Hemidactylium Research Articles

Caging Four-Toed Salamander Nests Reduces Nest Predation in Northeastern Tennessee

Abstract Four-toed salamanders Hemidactylium scutatum have disjunct populations in the United States and Canada. Increased predation could threaten their populations and cause them to become limited in certain areas. Installing cages to protect threatened populations can reduce the chance of predation. To test the efficacy of cages, we examined the effects of caging four-toed salamander nests in northeastern Tennessee. We randomly selected 65/120 nests to cage from our study sites at the South Holston Weir Dam and Bouton Tract in Sullivan County, Tennessee. Based on photos from a camera trapping survey, we found that raccoons Procyon lotor were the main predators of uncaged four-toed salamander nests. Raccoon damage resulted in portions of moss being ripped away and the disappearance of eggs. Twenty-six percent of uncaged nests were preyed upon, whereas none of the caged nests were preyed upon. Our findings provide a strategy for improving nest success of four-toed salamanders and could be used to protect threatened populations.

Advancing wildlife connectivity in land use planning: a case study with four‐toed salamanders

AbstractStable habitat connections that wildlife can safely traverse are essential to biodiversity conservation and healthy ecosystems. We developed high‐resolution landscape connectivity models to predict resistance to movement by a threatened wetland‐obligate amphibian, the four‐toed salamander (Hemidactylium scutatum), and identified priority management areas on the 13,000‐ha Department of Energy Oak Ridge Reservation (ORR) from 2019 to 2022. We developed a resistance surface based on aerial light detection and ranging data (LiDAR), >30 years of field‐based mapping of forest, hydrologic, and geologic features, and contemporary population surveys, alongside derived predictors at <1‐m resolution. We then modeled predicted movement corridors using a circuit theory‐based modeling approach. We worked closely with land management and natural resources personnel to integrate ecological modeling with broader land use priorities, monetary costs, and feasibility. We identified important terrestrial and aquatic areas on ORR and simulated management scenarios to promote stable connections for four‐toed salamanders. This approach allowed us to narrow down a list of 438 potential habitat manipulation sites to 10 sites where open‐bottomed culverts and buffers could be implemented. This smaller‐scale restoration approach produced a similar increase in landscape connectivity while costing <20% of a larger‐scale approach based on barrier removal. We successfully identified feasible, cost‐effective management strategies that integrated knowledge from a variety of sources. We offer a strategy that permitted integration of wildlife management goals into infrastructure upgrades wherein wildlife was not an initial consideration.

Reproductive Ecology and Nesting Site Characteristics of Four-Toed Salamanders (Hemidactylium scutatum) in Natural and Constructed Upland-Embedded Wetlands on the Appalachian Plateau, Kentucky

Many forested freshwater wetlands have been altered or destroyed, and wetlands are constructed to offset loss. However, they do not always replace the function of natural wetlands. It is important to understand how features of the habitat differ between types of wetlands and whether constructed wetlands provide an adequate habitat for species adapted to natural wetlands. Our objectives were to measure the characteristics of Four-toed Salamanders’ nesting habitat and determine which factors contribute to the abundance of eggs and nests in natural and constructed upland-embedded wetlands within a ridgetop ecosystem in eastern Kentucky. We located and examined characteristics for 207 nests in twelve wetlands and measured variables at the nest level and at the wetland level. The best predictor of the number of eggs and number of nests was amount of moss at the wetland. These measures of reproductive effort were similar between types of wetlands, but the number of eggs per nest was higher in constructed wetlands and inversely related to amount of moss, highlighting a deficit in nesting habitat. Research of embryonic and larval survival is needed but based on data from other amphibian species in this system, we predict that the survival of Four-toed Salamanders’ larvae is low in constructed wetlands with permanent hydrology. Restoration of constructed wetlands should address the need for moss as nesting substrate and drying of the wetland to reduce the abundance and diversity of predators of larvae.

