Capacity and establishment rules govern the number of nonnative species in communities of ground-dwelling invertebrates.

Nonnative species are a key agent of global change. However, nonnative invertebrates remain understudied at the community scales where they are most likely to drive local extirpations. We use the North American NEON pitfall trapping network to document the number of nonnative species from 51 invertebrate communities, testing four classes of drivers. We sequenced samples using the eDNA from the sample's storage ethanol. We used AICc informed regression to evaluate how native species richness, productivity, habitat, temperature, and human population density and vehicular traffic account for continent-wide variation in the number of nonnative species in a local community. The percentage of nonnatives varied 3-fold among habitat types and over 10-fold (0%-14%) overall. We found evidence for two types of constraints on nonnative diversity. Consistent with Capacity rules (i.e., how the number of niches and individuals reflect the number of species an ecosystem can support) nonnatives increased with existing native species richness and ecosystem productivity. Consistent with Establishment Rules (i.e., how the dispersal rate of nonnative propagules and the number of open sites limits nonnative species richness) nonnatives increased with automobile traffic-a measure of human-generated propagule pressure-and were twice as common in pastures than native grasslands. After accounting for drivers associated with a community's ability to support native species (native species richness and productivity), nonnatives are more common in communities that are regularly seasonally disturbed (pastures and, potentially deciduous forests) and those experiencing more vehicular traffic. These baseline values across the US North America will allow NEON's monitoring mission to document how anthropogenic change-from disturbance to propagule transport, from temperature to trends in local extinction-further shape biotic homogenization.

The role of Culex territans mosquitoes in the transmission of Batrachochytrium dendrobatidis to amphibian hosts.

BACKGROUND: Mosquitoes are the deadliest organisms in the world, killing an estimated 750,000 people per year due to the pathogens they can transmit. Mosquitoes also pose a major threat to other vertebrate animals. Culex territans is a mosquito species found in temperate zones worldwide that feeds almost exclusively on amphibians and can transmit parasites; however, little is known about its ability to transmit other pathogens, including fungi. Batrachochytrium dendrobatidis (Bd) is a topical pathogenic fungus that spreads through contact. With amphibian populations around the world experiencing mass die-offs and extinctions due to this pathogen, it is critical to study all potential modes of transmission. Because Cx. territans mosquitoes are in contact with their hosts for long periods of time while blood-feeding, we hypothesize that they can transmit and pick up Bd. METHODS: In this study, we first assessed Cx. territans ability to transfer the fungus from an infected surface to a clean one under laboratory conditions. We also conducted a surveillance study of Bd infections in frogs and mosquitoes in the field (Mountain Lake Biological station, VA, USA). In parallel, we determined Cx. territans host preference via blood meal analysis of field caught mosquitoes. RESULTS: We found that this mosquito species can carry the fungus to an uninfected surface, implying that they may have the ability to transmit Bd to their amphibian hosts. We also found that Cx. territans feed primarily on green frogs (Rana clamitans) and bullfrogs (Rana catesbeiana) and that the prevalence of Bd within the frog population at our field site varied between years. CONCLUSIONS: This study provides critical insights into understanding the role of amphibian-biting mosquitoes in transmitting pathogens, which can be applied to disease ecology of susceptible amphibian populations worldwide.

Integrating biogeography and behavioral ecology to rapidly address biodiversity loss.

Addressing climate change and biodiversity loss will be the defining ecological, political, and humanitarian challenge of our time. Alarmingly, policymakers face a narrowing window of opportunity to prevent the worst impacts, necessitating complex decisions about which land to set aside for biodiversity preservation. Yet, our ability to make these decisions is hindered by our limited capacity to predict how species will respond to synergistic drivers of extinction risk. We argue that a rapid integration of biogeography and behavioral ecology can meet these challenges because of the distinct, yet complementary levels of biological organization they address, scaling from individuals to populations, and from species and communities to continental biotas. This union of disciplines will advance efforts to predict biodiversity's responses to climate change and habitat loss through a deeper understanding of how biotic interactions and other behaviors modulate extinction risk, and how responses of individuals and populations impact the communities in which they are embedded. Fostering a rapid mobilization of expertise across behavioral ecology and biogeography is a critical step toward slowing biodiversity loss.

Temperature-habitat interactions constrain seasonal activity in a continental array of pitfall traps.

