Museum Genomics.

Natural history collections are invaluable repositories of biological information that provide an unrivaled record of Earth's biodiversity. Museum genomics-genomics research using traditional museum and cryogenic collections and the infrastructure supporting these investigations-has particularly enhanced research in ecology and evolutionary biology, the study of extinct organisms, and the impact of anthropogenic activity on biodiversity. However, leveraging genomics in biological collections has exposed challenges, such as digitizing, integrating, and sharing collections data; updating practices to ensure broadly optimal data extraction from existing and new collections; and modernizing collections practices, infrastructure, and policies to ensure fair, sustainable, and genomically manifold uses of museum collections by increasingly diverse stakeholders. Museum genomics collections are poised to address these challenges and, with increasingly sensitive genomics approaches, will catalyze a future era of reproducibility, innovation, and insight made possible through integrating museum and genome sciences.

What one genus of showy moths can say about migration, adaptation, and wing pattern.

The Ornate Moth, Utetheisa ornatrix, has served as a model species in chemical ecology studies for decades. Like in the widely publicized stories of the Monarch and other milkweed butterflies, the Ornate Moth and its relatives are tropical insects colonizing whole continents assisted by their chemical defenses. With the recent advances in genomic techniques and evo-devo research, it is becoming a model for studies in other areas, from wing pattern development to phylogeography, from toxicology to epigenetics. We used a genomic approach to learn about Utetheisa's evolution, detoxification, dispersal abilities, and wing pattern diversity. We present an evolutionary genomic analysis of the worldwide genus Utetheisa, then focusing on U. ornatrix. Our reference genome of U. ornatrix reveals gene duplications in the regions possibly associated with detoxification abilities, which allows them to feed on toxic food plants. Finally, comparative genomic analysis of over 100 U. ornatrix specimens from the museum with apparent differences in wing patterns suggest the potential roles of cortex and lim3 genes in wing pattern formation of Lepidoptera and the utility of museum-preserved collection specimens for wing pattern research.

Pathogen prospecting of museums: Reconstructing malaria epidemiology.

Malaria is a disease of global significance. Ongoing changes to the earth's climate, antimalarial resistance, insecticide resistance, and socioeconomic decline test the resilience of malaria prevention programs. Museum insect specimens present an untapped resource for studying vector-borne pathogens, spurring the question: Do historical mosquito collections contain Plasmodium DNA, and, if so, can museum specimens be used to reconstruct the historical epidemiology of malaria? In this Perspective, we explore molecular techniques practical to pathogen prospecting, which, more broadly, we define as the science of screening entomological museum specimens for human, animal, or plant pathogens. Historical DNA and pathogen prospecting provide a means of describing the coevolution of human, vector, and parasite, informing the development of insecticides, diagnostics, therapeutics, and vaccines.

Exon capture museomics deciphers the nine-banded armadillo species complex and identifies a new species endemic to the Guiana Shield.

The nine-banded armadillo (Dasypus novemcinctus) is the most widespread xenarthran species across the Americas. Recent studies have suggested it is composed of four morphologically and genetically distinct lineages of uncertain taxonomic status. To address this issue, we used a museomic approach to sequence 80 complete mitogenomes and capture 997 nuclear loci for 71 Dasypus individuals sampled across the entire distribution. We carefully cleaned up potential genotyping errors and cross contaminations that could blur species boundaries by mimicking gene flow. Our results unambiguously support four distinct lineages within the D. novemcinctus complex. We found cases of mito-nuclear phylogenetic discordance but only limited contemporary gene flow confined to the margins of the lineage distributions. All available evidence including the restricted gene flow, phylogenetic reconstructions based on both mitogenomes and nuclear loci, and phylogenetic delimitation methods consistently supported the four lineages within D. novemcinctus as four distinct species. Comparable genetic differentiation values to other recognized Dasypus species further reinforced their status as valid species. Considering congruent morphological results from previous studies, we provide an integrative taxonomic view to recognise four species within the D. novemcinctus complex: D. novemcinctus, D. fenestratus, D. mexicanus, and D. guianensis sp. nov., a new species endemic of the Guiana Shield that we describe here. The two available individuals of D. mazzai and D. sabanicola were consistently nested within D. novemcinctus lineage and their status remains to be assessed. The present work offers a case study illustrating the power of museomics to reveal cryptic species diversity within a widely distributed and emblematic species of mammals.

