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Objective Animals Studied Procedure Results Conclusion The aim of this study was to document ophthalmic examination findings and provide reference values for ocular examinations and diagnostics in clinically normal Western tiger salamanders (Ambystoma mavortium) and to evaluate the mydriatic effect of a topical rocuronium bromide and identify any adverse effects.Sixteen Western tiger salamanders.A complete ophthalmic examination, including intraocular pressure via rebound tonometry [iCare® TonoVet (dog‐setting) or iCare® TonoVet Plus (dog‐setting)], and fluorescein and Rose Bengal stains, was performed. Ocular surface flora culture swabs were collected for microbiology in four salamanders. Rocuronium bromide (50 μL; 0.01 mg) was administered into each eye in three salamanders. Pupil size and constriction ability were evaluated up to 60 min following administration of rocuronium bromide. Distant visual examination and Doppler heart rate were used to monitor for potential adverse effects of rocuronium bromide.Observed ocular abnormalities included lipid keratopathy/keratitis (19%; 3/16), parasitic ophthalmitis (19%; 3/16), and cataracts (6.3%; 1/16). A significant difference was found between tonometry with iCare® TonoVet (11.5 ± 2.7 mmHg) versus TonoVet Plus (15.4 ± 3.7 mmHg) (p = .025). Fluorescein and Rose Bengal stains were negative in all salamanders. Ocular surface microbiology revealed a high incidence of contamination from environmental flora, with the most common isolate being unidentified as Gram‐negative nonfermenters (75%; 3/4). No significant change in pupil size or constriction ability was observed with rocuronium bromide.This is the first study to document ocular examination findings and diagnostics in captive and wild urodeles and may serve as a reference for clinical assessment and future studies of the Western tiger salamander. [ABSTRACT FROM AUTHOR]

Recently metamorphosed amphibians transport substantial biomass and nutrients from wetlands to terrestrial ecosystems. Previous estimates (except 1) were limited to either a subset of the community or a single year. Our goal was to examine temporal variability in biomass export of all amphibians within breeding ponds and the composition of that export. We completely encircled ponds with drift fences to capture, count, and weigh emerging recently metamorphosed individuals in Maine (four wetlands, six years) and Missouri (eight wetlands, 2–4 years). We estimated total amphibian biomass export, export scaled by pond surface area, species diversity, and percentage of biomass from anurans. Biomass export and export composition varied greatly among ponds and years. Our estimates were of similar magnitude to previous studies. Amphibian biomass export was higher when species diversity was low and the proportion of anurans was higher. Biomass estimates tended to be highest for juvenile cohorts dominated by a single ranid species: green frogs (Missouri) or wood frogs (Maine). Ranid frogs made up a substantial proportion of amphibian biomass export, suggesting that terrestrial impacts will likely occur in the leaf litter of forests. Future studies should examine the impacts of ranid juveniles on terrestrial ecosystem dynamics. [ABSTRACT FROM AUTHOR]

Abstract Environmental DNA (eDNA), or DNA that is shed into the environment by an organism, can be used to detect the presence of cryptic species. However, eDNA methodology requires validation of an assay in both laboratory and field environments. Here, we describe the development of a quantitative PCR (qPCR) assay and field protocol for detecting a secretive amphibian, the Streamside Salamander (Ambystoma barbouri). This fossorial species is rarely encountered because adults are only active for several months during the winter when they breed and deposit eggs underneath rocks within intermittent streams. We designed and validated a qPCR assay for A. barbouri against five ambystomatid congeners and the Southern Two‐lined Salamander (Eurycea cirrigera), which occur within the range of A. barbouri in the focal study area. We detected DNA of A. barbouri in the laboratory to 0.0004 ng/µl, while all other congeners were rarely detected below 40 ng/µl. We collected 1‐L water samples from 45 streams from December 2016 to May 2017 and during April 2018 to validate our methods in the field. Our assay was effective at detecting A. barbouri in 24 of 45 streams. We confirmed physical presence of A. barbouri (adults, larvae, and/or eggs) at 21 out of 24 of these positive sites. The detection probability was 0.85 ± 0.05; CI: 0.75, 0.95 within a single sampling event that incorporated 5 water samples from each of 17 repeat‐visit sites. The per water sample sensitivity was 68% (163/238) and specificity was 100% (22/22). The per site visit sensitivity was 85% (46/54) and specificity was 100% (6/6). Lastly, the per site sensitivity was 95% (21/22) and specificity was 100% (3/3). Collectively, this protocol outlines an efficient and cost‐effective method to detect A. barbouri and provides a technique to rapidly identify sites where breeding is occurring and to determine the distribution of this species.

Abstract Aim For many endemic species with limited dispersal capacities, the relationship between landscape changes and species distributions is still unclear. We characterized the population structure of the endemic ringed salamander (Ambystoma annulatum) across its distribution in the Central Interior Highlands (CIH) of North America, an area of high species endemism, to infer the ecological and evolutionary history of the species. Methods We sampled 498 individuals across the species distribution and characterized the population genetic structure using nuclear microsatellite and mitochondrial DNA (mtDNA) markers. Results Ambystoma annulatum exist in two strongly supported nuclear genetic clusters across the CIH that correspond to a northern cluster that includes the Missouri Ozark populations and a southern cluster that includes the Arkansas and Oklahoma Ozarks and the Ouachita Mountains. Our demographic models estimated that these populations diverged approximately 2,700 years ago. Pairwise estimates of genetic differentiation at microsatellite and mtDNA markers indicated limited contemporary gene flow and suggest that genetic differentiation was primarily influenced by changes in the post‐Pleistocene landscape of the CIH. Main Conclusions Both the geologic history and post‐European settlement history of the CIH have influenced the population genetic structure of A. annulatum. The low mtDNA diversity suggests a retraction into and expansion out of refugial areas in the south‐central Ozarks, during temperature fluctuations of the Pleistocene and Holocene epochs. Similarly, the estimated divergence time for the two nuclear clusters corresponds to changes in the post‐Pleistocene landscape. More recently, decreased A. annulatum gene flow may be a result of increased habitat fragmentation and alteration post‐European settlement.

