Sci Nat (2016) 103: 31 DOI 10.1007/s00114-016-1353-4

ORIGINAL PAPER

Into the black and back: the ecology of brain investment in Neotropical army (Formicidae: Dorylinae)

S. Bulova1 & K. Purce1 & P. Khodak1 & E. Sulger 1 & S. O’Donnell1

Received: 13 October 2015 /Revised: 28 January 2016 /Accepted: 22 February 2016 /Published online: 8 March 2016 # Springer-Verlag Berlin Heidelberg 2016

Abstract Shifts to new ecological settings can drive evo- Keywords Eciton . Mosaic brain evolution . Mushroom lutionary changes in sensory systems and in the bodies . Optic lobes brain structures that process sensory information. We took advantage of the diverse habitat ecology of Neotropical army ants to test whether evolutionary transitions from Introduction below- to above-ground activity were associated with changes in brain structure. Our estimates of genus- Army ants as models for neuroecology typical frequencies of above-ground activity suggested a high degree of evolutionary plasticity in habitat use We measured the sizes of functionally distinct brain regions in a among Neotropical army ants. Brain structure consistently well-supported clade, Neotropical army ants (Dorylinae: Brady corresponded to degree of above-ground activity among et al. 2014), to test the hypothesis that species differences in genera and among species within genera. The most above- habitat use predict patterns of brain investment. The field of ground genera (and species) invested relatively more in neuroecology seeks to identify the relevant features of species’ visual processing brain tissues; the most subterranean spe- biotic and abiotic environments that impose selection on brain cies invested relatively less in central processing higher- region investment (Sherry 2006; Lefebvre and Sol 2008). brain centers (mushroom body calyces). These patterns Neuroecological analyses take advantage of structural speciali- suggest a strong role of sensory ecology (e.g., light levels) zations in the brain: anatomically distinct brain regions that pro- in selecting for army brain investment evolution and cess different cognitive functions (Farris 2008; Ilies et al. 2015). further suggest that the subterranean environment poses If brain investment is subject to constraints—either develop- reduced cognitive challenges to workers. The highly mental production costs or metabolic maintenance costs—then above-ground active genus Eciton was exceptional in hav- relative investment in a given brain region will be limited. ing relatively large brains and particularly large and struc- Species comparisons across many taxa demonstrate mosaic turally complex optic lobes. These patterns suggest that brain evolution: brain regions evolve in size and complexity at the transition to above-ground activity from ancestors that different rates (Barton and Harvey 2000; Smaers and Soligo were largely subterranean for approximately 60 million 2013). Therefore, selection favors brain architecture that bal- years was followed by re-emergence of enhanced visual ances the cognitive demands that a species faces (Laughlin function in workers. 2001; Niven and Laughlin 2008; Navarrete et al. 2011). When species evolve to occupy novel habitats, their sensory environ- Communicated by: Alain Dejean ments can shift, selecting for increased or decreased investment in brain structures that process different sensory modalities. * S. O’Donnell [email protected] Species differences in ecology: habitat use by army ants

1 Biodiversity, Earth & Environmental Science, Drexel University, Neotropical army ant species range between extremes from Philadelphia, PA 19104, USA largely subterranean to above-ground activity (Rettenmeyer 31 Page 2 of 11 Sci Nat (2016) 103: 31

