2006
892
197211
Original Article
GENETIC AND MORPHOLOGICAL DIFFERENTIATION IN DUNG BEETLE SISTER SPECIES
A. PIZZO
ET AL
Biological Journal of the Linnean Society, 2006, 89, 197211. With 8 figures
The present study investigated the morphological and genetic differentiation pattern between two sympatric dung
beetle sister species, Onthophagus taurus and Onthophagus illyricus. The geometric morphometric approach coupled
with the use of molecular markers allowed examination of the nature of interspecific relationships and an outline of
the evolutionary and geographical processes that might have led to interspecific differentiation and the present-day
partial sympatric and syntopic pattern of distribution. Geometric morphometrics failed to discrimininate the two
species on the ground of external morphological traits, but revealed interspecific differences when the shape of the
primary sexual traits was taken into account. The use of two different molecular markers (cytochrome oxidase subunit I gene and amplified fragment-length polymorphism) demonstrates that the two species are genetically well differentiated, forming two distinguishable lineages probably diverged during the Pliocene by allopatric speciation. No
evidence of past or recent introgression and no support for hybridization were found, suggesting that sympatry was
established subsequently, when speciation was accomplished. Both molecular markers and genital shape indicate
that phenotypically intermediate individuals, despite their ambiguous appearance, are members of O. illyricus species rather than hybrids. 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006,
89, 197211.
ADDITIONAL KEYWORDS: AFLP allopatric speciation COI gene external morphology genital
morphology geometric morphometrics interspecific shape variation mtDNA.
INTRODUCTION
Sister species, by definition, are derived from a common ancestor, shared by no other species and therefore
they are important models to study evolutionary
events that promote speciation. The importance of
identifying patterns of differentiation between closelyrelated species, was already recognized by Darwin
(1859) as a basis for understanding evolution by natural selection. Sister species often occur in sympatry
(Barraclough & Vogler, 2000), or share portions of the
distribution areas sometimes representing hybrid
zones, which provide an opportunity to investigate
genetic and ecological interactions between species
(Hardig et al., 2000). It has been suggested that sym.
*Corresponding author. E-mail: astrid.pizzo@unito.it
patric species which are also syntopic may have experienced the same environmental influences, at least
throughout their most recent evolutionary history
(Dawson et al., 2002).
The morphological similarity of sister species and
the existence of forms with intermediate morphological traits, together with their occurrence in sympatry
and in syntopy, have been interpreted in several ways.
Some authors consider that these conditions hint at
the possibility that speciation occurred in sympatry
(Seehausen, Witte & van Alphen; Barraclough &
Vogler, 2000; Barluenga & Meyer, 2004). Among the
several theories of speciation, sympatric speciation
has been the most controversial, but a growing body of
empirical data shows that a variety of evolutionary
processes can result in differentiation under sympatric conditions: the main accepted cause of divergence
2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211
197
198
A. PIZZO ET AL.
in sympatric populations is the ecological specialization to local environments (Kondrashov, Yampolsky &
Shabalina, 1998; Schluter, 2000), but sympatric
divergence trends on a large spatial scale have also
been hypothesized (Salewski, 2003). Sexual selection
through assortative mating (Seehausen et al., 1998;
Gavrilets & Waxman, 2002) and sexual conflict
(Arnqvist et al., 2000) have also been proposed as
mechanisms possibly promoting divergence between
sympatric populations.
However, it is believed that most species evolved in
allopatry by divergence of geographically-isolated populations from ancestral species (Mayr, 1963). Allopatric speciation, due to barriers that hinder gene flow,
may be followed by subsequent re-colonizations and
re-establishment of sympatry by the sister species
(Bernatchez & Dodson, 1990). In these cases, when
interspecific barriers are not completely developed,
hybridization can occur in the overlap zone.
Speciation has long been thought to involve a process of genetic divergence between populations coupled with a secondary acquisition of morphological
differences (Mayr, 1963). Nevertheless, species deriving from a common ancestor may show genetical
divergence without accompanying morphological
changes (Mathews et al., 2002). In these cases, the correct classification and interpretation of interspecific
relationships could be obtained only when genetic
data are considered (Parson & Shaw, 2001).
