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Fossils reveal the complex evolutionary history of the mammalian regionalized spine

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A unique characteristic of mammals is a vertebral column with anatomically distinct regions, but when and how this trait evolved remains unknown. We reconstructed vertebral regions and their morphological disparity in the extinct forerunners of mammals, the nonmammalian synapsids, to elucidate the evolution of mammalian axial differentiation. Mapping patterns of regionalization and disparity (heterogeneity) across amniotes reveals that both traits increased during synapsid evolution. However, the onset of regionalization predates increased heterogeneity. On the basis of inferred homology patterns, we propose a “pectoral-first” hypothesis for region acquisition, whereby evolutionary shifts in forelimb function in nonmammalian therapsids drove increasing vertebral modularity prior to differentiation of the vertebral column for specialized functions in mammals.
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MAMMALIAN EVOLUTION
Fossils reveal the complex
evolutionary history of the
mammalian regionalized spine
K. E. Jones
1
*, K. D. Angielczyk
2
, P. D. Polly
3
, J. J. Head
4
, V. Fernandez
5
,
J. K. Lungmus
6
, S. Tulga
7
, S. E. Pierce
1
*
A unique characteristic of mammals is a vertebral column with anatomically distinct regions,
but when and how this trait evolved remains unknown. We reconstructed vertebral regions
and their morphological disparity in the extinct forerunners of mammals, the nonmammalian
synapsids, to elucidate the evolution of mammalian axial differentiation. Mapping patterns
of regionalization and disparity (heterogeneity) across amniotes reveals that both traits
increased during synapsid evolution. However, the onset of regionalization predates increased
heterogeneity. On the basis of inferred homology patterns, we propose a pectoral-first
hypothesis for region acquisition, whereby evolutionary shifts in forelimb function in
nonmammalian therapsids drove increasing vertebral modularity prior to differentiation of
the vertebral column for specialized functions in mammals.
The evolution of the mammalian body plan
from the ancestral amniote condition is
one of the most iconic macroevolutionary
transitions in the vertebrate fossil record
(1,2). A unique feature of mammals is their
specialized vertebral column, which displays con-
strained vertebral counts but highly disparate
morphologies (24). In therian mammals, the
presacral vertebral column is traditionally divided
into cervical, rib-bearing thoracic, and ribless
lumbar regions (Fig. 1A). In contrast, the pre-
sacral vertebrae of basal amniotes are compar-
atively uniform and show little differentiation
(Fig.1B).Thetransitionfromanunregionalized
to a regionalizedpresacral column is an impor-
tant step in mammalian evolution and has been
linked to the origin of specialized gaits and
respiratory function (1,2,5,6).
Recent quantitative work has detected subtle
presacral regionalization in extant snakes and
limbed lizards, superficially unregionalized taxa
(7). It was hypothesized that the ancestral am-
niote condition is cryptic regionalization,in
which regions are present but are only subtly
expressed. The global-patterning Homeobox (Hox)
genes were implicated as underlying these con-
served regionalization patterns. Under this model,
thedegreeofregionalizationthe number of re-
gions presenthas remained constant through
mammalian evolution, whereas the amount of
morphological disparity between regions (here
termed heterogeneity) has increased. But this
evolutionary scenario is based solely on data
from extant species.
The two amniote cladesSynapsida (mammals
and their relatives) and Sauropsida (reptiles,
birds, and their relatives)diverged more than
320 million years ago and have independently
undergone substantial morphological transforma-
tions. Therefore, to document the evolution of the
mammalian vertebral column, we must examine
mammalsextinct forerunners, the nonmamma-
lian synapsids. Here, we examined the presacral
vertebral columns of 16 exceptionally preserved
nonmammalian synapsids (including pelycosaurs,
basal therapsids, and cynodonts), one extinct
amniote outgroup, and a broad range of extant
salamanders, reptiles, and mammals. Using mor-
phometric data, we qua ntified patterns of region-
alization and heterogeneity and compared their
evolution to elucidate when and how synapsid
presacral differentiation occurred.
