ARTÍCULO

Phylogenetic relationships between tuco-tucos (Ctenomys, Rodentia) of the Corrientes group and the C. pearsoni complex

 

Diego A. Caraballo¹, Ivanna H. Tomasco², Denise H. Campo¹, and María Susana Rossi¹

¹ IFIBYNE-CONICET. Laboratorio de Fisiología y Biología Molecular, Dep. Fisiología, Biología Molecular y Celular. Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires, Ciudad Universitaria, Pabellón II, 2do piso, EHA1428, Buenos Aires, Argentina. [Correspondence: María Susana Rossi <srossi@fbmc.fcen.uba.ar>], Tel. + 54 11 4576 3386/68
² Laboratorio de Evolución. Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay. Iguá 4225 esq. Mataojo. Montevideo, 11.400

Recibido 28 septiembre 2015.
Aceptado 17 marzo 2016.
Editor asociado: E Palma

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ABSTRACT.

Lineage delimitation is of extreme importance in evolutionary biology and constitutes an essential tool in basic and applied fields including conservation. We addressed the delimitation of two closely related species complexes of South American rodents of the genus Ctenomys (tuco-tucos), namely the C. pearsoni and Corrientes groups, whose relationships have been unclear or conflicting in previous phylogenetic studies. We performed a molecular phylogenetic analysis, increasing the number of representatives of each group and includ­ing sequences of three mitochondrial loci (the complete cytochrome b coding sequence, and partial regions of the cytochrome oxydase I gene and the D-loop of the control region). The trees obtained using Bayesian Infer­ence and Maximum Parsimony methods show the Corrientes group and the C. pearsoni complex as reciprocally monophyletic sister clades, with higher values of intergroup compared to intragroup genetic distances. These results indicate that the Corrientes group and the C. pearsoni complex are differentiated lineages. The resulting tree topology is in agreement with a scenario of independent chromosomal rearrangements from the ancestral karyomorph (2n = 70 FN = 84), leading to the considerable chromosomal diversity that characterizes both groups.

RESUMEN.

Relaciones filogenéticas entre los tuco-tucos (Ctenomys, Rodentia) del grupo Corrientes y del complejo C. pearsoni.

La delimitación de linajes tiene una gran importancia en biología evolutiva y constituye una herramienta fundamental en disciplinas básicas y aplicadas incluyendo la conservación. Abordamos la delimitación de dos complejos de especies de roedores sudamericanos del género Ctenomys (tuco-tucos), denominados grupos C. pearsoni y Corrientes, cuyas relaciones resultaron poco claras o conflictivas en estudios filogenéticos previos. Realizamos una filogenia molecular, aumentando el número de representantes de cada grupo e incluyendo secuencias de tres marcadores mitocondriales (la secuencia completa de la región codificante del gen del citocromo b y secuencias parciales del gen de la enzima citocromo oxidasa I, y del D-loop de la región control). Los árboles obtenidos mediante métodos de inferencia bayesiana y máxima parsimonia muestran que el grupo Corrientes y el complejo C. pearsoni son grupos hermanos recíprocamente monofiléticos con mayores valores de distancias genéticas entre grupos que dentro de los grupos. Estos resultados indican que el grupo Corrientes y el complejo C. pearsoni son linajes diferenciados. La topología resultante es consistente con un escenario de fijación de reordenamientos cromosómicos independientes a partir del cariomorfo ancestral (2n = 70 NF = 84), dando origen a la considerable diversidad cromosómica que caracteriza a ambos grupos.

Key words: Corrientes group; Ctenomys pearsoni; Mitochondrial phylogeny.

Palabras clave: Ctenomys pearsoni; Filogenia mitocondrial; Grupo Corrientes.