Onthophagus taurus Schreber 1759 and Onthophagus illyricus Scopoli 1763 are the only two European
species of the subgenus Onthophagus s.s. (Zunino,
1979), usually considered sister species for their
noticeable morphological similarity (Balthasar, 1963;
Baraud, 1992; Lohse & Lucht, 1992; Moczek & Emlen,
1999; Martin-Piera & Lopez-Colon, 2000). Onthophagus taurus shows a typical TuranicEuropean
Mediterranean distribution (Balthasar, 1963). The
chorology of O. illyricus is TuranicEuropean, and its
distribution greatly overlaps with that of O. taurus
but, because of the unreliability of some geographical
data, its actual distribution is still imprecise (MartinPiera & Lopez-Colon, 2000). However, in the extensive
overlap zone, the two species often occur in syntopy.
Males of both species exhibit alternative phenotypes
(male polyphenism); individuals larger than a critical
body size threshold develop a pair of disproportionated
head horns (major or horned males), whereas
smaller minor males develop only rudimental horns
or remain hornless (Moczek, 1996, 1998; Emlen &
Nijhout, 1999; Moczek & Emlen, 1999; Simmons,
Tomkins & Hunt, 1999; Hunt & Simmons, 2000;
Moczek & Emlen, 2000; Palestrini, Rolando & Laiolo,
2000; Hunt & Simmons, 2002).
According to the traditional classification criteria
(Janssens, 1960; Paulian & Baraud, 1982), differences
MORPHOMETRIC ANALYSIS
2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211
199
N (CO I)
N (AFLP)
Species
Sampling locality
Region, country
Females
Males
Females
Females
Onthophagus taurus
La Mandria
Brlog
Aosta
Modena
Caserta
Toulon
Doana
La Mandria
Brlog
Trieste
Caserta
La Mandria
Piemonte, Italy
Croatia
Western Alps, Italy
Emilia, Italy
Campania, Italy
France
Spain
Piemonte, Italy
Croatia
Friuli, Italy
Campania-Italy
Piemonte-Italy
79
81
20
70
70
17
5
2
2
2
2
2
2
5
2
3
2
4
9
7
5
9
Onthophagus illyricus
IUTS
For each species, sampling locality, region-country and number of individuals used in each analysis are given. COI,
cytochrome oxidase subunit I; AFLP, amplified fragment-length polymorphism; IUTS, individuals with intermediate
phenotypic condition.
Figure 1. Drawings of head of both sexes, pronotum, and elytra of Onthophagus taurus and Onthophagus illyricus.
Photographs of pronota and elytra were taken at 12.5 magnification, whereas heads are at 32 magnification The
landmarks for head (N = 5), pronotum (N = 4), and elytra (N = 7) were digitized on half of each structure to remove the
variability introduced by an eventual asymmetry.
IUTS) were found (Table 1). Images of each anatomical structure were captured using a digital camera
Olympus DP11 connected to a stereoscopic microscope
Leica MZ8, taking care to align the edges of each
structure on the same horizontal plane. Genitalia
2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211
200
A. PIZZO ET AL.
Figure 2. Female genitalia: the sclerotized area of the vagina and the landmarks ( N = 5) used in the geometric morphometric analysis. Genitalia were held with a pair of tweezers in a thin film of glycerol and photographed at 50 magnification. The two most extremely different morphologies are shown.
Figure 3 (left paramere of aedeagus). They were chosen for their relative ease of identification, their
homology in the two species, and the ability of the
suite of landmarks to capture the general shape of
each morphological structure.
To evaluate the confidence of the landmark configuration, a repeatability test was conducted by digitizing the landmarks ten times on the same specimen
and then calculating the ratio between the variance
on the same specimen and the variance of the total
sample (variance = (Procrustes distances) 2/n 1,
where n is the number of objects considered in each
set of measures). The landmark configuration was
accepted only if the ratio was less than or equal to
0.05.
MOLECULAR
MARKERS ANALYSES
2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211
201
(Villalba et al., 2002) referring to some European species of the genus Onthophagus and other two species
(Euoniticellus pallipes and Euoniticellus fulvus) used
as an outgroup.
