1

Department of Zoology, University of Graz, Graz, Austria;

2
Department of Research and Specialist Services, Fisheries Research
Division, Ministry of Agriculture, Food and Fisheries, Mpulungu, Zambia
Assessment of traditional versus geometric morphometrics for discriminating
populations of the Tropheus moorii species complex (Teleostei: Cichlidae),
a Lake Tanganyika model for allopatric speciation
M. Maderbacher
1
, C. Bauer
1
, J. Herler
1
, L. Postl
1
, L. Makasa
2
and C. Sturmbauer
1
Abstract
Lake Tanganyika harbours the oldest and ecologically, morphologically and behaviourally most diverse species ock of cichlid shes. Its species
are excellent subjects for the study of explosive speciation and adaptive radiation. Many species are subdivided into numerous genetically and
phenotypically distinct populations, often classied as distinct geographical races or colour morphs, which mostly dier in colour and much less
in terms of morphology. This study for the rst time quanties morphological dierences among such morphs by studying three populations of
Tropheus moorii. We compared traditional morphometrics (TM) and geometric morphometrics (GM) to explore their potential for
discriminating populations. So far species description and population discrimination are almost solely based on TM in the form of standardized
measurements, although specialists are aware of their lack of diagnostic power for discrimination of closely related entities. Moreover,
comprehensive TM measurements are time consuming and can best be done on dead specimens which have to be preserved in the case
re-measuring is necessary. In contrast, GM can also be based on photographs and computer scans of anaesthetized sh, so that the same
individual can be repeatedly analysed during its ontogeny. Here, we show that GM is more exible in data acquisition and more powerful in the
discrimination of species and closely related populations. While TM is restricted to distances and ratios of distances, GM not only includes these
measurements indirectly, but also allows for body shape analysis using a semi-landmark approach. It can be equally standardized as TM by
dening diagnostic landmarks. Data description by canonical variate analysis was most informative using GM data including semi-landmarks,
whereas dierences between populations were signicant (p < 0.05) based on both morphological approaches.
Key words: Cichlids comparative morphology landmark analysis semi-landmarks eastern Africa
Introduction
The Great Lakes of eastern Africa, Lake Tanganyika, Lake
Malawi and Lake Victoria are among the worlds most diverse
freshwater ecosystems and contain unique species ocks of
cichlid shes, each comprised of hundreds of endemic species
(Fryer and Iles 1972). With an estimated age of 912 Myr Lake
Tanganyika is the oldest of the Great Lakes (Cohen et al. 1993,
1997). Its species ock is of polyphyletic origin and can be traced
back to nine evolutionary lineages that once colonized the
emerging lake (Salzburger et al. 2002; Koblmu ller et al. 2005).
Despite Lakes Malawi and Victoria are younger, they harbour
more species of cichlid sh. However, the estimated 250 cichlid
species (Turner et al. 2001) in Lake Tanganyika are morpho-
logically, ecologically and behaviourally the most diverse (Fryer
and Iles 1972; Greenwood 1984; Chakrabarty 2005). Thus, Lake
Tanganyikas mature species ock is an excellent subject for the
study of explosive speciation and adaptive radiation. Following
the spatial segregation of the shore into rock- and sand-habitats,
many littoral species are subdivided into genetically and
phenotypically distinct populations.
The genus Tropheus, of which six nominal species (Poll 1986;
Snoeks et al. 1994) and about 120 distinctly coloured local
variants are currently described (Konings 1998; Schupke 2003),
represents an excellent example for this phenomenon. These
morphs occupy the same ecological niche all around Lake
Tanganyika, feeding on epilithic algae and keeping territories in
the upper littoral zone. Also, their overall morphology (except
coloration) is highly similar, and no dierences in mating
behaviour and breeding behaviour have been observed in
dierent morphs or species of Tropheus (Sturmbauer and Meyer
1992; Baric et al. 2003; Schupke 2003). This study is the rst to
quantify those small morphological dierences among three
populations of T. moorii. We applied traditional morphomet-
rics (TM) including all standard linear measurements in
combinationwithtraditional multivariate techniques, inparallel
with a geometric morphometrics (GM) approach. Geometric
morphometrics (Rohlf and Slice 1990; Bookstein 1991; Marcus
et al. 1996) is a landmark-based technique currently considered
to be the most rigorous morphometric technique, with more
powerful statistical analyses. This method also oers the chance
to observe the nature of shape dierences through visualization
of splines and by using sliding semi-landmarks to capture and
analyse curved outlines (Green 1996; Bookstein 1997).