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INTRODUCTION

Subterranean rodents of the genus Ctenomys that inhabit the southern cone of South America have one of the highest numbers of living species among mammals; at least 60 have been recognized (Woods and Kilpatrick, 2005). Although the relationship among dif­ferent groups of species is not fully resolved, chromosomal and molecular phylogenetic studies clustered related species into several species groups (Gallardo, 1979; Massarini et al., 1991; Ortells and Barrantes, 1994; Cook and Yates, 1994; Parada et al., 2011). One of these groups, named torquatus by Parada et al. (2011) includes the species that inhabit the eastern part of the distribution of the genus: C. torquatus, C. lami, C. minutus, C. pearsoni, and three species belonging to the Corrientes group, C. roigi, C. perrensi and C. dorbignyi. Recently a new member was described as a distinct species in the torquatus group: C. ibicuiensis (Freitas et al., 2012). However, the specific status or relationships among the species of tuco-tucos of the torquatus group are not fully resolved. In a phy­logeny based on complete cytochrome b (cyt-b) sequences that includes representatives from all the species of the group, four reciprocally monophyletic clades could be distinguished: C. ibicuiensis, C. torquatus, C. lami + C. minutus, and C. pearsoni + Corrientes group (Freitas et al., 2012). The relationship between C. pearsoni and the Corrientes group is poorly resolved in the published phylogenies, but these groups were never subjected to an exhaustive survey of molecular characters and taxon representation. Parada et al. (2011), using complete sequences of cyt-b, recovered a paraphyletic Corrientes group that included C. pearsoni, a result reaf­firmed by Freitas et al. (2012). In contrast, other phylogenies based on partial sequences of the same mitochondrial gene split C. pearsoni from the Corrientes group (Fernandes et al., 2009; Caraballo et al., 2012). In all these phylogenies, no more than four representatives of C. pearsoni were included.

The tuco-tucos of the Corrientes group in­habit a large area under the direct or indirect influence of the Iberá marsh and its lagoons and channels in the Province of Corrientes, Argentina (Fig. 1). Based on allozymes and chromosome banding, this group was delim­ited by the pioneering work of Ortells and Barrantes (1994). Ctenomys pearsoni inhabits the coastal plains of southern Uruguay (Lessa and Langguth, 1983; Tomasco and Lessa, 2007) and the western part of the Province of Entre Ríos, Argentina (García et al., 2000) (Fig. 1).

Fig. 1. Map showing sampling localities. The numbers correspond to the following localities: (1) Mbarigüí, (2) Paraje Angostura, (3) Manantiales, (4) Mburucuyá, (5) Loma Alta, (6) Pago Alegre, (7) Saladas Centro, (8) Estancia San Luis, (9) Rincón de Ambrosio, (10) Saladas Sur, (11) Colonia 3 de abril, (12) San Roque, (13) Santa Rosa, (14) Paraje Caimán, (15) San Miguel, (16) Curuzú Laurel, (17) Loreto, (18) Estancia La Tacuarita, (19) Contreras Cué, (20) San Alonso, (21) Chavarría, (22) Goya, (23) Paraje Sarandicito, (24) Médanos, (25) Limetas, (26) Arazatí, (27) Roosevelt, (28) Chihuahua, (29) José Ignacio, (30) Laguna de Rocha and (31) Valizas.

The Corrientes group and the C. pearsoni complex share extreme chromosomal variability, intraspecific in the case of C. pearsoni with chromosomal numbers (2n) ranging be­tween 56-58, 64-66 and 70 and fundamental numbers (FN) 80, 82 and 84 (Tomasco and Lessa, 2007, and references therein), and intra and interspecific in the case of the Corrientes group with chromosomal numbers that range between 41-44, 44-46, 48 and 50-70 and with FN 76, 78, 80 and 84, respectively (Caraballo et al., 2015, and references therein). The karyo­morph 2n = 70 FN = 84 is shared by the C. pearsoni complex and the Corrientes group and was proposed by Ortells et al. (1990) as the ancestral morph for both groups. These authors stated that the extant 2n = 70 FN = 84 karyomorphs found in Argentina and Uruguay are relicts of a more widespread distribution, from which lower chromosomal numbers could have originated by means of Robert­sonian rearrangements.

Lineage and species delimitation are non-trivial issues in evolutionary biology. Un­derestimation or inflation of the number of species in certain groups of organisms could adversely affect biodiversity conservation and management efforts (Zachos et al., 2013, and references therein). In addition, a reliable phylogeny is a crucial tool to understand the evolution of behavioural (Strier et al., 2014), morphological (Morgan and Álvarez, 2013) or genomic characters (Slamovits et al., 2001). In the case of the tuco-tucos of the Corrientes group and the C. pearsoni complex, tracking the conspicuous chromosomal evolution that characterizes both groups is not possible in the absence of clear delimitation of independent evolutionary lineages and knowledge of their historical relationships.