Recently metamorphosed amphibians transport substantial biomass and nutrients from wetlands to terrestrial ecosystems. Previous estimates (except 1) were limited to either a subset of the community or a single year. Our goal was to examine temporal variability in biomass export of all amphibians within breeding ponds and the composition of that export. We completely encircled ponds with drift fences to capture, count, and weigh emerging recently metamorphosed individuals in Maine (four wetlands, six years) and Missouri (eight wetlands, 2–4 years). We estimated total amphibian biomass export, export scaled by pond surface area, species diversity, and percentage of biomass from anurans. Biomass export and export composition varied greatly among ponds and years. Our estimates were of similar magnitude to previous studies. Amphibian biomass export was higher when species diversity was low and the proportion of anurans was higher. Biomass estimates tended to be highest for juvenile cohorts dominated by a single ranid species: green frogs (Missouri) or wood frogs (Maine). Ranid frogs made up a substantial proportion of amphibian biomass export, suggesting that terrestrial impacts will likely occur in the leaf litter of forests. Future studies should examine the impacts of ranid juveniles on terrestrial ecosystem dynamics.

Aim: For many endemic species with limited dispersal capacities, the relationship between landscape changes and species distributions is still unclear. We characterized the population structure of the endemic ringed salamander (Ambystoma annulatum) across its distribution in the Central Interior Highlands (CIH) of North America, an area of high species endemism, to infer the ecological and evolutionary history of the species. Methods: We sampled 498 individuals across the species distribution and characterized the population genetic structure using nuclear microsatellite and mitochondrial DNA (mtDNA) markers. Results: Ambystoma annulatum exist in two strongly supported nuclear genetic clusters across the CIH that correspond to a northern cluster that includes the Missouri Ozark populations and a southern cluster that includes the Arkansas and Oklahoma Ozarks and the Ouachita Mountains. Our demographic models estimated that these populations diverged approximately 2,700 years ago. Pairwise estimates of genetic differentiation at microsatellite and mtDNA markers indicated limited contemporary gene flow and suggest that genetic differentiation was primarily influenced by changes in the post‐Pleistocene landscape of the CIH. Main Conclusions: Both the geologic history and post‐European settlement history of the CIH have influenced the population genetic structure of A. annulatum. The low mtDNA diversity suggests a retraction into and expansion out of refugial areas in the south‐central Ozarks, during temperature fluctuations of the Pleistocene and Holocene epochs. Similarly, the estimated divergence time for the two nuclear clusters corresponds to changes in the post‐Pleistocene landscape. More recently, decreased A. annulatum gene flow may be a result of increased habitat fragmentation and alteration post‐European settlement. [ABSTRACT FROM AUTHOR]

Amphibians are one of the most vulnerable groups of tetrapods with 41% of species considered threatened with extinction. In amphibians, an increase in stress level is an early physiological response to factors associated with habitat degradation. In vertebrates, stress increases the susceptibility to parasite infections and is associated with changes in the number of blood cells, and therefore the proportion of the leukocytes, neutrophils to lymphocytes (N/L ratio) is used as a proxy measure of stress hormones. In this study, we used such leukocyte profiles to analyze stress levels and evaluate parasite load to elucidate the health condition of the endangered Mexican stream salamander, Ambystoma ordinarium. The habitat quality of streams inhabited by A. ordinarium was evaluated by Rapid Bioassessment Protocols (RBPs). We selected three streams with the highest RBPs scores as the undisturbed streams and three with the lowest scores as the disturbed streams. In each stream, we determined leukocyte profile and searched for ecto/endoparasites on sampled individuals. We report for the first time the leukocyte profile of A. ordinarium. A significantly higher N/L ratio was found in salamanders from disturbed vs. undisturbed streams. We also found a higher parasitic infection in salamanders from disturbed streams. Our results suggest that low habitat quality can increase stress levels and susceptibility to parasitic infections, thereby compromising the long-term persistence of populations of this species in disturbed habitats. [ABSTRACT FROM AUTHOR]

Background: Tissue regeneration is widely distributed across the tree of life. Among vertebrates, salamanders possess an exceptional ability to regenerate amputated limbs and other complex structures. Thus far, molecular insights about limb regeneration have come from a relatively limited number of species from two closely related salamander families. To gain a broader perspective on the molecular basis of limb regeneration and enhance the molecular toolkit of an emerging plethodontid salamander (Bolitoglossa ramosi), we used RNA-Seq to generate a de novo reference transcriptome and identify differentially expressed genes during limb regeneration. Results: Using paired-end Illumina sequencing technology and Trinity assembly, a total of 433,809 transcripts were recovered and we obtained functional annotation for 142,926 non-redundant transcripts of the B. ramosi de novo reference transcriptome. Among the annotated transcripts, 602 genes were identified as differentially expressed during limb regeneration. This list was further processed to identify a core set of genes that exhibit conserved expression changes between B. ramosi and the Mexican axolotl (Ambystoma mexicanum), and presumably their common ancestor from approximately 180 million years ago. Conclusions: We identified genes from B. ramosi that are differentially expressed during limb regeneration, including multiple conserved protein-coding genes and possible putative species-specific genes. Comparative analyses reveal a subset of genes that show similar patterns of expression with ambystomatid species, which highlights the importance of developing comparative gene expression data for studies of limb regeneration among salamanders. [ABSTRACT FROM AUTHOR]

In order to better understand the impacts and treatment of infection with Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) it is important to document host species, the effect of infection and response to treatment protocols. Here we report asymptomatic Bd infection detected through duplex qPCR screening of three Mexican ambystomatid salamanders; Ambystoma andersoni, Ambystoma dumerilii and Ambystoma mexicanum at three zoo collections, and A. andersoni and A. mexicanum in a private collection. Bsal was tested for but not detected. We also report the effectiveness and side effects of five treatment protocols in these species. Using the antifungal agent itraconazole, A. dumerilii were cleared of infection without side-effects using the granulated preparation (Sporanox). Morbidity and mortality occurred when A. dumerilii and A. andersoni were treated using a liquid oral preparation of the itraconazole (Itrafungol); infection was successfully cleared in surviving specimens of the latter species. Ambystoma mexicanum was successfully cleared without any side-effects using Itrafungol. Mortality and morbidity were likely caused by toxic effects of some component on the liquid preparation of itraconazole, but aspects of water quality and husbandry cannot be ruled out. [ABSTRACT FROM AUTHOR]

