Effects of endozoochory and diploendozoochory by captive mammals on Juniperus deppeana seeds

An overview of the experimental evaluation is given in this first section, which comprises two main stages, and the particular details of each stage are presented separately below. The experimental evaluation was conducted in March 2019 and consisted of two stages: I) to create the habit in mammals of ingesting J. deppeana seeds and II) the experimental evaluation of endozoochory and diploendozoochory. Prior to the two stages, the collection of seeded fruits of the plant component was carried out. The J. deppeana shrub produces globules that generally contain five seeds each, and the fruits of this species are consumed and dispersed by both birds and mammals (Livingston, 1972; Schupp et al., 1997). The animal component was also selected according to the dispersal system: gray fox, coati (omnivores), and rabbit (herbivore/feline prey) for endozoochory, and bobcat and cougar (predators) for diploendozoochory. It is documented that all selected mammals disperse seeds by either one system or the other. The selection of mammals was based on animal species (the gray fox, coati, and bobcat), where the dispersal of J. deppeana seeds by endozoochory and diploendozoochory had previously been verified in the forests of the SF-PNA (Rubalcava-Castillo et al., 2021). It had also been verified that rabbits can disperse Juniperus seeds by endozoochory in forests (Lezama-Delgado et al., 2016). The stage I lasted 10 days with the objective of adapting the animals to the ingestion of the fruits and seeds. After 10 days, the rabbits became accustomed to the fruit intake, as did all the other mammals to the seed intake, since the seed rations were eaten completely without any avoidance behavior. Once the animals had adapted, stage II began with the modeling, in which the animals were again offered seeds in their diets for 21 days. During the second stage, the seed retention time in the tracts was estimated by counting the time from seed ingestion to the time of excretion with seeds present where, in turn, the seeds were separated from the scats, collected, and counted. Subsequently, the scat-derived seeds and those collected from the canopy (control) were subjected to X-ray and SEM tests. Control group A control group was established to test the effects of endozoochory and diploendozoochory in seeds from mammals under evaluation versus seeds taken directly from the tree. During the spring of 2019, according to the calendar of the northern hemisphere meteorological stations, ripe fruits with seeds were collected from twelve random individual J. deppeana trees of basal diameter >5 cm and height >2 m. The trees selected according to these criteria corresponded to adult plants with a high probability of bearing ripe fruits (Rubalcava-Castillo et al., 2020). The fruits were collected when they presented a reddish-brown color in temperate forests of the SF-PNA. The collection was carried out in an area of 527 ha, within a temperate forest with a temperate subhumid climate and summer rainfall (Rzedowski, 1978), with an average annual precipitation of 650 mm (SEDESO, 1995). These same trees were also sampled for the collection of fruits with seeds for use in the feeding experiment. Mammal selection Seventeen domestic rabbits (Oryctolagus cuniculus; California breed, young adults, no detectable morphological or physiological alterations, five males and 12 females were chosen) were used to represent the primary disperser (prey) in the diploendozoochory process. The rabbits were kept in cages 1.60 m in width and 50 cm in height. In the morning, they were first fed with fruits and then with their usual diet consisting of a special mixture of alfalfa and standard commercial food. The mammals were selected from species in captivity at the Center for Environmental, Cultural, and Recreational Education of the Rodolfo Landeros Park in Aguascalientes City, Mexico. The selected species were females: a gray fox (Urocyon cinereoargenteus), a coati (Nasua narica), two bobcats, and a cougar. In the case of felines, it has been previously shown that the rabbit forms part of the diet of both the bobcat (Rubalcava-Castillo et al., 2020) and the cougar (de la Torre & de la Riva, 2009; Monroy-Vilchis et al., 2009) in the region. The animals were kept in zoo accommodations that were specially designed to meet the standards of space and habitability, in which they were fed their usual diet with seeds incorporated. The gray fox diet comprised fruits (papaya and melon) and chicken; the coati diet contained fruits (banana, papaya, watermelon, and