Breaking the constraint on the number of cervical vertebrae in mammals: On homeotic transformations in lorises and pottos.

Mammals almost always have seven cervical vertebrae. The strong evolutionary constraint on changes in this number has been broken in sloths and manatees. We have proposed that the extremely low activity and metabolic rates of these species relax the stabilizing selection against changes in the cervical count. Our hypothesis is that strong stabilizing selection in other mammals is largely indirect and due to associated pleiotropic effects, including juvenile cancers. Additional direct selection can occur due to biomechanical problems (thoracic outlet syndrome). Low metabolic and activity rates are thought to diminish these direct and indirect effects. To test this hypothesis within the primates, we have compared the number of cervical vertebrae of three lorisid species with particularly low activity and metabolic rates with those of more active primate species, including with their phylogenetically closest active relatives, the galagids (bushbabies). In support of our hypothesis, we found that 37.6% of the lorisid specimens had an abnormal cervical count, which is a higher percentage than in the other nine primate families, in which the incidence varied from zero to 2.2%. We conclude that our data support the importance of internal selection in constraining evolvability and of a relaxed stabilizing selection for increasing evolvability. Additionally, we discuss that there is no support for a role of the muscularized diaphragm in the evolutionary constraint.

Parthenogenesis and developmental constraints.

The absence of a paternal contribution in an unfertilized ovum presents two developmental constraints against the evolution of parthenogenesis. We discuss the constraint caused by the absence of a centrosome and the one caused by the missing set of chromosomes and how they have been broken in specific taxa. They are examples of only a few well-underpinned examples of developmental constraints acting at macro-evolutionary scales in animals. Breaking of the constraint of the missing chromosomes is the best understood and generally involves rare occasions of drastic changes of meiosis. These drastic changes can be best explained by having been induced, or at least facilitated, by sudden cytological events (e.g., repeated rounds of hybridization, endosymbiont infections, and contagious infections). Once the genetic and developmental machinery is in place for regular or obligate parthenogenesis, shifts to other types of parthenogenesis can apparently rather easily evolve, for example, from facultative to obligate parthenogenesis, or from pseudoarrhenotoky to haplodiploidy. We argue that the combination of the two developmental constraints forms a near-absolute barrier against the gradual evolution from sporadic to obligate or regular facultative parthenogenesis, which can probably explain why the occurrence of the highly advantageous mode of regular facultative parthenogenesis is so rare and entirely absent in vertebrates.

Fast running restricts evolutionary change of the vertebral column in mammals.

The mammalian vertebral column is highly variable, reflecting adaptations to a wide range of lifestyles, from burrowing in moles to flying in bats. However, in many taxa, the number of trunk vertebrae is surprisingly constant. We argue that this constancy results from strong selection against initial changes of these numbers in fast running and agile mammals, whereas such selection is weak in slower-running, sturdier mammals. The rationale is that changes of the number of trunk vertebrae require homeotic transformations from trunk into sacral vertebrae, or vice versa, and mutations toward such transformations generally produce transitional lumbosacral vertebrae that are incompletely fused to the sacrum. We hypothesize that such incomplete homeotic transformations impair flexibility of the lumbosacral joint and thereby threaten survival in species that depend on axial mobility for speed and agility. Such transformations will only marginally affect performance in slow, sturdy species, so that sufficient individuals with transitional vertebrae survive to allow eventual evolutionary changes of trunk vertebral numbers. We present data on fast and slow carnivores and artiodactyls and on slow afrotherians and monotremes that strongly support this hypothesis. The conclusion is that the selective constraints on the count of trunk vertebrae stem from a combination of developmental and biomechanical constraints.

When right differs from left: human limb directional asymmetry emerges during very early development.

The often observed directional asymmetry (DA) in human limb bones may have a genetic/developmental basis and/or could emerge from different mechanical loadings across sides due to handedness. Because behavioural lateralization in itself has a genetic basis, it has been suggested that DA in limbs could develop prenatally as a pre-adaptation to adult life. However, the presence of consistent differences in the size of left and right limb bones in early development is understudied. We study asymmetry in limb bones during early development (10-20 weeks of gestation) in a sample of 178 aborted foetuses. Statistically significant DA was found in several upper and lower limb bones, where the right-hand side was consistently larger than the left. We argue that this pattern is probably the consequence of developmental processes related to internal asymmetric positioning of organs.

Analysis of cervical ribs in a series of human fetuses.

