Chromosome-Level Reference Genome of the Ponza Grayling (Hipparchia sbordonii), an Italian Endemic and Endangered Butterfly.

Islands are crucial evolutionary hotspots, providing unique opportunities for differentiation of novel biodiversity and long-term segregation of endemic species. Islands are also fragile ecosystems, where biodiversity is more exposed to environmental and anthropogenic pressures than on continents. The Ponza grayling, Hipparchia sbordonii, is an endemic butterfly species that is currently found only in two tiny islands of the Pontine archipelago, off the coast of Italy, occupying an area smaller than 10 km2. It has been classified as Endangered (IUCN) because of the extremely limited area of occurrence, population fragmentation, and the recent demographic decline. Thanks to a combination of different assemblers of long and short genomic reads, bulk transcriptome RNAseq, and synteny analysis with phylogenetically close butterflies, we produced a highly contiguous, chromosome-scale annotated reference genome for the Ponza grayling, including 28 autosomes and the Z sexual chromosomes. The final assembly spanned 388.61 Gb with a contig N50 of 14.5 Mb and a BUSCO completeness score of 98.5%. Synteny analysis using four other butterfly species revealed high collinearity with Hipparchia semele and highlighted 10 intrachromosomal inversions longer than 10 kb, of which two appeared on the lineage leading to H. sbordonii. Our results show that a chromosome-scale reference genome is attainable also when chromatin conformation data may be impractical or present specific technical challenges. The high-quality genomic resource for H. sbordonii opens up new opportunities for the accurate assessment of genetic diversity and genetic load and for the investigations of the genomic novelties characterizing the evolutionary path of this endemic island species.

Ecological factors and parity mode correlate with genome size variation in squamate reptiles.

BACKGROUND: Evidence of correlation between genome size, the nuclear haploid DNA content of a cell, environmental factors and life-history traits have been reported in many animal species. Genome size, however, spans over three orders of magnitude across taxa and such a correlation does not seem to follow a universal pattern. In squamate reptiles, the second most species-rich order of vertebrates, there are currently no studies investigating drivers of genome size variability. We run a series of phylogenetic generalized least-squares models on 227 species of squamates to test for possible relationships between genome size and ecological factors including latitudinal distribution, bioclimatic variables and microhabitat use. We also tested whether genome size variation can be associated with parity mode, a highly variable life history trait in this order of reptiles. RESULTS: The best-fitting model showed that the interaction between microhabitat use and parity mode mainly accounted for genome size variation. Larger genome sizes were found in live-bearing species that live in rock/sand ecosystems and in egg-laying arboreal taxa. On the other hand, smaller genomes were found in fossorial live-bearing species. CONCLUSIONS: Environmental factors and species parity mode appear to be among the main parameters explaining genome size variation in squamates. Our results suggest that genome size may favour adaptation of some species to certain environments or could otherwise result from the interaction between environmental factors and parity mode. Integration of genome size and genome sequencing data could help understand the role of differential genome content in the evolutionary process of genome size variation in squamates.

A high-quality reference genome for the critically endangered Aeolian wall lizard, Podarcis raffonei.

The Aeolian wall lizard, Podarcis raffonei, is an endangered species endemic to the Aeolian archipelago, Italy, where it is present only in 3 tiny islets and a narrow promontory of a larger island. Because of the extremely limited area of occupancy, severe population fragmentation and observed decline, it has been classified as Critically Endangered by the International Union for the Conservation of Nature (IUCN). Using Pacific Biosciences (PacBio) High Fidelity (HiFi) long-read sequencing, Bionano optical mapping and Arima chromatin conformation capture sequencing (Hi-C), we produced a high-quality, chromosome-scale reference genome for the Aeolian wall lizard, including Z and W sexual chromosomes. The final assembly spans 1.51 Gb across 28 scaffolds with a contig N50 of 61.4 Mb, a scaffold N50 of 93.6 Mb, and a BUSCO completeness score of 97.3%. This genome constitutes a valuable resource for the species to guide potential conservation efforts and more generally for the squamate reptiles that are underrepresented in terms of available high-quality genomic resources.

How genomics can help biodiversity conservation.

