The evolution and genomic basis of beetle diversity.

The order Coleoptera (beetles) is arguably the most speciose group of animals, but the evolutionary history of beetles, including the impacts of plant feeding (herbivory) on beetle diversification, remain poorly understood. We inferred the phylogeny of beetles using 4,818 genes for 146 species, estimated timing and rates of beetle diversification using 89 genes for 521 species representing all major lineages and traced the evolution of beetle genes enabling symbiont-independent digestion of lignocellulose using 154 genomes or transcriptomes. Phylogenomic analyses of these uniquely comprehensive datasets resolved previously controversial beetle relationships, dated the origin of Coleoptera to the Carboniferous, and supported the codiversification of beetles and angiosperms. Moreover, plant cell wall-degrading enzymes (PCWDEs) obtained from bacteria and fungi via horizontal gene transfers may have been key to the Mesozoic diversification of herbivorous beetles-remarkably, both major independent origins of specialized herbivory in beetles coincide with the first appearances of an arsenal of PCWDEs encoded in their genomes. Furthermore, corresponding (Jurassic) diversification rate increases suggest that these novel genes triggered adaptive radiations that resulted in nearly half of all living beetle species. We propose that PCWDEs enabled efficient digestion of plant tissues, including lignocellulose in cell walls, facilitating the evolution of uniquely specialized plant-feeding habits, such as leaf mining and stem and wood boring. Beetle diversity thus appears to have resulted from multiple factors, including low extinction rates over a long evolutionary history, codiversification with angiosperms, and adaptive radiations of specialized herbivorous beetles following convergent horizontal transfers of microbial genes encoding PCWDEs.

Flight over the Proto-Caribbean seaway: Phylogeny and macroevolution of Neotropical Anaeini leafwing butterflies.

Our understanding of the origin and evolution of the astonishing Neotropical biodiversity remains somewhat limited. In particular, decoupling the respective impacts of biotic and abiotic factors on the macroevolution of clades is paramount to understand biodiversity assemblage in this region. We present the first comprehensive molecular phylogeny for the Neotropical Anaeini leafwing butterflies (Nymphalidae, Charaxinae) and, applying likelihood-based methods, we test the impact of major abiotic (Andean orogeny, Central American highland orogeny, Proto-Caribbean seaway closure, Quaternary glaciations) and biotic (host plant association) factors on their macroevolution. We infer a robust phylogenetic hypothesis for the tribe despite moderate support in some derived clades. Our phylogenetic inference recovers the genus Polygrapha Staudinger, [1887] as polyphyletic, rendering the genera FountaineaRydon, 1971 and Memphis Hübner, [1819] paraphyletic. Consequently, we transfer Polygrapha tyrianthina (Salvin & Godman, 1868) comb. nov. to Fountainea and Polygrapha xenocrates (Westwood, 1850) comb. nov. to Memphis. We infer an origin of the group in the late Eocene ca. 40 million years ago in Central American lowlands which at the time were separated from South America by the Proto-Caribbean seaway. The biogeographical history of the group is very dynamic, with several oversea colonization events from Central America into the Chocó and Andean regions during intense stages of Andean orogeny. These events coincide with the emergence of an archipelagic setting between Central America and northern South America in the mid-Miocene that likely facilitated dispersal across the now-vanished Proto-Caribbean seaway. The Amazonian region also played a central role in the diversification of the Anaeini, acting both as a museum and a cradle of diversity. We recover a diversification rate shift in the Miocene within the species-rich genus Memphis. State speciation and extinction models recover a significant relationship between this rate shift and host plant association, indicating a positive role on speciation rates of a switch between Malpighiales and new plant orders. We find less support for a role of abiotic factors including the progressive Andean orogeny, Proto-Caribbean seaway closure and Quaternary glaciations. Miocene host plant shifts possibly acted in concert with abiotic and/or biotic factors to shape the diversification of Anaeini butterflies.

