A Phylogenetic Characterization of the Microbiome of the Ponerine Ants (Hymenoptera: Formicidae: Ponerinae)

A Thesis Submitted to the Faculty of Drexel University by Narayan Hin-Lun Wong in partial fulfillment of the requirements for the degree of Master of Science in Biology December 2015

Acknowledgements

I would like to thank my academic advisor, Jacob Russell, for his invaluable intellectual guidance and oversight throughout the research process. Also, I would like to thank the other members of my Drexel thesis committee: Dan Marenda (Chair) and Sean O’Donnell. Andrew Suarez (University of Illinois at Urbana-Champaign) supplied the bulk of the ant samples, and graciously served as an external member of the thesis committee. Fredrick Larabee (Smithsonian Institution/University of Illinois) also contributed ant samples, taxonomic information of ant specimens, and a revised ponerine phylogeny used in this research. Piotr Łukasik (University of Montana, Missoula; formerly Drexel University) and Yi Hu (Drexel University) provided the modified quality-control pipeline used to analyze the amplicon sequencing results, and generously lent their expertise throughout the data analysis process. Work reported here was run on hardware supported by Drexel's University Research Computing Facility.

iii

Table of Contents

LIST OF TABLES ...... iv

LIST OF FIGURES ...... v

ABSTRACT ...... vi

1. BACKGROUND AND LITERATURE SURVEY ...... 1

1.1 Microbial symbionts: drivers of animal evolution across both ancient and recent time scales ...... 1

1.2 The ants (Hymenoptera: Formicidae) are a burgeoning study system for host- symbiont evolution...... 2

1.3 An overview of ponerine ecology and symbiosis in the ponerines ...... 5

2. THE PONERINE MICROBIOME: UNITING TARGETED AND COMMUNITY- WIDE ANALYSES ...... 6

2.1 Introduction ...... 6

2.2 Methods...... 9

2.3 Results ...... 16

2.4 Discussion ...... 26

3. CONCLUDING REMARKS AND RECOMMENDATIONS ...... 28

REFERENCES ...... 31

APPENDICES ...... 36 iv

LIST OF TABLES

Table 1: Diagnostic PCR results for ponerine ants ...... 10 Table 2: Wolbachia HVR allele analysis ...... 21

LIST OF FIGURES

Figure 1: Relative abundance of bacteria across ponerine genera and species ...... 16

Figure 2: Relative abundance of bacteria across the phylogeny of trap-jawed ponerines (genera Odnotomachus and Anochetus)...... 18

Figure 3: Maximum likelihood phylogeny of the Wolbachia endosymbiont in ponerine ants based on a 500 bp alignment ...... 20

Figure 4: Maximum likelihood phylogeny of Unclassified Firmicute 16S rRNA based on a ~250 bp alignment ...... 23

Figure 5: Maximum likelihood phylogeny of Unclassified Firmicutes rplB based on a ~600 bp alignment ...... 24

Figure 6: Maximum likelihood phylogeny of Entomoplasmatales 16S rRNA based on a ~650 bp alignment ...... 26

ABSTRACT

Ants (Hymenoptera: Formicidae) provide a range of ecosystem services across the world.

Understanding the composition of bacterial communities associated with this diverse family of insects is crucial for understanding ecological roles. Here, I present a review of the current state of host-symbiont research in the ants. My original research focuses on the subfamily Ponerinae, a clade of primarily carnivorous ants consisting of ~1,600 species found worldwide. By utilizing next-generation sequencing technology, I characterized the gut microbial communities of 48 individuals from seven of the 47 total ponerine genera. This 16S rRNA amplicon sequencing approach was complemented with phylogenetic analyses of several of the dominant bacterial taxa via sequencing of protein coding genes, as well as a newly revised phylogeny of the host ants. My main findings include: (1) a comparative 16S rRNA profile of gut microbiomes across the ponerines;

(2) the distribution of the symbiont known as Wolbachia across the ponerines; (3) the distributions and evolutionary histories of two ant-specific bacteria limited to carnivorous ants; and (5) phylogenetic analyses of the major bacterial taxa that make up the gut communities of the ponerines.

1. BACKGROUND AND LITERATURE SURVEY

1.1 Microbial symbionts: drivers of animal evolution across both ancient and recent time scales

Biologist are becoming increasingly aware of the importance of symbiosis in driving the evolution of plants (van Rhijn and Vanderleyden 1995), animals (McFall-Ngai et al.

2013), and fungi (Partida-Martinez and Hertweck 2005). Natural selection can act on symbiont-conferred phenotypes, thereby indirectly influencing fitness outcomes of the host. Thus, symbiosis can benefit both partners in a host-symbiont system by expanding the repertoire of genetic variation available to either party.

Broadly, symbiont-conferred functions can be classified into nutritional acquisition and host defense. Both of these functions have enabled symbiont possessing hosts to expand into otherwise inaccessible ecological niches. Microbial symbionts are partially responsible for allowing their hosts to colonize deep sea environments devoid of light

(Fisher et al. 1987), exploit diets poor in essential amino acids and vitamins (Douglas

1998; Wu et al. 2006), and defend against predators, pathogens, and parasitoids (Oliver,

Smith, and Russell 2014). Several particularly long-standing associations between hosts and symbionts have resulted in complete interdependence between symbiotic partners. In fact, organelles including mitochondria and chloroplasts are hypothesized to have originated from once free living organisms that were co-opted into their current-day functions through several symbiosis events (Gray and Burger 1999). These are extreme examples of symbiosis, where the microbial partner has been completely relegated to a lifestyle dependent on the host. Such obligate symbioses, where both host and symbiont 2 are mutually dependent on each other for survival, are often marked by a high degree of phylogenetic congruence across both ancient (parallel speciation) and recent (narrow- sense coevolution) time scales (De Vienne et al. 2013).

1.2 The ants (Hymenoptera: Formicidae) are a burgeoning study system for host- symbiont evolution

Animals owe much of their success to the functions conferred upon them by microbial partners, and several ant lineages possess well characterized beneficial symbionts. Microbial symbionts of the ants fall into diverse categories; we can find examples of ant symbionts in both nutritional (Feldhaar et al. 2007) and defensive roles

(Currie et al. 1999), and in extracellular (Currie et al. 2006) and intracellular lifestyles

(Feldhaar et al. 2007) .

