Polymorphism. Aaron Nielsen. April 27, Abstract. Eusociality, the highest level of social organization, has evolved few times.

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1 A Survey of Recent Genetic Developments in Ant Social Polymorphism Aaron Nielsen April 27, 2017 Abstract Eusociality, the highest level of social organization, has evolved few times. The groups of animals which evolved eusociality include humans and mole rats, along with insects such as ants, termites, and some species of bees and wasps. Since the evolution of eusociality isn t common, the dynamics of how it works are of great interest to sociobioligists. The social structure of ants typically has a queen (or queens), soldiers, workers, drones, and other specialized castes. Traditionally, it was believed that environmental cues lead to the division of labor. This project will look at three recent developments that suggest there is a genetic component to ant social polymorphism. 1

2 1 Introduction We see ants so often and in so many places that it is easy to overlook their immense success in conquering the world. One estimate is that there are insects on the planet and at least 1% of all insects are ants. This suggests there are ants on Earth right now. It is also estimated that as much as 25% of the land-based animal biomass is ants. There are an estimated 22,000 species of ants that inhabit environments as diverse as the tropical rain forests, the Russian tundra, the Mongolian steppe, and on occasion, even unsavory places like restaurant kitchens. The only known habitat that ants haven t conquered is Antarctica. Despite these astounding figures, few take the time to consider how ants became one of the most successful organisms on Earth. Why are ants so successful? This is certainly a complicated question to answer but any answer should include the eusociality of ants. Eusociality is defined as the highest level of animal sociality with three common characteristics: cooperative care of the young, overlapping generations in a colony, and a division of labor groups. This type of social complexity has evolved few times on Earth. Other insects such as wasps, bees, and termites can be eusocial. Humans are certainly eusocial. A couple of species of rodents (mole rats) are eusocial. Beyond those examples, there aren t any other known eusocial animals on Earth. Needless to say, few species have evolved such a complex social structure. It is believed that this complex social structure has allowed ants to conquer nearly every environment on the planet. Not all ant species share the same characteristics but many have some common characteristic. Typically, a colony of ants has a designated queen that lays eggs, a worker class that tends the eggs and collects or farms food (yes, ants have discovered agriculture!), and a soldier class that battles rival colonies and other predators. In addition to this social structure, many ant species have a peculiar sex-determination system called haplodiploidy, where males develop from unfertilized eggs while females develop from fertilized eggs. The 2

3 result of haplodiploidy is that males only have one set of chromosomes whereas females have two sets of chromosomes. Due to this unique system, males share 100% of their genes with their mother while females share 50% of their genes with their mother. This, in turn, gives seemingly counter-intuitive results like the proportion of shared genes between sisters is 3/4 Additionally, a female shares 1/4 of its genes with a brother, and a male shares 1/2 of its genes with a sister. Since this results in a stronger genetic relationship between members of a colony, it is theorized that this has helped eusociality in ants evolve. Social polymorphism is the existence of two or more clearly different social castes. At the forefront of the complex genetic system in ants is a queen. Some ant species have one queen while others have multiple queens. Regardless of the number of queens, it turns out that in most ant species the majority of females not only don t reproduce, they actually can t reproduce. From an evolutionary standpoint, it would seem that sterility would be quickly eliminated, but it hasn t. Understanding how ants evolved eusociality thus requires an explanation for the complex reproductive system. Traditional thought is that all females could potentially develop reproductive capabilities in the larvae stage but environmental cues lead to nearly all of the females losing this ability in time. One possible environmental cue is if the queen dies or is unable to further reproduce. It is likely that other members of a colony are at least partially responsible for these environmental cues. The established theory that ant queens are thus produced from environmental cues is well established and confirmed. Three case studies that utilize statistical genetics to better understand the social structure of ants will be examined. These studies suggest possible deviations from traditional beliefs about ant social structure. 3