The evolutionary causes and consequences of allopolyploidization, an exceptional pathway to instant hybrid speciation, are poorly investigated in animals. In particular, when and why hybrid polyploids versus diploids are produced, and constraints on sources of paternal and maternal ancestors, remain underexplored. Using the Palearctic green toad radiation (including bisexually reproducing species of three ploidy levels) as model, we generate a range-wide multi-locus phylogeny of 15 taxa and present four new insights: (i) at least five (up to seven) distinct allotriploid and allotetraploid taxa have evolved in the Pleistocene; (ii) all maternal and paternal ancestors of hybrid polyploids stem from two deeply diverged nuclear clades (6 Mya, 3.1--9.6 Mya), with distinctly greater divergence than the parental species of diploid hybrids found at secondary contact zones; (iii) allotriploid taxa possess two conspecific genomes and a deeply diverged allospecific one, suggesting that genomic imbalance and divergence are causal for their partly clonal reproductive mode; (iv) maternal versus paternal genome contributions exhibit asymmetry, with the maternal nuclear (and mitochondrial) genome of polyploids always coming from the same clade, and the paternal genome from the other.We compare our findings with similar patterns in diploid/polyploid vertebrates, and suggest deep ancestral divergence as a precondition for successful allopolyploidization. [ABSTRACT FROM AUTHOR]

Physiological condition linked to reproduction-related morphological traits ( e.g. , body mass, head width, tail height) is a key determinant of ecotypes related to fitness in ecological selection. In migratory salamanders such as the families Ambystomatidae (internal fertilization), Salamandridae (internal), and Hynobiidae (external), such morphological traits change at the transition between terrestrial and aquatic habitats. Unlike many other migratory salamanders, Hynobius kimurae (Hynobiidae) immigrates from land to water for aquatic hibernation during fall, and the immigration is not related to fall breeding. In this context, does fall immigration to water for winter dormancy induce changes in reproduction-related morphological traits? We analyzed changes in body shape and mass concurrent with physiological condition of 244 males and 131 females of H. kimurae with only data on first capture during fall and spring from 1996 to 2016. Although reproduction-related morphological traits in other migratory salamanders change simultaneously when they enter the water and subsequently initiate breeding during either season of fall–spring, such changes occurred both in male and female H. kimurae firstly during fall (shortly after entering the water) and secondly during spring (shortly after awaking from hibernation), except for head width being static throughout seasons. The differences suggest that these two-step changes during a prolonged period from fall to spring would be concentrated in a short period of breeding activity during either season of fall–spring in other migratory salamanders without hibernating in the water. As sexually selected, sexual dimorphism was detected in size and shape, especially shape of the tail, both height and length of which were greater in males than in females regardless of the sexes being terrestrial or aquatic. The male’s larger tail during the terrestrial phase may be a stock for a more developed tailfin (amount of reproductive output) in the aquatic-phase male potentially related to fitness. [ABSTRACT FROM AUTHOR]

Plasticity in life history strategies can be advantageous for species that occupy spatially or temporally variable environments. We examined how phenotypic plasticity influences responses of the mole salamander, Ambystoma talpoideum, to disturbance events at the St. Marks National Wildlife Refuge (SMNWR), FL, USA from 2009 to 2014. We observed periods of extensive drought early in the study, in contrast to high rainfall and expansive flooding events in later years. Flooding facilitated colonization of predatory fishes to isolated wetlands across the refuge. We employed multistate occupancy models to determine how this natural experiment influenced the occurrence of aquatic larvae and paedomorphic adults and what implications this may have for the population. We found that, in terms of occurrence, responses to environmental variation differed between larvae and paedomorphs, but plasticity (i.e. the ability to metamorphose rather than remain in aquatic environment) was not sufficient to buffer populations from declining as a result of environmental perturbations. Drought and fish presence negatively influenced occurrence dynamics of larval and paedomorphic mole salamanders and, consequently, contributed to observed short-term declines of this species. Overall occurrence of larval salamanders decreased from 0.611 in 2009 to 0.075 in 2014 and paedomorph occurrence decreased from 0.311 in 2009 to 0.121 in 2014. Although variation in selection pressures has likely maintained this polyphenism previously, our results suggest that continued changes in environmental variability and the persistence of fish in isolated wetlands could lead to a loss of paedomorphosis in the SMNWR population and, ultimately, impact regional persistence in the future. [ABSTRACT FROM AUTHOR]

The populations of ambystomatid salamanders around Mexico City are subject to a variety of threats, and some populations may be in decline. Three Ambystoma species found around Mexico City and in central Mexico are A. altamirani, A. leorae, and A. rivulare, and these three species are subject to a variety of conservation threats. We compiled a database of localities for these ambystomatid salamanders. The compiled observations of these three species of endangered salamanders suggest several patterns: 1) most localities for all three species are in the Estado de México, including several for A. altamirani within the borders of Mexico City; 2) there is little, if any, geographical overlap among these three species; 3) the relatively few documented sites for A. leorae and A. rivulare highlight their tenuous conservation status. Our hope is that this presentation of a map of documented locations of these three Mexican Ambystoma has created a starting point for future studies on these salamanders. [ABSTRACT FROM AUTHOR]

Ecological and evolutionary processes commonly result in morphological variation among larval amphibians. Variation in head shape plays a critical role in both food capture and predation risk in gape-limited salamanders, yet in situ studies of head shape variability are rare outside of cannibal morph assessments. We examined allometry differences in larval head width (HW) and snout-vent length (SVL) among three sympatric species of ambystomatid salamanders from 166 ponds in Missouri, USA: Ringed Salamander ( Ambystoma annulatum), Marbled Salamander ( A. opacum), and Spotted Salamander ( A. maculatum). We also tested whether several abiotic and biotic factors would predict HW after accounting for SVL. We found that larval HW and SVL were strongly correlated for all species but that the strength of this relationship varied among species. For early-stage larvae, Marbled Salamanders showed isometric scaling relationships, whereas both Spotted Salamanders and Ringed Salamanders were allometric. For late-stage larvae, all three species showed allometric patterns. At a small SVL, HW of small Ringed Salamanders was greater than the other species. As larvae increased in SVL, Marbled Salamander HW increased most rapidly and eventually exceeded both Ringed Salamanders and Spotted Salamanders of a similar size. We also found that both abiotic and biotic factors predicted significant differences in HW corrected for SVL among species, including predator density, competitor density, and hydroperiod. Overall, variability in scaling relationships may provide ecological advantages to each species at different points in ontogeny and different biotic and abiotic factors may induce such variation in asymmetric ways among species. [ABSTRACT FROM AUTHOR]

