Received: 16 April 2019 | Revised: 7 October 2019 | Accepted: 7 January 2020 DOI: 10.1111/btp.12772

ORIGINAL ARTICLE

The arboreal of a Neotropical rain forest show high species density and comprise one third of the fauna

John T. Longino1 | Robert K. Colwell2,3

1Department of Biology, The University of Utah, Salt Lake City, UT, USA Abstract 2Department of Ecology and Evolutionary In tropical rain forests, the ant community can be divided into ground and arboreal Biology, University of Connecticut, Storrs, faunas. Here, we report a thorough sampling of the arboreal ant fauna of La Selva CT, USA 3University of Colorado Museum of Natural Biological Station, a Neotropical rain forest site. Forty-five canopy fogging samples History, Boulder, CO, USA were centered around large trees. Individual samples harbored an average of 35 ant

Correspondence species, with up to 55 species in a single sample. The fogging samples yielded 163 John T. Longino, Department of Biology, observed species total, out of a statistically estimated 199 species. We found no rela- The University of Utah, Salt Lake City, UT 84112, USA. tionship between within-sample ant richness and focal tree species, nor were the ant Email: [email protected] faunas of nearby trees more similar to each other than the faunas of widely spaced

Funding information trees. Species density was high, and beta diversity was low: A single column of veg- National Science Foundation, Grant/Award etation typically harbors at least a fifth of the entire arboreal ant fauna. Considering Number: DEB-1354739 the entire fauna, based on 23,326 species occurrence records using a wide variety Associate Editor: Emilio Bruna of collecting methods, 182 of 539 observed species (196 of 605, estimated statisti- Handling Editor: Emilio Bruna cally) were entirely arboreal. The arboreal ant fauna is thus about a third of the total La Selva ant fauna, a robust result because inventory completeness was similar for ground and arboreal ants. The taxonomic history of discovery of the species that make up the La Selva fauna reveals no disproportionately large pool of undiscovered ant species in the canopy. The "last biotic frontier" for tropical ants has been the rot- ten wood, leaf litter, and soil of the forest floor. Abstract in Spanish is available with online material.

KEYWORDS , canopy, Costa Rica, fogging, formicidae

1 | INTRODUCTION the ground and canopy communities overlap very little (Longino & Colwell, 1997; Ryder Wilkie, Mertl, & Traniello, 2010; Yanoviak &

Tropical rain forests are renowned for their complex structure and Kaspari, 2000). One set is entirely ground-based, nesting in the soil, high diversity. Ants are a dominant biotic element in rain forests leaf litter, or rotten wood of the shaded forest floor and foraging (Davidson, Cook, Snelling, & Chua, 2003; Fittkau & Klinge, 1973; there or within a few meters of the ground. Another set is arboreal, Floren, Biun, & Linsenmair, 2002; Stork, 1988), and a growing body nesting and foraging mostly or entirely in the vegetation. Only a few of research documents the abundance, diversity, behavior, and hab- species routinely occur in both habitats. itat specificity of rain forest ants. Ant faunas are vertically strati- Arboreality is a derived trait in ants that has evolved repeatedly fied with respect to their nesting and foraging preferences, and in many ant lineages (Dejean, Corbara, Orivel, & Leponce, 2007;

© 2020 The Association for Tropical Biology and Conservation

 wileyonlinelibrary.com/journal/btp | 1 Biotropica. 2020;00:1–11. 2 | longino and colwell

