THE AMPHIBIAN TREE OF LIFE

The evidentiary basis of the currently accepted classification of living amphibians is discussed and shown not to warrant the degree of authority conferred on it by use and tradition. A new taxonomy of living amphibians is proposed to correct the deficiencies of the old one. This new taxonomy is based on the largest phylogenetic analysis of living Amphibia so far accomplished. We combined the comparative anatomical character evidence of Haas (2003) with DNA sequences from the mitochondrial transcription unit H1 (12S and 16S ribosomal RNA and tRNAValine genes, ø 2,400 bp of mitochondrial sequences) and the nuclear genes histone H3, rhodopsin, tyrosinase, and seven in absentia, and the large ribosomal subunit 28S (ø 2,300 bp of nuclear sequences; ca. 1.8 million base pairs; x¯ 5 3.7 kb/terminal). The dataset includes 532 terminals sampled from 522 species representative of the global diversity of amphibians as well as seven of the closest living relatives of amphibians for outgroup comparisons. The primary purpose of our taxon sampling strategy was to provide strong tests of the monophyly of all ‘‘family-group’’ taxa. All currently recognized nominal families and subfamilies were sampled, with the exception of Protohynobiinae (Hynobiidae). Many of the currently recognized genera were also sampled. Although we discuss the monophyly of genera, and provide remedies for nonmonophyly where possible, we also make recommendations for future research. A parsimony analysis was performed under Direct Optimization, which simultaneously optimizes nucleotide homology (alignment) and tree costs, using the same set of assumptions throughout the analysis. Multiple search algorithms were run in the program POY over a period of seven months of computing time on the AMNH Parallel Computing Cluster. Results demonstrate that the following major taxonomic groups, as currently recognized, are nonmonophyletic: Ichthyophiidae (paraphyletic with respect to Uraeotyphlidae), Caeciliidae (paraphyletic with respect to Typhlonectidae and Scolecomorphidae), Salamandroidea (paraphyletic with respect to Sirenidae), Leiopelmatanura (paraphyletic with respect to Ascaphidae), Discoglossanura (paraphyletic with respect to Bombinatoridae), Mesobatrachia (paraphyletic with respect to Neobatrachia), Pipanura (paraphyletic with respect to Bombinatoridae and Discoglossidae/Alytidae), Hyloidea (in the sense of containing Heleophrynidae; paraphyletic with respect to Ranoidea), Leptodactylidae (polyphyletic, with Batrachophrynidae forming the sister taxon of Myobatrachidae 1 Limnodynastidae, and broadly paraphyletic with respect to Hemiphractinae, Rhinodermatidae, Hylidae, Allophrynidae, Centrolenidae, Brachycephalidae, Dendrobatidae, and Bufonidae), Microhylidae (polyphyletic, with Brevicipitinae being the sister taxon of Hemisotidae), Microhylinae (poly/paraphyletic with respect to the remaining non-brevicipitine microhylids), Hyperoliidae (para/polyphyletic, with Leptopelinae forming the sister taxon of Arthroleptidae 1 Astylosternidae), Astylosternidae (paraphyletic with respect to Arthroleptinae), Ranidae (paraphyletic with respect to Rhacophoridae and Mantellidae). In addition, many subsidiary taxa are demonstrated to be nonmonophyletic, such as (1) Eleutherodactylus with respect to Brachycephalus; (2) Rana (sensu Dubois, 1992), which is polyphyletic, with various elements falling far from each other on the tree; and (3) Bufo, with respect to several nominal bufonid genera. A new taxonomy of living amphibians is proposed, and the evidence for this is presented to promote further