Brian T. Henen

Reference intervals and physiologic alterations in hematologic and biochemical values of free-ranging desert tortoises in the Mojave Desert.

Desert tortoise (Gopherus agassizii) populations have experienced precipitous declines resulting from the cumulative impact of habitat loss, and human and disease-related mortality. Evaluation of hematologic and biochemical responses of desert tortoises to physiologic and environmental factors can facilitate the assessment of stress and disease in tortoises and contribute to management decisions and population recovery. The goal of this study was to obtain and analyze clinical laboratory data from free-ranging desert tortoises at three sites in the Mojave Desert (California, USA) between October 1990 and October 1995, to establish reference intervals, and to develop guidelines for the interpretation of laboratory data under a variety of environmental and physiologic conditions. Body weight, carapace length, and venous blood samples for a complete blood count and clinical chemistry profile were obtained from 98 clinically healthy adult desert tortoises of both sexes at the Desert Tortoise Research Natural area (western Mojave), Goffs (eastern Mojave) and Ivanpah Valley (northeastern Mojave). Samples were obtained four times per year, in winter (February/March), spring (May/June), summer (July/August), and fall (October). Years of near-, above- and below-average rainfall were represented in the 5 yr period. Minimum, maximum and median values, and central 95 percentiles were used as reference intervals and measures of central tendency for tortoises at each site and/or season. Data were analyzed using repeated measures analysis of variance for significant (P < 0.01) variation on the basis of sex, site, season, and interactions between these variables. Significant sex differences were observed for packed cell volume, hemoglobin concentration, aspartate transaminase activity, and cholesterol, triglyceride, calcium, and phosphorus concentrations. Marked seasonal variation was observed in most parameters in conjunction with reproductive cycle, hibernation, or seasonal rainfall. Year-to-year differences and long-term alterations primarily reflected winter rainfall amounts. Site differences were minimal, and largely reflected geographic differences in precipitation patterns, such that results from these studies can be applied to other tortoise populations in environments with known rainfall and forage availability patterns.

Effects of climatic variation on field metabolism and water relations of desert tortoises

Abstract We used the doubly labeled water method to measure the field metabolic rates (FMRs, in kJ kg−1 day−1) and water flux rates (WIRs, in ml H2O kg−1 day−1) of adult desert tortoises (Gopherus agassizii) in three parts of the Mojave Desert in California over a 3.5-year period, in order to develop insights into the physiological responses of this threatened species to climate variation among sites and years. FMR, WIR, and the water economy index (WEI, in ml H2O kJ−1, an indicator of drinking of free water) differed extensively among seasons, among study sites, between sexes, and among years. In high-rainfall years, males had higher FMRs than females. Average daily rates of energy and water use by desert tortoises were extraordinarily variable: 28-fold differences in FMR and 237-fold differences in WIR were measured. Some of this variation was due to seasonal conditions, with rates being low during cold winter months and higher in the warm seasons. However, much of the variation was due to responses to year-to-year variation in rainfall. Annual spring peaks in FMR and WIR were higher in wet years than in drought years. Site differences in seasonal patterns were apparently due to geographic differences in rainfall patterns (more summer rain at eastern Mojave sites). In spring 1992, during an El Niño (ENSO) event, the WEI was greater than the maximal value obtainable from consuming succulent vegetation, indicating copious drinking of rainwater at that time. The physiological and behavioral flexibility of desert tortoises, evident in individuals living at all three study sites, appears central to their ability to survive droughts and benefit from periods of resource abundance. The strong effects of the El Niño (ENSO) weather pattern on tortoise physiology, reproduction, and survival elucidated in this and other studies suggest that local manifestations of global climate events could have a long-term influence on the tortoise populations in the Mojave Desert.