1963; Gotwald 1995;O’Donnell et al. 2007; Wilkie et al. 1971;Gotwald1995). We tested whether within- and 2007). We compared species from all five recognized between-species variation in worker morphology was associ- Neotropical army ant genera. Our subject species spanned ated with patterns of relative brain region investment. much of the range of habitat use in the clade, from exclusively Minimum brain size requirements can constrain the allometry above-ground active species to largely subterranean species, of brain/body size relationships: as minimum body size is and included species with intermediate habitat use patterns approached, brain size cannot decrease further for smaller in- (Table 1). Relationships among the five Neotropical army dividuals, leading to a steep brain/body size slope; as size ant genera have been reconstructed with a well-supported mo- increases, once brain size is sufficient to meet the species- lecular phylogeny, so the ordering and timing of trait transi- typical cognitive demands, further increases in brain invest- tions can be incorporated into analyses of brain evolution ment are disfavored by the high costs of neural tissue. Thus, (Brady 2003; Brady et al. 2014). Current hypothesized inter- the slope of the brain/body size relationship is expected to generic relationships of Neotropical army ants strongly sup- become shallower as body size increases (Seid et al. 2011; port their comprising a clade with predominantly subterranean Eberhard and Wcislo 2011). We tested these predictions by activity as the basal condition (Fig. 1; Brady et al. 2014). comparing brain size against body size across a wide range of We tested whether army ant brain investment covaried with worker body sizes within each army ant genus that we their exposure to high light levels using degree of above- sampled. versus below-ground activity as a proxy for light exposure (Jeffery 2001; Catania 2005). Species-typical ambient light Sampling strategy levels during behavioral activity are generally associated with the level of investment in sensory processing brain regions. For most of our analyses, we compared genera. Within-genus For example, the origins of nocturnal activity are associated species variation in brain structure was generally weak relative with reduced visual processing brain region size in verte- to differences among genera. Furthermore, the genera differed brates, paper wasps, and ants (Coody and Watkins 1986; consistently in habitat use (Table 1). Within the genera Eciton, Barton et al. 1995; Moser et al. 2004; Narendra et al. 2011; ,andNeivamyrmex, we sampled three species each, O’Donnell et al. 2013). We predicted that investment in visual and in these three genera, we further tested whether congeners processing brain regions would be positively associated with differed significantly in brain architecture (we sampled only species above-ground activity. We particularly focused on the one species each of Nomamyrmex and Cheliomyrmex). Data derived genus Eciton which exhibits the highest degree of on ecological and behavioral differences among army ant spe- above-ground activity (Table 1). Because Eciton is a derived cies are limited, but observations suggest that there are con- genus (Fig. 1), changes in brain architecture in Eciton could sistent, albeit often quantitative, habitat use differences among indicate the re-emergence of sensory and cognitive capacities species (Schneirla 1971;Gotwald1995; Baudier et al. 2015). that had been reduced or lost earlier in army ant evolution. Within the genus Eciton, Eciton hamatum and Eciton In addition to light levels, changes in other cognitive chal- burchellii parvispinum are recognized as the most fully lenges and opportunities such as natural enemies and prey surface-active species, while Eciton mexicanum more fre- species could be associated with increased above-ground ac- quently bivouacs below ground and workers often forage in tivity. We asked whether other aspects of army ant brain ar- sheltered/shaded locations (Rettenmeyer 1963;Schneirla chitecture, such as overall brain size and investment in olfac- 1971; personal observation). We expected E. b. parvispinum tory processing, covaried with habitat use. and E. hamatum to make higher visual brain tissue investment than E. mexicanum. In the genus Labidus, Labidus praedator Body size effects regularly forages in swarm raids that move above ground and are attended by birds, while Labidus coecus rarely or never We tested body size differences as an alternative to habitat use does so (Wheeler 1921; Weber 1941; Rettenmeyer 1963; in explaining brain architecture variation. Neotropical army Perfecto 1992; personal observation). Our observations in ant species differ in worker body size ranges, but the overlap Monteverde, Costa Rica, suggest that Labidus spinninodis in body sizes among species permits tests of species differ- forages above ground at least occasionally and their raids are ences in brain size for body-size-matched workers (Kaspari et attended by birds (O’Donnell et al. 2010). We expected visual al. 2011; Baudier et al. 2015). One common pattern among brain investment to be highest for L. praedator and lowest for is for smaller-bodied individuals and species to pos- L. coecus and that L. spinninodis would be intermediate. All sess relatively larger brains (Haller’s rule: Eberhard and three of our Neivamyrmex subject species are largely diurnal Wcislo 2011). We tested Haller’s rule by comparing brain size and regularly forage above ground; we did not have sufficient against head capsule volume within and across army ant gen- behavioral or ecological data to predict relative brain invest- era. Army ant colonies possess a complex caste system with ment among these species (Wheeler 1921; Rettenmeyer 1963; dramatic variation in worker size and morphology (Schneirla Dunn 2003;O’Donnell et al. 2009). Sci Nat (2016) 103: 31 Page 3 of 11 31

Table 1 Index of degree of above- versus below-ground activity for 11 species of Neotropical army ants, subject species for brain anatomy analyses

Species Raid substrate Raid Bivouac sites Bivouac Species Genus References score score index index

