The significance of diverse seed dispersal systems to biotic communities, especially concerning the important role of vertebrate animals as dispersers (zoochory), has received increasing attention from researchers over the past several decades (Correa et al., 2007; Traveset et al., 2007, 2008, 2014; Schupp et al., 2010; Hämäläinen et al., 2017; Coughlan et al., 2019; Beckman et al., 2021). In the dispersal system termed endozoochory, seeds are directly consumed (e.g., by bears, primates, rodents, birds, or turtles) and later voided via regurgitation or defecation (Traveset et al., 2001, 2007, 2008; Steyaert et al., 2019). Endozoochory is prevalent in many vertebrate lineages, including freshwater fishes (Galetti, 2007), anuran amphibians (Silva and de Britto-Pereira, 2006; Hocking and Babbitt, 2014), a variety of nonavian reptiles (Blake et al., 2012; Reiserer et al., 2018; Valido and Olesen, 2019; Falcón et al., 2020), birds (Nathan et al., 2008; Heleno et al., 2011; Padilla et al., 2012; Baños-Villalba et al., 2017; Bartel et al., 2018; Blanco et al., 2018; Coughlan et al., 2019; Bravo et al., 2021), and both placental and marsupial mammals (Traveset et al., 2014; Jaganathan et al., 2016; Hämäläinen et al., 2017).

Diploendozoochory, possibly first documented by Darwin (1859), differs from endozoochory in that it is a two-phase seed dispersal system with several key players. In this system, a secondary (P2) predator (e.g., carnivorous vertebrate) consumes a primary (P1) seed predator (granivore) such as a rodent or bird with seeds in its digestive tract (e.g., mouth, cheek pouch, crop, and stomach) which are subsequently eliminated in the feces of P2 (Hämäläinen et al., 2017). In diploendozoochorous systems, when a secondary predator (P2) such as a rattlesnake subjugates and consumes a primary seed predator (P1) that has intact seeds in its cheek pouches (e.g., in our system, a heteromyid and geomyid rodent), the seeds are transported to the digestive tract of P2 and ultimately excreted. Consequently, some or all excreted seeds from P2 potentially survive this journey and germinate; thus, they can be viewed as “rescued” from P1 (post-dispersal seed predation, see Gong et al., 2015). Unlike endozoochory, our present knowledge of seed germination and the fate of dispersed seeds by P2 in diploendozoochorous systems is limited (Vander Wall and Longland, 2004; Hämäläinen et al., 2017; van Leeuwen et al., 2017). Nonetheless, with increased knowledge of the trophic behavior and ecology of carnivorous vertebrates that feed on granivores, the role of seed rescue and secondary dispersal appears to be non-trivial (Vander Wall and Longland, 2004; Hämäläinen et al., 2017; van Leeuwen et al., 2017; Pérez-Méndez and Rodríguez, 2018; Reiserer et al., 2018). Importantly, diploendozoochory can influence plant fitness in a number of ways including (a) seed transport, (b) altering the viability of transported seeds, and (c) changing the quantity of seeds that are dispersed (Hämäläinen et al., 2017; Saldaña-Vázquez et al., 2019; Rubalcava-Castillo et al., 2020). Accordingly, numerous avenues of inquiry remain to be investigated in diploendozoochorous systems which include diversity of species as agents of seed rescue and dispersal (Hämäläinen et al., 2017; Reiserer et al., 2018).

Among the terrestrial vertebrates, snakes are the least studied group with respect to seed rescue and secondary dispersal (Engel, 1997; Reiserer et al., 2018). This deficiency is somewhat perplexing and lacks a clear explanation given that they can be among the most abundant predators (high population densities and biomass per hectare) of seed-eating mammals (e.g., rodents) and birds in temperate and tropical regions (Klauber, 1972; Greene, 1997; Bonnet et al., 2002; King et al., 2018; Reiserer et al., 2018; Martins and Lillywhite, 2019; Henderson et al., 2021). And recent work on rattlesnakes indicates their potential importance as agents in diploendozoochorous systems (Reiserer et al., 2018). Specifically, in a museum study of 50 preserved rattlesnake specimens, nearly 1000 seeds were found to be indirectly ingested by way of consuming rodents possessing seed pouches, particularly heteromyids and geomyids. Careful examinations of entire digestive tracts revealed that not only were rodent-derived seeds abundant, but that numerous seeds germinated in the snakes’ colons (Reiserer et al., 2018).