In humans, an increasing body of evidence has linked the frequency of cervical ribs to stillbirths, other malformations and early childhood cancers. However, the frequency of cervical ribs in a putatively healthy fetal population is not sufficiently known to assess the actual medical risks of these prenatal findings. We therefore analyzed the presence of skeletal anomalies in a series of 199 electively aborted fetuses, which were whole-mount stained with alizarin red specific for skeletal tissues. Results show that approximately 40% of the fetuses had cervical ribs, even though external congenital abnormalities such as craniofacial and limb defects were absent. A literature overview indicates that the observed frequency of cervical ribs is comparable to results previously obtained for deceased fetuses with no or minor congenital anomalies, and higher than expected for healthy fetuses. This unexpected result can probably in part be explained by a higher detection rate of small cervical ribs when using alizarin red staining instead of radiographs. Additionally, studies in the literature suggest that the size of a cervical rib may indicate the severity of abnormalities, but this possibility requires further research. Anomalies of the axial skeleton are known to be caused by a disturbance of early development, which alters Hox gene expression, but in this study the origin of the stress could not be verified as maternal medical data were not available. The co-occurrence of rudimentary or absent 12th ribs in 23.6% of the cases with cervical ribs indicates that in approximately 8% of the fetuses a homeotic shift occurred over a larger part of the vertebral column. This suggests that the expression of multiple Hox genes may have been affected in these fetuses. Together, the high incidence of cervical ribs and also their co-occurrence with rudimentary or absent 12th ribs suggests that there may have been a disturbance of early development such that the studied fetuses are probably not informative about the general population. Future studies determining the frequency of cervical ribs in a more healthy fetal population are therefore needed to evaluate their potential as an indicator of medical risks.

Sexual dimorphism in the prenatal digit ratio (2D:4D).

The second to fourth digit ratio (2D:4D) is smaller in human males than in females and hence this trait is sexually dimorphic. The digit ratio is thought to be established during early prenatal development under the influence of prenatal sex hormones. However, the general assumption of early establishment has hardly been studied. In our study, we analyzed the 2D:4D ratio in 327 deceased human fetuses. We measured digit lengths in 169 male and 158 female fetuses ranging from 14 to 42 weeks old. Our results showed a slight, but significant, sexual dimorphism in the expected direction, i.e., females had, on average, a ratio of 0.924 and males a ratio of 0.916. There was no significant relationship with the presence or absence of minor and major or single and multiple congenital abnormalities. There was a minimal, but significant difference between digit ratios based on digit lengths including and excluding the non-bony fingertip with the values being strongly correlated (r = .98). The prenatal 2D:4D ratio was lower than has thus far been reported for children and adults both for males and females. The extent of the sexual dimorphism in fetuses was similar to that found for children, but lower than for adults. The 2D:4D ratio, thus, seems to increase after birth in both men and women, with the second digit growing faster than the fourth digit (positive allometric growth of digit two) and perhaps more so in women than in men. Therefore, the sexual dimorphism is probably determined by prenatal as well as by postnatal developmental processes.

Exploring copy number variants in deceased fetuses and neonates with abnormal vertebral patterns and cervical ribs.

BACKGROUND: Cervical patterning abnormalities are rare in the general population, but one variant, cervical ribs, is particularly common in deceased fetuses and neonates. The discrepancy between the incidence in the general population and early mortality is likely due to indirect selection against cervical ribs. The cause for the co-occurrence of cervical ribs and adverse outcome remains unidentified. Copy number variations resulting in gain or loss of specific genes involved in development and patterning could play a causative role. METHODS: Radiographs of 374 deceased fetuses and infants, including terminations of pregnancies, stillbirths and neonatal deaths, were assessed. Copy number profiles of 265 patients were determined using single nucleotide polymorphism array. RESULTS: 274/374 patients (73.3%) had an abnormal vertebral pattern, which was associated with congenital abnormalities. Cervical ribs were present in 188/374 (50.3%) and were more common in stillbirths (69/128 [53.9%]) and terminations of pregnancies (101/188 [53.7%]), compared to live births (18/58, 31.0%). Large (likely) deleterious copy number variants and aneuploidies were prevalent in these patients. None of the rare copy number variants were recurrent or overlapped with candidate genes for vertebral patterning. CONCLUSIONS: The large variety of copy number variants in deceased fetuses and neonates with similar abnormalities of the vertebral pattern probably reflects the etiological heterogeneity of vertebral patterning abnormalities. This genetic heterogeneity corresponds with the hypothesis that cervical ribs can be regarded as a sign of disruption of critical, highly interactive stages of embryogenesis. The vertebral pattern can probably provide valuable information regarding fetal and neonatal outcome.