Artifactual Orthologs and the Need for Diligent Data Exploration in Complex Phylogenomic Datasets: A Museomic Case Study from the Andean Flora.

The Andes mountains of western South America are a globally important biodiversity hotspot, yet there is a paucity of resolved phylogenies for plant clades from this region. Filling an important gap in our understanding of the World's richest flora, we present the first phylogeny of Freziera (Pentaphylacaceae), an Andean-centered, cloud forest radiation. Our dataset was obtained via hybrid-enriched target sequence capture of Angiosperms353 universal loci for 50 of the ca. 75 spp., obtained almost entirely from herbarium specimens. We identify high phylogenomic complexity in Freziera, including the presence of data artifacts. Via by-eye observation of gene trees, detailed examination of warnings from recently improved assembly pipelines, and gene tree filtering, we identified that artifactual orthologs (i.e., the presence of only one copy of a multicopy gene due to differential assembly) were an important source of gene tree heterogeneity that had a negative impact on phylogenetic inference and support. These artifactual orthologs may be common in plant phylogenomic datasets, where multiple instances of genome duplication are common. After accounting for artifactual orthologs as source of gene tree error, we identified a significant, but nonspecific signal of introgression using Patterson's D and f4 statistics. Despite phylogenomic complexity, we were able to resolve Freziera into 9 well-supported subclades whose evolution has been shaped by multiple evolutionary processes, including incomplete lineage sorting, historical gene flow, and gene duplication. Our results highlight the complexities of plant phylogenomics, which are heightened in Andean radiations, and show the impact of filtering data processing artifacts and standard filtering approaches on phylogenetic inference.

Historic Museum Samples Provide Evidence for a Recent Replacement of Wolbachia Types in European Drosophila melanogaster.

Wolbachia is one of the most common bacterial endosymbionts, which is frequently found in numerous arthropods and nematode taxa. Wolbachia infections can have a strong influence on the evolutionary dynamics of their hosts since these bacteria are reproductive manipulators that affect the fitness and life history of their host species for their own benefit. Host-symbiont interactions with Wolbachia are perhaps best studied in the model organism Drosophila melanogaster, which is naturally infected with at least 5 different variants among which wMel and wMelCS are the most frequent ones. Comparisons of infection types between natural flies and long-term lab stocks have previously indicated that wMelCS represents the ancestral type, which was only very recently replaced by the nowadays dominant wMel in most natural populations. In this study, we took advantage of recently sequenced museum specimens of D. melanogaster that have been collected 90 to 200 yr ago in Northern Europe to test this hypothesis. Our comparison to contemporary Wolbachia samples provides compelling support for the replacement hypothesis. Our analyses show that sequencing data from historic museum specimens and their bycatch are an emerging and unprecedented resource to address fundamental questions about evolutionary dynamics in host-symbiont interactions. However, we also identified contamination with DNA from crickets that resulted in co-contamination with cricket-specific Wolbachia in several samples. These results underpin the need for rigorous quality assessments of museomic data sets to account for contamination as a source of error that may strongly influence biological interpretations if it remains undetected.

DNA sequences from type specimens and type strains - how to increase their number and improve their annotation in NCBI GenBank and related databases.

