Accounting for Errors in Data Improves Divergence Time Estimates in Single-cell Cancer Evolution.

Single-cell sequencing provides a new way to explore the evolutionary history of cells. Compared to traditional bulk sequencing, where a population of heterogeneous cells is pooled to form a single observation, single-cell sequencing isolates and amplifies genetic material from individual cells, thereby preserving the information about the origin of the sequences. However, single-cell data are more error-prone than bulk sequencing data due to the limited genomic material available per cell. Here, we present error and mutation models for evolutionary inference of single-cell data within a mature and extensible Bayesian framework, BEAST2. Our framework enables integration with biologically informative models such as relaxed molecular clocks and population dynamic models. Our simulations show that modeling errors increase the accuracy of relative divergence times and substitution parameters. We reconstruct the phylogenetic history of a colorectal cancer patient and a healthy patient from single-cell DNA sequencing data. We find that the estimated times of terminal splitting events are shifted forward in time compared to models which ignore errors. We observed that not accounting for errors can overestimate the phylogenetic diversity in single-cell DNA sequencing data. We estimate that 30-50% of the apparent diversity can be attributed to error. Our work enables a full Bayesian approach capable of accounting for errors in the data within the integrative Bayesian software framework BEAST2.

Phylogenomics of Anguis and Pseudopus (Squamata, Anguidae) indicates Balkan-Apennine mitochondrial capture associated with the Messinian event.

A dated phylogenetic hypothesis on the evolutionary history of the extant taxa of the Western Palearctic lizards Anguis and Pseudopus is revised using genome-wide nuclear DNA and mitogenomes. We found overall concordance between nuclear and mitochondrial DNA phylogenies, with one significant exception - the Apennine A. veronensis. In mitochondrial DNA, this species forms a common clade with the earliest diverging lineage, the southern Balkan endemic A. cephallonica, while it clusters together with A. fragilis in nuclear DNA. The nuclear phylogeny conforms to the morphology, which is relatively similar between A. veronensis and A. fragilis. The most plausible explanation for the mitonuclear discordance is ancient mitochondrial capture from the Balkan ancestor of A. cephallonica to the Apennine population of the A. fragilis-veronensis ancestor. We hypothesize that this capture occurred only in a geographically restricted population. The dating of this presumed mitochondrial introgression and capture coincides with the Messinian event, when the Balkan and Apennine Peninsulas were presumably largely connected. The dated nuclear phylogenomic reconstruction estimated the divergence of A. cephallonica around 12 Mya, while the sister clade representing the A. fragilis species complex consisting of the sister species A. fragilis-A. veronensis and A. colchica-A. graeca further diversified around 7 Mya. The depth of nuclear divergence among the evolutionary lineages of Pseudopus (0.5-1.2 Mya) supports their subspecies status.

The phylogeny of Dendropsophini (Anura: Hylidae: Hylinae).

The relationships of the hyline tribe Dendropsophini remain poorly studied, with most published analyses dealing with few of the species groups of Dendropsophus. In order to test the monophyly of Dendropsophini, its genera, and the species groups currently recognized in Dendropsophus, we performed a total evidence phylogenetic analysis. The molecular dataset included sequences of three mitochondrial and five nuclear genes from 210 terminals, including 12 outgroup species, the two species of Xenohyla, and 93 of the 108 recognized species of Dendropsophus. The phenomic dataset includes 46 terminals, one per species (34 Dendropsophus, one Xenohyla, and 11 outgroup species). Our results corroborate the monophyly of Dendropsophini and the reciprocal monophyly of Dendropsophus and Xenohyla. Some species groups of Dendropsophus are paraphyletic (the D. microcephalus, D. minimus, and D. parviceps groups, and the D. rubicundulus clade). On the basis of our results, we recognize nine species groups; for three of them (D. leucophyllatus, D. microcephalus, and D. parviceps groups) we recognize some nominal clades to highlight specific morphology or relationships and facilitate species taxonomy. We further discuss the evolution of oviposition site selection, where our results show multiple instances of independent evolution of terrestrial egg clutches during the evolutionary history of Dendropsophus.

