Intra- and interspecific skull variation in two sisterspecies of the subterranean rodent genus Ctenomys(Rodentia, Ctenomyidae): coupling geometricmorphometrics and chromosomal polymorphism
FABIANO ARAUJO FERNANDES1,2, RODRIGO FORNEL1,3,PEDRO CORDEIRO-ESTRELA1,3* and THALES RENATO O. FREITAS1,3
1Departamento de Genética – Instituto de Biociências – Universidade Federal do Rio Grande do Sul(UFRGS), Porto Alegre, Rio Grande do Sul, Brazil2Programa de Pós-Graduação em Biologia Animal – Universidade Federal do Rio Grande do Sul(UFRGS), Porto Alegre, Rio Grande do Sul, Brazil3Programa de Pós-Graduação em Genética e Biologia Molecular – Universidade Federal do RioGrande do Sul (UFRGS), Porto Alegre, Rio Grande do Sul, Brazil
Received 20 June 2007; accepted for publication 9 October 2007
How do species, or populations, differentiate fromeach other? Several authors have proposed that chro-mosomal rearrangements have played the primaryrole in speciation events (White, 1978; Patton & Sher-wood, 1983). As Rieseberg (2001) proposes, there aremany overlapping models of chromosomal speciationthat share at least one fundamental feature: chromo-
somal differences that have accumulated between theneospecies and its progenitors are assumed to impairthe fertility or viability of interspecific hybrids,thereby reducing gene flow. In the great majority ofthese models, post-zygotic isolation is achievedmainly through the reduced fitness of hybrids, origi-nating from the meiotic imbalance of chromosomalheterozygotes.
The great diversity of the fossorial rodent genusCtenomys (over 60 named species), commonly knownas tuco-tucos, can be inputted to this general mecha-nism. In fact, the great variation in the diploidnumber (2n), ranging from ten to 70 chromosomes,
*Corresponding author. E-mail: [email protected] of the authors have directly participated in the planning,execution, and analysis of results for the study.
Zoological Journal of the Linnean Society, 2009, 155, 220–237. With 7 figures
and fundamental number (FN), ranging from 16 to90, has been thought to arise from the chromosomalrearrangements favoured by their patchy distribu-tions, low vagility, territoriality, small effectivenumbers, and socially structured mating systems(Reig et al., 1990). Molecular data indicating verystructured populations, as well as basal irresolutionwithin the genus, seem to support such a model(Wlasiuk, Garza & Lessa, 2003; Castillo, Cortinas &Lessa, 2005). Contrasting with the genetic and karyo-typic differences, tuco-tucos show a relatively conser-vative cranial shape, despite their size ranging from90 g to 900 g (Vassallo, 1998; Mora, Olivares &Vassalo, 2003). Several authors have noted tuco-tucosas an excellent model for studying evolution andspeciation events (Ortells & Barrantes, 1994; Ortells,1995; Cook & Lessa, 1998; Lacey, Patton & Cameron,2000; Mascheretti et al., 2000), precisely because oftheir high karyotypic heterogeneity and low morpho-logical differentiation. One notable exception,however, is the highly variable angle of incisor proc-umbency among species (Mora et al., 2003), whichmight be an adaptive character related to the ‘chiseltooth’ mode of digging within Ctenomys. Nevertheless,few studies have focused on the coupling betweenskull morphology and chromosomal variation in thisgenus (Freitas & Lessa, 1984; Marinho & Freitas,2000; Freitas, 2005; Massarini & Freitas, 2005). Twomain aspects can be highlighted through this com-parison: one systematic and another evolutionary.
The description of patterns of variation in geneticand morphological characters within and amongpopulations is fundamental for defining boundaries ofindependent evolutionary units in nature. In system-atic mammalogy, information that allows the recog-nition of evolutionary units has classically beenderived from the analysis of the variation of the formof the skull, an important structure that harbours thetrophic apparatus, as well as the brain and the senseorgans (Voss, Marcus & Escalante, 1990; Hanken &Thorogood, 1993). An important initial step is theidentification of groups of populations that share mor-phological traits over geographic space(dos Reis et al., 2002). Once units are identified, onequestion of interest is how do observed patterns ofvariation in cranial shape yield insight into theorganization and structure of variation within andamong populations. Traditionally, this task has beenaccomplished by using multivariate statistical tech-niques applied to distance measurements. Recently,however, a powerful statistical framework for describ-ing the morphological organization and change incomplex morphological structures, such as the skull,has been provided by the development of the statis-tical formalism of geometric morphometrics (Rohlf &Marcus, 1993; Bookstein, 1996; Rohlf, 1996; Rohlf,
2000; Monteiro, Bordin & dos Reis, 2000; Cordeiro-Estrela et al., 2006).
The systematics within the genus Ctenomys can befairly simple, as some species posses a unique karyo-type and distinct skull size or other morphologicalcharacteristics. In contrast, other species show exten-sive intraspecific karyotypic variation (Ortells, Contr-eras & Reig, 1990; Gallardo, 1991; Massarini et al.,1991; Ortells, 1995; Giménez, Contreras & Bidau,1997; Freitas, 1997; Massarini, Dyzenchauz & Tiranti,1998; Garcia et al., 2000a, b; Freitas, 2001; Freygang,Marinho & Freitas, 2004), or variation in pelage colour,within populations (Reig, Contreras & Piantanida,1966; Langguth & Abella, 1970; D’Elia, Lessa & Cook,1998). In these cases, species delimitation can be anespecially important issue for the conservation oftuco-tucos, as many habitats of species are beingdestroyed (Altuna et al., 1999; Fernández-Stolz, Stolz& Freitas, 2007). Furthermore, the distinctionbetween chromosomal populations and species isimportant, as often we pay insufficient attention to theextinction of a local population, when in fact we aredealing with a new species containing invaluable vari-ability (Bidau, Giménez & Contreras, 1996).
