and are not homologous. The emerging picture sketched by these studies is thus one of great mechanistic and evolutionary diversity of elements that cheat during meiosis and are thereby preferentially inherited over sexual generations.

Pediobius Rotundatus (Fonscolombe, 1832) (Hymenoptera: Chalcidoidea: Eulophidae) New to Britain with First Reports of Native Parasitoids Reared from Plagiotrochus Quercusilicis (Fabricius, 1798) (Hymenoptera: Cynipidae) in England

Pediobius rotundatus (Fonscolombe) (Eulophidae) is reported for the first time from Britain, reared from sexual generation galls of Plagiotrochus quercusilicis (Fabricius) (Cynipidae) on Quercus ilex L ., collected in the counties of Suffolk and Kent, England. Additional first British host records of three species of Pteromalidae and one of Torymidae, from galls of P. quercusilicis, are also presented.

Pairing of sexual and asexual generations of Nearctic oak gallwasps, with new synonyms and new species names (Hymenoptera: Cynipidae, Cynipini).

Most oak gallwasps (Hymenoptera; Cynipidae, Cynipini) have lifecycles involving obligate alternation between a sexual and an asexual generation. Many species are currently known from only one of these generations, with the alternate generation either unknown or separately described with a different name. Here we describe previously unknown generations for 12 Nearctic oak cynipids. We describe the sexual generation for ten species previously known only from an asexual generation: Acraspis quercushirta (Bassett, 1864), Acraspis villosa Gillette, 1888, Amphibolips spinosa Ashmead, 1887, Andricus balanaspis (Weld, 1922), comb. nov., Antron quercusnubila (Bassett, 1881), Bassettia pallida Ashmead, 1896, Neuroterus floccosus (Bassett, 1881), comb. rev., Philonix fulvicollis Fitch, 1859, Phylloteras poculum (Osten Sacken, 1862) and Sphaeroteras carolina (Ashmead, 1887), comb. rev.. We describe the asexual generation of Bassettia flavipes (Gillette, 1889), comb. nov. and Melikaiella tumifica (Osten Sacken, 1865), both previously known only from a sexual generation. Three described taxa (Neuroterus vernus Gillette, 1889, syn. nov., Neuroterus papillosus Beutenmueller, 1910, syn. nov. and Neuroterus perminimus Bassett, 1900, syn. nov.) are all identified as the sexual generation of Neuroterus niger Gillette, 1888. We also comment on the presence of previously unknown non-functional males reared from galls of the asexual generation of Neuroterus niger. We identify two further synonyms: Acraspis macrocarpae Bassett, 1890 is syn. nov. of A. quercushirta (Bassett, 1864), and Bassettia archboldi Melika Abrahamson, 2007 is syn. nov. of Bassettia pallida Ashmead, 1896. In addition, we formally synonymise multiple varieties described by Kinsey within his Acraspis quercushirta, A. villosa, Antron quercusnubila, Neuroterus niger and Philonix fulvicollis species complexes. We provide descriptions and diagnoses for these taxa, and summarise what is known of their biology, including host associations. All taxa and relationships between alternate generations are supported by morphological and molecular data.

Re-establishment of the Nearctic oak cynipid gall wasp genus Druon Kinsey, 1937 (Hymenoptera: Cynipidae: Cynipini), with description of five new species.

The Nearctic cynipid oak gall wasp genus Druon Kinsey comb. rev. is re-established, with 5 new species and 10 species previously placed in the genus Andricus Hartig 1840: D. alexandri Melika, Nicholls Stone, sp. nov., D. flocculentum (Lyon), comb. nov., D. fullawayi (Beutenmller), comb. nov., D. garciamartinonae Pujade-Villar, sp. nov., D. gregori Melika, Nicholls Stone, sp. nov., D. hansoni Cuesta-Porta, Melika Pujade-Villar, sp. nov., D. ignotum (Bassett), comb. nov., D. linaria Kinsey, comb. rev., D. pattoni (Bassett), comb. nov., D. protagion Kinsey, comb. rev. (D. malinum Kinsey, syn. nov., D. polymorphae Kinsey, syn. nov.), D. quercusflocci (Walsh), comb. nov., D. quercuslanigerum (Ashmead), comb. nov., D. receptum Kinsey, comb. rev., D. rusticum Kinsey, comb. rev. and D. serretae Pujade-Villar, Cuesta-Porta Melika, sp. nov.. All species are known only from their asexual generation except for D. ignotum and D. quercuslanigerum, for which alternating asexual and sexual generations are known. The sexual generation of D. ignotum and a new morphological variety of D. quercuslanigerum from Mexico are both described for the first time. We provide descriptions, redescriptions, diagnoses, keys to species, DNA sequence data and analyses, and information on biology, phenology, and distribution. Additionally, four Andricus species with woolly galls and striato-reticulated metasomas are transferred to Striatoandricus Pujade-Villar: S. furnessulus, comb. nov., S. furnessae, comb. nov., S. guatemalensis, comb. nov., and S. tenuicornis, comb. nov.