investigation and data acquisition bearing on the evolutionary history of amphibians. The taxonomy provided is consistent with the International Code of Zoological Nomenclature (ICZN, 1999). Salient features of the new taxonomy are (1) the three major groups of living amphibians, caecilians/Gymnophiona, salamanders/Caudata, and frogs/Anura, form a monophyletic group, to which we restrict the name Amphibia; (2) Gymnophiona forms the sister taxon of Batrachia (salamanders 1 frogs) and is composed of two groups, Rhinatrematidae and Stegokrotaphia; (3) Stegokrotaphia is composed of two families, Ichthyophiidae (including Uraeotyphlidae) and Caeciliidae (including Scolecomorphidae and Typhlonectidae, which are regarded as subfamilies); (4) Batrachia is a highly corroborated monophyletic group, composed of two taxa, Caudata (salamanders) and Anura (frogs); (5) Caudata is composed of two taxa, Cryptobranchoidei (Cryptobranchidae and Hynobiidae) and Diadectosalamandroidei new taxon (all other salamanders); (6) Diadectosalamandroidei is composed of two taxa, Hydatinosalamandroidei new taxon (composed of Perennibranchia and Treptobranchia new taxon) and Plethosalamandroidei new taxon; (7) Perennibranchia is composed of Proteidae and Sirenidae; (8) Treptobranchia new taxon is composed of two taxa, Ambystomatidae (including Dicamptodontidae) and Salamandridae; (9) Plethosalamandroidei new taxon is composed of Rhyacotritonidae and Xenosalamandroidei new taxon; (10) Xenosalamandroidei is composed of Plethodontidae and Amphiumidae; (11) Anura is monophyletic and composed of two clades, Leiopelmatidae (including Ascaphidae) and Lalagobatrachia new taxon (all other frogs); (12) Lalagobatrachia is composed of two clades, Xenoanura (Pipidae and Rhinophrynidae) and Sokolanura new taxon (all other lalagobatrachians); (13) Bombinatoridae and Alytidae (former Discoglossidae) are each others’ closest relatives and in a clade called Costata, which, excluding Leiopelmatidae and Xenoanura, forms the sister taxon of all other frogs, Acosmanura; (14) Acosmanura is composed of two clades, Anomocoela (5 Pelobatoidea of other authors) and Neobatrachia; (15) Anomocoela contains Pelobatoidea (Pelobatidae and Megophryidae) and Pelodytoidea (Pelodytidae and Scaphiopodidae), and forms the sister taxon of Neobatrachia, together forming Acosmanura; (16) Neobatrachia is composed of two clades, Heleophrynidae, and all other neobatrachians, Phthanobatrachia new taxon; (17) Phthanobatrachia is composed of two major units, Hyloides and Ranoides; (18) Hyloides comprises Sooglossidae (including Nasikabatrachidae) and Notogaeanura new taxon (the remaining hyloids); (19) Notogaeanura contains two taxa, Australobatrachia new taxon and Nobleobatrachia new taxon; (20) Australobatrachia is a clade composed of Batrachophrynidae and its sister taxon, Myobatrachoidea (Myobatrachidae and Limnodynastidae), which forms the sister taxon of all other hyloids, excluding sooglossids; (21) Nobleobatrachia new taxon, is dominated at its base by frogs of a treefrog morphotype, several with intercalary phalangeal cartilages—Hemiphractus (Hemiphractidae) forms the sister taxon of the remaining members of this group, here termed Meridianura new taxon; (22) Meridianura comprises Brachycephalidae (former Eleutherodactylinae 1 Brachycephalus) and Cladophrynia new taxon; (23) Cladophrynia is composed of two groups, Cryptobatrachidae (composed of Cryptobatrachus and Stefania, previously a fragment of the polyphyletic Hemiphractinae) and Tinctanura new taxon; (24) Tinctanura is composed of Amphignathodontidae (Gastrotheca and Flectonotus, another