Nelson, Ree, & Moreau, 2018). A few of the arboreal lineages assessment of the arboreal fauna based on all collecting methods. have radiated spectacularly, forming the bulk of the abundance We use these results to estimate the total richness of arboreal ants and diversity of arboreal ants. The development of arboreal ant and to compare the richness of arboreal ants and ground ants. We communities is primarily a tropical phenomenon; the abundance of test for an effect of focal tree species on sample richness and species arboreal ants is orders of magnitude greater in the tropics than in composition, and we test for an effect of spatial proximity on species the temperate zone (Floren, Wetzel, & Staab, 2014). Tropical biolo- composition. The of the arboreal ant species is carefully gists have focused their research on assessing (a) the total richness considered, and species are linked to specimens with unique spec- of arboreal ants (LePonce et al., 2019; Majer, 1990; Wilson, 1987; imen identifiers in museum collections, insuring that the study can Yanoviak, Fisher, & Alonso, 2007); (b) how arboreal ant richness serve as a reference benchmark for future work. compares to richness of ground ants (Floren et al., 2014; Ryder Wilkie et al., 2010; Yanoviak & Kaspari, 2000); (c) whether arbo- real ants form "mosaics" of dominant and subdominant species 2 | METHODS (Dejean et al., 2016, 2019, 2007, 2018, 2015; Floren & Linsenmair, 2000; Jackson, 1984; LePonce et al., 2019; Leston, 1978; Majer & 2.1 | Study site and fogging methods Camer-Pesci, 1991; Room, 1971); and (d) whether ant communities are affected by habitat, tree species, tree size, or connectivity via The study was carried out at La Selva Biological Station, Heredia lianas (Adams, Schnitzer, & Yanoviak, 2017; Floren & Linsenmair, Province, Costa Rica (McDade, Bawa, Hespenheide, & Hartshorn, 1997, 2005; Klimes, Fibich, Idigel, & Rimandai, 2015; Klimes et al., 1993), from 1993 to 2000. Mean annual rainfall is approximately 2012; Osorio-Pérez, Barberena-Arias, & Aide, 2007; Philip, Fayle, 4 m. The habitat is a mosaic of mature lowland rain forest, second & Yusah, 2018; Ribas, Schoereder, Pic, & Soares, 2003; Schonberg, growth forest of various ages, and abandoned pastures. The study Longino, Nadkarni, Yanoviak, & Gering, 2004; Schulz & Wagner, area comprised approximately 1,500 ha, ranging from 50 m to 150 m 2002). in elevation. La Selva Biological Station in Costa Rica, one of the best-stud- Canopy fogging was carried out using the general procedures ied tropical rain forests, has been particularly well sampled for discussed by Erwin (1983), Adis, Lubin, and Montgomery (1984), and ants (Longino & Colwell, 1997; Longino, Colwell, & Coddington, Stork (1988). A total of 45 fogging samples were acquired (Figure 1, 2002). Ant sampling at La Selva was carried out over a period of Table 1). For 11 samples, the focal tree was Pentaclethra macroloba 13 years, with a combination of canopy fogging, Malaise traps, light (Willd.) O. Ktze. (Fabaceae), the most common tree species at La traps, Berlese samples, Winkler samples of sifted litter, baiting, and Selva. For 12 samples, the focal tree was Virola koschnyi Warb. hand-collecting. Some of the data have been previously published (Myristicaceae), a species of intermediate abundance. The remaining in the context of inventory efficiency (Longino & Colwell, 1997) and 22 samples targeted focal trees from a wide range of species and richness estimation of the entire ant fauna (Longino et al., 2002). The plant families. A stratified sampling design was used for the first 18 previous publications used only a portion of the fogging data. Here, fogging samples, with trees fogged in six sets of three closely spaced we present an analysis of the entire fogging dataset, along with an individuals (Figure 1). In each group of three samples, one tree was

FIGURE 1 Spatial distribution of fogging samples at La Selva Biological Station, Costa Rica. The first 18 samples are numbered, showing the triplet structure of the sampling. Latitudes and longitudes of each sample are in Table 1. The grid cells are 1 km2 longino and colwell | 3