Climate change and range restriction of common salamanders in eastern Canada and the United States

AbstractThe sensitivity of amphibian species to shifts in environmental conditions has been exhibited through long‐term population studies and the projection of ecological niche models under expected conditions. Species in biodiversity hotspots have been the focus of ample predictive modeling studies, while, despite their significant ecological value, wide‐ranging and common taxa have received less attention. We focused on predicting range restriction of the spotted salamander (Ambystoma maculatum), blue‐spotted salamander (A. laterale), four‐toed salamander (Hemidactylium scutatum), and red‐backed salamander (Plethodon cinereus) under future climate scenarios. Using bias‐corrected future climate data and biodiversity database records, we developed maximum entropy (MaxEnt) models under current conditions and for climate change projections in 2050 and 2070. We calculated positivity rates of species localities to represent proportions of habitat expected to remain climatically suitable with continued climate change. Models projected under future conditions predicted average positivity rates of 91% (89–93%) for the blue‐spotted salamander, 23% (2–41%) for the spotted salamander, 4% (0.7–9%) for the four‐toed salamander, and 61% (42–76%) for the red‐backed salamander. Range restriction increased with time and greenhouse gas concentration for the spotted salamander, four‐toed salamander, and red‐backed salamander. Common, widespread taxa that often receive less conservation resources than other species are at risk of experiencing significant losses to their climatic ranges as climate change continues. Efforts to maintain populations of species should be focused on regions expected to experience fewer climatic shifts such as the interior and northern zones of species' distributions.

Aquatic eDNA can advance monitoring of a small‐bodied terrestrial salamander and amphibian pathogen

AbstractSuccessful conservation efforts for threatened species depend on accurate characterization of their distribution, habitat use, and threats. However, surveillance can be challenging for species with small size, cryptic coloring, or elusive behavior. Environmental DNA (eDNA) monitoring can provide a sensitive and noninvasive alternative to traditional surveillance techniques by detecting trace DNA shed by a target species into their environment. We evaluated the efficacy of eDNA monitoring for the four‐toed salamander (Hemidactylium scutatum), a terrestrial salamander threatened throughout much of its range. Additionally, we integrated eDNA monitoring for ranavirus, a widespread pathogen of ectothermic vertebrates, to efficiently evaluate a potential disease threat to H. scutatum. We designed a novel species‐specific quantitative PCR (qPCR) assay for H. scutatum, multiplexed the assay with a previously developed ranavirus qPCR assay, and validated the multiplexed tests in silico, in vitro, and in situ. We collected aquatic eDNA weekly for 8 weeks from pools with historical reports of H. scutatum (n = 6) and contemporary reports of H. scutatum on associated uplands (n = 4). We identified H. scutatum eDNA at low quantities (< 12 copies/reaction) on multiple sampling days in two pools with historic species presence. Ranavirus eDNA was widespread; nine pools tested positive including the pools where H. scutatum eDNA was detected. Ranavirus eDNA quantities increased, peaked, then decreased (i.e., were not sporadic in time) in several pools but never rose in abundance to levels associated with ranavirosis in other studies. Our results suggest that H. scutatum eDNA is distributed sporadically at low quantities in pools; we recommend that future surveillance efforts prioritize increased frequency and quantity of sample collection per site visit and include repeat surveys of candidate sites. Further work to characterize Ranavirus eDNA movement, temporal trends, relationship to infectious virion, and decay is needed to help advance this tool.

Range‐wide phylogeography of the four‐toed salamander: out of Appalachia and into the glacial aftermath

AbstractAimWe assessed the phylogeography of Hemidactylium scutatum across the entire range of the species, quantifying the diversity of populations, and identifying extrinsic factors that may have generated observed patterns of genetic connectivity and distribution.LocationNorth America.MethodsWe performed DNA sequence analysis of the mitochondrial cytochrome oxidase 1 gene of 335 samples collected from 79 field locations distributed throughout the species range. Phylogenetic analyses, patterns of haplotype distribution, DNA mismatch distributions, and partial Mantel tests were performed to estimate divergence times of major clades, identify geographical barriers that determined phylogenetic breaks, and describe a potential scenario for the radiation of the species.ResultsPhylogenetic analyses showed deep sequence divergences with strong support for regional monophyletic groups. Patterns of haplotype distribution suggest that both ancient and modern river drainages acted as barriers to dispersal. Two distinct allopatric clades accounted for all sampling sites within glaciated areas of North America yet showed distinct patterns of recolonization. High levels of haplotype diversity were detected in the southern Appalachians, with several members of widely ranging clades as well as other unique, endemic, and highly divergent lineages. Bayesian analyses estimated the common ancestor of all living Hemidactylium at roughly 8 Ma, with the most basal splits confined to the Blue Ridge Mountains.Main conclusionsOur results are consistent with the ‘Out of Appalachia’ hypothesis for the origin of Hemidactylium scutatum, lending further support to the importance of this region as a generator of biodiversity in eastern North America.