In the present study, we analyse the skull variationof two sister species of Ctenomys, Ctenomys torquatusand Ctenomys pearsoni (D’Elia et al., 1999), with geo-metric morphometric methods. They both exhibitinter- and intraspecific karyotypic variation, andoccur in southern Brazil and Uruguay (Freitas &Lessa, 1984; Villar et al., 2005) in a contiguous bio-geographical region of grasslands, with an extensivecoastal plain of sand dunes.
Lichtenstein (1830) described C. torquatus withsamples from ‘borders of the Uruguay river andsouthern Brazil’. The type locality of this speciesremains uncertain because Nehring (1900) consideredMaldonado, Uruguay, as the type locality, but Lang-guth & Abella (1970) affirm that Sellow, who collectedthe individuals analysed by Lichtenstein, was not inMaldonado at the time the specimen was collected.
Karyotypes attributed to C. torquatus were firstreported by Reig et al. (1966), who found populationswith a diplod number of 2n = 68 at Maldonado andCarrasco, Uruguay, as well as in Medanos, Argentina.Reig & Kiblisky (1969), at the same localities,reported individuals with 2n = 68 (fundamentalnumber, FN = 96). Kiblisky et al. (1977) studied somesamples from Uruguay and described four differentkaryotypes: 2n = 44 at El Aguila, Salto Nuevo, andGuabiyú; 2n = 56 at Carrasco; 2n = 64 at Maldonado;and 2n = 70 at Barra de Santa Lucia, Playa Pascual,and Limetas river. They proposed two groups basedon the FN: the first composed by populations withFN = 82 and 84, from southern Uruguay, and theother including populations with FN = 76. Using a
classical morphological description methodology,Lessa & Langguth (1983) showed that the forms fromsouthern Uruguay (2n = 56, 64, and 70) also differ inskull morphology from populations with 2n = 44,showing, for example, a wider and reduced heightof the skull and a more elongated and depressedrostrum, and recognized them as a distinct entitynamed C. pearsoni. The name C. torquatus was main-tained for populations with 2n = 44, because they aresimilar in skull characters to the type specimen of C.torquatus from the Museum für Naturkunde, Berlin(Freitas & Lessa, 1984).
Now, Ctenomys torquatus Lichtenstien, 1830 has oneof the most widely known geographic distributionsamong the species of Ctenomys, ranging from central toeastern and southern regions of the Rio Grande do Sulstate, in Brazil, with karyotypic polymorphism:2n = 44, 46, and 40 (FN = 72). Excepting the karyotypepopulations with 2n = 44, the other two karyotypeshave narrow geographic distributions (Freitas, 1995;Freitas, 2006; Ximenes, Fernandes & Freitas, 2006). InUruguay, C. torquatus occurs in the northern andcentral regions, with only one diploid number: 2n = 44(FN = 72) (Freitas & Lessa, 1984; Villar et al., 2005;Novelo & Villar, 2006). Ctenomys pearsoni Lessa &Langguth, 1983 inhabits the costal plains of southernUruguay, and has seven known karyomorphs, repre-senting one of the largest range of diploid numbers inthe genus, from 2n = 56 to 70 (FN = 74–80) (Novello &Lessa, 1986; Novello et al., 1990, 1996; Novello &Altuna, 2002). In both species, the chromosomal formsdiffer by Robertsonian rearrangements (Freitas &Lessa, 1984; Villar et al., 2005).
In this paper, we analyze an example of the extra-ordinary chromosomal diversity that populations oftuco-tucos exhibit, and the intrinsic relationshipsbetween the sister species C. torquatus and C. pear-soni, using geometric morphometrics and pattern-recognition techniques. Shape and size variables arederived for dorsal, ventral, and lateral cranial views tostudy intraspecific sexual dimorphism, and to assessthe inter- and intraspecific variations with respect tochromosomal populations, particularly variationsbetween the C. pearsoni 2n = 70 and 2n = 66 popula-tions, and C. torquatus 2n = 44 populations from Braziland Uruguay. Furthermore, we determine if the use ofthe three views of the skull are more informativecombined together or separately, and which one ofthe three views is more informative for systematicpurposes.
MATERIAL AND METHODSSAMPLES
We examined 221 complete adult specimens of C.torquatus and C. pearsoni housed in the collections of
mammals of the Departamento de Genética, Institutode Biociências, Universidade Federal do Rio Grandedo Sul, Porto Alegre, Brazil and Museo Nacional deHistoria Natural y Antropología (MNHNA), Montev-ideo, Uruguay. The collection numbers of specimensare given in Appendix 1, and Figure 1 presents thecollecting areas. Our group karyotyped all specimensfrom Brazil, and diploid numbers from Uruguayanspecimens were established by their localitiesbased on the work of Villar et al. (2005). Chromo-somal populations employed in intraspecific analysiswere defined by the differences in the diploid number:C. pearsoni 2n = 66 and 70, C. torquatus 2n = 40, 46,and 44, from Brazil, and 2n = 44, from Uruguay (44u).Populations of C. torquatus with 2n = 44 fromUruguay were separated from those from Brazil,because in an analysis of geographic variation in theskull (Fernandes FA, Fornel R, Cordeiro-Estrela P &Freitas TRO, unpubl. data), only this populationappeared to be significantly distinct from the others.
