Outgroup Selection

Recent studies (Haas, 2003; Darst and Cannatella, 2004) have suggested that the Hylidae as traditionally understood is not monophyletic, with the Hemiphractinae displaced phylogenetically from the Hylinae, Phyllomedusinae, and Pelodryadinae. The aforementioned studies did not provide extensive outgroup comparisons. In order to avail ourselves of a strong test of hylid monophyly, we included 48 nonhylid outgroup taxa representing 14 neobatrachian families.

Basal Neobatrachians

Heleophrynidae, Sooglossidae, Limnodynastinae and Myobatrachinae1 have been related to each other by several authors (Lynch, 1973; Duellman and Trueb, 1986; Ford and Cannatella, 1993; Hay et al., 1995; Ruvinsky and Maxson, 1996; Biju and Bossuyt, 2003; Darst and Cannatella, 2004). While in the past they were considered part of Hyloidea, they were recently excluded by Darst and Cannatella (2004). The evidence indicates that they are basal neobatrachians distantly related to the apparently monophyletic Hyloidea; however, their exact positions and interrelationships are still unclear (Darst and Cannatella, 2004; Haas, 2003). We include one heleophrynid (Heleophryne purcelli), one myobatrachine (Pseudophryne bibroni), and two limnodynastines (Limnodynastes salmini and Neobatrachus sudelli) in our study. Furthermore, because some members of the Australopapuan hylids have been posited to be related to the Myobatrachidae (Lynch, 1971; Savage, 1973), their inclusion provides a strong test of hylid monophyly.

Ranoidea

Recent papers dealing with ranoid groups (Emerson et al., 2000; Vences et al., 2003a) or at least ranoid exemplars (Biju and Bossuyt, 2003; Darst and Cannatella, 2004) have suggested the existence of three major clades, although this remains to be elucidated. The three major clades are composed of (1) Arthroleptidae, Astylosternidae, Hemisotidae, and Hyperoliidae; (2) Microhylidae; and (3) the remaining ranoids (including Petropedetidae, Mantellidae, Rhacophoridae, and the paraphyletic Ranidae). We include exemplars of Astylosternidae, Hyperoliidae, Hemisotidae, Microhylidae, Ranidae, Mantellidae, and Rhacophoridae.

Hyloidea

The Hylidae has long been considered to be embedded within a poorly defined major group of neobatrachians (Nicholls, 1916; Noble, 1922; Lynch, 1971, 1973; Ford and Cannatella, 1993) for which no morphological evidence of monophyly exists, although molecular evidence (Hay et al., 1995; Ruvinsky and Maxson, 1996; Darst and Cannatella, 2004) does support its monophyly. As redefined by Darst and Cannatella (2004) Hyloidea includes the nonmonophyletic Leptodactylidae (Ford and Cannatella, 1993; Haas, 2003), Dendrobatidae, Hylidae, Bufonidae, Brachycephalidae, Centrolenidae, Rhinodermatidae, and the monotypic Allophrynidae. In order to have a strong test of the monophyly of hylids, we include representatives of most of the nonhylid hyloid families.

The monophyly of Dendrobatidae is not controversial (see Grant et al., 1997), although recent phylogenetic analyses using ribosomal mitochondrial sequences (Vences et al., 2000; Santos et al., 2003; Vences et al., 2003b) show that there is a serious need to redefine most of the currently recognized genera. According to the results of Vences et al. (2003b), there are two major clades of dendrobatids: (1) one composed of Dendrobates, Phyllobates, Cryptophyllobates, Epipedobates (paraphyletic), Minyobates, and several groups of the rampantly polyphyletic Colostethus; and (2) another clade composed of Mannophryne, Nephelobates, Allobates, and two separate clades of Colostethus (none of the aforementioned analyses included the apparently primitive genus Aromobates; see Myers et al. 1991). We include as exemplars of the first major clade Dendrobates auratus and Phyllobates bicolor, and as exemplar of the second clade, Colostethus talamancae.

Bufonidae is a monophyletic group (for a list of morphological synapomorphies, see Ford and Cannatella, 1993; Haas, 2003) for which no study addressing comprehensively its internal relationships has been published. Partial studies (Graybeal, 1997; Darst and Cannatella, 2004; Haas, 2003) support the idea that Melanophryniscus is one of its most basal clades in the family. As a rough sample of bufonid diversity we include representatives of Melanophryniscus, Dendropryniscus, Atelopus, Didynamipus, Schismaderma, Bufo, Pedostibes, and Osornophryne.

The notion that the presumably monophyletic Centrolenidae (Ruiz-Carranza and Lynch, 1991) is closely related to hylids (Lynch, 1973; Duellman and Trueb, 1986; Ford and Cannatella, 1993) was recently challenged by the phylogenetic analyses of Haas (2003), who used larval morphology, and Darst and Cannatella (2004), who used mitochondrial ribosomal genes. Austin et al. (2002) recently provided molecular evidence supporting a relationship between Centrolenidae and the monotypic Allophrynidae. The internal relationships of Centrolenidae remain virtually unstudied. As a rough representation of centrolenid diversity, in this study we include Centrolene prosoblepon, Cochranella bejaranoi, and Hyalinobatrachium eurygnathum. We also include Allophryne ruthveni.

Leptodactylid nonmonophyly has been accepted for some time (Lynch, 1971) and was confirmed in an explicit cladistic framework by analyses using morphology (Haas, 2003) and DNA sequences (Ruvinsky and Maxson, 1996; Darst and Cannatella, 2004; Vences et al., 2003b). In the analysis by Darst and Cannatella (2004), Leptodactylidae is rampantly paraphyletic because all the other hyloid exemplars are nested within it.

From the five currently recognized subfamilies of leptodactylids (Laurent, 1986) there is more or less convincing evidence of monophyly for two of them: Eleutherodactylinae (direct development, eggs relatively large, few in number; Lynch, 19712) and Leptodactylinae (presence of a bony element in the sternum; Lynch, 1971). The analysis of Darst and Cannatella (2004) corroborated the monophyly of these two groups, albeit with limited taxon sampling. In the analysis by Haas (2003), the exemplars of Leptodactylinae were not monophyletic. No demonstrable synapomorphies are known for Ceratophryinae, Cycloramphinae or Telmatobiinae. Considering this situation, we include several leptodactylid exemplars (see table 1); our poorest sampling is within Cycloramphinae, where we only have representation for one genus, Crossodactylus.

The single representative of Brachycephalidae included by Darst and Cannatella (2004) in their analysis was nested within the exemplars of the leptodactylid subfamily Eleutherodactylinae, as suggested earlier by Izecksohn (1988). We include Brachycephalus ephippium in our analysis.

Of the hyloid families, only Rhinodermatidae is not represented in our analysis. This group was suggested to be nested in the subfamily Telmatobiinae by Barrio and Rinaldi de Chieri (1971) based on a similar karyotype and by Manzano and Lavilla (1995a) based on the presence of the m. pelvocutaneus in Rhinoderma and Eupsophus. In the DNA sequences analyses by Ruvinsky and Maxson (1996) and Biju and Bossuyt (2003) Rhinoderma appears in different positions within Hyloidea.

The Ingroup: Hylidae

Inasmuch as the hylids are the primary focus of this study, our sampling is most dense for this taxon and requires substantially more detailed discussion than does our outgroup selection.

Duellman (1970) arranged the family in four subfamilies: Amphignathodontinae, Hemiphractinae, Hylinae (including both the Australian and American groups), and Phyllomedusinae. Trueb (1974) subsequently synonymized the Amphignathodontinae and Hemiphractinae. On the basis of evidence presented by Tyler (1971), Duellman (1977) placed the Australian hylids in their own subfamily, Pelodryadinae, although Savage (1973) had previously regarded it as a different family and suggested that it was derived from Myobatrachidae.

Duellman (2001), based mostly on da Silva's results (1998), presented a phylogenetic analysis where hylids, including the subfamily Pseudinae, were considered to be monophyletic. Synapomorphies suggested by Duellman (2001) as being common to his three most parsimonious trees are the possession of claw-shaped terminal phalanges and the three articular surfaces on metacarpal III. The possibility of hylid polyphyly has not been seriously considered by most frog systematists, even after Ruvinsky and Maxson's (1996) results (which showed their single Hemiphractinae exemplar not closely related with the other hylid exemplars), until the idea was suggested on morphological grounds by Haas (2003), followed by Darst and Cannatella (2004) on the basis of molecular evidence. These authors found no evidence of a relationship between Hemiphractinae and the other hylid subfamilies, which were thought to form a monophyletic group. Beyond this result, Haas (2003) presented evidence from larval morphology that suggested that Pelodryadinae is paraphyletic with respect to Hylinae (Hylinae not being demonstrably monophyletic), with Pseudinae and Phyllomedusinae possibly being imbedded within it.3

Hemiphractinae

Mendelson et al. (2000) and Duellman (2001) presented brief taxonomic histories of this taxon. The monophyly of Hemiphractinae is supported by the presence of bell-shaped gills in larvae and by female transport of eggs in a specialized depression or sac in the dorsum (Noble, 1927). Burton (2004) added the broad m. abductor brevis plantae hallucis. In Duellman's (2001) cladogram, Hemiphractinae is considered to be the sister group of Phyllomedusinae, with the evidence of this relationship being the proximal head of metacarpal II not between prepollex and distal prepollex, and the larval spiracle sinistral and ventrolateral.

Haas's (2003) exemplar4 of the Hemiphractinae was Gastrotheca riobambae. His results suggested that Hemiphractinae are unrelated to other hylids, although the position of Hemiphractinae within Neobatrachia is still unresolved. A similar result regarding Hemiphractinae as being unrelated to hylids was advanced by Ruvinsky and Maxson (1996) and corroborated by Darst and Cannatella (2004). These authors also did not recover the exemplars of Hemiphractinae that they used (Gastrotheca pseustes and Cryptobatrachus sp.) as forming a monophyletic group.5

Hemiphractinae includes five genera: Cryptobatrachus, Flectonotus, Gastrotheca, Hemiphractus, and Stefania. Mendelson et al. (2000) studied the relationships among these genera, performing a phylogenetic analysis using morphological and life-history characters, arriving at the topology (Cryptobatrachus Flectonotus (Stefania (“Gastrotheca” + Hemiphractus))). This analysis included five outgroups, all of which were representatives of the other hylid subfamilies. The results suggested that Cryptobatrachus and Flectonotus are each monophyletic, and that Gastrotheca is paraphyletic with Hemiphractus nested within it. As Hass (2003) noted, however, Mendelson et al.'s (2000) outgroup structure could not test the proposition of hylid diphyly.

Cryptobatrachus: In the analysis performed by Mendelson et al. (2000), the monophyly of the two representatives of Cryptobatrachus is supported by several osteological characters, among them the presence of an anteromedial process in the neopalatine.6 In their analysis, the relationship between Cryptobatrachus and the other Hemiphractinae is unresolved. This genus comprises three described species; in this study we include sequences of an unidentified species available from GenBank.

Gastrotheca: Mendelson et al. (2000) suggested that Hemiphractus is nested within Gastrotheca, a result that contrasts with the opinions of previous workers (Noble, 1927; Trueb, 1974; Duellman and Hoogmoed, 1984) who considered Hemiphractus basal among marsupial frogs because they lack a brooding pouch. However, Mendelson et al. (2000) continued to recognize Hemiphractus (the older of both names) and Gastrotheca pending a more complete phylogenetic study. Synapomorphies of the clade composed of these two genera are: cultriform process becoming distinctly narrow anteriorly; anterior process of vomer articulating only with maxilla; pre- and postchoanal process bifurcating at the level of the dentigerous process; nature of occipital artery pathway (a groove); brooding pouch with posterior opening; and bell-shaped gills fused distally.

Most of the 49 currently recognized species of Gastrotheca are placed in four species groups, the G. marsupiata, G. nicefori, G. plumbea, and G. ovifera groups (Duellman et al., 1988a). These groups are generally defined on the basis of overall similarity. The only character-based test of their monophyly, the analysis of Mendelson et al. (2000), included 17 exemplars and suggested that none of the three nonmonotypic groups is monophyletic.

Dubois (1987) placed the species within three subgenera: Gastrotheca, Duellmania (part of the G. plumbea group), and Opisthodelphys (G. ovifera as well as parts of the G. marsupiata and G. plumbea groups). Duellmania and Opisthodelphys were shown to be paraphyletic by Mendelson et al. (2000), although they did not test the monophyly of the subgenus Gastrotheca. In this study we include two species of the Gastrotheca marsupiata group (G. cf. marsupiata and G. pseustes) and two of the G. ovifera group (G. cornuta and G. fissipes).

Hemiphractus: Relationships of this genus were recently reviewed by Sheil et al. (2001), who provided a number of unambiguous synapomorphies to support its monophyly (such as the cultriform process of the parasphenoid that becomes distinctly narrow anteriorly, the presence of a zygomatic ridge, and the presence of a supraorbital ridge). Hemiphractus has six described species; we include Hemiphractus helioi in our study.

Stefania: Duellman and Hoogmoed (1984), Señaris et al. (“1996” [1997]), and MacCulloch and Lathrop (2002) reviewed this genus. Señaris et al. (“1996” [1997]) suggested that the zygomatic ramus of the squamosal being close to or in contact with the maxilla was a diagnostic character state of Stefania, “at least for the Venezuelan species” (translated from the Spanish). Mendelson et al. (2000) did not test the monophyly of Stefania since they included only one exemplar (S. evansi). It is unclear if any of the autapomorphies of S. evansi in that study are actually synapomorphies of Stefania.

Stefania was divided by Rivero (1970) into two species groups based on head shape (“as broad as long or longer than broad” in the S. evansi group; “much broader than long” in the S. ginesi group). The only test of the monophyly of these two groups is the phylogenetic analysis of the seven species then known, performed by Duellman and Hoogmoed (1984). In that analysis, the S. ginesi group was monophyletic and nested within the paraphyletic S. evansi group. Although Señaris et al. (“1996” [1997]) suggested the origin of the S. ginesi group from the S. evansi group, they continued to recognize of both groups.

Since the revision of Duellman and Hoogmoed (1984), another 11 species assigned to both species groups of Stefania have been named (see Barrio Amorós and Fuentes, 2003; MacCullogh and Lathrop, 2002). In our analysis we include one exemplar of the Stefania evansi group (S. evansi) and one of the S. ginesi group (S. schuberti).

Flectonotus: Duellman and Gray (1983) reviewed these frogs as two genera, Flectonotus and Fritziana, even though their phylogenetic analysis indicated that Flectonotus was paraphyletic with respect to Fritziana. Subsequently, Weygoldt and Carvalho e Silva (1991) placed Fritziana in the synonymy of Flectonotus to render a monophyletic taxonomy. Flectonotus is composed of five described species.

In the analysis by Mendelson et al. (2000), the position of Flectonotus remained unresolved with respect to Cryptobatrachus and the clade composed of Stefania plus Gastrotheca. Synapomorphies of Flectonotus in that analysis are: quadratojugal that does not articulate with maxilla; brooding pouch formed by dorsolateral folds of skin; overlap between m. intermandibularis and m. submentalis; and absence of supplementary elements of m. intermandibularis. Because of very limited availability of species of Flectonotus, in this study we include only Flectonotus sp., an unidentified species from southeastern Brazil, whose female has a brooding pouch with a middorsal slit.

Pelodryadinae

The monophyly of this Australopapuan group is supported by a single possible synapomorphy: presence of supplementary apical elements of m. intermandibularis (Tyler, 1971). Although relationships between Australian and New World hylids were recognized very early (most species of Litoria were named as Hyla), hypotheses regarding the relationships of Pelodryadinae with other groups have been rarely advanced. Trewavas (1933), Duellman (1970), and Bagnara and Ferris (1975) suggested a relationship between Pelodryadinae with Phyllomedusinae. Trewavas (1933) observed similarities in laryngeal structures in the limited data set at her disposal (only three pelodryadines and two phyllomedusines). Duellman (1970) referred to “similarities in vertebral characters” without further details and to identical number of chromosomes. Bagnara and Ferris (1975) noticed the presence in some species of Litoria of large melanosomes containing the red pigment rhodomelanochrome (later identified as pterorhodin; Misuraca et al., 1977), a character state previously known only for some species of Phyllomedusinae. Specifically, Bagnara and Ferris (1975) suggested that some species of Litoria could be related to the Phyllomedusinae, an implicit suggestion of Pelodryadinae paraphyly. This idea was discussed by Tyler and Davies (1978a), who rejected the possibility of pelodryadine paraphyly but did not address a possible sister-taxon relationship of Pelodryadinae and Phyllomedusinae. This alternative was suggested again, based on chromosome morphology, by Kuramoto and Allison (1991). In the phylogenetic analyses presented by Duellman (2001), Pelodryadinae was placed as the sister of a clade composed of the remaining subfamilies, which are united in having a distally bifid tendo superficialis. In this same cladogram, the only synapomorphy of Pelodryadinae is the anterior differentiation of the m. intermandibularis, although the monophyly of Pelodryadinae was assumed and not tested in that analysis.

The phylogenetic analysis performed by Haas (2003) presents the most extensive test of Pelodryadinae monophyly so far published; his Pelodryadinae exemplars form a paraphyletic series with respect to his Neotropical hylid exemplars. More recently, Darst and Cannatella (2004) and Hoegg et al. (2004) presented evidence from the ribosomal mitochondrial and nuclear genes supporting the monophyly of their Pelodryadinae sample and the monophyly of Pelodryadinae + Phyllomedusinae.

Litoria: The diagnosis and contents of Litoria were reviewed by Tyler and Davies (1978b). It is unclear whether any of the character states included in their extensive diagnosis are synapomorphic. However, considering subsequent comments by several authors (King et al., 1979; Tyler, 1979; Tyler and Davies, 1979; Maxson et al., 1985; Haas and Richards, 1998), the available evidence suggests that Litoria is paraphyletic with respect to the other genera of Pelodryadinae, Nyctimystes and Cyclorana, with the latter being the sister taxon of the L. aurea group.

The 132 currently recognized species (updated from Frost, 2004) placed in 37 species groups (Tyler and Davies, 1978b) make an exhaustive sampling of the group a goal beyond the present analysis.7 Relationships among some species groups of Litoria were addressed by means of albumin immunological distances as determined through microcomplement fixation (Maxson et al., 1982; Hutchinson and Maxson, 1986, 1987). From a character-based (as opposed to a distance-based) perspective, relationships among the species groups of Litoria remain unknown, and the monophyly of most of those groups with more than single species remains untested.

Tyler and Davies (1978b) tentatively divided the 37 species groups into three “Categories”, A (8 species groups), B (14 species groups), and C (7 species groups). Tyler (1982) added a fourth category (D), where he included the Litoria nannotis group. These groupings were criticized by King (1980) on karyotypic grounds. Besides, the monophyly of these four categories remain largely untested, with the only possible exception being the work by Cunningham (2002) on the L. nannotis group; however, his lack of sufficient outgroup sampling precluded a rigorous test.

In our analyses we include representatives of two species groups of category A, Litoria aurea (the L. aurea group) and L. freycineti (the L. freycineti group); three representatives of category B, L. caerulea (the L. caerulea group), L. infrafrenata (the L. infrafrenata group), and L. meiriana (the L. meiriana group); and one representative of category C, L. arfakiana (the L. arfakiana group).

Nyctimystes: This genus was rediagnosed by Tyler and Davies (1979). Among the list of characters provided by them, the synapomorphies of Nyctimystes seem to be the vertical pupil and the presence of palpebral venation. Tyler and Davies (1979) suggested that Nyctimystes was most closely related to some species groups of Litoria from New Guinea, implying that Nyctimystes is nested within Litoria. Specifically, they referred to the L. angiana, L. arfakiana, L. becki, L. dorsivena, L. eucnemis, and L. infrafrenata groups as the most likely to be related to Nyctimystes, because they share with Nyctimystes similarities in cranial structure (the L. infrafrenata and L. eucnemis groups) or the presence of large unpigmented ova and lotic tadpoles bearing large, ventral, suctorial mouths (the other groups). Nyctimystes currently comprises 24 described species, 5 of which (N. disruptus, N. oktediensis, N. trachydermis, N. tyleri, and N. papua) were included in the N. papua species group by Zweifel (1983) and Richards and Johnston (1993). We could not locate tissues of any members of the N. papua group, so we cannot test its monophyly. Nevertheless, we include the available species N. kubori, N. narinosus, and N. pulcher.

Cyclorana: This genus was thought to be related to the Australian leptodactylids (now Myobatrachidae) by Parker (1940), and was considered as such by Lynch (1971). Tyler (1972) first proposed its relationship to Australian hylids on the basis of the presence of a differentiated apical element of the m. intermandibularis. Subsequently, Tyler (1978) transferred Cyclorana to Hylidae. Tyler (1979), King et al. (1979), and Tyler et al. (1981) considered it to be related to the Litoria aurea group, a result that was coincident with the analyses of albumin immunological distances generated by microcomplement fixation (Hutchinson and Maxson, 1987). Burton (1996) suggested that having the m. palmaris longus divided into two segments (as opposed to three) is a synapomorphy supporting the monophyly of Cyclorana, L. dahlii, and L. alboguttata (two species of the L. aurea group). Based on sperm morphology, Meyer et al. (1997) transferred L. alboguttata to Cyclorana. The only morphological synapomorphy suggested for Cyclorana is the anterior ossification of the sphenethmoid to incorporate a portion of the tectum nasi (Tyler and Davies, 1993).

The 13 species of Cyclorana have been separated into different groups based on karyotypes, sperm morphology, and immunological distances (King et al., 1979; Maxson et al., 1982; Maxson et al., 1985; Meyer et al., 1997). These are the C. brevipes, C. australis, and C. platicephala “lineages”. In the present study, we include only C. australis.

Phyllomedusinae

Cruz (1990) and Duellman (2001) provided taxonomic histories of this group, mostly at the generic level. The monophyly of Phyllomedusinae has not been controversial; several character states have been advanced to support it. Some of the muscular character states include the supplementary posterolateral elements of the m. intermandibularis (Tyler, 1971); tendo superficialis pro digiti II (pes) arising from a deep, triangular muscle, which originates on the distal tarsal 2–3; tendo superficialis pro digiti III arising entirely from the aponeurosis plantaris; and m. extensor brevis superficialis digiti IV with a single slip (Burton, 2004). There are also several larval character states that support the monophyly of this group; for example, the arcus subocularis of larval chondrocranium with distinct lateral processes (Fabrezi and Lavilla; 1992, Haas, 2003); ultralow suspensorium (Haas, 2003); secondary fenestrae parietales (Haas, 2003); and absence of a passage between ceratohyal and ceratobranchial I (Haas, 2003).

The subfamily is comprised of six nominal genera: Agalychnis (8 species), Hylomantis (2 species), Pachymedusa (1 species), Phasmahyla (4 species), Phrynomedusa (5 species), and Phyllomedusa (32 species). Cruz (1990) discussed the taxonomic distribution of several character states shared by subsets of these genera. A cladistic analysis testing the monophyly of each of these and their interrelationships remains to be completed.

