The Nematalycidae (Acariformes): An exploration of large-scale morphological

variation and evolution using low-temperature scanning electron microscopy

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Samuel J. Bolton

Graduate Program in Evolution, Ecology and Organismal Biology

The Ohio State University

2016

Dissertation Committee:

Hans Klompen, Advisor

Norman F. Johnson

Meg Daly

Copyright by

Samuel John Bolton

2016

Abstract

The Nematalycidae are among the most bizarre looking arthropods; their extreme body elongation is an especially unusual modification within the Arachnida. However, there have been few attempts to study their morphology in detail. This is partly because there are few available specimens – they live in mineral regolith, which is sampled relatively rarely. But this is also due to their extremely small size and soft integument. The former makes them problematical for examination under a light microscope; the latter renders them inappropriate for conventional scanning electron microscopy. This dissertation is a body of work that attempts to address this knowledge gap via low-temperature scanning electron microscopy (LT-SEM). Chapter 2 is a description of a new genus and species.

Chapter 3 addresses how the two main different modes of locomotion, one of which is a novel discovery, correspond with modifications of the integument. Chapters 4 and 5 are concerned with hypotheses on adaptations of the gnathosomas (mouthparts) of three different genera. Chapter 4 includes a hypothesis on a novel form of microbivory, which could explain some of the gnathosomal features of Osperalycus and Gordialycus.

Chapter 5 addresses the evolutionary implications of the mouthparts of Cunliffea, which has a rudimentary sheath for chelate (‘biting’) chelicerae. This structure may help to explain how one of the unusual modifications of the Eriophyoidea, a stylet sheath,

ii originated. Chapter 6 concerns a phylogenetic analysis of morphological characters. The principal finding is that the Eriophyoidea, a diverse group of plant parasites, are more closely related to the Nematalycidae than any other lineage, and they may even be derived from within the Nematalycidae. Therefore, the unusual, vermiform bodies of these two taxa are shared because of their relatively recent common ancestry, and not because of evolutionary convergence. In the final chapter, the conclusion, I suggest that the Nematalycidae should be promoted from a family to a higher taxonomic rank. This is based on evidence from throughout the dissertation.

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Acknowledgments

First and foremost, I would like to thank my advisor, Dr. Hans Klompen. He not only put up with me, educated me, and mentored me, but he also gave me a much valued freedom that has allowed me to take ownership of my research. Whereas he was always available for advice, I am also very grateful for the complete trust and confidence that he has bestowed on me. This PhD would not have been possible without the collaboration of the

US Department of Agriculture, which provided the LT-SEM that was essential to this research. I am very thankful to Dr. Gary Bauchan, Dr. Ronald Ochoa and Christopher

Pooley, Beltsville Agricultural Research Center, for their technical collaboration. The

Smithsonian Institution provided me with a much needed pre-doctoral fellowship, which allowed me to embark on an immensely productive year of research. I would especially like to thank Dr. Jonathan Coddington for being my host while I was based there. Joseph

Cora deserves many thanks for his friendship, advice and constructive criticism, and for all the IT aid he provided me whenever I needed it. I also appreciate the help and advice of a number of other key research collaborators, including Dr. Pavel Klimov, University of Michigan, and Dr. Philipp Chetverikov, Saint Petersburg State University, Russia. And

I am very grateful for the advice, support and encouragement of Dr. Roy Norton, Dr.

Barry OConnor, Dr. David Walter and Dr. Evert Lindquist. Many thanks also go to my

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Acarology Lab mates, Orlando Combita, Dr. Kaitlin Uppstrom and Monica Farfan. Their support and friendship has helped to provide a fantastic setting for my PhD program at

The Ohio State University. I would like to thank the faculty of the Department of

Evolution, Ecology and Organismal Biology, who have been extremely helpful and supportive. Foremost among them are Dr Meg Daly, Dr Norman Johnson, Dr. John

Freudenstein and Dr. John Wenzel. Many thanks also to Dr. Steve Passoa for his help with microscopy. And my fellow graduate students at EEOB deserve many thanks for their support and encouragement (alas, there are too many of them to mention in name).

Last, but not least, I would like to thank my parents, Vera and John Bolton, who have had to endure my absence for much of the past six and a half years. I am ashamed to admit I have only been back to visit them, in the UK, four times since I began my PhD program, and I have not received the smallest complaint. This is not for shortage of love but because, as ever, they value my hopes and dreams above their own needs and desires.

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Vita

1996 to 2000 ...... BSc Bioarchaeology, University of Bradford, UK.

2000 to 2001 ...... MSc Applied Entomology, Imperial College, London, UK.

2002 to 2004 ...... Company Biologist, Microbee, London, UK. 2004 to 2007 ...... Freshwater Entomologist, National Museum Wales, Cardiff, UK. 2007 to 2008 ...... MSc Biosystematics, Imperial College, London, UK. 2009 to present ...... Graduate Teaching Associate & General Research Associate, Dept. Evol., Ecol., Org. Biol., The Ohio State University

Publications

Pfliegler, W.P. & Bolton, S.J. 2016. Two new families (Acari: Alicorhagiidae and

Platyhelminthes: Prorhynchidae) reported for Hungarian fauna from leaf litter in the

Bükk mountains. Opuscula Zoologica Budapest 47, 00-00.

Bolton, S.J., Bauchan, G.R., Ochoa, R. & Klompen, H. 2015. A novel fluid-feeding mechanism for microbivory in the Acariformes (Arachnida: Acari), Arthropod Structure and Development 44, 313-325.

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Bolton, S.J., Bauchan, G.R., Ochoa, R., Pooley, C. & Klompen, H. 2015. The role of the integument with respect to different modes of locomotion in the Nematalycidae (Endeostigmata). Experimental & Applied Acarology 65, 149-161.

Bolton, S.J. Klompen, H., Bauchan, G.R. & Ochoa, R. 2014. A new genus and species of Nematalycidae (Acari: Endeostigmata). Journal of Natural History 48, 1359-1373.

Spies, M. & Bolton, S.J. 2013. On the first record from Britain of Parachironomus elodeae (Diptera, Chironomidae). Dipterists digest 20, 79-85.

Walter, D.E., Bolton, S.J., Uusitalo, M. & Zhang, Z.-Q. 2011. Suborder Endeostigmata Reuter, 1909. In: Zhang Z-Q, editor. Animal biodiversity: An outline of higher-level classification and survey of taxonomic richness. Zootaxa, 139-140.

Bolton, S.J., Macleod, N. & Edgecombe, G.D. 2009. Geometric approaches to the taxonomic analysis of centipede gonopods (Chilopoda: Scutigeromorpha). Zoological Journal of the Linnean Society 156, 239-259.

Field of Study

Major Field: Evolution, Ecology and Organismal Biology

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Table of Contents

Abstract ...... ii

Acknowledgments ...... iv

Vita ...... vi

Table of Contents ...... viii

List of Figures ...... xii

List of Tables...... xiv

Chapter 1: Introduction ...... 1

Chapter 2 Summary: A new genus and species ...... 2

Chapter 3 Summary: The role of the integument in locomotion ...... 3

Chapter 4 Summary: A novel form of microbivory...... 3

Chapter 5 Summary: A sheath for biting chelicerae ...... 4

Chapter 6 Summary: Eriophyoidea and Nematalycidae form a clade ...... 4

Chapter 7 Summary: Conclusion ...... 5

Chapter 2: A new genus and species ...... 6

Introduction ...... 6

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Materials and methods ...... 7

Description ...... 11

Material examined ...... 23

Type material and depositor ...... 24

Etymology ...... 24

Systematic relationships ...... 24

Key to the genera of the Nematalycidae ...... 25

Chapter 3: The role of the integument in locomotion ...... 27

Introduction ...... 27

Materials and methods ...... 29

Results ...... 31

Discussion...... 37

Conclusion ...... 44

Chapter 4: A novel form of microbivory ...... 46

Introduction ...... 46

Method ...... 48

Morphology of mouthparts ...... 50

Discussion...... 62

Conclusion ...... 71

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Chapter 5: A sheath for biting chelicerae ...... 73

Introduction ...... 73

Method ...... 75

Morphology of mouthparts ...... 77

Discussion...... 80

Conclusion ...... 87

Chapter 6: Eriophyoidea and Nematalycidae form a clade ...... 88

Introduction ...... 88

Method ...... 91

Results ...... 96

Discussion...... 100

Conclusion ...... 108

Chapter 7: Conclusion ...... 110

References ...... 112

Appendix A: Specimens ...... 129

Chapter 3: Material examined under LT-SEM ...... 129

Chapter 4: Material examined under LT-SEM ...... 130

Chapter 5: Slide mounted voucher specimens of Cunliffea cf. strenzkei ...... 131

Chapter 6: Slide mounted specimens used for the phylogenetic analyses ...... 131

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Appendix B: Percent increase in mouthparts from larva to adult (Chapter 4) ...... 135

Appendix C: Character coding scheme and rationale (Chapter 6) ...... 136

General character treatment ...... 136

Body ratios...... 137

Tritonymph stage ...... 138

Sexual versus asexual ...... 138

Integument ...... 139

Prodorsum...... 139

Opisthosoma ...... 140

Gnathosoma ...... 144

Legs ...... 149

Characters that were not coded ...... 151

Appendix D: Character list (Chapter 6) ...... 154

Appendix E: Character matrix (Chapter 6) ...... 159

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List of Figures

Figure 1. Osperalycus tenerphagus sp. n. Female ...... 10

Figure 2. Osperalycus tenerphagus sp. n. Female, proterosoma ...... 12

Figure 3. Osperalycus tenerphagus sp. n. Integument ...... 13

Figure 4. Osperalycus tenerphagus sp. n. Tritonymph, opisthosoma ...... 14

Figure 5. Osperalycus tenerphagus sp. n. Female, tarsi and tibia ...... 16

Figure 6. Osperalycus tenerphagus sp. n. Female, solenidia on legs I and II ...... 17

Figure 7. Osperalycus tenerphagus sp. n. Larva ...... 19

Figure 8. Osperalycus tenerphagus sp. n. Nymphs ...... 20

Figure 9. Vermiform bodies of the Nematalycidae...... 29

Figure 10. The metapodosomal and genital region ...... 33

Figure 11. Ventrolateral views of the opisthosomal tip ...... 34

Figure 12. Close-up view of the anal valves of Gordialycus sp. A...... 34

Figure 13. Palettes and integument ...... 35

Figure 14. Live specimen of Osperalycus tenerphagus ...... 36

Figure 15. Different modes of locomotion ...... 39

Figure 16. Vermiform bodies of the Nematalycidae...... 48

Figure 17. Mouthparts of the Nematalycidae...... 50

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Figure 18. Dorsal views of the mouthparts of Osperalycus tenerphagus ...... 52

Figure 19. Pouch of Osperalycus tenerphagus ...... 53

Figure 20. Palps ...... 55

Figure 21. Ventral views of the lateral lips and rutella ...... 57

Figure 22. The mouthparts of Gordialycus sp. A ...... 58

Figure 23. Lateral view of the mouthparts of Gordialycus sp. B ...... 60

Figure 24. The three hypothesized stages of feeding on a microorganism...... 66

Figure 25. The mouthparts of an eriophyoid...... 74

Figure 26. Cunliffea cf. strenzkei, body and chelicera ...... 76

Figure 27. Cunliffea cf. strenzkei, antero-dorsal view of mouthparts ...... 78

Figure 28. Cunliffea cf. strenzkei, ventro-lateral view of mouthparts ...... 89

Figure 29. Cunliffea cf. strenzkei, 3D model of mouthparts ...... 80

Figure 30. Semischematic drawings of piercing-sucking mouthparts...... 83

Figure 31. Phylogram (random) from a heuristic search (unweighted characters)...... 98

Figure 32. Empodia and setae...... 143

Figure 33. Mouthparts...... 147

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List of Tables

Table 1. Osperalycus tenerphagus sp. n. Length (푥̅) of appendages and idiosoma across

instars ...... 21

Table 2. Osperalycus tenerphagus sp. n. Distinguishing attributes of instars...... 21

Table 3. Osperalycus tenerphagus sp. n. Setal addition pattern for legs across instars ..... 22

Table 4. Principal morphological features pertaining to locomotion ...... 32

Table 5. Scores of optimal trees in constrained analyses ...... 99

Table 6. Unambiguous synapomorphies that unite the E-N clade...... 100

Table 7. Distance along part of chelicera ...... 135

Table 8. Distance along part of palp ...... 135

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Chapter 1: Introduction

The Nematalycidae is an unusual family of soil mites with a vermiform body that can be extended and contracted via a combination of hydraulic pressure and a continuous muscle layer underlying a thin epidermis (Haupt & Coineau, 1999). Their unique physiology amongst mites is probably adapted for living in small interstitial spaces in mineral soil and sand.

The first species of Nematalycidae was described just over 60 years ago

(Strenzke, 1954). Since then, only four more species have been described. The most recent description is the second chapter of this dissertation (Bolton et al., 2014). It is very likely that the late discovery of this family can at least, in part, be attributed to their habitats, which are under sampled (most soil acarologists sample biotically rich habitats, e.g. top soil and litter layers, which contain many more species). This is also one of the reasons that the morphology of this strange-looking group of mites has been poorly studied; few specimens are available. But there are two other important reasons for our comparative ignorance of their morphology: 1) these mites have an extremely narrow body and, consequently, much of their morphology is difficult to observe and interpret using ordinary light microscopy; 2) they also have a very soft, pliable integument that completely shrivels up in the vacuum of a conventional scanning electron microscopy.

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This dissertation is a body of work that attempts to address the knowledge gap on the morphology of the Nematalycidae through the use of an advanced technique of microscopy – low-temperature scanning electron microscopy (LT-SEM). This technique involves freezing specimens before placing them in the vacuum chamber of an SEM.

Consequently, the integument of soft-bodied mites such as the Nematalycidae will not shrivel, resulting in superior close-up images.

The LT-SEM images were used to address a range of different topics: alpha taxonomy (Chapter 2), functional morphology (Chapters 3, 4 and 5), evolutionary morphology (Chapter 5), and phylogenetics (Chapter 6). The phylogenetic study (Chapter

6) does not directly concern LT-SEM. However, this study was largely prompted by a discovery that was made via LT-SEM (Chapter 5). Therefore, Chapter 6 tests a hypothesis leading from that discovery.

Chapter 2 Summary: A new genus and species

This chapter addresses the description of the fifth new species and genus. In addition to the adult stage, the larva and three nymphal stages are described. Included is a diagnostic key to the genera of the Nematalycidae. This chapter has been published as a separate paper (Bolton et al., 2014).

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Chapter 3 Summary: The role of the integument in locomotion

The high degree of idiosomal elongation has led to accompanying changes in the locomotion of the Nematalycidae. Peristalsis was the only mode of locomotion known for this family. Videographic recordings reveal a new mode of locomotion, involving the generation of a large degree of hydraulic pressure from the dorsoventral contraction of the metapodosomal region. LT-SEM shows integumental modifications that facilitate this contraction. LT-SEM also reveals that the anal valves are prominent in some genera.

Videographic recordings show how these anal valves are important to locomotion.

Finally, LT-SEM reveals noticeable differences in the shape of the integumental palettes between genera, which may be an adaption for increasing or decreasing friction. This chapter has been published as a separate paper (Bolton et al., 2015a).

Chapter 4 Summary: A novel form of microbivory

LT-SEM was undertaken on the mouthpart morphology of two different species of Gordialycus. Both genera have unusual mouthpart morphologies, which appear to be adapted for feeding on single-celled microorganisms. The functional morphology of this feeding apparatus is explained in detail. The ecological implications of these newly discovered mouthpart morphologies, which appear to involve delicately picking up microorganisms and placing them into food holders, are also addressed. This chapter has been published as a separate paper (Bolton et al., 2015b).

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Chapter 5 Summary: A sheath for biting chelicerae

LT-SEM revealed that Cunliffea has a very rudimentary sheath for enclosing chelate (‘biting’) chelicerae. The sheath, which is a modification of membranous extensions of the subcapitulum, extends the preoral cavity into the space between the cheliceral digits. This modification therefore allows the integration of the chelicerae with a food canal. The Eriophyoidea, a highly speciose lineage of plant feeding mites, have a subcapitular sheath that completely envelopes a stylet bundle. Therefore, Cunliffea may help to explain how the mouthparts of the Eriophyoidea originated from mouthparts with chelate chelicerae.

Chapter 6 Summary: Eriophyoidea and Nematalycidae form a clade

The phylogenetic position of the Nematalycidae has not been well investigated. It has been placed with particulate feeding mites in the superfamily Nematalycoidea

(Kethley, 1989). A phylogenetic investigation was undertaken using morphological characters. The discovery of Chapter 5 prompted the inclusion of the Eriophyoidea in the analysis. The Eriophyoidea was originally thought to belong with the Tydeoidea in the order Trombidiformes. The results of the analyses show that the Eriophyoidea are more closely related to the Nematalycidae than they are to any other mites, and that both lineages probably fall outside of the Trombidiformes. Therefore, the unusual, vermiform bodies of these two taxa are shared because of their relatively recent common ancestry, and not because of evolutionary convergence.

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Chapter 7 Summary: Conclusion

The evidence from throughout the dissertation indicates that the Nematalycidae is a major lineage of the Acariformes. Accordingly, the Nematalycidae should be promoted to a higher taxonomic rank.

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Chapter 2: A new genus and species

Introduction

The Nematalycidae is readily distinguished from all other Acari by a combination of a highly elongate idiosoma (adults ≥6 × width when fully extended), a much greater distance between legs II and III relative to legs I and II, and a genital opening much closer to legs IV than to the anal opening. The prodorsum of this family is also distinct from many others in lacking trichobothria and a naso, with fewer than four pairs of setae; the rostral seta is unpaired if present. The Nematalycidae was originally treated as belonging to the Endeostigmata (Sarcoptiformes) (Strenzke, 1954). It was later assigned to the Tydeoidea (Cunliffe, 1956; Wainstein, 1965; Krantz, 1970). Kethley (1982) placed it outside the Tydeoidea but proposed that derived characters suggested a closer relationship with the Tydeoidea than the Endeostigmata. However, since the discovery of two families – Micropsammidae (Coineau & Theron, 1983) and Proteonematalycidae

(Kethley, 1989) – that have a transitional morphology between Nematalycidae and other families within the Endeostigmata, the Nematalycidae have been more commonly regarded as belonging to the Endeostigmata (Walter, 2009; Walter et al., 2011).

Excluding this new genus (Bolton et al., 2014), four genera of Nematalycidae have been described (Walter et al., 2011). All four genera are monospecific. Nematalycus

6 was described from groundwater from the Algerian coast (Strenzke, 1954). Soon afterwards Cunliffea was described from pasture soil from the USA (Cunliffe, 1956) and has since been collected from sands in Germany (Russell & Alberti, 2009). Gordialycus was described from fine sands from southern France (Coineau et al., 1967). This genus has a world-wide distribution, having been found in southern Africa, Turkmenistan,

Venezuela, Cuba, Mauritania, New Caledonia, Australia, Brazil, Hungary and the USA,

(Coineau & Theron, 1983; Silva et al., 1989; Thibaud & Coineau, 1998; Norton &

Kinnear, 1999; Norton et al., 2008). Psammolycus was described from sandy soils from

Brazil (Schubart, 1973). This genus may also have been found in the USA (pers. obs.). A published record from Spain (Moraza, 2008) was in error and is instead Gordialycus

(pers. obs.).

A fifth genus and species of Nematalycidae is hereby described. The mites were collected from two different loam soils from relatively disturbed habitats in Ohio – a silty clay loam from a suburban prairie located on a university campus, and a sandy loam from a young chestnut plantation (see material examined for more details). Collecting took place throughout 2010 and 2011. As with all other species of Nematalycidae, all adults recovered were female, indicating thelytoky.

Materials and methods

For the collection of slide mounted specimens, soil samples were processed by soil washing in accordance with Kethley (1991). Mites that were recovered using this technique were slide mounted in Hoyers medium. Live mites were also collected for the 7 purpose of imaging with a low-temperature scanning electron microscope (LT-SEM) at the US Department of Agriculture, Electron & Confocal Microscopy Unit, Beltsville,

MD. This was accomplished by directly removing them from floated material that had not yet been sieved. Searching was undertaken with a dissection microscope.

Live specimens, for LT-SEM, were secured to 15 cm × 30 cm copper plates, using ultra smooth round (12 mm diameter), carbon adhesive tabs (Electron Microscopy

Sciences, Inc., Hatfield, PA, USA). The specimens were frozen conductively, in a

Styrofoam box, by placing the plates on the surface of a pre-cooled (-196°C) brass bar whose lower half was submerged in liquid nitrogen. After 20-30s, the holders containing the frozen samples were transferred to the Quorum PP2000 cryo-prep chamber (Quorum

Technologies, East Sussex, UK) attached to an S-4700 field emission scanning electron microscope (Hitachi High Technologies America, Inc., Pleasanton, CA, USA). The specimens were etched inside the cryotransfer system to remove any surface contamination (condensed water vapour) by raising the temperature of the stage to -90°C for 10-15 min. Following etching, the temperature inside the chamber was lowered below -130°C, and the specimens were coated with a 10nm layer of platinum using a magnetron sputter head equipped with a platinum target. The specimens were transferred to a pre-cooled (-130°C) cryostage in the SEM for observation. An accelerating voltage of 5kV was used to view the specimens. Images were captured using a 4pi Analysis

System (Durham, NC, USA). Images were sized and placed together to produce a single figure using Adobe® Photoshop CS 5.0.

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With the exception of the pre-larva – which, if existing, was not recovered – the different instars were carefully studied for ontogenetic variation. Slide mounted mites were drawn using a compound microscope (Zeiss Axioscope™) equipped with a camera lucida as well as phase contrast and differential interference contrast optical systems.

Many detailed features of this mite were not readily distinguishable without LT-SEM.

For example, the very small size of the mouthparts made it impossible to discern their true shape and structure without LT-SEM. LT-SEM was therefore used to make modifications and corrections to drawings where the light microscope provided insufficient detail.

Although the prodorsal, opisthosomal and genital setae tend to project straight out

(as revealed by LT-SEM), they are typically pushed into a flat 2D orientation by slide mounting. These setae were drawn as though slide mounted (with vertically orientated setae on the dorsum and ventrum projecting to the side) in order to reveal their actual lengths. LT-SEM also revealed minute barbs near the base of many setae. These setae are treated as simple because it is impossible to determine which setae do not have them

(being out of view in many LT-SEM images).

Measurements were made using an ocular micrometer and are given to the nearest micrometer. Legs were measured from the base of the trochanter to the tip of the empodium (adjusting for legs in which the empodium was not fully extended). All of the prodorsal and opisthosomal setae were measured (including setae from the genital region). Setae were measured from the centre of the base to the tip. All setal length

9 measurements in the main text are presented as ranges in parentheses with the symbol

“µm” omitted.

Figure 1. Osperalycus tenerphagus sp. n. Female: (A) dorsal view; (B) ventral view; (C) metapodosoma and genital region (D) genitalia – internal.

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In the adult female section, reference is occasionally made to images of nymphal instars to illustrate structures in which there is no apparent difference between instars

(e.g. integument, ventral furrow and solenidial grooves). Tables pertaining to the ontogeny section give abbreviations for instars: Larva= La; Protonymph= PN;

Deutonymph= DN; Tritonymph= TN; Female= F.

In the figures, almost all abbreviations follow conventions. Additional abbreviations are addressed in the figure caption where needed.

Description: Osperalycus tenerphagus Bolton & Klompen g. n., sp. n (Figures 1–8)

Diagnosis

Osperalycus is readily distinguished from other genera of Nematalycidae by the presence of long and simple setae of similar size along the opisthosoma (Figure 1A–B); the simple setae next to the anal opening of Gordialycus and Nematalycus are at least three times as long as those along the rest of the opisthosoma, although the opisthosoma of Gordialycus and Nematalycus is often almost completely nude. Cunliffea and

Psammolycus have bifurcate or trifurcate setae along the opisthosoma. Osperalycus is also readily distinguished from Gordialycus and Nematalycus by the baculiform solenidion on tarsus II – this solenidion has a distinctly swollen or bulbous tip in

Gordialycus and Nematalycus. The most distinct characters of Osperalycus are to be found in the mouthparts (Figure 2A–B). The pouch (Po) – modified from the lateral lips – is a very unusual structure that appears to be unique to this genus (Figure 2A). Markedly

11 short chelicerae (Ch) (<15 µm) are found in both Osperalycus tenerphagus and

Psammolycus delamarei, but not Nearctic cf. Psammolycus or any other genus of

Nematalycidae. Osperalycus is also distinct from all other genera in having rutella (Ru) that overlap at the midline (Figure 2A).

Figure 2. Osperalycus tenerphagus sp. n. Female, proterosoma: (A) dorsal view (chelicerae slanted downwards into the pouch, making them appear slightly shorter; broad bases of rutella partly behind chelicerae and pouch opening – shaded pale grey); (B) ventral view. Additional abbreviations: Pa = palp; Po = pouch; Ru = rutellum; Ch = chelicera.

Female

General morphology: Idiosoma ≈600 µm long (≈12 × longer than wide) when fully extended (Figure 1A–B). Cuticle with flat round projections, or palettes sensu Haupt

& Coineau (1999), lining the latitudinal annuli (Figure 3A–D). Trachea and peritremes absent. Lyrifissures absent from idiosoma. Podocephalic canals terminating anteriorly between the bases of the chelicerae; extending back beyond coxae I. Distinct glands

12 leading into podocephalic canals at coxae I. Long narrow oesophagus extends to proximity of metapodosoma.

Figure 3. Osperalycus tenerphagus sp. n. Integument: (A) extended region of body – vertical view (showing palettes as thin and flat); (B) contracted region of body – vertical view (showing interlocking palettes); (C) extended – diagonal view; (D) contracted – diagonal view.

Prodorsum: Trichobothria absent; naso absent; eyes absent. Three pairs of setae

(exa, exp and in) and one unpaired rostral (ro) seta. All setae simple and close to the midline of the prodorsum (Figure 2A). Setae ro, exa and in subequal and short (4–8); exp long (16–22).

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Opisthosoma: All setae simple. Setae associated with all nine opisthonotal segments: c1-4, d1-2, e1-2, f1-3, h1-2, ps1-3, ad1-3, an1-3, pa1-3. Frequent and noticeable asymmetry in the longitudinal positions of pairs of setae (Figure 1A–B). In segmental remnants F-PA, ancestrally dorsolateral setae get displaced to a ventrolateral or ventral position that is usually noticeably more anterior than the dorsomedial setae of the same segment. Segment C very long; position of c2 and c3 much more posterior than c1 and c4.

Figure 4. Osperalycus tenerphagus sp. n. Tritonymph, opisthosoma: (A) anal valves and ventral furrow – ventrolateral view; (B) anal valves – dorsal view; (C) anal valves – posterior view; (D) ventral furrow – close-up. 14

Setae on segments C-E generally shorter (5–15) than those on segments F-PA

(10–19). Setae c2 short (5–8); proximal setae c3 comparatively long (9–13). Longest opisthosomal setae h2 and ps3 (15–19). From segments H to PA, ventral setae (h2, ps3, ad3, an3, pa3) typically 1 to 4 µm longer than lateral and dorsal setae on the same segment; lateral and dorsal setae subequal.

