Burrowing Techniques, Behaviors, and Trace Morphologies of Extant Larval to Adult

Beetles

A thesis presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Master of Science

Joseph J. Wislocki

April 2021

This thesis titled

Burrowing Techniques, Behaviors, and Trace Morphologies of Extant Larval to Adult

Beetles

JOSEPH J. WISLOCKI

has been approved for

the Department of Geological Sciences

and the College of Arts and Sciences by

Daniel I. Hembree

Professor of Geological Sciences

Florenz Plassmann

Dean, College of Arts and Sciences 3

Abstract

WISLOCKI, JOSEPH J, M.S., April 2021, Geological Sciences

Burrowing Techniques, Behaviors, and Trace Morphologies of Extant Larval to Adult

Beetles

Director of Thesis: Daniel I. Hembree

Studies of the relationship between extant trace makers, known environmental conditions, and the morphology of their biogenic structures allow for the interpretation of continental ichnofossils. This study examined the burrowing techniques, behaviors, and trace morphologies of three extant species of burrowing beetles, Tenebrio molitor,

Zophobas morio, and Phyllophaga sp., from their larval to adult life stages under normal and stressed environmental conditions in a laboratory setting. Tenebrio molitor and Z. morio burrowed using their mandibles to compact the substrate, while Phyllophaga sp., burrowed by excavation and backfilling. The three species primary behaviors were locomotion, mobile deposit feeding, intermittent resting, and pupation. Larvae burrows of

T. molitor and Z. morio included open boxworks, while Phyllophaga sp. larvae generated elongate backfilled burrows which terminated in an open chamber. All three species created ovoid to ellipsoidal chambers when preparing for pupation. During their adult stage, T. molitor and Z. morio created conical traces and chambers, while Phyllophaga sp. produced loosely backfilled burrows. The environmental stresses tested were related to sediment sand and water content as well as sediment compactness. Higher trace abundance was produced in sediments with decreased sand content, increased water content, and low compactness, although trace morphologies did not change. Highly 4 compacted substrates had little activity, but distinct trace morphologies. The total level of bioturbation, quantified with the ichnofabric index, produced by multiple specimens of each species in large enclosures filled with layered sediment varied from 1 (T. molitor and Phyllophaga sp.) to 2-5 (Z. morio). Quantitative analyses of the quantitative properties of the different traces showed that, despite having similar morphologies, the traces produced by the three species were dissimilar, but also showed variation within species. Understanding extant traces of beetles can help in the recognition of their ichnofossils when body fossils are not found, which can allow for improved interpretations of paleoecosystems and paleoenvironments in continental settings. 5

Dedication

This is dedicated to my friends, family, supportive peers, and my professors both near and far. Geology is a pathway to many abilities, and I am glad you showed me the way.

Acknowledgments

I would like to thank the Geologic Society of America for the Graduate Student

Research Grant. I would like to thank the Ohio University Geological Science Alumni for their Graduate Research Grant. I would like to thank Ceara Purcell, Mary Reichle, and

Rachel Wawrzynski for assisting me in field collection and laboratory setup. I would like to thank my wonderful committee of Dr. Dan Hembree, Dr. Alycia Stigall, and Dr.

Xizhen Schenk for their support. I would like Dr. Dan Hembree for being an amazing advisor, and all-around great person.

Table of Contents

Page

Abstract ...... 3

Dedication ...... 5 Acknowledgments...... 6 List of Tables ...... 9 List of Figures ...... 10 Chapter 1: Introduction ...... 11 Chapter 2: Background ...... 14 Extant and Fossil Beetles ...... 14 Ichnology and Neoichnology of Beetles ...... 20 Chapter 3: Methods ...... 25 Experiment 1 ...... 31 Experiment 2 ...... 31 Experiment 3 ...... 31 Experiment 4 ...... 32 Experiment 5 ...... 32 Experiment 6 ...... 32 Burrow Analysis ...... 33 Comparison with Ichnofossils...... 35 Chapter 4: Results ...... 39 Burrow Techniques ...... 39 Larval Stage ...... 39 Adult Stage...... 41 Behavior ...... 42 Larval Stage ...... 42 Pupal Stage...... 44 Adult Stage...... 45 Burrow Morphology ...... 47 8

Larval Stage ...... 47 Pupal Stage...... 48 Adult Stage...... 51 Environmental Controls ...... 53 Experiment 3 ...... 53 Experiment 4 ...... 56 Experiment 5 ...... 59 Quantitative Analysis ...... 62 Comparison of Traces Between species ...... 62 Comparison of Traces Between Produced in Increased Sand vs Decreased Sand Substrate ...... 63 Comparison of Traces Produced in Increased Moisture vs Decreased moisture conditions ...... 64 Comparison of traces produced in firm vs loose substrate ...... 66 Comparison of extant traces with comparable ichnogenera ...... 68 Bioturbation and Ichnofabric ...... 72 Chapter 5: Discussion ...... 80 Controls on Beetle Trace Morphology ...... 80 Behaviors ...... 80 Body Morphology ...... 83 Specific Trace Maker ...... 85 Environmental Conditions ...... 87 Ichnofabric and Bioturbation Potential ...... 89 Preservation Potential ...... 90 Comparison to Ichnofossils...... 94 Chapter 6: Conclusion...... 99 References ...... 102 Appendix A ...... 114 Appendix B ...... 122

List of Tables Page

Table 1 Ichnofabric Index ...... 22 Table 2 Beetle Experimental Setup...... 26 Table 3 Bray-Curtis similary Indicies for Three Beetle Taxa, both Burrows and Chambers ...... 50 Table 4 Comparison of Traces Produced in Increased Sand vs Decreased Sand Substrate...... 64 Table 5 Comparison of Traces Produced in Increased Moisutre vs Decreased Moisture Substrate...... 66 Table 6 Comprison of Traces Produced in Firm and Loose Substrate ...... 68 Table 7 Comparison of Beetle Biogenic Structures to Ichnogenera ...... 71

List of Figures Page

Figure 1. Beetle Taxa Studied...... 17 Figure 2. Phylogenetic Tree...... 18 Figure 3. The Tools...... 28 Figure 4. The Enclosures...... 30 Figure 5. Burrow Descriptions...... 35 Figure 6. Known Beetle Ichnogenera ...... 37 Figure 7. Beetle Trace Behavior...... 40 Figure 8. Beetle Trace Morphology of Experiment 2...... 46 Figure 9. Adult Zophobas morio Chambers...... 52 Figure 10. Adult Phyllophaga sp. Distruption...... 53 Figure 11. Experiment 3...... 55 Figure 12. Experiment 4...... 58 Figure 13. Experiment 5...... 61 Figure 14. Experiment 6: Tenebrio molitor...... 73 Figure 15. Experiment 6: Phyllophaga sp...... 75 Figure 16. Experiment 6: Zophobas morio...... 77 Figure 17. Preservation of Burrows Over Time...... 93 Figure 18. Comparison of Beetle Burrows to Ichnogenera Visuals...... 97

Chapter 1: Introduction

Comparisons between ichnofossils and modern traces has led to the interpretation of their behavioral significance, the environmental conditions that controlled these behaviors, and even trace makers when body fossils are absent (Retallack, 1984; Genise et al., 2000; Counts and Hasiotis, 2009; Mikus and Uchman, 2013; Genise, 2017;

Hembree, 2016). However, burrows of most modern burrowing organisms are poorly understood scientifically, and further neoichnological studies of even common terrestrial animals are required to better understand the diversity of continental ichnofossils.

Although marine and continental depositional environments are similarly diverse (Genise et al., 2000), there are far fewer established ichnofacies for continental settings, and our knowledge of continental traces lags behind that of marine traces (Bromley, 1996;

Buatois and Mangano, 2011). Importantly, conditions affecting ichnofossil morphology in the marine are not always applicable to continental systems. Recent studies with continental organisms have improved our knowledge of these paleoecosystems (Hembree and Hasiotis, 2006; Smith and Hasiotis, 2008; Hembree, 2009; Counts and Hasiotis,

2009; Porkorny et al., 2015), but more work remains to be done.

While diverse and abundant today, beetles and their larvae are poorly represented in the fossil record, especially soil-dwelling groups. Despite being prolific burrowers most beetle traces are known only from Late Cretaceous to Neogene paleosols (Johnston et al., 1996; Counts and Hasiotis, 2009; Alonso-Zarza et al., 2014). This sparse record is likely a result of a lack of recognition of beetle-produced ichnofossils due to the limited 12 understanding of the range of biogenic structures produced by modern beetles over their full life cycle.

The purpose of this project was to examine the connections between three species of extant burrowing beetles, Tenebrio molitor, Zophobas morio, and Phyllophaga sp., their environmental conditions, and the morphology of their biogenic structures. The beetles were kept in a controlled laboratory setting to examine their behaviors, burrowing techniques, and resulting biogenic structures from their larval to adult life stages.

Environmental stresses that were applied included variations in sediment sand and water content and sediment compactness beyond those of their typical natural environments.

Five hypotheses were tested in this study: 1) T. molitor, Z. morio, and Phyllophaga sp. exhibit similar trace-making techniques and behaviors; 2) the beetles produce similar traces during each life stage due to similar body morphologies and behaviors; 3) changes in sediment composition, water content, and compactness result in new or altered behaviors and traces; 4) the beetle’s total bioturbation over their entire life cycle produces a moderate to high ichnofabric index; and 5) the beetles produce traces during each life stage comparable to known ichnogenera.

The results of this project provide valuable insights into several aspects of the trace morphologies of beetles. Direct connections were made between trace morphologies and observed behaviors and trace maker morphology. In addition, variations in trace morphology are linked to known substrate conditions. Finally, the total bioturbation potential of beetles over their life cycle is assessed to evaluate their role in substrate modification in continental environments. Descriptions of the beetles’ biogenic structures 13 can be used to identify and interpret similar ichnofossils when body fossils are absent, improving interpretations of the composition of ancient soil ecosystems and paleoenvironmental conditions.

Chapter 2: Background

Extant and Fossil Beetles

Beetles (Coleoptera) extend to the Carboniferous based on molecular genome dating but only to the Permian using body fossil data (McKenna et al., 2019). Beetles are the largest and most diverse order of insects, with roughly ~400,000 known species

(McKenna et al., 2019; Myer, 2020). Extant beetles have an environmental range that spans to nearly every landmass on Earth except modern Antarctica (Crowson, 1981), but recent studies indicate that their range did extend to Antarctica during the middle

Miocene (Ashworth and Erwin, 2016). Beetles are characterized by a head with strong mandibles, a thorax with three pairs of thoracic legs, and an abdomen. Adult beetles are covered in a dense, chitin exoskeleton. The elytra (frontal wings) cover their hind wings and abdomen forming a protective shield that forms a straight line, an easily identifiable feature of beetles (Lovei and Sunderland, 1996; Myer, 2020). Beetles are largely herbivores, but can also be omnivores, carnivores, detritivores, fungivores, carrion scavengers, or even coprophagous making them vital components of terrestrial food webs and ecosystems around the world (Cai et al., 2014; McKenna et al., 2019; Neita-Moreno,

2019). Their wide range of feeding styles, overall generalist behaviors, and life cycles which utilize a variety of feeding tiers has led beetles to occupy a variety of ecological niches within almost every type of terrestrial habit (Lovei and Sunderland, 1996;

Bradford et al., 2013; Ernst et al., 2016). Given this broad distribution, we might expect to find an abundance of body or trace fossils of beetles in a variety of sedimentary units. 15

Beetles undergo a complete metamorphosis during their life cycle, which consist of four stages (Keller and Cave, 2016). They start their life by emerging from an egg which is usually deposited in a substrate (Park, 1934; Axtell and Arends, 1990; Newton,

2008). After emerging, they enter their larval stage, which can last a few weeks to even years, during which their appearance is usually an elongate form, with a distinct head, thorax, and body segments on the abdomen (Axtell and Arends, 1990; Keller and Cave,

2016). The six legs of the larvae are located on the thorax which aid in movement and burrowing (Axtell and Arends, 1990; Counts and Hasiotis, 2008; Steiner Jr., 2014).

During this stage, the primary behavior of the larvae is to consume nutrients and prepare for metamorphosis, and intermitted resting (Axtell and Arends, 1990). Near the end of their larval stage, the larva forms a cavity or chamber in which to pupate (Axtell and

Arends, 1990); this pupal stage can last weeks to months (Axtell and Arends, 1990;

Keller and Cave, 2016). The pupa’s appearance varies between species, but overall, they are milky tan in color with six thoracic legs and three body sections, but no elytra. All the body parts and limbs undergo metamorphosis and development in this stage. After complete metamorphosis, the beetle emerges from its pupation chamber with their fully formed adult bodies. They immediately start searching for nutrients, and mates for reproduction (Axtell and Arends, 1990). During their different life stages, therefore, beetles display widely varying body morphologies and behaviors. As a result, there are unique trace morphologies that can be produced in each life stage. This provides the potential for a single beetle species to produce trace assemblages with both high ichnodiversity and ichnodisparity. 16

Two common groups of soil-dwelling beetles are the Tenebrionidae, which includes Tenebrio molitor and Zophobas morio, and Scarabaeidae, which includes

Phyllophaga sp. (Fig. 1) (Augustyn et al., 2016; Bousquet et al., 2018; Neita-Moreno et al., 2019). During their larval stages, members of the Tenebrionidae and Scarabaeidae have several morphological similarities as well as differences. Their worm-like larval forms are generally comparable, yet Scarabaeidae are much bulkier and larger with more pronounced mandibles, limbs, and head. Tenebrionidae are slender with smaller mandibles, limbs, and head. During the pupal stage, the beetles are the most comparable morphologically, yet Scarabaeidae pupae are usually squatter and rounder, while the

Tenebrionidae pupae are more elongated. During the adult stage they have comparable bulbous elytra and limbs, but the Scarabaeidae are more bulbous in appearance in comparison to the longer and sleeker Tenebrionidae. Despite their morphological similarities, Tenebrionoidea and Scarabaeoidea are not closely related (Fig. 2) (McKenna et al., 2019). Both families are first known from the Late Triassic (McKenna et al., 2019), although Scarabaeoidea is more common by the Late Jurassic (Krell, 2000; Counts and

Hasiotis, 2009). Members of the Tenebrionidae and Scarabaeidae also display differences in their ecological niches, life cycles, and behaviors. The extant members of these two families of beetles are semi-nomadic and semi-ground-dwelling with larval and pupal stages that inhabit the soil (McIntyre and Wiens, 1999; Brandhorst-Hubbard, 2001;

Counts and Hasiotis, 2009). In their larval stages, however, T. molitor is a near-surface to shallow-tier detritus feeder, Z. morio is a shallow to deep-tier detritus feeder, and

Phyllophaga sp. is a near-surface to deep-tier root and detritus feeder (McIntyre and 17

Wiens, 1999; Brandhorst-Hubbard, 2001). There is also variation in the duration of the life cycles between these species.

Figure 1

Beetle Taxa Studied

Note. Figure 1: A) Life stages of Phyllophaga sp. Left is larval stage, center the pupation stage (Cano, 2006), right the adult beetle. B) Life stages of Tenebrio molitor. Left is larval stage, center the pupation stage, right the adult beetle. C) Life stages of Zophobas morio. Left is larval stage, center the pupation stage, right the adult beetle. Scale bars represent 20 mm.

Figure 2

Phylogenetic Tree

Note. Figure 2: Phylogenetic tree based on the genome mapping depicting the relationships between

Tenebrionoidea, and Scarabaeoidea (Modified from McKenna et al., 2019).

The life cycle (larva to adult) of Tenebrio molitor is 6-8 months (Hill, 2002). This time is divided among 180-200 days of 16-24 larval instars, and 9-12 days during the pupal stage (Buttin et al., 199; Park et al., 2014). Specimens acquired were in the final instars, which shortened laboratory time significantly. Zophobas morio life cycle is roughly 27-99 days (Friederich and Volland, 2004). This is divided among 14-42 days of 19

8-12 larval instars, and 13-57 days of pupation (Delfosse, 2005). Specimens acquired were in the final instars, which shortened laboratory time significantly.

Phyllophaga sp. larval to adult cycle is 250-345 days (Reinhard, 1940). This is divided among 230-330 days of 3 instars, and 18-20 days of pupation (Reinhard, 1940).

Specimen were acquired in 2nd and 3rd instars, which shortened laboratory time. Overall, these three species are abundant, widely distributed, have short life cycles, and occupy a range of depths in the substrate, making them ideal candidates for experimental investigation of beetle biogenic structures.

Beetles have a generally poor preservation potential since they lack mineralized tissue (Doyen and Poinar Jr., 1994; Guerrero-Arenas et al., 2017). This low preservation potential of beetle fossils presents several problems since it creates a fossil record biased toward the Neogene and lacustrine and amber deposits, in particular (Smith et al 2006;

Smith and Marcot, 2015). Beetles experience rapid decay shortly after death, resulting in full disarticulation of the body (Smith et al., 2006). Increased preservation potential of beetles typically correlates with small size and robustness of the beetle’s exoskeleton, although waterlogging can increase resistance to transport and disarticulation (Smith et al., 2006). When beetles are preserved, it is primarily in amber (Doyen and Poinar Jr.,

1994; Kirejtshuk and Kernegger, 2008, Tarasov et al., 2016; Sadowski, 2017), but also in lacustrine, fluvial channels, and even volcanic deposits (Lubkin and Engel, 2005;

Sanchez and Genise, 2008; Kukalova-Peck and Beutel, 2012; Chang et al., 2016;

Ashworth and Erwin, 2016; Qin et al., 2019). Importantly, these environments of preservation are not the environments that these beetles inhabited in life. While the 20

Tenebrionidae is a large group of beetles, only a few body fossils have been described, mostly from Eocene Baltic amber (Doyen and Poinar Jr., 1994; Kirejtshuk and

Kernegger, 2008), as well as recent discoveries in the Jehol Biota of the

Jurassic/Cretaceous boundary in China (Chang et al., 2016). There are more fossil representatives of the Scarabaeidae but, compared to the overall diversity of the family they are still rare (Tarasov et al., 2016; Neita-Moreno et al., 2019). With ichnological studies, there is the potential to expand the temporal and paleogeographic distribution of different beetle groups like Tenebrionidae and Scarabaeidae since ichnofossils are preserved in situ and have a higher preservation potential than beetle body fossils

(Bromley, 1996; Buatois and Mangano, 2011; Smith and Marcot, 2015).

Ichnology and Neoichnology of Beetles

Ichnofossils are preserved biogenic structures that include burrows, borings, tracks, and trails (Bromley, 1996). Interpretations of ichnofossils can be made by comparing them to similar modern biogenic structures (Hasiotis and Bourke, 2006; Davis et al., 2007). Fundamentally, ichnofossil morphology can be used to interpret their behavioral significance (Bromley, 1996). In addition, ichnofossils have a direct connection to the environmental conditions in which they were produced, since they are preserved in situ (Bromley, 1996). Potential trace makers can also be identified through the comparison of ichnofossils to extant biogenic structures. Since extant trace makers are observed directly, these observations can give crucial insight into their specific trace- making techniques and behaviors and resulting trace morphologies which may then be tied to the specific organism. This can improve paleoecological interpretations because 21 while many organisms, such as beetles, have a low preservation potential, their presence can be indicated by their traces.