The specic goal of this study was to compare the eciency
of TM versus GM methods for describing dierences between
closely related entities, such as populations or colour morphs
of T. moorii in Lake Tanganyika. We also tested if the
inclusion of sliding semi-landmarks resulted in improvements
in shape discrimination on the population level.
Materials and Methods
In May 2005, 211 individuals of T. moorii were caught at three
locations in the southern part of Lake Tanganyika (Fig. 1a). Just after
capture specimens were sent to the University of Graz, Austria alive.
With about 70 individuals from each of the T. moorii-populations
Katoto, Mbita and Nakaku a near-balanced number of male, female
and juvenile individuals was obtained for each population.
To obtain digital images from each sh a atbed scanner was used
(Herler et al. 2007). Specimens were previously anaesthetized with
clove oil, placed on the scanner (HP Scanjet 4070) in a custom-made
plasticine pool, which was lled with water. Digital images were taken
from the left side of the specimens. The pictures shown in Fig. 1b were
obtained with this method. After scanning sh were killed by
overdosing clove oil and placed in a 10% formalin solution. After a
few days specimens were washed and transferred to 70% ethanol, to be
2008 The Authors
Journal compilation 2008 Blackwell Verlag, Berlin
Accepted on 4 June 2007
J Zool Syst Evol Res doi: 10.1111/j.1439-0469.2007.00447.x
J Zool Syst Evol Res (2008) 46(2), 153161
stored until further examination. In the present study, a subset of 120
adult individuals within a small size range was selected for morpho-
metric analysis [Table 1; KruskalWallis test (p 0.172) and Mann
Whitney U-test (p > 0.19) for pairwise comparisons of populations
revealed no signicant dierences in the standard length]. Details of
the analysed specimens with their corresponding standard length are
given in Table S1.
Traditional morphometrics
Body proportions
The following 19 characters were measured on dead, preserved
specimens: standard length (SL), head length (HL), head width
(HW), snout length (SNL), interorbital width (IOW), lower jaw width
(LJW), lower jaw length (LJL), cheek depth (CHD), eye diameter
(ED), dorsal n base length (DFB), anal n base length (AFB),
abdominal length (VAB), body depth at ventral n (BDV), body depth
at anal n (BDA), body width at anal n origin (AW), caudal n
length 1 and 2 (CFL 1, CFL 2), caudal peduncle length (CPL) and
caudal peduncle depth (CPD) (see Fig. 2a). Measurements are mainly
based on the publications of Barel et al. (1977) and Snoeks (1994). For
denitions of measurements see Table S2.
Measurements were taken with a digital calliper with an accuracy of
0.01 mm directly as the distance between one reference point and the
other. All measurements were taken from the left side of the specimens
unless this side was damaged in which case the right side was used.
There were some specimens with damaged caudal n, for which
measurement of the caudal n length had to be omitted. Head
measurements were taken under a binocular microscope. To account
for eects of body size, manova, canonical variate analysis (CVA) and
principal component analysis (PCA) were performed on log
10
-trans-
formed measurements. Statistical analysis was carried out with the
computer software past v.1.47 (PAleontological STatistics; Hammer
et al. 2001).
Geometric morphometrics
The same 120 specimens of T. moorii used for TM were used in the
following analysis. Seventeen landmarks and nine semi-landmarks
were collected for each individual: (1) Anterior tip of the snout, (2) and
(3) anterior and posterior insertion of the dorsal n, (4) and (6) upper
and lower insertion of caudal n, (5) midpoint of the origin of the
caudal n, (7) and (8) posterior and anterior insertion of the anal n,
(9) insertion of the ventral n, (10) most ventral point of the border
(a) (b)
Fig. 1. (a) Map of Lake Tangan-
yika with details on sampling
locations along the southern shore.