Agalychnis: Duellman (2001) presented a phylogenetic analysis of Agalychnis and Pachymedusa, using a vector of character states present in the Phyllomedusa buckleyi group as an outgroup (data taken from Cannatella, 1980). His analysis suggested no synapomorphies for Agalychnis. In our analysis we include all species available to us: A. calcarifer, A. callidryas, A. litodryas, and A. saltator (species not included are A. annae, A. craspedopus, A. moreletii, and A. spurrelli).

Hylomantis: This genus was resurrected by Cruz (1990) for the species formerly placed in the Phyllomedusa aspera group (Cruz, “1988” [1989]). From the extensive diagnosis presented by Cruz (1990), the only apparent synapomorphy of Hylomantis seems to be the lanceolate discs of fingers and toes. Cruz (1990: 725), however, considered likely that Hylomantis was paraphyletic with respect to Phasmahyla. Hylomantis has two described species, H. aspera and H. granulosa. Only H. granulosa was available for this study.

Pachymedusa: This monotypic genus was recognized by Duellman (1968a) to reflect his view that a remnant of the ancestral stock gave rise to the other Phyllomedusinae. However, Duellman (2001) found no evidence supporting the monophyly of Agalychnis independent of Pachymedusa. The single species Pachymedusa dacnicolor is included in our analysis.

Phasmahyla: This genus was erected by Cruz (1990) for the species formerly contained in the Phyllomedusa guttata group (Bokermann and Sazima, 1978; Cruz, 1982). Cruz (1990) provided an extensive definition of the genus based on adult and larval morphology. Probable synapomorphies of Phasmahyla are the lack of a vocal sac in adult males and larval modifications presumably associated with surface film feeding, such as the anterodorsal position of the oral disc, reduction in number and size of labial tooth rows, distribution and shape of submarginal papillae, and the upper jaw sheath with a medial projection (see Cruz, 1982, 1990). The genus is composed of four species, P. cochranae, P. exilis, P. guttata, and P. jandaia. We include Phasmahyla cochranae and P. guttata in our study.

Phrynomedusa: This genus was resurrected by Cruz (1990) for the species formerly placed in the Phyllomedusa fimbriata group (Izecksohn and Cruz, 1976; Cruz, 1982). From the extensive definition provided by Cruz (1990), possible synapomorphies of Phrynomedusa appear to be the presence of a bicolored iris and the complete marginal papillae in the oral disc of the larva. Phrynomedusa contains five described species; unfortunately, we could not secure any representatives of this taxon for the present study.

Phyllomedusa: No synapomorphies are known to support the monophyly of the 32 species of Phyllomedusa. This genus includes simply those species that are not included in the other five genera of Phyllomedusinae. There are currently five species groups recognized within Phyllomedusa: the P. buckleyi group (Cannatella, 1980), P. burmeisteri group (Lutz, 1950; Pombal and Haddad, 1992), P. hypochondrialis group (Bokermann, 1965a), P. perinesos group (Cannatella, 1982), and P. tarsius group (De la Riva, 1999). The monophyly of these groups had not been tested, and relationships among them remain unstudied. Furthermore, several species (e.g., P. bicolor, P. palliata, P. tomopterna, and P. vaillanti) have not been assigned to any species group. Some authors (Funkhouser, 1957; Duellman, 1968a, 1969; Cannatella, 1980; Jungfer and Weygoldt, 1994) have suggested that the Phyllomedusa buckleyi group deserves generic recognition.

Duellman et al. (1988b) posited the existence of a clade composed of most species of Phyllomedusa (excluding the P. buckleyi group and the P. guttata group, now Phasmahyla, but see below), implicitly including the species now placed in Hylomantis. Apparent synapomorphies of this clade are the well-developed parotoid glands; the presence of the slip of the m. depressor mandibulae that originates from the dorsal fascia at the level of the m. dorsalis scapulae; first toe longer than second; and the eggs wrapped in leaves. Duellman et al. (1988b) explicitly excluded the species then included in the P. guttata group (now Phasmahyla) from this apparent clade. However, Lutz (1954), Bokermann and Sazima (1978), Weygoldt (1984), and Haddad (personal obs.) reported P. guttata, P. jandaia, P. exilis, and P. cochranae, respectively, to oviposit in folded leaves, and Cruz (1990) reported in Hylomantis the presence of the slip of the m. depressor mandibulae that originates from the dorsal fascia at the level of the m. dorsalis scapulae. In this study we include representatives of the Phyllomedusa buckleyi group (P. lemur), P. burmeisteri group (P. tetraploidea), P. hypochondrialis group (P. hypochondrialis), and P. tarsius group (P. tarsius). We include also P. bicolor, P. palliata, P. tomopterna, and P. vaillanti, four species unassigned to groups.

Hylinae

This taxon is the primary focus of this study. Hyline monophyly is supported by two synapomorphies: the tendo superficialis digiti V with an additional tendon that arises ventrally from m. palmaris longus (da Silva, 1998, as cited by Duellman, 2001), and karyotype with 24 (or more) chromosomes (Duellman, 2001). The published molecular evidence is ambiguous regarding the issue.8

No comprehensive study of hyline systematics has been published, although the results of one unpublished dissertation (da Silva, 1998) have been widely circulated (e.g., Duellman, 2001). Although the problems of hylid systematics have been recognized for some time, almost all work has been done at the level of satellite genera (e.g., Duellmanohyla, Plectrohyla, Ptychohyla, Scinax) or species groups of Hyla.

For the purpose of our analysis we included representatives of all 27 genera of Hylinae. Within the genus Hyla, we included exemplars of 39 of the 41 species groups that had been recognized (we lack exemplars for the H. claresignata and H. garagoensis groups). We are not recognizing monotypic species group because (1) they do not represent testable hypothesis, and (2) they are not a rank in the Linnean taxonomy and therefore are not required for consistency and stability purposes.

Hyla is the most species-rich genus of hylid frogs. It is currently placed in 41 species groups, plus other species that had not been associated with any group. In turn, major clades composed of several of these species groups have been suggested. Because no study has ever suggested Hyla to be monophyletic and several have suggested that it is paraphyletic with respect to other hyline genera (Duellman and Campbell, 1992; Cocroft, 1994; Faivovich, 2002; Haas, 2003; Darst and Cannatella, 2004; Faivovich et al., 2004), we refer further discussion to the headings for the various genera and species groups. Comments about apparent major clades and proposed relationships among species groups are mostly reserved for the discussion section of this paper.

Gladiator Frogs

Kluge (1979: 1) referred to the frogs then placed in the Hyla boans group as “gladiator frogs” “in view of their extremely pugnacious behavior and the well developed pre-pollical spines that they use when fighting.” Following Duellman (1970, 1977), Kluge (1979) included in the group H. boans, H. circumdata, H. crepitans, H. faber, H. pardalis, and H. pugnax. Nevertheless, a prepollical spine has been reported for several species groups. Furthermore, territorial fighting has been reported or suspected to occur in several of these species.9 Because of this, and due to the lack of a better term, we prefer to use the term “Gladiator Frogs” to refer collectively to all the mostly South American species having a prepollical spine, as was done by Faivovich et al. (2004), instead of restricting its use to the H. boans group. The species groups that are currently referred to the Gladiator Frog clade are the H. albomarginata, H. albopunctata, H. boans, H. circumdata, H. claresignata, H. geographica, H. granosa, H. martinsi, H. pseudopseudis, H. pulchella, and H. punctata10 groups (Bokermann, 1972, Hoogmoed, 1979, Duellman et al., 1997, Eterovick and Brandão, 2001, Duellman, 2001, Faivovich et al., 2004).

Hyla albomarginata Group: The H. albomarginata group was first recognized formally by Cochran (1955) for a group of species (H. albomarginata, H. albosignata, H. albofrenata, H. musica) that Lutz (“1948” [1949]) referred to as the green species of Hyla of southeastern Brazil. Cochran (1955) included H. prasina (latter included in the H. pulchella group by Lutz, 1973) in this group. Duellman (1970) presented a definition of the group and included, besides the species considered by Cochran (1955), H. rufitela, H. pellucens, H. albopunctulata (now considered a member of the H. bogotensis group; see below), and H. albolineata (now considered a species of Gastrotheca; see Sachsse et al., 1999).

Cruz and Peixoto (“1985” [1987]) divided the Hyla albomarginata group into three “complexes”: the H. albomarginata complex, containing H. albomarginata and H. rufitela; the H. albofrenata complex, containing H. albofrenata, H. arildae, H. ehrhardti (as H. arianae; see Faivovich et al., 2002), H. musica, and H. weygoldti; and the H. albosignata complex, containing H. albosignata, H. callipygia, H. cavicola, H. fluminea, and H. leucopygia. Hyla pellucens should also be included in the albomarginata complex, because this species was included by Duellman (1970) but was overlooked by Cruz and Peixoto (“1985” [1987]); very likely the same applies for H. rubracyla, a species that was resurrected from the synonymy of H. pellucens by Duellman (1974) and included in the H. albomarginata group by Duellman in Frost (1985). The only possible synapomorphies that were proposed for these complexes were the red coloration of the webbing in the two species of the H. albomarginata complex studied by the authors, and presence of cloacal ornamentation in the H. albosignata complex (several instances of homoplasy within hylids). Haddad and Sawaya (2000) and Hartmann et al. (2004) further suggested that the H. albofrenata and H. albosignata complexes share a reproductive mode where the male constructs a subterranean nest in the muddy side of streams and pools that is completely concealed after spawning, a nest where larvae spend early stages of development; subsequent to flooding, the exotrophic larvae live in ponds or streams.

Cruz et al. (2003) added Hyla ibirapitanga and H. sibilata to the H. albosignata complex. Note that species included in both the H. albofrenata and H. albosignata complexes do not posses a prepollical spine, as do species in the H. albomarginata complex. On recent occasions, some authors (Haddad and Sawaya, 2000; Garcia et al., 2001a) referred directly to the H. albofrenata and H. albosignata groups without further comment. We include in the present analysis representatives of the three complexes: H. albosignata, H. callipygia, H. cavicola, and H. leucopygia as representatives of the H. albosignata complex; H. arildae, H. weygoldti, and a Hyla sp. 1, a new species similar to H. ehrhardti, as representatives of the H. albofrenata complex; and H. albomarginata, H. pellucens, and H. rufitela as exemplars of the H. albomarginata complex.

Hyla albopunctata Group: Cochran (1955) recognized the H. albopunctata group on the basis of the “more streamlined body shape, by lacking an outer metatarsal tubercle, and by having the fingers webbed only at the base …”. She included in the group H. albopunctata, H. raniceps, and several species from southeastern Brazil that are now in the H. claresignata and H. pulchella groups. Cochran and Goin (1970) recognized a H. lanciformis group (on the basis of large size, a white margin on the upper lip, pointed heads, and reduced webbing between the fingers) in which they included H. lanciformis, H. multifasciata, and H. boans (name applied incorrectly to H. albopunctata; see Duellman, 1971a). Duellman (1971a) implicitly united these two groups and considered the larger H. albopunctata group to be composed of H. albopunctata, H. lanciformis, H. multifasciata, and H. raniceps. De Sá (1995, 1996) stated that there was no evidence supporting the monophyly of the group. Caramaschi and Niemeyer (2003) added H. leucocheila to the group and suggested that it was monophyletic, but they presented no evidence to this effect. We include all species except H. leucocheila in our analysis.

Hyla boans Group: The constitution of the H. boans group as well as the definition of the group present a rather confusing history. Affinities between species of what is currently called the H. boans group were first recognized by Cochran (1955), who included in what she called the H. faber group the species H. crepitans, H. faber, H. langsdorffii (now a species of Osteocephalus, see Duellman, 1974), and H. pardalis. Some of the diagnostic characters of this group were large size and the presence of what she called a prominent spurlike prepollex in males. Cochran and Goin (1970) included H. faber, H. pardalis, H. rosenbergi, and H. maxima (now a junior synonym of H. boans; see Duellman, 1971a) in the H. maxima group, and they excluded H. crepitans, placing it in its own group. Duellman (1970) presented a formal definition of the H. boans group, in which he included H. boans, H. circumdata (now in the H. circumdata group), H. crepitans, H. faber, H. langsdorffii, H. pardalis, and H. rosenbergi. Lutz (1973) included the species in three different groups,11 in one of which she also included several species now included in the H. circumdata, H. pseudopseudis, and H. martinsi groups (the “species with long, sharp pollex rudiment”). Kluge (1979) resurrected H. pugnax from the synonymy of H. crepitans, including it also in the H. boans group. Martins and Haddad (1988) included in the group H. lundii (using the name H. biobeba, a junior synonym, see Caramaschi and Napoli, 2004), based on observations of nest construction done by Jim (1980). Implicitly, they also removed H. circumdata from the group. Hoogmoed (1990) resurrected H. wavrini from the synonymy of H. boans and retained it in the group. Duellman (2001) omitted H. lundii from the group without comment. Caramaschi and Rodrigues (2003) added H. exastis, suggesting that it was related to H. lundii and H. pardalis on the basis of its lichenous color pattern and the rugose skin texture. Caramaschi and Napoli (2004) presented a formal definition of the group. In summary, and following Caramaschi and Napoli (2004), we regard the H. boans group to be composed of H. boans, H. crepitans, H. exastis, H. faber, H. lundii, H. pardalis, H. pugnax, H. rosenbergi, and H. wavrini. The only synapomorphy that has ever been proposed for this group is the nest-building behavior of males, which has been observed in most species (see Martins and Moreira, 1991 for a review). Early reports of H. crepitans indicated that males do not construct nests; this was shown to be facultative by Caldwell (1992). This behavior is still unknown in H. pugnax. From this group we include in our analysis H. boans, H. crepitans, H. faber, H. lundii, and H. pardalis.

Hyla circumdata Group: This group was first mentioned by Bokermann (1967a, 1972), without providing any diagnosis. Heyer (1985) provided the first formal definition, diagnosing the group by the combination of a well-developed prepollex and the posterior face of the thigh having dark vertical stripes. The group was further discussed and expanded by Caramaschi and Feio (1990), Pombal and Haddad (1993), Napoli (2000), Caramaschi et al. (2001), and Napoli and Pimenta (2003). Three other species groups, the H. claresignata, H. martinsi, and H. pseudopseudis groups, as well as H. alvarengai, historically had been satellites of the H. circumdata group, with these species being alternatively included or excluded from the group. These groups and H. alvarengai are treated separately. With the recognition of these three groups being separate from the H. circumdata group, it is unclear which synapomorphies support its monophyly as currently defined.

Duellman et al. (1997) suggested that all species of the Hyla circumdata group should be transferred to the H. pulchella group. Faivovich et al. (2004) demonstrated by using DNA sequences from four mitochondrial genes that the two groups are not closely related. In the analysis of Faivovich et al. (2004), the three exemplars of the H. circumdata group then available (H. astartea, H. circumdata, and H. hylax) formed a monophyletic group that is the sister taxon of the remaining Gladiator Frogs they included in their analysis. Napoli and Pimenta (2003), Napoli and Caramaschi (2004), and Napoli (2005) recognized 15 species in the group: H. ahenea, H. astartea, H. caramaschii, H. carvalhoi, H. circumdata, H. feioi, H. gouveai, H. hylax, H. ibitipoca, H. izecksohni, H. lucianae, H. luctuosa, H. nanuzae, H. ravida, and H. sazimai. We include five species in our analysis: H. astartea, H. circumdata, H. hylax, as well as Hyla sp. 3 and Hyla sp. 4, two undescribed species from littoral areas of northern São Paulo (state) and southrn Rio de Janeiro (state), Brazil, respectively.

Hyla claresignata Group: A close relationship between H. clepsydra and H. claresignata was suggested by Bokermann (1972), who noticed striking similarities in larval and adult morphology. Bokermann (1972) suggested a possible relationship of these species with the H. circumdata group; following him, Jim and Caramaschi (1979) included H. clepsydra and H. claresignata in the H. circumdata group. However, subsequent workers (Caramaschi and Feio, 1990; Pombal and Haddad, 1993) who referred to the H. circumdata group did not include them in the group. The H. claresignata group was recognized in the restricted form by Duellman et al. (1997). Possible synapomorphies of the H. claresignata group are character states associated with the torrent-dwelling larvae of these species: oral disc completely surrounded by marginal papillae, and 7/12–8/13 labial tooth rows. We were not able to secure samples of either of the two species of this group.

Hyla geographica Group: This group was delimited by Cochran (1955: 180) as being characterized by its “extremely attenuate limbs”. Cochran and Goin (1970) characterized the species of this group as “moderate-sized tree frogs with elongate dermal appendages on the heels and reduced webbing between the fingers.” Duellman (1973a) presented an extensive characterization of the group (including vomers large with angular dentigerous processes, each bearing as many as 20 teeth; nuptial excrescences present in breeding males; projecting prepollices absent in both sexes; calcars present; palpebral membrane clear or reticulated). However, it is unclear from his account if any of these character states could be considered synapomorphic of the group.

Duellman (1973a) included in this group Hyla calcarata, H. fasciata, and H. geographica. Later, Pyburn (1977, 1984) added H. microderma and H. hutchinsi. Goin and Woodley (1969) considered H. kanaima related to the H. geographica group, and Pyburn (1984) included H. kanaima in the group. Lutz (1963, 1973) and Bokermann (1966a) stressed similarities between H. secedens and H. semilineata (as H. geographica), but Caramaschi et al. (2004a) suggested that actually this species is closer to H. bischoffi (of the H. pulchella group). Duellman (in Frost, 1985) and Duellman and Hoogmoed (1992), respectively, included H. picturata and H. roraima in the H. geographica group. D'Heursel and de Sá (1999) argued for the recognition of H. semilineata, a species that had previously been placed in the synonymy of H. geographica. Lescure and Marty (2000) included H. dentei, a species that Bokermann (1967b) considered to have character states of both H. raniceps (H. albopunctata group) and the H. geographica group. Caramaschi et al. (2004a) added H. pombali to the group. In summary, the H. geographica group is currently composed of 11 species: H. calcarata, H. dentei, H. fasciata, H. geographica, H. hutchinsi, H. kanaima, H. microderma, H. picturata, H. pombali, H. roraima, and H. semilineata. We include H. fasciata, H. calcarata, H. kanaima, H. microderma, H. picturata, H. roraima, and H. semilineata in our study.

Hyla granosa Group: This group was first defined by Cochran and Goin (1970) as green frogs that share the vomerine teeth being in rather heavy, arched series, and with males having a “protruding spine in the prepollex”. These authors included H. granosa, H. rubracyla (now in the H. albomarginata complex of the H. albomarginata group, see above), and H. guibei (now a junior synonym of H. pellucens; see Duellman, 1974). Previously, Rivero (1964) stated that H. alemani was allied with H. granosa. Rivero (“1971” [1972]) considered H. sibleszi to be related to H. granosa. Hoogmoed (1979) mentioned, without any diagnosis, the H. granosa group in the Guayanas, in which he included H. ornatissima. Mijares-Urrutia (1992a) considered H. alemani, H. granosa, H. ornatissima, and H. sibleszi to be the members of this group, and he provided a characterization based on larval features. We are not aware of any synapomorphies for this group. In this analysis we include H. granosa and H. sibleszi.

Hyla martinsi Group: This group was recognized by Bokermann (1965b) for two species, H. langei and H. martinsi, characterized by the presence of an extensive hooklike humeral crest and by a bifid prepollex. Bokermann (1964a) noticed “superficial similarities” of H. martinsi with H. circumdata. Caramaschi and Feio (1990) and Cardoso (1983) included H. martinsi in the H. circumdata group for having the diagnostic characters established by Heyer (1985). However, based on the presence of bifid prepollex and a humeral spine, Caramaschi et al. (2001) preferred to keep it as a separate species group. As a representative of this group we include H. martinsi in the analysis.

Hyla pseudopseudis Group: This group was recognized by Pombal and Caramaschi (1995) as closely related to the H. circumdata group, from which it was differentiated mostly by its color pattern. Eterovick and Brandão (2001) further differentiated both groups based on the presence of short, lateral irregular tooth rows and for having additional posterior tooth rows (6–8 rows) in the oral discs of the larvae of the H. pseudopseudis group (a maximum of 5 posterior rows in the H. circumdata group). Caramaschi et al. (2001) transferred H. ibitiguara from the H. circumdata group to the H. pseudopseudis group on the basis of its similar external morphology, color pattern, and habits. The group currently comprises three species, H. ibitiguara, H. pseudopseudis and H. saxicola, plus Hyla sp. 6 (aff. H. pseudopseudis), a new species from Bahia, Brazil, that is being described by Lugli and Haddad (in prep.). Only tissues of this new species were available for this study.

Hyla pulchella Group: The history of this group was recently reviewed by Faivovich et al. (2004). These authors also presented a phylogenetic analysis based on mitochondrial DNA sequences of four genes, and included 10 of the then 14 species included in the group, plus exemplars of the former H. polytaenia group and several outgroups. Their results indicate that the H. polytaenia group, as defined by Cruz and Caramaschi (1998), is nested within the H. pulchella group. Consequently, species included in the H. polytaenia group were transferred to the H. pulchella group, where they are recognized as the polytaenia clade. Considering this action and the species status given to H. cordobae and H. riojana, Faivovich et al. (2004) raised the number of species included in the H. pulchella group to 25. Carnaval and Peixoto (2004) recently added H. freicanecae to the group. Caramaschi et al. (2004a) suggested that H. secedens is related to H. bischoffi, therefore adding implicitly the species to the H. pulchella group. Caramaschi and Cruz (2004) added H. beckeri and H. latistriata to the H. polytaenia clade, adding two more species to the H. pulchella group. Faivovich et al. (2004) had doubts regarding the recognition of H. callipleura. Duellman et al. (1997) included this name as a junior synonym of H. balzani, but Köhler (2000) resurrected it using the combination H. cf. callipleura for some populations in Bolivia. We tentatively recognize H. callipleura as valid, but stress the necessity of further studies to clarify its status.

While the monophyly of this redefined Hyla pulchella group is supported by molecular evidence, no morphological synapomorphies have been proposed so far (see also comments for the H. circumdata and H. larinopygion groups). In summary, there are 30 species included in this group: H. albonigra; H. andina; H. balzani; H. beckeri; H. bischoffi; H. buriti; H. caingua; H. callipleura; H. cipoensis; H. cordobae; H. cymbalum; H. ericae; H. freicanecae; H. goiana; H. guentheri; H. joaquini; H. latistriata; H. leptolineata; H. marginata; H. marianitae; H. melanopleura; H. palaestes; H. phaeopleura; H. polytaenia; H. prasina; H. pulchella; H. riojana; H. secedens; H. semiguttata; and H. stenocephala. In this analysis we include the same species that were available to Faivovich et al. (2004) (H. andina; H. balzani; H. bischoffi; H. caingua; H. cordobae; H. ericae; H. guentheri; H. joaquini; H. leptolineata; H. marginata; H. marianitae; H. prasina; H. pulchella; H. riojana; H. semiguttata, and an undescribed species), plus H. polytaenia. The species that Faivovich et al. (2004) called Hyla sp. 2 corresponds to what Caramaschi and Cruz (2004) recently described as H. latistriata, and so is included under that name.