DNA Methylation Can Mediate Local Adaptation and Response to Climate Change in the Clonal Plant Fragaria vesca: Evidence From a European-Scale Reciprocal Transplant Experiment.

The ongoing climate crisis represents a growing threat for plants and other organisms. However, how and if plants will be able to adapt to future environmental conditions is still debated. One of the most powerful mechanisms allowing plants to tackle the changing climate is phenotypic plasticity, which can be regulated by epigenetic mechanisms. Environmentally induced epigenetic variation mediating phenotypic plasticity might be heritable across (a)sexual generations, thus potentially enabling rapid adaptation to climate change. Here, we assessed whether epigenetic mechanisms, DNA methylation in particular, enable for local adaptation and response to increased and/or decreased temperature of natural populations of a clonal plant, Fragaria vesca (wild strawberry). We collected ramets from three populations along a temperature gradient in each of three countries covering the southern (Italy), central (Czechia), and northern (Norway) edges of the native European range of F. vesca. After clonal propagation and alteration of DNA methylation status of half of the plants via 5-azacytidine, we reciprocally transplanted clones to their home locality and to the other two climatically distinct localities within the country of their origin. At the end of the growing season, we recorded survival and aboveground biomass as fitness estimates. We found evidence for local adaptation in intermediate and cold populations in Italy and maladaptation of plants of the warmest populations in all countries. Plants treated with 5-azacytidine showed either better or worse performance in their local conditions than untreated plants. Application of 5-azacytidine also affected plant response to changed climatic conditions when transplanted to the colder or warmer locality than was their origin, and the response was, however, country-specific. We conclude that the increasing temperature will probably be the limiting factor determining F. vesca survival and distribution. DNA methylation may contribute to local adaptation and response to climatic change in natural ecosystems; however, its role may depend on the specific environmental conditions. Since adaptation mediated by epigenetic variation may occur faster than via natural selection on genetic variants, epigenetic adaptation might to some degree help plants in keeping up with the ongoing environmental crisis.

Techniques for locating and analyzing subterranean Lycopodium and Diphasiastrum gametophytes in the field.

Homosporous club mosses have an archaic life cycle, alternating two locationally, nutritionally, and physiologically independent generations. The sexual generation of club mosses—the gametophytes (or prothallia)—are among the least researched botanical subjects. The gametophytes are responsible for not only sexual reproduction, but also the determination of recruitment of the new sporophyte generation, species habitat selection, migration, and evolution. Researchers often fail to find juvenile club moss populations and thus do not discover subterranean long‐lived achlorophyllous gametophytes. To date, the gametophytes of most club moss species remain undiscovered in nature and are not scientifically documented. Almost all researchers who have previously located subterranean club moss gametophytes declared that their first find was due to luck and that subsequently the researcher's intuition plays the most important role; however, intuition and good luck are not scientific methods. In our review, we combine our knowledge with data available in the literature and discuss the following questions using a methodical approach: (1) How can we locate a subterranean club moss gametophyte population? (2) How can we extract the gametophytes? and (3) What new knowledge about club moss population development can be gained by analyzing juvenile club moss populations?

Andricus cydoniae Giraud, 1859 Junior Synonym of Cynips conifica Hartig, 1843, as Experimentally Demonstrated (Hymenoptera: Cynipidae: Cynipini).

Simple SummaryPhytophagous members of the family Cynipidae induce a spectacular diversity of plant galls that are often complex in structure. Knowledge of the biology, life cycle, and life history of known cynipid species is largely fragmentary; gall wasps can exhibit an alternation of generations known as heterogony in which an all-female alternates with a bisexual generation. The unisexual generation produces eggs parthenogenetically, and these are usually inserted into a specific plant part. At the site of oviposition, galls are induced, within which a bisexual generation develops and later emerges. Emergent males and females mate, and females in turn induce galls from which the unisexual generation emerges. Females of the two generations may be morphologically dissimilar and may induce galls that differ greatly morphologically. Differences in the morphology of both wasps and galls between generations of the same species, coupled with incomplete knowledge of life cycles, have led to considerable taxonomic confusion. Alternating generations of numerous species have been described as separate species or even genera. Here, we demonstrate experimentally that two cynipid species, Cynips conifica (presently Andricus conificus) and Andricus cydoniae, which are morphologically different and produce very different galls on different host oaks, represent alternate generations of a single species.We demonstrated the life cycle closure of Cynips conifica Hartig, 1843 (presently Andricus conificus), previously supposed on the basis of molecular data, and the identity of the sexual generation, through laboratory experiments. As a consequence, Andricus cydoniae Giraud, 1859 became a junior synonym of A. conificus (Hartig, 1843). We provide illustrations and a diagnosis for adults and galls, observations on biology, and information on distribution. Moreover, as sexual galls of A. conificus cannot be distinguished from those of Andricus multiplicatus, a detailed comparison between sexual galls and adults of these two species is reported.