Amphibians in the Canaan Valley Drainage

Abstract I have been studying amphibians in Canaan Valley and its surrounding region since 1976. Most of my studies inventoried populations of Plethodon nettingi (Cheat Mountain Salamander). I sampled 29 upland sites on the mountains around Canaan Valley: (1) twelve sites on Canaan Mountain, which forms Canaan Valley's western rim, were distributed from Chimney Rock, at the southern end of Canaan Mountain, northwards to the Blackwater River at Camp 70; (2) on the eastern rim of Canaan Valley, 17 sites were on Cabin Mountain, from Snowy Point in the north to Weiss Knob in the south. At the montane sites, I observed 11 salamander species and 1 toad species. Salamanders found included Ambystoma maculatum (Spotted Salamander), Notophthalmus v. viridescens (Red-spotted Newt), Desmognathus monticola (Seal Salamander), Desmognathus ochrophaeus (Allegheny Mountain Dusky Salamander); Gyrinophilus p. porphyriticus (Northern Spring Salamander), Hemidactylium scutatum (Four-toed Salamander), Eurycea bislineata (North...

Enlarged Multilocus Data set Provides Surprisingly Younger Time of Origin for the Plethodontidae, the Largest Family of Salamanders.

Deep phylogenetic relationships of the largest salamander family Plethodontidae have been difficult to resolve, probably reflecting a rapid diversification early in their evolutionary history. Here, data from 50 independent nuclear markers (total 48,582 bp) are used to reconstruct the phylogeny and divergence times for plethodontid salamanders, using both concatenation and coalescence-based species tree analyses. Our results robustly resolve the position of the enigmatic eastern North American four-toed salamander (Hemidactylium) as the sister taxon of Batrachoseps + Tribe Bolitoglossini, thus settling a long-standing question. Furthermore, we statistically reject sister taxon status of Karsenia and Hydromantes, the only plethodontids to occur outside the Americas, leading us to new biogeographic hypotheses. Contrary to previous long-standing arguments that plethodontid salamanders are an old lineage originating in the Cretaceous (more than 90 Ma), our analyses lead to the hypothesis that these salamanders are much younger, arising close to the K-T boundary (~66 Ma). These time estimates are highly stable using alternative calibration schemes and dating methods. Our data simulation highlights the potential risk of making strong arguments about phylogenetic timing based on inferences from a handful of nuclear genes, a common practice. Based on the newly obtained timetree and ancestral area reconstruction results, we argue that (i) the classic "Out of Appalachia" hypothesis of plethodontid origins is problematic; (ii) the common ancestor of extant plethodontids may have originated in northwestern North America in the early Paleocene; (iii) origins of Eurasian plethodontids likely result from two separate dispersal events from western North America via Beringia in the late Eocene (~42 Ma) and the early Miocene (~23 Ma), respectively.

Current distribution and status of amphibians and reptiles in Will County, Illinois

The distribution of amphibians and reptiles in Will County, Illinois, was assessed using museum records and results from 58 surveys conducted between 1986 and 2009 on lands owned and managed by the Forest Preserve District of Will County (FPDWC) and Illinois Department of Natural Resources (IDNR). One hundred and twenty sources of information were examined including technical reports, theses, dissertations, and personal data belonging to the authors. Forty-eight species (19 amphibians, 29 reptiles) were documented by photos or specimens from Will County. Eleven species were determined widespread and common, 6 were categorized as patchy distributions and are uncommon or locally common, and 23 were found to have restricted distributions and are considered rare, including seven state-listed species. One of those state-listed species (Sistrurus catenatus) is likely extirpated. Eight species had undetermined distributions, four of them of questionable occurrence. One of the questionable species (Anaxyrus fowleri) is no longer included in the current list of Will County herpetofauna because it has been determined to have been an introduction and no known population exists. For three others Terrapene carolina, Pantherophis spiloides, Nerodia rhombifer) we were unable to conclusively interpret their records and they are assigned questionable status until further records confirm existence of viable, self sustaining populations. Of seven state-listed species (four threatened, three endangered), one may be extirpated (Sistrurus catenatus). Lithobates sylvaticus, Hemidactylium scutatum, Ambystoma texanum, and Eurycea cirrigera are glacial relicts that are highly vulnerable to extirpation due to pollution, isolation, and a warming climate. Presently, 43 species (18 amphibians, 25 reptiles) are known to occur in Will County, making it the most species-rich county in the Chicago region.

