RESEARCH ARTICLE
Ecophenotypic Variation and Developmental
Instability in the Late Cretaceous Echinoid
Micraster brevis (Irregularia; Spatangoida)
Nils Schlüter*
Georg-August University of Göttingen, Geoscience Centre, Department of Geobiology, Goldschmidtstr. 3,
37077, Göttingen, Germany
* nils.schluter@gmail.com
Abstract
The Late Cretaceous echinoid genusMicraster (irregular echinoids, Spatangoida) is one of
the most famous examples of a continuous evolutionary lineage in invertebrate palaeontol-
ogy. The influence of the environment on the phenotype, however, was not tested so far.
This study analyses differences in phenotypical variations within three populations ofMicra-
ster (Gibbaster) brevis from the early Coniacian, two from the Münsterland Cretaceous
Basin (Germany) and one from the North Cantabrian Basin (Spain). The environments of
the Spanish and the German sites differed by their sedimentary characteristics, which are
generally a crucial factor for morphological adaptations in echinoids. Most of the major phe-
notypical variations (position of the ambitus, periproct and development of the subanal fas-
ciole) among the populations can be linked to differences in their host sediments. These
phenotypic variations are presumed to be an expression of phenotpic plasticiy, which has
not been considered inMicraster in previous studies. Two populations (Erwitte area, Ger-
many; Liencres area, Spain) were tested for stochastic variation (fluctuating asymmetry)
due to developmental instability, which was present in all studied traits. However, differ-
ences in the amount of fluctuating asymmetry between both populations were recognised
only in one trait (amount of pore pairs in the anterior paired petals). The results strengthen
previous assumptions on ecophenotypic variations inMicraster.
Introduction
The environment plays a principal role in adaptive evolution by shaping through natural selec-
tion the means of population phenotypes and by evoking phenotypic variation without altering
the genetic background, e.g. through uncovering cryptic genetic variation (decanalization), or
phenotypic plasticity [1–4]. Phenotypic plasticity, however, is a property of a genotype and,
thus, its norm of reaction is influenced by genetic variation as well [5]. The developmental ori-
gins of observed phenotypic variation are often open to debate in palaeontological studies, rea-
soned in the complex interplay of genes and environment. Typically, the fossil record provides,
to some extent, only the factor environment in this equation. However, to decipher patterns in
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 1 / 26
OPEN ACCESS
Citation: Schlüter N (2016) Ecophenotypic Variation
and Developmental Instability in the Late Cretaceous
Echinoid Micraster brevis (Irregularia; Spatangoida).
PLoS ONE 11(2): e0148341. doi:10.1371/journal.
pone.0148341
Editor: Steffen Kiel, Naturhistoriska riksmuseet,
SWEDEN
Received: November 11, 2015
Accepted: January 15, 2016
Published: February 5, 2016
Copyright: © 2016 Nils Schlüter. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: The author acknowledges support by the
German Research Foundation and the Open Access
Publication Funds of the Göttingen University. This
research received in parts support from the
SYNTHESYS Project, http://www.synthesys.info/,
which is financed by European Community Research
Infrastructure Action under the FP7 „Capacities”
Program (FR-TAF-4917). The funders had no role in
study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
development, evolution and speciation in deep time, it is meaningful to address these mecha-
nisms in studies for fossil taxa as well [6]. However, another source of phenotypic variation—
developmental instability—is easy to explore but received limited attention by palaeontologists.
As a consequence of developmental instability, environmental or genetic stressors perturb
developmental pathways (within populations of similar genotypes and stable environments)
and lead to an increase in phenotypic variation [7, 8]. Developmental instability can be mea-
sured by fluctuating asymmetry, random (subtle) deviations from perfect symmetry, as the
genes on both sides of a symmetric organism are identical, barring somatic mutations [9].
Developmental stability, on the other hand, is the ability to buffer the development against
those perturbations. Developmental instability has ample origins and the processes leading to
it are still not well understood (reviewed in [10, 11]). Stochastic gene expression contributes to
developmental noise [12, 13], caused, for example, by environmental stressors, such as pollu-
tion, or mutations that reduce the activity of a gene.
Phenotypic variations also cause confusion, especially for the species concept in palaeontol-
ogy, exemplified by the Late Cretaceous fossil echinoidMicraster. According to their high vari-
ability, extensive taxonomic works resulted in vast names of species (compare [14]).Micraster
was geographically widespread (western Asia, Europe, northern Africa [15]) and inhabited a
wide spectrum of environments. Moreover,Micraster provides some of the most studied fossil
examples in speciation (e.g. [16–20]), due to their well-traceable shifts of phenotypic variations
in time. The influence of the environment on the phenotypic variation inMicraster, however,
was only vaguely assumed [17, 18, 21], and Ernst and Seibertz [22] suggested that species of
Micraster could have developed local ecophenotypism. However, it was assumed that the evo-
lution inMicraster would reflect a step-wise increase of adaptation towards a stable environ-
ment [23]. These ideas, however, have never been tested. To analyse and discuss the influence
of the environment on the phenotype ofMicraster, three populations ofMicraster (Gibbaster)
brevis (Fig 1, following:M. brevis) were compared, which was widespread during the early Con-
iacian in Europe (compare [24]). Villier and colleagues [25] demonstrated that well-elongated
pores in the frontal ambulacrum (associated to tube feet for gaseous exchange) in the Lower
Cretaceous spatangoid Heteraster indicate an adaptation to warm shallow waters, which was
also likely the case forM. brevis. Three populations were compared. Two samples come from
the Münsterland Cretaceous Basin (MCB: Grimberg (IV) mine shaft, Erwitte area, Germany),
and one comes from the North Cantabrian Basin (NCB: Liencres area, northern Spain) (Fig 2).
Palaeoenvironments can be deduced from the composition of the rocks. While the MCB is
characterised by wackestones with fine siliciclastic and calcareous ooze and a low content of
dispersed bioclasts (calcispheres and foraminifera) (S1A–S1C Fig), the facies of the NCB con-
tains silty packstones with abundant and coarser-grained, reworked silici- and bioclastics (e.g.
bivalves, foraminifera, hexactinellid sponges, echinoderms) (S1D and S1E Fig). More details on
the lithology can be found in [26] for the NCB and [27, 28] for the MCB. Additionally, stronger
sea currents and a shallower sea level can be inferred by the sediment nature (coarse grains,
angular silt) for Liencres [26] in comparison to the Grimberg (IV) mine shaft and the Erwitte
area (fine-grained matrix). Accordingly, a somewhat more proximal position is inferred for
Liencres than for the Erwitte area, which had a more basinal position in the inner shelf basin
(Münsterland Cretaceous Basin) [27, 28]. Furthermore, a somewhat higher palaeotemperature
for the Liencres area can be assumed, as a consequence of its lower palaeolatitude (compare
[29] for the Cenomanian age).
In order to assess the impact of the environment onM. brevis, shape variations between the
populations were analysed by a geometric morphometric approach, and additional compara-
tive semi-quantitative analyses of four further traits were conducted (ornamentation of the
periplastronal ambulacrals and the interradial area of the paired petals, projection of the
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 2 / 26
Competing Interests: The author has declared that
no competing interests exist.
labrum, development of the subanal fasciole) (see Table 1). Characters which had been previ-
ously assumed to be influenced by the sedimentary environment in spatangoid taxa were
included in the analyses (e.g. position of the periproct, development of the subanal fasciole,
Fig 2. Palaeogeographic map. Simplified palaeogeographic map of western Europe during the Late
Cretaceous (modified after [30]).
doi:10.1371/journal.pone.0148341.g002
Fig 1. Micraster brevis. (A-D) Specimen from the Liencres area, Spain (MB.E.8251) in apical, oral, lateral and posterior views. (E-H) Specimen from the
Erwitte area, Germany (GSUB E3867) in apical, oral, lateral and posterior views. Scale bar equals 1 cm.
doi:10.1371/journal.pone.0148341.g001
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 3 / 26
shape of the plastron, inflation of the test) [31–35], as well as traits in which modifications are
traditionally regarded as being the result of a continuous process in the evolution ofMicraster
(e.g. ornamentation of the periplastronal ambulacrals and the interradial area of the paired pet-
als, projection of the labrum, shape of the test) [16–18, 36, 37] (Table 1). In a second part, the
populations were tested for the presence and differences in the level of fluctuating asymmetry
(FA) to assess if both populations deviated in their degree of developmental stability. For FA
analyses, morphometric (periplastronal ambulacrals, paired petals) and meristic traits (the
amount of pore pairs in the anterior and posterior paired petals) were considered.