Scientific names permit humans and search engines to access knowledge about the biodiversity that surrounds us, and names linked to DNA sequences are playing an ever-greater role in search-and-match identification procedures. Here, we analyze how users and curators of the National Center for Biotechnology Information (NCBI) are flagging and curating sequences derived from nomenclatural type material, which is the only way to improve the quality of DNA-based identification in the long run. For prokaryotes, 18,281 genome assemblies from type strains have been curated by NCBI staff and improve the quality of prokaryote naming. For Fungi, type-derived sequences representing over 21,000 species are now essential for fungus naming and identification. For the remaining eukaryotes, however, the numbers of sequences identifiable as type-derived are minuscule, representing only 1,000 species of arthropods, 8,441 vertebrates, and 430 embryophytes. An increase in the production and curation of such sequences will come from (i) sequencing of types or topotypic specimens in museum collections, (ii) the March 2023 rule changes at the International Nucleotide Sequence Database Collaboration requiring more metadata for specimens, and (iii) efforts by data submitters to facilitate curation, including informing NCBI curators about a specimen's type status. We illustrate different type-data submission journeys and provide best-practice examples from a range of organisms. Expanding the number of type-derived sequences in DNA databases, especially of eukaryotes, is crucial for capturing, documenting, and protecting biodiversity.

Reduction of genetic diversity in 'Alala (Hawaiian crow; Corvus hawaiiensis) between the late 1800s and the late 1900s.

Genetic and genomic data are increasingly used to aid conservation management of endangered species by providing insights into evolutionary histories, factors associated with extinction risks, and potential for future adaptation. For the 'Alala, or Hawaiian crow (Corvus hawaiiensis), genetic concerns include negative correlations between inbreeding and hatching success. However, it is unclear if low genetic diversity and inbreeding depression are consequences of a historical population bottleneck, or if 'Alala had historically low genetic diversity that predated human influence, perhaps as a result of earlier declines or founding events. In this study, we applied a hybridization-based sequence capture to generate a genome-wide single nucleotide polymorphism (SNP) dataset for comparing historical specimens collected in the 1890s, when 'Alala were more numerous, to samples taken between 1973 and 1998, when 'Alala population densities were near the lowest documented levels in the wild, prior to all individuals being collected for captive rearing. We found low genome-wide diversity in both sample groups, however, the modern sample group (1973 to 1998 cohort) exhibited relatively fewer polymorphic alleles, a lower proportion of polymorphic loci, and lower observed heterozygosity, consistent with a population decline and potential bottleneck effects. These results combined with a current low population size highlight the importance of continued efforts by conservation managers to mitigate inbreeding and maintain founder representation to preserve what genetic diversity remains.

Dynamic evolution of size and colour in the highly specialized Zodarion ant-eating spiders.

Ecological specialists constitute relevant case studies for understanding the mechanisms, potential and limitations of evolution. The species-rich and strictly myrmecophagous spiders of the genus Zodarion show diversified defence mechanisms, including myrmecomorphy of different ant species and nocturnality. Through Hybridization Capture Using RAD Probes (hyRAD), a phylogenomic technique designed for sequencing poorly preserved specimens, we reconstructed a phylogeny of Zodarion using 52 (approx. a third of the nominal) species that cover its phylogenetic and distributional diversity. We then estimated the evolution of body size and colour, traits that have diversified noticeably and are linked to defence mechanisms, across the group. Our genomic matrix of 300 loci led to a well-supported phylogenetic hypothesis that uncovered two main clades inside Zodarion. Ancestral state estimation revealed the highly dynamic evolution of body size and colour across the group, with multiple transitions and convergences in both traits, which we propose is likely indicative of multiple transitions in ant specialization across the genus. Our study will allow the informed targeted selection of Zodarion taxa of special interest for research into the group's remarkable adaptations to ant specialization. It also exemplifies the utility of hyRAD for phylogenetic studies using museum material.

An Opiliones-specific ultraconserved element probe set with a near-complete family-level phylogeny.

