Data from: Baby snakes: Maternal investment and neonate sexing in smooth snakes

The smooth snake (Coronella austriaca Laurenti, 1768) is a non-venomous colubrid snake found throughout central, southern and northern-Europe and parts of Asia. Its main biotope in the Netherlands is heath and moorland. The species is ovoviviparous. All gravid females were collected in De Groote Peel (1500 ha; a national park), Deurnsche Peel (1400 ha) and the adjacent Mariapeel (1400 ha). During the summers of 2016 and 2017 a total of 15 gravid female smooth snakes were captured throughout the study area and kept in captivity to give birth. Females were collected shortly before parturition was expected to take place, to make sure that sex ratios were not influenced by handling or confinement. Exact capture locations were determined with a GPS device (Garmin eTrex 10, accuracy 3 m, RD coordinates). Terrariums (30 cm × 30 cm × 30 cm) with air vent grids and a removable frontal sliding window) were constructed and placed in a quiet room to prevent any disturbance for the gravid females. A heating cable was positioned underneath the terrariums to provide an opportunity for each individual to choose a preferred thermal spot. Conditions within the terrariums were sober: a sheet of kitchen paper was used to cover the floor, an empty egg carton (10-pack) provided a hiding place, while drinking water was available in a small water container. No food was supplied during captivity. Daily monitoring and cleaning was carried out, especially after birth of neonates occurred (removing remaining fluid, egg membranes etc.). A daily 16-hour light regime was applied in concurrence with natural light conditions during the summer season. Females typically rested on top off or inside the egg carton and selected spots close to the heating cable until parturition started. Neonates were kept in the same terrarium with their mother. Within two weeks after giving birth each female was released at exactly the same location where she was captured, together with their offspring.

Clutch size was determined for each adult female. The number of stillborn neonates and unfertilised eggs were counted as well. Snout-vent length (SVL) and tail length (TL) were measured for each neonate. Body mass of adult females was determined both before and after parturition using a scale-beam (Super Samson, 200 g max., increments 2 g). Body mass of all neonates was determined after first shedding, that usually occurred one week after parturition by using a Kern PCB 100-3 scale (in grams, accuracy three decimal places).

DNA samples were collected by buccal swabbing (Isohelix DNA/RNA buccal swabs, SK-1S) from all adult and subadult snakes. Since neonates are fragile, we used small round swabs for them (Puritan Pur Flock Ultra, Sterile Mini-Tip Flock Swab w/Polystyrene Hdl, REF 25-3316-U, Puritan Diagnostics L.L.C., Maine). Swabs were inserted in the mouth and were then slowly rotated a few times to collect DNA. Each sample was subsequently stored in test tubes with silica-gel capsules.

Three methods for identifying the sex of neonates were applied and compared: hemipenal eversion, morphometrics, and DNA analysis. Researchers involved in applying each method were not aware of the results of the other two methods of sex identification. For instance, DNA analyses were performed in a different lab, unaware of any other information other than a randomized snake ID, making it a double-blind study. The presence of hemipenes was established by hemipenal eversion or “popping” (Stahl, 2002). By applying gentle pressure with a finger on the snake below their vent one or both hemipenes will easily pop out (Fig. 1Q). This method of sexual identification is preferred over the use of a probe in the case of neonate snakes (Stahl, 2002) to prevent damage and is easiest to perform on neonate and juvenile individuals. However, successfully applying the hemipenal eversion procedure without injuring the young snakes requires skill and experience. One of the volunteers involved in our research had over 30 years of snake-breeding experience and performed this technique on all neonates born in captivity. Morphometric sexual identification is based on the ratio of total body length to tail length ((SVL+ TL)/TL) of neonates, a statistic that is often used (Käsewieter, 2002). Male neonates tend to have longer tails and therefore lower ratios.

Molecular sexing was done by amplification of two genes, CTNNB1 (NCBI gene ID 1499) and WAC (NCBI gene ID 51322), according to Laopichienpong et al. (2017) and Tawichasri et al. (2017). This has not been applied to C. austriaca. However, based on the studies of Laopichienpong et al. (2017) and Tawichasri et al. (2017) females were expected to show two amplicons and males a single amplicon for each marker after gel electrophoresis. 

The data are organized in two table, one for the 15 gravid females collected from the field, and one for the 107 offspring these females gave birth to in captivity.

gravidFemales.csv 

ID = unique ID per gravid female

momMassCapt = mass at the time of capture (in grams)

momMassAfter = mass after parturition (in grams)

clutchMass = summed mass of all neonates in a litter (in grams)

litterSize = number of neonates in a litter

nSons = number of sons

nDaughters = number of daugthers

neonates.csv

momID = unique ID of the gravid female collected in the field

DoB = date of birth of a neonate

mass = mass of a neonate (in grams)

tail = length of a neonate (in cm)

SVL = snout-vent-length of a neonate (in cm)

ratio = total-to-tail length ratio: (SVL+TL)/TL

wac = identified sex based on the wac primer

ctbb1 = identified sex based on the ctbb1 primer

eversion = identified sex based on hemipenal eversion

sex = identified sex, where DNA analysis was leading and hemipenal eversion only used when no DNA analysis was performed