(b) Representative specimens of the
three researched populations
Table 1. Mean, minimum (Min), maximum (Max) and standard deviation (SD) of traditional measurements on three populations of Tropheus
moorii from southern Lake Tanganyika
Katoto (n 38) Mbita (n 42) Nakaku (n 40)
Min Max Mean SD Min Max Mean SD Min Max Mean SD
Standard length (SL, mm) 57.9 85.9 74.8 6.11 65.3 82.4 73.6 4.39 61.2 87.9 75.7 7.9
Measurements (% SL)
Head length 30.56 34.12 32.29 0.77 30.50 34.45 32.57 0.81 31.68 34.98 32.87 0.80
Head width 16.08 17.46 16.80 0.36 16.09 17.71 16.85 0.38 16.38 18.57 17.17 0.44
Snout length 14.13 17.21 15.50 0.67 14.74 17.04 15.75 0.54 15.05 17.80 16.22 0.63
Interorbital width 11.13 12.40 11.72 0.32 10.90 12.48 11.69 0.37 10.99 13.29 12.11 0.46
Lower jaw width 13.56 15.97 14.70 0.64 14.16 16.09 14.94 0.40 13.86 16.43 15.11 0.54
Lower jaw length 7.92 9.59 8.72 0.40 8.14 9.62 8.96 0.38 8.26 10.08 8.88 0.36
Cheek depth 8.22 10.43 9.46 0.52 8.71 10.78 9.43 0.47 8.51 11.27 9.77 0.59
Eye diameter 7.66 9.81 8.52 0.44 8.02 9.32 8.55 0.31 7.65 9.40 8.45 0.48
Dorsal n base length 64.06 67.00 65.44 0.80 62.18 67.77 65.22 1.20 62.69 67.80 65.61 0.95
Anal n base length 23.20 26.76 24.83 0.88 23.22 26.43 25.08 0.77 22.74 26.27 24.59 0.75
Abdominal length 30.00 35.11 32.37 1.13 29.68 33.33 31.85 0.91 29.58 35.13 32.23 1.38
Body depth at ventral n 35.48 39.37 37.58 0.98 35.84 39.19 37.73 0.75 35.81 39.65 38.04 0.85
Body depth at anal n 28.34 33.26 31.17 0.98 28.82 32.82 31.32 0.83 29.05 33.43 31.50 0.95
Body width 13.68 18.21 16.57 1.10 15.28 17.88 16.79 0.64 14.69 17.73 16.52 0.73
Caudal n length 1 24.62 28.68 26.80 0.98 25.36 29.41 27.22 0.91 25.97 32.78 28.77 1.29
Caudal n length 2 24.15 28.84 26.37 1.16 25.40 28.83 26.86 0.81 25.53 30.29 28.03 1.19
Caudal peduncle length 13.66 15.87 14.79 0.53 13.92 16.70 14.76 0.52 13.52 16.49 14.95 0.77
Caudal peduncle depth 11.15 12.38 11.75 0.28 10.95 12.22 11.59 0.29 11.15 12.96 11.89 0.37
154 Maderbacher, Bauer, Herler, Postl, Makasa and Sturmbauer
2008 The Authors J Zool Syst Evol Res (2008) 46(2), 153161
Journal compilation 2008 Blackwell Verlag, Berlin
between interoperculum and sub-operculum, (11) the point where
praeoperculum, inter-operculum and suboperculum get in contact, (12)
upper insertion of the pelvic n, (13) dorsal origin of the operculum,
(14) dorsal end of the preopercular groove, (15) and (16) lie at the
extreme of the orbit along the anteroposterior body axis; capture the
width of the bony orbit, (17) most posterial point of the lips. For
positions of landmarks and semi-landmarks (19), see Fig. 2b.
For each specimen, the (X, Y) coordinates of 17 landmarks and 9
semi-landmarks were digitized using the software MakeFan (IMP
programs; Sheets 2003). The landmarks and semi-landmarks were rst
submitted to a generalized procrustes analysis (GPA; Dryden and
Mardia 1998; Rohlf 1999). This standard landmark-based morpho-
metric method removes all non-shape variation that can be attributed
to dierences in the location, orientation (or rotation) and scale of the
specimens. In this work, a partial procrustes superimposition, one
possibility to carry out generalized procrustes analyses, was used, in
which the centroid size (the square root of the summed squared
distances of landmarks from the centroid) is xed to the value 1
(Dryden and Mardia 1998; Rohlf 1999). The GPA iteratively estimates
a mean form and aligns all specimens to it. Due to the inclusion of
semi-landmarks we had to extend the standard procrustes superimpo-
sition procedure: in addition to translating, scaling and rotating
landmarks optimally, the semi-landmarks were slid along the outline
curve until they matched as well as possible the positions of
corresponding points along an outline in a reference conguration
(Adams et al. 2004). There are several criteria to slide points along an
outline. Two of the most widely used are minimum bending energy
(Bookstein 1996; Green 1996; Bookstein et al. 2002) and perpendicular
projection or minimum procrustes distance (Sampson et al. 1996;
Bookstein et al. 2002; Sheets et al. 2004). In our study we used both for
dierent applications. The comparisons below were based upon 17
landmarks alone, or on landmarks plus semi-landmarks.
Canonical variate analysis
The raw coordinates were aligned using the partial procrustes
superimposition (Rohlf 1999; Slice 2001) in CoordGen6f (IMP
programs, Sheets 2003). Semi-landmark alignment was carried out
using Semiland6 (IMP programs, Sheets 2003). This program allows
semi-landmark alignment based on perpendicular projection. The
components of the dierences in semi-landmark positions between the
reference form and the target form that are tangent to the curve were
mathematically removed. This procedure resulted in an alignment of
the semi-landmarks on the target form along lines perpendicular to the
curve passing through corresponding semi-landmarks on the reference
form (Sampson et al. 1996). As long as the contours lack abrupt
changes in curvature relative to semi-landmark spacing, this criterion
minimizes the distance between the semi-landmarks on the target and
the reference.
