In this study, we synthesize the vast taxonomic and systematic literature of the genus to identify the evidence supporting the recurrent taxonomic changes. We extend the review of Correa et al. (2017) to the whole genus, adding other lines of evidence that have been applied to the Eupsophus taxonomy, and provide a more complete compilation of geographic information. We also add new qualitative observations of external characters of live adults of selected populations and reassess the phylogenetic position of a putative new species from Tolhuaca (Andean foothills of Chile; Blotto et al. 2013 ), which was not included in the last species delimitation study ( Suárez-Villota et al. 2018b ). We aim not only to provide a complete and updated summary of the taxonomic, systematic and geographic information of the genus, but also to highlight the incongruences among different lines of evidence that should be addressed by future taxonomic and systematic studies.

The last complete review of the taxonomy and systematics of the genus Eupsophus was Nuñez (2003) , a doctoral dissertation that was not published in a peer-reviewed journal. That review presented a rather stable and uncontroversial view of the taxonomy of the genus, which at that time comprised eight species. Since that date, there have been several changes in the composition of the genus, specifically in the roseus group (summarized in Fig. 1 ). Correa et al. (2017) reviewed partially the taxonomy and geographic information of the genus, with a focus on the roseus group. These authors not only noted the weaknesses of the diagnoses of the species of that group, but also the problems that arise when comparing all the published chromosomal, bioacoustic and geographic information on the genus.

During the last six decades, morphometric, immunological, chromosomal, bioacoustic and molecular (allozymes, RFLPs and DNA sequences) approaches have been applied, separately or in combination, to the taxonomy and systematics of these frogs (reviewed by Nuñez 2003 ). Phylogenetic analyses with DNA sequences only have been performed since Nuñez (2003) , but they have had a profound influence on the estimation of species diversity and evolutionary patterns of the genus, particularly of the roseus group. Two of these studies ( Nuñez et al. 2011 , Blotto et al. 2013 ) suggested that the species diversity of that group may be underestimated. Nuñez et al. (2011) indicated that E. calcaratus would represent a species complex composed of six groups of mitochondrial haplotypes “diagnostic of species lineages”, and at least one of them would represent a new species (Villarrica population, foothills of Chilean Andes, 39° 20'S). Blotto et al. (2013) tested the monophyly of the genus and its species groups and investigated the relationships among species, including all the species recognized at that time. They synonymized E. queulensis with E. septentrionalis and suggested that the population from Tolhuaca, also located in the Chilean Andean foothills (38°13'S), would correspond to an undescribed species related to E. roseus . More recently, Correa et al. (2017) , applying several unilocus species delimitation analyses with mitochondrial sequences, proposed a new arrangement that reduced the species of the genus to six. Suárez-Villota et al. (2018b) rejected this arrangement using new samples, different molecular markers and several species delimitation analyses (unilocus and multilocus). They considered as valid the ten species recognized before 2017 and found support for recognizing the population of Villarrica as a putative species, although they did not include specimens from Tolhuaca. All these hypotheses, including the species status of Villarrica and Tolhuaca populations, have been supported exclusively by molecular phylogenetic evidence, without explicitly incorporating phenotypic characters.

