Insights into the molecular phylogeny of Rhaphidophoridae, an ancient, worldwide lineage of Orthoptera.

We investigated the molecular phylogenetic divergence and historical biogeography of cave crickets belonging to the family Rhaphidophoridae (Orthoptera, Ensifera). We used taxa representative of most of the regions embraced by the family, considering samples of Macropathinae from Gondwana land (i.e., Tasmania, Australia, New Zealand, South Africa, and South America); Aemodogryllinae and Rhaphidophorinae from Southern-eastern Asia (i.e., India, Bhutan, China, Philippines and the Sulawesi islands); Dolichopodainae and Troglophilinae from the Mediterranean region and Ceuthophilinae from North America. Based on previous papers, we carried out an analysis of both mitochondrial and nuclear DNA sequences considering the ribosomal RNA units 12S, 16S, 18S, and 28S. To reconstruct phylogeny, we use cladistics, Maximum Likelihood (ML), and Bayesian analyses. All phylogenetic analyses showed the same highly supported topology generally congruent with the classical systematic arrangement at the level of each sub-family but strongly disagree with previous affinity hypotheses between sub-families based on morphological characters. Our results reveal a close affinity between Asiatic and Gondwanian taxa from one hand and between North American and Mediterranean ones from the other hand. Dating estimates indicated that Rhaphidophoridae originated in the Cretaceous period during the Mesozoic era with the ancestral area located both in the northern and southern hemisphere. A possible biogeographic scenario, reconstructed using S-DEC with RASP software, suggested that the current distribution of Rhaphidophoridae might be explained by a combination of both dispersal and vicariance events occurred especially in the ancestral populations. The radiation of Rhaphidophoridae started within the Pangaea, where the ancestor of Rhaphidophoridae occurred throughout an ancestral area including Australia, North America, and the Mediterranean region. The opening of the Atlantic Ocean promoted the divergence of North American and Mediterranean lineages while the differentiation of the southern lineages, spread from Australia, appears to be related to the fragmentation of Gondwana land.

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.

The evolutionary jigsaw puzzle of the surviving trout (Salmo trutta L. complex) diversity in the Italian region. A multilocus Bayesian approach.

Mediterranean trout populations display a diversity of phenotypes, representing a valuable model for the study of adaptation and a puzzling dilemma for taxonomists and biogeographers, which is further entangled by the widespread introgression of allochthonous genes. In this paper we analysed DNA polymorphism at multiple loci (sequence variation of the mitochondrial control region and eight nuclear fragments and length variation at eleven nuclear microsatellite loci) in representative samples of the autochthonous taxonomic diversity described in Italian trout populations (Salmo marmoratus, S. carpio, S. cenerinus, S. cettii and S. fibreni) and in samples from hatchery-originated strains of Atlantic S. trutta. We employed model-based clustering and Approximate Bayesian Computation in order to: (i) describe the phylogeographic structure of Italian autochthonous trout populations; (ii) evaluate a set of evolutionary/biogeographic models. The inclusion of hatchery-originated strains allowed to account for man-mediated allochthonous introgression in Italian populations. Our results (i) showed that the analysed sample consists of two main autochthonous evolutionary lineages, including the marble trout populations on one side ('marble' lineage) and the three peninsular populations of S. cettii, S. cenerinus and S. fibreni on the other side ('peninsular' lineage); (ii) indicated that S. carpio originated from a 'peninsular' population, with a possible, limited contribution from the 'marble' lineage; (iii) pointed out that the 'marble' lineage started diverging before the separation of the 'peninsular' lineage from Atlantic S. trutta; (iv) suggested that a model of divergence involving gene flow from the 'peninsular' population into the ancestral gene pool of 'marble' trout is most consistent with the genetic data; (v) provided evidence that the autochthonous trout gene pools in the Tyrrhenian and Adriatic basins of the Italian peninsula started diverging very recently (most likely after the last glacial maximum).

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.

An overlooked pink species of land iguana in the Galapagos.

