Late embryos and bony skull development in Bothropoides jararaca (Serpentes, Viperidae)

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Zoology
116 (2013) 36–
63
Contents
lists
available
at
SciVerse
ScienceDirect
Zoology
jour
nal
homep
age
:
w
ww.e
l
sev
ier.c
om/locate/zool
Late
embryos
and
bony
skull
development
in
Bothropoides
jararaca
(Serpentes,
Viperidae)
Katja
M.
Polachowskia,
Ingmar
Werneburga,b,c,
aPaläontologisches
Institut
und
Museum
der
Universität
Zürich,
Karl-Schmid-Strasse
4,
CH-8006
Zürich,
Switzerland
bFachbereich
Geowissenschaften
der
Eberhard-Karls-Universität,
Hölderlinstraße
12,
D-72074
Tübingen,
Germany
cLaboratory
for
Evolutionary
Morphology,
RIKEN
Center
for
Developmental
Biology,
2-2-3
Minatojima-minami,
Chuo-ku,
Kobe,
Hyogo
650-0047,
Japan
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
23
March
2012
Received
in
revised
form
17
July
2012
Accepted
18
July
2012
Available online 22 January 2013
Keywords:
3D
morphometrics
Developmental
anatomy
SES-staging
Skeletogenesis
a
b
s
t
r
a
c
t
In
recent
years,
developmental
anatomy
received
increasing
interest
as
a
potential
new
source
for
phylo-
genetic
research.
For
skeletal
development,
studies
mainly
rely
on
the
first
appearance
of
ossification
centers.
However,
informative
events
occur
during
the
whole
course
of
skeletogenesis;
interactions
between
external
and
internal
development
occur
and
morphometric
changes
take
place
– all
of
which
present
potential
sources
for
phylogenetic
analyses.
Therefore,
the
Standard
Event
System
(SES)
was
used
to
traceably
describe
the
external
development
of
the
snake
species
Bothropoides
jararaca
and
exter-
nal
measurements
were
analyzed.
We
then
applied
micro-computed
tomography
(CT),
clearing
and
double-staining,
and
2D
and
3D
morphometric
methods
to
describe,
illustrate,
and
analyze
the
develop-
ment
of
the
head
in
great
detail.
We
found
a
3D
flattening
of
the
skull
during
ontogeny,
a
pattern
that
is
not
reflected
in
external
development.
This
may
be
explained
by
a
different
relationship
of
skeleto-
genesis
and
external
characters
to
the
developing
jaw
musculature
or
simply
by
the
different
type
of
data.
Clearing
and
double-staining
and
CT-scanning
revealed
a
broadly
similar
sequence
in
the
onset
of
ossification.
Minute
differences
may
be
due
to
the
treatment
of
embryos.
Bones
of
the
dermatocranium
are
among
the
first
to
ossify
and
the
development
of
the
calcified
endolymph
may
reflect
its
function
as
a
calcium
source
during
development.
The
value
of
phylogenetic
observations
using
the
sequence
of
first
ossifications
is
critically
discussed.
The
related
heterochronic
changes
are
interpreted
to
contribute
at
least
to
the
very
first
phase
of
divagating
skull
formation
among
taxa.
© 2012 Elsevier GmbH. All rights reserved.
1.
Introduction
Within
the
last
20
years,
developmental
comparative
anatomy
experienced
a
renaissance
among
evolutionary
development
(evo-
devo)
research
programs
(e.g.,
Minelli,
2003;
Olsson
et
al.,
2006;
Sánchez-Villagra,
2010).
Different
aspects,
such
as
novel
methodol-
ogy
(CT,
morphometry)
and
developing
conceptual
backgrounds
(phylogeny,
plasticity),
leverage
traditional
morphology
into
cur-
rent
scientific
discourse.
One
aspect
related
to
this
is
the
establishment
of
non-model
organisms
for
molecular
studies
(Tzika
and
Milinkovitch,
2008;
Werneburg
et
al.,
2012).
As
a
prerequi-
site,
however,
the
detailed
anatomy
has
to
be
known.
Thus,
the
developmental
variance
of
external
anatomy
and
ossification
pat-
terns
among
vertebrates
has
received
much
attention
(e.g.,
Maxwell
and
Harrison,
2008;
Werneburg
and
Sánchez-Villagra,
2009,
2011;
Germain
and
Laurin,
2009;
Werneburg
et
al.,
2009;
Hautier
et
al.,
2012).
Corresponding
author
at:
Fachbereich
Geowissenschaften
der
Eberhard-Karls-
Universität,
Hölderlinstraße
12,
D-72074
Tübingen,
Germany.
E-mail
address:
i.werneburg@gmail.com (I.
Werneburg).
The
scaled
reptiles
(Squamata)
consist
of
over
9000
extant
species
(Uetz,
2012).
They
are
very
diverse
and
include
snakes,
amphisbaenians,
and
lizards
(sensu
Evans
and
Jones,
2010).
Numerous
synapomorphies
support
the
monophyly
of
Squamata,
including
diverse
features
of
the
skull
(Mickoleit,
2004).
However,
the
phylogenetic
relationships
among
different
squamate
taxa
are
not
yet
fully
resolved
and
in
this
context,
the
position
of
snakes
is
one
of
the
most
discussed
issues.
Potential
sister
taxa
are
the
mon-
itor
lizards
(e.g.,
McDowell
and
Bogert,
1954;
Estes
et
al.,
1988;
Forstner
et
al.,
1995;
Lee,
1998,
2009)
or
diverse
limbless
groups
(e.g.,
Conrad,
2008).
For
phylogenetic
studies,
however,
ontoge-
netic
data
are
only
rarely
available
and
only
described
for
a
few
squamate
species
in
detail.
To
increase
this
knowledge,
we
here
describe
the
skeletogenesis
of
the
skull
and
the
external
morphol-
ogy
in
one
snake
species,
the
Jararaca
snake
Bothropoides
jararaca
(Viperidae,
Serpentes).
B.
jararaca
is
a
venomous,
semi-arboreal
pit
viper
(Crotalinae)
deeply
nested
within
Viperidae
(Carrasco
et
al.,
2012).
It
occurs
in
south-eastern
South
America
where
it
is
found
in
forests
but
also
in
disturbed,
open
habitats
and
even
in
cities.
