Neurocranium asymmetries in the Bottlenose Dolphin

Chapter

NEUROCRANIUM ASYMMETRIES IN THE

BOTTLENOSE DOLPHIN, TURSIOPS

TRUNCATUS (MONTAGU, 1821)

P. M. Parés-Casanova1,

and R. Bravi2,†

1Departmrnt of Animal Production, University

of Lleida, Lleida, Catalunya, Spain 2Department of Science; University

of Roma Tre, Rome, Italy

ABSTRACT

External evolutionary novelties of odontocete skulls (toothed whales,

including dolphins and porpoises) include a “telescoped” morphology,

which involves the posterior repositioning of anterior cranial bones and

postero-dorsal positioning of the nares to form the “blowhole”. Internal

cetacean skull architecture is also asymmetrical and shows the effects of

telescoping, too. The Bottlenose dolphin (Tursiops truncatus) is one of

this odontocete species. In this study we focused the attention on the skull

of Tursiops truncatus and assessed both size and shape asymmetry by

geometric morphometric method. Due to the difficulties related to data

collection and records in the field, the museum collections represented

P. M. Parés-Casanova: Department of Animal Production, University of Lleida, Av. Alcalde

Rovira Roure, 191, 25198 Lleida, Catalunya, Spain. E-mail: [email protected]. †

R. Bravi. Department of Science; University of Roma Tre, Viale Marconi, 446, 00146 Rome, Italy. E-mail: [email protected].

P. M. Parés-Casanova and R. Bravi 2

the primary source of information, which in this study consisted of 14

specimens (9 adults and 5 youngs) from the Natural History Museum of

Barcelona (Catalunya, Spain). Morphometric analysis was based on

unambiguous and repeatable anatomical 12 landmarks plotted on 2-D

images of dorsal views. For size, results revealed that there were

differences between right and left hemineurocranium. For shape, the

factor Individual explained the largest proportion of the total variation.

The second factor that contributed to the highest variation was fluctuating

asymmetry. The directional asymmetry had the lowest effect on shape variation. Bilateral trait measurements on dorsal aspect of skull were

found to be reliable indicators of between-subject differences and to

reflect true fluctuating asymmetry more than biologically significant

directional asymmetry.

These preliminary results show the need to include the other

projections of the skulls to better elucidate the degree and the pattern of

asymmetric variation. For further investigations, greater numbers of

specimens from known ontogenetic stages are encouraged.

Also, comparison between populations of different geographical

origin may be assessed. Such specimens and resultant information will

greatly improve our knowledge of the biology of Bottlenose dolphins.

Keywords: Delphinidae, morphological variation, odontocete, skull direction-

nal asymmetry, skull fluctuating asymmetry

INTRODUCTION

It is expected that the left and the right sides of a bilateral organism, or a

part of it which shows bilateral symmetry, are separate copies of the same

structure. They share the same genome, and in a homogeneous environment

these two parts develop as identical, as an effect of environmental pressure;

but in real developing systems small random perturbations could produce non

perfect symmetric organisms (McAdams and Arkin 1999; Klingenberg 2003).

In this context left-right asymmetries should emerge as expression of

developmental noise, and they accumulate through development (Palmer 1996,

2004; Klingenberg 2003).

Presence of asymmetries in bilateral organisms is now well known and

studied, and it is well known as well that among others, Fluctuating

Asymmetry (FA) and Directional Asymmetry (DA) are usually associated

with that developmental instability (Palmer and Strobeck 1986, Graham et al.

1993).

Neurocranium Asymmetries in the Bottlenose Dolphin … 3

FA is referred to non-directional deviation from perfect symmetry, and it

is expressed as individual differences between left and right sides. DA

outcomes every time there is a greater development of one side on the plane of

symmetry than the other; it is hypothesized that a proportion of DA has

genetic bases (Van Valen 1962, Carter et al. 2009).