4 2 Case Study 1: Temnothorax longispinosus 2.1 About Temnothorax longispinosus Temnothorax longispinosus is an ant species that inhabits the Eastern United States and Canada. This species has been found in forests, on plant material such as acorns, and under rocks. The number of queens in a nest can depend on the colony and season, as there is evidence of monogynous (one queen per colony) and polygynous (multiple queens per colony) nests of this species. Sometimes queens are produced in queenless nests. New Figure 1: Temnothorax longispinosus queens are also occasionally introduced into existing colonies that already have a queen in a process called pleometrosis. After workers begin to hatch, it is common for all but one queen to be killed. The gene expression patterns of the ant species Temnothorax longispinosus were examined by Feldmeyer et al [6] and were found to have significant differences between reproductive females and their worker castes. In particular, it was found that the most significant difference in gene expression was between the queen and the worker classes and the least significant differences in gene expression were between infertile brood-tenders and foragers. The fact that different castes express different genes differently is not surprising based on existing theory and modern technology is able to confirm this in Temnothorax longispinosus. 4

5 2.2 Model and Methods Colonies of Temnothorax longispinosus were collected at the E. N. Huyck Preserve, Rensselaerville, NY in the summer of This species of ant is primarily monogynous and queens of this ant species are singly inseminated, so all workers are full-sisters. As a result of this, full-sisters share 3/4 of their genes. This seemingly suggests that genotypic differences are unlikely to affect the assignment of workers to different castes. Instead, there appears to be phenotypic plasticity or a difference in how genes are expressed for different castes. Twenty workers from eleven colonies were isolated from the rest of the colony and were classified by their behavioral caste. It was also determined whether or not the workers were fertile. RNA was extracted from the various castes and were compared. A total of 11,016 significant gene expression differences were found in pairwise comparison between castes. Since the workers of the species are monomorphic (similar size and anatomy), the ants in the study were classified according to behavioral castes. The queen was removed from the colonies to potentially induce the workers to develop reproductive capabilities. Each ant was classified as a forager if they spent over 80% of the observations outside of the nest, or it was classified as a brood-worker if it spent 90% of observations with the young. In addition, workers that developed ovaries and eggs in the absence of the queen were classified as fertile while workers that developed short ovaries without eggs were classified as infertile. Gene expression for four groups (queens, fertile workers, infertile workers, and foragers) was compared. RNA from several individuals within each group was pooled together to obtain sufficient RNA for sequencing. Utilizing the pooled RNA from the various groups, nonmetric multidimensional scaling (NMDS) was used to estimate dissimilarity in gene expression patterns for the four groups. Differences in gene expression were noted to be statistically significant if the p-value was below 0.05 after a false discovery rate (FDR) correction. 5

6 2.3 Analysis and Results Prior to pre-processing, 55,000-97,000 contigs (overlapping portions of RNA) were available. Missing data was dealt with by pooling castes together and then averaging over the gene expression. Members of the same caste were potentially obtained from different colonies. Averaging over the gene expressions was beneficial because it was of interest how gene expression varied by caste rather than by individual. After meta-assembly, 44,797 contigs remained. A total of 11,016 statistically significant expression differences were found using a test for difference in proportions after a FDR correction. 5,346 of the these differences were found to correspond to single genes. Using nonmetric multidimensional scaling (NMDS) in two dimensions, there appears to be clear evidence of expression differences between queens, fertile workers, and infertile females (infertile workers and foragers). There appeared to be similar expression patterns for infertile workers and foragers. Figure 2: Nonmetric multidimensional scaling (NMDS) plot of differentially expressed genes in Temnothorax longispinosus 6

7 In addition to noting the overall differences in gene expression utilizing NMDS, the number of shared and private differentially expressed genes can be analyzed. In the proceeding figure, these differentially expressed genes are shown in a Venn diagram. Figure 3: Venn diagram depicting the patterns of private and shared differentially expressed genes among the female castes This analysis allows for the determination of genes that are exclusively regulated differentially for a specific caste. Queens were found to have the most caste-specific genes while infertile workers and foragers tended to express a majority of the genes analyzed in a similar manner. Previous studies had not identified many of these caste-specific gene expressions. It was also notable that there were a significant number of worker-specific genes. This study suggests that not only have the phenotypes of queens and non-reproducing females diverged over time but also that caste-specific genes have evolved. 7