melon) and chicken. The bobcat diet consisted only of chicken, and the cougar diet included veal and chicken. I) Creating the habit of ingesting J. deppeana seeds in mammals As part of the experimental evaluation, all mammals, including the rabbits, underwent a 10-day adaptation period for the ingestion of J. deppeana seeds. In the first part, the rabbits only were offered 3 whole fruits (equivalent to 15 seeds) daily in the morning to each of the rabbits to familiarize them with their taste, appearance, smell, and consumption. The rabbits were monitored to observe their behavior and any effects of the offered fruits. In the second part, at the same time as the rabbits, the gray fox, coati, and felines also underwent an adaptation period of eating the seeds. Only the seeds were offered without the whole fruit because these mammals in captivity have pre-established diets assigned by the institution (melon, banana, and watermelon pieces for gray fox and coati, and chicken breast for bobcat and cougar). The seeds were therefore placed within the usual diet of mammals. Small pieces of the fruits were broken for gray foxes and coatis, and pieces of chicken were provided for felines. Small pieces of fruit and chicken with the seeds inside were cut to enable the mammals to ingest the seeded portions in one bite to guarantee the intake of all the seeds and avoid choking problems. Five seeds (1.2 cm average diameter) were placed on each small piece of fruit and chicken (the seeds were inserted into each piece of chicken breast, making sure that the seeds were fixed inside the piece). Three pieces daily were offered to each mammal, and the animals were monitored to ensure they swallowed the food completely and consumed the whole ration. II) Experimental evaluation of endozoochory and diploendozoochory The evaluation consisted of two main parts: (1) endozoochory and (2) diploendozoochory. For endozoochory, seeds were offered to the gray foxes, coatis, and rabbits. Rabbits, in addition to carrying out the endozoochory as the primary disperser of Juniperus (Lezama-Delgado et al., 2016), also represent the prey of felines in diploendozoochory. Therefore, the diploendozoochory was divided into two stages: rabbits and felines. Part of the seeds dispersed by rabbits was used to analyze their endozoochory, and the other part was offered to felines to complete the transit of the seeds through both digestive tracts (prey and predator). Following the adaptation period, a period of four days (96 h.) was allowed to elapse to ensure that all seeds from the adaptation period had been defecated. Subsequently, for the experimental evaluation, the same procedure was repeated for the ingestion of seeds in all mammals for 21 days. When the seeds were offered, the exact time of ingestion was recorded. After ingestion, inspections were carried out in the rabbit cages and habitats of other mammals at 1-hour intervals to sweep and collect the scats present. All scats were collected from each habitat of each animal species over the entire plausible retention period, employing an exhaustive search of the entire area of the habitats, which had bare soil rendering visible all the scats deposited in that period. Thus, we ensured that the proportion of recovered seeds was an accurate reflection of the seeds lost to digestion rather than to the habitat. This monitoring was carried out continuously for 12 hours, during which visits were made to the habitats to check for the presence of scats or to observe whether the animal was defecating. Upon finding scats with seeds, the time of discovery was recorded to estimate the retention period in the mammalian tract, and the scats were placed in paper sacks. Until verifying the discovery of all seeds (entire or broken) of the ration in the scats of each species, the corresponding ration of seeds was offered to each one the following day. The scats were washed through a sieve system to extract the seeds, which were then dried and counted to estimate the percentage of recovered seeds for each of the different retention times for each animal species. For the second stage with felines, the seeds from the pellets were incorporated into the felines’ usual diet. The exact times of ingestion and excretion were recorded. In the same way, every hour, visits were made to the habitats of each feline for 10-15 minutes to check for scats and collect them if present. In this way, it was possible to complete the diploendozoochory system by obtaining in the scats of the felines the seeds that had initially passed through the digestive tract of the rabbits and then through the felines themselves.