Landscape genetics of the blotched tiger salamander (Ambystoma tigrinum melanostictum)

The field of landscape genetics has great potential to identify habitat features that influence population genetic structure. To identify landscape correlates of genetic differentiation in a quantitative fashion, we developed a novel approach using geographical information systems analysis. We present data on blotched tiger salamanders (Ambystoma tigrinum melanostictum) from 10 sites across the northern range of Yellowstone National Park in Montana and Wyoming, USA. We used eight microsatellite loci to analyse population genetic structure. We tested whether landscape variables, including topographical distance, elevation, wetland likelihood, cover type and number of river and stream crossings, were correlated with genetic subdivision (F(ST)). We then compared five hypothetical dispersal routes with a straight-line distance model using two approaches: (i) partial Mantel tests using Akaike's information criterion scores to evaluate model robustness and (ii) the BIOENV procedure, which uses a Spearman rank correlation to determine the combination of environmental variables that best fits the genetic data. Overall, gene flow appears highly restricted among sites, with a global F(ST) of 0.24. While there is a significant isolation-by-distance pattern, incorporating landscape variables substantially improved the fit of the model (from an r2 of 0.3 to 0.8) explaining genetic differentiation. It appears that gene flow follows a straight-line topographic route, with river crossings and open shrub habitat correlated with lower F(ST) and thus, decreased differentiation, while distance and elevation difference appear to increase differentiation. This study demonstrates a general approach that can be used to determine the influence of landscape variables on population genetic structure.     

Molecular characterization of major histocompatibility complex class II alleles in wild tiger salamanders (Ambystoma tigrinum)

Major histocompatibility complex (MHC) class II genes are usually among the most polymorphic in vertebrate genomes because of their critical role (antigen presentation) in immune response. Prior to this study, the MHC was poorly characterized in tiger salamanders (Ambystoma tigrinum), but the congeneric axolotl (Ambystoma mexicanum) is thought to have an unusual MHC. Most notably, axolotl class II genes lack allelic variation and possess a splice variant without a full peptide binding region (PBR). The axolotl is considered immunodeficient, but it is unclear how or to what extent MHC genetics and immunodeficiency are interrelated. To study the evolution of MHC genes in urodele amphibians, we describe for the first time an expressed polymorphic class II gene in wild tiger salamanders. We sequenced the PBR of a class II gene from wild A. tigrinum (n=33) and identified nine distinct alleles. Observed heterozygosity was 73%, and there were a total of 46 polymorphic sites, most of which correspond to amino acid positions that bind peptides. Patterns of nucleotide substitutions exhibit the signature of diversifying selection, but no recombination was detected. Not surprisingly, transspecies evolution of tiger salamander and axolotl class II alleles was apparent. We have no direct data on the immunodeficiency of tiger salamanders, but the levels of polymorphism in our study population should suffice to bind a variety of foreign peptides (unlike axolotls). Our tiger salamander data suggest that the monomorphism and immunodeficiencies associated with axolotl class II genes is a relict of their unique historical demography, not their phylogenetic legacy. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Behavioral determinations of thresholds for n-butyl acetate and nbutyl alcohol in the tiger salamander (Ambystoma tigrinum)

Two groups of tiger salamanders (Ambystoma tigrinum) were conditionedto respond to odorant-air mixtures of n-butyl acetate (8.9 x10^–5M) or n-butyl alcohol (6.7 x l0^–5M). They werethen given tests with various concentrations of the trainingodorants presented using a temporal forced-choice method ofascending limits. Results showed that reliable responses toodorant-air presentations were obtained with concentrationsof n-butyl acetate above 2.4 x l0^–7M and with concentrationsof n-butyl alcohol above 8.5 x 10^–8M. These results arein substantial agreement with previous dectrophysiological findings.     

Distribution of monoamine oxidase activity in tissues of the urodeles Ambystoma tigrinum (tiger salamander) and Necturus maculosus (mudpuppy)

1. Monoamine oxidase (MAO) activity was determined fluorometrically in tissues of adult mudpuppies, and pre- (young) and post- (adult) metamorphic tiger salamanders. 2. From responses to specific inhibitors it was determined that 95% activity was MAO type A in all tissues. 3. In young salamanders MAO activity was greater in brain and intestine of males than of females, and was considerably higher in kidney of both sexes and in intestine of males compared to adults. 4. MAO activity was distributed differently in the mudpuppy compared to the salamander. Intestine and liver contained high activity and brain had relatively little MAO activity compared to salamander. 5. The apparent Michaelis constant of MAO activity in the different groups and tissues was generally similar, suggesting a similarity of the MAO molecule.     

