Investigating the dietary niches of fossil Plio-Pleistocene European macaques: The case of Macaca majori Azzaroli, 1946 from Sardinia.

The genus Macaca includes medium- to large-bodied monkeys and represents one of the most diverse primate genera, also having a very large geographic range. Nowadays, wild macaque populations are found in Asia and Africa, inhabiting a wide array of habitats. Fossil macaques were also present in Europe from the Late Miocene until the Late Pleistocene. Macaques are considered ecologically flexible monkeys that exhibit highly opportunistic dietary strategies, which may have been critical to their evolutionary success. Nevertheless, available ecological information regarding fossil European species is very sparse, limiting our knowledge of their evolutionary history in this geographic area. To further our understanding of fossil European macaque ecology, we investigated the dietary ecology of Macaca majori, an insular endemic species from Sardinia. In particular, we characterized the dental capabilities and potential dietary adaptations of M. majori through dental topographic and enamel thickness analyses of two M2s from the Early Pleistocene site of Capo Figari (1.8 Ma). We also assessed its diet through dental microwear texture analysis, while the microwear texture of M. majori was also compared with microwear textures from other European fossil macaques from mainland Europe. The dental topographic and enamel thickness analyses suggest that M. majori frequently consumes hard/mechanically challenging and/or abrasive foods. The results of the dental microwear analysis are consistent with this interpretation and further suggest that M. majori probably exhibited more durophagous dietary habits than mainland Plio-Pleistocene macaques. Overall, our results indicate that M. majori probably occupied a different dietary niche compared to its mainland fossil relatives, which suggests that they may have inhabited different paleoenvironments.

Differential efficacies of Cas nucleases on microsatellites involved in human disorders and associated off-target mutations.

Microsatellite expansions are the cause of >20 neurological or developmental human disorders. Shortening expanded repeats using specific DNA endonucleases may be envisioned as a gene editing approach. Here, we measured the efficacy of several CRISPR-Cas nucleases to induce recombination within disease-related microsatellites, in Saccharomyces cerevisiae. Broad variations in nuclease performances were detected on all repeat tracts. Wild-type Streptococcus pyogenes Cas9 (SpCas9) was more efficient than Staphylococcus aureus Cas9 on all repeats tested, except (CAG)33. Cas12a (Cpf1) was the most efficient on GAA trinucleotide repeats, whereas GC-rich repeats were more efficiently cut by SpCas9. The main genetic factor underlying Cas efficacy was the propensity of the recognition part of the sgRNA to form a stable secondary structure, independently of its structural part. This suggests that such structures form in vivo and interfere with sgRNA metabolism. The yeast genome contains 221 natural CAG/CTG and GAA/CTT trinucleotide repeats. Deep sequencing after nuclease induction identified three of them as carrying statistically significant low frequency mutations, corresponding to SpCas9 off-target double-strand breaks.

Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions.

Microsatellites are short tandem repeats, ubiquitous in all eukaryotes and represent ~2% of the human genome. Among them, trinucleotide repeats are responsible for more than two dozen neurological and developmental disorders. Targeting microsatellites with dedicated DNA endonucleases could become a viable option for patients affected with dramatic neurodegenerative disorders. Here, we used the Streptococcus pyogenes Cas9 to induce a double-strand break within the expanded CTG repeat involved in myotonic dystrophy type 1, integrated in a yeast chromosome. Repair of this double-strand break generated unexpected large chromosomal deletions around the repeat tract. These deletions depended on RAD50, RAD52, DNL4 and SAE2, and both non-homologous end-joining and single-strand annealing pathways were involved. Resection and repair of the double-strand break (DSB) were totally abolished in a rad50Δ strain, whereas they were impaired in a sae2Δ mutant, only on the DSB end containing most of the repeat tract. This observation demonstrates that Sae2 plays significant different roles in resecting a DSB end containing a repeated and structured sequence as compared to a non-repeated DSB end. In addition, we also discovered that gene conversion was less efficient when the DSB could be repaired using a homologous template, suggesting that the trinucleotide repeat may interfere with gene conversion too. Altogether, these data show that SpCas9 may not be the best choice when inducing a double-strand break at or near a microsatellite, especially in mammalian genomes that contain many more dispersed repeated elements than the yeast genome.