Activity density (AD), the rate at which animals collectively move through their environment, emerges as the product of a taxon's local abundance and its velocity. We analyze drivers of seasonal AD using 47 localities from the National Ecological Observatory Network (NEON) both to better understand variation in ecosystem rates like pollination and seed dispersal as well as the constraints of using AD to monitor invertebrate populations. AD was measured as volume from biweekly pitfall trap arrays (ml trap-1 14 days-1 ). Pooled samples from 2017 to 2018 revealed AD extrema at most temperatures but with a strongly positive overall slope. However, habitat types varied widely in AD's seasonal temperature sensitivity, from negative in wetlands to positive in mixed forest, grassland, and shrub habitats. The temperature of maximum AD varied threefold across the 47 localities; it tracked the threefold geographic variation in maximum growing season temperature with a consistent gap of ca. 3°C across habitats, a novel macroecological result. AD holds potential as an effective proxy for investigating ecosystem rates driven by activity. However, our results suggest that its use for monitoring insect abundance is complicated by the many ways that both abundance and velocity are constrained by a locality's temperature and plant physiognomy.

Gut microbial ecology of Philippine gekkonids: ecoevolutionary effects on microbiome compositions.

Given the rapidly changing landscapes of habitats across the globe, a sound understanding of host-associated microbial communities and the ecoevolutionary forces that shape them is needed to assess general organismal adaptability. Knowledge of the symbiotic endogenous microbiomes of most reptilian species worldwide remains limited. We sampled gut microbiomes of geckos spanning nine species and four genera in the Philippines to (i) provide baseline data on gut microbiota in these host species, (ii) test for significant associations between host phylogenetic relationships and observed microbial assemblages, potentially indicative of phylosymbiosis, and (iii) identify correlations between multiple ecoevolutionary factors (e.g. species identity, habitat tendencies, range extents, and maximum body sizes) and gut microbiomes in Philippine gekkonids. We recovered no significant association between interspecific host genetic distances and observed gut microbiomes, providing limited evidence for phylosymbiosis in this group. Philippine gekkonid microbiomes were associated most heavily with host species identity, though marked variation among conspecifics at distinct sampling sites indicates that host locality influences gut microbiomes as well. Interestingly, individuals grouped as widespread and microendemic regardless of host species identity displayed significant differences in alpha and beta diversity metrics examined, likely driven by differences in rare OTU presence between groups. These results provide much needed insight in host-associated microbiomes in wild reptiles and the ecoevolutionary forces that structure such communities.

Generalizing Bayesian phylogenetics to infer shared evolutionary events.

Many processes of biological diversification can simultaneously affect multiple evolutionary lineages. Examples include multiple members of a gene family diverging when a region of a chromosome is duplicated, multiple viral strains diverging at a "super-spreading" event, and a geological event fragmenting whole communities of species. It is difficult to test for patterns of shared divergences predicted by such processes because all phylogenetic methods assume that lineages diverge independently. We introduce a Bayesian phylogenetic approach to relax the assumption of independent, bifurcating divergences by expanding the space of topologies to include trees with shared and multifurcating divergences. This allows us to jointly infer phylogenetic relationships, divergence times, and patterns of divergences predicted by processes of diversification that affect multiple evolutionary lineages simultaneously or lead to more than two descendant lineages. Using simulations, we find that the method accurately infers shared and multifurcating divergence events when they occur and performs as well as current phylogenetic methods when divergences are independent and bifurcating. We apply our approach to genomic data from two genera of geckos from across the Philippines to test if past changes to the islands' landscape caused bursts of speciation. Unlike previous analyses restricted to only pairs of gecko populations, we find evidence for patterns of shared divergences. By generalizing the space of phylogenetic trees in a way that is independent from the likelihood model, our approach opens many avenues for future research into processes of diversification across the life sciences.

Cryptic extinction risk in a western Pacific lizard radiation.