Phylogenomics of the superfamily Dytiscoidea (Coleoptera: Adephaga) with an evaluation of phylogenetic conflict and systematic error.

The beetle superfamily Dytiscoidea, placed within the suborder Adephaga, comprises six families. The phylogenetic relationships of these families, whose species are aquatic, remain highly contentious. In particular the monophyly of the geographically disjunct Aspidytidae (China and South Africa) remains unclear. Here we use a phylogenomic approach to demonstrate that Aspidytidae are indeed monophyletic, as we inferred this phylogenetic relationship from analyzing nucleotide sequence data filtered for compositional heterogeneity and from analyzing amino-acid sequence data. Our analyses suggest that Aspidytidae are the sister group of Amphizoidae, although the support for this relationship is not unequivocal. A sister group relationship of Hygrobiidae to a clade comprising Amphizoidae, Aspidytidae, and Dytiscidae is supported by analyses in which model assumptions are violated the least. In general, we find that both concatenation and the applied coalescent method are sensitive to the effect of among-species compositional heterogeneity. Four-cluster likelihood-mapping suggests that despite the substantial size of the dataset and the use of advanced analytical methods, statistical support is weak for the inferred phylogenetic placement of Hygrobiidae. These results indicate that other kinds of data (e.g. genomic meta-characters) are possibly required to resolve the above-specified persisting phylogenetic uncertainties. Our study illustrates various data-driven confounding effects in phylogenetic reconstructions and highlights the need for careful monitoring of model violations prior to phylogenomic analysis.

Stream flow alone does not predict population structure of diving beetles across complex tropical landscapes.

Recent theoretical advances have hypothesized a central role of habitat persistence on population genetic structure and resulting biodiversity patterns of freshwater organisms. Here, we address the hypothesis that lotic species, or lineages adapted to comparably geologically stable running water habitats (streams and their marginal habitats), have high levels of endemicity and phylogeographic structure due to the persistent nature of their habitat. We use a nextRAD DNA sequencing approach to investigate the population structure and phylogeography of a putatively widespread New Guinean species of diving beetle, Philaccolilus ameliae (Dytiscidae). We find that P. ameliae is a complex of morphologically cryptic, but geographically and genetically well-differentiated clades. The pattern of population connectivity is consistent with theoretical predictions associated with stable lotic habitats. However, in two clades, we find a more complex pattern of low population differentiation, revealing dispersal across rugged mountains and watersheds of New Guinea up to 430 km apart. These results, while surprising, were also consistent with the original formulation of the habitat template concept by Southwood, involving lineage-idiosyncratic evolution in response to abiotic factors. In our system, low population differentiation might reflect a young species in a phase of range expansion utilizing vast available habitat. We suggest that predictions of life history variation resulting from the dichotomy between lotic and lentic organisms require more attention to habitat characterization and microhabitat choice. Our results also underpin the necessity to study fine-scale processes but at a larger geographical scale, as compared to solely documenting macroecological patterns, to understand ecological drivers of regional biodiversity. Comprehensive sampling especially of tropical lineages in complex and threatened environments such as New Guinea remains a critical challenge.

Phylogeography and population genomics of a lotic water beetle across a complex tropical landscape.

The habitat template concept applied to a freshwater system indicates that lotic species, or those which occupy permanent habitats along stream courses, are less dispersive than lentic species, or those that occur in more ephemeral aquatic habitats. Thus, populations of lotic species will be more structured than those of lentic species. Stream courses include both flowing water and small, stagnant microhabitats that can provide refuge when streams are low. Many species occur in these microhabitats but remain poorly studied. Here, we present population genetic data for one such species, the tropical diving beetle Exocelina manokwariensis (Dytiscidae), sampled from six localities along a ~300 km transect across the Birds Head Peninsula of New Guinea. Molecular data from both mitochondrial (CO1 sequences) and nuclear (ddRAD loci) regions document fine-scale population structure across populations that are ~45 km apart. Our results are concordant with previous phylogenetic and macroecological studies that applied the habitat template concept to aquatic systems. This study also illustrates that these diverse but mostly overlooked microhabitats are promising study systems in freshwater ecology and evolutionary biology. With the advent of next-generation sequencing, fine-scale population genomic studies are feasible for small nonmodel organisms to help illuminate the effect of habitat stability on species' natural history, population structure and geographic distribution.

