Polymorphic microsatellite markers for the Mojave desert tortoise, Gopherus agassizii

We describe primers and polymerase chain reaction (PCR) conditions to amplify 14 tri- and tetranucleotide microsatellite loci for the Mojave desert tortoise (Gopherus agassizii). Across three populations (87 individuals) located in the Mojave Desert, USA, the markers yielded a range of four to 33 alleles and an average observed heterozygosity of 0.733 (range 0.433 to 0.933). We neither detected linkage disequilibrium between any pair of loci nor did we find a consistent pattern of deviation from Hardy-Weinberg equilibrium. These microsatellites are designed for PCR multiplexing, and provide higher throughput capacity to aid in conservation genetics studies for this threatened species.     

PCR primers for microsatellite loci in the tiger rattlesnake (Crotalus tigris,Viperidae)

The tiger rattlesnake (Crotalus tigris) is found only in the Sonoran Desert of North America, where it inhabits rocky foothills that are increasingly being affected by urban encroachment. In order to continue a long‐term study of the demography and movements of tiger rattlesnakes around Tucson, Arizona, we identified six novel polymorphic microsatellite loci for this species. All loci exhibited high variability (five to 41 alleles). These markers are useful for studies of paternity, population structure, and assessing appropriate destinations for translocated animals.     

Implications of Anthropogenic Landscape Change on Inter-Population Movements of the Desert Tortoise (Gopherus agassizii)

In the Sonoran Desert of North America, populations of the desert tortoise (Gopherus agassizii) occur in rocky foothills throughout southwestern Arizona and northwestern Mexico. Although tortoise populations appear to be isolated from each other by low desert valleys, individuals occasionally move long distances between populations. Increasingly, these movements are hindered by habitat fragmentation due to anthropogenic landscape changes. We used molecular techniques and radiotelemetry to examine movement patterns of desert tortoises in southern Arizona. We collected blood samples from 170 individuals in nine mountain ranges and analyzed variability in seven microsatellite loci to determine genetic differentiation among populations. Gene flow estimates between populations indicate that populations exchanged individuals historically at a rate greater than one migrant per generation, and positive correlation between genetic and geographic distance of population pairs suggests that the limiting factor for gene flow among populations is isolation by distance. Life history traits of the desert tortoise, a long-lived species with delayed sexual maturity, may severely constrain the ability of small populations to respond to disturbances that increase adult mortality. Historic gene flow estimates among populations suggests that recovery of declining populations may rely heavily on the immigration of new individuals from adjacent mountain ranges. Management strategies compatible with the evolutionary history of gene flow among disjunct populations will help ensure the long-term persistence of Sonoran desert tortoise populations.     

Flow cytometric method for quantifying viable Mycoplasma agassizii,an agent of upper respiratory tract disease in the desert tortoise (Gopherus agassizii)

Aims: Mycoplasma agassizii can cause upper respiratory tract disease in the threatened desert tortoise of the Southwestern United States. Two technical challenges have impeded critical microbiological studies of this microorganism: (i) its small size limits the use of light microscopy for cell counting and (ii) its extremely slow growth in broth and agar cultures impedes colony counting. Our aim was to develop a rapid and sensitive flow cytometric method using a vital fluorescent dye to enumerate viable M. agassizii cells. Methods and Results: Here, we demonstrate that the nonfluorescent molecule 5‐carboxyfluorescein (5‐CF) diacetate acetoxymethyl ester penetrates M. agassizii cell membranes and it is converted in the cytoplasm to the fluorescent molecule 5‐CF by the action of intracellular esterases. Labelled mycoplasma cells can be easily detected by flow cytometry, and cultures with as few as 100 viable mycoplasma cells ml^?1 can be labelled and counted in less than 1 h. Experiments using temperature‐induced cell death demonstrated that only viable M. agassizii cells are labelled with this procedure. Conclusions: A rapid and sensitive flow cytometric technique has been developed for enumerating viable M. agassizii cells. Significance and Impact of the Study: This technique should facilitate basic immunological, biochemical and pharmacological studies of this important pathogen which may lead to new diagnostic and therapeutic methods.     