To assess the variation between the three study populations, we used
CVA (Mardia et al. 1979). Canonical variate analysis is a method of
nding the set of axes that allows for the greatest possible ability to
discriminate between two or more groups. The program CVAGen
(IMP programs, Sheets 2003) computes partial warp scores to a
common reference and then does a manova followed by a CVA. It
determines how many distinct CVA axes there are in the data at a
p-value of 0.05, and computes the canonical variates scores of all the
specimens entered.
Relative warp analysis
Relative warp (RW) analysis (which is a PCA of shape variables) was
also performed using the software TPSRelw (version 1.42, Rohlf 2002).
This program provides sliding semi-landmarks based on minimum
bending energy. The positions of the semi-landmarks were allowed to
slide along the direction parallel to the contours to minimize the
bending energy necessary to produce the change in the contour relative
to the reference (the GPA-estimated mean form).
The RWs were computed to summarize the variation among the
specimens (with respect to their partial warp scores) in as few
dimensions as possible. For a 0 this is a PCA of the covariance
matrix of the partial warp scores. There is also a visualize-window that
shows the deformation in shape from the reference that corresponds to
selected positions in the ordination.
(a)
(b)
Fig. 2. (a) Schematic representa-
tion of measurements on lateral
side of the sh. (b) Positions of
landmarks (white numbers) and
semi-landmarks (black numbers).
For denitions see Materials and
Methods
Discriminating cichlid sh populations 155
2008 The Authors J Zool Syst Evol Res (2008) 46(2), 153161
Journal compilation 2008 Blackwell Verlag, Berlin
In addition to CVA and RW analyses, Goodalls F-tests were carried
out in TwoGroup6 (IMP programs, Sheets 2003). To test for shape
dierences that might be attributed to allometry, Pearsons correla-
tions between standard length and the rst two CVA and RW axes
were calculated. Statistical analysis was carried out with past v.1.47.
Results
Traditional morphometrics
Canonical variate analysis of body measurements plotting CV
2 against CV 1 could not clearly separate the three T. moorii
populations (Fig. 3a). Although population-specic groupings
are obvious, there were large overlaps between all three
groups. The scatter-plot of CV 3 against CV 2 did not separate
the groups at all. Likewise, separation among the three
populations was not clearly evident in the PCA plot of the
third against the second PC axes (Fig. 3b). In the PCA, all
character loadings onto PC 1 were positively correlated and
most had similar values. PC 1 explained 87.95% of total
variation but was not considered because of its strong
correlation with size (R
2
0.997). Top loading characters on
PC 2 were lower jaw length, eye diameter, abdominal length
and caudal peduncle length (Fig. 3c). Characters loading most
heavily onto PC 3 included lower jaw length, anal n base
length, body width at anal n origin, caudal n length 1 and 2.
Table 1 lists details on traditional measurements. Results from
manova showed signicant dierences (Table 2a).
Geometric morphometrics
A Goodalls F-test was computed to test for overall shape
dierences between groups. We used the Bonferroni method to
adjust for the lack of independence in the comparisons
between populations. Results from Goodalls F-test for 17
landmarks and 17 landmarks plus 9 semi-landmarks are shown
in Table 2b. Based on shape variables the three populations
Katoto, Mbita and Nakaku diered highly signicantly in
their average body shape.
Canonical variate analyses
Canonical variate analyses scatter-plot based upon 17 land-
marks showed three groups (Fig. 4). Only a few specimens
overlapped, with the strongest overlap between Katoto and
Mbita. Fig. 4 displays deformation grids (scaling fac-
tor 0.06) correlated with CV 1 and 2. Both deformation
grids demonstrated shape changes mainly in the head region.
CVA scatter-plot based on 17 landmarks and 9 semi-land-
marks revealed stronger separation (Fig. 5), in that the
Nakaku group was fully separated from the other two groups.
Deformation grids including semi-landmarks also indicated
changes in the head region.
Assignment test in CVAgen revealed slightly more correct
group assignments for data including sliding semi-landmarks
(for 17 landmarks: 111 of 120, for 17 landmarks + 9 semi-
landmarks: 114 of 120 correct assignments). However, those
dierences were not signicant (v
2
0.64; p 0.42).
Additional CVA was carried out considering dierent sexes.
