An effective method for preparing histological sections of blow fly intra-puparial stages for minimum PMI estimations.

Blow flies (Diptera: Calliphoridae) are generally early colonisers of fresh cadavers, enabling the estimation of a minimum post-mortem interval (minPMI) based on an accurate aging of the oldest immature stages associated with a cadaver. In blow flies, the pupal stage and the subsequent development of the adult take place inside a protective case, the puparium, formed from the hardened and darkened cuticle of the third instar larva. Because the puparium is an opaque structure that shows virtually no external changes, qualitative analyses of the internal tissues can be very informative for determining reliable age-specific morphological markers. Those analyses can be performed using either non-invasive but expensive and not widely accessible techniques, or traditional histological methods, which are invasive as they require the serial sectioning of the sample. Histological methods are often readily available for forensic researchers and practitioners; however, the histological study of blow fly intra-puparial stages has traditionally been hampered by the poor paraffin infiltration of tissues due to the abundance of fat bodies, resulting in usually fragmented sections and the subsequent loss of relevant information. We present here an effective method for the preparation of histological sections of blow fly intra-puparial stages, maximising the paraffin infiltration while enabling the production of clean and entire sections that allow for the use of reliable age-specific morphological markers, thus improving the accuracy of minPMI estimations when access to more costly techniques is not feasible.

Stolleagrion foghnielseni (Odonata, Cephalozygoptera, Dysagrionidae) gen. et sp. nov.: a new odonatan from the PETM recovery phase of the earliest Ypresian Fur Formation, Denmark.

We describe the new genus and species Stolleagrion foghnielseni n. gen. et sp. from the Fur Formation in northwestern Denmark based on a single fossil wing. This is the first odonatan described from the earliest part of the PETM recovery phase of the early Eocene. A combination of nine wing character states are considered to be diagnostic of the Dysagrionidae Cockrell only together with the cephalozygopteran head; however, the combination of these nine plus the presence of Ax0 is also diagnostic without the head. By this, we assign Stolleagrion foghnielseni to the Dysagrionidae and reassess the position of other odonates previously treated as cf. Dysagrionidae.

The damselfly genus Furagrion Petrulevicius et al. (Odonata, Zygoptera) from the early Eocene Fur Formation of Denmark and the dysagrionoid grade.

The earliest Eocene odonate genus Furagrion Petrulevicius et al. from the Danish Fur Formation is revised based on eighteen specimens, two of which apparently have been lost since their publication. The holotype of Phenacolestes jutlandicus Henriksen, type species of Furagrion, is incomplete and lacks the characters currently used to differentiate species, genera and higher taxa in Odonata. We, therefore, propose that the holotype is set aside and a recently discovered nearly complete Fur Formation fossil is designated as neotype. Furagrion possesses all of the nine wing character states currently used along with head shape for diagnosing the Dysagrionidae; however, Furagrion has a characteristically zygopteran head, not the distinctive head shape of the suborder Cephalozygoptera. We, therefore, treat it as a zygopteran unassigned to family. These nine wing character states appear in different combinations not only in various Zygoptera and Cephalozygoptera, but also in the Frenguelliidae, an Eocene family of Argentina that may represent an unnamed suborder. We recognise these taxa as constituting a dysagrionoid grade, in which these character states appear either convergently or as symplesiomorphies. Furagrion morsi Zessin is synonymized with Phenacolestes jutlandicus Henriksen, syn. nov. and Morsagrion Zessin with Furagrion Petrulevicius, Wappler, Wedmann, Rust, and Nel, syn. nov.

Formation of the Holarctic Fauna: Dated molecular phylogenetic and biogeographic insights from the Quedius-lineage of Ground-Dwelling Rove Beetles (Coleoptera, Staphylinidae).

Although the Holarctic fauna has been explored for centuries, many questions on its formation are still unanswered. For example, i) what was the impact of the uplift of the Himalaya and Tibetan Plateau?, ii) what were the timings and climate of the faunal bridges connecting the Nearctic and Palearctic regions?, and iii) how did insect lineages respond to the late Paleogene global cooling and regional aridification? To answer these, we developed a phylogenetic dataset of 1229 nuclear loci for a total of 222 species of rove beetles (Staphylinidae) with emphasis in the tribe Quediini, especially Quedius-lineage and its subclade Quedius sensu stricto. Using eight fossils for calibrating molecular clock, we estimated divergence times and then analysed in BioGeoBEARS paleodistributions of the most recent common ancestor for each target lineage. For each species we generated climatic envelopes of the temperature and precipitation and mapped them across the phylogeny to explore evolutionary shifts. Our results suggest that the warm and humid Himalaya and Tibetan Plateau acted as an evolutionary cradle for the Quedius-lineage originating during the Oligocene from where, in the Early Miocene, the ancestor of the Quedius s. str. dispersed into the West Palearctic. With the climate cooling from the Mid Miocene onwards, new lineages within Quedius s. str. emerged and gradually expanded distributions across the Palearctic. In Late Miocene, a member of the group dispersed to the Nearctic region via Beringia before the closure of this land bridge 5.3 Ma. Paleogene global cooling and regional aridification largely shaped the current biogeographic pattern for Quedius s. str. species, many of them originating during the Pliocene and shifting or contracting their ranges during Pleistocene.