Sequence capture of ultraconserved elements (UCEs) has transformed molecular systematics across many taxa, with arachnids being no exception. The probe set available for Arachnida has been repeatedly used across multiple arachnid lineages and taxonomic levels, however more specific probe sets for spiders have demonstrated that more UCEs can be recovered with higher probe specificity. In this study, we develop an Opiliones-specific UCE probe set targeting 1915 UCEs using a combination of probes designed from genomes and transcriptomes, as well as the most useful probes from the Arachnida probe set. We demonstrate the effectiveness of this probe set across Opiliones with the most complete family-level phylogeny made to date, including representatives from 61 of 63 currently described families. We also test UCE recovery from historical specimens with degraded DNA, examine population-level data sets, and assess "backwards compatibility" with samples hybridized with the Arachnida probe set. The resulting phylogenies - which include specimens hybridized using both the Opiliones and Arachnida probe sets, historical specimens, and transcriptomes - are largely congruent with previous multi-locus and phylogenomic analyses. The probe set is also "backwards compatible", increasing the number of loci obtained in samples previously hybridized with the Arachnida probe set, and shows high utility down to shallow population-level divergences. This probe set has the potential to further transform Opiliones molecular systematics, resolving many long-standing taxonomic issues plaguing this lineage.

Ultraconserved elements resolve phylogenetic relationships and biogeographic history of African-Malagasy bent-winged bats (Miniopterus).

African-Malagasy species of the bat genus Miniopterus are notable both for the dramatic increase in the number of newly recognized species over the last 15 years, as well as for the profusion of new taxa from Madagascar and the neighboring Comoros. Since 2007, seven new Malagasy Miniopterus species have been described compared to only two new species since 1936 from the Afrotropics. The conservative morphology of Miniopterus and limited geographic sampling in continental Africa have undoubtedly contributed to the deficit of continental species. In addition to uncertainty over species limits, phylogenetic relationships of Miniopterus remain mostly unresolved, particularly at deeper backbone nodes. Previous phylogenetic studies were based on limited taxon sampling and/or limited genetic sampling involving no more than five loci. Here, we conduct the first phylogenomic study of the Afrotropical Miniopteridae by analyzing up to 3772 genome-wide ultraconserved elements (UCEs) from historic and modern samples of 70 individuals from 25 Miniopterus species/lineages. We analyze multiple datasets of varying degrees of completeness (70, 90, and 100 percent complete) using partitioned concatenated maximum likelihood and multispecies coalescent methods. Our well-supported, species-level phylogenies resolved most (6/8 or 7/8) backbone nodes and strongly support for the first time the monophyly of the Malagasy radiation. We inferred the crown age of African Miniopteridae in the late Miocene (10.4 Ma), while the main lineages of Miniopterus appear to have contemporaneously diversified in two sister radiations in the Afrotropics and Madagascar. Species-level divergence of 23 of 25 African + Malagasy Miniopterus were estimated to have 95 % HPDs that overlap with the late Miocene (5.3-10.4 Ma). We present ancestral range estimates that unambiguously support a continental African radiation that originated in the Zambezian and Somalian/Ethiopian biogeographic regions, but we cannot rule out back colonization of Africa from Madagascar. The phylogeny indicates genetic support for up to seven new species.

Is it inappropriate to ask for your age? Evaluating parameter impact on tree dating in a challenging clade (Macroscelidea).

Sengis (order Macroscelidea) are small mammals endemic to Africa. The taxonomy and phylogeny of sengis has been difficult to resolve due to a lack of clear morphological apomorphies. Molecular phylogenies have already significantly revised sengi systematics, but until now no molecular phylogeny has included all 20 extant species. In addition, the age of origin of the sengi crown clade and the divergence age of its two extant families remain unclear. Two recently published studies based on different datasets and age-calibration parameters (DNA type, outgroup selection, fossil calibration points) proposed highly different divergent age estimates and evolutionary scenarios. We obtained nuclear and mitochondrial DNA from mainly museum specimens using target enrichment of single-stranded DNA libraries to generate the first phylogeny of all extant macroscelidean species. We then explored the effects of different parameters (type of DNA, ratio of ingroup to outgroup sampling, number and type of fossil calibration points) and their resulting impacts on age estimates for the origin and initial diversification of Macroscelidea. We show that, even after correcting for substitution saturation, both using mitochondrial DNA in conjunction with nuclear DNA or alone results in much older ages and different branch lengths than when using nuclear DNA alone. We further show that the former effect can be attributed to insufficient amounts of nuclear data. If multiple calibration points are included, the age of the sengi crown group fossil prior has minimal impact on the estimated time frame of sengi evolution. In contrast, the inclusion or exclusion of outgroup fossil priors has a major effect on the resulting node ages. We also find that a reduced sampling of ingroup species does not significantly affect overall age estimates and that terminal specific substitution rates can serve as a means to evaluate the biological likeliness of the produced temporal estimates. Our study demonstrates how commonly varied parameters in temporal calibration of phylogenies affect age estimates. Dated phylogenies should therefore always be seen in the context of the dataset which was used to produce them.

