SciELO - Scientific Electronic Library Online

 
vol.26 número1Locomoción en el roedor más veloz, la mara Dolichotis patagonum (Caviomorpha; Caviidae; Dolichotinae)Riqueza, endemismo y conservación de roedores sigmodontinos en Argentina índice de autoresíndice de assuntospesquisa de artigos
Home Pagelista alfabética de periódicos  

Serviços Personalizados

Journal

Artigo

Indicadores

  • Não possue artigos citadosCitado por SciELO

Links relacionados

Compartilhar


Mastozoología neotropical

versão impressa ISSN 0327-9383versão On-line ISSN 1666-0536

Mastozool. neotrop. vol.26 no.1 Mendoza jun. 2019

 

ARTÍCULO

How many species of grey foxes (Canidae, Carnivora) are there in Southern South America?

 

M. Amelia Chemisquy1, 2, Francisco J. Prevosti1, 2, Pablo Martínez3, Vanina Raimondi4, 5, Javier E. Cabello Stom6, Gerardo Acosta-Jamett7 and Juan I. Montoya-Burgos4

1 Centro Regional de Investigaciones Científicas y Transferencia Tecnológica de La Rioja (CRILAR - CONICET, UNLaR, UNCa, SEGEMAR), Anillaco, La Rioja, Argentina. [Correspondence: M. Amelia Chemisquy <amelych80@gmail.com>]

2 Departamento de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de La Rioja (UNLaR), La Rioja, Argentina.

3 PIBi Lab – Laboratorio de Pesquisas Integrativas em Biodiversidade, Universidade Federal de Sergipe, São Cristóvão, Sergipe, Brasil.

4 Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland.

5 División Mastozoología, Museo Argentino de Ciencias Naturales “Bernardino Rivadavia” (CONICET), Ciudad Autónoma de Buenos Aires, Argentina.

6 Facultad de Medicina Veterinaria, Universidad San Sebastián Sede de la Patagonia. Lago Panguipulli, Puerto Montt, Chile.

7 Instituto de Medicina Preventiva Veterinaria y Programa de Investigación Aplicada en Fauna Silvestre, Facultad de Ciencias Veterinarias Universidad Austral de Chile, Valdivia, Chile.


ABSTRACT

Two species of grey foxes are recognized in the Southern Cone of America: Lycalopex griseus, and L. gymnocercus, which were traditionally separated by size and some cranial differences. Morphometric analyses of the skull showed that both species could be the same and that they show clinal variation, while DNA studies based on one mitochondrial marker suggested that they belong to different species. Our objective is to evaluate the systematic status of these foxes using three mitochondrial markers (cytochrome B, cytochrome oxidase I, and control region), and a large sample covering a wide geographic range. The results indicate that there are two clades, that are not sister taxa, a finding that is more congruent with the hypothesis of two species, but their geographic distribution is not coincident with the accepted distribution of L. griseus and L. gymnocercus. Consequently, the distribution of L. griseus is extended eastern including north and center of Argentina, towards the west and south of the Paraná, Paraguay and Río de la Plata rivers. On the other hand, the clade that probably represents L. gymnocercus is restricted to the east of those rivers, except for a few specimens collected in Santa Fe, close to the Paraná river. However, an analysis of a wider sample using nuclear DNA is needed to confirm the taxonomic identity of these species of grey foxes.

RESUMEN

¿Cuántas especies de zorros grises (Canidae, Carnivora) hay en el sur de Sudamérica?

Dos especies de zorros grises se reconocen en el Cono Sur de América: Lycalopex griseus y L. gymnocercus, que tradicionalmente estaban separadas por tamaño y algunas diferencias craneales. Análisis morfométricos del cráneo mostraron que estas especies podrían ser una sola que muestra variación clinal, mientras que estudios de ADN basados en un marcador mitocondrial sugirieron que pertenecen a especies diferentes. Nuestro objetivo es evaluar el estado sistemático de estos zorros utilizando tres marcadores mitocondriales (citocromo B, citocromo oxidasa I y región control) y una muestra grande que cubre un amplio rango geográfico. Los resultados indican que hay dos clados, que no son taxones hermanos, un hallazgo que es más congruente con la hipótesis de dos especies, pero su distribución geográfica no es coincidente con la distribución aceptada de L.  griseus y L.  gymnocercus. Consecuentemente, la distribución de L. griseus se extiende hacia el este, incluyendo el norte y centro de la Argentina, hacia el este y el sur de los ríos Paraná, Paraguay y Río de la Plata. Por el otro lado, el clado que probablemente representa a L. gymnocercus está restringido hacia el este de esos ríos, excepto por algunos especímenes colectados en Santa Fe, cerca del río Paraná. Sin embargo, se necesita un análisis de una muestra más amplia que utilice ADN nuclear para confirmar la identidad taxonómica de estas especies de zorros grises.

Key words: Foxes; Mitochondrial DNA; Species delimitation; Systematics.

Palabras clave: ADN mitocondrial; Delimitación de especies; Sistemática; Zorros.

Recibido 13 junio 2018.
Aceptado 26 diciembre 2018.

Editor asociado: P. Teta


INTRODUCTION

Knowledge of the taxonomic limits and distribution of species is important not only for their conservation, but also for the study of their evolutionary history (e.g., Kutschera et al. 2014; vonHoldt et al. 2016). Recently in Carnivora, molecular studies have been changing these boundaries, both splitting species (e.g., Trigo et al. 2013; Helgen et al. 2013), as well as merging different taxa (e.g., Schiaffini et al. 2013). Also, molecular studies have been revealing complex patterns of evolution, such as hybridization and introgression, which also lead to a redefinition of species boundaries (e.g., Kutschera et al. 2014; vonHoldt et al. 2016). In this context, there are still some doubts regarding the identity and the species boundaries of South American canids, which have not been thoroughly evaluated using molecular data (e.g., Zunino et al. 1995).