Pairwise distances between haplotypes were
obtained under the assumption of the JukesCantor
model, and the tree was constructed by the Neighbourjoining (NJ) method (Saitou & Nei, 1987). Robustness
of the inferred trees was tested by bootstrapping
(Felsenstein, 1985) with 1000 replications. An
unweighted maximum parsimony (MP) analysis
(PAUP, version 4.0, Beta 10, Swofford, 1990) was performed using a heuristic search with 100 random
sequence additions and TBR branch swapping.
AFLP markers
The AFLP method was performed as described by Vos
et al. (1995) with a few modifications. Genomic DNA
was cut using the restriction enzymes EcoRI and MseI,
and double-stranded EcoRI and MseI adapters were
ligated to the sticky ends of the fragments. Preselective PCR was carried out with 1 : 5 template dilutions
and with an EcoRI primer (EcoRI adaptor sequence)
and MseI primer (MseI adaptor sequence + 1 nucleotide). Dilutions (1 : 20) of the preselective PCR
products were used as templates for selective amplification, which was performed with an EcoRI primer
containing two selective nucleotides and a MseI
primer containing three selective nucleotides. An initial screening using six selective primer combinations
was performed on 12 individuals (three O. taurus and
three O. illyricus from La Mandria and as many from
Brlog). The primer combinations giving clear, reproducible, and polymorphic electrophoretic patterns
were chosen for further studies. Analyses were performed on a total of 34 individuals, as indicated in
Table 1. The AFLP adapter sequences, preamplification primer sequences, and selective amplification
primer sequences were as previously reported by Carisio et al. (2004).
Selective amplification products were separated
with an AFLexpressII automated sequencer (AP Biotech). Fragments were automatically scored by the
program ALFwin Fragment Analyser 1.0 (AP Biotech).
When peak height exceeded the standard parametersetting threshold, a peak (i.e. a fragment) was scored
as present (1), otherwise it was scored as absent (0).
An additional visual check of gels was made to correct
possible misinterpretations of automated procedures.
For each combination, all loci showing a clear and
unambiguous banding pattern were scored, whereas
uncertain peaks were considered as missing data.
Results of scoring were exported to a presence/absence
matrix and used for further analyses. Band sizes were
estimated using a standard size marker (Gitelman &
Davis, 1997).
2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211
202
A. PIZZO ET AL.
RESULTS
GEOMETRIC
MORPHOMETRIC ANALYSIS
2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211
203
GENETIC
ANALYSES
Sequence analysis
Alignments of the 597-bp segments of the mitochondrial CO I gene obtained from 33 individuals (and one
O. taurus COI sequence taken from the GenBank)
were straightforward and all sequences were translated into amino acids, suggesting that they are functional. No indels or premature codons were found.
Onthophagus illyricus populations shared a single
haplotype, with a nucleotide composition of A = 30.8%,
C = 15.6%, G = 13% and T = 40.6%, whereas the alignment of O. taurus sequences yielded five different haplotypes with eight polymorphic nucleotide sites and a
mean base composition of A = 31.4%, C = 15.3%,
G = 12.6% and T = 40.7%, which is very similar to that
of O. illyricus.
2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211
204
A. PIZZO ET AL.
Table 2. Results of discriminant analysis on relative warps score of female and male genitalia
Classification after discriminant analysis
Female
II
Male
II
Onthophagus
Onthophagus
IUTS
Onthophagus
Onthophagus
Onthophagus
Onthophagus
IUTS
Onthophagus
Onthophagus
taurus
illyricus
taurus
illyricus
taurus
illyricus
taurus
illyricus
Onthophagus taurus
Onthophagus illyricus
IUTS
% correct
79
81
20
79
101
70
70
17
70
87
75
3
1
75
4
64
1
3
64
6
1
48
10
4
97
2
51
4
6
81
3
30
9
4
18
10
95
59
45
95
96
91
73
59
91
93
I: the three groups [O. taurus, O. illyricus and IUTS, individuals with intermediate phenotypic condition (IUTS)] are
analysed separately. II: the IUTS are included in the O. illyricus group, as suggested by the relative warp plot.
