Fatal amplexus: an invasive toad causes rapid mortality in a native frog through misdirected reproductive behavior

Pseudo-amplexus indicates individuals subjected to amplexus-like mechanical compression for five minutes; Control indicates individuals receiving no amplexus stimulation.</li><li>block: Pair identity (a–f). Each block consists of one pseudo-amplexus individual and one control individual.</li><li>Bodymass: Body mass of the experimental B. formosus individual, in grams (g).</li><li>Bodylength: Snout–vent length of the experimental B. formosus individual, in millimeters (mm).</li><li>Sex: Sex of the experimental B. formosus individual (male or female).</li><li>Area of lower body: Area of the lower dorsal body measured from the photograph using ImageJ.

The lower body was defined as the region posterior to the line connecting both knees when the toad was in its natural resting posture. Values are in image-area units and are used to calculate the proportional area covered by secretion.</li><li>White area: Area of the lower dorsal body on which whitish external secretion was visible, measured from the same photograph using ImageJ, in the same image-area units as Area of lower body.</li><li>Proportion of white area: Proportion of the lower dorsal body covered by visible whitish secretion, calculated as White area / Area of lower body.

Values range from 0 to 1.</li><li>The proportion of white area was logit-transformed and analyzed using a linear mixed-effects model, with treatment as a fixed effect and block (pair identity) as a random intercept.</li></ul><p>***********</p><p dir="ltr">Experiment 2: Forced amplexus experiment</p><p dir="ltr">File: Exp2_forced_amplexus_exp.xlsx<br></p><p dir="ltr">This experiment tested whether sustained forced amplexus-like contact with B. formosus causes mortality in male R. pirica.

Male R. pirica were gently restrained using plastic cable ties in one of two treatments: on the back of an adult male B. formosus (heterospecific treatment; n = 7) or on the back of an adult female R. pirica (conspecific treatment; n = 7). Individuals were monitored for up to 12 hours.</p><p dir="ltr">Each row represents one experimental male R. pirica.</p><p dir="ltr">Columns:</p><ul><li>Treatment: Species used as the amplexus partner.

B. formosus indicates the heterospecific treatment, in which the focal male R. pirica was positioned on an adult male B. formosus. R. pirica indicates the conspecific treatment, in which the focal male was positioned on an adult female R. pirica.<br>Body_mass: Body mass of the focal male R. pirica, in grams (g).</li><li>Snout-vent_length: Snout–vent length of the focal male R. pirica, in millimeters (mm).<br>time_to_death: Time in minutes from the onset of forced amplexus until death.

Individuals that remained alive throughout the 12-hour observation period are recorded as 720.</li><li>For survival analysis, individuals with time_to_death < 720 were treated as mortality events, whereas individuals recorded as 720 were treated as right-censored. Kaplan–Meier survival curves were generated for the two treatments and compared using a log-rank test.</li></ul><p dir="ltr"><br></p><p>**********</p><p dir="ltr">Experiment 3: Contact-transfer experiment<br></p><p dir="ltr">File: Exp3_contact-transfer_exp.xlsx<br></p><p dir="ltr">This experiment tested whether direct contact with B. formosus skin secretions is sufficient to cause mortality in male R. pirica in the absence of amplexus-related posture or mechanical pressure.

Anaesthetized male R. pirica were assigned to one of three treatments. Their ventral surfaces were gently rubbed approximately 10 times against (1) the unwashed dorsum of an adult B. formosus, (2) the thoroughly washed dorsum of an adult B. formosus, or (3) the dorsum of a conspecific R. pirica. Individuals were subsequently monitored for up to 12 hours.Each row represents one experimental male R. pirica.</p><p><br></p><p dir="ltr">Columns:</p><ul><li>Treatment: Contact-transfer treatment.

B. formosus–secretion present indicates contact with the unwashed dorsal skin of B. formosus, with surface skin secretions intact; B. formosus–secretion removed indicates contact with the dorsal skin of B. formosus after thorough rinsing with running water to remove surface secretions; R. pirica indicates contact with the dorsal skin of a conspecific and serves as the conspecific control.</li><li>Body_mass_(g): Body mass of the focal male R. pirica, in grams (g).</li><li>Snout-vent_length_(mm): Snout–vent length of the focal male R. pirica, in millimeters (mm).</li><li>Time_to_death: Time in minutes from the onset of contact transfer until death.

Individuals surviving throughout the 12-hour observation period are recorded as 720.</li></ul><p dir="ltr">For statistical analysis and graphical presentation, survival times were truncated at 120 minutes because all deaths in the secretion-present treatment occurred within this period. Individuals surviving beyond 120 minutes were therefore right-censored at 120 minutes for the analysis; all such individuals were nevertheless monitored for the full 12-hour observation period and remained alive.</p><p>************</p><p dir="ltr">R scriptFile: R_script Tokuda et al.docx</p><p dir="ltr">This file contains the R code used to analyze the three experiments and generate the associated figures.

Analyses were conducted in R version 4.4.3.</p><p dir="ltr">For Experiment 1, the script logit-transforms the proportional secretion data and fits a linear mixed-effects model with treatment as a fixed effect and pair identity as a random intercept.</p><p dir="ltr">For Experiments 2 and 3, the script performs Kaplan–Meier survival analyses and log-rank tests. In Experiment 2, individuals surviving the full 12-hour observation period are right-censored at 720 minutes.

In Experiment 3, survival data are right-censored at 120 minutes for statistical analysis and graphical presentation.</p><p dir="ltr">The script uses the R packages readxl, dplyr, janitor, lme4, survival, survminer, and ggplot2.</p>