Time-resolved microfluidics unravels individual cellular fates during double-strand break repair.

BACKGROUND: Double-strand break repair (DSBR) is a highly regulated process involving dozens of proteins acting in a defined order to repair a DNA lesion that is fatal for any living cell. Model organisms such as Saccharomyces cerevisiae have been used to study the mechanisms underlying DSBR, including factors influencing its efficiency such as the presence of distinct combinations of microsatellites and endonucleases, mainly by bulk analysis of millions of cells undergoing repair of a broken chromosome. Here, we use a microfluidic device to demonstrate in yeast that DSBR may be studied at a single-cell level in a time-resolved manner, on a large number of independent lineages undergoing repair. RESULTS: We used engineered S. cerevisiae cells in which GFP is expressed following the successful repair of a DSB induced by Cas9 or Cpf1 endonucleases, and different genetic backgrounds were screened to detect key events leading to the DSBR efficiency. Per condition, the progenies of 80-150 individual cells were analyzed over 24 h. The observed DSBR dynamics, which revealed heterogeneity of individual cell fates and their contributions to global repair efficacy, was confronted with a coupled differential equation model to obtain repair process rates. Good agreement was found between the mathematical model and experimental results at different scales, and quantitative comparisons of the different experimental conditions with image analysis of cell shape enabled the identification of three types of DSB repair events previously not recognized: high-efficacy error-free, low-efficacy error-free, and low-efficacy error-prone repair. CONCLUSIONS: Our analysis paves the way to a significant advance in understanding the complex molecular mechanism of DSB repair, with potential implications beyond yeast cell biology. This multiscale and multidisciplinary approach more generally allows unique insights into the relation between in vivo microscopic processes within each cell and their impact on the population dynamics, which were inaccessible by previous approaches using molecular genetics tools alone.

Feeding ecology of the last European colobine monkey, Dolichopithecus ruscinensis.

Currently, very little is known about the ecology of extinct Eurasian cercopithecids. Dietary information is crucial in understanding the ecological adaptations and diversity of extinct cercopithecids and the evolution of this family. For example, the colobine genus Dolichopithecus is represented by multiple large-bodied species that inhabited Eurasia during the Pliocene-Early Pleistocene. The available evidence, though limited, suggests semiterrestrial locomotion, which contrasts with most extant African and Asian colobines that exhibit morphological and physiological adaptations for arboreality and folivory. These differences raise questions regarding the dietary specialization of early colobine taxa and how/if that influenced their dispersion out of Africa and into Eurasia. To further our understanding of the ecology of Plio-Pleistocene cercopithecids, we characterized the dental capabilities and potential dietary adaptations of Dolichopithecus ruscinensis through dental topographic and enamel thickness analyses on an M1 from the locality of Serrat d'en Vacquer, Perpignan (France). We also assessed the feeding behavior of D. ruscinensis through dental microwear texture analysis on a broad sample of fossil molars from fossil sites in France, Greece, Bulgaria, and Romania. Dental topographic and enamel thickness analyses suggest that D. ruscinensis could efficiently process a wide range of foods. Results of the dental microwear texture analysis suggest that its diet ranged from folivory to the consumption of more mechanically challenging foods. Collectively, this suggests a more opportunistic feeding behavior for Dolichopithecus than characteristic of most extant colobines.

The landscape of tooth shape: Over 20 years of dental topography in primates.

Diet plays an incontrovertible role in primate evolution, affecting anatomy, growth and development, behavior, and social structure. It should come as no surprise that a myriad of methods for reconstructing diet have developed, mostly utilizing the element that is not only most common in the fossil record but also most pertinent to diet: teeth. Twenty years ago, the union of traditional, anatomical analyses with emerging scanning and imaging technologies led to the development of a new method for quantifying tooth shape and reconstructing the diets of extinct primates. This method became known as dental topography.

Trinucleotide repeat instability during double-strand break repair: from mechanisms to gene therapy.

