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Zoology 112 (2009) 3847

Cranial shape differentiation in three closely related delphinid cetacean

species: Insights into evolutionary history
Ana R. Amarala,, Maria M. Coelhoa, Jesus Marugan-Lobonb, F. James Rohlfc
Centro de Biologia Ambiental, Faculdade de Ciencias da Universidade de Lisboa, Campo Grande, 1749-016 Lisbon, Portugal
Unidad de Paleontologa, Departamento de Biologa, Universidad Autonoma de Madrid, 28049 Madrid, Spain
Department of Ecology and Evolution, Stony Brook University, Stony Brook, NY 11794-5245, USA

Received 21 September 2007; received in revised form 5 February 2008; accepted 6 March 2008

The present study investigates the pattern of differentiation of cranial shape in three closely related delphinid
cetacean species of the complex DelphinusStenellaTursiops: Delphinus delphis, Stenella coeruleoalba and Tursiops
truncatus. Dorsal and ventral aspects of the cranium were analysed using landmark-based geometric morphometric
methods. While there was no evidence of sexual dimorphism for shape or size, multivariate statistical analyses showed
that there were interspecic differences in skull morphology. Skull shape differences between the three studied species
were related with cranial width and differences in the length of the rostrum relative to the cranial portion of the skull.
D. delphis and S. coeruleoalba showed high cranial shape similarity, which is indicative of their evolutionary proximity
when compared with T. truncatus. Phenetic clusters based on cranial shape similarities were found to be concordant
with the molecular phylogenetic clades obtained from mitochondrial DNA genes. Geometric morphometric methods
can thus be an exceptionally useful tool for the study of differentiation of delphinid cetacean species and therefore
provide some insights into their evolutionary history.
r 2008 Elsevier GmbH. All rights reserved.

Keywords: Delphinidae; Geometric morphometrics; Morphological evolution

Introduction Heyning and Perrin 1994; Messenger and McGuire

1998; Buchholtz and Schur 2004). However, the few
The family Delphinidae comprises 17 genera and at attempts made to incorporate these characters into
least 33 species of dolphins, distributed in all oceans and cladistic analyses of the family were unsuccessful in fully
most seas of the world. There have been several resolving relationships at the genus level, since they
morphological studies based on osteological characters either focused on higher taxonomic levels or were unable
such as number of vertebrae, length and form of the to nd characters that could differentiate genera or
rostrum, number and size of the teeth, condition of the species (Mead 1975; Muizon 1988; Martins and Hansen
pterygoid bones and several external and skull measure- 1997; Messenger and McGuire 1998).
ments (Flower 1883; True 1889; Perrin et al. 1987; Patterns of pigmentation have also been studied as a
tool to understand evolutionary relationships among
Corresponding author. delphinid cetacean species. The evolutionary path of the
E-mail address: aramaral@fc.ul.pt (A.R. Amaral). pigmentation pattern between species in the group

0944-2006/$ - see front matter r 2008 Elsevier GmbH. All rights reserved.
A.R. Amaral et al. / Zoology 112 (2009) 3847 39