We obtained an alignment of 1304 nucleotide sites when the sequences of different length of both gene fragments were included (631 sites of cytb, 673 of COI), which was reduced to 998 when cutting ends with gaps (365 sites of cytb, 633 of COI). The four analyses (with or without sites with gaps, two or six partitions) recovered the two species groups and all the currently recognized nominal species of the roseus group as well-supported clades (posterior probability, pp > 0.97), but the topology within this group is variable among analyses, including some polytomies, and only partially congruent with previous phylogenetic studies (Fig. 2 ). Figure 6 shows the Bayesian consensus tree (15 002 sampled trees) of the analysis of the short alignment with six partitions. An important difference with respect to prior hypotheses is the position of E. insularis as the sister species of the all species of the roseus group, except for E. calcaratus ; though in the analysis of the short alignment with two partitions appears as the sister species of E. migueli + E. altor like in previous studies. Another difference with respect to the most recent hypothesis (Fig. 2F ) is the position of E. septentrionalis , recovered as the sister group of E. roseus , E. contulmoensis , E. nahuelbutensis and Villarrica and Tolhuaca populations, which is only consistent with the results of Suárez-Villota et al. (2018a) (Fig. 2E ). However, E. septentrionalis also formed a polytomy with E. roseus + Villarrica + Tolhuaca and E. contulmoensis + E. nahuelbutensis clades in both analyses with two partitions. The four analyses showed the close relationship of Villarrica and Tolhuaca populations with E. roseus , all of which comprise a clade with maximal support. However, the reciprocal relationship between Villarrica and Tolhuaca populations could not be resolved since in three of the four analyses both putative taxa form a tritomy with E. roseus (Fig. 6 shows the only analysis where this relationship is resolved, but with low support). This lack of resolution could be due to the low number of variable nucleotide sites with respect to other studies where more genes were included, but in no case the Villarrica or Tolhuaca specimens appear mixed with those of E. roseus . Therefore, Tolhuaca population also should be considered a candidate species under the current taxonomy.

One of the contributions of Correa et al. (2017) was the explicit recognition of the high level of intrapopulation variation in external characters considered diagnostic in the taxonomy of the genus. Here we show additional examples of intrapopulation variation in the three external characters most frequently included in the diagnoses of Eupsophus species (dorsal and ventral color patterns, iris color, and lateral and dorsal snout profile; Table 1 ; see also Correa et al. 2017 ), in live animals of two undescribed populations (Fig. 4 ) and two type localities (Fig. 5 ). Figure 4 illustrates the variation in dorsal coloration patterns in specimens from Pidenco (A, four adults randomly selected, from a total of 13, to show also the typical cryptic coloration of the genus and the variation of iris color and snout profile) and Las Lianas (B, five specimens chosen among 19 to represent contrasting dorsal coloration patterns, including one with a thin vertebral line). Most of specimens from Las Lianas had uniform brown eyes and only one had the upper part of the iris yellowish. Moreover, the length and profile of the snout varied among these specimens (data not shown). Figure 4 shows the variation of body coloration patterns (dorsal and ventral), iris coloration and shape of snout (both in dorsal and lateral profile) in the type localities of E. roseus (A, Valdivia, where it is the only species of the roseus group that has been reported; see Fig. 3 ) and E. migueli (B, Mehuín, where also E. roseus would be present, see above and Fig. 3 ). The six specimens of E. roseus were selected from 16, collected in two sessions, in order to exemplify the variation of iris color, which ranges from reddish to pale orange, and shape of the snout, which varies in length and form in lateral and dorsal profile. The three specimens of E. migueli (Fig. 5B ) were collected in two sessions (14 in total) and differ notably in dorsal and ventral coloration patterns and in snout profile. They also differ in coloration from the holotype, which had the dorsum grayish with two dark paravertebral areas and a thin light vertebral line ( Formas 1978a ). At Mehuín, where E. migueli and E. roseus supposedly coexist (see above), no specimens with the iris orange like E. roseus were observed.

Another of the six species of the roseus group endemic to Chile, which presents a restricted distribution according to Suárez-Villota et al. (2018b) . Eupsophus altor was reported originally from four localities (39°29' to 39°42'S, Nuñez et al. 2012a ), but a map by Nuñez et al. (2012b) shows six points without mentioning the localities (not included in Fig. 3B ). In any case, all these localities are between the two original ones of E. migueli , Mehuín and Los Molinos (localities 58 and 76 of Fig. 3B ). In one of the original localities, Alepúe, E. roseus has also been recorded ( Blotto et al. 2013 ). This last record can be added to the others mentioned above, which indicate the presence of E. roseus in several coastal locations where E. migueli and E. altor are found, but the map of the IUCN (2019) shows a continuous distribution of E. roseus that completely covers those of both species.