Trinucleotide repeats are a particular class of microsatellites whose large expansions are responsible for at least two dozen human neurological and developmental disorders. Slippage of the two complementary DNA strands during replication, homologous recombination or DNA repair is generally accepted as a mechanism leading to repeat length changes, creating expansions and contractions of the repeat tract. The present review focuses on recent developments on double-strand break repair involving trinucleotide repeat tracts. Experimental evidences in model organisms show that gene conversion and break-induced replication may lead to large repeat tract expansions, while frequent contractions occur either by single-strand annealing between repeat ends or by gene conversion, triggering near-complete contraction of the repeat tract. In the second part of this review, different therapeutic approaches using highly specific single- or double-strand endonucleases targeted to trinucleotide repeat loci are compared. Relative efficacies and specificities of these nucleases will be discussed, as well as their potential strengths and weaknesses for possible future gene therapy of these dramatic disorders.

A window into the early evolutionary history of Cercopithecidae: Late Miocene evidence from Chad, Central Africa.

Central Africa is known as a major center of diversification for extant Old World Monkeys (OWM) and yet has a poorly documented fossil record of monkeys. Here we report a new colobine monkey (Cercopithecoides bruneti sp. nov.) from the Central African hominin-bearing fossiliferous area of Toros-Menalla, Chad at ca. 7 Ma. In addition to filling a gap in the spatial and temporal record of early OWM evolutionary history, we assess the ecomorphological diversity of early OWM by providing evidence on the onset of a folivorous diet and a partial reacquisition of terrestrial locomotor habits among Miocene colobines. We also support the phylogenetic affinities of the genus Cercopithecoides among the stem group of the extant African colobine monkeys.

A comparison of relief estimates used in three-dimensional dental topography.

OBJECTIVES: Topographic estimates of dental relief are now commonly used to make dietary inferences from the teeth of extant and extinct primates. We thoroughly compared commonly used relief estimates in an effort to help researchers decide which variable best suits their objectives. MATERIALS AND METHODS: We combined a total of three datasets: five theoretical models built to compare the effect of tooth complexity and basin depth on relief estimates, a dataset of 110 lower molars of prosimians, and a dataset of 25 upper molars of apes. We investigated intra-mesh variation and tooth average relief, estimated from slope and three different relief indices, according to four criteria: (1) the ability to map relief on topographic maps, (2) the correlation with other relief estimates, (3) the ability to separate high-relief molars of folivores from deep-relief molars of insectivores in prosimians, and (4) the influence of surface complexity on relief estimates in apes. RESULTS: We found that polygon slope and relief index are linked by a mathematical relation. Tooth average slope and all relief indices are strongly correlated. In contrast, relief estimates are moderately correlated to cusp elevation. One relief index of four relief estimates had an excellent ability to separate high-relief from deep-relief molars in prosimians, whereas slope could not separate them. No significant effect of tooth complexity on dental relief could be detected in apes. CONCLUSIONS: Because slope and relief indices are highly correlated, it is strongly recommended not to combine them in multivariate analysis. Still, slope and relief indices show interesting differences in scaling, graphical representation, computation method, and ability to separate high-relief and deep-relief molars. Our results also suggest that slope and relief indices can vary independently of tooth complexity and are moderately affected by mean cusp elevation in apes.

Differential requirement of Srs2 helicase and Rad51 displacement activities in replication of hairpin-forming CAG/CTG repeats.

Trinucleotide repeats are a source of genome instability, causing replication fork stalling, chromosome fragility, and impaired repair. Specialized helicases play an important role in unwinding DNA structures to maintain genome stability. The Srs2 helicase unwinds DNA hairpins, facilitates replication, and prevents repeat instability and fragility. However, since Srs2 is a multifunctional protein with helicase activity and the ability to displace Rad51 recombinase, it was unclear which functions were required for its various protective roles. Here, using SRS2 separation-of-function alleles, we show that in the absence of Srs2 recruitment to PCNA or in helicase-deficient mutants, breakage at a CAG/CTG repeat increases. We conclude that Srs2 interaction with PCNA allows the helicase activity to unwind fork-blocking CAG/CTG hairpin structures to prevent breaks. Independently of PCNA binding, Srs2 also displaces Rad51 from nascent strands to prevent recombination-dependent repeat expansions and contractions. By 2D gel electrophoresis, we detect two different kinds of structured intermediates or joint molecules (JMs). Some JMs are Rad51-independent and exhibit properties of reversed forks, including being processed by the Exo1 nuclease. In addition, in a helicase-deficient mutant, Rad51-dependent JMs are detected, probably corresponding to recombination between sisters. These results clarify the many roles of Srs2 in facilitating replication through fork-blocking hairpin lesions.

