Download mechanical properties of the chitin-calcium

J OURNAL OF C RUSTACEAN B IOLOGY, 35(2), 123-131, 2015
MECHANICAL PROPERTIES OF THE CHITIN-CALCIUM-PHOSPHATE
“CLAM SHRIMP” CARAPACE (BRANCHIOPODA: SPINICAUDATA):
IMPLICATIONS FOR TAPHONOMY AND FOSSILIZATION
Timothy I. Astrop 1,∗ , Vasav Sahni 2 , Todd A. Blackledge 3 , and Alyssa Y. Stark 3
1 Department
of Biology and Biochemistry, The University of Bath, Claverton Down, Bath, Somerset, UK
2 Department of Polymer Science, The University of Akron, Akron, OH, USA
3 Department of Biology, Integrated Bioscience Program, The University of Akron, Akron, OH, USA
ABSTRACT
Spinicaudata (colloquially ‘the clam shrimp’) are freshwater branchiopod crustaceans that occur worldwide in lakes and temporary pools.
The spinicaudatans are easily recognizable by their bivalved carapace which is unusual among arthropods in that it is subject to only
partial molting. During ecdysis (molting), the outer surface of the carapace is not shed, resulting in the retention of the ontogenetic record
of an individual through distinct growth-rings representing each molt. When this unusual feature is considered alongside the interesting
chemical properties of the carapace, “clam shrimp” present an interesting biological material not seen anywhere else: a multi-laminar
calcium-phosphate-chitin composite. In addition, the carapace survives numerous destructive taphonomic processes (including transport,
decay, compaction, and desiccation) to become the dominant body component of Spinicaudata preserved in their 380 million year fossil
record. Understanding the mechanical properties and chemical composition of this structure may not only aid in a better understanding of
the evolutionary history of this group but also facilitate efforts to develop novel materials that retain functional material properties even
in harsh aquatic conditions. Therefore, this study aims to provide quantitative information about the composition and mechanics of this
unique and interesting biological material and help predict possible biases in the fossilization of different species of Spinicaudata to aid
future palaeontological research.
K EY W ORDS: arthropod cuticle, Branchiopoda, fracture mechanics, palaeobiology, taphonomy
DOI: 10.1163/1937240X-00002332
I NTRODUCTION
Arthropoda are without doubt the most diverse, successful,
and adaptable animals on earth (Giribet and Edgecombe,
2013). Originating in the Cambrian some 520 million years
ago (mya) (Legg et al., 2012), arthropods became essential
components of global ecosystems and are now ubiquitous
in aquatic, aerial, and terrestrial environments. The quality
of the fossil record for Arthropoda ranges from exquisitely
preserved amber encased specimens (Barden and Grimaldi,
2013), to isolated partial elements of the exoskeleton. Factors influencing the entrance of arthropodan remains into the
fossil record are of key importance to correctly interpreting
the evolutionary history of the group. Understanding the biotic and abiotic parameters that allow fossilization is the primary role of the science of taphonomy, and palaeobiologists
have long experimented with organismal remains to understand how processes such as decay, transport, predation, and
desiccation may ultimately shape the preservational record
of life on earth via the process of fossilization (Allison,
1986; Briggs, 1995; Behrensmeyer et al., 2000; Krause et
al., 2011).
The cuticle of the arthropods is a key adaptation that
provides both structural support and a barrier between
an organism’s internal biology and the harsh surrounding
∗ Corresponding
environment (Moussian, 2013). This cuticle is comprised
of several layers all containing the polysaccharide chitin;
a thin outer epicuticle, a mineralized exocuticle, and a less
rigid endocuticle (Gupta, 2011); it is the most commonly
represented component of arthropods in the fossil record.
Chitin is hardened via sclerotization in many arthropods,
such as insects, but chitin is also commonly found as a
composite material, as in the case with most crustaceans. In
many Crustacea, chitin is combined with calcium carbonate
into laminae in a unique helical pattern (Bouligand structure,
see Cheng et al., 2008) that provides great rigidity for
protection.
