How to extract and analyze pollen from internal organs and exoskeletons of fossil insects

This protocol explains how to extract pollen from fossil insects with subsequent descriptions of pollen treatment. We also describe how to document morphological and ultrastructural features with light-microscopy and electron microscopy. It enables a taxonomic assignment of pollen that can be used to interpret flower-insect interactions, foraging and feeding behavior of insects, and the paleoenvironment. The protocol is limited by the state of the fossil, the presence/absence of pollen on fossil specimens, and the availability of extant pollen for comparison. For complete details on the use and execution of this protocol, please refer to Wappler et al. (2015), Ulrich and Grímsson (2020), and Wedmann et al. (2021)

Fossilized Biophotonic Nanostructures Reveal the Original Colors of 47-Million-Year-Old Moths

Original structural colors reconstructed in fossil moths had a dual defensive function and illuminate the evolution of communication strategies in insects

Assessment of H2_{2}S in vivo using the newly developed mitochondria-targeted mass spectrometry probe MitoA

Hydrogen sulfide (H$_{2}$S) is produced endogenously in vivo and has multiple effects on signaling pathways and cell function. Mitochondria can be both an H$_{2}$S source and sink, and many of the biological effects of H$_{2}$S relate to its interactions with mitochondria. However, the significance of mitochondrial H$_{2}$S is uncertain, in part due to the difficulty of assessing changes in its concentration in vivo Although a number of fluorescent H$_{2}$S probes have been developed these are best suited to cells in culture and cannot be used in vivo To address this unmet need we have developed a mitochondria-targeted H$_{2}$S probe, MitoA, which can be used to assess relative changes in mitochondrial H$_{2}$S levels in vivo MitoA comprises a lipophilic triphenylphosphonium (TPP) cation coupled to an aryl azide. The TPP cation leads to the accumulation of MitoA inside mitochondria within tissues in vivo There, the aryl azido group reacts with H$_{2}$S to form an aryl amine (MitoN). The extent of conversion of MitoA to MitoN thus gives an indication of the levels of mitochondrial H$_{2}$S in vivo Both compounds can be detected sensitively by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the tissues, and quantified relative to deuterated internal standards. Here we describe the synthesis and characterization of MitoA and show that it can be used to assess changes in mitochondrial H$_{2}$S levels in vivo As a proof of principle we used MitoA to show that H$_{2}$S levels increase in vivo during myocardial ischemia.This work was supported in part by Medical Research Council UK Grant MC_U105663142, Wellcome Trust Investigator award 110159/Z/15/Z (to M. P. M.), Biotechnology and Biological Sciences Research Council Grant BB/I012826/1, Wellcome Trust Investigator award 110158/Z/15/Z (to R. C. H.), and a Consejo Nacional de Ciencia y Technología studentship (to C. B.-G.)

Fossil Carder Bee's nest from the Hominin locality of Taung, South Africa

The Buxton-Norlim Limeworks southwest of Taung, South Africa, is renowned for the discovery of the first Australopithecus africanus fossil, the ‘Taung Child’. The hominin was recovered from a distinctive pink calcrete that contains an abundance of invertebrate ichnofauna belonging to the Coprinisphaera ichnofacies. Here we describe the first fossil bee’s nest, attributed to the ichnogenus Celliforma, from the Plio-Pleistocene of Africa. Petrographic examination of a cell lining revealed the preservation of an intricate organic matrix lined with the calcitic casts of numerous plant trichomes–a nesting behaviour unique to the modern-day carder bees (Anthidiini). The presence of Celliforma considered alongside several other recorded ichnofossils can be indicative of a dry, savannah environment, in agreement with recent work on the palaeoenvironment of Plio-Pleistocene southern Africa. Moreover, the occurrence of ground-nesting bees provides further evidence that the pink calcrete deposits are of pedogenic origin, rather than speleogenic origin as has previously been assumed. This study demonstrates the potential value of insect trace fossils as palaeoenvironmental indicators

Phylogeographic Analysis Elucidates the Influence of the Ice Ages on the Disjunct Distribution of Relict Dragonflies in Asia

Unusual biogeographic patterns of closely related groups reflect events in the past, and molecular analyses can help to elucidate these events. While ample research on the origin of disjunct distributions of different organism groups in the Western Paleartic has been conducted, such studies are rare for Eastern Palearctic organisms. In this paper we present a phylogeographic analysis of the disjunct distribution pattern of the extant species of the strongly cool-adapted Epiophlebia dragonflies from Asia. We investigated sequences of the usually more conserved 18 S rDNA and 28 S rDNA genes and the more variable sequences of ITS1, ITS2 and CO2 of all three currently recognised Epiophlebia species and of a sample of other odonatan species. In all genes investigated the degrees of similarity between species of Epiophlebia are very high and resemble those otherwise found between different populations of the same species in Odonata. This indicates that substantial gene transfer between these populations occurred in the comparatively recent past. Our analyses imply a wide distribution of the ancestor of extant Epiophlebia in Southeast Asia during the last ice age, when suitable habitats were more common. During the following warming phase, its range contracted, resulting in the current disjunct distribution. Given the strong sensitivity of these species to climatic parameters, the current trend to increasing global temperatures will further reduce acceptable habitats and seriously threaten the existences of these last representatives of an ancient group of Odonata

