DOI: 10.5061/dryad.stqjq2c63
title: Effects of semi-constant temperature on embryonic and hatchling phenotypes of six-tubercled Amazon River turtles, Podocnemis sextuberculata

description: Purpose: We evaluated how constant incubation temperatures affect
 life-history traits pre-hatching and post-hatching of the six-tubercled
 Amazon River turtle, Podocnemis sextuberculata. Methods: We incubated eggs
 from natural nests at ten semi-constant temperatures between 22.26±1.01°C
 and 37.37±0.38°C (2013) and at six temperatures between 25.75±0.22°C and
 36.17±0.15°C (2016). In 2013, we raised hatchling for 90 days to evaluate
 effects of temperature on early hatchling growth. We evaluated maternal
 effects in 2016. Results: P. sextuberculata displays temperature-dependent
 sex determination and produces males at colder and females at warmer
 temperatures (TSD Ia). The estimated pivotal temperature was 33.73 ±
 0.15°C and the transitional range of temperatures (TRT) 1.16 ± 0.59°C.
 Semi-constant temperatures below 26°C and above 38°C were lethal.
 Intermediate temperatures (32.25°C and 31.5°C, respectively) were optimal
 for hatching success and produced larger hatchlings that grew slower early
 in life compared to colder or warmer conditions, which produced smaller
 hatchlings. Warmer incubation temperatures within the optimal range
 (28°C-37°C) accelerated embryonic development. In contrast, comparisons of
 30, 60 and 90 days-old suggests that warmer incubation temperatures
 reduced growth and mass gain rates post-hatching, such that incubation
 temperature effects on body size at emergence disappeared by 3 months of
 age. Conclusions: Six-tubercled Amazon River turtles showed the highest
 pivotal temperature reported for any turtle. The relatively narrow TRT may
 limit the evolutionary potential of this vulnerable turtle in the face of
 global warming. Future incubation experiments at a finer scale (33°C-36°C)
 are warranted to refine the sex-ratio reaction norm. Field studies that
 monitor natural nests are imperative to evaluate conservation measures and
 the effect of female-biased illegal hunting and climate change. By
 providing data about the thermal biology of an understudied lineage of
 non-model species, our study helps fill gaps in our understanding of the
 evolution of vertebrate sex determination and its potential adaptive
 value.
description: Two semi-constant-incubation-temperature experiments were
 conducted in 2013 and 2016. Freshly-laid eggs were collected at the Middle
 Solimoes River within the Mamiraua Sustainable Development Reserve,
 Central Amazon, Brazil and transported to the laboratory at the Mamiraua
 Sustainable Development Institute (Tefe, Amazonas State, Brazil). Egg
 incubation started &lt;24hrs from oviposition. Incubators were made
 from 120-liter Styrofoam boxes divided into two compartments, each with a
 60-watt lamp connected to a digital thermostat (AddTherm, model: A80).
 Eggs were buried in moist medium-size vermiculite purchased at a local
 garden store (in a proportion of 1g vermiculite:2g of water following
 Gallego-Garcia and Paez, 2016) inside plastic containers, along with the
 thermostat probe. Temperature was recorded hourly with a data logger with
 (±0.5°C accuracy). The thermostat probe and data logger were placed in the
 middle of the container. To maintain moisture levels, all boxes with
 vermiculite were incubated for a week, without eggs, at each temperature
 prior to the onset of the experiment to determine the rate of water loss
 per treatment by the change of weight of the box. Then, once the eggs were
 added and the experiment started, water was added at different intervals,
 between every 2 days (for warmer treatments with the highest rate water
 loss) to weekly (for colder treatments with lowest rate of water
 loss). In 2013, we collected 100 eggs (10 per clutch),
 and placed one egg per nest at 10 different target temperatures: 22°C,
 23°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 36°C and 37.5°C. However, the
 low-tech design of the incubators resulted in thermal regimes that
 fluctuated with varied amplitude around these target values (Figure S1)
 (22.86±1.01°C, 23.22±0.65°C, 27.89±0.19°C, 29.14±0.20°C, 29.99±0.23°C,
 31.11±0.21°C, 31.7±1.71°C, 33.18±0.40°C, 35.95±0.83°C and 37.37±0.38°C).
 In 2016, we collected 132 eggs from 11 different nests, and placed two
 eggs per nest at six different thermal regimes: 25.75±0.22°C,
 27.94±0.65°C, 29.83±0.26°C, 31.95±0.29°C, 34.01±0.67°C, 36.17±0.15°C.
 Because eggs from 22.86°C and 23.22°C did not hatch and lacked signs of
 embryo development, these temperatures were excluded from all further
 analyses. Incubation duration was measured from egg
 collection to full emergence of hatchlings from the eggshell (emergence).
 Unhatched eggs were opened at the end of the experiment, and those lacking
 traces of embryonic development excluded from further analyses, as they
 were assumed to be infertile or to have suffered embryonic mortality
 unrelated to temperature (this assumption was based on the fact that
 statistical tests did not detect any temperature effect on the number of
 undeveloped eggs: x<sup>2</sup> = 12, df = 9, p = 0.21) and
 excluded from further analyses. Straight carapace length (SCL), straight
 carapace width (SCW), plastron length (PL), plastron femoral width (PFW)
 and head width were measured on day one after emergence using a 150 mm
 caliper (Mitutoyo®), and hatchling mass was measured using a digital scale
 (Pesola®, MS500, precision = 0.1 g). To investigate the
 influence of incubation temperature on post-hatching growth, in 2013 we
 kept the hatchlings for eight days in a plastic box containing moist
 vermiculite and then raised them individually until day 90. We reared them
 in open plastic boxes (262x177x147 mm) filled with 40
 cm<sup>3</sup> of tap water per millimeter of hatchling SCL
 (adjusted when hatchlings were measured at 1, 8, 15, 30, 45, 60 and 90
 days of age), and provided with a rock as dry surface. Boxes were
 maintained indoors at 23 - 31°C under a ~12h daylight photoperiod and
 cleaned every two days. Hatchlings were fed commercial turtle food (Alcon®
 Reptolife: 34% protein, 10% fiber, 15% mineral, 1.6-3% calcium, 0.7%
 phosphor) ad libitum, starting at 8 days of age. At age 1, 8, 15, 30, 45,
 60 and 90 days, we measured and weighed hatchlings.
 Hatchlings were euthanized by intravenous (IV) injection of
 general anesthetics (association of xylazine 2 mg/kg, ketamine 50 mg/kg
 and acepromazine 0.25 mg/kg), followed by administration of potassium
 19.1% chloride (2 mg/kg, IV) after loss of corneal and muscles reflexes,
 as recommended by Resolution nº1000/2012 of the Brazilian Federal Council
 of Veterinary Medicine (CFMV, 2012). Gonads were dissected to assess sex
 by histology, and carcasses were fixed in 10% formalin, stored in 70%
 ethanol and deposited in the Mamiraua Sustainable Development Institute
 Reptile Collection in Tefe.
description: Data files can be opened using any spreadsheet reader, that opens
 .xls file format. All analyses were performed using R v. 3.5.2