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Combination of geometric morphometric and genetic approaches applied to a debated
taxonomical issue: the status of Onthophagus massai (Coleoptera, Scarabaeidae) as an endemic
species vicarious to Onthophagus fracticornis in Sicily
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21 November 2023
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Zoology, 2011, 114 (4), 10.1016/j.zool.2011.03.003
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Combination of geometric morphometric and genetic approaches applied to a
debated taxonomical issue: the status of Onthophagus massai (Coleoptera,
Scarabaeidae) as an endemic species vicarious to Onthophagus fracticornis in
Sicily.
Astrid Pizzo*, Fabio Mazzone, Antonio Rolando and Claudia Palestrini.
Dipartimento di Biologia Animale e dell’Uomo, Università degli Studi di Torino, via Accademia
Albertina 13, 10123 Torino, Italy
*Corresponding author: astrid.pizzo@gmail.com
Number of text pages: 22
Figures: 8
Tables: 5
Keywords: size, shape, static allometry, COI, male polyphenism.
Abstract
The present study deals with the phenomenon of insular speciation and discusses, as a case
study, the debated taxonomical issue of the status of Onthophagus massai (Coleoptera,
Sarabaeidae) as an endemic species vicarious to O. fracticornis in Sicily. The authors investigated
the differentiation patterns between an insular population belonging to the supposed species O.
massai (collected in its locus typicus, Piano Battaglia) and three Italian O. fracticornis populations
(collected along a N-S latitudinal gradient). These patterns have been described and analysed using
multiple approaches: the qualitative inspection of the microsculpture of elytral surfaces, considered
a diagnostic character for O. massai identification; the comparison of horn static allometries, known
to be a good indicator of divergence processes between closely related species or isolated
populations of the same species; the comparison of the patterns of shape and size difference of the
head, epipharynx and genitalia attained with a combination of traditional and geometric (landmark
and semilandmark) morphometric methods, and, finally, the estimation of the genetic relationships
between Sicilian and continental populations obtained by analysing COI mitochondrial gene
sequences. The integration of the results of these approaches indicates that there is not sufficient
evidence to vindicate the species status for O. massai, which should more likely be considered a
small-sized version of O. fracticornis (a possible case of insular dwarfism). However, the complex
pattern of shape, size and genetic variation observed between the populations analysed hinted at the
possibility that a diversification process is ongoing, but not only between insular and continental
populations; each population showed a tendency to evolve as an evolutionary independent unit.
Introduction
Islands cover a low percentage of the global land area and even if, in general, they show less
specific biodiversity with respect to continental areas, new species are continuously described and a
great part of them are endemics (Croft et al., 2006; Cucchi et al., 2006; Grill et al., 2007; Lohman et
al 2010; Perez-Gelabert, 2008). Due to their restricted habitat and vulnerability, endemic species
can easily become endangered or undergo extinction, and islands are among the ecoregions which
have the highest percentage of extinct and extant endemic taxa; for this reason, the scientific
community has been called upon to make a special effort in estimating, describing and preserving
island biodiversity (Dapporto and Dennis, 2008).
On islands, scientists have often documented the acquisition of adaptive features - e.g. a tendency
towards flightlessness or flight reduction, lifecycle acceleration and body size changes - which may
take place rapidly and could accelerate insular speciation. These phenomena, primarily described
for mammals, are not rare in birds (Grant and Grant, 1996; Trewick, 1997) and are also described
for insects (Vernon, 1981, Salomon, 2001). In some cases, especially when islands are very close
to, or have been in physical connection with, the continent at some stage, the endemic fauna
inhabiting the island could be the results of a secondary isolation, accompanied by the extinction of
continental populations.
In this study we consider the case of Onthophagus massai (Coleoptera, Scarabaeidae), a supposed
species endemic to Sicily belonging to the so-called “fracticornis-opacicollis-similis” species
complex (Martín-Piera and Boto, 1999); Onthophagus massai has been described as a separate
species, vicarious with respect to O. fracticornis, by Baraud in 1975 (Baraud, 1975, 1992), on the
basis of the study of material from Piano Battaglia, a locality in the Madonie natural park, but its
specific status is controversial (Palestrini, 1981).
In his works, Baraud (1975, 1992) considered the microsculpture of the elytral interstriae as a
diagnostic character to differentiate O. massai from O. fracticornis: O. fracticornis should show a
prominent granulation and a total absence of punctuation, whereas O. massai has small presetal
granules and marked perisetal punctuation (Baraud, 1992, Falahee and Angus, 2010). Additionally,
the two species should be different in the shape of the parameres of the aedeagus. Palestrini (1981)
refuted the species status for O. massai, ascertaining it could be considered a small-sized variant of
O. fracticornis. The slight differences in the apical region of parameres which, following Baraud,
are characteristic of this taxon, according to Palestrini (1981) may be found occasionally in other,
small individuals from Italian, Greek and Anatolian populations. Moreover, Palestrini (1981) did
not find any differences in the endophallus sclerites (i.e. the “lamella copulatrice”), nor in female
genitalia between Sicilian and continental populations.