Eciton hamatum Above ground 5 Above ground 4 9 7.7 3 Eciton burchellii Above ground 5 Often above ground 3 8 9 parvispinum Eciton mexicanum Often above ground 4 Sometimes above 26 3 ground Neivamyrmex cristatus Often above ground 4 Presumed subterranean 1 5 5.0 9 Neivamyrmex pilosus Often above ground 4 Subterranean 1 5 1, 3 Neivamyrmex sumichrasti Often above ground 4 Presumed subterranean 1 5 7 Labidus praedator Often above ground 4 Subterranean 1 5 3.7 1, 3 Labidus spinninodis Sometimes above 3 Presumed subterranean 1 4 9 ground Labidus coecus Subterranean 1 Subterranean 1 2 1, 2, 3, 4 Nomamyrmex esenbecki Rarely above ground 2 Subterranean 1 3 3.0 3 Cheliomyrmex andicola Subterranean 1 Presumed subterranean 1 2 2.0 1, 5, 6, 8

We arranged the species in descending order of estimated relative degree of above-ground activity for foraging (raid substrate) and nesting (bivouac sites) behavior. We ranked species from 5 (most above-ground) to 1 (least above-ground) for foraging and from 4 to 1 for nesting. To obtain the species score, we summed the two ranks; to obtain the genus score, we averaged species within the genus. Sources of behavioral data for ranking species are listed. 1 Wheeler (1921); 2 Weber (1941); 3 Rettenmeyer (1963); 4 Perfecto (1992); 5 Villareal et al. (1995); 6 O’Donnell et al. (2005); 7 Dunn (2003); 8 Berghoff and Franks (2007); 9 S. O’D. personal observation

Worker morphology and target brain regions for analysis Most brains include anatomically distinct peripheral processing lobes that receive olfactory inputs (antennal lobes, Army ant workers have particularly strong (allometric) henceforth AL: Kuebler et al. 2010) and visual inputs (optic variation in head capsule size (Gotwald 1995). We mea- lobes, henceforth OLs: Strausfeld 2005). Relative sizes of the sured head width and head length of all workers and eye OL correspond to light intensity typically encountered during height whenever possible, as indicators of body size var- behavioral activity (Gronenberg and Liebig 1999;O’Donnell iation. Head size and eye size are key morphological et al. 2013, 2015). Chemosensation is fundamental to commu- predictors of ecological variation among ants (Weiser nication and nest mate recognition in ants (Yamagata et al. and Kaspari 2006). 2006; Kuebler et al. 2010),andtheratioofOLtoAL