Danowhetaksa gen. nov. with two species from the early Eocene lst Formation from Denmark, the first Palearctic Whetwhetaksidae (Odonata: Cephalozygoptera).

We propose Danowhetaksa n. gen. (Odonata: Whetwhetaksidae) with two new species: D. birgitteae n. gen. et sp. and D. rusti n. gen. et sp. from the earliest Ypresian Stolleklint clay of the lst Formation in northwestern Denmark. Whetwhetaksidae has previously been known only from the Ypresian Okanagan Highlands of far-western North America, the new records are, therefore, the first from the Palearctic Region.

Comparative phylogeography uncovers evolutionary past of Holarctic dragonflies.

Here, we investigate the evolutionary history of five northern dragonfly species to evaluate what role the last glaciation period may have played in their current distributions. We look at the population structure and estimate divergence times for populations of the following species: Aeshna juncea (Linnaeus), Aeshna subarctica Walker, Sympetrum danae (Sulzer), Libellula quadrimaculata Linnaeus and Somatochlora sahlbergi Trybom across their Holarctic range. Our results suggest a common phylogeographic pattern across all species except for S. sahlbergi. First, we find that North American and European populations are genetically distinct and have perhaps been separated for more than 400,000 years. Second, our data suggests that, based on genetics, populations from the Greater Beringian region (Beringia, Japan and China) have haplotypes that cluster with North America or Europe depending on the species rather than having a shared geographic affinity. This is perhaps a result of fluctuating sea levels and ice sheet coverage during the Quaternary period that influenced dispersal routes and refugia. Indeed, glacial Beringia may have been as much a transit zone as a refugia for dragonflies. Somatochlora sahlbergi shows no genetic variation across its range and therefore does not share the geographic patterns found in the other circumboreal dragonflies studied here. Lastly, we discuss the taxonomic status of Sympetrum danae, which our results indicate is a species complex comprising two species, one found in Eurasia through Beringia, and the other in North America east and south of Beringia. Through this study we present a shared history among different species from different families of dragonflies, which are influenced by the climatic fluctuations of the past.

Natural history museum collection and citizen science data show advancing phenology of Danish hoverflies (Insecta: Diptera, Syrphidae) with increasing annual temperature.

We explore the phenological response by Danish hoverflies (Syrphidae) to continually rising annual temperatures by analysing >50.000 natural history collection and citizen science records for 37 species collected between 1900 and 2018, a period during which the annual average temperature in Denmark rose significantly (p << 0.01). We perform a simple linear regression analysis of the 10th percentile observation date for each species against year of observation. Fourteen of the species showed a statistically significant (p < 0.05) negative correlation between 10th percentile date and year of observation, indicating earlier emergence as a likely response to climatic warming. Eighteen species showed a non-significant (p ≥ 0.05) negative correlation between 10th percentile date and year of observation, while four species showed a non-significant (p ≥ 0.05) positive correlation, and one showed neither a positive nor a negative correlation. We explore the possible impact of the length of the data series on the regression analysis by dividing the species into four groups depending on how far back in time we have data: ultra-short series (with data from 2003-2018); short series (data from 1998-2018); medium series (data from 1980-2018); long series (data from 2018 to before 1980). The length of the series seems to have an effect on the results as 60% of the long series species (nine out of 15) showed a statistically significant negative correlation, while for the shorter series species less than 35% showed a statistically significant negative correlation. When we reduced the long series in length to short series, the proportion of statistically significant negative correlations fell to 33%, confirming this assumption. We conclude that northern temperate hoverflies generally react to the ongoing climatic warming by emerging earlier.

Ghosts from the past: a review of fossil Hepialoidea (Lepidoptera).