In North America, geomyid, heteromyid and some sciurid rodents have specialized cheek pouches for transporting seeds from plant source to cache sites, where they are often eliminated from the pool of plant propagules by consumption (Price et al., 2000; Kaufman et al., 2004; Hope and Parmenter, 2007). However, in some cases, seeds stored in these caches will germinate after a rainy season. Distances for seed dispersal in scatter-hoarding mammals (Morris, 1962) vary greatly and depend on species, size of seeds, nutrition value, season, and a host of other factors. For example, kangaroo rats, species with cheek pouches (heteromyids), typically predate, and cache seeds close to their home burrow systems (Jones, 1989; Daly et al., 1992; see Price et al., 2000; Lichti et al., 2017; Wang and Cortlett, 2017). Lifetime dispersal distances in Merriam’s kangaroo rat (Dipodomys merriami), a common rodent in the Sonoran Desert, range from 0 to 265 m in males, and from 0 to 158 m in females (Jones, 1989).

Seed-laden rodents are commonly consumed by snakes as they forage, but unlike raptors, coyotes, bobcats, and other endothermic predators which eat rodents and are known (or implicated) to be secondary seed dispersers (Sarasola et al., 2016), the role of snakes in seed dispersal in nature remains unexplored (Reiserer et al., 2018). Nevertheless, desert-dwelling rattlesnakes and other vipers can be abundant (e.g., western diamond-backed rattlesnake, Crotalus atrox, more than 50 adults per km²), and individuals are capable of consuming 12–20 rodent meals and potentially hundreds of seeds (Vander Wall et al., 1998) during an active season lasting 25–30 weeks. Moreover, individuals occupy large home ranges in which they sometimes travel more than 2 km within several days (Beaupre, 2016; Schuett et al., 2016; DeSantis et al., 2020). Consequently, vast numbers of seeds may potentially achieve exceptionally long secondary dispersal distances, perhaps several times greater than those distributed by the rodents themselves (Nathan et al., 2008; Wang and Cortlett, 2017). Despite the importance of the findings by Reiserer and colleagues (Reiserer et al., 2018) and the potential of snakes as seed dispersers outlined above, the most crucial ecological and evolutionary corollaries of this system hinge on the survival of voided (excreted) seeds and viability of seedlings (Hämäläinen et al., 2017).

Here, using live snake subjects, we experimentally tested germination properties in seeds of the Foothill Palo Verde (Parkinsonia microphylla), an ecologically important and dominant tree of the Sonoran Desert (Bowers, 1994, 2004). We first tested whether wild-collected seeds of P. microphylla placed in the abdomen of thawed laboratory mice and ingested by viperid and colubrid snakes would germinate and produce viable seedlings. Specifically, in our primary study, we tested the Western-Diamond-backed Rattlesnake, Crotalus atrox (Figure 1), a pitviper (viperid) species, based on results of a previous study (Reiserer et al., 2018). In a secondary study, we tested two species of sympatric colubrid snakes in which seed rescue and consumption are unknown but predicted to be present based on their diets, the Desert Kingsnake, Lampropeltis splendida, and the Sonoran Gopher Snake, Pituophis catenifer affinis (Figure 2). Second, in the primary study only, we tested whether the seeds exposed to gut transit germinated at a greater rate and frequency than those of the controls, viz. the accelerated germination hypothesis (Castilla, 2000; Traveset et al., 2001; Hämäläinen et al., 2017; Hanish et al., 2020; Salazar-Rivera et al., 2020; Yang et al., 2021).