DelphinusStenella was explored and a possible ecolo- analyses, the possibility to separate size and shape,
gical signicance for those patterns was suggested and the ability to produce graphical representations of
(Mitchell, 1970). the results, in which shape as a conguration of
Within the family Delphinidae, there is a problematic landmark points is visualised in addition to the common
group: the DelphinusStenellaTursiops clade. Members statistical scatterplots (Rohlf and Marcus 1993). This is
of these genera are very similar, closely related and thus possible since the geometry of the shape is preserved
were difcult to classify. One of the difculties was the throughout the analysis (Adams 2004).
dual nomenclature based upon osteological materials in In this study, dorsal and ventral aspects from skulls
the skull in some instances, or solely upon the external of three delphinid species, D. delphis (Linnaeus, 1758),
appearance of the animal in others (Flower 1883). S. coeruleoalba (Meyen, 1833) and Tursiops truncatus
For example, True (1889) revealed his uneasiness in (Montagu, 1821) were analysed using landmark-based
validating the characters that were being used to geometric morphometric methods and their associated
separate the genera Tursiops and Delphinus from the multivariate statistical procedures.
genus Prodelphinus ( Stenella). The only clear distinc- The aim of the study was: (i) to nd patterns of
tion between the skulls of Delphinus and Stenella differentiation in the cranial shape of the above-
specimens was the presence of deep longitudinal grooves mentioned species and compare them with patterns of
on the palate of the maxillary bone. Although the skulls genetic differentiation obtained from previous studies of
of Tursiops specimens showed some unique characters, mitochondrial DNA (mtDNA); (ii) to discuss whether
such as shorter rostrum, teeth that are stouter and fewer the study of cranial shape variation may provide some
in number and different overall cranial size, these insights into the evolutionary history of these species.
characters could not be successfully used in cladistic
More recently, several studies using DNA sequencing Material and methods
have tested the pattern of genetic variability of these
taxa (Escorza-Trevino et al. 2005; Adams and Rosel The 92 specimens (68 D. delphis, 40 males and 28
2006; Natoli et al. 2006; Parsons et al. 2006; Bourret females; 14 S. coeruleoalba, 5 males and 9 females; and
et al. 2007). However, few have focused on phylogenetic 10 Tursiops truncates, 4 males and 6 females) used in this
relationships between species, and those have not been study belong to collections held by the Aquario Vasco
successful in solving the systematics of the subfamily da Gama (Portugal) and the Museu Bocage (Lisbon,
Delphininae, particularly of the DelphinusStenella Portugal) (see online supplementary material at the
Tursiops clade (LeDuc et al., 1999; Dizon et al., 2000). Appendix). All specimens analysed were assessed as
Much of the taxonomic confusion comes from the fact physically mature, determined by the existence of rostral
that these genera are not monophyletic (LeDuc et al., fusion (Collet 1981; Murphy 2004). Although Perrin and
1999). For example, Stenella coeruleoalba seems to be Heyning (1993) reported that this method could be
more closely related with Delphinus delphis than with inadequate for assessing physical maturity, due to a lack
other species within the genus Stenella. Since these of age and total body length (TBL) data for most skulls
species occur in sympatry and have probably split analysed in the current study (and in the case of both
recently, they provide an excellent opportunity to study S. coeruleoalba and T. truncatus a lack of published
evolutionary processes responsible for species differen- articles on TBL ranges for adult males and females) the
tiation under sympatric conditions in the marine only available option was to assess maturity using this
environment (Barraclough and Nee 2001). However, it method. Although using rostral fusion may bring some
is crucial to nd new and reliable characters, either error in the identication of mature individuals, the
molecular or morphological, that enable a detailed study main objective of this study was to report interspecic
of species boundaries within the complex Delphinus differences and the geometric morphometrics technique
StenellaTursiops. can overcome differences in size, so the results presented
Geometric morphometrics (Rohlf and Marcus 1993) here would not be inuenced if other criteria had been
is based on the capture of shape through a set of adopted. The skulls were photographed in both dorsal
landmarks digitised on each specimen (Marcus 2000). and ventral views following some of the guidelines
This has proved useful in solving a variety of systematic/ presented by Zelditch et al. (2004). Ten landmarks were
taxonomic problems, principally when dealing with digitised on the dorsal view (DC) and eleven on the
closely related species (Dobigny et al. 2002; Cardini ventral view (VC) of the crania (Table 1; Fig. 1).
and OHiggins 2004; Pizzo et al. 2006). Two species of One of the characteristics of the skulls of odontocetes
the dolphin genus Sotalia could be distinguished based is the directional asymmetry in the right and left sides,
on these methods (Monteiro-Filho et al., 2002). Some of particularly in the elements associated with the airway
the advantages of landmark-based geometric morpho- (e.g. Ness 1967; Yurick and Gaskin 1988; MacLeod
metric methods are the more powerful statistical et al. 2007). However, as the main objective of this study
40 A.R. Amaral et al. / Zoology 112 (2009) 3847