Coope (2001) reported the identification in fossil materials from the Last Interglacial in England, at
Trafalgar Square, of at least 49 specimens of O. massai, on the basis of their small size, elytral
puncturation and tenebrosity. A recent study based on karyotype analysis (Falahee and Angus,
2010) evidenced significant differences between Sicilian and continental samples of O. fracticornis
(in RCL for autosome 1, 5, 8 and 9, in CI for autosome 1, 5, 7-9, and in centromere position for X
and Y chromosomes). These authors suggested that this comparison revealed a degree of difference
vindicating the placing of O. massai as a separate species.
The species complex to which O. massai should belong includes at least three different species well
recognisable in allopatry (O. fracticornis, O. opacicollis, O. similis) (Angus, 2008; Macagno et al.,
in press), and it might hide other taxa (Martìn-Piera and Boto, 1999; Falahee and Angus, 2010,
Pizzo et al., unpublished data). This is not a rare condition in this group, because Onthophagus
beetles have undergone a dramatic evolutionary radiation making them the largest genus of beetles,
and one of the most species-rich genera of life on Earth (Hanski and Cambefort, 1991; Davis,
Scholtz and Philips, 2002; Emlen et al., 2005). Their environmentally-mediated and conditiondependent male horn expression, an extreme case of phenotypic plasticity (Emlen, 1994; Moczek
and Emlen, 1999; Moczek, 2006; Moczek, Cruickshank and Shelby, 2006), is probably one of the
key reasons for this rapid radiation; the existence of many species-complexes and the evidence of
ongoing speciation processes further demonstrate the recent diversification of the genus. Closely
related Onthophagus sister species (see for example Pizzo et al., 2006a,b) and species-complexes
(Macagno et al., in press) are ideal models to investigate the micro-evolutionary dynamics
promoting speciation and represent excellent material for qualitative/quantitative analyses that
could provide additional clues to explain the evolution of morphology in the genus.
With the aim of resolving the species status of O. massai as vicarious to O. fracticornis in
Sicily, and underlining the complexity of the phenomenon of insular differentiation, we investigated
the pattern of diversification between Sicilian and other Italian populations of O. fracticornis with a
combination of different approaches, by assessing 1) the value of qualitative differences in the
microsculpture of elytral interstriae as a diagnostic character for O. massai identification; 2) the
pattern of horn static allometries: as horn allometries are known to diverge rapidly among extant
taxa, even between closely related species or isolated populations of the same species (Moczek et
al., 2002; Moczek and Nijhout, 2003), different allometries are often optimal indicators of a
divergence process.
Moreover, previous studies on Onthophagus beetles have hinted at the occurrence of a
certain degree of developmental correlation between male horns and genitalia (Moczek and Nijhout,
2004; Parzer and Moczek, 2008) and between male horns and head shape (Macagno et al., 2009;
Pizzo et al., 2006a,b). For this reason, we also explored 3) the pattern of shape and size difference
between head, epipharynx and male genitalia. To inspect the morphological differentiation between
Sicilian and continental populations, we used a combination of traditional and geometric (landmark
and semilandmark) morphometric methods, the latter being able to detect subtle shape differences
and also to quantify the two aspects of morphological variations, shape and size, as uncorrelated
traits (Zelditch et al., 2004). We therefore assessed the extent to which size and shape divergence
patterns of head and paramere -the part of the aedeagus directly involved in coupling with specific
female structures - were congruent with the pattern of horn expression. Additionally, we inspected
the divergence patterns of the epipharynx - one of the mouth parts - which is considered not to be
subject to costs associated with horn development (Pizzo et al., 2009), and whose morphology
could therefore be more free to evolve independently from other traits, possibly under selective
pressures for optimal feeding functions (Verdú and Galante, 2004). As mitochondrial markers can
provide fine details on the degree of genetic differentiation that may have resulted from historical
processes leading to speciation events, we investigated 4) the genetic relationships between Sicilian
and continental populations by comparing cytochrome oxidase subunit 1 (COI) mitochondrial gene
sequences.
Materials and Methods
Sample
The sample is composed of individuals from 3 populations of O. fracticornis collected in
Italy in three localities along a N-S latitudinal gradient (North Western Alps, Central Apennine, and
Southern Apennine) and by individuals collected in Sicily, in the locus typicus of the supposed
species O. massai, Piano Battaglia. The number and the geographic origin of specimens used in
each morphometric analysis are reported in Table 1.