Foxes of the Southern Cone of America include two genera, Cerdocyon that inhabits different kind of forested areas in the northern part of this area, and Lycalopex that covers the whole area with the exception of the Paranean and Atlantic forests (Macdonald & Sillero Zubiri 2004; Sillero Zubiri et al. 2004; Wilson & Mittermeier 2009). Lycalopex include several species: the culpeo fox (Lycalopex culpaeus) that is a large fox (ca. 10 kg) with a diet mostly composed of small mammals, and with an Andean and Patagonian distribution; the grey foxes that were commonly assigned to Lycalopex griseus and Lycalopex gymnocercus; and Darwin’s fox (Lycalopex fulvipes) (Macdonald & Sillero Zubiri 2004; Sillero Zubiri et al. 2004; Wilson & Mittermeier 2009). These last three species are smaller (ca. 3-7 kg) and have a more omnivorous diet and a wider distribution, that virtually covers the whole area, with the exception of L. fulvipes that is limited to the northwestern Chilean Patagonia (Macdonald & Sillero Zubiri 2004; Sillero Zubiri et al. 2004; Wilson & Mittermeier 2009). Lycalopex griseus, as currently defined, is distributed in dry habitats of Patagonia, western Argentina, central and northern Chile, and western Bolivia, while L. gymnocercus is present in wetter areas of central, northern and eastern Argentina, eastern Bolivia, Paraguay, southern Brasil and Uruguay (Zunino et al. 1995; Macdonald & Sillero Zubiri 2004; Sillero Zubiri et al. 2004; Wilson & Mittermeier 2009; Prevosti et al. 2013; Fig. 1). The distribution of these species is apparently overlapping along western parts of Argentina, and in northwestern Argentina, where supposedly they live in sympatry (Mares et al. 1989; Barquez et al. 1991; Díaz & Barquez 2002). Lycalopex fulvipes occurs in the Chiloé island and continental areas of Chile with Valdivian forests, in the regions of Biobío, La Araucanía and Los Lagos (Macdonald & Sillero Zubiri 2004; Sillero Zubiri et al. 2004; Wilson & Mittermeier 2009; Farías et al. 2016).


Fig. 1
. Geographical distribution of the samples analyzed. Stars: Lycalopex griseus; circles: L. gymnocercus. White circles: Clade B, black circles: Clade A. Dotted line indicates the Paraguay-Paraná river.

While the status of L. culpaeus, L. sechurae and L. fulvipes as valid species is clear, the separation of the other two species is disputed (Zunino et al. 1995; Prevosti et al. 2013). Some molecular, and “total evidence” phylogenetic analysis of canids showed that L.  griseus is the sister taxon of L. culpaeus instead of L.  gymnocercus (Bardeleben et al. 2005; Lindblad-Toh et al. 2005; Prevosti 2010; Austin et al. 2013; Tchaicka et al. 2016), something that could be interpreted as evidence of the separation of both species of grey foxes. Ruiz García et al. (2013), published a phylogenetic analysis based on CytB mitochondrial gene that includes a large sample of L. culpaeus, four specimens of L. griseus, and one of L.  gymnocercus, plus other species of the genus, that indicates that L. griseus is paraphyletic (in the maximum parsimony analysis) and the sister of a clade of L. culpaeus that contains the only specimen of L. gymnocercus. A recent study based on DNA control region (Tchaicka et al. 2016), which included a larger sample for both species of grey foxes (although geographically limited, including one locality in Argentina and Bolivia, seven localities from central and northern Chile, and four from southern Brazil), found that L. gymnocercus and L. griseus were not reciprocally monophyletic, since mitcochondrial DNA (mtDNA) lineages from three specimens originally determined as L. gymnocercus were nested in the L. griseus clade. The authors concluded that both species are valid, and proposed that secondary hybridization and mtDNA introgression are the best explanation for the position of the L.  gymnocercus samples within the L. griseus clade (Tchaicka et al. 2016).

On the other hand, morphological studies based on cranial and skin characters, using a morphometric approach (both traditional and 3D geometric morphometry), and the analysis of qualitative traits, failed to separate L. griseus from L. gymnocercus, supporting the hypothesis that these species form a cline from the north-east to the south-west of Argentina, in which L. griseus inhabits drier areas and has a smaller size (Zunino et al. 1995; Prevosti et al. 2013). These studies indicate that specimens were assigned to each species based on their geographic provenance, something that could introduce logic circularity. Moreover, the contradiction between morphological and DNA studies could be an artifact, because most of the studies based on DNA are focused in resolving the relationships of the Canidae family, and in consequence include few specimens for each species. On the other hand there is no way to corroborate the taxonomic assignation of the sequences due to the lack of voucher information, and even the assignation of some Genbank public sequences is wrong (see Prevosti et al. 2013).

The main objective of this article was to reassess the species limits between L. griseus and L. gymnocercus analyzing molecular data from three mitochondrial markers (CytB, COI, CR), from a large, widely distributed sample of specimens of both species and others of the genus Lycalopex.

MATERIALS AND METHODS

Biological samples and molecular methods

The sample comprised sequences from 25 specimens identified as L. griseus, 30 specimens identified as L.  gymnocercus, and 21 specimens of the following species used as outgroups: L. culpaeus (5), L. fulvipes (1), L. vetulus (1), L. sechurae (1), Cerdocyon thous (6), Speothos venaticus, Chrysocyon brachyurus (Table  1). Samples were obtained from road-killed animals. Most of the sequences were generated for these analyses, but we also included sequences from GenBank (see Table 1). Phylogenetic trees were rooted using Canis lupus. Since several papers showed that there is no way to separate L.  griseus and L. gymnocercus using skin or osteological characters (e.g., Zunino et al. 1995; Prevosti et al. 2013) we assigned each of these species using their current accepted distribution (e.g., Wilson & Mittermeier 2009).