Total bioturbation is also an important factor in evaluating trace-making communities (Taylor and Goldring, 1993). Bioturbation is the total disruption, displacement, and reworking of the substrate by the life activities of a community of organisms (Taylor and Goldring, 1993). Bioturbation is described by ichnofabric which includes all aspects of the texture and structure of a substrate that resulted from the effects of organism activity (Taylor and Goldring, 1993). Ichnofabric is quantified using the ichnofabric index, which quantifies the bioturbation within a viewable section of sedimentary rock (Taylor and Goldring, 1993) (Table 1). Ichnofabric does not require recognition of discrete ichnotaxa, but is a measure of the overall disruption of the sediment; with a high ichnofabric index, for example, it is often impossible to identify any ichnogenera within the substrate (Taylor and Goldring, 1993). Understanding the total potential bioturbation that different extant animals can produce is important in evaluating ichnofabric. Due to their changes in morphology and behavior through their life cycles, beetles have the potential to produce a significant amount of bioturbation per individual. However, this has not been fully tested. With this information, total bioturbation can be used to suggest the activity of beetles and even community size without discrete ichnofossils (Reineck, 1963; Taylor and Goldring, 1993).

Table 1

Ichnofabric Index

Percent Grade Classification Bioturbated 0 0 No bioturbation. Sparse bioturbation with 1 1-4 few traces and structures. Low bioturbation, low 2 5-30 trace density, with escape structures. Moderate bioturbation, 3 31-60 decrete traces, low overlap. High bioturbation, high 4 61-90 trace density, overlap common. Intense bioturbation, 5 91-99 limited reworking, low burrow visibility. Complete bioturbation, 6 100 repeated overprinting.

Note. Table 1: Ichnofabric Index where each grade describes the effects of disruption, displacement, and reworking within a substrate by organisms (Modified from Taylor and Goldring, 1993).

Previous neoichnological studies of extant beetles in the laboratory and field have revealed some of the diversity of trace morphologies that they can produce. The larval stage of masked chafer beetles (Cyclocephala lurida or C. borealis) studied by Counts and Hasiotis (2008) produced adhesive meniscate backfilled burrows similar to ichnofossils found in continental deposits as old as the Permian. Previously it was assumed that ichnofossils like these were only developed in lacustrine and fluvial 23 deposits by worms or aquatic arthropods (Smith and Hasiotis, 2006; Counts and Hasiotis,

2008). In another neoichnological study, Mikus and Uchman (2013) studied extant beetle biogenic structures in the field along the Dunajec River in Poland. Adult beetles produced vertical shafts with and without terminal chambers resembling ichnofossils found in marine and continental deposits as old as the Late Devonian. These studies illustrate the value of neoichnology in fully understanding the trace morphologies produced by beetles and other trace-making animals by comparing them to ichnofossils. Neoichnological studies have been critical in the direct association of several ichnogenera with beetles

(Hembree and Hasiotis, 2006; Counts and Hasiotis, 2009; Hembree, 2009; Hembree et al., 2012; Mikus and Uchman, 2013).

Beetles are largely associated with the continental ichnofacies Coprinisphaera, which is an ichnofossil assemblage characterized by dominance of traces produced by ants, bees, beetles, and termites, and containing a moderate to high ichnodiversity and abundance (Buatois and Mangano, 2011). Several ichnogenera in this ichnofacies, such as Taenidium, Naktodemasis, Coprinisphaera, Macanopsis, and Fictovichnus have been attributed to beetles (Retallack, 1984; Johnston et al., 1996; Sanchez and Genise, 2008;

Mikus and Uchman, 2013; Guerrero-Arenas et al., 2017). Taenidium is a meniscate backfilled burrow that can be sinuous to straight in subhorizontal to subvertical orientation and is associated with detritus feeding and locomotion (Genise, 2017).

Naktodemasis has distinct meniscate backfilled packets that are ellipsoid in shape and is usually associated with detritus feeding, locomotion, and dwelling behaviors (Smith and

Hasiotis, 2008; Genise, 2017). Coprinisphaera is a spherical to sub-spherical, ovoid 24 chamber with discrete, lined walls that may contain a hole, which is similar to dung-filled brood chambers produced by extant Scarabaeidae in association with pupation and nesting behaviors (Genise, 2004; Sanchez and Genise, 2009). Macanopsis is a simple vertical shaft that can end in a terminal chamber which is similar to burrows produced by a variety of arthropods in both continental and marine environments, and associated with pupation, nesting, and dwelling behaviors (Mikus and Uchman, 2013). Fictovichnus is a spheroid, ovoid, or ellipsoidal chamber, with a rough to smooth wall, and sometimes containing pellets, which is comparable to similar chambers occupied by extant beetles during pupation (Axtell and Arends, 1990; Genise, 2004; Guerrero-Arenas et al., 2018).

The high diversity of trace forms beetles produce over their entire life cycle suggests that they would be dominant trace makers in the ichnofossil record at least since the Permian, but this is not the case. Despite the number of ichnogenera attributed to beetles, most are associated with pupation or nesting behaviors (Genise et al., 2000; Laza, 2007; Sanchez and Genise, 2009; Sanchez et al., 2010). What is typically missing from the ichnofossil record is the full range of activities and behaviors through their entire life cycle. This gap in knowledge can be filled through direct observations of beetle trace production using experimental neoichnology.

Chapter 3: Methods

The hypotheses were tested using six experiments with three species of burrowing beetles, Tenebrio molitor, Zophobas morio, and Phyllophaga sp. The species were chosen based on later larval instars, ease of care, and availability. Tenebrio molitor and Z. morio were purchased in their larval stages from pet stores, whereas Phyllophaga sp. was acquired through field collection of larvae in Athens County, OH, USA. All specimens were housed in sediment-filled terraria simulating natural soil conditions according to their habitat as determined by a literature review (Brandhorst-Hubbard et al., 2001;

Hasiotis and Counts, 2009; Alonso-Zarza et al., 2014; Mckenna et al., 2019) with 9-hour

“light” and 15-hour “dark” cycles, temperatures between 25-30° C, and humidity between 30-40%. The sediment was composed of a varying mixture of silicate sand (S), sieved potting soil (PS) (clay and silt), and coconut fiber (CF) (Table 2). Soil moisture were maintained by daily spraying of the sediment with water for six seconds per 3.8- liters of sediment. Moisture was monitored and measured using Aquaterr EC-300

Multimeter (Fig. 3). Specimens were provided with various decomposing organic material as food items. After each experiment, adult specimens were placed in enclosures separated by species.

Table 2

Beetle Experimental Setup

Beetle Experimental setup Experiment 1: Trace Techniques Terrarium Sediment % Number of Sediment Temperature Duration Species Specimens per Trial Size Depth Water Compaction Trials Composition Range (oC) (Days) (liter) (mm) Content T. molitor 1 6 3.8 15% S + 50% PS +35% CF 20 30-32 35% 0.09 kg/cm2 60 Phyllophaga sp. 1 6 3.8 15% S + 50% PS +35% CF 20 30-32 35% 0.09 kg/cm2 60 Z. morio 1 6 3.8 15% S + 50% PS +35% CF 20 30-32 35% 0.09 kg/cm2 60 Experiment 2: Trace Morphology Terrarium Sediment % Number of Sediment Temperature Duration Species Specimens per Trial Size Depth Water Compaction Trials Composition Range (oC) (Days) (liter) (mm) Content T. molitor 1 6 3.8 15% S + 50% PS +35% CF 20 30-32 35% 0.09 kg/cm2 60 Phyllophaga sp. 1 6 3.8 15% S + 50% PS +35% CF 20 30-32 35% 0.09 kg/cm2 60 Z. morio 1 6 3.8 15% S + 50% PS +35% CF 20 30-32 35% 0.09 kg/cm2 60 Experiment 3: Sediment Composition Terrarium Sediment % Number of Sediment Temperature Duration Species Specimens per Trial Size Depth Water Compaction Trials Composition Range (oC) (Days) (liter) (mm) Content T. molitor 1 3 3.8 15% S + 50% PS +35% CF 20 30-32 35% 0.09 kg/cm2 60 T. molitor 1 3 3.8 55% PS + 43% CF 20 30-32 35% 0.09 kg/cm2 60 Phyllophaga sp. 1 3 3.8 15% S + 50% PS +35% CF 20 30-32 35% 0.09 kg/cm2 60 Phyllophaga sp. 1 3 3.8 55% PS + 43% CF 20 30-32 35% 0.09 kg/cm2 60 Z. morio 1 3 3.8 15% S + 50% PS +35% CF 20 30-32 35% 0.09 kg/cm2 60 Z. morio 1 3 3.8 55% PS + 43% CF 20 30-32 35% 0.09 kg/cm2 60 Experiment 4: Moisture Composition Terrarium Sediment % Number of Sediment Temperature Duration Species Specimens per Trial Size Depth Water Compaction Trials Composition Range (oC) (Days) (liter) (mm) Content T. molitor 1 3 3.8 15% S + 50% PS +35% CF 200 30-32 15% 0.09 kg/cm2 60 T. molitor 1 3 3.8 15% S + 50% PS +35% CF 200 30-33 65% 0.09 kg/cm2 60 Phyllophaga sp. 1 3 3.8 15% S + 50% PS +35% CF 200 30-34 15% 0.09 kg/cm2 60 Phyllophaga sp. 1 3 3.8 15% S + 50% PS +35% CF 200 30-35 65% 0.09 kg/cm2 60 Z. morio 1 3 3.8 15% S + 50% PS +35% CF 200 30-36 15% 0.09 kg/cm2 60 Z. morio 1 3 3.8 15% S + 50% PS +35% CF 200 30-37 65% 0.09 kg/cm2 60 Experiment 5: Compaction Composition Terrarium Sediment % Number of Sediment Temperature Duration Species Specimens per Trial Size Depth Water Compaction Trials Composition Range (oC) (Days) (liter) (mm) Content T. molitor 1 3 3.8 15% S + 50% PS +35% CF 200 30-32 35% 0.19 kg/cm2 60 T. molitor 1 3 3.8 15% S + 50% PS +35% CF 200 30-32 35% 0.03 kg/cm2 60 Phyllophaga sp. 1 3 3.8 15% S + 50% PS +35% CF 200 30-32 35% 0.19 kg/cm2 60 Phyllophaga sp. 1 3 3.8 15% S + 50% PS +35% CF 200 30-32 35% 0.03 kg/cm2 60 Z. morio 1 3 3.8 15% S + 50% PS +35% CF 200 30-32 35% 0.19 kg/cm2 60 Z. morio 1 3 3.8 15% S + 50% PS +35% CF 200 30-32 35% 0.03 kg/cm2 60

Experiment 6: Bioturbation Test Terrarium Sediment Sediment % Number of Temperature Duration Species Specimens per Trial Size Composition with Depth Water Compaction Trials Range (oC) (Days) (liter) alternating layers (L) (mm) Content

L1 [15% S + 50% PS +35% T. molitor 12 1 38 CF] 190 30-32 35% 0.09 kg/cm2 60 L2 [55% PS + 45% CF]

L1 [15% S + 50% PS +35% T. molitor 24 1 76 CF] 250 30-32 35% 0.09 kg/cm2 60

L2 [55% PS + 45% CF]

L1 [15% S + 50% PS +35% T. molitor 24 1 212 CF] 360 30-32 35% 0.09 kg/cm2 60

L2 [55% PS + 45% CF]

L1 [15% S + 50% PS +35% Phyllophaga sp. 12 1 38 CF] 190 30-32 35% 0.09 kg/cm2 60

L2 [55% PS + 45% CF]

L1 [15% S + 50% PS +35% Phyllophaga sp. 24 1 76 CF] 250 30-32 35% 0.09 kg/cm2 60

L2 [55% PS + 45% CF]

L1 [15% S + 50% PS +35% Phyllophaga sp. 24 1 212 CF] 360 30-32 35% 0.09 kg/cm2 60

L2 [55% PS + 45% CF]

L1 [15% S + 50% PS +35% Z. morio 12 1 38 CF] 190 30-32 35% 0.09 kg/cm2 60

L2 [55% PS + 45% CF]

L1 [15% S + 50% PS +35% Z. morio 24 1 76 CF] 250 30-32 35% 0.09 kg/cm2 60

L2 [55% PS + 45% CF]

L1 [15% S + 50% PS +35% Z. morio 24 1 212 CF] 360 30-32 35% 0.09 kg/cm2 60 L2 [55% PS + 45% CF]

Note. Table 2: Experimental setup for Experimental setup for Tenebrio molitor, Phyllophaga sp., and

Zophobas morio Experiments 1 to 6.

Figure 3

The Tools

Note. Figure 3: Left: Aquaterr EC-300 Multimeter, used to measure moisture levels in sediment. Right: Pocket Penetrometer, used to measure compaction of sediment.

Experiments 1-5 each involved six specimens of each species. In these experiments each specimen was placed in a single 3.8 L (17 cm l x 8 cm w 28 cm h), sediment-filled terrarium in their larval stage and remained until their adult stage or deemed expired (Fig. 4). Larvae of Z. morio and Phyllophaga sp. were placed on the sediment surface, whereas T. molitor larvae were buried at half the depth of the sediment to encourage burrowing since T. molitor had a tendency towards near surface conditions.

Experiments 1-2 tested hypotheses 1-2, examining the burrowing techniques, behaviors, and biogenic structures produced by each species under natural conditions. Experiments 29

3-5 tested hypothesis 3, examining how changes in sand content, water content, and firmness affected behavior and the morphology of biogenic structures. Experiment 6 tested hypothesis 4 and involved placing 12 larval specimens of each species into 38 L terraria (51 cm l x 26 cm w x 30 cm h), and 24 specimens of each species into a 76 L (60 cm l x 31 cm w x 42 cm h) and 212 L (77 cm l x 46 w x 63 cm h) terraria filled with layered sediment (Fig. 4). This experiment was used to document the total amount of bioturbation produced throughout the life cycle of each beetle species under different spatial constraints and described as an ichnofabric index. Due to Covid-19 and the late start to field collection, there were fewer specimens of Phyllophaga sp. available than planned. As a result, Experiments 4-5 involved only four specimens each and only one 38

L experiment was conducted for Phyllophaga sp. in Experiment 6.

Figure 4

The Enclosures

Note. Figure 4: A) 3.7-L enclosure with substrate composed of 15% sand, 50% sieved potting soil (clay), and

35% coconut fiber. Top is the side view, while the bottom is the front view. B) Enclosures for Experiment 6.

Top 37-L, middle 76-L, bottom 212-L from frontal view. Scale bars represent 20 cm.

Each experiment lasted approximately 60 days. Each enclosure was monitored daily during all six experiments. Photographs and digital recordings were made of active trace-making, biogenic structures, and associated behavioral activity. Traces were described qualitatively and quantitatively in two-dimensional cross-sectional views along 31 the sides of the terraria. Measurements included depth, length, width, angle of shafts or tunnels with respect to the horizontal, and branching angles. Complexity, the number of segments, surface openings, burrows, chambers, and their geometries were evaluated as well.

Experiment 1

Experiment 1 utilized a modified 1 cm wide, 3.8-liter terrarium in order to better observe each beetle burrowing techniques in all life stages. These 18 terraria were filled with substrate of moderate organic and sand content (15% S + 50% PS + 35% CF) to a depth of 20 cm (Table 2). Soil moisture was kept at 35% and compactness at 0.09 kg/cm2

(standard) (Table 2).

Experiment 2

Experiment 2 was used to determine the range of trace morphologies and associated behaviors of each species through all of their life stages under natural conditions. These 18, 3.8-liter terraria were filled with substrate of moderate organic and sand content (15% S + 50% PS + 35% CF) to a depth of 20 cm (Table 2), with a soil moisture of 35% and compactness of 0.09 kg/cm2 (Table 2).

Experiment 3

Experiment 3 evaluated the effect of sediment composition on burrowing behavior and resulting trace morphology by increasing (50% S + 35% PS + 15% CF) and decreasing (55% PS + 45% CF) sand content of the sediment from the initial composition

(Table 2). All other conditions were the same as Experiment 2 (Table 2). Half of the specimens of each species were tested with each substrate variation. 32

Experiment 4

Experiment 4 evaluated the effect of sediment water content on burrowing behavior and resulting trace morphology by increasing (65%) and decreasing (15%) water content of the sediment from the initial composition (Table 2). All other conditions were the same as Experiment 2 (Table 2). Half of the specimens of each species were tested with each substrate variation.

Experiment 5

Experiment 5 evaluated the effect of changing sediment compactness on burrowing behavior and resulting trace morphology by increasing sediment density to

0.19 kg/cm2 (maximum) and decreasing sediment density to 0.03 kg/cm2 (minimum)

(Table 2). Compactness was measured using a pocket penetrometer measuring in kg/cm2

(Fig. 3). Increased compactness of the substrate was achieved by pressing the substrate down with a heavy weight in the shape of terrarium surface. Decreased compactness was achieved by loosely filling the terraria with substrate. All other conditions were the same as Experiment 2 (Table 2). Half the specimens of each species were tested with each compactness variation.

Experiment 6

Experiment 6 evaluated the total amount of bioturbation produced throughout the life cycle of each beetle species based on available space quantified as an ichnofabric index. Zophobas morio was placed in the 38 L, 76 L, and 212 L terraria. Tenebrio molitor was placed in two 38 L and one 76 L terrarium due to their preference for near-surface

(<3 cm) conditions. Phyllophaga sp. was only placed in one 38 L terrarium as a result of 33 their low sample size. Each 38, 76, and 212-liter terrarium (Table 2) were filled to a depth of 19, 25, and 25 centimeters, respectively. The substrate in Experiment 6 consisted of ~1-centimeter thick, alternating layers of a sandy and an organic-rich substrate (Table

2). All other conditions were the same as Experiment 2. Twelve specimens of each species were used in the 38 L terrariums, whereas 24 specimens were used in the 76 L and 212 L terrariums.

Burrow Analysis

The beetles’ burrows were compared using the Bray-Curtis similarity test. This is a nonparametric test was used to evaluate the relative similarity of the burrows based on

14 properties simultaneously (e.g., Hembree, 2016). These properties include: depth, maximum width, minimum width, mean width, maximum length, minimum length, mean length, mean width/length ratio, maximum slope, minimum slope, mean slope, branching angles, complexity, and tortuosity all measured in millimeters. Measurements of width, length, and slope were combined to produce a maximum, minimum, and mean for each property. Width to length ratio was achieved by dividing the width by the length. This aspect ratio is important to understanding the space in which the trace inhabits. Slope was measured with a standard 360o protractor with a datum of the surface as 180o. Complexity is the sum of the segments within a burrow system, and tortuosity is found by dividing the length by a straight-line distance of a burrow segment (Fig. 5) (Hembree, 2016). The

Bray Curtis similarity matrix was performed with the Paleontological Statistics software package (PAST version 3) (Hammer et al., 2001) and used to compare burrows produced in Experiments 2-5 in order to test hypotheses 2, 3, and 5. This analysis evaluated the 34 similarity or dissimilarity of the morphologies of the burrows produced with and between the three species and between natural and altered substrate conditions. In the Bray Curtis similarity matrix, similarity is ranked from 1.0 to 0.0, with 1.0 indicating two samples have identical properties and 0.0 indicating two samples are completely dissimilar.