The most well characterized obligate nutritional symbiosis in the ant world involves the partnership between bacteria of the genus Blochmannia and the carpenter ant

(genus Camponotus). Blochmannia genomes possess capabilities for manufacturing essential amino acids, as well as nitrogen recycling abilities. Over the course of this roughly 30-40 million year old association (Feldhaar et al. 2007), the ant hosts have evolved specialized cells concentrated in the midgut that house most of the symbionts

(Sauer et al. 2002). Additionally, Blochmannia cells have been discovered in ovarial cells of reproductive females, suggesting that these symbionts are transmitted transovarially

(Sauer et al. 2002). Furthermore, Blochmannia is present across all camponotine species 3 that have been surveyed, as well as within individuals within colonies (He, Wei, and

Wheeler 2014). Further evidence for the obligate nature of this partnership comes from comparative genomic studies. The genomes of the two fully sequenced Blochmannia species, Blochmannia floridanus and Blochmannia pennsylvanicus, reveal that both have highly reduced genome size compared to that of free living ancestors (Gil et al. 2003;

Degnan, Lazarus, and Wernegreen 2005). Remarkably, much of this loss of genome size appears to be convergent between these two species. Both Blochmannia species exhibit synteny (orientation and ordering) of shared genes and functional retention of pathways such as amino acid biosynthesis, sulfur assimilation, and nitrogen recycling (Degnan,

Lazarus, and Wernegreen 2005). Furthermore, cross fostering experiments involving antibiotic cured workers and developing larvae suggest that despite a negative correlation between symbiont bacterial titer and host age, the presence of the Blochmannia symbiont contributes to ant ontogeny, thereby increasing the overall fitness of camponotine colonies (Zientz et al. 2006).

Another well-described example of microbial symbiosis in the ant world involves up to three partners: fungus farming ant hosts, mutualistic fungi, and a defensive cuticular bacterium. In this ~50 million year old association, ants from the tribe Attini (subfamily

Myrmicinae) cultivate basidiomycete fungal gardens typically using plant material as a substrate (Schultz and Brady 2008). The degree of specialization varies for the fungal partner across this lineage; among the so-called “higher” attines, the fungal cultivar is a highly derived lineage, relative to fungi cultivated by the “lower” attines (Mueller et al.

2001). The increased degree of specialization characteristic of fungi in the higher attines 4 is accompanied by a loss of independence; while more primitive fungal partners of the lower attines are capable of surviving independently, fungi from the higher attines are completely reliant upon their ant hosts for survival and competition against pathogens

(Schultz and Brady 2008). Fungal partners of higher attines display specialized hyphal structures called “gongylidia”. These are unique, carbohydrate and lipid rich structures on which the ant hosts preferentially feed, and are absent in free living fungal relatives (C R

Currie 2001). Leafcutter ant host lineages seem to maintain fungal strains with high fidelity, albeit through different means than those by which intracellular symbionts are transmitted. Foundress queens transfer the fungus to new nests on their nuptial flight by storing a small clump of inoculum in a specialized cavity within the mouthpart. Once a queen reaches her new nesting site, she initially tends to the growing garden until workers are produced (Mueller et al. 2001). Together, the combination of mutual dependence and transmission mode promote diffuse co-evolution of fungal symbiont and host.

The third player in the fungus farming ant symbiosis is a defensive bacterium from the genus Pseudonocardia. These bacteria produce antifungal compounds, providing an additional line of defense against pathogenic fungi from the genus

Escovopsis that may invade their hosts’ gardens (Currie et al. 1999; C R Currie 2001). In contrast to most other bacterial symbionts of insects, these bacteria persist in specialized structures on the exterior of the host cuticle (Currie et al. 2006). Like the leafcutter ant- fungus mutualism, the Pseudonocardia-attine ant association is similarly thought to be ancient. However, there is some controversy over the fidelity of the bacterium-host relationship, the specificity of bacterial-derived antifungal agents, and the diversity of 5 bacteria colonizing the cuticle of the ant hosts (Zhang, Poulsen, and Currie 2007).

Whether this relationship is as highly coevolved as was once thought is still a matter of controversy. However, several lines of evidence support a relatively specialized relationship, including (1) the stable persistence of dominant Pseudonocardia strains in lab-reared colonies of Acromyrmex echinator (Andersen et al. 2013), (2) the ability of

Acromyrmex workers to differentiate between native and foreign bacterial strains (Zhang,

Poulsen, and Currie 2007), and (3) the co-phylogeny of bacterium and host at both the broad (high vs low attine) and fine scale (at the level of host genus) (Cafaro et al. 2011).

1.3 An overview of ponerine ecology and symbiosis in the ponerines

The ponerines are an ancient lineage of the ants, with some debates about the timing of their divergence from other formicid lineages. Estimates place the birth of ponerines from other ant lineages between 85-102mya (Moreau 2006; Ward 2014). The ponerines contain 2 tribes, 47 genera, 1,203 species, making them the third most species- rich subfamily amongst extant ants (Wilson and Hölldobler 2005). Across the Ponerinae, we see a range of ecological traits considered more ancestral, when compared to other ant lineages. These properties include simplified caste structure, decreased morphological differentiation between castes, relatively solitary foraging habits, and small colony size

(Schmidt 2013).

The two genera Odontomachus and Anochetus, which comprise most of the samples used in this study, are known as “trap jawed” ants, a group united by the evolution of a set of mandibles capable of record-speed acceleration (Patek et al. 2006).

Ponerines are generally known to be generalist carnivorous/scavengers (Ehmer and 6

Holldobler 1995), but several species have been observed to specialize on certain arthropods (Schmidt 2013), collect hemipteran honeydew, and collect extrafloral nectar

(Larson et al. 2014).

Little is currently known about microbial symbionts amongst the ponerines.

Focused PCR-based screening assays have discovered the presence of Wolbachia symbionts in a handful of ponerine species, including a Leptogenys species, (J. A. Russell et al. 2009), Myopias emeryi and Odontomachus rixosus (Wenseleers et al. 1998). More recent efforts using 454 pyrosequencing further detected Wolbachia in an additional two species of Odontomachus, as well as a Spiroplasma symbiont in a species of

Pachycondyla (Kautz et al. 2013). Through other methods, microbial symbionts have been uncovered in three other ponerid lineages: Paraponera clavata, Odontomachus bauri and a Leptogenys species (Larsen et al. 2014; Caetano et al. 2009, Funaro et al.

2011). Paraponera clavata, an omnivorous relative of the ponerines, is host to a

Bartonella-like bacterium. This bacterium has been hypothesized to be involved in nitrogen metabolism, given the negative correlation between its presence in ants and host trophic level (Larson et al. 2014).

2. THE PONERINE MICROBIOME: UNITING TARGETED AND COMMUNITY-WIDE ANALYSES

2.1 Introduction

Advances in DNA sequencing technology, in terms of increased speed and decreased financial cost, have recently expanded our appreciation of the contributions 7 made by bacterial and other microbial symbionts to the diversity we see in the evolution of the animal kingdom (McFall-Nagai et al. 2013). The majority of multicellular life forms have evolved deeply intimate associations with bacteria, often blossoming into mutually dependent relationships between symbiont and host (Douglas 1998; Feldhaar et al. 2007, Gray and Burger 1999). Heritable symbiont genes that result in differential rates of survival and reproduction between infected hosts can be acted upon by natural selection. We can consider bacterial genes to be a source of genetic variability that augments the nuclear genome of the host (Bordenstein and Theis 2015).