Distinct anal valves projecting from the anus (Figure 4A–C). Ventral furrow, in which annular ridges terminate (Figure 4D), extending from proximity of anal valves to genital region (Figure 4A).

Three pairs of genital (g) setae and two pairs of aggenital (ag) setae (Figure 1C).

Genital and aggenital setae short (3–7) compared to most other opisthosomal setae. Two pairs of bilobed genital papillae (Figure 1D).

Podosoma: Large gap separating legs II and III (Figure 1B). Coxal fields I and II medially separated by intercoxal region (Figure 2B). Single pair of intercoxal setae

(between coxal fields II). Coxal fields III and IV medially fused (Figure 1C). Each coxal field I with one bifurcate seta and one simple seta; each coxal field II with one bifurcate seta; coxal fields III with one bifurcate seta and two simple setae on each side of the fused coxal plate; coxal fields IV with one simple seta on each side of the fused coxal plate.

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Figure 5. Osperalycus tenerphagus sp. n. Female, tarsi and tibia: (A–E) tarsi I-IV; (A) tarsus I; (B) tarsus II – ε obscured; (C) tarsus II – ε visible; (D) tarsus III; (E) tarsus IV. Deutonymph: (F) tibia I.

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Legs: Pretarsi I-IV each with two lateral claws and a ciliated empodium (Figure

5A–E). Solenidial formulae femora-tarsi for individual legs I-IV: 0-1-2-1; 0-0-0-1; 0-0-0-

0; 0-0-0-0. Longitudinal grooves of shortest solenidion phi (φ) (tibia I) restricted to bulbous tip (Figure 5F). Other solenidia baculiform with longitudinal grooves extending almost all the way to the base. Solenidia omega (ω) on tarsi I and II subequal and noticeably larger than solenidion phi (φ) and sigma (σ) on, respectively, tibia I and genu I

(Figure 6).

Figure 6. Osperalycus tenerphagus sp. n. Female: solenidia on legs I and II.

Setae absent from trochanters I-IV. Setal formulae femora-tarsi for individual legs

I-IV (excluding solenidia and famuli): 4-6-7-16; 2-2-2-9; 1-0-2-6; 1-0-2-5. All setae on 17 femora-tibiae I-IV simple. Tarsi I-IV with various setae (see below). Femur I sometimes lacking the dorsal seta normally added in post-protonymphal instars – some individuals with three setae on one femur I and four on the other.

Tarsus I (Figure 5A): Seven pairs of setae (p, tc, ft, u, a, pv and pl) and two unpaired setae (s and d). Proral (p) setae conical and extending from turbercle. One tectal

(tc″) and one fastigial (ft″) seta usually semi-bifurcate, with one branch short and barb- like – sometimes short and basal enough for the seta to qualify as simple (see aforementioned criteria in method), or rarely long enough to be bifurcate. Other fastigial

(ft') seta usually bifurcate, rarely semi-trifurcate or trifurcate. All other setae simple.

Subunguinal (s) seta centered between pair of anterolateral (a) setae. Dorsal (d) seta proximal and posterior to base of solenidion ω. Small stubby famulus (ε) near tc″.

Dorsolateral lyrifissure next to posterior margin of tarsus (obscured by overlying integument when viewed under LT-SEM).

Tarsus II (Figure 5B–C): Four pairs of setae (p, tc, ft and u) and one unpaired seta

(d). Setae p, tc and u bifurcate and projecting forward past base of ambulacrum; ft simple; d trifurcate. Long rod-like famulus near tc″. Base of solenidion ω next to posterior margin of tarsus.

Tarsus III (Figure 5D): Two pairs of setae (p and u) and two unpaired setae (tc″ and d). Setae p, u and tc″ bifurcate and projecting forward past base of ambulacrum; d bifurcate.

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Figure 7. Osperalycus tenerphagus sp. n. Larva: lateral view.

Tarsus IV (Figure 5E): Identical to tarsus III but with tc″ absent.

Gnathosoma: Gnathosoma including a pouch (Po) – modified from the lateral lips

– into which the chelae (opposing digits) slot (Figure 2A). Chelicerae (Ch) ≈12 µm long and slanting downwards, anteriorly, into the pouch. Single dorsal seta on each chelicera.

Rutella (Ru) consisting of a swollen base with a narrow extending digit. The rutella lie against the anterior surface of the pouch and overlap at the midline, appearing as an inverted v – more discernible, under a light microscope, when the chelae are not within the pouch. Palps (Pa) ≈10 µm long and three-segmented, designated femurogenu, tibia and tarsus; solenidia absent; setal formula femurogenu-tarsus for each palp: 0-1-6; distal tarsal seta with cup-shaped tip; all other setae simple. Palp tarsus also with three very small cuticular protuberances (only visible under LT-SEM and omitted from Figure 2A–

B). Subcapitulum with four pairs of setae: m, a, or1-2 (Figure 2B). Adoral setae (or1-2)

19 typically unevenly tapering (or2 sometimes evenly tapering); anterior pair (or1) vestigial and broad.

Figure 8. Osperalycus tenerphagus sp. n. Nymphs: lateral view: (A) protonymph; (B) tritonymph.

Ontogeny

Idiosoma and appendages: From larva to adult, the idiosoma increases greatly in length compared to the appendages (Table 1; Figures 7, 8A–B). Notably, there is no apparent increase in the length of the palp.

20

La PN DN TN F OA (%)

Palp 10 9 10 10 10 0

Leg I 43 43 48 54 57 33

Leg II 30 30 35 38 39 30

Leg III 29 30 32 34 34 17

Leg IV - 28 30 32 35 25

Idiosoma 334 363 458 526 611 83

Table 1. Osperalycus tenerphagus sp. n. Length (푥̅) of appendages and idiosoma across instars (µm). Measurements taken from slide mounted specimens rather than SEM, for which the body is generally not fully extended. OA = overall increase.

La PN DN TN F Number of genital setae 0 1 3 3 3 Number of aggenital setae 0 0 0 1 2 Number of genital papillae 0 1 2 2 2 Length of PS setae (µm) 3–7 ≥10 ≥10 ≥10 ≥10 Length of AD setae (µm) - 3–7 ≥10 ≥10 ≥10 Length of AN setae (µm) - - 3–7 ≥10 ≥10 Length of PA setae (µm) - - - 3–7 ≥10 Table 2. Osperalycus tenerphagus sp. n. Distinguishing attributes of instars.

Chaetotaxy: In addition to the legs and coxal fields (see below), setal additions occur in the genital region and segments AD to PA (Table 2). Adults readily distinguished from tritonymphs by the presence of setae ag2 (very posterior and close to f3) and long pa1-3 (Table 2).

New segmental additions of setae to the posterior tip of the opisthosoma always short (3–7), increasing 2 to 4 fold in length at the next instar (Table 2). Several other opisthosomal setae noticeably increase in length from the larval to adult instar (Figures

1A–B, 7): seta f3 increases approximately 4 fold (from 3–4) to be subequal to f2 and f1 21

(11–16); setae d2 and e2 increase by about 50% (from 7–10 to 10–15). All other opisthosomal and prodorsal setae increase only slightly (<30%) or not at all.

La PN DN TN F Tarsus I 10 (1) (2) 2 11 (1) (2) 2 11 (1) (2) 2 11 (1) (2) 2 11 (1) (2) 2 Tibia I 6 6 7 7 7 Genu I 4 6 6 6 6 Femur I 2 3 4 4 4 Trochanter I - - - - - Coxal field I 1 1 (1) 1 (1) 1 (1) 1 (1) Tarsus II 2 (6) ((1)) 2 (6) ((1)) 2 (6) ((1)) 2 (6) ((1)) 2 (6) ((1)) Tibia II 2 2 2 2 2 Genu II 2 2 2 2 2 Femur II 2 2 2 2 2 Trochanter II - - - - - Coxal field II (1) (1) (1) (1) (1) Tarsus III (6) (6) (6) (6) (6) Tibia III 2 2 2 2 2 Genu III - - - - - Femur III 1 1 1 1 1 Trochanter III - - - - - Coxal field III 1 (1) 2 (1) 2 (1) 2 (1) 2 (1) Tarsus IV n/a (5) (5) (5) (5) Tibia IV n/a 2 2 2 2 Genu IV n/a - - - - Femur IV n/a - 1 1 1 Trochanter IV n/a - - - - Coxal field IV n/a - 1 1 1 Table 3. Osperalycus tenerphagus sp. n. Setal addition pattern for legs (including coxal fields) across instars. Key: Unbracketed = simple seta; bracketed = bifurcate; bracketed & underscored = semi-bifurcate; double bracketed = trifurcate; angle bracketed = conical; hyphen = absence of setae; n/a = leg absent; highlighted grey = seta added in this instar.

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Leg setation complete (including coxal fields) by the deutonymphal instar (Table

3). Setation of tarsus I is completed by the addition of an unpaired dorsal (d) seta in the protonymph. Chaetotaxy unchanging for tarsi II-IV. Numbers of solenidia, famuli and lyrifissures also unchanging for all leg segments. Gnathosoma without additions of setae or any other structures.

Material examined

Holotype female (OSAL 0105134), U.S.A., Ohio, Franklin Co., Kinnear Road,

39.9990 N -83.0468 W, silty clay loam from suburban prairie (including shrubs, grasses and small trees); collector: Samuel Bolton, May-2011, 40 cm deep (SB11-05-I). Same data: 9 F (OSAL 0103239, 0105137, 0105138, 0105147, 0105148, 0105149, 0105153,

0105155, 0105156), 1 TN/F? (OSAL 0105135), 5 TN (OSAL 0105143, 0105146,

0105151, 0105152, 0105157), 1 DN (OSAL 0105145), 1 PN (OSAL 0105150), 3 L

(OSAL 0103237, 0105141, 0105142), 1 pharate (TN–F, OSAL 0105154). Same locality and collector, July-2010, 60 cm deep (SB10-0724-I): 4 F (OSAL 0103240, 0105136,

0105139, 0105140), 1 DN, (OSAL 0105144), 2 PN (OSAL 0103241, 0103242),1 L

(OSAL 0103245), 2 pharates (L-PN, OSAL 0103243, 0103244). August-2011 (LT-SEM material), 60 cm deep: 9 F, 1 TN/F?, 6 TN, 1 DN/TN?, 1 DN, 1 PN (used for SEM, not recovered). U.S.A., Ohio, Ashtabula Co., West Main Road, 41.9246 N -80.6138 W, sandy loam in small chestnut plantation, collector: Samuel Bolton, July-2011, 40 cm deep

(SB11-07-IV): 1 F (OSAL 0105133), 1 TN (OSAL 0103238), 1 DN (OSAL 0105132).

23

Type material and depositor

Holotype female (OSAL 0105134) at Ohio State University Acarology Collection

(OSAL), Columbus, Ohio, U.S.A. Paratypes: U.S. National Collection (USNM), housed at the Beltsville Agricultural Research Center, USDA, Beltsville, Maryland, USA: 2 F

(OSAL 0105137, 0105147); Natural History Museum (BMNH), London, United

Kingdom: 2 F (OSAL 0105140, 0105148); all other paratypes at OSAL.

Etymology

Binomial: Osperalycus tenerphagus. Ospera- is a combination of the Latin terms for ‘mouth’ (os) and ‘purse/bag’ (pera) in reference to the soft and unsclerotized pouch of the gnathosoma; -lycus is a Latinized Greek ending given to three of the four other genera of Nematalycidae. The species name, tenerphagus, combines the latin term for ‘tender’ with the Greek derived latin suffix for ‘feeding’, referring to the delicate mechanism hypothesized to explain how this mite may carefully pick up small micro-organisms and place them into its feeding pouch without rupturing them (Bolton et al. 2015b; Chapter

4).

Systematic relationships

Whereas rutella have been thought to be absent from the Nematalycidae (Walter

2009), they are clearly present in Osperalycus tenerphagus. Rutella also appear to be present in Gordialycus and Nearctic cf. Psammolycus (pers. obs.). The presence of rutella

24 combined with the absence of a tracheal system more firmly places the Nematalycidae within the Endeostigmata. Other newly observed characters also suggest a closer relationship with the Endeostigmata than the Tydeoidea, e.g. presence of setae for all nine opisthosomal segments and more than two pairs of setae on the C and F segments.

Key to the genera of the Nematalycidae

1a. Opisthosomal setae near the anal opening at least three times as long as those along

the rest of the opisthosoma, which is often almost completely nude; chelicerae long

(>15 µm).……………………………………………………………………………2

1b. Setae along the length of the opisthosoma of similar size; chelicerae can be short

(<15 µm) or long (>20 µm)…………………………………………………………3

2a. Legs III and IV noticeably smaller than leg II; body usually extremely elongated

(adult length >20 × width)………………………………………………Gordialycus

2b. Legs III and IV not noticeably smaller than leg II; body not extremely elongated

(adult length <20 × width)…………………………….……………….. Nematalycus

3a. Palps with two segments; pretarsi II to IV without claws; tritonymphs and adults

with three pairs of simple genital papillae; chelicerae long (>20 µm) but with very

short chelae (opposing digits); opisthosomal setae always trifurcate…….. Cunliffea

3b. Palps with three or four segments; lateral claws present on pretarsi II to IV;

tritonymphs and adults with two pairs of bilobed genital papillae; if chelicerae long

(>20 µm), chelae distinctly elongated; opisthosomal setae trifurcate, bifurcate or

simple……………………………………………………………………………….. 4 25

4a Opisthosomal setae distinctly bifurcate or trifurcate; rutella absent or never

overlapping at the midline when present; if chelicerae long (>20 µm), chelae

distinctly elongated…………………………………………………….. Psammolycus

4b. Opisthosomal setae long and simple (with very small basal barbs that are not visible

under a light microscope); rutella overlap at the midline; chelicerae short (<15 µm)...

…………………………………………………………………………………….Osperalycus

26

Chapter 3: The role of the integument in locomotion

Introduction

The Nematalycidae possess several idiosyncratic features that pertain to their locomotion: the length of the body is lined with annular ridges (annuli); legs I-II are separated from legs III-IV (the metapodosoma) by a long anterior region of the body; the genitalia are positioned directly behind the metapodosoma; finally, Nematalycidae possess a long posterior region behind the genitalia.

These highly elongated mites (Figure 9) move around like annelid worms by extending and contracting their bodies. The soft and flexible region of the integument between successive annuli folds or bends when that region of the body contracts, forcing the annuli together. Conversely, when the integument between successive annuli straightens, the annuli push apart. The contraction of the a body region is induced by underlying longitudinal muscles. The extension of the body occurs in regions in which longitudinal muscles relax. Although extension may be facilitated by some degree of elasticity in the folding of the integument (Haupt and Coineau, 1999), any large degree of extension requires the generation of hydraulic pressure in the haemocoel via the constriction of another region of the body. Notably, the Nematalycidae lack circular muscles for reducing the body diameter.

27

The annuli are lined with flat projections known as palettes, which are orientated so that their edges are perpendicular with respect to the annuli. These palettes have also been found in the Nanorchestidae (Alberti et al., 1981). Their function is not certain, but it has been hypothesized that they may be used to form a plastron to help avoid drowning

(Haupt and Coineau, 1999; Rounsevell and Greenslade, 1988). It is likely that the

Nematalycidae also (or instead) use them to improve the traction of the integument during movement (Coineau et al., 1978; Haupt and Coineau, 1999).

Knowledge of the locomotion of the Nematalycidae has been based solely on studies of Gordialycus, an exceptionally elongated genus that can move via a form of peristalsis (Coineau and Coineau, 1979; Coineau et al., 1978; Haupt and Coineau, 1999).

Given the lack of circular muscles for reducing the body diameter, peristalsis in

Gordialycus is incomplete and only involves longitudinal muscles (Haupt and Coineau,

1999).

The peristaltic motion of Gordialycus may only be suited to its unusually long body shape. We therefore undertook a comparative study of integumental characters associated with locomotion in the Nematalycidae using both light microscopy and low- temperature scanning electron microscopy (LT-SEM). The study was based on specimens of Cunliffea strenzke Cunliffe, cf. Psammolycus sp. A., Osperalycus tenerphagus Bolton and Klompen, and Gordialycus spp. n. A and B. In addition, the locomotion of live specimens of Osperalycus tenerphagus was filmed. The videos of the live specimens were used to help interpret how integumental features observed under LT-

SEM are used in locomotion. LT-SEM images of the other genera were examined to

28 determine if they shared some of these integumental features and, therefore, probably a similar mode of locomotion.

Figure 9. Vermiform bodies of the Nematalycidae: (a) Gordialycus sp. A (nymph/adult); (b) Cunliffea strenzkei (adult female).

Materials and methods

LT-SEM

LT-SEM was carried out at the US Department of Agriculture, Electron and

Confocal Microscopy Unit, Beltsville, MD. Specimens were collected via soil washing in accordance with Kethley (1991). The technique was modified for collecting live 29 specimens of Osperalycus tenerphagus for LT-SEM (see videography section of method). All other species were collected in accordance with the original protocol and then stored in 80 to 95% alcohol until they were mounted for LT-SEM. If not stated otherwise in the caption, the figured images were obtained using LT-SEM.

Five different species were collected and observed using LT-SEM: Gordialycus spp. n. A, B; Osperalycus tenerphagus; cf. Psammolycus sp. A; Cunliffea strenzkei.

Gordialycus sp. A and B are the same as Gordialycus sp. A and B in Bolton et al.

(2015b) and Chapter 4. Osperalycus tenerphagus was collected from a silty clay loam in

Columbus, Ohio. The other species were collected from sands in Indiana, Florida and

California. Vouchers of all taxa are deposited at the Ohio State University Acarology

Collection. Specimens were prepared for LT-SEM using the techniques described in

Chapter 2 and Bolton et al. (2014).

Videography

Live specimens of Osperalycus tenerphagus were collected using a modification of Kethley’s soil flotation method (Kethley, 1991). The specimens were removed from floated material that had not yet been sieved. The floated material was placed into a petri- dish and observed under a dissection microscope. Any live specimens, if present, were then delicately lifted out. Some were placed on a small chunk of silty clay loam from their native soil and filmed using a stereomicroscope (Nikon SMZ-1500) equipped with a digital camcorder (Sony HDR-HC7). Note that the interstitial pores of this type of soil are

30 too small to allow the passage of mites; Osperalycus tenerphagus must instead be living within the cracks and voids.

Other live specimens were placed directly in oil immersion fluid (Zeiss immersol

518 N) and very gently mounted on a conventional slide underneath a circular coverslip

(10 mm diameter). The mites were then filmed using a Zeiss Axioskop™ equipped with a phase contrast optical system and a digital camcorder (same make and model as above).

Videos are viewable online. See the following URLs:

Online resource 1 = http://www.edge-cdn.net/video_935424?playerskin=37016;

Online resource 2 = http://www.edge-cdn.net/video_935190?playerskin=37016;

Online resource 3 = http://www.edge-cdn.net/video_935426?playerskin=37016;

Online resource 4 = http://www.edge-cdn.net/video_935270?playerskin=37016.

Results

Integument and idiosomal elongation (LT-SEM)

The degree of elongation of the idiosoma varies dramatically throughout the

Nematalycidae because the length varies much more than the width (Table 4).

Gordialycus (idiosomal length >1.0 mm) is much longer than the two shortest genera – cf. Psammolycus and Cunliffea.

All of these taxa have an integument that is similar to the one described for

Gordialycus (Haupt and Coineau, 1999). Much of the integument of Nematalycidae consists of annuli. However, each side of the body of Osperalycus, Cunliffea and cf.

Psammolycus has three small patches of integument with longitudinal striations (Figure

31

10a–b). All three patches are located in the metapodosomal and genital region: two of the patches are directly above legs III and IV; the other is above the genitalia. Legs III and IV have enlarged coxal fields. Note that although the coxal fields have longitudinal striations, their annuli are not lined with palettes, and they appear to be comprised of the same type of relatively rigid integument that comprises the standard leg segments. It is therefore unlikely that the integument of the coxal fields is able to fold.

Longitudinal Adult idiosomal Adult idiosomal Protrusion of Shape of Genus striations on the length (mm) width (mm) the anal valves palettes metapodosoma

Gordialycus >1.0 0.05 Strong Circular Absent Osperalycus 0.6 0.04 Strong Circular Present Cunliffea 0.4 0.05 Weak Pointed (blunt) Present cf. Psammolycus 0.3 0.03 Weak Pointed (sharp) Present Table 4. Principal morphological features pertaining to locomotion

The patches of longitudinal striations are absent in Gordialycus (Figure 10c).

Instead, the annuli curve around legs III and IV. Legs III and IV, including their coxal fields, are also greatly reduced in Gordialycus compared to the other genera. In one specimen of Osperalycus, legs III and IV were shown to be able to contract very tightly into the body, causing an indentation of the body (Figure 10d).

In all five species, anal valves project from the anus (Figure 11, 12). The external surface of the anal valves is lined with striations that are similar to those found on other parts of the mite, including the legs and prodorsum.

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Figure 10. Lateral, lateroventral and ventral views of the metapodosomal and genital region (the anterior end is left in all images): (a) Cunliffea strenzkei; (b) Osperalycus tenerphagus; (c) Gordialycus sp. A; (d) Osperalycus tenerphagus – showing legs contracted into the metapodosoma. A= annular striations; G = genital region; L3 = leg 3; L4 = leg 4; C3 = coxal field 3; C4 = coxal field 4; I = indentation due to the contraction of the legs into the body.

The palettes are rounded in Gordialycus and Osperalycus (Figure 13a–b), whereas in Cunliffea and cf. Psammolycus they taper to a point. In Cunliffea, each palette tapers to a relatively short, blunt point (Figure 13c). By contrast, each palette of cf. Psammolycus consists of a long, narrow projection, tapering to a sharp point (Figure 13d).

33

Figure 11. Ventrolateral views of the opisthosomal tip, showing the anal valves: (a) Gordialycus sp. B; (b) Osperalycus tenerphagus; (c) cf. Psammolycus sp. A. – anal valves barely discernible; (d) Cunliffea strenzkei. V = anal valve; S = striation without palettes.

Figure 12. Close-up view of the anal valves of Gordialycus sp. A 34

Figure 13. Palettes and integument: (a) Osperalycus tenerphagus; (b) Gordialycus sp. B; (c) Cunliffea strenzkei; (d) cf. Psammolycus sp. A. P = palette.

Locomotion (videography)

Late nymphal and adult instars of Osperalycus would often move around by pushing off against the tip of their opisthosoma (the location of the anal valves). The posterior region of the opisthosoma initially bends and contracts along its longitudinal axis. The tip of the opisthosoma is then inserted into the soil, anchoring it in place. The posterior region of the opisthosoma subsequently extends and straightens, pushing the center and front of the mite forwards (Online Resource 1). This occurs via several distinct waves of extension that proceed anteriorly from the anus. As this is happening, the mite

35 is also crawling forwards using its legs. Once the posterior region is fully extended, the tip of the opisthosoma detaches from the soil substrate. This movement mechanism was also occasionally observed in the protonymph (Online Resource 2). However, the posterior region of the opisthosoma is more frequently dragged around by the protonymph (Online Resource 3).

Figure 14. Live specimen of Osperalycus tenerphagus, showing the constriction of the metapodosomal and genital region (freeze frame from video available through Online Resource 4). DC = dorsoventral constriction; G = genital region; L3 = leg 3; L4 = leg 4.

Longitudinal contractions along the main body are obviously essential to locomotion in the Nematalycidae. However, dorsoventral constriction was also observed in the metapodosomal and genital region of Osperalycus. The close-up views of live

Osperalycus (phase contrast microscopy) revealed waves of dorsoventral constriction along the metapodosomal and genital region (Online Resource 4; Figure 14). During the

36 progression of a wave, the anterior or posterior region of the body extends out longitudinally from the metapodosomal and genital region. Dorsoventral constrictions were not observed anywhere else along the body.

The body appears to be very pliable. When a small area of the idiosoma is flattened by an external force, it can readily re-inflate. This was observed when parts of the live specimens were accidentally squashed while maneuvering them under a stereomicroscope.

Discussion

Metapodosomal and genital body region

The results of this study indicate that the peristaltic locomotion of Gordialycus may be unique. Osperalycus, Cunliffea and cf. Psammolycus appear to employ a different mode of locomotion that involves dorsoventral constriction and expansion of the metapodosomal and genital region. The longitudinal striations that are present in this region (Figure 10a–b) allow the constriction of this body region (Figure 14). This is the same integumental mechanism by which the annular regions longitudinally contract—the striations move together and the palettes interdigitate. Whereas annular striations allow extension and contraction along a longitudinal axis, longitudinal striations that are located on the side of the body allow expansion and constriction of the dorsoventral axis. The constriction of the metapodosomal and genital region would help to generate the large amount of hydraulic pressure needed to extend the body in annular regions (or the posterior or anterior region), where there is no change in body diameter. This is 37 consistent with the videographic data of Online Resource 4, which shows the extension of the anterior region coinciding with the dorsoventral constriction of the metapodosomal and genital region.

The dorsoventral constrictions of the metapodosomal and genital region are almost certainly effected by dorsoventral muscles. In the annular regions, the longitudinal muscles underlie the integument, forcing the annuli together when the body region contracts. The longitudinal striations probably work in a similar way. Although no attempt has yet been made to observe the muscles of the metapodosomal and genital region, we hypothesize that dorsoventral muscles should be positioned at the sides of this region, just beneath the integument with the longitudinal striations. If the dorsoventral muscles were only near the midline, the integument—which is highly pliable—would likely bend into the body instead of fold around the edge of the body; longitudinal striations would have no obvious function.

Additional hydraulic pressure can also be generated from the center of the body by contracting legs III and IV into the body (Figure 10d). This would also enable the legs out of the way when the center of the body moves through tight gaps. Note that because legs III and IV project downwards, they impede movement by projecting away from the movement path of the main body, making the mite less streamlined. By contrast, legs I and II extend forwards, keeping within the movement path of the main body. Therefore, legs I and II do not appear to impede the movement of the main body when it is moving through tight spaces. For this reason, they would not be required to contract into the body.

38

Figure 15. Different modes of locomotion: (aI–III) hypothesized mode of locomotion for relatively short nematalycids moving through tight spaces; (b) peristalsis along the posterior region of Gordialycus. Zigzag lines indicate areas that are longitudinally contracted.

The ability to dramatically alter the volume of the metapodosomal and genital region allows the mite to use the center of its body to generate the pressure needed to extend or contract an annular region of the body. The action of each annular region can therefore be independent of the other, allowing for a relatively versatile form of locomotion (Figure 15a). While the annular regions extend or contract, the metapodosomal and genital region can be anchored in place by legs III and IV. But legs

III and IV can also be tucked into the body (see above) or used for crawling. This mode of locomotion could be especially useful when the mite is attempting to squeeze through tight gaps and crevices. The expansion of the metapodosomal and genital region can

39 enable the posterior region to be maneuvered and anchored into the soil before the anterior region of the mite then extends forwards via the contraction of the metapodosomal and genital region (Figure 15a). Dorsoventral expansion and contraction also means that the overall length of the body can change quite significantly.