GEOMETRIC MORPHOMETRICS
Each skull was photographed in dorsal, ventral, andlateral view with a digital camera in 3.1 megapixels ofresolution (2048 ¥ 1536) without zoom or flash. Thecamera was positioned at 17 cm from the photo-graphic plane. For the dorsal pictures, skulls werepositioned with both bullae and incisors touchingthe photographic plane, with the lens centered onthe frontals. For the ventral pictures, the skull wasplaced resting on its frontals with the lens centeredon the molariform teeth. For the lateral pictures theskull was placed on its right jugal on a modellingpaste base, and was then aligned with the photo-graphic plane using a ruler. The lens was centered onthe left jugal. Millimetre graph paper was used as abackground in each picture, thereby providing a scaleand assistance in the alignment of specimens, andshowing any possible deformation resulting fromthe photography. We defined 29, 30 and 21 two-dimensional morphological landmarks for dorsal,ventral, and lateral views of the skull, respectively(see Fig. 2 for landmark locations and Appendix 2 fortheir description). The coordinates of each landmarkwere obtained using tpsDig 1.40 software (Rohlf,2004) by one of us (FAF). The error in landmarkacquisition (operator variance) was assessed througha one-way analysis of variance of centroid size forthe repeated landmark acquisition of one image foreach species. The mean estimated measurement erroraveraged 0.08%. Coordinates were superimposedusing a generalized Procrustes analysis (GPA) algo-rithm (Dryden & Mardia, 1998). We obtained linearcombinations of the original landmark coordinatesafter standardizing for size and removing artefactual
variation resulting from different positioning of thespecimens in the process of data collection. The size ofeach skull was estimated using its centroid size: thesquare root of the sum of the squares of the distancesof each landmark from the centroid (Bookstein, 1991).
STATISTICAL ANALYSIS
Size was compared between sexes and across specieswith a two-way (sex ¥ species) analysis of variance(ANOVA), and differences between groups were
Figure 1. Map with sampled populations of Ctenomys torquatus from southern Brazil (1–17) and northern Uruguay(18–20), and for Ctenomys pearsoni (21–23) from southern Uruguay. Detailed information of voucher specimens are listedin Appendix 1, following the map numbering.
visualized through box plots. Principal componentanalysis (PCA) was carried out using the variance–covariance matrix of generalized least-squares super-imposition residuals. The PCs of the covariancematrix of superimposition residuals were used as newshape variables, to reduce the dimensionality of thedata set, as well as to work on independent variables.The matrices of PCA scores for each view of thecranium (dorsal, ventral, and lateral) were joined inone total matrix. Another PCA was carried out withthis final matrix to summarize information fromdorsal, lateral, and ventral views. The PCA andkernel density estimates (Wand & Jones, 1995) wereused as exploratory techniques, meaning that wewere interested in getting an overview of the struc-ture of the data (Baylac, Villemant & Simbolotti,
2003; Cordeiro-Estrela et al., 2006). If there is morethan one group based on skull shape variables, mul-tiple clusters should be visualized (Cordeiro-Estrelaet al., 2006). Shape differences between sexes andspecies, and their interactions, were tested throughmultivariate analysis of variance (MANOVA). A sepa-rate MANOVA was carried out between chromosomalpopulations. Linear discriminant analysis (LDA) wasused to classify specimens between sexes, species,and among chromosomal populations. To choose thenumber of PCs to be included in the LDA, we com-puted correct classification percentages with eachcombination of PCs (Baylac & Friess, 2005). Weselected the subset of PCs giving the highest overallgood classification percentage. We used a leave-one-out cross-validation procedure that allows anunbiased estimate of classification percentages(Baylac & Friess, 2005). Cross-validation was used toevaluate the performance of classification by LDA. Inthe leave-one-out cross-validation, all the data exceptone individual are used to calculate the discriminantfunction. The unused individual is then classified.The procedure is repeated to compute a mean classi-fication error and a probability of group membershipfor each individual. Mahalanobis distances were usedto compute a neighbour-joining tree to visualize themorphological relationship between chromosomalpopulations, as well as the measured Procrustesdistances.
The visualization of shape differences for the threeviews of the skull was obtained through multivariateregression of shape variables on discriminant axes.
INFORMATIVENESS OF THE DIFFERENT VIEWS
The acquisitions of landmarks for a geometric mor-phometric analysis are often performed by the calcu-lation of Cartesian coordinates from pictures of thespecimens. The reduction of the three-dimensionalstructure to two-dimensional planes has led research-ers to take different views of the skulls, and toanalyse them separately. Independent researchershave shown that the information conveyed by differ-ent views of rodent skulls is mostly similar, but notto the same extent for all views (Corti & Rohlf, 2001;dos Reis et al., 2002; Nicola et al., 2003; D’Anatro &Lessa, 2006). The lateral view of the skull was foundto be more informative than the dorsal and ventralviews in discriminating among some species ofrodents (dos Reis, 1990, dos Reis et al., 2002). But theinformation available in the analysis of these threeviews combined is still poorly investigated. Tocompare the different information conveyed by thethree views, we compared the correct classificationpercentages of LDA for sex, species, and chromosomalgroups for the three views, and for their combination.
Figure 2. Ctenomys torquatus skull, with indication ofmorphological landmarks for the dorsal (A), ventral (B),and lateral (C) views of the cranium. Appendix 2 gives thekey to the landmarks. Scale bar: 1 cm.
For all statistical analyses and to generate graphicswe used the ‘R’ language and environment for statis-tical computing (v2.2.1 for Linux; R DevelopmentCore Team, http://www.R-project.org) and the follow-ing libraries: MASS (Venables & Ripley, 2002), APEv1.8-2 (Paradis et al., 2006), and GenKern 1.1-0 (Lucy& Aykroid, 2004). Geometric morphometric proce-dures were carried with the Rmorph package (Baylac,2006), a geometric and multivariate morphometricslibrary.
RESULTSTESTS FOR SIZE AND SHAPE DIFFERENCES
The two-way ANOVA for the centroid size showedsignificant differences for sex in dorsal, ventral, andlateral views of the skull (F = 37.7, P << 0.001;F = 34.0, P << 0.001; F = 29.7, P << 0.001, respec-tively) with males, on average, being larger thanfemales in both species. For skull size differences atthe interspecific level, only the ventral view did notshow significant differences (P > 0.1). In the otherviews, C. pearsoni showed the largest skull sizebetween species (F = 16.3, P < 0.001 for the dorsalview; F = 9.0, P < 0.01 for the lateral view). A plot ofcentroid size distribution for the lateral view of thecranium between sexes and species is presented inFigure 3; for dorsal and ventral views the plots arevery similar (data not show).
When analyzing shape, the PCA and kernel densityestimates of principal components (Fig. 4) showedsome structure in the data, indicating the presenceof two groups corresponding to C. torquatus andC. pearsoni.