Hembree (2016) established a gradient of similarity in between these end members with

1.0-0.8 = similar, 0.7-0.6 = moderately similar, and 0.5-0.0 = dissimilar. Dendrograms based on the similarity and distance indices were produced to provide a visual representation of the relative similarity of the different biogenic structures (Appendix A).

In these diagrams, clusters of burrows that share common nodes are those that share similar sets of quantitative properties.

Figure 5

Burrow Descriptions

Note. Figure 5: Burrow description visualization. A) Burrows were described by their depth (D), maximum and minimum width (w), maximum and minimum length (l), slope, and branching angle (BA). B) Complexity is the sum of the number of segments (s) and chambers (h) within a single burrow system. C) Tortuosity is the average sinuosity of the segments within the burrow system. The tortuosity of a segment is found by dividing the total length (u) by the straight-line distance (v) (Hembree, 2016). (Modified from Hembree,

2012).

Comparison with Ichnofossils

The traces produced during each life stage of the beetles were compared to ichnofossils previously attributed to beetles and other arthropods (hypothesis 5). These comparisons were made to determine if specific ichnogenera were useful proxies for the burrows produced by the Tenebrionidae and Scarabidae, or if the biogenic structures produced in these experiments were unique and would be considered new ichnotaxa if found in the fossil record. Traces produced during the beetle’s larval stage were 36 compared to the ichnogenera Paleophycus, Planolites, Skolithos, Taenidium, and

Naktodemasis. Traces produced during their pupal stage were compared to Fictovichnus,

Pallichnus, and Rebuffoichnus and those produced during their adult stage they were compared to Conichnus and Macanopsis (Fig. 6). These ichnogenera were selected based off their similarity to extant beetle traces, their presence in continental sedimentary units

(Buatois and Mangano, 2011), and their previous association with beetle trace makers in different life stages. These ichnogenera were compared to the beetles’ biogenic structures with Bray-Curtis similarity test. This is a nonparametric test was used to evaluate the relative similarity of the burrows based on 10 properties simultaneously (e.g., Hembree,

2016). These properties include: maximum width, minimum width, mean width, maximum length, minimum length, mean length, mean width/length ratio, branching angles, complexity, and tortuosity all measured in millimeters (Fig. 5).

Figure 6

Known Beetle Ichnogenera

Note. Figure 6: A) Skolithos is a single, vertical shaft, unbranched, which can be lined for unlined. Straight to curved, in vertical to subvertical orientation (Alpert, 1974). B) Rebuffoichnus is a spheroid, ovoid, or ellipsoid chamber with horizonal to subhorizontal orientation with microrelief of low ridges on the walls

(Genise, 2017).C) Paleophycus is a horizontal, lined, branched or unbranched straight to sinuous, passively filled burrow (Pemberton and Frey, 1982). D) Pallichnus spheroid, ovoid chamber with vertical to subvertical orientation, and can contain a lining (Retallack, 1984). E) Macanopsis is a simple vertical shaft that can show a gradual thickening into a terminal chamber (Mikus and Uchman, 2013). F) Fictovichnus is a spheroid, ovoid, or ellipsoidal chamber with a rough or smooth wall and sometimes pellets (Genise, 2004). G)

Planolites is a horizontal, unlined, unbranched, and straight to sinuous, actively filled burrow (Frey and

Bromley, 1985). H) Taenidium is an unlined, straight to sinuous, meniscate backfilled burrow with secondary successive branching (Keighley and Pickerill, 1994). I) Naktodemasis is a varied orientation, unbranched burrows with meniscate backfill and thin discontinuous lining (Smith and Hasiotis, 2008). Scale bars represent 20 mm.

Chapter 4: Results

Burrowing Techniques

Larval Stage

Larva of Phyllophaga sp. burrowed by excavation of sediment with their mandibles after which sediment was passed to the limbs. The larvae would then rotate their body and pack the sediment behind themselves, creating a backfill. No linings were noticeable, but bioglyphs were produced from body compressions and burrow activity from the limbs and their head within the substrate (Fig. 7A).

Both Tenebrio molitor and Zophobas morio larvae burrowed using a combination of initial incision with their mandibles, followed by a forward thrust with their limbs, and final compaction with their heads (Fig. 7B, 7C). They also transported sediment backwards, passing it from their front limbs to their hind limbs (Fig. 7B). This sediment was deposited along the burrow walls and then compacted with their head to produce an extremely thin (< 0.05mm), compressional lining (Fig. 7F). Zophobas morio larvae produced noticeable bioglyphs from head compressions and body movement activity

(Fig. 7D, 7E, 7F). The bioglyphs were triangular and predominantly located on the dorsal side of tunnels. The bioglyphs were a good indicator of larva movement direction and tunnel orientation. A difference between the burrowing techniques of T. molitor and Z. morio larvae was that Z. morio larvae could efficiently burrow backwards using their tail end spine in a similar fashion to their head. Tenebrio molitor could also burrow in this fashion, but only to a limited extent.

Figure 7

Beetle Trace Behavior

Note. Figure 7: A) Phyllophaga sp. larva burrowing with time progression. Left is time 1, middle is time 2, right is time 3. B) Tenebrio molitor larva burrowing with time progression. Left is time 1, middle is time 2, right is time 3. C) Zophobas morio larva burrowing with time progression. Left is time 1, middle is time 2, right is time 3. All time progressions took place over several seconds to under one minute. D) Zophobas morio burrow with head compressions and compressional lining. E) Highlight of head compressions. F)

Highlight of compressional lining. Scale bars represent 20 mm.

Adult Stage

During the adult stage, the beetles primary burrowing technique was using their walking limbs to move upward through the substrate, either to the surface or just below the surface. Adult stages of Tenebrio molitor and Zophobas morio used their limbs to expand their pupal chamber and then generate vertical backfilled burrows. The pupal chambers were modified through compression from their dorsal side and some excavation with their limbs. The backfilled burrows were generated as the adult beetles used their front and middle limbs to slowly excavate the ceiling and walls of their open chamber, while standing and balancing on their hind pair of limbs and abdomen. This excavated material would fall and passed below them with their middle limbs. They would then stand on this excavated sediment and compress it using their hind limbs into the floor. In this way, the adults moved upward through the substrate. They would occasionally stop and “rest”, compressing the substrate around them using their elytra and limbs to produce a small spherical to ovoid chamber. When they continued their ascent from this secondary chamber, it would be infilled by excavated sediment. When adult beetles reached near surface depth (1-3 cm), the chamber ceiling would collapse from above filling it with sediment. The beetle would then exit their collapsed chamber to the surface using their walking limbs to “swim” through the substrate. Observation of

Phyllophaga sp. in adult stage was limited, but their burrowing technique also involved the use of their walking limbs to move through the substrate in a “swimming” manner.

Behavior

Larval Stage

Overall, during larval stages for all three species, the primary behavior observed was mobile deposit feeding on organic material in the substrate with intermittent resting.

Escape behaviors were also noted when dangers appeared, usually when attempting to extract the larvae from their enclosures. These escape behaviors entailed bursts of energy and rapid downward burrowing, with some individuals completely burrowing within seconds of the “danger” appearing. Within all three species, some individual larvae were able to produce high levels of bioturbation (Fig. 8E), while others displayed relatively sessile lifestyles and produced low levels of bioturbation (Fig. 8A).

Tenebrio molitor larvae preferred near-surface to shallow-tier depths, or a mean depth of ~2.5 cm, with an overall range of 1-7 cm. When initially buried at a depth of 10 centimeters or greater at the start of an experiment, they would immediately burrow upward to near-surface depths or even emerge on the surface. This behavior was most noticeable in Experiment 6. Tenebrio molitor larvae also tended to move to the surface during the dark hours in the laboratory, even amassing into ball swarms in experiments with multiple specimens. The larvae would then disperse at the start of the light hours to other areas of the sediment surface or back into the sediment. Overall, their burrowing behaviors were frequent resting with bursts of intermittent burrowing. These rests lasted up to or less than a day. Resting and even full life cycle completion at the surface was also observed, with at least one or more individuals going through their full cycle at the 43 surface during each of the experiments. Tenebrio molitor remained in the larval stage for

14 to 38 days, with a mean duration of 25 days.

Phyllophaga larvae preferred shallow- to deep-tier depths, or a mean depth of ~9 cm with a range of 1-20 cm. If substrate conditions were not ideal for the Phyllophaga larvae upon burrowing, they would move back to the surface and search for a location with more suitable conditions. If these conditions were not met, they would remain stationary at the surface in a shallow depression and would often die. This occurred with almost half of the Phyllophaga sp. specimens throughout all the experiments. Changes between light and dark hours did not alter their behaviors greatly, but during dark hours burrow generation was more noticeable, similar to the other species. Overall, the behaviors of the Phyllophaga sp. larvae were limited to locomotion and intermittent resting. These intermittent rests were typically short (less than five hours), but could last over twenty-four hours in some individuals. Phyllophaga sp. remained in the larval stage for 22 to 37 days, with mean duration of 30 days.

Zophobas morio larvae also preferred the shallow to deep tiers of the substrate, or a mean depth of ~8 cm, with a range of 1-25 cm. Light and dark hours of the laboratory had a moderate impact on Z. morio larva behavior; more activity was observed during dark hours, while light hours were dominated by escape from the sediment surface and minimal burrowing overall. Of all three beetle species examined, Z. morio displayed the highest levels of subsurface locomotion and burrow production (Fig. 8E, 8G). Overall, Z. morio behaviors were limited to quick bursts of burrow creation resulting from mobile deposit feeding, with prolonged resting periods of up to three days within their burrows. 44

Zophobas morio remained in the larval stage for 20 to 44 days, with a mean duration of

34 days.

Pupal Stage

Tenebrio molitor, Phyllophaga sp., Zophobas morio had comparable behaviors during the pupal stage, which consisted of stationary pupation (Fig. 8B, 8D, 8F), but occasionally the pupa would move about the chamber. There was also some deposit feeding along the walls of their chambers, but this was usually in the early stages of pupation before the larva metamorphosed completely. Tenebrio molitor and Z. morio preferred leaning against a rigid surface (i.e., the enclosure walls) while pupating, while

Phyllophaga sp. did not have this preference. Movement of the pupae could be inferred as possible chamber maintenance behaviors, compacting the sediment with their body motion. This behavior is unlikely since chambers in which pupae died, did not collapse until disturbed by outside forces. The pupation stage lasted 20-30 days (mean 25 days) with Tenebrio molitor and 29-50 days (mean 37) with Zophobas morio. The behavior of

Phyllophaga sp. larvae made it difficult to time their pupation chamber duration, and this data was not obtained. Phyllophaga sp. made multiple chambers throughout their larval stage, seldom made it to the full pupation stage, and often burrowed away from the observable area of the enclosure. The other two species were usually limited to a single chamber, although some individuals produced two as a result of an individual’s apparent change in location preference or faulty construction of the original chamber. 45

Adult Stage

The primary behaviors observed in the adult stages of all three species after leaving the pupation chamber were to feed on organic material within or on the surface and reproduction. During the adult life stages Tenebrio molitor and Zophobas morio would engage in escape behaviors to make their way to the surface; however, sometimes they would make new chambers and rest within the subsurface after leaving their pupation chamber (Fig. 8G). Adult T. molitor would usually surface within a 24-hour period after entering this life stage. Adult Z. morio would typically surface within minutes after entering the adult stage, but some individuals took up to seven days. Their previous pupation chambers would be actively filled by the exiting adult. Only one instance occurred where an adult of T. molitor left its chamber and did fill it in as it moved upward. However, the preservation potential of this chamber was extremely low, since after a week it passively filled. All adult Phyllophaga sp. moved and fed within the sediment rather than moving to the surface

Figure 8

Beetle Trace Morphology of Experiments 2

Note. Figure 8: Experiment 2: A) Phyllophaga sp. larvae which created an unlined meniscate burrow. The specimen can be seen within the leftmost chamber. This burrow is a typical representation of Phyllophaga sp. trace morphology and behavior, where the specimen can be seen resting after generating its backfilled burrow. B) Phyllophaga sp. specimen within pupation chamber. This was identified as a pupation chamber, since the specimen stayed within the chamber after several days of burrowing and did not leave. C) Tenebrio molitor burrow network within the upper 2 cm of the substrate. This is an important observed trace since it shows that they are able to generate large networks. D) Tenebrio molitor pupation chamber with specimen within. This picture also shows the vertical orientated chamber many T. molitor larvae constructed. E)

Zophobas morio burrow network. This is important since it displays that Z. morio could construct large networks across many different depths. F) Zophobas morio chamber with the specimen inside. G) Zophobas morio adult trace of which only a few were able to be documented. The specimen, after leaving its pupation molt, immediately began burrowing upwards over the course of several minutes. A’-G’ show highlighted burrows to give contrast. Arrows in A’ show previous resting chambers. Scale bars represent 20 mm.

Burrow Morphology

Larval Stage

Tenebrio molitor and Zophobas morio both generated boxworks composed of open, subhorizontal, horizontal, subvertical, and vertical tunnels and shafts with circular to ovoid cross sections (Fig. 8C, 8E). Branching within the boxworks consisted of simultaneous branching, secondary successive branching, and false branching (Fig. 8C,

8E). Primary successive branching was not observed, but it is not impossible based on the ability of these larvae to move both forward and backwards in the substrate with ease.

Most branches occurred at T- and Y-shaped junctions, with some individuals making X- shaped junctions (Fig. 8C, 8E). Branching angles ranged from 7-33o. The shafts and tunnels were straight to sinuous and were passively filled. The boxworks could be 48 extensive and continuous, with visible open tunnels and shafts having lengths of 3-5 cm, or they could be separated by one or several, passively filled sections to produce isolated tubular to ovoid burrows ranging in length from a few millimeters to a few centimeters.

Larvae of T. molitor and Z morio also generated J-, U-, and Y-shaped burrows both at the surface and within the sediment (Fig. 8C, 8E). Burrow walls contained small, wedged- shaped bioglyphs on the distal portion of the tunnels and very thin compressional linings

(< 0.05 mm) (Fig. 7D to 7F).

Larvae of Phyllophaga sp. did not produce any recognizable burrows during

Experiment 2, but they did generate several chambers. In other experiments, Phyllophaga sp. larvae produced backfilled subhorizontal, horizontal, subvertical, and vertical shafts and tunnels with circular to ovoid cross sections which terminated in open, subvertical to subhorizontal circular to ovoid chambers (Fig. 8A). These burrows originated at the surface, moving into the subsurface, and then back to the surface. The burrows were continuously backfilled, with breakage in the burrow length resulting from low cross- sectional visibility (Fig. 8A). The burrows of Phyllophaga larvae also displayed false and secondary successive branching in altered environmental conditions, along with movement into the substrate and back to the surface.

Pupal Stage

All three species produced comparable pupation chambers in terms of general morphology, but when analyzed quantitatively there were different (Table 3).

Morphologically, the chambers were subhorizontal to vertical, ovoid structures with no visible lining with widths from 8-25 mm and lengths of 4-45 mm (Appendix B1-B12) 49

When examining the chambers within the taxa, Phyllophaga sp. were overall slightly similar, with some variation. Chambers of Tenebrio molitor had high variation, with many of them being dissimilar to each other. Zophobas morio had the most consistent chambers of all three taxa The chambers were kept open during the entire pupal stage, then either passively or actively filled when exited by the adult. Phyllophaga sp. pupation chambers were usually connected to a backfilled burrow produced by the larva, whereas connections to T. molitor and Z. morio pupation chambers were not visible (Fig. 8A).

Table 3

Bray-Curtis Similary Indicies for Three Beetle Taxa, both Burrows and Chambers

Note. Table 3: Results of the Bray-Curtis similarity test comparing chambers of all three beetle taxa.

Phyllophaga sp. are denoted as P-1 to P-6. Tenebrio molitor are denoted as T-1 to T-6. Zophobas morio are denoted as Z-1 to Z-6. Most specimens are from Experiment 2. Those marked with an “*” are from

Experiments 3 and Experiments 4.

Adult Stage

Traces produced by adult Tenebrio molitor and Zophobas morio consisted of backfilled, vertical to subvertical shafts and tunnels with circular cross sections. These backfilled burrows were simple, vertical to subvertical shafts with no visible lining that originated from an infilled chamber (usually from pupation), and terminated in a series of multiple vertically orientated, V-shaped, conical laminations produced as the beetle exited to the surface (Fig. 8G). If the adult did not move to the surface, the backfilled burrow terminated in an open chamber (Fig. 9). The open chamber could contain bioglyphs resulting from burrowing activity (Fig. 9). Traces produced by adult

Phyllophaga sp. consisted of subhorizontal to subvertical orientated, chaotically arranged, tubular packets, with circular to ovoid cross sections (Fig. 10). These structures had disruption laminations as the beetles used their walking limbs to move through the substrate.

Figure 9

Adult Zophobas morio Chambers

Note. Figure 9: Zophobas morio trace progression from pupal stage to adult. Far left is the original pupation chamber, with the specimen inside. Middle is the adult beetle leaving the pupation chamber. Pupation chamber has now been actively filled. Middle image depicts a partially visible resting chamber. Right image shows the adult in a resting chamber. Within the resting chamber, bioglyphs can be seen along the floors and walls of the chamber. While the adult was burrowing upwards, it also crosscut an old larva trace seen in the left image, completely reworking it into the substrate. Scale bar represents 20 mm.

Figure 10

Adult Phyllophaga sp. Disruption

Note. Figure 10: A) Disruptive reworking of the substrate by an adult Phyllophaga sp. It was placed halfway beneath the surface, and buried with layered sediment. Each layer was 1-cm thick. A’ highlights the disruption to give contrast. Scale bar represents 20 mm.

Environmental Controls

Experiment 3

In Experiment 3, the beetles produced traces comparable to Experiment 2. There was no major difference in the trace morphologies produced by any of the life stages of

Tenebrio molitor and Zophobas morio with increased or decreased sand content (Fig.

11B-11E, 11G-11-I). In the sediment with increased sand content, the burrows did have a higher frequency of being passively filled. However, the burrows of T. molitor and Z. morio still consisted of mazework structures with tunnels and shafts, ovoid pupation chambers, and backfilled adult burrow. The larvae of Phyllophaga sp. produced 54 backfilled burrows (Fig. 11A) which terminated in an open, subvertical to subhorizontal, ovoid chamber in the sediment with decreased sand content. Overall, there was a higher abundance of traces produced by all three species in sediment with decreased sand content compared to high sand content. The beetle larvae of the three species also had a higher death rate in sediment with increased sand content.