Unveiling the diversification dynamics of Australasian predaceous diving beetles in the Cenozoic.

During the Cenozoic, Australia experienced major climatic shifts that have had dramatic ecological consequences for the modern biota. Mesic tropical ecosystems were progressively restricted to the coasts and replaced by arid-adapted floral and faunal communities. Whilst the role of aridification has been investigated in a wide range of terrestrial lineages, the response of freshwater clades remains poorly investigated. To gain insights into the diversification processes underlying a freshwater radiation, we studied the evolutionary history of the Australasian predaceous diving beetles of the tribe Hydroporini (147 described species). We used an integrative approach including the latest methods in phylogenetics, divergence time estimation, ancestral character state reconstruction, and likelihood-based methods of diversification rate estimation. Phylogenies and dating analyses were reconstructed with molecular data from seven genes (mitochondrial and nuclear) for 117 species (plus 12 outgroups). Robust and well-resolved phylogenies indicate a late Oligocene origin of Australasian Hydroporini. Biogeographic analyses suggest an origin in the East Coast region of Australia, and a dynamic biogeographic scenario implying dispersal events. The group successfully colonized the tropical coastal regions carved by a rampant desertification, and also colonized groundwater ecosystems in Central Australia. Diversification rate analyses suggest that the ongoing aridification of Australia initiated in the Miocene contributed to a major wave of extinctions since the late Pliocene probably attributable to an increasing aridity, range contractions and seasonally disruptions resulting from Quaternary climatic changes. When comparing subterranean and epigean genera, our results show that contrasting mechanisms drove their diversification and therefore current diversity pattern. The Australasian Hydroporini radiation reflects a combination of processes that promoted both diversification, resulting from new ecological opportunities driven by initial aridification, and a subsequent loss of mesic adapted diversity due to increasing aridity.

Comparative molecular species delimitation in the charismatic Nawab butterflies (Nymphalidae, Charaxinae, Polyura).

The charismatic tropical Polyura Nawab butterflies are distributed across twelve biodiversity hotspots in the Indomalayan/Australasian archipelago. In this study, we tested an array of species delimitation methods and compared the results to existing morphology-based taxonomy. We sequenced two mitochondrial and two nuclear gene fragments to reconstruct phylogenetic relationships within Polyura using both Bayesian inference and maximum likelihood. Based on this phylogenetic framework, we used the recently introduced bGMYC, BPP and PTP methods to investigate species boundaries. Based on our results, we describe two new species Polyura paulettae Toussaint sp. n. and Polyura smilesi Toussaint sp. n., propose one synonym, and five populations are raised to species status. Most of the newly recognized species are single-island endemics likely resulting from the recent highly complex geological history of the Indomalayan-Australasian archipelago. Surprisingly, we also find two newly recognized species in the Indomalayan region where additional biotic or abiotic factors have fostered speciation. Species delimitation methods were largely congruent and succeeded to cross-validate most extant morphological species. PTP and BPP seem to yield more consistent and robust estimations of species boundaries with respect to morphological characters while bGMYC delivered contrasting results depending on the different gene trees considered. Our findings demonstrate the efficiency of comparative approaches using molecular species delimitation methods on empirical data. They also pave the way for the investigation of less well-known groups to unveil patterns of species richness and catalogue Earth's concealed, therefore unappreciated diversity.

Anisomeriini diving beetles-an Atlantic-Pacific Island disjunction on Tristan da Cunha and Robinson Crusoe Island, Juan Fernández?