The MANOVA results indicate that skull shapediffers significantly between sexes (Wilks’ l = 0.46;
F = 3.6; P << 0.001) and between species (Wilks’l = 0.33; F = 6.3; P << 0.001). The interaction termbetween the sex and species was also significant(Wilks’ l = 0.64; F = 1.8; P < 0.01). For chromosomalpopulations, the MANOVA also indicated significantdifferences in skull shape (Wilks’ l = 0.63; F = 1.8;P < 0.01). A specific MANOVA performed on theresiduals of two intraspecific GPA, only includingC. torquatus 2n = 44 populations from Brazil andUruguay, and only C. pearsoni 2n = 66 and 2n = 70,also indicated significant shape differences betweenthe interspecific chromosomal groups (Wilks’ l = 0.39and 0.49; F = 6.2 and 3.8; P << 0.001 and P < 0.01,respectively).
INFERRED SHAPE DIFFERENCES BETWEEN GROUPS
The multivariate regression of discriminant axes onshape variables allowed us to characterize the majortrends of cranial shape differences between sexes,species, and chromosomal groups within the twostudied species (Fig. 5). The males showed a propor-tionally larger and longer rostrum than females, aswell as a wider zygomatic arch, whereas femaleshave a relatively larger neurocranium (Fig. 5). TheC. torquatus specimens showed relatively largernasals and frontals (dorsal view), a wider and higherrostrum, as well as a smaller angle of the rostrum(lateral view), i.e. less procumbency, and smaller tym-panic bulla (ventral view) than C. pearsoni. Intraspe-cific differences between chromosomal groups can besummarized as follows: C. torquatus individuals fromBrazil had a wider and higher rostrum than thoseform Uruguay. Furthermore, they had a proportion-ally larger jugal. On the other hand, C. torquatusfrom Uruguay showed wider pterygoid bones andlarger tympanic bullae than Brazilian specimens. TheC. pearsoni 2n = 66 individuals have a larger jugal, awider external auditory meatus, and a longer tym-panic bullae than individuals with 2n = 70.
MAGNITUDE OF DISCRIMINATION AND OF
SHAPE DIFFERENCES
The projection of specimens on the first and seconddiscriminant axes for karyotypic groups (Fig. 6) showsthe superimposition of populations of C. torquatus(2n = 40, 44, and 46) with the exception of 2n = 44 fromUruguay (44u). The populations 2n = 66 and 70 ofC. pearsoni appear separated, and the populations2n = 44 from Uruguay are partially superimposed withC. torquatus from Brazil and populations 2n = 70 of C.pearsoni from Uruguay. The C. pearsoni populationswith 2n = 66 are remarkably distinct from the otherCtenomys populations analysed.
Figure 3. Box-and-whisker plots showing the distributionof centroid size for the lateral view of the skull of twoCtenomys torquatus and Ctenomys pearsoni specimens,and for each sex. Upper and lower hinges correspond tothe first and third quartiles, and whiskers correspond tothe 95% confidence interval.
The Mahalanobis distances (Fig. 7) indicate the mor-phological distances computed from cranial shape vari-ables among the chromosomal groups. The first fivelarger distances indicate interspecific differences: fourof them are, in comparison with C. pearsoni, individu-als with 2n = 66. The largest intraspecific distances arebetween C. torquatus 2n = 44 from Uruguay and C.torquatus 2n = 46 from Brazil (25.9), and between2n = 66 and 2n = 70 for C. pearsoni (23.7). Distancesbetween the C. pearsoni 2n = 70 and 66 or C. pearsoni2n = 70 and all C. torquatus chromosomal groups (inaverage, 25.3) were much closer, meaning that thesetwo populations of C. pearsoni are as different as C.pearsoni 2n = 70 and C. torquatus chromosomal popu-lations. The smallest distances were among the C.torquatus populations that occur in Brazil (2n = 40, 44,and 46), but individuals of C. torquatus from Uruguay2n = 44 showed intermediate distances between Bra-zilian karyomorphs of this species and C. pearsonivalues. Procrustes distances show the same ordinationas the Mahalanobis distances (Table 1). In this analy-
sis, where the mean shape is equal to zero, the highestvalues were observed for C. pearsoni populations, andfor the lateral view of the skull.
The neighbour-joining phenogram in Figure 7,based on Mahalanobis distances, shows the relation-ship among chromosomal populations. The population2n = 44 from Uruguay is placed in an intermediateposition between the two species, C. torquatus and C.pearsoni, in both topology and branch lengths, whichare proportional to the Mahalanobis distances.
INFORMATIVENESS OF THE DIFFERENT VIEWS
The discriminant analysis of the dorsal view showedthe overall best discrimination percentages comparedwith the lateral and ventral views for sex, species,and karyotypic groups (Table 2). The combination ofthe three views did not show uniform results. For sex,the percentage of the three views combined wereequal to the one obtained for the dorsal view. Thepercentage of classification for females was higher
Figure 4. Kernel density estimates on principal components (PC) 1 and 2 of shape variables and convex hulls forspecimens of Ctenomys torquatus (�) and Ctenomys pearsoni (�). Variance percentages are given on the y axis. Dark areasindicate higher density regions.
than for males for the three views. The classificationpercentage of the dorsal view for the interspecificcomparisons was higher than with the three viewscombined (increase in 3.5%). Only for the comparisonamong the karyotypic groups did the combined viewsshow a higher classification percentage than any ofthe three views separately. In this case, the increasein classification percentage was 9.9% when combiningthe three views.
DISCUSSIONARE THE THREE VIEWS OF THE SKULL MORE
INFORMATIVE WHEN COMBINED TOGETHER OR
WHEN ISOLATED?
