Marta Novo

In the Iberian Peninsula, intensifying summer aridity poses a significant threat to soil fauna. Earthworms, as key ecosystem regulators, often rely on aestivation—a dormancy state involving metabolic depression—to survive these hostile conditions. However, little is known about this strategy in riparian species like Allolobophora molleri , whose reliance on waterlogged soils and its narrow distribution make it particularly vulnerable to climate change compared to cosmopolitan species like Aporrectodea trapezoides . Here, we investigate the aestivation response of A. molleri in a comparative framework with A. trapezoides , identifying the specific temperature and moisture thresholds that trigger dormancy. We further assessed the associated physiological costs through changes in weight, tissue water content and lipid peroxidation to understand their resilience to abiotic stress. Our results highlight soil moisture as the primary driver of aestivation, though temperature also acted as a constraint for A. molleri . Both species suffered significant biomass loss, however, lipid peroxidation levels remained stable, indicating no measurable oxidative damage. Aestivating individuals of A. molleri retained slightly higher tissue water content compared to active individuals. Morphological changes, such as a differentiated region in the posterior segments, were observed in aestivating individuals of both species. Finally, moisture restoration triggered a rapid recovery of activity, confirming the facultative nature of this dormancy. These findings show the different ecology of both species and provide key insights into their resilience under future climate change scenarios.

Earthworms thriving in naturally occurring geothermal soils offer rare insight into rapid adaptation to environmental extremes. Here, we show that the pantropical earthworm Amynthas gracilis survives and flourishes in soils of the Furnas Volcano (São Miguel Island, Azores), where conditions include elevated temperatures (up to 40 °C), high CO ₂ (88.6%), low O ₂ (10%), toxic metals, and mildly acidic pH. In a reciprocal-transplant, mesocosm-based experiment between soils overlying areas of active degassing volcanic gassing (hereafter active degassing soils) and reference soils, convergence of the epidermal thickness of the transplanted earthworms to the resident-soil phenotype (24 ± 3.9 µm active degassing soil, 43.8 ± 8 µm reference soil), was observed within 31 days. Combining RNA-Seq, DNA (5-cytosine) methylation mapping, and microRNA profiling, this phenotypic change results from coordinated transcriptional and epigenetic reprogramming. While gene-body methylation occurred at ∼98 % of loci, levels varied, and differentially methylated regions were enriched ffor genes with altered expression under volcanic stress. Multi-omics network analysis identified epithelial morphogenesis, circulatory system formation, and neural development as regulatory hubs, highlighted by a set of 41 epithelial-morphogenesis genes showing consistent methylation and miRNA patterns. Additional modules governing ion transport and signal transduction complemented the adaptive response. Collectively these findings demonstrate that A. gracilis employs dynamic DNA methylation and microRNA regulation alongside transcriptional reprogramming to generate a persistent phenotypic adjustment to a volcanic stress. This work advances our understanding of extremophile resilience and provides a scalable model for predicting organismal adaptive capacity in the face of environmental extremes.

Approximately, 40% of earthworm species can reproduce by parthenogenesis. This is the case for the cosmopolitan species, Aporrectodea trapezoides, although sexual forms have been described sporadically. We analyse the genotypes and microbiomes of 30 individuals from four localities where both forms appear in order to understand the evolutionary mechanisms related to parthenogenesis. In all sites, heterozygosity values were approximately 30% higher in parthenogenetic individuals. However, we detected a stronger genomic structuring due to reproduction than to the geographical setting only in the Algerian population, underpinned by 195 loci that were related to gametogenesis, symbiont‐like processes, and nitrate reduction. Similarly, statistical differences in the abundance of ZOTUs were only found between the Algerian sexual and parthenogenetic earthworms, with 754 ZOTUs that included the genus Romboutsia, which is involved in the production of nitric oxide, which enhances sperm motility. In summary, significant genomic and microbiome differences were found only between sexual and parthenogenetic lineages in a single locality. We hypothesise that obligate parthenogenesis evolved early, leaving traces at the genomic and microbiome levels in the Algerian parthenogens that were the earliest splitting lineage. Such obligate parthenogenesis was lost secondarily and individuals in the Iberian sites were facultative parthenogens, with the potential to copulate and therefore erase the genomic and microbial traces of obligate parthenogenesis. Our results indicate a hybrid origin of parthenogenesis in A. trapezoides and shed light on the complex interplay between genomic, microbiome, and reproductive mechanisms in A. trapezoides.