Low doses of Bisphenol S affect post-translational modifications of sperm proteins in male mice.

BACKGROUND: Bisphenol S (BPS) is increasingly used as a replacement for bisphenol A in the manufacture of products containing polycarbonates and epoxy resins. However, further studies of BPS exposure are needed for the assessment of health risks to humans. In this study we assessed the potential harmfulness of low-dose BPS on reproduction in male mice. METHODS: To simulate human exposure under experimental conditions, 8-week-old outbred ICR male mice received 8 weeks of drinking water containing a broad range of BPS doses [0.001, 1.0, or 100 µg/kg body weight (bw)/day, BPS1-3] or vehicle control. Mice were sacrificed and testicular tissue taken for histological analysis and protein identification by nano-liquid chromatography/mass spectrometry (MS) and sperm collected for immunodetection of acetylated lysine and phosphorylated tyrosine followed by protein characterisation using matrix-assisted laser desorption ionisation time-of-flight MS (MALDI-TOF MS). RESULTS: The results indicate that compared to vehicle, 100 µg/kg/day exposure (BPS3) leads to 1) significant histopathology in testicular tissue; and, 2) higher levels of the histone protein γH2AX, a reliable marker of DNA damage. There were fewer mature spermatozoa in the germ layer in the experimental group treated with 1 µg/kg bw (BPS2). Finally, western blot and MALDI-TOF MS studies showed significant alterations in the sperm acetylome and phosphorylome in mice treated with the lowest exposure (0.001 µg/kg/day; BPS1), although the dose is several times lower than what has been published so far. CONCLUSIONS: In summary, this range of qualitative and quantitative findings in young male mice raise the possibility that very low doses of BPS may impair mammalian reproduction through epigenetic modifications of sperm proteins.

Sonic Hedgehog and Triiodothyronine Pathway Interact in Mouse Embryonic Neural Stem Cells.

Neural stem cells are fundamental to development of the central nervous system (CNS)-as well as its plasticity and regeneration-and represent a potential tool for neuro transplantation therapy and research. This study is focused on examination of the proliferation dynamic and fate of embryonic neural stem cells (eNSCs) under differentiating conditions. In this work, we analyzed eNSCs differentiating alone and in the presence of sonic hedgehog (SHH) or triiodothyronine (T3) which play an important role in the development of the CNS. We found that inhibition of the SHH pathway and activation of the T3 pathway increased cellular health and survival of differentiating eNSCs. In addition, T3 was able to increase the expression of the gene for the receptor smoothened (Smo), which is part of the SHH signaling cascade, while SHH increased the expression of the T3 receptor beta gene (Thrb). This might be the reason why the combination of SHH and T3 increased the expression of the thyroxine 5-deiodinase type III gene (Dio3), which inhibits T3 activity, which in turn affects cellular health and proliferation activity of eNSCs.

Warfare induces post-marital residence change.

Post-marital residence is a sex-biased dispersal defined by the place where a newly-wed couple lives after marriage. Common choices for this practice include patrilocal residence, where the couple lives with the man's family, and matrilocal residence, where they live with the woman's family. Deviations from accepted practice typically invoke strong sanctions, but despite this pressure to conform to post-marital residence norms, residence states are unexpectedly dynamic over time. Theories have been proposed to explain the pressures, both internal and external, that drive these changes in post-marital residence state. Two of the most popular emphasize the importance of warfare, but are largely restricted to qualitative statements. Here, we develop an agent-based model that captures key features of these theories, with a particular focus on warfare. We show that warfare can change post-marital residence practices, but such change only propagates through a wider network of communities under a narrow set of conditions. Additional factors, potentially including a strong sex-bias in the division of labor, are required to induce change more widely. While warfare thus serves as an important trigger for residence change, multiple interacting forces appear to be necessary to shift communities between different post-marital residence states under most conditions.