Table 1. Denition and numbering of dorsal and ventral

cranial landmarks

Dorsal cranial view

8 11 1
Number Description 10
7 9
1 Rostral tip 5 0
2 Anteriormost point on the notch in the maxilla
3 Intersection between the frontal bone and zygomatic 6 2
process 4 3
4 Intersection between the parietal bone and frontal-
interparietal suture
5 Posteriormost point on the curve of the parietal bone
6 Posteriormost point on the curve of the occipital
8 9 1
7 Posteriormost point on the edge of the supraoccipital 7
bone 10
8 Anteriormost point of the suture between the frontal 6
and interparietal bones
9 Midpoint of the nasal bone suture 5 3
10 Posteriormost point in the premaxilla bone 4

Ventral cranial view Fig. 1. Landmark conguration for dorsal (above) and ventral
1 Rostral tip (below) aspects of the cranium for a D. delphis specimen.
2 Anteriormost point on the notch in the maxilla
3 Externalmost point in the suture below the
supraorbital process of the frontal bone Landmarks were superimposed using a generalised
4 Point in the suture between the frontal and Procrustes analysis superimposition (GPA), which
alisphenoid bones aligns, scales and rotates landmark congurations
5 Ventralmost point of the paraoccipital process removing any information unrelated to shape
6 Posteriormost point on the curve of the parietal bone (Rohlf and Slice 1990). The weight matrix (partial warp
7 Posteriormost point on the curve of the occipital
scores and the uniform component) and centroid sizes
(the square root of the sum of the squared distances
8 Posteriormost point on the edge of the supraoccipital
bone between all the landmarks and the centroid of the
9 Point in the suture between the basoccipital and conguration; Bookstein 1991) were obtained using
pterygoidal bones TpsRelw 1.44 (Rohlf 2006b) and used in statistical
10 Dorsalmost point in the petrygoidal notch analyses. A relative warp (RW) analysis, which is a
11 Anteriormost point on the palatine principal component analysis of the partial warp scores,
was conducted in order to summarise the variation in
shape among the specimens in the sample. Deformation
grids based on the use of the TPS were used to visualise
was to capture overall patterns of differentiation shape variation. Size was compared between sexes and
of cranial shape, landmarks were only digitised on across species with a two-way (sex  species) analysis of
the left side of the crania using TpsDig 2.05 variance (ANOVA). Multivariate analysis of variance
(Rohlf 2006a) so as to avoid the redundancy of sampling (MANOVA) and canonical variates analysis (CVA)
a bilateral structural topology. Additionally, this were performed to assess the probability of shape
approach provides the correct degrees of freedom for differentiation between groups (sexes and species).
statistical analyses and reduces the number of specimens Homoscedasticity in the ANOVA for centroid size was
required for testing statistical hypotheses (Zelditch et al. veried by means of Levenes test (P 0.458 for DC
2004). data and P 0.707 for VC data), a step unveriable in
To evaluate the reliability of landmark congurations, the multivariate analysis (MANOVA and CVA) since
a repeatability test was conducted on a sub-set of the number of variables is larger than the sample size for
individuals, which were photographed three times two of the studied species, a common problem in
on three consecutive days. Prole plots of the resul- taxonomic studies (Marcus, 1990). Multivariate regres-
tant landmark coordinates were used to detect sion of shape variables onto CVA scores was computed
landmarks with relatively large variation between to visualise shape changes associated with canonical
repetitions. Because there were no major inconsistencies, variates axes. Deformation grids for the extreme points
all analyses were conducted using the initial landmark along each axis were shown to illustrate variation along
conguration. these axes. Interspecic allometry was also investigated
A.R. Amaral et al. / Zoology 112 (2009) 3847 41

