PIEZO2 and perineal mechanosensation are essential for sexual function.

Despite the potential importance of genital mechanosensation for sexual reproduction, little is known about how perineal touch influences mating. We explored how mechanosensation affords exquisite awareness of the genitals and controls reproduction in mice and humans. Using genetic strategies and in vivo functional imaging, we demonstrated that the mechanosensitive ion channel PIEZO2 (piezo-type mechanosensitive ion channel component 2) is necessary for behavioral sensitivity to perineal touch. PIEZO2 function is needed for triggering a touch-evoked erection reflex and successful mating in both male and female mice. Humans with complete loss of PIEZO2 function have genital hyposensitivity and experience no direct pleasure from gentle touch or vibration. Together, our results help explain how perineal mechanoreceptors detect the gentlest of stimuli and trigger physiologically important sexual responses, thus providing a platform for exploring the sensory basis of sexual pleasure and its relationship to affective touch.

PIEZO2 in somatosensory neurons controls gastrointestinal transit.

The gastrointestinal tract is in a state of constant motion. These movements are tightly regulated by the presence of food and help digestion by mechanically breaking down and propelling gut content. Mechanical sensing in the gut is thought to be essential for regulating motility; however, the identity of the neuronal populations, the molecules involved, and the functional consequences of this sensation are unknown. Here, we show that humans lacking PIEZO2 exhibit impaired bowel sensation and motility. Piezo2 in mouse dorsal root, but not nodose ganglia is required to sense gut content, and this activity slows down food transit rates in the stomach, small intestine, and colon. Indeed, Piezo2 is directly required to detect colon distension in vivo. Our study unveils the mechanosensory mechanisms that regulate the transit of luminal contents throughout the gut, which is a critical process to ensure proper digestion, nutrient absorption, and waste removal.

Labeling PIEZO2 activity in the peripheral nervous system.

Sensory neurons detect mechanical forces from both the environment and internal organs to regulate physiology. PIEZO2 is a mechanosensory ion channel critical for touch, proprioception, and bladder stretch sensation, yet its broad expression in sensory neurons suggests it has undiscovered physiological roles. To fully understand mechanosensory physiology, we must know where and when PIEZO2-expressing neurons detect force. The fluorescent styryl dye FM 1-43 was previously shown to label sensory neurons. Surprisingly, we find that the vast majority of FM 1-43 somatosensory neuron labeling in mice in vivo is dependent on PIEZO2 activity within the peripheral nerve endings. We illustrate the potential of FM 1-43 by using it to identify novel PIEZO2-expressing urethral neurons that are engaged by urination. These data reveal that FM 1-43 is a functional probe for mechanosensitivity via PIEZO2 activation in vivo and will facilitate the characterization of known and novel mechanosensory processes in multiple organ systems.

The role of somatosensory innervation of adipose tissues.

Adipose tissues communicate with the central nervous system to maintain whole-body energy homeostasis. The mainstream view is that circulating hormones secreted by the fat convey the metabolic state to the brain, which integrates peripheral information and regulates adipocyte function through noradrenergic sympathetic output1. Moreover, somatosensory neurons of the dorsal root ganglia innervate adipose tissue2. However, the lack of genetic tools to selectively target these neurons has limited understanding of their physiological importance. Here we developed viral, genetic and imaging strategies to manipulate sensory nerves in an organ-specific manner in mice. This enabled us to visualize the entire axonal projection of dorsal root ganglia from the soma to subcutaneous adipocytes, establishing the anatomical underpinnings of adipose sensory innervation. Functionally, selective sensory ablation in adipose tissue enhanced the lipogenic and thermogenetic transcriptional programs, resulting in an enlarged fat pad, enrichment of beige adipocytes and elevated body temperature under thermoneutral conditions. The sensory-ablation-induced phenotypes required intact sympathetic function. We postulate that beige-fat-innervating sensory neurons modulate adipocyte function by acting as a brake on the sympathetic system. These results reveal an important role of the innervation by dorsal root ganglia of adipose tissues, and could enable future studies to examine the role of sensory innervation of disparate interoceptive systems.

Collagen Organization Within the Cartilage of Trpv4-/- Mice Studied with Two-Photon Microscopy and Polarized Second Harmonic Generation.