fragment of the polyphyletic Hemiphractinae) and Athesphatanura new taxon; (25) Athesphatanura is composed of Hylidae (Hylinae, Pelodryadinae, and Phyllomedusinae, and excluding former Hemiphractinae, whose inclusion would have rendered this taxon polyphyletic) and Leptodactyliformes new taxon; (26) Leptodactyliformes is composed of Diphyabatrachia new taxon (composed of Centrolenidae [including Allophryne] and Leptodactylidae, sensu stricto, including Leptodactylus and relatives) and Chthonobatrachia new taxon; (27) Chthonobatrachia is composed of a reformulated Ceratophryidae (which excludes such genera as Odontophrynus and Proceratophrys and includes other taxa, such as Telmatobius) and Hesticobatrachia new taxon; (28) Hesticobatrachia is composed of a reformulated Cycloramphidae (which includes Rhinoderma) and Agastorophrynia new taxon; (29) Agastorophrynia is composed of Bufonidae (which is partially revised) and Dendrobatoidea (Dendrobatidae and Thoropidae); (30) Ranoides new taxon forms the sister taxon of Hyloides and is composed of two major monophyletic components, Allodapanura new taxon (microhylids, hyperoliids, and allies) and Natatanura new taxon (ranids and allies); (31) Allodapanura is composed of Microhylidae (which is partially revised) and Afrobatrachia new taxon; (32) Afrobatrachia is composed of Xenosyneunitanura new taxon (the ‘‘strange-bedfellows’’ Brevicipitidae [formerly in Microhylidae] and Hemisotidae) and a more normal-looking group of frogs, Laurentobatrachia new taxon (Hyperoliidae and Arthroleptidae, which includes Leptopelinae and former Astylosternidae); (33) Natatanura new taxon is composed of two taxa, the African Ptychadenidae and the worldwide Victoranura new taxon; (34) Victoranura is composed of Ceratobatrachidae and Telmatobatrachia new taxon; (35) Telmatobatrachia is composed of Micrixalidae and a worldwide group of ranoids, Ametrobatrachia new taxon; (36) Ametrobatrachia is composed of Africanura new taxon and Saukrobatrachia new taxon; (37) Africanura is composed of two taxa: Phrynobatrachidae (Phrynobatrachus, including Dimorphognathus and Phrynodon as synonyms) and Pyxicephaloidea; (38) Pyxicephaloidea is composed of Petropedetidae (Conraua, Indirana, Arthroleptides, and Petropedetes), and Pyxicephalidae (including a number of African genera, e.g. Amietia [including Afrana], Arthroleptella, Pyxicephalus, Strongylopus, and Tomopterna); and (39) Saukrobatrachia new taxon is the sister taxon of Africanura and is composed of Dicroglossidae and Aglaioanura new taxon, which is, in turn, composed of Rhacophoroidea (Mantellidae and Rhacophoridae) and Ranoidea (Nyctibatrachidae and Ranidae, sensu stricto). Many generic revisions are made either to render a monophyletic taxonomy or to render a taxonomy that illuminates the problems in our understanding of phylogeny, so that future work will be made easier. These revisions are: (1) placement of Ixalotriton and Lineatriton (Caudata: Plethodontidae: Bolitoglossinae) into the synonymy of Pseudoeurycea, to render a monophyletic Pseudoeurycea; (2) placement of Haideotriton (Caudata: Plethodontidae: Spelerpinae) into the synonymy of Eurycea, to render a monophyletic Eurycea; (3) placement of Nesomantis (Anura: Sooglossidae) into the synonymy of Sooglossus, to assure a monophyletic Sooglossus; (4) placement of Cyclorana and Nyctimystes (Anura: Hylidae: Pelodryadinae) into Litoria, but retaining Cyclorana as a subgenus, to provide a monophyletic Litoria; (5) partition of ‘‘Limnodynastes’’ (Anura: Limnodynastidae) into Limnodynastes and Opisthodon to render monophyletic genera; (6) placement