One of the key innovations lent by symbiotic partners to their hosts is nutritional supplementation. This is a common symbiont-induced function employed by all walks of life, from plants (van Rhijn and Vanderleyen 1995) to insects (Munson et al 1991), to mammals (Bergen and Wu 2009). Recent studies aimed at characterizing the microbiomes of mammalian systems have discovered correlations between host diet and both composition and functions of symbiotic microbes (Muegge et al. 2011). Bacteria involved in the production of essential amino acids, vitamins and cofactors are found in many insect lineages, particularly in hosts that subsist on incomplete diets such as blood and plant sap (Douglas 2011). Specifically, across the ants (Order: Hymenoptera; Family:

Formicidae), researchers have found that the presence of symbiotic gut microbes negatively correlates with the trophic levels of hosts (Russell et al. 2009). Additionally, trophically similar but phylogenetically distant hosts harbor two ant-specific symbionts, echoing findings of microbiome convergence from mammalian systems (Andersen et al.

2012). The ants in particular make for an informative case study of diet-driven symbioses; behavioral studies have documented a diverse range of feeding habits, 8 including varying degrees of specialized, obligate carnivory (Ehmer and Holldobler

1995), opportunistic foraging and scavenging (Bennett and Breed 1985), cultivation of fungal gardens (Currie 2001), and macro-level symbiosis with honeydew producing hemipterans (Bluthgen et al. 2003).

An ideal ants for examining the relationship between feeding habits is the subfamily Ponerinae (Schmidt et al. 2013). The ponerine ants, together with the related subfamily Paraponerinae, are largely a group of generalist carnivores whose species span a wide trophic range (Schmidt et al. 2013). Some species are remarkably specialized predators of particular insect taxa while others are generalists who diet includes extrafloral nectar and seeds (Larson et al. 2014).

Despite a recent interest in the microbiology of ants, the ponerine microbiome has been relatively understudied. Limited information comes from PCR and 454 pyrosequencing –based assays across the ants; specifically, the ponerine genus

Leptogenys is infected with Wolbachia at a lower frequency than other ants (Kautz et al.

2013). Ponerines appear to have some tantalizing leads in terms of bacterial symbiosis, however; one species of Leptogenys was found to possess a bacterium from an otherwise host-specific lineage of army ant associates (Funaro et al. 2011). Additionally, studies using microscopy have detected the presence of bacterial cells concentrated both within and outside of host cells in the midgut of ants from the ponerine species Odontomachus bauri (Caetano et al. 2009). The range of feeding behaviors exhibited by the ponerine ants combined with the high degree of species richness points to these ants as a valuable system for elucidating the intersection between diet, phylogeny, and symbiosis.

In this work, I set out to determine the extent of microbiome convergence between ponerines and army ants, the composition of ponerine gut bacterial communities, and the evolutionary histories of microbes found in the ponerines. I accomplished these aims by utilizing a combination of Illumina-based 16S amplicon sequencing, PCR-based diagnostic screening, targeted protein-coding gene sequencing and a newly improved ponerine phylogeny (Larabee PhD Dissertation 2015).

2.2 Methods

Sample collection and DNA extraction

Adult worker ants were collected from field sites in 11 different countries. Live ants were immediately stored in 95% ethanol and kept at -20°C. Specimen identifiers, ant taxonomy, collection locale, and other demographic data for each ant in this survey are presented in Appendix 1. A subset of the total collection (n=133) was individually surface-sterilized in 6% bleach solution followed by a rinse in sterile deionized water.

After surface sterilization, bleach sterilized forceps were used to remove all abdominal segments 3 and higher. Each gaster was individually placed in a sterile 1.7mL tube, which was then briefly (~five seconds) immersed in liquid nitrogen. 180µL Enzymatic lysis buffer was then added to each tube, and a sterile pestle was used to pulverize each frozen ant gaster. Finally, extractions proceeded according to the protocol outlined in the

Qiagen DNeasy blood and tissue kit for gram negative bacteria.

Sample selection and screening 10

To determine presence of bacteria, we used the universal bacterial PCR primers 9Fa and

1513R to amplify a portion of the bacterial 16S ribosomal RNA gene. We also used universal mitochondrial DNA primers BEN and LCO1490 to determine the success of each DNA extraction, reasoning that failed amplification of the ant CO1 gene was evidence for poor template quality. PCR amplified product from each sample was run on a 2% agarose gel stained with ethidium bromide. Results from 16S rRNA and CO1 amplification are shown in Table 1. Samples with sufficient fluorescence for both CO1 and 16S were submitted for 16S rRNA amplicon sequencing.

Table 1: Diagnostic PCR screening for host mitochondrial genes, Wolbachia, Unclassified Firmicutes, and

Entomoplasmatales in Ponerine ant samples. “1” denotes positive amplification and “0” denotes failure to amplify, as determined via gel electrophoresis.

Sample ID COI WSP UncF UnE TW01 1 1 0 0 FL617 1 0 0 0 FL601 1 1 0 0 FL624 1 0 0 0 FL607 1 0 1 0 FL606 1 0 0 0 AVS4322 1 0 0 0 TW02 1 1 0 0 139-2 1 0 0 0 173-4 1 0 0 1 173-5 1 0 0 0 139-5 1 0 0 0 139-4 1 0 0 0 139-1 1 0 0 0 173-1 1 0 1 1 CSM0952-3 1 0 0 0 AVS4304 1 0 1 0 Hoffmann 1 0 0 0 Brian 1 0 0 0 AVS4315 1 0 0 0 ASMITH-07 1 0 0 0 ASMITH-06 1 0 0 0 11