The polymodal channel TRPV4 has been shown to regulate development and maintenance of cartilage. Here we investigate whether TRPV4 activity regulates the early deposition and structure of collagen matrix in the femoral head cartilage by comparing the 3D morphology and the sub-micrometer organization of the collagen matrix between wild type and Trpv4 -/- mice pups four to five days old. Two-photon microscopy can be used to conduct label-free imaging of cartilage, as collagen generates a second harmonic signal (second harmonic generation [SHG]) under pulsed infrared excitation. In one set of measurements, we use circularly polarized laser light to reconstruct the 3D morphology of the femoral head cartilage and to measure the tissue thickness. Second, by rotating the direction of the linearly polarized light and using polarized SHG detection, we investigate the sub-micrometer orientation of collagen fibers in the cartilage. At this developmental stage, we cannot detect statistically significant differences between the two mice strains, although a tendency toward a more random orientation of collagen fibers and a higher thickness of the whole cartilage seems to characterize the Trpv4 -/- mice. We discuss possible reasons for these observations. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Voltage gating of mechanosensitive PIEZO channels.

Mechanosensitive PIEZO ion channels are evolutionarily conserved proteins whose presence is critical for normal physiology in multicellular organisms. Here we show that, in addition to mechanical stimuli, PIEZO channels are also powerfully modulated by voltage and can even switch to a purely voltage-gated mode. Mutations that cause human diseases, such as xerocytosis, profoundly shift voltage sensitivity of PIEZO1 channels toward the resting membrane potential and strongly promote voltage gating. Voltage modulation may be explained by the presence of an inactivation gate in the pore, the opening of which is promoted by outward permeation. Older invertebrate (fly) and vertebrate (fish) PIEZO proteins are also voltage sensitive, but voltage gating is a much more prominent feature of these older channels. We propose that the voltage sensitivity of PIEZO channels is a deep property co-opted to add a regulatory mechanism for PIEZO activation in widely different cellular contexts.

Multilocus phylogenetic and geospatial analyses illuminate diversification patterns and the biogeographic history of Malagasy endemic plated lizards (Gerrhosauridae: Zonosaurinae).

Although numerous studies have attempted to find single unifying mechanisms for generating Madagascar's unique flora and fauna, little consensus has been reached regarding the relative importance of climatic, geologic and ecological processes as catalysts of diversification of the region's unique biota. Rather, recent work has shown that both biological and physical drivers of diversification are best analysed in a case-by-case setting with attention focused on the ecological and life-history requirements of the specific phylogenetic lineage under investigation. Here, we utilize a comprehensive analytical approach to examine evolutionary drivers and elucidate the biogeographic history of Malagasy plated lizards (Zonosaurinae). Data from three genes are combined with fossil information to construct time-calibrated species trees for zonosaurines and their African relatives, which are used to test alternative diversification hypotheses. Methods are utilized for explicitly incorporating phylogenetic uncertainty into downstream analyses. Species distribution models are created for 14 of 19 currently recognized species, which are then used to estimate spatial patterns of species richness and endemicity. Spatially explicit analyses are employed to correlate patterns of diversity with both topographic heterogeneity and climatic stability through geologic time. We then use inferred geographic ranges to estimate the biogeographic history of zonosaurines within each of Madagascar's major biomes. Results suggest constant Neogene and Quaternary speciation with divergence from the African most recent common ancestor ~30 million years ago when oceanic currents and African rivers facilitated dispersal. Spatial patterns of diversity appear concentrated along coastal regions of northern and southern Madagascar. We find no relationship between either topographic heterogeneity or climatic stability and patterns of diversity. Ancestral state reconstructions suggest that western dry forests were important centres of origin with recent invasion into spiny and rain forest. These data highlight the power of combining multilocus phylogenetic and spatially explicit analyses for testing alternative diversification hypotheses within Madagascar's unique biota and more generally, particularly as applied to phylogenetically and biologically constrained systems.

Small body size increases the regional differentiation of populations of tropical mantellid frogs (Anura: Mantellidae).

The processes affecting species diversification may also exert an influence on patterns of genetic variability within species. We evaluated the contributions of five variables potentially influencing clade diversification (body size, reproductive mode, range size, microhabitat and skin texture) on mtDNA divergence and polymorphism among populations of 40 species of frogs (Mantellidae) from two rainforest communities in Madagascar. We report an inverse association between body size and nucleotide divergence between populations but find no influence of other variables on genetic variation. Body size explained ca. 11% of the variation in nucleotide divergence between populations and was coupled with high F(ST) levels and an absence of haplotype sharing in small-bodied and medium-sized frogs. Low dispersal ability is likely the proximate mechanism producing higher population differentiation in small mantellids. The lack of genetic cohesion among populations establishes regional genetic fragmentation which in turn has the potential to accelerate rates of allopatric speciation in small frogs relative to large species. However, there is little evidence of increased speciation rates in these or other small-bodied organisms. We reconcile these contradictory observations by suggesting that lower dispersal ability also curbs colonization of new areas, decelerating diversification in weak dispersers. Our results imply that the intermediate dispersal model also applies to amphibians and may explain inconsistent previous results on the correlation of body size and speciation rate.

