Abstract Background Xenarthrans, comprising sloths, anteaters, and armadillos, represent one of the most morphologically and physiologically specialized mammalian clades, yet the genomic basis of their adaptations remains poorly understood. Here, we present chromosome-level genomes for the two-toed sloth (Choloepus didactylus) and the southern anteater (Tamandua tetradactyla) and investigate how retrotransposon-mediated gene duplications (retrocopies) have shaped genome evolution in these and other species in Xenarthra. Results Comparative analyses revealed that the xenarthran genomes analyzed here harbour the highest number of retrocopies reported among mammals, with lineage-specific insertion dynamics. Anteater and armadillo genomes contain older LINE1 repertoires and species-specific older retrocopy insertions. In contrast, sloths retain both an abundance of young LINE1s and thousands of young retrocopies, alongside a large shared set that originated from an evolutionary burst of retroduplication in the branch leading to their last common ancestor (~ 30 Mya). In C. didactylus, 49% of retrocopies were found to be expressed in five tissues, compared with 27% in Dasypus novemcinctus in three tissues. Evolutionary analyses identified 38 retrocopies with strong hallmarks of domestication in C. didactylus. Many of these retrocopies derive from parental genes involved in mitochondrial and metabolic processes, suggesting a potential genomic contribution to the physiological specializations of sloths. Conclusions Altogether, our findings identify retrotransposition as a major contributor to the genomic architecture of the xenarthrans presented here and highlight retrocopy origination as a mechanism for generating lineage-specific novelty and, possibly, distinctive biological specializations.

The Gambusia holbrooki (eastern mosquitofish) reference genome will offer a crucial resource for understanding the evolution and adaptation of invasive freshwater fish species. The genome of G. holbrooki was assembled into two haplotypes through a phased assembly approach; however, only the primary haplotype was designated as the reference genome for annotation and downstream analyses. The entirety of the genome sequence was assembled into 24 contiguous chromosomal pseudomolecules and 1 mitochondrial genome. This chromosome-level assembly encompasses 0.67 Gb, composed of 421 contigs and 318 scaffolds, with contig and scaffold N50 values of 15.9 Mb and 29.6 Mb, respectively.

Hirudo verbana Carena, 1820, commonly known as the southern medicinal leech, is one of several European medicinal leeches, whose full diversity has just recently started to be uncovered. Historically, it has been widely used as a medicinal leech and for centuries it was treated erroneously under the specific name of Hirudo medicinalis L. 1758. Recent molecular and taxonomic analyses have revealed subspecific diversity within the morphospecies H. verbana. Hirudo verbana is a blood-feeding species sucking blood from amphibians, fish, and mammals. It occupies freshwater habitats, typically shallow ponds and lakes. Studies show that this leech species has a "naturally limited microbiome", suggesting it may serve as a powerful model system for the study of gut microbiota. We expect this chromosome-level assembly of H. verbana to serve as a high-quality genomic resource for this most famous leech genus and to serve as a foundation to the study of the diversification and biodiversity of European medicinal leeches, as well as their gut-associated symbionts. The genome of H. verbana was assembled into two haplotypes through a phased assembly approach; however, only the primary haplotype was designated as the reference genome for annotation and downstream analyses. The entirety of the primary haplotype was assembled into 14 contiguous chromosomal pseudomolecules, including the mitogenome. This chromosome-level assembly encompasses 0.18 Gb, composed of 277 contigs and 27 scaffolds, with contig and scaffold N50 values of 1.3 Mb and 13.4 Mb, respectively.

Abstract Tropical rainforests, and Amazonia in particular, contain more tree species than anywhere else, most of which arose through rapid evolutionary radiations 1–3 . Rapid radiations are often catalysed by ecological opportunity 4–6 , which in rainforest trees is presented by intense insect herbivore pressure, spurring the evolution of novel plant defence chemistry to escape it 7 . However, we do not understand how long-lived trees can adapt quickly enough to keep pace with rapidly-evolving insect herbivores. Here we show that hybridisation in rainforest trees, which was considered rare, allows exchange of gene clusters used in chemical defence against herbivore attack, facilitating rapid adaptation and diversification. Using genome sequencing for 461 individuals from the genus Inga , a characteristic Amazonian tree radiation, we find that regional tree communities form syngameons - networks of closely related, co-occurring species connected by gene flow. Integrating these genomes with herbivore abundance data from the same communities across the tropical Americas, we show that herbivore compositional turnover coincides with local, recurrent interspecific transfer of defence gene clusters that are retained by balancing selection, consistent with fluctuating selective pressure imposed by shifting herbivore communities. Together, our results demonstrate that hybridisation allows long-lived tropical trees to rapidly evolve chemical defences, fuelling adaptation to the relentless insect herbivory that structures the world’s most species-rich forests.