Figure 11

Experiment 3

Note. Figure 11: Experiment 3: A) Phyllophaga sp. burrow in low sand substrate. The specimen can be seen within the lower chamber. Menisci can also be viewed in this burrow, along with a backfilled packet. B)

Tenebrio molitor burrow in low sand substrate, displaying secondary successive branching. C) Zophobas morio burrows in low sand substrate. These burrows have been highly reworked and disrupted, only leaving behind unconnected tunnels. D) Tenebrio molitor pupation chamber, including the specimen within. This was constructed in a low sand substrate, and the chambers’ vertical orientation is shown here. This morphology is common with T. molitor. E) Zophobas morio chamber in a low sand substrate. The specimen can be seen within. The high clay content of the substrate allowed for greater detail of bioglyphs within the chamber. These bioglyphs can be seen along the walls and ceiling of the chamber. The bioglyphs are from head and body compressions during chamber construction while the beetle was still a larva during early pupation stage. F) Phyllophaga sp. filled chamber in sand-rich substrate. The increased sand content did not allow for high preservation or visibility of Phyllophaga sp. burrows, except for chambers. G) Tenebrio molitor chamber in sand-rich substrate. The specimen can be seen within. Regardless of conditions of

Experiment 3, the pupation chamber of T. molitor were orientated vertically. H) Zophobas morio burrow in sand-rich substrate. The burrows of Z. morio had lower preservation, but did not display any morphological differences from the low sand substrate. I) Zophobas morio chamber in sand-rich substrate. The chamber has lower relief when compared to the low sand substrate. The specimen can be seen within. A’-I’ show highlighted burrows to give contrast. All scale bars represent 20 mm.

Experiment 4

During Experiment 4, Tenebrio molitor and Zophobas morio generated traces comparable to those of Experiment 2 in both substrate conditions. One of the main differences was that during this experiment Phyllophaga sp. created backfilled burrows with false branching in sediment with increased water content. In addition, in sediment with decreased water content two of the Phyllophaga sp. larvae produced long backfilled 57 burrows that showed initial movement into the subsurface followed by movement back to the surface. Once on the surface, the larvae generated a shallow depression in the sediment surface and died. There were no major differences between the trace morphologies produced in sediments with increased or decreased water content for all three species, although burrows produced by T. molitor and Z. morio in the drier sediment had a higher rate of being passively filled due to burrow collapse. Overall, there was a higher abundance of traces (Fig. 12E-12J ) produced by all three species in sediment with increased water content compared to decreased water content (Fig. 12A-

12D). As a result of decreased water content, the beetle larvae had a high mortality rate resulting from dehydration. Sediments with high water content had a higher frequency of fungal related deaths than normal, which occurred during all life stages, but mainly affected the pupae. However, the frequency of death from dehydration (2 Phyllophaga sp., 3 T. molitor, 1 Z. morio) was higher than fungal-related deaths (1 Phyllophaga sp., 2

T. molitor, 1 Z. morio).

Figure 12

Experiment 4

Note. Figure 12: Experiment 4: A) Phyllophaga sp. burrow in decreased water conditions. From left to right shows time progression over the course of several weeks from early June to late July. The specimen can often be seen in the terminating chamber. In the final time sequence, the larva can be seen on the surface, in a resting position. As the beetle larva burrowed, it created unlined meniscate. B) Zophobas morio pupation chamber with specimen in decreased water conditions. The substrate was difficult to compress and compact, but did not alter the overall morphology of their pupation chambers. C) Tenebrio molitor shaft leading into the surface. This specimen was stressed by the low moisture conditions, and moved back to the surface. D) Partially collapsed Zophobas morio burrows in decreased moisture conditions. E) Phyllophaga sp. burrows in increased water conditions. This example shows secondary successive branching while the beetle backfills. The specimen can be seen in the left corner in a resting chamber. F) Phyllophaga sp. burrows in increased water conditions. This also shows secondary successive branching. Overall, in increased water conditions, Phyllophaga sp. display higher disruption and overall bioturbation. G) Zophobas morio burrow in increased water conditions. The burrows here have partially collapsed. H) Adult Zophobas morio resting chamber in increased water conditions. This is a secondary chamber created by the adult. It can be partially traced back to its original pupation chamber, which has now been infilled. I) Tenebrio molitor burrows in increased water conditions. The specimen can be seen within its tunnel resting. The tunnels here show secondary successive branching. The specimen first went into the surface, then back down its previous shaft, and made a new tunnel. J) Zophobas morio pupation chamber in increased water conditions. The specimen can be seen within. A’-J’ show highlighted burrows to give contrast. Scale bars represent 20 mm.

Experiment 5

During Experiment 5 the beetles generally made traces comparable to those of

Experiment 2 in the loosely compacted substrate. In compacted substrates, burrow morphologies were unit from those produced in Experiment 2. Traces in the highly compacted substrates were either small, or none were produced at all. Among all three species, there was a higher abundance of traces in the loosely compacted substrate (Fig.

13A-13D) compared to the highly compacted substrate (Fig. 13E-13H). Burrows made by the three species had a lower visibility in the loose substrate due to their frequent collapse. Traces that could be produced in the firm substrate had a high visibility in all 60 three species. Phyllophaga sp. larvae in both the loose and firm substrate conditions made long, backfilled burrows which terminated in an open chamber. Tenebrio molitor larvae generated some structures U-shaped burrows in the firm substrate (Fig. 13 F), but most of their activity was concentrated in the loose substrate. Zophobas morio larvae exhibited the least diverse behaviors in the highly compacted substrate. Here, the larvae attempted to fracture the substrate surface and burrow into these limited areas. Some were able to do this and generate small tube-like traces, while others failed and died at the surface. One individual was able to go through a complete life cycle in this very limited space, but once it metamorphosed into an adult it became entombed within the substrate and died.

Figure 13

Experiment 5

Note. Figure 13: Experiment 5: A) Phyllophaga sp. burrow in loose conditions. This burrow shows movements from the surface into the subsurface, along with meniscate backfill. B) Zophobas morio burrow network in loose conditions. Since the substrate was loose, it was easily compressible. C) Tenebrio molitor burrow in loose conditions displaying T-junction with secondary successive branching. D) Zophobas morio chamber in loose conditions with specimen. E) Phyllophaga sp. burrow in firm conditions. The burrow shows movement from the surface into the subsurface. Despite the altered conditions, there was no difference in burrow morphology between loose and firm conditions. F) Tenebrio molitor U-shaped burrow in firm conditions with specimen. As a result of the highly compacted substrate, the larva was unable continue its burrow, and attempted to return to surface. G) Phyllophaga sp. pupation chamber in firm conditions. The larva remained in its pupation chamber for several weeks, unlike resting chambers. H) Zophobas morio burrow in firm conditions with specimen. The larva had difficulty burrowing, and only burrowed following a natural crack from the surface. A’- H’ show highlighted burrows to give contrast. Scale bars represent 20 mm. 62

Quantitative Analyses

Comparison of Traces Between Species

Burrows and chambers produced in each life stage were compared both within and between species using the Bray Curtis similarity matrix (Appendix A1-A2). These data primarily came from traces produced in Experiment 2 (Appendix B1-B3), with some supplemental data taken from traces produced in Experiments 3 and 4 to provide a larger sample size for the analyses (Table 3) (Appendix B4-B9). Larval burrows of Phyllophaga sp. were moderately similar (~0.66–0.78, n=2) to similar (~0.81–0.88, n=4) to each other.

Burrows of Tenebrio molitor were more variable in their level of similarity. The burrows of T. molitor were evenly dissimilar (~0.54–0.59, n=3) to moderately similar (~0.66–

0.78, n=3). Burrows of Zophobas morio were dissimilar (~0.36–0.59, n=2) to moderately similar (~0.62–0.76, n=4) to each other.

Burrows of Phyllophaga sp. and T. molitor were predominantly dissimilar

(~0.32–0.57, n=6). Burrows of Phyllophaga sp. and Z. morio were generally dissimilar

(~0.40–0.59, n=2), moderately similar (~0.63–0.79, n=2), similar (~0.80–0.88, n=2).

Burrows of T. molitor and Z. morio were evenly dissimilar (~0.41–0.59, n=3) to moderately similar (~0.60–0.79, n=3).

Chambers produced by Phyllophaga sp. were dissimilar (~0.43–0.52, n=3) to moderately similar (~0.62–0.79, n=3). Tenebrio molitor chambers were dissimilar

(~0.33–0.55, n=2) to moderately similar (~0.65–0.75, n=4) to each other. Chambers of

Zophobas morio were dissimilar (~0.38–0.50, n=3) to moderately similar (~0.69–0.77, n=3). 63

Chambers of Phyllophaga sp. and T. molitor were predominantly dissimilar

(~0.30–0.58, n=6). Phyllophaga sp. and Z. morio chambers were moderately similar

(~0.61–0.79, n=2) to dissimilar (~0.35–0.59, n=4). Tenebrio molitor and Z. morio chambers were mostly dissimilar (~0.29–0.58, n=6).

Comparison of Traces Produced in Increased Sand vs Decreased Sand Substrate

Comparisons were made between burrows and chambers produced in Experiment

3 (Appendix B4-B6) by each species using the Bray Curtis similarity matrix (Table 4)

(Appendix A3). Tenebrio molitor did not produce visible burrows in the increased sand conditions in any life stages, preventing their comparison. However, T. molitor did produce one visible chamber in the increased sand substrate and two in the decreased sand substrate. The analysis showed that the chambers produced under the two different environmental parameters were all dissimilar (~0.20–0.59, n=3). Phyllophaga sp. also did not produce visible burrows in Experiment 3, yet one chamber was visible in the increased sand substrate and two were visible in the decreased sand substrate which were all similar (~0.83–0.85, n=3). Zophobas morio produced two burrows within the increased sand conditions, and three in the decreased sand conditions which were moderately similar (~0.66–0.77, n=2) to similar (~0.85–0.89, n=3).

Table 4

Comparison of Traces Produced in Increased Sand vs Decreased Sand Substrate

Comparison of Traces Produced in Increased Sand vs Decreased Sand Substrate Zophobas morio burrows Zophobas morio chambers Z2-3 Z3-3 Z4-3 Z5-3 Z6-3 Z4-3 Z5-3 Z6-3 Z2-3 1.00 0.76 0.85 0.77 0.57 Z4-3 1.00 0.98 0.77 Z3-3 0.76 1.00 0.87 0.87 0.69 Z5-3 0.98 1.00 0.78 Z4-3 0.85 0.87 1.00 0.89 0.66 Z6-3 0.77 0.78 1.00 Z5-3 0.77 0.87 0.89 1.00 0.67 Z6-3 0.57 0.69 0.66 0.67 1.00 Tenebrio molitor chambers T3-3 T4-3 T5-3 T3-3 1.00 0.39 0.59 T4-3 0.39 1.00 0.20 T5-3 0.59 0.20 1.00

Phyllophaga sp. chambers P3-3 P4-3 P5-3 P3-3 1.00 0.83 0.78 P4-3 0.83 1.00 0.85 P5-3 0.78 0.85 1.00

Note. Table 4: Comparison of traces produced in Experiment 3 using the Bray Curtis similarity matrix.

Zophobas morio burrows: Z2-3, Z3-2 – high sand; Z4-3, Z5-3, Z6-3 – low sand. Zophobas morio chambers:

Z4-3 – high sand; Z5-3, Z6-3 – low sand. Tenebrio molitor chambers: T3-3 – high sand; T4-3, T5-3 – low sand. Phyllophaga sp. chambers: P3-3 – high sand; P4-3, P5-3 – low sand.

Comparisons of Traces Produced in Increased Moisture vs Decreased Moisture

Substrate

Comparisons were made between burrows and chambers produced in the two different substrates of Experiment 4 (Appendix B7-B9) by each species (Table 5)

(Appendix A4). All four Phyllophaga sp. specimens generated burrows in both substrates which were all similar (~0.83–0.92, n=4) regardless of moisture content. Phyllophaga sp. only produced one chamber in the increased moisture substrate and two in the decreased 65 moisture substrate which were found to be dissimilar (~0.46–0.58, n=3) to each other.

Tenebrio molitor produced three burrows in each of the substrates which were moderately similar (~0.64–0.76, n=6). Chambers of T. molitor included one in the increased moisture substrate and two in the decreased moisture substrate. Despite the difference in the substrates, they were also moderately similar (~0.62–0.73, n=3). A total of six burrows of Zophobas morio were also produced in both substrate types, and were moderately similar (~0.64–0.79, n=3) to similar (~0.81–0.93, n=3). The chambers of Z. morio included two in the high moisture and one in the low moisture substrate, all of which were similar (~0.87–0.91, n=3).

Table 5

Comparison of Traces Produced in Increased Moisutre vs Decreased Moisture Substrate

Comparisons of Traces Produced in Increased Moisture vs Decreased Moisture Substrate Zophobas morio burrows Zophobas morio chambers Z1-4 Z2-4 Z3-4 Z4-4 Z5-4 Z6-4 Z2-4 Z3-4 Z6-4 Z1-4 1.00 0.72 0.52 0.66 0.73 0.78 Z2-4 1.00 0.87 0.91 Z2-4 0.72 1.00 0.79 0.84 0.76 0.81 Z3-4 0.87 1.00 0.89 Z3-4 0.52 0.79 1.00 0.76 0.64 0.68 Z6-4 0.91 0.89 1.00 Z4-4 0.66 0.84 0.76 1.00 0.86 0.82 Z5-4 0.73 0.76 0.64 0.86 1.00 0.93 Z6-4 0.78 0.81 0.68 0.82 0.93 1.00

Tenebrio molitor burrows Tenebrio molitor chambers T1-4 T2-4 T3-4 T4-4 T5-4 T6-4 T2-4 T4-4 T6-4 T1-4 1.00 0.88 0.76 0.74 0.81 0.75 T2-4 1.00 0.73 0.62 T2-4 0.88 1.00 0.68 0.70 0.71 0.65 T4-4 0.73 1.00 0.67 T3-4 0.76 0.68 1.00 0.90 0.74 0.70 T6-4 0.62 0.67 1.00 T4-4 0.74 0.70 0.90 1.00 0.72 0.64 T5-4 0.81 0.71 0.74 0.72 1.00 0.88 T6-4 0.75 0.65 0.70 0.64 0.88 1.00

Phyllophaga sp. burrows Phyllophaga sp. chambers P1-4 P2-4 P3-4 P4-4 P1-4 P3-4 P4-4 P1-4 1.00 0.82 0.92 0.84 P1-4 1.00 0.60 0.46 P2-4 0.82 1.00 0.87 0.86 P3-4 0.60 1.00 0.58 P3-4 0.92 0.87 1.00 0.83 P4-4 0.46 0.58 1.00 P4-4 0.84 0.86 0.83 1.00

Note. Table 5: Comparison of traces produced in Experiment 4 using the Bray Curtis similarity matrix.

Zophobas morio burrows: Z1-4, Z2-4, Z3-4 – high moisture; Z4-4, Z5-4, Z6-4 – low moisture. Zophobas morio chambers: Z2-4, Z3-4 – high moisture; Z6-4 – low moisture. Tenebrio molitor burrows: T1-4, T2-4,

T3-4 – high moisture; T4-4, T5-4, T6-4 – low moisture. Tenebrio molitor chambers: T2-4 – high moisture;

T4-4, T6-4 – low moisture. Phyllophaga sp. burrows: P1-4, P2-4 – high moisture; P3-4, P4-4 – low moisture.

Phyllophaga sp. chambers: P1-4 – high moisture; P3-4, P4-4 – low moisture.

Comparison of Traces Produced in Firm vs Loose Substrate

Comparisons were made between burrows and chambers produced in Experiment

5 (Appendix B10-B12) by each species (Table 6) (Appendix A5). Phyllophaga sp. 67 generated one burrow each in both the firm and loose substrates which were similar

(~0.88, n=2). They produced one chamber in the firm substrate and two in the loose substrate, which were moderately similar (~0.60–0.85, n=1) to dissimilar (~0.51, n=2).

Tenebrio molitor generated one burrow in the firm substrate and three in the loose substrate which were moderately similar (~0.70–0.78, n=4). Tenebrio molitor did not produce chambers in either substrate type preventing comparison. Zophobas morio produced two burrows in the firm substrate and three in the loose substrate which were moderately similar (~0.62–0.77, n=5). Zophobas morio only produced a chamber in the loose substrate preventing comparison.

Table 6

Comprison of Traces Produced in Firm and Loose Substrate

Comparison of Traces Produced in Firm vs Loose Substrate Zophobas morio burrow Phyllophaga sp. burrows Z1-5 Z3-5 Z4-5 Z5-5 Z6-5 P3-5 P4-5 Z1-5 1.00 0.87 0.80 0.74 0.73 P3-5 1.00 0.88 Z3-5 0.87 1.00 0.74 0.62 0.62 P4-5 0.88 1.00 Z4-5 0.80 0.74 1.00 0.77 0.68 Z5-5 0.74 0.62 0.77 1.00 0.87 Z6-5 0.73 0.62 0.68 0.87 1.00

Tenebrio molitor burrows Phyllophaga sp. chambers T2-5 T4-5 T5-5 T6-5 P1-5 P3-5 P4-5 T2-5 1.00 0.78 0.72 0.70 P1-5 1.00 0.51 0.60 T4-5 0.78 1.00 0.73 0.76 P3-5 0.51 1.00 0.85 T5-5 0.72 0.73 1.00 0.76 P4-5 0.60 0.85 1.00 T6-5 0.70 0.76 0.76 1.00

Note. Table 6: Comparison of traces produced in Experiment 5 using Bray Curtis similarity matrix. Zophobas morio burrows: Z1-5, Z3-5 – firm; Z4-5, Z5-5, Z6-5 – loose. Tenebrio molitor burrows: T2-5 – firm; T4-5,

T5-5, T6-5 – loose. Phyllophaga sp. burrows: P3-5 – firm; P4-5 – loose. Phyllophaga sp. chambers: P1-5,

P3-5 – firm; P4-5 – loose.

Comparison of Extant Beetle Traces with Comparable Ichnogenera

Phyllophaga sp. larval burrows produced in Experiment 2 were compared to several known ichnogenera typically associated with beetle locomotion and deposit feeding (Macanopsis, Paleophycus, Planolites, Skolithos, Taenidium, and Naktodemasis)

(Table 7) (Appendix A6). Phyllophaga sp. larval burrows were dissimilar (~0.27–0.46, n=2) to moderately similar (~0.62–0.71, n=5) to Macanopsis. When compared to

Paleophycus they were dissimilar (~0.38–0.47, n=2) to similar (~0.92–0.95, n=5). When compared to Planolites, they were dissimilar (~0.27–0.33, n=2), to similar (~0.92, n=4). 69

When compared to Skolithos, they were dissimilar (~0.41–0.46, n=2), to moderately similar (~0.61–0.69, n=4). When compared to Taenidium, they were moderately similar

(~0.62–0.74, n=6). When compared to Naktodemasis, they were dissimilar (~0.34–0.40, n=2), moderately similar (~0.63–0.71, n=4).

Phyllophaga sp. chambers from Experiments 2–4 was compared to known ichnogenera typically associated with beetle pupation (Fictovichnus, Pallichnus, and

Rebuffoichnus) (Table 7). The chambers compared to Fictovichnus were similar (~0.81–

0.90, n=6). When compared to Pallichnus they were moderately similar (~0.61–0.76, n=6). When compared to Rebuffoichnus they were similar (~0.88–0.93, n=2) and moderately similar (~0.63–0.65, n=4).