by a multivariate regression of shape variables onto Table 3. ANOVA sex  species for cranium centroid size in
centroid size. These analyses were performed using delphinid cetacean species
TpsRegr 1.31 (Rohlf 2005).
Effect Sum of squares d.f. F P
Procrustes distances, which measure shape distances
between pairs of specimens computed directly in the Dorsal view
shape space, were obtained with TpsSmall 1.20 Sex 8.4033  1031 1 0.189 0.6652
(Rohlf 2003) and used to investigate phenetic relation- Species 1.9142  1034 2 21.503 o1  106
ships through cluster analyses in NTSYSpc 2.2. Sex  species 2.6940  1033 2 3.026 0.0547
(Rohlf 19982006). This program was also used to Error 3.2046  1034 72
compute the CVA. PAST (Hammer et al. 2001) was Ventral view
used to compute the rest of the statistical analyses. Sex 5.1811  1031 1 0.122 0.7383
Species 1.6495  1034 2 17.903 o1  105
Sex  species 3.7396  1033 2 4.059 0.0215
Results Error 3.2247  1034 70

Sexual dimorphism was not signicant for both shape

and size (Tables 2 and 3), although sample sizes
considering males and females separately were small, deformations associated with differences around land-
which suggests that males and females have, on average, marks 3, 4 and 5, which capture structures of the
similar cranial shape and the same cranial size in all zygomatic process, and a relatively longer rostrum
species. All further analyses were thus conducted with when compared to the cranial portion of the skull.
pooled sexes. S. coeruleoalba has a comparatively relatively larger
Cranial shape differences were found between the braincase and relatively shorter rostrum. T. truncatus
three studied species for both dorsal and VCs differs from the latter species by having a much larger
using MANOVA (Table 2) and CVA analyses (Fig. 2) braincase and an even shorter rostrum.
(Wilks L 0.0132, Po0.000001 for DC and Wilks Two main groups are visible in the plot along relative
L 0.0143, Po0.000001 for VC). Three main warp 1 (RW1) in any of the two analyses. One cluster
clusters are evident in the CVA ordination: D. delphis, corresponds to T. truncatus and the other one corre-
S. coeruleoalba and T. truncatus. Interspecic allometry, sponds to both D. delphis and S. coeruleoalba, which
although statistically signicant (Wilks L 0.3187, F77 shows the close morphological proximity between these
10.285, P 2.79  1013 for DC and Wilks L 0.2729, two latter species. The general pattern of morphological
F74 10.95, P 2.49  1014 for VC), explains a very differences outlined by the CVA is concordant with the
small percentage (7.7%, Goodalls F16.1472 9.8295, deformation grids obtained for the average DC and VC
Po0.0001 for DC and 10.6%, Goodalls F18.1638 of the two main groups.
6.8058, Po0.0001 for VC) of the variation in shape Phenetic clusters were obtained by both unweighted
among specimens of the three studied species. pair group method with arithmetic mean (UPGMA)
The rst two RWs explained 76.55% and 66.97% of and neighbour-joining (NJ) methods. However, only
the variance in the DC and VC, respectively (Fig. 3). UPGMA dendrograms are presented since this was the
Main shape differences between the three studied species method that better summarised the phenetic relation-
in both DC and VC can be illustrated using deformation ships among the studied taxa. Cophenetic correlations
grids obtained for the extremes of each canonical axis; of 0.858 for the DC and 0.905 for the VC were obtained
D. delphis has a narrower brain case, shown by versus only 0.786 (DC) and 0.811 (VC) obtained when
the NJ was correlated with the matrix of original
Table 2. MANOVA sex  species for the dorsal and ventral Procrustes distances. The dendrograms for both DC and
aspects of the cranium shape in delphinid cetacean species VC (Figs. 4 and 5) also highlight the close proximity of
D. delphis and S. coeruleoalba, which appeared as sister
Effect Wilks lambda d.f. P taxon in a cluster separated from T. truncatus, which
occupies a more dissimilar position.
Dorsal view
Sex  species 0.1136 16 o1  106
Sex 0.7340 16 0.2751
Species 0.0104 32 o1  106 Discussion
Ventral view The present study investigated the pattern of cranial
Sex  species 0.1792 18 o1  106 shape differentiation between three closely related
Sex 0.6566 18 0.0933
delphinid cetacean species within the species complex
Species 0.0203 36 o1  106
DelphinusStenellaTursiops. Our main purposes were
42 A.R. Amaral et al. / Zoology 112 (2009) 3847