Why did the savant syndrome not spread in the population? A psychiatric example of a developmental constraint.

A developmental constraint is a mechanism that limits the possibility of a phenotype to evolve. There is growing evidence for the existence of developmental constraints in the biological literature. We hypothesize that a developmental constraint prevents the savant syndrome, despite its positive aspects, from spreading in the population. Here, the developmental constraint is the result of the high interactivity among body parts in an early stage in embryological development, namely early organogenesis or the phylotypic stage. The interactivity during this stage involves all components of the embryo, and as a result mutations that affect one part of the embryo also affect other parts. We hypothesize that a mutation, which gives rise to the development of the positive aspects of the savant syndrome (e.g., an impressive memory capacity), will virtually always have a deleterious effect on the development of other phenotypic traits (e.g., resulting in autism and/or impaired motor coordination). Thus, our hypothesis states that the savant syndrome cannot spread in the population because of this developmental constraint. The finding that children with savant syndrome often have autism and physical anomalies, which are known to be established during early organogenesis, supports our hypothesis.

Increased prevalence of abnormal vertebral patterning in fetuses and neonates with trisomy 21.

PURPOSE: To assess the prevalence of an abnormal number of ribs in a cohort of fetuses and neonates with trisomy 21 and compare this with a subgroup of fetuses without anomalies. MATERIALS AND METHODS: Radiographs of 67 deceased fetuses, neonates, and infants that were diagnosed with trisomy 21 were reviewed. Terminations of pregnancy were included. The control group was composed of 107 deceased fetuses, neonates, and infants without known chromosomal abnormalities, structural malformations, infections or placental pathology. Cases in which the number of thoracic ribs or presence of cervical ribs could not be reliably assessed were excluded. The literature concerning vertebral patterning in trisomy 21 cases and healthy subjects was reviewed. RESULTS: Absent or rudimentary 12th thoracic ribs were found in 26/54 (48.1%) cases with trisomy 21 and cervical ribs were present in 27/47 (57.4%) cases. This prevalence was significantly higher compared to controls (28/100, 28.0%, Χ2(1) = 6.252, p = .012 and 28/97, 28.9%, Χ2(1) = 10.955, p < .001, respectively). CONCLUSIONS: Rudimentary or absent 12th thoracic ribs and cervical ribs are significantly more prevalent in deceased fetuses and infants with trisomy 21.

Conservation of the segmented germband stage: robustness or pleiotropy?

Gene expression patterns of the segment polarity genes in the extended and segmented germband stage are remarkably conserved among insects. To explain the conservation of these stages, two hypotheses have been proposed. One hypothesis states that the conservation reflects a high interactivity between modules, so that mutations would have several pleiotropic effects in other parts of the body, resulting in stabilizing selection against mutational variation. The other hypothesis states that the conservation is caused by robustness of the segment polarity network against mutational changes. When evaluating the empirical evidence for these hypotheses, we found strong support for pleiotropy and little evidence supporting robustness of the segment polarity network. This points to a key role for stabilizing selection in the conservation of these stages. Finally, we discuss the implications for robustness of organizers and long-term conservation in general.

High incidence of cervical ribs indicates vulnerable condition in Late Pleistocene woolly rhinoceroses.

Mammals as a rule have seven cervical vertebrae, a number that remains remarkably constant. Changes of this number are associated with major congenital abnormalities (pleiotropic effects) that are, at least in humans, strongly selected against. Recently, it was found that Late Pleistocene mammoths (Mammuthus primigenius) from the North Sea have an unusually high incidence of abnormal cervical vertebral numbers, approximately ten times higher than that of extant elephants. Abnormal numbers were due to the presence of large cervical ribs on the seventh vertebra, indicating a homeotic change from a cervical rib-less vertebra into a thoracic rib-bearing vertebra. The high incidence of cervical ribs indicates a vulnerable condition and is thought to be due to inbreeding and adverse conditions that may have impacted early pregnancies in declining populations. In this study we investigated the incidence of cervical ribs in another extinct Late Pleistocene megaherbivore from the North Sea and the Netherlands, the woolly rhinoceros (Coelodonta antiquitatis). We show that the incidence of abnormal cervical vertebral numbers in the woolly rhinoceros is unusually high for mammals (15,6%, n = 32) and much higher than in extant Rhinoceratidae (0%, n = 56). This indicates that woolly rhinoceros lived under vulnerable conditions, just like woolly mammoths. The vulnerable condition may well have contributed to their eventual extinction.