Tenebrio molitor larval burrows produced in Experiment 2 were compared to several known ichnogenera typically associated with beetle locomotion and deposit feeding (Macanopsis, Paleophycus, Planolites, Skolithos, Taenidium, and Naktodemasis)

(Table 7). When compared to Macanopsis, Tenebrio molitor larval burrows were dissimilar (~0.58–0.59, n=2), to moderately similar (~0.80–0.97, n=4). When compared to Paleophycus they were similar (~0.85–0.91, n=2) and moderately similar (~0.62–0.76, n=4). When compared to Planolites, they were moderately similar (~0.63–0.71, n=2) to similar (~0.84–0.91 n=4). When compared to Skolithos, they were dissimilar (~0.56–

0.57, n=2), moderately similar (~0.75–0.78, n=2), and similar (~0.94–0.96, n=2). When compared to Taenidium, they were dissimilar (~0.48–0.49, n=2) to moderately similar

(~0.63–0.75, n=4). When compared to Naktodemasis, they were dissimilar (~0.58–0.59, n=2) and similar (~0.81–0.96, n=4). 70

Tenebrio molitor chambers from Experiment 2 were compared to known ichnogenera typically associated with beetle pupation (Fictovichnus, Pallichnus, and

Rebuffoichnus) (Table 7). The chambers compared to Fictovichnus were similar (~0.86–

0.89, n=2) and moderately similar (~0.67–0.78, n=4). When compared to Pallichnus they were moderately similar (~0.61–0.76, n=6). When compared to Rebuffoichnus they were dissimilar (~0.42–0.51, n=3) and moderately similar (~0.62–0.70, n=3).

Zophobas morio larval burrows produced in Experiment 2 were compared to several known ichnogenera typically associated with beetle locomotion and deposit feeding (Macanopsis, Paleophycus, Planolites, Skolithos, Taenidium, and Naktodemasis)

(Table 7). When compared to Macanopsis they were dissimilar (~0.41–0.44, n=3) to similar (~0.92–94, n=3). When compared to Paleophycus they were moderately similar

(~0.60–0.72, n=6). When compared to Planolites, they were moderately similar (~0.63–

0.69, n=6). When compared to Skolithos, they were dissimilar (~0.40–0.43, n=3) to similar (~0.93–0.94, n=3). When compared to Taenidium, they were moderately similar,

(~0.76–0.79, n=2) and dissimilar (~0.36–0.56, n=4). When compared to Naktodemasis, they were dissimilar (~0.42–0.45, n=3) to similar (~0.86–0.90, n=3).

Zophobas morio chambers from Experiment 2 were compared to known ichnogenera typically associated with beetle pupation (Fictovichnus, Pallichnus, and

Rebuffoichnus) (Table 7). The chambers compared to Fictovichnus were moderately similar (~0.67–0.78, n=6). When compared to Pallichnus they were moderately similar

(~0.73, n=2) and similar (~0.80–0.89, n=4). When compared to Rebuffoichnus they were similar (~0.80–0.88, n=2), and moderately similar (~0.66–0.78, n=4). 71

Table 7

Comparison of Beetle Biogenic Structures to Ichnogenera

Comparison of Beetle Biogenic Structures to Ichnogenera Phyllophaga sp. burrows vs Ichnofossil burrows Phyllophaga sp. chambers vs Ichnofossil chambers P-1* P-2* P-3* P-4* P-5* P-6* M Pa Pt Sk TN P-1 P-2 P-3 P-4* P-5 P-6* F Pl R P-1* 1.00 0.78 0.70 0.70 0.43 0.47 0.92 0.76 0.75 0.90 0.74 0.93 P1 1.00 0.95 0.93 0.78 0.98 0.74 0.89 0.65 0.65 P-2* 0.78 1.00 0.92 0.91 0.38 0.43 0.71 0.95 0.92 0.69 0.65 0.71 P2 0.95 1.00 0.95 0.76 0.94 0.71 0.84 0.61 0.63 P-3* 0.70 0.92 1.00 0.96 0.35 0.47 0.63 0.94 0.92 0.61 0.66 0.64 P3 0.93 0.95 1.00 0.78 0.92 0.70 0.83 0.59 0.66 P-4* 0.70 0.91 0.96 1.00 0.34 0.42 0.63 0.92 0.92 0.61 0.62 0.63 P4* 0.78 0.76 0.78 1.00 0.76 0.91 0.86 0.78 0.88 P-5* 0.43 0.38 0.35 0.34 1.00 0.58 0.41 0.38 0.27 0.41 0.49 0.34 P5 0.98 0.94 0.92 0.76 1.00 0.72 0.90 0.66 0.63 P-6* 0.47 0.43 0.47 0.42 0.58 1.00 0.45 0.47 0.33 0.46 0.78 0.40 P6* 0.74 0.71 0.70 0.91 0.72 1.00 0.81 0.76 0.93 M 0.92 0.71 0.63 0.63 0.41 0.45 1.00 0.69 0.70 0.98 0.75 0.94 F 0.89 0.84 0.83 0.86 0.90 0.81 1.00 0.74 0.74 Pa 0.76 0.95 0.94 0.92 0.38 0.47 0.69 1.00 0.90 0.67 0.68 0.69 Pl 0.65 0.61 0.59 0.78 0.66 0.76 0.74 1.00 0.70 Pt 0.75 0.92 0.92 0.92 0.27 0.33 0.70 0.90 1.00 0.68 0.56 0.70 R 0.65 0.63 0.66 0.88 0.63 0.93 0.74 0.70 1.00 Sk 0.90 0.69 0.61 0.61 0.41 0.46 0.98 0.67 0.68 1.00 0.76 0.93 T 0.74 0.65 0.66 0.62 0.49 0.78 0.75 0.68 0.56 0.76 1.00 0.70 N 0.93 0.71 0.64 0.63 0.34 0.40 0.94 0.69 0.70 0.93 0.70 1.00

Tenebrio molitor burrows vs Ichnofossil burrows Tenebrio molitor chambers vs Ichnofossil chambers T-1 T-2 T-3 T-4 T-5 T-6 M Pa Pt Sk Ta N T-1 T-2 T-3 T-4 T-5 T-6 F Pl R T-1 1.00 0.95 0.78 0.79 0.78 0.70 0.77 0.91 0.91 0.75 0.63 0.78 T-1 1.00 0.92 0.83 0.87 0.63 0.87 0.75 0.71 0.51 T-2 0.95 1.00 0.75 0.82 0.74 0.73 0.80 0.85 0.86 0.78 0.63 0.81 T-2* 0.92 1.00 0.76 0.78 0.56 0.84 0.67 0.68 0.42 T-3 0.78 0.75 1.00 0.60 0.87 0.53 0.59 0.76 0.84 0.57 0.48 0.59 T-3 0.83 0.76 1.00 0.88 0.77 0.78 0.89 0.82 0.70 T-4 0.79 0.82 0.60 1.00 0.59 0.90 0.97 0.70 0.71 0.96 0.73 0.96 T-4 0.87 0.78 0.88 1.00 0.71 0.87 0.86 0.71 0.62 T-5 0.78 0.74 0.87 0.59 1.00 0.51 0.58 0.79 0.86 0.56 0.49 0.58 T-5* 0.63 0.56 0.77 0.71 1.00 0.59 0.73 0.76 0.82 T-6 0.70 0.73 0.53 0.90 0.51 1.00 0.92 0.62 0.63 0.94 0.75 0.88 T-6 0.87 0.84 0.78 0.87 0.59 1.00 0.78 0.61 0.51 M 0.77 0.80 0.59 0.97 0.58 0.92 1.00 0.69 0.70 0.98 0.75 0.94 F 0.75 0.67 0.89 0.86 0.73 0.78 1.00 0.74 0.74 Pa 0.91 0.85 0.76 0.70 0.79 0.62 0.69 1.00 0.90 0.67 0.68 0.69 Pl 0.71 0.68 0.82 0.71 0.76 0.61 0.74 1.00 0.70 Pt 0.91 0.86 0.84 0.71 0.86 0.63 0.70 0.90 1.00 0.68 0.56 0.70 R 0.51 0.42 0.70 0.62 0.82 0.51 0.74 0.70 1.00 Sk 0.75 0.78 0.57 0.96 0.56 0.94 0.98 0.67 0.68 1.00 0.76 0.93 T 0.63 0.63 0.48 0.73 0.49 0.75 0.75 0.68 0.56 0.76 1.00 0.70 N 0.78 0.81 0.59 0.96 0.58 0.88 0.94 0.69 0.70 0.93 0.70 1.00

Zophobas morio burrows vs Ichnofossil burrows Zophobas morio chambers vs Ichnofossil chambers Z-1* Z-2 Z-3 Z-4 Z-5 Z-6 M Pa Pt Sk TN Z-1 Z-2 Z-3 Z-4 Z-5 Z-6 F Pl R Z-1* 1.00 0.37 0.37 0.39 0.60 0.88 0.92 0.62 0.63 0.93 0.76 0.86 Z-1 1.00 0.48 0.47 0.38 0.34 0.49 0.63 0.44 0.38 Z-2 0.37 1.00 0.98 0.84 0.69 0.42 0.42 0.60 0.66 0.41 0.36 0.43 Z-2 0.48 1.00 0.90 0.84 0.77 0.88 0.80 0.80 0.88 Z-3 0.37 0.98 1.00 0.85 0.69 0.44 0.41 0.62 0.65 0.40 0.39 0.42 Z-3 0.47 0.90 1.00 0.85 0.80 0.96 0.78 0.88 0.80 Z-4 0.39 0.84 0.85 1.00 0.73 0.47 0.44 0.70 0.69 0.43 0.54 0.45 Z-4 0.38 0.84 0.85 1.00 0.88 0.82 0.65 0.81 0.73 Z-5 0.60 0.69 0.69 0.73 1.00 0.68 0.67 0.88 0.96 0.65 0.56 0.67 Z-5 0.34 0.77 0.80 0.88 1.00 0.77 0.59 0.73 0.66 Z-6 0.88 0.42 0.44 0.47 0.68 1.00 0.94 0.72 0.69 0.94 0.79 0.90 Z-6 0.49 0.88 0.96 0.82 0.77 1.00 0.79 0.89 0.78 M 0.92 0.42 0.41 0.44 0.67 0.94 1.00 0.69 0.70 0.98 0.75 0.94 F 0.63 0.80 0.78 0.65 0.59 0.79 1.00 0.74 0.74 Pa 0.62 0.60 0.62 0.70 0.88 0.72 0.69 1.00 0.90 0.67 0.68 0.69 Pl 0.44 0.80 0.88 0.81 0.73 0.89 0.74 1.00 0.70 Pt 0.63 0.66 0.65 0.69 0.96 0.69 0.70 0.90 1.00 0.68 0.56 0.70 R 0.38 0.88 0.80 0.73 0.66 0.78 0.74 0.70 1.00 Sk 0.93 0.41 0.40 0.43 0.65 0.94 0.98 0.67 0.68 1.00 0.76 0.93 T 0.76 0.36 0.39 0.54 0.56 0.79 0.75 0.68 0.56 0.76 1.00 0.70 N 0.86 0.43 0.42 0.45 0.67 0.90 0.94 0.69 0.70 0.93 0.70 1.00

Note. Table 7: Comparison of beetle biogenic structures compared to known ichnogenera using Bray Curtis similarity matrix. Phyllophaga sp. are denoted as P-1 to P-6. Tenebrio molitor are denoted as T-1 to T-6.

Zophobas morio are denoted as Z-1 to Z-6 . Many specimens are from Experiment 2, but those marked with

“*” are from Experiments 3 and 4. Comparable burrows are Macanopsis (M), Paleophycus (Pa), Planolites

(Pt), Skolithos (S), Taenidium (T), and Naktodemasis (N). Comparable chambers are Fictovichnus (F),

Pallichnus (Pl), and Rebuffoichnus (R).

Bioturbation and Ichnofabric

Tenebrio molitor specimens were placed in two 38-liter (L) terraria and one 76 L terraria. The 12 specimens in the first 38 L terrarium produced an ichnofabric index (ii) of 1 over 31 days (Fig. 14A-14C). The T. molitor in the second 38 L terrarium produced and ii of 2 over 48 days (Fig 14D and 14 E). The 24 specimens placed in the 76 L terrarium produced ii of 1 over 31 days (Fig 14F and 14G). During their larval stage, the

T. molitor specimens spent a significant amount of time on the surface during the dark hours (~15 hours) of the laboratory and would go into the subsurface during the light hours (~9 hours). Specimens of T. molitor experienced a high mortality rate in

Experiment 6 despite being under favorable environmental conditions. Most of the visible

T. molitor traces produced during the larval stage were present within the upper ~1-2 cm of the substrate. Many of the traces consisted of near-surface, horizontal to subhorizontal tunnels with no visible chambers. The observed behaviors and resulting traces were comparable to those produced in Experiments 2-4. As a result of their low bioturbation levels, the 212 L terrarium was not used for T. molitor.

Figure 14

Experiment 6: Tenebrio molitor

Note. Figure 14: Experiment 6 for Tenebrio molitor. A-C) 38-L terrarium. Bioturbation is low, and only near the surface. B and C highlight areas of activity within this terrarium. D-E) The second 38-L terrarium. D is the start of the experiment, with no activity. E shows the conclusion of the experiment after 48 days, displaying low levels of bioturbation, but higher than the previous 38-L. One specimen of T. molitor can be seen in E. F-G) 76-L terrarium. F shows the overall bioturbation, which was near the surface. G highlights low activity in the 76-L. Scale bars for A, D, and E equal 5 cm. Scale bars for B and C equal 20 mm. Scale bars for F and G equal 10 cm.

Due the limited number of available specimens, Phyllophaga sp. were only placed in a single 38 L terrarium. The 12 larvae burrowed to depths of ~1-5 cm and produced an ii of 2 over 51 days (Fig 15A). Many of the visible traces were V-shaped laminations produced by the larvae as they moved from the subsurface to the surface. Other noticeable traces were subhorizontal backfilled burrows and chambers. The observed behaviors and resulting traces were similar to those observed in Experiments 2-5. Despite a high mortality rate, three Phyllophaga sp. went through their entire life cycle in this experiment.

Figure 15

Experiment 6: Phyllophaga sp.

Note. Figure 15: Experiment 6 for Phyllophaga sp. A) Bioturbation in the 38-L terrarium. Several specimens of Phyllophaga sp. can be seen within. B) Pupation chamber containing the larvae. C) Resting chamber, with menisci backfills and the larvae. D) Cone-in-cone structure created by one of the specimens during the larval stage. The associated behavior was escape. E) Partially visible resting chamber with the larva visible. Overall activity was low, and concentrated in the first 1-5 cm. Scale bar for A represents 5 cm. Scale bar for B-E represents 20 mm.

Zophobas morio were placed in 38 L, 76 L, and 212 L terraria. In the 38 L terrarium the 12 specimens produced an ii of 2-3 over 74 days (Fig 16A). The 24 specimens in the 76 L terrarium produced an ii of 3-4 over 39 days (Fig. 16H), while the

24 specimens in the 212 L terrarium produced an ii of 4-5 over 69 days (Fig. 16O). Most of the visible traces and general sediment disruption were concentrated at depths of ~1-15 76 cm. Many biogenic structures in the larger terraria (76 and 212 L) were at depths of ~20-

25 cm. Most traces were subhorizontal to subvertical tunnels and shafts arranged in mazeworks, usually clustered within the upper portions of the substrate. Chambers were usually located at depths of ~10-20 cm, away from large cluster of larval burrows and sediment disruption. The observed behaviors and traces were similar to those observed in

Experiments 2-4.

Figure 16

Experiment 6: Zophobas morio

Note. Figure 16: Experiment 6 for Zophobas morio. A) 38-L terrarium. This displays the overall disruption which includes burrows and chambers. B) Close up of a sinuous burrow of Z. morio, scale bar is 5 cm. C)

Two pupation chambers, one of them has been slightly infilled but is still visible. The far left one is filled with fungus, which killed the pupa. Scalebar is 20 mm. D) An adult escape trace of Z. morio. The trace here is backfilled, originated from the pupation chamber, and crosscuts older larval burrows. Scale bar is 20 mm.

G) 76-L terrarium that has been highlighted. This documentation occurred within the first week of the experiment, and already displayed high activity. Scale bar is 5 cm. H) The end of the 76-L experiment, with few visible traces left, but overall high disruption of the substrate. Scale bar is 5 cm. I) Adult escape trace with cone-in-cone structure near the surface. This trace crosscuts several older larval burrows. Scale bar is

20 mm. J) High disruption during Experiment 6 for 76-L terrarium. One specimen can be seen within. Several burrows have crosscut each other. Scale bar is 20mm. K) High disruption of burrows at the end of Experiment

6 for 76-L terrarium. Scale bar is 5 cm. L) 212-L terrarium. This shows the start of the experiment displaying low activity. Scalebar is 10 cm. M) The first visible pupation chamber in the 212-L terrarium with the specimen inside. Bioglyphs can be seen within the chamber. These bioglyphs were a result of head and body compressions during the making of the chamber. Scale bar is 20 mm. N) Halfway point of Experiment 6 for the 212-L terrarium. Activity is high, but mostly concentrated to the upper 5 cm. Scale bar is 10 cm. O) End of Experiment 6 for 212-L terrarium. Activity is high. Several traces are not recognizable, but the amount of disruption is clear. Activity is throughout the entire terrarium, but concentrated in the upper 10 cm. Several adult traces can be seen with in highlighted area, since they crosscut old pupation burrows and originated from chambers. Scale bar is 10 cm.

Overall, the traces produced by each of the three different life stages were identifiable and distinguishable within these bioturbation experiments for each species.

The larval traces were limited to tunnels and shafts, largely concentrated at depths of ~1-

15 cm or uncommonly the maximum possible depth in that enclosure. Terraria which contained a higher ichnofabric index generally had fewer visible traces, but had highly 79 noticeable disruption of the substrate through intense burrowing activity from the different life stages of the beetles (Fig. 16O). Pupation chambers were not limited to specific depth ranges overall, but usually they were at depths below, or away from, larval activity. The morphology of pupation chambers was unique compared to the larval and adult traces. Adult traces infilled pupation chambers and were primarily vertical to subvertical in orientation. These adult traces crosscut larvae burrow on their progression to the surface.

Chapter 5: Discussion

Controls on the Beetle Trace Morphology

Trace morphology results from an amalgamation of behavior, the trace makers’ overall body morphology, and the environment (Fey, 1978; Seilacher, 1978; Bromley,

1996; Seilacher, 2007; Hembree, 2016). Various combinations of these parameters can lead to a multitude of different biogenic structures, even produced by individual organisms (Bromley, 1996; Hembree, 2016). However, identical biogenic structures may also be produced by entirely different species (Bromely, 1996; Hembree, 2016). As a result, successful recognition of the biogenic structures produced by beetles during their different life stages requires investigation of the effects of changing behavior, body morphology, and environmental conditions on the morphology their traces.