of Adenomera, Lithodytes, and Vanzolinius (Anura: Leptodactylidae) into Leptodactylus, to render a monophyletic Leptodactylus; (7) partition of ‘‘Eleutherodactylus’’ (Anura: Brachycephalidae) into Craugastor, ‘‘Eleutherodactylus’’, ‘‘Euhyas’’, ‘‘Pelorius’’, and Syrrhophus to outline the taxonomic issues relevant to the paraphyly of this nominal taxon to other nominal genera; (8) partition of ‘‘Bufo’’ (Anura: Bufonidae) into a number of new or revived genera (i.e., Amietophrynus new genus, Anaxyrus, Chaunus, Cranopsis, Duttaphrynus new genus, Epidalea, Ingerophrynus new genus, Nannophryne, Peltophryne, Phrynoidis, Poyntonophrynus new genus; Pseudepidalea new genus, Rhaebo, Rhinella, Vandijkophrynus new genus); (9) placement of the monotypic Spinophrynoides (Anura: Bufonidae) into the synonymy of (formerly monotypic) Altiphrynoides to make for a more informative taxonomy; (10) placement of the Bufo taitanus group and Stephopaedes (as a subgenus) into the synonymy of Mertensophryne (Anura: Bufonidae); (11) placement of Xenobatrachus (Anura: Microhylidae: Asterophryinae) into the synonymy of Xenorhina to render a monophyletic Xenorhina; (12) transfer of a number of species from Plethodontohyla to Rhombophryne (Microhylidae: Cophylinae) to render a monophyletic Plethodontohyla; (13) placement of Schoutedenella (Anura: Arthroleptidae) into the synonymy of Arthroleptis; (14) transfer of Dimorphognathus and Phrynodon (Anura: Phrynobatrachidae) into the synonymy of Phrynobatrachus to render a monophyletic Phrynobatrachus; (15) placement of Afrana into the synonymy of Amietia (Anura: Pyxicephalidae) to render a monophyletic taxon; (16) placement of Chaparana and Paa into the synonymy of Nanorana (Anura: Dicroglossidae) to render a monophyletic genus; (17) recognition as genera of Ombrana and Annandia (Anura: Dicroglossidae: Dicroglossinae) pending placement of them phylogenetically; (18) return of Phrynoglossus into the synonymy of Occidozyga to resolve the paraphyly of Phrynoglossus (Anura: Dicroglossidae: Occidozyginae); (19) recognition of Feihyla new genus for Philautus palpebralis to resolve the polyphyly of ‘‘Chirixalus’’; (20) synonymy of ‘‘Chirixalus’’ with Chiromantis to resolve the paraphyly of ‘‘Chirixalus’’; (21) recognition of the genus Babina, composed of the former subgenera of Rana, Babina and Nidirana (Anura: Ranidae); (22) recognition of the genera Clinotarsus, Humerana, Nasirana, Pelophylax, Pterorana, Pulchrana, and Sanguirana, formerly considered subgenera of Rana (Anura: Ranidae), with no special relationship to Rana (sensu stricto); (23) consideration of Glandirana (Anura: Ranidae), formerly a subgenus of Rana, as a genus, with Rugosa as a synonym; (24) recognition of Hydrophylax (Anura: Ranidae) as a genus, with Amnirana and most species of former Chalcorana included in this taxon as synonyms; (25) recognition of Hylarana (Anura: Ranidae) as a genus and its content redefined; (26) redelimitation of Huia to include as synonyms Eburana and Odorrana (both former subgenera of Rana); (27) recognition of Lithobates (Anura: Ranidae) for all species of North American ‘‘Rana’’ not placed in Rana sensu stricto (Aquarana, Pantherana, Sierrana, Trypheropsis, and Zweifelia considered synonyms of Lithobates); (28) redelimitation of the genus Rana as monophyletic by inclusion as synonyms Amerana, Aurorana, Pseudoamolops, and Pseudorana, and exclusion of all other former subgenera; (29) redelimitation of the genus Sylvirana (Anura: Ranidae), formerly a subgenus of Rana, with Papurana and Tylerana included as synonyms

Is The Amphibian Tree of Life really fatally flawed?