The genomic basis of cladogenesis and adaptive evolutionary change has intrigued biologists for decades. Here we show that the tectonics of genome evolution in clitellates, a clade composed of most freshwater and all terrestrial species of the phylum Annelida, is characterized by extensive genome-wide scrambling that resulted in a massive loss of macrosynteny between marine annelids and clitellates. These massive rearrangements included the formation of putative neocentromeres with newly acquired transposable elements and preceded a further period of genome-wide reshaping events, potentially triggered by the loss of genes involved in genome stability and homoeostasis of cell division. Notably, whereas these rearrangements broke short-range interactions observed between Hox genes in marine annelids, they were reformed as long-range interactions in clitellates. Our findings reveal extensive genomic reshaping in clitellates at both the linear (2D) and three-dimensional (3D) levels, suggesting that unlike in other animal lineages where synteny conservation constrains structural evolution, clitellates exhibit a remarkable tolerance for chromosomal rearrangements. Our study thus suggests that the genomic landscape of Clitellata resulted from a rare burst of genomic changes that ended a long period of stability that persists across large phylogenetic distances.

Although some earthworms can survive in permafrost and tolerate cold conditions, it is generally believed that most earthworms were eradicated from northern latitudes during the Last Glacial Maximum. The main goal of this study is to test this hypothesis using a cosmopolitan earthworm as model, Eiseniella tetraedra. We collected 1640 specimens in 294 localities from 19 different countries. We examined three molecular markers (COI, 16S and 28S) and their morphology. Furthermore, we investigated their potential niches through Species Distribution Models. Eleven genetic lineages were found, nested in five clades. Clade I was more prevalent in cold biogeographical regions such as the Continental, the Atlantic or even the Arctic, while clade II was prevalent in Mediterranean regions. Potential niches agreed with the distribution trends. The presence of restricted clades in the Iberian and Scandinavian peninsulas, as well as in Eastern Europe, suggests that these three regions served as refugia during the Last Glacial Maximum. Thus, nunatak and tabula rasa hypotheses were necessary to explain the actual distribution of this riparian earthworm.

The genomic basis of cladogenesis and adaptive evolutionary change has intrigued biologists for decades. The unique insights from a genome-level perspective have revealed a striking pattern of conserved macrosynteny across huge evolutionary distances in animals, yet progress in many lineages has been hampered by the absence of genome-level data. Here, we show that the origin of Clitellata, a clade composed of most freshwater and all terrestrial species of the phylum Annelida, coincided with an unprecedented genome-wide scrambling event that resulted in massive loss of macrosynteny between marine annelids and clitellates. This complete breakdown of chromosome-level relationships on just one short branch is unlike any other known pattern of evolutionary genome rearrangement, and instead resembles chromosome shattering and reassembly seen in chromoanagenesis. These massive punctuated rearrangements included the formation of neocentromeres with newly-acquired transposable elements, and preceded a further period of genome-wide reshaping events including whole genome duplications and massive macrosyntenic reshuffling between clitellate lineages, potentially triggered by the loss of genes involved in genome stability or homeostasis of cell division. Notably, while these rearrangements broke short-range interactions observed between genes in marine annelids (including Hox), they were reformed as long-range interactions in clitellates. Our study provides evidence of punctuated genomic change via chromoanagenesis leading not only to the origin of a new animal lineage - a ‘hopeful monster’ - but also to adaptive genomic changes facilitating the colonisation of new habitats.

