DOI: 10.5061/dryad.stqjq2ccd
title: Bimodal nesting season in Karoo dwarf tortoises (Chersobius boulengeri)

description: Tortoises in arid, unpredictable regions may use a bet-hedging strategy of
 regularly laying small clutches regardless of actual environmental
 conditions so that some hatchlings will emerge when conditions are
 favourable. Karoo dwarf tortoises (Chersobius boulengeri) are endangered
 in their arid, unpredictable range in South Africa, yet studies of
 reproduction are lacking. I used radiography and captive breeding to study
 reproduction in wild (n = 22, 2018–2022) and captive (n = 2, 2020–2023)
 individuals, expecting to find bet-hedging characteristics. My expectation
 was that females would regularly lay eggs in summer when most rain falls.
 In addition, I expected that females would produce consistently large eggs
 and hatchlings capable of surviving their harsh environment. Indeed,
 females produced multiple clutches of large single eggs per year, some
 even during drought. However, the egg-laying season had a unique bimodal
 pattern in autumn and spring, possibly dispersing hatching events
 throughout the unpredictable rainfall season. Fecundity was low,
 approximately 5 eggs per year when resources were unlimited. The large
 eggs were usually wider than pelvic width, but egg size was nevertheless
 constrained by female size. Hatchling size correlated positively with egg
 size, confirming a potential survival benefit. Surprisingly, egg size
 decreased in successive clutches in a season, apparently to increase
 offspring frequency while resources were limited. Reproduction of Karoo
 dwarf tortoises is unlikely to offset current predation levels that
 contribute to their endangered status, thus predation reduction should be
 a conservation priority. Projected regional aridification due to climate
 change may pose a previously underestimated threat because females lay
 fewer eggs during drought, females that lay eggs during drought may suffer
 increased mortality when body reserves cannot be restored, and hatchlings
 (particularly from late clutches) may have insufficient reserves for
 survival under aridified conditions. Conservation plans might focus on
 sites least affected by aridification.
description: <em>Study site and recordings in the
 wild</em> The study site consisted of a rocky
 mountain slope (altitude ~1,400 m) with adjacent plateaus, exposed
 dolerite sills, and dry river beds in the Northern Cape Province of South
 Africa (Loehr, 2023b; due to the sensitivity of this population the
 coordinates are withheld here, but are recorded on the biodiversity
 database of CapeNature, Western Cape Province, South Africa). Long-term
 modelled (30 years of hourly weather simulations) climatic conditions show
 that the site is arid (annual rainfall 162 mm, largely falling in summer),
 with mean minimum and maximum monthly temperatures ranging between 1–14
 and 14–30 °C, respectively (www.meteoblue.com). I placed data loggers
 (Hobo S-THB-M00x, RS3-B, and S-RGF-M002 connected to H21-USB; Onset
 Computer Corporation, Bourne, MA, USA) in the centre of the study site to
 record temperature and rainfall during this study.
 Between 20 February and 8 March 2018, I captured 16 female
 tortoises through inspection of holes under and among rocks. Females were
 equipped with radio transmitters (type RI-2B with internal antenna;
 Holohil Systems, Carp, ON, Canada), which were glued onto the posterior
 costal scutes (equipment mass ≤9.7% of body mass) to not obstruct mating.
 In summer (20 February and 8 March 2018, and 12 March 2019) and in spring
 (2 October, 19 October and 7 November 2018, and 9 October 2019), females
 were tracked, transported by hiking and driving to the nearest radiography
 facility (~185 km), radiographed dorso-ventrally at 50 kV for 0.25 s at 50
 mA with 6–10 tortoises placed on each radiograph, and released at the
 location of capture within 24 h after tracking. Prior to release, females
 were offered drinking water to replenish voided urine. On 8 March 2018,
 two opportunistically captured females were also radiographed, whereas two
 females with transmitters were found dead on 9 October 2019, and the
 signal of one female was lost on 7 November 2018. In autumn (13 April
 2022), after all transmitters had been removed in March 2020, I captured
 and radiographed four females, one of which had been radiographed
 previously. All radiographed females appeared mature, based on the lack of
 male characteristics (e.g., large tail, concave plastron) and straight
 carapace lengths that exceeded the minimum male straight carapace
 length. Each year in February, and for each female not
 captured by radiotelemetry, I used digital sliding calipers to record the
 nearest 0.01 mm straight carapace length (SCL), maximum shell width (SW),
 maximum shell height (SH), and plastron length (PL). Shell volume (SV,
 cm<sup>3</sup>) was calculated as π × SCL × SW × SH / 6,000.