Fig. 1 Top: same-scale 3-D reconstructions showing major brain neuropils in frontal view from head-size-matched workers for (left to right) Eciton burchellii parvispinum, Nomamyrmex esenbecki,andLabidus coecus. Dark blue: mushroom body calyces; light blue:antennal lobes; red: optic lobes; translucent green: brain size index structures (central complex, mushroom body peduncles, and protocerebral mass). Bottom: generic phylogeny of Neotropical army ants. Next to each genus name, the index of above-ground activity is shown (see Table 1); estimated divergence dates are shown at each node. Tree structure and divergence dates derived from Brady et al. (2014) 31 Page 4 of 11 Sci Nat (2016) 103: 31 investment may reflect species differences in social interac- For each subject species, we give sample size of workers used tions and pheromone use (Gronenberg and Riveros 2009). We for brain anatomy, sample size of workers for eye size, number tested whether genera and species differed in AL and OL of colonies sampled, and dates and geographic coordinates of investment relative to brain size and to each other and whether field collections: Cheliomyrmex andicola: brain n = 10, eye these differences corresponded to habitat use (above-ground size n = 10, two colonies: 26 Sep and 1 Oct 2003, Otonga, activity). We expected OL investment to covary positively Ecuador (79° 0′ W, 0° 25′ S). E. b. parvispinum:brain with above-ground activity (Gronenberg 1999). In contrast, n = 16, eye size n = 16, six colonies: Jul 2009, Jun 2010, Jul AL volume (as a percentage of total brain volume) is relatively 2014, Monteverde, Costa Rica (84° 48′ W, 10° 18′ N). E. invariant among sexes, castes, and taxa of ants—the AL are hamatum: brain n = 13, eye size n = 13, three colonies: Jun typically approximately 11 % of total brain volume 2007 and Jul 2014 Tiputini Station Ecuador and La Selva (Gronenberg 1999; Gronenberg and Hölldobler 1999). We Biological Station Costa Rica (76° 9′ W, 0° 37′ Sand84°1′ calculated the mean AL/total brain volume ratio for all sub- W, 10° 26′ N). E. mexicanum:brainn = 15, eye size n =15, jects and for each army ant species. We assumed that the AL three colonies: Jul 2014, Monteverde and Santa Elena, Costa and OL volumes that we measured accurately reflected spe- Rica (84° 49′ W, 10° 18′ N; 84° 48′ W, 10° 18′ N; 84° 48′ W, cies differences in brain tissue investment for processing their 10° 21′ N). L. praedator:brainn = 13, eye size n = 11, one respective sensory inputs, recognizing that there are other vi- colony: Jul 2010, San Gerardo Station, Costa Rica (84° 47′ sual and chemosensory processing regions in hymenopteran W, 10° 22′ N). L. coecus: brain n = 11, eye size n = 1, one brains that could not be differentiated by our histological colony: Jun 2010, Monteverde, Costa Rica (84° 48′ W, 10° methods. These include the lateral horn and the optic tubercle 18′ N). L. spinninodis:brainn = 14, eye size n =13,onecolo- (Mota et al. 2011; Nishikawa et al. 2012). ny: Jun 2007, Tiputini Station Ecuador (76° 9′ W, 0° 37′ S). The mushroom bodies (MB) are downstream brain Neivamyrmex pilosus:brainn = 16, eye size n =15,onecolo- neuropils that receive inputs from the peripheral lobes and ny: Sep 2010, La Selva Biological Station, Costa Rica (84° 1′ are involved in learning and memory and in sensory integra- W, 10° 26′ N). Neivamyrmex sumichrasti:brainn = 18, eye tion (Strausfeld et al. 1998; Fahrbach 2006;Farris2008). size n = 10, one colony: Jun 2010, Santa Elena, Costa Rica Mushroom body calyx size is positively associated with task (84° 48′ W, 10° 21′ N). Neivamyrmex cristatus:brainn =16, performance by social insect workers (Gronenberg et al. 1996; eye size n = 16, one colony: Jun 2007, Tiputini Station, Farris et al. 2001; Muscedere and Traniello 2012; Sulger et al. Ecuador (76° 9′ W, 0° 37′ S). Nomamyrmex esenbecki:brain 2014). Dendritic growth and connectivity in the mushroom n = 13, eye size n = 12, one colony: Jul 2012, Monteverde, body calyx may be necessary for cognitive processing related Costa Rica (10° 18′ N, 84° 49′ W). Vouchers of each species to complex task performance by workers (Farris et al. 2001; were deposited in the Entomology Collection of the Academy Jones et al. 2009). The mushroom body calyces of most hy- of Natural Sciences, Philadelphia, PA, USA. menopterans, including ants, are divided into anatomically distinct regions that process input from the eyes and antennae (Gronenberg 1999, 2001). As in some other ants, army ant Histology and neuroanatomical measurements mushroom body calyces are simplified in structure and the sensory sub-regions are not anatomically differentiated We cut each ant’s head capsule from the body at the narrow (Gronenberg 1999). We measured and analyzed the volume neck-like juncture with the thorax. We photographed each of the entire MB calyx. We asked whether genera differed in head capsule lying flat in a Petri dish from the frontal plane mushroom body calyx size relative to brain size. We expected using a digital camera mounted on a dissecting microscope at the relatively complex and variable sensory environments ex- ×24–56 magnification and digital image resolution of perienced by above-ground species to favor increased mush- 2048 × 1536 pixels. We measured head width at the widest room body calyx investment. point and head length along the midline from the photographs, using the ruler tool in ImageJ version 1.46 digital imaging analysis software (http://rsbweb.nih.gov/ij/). We estimated Materials and methods head depth as (0.7 ∗ head width), based on the average ratio from photographs of several large and small Eciton and Subject animal collections Labidus worker head capsules. We converted pixels to length in millimeter using photographs of a single 1-mm stage All ants were collected from foraging raids in the field directly micrometer taken at the same magnifications with the same into aldehyde-based fixative (Prefer, Anatech Ltd.). Ants were camera and microscope. We estimated head capsule volume stored in fix refrigerated at 4 °C until histological processing. (mm3) as an index of body size using the formula for the We collected brain anatomy and head size data from n =155 volume of an ellipsoid, 4/3 ∗ π ∗ (½ head width) ∗ (½ head army ant workers and eye size data from 132 of these workers. length) ∗ (½ head depth). Sci Nat (2016) 103: 31 Page 5 of 11 31

