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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Amniotic fluid deficiency and congenital abnormalities both influence fluctuating asymmetry in developing limbs of human deceased fetuses.

Fluctuating asymmetry (FA), as an indirect measure of developmental instability (DI), has been intensively studied for associations with stress and fitness. Patterns, however, appear heterogeneous and the underlying causes remain largely unknown. One aspect that has received relatively little attention in the literature is the consequence of direct mechanical effects on asymmetries. The crucial prerequisite for FA to reflect DI is that environmental conditions on both sides should be identical. This condition may be violated during early human development if amniotic fluid volume is deficient, as the resulting mechanical pressures may increase asymmetries. Indeed, we showed that limb bones of deceased human fetuses exhibited increased asymmetry, when there was not sufficient amniotic fluid (and, thus, space) in the uterine cavity. As amniotic fluid deficiency is known to cause substantial asymmetries and abnormal limb development, these subtle asymmetries are probably at least in part caused by the mechanical pressures. On the other hand, deficiencies in amniotic fluid volume are known to be associated with other congenital abnormalities that may disturb DI. More specifically, urogenital abnormalities can directly affect/reduce amniotic fluid volume. We disentangled the direct mechanical effects on FA from the indirect effects of urogenital abnormalities, the latter presumably representing DI. We discovered that both factors contributed significantly to the increase in FA. However, the direct mechanical effect of uterine pressure, albeit statistically significant, appeared less important than the effects of urogenital abnormalities, with an effect size only two-third as large. We, thus, conclude that correcting for the relevant direct factors allowed for a representative test of the association between DI and stress, and confirmed that fetuses form a suitable model system to increase our understanding in patterns of FA and symmetry development.

Evo-Devo of the Human Vertebral Column: On Homeotic Transformations, Pathologies and Prenatal Selection.

Homeotic transformations of vertebrae are particularly common in humans and tend to come associated with malformations in a wide variety of organ systems. In a dataset of 1,389 deceased human foetuses and infants a majority had cervical ribs and approximately half of these individuals also had missing twelfth ribs or lumbar ribs. In ~10 % of all cases there was an additional shift of the lumbo-sacral boundary and, hence, homeotic transformations resulted in shifts of at least three vertebral boundaries. We found a strong coupling between the abnormality of the vertebral patterns and the amount and strength of associated malformations, i.e., the longer the disturbance of the vertebral patterning has lasted, the more associated malformations have developed and the more organ systems are affected. The germ layer of origin of the malformations was not significantly associated with the frequency of vertebral patterns. In contrast, we find significant associations with the different developmental mechanisms that are involved in the causation of the malformations, that is, segmentation, neural crest development, left-right patterning, etc. Our results, thus, suggest that locally perceived developmental signals are more important for the developmental outcome than the origin of the cells. The low robustness of vertebral A-P patterning apparent from the large number of homeotic transformations is probably caused by the strong interactivity of developmental processes and the low redundancy of involved morphogens during early organogenesis. Additionally, the early irreversibility of the specification of the A-P identity of vertebrae probably adds to the vulnerability of the process by limiting the possibility for recovery from developmental disturbances. The low developmental robustness of vertebral A-P patterning contrasts with a high robustness of the A-P patterning of the vertebral regions. Not only the order is invariable, also the variation in the number of vertebrae per region is small. This robustness is in agreement with the evolutionary stability of vertebral regions in tetrapods. Finally, we propose a new hypothesis regarding the constancy of the presacral number of vertebrae in mammals.

Quantitative three-dimensional microtextural analyses of tooth wear as a tool for dietary discrimination in fishes.

Resource polymorphisms and competition for resources are significant factors in speciation. Many examples come from fishes, and cichlids are of particular importance because of their role as model organisms at the interface of ecology, development, genetics and evolution. However, analysis of trophic resource use in fishes can be difficult and time-consuming, and for fossil fish species it is particularly problematic. Here, we present evidence from cichlids that analysis of tooth microwear based on high-resolution (sub-micrometre scale) three-dimensional data and new ISO standards for quantification of surface textures provides a powerful tool for dietary discrimination and investigation of trophic resource exploitation. Our results suggest that three-dimensional approaches to analysis offer significant advantages over two-dimensional operator-scored methods of microwear analysis, including applicability to rough tooth surfaces that lack distinct scratches and pits. Tooth microwear textures develop over a longer period of time than is represented by stomach contents, and analyses based on textures are less prone to biases introduced by opportunistic feeding. They are more sensitive to subtle dietary differences than isotopic analysis. Quantitative textural analysis of tooth microwear has a useful role to play, complementing existing approaches, in trophic analysis of fishes-both extant and extinct.

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.

Breaking evolutionary and pleiotropic constraints in mammals: On sloths, manatees and homeotic mutations.

BACKGROUND: Mammals as a rule have seven cervical vertebrae, except for sloths and manatees. Bateson proposed that the change in the number of cervical vertebrae in sloths is due to homeotic transformations. A recent hypothesis proposes that the number of cervical vertebrae in sloths is unchanged and that instead the derived pattern is due to abnormal primaxial/abaxial patterning. RESULTS: We test the detailed predictions derived from both hypotheses for the skeletal patterns in sloths and manatees for both hypotheses. We find strong support for Bateson's homeosis hypothesis. The observed vertebral and rib patterns cannot be explained by changes in primaxial/abaxial patterning. Vertebral patterns in sloths and manatees are similar to those in mice and humans with abnormal numbers of cervical vertebrae: incomplete and asymmetric homeotic transformations are common and associated with skeletal abnormalities. In sloths the homeotic vertebral shift involves a large part of the vertebral column. As such, similarity is greatest with mice mutant for genes upstream of Hox. CONCLUSIONS: We found no skeletal abnormalities in specimens of sister taxa with a normal number of cervical vertebrae. However, we always found such abnormalities in conspecifics with an abnormal number, as in many of the investigated dugongs. These findings strongly support the hypothesis that the evolutionary constraints on changes of the number of cervical vertebrae in mammals is due to deleterious pleitropic effects. We hypothesize that in sloths and manatees low metabolic and activity rates severely reduce the usual stabilizing selection, allowing the breaking of the pleiotropic constraints. This probably also applies to dugongs, although to a lesser extent.