8 3 Case Study 2: Vollenhovia emeryi 3.1 About Vollenhovia emeryi Vollenhovia emeryi is an ant species found in North America (United States) and Asia (Korea, Japan, China, Thailand). This species of ant is queen-polymorphic, meaning that some colonies only produced longwinged queens while others only produced short-winged queens. Long-winged and Figure 4: Vollenhovia emeryi short-winged colonies can live in sympatry, that is, they can live in the same areas simultaneously. In addition, the species is polygynous, so multiple queens may be present in a nest. Vollenhovia emeryi have been found in forests and dead plant material such as branches. In Ohkawara et al (2006) [9], it was found that queens were homozygous at three microsatellite loci, whereas workers were mostly heterozygous at the same sites. The frequencies of genotypes at these three sites will be examined and statistical methods will be applied to see if there is a statistically significant difference in homozygous and heterozygous proportions between workers and reproductive females. This will either imply a complex genetic caste determination system or possibly the production of reproductive females from unfertilized eggs. 8

9 3.2 Model and Methods Colonies of Vollenhovia emeryi were collected in a mixed forest near the coast in Kanazawa City, Japan. Three DNA microsatellite loci (L-5, L-18, and Myrt-3) were amplified for the collected species and the three loci were chosen to use primers originally developed for Temnothorax nylanderi and Myrmica tahoensis. Microsatellites are tracts of repeated DNA and can be analyzed to a variety of ends. For this particular study, the goal was to study the direction of genetic flow between castes. The genotypes at the three loci were analyzed for the various castes and it was found that there was a significant difference between the various castes. DNA was extracted from the sampled ant heads and thoraces and the microsatellites at the three loci were amplified using PCR technology. Microsatellite alleles were identified using software and comparisons were made between castes using allele frequencies. A variety of statistical methods were utilized to analyze the data. First, an F-test was used within a hierarchical framework to analyze the molecular variance of the complete sample of genotypes. In addition, χ 2 test statistics were calculated to assess if there was a difference in allele frequencies between castes. 9

10 3.3 Analysis and Results Analysis of the DNA of Vollenhovia emeryi led to a number of discoveries. Queens and fertile females were overwhelmingly homozygous at the three loci. For instance, at L-5, long-winged colonies were 100% homozygous within the sample of queens and fertile females while shortwinged colonies produced only one example of a heterozygous fertile female. On the other hand, 87% of workers in L-colonies and 98.4% of workers in S-colonies were heterozygous. A χ 2 test found a statistically significant difference (p-value < ) in the frequency of homozygotes between queens and workers at the L-5 locus. Figure 5: Frequency of homozygous and heterozygous queens and workers from Vollenhovia emeryi colonies with long-winged queens (LQ, LQW) and colonies with short-winged queens (SQ, SQW) at the microsatellite loci (a) L-5; (b) L-18; and (c) Myrt-3 10

11 Using a hierarchical analysis of molecular variance, an F-statistic was also calculated and found to have a statistically significant excess of heterozygotes at L-5 for workers in comparison to fertile females and queens (95% CI: ( 0.578, 0.192)). Note that males are typically haploid and thus it doesn t make sense to discuss whether they were heterozygous or homozygous at the markers. Both the long-winged and the short-winged colonies exhibited statistically significant differences in molecular variance using the aforementioned method. These statistical methods along with some additional results gave three surprising results. The first result was that long-winged and the short-winged colonies were genetically differentiated. In particular, while the queens and fertile female were primarily homozygous at the three loci, they exhibited different alleles. At L-5, long-winged queens primarily had the genotype aa while the short-winged queens exclusively had the genotypes cc and dd. The second surprising result was that the workers were primarily heterozygous while the queens and fertile females were nearly exclusively homozygous. The final interesting result was that the males (at least in the short-winged colonies) appeared to carry the allele of the queen s mate. This last result is particularly surprising as male ants typically develop from an unfertilized egg but within this species, it appears that the genetic information that males receive is exclusively from the queen s mate. 11

12 4 Case Study 3: Pogonomyrmex rugosus and Pogonomyrmex barbatus 4.1 About Pogonomyrmex rugosus and Pogonomyrmex barbatus Pogonomyrmex rugosus is an ant species in the Southwestern United States and Mexico. It primar- ily feeds on seeds and dead insects while its predators include the black widow spider. Pogonomyrmex barbatus is a species highly related to Pogonomyrmex rugosus Figure 6: Pogonomyrmex rugosus and it is difficult to distinguish the two species. Pogonomyrmex barbatus also inhabits the southwestern United States and can live in sympatry (overlapping geography) or allopatry (non-overlapping geography) with Pogonomyrmex rugosus. Both species build large crater-like Figure 7: Pogonomyrmex barbatus mounds. In an interesting turn of events, these two species of ants in the American Southwest have been found to have a genetic element that contributed to production of queens. Genetic analysis of Pogonomyrmex rugosus and Pogonomyrmex barbatus [7] in areas of allopatry and sympatry suggest that hybridization of the species has led to queens being determined by genes rather than environmental cues. 12