Museomics and phylogenomics of lovebirds (Psittaciformes, Psittaculidae, Agapornis) using low-coverage whole-genome sequencing.

Natural history collections contain specimens that provide important insights into studies of ecology and evolution. With the advancement of high-throughput sequencing, historical DNA (hDNA) from museum specimens has become a valuable source of genomic data to study the evolutionary history of organisms. Low-coverage whole genome sequencing (WGS) has been increasingly applied to museum specimens for analyzing organelle genomes, but is still uncommon for genotyping the nuclear DNA fraction. In this study, we applied low-coverage WGS to phylogenomic analyses of parrots in the genus Agapornis by including both modern samples and historical specimens of âˆ¼100-year-old. Agapornis are small-sized African and Malagasy parrots with diverse characters. Earlier phylogenetic studies failed to resolve the positions of some key lineages, prohibiting a robust interpretation of the biogeography and evolution of these African parrots. Here, we demonstrated the use of low-coverage WGS for generating both mitochondrial and nuclear genomic data, and evaluated data quality differences between modern and historical samples. Our resolved Agapornis phylogeny indicates the ancestor of Agapornis likely colonized Madagascar from Australasia by trans-oceanic dispersal events before dispersing to the African continent. Genome-wide SNPs also allowed us to identify the parental origins of hybrid Agapornis individuals. This study demonstrates the potential of applying low-coverage WGS to phylogenomics and population genomics analyses and illustrates how including historical museum specimens can address outstanding questions regarding the evolutionary history of contemporary lineages.

First description of the mitochondrial genomes of the Central American brocket deer Mazama temama (Kerr, 1792) and the Yucatán Peninsula brocket deer Odocoileus pandora Merriam, 1901.

BACKGROUND: The Central American (Mazama temama) and the Yucatán Peninsula brocket deer (Odocoileus pandora) are deer species with cryptic habits, and little is known about their biology. Odocoileus pandora is listed as Vulnerable on the 2015 IUCN Red List of Threatened Species, while M. temama is considered Data Deficient; however, it currently faces a decreasing population trend. METHODS AND RESULTS: We assembled the complete mitochondrial genome for two M. temama specimens and one complete and one partial for O. pandora from Illumina 150 bp paired-end reads. The mitogenomes of M. temama and O. pandora have a length of 16,479-16,480 and 16,419 bp, respectively, AT-biased; they consist of 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and one non-coding control region, most of them follow a transcription direction in the heavy strand of the molecule. The mitochondrial genome of O. pandora shows some particularities compared to other deer species, like a shorter control region of 987-990 bp and a cytochrome b gene with a length of 1,143 bp. Our phylogenetic analyses confirm the close affinity of M. temama to South American M. americana and the nested position of the genus Odocoileus, including O. pandora, into the genus Mazama. CONCLUSIONS: Here, we described for the first time the complete mitochondrial genome for these two species. While our study provides additional information about the taxonomic status of the northern neotropical brocket deer, further research is needed to solve the complicated taxonomy of neotropical deer.

Large variance in inbreeding within the Iberian wolf population.