(-) CV 1 (+) (-) CV 1 (+)

(-) CV 2 (+) (-) CV 2 (+)

8 8

6 6

CV 2 (17.62%)

CV 2 (17.57%)
-6 -4 -2 0 2 4 6 8 10 12 14 -4 -2 0 2 4 6 8 10 12 14
CV 1 (82.38%) CV 1 (82.43%)

Fig. 2. Scatterplots of canonical variates (CV) 1 and 2 (percentage given in parentheses) obtained for (a) dorsal and (b) ventral
aspects of the cranium shape of delphinid species. White circles correspond to D. delphis specimens, grey circles to S. coeruleoalba
specimens, black circles to T. truncatus specimens. Thin plate spline deformation grids for the extreme points of each axis are shown.

to nd out if cranial morphology (shape), analysed with The absence of sexual dimorphism in cranial shape is
geometric morphometric procedures, would differenti- in contrast to traditional morphometric studies con-
ate the studied species, and if it would be informative to ducted so far. Sexual dimorphism has been described for
equate these differences with previously proposed D. delphis (Murphy and Rogan, 2006), T. truncatus
cladistic groups. (Wilson et al., 1999) and several other delphinid cetacean
Results from CVA and MANOVA analyses suggest species such as Stenella attenuata (Sanvicente-Anorve
that the three species are well differentiated and that et al., 2004) and Orcinus orca (Clark and Odell, 1999).
sexual dimorphism in shape is negligible when compared These studies showed that sexual dimorphism is gen-
with the interspecic differences. It is quite clear from erally expressed by sexual differences in overall body size
the results that the inter-taxon comparison performed and shape, shape and size of dorsal ns, postanal
surpasses any effects of sexual dimorphism, regardless if humps and pigmentation patterns. In the northeast
there is sexual dimorphism or not. The analyses would Atlantic and North Pacic Ocean, sexual dimorphism
not be affected at all by sample sizes since morpholo- was also found to be present in cranial features of
gical variation between taxa is much larger than any D. delphis, with males being larger than females
intraspecic shape difference (see e.g. CVA). In fact, (Heyning and Perrin 1994; Murphy et al. 2006).
sexual dimorphism is not crucial for this study, since One of the disadvantages of these studies was that
interspecic comparison was the main aim. However, size-free shape variables could not be extracted from the
patterns of morphological variation among taxa linear distance measurements used. This is a major
(RW analyses) do not show a clear separation between drawback of traditional morphometric approaches
D. delphis and S. coeruleoalba; only T. truncatus appears (Rohlf and Marcus 1993; Adams 2004), and hence
clearly differentiated. RW analyses preserve the geo- results were highly inuenced by differences in size
metric properties of shape space, whereas in the CVA, among samples. One main advantage of studying cranial
the space is based on statistical distances and differences shape with landmark-based geometric morphometric
between groups, not between individuals. Morphologi- methods is that size is discarded by the superimposition
cal similarity between D. delphis and S. coeruleoalba methods, thus shape differences are those that remain
should in principle be interpreted as high for both invariant to size (Rohlf and Slice 1990). Also, size
cranial views. (centroid size) in geometric morphometrics is computed
A.R. Amaral et al. / Zoology 112 (2009) 3847 43

Dorsal Ventral
(-) RW 1 RW 1
(+) (-) (+)

RW 2 RW 2
(-) (+) (-) (+)


RW 2 (15.51%)


RW 2 (19.18%)



-0.15 -0.10 -0.05 0.00 0.05 0.10 -0.15 -0.10 -0.05 0.00 0.05 0.10
RW 1 (61.04%) RW 1 (47.79%)

Fig. 3. Scatterplots of the two rst relative warps (RW) obtained from the relative warp analysis of the (a) dorsal and (b) ventral
aspects of the cranium shape of delphinid species. White circles correspond to D. delphis specimens, grey circles to S. coeruleoalba
specimens, black circles to T. truncatus specimens. Thin plate spline deformation grids for the extreme points of each axis are shown.