Material and Methods
Material
In total, 126 specimens were studied (see S1 File for individual collection numbers), which
originate from the coastline of Liencres (northern Cantabria, Spain, 49 specimens), from the
abandoned Grimberg (IV) mine shaft (27 specimens) close to Bergkamen (Westfalia, Ger-
many), and from the vicinity of Erwitte (Westfalia, Germany, 50 specimens). The material
from the Erwitte area was collected by Ekbert Seibertz (Wolfsburg, Germany) during the con-
struction of the highway A44 in the 1970s. The specimens from the Liencres and the Erwitte
areas originate from a quasi-isochronous short-term interval of 2 m thickness (equivalent to
approximately 30,000 years) from the basal Cremnoceramus crassus crassus/Cremnoceramus
deformis deformis Zone, early Coniacian (compare [26, 39]). The Grimberg material is from
the lower Cr. crassus crassus/Cr. deformis deformis Zone, collected between sinking depths of
286 and 282 m [40], corresponding to an interval of approximately 60,000 years. AlthoughM.
brevis is very abundant in each of the studied localities, specimens are often distorted or incom-
plete, which limited the sample size in this study.
Table 1. Investigated traits, their function, and evolutionary significance.
morphometric traits landmarks morphological function evolutionary significance
position of the apical shield 1 growth dependent in several taxa, possible
related to burrowing strategy [23, 36]
shift in position from anterior to posterior [15–20, 37]
shape of the paired petals 2–9 associated pore pairs are related to
gaseous exchange [38]
increase of length [15, 16, 20]
shape of the plastron (sternal
plates)
10, 12, 14–19,
21–22
related to locomotion behaviour, nature of
the substrate respectively [31, 35]
not considered so far
position of the periplastronal
ambulcral plates
11–15, 19–23 not known possible shift into the anterior direction, and elongation
of the perilabral plate, compare [20]
depth of the anterior notch 24–26 related to feeding strategies [36] a trend towards a deepening of the notch [36]
position of the ambitus 24–28 related to burrowing depth [32] lowered in the Gibbaster branch [17, 18]
position of the widest point of
the test
27–28 not known, possible related to burrowing
strategy [23]
shift towards the posterior direction [15–20, 37]
position of the periproct 30 related to burrowing depth [33] lowered in the Gibbaster branch [17, 18]
non-morphometric traits morphological function evolutionary significance
pore numbers in the paired
petals
associated tube feet are related to gaseous
exchange [38]
increase in numbers [15, 20]
structure in the interradial area
in the paired petals
not known accentuation of the interradial structure and
ornamentation [15–20, 36]
structure in the periplastronal
ambulcral plates
not known accentuation of the granulation [15–20, 37]
projection of the labrum not known, assumed to be related to a
change in feeding strategy
increase of the projection of the labrum, accordingly the
peristomal opening is completely covered [15–20,
37]
doi:10.1371/journal.pone.0148341.t001
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 4 / 26
For the comparison of shape variations, 86 specimens in total were analysed: 14 from the
Grimberg VI mine shaft, 37 from the surroundings of Erwitte, and 35 from the Liencres area.
To explore the shape variability of all populations, a Principal Component Analysis (PCA) was
conducted. For FA analysis, specimens that were not adequately preserved (slight deforma-
tions) were excluded, resulting in 33 specimens from Erwitte and 35 from the Liencres area.
The material from the Grimberg mine shaft was insufficient in numbers to give reliable results,
and omitted for this study. Semiquantitative analyses were conducted by inclusion of 121 speci-
mens in total (46 Liencres area, 49 Erwitte area, 26 Grimberg area) to examine the development
of the subanal fasciole, the variation of the interradial structure of the paired petals, and the
granulation of the periplastronal area.
For analysis of the projection of the labrum, data from 96 specimens were obtained (33
Liencres area, 46 Erwitte area, 17 Grimberg area). Due to the fragile nature of the labrum tip,
the extension of the labrum was not considered in the morphometric analyses, as it would have
reduced the available material.
To investigate for differences in variation of the average count and variation in FA of pore
pair numbers, 35 specimens were included for the Erwitte population and 36 specimens for the
Liencres population. The same material was used to test for FA in pore pair numbers in the
anterior and the posterior paired petals. No permits were required for the described study,
which complied with all relevant regulations.
Institutional abbreviations. To denote the repositories of specimens illustrated and/or
referred to in the text, the following abbreviations are used: (MB.E.) Museum für Naturkunde,
Leibniz-Institut für Evolutions- und Biodiversitätsforschung an der Humboldt-Universität zu Ber-
lin, Berlin, Germany; (BGR) Bundesanstalt für Geowissenschaften und Rohstoffe Berlin, Berlin,
Germany; (GSUB) Geowissenschaftliche Sammlung der Universität Bremen, Bremen, Germany.
Methods–geometric morphometrically based analyses
In order to realise 3D models of the material, specimens were mounted on a stick and posi-
tioned on a turntable, and photos were obtained from all available perspectives of the echinoids
by rotating the turntable across small angles. Overview photographs were supplemented by
close-ups for capturing highly detailed 3D models. For photogrammetric reconstructions
resulting in digital three-dimensional models, the images were elaborated with Autodesk1
123D1 Catch. Each 3D model (see S1 Multimedia) was reconstructed by implementing and
aligning 70 2D images. This process results in a mesh, which can be exported as a textured
object file that features the photographic detailed surfaces of the specimen. These files were
applied for further data collection for geometric morphometric purposes. For a more detailed
and practical guide for photogrammetry, it is referred to the works of Falkingham [41] and
Malison andWings [42].
Landmarks were digitised in the freeware MeshLab (Visual Computing Lab—ISTI—CNR;
http://meshlab.sourceforge.net/) from the textured 3D models. The Cartesian coordinates were
analysed with the morphometric software package MorphoJ [43]. Prior to each analysis a Pro-
crustes fit was performed, in which the shape of each specimen was rescaled to unit centroid
size, which removes information on size. Centroid size is the square root of the sum of squared
distances from a configuration of landmark to the centre of the shape configuration (centroid)
[44]. The rescaled shapes are then translated to the same position, and rotated to the best fitting
orientation of the landmark configurations. Shape variations within the whole sample were
investigated through a PCA by assessing a set of 30 landmarks (Fig 3 and S1 Multimedia).
A Procrustes ANOVA was performed to assess for the possible presence of FA and to quan-
tify the relative amounts of shape variation in asymmetry and measurement error [45]. Because
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 5 / 26
measurement error can inflate FA, it is preferred to test for its significance. The Procrustes
ANOVA contains the factor sides (fixed) and individuals (random). Directional asymmetry
(DA) is tested by the side factor, while FA is estimated by the individual-by-side interaction
term. Occurrence of antisymmetry was estimated by examining the scatter plots of shape asym-
metry for bimodality. Other asymmetric variations, e.g. DA, and antisymmetry are not suitable
for measuring developmental instability as they have a genetic component and should be there-
fore avoided in such studies [7]. As the studied traits here have object symmetry (objects which
are symmetric by themselves), tests for size dependency of FA are precluded [46]. Accordingly,
only shape FA can be considered here.
A set of replicated models of each specimen was used to quantify the measurement error.
For the analysis of FA, 21 landmarks, 18 paired and 3 median landmarks (Fig 3), were digitised.
Only landmarks of type 1 [44] were chosen, which were precisely defined by intersections with
other plate sutures, or very well locatable. The landmark configuration included the shape of
the periplastronal ambulacrals and paired petals.