Hyla punctata Group: This group was first recognized by Cochran and Goin (1970), who included H. punctata, H. rhodoporus, and H. rubeola (these last two were subsequently considered to be synonyms of H. punctata by Duellman, 1974). They characterized the group as “moderately small green tree frogs with small vomerine tooth patches, reduced webbing between the fingers, without spines on the pollex, and without ulnar or tarsal ridges.” Hoogmoed (1979) mentioned this group without defining it. No synapomorphies have been proposed for this group. Besides H. punctata, two other species could be included on this poorly defined group: H. hobbsi, a species resurrected from the synonymy of H. punctata by Pyburn (1978), and H. atlantica, a name recently applied by Caramaschi and Velosa (1996) for the populations on eastern Brazil previously considered as H. punctata. In this analysis we include H. punctata.

Species of Probable Gladiator Frogs Unassigned to Species Group: Hyla alvarengai: This bizarre species was said by Bokermann (1964a) to share some character states with H. martinsi and H. saxicola (now placed in the H. martinsi and H. pseudopseudis groups, respectively), such as the notable development of the prepollex and the shape of the sacral diapophyses. Lutz (1973) included it in the group of the “species with long, sharp pollex rudiment”, together with H. crepitans, H. faber, and species now included in the H. circumdata and H. martinsi groups. She referred to it as Hyla (Plectrohyla?) alvarengai and stated that it was “devoid of affinities with the very large species of Hyla”, suggesting instead a possible relationship with Plectrohyla. A similar opinion was presented by Sazima and Bokermann (1977), who noticed “superficial similarities” with Plectrohyla, but they argued that they differed in larval morphology, spawn, and vocalizations. Duellman et al. (1997) included H. alvarengai in the H. circumdata group, presumably because it shares the diagnostic characters of the group. Eterovick and Brandão (2001) and Caramaschi et al. (2001) did not consider it as a member of the H. circumdata group. Unfortunately, we could not secure this species for our analysis, although we include a new species similar to H. alvarengai that is in the process of being described by Lugli and Haddad (in prep.).

Hyla fuentei: This species was described by Goin and Goin (1968) based on a single adult female from Surinam. Hoogmoed (1979) mentioned the existence of two additional specimens collected close to the type locality. Since then, no author has referred to this species. From the original description, there are few characters that allow the association of this species with any other group of Hyla. The angulate dentigerous process of the vomer suggests that this species could be associated with certain Gladiator Frogs, as some species currently placed in the H. albopunctata, H. boans, and H. geographica groups have this character state. A study of the holotype and discovery of male specimens should clarify the matter.

Hyla heilprini: This West Indian hylid was associated with the H. albomarginata group by Duellman (1970) based on the presence of a “green” peritoneum (actually it is white parietal peritoneum, like in species of the H. albomarginata, H. bogotensis, H. granosa, and H. punctata groups; Lynch and Ruiz-Carranza [1991: 4]; Faivovich, personal obs.) and external pigmentation. This was followed by Trueb and Tyler (1974), who noticed that its morphology was “highly reminiscent” of those from that species group. While it seems clear that H. heilprini is a Gladiator Frog, we are not aware of any synapomorphy relating it to the H. albomarginata group. This species was included in the analysis.

Three species of Hyla from the Venezuelan Tepuis: There are three species of Hyla from the Venezuelan Tepuis that have not been posited to be related to any other species or group of species: H. benitezi, H. lemai, and H. rhythmicus. The presence of a prepollical spine (Rivero, “1971” [1972]; Donnelly and Myers, 1991; Señaris and Ayarzagüena, 2002) associates these species with Gladiator Frogs. Hyla benitezi and H. lemai were included in the analysis.

Hyla varelae: Carrizo (1992) described this species based on a single adult male. It was suggested to be related to H. raniceps in the description. No additional specimens have been collected since the description, and it was not included in this analysis.

Andean Stream-Breeding Hyla

Duellman et al. (1997) presented a phylogenetic analysis restricted to wholly or partially Andean species groups of Hyla. On their most parsimonious tree, the H. armata, H. bogotensis, and H. larinopygion groups together form a monophyletic group supported by three transformations in larval morphology: the enlarged, ventrally oriented oral disc; the complete marginal papillae; and labial tooth rows formula 4/6 or more.

Hyla armata Group: The H. armata group was first recognized by Duellman et al. (1997) for its single species, H. armata. Köhler (2000) and De la Riva et al. (2000) subsequently reported that H. charazani was a second member of the H. armata group. Duellman et al. (1997) described four synapomorphies for the H. armata group: the presence in males of keratin-covered bony spines on the proximal ventral surface of the humerus, on the expanded distal element of the prepollex, and on the first metacarpal; tadpole tail long with low fins and bluntly rounded tip; forearms hypertrophied; and the presence of a “shelf” on the larval upper jaw sheath. We include both species in our analysis.

Hyla bogotensis Group: This group was reviewed by Duellman (1970, 1972b, 1989) and Duellman et al. (1997). The only synapomorphy that has been suggested for this group is the presence in males of a mental gland.12 Hyla albopunctulata was redescribed by Duellman and Mendelson (1995), who rejected a possible relationship with the H. bogotensis group, as suggested by Goin in Rivero (1969) and Duellman (in Frost, 1985), and they simply stated that its relationships were unclear. Faivovich et al. (in prep.) studied two male syntypes (BMNH 1880.12.5159 and 1880.12.5160), which posses a noticeable mental gland. For this reason, we associate this species with the H. bogotensis group. The Hyla bogotensis group is then composed of 15 species: H. albopunctulata, H. alytolylax, H. bogotensis, H. callipeza, H. colymba, H. denticulenta, H. jahni, H. lascinia, H. lynchi, H. palmeri, H. phyllognata, H. piceigularis, H. platydactyla, H. simmonsi, and H. torrenticola. In this analysis we were able to include only H. colymba and H. palmeri.

Hyla larinopygion Group: Duellman and Hillis (1990) and Duellman and Coloma (1993) reviewed this group, and Duellman and Hillis (1990) and Duellman et al. (1997) provided a formal definition, although it is unclear whether any of the morphological character states employed in these characterizations is synapomorphic for the group. Duellman and Hillis (1990) performed a phylogenetic analysis using isozymes of five species of the group. In the phylogenetic analysis of Duellman et al. (1997), the authors did not identify any synapomorphy for the H. larinopygion group; it merely lacks the apparent synapomorphies of the H. armata and H. bogotensis groups. Because of problems with the limits of the H. larinopygion group, Kizirian et al. (2003) were uncertain about the placement of H. tapichalaca, a species that they considered most similar to the H. larinopygion, H. armata, and H. pulchella groups.13 Faivovich et al. (2004) showed that H. tapichalaca and H. armata (the only exemplars of Andean stream-breeding Hyla they included) were sister taxa, and only very distantly related to the H. pulchella group. The H. larinopygion group currently comprises nine species: H. caucana, H. larinopygion, H. lindae, H. pacha, H. pantosticta, H. psarolaima, H. ptychodactyla, H. sarampiona, and H. staufferorum. In this analysis, we include H. pacha, H. pantosticta, and H. tapichalaca.

The 30-Chromosome Hyla

Only the species of the Hyla microcephala group were initially reported to have 30 chromosomes (Duellman and Cole, 1965; Duellman, 1967). However, as species of other groups were reported to have 30 chromosomes (Duellman, 1970; Bogart, 1973), it became evident that this was a characteristic of several species groups. Currently, the H. columbiana, H. decipiens, H. garagoensis, H. labialis, H. leucophyllata, H. marmorata, H. microcephala, H. minima, H. minuta, H. parviceps, and H. rubicundula groups, plus several species unassigned to any group are believed to conform to a monophyletic group supported by this character state (Duellman, 1970; Duellman and Trueb; 1983; Duellman et al., 1997; Napoli and Caramaschi, 1998; Carvalho e Silva et al., 2003).

Hyla columbiana Group: This group was first proposed by Duellman and Trueb (1983) for three species: H. carnifex, H. columbiana, and H. praestans. Kaplan (1991, 1999) found no evidence of monophyly for the group. Kaplan (1997) resurrected H. bogerti from the synonymy of H. carnifex, adding a fourth species to the group. Kaplan (1999) suggested that “the presence of two close, triangular lateral spaces between the cricoid and arytenoids at the posterior part of the larynx” is a synapomorphy of the H. columbiana group excluding H. praestans, which he considered to be closely related to the H. garagoensis group. The group therefore is composed of H. bogerti, H. carnifex, and H. columbiana. In the present analysis, we include H. carnifex.

Hyla decipiens Group: While describing the tadpoles of H. oliveirai and H. decipiens, Pugliese et al. (2000) noticed that they have marginal papillae (unlike other known larvae of the H. microcephala group), and they pointed out that they may not be members of the H. microcephala group as considered by Bastos and Pombal (1996). Pugliese et al. (2000) noticed similarities in tadpole morphology with H. berthalutzae, with which these two species also share oviposition on leaves outside the water. Pugliese et al. (2000) also associated H. haddadi with these three species based on external similarity.

Carvalho e Silva et al. (2003) suggested the recognition of the Hyla decipiens group for these species. The group was defined by larval features that include one row of marginal papillae, an ovoid body in lateral view, eyes in the anterior third of the body, dorsal fin arising at the end of the body, tail with transverse dark stripes on a light background, and pointed tip without a flagellum. It is unclear which of these character states could be considered to be possible synapomorphies. Perhaps one apparent synapomorphy of the group, not mentioned by the authors, could be the oviposition on leaves outside the water. The group is composed of H. berthalutzae, H. decipiens, H. haddadi, and H. oliveirai. In our analysis, we include H. berthalutzae.

Hyla garagoensis Group: This species group was first recognized by Kaplan and Ruiz-Carranza (1997), who diagnosed it by the presence of alternated pigmented and unpigmented longitudinal stripes on the hindlimbs of larvae. The H. garagoensis group is currently composed of three species, H. garagoensis, H. padreluna, and H. virolinensis. Unfortunately, no species of this group was available for our analysis.

Hyla labialis Group: This group was first recognized by Cochran and Goin (1970) for H. labialis (including what is now H. platydactyla, a species of the H. bogotensis group). They characterized the group by the vomerine teeth being in two rounded patches and by the presence of a well-developed axillary membrane (referred to as a patagium) that is bright blue in life. Duellman (1989) presented a more extensive definition of the group, noting that the axillary membrane was absent, a point with which we agree. A similar definition was presented by Duellman et al. (1997). No synapomorphies were suggested for this species group. The group currently comprises three species, H. labialis, H. meridensis, and H. pelidna. In this study we include H. labialis.

Hyla leucophyllata Group: This group was defined and later partly reviewed by Duellman (1970, 1974). From Duellman's (1970) extensive definition, the only possible synapomorphy seems to be the presence of two glandular patches in the pectoral region (this character state, however, was ignored in every subsequent paper dealing with phylogenetic relationships of 30-chromosome Hyla). In the phylogenetic analysis presented by Duellman and Trueb (1983), the only synapomorphy they proposed for the group was violin larval body shape. This character state seems problematic in that it is present in larvae of some species associated with the H. microcephala and H. rubicundula groups (see Lavilla, 1990; Pugliese et al., 2001), and therefore the level of inclusiveness of the clade that is supported by this transformation is unclear. From the perspective of adult morphology, we suggest that glandular patches in the pectoral region is a synapomorphy of the H. leucophyllata group; we observed it clearly on specimens of both sexes in all species of the group. Another character state that is either a likely synapomorphy of the H. leucophyllata group or of a more inclusive clade is the oviposition on leaves hanging over water (this oviposition mode occurs also in other 30-chromosome species; see comments in the H. microcephala and H. parviceps groups).

The Hyla leucophyllata group is composed of seven species: H. bifurca, H. ebraccata, H. elegans, H. leucophyllata, H. triangulum, H. rossalleni, and H. sarayacuensis. In our analysis, we include H. ebraccata, H. sarayacuensis, and H. triangulum.

Hyla marmorata Group: This group was recognized by Cochran (1955) for H. marmorata, H. microps, and H. giesleri based on the presence of an axillary membrane, warty skin around the margin of the lower lip, short snout, crenulated margin of limbs, short hindlimbs, developed finger and toe webbing, dorsal marbled pattern, and orange coloration in thighs and webbings. Bokermann (1964b) further diagnosed the group by its possession of a very large vocal sac. Several of these character states are possible synapomorphies for the group (such as the warty skin around the margin of the lower lip, the crenulated margin of limbs, the dorsal marbled pattern). Duellman and Trueb (1983) suggested that this group could be diagnosed by the presence of a row of small marginal papillae in the larval oral disc.

Bokermann (1964b), like Cochran (1955), included in the Hyla marmorata group other species as well: H. parviceps, H. microps, H. schubarti, and H. moraviensis (now considered a synonym of H. lancasteri; see Duellman, 1966). Subsequent authors transferred these species to other groups. This group is now composed of eight species: H. acreana, H. dutrai, H. marmorata, H. melanargyrea, H. nahdereri, H. novaisi, H. senicula, and H. soaresi. In our analysis we include H. marmorata and H. senicula.

Hyla microcephala Group: This group was defined by Duellman and Fouquette (1968) and Duellman (1970). Despite their extensive characterization, the only character state mentioned by these authors that subsequently has been considered a possible synapomorphy of this group is the lack of marginal papillae in the oral disc. Later, Duellman and Trueb (1983) added the depressed body shape of the larvae, another likely synapomorphy.

While the study of Duellman and Fouquette (1968) was focused on Middle American species, Duellman (1970) referred a number of South American species to the Hyla microcephala group. Overall, he included in the group H. elongata (a junior synonym of H. rubicundula; see Napoli and Caramaschi, 1999), H. microcephala, H. minuta, H. nana, H. phlebodes, H. robertmertensi, H. sartori, and H. werneri. Duellman (1972a) also included H. mathiassoni and H. rhodopepla in the group, and he excluded H. minuta. Cochran and Goin (1970) also had excluded H. minuta by placing it in their H. minuta group. Duellman (1973b) included H. gryllata, and Langone and Basso (1987) added H. minuscula, H. sanborni, and H. walfordi.

Cruz and Dias (1991) placed Hyla bipunctata in the group on the basis of larval characters.14 Márquez et al. (1993) included H. leali in the H. microcephala group, without mentioning that it was included by Duellman (1982) in the H. minima group. Furthermore, Márquez et al. (1993) noticed that H. leali and H. rhodopepla share vocalizations with short, pulsatile notes with extremely low dominant frequencies. Because of these similarities in vocalizations of H. leali with a species of the H. microcephala group, in the absence of other evidence we prefer to keep H. leali in this group.

Bastos and Pombal (1996) suggested that Hyla branneri, H. decipiens, H. haddadi, and H. oliveirai were closely related species that could be tentatively associated with the H. microcephala group based on overall similarity of adult morphology, but this was questioned by Pugliese et al. (2000) and Carvalho e Silva et al. (2003), who excluded these species from the group (see comments for the H. decipiens group). Pombal and Bastos (1998) also added H. berthalutzae, H. cruzi, and H. meridiana. Cruz et al. (2000) added H. pseudomeridiana. Köhler and Lötters (2001a) tentatively included H. joannae, based on its similarities in vocalization and adult morphology with H. leali (but see comments regarding H. leali above). Carvalho e Silva et al. (2003) added H. studerae and excluded H. berthalutzae (see comments for the H. decipiens group).

Of the 20 species currently included in the Hyla microcephala group, tadpoles are only known for 9 species: H. bipunctata, H. meridiana, H. microcephala, H. nana, H. phlebodes, H. pseudomeridiana, H. rhodopepla, H. sanborni, and H. studerae (Bokermann, 1963, Duellman, 1970, 1972a, Lavilla, 1990, Cruz and Dias, 1991, Cruz et al., 2000, Pugliese et al., 2000, Carvalho e Silva et al., 2003). All of these species have the two apparent synapomorphies of the H. microcephala group (see comments for the H. rubicundula group).

The 20 species currently included in the Hyla microcephala group are H. bipunctata, H. branneri, H. cruzi, H. gryllata, H. joannae, H. leali, H. mathiassoni, H. meridiana, H. microcephala, H. minuscula, H. nana, H. phlebodes, H. pseudomeridiana, H. rhodopepla, H. robertmertensi, H. sanborni, H. sartori, H. studerae, H. walfordi, and H. werneri. In this analysis we include H. bipunctata, H. microcephala, H. nana, H. rhodopepla, H. sanborni, and H. walfordi.

Hyla minima Group: Duellman (1982) tentatively grouped together five species from the Upper and Middle Amazon Basin and eastern Andes for which data on larval morphology, vocalizations, and osteology were mostly absent. He based the grouping of these species on small body size and distribution. The species he included were H. aperomea, H. leali, H. minima, H. riveroi, and H. rossalleni. The group as such was not named until Duellman in Frost (1985) referred to it as the H. minima group. Vigle and Goberdhan-Vigle (1990) added H. miyatai to this group; Márquez et al. (1993) implicitly transferred H. leali to the H. microcephala group (see comments for the H. microcephala group above); De la Riva and Duellman (1997) redescribed H. rossalleni and placed it in the H. leucophyllata group.

Small size is a difficult criterion to apply for the Hyla minima group, considering that its constituent species are not smaller than several species of the H. microcephala group. Duellman (2001) suggested that the H. minima group should be associated with the H. parviceps group. Unfortunately, this does not improve the systematics of these frogs, because no synapomorphies are known for either of these two groups (see the H. parviceps group for more comments). In the present analysis, we could include only H. miyatai.

Hyla minuta Group: This group was first defined by Cochran (1955); most of the species then included subsequently were transferred by several authors to the H. leucophyllata, H. microcephala, or H. rubicundula groups. The character state Cochran (1955) used to distinguish the H. minuta group was the immaculate anterior and posterior surfaces of the thigh, a character state that is shared by several 30-chromosome Hyla.

Martins and Cardoso (1987) described Hyla xapuriensis and referred it to the H. minuta group without providing any definition. Köhler and Lötters (2001b) described H. delarivai and tentatively suggested that it is close to H. minuta. Duellman and Trueb (1983) stated that the H. minuta group contained two species, but they did not state which one was the second species, and they provided no evidence for its monophyly. The group is currently composed of H. minuta and H. xapuriensis, and, following Köhler and Lötters (2001b), we tentatively include H. delarivai. For the purpose of our analysis, only H. minuta was available.

Hyla parviceps Group: This group was first defined by Duellman (1970), and by Duellman and Crump (1974), who also reviewed it. According to Duellman and Crump (1974), the species in the H. parviceps group differ from other 30-chromosome Hyla species by having: (1) a pronounced sexual dimorphism in size; (2) shorter snout; (3) tympanic ring indistinct or absent; (5) small (or reduced) axillary membrane; (9) sexual dimorphism in width of dorsolateral stripes; (10) suborbital bars; (11) thighs marked with spots; (13) iris pale gray with red ring around pupil; (15) more perichondral ossification in the tectum nasi and solum nasi; and (17) squamosal articulating with prootics. Duellman and Crump (1974) characterized the tadpoles as having ovoid bodies and xiphicercal tails with moderately deep fins not extending into body; anteroventral oral disc, large marginal papillae, robust serrated jaw sheaths, and no more than one row of labial teeth. It is unclear which of these character states could be synapomorphies of the group. Duellman and Trueb (1983) did not provide any synapomorphy for the group.

Duellman and Crump (1974) included in the Hyla parviceps group H. bokermanni, H. brevifrons, H. luteoocellata, H. microps, H. parviceps, and H. subocularis. Heyer (1977) added H. pauiniensis. Heyer (1980) resurrected H. giesleri from the synonymy of H. microps. Weygoldt and Peixoto (1987) tentatively included H. ruschii in the group. Martins and Cardoso (1987) added H. timbeba. Duellman and Trueb (1989) added H. allenorum and H. koechlini. Duellman (2001) added H. grandisonae and H. schubarti. Lescure and Marty (2000) referred H. luteoocellata to a separate group, the H. luteoocellata group, which they characterized by the presence of a cream-colored suborbital stripe. Because they did not discuss differences with the H. parviceps group, we still consider H. luteoocellata a member of the H. parviceps group. However, the species they described, H. gaucheri, does not seem to have this stripe.

The Hyla parviceps group is currently composed of 15 species: H. allenorum, H. bokermanni, H. brevifrons, H. gaucheri, H. giesleri, H. grandisonae, H. koechlini, H. luteoocellata, H. microps, H. parviceps, H. pauiniensis, H. ruschii, H. schubarti, H. subocularis, and H. timbeba. In our analysis we include H. brevifrons, H. giesleri, and H. parviceps.

Hyla rubicundula Group: This group was recently defined by Napoli and Caramaschi (1998) and was diagnosed by small body size, patternless thighs, and green dorsum in life that changes to pinkish or violet when preserved. Bogart (1970), Rabello (1970) and Gruber (2002) reported a 30-chromosome karyotype in H. rubicundula and H. elianeae. Historically, species of this group were associated with species now placed in the H. microcephala group, such as H. nana and H. sanborni (Lutz, 1973). The group is currently composed of H. anataliasiasi, H. araguaya, H. cachimbo, H. cerradensis, H. elianeae, H. jimi, H. rhea, H. rubicundula, and H. tritaeniata. In our analysis, we include H. rubicundula.

Species Known or Presumed to Have 30 Chromosomes Not Assigned to Any Group: Hyla anceps: Lutz (1948, 1973) associated H. anceps with H. leucophyllata based on it having an axillary membrane, tadpoles with xiphicercal tail, and vivid flash colors. Cochran (1955) considered this species to have no known close relatives and placed it on its own species group. Bogart (1973) reported a 30-chromosome karyotype for this species, and he tentatively associated it with H. microcephala, H. bipunctata, H. elongata (a junior synonym of H. rubicundula), and H. rhodopepla for sharing a single pair of telocentric chromosomes. Regardless of having been reported to have 30 chromosomes, Hyla anceps has been ignored in all subsequent literature dealing with phylogenetic relations of 30-chromosome species of Hyla. Wogel et al. (2000) redescribed the tadpole of H. anceps and stated that its morphology supported the idea of this species belonging to its own species group, without giving further details. We include this species in the analysis.

Hyla amicorum: This species was considered to be similar to H. battersbyi and H. minuta (Mijares-Urrutia, 1998). On this basis we consider it a 30-chromosome species. We could not include this species in the analysis.

Hyla battersbyi: Since its original description (Rivero, 1961) this species has been scarcely mentioned in the literature. We consider it tentatively as a 30-chromosome species based on the association made by Mijares-Urrutia (1998) of this species with H. minuta based on overall similarity. We could not include this species in the analysis.

Hyla haraldschultzi: Bokermann (1962) stated that the relationships of this species were uncertain. Lutz (1973), while treating the “small to minute forms”, where she included most of the 30-chromosome Hyla, considered H. haraldschultzi to be insufficiently known. We could not include this species in the analysis.

Hyla limai: In the original description, Bokermann (1962) associated this species with H. minuta and H. werneri. Apart from a brief comment by Lutz (1973), it has not been referred to again in the literature. Haddad (unpubl. data), based on morphological variation in populations of H. minuta, finds that H. limai could be a junior synonym of this species. We could not include this nominal species in the analysis.