Proteins adsorbed to a polysulfone hemodialysis membrane under heparin and citrate anticoagulation regimens.

The study aim was to compare molecular-level effects (blood-dialyzer interactions) of heparin and citrate anticoagulation using proteome-wide analysis of biofilm adsorbed to dialysis membrane. Ten patients receiving maintenance hemodialysis were examined in a crossover design under three different anticoagulation regimens, namely citrate, heparin, and anticoagulation-free (control). Following a regular hemodialysis session (4 hours, polysulfone membrane), dialyzers were flushed and the surface biofilm eluted by acetic acid. Protein composition of the eluates was determined by 2-dimensional gel electrophoresis and resulting patterns compared between regimens. Proteins responsible for the difference were identified by mass spectrometry. Citrate anticoagulation was associated with significantly less protein adsorption to the membrane than heparin (2.2 [1.1-2.9] mg vs. 6.5 [2.9-11.6] mg, P = 0.009). Among the proteins identified as major discriminators between citrate and the other regimens, fibrin α-chain fragments of molecular weight below 40 kDa prevailed. In these fragments, an analysis of the amino acid sequence has been performed by comparison with the UniProt database. It showed missing α-chain cross-links. On the contrary, heparin prevented adsorption and cleavage of several heparin-binding proteins; especially complement factor H-related protein 3, insulin-like growth factor binding proteins (2, 4, and 5), and chemerin. Compared to heparin, citrate is associated with less protein adsorption and imperfectly crosslinked fibrin clot formation. Membrane adsorptive properties are significantly modified by the anticoagulation regimen.

Long-term exposure to very low doses of bisphenol S affects female reproduction.

Bisphenols belong to the endocrine disruptors, affecting reproduction even in extremely low doses. Bisphenol S (BPS) has become widely used as a substitute for the earlier-used bisphenol A; however, its harmlessness is questionable. The aim of this study was to evaluate the effect of BPS on folliculogenesis and oocyte quality after in vivo exposure to low doses of BPS. Four-week-old ICR females (n = 16 in each experimental group) were exposed to vehicle control (VC), BPS1 (0.001 ng BPS.g/bw/day), BPS2 (0.1 ng.g/bw/day), BPS3 (10 ng.g/bw/day) and BPS4 (100 ng.g/bw/day) for 4 weeks. Ovaries were subjected to stereology and nano liquid chromatography-mass spectrometry (LC/MS). Simultaneously, metaphase II oocytes were obtained after pregnant mare serum gonadotrophin and human chorionic gonadotrophin administration, followed by immunostaining. In particular, mating and two-cell embryo flushing were performed. We observed that BPS decreases the amount of ovarian follicles and BPS2 (0.1 ng.g/bw/day) affects the volume of antral follicles. Accordingly, ovarian proteome is affected after BPS2 treatment. While BPS2 dosing results mainly in cytoskeletal damage in matured oocytes, the effects of BPS3 and BPS4 seem to be due instead to epigenetic alterations in oocytes. Arguably, these changes lead to observed affection of in vivo fertilization rate after BPS3 and BPS4 treatment. BPS significantly affects female reproduction astoundingly in extremely low doses. These findings underline the necessity to assess the risk of ongoing BPS exposure for public health.

A simple, time-saving, microwave-assisted periodic acid-Schiff´s staining of glycoproteins on 1D electrophoretic gels.

We introduce an optimized periodic acid-Schiff´s staining of glycoproteins on 1D electrophoretic gels. Thanks to heating in a household microwave oven the protocol of standard periodic acid-Schiff´s staining has been accelerated from 6 h to below 10 min employing standard chemistry. At the same time, we show that the microwave-assisted glycoprotein staining is at least as sensitive as the conventional approach. All glycoproteins stained by the microwave-accelerated procedure were successfully identified using MALDI TOF/TOF mass spectrometry. The ensuing reduction in gel staining time and simplification of the staining protocol should significantly increase laboratory throughput when glycoprotein detection on electrophoretic gels is required in large numbers.