Gordialycus lacks the longitudinal striations needed to dorsoventrally constrict the metapodosomal and genital region. Instead, this mite is probably only able to move around through peristalsis, whereby a wave of contraction is closely followed by a wave of extension (Figure 15b). Longitudinal striations would cause the interruption of peristaltic waves along the length of the body. The dorsoventral expansion of the metapodosomal and genital region would therefore appear to be an impediment to locomotion via peristalsis. With respect to squeezing through tight gaps, the extremely long posterior region would help to anchor the mite in place while the front of the mite extends forwards at the end of a peristaltic wave. Because peristalsis involves the movement of short regions of contraction along the body, there is little overall change in body length.

Legs III and IV of Gordialycus may be reduced for several different reasons.

Firstly, their role in anchorage would be less important. Because the metapodosomal and genital region does not constrict or expand dorsoventrally, the legs are not needed to anchor this region of the body in place during the longitudinal contraction or extension of other body regions. Secondly, like the longitudinal striations, the legs would interfere with the completion of peristaltic waves along the length of the body. This is most obvious with respect to the coxal fields, which are all very large in the other genera. In

40

Gordialycus, the legs have moved in towards the midline as a result of the dramatic reduction in size of the coxal fields. Once legs III and IV become less important with respect to anchorage, there should be strong selective pressure towards reduction. As mentioned above, legs III and IV make the mite less streamlined by projecting away from the movement path. Their reduction would therefore help the locomotion of this region of the body through tight spaces.

In the video footage of Osperalycus tenerphagus, no peristaltic waves were observed. It could be that peristalsis is reserved for particular situations. Alternatively, it may be that peristalsis is not compatible with the dorsoventral expansion and constriction of the metapodosomal and genital region. Furthermore, it may be that Gordialycus, which reaches over 1.0 mm in length, is the only known genus of the Nematalycidae that is long enough to be able to make effective use of peristalsis.

Anal valves

The anal valves appear to play an important role in locomotion by providing an anchor from which the rest of the body can push off. They may be especially important to the locomotion of the genera with long posterior regions – Gordialycus and Osperalycus.

The genera with short posterior regions would appear to be able to drag them along.

The videographic data shows that Osperalycus is able to move around by pushing off against the tip of its opisthosoma, which is first anchored or wedged into the soil

(Online Resource 1). The anal valves (Figure 11, 12), which protrude out strongly in

Osperalycus and Gordialycus, are appropriately structured and positioned to help anchor

41 the tip of the opisthosoma. It is not known whether the anchorage of the tip is accomplished through a wedging or clasping action.

The anal valves are especially prominent in Gordialycus relative to the size of the opisthosomal tip (Figure 11a). It is likely that they are important in the initiation of peristalsis, which begins at the opisthosomal tip. When a wave of contraction starts, the tip of the opisthosoma is pulled inwards and dislodged from the soil. A subsequent wave of extension would then closely follow, causing the tip to extend outwards until it is lodged into a new position in the soil substrate. As the wave of extension extends further forwards through the body, the corresponding region of the body initially moves forwards through the soil because it is extending away from the anchored anus. This wave continues to proceed forwards into the contracted portion of the body in front of it, causing peristaltic motion of the entire body (Figure 15b). The relatively long setae near to the anal valves may be used to locate a suitable anchorage point. The setae along the rest of the opisthosoma are short enough to be barely, if at all, noticeable (Figure 9a).

This is possibly an adaptation for reducing friction during the locomotion of such a long body.

The posterior region of Osperalycus is typically dragged around by the protonymph (Online Resource 3). The adult stages of the short bodied genera of

Nematalycidae – Cunliffea and cf. Psammolycus – have a posterior region that is similar in length to that of the protonymph of Osperalycus. It is therefore possible that the posterior region is commonly dragged around by all of the active instars of Cunliffea and cf. Psammolycus. Moreover, the weakly projecting anal valves of Cunliffea and cf.

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Psammolycus (Figure 11c–d) indicate that the anal region has a less significant role with respect to anchorage. However, it is also possible that the anal region is still used to facilitate anchorage under certain circumstances, such as when these mites are squeezing through tight gaps (Figure 15a).

Palette shape

Variation in the shape of the palettes between genera may be an adaptive trait that accommodates the significant intergeneric differences in the length of the posterior regions. The ability of these mites to maneuver through tight spaces requires anchoring one region of the body firmly in place while another extends or contracts. The secure placement of the static region is essential to success (Figure 15a). The short posterior regions of some nematalycids cause them to encounter little overall friction when moving around. For this reason, they are more likely to have problems pertaining to slippage.

Pointed palettes may therefore be an adaptation for improving the grip of any region of the body that is placed firmly against a surface. This is consistent with the correlation between the shape of the palettes and the relative length of the body. Nematalycids with short posterior regions – Cunliffea and cf. Psammolycus – appear to have pointed palettes that increase anchorage (Figure 13c–d). The shortest genus (cf. Psammolycus) has the palettes with the sharpest points.

The more elongated bodies of Gordialycus and Osperalycus would encounter a lot of friction when contracting, extending or being dragged along. Their mobility depends on waves of extension and contraction along the long posterior regions of their

43 bodies; they would probably not be able to move around using only their legs. Pointed palettes would likely present a serious encumbrance to the locomotion of these mites.

Instead they have round palettes that are orientated in the direction of movement, enabling them to slide their long posterior regions over surfaces with relative ease (Figure

13a–b). Although their integument has less grip or traction, anchorage is facilitated by their longer bodies.

Conclusion

The Nematalycidae appear to be well adapted for life in the mineral regolith, having originated a number of important modifications that seem to be beneficial for this habitat. The integument of the Nematalycidae appears to be well suited to movement in tight spaces. These mites can contract one annular region in order to generate the hydraulic pressure needed to extend another. Most significantly, the mode of locomotion and associated morphology appears to be largely determined by the relative degree of elongation. Peristalsis is clearly essential to Gordialycus, which is the only known genus of Nematalycidae with an incredibly long body. But peristalsis was not observed in

Osperalycus, and it may be that the use of peristalsis is restricted to Gordialycus.

Cunliffea and cf. Psammolycus are probably too short to be able to make effective use of peristalsis. Instead, these mites appear to use a mode of locomotion that involves the dorsoventral expansion and constriction of their central region.

Interestingly, the functional morphology of Osperalycus appears to be transitional between Gordialycus and the other genera of Nematalycidae (Table 4). Osperalycus 44 resembles Gordialycus in having round palettes and a strongly projecting anal vale. The posterior region of Osperalycus is much shorter than the posterior region of Gordialycus but much longer than the posterior region of the other genera. This mite resembles

Cunliffea and cf. Psammolycus in having a metapodosomal and genital region that can dorsoventrally constrict and expand; this feature is clearly absent in Gordialycus.

Peristalsis likely represents a derived mode of locomotion within the

Nematalycidae. The primitive mode of locomotion – the dorsoventral expansion and constriction of the central region – provides short bodied nematalycids with a versatile way of contracting and extending annular regions. The transition to peristalsis allowed the evolution of a much longer body length. This is because peristaltic waves allow many short portions of a very long body to be moved along in quick succession; there is no need to extend or contract the entirety of a long body region.

Once the switch to peristalsis was made, the old mode of locomotion was probably not retained for very long as a supplementary form of movement. Instead, key features of the old mode of locomotion were likely to have been quickly jettisoned or modified because they would have interrupted peristaltic motion (viz. large coxal fields and longitudinal striations). Therefore, the successful transition to peristalsis probably manifested as a large scale and dramatic evolutionary event, one mode of locomotion being rapidly and completely replaced by another.

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Chapter 4: A novel form of microbivory

Introduction

Fluid-feeding clearly predominates within the Arachnida; only the Opiliones, some Solifugae and some Acari feed on solids (Muma, 1966; Walter, 1988; Hillyard and

Sankey, 1989; Walter and Proctor, 1998; Acosta and Machado, 2007; Heethoff and

Norton, 2009). Whereas a large number of species of fluid feeding Acariformes have piercing-sucking mouthparts, in which the chelicerae are usually highly modified (Krantz and Lindquist, 1979; Di Palma et al., 2009; Beard et al., 2012; Krenn and Aspöck, 2012), most other arachnids use chelate chelicerae to macerate their prey for the extraction of fluids. This is often accompanied by extra-oral digestion in which enzymes are introduced into the food (Cohen, 1995, 1998). Other fluid-feeding adaptations have arisen in association with chelate chelicerae. For example, the Mesostigmata (Parasitiformes) use a specialized structure – the tritosternum – to prevent loss of fluid after opening up a prey item with their chelicerae (Wernz and Krantz, 1976).

Although the majority of fluid-feeding arachnids feed on other animals or, more rarely, vascular plants, many species of mites are small enough to be able to extract the fluid contents of microorganisms such as protists and fungi. Some mites within the

Mesostigmata appear to have specially adapted chelate chelicerae for squeezing the liquid

46 contents out of mycelial masses and into their prebuccal cavity (Walter and Lindquist,

1989), and some species of Prostigmata have fine enough stylet-like chelicerae to pierce the hyphae of fungi (Kaliszewski et al., 1995). The Nanorchestidae (Endeostigmata) have evolved a fine labral process beneaththe chelicerae, which has been hypothesized to act as a piercing structure for feeding on the fluid contents of algae (Krantz and Lindquist,

1979).

Although these feeding mechanisms are suitable for some of the larger microorganisms, they may be of little use for very small single-celled eukaryotes and prokaryotes (<10 μm). The chelate chelicerae of many mites are likely to be too messy and inefficient for dealing with such a low volume of fluid, and would require a large aggregation of the food items in order to adequately process them. Microorganisms also present a problem for piercing-sucking mouthparts; the mouthparts may be too broad, crushing instead of piercing the organism, or too long, penetrating all the way through the organism. It would also be challenging for a mite to accurately direct a stylet-like structure into such a small target.

A minute mouth opening and a very narrow esophagus suggests that the

Nematalycidae feeds exclusively on fluids (Haupt and Coineau, 1999; pers. obs.).

However, it is not known what specific types of food the Nematalycidae feed on. Distinct differences in the morphology of the mouthparts between genera indicate that diet is likely to vary across the family. With the aid of low-temperature scanning electron microscopy (LT-SEM), we demonstrate that Osperalycus and Gordialycus (Acariformes:

47

Nematalycidae) have unusual mouthparts that appear to be adapted for feeding on the fluid contents of small microorganisms (<5 μm).

Figure 16. Vermiform bodies of the Nematalycidae: (a) Gordialycus sp. A (nymph/adult); (b) Osperalycus tenerphagus (adult female).

Method

LT-SEM was undertaken at the US Department of Agriculture, Electron &

Confocal Microscopy Unit, Beltsville, MD. Most species of Nematalycidae were collected via soil washing in accordance with Kethley (1991). This meant having to

48 mount specimens that had been kept in alcohol (80% or 95%). However, live specimens of Osperalycus tenerphagus Bolton & Klompen were collected and mounted for LT-

SEM by directly removing them from floated material that had not yet been sieved. This was accomplished by placing the floated material directly under a dissection microscope

(see also Bolton et al., 2015a; Chapter 3). If not stated otherwise in the caption, the figured images were obtained using LT-SEM.

Five different species were collected and observed in an LT-SEM: Gordialycus sp. A (absence of lateral claws) & B (two lateral claws on legs I, II and III), Osperalycus tenerphagus, cf. Psammolycus sp. A and Cunliffea strenzkei Cunliffe. The mites were collected from four different locations across the USA (Appendix A). Osperalycus tenerphagus was collected from a silty clay loam in Columbus, Ohio. All of the other species were collected from sands. Voucher specimens of all taxa are deposited at the

Ohio State University Acarology Collection. Specimens were prepared for LT-SEM using the same techniques as described in Bolton et al. (2014) and Chapter 2.

Measurements were based on LT-SEM images of multiple specimens. It was not always possible to distinguish nymphs and adults, especially for Gordialycus. Therefore, mean values for sizes of mouthpart structures almost certainly included specimens from different developmental stages (excluding the readily recognizable stages of larva and pre-larva). However, there is very little growth in the mouthparts from the larval to adult stage. Light microscope measurements, where developmental stages could be confidently determined, showed that the size of the chelicerae and palps increased by less than 10% in Osperalycus, and less than 20% in Gordialycus from larva to adult (Appendix B).

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Figure 17. Mouthparts of the Nematalycidae (drawings are differently scaled): (a) general schematic for the mouthparts of the Nematalycidae, representing a transverse section of the gnathosoma between the mouth and the tip of the labrum; (b) chelicera of Osperalycus tenerphagus; (c) chelicera of Gordialycus sp. A. La = labrum; Pc = preoral cavity; LL = lateral lip; Pa = palp; Ru = rutellum; Fd = fixed digit; Md = movable digit; Cs = cheliceral shaft; Tr = trochanter; Sc = cheliceral seta.

Morphology of mouthparts

General mouthpart structure and function

The Nematalycidae have a mouthpart plan that is similar to that of other mites in the Endeostigmata – the chelicerae are chelate and the subcapitulum, which is a ventral structure formed from the fusion of the palp coxae, includes a pair of lateral lips

(subcapitular extensions) and rutella (sclerotized structures that project from the lateral lips) (Figure 17a). The rutella have been considered to be absent in the Nematalycidae

(Walter, 2009). However, they are present but minute (Kethley, 1990; Bolton et al., 2014;

Chapter 2). The function of the rutella is to facilitate the manipulation and ingestion of 50 foods (Alberti, 2008). The diversity of shapes that have been found in these structures appears to be associated with different feeding methods (Akimov, 1979; Alberti and

Coons, 1999). In the majority of cases they are associated with particulate feeding, where they appear to be used to break up food particles that are being pulled into the preoral channel by the chelicerae (Grandjean, 1957; Dinsdale, 1974; Théron, 1979; Evans, 1992).

The general structure of the mouthparts of the Nematalycidae is quite compact

(Figure 17a). In common with almost all other mites, this family lacks a labium. The lateral lips tend to be tightly adjoined, forming a single structure (Figure 2a); a suture runs between them. The labrum is usually long and narrow. It lies over the surface of the lateral lips, creating a tight channel that leads into a small mouth at the base of the labrum.

The number of palp segments varies between genera (1 to 4 segments). There is also considerable intergeneric variation in the length of the chelicerae and cheliceral digits, and in the form of the rutella and lateral lips.

Each chelicera comprises a trochanter, a shaft and two digits (Figure 17b–c) – the fixed digit and the movable digit (the term ‘digit’ is treated here as a synonym of ‘chela’, i.e. it excludes the large cheliceral shaft). The fixed digit is a dorsal extension of the cheliceral shaft. The movable digit articulates against the fixed digit, allowing the chelicerae to “grab” or break up food items.

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Figure 18. Dorsal views of the mouthparts of Osperalycus tenerphagus: (a) digits inserted into the pouch; (b) digits withdrawn from the pouch – right chelicera twisted out of its natural orientation due to damage. Po = pouch; Ru = rutellum; Cs = cheliceral shaft; Pt = Palp tarsus; Pg = tibia; Pf = palp femurogenu; Fd = fixed digit; Md = movable digit; Sc = cheliceral seta; Le = leaf (flap-like extension of the lateral lips).

Osperalycus

LT-SEM revealed that the anterior region of the lateral lips has been modified into a small pouch (Figure 18a–b). The pouch is 5½ µm wide at the rim, although the internal width is only 4 μm due to the thickness of the wall. The wall of the pouch is composed of smooth or non-striated integument. Non-striated integument also makes up some of the venter of the gnathosoma (Figure 19c). The dorsal surface of the pouch appears to be composed of several overlapping integumental sheets or leaves (Figure 18b).

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Figure 19. Pouch of Osperalycus tenerphagus. (a) dorsal view (chelicerae inserted) – rutellum highlighted by white outline; (b) dorsal view (chelicerae withdrawn) under phase contrast microscope – rutellum highlighted with the white outline from (a), showing sclerotization. Labrum (left side only) demarcated by dashed line; (c) anteroventral view. Cs = cheliceral shaft; Fd = fixed digit; Po = pouch; Rb = rutellum base; Rd = rutellum digit; Su = suture adjoining the lateral lips; La = labrum; LL = lateral lips; Pt = palp tarsus; Cu = cup shaped seta-tip (uneven rim); S = seta of the palp tarsus (trichoid); Sc = cheliceral seta (trichoid); Cp = cuticular protuberance.

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We do not know how each of these leaves correspond with each of the two lateral lips. The leaves appear to represent flap-like dorsal extensions of the lateral lips, although their developmental origin is far from clear. The venter of the pouch is simpler and reveals a single suture between the lateral lips (Figure 19c), which runs posteriorly from where the rutella overlap at the midline. . The pouch is invisible under a light microscope due to low levels of sclerotization. The much darker and more sclerotized rutella are convergent and are pressed up firmly against the outside of the pouch (Figure 19a–b).

The labrum is relatively long and narrow, projecting into the pouch at an angle that is nearly horizontal (Figure 19b). Consequently, the preoral cavity (the channel lying between the labrum and the lateral lips) runs from the inside of the pouch into the mouth.

The pouch therefore forms a separate and additional cavity that contains the terminus of the preoral cavity.

The chelicerae are short (~12 µm), each with a single seta located on the dorsum of the shaft, proximal to the fixed digit. When the chelicerae are extended forwards, the dorsoventrally narrow digits completely slot into the pouch (Figure 18a). Whereas the fixed digits are pressed together above the labrum when the chelicerae are slotted into the pouch (Figure 19a), the movable digits slant outwards and away from one another in order to slot in on either side of the labrum.

The chelicerae are slanted downwards into the pouch – the dorsal views of the chelicerae and pouch (Figure 18, 19) would therefore be anterodorsal with respect to the main body if the main body was in the field of view.

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Figure 20. Palps: (a) Osperalycus tenerphagus – close-up lateral view of the palp seta with cup-shaped tip; (b) Osperalycus tenerphagus – lateral view of the palp tarsus; (c) Gordialycus sp. A – ventral view of the palp tarsus; (d) Gordialycus sp. B – lateral view of the palp tarsus; (e) Cunliffea strenzkei – lateral view of the palp tarsus; (f) cf. Psammolycus sp. A. – lateral view of the palp tarsus. Pt = palp tarsus; Pg = palp tibia; Pf = palp femurogenu; Cu = cup shaped seta-tip; Cp = cuticular protuberance; S = seta of the palp tarsus (trichoid); Spg = seta of the palp tibia (trichoid); Sc = cheliceral seta (trichoid). 55

The rutella are pressed up against the outside of the pouch. The bases of the rutella are broad and intrude into the sides of the pouch. This produces a narrow slot inside the pouch, between the bases, into which the digits neatly fit when they are extended (Figure 19a, c). The rutellum distally narrows into a dorsoventrally deep digit that lies firmly against the front of the pouch. The tips of both digits are flanged and clearly overlap one another, buttressing the anterior corner of the pouch at the midline

(Figure 19c, 21a).

The palps are short (~10 µm) and three-segmented, tentatively designated femurogenu, tibia and tarsus (Bolton et al., 2014; Chapter 2). Each of the palps bear six setae on the tarsus (three ventral, two dorsal and one anterior) and a single dorsal seta on the tibia. The anterior/distal seta, on the palp tarsus, has a tip that has been modified into a shallow cup-like or concave structure (Figure 20a). The cup has a diameter of 0.8 μm.

The length and minimal thickness of the stem is 1.5 and 0.3 µm, respectively. There are two very short cuticular protuberances at the base of this modified seta (Figure 20a: Cp).

A similarly shaped protuberance is also present on the dorsum of the palp tarsus. Four of the five other setae on the palp tarsus have tips that reach near to the modified seta

(Figure 20b).

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Figure 21. Ventral views of the lateral lips and rutella: (a) Osperalycus tenerphagus; (b) Gordialycus sp. A; (c) Gordialycus sp. B. Ru = rutellum; Su = suture adjoining the lateral lips; or = adoral seta; a + m = subcapitular seta.

Gordialycus

As with Osperalycus, the lateral lips of both species of Gordialycus tightly adjoin.

But instead of forming a pouch, they are shaped into a narrow projection in between the rutella (Figure 21b–c). The front of the lateral lips appears to be composed of the same type of smooth integument that forms the pouch in Osperalycus (Figure 21a). A ventral suture (s) runs along the midline of the subcapitulum in both genera. This is where the smooth integument of the lateral lips adjoin (Figure 21). The rutella are large and

57 convergent (Figure 22b–d, 23). The rutella meet, but unlike Osperalycus, there is very little, if any, overlap.

Figure 22. The mouthparts of Gordialycus sp. A: (a) dorsolateral view of the chelicerae; (b) anterior view of the mouthparts – chelicerae partially retracted; (c) ventral view of the lateral lips and rutella, showing connection between lateral lips and rutella (large arrowhead) – chelicerae withdrawn so that there is a gap between the lateral lips and the rutella); (d) anterior view of the mouthparts – chelicerae extended forwards so that the movable digit (Md) fills in the gap between the lateral lips and the rutella. Ru = rutellum; Cs = cheliceral shaft; Md = movable digit; Fd = fixed digit; LL = lateral lips; Sc = cheliceral seta; or = adoral seta; Pt = palp tarsus; Pg = palp tibia.

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The rutella of Gordialycus sp. A are dorsoventrally expanded, creating a barrier in front of the chelicerae (Figure 21b). The dorsal and ventral edges of the rutella curve back under and over the space between the lateral lips and the rutellum (Figure 22b, d).

But the ventral edges of the rutella do not extend all the way in to seal the gap between the rutella and the lateral lips (Figure 21b, 22c). The front of the lateral lips adjoins the meeting point of the rutella (Figure 22c).

By contrast, the ventral margins of the rutella of Gordialycus sp. B form a contiguous boundary with the lateral lips (Figure 21c). The opening that is present in the gnathosomal venter of Gordialycus sp. A is sealed by the expansion of the rutella in

Gordialycus sp. B. The rutella of this species also have narrow hair-like extensions protruding from the front (Figure 21c, 23). These are clearly absent in Gordialycus sp. A.

The labrum of Gordialycus is relatively long and narrow, and always projects diagonally down in between the rutella.

The chelicerae of both species of Gordialycus are almost twice as long as those of

Osperalycus (~22 µm), each with a single seta located dorsolaterally, proximal to the fixed and movable digit. In common with Osperalycus, the movable digits slant outwards and away from one another (Figure 22a–b). This allows them to fit on either side of the lateral lips and the overlying labrum when the chelicerae are placed down against them.

When a chelicera of Gordialycus sp. A and B is pushed up against the rutellum, the movable digit slots into the gap between the rutellum and the lateral lips (Figure 22d: Md and Ru). In Gordialycus sp. A, the movable digits can be clearly seen to be proximal to the adoral setae (or) (Figure 22d). But the movable digits cannot be viewed from the

59 venter of the gnathosoma in Gordialycus sp. B because of the contiguous boundary between the lateral lips and the rutella (Figure 21c); the movable digit slots into a cavity on either side of lateral lips (Figure 23).

Figure 23. Lateral view of the mouthparts of Gordialycus sp. B – showing that the large cup-shaped seta could be maneuvered to hold a microorganism into the bowl shaped rutella. There is obvious visible damage (probably due to desiccation prior to mounting) – the rutella have pulled away from one another and the lateral lips. They terminate in a hairlike extension. Cs = cheliceral shaft; Md = movable digit; Fd = fixed digit; Ru = rutellum; LL = lateral lips; Pt = palp tarsus; Pg = palp tibia; Pf = palp femurogenu; Ptr = palp trochanter; Cu = cup shaped seta-tip; S = seta of the palp tarsus (trichoid); Spg = seta of the palp tibia (trichoid); Sc = cheliceral seta; Cp = cuticular protuberance.

The palps of both species of Gordialycus are very similar in length to those of

Osperalycus (~10 µm), with the same number and arrangement of setae as for 60

Osperalycus (tarsus = three ventral, two dorsal and one anterior; tibia = one dorsal). The anterior seta also has a cup-shaped or concave tip (Figure 20c–d). Similar to Osperalycus, the trichoid setal tips of the palp tarsus reach near to the seta with the cup-shaped tip, a difference being that all five setae do this instead of just four, as in Osperalycus. The palps are distinguishable from Osperalycus in having an additional segment at the base – a trochanter. Whereas Gordialycus also has two cuticular protuberances at the base of the cup-shaped seta, it lacks the posterior cuticular protuberance at the dorsum of the palp tarsus. The cup of the cup-shaped seta of Gordialycus sp. A has a diameter of 1.3 µm.

The length and minimal thickness of the stem of the cup-shaped seta is 1.5 and 0.4 µm, respectively. The cup of Gordialycus sp. B is slightly oval (length = 2.6 µm; width = 2.2

µm). It is also distinctly larger than the cup of both Osperalycus and Gordialycus sp. A.

The cup-shaped seta of Gordialycus sp. B has a short and thick stem (length = 1.1 µm; minimal thickness = 0.7 µm).

Other genera

The mouthparts of Cunliffea and cf. Psammolycus are clearly distinct from

Osperalycus and Gordialycus. In Cunliffea and cf. Psammolycus, the rutella are more attenuated than in Gordialycus and Osperalycus. These rutella do not meet one another and they do not reinforce a pouch. The palps of both Cunliffea and cf. Psammolycus do not have any highly modified setae (Figure 20e–f).

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Discussion

A rupturing mechanism for microbivory

Particulate feeding mites that possess rutella will typically use their chelicerae to pull their food substrate (e.g. a hyphal strand) between the sharp, and often dentate, anterior edges of the rutella, causing the food substrate to break up for the purposes of ingestion (Théron, 1979). In both Osperalycus and Gordialycus the rutella have converged so that their anterior edges now meet or overlap. Their anterior edges therefore do not project out for the purpose of cutting up food particles. Furthermore, in

Osperalycus the rutella are pressed up against the outside of a pouch, whereas in

Gordialycus the dorsal edges of the rutella project up or back rather than forward. The rutella are therefore not positioned or angled to present a cutting edge for food items that are being pulled back and into the gnathosoma. But this is to be expected, considering that the Nematalycidae appear to be fluid feeders rather than particulate feeders.

We hypothesize that the unusual mouthpart morphology of Osperalycus and

Gordialycus is an adaptation for feeding on the fluid contents of small microorganisms

(diameter <5 μm). A rupturing mechanism is indicated by a barrier in front of the chelicerae (a rutella or a pouch), which appears to be an adaptation for crushing microorganisms that can be placed between this barrier and the chelicerae when they are not fully extended. A rupturing mechanism is also suggested by the way that the chelicerae can be inserted tightly between convergent rutella, or within a reinforced pouch, while also being in the immediate proximity of the mouth. A tight slotting mechanism helps to ensure that the food organism is completely squeezed for the

62 purposes of extracting its fluid contents. Once a microorganism is ruptured, the fluids would be contained by the modified rutella or pouch before they are drawn up underneath a narrow labrum that is inserted between the rutella or within the pouch. The labrum forms the roof of the preoral cavity, whereas the lateral lips form the floor. The preoral cavity, which opens into the rupturing structure (pouch/rutella), is therefore used to draw fluid out of the rupturing structure and into the mouth. .