The
snout–vent
length
(SVL)
of
mature
adults
ranges
from
about
65
cm
to
100
cm.
B.
jararaca
is
an
ovoviviparous
species
with
clutch
size
extremes
0944-2006/$
see
front
matter ©
2012 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.zool.2012.07.003
K.M.
Polachowski,
I.
Werneburg
/
Zoology
116 (2013) 36–
63 37
Table
1
The
various
specimens
of
Bothropoides
jararaca
and
the
techniques
used
to
study
them.
Arranged
according
to
external
morphology
and
total
length.
Collection
No.
Abbreviation
External
measurements
SES
staging
SES
with
photographs
(Appendix
1)
Clearing
and
double-staining
CT-scan
Morphometric
analysis
PIMUZ
lab#2010.IW020 B0 X
X
X
(X)
PIMUZ
lab#2010.IW029 B1
X
X
X
PIMUZ
lab#2010.IW001
B2
X
X
X
X
X
PIMUZ
lab#2010.IW003
B3
X
X
X
PIMUZ
lab#2010.IW004
B4
X
X
X
X
PIMUZ
lab#2010.IW059
B5
X
X
X
X
PIMUZ
lab#2010.IW065 B6 X X X X
PIMUZ
lab#2010.IW064 B7 X
X
X
X
PIMUZ
lab#2010.IW062 B8
X
X
(X)
PIMUZ
lab#2010.IW068
B9
X
X
X
X
PIMUZ
lab#2010.IW109
B10
X
X
X
PIMUZ
lab#2010.IW096
B11
X
X
X
PIMUZ
lab#2010.IW099 B12 X X X X X
PIMUZ
lab#2010.IW101
B13
X
X
X
PIMUZ
lab#2010.IW006 B14
X
X
X
X
X
X
PIMUZ
lab#2010.IW083
B15
X
X
X
X
PIMUZ
lab#2010.IW113 B16 X X X
PIMUZ
lab#2010.IW010
B17
X
X
X
PIMUZ
lab#2010.IW008 B18
X
X
X
X
(X)
PIMUZ
lab#2010.IW145
B19
X
X
X
X
X
X
PIMUZ
lab#2010.IW135
B20
X
X
X
PIMUZ
lab#2010.IW142
B21
X
X
X
PIMUZ
lab#2010.IW015
B22
X
X
X
PIMUZ
lab#2010.IW123 B23 X X X
PIMUZ
lab#2010.IW016
B24
X
X
X
(X)
X
PIMUZ
lab#2010.IW151 B25
X
X
X
X
X
PIMUZ
lab#2010.IW155
B26
X
X
X
X
X
X
PIMUZ
lab#2010.IW017
B27
X
X
X
X
Remaining
specimens
(see
Table
2)X
X
=
corresponding
technique
was
successfully
applied
in
this
specimen.
(X)
=
data
only
partly
usable
due
to
destroyed
or
inadequate
quality
of
the
specimen
or
due
to
problems
during
CT-scanning.
from
2
to
34.
Average
neonate
SVL
is
24.5
cm
in
males
and
25.3
cm
in
females,
and
weight
is
9.31
g
in
males
and
10.61
g
in
females
(Sazima,
1992).
B.
jararaca
is
a
diet
generalist
and
feeds
mainly
on
small
mammals
and
birds,
but
also
on
lizards,
frogs,
and
centipedes
(Martins
et
al.,
2002).
Descriptions
of
the
adult
skull
of
different
Bothropoides
species
are
available
(e.g.,
Radovanovi´
c,
1938;
Moro,
1996;
Zanella,
1999).
Additionally,
there
exist
some
descriptive
studies
of
the
embryonic
development
of
different
pit
vipers
(Savitzky,
1992;
Hofstadler-
Deiques,
1997).
Developmental
aspects
of
the
primordial
cranium
in
B.
jararaca
were
studied
by
Hofstadler-Deiques
(1997,
2002,
2004)
and
Hofstadler-Deiques
et
al.
(2005)
using
serial
sections.
In
those
studies,
however,
the
description
of
the
ossification
and
of
the
external
morphology
is
rather
short
and
the
resolution
of
the
ossification
sequence
is
rather
low
to
be
sufficient
for
future
phylogenetic
analyses.
Our
new
data
set
will
expand
those
earlier
studies.
The
external
morphology
of
the
embryos
was
examined
using
the
Standard
Event
System
(Werneburg,
2009;
Tables
1–4,
Appendix
1).
Additionally,
different
measurements
of
body
and
head
parameters
were
taken
in
136
embryos.
To
study
the
skele-
togenesis
of
the
skull,
micro-computed
tomography
(CT)
scans
(14
specimens)
and
clearing
and
double-staining
of
skeletons
were
performed
(28
specimens).
To
calculate
shape
changes
dur-
ing
development,
3D
morphometric
analyses
using
a
multivariate
approach
were
performed
for
6
specimens.
2.
Materials
and
methods
2.1.
Specimens
We
had
access
to
136
embryonic
specimens
of
B.
jararaca
(Table
1)
provided
by
Prof.
Dr.
Wolfgang
Maier,
Universität
Tübin-
gen,
Germany,
now
housed
in
Paläontologisches
Institut
und
Museum
der
Universität
Zürich
(PIMUZ;
laboratory
of
Prof.
Dr.
Marcelo
R.
Sánchez-Villagra).
Originally,
the
collection
consisted
of
11
tubes,
each
of
which
contained
3–24
embryos
of
approximately
the
same
size.
Besides
these,
there
were
15
additional
embryos
of
different
sizes
available.
The
embryos
were
preserved
in
a
70%
ethanol
(EtOH)
solution.
A
few
tubes
were
labelled
with
São
Paulo,
or
more
specifically
Espírito
Santo,
which
apparently
represents
the
collection
sites
in
Brazil.
Most
tubes
were
labelled
with
dates
ranging
from
08.12.[19]92
to
04.03.[19]93,
presumably
the
collec-
tion
dates.
More
information
about
the
embryos
was
not
available.
However,
we
assume
that
the
specimens
were
collected
by
Clarice
Hofstadler-Deiques,
who
did
her
PhD
thesis
in
the
laboratory
of
Dr.
Maier,
and
the
embryos
used
for
that
study
were
dissected
out
of
the
mother
(Hofstadler-Deiques,
1997).