The gray wolf (Canis lupus) population on the Iberian Peninsula was the largest in western and central Europe during most of the 20th century, with its size apparently never under a few hundred individuals. After partial legal protection in the 1970s in Spain, the northwest Iberian population increased to about 300-350 packs and then stabilized. In contrast to many current European wolf populations, which have been connected through gene flow, the Iberian wolf population has been isolated for decades. Here we measured changes on genomic diversity and inbreeding through the last decades in a geographic context. We find that the level of genomic diversity in Iberian wolves is low compared to other Eurasian wolf populations. Despite population expansion in the last 50 years, some modern wolves had very high inbreeding, especially in the recently recolonized and historical edge areas. These individuals contrast with others with low inbreeding within the same population. The high variance in inbreeding despite population expansion seems associated with small-scale fragmentation of the range that is revealed by the genetic similarity between modern and historical samples from close localities despite being separated by decades, remaining differentiated from other individuals that are just over 100 km away, a small distance for a species with great dispersal capacity inhabiting a continuous range. This illustrates that, despite its demographically stable condition, the population would probably benefit from favoring connectivity within the population as well as genetic exchange with other European wolf populations to avoid excessive fragmentation and local inbreeding depression.

Resolution of eleven reported and five novel Podaxis species based on ITS phylogeny, phylogenomics, morphology, ecology, and geographic distribution.

The genus Podaxis was first described from India by Linnaeus in 1771, but several revisions of the genus have left the taxonomy unclear. Forty-four Podaxis species names and nine intraspecific varieties are currently accepted, but most fungarium specimens are labelled Podaxis pistillaris. Recent molecular analyses based on barcoding genes suggest that the genus comprises several species, but their status is largely unresolved. Here we obtained basidiospores and photographs from 166 fungarium specimens from around the world and generated a phylogeny based on rDNA internal transcribed spacer ITS1,5.8S and ITS2 (ITS), and a phylogenomic analysis of 3 839 BUSCO genes from low-coverage genomes for a subset of the specimens. Combining phylogenetics, phylogenomics, morphology, ecology, and geographical distribution, spanning 250 years of collections, we propose that the genus includes at least 16 unambiguous species. Based on 10 type specimens (holotype, paratype, and syntype), four recorded species were confirmed, P. carcinomalis, P. deflersii, P. emerici, and P. farlowii. Comparing phylogenetic analysis with described species, including morphology, ecology, and distribution, we resurrected P. termitophilus and designated neotypes, epitypes, or lectotypes for five previously described species, P. aegyptiacus, P. africana, P. beringamensis, P. calyptratus, and P. perraldieri. Lastly, based on phylogenies and morphology of type material, we synonymized three reported species, P. algericus, P. arabicus, and P. rugospora with P. pistillaris, and described five new species that we named P. desolatus, P. inyoensis, P. mareebaensis, P. namaquensis, and P. namibensis. Citation: Li GS, Leal-Dutra CA, Cuesta-Maté A, et al. 2023. Resolution of eleven reported and five novel Podaxis species based on ITS phylogeny, phylogenomics, morphology, ecology, and geographic distribution. Persoonia 51: 257-279. doi: 10.3767/persoonia.2023.51.07.

Genomics Reveals the Origins of Historical Specimens.

Centuries of zoological studies have amassed billions of specimens in collections worldwide. Genomics of these specimens promises to reinvigorate biodiversity research. However, because DNA degrades with age in historical specimens, it is a challenge to obtain genomic data for them and analyze degraded genomes. We developed experimental and computational protocols to overcome these challenges and applied our methods to resolve a series of long-standing controversies involving a group of butterflies. We deduced the geographical origins of several historical specimens of uncertain provenance that are at the heart of these debates. Here, genomics tackles one of the greatest problems in zoology: countless old specimens that serve as irreplaceable embodiments of species concepts cannot be confidently assigned to extant species or population due to the lack of diagnostic morphological features and clear documentation of the collection locality. The ability to determine where they were collected will resolve many on-going disputes. More broadly, we show the utility of applying genomics to historical museum specimens to delineate the boundaries of species and populations, and to hypothesize about genotypic determinants of phenotypic traits.

Target sequence capture of Barnadesioideae (Compositae) demonstrates the utility of low coverage loci in phylogenomic analyses.

