Draft genome and multi-tissue transcriptome assemblies of the Neotropical leaf-frog Phyllomedusa bahiana.

Amphibians are increasingly threatened worldwide, but the availability of genomic resources that could be crucial for implementing informed conservation practices lags well behind that for other vertebrate groups. Here, we describe draft de novo genome, mitogenome, and transcriptome assemblies for the Neotropical leaf-frog Phyllomedusa bahiana native to the Brazilian Atlantic Forest and Caatinga. We used a combination of PacBio long reads and Illumina sequencing to produce a 4.74-Gbp contig-level genome assembly, which has a contiguity comparable to other recent nonchromosome level assemblies. The assembled mitogenome comprises 16,239 bp and the gene content and arrangement are similar to other Neobratrachia. RNA-sequencing from 8 tissues resulted in a highly complete (86.3%) reference transcriptome. We further use whole-genome resequencing data from P. bahiana and from its sister species Phyllomedusa burmeisteri, to demonstrate how our assembly can be used as a backbone for population genomics studies within the P. burmeisteri species group. Our assemblies thus represent important additions to the catalog of genomic resources available from amphibians.

Phylogenetic relationships of the Boana pulchella Group (Anura: Hylidae).

In this paper we present a phylogenetic analysis of the treefrogs of the Boana pulchella Group with the goals of (1) providing a rigorous test of its monophyly; (2) providing a test of relationships supported in previous studies; and (3) exploring the relationships of the several species not included in previous analyses. The analyses included>300 specimens of 37 of the 38 species currently included in the group, plus 36 outgroups, exemplars of the diversity of Boana and the other genera of the hylid tribe Cophomantini. The dataset included eight mitochondrial genes (12S, 16S, CytB, COI, ND1, tRNAIle, tRNALeu, and tRNAVal) and five nuclear genes (RHO, TYR, RAG-1, CXCR4, SIAH1). The phylogenetic analyses recover the monophyly of the B. pulchella Group with lower support than previous studies, as a result of the inclusion of the B. claresignata Group, which is recovered as its sister taxon. Within the B. pulchella Group, the inclusion of almost all species of the group had little impact on previous notions of its phylogeny, except for the rejection of the hypothesized B. polytaenia Clade (B. goiana and B. phaeopleura are nested in the clade here called the B. prasina Clade), which is redefined. Phylogenetic support is strong for five major clades, which collectively include all but three of the species sampled: the B. balzani Clade (B. aguilari, B. balzani, B. gladiator, B. melanopleura, B. palaestes), the redefined B. polytaenia Clade (B. botumirim, B. buriti, B. cipoensis, B. jaguariaivensis, B. leptolineata, B. polytaenia, B. stenocephala, and two undescribed species), the B. prasina Clade (B. bischoffi, B. caingua, B. cordobae, B. goiana, B. guentheri, B. marginata, B. phaeopleura, B. prasina, B. pulchella, and one undescribed species), the B. riojana Clade (B. callipleura, B. marianitae, B. riojana), and the B. semiguttata Clade (B. caipora, B. curupi, B. joaquini, B. poaju, B. semiguttata, B. stellae, and two undescribed species). The monophyly of the B. prasina + B. riojana Clades, and that of the B. polytaenia + B. semiguttata Clades are well-supported. The relationships among these two clades, the B. balzani Clade, B. ericae + B. freicanecae, and B. cambui (representing the deepest phylogenetic splits within the B. pulchella Group) are recovered with weak support. We discuss the phenotypic evidence supporting the monophyly of the B. pulchella Group, and the taxonomy of several species, identifying three new synonyms of Boana polytaenia, one new synonym of Boana goiana, and one new synonym of B. riojana.

Lost and found: Frogs in a biodiversity hotspot rediscovered with environmental DNA.

Declines and extinctions are increasing globally and challenge conservationists to keep pace with biodiversity monitoring. Organisms leave DNA traces in the environment, e.g., in soil, water, and air. These DNA traces are referred to as environmental DNA (eDNA). The analysis of eDNA is a highly sensitive method with the potential to rapidly assess local diversity and the status of threatened species. We searched for DNA traces of 30 target amphibian species of conservation concern, at different levels of threat, using an environmental DNA metabarcoding approach, together with an extensive sequence reference database to analyse water samples from six montane sites in the Atlantic Coastal Forest and adjacent Cerrado grasslands of Brazil. We successfully detected DNA traces of four declined species (Hylodes ornatus, Hylodes regius, Crossodactylus timbuhy, and Vitreorana eurygnatha); two locally disappeared (Phasmahyla exilis and Phasmahyla guttata); and one species that has not been seen since 1968 (putatively assigned to Megaelosia bocainensis). We confirm the presence of species undetected by traditional methods, underscoring the efficacy of eDNA metabarcoding for biodiversity monitoring at low population densities, especially in megadiverse tropical sites. Our results support the potential application of eDNA in conservation biology, to evaluate persistence and distribution of threatened species in surveyed habitats or sites, and improve accuracy of red lists, especially for species undetected over long periods.