Hyla praestans: Duellman and Trueb (1983) originally placed this species in the H. columbiana group. Kaplan (1999), based on the presence of a small medial depression in the internal surface of each arytenoid, suggested that H. praestans was related to the H. garagoensis group and possibly its sister taxon. We could not secure samples for the analysis.

Hyla stingi: This species, externally similar to H. minuta, has been suggested to be the sister group of a clade composed of the H. minuta, H. marmorata, H. parviceps, H. leucophyllata, and H. microcephala groups (this being supported by the reduction in the labial tooth row formula from 1/2 to 0/1; Kaplan, 1994). The apparent synapomorphy uniting H. stingi with this clade is the anterior position of the oral disc. This species could not be included in the analysis.

Hyla tintinnabulum: This species was associated with H. branneri and H. rubicundula by Lutz (1973). This fact and our study of one of the syntypes (NHMG 473; adult male) suggest that it is a 30-chromosome Hyla. We could not include this species in the analysis.

Hyla yaracuyana: This species was associated with the 30-chromosome Hyla by Mijares-Urrutia and Rivero (2000), but it was not included in any species group. We could not include this species in the analysis.

Middle American/Holarctic Clade

The monophyly of most Middle American and Holarctic Hylinae was suggested by Duellman (1970, 2001) based on biogeographic grounds. This clade would include most Middle American and Holarctic species groups of Hyla plus the genera Acris, Anotheca, Duellmanohyla, Plectrohyla, Pseudacris, Pternohyla, Ptychohyla, Smilisca, and Triprion.

Acris: This very distinctive taxon was reviewed by Duellman (1970) and was included in several studies of Holarctic hylids (Gaudin, 1974; Hedges, 1986; Cocroft, 1994; da Silva, 1997). In the strict consensus of the analysis by Cocroft (1994), the position of Acris was unresolved with respect of the other Holarctic hylids. In the reanalysis done by da Silva (1997), it is sister to a clade composed of the species of the Hyla cinerea and H. versicolor groups. The two species A. crepitans and A. gryllus are included in our analysis.

Anotheca: This monotypic genus was reviewed by Duellman (1970, 2001). Autapomorphies of this taxon include the unique skull ornamentation composed of sharp, dorsally pointed spines in the margins of frontoparietal, maxilla, nasal (including canthal ridge), and squamosal, and character states that result in its reproductive mode, including the female feeding tadpoles with trophic eggs (see Jungfer, 1996). We include the single species Anotheca spinosa in this analysis.

Duellmanohyla: This genus was reviewed by Duellman (2001). Duellmanohyla was proposed by Campbell and Smith (1992), based on four suggested synapomorphies involving larval morphology: a greatly enlarged, pendant oral disc (referred to as funnel-shaped mouth by Duellman, 2001); long and pointed serrations on the jaw sheaths; upper jaw sheath lacking lateral processes; and greatly shortened tooth rows. Duellman (2001) added the bright red iris and the labial stripe expanded below the orbit. Duellmanohyla contains the following eight species: D. chamulae, D. ignicolor, D. lythrodes, D. rufioculis, D. salvavida, D. schmidtorum, D. soralia, and D. uranochroa. In our analysis, we include D. rufioculis and D. soralia.

Hyla arborea Group: All of the species of Hyla of Europe, North Africa, and Asia have long been recognized as composing a single species group (Stejneger, 1907; Pope, 1931). Probably because species of this group have generally been discussed in isolation of other hylids, we are not aware of any author having provided a diagnosis of this group that could differentiate them, at least phenotypically, from the Nearctic species placed in the H. cinerea and H. eximia groups. No synapomorphy has ever been suggested; the monophyly of the group has apparently been assumed based on geography and the external similarity of most species (9 of the 16 currently recognized species have been considered at some point of it taxonomic history to be subspecies or varieties of H. arborea).

Various authors considered the Hyla arborea group to be closely related with some North American species. Based on similarities in advertisement calls, Kuramoto (1980) suggested that the H. arborea group is closely related to the H. eximia group of temperate Mexico and southwestern United States.

The assumed monophyly of the Hyla arborea group was challenged by Anderson (1991) on karyotypic grounds, and this was supported by Borkin (1999). These authors suggested the presence of at least two lineages resulting from two independent invasions to Eurasia. According to Anderson (1991), at least H. japonica and H. suweonensis (she did not include other eastern Asian species) share the presence of a NOR in chromosome 6 with the representatives she studied of the H. eximia group (H. arenicolor, H. euphorbiacea, H. eximia), some of the H. versicolor group (H. avivoca, H. chrysoscelis, H. versicolor; H. femoralis), and with H. andersonii. Hyla arborea, H. chinensis, H. meridionalis, and H. savignyi share with most Nearctic species of Hyla, as well as several of the outgroups that Anderson (1991) included, the NOR located in chromosomes 10/11 (she considered the chromosome pairs to be homologous in these species, with the shift in NOR position presumably corresponding to modulation of heterochromatin material and not to a translocation event; Anderson 1991: 323).

The Hyla arborea group as currently understood is composed of the following 16 species: H. annectans, H. arborea, H. chinensis, H. hallowellii, H. immaculata, H. intermedia, H. japonica, H. meridionalis, H. sanchiangensis, H. sarda, H. savignyi, H. simplex, H. suweonensis, H. tsinlingensis, H. ussuriensis, and H. zhaopingensis. In this analysis we include H. arborea, H. annectans, H. japonica, and H. savignyi.

Hyla bistincta Group: This group was reviewed by Duellman (2001). The lack of evidence supporting the monophyly of the H. bistincta group, together with the possibility that Plectrohyla is nested within it, has been repeatedly noted (Duellman and Campbell: 1992; Toal, 1994; Wilson et al., 1994a; Mendelson and Toal, 1995; Ustach et al., 2000; Canseco-Márquez et al., 2002).

Duellman (2001) presented a cladistic analysis of the Hyla bistincta group that included the 17 species known at that time, using a vector of character states of Plectrohyla and H. miotympanum as the root. He reported that the analysis resulted in 89 most parsimonious trees, from which he chose one that is presented in his figure 400 (Duellman 2001: 952). In this tree, the H. bistincta group is monophyletic, being supported by a single transformation, the loss of vocal slits. A reanalysis of his data set15 indicates that Plectrohyla is nested within the H. bistincta group on several of the most parsimonious trees, and that the strict consensus tree is almost entirely collapsed. Therefore, Duellman's analysis provides no evidence for the monophyly of the H. bistincta group.

The Hyla bistincta group currently comprises the following 18 described species: H. ameibothalame, H. bistincta, H, calthula, H. calvicollina, H. celata, H. cembra, H. charadricola, H. chryses, H. crassa, H. cyanomma, H. labedactyla, H. mykter, H. pachyderma, H. pentheter, H. psarosema, H. robertsorum, H. sabrina, and H. siopela. In this analysis we include H. bistincta and H. calthula.

Hyla bromeliacia Group: The taxonomy, composition, and history of this group were reviewed by Duellman (1970, 2001). Possible synapomorphies suggested by Duellman (2001) are those modifications of the phytotelmic larvae, mostly the depressed body and the elongated tail. The other characters he used to separate this group from the bromeliad-dwelling species of the H. pictipes group are likely plesiomorphic, such as the lack of massive temporal musculature (present only in H. zeteki and H. picadoi), the presence of more than one tooth row (only one in H. picadoi and H. zeteki), and the oral disc not entirely bordered by a single row of papillae (as in H. zeteki and H. picadoi). The group comprises two species, H. bromeliacia and H. dendroscarta. In this study we include only H. bromeliacia.

Hyla cinerea Group: This group was first recognized by Blair (1958) based on advertisement call structure. For a review of its history see Anderson (1991). The group is currently composed of four species, H. cinerea, H. femoralis, H. gratiosa, and H. squirella, and the group is monophyletic in the analyses of Hedges (1986), Cocroft (1994), and da Silva (1998), being supported by allozyme data (taken in the last two studies from Hedges, 1986). We included all four species of the group in the present analysis.

Hyla eximia Group: The taxonomy, composition, and history of this group were thoroughly reviewed by Duellman (1970, 2001). Duellman (2001) presented an extensive definition; however, it is unclear which of the character states could be taken as evidence of monophyly of the group. Hedges (1986) and Cocroft (1994) included two species, H. arenicolor and H. eximia, in their analyses. While in Hedges's (1986) results these two species form a monophyletic group, on the strict consensus of Cocroft's (1994) most parsimonious trees, both species are not monophyletic and form a basal polytomy with several other species of Hyla. da Silva's (1997) reanalysis of Cocroft's (1994) data set yielded on its strict consensus H. arenicolor and H. eximia as a monophyletic group, which appears to be supported by the allozyme data of Hedges (1986). Besides the seven species included by Duellman (2001) in the group, Eliosa León (2002) resurrected H. arboricola from the synonymy of H. eximia, adding an eigth species to the group. See the H. arborea group for additional comments. The H. eximia group is currently composed of eight species: Hyla arboricola, H. arenicolor, H. bocourti, H. euphorbiacea, H. eximia, H. plicata, H. walkeri, and H. wrightorum. In this analysis we include H. arenicolor, H. euphorbiacea, H. eximia, and H. walkeri.

Hyla godmani Group: The taxonomy, composition, and history of this group were reviewed by Duellman (1970, 2001). Duellman (2001) included in this group the only lowland pond-breeders from Middle America that do not have 30 chromosomes. He suggested as tentative evidence of monophyly the weakly ossified skulls and the presence of an axillary membrane. This group currently comprises four species: H. godmani, H. loquax, H. picta, and H. smithii. We include H. picta and H. smithii in our analysis.

Hyla miotympanum Group: The taxonomy, composition, and history of this group were thoroughly reviewed by Duellman (1970, 2001). Duellman (2001), using a hypothetical outgroup, suggested eight synapomorphies for the group16: abbreviated axillary membrane; indistinct fold on wrist; fingers less than one-half webbed; tarsal fold present through the entire length of the tarsus; cloacal opening directed posteroventrally; nuptial excrescences present; medial ramus of pterygoid short, not in contact with prootic; and larval oral disc in ventral position. It seems unlikely that any of these character transformations will hold as synapomorphies of the group in the context of a more inclusive analysis. The group currently contains 11 species: H. abdivita, H. arborescandens, H. bivocata, H. catracha, H. cyclada, H. hazelae, H. juanitae, H. melanomma, H. miotympanum, H. perkinsi, and H. pinorum. Our analysis includes H. arborescandens, H. cyclada, H. melanomma, H. miotympanum, and H. perkinsi.

Hyla mixomaculata Group: Duellman (1970) reviewed the group and listed several character states that define it. Some of these are probably synapomorphies, such as (known) larvae with an enlarged oral disc with 7/10 or 11 labial tooth rows (the largest number of posterior tooth rows known for a Middle American hylid group); the taxonomic distribution of other character states indicate that they are likely synapomorphies of a more inclusive clade, such as the large frontoparietal fontanelle and the absence (or reduction, unclear on fig. 211 of Duellman, 1970) of the quadratojugal. The group is currently composed of four species, H. mixe, H. mixomaculata, H. nubicola, and H. pellita. Only H. mixe was available for this analysis.

Hyla pictipes Group: The taxonomy, composition, and history of this group were reviewed by Duellman (1970, 2001). Duellman (2001) provided a phylogenetic analysis17 of the montane species of Hyla of lower Central America, where he included the H. pseudopuma and H. pictipes groups.

Duellman (2001) suggested six synapomorphies for the group: slender nasals in adults; tadpoles with stream-dwelling habits; oral disc ventral; complete marginal papillae; only one row of marginal papillae; and presence of submarginal papillae. Note that with the possible exception of slender nasals in adults, the other three character states as defined by Duellman (2001) are also present in several other groups of Middle American stream-breeding frogs (i.e., the Hyla bistincta group, H. mixomaculata group, H. sumichrasti group, and Plectrohyla).

The Hyla pictipes group includes 11 species: H. calypsa, H. debilis, H. insolita, H. lancasteri, H. picadoi, H. pictipes, H. rivularis, H. thorectes, H. tica, H. xanthosticta, and H. zeteki. Considering the uncertainties regarding the monophyly of this group, an appropriate taxon sampling should ideally include representatives of the four former species groups currently combined into the H. pictipes group. Unfortunately, the only representatives available are H. rivularis and Hyla sp. 5 (aff. H. thorectes), an undescribed species from Mexico similar to H. thorectes.

Hyla pseudopuma Group: The taxonomy, composition, and history of this group were reviewed by Duellman (1970, 2001), who could not provide a synapomorphy for it. In his phylogenetic analysis of the H. pseudopuma and H. pictipes groups, the H. pseudopuma group appears as a basal unresolved grade, although this is a consequence of constraining the nonmonophyly of the H. pseudopuma group by using H. angustilineata as the root. The H. pseudopuma group includes four species: H. angustilineata, H. graceae, H. infucata, and H. pseudopuma. In this study we include only H. pseudopuma.

Hyla sumichrasti Group: The taxonomy, composition, and history of this group were reviewed by Duellman (1970, 2001). Possible synapomorphies for the group (Duellman, 2001) are the presence of massive nasals, and tadpoles with immense oral discs, with 3/6 to 3/7 labial tooth rows instead of the 2/3 to 2/6 of the H. miotympanum group. The group currently includes four species: H. chimalapa, H. smaragdina, H. sumichrasti, and H. xera. In this study we include H. chimalapa and H. xera.

Hyla taeniopus Group: This group was reviewed by Duellman (1970, 2001), who defined it as having well-ossified quadratojugals in contact with maxillaries, and tadpoles that have ventral mouths with two or three anterior rows of teeth and three or four posterior rows. While the presence of enlarged testes is a possible synapomorphy of a subgroup composed of H. altipotens, H. trux, and H. taeniopus, there is no evidence for the monophyly of the entire group (Mendelson and Campbell, 1999; Duellman, 2001). Five species are currently included in this group: H. altipotens, H. chaneque, H. nephila, H. taeniopus, and H. trux. In this analysis, we include H. nephila and H. taeniopus.

Hyla tuberculosa Group: Affinities among species of fringe-limbed treefrogs were first suggested by Dunn (1943) and by Taylor (1948, 1952) based on their overall appearance. Firschein and Smith (1956) suggested that the presence of a “prepollex” (presumably referring to an enlarged prepollex) and similarity of external habitus, size, skin texture, and fringed limbs were indicative of a common origin. Duellman (1970, 2001) presented a formal definition of the group, asserting that these frogs be placed in the same group based on their large size, presence of dermal fringes on the limbs (although absent in H. dendrophasma), extensive webbing on hand and feet, and modified prepollices. (Importantly, note that apparently the modified prepollices involve three different morphologies: modification into a projecting spine or a spadelike blade or a clump of spines; Duellman, 1970.) Duellman (2001) added that there is no compelling evidence that the group is monophyletic, an opinion that we share. Furthermore, he tentatively suggested that this group could be related with the Gladiator Frogs rather than with the Middle American/ Holarctic clade. The Hyla tuberculosa group has been referred to as the H. miliaria group (Campbell et al., 2000; Duellman, 1970, 2001; Savage and Heyer, “1968” [1969]) and is composed of H. dendrophasma, H. echinata, H. fimbrimembra, H. miliaria, H. minera, H. phantasmagoria, H. salvaje, H. thysanota, H. tuberculosa, and H. valancifer. In this analysis, we include H. dendrophasma and H. miliaria.

Hyla versicolor Group: This group was first recognized by Blair (1958) on the basis of advertisement call structure. See Duellman (1970) and Anderson (1991) for a brief taxonomic history. Both Blair (1958) and Duellman (1970) included H. arenicolor in the group until Hedges (1986), Cocroft (1994), and da Silva (1997) showed on successive analyses that this species was more closely related to H. eximia (see the H. eximia group for further comments), always on the basis of the allozyme data collected by Hedges (1986).

Although placed originally in the Hyla cinerea group by Blair (1958), H. andersonii was transferred to the H. versicolor group by Wiley (1982), with this being supported by Hedges (1986), Cocroft (1994), and da Silva (1997) on the basis of the allozyme data collected by Hedges (1986). Similarly, H. femoralis was considered a member of the H. versicolor group until Hedges (1986) transferred it to the H. cinerea group, an assignment also corroborated by da Silva (1997). The group currently comprises four species: H. andersonii, Hyla avivoca, H. chrysoscelis, and H. versicolor. In this study we include all but H. chrysoscelis.

Pseudacris: The phylogenetic relationships of this genus were reviewed by Hedges (1986), Cocroft (1994), da Silva (1997), and Moriarty and Cannatella (2004). Hedges (1986) presented an electrophoretic analysis of 33 presumed genetic loci, where all species of Pseudacris were monophyletic, and which provided evidence to include the former Hyla cadaverina, H. crucifer, and H. regilla in Pseudacris. Cocroft (1994) performed a phylogenetic analysis of Pseudacris, including several Holarctic hylids as outgroups, with characters from various sources (osteology, vocalizations, karyotypes, allozymes, sperm morphology) and previous analyses (Hedges, 1986). The strict consensus of his most parsimonious trees shows P. crucifer as the sister taxon of the remaining species of Pseudacris; the two classically recognized species groups (the P. ornata and the P. nigrita groups) each being monophyletic; and P. ocularis being the sister taxon of the P. nigrita group. However, Hedges (1986) found no evidence supporting the inclusion of P. cadaverina and P. regilla in Pseudacris, and for this reason he treated them as Hyla. Furthermore, the relationships of the remaining, monophyletic species of Pseudacris with the other Holarctic hyline are unresolved. In the modified reanalysis performed by da Silva (1997), the strict consensus shows that the clade composed of H. regilla and H. cadaverina is sister to Pseudacris and is supported apparently by allozyme data. Because of this, these two species are considered again to be within Pseudacris (da Silva, 1997).

Moriarty and Cannatella (2004) presented a phylogenetic analysis of the mitochondrial ribosomal genes 12S, tRNA valine, and 16S that included all species of Pseudacris and three outgroups, Hyla andersonii, H. chrysoscelis, and H. eximia. The analysis identified four major clades: (1) the P. regilla clade (P. cadaverina and P. regilla; Moriarty and Cannatella referred to it as the West Coast clade); (2) the Pseudacris ornata clade (P. ornata, P. streckeri, and P. illinoiensis; Moriarty and Cannatella called it fat frogs clade); (3) the P. crucifer clade (P. crucifer and P. ocularis); and (4) the P. nigrita clade (including P. brimleyi, P. brachyphona, P. clarkii, P. feriarum, P. maculata, and P. triseriata; Moriarty and Cannatella called it Trilling Frogs clade). In our study we include Pseudacris cadaverina, P. crucifer, P. ocularis, P. regilla, and P. triseriata.

Pternohyla: This Mexican casque-headed frog genus was reviewed by Trueb (1969) and Duellman (1970, 2001). In Duellman's (2001) phylogenetic analysis of Pternohyla, Smilisca, and Triprion, the monophyly of Pternohyla is supported by four synapomorphies: small discs on fingers; supernumerary tubercles diffuse or absent; large inner metatarsal tubercle18; and large marginal papillae in the larval oral disc. Unfortunately, small discs on fingers are a synapomorphy only under an accelerated optimization (ACCTRAN), and therefore they have no evidential value for the clade.

In Duellman's (2001) analysis, Triprion plus Pternohyla forms a monophyletic group nested within Smilisca. The synapomorphies supporting Triprion plus Pternohyla are19: nasals with broad medial contact; median ramus of pterygoid not in contact with prootic; maxilla moderately expanded laterally; cranial-integumentary co-ossification present; webbing between fingers absent; and inner metatarsal tubercle small. Pternohyla is composed of two similar species, P. dentata, and P. fodiens. In this analysis we include P. fodiens.

Plectrohyla: This genus was reviewed by Duellman (2001) and discussed by McCranie and Wilson (2002). Its phylogenetic relationships were addressed by Duellman and Campbell (1992), Wilson et al. (1994a), and Duellman (2001). The monophyly of Plectrohyla does not appear to be controversial. Duellman and Campbell (1992) listed six synapomorphies: bifurcated alary process of premaxilla; sphenethmoid ossified anteriorly, incorporating the septum nasi and projecting forward to the leading margins of the nasals; frontoparietals abutting broadly anteriorly and posteriorly, exposing a small area of the frontoparietal fontanelle; hypertrophied forearms; and absence of lateral folds in the oral disc. Wilson et al. (1994a) added “prepollex enlarged, elongated, ossified, flat, terminally blunt.” Duellman (2001) interpreted that this definition corresponded to more than one character, and so he divided it into two characters, the derived state of the first one being “enlarged and ossified prepollex in both sexes”, and the derived state of the second one being “enlarged and truncate prepollex.” See comments under the Hyla bistincta group.

Plectrohyla currently contains 18 species: P. acanthodes, P. avia, P. chrysopleura, P. dasypus, P. exquisitia, P. glandulosa, P. guatemalensis, P. hartwegi, P. ixil, P. lacertosa, P. matudai, P. pokomchi, P. psiloderma, P. pycnochila, P. quecchi, P. sagorum, P. tecunumani, and P. teuchestes. In this analysis we include P. guatemalensis, P. glandulosa, and P. matudai.

Ptychohyla: This group was reviewed by Duellman (2001). Campbell and Smith (1992) suggested three synapomorphies for Ptychohyla: the presence of two rows of marginal papillae, an increased number of tooth rows in larvae (from 3/5 to 6/9), and a strongly developed lingual flange of the pars palatina of the premaxilla. Duellman (2001) also suggested as synapomorphies the presence of ventrolateral glands in breeding males, and the coalescence of tubercles to form a distinct ridge on the ventrolateral edge of the forearm. The increase in the number of tooth rows could actually be a synapomorphy not of Ptychohyla but for a more inclusive clade containing Ptychohyla plus other species of stream-breeding hylids that also have a tooth row formula larger than 2/3. Similarly, ventrolateral glands are present also in some species of Duellmanohyla (Campbell and Smith, 1992; Duellman, 2001).

For an unstated reason, Savage (2002a) excluded Ptychohyla legleri and P. salvadorensis from Ptychohyla, placing them back in Hyla. These two species were originally in Hyla (former H. salvadorensis group; see Duellman, 1970) until Campbell and Smith (1992) transferred them to Ptychohyla. Because we are not aware of any evidence supporting Savage's action, we consider them to be members of Ptychohyla.

Ptychohyla is composed of 12 species: P. acrochorda, P. erythromma, P. euthysanota, P. hypomykter, P. legleri, P. leonhardschultzei, P. macrotympanum, P. panchoi, P. salvadorensis, P. sanctaecrucis, P. spinipollex, and P. zophodes. In this analysis we include P. euthysanota, P. hypomykter, P. leonhardschultzei, P. spinipollex, P. zophodes, and Ptychohyla sp., an undescribed species from Oaxaca, Mexico.

