In recent years, progresses in imaging and musculoskeletal modelling 
have greatly advanced how we can study the movement capacities of 
an extinct species. This talk will provide an overview of the challenges 
we face in evolutionary biomechanics, from discovery to simulation. 
To develop a musculoskeletal model, a scientist must first create a 
representation of the specimen’s skeleton, which might be heavily 
distorted and fragmentary. This can require extensive reconstruction 
and retrodeformation, and also rearticulation of disarticulated remains. 
After reassembling the skeleton, muscles must be added to the model. 
Soft tissues do not preserve in the fossil record, and thus assumptions 
must be made using a phylogenetic bracket of living, comparative 
species. However, the muscles’ shapes, sizes and configurations 
are not sufficient to drive the model forward. Rather, the muscles 
require estimation of their architectural data, such as fibre lengths 
and physiological cross-sectional areas, which do not preserve in the 
fossil record. The finalised model can be used to investigate movement 
through simulations of performance. The kinematics and kinetics of 
a specimen can be computed and biomechanical information such 
as moment arms, moments and muscle activations can be extracted 
so that we can model how an extinct species might have moved. 
Here, we present pelvic and lower limb musculoskeletal models of 
extinct hominin species, ranging from Australopithecus afarensis to 
Homo erectus, in which soft tissues were reconstructed, muscular 
parameters were estimated and preliminary simulations have begun 
to unravel changes in the movement capability within the human 
evolutionary lineage.

From discovery to simulation: The challenges of musculoskeletal modelling in fossil specimens

Analogous to the situation in several lepidopteran and dipteran 
insects also the larval stages of some echinoderms and hemichordates 
(ambulacraria superphylum) evolved highly alkaline midguts up to pH 
10.5. Despite its pivotal role in species´ immunity, digestion physiology 
and sensitivity to changes in seawater pH, the underlying epithelial 
transport mechanisms remain largely unknown. 
We measured intracellular pH regulation of midgut cells and pH 
maintenance of midgut fluids in the sea urchin larva in the presence 
of different inhibitors targeting acid-base transporters.Measurements of midgut pHi regulatory capacities under different 
inhibitor treatments demonstrated that vacuolar H+ -ATPase 
(bafilomycin a1), carbonic anhydrase (acetazolamide), anion-exchangers 
(4,4′-Diisothiocyano-2,2′-stilbendisulfonsäure (DIDS)) and soluble 
adenylyl cyclase (KH7) play an important role in cellular acid-base 
regulation. 
Using ion-selective microelectrodes (ISMs) we found that midgut 
alkalization is reduced by bafilomycin a1 and completely abolished 
by KH7 while DIDS only inhibited alkalization when applied from 
the luminal side. Interestingly, high external ammonia (HEA; 1.5 to 5 
mM) also decreased midgut pH suggesting a coupling of NH3 /NH4 + 
transport to midgut alkalization. Single cell transcriptomics revealed 
the presence of a RhP expressed in midgut cells that is stimulated on 
the mRNA level under acidic conditions, indicating an involvement in 
defending midgut pH. 
Ammonia excretion rates are affected by bafilomycin a1 and KH7 
suggesting an involvement of sAC and H+ -ATPase in the excretion 
of nitrogenous waste products. 
This comprehensive pharmacological analysis has led to a new 
working model of midgut

Pharmacological studies reveal ammonia-dependent pH regulatory mechanisms in the alkaline midgut of the sea urchin larva

A variety of factors (e.g. climate change, urbanization) are leading 
to increases in water temperatures in streams and rivers and is 
hypothesized to be contributing to declines of many freshwater 
fishes. However, factors that influence individual variation in thermal 
tolerance, and how these may change seasonally and how they are 
controlled mechanistically, are not well understood. To address this 
knowledge gap, we studied redside daceClinostomus elongatus, an 
imperilled stream fish native to rapidly urbanizing areas of eastern North 
America. We conducted field studies to examine thermal tolerance 
(using CTMax) in the wild, used laboratory studies to experimentally 
manipulate individual condition and employed transcriptomics to provide a mechanistic understanding of an organism’s response to 
increasing temperatures. We found that (1) thermal safety margins 
(CTmax − ambient water temperature) were less than the increases 
in peak water temperature predicted for many streams due to the 
combined effects of climate change and urbanization., (2) thermal 
tolerance was higher in fish fed a high- vs. low-ration diet, indicating 
a causal link between nutrition and thermal tolerance and (3) found 
that thermally stressed fish showed a transcriptional response related 
to transcription regulation and responses to unfolded proteins, and 
alternatively spliced genes related to gene expression regulation and 
metabolism. Our results can inform conservation captive breeding 
efforts, translocation and reintroduction programs for redside dace 
and other imperilled species by helping to identify appropriate source 
populations.