Another of the six species of the roseus group endemic to Chile, specifically to the Nahuelbuta Range, which would have a restricted distribution according to Suárez-Villota et al. (2018b) . There are few records of this species in the literature (see Appendix 1 ). However, Ortiz and Ibarra-Vidal (2005) pointed out that this species has a wider distribution on the western slopes of the Nahuelbuta Range, between the south of the Biobío River (~37°10'S) and the latitude of the town of Tirúa (~38°20'S). On the other hand, the maps of Nuñez (2003) and Rabanal and Nuñez (2008) restrict the distribution of this species to its type locality and surroundings.

†We add the diagnosis of E. queulensis because it includes a greater number of characters. ‡The original diagnosis of E. vertebralis ( Grandison 1961 ) is very extensive, but based exclusively on external morphology (e.g., characteristics of the skin, ears, and limbs), so here we only included those characters comparable with other species; Nuñez (2003) indicated that the diagnosis of Grandison (1961) also included specimens of Alsodes , but he did not provide further details to support this assertion. § Formas (1989) included the adult size in its diagnosis to differentiate it from its sister species E. vertebralis ; however, there is a high degree of superposition in male and female sizes between both species (Table 1 of Formas 1989 ). | Formas and Vera (1982) used this character to differentiate E. calcaratus from E. roseus , but they did not describe the snout profile of E. roseus (they only showed a drawing of the head in lateral profile). ¶To simplify the table, we reunite in this miscellaneous category a series of osteological details of the skull that have been included occasionally in the diagnoses. #These are not character states, but we transcribed them as they appear in the original diagnosis.

Discussion

During the last six decades, the taxonomic and systematic research on ground frogs, beyond of species descriptions and estimations of phylogenetic relationships, has focused on solving three fundamental issues: the delimitation of the genus, its division into species groups and the estimation of its species diversity. The monophyly and distinction of Eupsophus with respect to its sister genus, Alsodes, is now well established based on morphological, chromosomal, bioacoustic, developmental and molecular phylogenetic evidence (Gallardo 1970, Lynch 1978, Nuñez 2003, Vera Candioti et al. 2011, Blotto et al. 2013). Likewise, the subdivision of the genus into two groups is supported by cumulative morphological, chromosomal, bioacoustic, genetic, immunological, and molecular phylogenetic evidence (see references in Results; reviewed in Nuñez 2003, although this author suggested that each group could represent a different genus). However, the number of species, which progressively increased from two (Lynch 1978) to a maximum of 11 (Nuñez et al. 2012a), decreased to six in the following five years (Blotto et al. 2013, Correa et al. 2017) and more recently, raised again to 11 (Suárez-Villota et al. 2018b; Fig. 1). This recent instability is due to two opposing views about the species diversity of the roseus group. Correa et al. (2017) used only unilocus species delimitation methods, but their proposal took into account the scarce chromosomal and bioacoustic differentiation within the group. Instead, Suárez-Villota et al. (2018b), using a bigger dataset and more sophisticated (multilocus) analyses, ratified the validity of the same nominal species recognized by 2013 and provided support for a new putative species. This last proposal implies the consolidation of the taxonomic work of the last decades and reinforces the idea that the species diversity of the genus could be underestimated (Nuñez et al. 2011, Blotto et al. 2013). Logically, this advance depends on the robustness of the previous taxonomy, but as shown in Correa et al. (2017) and here, there are enough precedents in the literature that allow to question the “traditional” taxonomy, something that was not considered by Suárez-Villota et al. (2018b). Most of these precedents were developed in Results, so below we only discuss the main problems that emerged from the comparison and critical analysis of all that information.