The dazed and confused identity of Agassiz's land tortoise, Gopherus agassizii (Testudines, Testudinidae) with the description of a new species, and its consequences for conservation

We investigate a cornucopia of problems associated with the identity of the desert tortoise, Gopherus agassizii (Cooper). The date of publication is found to be 1861, rather than 1863. Only one of the three original cotypes exists, and it is designated as the lectotype of the species. Another cotype is found to have been destroyed in the 1906 San Francisco earthquake and subsequent fire. The third is lost. The lectotype is genetically confirmed to be from California, and not Arizona, USA as sometimes reported. Maternally, the holotype of Gopherus lepidocephalus (Ottley & Velázques Solis. 1989) from the Cape Region of Baja California Sur, Mexico is also from the Mojavian population of the desert tortoise, and not from Tiburon Island, Sonora, Mexico as previously proposed. A suite of characters serve to diagnose tortoises west and north of the Colorado River, the Mojavian population, from those east and south of the river in Arizona, USA, and Sonora and Sinaloa, Mexico, the Sonoran population. Species recognition is warranted and because Gopherus lepidocephalus is from the Mojavian population, no names are available for the Sonoran species. Thus, a new species, Gopherus morafkaisp. n., is named and this action reduces the distribution of Gopherus agassizii to only 30% of its former range. This reduction has important implications for the conservation and protection of Gopherus agassizii, which may deserve a higher level of protection.     

Quantitative PCR method for detection of mycoplasma spp. DNA in nasal lavage samples from the desert tortoise (Gopherus agassizii)

Mycoplasma agassizii and M. testudineum have been associated with upper respiratory tract disease (URTD) in the threatened desert tortoise (Gopherus agassizii). Because microbiological culture methods have proven difficult to employ in wild desert tortoises, our goal was to develop a sensitive and specific qPCR method for detecting and quantifying mycoplasma DNA in nasal lavage fluid collected in the field. Primers for 16S ribosomal RNA gene sequences specific for M. agassizii and M. testudineum were designed, together with primers that recognize conserved sequences of both microorganisms. Standard curves generated with DNA extracted from known numbers of mycoplasma cells revealed a lower detection limit of approximately 5 fg. The qPCR method did not recognize normal flora DNA, and nasal lavage fluid contained no interfering substances. Nasal lavage samples collected from 20 captive desert tortoises housed at the Desert Tortoise Conservation Center (Clark County, Nevada, USA) revealed the presence of M. agassizii DNA in 100% of the tortoises. Concentrations ranged from a low of 6 pg ml^− 1 to a high of 72,962 pg ml^− 1. Only one of the tortoises was positive for M. testudineum. Interestingly, not all of the qPCR positive tortoises showed evidence of seroconversion, suggesting that they were colonized but not infected. This new quantitative method will provide a critical tool for managing threatened populations of the desert tortoise.     

PCR primers for polymorphic microsatellite loci in the desert locust,Schistocerca gregaria (Orthoptera: Acrididae)

Nine pairs of polymerase chain reaction (PCR) primers that amplify polymorphic microsatellite loci in the desert locust, Schistocerca gregaria (Forskal), were developed using a magnetic bead‐based enrichment protocol. A sample of 48 locusts collected during the 1993 and 1995 upsurge periods in Eritrea, East Africa, were genotyped. The number of alleles per locus ranged from six to 20; the average was 12.67. Allelic distributions were significantly different between samples from different localities.     

Identification and characterization of microsatellite markers in Hermann's tortoise (Testudo hermanni,Testudinidae)

The isolation and characterization of six polymorphic loci from a Testudo hermanni genomic library is reported. Enrichment was performed for AC but four of the characterized microsatellites present also an additional motif. Variability was tested on populations of the two recognized subspecies, Testudo hermanni hermanni and Testudo hermanni boettgeri. For one locus, a size range specific for the subspecies T. h. hermanni was observed. These are the first primers identified directly in the genome of T. hermanni     

Characterization of polymorphic microsatellite markers for the endangered Malagasy radiated tortoise (Geochelone radiata)

The radiated tortoise (Geochelone radiata) is an endangered endemic species from Madagascar that inhabits the semiarid spiny forest of the southern part of the island. Habitat destruction and illegal harvesting greatly threaten this species, as attested by the significant reduction of its distribution area in the past 30 years. In order to undertake appropriate conservation actions, it is essential to acquire a better knowledge of its genetic structure. For this study, 145 blood samples were collected from three populations of radiated tortoises in southwestern Madagascar. Eight microsatellite loci were found to be polymorphic, and allelic diversity and observed heterozygosity were high for all markers.     