The
specimens
used
in
our
study
were
treated
with
dif-
ferent
morphological
methods
and
except
for
external
mea-
surements
the
specimens
were
progressively
numbered
B0–B27
(Table
1).
2.2.
General
measurements
In
all
136
embryos,
total
length
(TL)
was
measured
as
the
distance
between
snout-tip
and
tail-tip
along
the
dorsal
line
of
the
body,
using
a
ruler
(Table
2).
Because
the
pos-
ture
of
the
embryos
was
curved,
they
were
turned
around
along
the
ruler.
The
measurement
had
a
variance
of
±2
mm.
This
variance
was
taken
into
account
when
interpreting
the
results.
A
more
accurate
measurement
was
not
attempted,
so
as
not
to
risk
damaging
the
embryos
whilst
measur-
ing.
The
weight
of
all
embryos
was
measured
with
a
precision
balance
(Kern
EW
220-3NM;
Kern
und
Sohn
GmbH,
Balingen,
Germany)
(Table
2).
For
weighing,
the
embryos
were
taken
out
of
the
70%
EtOH
solution
and
placed
on
a
tissue.
They
were
38 K.M.
Polachowski,
I.
Werneburg
/
Zoology
116 (2013) 36–
63
Table
2
Detailed
values
of
the
different
measurements
in
all
analysed
specimens
of
Bothropoides
jararaca.
Specimen
Total
length
(mm)
Weight
(g)
Head
length
(mm)
Head
breadth
(mm)
Head
height
(mm)
Eye-to-
snout
distance
(mm)
Distance
between
the
eyes
(mm)
Eye
diameter
(mm)
Upper
jaw
(mm)
Lower
jaw
(mm)
PIMUZ
lab#2010.IW001 7.6
0.34
7.62
4.75
5.46
3.26
5.38
1.95
4.52
3.94
PIMUZ
lab#2010.IW002
7.5
0.39
7.73
4.06
5.9
3.97
4.45
2.65
4.72
4.52
PIMUZ
lab#2010.IW003
7.9
0.462
8.12
4.99
5.83
3.74
5.75
2.38
5.62
5.38
PIMUZ
lab#2010.IW004
8.2
0.42
8.3
4.83
5.75
3.4
5.9
2.55
5.33
4.86
PIMUZ
lab#2010.IW005 14.4
1.16
10.99
5.6
6.1
4.37
5.61
2.46
7.72
7.29
PIMUZ
lab#2010.IW006 14.7
1.33
11.51
6.49
6.8
4.86
7.23
2.56
7.9
7.8
PIMUZ
lab#2010.IW007 17.3
2.31
13.25
7.65
6.94
4.9
6.79
2.93
9.55
9.37
PIMUZ
lab#2010.IW008
19.1
2.82
13.55
7.17
6.04
3.85
6.87
2.3
8.68
8.58
PIMUZ
lab#2010.IW009
18.5
1.951
12.67
6.9
5.12
4.88
6.7
2.79
9.02
8.54
PIMUZ
lab#2010.IW010
18.6
2.85
13.16
8.85
6.79
4.43
7.92
2.55
8.82
8.76
PIMUZ
lab#2010.IW011
18.9
2.53
13.34
6.62
6.22
4.66
6.26
2.81
7.97
7.89
PIMUZ
lab#2010.IW012
19.3
3.01
13.51
8.64
6.29
5.35
6.81
2.48
8.93
8.73
PIMUZ
lab#2010.IW013 22.6
4.13
14.96
8.74
6.9
5.41
8.22
2.82
11.23
11.2
PIMUZ
lab#2010.IW014
22.6
4.59
14.34
8.46
8.03
4.85
8.05
2.63
10.9
11.11
PIMUZ
lab#2010.IW015 23.9
4.72
15.64
9.51
6.67
5.14
8.13
2.51
11.51
11.47
PIMUZ
lab#2010.IW016
24.7
5.56
14.61
9.9
7.53
4.3
8.61
2.3
9.64
9.64
PIMUZ
lab#2010.IW017 31.2
14.54
18.88
11.87
8.48
6.08
10.06
2.86
14.57
14.47
PIMUZ
lab#2010.IW020
4.6
0.072
4.29
2.6
3.4
1.67
3.03
1.93
1.99
1.44
PIMUZ
lab#2010.IW022
4.5
0.086
4.68
2.58
3.87
1.58
3.33
1.86
1.96
1.84
PIMUZ
lab#2010.IW024
5.1
0.078
4.48
2.6
3.86
2
3
1.82
2.55
1.94
PIMUZ
lab#2010.IW025
5.1
0.077
4.79
2.42
3.86
1.5
2.99
1.89
2.11
1.66
PIMUZ
lab#2010.IW029 4.7
0.079
4.52
2.48
3.8
1.5
3.2
1.88
2.6
1.92
PIMUZ
lab#2010.IW030
4.7
0.085
4.568
2.78
3.9
1.93
3.5
2
2.64
1.93
PIMUZ
lab#2010.IW031 5.1
0.083
4.43
2.62
3.75
1.93
2.72
1.84
2.15
1.69
PIMUZ
lab#2010.IW033
4.6
0.071
4.65
2.56
4.08
1.93
3.06
1.9
2.46
1.92
PIMUZ
lab#2010.IW034
5.1
0.099
4.46
2.82
4.16
1.2
3.81
1.6
2.12
1.77
PIMUZ
lab#2010.IW036 5.4
0.084
4.58
3.19
4.5
1.47
3.79
1.78
2.47
1.79
PIMUZ
lab#2010.IW038
5
0.117
5.16