Hirudo verbana Carena, 1820, commonly known as the southern medicinal leech, is one of several European medicinal leeches, whose full diversity has just recently started to be uncovered. Historically, it has been widely used as a medicinal leech and for centuries it was treated erroneously under the specific name of Hirudo medicinalis L. 1758. Recent molecular and taxonomic analyses have revealed subspecific diversity within the morphospecies H. verbana. Hirudo verbana is a blood-feeding species sucking blood from amphibians, fish, and mammals. It occupies freshwater habitats, typically shallow ponds and lakes. Studies show that this leech species has a "naturally limited microbiome", suggesting it may serve as a powerful model system for the study of gut microbiota. We expect this chromosome-level assembly of H. verbana to serve as a high-quality genomic resource for this most famous leech genus and to serve as a foundation to the study of the diversification and biodiversity of European medicinal leeches, as well as their gut-associated symbionts. The genome of H. verbana was assembled into two haplotypes through a phased assembly approach; however, only the primary haplotype was designated as the reference genome for annotation and downstream analyses. The entirety of the primary haplotype was assembled into 14 contiguous chromosomal pseudomolecules, including the mitogenome. This chromosome-level assembly encompasses 0.18 Gb, composed of 277 contigs and 27 scaffolds, with contig and scaffold N50 values of 1.3 Mb and 13.4 Mb, respectively.

The Gambusia holbrooki (eastern mosquitofish) reference genome will offer a crucial resource for understanding the evolution and adaptation of invasive freshwater fish species. The genome of G. holbrooki was assembled into two haplotypes through a phased assembly approach; however, only the primary haplotype was designated as the reference genome for annotation and downstream analyses. The entirety of the genome sequence was assembled into 24 contiguous chromosomal pseudomolecules and 1 mitochondrial genome. This chromosome-level assembly encompasses 0.67 Gb, composed of 421 contigs and 318 scaffolds, with contig and scaffold N50 values of 15.9 Mb and 29.6 Mb, respectively.

Understanding the genetic basis of widespread phenotypic convergence, particularly for complex morphological traits, remains a major challenge in evolutionary biology. The Mediterranean gravel beach clingfishes of the genus Gouania provide an excellent system to study this phenomenon. Within this genus, two distinct morphotypes, "slender" and "stout," have repeatedly evolved, adapting to different microhabitats. These morphotypes differ in multiple complex traits, including body elongation, head compression, vertebral number, eye size, and the structure of the adhesive disc. First, to scrutinize phylogenetic convergence, we combined 3D morphometrics of the pelvic girdle and skull, with molecular species delimitation based on >660 DNA barcodes, and a phylogenomic framework based on more than 3,400 single-copy orthologs. Second, by employing whole-genome resequencing and a novel "convergence score" statistic, we examined genomic convergence across multiple levels: nucleotides, sequences, genes, and functional pathways. While we found no evidence of large-scale genomic or protein-level convergence, we identified promising candidate regions at the level of single variants, genes, and biological pathways. Notably, a longer shared (but interrupted) haplotype around the candidate gene adam12 was associated with convergent traits. The lack of simple genomic patterns may reflect the radiation's age and the complex genetic basis of the underlying morphological traits (eg eye size, neurocranium shape). Altogether, our findings highlight the importance of assessing genomic convergence at multiple molecular levels to uncover diagnostic signals across varying evolutionary processes and timescales.

Scleractinia (stony corals) are a diverse taxonomic order within the phylum Cnidaria, comprising more than 1,600 species described to date. Most of these corals play a key role as reef builders by secreting calcium carbonate, forming rigid skeletons that provide the structural foundation of tropical coral reef ecosystems. Through colonial growth and symbioses with their photosynthetic microalgae (Symbiodiniaceae) and a suite of other microorganisms, scleractinian corals support high primary productivity and underpin the vast biodiversity of coral reefs. Corals are under severe pressure from climate change, including ocean warming and acidification, which threaten their survival and, consequently, the persistence of coral reef systems globally. In recent years, significant efforts have been made to increase the number of sequenced genomes from scleractinian corals, thereby providing crucial insights into their biology, evolution, resilience, and vulnerability. However, there are few high-quality reference genomes for Scleractinia, and many available genomes remain unannotated, creating barriers to collaboration and scientific insight. Annotation outcomes also vary depending on the methods and software utilised. To address these issues, we applied a standardised pipeline for generating high-quality gene models to 40 scleractinian genomes, spanning 22 genera across 13 families. These genomes, produced through the Aquatic Symbiosis Genomics (ASG) Project, are publicly available. This curated resource of annotated quality genomes will provide essential molecular tools at a critical time for coral reef conservation.

