20-Month monitoring of SARS-CoV-2 in wastewater of Curitiba, in Southern Brazil.

The COVID-19 pandemic resulted in the collapse of healthcare systems and led to the development and application of several approaches of wastewater-based epidemiology to monitor infected populations. The main objective of this study was to carry out a SARS-CoV-2 wastewater based surveillance in Curitiba, Southern Brazil Sewage samples were collected weekly for 20 months at the entrance of five treatment plants representing the entire city and quantified by qPCR using the N1 marker. The viral loads were correlated with epidemiological data. The correlation by sampling points showed that the relationship between the viral loads and the number of reported cases was best described by a cross-correlation function, indicating a lag between 7 and 14 days amidst the variables, whereas the data for the entire city presented a higher correlation (0.84) with the number of positive tests at lag 0 (sampling day). The results also suggest that the Omicron VOC resulted in higher titers than the Delta VOC. Overall, our results showed that the approach used was robust as an early warning system, even with the use of different epidemiological indicators or changes in the virus variants in circulation. Therefore, it can contribute to public decision-makers and health interventions, especially in vulnerable and low-income regions with limited clinical testing capacity. Looking toward the future, this approach will contribute to a new look at environmental sanitation and should even induce an increase in sewage coverage rates in emerging countries.

A review of the diagnosis and geographical distribution of the recently described flea toad Brachycephalus sulfuratus in relation to B. hermogenesi (Anura: Brachycephalidae).

BACKGROUND: The flea toad Brachycephalus sulfuratus was recently described from southeastern and southern Brazil. In its description, the authors overlooked previous records of flea toads that had been identified as "Brachycephalus sp. nov." and B. hermogenesi occurring in the same regions, which could suggest the possibility of up to three flea toads coexisting in southern Brazil. In addition, B. sulfuratus is characterized by substantial phenotypic variability, to an extent that compromises its current diagnosis with respect to its congener B. hermogenesi. Therefore, the current state-of-affairs regarding the geographical distribution of these two species and the identification of previously known populations is hitherto uncertain. Our goals are to reassess previous records of flea toads attributable to B. hermogenesi, B. sulfuratus and "Brachycephalus sp. nov.", considering the description of B. sulfuratus, and to review the diagnosis of B. sulfuratus. METHODS: A critical analysis of the species identity of flea toad specimens attributable to B. hermogenesi, B. sulfuratus, or to a potentially undescribed species from southeastern and southern Brazil was based either on the analysis of morphology or on their advertisement calls. These analyses include our independent examinations of specimens and, when not possible, examinations of published descriptions. To allow for a consistent comparison of advertisement calls between B. hermogenesi and B. sulfuratus, we made recordings of both species, including in the type locality of the former. RESULTS: We found that morphological and call characters originally proposed as diagnostic for B. sulfuratus in relation to B. hermogenesi vary intraspecifically. Live individuals with ventral yellow spots correspond to B. sulfuratus; individuals without yellow spots can be either B. sulfuratus or B. hermogenesi. In preservative, they are indistinguishable. Previous records of Brachycephalus sp. nov. correspond to B. sulfuratus. We propose that the reduced number of notes per call and the presence of only isolated notes in the call of B. sulfuratus, as opposed to a high number of notes per call with isolated notes and note groups in the call of B. hermogenesi, as the only diagnostic characters between them. Regarding their distributions and based in our assessment, only B. sulfuratus occurs in southern Brazil, without any overlap with B. hermogenesi. There is a narrow gap between the distributions of these species around the southeast of the city of São Paulo. Our revision also revealed that some records previously attributed to B. hermogenesi in Rio de Janeiro and north São Paulo represent a distinct, unidentified flea toad that is not B. sulfuratus. Both species occur side by side in Corcovado, São Paulo, a locality from where five paratypes of B. hermogenesi were obtained. Biogeographic events that might have led to vicariance between B. hermogenesi and B. sulfuratus are discussed.