We critically re-examine nine of the ten fossil specimens currently assigned to Hepialidae. Three fossils with impressions of wing veins and scales placed in the fossil genus Prohepialus Piton, 1940, and two mummified larvae that show apomorphic characters, have features that support placement in Hepialidae. The other four fossils that we evaluate, Prohepialus incertus Piton, 1940; Oiophassus nycterus Zhang, 1989; Protohepialus comstocki Pierce, 1945; and a fossil scale, lack definitive hepialid characters. One of these, Prohepialus incertus Piton, 1940, appears to represent a symphytan (Hymenoptera), and is excluded from Lepidoptera. The fossilized wings placed in Prohepialus by Jarzembowski display numerous features that indicate a proximate phylogenetic relationship to extant members of the hepialid genus Sthenopis Packard and related genera.

Reassessment of known fossil Pyraloidea (Lepidoptera) with descriptions of the oldest fossil pyraloid and a crambid larva in Baltic amber.

The identifications of known fossils currently placed in the lepidopteran superfamily Pyraloidea are critically re-examined. Of the eleven fossils examined, only three are confirmed to show morphological characters supporting placement in the superfamily. These fossils include a crambid larva in Baltic Amber, Baltianania yantarnia, Solis gen. n. et sp. n. and the oldest known fossil pyraloid, Eopyralis morsae Simonsen, gen. n. et sp. n. The third fossil, Glendotricha olgae Kusnezov, 1941, displays apomorphic characters for Pyraloidea, but is shown to be an inclusion in copal, not Baltic amber as had been reported. Seven fossil specimens lack reliable characters and cannot be assigned to Pyraloidea with certainty: Pyralites obscurus Heer, 1856; Pyralites preecei Jarzembowski, 1980; Petisca dryellina Martins-Neto, 1998; three fossil larvae tentatively identified as Pyralidae by Zeuner (1931); and Gallerites keleri Kernbach, 1967. A possible fossil pyraloid in Mizunami amber could not be located in museum collections and available literature does not provide details to assess the validity of the identification. We discuss the contribution of the reliably identified fossils towards better understanding the evolutionary history of Pyraloidea.

Documenting diversity in the Amazonian butterfly genus Bia (Lepidoptera, Nymphalidae).

A comparative study of over 1,000 specimens allowed us to revise the taxonomy of the Amazonian butterfly genus Bia. We redescribed the genus, and used a selected set of characters to define and describe new species and subspecific taxa. We found that male genitalia showed little variation among taxa, and that wing and genitalia characters varied independently across the range of the genus. We also noted that species diversification seems to follow a north-south Amazonian divide, with Bia actorion and decaerulea occupying the northern portion of the genus range while the remaining four species are found in southern Amazonia. As defined here Bia includes six species: B. actorion (Linnaeus), B. actorion ecuatoria DeVries & Penz, NEW SSP., B. actorion occulta Casagrande & Penz, NEW SSP., B. decaerulea Weymer STAT. NOV., B. decaerulea cayana Simonsen & Penz, NEW SSP., B. decaerulea pallida Casagrande & Penz, NEW SSP., B. rebeli Bryk STAT. NOV., B. rebeli aegina Penz & Simonsen, NEW SSP., B. rebeli acreana Casagrande & Penz, NEW SSP., B. rebeli arikeme Penz & Casagrande, NEW SSP., B. rebeli pareci Penz & DeVries, NEW SSP., B. rebeli cuprea Penz & Casagrande, NEW SSP., B. rebeli tapajos Penz & Simonsen, NEW SSP., B. caelestis Penz & DeVries, NEW SP., B. pucallpa Casagrande & Penz, NEW SP., and B. peruana Röber.

Age estimation during the blow fly intra-puparial period: a qualitative and quantitative approach using micro-computed tomography.

Minimum post-mortem interval (minPMI) estimates often rely on the use of developmental data from blow flies (Diptera: Calliphoridae), which are generally the first colonisers of cadavers and, therefore, exemplar forensic indicators. Developmental data of the intra-puparial period are of particular importance, as it can account for more than half of the developmental duration of the blow fly life cycle. During this period, the insect undergoes metamorphosis inside the opaque, barrel-shaped puparium, formed by the hardening and darkening of the third instar larval cuticle, which shows virtually no external changes until adult emergence. Regrettably, estimates based on the intra-puparial period are severely limited due to the lack of reliable, non-destructive ageing methods and are frequently based solely on qualitative developmental markers. In this study, we use non-destructive micro-computed tomography (micro-CT) for (i) performing qualitative and quantitative analyses of the morphological changes taking place during the intra-puparial period of two forensically relevant blow fly species, Calliphora vicina and Lucilia sericata, and (ii) developing a novel and reliable method for estimating insect age in forensic practice. We show that micro-CT provides age-diagnostic qualitative characters for most 10% time intervals of the total intra-puparial period, which can be used over a range of temperatures and with a resolution comparable to more invasive and time-consuming traditional imaging techniques. Moreover, micro-CT can be used to yield a quantitative measure of the development of selected organ systems to be used in combination with qualitative markers. Our results confirm micro-CT as an emerging, powerful tool in medico-legal investigations.