The aim of the present work is to examine the relationship between C. pearsoni and the tuco-tucos that inhabit the Corrientes Province with an expanded taxon and character sam­pling. We included complete cyt-b sequences of 42 representatives of the Corrientes group, 15 of C. pearsoni and 15 additional specimens, including representatives of the torquatus group and other Ctenomys species. We also added to the analysis partial sequences from two additional mitochondrial loci, the control region (D-loop) and the cytochrome oxidase I coding gene (COI).

MATERIALS AND METHODS

Specimens

Animals were captured with modified Oneida Victor Number 0 snap traps. Guidelines of the American Society of Mammalogists (Gannon and Sikes, 2007) were followed. Well-preserved skulls were submitted to the Colección de Mastozoología of the MuseoAr­gentino de Ciencias Naturales "Bernardino Rivada­via" for Argentinean specimens, and to the collection of the Laboratorio de Evolución in the Facultad de Ciencias of the Universidad de la República, Mon­tevideo, for the Uruguayan specimens. Trapping localities with their geographical location (Fig. 1) as well as field / catalog numbers (when available) were recorded (Table 1).

Table 1 Sampling localities, specimen field codes, voucher numbers (when available), geographical location and Gen­Bank accession numbers of the samples of the Corrientes group and the C. pearsoni complex included in this study. Acronym MACN-Ma corresponds to vouchers deposited at the Colección de Mastozoología of the Museo Argentino de Ciencias Naturales "Bernardino Rivadavia", while acronyms CA and EV correspond to the collection of the Laboratorio de Evolución in the Facultad de Ciencias of the Universidad de la República, Montevideo, Uruguay.

This study included a total of 73 samples; 42 individuals were from 23 localities of the Corrientes Province (Argentina), two samples of C. pearsoni are from Médanos, Entre Ríos Province (Argentina), while 13 were sampled at 7 Uruguayan localities (Table 1). The rationale of the sampling strategy was to cover the overall haplotypic diversity ob­served among these two groups along their known geographic distributions, based on previous mtDNA studies (Tomasco and Lessa, 2007; Caraballo et al., 2012). To test the Corrientes group + C. pearsoni complex monophyly, we also included sequences from other members of the torquatus group: 4 specimens of C. torquatus (Fernandes et al., 2009), 3 specimens of C. minutus (Lopes et al., 2013), 1 C. lami (Lopes and Freitas, 2012) and 1 C. ibicuiensis (Freitas et al., 2012). As outgroup for the torquatus group, we included sequences of the species: C. conoveri (1), C. rionegrensis (3), C. sociabilis (1), C. leucodon (1) and Octodon degus (1) (Tomasco and Lessa, 2011; Caraballo et al., 2012).

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega). A 482 bp partial fragment encompassing the 5' end of the mitochondrial D-loop, plus a 775 bp fragment cover­ing a 53 bp non-coding flanking sequence and 722 bp of the 5' end of the COI coding sequence were amplifyied using primers and protocols detailed in Caraballo et al. (2012). Complete cyt-b sequences were obtained by PCR amplification using the fol­lowing primer pairs: MVZ05TUCO and tuco06, tuco07 and tuco14a, TUTU-F and TUTU-R, MVZ06 and tuco16. Primers tuco06, tuco07, tuco14a and tuco16 were designed by Wlasiuk et al. (2003) while MVZ06, by Smith and Patton (1999). MVZ05TUCO is a modification of MVZ05 (Smith and Patton, 1999) designed by us: 5'CAAGACTAATGATAT­GAAAAACCATTGTT 3'. Primers TUTU-F and TUTU-R were designed by Caraballo et al. (2012).