3.3
4.69
1.98
4.09
1.9
2.28
1.75
PIMUZ
lab#2010.IW039 5.6
0.096
4.67
3.45
4.28
1.49
4
1.8
2.51
1.78
PIMUZ
lab#2010.IW040
4.8
0.087
4.34
2.95
4.13
1.8
3.5
1.7
2.34
1.82
PIMUZ
lab#2010.IW041 5.2
0.1
5.11
3.03
4.5
1.83
4.12
1.87
2.57
1.88
PIMUZ
lab#2010.IW042
5.8
0.103
4.55
3.44
4.51
1.88
3.9
1.76
2.6
1.73
PIMUZ
lab#2010.IW043
6
0.175
5.12
3.39
4.7
2.72
3.88
2.05
3.22
2.85
PIMUZ
lab#2010.IW046 6.5
0.188
5.74
2.99
4.97
2.75
3.68
2.13
2.97
2.33
PIMUZ
lab#2010.IW047
6.4
0.204
5.99
3.05
5.47
2.47
3.83
2.04
3.05
2.57
PIMUZ
lab#2010.IW048 6.4
0.192
5.71
3.27
4.7
2.63
4.18
2.02
2.74
2.31
PIMUZ
lab#2010.IW050
6.3
0.195
5.89
2.91
4.88
2.74
3.96
2.09
2.86
2.61
PIMUZ
lab#2010.IW051
6.3
0.193
5.98
3.42
5.07
2.79
4.36
2.33
3.47
2.88
PIMUZ
lab#2010.IW052
6.2
0.182
5.53
3.4
4.46
2.1
4.27
1.87
2.73
2.45
PIMUZ
lab#2010.IW053
7.2
0.215
6.02
3.21
4.95
2.98
4.4
1.84
3.02
2.61
PIMUZ
lab#2010.IW054 6.5
0.19
6.06
3.27
4.93
2.91
4.34
2.15
3.57
3.05
PIMUZ
lab#2010.IW055
7
0.182
5.8
3.1
4.72
2.81
3.97
1.9
2.7
2.24
PIMUZ
lab#2010.IW056 7
0.171
5.239
3.36
4.83
1.73
3.56
1.76
2.57
2.14
PIMUZ
lab#2010.IW059
9
0.5
9.3
5.15
6.35
3.64
6.2
2.7
6.19
5.87
PIMUZ
lab#2010.IW060
10.5
0.6
9.23
5.33
6.02
3.74
6.45
2.7
5.95
5.76
PIMUZ
lab#2010.IW061
11.3
0.53
10
5.61
6.25
3.64
6.72
2.74
6.39
6.23
PIMUZ
lab#2010.IW062
10.3
0.63
9.74
5.51
5.78
4.29
6.77
2.59
6.71
6.37
PIMUZ
lab#2010.IW063
10.8
0.67
10.26
5.03
6.16
4.15
6.2
2.89
6.8
6.73
PIMUZ
lab#2010.IW064
10.1
0.68
9.65
5.66
5.92
4.81
6.18
2.63
6.11
5.64
PIMUZ
lab#2010.IW065
9.6
0.66
9.43
5.43
6.22
3.82
6.45
2.58
6.18
5.47
PIMUZ
lab#2010.IW068
10.9
0.69
9.44
5.65
6.35
4.06
6.14
2.71
6.7
6.72
PIMUZ
lab#2010.IW069
13.5
0.87
11.24
4.87
6.44
4.74
4.71
2.84
7.88
7.68
PIMUZ
lab#2010.IW070
16.1
1.38
10.23
6.45
4.85
3.27
6.46
1.83
7.06
6.92
PIMUZ
lab#2010.IW071
15.2
1.29
11.98
6.25
6.11
4.58
5.5
2.23
8.61
8.3
PIMUZ
lab#2010.IW072
15.7
1.36
12.46
6.22
5.68
5.01
5.66
2.7
8.43
8.17
PIMUZ
lab#2010.IW073
14.9
1.2
12.5
6.06
5.29
5.04
6.25
2.71
9.25
9.25
PIMUZ
lab#2010.IW074
16.2
1.34
12.48
6.81
5.28
4.92
6.37
2.63
8.45
8.64
PIMUZ
lab#2010.IW075
16.4
1.37
12.46
6.27
5.84
5.02
6
2.74
9.36
9.32
PIMUZ
lab#2010.IW076
16.7
1.47
11.61
6.82
5.47
4.09
6.57
2.38
9.02
8.72
PIMUZ
lab#2010.IW077
15.9
1.24
11.65
6.01
5.89
4.94
6.35
2.67
8.9
8.9
PIMUZ
lab#2010.IW078
16.1
1.37
12
6.07
5.73
4.48
6
2.63
8.71
8.53
PIMUZ
lab#2010.IW079
16.6
1.38
12.92
6.57
5.69
5.15
6.2
2.49
9.6
9.43
PIMUZ
lab#2010.IW080 16.1
1.34
12.24
6.21
5.08
4.55
6.06
2.64
8.86
8.63
PIMUZ
lab#2010.IW081
16.2
1.32
12.17
7.04
5.34
4.62
6.74
2.3
8.68
8.72
PIMUZ
lab#2010.IW082
16.1
1.53
12.17
6.88
6.14
4.81
6.35
2.6
9.08
9.12
PIMUZ
lab#2010.IW083
15.2
1.27
12.54
6.66
5.36
4.68
6.15
2.35
9.03
8.98
PIMUZ
lab#2010.IW084
15.2
1.41
12.56
6.73
6.79
5.06
6.41
2.32
9.12
8.17
PIMUZ
lab#2010.IW085 16 1.43
12.34
7.25
5.56
5.12
6.63
2.7
9.71
9.65
PIMUZ
lab#2010.IW086 16.5
1.34
13
6.39
5.46
4.72
5.88
2.7
9.64
9.69
PIMUZ
lab#2010.IW087
16.1
1.42
11.64
6.92
4.86
4.45
6.46
2.52
9.01
8.65
PIMUZ
lab#2010.IW088 16.3
1.48
12.12
6.6
6.13
4.18
5.78
2.1
8.86
7.61
PIMUZ
lab#2010.IW089
16.3
1.35
12.77
6.41
6.29
5.19
6.03
2.62
9.63
9.58
PIMUZ
lab#2010.IW090 16.1
1.39
11.58
6.45
5.82
4.39
6.21
2.48
8.1
7.89
K.M.
Polachowski,
I.