The tadpole of Physalaemus orophilus from the Atlantic rainforest of southeastern Brazil (Amphibia, Anura, Leptodactylidae).

The neotropical genus Physalaemus Fitzinger is currently composed by 48 species (Cardozo Pereyra 2018). Recently, a phylogenetic analysis aiming to investigate the internal relationships of the genus recovered two major clades, the Physalaemus cuvieri and P. signifer clades (Lourenço et al. 2015). The following species groups were retrieved in the first clade: P. biligonigerus, P. cuvieri, P. gracilis, P. henselii, and P. olfersii groups (Lourenço et al. 2015). This proposal redefined the P. olfersii group including P. olfersii (Lichtenstein Martens), P. soaresi Izecksohn, P. maximus Feio, Pombal, Caramaschi, P. feioi Cassini, Cruz, Caramaschi and P. lateristriga (Steindachner). The authors also allocated tentatively P. orophilus Cassini, Cruz, Caramaschi, and P. insperatus Cruz, Cassini, Caramaschi in the P. olfersii group due to their morphological similarity with the other species (Cruz et al. 2008; Cassini et al. 2010). Otherwise, Physalaemus aguirrei Bokermann was not recovered nested within this group, contradicting what was suggested in a previous phenetic analysis (Nascimento et al. 2005). Members of the P. olfersii group inhabit the Atlantic rainforest and most of them have a similar advertisement calls with pulsed notes, without frequency modulation and harmonic structure (Giaretta et al. 2009; Cassini et al. 2010; Lourenço et al. 2015). Regarding their larval stage, only P. soaresi, P. maximus, and P. olfersii have their tadpoles described (Weber et al. 2005; Baêta et al. 2007; Giaretta et al. 2009). Physalaemus orophilus occurs in montane Atlantic Forest sites at the eastern slope of the Espinhaço Range in the State of Minas Gerais, southeastern Brazil (Cassini et al. 2010). Herein, we describe the tadpole of P. orophilus from Quadrilátero Ferrífero mountain region, southern limit of the Espinhaço Range and compared it to the known tadpoles of the P. olfersii group.

Cryptic diversity in Brazilian endemic monkey frogs (Hylidae, Phyllomedusinae, Pithecopus) revealed by multispecies coalescent and integrative approaches.

Even though Brazil is the world leader in amphibian diversity, a significant part of its richness remains unknown or hidden under cryptic taxa. Here, we used model-based species delimitation in an integrative taxonomic approach, by gathering molecular and morphometric data to assess cryptic taxa within the monkey frogs Pithecopus rohdei, from the Atlantic Forest, and P. megacephalus, from campos rupestres ecosystem. We sampled one mitochondrial, five nuclear loci, and 18 morphometric variables. Using species-delimitation methods with genetic and morphometric data, we recovered five divergent lineages within P. rohdei and no cryptic lineages were recovered for P. megacephalus. Morphometric data show differentiation only for one of the candidate species revealed by the delimitation approaches, suggesting that individuals from Doce River basin constitute a putative species for formal taxonomic description. The time-calibrated mtDNA tree shows that P. rohdei complex lineages began to diverge in late Miocene. However, dates from the multilocus species tree are more recent, occurring in Pleistocene, and suggesting their persistence in refuges of forest and sky islands within the Atlantic Forest biome. The existence of cryptic taxa within P. rohdei is, therefore, relevant for planning conservation strategies for this species complex in the Atlantic Forest biodiversity hotspot.

Amphibian parasites of the Order Dermocystida (Ichthyosporea): current knowledge, taxonomic review and new records from Brazil.