These mites can probably only feed on what they can pick up and fit into their holding structure – small single celled organisms such as bacteria or yeasts. The chelicerae, which are pointed and chelate, would appear to break up food in the conventional ways – piercing and slicing – while the microorganism is being held in place. However, it may be that the movable digits have to close against the fixed digits in order for the cheliceral digits to completely slot into the containing structure.

Modifications of the palp

This mode of feeding is obviously only possible if the mite can pick up a microorganism without puncturing or crushing it before placing it into the holding structure. Both genera have very similar palps, with modified setae that appear to be adapted for this function. The short length of the palps (~10 µm) makes them suitable for this task. A seta on the palp tarsus has a tip that has been modified into a cup-like structure that has the appropriate shape, size and location for picking up small microorganisms and then delicately maneuvering them (Figure 20a–d, 24). The smooth and concave surface of the cup should provide a much greater surface area of attachment

63 to the microorganism than between the microorganism and the uneven and convex surfaces of the soil particles or other microorganisms contacting it. Attractive intermolecular forces, such as capillarity and van der Waals, should then be sufficient to pick the microorganism up. The cups also appear pliable and can be seen to bend when pressed up against other surfaces. This means that they can probably accommodate some variation in the size or shape of the microorganism they are attached to. Although the cups look like they are adapted for suction, some specimens appear to have cups with uneven rims that would greatly compromise their ability for that function (Figure 19c).

If the surface of both the microorganism and the cup are smooth enough to enable a sufficiently close contact (<5 nm), van der Waals forces are likely to be important to the attachment. At greater distances, retardation effects cause the van der Waals force to rapidly decay (Israelachvili, 2011), and the relative strength of other intermolecular forces, including capillarity, become more important. The species of Nematalycidae with the smallest cup is Osperalycus tenerphagus. The cup of this mite has an approximately 5

× 10-13 m2 area of contact (diameter = 0.8 μm) – assuming every part of the cup’s interior surface is in contact with the substrate. At a contact distance of 5 nm, the van der Waals adhesion (detachment) force between the cup and a sphere with a diameter of 3.5 μm – the typical size and shape of a yeast cell that would fit into the feeding pouch – is 18.3 times as great as the adhesion force between the sphere and a flat surface (Leckband and

Israelachvili, 2001). When the weak force of gravity is also accounted for (weight based on the mass of water contained within the yeast cell), the difference decreases to a factor

64 of 6.3 (Hamaker constant = 1 × 10-20). The larger cups of Gordialycus may be used to pick up larger microorganisms (but see the explanation for Gordialycus sp. B below).

Note that capillarity might be important to the attachment if greater contact distances cause retardation effects with respect to the van der Waals forces. Under wet or very moist conditions, large meniscus bridges between a microorganism and another surface would require the seta to have a cup with a larger contact area in order for the setal cup to pick up the microorganism. This feeding mechanism might therefore be more effective under relatively dry conditions.

The location of the cup at the tip of a flexible seta helps to avoid puncturing or crushing the microorganism (preventing any potential waste of the fluid contents). The form and flexibility may also assist in the attachment – the stem of the seta should have sufficient flexibility to cause the cup to align squarely with the surface of the microorganism as it is being pressed against it, therefore enabling the necessary angle for a good attachment. The palp tarsal setae that curve into the proximity of the cup could be chemosensory, being well positioned to detect any material that is either attached to the cup or very close to it (Figure 20b–d). But this would require the setae to have terminal pores, which is not yet known. These setae may also, or instead, function as basic mechanoreceptors for sensing if the cup has successfully attached to or detached from a microorganism. The two short cuticular protuberances present at the base of the seta with the cup (Figure 20a, c–d), may be used as scrapers for dislodging microorganisms that are strongly attached to any surface via extracellular polymeric adhesives. Such substances are known to be secreted by many different types of microorganisms (Sutherland, 1982;

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McCourtie and Douglas, 1985; Hokputsa et al., 2003; Long et al., 2009). These protuberances may also or instead function to prevent the seta with the cup from flexing back too far when it is being used to pick up food items, therefore preventing the damage of a crucial feeding structure. The dorsal tarsal protuberance, which is exclusive to

Osperalycus tenerphagus, has a more posterior position and may therefore have a different function.

Figure 24. Dorsal views of the mouthparts of Osperalycus for the three hypothesized stages of feeding on a microorganism (pouch shown as transparent): (a) cup on the palp tarsus attaches to a microorganism; (b) palp bends back and over the pouch opening – chelicerae retract to make space for the microorganism; (c) chelicerae extend forwards, pushing the microorganism into and against the anteriorly narrowing pouch until it ruptures. The fluid contents can then be channeled into the mouth (inside the pouch). Rutella = dark grey; pouch wall = medium grey.

Osperalycus tenerphagus

The pouch of Osperalycus tenerphagus appears to be soft based on its complete lack of visibility under a light microscope (Figure 19b). The rutella give support and shape to the pouch, restricting its movement during the puncturing of the food contents.

The hard bases of the rutella, which intrude into the sides of the pouch, cause the pouch to narrow distally (Figure 19). The convergent and narrow digits of the rutella reinforce 66 and shape the front of the pouch as it increasingly narrows towards the terminus. A microorganism would therefore be channeled into a firmly held position by simply pushing it into the reinforced pouch with the chelicerae. Once the microorganism is tightly in place, further extension of the chelicerae would crush or puncture the microorganism, causing the liquid contents to spill out into the pouch (Figure 24). The extension of the chelicerae would also channel any solid or liquid inside the pouch into the corner of the pouch, which is buttressed by the flanged and overlapping tips of the rutella (Figure 19c). The rutella therefore also appear to be shaped and situated for the reinforcement of the most vulnerable part of the pouch.

Gordialycus sp. A

The gap between the rutella and the lateral lips (Figure 22c) provides a slot for the movable digit of each chelicera (Figure 22d). This would enable Gordialycus sp. A to effectively rupture any small microorganism that is placed in between the rutellum and the lateral lips. As the chelicerae extend forwards into the microorganism, the fixed digit would pin it in place against the rutellum. The movable digit would then be able to slice into it from the side. The ventral opening (the gap between the ventral edge of the rutellum and the lateral lips) may provide space for the movable digit to articulate during or after the extension of the chelicera.

As both piercing and slicing occurs, the microorganism is tightly held between the lateral lips and the rutellum. The rutellum is appropriately shaped to hold a

67 microorganism firmly in place due to the way that the front of the rutellum curves back over and under the slot for the movable digit (Figure 22b, d).

Gordialycus sp. B

The gap in the venter that is present in Gordialycus sp. A is sealed by the expansion of the rutella in Gordialycus sp. B, which would prevent the loss of fluid via the venter. Therefore, although the rutella of Gordialycus sp. B would be able to hold microorganisms in place, they may also function as an effective container of the fluid that is ejected from ruptured microorganisms (Figure 23). The movable digits would slot in between the rutella on either side of the lateral lips. Anything in between the rutella would therefore probably be ruptured in a similar way to the mechanism described for

Gordialycus sp. A. However, it may be that the absence of a ventral opening between the rutellum and the lateral lips reduces the maneuverability of the movable digits during the rupturing process.

Although the subcapitulum of Gordialycus sp. B functions as a suitable fluid container, it would be susceptible to the loss of microorganisms over the edge as the chelicerae push into them. This is because the front of the rutellum does not curve back dorsally, in contrast to Gordialycus sp. A. The rutella therefore appear to have reduced functionality with respect to holding the microorganism in place. This function may be instead partially served by the cup-shaped palp seta (Figure 20d, 23). In this species the cup is much larger (roughly double the diameter of the setal cup of Gordialycus sp. A and triple that of Osperalycus tenerphagus). The stem of the seta is also much thicker and

68 shorter than that of Osperalycus tenerphagus or Gordialycus sp. A. These would be useful modifications if, in addition to picking up microorganisms, this seta functioned to hold the microorganisms down while they are in between the rutella during the rupturing process (Figure 23). The reduced length and increased width of the stem would be important in giving the seta additional rigidity when it is pushed down against the microorganism (a long, thin and highly flexible stem would be useless in this regard).

The increased contact area of the cup would provide a larger and stronger attachment.

There is no other obvious reason for the modified seta to have a much larger cup while also having a much shorter and thicker stem. Furthermore, the modified seta appears to be the only structure that could prevent the microorganism from slipping over the edge of the rutella during the extension of the chelicerae.

The hair-like extensions at the front of the rutella may function to help reduce spillage or loss of fluid (Figure 21c, 23). The extensions would cause fluid to accumulate at the dorsal edge of the rutella instead of spilling over. They may therefore have a similar function to the tritosternum of the Mesostigmata, which has hair-like extensions for helping to trap fluid against the venter of the gnathosoma (Wernz and Krantz, 1976).

Ecological relevance of the rupturing mechanism

Like other nematalycids, Osperalycus and Gordialycus appear to live exclusively in mineral soils or sands. Their unusual integumental morphology appears to be adapted for moving around in these habitats (Bolton et al., 2015a; Chapter 3). Gordialycus is frequently found in deserts, beaches and sand dunes (Appendix A; Coineau et al., 1967;

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Silva et al., 1989; Norton et al., 2008). Osperalycus has been collected from a deep mineral soil from a prairie and a young chestnut plantation (Bolton et al., 2014; Chapter

2). The soft integument and vermiform bodies of the Nematalycidae would make them vulnerable to the many predatory mites and other arthropods that live at relatively high densities in organically rich soils and plant litters. Organically impoverished soils therefore likely provide a refuge from predation and perhaps also competition.

If our functional-morphology argument is correct, by allowing Osperalycus and

Gordialycus to feed on individual microorganisms, their cell-rupturing mouthparts appear to provide a novel solution for the main challenge presented by the adverse and organically impoverished habitats in which they live. Microorganisms are ubiquitous.

They can provide a reliable source of nutrients in adverse and organically impoverished habitats such as deserts and subsurface environments, where they make up an important component of their ecosystems (Skujinš, 1984; Dobbins et al., 1992; Vishniac, 2006;

Pointing & Belnap, 2012). It should therefore be expected that many of the microarthropods that live in these habitats are specialized for feeding on microorganisms, especially as many other types of potential food organisms are either absent from these habitats or less evenly distributed within them.

Furthermore, because food of any kind is relatively scarce in organically impoverished environments, there should be strong selection for efficient feeding.

Cutting or grinding up small microorganisms with chelate mandibles or chelicerae should normally be wasteful, with a large proportion of the relatively small volume of fluid adhering to surfaces from which it cannot be readily channeled into the mouth. By

70 rupturing the microorganism in a structure that is proximal to the mouth, a large amount of waste is avoided.

The evolution of these rupturing mechanisms raises the question of why these mites did not instead switch to particulate feeding, where microorganisms would be swallowed whole and digested in the gut. Particulate feeding is unusual within the

Arachnida, and has only evolved in a small number of lineages. Part of the reason for this may be that this mode of feeding is relatively costly due to the enzymes needed to either penetrate or digest the cell walls of microorganisms (Hubert et al., 2001; Hubert et al.,

2004). These costs are normally met through the direct metabolism of the enzymes, or indirectly by hosting microbial symbionts (Smrž and Čatská, 2010). In an organically impoverished environment it may be more efficient to circumvent these costs by mechanically separating the fluid contents from the cell wall prior to ingestion.

Conclusion

Although it is not yet certain how the mouthparts of Gordialycus and Osperalycus function, a rupturing mechanism is consistent with many different features of these mites.

A hard or reinforced structure is present directly in front of the chelicerae, providing an obvious way to secure a microorganism during its rupture. Furthermore, in both genera the preoral cavity extends directly into the space between the rutella, where the fluid would be released from the ruptured microorganism.

A collecting mechanism also appears to be present. There is a remarkable degree of resemblance between the palps – the hypothesized collecting structures – of the two 71 genera. In the other genera that were observed with LT-SEM (Cunliffea and

Psammolycus), neither a holding structure nor a collecting mechanism is apparent. This is fully consistent with the rupturing mechanism hypothesis, which requires a means of maneuvering microorganisms into a holding structure during the puncturing process.

A couple of issues remain unsolved. It is not known how the cell wall of the microorganism is removed from the feeding apparatus after it has been ruptured. It is also not yet clear exactly how the simple setae and cuticular protuberances on the palps function.

As these mouthparts could be adapted for feeding on very small food items, they may represent one of the most unusual and extreme adaptations to microbivory throughout the Arachnida – a possible result of strong selective pressure for more efficient ways of feeding on small microorganisms in organically impoverished environments.

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Chapter 5: A sheath for biting chelicerae

Introduction

The earliest fossils of the eurypterids show that the chelate (‘biting’) form of chelicerae originated no later than the Ordovician (c. 460 Ma) (Lamsdell et al., 2015).

This form of chelicerae would therefore appear to represent an extremely ancient plesiomorphy that has been retained by most of the major lineages of the extant

Chelicerata. However, a few very successful and speciose lineages of the Arachnida, including the Araneae and some of the Acariformes, have chelicerae that have deviated from the chelate form (Krantz & Lindquist, 1979; de Lillo et al., 2001; Zonstein, 2003).

Different lineages of the Acariformes have evolved styliform chelicerae (de Lillo et al.,

2001), which allows for the penetration of deeper surfaces such as plant and animal tissue. Typically, styliform chelicerae have originated in conjunction with gnathosomal integration, i.e. the integration of the chelicerae with the subcapitulum. Gnathosomal integration allows the chelicerae to be incorporated into the preoral cavity (preoral groove), enabling the ready passage of fluid from a puncture through the wall of a food organism, made by the styliform chelicerae, to the pharynx (the cheliceral stylets rarely, if ever, conduct food). The subcapitulum can also function as a guide for styliform chelicerae.

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The Eriophyoidea make up a major component of what is probably the most ancient lineage of mites to have evolved styliform chelicerae for fluid-feeding on higher plants. The Eriophyoidea have a highly complex and unusual form of gnathosomal integration: the styliform chelicerae are part of a stylet bundle that is enveloped within a large, membranous sheath (Figure 25). The Tetrapodili (comprising extant Eriophyoidea and fossilized, eriophyoid-like mites) had already evolved a stylet sheath by the Triassic

(c. 230 Ma) (Sidorchuk et al., 2014).

Figure 25. The mouthparts of an eriophyoid – modified from Figure 21 of Nuzzaci, 1979. Ap = apodeme; Ch = cheliceral stylets; Ig&As = infracapitular guide and auxiliary stylets; La = labrum; Ph = pharynx; Sh = stylet sheath.

Until very recently, it was widely thought that the Eriophyoidea belong in the

Trombidiformes (Baker & Wharton, 1952; Lindquist, 1996b; Lindquist et al., 2009;

Zhang et al., 2011). However, the results of a recent set of phylogenetic analyses indicate that the Eriophyoidea falls outside the Trombidiformes (Chapter 6). These analyses also 74 show strong support for a clade that only contains the Eriophyoidea and the

Nematalycidae. With respect to their chelicerae, all of the Nematalycidae have retained the primitive, chelate form. But at least some, and possibly all, of the Nematalycidae have undergone some form of gnathosomal integration. For example, the chelicerae of

Osperalycus and Gordialycus neatly slot into subcapitular modifications such as a pouch or modified rutella (Bolton et al., 2015b; Chapter 4).

The somewhat more primitive mouthpart morphology of the Nematalycidae may lead to a better understanding of the origin of the Eriophyoidea’s complex and unusual mouthparts. This chapter chiefly addresses the gnathosomal morphology of Cunliffea cf. strenzkei Cunliffe (Acariformes: Nematalycidae), which has subcapitular modifications that provide a new possible explanation for how the stylet sheath of the Eriophyoidea originated. This discovery, which is addressed in this chapter in light of the findings of the phylogenetic analyses (Chapter 6), was the primary reason for including the

Eriophyoidea in those phylogenetic analyses.

Method

Cunliffea cf. strenzkei (Figure 26a) was collected from sand from the Indiana

Dunes National Lakeshore, USA. Extraction was via washing in accordance with Kethley

(1991). Before their examination, specimens were stored in alcohol (90%). Low- temperature scanning electron microscopy (LT-SEM) was undertaken at the US

Department of Agriculture, Electron & Confocal Microscopy Unit, Beltsville, MD.

Specimens were prepared for LT-SEM using the same techniques as described in Bolton 75 et al. (2014) and Chapter 2. If not stated otherwise in the caption, the figured images were obtained using LT-SEM. LT-SEM voucher specimens are deposited at the Ohio State

University Acarology Collection (Appendix A).

Figure 26. Cunliffea cf. strenzkei: (a) lateral view of body (adult); (b) lateral view of chelicera with the sheath removed due to damage (nymph/adult). Cs = cheliceral shaft; Fd = fixed digit; Md = movable digit.

Confocal laser scanning microscopy (CLSM) was undertaken in order to observe internal features of the mouthparts, e.g. the pharynx, preoral cavity and labrum. A specimen of Cunliffea cf. strenzkei was slide mounted in Hoyer’s medium and then imaged using a laser at the blue-green part of the visible light spectrum (488 nm). The band detector range was extended out broadly, and the power of the laser was increased over the course of a z-stack in order to compensate for the effects of bleaching.

Throughout the scan, the range indicator was used to ensure that the brightness of the 76 resulting 3D model was distributed as evenly as possible through the z axis (saturation was kept to a minimum). Confocal voucher specimens are deposited at the Ohio State

University Acarology Collection (Appendix A).

Morphology of mouthparts

The main body of the subcapitulum comprises adjoining but not fused malapophyses. The overlying chelicerae are chelate and their shafts are relatively elongated (Figure 26b). The shafts are tightly pressed up against one another, as are the fixed digits (Figure 27). The movable digits slot very neatly into an indentation on either side of the subcapitulum (Figure 28).

The front of the subcapitulum is shaped into a pad with a soft, membranous rim

(Figure 27). Flaps extend out from this pad, forming a rudimentary sheath for the cheliceral digits (Figure 27, 28). The sheath, which leaves much of the digits exposed, forms a lateral and external cover over the empty space between the fixed and movable digits. The anterior edges of this sheath form the opening of the preoral cavity. There is lateral and outward flaring along those edges (Figure 27, 28). The rutella are extremely slender and extend across the movable digits (Figure 28). The tips of the rutella abut the subcapitular flaps that form the sheath.

Under LT-SEM, the labrum is not normally visible from above because it is concealed beneath the chelicerae. Confocal microscopy reveals that this structure is relatively long and thin, and extends into the proximity of the cheliceral digits (Figure

29). 77

Figure 27. Cunliffea cf. strenzkei (nymph/adult): antero-dorsal view of mouthparts. Fd = fixed digit; Md = movable digit; Of = outward flaring at the front of the cheliceral sheath; Pd = pad at the front of the subcapitulum; Ru = rutellum; Sh = cheliceral sheath.

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The palps, which are dorsoventrally deep (Figure 28), are two-segmented and have seven setae – all of the setae are on the terminal segment. One seta is very thick and may be eupathidial.

Figure 28. Cunliffea cf. strenzkei (nymph/adult): ventro-lateral view of mouthparts (different specimen to Figure 27). Cheliceral digits are artificially colored as green to improve visibility; movable digits are slotted on either side of the subcapitulum. Pt = palp tibiotarsus; Ru = rutellum (highlighted brown); Sh = cheliceral sheath; Sub = subcapitulum.

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Figure 29. Cunliffea cf. strenzkei (adult): (a), dorsal view of 3D model of mouthparts (shadow/light source mode), generated using confocal laser scanning microscopy; (b), single section from the z-stack (dorso- ventral view) used to build the 3D model in (a). In (a), it is clear that the chelicerae have dislodged from the sheath and they no longer press against one another – both of these features are probably attributable to the slide mounting process. The space between the chelicerae reveals that the labrum is relatively attenuated. In b, the section shows the narrow pre-oral cavity (Pc) underlying the labrum. Ch = chelicera; La = labrum; Pc = preoral cavity; Ph = pharynx; Pt = palp tibiotarsus; Sh= cheliceral sheath.

Discussion

Hypothesis

The stylet sheath of the Eriophyoidea originated from a rudimentary sheath

(=protosheath) for chelate chelicerae, which was formed from subcapitular flaps. The original function of this sheath was to extend the preoral cavity (a hermetically sealed channel) into the area between all four of the cheliceral digits. Evidence for this hypothesis comes from the relatively primitive mouthparts of Cunliffea strenzkei.

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Functional morphology of the mouthparts of Cunliffea strenzkei

By extending over the space between the fixed and movable digits (Figure 28), the cheliceral sheath of Cunliffea forms a hermetically sealed chamber, thereby extending the preoral cavity anteriorly, beyond the labrum. The floor of the chamber is formed from the subcapitulum (Figure 28, 30d). This chamber is also sealed by modifications of the cheliceral digits. The fixed digits press together tightly over the top of the chamber

(Figure 27, 30d), and the movable digits neatly interlock with the edge of the subcapitulum (Figure 28, 30d). Therefore, the cheliceral digits form part of the boundary of the chamber. The hermetic seal of the chamber may be compromised while the mite contracts its movable digits in order to ‘bite’ into food. But if this is the case, the movable digits would return to a position that would ensure a good hermetic seal before the expansion of the pharynx, which would draw fluid through the puncture formed by the cheliceral digits. The rutella, which abut the cheliceral sheath (Figure 28), appear to have the function of holding the sheath in position. The rutella may also play an important role in preventing the sheath from being drawn inwards by the suction that accompanies the expansion of the pharynx. This would require some form of adhesion between the sheath and the tips of the rutella.

The pad at the front of the subcapitulum is likely to be involved in the formation of a hermetic seal around any puncture that is made by the chelicerae through the wall of a food organism. The soft, fleshy rim of the pad (Figure 27, 28) should provide an effective seal. Other acariform mites also use soft, membranous modifications near the tip of the subcapitulum to form a hermetic seal around punctures. For example, the

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Tetranychidae use a soft, flap-like extension of the rostrum to form this seal (Hislop &

Jeppson, 1976; Razaq et al., 2000; Beard et al., 2012). In Cunliffea, the outward flaring at the front of the cheliceral sheath provides the opening of the preoral cavity with a fleshy, lip-like quality (Figure 27), which would further aid the formation of the seal. The tip of the subcapitulum also flares outwards in some other fluid-feeding mites, which are likely to use this convergent feature for the same function (e.g. Figure 77B, C Allothrombium fulginosum in Alberti & Coons, 1999). These flares are always anteriorly flattened, which provides a good and even contact with the smooth surface around the puncture.

A distinct mode of gnathosomal integration from the Trombidiformes

In the Trombidiformes, gnathosomal integration has arisen via the loss of digital symmetry, i.e., the opposing digits of the chelicerae have been modified so that they no longer resemble one another in shape and/or size. Only the movable digits have become completely styliform. The movable digits protract and contract via muscles attached to the shafts. The shafts have sometimes fused with one another and with the subcapitulum, resulting in a gnathosomal capsule (de Lillo et al., 2001; Nuzzaci et al., 2002; Di Palma et al., 2009). Because there is no attenuation to the shaft, the movable digits have retained a relatively ventral position. The movable digits have integrated with the subcapitulum via the formation of a channel or gutter through the main body of the subcapitulum. The movable digits run through the channel, which has relatively thick walls (Figure 30a–b).

The fixed digits have remained broad and either they form the roof of the preoral cavity

(Figure 30b) or they are much shorter than the movable digits. In the latter case, the

82 subcapitulum extends all the way around the movable digits, forming the floor, walls and roof of the preoral cavity (Figure 30a). In some or all cases, the styliform movable digits could have originated via the formation of a relatively shallow subcapitular groove, which may have initially only functioned as a guide for the movable digits as they attenuated. This groove could have gradually enlarged until the movable digits became completely enveloped within a deep channel.

Figure 30. Semischematic drawings that illustrate transverse and highly distal sections of the main different forms of piercing-sucking mouthparts in the Acariformes: (a) Cheyletidae (based on Figure 2 of Di Palma et al., 2009); (b) Tarsonemidae (based on Figure 4.5 of de Lillo et al., 2001); (c) Eriophyoidea (based on Figure 1.2.17 of Nuzzaci, G. & Alberti, 1996); (d) Cunliffea (based on LT-SEM and confocal images). Cheliceral digits = black; labrum = light grey; other styliform components = dark grey; subcapitulum = white with black outline; As = auxiliary stylet; Fd = fixed digit (in b the fixed digits have fused); Ig = infracapitular guide; Md = movable digit; Me = membranous extension of the subcapitulum; Pc = preoral cavity; Ru = rutellum.

In contrast to the Trombidiformes, both the Eriophyoidea and Cunliffea have retained their digital symmetry. They have both undergone gnathosomal integration via membranous extensions of the subcapitulum (Figure 30c–d). These membranous extensions extend upwards from the edge of the subcapitulum. In the case of Cunliffea,

83 the chelate form of chelicerae has been retained. Also, the subcapitular sheath is very rudimentary and only covers the space between the opposing cheliceral digits. In

Eriophyoidea, the entire chelicerae (shafts and both pairs of digits) have attenuated into stylets (Keifer, 1975; Nuzzaci, 1979; Nuzzaci & de Lillo, 1991; Lindquist, 1996a;

Lindquist, 1998; Nuzzaci & Alberti, 1996). The sheath of the Eriophyoidea is much more extensive and more or less completely envelopes the chelicerae and other stylets. One other distinction between Eriophyoidea and Cunliffea is that in the latter the malapophyses are tightly adjoined, whereas in former they have probably completely fused.

The origin of the stylet sheath from a protosheath for chelate chelicerae

The stylet-sheath of Eriophyoidea could have originated via a protosheath that closely resembles the cheliceral sheath of Cunliffea. Like Cunliffea, this protosheath may have comprised small, lateral flaps that covered the gap between the movable and fixed digits of chelate chelicerae (Figure 30d). Consequently, the area between all four of the digits was incorporated into a hermetically sealed chamber, thus extending the preoral cavity into the area in front of the labrum. Because the cheliceral digits form part of the wall of this chamber, the large-scale attenuation of the chelicerae into stylets would have compromised the hermetic seal unless the sheath expanded in a compensating way.

Accordingly, the sheath would have gradually expanded over the attenuating chelicerae until it almost completely enveloped them (the bases of the cheliceral shafts are often dorsally exposed in the Eriophyoidea). If, like Cunliffea, the rutella were originally

84 pressed up against the outside of the sheath, they may have migrated inwards until they were also enveloped by the sheath. They may have then been modified into the infracapitular guides.

One important implication of this new hypothesis is that gnathosomal integration could have initially arisen in the Tetrapodili, as it has done in Cunliffea, without any cheliceral attenuation. The small, robust and chelate digits of the Nematalycidae are poorly adapted for penetrating the relatively deep surfaces of vascular plants. Without any cheliceral attenuation, the earliest members of the Tetrapodili would have been equally ineffective at that task. Therefore, their protosheath may have originated as an adaptation for feeding on the fluid contents of thin walled organisms, such as, perhaps, fungi. In this early version, the function of the sheath would have been to form a hermetically sealed channel that led from a puncture, made by the cheliceral digits, to the part of the preoral cavity underlying the labrum. This mode of feeding could have been modified for feeding on higher plants via the gradual attenuation of the chelicerae.

Accordingly, the rudimentary version of one of the most distinctive features of the

Eriophyoidea, the stylet sheath, may have provided an evolutionary pathway to styliform chelicerae, and without involving the loss of digital symmetry. It may be that digital symmetry is an evolutionary constraint that the Trombidiformes overcame.