In the present work the three views of the skullprovided distinct perspectives on cranial shape varia-tion, as was observed among Thrichomys apereoidespopulations (dos Reis et al., 2002). However, in ourcase, the dorsal view provided the most informationcontent for discrimination between sexes, species, andamong karyotypic groups. D’Anatro & Lessa (2006)considered that the three views of the skull ofCtenomys rionegrensis showed approximately equalresults, but that the differentiation among local popu-lations was particularly evident when data fromlateral views were used. Similarly, the lateral viewwas the most informative in terms of populations thatshared similarity in cranial shape and continuity overgeographic space (dos Reis et al., 2002). Conversely tothese works, we combined the three different views ofthe skull. This integrative analysis for the three skullviews provided an increase in almost 10% of classifi-cation percentages in the specific case of karyotypicgroups. But, as this result was not generalized to sexand interspecific comparisons, we advise concomitantanalysis of the views separately, before combiningthem, to obtain a precise picture of the patternshighlighted by each view.
SEXUAL DIMORPHISM
Sexual dimorphism is highly significant for both thesize and shape of the skull, but the size differences
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Discriminant Axis−1 (29.8%)
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4044467044u66
Figure 6. Plot of the six chromosomal populations for the first two axes of the linear discriminant analysis (LDA) forthree integrated views. Ctenomys torquatus, 2n = 40, 44, 44u, and 46; Ctenomys pearsoni, 2n = 66 and 70.
Ctenomys pearsoni
4446
40
66
70
44uCtenomys torquatus
BrazilUruguay
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Figure 7. Phenogram computed from the Mahalanobisdistances between chromosomal groups for Ctenomystorquatus from Brazil (2n = 40, 44, and 46), C. torquatusfrom Uruguay (2n = 44u), and Ctenomys pearsoni (2n = 66and 70). Tree made by using the neighbour-joining methodwith branch lengths proportional to morphological dis-tances. Scale bar: 4 units.
appear to be more intense for sexual dimorphism,whereas shape differences are intense both betweensexes and species. There are at least three hypothesesproposed to explain the origin and maintenance ofsexual size dimorphism in mammals: sexual selection(especially in mate choice or mating system), selectionon reproductive life history traits, and intersexualecological divergence (Hood, 2000). All of these couldbe invoked in the case of tuco-tucos, because of theirpolygynous mating systems, the phylopatric charac-teristics of the females, the dispersal patterns, andthe agonistic behaviour of the males (Lacey et al.,2000). The sexual dimorphism generally observed fortuco-tuco species (Ctenomys minutus, Gastal, 1994;Marinho & Freitas, 2006; Ctenomys lami, El Jundi &Freitas, 2004; Ctenomys flamarioni, Fernández-Stolzet al., 2007; Ctenomys mendocinus, Rosi, Cona &Roig, 2002; Ctenomys opimus, Pearson, 1959; Cook,Anderson & Yates, 1990; and Ctenomys talarum andCtenomys australis, Malizia, Vassalo & Busch, 1991)was also recorded for C. torquatus and C. pearsoni inthis study, and in all cases males were longer, larger,or heavier than females.
INTER- AND INTRASPECIFIC VARIATIONS
Exploratory analysis such as PCA and kernel densityhas demonstrated that C. pearsoni and C. torquatushave a similar skull morphology, with a smooth ten-
dency for a separation into two distinct morphologicalgroups, despite the difference in sample size betweenthe two species. Statistical tests for shape and sizedifferences (MANOVA and ANOVA) confirm highlysignificant interspecific skull differences. The difficultyin morphologically characterizing the two species withexploratory techniques is in accordance with phyloge-netic studies demonstrating their sister-species rela-tionship (Castillo et al., 2005), which has already beensuggested by synapomorphic characters, such as thesame spermatozoid type (Freitas, 1995). Geometricmorphometric methods also quantitatively confirm thedescription made by Lessa & Langguth (1983), whichwas based on a qualitative traditional morphologicalcharacterization. The specimens of C. pearsoni are, onaverage, larger than those of C. torquatus, and theangle of procumbency and the length of the rostrumare larger in C. pearsoni than in C. torquatus (Fig. 5).Other morphological distinctions between C. torquatusand C. pearsoni were also observed in phallic charac-ters (Lessa & Cook, 1989), in the mean length ofspikes, and in the spiny bulb of the baculum, which ischaracteristic of C. pearsoni and absent in C. torquatus(Altuna & Lessa, 1985). Both of these differences couldbe proposed as a pre-zygotic reproductive barrier asso-ciated with a chromosomal speciation mode. However,these characters were not systematically described forchromosomal populations. Mora et al. (2003) proposedthat differences in rostrum angle could result fromadaptations to different fossorial behaviours. In spiteof both species inhabiting a wide geographical range(Freitas & Lessa, 1984), using claws and incisors to digtheir tunnel systems (Vassallo, 1998), and havingbroadly homogeneous ecological niches and behav-iours, the putative adaptative hypothesis for differen-tiation between these tuco-tuco species could resultfrom differences in the soil type in which they buildtheir tunnel systems. In fact, C. pearsoni inhabitscoastal dunes, with predominantly sandy soils with avariable clay-loam fraction, whereas C. torquatus occu-pies harder soils.
In this adaptive hypothesis, one prediction wouldbe that C. torquatus 2n = 46 populations living in theRio Grande do Sul sandy coastal plain would show
Table 1. Procrustes distances from the mean shape for each chromosomal form, and for each view, of Ctenomys torquatusand Ctenomys pearsoni
Table 2. Percentage of correct classification from dis-criminant analysis for three views of the cranium sepa-rated and unified for the categorical variables species, sex,and chromosomal groups for Ctenomys torquatus fromBrazil (2n = 40, 44, and 46), C. torquatus from Uruguay(2n = 44u), and Ctenomys pearsoni (2n = 66 and 70)
convergence in skull shape with C. pearsoni popula-tions inhabiting similar habitats. In fact, thesehabitat-related populations were the most distant interms of shape distance (Fig. 7). It is more probablethat the differentiation observed in C. torquatus2n = 46 populations, in relation to the more closelyrelated C. torquatus 2n = 44 populations, would beexplained by genetic drift. Although this hypothesisis very likely, current 2n = 46 populations are com-pletely isolated by a complex of marshes and the SãoGonçalo channel, with the closing of the channeldating back to only 2600 years ago (Freitas & Lessa,1984). Therefore, under the drift hypothesis, thispopulation would have accumulated an enormousnumber of morphological differences in a very shortperiod of time. Adding to the morphological differen-tiation, Moreira et al. (1991), using biochemicalpolymorphisms for the same populations (2n = 44 and2n = 46), presented high coefficients of genetic simi-larity, of magnitudes generally found between species.