The 'dance' of life: visualizing metamorphosis during pupation in the blow fly Calliphora vicina by X-ray video imaging and micro-computed tomography.

The dramatic metamorphosis from larva to adult of insect orders such as Diptera cannot usually be witnessed because it occurs within an opaque structure. For the cyclorrhaphous dipterans, such as blow flies, this structure is the puparium, formed from the larval cuticle. Here, we reveal metamorphosis within the puparium of a blow fly at higher temporal resolution than previously possible with two-dimensional time-lapse videos created using the X-ray within a micro-computed tomography scanner, imaging development at 1 min and 2 min intervals. Our studies confirm that the most profound morphological changes occur during just 0.5% of the intrapuparial period (approx. equivalent to 1.25 h at 24°C) and demonstrate the significant potential of this technique to complement other methods for the study of developmental changes, such as hormone control and gene expression. We hope this will stimulate a renewed interest among students and researchers in the study of morphology and its astonishing transformation engendered by metamorphosis.

Looking into the puparium: Micro-CT visualization of the internal morphological changes during metamorphosis of the blow fly, Calliphora vicina, with the first quantitative analysis of organ development in cyclorrhaphous dipterans.

Metamorphosis of cyclorrhaphous flies takes place inside a barrel-like puparium, formed by the shrinking, hardening and darkening of the third-instar larval cuticle. The opacity of this structure hampers the visualization of the morphological changes occurring inside and therefore a full understanding of the metamorphosis process. Here, we use micro-computed tomography (micro-CT) to describe the internal morphological changes that occur during metamorphosis of the blow fly, Calliphora vicina Robineau-Desvoidy 1830 (Diptera: Calliphoridae) at a greater temporal resolution than anything hitherto published. The morphological changes were documented at 10% intervals of the total intra-puparial period, and down to 2.5% intervals during the first 20% interval, when the most dramatic morphological changes occur. Moreover, the development of an internal gas bubble, which plays an essential role during early metamorphosis, was further investigated with X-ray images and micro-CT virtual sections. The origin of this gas bubble has been largely unknown, but micro-CT virtual sections show that it is connected to one of the main tracheal trunks. Micro-CT virtual sections also provided enough resolution for determining the completion of the larval-pupal and pupal-adult apolyses, thus enabling an accurate timing of the different intra-puparial life stages. The prepupal, pupal, and pharate adult stages last for 7.5%, 22.5%, and 70% of the total intra-puparial development, respectively. Furthermore, we provide for the first time quantitative data on the development of two organ systems of the blow fly: the alimentary canal and the indirect flight muscles. There is a significant and negative correlation between the volume of the indirect flight muscles and the pre-helicoidal region of the midgut during metamorphosis. The latter occupies a large portion of the thorax during the pupal stage but narrows progressively as the indirect flight muscles increase in volume during the development of the pharate adult.

Haplopacha (Lepidoptera: Lasiocampidae) reviewed: four new species, first descriptions of the genitalia of both sexes, and unique alar scale organs.

The collection of five specimens of Haplopacha at Ndumo Nature Reserve in South Africa highlights unknown species diversity in the monotypic genus. A total of four new species; H. riftensis sp. nov., H. tangani sp. nov., H. lunata sp. nov. and H. ndoumoi sp. nov., were identified in the Natural History Museum of London collection and the study of fresh specimens also revealed the presence of uniquely characteristic alar scale patches absent from the original species description of the genus type species H. cinerea. Based on the new species the descriptions of Haplopacha is amended. The identification of the new species was done using conventional light microscopy and scanning electron microscopy of the general morphology, wing venation and genitalia observations.

Elhamma Walker (Lepidoptera: Hepialidae) revisited: adult morphology, assessment of recently proposed synonyms and descriptions of two species.

