Our literature review was focused on (but not restricted to) taxonomic, genetic and phylogenetic studies in which phenotypic and/or genetic variation within and among Eupsophus species is described. As starting point, we considered the first reviews exclusively dedicated to the taxonomy of Chilean Eupsophus, Cei (1960), Grandison (1961) and Cei (1962a), because they combined several problematic taxa (e.g., the forms described by Philippi 1902) under that genus name. Although those reviews (and some previous ones, such as Capurro 1958 and Cei 1958) included some species currently considered members of other South American genera (Alsodes, Batrachyla, Phrynopus, Thoropa), information about the genus, in its current definition (e.g., Lynch 1978), is easily retrievable. The last complete review of the taxonomy and systematics of Eupsophus is the unpublished doctoral dissertation of Nuñez (2003), but recently Correa et al. (2017) partially reviewed the chromosome, bioacoustic and geographic information on the genus. Other taxonomic and/or systematic studies with wider taxonomic coverage (but that include several species of Eupsophus) are Díaz (1986), Correa et al. (2006), and Blotto et al. (2013). The latter also contains a synthesis of the recent systematics of Eupsophus and was the most comprehensive molecular phylogenetic study of the genus until Correa et al. (2017) and Suárez-Villota et al. (2018b). Descriptions and redescriptions of the ten nominal species recognized by Suárez-Villota et al. (2018b) are included in Duméril and Bibron (1841) (E. roseus as Cystignathus roseus), Günther (1881) (E. calcaratus as Cacotus calcaratus), Philippi (1902) (E. insularis as Borborocoetus (Cystignathus) insularis), Grandison (1961) (E. vertebralis and E. roseus, the latter as E. grayi), Capurro (1963) (who proposed to recognize E. insularis as subspecies of E. grayi), Formas (1978a) (E. migueli), Formas and Vera (1982) (revalidation of E. calcaratus and E. insularis), Formas (1989) (E. emiliopugini), Ortiz et al. (1989) (E. contulmoensis), Ortiz and Ibarra-Vidal (1992) (E. nahuelbutensis), Nuñez (2003) (which includes somewhat different descriptions of the aforementioned eight species), Ibarra-Vidal et al. (2004) (E. septentrionalis), Veloso et al. (2005) (E. queulensis, synonymized with E. septentrionalis by Blotto et al. 2013), and Nuñez et al. (2012a) (E. altor). Other studies of Eupsophus with a taxonomic and/or systematic focus have used different approaches: Capurro (1963) (morphology), Formas (1978b) (karyotypes), Formas (1980) (karyotypes), Iturra and Veloso (1981) (karyotypes), Formas et al. (1983) (allozymes), Formas (1985) (calls), Fernández de la Reguera (1987) (morphometrics), Iturra and Veloso (1989) (karyotypes), Formas (1991) (karyotypes), Formas et al. (1991) (allozymes), Formas et al. (1992) (allozymes and morphometrics), Formas (1992) (karyotypes), Formas and Brieva (1992) (immunology), Formas (1993) (allozymes and morphometrics), Formas and Brieva (1994) (calls), Cuevas and Formas (1996) (karyotypes), Nuñez et al. (1999) (morphometrics and RFLPs), Cárdenas-Rojas et al. (2007) (larval morphology), Nuñez and Úbeda (2009) (larval morphology), Opazo et al. (2009) (calls), Lavilla et al. (2010) (morphology), Nuñez et al. (2011) (phylogeography using mitochondrial sequences), and Vera Candioti et al. (2011) (larval morphology).

We compiled literature records to define the geographic ranges of the 11 species recognized by Suárez-Villota et al. (2018b) and compared them with the most recent maps (Nuñez 2003, Rabanal and Nuñez 2008, Correa et al. 2017, and IUCN 2019). Locality data were obtained from the publications in which the species were described (see above) and from other sources (e.g., Webb and Greer 1969, Formas and Vera 1980, 1982, Formas et al. 1991, Nuñez et al. 1999, Úbeda 2000, Díaz-Páez and Nuñez 2002, Méndez et al. 2005, Ortiz and Ibarra-Vidal 2005, Asencio et al. 2009, Nuñez et al. 2011, Blotto et al. 2013, Núñez and Gálvez 2015, Correa et al. 2017, Suárez-Villota et al. 2018b). Distribution data and/or maps of older reviews (Cei 1960, 1962a, 1962b, Grandison 1961, Formas 1979) were carefully considered because the delimitations of the species at that time were quite different from the present. In addition, we reviewed all biological studies of the genus and other relevant sources about Chilean amphibians to collect additional geographic data.

Correa et al. (2017) showed that the four characters most frequently included in the diagnoses of the species of the roseus group (body coloration pattern, iris color, lateral and dorsal snout profile, and shape of the end of the xiphisternum) vary at the intrapopulation level. Here, we provide additional examples of intrapopulation variation in the first three characters. The observations were made in two undescribed and two type localities (Valdivia, E. roseus, and Mehuín, E. migueli), including less than 20 live specimens per locality. All specimens were released at the same capture site after being photographed.