Normal table of embryonic development in the four-toed salamander, Hemidactylium scutatum

We present a complete staging table of normal development for the lungless salamander, Hemidactylium scutatum (Caudata: Plethodontidae). Terrestrial egg clutches from naturally ovipositing females were collected and maintained at 15 °C in the laboratory. Observations, photographs, and time-lapse movies of embryos were taken throughout the 45-day embryonic period. The complete normal table of development for H. scutatum is divided into 28 stages and extends previous analyses of H. scutatum embryonic development (Bishop, 1920; Humphrey, 1928). Early embryonic stage classifications through neurulation reflect criteria described for Xenopus laevis, Ambystoma maculatum and other salamanders. Later embryonic stage assignments are based on unique features of H. scutatum embryos. Additionally, we provide morphological analysis of gastrulation and neurulation, as well as details on external aspects of eye, gill, limb, pigmentation, and tail development to support future research related to phylogeny, comparative embryology, and molecular mechanisms of development.

Myf-5 and the molecular analysis of tail development in the four-toed salamander, Hemidactylium scutatum

Myf-5 and the molecular analysis of tail development in the four-toed salamander, Hemidactylium scutatum

Antifungal skin bacteria, embryonic survival, and communal nesting in four-toed salamanders, Hemidactylium scutatum

We examined a novel hypothesis for the maintenance of communal nesting in the salamander, Hemidactylium scutatum, namely that communal nests are more likely than solitary nests to be associated with cutaneous antifungal bacteria, which can inhibit fungal infections of embryos. A communal nest contains eggs of two or more females of the same species. The nesting behavior of H. scutatum females and survival of embryos were determined by frequent nest surveys at three ponds. For communal nests, embryonic survival tended to be higher and catastrophic nest failure was lower. Pure bacterial cultures of resident species were obtained from the salamanders' skins by swabbing and tested against a fungal pathogen of embryos (Mariannaea sp.) in laboratory assays. We found that 27% of females had skin bacteria inhibitory to Mariannaea sp. Communal nests were more likely to have at least one female with antifungal bacteria than were solitary nests. Using a culture-independent assay (denaturing gradient gel electrophoresis of 16S rRNA gene fragments), we found that bacterial species on females and embryos were more similar to each other than they were to bacterial species found in soil within the nest, suggesting that females transmitted skin bacteria to embryos. The presence of anti-Mariannaea skin bacteria identified from the laboratory assays did not prevent fungal presence in field nests. However, once a nest was visibly infected with fungi, presence of anti-Mariannaea bacteria was positively correlated with survival of embryos. Microbe transmission is usually thought to be a cost of group living, but communal nesting in H. scutatum may facilitate the transmission of antifungal bacteria to embryos.

NEST SITE SELECTION AND EMBRYONIC SURVIVAL IN FOUR-TOED SALAMANDERS, HEMIDACTYLIUM SCUTATUM (CAUDATA: PLETHODONTIDAE)

Nest site selection occurs when females place eggs at sites that differ from random sites within a delimited area. Selection of components of the microhabitat that increase embryonic survival is expected. Female four-toed salamanders, Hemidactylium scutatum, at two montane study ponds in Virginia selected nesting locations that had more moss, grass, and other living vegetation than found in other locations around the pond. In addition, selected nest sites had steeper bank angles and average lower pH's than other sites and were more often found facing north than expected by chance. Of the variables selected by H. scutatum when choosing a nest site, bank angle, nest aspect, and pH were correlated with embryonic survival. We found that north-facing nests were cooler than south-facing nests. Embryonic survival was highest in nests on steep slopes that faced north with lower average temperatures and that had lower pH's. A third pond that historically had fewer nesting females and was characterized by low embryonic survival did not have nesting habitats that combined steep bank angles with a northerly aspect. When constructing ponds for wildlife in areas where H. scutatum is of special concern, we suggest that steep bank angles be situated so that they face north.