A caged progesterone analog alters intracellular Ca2+ and flagellar bending in human sperm.

Progesterone is a physiological agonist for mammalian sperm, modulating its flagellar movement and facilitating the acrosome reaction. To study the initial action of progesterone, we developed a caged analog with a photosensitive group: nitrophenylethanediol, at position 20. Using this compound combined with stroboscopic illumination, we performed Ca(2)(+) imaging of human spermatozoa and analyzed the effects of progesterone on the intracellular Ca(2)(+) concentration ([Ca(2)(+)](i)) of beating flagella for the first time. We observed a transient [Ca(2)(+)](i) increase in the head and the flagellum upon photolysis of the caged progesterone and an increase in flagellar curvature. Detailed kinetic analysis revealed that progesterone elicits an increase in the [Ca(2)(+)](i) immediately in the flagellum (mid-piece and principal piece), thereafter in the head with a short time lag. This observation is different from the progesterone-induced Ca(2)(+) mobilization in mouse spermatozoa, where the Ca(2)(+) rise initiates at the base of the sperm head. Our finding is mostly consistent with the recent discovery that progesterone activates CatSper channels in human spermatozoa, but not in mouse spermatozoa.

Aligning conservation priorities across taxa in Madagascar with high-resolution planning tools.

Globally, priority areas for biodiversity are relatively well known, yet few detailed plans exist to direct conservation action within them, despite urgent need. Madagascar, like other globally recognized biodiversity hot spots, has complex spatial patterns of endemism that differ among taxonomic groups, creating challenges for the selection of within-country priorities. We show, in an analysis of wide taxonomic and geographic breadth and high spatial resolution, that multitaxonomic rather than single-taxon approaches are critical for identifying areas likely to promote the persistence of most species. Our conservation prioritization, facilitated by newly available techniques, identifies optimal expansion sites for the Madagascar government's current goal of tripling the land area under protection. Our findings further suggest that high-resolution multitaxonomic approaches to prioritization may be necessary to ensure protection for biodiversity in other global hot spots.

Opening the black box: phylogenetics and morphological evolution of the Malagasy fossorial lizards of the subfamily "Scincinae".

The island of Madagascar harbors a highly endemic vertebrate fauna including a high diversity of lizards of the subfamily "Scincinae," with about 57 species in eight genera. Since limb reduction seems to have been a common phenomenon during the evolution of Malagasy "scincines," diagnosing evolutionary relationships based on morphology has been difficult. Phylogenetic analyses of multiple mitochondrial DNA sequences including the entire ND1, tRNA(LEU), tRNA(ILE), tRNA(GLN) genes, and fragments of the 12S and 16S rRNA and tRNA(MET) genes were conducted to test the monophyly of the largest genus Amphiglossus, and to evaluate the various formal and informal species groupings previously proposed for this species-rich group. A further objective was to determine the phylogenetic placements of the several greatly limb-reduced and limbless Malagasy "scincines" and ascertain whether any of these are derived from within the morphologically plesiomorphic Amphiglossus. As limb reduction in skinks is mostly associated with body elongation via an increase in the number of presacral vertebrae, we evaluate the pattern of evolution of the numbers of presacral vertebrae in the context of our phylogeny. We demonstrate that Amphiglossus as currently diagnosed is non-monophyletic, and the species fall into two major groups. One of these groups is a clade that contains the included species of the subgenus Amphiglossus (Madascincus) among other species and is a member of a larger clade containing Paracontias and Pseudoacontias. In the second group, the nominate subgenus Amphiglossus (Amphiglossus) forms several subclades within a larger clade that also contains Androngo crenni and Pygomeles braconnieri, and is sister to Voeltzkowia. All analyses provide strong support for the monophyly of Paracontias and Voeltzkowia. Based on the preferred phylogenetic hypothesis and weighted squared-change parsimony we show that the ancestor of the Malagasy clade was already elongated and had a moderately high number of presacral vertebrae (46-48), which is hypothesized to be the ancestral condition for the whole Malagasy "scincine" clade. We further demonstrate that both multiple increases and reductions of presacral vertebrae evolved in many clades of Malagasy "scincines" and that the use of presacral vertebrae as a major character to diagnose supraspecific units is dubious. Based on our results and published morphological evidence we consider Scelotes waterloti Angel, 1930 to be a junior synonym of Amphiglossus reticulatus (Kaudern, 1922).