Cryptic ecologies, the Wallacean Shortfall of undocumented species' geographical ranges and the Linnaean Shortfall of undescribed diversity, are all major barriers to conservation assessment. When these factors overlap with drivers of extinction risk, such as insular distributions, the number of threatened species in a region or clade may be underestimated, a situation we term 'cryptic extinction risk'. The genus Lepidodactylus is a diverse radiation of insular and arboreal geckos that occurs across the western Pacific. Previous work on Lepidodactylus showed evidence of evolutionary displacement around continental fringes, suggesting an inherent vulnerability to extinction from factors such as competition and predation. We sought to (1) comprehensively review status and threats, (2) estimate the number of undescribed species, and (3) estimate extinction risk in data deficient and candidate species, in Lepidodactylus. From our updated IUCN Red List assessment, 60% of the 58 recognized species are threatened (n = 15) or Data Deficient (n = 21), which is higher than reported for most other lizard groups. Species from the smaller and isolated Pacific islands are of greatest conservation concern, with most either threatened or Data Deficient, and all particularly vulnerable to invasive species. We estimated 32 undescribed candidate species and linear modelling predicted that an additional 18 species, among these and the data deficient species, are threatened with extinction. Focusing efforts to resolve the taxonomy and conservation status of key taxa, especially on small islands in the Pacific, is a high priority for conserving this remarkably diverse, yet poorly understood, lizard fauna. Our data highlight how cryptic ecologies and cryptic diversity combine and lead to significant underestimation of extinction risk. Supplementary Information: The online version contains supplementary material available at 10.1007/s10531-022-02412-x.

Gut microbial ecology of the Critically Endangered Fijian crested iguana (Brachylophus vitiensis): Effects of captivity status and host reintroduction on endogenous microbiomes.

Animals often exhibit distinct microbial communities when maintained in captivity as compared to when in the wild. Such differentiation may be significant in headstart and reintroduction programs where individuals spend some time in captivity before release into native habitats. Using 16S rRNA gene sequencing, we (i) assessed differences in gut microbial communities between captive and wild Fijian crested iguanas (Brachylophus vitiensis) and (ii) resampled gut microbiota in captive iguanas released onto a native island to monitor microbiome restructuring in the wild. We used both cloacal swabs and fecal samples to further increase our understanding of gut microbial ecology in this IUCN Critically Endangered species. We found significant differentiation in gut microbial community composition and structure between captive and wild iguanas in both sampling schemes. Approximately two months postrelease, microbial communities in cloacal samples from formerly captive iguanas closely resembled wild counterparts. Interestingly, microbial communities in fecal samples from these individuals remained significantly distinct from wild conspecifics. Our results indicate that captive upbringings can lead to differences in microbial assemblages in headstart iguanas as compared to wild individuals even after host reintroduction into native conditions. This investigation highlights the necessity of continuous monitoring of reintroduced animals in the wild to ensure successful acclimatization and release.

Hidden limbs in the "limbless skink" Brachymeles lukbani: Developmental observations.

Reduced limbs and limblessness have evolved independently in many lizard clades. Scincidae exhibit a wide range of limb-reduced morphologies, but only some species have been used to study the embryology of limb reduction (e.g., digit reduction in Chalcides and limb reduction in Scelotes). The genus Brachymeles, a Southeast Asian clade of skinks, includes species with a range of limb morphologies, from pentadactyl to functionally and structurally limbless species. Adults of the small, snake-like species Brachymeles lukbani show no sign of external limbs in the adult except for small depressions where they might be expected to occur. Here, we show that embryos of B. lukbani in early stages of development, on the other hand, show a truncated but well-developed limb with a stylopod and a zeugopod, but no signs of an autopod. As development proceeds, the limb's small size persists even while the embryo elongates. These observations are made based on external morphology. We used florescent whole-mount immunofluorescence to visualize the morphology of skeletal elements and muscles within the embryonic limb of B. lukabni. Early stages have a humerus and separated ulna and radius cartilages; associated with these structures are dorsal and ventral muscle masses as those found in the embryos of other limbed species. While the limb remains small, the pectoral girdle grows in proportion to the rest of the body, with well-developed skeletal elements and their associated muscles. In later stages of development, we find the small limb is still present under the skin, but there are few indications of its presence, save for the morphology of the scale covering it. By use of CT scanning, we find that the adult morphology consists of a well-developed pectoral girdle, small humerus, extremely reduced ulna and radius, and well-developed limb musculature connected to the pectoral girdle. These muscles form in association with a developing limb during embryonic stages, a hint that "limbless" lizards that possess these muscles may have or have had at least transient developing limbs, as we find in B. lukbani. Overall, this newly observed pattern of ontogenetic reduction leads to an externally limbless adult in which a limb rudiment is hidden and covered under the trunk skin, a situation called cryptomelia. The results of this work add to our growing understanding of clade-specific patterns of limb reduction and the convergent evolution of limbless phenotypes through different developmental processes.

