The Andes of South America hosts perhaps the highest amphibian species diversity in the world, and a sizable component of that diversity is comprised of direct-developing frogs of the genus Pristimantis (Anura: Craugastoridae). In order to better understand the initial stages of species formation in these frogs, this study quantified local-scale spatial genetic structuring in three species of Pristimantis. DNA sequences of two mitochondrial gene fragments (16S and COI) were obtained from P. brevifrons, P. palmeri and P. jubatus at different locations in the Cordillera Occidental. We found high levels of genetic diversity in the three species, with highly structured populations (as measured by F_ST) in P. brevifrons and P. palmeri while P. jubatus showed panmixia. Large effective population sizes, inferred from the high levels of genetic diversity, were found in the three species and two highly divergent lineages were detected within P. jubatus and P. palmeri. Estimated divergence times among populations within P. brevifrons and P. palmeri coincide with the Pleistocene, perhaps due to similar responses to climatic cycling or recent geological history. Such insights have important implications for linking alpha and beta diversity, suggesting regional scale patterns may be associated with local scale processes in promoting differentiation among populations in the Andes.

Long‐term monitoring of frog populations is needed to understand the effects of global change. To better understand the relationships between climate variation and calling activity, we monitored an anuran assemblage in a Puerto Rican wetland by sampling the acoustic environment for one minute every 10 minutes, for 41 months. By automating data collection using passive acoustic monitoring hardware, we collected more than 110,000 recordings. These recordings were analyzed using species‐specific identification algorithms of fourEleutherodactylusspecies. The peak calling activity ofE. coqui(>0.3 detection frequency) andE. cochranae(>0.2) occurred between April and September, and there was a clear decline in activity during the dry months of January–March. There was no clear annual pattern inE. brittoniorE. juanariveroi, butE. juanariveroidid show a significant decline in calling activity over the 41‐month study (∼0.5 to ∼0.35). Calling activity ofE. coquiandE. cochranaewas positively correlated with temperature, whileE. brittoniandE. juanariveroiresponded negatively to temperature and precipitation. This difference in response to temperature and precipitation could be related to differences in body size and the location of calling sites among the four species. For example,E. brittoniandE. juanariveroiare small species that call from the tips of the vegetation. High temperatures and intense precipitation may cause them to retreat into the vegetation, and reduce calling activity. In contrast,E. coquiandE. cochranaecall lower in the vegetation and from leaf axils where they are more protected. Based on these findings, future scenarios of climate change could pose a threat for the survival of the populations of these four species. New climate regimes could negatively affect the calling activity, and thus diminish reproductive events.

Immune modulation is considered a hallmark of cancer initiation and progression. The recent development of immunotherapies has ushered in a new era of cancer treatment. These therapeutics have led to revolutionary breakthroughs; however, the efficacy of immunotherapy has been modest and is often restricted to a subset of patients. Hence, identification of which cancer patients will benefit from immunotherapy is essential. Multiplex immunofluorescence (mIF) microscopy allows for the assessment and visualization of the tumor immune microenvironment (TIME). The data output following image and machine learning analyses for cell segmenting and phenotyping consists of the following information for each tumor sample: the number of positive cells for each marker and phenotype(s) of interest, number of total cells, percent of positive cells for each marker, and spatial locations for all measured cells. There are many challenges in the analysis of mIF data, including many tissue samples with zero positive cells or “zero-inflated” data, repeated measurements from multiple TMA cores or tissue slides per subject, and spatial analyses to determine the level of clustering and co-localization between the cell types in the TIME. In this review paper, we will discuss the challenges in the statistical analysis of mIF data and opportunities for further research.

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer with a ∼50% response rate to immune checkpoint blockade (ICB) therapy. To identify predictive biomarkers, we integrated bulk and single-cell RNA sequencing (RNA-seq) with spatial transcriptomics from a cohort of 186 samples from 116 patients, including bulk RNA-seq from 14 matched pairs pre- and post-ICB. In nonresponders, tumors show evidence of increased tumor proliferation, neuronal stem cell markers, and IL1. Responders have increased type I/II interferons and preexisting tissue resident (Trm) CD8 or Vδ1 γδ T cells that functionally converge with overlapping antigen-specific transcriptional programs and clonal expansion of public T-cell receptors. Spatial transcriptomics demonstrated colocalization of T cells with B and dendritic cells, which supply chemokines and costimulation. Lastly, ICB significantly increased clonal expansion or recruitment of Trm and Vδ1 cells in tumors specifically in responders, underscoring their therapeutic importance. These data identify potential clinically actionable biomarkers and therapeutic targets for MCC. Significance: MCC serves as a model of ICB response. We utilized the largest-to-date, multimodal MCC dataset (n = 116 patients) to uncover unique tumor-intrinsic properties and immune circuits that predict response. We identified CD8 Trm and Vδ1 T cells as clinically actionable mediators of ICB response in major histocompatibility complex–high and –low MCCs, respectively.