Thermal tolerance as it relates to season, body condition and gene expression in imperilled redside dace, Clinostomus elongatus

Trehalose 6-phosphate (Tre6P) is a sucrose specific signalling metabolite 
that has been implicated in the broad regulation of metabolism as 
well as developmental processes like flowering and shoot branching. 
Expressing tagged forms of TREHALOSE-6-PHOSPHATE SYNTHASE1 
(TPS1), the main Tre6P-synthesizing enzyme in arabidopsis, revealed 
that Tre6P synthesis occurs predominantly in the vasculature and 
meristematic tissues of the whole plant. The vascular expression of 
TPS1 on both sides of the apoplastic barrier (in phloem parenchyma as 
well as in companion cells and sieve elements) ideally places Tre6P at 
the interface between source and sink and at a highly strategic site for 
systemic signalling of sucrose status. To test this potential of Tre6P as 
a hormone-like signal, we investigated the impact of localized changes 
in Tre6P levels in axillary buds and found that lowering Tre6P strongly 
inhibited bud growth. We further found that changing Tre6P levels in 
the vasculature modulated flowering and branching. These effects on 
plant development involve interaction with photoperiodic signalling, 
alteration in sucrose allocation, and interaction with strigolactone 
signalling. As phytohormone signalling components including cytokinin 
and strigolactones are also located in the vasculature, the vasculature 
may be considered as a hub for developmental decision making. 
Combinatorial mutant analyses, metabolic profiling and RNA sequencing 
approaches revealed multiple connections between Tre6P and other 
phytohormone signalling pathways, especially strigolactones, allowing 
us to place Tre6P signalling in the network of hormonal regulation of 
plant development.

The signalling metabolite trehalose 6-phosphate is a key signal in the regulation of development

fly’, i.e. without any significant interruption in their forward movement. 
Using an inverse approach, planar dynamics are deduced from (lateral 
view) video-recordings. Starting from the momentum-impulse 
theorem, possible strategies for such dynamical mode transitions are 
evaluated, and applied, first to an example sequence. The strategy used 
in this worked example boils down to: crouch the hind parts and sprint 
them underneath the rising body centre of mass. Forward accelerations 
are not in play. Key characteristics of this transition strategy were 
extracted: progression speed, hip height, step duration(frequency), foot 
positioning at touchdown with respect to the hip and the body centre 
of mass (BCoM), and congruity between the moments of the ground 
reaction force about the BCoM and the rate of change of the total 
angular moment. Statistical analyses across all available recordings of 
transitions (15 by 10 individuals) confirm this strategy is always used 
and is shared across individuals. The costs (in J kg−1 m−1 ) linked 
to such ‘on the fly’ transitions were estimated. These approximately 
only double those of both the preceding quadrupedal and subsequent 
bipedal walking. Given the short duration of the transition as such (<1 
s), it is argued that the energetic costs to change walking posture ‘on 
the fly’ are negligible when considered in the context of the entire 
locomotor repertoire of baboons.

Dynamics of 'on the fly' quadrupedal-to-bipedal walking transitions in baboons.

As habitat loss and climate change threaten global biodiversity, we 
need a theoretical framework for predicting the combined impacts of 
these two threats on the performance of organisms. However, current 
models simplify microhabitats' diversity of natural landscapes and are 
prone to biased predictions. By combining empirical and modeling 
approaches, we show the cascading effect of habitat loss and climate 
change on activity time and thermoregulatory accuracy in a diurnal 
desert lizard. Our model predicted a severe loss of summer activity in 
the absence of medium and large rocks. Future warming will gradually 
decrease summer activity as even large rocks become thermally 
stressful. Warmer winters will enable more activity and require bushes 
and small rocks as shade retreats. Hence, microhabitats that may seem 
unimportant today may become important under climate change. 
Such modeling frameworks may be crucial to understanding species’ 
microhabitat requirements and developing conservation programs.