Individuals for morphometric analyses were cleaned in 100°C distilled water for 10 min and
dissected. Genitalia (parameres) were extracted and cleared in boiling 5% Potassium hydroxide
(KOH) for 5 min. For the lateral view they were positioned on wet cotton wool, taking care to align
their edges on the same horizontal plane. Heads, pronota, elytra and parameres(dorsal view), were
fixed separately on horizontally-levelled plasticine supports. Epipharynges were treated following
the protocol described in Pizzo et al. (2009), mounted on microscope slides and covered with
coverslips.
Two dimensional images of each structure were taken using a Leica Z16Apo stereoscopic
dissecting scope (Leica Microsystems AG, Wetzlar, Germany) at magnifications of 57.5x
(epipharynx, dorsal side; phalloteca, lateral side), 50x (paramere, dorsal side), 20x (pronotum,
dorsal side), 40x (elytra, lateral side), 200x (elytra, detail of the dorsal surface), 31.3x (O.
fracticornis horn, lateral side), 39.4x (O. similis and O. opacicollis head, dorsal side), 25x (O.
fracticornis head, dorsal side). After calibration, linear measurements of pronotum width (used as a
proxy for body size: Eberhard and Gutierrez, 1991) and horn length (taken as described in Macagno
et al., 2009) were taken with the software LAS v 2.5.0 (Leica Application Suite).
Molecular analyses were carried out on a subsample of 14 specimens, as shown in Table 2. .
Analyses
1. Qualitative analyses of the elytral microsculpture
Photographs of elytral surfaces were made at two different magnifications in two alternating
views (lateral, at 40x, and dorsal, at 200x magnification) to inspect the pattern of presetal
granulation and perisetal punctuation between populations, looking for the diagnostic differences
between O. fracticornis and O. massai.
2. Horn static allometry
An earlier study on O. fracticornis (Macagno et al., 2009) found that a sigmoidal model was
a good fit for male horn length-body size data. For each taxa, the horn static allometry was therefore
determined by fitting to the horn length-body size data a four-parameter sigmoidal regression in the
y = yo +
form
a ⋅ xb
c b + x b , where x is pronotum width (used as a proxy for body size), y is horn length,
y0 specifies minimum horn length, a defines the horn length range in the sample, b is a slope
coefficient and c is body size at the point of inflection of the sigmoid curve (commonly used as an
estimate of the average body size threshold at which Onthophagus males switch from the hornless
to the horned phenotype: Moczek et al., 2002; Moczek and Nijhout 2003; Moczek, Brühl and Krell,
2004; Macagno et al., 2009). Parameter values of the regression were obtained via iterations using
Sigma PlotTM (Systat Software Inc.) curve-fitting procedures.
The differences between the four sigmoid curves were inspected by first testing interspecific differences in mean pronotum width (observed values) with a one-way ANOVA and LSD
post-hoc tests, and in horn length (observed values) with a Kruskal-Wallis non-parametric ANOVA.
Additionally, to test difference between curves, we generated a best fit sigmoid regression curve
based on a reference population (a population obtained combining all samples) using Sigma Plot.
Using this curve, we computed the expected horn length for each individual given its body size, and
then the difference between observed and expected horn length (= residual horn length). We used
T-tests to compare residuals across populations. To know which parameter, if any, accounted for
these differences, we used parameter estimates as derived from Sigma Plot (means plus standard
errors), and used Welch T-test with sequential Bonferroni corrections to compare each parameter
across populations.
3. Morphometric analyses
Shape variation
A landmark- (for the heads, the epipharynges and the dorsal side of parameres) and
semilandmark- (for the lateral view of parameres) based on a geometric morphometric approach
(Bookstein, 1991; Rohlf and Marcus, 1993; Adams, Slice and Rohlf, 2004) was used to characterise
the shape of the structures analysed. All landmarks and semilandmarks were digitised by the same
person using TpsDig 2.10 (Rohlf, 2010) on the left side of the structures, with the aim of removing
any bias possibly caused by bilateral asymmetry. For each structure, the landmark or semilandmark
configuration (Fig. 1) was chosen following criteria of homology (Bookstein, 1991), detection ease,
and on the basis of the available sample sizes (i.e., taking care that the sample size was larger than
the number of landmark coordinates). To digitise semilandmarks on the lateral view of parameres
(Fig. 1e) with a satisfying degree of accuracy, we manipulated the images with MakeFan6 (Sheets,
2003) software, superimposing a regularly spaced grid on the curve between landmarks 1 and 4
(comb option).