The ‘clam shrimp,’ Order Spinicaudata, are seemingly
inconspicuous crustaceans that inhabit temporary freshwater
habitats world-wide and are easily recognized as adults
due to their chitinous bivalved carapace, which completely
encapsulates them (Fig. 1). This carapace is unique among
invertebrates in that molts are partially retained during
ecdysis. The outer surface of the carapace is retained after
each molt causing distinctive growth lines that record the
organism’s ontogeny (Rieder et al., 1984). This results in
the carapace forming a ‘layered’ structure, superficially
similar to molluscan bivalves, in contrast to the complete
discarding of exuviae seen in more familiar arthropods
such as crabs, lobsters, and other members of Ecdysozoa.
author; e-mail: [email protected]
© The Crustacean Society, 2015. Published by Brill NV, Leiden
DOI:10.1163/1937240X-00002332
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JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 35, NO. 2, 2015
Fig. 1. Taxa investigated in this study. A, Leptestheria compleximanus; B, Eulimnadia feriensis; C, Cyzicus gynecia. Note the accretional growth rings.
Scale bar is 2 mm.
Additionally, the chemical composition of spinicaudatan
carapace itself is unique in that, rather than forming a chitincalcium complex, the carapace of Spinicaudata is believed
to be primarily chitin-phosphate, possibly a product of their
freshwater environment that is calcium deficient (Stigall and
Hartmann, 2008). Whether there are differences in the mode
and level of mineralization among spinicaudatan families is
unknown but could lead to differential preservation bias in
the spinicaudatan fossil record (Fig. 2).
The strong arthropod cuticle plays a significant role in
determining their preservation potential (Edgecombe and
Legg, 2013) so that they have an excellent fossil record, extending back to the Devonian (380 mya). The natural ecology confines Spinicaudata to freshwater, making them useful as an indicator of historical freshwater environments. In
addition, the accretional carapace growth of spinicaudatan
also records information about environmental stresses, fluctuations and seasonality in successive growth ‘rings.’ Several
studies explored the possibility of carapace morphology reflecting sexual dimorphism within fossil Spinicaudata popu-
lations, in turn allowing the diagnoses of particular sexual
systems in geologic time (Astrop et al., 2012; Gallego et al.,
2013; Monferran et al., 2013; Stigall et al., 2013). Understanding taphonomic influences of preservation within different lineages of Spinicaudata is of great importance to understanding the reliability of data used for such studies.
The mineralized carapace of Spinicaudata is the main constituent of their excellent fossil record, but the lack of chemical and mechanical characterization of this unique material
results in tentative use of this arthropod in palaeobiological
studies. We hypothesize that family-level differences in the
composition and mechanical properties of the spinicaudatan
carapace affect susceptibility to taphonomic processes. In
this study, we utilize material testing method to investigate
the strength of the carapace of representatives of spinicaudatan families as a proxy for taphonomic susceptibility. Such
a preservational bias, should it exist, could have a great numerical effect on the abundance of different taxa and would
need to be considered when utilizing the fossil record of this
order.
Fig. 2. Generic diversity of Spinicaudata over geologic time based on multiple literature sources (primarily Tasch, 1969; Zhang et al., 1976). Note the
periodic disappearance of Limnadiidae from the record, the persistence of Cyzicidae, and the recent occurrence of Leptestheriidae. Extinct genera are
included for reference.
ASTROP ET AL.: CHITIN IN SPINICAUDATANS
M ATERIALS AND M ETHODS
Sample Preparation
Three species of Spinicaudata were used for compositional assessment and
materials testing, these three species were chosen to represent the three
extant families within the spinicaudatans, Cyzicus gynecia (Mattox, 1950),
Leptestheria compleximanus (Packard, 1877), Eulimnadia feriensis Dakin,
1914 (Fig. 1). All populations were reared in the laboratory following the
protocol outlined in (Weeks and Zucker, 1999). To control for differences in
age, and thus number of molts or layers of cuticle, we only used individuals
with the same hatch date. Live samples were refrigerated for sedation and
subsequently removed from their carapaces. Valves were stored at −10°C
for no longer than 3 days while trials where in progress. Samples used for
elemental assessment using energy dispersive X-ray spectroscopy (EDAX)
were immediately removed from their carapaces and quickly dried via
absorption before embedding in epoxy.
Elemental Composition
EDAX was performed using a Princeton Gamma Tech (Princeton, NJ,
USA) energy dispersive X-ray spectrometry (EDS) system within an FEI
XL-30 environmental scanning electron microscope with a Ge detector.
Uncoated samples were embedded in epoxy resin on a glass slide before
being ground with an electric rock grinder in order to expose a transverse
section through the carapace. Samples were washed with alcohol and
dried before being placed in the ESEM chamber at 90 ± 2 kPa. EDAX
analyses were performed using a voltage of 25 keV. Two individuals of L.
compleximanus and C. gynecia were used and one individual was used to
represent E. feriensis. Quantitative information was obtained using ZAF
Quantification (Standardless) via the EDAX Genesis software package
V4.52.