Fig 3. Landmark configurations. (A) Plating drawings (after specimen GSUB E3867) depicting the landmark configurations for the specific analyses. (B)
Global shape variation, shown as wireframe graph. (C) Fluctuating asymmetry analysis. For FA analysis: red circles represent paired landmarks, green
circles represent median landmarks.
doi:10.1371/journal.pone.0148341.g003
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 6 / 26
The Procrustes ANOVA was independently applied for the Erwitte and the Liencres popu-
lations. In both samples a Procrustes ANOVA was computed for the global shape and for each
trait separately (periplastronal ambulacral plates, paired petals). Levene's test was performed to
test for differences in FA among populations, which is more robust to departures from normal-
ity [7]. To visualise and investigate shape variations, a PCA was conducted for each population,
which took symmetric and asymmetric components into account.
Methods–variation in non-morphometric characters
Subanal fasciole, projection of the labrum, interradial structure of the paired petals, and
granulation of the periplastronal area. To define the degree of coverage by the labrum and
the projection of the labrum, three descriptive states were used: i) open peristome (Fig 4A), ii)
peristome is completely covered (labrum reachs the frontal margin of the peristome), and iii)
the labrum exceeds significantly the margin of the peristome (Fig 4B). In terminology for the
development of the subanal fasciole, Néraudeau and colleagues [47] was followed. According
to the terminology and classification of Rowe [16], Fouray [37] and Olszewska-Nejbert [24],
the populations were studied to compare the development of the interradial structure of the
paired petals and the granulation of the periplastronal area.
Pore pair numbers versus test length. The number of pore pairs from the Erwitte and the
Liencres populations were counted with the help of the image analysis software ImageJ [48].
The pore number averages of the anterior and the posterior paired petals of each specimen
[(R+L)/2] were plotted against the test length. An ANCOVA was computed to assess for signif-
icance of differences between slopes among the populations.
FA analysis of pore pair numbers in the anterior and the posterior paired petals. The
counts of the pore pairs were not repeated, as they can be confidently performed without any
measurement error. Grubb’s test was applied to check for outliers, as outliers otherwise could
artificially inflate the results of FA. Prior to FA analyses, a size dependency of FA was tested by
calculating a Spearman rank correlation of unsigned values and averaged trait size. FA, which
varies with trait size, could otherwise obscure the analysis [49]. Normal distribution was
assessed with a Shapiro–Wilk test, to test for ideal FA, since the property of FA is a normal dis-
tribution of right side–left side differences with a mean of zero, and hence to exclude the possi-
ble occurrence of antisymmetry [49]. A one-sample t-test was performed to test if the data sets
deviate from a mean of zero, in which case DA would be present.
Following Palmer and Strobeck [49, 50], two indices were computed to estimate develop-
mental instability. FA1 displays the mean of unsigned differences between the right and left
sides [mean |R-L|], and FA4a assesses the variance within a given trait [0.798
p
var (R–L)]. The
FA4a index is a modified version of the FA4 index, which has the advantage (compared to
FA1) of not being biased by the presence of DA [7]. If DA is present, the influence of DA on
Fig 4. Variation in the projection of the labral plate. (A) Weakly projecting, not covering the peristomal
opening (GSUB E3847, Erwitte area, Germany), (B) strongly projecting, covering completely the peristomal
opening (MB.E.8251, Liencres area, Spain). Scale bars equal 1 cm.
doi:10.1371/journal.pone.0148341.g004
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 7 / 26
the asymmetry values could be assessed by comparing DA, as the mean (R–L), with the value
of FA4a. In the case of DA not exceeding FA4a, the variation in the trait is mainly due to devel-
opmental instability [50]. Levene’s test was conducted by using the unsigned values to evaluate
differences of FA among populations.
Results
Morphometric variation
Shape analysis. The first four PCs account for 66.53% of the total shape variance (Figs 5
and 6). PC 1 reveals the best separation between the German populations and the Spanish pop-
ulation (Figs 7 and 8); they reveal, however, some overlap, but a larger dispersion of the Span-
ish population is found along the positive direction of PC 1—both German populations
disperse rather in a negative direction. The latter both have in any of the describing principal
components large overlaps.
PC 1 (33.92% of total variance) is linked in a positive direction with a higher positioned
periproct and ambitus, and the widest point of the test is set in the posterior position. The ster-
nal plates are generally slimmer and shorter, the periplastronal ambulacral plates are set more
in the anterior direction, and the plastronal area is more inflated. Furthermore, the shape of
the test is more tumid, less elongated, and taller with a strongly positive PC 1.
PC 2 (15.66% of total variance) is associated in a positive direction with a posterior shift of
the periplastronal ambulacral plates, combined with shorter sternal plates. The paired petals
are less extended.
PC 3 (10.27% of total variance) pertains to shape variations with a change in a positive
direction in a lower-levelled ambitus and periproct. Furthermore, the widest point is more
anteriorly situated, and the more asymmetric sternal plates are found in a rather anterior posi-
tion and have a broader contact to the labrum. The periplastronal ambulacral plates moved in
the anterior direction, and the paired petals are shorter by a concurrent shift of the apical shield
closer to the anterior margin than in relation to a negative direction.
In the positive direction of PC 4 (6.67% of total variance) a change in shape is illustrated
mainly by a lower test with a higher position of the deepest impression in the notch, and the
maximum width is found in the posterior direction. The apical shield is closer to the anterior
notch, combined with longer anterior paired petals. The sternal plates are more asymmetric
than in a change in the negative direction, and are in a broader contact to the labral plates; the
periplastronal ambulacral plates are positioned in the anterior direction.
Fluctuating asymmetry analysis. Directional asymmetry is statistically significant in the
Erwitte population for the global shape variation and for the separately analysed periplastronal
ambulacrals and paired petals; in the Liencres population, DA is significant in the paired petals,
in the global shape variations, and in the periplastronal ambulacrals it is significant only at a P-
value level of 0.05. FA is significant for all analysed shape variations in the Erwitte and in the
Liencres populations (Table 2). The measurement error is compared to the interaction term
(individual-by-side) is only minor throughout and is therefore negligible. In both populations,
FA is most conspicuous in the oral ambulacral plates and shape changes refer mainly to shifts
of the periplastronal ambulacral plates along the posterior–anterior axis (Fig 9, see S2–S4 Figs
for PC2, PC3, PC4 respectively), whereas variations in the symmetric component are largely
associated with the shape of the plastron. Differences in the amount of FA among both popula-
tions (see S1 Table for individual FA scores) were for all shape variations not significant, as
revealed by Levene’s test (global shape: P = 0.49; periplastronal ambulacrals: P = 0.87, paired
petals: P = 0.37).
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 8 / 26
Variation in non-morphometric characters
Subanal fasciole, projection of the labrum, interradial structure of the paired petals, and
granulation of the periplastronal area. An obvious trend is seen in the development of the
subanal fasciole, which is most pronounced in the Liencres population (Fig 10A, S2 Table),
where a trace of a subanal fasciole is always present. In a large proportion of the Grimberg and
Erwitte populations, on the other hand, no subanal fasciole could be detected (46% and 41%
respectively, Figs 10A and 11A). Likewise, more complete types of fasciole development are
found in specimens from the Liencres area (parafasciole: 41%, Fig 11C; orthofasciole: 7%,
Fig 5. Global shape variation.Wireframe graphs show the shape changes from the mean shape (red) to shape changes associated within PC1 and PC2
with a negative and positive direction.
doi:10.1371/journal.pone.0148341.g005
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 9 / 26
Fig 11D, S3 Table). In both populations, specimens having a parafasciole were only found in
the Grimberg material; orthofascioles are completely missing.
A similar tendency is found for the projection of the labrum. A minor part of the Liencres
material has an uncovered peristome. Predominantly, the labrum is covering and/or even
exceeding the margin of the peristome. This is in contradiction to the observations made in the
German populations, in which the majority of the specimens have only weakly projecting labral
plates; the peristomes are, to a large extent, uncovered. No significant differences between all
populations were observed in the development interradial structure of the paired petals (Fig
12) and the granulation of the periplastronal area (results are not shown).