If the stylet sheath of the Eriophyoidea is derived from a sheath like that of

Cunliffea, the mouthparts of the Eriophyoidea have clearly undergone a number of dramatic modifications, some of which may have led to some important alterations to the precise function of the sheath. It has been hypothesized that the infracapitular guides and

85 the labrum are used to conduct the fluid contents of plant cells into the pharynx from inside a pierced cell (Nuzzaci & Alberti, 1996). If these stylets form an adequately sealed channel, the sheath would no longer be needed to seal the puncture made by the cheliceral stylets.

The precise phylogenetic relationship between the Eriophyoidea and the

Nematalycidae is not yet clear – they may be sister taxa, or the Eriophyoidea may be nested within the Nematalycidae. The results of the morphological component of a set of phylogenetic analyses (Chapter 6) place Cunliffea in a clade with Gordialycus and the

Eriophyoidea. Two potentially important synapomorphies may unite Cunliffea,

Gordialycus and the Eriophyoidea: 1) coxal fields I have fused; 2) the lateral claws have been lost from some or all of the leg tarsi. If it turns out that the Eriophyoidea is nested within the Nematalycidae, and it is also relatively closely related to Cunliffea, it is much more likely that the cheliceral sheath of Cunliffea is a true transitional form, i.e. a synapomorphy that was further modified into a large stylet sheath in the Tetrapodili.

Alternatively, the Eriophyoidea and the Nematalycidae may be sister taxa. This would mean that the pouch of Osperalycus (Bolton et al., 2015b; Chapter 4), the cheliceral sheath of Cunliffea, and the stylet sheath of the Eriophyoidea are different variants of a shared mode of gnathosomal integration, i.e. membranous extensions of the subcapitulum. This shared mode of gnathosomal integration would, therefore, appear to be a synapomorphy that originated before the evolution of styliform chelicerae. This is consistent with the hypothesis that the Eriophyoidea’s stylet sheath originated through a protosheath for chelate chelicerae.

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Conclusion

The cheliceral sheath of Cunliffea may resemble the protosheath of the earliest members of the Tetrapodili. A protosheath would have been preadapted for feeding on plants because it possesses a mode of feeding that involves the formation of a hermetic seal around a puncture site. Plant-feeding would have arisen through the modification of the chelate chelicerae into attenuated and non-biting chelicerae. At the same time the sheath would have expanded, preventing the hermetically sealed preoral cavity from being compromised by the attenuation of the chelicerae.

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Chapter 6: Eriophyoidea and Nematalycidae form a clade

Introduction

The Nematalycidae have been placed in a basal, paraphyletic lineage –

Endeostigmata – within the order Sarcoptiformes (Kethley, 1989; Lindquist et al., 2009;

Walter et al., 2011), which is one of the two orders of the superorder Acariformes; the other order is Trombidiformes. There is some evidence to support the placement of the

Nematalycidae within a basal lineage of the Acariformes (Pepato and Klimov, 2015).

Whereas phylogenetic relationships among some of the more derived lineages of the

Acariformes have become clearer (OConnor, 1984; Maraun et al., 2004; Domes et al.,

2007; Bochkov et al., 2008; Klimov and OConnor, 2008), relationships among the basal lineages have been largely neglected. For example, Dabert et al. (2010) only included five different genera of the “Endeostigmata” in their study of the phylogeny of the

Acariformes. A very recent study on the Acariformes phylogeny, using whole mitochondrial genomes, fails to include a single taxon that is considered to belong within the Endeostigmata (Xue et al., 2016).

However, the phylogenetic relationships of the Trombidiformes, the largest order of the Acariformes, have also been relatively neglected, and the positions of some of the lineages of the Trombidiformes are in flux. For example, the results of a recent

88 phylogenetic study indicate that the Eupodina (Eupodes) is an artificial group (Pepato and

Klimov, 2015). Notably, the Eriophyoidea, which was placed in the Eupodina relatively recently (Nuzzaci & de Lillo, 1991; Norton et al., 1993; Lindquist, 1996b; Lindquist et al., 2009; Zhang et al., 2011), were not included in this study. The phylogenetic position of the Eriophyoidea is of particular interest to this chapter on the phylogenetic position of the Nematalycidae because the Eriophyoidea share a number of unusual features with the

Nematalycidae, including an elongated body that is lined with annuli. But similarities between these two taxa have generally been considered to be the result of convergence

(Lindquist, 1996b).

The phylogenetic position of the Eriophyoidea is of general interest because of their hyperdiversity and economic importance as a plant feeding lineage (Lindquist et al.,

1996). This group contains the largest family of mites, Eriophyidae (>3,500 species).

New species of this family are being described at a much faster rate than any other family of mites (Liu et al., 2013), and known species represent only a very small fraction of the total number (Amrine & Stasny, 1994; Amrine et al., 2003). Their species richness is explained, at least in part, by their high level of host specificity – most species feed on only one species (Skoracka et al., 2010). Their species richness is further promoted by a large range of possible hosts. They feed on all vascular plants, which comprise approximately 300,000 species. Therefore, the total number of species of the

Eriophyoidea may number in the hundreds of thousands. The economic importance of the

Eriophyoidea, as plant feeding mites, is compounded by their ability to transmit a range of different viruses to their hosts (Oldfield & Proeseler, 1996).

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Although we are beginning to resolve phylogenetic relationships within the

Eriophyoidea (Chetverikov et al., 2015; Li et al., 2014; Lewandowski et al., 2014), the phylogenetic position of the Eriophyoidea, as a group, has received less attention. Over the past sixty or so years, the Eriophyoidea has been widely regarded as belonging within the Trombidiformes (Baker & Wharton, 1952), and more specifically within the

Prostigmata (Evans, 1992; Lindquist, 1996b; Lindquist et al., 2009; Zhang et al., 2011).

However, some aberrant features of the Eriophyoidea provide a basis for doubting this placement. They lack prosomal stigmata, and both the movable and fixed digits (chelae) of the Eriophyoidea’s chelicerae are styliform (Keifer, 1975; Nuzzaci, 1979; Nuzzaci & de Lillo, 1991; Lindquist, 1996a; Nuzzaci & Alberti, 1996). In other Trombidiformes, evolutionary transitions towards styliform chelicerae only involve the dramatic attenuation of the movable digits. This presents a potential obstacle to any explanation for the evolution of eriophyoid mouthparts from those of the Trombidiformes (Lindquist,

1998). A placement within the Trombidiformes has also been undermined by a recent study using the whole mitochondrial genomes, which places Eriophyoidea as the sister group to the rest of the Acariformes (Xue et al., 2016). However, this study suffers from a severe lack of taxonomic sampling, and it is therefore unclear whether any other mite taxa, including supposed members of the Trombidiformes, belong with the Eriophyoidea at the base of the Acariformes.

In addition to the attenuation of the complete chelicerae, the mouthparts of the

Eriophyoidea include another unusual apomorphy – the cheliceral stylets are enveloped by a sheath (Nuzzaci & de Lillo, 1991). This sheath is formed from membranous

90 extensions of the subcapitulum. In some species of the Nematalycidae the cheliceral digits are also covered by highly modified, membranous extensions of the subcapitulum

(Bolton et al. 2015b; Chapter 4; Chapter 5). In particular, Cunliffea has a rudimentary sheath that somewhat resembles the stylet sheath of the Eriophyoidea (Chapter 5).

Until relatively recently, the Nematalycidae were widely thought to be within or closely related to the Tydeoidea (Cunliffe, 1956; Wainstein, 1965; Krantz, 1970; Kethley,

1982; Evans, 1992), and the Eriophyoidea were not yet regarded as close relatives of the

Tydeoidea or the Nematalycidae (Krantz, 1970; Krantz & Lindquist, 1979; Evans, 1992).

But by the time that the Tydeoidea were considered to be the probable sister group of the

Eriophyoidea (Nuzzaci & de Lillo, 1991; Norton et al., 1993; Lindquist, 1996b), the

Nematalycidae had already been informally relocated to the Endeostigmata in light of the discovery of Proteonematalycus (Kethley, 1989). Consequently, nobody has ever proposed a close relationship between the Eriophyoidea and the Nematalycidae.

The resemblance between the mouthparts of the Nematalycidae and the Eriophyoidea led to the latter’s inclusion in a phylogenetic investigation, the subject of this final chapter. The primary goal of this investigation is to determine the phylogenetic position of the Nematalycidae.

Method

Taxon selection

A total of 48 taxa were selected for the analyses. Ingroup taxa include representatives of all groups of Endeostigmata, a variety of Eriophyoidea (all families), 91 and representatives of most recognized lineages of Trombidiformes. More derivative lineages of the Trombidiformes (e.g. Heterostigmata and Parasitengona) were excluded because of their greater phylogenetic certainty. Sampling of Oribatida s.l. was much sparser (2 basal taxa) because none of the target groups has ever been associated with an oribatid lineage. A solifugid and a ricinuleid – Mummucia ibirapemussu Carvalho et al. and Cryptocellus iaci Tourinho et al. – were selected for the outgroup taxa (both

Solifugae and Ricinulei come out relatively close to the Acariformes in a recent and unpublished molecular phylogenetic analysis (Pavel Klimov pers. comm.)). Solifugae has also commonly come out as the sister taxon to the Acariformes in nuclear rDNA based analyses (Dabert et al., 2010; Pepato et al., 2010; Pepato and Klimov, 2015).

Character selection and scoring

A total of 112 characters were selected for the analyses (throughout the text, specific characters are abbreviated to ch. #). We included as many characters as possible that could be homologized confidently across taxa. Characters lacking a good basis for homology, e.g. setal counts, were excluded (Appendix C). Individual sensilla (setae, solenidia) were included as separate characters if, and only if, they could be homologized across taxa with a good degree of confidence. Finally, a number of characters have been used to support a sister group relationship between the Tydeoidea and the Eriophyoidea

(Lindquist, 1996b). An attempt was made to include as many of these characters as possible. A decision to exclude a few was again based on difficulties associated with the homology of those characters across the taxa (Appendix C).

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Data on morphological characters were obtained by combining examination of specimens (Appendix A) and study of descriptions. Specimens were observed using a compound microscope equipped with phase contrast (Zeiss Axioskop 3) and/or differential interference contrast (Nikon eclipse 90i).

Three of the morphological characters are ratios (ch. 1-3). These characters, which capture basic information about body shape and the relative distance between certain structures, are ratios that are based on two of the four following measurements: 1) idiosomal length – measurement along the midline (including anus); 2) maximal idiosomal width; 3) distance between the anal opening (most anterior point along the venter) and genital opening (most posterior point) – measurement along the midline; 4) minimum distance between coxae I. Measurements were made with the NIS-Elements

BR software, version 3.22.01 (Nikon). Measurements were only taken from adult females. In cases where there were no specimens available for measurements, data were obtained from measurements of figures from the descriptions. The ratios were log transformed and then discretized into integers from 0 to 9 in accordance with Thiele

(1993). The resulting characters were treated as ordered. All other multistate characters were treated as unordered (Appendix C).

Maximum parsimony analyses of morphological data

Maximum parsimony analyses were undertaken using PAUP, version 4.0a146

(Swofford, 2002). For all of the analyses, stepwise addition was set to random, and a tree- bisection-reconnection branch swapping algorithm was applied (reconnection limit = 8).

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A heuristic search of the complete morphological dataset was undertaken using the following settings: all characters unweighted; stepwise addition = 500 reps; the maximum number of saved trees = limitless. A strict consensus tree was generated from the optimal trees, which was used to interpret the results. Therefore, unless otherwise stated, the results section highlights congruent relationships from across all optimal trees. A phylogram was randomly selected for Figure 31 from the pool of optimal trees in order to represent the relative number of apomorphies among branches. The strict consensus tree was used to determine synapomorphies and their associated consistency (CI), retention

(RI) and rescaled consistency (RC) indices (Table 6).

Using the same settings as the original heuristic search, Bremer support was determined through searches for trees that are suboptimal, and through searches in which constraints were imposed against monophyly. Bootstrap support analyses were undertaken using the following settings: number of bootstrap replicates = 5000; stepwise addition = 1 rep; the maximum number of saved trees = 1000. An additional bootstrap support analysis was undertaken using the same settings, but with characters scaled for equal weighting. Under this scheme the three Thiele converted ratio characters (ch. 1-3), which each comprise 10 character states, are dramatically deemphasized. This additional analysis was undertaken in order to determine if these three ratio characters drown out conflicting signal from other characters. Bootstrap was chosen over jackknife because scaling for equal weighting creates non-integer values that are not compatible with jackknife analysis in PAUP.

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A number of additional heuristic searches (settings were identical to the unconstrained search using unweighted characters) were undertaken using constraint schemes: 1) Eriophyoidea, Nematalycidae and Trombidiformes together monophyletic;

2) Eriophyoidea and Trombidiformes together monophyletic; 3) Eriophyoidea and

Rhaphignathina together monophyletic; 4) Eriophyoidea and Tydeoidea together monophyletic; 5) Nematalycidae and Proteonematalycus together monophyletic; 6)

Nematalycidae and Micropsammus together monophyletic; 7) Proteonematalycus and

Micropsammus together monophyletic, and in a sister relationship with the

Nematalycidae. All of these constraints are consistent with current or previously held views on phylogeny (Kethley, 1989; Evans, 1992; Lindquist, 1996b; Lindquist et al.,

2009). The constraints that pertain to Proteonematalycus and Micropsammus were included in order to address the feasibility of three different relationships of the

Nematalycidae that are all consistent with the Nematalycoidea as hypothesized by

Kethley (1989), although it is worth noting that a close relationship between

Nematalycidae and Micropsammidae has already been undermined by the results of the phylogenetic analysis undertaken by Pepato and Klimov (2015). The constraint for the monophyly of the Eriophyoidea and the Rhaphignathina is consistent with the older and now widely disregarded hypothesis that the Eriophyoidea belong in the Raphignathina

(Krantz & Lindquist, 1979).

The Eriophyoidea-Nematalycidae clade is abbreviated to E-N throughout the results, discussion and conclusion.

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Results

The heuristic search using the unweighted scheme generated 42 minimum length trees (tree length, 531 steps; consistency index =0.28; retention index =0.67; rescaled consistency index =0.19). Six nodes have Bremer support >3 and bootstrap support >50%

(Figure 31). The Sarcoptiformes are paraphyletic with respect to Trombidiformes. Almost all of the fluid feeders within the Acariformes (but not Proterorhagia) unite into a large clade: Trombidiformes; Alycidae; Nanorchestidae, Nematalycidae and Eriophyoidea.

Another large clade was recovered that includes all of the Acariformes except

Proterorhagia oztotloica Lindquist & Palacios-Vargas. And one of the two species of the

Oribatida came out as the sister taxon to the rest of Acariformes (not including

Proterorhagia). However, all basal relationships (very large clades) are weakly supported

(Bremer<3; bootstrap <50%).

Some traditional taxa were recovered with strong support (Bremer >3; bootstrap

>50%), namely Raphignathina, Nanorchestidae and Eriophyoidea. However, the heuristic search did not recover Eupodina (Eupodides) as a clade. Some of the commonly accepted members of Eupodina, i.e. the Tydeoidea, form a grade with respect to the

Raphignathina, and Eriophyoidea falls outside of the Trombidiformes. Aside from these features, the structure and composition of the Trombidiformes largely accords with current classification schemes (Lindquist et al., 2009; Zhang et al., 2011). Notably, the

Sphaerolichida (Hybalicus and Sphaerolicha) comes out of the base of the

Trombidiformes. However, support for a Trombidiformes clade that includes the 96

Sphaerolichida is very weak (Bremer =1; bootstrap <50%), and a clade that contains the remainder of the Trombidiformes (the Prostigmata) only has slightly stronger support

(Bremer =2; bootstrap <50%).

The Eriophyoidea was found nested within an exclusive clade with the

Nematalycidae. A larger clade unites Eriophyoidea-Nematalycidae (E-N) with

Nanorchestidae, although support for this clade is not as strong (Bremer =4; bootstrap

<50%). By contrast, E-N is one of the strongest supported clades across the whole tree:

Bremer =9; bootstrap =84%. With respect to the Bremer value, the only other clade to exceed that degree of support is the Eriophyoidea (Bremer =11). Alternative hypotheses are poorly supported. There is a cost of at least 12 additional steps for all of the constraints that do not allow for an E-N clade (constraints 2 to 7, Table 5). When the

Eriophyoidea are constrained to be monophyletic with the Tydeoidea, there is a cost of 15 additional steps. And constraints that are consistent with the Nematalycoidea (Kethley,

1989) cost at least 18 steps. When the tree has been constrained so that the

Nematalycidae, the Eriophyoidea and the Trombidiformes together form a clade, 5 additional steps are needed. Under this constraint, the Eriophyoidea and the

Nematalycidae form an exclusive clade within the Trombidiformes.

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Figure 31. Phylogram (random) from a heuristic search (unweighted characters). The light gray branches of the tree collapse under strict consensus (Bremer = 0). Strongly supported nodes (Bremer >3; bootstrap >50%) are labelled as black opaque circles with associated values – Bremer above, bootstrap below. Moderately supported nodes (Bremer = 1 to 3; bootstrap >50%) are labelled with black circles with white backgrounds. All other nodes are weakly supported (bootstrap <50%). 98

The bootstrap support analysis in which the characters were scaled for equal weighting caused the support for E-N to drop from 84% to 70%. This is readily explained by the relative importance of the ratio characters to the unification of this group.

However, a support value of 70% is still relatively strong. Therefore, under an unweighted scheme, ratio characters do not appear to be drowning out conflicting signal from other characters.

Constraint Score of optimal tree(s) 1) (Eriophyoidea, Nematalycidae, Trombidiformes) 536 (+5) 2) (Eriophyoidea, Trombidiformes) 543 (+12) 3) (Eriophyoidea, Rhaphignathina) 545 (+14) 4) (Eriophyoidea, Tydeoidea) 546 (+15) 5) (Nematalycidae, Proteonematalycus) 550 (+19) 6) (Nematalycidae, Micropsammus) 550 (+19) 7) (Nematalycidae (Micropsammus, Proteonematalycus)) 549 (+18) Table 5. Scores of optimal trees in constrained analyses (heuristic analysis – characters unweighted). Numbers in brackets indicate the additional number of steps relative to the optimal unconstrained trees (length 531).

The phylograms show that Eriophyoidea is one of the most apomorphy rich lineages in the tree (Figure 31). A number of synapomorphies unite E-N (Table 6), and five unambiguous synapomorphies unite the Nanorchestidae, the Nematalycidae and the

Eriophyoidea: 1) absence of opisthosomal lyrifissures (ch. 26); 2) absence of solenidia from the palp tarsus (ch. 67); 3) v prime absent on trochanters II (ch. 80); 3) absence of solenidia from tibiae II (ch. 94); 4) absence of solenidia from genua I (ch. 97); 5) absence of solenidia from genua II (ch. 98).

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Synapomorphy CI RI RC Idiosomal elongation = 2 to 9 (1) 0.31 0.64 0.20 Relative distance between anus and genitalia = 5 to 9 (2) 0.27 0.72 0.19 Relative distance between coxal fields I = 0 to 1 (3) (R?) 0.20 0.61 0.12 Annuli present (6) 1.00 1.00 1.00 Naso absent (9) (R) * 0.11 0.64 0.07 Unpaired vi seta (16) 0.33 0.75 0.25 Females lacking eugenital setae (49) 0.11 0.53 0.06 Palp trochanter fused with palp femur (62) (R) 0.25 0.63 0.16 Palp femur fused with palp genu (63) 0.17 0.75 0.13 Table 6. Unambiguous synapomorphies that unite the E-N clade. First column: character number in parentheses. The remaining columns are values associated with the strict consensus tree: CI = consistency index; RI = retention index; RC = rescaled consistency index. Note that the values associated with idiosomal elongation and the distance between the anus and genitalia (left column) refer to discretized ratios (Thiele, 1993) that range from 0 to 9. (R) indicates that the trait has reversed to the plesiomorphic state among some members of the clade. *The naso may have re-evolved in some of the Eriophyoidea, but it is not obvious that this structure is truly homologous with the naso found in other mites.

Discussion

Nematalycidae and Eriophyoidea

The results do not support the widely held view that similarities between the unusual morphologies of the Eriophyoidea and the Nematalycidae are the result of convergent evolution. High support values from Bremer (9) and bootstrap analyses (84%) indicate that these two taxa appear to belong within the same monophyletic clade. All of the constraints that cause E-N to be pulled apart cost at least 12 extra steps. Furthermore, some of the syanpomorphies that unite E-N are very distinct and unusual: 1) an elongated idiosoma (ch. 1) lined with many annuli (ch. 6), which allows the body to extend and contract; 2) a very large relative distance between the anus and genitalia (ch. 2); 3) vi, on

100 the prodorsum, is unpaired if present (ch. 16); 4) the use of the anal valves (modified into lobes in Eriophyoidea) to anchor the idiosoma in place (Baker et al., 1987; Lindquist,

1996a; Bolton et al., 2015a; Chapter 3).

The results of the analyses do not support a very close relationship between the

Nematalycidae and either Proteonematalycus or Micropsammus. Constraining the tree search so that the Nematalycidae forms one of the possible relationships that are consistent with Nematalycoidea (Kethley, 1989) costs at least 18 additional steps (Table

5). And the constraint that places either Proteonematalycus or Micropsammus in an exclusive clade with the Nematalycidae was extremely costly, requiring 19 additional steps (Table 5).

Although this result is not in accordance with recent classification schemes, it was not that unexpected. Compared to the Eriophyoidea, both Proteonematalycus and

Micropsammus share few features with the Nematalycidae. They lack annuli (ch. 6) – their opisthosomal striations are predominantly longitudinal, which prevents the extension or contraction of their bodies. Furthermore, they have a genital region that is near to their anus (ch. 2), and they have a prodorsum that has either a complete or near- complete set of setae (ch. 15-22). They also have opisthosomal lyrifissures (ch. 26) and at least one post-podosomal constriction (ch. 34), which are features that are completely absent from the Nematalycidae and the Eriophyoidea. Perhaps most importantly,

Proteonematalycus and Micropsammus are particulate feeding mites (ch. 50) like almost all of the other Sarcoptiformes, whereas Nematalycidae and Eriophyoidea are fluid feeding mites. This is significant because the transition to particulate feeding is clearly a

101 rare event. Throughout the the Arachnida – particulate feeding is only found in the

Opiliones, some solifugids, and some Acari (Muma, 1966; Walter, 1988; Hillyard and

Sankey, 1989; Walter and Proctor, 1998; Acosta and Machado, 2007; Heethoff and

Norton, 2009).

Lindquist based his argumentation for a sister relationship between the Tydeoidea and the Eriophyoidea on a relatively large number of different characters (Lindquist,

1996b). Despite the inclusion of the majority of these characters in the analyses, the results do not support a close relationship between the two taxa. Furthermore, constraining the tree search so that these taxa always form a clade requires 15 additional steps (Table 5). With the exception of the constraints that are compatible with the

Nematalycoidea (Kethley, 1989), no constraint was as costly. Almost all of the characters that were highlighted by Lindquist (1996b), but not included in the analyses, do not show a greater resemblance between Eriophyoidea and Tydeoidea than between Eriophyoidea and Nematalycidae (Appendix C – section on Tydeoidea and Eriophyoidea).

The Tydeoidea and the Eriophyoidea are clearly very different with respect to body proportions. A number of similarities between these taxa, which include cheliceral attenuation (ch. 55-57) and reduced segmental anamorphosis (ch. 43), are likely to be the result of convergence. Sexually dimorphic suppression of eugenital setae has been used as an argument for linking the Eriophyoidea and the Tydeoidea as sister groups

(Lindquist, 1996b), but this feature is also present in the Nematalycidae (Appendix C – section on Tydeoidea and Eriophyoidea). Indeed, the absence of eugenital setae in the females is one of the synapomorphies that unites E-N (Table 6).

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Two other features, fusion of palp segments (ch. 62-65) and the small size of the gap between coxae I (ch. 3), have also been suggested as possible evidence of a sister relationship between the Eriophyoidea and Tydeoidea (Lindquist, 1996b). But with respect to these features there is a much greater similarity between the Nematalycidae and the Eriophyoidea. In both of these taxa, coxae I (ch. 3) are adjoined or extremely close together (character state = 0 to 3). In the Tydeoidea, this character is usually much higher and never below 2. With regard to the palps, the palp trochanter (ch. 62) of the

Eriophyoidea and the Nematalycidae is usually fused with the palp femur. This is never the case in the Tydeoidea.

These results agree with unpublished molecular data (5 genes) generated by Pavel

Klimov, University of Michigan, which show greater support for E-N than a clade that unites Tydeoidea and Eriophyoidea.

The Eriophyoidea are a very strongly supported monophyletic group; bootstrap values are 99% and bremer support is 11. In contrast, the monophyly of Nematalycidae has no support. In all of the optimal trees from the main heuristic search, the

Nematalycidae is paraphyletic with respect to the Eriophyoidea; Gordialycus is the sister taxon to Eriophyoidea. But support for a clade that includes Eriophyoidea and only

Gordialycus is not strong (Bremer = 2; bootstrap =<50%).

Paraphyly would mean that the Nematalycidae is even more ancient than the

Tetrapodili (comprising Eriophyoidea and extinct eriophyoid-like mites), which have been recovered as fossils from the Triassic (c. 230 Ma) (Schmidt et al., 2012; Sidorchuk et al., 2014). The highly elongated body and unusual modes of locomotion (Bolton et al.,

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2015a; Chapter 3) of the Nematalycidae may therefore represent a very early departure from the primitive body form and mode of locomotion of the Acariformes. A possible sister relationship between the Eriophyoidea and Gordialycus would suggest that the dramatic reduction of the rear legs in the latter may be a synapomorphy that the

Eriophyoidea took to completion with the total loss of those legs. It would also mean that the loss of claw-like empodia (ch. 74) is a synapomorphy that unites Gordialycus and the

Eriophyoidea.

The phylogenetic position of the Eriophyoidea-Nematalycidae clade

E-N falls outside of the Trombidiformes, and into a clade with the Nanorchestidae

(Figure 31), although support for that clade does not have very strong support (Bremer =

4; bootstrap <50%). Constraining E-N to be part of the Trombidiformes requires 5 additional steps (Table 5). Additional evidence against the placement of E-N in the

Trombidiformes comes from the results of Pepato and Klimov (2015), which places the

Nematalycidae in an early derivative position that is distantly removed from the

Trombidiformes.

There are also particular morphological characters that would appear to undermine any case for the placement of E-N within the Trombidiformes (Walter et al.,

2009). Both the Eriophyoidea and the Nematalycidae lack prosomal stigmata (ch. 68), and the Nematalycidae possess rutella (ch. 51). Modified rutella may also be present in the Eriophyoidea, possibly as one pair of the auxiliary elements in the styliform bundle

(Lindquist, 1998; Appendix C).

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The placement of E-N with Nanorchestidae, albeit weakly supported, could possibly be interpreted as evidence for a relatively basal position within the Acariformes

– Nanorchestidae is a long accepted member of the Endeostigmata. However, the results also suggest E-N occupies a relatively derived position within the Endeostigmata (Figure

31), and that E-N and Nanorchestidae together form a clade of equal rank to the

Trombidiformes. But support for these relationships is far too weak to settle the position of E-N.