Anisomeriini diving beetles contain only two enigmatic species, representing a remarkable disjunction between the Pacific Juan Fernández Islands (Anisomeria bistriata) and the South Atlantic Tristan da Cunha Archipelago (Senilites tristanicola). They belong to the Colymbetinae, which contain 140 species worldwide. Here we aim to reconstruct the evolutionary history of the Anisomerinii and use > 9000 bp DNA sequence data from 13 fragments of 12 loci for a comprehensive sampling of Colymbetinae species. Analyses under different optimization criteria converge on very similar topologies, and show unambiguously that Anisomeria bistriata and Senilites tristanicola belong to the Neotropical Rhantus signatus species group, a comparatively recent clade within Colymbetinae. Anisomeriini therefore are synonomized with Colymbetini and both species are transferred to Rhantus accordingly, resulting in secondary homonymy of Rhantus bistriatus (Brullé, 1835) with Rhantus bistriatus (Bergsträsser, 1778). We propose the replacement name Rhantus selkirki Jäch, Balke & Michat nom. nov. for the Juan Fernández species. Presence of these species on remote islands is therefore not relictary, but the result of more recent range expansions out of mainland South America. Finally, we suggest that Carabdytini should be synonymized with Colymbetini. Our study underpins the Hennigian principle that a natural classification can be derived only from the search for shared apomorphies between species, not from differences.

Review of the genus Sekaliporus Watts, 1997 with description of a new species from northern Australia (Coleoptera: Dytiscidae, Hydroporinae).

Sekaliporus davidi sp. n. is described from northern Australia. It is the second species in the genus. It occurs from the Kimberley region in the northwest of Western Australia to north-eastern Queensland. The new species is morphologically similar to S. kriegi Watts, 1997 described from the Kakadu area in the Northern Territory but well characterized by its constantly larger size, six yellowish markings on elytra, the different form of the apical triangular spine of elytra, and the form of the median lobe. Sekaliporus davidi sp. n. is a lotic species from intermittent creeks and slow flowing rivers and their residual pools. Important species characters (median lobes, parameres and colour patterns) of the two species are figured, and notes on their habitats and distribution are given. Both species are capable of flight and were also collected at light.

Multiple transgressions of Wallace's Line explain diversity of flightless Trigonopterus weevils on Bali.

The fauna of Bali, situated immediately west of Wallace's Line, is supposedly of recent Javanese origin and characterized by low levels of endemicity. In flightless Trigonopterus weevils, however, we find 100% endemism for the eight species here reported for Bali. Phylogeographic analyses show extensive in situ differentiation, including a local radiation of five species. A comprehensive molecular phylogeny and ancestral area reconstruction of Indo-Malayan-Melanesian species reveals a complex colonization pattern, where the three Balinese lineages all arrived from the East, i.e. all of them transgressed Wallace's Line. Although East Java possesses a rich fauna of Trigonopterus, no exchange can be observed with Bali. We assert that the biogeographic picture of Bali has been dominated by the influx of mobile organisms from Java, but different relationships may be discovered when flightless invertebrates are studied. Our results highlight the importance of in-depth analyses of spatial patterns of biodiversity.

Revision of the Austrelatuspapuensis group with descriptions of 42 new species from New Guinea (Coleoptera, Dytiscidae, Copelatinae).