Cryptic sex has been argued to explain the exceptional longevity of certain parthenogenetic vertebrate lineages, yet direct measurements of genetic exchange between sexual and apparently parthenogenetic forms are rare. Female unisexual mole salamanders ( Ambystoma sp.) are the oldest known unisexual vertebrate lineage (~5 million years), and one hypothesis for their persistence is that allopolyploid female unisexuals periodically exchange haploid genomes 'genome exchange' during gynogenetic reproduction with males from sympatric sexual species. We test this hypothesis by using genome-specific microsatellite DNA markers to estimate the rates of genome exchange between sexual males and unisexual females in two ponds in NE Ohio. We also test the prediction that levels of gene flow should be higher for 'sympatric' (sexual males present) genomes in unisexuals compared to 'allopatric' (sexual males absent) unisexual genomes. We used a model testing framework in the coalescent-based program MIGRATE-N to compare models where unidirectional gene flow is present and absent between sexual species and unisexuals. As predicted, our results show higher levels of gene flow between sexuals and sympatric unisexual genomes compared to lower (likely artefactual) levels of gene flow between sexuals and allopatric unisexual genomes. Our results provide direct evidence that genome exchange between sexual and unisexual Ambystoma occurs and demonstrate that the magnitude depends on which sexual species are present. The relatively high levels of gene flow suggest that unisexuals must be at a selective advantage over sexual forms so as to avoid extinction due to genetic swamping through genome exchange. [ABSTRACT FROM AUTHOR]

We examined how variation in food availability and temperature influences patterns of energy allocation to growth, storage, metamorphosis, and reproduction in larval Ambystoma maculatum and A. tigrinum. In each species, an allocation vector consisting of metamorphic size, duration of larval period, fat body mass, and gonad mass was influenced by both food and temperature. Larvae grew rapidly at high food levels and high temperature, and delayed metamorphosis when grown at low temperature. High food levels resulted in larger fat bodies and decreased time to metamorphosis in A. tigrinum, and larger gonads in both species. The allocation vector collectively representing growth rate, storage, reproduction and metamorphosis differed between species: in A. maculatum, only the correlation between metamorphic size and fat body mass was significant, whereas all four variables were influenced in A. tigrinum. An interaction of food and temperature on the allocation vector was only detected for A. tigrinum. Our results indicate that interaction among allocation variables is stronger in some species than others and that these associations might be important in understanding life history variation between these species. [ABSTRACT FROM AUTHOR]

The early evolution of salamanders, which are one of the three living groups of lissamphibians, is not well known. Both stem- and crown-group salamanders first appeared in the Middle Jurassic (Bathonian), but subsequently had different evolutionary histories: stem salamanders were thought to have gone extinct in the Late Jurassic, while crown salamanders persist to the present day. Here, I report the discovery of an indeterminate stem salamander in the Lower Cretaceous (Aptian-Albian) Ilek Formation of Western Siberia. This is new evidence that the most basal salamanders survived beyond the Jurassic-Cretaceous boundary and co-existed with crown-group salamanders during approximately the first 40 million years of the known history of salamanders. The recognition of stem salamanders in the Early Cretaceous of Western Siberia adds to the inventory of taxa that suggest this area was a refugium for various groups of vertebrates with Jurassic affinities. [ABSTRACT FROM AUTHOR]

Quantifying the relationship between demographic patterns and genetic diversity are important in predicting temporal population genetic changes. To make predictions there must be an understanding of the relationship between census size, the effective number of breeders (N) and effective population size (N). We evaluate the temporal genetic variation between three populations of Ambystoma salamanders, and compare genetic and demographic estimates of N and N. We sampled two wetlands, RB and GB for A. opacum and sampled A. talpoideum at RB, over a 20-year period. Ambystoma opacum colonized the RB wetland in 1980 and the population has steadily expanded as A. talpoideum has declined towards local extinction; the GB population of A. opacum has remained relatively large and stable over this same time period. Genetic variation at 10 microsatellites remained stable at each population over the sampled time frame, and did not reflect changes in population size. Genetic estimates of N also did not reflect demographic trends, and were lower than demographic estimates of N. Genetic methods of determining N gave similar estimates to demographic methods. Our findings indicate that sample sizes and number of markers typically used in genetic studies do not provide enough precision to monitor population size changes in amphibians, which likely violate many of the assumptions of N and N estimation models. These findings should be considered when using N in conservation and management assessments of amphibian populations. [ABSTRACT FROM AUTHOR]

A fundamental goal of evolutionary ecology is to understand how asymmetric competition influences phenotype expression, yet few studies have quantified the relative effects of intra- and interspecific competition on phenotypes. We examined the effects of intra- and interspecific larval competition on both discrete and continuous phenotype expression of the facultatively paedomorphic mole salamander (Ambystoma talpoideum). We manipulated the density and frequency of larval A. talpoideum and a common competitor, the spotted salamander (A. maculatum), in experimental mesocosms within a response surface design. The production of discrete ontogenetic morphs (metamorphs, paedomorphs, and overwintering larvae) was affected more by intraspecific than interspecific competition, but the effect varied between morphs. Paedomorph and metamorph production were more strongly affected by intraspecific than interspecific competition, while the production of overwintering larvae was affected by each type of competition approximately equally. Paedomorphs largely occurred only at low conspecific densities, overwintering larvae primarily occurred at high overall densities, and metamorph production peaked at intermediate densities of con- and heterospecifics. Density-dependent intraspecific competition had stronger effects on body size and growth rates than interspecific competition for both metamorphs and overwintering larvae, whereas interspecific competition more strongly affected paedomorph size. Overall, these results support the hypothesis that intraspecific competition influences both continuous and discrete phenotypic expression more strongly than interspecific competition. Fluctuating strengths of each type of competition can affect variation in life history strategies by influencing discrete ontogenetic pathways via individual body sizes, leading to differential morph production and fitness within and among morphs. [ABSTRACT FROM AUTHOR]

Ambystoma altamiranoi is an endangered endemic salamander found in the Transvolcanic Belt of Mexico. Unfortunately, relatively little is known about its ecology. Here we report on the diet of larval A. altamiranoi from a population in Llano de los Axolotes, Sierra de las Cruces, State of México, Mexico. Empty stomachs were found in 13.3% of individuals. Ostracods and gastropods dominated the diet of A. altamiranoi, together accounting for 89.9% of prey items consumed. The remainder of the diet consisted primarily of insects. Our observations suggest that the diet of A. altamiranoi is relatively narrow and that resources may be limited (relatively high frequency of empty stomachs), suggesting that factors that could impact the availability of ostracods and gastropods might have serious consequences for these endangered salamanders. [ABSTRACT FROM AUTHOR]