Egg Size and Annual Egg Production by Female Desert Tortoises (Gopherus agassizii): The Importance of Food Abundance, Body Size, and Date of Egg Shelling

We studied egg production in two Californian populations of desert tortoises, (Gopherus agassizii) in 1992 and 1993. One population inhabited the Desert Tortoise Research Natural Area (DTNA) in the western Mojave Desert, where most of the rain falls in the winter. The second population lived near Goffs, in the eastern Mojave, where annual precipitation is divided more evenly between winter and sum- mer. Due to El Niio conditions, heavy winter rains fell at both sites in both years (1991-1992 and 1992- 1993). Consequently, the biomass of spring annuals and annual egg production by tortoises were high in both years at both sites. There were no differences in reproductive output between years so we pooled data for both years to examine the relationship between egg-laying parameters (clutch size and frequency, annual egg production, egg size, etc.) and female size. Variation in annual egg production was due mainly to variation in clutch size, not clutch frequency. Annual egg production per female was lower at DTNA than at Goffs, because some adult females at DTNA did not produce eggs in some years. Females that did lay eggs produced the same number of eggs per year at both sites, even though females at Goffs were smaller (midline carapace length = 214 mm) than females at DTNA (MCL = 234 mm). Despite correction for these body size differences, the eggs produced at Goffs were smaller in all dimensions than eggs produced at DTNA. Smaller eggs and presumably smaller neonates may be related to the greater predictability of sum- mer rain and consequent greater food supply for emergent hatchlings at Goffs. For adult females, food supply probably limits reproduction only during drought years. How can individual females vary their annual reproductive output? Our more extensive data for DTNA tortoises showed that larger females pro- duced larger clutch sizes. In addition, by statistically removing the effects of body size we showed that larger clutches contained smaller eggs. Moreover, larger females produced eggs earlier in the year giving them a better opportunity to produce a second clutch that year. Thus, timing of first clutch was important Still, much of the variation in reproductive output was not explained. Other characteristics of individuals (e.g, age, genetics, physiological maturity, home range quality, or forage selection) may explain some of the variation in reproductive output

Clinical disease and laboratory abnormalities in free-ranging desert tortoises in California (1990-1995).

Desert tortoise (Gopherus agassizii) populations have experienced precipitous declines resulting from the cumulative impact of habitat loss and human and disease-related mortality. Diagnosis of disease in live, free-ranging tortoises is facilitated by evaluation of clinical signs and laboratory test results but may be complicated by seasonal and environmental effects. The goals of this study were: 1) to describe and monitor clinical and laboratory signs of disease in adult, free-ranging desert tortoises at three sites in the Mojave Desert of California (USA) between October 1990 and October 1995; 2) to evaluate associations between clinical signs and hematologic, biochemical, serologic, and microbiologic test results; 3) to characterize disease patterns by site, season, and sex; and 4) to assess the utility of diagnostic tests in predicting morbidity and mortality. Venous blood samples were obtained four times per year from tortoises of both sexes at the Desert Tortoise Research Natural Area (DTNA), Goffs/Fenner Valley, and Ivanpah Valley. Tortoises were given a physical examination, and clinical abnormalities were graded by type and severity. Of 108 tortoises, 68.5% had clinical signs of upper respiratory tract disease consistent with mycoplasmosis at least once during the study period. In addition, 48.1% developed moderate to severe shell lesions consistent with cutaneous dyskeratosis. Ulcerated or plaque-like oral lesions were noted on single occasions in 23% of tortoises at Goffs and 6% of tortoises at Ivanpah. Tortoises with oral lesions were significantly more likely than tortoises without lesions to have positive nasal cultures for Mycoplasma agassizii (P=0.001) and to be dehydrated (P=0.0007). Nine tortoises had marked azotemia (blood urea nitrogen [BUN] >100 mg/dl) or persistent azotemia (BUN 63–76 mg/dl); four of these died, three of which had necropsy confirmation of urinary tract disease. Laboratory tests had low sensitivity but high specificity in assessing morbidity and mortality; there was marked discrepancy between serologic and culture results for M. agassizii. Compared with tortoises at other sites, tortoises at DTNA were more likely to be seropositive for M. agassizii. Tortoises at Goffs were significantly more likely to have moderate to severe shell disease, oral lesions, positive nasal cultures for M. agassizii, and increased plasma aspartate aminotransferase activity. The severe disease prevalence in Goffs tortoises likely contributed to the population decline that occurred during and subsequent to this study.