Early urinary biomarkers of diabetic nephropathy in type 1 diabetes mellitus show involvement of kallikrein-kinin system.

BACKGROUND: Additional urinary biomarkers for diabetic nephropathy (DN) are needed, providing early and reliable diagnosis and new insights into its mechanisms. Rigorous selection criteria and homogeneous study population may improve reproducibility of the proteomic approach. METHODS: Long-term type 1 diabetes patients without metabolic comorbidities were included, 11 with sustained microalbuminuria (MA) and 14 without MA (nMA). Morning urine proteins were precipitated and resolved by 2D electrophoresis. Principal component analysis (PCA) and Projection to latent structures discriminatory analysis (PLS-DA) were adopted to assess general data validity, to pick protein fractions for identification with mass spectrometry (MS), and to test predictive value of the resulting model. RESULTS: Proteins (n = 113) detected in more than 90% patients were considered representative. Unsupervised PCA showed excellent natural data clustering without outliers. Protein spots reaching Variable Importance in Projection score above 1 in PLS (n = 42) were subjected to MS, yielding 33 positive identifications. The PLS model rebuilt with these proteins achieved accurate classification of all patients (R2X = 0.553, R2Y = 0.953, Q2 = 0.947). Thus, multiple earlier recognized biomarkers of DN were confirmed and several putative new biomarkers suggested. Among them, the highest significance was met in kininogen-1. Its activation products detected in nMA patients exceeded by an order of magnitude the amount found in MA patients. CONCLUSIONS: Reducing metabolic complexity of the diseased and control groups by meticulous patients' selection allows to focus the biomarker search in DN. Suggested new biomarkers, particularly kininogen fragments, exhibit the highest degree of correlation with MA and substantiate validation in larger and more varied cohorts.

Contrasting evolutionary histories of the legless lizards slow worms (Anguis) shaped by the topography of the Balkan Peninsula.

BACKGROUND: Genetic architecture of a species is a result of historical changes in population size and extent of distribution related to climatic and environmental factors and contemporary processes of dispersal and gene flow. Population-size and range contractions, expansions and shifts have a substantial effect on genetic diversity and intraspecific divergence, which is further shaped by gene-flow limiting barriers. The Balkans, as one of the most important sources of European biodiversity, is a region where many temperate species persisted during the Pleistocene glaciations and where high topographic heterogeneity offers suitable conditions for local adaptations of populations. In this study, we investigated the phylogeographical patterns and demographic histories of four species of semifossorial slow-worm lizards (genus Anguis) present in the Balkan Peninsula, and tested the relationship between genetic diversity and topographic heterogeneity of the inhabited ranges. RESULTS: We inferred phylogenetic relationships, compared genetic structure and historical demography of slow worms using nucleotide sequence variation of mitochondrial DNA. Four Anguis species with mostly parapatric distributions occur in the Balkan Peninsula. They show different levels of genetic diversity. A signature of population growth was detected in all four species but with various courses in particular populations. We found a strong correlation between genetic diversity of slow-worm populations and topographic ruggedness of the ranges (mountain systems) they inhabit. Areas with more rugged terrain harbour higher genetic diversity. CONCLUSIONS: Phylogeographical pattern of the genus Anguis in the Balkans is concordant with the refugia-within-refugia model previously proposed for both several other taxa in the region and other main European Peninsulas. While slow-worm populations from the southern refugia mostly have restricted distributions and have not dispersed much from their refugial areas, populations from the extra-Mediterranean refugia in northern parts of the Balkans have colonized vast areas of eastern, central, and western Europe. Besides climatic historical events, the heterogeneous topography of the Balkans has also played an important role in shaping genetic diversity of slow worms.

Conservation of sex chromosomes in lacertid lizards.