Despite the attention given to them, the Galápagos have not yet finished offering evolutionary novelties. When Darwin visited the Galápagos, he observed both marine (Amblyrhynchus) and land (Conolophus) iguanas but did not encounter a rare pink black-striped land iguana (herein referred to as "rosada," meaning "pink" in Spanish), which, surprisingly, remained unseen until 1986. Here, we show that substantial genetic isolation exists between the rosada and syntopic yellow forms and that the rosada is basal to extant taxonomically recognized Galápagos land iguanas. The rosada, whose present distribution is a conundrum, is a relict lineage whose origin dates back to a period when at least some of the present-day islands had not yet formed. So far, this species is the only evidence of ancient diversification along the Galápagos land iguana lineage and documents one of the oldest events of divergence ever recorded in the Galápagos. Conservation efforts are needed to prevent this form, identified by us as a good species, from extinction.

Tempo and mode of species diversification in Dolichopoda cave crickets (Orthoptera, Rhaphidophoridae).

This study focuses on the phylogenetic relationships among ninety percent of known Dolichopoda species (44 out of 49); primarily a Mediterranean genus, distributed from eastern Pyrenees to Caucasus. A total of 2490 base pairs were sequenced corresponding to partial sequences of one nuclear (28SrRNA) and three mitochondrial genes (12S, 16S and COI). A relaxed molecular clock, inferred from Bayesian analysis was applied to estimate the divergence times between the lineages using well dated palaeoevents of the study areas. Molecular substitution rates per lineage per million years were also obtained for each analyzed gene. Based on the nearly complete species phylogeny, temporal patterns of diversification were analyzed using Lineage-Through-Time plots and diversification statistics. Alternative hypotheses about the colonization of present range by Dolichopoda species were tested by means of Approximate Bayesian Computation analysis. Results from this analysis carried out on the 90% of known Dolichopoda species confirmed the previous ones based on subgroups of species, suggesting the ABC analysis as a remarkable tool in biogeographic studies. Based on these results, the distribution of Dolichopoda species appears to have been shaped by the palaeogeographic and climatic events that occurred from Late Miocene up to the Plio-Pleistocene. Both vicariance and dispersal events appear to have influenced Dolichopoda species distributions, with many processes occurring in ancestral epigean populations before the invasion of the subterranean environment.

Phylogeography of Helleria brevicornis (Crustacea, Oniscidea): old and recent differentiations of an ancient lineage.

Helleria brevicornis has a disjunct, peri-Tyrrhenian distribution that mirrors that of several organisms, for which geographic vicariance is invoked, due to the geological events started with the Oligocene split of the Sardo-Corsican microplate from the Pyrenees, and successive separation between Sardinia and Corsica. Molecular phylogenetic analyses demonstrated that such a biogeographic model does not apply to Helleria. The original split of the Sardinian and Corsican lineages originated in the Early Pliocene. Further diversification occurred later. The colonization of the Tuscan archipelago, French, and Italian mainland occurred most recently, but a possible time dependency bias suggests that such colonization was driven by human-transport.

Unveiling an ancient biological invasion: molecular analysis of an old European alien, the crested porcupine (Hystrix cristata).

BACKGROUND: Biological invasions can be considered one of the main threats to biodiversity, and the recognition of common ecological and evolutionary features among invaders can help developing a predictive framework to control further invasions. In particular, the analysis of successful invasive species and of their autochthonous source populations by means of genetic, phylogeographic and demographic tools can provide novel insights into the study of biological invasion patterns. Today, long-term dynamics of biological invasions are still poorly understood and need further investigations. Moreover, distribution and molecular data on native populations could contribute to the recognition of common evolutionary features of successful aliens. RESULTS: We analyzed 2,195 mitochondrial base pairs, including Cytochrome b, Control Region and rRNA 12S, in 161 Italian and 27 African specimens and assessed the ancient invasive origin of Italian crested porcupine (Hystrix cristata) populations from Tunisia. Molecular coalescent-based Bayesian analyses proposed the Roman Age as a putative timeframe of introduction and suggested a retention of genetic diversity during the early phases of colonization. The characterization of the native African genetic background revealed the existence of two differentiated clades: a Mediterranean group and a Sub-Saharan one. Both standard population genetic and advanced molecular demography tools (Bayesian Skyline Plot) did not evidence a clear genetic signature of the expected increase in population size after introduction. Along with the genetic diversity retention during the bottlenecked steps of introduction, this finding could be better described by hypothesizing a multi-invasion event. CONCLUSION: Evidences of the ancient anthropogenic invasive origin of the Italian Hystrix cristata populations were clearly shown and the native African genetic background was preliminary described. A more complex pattern than a simple demographic exponential growth from a single propagule seems to have characterized this long-term invasion.