Punch Test
Biological materials testing is generally challenging due to their anisotropic
and composite nature and because most biomaterials are viscoelastic (Strait
and Vincent, 1998; Aranwela et al., 1999; Edwards et al., 2000). We
measured the material properties of individual spinicaudatan shells using
a modified punch test or punch-and-die test (Choong et al., 1992; Aranwela
et al., 1999; Sanson et al., 2001). In these tests, a punch is lowered at a
controlled rate normal to the sample until fracture occurs. To control the
region of the sample loaded, punches are small enough to fit through a die,
which holds the sample (see Fig. 3 for a schematic of our experimental
set-up).
Due to the nature of material failure, the area of fracture can be larger
than the die, thus punch tests sometimes inflate measured values, especially
for complex biological materials (Lucas et al., 1991). Furthermore, when
125
punching through a material, resistance to penetration involves multiple
material properties, such as shear and compressive strength and resistance
to crack propagation (Vincent, 1992; Aranwela et al., 1999; Edwards et al.,
2000) so that the actual material property measured is not clear. However,
the test clearly mimics real physical trauma both from predation and
taphonomic process. Moreover, other authors argue that the punch test is
a good indicator of both material and structural properties and suggest that
the variation in sample fracture can actually enhance our understanding of
the material’s performance (Evans and Sanson, 2005; Freeman and Lemen,
2007).
As a result of the inherent challenges of both biological materials testing
and punch tests in particular, some suggest this test method should not
be adopted (Aranwela et al., 1999; Edwards et al., 2000). Compressive
properties of some biomaterials like bone and wood can instead be
characterized from milled samples (Vincent, 1992). However, this was not
deemed feasible with the small, thin, and fragile spinicaudatan carapaces.
Spinicaudata are aquatic and their shells are highly sensitive to desiccation.
In preliminary investigations we observed the shells curling and noted
qualitatively different material properties after only a few minutes in air.
Our use of the punch test addresses these problems as samples can be
quickly mounted as whole shells and tested before extensive drying or
damage occurs. We also chose the punch test as it investigates material
and structural properties (Evans and Sanson, 2005), both of which may
be relevant for taphonomic and fossilization processes. Finally, the curved
nature of our samples provided additional complexity which seemed best
controlled by a punch test (see methodology below).
Several material properties are commonly reported when using a punch
test and these are outlined in Table 1 (Edwards et al., 2000; Sanson et al.,
2001; Evans and Sanson, 2005). Force and displacement were measured
during experimental trials and further used to calculate specific material
properties (Table 1). The force-displacement curve gives the maximum
force and displacement at break (Fig. 4). The area under the forcedisplacement curve (grey in Fig. 4) represents the total work to break
the specimen. To calculate additional material properties, we normalized
force at break to the area of the punch to estimate the compressive “punch
strength” of the carapace and then expressed punch strength relative to
shell thickness to account for differences among samples as “specific punch
strength” (Table 1). How much work is required to punch through a surface
depends in part on how much material is under the area of impact. To
address this we normalized work to break in two ways (Table 1). Work
to punch, expressed as work relative to the area of the probe impacting
the shell, while specific work to punch is expressed as work relative to the
volume of material directly under the impact site (Table 1) and is analogous
to many common measures of toughness. In reality, while our definitions
allow clear, repeatable comparison across species, these values probably
Fig. 3. A, Schematic of the experimental set-up showing the spinicaudatan shell suspended across a nylon lock nut. B, Location of the glass rod (punch) as
it makes contact with the center of the spinicaudatan shell. A glass rod was attached to a movable cross head grip which was lowered at a speed of 1.00 ×
10−2 mm/s until it punctured the shell to failure. The resulting load on the sample prior to failure was recorded by the bottom force sensor. To allow for
complete puncture of the shell, it was positioned and glued to lie across a nylon lock nut. The lock nut was held on to the bottom force sensor grip by a
standard push pin. C, picture of sample set up (carapace on a lock nut and push pin).
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Table 1. Elemental composition of six individual Spinicaudatan carapace
valves. Two specimens for L. compleximanus and C. gynecia and one
specimen for E. ferriensis were used to investigate intra-specific variation.