Skulls are routinely studied as part of investigations of mammalian

biology. Both qualitative and quantitative features are examined to discern the

presence and degree of sexual dimorphism, ontogenetic and individual

variation, as well as geographic or oceanographic variation within and among

populations (Perrin 1975). Such data contribute substantially towards the

answers of taxonomic, ecological and evolutionary questions. Because these

characters typically change during growth, an adult skull with relatively fixed

characteristics is preferred for non-developmental aspects of these examina-

tions (Perrin and Heyning 1993). Geometric morphometrics (GM) has been

used to distinguish between confusing cases of living species or subspecies of

Delphinidae (Monteiro-Filho et al. 2002; Paola 2010) and a previous study

assessing the presence of symmetries has been done in Bottle-nose (Parés and

Fabre 2013).

Odontocete skulls are drastically modified relative to the general mamma-

lian condition (Colbert et al. 2005). External evolutionary novelties of

odontocetes include a ‘telescoped’ morphology (Miller 1923), which involves

the posterior repositioning of anterior cranial bones and postero-dorsal

positioning of the nares to form the ‘blowhole’, and bilateral asymmetry. The

relationship between these cranial modifications and evolution of an aquatic

life history has been widely discussed (e.g. Moore 1981; Novacek 1993;

Fordyce and de Muizon 2001). Internal cetacean skull architecture is also

asymmetrical and shows the effects of telescoping. Additionally, internal

osteology reflects their large brain size and provides evidence of evolutionary

transformations in relative importance of the different peripheral sensory

inputs (Breathnach 1960; Morgane and Jacobs 1972; Morgane et al. 1980;

Ridgway 1990). The taxonomic Order Cetacea includes three suborders: the

Mysticeti, or baleen whales; the Odontoceti, or toothed whales; and the

Archaeoceti, the now extinct ancestral forms of some modern whales and

dolphins. Tursiops truncatus (Montagu, 1821), the Bottlenose dolphin, is a

toothed whale of the Family Delphinidae, which includes approximately 30

species of dolphins, pilot whales, killer whales and false killer whales. The

Bottlenose dolphin is the most common dolphin species held in marine parks

worldwide, and it has been bred successfully in captivity (Amundin 1986;

Yoshioka et al. 1986; Cornell et al. 1987).


The Bottlenose dolphin is around 2.5 m long and weighs between 200 and

300 kg. It has a worldwide distribution, ranging from the Mediterranean Sea to

the Atlantic and Pacific Oceans. This wide-ranging distribution, together with

high levels of polymorphism and clinal variation, has made the taxonomic

status of Tursiops difficult to resolve.

Moreover, skulls held in museum collections frequently lack accompa-

nying vital data of body size, reproductive status and other age-related infor-

mation.

In the present study skull asymmetries in T. truncatus are analysed with

the technique of geometric morphometrics with the aim to depict and quantify

asymmetries, and moreover to relate them with telescoping and echoimaging

capabilities.

MATERIALS AND METHODS

A total of 14 skulls (9 adults and 5 youngs) belonging to Tursiops

truncatus were examined. Skulls were deposited in the Natural History

Museum of Barcelona (Table 1). Sex was available only for one specimen. All

pieces showed no gross pathological appearance that might lead to errors in

measurement.

Image capture was performed with a Nikon® D70 digital camera (image

resolution of 2,240 x 1,488 pixels) equipped with a Nikon AF Nikkor® 28-200

mm telephoto lens and using a standard procedure to avoid the effects of

distortion. The focal axis of the camera was parallel to the horizontal plane of

reference and centred on the dorsal view of each skull.

A ruler was used in this process (scale: 100 mm). 12 homologous and

topologically equivalent landmarks (LMs) were plotted on the dorsal skull (to

obtain the Cartesian x and y coordinates), including 5 paired points (3-4, 5-6,

7-8, 9-10 and 11-12) on each side and two on the midline (Figure 1).

All these LMs are considered to encompass elements of neurocranium.

Landmarks were digitized twice using tpsDig 2.16 (Rohlf 2010) by the same

person (PMPC) on two different days in the same order, for assessing

measurement error.

In order to compare Procrustes to tangent space distances between

individuals, a Generalized Procrustes Analysis superimposition (equivalent to

generalized least squares) procedure of Rohlf and Slice (1990) was performed

on each data set using TPS-Small 1.20 (Rohlf 2003). The approximation of

shape space by tangent space presented a high correlation (0.999).