The most important outcome in all diploendozoochorous systems, from ecological and evolutionary perspectives, is that seeds survive all steps of the process—harvested or swallowed by a seed predator, swallowed whole or stored undamaged (e.g., cheek pouches and crop), swallowed undamaged by a secondary predator, and retention of germination viability after elimination from the gut of the secondary predator (Traveset et al., 2008, 2019; Hämäläinen et al., 2017). In a study involving preserved museum specimens of rattlesnakes (Reiserer et al., 2018), seeds found in the guts of snakes were derived from heteromyid and geomyid rodents, which were likely harvested and stored unharmed in their seed pouches. Uniquely, rattlesnakes and most other snake species subjugate and consume these rodents whole and do not chew (crush); consequently, most seeds transmitted through the GI tracts of snakes from these sources do not appear to be mechanically damaged. Furthermore, owing to long retention times in the digestive tracts of rattlesnakes, some seeds are capable of germination during gut transit (Reiserer et al., 2018; see Cabral et al., 2019; Carbajal-Márquez et al., 2020).

In support of our main hypothesis, we provide the first experimental evidence, to our knowledge, that seeds of the Foothill Palo Verde (P. microphylla) retain germination performance (e.g., viability) when indirectly consumed by snakes—one species of rattlesnake and two species of colubrids— and subsequently recovered from their excreta (Figures 1, 2 and Table 1). Importantly, seeds found to be imbibed and germinated in excreta (feces/urates) produced healthy seedlings (Figure 6). In the primary study, all germinated seeds subsequently tested for seedling viability grew normally and developed into healthy plants.

Under the conditions of the primary study, both aspects of our second hypothesis (accelerated germination) were not statistically supported. The mature seeds of P. microphylla (located in pods) are dry and have physical dormancy (“hardseededness”) resulting from densely packed layers of palisade cells impregnated with water-repellent substances which constrains imbibition of water (Baskin et al., 2000; Baskin and Baskin, 2014), a necessary step for germination. Thus, based on studies of other organisms (reviewed in Hämäläinen et al., 2017), we anticipated that gut-passage in the snakes would aid in chemical and mechanical scarification and accelerate germination frequency and rate. In other studies involving reptiles, the effects of gut passage on germination rate and frequency are mixed; in some cases both germination rate and frequency are accelerated (e.g., Yang et al., 2021), yet in others neither of these effects are observed (e.g., Castilla, 2000). Clearly, for reptiles, studies of diploendozoochory are in their infancy and it would be premature to make hard conclusions at this time based on the little available information (Hämäläinen et al., 2017; Reiserer et al., 2018). Nonetheless, the importance of positive effects of gut passage, such as described in other studies (Hämäläinen et al., 2017), cannot be overlooked and should encourage further research in this understudied area of ecology.

We provide several possible explanations for our results with respect to the second hypothesis tested in the primary study. Foothill Palo Verde (P. microphylla) is a species in which the seed crop is comprised of both non-dormant and dormant seeds and thus diversified germination behavior is exhibited (Bowers and Turner, 2002; Bowers, 2004). Consequently, a variable proportion (e.g., 20 to 34%) of them can germinate shortly after dispersal from their pods and, importantly, without scarification (Bowers, 2004). The dormant seeds can persist into a second season (or even longer) and require exposure to environmental scarification (high heat, rain, wind, and abrasion) for germination to occur (Bowers and Turner, 2002; Bowers, 2004). Second, in germination studies of P. microphylla, we suspect capturing subtle differences in germination rate and frequency will likely require a much larger sample size of seeds to achieve statistical significance for a potentially small effect. Perhaps a 10-fold increase of seeds (e.g., 1000) would be required to overcome the abovementioned problems associated with dormant and non-dormant seed types (see Figure 5). Finally, because wet heat appears to be an important release of dormancy in a closely related species of tree from Mexico (Parkinsonia aculeate), and thus likely others (Van Klinken et al., 2006), subjecting P. microphylla seeds to warmer conditions (e.g., 38–45°C) during germination tests in future studies is warranted.