Using biophysical models to predict how habitat loss may render the ability of animals to cope with climate change

Birds can morph their wings with exceptional precision for a variety 
of tasks. They can change their wing shape and area to generate lift, 
adjust air flow for manoeuvrability, or to fold and store the wings 
when on the ground. A strong link exists between wing joint mobility 
and the flight behaviour of modern birds, with different flight styles exhibiting different constraints on joint range of motion (ROM). 
We can investigate ex vivo joint movement and ROM in extant taxa 
with high fidelity using X-ray Reconstruction of Moving Morphology 
(XROMM). However, for extinct taxa we are often left with bare bones 
and no connective or other soft tissues, thus making it impossible 
to measure potential joint movement directly. Recent advances in 
implementing and inferring soft-tissue constraints on ROM estimates 
can overcome these longstanding challenges. Here we present a 
workflow of how a functionally informative ROM estimate can be 
obtained for a fossil taxon and how it can be contextualised to draw 
functional and ecological inferences. We estimate ROM of the wing 
joints of the crownward stem-bird Ichthyornis dispar and calculate 
ligamentous constraints on its joint mobility. We then project our 
Ichthyornis joint mobility data into the ‘EcoPhyloMobilitySpace’ 
generated from a phylogenetically broad ex vivo XROMM dataset 
of extant birds, with specimens representing most major clades and 
a wide range of ecological niches and flight styles. This allows us to 
infer the flight behaviour of this stem-bird, which has implications for 
the ancestral flight capabilities of all crown-birds.

Wing Joint Mobility Across Ornithurae - How did Stem-Birds Fly?

Equine back pain is prevalent in at least 35% of ridden horses and often 
attributed to poor saddle fit. Saddles are designed and assessed with 
a stationary horse. However, the saddle is placed on, or adjacent to, 
epaxial muscles and extrinsic muscles of the forelimb which activate 
in temporally and spatially distinct patterns, signifying that, despite 
a relatively rigid skeletal frame, the overlaying soft tissue supporting 
the saddle is dynamic. Here our aim was to measure the regional 
morphing of the equine back during locomotion to understand how soft 
tissue movement could lead to high pressures and tissue damage. We 
measured shape change across the equine back in walk and trot, using 
photogrammetric high-speed surface reconstructions to create a map 
of the most and least mobile areas of the equine back for the two gaits. 
This map may be critical for future saddle design as the least mobile 
areas of the back are the most appropriate for distributing pressure 
and, thus, the regions best justified for supporting the compressive 
forces of the saddle and rider.

The dynamic surface of the saddle region in horses

Enhancer clusters (ECs) greater than 1.25 megabases upstream of 
SOX9 are frequently ablated in Pierre Robin sequence (PRS) patients 
exhibiting lower jaw hypoplasia. We previously characterised the 
activity of these ECs in facial progenitor cells (cranial neural crest cells, 
CNCCs) and quantified the impact of EC ablation on gene expression 
and craniofacial morphological development. To explore mechanisms 
facilitating extreme long-range regulation at this locus, we utilised 
optical reconstruction of chromatin architecture (ORCA) imaging 
to trace 3D locus topology during PRS EC activation. We observed 
pronounced changes in locus topology during CNCC differentiation from 
pluripotent stem cells and determined that these ensemble-average 
differences arise through changes in the frequency of commonly 
sampled topologies – an observation made possible through analysis 
of ORCA single chromatin fibre traces. We further identified two CTCFbound 
elements, internal to the SOX9 topologically associating domain, 
which promote stripe formation, are positioned near the domain’s 3D 
geometric centre and bridge enhancer-promoter contacts in a series 
of chromatin loops. Ablation of these elements results in diminished 
SOX9 expression and altered domain-wide contacts. Polymer models 
with uniform loading across the domain and frequent cohesin collisions 
recapitulate this multiloop, centrally clustered geometry. Together, 
we provide mechanistic insights into gene regulation over ultra-long 
genomic ranges, involving distal enhancer clusters and structural 
elements that mediate architectural stripe formation.