Table 1
Specimens and sequences used in our study. COX and COI represent two fragments of Cytochrome Oxidase I (see Materials and Methods). GB indicate sequences obtained from GenBank.Voucher number corresponds to the tissue collection of the Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Buenos Aires, Argentina (MACN-Ma-CT). Specimens without voucher correspond to tissues from personal collections of some of the authors (VR and PM).

DNA extractions from fresh tissue (muscle) were performed using a SDS-proteinase K-ClNa protocol (modified from Miller et al. 1988). Three different fragments were amplified by the polymerase chain reaction (PCR): (i) the complete cytochrome b gene (cytB) using primers CytBDF1 and CytBDR1 (Tchaika et al. 2006); (ii) the cytochrome c oxidase I (COI) in two fragments using universal primers LCO1490 and HCO2198 (COX; Folmer et al. 1994), and primers L6569 and H7227 (COI; Wayne et al. 1997); (iii) the 5¢ portion of the mtDNA control region (CR), containing the first hypervariable segment (HVS-I), was amplified using primers MTLPRO2 and CCR-DR1 (Tchaika et al. 2006). Polymerase chain reactions (PCR) were performed in a final volume of 15 μl. Each reaction contained between 50 and 100  ng of DNA, 1.5 units of Taq polymerase, 1x PCR Buffer, 5 mM MgCl2, 0.2 μM of each primer and 0.025 mM dNTP each. BSA 0.4% was included as additive and enhancing agent to increase the yield of PCR reactions. PCR amplifications were carried out as follows: a first denaturation period at 94  ºC for 5 min, followed by 35 cycles of denaturation at 94 ºC for 45 s, annealing at 50-56  ºC for 1 min, and extension at 72  ºC for 1 min. Final extension at 72  ºC for 6 min terminated the reactions. A negative control with no template was included for each series of amplifications to test for contamination. PCR products were electrophoresed on a 1% TBE agarose gel stained with ethidium bromide. Sequencing was performed in MACROGEN (Korea).

Phylogenetic relationships

Sequences were edited and hand-aligned (since the alignments were trivial) using the software BioEdit (Hall 1999). Maximum Parsimony (MP) analyses were performed using the software TNT (Goloboff et al. 2008), using 1000 series of random addition of sequences (RAS), swapping the trees with tree bisection-reconnection (TBR), plus an additional rearrangement of all the most parsimonious trees found using TBR. A strict consensus was calculated using all the most parsimonious trees found. Branch support was evaluated with 10 000 pseudoreplicates of jackknife (JK; Farris et al. 1996). Maximum likelihood analyses were conducted using RAxML GUI (Silvestro & Michalak 2012), a graphical front-end for RAxML-VI-HPC (Randomized Accelerated Maximum Likelihood; Stamatakis 2006). Maximum likelihood with the thorough bootstrap (BS) option was run from a starting random seed to generate 1000 nonparametric bootstrap replicates. Analyses were performed for each marker separately and also combining the three markers in a single matrix. In the combined matrix, we excluded specimens that only had information for one marker. The analyses were performed using a GTR+G+I model as selected by using jModeltest (Posada 2008) available online on the server Phylemon (http//phylemon.bioinfo.cipf.es).

Inter- and intraspecific genetic distances were estimated with the Tamura 3-parameter model (Tamura 1992) implemented in the software MEGA6 (Tamura et al. 2013) for each marker separately. The variation rate among sites was modeled with a gamma distribution (shape parameter = 1). Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair. Other parameters were used following the default option of the software. The Kimura 2-parameter model is frequently used without justification, but recent analyses showed that is not always the best model for the data being analyzed (Srivathsan & Meier 2012). Consequently, the model used for estimating distances was the model that best fit our dataset, as chosen by the software MEGA6.

For the grey foxes, analyzing each marker separately, a Mantel test was performed between genetic distances and geographic (Euclidean) distances using the package ade4 for R (Dray & Dufour 2007; R Development Core Team 2016). For each analysis, 9999 replicates were performed.

RESULTS

In Table 2 there is a summary of the characteristics of the sequences and data sets analyzed in this article. All the new sequences were deposited in GenBank (codes in Table  1). The MP analysis of the combined dataset (three mitochondrial markers) resulted in 3726 trees of 1424 steps. The strict consensus tree showed Lycalopex as a well-supported monophyletic group (JK 99), sister to Cerdocyon (Fig. 2). Lycalopex sechurae is the sister taxon of the other species of Lycalopex, which are in a polytomy that include well to moderately-supported clades including the remaining species of Lycalopex: L. fulvipes, L. vetulus (JK 98), L. culpaeus (JK 87), and the specimens of L. gymnocercus and L. griseus grouped in two clades (A and B) that include haplotypes coming from individuals of both species (i.e., the traditional concept of these species is not monophyletic). Clade A (JK 85) includes haplotypes from specimens of L. gymnocercus from the northeast of Argentina (Entre Ríos and Santa Fe Provinces), northeastern Paraguay (Amambay) and southern Brazil. Clade B (JK 73) includes sequences of specimens of L. gymnocercus from Buenos Aires, Santa Fe and Córdoba (Argentina) and western Paraguay (Boquerón), and of specimens assigned to L. griseus, from Coquimbo, Araucanía and Magallanes (Chile), and Southern Argentina (La Pampa, Mendoza, Río Negro, Chubut and Santa Cruz). None of the clades that group specimens of L. gymnocercus and L. griseus show any phylogeographic pattern in the arrangement of the specimens.