Werneburg
/
Zoology
116 (2013) 36–
63 39
Table
2
(Continued)
Specimen
Total
length
(mm)
Weight
(g)
Head
length
(mm)
Head
breadth
(mm)
Head
height
(mm)
Eye-to-
snout
distance
(mm)
Distance
between
the
eyes
(mm)
Eye
diameter
(mm)
Upper
jaw
(mm)
Lower
jaw
(mm)
PIMUZ
lab#2010.IW091
15.3
1.3
11.88
6.29
6.2
4.88
5.1
2.19
8.43
7.71
PIMUZ
lab#2010.IW092 12.8
1.01
10.38
6.12
5.95
3.65
6.94
2.57
7.08
6.86
PIMUZ
lab#2010.IW093
13.1
0.96
10.96
5.84
5.97
4.33
6.71
2.62
7.37
7.44
PIMUZ
lab#2010.IW094
13.1
0.94
10.1
6.14
6.11
4.03
6.73
2.63
7.22
7.16
PIMUZ
lab#2010.IW095
11.9
1.06
10.23
6.03
5.95
3.76
6.9
2.74
7.07
7.04
PIMUZ
lab#2010.IW096 12.5
1.03
10.92
6.06
6.23
4.72
6.96
3
7.65
7.46
PIMUZ
lab#2010.IW097 13.3
1.12
10.91
6.27
6
4.67
6.96
2.87
7.79
7.65
PIMUZ
lab#2010.IW098 12.5
0.98
10.72
6.09
6.38
4.51
6.88
2.8
7.41
7.11
PIMUZ
lab#2010.IW099
12.9
0.99
9.65
5.89
6.14
3.36
6.91
2.4
7.02
6.58
PIMUZ
lab#2010.IW100
12.6
1.01
10.6
5.54
6
4.32
6.72
2.71
7.44
7.5
PIMUZ
lab#2010.IW101
13.2
1.04
10.17
6
6.32
3.75
6.9
2.46
6.94
6.53
PIMUZ
lab#2010.IW102 13.1
0.95
10.18
6.2
6.23
4.01
6.94
2.61
6.95
6.58
PIMUZ
lab#2010.IW103
12.6
1.03
10.72
6.38
6.04
4.29
7.56
2.83
7.31
7.23
PIMUZ
lab#2010.IW104 12.6
1.02
10.89
6.07
6.13
4.59
6.77
2.79
7.48
7.38
PIMUZ
lab#2010.IW105
12.5
0.91
9.92
5.51
5.98
3.47
6.4
2.37
6.72
6.17
PIMUZ
lab#2010.IW106 12.7
1.01
10.5
5.81
6.39
3.99
6.82
2.59
7.56
7.76
PIMUZ
lab#2010.IW107
12.8
0.95
10.26
5.74
5.4
3.71
6.49
2.48
6.93
6.63
PIMUZ
lab#2010.IW109 12.3
0.87
10.01
5.26
5.7
3.82
5.81
2.5
6.69
6.6
PIMUZ
lab#2010.IW110
17.1
2.26
11.64
7.61
7.24
4.3
7.76
2.47
7.86
7.86
PIMUZ
lab#2010.IW111
14.6
1.61
11.68
8.09
5.81
3.83
7.26
2.08
7.57
7.44
PIMUZ
lab#2010.IW113
16.8
2.15
13.05
8.81
5.75
4.84
7.62
2.54
9.49
9.32
PIMUZ
lab#2010.IW114
17.1
2.17
12.74
7.82
5.97
4.82
7.79
2.7
9.21
8.73
PIMUZ
lab#2010.IW115 17 2.24
12.68
7.43
6.03
4.45
7.4
2.43
8.51
8.27
PIMUZ
lab#2010.IW116
16.1
1.87
12.79
7.03
6.36
4.84
7.21
2.83
9.44
9.13
PIMUZ
lab#2010.IW118 16.9
1.71
12.86
7.22
6.21
4.97
7
2.37
8.97
8.84
PIMUZ
lab#2010.IW119
22.7
3.33
14.69
8.11
6.59
5.8
7.29
2.94
11.24
11.03
PIMUZ
lab#2010.IW120
23.6
3.34
15.38
8.44
6.51
5.73
7.25
2.84
11.09
10.65
PIMUZ
lab#2010.IW121 24.8
3.48
14.8
7.13
6.93
5.14
6.43
2.68
10.67
10.58
PIMUZ
lab#2010.IW122
23.6
3.32
14.34
8.7
5.82
4.89
7.81
2.96
10.51
10.18
PIMUZ
lab#2010.IW123 24.5
3.28
13.99
7.85
6.3
5.28
7.02
2.72
10.78
10.8
PIMUZ
lab#2010.IW124
23.1
3.16
13.8
7.48
6.36
5.03
7
2.81
10.65
10.44
PIMUZ
lab#2010.IW125 23.5
3.15
14.48
8.9
5.28
5.24
8.36
2.75
10.49
10.54
PIMUZ
lab#2010.IW126
22.8
3.51
14.3
7.94
6.6
4.9
6.99
3.16
11.1
10.98
PIMUZ
lab#2010.IW127
21.4
3.42
15.89
8.83
7.25
5.57
8.01
2.9
11.24
11.44
PIMUZ
lab#2010.IW128 22.9
3.51
13.89
8.02
6.45
4.54
8.02
2.84
10.36
10.48
PIMUZ
lab#2010.IW129
22.3
3.72
15.53
9.56
6.58
5.95
9.28
3.2
11.7
11.72
PIMUZ
lab#2010.IW130 22.5
3.4
14.35
8.22
5.76
5.41
7.75
3.04
10.6
10.88
PIMUZ
lab#2010.IW131
22.9
3.89
14.76
9.15
6.74
4.91
8.66
2.93
10.82
10.73
PIMUZ
lab#2010.IW132
22.6
3.49
14.08
8.86
6.25
5.14
8.13
2.72
10.79
10.87
PIMUZ
lab#2010.IW133
21.6
3.13
13.64
7.82
5.93
4.44
8.17
2.76
9.8
9.69
PIMUZ
lab#2010.IW134
22.3
3.1
14.85
7.55
7
5.17
7.43
2.74
10.55
10.28
PIMUZ
lab#2010.IW135 22.8
3.5
15.32
8.79
6.26
5.99
8.44
2.98
12.05
11.92
PIMUZ
lab#2010.IW136
28.6
8.09
14.97
9.93
6.82
4.82
8.85
2.78
10.95
10.67
PIMUZ
lab#2010.IW137 21.9
3.04
12.9
9.02
5.39
4.51
8.31
2.79
9.5
9.47
PIMUZ
lab#2010.IW138
21.6
3.03
14.35
8.1
6.93
5.39
6.7
2.67
10.64
10.54
PIMUZ
lab#2010.IW139
22.4
3.89
15.39
8.97
6.42
5.89
7.83
3.08
11.51
11.54
PIMUZ
lab#2010.IW140
23.5
3.61
14.74
8.38
6.56
4.78
8.47
3.05
10.84
10.91
PIMUZ
lab#2010.IW141
22.6
3.2
14.14
7.39
6.76
5.8
6.93
2.9
10.38
10.3
PIMUZ
lab#2010.IW142
23
3.65
15.08
8.55
6.49
4.97
8.03
2.67
10.6
10.45
PIMUZ