Behavior

Animal behavior is often difficult to evaluate, even with continuous direct observations as noted in other neoichnological projects (Davis et al., 2007; Counts and

Hasiotis, 2009; Hembree, 2016). Despite this difficulty, multiple behaviors were observed or inferred in each of the life stages of the three studied species. The behaviors observed in the larval stage related to trace production were locomotion, escape, feeding, resting, and pupation chamber construction. During the pupal stage, the observed behaviors linked to discrete traces (the pupal chamber) were feeding (before complete metamorphosis) and pupation. The observed behaviors during the adult stage associated with trace production were escape, resting, locomotion, and feeding. 81

During the larval stage, all three beetle species produced unique trace morphologies when compared to the adult stage. The trace morphologies were heavily influenced by behavior. During this stage, the primary behavior associated to these traces was locomotion and active deposit feeding. The traces resulting from these behaviors were indistinguishable, since all three beetle species fed on materials in the substrate while actively moving within the substrate. Any movement with accompanying trace production without feeding was accomplished with similar mechanical processes. This is typical of deposit feeding organisms, since they continuously move through the substrate in search of nutrients (Gingras et al., 2008). In areas mobile deposit feeding was not directly observed, it was inferred from the presence of sediment disruption and general bioturbation. Mobile deposit feeding traces of Tenebrio molitor and Zophobas morio were diagnosed as partially lined, partially open tunnels and shafts which were usually part of a larger boxwork structure produced from false and secondary successive branching. Phyllophaga sp. mobile deposit feeding traces were diagnosed as long backfilled burrows which could contain secondary successive branching and terminated in an open chamber.

Adult beetle traces, despite being produced from similar behaviors, had distinct morphologies. Adults of T. molitor and Z. morio did not make any recognizable active deposit feeding traces, but locomotion was visible as they burrowed upward to the surface. This is a result of a change from primarily depositing feeding to surface detritus feeding behaviors. Direct observation of the adults of Phyllophaga sp. were limited, but they were still mobile deposit feeding since they did not venture into the surface after 82 leaving the pupal stage. The adult Phyllophaga sp. biogenic structures were dissimilar to the larvae burrows. Rather than distinct backfilled burrows, the adults produced undulating disruption patterns within the substrate.

Both larval and adult beetles engaged in escape behaviors. These behaviors, despite both dealing with a need to escape, had different motivations. During the larval stage, it was observed that escape was different from basic locomotion or even active deposit feeding in terms of the rate of burrowing into/within the substrate. Normal locomotion for any of the three beetle species was very slow, usually moving at the 10s of minutes to even hours. This was likely a result of conserving energy. When disturbed or aggravated at the surface they would often flee at much greater rates, usually moving entirely below the surface within seconds of their perceived danger. These traces would often passively fill shortly after production, and leave partially visible disruption features in the substrate. Adult beetle escape behavior was related to venturing to the surface. This was different from their locomotion within the substrate, which usually produced backfilled burrows terminating in an open chamber. As they neared the surface, this open chamber would collapse, and infill with surrounding sediment. The beetles would then burrow their way out, leaving behind a series of vertically nested cone-in-cone structures.

These structures were unique to adult Tenebrio molitor and Zophobas morio. Adults of T. molitor and Z. morio would not reenter the substrate, so this was a unique biogenic structure they would not make again during their adult stage. Once on the surface, escape behaviors were limited to surface locomotion, and were now a flight response, instead of 83 a life stage behavior. Phyllophaga sp. adults were not observed in any escape behaviors in this project.

Chambers were constructed by larvae and adults, but not observed in the adults of

Phyllophaga sp. The associated behavior for larvae was to generate pupation chambers, while for adults’ chambers were sites for temporary occupation while they moved to the surface (i.e., resting). Adult chambers usually had connecting backfilled burrows, which could be traced to and an activity filled pupation chamber. Pupation chambers had no visible connecting burrows from the larvae. The relative dimensions of these chambers were similar, but their orientation was different. Adult chambers were usually more vertically orientated, while pupation chambers were more horizontally orientated.

However, Tenebrio molitor pupation chambers were also often vertical to subvertical and near the surface. This allowed them to reach the surface after leaving their pupation chamber with little effort. This resulted in the production of escape traces of nested cone- in-cone structures and no backfilled burrows similar to Z. morio.

Body Morphology

Trace morphologies were also controlled by the beetles’ changing body morphology. At each life stage the beetles had a unique form which could only create a limited number of trace morphologies. The larval forms could not physically construct the traces of the adults and vice versa. For example, the burrows of Tenebrio molitor and

Zophobas morio larvae were open, branching tunnels and shafts, while the adults produced backfilled burrows and vertical cone-in-cone structures. The larvae of

Phyllophaga sp. generated distinct backfilled burrows which terminated with an open 84 chamber, whereas the adults usually generated chaotic and loose undulating patterns while moving within the substrate.

The reason for these dramatic changes in trace morphology was their new body form at these different life stages. The larvae of T. molitor and Z. morio had an elongate, wormlike bodies with short appendages, which was reflected in the biogenic structures they generated. These structures were tubular in form, which reflected their form. They were also usually straight to sinuous, and had an overall linear direction dictated by their bodies ability to move in the substrate. The larvae of Phyllophaga sp. had an elongate, but more robust and curved form, with longer appendages when compared to the other species. This form was ideal for its backfilled burrow generation. Its curved form and longer appendages aided in its manipulation and compaction of the substrate to generate its’ burrows.

When the beetles metamorphosed into adults, they all lost this elongate wormlike form. They now had a more bulbous and cumbersome body, but comparably longer appendages. Tenebrio molitor and Z. morio adults had much longer bodies when compared to the adults of Phyllophaga sp. They would use this new, longer form to burrow by backfilling within the substrate, using their elytra and longer limbs to compress and excavate sediment. Phyllophaga sp. had a shorter and more bulbous body when compared to the other taxa. This hindered it from generating any backfilled structures, but allowed it to effortlessly burrow by forcing its way in and about the sediment through intrusion, leaving behind disruption patterns.

Specific Trace Maker

Quantitative properties were used to mathematically compare the biogenic structures produced by the three taxa during their different life stages to determine if they were unique. Despite simply belonging to different families or genera, and having many qualitatively similar trace morphologies, there was substantial variation in the quantitative properties of both burrows and chambers produced by the three species.

However, there was variation among traces produced by individual species as well.

Variations in the quantitative properties of the larval and adult burrows included length, slope, width, mean length/width, complexity, and tortuosity. Depth and branching angles were also important factors, but not as significant. Among these factors, the dominant control on differentiating burrows by species was width. This factor was related to the overall size of the species. Zophobas morio was on average the largest during the larval stage, usually 5-8 mm wide and 1.5-2.0 cm long. The next largest was Phyllophaga sp., which had considerable size variation. Some specimens of Phyllophaga sp. larvae were 3-4 mm in width, while others were 5-8 mm wide, and ranged from 1.0-2.5 cm in length. Tenebrio molitor was on average the smallest of the taxa. Their larvae were usually between 1.0-1.5 cm long and 3-5 mm wide. Due to their burrowing techniques, these size factors determined the general dimensions of the burrows they could create, with its greatest effect on the overall width of the burrow.

Variation in the quantitative properties of chambers included their depth, slope, length, and width. The mean length/width ratio and tortuosity were secondarily important factors. Among these factors, depth was the primary control on chamber differentiation 86 by species, followed by slope. Depth was related to the species primary trophic levels within the substrate. Zophobas morio and Phyllophaga sp. inhabited deeper depths (10-

15 cm) while they deposit fed. Tenebrio molitor usually inhabited shallow to near surface depths (1-5 cm) while they deposit fed. These species would usually construct their chambers within or slightly below these depth ranges. This was one reason why

Phyllophaga sp. and Z. morio chambers were quantitatively more similar to each other than to T. molitor. The second major factor was the slope, or orientation, of the chambers.

The majority of T. molitor chambers were subvertical to vertical, while the other taxa were subhorizontal to horizontal. The cause of this vertical orientation could be related to their body morphology since T. molitor pupae were narrower than those of the other taxa.

Other factors could be T. molitor’ near surface location. While in an upright position, this could give them an advantage in leaving their chamber in case of accidental collapse before pupation is finished. While being in a leaning vertical position, it could be easier for them to leave a collapsed chamber. It was observed in this study that any of the species could pupate outside the chamber on the substrate surface. It is not like a cocoon, which is required for metamorphosis. The chambers are primarily for safety while undergoing metamorphosis since the beetles are in a vulnerable state. It was also observed that the vertically oriented structures were less prone to collapse. This could aid in the longevity of the chamber until the adult stage is reached, especially at shallower depths.

Variation in quantitative properties of both burrows and chambers can be identified and recognized for beetles. The use of quantitative analyses, such as the Bray 87

Curtis similarity matrix, is useful in recognizing differences in otherwise visually similar looking traces (Hembree, 2016). Therefore, while some beetles may produce a multitude of traces, these analyses can aid the interpretation of more specific trace makers.

Environmental Conditions

The behaviors of beetles, along with many other trace-making organisms, are affected by variations in their environment (Hembree, 2016). The three beetle taxa are native to temperate to subtropical climates (Park, 1934; Reinhard, 1940; Lovei and

Sunderland, 1996; Park et al., 2014), but are generalists and can, therefore, be found in a variety of different environments across these broad regions (Lovei and Sunderland,

1996). As a result, major to minor differences in the conditions within these different environments have the potential to alter beetle behavior and resulting trace morphologies.

In the sediments with a high sand content, all three beetle species displayed an observable change in behavior. When in a high sand substrate, the beetle larvae would either return to the surface immediately after burrowing or they would stay in upper 1-3 cm. Both resulted in the larvae expiring. This return to the surface by the larvae was likely related to an attempt to locate a more favorable burrowing location for feeding and eventual pupation. The prolonged stay and low levels of activity in near surface conditions was likely related to waiting for conditions to become favorable. The traces that resulted from this activity were not morphologically different from those produced under ideal conditions, but among all three taxa there was a lower abundance of traces produced during all life stages and preservation after production was low. This was a result of the high sand substrate not having the ability to compress well. Compression 88 was a primary means of trace construction by the larvae. Therefore, the inability of the larvae to compress the sediment around their burrows likely factored into the decreased stability of the burrows. The lower abundance of burrows likely resulted from the higher energy expenditure needed to move through continuously collapsing passages.

Sediment water content also affected trace abundance and preservation. As with changes in sand concentration, the trace morphologies were not affected. However, sediments with a high-water content had a higher abundance and preservation potential of traces, whereas sediments with low water content resulted in the collapse of most of the burrows. The beetles could still burrow in the sediments with low moistures levels, but the overall behavioral activity appeared to be lower. Larvae of Tenebrio molitor and

Zophobas morio displayed minimal movement in very dry conditions, and remained within the upper 1-3 cm of the sediment where moisture levels were highest.

Highly compacted sediment had the greatest impact on beetle behavior and trace morphology. Tenebrio molitor and Zophobas morio demonstrated completely altered behaviors and produced different trace morphologies. The highly compacted substrate limited what burrows could be made and even to what capacity they could be created.

This limitation was usually expressed in the width and depth of the burrows as well as the overall abundance of traces. This difficult condition forced several specimens to remain on the surface or within the upper 1-3 cm of the sediment. Some specimens of Z. morio displayed a completely new behavior, where they attempted to fracture the substrate surface and burrow in these limited, shallow areas. This fracturing attempt for burrowing required high energy expenditures, and in the end resulted in death. 89

Ichnofabric and Bioturbation Potential

All three beetle taxa generated an abundance of traces throughout their life stages.

The number of traces which individuals could produce varied between and within beetle taxa. For example, some individuals produced high levels of bioturbation from larvae to adults resulting in significant disruption of the substrate, while other individuals of the same species under the same environmental parameters displayed very low activity and produced little bioturbation.

The life stage and behavior of the beetles caused the greatest variation in the levels of bioturbation produced by each species. In general, each individual would generate moderate to low levels of bioturbation during the larval stage. This bioturbation would mostly be the product of the construction of tunnels and shafts while engaged in mobile deposit feeding which would become overprinted and reworked by the tunnels and shafts produced by the other larvae in the community. Pupation chambers were constructed away from areas of larval activity and contributed little to the overall level of bioturbation. After pupation, the adult beetles would infill the chamber on their ascent towards the surface, typically crosscutting and disrupting old larval burrows. This entire process resulted in significant disruption of the original sediment structure, with some original structures still visible. These remaining structures were discrete traces produced by each life stage, and could be recognized within the final ichnofabric.

These results indicate that beetles have a high potential to leave recognizable signs of bioturbation through their entire life stages in natural settings. Since the biogenic structures produced by each life stage are recognizable on both a qualitative and 90 quantitative level, beetle ichnofossils of all types should common and abundant in the ichnofossil record. Despite this, most investigations into beetle ichnology have focused solely on pupation or nesting chambers (Genise et al., 2000; Laza, 2007; Guerrero-

Arenas et al., 2017; Guerrero-Arenas et al., 2018). This results in a biased view on the types and levels of bioturbation which beetles are capable of producing. The absence of abundant documented beetle ichnofossils may simply be from a lack of recognition, which neoichnological studies such as this can help to improve (Hembree, 2016).

However, beetle bioturbation may also be associated with several other burrowing organisms which inhabit their same tiers and trophic levels. This co-occurrence of bioturbators can causing extreme overprinting, which has been observed in the modern

(Song et al., 2010). This overprinting likely leads to more misidentification or lack of preservation of discrete ichnofossils.

Preservation Potential

The burrows and chambers which beetles created in this experiment likely have a moderate to low potential for preservation. The burrows were shallow to deep-tier structures and, in a natural setting, would be generated in environments with low energy

(Brandhorst-Hubbard et al., 2001; Hasiotis and Counts, 2009; Alonso-Zarza et al., 2014;

Mckenna et al., 2019). Their burrows were not resistant to collapse, but their chambers were more stable. In addition, the burrows only received indirect maintenance from reuse, while the chambers were continually maintained while they were occupied. The growth of fungus or mold around the chambers also provided added rigidity to their walls. Successful preservation of the burrows and chambers would require rapid burial 91

(Fig. 17). Rapid burial would not fill the burrows with sediment, however, since they were not connected to the surface; but rapid burial could aid in preservation by moving the burrows and chambers to depths below the reach of other burrowing organisms.

These rapid influxes of sediment would require that these traces were produced in floodplain soils or similar near-channel alluvial settings, which has been observed in the modern (Mikus and Uchman, 2013). In these settings, sediment would rapidly cover the landscape during floods, burying the soil surface with a thick layer of new sediment.

The lack of abundant, documented ichnofossils for beetles through all of their life stages in the sedimentary record could be a result of their burrowing techniques and the tiers they inhabit. In this project, it was observed that beetle larvae and adults predominantly generated tunnels and shafts. These traces would usually passively collapse fully or partially shortly after their production, or be reworked by further burrowing activity. If these traces were able to survive though the entire beetles’ life cycle, the slightest vibrations of the enclosures would collapse these burrows. Subvertical shafts and tunnels had higher preservation potential in this regard than horizontal to subhorizontal tunnels. The burrows with the highest preservation potential were the backfilled burrows of Phyllophaga sp. larvae and adult Zophobas morio. These structures had high contrast from the surrounding sediment in cross-sectional view and, because they were actively filled, could not collapse. If these burrows were rapidly buried to a depth which others bioturbators could not reach, and later subjected to diagenesis, they could easily be preserved as recognizable backfilled burrows (Bertling et al., 2006). 92

The most common ichnofossils associated with beetles include pupation and nesting chambers (Genise et al., 2000; Laza, 2007; Guerrero-Arenas et al., 2017;

Guerrero-Arenas et al., 2018). In these laboratory experiments, however, pupation chambers were almost always infilled and difficult to recognize when the beetles emerged as adults. The only time these chambers were preserved as voids was when the pupating beetle died, and the chamber was lined by fungus or mold. These pupation structures held their shape well after the conclusion of experiments even with additional sediment placed on the substrate surface (Fig 16C ).

Despite being semi-nomadic, several beetle taxa will cluster near each other in large communities within the same habitat, along with sharing these spaces with many other burrowing animals (Song et al., 2010). This will lead to dense levels of bioturbation

(Song et al., 2010), making it difficult to recognize discrete traces and interpret their behavioral significance or potential trace makers. This is a potential reason why beetles are not recorded as the dominant trace makers more often in documented ichnofossil assemblages since they are removed by the disruption of their own and others’ burrowing activity.

Figure 17

Preservation of Burrows Over Time

Note. Figure 17: Burrow preservation potential was investigated on the 76-L Experiment 6 terrarium for

Zophobas morio. A) Time 1, shortly after the experiment was concluded. B) 38-L of coarse-grained sediment was placed on top of the experiment shortly after Time 1. C) 5 months after the placement of sediment in

Time 1, overall trace morphology has not changed. Scale bar represents 10 cm.

Comparisons to Ichnofossils

Several common ichnogenera are typically interpreted as beetle burrows (Genise,

2004; Buatois and Mangano, 2011) (Fig. 18). When examined quantitatively and qualitatively there were many differences and similarities between the burrows produced in these experiments and these ichnogenera. Of these known ichnogenera, the most quantitatively comparable to the extant beetle larvae burrows were Paleophycus and

Planolites. Still, despite being quantitatively comparable, there were many qualitative differences with the extant biogenic structures.

In comparison to Paleophycus, the beetle larvae lack a discrete and continuous lining. The larva burrows only contained intermittently visible compressional linings.

These linings were mainly observed in Zophobas morio, and only seldom or not seen in the other taxa. In comparison to Planolites, the beetle larvae burrows had two main differences. Planolites are passively filled with sediment that is different from host substrate (Pemberton and Frey, 1982). The beetle larvae burrows had no surface connections, and were too sporadically passively filled with host sediment form a fully connected burrow network. This would make it nearly impossible to be filled the same as

Planolites. Therefore, the extant beetle larvae burrows resemble a combination of both

Paleophycus and Planolites.

The beetle larvae burrows were very different from Skolithos. The larval burrows could be long, vertical to subvertical shafts, but these were usually connected to, or could be traced to, a larger burrow. The backfilled burrow Taenidium had many similarities with the larval burrows of Phyllophaga sp. Like Taenidium, they were unlined meniscate 95 burrows that were straight, curved, or sinuous, and possessed secondary successive branching (Keighley and Pickerill, 1994). Unlike Taenidium, the larval burrows of

Phyllophaga sp. terminated with an open chamber. In their larval stage the other species did not produce backfilled burrows comparable to Taenidium. However, the backfilled burrows produced by adult Zophobas morio were similar to Taenidium, but had differences. Adult Z. morio burrows terminated in either an open chamber, or a cone-to- cone structure, along with originating from an actively filled chamber. Any other similarities between Taenidium and the other taxa, were superficial. Overall, larval

Phyllophaga sp. burrows and adult Zophobas morio burrows were comparable at the ichnogenus level to Taenidium.