Mating and aggregative behaviors among basal hexapods in the Early Cretaceous

Among the many challenges in paleobiology is the inference and reconstruction of behaviors that rarely, if ever, leave a physical trace on the environment that is suitable for fossilization. Of particular significance are those behaviors tied to mating and courtship, individual interactions critical for species integrity and continuance, as well as those for dispersal, permitting the taxon to expand its distribution as well as access new habitats in the face of local or long-term environmental change. In this context, two recently discovered fossils from the Early Cretaceous amber of Spain (ca. 105 mya) give a detailed view of otherwise fleeting ethologies in Collembola. These occurrences are phylogenetically spaced across the class, and from species representing the two major clades of springtailsÐSymphypleona and Entomobryomorpha. Specifically, we report unique evidence from a symphypleonan male (Pseudosminthurides stoechus SaÂnchez-GarcõÂa & Engel, 2016) with modified antennae that may have functioned as a clasping organ for securing females during mating on water's surface, and from an aggregation of entomobryomorphan individuals (Proisotoma communis Sánchez-García & Engel, 2016) purportedly representing a swarming episode on the forest floor. We demonstrate that the mating behavioral repertoire in P. stoechus, which is associated with considerable morphological adaptations, likely implied elaborate courtship and maneuvering for guarantee sperm transfer in an epineustic species. These discoveries reveal significant behaviors consistent with modern counterparts and a generalized stasis for some ancient hexapod ethologies associated with complex mating and courtship and social or pre-social aggregations, so critical to specific constancy and dispersal

Changes to the Fossil Record of Insects through Fifteen Years of Discovery

The first and last occurrences of hexapod families in the fossil record are compiled from publications up to end-2009. The major features of these data are compared with those of previous datasets (1993 and 1994). About a third of families (>400) are new to the fossil record since 1994, over half of the earlier, existing families have experienced changes in their known stratigraphic range and only about ten percent have unchanged ranges. Despite these significant additions to knowledge, the broad pattern of described richness through time remains similar, with described richness increasing steadily through geological history and a shift in dominant taxa, from Palaeoptera and Polyneoptera to Paraneoptera and Holometabola, after the Palaeozoic. However, after detrending, described richness is not well correlated with the earlier datasets, indicating significant changes in shorter-term patterns. There is reduced Palaeozoic richness, peaking at a different time, and a less pronounced Permian decline. A pronounced Triassic peak and decline is shown, and the plateau from the mid Early Cretaceous to the end of the period remains, albeit at substantially higher richness compared to earlier datasets. Origination and extinction rates are broadly similar to before, with a broad decline in both through time but episodic peaks, including end-Permian turnover. Origination more consistently exceeds extinction compared to previous datasets and exceptions are mainly in the Palaeozoic. These changes suggest that some inferences about causal mechanisms in insect macroevolution are likely to differ as well

New middle Eocene formicid species from Germany and the evolution of weaver ants

Two new species of weaver ant are described from the Eocene of Germany. Males and gynes of Oecophylla longiceps Dlussky sp. nov. are found in the middle Eocene (about 47 Ma) of Grube Messel, Germany. Males, gynes and two workers of O. eckfeldiana Dlussky sp. nov. are recorded from the middle Eocene (about 43 Ma) of Eckfeld maar, Germany. The two new species are among the oldest records of the extant genus Oecophylla. First adaptations for moving in the forest canopy are present in the workers of O. eckfeldiana. Even more specialized adaptations for arboreal life are found in the workers of O. brischkei from Baltic amber. The coexistence of two species in Baltic amber and in the Bembridge marls suggests that in these cases different ecological niches were realised by sympatric species. Comparisons of the queens from different fossil and extant species indicate morphological trends, partly connected with increasing fertility. Most likely Oecophylla originated in the early Paleogene in the Palaearctic realm, radiating strongly during the climatic changes of the Eocene–Oligocene transition

The first fossil leaf insect: 47 million years of specialized cryptic morphology and behavior

Stick and leaf insects (insect order Phasmatodea) are represented primarily by twig-imitating slender forms. Only a small percentage (≈1%) of extant phasmids belong to the leaf insects (Phylliinae), which exhibit an extreme form of morphological and behavioral leaf mimicry. Fossils of phasmid insects are extremely rare worldwide. Here we report the first fossil leaf insect, Eophyllium messelensis gen. et sp. nov., from 47-million-year-old deposits at Messel in Germany. The new specimen, a male, is exquisitely preserved and displays the same foliaceous appearance as extant male leaf insects. Clearly, an advanced form of extant angiosperm leaf mimicry had already evolved early in the Eocene. We infer that this trait was combined with a special behavior, catalepsy or “adaptive stillness,” enabling Eophyllium to deceive visually oriented predators. Potential predators reported from the Eocene are birds, early primates, and bats. The combination of primitive and derived characters revealed by Eophyllium allows the determination of its exact phylogenetic position and illuminates the evolution of leaf mimicry for this insect group. It provides direct evidence that Phylliinae originated at least 47 Mya. Eophyllium enlarges the known geographical range of Phylliinae, currently restricted to southeast Asia, which is apparently a relict distribution. This fossil leaf insect bears considerable resemblance to extant individuals in size and cryptic morphology, indicating minimal change in 47 million years. This absence of evolutionary change is an outstanding example of morphological and, probably, behavioral stasis