Eleven female Jefferson Salamanders (Ambystoma jeffersonianum) and five female Spotted Salamanders (A. maculatum) were collected during 28 February-17 March 2011 from the same vernal pool in south-central Pennsylvania for comparison of reproductive characteristics. Mean clutch size (169.5 eggs) and relative clutch mass (12.9%) of A. jeffersonianum was larger than those values of A. maculatum (130.8 eggs and 9.4%, respectively) despite an 8.9% smaller body size of the former. However, mean egg diameter was larger in A. maculatum. Greater fecundity, even if at the cost of egg size, could provide A. jeffersonianum with an added reproductive advantage in areas of syntopy with A. maculatum, where it is known to be a superior competitor and predator of A. maculatum at the larval stage. [ABSTRACT FROM AUTHOR]

Introduction Many species of ambystomatid salamanders are dependent upon highly variable temporary wetlands for larval development. High larval densities may prompt the expression of a distinct head morphology that may facilitate cannibalism. However, few studies have characterized structural cannibalism within natural populations of larval salamanders. In this study we used two species of larval salamanders, long-toed (Ambystoma macrodactylum) and ringed salamanders (A. annulatum). Head morphometrics and stable isotopic values of carbon (δ13C) and nitrogen (δ15N) were used to identify the presence or absence of structural cannibalism. Weather conditions were also analyzed as a potential factor associated with the expression of cannibalistic morphology. Results Populations of salamander larvae did not consistently exhibit cannibalistic morphologies throughout collection periods. Larval long-toed salamanders exhibited trophic polymorphisms when relatively lower precipitation amounts were observed. Larval ringed salamanders were observed to be cannibalistic but did not exhibit polymorphisms in this study. Conclusions Structural cannibalism may be transient in both species; however in long-toed salamanders this morphology is necessary for cannibalism. Ringed salamanders can be cannibalistic without morphological adaptations; however the cannibal morph may prolong the viable time period for cannibalism. Additionally, weather conditions may alter pond hydroperiod, subsequently influencing head morphology and cannibalism. [ABSTRACT FROM AUTHOR]

The reduced immigration and emigration rates resulting from the lack of landscape connectivity of patches and the hospitality of the intervening matrix could favor the loss of alleles through genetic drift and an increased chance of inbreeding. In order for isolated populations to maintain sufficient levels of genetic diversity and adapt to environmental changes, one important conservation goal must be to preserve or reestablish connectivity among patches in a fragmented landscape. We studied the last known population of Ambystoma leorae, an endemic and critically threatened species. The aims of this study were: (1) to assess the demographic parameters of A. leorae and to distinguish and characterize the microhabitats in the river, (2) to determine the number of existing genetic groups or demes of A. leorae and to describe possible relationships between microhabitats types and demes, (3) to determine gene flow between demes, and (4) to search for geographic locations of genetic discontinuities that limit gene flow between demes. We found three types of microhabitats and three genetically differentiated subpopulations with a significant level of genetic structure. In addition, we found slight genetic barriers. Our results suggest that mole salamander’s species are very sensitive to microhabitat features and relatively narrow obstacles in their path. The estimates of bidirectional gene flow are consistent with the pattern of a stepping stone model between demes, where migration occurs between adjacent demes, but there is low gene flow between distant demes. We can also conclude that there is a positive correlation between microhabitats and genetic structure in this population. [ABSTRACT FROM AUTHOR]

The article describes a case study that presents a new species of Aplectana in Ambystoma velasci found in Mexico. Topics covered include the identified species of Ambystoma, the materials and methods used to examine the specimens of Ambystoma velasci collected from Lago de Tecocomulco in Hidalgo, Mexico, and the description of the new species of Aplectana. It also discusses the taxonomic summary relating to Aplectana found in Ambystoma velasci.

Distinct genetic markers should show similar patterns of differentiation between species reflecting their common evolutionary histories, yet there are increasing examples of differences in the biogeographic distribution of species-specific nuclear (nu DNA) and mitochondrial DNA (mt DNA) variants within and between species. Identifying the evolutionary processes that underlie these anomalous patterns of genetic differentiation is an important goal. Here, we analyse the putative mitonuclear discordance observed between sister species of mole salamanders ( Ambystoma barbouri and A. texanum) in which A. barbouri-specific mt DNA is found in animals located within the range of A. texanum. We test three hypotheses for this discordance (undetected range expansion, mt DNA introgression, and hybridization) using nu DNA and mt DNA data analysed with methods that varied in the parameters estimated and the timescales measured. Results from a Bayesian clustering technique ( structure), bidirectional estimates of gene flow ( migrate- n and IMa2) and phylogeny-based methods (* beast, bucky) all support the conclusion that the discordance is due to geographically restricted mt DNA introgression from A. barbouri into A. texanum. Limited data on species-specific tooth morphology match this conclusion. Significant differences in environmental conditions exist between sites where A. texanum with and without A. barbouri-like mt DNA occur, suggesting a possible role for selection in the process of introgression. Overall, our study provides a general example of the value of using complimentary analyses to make inferences of the directionality, timescale, and source of mt DNA introgression in animals. [ABSTRACT FROM AUTHOR]

Loss or alteration of natural wetland habitats is a near ubiquitous global phenomenon. In the US, legislation mandates that all lost wetland habitats be replaced; manmade wetland habitats rarely have the same structural form or ecological function as natural wetlands. In the eastern US, these manmade pond habitats often serve as water sources for wildlife, but many are also utilized by amphibians for reproduction. Understanding the features that maximize species' abundance and diversity is critical to effective management. In this study, we surveyed for ambystomatid salamander larvae at 169 manmade ponds in a military training installation. Three species were present: Ambystoma maculatum, A. opacum and the regionally endemic A. annulatum. We estimated larval densities in each pond in relation to landscape- and pond-level covariates. Important factors relating to larval density were forest habitat surrounding each pond, canopy cover over a pond, the number of ponds within 300 m of the focal pond, presence of fish, slope of the pond basin, hydroperiod and amount of vegetation within the pond. Density estimates for each species were best predicted by different combinations of these factors, underscoring the need to provide a range of pond habitats to promote species diversity on the landscape. Our results indicate that manmade ponds are providing a valuable reproductive resource, but that future construction of ponds on the landscape will best serve the salamander and broader amphibian community if different combinations of hydroperiod and slope are utilized. [ABSTRACT FROM AUTHOR]