After taking the photographs for morphology and prior to also performed planned comparisons of differences in means histological processing, we removed the antennae and mouth- comparing Eciton pairwise to all other genera, with critical parts by cutting with fine scissors or Teflon-coated razor alpha set at p < 0.05. We used GLM with Tukey post hoc tests blades. (critical alpha = 0.05) (SPSS v. 23 software) to test whether We then dehydrated the head capsules through an ethanol genera (or species) differed in the distribution of the sizes series, acetone, then increasing concentrations of plastic resin. (relative to brain size) of selected brain structures. Resin comprised 45 % (by weight) Embed812 (a mixture of Bisphenol A/Epichchlorohydrin Epoxy Resin (CAS#25068- 38-6) and Epoxy Modifier (CAS#2425-79-8)), 47 % dodecenyl succinic anhydride (DDSA), 5.5 % dibutyl phthalate (DBP), Results and 2.5 % DMP-30 (2,4,6-(tri(dimethylaminoethyl)phenol)). Individual ant heads were incubated in 0.2 ml resin in Genus differences: eye size allometry pyramid-shaped flexible molds at 60 °C for 72 h. The solid resin pyramids were mounted on acrylic posts. Each head Eye size (height) was positively correlated with head size was sectioned into 12–14-μm-thick slices using a rotary micro- (width) (ANCOVA F1,121 =13.64, p < 0.001). The allometric tome with disposable steel histology blades. We placed the slopes of the eye/head relationships did not differ significantly sections on gelatin-coated microscope slides and stained the among genera (ANCOVA equality of slopes test F4,121 =1.19, tissue with toluidine blue. We cleared in an ethanol series and p = 0.32). Genera differed significantly in relative eye size cover slipped under transparent mounting medium (Permount). (Fig. 2; ANCOVA F4,121 =88.57,p < 0.001), and planned con- We used a digital camera mounted on a compound light trasts of means showed that Eciton had relatively larger eyes microscope using the ×10 objective to photograph the tissue than all other genera (all contrasts p <0.05). sections, with a digital image resolution of 2048 × 1536 pixels. For each ant worker, we photographed every section starting at the section where brain tissue first became visible. ImageJ v. Genus differences: allometry of total brain volume 1.46 software was used to quantify the volumes of brain struc- tures. To quantify brain regions on each section, we outlined Total measured brain volume increased with head cap- the target brain regions and used ImageJ to count the number sule volume (ANCOVA F1,145 = 33.02, p <0.001) but at of image pixels in the structure. We converted the pixel counts a lower rate than expected with an isometric rate of vol- to area using a photograph of a stage micrometer taken at the ume increase (Fig. 3; linear regression slopes for ln(brain same magnification with the same microscope and camera as a volume) versus ln(head capsule volume) ranged from size reference and then multiplied the areas by section thick- 0.17 to 0.30 among genera; all slopes were significantly ness to yield a volume estimate. lower than 1.0 at p < 0.001). Genera did not differ signif- Only brain neuropils (dendritic and axonal fields) were icantly in the slopes of their brain volume/head volume measured; we did not measure the adjacent cell body regions. relationships (ANCOVA equality of slopes test F4, We measured and analyzed the volumes of the following brain 145 = 1.10, p = 0.36). Genera differed significantly in rela- sub-regions: the OL, the AL, and the mushroom body calyces. tive brain size (Fig. 3;ANCOVAF4,145 = 58.37, Volumes of other brain regions were pooled and are hence- p < 0.001), and planned contrasts of means showed that forth referred to as the Bbrain size index^:mushroombody Eciton had relatively larger brains than all other genera peduncle and lobes; central complex; and the remainder of the (all contrasts p <0.05). protocerebrum, deutocerebrum, and tritocerebrum. For some Relative brain investment (brain volume/head volume) comparisons to previously published data, we also calculated was strongly dependent on body size; relative brain in- investment in some structure (e.g., AL) relative to total brain vestment increased sharply at smaller head sizes in sup- volume. port of Haller’srule(Fig.3). Analysis of ln-transformed data showed that the brain volume/head volume ratio Statistical analyses decreased significantly with head volume (ANCOVA