13 4.2 Model and Methods Samples of each species were collected during mating season in areas of sympatry and allopatry within the states of Arizona, New Mexico, and Texas. DNA was extracted from the abdomens of the ants after their caste was determined. Randomly amplified polymorphic DNA (RAPD) genetic markers were used to examine genotypic differences between workers, queens, and males. Previous data has suggested hybridization occurs between the two species in areas of overlap and studies have shown that both species often have mtdna (mitochondrial DNA) of the other species. A specific primer, C9, was chosen as it was found to be particularly useful for identifying heterozygotes. 4.3 Analysis and Results Genotyping was completed using the genetic marker C9 for queens, workers, and males. χ 2 tests were completed to determine if there was statistically significant evidence of a difference in genotypes between the castes. At the C9 marker, there were two possible alleles, one of length 510 kb and one of length 550 kb. The results of the data collection are given in the following table. Figure 8: Genotypes based on C9 marker with two different alleles, one at 510 bp and one at 550bp 13

14 Pogonomyrmex rugosus has mostly homozygous in areas of overlap with Pogonomyrmex barbatus and there was a higher frequency of heterogyzotes in the sympatric colonies. In Pogonomyrmex barbatus sympatric colonies, queens were exclusively homozygotes while workers were exclusively heterozygotes. In allopatric colonies, Pogonomyrmex barbatus queens and workers had a more diverse division of heterozygotes and homozygotes. A χ 2 test was completed and found that there is a significant difference in the proportion of homozygotes between Pogonomyrmex rugosus allopatric and sympatric colonies (χ 2 test = 26.1, p-value < ). In addition, there was a statistically significant difference in the proportion of homozygotes in Pogonomyrmex barbatus allopatric and sympatric colonies (χ 2 test = 40, p-value < ). These statistical results give some interesting conclusions. First of all, whether the C9 marker was heterozygous or homozygous is a very good indicator of whether the ant became a queen or a worker. This shouldn t be interpreted to mean that this gene is causal in determining caste but rather that some structure of interbreeding is occurring. Unlike the previous case study of Vollenhovia emeryi, the males in these colonies appeared to inherit their genes exclusively from the queen mother. The alleles present in the workers that were not present in the males suggest they were inherited from the queen s mate. It is hypothesized that the difference in the frequency of homozygotes and heterzygotes in the sympatric and allopatric populations is the result of hybridization. Queens of these species mate with multiple males, so it s possible that workers of the colony are sterile hybrids, whereas reproductive females are produced from matings of like species. Since hybrids would be likely be sterile, there could be selection pressures on males and queens. Males that mate within the species would be much more likely to produce reproductive females. As a result, seleciton would favor males that are more likely to mate within the 14

15 species and this could result in a push towards a higher frequency of homozygotes. These results taken together suggest that eggs fertilized with semen from the sister species become infertile workers and eggs fertilized with semen from the same species are disproportionately likely to become queens. 5 Discussion The three studies presented in this paper utilized modern methods in statistical genetics to find some curious results in ant social dynamics that weren t previously known. In the first study of Temnothorax longispinosus, a North American ant, it was found that genes were differentially expressed by different castes. Not only were some genes differentially expressed by the various castes, some genes were only expressed by particular castes. Over 1,000 genes were identified as only being expressed by queens. A nonmetric multidimensional scaling analysis showed a significant difference in gene expression between queens and workers and between fertile and infertile workers. It showed a relative similarity in gene expression between infertile workers and foragers. The second study analyzing Vollenhovia emeryi found a number of interesting results. First of all, queens tended to be homozygous while workers tended to be heterozygous. Next, while long-winged queens and short-winged queens tended to be homozygous at the markers of interest, they exhibited different alleles. This suggests a lack of mating between the two types of Vollenhovia emeryi. Finally and probably most surprising is that males appeared to inherit genetic information from the queen s mate rather than the queen. It was previously assumed that males only received genetic information from the mother after hatching from an unfertilized egg. The final study involving Pogonomyrmex rugosus and Pogonomyrmex barbatus found ev- 15