ASMITH-05 1 0 0 0 ASMITH-04 1 0 0 0 ASMITH-15 1 0 0 0 ASMITH-13 1 1 0 0 ASMITH-10 1 0 0 0 ASMITH-09 1 1 0 0 AVS4188 1 0 0 0 AVS4202 1 1 0 0 AVS4278 1 0 0 0 AVS4273 1 0 0 0 AVS2997 1 1 0 0 AVS4155 1 0 0 0 AVS4189 1 0 1 0 AVS4017 1 1 0 0 AVS3069 1 0 1 1 AVS4083 1 1 0 0 AVS4153 1 0 1 0 AVS4124 1 1 1 0 AVS4202-1 1 1 1 0 AVS2485 1 1 0 0 AVS2899 1 0 1 0 AVS4021 1 1 1 0 AVS2766 1 1 0 0 BLF22672 1 0 0 0 AVS4359 1 0 0 1 AVS4358 1 0 0 0 FL607_2 1 0 0 0 FL624_2 1 0 0 0 FL606_2 1 0 0 0 FL617_2 1 0 1 0 FL601_2 1 1 0 0 TW01_2 1 1 0 0 MAL26_2 1 1 0 0 AVS4304_2 1 0 0 0 AVS4202_2 1 0 0 0 AVS2997_2 1 1 0 0 AVS4017_2 1 0 0 0 AVS3017_2 1 0 0 0 AVS4021_2 1 1 1 0 FL713 1 1 0 0 FL607 1 0 0 0 FL660 1 0 0 0 12

EIZ0-16 1 1 0 0 FL554 1 1 1 1 FL590 1 0 0 0 AVS4267 1 1 0 0 AVS4257 1 0 0 0 TH016 1 0 0 0 AVS4001 1 0 0 0 AVS3032 1 1 1 1 AVS2472 1 1 1 0 AVS2441 1 0 0 0 CPXXX 1 0 0 0 FL671 1 0 0 0 AVS4339 1 0 1 0 361H 2010 Hawaii HILO 1 0 GAN 0 0 AVS4190 1 1 0 0 AVS4184 1 0 0 0 4180 1 1 0 0 PH001 1 0 1 0 CdB120110 1 0 0 0

16S rRNA amplicon sequencing

Fifty-two DNA extractions, consisting of 49 individually extracted ponerine gasters and three blank extractions (i.e. with no added biological material), were submitted to the Argonne National Laboratory for Illumina MiSeq paired-end sequencing. This generated a total of 1,822,087 raw reads across all 52 libraries. These sequences were analyzed using mothur version 1.36.0 (Schloss et al. 2009). Each sequence was trimmed to a minimum length of 251 bp. In order to expedite downstream contamination removal, redundant sequences were grouped into 405,831 unique sequences. Rare unique sequences were discarded using the split.abund function in mothur with a threshold of 15 reads. 13

A count table consisting of unique sequence indexes and their corresponding representation in each library was then exported to Microsoft Excel, where contaminant sequences and singletons were manually removed. This was accomplished by converting the values in the unique sequence count table into relative abundances (fraction represented in each library), and comparing the relative abundances of each unique sequence in the blank extraction libraries to the ponerine extraction libraries. Unique sequences were deemed to be contaminants if their maximum relative abundance across blank libraries was more than ten times greater than their maximum relative abundance across ponerine libraries. One ponerine library, “CPXXX” lost over 75% of its total read count through this process and was discarded from further analysis, as were blank libraries. This count table is supplied in Appendix 2.

Remaining unique sequences (3,338 unique sequences representing 1,002,373 total reads) were aligned against the SILVA database (version 119) in mothur. Unique sequences that failed to overlap with nucleotide positions 13,862-23,444 were removed from the analysis, and remaining sequences were trimmed again to fall within this region of the 16S rRNA gene. The trimming process generated new duplicate sequences, which were again condensed into unique sequences. Chimeras were detected and removed using

UCHIME in mothur. UCHIME is a program that analyzes short sequences against either a pre-existing database or by using abundance data to find “chimeras”, artificial sequences resulting from PCR error (Edgar et al. 2011). The remaining non-chimeric, unique sequences were then classified using the Ribosomal Database Project’s rpd10 reference alignment (Cole et al. 2014). On the basis of this taxonomic classification, 14 sequences originating from eukaryotic nuclear genomes, mitochondrial and chloroplast

DNA were removed.

Using a distance matrix of nucleotide dissimilarity, the final pool of 3,614 unique sequences was binned into 97% Operational Taxonomic Units (OTU’s) with mothur’s average neighbor clustering method. With these data, a file complete with read numbers, representative sequences and taxonomic classification was generated. The taxonomic assignments of the top 20 most abundant OTU’s were verified by uploading the representative FASTA-formatted file to the SeqMatch tool from the Ribosomal Database

Project.

PCR Screening and sequencing: Wolbachia, Unclassified Firmicutes, and

Unclassified Entomoplasmatales

I used a diagnostic PCR-based approach to screen for and sequence DNA from several groups of known, ant-associated bacterial symbionts in ponerine samples.

For all PCR screening reactions, per sample I used 5 µl of MyTaq HS Red Mix (Bioline

Ltd.); 1 µl of DNA; primers at 500 nM each; and added water to 10 µl. For all PCR sequencing reactions, I used 12.5 µl of MyTaq HS Red Mix (Bioline Ltd.); 1.5 µl of

DNA; primers at 500 nM each; and added water to reach a total volume of 25 µl.

Wolbachia screening was done using primers (wsp81F and wsp691R) targeting an antigen protein-coding gene, wsp (Zhou et al. 1998). PCR cycling conditions for wsp are as follows: 95°C for 1 minute, 40 cycles of 95°C for 15 seconds, 55°C for 15 seconds,

72°C for 20 seconds; 72°C for 2 minutes. 15

For Unclassified Firmicutes, screening was done using primers (rplB-Ento1F and rplB-UNF1R) targeting a gene that encodes for the 50S ribosomal protein L2, rplB. PCR cycling conditions for Firmicute rplB are as follows: 95°C for 2 minutes, 15 cycles of

95°C for 15 seconds, 58°C-->53.8°C (decreasing by 0.3°C each cycle) for 15 seconds,

72°C for 30 seconds; 35 cycles of 95°C for 15 seconds, 48°C for 15 seconds, 72°C for 30 seconds; 72C° for 1 minute

For Entomoplasmatales, screening was done using primers (UNE-Forward5 and

UNE1R) specific bacteria from the order Entomoplasmatales. PCR cycling conditions for

Entomoplasmatales PCR were as follows: 95°C for 1 minute, 45 cycles of 95°C for 15 seconds, 54°C for 15 seconds, 72°C for 20 seconds; 72°C for 2 minutes.

Phylogenetic Tree Construction

Unclassified Firmicute rplB nucleotide sequences from ponerines were compiled and manually edited in CodonCode Aligner version 4.2.7. Edited sequences were trimmed and exported into SeaView version 4.5.3. Ponerine nucleotide rplB sequences, rplB sequences from army ants and several outgroup sequences from Genbank were translated into amino acid sequences and aligned using the MUSCLE algorithm. The resulting amino acid alignment was converted back into nucleotides, and we inferred a maximum likelihood phylogeny using the model GTR with 100 bootstrap replicates

(Gouy et al. 2010). The most likely tree was uploaded to iToL to facilitate annotation of demographic information including host classification and location data (Letunic & Bork

2007, 2011).