Wiens (2007, Q. Rev. Biol. 82, 55–56) recently published a severe critique of Frost et al.\u27s (2006, Bull. Am. Mus. Nat. Hist. 297, 1–370) monographic study of amphibian systematics, concluding that it is “a disaster” and recommending that readers “simply ignore this study”. Beyond the hyperbole, Wiens raised four general objections that he regarded as “fatal flaws”: (1) the sampling design was insufficient for the generic changes made and taxonomic changes were made without including all type species; (2) the nuclear gene most commonly used in amphibian phylogenetics, RAG-1, was not included, nor were the morphological characters that had justified the older taxonomy; (3) the analytical method employed is questionable because equally weighted parsimony “assumes that all characters are evolving at equal rates”; and (4) the results were at times “clearly erroneous”, as evidenced by the inferred non-monophyly of marsupial frogs. In this paper we respond to these criticisms. In brief: (1) the study of Frost et al. did not exist in a vacuum and we discussed our evidence and evidence previously obtained by others that documented the non-monophyletic taxa that we corrected. Beyond that, we agree that all type species should ideally be included, but inclusion of all potentially relevant type species is not feasible in a study of the magnitude of Frost et al. and we contend that this should not prevent progress in the formulation of phylogenetic hypotheses or their application outside of systematics. (2) Rhodopsin, a gene included by Frost et al. is the nuclear gene that is most commonly used in amphibian systematics, not RAG-1. Regardless, ignoring a study because of the absence of a single locus strikes us as unsound practice. With respect to previously hypothesized morphological synapomorphies, Frost et al. provided a lengthy review of the published evidence for all groups, and this was used to inform taxonomic decisions. We noted that confirming and reconciling all morphological transformation series published among previous studies needed to be done, and we included evidence from the only published data set at that time to explicitly code morphological characters (including a number of traditionally applied synapomorphies from adult morphology) across the bulk of the diversity of amphibians (Haas, 2003, Cladistics 19, 23–90). Moreover, the phylogenetic results of the Frost et al. study were largely consistent with previous morphological and molecular studies and where they differed, this was discussed with reference to the weight of evidence. (3) The claim that equally weighted parsimony assumes that all characters are evolving at equal rates has been shown to be false in both analytical and simulation studies. (4) The claimed “strong support” for marsupial frog monophyly is questionable. Several studies have also found marsupial frogs to be non-monophyletic. Wiens et al. (2005, Syst. Biol. 54, 719–748) recovered marsupial frogs as monophyletic, but that result was strongly supported only by Bayesian clade confidence values (which are known to overestimate support) and bootstrap support in his parsimony analysis was \u3c 50%. Further, in a more recent parsimony analysis of an expanded data set that included RAG-1 and the three traditional morphological synapomorphies of marsupial frogs, Wiens et al. (2006, Am. Nat. 168, 579–596) also found them to be non-monophyletic. Although we attempted to apply the rule of monophyly to the naming of taxonomic groups, our phylogenetic results are largely consistent with conventional views even if not with the taxonomy current at the time of our writing. Most of our taxonomic changes addressed examples of non-monophyly that had previously been known or suspected (e.g., the non-monophyly of traditional Hyperoliidae, Microhylidae, Hemiphractinae, Leptodactylidae, Phrynobatrachus, Ranidae, Rana, Bufo; and the placement of Brachycephalus within “Eleutherodactylus”, and Lineatriton within “Pseudoeurycea”), and it is troubling that Wiens and others, as evidenced by recent publications, continue to perpetuate recognition of non-monophyletic taxonomic groups that so profoundly misrepresent what is known about amphibian phylogeny

Multiple connections between Amazonia and Atlantic Forest shaped the T phylogenetic and morphological diversity of Chiasmocleis Mehely, 1904 (Anura: Microhylidae: Gastrophryninae)