New evidence for parallel evolution of colour patterns in Malagasy poison frogs (Mantella).

Malagasy poison frogs of the genus Mantella are diurnal and toxic amphibians of highly variable and largely aposematic coloration. Previous studies provided evidence for several instances of homoplastic colour evolution in this genus but were unable to sufficiently resolve relationships among major species groups or to clarify the phylogenetic position of several crucial taxa. Here, we provide cytochrome b data for 143 individuals of three species in the Mantella madagascariensis group, including four newly discovered populations. Three of these new populations are characterized by highly variable coloration and patterns but showed no conspicuous increase of haplotype diversity which would be expected under a scenario of secondary hybridization or admixture of chromatically uniform populations. Several populations of these variable forms and of M. crocea were geographically interspersed between the distribution areas of Mantella aurantiaca and Mantella milotympanum. This provides further support for the hypothesis that the largely similar uniformly orange colour of the last two species evolved in parallel. Phylogenies based on over 2000 bp of two nuclear genes (Rag-1 and Rag-2) identified reliably a clade of the Mantella betsileo and Mantella laevigata groups as sister lineage to the M. madagascariensis group, but did not support species within the latter group as monophyletic. The evolutionary history of these frogs might have been characterized by fast and recurrent evolution of colour patterns, possibly triggered by strong selection pressures and mimicry effects, being too complex to be represented by simple bifurcating models of phylogenetic reconstruction.

Climatic oscillations triggered post-Messinian speciation of Western Palearctic brown frogs (Amphibia, Ranidae).

Oscillating glacial cycles over the past 2.4 million years are proposed to have had a major impact on the diversity of contemporary species communities. We used mitochondrial and nuclear DNA sequence data to infer phylogenetic relationships within Western Palearctic brown frogs and to test the influence of Pliocene and Pleistocene climatic changes on their evolution. We sequenced 1976bp of the mitochondrial genes 16S rRNA and cytochrome b and of the nuclear rhodopsin gene for all current species and subspecies. Based on an established allozyme clock for Western Palearctic water frogs and substitution rate constancy among water frogs and brown frogs, we calibrated a molecular clock for 1425bp of the 16S and rhodopsin genes. We applied this clock to date speciation events among brown frogs. Western Palearctic brown frogs underwent a basal post-Messinian radiation about 4 million years ago (mya) into five major clades: three monotypic lineages (Rana dalmatina, Rana latastei, Rana graeca), an Anatolian lineage, and a lineage comprising Rana italica, Rana arvalis, and all Iberian taxa. Polytypic lineages radiated further in concordance with the onset of climatic oscillations ca. 3.2, 2.0, and 1.0-0.6 mya, respectively. The dated fossil record corroborates our paleobiogeographic scenario. We conclude that drastic climatic changes followed by successive temperature oscillations "trapped" most brown frog species in their southern European glacial refugia with enough time to speciate. Substantial dispersal was only possible during extensive interglacial periods of a constant subtropical climate.

Exploring the potential of life-history key innovation: brook breeding in the radiation of the Malagasy treefrog genus Boophis.

The treefrog genus Boophis is one of the most species-rich endemic amphibian groups of Madagascar. It consists of species specialized to breeding in brooks (48 species) and ponds (10 species). We reconstructed the phylogeny of Boophis using 16S ribosomal DNA sequences (558 bp) from 27 species. Brook-breeders were monophyletic and probably derived from an ancestral pond-breeding lineage. Pond-breeders were paraphyletic. The disparity in diversification among pond-breeders and brook-breeders was notable among endemic Malagasy frogs, although it was not significant when considering Boophis alone. Sibling species which have different advertisement calls but are virtually indistinguishable by morphology were common among brook-breeders; genetic divergence between these species was high (modal 8% total pairwise divergence). Substitution rates in brook-breeding species were significantly higher than in pond-breeders. Speciation of pond-breeders may be hindered by their usually more synchronous reproduction and a higher vagility which enhances gene flow, while a higher potential of spatial segregation and speciation may exist along brooks.

Out of Asia: mitochondrial DNA evidence for an Oriental origin of tiger frogs, genus Hoplobatrachus.