Molecular evolution of the pDo500 satellite DNA family in Dolichopoda cave crickets (Rhaphidophoridae).

BACKGROUND: Non-coding satellite DNA (satDNA) usually has a high turn-over rate frequently leading to species specific patterns. However, some satDNA families evolve more slowly and can be found in several related species. Here, we analyzed the mode of evolution of the pDo500 satDNA family of Dolichopoda cave crickets. In addition, we discuss the potential of slowly evolving satDNAs as phylogenetic markers. RESULTS: We sequenced 199 genomic or PCR amplified satDNA repeats of the pDo500 family from 12 Dolichopoda species. For the 38 populations under study, 39 pDo500 consensus sequences were deduced. Phylogenetic analyses using Bayesian, Maximum Parsimony, and Maximum Likelihood approaches yielded largely congruent tree topologies. The vast majority of pDo500 sequences grouped according to species designation. Scatter plots and statistical tests revealed a significant correlation between genetic distances for satDNA and mitochondrial DNA. Sliding window analyses showed species specific patterns of variable and conserved regions. The evolutionary rate of the pDo500 satDNA was estimated to be 1.63-1.78% per lineage per million years. CONCLUSIONS: The pDo500 satDNA evolves gradually at a rate that is only slightly faster than previously published rates of insect mitochondrial COI sequences. The pDo500 phylogeny was basically congruent with the previously published mtDNA phylogenies. Accordingly, the slowly evolving pDo500 satDNA family is indeed informative as a phylogenetic marker.

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.

New data on Weddell seal (Leptonychotes weddellii) colonies: A genetic analysis of a top predator from the Ross Sea, Antarctica.

In this paper, we studied the genetic variability in Weddell seal from colonies in Terra Nova Bay and Wood Bay, both sites located in the Ross Sea area, Antarctica. Two mitochondrial genes and one nuclear gene, with different mutation rates, were sequenced to investigate the haplotype diversity of the colonies and to test for a possible recent expansion. Fifteen microsatellites were used to analyze their genetic structure. Sequenced genes and microsatellites were also used to estimate the effective population size of the studied colonies and the Ross Sea seal population. The Ross Sea has a high density population of Weddel seals, with an estimated effective number of 50,000 females, and 1,341 individuals for the sampling area, possibly due to its high primary production. The colonies showed high diversity (Hd > 0.90) and many exclusive haplotypes (> 75%), likely a consequence of the surprisingly high site fidelity of Weddell seals, despite the proximity of the colonies. Nevertheless, there was low microsatellite differentiation between colonies, suggesting that they are part of a single larger population. Their expansion seemed to have started during the last glacial cycle (around 58,000 years ago), indicating that the Ross Sea seal populations have been present in the area for long time, probably due to the lack of hunting by humans and terrestrial predation. As a top predator, the role of Weddell seals in the Ross Sea ecology is crucial, and its demographic dynamics should be monitored to follow the future changes of such an important ecosystem.

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.

Phylogeography and systematics of the westernmost Italian Dolichopoda species (Orthoptera, Rhaphidophoridae).

The genus Dolichopoda (Orthoptera; Rhaphidopohoridae) is present in Italy with 9 species distributed from northwestern Italy (Piedmont and Liguria) to the southernmost Apennines (Calabria), occurring also in the Tyrrhenian coastal areas and in Sardinia. Three morphologically very close taxa have been described in Piedmont and Liguria, i.e., D. ligustica ligustica, D. ligustica septentrionalis and D. azami azami. To investigate the delimitation of the northwestern species of Dolichopoda, we performed both morphological and molecular analyses. Morphological analysis was carried out by considering diagnostic characters generally used to distinguish different taxa, as the shape of epiphallus in males and the subgenital fig in females. Molecular analysis was performed by sequencing three mitochondrial genes, 12S rRNA, 16S rRNA, partially sequenced and the entire gene of COI. Results from both morphological and molecular analyses highlighted a very homogeneous group of populations, although genetically structured. Three haplogroups geographically distributed could be distinguished and based on these results we suggest a new taxonomic arrangement. All populations, due to the priority of description, should be assigned to D. azami azami Saulcy, 1893 and to preserve the names ligustica and septentrionalis, corresponding to different genetic haplogroups, we assign them to D. azami ligustica stat. n. Baccetti & Capra, 1959 and to D. azami septentrionalis stat. n. Baccetti & Capra, 1959.