Landmark configurations of each structure were superimposed with a full Procrustes fit (Rohlf
and Slice, 1990; Goodall, 1991: for semilandmark superimposition we used the slide method as
“chord= min bending energy” and 3 iterations), i.e., they were translated to a common origin, scaled
to unit centroid size and rotated to best fit using a least-squares criterion. The resulting coordinate
configurations in a non-Euclidean (Kendall's) shape space (Kendall, 1981, 1984; Rohlf, 1996) were
then projected into a linear tangent space by orthogonal projection (Dryden and Mardia, 1998). To
inspect patterns of shape variation in the sample, we performed a Principal Components Analysis
(PCA ) on shape coordinates by using TpsRelw (Rohlf, 2008). Visualisation of the resultant sample
distribution in morphospace was made with SPSS 18.0. Using the same software, we performed
canonical variate analyses (CVA) on shape variables of each structure separately. The reliability of
the discrimination was assessed by leave-one-out cross-validation (e.g. Lachenbruch 1967).
Size variation and static allometry of head, epipharynx and parameres
The centroid sizes (CS) of each landmark and semilandmark configuration (computed in Tps Relw,
Rohlf, 2008) were used as estimates of head, epipharynx and paramere size. A preliminary
inspection of scatterplots of pronotum width vs head, epipharynx and paramere size did not reveal
any significant deviation from linearity. Therefore, linear regression models were used to fit these
distributions. Kolmogorov-Smirnov tests were used to check the assumption of normality, and the
assumption of linear scaling was further tested by checking the absence of visible trends in the
scatterplot of standardised predicted values vs residuals (Sokal and Rohlf, 1995).
All analyses were conducted in SPSS 18.0.
4. Molecular analyses (COI sequencing)
DNA was extracted using Quiagen Dneasy columns from heads ground up with a Tissue
Lyser (Quiagen). A fragment of the mitochondrial gene COI was amplified, purified and sequenced
using the primers TL2-N-3014 (alias Pat) and C1-J-2183 (alias Jerry) (Simon et al., 1994).
Sequencing was performed on both strands using a CEQ8000 automated sequencer
(Beckman Coulter). Sequences were assembled, edited and aligned with Geneious Pro 4.7.6
software (Rozen and Skaletsky, 2000). Sequences of the two closest related species (O.similis and
O. opacicollis, Pizzo et al., in press) and a sequence of Onthophagus (Palaeonthophagus) vacca,
courteously provided by Dirk Ahrens as an outgroup, were added to the alignment.
Pairwise distances between haplotypes were estimated under the assumptions of the Jukes–Cantor
model in MEGA 2.1 (Kumar et al., 2001). Phylogenetic trees were constructed by the neighbour
joining (NJ), minimum evolution (ME) and Parsimony (MP) methods in MEGA 2.1. A maximum
parsimony (MP) phylogenetic tree was based on close-neighbour-interchange (CNI). Robustness of
the inferred trees was tested by bootstrapping (Felsenstein, 1985) with 1000 replications. Maximum
likelihood (ML) analyses used the default search parameters in RAxML VI-HPC v2.2.0 (Stamatakis
2006) with 25 replicates. A Bayesian analysis was also performed using MrBayes version 3.2
(Ronquist and Huelsenbeck, 2003). The substitution model for Bayesian analyses was selected
using the Akaike Information Criterion test implemented in the software MrModeltest v. 2.3
(Nylander, 2004). The test suggested GTR+G+I as the best fitting model. Four independent Markov
chain Monte Carlo runs, with one cold and three incrementally heated chains each, were performed
for 10 million generations, sampling trees every 1000th generation. The first 2 million generations
were discarded as burn-in, and the remaining trees used to construct the Bayesian consensus tree.
We used the TCS 1.06 software of Clement et al. (2000) to generate haplotype network (Templeton
et al., 1992).
Results
1. Qualitative inspection of the elytral surface
Dorsal and lateral views of elytral surface (Fig. 2) showed the presence of marked presetal
granules and the absence of punctuation in the samples from the N-W Alps and Southern
Apennines. We also highlighted this microsculptural pattern in some Sicilian specimens (as in the
ind. 1, Fig. 2), coexisting with individuals showing evident punctuation and virtually absent presetal
granules, the typical pattern described for O. massai (as in the ind. 2, Fig. 2). This last configuration
can be unexpectedly found also in the Central Apennines sample (fig. 2) .
2. Horn allometry
The ANOVA on mean pronotum width and subsequent LSD post-hoc tests for multiple
comparisons showed that, with the exception of the Southern Apennines and the NW Alps having
similar size (P=0.068), all populations differed significantly with respect to their pronotum width
(F3,171=26.539, P<0.001). On the other hand, a Kruskal-Wallis non-parametric ANOVA failed to
detect any difference in horn length across the four populations (P=0.162). T-tests performed to
compare the residuals derived from the unique sigmoidal regression across samples showed that
they were significantly different between the Sicilian and the other three populations, and between
the Central Apennines and the NW Alps, but not between the Southern and the Central Apennines
nor between the Southern Apennines and the NW Alps: four separated sigmoidal regressions, one
for each population (Fig. 3), probably describe the allometric structure of the sample better than a
single one. Parameters significantly differing between curves are shown in Table 3.