Phylogenetic relationships of diurnal, phytotelm-breeding Melanophryniscus (Anura: Bufonidae) based on mitogenomic data.

Melanophryniscus is a bufonid frog genus with a broad geographic distribution over southeastern South America. In recent years, several new species of Melanophryniscus have been discovered in southern Brazil showing a distinctive life-history strategy for the genus - breeding in phytotelmata - as well as a strong association with high-altitude regions. In this study, we use mitogenomic data to infer the phylogenetic relationships among diurnal, phytotelm-breeding Melanophryniscus and to determine the timing of their divergence. We obtained the mitochondrial genomes (not including the control region) for eight individuals of Melanophryniscus representing all three described species (M. alipioi, M. milanoi, and M. xanthostomus), as well as some recently-discovered and potentially new species. Gene order was conserved in all species and corresponded to the general order found in bufonids. Although the phylogenetic relationships among the studied species was poorly supported, dating confirmed that they diverged during the Pleistocene, suggesting that phytotelm breeding could have arisen during drier periods in the glacial/interglacial cycles due to a decrease in the availability of permanent streams or ephemeral/temporary streams or ponds in which Melanophryniscus species commonly breed.

Development of a real-time PCR assay for the detection of the golden mussel ( Limnoperna fortunei , Mytilidae) in environmental samples.

The golden mussel, Limnoperna fortunei, is among the most devastating invasive species in freshwater habitats worldwide, leading to severe environmental disturbances and economic losses. Therefore, management efforts would be greatly improved by methods that efficiently detect and quantify the abundance of the golden mussel in freshwater habitats, particularly in early stages of colonization. In this study, we describe a highly-sensitive real-time PCR assay targeting a 100-bp region of the COI mitochondrial gene of the golden mussel. The method was able to detect as little as 0.225 pg of target DNA. This assay represents an important contribution to surveillance methods, as well as to optimize field measures to contain and manage populations of the golden mussel in its introduced range.

Development of a real-time PCR assay for the detection of the golden mussel ( Limnoperna fortunei, Mytilidae) in environmental samples

ABSTRACT The golden mussel, Limnoperna fortunei, is among the most devastating invasive species in freshwater habitats worldwide, leading to severe environmental disturbances and economic losses. Therefore, management efforts would be greatly improved by methods that efficiently detect and quantify the abundance of the golden mussel in freshwater habitats, particularly in early stages of colonization. In this study, we describe a highly-sensitive real-time PCR assay targeting a 100-bp region of the COI mitochondrial gene of the golden mussel. The method was able to detect as little as 0.225 pg of target DNA. This assay represents an important contribution to surveillance methods, as well as to optimize field measures to contain and manage populations of the golden mussel in its introduced range.

Geographical and altitudinal distribution of Brachycephalus (Anura: Brachycephalidae) endemic to the Brazilian Atlantic Rainforest.

Mountains of the Brazilian Atlantic Forest can act as islands of cold and wet climate, leading to the isolation and speciation of species with low dispersal capacity, such as the toadlet species of the genus Brachycephalus. This genus is composed primarily by diurnal species, with miniaturized body sizes (<2.5 cm), inhabiting microhabitats in the leaf litter of montane forests. Still, little is known about the geographical distribution, altitudinal range, and ecological limits of most Brachycephalus species. In this study, we review the available data on the geographical and altitudinal distribution of Brachycephalus based on occurrence records compiled from literature and museums, both for the genus as a whole and separately for the three recently proposed groups of species (ephippium, didactylus, and pernix). The final ensemble dataset comprised 333 records, 120 localities, 28 described species, and six undescribed ones. Species were recorded in six relief units, the richest of which being the Serra do Mar, with 30 species. When the Serra do Mar is subdivided into three subunits, Northern, Central and Southern Serra do Mar, the number of species increase from north to the south, with records of six, nine, and 16 species, respectively. We were able to estimate the extent of occurrence of nearly half of the described species, and the resulting estimates indicate that many of them show remarkably small ranges, some of which less than 50 ha. Brachycephalus species are present from sea level to roughly 1,900 m a.s.l., with the highest richness being found between 751 and 1,000 m a.s.l. (21 spp.). The species with the broadest altitudinal range were B. didactylus (1,075 m) and Brachycephalus sp. 1 (1,035 m), both in the didactylus group, and B. ephippium (1,050 m), of the ephippium group. The broadest altitudinal amplitude for species of the pernix group was recorded for B. brunneus (535 m). The lowest altitudinal records for the pernix group were at 845 m a.s.l. in the state of Paraná and at 455 m a.s.l. in the state of Santa Catarina. The altitudinal occurrence in the pernixspecies group seems to decrease southward. Syntopy between species is also reviewed.