Evolutionary implications for the Eriophyoidea

The placement of E-N obviously has important implications with respect to our understanding of the evolution of the Eriophyoidea. The Eriophyoidea was placed in the

Trombidiformes because they share many of the same apomorphies as some of the

Trombidiformes, including the loss of nymphal stages (ch 4, but see Appendix C), setal losses from the opisthosoma (ch. 37-40), reduced segmental anamorphosis (ch. 43), cheliceral attenuation (ch. 55-57) and phytophagy on vascular plants. In light of E-N’s probable placement outside of the Trombidiformes, all of the Eriophyoidea’s apomorphies now appear to be the result of convergent evolution. Although this may seem unexpected, lineages that are independently rich in apomorphies can end up sharing a number of apomorphies as an effect of chance (long-branch attraction). This is compounded by the tendency of many apomorphies to evolve in a single direction. Such is the case with characters that pertain to ontogeny. For example, early derivative acariform mites commonly have the complete set of segmental remnants (ch. 43),

105 whereas highly apomorphy rich lineages such as the Astigmata and the Heterostigmata have independently lost segmental remnants. In this respect, the Eriophyoidea are no different.

Cheliceral attenuation (ch. 55-57) is an apomorphy that has proceeded very differently between E-N and the Trombidiformes. In the Eriophyoidea, the whole of the chelicerae, including the shafts (ch. 55) and fixed digits (ch. 56), have become styliform, whereas only the movable digits (ch. 57) are styliform in the Trombidiformes (Lindquist,

1998). Therefore, cheliceral attenuation is neither truly convergent nor synapomorphic; it manifests in two very different ways.

The Eriophyoidea and the Nematalycidae clearly share many characters in addition to their unusually vermiform bodies. Indeed, other acarologists have long been aware of the similarities between these taxa. Keiffer (1975) suggested that the disappearance of the rear pair of legs of the Eriophyoidea may be attributed to the anterior movement of the genitalia that is also evident in the Nematalycidae (ch. 2), but this hypothesis was not used to suggest a close relationship between the two groups.

Given the strong resemblance between these taxa, it is surprising that a close relationship has not been seriously considered until now. The probable reason for this is that the

Nematalycidae have retained a number of plesiomorphies that are shared with other early derivative acariform mites, whereas the Eriophyoidea are rich in apomorphies (Figure

31). Perhaps most significantly, the Eriophyoidea live and feed on vascular plants, whereas the Nematalycidae are extremophiles that inhabit mineral regolith – deep soil and sand. The scale of this difference is brought into context by the Trombidiformes. In

106 this group, mites that feed on vascular plants are often closely related to mites that live, but do not feed, on vascular plants.

However, the many apomorphies of the Eriophyoidea can probably be attributed to the long period of time that is likely to have elapsed since they diverged from the

Nematalycidae. Fossil evidence indicates that Tetrapodili, which includes Eriophyoidea, dates back to at least the Triassic (c. 230 Ma) (Schmidt et al., 2012; Sidorchuk et al.,

2014). A placement outside of the Trombidiformes indicates that E-N may have an extremely ancient origin, as is the case with other early derivative members of the

Acariformes, which have fossils that date back to the Devonian (Hirst, 1923; Dubinin,

1962).

Large-scale modifications of the subcapitulum are a key component of the evolution of both the Eriophyoidea and the Nematalycidae. As highlighted in the introduction of this chapter, both of these lineages have a subcapitulum with membranous extensions. In the case of Osperalycus, a recently discovered genus (Bolton et al., 2014;

Chapter 2), the lateral lips have been modified into a pouch, which may be an adaptation for microbivory (Bolton et al., 2015b; Chapter 4). The chelicerae slide directly into this pouch when they are extended. In Cunliffea, the membranous extensions form a rudimentary sheath for the chelicerae (Chapter 5), whereas in the Eriophyoidea the sheath has expanded and the chelicerae are dramatically attenuated. In all of these cases, the edges of the subcapitulum have flared upwards and over the chelicerae, forming a pouch or a sheath. It may be that these are parallel modifications that represent variants of a more general evolutionary trend, within E-N, towards the integration of the chelicerae

107 with the subcapitulum. Alternatively, it is possible that the different modifications of the subcapitulum (pouch, sheath, etc.) have been coopted among these groups following gnathosomal integration, which could have happened before the divergence of the

Eriophyoidea and the Nematalycidae.

The new phylogenetic placement of the Eriophyoidea can also explain features of their mouthparts that were previously problematical when they were treated as trombidiform mites. Although the Eriophyoidea have styliform chelicerae, the fixed digits are not reduced (ch. 54). Lindquist (1998) suggested that this was a potential obstacle to the placement of the Eriophyoidea within the Trombidiformes. And unlike the

Trombidiformes, the rutella (ch. 51) have been retained by some or all members of E-N.

These structures are clearly present in the Nematalycidae (Bolton et al., 2015b; Chapter

4), and may also be present as one pair of the additional stylets of the Eriophyoidea, possibly the infracapitular guides (Lindquist, 1998).

Conclusion

There is strong support for the placement of the Nematalycidae with the

Eriophyoidea. This does not mean that the Nematalycidae belongs to the

Trombidiformes, which was a widely supported hypothesis for some time (Cunliffe,

1956; Wainstein, 1965; Krantz, 1970; Kethley, 1982; Evans, 1992). Instead, E-N probably falls outside of the Trombidiformes, although it is not yet clear where E-N belongs among the basal lineages of the Acariformes. Indeed, E-N may form one of the major lineages of the Acariformes. This is consistent with the recent discovery of fossils 108 that reveal the Eriophyoidea is very ancient (c. 230 Ma) (Schmidt et al., 2012; Sidorchuk et al., 2014). If the Eriophyoidea are nested within the Nematalycidae, E-N is almost certain to date back to the Paleozoic.

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Chapter 7: Conclusion

The Nematalycidae may represent a major lineage of the Acariformes.

Traditionally, the Nematalycidae have been ranked as a family within the suborder

Endeostigmata (Walter, 2009; Walter et al., 2011). However, the evidence presented throughout this dissertation indicates that a different rank may be more appropriate.

Chapters 3 to 5 reveal that the Nematalycidae contains a very large amount of morphological disparity. Different genera have evolved completely different morphologies associated with very distinct modes of locomotion (Chapter 3) and feeding

(Chapters 4 & 5). This is not at all common at the level of family.

Moreover, the results of the phylogenetic chapter (Chapter 6) place the

Nematalycidae in paraphyly with respect to the Eriophyoidea. This could mean that the

Nematalycidae is extremely ancient. Fossils of the Eriophyoidea date back to the Triassic

(Schmidt et al., 2012; Sidorchuk et al., 2014). And fossils of the Nanorchestidae, which came out as the closest related clade to Nematalycidae-Eriophyoidea clade, suggest that both clades had already diverged by the beginning of the Devonian (410 mya) (Hirst,

1923; Dubinin, 1962).

The results of the phylogenetic analysis also indicate that Nematalycidae,

Nanorchestidae and Eriophyoidea combine to form a clade that is equal in rank to the

110 order Trombidiformes. If the Nematalycidae is extremely ancient and also contains the

Eriophyoidea, the current rank of family is obviously is in error. It would be unwise to lower the rank of the Eriophyoidea given that this lineage is both very ancient (Schmidt et al., 2012; Sidorchuk et al., 2014) and hyper-speciose (Liu et al., 2013; Amrine &

Stasny, 1994; Amrine et al., 2003). It may be more appropriate to rank Nematalycidae as a suborder. If Nematalycidae and Eriophyoidea proved to be sister taxa, a rank of infraorder or superfamily would be appropriate for each of these taxa.

111

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Wernz, J.G. & Krantz, G.W. 1976. Studies on the function of the tritosternum in selected Gamasida (Acari). Canadian Journal of Zoology 54, 202-213.

Xue, X.-F., Guo, J.-F., Dong, Y., Hong, X.-Y. & Shao, R. 2016. Mitochondrial genome evolution and tRNA truncation in Acariformes mites: new evidence from eriophyoid mites. Scientific reports 6, 18920.

Zhang, Z.-Q., Fan, Q.-H., Pesic, V., Smit, H., Bochkov, A.V., Khaustov, A.A., Baker, A., Wohltmann, A., Wen, T.-H., Amrine, J.W., Jr., Beron, P., Lin, J.-Z., Gabrys, G. & Husband, R. 2011. Order Trombidiformes Reuter, 1909. In: Zhang, Z-Q., editor. Animal

127 biodiversity: an outline of higher-level classification and survey of taxonomic richness. Zootaxa 3148, p. 129-138.

Zonstein, Z. 2003. The spider chelicerae: some problems of origin and evolution. In: Logunov, D.V. & Penney, D., editors. European Arachnology: Proceedings of the 21st European Colloquium of arachnology. St.-Petersburg, 4-9 August, p. 349-366.

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Appendix A: Specimens

Chapter 3: Material examined under LT-SEM

Osperalycus tenerphagus – U.S.A., Ohio, Franklin Co., Kinnear Road, 39.9990 N - 83.0468 W, silty clay loam from suburban prairie (including shrubs, grasses and small trees); collector: Samuel Bolton, August-2011, 50 cm deep: Protonymph×1; Deutonymph×1; Tritonymph×6; Adult×9.

Gordialycus sp. A – U.S.A., California, Imperial County, Algodones Dunes, Imperial Sand Dunes Recreation Area, 32.9811 N -115.1317 W, bottom of sand dune; collector: Samuel Bolton, October-2013, 10 cm deep: larva×3; nymph/adult×7.

Gordialycus sp. B – U.S.A., Indiana, Lake County, Marquette Park, 41.6156 N -87.2743 W, top of sand dune; collector: Samuel Bolton, May-2013, 10 cm deep: nymph/adult×5.

Cunliffea strenzkei – U.S.A., Indiana, Lake County, Marquette Park, 41.6156 N -87.2743 W, top of sand dune; collector: Samuel Bolton, May-2013, 10 cm deep: nymph/adult×6. U.S.A., Florida, Highlands Co., Highlands Hammock Park, 27.4713 N 81.5646 W, sandy soil from sparsely wooded area; collector: Samuel Bolton, April-2011, 30 cm deep: tritonymph/adult ×2. cf. Psammolycus sp. A – U.S.A., Florida, Highlands Co., Highlands Hammock Park, 27.4713 N 81.5646 W, sandy soil from sparsely wooded area; collector: Samuel Bolton, April-2011, 30 cm deep: post-larva ×2. 129

Note that all adults were female.

Chapter 4: Material examined under LT-SEM

Osperalycus tenerphagus – U.S.A., Ohio, Franklin Co., Kinnear Road, 39.9990 N - 83.0468 W, silty clay loam from suburban prairie (including shrubs, grasses and small trees); collector: Samuel Bolton, August-2011, 50 cm deep: deutonymph/tritonymph×1, tritonymph×2, adult×7.

Gordialycus sp. A – U.S.A., California, Imperial County, Algodones Dunes, Imperial Sand Dunes Recreation Area, 32.9811 N -115.1317 W, bottom of sand dune; collector: Samuel Bolton, October-2013, 10 cm deep: larva×3; nymph/adult×5.

Gordialycus sp. B – U.S.A., Indiana, Lake County, Marquette Park, 41.6156 N -87.2743 W, top of sand dune; collector: Samuel Bolton, May-2013, 10 cm deep: nymph/adult×6.

Cunliffea strenzkei – U.S.A., Florida, Highlands Co., Highlands Hammock Park, 27.4713 N 81.5646 W, sandy soil from sparsely wooded area; collector: Samuel Bolton, April- 2011, 30 cm deep: tritonymph/adult ×2. cf. Psammolycus sp. A – U.S.A., Florida, Highlands Co., Highlands Hammock Park, 27.4713 N 81.5646 W, sandy soil from sparsely wooded area; collector: Samuel Bolton, April-2011, 30 cm deep: post-larva ×2.

Note that all adults were female.

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Chapter 5: Slide mounted voucher specimens of Cunliffea cf. strenzkei

LT-SEM – U.S.A., Indiana, Indiana Lake Co., Marquette Park, 41.6173 N 87.2712 W, sand dune (10 cm deep); collector: Samuel Bolton, 25 May, 2013. 1 adult (OSAL 0114171), 2 adults/nymphs (OSAL 0114172, 0114173).

Confocal – U.S.A., Florida, Highlands Co., Highlands Hammock Park, 27.4713 N 81.5646 W, sandy soil from sparsely wooded area (30 cm deep); collector: Samuel Bolton, April-2011, 1 adult (OSAL 0114118). U.S.A., Indiana, Indiana Lake Co., Marquette Park, 41.6159 N 87.2742 W, sand dune (10 cm deep); collector: Samuel Bolton, 25 May, 2013. 1 adult (OSAL 0114119).

Note that all adults were female.

Chapter 6: Slide mounted specimens used for the phylogenetic analyses

Metatydaeolus sp. – USA, Ohio, Hocking Co., Deep Woods Farm, 39.4088 N, 82.5766 W, loam, 20-30 cm deep; collector: Samuel Bolton, May, 2010, (SB10-0516-III), 1 female (OSAL 0116344).

Brachytydeus sp. – USA, Ohio, Fairfield Co., Carroll, 39.7986 N, 82.70709 W, ex under apple bark; collector: D. W. Davis, 8 April 1966, 1 female (OSAL 0065954).

Riccardoella triodopsis – U.S.A. Alabama, Lawrence Co., Bankhead National Forest, 34.1334 N, 87.2583 W, ex Triodopsis obstricta; collector J. Petranka, 27 Sep 1984 (HK 85-0205-2) 2 males, 1 larva (OSAL 0065202, 0065203, 0065204).

Triophtydeus sp. – USA, Ohio, Fairfield Co., Carroll, 39.7986 N, 82.70709 W, ex under apple bark; collector: D. W. Davis, 8 April 1966, 1 female (OSAL 0065963). 131

Proctotydaeus sp. – Mexico, Yucatan, 30 km north of Santa Elena, 20.3279 N, 89.64409 W, ex Schistocerca; collector: Mario Poot, 10 Sept 2009, 1 female (OSAL 0103884).

Stereotydeus sp. – unknown location; collector J. Robillard, 5 Sep 1968, 1 female (OSAL 0065522).

Claveupodes sp. – Italy, Tuscany, Florence, Boboli Gardens, 43.7627 N, 11.2481 E; collector: D. Wrensch, 2 Nov 1974, 1 female (OSAL 0065214).

Neoscirula reticulata – USA, Arkansas, Newton Co., Buffalo National River, Boen Gulf transect, 35°52.040 N 93°24.099W, ex litter old growth beech, flag 7; collectors: J.R. Fisher & M. J. Skvarla, 5 May 2010, 1 female (OSAL 0104763) (APGD 10-0506-011).

Trachymolgus purpureus – USA, Arkansas, Newton Co., Rock Bridge Creek, 2.5km N of Mount Sherman, N36.0566 N, 93.2570 W, ex litter in & near rotten stumps; collectors: W. C. & J. M. Welbourn, 20 July 1986, 1 protonymph (measurements based on description) (OSAL 0061852).

Apomerantzia kethleyi – USA, Ohio, Hocking Co., Deep Woods Farm, 39.4088 N, 82.5766 W, 20-30 cm deep; collector: S.J. Bolton, 1 May 2010, 1 female (OSAL 0116332).

Labidostomma sp. – Guatemala, El Progresso, Cerro Pinalón, 15.0847 N, 89.9499 W, ex: sifted leaf litter, cloud forest; collector: LLAMA, 02 May 2009, 1 male (measurements based on SEM images) (OSAL 0116345).

Gordialycus sp. A – USA, New Mexico, 45-60 cm deep; collector, J.B. Kethley, 1985 (FMHD 85-289), 1 female (OSAL 0116330).

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Gordialycus sp. B – USA, New Mexico; collector, J.B. Kethley, 1983 (FMHD 83-586), 1 female (OSAL 0116331).

Osperalycus tenerphagus – USA, Ohio, Franklin Co., Kinnear Road, 39.9990 N, 83.0468 W, silty clay loam from suburban prairie (including shrubs, grasses and small trees), 40 cm deep; collector: Samuel Bolton, May 2011, (SB11-05-I), 3 females (OSAL 015134, 0103239, 0105138).

Cunliffea cf. strenzkei – USA, Indiana, Lake Co., Marquette Park, 41.6175 N, 87.2711 W; collector, S.J. Bolton, 25 May 2013, 2 females (OSAL 0114124, 0116329). cf. Psammolycus sp. A – USA, Florida, Highlands Co., Highlands Hammock State Park, 27.4713 N 81.5646 W, sand, 30 to 40 cm deep; collector: S.J. Bolton, April 2011, 1 female (OSAL 0116328). cf. Psammolycus sp. B – USA, Indiana, Lake Co., Marquette Park, 41.6175 N, 87.2711 W; collector, S.J. Bolton, 25 May 2013, 1 female (OSAL 0114146).

Alycus cf. denasutatus – USA, Illinois, Carroll Co., Miss. Palisades St. Pk., 2 mi N Savanna, ravine litter with interrupted fern; collectors: J. Wagner & J. Kethley, 11 April 1983 (FMHD 83-61), 1 female, 1 male (OSAL 0116334, 0116395).

Pachygnathus sp. – USA, Nebraska, Washington Co., N. Fort Calhoun, Desoto Natl. Wildlife | Refuge, floodplain litter; collector: W. Suter, 15 May 1982 (FMHD 82-144), female (OSAL 0116337).

Bimichaelia nr. campylognatha – USA, Wisconsin, Kenosha Co., Silver Lake Bog, Sphagnum, u. Larix & poison sumac; collector: W. Suter, 28 April 1985 (FMHD 85-

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173), 1 female (OSAL 0116338).

Petralycus sp. – USA, Florida, Franklin Co., Ochlockonee Bay, algal drift near river; collector: W.S. Suter (FMHD 82-105), 27 March 1982, 1 female, 1 male (OSAL 0116335, 0116336).

Proteonematalycus wagneri – USA, Indiana, Indiana Dunes State Park, Marquette Park, sand, upper 10 cm; collector: J.B. Kethley, 14 May 1986 (FMHD 86-203), 1 female (OSAL 0116343).

Micropsammus sp. – 1) USA, Florida, Pinellas Co., Clearwater Beach Island, 27.9845 N, 82.8280 W, sand among marram grass, upper 10 cm; collector: S.J. Bolton, April 2011, 1 female, 2 males (OSAL 0116339, 0116340, 0116341). 2) USA, Florida, Archer; collector: J.B. Kethley, 1987 (FMHD 87-129), 1 female (OSAL 116342 ).

Oehserchestes humicolus (=spathatus) – unknown location and collector, 1985 (FMHD 85-7), 1 female (OSAL 0116333).

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Appendix B: Percent increase in mouthparts from larva to adult (Chapter 4)

Species Larva Adult Percent

n 푥̅ σ n 푥̅ σ increase Osperalycus tenerphagus 5 8.6 0.2 7 8.7 0.2 1

Gordialycus sp. C 4 20.6 1.1 4 24 1.5 19

Table 7. Distance from the base of the chelicera (excluding trochanter) to the dorsal seta of the fixed digit (µm).

Species Larva Adult Percent

n 푥̅ σ n 푥̅ σ increase Osperalycus tenerphagus 5 3.5 0.3 7 3.7 0.2 7 Gordialycus sp. C 4 4.0 0.1 4 4.6 0.2 14

Table 8. Distance between the dorsocentral seta on the palp-tarsus and the dorsocentral seta on the palp- tibia (µm).

Measurements were obtained from slide mounted specimens using a Zeiss Axioskop™ equipped with a phase contrast optical system. Osperalycus tenerphagus was collected by Samuel Bolton (2010 to 2011) from Ohio (USA), Franklin Co., Kinnear Road, 39.9990 N -83.0468 W. Gordialycus sp. C (two lateral claws on legs I and II and single lateral claws on legs III and IV) was collected by John Kethley (1985) from an unknown location. The Gordialycus sp. C specimens were borrowed from the Field Museum, Chicago, USA.

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Appendix C: Character coding scheme and rationale (Chapter 6)

General character treatment

Characters are selected on the basis of structures or setae that can be homologized with a reasonable degree of confidence. Ambiguous and problematical characters were, therefore, excluded. Counts of setae were also excluded because they are not based on the homology of specific structures; the same character state may comprise a different setal composition. Some of the idiosomal and all of the leg trochanteral setae were used as characters because their homologues can be identified with a reasonable degree of confidence. Many setae were excluded because across the combined taxa of the Acariformes, it is impossible to confidently determine their homology. Schemes that work fine at low levels of taxonomy (within and between genera and families) should not be assumed to readily translate to high levels of taxonomy (between orders and suborders). The early derivative lineages of the Acariformes, the focal group of this study, form a number of extremely ancient lineages that may each be as ancient as the orders Trombidiformes and Sarcoptiformes. When characters did not appear independent, due to identical patterns of expression, they were amalgamated into a single character. For example, the pattern of empodial expression (ch 73) is identical for legs II to IV across all of the taxa. Each of the legs is not, therefore, treated as a single character with respect to the presence/absence of empodia. A total of 102 characters, the greater majority, are binary. Ten characters are multistate. All of them are unordered except the three ratio characters, which were discretized into ten character states (Thiele, 1993). These should be treated as ordered because the integers (0-9) are arbitrary divisions along a continuum. The seven remaining 136 multistate characters are unordered because this forces no prior assumptions with respect to their transition between states. Some of these characters represent probable ontogenetic sequences, e.g. identity of the terminal segment (ch. 43), no. of genital papillae (ch. 48), fusion of femora (ch. 106-109), and therefore they could, in reality, be ordered. But their ontogenetic status also means that the switching off and on of key developmental pathways could allow the sudden loss or recovery of more than one stage along an ontogenetic sequence. For this reason, they were treated as unordered.

Body ratios (ch. 1 to 3 – multistate, ordered) Three body ratios were included: 1) the degree of elongation (ch. 1) (idiosomal length/width); 2) the proportion of the idiosomal length that is between the anus and genitalia (ch. 2); 3) the distance between coxae I relative to the maximal width (ch. 3). The first of these ratios represents the basic shape of the mite. The second of these ratios is largely indicative of the relative position of the genitalia along the body (the anus tends to remain in a highly posterior position). The third ratio was included because Lindquist has suggested that the relatively small gap between coxae I in the Ereynetidae may be an ancestral trait of the Tydeoidea, and might therefore be a synapomorphy that unites the Tydeoidea and Eriophyoidea (Lindquist, 1996b). In all of the Eriophyoidea that were included in the analysis, coxae I have consolidated (ch. 3). In the Ereynetidae (a family within the Tydeoidea) the gap between the coxae is often reduced. Coxae I therefore appear to be fairly close to a state of consolidation. A raw or absolute measurement of the gap between coxae I could mean that similarities are an artifact of having a small or narrow body. In this regard, any resemblance between the Nematalycidae and the Eriophyoidea would be inflated; both taxa have narrow bodies. Therefore, a ratio that used the coxa I gap size relative to the maximal width of the idiosoma was used in an attempt to exclude this effect. However, despite the use of this ratio, the Nematalycidae clearly bear a much closer resemblance to the Eriophyoidea. In the Nematalycidae, it is also quite common for coxae I to be fused into a single plate, whereas in the Tydeoidea, coxae I are always separate and often

137 relatively far apart. Of all the taxa that were included in the morphological character matrix, Triophtydeus sp., which falls within the Tydeoidea, had the largest gap of any species between coxae I relative to the body width.

Tritonymph stage (ch. 4 – binary) Among the taxa that were included in the analysis, the tritonymph is the only developmental stage that is sometimes suppressed. Therefore, the presence/absence of the other instars was excluded because they are uninformative. Although it was believed that the Eriophyoidea normally suppress the deutonymph and tritonymph, all three nymphal stages have been found in a species of Phytoptus following a detailed study of the development of the mite (Ozman, 2000). It is uncertain whether the deutonymph and tritonymph is present in some or perhaps even all of the Eriophyoidea. The tritonymph will obviously be present if all three stages are normally present. If only one additional nymph is normally present, it may be a deutonymph or tritonymph. Consequently, the presence of the tritonymph was coded as uncertain throughout the Eriophyoidea. The presence of the tritonymph is sometimes designated as unknown in other taxa when the number of specimens is deemed inadequate to rule out its presence. However, because the number of nymphal stages is often very stable across taxonomic groups, the presence or absence of this stage can be readily inferred from closely related taxa.

Sexual versus asexual (ch. 5 – binary) Males are unknown for a large proportion of the species included in this analysis. As with the detection of nymphal stages, this may be due to the relatively low numbers of available specimens of many species. Therefore, the status of those species was sometimes designated as unknown with regard to sexual reproduction. But in other cases a status was not designated as unknown when all known species from the same taxonomic group are either sexual or asexual, and when the few available specimens of the target species do not demonstrate otherwise.

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Integument (ch. 6 to 8 – binary) Three integumental characters were used in the analysis: annuli (ch. 6); integumental protrusions (ch. 7); reticulated regions (ch. 8). When present, these features comprise a large proportion of the idiosomal integument. Annuli (ch. 6) are ridges that run completely around the circumference of the opisthosoma. Integumental protrusions (ch. 7) include any projecting structures that are not striae, e.g. protubercles. Reticulation (ch. 8) is present when striae interlink to form a network across the integument; in most mites these striae run parallel but never meet.

Prodorsum (ch. 9 to 25) Non-setal characters (ch. 9 to 14 – binary): A number of different prodorsal characters were used for the analysis, including a naso (ch. 9, 11), a central eye (ch. 10), lateral eyes (ch. 12), and post-ocular bodies (ch. 14). The eyes of solifugids are probably homologous with the central eye (ch. 10) of the acariformes, which is indicated by their anteromedial position. A central eye was therefore coded as present in the single solifugid that was used for the outgroup. The Eriophyoidea also have eye-like structures that may be true eyes (Smith, 1977). These were therefore coded as unknown for the presence/absence of lateral eyes. Some mites have post-ocular bodies (ch. 14), which are eye-like structures posterior to the lateral eyes. These structures have sometimes been treated as an extra pair of eyes (e.g. Uusitalo, 2010a, b). However, they do not appear to be true eyes because they lack the smooth integumental covering that characterizes eye lenses. They were, therefore, not treated as an extra pair of eyes in the coding scheme. Based on their ultrastructure, the pustules of the Labidostommatidae are regarded as glands (Alberti & Coons, 1999), and are therefore very unlikely to be homologous with eyes or post-ocular bodies. This structure was excluded from the analysis because it is uninformative (it is an autapomorphy for the single labidostommatid that was included in the analysis). Setae (ch. 15 to 25 – binary): Kethley (1990) formulated a prodorsal scheme that homologized the setae of the Trombidiformes with the Sarcoptiformes. His scheme was

139 adopted for the phylogenetic analysis. Accordingly, exp (ch. 21) and in (ch. 22) are missing from the prodorsum in most trombidiform mites, and also in the Nematalycidae. The Eriophyoidea, which are widely regarded as prostigmatid mites, are also generally treated as missing exp and in setae (Lindquist, 1996a). We did not deviate from this convention. The Eriophyoidea are missing a pair of scapular setae (ch. 19, 20), although the exact seta (sce or sci) is not usually designated. We treated sce (ch. 20) as missing in the Eriophyoidea, but this choice would not have made any difference to the outcome of the analysis because both of the scapular setae are present in all of the other mites that were included in the analysis. However, if sce have been lost, the sci of the Eriophyoidea cannot be trichbothrial (ch. 25), whereas the trichbothrial status of sci would be unknown if sci were absent. For the sake of caution, the trichbothrial status of sci was treated as unknown in the Eriophyoidea (it is inapplicable in Rhynacus because both scapular setae are absent). A single deviation from Kethley’s scheme occurred in the setal coding of Labidostomma sp.; exp (ch. 21) and in (ch. 22) are treated as present. This is in accordance with Walter et al. (2009). This modification arose because exp, which was previously treated as opisthosomal, is anterior to the lateral ocellus and must therefore be prodorsal.