3. Morphometrics analyses
Shape variation
While PCA conducted on male head shape variables was not able to evidence any differences
between populations (results were the same when male morphs were analysed separately or
together), female head shape analysis revealed a certain degree of differentiation: the NorthWestern Alps seemed to be the most differentiated population and shared with those from the
Central Apennines one the upper left part of the morphospace; Sicilian specimens were mainly
located in the right lower part of the morphospace, while Southern Apennine specimens appeared to
connect the two groups (Fig. 4). As the epipharynx is not a sexual dimorphic structure (Pizzo et al,
2009), a PCA of shape variables was carried out taking the two sexes together which showed a high
level of overlap between populations; however, it was possible to detect a little differentiation with
a pattern similar to that evident for female head shape (Fig. 5). Paramere shape described by
landmarks collected on the lateral view showed an evident differentiation of the North-Western
Alps population with respect to the other three, which showed some degree of overlap (Fig. 6, left).
The dorsal view of parameres (Fig. 6, right) showed different relationships between populations: in
the PC plot of the two first principal components, the two most differentiated populations seemed
to be those from Sicily and the Central Apennines. The dorsal view of parameres, as already
evidenced by Baraud (1992), showed a higher diagnostic power than the lateral view in evidencing
differences between populations, as indicated also by the higher percentage of variance explained of
the first principal component; however, the pattern highlighted with the analysis of the dorsal view
of parameres did not allow consideration of the Sicilian population as a differentiated species, as its
divergence, although extant, was of the same range as the Central Apennine population. Singular
values, percentages of variance explained and cumulative variance for all principal components,
resulting from each PCA are shown in Table 4. Cross-validated results of CVA on shape variables
are summarized in Table 5.
Size variation and static allometry of head, epipharynx and paramere
Kruskal-Wallis tests on female and male head centroid size revealed that the Central Apennines had
significantly larger head size (females: P<0.01 in all contrasts; males: P< 0.001 in all contrasts);
static allometries of female head had similar slopes between populations (P=0.854), suggesting the
same onthogenetic trajectories in all populations, but the intercept value for Central Apennine
population was different with respect to the others (P<0.004), indicating the larger head size at the
same body size for the individuals of this population with respect to the others. On the contrary,
male head static allometry indicated that the allometry of the NW Alps differed significantly from
the others in the slope (P<0.001), a result in agreement with the differentiation pattern observed for
shape.
Epipharynx size analyses indicated a subdivision of the sample in two groups, NW Alps and Sicily
on one side, having lower intercept values in allometries (P<0.001) and centroid size values
significantly lower (P<0.001 in all contrasts), and the Southern and Central Apennines on the other.
The non-parametric Kruskal-Wallis test on paramere centroid size (lateral view) revealed
significant differences in dimensions between the four populations (P<0.001), with the Southern
Apennine population having the largest paramere (lateral view) and the Sicilian one the smallest.
Allometric slopes differed, even weakly, between populations (P=0.046) except for the contrast
North Western Alps-Central Apennine (P=0.108). R2 values of the paramere (lateral, view)
allometric regressions (N-W Alps R2=0.183; Central Apennine R2=0.429; Southern Apennine
R2=0.083; Sicily R2=0.072) were very low compared to the R2 of the other morphological trait
allometries, which approached one. The non-parametric Kruskal-Wallis test on paramere centroid
size (dorsal view) revealed again significant differences in dimensions between the four
populations, but Sicily and NW Alps are dimensionally comparable (P= 0.241), and the same is for
Central and Southern Apennines (P= 0.613). This result fitted with the results of the comparison of
the epipharynx allometries. Allometric slopes of paramere (dorsal view) differed between
populations except for the contrast North Western Alps-Sicily (P=0. 213). R2 values of the
allometric regressions of the dorsal view of paramere were, again, very low, and very similar to
those calculated from the allometries of the paramere lateral view (N-W Alps R2= 0.284; Central
Apennine R2= 0.320; Southern Apennine R2=0.081; Sicily R2=0.386).
4. Molecular analyses
We aligned 601 bp of COI resulting in a combined matrix with 66 parsimony informative
characters. Thirteen haplotypes were identified, grouped coherently with their geographic origin.
The mean uncorrected p-distance between any two sequences was 0.049. The two closest
populations were NW Alpine and Southern Apennine (p-distance = 0.0277), whereas the most
differentiated (p-distance = 0.0374) were Central and Southern Apennine populations; the Sicilian
population was as distant from the Southern Apennine population (p-distance = 0.0295) as from the
Alpine population (p-distance = 0.0294). Distances of each population with respect to the out-group
were all in the same range of magnitude (0.1) and intra-population distances (p-distances < 0.007)
were always lower than inter-population distances.