Diversity of cutaneous bacteria with antifungal activity isolated from female four-toed salamanders

Among the microbiota of amphibian skin are bacteria that produce antifungal compounds. We isolated cutaneous bacteria from the skins of three populations of the nest-attending plethodontid salamander Hemidactylium scutatum and subsequently tested the bacterial isolates against two different fungi (related to Mariannaea elegans and Rhizomucor variabilis) that were obtained from dead salamander eggs. The culturable antifungal bacteria were phylogenetically characterized based on 16S rRNA phylogeny, and belonged to four phyla, comprising 14 bacterial families, 16 genera and 48 species. We found that about half of the antifungal bacterial genera and families were shared with a related salamander species, but there was virtually no overlap at the species level. The proportion of culturable antifungal bacterial taxa shared between two large populations of H. scutatum was the same as the proportion of taxa shared between H. scutatum and Plethodon cinereus, suggesting that populations within a species have unique antifungal bacterial species. Approximately 30% of individuals from both salamander species carried anti-M. elegans cutaneous bacteria and almost 90% of P. cinereus and 100% of H. scutatum salamanders carried anti-R. variabilis cutaneous bacteria. A culture independent method (PCR/DGGE) revealed a shared resident bacterial community of about 25% of the entire resident bacterial community within and among populations of H. scutatum. Thus, the culturable antifungal microbiota was far more variable on salamander skins than was the bacterial microbiota detected by PCR/DGGE. The resident cutaneous antifungal bacteria may play an important role in amphibians' innate defense against pathogens, including the lethal chytrid fungus Batrachochytrium dendrobatidis.

Body condition and order of arrival affect cooperative nesting behaviour in four-toed salamanders Hemidactylium scutatum

Joint nesting is defined as two or more conspecific females laying eggs in the same nest. Female four-toed salamanders may lay and brood eggs in a solitary nest, lay eggs in a joint nest and brood them along with eggs of other females, or lay eggs in a joint nest that is brooded by another female. Using data from a 9-year study in mesocosms, I found that body condition index scores were associated with nesting behaviour. Joint nesters who stayed with the nest were in the best condition, whereas joint nesters who deserted the nest were in the worst condition. Solitary nesters, who almost always stayed with their nests, were in intermediate condition. Nesting behaviour was also a function of whether a female founded or joined a nest. Females that laid eggs first in what ultimately became a joint nest tended to stay regardless of relative body condition. Nest joiners stayed if they were in relatively good condition, but deserted if they were in relatively poor condition. Nest founders may tolerate nest joiners because there is no cost to doing so, they cannot nest again within a season, and the costs of fighting to exclude a potential nest joiner are prohibitive. Body condition of individuals varies in populations and may often affect costs and benefits of nesting decisions. Therefore, the inclusion of body condition variation into models of joint nesting will lead to a more predictive theory of nesting and breeding decisions.

A Review of the Amphibians of the Jim Timmerman Natural Resources Area, Oconee and Pickens Counties, South Carolina

Thirteen species of salamanders and nine species of frogs and toads are recorded from the Jim Timmerman Natural Resources Area in South Carolina. Additionally, eight species of salamanders and five species of frogs are listed as potentially occurring within the boundaries of the property. Pseudotriton montanus (Mud Salamander) is considered rare on the property due to the scarcity of suitable habitat and is known only from historical records. Ambystoma maculatum (Spotted Salamander) may utilize non-breeding habitat on the property, but known breeding sites are located off the property near the southern boundary. Desmognathus fuscus (Northern Dusky Salamander) is apparently absent from the property, and perhaps from the entire escarpment and mountain region in South Carolina. Current range maps of D. fuscus, based on historical records, suggest a continuous distribution through this region, but a re-examination of pertinent museum specimens reveals that all records from the escarpment and mountains of South Carolina are based on erroneous identifications. Hemidactylium scutatum is known from only two localities in Pickens County near the southern boundary of the property. It is considered rare and patchily distributed in the upstate. Cryptobranchus alleganiensis is apparently absent from the property as well as the entire upstate. Its purported presence in South Carolina is based on one specimen record from the Lake Tugaloo section of the Savannah drainage system. Rana palustris (Pickerel Frog) is uncommon, and Rana sphenocephala (Southern Leopard Frog) is known only from historical records, and may now be extinct within the property. The reported isolate of Bufo terrestris in Pickens and Greenville counties has not been verified following field surveys. It is hypothesized that the several upstate records are based on anomalous specimens of Bufo americanus. The diverse amphibian fauna of the Jocassee Valley was extirpated when the valley was flooded following completion of the Jocassee Dam in 1972.