Fifteen novel universal primer pairs for sequencing whole chloroplast genomes and a primer pair for nuclear ribosomal DNAs

Chloroplast genome information helps improve the phylogenetic resolution and can act as organelle-scale barcodes in recently radiated plant groups. Previously we reported that nine universal primer pairs could amplify angiosperm whole chloroplast genomes by long-range polymerase chain reaction and using next-generation sequencing. Although these primers show high universality and efficiency for sequencing whole chloroplast genomes in angiosperms, they did not fully resolve the following two issues surrounding sequencing angiosperm chloroplast genomes: (i) approximately 30% of angiosperms cannot be amplified successfully; and (ii) only fresh leaves can be applied. In this study, we designed another set of 15 universal primer pairs for amplifying angiosperm whole chloroplast genomes to complement the original nine primer pairs. Furthermore, we designed a primer pair for nuclear ribosomal DNAs (nrDNAs). To validate the functionality of the primers, we tested 44 species with silica gel-dried leaves and 15 species with fresh leaves that have been shown to not be amplified with the original nine primer pairs. The result showed that, in 65.9% and 88.6% of the 44 species with silica gel-dried leaves, the whole chloroplast genome and nrDNAs could be amplified, respectively. In addition, all 15 fresh leaf samples could have the whole chloroplast genome successfully amplified. The nrDNAs comprise partial sequences of 18S and 26S, along with the complete sequence of 5.8S and the internal transcribed spacers ITS1 and ITS2. The mean size of nrDNA was 5800 bp. This study shows that the 15 universal primer set is an indispensable tool for amplifying whole chloroplast genomes in angiosperms, and these are an important supplement to the nine reported primer pairs.     

A three-gene phylogeny of the Mycena pura complex reveals 11 phylogenetic species and shows ITS to be unreliable for species identification

Phylogenetic analyses of Mycena sect. Calodontes using ITS previously suggested ten cryptic monophyletic ITS lineages within the Mycena pura morphospecies. Here, we compare ITS data (645 bp incl. gaps) from 46 different fruit bodies that represent the previously described ITS diversity with partial tEF-1-α (423 bp) and RNA polymerase II (RPB1) (492 bp) sequence data to test the genealogical concordance.While neither of the markers were in complete topological agreement, the branches differing between the tEF and RPB1 trees had a low bootstrap (<50) support, and the partition homogeneity incongruence length difference (ILD) tests were not significant. ILD tests revealed significant discordances between ITS and the tEF and RPB1 markers in several lineages. And our analyses suggested recombination between ITS1 and ITS2, most pronounced in one phylospecies that was identical in tEF and RPB1. Based on the agreement between tEF and RPB1, we defined 11 mutually concordant terminal clades as phylospecies inside the M. pura morphospecies; most of them cryptic. While neither of the markers showed an unequivocal barcoding gap between inter- and intraspecific diversity, the overlap was most pronounced for ITS (intraspecific diversity 0-3.5 %, interspecific diversity 0.4 %-8.8 %). A clustering analysis on tEF separated at a 1.5 % level returned all phylogenetic species as Operational Taxonomic Units (OTUs), while ITS at both a 1.5 % level and at a 3 % threshold level not only underestimated diversity as found by the tEF and RPB1, but also identified an OTU which was not a phylogenetic species. Thus, our investigation does not support the universal suitability of ITS for species recognition in particular, and emphasises the general limitation of single gene analyses combined with single percentage separation values.     

Analysis of a hybrid zone between subspecies of the tiger salamander (Ambystoma tigrinum) in central New Mexico,USA

We analyzed patterns of variation in polymorphic loci across a contact zone between two subspecies of tiger salamander, Ambystoma tigrinum nebulosum and A. t. mavortium, in west-central New Mexico. We found no fixed differences between A. t. mavortium and A. t. nebulosum. Allele frequencies across the contact zone did not vary concordantly among loci, but frequencies at two loci varied with elevation. Gene flow extends through the contact zone from A. t. mavortium into A. t. nebulosum, but not conversely. A. t. mavortium and A. t. nebulosum have no obvious impediments to gene flow within this contact zone, but some populations differ in time of reproduction. The contact zone comprises a variety of habitats, approximating typical A. t. mavortium or A. t. nebulosum habitats. There is an irregular pattern of differences in allelic frequencies among populations that covaries with habitats and is consistent with a model of a mosaic contact zone. Our results contrast with those of a study along the Front Range in Colorado, where allelic composition at some loci changed abruptly across a narrow zone of contact. These contrasting results suggest that the dynamics shaping the contact zone between the same taxa may differ. Absence of a similar pattern of genetic differentiation between A. t. mavortium and A. t. nebulosum in New Mexico and in Colorado suggests a complex set of evolutionary interactions along the eastern front of the Rocky Mountain axis.     