Smilisca: This genus was reviewed by Duellman and Trueb (1966) and Duellman (1970, 2001). Duellman (2001) could advance no evidence for the monophyly of Smilisca. He presented a phylogenetic analysis rooted with a hypothetical ancestor, whose strict consensus showed Pternohyla plus Triprion nested within Smilisca, being more closely related to S. baudinii and S. phaeota. The synapomorphies supporting Pternohyla + Triprion + “Smilisca” are the presence of lateral flanges on the frontoparietals, and the unexposed frontoparietal fontanelle. The species of Smilisca have been divided (Duellman and Trueb, 1966) into the S. sordida group (S. puma and S. sordida), the S. baudinii group (S. baudinii, S. cyanosticta, and S. phaeota), and S. sila, a form considered intermediate between these two groups. In Duellman's (2001) phylogenetic analysis, S. sila plus the S. sordida group is monophyletic, with its synapomorphy being the short maxillary process of the nasal. Smilisca contains six species: S. baudinii, S. cyanosticta, S. phaeota, S. puma, S. sila, and S. sordida. In this analysis we include the three species in the S. baudinii group, S. baudinii, S. cyanosticta, S. phaeota, and one species of the S. sordida group, S. puma.

Triprion: This genus was reviewed by Trueb (1969) and Duellman (1970, 2001). In Duellman's (2001) phylogenetic analysis of Pternohyla, Smilisca, and Triprion, the monophyly of Triprion is supported by three synapomorphies20: maxilla greatly expanded laterally, prenasal bone present, and presence of parasphenoid odontoids. See comments for Smilisca and Pternohyla. Triprion is composed of two species, T. petasatus and T. spatulatus. In the analysis we include T. petasatus.

Casque-Headed Frogs and Related Genera

Duellman's (2001) suggestion of Middle American/Holarctic frogs being monophyletic clearly separates the Middle American casque-headed frogs (Triprion, Pternohyla) from the South American and West Indian casque-headed frogs. This is not surprising considering that traditionally the group known as the casque-headed frogs was considered to be nonmonophyletic (Trueb, 1970a, 1970b). However, the position of the South American and West Indian casque-headed frogs remains controversial, and no author has presented evidence indicating whether they form a monophyletic group.

Aparasphenodon: This genus of casque-headed frogs was reviewed and characterized by Trueb (1970a) and Pombal (1993). The presence of a prenasal bone is a likely synapomorphy of Aparasphenodon (with a known homoplastic occurrence in Triprion, as reported by Trueb, 1970a). This genus currently comprises three species, A. bokermanni, A. brunoi, and A. venezolanus. We include A. brunoi in the analysis.

Argenteohyla: This monotypic genus was described and reviewed by Trueb (1970b), who segregated it from Trachycephalus, where it had been placed by Klappenbach (1961). Motives for this segregation were the absence in Argenteohyla of several character states of Trachycephalus as redefined by Trueb (1970a), such as the dermal sphenethmoid, the poorer development of ossification and cranial sculpturing, and vocal sacs that when inflated protrude posteroventrally to the angles of the jaw. Possible autapomorphies of this taxon include the fusion of the zygomatic ramus of the squamosal with the pars facialis of the maxilla. The genus comprises a single species, A. siemersi, for which a northern subspecies, A. s. pederseni, was described by Williams and Bosso (1994). In this analysis we included a specimen that corresponds to the northern form.

Corythomantis: This monotypic genus was reviewed by Trueb (1970a). Autapomorphies of this genus include the absence of palatines, and nasals that conceal the allary processes of premaxillaries (Trueb, 1970a). We include the single species Corythomantis greeningi in this analysis.

Osteocephalus: This genus was diagnosed by Goin (1961) and Trueb (1970a) and studied in detail by Trueb and Duellman (1971). These authors recognized five species: Osteocephalus verruciger, O. taurinus, O. buckleyi, O. leprieurii, and O. pearsoni. In the last 20 years, several new species were described, adding to a total of 18 currently recognized species (see Jungfer and Hödl, 2002; Lynch, 2002). Trueb and Duellman (1971) employed 20 character states to characterize Osteocephalus. Jungfer and Hödl (2002) modified some of these characters to take into account subsequently discovered species. As stated by Ron and Pramuk (1999), referring to the diagnostic states employed earlier by Trueb and Duellman (1971), it is unclear which, if any, of the character states are synapomorphic for the genus. Trueb (1970a) and Trueb and Duellman (1971) suggested, based on the presence of paired lateral vocal sacs in the five species then recognized, that Osteocephalus was related to a group composed of Argenteohyla, Trachycephalus, and Phrynohyas.

Martins and Cardoso (1987) described Ostecephalus subtilis that, unlike the other species known at that time, is characterized by a single, subgular vocal sac that expands laterally; a similar morphology was described by Smith and Noonan (2001) in O. exophthalmus. Jungfer and Schiesari (1995) described O. oophagus, a species with a single, median vocal sac, a reproductive mode involving oviposition in bromeliads, and phytotelmous oophagous larvae. Jungfer et al. (2000), Jungfer and Lehr (2001), and Lynch (2002) described four species, O. deridens, O. fuscifacies, O. leoniae, and O. heyeri, which also have a single, median vocal sac. According to Lynch (2002), O. cabrerai also shares this characteristic. Reproductive modes are unknown for O. cabrerai, O. exophthalmus, O. heyeri, O. leoniae, and O. subtilis; spawning in bromeliads is suspected for O. deridens and O. fuscifacies (Jungfer et al., 2000). Note that Lynch (2002) doubted a possible relationship between O. heyeri and what he called the “presumed clade of oophagous species” (where he included O. deridens, O. fuscifacies, and O. oophagus), suggesting instead that it could be related to what he called O. rodriguezi (at that time already transferred to the new genus Tepuihyla by Ayarzagüena et al., “1992” [1993b]). While the species known or suspected to spawn in bromeliads could be monophyletic, we are not aware of any synapomorphy supporting the monophyly of all remaining species of Osteocephalus.

The species currently included in Osteocephalus are O. buckleyi, O. cabrerai, O. deridens, O. elkejungingerae, O. exophthalmus, O. fuscifacies, O. heyeri, O. langsdorffii, O. leoniae, O. leprieurii, O. mutabor, O. oophagus, O. pearsoni, O. planiceps, O. subtilis, O. taurinus, O. verruciger, and O. yasuni. Considering the uncertainties regarding Osteocephalus, we attempted to include representatives of the morphological and reproductive diversity within the genus: O. cabrerai, O. langsdorffii, O. leprieurii, O. oophagus, and O. taurinus.

Osteopilus: The genus Osteopilus was resurrected by Trueb and Tyler (1974) for three apparently related species that were often referred to collectively as the Hyla septentrionalis group (see Dunn, 1926; Trueb, 1970a). Trueb and Tyler (1974) provided a diagnostic definition of the genus; a possible synapomorphy is the differentiation of the m. intermandibularis to form supplementary apical elements. Trueb and Tyler (1974) also maintained, due to the impressive morphological divergence, that Osteopilus, the other Antillean groups then considered to be in Hyla (H. heilprini, H. marianae, H. pulchrilineata, H. vasta, H. wilderi), and the new genus they erected, Calyptahyla, represented several independent invasions from the mainland.

Maxson (1992) and Hass et al. (2001), using albumin immunological distances, suggested that Osteopilus is paraphyletic with respect to most other West Indian hylids (with the exception of Hyla heilprini, a Gladiator Frog). Hedges (1996) mentioned that unpublished DNA sequence data confirmed these findings. Anderson (1996) presented a karyological study of the three species of Osteopilus, indicating that her data were compatible with a monophyletic Osteopilus. Based on the comments by Hedges (1996), and immunological results of Hass et al. (2001), Franz (2003), Powell and Henderson (2003a, 2003b), and Stewart (2003) transferred Calyptahyla crucialis, H. marianae, H. pulchrilineata, H. vasta, and H. wilderi to Osteopilus that now includes eight species. Osteopilus is grouped together only on the basis of the immunological distance results, as no discrete character data set supporting its monophyly has yet been published. The species of Osteopilus available for our study were O. crucialis, O. dominicensis, O. septentrionalis, and O. vastus.

Phrynohyas: This genus was reviewed by Duellman (1971b). Although very distinctive externally, the only seeming synapomorphy in the diagnostic definition of Phrynohyas provided by Duellman (1971b) is the extensively developed parotoid glands in the occipital and scapular regions. Likely related to this character state, the viscous, milky secretions of the species of this genus could also be considered synapomorphic. Lescure and Marty (2000) transferred Hyla hadroceps to this genus; this was confirmed in a phylogenetic analysis using the mitochondrial ribosomal gene 12S by Guillaume et al. (2001). Pombal et al. (2003) described P. lepida. See Osteocephalus for further comments. Phrynohyas currently contains seven species: P. coriacea, P. hadroceps, P. imitatrix, P. lepida, P. mesophaea, P. resinifictrix, and P. venulosa. In our analysis we include P. hadroceps, P. mesophaea, P. resinifictrix, and P. venulosa.

Tepuihyla: This genus was defined by Ayarzagüena et al. (“1992” [1993b]) for five species of Osteocephalus previously considered to constitute the O. rodriguezi species group (Duellman and Hoogmoed, 1992; Ayarzagüena et al., “1992” [1993a]). Ayarzagüena et al. (“1992” [1993b]) differentiated Tepuihyla from Osteocephalus using the following character states present in Tepuihyla: subgular vocal sac, absence or extreme reduction of hand webbing, more reduced toe webbing, smaller size, absence of cranial co-ossification, large frontoparietal fontanelle, shorter nasals, and shorter frontoparietals. It is unclear which, if any, of these character states are apparent synapomorphies of Tepuihyla. There are eight species currently included in this genus: T. aecii, T. celsae, T. edelcae, T. galani, T. luteolabris, T. rimarum, T. talbergae, and T. rodriguezi. In this analysis we include only T. edelcae.

Trachycephalus: The relationships of this casque-headed taxon were discussed by Trueb (1970a) and Trueb and Duellman (1971). They diagnosed Trachycephalus from Argenteohyla, Osteocephalus, and Phrynohyas for having heavily casqued and co-ossified skulls, a medial ramus of pterygoid that does not articulate with the prootic, and a parasphenoid having odontoids. A likely synapomorphy of Trachycephalus is the presence of exostosis on the alary process of the premaxillae (Trueb, 1970a). Trachycephalus contains three species: T. atlas, T. jordani, and T. nigromaculatus. In this analysis we include T. jordani and T. nigromaculatus.

Species and Species Groups of Hyla Not Associated with Any Major Clade

Hyla aromatica Group: This group was proposed by Ayarzagüena and Señaris (“1993” [1994]) for two species from the Venezuelan Tepuis, H. aromatica and H. inparquesi, which they could not associate with any of the species groups known from the Guayanas. Ayarzagüena and Señaris (“1993” [1994]) noticed that the H. aromatica group shares some characters with the H. larinopygion group; however, they preferred to retain it as a separate group. They justified this decision based on the smaller size of members of the H. aromatica group, different coloration pattern, supraorbital cartilaginous process, vomerine odontophores smoothly S-shaped and with more odontophores, small nasals, and large prepollex. They included as well other character states, that actually, like some of these just mentioned, are either shared with several neotropical groups (supraorbital cartilaginous process; Faivovich, personal obs.), or some species of the H. larinopygion group (vomerine odontophores smoothly S-shaped; Duellman and Hillis, 1990: 5), or with the H. armata group (labial tooth row formula; see Cadle and Altig, 1991), or they support the monophyly of the H. aromatica group (adults with strong odor). Considering the lack of evidence of monophyly for the H. larinopygion group, Ayarzagüena and Señaris (“1993” [1994]) cannot be questioned for recognizing a separate species group.

Ongoing research by Faivovich and McDiarmid suggests that Hyla loveridgei should also be considered part of the H. aromatica group. For this analysis, we include H. inparquesi.

Hyla uruguaya Group: This group has never been mentioned as such in the literature. However, clear similarities had been shown by Langone (1990) between H. uruguaya and H. pinima (these species being almost undistinguishable). Possible synapomorphies of the H. uruguaya group are the bicolored iris (also shared with Aplastodiscus; see Garcia et al., 2001a), the presence in tadpoles of two small, keratinized plates below the lower jaw sheath, and a reduction in the size of the marginal papillae of the posterior margin of the oral disc relative to the other papillae (Kolenc et al., “2003” [2004]). From this apparent clade we include H. uruguaya in our analysis.

Hyla chlorostea: Duellman at al. (1997) proposed the recognition of a species group to include the enigmatic Hyla chlorostea, a species known only from its holotype (a subadult male), which could not be associated with any known group of Hyla after its description (Reynolds and Foster, 1992). Unfortunately, we were unable to include this taxon in our analysis.

Hyla vigilans: Different perspectives concerning this enigmatic species were summarized by Suarez-Mayorga and Lynch (2001a). These authors rejected the possibility of a relationship with Scinax (as suggested by La Marca in Frost, 1985). Instead, they asserted that they suspected a possible relationship with Sphaenorhynchus or with H. picta (from the H. godmani species group) based on “oral disc and mouth position” of the tadpoles. We could not obtain samples of this species for our analysis.

Hyla warreni: This species, known only from two adult females, was described by Duellman and Hoogmoed (1992), who did not associate it with any other species or species group. Unfortunately, we could not obtain samples of this species for our analysis.

Other Genera

Aplastodiscus: The taxonomy and history of this genus was recently reviewed thoroughly by Garcia et al. (2001a). According to these authors the monophyly of the genus is supported by four putative synapomorphies: (1) the absence of webbing between toes I and II and basal webbing between the other toes; (2) bicolored iris; (3) females with unpigmented eggs; and (4) great development of internal metacarpal and metatarsal tubercles. Based on overall morphological and advertisement call similarities B. Lutz (1950) suggested a close relationship of this genus with Hyla albosignata. Garcia et al. (2001a) suggest that Aplastodiscus could be related with the H. albofrenata and H. albosignata complexes of the H. albomarginata group, as defined by Cruz and Peixoto (1984), based on the presence of enlarged internal metacarpal and metatarsal tubercles, and unpigmented eggs. Haddad et al. (2005) described the reproductive mode of A. perviridis and noticed that it was the same as that described by Haddad and Sawaya (2000) and Hartmann et al. (2004) in species included in H. albofrenata and H. albosignata complexes. Based on this, Haddad et al. (2005) suggested a possible relationship between these two species complexes and Aplastodiscus. Aplastodiscus is composed of two species, Aplastodiscus cochranae and A. perviridis; we include both in our analysis.

Nyctimantis: This monotypic Neotropical genus was considered a member of the Hemiphractinae by Duellman (1970) and Trueb (1974). Duellman and Trueb (1976) reviewed the taxon and placed it in Hylinae. Duellman and Trueb (1976) considered Nyctimantis to be related with Anotheca spinosa because both share the medial ramus of the pterygoid that is juxtaposed squarely against the anterolateral corner of the ventral ledge of the otic capsule. Also, frogs of both genera are known (Anotheca; Taylor, 1954; Jungfer, 1996) or suspected (Nyctimantis; Duellman and Trueb, 1976) to deposit their eggs in water-filled tree cavities. However, Duellman (2001) latter placed Anotheca in the Middle American/Holarctic clade, implicitly suggesting no relationship with Nyctimantis. Considering the uncertainty of the position of Nyctimantis within hylines, at this stage it is difficult to interpret which character states are autapomorphic. We include the single species Nyctimantis rugiceps in this analysis.

Phyllodytes: The history of this genus was reviewed by Bokermann (1966b). Possible synapomorphies of the taxon are the presence of odontoids on the mandible and on the cultriform process of the parasphenoid (Peters, “1872” [1873]), something unique within the Hylinae. Peixoto and Cruz (1988) noticed that among the six species recognized at that time, four species (P. acuminatus, P. brevirostris, P. luteolus, and P. tuberculosus) share the presence of series of enlarged tubercles on the venter and an enlarged tubercle on each side at the origin of the thigh (Bokermann, 1966b: fig. 6). The other two species, P. auratus and P. kautskyi, have uniform granulation on the venter and lack enlarged tubercles on the thighs, as also seems to be the case in P. melanomystax, a species described later (see Caramaschi et al., 1992). Peixoto et al. (2003) described two additional species, P. edelmoi and P. gyrinaethes; both have a tubercle on each side at the origin of the thigh. Phyllodytes edelmoi has a series of indistinct tubercles on the venter; in P. gyrinaethes they do not form series. Caramaschi and Peixoto (2004) added P. punctatus, which has two medial, poorly distinct rows of tubercles. Caramaschi et al. (2004a) resurrected P. wuchereri. Peixoto et al. (2003) suggested three different species groups based on color pattern, and Caramaschi et al. (2004) further expanded the definitions. The P. luteolus group is characterized by a plain pattern with a variably defined dorsolateral dark brown to black line on canthus rostralis and/or behind the corner of eye. This group includes P. acuminatus, P. brevirostris, P. edelmoi, P. kautskyi, P. luteolus, and P. melanomystax. The P. tuberculosus group has a pale brown dorsum with scattered dark brown dots and includes P. punctatus and P. tuberculosus. The P. auratus group has a dorsal pattern of two dorsolateral, longitudinal white or yellowish stripes, with each stripe being bordered by a dark brown or black line from posterior corner of eye to groin. This group includes P. auratus and P. wuchereri. Finally, P. gyrinaethes is placed in its own group for having red color on hidden surfaces of thighs and a highly modified tadpole. It is unclear if any of these groups is monophyletic. Tissues were available for P. luteolus and an unidentified species, Phyllodytes sp., from Bahia, Brazil.

Lysapsus and Pseudis: The monophyly of the former subfamily Pseudinae has not been historically controversial; it is supported by the presence of a long, ossified intercalary element between the ultimate and penultimate phalanges. Haas (2003) added several synapomorphies from larval morphology, based on the study of larvae of two species of Pseudis. The limits and definitions of Pseudis and Lysapsus were reviewed by Savage and Carvalho (1953) and by Klappenbach (1985). From their observations it is unclear which character states support the monophyly of either genus. Savage and Carvalho (1953: 199) implicitly proposed the paraphyly of Pseudis, when they suggested that Lysapsus “seems to have arisen from Pseudis.” Garda et al. (2004) recently distinguished both genera on the basis of sperm morphology. In Lysapsus laevis (the only species of Lysapsus available to them) the subacrosomal cone is nearly absent, but it is clearly present in the four species of Pseudis they studied. Regardless, the monophyly of either genus has not been satisfactorily documented.

Morphological diversity within Pseudis includes large species, several of which were included in the past in the synonymy of P. paradoxa and were recently resurrected (Caramaschi and Cruz, 1998), and smaller species with a double vocal sac, P. cardosoi and P. minuta (Klappenbach, 1985; Kwet, 2000). Lysapsus includes three species, L. caraya, L. laevis and L. limellum; we include in our analysis the last two. Pseudis is composed of six species: P. bolbodactyla, P. cardosoi, P. fusca, P. minuta, P. paradoxa, and P. tocantins, of which we include in our analysis P. minuta and P. paradoxa.

Scarthyla: Duellman and de Sá (1988) and Duellman and Wiens (1992) suggested that this monotypic genus was sister to Scinax, but more recently, Darst and Cannatella (2003) presented evidence supporting a sister group relationship between Scarthyla and “pseudids”. The single species, Scarthyla goinorum, is included in our analysis.

Scinax: With roughly 86 recognized species, Scinax is the second largest genus within Hylinae. This genus includes the species formerly placed in the Hyla catharinae and H. rubra groups; a taxonomic history was presented by Faivovich (2002). The relationships among the species of Scinax were recently addressed by Faivovich (2002), who performed a phylogenetic analysis using 38 species representing the five species groups then recognized. Although he employed eight outgroups, the analysis is not a strong test of the monophyly of Scinax nor of the relationships of Scinax with other hylines. Duellman and Wiens (1992) suggested that Scinax is the sister group of Scarthyla and that this clade is sister to Sphaenorhynchus. Faivovich (2002) did not test this assertion because his selection of outgroups was heavily influenced by da Silva's results (1998), which did not suggest a close relationship between Scinax and these two genera. Taxon choice in the present study will test more appropriately the hypothesis of (Scinax + Scarthyla) + Sphaenorhynchus.

Faivovich's (2002) results suggested that Scinax contains two major clades: (1) a S. ruber clade composed of species that had been previously grouped into the S. rostratus, S. ruber, and S. staufferi groups; and (2) a S. catharinae clade composed of the species that were included in the S. catharinae and S. perpusillus groups. Faivovich (2002) continued recognition of these two species groups within the S. catharinae clade, as well as the S. rostratus group within the S. ruber clade, as the individual monophyly of the S. catharinae and S. rostratus groups were corroborated by his analysis. The S. perpusillus group is recognized because its monophyly could not be tested, and it still awaits a rigorous test. All species previously included in the nonmonophyletic groups of S. ruber and S. staufferi are included in the larger S. ruber clade, without being assigned to any group. For a list of the species currently included in Scinax, see page 95.

In anticipation of a forthcoming study of the phylogeny of Scinax by Faivovich and associates, we include only S. berthae and S. catharinae as exemplars of the S. catharinae clade, and S. acuminatus, S. boulengeri, S. elaeochrous, S. staufferi, S. fuscovarius, S. ruber, S. squalirostris, and S. nasicus as exemplars of the S. ruber clade.

Sphaenorhynchus: More has been written about nomenclatural confusion surrounding Sphaenorhynchus than about its systematics (see Frost, 2004). This genus has been reviewed by Caramaschi (1989). Duellman and Wiens (1992) proposed the following synapomorphies for Sphaenorhynchus: posterior ramus of pterygoid absent; zygomatic ramus of squamosal absent or reduced to a small knob; pars facialis of maxilla and alary process of premaxilla reduced; postorbital process of maxilla reduced, not in contact with quadratojugal; neopalatine reduced to a sliver or absent; pars externa plectri entering tympanic ring posteriorly (rather than dorsally); pars externa plectri round; hyale curved medially; coracoids and clavicle elongated; transverse process of presacral vertebra IV elongate, oriented posteriorly; and prepollex ossified, bladelike. The genus is composed of 11 species: S. bromelicola, S. carneus, S. dorisae, S. lacteus, S. orophilus, S. palustris, S. pauloalvini, S. planicola, S. prasinus, S. platycephalus, and S. surdus. In our analysis we include S. dorisae and S. lacteus.

Xenohyla: This genus was named by Izecksohn (1996) for the bizarre frog Hyla truncata, which had previously been suggested to be related to Sphaenorhynchus by Izecksohn (1959, 1996) and Lutz (1973). According to Izecksohn (1996), Xenohyla shares with Sphaenorhynchus the reduced number of maxillary teeth, a relatively short urostyle, and the development of the transverse processes of presacral vertebra IV; furthermore, Xenohyla shares with Sphaenorhynchus the quadratojugal not in contact with the maxilla. Izecksohn (1996) suggested also a close relationship with Scinax based on the presence in Xenohyla of a coracoid ridge and an internal, subgular vocal sac. While the coracoid ridge is present in Scinax, it is also present in several other hylines (e.g., see Faivovich, 2002). The internal, subgular vocal sac is not a synapomorphy of all Scinax, but only of the S. catharinae clade. Caramaschi (1998) added X. eugenioi, a second species for the genus. We include X. truncata in our study.