Whole genome data are invaluable resources for both conservation and adaptation studies, especially for endemic species, providing insights into the evolution of genes involved in genomic adaptation across different environments. We compare the newly generated genomic and transcriptomic data of the Cretan endemic lizard species Podarcis cretensis to other Podarcis species to obtain an overview of gene family evolution and genome structure within the genus. Comparative genomic and transcriptomic analyses were performed using the newly published genome of P. cretensis. A gene set was predicted using RNA-seq data from 36 samples, comprising three tissues (liver, brain, and muscle) from both male and female individuals across three distinct habitats. The main findings revealed that P. cretensis and P. raffonei present the best genome assemblies and the most syntenic among the Podarcis species examined. Moreover, P. cretensis displayed the highest percentage of single-copy genes and the lowest percentage of duplicated genes. These duplicated genes are primarily associated with immune and sensory-related gene families, including chemokines, interleukins, immunoglobulin-like domain proteins, secreted proteins, and vomeronasal type-2 receptors. This study deepens our understanding of chromosome structure, gene expression, and genome evolution in the Podarcis genus, representing the most extensive comparative analysis to date. The newly predicted gene set of the insular endemic species P. cretensis offers initial insights into gene expression related to adaptation across environments and tissues. Comparative genomic analyses further revealed gene families potentially involved in environmental adaptation.

Major depressive disorder (MDD) is a leading cause of disability worldwide. Risk for MDD is heritable, and the genetic structure of founder populations enables investigation of rare susceptibility alleles with large effect. In an extended Old Order Mennonite family cohort, we identified a rare missense variant in GPR156 (c.1599G>T, p.Glu533Asp) associated with a two-fold increase in the relative risk of MDD. GPR156 is an orphan G protein-coupled receptor localized in the medial habenula, a region implicated in mood regulation. Insertion of a human sequence containing c.1599G>T into the murine Gpr156 locus induced medial habenula hyperactivity and abnormal stress-related behaviors. This work reveals a human variant that is associated with depression, implicates GPR156 as a target for mood regulation, and introduces informative murine models for investigating the pathophysiology and treatment of affective disorders.

PURPOSE:An evaluation of the accuracy, safety, and efficiency of the Halcyon ring delivery system (RDS) for stereotactic radiosurgery (SRS) treatment to relatively small (1-3 cm) brain lesions. METHODS:After completing the extensive in-house quality assurance checks including Winston-Lutz test and independent dose verification via MD Anderson IROC SRS head phantom irradiation on Halcyon, fifteen brain SRS patients previously treated with a single dose of 20 Gy on TrueBeam (6MV-FFF) with HyperArc geometry were retrospectively replanned on Halcyon (6MV-FFF). Plan quality metrics including conformity index (CI), gradient index (GI), gradient distance (GD), PTV coverage, gross tumor volume (GTV) dose, heterogeneity index (HI), and doses to organs-at-risk (OAR) including normal brain dose were evaluated. Patient-specific quality assurance (PSQA) and independent dose verification via in-house Monte Carlo (MC) 2nd checks were performed. RESULTS:The Halcyon was able to provide highly conformal brain SRS plans. When compared to TrueBeam, CI, planning target volume (PTV) coverage, GTV dose (mean and minimum), HI, and doses to brainstem, optic pathway, and cochlea were statistically insignificant. Statistically significant increases in GI (3.76 vs. 3.25, p < 0.001), GD (0.56 cm vs. 0.48 cm, p = 0.001), and V12Gy (5.5 cc vs. 4.6 cc, p = 0.014), on average using Halcyon versus TrueBeam was found, albeit clinically acceptable values for the majority of brain SRS cases. Halcyon plans provided statistically insignificant maximum dose to most adjacent OARs, though there was a statistically significant decrease in the maximum dose to the spinal cord (0.1 Gy vs. 0.4 Gy, p = 0.009). Halcyon beam-on time increases by a factor of ∼2 (p < 0.001). However, the faster patient setup on Halcyon results in a comparable estimated overall treatment time for both platforms. Plan deliverability and accuracy was ensured with PSQA (> 95% pass rate for 2%/2 mm clinical gamma criteria) results and MC 2nd check agreement within ± 5.0%. CONCLUSIONS:Halcyon brain SRS plans provided a similar plan quality compared to HyperArc plans, although it demonstrated an inferior intermediate dose fall off thus slightly higher V12Gy. This study suggests that Halcyon provides acceptable treatment for solitary relatively small brain lesions of 1-3 cm in diameter. Treatment of select patients on Halcyon will be started at our clinic and it is recommended that other clinics complete an end-to-end test, validate, and implement Halcyon SRS treatments at their practices, especially community cancer centers to provide high-quality service to an underserved patient cohort.