Western blot can distinguish natural and acquired antibodies to Mycoplasma agassizii in the desert tortoise (Gopherus agassizii)

Mycoplasma agassizi has been identified as a cause of upper respiratory tract disease (URTD) in the threatened Mojave population of the desert tortoise (Gopherus agassizii), and anti-M. agassizii antibodies have been found by ELISA in as many as 15% of these animals across their geographic range. Here we report that a cohort of 16 egg-reared desert tortoises never exposed to M. agassizii had ELISA antibody titers to this organism that overlapped with titers obtained from some M. agassizii-infected tortoises. These natural antibodies were predominantly of the IgM class. Western blots of plasma from these non-infected tortoises produced a characteristic banding pattern against M. agassizii antigens. A group of 38 wild-caught desert tortoises was tested by ELISA, and although some of these tortoises had antibody titers significantly higher than the non-infected tortoises, there was considerable overlap at the lower titer levels. However, Western blot analysis revealed distinct banding patterns that could readily distinguish between the non-infected tortoises and tortoises with acquired antibodies, regardless of ELISA antibody titers. We conclude that desert tortoises have natural antibodies to M. agassizii that can compromise the determination of infection status by ELISA. However, the Western blot technique can distinguish between natural and acquired antibody patterns and can be used to confirm the diagnosis of M. agassizii infections in the desert tortoise.     

Histomorphometric studies of dermal bone in the desert tortoise, Gopherus agassizii.

Dermal bone biopsies were collected from the periphery of the carapaces of adult desert tortoises (Gopherus agassizii) from grazed and ungrazed habitats near the Arizona/Utah border (USA). Quantitative bone histomorphometry was performed on these biopsies as well as on dermal bone biopsies collected from captive juvenile desert tortoises. Except for mild osteomalacia, carapaces of adult desert tortoises from the grazed habitat were relatively normal. No signs of osteopenia were observed. Based on the low numbers of osteoblasts and osteoclasts in dermal bone of both populations of adult desert tortoises, it appears that the peripheral carapace is relatively inert with very low levels of dermal bone turnover. Bone cells and osteoid were more common in dermal bone biopsies from the carapace and plastron of captive juvenile desert tortoises than in adult desert tortoises. However, the great variability in the incidence of bone cells among individuals and the difficulty in collecting juvenile desert tortoises in the field limit the usefulness of dermal bone biopsies from animals of this age group. Based on these results, we propose that dermal bone of the peripheral carapace is a poor sample site for evaluating the effects of dietary or environmental conditions on calcified tissues in desert tortoises.     

Western blot can distinguish natural and acquired antibodies to Mycoplasma agassizii in the desert tortoise (Gopherus agassizii)

Mycoplasma agassizi has been identified as a cause of upper respiratory tract disease (URTD) in the threatened Mojave population of the desert tortoise (Gopherus agassizii), and anti-M. agassizii antibodies have been found by ELISA in as many as 15% of these animals across their geographic range. Here we report that a cohort of 16 egg-reared desert tortoises never exposed to M. agassizii had ELISA antibody titers to this organism that overlapped with titers obtained from some M. agassizii-infected tortoises. These natural antibodies were predominantly of the IgM class. Western blots of plasma from these non-infected tortoises produced a characteristic banding pattern against M. agassizii antigens. A group of 38 wild-caught desert tortoises was tested by ELISA, and although some of these tortoises had antibody titers significantly higher than the non-infected tortoises, there was considerable overlap at the lower titer levels. However, Western blot analysis revealed distinct banding patterns that could readily distinguish between the non-infected tortoises and tortoises with acquired antibodies, regardless of ELISA antibody titers. We conclude that desert tortoises have natural antibodies to M. agassizii that can compromise the determination of infection status by ELISA. However, the Western blot technique can distinguish between natural and acquired antibody patterns and can be used to confirm the diagnosis of M. agassizii infections in the desert tortoise.     

Characterization of 20 microsatellite markers in the plowshare tortoise,Astrochelys yniphora

The plowshare tortoise, Astrochelys yniphora, or Angonoka is one of Madagascar’s land tortoises, living in the vicinity of Baly Bay bamboo scrub in northwest Madagascar. Primary threats to this endangered species are due to poaching for pet trade and the loss and fragmentation of its natural habitat. The number of wild Angonoka is estimated to be ~400. Twenty polymorphic nuclear microsatellite loci were isolated from a genomic DNA on individuals from Andrafiafaly in Baly Bay National Park, Madagascar. Results from this study will help for future studies of the social structure and population dynamics of this species as well as identification of confiscated individuals from the illegal pet trade.     