The availability of public genomic resources can greatly assist biodiversity assessment, conservation, and restoration efforts by providing evidence for scientifically informed management decisions. Here we survey the main approaches and applications in biodiversity and conservation genomics, considering practical factors, such as cost, time, prerequisite skills, and current shortcomings of applications. Most approaches perform best in combination with reference genomes from the target species or closely related species. We review case studies to illustrate how reference genomes can facilitate biodiversity research and conservation across the tree of life. We conclude that the time is ripe to view reference genomes as fundamental resources and to integrate their use as a best practice in conservation genomics.

A chromosome-level reference genome and pangenome for barn swallow population genomics.

Insights into the evolution of non-model organisms are limited by the lack of reference genomes of high accuracy, completeness, and contiguity. Here, we present a chromosome-level, karyotype-validated reference genome and pangenome for the barn swallow (Hirundo rustica). We complement these resources with a reference-free multialignment of the reference genome with other bird genomes and with the most comprehensive catalog of genetic markers for the barn swallow. We identify potentially conserved and accelerated genes using the multialignment and estimate genome-wide linkage disequilibrium using the catalog. We use the pangenome to infer core and accessory genes and to detect variants using it as a reference. Overall, these resources will foster population genomics studies in the barn swallow, enable detection of candidate genes in comparative genomics studies, and help reduce bias toward a single reference genome.

Global phylogeography of ridley sea turtles (Lepidochelys spp.): evolution, demography, connectivity, and conservation.

Globally distributed marine taxa are well suited for investigations of biogeographic impacts on genetic diversity, connectivity, and population demography. The sea turtle genus Lepidochelys includes the wide-ranging and abundant olive ridley (L. olivacea), and the geographically restricted and 'Critically Endangered' Kemp's ridley (L. kempii). To investigate their historical biogeography, we analyzed a large dataset of mitochondrial DNA (mtDNA) sequences from olive (n = 943) and Kemp's (n = 287) ridleys, and genotyped 15 nuclear microsatellite loci in a global sample of olive ridleys (n = 285). We found that the ridley species split ~ 7.5 million years ago, before the Panama Isthmus closure. The most ancient mitochondrial olive ridley lineage, located in the Indian Ocean, was dated to ~ 2.2 Mya. Both mitochondrial and nuclear markers revealed significant structure for olive ridleys between Atlantic (ATL), East Pacific (EP), and Indo-West Pacific (IWP) areas. However, the divergence of mtDNA clades was very recent (< 1 Mya) with low within- clade diversity, supporting a recurrent extinction-recolonization model for these ocean regions. All data showed that ATL and IWP groups were more closely related than those in the EP, with mtDNA data supporting recent recolonization of the ATL from the IWP. Individual olive ridley dispersal between the ATL, EP, and IN/IWP could be interpreted as more male- than female-biased, and genetic diversity was lowest in the Atlantic Ocean. All populations showed signs of recent expansion, and estimated time frames were concordant with their recent colonization history. Investigating species abundance and distribution changes over time is central to evolutionary biology, and this study provides a historical biogeographic context for marine vertebrate conservation and management. Supplementary Information: The online version contains supplementary material available at 10.1007/s10592-022-01465-3.

The era of reference genomes in conservation genomics.

Progress in genome sequencing now enables the large-scale generation of reference genomes. Various international initiatives aim to generate reference genomes representing global biodiversity. These genomes provide unique insights into genomic diversity and architecture, thereby enabling comprehensive analyses of population and functional genomics, and are expected to revolutionize conservation genomics.

Long-lasting effects of chronic exposure to chemical pollution on the hologenome of the Manila clam.

Chronic exposure to pollutants affects natural populations, creating specific molecular and biochemical signatures. In the present study, we tested the hypothesis that chronic exposure to pollutants might have substantial effects on the Manila clam hologenome long after removal from contaminated sites. To reach this goal, a highly integrative approach was implemented, combining transcriptome, genetic and microbiota analyses with the evaluation of biochemical and histological profiles of the edible Manila clam Ruditapes philippinarum, as it was transplanted for 6 months from the polluted area of Porto Marghera (PM) to the clean area of Chioggia (Venice lagoon, Italy). One month post-transplantation, PM clams showed several modifications to its resident microbiota, including an overrepresentation of the opportunistic pathogen Arcobacter spp. This may be related to the upregulation of several immune genes in the PM clams, potentially representing a host response to the increased abundance of deleterious bacteria. Six months after transplantation, PM clams demonstrated a lower ability to respond to environmental/physiological stressors related to the summer season, and the hepatopancreas-associated microbiota still showed different compositions among PM and CH clams. This study confirms that different stressors have predictable effects in clams at different biological levels and demonstrates that chronic exposure to pollutants leads to long-lasting effects on the animal hologenome. In addition, no genetic differentiation between samples from the two areas was detected, confirming that PM and CH clams belong to a single population. Overall, the obtained responses were largely reversible and potentially related to phenotypic plasticity rather than genetic adaptation. The results here presented will be functional for the assessment of the environmental risk imposed by chemicals on an economically important bivalve species.

