An illustrated guide to seeds found in nests of the Florida harvester ant, Pogonomyrmex badius

The Florida harvester ant, Pogonomyrmex badius collects the seeds of many plant species and stores them in underground nest chambers for later consumption. Seeds taken from multiple nests in 1989, 2014 and 2015 were separated by size and species and identified through published keys, comparison with herbarium specimens and with identified seed collections. Harvester ants stored at least 58 species of seeds from 20 plant families in their chambers. This paper presents images of each seed species in several aspects, their relative abundance in P. badius nests, their size relative to the smallest, and links to online data and images of the parent plant species, as well as to herbarium specimens. A number of seeds and plant families present at the site were not found in ant nests. These data and images will be valuable for future studies and experiments to untangle the choices the ants make in relation to what the plants and the seasons offer them.


Introduction
The ability to collect, store and consume seeds has evolved independently more than 18 times among the genera of ants [1,2]. Seed harvesting ants are often associated with arid or semi-arid habitats. Most of the 29 North American species of Pogonomyrmex harvest and consume seeds [2], some to such an extent that it was claimed that they compete with seed-eating mammals [3], although later work cast doubt on this claim (reviewed in [2]). Usually, ants collect multiple seed species, ranging from a few to about 45 [4][5][6]. For most harvesting ant species, seeds form only part of their diet, the remainder consisting of insects, fungus, plant material etc., but a few feed almost exclusively on seeds [1,[7][8][9][10]. Seed preferences have been reported to be correlated to body size, abundance, nutritional content, toxicity and novelty [11][12][13][14][15][16].
Most harvesting ants store the seeds in underground chambers. For P. badius, these chambers are located between about 40 and 100 cm below ground, and may contain (in the aggregate) up to 300,000 seeds weighing half a kg [17,18]. Seed collection is usually seasonal [9,19,20], and while it has been suggested that some species may overwinter without seeds [7], this claim is contradicted by Lavigne [5]. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Although early observers sometimes noted germinating seeds in the nests of harvesting ants, the possibility that the ants might actually require some seeds to germinate in order to be able to eat them was never carefully investigated until the recent work of Tschinkel and Kwapich [21]. Through experiments and observations, Tschinkel and Kwapich [21] showed that P. badius workers cannot open seeds larger than 1 to 1.4 mm wide, that they readily use germinated seeds, feeding these to larvae, that seeds actually germinate within the subterranean storage chamber are rapidly removed by workers to be fed to larvae, and that seed germination rates are related to the seed species, the season and temperature.
This paper is a companion paper to that of Tschinkel and Kwapich [21] for the purpose of providing identifications and images of the 58 species of seeds taken from P. badius nests. In addition, we also provide identifications and images of some other seeds found at the study site, Ant Heaven, but not within P. badius nests.

Study site
The study population of Florida harvester ant, P. badius, is located in a 23 ha site (latitude 30.3587, longitude -84.4177) about 16 km southwest of Tallahassee, Florida, USA, within the sandhills ecotype of the Apalachicola National Forest. The site, Ant Heaven, consists of excessively drained sandy soil occupying a slope to a wetland and stream, causing its water table to be depressed (>5 m at the maximum), thereby making it suitable for P. badius and Solenopsis geminata, as well as several drought-resistant species in the genera Opuntia and Nolina. The forest consists of longleaf pines (Pinus palustris) planted ca. 1975, turkey oak (Quercus laevis), bluejack oak (Quercus incana), occasional sand pines (Pinus clausa) and sand live oak (Quercus geminata). Because the soil had been disturbed in the early 1970s, the natural ground cover of wiregrass (Aristada stricta) is absent, replaced by broomsedge (Andropogon spp.) and several other successional species of grasses, herbs and shrubs. The same disturbance may have helped establish this dense population of P. badius, whose nests are easily spotted because the ants decorate the excavated soil disc with a layer of charcoal bits (mostly the ends of burned pine needles). The black charcoal contrasts sharply with the light-colored sand or litter.
This project was carried out under US Forest Service, Apalachicola National Forest permit number APA56302, Expiration Date: 12/31/2017. Pogonomyrmex badius is not a protected species.