This CVA scatter-plot based on 17 landmarks (Fig. 6a) and 17
landmarks and 9 semi-landmarks (Fig. 6b) showed that there
are morphological dierences between males and females,
particularly between Nakaku males and females.
The CVA including semi-landmarks displayed solid
male and female groupings in the Nakaku and Mbita
population, while within Katoto population sexual dimor-
phism was not indicated. It showed that sexual dimorphism
was mainly caused by morphological dierences in head
shape. When only males or only females of the three
populations were compared based upon the 17 landmark
system, the clearest non-overlapping separation was found
(see Fig. S1a,b).
(a)
(b)
(c)
Fig. 3. Statistical analyses of body proportions (traditional morpho-
metrics) of three populations of Tropheus moorii (Nakaku m, Katoto
, Mbita h) from southern Lake Tanganyika. (a) Canonical
variate analysis. (b) Principal component analysis. (c) Loadings of
characters on PC 2 and 3
156 Maderbacher, Bauer, Herler, Postl, Makasa and Sturmbauer
2008 The Authors J Zool Syst Evol Res (2008) 46(2), 153161
Journal compilation 2008 Blackwell Verlag, Berlin
Relative warp analyses
The RW analysis on 120 male and female specimens, based on
17 landmarks (see Fig. S2a) separated most individuals of the
Nakaku group from the two other populations. Most Nakaku
specimens were located on the negative axis of RW 1. The rst
two RWs explained 29% and 10% of the total variation
among the three populations. Mbita and Katoto could not be
distinguished at all. As shown by the deformation grid for
negative RW 1 (Fig. 7a), Nakaku specimens have a steeper
dorsal head prole and the orientation of the mouth was not
the same as in specimens from Katoto and Mbita. Mouth
orientation of Nakaku appeared to be more in level. Also,
placement towards the negative RW 1 and RW 2 reected
slightly slimmer body form.
Relative warp analysis was also carried out for the 17
landmarks and 9 semi-landmarks system. Results did not dier
much from results of analyses based upon the 17 landmarks
only (see Fig. S2b). The same general shape variation was
observed, but by including semi-landmarks the steeper dorsal
head prole and the dierent mouth orientation of the Nakaku
population was clearer (Fig. 7b). Relative warp analysis for
males and females did not increase population discrimination
in either sex. None of the shape dierences were attributable to
allometry based on Pearsons correlations calculated between
standard length and the rst two CVA and RW axes.
Discussion
A large body of literature on comparative morphological traits
and their variation in shape exists for cichlid shes (e.g. Liem
1973, 1980; Barel 1983; Yamaoka 1983, 1997; Smits et al.
1996; Stiassny 1997; De Visser and Barel 1998; Bouton et al.
2002; Chakrabarty 2005). These works demonstrated that
structures of the oral jaw apparatus (OJA) are most signicant
in cichlid diversication. However, it was also argued that the
Fig. 4. Canonical variate analysis
on shape variables of 120 speci-
mens of three populations of
Tropheus moorii (Nakaku n,
Katoto , Mbita h) from
southern Lake Tanganyika based
on 17 landmarks and changes in
shape that are correlated with CV 1
and 2
Table 2. (a) Pairwise comparisons from manova of body proportions
of three populations of Tropheus moorii from southern Lake
Tanganyika. (b) Goodalls F-scores (100 bootstraps) and p-values
from landmark analysis
KatotoMbita MbitaNakaku KatotoNakaku
(a)
Wilks Lambda 0.5478 0.4205 0.3577
F-score 2.6 4.5 5.5
p* 7.5 10
)3
1.02 10
)5
1.47 10
)6
(b)
17 landmarks
F-score 3.3 16.1 17
p* 1.2 10
)3
0 0
17 + 9 landmarks
F-score 2.7 18.8 20.1
p* 7.98 10
)9
0 0
*Bonferroni adjusted p-value.
Fig. 5. Canonical variate analysis
on shape variables of 120 speci-
mens of three populations of Tro-
pheus moorii (Nakaku n, Katoto
, Mbita h) from southern
Lake Tanganyika based on 17
landmarks and 9 semi-landmarks
and changes in shape that are cor-
related with CVA axis 1 and 2 are
shown
Discriminating cichlid sh populations 157
2008 The Authors J Zool Syst Evol Res (2008) 46(2), 153161
Journal compilation 2008 Blackwell Verlag, Berlin
enormous eco-morphological diversity of cichlid sh was
achieved mainly by allometric changes of the same bony
structures and very rarely by de-novo evolution of characters
(Stiassny 1991). Recent studies have located regions in the
cichlid genome responsible for allometric shape dierences in
the OJA that could be linked to particular trophic specializa-
tions (Albertson and Kocher 2001; Albertson et al. 2003a,b).