I revise the Australian-New Guinean ghost moth genus Elhamma. Two recent synonymies are assessed, and two new species from New Guinea, E. grehani sp. nov. and E. viettei sp. nov. are described. I provide an updated diagnosis for the genus and conclude that the presence of only 2 M-veins in the hind wing in both sexes (when females are known) and a strongly cup-shape juxta in the male genitalia are unique diagnostic characters among Hepialidae. I give a detailed description of the adult morphology based on male E. australasiae, and provide a key to all known species based on adult male characters.

Towards a mitogenomic phylogeny of Lepidoptera.

The backbone phylogeny of Lepidoptera remains unresolved, despite strenuous recent morphological and molecular efforts. Molecular studies have focused on nuclear protein coding genes, sometimes adding a single mitochondrial gene. Recent advances in sequencing technology have, however, made acquisition of entire mitochondrial genomes both practical and economically viable. Prior phylogenetic studies utilised just eight of 43 currently recognised lepidopteran superfamilies. Here, we add 23 full and six partial mitochondrial genomes (comprising 22 superfamilies of which 16 are newly represented) to those publically available for a total of 24 superfamilies and ask whether such a sample can resolve deeper lepidopteran phylogeny. Using recoded datasets we obtain topologies that are highly congruent with prior nuclear and/or morphological studies. Our study shows support for an expanded Obtectomera including Gelechioidea, Thyridoidea, plume moths (Alucitoidea and Pterophoroidea; possibly along with Epermenioidea), Papilionoidea, Pyraloidea, Mimallonoidea and Macroheterocera. Regarding other controversially positioned higher taxa, Doidae is supported within the new concept of Drepanoidea and Mimallonidae sister to (or part of) Macroheterocera, while among Nymphalidae butterflies, Danainae and not Libytheinae are sister to the remainder of the family. At the deepest level, we suggest that a tRNA rearrangement occurred at a node between Adeloidea and Ditrysia+Palaephatidae+Tischeriidae.

Metamorphosis revealed: time-lapse three-dimensional imaging inside a living chrysalis.

Studies of model insects have greatly increased our understanding of animal development. Yet, they are limited in scope to this small pool of model species: a small number of representatives for a hyperdiverse group with highly varied developmental processes. One factor behind this narrow scope is the challenging nature of traditional methods of study, such as histology and dissection, which can preclude quantitative analysis and do not allow the development of a single individual to be followed. Here, we use high-resolution X-ray computed tomography (CT) to overcome these issues, and three-dimensionally image numerous lepidopteran pupae throughout their development. The resulting models are presented in the electronic supplementary material, as are figures and videos, documenting a single individual throughout development. They provide new insight and details of lepidopteran metamorphosis, and allow the measurement of tracheal and gut volume. Furthermore, this study demonstrates early and rapid development of the tracheae, which become visible in scans just 12 h after pupation. This suggests that there is less remodelling of the tracheal system than previously expected, and is methodologically important because the tracheal system is an often-understudied character system in development. In the future, this form of time-lapse CT-scanning could allow faster and more detailed developmental studies on a wider range of taxa than is presently possible.

Deceptive single-locus taxonomy and phylogeography: Wolbachia-associated divergence in mitochondrial DNA is not reflected in morphology and nuclear markers in a butterfly species.

The satyrine butterfly Coenonympha tullia (Nymphalidae: Satyrinae) displays a deep split between two mitochondrial clades, one restricted to northern Alberta, Canada, and the other found throughout Alberta and across North America. We confirm this deep divide and test hypotheses explaining its phylogeographic structure. Neither genitalia morphology nor nuclear gene sequence supports cryptic species as an explanation, instead indicating differences between nuclear and mitochondrial genome histories. Sex-biased dispersal is unlikely to cause such mito-nuclear differences; however, selective sweeps by reproductive parasites could have led to this conflict. About half of the tested samples were infected by Wolbachia bacteria. Using multilocus strain typing for three Wolbachia genes, we show that the divergent mitochondrial clades are associated with two different Wolbachia strains, supporting the hypothesis that the mito-nuclear differences resulted from selection on the mitochondrial genome due to selective sweeps by Wolbachia strains.

No specimen left behind: industrial scale digitization of natural history collections.

Traditional approaches for digitizing natural history collections, which include both imaging and metadata capture, are both labour- and time-intensive. Mass-digitization can only be completed if the resource-intensive steps, such as specimen selection and databasing of associated information, are minimized. Digitization of larger collections should employ an "industrial" approach, using the principles of automation and crowd sourcing, with minimal initial metadata collection including a mandatory persistent identifier. A new workflow for the mass-digitization of natural history museum collections based on these principles, and using SatScan® tray scanning system, is described.