Diagnoses are fundamental in taxonomy, since diagnostic characters summarize the differences among closely related taxa (Winston 1999). However, we detected two general problems with the quality of diagnoses of Eupsophus species: the heterogeneity in the number and type of characters included and the use of very variable characters for distinguishing species of the same group. The heterogeneity can be clearly seen in Table 1 and implies that, over time, very different criteria have been applied to define which and how many characters are sufficient to diagnose the species. Indeed, only four characters have been included in four or more diagnoses (the first four characters of Table 1). Regarding character variation, Correa et al. (2017) showed, with examples from the literature and observations of live animals, that these same four characters vary intraspecifically. In fact, body coloration patterns, which are included in most diagnoses, vary even in the type series (Correa et al. 2017). These observations of the type material have been corroborated with examples of live specimens from the type localities of E. roseus and E. altor (Correa et al. 2017), and E. roseus and E. migueli (this study). These and additional examples from other populations show that variation in body coloration is widespread in the genus, but this phenomenon has rarely been recognized in the literature (Cei 1962a, 1962b, Nuñez 2003, Nuñez et al. 2012a) and its implications for the taxonomy never have been addressed. The other two external characters, iris color and snout shape (Correa et al. 2017; this study), and the shape of the xiphisternum (Díaz 1986) also vary extensively within species. Taken together, all this information weakens the evidence used to distinguish some species, particularly those whose diagnoses rest almost exclusively on these characters (e.g., E. insularis and E. migueli ). These high levels of variation in diagnostic characters have deep consequences for the current taxonomy (Suárez-Villota et al. 2018b), since that proposal is based on material only from the type locality for several species and according to its proponents is concordant with the taxonomic work of the last decades.

Our review of the literature showed that, apart from external and internal morphology, morphometrics, karyotypes, and calls have been the main lines of evidence applied to the taxonomy and systematics of Eupsophus. Although these kinds of data have been rarely incorporated into diagnoses, they have been included in the descriptions of several species (Formas 1978a, 1989, Veloso et al. 2005, Nuñez et al. 2012a). Each of those three lines of evidence support the distinction between the two species groups, though they have limited utility to differentiate species within groups. Except in the case of the two species of the vertebralis group, E. vertebralis and E. emiliopugini , which are clearly differentiated by their karyotypes and to a lesser extent by their advertisement calls (Formas 1989), few species of the genus can be differentiated with these data. In fact, none of the species of the roseus group can be distinguished by their advertisement calls, since all the parameters used to describe them overlap extensively and the descriptions of the calls of some species differ among studies (Correa et al. 2017). The karyotypic evidence deserves an additional commentary, since it has been explicitly (Formas 1978b) or implicitly (Veloso et al. 2005) assumed that species of this genus have characteristic karyotypes. The comparison of all published karyotypes shows that this is not the case and that different karyotypes were described for the same species and locality by different authors ( E. roseus , E. migueli , and E. vertebralis ), suggesting strongly observer biases (Correa et al. 2017). Even though these differences were real, the level of intrapopulation and intraspecific variation in chromosome morphology and position of secondary constrictions would be as high as the variation at interspecific level (see Table 2), so that this type of evidence would not be useful in the taxonomy of the roseus group.

The review of the geographic information also revealed difficulties in establishing the spatial boundaries of the species of the genus. Recently, Correa et al. (2017) compiled records of the literature (that we expand here), showing a high degree of overlap of distribution ranges and cases of sympatry among species of the same group that had not been recognized in previous studies and reviews (e.g., Nuñez 2003, Blotto et al. 2013). These compilations of records differ from the most recent published maps (Nuñez 2003, Rabanal and Nuñez 2008, IUCN 2019), which show mainly allopatric distributions for species of the same group and do not coincide with each other for some species. These discrepancies between available maps and the points collected are closely linked to the four species ( E. migueli , E. contulmoensis , E. nahuelbutensis and E. altor ) described within of the distribution range of E. roseus , whose limits and degree of sympatry have been never precisely established. The records compiled here also show an overlap between the distribution ranges of E. roseus and E. calcaratus , which is partially supported by molecular evidence but does not coincide with the previously established limits (e.g., Nuñez 2003). The proposal of Correa et al. (2017), by expanding the taxonomic limits of E. roseus and E. migueli , resulted in a considerable reduction in the levels of overlap of the distribution ranges, but the rebuttal of Suárez-Villota et al. (2018b) implicitly meant returning to the confusing situation derived of the geographic information of the literature. Moreover, they added one more factor of uncertainty when affirming that some species ( E. migueli , E. altor , E. contulmoensis , E. nahuelbutensis , Eupsophus sp. and E. septentrionalis ) have “restricted distributions”, which implies that the genus would have a highly fragmented distribution at present. This pattern is incompatible with the information available since there are historical records of E. roseus (see map of Fig. 3) and taxonomically undetermined populations (Correa et al. 2017) between the localities assigned to these species. Currently, it is not clear how these intermediate populations would fit into the taxonomic scheme of Suárez-Villota et al. (2018b). The problems to define the boundaries between species are not only limited to Chile, where the greatest diversity of species is found, but also extend to Argentina where the boundary between E. roseus and E. calcaratus is not clear.