Target sequence capture has emerged as a powerful method to sequence hundreds or thousands of genomic regions in a cost- and time-efficient approach. In most cases, however, targeted regions lack full sequence information for certain samples, due to taxonomic, laboratory, or stochastic factors. Loci lacking molecular data for a large number of samples are commonly excluded from downstream analyses, even though they may still contain valuable information. On the other hand, including data-poor loci may bias phylogenetic analyses. Here we use a target sequence capture dataset of an ecologically and taxonomically diverse group of spiny sunflowers (Asteraceae, or Compositae: Barnadesioideae) to test how the inclusion or exclusion of such data-poor loci affects phylogenetic inference. We investigate the sensitivity of concatenation and coalescent approaches to missing data with matrices of varying taxonomic completeness by filtering loci with different proportions of missing samples prior to data analysis. We find that missing data affect both the topology and branch support of the resulting phylogenies. The matrix containing all loci yielded the overall highest node support values, independently of the amount of missing nucleotides. These results provide empirical support to earlier suggestions based on single genes and data simulations that taxa with high amounts of missing data should not be readily dismissed as they can provide essential information for phylogenomic reconstruction.

High-throughput degraded DNA sequencing of subfossil shells of a critically endangered stenoendemic land snail in the Aegean.

High-throughput sequencing has enabled the comprehensive genetic exploration of biological diversity, especially by using natural history collections to study hard-to-find, threatened or even extinct-in-the-wild taxa. Mollusk shells are under-exploited as a source for DNA-based approaches, despite their apparent advantages in the field of conservation genetics. More particularly, degraded DNA techniques combined with high-throughput sequencing have never been used to gain insights about the DNA preservation in land snail subfossil or historical shells. Here, we applied degraded DNA analysis on two historical shells of Levantina rechingeri, a stenoendemic Critically Endangered species that has never been found alive, in order to explore the patterns of DNA preservation on land snail shells originating from the eastern Mediterranean, as well as to infer its molecular phylogenetic placement. Our results showed that centuries to decades-old DNA from an empty shell collected in an Aegean island exhibits characteristic post-mortem damage patterns similar to those observed in ancient DNA from eastern Mediterranean terrestrial animals, setting a precedent for future museomics studies on taxa distributed in areas with similar climate. Finally, genome skimming of the empty shell allowed high coverage of multiple nuclear and mitochondrial loci, enabling the phylogenetic placement of the focal taxon, the re-evaluation of its taxonomic classification, and the revealing of a new Aegean land snail lineage, Aristena genus novum. This approach is a non-invasive way to sample DNA from threatened land snail species and suitable to study the evolutionary history of taxa with cryptic ecology, stenoendemics, or extinct-in-the-wild, as well as old museum specimens.

Out of Asia: Intercontinental dispersals after the Eocene-Oligocene transition shaped the zoogeography of Limenitidinae butterflies (Lepidoptera: Nymphalidae).

Most members of the nymphalid subfamily Limenitidinae are distributed in tropical regions of Africa, Asia, and the Americas. Previous studies have inferred their higher-level phylogeny and found that Southeast Asia seems to be the center of origin, with numerous dispersal events to other continents. However, the complete biogeographic history of Limenitidinae butterflies is still largely unknown. We sampled 181 taxa from 164 species and used a metagenomic method to obtain 40 genes (mitogenomes and three nuclear ribosomal loci) for inferring the historical biogeography of the group. We find that Limenitidinae originated in eastern Asia during the early Eocene (ca. 52 Ma) and started to diversify and disperse into Africa before the end of Eocene. Intercontinental exchanges between Africa and eastern Asia continued in the early Miocene: Asian Adoliadini and Asian endemic taxa Bhagadatta had African origins in the Oligocene, whereas African Neptini dispersed in the opposite direction from Asia in the early Miocene. In addition, ancestors of the tribes Limenitidini and Adoliadini dispersed into the Neotropics and Australasia multiple times during the early-to-middle Miocene. Eastern Asia is the center of origin of the tribe Limenitidini, with several taxa disjunctly distributed in eastern Asia and the Americas. Our work provides a robust phylogenetic hypothesis of relationships in the tribe Limenitidini and suggests that the alala-species group of Adelpha should be placed in the genus Limenitis. Renamed taxa comb. nov. based on our findings are listed in the text.