2.3 Results

16S rRNA profile of gut microbiomes across the ponerines

The 3,614 unique sequences remaining after quality control were binned into a total of 158 97% OTUs. Each library consisted of between 1 and 24 OTUs with a mean of 6.4. A graphical representation of the relative abundance of 97% OTUs within individual ant libraries is shown in Figure 1.

Figure 1: Relative abundance of bacteria across ponerine genera and species. Each bar displays the percentage of

Illumina reads classified to 97% OTU’s. Taxonomic assignment of important OTU’s are provided on the right-hand side vertical axis. Host genus is supplied on the bottom horizontal axis.

Libraries from ants in the genus Odontomachus were mostly dominated by OTU’s

1 and 2-4 which correspond to the genus Wolbachia (Alphaproteobacteria: Rickettsiales) and phylum Firmicutes (could not be classified any more specificallly), respectively. The unclassified Firmicutes corresponding to OTU 2-4 appeared only in Odontomachus 17 samples, and in fourteen out of seventeen libraries containing unclassified Firmicutes, were present at greater than 70% relative abundance. Overall, unclassified Firmicutes were constrained to Odontomachus and Paraponera libraries, with the exception of a single occurrence in one Anochetus library. Similarly, seven out of eight Anochetus libraries contained an OTU corresponding to Wolbachia.

Wolbachia sequences were present in 28 out of 48 libraries, and appeared at least once in every host genus except for the genus Neoponera. In 21 out of the 28 libraries containing Wolbachia, Wolbachia reads exceeded 75% relative abundance. Other OTU’s with high sequence similarity to previously described bacterial ant associates are OTU 6

(Order Actinomycetales; 99% nucleotide sequence identity to an unclassified

Actinomycetales found in an Aphaenogaster (Russell et al. 2012)), and OTU 11 (family

Enterobacteriaceae; 99% nucleotide similarity to a Blochmannia symbiont of a

Camponotus species). Lastly, OTU 16 (family Entomoplasmataceae), found in two

Odontomachus libraries and one Paraponera library, matched with 99% nucleotide sequence identity to a group of unclassified Entomoplasmatales that has previously shown to form host-specific clades within two major unrelated army ant clades, the

Aenictinae and Ecitoninae (Funaro et al. 2011).

Libraries corresponding to ants belonging to the genera Odontomachus and

Anochetus were also analyzed with respect to a newly revised host phylogeny (Larabee

PhD Dissertation 2015). 18

jawed ponerinesjawed

side. Bacterial OTU's are color coded with respect to taxonomic are taxonomic colorOTU's to respect Bacterial with coded side.

). Host phylogeny is displayed on left hand side, while bacterial 97% OTU is 97% OTU bacterial hand is displayed while on left side, phylogeny ). Host

Anochetus

and

Host phylogeny adapted from Larabee PhD 2015. adaptedLarabee Thesis from Hostphylogeny

Relative abundance of bacteria across the phylogeny of trap the across phylogeny bacteria abundance of Relative

Odnotomachus

Figure 2 Figure (genera horizontalon hand indicatedbarsby right assignment.

The Wolbachia OTU, shown in purple, is found in all three Odontomachus clades

(Figure 2). OTU 5, which corresponds to Rhizobiales, appears in two libraries and is limited to one of the three Odontomachus clades. Unclassified Firmicutes OTU’s, shown in shades of blue, are scattered across the three Odontomachus clades. Two of the

Unclassified Firmicutes OTU’s are limited to host clade C, while the remainder of the

Unclassified Firmicutes OTU’s are not confined to a single host clade.

Wolbachia (Alphaproteobacteria:Rickettsiales) screening and analysis

While Illumina sequencing gave insight into bacterial prevalence, I also measured the infection rates for Wolbachia across ponerine ants using PCR primers specific for protein coding genes. Out of a total of 132 ponerine worker DNA extractions, 84 were determined to contain sufficient DNA via amplification of CO1. A total of 29 ponerine ant DNA extractions (34.5%) yielded band sizes consistent with Wolbachia wsp on a 2% agarose gel (Table 1).

PCR products amplified with wsp primers were submitted to Eurofins MWG

Operon for sequencing. Based on visual determination in the program CodonCode, 14 Of the 29 wsp positive samples were determined to contain single infections of Wolbachia.

A Maximum-Likelihood phylogenetic tree containing the 14 clean wsp sequences and their 40 closest BLAST hits is displayed in Figure 3. 20

Figure 3: Maximum-Likelihood phylogeny of ant-associated Wolbachia wsp sequences based on a ~500bp alignment. Host order is indicated by colored nodes, while host geography is indicated by the outer ring.

Each wsp sequence was also analyzed with respect to the amino acid motifs of four previously characterized hypervariable regions of wsp, per instructions in Baldo et al. 2006. HVR analysis of the 14 wsp ponerine sequences recovered three previously characterized wsp allelles. Five ponerine wsp sequences grouped into a single wsp allele type, wsp18. Results of wsp HVR allele characterization are displayed in Table 2.

wsp-18

wsp-50

wsp-18

wsp-28 wsp Allelewsp

HVR4

HVR4-21

Unidentified

HVR4-9; HVR4-271 HVR4-9;

HVR4-267; HVR4-256 HVR4-267;

HVR4-25 HVR4-269 HVR4-272 HVR4-269 HVR4-25

HVR4-14; HVR4-267; HVR4-256 HVR4-267; HVR4-14;

HVR4-14; HVR4-267; HVR4-256 HVR4-267; HVR4-14; HVR4-14; HVR4-267; HVR4-256 HVR4-267; HVR4-14;

HVR4-256 HVR4-267; HVR4-14;

HVR3 HVR3

HVR3-9

HVR3-17

HVR3-15

HVR3-17

HVR3-76

HVR3-17 HVR3-25

HVR3-17

Unidentified

HVR2 HVR2

HVR2-9

HVR2-15

HVR2-43

HVR2-76

HVR2-15 HVR2-21

HVR2-175

Unidentified

Unidentified HVR1

HVR1-9

HVR1-13

HVR1-42

HVR1-13

HVR1-21

HVR1-13

HVR1-238

HVR1-229

Unidentified

Unidentified allele 18, the most commonly found HVR allele, allele, HVR found mostcommonly 18, the allele

Old

New

wsp OldNewvs World

Peru

Taiwan

Uganda

Country

Malaysia

Australia

Malaysia

Australia Costa Rica Costa

: Wolbachia wsp allele analysis. Each row corresponds to a clean wsp clean tocorresponds Each a row analysis. allele : Wolbachiawsp

Species

africanus

monticola troglodytes

erythrocephalus

highlighted in yellow. highlighted in

Table2 ant. sequence from ponerine a is

Genus

Anochetus

Leptogenys Paraponera

Leptogenys

Odontomachus

TW02

FL713

173-4

TW01

EIZ0-16

Sample

AVS4021

AVS4184

AVS4017 AVS4190

AVS2485

AVS4202-1

AVS4202-2

AVS4021-2 AVS2997-2

Unclassified Firmicutes screening and phylogeny

Unclassified Firmicutes were present at a lower infection frequency relative to

Wolbachia; out of the 84 ants screened for unclassified Firmicutes, 17 tested positive for unclassified Firmicute DNA via PCR screening, netting an infection rate of 20.2% (Table

1). Based on host phylogenetic information, among the samples used for amplicon sequencing, Unclassified Firmicutes in the ponerines are enriched in the genus

Odontomachus.