Many meristic counts and measurements are often plastic and
overlap between species. As a consequence, comparative
morphology based on those standard measurements, here
called traditional morphometrics, often is at its limit when
closely related entities are analysed. This study compared TM
and GM techniques with respect to their potential to discrim-
inate three closely related populations of T. moorii. About 120
distinct populations are known, some of which live in
sympatry. This makes the genus to an ideal model to study
allopatric divergence and speciation. Populations can be
subdivided into nine major mtDNA lineages of about the
same age as the entire Lake Malawi cichlid ock (Sturmbauer
and Meyer 1992; Sturmbauer et al. 2005). Given the long time
of separate evolution of many populations and the great
degree of overall morphological similarity except for colora-
tion, it was suggested that their morphology is constrained by
stabilizing selection, but no systematic survey was attempted to
date.
Concerning data acquisition, one can state that the neces-
sary measurements for traditional comparative morphology,
such as measuring body proportions and counting scales, are
time consuming. Nineteen standardized measurements were
carried out in this study and it was impossible to do such
extensive measuring on life sh, because anaesthetization time
could not be extended long enough to carry out all 19
measurements in sucient precision. Thus, the necessity of
killing specimens is the rst major drawback of traditional
methods. Measurements had to be done on formalin-xed and
subsequently ethanol-preserved specimens. In contrast, GM
methods can be based on photographs and computer scans of
anaesthetized sh, so that killing and preserving of specimens
is unnecessary, unless voucher specimens are needed. More-
over, the same individual can be repeatedly analysed during its
ontogeny, even in the eld. Geometric morphometrics imply
the availability of more technical resources, such as computer,
camera or scanner during data acquisition, which might be
problematic in the eld. For type specimens from museum
collections, GM methods can be easily carried out, if there are
photos or scans available, so that fragile type specimens are
not touched. However, it is useless to compare data of fresh
and preserved specimens, as preservation aects shape (Ma-
derbacher and Herler, personal observations), so that each set
of comparisons needs to be carried out either on fresh or
preserved specimens.
Traditional morphometric data are measurements of
lengths, depths and widths. Such a data set contains relatively
little information about shape because many of the measure-
ments overlap or run in similar directions. Several of the
measurements originate from a single point, so some values
cannot be considered as completely independent (Zelditch
et al., 2004). In this study such a point was the anterior tip of
the snout. Standard length, head length, snout length and
lower jaw length had their origin at that position. It was
recently upheld that correlation of measurements complicates
the problem of describing shape dierences of particular body
parts (Zelditch et al. 2004).
An advantage of TM is the fact that measurements can be
done in three dimensions, while scans are two-dimensional
reductions of shape. Since our study object T. moorii is quite
deep-bodied, most informative structures and points could be
captured on the lateral view. Of course, additional scans
viewing the dorsal and/or ventral side are possible. An
alternative method for capturing three-dimensional images
would be the application of computer tomographic techniques
or three-dimensional scans.
Quality and interpretation of results
Both approaches showed that the three T. moorii populations
Katoto, Mbita and Nakaku varied in terms of morphology.
Statistical tests of both data sets determined that populations
signicantly diered from each other. A closer similarity of
Katoto to Mbita rather than to Nakaku was indicated
(Table 2). The CVA and PCA on TM data did not provide
any useful results for separating the populations. A slight
segregation of the Nakaku population along the third PC axis
could be associated with caudal n length and body width at
(a)
(b)
Fig. 6. Canonical variate analysis on shape variables of 120 specimens
of three populations of Tropheus moorii (Nakaku males n, Nakaku
females Katoto males , Katoto females Mbita males h,
Mbita females h) from southern Lake Tanganyika based on (a) 17
landmarks and (b) 17 landmarks and 9 semi-landmarks, divided into
populations and sexes
158 Maderbacher, Bauer, Herler, Postl, Makasa and Sturmbauer
2008 The Authors J Zool Syst Evol Res (2008) 46(2), 153161
Journal compilation 2008 Blackwell Verlag, Berlin
anal n origin, as suggested by characters loadings (Fig. 3c).
However, the third component only accounted for 2.18% of
total variance and not even the Nakaku specimens were clearly
separated. Clear-cut conclusions about dierences in length of
caudal n or body width between Nakaku and the other two
groups could not be drawn by PCA on TM data.