PCR products were amplified and directly se­quenced on one strand, since previous contigs of both strands did not reveal differences in these loci (Caraballo et al., 2012). Cytochrome oxydase I, D-loop and the 3' end of cyt-b sequences from the Corrientes group were previously obtained and deposited in GenBank under the accession numbers JX275502 to JX275655 (Caraballo et al., 2012). The remaining cyt-b partial sequences of the Corrientes group, as well as the complete dataset of C. pearsoni and the samples CA 426 (C. rionegrensis), EV 1169 (C. rionegrensis), CA 783 (C. torquatus) and NK 12607 (C. conoveri) were generated in this study and deposited in GenBank (accession numbers KT818638 - KT818684 and KT900 - KT900168). The sequences corresponding to an additional C. rionegrensis, C. ibicuiensis, C. lami, C. minutus, 3 additional C. torquatus, C. sociabilis, C. leucodon and Octodon degus specimens were retrieved from GenBank (see Table 2, Supplementary Material).

Sequence alignment and analyses

Sequence electropherograms were visually inspected using Bioedit (Hall, 1999) and aligned with CLUST­AL X2 (Larkin et al., 2007). Ambiguous alignments were not evident in any of the sequenced loci and four indels were postulated only for D-loop. Nucleotide variability indexes (number of variable sites, parsimony informative sites, nucleotide and haplotype diversity indexes) were computed for each locus using DNASP 5.10.01 (Librado and Rozas, 2009). Substitution saturation was assessed with DAMBE (Xia and Xie, 2001), performing a test introduced by Xia et al. (2003, 2009) in 5 different partitions: 1^st and 2^nd cyt-b codon positions, 3^rd cyt-b codon position, 1^st and 2^nd COI codon position, 3rd COI codon position, D-loop. P-distances and net divergence among pairs of species and complexes were calculated with MEGA (Tamura et al., 2013).

Phylogenetic inference

Phylogenetic analyses were performed with the concatenated data set since all 3 loci are linked in the mitochondrial genome. Trees were rooted on the branch leading to Octodon degus. Different schemes and models were assayed by Bayesian Inference and Maximum Parsimony.

Maximum parsimony (MP) searches were per­formed in PAUP 4.0 (Swofford, 2003) using equal transformation costs and weights while internal gaps were treated as a fifth character. Heuristic searches were performed running 1000 independent Ran­dom Addition Sequences (RAS) with hold 10 and using TBR swapping. Clade support was assessed running 10 000 bootstrap replicates with the same search parameters.

Bayesian inference (BI) analyses were carried out using MrBayes 3.2.5 (Ronquist et al., 2012) under 3 different data treatments: (1) fixing a substitution model for each of the three mitochondrial loci, (2) estimating a substitution model for each locus and (3) estimating a substitution model discrimi­nating codon positions and non-coding sequences. For (1), the corrected Akaike Information Criterion (AICc) was used to find the simplest substitution model that explained the data, using the program MrModeltest v2 (Nylander, 2004). In the first two treatments, the dataset was partitioned into three subsets, D-loop, cyt-b and COI, while in the third treatment the dataset was partitioned into 5 subsets: D-loop, 1^st and 2^nd codon positions for cyt-b, 3^rd codon position for cyt-b, 1^st and 2^nd codon positions for COI and 3^rd codon position for COI.

For each data treatment, two simultaneous runs with four Markov chains each were run for 2 000 000 generations, and trees were sampled every 500 generations. The standard deviation of split frequencies was analyzed to determine when the Markov chain reached stationarity and to determine the number of samples to be discarded as "burn-in". Additionally, the effective sample sizes (ESS) of parameters sampled from Markov chain Monte Carlo were analyzed using TRACER 1.6 (Rambaut and Drummond, 2013). We stopped the analyses when we verified that the standard deviation of split frequencies was smaller or equal to 0.01, ESS for all estimated parameters were higher than 200 and the "burn-in" phase corresponded at most to one third of total generations. These conditions were accomplished with the initial settings for data treat­ments 1 and 2, while we needed to run 12 000 000 additional generations for data treatment 3. A 50% majority rule consensus tree was constructed with the post "burn-in" distribution and the percentage of samples recovering each clade was used to estimate its posterior probability. These consensus trees were visualized in FigTree 1.4.2 (Rambaut, 2014).