Using a 250bp alignment generated from representative sequences from each ponerine Unclassified Firmicute OTU, army ant Unclassified Firmicutes and top BLAST hits from environmental sources was generated in Seaview.

Figure 4: Maximum likelihood phylogeny of ant-associated Unclassified Firmicutes 16S sequences from ponerids, army ants and related environmental bacteria based on a ~250 bp alignment. Host taxonomy is indicated by colors on right hand side of bacterial phylogeny.

The resulting Maximum-Likelihood tree (Figure 4) shows an ant-specific clade containing 18 of the 20 ponerine Unclassified Firmicute 16S sequences with army ant

Unclassified Firmicute sequences. All army ant Unclassified Firmicute sequences, a ponerine sequence from a previous study, and two of the 18 ant-specific ponerine sequences form a subgroup with 79% bootstrap support within this ant-specific clade.

The two remaining Unclassified Firmicute 16S sequences originating from ponerine libraries group more closely with environmental bacteria. 24

With PCR primers used in the diagnostic screening for Unclassified Firmicutes, rplB sequences were obtained for samples that tested positive in the PCR screen. Samples with sufficient template quality for rplB sequencing are indicated in Table 2. A maximum likelihood tree displaying ponerine rplB sequences alongside rplB sequences from army ants and free living relatives is shown in Figure 5.

Figure 5: Maximum-Likelihood phylogeny of army-ant and ponerine associated unclassified Firmicutes rplB gene based on a ~580 bp nucleotide alignment. Branch tip colors correspond to host subfamily. Outer ring corresponds to geography.

This tree shows ponerine Unclassified Firmicute rplB sequences grouping together within an ant-specific clade which excludes environmental bacteria. One 25 ponerine rplB sequence did not group within the mostly-ponerine sub-group of the ant- specific lineage, but grouped with other rplB sequences from army ants.

Unclassified Entomoplasmatales analysis

Three ponerine libraries contained OTU 16, which matched with 99% sequence similarity to an Entomoplasmatales lineage originally found to be exclusive to army ants

(but see Kautz et al. 2013). Additionally, two more libraries contained OTU’s 12 and 49 which matched with 99% sequence ID to an Entomoplasma and a Spiroplasma, respectively. All OTU’s from the amplicon submission containing bacteria from the order

Entomoplasmatales were amplified with a set of PCR primers designed to provide longer

(~600bp) 16S rRNA sequences. The extended 16S representative sequence from OTU16 was placed on a maximum likelihood phylogeny, along with 16S sequences from the top

50 non-redundant BLAST hits containing ant-associated Spiroplasmas, Mesoplasmas and

Entomoplasmas. 26

Figure 6: Maximum Likelihood phylogeny of Ant-associated Entomoplasmatales 16S rRNA sequences based on a ~600bp alignment. Colors correspond to host subfamily, and are indicated in the key provided. Outer circle indicates old world vs new world origin.

This tree nests a ponerine-specific group within a larger ant-specific lineage of

Unclassified Entomoplasmatales. The ponerine-specific Entomoplamatales lineage has a strong (95%) bootstrap support value. The ant-specific group consists of both Old World and New World ants, without segregation based on host geography.

2.4 Discussion

The finding of Wolbachia in the ponerine ants was not unexpected; Wolbachia is estimated to infect 66% of all insect species (Hilgenboecker et al. 2008), and several 27 studies have demonstrated the presence of Wolbachia in ants, including members of the ponerines (Russell et al. 2009; Kautz, Rubin, and Moreau 2013). It is important to cautiously interpret the 34.5% infection frequency of Wolbachia; Wolbachia infection frequencies are known to be highly variable both within colonies and within species.

Also, further taxonomic assignment about Wolbachia infected and uninfected hosts is required before we can make clearer conclusions of the prevalence of Wolbachia infections within species and genera across the ponerine ants.

Wsp sequences from ponerine ants did not seem to cluster strongly with each other compared to wsp sequences from other ant subfamilies. Overall, host geography appears to better match the wsp phylogeny compared to host taxonomy. Old world and new world ants tend to cluster separately, with some exceptions; major old world/new world clades are interrupted occasionally by a few new world/old world wsp sequences

(Figure 3).

We discovered intermediate frequencies of Unclassified Firmicute infection across the ponerines, with evidence of enrichment in the genus Odontomachus. These unclassified Firmicutes match closely at both the 16S rRNA and RplB sequence level to associates of unrelated army ant lineages. These sequences form an ant-specific clade both at the 16S and at the protein coding gene level, with ponerine sequences mostly clustering together. Similarly, the unclassified Entomoplasmatales OTU recovered from three ponerine libraries closely matches that of unclassified Entomoplasmatales associates that have previously been found to be limited to ponerines and army ants

(Funaro et al. 2011, Kautz et al. 2013). Using the phylogeny based on the extended 600 28 bp 16S Entomoplasmatales sequences, the ponerine unclassified Entomoplasmatales groups into the ant-specific clade consisting strictly of army ant and ponerine associates.

This is especially exciting; it appears that these three lineages of unrelated ants all possess two closely related, ant-specific bacterial lineages. When present, ant-associated

Entomoplasmatales dominate the library. This is consistent with the findings from the

2011 Funaro study, where the rank abundance of Entomoplasmatales was either first or second (Funaro et al. 2011).

Finally, we found evidence of two more bacteria closely related to known ant associates: an unclassified Enterobacteriaceae and an unclassified Rhizobiales. The unclassified Enterobacteriaceae was found in high abundance in one Paraponera library and one Odontomachus library, and at low abundance in a Leptogenys library. This bacterium matched with 99% sequence similarity to Blochmannia, the obligate symbiont of carpenter ants. Given the highly coevolved relationship between carpenter ants and

Blochmannia, this sequence is probably a result of a ponerine ant consuming a carpenter ant, rather than being an actual, functional symbiont of a ponerine. The unclassified

Rhizobiales was found in one Paraponera library and three Odontomachus libraries, and closely resembles the Bartonella-like bacterium formerly found in Paraponera clavata

(Larson et al 2014).