Maximum Parsimony (MP) analysis generated nine equally parsimonious trees resulting in a
consensus tree with main branches supported with high bootstrap values; for parsimony informative
sites, the consistency index (CI) was 0.75, the retention index (RI) 0.84 and the rescaled consistency
index (RCI) 0.63; tree length was 161. The sum of the branch Lengths (SBL) for the ME tree was
0.2440441 and for the NJ tree was 0.27080580. The trees generated by these three different
methods showed the same topology, with no differences even in the resolution of terminal clades
(Fig. 7); in these phylogenetic reconstructions, individuals of each population were clearly grouped
in different clades, with the Central Apennine population as the most differentiated. The ML and
Bayesian tree showed a different topology, indicating the Sicilian population as the most
differentiated. However, the phylograms (Fig. 7 and Fig. 8, right) clearly indicates that the Sicilian
population belonged to the O. fracticornis lineage, the distance being short with respect to the other
Italian populations , if compared with the range of interspecific distance between O. fracticornis
and the other species of the complex (O. similis and O. opacicollis).
The analysis made with TCS showed that the maximum number of mutational steps between
haplotypes allowing parsimonious connections in the same network with a probability higher than
95% was 10 steps. Haplotype network estimation using parsimony within this probability limit
resulted in four separate networks, one for each population. To connect the networks in the same
cladogram (as in Fig. 8, left), we had to use at least 16 mutational steps, which is below the
accepted 95% probability limit.
Discussion
In his works, Baraud (Baraud, 1975, 1992) asserted that O. massai is a good species, endemic
and vicarious to O. fracticornis in Sicily, and that there are some diagnostic characters allowing
unambiguous identification of the species, in particular the elytral microsculptural pattern and the
paramere shape. Falahee and Angus (2010) confirmed these arguments, presenting a comparison of
the microsculptural details of the elytra of modern and fossil individuals of O. massai and O.
fracticornis (Coope, 2001; Osborne, 1969). They showed the marked puncturation in the interstrial
surface in the first taxon and evident presetal granulation in the second, and supported this
morphological evidence with karyotypic analyses. Validation of O. massai as a good species would
indicate that this taxon has substantially altered its geographical distribution as a consequence of
dramatic glacial/interglacial climatic changes, as its presence would have been recorded in England
in the warm Ipswichian period (about 130.000-110.000 years ago) (Coope, 2001). In this case, its
insular endemism should have to be interpreted as the result of isolation in a refugial area during the
last ice age, accompanied by the complete extinction of continental and peninsular populations.
From our extensive microscopic inspections of the elytral surfaces, it has unexpectedly emerged
that the puncturation pattern typical of O. massai is detectable even in Central Apennine
individuals, and that O. massai specimens are not totally homogeneous for this character. At least
three different explanations could follow from this result: a) the elytral microsculptural pattern is
not a diagnostic character for O. massai identification, and another trait should be found to
unambiguously identify this species; b) the microsculptural pattern would be just a phenotypic trait
with variable frequencies in different populations of O. fracticornis and therefore not a diagnostic
character: in this case, Sicilian individuals could be simply insular members of O. fracticornis
which more frequently exhibit this character, and not a vicarious species; c) O. massai is a real
species but it would not be endemic and would coexist in microsympatry with O. fracticornis in
continental localities, such as the Central Apennines. In trying to validate these hypotheses, we
proceeded with a thorough inspection of morphological, allometric and genetic patterns between
continental and insular populations.
The ANOVA on pronotum width revealed that the NW Alps and Southern Apennine populations
had similar body size whereas the Sicilian and Central Apennine populations were the two most
differentiated with respect to their general size (the smallest was Sicily, as expected, and the largest
was the Central Apennines): these results leads us to presume that it is very unlikely that O. massai
individuals, considered to be decidedly smaller with respect to O. fracticornis, could be present in
the Central Apennines sample. However the size is not considered a good character to define a
species by. The literature contains many examples showing that the mean body size of an insect
species may change along an altitudinal (or latitudinal) gradient, but some species show
increasing size with increased altitude while others show the reverse (or a more complicated) trend
(Hawkins and deVries, 1996; Chown and Klok, 2003; Krasnov et al., 1996). For the present, an
exhaustive information of the altitudinal/latitudinal size variation pattern of O. fracticornis is not
available, so we can’t interpret our results in this framework. Horn static allometries (Fig. 3, Table
3) reflected the results come out from size comparison, showing that the Central Apennines and
Sicily had the two most divergent allometries, both with respect to the body size threshold at which
males switch from the hornless to the horned phenotype, and to the horn size range, which was
wider for the Central Apennines and narrower for Sicily: the switch point between majors and
minors in the Central Apennines occured at the same body size at which we observed the largest
major male in Sicily.