Phylogeny and species diversity of the genus Herichthys (Teleostei: Cichlidae)

We provide a review of the systematics of Herichthys by evaluating the usefulness of several mitochondrial and nuclear genetic markers together with morphological data. The nDNA next‐generation sequencing ddRAD analysis together with the mtDNA cytochrome b gene provided well‐resolved and well‐supported phylogenies of Herichthys. On the other hand, the nDNA S7 introns have limited resolution and support and the COI barcoding analysis completely failed to recover all but one species of Herichthys as monophyletic. The COI barcoding as currently implemented is thus insufficient to distinguish clearly distinct species in the genus Herichthys that are supported by other molecular markers and by morphological characters. Based on our results, Herichthys is composed of 11 species and includes two main clades (the H. labridens and H. cyanoguttatus species groups). Herichthys bartoni is in many respects the most plesiomorphic species in the genus and has a conflicting phylogenetic position between mtDNA and nDNA markers, where the robust nDNA ddRAD data place it as a rather distant basal member of the H. labridens species group. The mtDNA of H. bartoni is on the other hand only slightly divergent from the sympatric and syntopic H. labridens, and the species thus probably have hybridized in the relatively recent past. The sympatric and syntopic Herichthys steindachneri and H. pame are supported as sister species. The Herichthys cyanoguttatus species group shows two well‐separated basal species (the northernmost H. minckleyi and the southernmost H. deppii) followed by the closely related and centrally distributed species H. cyanoguttatus, H. tepehua, H. carpintis, and H. tamasopoensis whose relationships differ between analyses and show likely hybridizations between themselves and the two basal species as suggested by conflicts between DNA analyses. Several instances of introgressions/hybridizations have also been found between the two main clades of Herichthys.     

High‐throughput SNP discovery in the rabbit (Oryctolagus cuniculus) genome by next‐generation semiconductor‐based sequencing

The European rabbit (Oryctolagus cuniculus) is a domesticated species with one of the broadest ranges of economic and scientific applications and fields of investigation. Rabbit genome information and assembly are available (oryCun2.0), but so far few studies have investigated its variability, and massive discovery of polymorphisms has not been published yet for this species. Here, we sequenced two reduced representation libraries (RRLs) to identify single nucleotide polymorphisms (SNPs) in the rabbit genome. Genomic DNA of 10 rabbits belonging to different breeds was pooled and digested with two restriction enzymes (HaeIII and RsaI) to create two RRLs which were sequenced using the Ion Torrent Personal Genome Machine. The two RRLs produced 2 917 879 and 4 046 871 reads, for a total of 280.51 Mb (248.49 Mb with quality >20) and 417.28 Mb (360.89 Mb with quality >20) respectively of sequenced DNA. About 90% and 91% respectively of the obtained reads were mapped on the rabbit genome, covering a total of 15.82% of the oryCun2.0 genome version. The mapping and ad hoc filtering procedures allowed to reliably call 62 491 SNPs. SNPs in a few genomic regions were validated by Sanger sequencing. The Variant Effect Predictor Web tool was used to map SNPs on the current version of the rabbit genome. The obtained results will be useful for many applied and basic research programs for this species and will contribute to the development of cost‐effective solutions for high‐throughput SNP genotyping in the rabbit.     

Landscape genetics and least-cost path analysis reveal unexpected dispersal routes in the California tiger salamander (Ambystoma californiense)

A major goal of landscape genetics is to understand how landscapes structure genetic variation in natural populations. However, landscape genetics still lacks a framework for quantifying the effects of landscape features, such as habitat type, on realized gene flow. Here, we present a methodology for identifying the costs of dispersal through different habitats for the California tiger salamander ( Ambystoma californiense ), an endangered species restricted to grassland/vernal pool habitat mosaics. We sampled larvae from all 16 breeding ponds in a geographically restricted area of vernal pool habitat at the Fort Ord Natural Reserve, Monterey County, California. We estimated between-pond gene flow using 13 polymorphic microsatellite loci and constructed GIS data layers of habitat types in our study area. We then used least-cost path analysis to determine the relative costs of movement through each habitat that best match rates of gene flow measured by our genetic data. We identified four measurable rates of gene flow between pairs of ponds, with between 10.5% and 19.9% of larvae having immigrant ancestry. Although A. californiense is typically associated with breeding ponds in grassland habitat, we found that dispersal through grassland is nearly twice as costly as dispersal through chaparral and that oak woodland is by far the most costly habitat to traverse. With the increasing availability of molecular resources and GIS data, we anticipate that these methods could be applied to a broad range of study systems, particularly those with cryptic life histories that make direct observation of movement challenging.     

