Botulinum toxin intoxication requires retrograde transport and membrane translocation at the ER in RenVM neurons.

Botulinum neurotoxin A (BoNT/A) is a highly potent proteolytic toxin specific for neurons with numerous clinical and cosmetic uses. After uptake at the synapse, the protein is proposed to translocate from synaptic vesicles to the cytosol through a self-formed channel. Surprisingly, we found that after intoxication proteolysis of a fluorescent reporter occurs in the neuron soma first and then centrifugally in neurites. To investigate the molecular mechanisms at play, we use a genome-wide siRNA screen in genetically engineered neurons and identify over three hundred genes. An organelle-specific split-mNG complementation indicates BoNT/A traffic from the synapse to the soma-localized Golgi in a retromer-dependent fashion. The toxin then moves to the ER and appears to require the Sec61 complex for retro-translocation to the cytosol. Our study identifies genes and trafficking processes hijacked by the toxin, revealing a new pathway mediating BoNT/A cellular toxicity.

Drivers of morphological evolution in the toothed whale jaw.

Toothed whales (odontocetes) emit high-frequency underwater sounds (echolocate)-an extreme and unique innovation allowing them to sense their prey and environment. Their highly specialized mandible (lower jaw) allows high-frequency sounds to be transmitted back to the inner ear. Echolocation is evident in the earliest toothed whales, but little research has focused on the evolution of mandibular form regarding this unique adaptation. Here, we use a high-density, three-dimensional geometric morphometric analysis of 100 living and extinct cetacean species spanning their ∼50-million-year evolutionary history. Our analyses demonstrate that most shape variation is found in the relative length of the jaw and the mandibular symphysis. The greatest morphological diversity was obtained during two periods of rapid evolution: the initial evolution of archaeocetes (stem whales) in the early to mid-Eocene as they adapted to an aquatic lifestyle, representing one of the most extreme adaptive transitions known, and later on in the mid-Oligocene odontocetes as they became increasingly specialized for a range of diets facilitated by increasingly refined echolocation. Low disparity in the posterior mandible suggests the shape of the acoustic window, which receives sound, has remained conservative since the advent of directional hearing in the aquatic archaeocetes, even as the earliest odontocetes began to receive sounds from echolocation. Diet, echolocation, feeding method, and dentition type strongly influence mandible shape. Unlike in the toothed whale cranium, we found no significant asymmetry in the mandible. We suggest that a combination of refined echolocation and associated dietary specializations have driven morphology and disparity in the toothed whale mandible.

A heavyweight early whale pushes the boundaries of vertebrate morphology.

The fossil record of cetaceans documents how terrestrial animals acquired extreme adaptations and transitioned to a fully aquatic lifestyle1,2. In whales, this is associated with a substantial increase in maximum body size. Although an elongate body was acquired early in cetacean evolution3, the maximum body mass of baleen whales reflects a recent diversification that culminated in the blue whale4. More generally, hitherto known gigantism among aquatic tetrapods evolved within pelagic, active swimmers. Here we describe Perucetus colossus-a basilosaurid whale from the middle Eocene epoch of Peru. It displays, to our knowledge, the highest degree of bone mass increase known to date, an adaptation associated with shallow diving5. The estimated skeletal mass of P. colossus exceeds that of any known mammal or aquatic vertebrate. We show that the bone structure specializations of aquatic mammals are reflected in the scaling of skeletal fraction (skeletal mass versus whole-body mass) across the entire disparity of amniotes. We use the skeletal fraction to estimate the body mass of P. colossus, which proves to be a contender for the title of heaviest animal on record. Cetacean peak body mass had already been reached around 30 million years before previously assumed, in a coastal context in which primary productivity was particularly high.

Ecological signal in the size and shape of marine amniote teeth.

Amniotes have been a major component of marine trophic chains from the beginning of the Triassic to present day, with hundreds of species. However, inferences of their (palaeo)ecology have mostly been qualitative, making it difficult to track how dietary niches have changed through time and across clades. Here, we tackle this issue by applying a novel geometric morphometric protocol to three-dimensional models of tooth crowns across a wide range of raptorial marine amniotes. Our results highlight the phenomenon of dental simplification and widespread convergence in marine amniotes, limiting the range of tooth crown morphologies. Importantly, we quantitatively demonstrate that tooth crown shape and size are strongly associated with diet, whereas crown surface complexity is not. The maximal range of tooth shapes in both mammals and reptiles is seen in medium-sized taxa; large crowns are simple and restricted to a fraction of the morphospace. We recognize four principal raptorial guilds within toothed marine amniotes (durophages, generalists, flesh cutters and flesh piercers). Moreover, even though all these feeding guilds have been convergently colonized over the last 200 Myr, a series of dental morphologies are unique to the Mesozoic period, probably reflecting a distinct ecosystem structure.