lab#2010.IW143
21.1
3.14
14.54
8.83
6.45
5.65
7.55
3.21
10.84
10.82
PIMUZ
lab#2010.IW144
21.2
3.01
13.67
8.48
5.63
4.41
7.72
3.09
9.85
9.79
PIMUZ
lab#2010.IW145
21.1
3.01
14.18
8.55
6.24
6.08
7.47
3.26
10.96
10.86
PIMUZ
lab#2010.IW146
22.8
3.33
13.48
8
6.58
4.55
8.13
3.08
9.03
9.11
PIMUZ
lab#2010.IW147
21.9
3.21
14.34
8.28
5.6
5.34
8.34
2.95
10.51
10.91
PIMUZ
lab#2010.IW148
22.2
3.45
15.03
8.66
6.58
6.2
8.34
2.98
11.1
11.29
PIMUZ
lab#2010.IW149
22.5
3.34
14.73
8.52
7.22
5.1
7.73
2.71
10.04
9.74
PIMUZ
lab#2010.IW150
20.5
2.98
15.03
8.51
6.04
5.79
7.52
3.05
11.29
11.32
PIMUZ
lab#2010.IW151
27.2
7.97
17.26
10.67
6.77
6.15
9.02
2.98
13.17
13.12
PIMUZ
lab#2010.IW152
28.2
9.63
17.87
10.66
6.93
5.46
9.03
2.83
12.51
12.64
PIMUZ
lab#2010.IW153
29.5
9.47
18.77
10.6
6.83
5.98
9.36
3.28
12.83
12.78
PIMUZ
lab#2010.IW154
28.8
8.92
17.86
9.54
6.71
5.13
8.89
2.67
11.36
11.28
PIMUZ
lab#2010.IW155
29.7
9.44
17.68
10.2
6.84
6.06
8.83
2.37
12.34
12.29
PIMUZ
lab#2010.IW156
28.9
8.65
17.93
10.34
6.69
6.21
8.44
3.01
13.03
13.17
superficially
dried
with
the
tissue
until
they
were
no
longer
glossy
and
were
immediately
placed
on
the
balance.
As
EtOH
constantly
evaporated
out
of
the
embryos,
there
was
no
constant
value.
To
enable
comparability,
the
first
value
was
always
recorded.
It
was
measured
to
the
nearest
0.01
g.
It
was
not
possible
to
measure
SVL
due
to
the
fragile
condition
of
many
of
the
preserved
specimens.
2.3.
Head
measurements
Different
external
head
measurements
were
taken
(Fig.
1B
and
C
and
Table
2).
As
the
specimens
were
too
small
to
take
accurate
measurements
with
a
calliper,
their
heads
were
first
photographed
through
a
stereo
microscope
(Leica
M165C;
Leica
Microsystems,
Wetzlar,
Germany)
mounted
to
a
digital
camera
(Leica
DFC420C)
40 K.M.
Polachowski,
I.
Werneburg
/
Zoology
116 (2013) 36–
63
Table
3
Regression
coefficients
(R2)
resulting
from
different
regression
models
for
all
measurement
data
sets.
The
highest
regression
coefficient
is
highlighted
in
bold
for
each
measurement
separately.
The
linear
regression
of
total
length
(mm)
correlates
best
with
the
actual
development
of
Bothropoides
jararaca.
Regression
models Total
length
(mm)
Weight
(g)
Head
length
(mm)
Head
breadth
(mm)
Head
height
(mm)
Eye-to-
snout
distance
(mm)
Distance
between
the
eyes
(mm)
Eye
diameter
(mm)
Upper
jaw
(mm)
Lower
jaw
(mm)
Exponential
0.9252
0.9152
0.8524
0.8988
0.8678
0.7863
0.8634
0.9050
0.8164
0.7954
Linear
0.9784
0.6706
0.9400
0.9651
0.9092
0.9090
0.9363
0.9409
0.9395
0.9398
Logarithmic
0.7750
0.3940
0.8555
0.825
0.9060
0.9223
0.8863
0.8939
0.8599
0.8585
Polynomic 0.9784
0.8056
0.9607
0.9679
0.9344
0.9598
0.9569
0.9697
0.9661
0.9669
Potential 0.9005
0.8926
0.9102
0.9129
0.9549
0.9619
0.9452
0.9285
0.9125
0.9082
Table
4
Regressions
of
various
measurements
against
total
length
in
Bothropoides
jararaca,
with
linear
regression
lines.
For
all
measurements
N
=
136
and
p
<
0.0001.
Log
weight
Head
height
Eye
diameter
Head
length
Head
breadth
Eye-to-
snout
distance
Distance
between
the
eyes
Lower
jaw
Upper
jaw
Formula
of
linear
regression
(y=)
0.08x
1.18
0.1x
+
4.3
0.04x
+
1.89
0.052x
+
3.19
0.31x
+
1.71
0.16x
+
1.79
0.21x
+
3.15
0.45x
+
0.6
0.43x
+
1.18
R20.93
0.58
0.53
0.94
0.92
0.75
0.79
0.91
0.91
using
the
Leica
Application
Suite
version
2.1.8.
Measurements
were
then
taken
on
the
photographs
(using
the
same
software)
to
the
nearest
0.01
mm.
The
head
parameters
were
chosen
according
to
a
previous
paper
using
head
measurements
in
embryos
of
the
Nile
crocodile
Crocodylus
niloticus
niloticus
(Peterka
et
al.,
2010),
with
the
addition
of
head
height
and
head
breadth
(Fig.
1B
and
C).
Mea-
surements
of
the
head
included
the
following.
Head
length.
Measurement
of
the
maximum
anterior–posterior
head
extension,
measured
in
left
lateral
view.