During fieldwork conducted between end of February and mid-April 2012 14 new localities were ascertained for four different taxa of the amphibian genus Lyciasalamandra (L. l. basoglui, L. l. finikensis, L. arikani and L. atifi). This paper represents the first record of L. l. basoglui from Muğla province (Saklıkent, Fethiye). Unlike the common belief in previous researches based on literature we determined that there are almost no gaps between the distributional areas of the known subspecies of L. luschani, moreover the subspecies were found in contact. The recently discovered populations of L. arikani and L. atifi were found to have some distinctive morphological differences compared to other populations of the respective species. [ABSTRACT FROM AUTHOR]

The movements of 52 cave salamanders (Speleomantes strinatii) were studied from May to October 2012, in NW Italy. Cave salamanders, caught inside an artificial cave, were individually marked with fluorescent visible implant elastomers and displaced 20 m from the capture site. A total of 27 (52% of displaced individuals) were recaptured in the cave by the end of the experiment. Six (11.5%) of these were recaptured inside the cave within two nights and ten (19%) within five nights. There were no sex or body size differences between returning or non-returning salamanders. These preliminary results suggest the existence of homing behaviour in S. strinatii [ABSTRACT FROM AUTHOR]

Branchiate Ambystoma talpoideum (Mole Salamanders) in fishless ponds can be large enough to act as predators, rather than competitors, during the spring breeding season of other amphibians. Food habits of high-density Mole Salamander populations from 2 proximate woodland ponds in Clark County, AR were examined before and after egg-laying by frogs, with an expectation that the salamanders likely would consume hatching tadpoles. However, salamanders instead commonly fed on the novel item of freshly-laid frog eggs. Results from both ponds indicated that the salamanders, perhaps due to food limitation, consumed smaller prey items than would be expected and heavily consumed frog eggs, a novel item. [ABSTRACT FROM AUTHOR]

Highlights: [•] We have made the first attempt at reconstructing the species tree for Ambystoma using multiple nuclear markers. [•] We used coalescent and concordance methods for phylogenetic reconstruction. [•] We recovered a well-resolved species tree that differs from previous phylogenetic hypotheses for this group. [•] The phylogeny was not significantly altered by removal of different loci from analyses. [ABSTRACT FROM AUTHOR]

Predation risk is an important contributor to community structure that varies in response to abiotic and biotic factors. In aquatic habitats, predation risk is often linked to hydroperiod as the latter directly influences predator identity within these ecosystems. For pond-breeding salamanders, intraguild predation (IGP) and cannibalism are prevalent interactions in larval communities, but the frequency of each type of agonistic interaction, as well as changes in their pervasiveness along hydroperiod gradients are not well understood. Size-structured populations of aquatic life stages (paedomorphic adults and overwintered larvae) of mole salamanders (Ambystoma talpoideum) have the potential to be dominant IG predators and cannibals in permanent ponds because of large size advantages over successive cohorts, but the species exists as only a single larval cohort in temporary ponds with reduced predatory abilities on guild members. Thus, both the potential for and intensity of predation by this species is linked to hydroperiod; yet, the predatory abilities of aquatic life stages of mole salamanders have not been evaluated. This study examined the extent to which larger size classes (paedomorphs and overwintered larvae) of mole salamanders preyed upon conspecifics and a congeneric competitor, the spotted salamander (Ambystoma maculatum). Predation trials occurred in indoor microcosms and were executed under two habitat treatments that included pseudo-natural conditions containing alternate prey and refugia, as well as simplified environments (no alternative food or cover). Embryos or hatchlings of mole and spotted salamanders were exposed either separately or concurrently to paedomorphic and overwintered larval mole salamanders in each habitat treatment. Additionally, overwintered larvae were offered as prey to paedomorphic adults. No significant differences in mortality were observed between habitat treatments (complex versus simple) for either embryo or hatchling predation experiments. Mortality of mole and spotted embryos did not differ when they were offered separately or together, but paedomorphs caused significantly higher mortality among mole embryos compared to spotted embryos. Hatchlings of both species were heavily preyed upon (nearly 100% in all trials), but overwintered larvae exhibited 100% survival with paedomorph predators, indicating size-thresholds of predation risk that were not species specific. Paedomorphs and overwintered larvae therefore are most likely generalist predators that feed equally and effectively on hatchlings of co-occurring species, but are more likely to consume egg masses that do not incur significant handling costs. When present, paedomorphs or overwintered larvae have the potential to alter population dynamics of both guild members and conspecifics by being dominant predators on early life stages. As the prevalence of overwintering and paedomorphosis varies along hydroperiod gradients, predation risk from this species is likely to occur discontinuously and thereby may be an important, yet variable, structuring force. [ABSTRACT FROM AUTHOR]

Abstract: Ability to regenerate limbs and central nervous system (CNS) is unique to few vertebrates, most notably the axolotl (Ambystoma sp.). However, despite the fact the neurotransmitter receptors are involved in axonal regeneration, little is known regarding its expression profile. In this project, RT-PCR and qPCR were performed to gain insight into the neurotransmitter receptors present in Ambystoma. Its functional ability was studied by expressing axolotl receptors in Xenopus laevis oocytes by either injection of mRNA or by direct microtransplantation of brain membranes. Oocytes injected with axolotl mRNA expressed ionotropic receptors activated by GABA, aspartate+glycine and kainate, as well as metabotropic receptors activated by acetylcholine and glutamate. Interestingly, we did not see responses following the application of serotonin. Membranes from the axolotl brain were efficiently microtransplanted into Xenopus oocytes and two types of native GABA receptors that differed in the temporal course of their responses and affinities to GABA were observed. Results of this study are necessary for further characterization of axolotl neurotransmitter receptors and may be useful for guiding experiments aimed at understanding activity-dependant limb and CNS regeneration. [Copyright &y& Elsevier]