Blotto et al. (2013) identified one specimen from Tolhuaca (foothills of Chilean Andes, ~38°S) as a probable undescribed species, sister to E. roseus. Correa et al. (2017) included the same specimen and other samples from near Villarrica (as representatives of the area where there would be another undescribed species according to Nuñez et al. 2011) in their phylogenetic and species delimitation analyses, finding support for the inclusion of all of them into a redefined E. roseus. Suárez-Villota et al. (2018b) included specimens from Villarrica, but not from Tolhuaca in their species delimitation analyses, so the reciprocal relationships between both populations and the taxonomic status of the latter currently are not clear. Here we address both issues, using the two coding mitochondrial fragments included in common by Blotto et al. (2013), Suárez-Villota et al. (2018a, b): cytochrome b (cytb) and cytochrome c oxidase subunit I (COI). We concatenated the sequences of both fragments, totaling 147 specimens representing the ten currently recognized species and the two undescribed taxa (Villarrica and Tolhuaca). The sequences of both genes differ in length between studies, so an initial alignment was obtained with blocks of gaps at the ends of the genes. We obtained an alternative alignment by cutting those extremes. Two schemes to apply nucleotide evolution models were used in both alignments: considering each gene fragment as a partition or each position of the codons as a distinct partition within each fragment (six partitions). Sequences were aligned with Muscle v3.5 (Edgar 2004) and then inspected by eye. Phylogenetic relationships were estimated through a Bayesian inference (BI) method with a Markov Chain Monte Carlo algorithm, performed with the program MrBayes v3.2.6 (Ronquist et al. 2012). A General Time Reversible, plus gamma and proportion of invariable sites model was independently applied to each fragment/partition, using also a reversible jump method. Two independent BI analyses (each consisting of two groups of four chains that ran independently) applying that method were run for 10 million generations, sampling every 1000^th generation. The first 25% of generations were conservatively discarded as burn-in after observing the stationarity of ln-likelihoods of trees in Tracer v1.7 (Rambaut et al. 2018). Convergence and mixing of chains were assessed examining values of average standard deviation of split frequencies, and expected sampling sizes and potential scale reduction factors for all parameters. One specimen of Alsodes norae of Suárez-Villota et al. (2018b) was used as outgroup (MK180951, cytb; MK181499, COI).

The reviews by Cei (1960, 1962a) and Grandison (1961) are fundamental for the recent taxonomy of Eupsophus, because they combined several invalid (for example, several forms of Cystignathus and Borborocoetus of Philippi 1902) and now valid species (E. calcaratus and E. insularis) into two taxa, which represent the two species groups currently recognized (Fig. 1; see below). However, since the description of E. migueli (Formas 1978a), the number of species increased from three to eleven (with E. altor), most of them derived from or closely related to E. roseus. One additional species from Isla Wellington (southern Chile), closely related to E. calcaratus, was proposed by Nuñez (2003), though it was never named or formally described (Fig. 1; Blotto et al. 2013 included specimens from Isla Wellington, showing that they belong to E. calcaratus). All descriptions and revalidations (in the case of E. calcaratus and E. insularis) were primarily motivated by observations of differences in external morphological characters and in some cases also internal ones. Other types of characters were added in some descriptions and diagnoses (see Table 1), but only exceptionally additional evidence was obtained subsequently to reinforce the distinction of some species (e.g., the karyotype of E. migueli, Iturra and Veloso 1981). Another important change was the synonymization of E. queulensis with E. septentrionalis (Blotto et al. 2013), which resulted in ten formally recognized species until 2017. That year, Correa et al. (2017) proposed to synonymize E. contulmoensis, E. nahuelbutensis and E. septentrionalis with E. roseus, and E. altor with E. migueli, thus reducing from ten to six the species of the genus (Fig. 1). These authors suggested that part of the diversity of species previously recognized was due to the excessive importance attributed to non-fixed morphological differences in certain populations. These last synonymizations were reverted by Suárez-Villota et al. (2018b), who revalidated the same ten species recognized by 2017 plus one not described from Villarrica, Chile (Fig. 1), although they did not include specimens from Tolhuaca, Chile (Eupsophus sp. 2 of Blotto et al. 2013, Fig. 1). The division of Eupsophus into two species groups, roseus and vertebralis (Fig. 1), already implicit in the reviews of Cei (1960, 1962a) and Grandison (1961), it was first formally proposed by Formas (1991) based on karyotype differences. This division has been supported by cumulative morphological (Fernández de la Reguera 1987, Nuñez 2003), chromosomal (Formas 1980, Formas 1991), bioacoustic (advertisement calls; Formas 1985, Formas and Brieva 1994), genetic (allozymes; Formas et al. 1983) and immunological evidence (Formas and Brieva 1992). More recently, molecular phylogenetic analyses with DNA sequences have ratified the reciprocal monophyly and high genetic divergence between those groups (Nuñez 2003, Correa et al. 2006, Blotto et al. 2013, Correa et al. 2017, Suárez-Villota et al. 2018a, b).