Molecular phylogeography of Dolichopoda cave crickets (Orthoptera, Rhaphidophoridae): a scenario suggested by mitochondrial DNA.

This study focuses on the phylogenetic relationships among a number of West-Mediterranean cave crickets species belonging to Dolichopoda; primarily a Mediterranean genus, distributed from eastern Pyrenees to Caucasus. In this paper, 11 Dolichopoda species from the French Pyrenees (D. linderi), the island of Corsica (D. bormansi and D. cyrnensis), and northern, central, and southern Italy (D. ligustica, D. schiavazzii, D. aegilion, D. baccettii, D. laetitiae, D. geniculata, D. capreensis, and D. palpata) were studied. Two more species, one from the Caucasus, D. euxina, and one from Greece, D. remyi, were also included in the analyses, together with more distant species within the same family to be used as outgroups. Fifteen hundred base pairs of mitochondrial DNA, corresponding to the small subunit of the ribosomal RNA (16S rRNA) and to the subunit I of the cytochrome oxidase I (COI), were sequenced in order to clarify the phylogenetic relationships and biogeography of this group of Mediterranean cave crickets. The molecular data are congruent with a phylogeographic pattern; with the geographically close species also the most related ones. Based on mtDNA divergence, the present-day distribution of genetic diversity seems to have been impacted by climatic events due to glacial and interglacial cycles that have characterized the Pleistocene era.

MOLECULAR EVOLUTIONARY DIVERGENCE AMONG NORTH AMERICAN CAVE CRICKETS. I. ALLOZYME VARIATION.

Forty-nine populations of nine species of North American cave crickets (genera Euhadenoecus and Hadenoecus) have been studied for genetic variation at 41 loci by electrophoresis. Wright's FST , Slatkin's Nm* gene-flow estimator, and Nei's genetic distances (D) have been used to compare closely related species that have different ecological requirements (cave vs. forest species), distribution patterns, and/or different degrees of geographic isolation among populations. Cave and epigean (noncave) species differ greatly in their levels of genetic differentiation. Cave species have lower rates of gene exchange (low Nm, high D, and FST ) than epigean species. Within cave species the degree of genetic differentiation among populations is correlated with the limestone structure of the area where the species occur. Species or groups of populations inhabiting areas where the limestone is continuous and highly fissured (e.g., H. subterraneus populations in the Mammoth Cave region) are genetically less differentiated than are populations occurring in regions where the limestone distribution is more fragmented, such as the Appalachian Ridge where E. fragilis occurs; this effect is more extreme in Central Tennessee where genetically differentiated E. insolitus populations occur only a few kilometers apart. This suggests that epigean dispersal through forest habitat in cave-dwelling species is negligable. For forest species, the data indicate relatively recent radiation with ongoing gene exchange among populations. For cave species, the distribution of protein polymorphisms is apparently more a function of historical patterns of gene exchange rather than current gene exchange. Phylogenetic relationships were studied using cluster analyses (UPGMA and Wagner algorithms) of Nei's and Edwards' genetic distances and multivariate analysis (correspondence analysis) of the raw allele frequencies. Different algorithms result in branching patterns that are similar but not entirely concordant with one another or with the phylogeny based on morphology.

INDIRECT METHODS TO ESTIMATE GENE FLOW IN CAVE AND SURFACE POPULATIONS OF ANDRONISCUS DENTIGER (ISOPODA: ONISCIDEA).

We estimateci gene flow among several populations of the troglophilic woodlouse Androniscus dentiger from central Italy using allozyme data. Estimates of gene flow were obtained from GST , θ, and the private alleles method, after being tested whether the assumption of the population genetic model and the assumption of neutrality of alleles had been met. Hierarchical analysis of gene flow has been used to investigate the geographic scale at which gene exchange can actually occur. Results showed that, independent of the methods, no ongoing gene flow can be detected among populations, even among geographically proximate ones. Genetic drift is likely the main agent shaping the pattern of genetic divergence among these populations. Patterns of past and ongoing gene flow were considered, as surface populations have become genetically isolated more recently than cave populations. In general, all three methods to estimate gene flow provided Nm estimates of the same magnitude.