67.38 39.31 65.04 18.00 50.66 28.99 50.05 28.52 54.58 54.55 36.24 38.10 46.09 68.42 46.75 44.31 48.14 iChao2 (Continues) 57.88 37.97 14.88 24.66 24.68 45.71 52.72 42.86 25.73 28.07 54.39 34.30 63.44 43.50 43.56 34.78 46.51 64.60 40 S 8 obs 51 17 17 19 19 49 28 28 27 21 32 21 26 28 26 26 15 28 28 50 33 20 35 35 13 13 18 18 28 28 35 35 33 30 48 32 32 36 36 S 34 34 44 34 34 34 34 1 Processing Processing method 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 17 11 11 11 10 10 10 10 10 10 10 10 10 10 10 10 20 40 Funnels Funnels processed 40 40 40 40 40 40 40 40 40 40 40 40 40 40 (Euphorbiaceae) (Fabaceae) (Fabaceae) (Fabaceae) (Fabaceae) (Fabaceae) (Fabaceae) (Fabaceae) (Fabaceae) (Fabaceae) (Fabaceae) (Araliaceae) (Humiriaceae) (Sapotaceae) (Tiliaceae) (Anacardiaceae) (Meliaceae) (Burseraceae) (Myristicaceae) (Myristicaceae) (Myristicaceae) (Myristicaceae) (Myristicaceae) (Myristicaceae) (Myristicaceae) (Myristicaceae) (Myristicaceae) (Myristicaceae) (Myristicaceae) (Aquifoliaceae) sp-d (Hernandiaceae) sp-d Virola koschnyi Species Carapa guianensis Carapa Pentaclethra macroloba Pentaclethra Conceveiba pleiostemona Conceveiba Pentaclethra macroloba Pentaclethra Virola koschnyi Goethalsia meiantha Pentaclethra macroloba Pentaclethra Virola koschnyi Tapirira guianensis Tapirira Pentaclethra macroloba Pentaclethra Virola koschnyi macroloba Pentaclethra Sacoglottis trichogyna Virola koschnyi Vitex cooperi (Verbenaceae) Virola koschnyi Pentaclethra macroloba Pentaclethra Virola koschnyi Ilex skutchii Ilex Hernandia Pentaclethra macroloba Pentaclethra Virola koschnyi Pentaclethra macroloba Pentaclethra Pouteria standleyana Pouteria Virola koschnyi Pentaclethra macroloba Pentaclethra Virola koschnyi Protium glabrum Protium Virola koschnyi Pentaclethra macroloba Pentaclethra Dendropanax arboreus Dendropanax −84.00894 Longitude −84.0127 −84.01195 −84.00395 −84.00379 −84.00432 −84.00792 −84.00746 −84.0073 −84.0133 −84.00488 −84.01268 −84.01129 −84.00512 −84.0048 −84.01293 −84.01245 −84.01283 −84.01137 −84.01087 −84.00937 −84.00968 −84.00939 −84.00841 −84.00793 −84.00844 −84.0144 −84.01444 −84.01378 −84.01042 −84.00973 −84.00946 10.43278 Latitude 10.43486 10.43454 10.41274 10.41317 10.41406 10.42411 10.42461 10.42414 10.42582 10.42543 10.42663 10.42685 10.42519 10.42487 10.43012 10.43076 10.4315 10.4203 10.4214 10.42268 10.42302 10.42349 10.42604 10.42662 10.42696 10.43156 10.43221 10.43204 10.43137 10.43177 10.43214 14-Jan−93 Date 5-Mar−93 6-Mar−93 5-May−93 6-May−93 7-May−93 3-Jul−93 4-Jul−93 5-Jul−93 3-Sep−93 6-Nov−93 4-Sep−93 5-Sep−93 8-Nov−93 9-Nov−93 5-Jan−94 7-Jan−94 8-Jan−94 8-Oct−94 10-Oct−94 13-Oct−94 14-Oct−94 15-Oct−94 18-Oct−94 19-Oct−94 20-Oct−94 2-Nov−94 3-Nov−94 4-Nov−94 10-Nov−94 11-Nov−94 12-Nov−94 VK/24 VK/27 VK/32 VK/34 PM/23 OT/21 OT/22 PM/25 PM/31 OT/26 PM/35 OT/33 OT/36 FVK/01 Sample code Sample FOT/02 FPM/03 FOT/04 FPM/05 FVK/06 FOT/07 FPM/08 FVK/09 FOT/10 FPM/13 FVK/11 FPM/12 FOT/14 FVK/15 FOT/16 FVK/17 FPM/18 FVK/19 F F F F F F F F F F F F F TABLE 1 FoggingTABLE samples. See text for explanation of processing methods 4 | longino and colwell