Sex chromosomes are believed to be stable in endotherms, but young and evolutionary unstable in most ectothermic vertebrates. Within lacertids, the widely radiated lizard group, sex chromosomes have been reported to vary in morphology and heterochromatinization, which may suggest turnovers during the evolution of the group. We compared the partial gene content of the Z-specific part of sex chromosomes across major lineages of lacertids and discovered a strong evolutionary stability of sex chromosomes. We can conclude that the common ancestor of lacertids, living around 70 million years ago (Mya), already had the same highly differentiated sex chromosomes. Molecular data demonstrating an evolutionary conservation of sex chromosomes have also been documented for iguanas and caenophidian snakes. It seems that differences in the evolutionary conservation of sex chromosomes in vertebrates do not reflect the distinction between endotherms and ectotherms, but rather between amniotes and anamniotes, or generally, the differences in the life history of particular lineages.

Out of Africa: Phylogeny and biogeography of the widespread genus Acanthodactylus (Reptilia: Lacertidae).

Acanthodactylus lizards are among the most diverse and widespread diurnal reptiles in the arid regions spanning from North Africa across to western India. Acanthodactylus constitutes the most species-rich genus in the family Lacertidae, with over 40 recognized species inhabiting a wide variety of dry habitats. The genus has seldom undergone taxonomic revisions, and although there are a number of described species and species-groups, their boundaries, as well as their interspecific relationships, remain largely unresolved. We constructed a multilocus phylogeny, combining data from two mitochondrial (12S, cytb) and three nuclear (MC1R, ACM4, c-mos) markers for 302 individuals belonging to 36 known species, providing the first large-scale time-calibrated molecular phylogeny of the genus. We evaluated phylogenetic relationships between and within species-groups, and assessed Acanthodactylus biogeography across its known range. Acanthodactylus cladogenesis is estimated to have originated in Africa due to vicariance and dispersal events from the Oligocene onwards. Radiation started with the separation into three clades: the Western and scutellatus clades largely distributed in North Africa, and the Eastern clade occurring mostly in south-west Asia. Most Acanthodactylus species diverged during the Miocene, possibly as a result of regional geological instability and climatic changes. We support most of the current taxonomic classifications and phylogenetic relationships, and provide genetic validity for most species. We reveal a new distinct blanfordii species-group, suggest new phylogenetic positions (A. hardyi, A. masirae), and synonymize several species and subspecies (A. lineomaculatus, A. boskianus khattensis and A. b. nigeriensis) with their phylogenetically closely-related species. We recommend a thorough systematic revision of taxa, such as A. guineensis, A. grandis, A. dumerilii, A. senegalensis and the pardalis and erythrurus species-groups, which exhibit high levels of intraspecific variability, and clear evidence of phylogenetic complexity.

A Skull Might Lie: Modeling Ancestral Ranges and Diet from Genes and Shape of Tree Squirrels.

Tropical forests of Central and South America represent hotspots of biological diversity. Tree squirrels of the tribe Sciurini are an excellent model system for the study of tropical biodiversity as these squirrels disperse exceptional distances, and after colonizing the tropics of the Central and South America, they have diversified rapidly. Here, we compare signals from DNA sequences with morphological signals using pictures of skulls and computational simulations. Phylogenetic analyses reveal step-wise geographic divergence across the Northern Hemisphere. In Central and South America, tree squirrels form two separate clades, which split from a common ancestor. Simulations of ancestral distributions show western Amazonia as the epicenter of speciation in South America. This finding suggests that wet tropical forests on the foothills of Andes possibly served as refugia of squirrel diversification during Pleistocene climatic oscillations. Comparison of phylogeny and morphology reveals one major discrepancy: Microsciurus species are a single clade morphologically but are polyphyletic genetically. Modeling of morphology-diet relationships shows that the only group of species with a direct link between skull shape and diet are the bark-gleaning insectivorous species of Microsciurus. This finding suggests that the current designation of Microsciurus as a genus is based on convergent ecologically driven changes in morphology.

SigHunt: horizontal gene transfer finder optimized for eukaryotic genomes.