Xenarthrans, comprising sloths, anteaters, and armadillos, represent one of the most morphologically and physiologically specialised mammalian clades, yet the genomic basis of their adaptations remains poorly understood. Here, we present chromosome-level genomes for the two-toed sloth ( Choloepus didactylus ) and the southern anteater ( Tamandua tetradactyla ), and investigate how retrotransposon-mediated gene duplications (retrocopies) have shaped xenarthran genome evolution. Comparative analyses revealed that xenarthran genomes harbor the highest number of retrocopies reported among mammals, with lineage-specific insertion dynamics. Anteater and armadillo genomes contain older LINE1 repertoires and species-specific older retrocopy insertions. In contrast, sloths retain both an abundance of young LINE1s and thousands of young retrocopies, alongside a large shared set that originated from an evolutionary burst of retroduplication in the branch leading to their last common ancestor (∼30 Mya). In C. didactylus, approximately 50% of retrocopies are expressed, compared with 24% in Dasypus novemcinctus . Evolutionary analyses identified 38 retrocopies with strong hallmarks of domestication in C. didactylus . Many of these retrocopies derive from parental genes involved in mitochondrial and metabolic processes, suggesting a genomic mechanism underlying the physiological specialisations of sloths. Altogether, our findings identify retrotransposition as a major contributor to the genomic architecture of Xenarthra and highlight retrocopy origination as a mechanism for generating lineage-specific novelty and distinctive biological specialisations. ### Competing Interest Statement The authors have declared no competing interest. European Union’s Horizon 2020, Marie Skłodowska-Curie, 750747 Wellcome Trust, https://ror.org/029chgv08, Grant 220540/Z/20/A, Wellcome Sanger Institute Quinquennial Review 2021-2026 São Paulo Research Foundation (FAPESP) awarded to PAFG, HBC, and RLVM, 2018/15579-8

A central question in evolutionary biology is what drives the diversification of lineages. Rapid, recent radiations are ideal systems for this question because they still show key morphological and ecological adaptations associated with speciation. While most research on recent radiations focuses on those occurring in insular environments, less attention has been given to continental radiations with complex species interactions. Here, we study the drivers of continental radiations of Melinaea and Mechanitis butterflies (Nymphalidae: Ithomiini), which have rapidly radiated in the continental Neotropics. They are classical models for Amazonian biogeography and color pattern mimicry and have been proposed as biodiversity indicators. We generated reference genomes for five species of each genus and whole-genome resequencing data of most species and subspecies covering a wide geographic range to assess phylogeographic relationships, hybridization patterns, and chromosomal rearrangements. Our data help resolve the classification of these taxonomically challenging butterflies and reveal very high diversification rates. We find rampant evidence of historical hybridization and putative hybrid species in both radiations, which may have facilitated their rapid diversification by enriching the genetic diversity. Moreover, we identified dozens of chromosomal fusions and fissions between congeneric species that have likely expedited reproductive isolation. We conclude that interactions between geography, hybridization and chromosomal rearrangements have contributed to these rapid radiations in the highly diverse Neotropical region. We hypothesize that rapid radiations may be spurred if repeated periods of geographic isolation are combined with lineage-specific rapid accumulation of incompatibilities, followed by secondary contact with some gene exchange.

The reference genome of Cheirolophus tagananensis, locally known as the Cabezon de Taganana, will provide an exceptional opportunity to establish a new framework to develop comparative genomic tools. These tools will help uncover the genetic basis of rapid plant radiations and microevolutionary adaptation processes of insular species on oceanic islands. This genomic resource will also serve as a resource to facilitate the establishment of better informed in situ and ex-situ conservation strategies for this narrow endemic in the face of potential habitat degradation and support taxonomic studies to better understand genetic diversity at the population, species, and genus levels. A total of 16 contiguous chromosomal pseudomolecules were assembled from the genome sequence. This chromosome-level assembly encompasses 0.62 Gb, composed of 421 contigs and 235 scaffolds, with contig and scaffold N50 values of 4.0 Mb and 36.5 Mb, respectively. ### Competing Interest Statement The authors have declared no competing interest.

