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.

Reinforcing the idea of an early dispersal of Hippopotamus amphibius in Europe: Restoration and multidisciplinary study of the skull from the Middle Pleistocene of Cava Montanari (Rome, central Italy).

A skull of Hippopotamus recovered from the area of Tor di Quinto, within the urban area of Rome (central Italy) is here redescribed. Despite being one of the most complete specimens of hippopotamuses of the European Pleistocene, the Tor di Quinto skull did not attract much research interest, due to long-standing uncertainties on its provenance. This work begun in 2021, when the skull was restored, within a large renovation project on the vertebrate exposed at the Earth Science University Museum of Sapienza University of Rome. Original sediments were found inside the cranial and mandible cavities during the restoration work, which were sampled for petrographic analyses. By combining a review of the old paleontological, archeological and geological literature published during the 19th and 20th century on the Rome basin and the correlation of these new sedimentological and petrographic information with the lithostratigraphic and synthemic units of the national geological cartography, we clarify that the Hippopotamus skull was most likely to have been collected from a quarry called Cava Montanari, from a formation dated between 560 and 460 ka. Morphological and biometric analyses clearly support an attribution of the Cava Montanari specimen to the extant species Hippopotamus amphibius. The reassessment of the stratigraphic and geological data on Cava Montanari implies that the studied specimen is the earliest confirmed occurrence of Hippopotamus amphibius in the European fossil record.

Species-specific gill's microbiome of eight crab species with different breathing adaptations.

Transitions to physically different environments, such as the water-to-land transition, proved to be the main drivers of relevant evolutionary events. Brachyuran crabs evolved remarkable morphological, behavioral, and physiological adaptations to terrestrial life. Terrestrial species evolved new respiratory structures devoted to replace or support the gills, a multifunctional organ devoted to gas exchanges, ion-regulation and nitrogen excretion. It was hypothesized that microorganisms associated with respiratory apparatus could have facilitated the processes of osmoregulation, respiration, and elimination of metabolites along this evolutionary transition. To test if crab species with different breathing adaptations may host similar microbial communities on their gills, we performed a comparative targeted-metagenomic analysis, selecting two marine and six terrestrial crabs belonging to different families and characterised by different breathing adaptations. We analysed anterior and posterior gills separately according to their different and specific roles. Regardless of their terrestrial or marine adaptations, microbial assemblages were strongly species-specific indicating a non-random association between the host and its microbiome. Significant differences were found in only two terrestrial species when considering posterior vs. anterior gills, without any association with species-specific respiratory adaptations. Our results suggest that all the selected species are strongly adapted to the ecological niche and specific micro-habitat they colonise.

Chemosensory proteins as putative semiochemical carriers in the desert isopod Hemilepistus reaumurii.

The order Isopoda contains both aquatic and terrestrial species, among which Hemilepistus reaumurii, which lives in arid environments and is the most adapted to terrestrial life. Olfaction has been deeply investigated in insects while it has received very limited attention in other arthropods, particularly in terrestrial crustaceans. In insects, soluble proteins belonging to two main families, Odorant Binding Proteins (OBPs) and Chemosensory Proteins (CSPs), are contained in the olfactory sensillar lymph and are suggested to act as carriers of hydrophobic semiochemicals to or from membrane-bound olfactory receptors. Other protein families, namely Nieman-Pick type 2 (NPC2) and Lipocalins (LCNs) have been also reported as putative odorant carriers in insects and other arthropod clades. In this study, we have sequenced and analysed the transcriptomes of antennae and of the first pair of legs of H. reaumurii focusing on soluble olfactory proteins. Interestingly, we have found 13 genes encoding CSPs, whose sequences differ from those of the other arthropod clades, including non-isopod crustaceans, for the presence of two additional cysteine residues, besides the four conserved ones. Binding assays on two of these proteins showed strong affinities for fatty acids and long-chain unsaturated esters and aldehydes, putative semiochemicals for this species.

Conserved organ-specific microbial assemblages in different populations of a terrestrial crab.

Microorganisms are ubiquitous in the environment and provide genetic and physiological functions to multicellular organisms. Knowledge on the associated microbiota is becoming highly relevant to understand the host's ecology and biology. Among invertebrates, many examples of endosymbiosis have been described, such as those in corals, ants, and termites. At present, however, little is known on the presence, diversity, and putative roles of the microbiota associated to brachyuran crabs in relation to their environment. In this work we investigated the associated microbiota of three populations of the terrestrial brachyuran crab Chiromantes haematocheir to find evidence of a conserved organ-specific microbiome unrelated to the population of origin and dissimilar from environmental microbial assemblages. Bacterial 16S rRNA gene and fungal ITS sequences were obtained from selected crab organs and environmental matrices to profile microbial communities. Despite the presence of truly marine larval stages and the absence of a gregarious behaviour, favouring microbiota exchanges, we found common, organ-specific microbiota, associated with the gut and the gills of crabs from the different populations (with more than 15% of the genera detected specifically enriched only in one organ). These findings suggest the presence of possible functional roles of the organ-specific microbiota.

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.