MOTIVATION: Genomic islands (GIs) are DNA fragments incorporated into a genome through horizontal gene transfer (also called lateral gene transfer), often with functions novel for a given organism. While methods for their detection are well researched in prokaryotes, the complexity of eukaryotic genomes makes direct utilization of these methods unreliable, and so labour-intensive phylogenetic searches are used instead. RESULTS: We present a surrogate method that investigates nucleotide base composition of the DNA sequence in a eukaryotic genome and identifies putative GIs. We calculate a genomic signature as a vector of tetranucleotide (4-mer) frequencies using a sliding window approach. Extending the neighbourhood of the sliding window, we establish a local kernel density estimate of the 4-mer frequency. We score the number of 4-mer frequencies in the sliding window that deviate from the credibility interval of their local genomic density using a newly developed discrete interval accumulative score (DIAS). To further improve the effectiveness of DIAS, we select informative 4-mers in a range of organisms using the tetranucleotide quality score developed herein. We show that the SigHunt method is computationally efficient and able to detect GIs in eukaryotic genomes that represent non-ameliorated integration. Thus, it is suited to scanning for change in organisms with different DNA composition. AVAILABILITY AND IMPLEMENTATION: Source code and scripts freely available for download at http://www.iba.muni.cz/index-en.php?pg=research-data-analysis-tools-sighunt are implemented in C and R and are platform-independent. CONTACT: 376090@mail.muni.cz or martinkova@ivb.cz.

Hidden relationships and genetic diversity: Molecular phylogeny and phylogeography of the Levantine lizards of the genus Phoenicolacerta (Squamata: Lacertidae).

The Levant region witnessed dramatic tectonic events and climatic fluctuations that changed the historical landscape of the area and consequently influenced the cladogenesis and distribution of the local biota. In this study we use information from two mitochondrial and two nuclear genes and species delimitation methods in order to obtain the first robust time-calibrated molecular phylogeny of the Levantine rock lizards of the genus Phoenicolacerta. We sampled from across its distributional range with the aim to clarify its systematics, biogeography and evolution. Our results suggest that the genus includes two well-supported clades, one comprising solely the montane species Phoenicolacerta kulzeri, and the other including the three remaining species, the relatively widespread, P. laevis, the Syrian-Turkish P. cyanisparsa and the Cypriot endemic P. troodica. We found that both P. laevis and P. cyanisparsa are not monophyletic, as the Turkish populations of P. laevis branch within P. cyanisparsa. We found high levels of undescribed diversity within P. laevis which necessitate a thorough revision. We suggest that Phoenicolacerta started radiating during the mid-late Miocene, and that both vicariance and dispersal events shaped the diversification and distribution of the genus concomitantly with the formation of major geological structures and climatic fluctuations in the Levant. These results highlight the region as an important center of speciation, contributing to the species diversity of the eastern Mediterranean.

Speciation history and widespread introgression in the European short-call tree frogs (Hyla arborea sensu lato, H. intermedia and H. sarda).

European tree frogs (Hyla) characterized by short temporal parameters of the advertisement call form six genetically differentiated but morphologically cryptic taxa, H. arborea sensu stricto, H. orientalis and H. molleri from across Europe to western Asia (together referred to as H. arborea sensu lato), two putative taxa within H. intermedia (Northern and Southern) from the Italian Peninsula and Sicily, and H. sarda from Sardinia and Corsica. Here, we assess species limits and phylogenetic relationships within these 'short-call tree frogs' based on mitochondrial DNA and nuclear protein-coding markers. The mitochondrial and nuclear genes show partly incongruent phylogeographic patterns, which point to a complex history of gene flow across taxa, particularly in the Balkans. To test the species limits in the short-call tree frogs and to infer the species tree, we used coalescent-based approaches. The monophyly of H. arborea sensu lato is supported by the mtDNA as well as by the all-gene species tree. The Northern and Southern lineages of H. intermedia have been connected by nuclear gene flow (despite their deep mtDNA divergence) and should be treated as conspecific. On the contrary, the parapatric taxa within H. arborea sensu lato should be considered distinct species (H. arborea, H. orientalis, H. molleri) based on the coalescent analysis, although signs of hybridization were detected between them (H. arborea×H. orientalis; H. arborea×H. molleri). A mitochondrial capture upon secondary contact appears to explain the close mtDNA relationship between the geographically remote Iberian H. molleri and H. orientalis from around the Black Sea. Introgressive hybridization occurred also between the Balkan H. arborea and northern Italian H. intermedia, and between the Minor Asiatic H. orientalis and Arabian H. felix arabica (the latter belonging to a different acoustic group/clade). Our results shed light on the species limits in the European short-call tree frogs and show that introgression played an important role in the evolutionary history of the short-call tree frogs and occurred even between taxa supported as distinct species.