Species
C (%) O (%)
Na (%) P (%) Ca (%)
Leptestheria
compleximanus
Leptestheria
compleximanus
Cyzicus gynecia
Cyzicus gynecia
Eulimnadia ferriensis
67.6
27.52
0
1.94
2.94
78.87
16.49
0
1.56
3.09
65.6
54.38
54.30
17.45
24.24
29.16
0
0
3.15
5.34
7.35
0.23
11.62
14.03
1.24
underestimate the total amount of material involved in breaking as fractures
likely propagated longitudinally from the impact site.
We chose a fixed speed of 1.00 × 10−2 mm/s for the probe because
high speeds generally result in brittle fracture of the material (Aranwela
et al., 1999). Aranwela et al. (1999) argued that punch diameter and hole
size of the die should be similar so that shear is enhanced and deformation
of the surrounding area is minimized (Aranwela et al., 1999). In contrast,
we used a 0.2 mm diameter glass rod for our punch while the shells were
mounted on a die with an internal hole diameter of 2.5 mm. We chose a
small diameter punch so that it would make full contact with the curved
shells and we used the large die hole diameter to minimize the effects of
that curvature (Fig. 3b), which was not consistent across species and also
not easy to characterize at smaller spatial scales. Moreover, the large die size
allows the shape of the shell (i.e., curvature) to influence punch resistance,
similar to the way that curvature would influence resistance to forces during
taphonomy.
Sampling Method
Ten valves from the purely selfing-hermaphrodite species C. gynecia and
E. feriensis were used for materials testing. Each individual was dissected
to provide up to two valves, however not all valves were used due to
premature damage during dissection or material testing. Therefore, of the
ten valves for each species, six different individuals contributed valves to
the C. gynecia sample group and eight different individuals contributed to
the E. feriensis sample group. Fourteen valves were used in the dioecious
(male/female) species L. compleximanus. Of the fourteen valves from L.
compleximanus, six valves from three different females and eight valves
from five different males were used.
Immediately before testing, sample valves were removed from vials
filled with water and blotted dry to remove excess water. Samples that
had been removed from water for over five minutes were not used in data
analysis due to clear structural changes observed in the valve (i.e., curling)
and subsequent inflated material property values. Samples were then
mounted on a Nano Bionix tensile tester (Agilent Technology, Amstelveen,
The Netherlands) and positioned for the punch test. In order to load the
sample to failure at the punch site we suspended the samples on a nylon
lock nut (No. 6-32). Ethyl cyanoacrylate glue was brushed lightly around
the top rim of the nut and allowed to cure slightly to avoid wicking of glue
onto the sample. After curing the sample was placed on the glued edge of
the nut and pressed lightly into place along the circumference of the nut. All
samples were larger than the nut diameter to ensure consistent attachment
of the sample to the testing apparatus and to control for variations in size of
the individual. Although there was some size variation within and across
species, the standard nut diameter controlled for variations in shell size
by limiting the sample testing area to 4.91 mm2 for all samples. Prior to
removing the sample valve, the base of the nut was glued firmly to the
plastic top of a standard push pin mounted in the bottom grip of the Nano
Bionix tensile tester (Fig. 3). This grip is attached to a force sensor with a
force and displacement resolution of 1-2 μN and 1 μm, respectively. The
glass rod was positioned in the top movable crosshead grip normal to the
sample (Fig. 3). The rod was then lowered at a fixed speed (0.01 mm/s).
As the rod contacted and pressed the sample, displacement occurred and
force and displacement was recorded using Testworks 4.0 software (MTS,
Eden Prairie, MN, USA). Samples were punctured to failure. In some cases,
the force-displacement curve showed a jump in data that we associated
with the glass rod sliding down the concave shell because we often could
visually observe the glass rod jumping or scraping along the shell. While
the rod was positioned above the most concave portion of the shell, it was
not possible to confirm ideal placement of the rod by eye alone (ideal
would be placement in the center of the shell so that none of the shell
curvature is directly experienced by the punch (Fig. 3b)). We determined
that an aberrantly fast change in force indicated sliding of the rod down the
concave wall of the sample. All samples that had this “jump” in force were
removed from analysis. Jumps ranged from about 0.6mN to about 3.5mN
and generally occurred within 0.025 mm displacement, though in one test
the jump occurred over 0.5 mm displacement. By removing jumps we were
able to control for the puncture location in test samples as force curves that
show a smooth displacement, without jumps, signifies that the rod was in
the most concave portion of the shell. For this reason the samples could not
be tested in the convex position, where slipping and uncontrolled structural
deformations dominated preliminary tests in this orientation. Total area
under the force-displacement curve was integrated using a sigmoid function
to fit the data and is reported as “toughness.” An example of the fitting
function overlaid on the raw data values is shown in Fig. 5. Due to the
fragility of the samples and rapid rate of desiccation, we were unable to
measure thickness of each sample prior to testing. In order to estimate
sample thickness we averaged the measured thickness of ten additional
valves from six individuals from E. feriensis and five individuals from C.