). 47.53 27.62 74.89 52.63 56.28 38.18 64.64 48.20 iChao2 104.53 40 S obs 19 19 55 55 27 27 28 28 15 15 39 39 32 32 39 39 33 33 38 38 30 30 34 34 30 30 S 2 Processing Processing method 2 2 2 3 3 3 3 3 3 3 3 3 40 Funnels Funnels processed 40 40 40 40 40 40 40 40 40 40 40 40 extrapolated to 40 funnels using iNEXT if fewer than 40 funnels processed; see Methods est (Fabaceae) S (Fabaceae) (Olacaceae)

= (Tiliaceae) (Tiliaceae) (Tiliaceae)

(Sabiaceae) 40 S (Fabaceae) (Cecropiaceae) (Myristicaceae) ? (Meliaceae) ? sp (Myrtaceae) sp (Meliaceae) sp Virola koschnyi Species Guarea guara Pentaclethra macroloba Pentaclethra Guarea Tachigali costaricensis Tachigali Inga leiocalycina Inga Minquartia guianensis Meliosma vernicosa Meliosma Pouruma minor Pouruma Eugenia Goethalsia meiantha Goethalsia meiantha Goethalsia meiantha −84.01352 Longitude −84.01226 −84.01153 n.d. −84.01281 −84.01227 −84.01245 −84.01222 −84.01204 −84.01221 −84.01484 −84.01439 −84.01415 : observed or estimated richness in 40 funnels ( 40 10.43541 Latitude 10.43577 10.43606 n.d. 10.41284 10.41338 10.41365 10.41445 10.41472 10.41479 10.43252 10.43301 10.43313 15-Nov−94 Date 16-Nov−94 17-Nov−94 13-Jan−96 28-Dec−99 29-Dec−99 30-Dec−99 3-Jan−00 4-Jan−00 5-Jan−00 9-May−00 10-May−00 11-May−00 : observed species richness in sample. S obs : improved Chao2 lower-bound asymptotic richness estimator (Chiu et al., 2014). PM/39 OT/38 OT/40 OT/41 FVK/37 Sample code Sample F F F FOT/42 F FOT/43 FOT/44 FOT/45 FOT/46 FOT/47 FOT/48 FOT/49 iChao2 TABLE 1 (Continued) TABLE Note: S longino and colwell | 5

Pentaclethra macroloba, one was Virola koschnyi, and one was a third separated all ants from other specimens in the catch species, different in each triplet. from each funnel and placed them in a separate vial for each fun- We chose focal trees that had large crowns (extending beyond nel; Longino identified the ants in each vial, resulting in a species the funnels below), little overlap with adjacent crowns, and good list for each funnel. access for climbing. On the day prior to fogging a tree, the tree was For each species encountered in the study, at least one specimen rigged with mountain-climbing ropes and a pulley system, so that was mounted, labeled, tagged with a unique specimen code, and the following day an operator could climb to the lower branches databased. All occurrence records for the fogging study, and per- of the crown and the fogging machine could be hoisted on pulleys. manent, unique specimen codes for mounted specimens, are openly Ropes were strung from trunk to trunk between the focal tree available in Dryad Digital Repository at https​://doi.org/10.5061/ and neighboring trees to form an irregular network, 2–3 m above dryad.ncjsx​ksr0. Data and current identifications for the voucher ground level. Forty 1-m2 funnels were suspended from these ropes, specimens are deposited in the curated online repository AntWeb distributed as evenly as possible in the area beneath the crown of (www.antweb.org). Future name and classification changes can thus the focal tree, and within 10 m of the focal tree trunk (Figure 2). be tracked with the specimen codes. The funnels were constructed of ripstop nylon mounted on a cir- Among the species encountered in the fogging samples, 26 were cular metal hoop, with a threaded ring at the bottom for the at- excluded as "tourists" (Longino et al., 2002). Two of these species tachment of a plastic sample-collection bottle. Palm leaves and were laboratory pests assumed to be contaminants. The others were other understory vegetation immediately above each funnel were very rare in the fogging samples but known from other records and clipped or bent back out of the way, but otherwise the understory observations to be exclusively ground-nesting and ground-foraging; vegetation was left intact. it is possible that they were clinging to the funnels when they were Before dawn on the day of fogging, the funnels were suspended lifted from the ground. and the bottles filled with 70% ethanol. An operator climbed to the first branches at the base of the crown, 15 m to 20 m above ground level, and commenced fogging at about 0600 hrs. We 2.3 | Richness used a Golden Eagle DynaFogger®, on setting 6, to fog 3.8 L of Pyrethrins 123® insecticide (Summit Chemical Co.). This is a When fewer than 40 funnels were processed from a fogging event, 3% solution of a natural pyrethrin insecticide with synergists, in the expected sample richness for 40 funnels was estimated by ex- a petroleum distillate carrier. The operator gradually fogged in a trapolating to 40 funnels following models developed by Colwell et 360˚circle, attempting to cover the crown evenly. Following fog- al. (2012) and Chao et al. (2014) and implemented in the R pack- ging, a two-hour drop time was allowed, after which the sides of age iNEXT (Hsieh, Ma, & Chao, 2016). When funnels were individu- the funnels were washed down with ethanol into the collecting ally processed, yielding incidence frequencies within samples, the bottles, to capture any adhering to the funnels, and the iChao2 lower-bound asymptotic richness estimate (Chiu, Wang, bottles were capped and collected. Walther, & Chao, 2014) was calculated, as implemented in SpadeR (Chao, Ma, Hsieh, & Chiu, 2015).