Correa et al. (2017) summarized the diagnostic characters of nine species of the roseus group (the eight species currently recognized plus E. queulensis). They extracted the information mainly from the original diagnoses, but also used other two sources for E. roseus, E. calcaratus and E. insularis, since the original descriptions and diagnoses of these species are very brief and were made under generic names no longer used. The two additional sources are Formas and Vera (1982), where E. calcaratus and E. insularis are revalidated, and Nuñez (2003), which contains partially different diagnoses for the eight species recognized at that date. The summary of Correa et al. (2017) highlighted several general deficiencies of the diagnoses of the species of the roseus group: 1) in some cases, characters that varied in the type series were used; 2) the great heterogeneity in number and type of characters used, which makes it difficult to identify the differences among the species; and 3) the four characters most frequently included in the diagnoses vary widely at the intraspecific level. Here (Table 1), we expand the summary table of Correa et al. (2017) to include the species of the vertebralis group and reorder the species according to the taxonomy and phylogenetic hypothesis of Suárez-Villota et al. (2018b). Table 1 allows to compare the diagnostic differences between species within groups, showing that the diagnoses are heterogeneous in the number of characters and level of detail, so they are scarcely comparable, regardless of the taxonomic scheme used (Correa et al. 2017 or Suárez-Villota et al. 2018b). In particular, diagnoses of sister species do not contain characters in common (E. migueli and E. altor) or these could be differentiated only by the body coloration pattern (E. contulmoensis and E. nahuelbutensis, E. vertebralis and E. emiliopugini), which has been described as variable in most species (see Correa et al. 2017 and the section Phenotypic observations).

Correa et al. (2017) showed, using literature information and observations of live specimens, that the four characters most frequently included in diagnoses (body coloration, color of upper part of iris, shape of snout and shape of the end of the xiphisternum) vary within species. Here we summarize the information used by those authors and add some additional details from the literature. The first comprehensive reviews of the genus (Cei 1960, 1962a, Grandison 1961) already mentioned, although briefly, that body coloration patterns vary at intrapopulation level in species of the roseus group. However, these type of observations did not prevent the coloration pattern (dorsal and/or ventral) from being later included as a diagnostic character for several species of the group (Table 1). Moreover, according to their descriptions, body coloration varies in E. calcaratus (Formas and Vera 1982), E. emiliopugini (Formas 1989) and E. altor (Nuñez et al. 2012a; see their fig. 5). Another characteristic that contributes to the variation of the dorsal coloration patterns is a mid-dorsal (vertebral) line of whitish or yellowish color, which may be present or absent, and vary in length and width. This vertebral line is more frequent in the two species of the vertebralis group (Cei 1962b, Grandison 1961, Formas 1989), but also has been reported in some specimens of E. migueli (Formas 1978a), E. calcaratus (Formas and Vera 1982), E. contulmoensis (Ortiz et al. 1989), E. nahuelbutensis (Ortiz and Ibarra-Vidal 1992) and E. septentrionalis (Ibarra-Vidal et al. 2004, Veloso et al. 2005; see also Fig. 4B). Correa et al. (2017) discussed the possible causes and practical consequences of the variation of the body coloration patterns, adding several examples with live specimens of the roseus group (see their Supporting Information). There are also previous literature records of variation in the other three characters mentioned. The coloration of the iris has been included recurrently in the descriptions and diagnoses of the species of the roseus group, so it was considered a useful character to distinguish certain species (Table 1). In contrast, the iris of both species of the vertebralis group is very similar, uniformly reticulated in black and yellowish (Nuñez 2003). Iris coloration appears to be a less variable trait, because there are only a couple of references of intraspecific variation in the literature. Nuñez (2003) suggested indirectly that there is variation in this trait: the iris color of E. calcaratus and E. nahuelbutensis is “generally” yellow, whereas that of E. roseus, E. migueli, and E. contulmoensis “can be” orange. Moreover, Nuñez et al. (1999) mentioned that the typical copper-colored upper part of the iris of E. roseus is also observed occasionally in specimens of E. calcaratus, which otherwise is bronze-yellow. The snout profile also has been included in several diagnoses of species of both groups (Table 1). For example, the snout profile, both in dorsal and lateral view, was one of the few characters used by Formas and Vera (1982) to differentiate E. calcaratus from E. roseus. Only in the case of E. nahuelbutensis this character was described as variable in the type series (some paratypes had the snout rounded, Ortiz and Ibarra-Vidal 1992). Another instance of intraspecific variation stems from the synonymy of E. queulensis with E. septentrionalis, since the shape of the snout was described as truncate in the former (Veloso et al. 2005) and short and rounded in lateral profile in the latter (Ibarra-Vidal et al. 2004; Table 1). Correa et al. (2017) gave examples of intrapopulation variation of iris coloration and snout profile in live specimens of several populations, including individuals of the type localities of E. roseus and E. altor, showing that these characters are not useful to diagnose the species of the roseus group. We provide additional examples of variation of body and iris coloration and snout profile with specimens of four localities, including the type localities of E. roseus and E. migueli (section Phenotypic observations). The shape of the distal end of the xiphisternum is the osteological character most frequently included in descriptions and diagnoses (Table 1), where it has been implicitly considered as fixed. According to the literature, the xiphisternum of most species is rounded and unnotched (E. roseus, E. calcaratus, E. vertebralis, E. contulmoensis, E. nahuelbutensis, E. septentrionalis, E. queulensis, and E. altor), but in E. insularis it is truncated and slightly notched (Capurro 1963, Formas and Vera 1982; although in this last study it was drawn as unnotched), and in E. migueli it is notched (Formas 1978a) (this character has not been described in E. emiliopugini). However, one study (Díaz 1986) examined the form of the xiphisternum in a significant number of specimens from the type localities of E. roseus (Valdivia, N = 37) and of E. migueli (Mehuín, N = 45), finding four types of xiphisternum (rounded, pointed, notched and seminotched) in E. migueli and three in E. roseus (notched condition was not found). Although in both species the rounded xiphisternum was the most frequent condition, this example demonstrates that intrapopulational variation in osteological characters may be detected when a large number of specimens is examined. Nuñez (2003) mentioned that some osteological characters vary at intra- and interspecific levels (for example, the relative position of epicoracoids, which has been included in the diagnoses of two species, Table 1), though which species display the variation were not specified by the author.