In
small
specimens,
the
measurements
were
taken
from
the
most
posterior
part
of
the
occipital
region
to
the
snout.
In
embryos
larger
than
6
mm,
the
most
posterior
part
of
the
head
is
represented
by
the
posterior
end
of
the
lower
jaw.
The
measurements
were
therefore
taken
from
the
posterior
end
of
the
lower
jaw
to
the
snout.
It
was
not
possible
to
always
take
the
same
measurements
in
all
specimens
because
it
was
not
possible
to
find
the
homologous
points
in
all
specimens.
This
fact
was
taken
into
account
when
interpreting
the
results.
Head
breadth.
Measurement
of
the
maximum
head
breadth,
measured
in
dorsal
view
and
not
including
the
eyes.
Head
height.
Measurement
of
the
maximum
head
height,
mea-
sured
in
left
lateral
view.
Head
height
is
defined
as
the
distance
from
the
dorsal-most
point
of
the
head
down
to
the
ventral-most
border
of
the
lower
jaw
in
a
vertical
orientation.
In
specimens
with
an
open
mouth,
the
measurements
were
not
taken
at
the
ventral
border
of
the
lower
jaw,
because
this
would
have
overestimated
the
real
head
height.
Instead,
the
measurements
were
taken
at
an
estimated
ventral
line
of
the
lower
jaw,
assuming
the
mouth
to
be
closed.
This
fact
was
taken
into
account
when
interpreting
the
results.
Table
5
Description
of
the
additional
SES
characters
with
codings
as
suggested
by
Werneburg
(2009).
CC
SEC
CN
Description
of
the
characters
Character
illustration
Somites
(V04) V04n
61
and
more
somite
pairs
In
species
with
extremely
elongated
bodies,
61
and
more
somite
pairs
are
present.
Hemipenes
(Sq01)
Sq01a
Hemipenes
visible
The
everted
hemipenes
are
seen
as
paired
structures
sticking
out
on
each
side
of
the
cloaca.
Depending
on
the
taxa,
different
forms
are
possible.
The
hemipenes
are
only
found
in
males.
Sq01b
Hemipenes
inverted
The
hemipenes
are
inverted
and
thus
no
longer
visible
from
the
outside.
K.M.
Polachowski,
I.
Werneburg
/
Zoology
116 (2013) 36–
63 41
specimens ordered by total length
weight
y = 0,1741x + 3,679
R² = 0,9784
total length (cm)
percental growth of head measurements (A-H),
in total = 100% / total length (cm) / weight (g)
head measurements in mm
specimens ordered by total length
AB
C
D
E
F
G
H
I
a
b
c
f
d
e
gh
14.54g
31.2cm
0.086g
4.5cm
0
2
4
6
8
10
12
14
16
18
20
e
f
d
a
h
g
b
c
a
h
g
f
e
d
c
b
y = 0,0664e
0,1815x
R² = 0,9301
0
5
10
15
20
25
0
10
20
30
40
weight (gramm)
total length (cm)
y = 2,4949x
0,3572
R² = 0,7311
0
1
2
3
4
5
6
7
8
9
0
5
10
15
20
head heigth (mm) (b)
head length (mm) (a)
0
2
4
6
8
10
12
14
16
18
20
0
5
10
15
20
25
30
35
head length (mm) (a)
total length (cm)
y = 6,8768ln(x) -6,7533
R² = 0,964
0
2
4
6
8
10
12
14
0
5
10
15
20
head breadth (mm ) (h)
head length (mm) (a)
y = 0,6235x
0,9643
R² = 0,9488
0
2
4
6
8
10
12
14
0
2
4
6
8
10
y = 0,1843x
1,9878
R² = 0,7037
head heigth (b)
head breadth (mm)
(h)
Fig.
1.
Comparison
of
different
head
measurements
of
Bothropoides
jararaca
specimens.
(A)
Total
length
with
regression
line
and
the
weight
of
all
123
specimens,
arranged
according
to
their
total
length.
(B
and
C)
Measurements
of
the
head
in
specimen
B13
in
left
lateral
(B)
and
dorsal
(C)
view.
Note
the
white
calcified
endolymph
in
the
occipital
region.
The
percent
relative
growth
of
head
parameters
is
shown
in
(A).
(D)
Different
head
measurements
plotted
against
specimens,
which
are
ordered
by
total
length.
(E–I)
Comparison
of
selected
head
measurements
plotted
against
each
other.
For
discussion
see
text.
Head
measurements:
a
=
head
length,
b
=
head
height,
c
=
eye-to-snout
distance,
d
=
eye
diameter,
e
=
upper
jaw,
f
=
lower
jaw,
g
=
head
breadth,
h
=
distance
between
the
eyes.
Scale
bar
for
(B)
and
(C)
=
2
mm.
42 K.M.
Polachowski,
I.
Werneburg
/
Zoology
116 (2013) 36–
63
A
B
D
F
E
G
C
B0 (SES-stage 1)
B2 (SES-stage 2)
B5 (SES-stage 3)
B14 (SES-stage 4)
B18 (SES-stage 5)
B19 (SES-stage 6)
B26 (SES-stage 7)
Fig.
2.
Lateral
views
of
the
heads
of
selected
Bothropoides
jararaca
embryos.
(A)
Specimen
B0
(SES-stage
1)
(mirrored),
(B)
specimen
B2
(SES-stage
2),
(C)
speci-
men
B5
(SES-stage
3),
(D)
specimen
B14
(SES-stage
4),
(E)
specimen
B18
(SES-stage
5),
(F)
specimen
B19
(SES-stage
6),
(G)
specimen
B26
(SES-stage
7).
Scale
bars:
(A
and
B)
=
2
mm;
(C–G)
=
5
mm.
The
figure
corresponds
to
the
supplementary
online
Appendix
6.
For
photographs
and
more
specimens
see
Appendix
1.
Eye-to-snout
distance.
Measurement
from
the
centre
of
the
eye
to
the
most
anterior
part
of
the
snout,
measured
in
left
lateral
view.
Distance
between
the
eyes.
Measurement
of
the
maximal
dis-
tance
between
the
eyes,
measured
in
dorsal
view.
Several
embryos
had
crushed
eyes,
probably
due
to
storing
circumstances
before
the
start
of
the
study.
This
fact
led
to
an
underestimation
of
this
mea-
surement
in
embryos
with
crushed
eyes,
which
was
considered
when
interpreting
the
results.