2.2 | Sample processing 2.4 | Tree species effect Different sample processing methods were used during successive phases of a larger arthropod inventory project (Project ALAS, the To test for an effect of focal tree species on sample richness (ob- Arthropods of La Selva, Longino & Colwell, 1997), of which the served and estimated), samples were grouped into three categories: fogging samples were a part. Three methods were used (Table 1). Virola koschnyi (n = 12), Pentaclethra macroloba (n = 11), and "other" Method 1: For 18 samples, project staff exhaustively selected (a diverse assemblage of species from multiple families, n = 22; examples of each putative morphospecies from each funnel for see Table 1). One-way ANOVA was used to examine differences dry-mounting; Longino identified the dry-mounted specimens, re- in mean sample richness. Differences in community composition sulting in a species list for each processed funnel; a variable num- were tested with PERMANOVA using the R package Vegan 2.5–4 ber of funnels per sample were processed, typically 10 of the 40 (Oksanen et al., 2019). The data matrix consisted of species pres- funnels; when fewer than 40 funnels were processed, total sam- ence/absence (incidence) values within samples and the Jaccard ple richness was estimated (see below). Method 2: For 18 samples, distance metric. We also analyzed a subset of the data using just specimens were pooled among all 40 funnels; project staff se- the first 18 samples, which were distributed in closely spaced tri- lected putative morphospecies from the pooled sample and placed plets and processed with Method 1. For samples with more than them in a single vial; Longino identified the selected specimens 10 processed funnels, we randomly selected 10. We thus gener- in ethanol, mounting problematic specimens, resulting in a spe- ated a replicated incidence matrix (sample × species) and enumer- cies list for each sample. Method 3: For 9 samples, project staff ated the incidence frequency—the number of funnels in which a 6 | longino and colwell

FIGURE 2 Typical layout of funnels for a fogging sample. Clockwise from upper left: funnels suspended from network of ropes; removing sample bottle from single funnel (left to right Ronald Vargas and Danilo Brenes); view from above of suspended funnels. Mouth of each funnel is 1 m2

species occurred, a value from 1 to 10 (Colwell et al., 2012). We 3 | RESULTS again tested for a tree species effect, treating these frequencies as indicators of abundance, and also tested for a "triplet" effect, using 3.1 | Richness PERMANOVA. For these analyses, with abundance data, we used the Bray–Curtis distance metric. Overall, considering both observed values in 40 funnels and values extrapolated to 40 funnels, mean sample richness was 34.5 species

(S40 in Table 1, 95% C.I. 31–38, n = 45). The mean iChao2 estimate 2.5 | Proportion of total ant fauna of sample richness was 49.05 species (iChao2 in Table 1, 95% C.I. 44–54, n = 27). Processing method had a significant effect on sam-