Morphometric approaches have usually been used to infer, implicitly or explicitly, the relationships among species or to discriminate (or validate) them. Also, they have been used in conjunction with allozymes (see below) to evaluate explicitly the agreement between morphological and genetic evolution in the genus (Formas et al. 1983, Formas et al. 1992, Formas 1993). The first comprehensive reviews (Grandison 1961, Cei 1962a) contain measurements and/or indices (ratios) of body, head and hind legs of adults of only two species of Eupsophus (equivalent to the two species groups) and the other species (Alsodes spp., Batrachyla taeniata) that the genus contained at that time. Cei (1962a) described morphometric differences between continental and insular (Chiloé Island) populations of E. grayi (equivalent to the current roseus group), but in those groups of populations he mixed several species that were described later. Subsequent studies on adults have applied multivariate statistical techniques (mainly principal components and discriminant analyses), but they have been carried out with a small number of species (no more than four species per study; E. nahuelbutensis and E. septentrionalis have not been included in any study) and populations (no study included more than one population per species). Despite these limitations, morphometric differences have been observed between the species groups (Fernández de la Reguera 1987), and not within them (Formas et al. 1983, Díaz 1986, Fernández de la Reguera 1987, Formas et al. 1992, Formas 1993, Nuñez et al. 1999, Nuñez et al. 2012a). In particular, some species of the roseus group are morphometrically indistinguishable from each other (E. roseus, E. migueli, and E. altor; Díaz 1986, Nuñez et al. 2012a). Similarly, the only comparative morphometric study of tadpoles, Nuñez and Úbeda (2009), showed a clear differentiation between species groups (E. vertebralis and E. emiliopugini versus E. roseus and E. nahuelbutensis), but scarce differences within them.

The karyotypes of nine of the ten species of Eupsophus currently recognized are shown in Table 2, ordered by species group and date of description (that of E. nahuelbutensis has not been described, although Nuñez 2003 pointed out that it has 30 chromosomes). Species groups are characterized by different numbers of chromosomes (30 in the roseus group, 28 in the vertebralis group; Nuñez 2003, Veloso et al. 2005) and three species present heteromorphic sex chromosomes (E. migueli, Iturra and Veloso 1981; E. insularis, Cuevas and Formas 1996; and E. septentrionalis, Veloso et al. 2005). In E. roseus the sex chromosomes do not differ in form, but can be distinguished by their constitutive heterochromatin patterns (Iturra and Veloso 1989). Correa et al. (2017) noted that different authors described different karyotypes for the same population in several species, without reporting variation among the specimens used, even though in most studies more than one was included (in some cases more than ten, e.g., Formas 1978a, 1978b, Cuevas and Formas 1996). Correa et al. (2017) argued that these differences are due to observer biases, which is consistent with the information of the karyotypes summarized in Table 2, where karyotypes of the same species obtained by several authors, from the same (e.g., E. roseus, E. migueli) or several localities (e.g., E. roseus, E. calcaratus) can be compared. Almost all karyotypes of the same species described by different authors differ in chromosomal morphology and position of the secondary constriction, and even in the presence or absence of this last structure (E. vertebralis and E. emiliopugini), so that intrapopulation and/or intraspecific variation is revealed only when different studies are compared. The levels of variation in chromosome morphology and position of the secondary constriction within a same species (considering all studies by different authors) are as high as the levels of variation observed among species of the same group (e.g., between E. migueli and E. insularis, or between E. roseus and E. contulmoensis; Table 2). The discovery of heteromorphic sex chromosomes in E. migueli (Iturra and Veloso 1981) is another example of inconsistent descriptions of karyotypes of the same population and species, since they were not observed in previous studies of the species (Bogart 1970, Formas 1978a, 1978b; Table 2). Differences in chromosome morphology are not due to methodological issues, since all studies followed Levan et al. (1964) to determine the position of the centromere and Bogart (1970, 1973) to determine the relative lengths of the chromosomes, so we agree with the suggestion of Correa et al. (2017) that many of the differences among studies are observer-dependent.