The Austrelatuspapuensis group is the second species group of the New Guinean representatives of the recently described genus AustrelatusShaverdo et al., 2023. The group is mainly defined by distinct scale- and/or spinula-like surface structures of the dorsal sclerite of the median lobe. The species group already contains four described species and 42 new species and one subspecies treated here: Austrelatusaiyurensissp. nov., A.asteiossp. nov., A.bewaniensissp. nov., A.bosaviensissp. nov., A.bundunensissp. nov., A.centralensissp. nov., A.craterensissp. nov., A.decorissp. nov., A.dekaisp. nov., A.epicharissp. nov., A.flavocapitatussp. nov., A.fuscussp. nov., A.herzogensissp. nov., A.inconstanssp. nov., A.iriatoisp. nov., A.kalibumisp. nov., A.kebarensissp. nov., A.kokodensissp. nov., A.leptossp. nov., A.lolokisp. nov., A.lopintolensissp. nov., A.madangensissp. nov., A.maindaisp. nov., A.mamberamosp. nov., A.mianminensissp. nov., A.miltokarenossp. nov., A.noiadisp. nov., A.normanbyensissp. nov., A.ohusp. nov., A.posmanisp. nov., A.procerussp. nov., A.pseudogestroisp. nov., A.pseudomianminensissp. nov., A.robustussp. nov., A.sarartisp. nov., A.sumokedisp. nov., A.wanangensissp. nov., A.wasiorensissp. nov., A.wasurensissp. nov., A.weigelisp. nov., A.yamurensissp. nov., A.yeretuarsp. nov., A.xanthocephalusnabirensisssp. nov. A checklist and identification key to New Guinean species of the group are provided and important diagnostic characters are illustrated. Data on the species distributions and habiat preferences are given.

A molecular phylogeny of Alpine subterranean Trechini (Coleoptera: Carabidae).

BACKGROUND: The Alpine region harbours one of the most diverse subterranean faunas in the world, with many species showing extreme morphological modifications. The ground beetles of tribe Trechini (Coleoptera, Carabidae) are among the best studied and widespread groups with abundance of troglobionts, but their origin and evolution is largely unknown. RESULTS: We sequenced 3.4 Kb of mitochondrial (cox1, rrnL, trnL, nad1) and nuclear (SSU, LSU) genes of 207 specimens of 173 mostly Alpine species, including examples of all subterranean genera but two plus a representation of epigean taxa. We applied Bayesian methods and maximum likelihood to reconstruct the topology and to estimate divergence times using a priori rates obtained for a related ground beetle genus. We found three main clades of late Eocene-early Oligocene origin: (1) the genus Doderotrechus and relatives; (2) the genus Trechus sensu lato, with most anisotopic subterranean genera, including the Pyrenean lineage and taxa from the Dinaric Alps; and (3) the genus Duvalius sensu lato, diversifying during the late Miocene and including all subterranean isotopic taxa. Most of the subterranean genera had an independent origin and were related to epigean taxa of the same geographical area, but there were three large monophyletic clades of exclusively subterranean species: the Pyrenean lineage, a lineage including subterranean taxa from the eastern Alps and the Dinarides, and the genus Anophthalmus from the northeastern Alps. Many lineages have developed similar phenotypes independently, showing extensive morphological convergence or parallelism. CONCLUSIONS: The Alpine Trechini do not form a homogeneous fauna, in contrast with the Pyrenees, and show a complex scenario of multiple colonisations of the subterranean environment at different geological periods and through different processes. Examples go from populations of an epigean widespread species going underground with little morphological modifications to ancient, geographically widespread lineages of exclusively subterranean species likely to have diversified once fully adapted to the subterranean environment.

The effect of geographical scale of sampling on DNA barcoding.