Opisthosoma (ch. 26 to 50) Lyrifissures (ch. 26 to 33 – binary): Many acariform mites bear a pair of lyrifissures (cupules) (ch. 26) on their segments. The complete set of lyrifissures is as follows: segment D–ia (ch. 27); E–im (ch. 28); F–ip (ch. 29); H–ih (ch. 30); PS–ips (ch. 31); AD–iad (ch. 32). The Proteonematalycidae and Micropsammidae also have an additional pair of lyrifissures on their genitalia (ch. 33). The presence/absence of each particular pair of lyrifissures is treated as a separate character. (ch. 27-33). Many mites have a complete or near complete set of lyrifissures, or else they tend to be completely absent. This suggests that the absence of any particular lyrifissures could be because the ability to express any lyrifissures has been completely

140 suppressed or lost. Therefore, the presence/absence of any lyrifissures is also used as an additional character (ch. 26), and the presence of any particular lyrifissure is treated as unknown for taxa in which lyrifissures are absent from all segments. The presence/absence of post-podosomal constriction (ch. 34, binary): Some mites have a constriction in the opisthosoma directly behind the podosoma. Setae on the segments (ch. 35 to 42, binary): Near the posterior region of the opisthosoma, many of the opisthosomal setae cannot be confidently homologized. For this reason, these setae were excluded from the analysis. But setae from segments C (ch. 35-38), D (ch. 39, 40) and E (ch. 41, 42) were used in the analysis because there are few enough to homologize. These setae have also been used because some of them have been lost by the Trombidiformes (OConnor, 1984). They are, therefore, important with respect to synapomorphies that unite that group. The internal setae from some of these segments are uninformative because they are universally present among taxa. They were, therefore, excluded from the analysis. The identity of the terminal segmental remnant of the adult stage (ch. 43 – multistate, unordered): This character comprises five states: 1) PS; 2) AD; 3) AN; 4) PA; 5) PA+1. The last of these states reflects the existence of an additional terminal body segment in almost all arachnids, and which is therefore needed for the outgroup taxa used in this analysis. The identity of the terminal segmental remnant is usually indicated by the associated setae – the anal shields represent the terminal segment, which may sometimes be nude. In the Eriophyoidea, no segmental remnants appear to be added after PS. The suppression of anamorphosis has been considered to be possible evidence of a close relationship between the Tydeoidea and Eriophyoidea (Lindquist, 1996b), but the Tydeoidea also appear to add segment AD even though setae ad and lyrifissures ips are absent (André, 1981; Kazmierski, 1989; Kethley, 1990). Structure and shape of opisthosomal setae on the dorsum (ch. 44 & 45 – binary): Setae from the dorsum of the opisthosoma were used for characters pertaining to setal shape and structure. This is because they are large and, therefore, it is easy to distinguish the different character states. Two characters were used: presence/absence of branching

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(Figure 32B, C) (ch. 44); shape of setal body (thin/swollen) (ch. 45). Any setae that possess setules (short terminal branches) are coded as branched (Figure 32D-G). In the case of Proctotydaeus, the lengths of the setules are highly reduced, causing the profile of the seta to look serrate (Figure 32E). In all other Tydeoids, the setules tend to be more prominent, although in some cases the form is very close to that of Proctotydaeus. Across the range of taxa studied, the length of terminal branches varies dramatically. However, the lengths of terminal branches do not fall into readily defined and discrete character states. Characters for branch length were therefore not developed for this analysis. The second setal character – shape of setal body (ch. 45) – excludes the setules. The setae of most mites have a thin setal body (Figure 32B-E). But in some mites the setal body is distinctly swollen (Figure 32F, G). Hypertrichy of the setae (ch. 46 – binary): Some mites have a dense covering of setae on the opisthosoma that is readily distinguishable from holotrichy. Presence of hypertrichy does not include mites that exceed the holotrichous state by merely a few setae. Genital papillae in the adult stage (ch. 47 – binary; ch. 48 multistate, unordered): As with other characters, if genital papillae are absent (ch. 47), the number of genital papillae (ch. 48), which varies from 1 to 3 pairs, is coded as unknown. Genital setae (ch. n/a): Genital and aggenital setae were not included because it was too difficult to confidently determine the homology of each seta. Furthermore, establishing which setae are genital or aggenital is also often difficult or based on somewhat arbitrary criteria. And in some cases an apparent aggenital seta has migrated to the extreme venter from the dorsum, and should instead be designated as a seta from a segment.

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Figure 32. Empodia and setae. Palp and setal claw of palp-tibia: A, Apomerantzia kethleyi Price (copied from Price, 1975). Setae: B, branched; C, simple; D, thin with long setules; E, thin with short setules; F, swollen - spatulate; G, swollen – globose; Empodia (figures exclude setules and claws): H, Elongated and straight – lateral view; I, Elongated and curved (claw-like) – lateral view; J, Bulbous – lateral view; K, Flat and rounded (pad-like) – dorsal view. S = solenidion; C = claw-like seta.

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Presence/absence of eugenital setae in females (ch. 49 – binary): Eugenital setae are the setae next to the opening of the genitalia, and can be readily distinguished from the genital and aggenital setae. The complete absence of these setae from females was hypothesized as a possible synapomorphy for the Nematalycoidea (Kethley, 1989). In Oribatida and Speleorchestes, the opening of the genitalia is at the tip of an ovipositor, which has setae that appear to be homologous with the eugenital setae of mites in which the ovipositor is vestigial or absent. Accordingly, the coding scheme treats these setae as eugenital. Gut boluses (ch. 50 – binary): A gut bolus can often be observed in the opisthosoma of particulate feeding mites. The results of Walter (1988) were used to help determine which genera contain gut boluses in cases where specimens are unavailable, i.e. where descriptions did not reveal this information. Observations of Proteonematalycus wagneri Kethley, which was not included in Walter (1988), showed that this species also often contains gut boluses. Species which are scored as ‘absent’ have never been shown to contain gut boluses, whereas species that are scored ‘present’ have boluses in 10% or more of specimens.

Gnathosoma (ch. 51 to 68) Presence/absence of rutella (ch. 51 – binary): The presence of rutella is usually relatively obvious. However, in several cases their presence is ambiguous. Although the Sphaerolichida are often regarded as lacking rutella (e.g. Walter et al., 2009), OConnor (1984) hypothesized that a pair of subcapitular setae on Hybalicus may be homologous with rutella. This was based on the position of the setae. To minimize ambiguity, ordinary setae that have no evidence of thickening are never coded as rutella, regardless of their position. Rutella are, therefore, treated as absent in all of the Trombidiformes. It is not clear if rutella are present or absent in the Eriophyoidea, which contains a number of mouthpart elements of unknown origin, including auxiliary and infracapitular stylets. It is possible that some of these structures are derived from the rutella (Lindquist,

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1998). However, there is no confident basis for homology. This character has therefore been coded as unknown in the Eriophyoidea. The single described species of Proterorhagiidae – Proterorhagia oztotloica Lindquist & Palacios-Vargas – appears to have very small, possibly vestigial pair of rutella (Lindquist & Palacios-Vargas, 1991). Therefore, this species was also coded as unknown for the presence/absence of rutella. Rutella shape and arrangement (ch. 52 & 53 – binary): Many mites have rutella with teeth. Others have lobes (Figure 33A) or long spine-like projections. These all represent different types of projections from the main body of the rutellum. They also represent forms that are part of a continuum, and it is therefore not always possible to confidently characterize a projection as one type or another. For example, teeth are very small lobes, and spine-like projections are long and attenuated lobes. Rutella often have highly complex shapes and may possess all three types of projections (Figure 33D). Homologizing each of these individual projections is probably not possible across disparate and distantly diverged taxa. Because of this, and because the characterization of shape is not robust, we used a simple shape-based character (ch. 52) for the presence or absence of multiple projections. Projections can include teeth, lobes and spines. Multiple projections include all rutella that branch into two or more projections. The setiform rutella of Nanorchestes were, therefore, coded for the presence of multiple projections (Figure 33C). Multiple projections are present in almost all mites that possess rutella. They are only absent in some Nematalycidae (Figure 33E). In most mites the rutella never touch, but in some of the Nematalycidae they meet at the midline (Figure 33B). This feature was therefore used as an additional character (ch. 53). Cheliceral shape (ch. 54 to 57 – binary): Four characters pertain to the shape of the chelicerae: 1) reduction of the fixed digits (ch. 54); 2) attenuation of the cheliceral shafts (ch. 55); 3) attenuation of the fixed digits (ch. 56); 4) attenuation of the movable digits (ch. 57). The first of these characters pertains to the dramatic reduction of the fixed digits in some trombidiform mites (Figure 33F). The other characters address the

145 styliform/substyliform chelicerae of some mites (Figure 33F, G). The Eriophyoidea have styliform shafts and digits (Figure 33G), whereas in the trombidiform mites only the movable digits are styliform (Figure 33F). In all of the other mites that were included in the morphological matrix, the chelicerae, including the digits, are comparatively robust and chelate (Figure 33H), and are therefore easy to distinguish from styliform mouthparts. Fusion of the cheliceral shafts (ch. 58 – binary): In some cases the chelicerae fuse along their cheliceral shafts. In the Tydeoidea and the Rhaphignathina, where this feature is sometimes present, it is easy to identify. The Eriophyoidea possess a unique and specialized structure at the base of their stylets, the motivator. It has been suggested that the motivator may be homologous with the cheliceral shafts (sometimes termed cheliceral bases) (Lindquist, 1996a; Lindquist, 1996b; Nuzzaci & Alberti, 1996). However, the issue of homologizing the motivator is problematical (Lindquist, 1996a; Nuzzaci & Alberti, 1996). The Eriophyoidea possess long and attenuated cheliceral shafts that project from the motivator (Figure 33G). The motivator would therefore have to be a highly unusual modification at the base of the shafts. In the Tydeoidea in which the shafts are fused, fusion takes place along a large proportion of the length of each shaft, and there is no dramatic modification of the shaft base. If the motivator represents the amalgamation of the cheliceral shafts, fusion proceeded in a very different way between the Eriophyoidea and the Tydeoidea. The likelihood that the motivator does represent the fusion of the shafts seems further diminished by the fact that the motivator functions as a fulcrum, therefore enabling the alternating movement of the cheliceral stylets (Nuzzaci & Alberti, 1996). In order to function as a fulcrum the motivator has to articulate with the rest of the chelicerae. The shaft would therefore have to have divided into an additional segment (fusion only occurring between the basal segments). Although this is not impossible, this is clearly not a strong basis for homologizing the fused shafts of the Tydeoidea with the motivator. Therefore, the chelicerae are coded as separate along the shafts in the Eriophyoidea.

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Figure 33. Mouthparts. Ventral views of subcapitulum: A, Stigmalychus nr. veretrum; B, Gordialycus sp. A; C, Nanorchestes globosus Theron & Ryke (copied from Theron & Ryke, 1969). Lateral views of rutella: D, Petralycus unicornis Grandjean (copied from Grandjean, 1943); E, cf. Psammolycus sp. A. Lateral views of chelicera: F, Proctotydaeus galapagosensis Fain & Evans (copied from Fain & Evans, 1966); G, Eriophyidae (copied from Lindquist, 1996a); H, Cunliffea strenzkei. Dorsal view of epistome and chelicerae: I, Neonanorchestes sp. (Ohio, USA). Ru = rutellum; LL = Lateral lip; FD = fixed digit; MD = movable digit; CS = cheliceral shaft; Ch = chelicera; Ep = epistome. 147

Cheliceral setae (ch. 59 & 60 – binary): The cheliceral setae can be fairly confidently homologized across the taxa. There is usually only a single seta on each chelicera, which is often near the fixed digit (ch. 59). Sometimes there is an additional seta, which is noticeably posterior relative to the other cheliceral seta (ch. 60). A cheliceral seta may be routinely present in the Eriophyoidea. A detailed study of their mouthparts has revealed protuberances (Chetverikov & Craemer, 2015 – Figure 1A, Figure 3ABCD indicated as “a”, Figure 4 two white arrows) that closely resemble cheliceral setae or perhaps vestiges of setae. These structures may be present on most or all of the Eriophyoidea that were included in the dataset. It is not known whether they are true setae, or which particular cheliceral setae they may represent (they are not strongly posterior or anterior). For these reasons, both the anterior and posterior setae were coded as unknown for all of the Eriophyoidea. Presence/absence of an epistome (ch. 61 – binary): The epistome is the triangular structure between the bases of the chelicerae of the Nanorchestidae (Figure 33I). This triangular structure is not found in any other taxa included in this analysis. It is not obvious that the epistome of the Nanorchestidae is a true homologue of the epistome of other arachnids. Palp (ch. 62 to 67 – binary): In the Acari, the palp segments can fuse in a number of different places. Fusions in acariform mites typically appear to involve the femur, genu and trochanter. The first segments to fuse are often the femur and genu (ch. 63). The result is that a relatively long palp segment – a femurogenu – is directly anterior to a short trochanteral segment at the base of the palp. This is consistent with generally accepted hypotheses on the fusion of the palp segments. For example, Lindquist (1996b) has suggested that the palp femur and genu has fused in the Tydeoidea and the Eriophyoidea. For the analysis, the state of consolidation of each pair of adjoining segments is treated as a binary character – fused or separate. Several palp structures were also incorporated into the character matrix. Some trombidiform mites have a claw-like seta on the palp tibia (Figure 32a) (ch. 66). Among the taxa included in this analysis, this seta is easy to distinguish from ordinary setae. The

148 trichoid setae of the palp can be numerous, and are therefore difficult to homologize. For this reason, they were excluded from the analysis. However, a very large proportion of acariform mites have a single solenidion on the palp tarsus. This is easy to homologize because no additional solenidia are apparent. Therefore, the presence/absence of the palp solenidion was included in the analysis (ch. 67). Gnathosomal stigmata (ch. 68 – binary): The presence of gnathosomal stigmata is considered to be an important synapomorphy for most of the Trombidiformes. In some members of this taxonomic group, the stigmata have repositioned onto the prodorsum, but no examples of these mites were included in the matrix.

Legs (ch. 69 to 112) Presence/absence of lateral claws (ch. 69 to 71 – binary): The pre-tarsi of mites usually bear lateral claws. But in some or all legs they are suppressed. The taxa showed an identical pattern of suppression in legs II and III (ch. 70). These legs were, therefore, amalgamated into the same character. But legs I (ch. 69) and IV (ch. 71) were kept as separate characters because they can sometimes differ from all of the other legs. Presence/absence of empodia (ch. 72 & 73 – binary): Empodia can also vary in their expression. The taxa showed an identical pattern of empodial suppression in legs II to IV. Therefore, these legs were amalgamated into a single character (ch. 73). Empodial shape (ch. 74 – multistate, unordered; ch. 75 & 76 – binary): If the cilia (setules) are excluded, the shape of the empodium can be characterized as four basic character states: elongated and straight (Figure 32H); elongated and curved – claw-like (Figure 32I); bulbous (Figure 32J); flat and rounded – pad-like (Figure 32K). The shape of the main empodial body (empodium minus cilia) was therefore coded as a separate character (ch. 74) to the presence/absence of cilia/setules (ch. 75). The empodial shape and presence/absence of cilia, including tenent hairs, is always identical across all 4 pairs of legs. The Rhaphignathina have tenent hairs, which are cilia with distinct T-shaped tips. These are treated as ordinary cilia, but an additional character is also used for the presence/absence of tenent hairs (ch. 76).

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Setae (ch. 77 to 88 – binary): Setal counts were not used because they do not represent homologous states; identical counts can comprise different setae. Among early derivative mites, most of leg segments typically have too many setae and too much variation in the number of setae across taxa to confidently determine their homology. A presence/absence scheme for individual setae was, therefore, only undertaken for the trochanteral setae, where numbers of setae per segment remain low enough (3 or less) across all taxa to make it possible to fairly confidently determine setal homology between families, infraorders and suborders. These trochanteral setae also appear to occupy a relatively stable position, and numbers of setae vary very little, if at all, within families and superfamilies. Presence/absence of solenidia (ch. 89 to 105 – binary): For each leg segment, the presence/absence of solenidia was coded, but not the number (for the reason mentioned above). These characters are informative because many leg segments do not have solenidia, and also because it is likely that one or more of the solenidia that are on homologous leg segments of different taxa are also homologous. The presence/absence of rhagidial solenidia was also included as an additional character (ch. 105). Rhagidial solenidia are recumbent structures that lie within tight and shallow integumental depressions on the tarsi of legs I and II. The fusion of leg segments (ch. 106-109 – multistate, unordered; ch. 110 & 111 – binary): In many arachnids, the femora comprise two segments (ch. 106-109). These segments often form a single, consolidated segment in the acariformes. Femoral fusion for each pair of legs comprises three character states: 1) no fusion; 2) partial fusion; 3) complete fusion. Partial fusion is indicated by the presence of a suture, which may or may not completely encircle the leg. Complete fusion indicates that no trace of any segmentation could be detected across each femur. The fusion of the femora varies among the legs. Therefore, each pair of legs represents a distinct character with respect to femoral fusion. In Gordialycus, another fusion event has arisen – the genua of legs III and IV have fused with the femora. A separate and additional character addresses this

150 synapomorphy (ch. 110). In the non-acarine outgroup taxa, all of the tarsi are divided into two or more segments; this was treated as a single character (ch. 111).

Presence/absence of legs III and IV (ch.112 – binary): Legs III and IV are lost in all of the eriophyoidea. This character is therefore an obvious and robust synapomorphy for that group.

Characters that were not coded, but which have been treated as evidence for a sister relationship between the Tydeoidea and the Eriophyoidea. Lindquist (1996b) has suggested that a number of similarities between the Eriophyoidea and the Tydeoidea may be possible synapomorphies that support a sister relationship between these two groups. For this reason, an attempt was made to include those hypothesized syanpomorphies in the phylogenetic analysis. However, a small number of those characters were excluded because they are problematical with respect to character coding. There is little reason to suggest that those characters undermine a case for a close relationship between the Eriophyoidea and the Nematalycidae. In the case of most characters, the Eriophyoidea are no more similar to the Tydeoidea than they are to the Nematalycidae. Characters are generally excluded because of difficulties with respect to determining the character state, e.g. lack of confidence in homology, or because the character is uninformative. Coxisternal setae: Lindquist (1996b) used coxisternal setae to make a case against a sister relationship between the Eriophyoidea and any of the Eleutherengone superfamilies. There is a shared retention of fundamental setae 2a on coxisternae II in Tydeoidea and Eriophyoidea. However, Lindquist is careful to point out that this is a symplesiomorphy, and therefore not a reliable indicator of a close relationship between Tydeoidea and Eriophyoidea – the Nematalycidae also possess a fundamental seta on coxisterna 2. We have not coded the coxisternal setae due to issues of homology. These setal characters do not appear to be robust enough for use in a phylogenetic analysis that includes different suborders. Setae that are present on a coxa can readily shift into the

151 intercoxal region within and between families. Sometimes this appears to be caused by the reduction or movement of the coxal fields. Sexually dimorphic suppression of eugenital setae: Lindquist also suggests that the suppression of eugenital setae in females is a potential synapomorphy for linking Tydeoidea and Eriophyoidea as sister groups (Lindquist, 1996b). However, whereas there is complete suppression of eugenital setae in Eriophyoids (Loboquintus Chetverikov et al. may be an exception (Chetverikov et al., 2013)), there is noticeable variation in this character in the Tydeoidea. For example, there is complete suppression in both males and females in the subfamily Pronematinae, and females have fewer pairs of eugenital setae in the Triophtydeinae, but they are not completely suppressed. But the Nematalycidae also show evidence of the dimorphic suppression of eugenital setae. Like the Eriophyoidea, female nematalycids completely lack eugenital setae. Almost all known nematalycids are thelytokous, and spanandric males are, so far, unknown. However, an undescribed sexual species of Nematalycidae from New Mexico clearly shows that the males possess eugenital setae. There is, therefore, no stronger basis for interpreting this character as a synapomorphy for linking the Eriophyoidea with the Tydeoidea than there is for interpreting this character as a synapomorphy for linking the Eriophyoidea with the Nematalycidae. In this analysis, the presence/absence of eugenital setae in females was coded (see above). But sexually dimorphic suppression was not coded because there are too many missing data for other taxa included in the analysis. For a number of species this information was not available due to absence of specimens of one of the sexes. Furthermore, dimorphic suppression is impossible to determine in thelytokous lineages in which spandandric males are absent. Sex determination mechanisms: This character is based on karyotypic studies, which indicate that both Tydeoidea and the Eriophyoidea are haplo-diploid (Helle and Wysoki, 1983). This character is excluded because there is no data on the karyotype of any of the traditional Endeostigmata, including the Nematalycidae (Helle et al., 1984;

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Norton et al., 1993). The status of all of the members of this order is therefore unknown with respect to haplodiplody. Calyptostatic nymphs: A few of the Tydeoidea have calyptostatic nymphs. It has been hypothesized that this represents a stage of ontogenetic reduction that is approaching the complete loss of the nymphal stages (Lindquist, 1996b). Accordingly, the Eriophyoidea have taken this ontogenetic reduction to completion by eliminating some of those nymphal stages altogether. But this argument rests on the assumption that calyptostases is a transitional step towards the loss of a nymphal stage. There does not appear to be evidence to support that argument; none of the Eriophyoidea has calyptostatic nymphs. Furthermore, none of the taxa included in the analysis appear to have calyptostatic nymphs. Therefore, this character is uninformative and has been excluded from the analysis. Suppression of nymphal progenital chamber: In the Eriophyoidea and the Tydeoidea, a genital chamber does not develop until adulthood (André, 1981). This was hypothesized as a synapomorphy for the Eriophyoidea and the Tydeoidea because in the more early derivative taxa, a progenital chamber is formed in the nymphal stages (Lindquist, 1996b). This character was excluded because its presence/absence could not be confidently determined for many of the taxa included in the analysis.

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Appendix D: Character list (Chapter 6)

No. Character and states 1 Idiosomal elongation (length/maximal width): 0-9 2 Distance between anus and genitalia as proportion of idiosomal length: 0-9 3 Distance between coxae I as a proportion of maximal width: 0-9 4 Tritonymph: [0] not suppressed; [1] suppressed 5 Mode of reproduction: [0] sexual; [1] asexual 6 Annuli: [0] absent; [1] present 7 Integumental protrusions/microturbercles: [0] absent; [1] present 8 Reticulation of the integument: [0] absent; [1] present 9 Naso: [0] absent; [1] present 10 Central eye: [0] absent; [1] present 11 Naso position: [0] naso part of anterior margin of prodorsum; [1] naso receded back from margin of prodorsum 12 Lateral eyes: [0] absent; [1] present 13 Number of lateral eyes: [0] 1 pair; [1] 2 pairs 14 Postocular bodies: [0] absent; [1] present 15 Seta(e) vi: [0] absent; [1] present 16 Number of vi setae: [0] paired; [1] unpaired 17 Seta(e) vi and naso: [0] if vi and naso present, naso without vi; [1] if vi and naso present, naso bearing vi 18 Setae ve: [0] absent; [1] present 19 Setae sci: [0] absent; [1] present 20 Setae sce: [0] absent; [1] present

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21 Setae exp: [0] absent; [1] present 22 Setae in: [0] absent; [1] present 23 Trichobothria 1: [0] vi is trichobothrial; [1] vi is not trichobothrial 24 Trichobothria 2: [0] ve is trichobothrial; [1] ve is not trichobothrial 25 Trichobothria 3: [0] sci is trichobothrial; [1] sci is not trichobothrial 26 Opisthosomal lyrifissures; [0] absent; [1] present 27 Lyrifissures ia: [0] absent; [1] present 28 Lyrifissures im: [0] absent; [1] present 29 Lyrifissures ip: [0] absent; [1] present 30 Lyrifissures ih: [0] absent; [1] present 31 Lyrifissures ips: [0] absent; [1] present 32 Lyrifissures iad: [0] absent; [1] present 33 Lyrifissures ig: [0] absent; [1] present 34 Body constriction: [0] body not constricted behind podosoma; [1] body constricted behind podosoma 35 Setae c1: [0] absent; [1] present 36 Setae c2: [0] absent; [1] present 37 Setae c3: [0] absent; [1] present 38 Setae c4: [0] absent; [1] present 39 Setae d2: [0] absent; [1] present 40 Setae d3: [0] absent; [1] present 41 Setae e2: [0] absent; [1] present 42 Setae e3: [0] absent; [1] present 43 Identity of terminal segment in adult: [0] PS; [1] AD; [2]; AN; [3] PA; [4] PA+1 44 Setal branching: [0] absent; [1] present 45 Shape of body of setae: [0] thin; [1] swollen 46 Hyertrichy of the opisthosoma [0] absent; [1] present 47 Genital papillae in the adult stage: [0] absent; [1] present 48 Number of pairs of genital papillae in the adult stage: 1-3

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49 Eugenital setae: [0] absent; [1] present 50 Gut boluses: [0] absent; [1] present 51 Rutella: [0] absent; [1] present 52 Rutella structure: [0] rutella without multiple projections; [1] rutella with multiple projections 53 Rutella convergence: [0] rutella do not meet at midline; [1] rutella meet at the midline (fully convergent) 54 Fixed digit reduction: [0] fixed digit not reduced relative to movable digit; [1] fixed digit reduced relative to movable digit 55 Checlieral shaft attenuation: [0] cheliceral shaft robust; [1] cheliceral shaft styliform/substyliform 56 Fixed digit attenuation: [0] fixed digit robust and chelate; [1] fixed digit styliform/substyliform 57 Movable digit attenuation: [0] movable digit robust and chelate; [1] movable digit styliform/substyliform 58 Fusion of cheliceral shafts: [0] separate; [1] fused 59 Anterior cheliceral seta: [0] absent; [1] present 60 Posterior cheliceral seta: [0] absent; [1] present 61 Epistome: [0] absent; [1] present 62 Palp segmental fusion 1: [0] trochanter separate from femur; [1] trochanter fused with femur 63 Palp segmental fusion 2: [0] femur separate from genu; [1] femur fused with genu 64 Palp segmental fusion 3: [0] genu separate from tibia; [1] genu fused with tibia 65 Palp segmental fusion 4: [0] tibia separate from tarsus [1] tibia fused with tarsus 66 Claw-like seta on tibia: [0] absent; [1] present 67 Solenidion on palp tarsus: [0] absent; [1] present 68 Gnathosomal stigmata: [0] absent; [1] present 69 Legs I lateral claws: [0] absent; [1] present 70 Legs II & III lateral claws: [0] absent; [1] present