 Because all females appeared mature, I assumed that growth would be
 negligible within years. Body mass (BM) was recorded on each radiography
 date and used to calculate body condition (BC) as BM / SV. Radiographs
 were inspected for females gravid with shelled eggs, and successive
 clutches were distinguished based on the level of egg calcification (e.g.,
 successive radiographs of a female showing a heavily and lightly calcified
 egg indicated different clutches) and egg dimensions. For each female on
 the radiographs, I measured PL and pelvic width (PW, mm, at the largest
 gap between the ilia), and for each egg, I measured egg length (EL, mm)
 and egg width (EW, mm), to the nearest 0.01 mm. Egg volume (EV,
 cm<sup>3</sup>) was calculated as π × EL ×
 EW<sup>2</sup> / 6,000. I estimated egg mass (EM) based on the
 egg density of captive eggs in this study and subtracted EM from female BM
 to calculate BC of gravid females excluding egg mass. Measurements of
 radiographs were performed using either DICOM files (MiViewer 10.25.0,
 MillenSys, Cairo, Egypt) or bitmaps with scale bar (ImageJ v1.50i, LOCI,
 University of Wisconsin, Madison, WI, USA). I verified recordings by
 comparing PL on radiographs with actual PL. One radiograph (13 April 2022)
 was not available as DICOM or bitmap with a scale bar, so I used the ratio
 between PL on the radiograph and actual PL to determine the
 scale. <em>Husbandry and recordings in
 captivity</em> In March 2019, I captured two
 females and two males outside the core of the study site and transported
 them to indoor facilities in the northern hemisphere (Netherlands), for
 study until 2023. Both females had very low body masses and appeared
 emaciated by drought. Consequently, I provided a northern hemisphere
 seasonal cycle (i.e., March representing spring), delaying unfavourable
 winter conditions (Loehr, 2023a). Females were individually housed in
 well-structured enclosures, fed <em>ad libitum</em> on a
 fibre-rich diet low in sugars and proteins, and seasonal cycles of
 temperatures and photoperiod mimicked those in the wild. Enclosure
 temperatures were monitored with Pt100 sensors (Yageo Nexensos, New Taipei
 City, Taiwan) connected to a controller (Siemens LOGO!, Munich, Germany).
 Males were occasionally introduced in female enclosures to mate.
 When an egg had been produced, it was excavated immediately. I
 measured EL and EW to the nearest 0.01 mm with digital sliding calipers,
 and recorded egg mass (EM) to the nearest 0.1 g with a digital balance.
 Egg dimensions were used to calculate EV. Eggs were transferred to an
 incubator (IPP30plus, Memmert, Schwabach, Germany), where they incubated
 at a diurnal temperature cycle, interrupted by a 20-day period in a
 constant-temperature incubator (INCU-Line IL 10, VWR International,
 Amsterdam, Netherlands, retrofitted with a CAREL ir33 temperature
 controller, CAREL Industries, Padova, Italy) to control offspring sex
 assuming temperature-dependent sex determination. Initially, the diurnal
 temperature cycle ranged from 28.0–33.0 °C, but this was changed to
 26.0–31.0 °C from 10 September 2021 onwards to reduce the development of
 supernumeral scutes. During the constant-temperature phase (incubation
 days 30–50 for the first two eggs, 23–43 for all other eggs), eggs were
 incubated at 30 °C (to produce males) or 33.0 °C (to produce females), but
 the latter temperature was reduced to 32.5 °C from 4 April 2023 onwards. I
 ensured that incubation treatments were evenly distributed among eggs from
 the two females. After hatching, hatchlings were placed on wet tissue for
 12–24 h, after which I measured SCL, SW, SH, and BM, enabling calculation
 of SV using the equation that had been used for adults. At the end of this
 study, husbandry and breeding were continued in a studbook under the
 auspices of Dwarf Tortoise Conservation (IJsselstein, Netherlands) and the
 European Studbook Foundation (Sneek, Netherlands), with the aim to develop
 an <em>ex-situ</em> assurance population.
description: # Bimodal nesting season in Karoo dwarf tortoises (*Chersobius
 boulengeri*)
 [https://doi.org/10.5061/dryad.stqjq2ccd](https://doi.org/10.5061/dryad.stqjq2ccd) ## Description of the data and file structure Female Karoo dwarf tortoises (*Chersobius boulengeri*) were sampled in the wild and radiographed, and two male and two female individuals were transferred to captivity for captive breeding. Captive-produced eggs and hatchlings were recorded. The Methods field of this submission contains details on how data were collected. All data are gathered in one xlsx file that contains four tabs. ### Files and variables #### File: Reproduction\_raw\_data.xlsx **Description:** Characteristics of wild females, recordings from radiographs, characteristics of captive females, characteristics of captive eggs and hatchlings, captive temperatures, and wild temperatures and rainfall. ##### Variables * All variables in the xlsx file are spelled out in full and mention units of measurement. * Cells without data contain a dash. ## Code/software Microsoft Excel 365