Bioacoustic studies

Vocalizations of nine nominal species of both species groups have been described (Table 3; summarized by Nuñez 2003 and Correa et al. 2017). The vocalizations emitted more frequently by males are advertisement calls (called type A or short calls; Formas 1985, Formas and Brieva 1994, Penna and Veloso 1990), which have been described for most species. The difference in the temporal and spectral (frequencies) structure of the advertisement calls is one of the lines of evidence that has been used to support the division of the genus into two groups (Formas 1985, Formas and Brieva 1994, Nuñez 2003). Also, long calls (> 2.7 seconds; type B of Formas 1985) are emitted by males of some species of the roseus group, which could correspond to territorial or encounter calls (Formas 1985, Penna and Veloso 1990), but these calls have been described only in E. migueli (Formas 1985, Penna and Veloso 1990) and E. roseus (Penna and Veloso 1990) (Table 3). Another type of call described in the roseus group is an aggressive call recorded occasionally in E. calcaratus and E. roseus (Márquez et al. 2005). Short advertisement calls are structurally very similar among species of the roseus group: all calls consist of only one note and ranges of temporal and spectral parameters overlap extensively among species (Table 3; see comments in Formas and Brieva 1994 and Correa et al. 2017). Formas and Brieva (1994) noted only differences in the intervals among harmonics among species of the roseus group: E. contulmoensis and E. insularis have harmonics at about 500 Hz, while E. calcaratus, E. migueli and E. roseus show harmonics at about 1000 Hz intervals. Instead, the advertisement calls of both species of the vertebralis group differ in notes per call, although the other parameters show a high degree of overlap (Formas 1989, Nuñez 2003). Table 3 contains the parameters most commonly used in the descriptions of Eupsophus vocalizations, but other parameters have been reported in some species: for example, pulses per second in E. roseus and E. vertebralis (as E. vittatus, Formas and Vera 1980), and notes per second and note duration in E. vertebralis and for long calls of E. roseus and E. migueli (Penna and Veloso 1990). More recently, the maximum frequency was included in the diagnosis of E. altor (Nuñez et al. 2012a) to differentiate it from E. roseus and E. migueli: this parameter surpasses 20 kHz in E. altor, while in the other two species it does not exceed 15 kHz. Correa et al. (2017) argued that this parameter would be the only diagnostic difference to distinguish E. altor from E. migueli, but they considered it insufficient to support the validity of E. altor. Variation in frequency modulation patterns of short advertisement calls have been described in E. calcaratus (Márquez et al. 2005), E. roseus (Márquez et al. 2005) and E. septentrionalis (as E. queulensis; Opazo et al. 2009).

Immunological, allozyme and RFLPs studies

Since the mid-1970s, several immunological techniques and enzymatic systems (e.g., lactate dehydrogenases, hepatic hexokinases) were used to solve taxonomic and systematic problems of the anurans of the temperate forests of South America, including the genus Eupsophus. However, the earliest studies with enzymes (Díaz and Veloso 1979, Díaz 1981, 1986) had a more systematic orientation at the genus level and included only a few species of Eupsophus. Here we consider only those molecular studies focused on estimating genetic differentiation and relationships among the species of the genus. Similarly to morphometric analyses, allozyme studies revealed greater genetic differentiation between species groups (Formas et al. 1983) than within groups (Formas et al. 1983; Díaz 1986; Formas et al. 1992; Formas 1993; Ibarra-Vidal et al. 2004). In fact, some species such as E. roseus and E. migueli (Díaz 1986), and E. contulmoensis and E. nahuelbutensis (Ibarra-Vidal et al. 2004) are almost genetically indistinguishable according to this technique. The comparative studies of morphometry and allozymes showed that in general there is more disagreement (Formas et al. 1983; Formas et al. 1991, within E. roseus; Formas et al. 1992) than concordance (Formas 1993) between the morphological and genetic differentiation within the genus. Ibarra-Vidal et al. (2004) was the last study in which these markers were used in the genus, where two diagnostic loci between E. septentrionalis and E. roseus (among 19 putative loci), and less differentiation between E. septentrionalis and its geographically closest congeners, E. contulmoensis and E. nahuelbutensis, were reported. These allozyme patterns, particularly the almost fixed differences between E. septentrionalis and E. roseus, were used to support the specific status of E. septentrionalis (Ibarra-Vidal et al. 2004; Table 1). Only one study investigated intraspecific genetic variation using these markers: Formas et al. (1991) analyzed the allozyme variation among seven populations of E. roseus, representing a substantial part of its distribution. These authors found low levels of genetic differentiation among populations and interpreted that in support of its taxonomic status. It should be noted that in that study, the population of P.N. Nahuelbuta (type locality of E. nahuelbutensis; Ortiz and Ibarra-Vidal 1992) was included as part of E. roseus. The only immunological study focused exclusively on the relationships of the genus Eupsophus was Formas and Brieva (1992), who used precipitin tests in agar-gel. Although the focus of that study was mainly to examine the relationships of Eupsophus with other genera, they found a great affinity among some species of the roseus group and ratified the differentiation of the genus into two groups previously observed with chromosomal (Formas 1991) and bioacoustic (Formas 1985) evidence. Regarding RFLP markers, a single taxonomic study (Nuñez et al. 1999) used this technique to distinguish between the morphologically similar species E. calcaratus and E. roseus. They found identical restriction patterns of mitochondrial DNA within each species (two localities each) using two restriction enzymes.