3. Concluding remarks and recommendations

By using Illumina amplicon sequencing of 16S rRNA, diagnostic PCR and focused protein-coding gene sequencing, I have taken some of the first steps toward the characterization of the microbes that associate with ants from the subfamily Ponerinae. 29

Our results of Wolbachia screening and sequencing were not unexpected; the rates of

Wolbachia infection across ponerine ants surveyed did not deviate significantly from previously published figures, which place Wolbachia infections at roughly 30% of ant species (Russell et al. 2012). In order to conclude that the screening results were reflective of real-world Wolbachia infections across the ponerines, further screening needs to be done within colonies of conspecifics. The numbers that I generated here are at best a conservative underestimate of true Wolbachia/unclassified Firmicute abundance.

The finding of prevalent unclassified Firmicute and intermediate frequencies of unclassified Entomoplasmatales across the Odontomachus (and possibly other ponerine genera) is strong evidence of symbiont convergence between unrelated ants at one end of the food chain, given the overall tendency of ponerine ants and army ants to live relatively carnivorous lifestyles. It is also intriguing that several ponerine species such as those from the genus Simopelta exhibit army-ant like raiding behavior (Schmidt and

Shattuck 2014); however, none of our samples with full species definitions harboring either unclassified Firmicutes or unclassified Entomoplasmatales are known to exhibit army ant-like behavior. Even if the presence of these two carnivorous ant-specific groups of bacteria are not limited to ants specifically exhibiting raiding behavior, the fact that they are both limited exclusively to army ants and ponerines suggests that host diet may be at play.

All together, these results add to our current knowledge of ant-associated symbionts. We recapitulated some previous findings, including the presence of

Rhizobiales in the omnivorous Paraponera clavata, as well as overall intermediate 30

Wolbachia infection frequencies. The discovery that army-ant associated unclassified

Firmicutes and Entomoplasmatales are not just present in a single ponerine species hints at the need to conduct wider PCR-based surveys across other ant taxa that may exhibit similar diets or overall lifestyles.

Given the presence of two dominant, ant-specific bacterial taxa in army ants and ponerines, the unclassified Entomoplasmatales and unclassified Firmicutes, future work should be focused on determining the functions that these bacteria may perform in their respective hosts. Previous PCR-based screening within army ant colonies found that the unclassified Entomoplasmatales are present at intermediate frequencies within colonies and across life stages, suggesting that, at least in army ants, the unclassified

Entomoplasmatales are not strictly required for survival (Funaro et al. 2011).

Additionally, the unclassified Entomoplasmatales were not found in reproductive regions of army ants via tissue-specific PCR screenings, suggesting that transovarial transmission is unlikely as a method of transmission (Funaro et al. 2011). Within-colony PCR screening of unclassified Entomoplasmatales in ponerine ants would therefore be useful in determining the necessity of this symbiont in ponerine ants, as well as the necessity of the unclassified Firmicutes toward host survival. Future PCR-based screenings in general should be performed on ants that have been starved before preservation in ethanol. This is especially apparent, given our finding of Blochmannia, which is almost certainly not a true symbiont of ponerines. By allowing food items to pass through completely before screening, we would be able to conclusively separate symbionts of prey items and ambient bacteria from true symbionts. 31

Functions of the unclassified Firmicutes and unclassified Entomoplasmatales could be determined through several means; RNA probe-based microscopy that targets either of these bacteria in host tissues would indicate the locations of these symbionts with respect to host organs. Additionally, metagenomic or metatranscriptomic sequencing of infected hosts could also yield information about metabolic pathways that these bacteria may be complementing, if they are indeed involved in nutrition.

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Appendix 1: List of samples used in this study with collection ID's, taxonomic assignments, and demographic information