Similar to the results of the primary study involving Western Diamond-backed Rattlesnakes (C. atrox) mice impregnated with seeds of P. microphylla were consumed by the Sonoran Gophersnake (P. catenifer) and Desert Kingsnake (L. splendida). Some of those seeds that transited the digestive tracts retained viability and germinated (see Supplementary Figure 3). Nonetheless, when compared to the primary study, germination frequency was significantly greater in the primary study (z-test, two-tailed: z = 7.140, p < 0.001). This result was likely due to greater trial duration in the primary study (trials were run for 128 days) and germination conditions were likely superior (e.g., less moisture, warmer, and better air circulation) for P. microphylla. Mold was commonly associated with seeds kept in their feces in trials 1 and 2 of the secondary study. However, we suspect that the highly moist conditions and lack of air flow in petri dishes contributed to this issue. In the Sonoran Desert, similar condition would rarely be encountered. Clearly, in the secondary study, better results were obtained when seeds were removed from the feces and urates. Germination conditions that more closely mimic natural situations (e.g., drier and warmer) are warranted in future studies, which we envision to include performing tests under a range of natural conditions (e.g., outdoor plots).

We provide herein support for the role of viperid and colubrid snakes as important agents of seed rescue and dispersal in nature, highlight their importance as potential ecosystem engineers, and offer sound justification for future field-based studies (Reiserer et al., 2018). Clearly, more research is needed, especially studies that are field-based, to provide context and to better understand the ecological and evolutionary ramifications of this fascinating, yet mostly understudied, diploendozoochorous system (Franklin et al., 2016). Although our current focus has been on organisms of North American deserts, clearly other regions (e.g., tropics) should be explored (Reiserer et al., 2018; Dugger et al., 2019). Furthermore, other plant and snake species would be desirable to study purely for inclusion of greater biodiversity and exploration of potential variation (Hämäläinen et al., 2017).

Topics closely related to seed dispersal and their fates will need to be investigated in field-based studies of diploendozoochory in snakes. For example, despite an abundance of research on defecation sites of lizards, the closest extant relatives of snakes, similar detailed studies are nonexistent, to the best of our knowledge, for wild snakes. Unquestionably, the fate of eliminated seeds in instances of diploendozoochory by snakes and other vertebrates may be further influenced, both positively and negatively, by abiotic (e.g., rain and temperature) and biotic factors (Vander Wall and Longland, 2004). Invertebrates, for instance, such as ants (Pascov et al., 2015; Luna et al., 2018; Anjos et al., 2020) and dung beetles (D’hondt et al., 2007; Midgley et al., 2015), are common vectors in several ecosystems that disperse and alter ground-based seeds both mechanically and chemically (Franklin et al., 2016). Given that only anecdotal data are available, the ecology of defecation sites in snakes and the fate of seeds present in their feces remain open research questions. Indeed, as stated by Anni Hämäläinen and her colleagues (Hämäläinen et al., 2017, p. 13), “It is currently unknown how important the phenomenon (of diploendozoochory) is ecologically, but given its potentially vast prevalence and the possible implications, it is possible that ignoring it could impair the interpretation of broad ecological patterns or hinder conservation efforts.” Importantly, it is incumbent upon us to comment that many of the vipers and other snake species that are candidate ecosystem engineers are themselves endangered for various reasons, including from direct persecution by humans (Maritz et al., 2016; Birskis-Barros et al., 2019; Fathinia et al., 2020). It is hoped that highlighting their potential new role as agents of seed rescue and secondary dispersal for deserts and other ecosystems will encourage both academic and public involvement (e.g., citizen scientists) in generating interest and legislature for their protection and long-term conservation.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Dryad Digital Repository: https://doi.org/10.5061/dryad.65gr2.

Ethical review and approval was not required for the animal study because our study involved collecting seeds from the fecal wastes of museum and zoo snakes. No animals were harmed in this study. Written informed consent was obtained from the owners for the participation of their animals in this study.

GS and RR conceived the study. GS, AS, WH, SB, JH, RZ, and CF collected the data. RR, GS, AS, and MD analyzed data. GS, RR, AS, WH, MD, and HG wrote the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.