To place the fogging results in the context of the entire La Selva ant ple richness (1-way ANOVA, F1,42 = 7.524, p < .01), with Method 1 fauna, a comprehensive species list for the site was compiled from being highest and Method 2 lowest. Mean richness for Method 3, the a database of species occurrence records. The database contained most accurate method, was 35.4 (95% C.I. 29–42, n = 9), similar to 23,326 individual species occurrence records in 3,968 collection the mean for all 45 samples (34.5). For fully processed samples, the events. Using accumulated natural history knowledge of the ant spe- range of observed richness was 13 to 55 species. cies, they were categorized as (a) entirely arboreal, nesting and for- A total of 163 species were found in the 45 fogging samples. The aging in the vegetation; (b) entirely ground, nesting in soil, the litter randomized species accumulation (rarefaction) curve was strongly layer, or rotten wood, and rarely foraging more than a meter above convex, showing that relatively few fogging samples capture a large ground level; (c) arboreal foragers, nesting at or below the ground fraction of the arboreal community (Figure 3). The lower-bound as- but foraging arboreally; (d) generalists, nesting and foraging in both ymptotic richness estimate (iChao2, Chiu et al., 2014), based on the habitats; or (e) pest ants, found mostly in and around buildings. Most fogging samples alone, was 199 (95% confidence interval 183–228) species were either entirely arboreal or entirely ground. Inventory species. Thirty-two species (20%) were uniques (each found in only completeness for these two categories was assessed by the pro- one sample) in the fogging dataset, implying a still-rising species ac- portion of uniques (the proportion of species known from a single cumulation curve. If tourists had not been excluded (see Methods), sample) and by coverage (the proportion of the total number of in- the proportion of uniques would have been even higher. However, dividuals in an assemblage that belong to species represented in the the 32 uniques in the fogging samples can be evaluated, and in some sample; Chao & Jost, 2012). cases, their rarity explained as "edge effects" (Longino et al., 2002). To assess whether the canopy has been historically undersam- Atta cephalotes is the common leaf-cutter ant, but it appears to be pled, relative to the ground, and whether it holds a disproportionate rare in fogging samples. Pheidole passivaeferox is a specialized plant number of new species, the rates of discovery of ground and arbo- ant. Two species of Brachymyrmex, a species of Pseudoponera, and real species were compared. The distribution of discovery year was Strumigenys biolleyi are primarily ground ants. Of the 32 uniques, 28 plotted separately for ground and arboreal ants. Year of discovery species are known from additional collections at La Selva (hand-col- was assumed to be year of publication for described species and was lecting, Malaise traps, etc.). Only four species are uniques for the en- set to 2019 for currently undescribed morphospecies. tire La Selva faunal inventory. longino and colwell | 7

We found no evidence that Pentaclethra macroloba, Virola koschnyi, and the "Other" category (multiple species) have distinctive

communities of arboreal ants (PERMANOVA, F2,42 = 1.320, p = .09). Similarly, for the reduced dataset of the first 18 triplet-grouped sam- ples (the grouping of samples into six spatial and temporal clusters of three trees each), there was no effect of tree species on arboreal ant

fauna (PERMANOVA, F2,15 = 1.018, p = .43). There was also no effect of the spatio-temporal specification among triplets (PERMANOVA,

F5,12 = 0.917, p = .71). Thus, beta diversity was minimal in this study.