gynecia. The valves were harvested on a similar timescale before being
measured, separated and refrigerated in a manner similar to those used for
material analysis. Thickness was measured using a flat digital caliper which
measured the whole shell. Samples for L. compleximanus were more limited
and we were unable to measure a standard thickness value for this group,
therefore properties normalized by thickness could only be estimated for
L. compleximanus. See the Results section for a detailed description of the
thickness estimation.
Statistical Analysis
Fig. 4. Representative force-displacement curves for all three species.
(light grey, Cyzicus gynecia; dark grey, Eulimnadia feriensis; black,
Leptestheria compleximanus). Curves were similar across all samples. The
last point of the curve is the point where the sample broke and force dropped
to zero.
To determine if using two valves from the same individual had any effect
on statistical conclusions (lack of independence), we randomly picked one
valve per individual (if two were available) from the final data set and
performed statistical tests on this subset similar to Evans and Sanson (2005).
Force and displacement data were analyzed using Igor Pro software version
6.2.2.2 (WaveMetrics, Lake Oswego, OR, USA). An analysis of variance
(ANOVA) and a Tukey HSD post hoc test of all species combinations
for each measurement and material property was performed. We also
performed a Student’s t-test to test for a difference between sexes in L.
compleximanus. Statistical analyses were performed with JMP version 10
(SAS Institute, Cary, NC, USA). All errors are reported as mean ± 1 SEM.
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ASTROP ET AL.: CHITIN IN SPINICAUDATANS
Fig. 5. Example fitting curve. Data were fit using a sigmoid function
and area under the fitted function (grey) was used to determine material
‘toughness’ (Force × Displacement). Black cross bars represent the fit and
dots are the measured data points. The point of maximum extension and
maximum load is denoted with a circle.
R ESULTS
Energy Dispersive X-ray Spectroscopy (EDAX)
We undertook energy dispersive X-ray spectrographic analyses of three spinicaudatan taxa (Fig. 6). Regions of ele-
mental signal are clearly localized to the thin section of the
spinicaudatan carapace (as indicated by color), rather than
the resin matrix. These results confirm that the spinicaudatan carapace is mainly composed of a calcium-phosphate
complex (Ca, P) that most likely represents mineralization
of the chitinous component (C, O) of the cuticle. Trace
amounts of zinc, sulfur and other elements also occurred in
very low quantities. A weak signal for silicon was also reported but this is likely an artifact of the abrasive used in
the preparation of resin-embedded specimens. Only relevant
elemental percentages are reported in Table 1. Percentage
of C and O, the chitinous component, is relatively similar
in all three species. Quantitative differences are clear, however, in the mineralized component of the carapace where
C. gynecia has a relatively high calcium and phosphate percentage, and both E. feriensis and L. compleximanus have
considerably less strongly mineralized carapaces (lower calcium/phosphate percentage). Interestingly, the lowest calcium/phosphate signal comes from E. feriensis, which has a
sodium component (the only one observed) that is equivalent
in magnitude to the calcium-phosphate signal in L. compleximanus (about 3-4%).
Materials Testing
Our data include 12 individuals where both sides of their
valves were tested and 10 individuals where only a single
valve was tested. We found no difference in the outcome of
Fig. 6. Chemical analysis using EDAX. Scanning electron micrographs and subsequent element maps (Ca and P) of the carapace cross-sections (R, resin
matrix; C, carapace section).
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Table 2. Measured and calculated mechanical properties for the punch test, adapted from Edwards et al. (2000), Sanson et al. (2001) and Evans and Sanson
(2005). F is the maximum force measured during testing, T is the thickness of the sample, A is the area of punch and D is the displacement of the punch
mounted in the moving crosshead of the test machine.
Force at break (Fb )
Work to break (Wb )
Punch strength (Sp )
Specific punch strength
Work to punch (Wp )
Specific work to punch
Calculation
Definition
Recorded during test
Area under force-displacement curve
Fb /A
Sp /T
Wb /A
Wp /T
Final load on specimen
Total work done by the punch during the test
Load normalized to area of carapace that was punched
Strength normalized to carapace thickness
Total work done normalized to area of the punch
Total work done normalized to volume of material under the punch
our results using a randomly selected subset of valves (n =
22), where each individual was only represented once, thus
we treated the complete data set (n = 34) as independent in
our analyses. The overall ANOVA models which compare
all three species, C. gynecia, L. compleximanus and E.
feriensis, in each of the measured mechanical properties
(Table 2) shows that there is a significant difference between
the three groups in all of our measured material properties
(Table 3). Specifically, when testing each species pairing
we found that L. compleximanus was statistically lower than
both C. gynecia and E. feriensis in all measured properties.