A major question in evolutionary biology is what drives the diversification of lineages. Rapid, recent radiations are ideal systems for addressing how new species arise because they may preserve key morphological and ecological adaptations associated with speciation. Melinaea and Mechanitis are two classic examples of rapidly radiating Neotropical butterfly genera of the tribe Ithomiini. They were models for early studies of Amazonian biogeography and colour pattern mimicry and have been proposed as biodiversity indicators. We generated reference genomes for five species of each genus, and whole-genome resequencing data of most species and subspecies covering a wide geographic range to assess phylogeographic relationships, patterns of hybridisation and chromosomal rearrangements. We find rampant evidence of hybridisation within both radiations, which may have facilitated their rapid diversification. Our data also provide evidence for a putative hybrid species that combines traits of both parental species. Moreover, many chromosomal fusions and fissions were identified, even between sister species. Our data also help resolve the classification of these notoriously taxonomically challenging butterflies. We conclude that interactions between geography, hybridisation and chromosomal rearrangements have contributed to these two rapid radiations in the highly diverse Neotropical region. ### Competing Interest Statement The authors have declared no competing interest.

We present genome assembly from individual female An. coustani (African malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae) from Lopé, Gabon. The genome sequence is 270 megabases in span. Most of the assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled for both species. The complete mitochondrial genome was also assembled and is 15.4 kilobases in length.

We present a genome assembly from an individual female Anopheles marshallii (the malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae) from Lopé, Gabon. The genome sequence is 225.7 megabases in span. Most of the assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled. The complete mitochondrial genome was also assembled and is 15.4 kilobases in length.

A genomic database of all Earth's eukaryotic species could contribute to many scientific discoveries; however, only a tiny fraction of species have genomic information available. In 2018, scientists across the world united under the Earth BioGenome Project (EBP), aiming to produce a database of high-quality reference genomes containing all ~1.5 million recognized eukaryotic species. As the European node of the EBP, the European Reference Genome Atlas (ERGA) sought to implement a new decentralised, equitable and inclusive model for producing reference genomes. For this, ERGA launched a Pilot Project establishing the first distributed reference genome production infrastructure and testing it on 98 eukaryotic species from 33 European countries. Here we outline the infrastructure and explore its effectiveness for scaling high-quality reference genome production, whilst considering equity and inclusion. The outcomes and lessons learned provide a solid foundation for ERGA while offering key learnings to other transnational, national genomic resource projects and the EBP.

PurposeTo demonstrate the ease and feasibility that hippocampal sparing whole brain (WB) simultaneous integrated boost (HSWB-SIB) plans can be generated using knowledge-based planning and Eclipse Scripting Application Programming Interface (ESAPI) for three different modalities, HyperArc on TrueBeam (TB-HA), a coplanar beam arrangement on TrueBeam (TB-Co), and the ring-mounted Halcyon LINAC (Hal).MethodsTwelve patients with 2-14 brain metastases were retrospectively replanned for HSWB-SIB using a published HSWB RapidPlan model with modifications for the automated addition of SIB to metastases. Prescribed dose was 30 Gy to the WB planning target volume (PTV) and 50 Gy to the metastases in 10 fractions. Eclipse treatment planning system (v16.1) was used with a 6 MV-FFF beam and Acuros XB dose algorithm.ResultsThe methodology was successfully used for all modalities, generating plans in under 30 min. The plan doses were normalized to the WB PTV D95% receiving 30 Gy. Reporting values in the order of Hal, TB-Co, and TB-HA: The WB PTV received a V48 Gy of 4.58, 3.98, and 4.45 cc with statistically insignificant differences (p = 0.806). The boost PTVs received a D95% of 50.60, 50.43, and 51.13 Gy with statistically significant comparisons between TB-HA and the other two modalities (p = 0.005). The hippocampus maximum dose was 11.81, 11.51, and 11.13 Gy with no statistically significant comparisons (p = 0.105). All other oragns-at-risk (OAR) doses were clinically acceptable. The modalities were evaluated using a dosimetric scorecard, achieving average scores of 84.85%, 86.45%, and 87.39%. End-to-end testing ensured the deliverability of the HSWB-SIB plans for all modalities.ConclusionThe novel modification of the preexisting HSWB RapidPlan model with the automated inclusion of SIB objectives allows for easy, intuitive planning of complex HSWB-SIB treatments. All modalities demonstrated can be used with clinically comparable results. Other institutions are recommended to pursue and validate this HSWB-SIB technique to increase the accessibility of a single-course of high-quality treatment for patients with multiple brain lesions.