No relationship between vertebral column shifts and limb fluctuating asymmetry in human foetuses.

Disturbance from the normal developmental trajectory of a trait during growth-the so-called developmental instability-can be observed morphologically through phenodeviants and subtle deviations from perfect symmetry (fluctuating asymmetry). This study investigates the relationship between phenodeviance in the human vertebral column (as a result of axial patterning defects) and limb fluctuating asymmetry. Since both types of markers of developmental instability have been found associated with congenital abnormalities in humans, we anticipate a relationship between them if the concept of developmental instability, measured through either phenodeviants or asymmetry, would reflect an organism-wide process. Yet we did not find any support for this hypothesis. We argue that the vast differences in the developmental processes involved in both systems renders these two markers of developmental instability unrelated, in spite of their associations with other congenital abnormalities. Our results thus contribute to the growing awareness that developmental instability is not an organism-wide property.

Extreme selection in humans against homeotic transformations of cervical vertebrae.

Why do all mammals, except for sloths and manatees, have exactly seven cervical vertebrae? In other vertebrates and other regions, the vertebral number varies considerably. We investigated whether natural selection constrains the number of cervical vertebrae in humans. To this end, we determined the incidence of cervical ribs and other homeotic vertebral changes in radiographs of deceased human fetuses and infants, and analyzed several existing datasets on the incidence in infants and adults. Our data show that homeotic transformations that change the number of cervical vertebrae are extremely common in humans, but are strongly selected against: almost all individuals die before reproduction. Selection is most probably indirect, caused by a strong coupling of such changes with major congenital abnormalities. Changes in the number of thoracic vertebrae appear to be subject to weaker selection, in good correspondence with the weaker evolutionary constraint on these numbers. Our analysis highlights the role of prenatal selection in the conservation of our common body plan.

Adverse Fetal and Neonatal Outcome and an Abnormal Vertebral Pattern: A Systematic Review.

IMPORTANCE: The human vertebral column has a stable number of vertebrae and ribs, which is presumably the result of evolutionary selection. An association between an abnormal vertebral pattern, especially in the cervical region, and congenital anomalies or adverse fetal outcome has been reported. OBJECTIVE: The aim of this study was to review the current literature concerning an abnormal vertebral pattern and prevalence of cervical ribs in healthy subjects and in subjects with adverse outcome. EVIDENCE ACQUISITION: Scientific databases were searched systematically. Studies assessing the number of vertebrae and/or ribs were included, and data concerning anomalies and outcome were extracted. RESULTS: Thirty-nine studies including 75,018 healthy subjects and 6130 subjects with structural or chromosomal anomalies or adverse outcome were selected. The majority of these studies focused on the prevalence of cervical ribs. The prevalence of cervical ribs was considerably higher in fetuses with adverse outcome, including aneuploidies, compared with healthy individuals in the vast majority of studies. Studies suggest an association between cervical ribs and other structural anomalies. CONCLUSIONS: These results demonstrate that detailed assessment of the fetal vertebral column, especially of the cervicothoracic region, could provide valuable information regarding fetal and neonatal prognosis. Based on the available evidence, the application of 3-dimensional (3D) ultrasound to assess the vertebral column and ribs, in particular the cervical region, warrants further research. RELEVANCE: Prenatal assessment of the ribs and vertebral pattern by 3D ultrasound, which is currently not routinely performed, might be useful in the assessment of the fetus, because this can predict fetal and neonatal outcome in some cases.

Homeotic transformations and number changes in the vertebral column of Triturus newts.