Venomous Snakes Reveal Ecological and Phylogenetic Factors Influencing Variation in Gut and Oral Microbiomes.

The gastrointestinal tract (GIT) of vertebrates contains a series of organs beginning with the mouth and ending with the anus or cloacal opening. Each organ represents a unique environment for resident microorganisms. Due to their simple digestive anatomy, snakes are good models for studying microbiome variation along the GIT. Cloacal sampling captures the majority of the microbial diversity found in the GIT of snakes-yet little is known about the oral microbiota of snakes. Most research on the snake mouth and gut microbiota are limited to studies of a single species or captive-bred individuals. It therefore remains unclear how a host's life history, diet, or evolutionary history correlate with differences in the microbial composition within the mouths and guts of wild snakes. We sampled the mouth and gut microbial communities from three species of Asian venomous snakes and utilized 16S rRNA microbial inventories to test if host phylogenetic and ecological differences correlate with distinct microbial compositions within the two body sites. These species occupy three disparate habitat types: marine, semi-arboreal, and arboreal, our results suggest that the diversity of snake mouth and gut microbial communities correlate with differences in both host ecology and phylogeny.

Two New Species of Diploderma Hallowell, 1861 (Reptilia: Squamata: Agamidae) from the Hengduan Mountain Region in China and Rediscovery of D. brevicaudum (Manthey, Wolfgang, Hou, Wang, 2012).

Recent studies have highlighted the underestimated diversity of the genus Diploderma Hallowell, 1861 in the Hengduan Mountain Region in Southwest China, but much of the region remains poorly surveyed for reptile diversity. In this study we describe two new species of Diploderma from the upper Jinsha and middle Yalong River Valley, based on evaluations of morphological, genetic, and distribution data. The two new species are morphologically most similar to D. angustelinea and D. vela, but they can be diagnosed from both recognized taxa and all remaining congeners by a suite of morphological features, particularly the distinct coloration of gular spots. Additionally, both new species either render other recognized species paraphyletic or are allopatric with respect to their morphologically similar congeners. Furthermore, we rediscover D. brevicaudum in the wild for the first time, which was known from historical museum specimens only. We estimate the phylogenetic position of D. brevicaudum within the genus Diploderma based on mitochondrial genealogy, and we provide an expanded diagnosis and comparisons against closely related congeners and provide a detailed description of coloration in life based on newly collected specimens. Our discoveries of the new Diploderma species further highlight the urgent conservation needs of the currently neglected hot-dry valley ecosystems in the Hengduan Mountain Region of China.

Locomotion and palaeoclimate explain the re-evolution of quadrupedal body form in Brachymeles lizards.

Evolutionary reversals, including re-evolution of lost structures, are commonly found in phylogenetic studies. However, we lack an understanding of how these reversals happen mechanistically. A snake-like body form has evolved many times in vertebrates, and occasionally a quadrupedal form has re-evolved, including in Brachymeles lizards. We use body form and locomotion data for species ranging from snake-like to quadrupedal to address how a quadrupedal form could re-evolve. We show that large, quadrupedal species are faster at burying and surface locomotion than snake-like species, indicating a lack of expected performance trade-off between these modes of locomotion. Species with limbs use them while burying, suggesting that limbs are useful for burying in wet, packed substrates. Palaeoclimatological data suggest that Brachymeles originally evolved a snake-like form under a drier climate probably with looser soil in which it was easier to dig. The quadrupedal clade evolved as the climate became humid, where limbs and large size facilitated fossorial locomotion in packed soils.

Resurrection of Amolops nepalicus Yang, 1991 (Amphibia: Anura: Ranidae), with Comments on the Record of A. cf. afghanus in Nepal and China and the Validity of Two Other Junior Synonyms of A. marmoratus (Blyth, 1855).