Seed collection and preparation
Colonies of P. badius were excavated by digging a pit next to the nest and exposing the horizontal chambers one by one by lifting off the soil with a large trowel, whereupon seeds, ants and other contents were collected [17,18]. Seeds were mostly found in dedicated chambers between about 30 and 100 cm below ground. The seeds used in this study were collected from 31 nests excavated in 1989 by WRT, nine in 2014 and eleven in 2015. All seeds were stored dry in the laboratory.
Seeds were separated into size classes with U.S. Standard Testing sieves No. 8-35, and the proportion of the total weight in each size class was computed. The more common seed species of all size classes were separated for determination of their mean weights. Four size classes of the 2014 seeds were used in a series of experiments on the consumption of germinating and non-germinating seeds. The results of these studies can be found in Tschinkel and Kwapich [21].

Seed identification and imaging
Several inputs helped identify seeds. Two illustrated manuals with keys for identifying seeds important as food for wild quail [22,23] were primary sources. Initial identifications were checked by comparing with seeds taken from identified herbarium specimens in the R.K. Godfrey Herbarium at Florida State University. Some were also compared with seeds in the seed collection of the University of Florida Herbarium in Gainesville, Florida. In addition, seedbearing plants were collected at the study site, identified in the Godfrey Herbarium, and their seeds compared with unknowns from ant nests. In some cases, online images were helpful. Finally, we planted some seeds in order to grow them to an identifiable size. Through these multiple inputs, we were able to identify 48 of the 58 seeds in P. badius nests. Some seeds remain unidentified, though some of these can be assigned to family.
Seeds were placed in several aspects onto a glass plate above a neutral background and photographed with a DinoCapture 2.0 digital microscope. The microscope added a scale to each image, and these are included on all figures.

Results
Of the 58 types of seeds that were readily separated in the 1989 sample, we identified 48 with confidence. Ten species remain unidentified, but none occurred at greater than 0.1% of the total. All seeds are listed in order of their 1989 frequency in P. badius nests in Table 1, along with their proportion of the total number of seeds in the 1989 sample, and a hyperlink to the image of each seed in this paper. A second column reports the frequency of the seeds in the 2014-2015 sample. The table also presents the weight of each seed relative to the smallest seeds found in P. badius nests. Figs 1 to 58 present images of the seeds from P. badius nests in several aspects, along with two links to online images of the parent plant, one to the Atlas of Florida Plants (AOFP) and one to the Florida State Herbarium specimen images (FSU Herbarium). Figs 1-58 are in alphabetical order. For visual ease, Table 2 presents the seed names in alphabetical order. Table 1. Seed species found in the nests of the Florida harvester ant, Pogonomyrmex badius. Each species is linked to its image below. Seeds were collected from 31 nests in 1989 and 9 in 2014. The frequency of each is shown in the columns as percent of total number of seeds. Each seed image has a link to return to this table, a link to the Atlas of Florida Plants at the University of South Florida (AOFP) and a link to the images of the plant specimens in the R.K. Godfrey Herbarium at Florida State University (FSU herbarium).   Table 1, and are shown in multiple aspects with a scale. Relative weight is also presented for many seeds in Table 1. Table 3 lists species of seeds found at the study site, Ant Heaven, but not found in P. badius nests. Images for these seeds can be found in alphabetical order in Figs 59-78. Whether this absence resulted from choices made by ant foragers, seed abundance, season of seed ripening or some other chance factor is unknown. We have made no attempts to estimate the relative availability of ant and non-ant seeds in the landscape (a difficult task, without doubt).  Table 3. Seeds found at the study site, Ant Heaven, but not in the nests of harvester ants. Each species name is linked to its image below. These species are only a small sample of those found at Ant Heaven.  Figs 59-78. Seeds not found in harvester ant nests. Field seeds were collected at the study site, Ant Heaven. Herbarium seeds were taken from dried herbarium specimens of plants occurring at Ant Heaven. (R.K. Godfrey Herbarium at Florida State University).