3.3 | Proportion of total ant fauna

The results for the entire La Selva inventory, which included Malaise traps and abundant hand-collecting in addition to fogging, appear in Table 2. The observed total ant fauna of La Selva is 539 species, 182 (34%) of which are entirely arboreal. Estimated richness is 605 spe- cies for all ants (iChao1, 95% CI 584–636, Table 2) and 196 species (33% of estimated total) for arboreal ants (95% CI 189–210). The fogging samples captured 30% of the observed fauna, and the estimated richness from fogging samples was 33% of the estimated total fauna. However, the species captured by fogging were not entirely congruent with the "entirely arboreal" category in Table 2. Among the FIGURE 3 Randomized ant species accumulation (rarefaction) 163 species in the fogging dataset, 138 were entirely arboreal, 17 were curve for fogging samples at La Selva Biological Station, Costa Rica. entirely ground, 4 were generalist, and 4 were ground-nesting spe- Curve and 95% confidence interval follow methods of Colwell et al. cies with arboreal foragers. The 17 entirely ground species captured (2012) and Chao et al. (2014) as implemented in iNEXT (Hiseh et al., by fogging were species known to be in a "gray zone" with respect to 2016) stratification, nesting at or near ground level, but often foraging several 3.2 | Tree species effect meters above ground in shaded understory. Even though these species were classified as "entirely ground" in Table 2, they were not excluded Focal tree species had no significant effect on sample richness (1- as tourists (see Methods) because they were moderately abundant in way ANOVA, F2,42 = 0.503, p = .61) or the iChao2 estimate of rich- samples and considered a legitimate component of the aboveground ness (1-way ANOVA, F2,22 = 0.389, p = .68). Standard deviations fauna. Thus, the fogging samples appeared to slightly underestimate for sample richness of Pentaclethra macroloba, Virola koschnyi, and the number of entirely arboreal species. This underestimation may in "Other" were 12, 13, and 12, respectively, indicating that richness part be due to targeted collecting of specialized arboreal ants in the full in single species of trees was no less variable than richness across faunal dataset. Only three of nine known specialized plant ants were multiple species. captured by fogging. None of the arboreal Cylindromyrmex were

TABLE 2 Total richness of the ant fauna of La Selva Biological Station, by nesting and foraging categories

Occurrences Species Uniques Duplicates Coverage iChao1

Entirely arboreal 5,077 182 20 (11%) 17 0.996 196 (189–210) Entirely ground 17,055 340 47 (14%) 24 0.997 395 (374–428) Arboreal foraging 275 5 0 0 Generalist 831 6 0 0 Pest ant 88 6 1 1 Total 23,326 539 68 42 0.997 605 (584–636)

Note: See Methods for description of categories. Uniques are species that occurred in only a single collection event, regardless of abundance within the event (e.g., multiple individuals in a single fogging sample); duplicates are species occurring in exactly two collection events. Coverage is a measure of sample completeness and is defined as the proportion of the total number of individuals in an assemblage that belong to species represented in the sample (Chao & Jost, 2012). iChao1 is the iChao1 lower-bound asymptotic richness estimator and 95% confidence interval of the estimate (Chiu et al., 2014). 8 | longino and colwell captured, ants that nest deep in dead wood and are mostly known epiphytes, hemiepiphytes, lianas, smaller understory trees beneath from alates in Malaise traps. In some cases, morphologically similar ar- the crown, and overlapping crowns of nearby large trees. For exam- boreal species could be more readily identified with nest collections, ple, Wilson's (1987) study of fogging samples from a lowland rain with greater likelihood of misidentification of scattered workers in fog- forest in Peru reported 43 ant species "in a single tree," and a total of ging samples. Regardless, the results suggest that, at most, a third of 135 species for the study as a whole, revealing the tight spatial prox- the La Selva ant fauna is entirely arboreal. imity of species. Other rain forest canopy fogging studies include Eleven percent of arboreal species are uniques (known from Floren and Linsenmeier (1997), averaging 36 species per tree with only one collection), and 14% of ground species are uniques. a maximum of 56 (Malaysia); Schulz and Wagner (2002), averaging Coverage, a measure of inventory completeness, was nearly iden- 18 species per tree with a maximum of 37 (Uganda); and Floren et tical for arboreal and ground species (Table 2). Thus, the observed al. (2014), averaging 24.4 species per tree with a maximum of 53 proportions present a robust finding and not an artifact of unequal (Malaysia). sampling. Other methods are more rigorously focused on individual trees Most of the ant species in the inventory already have published and use baiting or manual collecting to target individual foragers and names, but 10% of arboreal species and 14% of ground species are nests. Klimes et al. (2015) felled entire trees to sample the arboreal undescribed morphospecies. Names of ground and arboreal species ant fauna of lowland rain forest in New Guinea, allowing the careful were published at similar rates until the early 1900s, after which dissection of individual trees of all sizes. Adams et al. (2017) climbed ground species were discovered at a higher rate (Figure 4). There into the canopy of individual large trees and used a mixture of bait- is no evidence for a large reservoir of undiscovered species in the ing and hand sampling to characterize the fauna (Panama). Yanoviak canopy. et al. (2007) used similar methods to sample arboreal ants in Gabon. These studies generally yield fewer species per sample than fog- ging, but they allow a more nuanced treatment of fine-scale species 4 | DISCUSSION density. Regardless of sampling method, surveys of the arboreal fauna La Selva Biological Station, a 1,500 ha area of mature lowland rain of tropical rain forests throughout the world yield a relatively nar- forest in Costa Rica, has an estimated ant fauna of about 600 spe- row range of values for total richness. The above-referenced studies cies. An extensive canopy fogging program, combined with other ranged from 128 to 218 species. The fogging studies in particular forms of collecting, showed that about a third of the fauna is arbo- reveal that the vegetation in the vicinity of a single large rain forest real—nesting and foraging in the vegetation. The arboreal species tree can house nearly a third of the total arboreal fauna. are locally very rich, averaging 35 species in samples encompassing We found no evidence of a “tree species effect” on community columns of vegetation that are about 20 m in diameter. The highest composition in the La Selva fauna. Most other studies of canopy ants observed richness in a single sample was 55 species—nearly a third that address tree specificity have likewise shown little or no tree of the entire arboreal fauna. species effect (Schulz & Wagner, 2002; Stork, 1987a, 1987b; Wilson, Methods for sampling arboreal ant diversity vary among re- 1987), although tree species may influence the ant mosaic in the search groups, influencing the observed richness in the sampling forest canopy in Gabon (Dejean et al., 2015). In contrast, structural units of different studies. Canopy fogging often produces high rich- complexity has often been cited as a driver of ant diversity. Although ness per sample (e.g., Yusah, Fayle, Harris, & Foster, 2012). Fogging our study did not measure structural complexity, other studies have samples, though often centered around individual trees, do not dif- shown that ant diversity is higher on trees with epiphytes (Yanoviak, ferentiate between species nesting in the tree and foragers entering Berghoff, Linsenmair, & Zotz, 2011), on trees with greater connectiv- the vegetation column from nearby parts of the canopy. Often, large ity to adjacent trees (Klimes et al., 2015), and on larger trees (Adams trees are selected for fogging, which are complex environments with et al., 2017).