Chiasmocleis is the most species-rich genus of Neotropical microhylids. Herein, we provide the first comprehensive multilocus phylogeny for the genus, including all but 3 of the 34 recognized species and multiple individuals per species. We discuss cryptic speciation, species discovery, patterns of morphological evolution, and provide a historical biogeographic analysis to account for the current distribution of the genus. Diversification of Chiasmocleis from other New World microhylids began during the Eocene, app. 40 mya, in forested areas, and current diversity seems to be a product of recurrent connections between the Atlantic Forest and Amazonia. Small-sized species evolved independently three times in Chiasmocleis. Furthermore, the extremely small-bodied (i.e. miniaturized) species with associated loss of digits, phalanges, and pectoral girdle cartilages evolved only once and are restricted to Amazonia. Using the phylogeny, we recognized three subgenera within Chiasmocleis: Chiasmocleis Méhely, 1904, Relictus subg. nov., and Syncope Walker, 1973. The recognition of the subgenus Syncopeinforms future research on patterns of miniaturization in the genus, and the subgenus Relictus highlights isolation of an endemic and species-poor lineage to the Atlantic Forest, early (about 40 mya) in the history of Chiasmocleis

LÍQUIDO FOLICULAR EQÜINO NA MATURAÇÃO IN VITRO DE OÓCITOS BOVINOS

O desenvolvimento embrionário de oócitos bovinos maturados in vitro (MIV) foi avaliado em meio suplementado com líquido folicular eqüino (Lfe). Foram distribuídos 1045 oócitos em 11 repetições formando três grupos tratamentos (T1, T2, T3) e um controle (C). O meio de maturação utilizado foi o TCM-199 acrescido de piruvato de sódio, hormônio folículo estimulante recombinante (rFSHh) e hormônio luteinizante equino (LHe). Suplementou-se esse meio com 10% de soro de égua em estro para o grupo controle e para T1, T2 e T3, o meio foi suplementado com 5, 10, e 20% de LFe, respectivamente. Os oócitos foram maturados in vitro (MIV) por 24h. A fecundação in vitro (FIV) foi realizada em meio Talp-Fert. A MIV e a FIV foram realizadas em estufa a 39ºC com 5% de CO2 em ar e umidade saturada. Os zigotos foram cultivados em meio SOFaaci, sob óleo mineral no interior de bolsas plásticas gaseificadas. As taxas de clivagem e de blastocistos foram observadas diariamente (D), e em D7, foram superiores (P0,05) às do grupo controle. Em D9, a taxa de blastocistos do T2 foi superior (P0,05). O LFe, na concentração de 10% pode ser utilizado, em substituição ao soro de égua em estro para suplementar o meio de MIV de oócitos bovinos. Equine follicular fluid on in vitro maturation of bovine oocytes Abstract Embryo development of bovine oocytes was evaluated using maturation medium supplemented with equine follicular fluid (eFF). One thousand and forty five (1045) oocytes were distributed in 11 replications forming three treatment groups (T1, T2 e T3) and one Control (C). TCM-199 added with sodium pyruvate, rFSHh and LHe was used as maturation medium. This medium was supplemented with 10% estrous mare serum for Control group, and 5, 10, and 20% eFF, respectively, for T1, T2 e T3 groups. In vitro maturation (IVM) of all groups was performed during 24h. In vitro fertilization (IVF) was performed in TALP-FERT medium. IVM and IVF were carried out in an incubator at 39ºC with 5% CO2 in air and saturated humidity. Zygotes were cultured in SOFaaci medium, under mineral oil in gasified bags. Cleavage and blastocyst rates were daily observed (D), and at D7, were higher (P0.05) for those from control group. At D9, blastocyst rate of T2 was higher (P0.05). The eFF, at a 10% concentration, can replace the use of estrous mare serum to supplement the IVM medium of bovine oocytes

The Advertisement Calls and Distribution of Two Sympatric Species of \u3cem\u3eChiasmocleis\u3c/em\u3e (Méhely 1904) (Anura, Microhylidae, Gastrophryninae) from the Atlantic Forest

The advertisement calls of Chiasmocleis cordeiroi and C. crucis are described for populations from the municipalities of Igrapiúna and Camacan, respectively, state of Bahia, Brazil. Both calls consist of multipulsed notes produced in series. Differences between the two calls are: dominant frequency, higher in C. cordeiroi (range 4500-4898 Hz; C. crucis range 4069-4435 Hz); note rate, higher in C. cordeiroi (range 6.20--7.46 s/note; C. crucis range 5.17-5.59 s/note); pulse rate, higher in C. cordeiroi (151.82-194.83 s/note; C. crucis range 125.30- 142.12 s/note); and the structure of the modulation patterns of the notes. Moreover, the advertisement calls of C. crucis and C. cordeiroi are more similar than the calls of all syntopic congeners. Furthermore, the current distribution of both species was extended