PCA of shape variables showed a pattern coherent among all the morphological structures
considered in the analysis: the four populations largely overlapped in the morphospace, even if it
was possible to single out each population taking up predominantly specific areas in the
morphospace; a biplot of the first two principal components of males and females head shapes
showed a similar profile (Fig. 4), with shape diversification between populations more evident in
females: the NW Alps appeared to be the most differentiated population, with a very weak overlap
with the Central Apennine population, which mainly occuppied the upper part of the morphospace;
Southern Apennine specimens mainly overlapped with the Sicilian ones. This morphospatial
arrangement of populations seems to reflect a clinal variation along a north-south latitudinal
gradient. Since the epipharynx is placed in a cavity of the head (anterior pharynx wall), one might
have expected variation in its shape to be reflected in that of the head. Epipharynx shape was
instead more homogeneous, level of diversification between populations being hardly perceptible;
however, a trend of variation similar to that of the head could be summised (Fig. 5). The greater
homogeneity is can be attributable to the strict association of this species with particular feeding
resources (Halffter and Matthews 1966, Verdù and Galante, 2004); stabilising selective pressures
are likely to act on this structure, so that optimal functionality is maintained in each population and
this probably reflects a lack of differences in trophic habits and food selection between the
populations along the latitudinal gradient.
Paramere (lateral view) shape variation along the first two principal axes (Fig. 6, left) showed a
pattern in which, again, the NW Alps population was clearly identifiable from the others: however,
in this case the Central Apennine population appeared as the most differentiated from the NW Alps
and the most overlapping with the Sicilian population. Southern Apennine paramere shape was
more similar to that of the NW Alps. However, paramere (dorsal view) shape variation along the
first two principal axes (Fig. 6, right) shows that the two most different populations, with a similar
range of diversification with respect to the average shape, are those from Sicily and Central
Apennine; however, some Sicilian individuals overlap with those from NW Alps and Southern
Apennine; These results are in agreement with the qualitative observations of Palestrini (1981),
who found that the shape of the apical region of parameres of the supposed O. massai was similar to
that of specimens from other Italian and foreign populations. The two main diagnostic characters
for O. massai identification (elytral microsculptures and paramere shape, Baraud, 1975, 1992) seem
to be unreliable. In cross-validated results of CVA, the NW Alps was the best identifiable
population, both for the head and for epipharynx shape, with high percentages of correct
classifications; cross-validated CVA results of paramere shape variables indicated that even if the
NW Alps was not the population with the best classification percentages, it was rarely confused
with other populations.
None of these shape analyses indicated the Sicilian population as the most differentiated, leading us
to suppose that O. massai is an invalid species. Sicilian individuals almost always overlapped the
morphospace of the Southern Apennine individuals, the closest geographic population, except for
the paramere shape (lateral view), which was more similar to that of the Central Apennines.
Interpopulational differences, although present, were weak and seemed to be in the range of the
differences detectable among populations of the same species.
Individuals from the Central Apennine population can be described as the largest not only in terms
of mean body size, but also as they can be distinguished from all other populations for having,
proportionally, the largest heads in females, an epipharynx proportionally bigger - even if
comparable with those of the Southern Apennine population - and a horn static allometry indicating
that the switch point between majors and minors in this population comes at the largest body size
value. Surprisingly, the Central Apennines didn’t show a copulatory structure proportionally larger
than other populations, a primacy set by the Southern Apennine population instead, which largely
invests in the development of male genitalia. Paramere allometry suggested that the complete
independence of the allometric trajectories between populations, with significantly different slopes,
could express the incipient reproductive isolation of the populations. The only allometric result in
agreement with information collected from the shape analyses was the male head allometry, in
which the NW Alps emerged as the only differentiated population. Epipharynx allometries also
showed a perfect superimposition of the allometric trajectories between the NW Alps and Sicily. In
conclusion, from the analyses of allometries there is no evidence of a specific divergence in the
onthogenetic trajectory of the Sicilian population from the others.
Phylogenetic reconstruction showed the same general topology evidenced in a recent study
on the phylogenetic relationships within the complex (Macagno et al., in press), where O.