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.

A multidisciplinary integrated approach using Pachygrapsus marmoratus to assess the impact of port activities on mediterranean marine protected areas.

The establishment of marine protected areas is considered the main global strategy to halt the loss of marine biodiversity. Since most of marine areas are open systems, this form of habitat protection cannot prevent their contamination due to human activities performed outside of their borders. Innovative approaches to assess the health status of protected marine habitats are therefore needed. Here we developed a multidisciplinary approach that combines ecological characteristics, bioaccumulation of inorganic and organic pollutants, cell damage (micronuclei frequency, nuclear alterations and LPO) and enzymatic (AChE, CAT, IDH, LDH, GST and CAT) markers focused on an intertidal brachyuran crab, Pachygrapsus marmoratus, to assess the impacts of contaminant exposure on Mediterranean coastal habitats. As study sites we selected two protected areas and two sites within industrial ports of the Ligurian Sea. Our results showed that the selected crab species is an excellent bioindicator. Individuals collected in sites with the highest levels of heavy metal pollution showed the highest signals of stress responses at both cellular and enzymatic levels, coupled with a high incidence of the parasite Sacculina carcini, a signal of impairment of their standard development and reproduction cycle. We could also prove that one of the selected marine protected areas showed the same intensity of impact as its adjacent port site. Our multidisciplinary approach proved to be a valuable tool to assess the environmental quality and health of protected and disturbed Mediterranean coastal environments and to inform efficient management and protection schemes for such habitats.

Stratigraphic reassessment of Grotta Romanelli sheds light on Middle-Late Pleistocene palaeoenvironments and human settling in the Mediterranean.

During the last century, Grotta Romanelli (Southern Italy) has been a reference site for the European Late Pleistocene stratigraphy, due to its geomorphological setting and archaeological and palaeontological content. The beginning of the sedimentation inside the cave was attributed to the Last Interglacial (MISs 5e) and the oldest unearthed evidence of human occupation, including remains of hearths, was therefore referred to the Middle Palaeolithic. Recent surveys and excavations produced new U/Th dates, palaeoenvironmental interpretation and a litho-, morpho- and chrono-stratigraphical reassessment, placing the oldest human frequentation of the cave between MIS 9 and MIS 7, therefore embracing Glacial and Interglacial cycles. These new data provide evidence that the sea reached the cave during the Middle Pleistocene and human occupation occurred long before MISs 5e and persisted beyond the Pleistocene- Holocene boundary.

A Middle Pleistocene wolf from central Italy provides insights on the first occurrence of Canis lupus in Europe.

Here, we describe a partial cranium of a large canid dated at 406.5 ± 2.4 ka from the Middle Pleistocene of Ponte Galeria (Rome, Italy). The sample represents one of the few Middle Pleistocene remains of a wolf-like canid falling within the timeframe when the Canis mosbachensis-Canis lupus transition occurred, a key moment to understand the spread of the extant wolf (Canis lupus) in Europe. CT-based methods allow studying the outer and inner cranial anatomy (brain and frontal sinuses) of a selected sample of fossil and extant canids. Morphological and biometric results allowed to: (I) ascribe the cranium from Ponte Galeria to an adult Canis lupus, representing the first reliable occurrence of this taxon in Europe; (II) provide the content for a biochronological revision of the Middle Pleistocene record of European wolves.

Genetic load: genomic estimates and applications in non-model animals.

Genetic variation, which is generated by mutation, recombination and gene flow, can reduce the mean fitness of a population, both now and in the future. This 'genetic load' has been estimated in a wide range of animal taxa using various approaches. Advances in genome sequencing and computational techniques now enable us to estimate the genetic load in populations and individuals without direct fitness estimates. Here, we review the classic and contemporary literature of genetic load. We describe approaches to quantify the genetic load in whole-genome sequence data based on evolutionary conservation and annotations. We show that splitting the load into its two components - the realized load (or expressed load) and the masked load (or inbreeding load) - can improve our understanding of the population genetics of deleterious mutations.

A DNA barcode library for mangrove gastropods and crabs of Hong Kong and the Greater Bay Area reveals an unexpected faunal diversity associated with the intertidal forests of Southern China.

BACKGROUND: Mangroves are tropical and subtropical intertidal forests colonising sheltered coasts across the world. They host a unique faunal community, dominated by brachyuran crabs and gastropods. These invertebrates strongly contribute to the functionality of the entire forest. The reliable assessment of mangrove faunal diversity is, thus, a crucial step for efficient management and conservation plans, but it is hindered by difficulties in species identification. Here we provide a verified DNA barcode library for brachyuran crabs and gastropods inhabiting the mangroves of the Greater Bay Area, Southern China. In particular, we collected and morphologically identified 1100 specimens of mangrove associated brachyuran crabs and gastropods. The partial sequences of the mtDNA cytochrome oxidase subunit I gene were obtained from 275 specimens. Barcode sequences were then used to delineate Molecular Operational Taxonomic Units (MOTUs), employing three different delimitation methods: the automatic barcode gap discovery (ABGD) method, the general mixed Yule coalescent (GMYC) model and a Bayesian implementation of the Poisson tree processes (bPTP) model. RESULTS: By integrating DNA barcodes with morphology, we identified 44 gastropod species and 58 brachyuran species associated with Hong Kong mangroves, with five and seven new records, for gastropods and crabs, respectively, for the Greater Bay Area. The delineation of MOTUs based on barcode sequences revealed a strong congruence between morphological and molecular identification for both taxa, showing the high reliability of the barcode library. CONCLUSIONS: This study provides the first reference barcode library for mangrove-associated macrobenthic fauna in the Greater Bay Area and represents a reliable tool to management and conservation plans. Our molecular analyses resolved long lasting taxonomic misidentifications and inconsistencies and updated the knowledge on the geographical distribution of Asian mangrove associated fauna, ultimately highlighting a level of biodiversity higher than previously thought for Southern China.