Table 2
Characteristics of the datasets analyzed


Fig. 2
. Strict consensus tree obtained from the maximum parsimony analysis of the three mitochondrial markers. Numbers below the branches indicate jackknife support values. Numbers associated to taxon names refer to sequence codes (see Table  1). Amam: Amambay Department (Paraguay); Arau: Araucanía Region (Chile); BA: Buenos Aires Province (Argentina); Boq: Boquerón Department (Paraguay); Cba: Córdoba Province (Argentina); Chu: Chubut Province (Argentina); Coqui: Coquimbo Region (Chile); ER: Entre Ríos Province (Argentina); Juj: Jujuy Province (Argentina); LP: La Pampa Province (Argentina); Maga: Magallanes Region (Chile); Mza: Mendoza Province (Argentina); RN: Río Negro Province (Argentina); SC: Santa Cruz Province (Argentina); SF: Santa Fe Province (Argentina); SL: San Luis Province (Argentina).

The ML analysis of the combined dataset resulted in a tree (likelihood -11538.03797; Fig.  3) with more resolution inside the Lycalopex clade. In this case, L. vetulus is the most basal species, and all the clades that were collapsed in a polytomy in the MP tree show clearer relationships (although with low support; Fig. 3). Clades A and B are also present, with moderate to high support, but in this analysis, L. culpaeus is the sister taxon of Clade B (BS of Clade B + L.  culpaeus: 51; Fig. 3), while Clade A is placed as sister clade of L. fulvipes + Clade B + L. culpaeus (BS 61; Fig. 3).


Fig. 3
. Maximum likelihood phylogram based on the analysis of the three mitochondrial markers. Numbers below the branches indicate bootstrap support values. Numbers associated to taxon names refer to sequence codes (see Table 1). Amam: Amambay Department (Paraguay); Arau: Araucanía Region (Chile); BA: Buenos Aires Province (Argentina); Boq: Boquerón Department (Paraguay); Cba: Córdoba Province (Argentina); Chu: Chubut Province (Argentina); Coqui: Coquimbo Region (Chile); ER: Entre Ríos Province (Argentina); Juj: Jujuy Province (Argentina); LP: La Pampa Province (Argentina); Maga: Magallanes Region (Chile); Mza: Mendoza Province (Argentina); RN: Río Negro Province (Argentina); SC: Santa Cruz Province (Argentina); SF: Santa Fe Province (Argentina); SL: San Luis Province (Argentina).

The analysis of each marker separately resulted in trees that were inconclusive, with little resolution and low support. The results of these analyses are presented as supplementary data (Supplement 1).

Distance between Clades A and B was 0.039, which is similar (or even larger) than the distances between other species of Lycalopex, and larger than the intra-clade distance (Table 3). The Mantel test showed that there is no correlation between the genetic and the geographic matrix for any of the matrices (R -0.1012; -0.0036; 0.0393 for CR, cytB and COI respectively, p > 0.1 in all the cases), which is congruent with the lack of correlation between geography and topology obtained in the phylogenetic trees. The results did not change when analyzing Clade B separately.

Table 3
Estimates of evolutionary divergence over sequence pairs between groups. The number of base substitutions per site from averaging over all sequence pairs between groups are shown.

DISCUSSION

Our results based on three mitochondrial genes show that South American grey foxes (L. griseus and L. gymnocercus) do not form a monophyletic group, and instead could be separated in two clades (A and B), with moderate node support (Figs. 2-3), that are not always recovered in individual gene analyses (Supplement 1). Also it should be noted that the relationship of Clades A and B to other Lycalopex species (L.  culpaeus and L. fulvipes), is ambiguous since in the MP analysis the four species are in a politomy, while in the ML analysis the nodes that support the relationships among them are poorly supported (Figs. 2-3). This lack of resolution in the phylogenetic placement of grey foxes should be taken into consideration, since the distribution of both species is not ideally sampled, mainly in the area where they are in contact as traditionally considered, and also in the area where clades A and B are in contact (at least as evidenced from our sample; see Fig.  1). This apparent recognition of two clades that do not form a monophyletic group is congruent with previous phylogenetic works (Wayne et al. 1997; Zrzavý & Řičánková 2004; Bardeleben et al. 2005; Prevosti 2006, 2010; Perini et al. 2010; Fuentes González & Muñoz Durán 2012; Austin et al. 2013; Tchaicka et al. 2016) that found that L.  griseus and L.  gymnocercus do not form a monophyletic group. However, in other analyses based on nuclear genes (Bardeleben et al. 2005; Lindblad-Toh et al. 2006) or in some combining nuclear and mitochondrial genes (Bardeleben et al. 2006) both species form a single clade, suggesting that the non-monophyletic relationship between L. griseus and L.  gymnocercus is a signal that comes from mitochondrial data. It should be noted that some level of incongruence between nuclear and mitochondrial genes in the canid phylogeny was detected by Prevosti (2010) using different topological measurements. In this context it is interesting that the relationships found with nuclear data are more in agreement with morphological studies, which in the case of these species failed to find differences between L. griseus and L. gymnocercus (Zunino et al. 1995; Prevosti et al. 2013).

The geographic distribution of Clades A and B do not agree with the traditionally accepted distribution of L. griseus and L. gymnocercus (the first in Chile, Patagonian region and west part of Argentina and Bolivia, and the second on central, northern and eastern Argentina, part of Bolivia, Paraguay, Uruguay and southern Brazil; Zunino et al. 1995; Macdonald & Sillero Zubiri 2004; Sillero Zubiri et al. 2004; Wilson & Mittermeier 2009). In this sense, our results are more congruent with Tchaicka et al.’s (2016) recent work, that although it has a limited geographic sample, mainly in central areas where both species are in contact, found that grey foxes do not form a monophyletic group and could be separated in clades that are similar to our clades A and B. It must be noted that the traditional interpretation about the distribution of these supposed valid species (i.e., L. griseus and L. gymnocercus) is arbitrary, since there is no clear way to separate them on the basis of morphological features (Zunino et al. 1995; Prevosti et al. 2013). This arbitrary taxonomic assignation becomes apparent in the different geographic distribution of both species established by different authors. For example, Kraglievich (1930) considered that the foxes from the La Pampa province, in central Argentina, belong to L. griseus, while Cabrera (1957) considered that they belong to L. gymnocercus. Based on all these evidences, the most recent list of mammals of Argentina consider both species as synonyms under Lycalopex gymnocercus (Teta et al. 2018).

