DOI: 10.5061/dryad.pnvx0k6td
title: Effects of endozoochory and diploendozoochory by captive mammals on Juniperus deppeana seeds

description: Carnivorous mammals disperse seeds through endozoochory and
 diploendozoochory. The former consists of ingestion of the fruit, passage
 through the digestive tract, and expulsion of the seeds, a process that
 allows scarification and dispersal of the seeds over long or short
 distances. The latter is typical of predators that expel seeds that were
 contained in the prey and the effects of which may differ from those of
 endozoochory with respect to the retention time of the seeds in the
 tracts, as well as their scarification and viability. The objective of
 this study was to conduct an experimental evaluation comparing the
 capacity of each mammal species in terms of the dispersal of Juniperus
 deppeana seeds and, at the same time, to compare this capacity through the
 two dispersal systems: endozoochory and diploendozoochory. We measured
 dispersal capacity using indices of recovery, viability, changes in
 testas, and retention time of seeds in the digestive tract. Juniperus
 deppeana fruits were collected in the Sierra Fría Protected Natural Area
 in Aguascalientes, Mexico, and were administered in the diet of captive
 mammals: gray fox (Urocyon cinereoargenteus), coati (Nasua narica) and
 domestic rabbits (Oryctolagus cuniculus). These three mammals represented
 the endozoochoric dispersers. For the diploendozoochoric treatment, seeds
 excreted by rabbits were incorporated into the diets of captive mammals:
 bobcat (Lynx rufus) and cougar (Puma concolor), in a local zoo. Seeds
 present in the scats were then collected, and recovery rates and retention
 times were estimated. Viability was estimated by X-ray optical
 densitometry and testa thicknesses were measured and surfaces checked
 using scanning electron microscopy. The results showed a recovery of seeds
 greater than 70% in all the animals. The retention time was &lt; 24 h
 in the endozoochory, but longer at 24-96 h in the diploendozoochory (P
 &lt; 0.05). Seed viability ( ± SD) was decreased in rabbits
 (74.0 ± 11.5 %), compared to fruits obtained directly from the canopy
 (89.7 ± 2.0 %), while gray fox, coati, bobcat, and cougar did not affect
 seed viability (P &lt; 0.05). An increase in the thickness of the
 testas was also observed in seeds excreted from all mammals (P &lt;
 0.05). Through evaluation, our results suggest that mammalian endozoochory
 and diploendozoochory contribute to the dispersal of J. deppeana by
 maintaining viable seeds with adaptive characteristics in the testa to
 promote forest regeneration and restoration. In particular, feline
 predators can provide an ecosystem service through scarification and seed
 dispersal.
description: 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 <em>J. deppeana</em> 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 <em>J. deppeana </em>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 <em>J. deppeana</em> 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. <strong>Control
 group</strong> 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 <em>J. deppeana</em> trees of basal diameter
 &gt;5 cm and height &gt;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.
 <strong>Mammal
 selection</strong> Seventeen domestic rabbits
 (<em>Oryctolagus cuniculus</em>; 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 (<em>Urocyon
 cinereoargenteus</em>), a coati (<em>Nasua narica</em>),
 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 <em>et al</em>., 2020) and the cougar (de
 la Torre & de la Riva, 2009; Monroy-Vilchis <em>et
 al</em>., 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.
 <strong><em>I)
 Creating the habit of ingesting J. deppeana seeds in
 mammals</em></strong> As part of the experimental evaluation, all
 mammals, including the rabbits, underwent a 10-day adaptation period for
 the ingestion of <em>J. deppeana</em> 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. <em><strong>II) Experimental evaluation of endozoochory and
 diploendozoochory</strong></em>
 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 <em>Juniperus</em> (Lezama-Delgado <em>et
 al</em>., 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.