Studies with DNA sequences

These studies have aimed to estimate the phylogenetic relationships within Eupsophus, its phylogenetic position with respect to other anuran groups, the phylogeographic history of one of its species (E. calcaratus) and its species diversity with species delimitation approaches (Fig. 2). Nuñez (2003) was the first study in which DNA sequences were incorporated to investigate the phylogenetic relationships of the genus. Nuñez (2003) included only one specimen per species (eight), obtaining a high support for the monophyly of the genus and its division into two groups, with E. calcaratus as sister of the rest of the species of the roseus group (Fig. 2A). Two later studies including more than one species (but still only one specimen of each) defined the phylogenetic position of the genus with respect to other anuran taxa. Correa et al. (2006), although including only five species of the genus, obtained a topology within Eupsophus congruent with that of Nuñez (2003) and found a close relationship of this genus with Alsodes, while Pyron and Wiens (2011) also recovered a well-supported sister relationship between Eupsophus and Alsodes, but with specimens wrongly labeled as Batrachyla and Hylorina nested within a monophyletic Eupsophus (confusion clarified by Blotto et al. 2013). Subsequent studies have included more than one specimen per species, so they have also allowed to assess the phylogenetic relationships among populations. Nuñez et al. (2011) reconstructed the phylogeographic history of E. calcaratus with mitochondrial sequences, including samples of most of its distribution range. They considered the six main groups identified in their phylogenetic analyses (labeled A to F) as “diagnostic of species lineages” (Fig. 2B), highlighting the great divergence between lineage A (locality of Villarrica) and the rest of the lineages (which they recovered as the sister taxon to E. calcaratus; see comment below). Nuñez et al. (2012a), in the description of E. altor, performed a phylogenetic analysis with a fragment of the control region (including samples of E. calcaratus, E. roseus and E. migueli), in which a sister relationship between E. altor (samples only from the type locality) and E. migueli was recovered (not included in Fig. 2). They included the molecular divergence between both species in the diagnosis of E. altor (nine nucleotide substitutions, according to the paper), but an examination of the sequences of Nuñez et al. (2012a) shows that this figure is higher (22 sites with fixed differences between both species and seven additional variable sites within E. altor; see comment in Correa et al. 2017). Blotto et al. (2013) performed a phylogenetic analysis of Eupsophus and Alsodes with mitochondrial and nuclear genes, including the 11 nominal species of Eupsophus recognized at that time, and in some cases more than one locality per species (Fig. 2C). They recovered the two species groups and ten of the eleven species as well-supported lineages, except for E. queulensis and E. septentrionalis, which were sympatric and had an extremely low sequence divergence (and consequently they were synonymized). Blotto et al. (2013) also suggested that one specimen from Tolhuaca probably represents an undescribed taxon, sister to E. roseus (Fig. 1). Correa et al. (2017) reassessed the species diversity of Eupsophus, specifically of the roseus group (see the next section), and estimated the phylogenetic relationships within the genus, using mitochondrial and nuclear sequences and including a greater number of specimens and localities than Blotto et al. (2013). Correa et al. (2017) found support for both species groups and for a topology within the roseus group consistent with that of Blotto et al. (2013) (although reduced to only four species; Fig. 2D). Suárez-Villota et al. (2018a) used a novel combination of mitochondrial sequences for reconstructing the relationships within the genus with a few specimens per species, but following the same taxonomy of Blotto et al. (2013). They obtained a high support for both species groups and recovered E. calcaratus in a different position with respect to previous studies (Nuñez 2003, Blotto et al. 2013, Correa et al. 2017; Fig. 2E). More recently, Suárez-Villota et al. (2018b) used a set of mitochondrial and nuclear genes and several phylogenetic approaches to reconstruct the relationships within the genus and estimate its species diversity with species delimitation approaches (see next section). They included an even greater number of specimens than Correa et al. (2017) (although a similar number of localities), obtaining a strong support for the species groups, but different positions for E. calcaratus depending on the analysis: the same position as in the hypothesis of Suárez-Villota et al. (2018a) (in a maximum likelihood analysis with concatenated sequences) or as the sister species of all the other species of the roseus group (in their species tree analyses). They also obtained a weak support for an alternative position of E. septentrionalis, which is congruent with previous hypothesis (Blotto et al. 2013, Suárez-Villota et al. 2018a), and strong support for recognizing the Villarrica lineage as a new putative species, although as the sister taxon to E. roseus (differing from the position found by Nuñez et al. 2011). Furthermore, Suárez-Villota et al. (2018b) estimated diversification times within the genus, finding that their delimited species diverged from 0.396 to 0.023 Mya (means). In summary, the relationships among the most of nominal species of the roseus group are well-supported by several studies (the clades E. insularis + (E. migueli + E. altor) and E. contulmoensis + E. nahuelbutensis, the position of E. calcaratus as sister taxon of all the other species of the roseus group), with the notable exception of E. septentrionalis, whose position fluctuates between studies (e.g., Blotto et al. 2013, Suárez-Villota et al. 2018a, b). Also, the position of the two putative species with respect to E. roseus (Villarrica and Tolhuaca populations) is uncertain, since both have not been included simultaneously in any study (Correa et al. 2017 included specimens from the surroundings of Villarrica, but not from the exact location where the new species would be found). Finally, a series of populations included by Correa et al. (2017) (Eupsophus sp. = Esp of Fig. 2D), whose geographic and phylogenetic position is intermediate with respect to E. roseus, E. septentrionalis, E. contulmoensis and E. nahuelbutensis, currently cannot be assigned to any of these species since they were not included in the species delimitation analyses of Suárez-Villota et al. (2018b). With respect to the two species of the vertebralis group, they show a very low degree of genetic divergence and are not always recovered as reciprocally monophyletic groups (Suárez-Villota et al. 2018a) or with high support values (Blotto et al. 2013). This low degree of divergence is reflected in the estimated time of separation of both species, which is the lowest in the genus (mean of 23 kya).