We explored intraspecific variation in vertebral formulae, more specifically the variation in the number of thoracic vertebrae and frequencies of transitional sacral vertebrae in Triturus newts (Caudata: Salamandridae). Within salamandrid salamanders this monophyletic group shows the highest disparity in the number of thoracic vertebrae and considerable intraspecific variation in the number of thoracic vertebrae. Triturus species also differ in their ecological preferences, from predominantly terrestrial to largely aquatic. Following Geoffroy St. Hilaire's and Darwin's rule which states that structures with a large number of serially homologous repetitive elements are more variable than structures with smaller numbers, we hypothesized that the variation in vertebral formulae increases in more elongated species with a larger number of thoracic vertebrae. We furthermore hypothesized that the frequency of transitional vertebrae will be correlated with the variation in the number of thoracic vertebrae within the species. We also investigated potential effects of species hybridization on the vertebral formula. The proportion of individuals with a number of thoracic vertebrae different from the modal number and the range of variation in number of vertebrae significantly increased in species with a larger number of thoracic vertebrae. Contrary to our expectation, the frequencies of transitional vertebrae were not correlated with frequencies of change in the complete vertebrae number. The frequency of transitional sacral vertebra in hybrids did not significantly differ from that of the parental species. Such a pattern could be a result of selection pressure against transitional vertebrae and/or a bias towards the development of full vertebrae numbers. Although our data indicate relaxed selection for vertebral count changes in more elongated, aquatic species, more data on different selective pressures in species with different numbers of vertebrae in the two contrasting, terrestrial and aquatic environments are needed to test for causality.

Determination of hip-joint loading patterns of living and extinct mammals using an inverse Wolff's law approach.

It is well known that bone adapts its microstructure in response to loading. Based on this form-follows-function relationship, we previously developed a reverse approach to derive joint loads from bone microstructure as acquired with micro-computed tomography. Here, we challenge this approach by calculating hip-joint loading patterns for human and dog, two species exhibiting different locomotion, and comparing them to in vivo measurements. As a proof of concept to use the approach also for extinct taxa, we applied it to a cave lion fossil bone. Calculations were in close agreement with in vivo measurements during walking for extant species, showing distinguished patterns for bipedalism and quadrupedalism. The cave lion calculations clearly revealed its quadrupedal locomotion and suggested a more diverse behaviour compared to the dog, which is in agreement with extant felids. This indicates that our novel approach is potentially useful for making inferences about locomotion in living as well as extinct mammals and to study evolutionary joint development.

Extraordinary incidence of cervical ribs indicates vulnerable condition in Late Pleistocene mammoths.

The number of cervical vertebrae in mammals is highly conserved at seven. We have shown that changes of this number are selected against due to a coupling with major congenital abnormalities (pleiotropic effects). Here we show that the incidence of abnormal cervical vertebral numbers in Late Pleistocene mammoths from the North Sea is high (33.3%) and approximately 10 times higher than that of extant elephants (3.6%). Abnormal numbers were due to the presence of large cervical ribs on the seventh vertebra, which we deduced from the presence of rib articulation facets on sixth (posterior side) and seventh (anterior side) cervical vertebrae. The incidence of abnormal cervical vertebral numbers in mammoths appears to be much higher than in other mammalian species, apart from exceptional sloths, manatees and dugongs and indicates a vulnerable condition. We argue that the increased incidence of cervical ribs in mammoths is probably caused by inbreeding and adverse conditions that impact early pregnancies in declining populations close to extinction in the Late Pleistocene.

Higher limb asymmetry in deceased human fetuses and infants with aneuploidy.

Aneuploidies cause gene-dosage imbalances that presumably result in a generalized decreased developmental homeostasis, which is expected to be detectable through an increase in fluctuating asymmetry (FA) of bilateral symmetric traits. However, support for the link between aneuploidy and FA is currently limited and no comparisons among different aneuploidies have been made. Here, we study FA in deceased human fetuses and infants from a 20-year hospital collection. Mean FA of limb bones was compared among groups of aneuploidies with different prenatal and postnatal survival chances and two reference groups (normal karyogram or no congenital anomalies). Limb asymmetry was 1.5 times higher for aneuploid cases with generally very short life expectancies (trisomy 13, trisomy 18, monosomy X, triploidy) than for trisomy 21 patients and both reference groups with higher life expectancies. Thus, FA levels are highest in groups for which developmental disturbances have been highest. Our results show a significant relationship between fluctuating asymmetry, human genetic disorders and severity of the associated abnormalities.

No sexual dimorphism in human prenatal metacarpal ratios.

BACKGROUND: Ratios of digit lengths are studied intensively as markers of prenatal sex hormone levels. AIM: Study sexual dimorphism in ratios of metacarpals, which received less attention. METHODS: We studied six metacarpal ratios in deceased human fetuses of ages 10 to 42weeks. RESULTS AND CONCLUSION: We found no indication of a sexual dimorphism at this early stage of development.