Using a likelihood-based segmented regression
approach, we calculated a regionalization score
for each taxon [an Akaike information criterion
(AIC)weighted average of the relative fit of one-
to six-region hypotheses], producing a continu-
ousvariablethatreflectstheestimatednumber
of vertebral regions (fig. S2). Similar to prior
work (7), most reptiles and some extant mam-
mals (e.g., monotremes) have regionalization
scores indicating the presence of four regions
(Fig. 2A), whereas therians (marsupials and pla-
centals) most frequently display five regions.
Therian regionalization scores are also more
variable, probably reflecting high ecomorpho-
logical diversification of their axial system (4).
Thus, data from extant amniotes alone support
the null hypothesis of conserved regionalization.
However, both salamanders and basal synapsids
have lower regionalization scores than extant
amniotes (Fig. 2A, cool colors), which demon-
strates that regionalization increased indepen-
dently in the sauropsid and synapsid lineages.
Accordingly, we reject the hypothesis of con-
served regionalization patterns in amniotes, and
instead propose the hypothesis of increasing re-
gionalization in synapsid evolution.
Heterogeneity, expressed as the logarithm of
themeanvarianceofthemorphologicalmeasures
for each column, also increased during synapsid
evolution (Fig. 2B). Lepidosaurs and salaman-
ders have low heterogeneity, denoting relative
uniformity of the axial column; therians have
much higher values, reflecting their extreme
disparity; and crocodilians have intermediate
levels. Most nonmammalian synapsids also have
intermediate levels of heterogeneity. The out-
group Diadectes and the ophiacodontids display
particularly low values, reinforcing previous as-
sertions of homoplastic increases in mammals
and archosaurs from a homogeneous ancestral
condition (7). The cynodont Kayentatherium
has more heterogeneous morphologies than the
RESEARCH
Jones et al., Science 361, 12491252 (2018) 21 September 2018 1of4
1
Museum of Comparative Zoology and Department of
Organismic and Evolutionary Biology, Harvard University,
Cambridge, MA 02138, USA.
2
Integrative Research Center,
Field Museum of Natural History, Chicago, IL 60605, USA.
3
Department of Earth and Atmospheric Sciences, Indiana
University, Bloomington, IN 47405, USA.
4
Department of
Zoology and University Museum of Zoology, University of
Cambridge, Cambridge CB2 3EJ, UK.
5
European Synchrotron
Radiation Facility, 38000 Grenoble, France.
6
Department of
Organismal Biology and Anatomy, University of Chicago,
Chicago, IL 60637, USA.
7
Department of Geophysical
Sciences, University of Chicago, Chicago, IL 60637, USA.
*Corresponding author. Email: katrinajones@fas.harvard.edu
(K.E.J.); spierce@oeb.harvard.edu (S.E.P.)
0
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4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
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3 4 5 6 7 8 910111213141516171819202122232425
Angle of Zygapophyses
Angle of Zygapophyses
Cervical Thoracic Lumbar Cervical Dorsal
AB
Heterogeneity
Regionalization
Fig. 1. Regionalization and heterogeneity. (A) The therian presacral column is highly regionalized
and morphologically differentiated (Mus musculus). (B) Basal synapsids display a homogeneous
dorsal region with little differentiation (Ophiacodon).
on September 20, 2018 http://science.sciencemag.org/Downloaded from
other fossil taxa, reflecting its position close to
the mammal radiation. Given the association
between heterogeneity and functional special-
ization of the axial skeleton in therians, the
more homogeneous morphologies of most non-
mammalian synapsids point toward functional
conservatism.
Although regionalization and heterogeneity
increased during synapsid evolution, they are
not significantly related (fig. S7 and table S6,
P= 0. 73) , meaning that simple linear change is
insufficient to explain these patterns. Instead,
quantitative trait modeling supports evolution
toward shifting adaptive optima (multiple opti-
mum Ornstein-Uhlenbeck models) for these data
(table S7). On the basis of AIC fitting, we recon-
structed two major adaptive shifts in each trait
during synapsid evolution (Fig. 3 and fig. S8). The
adaptive optimum for regionalization increases
from around three regions in pelycosaursto
around four regions at the base of Therapsida, with
a later shift to five regions occurring in Theria. The
adaptive optimum for heterogeneity increases first
at Cynodontia and subsequently within therians.
Taken together, our data reveal that vertebral
regionalization increased before heterogeneity
increased, hence these two measures of axial dif-
ferentiation evolved independently.