Figure 2. 

Phylogenetic hypotheses of Eupsophus obtained with DNA sequences. In some of these studies several phylogenetic analyses were made but here we show the hypotheses preferred by the authors. The trees were simplified by merging the terminal nodes by species or other relevant groupings and uniforming the branch lengths, but maintaining the original topologies. The numbers next to the nodes indicate the bootstrap or jackknife support values for the maximum parsimony (MP) analyses or posterior probability for those of Bayesian inference (BI). Black circles over the nodes indicate maximum support. The number of specimens included for each taxon or population is indicated in parentheses (omitted when only one was included). When relevant, the localities of origin of some specimens are indicated in parentheses. For simplicity, some names were abbreviated (for example, Esep = E. septentrionalis; Esp = Eupsophus sp.). Below the trees are indicated the gene fragments used, whether they are mitochondrial (mt) or nuclear (nuc), the analysis strategy (concatenated: ctd; species tree: st) and the phylogenetic reconstruction method used. A Nuñez (2003); this is the only tree of those shown where morphological characters (15) were included to build it B Nuñez et al. (2011); the only one of these studies where not all species of the genus were included; lineages A-F were considered a priori as E. calcaratus C Blotto et al. (2013); the alternative position of E. septentrionalis (with its respective support value) obtained with a Bayesian analysis of the same data set is shown in red; the method used was MP with direct optimization (do); the support values correspond to jackknife absolute frequencies D Correa et al. (2017); note that several undescribed populations (Eupsophus sp. = Esp) appear intermixed with some nominal species of the roseus group; in this analysis E. contulmoensis (Econ) and E. nahuelbutensis (Enah) make up a clade but they are not reciprocally monophyletic E Suárez-Villota et al. (2018a); in this analysis E. vertebralis (Ever) and E. emiliopugini (Eemi) are not reciprocally monophyletic F Suárez-Villota et al. (2018b); they obtained a different topology within the roseus group in maximum likelihood and BI analyses of the same concatenated data set (not shown).

Recently, two studies have focused explicitly on the delimitation of species, particularly in the roseus group (Correa et al. 2017, Suárez-Villota et al. 2018b). These two studies present contrasting views of the diversity of the genus (six and eleven species, respectively), so it is pertinent to review the evidence and methodology that supports both proposals, and their taxonomic and biogeographic implications. Correa et al. (2017) used one mitochondrial and two nuclear fragments of relatively conserved genes to reassess the species diversity of the roseus group, applying three unilocus species delimitation approaches. The sampled populations, many of them not described, cover the whole distribution of the genus, but are concentrated between 36 and 40°S, where the greatest diversity of species of the roseus group is found. In addition, they reviewed the chromosomal and bioacoustic evidence of the genus, which was used to choose between different delimitation scenarios. The proposal of Correa et al. (2017) represents a novel view of the diversity of species of the genus, recognizing only four species in the roseus group (Fig. 1). The proposed synonymizations were also supported by non-molecular arguments. Biogeographically, these changes imply a more simplified scenario since three of the synonymized species (E. contulmoensis, E. nahuelbutensis and E. altor) had distributions surrounded by populations of other species according to literature records. On the other hand, Suárez-Villota et al. (2018b) used three mitochondrial fragments (more variable) and two nuclear regions analyzed with several unilocus and multilocus species delimitation methods. The number of samples was double, but the number of localities was roughly the same as that of Correa et al. (2017). Their sampling scheme also covered the entire distribution range of the genus, but most of sampled populations are located between 39 and 46°S (and half of the localities included belong to E. calcaratus). Although Suárez-Villota et al. (2018b) used more sophisticated methods (multilocus), making use of mitochondrial and nuclear sequences, they did not explicitly consider non-molecular evidence to support their proposal. From a taxonomic point of view, Suárez-Villota et al. (2018b) reverted the changes proposed by Correa et al. (2017), returning to the previous classification of ten species, to which a new one not described would be added (Fig. 1). Biogeographically, this proposal implies that several species of the roseus group have restricted distributions, maintaining the same pattern of overlap between some species that is derived from the accumulated information of the literature (see fig 2 of Correa et al. 2017, and the collection of localities below).