F1,145 =290.09, p < 0.001). Genera did not differ signifi- We tested for genus differences in allometry using analysis of cantly in the slopes of this relationship (ANCOVA equal- covariance (ANCOVA) models on natural-log transformed ity of slopes test F4,145 = 1.10, p =0.36), but the genera data (SPSS v. 23 software). The ANCOVA models tested differed in ln(brain volume/head volume) ratio (F4, effects on eye size or brain investment of body size covariates 145 = 58.36, p < 0.001). Planned contrasts of means (head volume), genus identity, and the interaction term tested showed that Eciton had relatively higher ln(brain whether genera differed in the slopes of the regressions be- volume/head volume) ratios than all other genera (all tween the response and predictor (body size) variables. We contrasts p <0.05). 31 Page 6 of 11 Sci Nat (2016) 103: 31

Fig. 2 Scatter plots showing the -1.0 relationship between eye height Cheliomyrmex and head capsule width for army Eciton ant workers from five Neotropical -1.5 Labidus genera (data were natural log Neivamyrmex transformed) Nomamyrmex -2.0

-2.5

-3.0 ln(eye height, mm) ln(eye

-3.5

-4.0

-0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 ln(head width, mm)

Genus differences: relative sizes of sensory lobes to 15.8 % (E. mexicanum). AL was not significantly correlated with the investment in AL was not significantly correlated The OLs were relatively small in all species that we with the species above-ground behavior index (r =0.44, sampled, comprising from approximately 10 % to less df =9,p =0.18). than 1 % of the volume of the AL (Figs. 1 and 4). However, neuroanatomy suggested that the OLs were not vestigial and functioned in visual processing, at least Genus differences: relative size of mushroom body calyces in the genus Eciton: the OLs were connected by neural tracts to the layer of sensory cells associated with the Genera differed significantly in the MB calyx to brain external eye facet (Fig. 5). The genus Eciton was excep- size index ratio (Fig. 6; F4,149 = 24.72, p <0.001). tional in having large OL relative to brain size (genus Relative calyx size corresponded to habitat use: subterra- differencesinOLtobrainsizeindexratio:F4,150 = 42.70, nean species tended to have smaller MB calyces. The p < 0.001; Tukey post hoc tests showed that Eciton dif- most subterranean genus Cheliomyrmex had the smallest fered from all other genera; no other genera differed). relative MB calyx size among the genera, and the rela- Eciton OLs were distinctly bipartite, suggesting division tive MB calyx size of the next most subterranean genus into medulla and lobula regions, while the OLs of all Nomamyrmex was significantly lower than in the remain- other genera were reduced to a single anatomically dis- ing genera (Table 1;Fig.6). tinct structure (Figs. 1 and 5). Genera also differed in relative AL size (genus differences in AL to brain size index ratio: F4,150 = 26.23, p < 0.001; Species differences: relative sizes of sensory lobes Tukey post hoc groupings of genera from lowest to highest mean AL/brain size index ratio were [Cheliomyrmex, For each of the three genera with samples from multiple spe- Nomamyrmex, Labidus] and [Neivamyrmex, Eciton]). The cies, species differed significantly in brain architecture (Fig. 7; genera differed significantly in the OL/AL ratio (Fig. 4; F4, Eciton: F2,41 = 21.98, p <0.001; Labidus: F2,35 =4.69, 150 =37.63); Eciton differed from all other genera in having p =0.016; Neivamyrmex: F2,47 =7.18, p = 0.002). In the case the highest OL/AL ratio (Fig. 4). Although the Eciton OL/AL of Eciton, E. hamatum and E. b. parvispinum (the most ratio was highest, this genus had both the largest OL and the surface-active species) had higher OL/AL ratios than E. largest AL among the genera (Fig. 4). mexicanum;inLabidus,theL. praedator (most surface- Army ant AL were relatively large: across all subjects, the active) OL/AL ratio was higher than the L. coecus ratio, and AL accounted for 14.1 % of total brain volume (99.9 % CI L. spinninodis was intermediate; in Neivamyrmex,the 13.65 to 14.60 %, highly significantly greater than the mean of Neivamyrmex pilosus OL/AL ratio was higher than both 11 % for 13 ant species; Gronenberg and Hölldobler 1999); Neivamyrmex cristatus and Neivamyrmex sumichrasti species means ranged from 11.1 % (Cheliomyrmex anidicola) (Fig. 7; Tukey post hoc tests, critical alpha = 0.05). Sci Nat (2016) 103: 31 Page 7 of 11 31