0.13 0.11 0.08 0.05 0.02

Procrustes Distance

Fig. 4. UPGMA phenogram computed on the matrix of Procrustes distances among mean dorsal aspects of the cranium (DC) shape
of delphinid species.
44 A.R. Amaral et al. / Zoology 112 (2009) 3847


0.13 0.10 0.07 0.04 0.00

Procrustes Distance

Fig. 5. UPGMA phenogram computed on the matrix of Procrustes distances among mean ventral aspects of the cranium (VC)
shape of delphinid species.

as an independent variable capturing the scale of the only the topology of the UPGMA dendrograms where
conguration of landmarks. the two species appear in the same cluster (as the RW
In the interspecic comparisons depicted by the analyses also suggest), but also the inconsistencies
phenograms the topology obtained is very similar to that have been described in both molecular and
the one obtained in phylogenetic analyses conduc- morphological data (e.g. Amaral et al. 2007b; Stockin
ted with mtDNA genes in the three studied species and Visser 2005).
(Amaral et al. 2007a). These authors compared the Skull-shape evolution in delphinid species may have
phylogenetic signal given by the mitochondrial genes for been indirectly inuenced by several evolutionary
cytochrome b and cytochrome c oxidase I (COI). The changes related to adaptation to particular niches.
obtained phylogenies differed in the level of divergence Feeding specialisations may have occurred and these
between D. delphis and S. coeruleoalba. While for the might explain modications in the form and length of
cytochrome b gene two well-distinct clades were the rostrum and also in the number of teeth. Braincase
obtained, for the COI gene, no differentiation was size, which is related to the development of the
evident. T. truncatus occupied a basal position in both communication and echolocation systems in delphinids,
analyses. The difference in phylogenetic resolution so important for their survival (Berta and Summich
between different mtDNA genes was explained to be 1999), may also have been subjected to evolutionary
due to the different molecular evolutionary rates of the forces according to the characteristics of the habitat and
two genes. However, an incomplete lineage sorting the social structure inherent to each species. Unfortu-
process may also account for the lack of differentiation nately, current understanding of dolphin niches does not
between D. delphis and S. coeruleoalba when mitochon- allow a detailed discussion of how and why these species
drial DNA genes are used, since the time of divergence have evolved (Gygax 2002). It could have been a matter
between them could have been insufcient for the of feeding specialisation or exploration of different
lineages to split completely (LeDuc et al., 1999; Amaral resources leading to different foraging behaviour, which
et al., 2007b). An incomplete lineage sorting process is one of the strategies adopted by sympatric species that
between the two species would mean that the speciation co-exist (Bearzi 2005). Recent studies on the feeding
process may not yet have been completed and that some ecology of D. delphis and S. coeruleoalba in the
level of interbreeding between the two species may still northeast Atlantic region have concluded that these
exist, since they occur in sympatry and mixed groups are species rely on different preys and have different feeding
frequently seen. If the rst generation of hybrids existed, strategies (Lahaye et al., 2005; Spitz et al., 2006; Pusineri
they would be quite hard to identify since morphologi- et al., 2007). Assortive mating leading to reproductive
cally they could resemble one of the parental species isolation could be another explanation for the occur-
(Mallet 2005). However, hybridisation could explain not rence of speciation in such closely related species.
A.R. Amaral et al. / Zoology 112 (2009) 3847 45

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gating phylogeographic patterns funded by FCT whales, dolphins, and porpoises. In: Proceedings of a
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