Spatial autocorrelation (SAC) is often observed in species distribution data, and can be caused by exogenous, autocorrelated factors determining species distribution, or by endogenous population processes determining clustering such as dispersal. However, it remains debated whether SAC patterns can actually reveal endogenous processes. We reviewed studies measuring dispersal of the salamander Salamandra salamandra, to formulate a priori hypotheses on the scale at which dispersal is expected to determine population distribution. We then tested the hypotheses by analysing SAC in distribution data, and evaluating whether controlling for the effect of environmental variables can reveal endogenous processes. We surveyed 565 streams to obtain species distribution data; we also recorded landscape and microhabitat features known to affect the species. We used multiple approaches to tease apart endogenous and exogenous SAC: the analysis of residuals of logistic regression models considering different environmental variables; the analysis of eigenvectors extracted by several implementations of spatial eigenvector mapping. In capture-mark-recapture studies, 98% of individuals moved 500 m or less. Both species distribution and environmental features were strongly autocorrelated. The residuals of logistic regression relating species to environmental variables were autocorrelated at distances up to 500 m; analyses considering different sets of environmental variables, or assuming non-linear species habitat relationships, yielded identical results. The results of spatial eigenvector mapping strongly depended on the matrix of distances used. Nevertheless, the eigenvectors of models with best fit were autocorrelated at distances up to 200-500 m. The concordance between multiple approaches suggests that 500 m is the scale at which dispersal connects breeding localities, increasing probability of occurrence. If exogenous variables are correctly identified, the analysis of SAC can provide important insights on endogenous population processes, such as the flow of individuals. SAC analysis can also provide important information for conservation, as the existence of metapopulations or population networks is essential for long term persistence of amphibians. [ABSTRACT FROM AUTHOR]

The achójki is an Ambystomatidae salamander, this species is endemic and inhabit exclusively at lake Pátzcuaro, Michoacán; it presents paedogenesis, and is a generalist carnivorous that probably has interspecific competition with some freshwater fish, such as: Bulldog Goodeid (Alloophorus robustus), Mexican Silverside (Menidia estor) and Largemouth Black Bass (Micropterus salmoides). For p'urhépecha people, this species has an alimentary use, cooking by broth or fried, by their high protein content and also is considered medicinal, prescripted for respiratory diseases and supplying energy for anemic children and breastfeeding women. This paper is divided in two issues, ecology and cultural use of this species, based on bibliographical search and interviews on field work. Most of the adults had knowledge of use of achójki, and claim the population reduction. Currently is considered a threatened species and is registered in national and international lists of conservation. The main factors that have contributed to the species decrease are: lake pollution, decrease of the water level, introduction of exotic species and overexploitation of resources. Were founded are three UMAs or reproduction sites: UMA Jimbani Erandi, PIMVS Jimbani Tzípekua and UMA Dumerilii. Development programs that involve local initiatives should be done for the survival of the species with a sustainable perspective. The general aim of the present paper is to recognize the ecological and cultural importance of the achójki (Ambystoma dumerilii) in Patzcuaro's lake. [ABSTRACT FROM AUTHOR]

Abstract: Axolotls (Ambystoma mexicanum) are aquatic salamanders that are widely used in research. Axolotls have been bred in laboratories for nearly 150years, yet little is known about the basic biology of reproduction in these animals. We investigated the effects of changing day length, time of year, and food availability on levels of circulating estradiol and androgens in adult female and male axolotls, respectively. In addition, we examined the effects of these variables on the mass of ovaries, oviducts, and eggs in females and on mass of testes in males relative to each individual’s body weight, to calculate a form of gonadosomatic index (GSI). In both sexes, GSI was not correlated with levels of circulating steroids. In female axolotls, estradiol levels were influenced by food availability, changes in day length, and season, even when animals were held at a constant temperature and day length was decorrelated with calendar date. In addition, the mass of ovaries, oviducts, and eggs varied seasonally, peaking in the winter months and declining during the summer months, even though our animals were not breeding and shedding eggs. In males, levels of androgens appeared to vary independently of external conditions, but GSI varied dramatically with changes in day length. These results suggest that reproduction in axolotls may vary seasonally, as it does in many other ambystomid species, although both male and female axolotls are capable of reproducing several times each year. The physiological basis of this ability remains enigmatic, given the indications of seasonality contained in our data. [Copyright &y& Elsevier]

Literary data on the peculiarities of the egg cleavage process in various representatives of the order of caudate amphibians consisting of 10 families have been considered. It has been recognized that in considerable number of species of Plethodontidae, Cryptobranchidae, and some other families, the synchrony of divisions is lost already after the 8-celled stage of the cleavage in large, yolk-rich and unpigmented eggs. A 'standard' cleavage of early embryos of caudate amphibians, which had been described in the text-books on developmental biology and consists approximately of 10 synchronous divisions of comparatively small eggs, is characteristic only of the families Ambystomatidae and Salamandridae including 19.3% of species within the order Caudata. However, within each of these families there seems to be a number of species with a 'non-standard' type of early cleavage. The evolutionary relationships between two main types of early embryogenesis within the order Caudata are discussed. [ABSTRACT FROM AUTHOR]

We observed agonistic behavior among larval Ambystoma opacum, A. jeffersonianum, and A. maculatum in intra- and interspecific pairwise comparisons to determine if sympatric species exhibit unique behavioral responses to the presence of con- or heterospecific larvae, if they respond similarly to variation in competitor size, and if species identity is a reliable predictor of predation and cannibalism versus strictly nonlethal competitive interactions as an outcome of larval aggression. Each species exhibited a unique suite of behavioral responses associated with species and competitor size. Ambystoma opacum displayed high levels of aggression, most often towards conspecifics, but these behaviors rarely resulted in predation. Conversely, A. jeffersonianum displayed aggression frequently and often consumed both con- and heterospecific larvae. Furthermore, this species did not reduce aggression even in the presence of larger larvae. Ambystoma maculatum displayed relatively low levels of aggression under most circumstances. We conclude that these characteristic responses are associated with species-specific morphological and developmental features and the temporally staggered pattern in which these species appear in ponds. These observations highlight the importance of these traits to behavioral divergence among ecologically similar taxa occurring in sympatry. [ABSTRACT FROM AUTHOR]