C. gynecia and E. feriensis did not differ significantly in
any of the measured properties except when standardized by
thickness of the sample, which is not surprising as the punch
test is strongly dependent on sample thickness (Choong et
al., 1992) (Table 3).
Our results are complicated by our measurement of
sample thickness. Thickness measurements were collected
from a subset of samples that were not used in testing
due to the fragility of the samples and their propensity to
desiccate rapidly. This was also done for the whole valve and
not for the specific punch site. For both C. gynecia and E.
feriensis we measured 10 valves each and took the average
to represent the thickness of their respective species groups
(1.81 ± 0.18 × 10−5 m for C. gynecia and 0.83 ± 0.07 ×
10−5 m for E. feriensis). Although sample thickness was
determined in this way, we do not believe it has a significant
impact on our results as the differences between the groups
were very large and thickness was measured the same way
across species. In contrast, we were unable to measure
fresh samples from L. compleximanus, instead measuring an
average thickness (1.80 ± 0.19 × 10−5 m) from only four.
When we compare fresh and slightly decayed samples in
C. gynecia and E. feriensis we found that decay seems to
increase thickness. Thus our report of estimated thickness
for L. compleximanus, and subsequently material properties
that take into account sample thickness, are likely lower than
their true values. Interestingly, we found no difference in
any of the measured material properties based on sex in L.
compleximanus (p > 0.5 for all).
D ISCUSSION
The ultrastructure of the spinicaudatan carapace is a multilayered cuticle composed of successive layers of the endocuticle and exocuticle retained during the partial molt
(Rieder et al., 1984; Martin, 1991; Olempska, 2004). The
accreted, multi-laminar ‘macro-structure’ of the spinicaudatan carapace is resilient to taphonomic processes such as
decay, desiccation, and compaction compared to the nonmineralized cuticle of the organism within the carapace (Krishnan, 1958). Our analysis of the spinicaudatan carapace
clearly shows differences in both chemical composition and
specific mechanical properties between the three extant families of Spinicaudata. Our results from elemental analysis
show that the proportion of mineralized cuticle is highest
in C. gynecia, containing some 16-21% (approx.) calciumphosphate, considerably more than the 5% (approx.) seen
in representatives of Leptestheriidae (Daday, 1923) (L. compleximanus), and 2% (approx.) in the representative of Limnadiidae (Burmeister, 1843) (E. feriensis). Results from the
materials testing, however, show that C. gynecia and E. feriensis are no different from each other in all mechanical
Table 3. Measured and calculated mechanical properties from the punch test for three species of Spinicaudatan. Degrees of freedom (df), F -ratios and
p-values are reported for an ANOVA which tests for differences in a particular material property across species. Pair-wise comparisons between species are
reported below the associated material property. Species with the same letter (i.e., A, B or C) do not differ from one another. Error is reported as mean ± 1
SEM.
×10−3
Force at break (Fb ,
N)
Work to break (Wb , ×10−3 J)
Punch strength (Sp , ×102 N/m2 )
Specific punch strength
(×107 N/m2 per m)
Work to punch (Wp , ×10 J/m2 )
Specific work to punch
(×106 J/m2 per m)
Cyzicus gynecia
Eulimnadia ferriensis
Leptestheria compleximanus
df
F -ratio
p-value
111.51 ± 6.25 (A)
8.49 ± 0.61 (A)
37.17 ± 2.08 (A)
20.54 ± 1.15 (A)
91.88 ± 5.92 (A)
7.39 ± 0.67 (A)
30.63 ± 1.97 (A)
36.90 ± 2.38 (B)
63.35 ± 5.77 (B)
4.71 ± 0.54 (B)
21.12 ± 1.92 (B)
11.73 ± 1.07 (C)
2
2
2
2
17.10
11.21
17.10
68.89
<0.0001∗
0.0002∗
<0.0001∗
<0.0001∗
28.30 ± 2.03 (A)
15.64 ± 1.12 (A)
24.65 ± 2.23 (A)
29.70 ± 2.69 (B)
15.71 ± 1.81 (B)
8.73 ± 1.00 (C)
2
2
11.21
42.35
0.0002∗
<0.0001∗
ASTROP ET AL.: CHITIN IN SPINICAUDATANS
properties except those that take into account the distinct
difference in valve thickness. Leptestheria compleximanus
on the other hand is significantly lower in all measured material properties (Table 3). We conclude that, despite the variation in cuticle chemistry, the degree of mineralization alone
does not predict the mechanical properties of the valves.