Eight years after DNA barcoding was formally proposed on a large scale, CO1 sequences are rapidly accumulating from around the world. While studies to date have mostly targeted local or regional species assemblages, the recent launch of the global iBOL project (International Barcode of Life), highlights the need to understand the effects of geographical scale on Barcoding's goals. Sampling has been central in the debate on DNA Barcoding, but the effect of the geographical scale of sampling has not yet been thoroughly and explicitly tested with empirical data. Here, we present a CO1 data set of aquatic predaceous diving beetles of the tribe Agabini, sampled throughout Europe, and use it to investigate how the geographic scale of sampling affects 1) the estimated intraspecific variation of species, 2) the genetic distance to the most closely related heterospecific, 3) the ratio of intraspecific and interspecific variation, 4) the frequency of taxonomically recognized species found to be monophyletic, and 5) query identification performance based on 6 different species assignment methods. Intraspecific variation was significantly correlated with the geographical scale of sampling (R-square = 0.7), and more than half of the species with 10 or more sampled individuals (N = 29) showed higher intraspecific variation than 1% sequence divergence. In contrast, the distance to the closest heterospecific showed a significant decrease with increasing geographical scale of sampling. The average genetic distance dropped from > 7% for samples within 1 km, to < 3.5% for samples up to > 6000 km apart. Over a third of the species were not monophyletic, and the proportion increased through locally, nationally, regionally, and continentally restricted subsets of the data. The success of identifying queries decreased with increasing spatial scale of sampling; liberal methods declined from 100% to around 90%, whereas strict methods dropped to below 50% at continental scales. The proportion of query identifications considered uncertain (more than one species < 1% distance from query) escalated from zero at local, to 50% at continental scale. Finally, by resampling the most widely sampled species we show that even if samples are collected to maximize the geographical coverage, up to 70 individuals are required to sample 95% of intraspecific variation. The results show that the geographical scale of sampling has a critical impact on the global application of DNA barcoding. Scale-effects result from the relative importance of different processes determining the composition of regional species assemblages (dispersal and ecological assembly) and global clades (demography, speciation, and extinction). The incorporation of geographical information, where available, will be required to obtain identification rates at global scales equivalent to those in regional barcoding studies. Our result hence provides an impetus for both smarter barcoding tools and sprouting national barcoding initiatives-smaller geographical scales deliver higher accuracy.

Integrative taxonomy on the fast track - towards more sustainability in biodiversity research.

BACKGROUND: A so called "taxonomic impediment" has been recognized as a major obstacle to biodiversity research for the past two decades. Numerous remedies were then proposed. However, neither significant progress in terms of formal species descriptions, nor a minimum standard for descriptions have been achieved so far. Here, we analyze the problems of traditional taxonomy which often produces keys and descriptions of limited practical value. We suggest that phylogenetics and phenetics had a subtle and so far unnoticed effect on taxonomy leading to inflated species descriptions. DISCUSSION: The term "turbo-taxonomy" was recently coined for an approach combining cox1 sequences, concise morphological descriptions by an expert taxonomist, and high-resolution digital imaging to streamline the formal description of larger numbers of new species. We propose a further development of this approach which, together with open access web-publication and automated pushing of content from journal into a wiki, may create the most efficient and sustainable way to conduct taxonomy in the future. On demand, highly concise descriptions can be gradually updated or modified in the fully versioned wiki-framework we use. This means that the visibility of additional data is not compromised, while the original species description -the first version- remains preserved in the wiki, and of course in the journal version. A DNA sequence database with an identification engine replaces an identification key, helps to avoid synonyms and has the potential to detect grossly incorrect generic placements. We demonstrate the functionality of a species-description pipeline by naming 101 new species of hyperdiverse New Guinea Trigonopterus weevils in the open-access journal ZooKeys. SUMMARY: Fast track taxonomy will not only increase speed, but also sustainability of global species inventories. It will be of great practical value to all the other disciplines that depend on a usable taxonomy and will change our perception of global biodiversity. While this approach is certainly not suitable for all taxa alike, it is the tool that will help to tackle many hyperdiverse groups and pave the road for more sustainable comparative studies, e.g. in community ecology, phylogeography and large scale biogeographic studies.

Biodiversity into your hands - A call for a virtual global natural history 'metacollection'.