Regarding nomenclatural issues, Clade B includes one specimen from the topotypic area of L. griseus, that is the coast of the Magellan Strait (Cabrera 1957), but we do not have access to samples from the type locality of L. gymnocercus (around Asunción, Paraguay; Cabrera 1957). The absence of samples from the type locality of L. gymnocercus prevents us to confirm the assignation of Clade A to L. gymnocercus. Moreover, the two specimens sampled from Paraguay are nested in the different clades, and none is geographically close to Asunción to make any inference (see Fig. 1). In fact, the clade that is assigned by Tchaicka et al. (2016), to L. gymnocercus, cannot be confirmed as belonging to this species due to the same issue.

Although the distribution of these mitochondrial clades do not match with the traditionally delimited distribution of L. griseus and L.  gymnocercus, they present an interesting pattern, since they are separated by the Paraguay and Paraná rivers (Fig. 1), Clade A being limited to northeastern Argentina and Paraguay, and southern Brazil, while Clade B includes specimens from the rest of Argentina, Chile and the western Paraguay (Figs. 2-3). A similar pattern of species separation has been already described for other animals and plants (e.g., Parodi 1934; Ringuelet 1961; Cabrera & Willink 1980; Myers 1982; Pennington et al. 2000; Giraudo & Arzamendia 2004; Arzamendia & Giraudo 2009, 2012; De la Sancha et al. 2011; Chemisquy & Flores 2012). Athough the rivers themselves do not strictly limit the distribution of Clade A to the west of Paraguay and Paraná rivers (Fig. 1), specimens are restricted to an area close to these rivers, as happens with other mammals, vertebrates and plants (Myers 1982; Pennington et al. 2000; Giraudo & Arzamendia 2004; Arzamendia & Giraudo 2009, 2012). In fact, due to historical (e.g., geologic, edaphic) and ecological (environmental conditions) factors, these rivers appear to act as a dispersal root and the generator of special environments that facilitate the spread of some species that are distributed to their east (Myers 1982; Giraudo & Arzamendia 2004; Arzamendia & Giraudo 2009, 2012). The annual precipitation also diminishes from east to west in this area (Parodi 1934; García 1991; Cabrera & Willink 1980; Pennington et al. 2000), and in the Santa Fe province (Argentina), where Clade A is found west of the Paraná river; it is found in an area that from a geologic point of view belongs to the zone of influence of the Paraná river (Iriondo 2010). Thus historical and ecological factors related to this major river could explain why Clade B is limited to the south and west of the Paraná and Paraguay rivers, while Clade A is distributed to the east of these rivers or very close to them.

It should be noted that specimens of Clades A and B are very closely distributed and even overlap in the Santa Fe province (Argentina; Fig. 1), where they are probably in sympatry, since specimens of each clade came from localities located less than 40 km apart from each other, and are in similar environments (areas transformed in agroecosystems) without any barrier between them (Fig. 1). This potential sympatric distribution in this area could be a recent phenomenon, generated by the strong environmental modifications caused by humans since the nineteenth century, where large areas where transformed in crop fields or occupied by livestock farming. Also, this area could be considered a hybrid zone, which could be facilitated by the fact that both species were separated recently, as suggested by molecular dating analysis (~500 000 ybp; Tchaicka et al. 2016).

We think that two competing hypotheses could be discussed regarding the biological meaning of our results, and previously published evidence using morphology and nuclear DNA: 1) grey foxes belong to two different species, as is traditionally supported, and as suggested by recent works (e.g., Tchaicka et al. 2016); 2) they belong to the same species as morphology (Zunino et al. 1995; Prevosti et al. 2013), and the limited published nuclear data (Lindblad-Toh et al. 2005) indicate. The first hypothesis is the best supported from our results, although not all the analyses recovered Clades A and B (Supplement 1), and when present, they showed moderate branch support values (Figs. 2, 3). Consequently, one can consider that L. griseus (= Clade B) is widely distributed in Argentina, excluding the Mesopotamia (i.e., Entre Ríos, Corrientes and Misiones) and apparently is present western to the Paraguay river in Paraguay. “Lycalopex gymnocercus” (= Clade A), on the other hand, is present eastern to the Paraná-Paraguay river, with some specimens also present in Santa Fe. It is important to note that until a specimen from the type locality of the species is included in the analysis, we cannot be sure that Clade A is assignable to L. gymnocercus, and that is why we are using inverted commas in the name of the species.

FUTURE CONSIDERATIONS

A limitation of the interpretation that clades A and B represent two different species is that our results are based only on mitochondrial genes, and since they are inherited only from the mother they only tell part of the genetic history of these foxes (Funk & Omland 2003), and could be interpreted as a linkage-group tree because the three genes we used are not independent (Moore 1995; Giannasi et al. 2001). Moreover, in the last years several papers were published showing a difference in species delimitation based on nuclear and mitochondrial genes. Analyses published in bears (Hailer et al. 2012; Cronin et al. 2014; Cahill et al. 2013; Kutschera et al. 2014), monkeys (Zinner & Ross 2014), cricetids (Cañón et al. 2014) and goats (Ropiquet & Hassanin 2006) showed that morphology is more in agreement with nuclear DNA than with mitochondrial genes, which has important implications in species delimitation (Cahill et al. 2013; Cañón et al. 2014; Zinner & Ross 2014; vonHoldt et al. 2016). These studies showed the relevance of hybridization, introgression and incomplete lineage sorting, and that multiple independently inherited loci are needed to resolve complex evolutionary patterns (Kutschera et al. 2014; vonHoldt et al. 2016). In this context, and without information from other lines of DNA evidence (i.e., nuclear markers) that could discard the presence of introgression or incomplete lineage sorting, we must consider that our results can change with future evidence, and that both species could end up being synonymized, as morphology suggests.