Population structure, genomic diversity and demographic history of Komodo dragons inferred from whole-genome sequencing.

Population and conservation genetics studies have greatly benefited from the development of new techniques and bioinformatic tools associated with next-generation sequencing. Analysis of extensive data sets from whole-genome sequencing of even a few individuals allows the detection of patterns of fine-scale population structure and detailed reconstruction of demographic dynamics through time. In this study, we investigated the population structure, genomic diversity and demographic history of the Komodo dragon (Varanus komodoensis), the world's largest lizard, by sequencing the whole genomes of 24 individuals from the five main Indonesian islands comprising the entire range of the species. Three main genomic groups were observed. The populations of the Island of Komodo and the northern coast of Flores, in particular, were identified as two distinct conservation units. Degrees of genomic divergence among island populations were interpreted as a result of changes in sea level affecting connectivity across islands. Demographic inference suggested that Komodo dragons probably experienced a relatively steep population decline over the last million years, reaching a relatively stable Ne during the Saalian glacial cycle (400-150 thousand years ago) followed by a rapid Ne decrease. Genomic diversity of Komodo dragons was similar to that found in endangered or already extinct reptile species. Overall, this study provides an example of how whole-genome analysis of a few individuals per population can help define population structure and intraspecific demographic dynamics. This is particularly important when applying population genomics data to conservation of rare or elusive endangered species.

Bridging the Gap between Vertebrate Cytogenetics and Genomics with Single-Chromosome Sequencing (ChromSeq).

The study of vertebrate genome evolution is currently facing a revolution, brought about by next generation sequencing technologies that allow researchers to produce nearly complete and error-free genome assemblies. Novel approaches however do not always provide a direct link with information on vertebrate genome evolution gained from cytogenetic approaches. It is useful to preserve and link cytogenetic data with novel genomic discoveries. Sequencing of DNA from single isolated chromosomes (ChromSeq) is an elegant approach to determine the chromosome content and assign genome assemblies to chromosomes, thus bridging the gap between cytogenetics and genomics. The aim of this paper is to describe how ChromSeq can support the study of vertebrate genome evolution and how it can help link cytogenetic and genomic data. We show key examples of ChromSeq application in the refinement of vertebrate genome assemblies and in the study of vertebrate chromosome and karyotype evolution. We also provide a general overview of the approach and a concrete example of genome refinement using this method in the species Anolis carolinensis.

Identifying island safe havens to prevent the extinction of the World's largest lizard from global warming.

The Komodo dragon (Varanus komodoensis) is an endangered, island-endemic species with a naturally restricted distribution. Despite this, no previous studies have attempted to predict the effects of climate change on this iconic species. We used extensive Komodo dragon monitoring data, climate, and sea-level change projections to build spatially explicit demographic models for the Komodo dragon. These models project the species' future range and abundance under multiple climate change scenarios. We ran over one million model simulations with varying model parameters, enabling us to incorporate uncertainty introduced from three main sources: (a) structure of global climate models, (b) choice of greenhouse gas emission trajectories, and (c) estimates of Komodo dragon demographic parameters. Our models predict a reduction in range-wide Komodo dragon habitat of 8%-87% by 2050, leading to a decrease in habitat patch occupancy of 25%-97% and declines of 27%-99% in abundance across the species' range. We show that the risk of extirpation on the two largest protected islands in Komodo National Park (Rinca and Komodo) was lower than other island populations, providing important safe havens for Komodo dragons under global warming. Given the severity and rate of the predicted changes to Komodo dragon habitat patch occupancy (a proxy for area of occupancy) and abundance, urgent conservation actions are required to avoid risk of extinction. These should, as a priority, be focused on managing habitat on the islands of Komodo and Rinca, reflecting these islands' status as important refuges for the species in a warming world. Variability in our model projections highlights the importance of accounting for uncertainties in demographic and environmental parameters, structural assumptions of global climate models, and greenhouse gas emission scenarios when simulating species metapopulation dynamics under climate change.