Eye
diameter.
Measurement
of
the
maximal
eye
diameter,
mea-
sured
in
left
lateral
view.
Upper
and
lower
jaw.
Measurements
of
the
upper
and
lower
jaw
lengths,
measured
in
left
lateral
view.
Measurements
were
taken
from
the
corner
of
the
mouth
to
the
anterior-most
end
of
the
upper
or
lower
jaw.
While
in
some
cases
the
jaws
were
curved,
the
measurements
were
always
taken
in
a
direct
line.
The
corner
of
the
mouth
and
the
anterior
ending
of
the
upper
jaw
were
not
always
clearly
identifiable,
however,
the
measurements
were
taken
as
accurately
as
possible.
2.4.
External
morphology
The
description
of
the
external
morphology
(Fig.
2,
Appendix
1)
is
based
on
the
Standard
Event
System
(SES)
(Werneburg,
2009).
This
system
provides
a
standardised
protocol
to
docu-
ment
developmental
characters
in
embryos.
Some
extensions
were
added
to
the
character
list
of
the
original
SES
protocol
using
new
data
from
another
study
(Werneburg
and
Sánchez-Villagra,
2011)
and
our
own
examinations
(Table
5).
Species-specific
characters,
such
as
pigmentation
and
the
development
of
the
heat-sensitive
pit
organ
(Barrett
et
al.,
1970;
Newman
and
Hartline,
1982;
Hofstadler-Deiques,
2002),
and
problematic
characters
with
an
unclear
phylogenetic
significance,
such
as
the
calcified
endolymph,
were
not
coded
as
SES
characters
but
written
in
italics
as
proposed
by
Werneburg
(2009).
Total
length
was
used
to
order
the
embryos
according
to
their
developmental
progress.
External
morphology
was
studied
with
a
stereo
microscope
Leica
MZ16.
Photographs
were
taken
with
a
digital
camera
GE
A1150
(General
Imaging,
Fair-
field,
CT,
USA)
or
with
a
digital
camera
Leica
DFC420C
and
the
image
manager
software
Leica
IM50.
Out
of
the
27
specimens
of
B.
jararaca
analysed
for
this
part
of
the
study
(Table
1),
only
those
specimens
that
show
newly
developing
characters
in
the
embryonic
series
were
photographed,
coded
(supplementary
online
Appendix
1),
and
described
in
detail.
2.5.
Skeletogenesis
2.5.1.
Micro-computed
tomography
scanning
Micro-computed
tomography
scanning
(CT-scanning)
is
a
technique
for
studying
skeletal
development
and
morphology
without
destroying
the
material.
It
is
not
suitable
for
the
study
of
cartilaginous
structures.
Around
an
axis
of
rotation,
two-dimensional
X-ray
images
are
taken
and
processed
into
a
three-dimensional
image
(Figs.
3–6,
Table
6).
In
total,
14
selected
specimens
(Table
1)
were
scanned
with
a
CT-scanner
(CT
80;
Scanco
Medical
AG,
Brüttisellen,
Switzerland)
at
Anthropologisches
Institut
und
Museum
der
Universität
Zürich,
covering
the
length
spectrum
as
well
as
pos-
sible.
For
CT-scanning,
the
specimens
were
tightly
wrapped
in
bubble
wrap.
To
prevent
high
evaporation
and
thus
shrinkage
of
the
tissue,
the
scanning
tubes
were
closed
with
tape.
In
every
scan
several
specimens
were
scanned
simultaneously
in
the
same
tube.
The
following
scanning
parameters
were
used:
voltage
=
70
kV;
current
=
114
A;
image
resolution
=
1024
pixels.
Slice
thickness
was
39
m.
Data
sets
were
rendered
using
Avizo
Standard,
ver-
sion
6.2
(VSG,
Burlington,
MA,
USA).
All
descriptions
are
based
on
the
study
of
slices
and
different
isosurface
renderings
made
with
K.M.
Polachowski,
I.
Werneburg
/
Zoology
116 (2013) 36–
63 43
mx pal ect pt
mx pal ect pt
c?
c?
mx pal ect pt c
ce
eo
ce
c
st
pt
p
ect
pal
mx
prf
f
vsm
pm
ce
st
eo
p
ect
f
v
prf
sm
pm
pal
mx
dc
pt
ce
pt
ect
pal
mx
c
ce
st
eo
bo
bs
p
vpal
ect
pt
f
n
prf
sm
pm
mx
fa
dsp ang
c
f
prf mx
ect
p
c
st
q
eo
ce
bo
bs
pal
sm
pm
n
ang
sp
d
v
pt
G)
AB
CD
E
F
G
H
B4 (SES-stage 2)
B6 (SES-stage 3)
B9 (SES-stage 3)
B12 (SES-stage 4)
Fig.
3.
Skulls
of
Bothropoides
jararaca
specimens
of
different
total
lengths.
Pictures
are
based
on
isosurface
renderings
with
a
density
threshold
of
2500,
obtained
by
CT-
scanning.
In
all
left
lateral
views
the
right
side
of
the
skull
was
removed.
Individual
bones
are
highlighted
with
different
colours.
Specimen
B4
in
(A)
left
lateral
and
(B)
dorsal
view
(cf.
Appendix
2).
Specimen
B6
in
(C)
left
lateral
and
(D)
dorsal
view
(cf.
Appendix
3).
Specimen
B9
in
(E)
left
lateral
and
(F)
dorsal
view
(cf.
Appendix
4).
Specimen
B12
in
(G)
left
lateral
and
(H)
dorsal
view
(cf.
Appendix
5).
Abbreviations:
ang
=
angular;
bo
=
basioccipital;
bs
=
basisphenoid;
c
=
composite
bone;
ce
=
calcified
endolymph;
d
=
dentary;
ect
=
ectopterygoid;
eo
=
exoccipital;
f
=
frontal;
fa
=
fang;
mx
=
maxilla;
n
=
nasal;
p
=
parietal;
pal
=
palatine;
pm
=
premaxilla;
prf
=
prefrontal;
pt
=
pterygoid;
q
=
quadrate;
sm
=
septomaxilla;
sp
=
splenial;
st
=
supratemporal;
v
=
vomer.
Scale
bars
=
5
mm.