This review summarizes six decades of taxonomy and systematic research on Eupsophus (partially reviewed by Correa et al. 2017), but unlike the last comprehensive review treating these topics (Nuñez 2003) the information from various sources is compared. Only this retrospective and comparative approach allowed to reveal the high degree of variation described in some morphological characters used for the descriptions and diagnoses, the lack of significant differentiation in morphometrics and advertisement calls, and the incongruences in the chromosomal evidence and geographic data (see also Correa et al. 2017). These patterns agree with the general decoupling between the morphological and phylogenetic differentiation implied for the last phylogenetic studies (Blotto et al. 2013, Correa et al. 2017, Suárez-Villota et al. 2018b), which had already been suggested by the comparative studies with allozymes and morphometry (Formas et al. 1983, Formas et al. 1991, Formas et al. 1992). Moreover, a practical issue emerged from this comparative synthesis. Since the levels of intra/interspecific morphological variation and divergence among species are high but poorly known, especially in the roseus group (regardless of the taxonomy adopted), field identification would be reliable only within the assumed distribution ranges and, as we have demonstrated, there has not been consensus about them. Therefore, inconsistent diagnoses, field misidentifications and misleading geographic data might be intimately linked, explaining most cases of sympatry and range overlap inferred from the compilation of localities. In turn, erroneous geographic data might influence the identification of atypical specimens, particularly in the distribution limits and unexplored zones. The problem of field misidentification is expected to persist under the most recent taxonomic arrangement (Suárez-Villota et al. 2018b) since that proposal is mainly based on material from the type localities or surroundings (except for E. calcaratus ) and, as we pointed out above, the diagnoses of Eupsophus species are unreliable and their geographic boundaries are still poorly defined.

In this context, phylogenetic and species delimitation studies with DNA sequences have emerged as an independent and powerful way to reassess the taxonomy of Eupsophus. However, except for Correa et al. (2017), those studies (Nuñez et al. 2011, 2012a, Blotto et al. 2013, Suárez-Villota et al. 2018a, b) have progressively reinforced the previous taxonomic work, without questioning the bases that support it. In addition, they have installed the idea that diversity at the species level would be underestimated by identifying two candidate species (Villarrica and Tolhuaca populations). Apparently, these advances constitute the consolidation of decades of taxonomic research based on other types of evidence, but the critical examination of the taxonomic literature done here allows us to outline two issues that weaken this assertion. First, there is scarce morphometric, karyotypic and bioacoustic differentiation and a very high level of intrapopulation variation in some external and internal characters (e.g., shape of the head, body coloration, shape of the xiphisternum) in the roseus group (patterns already noted by Correa et al. 2017), which excludes them as reliable sources of characters to distinguish the species. Taken together, these types of characters, which support most of the descriptions and diagnoses of the species, suggest that the diversity of the genus at species level is not well described so it is not clear how a delimitation approach based exclusively on molecular evidence can ratify such taxonomic scheme. Second, the claim that most species of the roseus group have “restricted distributions” (see above) has important consequences for the biogeography and taxonomy of the genus. Historical records and intermediate undetermined populations show that this pattern of isolated species does not adequately reflect the distribution of the genus, but more importantly, some of these populations occupy intermediate phylogenetic positions between some narrow-range species of the roseus group, decreasing the genetic divergence among them (Correa et al. 2017). The latter implies that the populations that make up these species with restricted distributions do not represent well the overall phylogenetic diversity of the genus, so that this dimension of its diversity is not adequately reflected by the current taxonomy. Therefore, future taxonomic and systematic studies of Eupsophus, whether molecular or not, should take into account the incongruities between the patterns of molecular, morphological, bioacoustic and chromosomal divergence and incorporate more intermediate populations to obtain a more accurate estimate of its species diversity.