Testing Classical Species Properties with Contemporary Data: How "Bad Species" in the Brassy Ringlets (Erebia tyndarus complex, Lepidoptera) Turned Good.

All species concepts are rooted in reproductive, and ultimately genealogical, relations. Genetic data are thus the most important source of information for species delimitation. Current ease of access to genomic data and recent computational advances are blooming a plethora of coalescent-based species delimitation methods. Despite their utility as objective approaches to identify species boundaries, coalescent-based methods (1) rely on simplified demographic models that may fail to capture some attributes of biological species, (2) do not make explicit use of the geographic information contained in the data, and (3) are often computationally intensive. In this article, we present a case of species delimitation in the Erebia tyndarus species complex, a taxon regarded as a classic example of problematic taxonomic resolution. Our approach to species delimitation used genomic data to test predictions rooted in the biological species concept and in the criterion of coexistence in sympatry. We (1) obtained restriction-site associated DNA (RAD) sequencing data from a carefully designed sample, (2) applied two genotype clustering algorithms to identify genetic clusters, and (3) performed within-clusters and between-clusters analyses of isolation by distance as a test for intrinsic reproductive barriers. Comparison of our results with those from a Bayes factor delimitation coalescent-based analysis, showed that coalescent-based approaches may lead to overconfident splitting of allopatric populations, and indicated that incorrect species delimitation is likely to be inferred when an incomplete geographic sample is analyzed. While we acknowledge the theoretical justification and practical usefulness of coalescent-based species delimitation methods, our results stress that, even in the phylogenomic era, the toolkit for species delimitation should not dismiss more traditional, biologically grounded, approaches coupling genomic data with geographic information.

COI Barcodes combined with multilocus data for representative Aporia taxa shed light on speciation in the high altitude Irano-Turanian mountain plateaus (Lepidoptera: Pieridae).

Even though the high plateaus of Qinghai-Tibet and Iran share many faunal elements, the historical biogeography of the species present in this area are not very well understood. We present a complete COI barcode library for Aporia Hübner and a first comprehensive phylogeny for the genus including all known species and majority of subspecies using ten available genes (COI-COII, ND1, ND5, Cytb, EF-1a, Wg, 16S, 28S-D2/D3 and 28S-D8). We then focus on A. leucodice (Eversmann, 1843) and related taxa in order to resolve some long-standing taxonomic issues in this species-group. Based on DNA sequence data as well as morphology, we raise Aporia illumina (Grum-Grshimailo 1890) stat. nov. (= pseudoillumina Tshikolovets 2021 syn. nov.) as a distinct species and designate a lectotype; synonymize Aporia leucodice leucodice Eversmann, 1843 (= A. l. morosevitshae Sheljuzhko, 1908 syn. nov.); and describe a new species, Aporia ahura sp. nov., from the Central Alborz Mountains in northern Iran.

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.

ClimCKmap, a spatially, temporally and climatically explicit distribution database for the Italian fauna.

Understanding and counteracting biodiversity losses requires quantitative knowledge on species distribution and abundance across space and time, as well as integrated and interoperable information on climate conditions and climatic changes. In this paper we developed a new biodiversity-climate database for Italy, ClimCKmap, based on the critical analysis, quality estimation and subsequent integration of the CKmap database with several high-resolution climate datasets. The original database was quality-checked for errors in toponym, species name and dating; the retained records were georeferenced and their distribution polygonised via Voronoi tessellation. We then integrated the species distribution information with several high-resolution climatic datasets: average monthly minimum and maximum temperature and total monthly precipitation were reconstructed for each Voronoi cell and year. The resulting database contains 268,977 occurrence records from 8,445 binomials and 16,332 localities, dating between 1680 and 2006 CE. This dataset, fully available at https://doi.org/10.6084/m9.figshare.7906739.v4 and http://hdl.handle.net/21.11125/a91f85cb-befd-4e14-8e83-24f17c4a0491 , represents the largest, fully quality-checked, spatially, temporally and climatically explicit distribution database ever assembled for the Italian fauna, now ready for scientific exploitation.