Table 1. References of studied specimens (n=14)

Code Element Sex Age Site

MZB 82-7967 Skull Unknown Adult Unknown

MZB 82-7968 Skull Unknown Adult Alacant (Spain)

MZB 82-7969 Disarticulated

skeleton Unknown Adult Unknown

MZB 82-7982 Skull Male Adult Oropesa (Spain)

MZB 82-8007 Skull Unknown Young Unknown

MZB 82-8011 Skull Unknown Young Tarragona (Spain)



MZA 91-0440 Skull Unknown Young Unknown

MBZ 93-0254 Skull Unknown Adult Unknown




MZB 94-0416 Disarticulated

skeleton Unknown Adult Zoo

This high degree of approximation of shapes in the sample (=shape space)

by the reference shape (=tangent space) allowed an accurate capturing of the

nature and extent of shape deformations in subsequent statistical analyses.

So, once obtained the Cartesian x-y coordinates for all points, the shape

information was extracted from the coordinate data by using a full Procrustes

fit, which removes information on location orientation and rotation, and

standardizes each specimen to unit centroid size (CS -that is, the square root of

the summed squared Euclidean distances from each landmark to the specimen

centroid that provides an estimate of the size of the studied structure- Rohlf

and Slice 1990; Dryden and Mardia 1998). CS was used as a measure of size,

while shape information was extracted from Procrustes superimposition. Due

to the object symmetry in the dataset reflection is removed by including the

original and mirror image of all configurations in the analysis, and superimpo-

sing all of them simultaneously (Klingenberg et al. 2002).

Variation of shape in the whole dataset was analysed with principal

component analysis (PCA), based on the covariance matrix of symmetric and

asymmetric components of shape variation.


Figure 1. Schematic drawing of ventral skull of Tursiops truncatus with 12 landmarks

used. Landmarks are marked with circles (left). Projection of landmark locations for all

specimens, after general least square alignment (right). Five paired points (3-4, 5-6, 7-

8, 9-10 and 11-12) on each side and two (1 and 2) on the midline.

These two components were extracted from the raw data during Procrustes

fit, and represented respectively the average of left and right sides (i.e. the

shape variation component), and the individual left-right differences (Klingen-

berg et al. 2002). Shape variation was described for both symmetric and

asymmetry components.

Asymmetries (DA and FA) were analysed and quantified with Procrustes

ANOVA for size and shape and MANOVA analyses assessed for studied on

object symmetry.

After assessing asymmetries we performed a discriminant function

analysis (DFA) to evaluate if these asymmetries were affected by ontogenetic

allometry. DFA evaluates differences between two groups of observations, and

it is implemented in MorphoJ to include a T-square test (parametric) and a

permutation test; results are given as Procrustes distance, Mahalanobis

distance and T-square values, and associated p-values for permutation test.

All analyses were performed using MorphoJ 10.6a (Klingenberg 2011),

and permutation test with 10,000 permutation runs.


RESULTS

Firstly the LM at the tip of the rostrum (L1) was removed because

introducing some noise in data interpretation, as we were interested mostly in

depicting asymmetries in the neurocranium, not in the entire skull.

After that we inferred measurement error with Procrustes ANOVA, and it

resulted reasonably lesser than individual variation and fluctuating asymmetry

(Table 2). Consequently it didn’t affect the estimation of asymmetries in the

data set, and we could proceed with subsequent analyses. The entire dataset

was then averaged by individual, to remove every possible (even subtle) noise

due to replica repetition.

Assessing asymmetries was the next step. The dataset resulted affected by

both DA and FA (see Table 3) that even subtle were both highly significative

(see Table 1 for details), and partially confirmed by MANOVA (DA for

asymmetric component didn’t result significative) (Table 3).

Shape variation was then assessed by a PCA, and because we were

interested in describing the asymmetries, we considered the asymmetric

component of variation, and left the symmetry component for describing the

whole shape variation only.