We hypothesized that more strongly mineralized valves
would resist force better and require more work to fracture.
This was not entirely the case (Table 3). While the highly
mineralized C. gynecia reported high values for the material
properties measured, the weakly mineralized E. feriensis
was surprisingly no different from C. gynecia. In fact, once
the thicker shells of C. gynecia were taken into account,
the specific punch strength and specific work to punch of
E. feriensis were both almost twice as high as C. gynecia.
Thus, it is possible that the low mineralization of E. feriensis
and their thin carapace actually help to resist puncture in
terms of strength and work to punch. For instance the forcedisplacement curves for C. gynecia and E. feriensis (Fig. 4)
clearly show that the thin-shelled E. feriensis failed at a
similar force (not normalized by area or thickness) as C.
gynecia but only after deforming over about a 25% greater
distance. Thence, E. feriensis are more deformable at high
loads over longer distances than the thick, mineralized shells
of C. gynecia. We believe that E. feriensis and C. gynecia
behave similarly in all material properties except those
that normalize by thickness via two different approaches;
high mineralization and likely stiffness (steep slope of
the force-displacement curve) verses higher extensibility
(longer displacement or ‘stretching’ in colloquial terms)
of the sample prior to fracture. The result of these two
approaches causes these two species with different material
compositions to have similar strength and the carapace
requires more work to punch. It is important to remember
here that when we take into account valve thickness, which
is much lower in E. feriensis than C. gynecia, E. feriensis
has significantly higher strength and work required to punch
thus valve thickness clearly also has a significant effect
on fracture resistance. Leptestheria complexiamnus on the
other hand does not reach high maximum force values
seen in C. gynecia and E. feriensis nor does it reach high
displacement values characteristic of E. feriensis (Fig. 4)
and thus performs lower than both C. gynecia and E.
feriensis in all materials tests. Interestingly this is despite
being the intermediately mineralized example of the three
and what we expect to be only slightly less thick than C.
gynecia. The direct comparison between C. gynecia and L.
compleximanus, which has similar thickness (approximated)
yet different levels of mineralization suggests that material
properties can be directly related to mineralization however,
mineralization requirements can be overcome by producing
a thinner, less mineralized, and therefore more deformable
carapace like E. feriensis.
Interestingly, we found a sodium signature in E. feriensis in our chemical analysis. To our knowledge sodium has
not been reported as a component of the spinicaudatan carapace previously, and intriguingly, this was only found in one
of the three species analyzed. The Na-Ca-P total percentage from the elemental analysis of E. feriensis was similar to the Ca-P total percentage for L. compleximanus, sug-
129
gesting the possibility of an alternate method for mineralization in Spinicaudata. This species was also more extensible and had similar material properties as the highly mineralized C. gynecia when thickness was not taken into account. When we normalized for thickness, these extremely
thin shelled species (E. feriensis) still had higher specific
punch strength and specific work to punch than the thicker
shelled L. compleximanus. Further investigation using multiple specimens from this species and contemporaneous taxa
within Limnadiidae is required to deduce whether or not this
is a particular anomaly or a feature of the species or clade. It
is interesting to consider, however, an alternative approach
to building a strong yet flexible shell in a calcium deficient
environment.
The spinicaudatan carapace readily enters the fossil record
where conditions allow and seems much more resistant to
taphonomic processes than the soft parts of branchiopod
crustaceans, as is evidenced from the thousands of instances
of carapace preservation (Astrop and Hegna, 2015), and the
relatively few (Zhang et al., 1990) instances of fossilized
spinicaudatan soft tissue. When considering the fossil record
of proposed members of these lineages there are far more
fossil taxa with proposed affiliations to the extant Cyzidae
than there are to the extant Limnadiidae (Novojilov, 1961;
Tasch, 1969), despite both families having a similar diversity in the present (Brendonck et al., 2008) (Fig. 2). To
avoid potential bias, the materials testing performed in this
analysis allows us to take a unique empirical look at how
mechanical forces associated with naturally occurring, destructive taphonomic processes (post-mortem transportation,
abrasion, predation and scavenging) may affect the entrance
of spinicaudatan remains into the fossil record. More specifically, if major differences in material properties existed between spinicaudatan taxa, it would not be unreasonable to
predict subsequent differences in the proportional representation of associated taxa in the fossil record.