Assessment of seasonal variation of diet composition in rodents using DNA barcoding and Real-Time PCR.

The study of animal diet and feeding behaviour is a fundamental tool for the illustration of the ecological role of species in the ecosystem. However, size and quality of food intake samples make it hard for researchers to describe the diet composition of many small species. In our study, we exploited genomic tools for the analysis of the diet composition of the Savi's pine vole (Microtus savii) using DNA barcoding and qPCR techniques for the identification of ingested plant species retrieved from stomach contents. In contrast with previous studies, we found that, despite being a fossorial species, the Savi's pine vole is a selective feeder that undergoes intense superficial activity in search for food. In addition, our study shows that with a a priori knowledge of the candidate plant species included in animal diet, qPCR is a powerful tool to assess presence/absence, frequency of occurrence and electivity of ingested species. We conclude that this approach offers new opportunities to implement the analysis of food selection in small animals, thereby revealing a detailed picture of plant-animal interactions.

Host-microbiota interactions shed light on mortality events in the striped venus clam Chamelea gallina.

Mass mortalities due to disease outbreaks have recently affected a number of major taxa in marine ecosystems. Climate- and pollution-induced stress may compromise host immune defenses, increasing the risk of opportunistic diseases. Despite growing evidence that mass mortality events affecting marine species worldwide are strongly influenced by the interplay of numerous environmental factors, the reductionist approaches most frequently used to investigate these factors hindered the interpretation of these multifactorial pathologies. In this study, we propose a broader approach based on the combination of RNA-sequencing and 16S microbiota analyses to decipher the factors underlying mass mortality in the striped venus clam, Chamelea gallina, along the Adriatic coast. On one hand, gene expression profiling and functional analyses of microbial communities showed the over-expression of several genes and molecular pathways involved in xenobiotic metabolism, suggesting potential chemical contamination in mortality sites. On the other hand, the down-regulation of several genes involved in immune and stress response, and the over-representation of opportunistic pathogens such as Vibrio and Photobacterium spp. indicates that these microbial species may take advantage of compromised host immune pathways and defense mechanisms that are potentially affected by chemical exposure, resulting in periodic mortality events. We propose the application of our approach to interpret and anticipate the risks inherent in the combined effects of pollutants and microbes on marine animals in today's rapidly changing environment.

Genome of the Komodo dragon reveals adaptations in the cardiovascular and chemosensory systems of monitor lizards.

Monitor lizards are unique among ectothermic reptiles in that they have high aerobic capacity and distinctive cardiovascular physiology resembling that of endothermic mammals. Here, we sequence the genome of the Komodo dragon Varanus komodoensis, the largest extant monitor lizard, and generate a high-resolution de novo chromosome-assigned genome assembly for V. komodoensis using a hybrid approach of long-range sequencing and single-molecule optical mapping. Comparing the genome of V. komodoensis with those of related species, we find evidence of positive selection in pathways related to energy metabolism, cardiovascular homoeostasis, and haemostasis. We also show species-specific expansions of a chemoreceptor gene family related to pheromone and kairomone sensing in V. komodoensis and other lizard lineages. Together, these evolutionary signatures of adaptation reveal the genetic underpinnings of the unique Komodo dragon sensory and cardiovascular systems, and suggest that selective pressure altered haemostasis genes to help Komodo dragons evade the anticoagulant effects of their own saliva. The Komodo dragon genome is an important resource for understanding the biology of monitor lizards and reptiles worldwide.

Insights into Emydid Turtle Cytogenetics: The European Pond Turtle as a Model Species.

Our knowledge of Testudines evolution is limited by the lack of modern cytogenetic data. Compared to other reptiles, there is little information even on chromosome banding, let alone molecular cytogenetic data. Here, we provide detailed information on the karyotype of the European pond turtle Emys orbicularis, a model Emydidae, employing both chromosome banding and molecular cytogenetics. We provide a high-resolution G-banded karyotype and a map of rDNA genes and telomeric sequences using fluorescence in situ hybridization. We test hypotheses of sex-determining mechanisms in Emys by comparative genomic hybridization to determine if Emys has a cryptic sex-specific region. Our results provide valuable data to guide future efforts on genome sequencing and anchoring in Emydidae and for understanding karyotype evolution in Testudines.