Gene Selection

Because this study involves the simultaneous analysis of taxa of disparate levels of divergence, we assembled a large data set, including four mitochondrial and five nuclear genes, spanning a broad range of variation, from the fast-evolving cytochrome b (Graybeal, 1993) to the much conserved nuclear genes such as 28S (Hillis and Dixon, 1991).

Ribosomal mitochondrial genes and cytochrome b have been employed recently in several phylogenetic studies of various anuran groups at various levels of divergence (Read et al., 2001; Vences and Glaw, 2001; Cunningham, 2002; Salducci et al., 2002). Nuclear genes have been poorly explored for their use in anuran phylogenetics. The 28S ribosomal nuclear gene has been used in amphibians by Hillis et al. (1993). The protein-coding genes rhodopsin, tyrosinase, RAG-1, and RAG-2 were used to study problems at different levels by Bossuyt and Milinkovitch (2000), Biju and Bossuyt (2003), and Hoegg et al. (2004). In this study we include 12S, tRNA valine, 16S, and fragments of cytochrome b, rhodopsin, tyrosinase, 28S, RAG-1, and seventh in absentia. The last gene is used here for the first time in amphibians.

DNA Isolation and Sequencing

Whole cellular DNA was extracted from frozen and ethanol-preserved tissues (usually liver or muscle) using either phenol-chloroform extraction methods or the DNeasy (QIAGEN) isolation kit. See table 2 for a list and sources of the primers employed.

Amplification was carried out in a 25-μl-volume reaction using either puRe Taq Ready-To-Go PCR beads (Amersham Biosciences, Piscataway, NJ) or Invitrogene PCR SuperMix. For all the amplifications, the PCR program included an initial denaturing step of 30 seconds at 94°C, followed by 35 or 38 cycles of amplification (94°C for 30 seconds, 48–60°C for 60 seconds, 72°C for 60 seconds), with a final extension step at 72°C for 6 min.

Polymerase chain reaction (PCR)-amplified products were cleaned either with a QIAquick PCR purification kit (QIAGEN, Valencia, CA) or with ARRAY-IT (TeleChem International, Sunnyvale, CA) and labeled with fluorescent-dye labels terminators (ABI Prism Big Dye Terminators v. 3.0 cycle sequencing kits; Applied Biosystems, Foster City, CA). Depending on whether the cleaned product was purified with QIAquick or Array-It, the sequencing reaction was carried out in either 10 μl or 8 μl volume reaction following standard protocols. The labeled PCR products were isopropanol-precipitated following the manufacturer's protocol. The products were sequenced either with an ABI 3700 or with an ABI Prism 377 sequencer. Most samples were sequenced in both directions.

Chromatograms obtained from the automated sequencer were read and contigs made using the sequence editing software Sequencher 3.0. (Gene Codes, Ann Arbor, MI). Complete sequences were edited with BioEdit (Hall, 1999).

Morphology

Because the present study is mostly based on molecular data, the failure to include a thorough morphological data set doubtless is its weakest point. As trained morphologists, most of the authors of this paper think that a phylogenetic hypothesis that explains all the available data is the best hypothesis that we can aspire to, and that no class of data is better than any other. Apart from da Silva's unpublished dissertation, which is commented upon below, published comparative studies involving a diversity of hylid exemplars are rare. Major exceptions are the thorough osteological studies by Trueb (1970a) and those on hand muscles of Pelodryadinae (Burton, 1996), distal extensor muscles of anurans (Burton, 1998a), and foot muscles of Hylidae (Burton, 2004). Explicit character descriptions in the context of phylogenetic comparisons include those by Duellman and Trueb (1983), Campbell and Smith (1992), Duellman and Campbell (1992), Duellman and Wiens (1992), Fabrezi and Lavilla (1992), Kaplan (1994, 1999), Cocroft (1994), Burton (1996, 1998a, 2004), Haas (1996, 2003), da Silva (1997), Duellman et al. (1997), Kaplan and Ruiz-Carranza (1997), Mendelson et al. (2000), Sheil et al. (2001), Faivovich (2002), and Alcalde and Rosset (“2003” [2004]). Most of these studies were targeted in general to very specific apparent clades or to very large clades using very few terminals, which leaves particular sets of characters known for very few terminals. Unfortunately, for the inclusion of the characters employed in these studies to be informative, detailed anatomic work would be required on a very large number of terminals (besides the potentially serious need to redefine several characters), a task that we find impossible to pursue at this time. Much to our regret, we find that there are almost no published studies from which we could derive character scorings to enrich our data set without extensive work. The only data set that we thought could be included, due to its relatively dense taxon sampling, is the one resulting from the collection of observations presented by Burton (2004). Although its sampling of nonhylid taxa that match our sampled taxa is particularly sparse, we consider Burton's study to be an important addition to this analysis. Characters are listed and discussed in appendix 3.

da Silva's (1998) Dissertation

da Silva (1998) presented his Ph.D. dissertation on phylogeny of hylids with emphasis on Hylinae. Although da Silva's dissertation has not been published, some of its results and conclusions were described and commented in detail by Duellman (2001). Because the present paper deals specifically with the phylogeny of Hylinae, we cannot avoid a few comments dealing with da Silva's work. Considering the mostly coincident scope of both da Silva's dissertation and this paper, it is evident that a thorough discussion and comparison of his results with ours would almost amount to the publication of his chapter on Hylinae relationships. This is a situation with which we feel most uncomfortable, because we think that this is a responsibility that rests on Helio R. da Silva.

From a purely practical perspective, at this point the integration of da Silva's data set with ours is impracticable for two reasons: (1) The data matrix as printed in the dissertation distributed by the University of Michigan is incomplete, as it lacks the scorings for 10 characters (chars. 110–120) for all taxa. This is also the situation with the thesis that is deposited at the Department of Herpetology library of the University of Kansas, Natural History Museum (Faivovich, personal obs.). (2) A few scorings for groups that we are familiar with are not coincident with our observations on the same species, something suggestive either of polymorphism in those characters or mistaken scorings.21 If this were the case, it would not be surprising, as scoring mistakes are to be expected in such an impressive data set. The problem with them is that once detected, they have to be corrected and the analysis has to be redone. It is evident that a revision of the data set is necessary before any integration can take place.

Pelodryadinae

As stated previously, our analysis does not include enough of a comprehensive taxon sampling of Pelodryadinae to address its internal relationships in a meaningful way. Nonetheless, our results corroborate the long-held idea (see previous discussions) that Cyclorana and Nyctimystes are nested within Litoria. For the reasons detailed earlier, our analysis is not an overly strong test of the positions of the former two genera within Litoria. Nevertheless, the single exemplar of Cyclorana is the sister taxon of L. aurea, an exemplar of the L. aurea group with which Cyclorana is supposed to be related based on various sources of evidence (King et al., 1979; Tyler, 1979; Tyler et al., 1981). Nyctimystes is the sister taxon of L. infrafrenata, one of the groups of Litoria that Tyler and Davies (1979) considered as possibly related to Nyctimystes based on morphological similarities of its skull with that of N. zweifeli. The other groups they considered are mostly the montane species of Litoria; from these we included a single exemplar, L. arfakiana, that is quite distant from Nyctimystes, being the sister taxon of L. meiriana (with which, incidentally, it also shares the presence of a flange in the medial surface of metacarpal III; Tyler and Davies, 1978b). In our analysis, the fibrous origin of the m. extensor brevis superficialis digiti III on the distal end of the fibulare is a synapomorphy of Pelodryadinae; we are skeptical, however, that this optimization will hold with better sampled outgroups for muscular characters, because Burton (2004) found the same character state in several leptodactylids, none of which is included in our outgroup sample.

Phyllomedusinae

Several authors (Funkhouser, 1957; Duellman, 1970; Donnelly et al., 1987; Hoogmoed and Cadle, 1991) noticed the distinctiveness of Agalychnis calcarifer and its presumed sister taxon, A. craspedopus, from the other species of Agalychnis. Corroborating the results of Duellman (2001), we found no evidence for the monophyly of Agalychnis. Our results indicate that A. calcarifer is the sister group of the remaining Phyllomedusinae, and it has no close relationship with the other exemplars of Agalychnis.

Phyllomedusa lemur, the only exemplar of the P. buckleyi group available for this analysis, is recovered, although with low Bremer support (3), as the sister group of Hylomantis, and it is only distantly related with the other exemplars of Phyllomedusa. This situation corroborates previous suggestions (Funkhouser, 1957; Cannatella, 1980; Jungfer and Weygoldt, 1994) that the P. buckleyi group should not be included in Phyllomedusa. Cruz (“1988” [1989]) suggested, on the basis of iris coloration, skin texture, poor development of webbing, and slender body, that Hylomantis is related to two species of the P. bukleyi group, P. buckleyi and P. psilopygion. On the basis of the same character states, Cruz (1990) associated the P. buckleyi group with both Hylomantis and Phasmahyla. Our results support these ideas only in part, because while our only exemplars of Hylomantis and the P. buckleyi group are each monophyletic, Phasmahyla is more closely related to Phyllomedusa (excluding the P. buckleyi group).

We had no clear idea regarding the position of Hylomantis and Phasmahyla. Morphologically, the evidence is conflicting in that each one shares at least one different possible synapomorphy with the restricted Phyllomedusa (Phyllomedusa excluding the P. buckleyi group). Phasmahyla has the same type of nest where the eggs are wrapped in a leaf; nests are unknown in Hylomantis, but species of this genus share with Phyllomedusa (excluding the P. buckleyi group) the presence of the slip of the m. depressor mandibulae that originates from the dorsal fascia at the level of the m. dorsalis scapulae (Cruz, 1990; Duellman et al., 1988b), a character state that is absent in all other Phyllomedusinae. Our analysis recovers Phasmahyla as the sister group of the restricted Phyllomedusa, suggesting that the eggs wrapped in a leaf are a synapomorphy of this clade.

South American Clade I

This clade is composed of all Gladiator Frogs, the Andean stream-breeding Hyla, the genus Aplastodiscus, and a Tepuian clade of Hyla. It contains five major clades (fig. 3). The first of these is called the Tepuian clade, and is composed solely of two exemplars of the H. aromatica and H. geographica groups. The second clade is composed of all Andean stream-breeding Hyla. The third is composed of all the exemplars of the H. circumdata, H. martinsi, and H. pseudopseudis groups, from southeastern Brazil, and we are calling it informally the Atlantic/Cerrado clade. The fourth is composed of the southeastern Brazilian H. albosignata and H. albofrenata complexes of the larger, nonmonophyletic H. albomarginata group plus the two species of Aplastodiscus, and we are calling it informally the Green clade. The fifth clade is composed of all the remaining species groups (H. geographica, H. pulchella, H. boans, H. granosa, H. punctata, H. albomarginata complex of the H. albomarginata group) and unassigned species associated in the past with the Gladiator Frogs, and we are calling it informally the TGF clade (for True Gladiator Frogs.)

Six currently recognized species groups within the South American clade I are not monophyletic. The Hyla albomarginata group is not monophyletic because its three “complexes” defined by Cruz and Peixoto (“1985” [1987]) are spread throughout the Green clade and the TGF clade. The H. albomarginata complex is not monophyletic, with its species being related with different groups in the TGF clade (see below). The H. albosignata complex is monophyletic, as is the H. albofrenata complex. These two complexes, however, do not form a monophyletic group, because Aplastodiscus is the sister group to the H. albosignata complex, and this clade is sister to the H. albofrenata complex. Within the TGF Clade, the only group that is not represented by a single exemplar (the Hyla punctata group) that results as monophyletic is the H. pulchella group. The H. albomarginata complex, H. albopunctata, H. boans, H. geographica, and H. granosa groups are nonmonophyletic.

Hyla pellucens and H. rufitela are the sister group of the clade composed of H. heilprini and the paraphyletic H. albopunctata group (see below); H. albomarginata is the sister taxon of a fragment of the H. boans group (see below). The H. albopunctata group is paraphyletic inasmuch as H. fasciata plus H. calcarata is nested within it. The H. boans group is polyphyletic because the mostly southeastern Brazil/northeastern Argentina exemplars (H. faber, H. lundii, H. pardalis, H. crepitans, and H. albomarginata) together with H. albomarginata are the sister taxon of the H. pulchella group and are only distantly related to H. boans. Hyla boans is the sister taxon of H. geographica plus H. semilineata. The H. geographica group is rampantly polyphyletic, with its exemplars partitioned into five different clades within the South American clade I: (1) H. kanaima is the sister taxon of H. inparquesi, the single exemplar of the H. aromatica group; (2) H. roraima and H. microderma form a monophyletic group with four Guayanese and one Amazonian species; (3) Hyla picturata is related to the exemplars of the H. punctata and H. granosa groups; (4) Hyla semilineata + H. geographica are related to H. boans; and (5) H. fasciata + H. calcarata are nested within the H. albopunctata group as detailed above. The H. granosa group is paraphyletic by having H. picturata and H. punctata nested within it.

Andean Stream-Breeding Hyla and the Tepuian Clade

The monophyly of the Andean stream-breeding Hyla is congruent with suggestions presented by Duellman et al. (1997) and Mijares-Urrutia (1997), who noticed similarities in larval morphology of the H. bogotensis and H. larinopygion groups. Duellman et al. (1997) presented a phylogenetic analysis restricted to wholly or partially Andean species groups of Hyla. In their most parsimonious tree, the H. armata, H. bogotensis, and H. larinopygion groups formed a monophyletic group supported by three transformations in tadpole morphology: the enlarged, ventrally oriented oral disc; the complete marginal papillae; and a labial tooth row formula 4/6 or higher.

Duellman et al. (1997) suggested a close relationship between the H. armata and H. larinopygion groups based on the presence in males of a greatly enlarged prepollex lacking a projecting spine. While our results are congruent with this, note that males of the H. bogotensis group also have a prepollex with the same external morphology as those of the H. armata and H. larinopygion groups. Kizirian et al. (2003) suggested that the H. armata group was nested in the H. larinopygion group. Our results do not support this suggestion. However, this could be a consequence of the few exemplars of the H. larinopygion group available for our study.

Kizirian et al. (2003) had doubts about the placement of Hyla tapichalaca. Faivovich et al. (2004) showed that this species is related to the H. armata–H. larinopygion groups (although they only included H. armata in their analysis). Our results go a step further, indicating a closer relationship with the H. larinopygion group.

Hyla inparquesi and H. kanaima forming the sister taxon of all the remaining South American clade I is an unexpected result. On the basis of morphology, we expected our only exemplar of the H. aromatica group, H. inparquesi22, to be related to the Andean stream-breeding clade of Hyla, because both share the character states that Duellman et al. (1997) suggested as synapomorphies in support of the monophyly of the Andean stream-breeding Hyla: (1) known larvae with ventral, enlarged oral discs; (2) complete marginal papillae; and (3) with a minimum labial tooth row formula of 4/6. Furthermore, adults of the H. aromatica group share a greatly enlarged prepollex without a projecting spine in males that, as we mentioned above, is present in most species of Andean stream-breeding Hyla (the only known exception being H. tapichalaca; Kizirian et al., 2003).

This sister-group relationship between the Tepuian clade and all the remaining groups of the South American clade I has further implications. Based on the available material, Duellman et al. (1997) considered the prepollex greatly enlarged without a projecting spine to be an intermediate state in an ordered transformation series from prepollex not greatly enlarged to prepollex greatly enlarged with a projecting spine. Our topology implies that the greatly enlarged prepollex without a projecting spine is a synapomorphy of the whole South American clade I (with subsequent transformations, including the development of a projecting spine). However, H. kanaima does not have an enlarged prepollex as prominent as that found in the H. armata, H. aromatica, H. bogotensis, and H. larinopygion groups. In order to clarify this situation, it would be necessary to (1) define the prepollex character states osteologically, and (2) include a denser sampling of the H. aromatica group, to better understand its relationship with H. kanaima. (Perhaps the character state of H. kanaima could be interpreted as a reversal.)

Our topology implies an interesting scenario regarding the evolution of larval morphology in the South American clade I; that is, that larval morphology of the Atlantic/ Cerrado, Green, and TGF clades evolved from an ancestor with the highly modified morphology typical of stream larvae (including large numbers of labial tooth rows, a large oral disc with complete marginal papillae, and relatively low fins) that during evolution, underwent a transformation of these character states (specifically, reduction in labial tooth row formulae, a reduced oral disc, and formation of an anterior gap in the marginal papillae). These transformations were coincident with distributional shifts from high-elevation mountain streams (as in the Tepuian and Andean stream-breeding clades) toward lower elevation forest mountain streams (the cases of the Atlantic/Cerrado clade, the Green clade, and, and some taxa of the TGF clade), and Amazonian lowlands and the Cerrado-Chaco (several taxa of the TGF clade).

Gladiator Frogs

Duellman et al. (1997) suggested the existence of a clade composed of the Hyla albomarginata, H. albopunctata, H. boans, H. circumdata, H. geographica, and H. pulchella groups. The synapomorphy supporting this clade is, according to these authors, the presence of “an enlarged prepollical spine lacking a quadrangular base”. Faivovich et al. (2004), based on the analysis of mitochondrial DNA sequences and on the observation (Garcia and Faivovich, personal obs.) that H. punctata and the H. polytaenia group show the same morphology of the prepollical spine, argued that these additional groups also belong to that clade (these authors further included the H. polytaenia group within the H. pulchella group), which was called earlier in this paper “Gladiator Frogs”. Our results indicate that a clade with the composition suggested by Duellman et al. (1997) and Faivovich et al. (2004) is paraphyletic because Aplastodiscus is nested within it; furthermore, H. kanaima of the H. geographica group is only distantly related to this clade.

Atlantic/Cerrado Clade

Our results corroborate the long-suspected association of the Hyla circumdata group with the groups of H. pseudopseudis and H. martinsi (Bokermann, 1964a; Cardoso, 1983; Caramaschi and Feio, 1990; Pombal and Caramaschi, 1995), even though the monophyly of these two groups could not be tested by our analysis. We also consider as corroborated the suspected relationship of the Hyla circumdata and H. pseudopseudis groups with H. alvarengai (Bokermann, 1964a; Duellman et al., 1997), because Hyla sp. 9 (aff. H. alvarengai) is nested in this clade.

Unfortunately, due to the unavailability of samples, we could not test the relationships of the Hyla claresignata group with this clade. Considering the phylogenetic context of the Atlantic/Cerrado clade within the South American clade I, we must revisit the apparent synapomorphies of the H. claresignata group mentioned earlier (oral disc completely surrounded by marginal papillae, and 7/12–8/ 13 labial tooth rows). The presence and distribution of these character states in the Tepuian and Andean stream-breeding Hyla clades is suggestive, not of a closer relationship of the H. claresignata group with any of them (these character states are plesiomorphies in this context), but of the nature of its relationship with the Atlantic/Cerrado clade. Perhaps the H. claresignata group is not a member of the Atlantic/Cerrado clade, but is a basal group related either to the Tepuian or Andean stream-breeding clade, or perhaps it is even the sister taxon of the clade composed of the Atlantic/Cerrado, Green, and TGF clades. At this point we are not aware of evidence favoring any of these alternatives.

Green Clade

Lutz (1950) was the first to suggest that Aplastodiscus was related to species latter included in the Hyla albosignata complex, as defined by Cruz and Peixoto (“1985” [1987]). This was supported by Garcia et al. (2001a) based on the presence of enlarged internal metacarpal and metatarsal tubercles and unpigmented eggs. More recently, Haddad et al. (2005) described the reproductive mode of Aplastodiscus perviridis, which includes egg deposition in a subterranean nest excavated by the male, where exotrophic larvae spend the early stages of development until flooding releases them to a nearby water body. This mode is the same as that observed in one species of the H. albosignata complex, H. leucopygia (Haddad and Sawaya, 2000), and for the undescribed species of the H. albofrenata complex included in our analysis, Hyla sp.1 (aff. H. ehrhardti) (Hartmann et al., 2004); this mode is further suspected to occur in all species of both the H. albosignata and H. albofrenata complexes (Haddad and Sawaya, 2000, Hartmann et al., 2004). Species included in the H. albomarginata complex instead lay their eggs on the water film surface (Duellman, 1970). Based on the shared reproductive mode, Haddad et al. (2005) suggested that Aplastodiscus could be related to the H. albofrenata and H. albosignata complexes. Our results support the monophyly of both the H. albofrenata and H. albosignata complexes and their close relationship with Aplastodiscus, that is the sister taxon of the H. albosignata complex.

While we are not aware of nonmolecular synapomorphies for several nodes supported by molecular evidence, the few osteological data available for the South American clade I indicate that the Green clade and the TGF clade share the presence of transverse processes of the sacral diapophyses notably expanded distally, while species of the Atlantic/ Cerrado clade (Bokermann, 1964a; Garcia 2003) and H. tapichalaca (Kizirian et al., 2003), the only Andean stream-breeding Hyla with any described postcranial osteology, have the transverse processes poorly expanded or not expanded at all. However, the distribution of this character state is in conflict with the presence of a prepollical spine in the Atlantic/Cerrado clade and the TGF clade, which is absent in the Green clade, where there is a fairly enlarged prepollex but no spine. A detailed study of prepollex morphology in these taxa would help to better define the relevant transformation series and it transformation sequences in the tree.

True Gladiator Frog Clade

The results imply the nonmonophyly of several species groups within this clade, as described earlier. This situation is not unexpected considering the paucity of evidence of monophyly previously available for most of them.

The monophyly of the group composed of the two unassigned species from the Guayana Highlands, Hyla benitezi, H. lemai, Hyla sp. 2, two members of the H. geographica group (H. microderma and H. roraima), and Hyla sp. 8 is further supported by the presence of a mental gland in males (Faivovich et al., in prep.)

In the DNA-based phylogenetic analysis of the Hyla pulchella group performed by Faivovich et al. (2004), H. punctata is the sister species of H. granosa, with this overall clade forming the sister taxon of a clade composed of the H. albopunctata, H. geographica, H. albomarginata, H. boans, and H. pulchella groups. In our analysis, H. punctata, our only exemplar of the group, is the sister taxon of H. granosa, and this taxon is at the apex of a pectinated series that includes H. sibleszi and, curiously, H. picturata. Prior to the analysis, we had no idea as to with which species group H. picturata would be related, but certainly we did not expect this colorful frog to be nested within a group of green species.

Our results lend only partial support for a relationship between Hyla heilprini and the H. albomarginata group, as tentatively suggested by Duellman (1974) and Trueb and Tyler (1974) based on overall pigmentation and the white peritoneum, because H. heilprini is the sister taxon of the H. albopunctata group (including a fragment of the H. geographica group), with H. heilprini plus this unit being the sister taxon of a fragment of the H. albomarginata group. The nonmonophyly of the H. albopunctata group corroborates comments advanced by de Sá (1995, 1996) as to the lack of evidence for its monophyly.

Hyla crepitans, H. faber, H. lundii, and H. pardalis form, together with H. albomarginata, a monophyletic group only distantly related to H. boans, the species that gives the name to the former group. Within this clade, the nest builders23 H. faber, H. lundii, and H. pardalis, are monophyletic.