We found that the material properties of the carapace of
L. compleximanus to be consistently weaker in all measurements taken. In the fossil record we see that Leptestheriidae are represented less frequently, consistent with their
weak carapace when compared to E. feriensis and C. gynecia. In contrast, the lack of significant differences between
E. feriensis and C. gynecia with respect to resistance to
the mechanical forces generated in this experiment insinuates that they are equally as likely to resist physically destructive taphonomic effects despite substantial differences
in their carapace composition. By extension, one would not
expect mechanical forces associated with destructive taphonomic processes to produce severe numerical bias in the fossil record of either Cyzicidae (Stebbing, 1910), Limnadiidae,
or related ancestral groups assuming similar carapace compositions in the past. This is not the case however, as we see
that presence of Cyzicidae is consistent throughout the fossil record whereas Limnadiidae is patchy, “appearing” and
“disappearing” throughout time (Fig. 2). In this case, perhaps the low level of mineralization compared to Cyzicidae
contributes more significantly to the very poor fossil record
for this family than the equally resistant material properties (when considering overall structure and material components). This would suggest that members of Cyzicidae are
130
JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 35, NO. 2, 2015
not inherently less resistant to destructive taphonomic effects
such as post-mortem transport and abrasion, scavenging, or
predation, rather they are perhaps less likely to promote positive effects such as permineralization that would help them
become more prevalent in the fossil record (Briggs, 2003;
Astrop and Hegna, 2015).
When considering phylogeny and ontogeny, we find that
the poorly performing (thin, weak shells) representatives of
Leptestheriidae (L. compleximanus) are more closely related
to the better performing Cyzicidae (C. gynecia) (thick,
strong shells). Although the thinner shelled representative of
Limnadiidae (E. feriensis) performs similarly to Cyzicidae,
and when normalized for thickness they perform better,
interestingly, they are more distantly related to either of
the two other groups with which they share either similar
thickness, or similar (or better) material properties. This
lack of phylogenetic bias in material performance suggests
there is another factor driving the mechanical properties
of these three species. In terms of ontogeny, the results of
this study indicate that the thinner carapace of Limnadiidae
(represented by E. feriensis) is less mineralized and is
more extensible, conferring a different dimension of material
strength without the need for costly investment in mineral
deposition. This could be due to ontogenetic differences,
in that limnadiids are often characterized by their quick
growth to sexual maturity (Brown et al., 2014). Cyziciidae
on the other hand molt at more infrequent intervals for
longer periods of time and often inhabit less ephemeral water
bodies. The longer lived, slower growing cyziciids do not
exhibit the ‘rush’ to sexual maturity seen in limnadiids.
Further investigation on evolutionary factors driving the
material properties of these species, such as environment,
behavior and time to sexual maturity are necessary to fully
understand why these differences arise.
The less mineralized, extensible limnadiid carapace and
the thicker, more mineralized carapace of cyzicids are both
likely to resist the physical stresses associated with postmortem transport of biological remains in aqueous environments, implying that pre-depositional processes might not
play a large role in producing taxonomic bias in the fossil
record of either group. However, the relative weakness of the
leptestheriid carapace could explain their near-absence from
the fossil record. Further integrated studies testing different
aspects of how this material behaves are required for both a
better understanding of the spinicaudatan carapace as a biological structure and how such a structure behaves when subjected to the destructive forces of other taphonomic process
than those mentioned here, specifically post-depositional effects such as permineralization and dissolution. Regardless,
this study provides a unique approach in understanding the
forces involved in organismal remains entering the fossil
record and how different parameters of the ‘taphonomic window’ may vary for different taxa as a result of differing biogenic composition. Using modern analogues and heuristic
integrative approaches to understand how organic remains
of different taxa may enter the fossil record is an extremely
useful and an underutilized approach that has the potential
to open up areas of palaeobiological and biological material
science that were previously unreachable.
ACKNOWLEDGEMENTS
We would like to thank Thomas Quick, Ali Dhinojwala, Stephen Weeks
and Lisa Park for the helpful advice and technical expertise. This study was
funded in part by an NSF Doctoral Dissertation Improvement Grant (DDIG)
awarded to TIA.
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R ECEIVED: 25 November 2014.
ACCEPTED: 10 February 2015.