An ancient lineage of slow worms, genus Anguis (Squamata: Anguidae), survived in the Italian Peninsula.

Four species of legless anguid lizard genus Anguis have been currently recognized: A. fragilis from western and central Europe, A. colchica from eastern Europe and western Asia, A. graeca from southern Balkans, and A. cephallonica from the Peloponnese. Slow worms from the Italian Peninsula have been considered conspecific with A. fragilis, despite the fact that the region served as an important speciation center for European flora and fauna, and included some Pleistocene glacial refugia. We used mitochondrial and nuclear DNA sequences to investigate the systematic and phylogenetic position of the Italian slow-worm populations and morphological analyses to test for phenotypic differentiation from A. fragilis from other parts of Europe. Our phylogenetic analyses revealed that Italian slow worms form a distinct deeply differentiated mtDNA clade, which presumably diverged during or shortly after the basal radiation within the genus Anguis. In addition, the specimens assigned to this clade bear distinct haplotypes in nuclear PRLR gene and show morphological differentiation from A. fragilis. Based on the differentiation in all three independent markers, we propose to assign the Italian clade species level under the name Anguis veronensisPollini, 1818. The newly recognized species is distributed throughout the Italian Peninsula to the Southern Alps and south-eastern France. We hypothesize that the Tertiary Alpine orogeny with subsequent vicariance might have played a role in differentiation of this species. The current genetic variability was later presumably shaped in multiple glacial refugia within the Italian Peninsula, with the first splitting event separating populations from the region of the Dolomite Mountains.

Calochroa miroklichai sp. nov., a new species from Thailand with rectification of different concepts of Calochroa tritoma (Schmidt-Goebel) and comparisons to similar species. (Coleoptera: Cicindelidae).

A new species of the genus Calochroa Hope, 1838 is described as new to science from Thailand. Two different concepts of Calochroa tritoma (Schmidt-Goebel, 1846) are rectified here, stating that the correct concept of C. tritoma (fully corresponding to the holotype in NMPC) is that presented by Naviaux (1991), Naviaux & Pinratana (2004) and Klícha & Durícek (2021), while C. tritoma sensu Pearson et al. (2020) is described here as Calochroa miroklichai sp. nov. Accordingly, C. tritoma sensu Acciavatti & Pearson (1989) represents a different, probably an undescribed species. C. anometallescens (W. Horn, 1893) and Calochroa laurae (Gestro, 1893), as well as the new species, have been partly misidentified as C. tritoma in collections. The occurrence of C. tritoma is newly restricted to Thailand and Myanmar only. The above-mentioned species, as well as Calochroa fumikoae Wiesner & Phyu, 2019 are compared to C. miroklichai sp. nov. Calochroa goebeli (W. Horn, 1895), treated in literature either as a "variety" or junior synonym of C. anometallescens (or confused with C. tritoma), proved to represent a separate species. Some other taxa and discrepancies within the genus are discussed. Habitats and characters of several species versus their relations to the genus Cosmodela Rivalier, 1961 also are annotated. Habitus and diagnostic characters are illustrated in colour photographs, and photographs of the habitus and labels of type specimens of C. tritoma, C. laurae and C. goebeli are presented here for the first time.