Fig 6. Global shape variation.Wireframe graphs show the shape changes from the mean shape (red) to shape changes associated within PC3 and PC4
with a negative and positive direction.
doi:10.1371/journal.pone.0148341.g006
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 10 / 26
Pore numbers versus test length. The slope for the anterior paired petals of the Liencres
population differs somewhat from the Erwitte population (0.93 and 0.90 respectively, Fig 13).
The ANCOVA, however, revealed no significant differences among both populations
(P = 0.57). Similar results are found for the posterior paired petals, and the slope in the Liencres
population is larger (0.93) than in the Erwitte population (0.82), but insignificantly different
(P = 0.45).
Fluctuating asymmetry analysis for the pore numbers in the paired petals. The values
of each FA index and mean asymmetries are given in Table 3 (see S4 Table for individual FA
values). One outlier from the set of posterior paired petals was excluded from the following
Fig 7. Principal component analysis scatter plots. (A) PC1 versus PC2. (B) PC1 versus PC3.
doi:10.1371/journal.pone.0148341.g007
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 11 / 26
analyses according to Grubb's test. All studied traits reveal a normal distribution (P>0.05);
accordingly, antisymmetry is not present in the studied traits.
A correlation of FA with the trait size could neither be detected in the anterior nor in the
posterior paired petals. The differences of FA between the Erwitte and Liencres populations in
the posterior petals are not significant, as concluded by Levene’s test (P = 0.84); DA could not
be detected. The presence of DA in the anterior petals was confirmed by a t-test in both sam-
ples. Estimated by a comparison of the mean (R–L) and FA4a for each sample, the values of the
FA4a are larger than the mean (R–L) in both cases; accordingly, the asymmetry variation
accounts largely for FA. The FA scores (FA1 & FA4a) for the anterior petals in the Liencres
population are significantly higher (P<0.001) than in the Erwitte population (Table 3 and
Fig 8. Principal component analysis scatter plots. (A) PC1 versus PC3. (B) PC2 versus PC3.
doi:10.1371/journal.pone.0148341.g008
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 12 / 26
Fig 14). The range in between-sides differences in this trait is sufficient for avoiding biased esti-
mates of FA, as suggested by Swain [51] for the reliability of meristic traits to assess develop-
mental instability.
Discussion
Shape variation
The Spanish and the German populations ofM. brevis are distinguished by the displacement of
the periproct and the ambitus, which are generally in a higher position in the Spanish speci-
mens. Furthermore, the shape of the plastron is slimmer, shorter and more inflated in the
Liencres population than in the German populations. The latter populations show a large over-
lap in their morphospace and are virtually indistinguishable. The morphological differences
between the German and the Liencres populations argue for an adaption to the grain size of
their host sediments. With regard to the burrowing behaviour, the Spanish and the German
populations must have been different. An inflated plastron, like in the Liencres populations, is
found in deeper burrowing spatangoids [35]. The position of the periproct is related to the bur-
rowing depth of the animal, and deeper burrowing species have a higher positioned periproct
than shallow or epifaunal burrowers [24, 33]. The generally higher situated periproct in the
Liencres sample supports the interpretation of a deeper burrowing behaviour.
Table 2. Procrustes ANOVA results for the populations from Erwitte and Liencres area.
population trait Effect SS MS df F P (param.)
Erwitte area
general shape Individual 0.26086047 0.00054346 480 6.76 <0.0001
Side 0.0053173 0.00037981 14 4.72 <0.0001
Individual x Side 0.03602375 8.041E-05 448 38.12 <0.0001
Measurement error 0.00201864 2.1093E-06 957
periplastronal ambulacralia Individual 0.28937101 0.00100476 288 3.86 <0.0001
Side 0.01070259 0.00133782 8 5.14 <0.0001
Individual x Side 0.06665542 0.00026037 256 68.21 <0.0001
Measurement error 0.00214141 3.8171E-06 561
paired petals Individual 0.18856263 0.0009821 192 10.21 <0.0001
Side 0.0028151 0.00056302 5 5.85 <0.0001
Individual x Side 0.01538751 9.6172E-05 160 16.46 <0.0001
Measurement error 0.00212048 5.8416E-06 363
Liencres area
general shape Individual 0.21060018 0.00041294 510 5.68 <0.0001
Side 0.00283036 0.00020217 14 2.78 0.0005
Individual x Side 0.03462216 7.2736E-05 476 36.89 <0.0001
Measurement error 0.00200114 1.9716E-06 1015
periplastronal ambulacralia Individual 0.26783721 0.00087529 306 3.52 <0.0001
Side 0.00539528 0.00067441 8 2.71 0.0069
Individual x Side 0.06765032 0.00024871 272 60.98 <0.0001
Measurement error 0.00242686 4.0788E-06 595 -0.01
paired petals Individual 0.1466068 0.00071866 204 7.31 <0.0001
Side 0.00283686 0.00056737 5 5.77 <0.0001
Individual x Side 0.01671819 9.8342E-05 170 17.41 <0.0001
Measurement error 0.00217429 5.6475E-06 385
doi:10.1371/journal.pone.0148341.t002
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 13 / 26
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 14 / 26
A similar pattern for the variation in the plastron shape in relation to the sediment was
found in the extant spatangoid Echinocardium cordatum among populations from the waters
of Great Britain and New Zealand [31]. Curiously, however, the relation to the grain size is in
contradiction to the pattern which can be observed inM. brevis. In E. cordatum, however,
broader shapes are related to mud and clay, and narrow shapes to sand and gravel. Only the
degree in inflation of the plastronal area is in both species linked to coarser substrates. These
contradictory outcomes are probably related to differences in locomotion and burrowing
mechanisms among both species, which demonstrates interspecific variations [33, 52]. Echino-
cardium cordatum, for example, is a deeply burrowing species [33], which is in contrast with
the assumed shallow burrowing depth ofM. brevis. These different patterns in plastron shape
are, however, worth to be studied in more detail elsewhere. Regardless of these deviations, both
studies show a strong linkage between the substrate and plastron shape. Egea and colleagues
[53] mention that phenotypic plasticity has an influence on the morphology of E. cordatum,
unfortunately without giving specific examples. Another example of how the morphology in
spatangoid echinoids changed in relation to the sediment is given by the fossil Toxaster grano-
sus kiliani (Lower Cretaceous) [34]. It responded to an increase in the abundance of clasts in
the sediment by an inflation of the test and by a higher positioned periproct. This pattern was
also observed in E. cordatum from northern France [34]. These observations correspond to the
results found inM. brevis.
Fig 9. Shape variation of the asymmetric and symmetric component. Shape variation on PC1 for the
population from the Liencres and the Erwitte area.
doi:10.1371/journal.pone.0148341.g009
Fig 10. Variation in development of the subanal fasciole. Bar charts indicating the percentage for the
particular populations in (A) the development of the subanal fascioles, (B) the development of the projection
of the labrum (Li = Liencres area; Er = Erwitte area; Gr = Grimberg).
doi:10.1371/journal.pone.0148341.g010
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 15 / 26
The phenotypical differences withinM. brevis either relied on genetic differentiation or
were reasoned in phenotypic plasticity. Although phenotypic plasticity is ubiquitous in organ-
isms, it is challenging to draw conclusions about phenotypic plasticity in the fossil record (dis-
cussed for the case in ammonoids [54]). McNamara and McKinney [55], Chauffe and Nichols
[56], and West-Eberhard [3], however, proposed criteria to evaluate phenotypic plasticity in
the fossil record. It was argued that phenotypic plasticity can be assumed, for instance, if the
modified trait is not occurring uniquely; all forms of (isogenic) taxa would modify in the same
way, and other related taxa would modify in a similar way (if being exposed to the same sti-
muli). This evaluation can be hampered by the fact that phenotypic plasticity, however, is a
property of a genotype. Accordingly, the reaction norm (the range of phenotypic expression of
a genotype across an environmental variable) can vary within or between populations [57]. In
addition, initially, plastic traits can change by genetic accommodation, hence being fixed [3,
58, 59]. Nevertheless, the relationship between the position of the ambitus, the periproct
Fig 11. Photographs illustrating variation in development of the subanal fasciole. (A) Not present (GSUB E3847, Erwitte area, Germany). (B)
Incomplete (GSUB E3850, Erwitte area, Germany). (C) Protofasciole (BGR X 06195, Grimberg IV shaft, Germany). (D) Orthofasciole (MB.E.3873, Liencres
area, Spain). Scale bars equal 0.5 cm.
doi:10.1371/journal.pone.0148341.g011
Fig 12. Development in the structure of periplastronal area and the interporiferous area of the paired
petals. (A) Granular periplastronal area (MB.E.8196, Liencres, Spain). (B) Subdivided interporiferous area of
the paired petals (GSUB E3867, Erwitte area, Germany). Scale bars equal 0.5 cm.
doi:10.1371/journal.pone.0148341.g012
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 16 / 26
respectively, and the grain size in fossils T. granosus kiliani,M. brevis and the extant E. corda-
tum, as mentioned above, suggests an influence of phenotypic plasticity. This is possibly true
for the development of the plastron shape as well, but further confirmation is needed.