To understand how vertebral regionalization
increased in synapsids, we reconstructed region
boundaries recovered in the best-fit segmented
regression models (Fig. 4A). Region boundaries
were then cross-referenced with developmental
data, anatomical landmarks, and variation in
extant species to identify homologies (Fig. 4B).
In extant tetrapods, the cervicothoracic transi-
tion is correlated with Hox6 expression, rib mor-
phology, and the position of the forelimb and
brachial plexus (8). Therefore, the cervicothoracic
boundary was identified by (i) the position of the
posterior branch of the brachial plexus, and
(ii) the location of the anterior sternal articulation
or first long rib. Functional studies in Mus also
show that Hox9 patterns the transition from
sternal to nonarticulating ribs and that Hox10
controls the suppression of ribs altogether in
the lumbar region (Fig. 4B) (9,10). In keeping
with this association, dorsal regions were defined
relative to their proximity to long ribs (anterior
dorsal), short ribs (posterior dorsal), or absent
ribs (lumbar).
Using these criteria, region homology hypothe-
ses were constructed in key taxa for which rib
or neural anatomy are known (Fig. 4B). In sal-
amanders (and the stem amniote Diadectes;see
supplementary text), three regions are recovered.
The anterior break correlates with the posterior
branch of the brachial plexus in Ambystoma,im-
plying homology with the cervical region despite
the lack of a true neck(Fig. 4B, red region). Al-
though salamanders have poorly developed ribs,
the position of the posterior break in the mid-
trunk is consistent with the anterior-posterior
dorsal transition in other taxa (Fig. 4, yellow and
pale blue regions). This ancestral three-region
pattern is retained in the most basal synapsids.
In pelycosaurs,the first break corresponds
to the inferred cervicothoracic transition based
on rib length and forelimb position (e.g., v5 in
Edaphosaurus,v7inDimetrodon), whereas the
second break corresponds to the gradual tran-
sition from longer to shorter dorsal ribs, signifying
cervical, anterior dorsal, and posterior dorsal
homologies (Edaphosaurus;Fig.4B).
Our data point to the convergent addition of a
fourth region in distinct locations in sauropsids
and synapsids. In sauropsids, a fourth region is
detected anterior to the brachial plexus, suggest-
ing a novel cranial region within the neck (Iguana;
Fig. 4B, purple region). Sauropsids exhibit more
variationincervicalcountthandosynapsids(11),
providing a potential connection between neck
plasticity and cervical modularity in this lineage.
Conversely, in basal therapsids and cynodonts,
afourth region is detected posterior to the cer-
vicothoracic transition (Thrinaxodon; Fig. 4B,
orange region). In Thrinaxodon, the first break
corresponds to the cervicothoracic transition
and first full-length rib (v78), the second break
liesinthemiddleofthelongribseries(v1213),
and the anterior-posterior dorsal boundary falls
at the transition from long to short ribs (v1920).
These regions conform to the ancestral cervi-
cal region (red), a novel pectoral region (orange),
and the ancestral anterior dorsal (yellow) and
posterior dorsal (pale blue) regions. Therian
mammals display an additional break within the
posterior dorsal region that differentiates the
ribless lumbar region (Mus;Fig.4B,blueregion).
Considering the pattern ofregionacquisition,
we propose a pectoral-firsthypothesis for the
evolution of mammalian presacral regionaliza-
tion (Fig. 4). Under this hypothesis, pelycosaurs
retained the three-region ancestral amniote con-
dition. In basal therapsids, addition of a fourth
pectoral moduleaccompanied the reorganiza-
tion of the pectoral girdle and forelimb. Unlike
Jones et al., Science 361, 12491252 (2018) 21 September 2018 2of4
Time (Ma)
-5
Time (Ma)
Amniota
Mammalia
'Pelycosaurs'
Therapsida
Cynodontia
AB
-5 1
35
1
Heterogeneity
Regionalization
35
Fig. 2. Evolution of presacral differentiation in amniotes. (A) Regionalization score. (B) Heteroge-
neity [log(mean variance)]. Warmer colors reflect more regions and greater morphological heterogeneity,
respectively. Black circles, mammals; gray circles, fossil taxa; triangles, reptiles; stars, amphibians;
grayed tips in (A), fossil taxa excluded because of <0.75 r
2
of regionalization model. See table S5 for
full taxonomic names. Ma, millions of years.