Using motion-sensor camera technology to infer seasonal activity and thermal niche of the desert tortoise (Gopherus agassizii)

Understanding the relationships between environmental variables and wildlife activity is an important part of effective management. The desert tortoise (Gopherus agassizii), an imperiled species of arid environments in the southwest US, may have increasingly restricted windows for activity due to current warming trends. In summer 2013, we deployed 48 motion sensor cameras at the entrances of tortoise burrows to investigate the effects of temperature, sex, and day of the year on the activity of desert tortoises. Using generalized estimating equations, we found that the relative probability of activity was associated with temperature (linear and quadratic), sex, and day of the year. Sex effects showed that male tortoises are generally more active than female tortoises. Temperature had a quadratic effect, indicating that tortoise activity was heightened at a range of temperatures. In addition, we found significant support for interactions between sex and day of the year, and sex and temperature as predictors of the probability of activity. Using our models, we were able to estimate air temperatures and times (days and hours) that were associated with maximum activity during the study. Because tortoise activity is constrained by environmental conditions such as temperature, it is increasingly vital to conduct studies on how tortoises vary their activity throughout the Sonoran Desert to better understand the effects of a changing climate.     

Multiplex PCR system applied for analysing microsatellite loci of Schlegel's black rockfish,Sebastes schlegeli

Two multiplex PCR amplifications were performed to analyse six microsatellite loci of Schlegel's black rockfish, Sebastes schlegeli, an important commercial fish in the northern part of Japan and an important species for the stock enhancement program in this area. We analysed 67 wild samples from Yamada Bay, Iwate Prefecture, Japan. The observed genotype frequencies agreed with the Hardy–Weinberg expectations at all loci, and the observed heterozygosities ranged from 0.072 to 0.897.     

Estimating the probability of illegal desert tortoise collection in the Sonoran Desert

The expansion of road networks in desert tortoise (Gopherus agassizii) habitat in the Sonoran Desert has raised questions concerning appropriate mitigation to reduce impacts at the population level. Although some effects, namely road-kill and habitat loss, have been well documented, illegal tortoise collection has been insufficiently addressed. It has become increasingly important for wildlife and land-use managers to understand the cumulative impacts of roads on tortoises and the effect that those impacts have on population persistence. We estimated the probability of desert tortoise detection and collection along 2-lane paved, maintained gravel, and non-maintained gravel roads to evaluate whether collection probabilities were related to road type. Although collection probability did not vary by road type, the probability of desert tortoise detection by passing motorists was greatest on maintained gravel roads and fewest on non-maintained gravel and paved roads. These results have implications for effectively mitigating the impacts of roads on desert tortoises. Published 2011. This article is a U.S. Government work and is in the public domain in the USA.     

Newly developed PCR primers for polymorphic microsatellite loci from the marine isopod Limnoria sp.

Nine microsatellite loci are isolated and characterized for the marine isopod Limnoria sp., a model invertebrate species for the study of connectivity of marine brooders with high potential of dispersal through rafting. Analysis of 35 individuals of one population revealed a mean number of alleles of 12.66 and mean expected heterozygosity of 0.83, indicating a high level of variability at most loci. These polymorphic microsatellite loci should be useful to the study of the geographical distribution of the genetic diversity of this species of Limnoria.     

Translocation as a conservation tool for Agassiz's desert tortoises: Survivorship,reproduction, and movements

We translocated 120 Agassiz's desert tortoises to 5 sites in Nevada and Utah to evaluate the effects of translocation on tortoise survivorship, reproduction, and habitat use. Translocation sites included several elevations, and extended to sites with vegetation assemblages not typically associated with desert tortoises in order to explore the possibility of moving animals to upper elevation areas. We measured survivorship, reproduction, and movements of translocated and resident animals at each site. Survivorship was not significantly different between translocated and resident animals within and among sites, and survivorship was greater overall during non-drought years. The number of eggs produced by tortoises was similar for translocated and resident females, but differed among sites. Animals translocated to atypical habitat generally moved until they reached vegetation communities more typical of desert tortoise habitat. Even within typical tortoise habitat, tortoises tended to move greater distances in the first year after translocation than did residents, but their movements in the second or third year after translocation were indistinguishable from those of resident tortoises. Our data show that tortoises translocated into typical Mojave desert scrub habitats perform well; however, the large first-year movements of translocated tortoises have important management implications. Projects that employ translocations must consider how much area will be needed to contain translocated tortoises and whether roads need fencing to prevent the loss of animals. © 2012 The Wildlife Society.