The recent description of the mitochondrial genomes of two species (E. vertebralis and E. emiliopugini) (Suárez-Villota et al. 2018a) marks the beginning of the genomic studies in the genus. Both species exhibit the same mitochondrial gene order as other neobatrachian frogs, and their mitogenomes are composed by 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and a non-coding control region. Both genomes share 94.5% identity, which agrees with the low genetic divergence observed between the two species in several phylogenetic studies (e.g., Blotto et al. 2013, Correa et al. 2017, Suárez-Villota et al. 2018b).

The genus is distributed approximately between 35°28'S (Núñez and Gálvez 2015) and 49°25'S (Asencio et al. 2009) in Chile, and between 39°20'S and 43°S in Argentina (Úbeda 2000, Vaira et al. 2012, Blotto et al. 2013) (Fig. 3). The distribution range of the roseus group is the same as that of the genus (Fig. 3A–C), but that of the vertebralis group is more restricted (37°19' to 45°30'S, approximately; Fig. 3D). The most recent sources of range maps of Eupsophus species are Nuñez (2003), Rabanal and Nuñez (2008), Correa et al. (2017) and IUCN (2019). Nuñez (2003) and Rabanal and Nuñez (2008) contain highly congruent maps of eight species (E. roseus, E. calcaratus, E. insularis, E. vertebralis, E. migueli, E. contulmoensis, E. emiliopugini, and E. nahuelbutensis) generated with point occurrences and areas, respectively. Correa et al. (2017) reviewed the geographic information of the genus and compiled literature records to define the distribution ranges of the ten species recognized until that date, with an emphasis on the roseus group and the Chilean portion of the distribution. However, their maps (their fig. 2) were only intended to represent the boundaries among species that can be inferred by combining all the occurrence points collected from the literature. Correa et al. (2017) showed that the eight species of the roseus group exhibited a high degree of overlap, including several cases of the presence of more than one species in the same locality reported in the same or different publications (see details in S4 File of Correa et al. 2017 and Appendix 1). These instances of sympatry were not considered in the previous reviews or map sources, where a general pattern of allopatry among species of the same group was assumed (e.g., Formas 1989, Formas and Brieva 1994, Nuñez et al. 1999). Recently, the IUCN (2019) updated the assessments of Eupsophus species, adopting the taxonomy of Correa et al. (2017) (six species, Fig. 1), so its maps (areas representing the extent of occurrence) incorporated the synonymizations proposed by those authors. Despite being the most recent, the maps of IUCN (2019) do not adequately reflect the distribution limits of some species according to the literature (see details below). Here we update and complement the compilation of localities made by Correa et al. (2017) (Fig. 3 and Appendix 1), considering the current taxonomy (ten nominal species plus several undescribed populations), and highlight the inconsistencies that arise when all the available geographic information of the genus is compared.

Figure 3. 

Compilation of localities of Eupsophus species gathered from the literature (see the complete list of localities in Appendix 1). Multicolored circles and the star indicate localities where two or three species of the same group have been reported in the same or different sources. White circles indicate the localities where two undescribed species have been identified (Villarrica and Tolhuaca), two undetermined populations included in this study (Fig. 4) and several ones considered by Correa et al. (2017) as E. roseus, whose taxonomic status is uncertain according to the current taxonomy (Suárez-Villota et al. 2018b). Thin gray lines within Chile represent boundaries of Administrative Regions.

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.

Figure 4. 

Cryptic coloration and variation of coloration patterns in two undetermined populations of the Eupsophus roseus group A adult females from Pidenco, showing cryptic coloration resembling the forest ground; insets show head profiles of the same individuals B adults and juveniles from Las Lianas exemplifying variation in coloration patterns. Both localities were included as Eupsophus sp. in the map of Fig. 3.

Figure 5. 

Examples of intrapopulation external variation in adult specimens of the type localities of two species of the Eupsophus roseus group A Eupsophus roseus from Valdivia B Eupsophus migueli from Mehuín. Both examples illustrate the variation in dorsal and ventral (B) coloration, iris color and snout shape.

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.

Figure 6. 

Consensus phylogram (50% mayority-rule) of the Bayesian analysis of the mitochondrial fragments cytochrome c oxidase subunit I and cytochrome b. For simplicity, the outgroup (Alsodes norae) is not shown. Colored branches indicate the specimens of the two putative species: Villarrica (green) and Tolhuaca (red). The values next to the nodes are the posterior probabilities (pp); asterisks represent maximum values (pp = 1). Note that all species currently recognized (Suárez-Villota et al. 2018b) are supported by high pp values (> 0.97), except for both of the vertebralis group, wich are not reciprocally monophyletic. The scale bar under the tree represents the expected substitutions per site.