Shortening trinucleotide repeats using highly specific endonucleases: a possible approach to gene therapy?

Trinucleotide repeat expansions are involved in more than two dozen neurological and developmental disorders. Conventional therapeutic approaches aimed at regulating the expression level of affected genes, which rely on drugs, oligonucleotides, and/or transgenes, have met with only limited success so far. An alternative approach is to shorten repeats to non-pathological lengths using highly specific nucleases. Here, I review early experiments using meganucleases, zinc-finger nucleases (ZFN), and transcription-activator like effector nucleases (TALENs) to contract trinucleotide repeats, and discuss the possibility of using CRISPR-Cas nucleases to the same end. Although this is a nascent field, I explore the possibility of designing nucleases and effectively delivering them in the context of gene therapy.

A 2Ma old baboon-like monkey from Northern Greece and new evidence to support the Paradolichopithecus - Procynocephalus synonymy (Primates: Cercopithecidae).

A new fossil cranium of a large papionin monkey from the Lower Pleistocene site of Dafnero-3 in Western Macedonia, Greece, is described by means of outer and inner morphological and metric traits using high-resolution micro-computed tomography. Comparisons with modern cercopithecids and contemporaneous Eurasian fossil taxa suggest that the new cranium could equally be ascribed to either the Eurasian Paradolichopithecus or to the East Asian Procynocephalus. The combination of the available direct and indirect fossil evidence, including the new cranium from Dafnero, revives an earlier hypothesis that considers these two sparsely documented genera as synonyms. The timing and possible causes of the rise and demise of Paradolichopithecus - Procynocephalus are discussed.

Was Mesopithecus a seed eating colobine? Assessment of cracking, grinding and shearing ability using dental topography.

Extant colobine monkeys have been historically described as specialized folivores. However, reports on both their behavior and dental metrics tend to ascribe a more varied diet to them. In particular, several species, such as Pygathrix nemaeus and Rhinopithecus roxellana, are dedicated seasonal seed eaters. They use the lophs on their postcanine teeth to crack open the hard endocarp that protects some seeds. This raises the question of whether the bilophodont occlusal pattern of colobine monkeys first evolved as an adaptation to folivory or sclerocarpic foraging. Here, we assess the sclerocarpic foraging ability of the oldest European fossil colobine monkey, Mesopithecus. We use computed microtomograpy to investigate the three-dimensional (3D) dental topography and enamel thickness of upper second molars ascribed to the late Miocene species Mesopithecus pentelicus from Pikermi, Greece. We compare M. pentelicus to a sample of extant Old World monkeys encompassing a wide range of diets. Furthermore, we combine classic dietary categories such as folivory with alternative categories that score the ability to crack, grind and shear mechanically challenging food. The 3D dental topography of M. pentelicus predicts an ability to crack and grind hard foods such as seeds. This is consistent with previous results obtained from dental microwear analysis. However, its relatively thin enamel groups M. pentelicus with other folivorous cercopithecids. We interpret this as a morphological trade-off between the necessity to avoid tooth failure resulting from hard food consumption and the need to process a high amount of leafy material. Our study demonstrates that categories evaluating the cracking, grinding or shearing ability, traditional dietary categories, and dental topography combine well to make a powerful tool for the investigation of diet in extant and extinct primates.

Relationship between foramen magnum position and locomotion in extant and extinct hominoids.