RESULTS

Sequence variability

Different measures of polymorphism and nucleotide diversity were computed for the Corrientes group and the C. pearsoni complex. The 697 bp sequence alignment obtained for COI yielded 70 polymorphic sites (48 of which are parsimony informative sites) defining 37 different haplotypes for 55 of the 57 indi­viduals (samples EV1471 and CA 455 did not yield PCR products). Uncorrected nucleotide diversity (Pi) and haplotype diversity (HD) indexes were 0.015 (SD 0.001) and 0.980 (SD 0.008), respectively.

The 1140 bp sequence alignment obtained for cyt-b yielded 102 polymorphic sites (77 of which are parsimony informative) defining 35 different haplotypes for the 57 studied specimens. Uncorrected nucleotide diversity (Pi) and haplotype diversity (HD) indexes were 0.014 (SD 0.002) and 0.982 (SD 0.006), respectively.

The 438 bp sequence alignment obtained for D-loop yielded 39 polymorphic sites (26 of which were parsimony informative) defin­ing 38 different haplotypes for the 57 studied specimens. Uncorrected nucleotide diversity (Pi) and haplotype diversity (HD) indexes were 0.019 (SD 0.001) and 0.986 (SD 0.006), respectively.

Table 3 shows net distances within species and groups of species of the torquatus group. As expected, distances between any member of the torquatus group and outgroups were higher than between any of its members. Within the torquatus group, with the exception of the low distance between C. minutus and C. lami (0.002) that reflects the close relationship between these two hybridizing species (Gava and Freitas, 2003), net distance values are comparable. However, the net distances between C. torquatus and C. lami or C. minutus are lower (0.011 and 0.016, respectively) than the distances between C. torquatus and C. pearsoni or the Corrientes group (0.025 and 0.019, respectively), confirming again that C. torquatus is most closely related to these two Brazilian coastal species (Freitas, 2012). The Ctenomys pearsoni complex and the Corrientes group showed a net distance value (0.012), comparable with the C. torquatus-C. lami or C. torquatus- C. minutus distances, verifying also their close relatedness. However, the intragroup distances are lower (0.014 for C. pearsoni complex and 0.011 for the Corrientes group) than the inter­group distance (0.024), which indicates these two groups are genetically distinctive.

Table 3 Net divergence among pairs of species and complexes within the torquatus group and between them and outgroups.

No saturation was revealed (Iss<Iss.c, p<0.0001) for any of the five partitions (COI 1^st and 2^nd codon positions, COI 3^rd codon position, cyt-b 1^st and 2^nd codon position, cyt-b 3^rd codon position, and D-loop), indicating they are usable for phylogenetic analyses.

Phylogenetic relationships

The concatenated matrix (D-loop + COI + com­plete cyt-b) is 2282 bp long; 733 characters are variable, 401 of which are parsimony informa­tive. For the Corrientes group + C. pearsoni complex, 141 characters are variable, 103 of which are parsimony informative, defining a total of 40 haplotypes.

The trees obtained by BI under the three conditions and by MP were congruent and the main clades (CP, C and P, see below) received high bootstrap support in all cases (see Table 2, Supplementary Material). Fig. 2 shows the BI consensus tree resulting from the estimation of independent substitution models distinguish­ing between different codon positions as well as non-coding sequences (see Materials and Methods; model parameters estimated by MrBayes, as well as fixed model parameters obtained by MrModeltest are shown in Table 2, Supplementary Material). As expected, the torquatus group divergence from the remain­ing Ctenomys species is represented by a well-supported basal node (node O). The torquatus group (node T) splits into three well supported lineages: the recently described C. ibicuiensis (Freitas et al., 2012), C. minutus + C. lami + C. torquatus and the Corrientes group + C. pear­soni (node CP). This basal splitting within the torquatus group coincides with the phylogeny previously published by Freitas et al. (2012). However, while the individual of C. pearsoni grouped to a representative from Paraje Sa­randicito (a member of the Corrientes group, which turned out paraphyletic) in Freitas et al. (2012), in our analysis all the 15 representa­tives of C. pearsoni, including two individuals from the locality of Médanos in the Province of Entre Ríos, Argentina, clustered above a well supported node (node P). On the other hand, the 42 individuals from 23 Correntinean tuco-tucos populations, including those from Paraje Sarandicito, formed a well-supported clade (C). Thus, C. pearsoni and the Corrientes group are monophyletic sister clades that belong to the torquatus group.