Conserved sex chromosomes and karyotype evolution in monitor lizards (Varanidae).

Despite their long history with the basal split dating back to the Eocene, all species of monitor lizards (family Varanidae) studied so far share the same chromosome number of 2n = 40. However, there are differences in the morphology of the macrochromosome pairs 5-8. Further, sex determination, which revealed ZZ/ZW sex microchromosomes, was studied only in a few varanid species and only with techniques that did not test their homology. The aim of this study was to (i) test if cryptic interchromosomal rearrangements of larger chromosomal blocks occurred during the karyotype evolution of this group, (ii) contribute to the reconstruction of the varanid ancestral karyotype, and (iii) test homology of sex chromosomes among varanids. We investigated these issues by hybridizing flow sorted chromosome paints from Varanus komodoensis to metaphases of nine species of monitor lizards. The results show that differences in the morphology of the chromosome pairs 5-8 can be attributed to intrachromosomal rearrangements, which led to transitions between acrocentric and metacentric chromosomes in both directions. We also documented the first case of spontaneous triploidy among varanids in Varanus albigularis. The triploid individual was fully grown, which demonstrates that polyploidization is compatible with life in this lineage. We found that the W chromosome differs between species in size and heterochromatin content. The varanid Z chromosome is clearly conserved in all the analyzed species. Varanids, in addition to iguanas, caenophidian snakes, and lacertid lizards, are another squamate group with highly conserved sex chromosomes over a long evolutionary time.

Isolating Chromosomes of the Komodo Dragon: New Tools for Comparative Mapping and Sequence Assembly.

We developed new tools to build a high-quality chromosomal map of the Komodo dragon (Varanus komodoensis) available for cross-species phylogenomic analyses. First, we isolated chromosomes by flow sorting and determined the chromosome content of each flow karyotype peak by FISH. We then isolated additional Komodo dragon chromosomes by microdissection and amplified chromosome-specific DNA pools. The chromosome-specific DNA pools can be sequenced, assembled, and mapped by next-generation sequencing technology. The chromosome-specific paint probes can be used to investigate karyotype evolution through cross-species chromosome painting. Overall, the set of chromosome-specific DNA pools of V. komodoensis provides new tools for detailed phylogenomic analyses of Varanidae and squamates in general.

Giant tortoise genomes provide insights into longevity and age-related disease.

Giant tortoises are among the longest-lived vertebrate animals and, as such, provide an excellent model to study traits like longevity and age-related diseases. However, genomic and molecular evolutionary information on giant tortoises is scarce. Here, we describe a global analysis of the genomes of Lonesome George-the iconic last member of Chelonoidis abingdonii-and the Aldabra giant tortoise (Aldabrachelys gigantea). Comparison of these genomes with those of related species, using both unsupervised and supervised analyses, led us to detect lineage-specific variants affecting DNA repair genes, inflammatory mediators and genes related to cancer development. Our study also hints at specific evolutionary strategies linked to increased lifespan, and expands our understanding of the genomic determinants of ageing. These new genome sequences also provide important resources to help the efforts for restoration of giant tortoise populations.

Exploring mechanisms and origins of reduced dispersal in island Komodo dragons.

Loss of dispersal typifies island biotas, but the selective processes driving this phenomenon remain contentious. This is because selection via, both indirect (e.g. relaxed selection or island syndromes) and direct (e.g. natural selection or spatial sorting) processes may be involved, and no study has yet convincingly distinguished between these alternatives. Here, we combined observational and experimental analyses of an island lizard, the Komodo dragon (Varanus komodoensis, the world's largest lizard), to provide evidence for the actions of multiple processes that could contribute to island dispersal loss. In the Komodo dragon, concordant results from telemetry, simulations, experimental translocations, mark-recapture, and gene flow studies indicated that despite impressive physical and sensory capabilities for long-distance movement, Komodo dragons exhibited near complete dispersal restriction: individuals rarely moved beyond the valleys they were born/captured in. Importantly, lizard site-fidelity was insensitive to common agents of dispersal evolution (i.e. indices of risk for inbreeding, kin and intraspecific competition, and low habitat quality) that consequently reduced survival of resident individuals. We suggest that direct selection restricts movement capacity (e.g. via benefits of spatial philopatry and increased costs of dispersal) alongside use of dispersal-compensating traits (e.g. intraspecific niche partitioning) to constrain dispersal in island species.