From the Miocene Sahelanthropus tchadensis to Pleistocene Homo sapiens, hominins are characterized by a derived anterior position of the foramen magnum relative to basicranial structures. It has been previously suggested that the anterior position of the foramen magnum in hominins is related to bipedal locomotor behavior. Yet, the functional relationship between foramen magnum position and bipedal locomotion remains unclear. Recent studies, using ratios based on cranial linear measurements, have found a link between the anterior position of the foramen magnum and bipedalism in several mammalian clades: marsupials, rodents, and primates. In the present study, we compute these ratios in a sample including a more comprehensive dataset of extant hominoids and fossil hominins. First, we verify if the values of ratios can distinguish extant humans from apes. Then, we test whether extinct hominins can be distinguished from non-bipedal extant hominoids. Finally, we assess if the studied ratios are effective predictors of bipedal behavior by testing if they mainly relate to variation in foramen magnum position rather than changes in other cranial structures. Our results confirm that the ratios discriminate between extant bipeds and non-bipeds. However, the only ratio clearly discriminating between fossil hominins and other extant apes is that which only includes basicranial structures. We show that a large proportion of the interspecific variation in the other ratios relates to changes in facial, rather than basicranial, structures. In this context, we advocate the use of measurements based only on basicranial structures when assessing the relationship between foramen magnum position and bipedalism in future studies.

Investigating the dental toolkit of primates based on food mechanical properties: Feeding action does matter.

Although conveying an indisputable morphological and behavioral signal, traditional dietary categories such as frugivorous or folivorous tend to group a wide range of food mechanical properties together. Because food/tooth interactions are mostly mechanical, it seems relevant to investigate the dental morphology of primates based on mechanical categories. However, existing mechanical categories classify food by its properties but cannot be used as factors to classify primate dietary habits. This comes from the fact that one primate species might be adapted to a wide range of food mechanical properties. To tackle this issue, what follows is an original framework based on action-related categories. The proposal here is to classify extant primates based on the range of food mechanical properties they can process through one given action. The resulting categories can be used as factors to investigate the dental tools available to primates. Furthermore, cracking, grinding, and shearing categories assigned depending on the hardness and the toughness of food are shown to be supported by morphological data (3D relative enamel thickness) and topographic data (relief index, occlusal complexity, and Dirichlet normal energy). Inferring food mechanical properties from dental morphology is especially relevant for the study of extinct primates, which are mainly documented by dental remains. Hence, we use action-related categories to investigate the molar morphology of an extinct colobine monkey Mesopithecus pentelicus from the Miocene of Pikermi, Greece. Action-related categories show contrasting results compared with classical categories and give us new insights into the dietary adaptations of this extinct primate. Finally, we provide some possible directions for future research aiming to test action-related categories. In particular, we suggest acquiring more data on mechanically challenging fallback foods and advocate the use of other food mechanical properties such as abrasiveness. The development of new action-related dental metrics is also crucial for primate dental studies.

Genome-wide replication landscape of Candida glabrata.

BACKGROUND: The opportunistic pathogen Candida glabrata is a member of the Saccharomycetaceae yeasts. Like its close relative Saccharomyces cerevisiae, it underwent a whole-genome duplication followed by an extensive loss of genes. Its genome contains a large number of very long tandem repeats, called megasatellites. In order to determine the whole replication program of the C. glabrata genome and its general chromosomal organization, we used deep-sequencing and chromosome conformation capture experiments. RESULTS: We identified 253 replication fork origins, genome wide. Centromeres, HML and HMR loci, and most histone genes are replicated early, whereas natural chromosomal breakpoints are located in late-replicating regions. In addition, 275 autonomously replicating sequences (ARS) were identified during ARS-capture experiments, and their relative fitness was determined during growth competition. Analysis of ARSs allowed us to identify a 17-bp consensus, similar to the S. cerevisiae ARS consensus sequence but slightly more constrained. Megasatellites are not in close proximity to replication origins or termini. Using chromosome conformation capture, we also show that early origins tend to cluster whereas non-subtelomeric megasatellites do not cluster in the yeast nucleus. CONCLUSIONS: Despite a shorter cell cycle, the C. glabrata replication program shares unexpected striking similarities to S. cerevisiae, in spite of their large evolutionary distance and the presence of highly repetitive large tandem repeats in C. glabrata. No correlation could be found between the replication program and megasatellites, suggesting that their formation and propagation might not be directly caused by replication fork initiation or termination.

Integration between the face and the mandible of Pongo and the evolution of the craniofacial morphology of orangutans.