Fig. 2. Bayesian Inference phylogenetic tree based on concatenated sequences of cyt-b, COI and D-loop, estimating different model parameters for codon positions (1st + 2nd, and 3rd position separately) and non-coding sequences (see Material and Methods). The numbers in the nodes indicate posterior probability. T: torquatus group, C + P: Corrientes group + C. pearsoni complex group, C: Corrientes group and P: C. pearsoni complex group. Branch lengths are proportional to the number of changes.

DISCUSSION

We reanalyzed the evolutionary relationship between the C. pearsoni complex and the Corri­entes group of tuco-tucos that was controversial in the literature. We constructed a molecular phylogeny by enlarging the number of represen­tatives of each group and including sequences of three mitochondrial loci, resulting in the largest data matrix for these groups to date: 2275 characters and 57 taxa within the Corri­entes group + C. pearsoni complex. Concordant with previous analyses (Freitas et al., 2012), the tree recovered three main basal lineages of the torquatus group: C. ibicuiensis, C. tor­quatus + C. lami + C. minutus and C. pearsoni complex + Corrientes group. The addition of a higher number of representatives and characters clearly split the C. pearsoni complex and the Corrientes group into two sister clades. These two divergent groups of tuco-tucos should be recognized by biodiversity management programs, as they are independent lineages. However, these mitochondrial data should be supplemented with nuclear markers.

Within the Corrientes group, most popula­tions are monophyletic or (as in the cases of Loma Alta and San Miguel) are included in clades with closely related karyomorphs (Cara­ballo et al., 2015). This may be the case in the C. pearsoni complex, although there is evidence of maintenance of ancestral polymorphisms, as shown by Tomasco and Lessa (2007).

These two groups of tuco-tucos have un­dergone high rates of chromosomal evolution. The 2n = 70 FN = 84 karyomorph is the only form common to both clades, and hence it is likely to be ancestral. The ample chromosomal variability observed in both groups would be the result of the fixation of independent rear­rangements across the lineages. Although most chromosomal rearrangements postulated are Robertsonian fusions/fissions, which involve changes in chromosomal number without altering the fundamental number, inversions and/or translocations have been postulated as responsible of the changes in the fundamental number (Ortells and Barrantes, 1994; Caraballo, 2013), and hence are likely to constitute reproductive barriers.

Finally, we would note that although the population of Médanos falls into the C. pearsoni clade (node P) and the 2n = 70 FN = 84 karyo­morph of specimens of this population was indistinguishable from others of C. pearsoni (García et al., 2000), this is the only population of C. pearsoni of Entre Ríos included in phylo­genetic analyses to date. An expanded survey in the Province of Entre Ríos should be performed to further document the phylogenetic position of these tuco-tucos. Ctenomys pearsoni is not the only tuco-tuco species on both sides of the Uruguay River: populations of C. rionegrensis are distributed in both Uruguay and Argentina at the 31° 44' S - 34° 56' S latitude (Bidau et al., 2008 a,b). The association of these species with the history of the Uruguay River remains to be explored.

ACKNOWLEDGMENTS

This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 11220110100910CO) and the Linnean Society (Systematics Research Fund) from Argentina and the United Kingdom, respectively. D.A.C. is supported by a postdoctoral fellow­ship awarded by CONICET. M. S. R. is career investigator of CONICET. We are grateful to Thales de Freitas for assistance in matching individual sequences of Brazilian tuco-tucos. Finally, we would like to thank J.A.C., an anonymous reviewer and the editor whose comments improved the manuscript.

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SUPLEMENTARY MATERIAL ON-LINE

Fig 2. Maximum Parsimony consensus tree computed for 10 000 bootstrap replicates (node values indicate bootstrap support) of heuristic searches consisting in 1000 RAS with hold 10. https://www.sarem.org.ar/wp-content/uploads/2016/06/SAREM_MastNeotrop_23-1_Caraballo-sup1.docx