Naktodemasis had many similarities the burrows of Phyllophaga sp. Burrows of

Phyllophaga sp. consisted of a straight to sinuous, unlined series of ellipsoidal shaped packets of menisci with variable orientations (Counts and Hasiotis, 2009). However, burrows of Phyllophaga sp. also possessed branching, which is not diagnostic of

Naktodemasis. The burrows of Z. morio and T. molitor were not similar to Naktodemasis in any life stage. The adult burrows of Z. morio were backfilled, but to the form and arrangement of the backfills were distinct from Naktodemasis. Overall, larval burrows of

Phyllophaga sp. were comparable to Naktodemasis at the ichnogenus level.

Phyllophaga sp. larvae burrows were comparable to Macanopsis. Unlike

Macanopsis which are described as long, subvertical shafts with elongated terminal chambers (Mikus and Uchman, 2013), however, Phyllophaga sp. had branches and was backfilled. Adult burrows of Z. morio also resembled Macanopsis, but were completely 96 backfilled. Any similarities with burrows of T. molitor were superficial, since those beetles did not generate any biogenic structures resembling the morphology of

Macanopsis. The burrows of the taxa were not comparable at the genus or species level for Macanopsis. The closest are adult Z. morio, but this would require a backfilled

Macanopsis variant ichnofossil to be discovered.

Several common ichnofossils have also been interpreted as beetle pupation or nesting chambers (Genise, 2004; Buatois and Mangano, 2011)(Fig. 18). When the chambers produced by the three species in this study were compared quantitatively and qualitatively to these ichnogenera (Fictovichnus, Pallichnus, and Rebuffoichnus), they are overwhelming similar across all of the tested properties. The major difference between the chambers of the extant beetles and the different ichnogenera was how they were preserved. The ichnogenera are usually preserved by passive fill, from material different from the host sediment typically possess a lining (Genise et al., 2007). This material has included volcanic ash, non-recrystallized clay, or other fine-grained material (Genise et al., 2007). However, none of the chambers in this project were infilled in this way, or were capable of such preservation since they were cut off from the surface. The only similarity is that some chambers had the potential to produce chemical linings when the beetle died within resulting in the growth of bacteria, mold, or fugus around and within the chamber. The chambers in this study were most often actively filled when the adults moved to the surface. Despite their strong quantitative similarities, these modern biogenic chambers have different preservation properties not seen in the ichnofossils, which means that these chambers are not directly comparable to already established ichnotaxa. 97

Figure 18

Comparison of Beetle Burrows to Ichnogenera Visuals

Note. Figure 18: Qualitative comparison of beetle biogenic structures to known ichnogenera. A) Subvertical shaft is similar in appearance to Skolithos (A’). B) Large network of tunnels created by several larvae is similar to Planolites (B’). C) Partially lined tunnels are similar to Paleophycus (C’). D) Vertical backfilled burrow connected to a chamber is similar to Macanopsis (D’). E) Chamber constructed for pupation similar to Pallichnus (E’). F) Pupation chamber is similar to Fictovichnus (F’). G) Pupation chamber similar to

Rebuffoichnus (G’). H) Backfilled burrows composed of ellipsoidal sediment packets similar to

Naktodemasis (H’). I) Backfilled burrow with sinuous to straight series of overlapping semicircular sediment packets similar to Taenidium (I’). Scale bars represent 20 mm.

Chapter 6: Conclusion

By understanding beetle trace morphologies in a laboratory setting, we can properly assign beetles as trace makers in continental ichnofossil assemblages. Before this can be done, it is necessary to understand the qualitative and quantitative properties of beetle biogenic structures through all of their life stages. It is also important to understand potential variation in beetle trace morphology under different environmental conditions, as well as their total potential for bioturbation and the preservation potential of their traces. By doing this, we can further improve the paleoenvironmental and paleoecological significance of beetle ichnofossils.

Experiments with the three species of beetles in this project have provided a set of distinct burrow morphologies associated with their larval, pupal, and adult life stages.

Descriptions of these different beetle traces will be helpful in interpreting a variety of continental ichnofossils. Despite having similarities in body morphology during their life stages, each beetle species had quantitatively unique burrow morphologies in their larval and adult life stages. Pupation and resting chamber morphologies were also quantitatively unique between taxa; however, they were very similar to each other in terms of general morphology. The beetles’ behavior and body morphology had a fundamental influence on trace morphology. The three environmental parameters that were tested were important in altering behavior when extreme stresses were applied, generally prohibiting burrowing, but did not change the overall trace morphologies. As a result, variations in beetle trace morphology are not likely good paleoenvironmental indicators. 100

The total bioturbation potential of beetles was fundamental to their interpreted moderate to low preservation potential. Since beetles inhabit tiers in the soil with many other burrowing organisms, their traces can be easily reworked by themselves or other animals within the substrate. Therefore, understanding and recognizing general beetle disruption patterns as well as distinct traces can help to identify beetle bioturbation in the ichnofossil record. However, beetles alone would likely not be the sole producers of bioturbation in their ecosystems. Through recognition of the minute patterns and structures that might be produced and preserved, the entire life cycle of beetles could be observed in the ichnofossil record. This could help expand beetle ichnology beyond the description of their chambers. Pupation and nesting chambers are indicative of beetles, but only express a single life stage, in an otherwise complicated multistage organism.

Based on the observations in this study, a number of paleoenvironmental and paleoecological interpretations may be based on the occurrence of beetle ichnofossils.

Since beetles are abundant in soils, the occurrence of their traces can help interpret continental deposits as paleosols. In order to produce burrows in their larval stage, these paleosols would have needed to be generally loose, moist, and with moderate clay content. Finally, the occurrence of abundant pupation chambers is suggestive of well- drained soil conditions as excessive moisture typically results in the death of pupae. The presence of beetle ichnofossils can also provide insight into the reconstruction of ancient food webs. Extensive bioturbation by mobile deposit-feeding beetle larvae is suggestive of nutrient-rich soils. Several species of beetles are also root feeders, so their presence can be used to make inferences on the abundance of plants in paleoecosystems, even if no 101 plant fossils are preserved. Beetles, in all of their life stages, are also important prey items for various invertebrate and vertebrate carnivores.

By understanding the traces of extant beetles, we can compare them to continental ichnofossils to improve the interpretation of their behavioral, paleoenvironmental, and paleoecological significance, along with their potential trace makers. This is especially important for beetles considering the variety of trace morphologies they produce throughout their different life stages. The results of this study show that beetle biogenic structures can be distinguished using a variety distinctive qualitative and quantitative properties. However, ichnogenera that have been previously attributed to beetles do not fully match the morphologies of the burrows and chambers produced in these experiments. Therefore, if recognized in the fossil record, many of these traces would require description as new ichnospecies or ichnogenera.

102

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114

Appendix A

Appendix A has dendrograms created during the Bray-Curtis similarity test.

Order of Appendix A:

1) Burrow analysis between beetle species.

2) Chamber analysis between beetle species.

3) Experiment 3 analysis.

4) Experiment 4 analysis.

5) Experiment 5 analysis.

6) Beetles biogenic structures vs known ichnogenera.

115

Appendix: A1

Burrow Analysis Between Beetle Species

1) Dendrogram showing the relationship between the burrows produced by the three beetle species based on the Bray Curtis similarity matrix. T = Tenebrio molitor; Z = Zophobas morio; P = Phyllophaga sp. 116

Appendix: A2

Chamber Analysis Between Beetle Species

2) Dendrogram showing the relationship between the chambers produced by the three beetle species based on the Bray Curtis similarity matrix. T = Tenebrio molitor; Z = Zophobas morio; P = Phyllophaga sp. 117

Appendix: A3

Analysis within Beetle Species

3) Dendrogram showing the relationship between the different conditions of high sand vs low sand in Experiment 3 based on the Bray Curtis similarity matrix. T = Tenebrio molitor; Z = Zophobas morio; P = Phyllophaga sp.

118

Appendix: A4

Analysis within Beetle Species

4) Dendrogram showing the relationship between the different conditions of high moisture vs low moisture in Experiment 4 based on the Bray Curtis similarity matrix. T = Tenebrio molitor; Z = Zophobas morio; P = Phyllophaga sp. 119

Appendix: A5

Analysis within Beetle Species

5) Dendrogram showing the relationship between the different conditions of firm vs loose substrate in Experiment 5 based on the Bray Curtis similarity matrix. T = Tenebrio molitor; Z = Zophobas morio; P = Phyllophaga sp. 120

Appendix: A6

Analysis Comparing Beetle Biogenic Structures vs Known Ichnogenera

121

6) Dendrogram showing the relationship between the different beetle biogenic structures vs known ichnogenera based on the Bray Curtis similarity matrix. T = Tenebrio molitor; Z = Zophobas morio; P = Phyllophaga sp. Macanopsis (M), Paleophycus (Pa), Planolites (Pt), Skolithos (S), Taenidium (T), and Naktodemasis (N). Comparable chambers are Fictovichnus (F), Pallichnus (Pl), and Rebuffoichnus (R).

122

Appendix B

Appendix B: Tables displaying the fourteen properties listed in the methods, along with time duration of larval and pupal stages, and mortality status.

Order of Appendix B:

Experiment 2:

1) Phyllophaga sp. chambers.

2) Tenebrio molitor chambers and burrows.

3) Zophobas morio chambers and burrows.

Experiment 3:

4) Phyllophaga sp. chambers and burrows.

5) Tenebrio molitor chambers and burrows.

6) Zophobas morio chambers and burrows.

Experiment 4:

7) Phyllophaga sp. chambers and burrows.

8) Tenebrio molitor chambers and burrows.

9) Zophobas morio chambers and burrows.

Experiment 5:

10)Phyllophaga sp. chambers and burrows.

11) Tenebrio molitor chambers and burrows.

12) Zophobas morio chambers and burrows.

123

Appendix: B1

Experiment 2 Averages: Phyllophaga sp.

Experiment 2 Average Phyllophaga sp. Chambers P1-2 P2-2 P3-2 P4-2 P5-2 P6-2 Depth 106.10 13.00 44.08 N/A 166.67 N/A Max Width 7.90 8.46 9.45 N/A 27.25 N/A Min Width 6.93 6.52 7.96 N/A 21.12 N/A Mean Width 7.08 7.49 8.70 N/A 24.18 N/A Max Length 5.55 7.16 6.38 N/A 12.43 N/A Min Length 3.55 5.18 4.33 N/A 10.05 N/A Mean Length 4.47 6.17 5.36 N/A 11.24 N/A Mean W/H ratio 1.96 1.15 1.62 N/A 1.88 N/A Max slope 50.17 21.75 32.17 N/A 14.83 N/A Min slope 37.43 4.50 19.58 N/A 9.33 N/A Mean slope 43.80 13.13 25.88 N/A 12.08 N/A Branching angles 0.00 0.00 0.00 N/A 0.00 N/A Complexity 1.00 1.00 1.00 N/A 1.00 N/A Tortuosity 1.78 1.59 1.65 N/A 1.56 N/A Larval stage (Days) 12 10 12 N/A 12 N/A Pupal stage (Days) Dead Dead Dead Dead Dead Dead Total Life Cycle (Days) N/A N/A N/A N/A N/A N/A

Experiment 2 Average Phyllophaga sp. Burrows P1-2 P2-2 P3-2 P4-2 P5-2 P6-2 Depth N/A N/A N/A N/A N/A N/A Max Width N/A N/A N/A N/A N/A N/A Min Width N/A N/A N/A N/A N/A N/A Mean Width N/A N/A N/A N/A N/A N/A Max Length N/A N/A N/A N/A N/A N/A Min Length N/A N/A N/A N/A N/A N/A Mean Length N/A N/A N/A N/A N/A N/A Mean W/H ratio N/A N/A N/A N/A N/A N/A Max slope N/A N/A N/A N/A N/A N/A Min slope N/A N/A N/A N/A N/A N/A Mean slope N/A N/A N/A N/A N/A N/A Branching angles N/A N/A N/A N/A N/A N/A Complexity N/A N/A N/A N/A N/A N/A Tortuosity N/A N/A N/A N/A N/A N/A

1) Experiment 2 averages for Phyllophaga sp. chambers and burrows in mm. 124

Appendix: B2

Experiment 2 Averages: Tenebrio molitor.

Experiment 2 Average Tenebrio molitor Chambers T1-2 T2-2 T3-2 T4-2 T5-2 T6-2 Depth N/A 4.26 24.62 16.80 N/A 9.71 Max Width N/A 16.75 17.12 15.27 N/A 14.76 Min Width N/A 11.56 13.16 10.35 N/A 6.81 Mean Width N/A 14.16 15.14 12.81 N/A 10.79 Max Length N/A 12.50 9.58 20.07 N/A 16.75 Min Length N/A 7.73 5.25 15.19 N/A 9.24 Mean Length N/A 10.11 7.42 17.63 N/A 12.99 Mean W/L ratio N/A 1.70 2.04 0.77 N/A 0.94 Max Slope N/A 17.33 5.00 1.50 N/A 26.93 Min Slope N/A 15.34 8.00 0.77 N/A 13.14 Mean Slope N/A 16.10 6.50 1.13 N/A 20.04 Branching angles N/A 0.00 0.00 0.00 N/A 3.10 Complexity N/A 1.00 1.00 1.00 N/A 1.21 Tortuosity N/A 1.60 1.43 2.32 N/A 2.16 Larval stage (Days) N/A 26 31 38 N/A 14 Pupal stage (Days) Dead 27 24 20 Dead 30 Total Life Cycle (Days) N/A 53 55 58 N/A 44

Experiment 2 Average Tenebrio molitor Burrows T1-2 T2-2 T3-2 T4-2 T5-2 T6-2 Depth 6.81 10.67 17.64 9.30 8.34 12.05 Max Width 6.55 5.18 5.96 4.60 7.64 4.05 Min Width 3.68 2.84 2.68 2.80 3.54 2.65 Mean Width 5.11 4.01 4.32 3.70 5.59 3.35 Max Length 46.45 46.03 99.88 30.60 74.70 24.20 Min Length 43.53 39.66 42.75 27.10 70.24 22.85 Mean Length 44.99 40.18 71.32 28.85 72.47 23.53 Mean W/L ratio 0.11 3.20 0.07 0.13 0.10 0.15 Max Slope 14.67 27.89 9.83 31.00 18.40 19.00 Min Slope 8.33 12.33 4.33 19.00 8.87 6.00 Mean Slope 11.50 20.11 7.08 25.00 13.53 12.50 Branching angles 0.00 23.67 27.63 74.00 17.48 0.00 Complexity 1.00 4.83 2.50 2.00 2.33 1.00 Tortuosity 1.28 1.58 1.63 1.42 1.28 0.89

2) Experiment 2 averages for Tenebrio molitor. chambers and burrows in mm. 125

Appendix: B3

Experiment 2 Averages for Zophobas morio

Experiment 2Average Zophobas morio Burrows Z1-2 Z2-2 Z3-2 Z4-2 Z5-2 Z6-2 Depth N/A 64.31 23.44 58.91 7.99 71.99 Max Width N/A 5.19 5.24 9.29 26.43 6.62 Min Width N/A 2.83 3.54 5.29 11.73 4.42 Mean Width N/A 4.01 4.39 7.29 19.08 5.52 Max Length N/A 25.24 118.53 120.43 93.25 60.48 Min Length N/A 21.02 109.95 107.92 90.46 55.25 Mean Length N/A 23.13 114.24 114.17 91.86 57.87 Mean W/L ratio N/A 0.22 0.06 0.09 0.24 0.14 Max Slope N/A 41.50 19.86 42.42 8.50 26.00 Min Slope N/A 31.00 7.29 24.25 4.00 14.83 Mean Slope N/A 36.25 13.57 33.33 6.25 20.42 Branching angles N/A 0.00 12.25 33.34 0.00 12.50 Complexity N/A 1.00 2.00 4.25 1.00 1.33 Tortuosity N/A 0.95 1.32 1.92 3.81 1.30

Experiment 2 Average Zophobas morio Chambers Z1-2 Z2-2 Z3-2 Z4-2 Z5-2 Z6-2 Depth 16.82 151.28 70.45 64.31 69.82 N/A Max Width 9.86 12.11 17.29 25.23 18.80 N/A Min Width 6.01 9.60 12.99 11.24 13.59 N/A Mean Width 7.94 10.86 15.14 18.24 16.19 N/A Max Length 4.65 27.01 28.75 39.41 49.38 N/A Min Length 3.96 28.00 21.76 30.13 40.63 N/A Mean Length 4.31 27.50 25.25 34.77 45.00 N/A Mean W/L ratio 1.84 0.43 0.61 0.52 0.36 N/A Max Slope 7.00 6.73 10.50 25.00 9.33 N/A Min Slope 6.00 2.09 3.83 11.00 3.17 N/A Mean Slope 6.50 4.41 7.17 18.00 6.25 N/A Branching angles 0.00 0.00 0.00 0.00 0.00 N/A Complexity 1.00 1.00 1.00 1.00 1.00 N/A Tortuosity 3.34 1.21 1.47 1.83 1.24 N/A Larval stage (Days) 39 32 44 37 20 N/A Pupal stage (Days) Dead 50 31 Dead 29 Dead Total Life Cycle (Days) N/A 82 75 N/A 49 N/A

3) Experiment 2 averages for Zophobas morio chambers and burrows in mm 126

Appendix: B4

Experiment 3 Averages: Phyllophaga sp.

Experiment 3 Averages Phyllophaga sp. Chambers P1-3 P2-3 P3-3 P4-3 P5-3 P6-3 Depth N/A N/A 73.57 136.19 110.61 N/A Max Width N/A N/A 7.91 6.79 6.89 N/A Min Width N/A N/A 6.43 5.21 5.89 N/A Mean Width N/A N/A 7.17 6.00 6.39 N/A Max Length N/A N/A 12.26 13.25 23.05 N/A Min Length N/A N/A 10.96 11.09 21.15 N/A Mean Length N/A N/A 11.61 12.17 22.10 N/A Mean W/H ratio N/A N/A 0.62 0.53 0.30 N/A Max slope N/A N/A 27.00 23.50 18.18 N/A Min slope N/A N/A 15.00 12.75 7.27 N/A Mean slope N/A N/A 21.00 18.13 12.73 N/A Branching angles N/A N/A 0.00 0.00 0.00 N/A Complexity N/A N/A 1.00 1.00 1.00 N/A Tortuosity N/A N/A 1.56 1.64 1.23 N/A Larval stage (Days) N/A N/A 25 22 37 N/A Pupal stage (Days) Dead Dead Dead Dead Dead Dead Total Life Cycle (Days) N/A N/A N/A N/A N/A N/A

Experiment 3 Averages Phyllophaga sp. Burrows P1-3 P2-3 P3-3 P4-3 P5-3 P6-3 Depth N/A N/A N/A 121.48 88.46 N/A Max Width N/A N/A N/A 6.56 11.43 N/A Min Width N/A N/A N/A 4.46 5.76 N/A Mean Width N/A N/A N/A 5.51 8.59 N/A Max Length N/A N/A N/A 31.98 53.43 N/A Min Length N/A N/A N/A 30.57 45.27 N/A Mean Length N/A N/A N/A 31.28 49.35 N/A Mean W/H ratio N/A N/A N/A 0.21 0.21 N/A Max slope N/A N/A N/A 40.00 55.33 N/A Min slope N/A N/A N/A 37.33 45.33 N/A Mean slope N/A N/A N/A 38.67 50.33 N/A Branching angles N/A N/A N/A 0.00 0.00 N/A Complexity N/A N/A N/A 1.00 1.00 N/A Tortuosity N/A N/A N/A 1.33 1.46 N/A

4) Experiment 3 averages for Phyllophaga sp. chambers and burrows in mm

127

Appendix: B5

Experiment 3 Averages: Tenebrio molitor.