The systematic status of the chromosomal races ofC. pearsoni identified along the Uruguayan coast ofthe La Plata River and Atlantic Ocean (with diploidnumbers ranging from 2n = 56 to 70) has remaineduncertain since its original description by Kibliskyet al. (1977). At that time, all of these forms wereassigned to C. torquatus, before Lessa & Langguth(1983) designated them as C. pearsoni, based on thewestern populations (2n = 70) typical form for thespecies. The other karyomorphs have been referred toas members of the ‘pearsoni complex’ (Altuna & Lessa,1985; Tomasco & Lessa, 2007), or as yet-to-be-described independent evolutionary units (Altunaet al., 1999; González, 2001). Our analyses partiallycorroborate these considerations by showing clearmorphological differentiation among chromosomalpopulations. Particularly, the results indicate a verydistinct cranial shape for 2n = 66 populations, in rela-tion to the 2n = 70 ones and to C. torquatus chromo-somal populations. The magnitude of the interspecificmorphological difference, measured by both Pro-crustes and Mahalanobis distances, between theC. pearsoni 2n = 70 population and the C. torquatus2n = 44 population is very similar to the intraspecificone between the C. pearsoni 2n = 70 and 66 popula-tions. In spite of these intraspecific differences inskull shape, in karyomorphs (Villar et al., 2005) andthe probable distinctions based on penial morphology(Altuna & Lessa, 1985) there is no reason at this timeto recognize different species among the coastal popu-lations of tuco-tucos in Uruguay. There are mainlythree reasons for this. First, the range of chromo-somal polymorphisms found in C. pearsoni is alsopresent within other species of tuco-tucos (Freitas,2006, 2007). Second, we have sampling gaps betweenthe studied populations of C. pearsoni and along the
geographic distribution of the species that presentother chromosomal numbers not included in thepresent study (2n = 58, 64, 70b, and 70c). Finally,Tomasco & Lessa (2007) have shown, using the mito-chondrial control region, that C. pearsoni chromo-somal populations are polyphyletic, indicating onlyone biological species.
Chromosomal populations 2n = 46 and 66 present aparadox because they are likely to be the most recent,either inferred by the number of rearrangements andbiogeographical information in the first case (Freitas& Lessa, 1984), or by a molecular phylogeny in thesecond (Tomasco & Lessa, 2007), and both showedhigher morphological distances from the other popu-lations. The accelerated accumulation of differencesmight result from either strong selective pressuredriving morphological differentiation or strong driftcaused by small population sizes. All of these sce-narios of chromosomal and skull evolution show thatthe adaptive hypothesis is unlikely to be true for bothtuco-tucos species, and events of recent colonizationcoupled with the life strategy are the main factorsthat could explain these patterns.
The intraspecific difference between C. torquatus2n = 44 populations from Brazil and Uruguay wasalso remarkable. Despite Reig et al. (1990) havingproposed that the Ctenomys genus is a paradigm ofhigh chromosomal variation and quick speciation,with poor morphological differentiation, we find acoupling between morphometric and chromosomalvariation at both the inter- and the intraspecific level.Even more interestingly, we document a unique casein the genus of skull morphometric differentiationbetween Brazilian and Uruguayan populations of C.torquatus that present karyotypic uniformity: 2n = 44and FN = 72 (Freitas & Lessa, 1984). Even thoughthese populations showed differences in skull shape,a clear effective geographic barrier between the twocountries or ecological differences are absent. Prob-ably, the intrinsic patchy distribution and low vagilityof tuco-tucos (Lacey et al., 2000) can explain theisolation of some populations, and generated the mor-phological differentiation between these two groupsof populations of C. torquatus by drift, without anychromosomal rearrangement.
SPECIATION MODE AND MORPHOLOGICAL
EVOLUTION IN CTENOMYS
Speciation is a central theme in evolutionary biology(Berlocher, 1998). Chromosomal speciation can berapid, occurring over a brief time span, and oftenresults in numerous new species (King, 1993). Popu-lation size and structure, reproductive strategy,vagility, and the breeding system of taxa have beensuggested as determining their dominant speciation
modes (Bush, 1975). Empirical evidence and numericalsimulations (Gravilets, Li & Vose, 1998) haveconfirmed these predictions. The fossorial genusCtenomys, with its low population densities and patchydistribution, low vagility, and probably high inbreed-ing are supposed to speciate mainly with parapatricand microallopatric modes. Furthermore, the greatvariability in fundamental numbers within the genuspoint to chromosomal rearrangements as the maincandidate for reproductive barriers. It is well estab-lished that chromosome structure and reorganizationmay sometimes be involved in speciation (White,1978). On the whole, chromosomal variation has beenconsidered to be the main factor associated with spe-ciation in Ctenomys (Reig & Kiblisky, 1969; Reig et al.,1990), based on the widespread notion that within-genus chromosomal variation in Ctenomys is species-specific, and that chromosomal rearrangements areeither neutral or weakly under-dominant. However,the role of chromosomal rearrangements as a repro-ductive barrier in speciation is still controversial. Infact, the chromosomal literature shows a growingnumber of chromosomal polymorphisms that seem tofail to cause sterility in heterozygous individuals, aswell as several hybrid zones that showed probably noevidence of negative heterosis (Freitas, 1997; Braggioet al., 1999; Gava & Freitas, 2002; Gava & Freitas,2003). Moreover, the high frequency of chromosomalrearrangements in Ctenomys might indicate that inmost cases they are not intrinsically deleterious. As aconsequence, in the absence of the postulated mainisolating mechanism, not only does a sympatric modeof speciation seem unlikely, but more attention willalso have to be given either to possible isolatingcharacters or to the importance of genetic drift.