ACKNOWLEDGMENTS

For partial financial support, to CONICET (PUE 2015-0125) and ANPCyT (PICT 2015-966, PICT 2016-3151). To D.  Cossios and W. Molina for contributing with some of the molecular analyses. To A. Forasiepi, A. Fameli, G. Martin, M. Schiaffini, A. Pautaso and J. Pereira for kindly giving us tissues of Lycalopex. To G. Cassini and the anonymous reviewers who contributed to improve the manuscript. Vanina Raimondi was supported by an excellence grant of the Swiss Confederation.

LITERATURE CITED

1. Arzamendia, V., & A. R. Giraudo. 2009. Influence of large South American rivers of the Plata Basin on distributional patterns of tropical snakes: a panbiogeographical analysis. Journal of Biogeography 36:1739‒1749. https://doi.org/10.1111/j.1365-2699.2009.02116.x        [ Links ]

2. Arzamendia, V., & A. R. Giraudo. 2012. A panbiogeographical model to prioritize areas for conservation along large rivers. Diversity and Distributions 18:168‒179. https://doi.org/10.1111/j.1472-4642.2011.00829.x         [ Links ]

3. Austin, J. J. et al. 2013. The origins of the enigmatic Falkland Islands wolf. Nature Communications 4:1552.         [ Links ]

4. Bardeleben, C., R. L. Moore, & R. K. Wayne. 2005. A molecular phylogeny of the Canidae based in six nuclear loci. Molecular Phylogenetics and Evolution 37:815‒831. https://doi.org/10.1016/j.ympev.2005.07.019        [ Links ]

5. Barquez, R. M., M. Mares, & R. Ojeda. 1991. Mamíferos de Tucumán - Mammals of Tucumán. Oklahoma Museum of Natural History - University of Oklahoma, Norman, Oklahoma.         [ Links ]

6. Cabrera, A. 1957. Catálogo de los Mamíferos de América del Sur. Parte I. Revista del Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Zoología 4:1-307.

7. Cabrera, A. L. & A. Willink. 1980. Biogeografía de América Latina. Monografía 13, Serie de Biología OEA, Washington, D.C.         [ Links ]

8. Cahill, J. A. et al. 2013. Genomic evidence for island population conversion resolves conflicting theories of polar bear evolution. PLoS Genetics 9: e1003345. https://doi.org/10.1371/journal.pgen.1003345        [ Links ]

9. Cañón, C., D. Mir, U. F. J. Pardiñas, E. P. Lessa, & G. D’Elía. 2014. A multilocus perspective on the phylogenetic relationships and diversification of rodents of the tribe Abrotrichini (Cricetidae: Sigmodontinae). Zoologica Scripta 43:443‒454. https://doi.org/10.1111/zsc.12069

10. Chemisquy, M. A., & D. A. Flores. 2012. Taxonomy of the southernmost populations of Philander (Didelphimorphia, Didelphidae), with implications for the systematics of the genus. Zootaxa 3481:60-72. https://doi.org/10.11646/zootaxa.3481.1.5        [ Links ]

11. Cronin, M. et al. 2014. Molecular phylogeny and SNP variation of polar bears (Ursus maritimus), brown bears (U. arctos), and black bears (U. americanus) derived from genome sequences. Journal of Heredity 105:312‒323. https://doi.org/10.1093/jhered/est133        [ Links ]

12. De la Sancha, N. U., G. D’Elía, & P. Teta. 2011. Systematics of the subgenus of mouse opossums Marmosa (Micoureus) (Didelphimorphia, Didelphidae) with noteworthy records from Paraguay. Mammalian Biology 77:229‒236. https://doi.org/10.1016/j.mambio.2011.10.003

13. Díaz, M., & R. Barquez. 2002. Los mamíferos de Jujuy, Argentina. LOLA, Buenos Aires.         [ Links ]

14. Dray, S., & A. B. Dufour. 2007. The ade4 package: implementing the duality diagram for ecologists. Journal of Statistical Software 22:1‒20. https://doi.org/10.18637/jss.v022.i04        [ Links ]

15. Farias, A., J. Jimenez, D. Moreira, J. Cabello, &  E. Silva, E. 2016. Lycalopex fulvipes. The IUCN Red List of Threatened Species. Version 2018-1. https://doi.org/10.2305/iucn.uk.2016-1.rlts.t41586a85370871.en        [ Links ]

16. Farris, J. S., V. A. Albert, M. Källersjö, D. Lipscomb, & A. G. Kluge. 1996. Parsimony Jackknifing outperforms Neighbor-Joining. Cladistics 12:99‒124. https://doi.org/10.1111/j.1096-0031.1996.tb00196.x        [ Links ]

17. Folmer, O., M. Black, W. Hoeh, R. Lutz, &  R.  Vrijenhoek. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3(5):294-9        [ Links ]

18. Fuentes-González, J., & J. Muñoz-Durán. 2012. Filogenia de los cánidos actuales (Carnivora: Canidae) mediante análisis de congruencia de caracteres bajo parsimonia. Actualidades Biológicas 34:85‒102.         [ Links ]

19. Funk, D. J., & K. E. Omland. 2003. Species-level paraphyly and polyphyly: frequency, causes, and consequences, with insights from animal mitochondrial DNA. Annual Review of Ecology, Evolution and Systematics 34:397‒423. https://doi.org/10.1146/annurev.ecolsys.34.011802.132421        [ Links ]

20. García, N. O. 1991. Síntesis climatográfica de la República Argentina. Publicación de la Facultad de Ingeniería y Ciencias Hídricas - Universidad Nacional del Litoral 36:1-33.         [ Links ]