Many animals, such as crustaceans, insects, and salamanders, package their sperm into spermatophores, and the number of spermatozoa contained in a spermatophore is relevant to studies of sexual selection and sperm competition. We used two molecular methods, real-time quantitative polymerase chain reaction (RT-qPCR) and spectrophotometry, to estimate sperm numbers from spermatophores. First, we designed gene-specific primers that produced a single amplicon in four species of ambystomatid salamanders. A standard curve generated from cloned amplicons revealed a strong positive relationship between template DNA quantity and cycle threshold, suggesting that RT-qPCR could be used to quantify sperm in a given sample. We then extracted DNA from multiple Ambystoma maculatum spermatophores, performed RT-qPCR on each sample, and estimated template copy numbers (i.e. sperm number) using the standard curve. Second, we used spectrophotometry to determine the number of sperm per spermatophore by measuring DNA concentration relative to the genome size. We documented a significant positive relationship between the estimates of sperm number based on RT-qPCR and those based on spectrophotometry. When these molecular estimates were compared to spermatophore cap size, which in principle could predict the number of sperm contained in the spermatophore, we also found a significant positive relationship between sperm number and spermatophore cap size. This linear model allows estimates of sperm number strictly from cap size, an approach which could greatly simplify the estimation of sperm number in future studies. These methods may help explain variation in fertilization success where sperm competition is mediated by sperm quantity. [ABSTRACT FROM AUTHOR]

The article presents a study which identifies salamanders that act as hosts of metacercariae of Clinostomum or trematoda species in Arkansas and Illinois. The study collected various salamander species like the Ambystoma jeffersonianum, maculatum, and texanum which were examined if they are infected with metacercariae. Results reveal that 13 out of Ambystoma jeffersonianum, 1 out 72 of Ambystoma texanum, and 4 of 69 Ambystoma maculatum are infected with metacercariae of Clinostomum species.

Background: Loss of pond habitat is catastrophic to aquatic larval amphibians, but even reduction in the amount of time a breeding site holds water (hydroperiod) can influence amphibian development and limit reproductive success. Using the landscape variation of a glacial valley in the Greater Yellowstone Ecosystem as the context for a natural experiment, we examined variation in growth pattern and life history of the salamander Ambystoma tigrinum melanostictum and determined how these developmental characteristics varied with hydroperiod over several summers. Results: In ponds that dried early in the season, maximum larval size was reduced relative to the sizes achieved in permanent ponds. Ephemeral ponds were associated with early metamorphosis at small body sizes, while permanent ponds facilitated longer larval periods and later metamorphosis. Paedomorphosis resulted from indefinite metamorphic postponement, and was identified only in the most permanent environments. Patterns of growth and allometry were similar between ponds with different hydroperiods, but considerable life history variation was derived from modulating the timing of and size at metamorphosis. Considering maximum rates of growth and inferring the minimum size at metamorphosis across 25 ponds over the course of three years, we calculated that hydroperiods longer than three months are necessary to support these populations through metamorphosis and/or reproductive maturity. Conclusions: Landscape heterogeneity fosters life history variation in this natural population. Modulation of the complex ambystomatid life cycle allows this species to survive in unpredictable environments, but current trends towards rapid pond drying will promote metamorphosis at smaller sizes and could eliminate the paedomorphic phenotype from this region. Metamorphosis at small size is has been linked to altered fitness traits, including reduced survival and fecundity. Thus, widespread environmental truncation of larval periods may lead to decreased population persistence. We found that the hydroperiods of many ponds in this region are now shorter than the developmental period required for larvae to reach the minimum size for metamorphosis; these locations serve as reproductive sinks that may be detrimental for persistence of the species in the region. [ABSTRACT FROM AUTHOR]

Background: Loss of pond habitat is catastrophic to aquatic larval amphibians, but even reduction in the amount of time a breeding site holds water (hydroperiod) can influence amphibian development and limit reproductive success. Using the landscape variation of a glacial valley in the Greater Yellowstone Ecosystem as the context for a natural experiment, we examined variation in growth pattern and life history of the salamander Ambystoma tigrinum melanostictum and determined how these developmental characteristics varied with hydroperiod over several summers. Results: In ponds that dried early in the season, maximum larval size was reduced relative to the sizes achieved in permanent ponds. Ephemeral ponds were associated with early metamorphosis at small body sizes, while permanent ponds facilitated longer larval periods and later metamorphosis. Paedomorphosis resulted from indefinite metamorphic postponement, and was identified only in the most permanent environments. Patterns of growth and allometry were similar between ponds with different hydroperiods, but considerable life history variation was derived from modulating the timing of and size at metamorphosis. Considering maximum rates of growth and inferring the minimum size at metamorphosis across 25 ponds over the course of three years, we calculated that hydroperiods longer than three months are necessary to support these populations through metamorphosis and/or reproductive maturity. Conclusions: Landscape heterogeneity fosters life history variation in this natural population. Modulation of the complex ambystomatid life cycle allows this species to survive in unpredictable environments, but current trends towards rapid pond drying will promote metamorphosis at smaller sizes and could eliminate the paedomorphic phenotype from this region. Metamorphosis at small size is has been linked to altered fitness traits, including reduced survival and fecundity. Thus, widespread environmental truncation of larval periods may lead to decreased population persistence. We found that the hydroperiods of many ponds in this region are now shorter than the developmental period required for larvae to reach the minimum size for metamorphosis; these locations serve as reproductive sinks that may be detrimental for persistence of the species in the region. [ABSTRACT FROM AUTHOR]

The article reports on the distribution records for herpetofauna of Louisiana as of December 2015. These include Brown Anole at Buckhorn Wildlife Management Area (WMA), Rio Grande Chirping Frog found at Alexander State Forest WMA, Mole Salamander at Boeuf WMA, and Timber Rattlesnake at Sicily Island Hills WMA.

Habitat loss is the single greatest threat to persistence of the critically threatened California tiger salamander (Ambystoma californiense). To aid management plans that designate critical habitat for this species, I developed and characterized 21 tetranucleotide microsatellite markers using two native populations in Santa Barbara and Alameda Counties. Allelic variation and average heterozygosities were lower in the endangered Santa Barbara population (allele range 1-4, mean 2.4; HO = 0.308 HE = 0.288) compared with the threatened Alameda population (allele range 2-10, mean 6.7; HO = 0.712, HE = 0.722). In-depth population studies using these markers will provide vital information for plans to assign critical habitat that optimize gene flow among breeding populations, as well as for identifying non-native hybrid genotypes that threaten native A. californiense stocks. Beyond the conservation goals for A. californiense, the close phylogenetic relationships within the tiger salamander complex also suggest a broad utility for population studies using these markers. [ABSTRACT FROM AUTHOR]