The polyphyly of the Hyla boans group has implications for the evolution of reproductive modes, in that it implies independent origins of nest-building behavior by males. Although theoretically possible, certainly no author had ever suggested that such a characteristic behavior as the nest building could be a homoplastic feature.24 However, the molecular evidence points that way, and further evidence indicates that this or a similar reproductive mode also occurs in at least some species of the H. circumdata group (Pombal and Haddad, 1993, Pombal and Gordo, 2004), implying then at least three independent occurrences within the South American clade I. In a wider context, nest building was reported as well in Pelodryadinae in males of Litoria jungguy (Richards, 1993; using the name L. lesueuri, see Donnellan and Mahony [2004]), thus implying a fourth instance of homoplasy within Hylidae.

Our topology for the Hyla pulchella group is identical to that of Faivovich et al. (2004), including the exemplars of the former H. polytaenia group nested within it. Following Faivovich et al. (2004), we continue to recognize a H. polytaenia clade within the H. pulchella group. These authors stated that the lack of any pattern on the hidden surfaces of thighs was one of two possible morphological synapomorphies of this clade (with the other being the mostly striped dorsal pattern). This observation is mistaken, because the same character state occurs in H. ericae (Caramaschi and Cruz, 2000), H. joaquini, H. marginata, H. melanopleura, H. palaestes, and H. semiguttata (Duellman et al., 1997; Garcia et al., 2001b), suggesting that it actually may be a synapomorphy of a more inclusive clade whose contents are still undefined.

South American II Clade

The South American II clade (fig. 4) is composed of the 30-chromosome Hyla, the Hyla uruguaya group, Lysapsus, Pseudis, Scarthyla, Scinax, Sphaenorhynchus, and Xenohyla. It contains two main clades: one composed of Lysapsus, Pseudis, Scarthyla, and Scinax (including the H. uruguaya group), and the other composed of Sphaenorhynchus, Xenohyla, and all the exemplars of 30-chromosome Hyla species groups.

Within this clade, Scinax and the Hyla microcephala group are not monophyletic. Scinax has H. uruguaya, an exemplar of the H. uruguaya group, nested within it. The H. microcephala group is paraphyletic with respect to the available exemplars of the H. decipiens and H. rubicundula groups.

Relationships of Scinax

Several hypotheses have been advanced on the relationships of Scinax. Aparasphenodon was considered by Trueb (1970a) to be closely related to Corythomantis and, in turn, she considered these two genera to be nested within the (then) Hyla rubra group (currently the genus Scinax), based on overall similarities in cranial morphology. Scarthyla was considered to be related to Scinax by Duellman and de Sá (1988). Duellman and Wiens (1992) extended this to suggest that Scarthyla is the sister taxon of Scinax, which together form the sister taxon of Sphaenorhynchus. According to Duellman and Wiens (1992), character states supporting the monophyly of these three genera are narrow sacral diapophyses, anteriorly inclined alary processes of the premaxillae, and tadpoles with large, laterally placed eyes. Duellman and Wiens (1992) suggested that possible morphological synapomorphies of Scinax and Scarthyla are reduced webbing on the hand and the presence of an anterior process of the hyale. Tepuihyla was suggested to be closely related with Scinax, this being supported by the absence or extreme reduction of webbing between toes I and II, adhesive discs wider than long, and the presence of double-tailed sperm (Ayarzagüena et al., “1992” [1993b]). This association was questioned by Duellman and Yoshpa (1996) on the grounds that the absence or extreme reduction of webbing between toes I and II was homoplastic among hylids (although Duellman and Wiens [1992] suggested this same character state to be a synapomorphy of Scinax). These authors suggested that the only evidence uniting Tepuihyla with Scinax could be the double-tailed sperm reported by Ayarzagüena et al. (“1992” [1993b]) for Tepuihyla and by Fouquette and Delahoussaye (1977) for Scinax.25

Mijares-Urrutia et al. (1999) again suggested a close relationship of Tepuihyla with Scinax, but also with Scarthyla and Sphaenorhynchus– Scinax relatives, as suggested by Duellman and Wiens (1992). Mijares-Urrutia et al. (1999) also noted that Tepuihyla has rounded sacral diapophyses as found in these three genera (Duellman and Wiens, 1992).

Our results do not support a relationship of Scinax with Aparasphenodon, Corythomantis, or Tepuihyla because these three genera are nested within the South American/ West Indies Casque-headed Frog clade. Furthermore, Scinax is not the sister group of Scarthyla but of a clade composed of Scarthyla plus Lysapsus and Pseudis (in the 1:1: 1 analysis) or of all the remaining genera included in the South American II clade (in the 3:1:2 analysis).

Scinax is also paraphyletic with respect to the Hyla uruguaya group, for which Bokermann and Sazima (1973a) and Langone (1990) could not suggest affinities with any other hylids. Most recently, Kolenc et al. (“2003” [2004]) observed in the larvae of H. uruguaya and H. pinima the morphological synapomorphies of the larvae of the Scinax ruber clade that were reported by Faivovich (2002), and they suggested a possible relationship between the H. uruguaya group and the Scinax ruber clade. Adults of the H. uruguaya group are quite characteristic morphologically, and perhaps as a consequence this group was never associated with any species of Scinax prior to Kolenc et al. (“2003” [2004]).

Our results reveal that none of the outgroups employed by Faivovich (2002) in the phylogenetic analysis of Scinax is particularly close to Scinax; instead, all other components of the South American II clade are much more suitable to establish character-state polarities in this genus. Consequently, exemplars of these closer neighbors of Scinax need to be added, and the synapomorphies of Scinax resulting from that analysis need to be reevaluated.

Lysapsus, Pseudis, and Scarthyla

The sister-group relationship between Scarthyla goinorum and “pseudids” (Lysapsus + Pseudis), and this group being nested within Hylinae, corroborates recent findings by Darst and Cannatella (2004) and Haas (2003). Burton (2004) reported a likely synapomorphy for the Scarthyla plus the “pseudid” clade that is corroborated in the present analysis; that is, the m. transversus metatarsus II oblique, with a narrow, proximal connection to metatarsus II, and a broad, distal connection to metatarsus III. Another character state described by Burton (2004), the undivided tendon of the m. flexor digitorum brevis superficialis, optimizes in this analysis as a synapomorphy of this clade plus Scinax.

Besides the molecular data, the monophyly of Lysapsus plus Pseudis is further supported by the tendo superficialis pro digiti III arising from the m. flexor digitorum brevis superficialis, with no contribution from the aponeurosis plantaris; the origin of m. flexor ossis metatarsi IV and the joint tendon of origin of mm. flexores ossum metatarsorum II and III crossing each other; the m. flexor ossis metatarsi IV very short, inserting on the proximal two-thirds of metatarsal IV or less; absence of a tendon from the m. flexor digitorum brevis superficialis to the medial slip of the medial m. lumbricalis brevis digiti V; and m. transversus metatarsus III oblique, with a narrow, proximal connection onto metatarsal III, and a broad, distal connection to metatarsal IV. Another likely morphological synapomorphy is the elongated intercalary elements.

Vera Candioti (2004) noticed that Lysapsus limellum and most of the 30-chromosome Hyla (H. nana and H. microcephala) studied by her and Haas (2003) share two of the synapomorphies that Haas (2003) reported for Pseudis paradoxa and P. minuta: insertion of the m. levator mandibulae lateralis in the nasal sac, and a distinct gap in the m. subarcualis rectus II–IV. Haas (2003) observed different character states for H. ebraccata (the m. levator mandibulae lateralis inserts in tissue close to posterodorsal process of suprarostral cartilage or adrostral tissue; continuous m. subarcualis rectus II–IV), suggesting the need for additional studies on its taxonomic distribution within the 30-chromosome Hyla and in the other genera of the South American II clade.

Sphaenorhynchus, Xenohyla and the 30-Chromosome Hyla

Izecksohn (1959, 1996) suggested possible relationships of Xenohyla with Sphaenorhynchus and Scinax (Izecksohn, 1996). These ideas are partially corroborated by our 1:1:1 results, with the exception that they also suggest that Xenohyla is the sister group of the 30-chromosome Hyla. The karyotype is still unknown in Xenohyla, and this poses an obstacle to our understanding of the limits of the 30-chromosome Hyla. Interestingly, while both Sphaenorhynchus and Xenohyla do have a quadratojugal, in both cases it does not articulate with the maxilla (Duellman and Wiens, 1992; Izecksohn, 1996), which could be seen as an intermediate step before the extreme reductions of the quadratojugal seen in the 30-chromosome Hyla (Duellman and Trueb, 1983). Tadpoles of Xenohyla and several species of 30-chromosome Hyla share the presence of the tail tip extended into a flagellum, as well as the presence of high caudal fins (e.g., see Bokermann, 1963; Kenny, 1969; Gomes and Peixoto, 1991a, 1991b; Izecksohn, 1996; Peixoto and Gomes, 1999).

Phylogenetic hypotheses of the 30-chromosome Hyla species groups using morphological characters were presented by Duellman and Trueb (1983), Duellman et al. (1997), Kaplan (1991, 1994), and Kaplan and Ruíz (1997); none of these tested the monophyly of the contained species groups. A summary of their proposals and the supporting evidence are depicted in figure 6.

Chek et al. (2001) presented a phylogenetic analysis using partial 16S and cytochrome b sequences of the Hyla leucophyllata group, including exemplars of other 30-chromosome Hyla species groups. Because they did not include non-30-chromosome hylids, they did not test the monophyly of this clade.

The distribution of certain characters in several species associated with the currently recognized species groups suggests problems in our phylogenetic understanding of these frogs. The monophyly of a group composed of the Hyla leucophyllata, H. marmorata, H. microcephala, and H. parviceps groups is currently supported by the absence of labial tooth rows in their larvae (Duellman and Trueb, 1983). However, within the H. parviceps group, H. microps (Santos et al., 1998) and H. giesleri (Bokermann, 1963; Santos et al., 1998) have at least one labial tooth row. Similarly, Gomes and Peixoto (1991a) and Peixoto and Gomes (1999) noticed in the H. marmorata group the presence of one labial tooth row in the larvae of H. nahdereri, H. senicula, and H. soaresi. Based on these facts and similarities in tail depth, tail color, general body shape, and predatory habits, they suggested that the H. marmorata group could instead be more closely related to H. minuta than to the groups suggested by Duellman and Trueb (1983). Gomes and Peixoto (1991b) pointed out the presence of a labial tooth row in the larva of H. elegans. Wild (1992) further noted the absence of marginal papillae (an apparent synapomorphy of the H. microcephala group) in the larva of H. allenorum (a species of the H. parviceps group).

The reproductive modes of the different species are also informative. According to Duellman and Crump (1974), Hyla parviceps deposits its eggs directly in the water, as does H. microps (Bokermann, 1963), whereas H. bokermanni and H. brevifrons oviposit on leaves overhanging ponds; upon hatching, the tadpoles drop into the water where they complete development. Hyla ruschii oviposits on leaves overhanging streams (Weygoldt and Peixoto, 1987). The oviposition on leaves occurs in most species of the H. leucophyllata group, whereas both reproductive modes occur in the H. microcephala group.

Our results recover the 30-chromosome Hyla species as monophyletic; however, our topology differs from previous hypotheses. In our topology, the root is placed between the H. marmorata group and the other exemplars, instead of between the H. labialis group and the other exemplars, as was assumed in previous analyses (Duellman and Trueb, 1983; Kaplan, 1991, 1994, 1999; Chek et al., 2001).

Topological differences from previous hypotheses are not due merely to a re-rooting of the previously accepted tree; the relationships obtained by our analysis are quite different from previous proposals. Our analysis does not recover as monophyletic the exemplars of the three species groups once thought to be monophyletic on the basis of lacking labial tooth rows, that is, the Hyla leucophyllata, H. microcephala, and H. parviceps groups. Instead, the H. microcephala group (including the taxa imbedded within it) is the sister taxon of a clade composed of H. anceps and the exemplars of the H. leucophyllata group. The exemplars of the H. parviceps group are the sister taxon of a clade composed of the exemplars of the H. columbiana and H. labialis groups. This shows that the scenario of labial tooth row evolution is more complex than previously thought, because it implies several transformations in both directions between presence and absence of labial teeth within the clade.

Observations by Wassersug (1980), Spirandeli Cruz (1991), and Kaplan and Ruiz-Carranza (1997) on the internal oral features of larvae of representatives of the Hyla leucophyllata (H. ebraccata and H. sarayacuensis), H. microcephala (H. microcephala, H. nana, H. phlebodes, and H. sanborni), and H. garagoensis (H. padreluna and H. virolinensis) groups revealed a reduction of internal oral structures (including reduction of most internal papillation, reduction of branchial baskets, reduction or absence of secretory ridges and secretory pits) that is most extreme in the representatives of the H. microcephala group. Hyla minuta does not show the reductions seen in these species groups (Spirandeli Cruz, 1991). This species also shares with representatives of the H. leucophyllata group described by Wassersug (1980) a reduction in the density of the filter mesh of the branchial baskets in comparison with other hylid tadpoles. It is clear that the study of internal oral features will provide several additional characters relevant for the study of the 30-chromosome species of Hyla.

The exemplars of the Hyla parviceps group obtain as monophyletic. However, we included only 3 of the 15 species currently included in this problematic group. We are not confident that the monophyly of the H. parviceps group will be maintained as more taxa are added. Regarding the exemplars of the H. leucophyllata group, their relationships are equivalent to those obtained by Chek et al. (2001).

The sister-group relationship of Hyla anceps and the H. leucophyllata group corroborates early suggestions by Lutz (1948, 1973) that these could be related on the basis of sharing a large axilar membrane and flash coloration.

While we could not test the monophyly of the Hyla minima and H. minuta groups, our exemplars of these groups are sister taxa, and they are only distantly related with the exemplars of the H. parviceps group. This position does not support Duellman's (2001) tentative suggestion that the species of the H. minima group should be included in the H. parviceps group.

The paraphyly of the Hyla microcephala group with respect to the H. rubicundula group is an expected result, as historically its species were associated with H. nana and H. sanborni (Lutz, 1973). Nevertheless, the association of the H. microcephala and H. rubicundula groups were reinforced by Pugliese et al. (2001), who described the larva of H. rubicundula and noted similarities (like the lack of marginal papillae) with the larvae of members of the H. microcephala group. In particular, these authors noticed similarities with H. nana and H. sanborni, with the latter being the sister taxon of H. rubicundula in our analysis.

Carvalho e Silva et al. (2003) segregated the Hyla decipiens group from the H. microcephala group on the basis that the larvae of these species lack the possible morphological synapomorphies currently diagnostic of the H. microcephala group (body of tadpole depressed, labial papillae absent in tadpoles) and the putative clade composed of the H. leucophyllata, H. microcephala, and H. parviceps groups (absence of labial tooth rows). However, as mentioned above, our results imply a complex scenario for labial tooth row transformations and place the H. decipiens group within the H. microcephala group. The available taxon sampling did not allow testing the monophyly of the H. decipiens group. The fact that its known species share the oviposition on leaves above the water, and the reversals in larval morphology that led Carvalho e Silva et al. (2003) to consider them unrelated to the H. microcephala group, probably indicates that, even if nested inside this group, the species assigned to the H. decipiens group could be a monophyletic unit.

The relationships of the Hyla garagoensis group, from which no exemplar was available for this study, were discussed by Kaplan and Ruiz-Carranza (1997). Based on the absence of labial tooth rows, they placed the H. garagoensis group in a polytomy together with the H. marmorata group and the clade composed of the H. microcephala, H. parviceps, and H. leucophyllata groups. Duellman et al. (1997) presented a cladogram for most of the 30-chromosome Hyla groups, where the H. garagoensis and H. marmorata groups appear together as a clade supported by the presence of one ventral row of small marginal papillae in larvae. This character state needs further assessment, as indicated by Gomes and Peixoto (1991a) and Peixoto and Gomes (1999), because tadpoles of the H. marmorata group have either one (H. nahdereri) or two rows of marginal papillae (H. senicula; H. soaresi); the tadpoles of H. padreluna, a species of the H. garagoensis group, also has a double row (Kaplan and Ruiz-Carranza, 1997). Considering this and earlier comments, we do not see evidence that associates the H. garagoensis group with the H. marmorata group more than with any other group within the 30-chromosome Hyla clade.

Middle American/Holarctic Clade

This clade is composed of most of the Middle American/Holarctic genera and species groups of treefrogs (fig. 5). For the purposes of discussion, we divide it into four large clades. The first of these includes Acris and Pseudacris. The second includes Plectrohyla, the Hyla bistincta group, the H. sumichrasti group, and various elements of the H. miotympanum group. The third clade includes Duellmanohyla, Ptychohyla, H. miliaria, H. bromeliacia (the sole exemplar of the H. bromeliacia group), and one element of the H. miotympanum group. The fourth clade includes Smilisca, Triprion, Anotheca, and the exemplars of the H. arborea, H. cinerea, H. eximia, H. godmani, H. mixomaculata, H. pictipes, H. pseudopuma, H. taeniopus, and H. versicolor groups.

Within the Middle American/Holarctic clade, the genera Ptychohyla and Smilisca, as well as the Hyla arborea, H. cinerea, H. eximia, H. miotympanum, H. tuberculosa, and H. versicolor groups, are not monophyletic. The H. miotympanum group is polyphyletic; its exemplars split among three different clades: H. miotympanum is the sister taxon of one of the exemplars of the H. tuberculosa group, H. miliaria; H. arborescandens and H. cyclada are related to an undescribed species close to H. thorectes, and together are related to exemplars of the H. bistincta group; H. melanomma and H. perkinsi are at the base of the H. sumichrasti group. Smilisca is not monophyletic, having Pternohyla fodiens nested within it. Ptychohyla is paraphyletic with respect to H. dendrophasma (H. tuberculosa group). The H. arborea group is polyphyletic, with H. japonica nested within the H. eximia group. The H. cinerea group is not monophyletic, with H. femoralis being more closely related to members of the H. eximia and H. versicolor groups than to H. cinerea, H. gratiosa, and H. squirella. The H. versicolor group is not monophyletic because H. andersonii is more closely related to the H. eximia group.

The monophyly of all genera and species groups of Hyla contained in this clade was maintained by Duellman (1970, 2001) based mostly on biogeographic grounds, because morphological evidence of monophyly was lacking. Duellman (2001) further presented a diagram depicting “suggested possible evolutionary relationships” among Middle and North American Hylinae, using as terminals the species groups of Hyla and the different genera (redrawn here as fig. 7). Duellman (2001) envisioned a North American basal lineage being the sister taxon of what he called the Middle American basal lineage. This Middle American basal lineage is further divided into a lower Central American lineage (itself divided into an isthmian highland lineage and a lowland lineage) and the Mexican-Nuclear Central American lineage (in turn divided into a Mexican-Nuclear Central American highland lineage and a lowlands lineage).

While the basal position of Acris and Pseudacris in this clade is consistent with Duellman's (2001) intuitive suggestion of a North American basal lineage, it differs in that the Holarctic species groups of Hyla are only distantly related to them.

We found no evidence supporting Duellman's (2001) exclusively Mexican-Nuclear Central American lineage, nor of his Mexican-Nuclear Central American highland lineage. The latter has nested within it the Mexican-Nuclear Central American lowland clade (the H. godmani group), a clade reminiscent of Duellman's (2001) isthmian highland-lowlands lineage, and also all species groups of Holarctic Hyla. Biogeographic implications of the discordant nature of our results with Duellman's suggested relationships between Middle- and North American Hylinae will be dealt with from a biogeographic perspective later in this paper.

The nonmonophyly of North American Hylinae does not agree with previous analyses (Hedges, 1986; Cocroft, 1994; da Silva, 1997; Moriarty and Cannatella, 2004) because those analyses assumed implicitly that Acris, the Holarctic Hyla, and Pseudacris are monophyletic. In spite of this, the internal relationships of Pseudacris recovered in this analysis are consistent to those obtained by Moriarty and Cannatella (2004).

Previous analyses either did not find evidence that Acris was particularly close to any group of North American hylids (Cocroft, 1994), or else suggested relationships with different species groups of North American Hyla (Hedges, 1986; da Silva, 1997). Two morphological synapomorphies of this Acris + Pseudacris clade could be the spherical or ovoid testes (as opposed to elongate testes) and the presence of dark pigmentation in the peritoneum surrounding the testes (Ralin, 1970, as cited by Hedges, 1986).

The nonmonophyly of the Hyla miotympanum group is not surprising because, as discussed in the section on taxon sampling, it did not appear that any of its putative synapomorphies could withstand a test with a broader taxon sampling. Duellman (2001) merged the formerly recognized H. pinorum group (Duellman, 1970) with the H. miotympanum group. With the notable exception of H. miotympanum, our results are close to recovering both groups as originally envisioned by Duellman (1970), because H. cyclada and H. arborescandens (H. miotympanum group) are recovered as monophyletic, and the former H. pinorum group is recovered as a paraphyletic assemblage that includes the H. sumichrasti group nested within it. In his analysis (Duellman, 2001: 912), the two species included by Duellman (1970) in the H. pinorum group (H. melanomma and H. pinorum), plus the two species that were later associated with this group (H. perkinsi and H. juanitae), form a monophyletic group supported by a single synapomorphy, the presence of an extensive (equal to or more than one-half length of upper arm) axillary membrane.

The dubious monophyly of the 17 species assigned to the Hyla bistincta group was not seriously tested in our analysis, because only 2 species were available. These two exemplars form the sister group of two taxa previously associated with the H. miotympanum group, and together they form the sister taxon of Plectrohyla. According to Duellman and Campbell (1992), character states supporting a monophyletic H. bistincta group plus Plectrohyla are: medial ramus of pterygoid long, in contact with otic capsule; dorsal skin thick (but see Mendelson and Toal [1995] and Duellman [2001] for discussions of this character); complete marginal papillae of the oral disc; and presence of at least one row of submarginal papillae (called by these authors accessory labial papillae) on the posterior labium (but see Wilson et al. [1994a] for discussion of this character state). From these, the complete marginal papillae of the oral disc occur in all known larvae of the clade containing Plectrohyla, and the H. bistincta, H. sumichrasti, and fragments of the H. miotympanum group, as well as in larvae of several other nearby clades (Ptychohyla, Duellmanohyla, the H. mixomaculata, and H. taeniopus groups; see Duellman 1970, 2001). Furthermore, the row of submarginal papillae in the larval oral disc presents a fair amount of variation in the extent and distribution of the papillae, within which could probably be subsumed the morphology seen in all known larvae of the clade mentioned above (see illustrations of all these oral discs in Duellman, 1970, 2001). Besides discussions provided by Mendelson and Toal (1995) and Duellman (2001) regarding the definition of the character state “thick skin”, it does not occur in the following species currently assigned to the H. bistincta group: H. calvicollina, H. charadricola, H. chryses, H. labedactyla, and H. sabrina (Duellman, 2001). Considering that 15 of 17 species currently included in the H. bistincta group and 15 of 18 included in Plectrohyla could not be included in the analysis, we do not consider our results a strong test of their intrarelationships, particularly when several species of the H. bistincta group that present suspicious character state combinations, like the ones mentioned above, were not available. Our results relating H. arborescandens with species of the H. bistincta group corroborate earlier suggestions by Caldwell (1974) that relate this species to species currently placed in the H. bistincta group (H. mykter, H. robertsorum, and H. siopela.). Mendelson and Toal (1996) also suggested affinities of H. arborescandens and H. hazalae with the H. bistincta group on the basis of unpublished osteological data.