The populations of E. cordatum from New Zealand and Great Britain, analysed by Higgins
[31] with respect to the plastron shape, are yet genetically divergent [53], but whether these
genetic differentiations contributed to the shape variations is unclear. The high and rapid dis-
persal potential inMicraster, due to their planktotrophic larvae [60], would have enabled gene
flow between the Spanish and German populations. However, gene flow between both areas
may have been asymmetric; several examples suggest that a north to south directed migration
of taxa existed during the Late Cretaceous [61]. In the case of the cryptic species complex E.
cordatum [53, 62], planktotrophic larvae allowed for widespread settlement of genetically
homogeneous clades. However, the possibility of genetic variation inM. brevis cannot be totally
excluded. Other shape variations are shared by all three populations and cannot be referred to
distinct habitats. Thus, insofar as it is not possible at present to associate these variations with
Fig 13. Bivariate scatter plot of the numbers of pore pairs against test length.
doi:10.1371/journal.pone.0148341.g013
Table 3. Results of the FA analysis considering the pore pair numbers.
FA1 FA4a mean (R-L) t-test (P-values)
Liencres
anterior petals 2.78 2.26 1.89 0.00031
posterior petals 1.75 1.63 -0.26 0.46
Erwitte
anterior petals 1.2 1.16 0.57 0.026
posterior petals 1.57 1.6 -0.46 0.1863
doi:10.1371/journal.pone.0148341.t003
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 17 / 26
an environmental gradient, their development may be attributed to genetically influenced
variation.
Fluctuating asymmetry
Environmental stressors, like assumable temperature clines, potentially could have contributed
only to a negligible amount of FA. Moreover, shared inheritable factors (e.g. effects of non-
additive gene–gene interactions, see [63]) could have contributed to similar patterns and
degrees of FA in the periplastronal ambulacrals found in both populations. This is, of course,
very speculative and needs to be discussed further by inclusion of more populations of different
habitats. Additionally, assessments on phenotypic variance can be biased by time averaging,
which potentially inflates phenotypic variance within larger time intervals [64]. However, it
was tried to limit this bias by restricting the time interval under study as much as possible.
Remarkably, variation within individuals can exhibit conspicuous variability in the periplas-
tronal ambulacral plates, in which one plate can be very shortened, while the opposing plate
can show a very extended shape (Fig 15). These within-individual variations reflect intra-popu-
lational variation in the development of these plates, as described by the first four principal
component factors for global shape variations. The pattern of the symmetric shape and the
asymmetric shape variation, however, are not congruent. This disagreement is possibly
explained by the fact that a large amount of variation is attributed to the shape variation in the
plastron, which is associated with the position of the periplastronal ambulacral plates.
These stochastic variations might give some clues to the developmental process of this trait.
Inferred from the probability that these extreme asymmetries are a result of stochastic gene
expression, i.e. fluctuations in the amount of a gene product [65], it suggests that the (symmet-
ric) variation in these plates was a result of homometry (changes in the amount of gene prod-
ucts, see [66]). McNamara [67], however, suggested that displacement in the periplastronal
ambulacral plates of spatangoid echinoids is reasoned in allometry. Studies on allometric varia-
tion in this trait ofM. brevis are needed to give better ideas on their development.
A study on FA of two populations of Echinocardium flavescens [68] has shown that varia-
tions in the periplastronal ambulacrals are most intense along the main growth (longitudinal)
axis and are, to some extent, constrained to this direction. This suggests that shape variations
of FA in the periplastronal ambulacrals are, to some degree, under common developmental
constraints. The amount of FA in E. flavescens, however, generally increased along the
Fig 14. FA1 scores of the pore pairs numbers. Bar charts of the FA scores (FA1 index) for pore counts of
the anterior and the posterior paired petals of the Liencres and the Erwitte population.
doi:10.1371/journal.pone.0148341.g014
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 18 / 26
anterior–posterior axis, unlike inM. brevis, where the anterior landmarks are more influenced
by FA. This assumes that regulatory mechanisms betweenM. brevis and E. flavescens are par-
tially different. DA is thought to be ubiquitous in the skeleton of irregular echinoids, which is
assumed to be related to the asymmetry of the digestive system [69, 70]. Accordingly, the
occurrence of DA in at least one sample (Erwitte population) is not surprising.
Variation in non-morphometric characters
Subanal fasciole and peristome coverage by the labrum. The subanal fasciole is better
developed in the Liencres specimens, while it is often totally absent in the German populations.
The task of the subanal fasciole is to provide a water current, by movement of the ciliated
spines, and thus to sweep the faeces away from the body [23]. A sustainment of such a feature
is more reasonable at a deeper burrowing depth. The development of the fasciole had been
linked to the nature of the sediment in several taxa of fossil spatangoids by previous authors
[32, 47], which is in agreement with the present observations onM. brevis. Néraudeau [71]
already stated that the occurrence and development of fascioles in general are influenced by
phenotypic plasticity. Fascioles (e.g. subanal, or lateral fascioles) in fossil spatangoids are better
developed if these inhabited finer grained sediments, whereas in coarse grained sediments fas-
cioles tend to be lesser developed, or even become lost. As this pattern is in an inverse relation
to the here recognised variation, it has to be considered that the need for fascioles is influenced
by the burrowing behaviour [23], fascioles are generally rather found in deeper burrowing taxa.
Accordingly, a need for fascioles corresponds to the permeability of the sediments (governed
by grain size) and burrowing depth. According to the tendency for better-developed subanal
fascioles in the Liencres population, it is probably related to the foregoing burrowing depth,
due to coarser-grained sediment.
Fig 15. Sketches illustrating the variation in the periplastronal ambulacral plates. (A-B) Conspicuous
asymmetric development. (C-D) Symmetric development, with longer ambulacral plates (C) and shorter
plates (D) next to the labrum (A: MB.E.8196, Liencres area, Spain; B: GSUB E3847, Erwitte area, Germany;
C: GSUB E3867, Erwitte area, Germany; D: MB.E.8257, Liencres area, Spain). Scale bars equal 1 cm.
doi:10.1371/journal.pone.0148341.g015
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 19 / 26
It is unclear to which function the projection of the labrum is related [72]. Nichols [36]
interpreted this development as being associated with a change in feeding habits, from a food
supply from underneath the peristome towards a transport of particles from the surface to the
peristome via the frontal notch. However, so far there is no clear evidence in support of this
idea. A stronger projection of the labrum can be observed within the evolution fromM. leskei
(late Turonian) toM. coranguinum (late Coniacian), e.g. [23].Micraster brevis is assumed to be
a side branch of this lineage [17, 18]; thus, it is remarkable that a change of the labrum projec-
tion apparently took place independently in different species ofMicraster. Accordingly, the
development of the labrum inMicraster would then be homoplastic, as a result of either paral-
lelism or convergence. An increase in the projection of the labrum also appeared in other spa-
tangoid lineages, e.g. [73], which supports the idea of homoplastic development in this trait.
Moreover, the here-observed differences in the degree of projection in the labrum between the
populations argue for a large influence of the environment on the development of the labrum.
To what degree this development was mediated through phenotypic plasticity or genetically
determined must remain unclear without comparable case studies.
Interestingly, the interradial structure of the paired petals and the granulation of the peri-
plastronal area are the most invariant traits considered here. This finding could indicate low
genetic variation, and/or low environmental influences, or high developmental stability, or can-
alization, which buffers against any perturbations.