Seeds present in field but not found/confirmed in nests
The number of species per plant family ranged from ten (Fabaceae) to one (10 families) ( Table 4). Ten families were represented by two to five species, and another ten by a single species. Some of the singleton families are either rare at the site, or represented by only one or two species (e.g. Cactaceae, Smilacaceae), but others are common and/or speciose at the site, yet infrequent or absent in the nest stores. For example, the Asteraceae are represented by a substantial number of species, some fairly common, yet seeds of Asteraceae are uncommon in the seed stores. On the other hand, Ilex glabra (gallberry) and Gaylussacia dumosa (shiny blueberry) are common shrubs at the site, but their seeds are infrequent in P. badius nests. Table 4. Species distribution among families. Seed species represented 20 plant families, with the most species from the Fabaceae, followed by Poaceae. Ten families were represented by more than one species, and 12 by a single species each.

Discussion
The seeds of more than a dozen plant species occur frequently in the nests of the Florida harvester ant, with another almost four dozen occurring occasionally to rarely. The 58 species we found in nests represent an important food resource for the ants, but the presence at Ant Heaven of seed species that were not found in P. badius nests suggests some degree of either selectivity by foragers or availability in the habitat. Whereas cafeteria experiments with Ant Heaven seeds might reveal forager preferences among seed species, it is difficult to test if the relative abundance in nest chambers reflects availability, for to do so, one must see seeds through the eyes of foragers, not humans. What may seem available to a human may or may not be available to an ant and vice versa. The 58 species found in nests belonged to 20 plant families, several represented by multiple species, but an additional seven families were found at the site but not in nests. Because our cataloguing was opportunistic, these seven families represent only a fraction of the plant families occurring at the site. In contrast, the 20 families represented in the nest stores are probably a large fraction of the total that an enlarged search would reveal. This uneven representation of seeds from a range of plant families poses questions about the suitability of seeds as ant food, and subsequently, whether the ants have evolved a preference for some species and an avoidance of others. Considering the importance of germination for gaining access to seeds as food, the phenology of production and germination may be important. It is particularly striking that ten species of Fabaceae and five Poeaceae were found in ant nests, with only two and zero (respectively) found at the site but not in nests. On the other hand, only a single species of Asteraceae occurred in nests, but six were present at the site but not in nests. Also striking is that although both gallberry (Ilex glabra) and shiny blueberry (Gaylussacia dumosa) were extremely abundant at the site, they were infrequent to rare in the nest stores. The tiny seeds of the very abundant dog fennel (Eupatorium capillifolium) were completely absent from nest stores, as were those of beardgrass (Andropogon sp.).
A few species of seeds were obviously not within the foraging range of the P. badius nests at Ant Heaven. Neither of the species of Magnolia grew there, nor did Nyssa sylvatica, Phytolacca americana or Ilex myrtifolia. A possible source of importation might be animal feces, but this remains to be evaluated.
This study was carried out on one population at one site. Vegetation, and therefore seed inventory, almost surely differs among sites. To the degree to which seed stores show or do not show consistency across sites, site differences could illuminate the question of availability vs. preference. No doubt, other researchers will add to the list of seeds stored in P. badius nests, along with geographic and ecological information. The high quality, multiple-aspect images we present here will make future identification of seeds from harvester ant nests much easier, and may stimulate more studies.
Because the seeds are ant food, their size and nutritional quality are important attributes. We lack information on quality, but Fig 79 dramatizes the nutritional, caloric benefit of exploiting larger seeds. Relative to the smallest seeds found in P. badius nests, larger seeds provide up to 90-fold more food. However, the ants cannot open seeds larger than about 1 to 1.4 mm across [21]. Tschinkel and Kwapich [21] showed that large seeds are used once they germinate in the nest, and the ants benefit greatly from this strategy. For example, a germinating Smilax auriculata seed (which germinate fairly regularly) would yield 70 times as much food as an ant-opened Polygonella gracilis seed. In view of the benefits of this strategy, it seems inevitable that it would evolve.
P. badius is only one of many ant species that harvest seeds and store them for later consumption. Several species of ants collect multiple species of seeds, but it remains to be determined whether other species also exploit seed germination to increase the range of seed sizes and species they are able to consume.  [21]. The seed list is not complete, but is representative of the total. Seed silhouette positions on the abscissa are in approximate order of their relative weights. The number in each seed silhouette is the U.S. Standard testing sieve on which the seed was retained. Ant images modified from antweb.org. Photos by April Nobile: minor worker:URL: https://www.antweb.org/bigPicture.do?name=casent0104423&shot=p&number=1. Major worker: https://www.antweb.org/ bigPicture.do?name=casent0103057&shot=p&number=1.