A general problem of TM data sets is that information
about shape can only be derived from ratios among
particular measurements. Consequently, more complex as-
pects of shape dierences are not addressable. In contrast,
GM methods are highly eective in capturing information
about shape, as geometry of the whole organism is captured
by the landmarks, and even more directly by sliding semi-
landmarks. Canonical variate analyses of landmark-based
data conrmed a closer position of Katoto specimens to
Mbita rather than to Nakaku, as indicated by Goodalls
F-test. CVA on data set including semi-landmarks fortied
these ndings as Nakaku specimens were totally separated
from the other two groups in scatter-plot. Sturmbauer et al.
(2005) and Sefc et al. (2006) recently showed that the
Katoto population goes back to natural hybrids between the
population at Nakaku (it belongs to the Chaitika Mountain
range colour morph) and Mbita (Mpulungu morph). It is
thus an interesting nding that Katoto individuals are
morphologically more similar to the Mbita population than
to that at Nakaku. The potential to visualize shape changes
implied by CVA provided the opportunity to localize
dierences between populations. Deformation grids showed
that most morphological variation occurred in the head
region. Compared to overall body shape, Fryer and Iles
(1972) observed that the head region, particularly the
mouth, is the most diverse in cichlid shes. Cichlid shes
diversied by trophic specialization, so that structures of the
trophic apparatus dier most among species. Indeed, this is
also the case in the analysed Tropheus populations, albeit
dierences are small and sh occupy exactly the same
trophic niche.
To check for sexual dimorphism an additional CVA was
carried out for males and females separately. This demon-
strated morphological dierences between males and females,
particularly visible in scatter-plot based on landmarks and
semi-landmarks. By adding semi-landmarks to our landmark
system we were able to detect shape variation in curved body
parts, such as the forehead. The dierences between males and
females (see deformation grid in Fig. 7) occurred in the head
region, especially in the forehead. Other studies, on more
comprehensive samples, are intended to denitely quantify
morphological dierences between males and females. Another
interesting observation from the CVA scatter-plot shown in
Fig. 7 is the fact that Katoto males and females are positioned
between Mbita males and Mbita females. Mbita females are
morphologically more similar to Katoto males than to their
own males. This unexpected result needs to be addressed in
further studies concerning the Katoto hybrid status.
Concerning potential inuences of allometric changes dur-
ing growth, we selected adult individuals of the same size range
of all three populations. To test whether it is better to pool the
sexes or to dierentiate between males and females for
discrimination of populations, a separate CVA for males and
females (17 landmarks were analysed) was carried out. These
sex-specic scatter-plots separated the populations much
clearer, in that specimen clusters did not overlap between
populations. Also, it is not so easy to sex life juvenile Tropheus,
so that only mature individuals can be analysed by sex without
killing the sh.
In addition to CVA we carried out an RW analysis. Again,
scatter-plots showed a closer position of Katoto specimens to
Mbita than to Nakaku. Deformation grids indicated that mouth
orientation of Nakaku specimens is more in level than that of
Katoto and Mbita individuals. The individuals of the Nakaku
Fig. 7. (a) TPS deformation grids
for the extreme points (see Fig. -
S2a) of each relative warp axis (17
landmarks). (b) TPS deformation
grids for the extreme points (see
Fig. S2b) of relative warp axis 1
and 2 (17 landmarks and 9 semi-
landmarks)
Discriminating cichlid sh populations 159
2008 The Authors J Zool Syst Evol Res (2008) 46(2), 153161
Journal compilation 2008 Blackwell Verlag, Berlin
population had a steeper dorsal head prole. Interestingly, the
sex-specic RW analysis did not result in clearer separation of
the populations as opposed to the sex-specic CVA.
To conclude, both TM and GM methods are able to
discriminate populations in the genus Tropheus. However, the
dierences can much better be visualized and quantied by
coordinate-based methods, which should be especially pre-
ferred for dierentiation of closely related entities. The inclu-
sion of sliding semi-landmarks led to a clearer albeit not
signicantly better discrimination of the three study popu-
lations. However, it was possible to address more complex
aspects of body shape such as the dorsal head prole that might
be of great relevance for the assessment of sexual dimorphism.
Acknowledgements
We would like to thank P. Ngalande, H. Phiri and D. Sinyinza,
Department of Fisheries, Ministry of Agriculture and Co-
operatives, Republic of Zambia, for their support during eld
work. This study was nanced by the Austrian Science fund
project number P17680-B06.
Zusammenfassung
Quantitativ morphologische Diskriminierung von drei Tropheus-Popu-
lationen (Teleostei: Cichlidae) des Tanganyikasees
Der Tanganyikasee geho rt zu den komplexesten und mannigfaltigsten
Su wassero kosystemen der Welt. Er bietet Lebensraum fu r einzigar-
tige Buntbarschartenschwa rme, welche, jeder fu r sich, aus hunderten
endemischen Arten bestehen (Fryer and Iles 1972). Viele Arten sind
wiederum unterteilt in zahlreiche genetisch und pha notypisch unter-
schiedliche Populationen. Diese Studie quantiziert morphologische
Unterschiede zwischen drei Populationen der Art Tropheus moorii.