The taxonomic validity of the controversial taxon, Amolops nepalicus Yang, 1991, is evaluated based on the examination of its holotype, along with topotypic A. marmoratus (Blyth, 1855) and A. afghanus (Günther, 1858), and other related congeners. Morphological comparison shows the holotype of A. nepalicus differs from its senior synonym A. marmoratus and all recognized species, supporting its valid species status. We provide a detailed redescription of the holotype based on a robust morphometric dataset and expanded comparisons with recognized congeners from the Pan-Himalayas and Southeast Asia. In addition, we discuss the remaining suspicious records of A. cf. afghanus in Nepal and China based on available specimens, and comment on the validity of two remaining junior synonyms of A. marmoratus, namely Ixalus argus Annandale, 1912 and Rana latopalmata Boulenger, 1882, suggesting that they may represent subjective synonyms of A. gerbillus (Annandale, 1912) and A. panhai Matsui and Nabhitabhata, 2006, respectively.

A new forest frog of the genus Platymantis (Amphibia: Anura: Ceratobatrachidae: subgenus Tirahanulap) from Leyte and Samar islands, eastern Philippines.

We describe a new species of frog of the genus Platymantis Günther (subgenus Tirahanulap), from the east-central regions of the Philippines. It belongs to the the previously-defined P. hazelae Group) based on morphological and bioacoustic datasets. The new species is phenotypically and ecologically most similar to members of Tirahanulap, an assemblage of small-bodied arboreal frogs inhabiting montane forests of the central and northern islands of the Philippine archipelago. The new species represents the first taxon in the Cloud Frog species known from the biogeographically unique Mindanao Pleistocene Aggregate Island Complex. Particularly susceptible to local extirpation following deforestation, all known species of Tirahanulap are important indicator species for environmental and conservation assessments, making this new species not only an exceptional addition to Philippine biodiversity but also an important symbol for conservation initiatives in the region.

Using 3D-digital photogrammetry to examine scaling of the body axis in burrowing skinks.

Three-dimensional (3D) modeling techniques have been increasingly utilized across disciplines for the visualization and analysis of complex structures. We employ 3D-digital photogrammetry for understanding the scaling of the body axis of 12 species of scincid lizards in the genus Brachymeles. These skinks represent a diverse radiation which shows tremendous variation in body size and degree of axial elongation. Because of the complex nature of the body axis, 3D-methods are important for understanding how the body axis evolves. 3D-digital photogrammetry presents a flexible, inexpensive, and portable system for the reconstruction of biological forms. As body size increased among species, the cross-sectional area and circumference of the head and other portions of the body axis increased isometrically, which indicates that species of differing sizes possess proportionally similar head and body shapes. These results suggest that there are no substantial head and body shape changes with body size among the sampled species, but further comparative studies with larger sample sizes and functional studies of size and morphology effects on burrowing or above-ground locomotion are needed.

Convergent Evolution of Elongate Forms in Craniates and of Locomotion in Elongate Squamate Reptiles.

Synopsis Elongate, snake- or eel-like, body forms have evolved convergently many times in most major lineages of vertebrates. Despite studies of various clades with elongate species, we still lack an understanding of their evolutionary dynamics and distribution on the vertebrate tree of life. We also do not know whether this convergence in body form coincides with convergence at other biological levels. Here, we present the first craniate-wide analysis of how many times elongate body forms have evolved, as well as rates of its evolution and reversion to a non-elongate form. We then focus on five convergently elongate squamate species and test if they converged in vertebral number and shape, as well as their locomotor performance and kinematics. We compared each elongate species to closely related quadrupedal species and determined whether the direction of vertebral or locomotor change matched in each case. The five lineages examined are obscure species from remote locations, providing a valuable glimpse into their biology. They are the skink lizards Brachymeles lukbani, Lerista praepedita, and Isopachys anguinoides, the basal squamate Dibamus novaeguineae, and the basal snake Malayotyphlops cf. ruficaudus. Our results support convergence among these species in the number of trunk and caudal vertebrae, but not vertebral shape. We also find that the elongate species are relatively slower than their limbed counterparts and move with lower frequency and higher amplitude body undulations, with the exception of Isopachys. This is among the first evidence of locomotor convergence across distantly related, elongate species.

A taxonomic conundrum: Characterizing a cryptic radiation of Asian gracile skinks (Squamata: Scincidae: Riopa) in Myanmar.