RESEARCH |REPORT
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pelycosaurs,therapsids are characterized by
reduction of the pectoral girdle dermal bones
and increased shoulder mobility (1,12). Medial
extrinsic shoulder muscles (e.g., levator scapulae,
serratus ventralis) originating on the scapula
are thought to have expanded their axial inser-
tions during synapsid evolution (12). As these
vital body-support muscles attach directly onto
the underlying vertebrae and ribs, shifts in pec-
toral morphology and function likely drove di-
vergent neck- and shoulder-selective regimes
in the axial skeleton, providing impetus for in-
creased regionalization (1,12,13). Further, the
vertebrae, medial extrinsic shoulder muscles,
and dorsal border of the scapula all develop
directly from somitic mesoderm (primaxial),
signifying strong developmental ties between
these structures (14).
It has been proposed that the muscular dia-
phragm evolved from an unmuscularized septum
or proto-diaphragmvia co-option of shoulder
muscle precursor cells that were later canalized
into a distinct cell population by repatterning of
the posterior neck (15). Reorganization of the
anterior column and pectoral girdle in therapsids
may have facilitated this transition by increas-
ing cervicothoracic modularity and remodeling
shoulder musculature. Subsequent fixation of
the cervical count at seven in nonmammalian
cynodontsishypothesizedtorepresenttheap-
pearance of the mammalian-style muscular dia-
phragm (6). Thus, anterior regionalization that
had initially been associated with shoulder evo-
lution in early therapsids was likely later exapted
in cynodonts in response to selection for in-
creased ventilatory efficiency (5,15).
Alumbar moduleevolved later in therian
mammals. Evolution of the lumbar region in
mammals is associated with Hox10,whichfunc-
tions to repress rib formation and patterns lum-
bar identity in Mus (10) (Fig. 4B). Convergent
loss or gain of lumbar ribs in multiple fossil
theriiform clades suggests high plasticity of this
character early in therian evolution (16). Within
therians, lumbar count and morphology vary,
and this is reflected by translocation of the (mor-
phometrically defined) region boundary in our
sample. Because the lumbar region plays a crit-
ical role in mammalian locomotion, it is predicted
that region variability is related to ecological
specialization caused by clade-specific function-
al overprinting.
Regional differentiation is the major structural
difference between reptilian and mammalian
vertebral columns(13), yet its evolution has never
been quantitatively examined. Our findings show
that regionalization and heterogeneitythe two
aspects of vertebral differentiationevolved
independently. Forelimb reorganization in the-
rapsids drove initial increases in regionalizatio n
as a result of developmental and functional
connections between the pectoral girdle and
underlying vertebrae. High heterogeneity and
presumed functional diversity did not appear
until crown mammals. The combination of a
regionalized axial skeleton with heterogeneous
vertebral morphologies ultimately enabled mam-
mals to become specialized for a remarkable di-
versity of ecologies and behaviors.
Jones et al., Science 361, 12491252 (2018) 21 September 2018 3of4
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Alligator
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Gekko
Varanosaurus
Ophiacodon
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Edaphosaurus
Dimetrodon
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Eosimops
Lystrosaurus
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0100
Fig. 4. Best-fit region models, region homologies, and evolutionary hypothesis. (A) Best-fit
region models for select taxa. Colors represent inferred region homologies; St. Dev., standard
deviation of break locations; PS count, presacral count; R-sq, adjusted r
2
; % complete, total
completeness; shaded region models reflect taxa with <0.75 r
2
fit (excluded from evolutionary
reconstructions). (B)Pectoral-firsthypothesis for the evolution of synapsid presacral region-
alization. Taxa (left to right): Ambystoma,Iguana,Edaphosaurus [redrawn from (17)], Thrinaxodon
[redrawn from (2)], Mus. Width of gray bars reflects relative rib lengths and/or connection to
sternum; vertical dashed lines denote cervicothoracic transition. For Mus, Hox bands correspond to
vertebrae affected by functional gene manipulation (18).