2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211
205
Figure 6. Phylogenetic tree resulting from Neighbour-joining reconstruction. Robustness was tested by bootstrapping
with 1000 replications. Only bootstrap values higher than 70% are shown in the figure. Five different haplotypes were
found in Onthophagus taurus (A1A5) and only one in Onthophagus illyricus. Names of the species are followed by the
code of the sequence in GenBank Database.
Table 3. Statistics of genetic differentiation among population in each species (A); statistics of genetic differentiation
among species in each site where the two species are found in syntopy (B); and statistics of genetic differentiation among
species in the whole sample (C)
A
B
C
Onthophagus taurus
Onthophagus illyricus
La Mandria
Brlog
Whole sample
HT
HW SE
HB SE
FST SE
0.243
0.196
0.410
0.441
0.417
0.236 0.012
0.182 0.012
0.197 0.027
0.221 0.028
0.206 0.022
0.007 0.000
0.014 0.000
0.213 0.000
0.22 0.000
0.211 0.000
0.027 0.051*
0.075 0.059**
0.517 0.065***
0.497 0.062***
0.504 0.052***
HT, total gene diversity; HW, average gene diversity within populations; HB, average gene diversity among populations in
excess of that observed within populations; FST, Wrights t fixation index for which significance was tested with 1000 random
permutations.
*P < 0.05; **P < 0.005; ***P < 0.001.
DISCUSSION
The present study investigated the morphological and
genetic differentiation pattern between two sympatric
2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211
206
A. PIZZO ET AL.
2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211
207
2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211
208
A. PIZZO ET AL.
The degree of genetic divergence between these sister species allowed us to estimate that they originated
in a time frame of 34 Mya, in the Pliocene, corresponding to an important frequency peak of speciation
for several beetle taxa (Davis, Scholtz & Philips, 2002;
Carisio et al., 2004; Ribera & Vogler, 2004). The
Pliocene climate changes could have fragmented the
distribution of the ancestral species in small isolated
populations, promoting speciation. Palaeoclimatic
fluctuations of the Plio-Pleistocene period are thought
to have heavily influenced the distribution of intraspecific genetic variation in numerous palearctic plants
and animals (Hewitt, 1996, 2000; Taberlet et al.,
1998). Many cycles of contraction/expansion of geographical ranges according to Pleistocene climatic
oscillations have led to the present genetic structure of
populations, species, and communities (Hewitt, 2000).
A population geographical structure is frequently
found in beetles as a result of extinctions, recolonization, and migration events during and after
the Pleistocene cold period (Reiss, Schwert & Ashworth, 1999; Carisio et al., 2004; Ribera & Vogler,
2004). Unexpectedly, the distribution of the genetic
variability observed in both O. taurus or O. illyricus
did not show appreciable geographical structure. Dispersal ability could have determined this unusual
absence of geographical population structure but
other factors, such as regional patterns of extinction or
recolonization, might have had important role
(Palumbi, 1995; Vogler, 1998). An elevated gene flow
among populations could be plausible for O. taurus,
which is known to be an extremely eurytopic species.
By contrast, O. illyricus is known as a more oligotopic
species (Lumaret, 1990; Borghesio, Palestrini & Passerin dEntreves, 2001; G. Dellacasa, pers. comm.) and
populations showed a unique mtDNA haplotype and
very similar AFLP profiles. In this case, dispersal ability and gene flow cannot exhaustively explain the
absence of geographical structure. However, a similar
marked interspecific and low intraspecific differentiation is described for two rodent sister species of the
genus Peromyscus (Zheng, Arbogast & Kenagy, 2003).
It has been suggested that this kind of pattern can
result from a recent expansion in population size and
geographical range. Nevertheless, further analyses
using other molecular markers must be conducted to
confirm this lack of geographical structure.
REFERENCES
Adams DC, Slice DE, Rohlf FJ. 2004. Geometric morphometrics: Ten years of progress following the revolution.
Italian Journal of Zoology 71: 516.
Albertson RC, Markert JA, Danley PD, Kocher TD. 1999.
Phylogeny of a rapidly evolving clade: the cichlid fishes of
Lake Malawi, East Africa. Proceedings of the National Acad-
2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211
209
2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211
210
A. PIZZO ET AL.
2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211
211
2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 197211