Collection Code Genus Species Date Country State/Prov. County/Location 1 City/Location 2 Latitude Longitude Elev (m) Habitat Hoffmann Anochetus Australia Northern Terr Nhulunbuy TW01 Anochetus Taiwan MAL26 Anochetus Malaysia Sabah Malaui Basin Flavia Anochetus Malaysia Sabah Malaui Basin RSB352 Anochetus Malaysia AVS4188 Anochetus 10.viii.2012 Uganda Kibale N.P. Kenyawara Bio. Stn. N 0.56437 E 30.36059 1510m evergreen forest AVS4189 Anochetus 10.viii.2012 Uganda Kibale N.P. Kenyawara Bio. Stn. N 0.56437 E 30.36059 1510m evergreen forest AVS4268 Anochetus 6.viii.2013 Peru Cusco Est. Biol. Villa Carmon S 12.89474 W 71.403850 520m successional vegetation AVS4278 Anochetus 8.viii.2013 Peru Cusco Est. Biol. Villa Carmon across riverfrom station 650m successional vegetation AVS4202 Anochetus 13.viii.2012 Uganda Kibale N.P. Kenyawara Bio. Stn. N 0.56437 E 30.36059 1510m evergreen forest AVS3017 Anochetus 13.viii.2006 Australia Queensland Cairns nr. Mareeba open woodland AVS4359 Diacamma Malaysia Sabah AVS4358 Haregnathus Malaysia Sabah AVS4315 Leptogenys Taiwan AVS2997 Leptogenys 11.viii.2006 Australia Queensland Cairns James Cook Univ, Smithfield rainforest AVS4146 Leptogenys 27.vi.2012 Cambodia Pursat Prov. Cardamom Mountains "bird trail" N 12.04708 E 103.20239 560m selectively logged forest FL601 Odontomachus Australia FL606 Odontomachus Australia FL607 Odontomachus Australia FL617 Odontomachus Australia FL618 Leptogenys 22.vii.2014 Australia Queensland Lockhart River -12.70693 143.29367 FL624 Odontomachus Australia FL716 Odontomachus Australia Brian Odontomachus monticola Apr-10 Taiwan New Taipei City AVS4304 Odontomachus turneri Australia Northern Terr Nhulunbuy TW02 Odontomachus monticola Taiwan A.Smith-01 Odontomachus relictus Jun-10 USA Florida Withlacoochi State Forest A.Smith-02 Odontomachus brunneus 2013-3 USA Florida Archbold Ranch colony 2013-3 A.Smith-03 Odontomachus brunneus 2013-2 USA Florida Withlacoochi State Forest colony 2013-2 A.Smith-04 Odontomachus haematodus Jan-10 USA Alabama Week's Bay colony 12 A.Smith-05 Odontomachus haematodus Jan-10 USA Alabama Week's Bay colony 6 A.Smith-06 Odontomachus haematodus Feb-10 USA Alabama Week's Bay colony 5 A.Smith-07 Odontomachus haematodus Feb-10 USA Alabama Week's Bay colony 11 A.Smith-08 Odontomachus ruginoides 2014-4 USA Florida Land O Lakes colony 4 A.Smith-09 Odontomachus brunneus 2013-2 USA Florida Archbold Ranch colony 2013-2 A.Smith-10 Odontomachus haematodus Feb-10 USA Alabama Week's Bay colony 4 A.Smith-11 Odontomachus ruginoides 2013-3 USA Florida Archbold Ranch colony 2013-3 A.Smith-12 Odontomachus ruginoides 2014-2 USA Florida Archbold Ranch colony 2014-2 A.Smith-13 Odontomachus ruginoides 2014-1 USA Florida Land O Lakes colony 2014-1 A.Smith-14 Odontomachus relictus 2013-2 USA Florida Station colony 2013-2 A.Smith-15 Odontomachus ruginoides 2013-2 USA Florida Archbold Ranch colony 2013-2 A.Smith-16 Odontomachus brunneus 2013-1 USA Florida Withlacoochi State Forest colony 2013-1 A.Smith-17 Odontomachus brunneus 2014-1 USA Florida Plant City colony 2014-1 A.Smith-18 Odontomachus relictus 2013-2 USA Florida Withlacoochi State Forest colony 2013-2 A.Smith-19 Odontomachus relictus 2013-1 USA Florida Station colony 2013-1 A.Smith-20 Odontomachus relictus 2013-1 USA Florida Withlacoochi State Forest colony 2013-1 AVS4273 Odontomachus 6.viii.2013 Peru Cusco Est. Biol. Villa Carmon S 12.89474 W 71.403850 520m successional vegetation AVS4202-1 Odontomachus 13.viii.2012 Uganda Kibale N.P. Kenyawara Bio. Stn. N 0.56437 E 30.36059 AVS4252 Odontomachus 11.ii.2013 Panama Gamboa STRI neighborhood urban / tropical forest AVS4113 Odontomachus rixosus 25.vi.2012 Cambodia Pursat Prov. Cardamom Mountains "riverbed" N 12.37569 E 103.09410 300m riparian forest AVS4124 Odontomachus 26.vi.2012 Cambodia Pursat Prov. Cardamom Mountains "near leech trail" N 12.15066 E 103.19118 525m secondary forest AVS4153 Odontomachus 28.vi.2012 Cambodia Pursat Prov. Cardamom Mountains "bird trail" N 12.04708 E 103.20239 560m selectively logged forest AVS4083 Odontomachus 24.vi.2012 Cambodia Koh Kong Prov. Cardamom Mountains "meadow site" N 11.88546 E 103.07561 475m dipterocarp forest AVS3069 Odontomachus 27.i.2008 Argentina Misiones P.N. Iguazu Macuco Trail S 25o40.503 W 54o26.893 173m forgers by station AVS4017 Odontomachus 20.viii.2010 Malaysia Sabah Danum Valley West Side Trail AVS4021 Odontomachus 21.viii.2010 Malaysia Sabah Danum Valley West Side Trail AVS2957 Odontomachus laticeps 3.vii.2006 Costa Rica Heredia Est. Biol. La Selva N 10 26 W 84 01 100m tropical lowland rainforest AVS3019 Odontomachus ruficeps 13.viii.2006 Australia Queensland Cairns nr. Mareeba open woodland AVS2858 Odontomachus haematodus 6.xii.2005 Argentina Santa Fe Ocampo fish camp along river S 26o31' W 58o17' 50m along river at base of tree AVS2899 Odontomachus chelifer 11.xii.2005 Argentina Misiones P.N. Iguazu Macuco Trail S 25o40.503 W 54o26.893 173m on ground AVS2485 Odontomachus erythrocephalus 6.viii.2004 Costa Rica Heredia Est. Biol. La Selva N 10 26 W 84 01 100m tropical lowland rainforest AVS2766 Odontomachus clarus 5.viii.2005 USA Arizona Portal South West Research Station BLF22672 Odontomachus coquerelli Madagascar AVS4155 Odontoponera 26.vi.2012 Cambodia Pursat Prov. Cardamom Mountains "near leech trail" N 12.15066 E 103.19118 525m secondary forest AVS4322 Pachycondyla Taiwan AVS4269 Pachycondyla 6.viii.2013 Peru Cusco Est. Biol. Villa Carmon S 12.89474 W 71.403850 520m successional vegetation EMS2895 Pseudoneoponera Malaysia Sabah FL671 Odontomachus cephalotes? 27.vii.2014 Australia Queensland Lockhart River -12.71315 143.29843 37 m forest edge AVS4339 Odontomachus Australia Queensland AVSHilo Leptogenys USA Hawaii AVS4190 Anochetus 10.viii.2012 Uganda Kibale N.P. Kenyawara Bio. Stn. N 0.56437 E 30.36059 1510m evergreen forest AVS4184 Anochetus africanus 10.viii.2012 Uganda Kibale N.P. Kenyawara Bio. Stn. N 0.56437 E 30.36059 1510m evergreen forest FL360 Anochetus rugosus AVS4180 9.viii.2012 Uganda Kibale N.P. Kenyawara Bio. Stn. N 0.56437 E 30.36059 1510m evergreen forest PH0011 Odontomachus phillipinus CJB120/10 Anochetus AVS4267 Odontomachus haematodus Peru Est. Biol. Villa Carmon S 12.89474 W 71.403850 AVS4257 Odontomachus 13.ii.2013 Panama BCI TH16 Odontomachus opaciventris vi.2006 Costa Rica Las Cruces AVS3044 Odontomachus desertorum USA Arizona class trip with Bob desert AVS4001 Odontoponera 18.viii.2010 Malaysia Sabah Danum Valley station AVS3032 Odontomachus ruficeps (c.f.) 15.viii.2006 Australia Queensland Cairns Cape Tribulation rain forest AVS2472 Odontomachus hastatus 4.viii.2004 Costa Rica Heredia Est. Biol. La Selva N 10 26 W 84 01 100m tropical lowland rainforest AVS2441 Odontomachus bauri 3.viii.2004 Costa Rica Heredia Est. Biol. La Selva N 10 26 W 84 01 100m tropical lowland rainforest CPXXX Harpegnathos FL713 Leptogenys 30.vii.2014 Australia Queensland Lockhart River Portland Road -12.87743 S 143.00748 E FL600 Ponera FL607 Odontomachus FL660 Pachycondyla JTL6469-3 Anochetus simoni E12-0-16 Odontomachus troglodytes FL554 Odontomachus latidens FL590 Platythyrea punctata 139-4 Neoponera Peru 139-5 Neoponera Peru 173-1 Paraponera Peru 173-4 Paraponera Peru