Region score
Log Variance
Time (Ma)
θ: -3.94
θ: -1.57
θ: -0.48
Therapsida
Theria
Cynodontia
Boreoeutheria
θ: 3.01
θ: 3.89
θ: 4.80
Pz T JKCz
Regionalization
Heterogeneity
A
B
Fig. 3. Adaptive regime shifts in vertebral
evolution. (A) Regionalization. (B) Heterogene-
ity. Theta denotes adaptive optima of each
regime. Pz, Paleozoic; T, Triassic; J, Jurassic;
K, Cretaceous; Cz, Cenozoic.
RESEARCH |REPORT
on September 20, 2018 http://science.sciencemag.org/Downloaded from
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ACKNOW LEDGMEN TS
We thank R. Asher, B. Brainerd, D. Brinkman, T. Capellini,
C. Capobianco, J. Chupasko, J. Cundiff, K. Jakata, T. Kemp,
C. Mehling, A. Millhouse, M. Omura, A. Resetar, J. Rosado,
B. Rubidge, R. Smith, K. Smithson, C. Tabin, R. Tykoski, I. Werneburg,
and B. Zipfel.Funding: Supported by NSF grants EAR-1524523
(S.E.P.) and EAR-1524938 (K.D.A.) and by an AAA Postdoctoral
Fellowship (K.E.J.). Author contributions: Study design, K.E.J.,
K.D.A., and S.E.P.; methods, K.E.J., P.D.P., and S.E.P.; data
collection, K.E.J., K.D.A., S.E.P., J.J.H., V.F., J.K.L., and S.T.; data
analysis, K.E.J.; manuscript preparation, K.E.J., K.D.A., and S.E.P.
Competing interests: The authors have no competing interests. Data
and materials availability: Data are available in table S5 and Dryad
(doi:10.5061/dryad.jm820mg). Code is available via github (https://
github.com/katrinajones/regions).
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/361/6408/1249/suppl/DC1
Materials and Methods
Supplementary Text
Figs. S1 to S8
Tables S1 to S7
References (1949)
30 October 2017; resubmitted 27 April 2018
Accepted 25 July 2018
10.1126/science.aar3126
Jones et al., Science 361, 12491252 (2018) 21 September 2018 4of4
RESEARCH |REPORT
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... Although their cranial osteology has been relatively well documented (e.g., Bardua et al., 2019;Lowie et al., 2021;Sherratt et al., 2014;Wake, 1993;Wilkinson & Nussbaum, 1997), few studies have focused on the vertebral morphology of adult extant caecilians (e.g., Peter, 1894;Renous et al., 1993;Renous & Gasc, 1989;Taylor, 1977;Wake, 1980;Wiedersheim, 1879). In general, vertebrate axial skeletons are not a homogenous series of vertebrae and two types of morphological variation have been documented in previous studies: regionalization and heterogeneity (Jones et al., 2018). Whereas regionalization defines the number of regions along the vertebral column and relies on the expression of Homeobox genes (Head & Polly, 2015), heterogeneity defines the degree of morphological disparity observed among regions (Jones et al., 2018). ...
... In general, vertebrate axial skeletons are not a homogenous series of vertebrae and two types of morphological variation have been documented in previous studies: regionalization and heterogeneity (Jones et al., 2018). Whereas regionalization defines the number of regions along the vertebral column and relies on the expression of Homeobox genes (Head & Polly, 2015), heterogeneity defines the degree of morphological disparity observed among regions (Jones et al., 2018). Although these two types of axial differentiation evolve independently, both increased during synapsid evolution and led to well-defined vertebral regions in mammals (Jones et al., 2018). ...
... Whereas regionalization defines the number of regions along the vertebral column and relies on the expression of Homeobox genes (Head & Polly, 2015), heterogeneity defines the degree of morphological disparity observed among regions (Jones et al., 2018). Although these two types of axial differentiation evolve independently, both increased during synapsid evolution and led to well-defined vertebral regions in mammals (Jones et al., 2018). This combination of morphologically specialized vertebrae and regionalization allowed mammals to become functionally specialized for locomotion (Jones et al., 2020). ...