On the whole, for symmetric component of shape variation, the first two

PCs (88.0% of the total observed variation) were sufficient to describe the

majority of variation, but the higher contribute to shape variation was due to

first component only (59.0% of the total observed variation).

Table 2. Procrustes ANOVA for both centroid size (CS) and shape (SH).

Sums of squares (SS) and mean squares (MS) are in units of Procrustes

distances (dimensionless). Distances between young and adult (age) are

reported, as well as individual variation

Effect SS MS Df F P (param.)

CS

Age 6.69259E+04 6.69259E+04 1 4.56 0.054

Individual 1.75980E+05 1.46650E+04 12 1038.39 <0.0001

Error 1.97719E+03 14.12 14

SH

Age 0.0069 0.0014 5 1.55 0.19

Individual 0.054 0.00089 60 2.38 0.0004

DA 0.012 0.0024 5 6.35 <0.0001

FA 0.024 0.00038 65 46.53 <0.0001

Error 0.0011 0.0000081 140

DA = directional asymmetry; FA = fluctuating asymmetry. Both DA and FA were

highly significative.


Table 3. MANOVA test for directional asymmetry (DA) and fluctuating

asymmetry (FA) for both symmetric (SYMM) and asymmetry (ASYMM)

components of the variation. Differences between the young and adult

(Age) are reported, as well as individual variation. DA for asymmetric

component didn’t result significative)

Effect Pillai tr. P (param.)

SYMM Age 0.75 0.027

Individual 4.75 <0.0001

ASYMM

Age 0.58 0.15

DA 0.58 0.12

FA 4.03 <0.0001

Regarding the asymmetries, the very higher part of variation was

concentrated on the first dimension only (86.4% of the total observed

variation), the other factors counting for the very subtle part (see Table 4 for

details). Age differences, although present (T-square = 35.30, P = 0.027;

Mahalanobis distance = 3.31; Procrustes distance = 0.21, P = 0.024) didn’t

affect asymmetries (T-square = 16.63, P = 0.15; Mahalanobis distance = 2.27;

Procrustes distance = 0.064, P = 0.15), even if some differences in shape are

well evident between young and adult individual (Figure 2).

DISCUSSION

Most odontocetes (but not mysticetes) have asymmetrical skulls and faces

(Colbert et al. 2005; Ness 1967), which is considered fundamentally associated

with the development of their echoimaging capability (Cranford et al. 1996),

although recently this directional asymmetry has been related to an aquatic

adaptation enabling swallowing large, whole prey while maintaining respire-

tory tract protection (MacLeod et al. 2007). The distribution and degree of

asymmetry varies interspecifically and intraspecifically among odontocetes,

being less pronounced in the more basally diverging species and non-existent

in some early whales (Berta and Sumich 1999).

Different brain waves have been detected in right and left dolphin

hemispheres, too (Ridgway 2002). This asymmetry always occurs on the facial

region (Berta and Sumich 1999). Truly speaking DA in our data is present and

statistically significant (although not so high as FA was), so we should

conclude it could be consistent with MacLeod’s statement.


a

b

Figure 2. Discriminant function for asymmetric component, for young (n=4) and adult

(n=9) specimens. A) histogram with discriminant scores; B) deformation grids repre-

senting shape deformations at the extremities of the graph, for young and adult.

Our preliminary results show also the need to include the other projections

of the skulls to better elucidate the degree and the pattern of asymmetric

variation. The strength of conclusions is restricted, however, by the small

number of skulls. Moreover, the bias related to the origin of death would be

taken into account.

Asymmetries are not influenced by ontogeny, since differences between

young and adult did not emerged. Further investigations with greater numbers

of specimens from known ontogenetic stages are encouraged.

Also, comparison between populations of different geographical origin

may be assessed.

Such specimens and resultant information will greatly improve our

knowledge of the biology of Bottlenose dolphins.


ACKNOWLEDGMENTS

Thanks to Natural History Museum of Barcelona, especially its mammals

curator Dr Javier Quesada for loaning the specimens and the associated

information.

Letícia Fabre and Lluís Alonso helped to photograph the specimens.

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Neurocranium asymmetries in the Bottlenose Dolphin

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