Pore numbers versus test length. Elongated pores such as those in the paired petals of
Micraster are linked to ambulacral tube feet, which are specialised for gaseous exchange [38].
Accordingly, as suggested by the works of Stokes [15] and Zaghbib-Turki [32], an adaptation
towards higher temperatures in southern palaeolatitudes, by an increase in the petaloid pore
pair numbers, could have been expected in the population from the Liencres area. However, no
significant differences between the populations could be found. This could argue either for
insufficient statistical power or for similar temperatures in both realms. Unfortunately, recon-
structions of palaeotemperatures of the early Coniacian of these areas are not available.
Fluctuating asymmetry analysis for the pore pair numbers in the paired petals. The
pore numbers in the anterior paired petals show a higher amount of FA in the Liencres popula-
tion, which indicates a higher level of developmental instability, caused by either environmen-
tal factors or genetic stressors. For instance, possible higher sea temperatures could have posed
an environmental stressor in this case, as water temperatures and oxygen content are inti-
mately linked, and must have served as a selective force for this trait. It is suggested [74, 75]
that functionally important traits are more stable in development and, thus, reveal only low lev-
els of FA, as they would be subjected more to stabilising selection. As the pore pairs in the
paired petals have a highly important function, however, it could therefore be assumed that the
Liencres population was exposed to non-stabilising selection.
Metric FA analysis of the ambulacral plates, including the paired petals, revealed no signifi-
cant differences among both populations. This is in contradiction to the results of the meristic
analysis, which suggests either insufficient sample sizes to detect significant differences in FA
of shape variation, or that different processes in development of the shape and the pore pair
numbers were involved. The latter idea is supported by comparisons of individual metric FA
scores (computed only for the anterior paired petals) with the meristic unsigned asymmetry
values of the paired petals, in which values of either analysis are only weakly to moderately
related (see S5 Fig). However, comparisons of FA analysis considering the position and the
numerical values of the same trait had different outcomes, in which positional FA was more
sensitive to developmental instability [76], which is inconsistent with findings of the current
study.
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 20 / 26
Conclusions
This study confirms the assumption thatMicraster developed local ecophenotypism [22]. It is
most likely that especially the nature of the sediment (e.g. grain size) had a large influence on
the morphology ofM. brevis. The phenotypic variations suggest different burrowing behav-
iours of the populations in their respective environment. The morphological features in the
Liencres population indicate a greater burrowing depth than in the German populations,
which likely was attributed to the coarser sediment in Liencres.
Morphological variations in the position of the ambitus, the periproct and the subanal fas-
ciole were most likely influenced by phenotypic plasticity, and potentially also the shape of the
plastron. The projection of the labrum was achieved independently in different species of
Micraster. The findings of more pronounced projecting labral plates in the Spanish sample,
however, raise the question to which degree the environment played a shaping force and what
kind of factors were involved. Further comparisons to distinctMicraster populations/species
are required to gain more insights into the dependence between environmental factors and the
development of this trait.
Variations due to developmental instability exist in the Liencres and in the Erwitte popula-
tions. Differences in the amount of FA, however, were only significant for the numbers of pore
pairs in the anterior paired petals. Differences in environmental factors, which could have pro-
voked these higher FA values, are unclear. Temperature gradients, for instance, to which this
trait would have most reasonably responded, could not be detected, since data on palaeotem-
peratures are not available. Similarities among the populations in FA levels of the periplastro-
nal ambulacral plates could have resulted from common perturbations in their developmental
regulatory mechanisms.
Other shape modifications (asymmetry in the sternal plates, contact of the labrum and the
sternal plates, position in the periplastronal ambulacral plates, position of the apical shield, and
the widest point of the test), which were traditionally regarded as being involved in a continu-
ous process of evolution inMicraster, revealed no distinct relation to specific populations and,
hence, to environmental differences.
It is noteworthy that two of the here-studied traits (interradial structure of the paired petals,
granulation of the periplastronal) were very robust in their development, as they reveal appar-
ently no variation. The influence of environmental variation, however, was able to create an
increase in morphological diversity, which is worth studying in the evolutionary context of the
Micraster lineage.
Concepts and mechanisms of variability, such as phenotypic plasticity and developmental
robustness, are important topics and are of great interest for evolutionary development. The
majority of works addressing these concepts, however, relied on extant organisms. Data from
the fossil record, however, are invaluable to our understanding of evolution in nature. Accord-
ingly, works on these topics are worth to be extended to the fossil record and have the potential
to provide important insights into trends and patterns in evolutionary history, which can be
incorporated into ideas of great interest in evolutionary biology.
Supporting Information
S1 Fig. Thin sections. (A) Grimberg. (B, C) Erwitte. (D-F) Liencres. (A-C) Wackestones con-
tain clay and silt, with relatively low content of bioclasts. (D-F) Silty packstones with abundant
bioclasts and siliciclasts.
(TIF)
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 21 / 26
S2 Fig. Shape variation of the asymmetric and symmetric component (PC2) for the popula-
tion from the Liencres and the Erwitte area.
(TIF)
S3 Fig. Shape variation of the asymmetric and symmetric component (PC3) for the popula-
tion from the Liencres and the Erwitte area.
(TIF)
S4 Fig. Shape variation of the asymmetric and symmetric component (PC4) for the popula-
tion from the Liencres area and the Erwitte area.
(TIF)
S5 Fig. Comparison of the individual metric and meristic FA values for the specimens from
Liencres area (A) and from the Erwitte area (B).
(TIF)
S1 File. Material included for particular analysis.
(DOC)
S1 Multimedia. A 3Dmodel of GSUB E3840 (Erwitte area) shows the landmark configura-
tion. This file (.obj file)can be opened (import mesh) in the open-source software MeshLab
(Visual Computing Lab—ISTI—CNR), available at: http://meshlab.sourceforge.net/. The land-
mark coordinates (GSUB E3840_picked_points.pp) can be loaded via the PickPoints function.
(ZIP)
S1 Table. Individual FA scores for the metric analysis.
(XLS)
S2 Table. Variation in the projection of the labrum for each population.
(XLS)
S3 Table. Variation in the development of the subanal fasciole for each population.
(XLS)
S4 Table. Individual values for the pore pair numbers.
(XLS)
Acknowledgments
I am very grateful to Ekbert Seibertz (Wolfsburg) for informing and pointing me to the material
collected from him. I thankMartin Krogman, Jens Lehman (both Bremen) and Angela Ehling
(Berlin) for loans of material. Tim Ewin (London) and Christian Neumann (Berlin) kindly loaned
additional specimens. I much appreciated the comments and suggestions of Loïc Villier, Andreas
Kroh and Steffen Kiel on an earlier draft and for that I thank them. Further, I thank FrankWiese
for comments and discussions, Esra Ertan Schlüter for helpful suggestions and comments. Addi-
tionally, I thank Alessandro Airo for introducing me into the world of photogrammetry.
Author Contributions
Conceived and designed the experiments: NS. Performed the experiments: NS. Analyzed the
data: NS. Contributed reagents/materials/analysis tools: NS. Wrote the paper: NS.
References
1. Pigliucci M. Developmental phenotypic plasticity: where internal programming meets the external envi-
ronment. Curr Opin Plant Biol. 1998; 1: 87–91. doi: 10.1016/S1369-5266(98)80133-7 PMID: 10066552
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 22 / 26
2. Gibson G, Wagner G. Canalization in evolutionary genetics: a stabilizing theory? BioEssays. 2000; 22:
372–80. doi: 10.1002/(SICI)1521-1878(200004)22:4<372::AID-BIES7>3.0.CO;2-J PMID: 10723034
3. West-Eberhard MJ. Developmental Plasticity and Evolution. New York: Oxford University Press;
2003.
4. Gibson G, Dworkin I. Uncovering cryptic genetic variation. Nat Rev Genet. 2004; 5: 681–90. doi: 10.
1038/nrg1426 PMID: 15372091
5. DeWitt TJ, Scheiner SM. Phenotypic variation from single genotypes. In: DeWitt TJ Scheiner SM, edi-
tors. Phenotypic Plasticity. Functional and Conceptual Approaches. Oxford: Oxford University Press;
2004. pp. 1–9.