Experiment 3 Averages Tenebrio molitor Chambers T1-3 T2-3 T3-3 T4-3 T5-3 T6-3 Depth N/A N/A 44.93 18.97 42.71 N/A Max Width N/A N/A 15.47 11.71 13.41 N/A Min Width N/A N/A 6.60 8.74 6.32 N/A Mean Width N/A N/A 11.03 10.23 9.87 N/A Max Length N/A N/A 33.53 9.94 31.08 N/A Min Length N/A N/A 31.47 8.06 22.96 N/A Mean Length N/A N/A 32.50 9.00 27.02 N/A Mean W/H ratio N/A N/A 0.34 1.14 0.37 N/A Max slope N/A N/A 83.00 10.00 271.50 N/A Min slope N/A N/A 87.00 9.00 260.00 N/A Mean slope N/A N/A 85.00 9.50 265.75 N/A Branching angles N/A N/A 0.00 0.00 0.00 N/A Complexity N/A N/A 1.00 1.00 1.00 N/A Tortuosity N/A N/A 1.44 2.44 1.44 N/A Larval stage (Days) N/A N/A 10 15 1 N/A Pupal stage (Days) Dead Dead Dead Dead Dead Dead Total Life Cycle (Days) N/A N/A N/A N/A N/A N/A

Experiment 3 Averages Tenebrio molitor Burrows T1-3 T2-3 T3-3 T4-3 T5-3 T6-3 Depth N/A N/A N/A N/A 40.88 N/A Max Width N/A N/A N/A N/A 9.47 N/A Min Width N/A N/A N/A N/A 7.72 N/A Mean Width N/A N/A N/A N/A 8.60 N/A Max Length N/A N/A N/A N/A 28.07 N/A Min Length N/A N/A N/A N/A 14.40 N/A Mean Length N/A N/A N/A N/A 21.24 N/A Mean W/H ratio N/A N/A N/A N/A 0.40 N/A Max slope N/A N/A N/A N/A 16.00 N/A Min slope N/A N/A N/A N/A 4.00 N/A Mean slope N/A N/A N/A N/A 10.00 N/A Branching angles N/A N/A N/A N/A 0.00 N/A Complexity N/A N/A N/A N/A 1.00 N/A Tortuosity N/A N/A N/A N/A 1.27 N/A

5) Experiment 3 averages for Tenebrio molitor. chambers and burrows in mm.

128

Appendix: B6

Experiment 3 Averages: Zophobas morio.

Experiment 3 Averages Zophobas morio Chambers Z1-3 Z2-3 Z3-3 Z4-3 Z5-3 Z6-3 Depth N/A N/A N/A 161.64 165.61 85.52 Max Width N/A N/A N/A 30.13 27.56 16.72 Min Width N/A N/A N/A 25.22 23.10 14.26 Mean Width N/A N/A N/A 27.68 25.33 15.49 Max Length N/A N/A N/A 26.12 27.23 26.75 Min Length N/A N/A N/A 24.64 22.58 19.70 Mean Length N/A N/A N/A 25.38 24.91 23.23 Mean W/H ratio N/A N/A N/A 1.07 1.02 0.67 Max slope N/A N/A N/A 19.25 18.67 9.00 Min slope N/A N/A N/A 6.00 7.67 6.00 Mean slope N/A N/A N/A 12.63 13.17 7.50 Branching angles N/A N/A N/A 0.00 0.00 0.00 Complexity N/A N/A N/A 1.00 1.00 1.00 Tortuosity N/A N/A N/A 1.82 1.70 1.69 Larval stage (Days) N/A N/A N/A 11 5 25 Pupal stage (Days) Dead Dead Dead Dead 21 Dead Total Life Cycle (Days) N/A N/A N/A N/A 26 N/A

Experiment 3 Averages Zophobas morio Burrows Z1-3 Z2-3 Z3-3 Z4-3 Z5-3 Z6-3 Depth N/A 106.01 145.06 134.28 126.73 188.19 Max Width N/A 6.90 13.59 4.93 8.49 35.77 Min Width N/A 3.21 11.60 3.26 7.28 28.97 Mean Width N/A 5.06 12.59 4.10 7.88 32.37 Max Length N/A 42.68 17.55 22.00 16.87 26.63 Min Length N/A 37.13 15.71 20.28 14.93 21.47 Mean Length N/A 39.91 16.63 21.14 15.90 24.05 Mean W/H ratio N/A 0.13 0.76 0.19 0.48 1.35 Max slope N/A 43.00 52.00 44.00 33.00 12.00 Min slope N/A 37.00 45.00 32.50 28.00 7.00 Mean slope N/A 40.00 48.50 38.25 30.50 9.50 Branching angles N/A 29.00 0.00 12.00 0.00 0.00 Complexity N/A 2.00 1.00 1.50 1.00 1.00 Tortuosity N/A 1.93 1.22 1.68 1.37 1.73

6) Experiment 3 averages for Zophobas morio chambers and burrows in mm.

129

Appendix: B7

Experiment 4 Averages: Phyllophaga sp.

Experiment 4 Averages Phyllophaga sp. Chambers P1-4P2-4P3-4P4-4 Depth 99.12N/A76.32 14.26 Max Width 9.76N/A8.659.75 Min Width 7.58N/A5.913.76 Mean Width 8.67N/A7.286.75 Max Length 13.69N/A28.396.24 Min Length 11.41N/A25.075.03 Mean Length 12.55N/A26.735.63 Mean W/H ratio 0.77N/A0.481.20 Max slope 9.50N/A58.00 31.00 Min slope 4.50N/A49.40 27.00 Mean slope 7.00N/A53.70 29.00 Branching angles 0.00N/A0.000.00 Complexity 1.00N/A1.001.00 Tortuosity 1.38N/A1.241.79 Larval stage (Days) 30 N/A 34 Dead Pupal stage (Days) Dead DeadDeadDead Total Life Cycle (Days) N/AN/AN/AN/A

Experiment 4 Averages Phyllophaga sp. Burrows P1-4 P2-4 P3-4 P4-4 Depth 127.45 52.56 87.54 116.74 Max Width 16.59 13.86 12.25 20.40 Min Width 9.08 12.22 9.03 12.96 Mean Width 12.84 13.04 10.64 16.68 Max Length 58.13 80.85 60.89 77.60 Min Length 53.51 70.68 55.98 73.18 Mean Length 55.82 75.77 58.44 75.39 Mean W/H ratio 0.28 0.17 0.24 0.26 Max slope 72.59 82.00 70.80 54.67 Min slope 64.94 40.00 55.20 27.33 Mean slope 68.76 61.00 63.00 41.00 Branching angles 0.00 0.00 8.20 0.00 Complexity 1.24 1.00 1.20 1.00 Tortuosity 1.44 1.41 1.26 2.12

7) Experiment 4 averages for Phyllophaga sp. chambers and burrows in mm.

130

Appendix: B8

Experiment 4 Averages: Tenebrio molitor.

Experiment 4 Averages Tenebrio molitor Chambers T1-4 T2-4 T3-4 T4-4 T5-4 T6-4 Depth N/A 45.83 N/A 21.73 N/A 89.73 Max Width N/A 5.56 N/A 3.02 N/A 5.38 Min Width N/A 4.19 N/A 2.47 N/A 3.48 Mean Width N/A 4.88 N/A 2.75 N/A 4.43 Max Length N/A 12.44 N/A 8.07 N/A 7.93 Min Length N/A 10.86 N/A 6.93 N/A 6.90 Mean Length N/A 11.65 N/A 7.50 N/A 7.42 Mean W/H ratio N/A 0.42 N/A 0.37 N/A 0.60 Max slope N/A 69.00 N/A 40.00 N/A 31.00 Min slope N/A 61.00 N/A 35.00 N/A 14.00 Mean slope N/A 65.00 N/A 37.50 N/A 22.50 Branching angles N/A 0.00 N/A 0.00 N/A 0.00 Complexity N/A 1.00 N/A 1.00 N/A 1.00 Tortuosity N/A 1.77 N/A 1.26 N/A 1.75 Larval stage (Days) 35 16 35 5 35 1 Pupal stage (Days) Dead 10 Dead Dead Dead Dead Total Life Cycle (Days) N/A 26 N/A N/A N/A N/A

Experiment 4 Averages Tenebrio molitor Burrows T1-4 T2-4 T3-4 T4-4 T5-4 T6-4 Depth 60.89 47.90 77.77 79.02 25.85 40.92 Max Width 5.25 6.38 4.21 4.00 4.92 4.22 Min Width 4.28 4.45 2.89 2.96 2.75 2.81 Mean Width 4.76 5.41 3.55 3.48 3.83 3.52 Max Length 44.35 59.97 26.09 25.73 24.00 10.48 Min Length 40.23 43.46 22.18 21.91 22.21 10.07 Mean Length 42.29 51.72 24.14 23.82 23.11 10.28 Mean W/H ratio 0.14 0.10 0.15 0.15 0.17 0.42 Max slope 65.50 47.50 77.00 47.00 64.67 73.33 Min slope 46.25 39.00 3.00 12.50 61.00 62.67 Mean slope 55.88 43.25 40.00 29.75 62.83 68.00 Branching angles 11.25 24.00 0.00 0.00 0.00 0.00 Complexity 2.00 1.50 1.00 1.00 1.00 1.00 Tortuosity 1.56 1.33 1.36 1.40 1.42 1.32

8) Experiment 4 averages for Tenebrio molitor chambers and burrows in mm.

131

Appendix: B9

Experiment 4 Averages: Zophobas morio

Experiment 4 Averages Zophobas morio Chambers Z1-4 Z2-4 Z3-4 Z4-4 Z5-4 Z6-4 Depth N/A 163.48 134.84 N/A N/A 146.63 Max Width N/A 16.25 17.83 N/A N/A 10.66 Min Width N/A 11.88 12.54 N/A N/A 8.13 Mean Width N/A 14.06 15.19 N/A N/A 9.40 Max Length N/A 28.66 28.79 N/A N/A 25.60 Min Length N/A 25.09 26.63 N/A N/A 29.94 Mean Length N/A 26.88 27.71 N/A N/A 27.77 Mean W/H ratio N/A 0.52 0.56 N/A N/A 0.34 Max slope N/A 7.00 24.00 N/A N/A 13.00 Min slope N/A 3.00 18.00 N/A N/A 6.00 Mean slope N/A 5.00 21.00 N/A N/A 9.50 Branching angles N/A 0.00 0.00 N/A N/A 0.00 Complexity N/A 1.00 1.00 N/A N/A 1.00 Tortuosity N/A 1.32 1.66 N/A N/A 1.39 Larval stage (Days) N/A 11 15 N/A N/A 25 Pupal stage (Days) Dead Dead 32 Dead Dead Dead Total Life Cycle (Days) N/A N/A 47 N/A N/A N/A

Experiment 4 Averages Zophobas morio Burrows Z1-4 Z2-4 Z3-4 Z4-4 Z5-4 Z6-4 Depth 19.47 69.49 90.14 119.20 115.75 88.67 Max Width 5.32 7.94 6.87 7.30 5.15 8.07 Min Width 4.15 4.27 4.66 4.01 3.85 5.30 Mean Width 4.74 6.11 5.77 5.66 4.50 6.69 Max Length 26.84 50.42 79.52 43.76 28.55 29.16 Min Length 24.74 48.00 75.78 38.90 26.55 25.78 Mean Length 25.79 49.21 77.65 41.33 27.55 27.47 Mean W/H ratio 0.18 0.13 0.08 0.14 0.16 0.24 Max slope 23.00 25.43 14.00 25.00 32.50 33.00 Min slope 17.00 14.71 9.00 18.00 23.00 19.00 Mean slope 20.00 20.07 11.50 21.50 27.75 26.00 Branching angles 0.00 2.14 16.50 22.50 0.00 0.00 Complexity 1.00 1.14 1.50 2.00 1.00 1.00 Tortuosity 1.14 1.15 1.26 1.72 1.09 1.35

9) Experiment 4 averages for Zophobas morio chambers and burrows in mm.

132

Appendix: B10

Experiment 5 Averages: Phyllophaga sp.

Experiment 5 Averages Phyllophaga sp. Chambers P1-3 P2-3 P3-3 P4-3 Depth 28.00 N/A 154.82 115.70 Max Width 8.98 N/A 7.04 11.42 Min Width 7.09 N/A 6.05 10.27 Mean Width 8.04 N/A 6.54 10.84 Max Length 13.63 N/A 10.49 17.84 Min Length 11.84 N/A 9.63 12.72 Mean Length 12.74 N/A 10.06 15.28 Mean W/H ratio 0.63 N/A 0.65 0.71 Max slope 6.00 N/A 24.00 21.75 Min slope 4.00 N/A 17.00 13.75 Mean slope 5.00 N/A 20.50 17.75 Branching angles 0.00 N/A 0.00 0.00 Complexity 1.00 N/A 1.00 1.00 Tortuosity 1.34 N/A 1.41 1.32 Larval stage (Days) 21 N/A 21 21 Pupal stage (Days) Dead Dead Dead Dead Total Life Cycle (Days) N/A N/A N/A N/A

Experiment 5 Averages Phyllophaga sp. Burrows P1-3 P2-3 P3-3 P4-3 Depth N/A N/A 136.45 105.93 Max Width N/A N/A 7.96 11.46 Min Width N/A N/A 6.71 9.66 Mean Width N/A N/A 7.34 10.56 Max Length N/A N/A 111.51 130.01 Min Length N/A N/A 108.55 118.58 Mean Length N/A N/A 110.03 124.29 Mean W/H ratio N/A N/A 0.07 0.09 Max slope N/A N/A 81.00 45.50 Min slope N/A N/A 56.00 36.50 Mean slope N/A N/A 68.50 41.00 Branching angles N/A N/A 0.00 0.00 Complexity N/A N/A 1.00 1.00 Tortuosity N/A N/A 0.80 0.70

10) Experiment 5 averages for Phyllophaga sp. chambers and burrows in mm. 133

Appendix: B11

Experiment 5 Averages: Tenebrio molitor

Experiment 5 Averages Tenebrio molitor Chambers T1-5 T2-5 T3-5 T4-5 T5-5 T6-5 Depth N/A N/A N/A N/A N/A N/A Max Width N/A N/A N/A N/A N/A N/A Min Width N/A N/A N/A N/A N/A N/A Mean Width N/A N/A N/A N/A N/A N/A Max Length N/A N/A N/A N/A N/A N/A Min Length N/A N/A N/A N/A N/A N/A Mean Length N/A N/A N/A N/A N/A N/A Mean W/H ratio N/A N/A N/A N/A N/A N/A Max slope N/A N/A N/A N/A N/A N/A Min slope N/A N/A N/A N/A N/A N/A Mean slope N/A N/A N/A N/A N/A N/A Branching angles N/A N/A N/A N/A N/A N/A Complexity N/A N/A N/A N/A N/A N/A Tortuosity N/A N/A N/A N/A N/A N/A Larval stage (Days) N/A 2 N/A 2 8 6 Pupal stage (Days) Dead Dead Dead Dead Dead Dead Total Life Cycle (Days) N/A N/A N/A N/A N/A N/A

Experiment 5 Averages Tenebrio molitor Burrows T1-5 T2-5 T3-5 T4-5 T5-5 T6-5 Depth N/A 18.12 N/A 44.26 36.49 64.52 Max Width N/A 4.03 N/A 3.83 3.70 4.34 Min Width N/A 2.82 N/A 2.89 2.60 2.25 Mean Width N/A 3.42 N/A 3.36 3.15 3.30 Max Length N/A 42.95 N/A 55.79 54.07 78.37 Min Length N/A 28.19 N/A 54.44 44.76 77.01 Mean Length N/A 35.57 N/A 55.12 49.42 77.69 Mean W/H ratio N/A 0.10 N/A 0.11 0.15 0.04 Max slope N/A 57.00 N/A 85.00 29.75 50.00 Min slope N/A 48.00 N/A 72.00 13.00 27.67 Mean slope N/A 52.50 N/A 78.50 21.38 38.83 Branching angles N/A 0.00 N/A 0.00 0.00 0.67 Complexity N/A 1.00 N/A 1.00 1.25 1.33 Tortuosity N/A 1.10 N/A 1.05 0.99 0.84

11) Experiment 5 averages for Tenebrio molitor chambers and burrows in mm. 134

Appendix B12

Experiment 5 Averages: Zophobas morio.

Experiment 5 Averages Zophobas morio Chambers Z1-5 Z2-5 Z3-5 Z4-5 Z5-5 Z6-5 Depth N/A N/A N/A N/A N/A 136.66 Max Width N/A N/A N/A N/A N/A 18.50 Min Width N/A N/A N/A N/A N/A 13.91 Mean Width N/A N/A N/A N/A N/A 14.85 Max Length N/A N/A N/A N/A N/A 24.44 Min Length N/A N/A N/A N/A N/A 23.84 Mean Length N/A N/A N/A N/A N/A 24.40 Mean W/H ratio N/A N/A N/A N/A N/A 5.23 Max slope N/A N/A N/A N/A N/A 15.93 Min slope N/A N/A N/A N/A N/A 11.20 Mean slope N/A N/A N/A N/A N/A 13.20 Branching angles N/A N/A N/A N/A N/A 1.80 Complexity N/A N/A N/A N/A N/A 0.80 Tortuosity N/A N/A N/A N/A N/A 1.75 Larval stage (Days) N/A N/A 1 8 14 12 Pupal stage (Days) N/A Dead Dead Dead Dead 8 Total Life Cycle (Days) 21 N/A N/A N/A N/A Dead

Experiment 5 Averages Zophobas morio Burrows Z1-5 Z2-5 Z3-5 Z4-5 Z5-5 Z6-5 Depth 35.78 N/A 23.14 28.48 39.15 41.38 Max Width 7.52 N/A 6.39 6.81 7.53 9.57 Min Width 4.36 N/A 3.35 3.71 4.06 4.95 Mean Width 5.94 N/A 4.87 5.26 5.80 7.26 Max Length 37.65 N/A 29.38 31.14 66.21 84.35 Min Length 31.61 N/A 26.70 29.91 61.50 78.27 Mean Length 34.63 N/A 28.04 30.52 63.86 81.31 Mean W/H ratio 0.17 N/A 0.17 0.19 0.13 0.13 Max slope 56.00 N/A 79.00 30.67 31.00 41.00 Min slope 27.00 N/A 28.00 11.67 13.00 24.00 Mean slope 41.50 N/A 53.50 21.17 22.00 32.50 Branching angles 0.00 N/A 0.00 19.67 6.80 16.75 Complexity 1.00 N/A 1.00 1.67 1.40 1.75 Tortuosity 1.34 N/A 0.99 1.50 1.80 1.60

12) Experiment 5 averages for Zophobas morio. chambers and burrows in mm. ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

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