Most examples of intercontinental dispersal events after the Miocene contact between Africa and Asia involve mammal lineages. Among amphibians, a number of probably related groups are known from both continents, but their phylogenies are so far largely unresolved. To test the hypothesis of Miocene dispersal against a Mesozoic vicariance scenario in the context of Gondwana fragmentation, we analyzed fragments of the mitochondrial 16S rRNA gene (572 bp) in 40 specimens of 34 species of the anuran family Ranidae. Results corroborated the monophyly of tiger frogs (genus Hoplobatrachus), a genus with representatives in Africa and Asia. The African H. occipitalis was the sister group of the Asian H. crassus, H. chinensis, and H. tigerinus. Hoplobatrachus was placed in a clade also containing the Asian genera Euphlyctis and Nannophrys. Combined analysis of sequences of 16S and 12S rRNA genes (total 903 bp) in a reduced set of taxa corroborated the monophyly of the lineage containing these three genera and identified the Asian genus Fejervarya as its possible sister group. The fact that the African H. occipitalis is nested within an otherwise exclusively Asian clade indicates its probable Oriental origin. Rough molecular clock estimates did not contradict the assumption that the dispersal event took place in the Miocene. Our data further identified a similar molecular divergence between closely related Asian and African species of Rana (belonging to the section Hylarana), indicating that Neogene intercontinental dispersal also may have taken place in this group and possibly in rhacophorid treefrogs.

First data on the molecular phylogeography of scincid lizards of the genus Mabuya.

A 487-bp fragment of the mitochondrial 16S rRNA gene was sequenced in 26 species of the circumtropical lizard genus Mabuya and used to analyze phylogenetic relationships within the genus. The species from Africa and Madagascar formed a monophyletic group relative to the included Asian and South American taxa. The Malagasy species included (M. elegans, M. cf. dumasi, and M. comorensis) did not appear as a monophylum. Combined and separate analysis of the 16S data and additional sequences of the mitochondrial 12S rRNA, ND4, and cytochrome b genes (a total of 2255 bp) in one Asian, two Malagasy, and two African species also did not result consistently in a monophyletic grouping of the Malagasy taxa. However, a monophylum containing African and Malagasy taxa was strongly supported by the combined analysis. These preliminary results indicate that Mabuya probably colonized Madagascar from Africa through the Mozambique Channel.

Cytological and molecular analysis in the rare discoglossid species, Alytes muletensis (Sanchiz & Adrover 1977) and its bearing on archaeobatrachian phylogeny.

Cytogenetic and molecular data on Alytes muletensis (Amphibia: Discoglossidae) are compared with other representatives of archaeobatrachian frogs: Bombina variegata pachypus, Pelobates cultripes, Pelodytes punctatus, Xenopus laevis, and Discoglossus. A. muletensis has the karyotype typical for the genus Alytes, 38 elements with either one or two arms, some of which can be considered as 'microchromosomes'. The NORs are located on the telomeres of the tenth chromosome pair which agrees with the state in A. obstetricians but differs from A. cisternasii reflecting phylogenetic affinities. C-banding and staining with DAPI and chromomycin A3 revealed important blocks of telomeric CMA-positive heterochromatin on the smaller chromosomes of Alytes, similar to the state found in Discoglossus. Phylogenetic analysis of 750 bp of fragments of the mitochondrial 16S and 12S rRNA genes corroborated that Discoglossus and Alytes are sister taxa which together probably form the sister group of the Bombinatorinae. Centromeric heterochromatin in Alytes may be responsible for the retention of a plesiomorphic asymmetric karyotype which independently has evolved into a symmetric karyotype through centric fusions in Bombina and Discoglossus. The HindIII satellite DNA family was present in all archaeobatrachians studied but absent in hyloid and ranoid neobatrachians.

Phylogeny and classification of poison frogs (Amphibia: dendrobatidae), based on mitochondrial 16S and 12S ribosomal RNA gene sequences.

An analysis of partial sequences of the 16S ribosomal rRNA gene (582 bp) of 20 poison frog species (Dendrobatidae) confirmed their phylogenetic relationships to bufonid and leptodactylid frogs. Representatives of the ranoid families and subfamilies Raninae, Mantellinae, Petropedetinae, Cacosterninae, Arthroleptidae, Astylosternidae, and Microhylidae did not cluster as sister group of the Dendrobatidae. Similar results were obtained in an analysis using a partial sequence of the 12S gene (350 bp) in a reduced set of taxa and in a combined analysis. Within the Dendrobatidae, our data supported monophyly of the genus Phyllobates but indicated paraphyly of Epipedobates and Colostethus. Minyobates clustered within Dendrobates, contradicting its previously assumed phylogenetic position. Phobobates species clustered as a monophyletic unit within Epipedobates. Allobates was positioned in a group containing two Colostethus species, indicating that lack of amplexus, presence of skin alkaloids, and aposematic coloration evolved independently in Allobates and the remaining aposematic dendrobatids.