21. Giannasi, N., Malhotra, A., & R.S. Thorpe. 2001. Nuclear and mtDNA phylogenies of the Trimeresurus complex: implications for the gene versus species tree debate. Molecular Phylogenetics and Evolution 19:57‒66. https://doi.org/10.1006/mpev.2001.0899        [ Links ]

22. Giraudo, A. R., & V. Arzamendia. 2004. ¿Son los humedales fluviales de la Cuenca del Plata, corredores de biodiversidad? Los amniotas como ejemplo. Humedales de Iberoamérica (J. J. Neiff, ed.). CYTED, Programa Iberoamericano de Ciencia y Tecnología para el desarrollo - Red Iberoamericana de Humedales (RIHU), La Habana.         [ Links ]

23. Goloboff, P. A., J. S. Farris, & K. C. Nixon. 2008. TNT, a free program for phylogenetic analysis. Cladistics 24:74‒786. https://doi.org/10.1111/j.1096-0031.2008.00217.x        [ Links ]

24. Hailer, F. et al. 2012. Nuclear genomic sequences reveal that polar bears are an old and distinct bear lineage. Science 336:344‒347. https://doi.org/10.1126/science.1216424        [ Links ]

25. Hall, T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41:95‒98.         [ Links ]

26. Helgen, K. M. et al. 2013. Taxonomic revision of the olingos (Bassaricyon), with description of a new species, the Olinguito. ZooKeys 324:1‒83. https://doi.org/10.3897/zookeys.324.5827        [ Links ]

27. Kutschera, V. E., T. Bidon, F. Hailer, J. L. Rodi, S. R. Fain, & A. Janke. 2014. Bears in a forest of gene trees: phylogenetic inference is complicated by incomplete lineage sorting and gene flow. Molecular Biology and Evolution 31:2004‒2017. https://doi.org/10.1093/molbev/msu186        [ Links ]

28. Iriondo, M. I. 2010. Geología del Cuaternario en Argentina. Museo Provincial de Ciencias Naturales Florentino Ameghino, Santa Fe.         [ Links ]

29. Kraglievich, L. 1930. Craneometría y clasificación de los cánidos sudamericanos, especialmente los argentinos actuales y fósiles. Physis 10:35-73.         [ Links ]

30. Lindblad-Toh, K. et al. 2005. Genome sequence, comparative analysis and haplotype structure of the domestic dog. Nature 438:803-819.         [ Links ]

31. MacDonald, D. W., & C. Sillero Zubiri. 2004. Biology and conservation of wild canids. Oxford University Press, Oxford. https://doi.org/10.1093/acprof:oso/9780198515562.001.0001        [ Links ]

32. Mares, M. A., R. A. Ojeda, & R. M. Barquez. 1989. Guía de los mamíferos de la provincia de Salta, Argentina. University of Oklahoma Press, Norman, Oklahoma.         [ Links ]

33. Miller, S., D. Diker, & H. Polesky. 1988. A Simple Salting out procedure for extracting DNA from human nucleated cells. Nucleic Acid Research 6:1215. https://doi.org/10.1093/nar/16.3.1215        [ Links ]

34. Moore, W. S. 1995. Inferring phylogenies from mtDNA variation: mitochondrial gene trees versus nuclear gene trees. Evolution 49:718-726. https://doi.org/10.2307/2410325        [ Links ]

35. Myers, P. 1982. Origins and affinities of the mammal fauna of Paraguay. Special Publication of the Pymatuning Laboratory of Ecology 6:85-93.         [ Links ]

36. Parodi, L. R. 1934. Las plantas indígenas no alimenticias cultivadas en la Argentina. Revista Argentina de Agronomía. 1:165-212.         [ Links ]

37. Pennington, R. T., D. E. Prado, C. A. Pendry, & R.  Botanic. 2000. Neotropical seasonally dry forests and Quaternary vegetation changes. Journal of Biogeography 27:261‒273. https://doi.org/10.1046/j.1365-2699.2000.00397.x        [ Links ]

38. Perini, F. A., C. A. M. Russo, & C. G. Schrago. 2010. The evolution of South American endemic canids: a history of rapid diversification and morphological parallelism. Journal of Evolutionary Biology 23:311‒22. https://doi.org/10.1111/j.1420-9101.2009.01901.x        [ Links ]

39. Posada, D. 2008. jModelTest: phylogenetic model averaging. Molecular Biology and Evolution 25:1253‒1256. https://doi.org/10.1093/molbev/msn083        [ Links ]

40. Prevosti, F. J. 2006. Grandes cánidos (Carnivora, Canidae) del Cuaternario de la República Argentina: sistemática, filogenia, bioestratigrafía y paleoecología. Tesis de doctorado, Universidad Nacional de La Plata, La Plata, Argentina.         [ Links ]

41. Prevosti, F. J. 2010. Phylogeny of the large extinct South American Canids (Mammalia, Carnivora, Canidae) using a ‘total evidence’ approach. Cladistics 26:456-481. https://doi.org/10.1111/j.1096-0031.2009.00298.x

42. Prevosti, F. J., V. Segura, G. Cassini, & G. M. Martin. 2013. Revision of the systematic status of Patagonian and Pampean gray foxes (Canidae: Lycalopex griseus and L. gymnocercus) using 3d geometric morphometrics. Mastozoología Neotropical 20:289-300.         [ Links ]

43. R Development Core Team. 2016. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. http://www.r-project.org        [ Links ]

44. Ringuelet, R. A. 1961. Rasgos fundamentales de la Zoogeografía de la Argentina. Physis 22:151-170.         [ Links ]

45. Ropiquet, A., & A. Hassanin. 2006. Hybrid origin of the Pliocene ancestor of wild goats. Molecular Phylogenetics and Evolution 41:395‒404. https://doi.org/10.1016/j.ympev.2006.05.033        [ Links ]