16 idence of hybridization of the two species in ares of sympatry. In these areas, it appears that workers develop from eggs fertilized using sperm from the sister species while reproductive females develop from eggs fertilized from the same species. As in the case of Temnothorax longispinosus, there is additional speculation that the high rate of homozygosity in fertile females could be the result of thelytokous parthenogenesis or the production of females from unfertilized eggs. Taken together these three studies suggest that there is an abundance of potential research in ant social polymorphy using the methods of modern statistical genetics. While none of these studies fundamentally change knowledge of ants taken as a whole, they do suggest that individual species of ants may have modes of reproduction even more complex than most ants. Future studies could be designed similar to the ones presented but with different species. Additionally, new studies could examine other aspects of ants such as the phylogeny using genetic methods. 16

17 6 Appendix References [1] Hasere Ilaclama Antalya. Ants. wp-content/uploads/2015/05/images-11.jpg, [Online; accessed 05-December- 2015]. [2] Antwiki. Pogonomyrmex barbatus. barbatus, [Online; accessed 05-December-2015]. [3] Antwiki. Pogonomyrmex rugosus. rugosus, [Online; accessed 05-December-2015]. [4] Antwiki. Temnothorax longispinosus. longispinosus, [Online; accessed 05-December-2015]. [5] Antwiki. Vollenhovia emeryi [Online; accessed 05-December-2015]. [6] B. Feldmeyer, D. Elsner, and S. Foitzik. Gene expression patterns associated with caste and reproductive status in ants; worker-specific genes are more derived than queenspecific ones. Molecular Biology, 23: , [7] Glennis E. Julian, Jennifer H. Fewell, Jurgen Gadau, Robert A. Johnson, and Debbie Larrabee. Genetic determination of the queen caste in an ant hybrid zone. Proceedings of the National Academy of Sciences, 99(12): , [8] Michael Lynch and Bruce Walsh. Genetics and Analysis of Quantitative Traits. Sinauer Associates,

18 [9] Kyohsuke Ohkawara, Megumi Nakayama, Atsumi Satoh, Andreas Trindl, and Jurgen Heinze. Clonal reproduction and genetic caste differences in a queen-polymorphic ant, vollenhovia emeryi. Biology Letters, 2: , [10] Daniel Stram. Design, Analysis, and Interpretation of Genome-Wide Association Scans (Statistics for Biology and Health). Springer, [11] Wikipedia. Haplodiploidy [Online; accessed 05-December-2015]. [12] Edward O. Wilson. Sociobiology: The New Synthesis. Belknap Press, [13] Edward O. Wilson and Bert Hlldobler. Journey to the Ants: A Story of Scientific Exploration. Belknap Press,

19 6.1 Motivation My dissertation research with Dr. Burt Simon will involve modeling the evolution of cooperation using a multilevel selection framework (individiual selection, group selection, etc). A lot of research in the specific modes of selection today involves ants due to their eusociality. Traditional models have used kin selection to explaining the complexity of ant societies, but in recent years there has been a renewal in theories explain the evolution of ants in terms of group selection. I chose to study ant genetics in this project to familiarize myself with some of the vocabulary and ideas currently being published on ants, specifically using genetic material. 6.2 Project Evolution This project was initially intended to analyze a data set involving ant genetics. I attempted to contact the authors of a couple of papers for their raw data, but in both cases I was either unable to get in touch with the author or wasn t able to obtain the data sets. As a result of this, I decided to focus on exploring the results of three studies that challenged traditional beliefs about ants. The results used in this report were from the papers rather than being explicitly calculated. 6.3 Project Requirements Where possible, I tried to address the requirements laid out by the project requirements. Unfortunately, this wasn t always possible. Missing data was addressed in one of the data sets and likewise noted, but it wasn t addressed in the other studies. Most of the models utilized were assessed using simple χ 2 tests, so further details of the test were not provided. The types of data were noted and were typically just the genotypes at specific loci. Not a lot of evidence of pre-filtering was discussed in the individual papers save for one paper. 19