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The origin of amniotes 320 million years ago signalled independence from water in vertebrates and was closely followed by divergences within the mammal and reptile stem lineages (Synapsida and Reptilia). Early members of both groups had highly similar morphologies, being superficially 'lizard-like' forms with many plesiomorphies. However, the extent to which they might have exhibited divergent patterns of evolutionary change, with potential to explain the large biological differences between their living members, is unresolved. We use a new, comprehensive phylogenetic dataset to quantify variation in rates and constraints of morphological evolution among Carboniferous-early Permian amniotes. We find evidence for an early burst of evolutionary rates, resulting in the early origins of morphologically distinctive subgroups that mostly persisted through the Cisuralian. Rates declined substantially through time, especially in reptiles. Early reptile evolution was also more constrained compared to early synapsids, exploring a more limited character state space. Postcranial innovation in particular was important in early synapsids, potentially related to their early origins of large body size. In contrast, early reptiles predominantly varied the temporal region, suggesting disparity in skull and jaw kinematics, and foreshadowing the variability of cranial biomechanics seen in reptiles today. Our results demonstrate that synapsids and reptiles underwent an early divergence of macroevolutionary patterns. This laid the foundation for subsequent evolutionary events and may be critical in understanding the substantial differences between mammals and reptiles today. Potential explanations include an early divergence of developmental processes or of ecological factors, warranting cross-disciplinary investigation.
... hox genes j regionalization j chondrichthyan j vertebral column T he axial skeleton (vertebrae and ribs) is a defining feature of the vertebrate body form, and the existence of distinct axial skeletal regions along the anteroposterior (AP) axis of the body is thought to have facilitated evolutionary radiations and ecological specializations across vertebrate phylogeny (1)(2)(3). Tetrapod vertebral columns include at least four axial regions: cervical, trunk (further divided into thoracic and lumbar in most mammals and some reptiles), sacral, and caudal, the lengths of which have been modified in different tetrapod groups to suit various ecologies and lifestyles. In mammals and birds, vertebral regions are patterned by Hox gene expression within the paraxial mesoderm, and experimental manipulations to anterior Hox expression boundaries result in corresponding shifts in vertebral regional boundaries (4)(5)(6)(7)(8). ...
... We quantified vertebral morphology as homologous landmarks represented by three-dimensional (3D) Cartesian grid coordinates ( Fig. 1B and SI Appendix, Table S1) and used principal-component (PC) scores of realigned landmark coordinates as shape variables. To test for region numbers and boundaries, we performed segmented linear regression on PC scores and used maximum-likelihood model selection to determine the best fit number of regions within the quantified axial skeleton, where individual regression slopes indicate regions and slope breaks represent region boundaries (SI Appendix, Fig. S1 and Table S2) (3,38). PC analysis revealed a shape gradient along the AP axis of the vertebral column in the skate ( Fig. 1C and SI Appendix, Fig. S1 A-C), with a four-region model having the best fit to the gradient, and an overall best model of five regions with boundaries at approximately vertebrae 26, 31, 46, and 51 when we included the synarcual as the first region ( Fig. 1 D and E and SI Appendix, Fig. S1 A 0 -C 0 and D and Table S2). ...
... While the tail region of the skate axial column is easily recognizable by the change in morphology from laterally projecting transverse processes to ventrally directed hemal arches, the more rostral vertebral regions vary more subtly in morphology. The morphometric approaches used here have recently resolved cryptic anatomical boundaries in the vertebral skeletons of tetrapod taxa previously thought to have lost axial regionalization (38), and have allowed for finer-scale resolution of regional boundaries in fossil lineages (3). If applied more widely, comparative morphometric approaches can help to detect cryptic regionalization in a much broader array of taxa, providing a more comprehensive picture of the evolutionary history of axial regionalization. ...
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... To test whether there were any differences in wholehindlimb muscle architecture among species, we first performed a principal coordinate analysis (PCO) on the entire dataset using the 'regions' package (Jones et al. 2018) in R (R Core Team 2021); PCO is a distance-based ordination that allows for missing data in the distance matrix. We then performed a non-parametric multivariate analysis of variance (MANOVA) on the PCO axes that individually explained at least 3% of the variance (5 axes) in the dataset with species as the grouping factor. ...
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Thesis
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Mammals are the dominant large animals of today, occurring in virtually every environment. This book is an account of the remarkable fossil records that document their origin since the extinction of the dinosaurs. Tracing their evolution over the last 35 million years. For the first time presented in one single volume Kemp unveils the exciting DNA sequence evidence which coupled with fossil evidence challenges current thinking on the relationships amongst mammal and their inferred history.
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