Fig. 3 a Scatter plots showing A the relationship of total brain -2.0 volume with body size (head Cheliomyrmex Eciton capsule volume) for workers from -2.5

) Labidus

five Neotropical army ant genera 3 Neivamyrmex (data were natural log Nomamyrmex transformed). The linear best-fit -3.0 lines for least-squares linear regression are shown for each -3.5 genus for illustration purposes; the regression lines are solid expect that Eciton has a dashed -4.0 line. b Raw data scatter plots showing the relationship between body size relative brain -4.5 investment and body size (head capsule volume) for workers from ln(total brain volume, mm -5.0 five Neotropical army ant genera

-5.5 -3 -2 -1 0 1 2

ln(head capsule volume, mm3) B 0.14

0.12

0.10

0.08

0.06

Total brain volume/ brain volume/ Total 0.04 Head capsule volume

0.02

0.00 0246810 Head capsule volume, mm3

Discussion workers (Gronenberg 1999; Gronenberg and Hölldobler 1999). We found that Neotropical army ants had larger AL Comparisons of army ant brain structure to other ants than most other ants studied to date, with army ant AL ac- counting for approximately 14 % of their total brain volume. A recent review of brain size variation showed that doryline army ants overlapped broadly in relative brain size (brain Army ant body size and the allometry of brain investment mass/body mass) with ants from several other subfamilies (Seid et al. 2011). Relative OL size varies widely in ants, from We found strong general support for Haller’s rule: relative 0 to 33 % of total brain volume among 13 species from diverse brain size decreased with body size in all army ant genera, taxa (Gronenberg and Hölldobler 1999), but OL size is gen- as seen in other ants (Eberhard and Wcislo 2011). However, erally reduced in ants compared to other ; some Eciton workers had larger brains relative to body size than the subterranean ants may lack OL altogether (Gronenberg 1999). other genera. Although total brain size was positively corre- In contrast, relative AL volume is less variable, with AL com- lated to worker body size, brains increased in size at a signif- prising approximately 11 % of total brain volume in ant icantly lower than isometric rate. As found previously for 31 Page 8 of 11 Sci Nat (2016) 103: 31

Fig. 5 Light micrograph of stained thin section (frontal plane) of Eciton burchellii parvispinum worker brain, showing major brain regions and the neural connection of the optic lobes to the external eye facet. MBc mushroom body calyx, Pr protocerebral mass, AL glomeruli of the antennal lobe, OL optic lobe

Habitat complexity, brain size, and mushroom body investment

Army ant genus- and species-typical brain investment appeared to be influenced heavily by ecology, indepen- dently of body size and overall brain size effects. The increase in total brain volume (relative to body size) in Eciton was likely related to their shift to a more above- ground ecology. Abiotic (e.g., light levels, discussed be- low) and biotic factors may contribute to increased envi- ronmental complexity. Potential biotic sensory opportuni- ties and challenges that increase above-ground include diversity of prey species and the range of natural ene- mies (predators, parasites, and competitors), both of Fig. 4 a Bar graph showing mean ± SD values of the ratio of optic lobe to antennal lobe volumes for five genera of Neotropical army ants, ordered from lowest to highest OL/AL ratio. Bars with different shading indicate significantly different means according to Tukey post hoc analysis. b Bar graphs showing mean ± SD values of the relative volumes of the optic and antennal lobes for five genera of Neotropical army ants, ordered from 0.3 lowest to highest mean optic lobe investment. Bars with different shading indicate significantly different means according to Tukey post hoc analysis 0.2 fungus-gardening ants with polymorphic workers (Seid et al. 2011), the slope of the brain-body size relationship 0.1

decreased sharply at larger body sizes within species. brain size index volume)

This pattern may be due to constraints on minimum brain Mean +/- SD (MB calyx volume/ 0.0 size: within a taxon, a minimal brain investment may be x s yrme mex abidu Eciton mam vamyr L necessary to achieve the cognitive processing necessary CheliomyrmexNo Nei for meeting that species’ ecological challenges. If brains Genus cannot fall below a minimum critical volume in small- bodied individuals, the brain/body size slope becomes Fig. 6 Top: bar graphs showing mean ± SD ratios of mushroom body calyx to brain size index volume for workers from five genera of very steep when approaching the minimum-size boundary Neotropical army ants. Bars with different shading indicate (Seid et al. 2011; Eberhard and Wcislo 2011). significantly different means according to Tukey post hoc analysis Sci Nat (2016) 103: 31 Page 9 of 11 31