fracticornis has been indicated as the most differentiated species within the fracticornis-opacicollissimilis complex and O. similis and O. opacicollis were grouped as sister species. The weak
bootstrap value (0.85, Fig. 7) and posterior probability (0.65) found at this bifurcation in our
analyses likely depend from the fact that this two species are represented by a single individual; in
Macagno et al., in press, this same phylogenetic relationship, obtained from a higher number of
individuals form each species, is better supported. Each population of O. fracticornis was perfectly
monophyletic. In ME, MP and NJ phylogenetic reconstruction (Fig. 7), the resulting tree topologies
showed the Central Apennines as the most differentiated population, showing its sister relationships
with respect to the other populations supported by very high bootstrap values. Otherwise, ML and
Bayesian analysis (Fig. 8, right) suggested a greater differentiation of the Sicilian population. The
lineages within O. fracticornis could be considered as potential speciation events, but they are not
supported as species (low genetic distances with respect to the other species of the complex, partial
inconsistency of morphometric data with respect to molecular data); at most, they could be
considered as sub-species, or just simply genetically distinct units within O. fracticornis. Overall,
these results indicate that the Sicilian population is not sufficiently and unambiguously
differentiated from the populations belonging to O. fracticornis from both genetic and phenotypic
point of view, and it therefore cannot be considered a good species (O. massai). This result partly
contrasts with previous karyotypic analyses (Falahee and Angus, 2010), where the authors found
differences in RCLs of four of the nine pairs of autosomes between O. fracticornis and the
supposed O. massai. When chromosomal and genic data show two distinct patterns of variation, the
lower level of genic variation between populations may be due to homoselection in a constant
microenvironment (Nevo et al., 1984; Nevo and Shaw, 1972), whereas extensive chromosomal
differences might coincide with an incipient reproductive isolation, maybe due to the physiographic
barrier of the Strait of Messina. Extensive reorganisation of euchromatic chromosomal segments
can produce genetic isolation via hybrid sterility (Patton and Sherwood, 1983), thus it follows that
we cannot at present exclude an onoging speciation process , occurring without concomitant genetic
modification (Selander et al., 1974, Sage et al., 1986).
Network analyses indicated each population as an independent network (Fig. 8, left); the
connection of networks is possible only when significantly lowering the probability of
parsimonious connections below 95%, suggesting that they appear to be “genetic islands”, with
little genetic flow between them; this particular pattern of isolation could be indicative of a low
dispersal capacity and a high degree of habitat specialisation (this oligophagous species seems to be
restricted to pastures in cacuminal areas in the Alps and Apennines, and many authors have
emphasised recors in mountainous regions (Petrovitz 1956, Avila and Pascual 1988, Baum 1989,
Gangloff 1991, Sowig, 1995). However, this pattern can arise from neutral processes, particularly
where there is long-term historical isolation (Moritz 2002; Tregenza, 2002). The importance of
quaternary glaciations in moulding the population structure is, indeed, well established (Avise and
Walker 1998; Hewitt 2000); however, because it was impossible to perform a true phylogeographic
analysis on separate networks, nothing can be hypothesised on the specific dynamics of habitat
fragmentation, isolation and recolonisation of the Italian region made in the past by O. fracticornis,
except that, maybe, the Apennine range has been one of the first geographic barriers separating a
western and an eastern lineage of the species. The presence of evident shape differences in the NW
Alps population only, not accompanied by strong genetic diversification, could also suggest an
ancient isolation of this population in a habitat with strong selective pressures, maybe an alpine
nunatakker (an “insular mountain” maintaining a suitable habitat for a species surrounded by ice
during the glacial period). It has been acknowledged that mitochondrial DNA markers are more
susceptible to the effects of genetic drift (Moritz et al., 1987; Sunnucks, 2000; Avise, 2004) and
thus completely reliable inferences on species’ genetic diversity and structure should ideally be
based on multiple types of molecular markers (Canestrelli et al., 2007); moreover, a more abundant
sampling for each population and the collection of samples from other parts of the distribution
range would allow to deeper examine, with the help of molecular markers, the extent of the
geographical isolation, and the possibility of traces of other cryptic species; this could be a further
development of this research.
To summarise, the results of this study seem to indicate that there is not sufficient evidence
for O. massai to be considered as a good species, differentiated from O. fracticornis and vicarious
to it in Sicily, but that speciation may be taking place; moreover, from our analyses it clearly
emerges that the Sicilian population is significantly smaller than the others, which could be the
result of insular evolution toward dwarfism (Vernon, 1981).
The Central Apennine population deserves particular attention: it was differentiated in terms
of genetics and overall body size; additionally, it appeared similar to the Sicilian population for
both characters considered diagnostic for O. massai identification, paramere shape and elytral
microsculptures. However, no other character (shape, size, allometries, genetics) suggested a
particular relationship between Sicilian and Central Apennine populations.
Overall, each population examined presents peculiar aspects of differentiation; in particular, genetic
analyses seem to suggest that the populations are evolving as evolutionary independent units.
Acknowledgements
Authors thank Prof. Massimo Maffei and Dr. Cinzia Bertea for their kind welcome at the
CEBIOVEM laboratories of the Turin University and for their help with genetic manipulations.
Onthophagus vacca COI sequences have been courteously provided by Dr. Dirk Ahrens
(Zoologische Staatssammlung München, Germany). Daniele Silvestro (Senckenberg
Forschungsinstitut, Frankfurt, Germany) helped us in the ML and Bayesian phylogenetic
reconstruction. Dr. Dan Chamberlain (University of Turin) revised the English. Authors are grateful
to their two anonymous referees for their suggestions and careful corrections.
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