Targeted multiplex next‐generation sequencing: advances in techniques of mitochondrial and nuclear DNA sequencing for population genomics

Next‐generation sequencing (NGS) is emerging as an efficient and cost‐effective tool in population genomic analyses of nonmodel organisms, allowing simultaneous resequencing of many regions of multi‐genomic DNA from multiplexed samples. Here, we detail our synthesis of protocols for targeted resequencing of mitochondrial and nuclear loci by generating indexed genomic libraries for multiplexing up to 100 individuals in a single sequencing pool, and then enriching the pooled library using custom DNA capture arrays. Our use of DNA sequence from one species to capture and enrich the sequencing libraries of another species (i.e. cross‐species DNA capture) indicates that efficient enrichment occurs when sequences are up to about 12% divergent, allowing us to take advantage of genomic information in one species to sequence orthologous regions in related species. In addition to a complete mitochondrial genome on each array, we have included between 43 and 118 nuclear loci for low‐coverage sequencing of between 18 kb and 87 kb of DNA sequence per individual for single nucleotide polymorphisms discovery from 50 to 100 individuals in a single sequencing lane. Using this method, we have generated a total of over 500 whole mitochondrial genomes from seven cetacean species and green sea turtles. The greater variation detected in mitogenomes relative to short mtDNA sequences is helping to resolve genetic structure ranging from geographic to species‐level differences. These NGS and analysis techniques have allowed for simultaneous population genomic studies of mtDNA and nDNA with greater genomic coverage and phylogeographic resolution than has previously been possible in marine mammals and turtles.     

Environmental DNA detection of rare and invasive fish species in two Great Lakes tributaries

The extraction and characterization of DNA from aquatic environmental samples offers an alternative, noninvasive approach for the detection of rare species. Environmental DNA, coupled with PCR and next‐generation sequencing (“metabarcoding”), has proven to be very sensitive for the detection of rare aquatic species. Our study used a custom‐designed group‐specific primer set and next‐generation sequencing for the detection of three species at risk (Eastern Sand Darter, Ammocrypta pellucida; Northern Madtom, Noturus stigmosus; and Silver Shiner, Notropis photogenis), one invasive species (Round Goby, Neogobius melanostomus) and an additional 78 native species from two large Great Lakes tributary rivers in southern Ontario, Canada: the Grand River and the Sydenham River. Of 82 fish species detected in both rivers using capture‐based and eDNA methods, our eDNA method detected 86.2% and 72.0% of the fish species in the Grand River and the Sydenham River, respectively, which included our four target species. Our analyses also identified significant positive and negative species co‐occurrence patterns between our target species and other identified species. Our results demonstrate that eDNA metabarcoding that targets the fish community as well as individual species of interest provides a better understanding of factors affecting the target species spatial distribution in an ecosystem than possible with only target species data. Additionally, eDNA is easily implemented as an initial survey tool, or alongside capture‐based methods, for improved mapping of species distribution patterns.     

Isolation and characterization of microsatellite markers for Viola mirabilis (Violaceae) using a next‐generation sequencing platform

We isolated and characterized microsatellite loci in Viola mirabilis (Violaceae), an endangered species from South Korea. Twenty‐three polymorphic microsatellite loci were developed and tested in Korean, Chinese and Japanese populations. The number of alleles per locus varied from two to eight. The observed and expected heterozygosities within the three populations were 0.000–0.625 and 0.469–0.695, respectively. A total of six loci in the Korean population, one locus in the Chinese population and seven loci in the Japanese population deviated from Hardy–Weinberg equilibrium. We expect that these newly developed microsatellite markers will contribute to understanding the phylogeography and population genetics of V. mirabilis, which will aid in developing conservation strategies for this species.     

Evaluation of two approaches to genotyping major histocompatibility complex class I in a passerine-CE-SSCP and 454 pyrosequencing

Genes of the highly dynamic major histocompatibility complex (MHC) are directly linked to individual fitness and are of high interest in evolutionary ecology and conservation genetics. Gene duplication and positive selection usually lead to high levels of polymorphism in the MHC region, making genotyping of MHC a challenging task. Here, we compare the performance of two methods for MHC class I genotyping in a passerine with highly duplicated MHC class I genes: capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) analysis and 454 GS FLX Titanium pyrosequencing. According to our findings, the number of MHC variants (called alleles for simplicity) detected by CE-SSCP is significantly lower than detected by 454. To resolve discrepancies between the two methods, we cloned and Sanger sequenced a MHC class I amplicon for an individual with high number of alleles. We found a perfect congruence between cloning/Sanger sequencing results and 454. Thus, in case of multi-locus amplification, CE-SSCP considerably underestimates individual MHC diversity. However, numbers of alleles detected by both methods are significantly correlated, although the correlation is weak (r = 0.32). Thus, in systems with highly duplicated MHC, 454 provides more reliable information on individual diversity than CE-SSCP.