Multiple evolutionary units and demographic stability during the last glacial maximum in the Scytalopus speluncae complex (Aves: Rhinocryptidae).

The Atlantic Forest (AF) of South America harbors one of the world's highest bird species richness, but to date there is a deficient understanding of the spatial patterns of genetic diversity and the evolutionary history of this biome. Here we estimated the phylogenetic and populational history of the widespread Mouse-colored Tapaculo (Scytalopus speluncae) complex across the Brazilian AF, using data from two mitochondrial genes and 12 microsatellite loci. Both markers uncovered several cryptic, mostly allopatric and well-supported lineages that may represent distinct species-level taxa. We investigated whether diversification in S. speluncae is compatible with the Carnaval-Moritz model of Pleistocene refugia. We found that northern lineages have high levels of genetic diversity, agreeing with predictions of more stable forest refugia in these areas. In contrast, southern lineages have lower levels of mtDNA diversity with a signature of population expansion that occurred earlier (∼0.2Mya) than the last glacial maximum. This result suggests that the AF may be stable enough to maintain endemic taxa through glacial cycles. Moreover, we propose that the "mid-Pleistocene climate transition" between 1.2 and 0.7million years ago, from a warmer to a colder climate, may have played an important but mostly overlooked role in the evolution of AF montane taxa.

Three New Species of Phytotelm-Breeding Melanophryniscus from the Atlantic Rainforest of Southern Brazil (Anura: Bufonidae).

Three new species of Melanophryniscus are described from the Serra do Mar mountain range of the state of Santa Catarina, southern Brazil. All species are found at intermediate to high altitudes and share phytotelm-breeding as their reproductive strategy. The new species are distinguished from other phytotelm-breeding Melanophryniscus based on different combinations of the following traits: snout-vent length, presence of white and/or yellow spots on forearms, mouth, belly and cloaca, pattern and arrangement of warts, and presence and number of corneous spines. The discovery of these species in a rather restricted geographical area suggests that the diversity of phytotelm-breeding species of Melanophryniscus might be severely underestimated. The conservation status of these species is of particular concern, given that one of them is at risk of extinction not only due to its restricted habitat, but also because of anthropogenic disturbances.

Seven new microendemic species of Brachycephalus (Anura: Brachycephalidae) from southern Brazil.

Brachycephalus (Anura: Brachycephalidae) is a remarkable genus of miniaturized frogs of the Brazilian Atlantic Rainforest. Many of its species are highly endemic to cloud forests, being found only on one or a few mountaintops. Such level of microendemism might be caused by their climatic tolerance to a narrow set of environmental conditions found only in montane regions. This restriction severely limits the chance of discovery of new species, given the difficulty of exploring these inaccessible habitats. Following extensive fieldwork in montane areas of the southern portion of the Atlantic Rainforest, in this study we describe seven new species of Brachycephalus from the states of Paraná and Santa Catarina, southern Brazil. These species can be distinguished from one another based on coloration and the level of rugosity of the skin in different parts of their body. These discoveries increase considerably the number of described species of Brachycephalus in southern Brazil.