6. Jablonski D. The future of the fossil record. Science. 1999; 284: 2114–6. doi: 10.1126/science.284.
5423.2114 PMID: 10381868
7. Palmer AR. Fluctuating asymmetry analyses: a primer. In: Markow T, editor. Developmental Instability:
Its Origins and Evolutionary Implications. Dordrecht: Kluwer; 1994. pp. 335–364.
8. Willmore KE, Young NM, Richtsmeier JT. Phenotypic variability: its components, measurement and
underlying developmental processes. Evol Biol. 2007; 34: 99–120. doi: 10.1007/s11692-007-9008-1
9. Van Valen L. A study of fluctuating asymmetry. Evolution. 1962; 16: 125–142. doi: 10.2307/2406192
10. Klingenberg CP. A developmental perspective on developmental instability: theory, models and mech-
anisms. In: Polak M, editor. Developmental Instability: Causes and Consequences. New York, Oxford
University Press; 2003. pp. 14–34.
11. Willmore KE, Hallgrímsson B. Within individual variation: developmental noise versus developmental
stability. In: B. Hallgrímsson B, Hall BK, editors. Variation: A Central Concept in Biology. Burlington,
MA: Elsevier; 2005. pp. 191–218.
12. McAdams HH, Arkin A. Stochastic mechanisms in gene expression. Proc Natl Acad Sci. 1997; 94:
814–819. PMID: 9023339
13. Kaern M, Elston TC, BlakeWJ, Collins JJ. Stochasticity in gene expression: from theories to pheno-
types. Nat Rev Genet. 2005; 6: 451–64. doi: 10.1038/nrg1615 PMID: 15883588
14. Lambert J, Thiéry PP. Essai de nomenclature raisonnée des échinides. Chaumont: Librairie L. Fer-
rière; 1909–1925
15. Stokes R. B. Royaumes et provinces fauniques du Crétacé établis sur la base d’une étude systéma-
tique du genreMicraster. MemMus Nat Hist Nat Nouv Ser. 1975; C 31: 1–94.
16. Rowe AW. An analysis of the genusMicraster, as determined by rigid zonal collecting from the zone of
Rhynchonella Cuvieri to that ofMicraster cor-anguinum. Q J Geol Soc London. 1899; 55: 494–547.
doi: 10.1144/GSL.JGS.1899.055.01–04.34
17. Ernst G. Zur Stammesgeschichte und stratigraphischen Bedeutung der Echiniden-GattungMicraster in
der nordwestdeutschen Oberkreide. Mitt Geol-Paläont Inst Hamburg. 1970; 39: 117–135.
18. Ernst G. Grundfragen der Stammesgeschichte bei irregulären Echiniden der nordwesteuropäischen
Oberkreide. Geol Jb A. 1972; 4: 63–175.
19. David B, Fouray M. Variabilité et disjonction évolutive des caractères dans des populations deMicra-
ster (Echinoidea, Spatangoidea) du Crétacé supérieur de Normandie. Geobios. 1984; 17: 447–476.
20. Smith AB, Wright CV. British Cretaceous echinoids. Part 9, Atelostomata, 2. Spatangoida (2). Palaeon-
togr Soc Monogr. 2012; 166: 635–754.
21. Drummond PVO. TheMicraster biostratigraphy of the Senonian white chalk of Sussex, southern
England. Geol Mediterr. 1983; 10: 177–182.
22. Ernst G, Seibertz E. Concepts and methods of echinoid biostratigraphy. In: Kauffman EG, Hazel JE,
editors. Concepts and Methods of Biostratigraphy. Stroudsburg: Hutchinson & Ross; 1977. pp. 541–
563.
23. Smith AB. Echinoid Palaeobiology. London: Allen & Unwin; 1984.
24. Olszewska-Nejbert D. Late Cretaceous (Turonian—Coniacian) irregular echinoids of western Kazakh-
stan (Mangyshlak) and southern Poland (Opole). Acta Geol Pol. 2007; 57: 1–87.
25. Villier L, David B, Néraudeau D. Ontogenetic and morphological evolution of the ambulacral pores in
Heteraster (early spatangoids). In: Barker M, editor. Echinoderms 2000. Lisse: Swets & Zeitlinger;
2001. pp. 563–567.
26. Wiese F. Das Turon und Unter-Coniac im Nordkantabrischen Becken (Provinz Kantabrien, Nordspa-
nien): Faziesentwicklung, Bio-, Event- und Sequenzstratigraphie. Berliner geowiss Abh E. 1997; 24: 1–
131.
27. Seibertz E. Stratigraphie, Fazies und Paläogeographie der „Mittel"-Kreide zwischen Rüthen und Erwitte
(Alb-Coniac, SE-Münsterland). Aufschluß Sonderbd. 1979; 29: 85–92.
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 23 / 26
28. Kaplan U, Skupin K. Coniacian near Erwitte. In: Mutterlose J; Bornemann A, Rauer S; Spaeth C, Wood
CJ, editors. Key localities of the northwest European Cretaceous. Bochumer geol u geotechn Arb.
1998;48: 184–185.
29. Voigt S, Wilmsen M, Mortimore RN, Voigt T. Cenomanian paleotemperatures derived from the oxygen
isotopic composition of brachiopods and belemnites: evaluation of Cretaceous paleotemperature prox-
ies. Int J Earth Sci. 2003; 92: 285–299. doi: 10.1007/s00531-003-0315-1
30. Ziegler PA. Evolution of the Arctic-North Atlantic and theWestern Tethys. Am Assoc Pet Geol Mem.
1988; 43: 198 pp.
31. Higgins R. C. Specific status of Echinocardium cordatum, E. australe and E. zealandicum (Echinoidea:
Spatangoida) around New Zealand, with comments on the relation of morphological variation to envi-
ronment. J Zool. 1974; 173: 451–47. doi: 10.1111/j.1469-7998.1974.tb04127.x
32. Zaghbib-Turki D. Stratégie adaptive desHemiaster et des Periaster du Crétacé supérieur (Cénoma-
nien-Coniacien) de la Plate-forme carbonatée de Tunisie. In: De Ridder C, Dubois P, Lahaye MC, Jan-
goux M, editors. Echinoderm Research. Rotterdam: Balkema; 1990. pp. 49–56.
33. Kanazawa K. Adaptation of test shape for burrowing and locomotion in spatangoid echinoids. Palaeon-
tology. 1992; 35: 733–750.
34. François É, David B. Variations morphologiques des Toxaster (Echinoida: Spatangoida) en regard des
fluctuations spatiales (Arc de Castellane, SE France) et temporelles (Valanginien-Hauterivien) du
milieu sédimentaire: expression d’un potentiel adaptatif restreint. Geobios. 2006; 39: 355–371. doi: 10.
1016/j.geobios.2005.01.002
35. Saitoh M, Kanazawa K. Adaptive morphology for living in shallow water environments in spatangoid
echinoids. In: Kroh A, Reich M, editors. Echinoderm Research 2010: Proceedings of the Seventh Euro-
pean Conference on Echinoderms, Göttingen, Germany, 2–9 October 2010. Zoosymposia. 2012;7:
255–265.
36. Nichols D. Changes in the chalk heart-urchinMicraster interpreted in relation to living forms. Philos
Trans R Soc Lond B Biol Sci. 1959; 242: 347–437. doi: 10.1098/rstb.1959.0007
37. Fouray M. L’evolution desMicraster (Echinides, Spatangoida) dans le Turonien-Coniacien de Picardie
occidentale (Somme). Interets biostratigraphiques. Ann Palaeontol (Invert). 1981; 67: 81–134.
38. Smith AB. The structure, function and evolution of tube feet and ambulacral pores in irregular echinoids.
Palaeontology. 1980; 23: 39–83.
39. Kaplan U, KennedyWJ. Die Ammoniten des westfälischen Coniac. Geol Palaeont Westf. 1994; 31: 1–
155.