Duellman (2001) noted the lack of evidence for the monophyly of the Hyla tuberculosa group. Although poor, our taxon sampling does not recover it as monophyletic, because H. dendrophasma is nested within Ptychohyla, and H. miliaria is the sister taxon of H. miotympanum. Duellman (2001) also referred to the possibility advanced by da Silva (1997) of a relationship of the H. tuberculosa group with the Gladiator Frogs; at this point the evidence presented herein does not support this idea, but in case a denser sampling of the group still corroborates its polyphyly, we would not be surprised if some of its elements (particularly H. tuberculosa26) are shown to be related with the TGF clade.

Hyla miotympanum has repeatedly been considered a generalized Middle American hyline (Duellman, 1963, 1970; Campbell and Smith, 1992; Duellman, 2001), largely because the larva of H. miotympanum exhibits a labial tooth row formula of 2/3 and a relatively small oral disc with an anterior gap in the marginal papillae. We are not aware of any morphological synapomorphy supporting its relationship with H. miliaria, although our molecular data firmly place it there. According with our results, there is a morphological synapomorphy supporting the monophyly of the clade composed of these two species plus Duellmanohyla, the H. bromeliacia group, and Ptychohyla (including H. dendrophasma): the tendo superficialis hallucis that tapers from an expanded corner of the aponeurosis plantaris, with fibers of the m. transversus plantae distalis originating on distal tarsal 2–3 that insert on the lateral side of the tendon.

Considering its overall external appearance, we are surprised by the position of the poorly known Hyla dendrophasma. This species was originally considered to be a member of the H. tuberculosa group (Campbell et al., 2000) based on its large snout–vent length and extensive hand webbing, although with the caveat that it lacks dermal fringes, the only character state shared by all other species placed in the H. tuberculosa group. DNA was isolated and sequenced twice from tissues of the female holotype, the only known specimen (Campbell et al., 2000). Inasmuch as most previous notions of relationships among species of Ptychohyla derive from adult male morphology and tadpoles, the discovery of at least one male specimen of H. dendrophasma could hopefully allow us to better understand its relationships within Ptychohyla.

Campbell and Smith (1992) and Duellman (2001) suggested five morphological synapomorphies for Ptychohyla. One of these is apparently unique to Ptychohyla (pars palatina of the premaxilla with well-developed lingual flange), while the other four show a more extensive taxonomic distribution. (1) The cluster of ventrolateral mucous glands in breeding males is present in Duellmanohyla chamulae, D. ignicolor, and D. schmidtorum (Campbell and Smith, 1992; see also Thomas et al., 1993). (2) The presence in the ventrolateral edge of forearm of tubercles coalesced into a ridge (as opposed to the absence of tubercles) was reported for D. lythrodes, D. salvavida, D. schmidtorum, and D. soralia (Duellman, 1970; 2001); H. bromeliacia has an indistinct row of tubercles that do not coalesce into a ridge (absent in H. dendroscarta) (Duellman, 1970). (3) The double row of marginal papillae is present as well in larvae of the H. bromeliacia group (Duellman, 1970). Finally, (4) larvae of the H. bromeliacia group have a labial tooth row formula of 2/4 or 2/5, and all known larvae of Duellmanohyla have a labial tooth row formula of 3/3; the minimum known for a species of Ptychohyla is 3/5 (P. legleri and P. salvadorensis) (Duellman, 1970, 2001; Campbell and Smith, 1992).

The monophyly of the group composed of Ptychohyla euthysanota, P. hypomykter, P. leonhardschultzei, and P. zophodes is congruent with the results of Duellman (2001), who supported the monophyly of these taxa based on the presence of a thick, rounded tarsal fold. These species further share the presence of hypertrophied ventrolateral glands in breeding males with two species we could not include in our analysis: P. macrotympanum and P. panchoi. Furthermore, all these species also share with P. spinipollex the presence of the nuptial excrescences composed of enlarged individual spines. The states of these characters are unknown in Ptychohyla sp. and Hyla dendrophasma because the only available specimens are females. The nonmonophyly of P. hypomykter plus P. spinipollex is most surprising, considering that both were considered to be a single species (Wilson and McCranie, 1989; see also McCranie and Wilson, 1993).

Although we included several species of Ptychohyla in our analysis, we do not think that we have apprehended a good representation of the morphological diversity of the group, and the absence of species like P. erythromma, P. legleri, and P. sanctaecrucis certainly weakens the test of monophyly of Ptychohyla. This is more so considering the fact that several of the putative morphological synapomorphies of Ptychohyla are actually shared with some species of its sister taxon, as discussed earlier, and that the monophyly of our exemplars of Ptychohyla is weakly supported. The low Bremer support (3) for Ptychohyla also suggests that the evidence for its monophyly deserves further attention.

The Hyla bromeliacia group was tentatively associated with the polyphyletic H. miotympanum group by Duellman (2001: 779). Other than this, we are not aware of it being associated with any other group. Besides the molecular evidence, we are aware of at least one likely morphological synapomorphy supporting the monophyly of Duellmanohyla plus the Hyla bromeliacia group: the presence of pointed serrations of the larval jaw sheaths (Campbell and Smith, 1992; Duellman, 1970; 2001). These are apparently longer in some species of Duellmanohyla than in the H. bromeliacia group, but both seem to be notably more pointed than in Ptychohyla (see descriptions and illustrations in Duellman, 1970).

We share with Duellman (2001) and Mendelson and Campbell (1999) doubts regarding the monophyly of the Hyla taeniopus group. Nevertheless, our two exemplars are recovered as monophyletic in the analysis. Duellman (2001) examined the possibility of a relationship between this group and the H. bistincta group, based on the fact that both have large stream-adapted tadpoles with small, ventral oral discs with complete marginal papillae and bear a labial tooth row formula of 2/3 (but noting that the tooth-row formula is slightly higher for H. nephila and H. trux). Our results suggest instead that the H. taeniopus group is the sister taxon of a clade composed of H. mixe (the only available exemplar of the H. mixomaculata group) plus the clade composed of the Holarctic Hyla groups, the H. godmani, H. pictipes, and H. pseudopuma groups, and Anotheca, Smilisca (including Pternohyla), and Triprion. Furthermore, in the context of our results, the ventral oral disc with complete marginal papillae seems to be a synapomorphy of the whole Middle American clade, with subsequent transformations in the clade just mentioned and in other points of the tree.

We were unable to test the monophyly of the Hyla mixomaculata group, because H. mixe was the only taxon available. Regardless, and until a rigorous test is possible, the monophyly of this group could be reasonably assumed based on the presence of the enlarged oral disc with 7/10 or 11 labial tooth rows. At this point it should be stressed that the sequenced sample comes from a tadpole that was assigned to the H. mixomaculata group based that on that characteristic, and that it was tentatively assigned to H. mixe for being the only species of the group known from the region where the larva was collected; thus, considering the uncertainty in its determination, its position in the tree should be viewed cautiously.

Duellman (2001) included the species of the former Hyla picta group in the H. godmani group. Unfortunately, the two exemplars available to us for this analysis are only the two members of the former H. picta group and none of the restricted H. godmani group; therefore, this is not a satisfactory test of the monophyly of the H. godmani group (sensu lato).

The Lower Central American Lineage

The monophyly of the included exemplars of the Hyla pictipes and H. pseudopuma groups, and its relationship with a clade composed of Anotheca, Smilisca, Triprion, and Pternohyla, is quite consistent (in the sense that it contains almost the same groups) with Duellman's intuitive proposal of a lower Central American clade that contains an Isthmian Highland lineage and a Lowland lineage (fig. 7), with the only exception being that he tentatively considered the H. miliaria group related to Anotheca.

The monophyly of a group composed of Hyla pseudopuma and H. rivularis, the only two exemplars available from the H. pseudopuma and H. pictipes groups, could be suggestive of a lineage of highland isthmian Hylinae as suggested by Duellman (2001). Although the monophyly of each of these two groups has not been tested here, the position of the undescribed Mexican species Hyla sp. 5 (aff. H. thorectes) deserves some comments.

Duellman (1970) recognized the Hyla hazelae group in which he included the nominal species and H. thorectes. Reasons for recognizing this group were “the combination of large hands with vestigial webbing, half webbed feet … and presence of a tympanum are external features which separate these species from other small stream-breeding Mexican Hyla. Furthermore both species have small, relatively narrow tongues and large tubercles below the anal opening. The nature of the nasals and sphenethmoid are unique among northern Middle American hylids” (Duellman, 1970: 384). It is unclear if any of these character states could have been considered as evidence of monophyly of the group. It is also unclear on what basis Duellman (2001) dismantled the group and placed H. hazelae in the H. miotympanum group, while H. thorectes was transferred to the H. pictipes group. Wilson et al. (1994b) suggested that H. thorectes could be related to H. insolita and H. calypsa (under the name H. lancasteri; see Lips, 1996) because they share oviposition on leaves overhanging streams and have dark ventral pigmentation; these character states were not included in Duellman's (2001) analysis of the group. Although we do not have data to take a position regarding these actions, the fact that Hyla sp. 5 (aff. H. thorectes) is unrelated to H. rivularis is here taken as evidence that H. thorectes should not be included in the H. pictipes group. Furthermore, all character states advanced by Duellman (2001) as shared by H. thorectes and the species of the H. pictipes group are also shared by H. thorectes and the taxa to which Hyla sp. 5 (aff. H. thorectes) appears to be closely related in our analysis. Because we were unable to include H. calypsa, H. insolita, or H. lancasteri, we do not have elements to test the hypothesis of Wilson et al. (1994b) regarding the close relationship of H. calypsa, H. insolita, and H. thorectes.

In order to better understand the relationships of the Hyla pictipes group, it would be important to add to this analysis exemplars of the former H. zeteki and H. lancasteri groups, because together with the original H. pictipes and H. rivularis groups (as defined by Duellman, 1970) they represent the three main morphological extremes of the group. The fact that so few exemplars of these two groups were available is one of the weaker points of our analysis.

The paraphyly of Smilisca is partly consistent with Duellman's (2001) phylogenetic analysis of this genus in that Pternohyla is nested within it. However, we did not recover Triprion nested within Smilisca, as did Duellman (2001).

A possible relationship between Triprion and Anotheca was first advanced by Lutz (1968) because she considered them to be the extreme of one specialization consisting “in excessive ossification of the head, accompanied at some stages by extra dentition” (Lutz, 1968: 10). Within this same line, she included all casque-headed frogs, including together South American and Middle American forms. Duellman and Trueb (1976) suggested a possible link of Anotheca with Nyctimantis (discussed below). Duellman (2001: 332) proposed a tentative relation of Anotheca with the Hyla miliaria group based on the oophagous tadpoles that develop in bromeliads or tree-holes (known for the only species of the H. tuberculosa group with a known tadpole, H. salvaje; see Wilson et al., 1985). While our results support a sister-group relationship of Anotheca and Triprion as suggested by Lutz (1968), it occurs within the Holarctic/Middle American clade and not within a group composed of all casque-headed frogs as she suggested. In the context of this analysis, both Triprion and Anotheca share the posterior expansion of the frontoparietals that cover almost all the otoccipital dorsally (see figures in Duellman, 1970).

Our analysis indicates that the insertion of m. extensor digitorum comunis longus on metatarsal II is a synapomorphy of a group composed of Anotheca, Triprion, and the paraphyletic Smilisca. Furthermore, Smilisca (including Pternohyla) and Triprion share the type I septomaxillary (see Trueb, 1970a) and bifurcated cavum principale of the olfactory capsule (Trueb, 1970a); the distribution of these character states should be studied in Anotheca and nearby groups to determine the level of inclusiveness of these possible synapomorphies.

Real Hyla

Our results concerning the relationships between the North American and Eurasiatic species groups of Hyla differ from previous analyses, in part likely because of the previous assumption of monophyly of North American/Holarctic Hylinae. In the first place, the molecular evidence supports a clade containing all North American and Eurasiatic species groups of Hyla, a result that differs from previous analyses where relationships either were unresolved (Cocroft, 1994) or were paraphyletic with respect to Acris and/or Pseudacris (Hedges, 1986; da Silva, 1997). The polyphyly of the Hyla arborea group and the paraphyly of the H. eximia group corroborate previous ideas by Anderson (1991) and Borkin (1999) regarding their nonmonophyly and the closer relationship of H. japonica with the H. eximia and H. versicolor groups. A likely synapomorphy of the H. eximia and H. versicolor groups, including H. japonica and H. andersonii, is the nucleolar organizer region (NOR) present in chromosome 6 instead of chromosome 10 (Anderson, 1991).

South American/West Indian Casque-Headed Frogs

This clade (fig. 5) is composed of Phyllodytes, Phrynohyas, Nyctimantis, and all South American/West Indian casque-headed frogs: Argenteohyla, Aparasphenodon, Corythomantis, Osteopilus, Osteocephalus, Trachycephalus, and Tepuihyla. It is divided basally in a group composed of the two exemplars of Phyllodytes, and another group composed of all casque-headed frog genera, including Phrynohyas and Nyctimantis.

Within this clade, other than those genera that are not monotypic (Argenteohyla, Nyctimantis, Corythomantis) or represented in this analysis by a single species (Aparasphenodon, Tepuihyla), Phyllodytes and Osteopilus are monophyletic, and Osteocephalus, Phrynohyas, and Trachycephalus are not monophyletic. Osteocephalus is not monophyletic because O. langsdorffii (the only species of the genus distributed in the Atlantic forest) is not related to the remaining exemplars of Osteocephalus, which form a monophyletic group that is the sister taxon of Tepuihyla. Phrynohyas is not monophyletic, having Trachycephalus nigromaculatus nested within it, and Trachycephalus is not monophyletic, with T. jordani forming the sister taxon of “Phrynohyas” + T. nigromaculatus.

With respect to Phyllodytes, we were unable to find any published hypothesis regarding its relationships, and considering the scant information available on its morphology, we had no previous clue as to other groups of Hylidae with which it might be related. The only morphological character state of which we are aware that Phyllodytes shares with several members of the South American/West Indian Casque-headed Frogs clade is the presence of at least four posterior labial tooth rows in the tadpole oral disc (see below, “Taxonomic Conclusions: A New Taxonomy of Hylinae and Phyllomedusinae”, for further details).

The polyphyly of Osteocephalus was not unexpected considering the lack of any evidence of its monophyly. This polyphyly results because of the position of O. langsdorffii. This is the only species of the genus present in the Atlantic forest and a species that had been particularly poorly discussed in the context of the systematics of Osteocephalus (Duellman, 1974). While we do not test the monophyly of the bromeliad-breeding/single vocal sac species (here represented by a O. oophagus), our results show that our exemplars with lateral vocal sacs are paraphyletic with respect to O. oophagus.

The monophyly of Tepuihyla was not tested in this analysis. Its sister-group relationship with Osteocephalus (excluding O. langsdorffii) is supported by our data, instead of with Scinax as first suggested (Ayarzagüena et al., “1992” [1993b]; see earlier discussion on the relationships of Scinax). This situation requires changes in the original interpretation of two character states that provided evidence of the relationships of Tepuihyla with other hylids. The presence of spicules in the dorsum of males is more parsimoniously interpreted as a putative synapomorphy of Tepuihyla plus Osteocephalus, instead of a homoplasy, as advanced by Ayarzagüena et al. (“1992” [1993b]). Similarly, the reduction of webbing between toes I and II is more parsimoniously interpreted as a putative synapomorphy of Tepuihyla (homoplastic with Scinax) instead of a synapomorphy of Scinax + Tepuihyla.

The monophyly of the four exemplars of Osteopilus corroborates in a much broader taxonomic context the results of Maxson (1992), Hedges (1996), and Hass et al. (2001), based on albumin immunological distances and still unpublished sequence data regarding its monophyly, and the recent taxonomic changes summarized by Powell and Henderson (2003b). Unfortunately, we could not include in our analysis O. marianae, O. pulchrilineatus, and O. wilderi, and we are not aware of any possible morphological synapomorphy supporting their monophyly. In the absence of other evidence, it could be suggested that the oviposition and development in bromeliads, which occurs in these three species and O. brunneus, is a possible synapomorphy uniting these with O. crucialis, which apparently also has this reproductive mode (Hedges, 1987).

The presence of paired lateral vocal sacs and biogeographic considerations led Trueb (1970b) and Trueb and Duellman (1971) to suggest the collective monophyly of Argenteohyla, Osteocephalus, Phrynohyas, and Trachycephalus (at that time the species of Osteocephalus having a single subgular vocal sac were still unknown). Furthermore, these authors considered Trachycephalus and Phrynohyas to be a monophyletic group on the basis of sharing vocal sacs that are more lateral and protrude posteriorly to the angles of the jaws when inflated. Trueb and Tyler (1974) suggested that the West Indian Osteopilus and the former Calyptahyla crucialis (now Osteopilus crucialis) were also related to this clade, although they exhibit a single, subgular vocal sac. Our results corroborate the monophyly of Phrynohyas plus Trachycephalus (see below), but they also suggest a more complex situation where the casque-headed frogs with double vocal sacs are paraphyletic, with all of the genera of casque-headed frogs that have a single subgular sac being nested within them.

Duellman and Trueb (1976) suggested that Nyctimantis was related to Anotheca, because both share the medial ramus of the pterygoid being juxtaposed squarely against the anterolateral corner of the ventral ledge of the otic capsule. Also, frogs of both genera are known (Anotheca; Taylor, 1954; Jungfer, 1996) or suspected (Nyctimantis; Duellman and Trueb, 1976) to deposit their eggs in water-filled tree cavities. Our results suggest a radically different picture, with Nyctimantis nested within the South American/West Indian Casque-headed Frogs, while Anotheca is nested within the Middle American/Holarctic clade, being the sister taxon of Triprion.

The topology has some discrepancies with previous suggestions as to the relationships of Argenteohyla, Aparasphenodon, and Corythomantis (for the latter two genera, see also comments for Scinax above). Argenteohyla siemersi was segregated by Trueb (1970b) from Trachycephalus, where it had been placed by Klappenbach (1961), because it lacks the diagnostic character states of Trachycephalus established by Trueb (1970a). Further, she suggested that Argenteohyla is a close ally of Osteocephalus based on the presence of paired lateral vocal sacs. Although Trueb (1970a) suggested that Aparasphenodon and Corythomantis are sister taxa, our evidence suggests that both Argenteohyla and Nyctimantis are closer to Aparasphenodon than to Corythomantis.

Most species in the South American/West Indies Casque-headed Frog clade frequently live in or seek refuge in bromeliads or treeholes. This has been reported for Aparasphenodon (Paolillo and Cerda, 1981; Teixeira et al., 2002), Argenteohyla (Barrio and Lutz, 1966; Cespedez, 2000), Corythomantis (Jared et al., 1999), Nyctimantis (Duellman and Trueb, 1976), Osteocephalus langsdorffii (Haddad, personal obs.), Phrynohyas (Goeldi, 1907; Prado et al., 2003), Tepuihyla (Ayarzagüena et al., “1992” [1993b]), and Trachycephalus (Lutz, 1954; Bokermann, 1966c). Furthermore, all species of Phyllodytes (Peixoto et al., 2003), some species of Osteocephalus (Jungfer and Schiesari, 1995; Jungfer and Weygoldt, 1999; Jungfer and Lehr, 2001), some species of Osteopilus (Hedges, 1987; Lannoo et al., 1987), and at least two species of Phrynohyas (Goeldi, 1907; Lescure and Marty, 2000) even lay their eggs in phytotelmata or treeholes where their exotrophic larvae develop. While bromeliads and treeholes are used as refuges or for reproduction in other groups of hylids (e.g., Anotheca spinosa, the Hyla bromeliacia group, the two bromeliad breeding frogs of the H. pictipes group, H. astartea, the H. tuberculosa group, Scinax alter, the Scinax perpusillus group of the S. catharinae clade), the South American/West Indian Casque-headed Frog clade seems to be the largest clade of hylids that consistently makes use of bromeliads or treeholes.

The phylogenetic structure of the South American/West Indies Casque-headed Frog clade implies a minimum of one instance of reversal from presence of heavily exostosed and co-ossified skulls to normal looking, albeit heavily built skulls (the case of Phrynohyas), and at least a possible second and third instance involving reversals from exostosed skulls (the cases of Tepuihyla and Osteopilus vastus).

From a morphological perspective, the paraphyly of Phrynohyas with respect to Trachycephalus nigromaculatus and the concomitant nonmonophyly of Trachycephalus are most interesting and surprising. Herpetologists have been noticing for years that T. nigromaculatus and Phrynohyas mesophaea produce hybrids throughout their overlapping ranges of distribution (Haddad, personal obs.; Pombal, personal commun.; Ramos and Gasparini, 2004). The possibility of hybridization leads us to think that perhaps the introgression of P. mesophaea mitochondria could actually be the reason for the recovered paraphyly of Trachycephalus. However, phylogenetic analyses using either tyrosinase or rhodopsin alone (the only two nuclear genes that were successfully sequenced in T. nigromaculatus) still recover a paraphyletic Trachycephalus (results not shown).

Aplastodiscus albofrenatus Group

Aplastodiscus albosignatus Group

Aplastodiscus perviridis Group

Bokermannohyla circumdata Group

Bokermannohyla claresignata Group

Bokermannohyla martinsi Group

Bokermannohyla pseudopseudis Group

Hyloscirtus armatus Group

Hyloscirtus bogotensis Group

Hyloscirtus larinopygion Group

Hypsiboas albopunctatus Group

Hypsiboas benitezi Group

Hypsiboas faber Group

Hypsiboas pellucens Group

Hypsiboas pulchellus Group

Hypsiboas punctatus Group

Hypsiboas semilineatus Group

Species of Hypsiboas Unassigned to Group

There are two species of Hypsiboas that we do not assign to any group because we do not have evidence favoring a relationship with any of the species groups that we are recognizing for the genus. These species are Hypsiboas fuentei (Goin and Goin, 1968), new comb., and Hypsiboas varelae (Carrizo, 1992), new comb.

Dendropsophus columbianus Group

Dendropsophus garagoensis Group

Dendropsophus labialis Group

Dendropsophus leucophyllatus Group

Dendropsophus marmoratus Group

Dendropsophus microcephalus Group

Dendropsophus minimus Group

Dendropsophus minutus Group

Dendropsophus parviceps Group

Species of Dendropsophus Unassigned to Group

There are several species of Dendropsophus that have not been associated with any group. These are: Dendropsophus amicorum (Mijares-Urrutia, 1998), new comb.; Dendropsophus battersbyi (Rivero, 1961), new comb.; Dendropsophus haraldschultzi (Bokermann, 1962), new comb.; Dendropsophus stingi (Kaplan, 1994), new comb.; Dendropsophus tintinnabulum (Melin, 1941), new comb.; Dendropsophus yaracuyanus (Mijares-Urrutia and Rivero, 2000), new comb.

Scinax catharinae Clade

Scinax catharinae Group

Scinax perpusillus Group

Scinax ruber Clade

Scinax rostratus Group

Scinax uruguayus Group

Species of the Scinax ruber Clade Unassigned to a Species Group