Regulatory and topological perturbation at a human disease locus

The origin of mammals from non-mammalian synapsids (NMS) 
represents an iconic locomotor transition, characterized by a change 
from reptile-like abducted limbs and lateral movements of the 
backbone to mammal-like adducted limbs and sagittal backbone 
movements. However, recent research has called into question the 
lateral-to-sagittal functional shift in NMS, instead suggesting that basal 
synapsids displayed a distinct functional regime and ancestral condition 
not observed in extant reptiles. To investigate this idea further, we 
usedAutobend,a technique for estimating vertebral osteological 
range of motion (oROM) from skeletons using digital modeling.We 
appliedAutobendto seven extant mammal and reptile species and 
8 exceptionally preserved non-mammalian synapsids to estimate 
vertebral oROM and intervertebral joint stiffness. Results revealed 
a clear distinction between extant mammals and reptiles in oROM, 
with reptiles emphasizing lateral bending and mammals sagittal 
bending as expected. While most extant taxa exhibited relatively similar 
levels of stiffness in lateral and sagittal directions, extant lizards and 
salamanders displayed much more compliance in lateral bending and 
lower stiffness overall, in keeping with their observed axial kinematics. 
The mammalian condition of sagittal mobility accompanied by axial 
twisting in the anterior column is first observed in the crownward 
tritylodontid cynodontKayentatherium. Given the strong association 
between sagittal bending and asymmetric gaits in mammals, these 
results provide an important first step in reconstructing the evolution 
of synapsid locomotor patterns.

Evolutionary origins of mammalian axial function revealed through digital bending experiments

Changes in surface temperature under climate change predict sea 
level rise and heat wave risk, but not responses of animals to altered 
thermal environments. Local air temperature plays a minor role in heat 
stress on terrestrial animals outdoors, so the conventional practice of 
projecting responses from future air temperature is inadequate. Solar 
radiation, direct and indirect, is the main source of heat stress, and 
will be under climate change too. The calculation of radiant heat load 
from ambient radiant flux is possible, although difficult. Black globe 
thermometers provide a simple alternative to assess radiant heat load, 
and have been used successfully to predict heat stress across a wide 
range of animal taxa in their natural environments. Miniature globes, 
carried by animals themselves, provide an index of dry heat stress in 
the microclimate selected. Diurnally active animals can escape solar 
radiation by switching to nocturnal activity, or by burrowing, but to 
only a limited extent by seeking shade; all those behaviours incur 
costs. Animals that sweat or pant have the capacity to dissipate all the 
heat load expected under global warming, even with metabolic heat 
load increased by exercise, but the body water evaporated has to be 
replaced. Evaporative cooling depends on skin temperature, ambient 
water vapour pressure, and local wind speed. We recommend that globe 
thermometry should become routine in studies of animal responses 
to global warming, and that those studies should report surface wind 
speeds, and water vapour pressures, not relative humidity.

Solar radiation, humidity and climate change

Here we address knowledge gaps regarding the evolution of glucose
acquisition in vertebrates by examining glucose uptake in the
gastrointestinal tract (GIT) of ancient fish, including lake sturgeon
(Acipenser fulvescens) and Pacific spiny dogfish (Squalus suckleyi).
Within the GIT of these “living fossils” evolved an organ called the spiral
valve. Due to its scroll-like internal folds, the spiral valve presumably
increases gastrointestinal surface area and slows down chyme
passage to maximize nutrient absorption, suggesting it to be primarily
responsible for nutrient acquisition. However, there is currently minimal
functional data to support this claim. This study aims to determine
the functional role of the spiral valve in carbohydrate breakdown and
glucose acquisition during different feeding states and determine if also took into account different sex: males, females and neomales.
This exploration was performed using UV-LC allowing to monitor all
C5-cytosine variants (5-mC, 5-hmC, 5-caC, 5-fC, C) in the context
of CpG and non-CpG. CHO nutrition induced a modification of 5-mC
levels mostly in neomales and preferentially at early stages of life.
Of huge interest, this epigenetic phenotype was found in all-sex
alevins born from broodstock fed during a whole reproductive period
with a high CHO diet suggesting an intergenerational transmission
of the phenotype.
this role changes in organisms with different life histories. To test this,
we assessed glucose digestibility and transport capacity along the GIT
of A. fulvescens and S. suckleyi in fed and fasted fish. Digestibility was
assessed through biochemical assays for the carbohydrate digesting
enzyme maltase, while transport capacity was determined via i) mRNA
abundance of the primary dietary glucose transporter (SGLT1), and ii)
through in vitro intestinal fluxes. In comparison to A. fulvescens, the
spiral valve in S. suckleyi has an elevated role in glucose digestion and
transport. There were no significant postprandial changes in SGLT1
mRNA abundance nor maltase activity in A. fulvescens in comparison
to S. suckleyi, supporting their feeding strategies with A. fulvescens
being continuous feeders and S. suckleyi being opportunistic feeders.

Comparing glucose acquisition strategies between two ancient fish species: Lake sturgeon (Acipenser fulvescens) and Pacific spiny dogfish (Squalus suckleyi)

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