Several approaches are necessary to tackle the pos-sible relationship between chromosomal change andspeciation in Ctenomys. Recent research has focusedon the relationship between molecular and chromo-somal evolution (Slamovits et al., 2001; Tomasco &Lessa, 2007), and several studies have detailed chro-mosomal polymorphism (Kiblisky et al., 1977; Reiget al., 1990; Massarini et al., 1995; Braggio et al., 1999;García et al., 2000a; Freitas, 2001, 2006, 2007), or haveexamined breeding patterns in contact zones betweendifferent chromosomal races (Gava & Freitas, 2002;Gava & Freitas, 2003). Interestingly, studies haveshown the absence or very low levels of allozyme(Apfelbaum et al., 1991; Ortells & Barrantes, 1994),nucleotide (Mascheretti et al., 2000; Giménez et al.,2002; Tomasco & Lessa, 2007), and morphological(Ortells et al., 1990) differentiation among populationsand named species with different karyotypes.However, there has been no study coupling geometricmorphometric shape descriptors to chromosomalvariations to infer differentiation modes in Ctenomys.
Only one work has investigated the geographic varia-tion of the C. rionegrensis skull shape, and found nocorrelation between the morphological, geographic,and genetic distances (D’Anatro & Lessa, 2006). Ouranalyses quantified the size and shape variations inthe skull of C. torquatus and C. pearsoni, and alsoamong their chromosomal populations along its geo-graphical distribution in Brazil and Uruguay. Theeffects of chromosomal changes on both skull size andshape have already been investigated in rodents, andmany studies have shown that chromosomal varia-tions can be accompanied by morphological ones, some-times in correlation with ecological parameters (e.g.Mus, Corti, Ciabatti & Capanna, 1990; Chatti et al.,1999; Saïd et al., 1999; Spalax, Corti et al., 1996; andSouth African Murinae and Gerbillinae, Taylor, 2000).In other examples, few morphological differences havebeen observed in Taterillus species and Otomys irrora-tus karyomorphs (Muridae, Otomyinae), in spite ofbeing highly differentiated in terms of chromosomes(Viegas-Péquignot et al., 1986; Taylor, 2000; Dobigny,Baylac & Denys, 2002). Our study demonstrated andquantified morphological differences between speciesand between chromosomal forms within both species.At the interspecific level, the allopatric distribution,chromosomal distinctness, and sister-species relation-ship between C. torquatus and C. pearsoni, coupledwith morphological differences in their digging appa-ratus related to their habitat, suggests a parapatricspeciation mode with an invasion of a new ecologicalniche: sand dune habitats. The basal position of tuco-tucos from the inland (Slamovits et al., 2001) confirmsthat the invasion of dune habitats is derived. Within C.torquatus, this same mode of speciation might be atwork, as we confirmed significant morphometric andkaryotype difference in peripheral populations sur-rounding the widespread 2n = 44 karyotype (Fig. 1).Adding to that, the morphometric distinction of2n = 44 Uruguayan populations further corroboratesthis hypothesis. The evolutionary force driving bothmorphological and karyotypic evolution in this caseseems to be genetic drift, as we detected no morpho-logical convergence in the 2n = 46 population inhabit-ing dune habitats. Genetic drift seems to be especiallystrong, as morphological differences accumulatedrapidly in the 2n = 46 populations (over c. 2600 years).Isolation processes in C. pearsoni are likely to bedifferent. The distribution of different karyotypes islinear along the Uruguayan coast, and might havebeen the product of phylogeography (Avise, 2000), aswell as of recent geological processes such as sea levelchanges and Holocene marine transgressions thathave probably shifted their distribution (see Tomasco& Lessa, 2007). These processes are likely to haveinfluenced the eastern part of the distribution ofC. pearsoni, which consists of sand dunes along the
Atlantic coast interspersed with lagoons and wetlands,acting as isolating mechanisms. Genetic drift underperiods of geographic isolation might explain themorphological differences of interspecific magnitudebetween actual C. pearsoni populations.
The magnitude of morphological differences foundat the intraspecific level is surprising in at least twoaspects. First, because morphological differences seemto have accumulated extremely rapidly in regards topaleogeographical events that confirm populationisolation (2n = 66 and 70), or in the absence of suchisolating mechanisms (2n = 40 and 44 from Uruguay).Second, because tuco-tucos are fossorial organisms,and probably undergo strong stabilizing selective pres-sures for optimally fit shapes for excavation. The firstassertion seems to be confirmed by the polyphily of C.pearsoni (Tomasco & Lessa, 2007). The second asser-tion is confirmed by Mora et al. (2003), where only theangle of incisor procumbency is found to be variableamong species of Ctenomys. An apparent contradictionarises: on one hand there are significant morphologicaldifferences between chromosomal populations thatwould be driven by strong genetic drift forces andgeographical isolation, which are both products ofintrinsic organism properties (Reig et al., 1990) andpaleogeographical events. On the other hand, interspe-cific differences are minor, and when found are locatedin potentially adaptive regions of the skull, such asthe rostrum (Mora et al., 2003; this study). A possibleexplanation might be that drift and selection may beacting separately or in combination within differentmodules (see Klingenberg et al., 2001), although thishypothesis has to be formally tested (Fornel R,Cordeiro-Estrela P, Sanfelice D & Freitas TRO,unpubl. data). Directional selection might be acting onthe rostrum, with marked allometry, because it mightbe along the line of least evolutionary resistance, ascan be interpreted for other mammals (Voss et al., 1990for Sigmodontines; Marroig & Cheverud, 2001 forPrimates). But, as it is an axis of major variation, it isalso subjected to an increase in variance under drift.However, the functional interpretation of evolutionarymorphometric pattern is problematic in the contextof the microevolutionary events characterizing specia-tion in species of tuco-tucos.