Corsica and Sardinia are amongst the largest islands of the Western Mediterranean. Their complex geological history included belonging to the European–Iberian continental margin (close to current-day Catalonia and Provence) and varying degrees of isolation for the last 30 million years, leading to peculiar, highly endemic faunas and floras. This is especially true for their earthworm faunas, which include endemic species of several Lumbricoidea genera and the endemic family (or subfamily) Diporodrilidae. Only three species have been described for the morphologically unique Diporodrilus, but there exists evidence for wide morphological variability within them and the existence of several species-level genetic lineages within Corsica. This work aimed to investigate the genetic diversity and phylogenetic relationships between the genetic lineages of Diporodrilus from Corsica and Sardinia (based on the sequences of 5 mito-nuclear markers), to perform an integrative systematics revision combining species delimitation techniques and morphological data, and to obtain a time-calibrated phylogeny of Diporodrilus and other Corso-Sardinian Lumbricoidea. Within 15 populations of the morphospecies Diporodrilus omodeoi and Diporodrilus pilosus across Corsica and Sardinia, 10 species-level genetic lineages were detected. Phylogenetic independence, high genetic divergence and morphological differences provided the support for the description of five new pseudocryptic species: Diporodrilus rotundus sp. nov., Di. jorgei sp. nov., Di. minor sp. nov., Di. meridionalis sp. nov. and Di. telti sp. nov. Time-calibrated phylogenetic inference estimated the age for genus Diporodrilus at 65.9 Mya; even if other Corso-Sardinian genera were significantly younger, all of them presented deep divergences predating the break-off of the microplate from the continent. The almost threefold increase in the known diversity of Diporodrilus stresses the need for comprehensive earthworm diversity inventories in both Sardinia and Corsica, and for studies on their ecological role and conservation status. The close correspondence between some geological and cladogenetic events suggest that the distribution of Corso-Sardinian earthworms could be used to inform standing geological controversies.

Earthworms have a central role in ministering the terrestrial ecosystems. Aestivation is a form of dormancy employed by the organisms living in deserts and arid environments, when confronted with prolonged periods of drought. As a survival tactic, aestivation has been studied in sea cucumbers, land snails and frogs, however little is known about aestivating earthworms. To understand the global metabolic adjustments required for withstanding the harsh conditions of the ever more severe Iberian drought, we performed a global transcriptomic exploration of the endogeic earthworm Carpetania matritensis during aestivation. There were a total of 6,352 differentially expressed transcripts in the aestivating group, with 65% being downregulated. Based on GO and KEGG enrichment analyses, downregulated genes seem to indicate an overall metabolic depression during aestivation. We noted a reduction of protein turnover and macromolecule metabolism coupled with suppression of digestion. Upregulated genes, namely antioxidant genes and DNA repair genes showed signs of abiotic stress caused by ROS generation. Abiotic stress led to transcriptomic changes of genes involved in immune response, mostly affecting the NF-κB signalling pathway and changes in apoptotic genes, indicating the necessity of investigating these processes in a tissue specific manner. Lastly we uncovered a possible mechanism for water retention by nitrogenous waste accumulation. This study provides the first ever transcriptomic investigation done on earthworms in condition of prolonged drought and as such serves as a general framework for investigation on other earthworm species and other soil invertebrates, which is becoming increasingly important with the current scenario of climate change.

Hormogastrid earthworms are found in the diversity hotspot of the Franco-Iberian domain, together with the better-known family Lumbricidae. Integrative systematics (the combination of morphological, molecular and ecological data) have increased our knowledge of the diversity and evolutionary history of these earthworms, highlighting unresolved taxonomic conflicts. One example of a species group in need of integrative taxonomic revision is the genus Boucheona in France. In this work, we analyzed their diversity using previously published data together with additional data obtained from recently sampled localities. Molecular data including DNA barcodes and additional markers enabled us to reconstruct Bayesian and time-calibrated phylogenies to discuss the evolutionary relationships among the different taxa, and to propose hypotheses regarding their biogeographical history. Based on our results, four species of Boucheona are present in Southern France, including two new taxa. Morphological distinctness and molecular phylogenetics results supported the status of four populations as the newly described Boucheona corbierensis sp. nov., as well as the status of “Hormogaster pretiosa var. nigra” as an independent species, redescribed as Boucheona tenebrae sp. nov. These results provide a new perspective of the importance of the genus Boucheona in southern France, as the possible evolutionary origin of a clade of giant anecic earthworms with unknown (but probably remarkable) impact on ecosystem functioning across their range.