BACKGROUND: Many scientific disciplines rely on correct taxon delineations and identifications. So does a great part of the general public as well as decision makers. Researchers, students and enthusiastic amateurs often feel frustrated because information about species remains scattered, difficult to access, or difficult to decipher. Together, this affects almost anyone who wishes to identify species or verify identifications. Many remedies have been proposed, but we argue that the role of natural history collections remains insufficiently appreciated. We suggest using state-of-the-art mass imaging technology and to join forces to create a global natural history metacollection on the internet, providing access to the morphology of tens of millions of specimens and making them available for automated digital image analysis. DISCUSSION: Robotic high-resolution imaging technology and fast (high performance) computer-based image stitching make it now feasible to digitize entire collection drawers typically used for arthropod collections, or trays or containers used for other objects. Resolutions of 500 megapixels and much higher are already utilized to capture the contents of 40x50 cm collection drawers, providing amazing detail of specimens. Flanked by metadata entry, this helps to create access to tens of thousands of specimens in days. By setting priorities and combining the holdings of the most comprehensive collections for certain taxa, drawer digitizing offers the unique opportunity to create a global, virtual metacollection.The taxonomic and geographic coverage of such a collection could never be achieved by a single institution or individual. We argue that by joining forces, many new impulses will emerge for systematic biology, related fields and understanding of biodiversity in general.Digitizing drawers containing unidentified, little-curated specimens is a contribution towards the beginning of a new era of online curation. It also will help taxonomists and curators to discover and process the millions of "gems" of undescribed species hidden in museum accessions. SUMMARY: Our proposal suggests creating virtual, high-resolution image resources that will, for the first time in history, provide access for expert scientists as well as students and the general public to the enormous wealth of the world's natural history collections. We foresee that this will contribute to a better understanding, appreciation and increased use of biodiversity resources and the natural history collections serving this cause.

Limbodessus moni sp. nov., a new high altitudinal diving beetle from the Grasberg in West Papua, Indonesia (Coleoptera: Dytiscidae, Bidessini).

Limbodessus moni sp. nov. is described from Lake Anderson and from small, richly vegetated swampy areas around alpine lakes at 3,970 m a.s.l. near the Grasberg Mine (Carstensz Pyramid) in the Central Mountain Range of New Guinea. The record of the new species marks the altitudinal maximum of a diving beetle in New Guinea. The new species is morphologically similar to L. alexanderi Balke & Hendrich, 2015 in terms of body size and dark coloration; however, both species can be easily separated by the shape of the median lobe and the more moniliform female antennomeres, not forming a conspicuous club as in L. alexanderi. A modified key for all five Limbodessus species from New Guinea and adjacent islands is presented.

Radiographic Outcome of Vital Teeth Treated with Prosthodontic Crowns in Dogs: 26 Cases (2015-2017).

This retrospective study evaluated the effects of prosthodontic crown placement on tooth vitality. Prosthodontic crown placement may be indicated for vital teeth affected by attrition, abrasion, uncomplicated crown fractures, enamel defects, and enamel hypoplasia. This study evaluated 26 vital teeth in 17 patients at the time of crown placement and after 1-year following crown placement. Dental radiographs were used to determine vitality of the 26 teeth. Twenty-five teeth were found to be vital and 1 tooth was non-vital 1-year after crown placement. These results demonstrated that tooth vitality was maintained after titanium alloy crown placement to treat crown attrition, abrasion, uncomplicated crown fractures, and enamel defects.

Bunites distigma (Brull, 1837): discovery of the second- and third-instar larvae and phylogenetic relationships within the Colymbetinae (Coleoptera: Dytiscidae).

The second- and third-instar larvae of the diving beetle Bunites distigma (Brull, 1837) are described and illustrated for the first time, including detailed morphometric and chaetotaxic analyses of selected structures, and their phylogenetic relationships within the Colymbetinae are re-evaluated. The results support previous hypotheses on the position of this genus based on first-instar characters, as Bunites Spangler, 1972 shares a common origin with Meladema Laporte, 1835, Hoperius Fall, 1927 and Neoscutopterus J. Balfour-Browne, 1943, and within this clade, it is sister to Meladema. Instars II and III of Bunites differ from other colymbetine genera by the presence of a basal suture on the urogomphi combined with the presence of posteroventral secondary setae on the protarsus. Some information on the habitat of the species is also provided.