REPRODUCTION OF THE SMALLEST TORTOISE, THE NAMAQUALAND SPECKLED PADLOPER, HOMOPUS SIGNATUS SIGNATUS

The smallest tortoise, Homopus signatus, is inadequately protected, and information on its reproductive ecology can facilitate effective conservation. We combined X-ray radiography and ultrasonography to assess the reproductive status of free-ranging female Homopus signatus signatus during August–September 2000 (n = 30) and September–October 2001 (n = 29). Females produced only single-egg clutches but can produce more than one clutch in a season. Most (ca. 75%) of the females were gravid each spring, so H. s. signatus probably has a seasonal pattern of egg production. This pattern may be related to the seasonal climate; summers are hot and dry, yet winter rainfall is moderately predictable. Females were gravid from August through October, but further analyses are necessary to characterize the entire reproductive season and quantify clutch frequency. Large females produced large eggs, compared to eggs of small females. The smallest gravid female had a straight-line carapace length of 84.1 mm while the largest female measured 110.0 mm. Compared to gravid females, nongravid females were in poorer body condition (body mass and mass relative to carapace length) only in 2000, when there was lower rainfall and plant availability than in 2001. The difference in body mass approximated the mass of one egg. The low clutch size and fecundity suggest that populations have low intrinsic rates of natural increase. This plus their limited range and specific habitat requirements make H. s. signatus vulnerable to anthropogenic impacts.

Reproductive Effort and Reproductive Nutrition of Female Desert Tortoises: Essential Field Methods

Abstract I used three innovative, nondestructive field methods (gas dilution, doubly labeled water and radiography) to measure individual energy and water budgets of wild, female desert tortoises (Gopherus agassizii). With these budgets, I evaluated whether body reserves help females produce eggs independent of rainfall and food availability. Female desert tortoises used large seasonal and annual changes in metabolism and body water, protein and energy reserves to survive and produce eggs. Although lipid reserves are important to female desert tortoises, nitrogen or crude protein appears to be the primary limiting resource for producing eggs. By reducing metabolic rates 90%, females conserved enough body reserves to produce eggs during extreme drought conditions; this is an effective bet-hedging reproductive pattern in an extreme and unpredictable environment.

Shell Characteristics and Sexual Dimorphism in the Namaqualand Speckled Padloper, Homopus signatus signatus

Abstract There is little quantitative information regarding the two subspecies of the worlds smallest tortoise, Homopus signatus. To help characterise the northern subspecies H. s. signatus and evaluate the development of shell size and colour patterns, we measured shell characteristics of wild juvenile, male and female H. s. signatus. When scaling shell dimensions on carapace length, male shell size represented an extension of juvenile shell size for shell height, width and volume, but not plastron length. The slope of plastron length scaled on carapace length was smaller for males than for juveniles or females, suggesting differential growth of the plastron. The smaller male plastron translates to large shell openings, perhaps to improve locomotion and to facilitate tail movement during copulation. Conversely, the slope of female shell height, width and volume (scaled) was larger than for juveniles or males, presumably providing more space to accommodate follicles and the large egg. Serration of the marginal scutes and shell colour changed with body size, and shell colour pattern differed between sexes. The carapace was darker at intermediate body sizes (large juveniles and small adults); the increase in dark appearance resulted primarily from widening of the dark pigment band around scute margins as growth laminae were added. Both the lighter shell colour and reduced serration of large adults may be due to shell wear; large adults may produce less dark pigment, and older laminae disappear due to flaking or peeling. Females had a darker overall colour, more rays and fewer speckles than did males. This dimorphism may relate to thermoregulation, gamete protection or intraspecific communication. Both male and female patterns may confer crypsis if the sexes use microhabitats differently.