OBJECTIVES: Extant Pongo diverges from other hominids by a series of craniofacial morphological features, such as a concave face, a reduced supraorbital torus, or an upwardly orientated palate. These traits are not independent because the skull is a complex integrated structure. The aim of this study is to describe the relationship between the face and mandible of Pongo, in order to examine the link between mandibular structures and the set-up of the unique facial features of orangutans. MATERIALS AND METHODS: Using 3D geometric morphometrics, the morphological integration between face and mandible of Pongo is compared to that of the three extant hominids: Homo, Pan, and Gorilla. Pooled within-species partial least squares analyses are computed in order to quantify the patterns and levels of integration. RESULTS: The covariation analyses show unique patterns of integration and levels of correlation in Pongo when compared to other hominids. This study shows that the craniofacial features distinguishing Pongo from African great apes are related to differences in the patterns of integration and levels of correlation between facial and mandibular shape. DISCUSSION: Changes in important functions may play a part in these modifications of craniofacial integration. This study underlines the importance of the mandible and of the mandibular functions in the development of the unique craniofacial features of Pongo.

Purification of G1 daughter cells from different Saccharomycetes species through an optimized centrifugal elutriation procedure.

Centrifugal elutriation discriminates cells according to their sedimentation coefficients, generating homogeneous samples well suited for genomic comparative approaches. It can, for instance, isolate G1 daughter cells from a Saccharomyces cerevisiae unsynchronized population, alleviating ageing and cell-cycle biases when conducting genome-wide/single-cell studies. The present report describes a straightforward and robust procedure to determine whether a cell population of virtually any yeast species can be efficiently elutriated, while offering solutions to optimize success. This approach was used to characterize elutriation parameters and S-phase progression of four yeast species (S. cerevisiae, Candida glabrata, Lachancea kluyveri and Pichia sorbitophila) and could theoretically be applied to any culture of single, individual cells.

Detection and characterization of megasatellites in orthologous and nonorthologous genes of 21 fungal genomes.

Megasatellites are large DNA tandem repeats, originally described in Candida glabrata, in protein-coding genes. Most of the genes in which megasatellites are found are of unknown function. In this work, we extended the search for megasatellites to 20 additional completely sequenced fungal genomes and extracted 216 megasatellites in 203 out of 142,121 genes, corresponding to the most exhaustive description of such genetic elements available today. We show that half of the megasatellites detected encode threonine-rich peptides predicted to be intrinsically disordered, suggesting that they may interact with several partners or serve as flexible linkers. Megasatellite motifs were clustered into several families. Their distribution in fungal genes shows that different motifs are found in orthologous genes and similar motifs are found in unrelated genes, suggesting that megasatellite formation or spreading does not necessarily track the evolution of their host genes. Altogether, these results suggest that megasatellites are created and lost during evolution of fungal genomes, probably sharing similar functions, although their primary sequences are not necessarily conserved.

Morphology of the Bony Labyrinth Supports the Affinities of Paradolichopithecus with the Papionina.

Discoveries in recent decades indicate that the large papionin monkeys Paradolipopithecus and Procynocephalus are key members of the Late Pliocene - Early Pleistocene mammalian faunas of Eurasia. However, their taxonomical status, phylogenetic relationships, and ecological profile remain unclear. Here we investigate the two latter aspects through the study of the inner ear anatomy, as revealed by applying micro-CT scan imaging techniques on the cranium LGPUT DFN3-150 of Paradolichopithecus from the lower Pleistocene (2.3 Ma) fossil site Dafnero-3 in Northwestern Greece. Using geometric morphometric methods, we quantified shape variation and the allometric and phylogenetic signals in extant cercopithecines (n = 80), and explored the morphological affinities of the fossil specimen with extant taxa. LGPUT DFN3-150 has a large centroid size similar to that of baboons and their relatives. It shares several shape features with Macacina and Cercopithecini, which we interpret as probable retention of a primitive morphology. Overall, its inner ear morphology is more consistent with a stem Papionini more closely related to Papionina than Macacina, or to a basal crown Papionina. Our results, along with morphometrical and ecological features from previous studies, call into question the traditional hypothesis of a Paradolichopithecus-Macacina clade, and provide alternative perspectives in the study of Eurasian primate evolution during the late Neogene-Quaternary. Supplementary Information: The online version contains supplementary material available at 10.1007/s10764-022-00329-4.