40. Tröger K- A. Zur Biostratigraphie des Ob.-Turons bis Unt.-Santons aus dem Schachtaufschluß der
Zeche Grimberg IV bei Bergkamen (BRD). Freiberg Forschungsh C. 1974; 298: 109–138.
41. Falkingham PL. Acquisition of high resolution 3Dmodels using free, open-source, photogrammetric
software. Palaeontol Electron. 2012; 15: 15p. palaeo-electronica.org/content/issue-1-2012-technical-
articles/92-3d-photogrammetry
42. Mallison H, Wings O. Photogrammetry in paleontology–a practical guide. J Paleontol Tech. 2014; 12:
1–3.
43. Klingenberg CP. MorphoJ: an integrated software package for geometric morphometrics. Mol Ecol
Resour. 2010; 11: 353–357. doi: 10.1111/j.1755-0998.2010.02924.x PMID: 21429143
44. Bookstein FL. Morphometric tools for landmark data: geometry and biology. Cambridge: Cambridge
University Press; 1991.
45. Klingenberg CP, McIntyre GS. Analyzing patterns of fluctuating asymmetry with Procrustes methods.
Evolution. 1998; 52: 1363–1375.
46. Klingenberg CP, BarluengaM, Meyer A. Shape analysis of symmetrical structures: quantifying variation
among individuals and asymmetry. Evolution. 2002; 56: 1909–1920. PMID: 12449478
47. Néraudeau D, David B, Madon C. Tuberculation in spatangoid fascioles: Delineating plausible homolo-
gies. Lethaia. 1998; 31: 323–334.
48. Schneider CA, RasbandWS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Meth-
ods. 2012; 9: 671–675. PMID: 22930834
49. Palmer AR, Strobeck C. Fluctuating asymmetry: measurement, analysis, patterns. Annu Rev Ecol
Syst. 1986; 17: 391–421. doi: 10.1146/annurev.es.17.110186.002135
50. Palmer AR, Strobeck C. Fluctuating asymmetry revisited. In: Polak M, editor. Developmental Instability
(DI): Causes and Consequences. Oxford: Oxford University Press; 2003. pp. 279–319.
51. Swain DP. A Problem with the use of meristic characters to estimate developmental stability. Am Nat.
1987; 129: 761–768.
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 24 / 26
52. Walker DE, Gagnon J- M. Locomotion and functional spine morphology of the heart urchin Brisaster fra-
gilis, with comparisons to B. latifrons. J Mar Biol. 2014;Article ID 297631, 9 pages. doi: 10.1155/2014/
297631
53. Egea E, David B, Choné T, Laurin B, Féral JP, Chenuil A. Morphological and genetic analyses reveal a
cryptic species complex in the echinoid Echinocardium cordatum and rule out a stabilizing selection
explanation. Mol Phylogenet Evol. 2015; 94: 207–220. doi: 10.1016/j.ympev.2015.07.023 PMID:
26265259
54. De Baets K, Bert D, Hoffmann R, Monnet C, Yacobucci MM, Klug C. Chapter 9. Ammonoid intraspecific
variability. In: Klug C, Korn D, De Baets K, Kruta I, Mapes RH, editors. Ammonoid Paleobiology: From
Anatomy to Ecology. Topics In Geobiology. 2015;43: 359–426.
55. McKinney ML, McNamara KJ. Heterochrony. The Evolution of Ontogeny. New York, London: Plenum
Press; 1991.
56. Chauffe KM, Nichols PA. Differentiating evolution from environmentally induced modifications in mid-
Carboniferous conodonts. Palaeontology. 1995; 38: 875–895.
57. Scheiner SM. Genetics and Evolution of Plasticity. Annu Rev Ecol Syst. 1993; 24: 35–68.
58. Braendle C, Flatt T. A role for genetic accommodation in evolution? BioEssays. 2006; 28: 868–873.
PMID: 16937342
59. Suzuki Y, Nijhout HF. Evolution of a polyphenism by genetic accommodation. Science. 2006; 311:
650–652. PMID: 16456077
60. Cunningham JA, Jeffery Abt CH. Coordinated shifts to non-planktotrophic development in spatangoid
echinoids during the Late Cretaceous. Biol Lett. 2009; 5: 647–650. doi: 10.1098/rsbl.2009.0302 PMID:
19515650
61. Wiese F, Voigt S. Late Turonian (Cretaceous) climate cooling in Europe: faunal response and possible
causes. Geobios. 2002; 35: 65–77. doi: 10.1016/S0016-6995(02)00010-4
62. Chenuil A, Féral JP. Sequences of mitochondrial DNA suggest that Echinocardium cordatum is a com-
plex of several sympatric or hybridizing species. A pilot study. In: Féral J-P, David B, editors. Echino-
derm Research 2001: proceedings of the 6th European Conference on Echinoderm Research,
Banyuls-sur-mer, 3–7 September 2001. Lisse: Swets & Zeitlinger; 2003. pp. 15–21.
63. Klingenberg CP, Nijhout HF. Genetics of fluctuating asymmetry: a developmental model of develop-
mental instability. Evolution. 1999; 53: 358–375. doi: 10.2307/2640773
64. Hunt G. Phenotypic variation in fossil samples: modeling the consequences of time-averaging. Paleobi-
ology. 2004; 30: 426–443. doi: 10.1666/0094-8373(2004)030<0426:PVIFSM>2.0.CO;2
65. Chalancon G, Ravarani CNJ, Balaji S, Martinez-arias A, Aravind L, Jothi R, Madan Babu M. Interplay
between gene expression noise and regulatory network architecture. Trends Genet. 2012; 28: 221–
232. doi: 10.1016/j.tig.2012.01.006 PMID: 22365642
66. Arthur W. The concept of developmental reprogramming and the quest for an inclusive theory of evolu-
tionary mechanisms. Evol Dev. 2000; 2: 49–57. doi: 10.1046/j.1525-142X.2000.00028.x PMID:
11256417
67. McNamara KJ. Plate translocation in spatangoid echinoids: its morphological, functional and phyloge-
netic significance. Paleobiology. 1987; 13: 312–325.
68. Saucède T, Alibert P, Laurin B, David B. Environmental and ontogenetic constraints on developmental
stability in the spatangoid sea urchin Echinocardium (Echinoidea). Biol J Linn Soc. 2006; 88: 165–177.
69. Lawrence JM, Pomory CM, Sonnenholzner J, Chao C- M. Bilateral symmetry of the petals inMellita
tenuis, Encopemicropora, and Arachnoides placenta (Echinodermata: Clypeasteroida). Invert Biol.
1998; 117: 94–100. doi: 10.2307/3226855
70. Stige LC, David B, Alibert P. On hidden heterogeneity in directional asymmetry—can systematic bias
be avoided? J Evol Biol. 2006; 19: 492–499. doi: 10.1111/j.1420-9101.2005.01011.x PMID: 16599925
71. Néraudeau D. La variabilité morphologique est-elle un obstacle à la définition des espèces paléontolo-
giques? Le cas des échinides spatangues, Biosystema. 2001; 19: 93–107.
72. De Ridder C, Lawrence J. Food and feeding mechanisms: Echinoidea. In: Jangoux M, Lawrence J, edi-
tors. Echinoderm Nutrition. Rotterdam: A.A. Balkema Press; 1982. pp. 97–115.
73. McNamara KJ. Philip GM. Australian tertiary schizasterid echinoids. Alcheringa. 1980; 4: 47–65. doi:
10.1080/03115518008558980
74. Møller AP, Swaddle JP. Asymmetry, Developmental Stability, and Evolution. Oxford: Oxford University
Press; 1997.
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 25 / 26
75. Karvonen E, Merilä J, Rintamäki PT, Van Dongen S. Geography of fluctuating asymmetry in the green-
finch, Carduelis chloris. Oikos. 2003; 100: 507–516.
76. Polak M. Ectoparasitism in mothers causes higher positional fluctuating asymmetry in their sons : impli-
cations for sexual selection. Am Nat. 1997; 149: 955–974. PMID: 18811257
Phenotypic Variation in the Cretaceous EchinoidMicraster brevis
PLOSONE | DOI:10.1371/journal.pone.0148341 February 5, 2016 26 / 26