Above-ground activity and visual processing investment 0.06 Eciton We found that the most above-ground active genus (Eciton) 0.04 had relative large external eye facets, and this difference was

0.02 reflected in brain investment patterns: Eciton OLs were sig- nificantly larger than OL of the other genera relative to brain 0.00 size. OLs were present (albeit small in volume) in all army ant um um um ican mat spin mex E. ha arvi species that we examined, and the OL varied in relative size E. . b. p E among species. Our anatomical analyses show that the OLs 0.016 Labidus are connected via neural tracts to receptors underlying the external eye facets, suggesting the OL function in visual pro- 0.012 cessing (Werringloer 1932). There were both inter-generic and 0.008 inter-specific differences in brain architecture consistent with habitat use differences. The diversity of above- versus below- 0.004 ground habitat use among Neotropical army ants is associated 0.000 with species differences in typical ambient light levels and us dis tor antennal lobe volume ec no da L. co inni prae provides an interesting analogy to other ant taxa where light- L. sp L. level variation is associated with diurnal vs. nocturnal activity, Neivamyrmex including differences among castes within species (Coody and Species mean optic lobe volume/ 0.020 Watkins 1986; Moser et al. 2004; Narendra et al. 2011). 0.015 Neotropical army ants vary in diel activity patterns, with for- 0.010 aging activity ranging from species restricted to daylight, nighttime only, and activity throughout day (Schneirla 1971; 0.005 O’Donnell et al. 2009). Future studies are needed to assess 0.000 whether diel activity differences are reflected in brain anatomy rasti atus sus mic crist . pilo N. su N. N variation. Unlike the OL, relative AL size was not consistently related to army ant species ecology. Species How well do Eciton workers see? The external eyes of Fig. 7 Bar graphs showing mean ratios of optic to antennal lobe volumes Eciton workers comprise a single facet, and their eyes cannot for three species in each of three genera of Neotropical army ants. Bars form images. E. burchellii workers appeared to be unrespon- with different shading indicate significantly different means according to Tukey post hoc analysis sive to artificial changes in light levels, including illumination by stage lights for filming, during the day and at night, when on raids, while emigrating and at bivouacs (S. O’D. personal which can select for increased brain investment (Mace observation), but Rettenmeyer (1963) reported that evening E. and Eisenberg 1982;Allman2000;Molleretal.2005). mexicanum raid columns could be disturbed by flashlight il- Army ant mushroom body investment was related to lumination. Many species of Eciton show evidence of coordi- habitat use, but unlike the OL, the MB calyces varied nating colony activities (raiding, emigrating) to specific times most strongly in the highly subterranean genera. The rel- of the day (O’Donnell et al. 2007, 2009), and they may re- ative size of the MB calyces of social insect workers in- spond in part to changes in ambient light levels to trigger creases with task performance, such as foraging, presum- changes in colony activity (Schneirla 1971). At least some ably in response to associated cognitive challenges such partly or fully subterranean army ant species show less diel as orientation and navigation and assessing resource qual- patterning of their activities, for example, raiding at similar ity (Farris et al. 2001; Muscedere and Traniello 2012; rates across the 24-h day (Rettenmeyer 1963;O’Donnell et Sulger et al. 2014; Rehan et al. 2015). The reduced MB al. 2009). Our data on brain investment patterns suggest the calyx investment of the most subterranean army ants sug- Eciton visual sensory system is functional and has enhanced gests relaxed cognitive demands in the underground eco- capacity relative to other Neotropical army ant genera. system. Both AL and OL investment were also reduced in Psychophysical tests are needed to further assess Eciton visual the most subterranean genera, suggesting that life below abilities. ground provides a simplified sensory environment and not Increased OL investment in Eciton is a potential example merely a different (e.g., more olfactory) one. Further sup- of a partial regain of sensory function after loss or reduction to port for this assertion comes from the fact that although a vestigial condition. The dramatically expanded and bi-lobed Eciton had a high OL/AL ratio, their relative AL size was Eciton OLs are particularly striking in comparison to their also the highest among the five genera. highly subterranean sister genus Nomamyrmex (Fig. 1). 31 Page 10 of 11 Sci Nat (2016) 103: 31

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