Amphibian parasites of the Order Dermocystida (Ichthyosporea) are widespread pathogens known mainly from Europe and North America, which cause primarily a disease of skin and subcutaneous tissue in their hosts. The taxonomy of these organisms has been problematic given their conserved morphology, similar clinical disease and pathology. Currently recognized taxa belong to the three closely related genera, Amphibiocystidium, Amphibiothecum, and Rhinosporidium, whereas species of Dermocystidium and Sphaerothecum destruens include fish parasites. Here, we review the taxonomy of Dermocystida based on a molecular phylogenetic analysis, principally of amphibian parasites, including DNA sequences obtained from amphibian hosts collected in the central-eastern region of South America. A new taxonomic arrangement is proposed, which includes the designation of type material for Dermocystidium pusula, synonymization of Amphibiothecum with Dermocystidium, and the restriction of Amphibiocystidium to its type species A. ranae. We also review the taxonomic status of Dermosporidium hylarum until the present work included in the synonymy of the human and animal pathogen R. seeberi, and considered herein as a valid taxon, however. In addition, a new species of Sphaerothecum parasitic to amphibians is described, being the first record of this genus in the southern hemisphere and in an amphibian host.

A new species of the Scinax catharinae group (Anura, Hylidae) from Serra da Canastra, southwestern state of Minas Gerais, Brazil.

We describe Scinax pombali sp. n. a new species of treefrog of the Scinax catharinae group from Serra da Canastra, municipality of Capitólio (20°36'03''S, 46°17'34.9''W, 987 m a.s.l.), located in the Cerrado domains of the State of Minas Gerais, Southeastern Brazil. The new species is characterized by its small size, blotches and color pattern on dorsal surface and hidden regions of flanks and thighs, canthus rostralis lightly concave and well marked, absent nuptial pad, and lack of externally differentiated inguinal gland. Additionally, we describe the tadpole of this new species, which is characterized by the large-sized oral disc and presence of a large number of marginal papillae (two to three rows on its dorsal portion and some rows in unorganized arrangement on its lateroventral portion).

Structural diversity of nuptial pads in Phyllomedusinae (Amphibia: Anura: Hylidae).

We studied the morphological variation of the nuptial pads using light microscopy and scanning electron microscopy (SEM) in 26 species of phyllomedusines (Anura: Hylidae), representing the five currently recognized genera. All phyllomedusines have single nuptial pads with dark colored epidermal projections (EPs). Spine-shaped EPs occur in Cruziohyla calcarifer, Phrynomedusa appendiculata and in one species of Phasmahyla. The other species have roundish EPs. The density of the EPs on the pad is variable. Species in the Phyllomedusa hypochondrialis Group have EPs with a density that varies between 764 ± 58/mm(2) and 923 ± 160/mm(2). In all other studied species (including the Phyllomedusa burmeisteri and Phyllomedusa perinesos groups, Phyllomedusa camba, Phyllomedusa boliviana, Phyllomedusa sauvagii, Phyllomedusa bicolor, and Phyllomedusa tomopterna) the density of EPs varies between 108 ± 20/mm(2) and 552 ± 97/mm(2). Pores were observed with SEM in C. calcarifer, Agalychnis lemur, Agalychnis moreletii, but its presence is confirmed through histological sections on several other species. Its visibility using SEM seems to be related with the level of separation between adjacent EPs. The pores in the four studied species of Agalychnis are shown with SEM and histological sections to have a characteristic epidermal rim, that is absent in the otherphyllomedusines. Unlike most previous reports on breeding glands, those of phyllomedusines are alcian blue positive, indicating the presence of acidic mucosubstances on its secretions.

The phylogenetic relationships of the charismatic poster frogs, Phyllomedusinae (Anura, Hylidae).

The leaf or monkey frogs of the hylid subfamily Phyllomedusinae are a unique group of charismatic anurans. We present a molecular phylogenetic analysis that includes 45 of the 60 species of phyllomedusines using up to 12 genes and intervening tRNAs. The aims were to gain a better understanding of the phylogenetic position of Phrynomedusa, test the monophyly and explore the relationships among several putative lineages (Hylomantis, the H. buckleyi Group, Phasmahyla, the four species groups of Phyllomedusa, and the species of Phyllomedusa that remain unassigned to any group), and to examine the implications of our phylogeny for the evolution of several characters in phyllomedusines. The analyses resulted in a well-supported phylogenetic hypothesis that provides a historical framework for a discussion of the evolution of characters associated with reproductive biology, gliding behaviour, the physiology of waterproofing, and bioactive peptides. Implications include an earlier origin for eggless capsules than for leaf-folding behaviour during amplexus, two independent origins of gliding, and an earlier origin of reduction in evaporative water loss than uricotelism, which is a result that originally was predicted on the basis of physiology alone. Furthermore, our results support the prediction that bioactive peptides from different peptide families are to be expected in all species of Phyllomedusinae. Hylomantis (as recently redefined) is shown to be paraphyletic and the synonymy of Agalychnis is revised to remedy this problem by including both Hylomantis and Pachymedusa. © The Willi Hennig Society 2009.