To test these hypotheses, a complex researchprogram is necessary. A formal test of genetic driftversus selection (Ackeman & Cheverud, 2002; Marroig& Cheverud, 2004; Harmon & Gibson, 2006) on multi-variate data must be carried out at the interspecificlevel. Population genetic studies are necessary toquantify effective population sizes and drift. Phyloge-netic studies will have to be carried out on the samespecies to compare morphological and molecular ratesof evolution (Bromham et al., 2002). One of the impli-cations of this fact is that, until research programs
reach those objectives, inferences from this studybased on collection material are of crucial importance.
ACKNOWLEDGEMENTS
The Uruguayan samples were kindly provided by DrEnrique González from the Museo Nacional de Histo-ria Natural y Antropología, Montevideo, Uruguay. RF,PCE and TROF were funded by CNPq grants. FAF wasfunded by a CAPES grant. The laboratory work wassupported by Projeto tuco-tuco, CNPq and FAPERGS.
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APPENDIX 1
List of Ctenomys specimens examined.
Numberin map Species 2n Country Municipality/department Sex and collection number
4 C. torquatus 44 Brazil Butiá F: TR289, 594, J131, 170, 199, 201, 204,205, JR591–595
5 C. torquatus 44 Brazil Candelária M: MNHNA18856 C. torquatus 44 Brazil Cachoeira do Sul F: TR921–9247 C. torquatus 44 Brazil Dom Pedrito F: TR903, 904/M: TR9068 C. torquatus 44 Brazil General Câmara F: TR902, J238, 239/M:TR900, 901, J2379 C. torquatus 44 Brazil Itaqui F: TR592, 593, 597/M: TR956
11 C. torquatus 44 Brazil Quaraí F: TR912/M: TR91112 C. torquatus 44 Brazil Rosário do Sul F: TR931, 932, 935, 936, 938/M: TR933,
934, 93713 C. torquatus 44 Brazil Santa Maria F: TR926–93014 C. torquatus 44 Brazil Santana do Livramento F: TR908, 909/M: TR91015 C. torquatus 44 Brazil Uruguaiana F: TR954/M:TR95516 C. torquatus 46 Brazil Rio Grande F: TR212, 582, 589, 591, 593, 603, 605,
609, 946, 948, 949/M: TR404, 595, 947,950, 951
17 C. torquatus 46 Brazil Taim F: TR 57, 71–73, 87, 577–579, 583, 584,587, 592, 602, 604, 608, 614/M: TR70,86, 575, 617
18 C. torquatus 44 Uruguay Cerro Largo F: MNHNA 1809, 2474/M: MNHNA1810, 2026, 2623
23 C. pearsoni 70 Uruguay San José F: MNHNA1806, 1807, 1951, 2795, 2796
F, females; M, males.The collections of mammals were from Departamento de Genética, Instituto de Biociências, Universidade Federal do RioGrande do Sul, Porto Alegre, Brazil (TR, J, and JR) and Museo Nacional de Historia Natural y Antropología, Montevideo,Uruguay (MNHINA).
Key to the landmarks, with numbers and locations,indicated in the dorsal, lateral, and ventral views ofthe skull of the Ctenomys species shown in Figure 2.
Dorsal view of cranium: 1, anterior tip of the suturebetween premaxillaries; 2–3, anterolateral extremityof incisive alveolus; 4, anterior extremity of the suturebetween nasals; 5–6, anteriormost point of the suturebetween nasals and premaxillaries; 7–8, anteriormostpoint of the root of the zygomatic arch; 9, suturebetween nasals and frontals; 10–11, anterolateralextremity of lacrimal bone; 12–13, point of minimumbreadth between frontals; 14–15, terminal extremity ofpostorbital process; 16–17, anterior extremity of thesuture between frontal and squamosal; 18–19, poste-rolateral extremity of postorbital process; 20–21, tip ofposterior process of jugal; 22, suture between frontalsand parietals; 23–24, anterolateral extremity of thesuture between parietal and squamosal; 25–26, ante-rior tip of the external auditory meatus; 27–28, point ofmaximum curvature on mastoid apophysis; 29, poste-riormost point of occipital.
Ventral view of cranium: 1, anterior tip of thesuture between premaxillaries; 2–3, anterolateralextremity of incisive alveolus; 4–5, lateral edge ofincisive foramen at the suture between premaxillaryand maxillary; 6–7, anteriormost point of the root ofthe zygomatic arch; 8–9, anteriormost point of theorbit; 10–11, anteriormost point of the premolaralveolus; 12–13, posterior extremity of third molaralveolus; 14, posterior extremity of suture between
palatines; 15–16, anteriormost point of intersectionbetween jugal and squamosal; 17–18, posteriormostpoint of pterygoid; 19–20, anterior extremity oftympanic bulla; 21–22, anterior tip of the externalauditory meatus; 23–24, posterior extremity ofthe mastoid apophysis; 25–26, posterior extremity ofthe paraoccipital process; 27, anteriormost pointof foramen magnum; 28–29, posterior extremity ofoccipital condyle; 30, posteriormost point of foramenmagnum.
Lateral view of cranium: 1, anteriormost point ofpremaxillary; 2, posteriormost point of incisive alveo-lus; 3, inferiormost point of incisive alveolus; 4, ante-rior tip of nasal; 5, anteriormost point of the suturebetween nasal and premaxillary; 6, suture betweenpremaxilla, maxilla and frontal; 7, inferiormost pointof suture between lacrimal and maxillary; 8, inferior-most point of infraorbital foramen; 9, inferiormostpoint of suture between premaxillary and maxillary;10, anteriormost point of premolar alveolus; 11, supe-rior extremity of postorbital process; 12, inferiorextremity of jugal process; 13, tip of posterior processof jugal; 14, medial point of suture between parietaland squamosal; 15, superior extremity of lambdoidalcrest; 16, superiormost point of suture between squa-mosal and tympanic bulla; 17, posteriormost point ofpterygoid; 18, inferior extremity of mastoid process;19, anteriormost margin of paraoccipital process; 20,posteriormost margin of paraoccipital process; 21,posterior extremity of intersection between occipitaland tympanic bulla.