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.

Lipocalins in Arthropod Chemical Communication.

Lipocalins represent one of the most successful superfamilies of proteins. Most of them are extracellular carriers for hydrophobic ligands across aqueous media, but other functions have been reported. They are present in most living organisms including bacteria. In animals they have been identified in mammals, molluscs, and arthropods; sequences have also been reported for plants. A subgroup of lipocalins, referred to as odorant-binding proteins (OBPs), mediate chemical communication in mammals by ferrying specific pheromones to the vomeronasal organ. So far, these proteins have not been reported as carriers of semiochemicals in other living organisms; instead chemical communication in arthropods is mediated by other protein families structurally unrelated to lipocalins. A search in the databases has revealed extensive duplication and differentiation of lipocalin genes in some species of insects, crustaceans, and chelicerates. Their large numbers, ranging from a handful to few dozens in the same species, their wide divergence, both within and between species, and their expression in chemosensory organs suggest that such expansion may have occurred under environmental pressure, thus supporting the hypothesis that lipocalins may be involved in chemical communication in arthropods.

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.

To the Land and Beyond: Crab Microbiomes as a Paradigm for the Evolution of Terrestrialization.

The transition to terrestrial environments by formerly aquatic species has occurred repeatedly in many animal phyla and lead to the vast diversity of extant terrestrial species. The differences between aquatic and terrestrial habitats are enormous and involved remarkable morphological and physiological changes. Convergent evolution of various traits is evident among phylogenetically distant taxa, but almost no information is available about the role of symbiotic microbiota in such transition. Here, we suggest that intertidal and terrestrial brachyuran crabs are a perfect model to study the evolutionary pathways and the ecological role of animal-microbiome symbioses, since their transition to land is happening right now, through a number of independent lineages. The microorganisms colonizing the gut of intertidal and terrestrial crabs are expected to play a major role to conquer the land, by reducing water losses and permitting the utilization of novel food sources. Indeed, it has been shown that the microbiomes hosted in the digestive system of terrestrial isopods has been critical to digest plant items, but nothing is known about the microbiomes present in the gut of truly terrestrial crabs. Other important physiological regulations that could be facilitated by microbiomes are nitrogen excretion and osmoregulation in the new environment. We also advocate for advances in comparative and functional genomics to uncover physiological aspects of these ongoing evolutionary processes. We think that the multidisciplinary study of microorganisms associated with terrestrial crabs will shed a completely new light on the biological and physiological processes involved in the sea-land transition.

The origin of early Acheulean expansion in Europe 700 ka ago: new findings at Notarchirico (Italy).

Notarchirico (Southern Italy) has yielded the earliest evidence of Acheulean settlement in Italy and four older occupation levels have recently been unearthed, including one with bifaces, extending the roots of the Acheulean in Italy even further back in time. New 40Ar/39Ar on tephras and ESR dates on bleached quartz securely and accurately place these occupations between 695 and 670 ka (MIS 17), penecontemporaneous with the Moulin-Quignon and la Noira sites (France). These new data demonstrate a very rapid expansion of shared traditions over Western Europe during a period of highly variable climatic conditions, including interglacial and glacial episodes, between 670 and 650 (i.e., MIS17/MIS16 transition). The diversity of tools and activities observed in these three sites shows that Western Europe was populated by adaptable hominins during this time. These conclusions question the existence of refuge areas during intense glacial stages and raise questions concerning understudied migration pathways, such as the Sicilian route.

First report of Sus strozzii (Suidae, Mammalia) from the Early Pleistocene of Hungary (Dunaalmás) and species distinction based on deciduous teeth.

Herein we describe a fragment of a mandible with a deciduous fourth premolar (dp4) from the Early Pleistocene locality of Dunaalmás, representing the first confirmed report of Sus strozzii from Hungary. The comparison of dp4 measurements supports a statistically significant distinction between S. strozzii and Sus scrofa. The two species overlap in time during the late Early Pleistocene of Europe (Epivillafranchian), but suid remains of this time-span are seldom classified at a species level. The correct taxonomic identification of the Epivillafranchian suids, which are often associated with evidences of hominin presence, is of great palaeoenvironmental value because S. scrofa and S. strozzii possess different ecological requirements.