Polyandry and multiple paternities in the threatened Agassiz's desert tortoise, Gopherus agassizii

We used data from 17 to 20 microsatellite markers to investigate the incidence of multiple paternities in wild Agassiz’s desert tortoises, Gopherus agassizii. Neonates were sampled from clutches of eggs laid by wild mothers in nesting enclosures at Edwards Air Force Base and at the Marine Corps Air Ground Combat Center, California. We genotyped 28 clutches from 26 females sampling an average of six neonates per clutch. The number of paternal alleles was used to determine the minimum number of sires for each clutch. Based on conservative criteria requiring evidence from at least two loci to determine multiple paternity, a minimum of 64% of females were polyandrous, while a minimum of 57% of clutches were sired by multiple males. This formed one of the highest incidences of multiple paternities recorded to date in any species of tortoise. The high number of microsatellite loci involved in the analyses allowed detection of multiple paternities in clutches where this may have been missed if fewer loci were used. Our results highlighted the potential pitfalls of quantitatively comparing paternity studies based on differing sampling strategies. Finally, we summarized the conservation implications of the high rate of multiple paternities in this threatened species.

Plasma steroid and nutrient levels during the active season in wild Testudo horsfieldi

Plasma concentrations of sex steroids (testosterone and progesterone), proteins (total protein and albumin), lipids (phospholipids, cholesterol, and triglycerides), and minerals (calcium and phosphorus) were measured in wild Testudo horsfieldi in Uzbekistan, during the short, 3-month activity period (March-May, 1998). Testosterone concentration in males was highest (52 ng/ml) when they had just emerged from brumation (hibernation) in mid-March, which was also the beginning of the mating period, and fell in April. In females, progesterone peaked in mid-April (at 10 ng/ml), just before ovulation of the first clutches at the end of April and beginning of May. Testosterone levels in females and progesterone levels in males were low (<3 ng/ml) throughout the activity period. In general, the plasma concentrations of proteins, lipids, and phosphorus increased slowly in males, but more rapidly in females, during the activity season. These increases were particularly strong in females in the second half of April, coinciding with the peaks in female hormone levels. The changes in plasma hormones and nutrients reflected the timing of the different behaviours. The four first weeks of above-ground activity (mid-March to mid-April) by males, when they had high testosterone levels, were primarily allocated to fighting other males, courtship, and mating, while females spent much of that time feeding. Thereafter, both sexes concentrated on feeding. Females were probably preparing to ovulate in late-April, when their progesterone levels were highest and when plasma nutrient levels increased considerably.

Small and sensitive to drought : consequences of aridification to the conservation of Homopus signatus signatus : short communication

Abstract Several climate models predict that the western Succulent Karoo in South Africa will aridi-fy. This region includes the range of the smallest tortoise, Homopus signatus signatus. Although the effects of rainfall on the physiology and ecology of H. s. signatus received attention in recent years, the results of these studies have not been integrated to facilitate conservation planning. Here we evaluate the importance of body size and the responses of H. s. signatus to rainfall variation to make recommendations for the taxons conservation. The small body of H. s. signatus offers one solution to its habitat of low primary productivity and rocky slopes. Nevertheless, female fecundity and egg size increase with female size, and large eggs result in large hatchlings capable of surviving their harsh environment. Females accumulate nutrients in the rainfall season, winter, but also in the dry season, to enable the production of large eggs. Egg production decreases during drought, although some females continue to channel resources to reproduction, apparently at the cost of their own growth. Reduced fecundity and growth, a result of aridification, would likely lower the production of large eggs and hatchlings. Therefore, conservation measures that reduce the mortality of large females may aid population sus-tainability. Because egg and hatchling size might drop below a minimum viable size in an aridified environment, H. s. signatus conservation would benefit from the development of suitable habitat corridors to enable tortoise movements to regions that will receive sufficient rainfall in the future.