Auerdem werden die Methoden der Traditionellen Morphometrie
(TM) und der Geometrischen Morphometrie (GM) angewandt und
hinsichtlich ihrer Mo glichkeiten verglichen. Bislang beruhten Arten-
beschreibungen und die Dierenzierung von Populationen hauptsa ch-
lich auf traditionellen Methoden, wie standardisierten Messungen und
meristischen Daten. Diese Verfahrensweise ist mit zahlreichen Ma n-
geln behaftet und die Ergebnisse sind oft zweideutig und umstritten.
Messungen beanspruchen sehr viel Zeit und ko nnen nur an toten
Individuen durchgefu hrt werden. Diese mu ssen, fu r den Fall einer
erneuten Messung, fu r la ngere Zeit aufbewahrt werden. Im Gegensatz
dazu basiert die geometrische Morphometrie auf Photos oder Com-
puter-Scans von z.B. nur beta ubten Tieren. Somit ist es mo glich,
denselben Fisch mehrmals im Verlauf seiner Ontogenie zu analysieren.
Die Geometrische Morphometrie (Rohlf and Bookstein 1990 ; Rohlf
and Slice 1990; Bookstein 1991; Marcus et al. 1996) arbeitet mit
homologen anatomischen Punkten (Landmarken), mit deren Hilfe die
Form und Proportionen eines Objektes, in unserem Fall eines Fisches,
beschrieben werden kann. Komplexe Ko rperkonturen ko nnen u ber-
dies mit Hilfe von sliding Semi-Landmarks in die Analyse einbezo-
gen werden.
Ergebnis der Studie ist, dass man mit beiden Methoden signikante
Unterschiede zwischen den Populationen nden kann. Aufgrund von
Vorteilen in der Datenaufnahme, der vielen Mo glichkeiten der
Datenbeschreibung und der besseren Visualisierbarkeit der Ergebnisse
ist geometrische Morphometrie in Summe die aussagekra ftigere
Methode, besonders deutlich unter Einbeziehung von Semi-Land-
marks. Fu r die Dierenzierung von sich sehr nahe stehenden
Gruppen, wie Populationen oder Schwesternarten, erscheint geome-
trische Morphometrie besser geeignet als traditionelle Methoden.
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Authors addresses: Michaela Maderbacher, Department of Zoology,
University of Graz, Universita tsplatz 2, 8010 Graz, Austria. E-mail:
michaela.maderbacher@edu.uni-graz.at; Christian Bauer, Department
of Zoology, University of Graz, Universita tsplatz 2, 8010 Graz,
Austria. E-mail: cyprinus@gmx.at; Ju rgen Herler, Department of
Zoology, University of Graz, Universita tsplatz 2, 8010 Graz, Austria.
E-mail: juergen.herler@univie.ac.at; Lisbeth Postl, Department of
Zoology, University of Graz, Universita tsplatz 2, 8010 Graz, Austria.
E-mail: lisbeth.postl@edu.uni-graz.at; Lawrence Makasa, Department
of Research and Specialist Services, Fisheries Research Division,
Ministry of Agriculture, Food and Fisheries, PO Box 55, Mpulungu,
Zambia; Christian Sturmbauer (for correspondence), Department of
Zoology, University of Graz, Universita tsplatz 2, 8010 Graz, Austria.
E-mail: christian.sturmbauer@uni-graz.at
Supplementary Material
The following supplementary material is available for this
article online:
Fig. S1. Canonical variate analysis on shape variables based
on 17 landmarks including only 60 males and only 60 females
of three populations of Tropheus moorii from southern Lake
Tanganyika
Fig. S2. Relative warp analysis of 120 specimens of three
populations of Tropheus moorii from southern Lake Tanga-
nyika based on 17 landmarks and 17 landmarks and 9 semi-
landmarks
Table S1. Specimens and their corresponding standard
length included in analysis
Table S2. Denitions of measurements
This material is available as part of the online article from:
http://www.blackwell-synergy.com/doi/abs/10.1111/j.1439-0469.
2007.00447.x
(This link will take you to the article abstract).
Please note: Blackwell Publishing are not responsible for the
content or functionality of any supplementary materials
supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author for
the article.
Discriminating cichlid sh populations 161
2008 The Authors J Zool Syst Evol Res (2008) 46(2), 153161
Journal compilation 2008 Blackwell Verlag, Berlin