Global ecomorphological restructuring of dominant marine reptiles prior to the Cretaceous-Palaeogene mass extinction.

Mosasaurid squamates were the dominant amniote predators in marine ecosystems during most of the Late Cretaceous. Here, we use a suite of biomechanically rooted, functionally descriptive ratios in a framework adapted from population ecology to investigate how the morphofunctional disparity of mosasaurids evolved prior to the Cretaceous-Palaeogene (K/Pg) mass extinction. Our results suggest that taxonomic turnover in mosasaurid community composition from Campanian to Maastrichtian is reflected by a notable global increase in morphofunctional disparity, especially driving the North American record. Ecomorphospace occupation becomes polarized during the Late Maastrichtian, with morphofunctional disparity plateauing in the Southern Hemisphere and decreasing in the Northern Hemisphere. We show that these changes are not strongly associated with mosasaurid size, but rather with the functional capacities of their skulls. Our novel approach indicates that mosasaurid morphofunctional disparity was in decline in multiple provincial communities before the K/Pg mass extinction, highlighting region-specific patterns of disparity evolution and the importance of assessing vertebrate extinctions both globally and locally. Ecomorphological differentiation in mosasaurid communities, coupled with declines in other formerly abundant marine reptile groups, indicates widespread restructuring of higher trophic levels in marine food webs was well underway when the K/Pg mass extinction took place.

The tempo of cetacean cranial evolution.

The evolution of cetaceans (whales and dolphins) represents one of the most extreme adaptive transitions known, from terrestrial mammals to a highly specialized aquatic radiation that includes the largest animals alive today. Many anatomical shifts in this transition involve the feeding, respiratory, and sensory structures of the cranium, which we quantified with a high-density, three-dimensional geometric morphometric analysis of 201 living and extinct cetacean species spanning the entirety of their ∼50-million-year evolutionary history. Our analyses demonstrate that cetacean suborders occupy distinct areas of cranial morphospace, with extinct, transitional taxa bridging the gap between archaeocetes (stem whales) and modern mysticetes (baleen whales) and odontocetes (toothed whales). This diversity was obtained through three key periods of rapid evolution: first, the initial evolution of archaeocetes in the early to mid-Eocene produced the highest evolutionary rates seen in cetaceans, concentrated in the maxilla, frontal, premaxilla, and nasal; second, the late Eocene divergence of the mysticetes and odontocetes drives a second peak in rates, with high rates and disparity sustained through the Oligocene; and third, the diversification of odontocetes, particularly sperm whales, in the Miocene (∼18-10 Mya) propels a final peak in the tempo of cetacean morphological evolution. Archaeocetes show the fastest evolutionary rates but the lowest disparity. Odontocetes exhibit the highest disparity, while mysticetes evolve at the slowest pace, particularly in the Neogene. Diet and echolocation have the strongest influence on cranial morphology, with habitat, size, dentition, and feeding method also significant factors impacting shape, disparity, and the pace of cetacean cranial evolution.

The macroevolutionary landscape of short-necked plesiosaurians.

Throughout their evolution, tetrapods have repeatedly colonised a series of ecological niches in marine ecosystems, producing textbook examples of convergent evolution. However, this evolutionary phenomenon has typically been assessed qualitatively and in broad-brush frameworks that imply simplistic macroevolutionary landscapes. We establish a protocol to visualize the density of trait space occupancy and thoroughly test for the existence of macroevolutionary landscapes. We apply this protocol to a new phenotypic dataset describing the morphology of short-necked plesiosaurians, a major component of the Mesozoic marine food webs (ca. 201 to 66 Mya). Plesiosaurians evolved this body plan multiple times during their 135-million-year history, making them an ideal test case for the existence of macroevolutionary landscapes. We find ample evidence for a bimodal craniodental macroevolutionary landscape separating latirostrines from longirostrine taxa, providing the first phylogenetically-explicit quantitative assessment of trophic diversity in extinct marine reptiles. This bimodal pattern was established as early as the Middle Jurassic and was maintained in evolutionary patterns of short-necked plesiosaurians until a Late Cretaceous (Turonian) collapse to a unimodal landscape comprising longirostrine forms with novel morphologies. This study highlights the potential of severe environmental perturbations to profoundly alter the macroevolutionary dynamics of animals occupying the top of food chains.