46. Ruiz García, M., D. Rivas-Sánchez, & N. Lichilín-Ortiz. 2013. Phylogenetic relationships among four putative taxa of foxes of the Pseudalopex Genus (Canidae, Carnivora) and molecular population genetics of Ps. culpaeus and Ps. sechurae. Molecular population genetics, evolutionary biology and biological conservation of the Neotropical carnivores (M. Ruiz García & J.M. Shostell, eds.). Nova Publishers, New York.         [ Links ]

47. Schiaffini, M. I. et al. 2013. Taxonomic status of southern South American Conepatus (Carnivora: Mephitidae). Zoological Journal of the Linnean Society 167:327-344. https://doi.org/10.1111/zoj.12006        [ Links ]

48. Sillero Zubiri, C., M. Hoffmann, & D. W. Macdonald. 2004. Canids: foxes, wolves, jackals and dogs. Status survey and conservation action plan. IUCN Species Programme, Gland.         [ Links ]

49. Silvestro, D., & I. Michalak. 2012. raxmlGUI: a graphical front-end for RAxML. Organisms Diversity and Evolution 12:335‒337. https://doi.org/10.1007/s13127-011-0056-0        [ Links ]

50. Srivathsan, A., & R. Meier. 2012. On the inappropriate use of Kimura-2-parameter (K2P) divergences in the DNA-barcoding literature. Cladistics 28:190-194. https://doi.org/10.1111/j.1096-0031.2011.00370.x        [ Links ]

51. Stamatakis, A. 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688-2690. https://doi.org/10.1093/bioinformatics/btl446        [ Links ]

52. Tamura, K. 1992. Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G +C-content biases. Molecular Biology and Evolution 9:678‒687. https://doi.org/10.1093/oxfordjournals.molbev.a040752        [ Links ]

53. Tamura, K., G. Stecher, D. Peterson, A. Filipski, & S. Kumar. 2013. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution 30:2725‒2729. https://doi.org/10.1093/molbev/mst197        [ Links ]

54. Tchaicka, L. et al. 2016. Molecular assessment of the phylogeny and biogeography of a recently diversified endemic group of South American canids (Mammalia: Carnivora: Canidae). Genetics and Molecular Biology 39:442‒451. https://doi.org/10.1590/1678-4685-gmb-2015-0189        [ Links ]

55. Teta, P. et al. 2018. Lista revisada de los mamíferos de Argentina. Mastozoología Neotropical 25:163-198. https://doi.org/10.31687/saremmn.18.25.1.0.15        [ Links ]

56. Trigo, T. C. et al. 2013. Molecular data reveal complex hybridization and a cryptic species of Neotropical wild cat. Current Biology 23:2528‒2533. https://doi.org/10.1016/j.cub.2013.10.046        [ Links ]

57. vonHoldt, B. et al. 2016. Whole-genome sequence analysis shows that two endemic species of North American wolf are admixtures of the coyote and gray wolf. Science Advances 2:e1501714. https://doi.org/10.1126/sciadv.1501714        [ Links ]

58. Wayne, R. K., E. Geffen, D. J. Girman, K. P. Koepfli, L. M. Lau, & C. R. Marshall. 1997. Molecular systematics of the Canidae. Systematics Biology 46:622‒653. https://doi.org/10.1093/sysbio/46.4.622        [ Links ]

59. Wilson, D. E., & R. A. Mittermeier. 2009. Handbook of the mammals of the world, Vol. 1, Carnivores. Lynx editions in association with Conservation International and IUCN, Barcelona.         [ Links ]

60. Zinner, D., & C. Roos. 2014. So what is a species anyway? A primatological perspective. Evolutionary Anthropology 23:21-23. https://doi.org/10.1002/evan.21390        [ Links ]

61. Zrzavý, J., & V. Řičanková. 2004. Phylogeny of recent Canidae (Mammalia, Carnivora): relative reliability and the utility of morphological and molecular datasets. Zoologica Scripta 33:311‒333. https://doi.org/10.1111/j.0300-3256.2004.00152.x        [ Links ]

62. Zunino, G. E., O. B. Vaccaro, M. Canevari, & A. L. Gardner. 1995. Taxonomy of the genus Lycalopex (Carnivora: Canidae) in Argentina. Proceedings of the Biological Society of Washington 108:729-747.         [ Links ]

SUPPLEMENTARY ONLINE MATERIAL

Supplement 1

https://www.sarem.org.ar/wp-content/uploads/2019/07/SAREM_MastNeotrop_26-1_Chemisquy-sup1.pdf

Phylogenetic analysis of the individual markers. The COI analyses (both MP and ML) were the ones that best resemble the combined analyses, recovering Clades A and B (Supp. Fig. S1 a, b). The MP analysis of cytB failed to recover Clade A (Supp. Fig. S1 c), and although that clade is present in the ML analysis, its support is extremely low (BS 25; Supp. Fig. S1 d). Moreover, in the ML analysis L. culpaeus is nested inside Clade B (Fig. S1 d). Finally, the MP analysis of the CR dataset did not recover Clade B, and most specimens of that clade are in a large polytomy (individually or in small clades) that also include Clade A, and the remaining species of Lycalopex (Supp. Fig. S1 e). In the ML analysis of CR, clade B was recovered (but with low support; BS 45), and all the internal nodes that separate the different Lycalopex species have almost no support (BS > 30; Supp. Fig. S1 f).

Fig. S1. Schematic representation of the results of the maximum parsimony (MP) and maximum likelihood (ML) analyses of each marker separately. a, MP analysis of COI; b, ML analysis of COI; c, MP analysis of cytB; d, ML analysis of cytB; e, MP analysis of CR; f, ML analysis of CR. Numbers above the branches represent jackknife and bootstrap values respectively.

 

 

 

 

 

Creative Commons License Todo o conteúdo deste periódico, exceto onde está identificado, está licenciado sob uma Licença Creative Commons