Recognizing species-level diversity is important for studying evolutionary patterns across biological disciplines and is critical for conservation efforts. However, challenges remain in delimiting species-level diversity, especially in cryptic radiations where species are genetically divergent but show little morphological differentiation. Using multilocus molecular data, phylogenetic analyses, species delimitation analyses, and morphological data, we examine lineage diversification in a cryptic radiation of Riopa skinks in Myanmar. Four species of Riopa skinks are currently recognized from Myanmar based on morphological traits, but the boundaries between three of these species, R. anguina, R. lineolata, and R. popae, are not well-defined. We find high levels of genetic diversity within these three species. Our analyses suggest that they may comprise as many as 12 independently evolving lineages, highlighting the extent to which species diversity in the region is underestimated. However, quantitative trait data suggest that these lineages have not differentiated morphologically, possibly indicating that this cryptic radiation represents non-adaptive evolution, although additional data is needed to corroborate this.

Parachute geckos free fall into synonymy: Gekko phylogeny, and a new subgeneric classification, inferred from thousands of ultraconserved elements.

Recent phylogenetic studies of gekkonid lizards have revealed unexpected, widespread paraphyly and polyphyly among genera, unclear generic boundaries, and a tendency towards the nesting of taxa exhibiting specialized, apomorphic morphologies within geographically widespread "generalist" clades. This is especially true in Australasia, where monophyly of Gekko proper has been questioned with respect to phenotypically ornate flap-legged geckos of the genus Luperosaurus, the Philippine false geckos of the genus Pseudogekko, and even the elaborately "derived" parachute geckos of the genus Ptychozoon. Here we employ sequence capture targeting 5060 ultraconserved elements (UCEs) to infer phylogenomic relationships among 42 representative ingroup gekkonine lizard taxa. We analyze multiple datasets of varying degrees of completeness (10, 50, 75, 95, and 100 percent complete with 4715, 4051, 3376, 2366, and 772 UCEs, respectively) using concatenated maximum likelihood and multispecies coalescent methods. Our sampling scheme addresses four persistent systematic questions in this group: (1) Are Luperosaurus and Ptychozoon monophyletic, and are any of these named species truly nested within Gekko? (2) Are prior phylogenetic estimates of Sulawesi's L. iskandari as the sister taxon to Melanesian G. vittatus supported by our genome-scale dataset? (3) Is the high-elevation L. gulat of Palawan Island correctly placed within Gekko? (4) And, finally, where do the enigmatic taxa P. rhacophorus and L. browni fall in a higher-level gekkonid phylogeny? We resolve these issues; confirm with strong support some previously inferred findings (placement of Ptychozoon taxa within Gekko; the sister taxon relationship between L. iskandari and G. vittatus); resolve the systematic position of unplaced taxa (L. gulat, and L. browni); and transfer L. iskandari, L. gulat, L. browni, and all members of the genus Ptychozoon to the genus Gekko. Our unexpected and novel systematic inference of the placement of Ptychozoon rhacophorus suggests that this species is not grouped with Ptychozoon or even Luperosaurus (as previously expected) but may, in fact, be most closely related to several Indochinese species of Gekko. With our resolved and strongly supported phylogeny, we present a new classification emphasizing the most inclusive, original generic name (Gekko) for these ~60 taxa, arranged into seven subgenera.

Two new species of Hemiphyllodactylus (Reptilia: Gekkonidae) from Laos.

Two new species of the gekkonid lizard genus Hemiphyllodactylus are described based on specimens collected from Champasak Province in southern Laos and Houaphanh Province in northern Laos. Phylogenetic analyses recover H. indosobrinus sp. nov. from Champasak Province as the sister lineage to the Thai endemic H. flaviventris. Hemiphyllodactylus indosobrinus sp. nov. can be diagnosed by having 15 supralabial scales, eight chin scales, 4­5­5­4 subdigital lamellae on Fingers II-V, 4­5­5­5 subdigital lamellae on Toes II-V, and 18 continuous precloacofemoral pores. Phylogenetic analyses recover H. serpispecus sp. nov. from Houaphanh Province to be most closely related to the Vietnamese endemic H. banaensis. Hemiphyllodactylus serpispecus sp. nov. differs from congeners by having seven chin scales; 3­4­4­4 subdigital lamellae on Fingers II-V; 3­4­4­5 subdigital lamellae on Toes II-V; and 11 continuous precloacofemoral pores. The recognition of these two new species doubles the number of Hemiphyllodactylus known from Laos.