FIGURE 4 Year of discovery (species new to science) for the La Selva ant fauna. Year of discovery is year of taxonomic publication for named species, or 2019 for currently undescribed morphospecies longino and colwell | 9

The high canopy has been referred to as "the last biotic frontier" ACKNOWLEDGMENTS (Erwin, 1983) because of the high number of previously unknown First and foremost, we thank the ALAS staff who made the pro- beetles found in fogging samples from the tropics. Floren et al. ject possible: Danilo Brenes, Flor Cascante, Carolina Godoy, Nelci (2014) make a similar claim for tropical ants, suggesting that difficul- Oconitrillo, Maylin Paniagua, and Ronald Vargas. The Directors and ties of canopy access have slowed discovery of canopy ant species. staff of the La Selva Biological Station were extremely helpful. We Erwin's conclusions were tempered by Stork (1988) and Novotny et thank four anonymous reviewers for greatly improving the manu- al. (2007), who lowered estimates of canopy richness by question- script. This work was supported most recently by National Science ing assumptions of high host specificity in canopy insects. Similar Foundation grant DEB-1932405 (Ants of the World). tempering might be warranted for canopy ants. There is no large reservoir of undiscovered canopy ant species at La Selva, and the DATA AVAILABILITY STATEMENT proportion of new ant species from the canopy is not proportion- Data available from the Dryad Digital Repository: https​://doi. ately larger than the new species found on the ground. If anything, org/10.5061/dryad.ncjsx​ksr0 (Longino & Colwell, 2020). the reverse is true, and the true trove of unrecognized diversity has been for cryptic species hidden in the rotten wood, leaf litter, and ORCID soil of the forest floor. Early taxonomists described arboreal species John T. Longino https://orcid.org/0000-0001-5465-0293 at a similar rate to ground species (Figure 4). Arboreal species were readily collected from felled trees or recent treefalls, and many arbo- REFERENCES real ants were large and active. Starting in the 1900s, relatively more Adams, B. J., Schnitzer, S. 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