Chemically mediated species recognition in two sympatric Grayling butterflies: Hipparchia fagi and Hipparchia hermione (Lepidoptera: Nymphalidae, Satyrinae).

Pheromones are known to play an important role in butterfly courtship and may influence both individual reproductive success and reproductive isolation between species. Recent studies have focused on courtship in Hipparchia butterflies (Nymphalidae: Satyrinae) emphasizing morphological and behavioural traits, as well as genetic differences. Behavioural observations suggested a role for chemical cues in mate and species recognition, where the androconial scales on the forewings of these species may be involved in chemical communication between individuals. Cchemical-mediated signals have received relatively little attention in this genus. Here, we report the results of a three-year investigation of the volatile organic compounds (VOCs) released by Hipparchia fagi and H. hermione in order to identify differences in VOCs between these species where they live in syntopy. Our study was carried out using an array of cross-selective sensors known as an "Electronic Nose" (EN) that operates by converting chemical patterns into patterns of sensor signals. While the identity of volatile compounds remained unknown, sensor signals can be compared to identify similar or dissimilar chemical patterns. Based on the EN signals, our results showed that: 1) the two sexes have a similar VOCs pattern in H. fagi, while they significantly diverge in H. hermione; 2) VOCs patterns were different between females of the two species, while those of males were not.

Tackling critical parameters in metazoan meta-barcoding experiments: a preliminary study based on coxI DNA barcode.

Nowadays DNA meta-barcoding is a powerful instrument capable of quickly discovering the biodiversity of an environmental sample by integrating the DNA barcoding approach with High Throughput Sequencing technologies. It mainly consists of the parallel reading of informative genomic fragment/s able to discriminate living entities. Although this approach has been widely studied, it still needs optimization in some necessary steps requested in its advanced accomplishment. A fundamental element concerns the standardization of bioinformatic analyses pipelines. The aim of the present study was to underline a number of critical parameters of laboratory material preparation and taxonomic assignment pipelines in DNA meta-barcoding experiments using the cytochrome oxidase subunit-I (coxI) barcode region, known as a suitable molecular marker for animal species identification. We compared nine taxonomic assignment pipelines, including a custom in-house method, based on Hidden Markov Models. Moreover, we evaluated the potential influence of universal primers amplification bias in qPCR, as well as the correlation between GC content with taxonomic assignment results. The pipelines were tested on a community of known terrestrial invertebrates collected by pitfall traps from a chestnut forest in Italy. Although the present analysis was not exhaustive and needs additional investigation, our results suggest some potential improvements in laboratory material preparation and the introduction of additional parameters in taxonomic assignment pipelines. These include the correct setup of OTU clustering threshold, the calibration of GC content affecting sequencing quality and taxonomic classification, as well as the evaluation of PCR primers amplification bias on the final biodiversity pattern. Thus, careful attention and further validation/optimization of the above-mentioned variables would be required in a DNA meta-barcoding experimental routine.

Is radon emission in caves causing deletions in satellite DNA sequences of cave-dwelling crickets?

The most stable isotope of radon, 222Rn, represents the major source of natural radioactivity in confined environments such as mines, caves and houses. In this study, we explored the possible radon-related effects on the genome of Dolichopoda cave crickets (Orthoptera, Rhaphidophoridae) sampled in caves with different concentrations of radon. We analyzed specimens from ten populations belonging to two genetically closely related species, D. geniculata and D. laetitiae, and explored the possible association between the radioactivity dose and the level of genetic polymorphism in a specific family of satellite DNA (pDo500 satDNA). Radon concentration in the analyzed caves ranged from 221 to 26,000 Bq/m3. Specimens coming from caves with the highest radon concentration showed also the highest variability estimates in both species, and the increased sequence heterogeneity at pDo500 satDNA level can be explained as an effect of the mutation pressure induced by radon in cave. We discovered a specific category of nuclear DNA, the highly repetitive satellite DNA, where the effects of the exposure at high levels of radon-related ionizing radiation are detectable, suggesting that the satDNA sequences might be a valuable tool to disclose harmful effects also in other organisms exposed to high levels of radon concentration.