Amphibian Pathogen Batrachochytrium dendrobatidis Is Inhibited by the Cutaneous Bacteria of Amphibian Species

Population declines of amphibian species in many parts of the world are associated with a lethal fungal pathogen, Batrachochytrium dendrobatidis. Using laboratory challenge assays, we describe the inhibition of B. dendrobatidis by members of eight genera of bacteria isolated from the skin of two amphibian species that exhibit parental care behavior (Plethodon cinereus and Hemidactylium scutatum). We found that members of three genera of bacteria isolated from the skins of the salamander P. cinereus and members of seven genera isolated from the salamander H. scutatum inhibited the growth of B. dendrobatidis. Understanding how B. dendrobatidis interacts with an ecological community of cutaneous flora may be important in explaining and preventing amphibian population declines.

Wetland and Microhabitat Use by Nesting Four-Toed Salamanders in Maine

Little is known of Four-Toed Salamander (Hemidactylium scutatum) habitat use, despite the species' extensive range and elevated conservation status. We investigated species-habitat relationships that predict H. scutatum nesting presence in Maine at wetland and microhabitat scales by comparing microhabitats with and without nests. We created logistic regression models, selected models with AIC, and evaluated models with reserve data. Wetlands with nests were best predicted by shoreline microhabitat of Sphagnum spp., wood substrate, water flow, blue-joint reed grass (Calamagrostis canadensis), meadowsweet (Spiraea alba), steeplebush (Spiraea tomentosa), sensitive fern (Onoclea sensibilis), and absence of sheep laurel (Kalmia angustifolia) or deciduous forest canopy. Within occupied wetlands, shoreline microhabitat where nests occurred was best distinguished from available, unoccupied shoreline microhabitat by steeper shore, greater near-shore and basin water depth, deeper nesting vegetation, presence of moss spp. and winterberry (Ilex verticillata), and a negative association with S. alba, leatherleaf (Chamaedaphne calyculata), and K. angustifolia. These models of wetland and microhabitat use by H. scutatum may assist ecologists and managers in detecting and conserving this species.

The Herpetofauna of Arnold Air Force Base in the Barrens of South-Central Tennessee

Arnold Air Force Base (AFB) comprises ca. 15,800 ha in the Barrens region of south-central Tennessee. General herpetofaunal field collecting tech- niques were used to survey low-lying forests subject to seasonal flooding, dry forested slopes, old fields, intermittent and permanent streams, and a 1611-ha reservoir. Sixty species were found, comprising 46% of the known state- and 75% of the suspected Barrens-herpetofaunal species. Several species listed as needing special protection by state or federal agencies occur at the site, includ- ing Rana capito, Hyla gratiosa, Ambystoma talpoideum, Hemidactylium scutatum, Pituophis melanoleucus, and Ophisaurus attenuatus. Significant ex- tensions or clarifications of distributions were noted for R. capito, Ambystoma texanum, Nerodia erythrogaster, Virginia valeriae, Thamnophis sauritus, Pseudemys concinna, and Kinosternon subrubrum.

RESOURCE LEVEL AND REPRODUCTIVE FREQUENCY IN FEMALE FOUR-TOED SALAMANDERS, HEMIDACTYLIUM SCUTATUM

Many species of vertebrate animals skip reproduction in some years. We studied the pattern of reproduction exhibited by female four-toed salamanders (Hemidactylium scutatum). Data from a 10-year field study showed that females reproduced in sequential years with a frequency of 0.56 but skipped one or more years of reproduction with a frequency of 0.44. This pattern was not correlated with whether a female attended or deserted her nest. When females skipped reproduction, they had higher growth, which can translate into higher fecundity. We experimentally examined whether female four-toed salamanders reproduced in sequential years if they had adequate resources after nesting and before the onset of winter. A manipulation of food level after the nesting period revealed that females fed a high food ration were twice as likely to return the next year to oviposit as females fed a lower food level. This study is the first to document experimentally that reproductive frequency varies with resource level in a vertebrate species.