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71 Legs IV lateral claws: [0] absent; [1] present 72 Legs I empodia: [0] absent; [1] present 73 Legs II to IV empodia: [0] absent; [1] present 74 Empodial shape (excluding setules/cilia): [0] elongated and straight; [1] elongated and curved – claw-like; [2] flat and rounded – pad-like; [3] bulbous 75 Empodial ciliation: [0] nude; [1] ciliated (covered in setules – fine hairs) 76 Empodial tenent hairs: [0] cilia, if present, are not tenent hairs; [1] cilia, if present, are tenent hairs 77 v’ on trochanters 1: [0] absent; [1] present 78 l’ on trochanters 1: [0] absent; [1] present 79 v’’ on trochanters 1: [0] absent; [1] present 80 v’ on trochanters 2: [0] absent; [1] present 81 l’ on trochanters 2: [0] absent; [1] present 82 v’’ on trochanters 2: [0] absent; [1] present 83 v’ on trochanters 3: [0] absent; [1] present 84 l’ on trochanters 3: [0] absent; [1] present 85 v’’ on trochanters 3: [0] absent; [1] present 86 v’ on trochanters 4: [0] absent; [1] present 87 l’ on trochanters 4: [0] absent; [1] present 88 v’’ on trochanters 4: [0] absent; [1] present 89 Solenidia on tarsi I: [0] absent; [1] present 90 Solenidia on tarsi II: [0] absent; [1] present 91 Solenidia on tarsi III: [0] absent; [1] present 92 Solenidia on tarsi IV: [0] absent; [1] present 93 Solenidia on tibiae I: [0] absent; [1] present 94 Solenidia on tibiae II: [0] absent; [1] present 95 Solenidia on tibiae III: [0] absent; [1] present 96 Solenidia on tibiae IV: [0] absent; [1] present 97 Solenidia on genua I: [0] absent; [1] present

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98 Solenidia on genua II: [0] absent; [1] present 99 Solenidia on genua III: [0] absent; [1] present 100 Solenidia on genua IV: [0] absent; [1] present 101 Solenidia on femora I: [0] absent; [1] present 102 Solenidia on femora II: [0] absent; [1] present 103 Solenidia on femora III: [0] absent; [1] present 104 Solenidia on femora IV: [0] absent; [1] present 105 Rhagidial solenidia: [0] absent; [1] present 106 Femur I subdivision: [0] not subdivided; [1] partially subdivided; [2] completely subdivided 107 Femur II subdivision: [0] not subdivided; [1] partially subdivided; [2] completely subdivided 108 Femur III subdivision: [0] not subdivided; [1] partially subdivided; [2] completely subdivided 109 Femur IV subdivision: [0] not subdivided; [1] partially subdivided; [2] completely subdivided 110 Fusion state of femora III and IV with genu III and IV: [0] femora III and IV distinct segments from genua III and IV; [1] femora III and IV fused with genua III and IV 111 tarsi subdivision: [0] not subdivided; [1] subdivided 112 legs III and IV of adults: [0] absent; [1] present

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Appendix E: Character matrix (Chapter 6)

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0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 3 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 Mummucia ibirapemussu 3 ? ? 0 0 0 ? ? 0 1 - 0 - 0 1 ? - 1 1 1 1 1 1 1 1 ? ? ? ? ? Cryptocellus iaci 1 8 5 0 0 0 ? ? 0 0 - 1 0 0 1 ? - 1 1 1 1 1 1 1 1 ? ? ? ? ? Metatydaeolus sp. 2 1 4 ? ? 0 1 0 0 0 - 0 - 0 1 0 - 1 1 1 0 0 1 1 0 1 1 1 1 1 Brachytydeus sp. 0 1 8 0 ? 0 1 1 0 0 - 0 - 0 1 0 - 1 1 1 0 0 1 1 0 1 1 1 0 0 Riccardoella triodopsis 0 1 2 ? 0 0 1 0 0 0 - 0 - 0 1 0 - 1 1 1 0 0 1 1 0 1 1 1 1 1 Triophtydeus sp. 1 2 9 ? ? 0 1 0 0 0 - 0 - 0 1 0 - 1 1 1 0 0 1 1 0 1 1 1 1 1 Proctotydaeus sp. 1 2 5 ? 0 0 1 0 0 0 - 0 - 0 1 0 - 1 1 1 0 0 1 1 0 1 1 1 1 1 Halotydeus destructor 1 4 6 0 0 0 1 0 0 0 - 1 0 0 1 0 - 1 1 1 0 0 1 1 0 1 1 1 1 1 Stereotydeus sp. 0 2 0 0 ? 0 1 0 1 0 0 0 - 0 1 0 1 1 1 1 0 0 1 1 0 1 1 1 1 1 Arhagidia monothrix 2 1 0 0 ? 0 1 0 1 0 0 0 - 0 1 1 1 1 1 1 0 0 1 1 0 1 1 1 1 1 Claveupodes sp. 1 2 6 0 ? 0 1 0 1 0 0 0 - 0 1 0 0 1 1 1 0 0 1 1 0 1 1 1 1 1 Neoscirula reticulata 0 1 0 0 ? 0 1 0 0 0 - 0 - 0 1 0 - 1 1 1 0 0 0 1 0 1 0 1 0 1 Trachymolgus purpureus 1 0 0 0 0 0 0 0 0 0 - 1 1 0 1 0 - 1 1 1 0 0 0 1 0 1 1 1 1 1 Apomerantzia kethleyi 2 0 2 0 1 0 0 0 0 0 - 0 - 0 0 - - 1 1 1 0 0 - 1 1 0 - - - - Neognathus ozkani 1 ? 9 1 ? 0 0 0 0 0 - 0 - 0 1 0 - 1 1 1 0 0 1 1 1 1 1 1 1 1 Stigmaeus cariae 1 0 6 1 ? 0 0 0 0 0 - 0 - 0 1 0 - 1 1 1 0 0 1 1 1 0 - - - - Aegyptobia pirii 1 0 4 1 ? 0 0 1 0 0 - 1 1 0 0 - - 1 1 1 0 0 - 1 1 0 - - - - Labidostomma sp. 1 0 0 0 ? 0 1 1 0 0 - 0 - 0 1 0 - 1 1 1 1 1 0 1 0 0 - - - - Sphaerolichus lekprayoonae 0 2 ? ? 0 0 1 0 1 1 0 1 0 0 1 0 1 1 1 1 1 1 0 1 0 1 1 0 0 1 Hybalicus multifurcatus 1 1 5 ? 1 0 1 0 1 0 0 0 - 0 0 - 0 1 1 1 0 0 - 0 0 0 - - - - Loboquintus subsquamatus 2 9 0 ? 0 1 1 0 1 0 0 0 - 0 1 1 1 1 1 0 0 0 1 1 ? 0 - - - - Pentasetacus araucariae 3 8 0 ? 0 1 1 0 1 0 0 0 - 0 1 1 0 1 1 0 0 0 1 1 ? 0 - - - - Oziella sibirica 4 9 0 ? 0 1 1 0 0 0 - ? 0 0 0 - - 1 1 0 0 0 - 1 ? 0 - - - - Notostrix macrothrix 3 8 0 ? 0 1 ? 0 1 0 0 0 - 0 0 - - 0 1 0 0 0 - - ? 0 - - - - Rhynacus acerioides 3 8 1 ? 0 1 1 0 0 0 - 0 - 0 1 1 - 0 0 0 0 0 1 - - 0 - - - - Gordialycus sp. A 9 8 0 ? 1 1 1 0 0 0 - 0 - 0 1 1 - 1 1 1 0 0 1 1 1 0 - - - - Gordialycus sp. B 9 9 0 ? 1 1 1 0 0 0 - 0 - 0 0 - - 1 1 1 0 0 - 1 1 0 - - - - Osperalycus tenerphagus 6 7 1 0 1 1 1 0 0 0 - 0 - 0 1 1 - 1 1 1 0 0 1 1 1 0 - - - - Cunliffea cf. strenzkei 4 5 0 0 1 1 1 0 0 0 - 0 - 0 0 - - 1 1 1 0 0 - 1 1 0 - - - - cf. Psammolycus sp. A 4 5 ? ? 1 1 1 0 0 0 - 0 - 0 1 1 - 1 1 1 0 0 1 1 1 0 - - - - cf. Psammolycus sp. B 5 7 3 0 1 1 1 0 0 0 - 0 - 0 1 1 - 1 1 1 0 0 1 1 1 0 - - - - Alycus cf. denasutatus 0 1 5 0 0 0 1 0 0 0 - 1 0 1 1 0 - 1 1 1 1 1 1 0 0 1 1 1 1 1 Pachygnathus sp. 0 2 4 0 0 0 1 0 1 0 1 1 0 1 1 0 0 1 1 1 1 1 1 0 0 1 1 ? 1 1 Bimichaelia nr. campylognatha 0 1 5 ? ? 0 1 1 1 1 1 0 - 0 1 0 0 1 1 1 1 1 1 0 0 ? ? ? ? ? Petralycus sp. 0 1 4 0 0 0 1 0 1 0 1 0 - 0 1 0 0 1 1 1 0 1 1 0 0 1 ? ? ? 1 Nanorchestes globosus 0 2 2 0 0 0 1 0 1 0 1 1 0 1 1 0 0 1 1 1 1 1 1 0 0 0 - - - - Speleorchestes nylsvleyensis 2 ? ? 0 0 0 0 0 1 1 0 1 0 1 1 0 0 1 1 1 1 1 1 0 0 0 - - - - Speleorchestes potchefstroomensis 1 2 ? 0 ? 0 0 0 1 1 0 1 0 1 1 0 0 1 1 1 1 1 1 0 0 0 - - - - Proterorhagia oztotloica 2 2 6 ? ? 0 1 0 0 1 - 0 - 0 1 0 - 1 1 1 1 1 1 0 0 1 1 1 1 1 Proteonematalycus wagneri 2 0 4 1 1 0 0 0 1 1 0 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Micropsammus sp. 2 0 5 0 0 0 0 0 1 0 0 0 - 0 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Terpnacarus glebulentus 0 1 6 0 1 0 1 0 1 1 0 1 0 0 1 0 1 1 1 1 1 1 1 1 0 1 ? ? ? ? Alicorhagia usitata 1 1 5 1 1 0 0 1 1 0 0 0 - 0 1 0 1 1 1 1 1 1 1 1 0 0 - - - - Stigmalychus veretrum 2 3 6 0 1 0 1 1 1 0 0 0 - 0 1 0 1 1 1 1 1 1 1 1 0 1 0 1 1 1 Grandjeanicus uncus ? ? ? 0 1 0 1 0 1 0 0 0 - 0 1 0 1 1 1 1 0 1 1 1 0 0 - - - - Oehserchestes humicolus 0 1 6 0 1 0 1 0 1 0 1 0 - 0 1 0 1 1 1 1 0 1 1 1 0 0 - - - - Aphelacarus acarinus 2 2 6 0 ? 0 0 0 1 0 0 0 - 0 1 0 0 1 1 1 1 1 1 1 0 0 - - - - Psammochthonius kethleyi 2 0 0 0 1 0 ? 0 1 0 0 0 - 0 1 0 1 1 1 1 1 1 1 1 0 1 1 1 1 1

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0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 6 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 Mummucia ibirapemussu ? ? ? 1 1 1 1 1 1 1 1 1 4 0 0 1 0 - ? ? 0 - - 0 0 0 0 0 1 1 Cryptocellus iaci ? ? ? 0 1 1 1 1 1 1 1 1 4 0 0 1 0 - ? 0 0 - - 1 0 0 0 0 1 1 Metatydaeolus sp. 0 0 0 0 1 1 0 0 0 0 0 0 1 1 0 0 1 0 0 0 0 - - 1 0 0 1 1 0 0 Brachytydeus sp. 0 0 0 0 1 1 0 0 1 0 1 0 1 1 0 0 1 0 0 0 0 - - 1 0 0 1 1 0 0 Riccardoella triodopsis 0 0 0 0 1 1 0 0 0 0 0 0 1 1 0 0 1 1 0 0 0 - - 1 0 0 1 0 0 0 Triophtydeus sp. 0 0 0 0 1 1 0 0 0 0 0 0 1 1 0 0 1 0 1 0 0 - - 1 0 0 1 0 0 0 Proctotydaeus sp. 0 0 0 0 1 1 0 0 0 0 0 0 1 1 0 0 1 0 0 0 0 - - 1 0 0 1 0 0 0 Halotydeus destructor 0 0 0 0 1 1 0 0 0 0 0 0 1 1 0 0 1 1 1 0 0 - - 1 0 0 1 0 0 0 Stereotydeus sp. 0 0 0 0 1 1 0 0 0 0 0 0 1 1 0 0 1 1 1 0 0 - - 1 0 0 0 0 1 0 Arhagidia monothrix 0 0 0 0 1 1 0 0 0 0 0 0 1 1 0 0 1 1 1 0 0 - - 0 0 0 0 0 1 0 Claveupodes sp. 0 0 0 0 1 1 0 0 0 0 0 0 1 1 0 0 1 1 1 0 0 - - 0 0 0 0 0 1 0 Neoscirula reticulata 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 1 1 ? 0 0 - - 1 0 0 0 0 0 0 Trachymolgus purpureus 0 0 0 0 1 1 0 0 0 0 0 0 1 1 0 0 1 2 1 0 0 - - 0 0 0 0 0 1 1 Apomerantzia kethleyi - - - 0 1 1 0 0 0 0 0 0 0 0 0 0 1 2 1 0 0 - - 1 0 0 0 0 1 0 Neognathus ozkani ? ? ? 0 1 1 0 0 0 0 0 0 0 0 0 0 0 - 0 0 0 - - 1 0 0 0 1 0 0 Stigmaeus cariae - - - 0 1 1 0 0 1 0 1 0 0 0 0 0 0 - 0 0 0 - - 1 0 0 1 0 0 0 Aegyptobia pirii - - - 0 1 1 1 0 1 1 1 1 0 1 1 0 0 - 0 0 0 - - 1 0 0 1 1 0 0 Labidostomma sp. - - - 0 1 1 0 0 1 0 1 0 1 0 0 0 1 1 0 0 0 - - 0 0 0 0 0 1 1 Sphaerolichus lekprayoonae 0 0 0 0 1 1 0 0 1 0 1 0 1 1 0 0 1 1 0 ? 0 - - 0 0 0 0 0 1 1 Hybalicus multifurcatus - - - 0 1 1 1 0 1 0 1 0 1 1 0 0 1 2 0 ? 0 - - 1 0 0 0 0 1 1 Loboquintus subsquamatus - - - 0 1 1 0 0 0 0 0 0 0 0 0 0 0 - ? 0 ? ? ? 0 1 1 1 0 ? ? Pentasetacus araucariae - - - 0 1 1 0 0 0 0 0 0 0 0 0 0 0 - 0 0 ? ? ? 0 1 1 1 0 ? ? Oziella sibirica - - - 0 1 1 0 0 0 0 0 0 0 0 0 0 0 - 0 0 ? ? ? 0 1 1 1 0 ? ? Notostrix macrothrix - - - 0 0 1 0 0 0 0 0 0 0 0 0 0 0 - 0 0 ? ? ? 0 1 1 1 0 ? ? Rhynacus acerioides - - - 0 0 0 0 0 0 0 0 0 0 0 0 0 0 - 0 0 ? ? ? 0 1 1 1 0 ? ? Gordialycus sp. A - - - 0 1 1 1 1 0 0 1 0 ? 1 0 0 1 1 0 0 1 0 1 0 0 0 0 0 1 0 Gordialycus sp. B - - - 0 1 1 1 1 0 0 1 0 ? 1 0 0 1 1 0 0 1 0 1 0 0 0 0 0 1 0 Osperalycus tenerphagus - - - 0 1 1 1 1 1 0 1 0 3 1 0 0 1 1 0 0 1 1 1 0 0 0 0 0 1 0 Cunliffea cf. strenzkei - - - 0 1 1 1 1 1 0 1 ? 3 1 0 0 1 1 0 0 1 0 0 0 0 0 0 0 1 0 cf. Psammolycus sp. A - - - 0 1 1 1 1 1 0 1 ? ? 1 0 0 1 1 0 0 1 0 0 0 0 0 0 0 0 0 cf. Psammolycus sp. B - - - 0 1 1 1 1 1 0 1 ? 3 1 0 0 1 1 0 0 1 1 0 0 0 0 0 0 1 0 Alycus cf. denasutatus 1 1 0 0 1 1 1 1 1 1 1 1 3 1 0 1 1 2 1 0 1 1 0 0 0 0 0 0 1 0 Pachygnathus sp. 1 ? 0 0 1 1 1 1 1 1 1 1 3 1 0 1 1 2 1 0 1 1 1 0 0 0 0 0 0 0 Bimichaelia nr. campylognatha ? ? 0 0 1 1 1 1 1 1 1 1 3 1 0 1 1 2 1 0 0 - - 0 0 0 0 0 0 0 Petralycus sp. 1 ? 0 0 1 1 1 1 1 0 1 0 3 1 0 0 1 2 1 0 1 1 1 0 0 0 0 0 0 0 Nanorchestes globosus - - - 0 1 1 1 1 1 1 1 1 ? 1 0 1 1 1 1 0 1 1 0 0 0 0 0 0 ? 1 Speleorchestes nylsvleyensis - - - 1 1 1 1 1 1 0 1 0 ? 1 1 1 1 2 1 0 1 1 0 0 0 0 0 0 1 1 Speleorchestes potchefstroomensis - - - 0 1 1 1 1 1 0 1 0 ? 1 1 1 1 2 1 0 1 ? ? 0 0 0 0 0 1 1 Proterorhagia oztotloica 1 1 0 1 1 1 1 1 1 0 1 0 2 1 0 0 1 2 0 0 ? ? ? 0 0 0 0 0 1 0 Proteonematalycus wagneri 1 1 1 1 1 1 1 1 1 0 1 0 2 1 0 0 1 2 0 1 1 1 0 0 0 0 0 0 1 0 Micropsammus sp. 1 1 1 1 1 1 1 1 1 0 1 0 3 1 0 0 1 1 0 1 1 1 0 0 0 0 0 0 1 0 Terpnacarus glebulentus ? 1 0 0 1 1 1 1 1 0 1 0 3 1 0 0 1 2 1 1 1 1 0 0 0 0 0 0 1 1 Alicorhagia usitata - - - 0 1 1 1 1 1 0 1 0 2 1 0 0 1 1 0 1 1 1 0 0 0 0 0 0 1 0 Stigmalychus veretrum 1 1 0 1 1 1 1 1 1 0 1 0 2 1 0 0 1 2 0 1 1 1 0 0 0 0 0 0 1 0 Grandjeanicus uncus - - - 0 1 1 ? ? ? ? ? ? ? 1 0 0 1 2 0 1 1 1 0 0 0 0 0 0 1 1 Oehserchestes humicolus - - - 0 1 1 1 0 1 0 1 0 2 1 1 0 1 2 1 1 1 1 0 0 0 0 0 0 1 1 Aphelacarus acarinus - - - 1 1 1 1 1 1 0 1 0 2 0 0 0 1 1 1 1 1 1 0 0 0 0 0 0 1 1 Psammochthonius kethleyi 1 0 0 1 1 1 1 1 1 0 1 0 1 ? 0 0 1 2 1 1 1 1 0 0 0 0 0 0 1 0

161

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8 9 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 Mummucia ibirapemussu 0 0 0 0 0 0 ? 0 1 1 1 ? ? ? ? ? 1 1 1 1 1 1 1 1 1 1 1 1 ? ? Cryptocellus iaci 0 0 0 1 0 0 ? 0 1 1 1 0 0 - - - 1 1 1 1 1 1 1 1 1 1 1 1 ? ? Metatydaeolus sp. 0 0 1 0 0 0 1 1 1 1 1 1 1 2 1 0 1 0 0 1 0 0 1 0 0 0 0 0 1 1 Brachytydeus sp. 0 0 1 0 0 0 1 1 1 1 1 1 1 2 1 0 1 0 0 0 0 0 1 0 0 0 0 0 1 1 Riccardoella triodopsis 0 0 1 ? ? 0 1 1 1 1 1 1 1 2 1 0 1 0 0 1 0 0 1 0 0 0 0 0 1 1 Triophtydeus sp. 0 0 1 0 0 0 1 1 1 1 1 1 1 2 1 0 1 0 0 1 0 0 1 0 0 0 0 0 1 0 Proctotydaeus sp. 0 0 1 0 0 0 1 1 0 1 1 0 1 - 1 0 1 0 0 1 0 0 1 0 0 0 0 0 1 1 Halotydeus destructor 0 0 1 0 0 0 1 ? 1 1 1 1 1 2 1 0 1 0 0 1 0 0 1 0 0 1 0 0 1 1 Stereotydeus sp. 0 0 1 0 0 0 1 1 1 1 1 1 1 2 1 0 1 0 0 1 0 0 1 0 0 1 0 0 1 1 Arhagidia monothrix 0 0 1 0 0 0 1 ? 1 1 1 1 1 2 1 0 1 0 0 1 1 0 1 1 0 1 1 0 1 1 Claveupodes sp. 0 0 0 0 0 0 1 1 1 1 1 1 1 2 1 0 1 0 0 1 0 0 1 0 0 1 0 0 1 1 Neoscirula reticulata 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 0 1 0 0 1 0 0 1 1 0 1 0 0 1 1 Trachymolgus purpureus 0 0 1 0 0 0 1 1 1 1 1 1 1 2 1 0 1 1 0 1 1 0 1 1 0 1 1 0 1 1 Apomerantzia kethleyi 0 0 0 0 0 1 1 1 1 1 1 0 0 - - - 1 0 0 1 0 0 1 1 0 1 0 0 1 1 Neognathus ozkani 0 0 0 0 0 1 1 1 1 1 1 1 1 0 1 1 1 0 0 1 0 0 1 1 0 1 0 0 1 1 Stigmaeus cariae 0 0 0 0 0 1 1 0 1 1 1 1 1 0 1 1 1 0 0 1 0 0 1 1 0 1 0 0 1 1 Aegyptobia pirii 0 0 0 0 0 0 1 1 1 1 1 1 1 3 1 1 1 0 0 1 0 0 1 1 0 1 0 0 1 1 Labidostomma sp. 0 1 0 1 0 0 0 1 1 1 1 0 1 - 0 - 1 0 0 1 0 0 1 0 0 1 0 0 1 1 Sphaerolichus lekprayoonae 0 0 0 0 0 0 1 0 1 1 1 0 1 - 0 - 0 0 0 1 0 0 1 0 0 1 0 0 1 1 Hybalicus multifurcatus 0 0 0 0 0 0 1 0 1 1 1 1 1 2 1 0 0 0 0 0 0 0 1 0 0 1 0 0 1 1 Loboquintus subsquamatus 0 1 1 0 0 0 0 0 0 0 - 1 1 0 1 0 0 0 0 0 0 0 ------1 1 Pentasetacus araucariae 0 1 1 0 0 0 0 0 0 0 - 1 1 0 1 0 0 0 0 0 0 0 ------1 1 Oziella sibirica 0 1 1 0 0 0 0 0 0 0 - 1 1 0 1 0 0 0 0 0 0 0 ------1 1 Notostrix macrothrix 0 1 1 0 0 0 0 0 0 0 - 1 1 0 1 0 0 0 0 0 0 0 ------1 1 Rhynacus acerioides 0 0 1 0 0 0 0 0 0 0 - 1 1 0 1 0 0 0 0 0 0 0 ------1 1 Gordialycus sp. A 0 0 1 0 0 0 0 0 0 0 0 1 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 Gordialycus sp. B 0 0 1 0 0 0 0 0 1 1 0 1 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 Osperalycus tenerphagus 0 1 1 0 0 0 0 0 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 Cunliffea cf. strenzkei 0 1 1 0 1 0 0 0 1 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 cf. Psammolycus sp. A 0 1 1 0 0 0 0 0 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 cf. Psammolycus sp. B 0 1 1 0 0 0 0 0 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 Alycus cf. denasutatus 0 0 0 0 0 0 1 0 1 1 1 1 1 1 1 0 1 0 0 1 0 0 1 1 0 1 0 0 1 1 Pachygnathus sp. 0 0 0 0 0 0 1 0 1 1 1 1 1 1 1 0 1 0 0 1 0 0 1 1 0 0 0 0 1 1 Bimichaelia nr. campylognatha 0 0 0 0 0 0 1 0 1 1 1 1 1 0 1 0 1 0 0 1 0 0 1 0 0 1 0 0 1 1 Petralycus sp. 0 0 0 0 0 0 1 0 1 1 1 1 1 0 1 0 1 0 0 1 0 0 1 1 0 1 1 1 1 1 Nanorchestes globosus 1 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 Speleorchestes nylsvleyensis 1 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Speleorchestes potchefstroomensis 1 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Proterorhagia oztotloica 0 0 0 0 0 0 1 0 1 1 1 1 1 1 1 0 1 0 0 1 1 1 1 1 0 1 1 0 1 1 Proteonematalycus wagneri 0 0 0 0 0 0 1 1 1 1 1 1 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 Micropsammus sp. 0 0 1 0 0 0 1 0 1 1 1 1 1 0 1 0 0 0 0 0 0 0 1 1 0 1 0 0 1 1 Terpnacarus glebulentus 0 0 0 0 0 0 1 0 1 1 1 1 1 1 1 0 0 0 0 0 0 0 1 1 0 0 0 0 1 1 Alicorhagia usitata 0 0 0 0 0 0 1 0 0 0 0 1 1 1 0 - 0 0 0 0 0 0 1 1 0 1 0 0 1 1 Stigmalychus veretrum 0 0 0 0 0 0 1 0 0 0 0 1 1 1 0 - 0 0 0 0 0 0 1 1 0 1 0 0 1 1 Grandjeanicus uncus 0 0 0 0 0 0 1 0 0 1 1 0 1 - 0 - 0 0 0 0 0 0 1 1 0 0 0 0 1 1 Oehserchestes humicolus 0 0 0 0 0 0 1 0 0 1 1 1 1 1 0 - 0 0 0 0 0 0 1 1 0 0 0 0 1 1 Aphelacarus acarinus 0 0 0 0 0 0 1 0 1 1 1 1 1 1 0 - 0 0 0 1 0 0 1 1 0 1 1 0 1 1 Psammochthonius kethleyi 0 0 0 0 0 0 1 0 1 1 1 1 1 1 0 - 0 0 0 0 0 0 1 0 0 1 0 0 1 1

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0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 9 9 9 9 9 9 9 9 9 0 0 0 0 0 0 0 0 0 0 1 1 1 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 Mummucia ibirapemussu ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 2 2 2 2 0 1 0 Cryptocellus iaci ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 0 0 0 0 0 1 0 Metatydaeolus sp. 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Brachytydeus sp. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Riccardoella triodopsis 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 Triophtydeus sp. 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 Proctotydaeus sp. 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Halotydeus destructor 0 0 1 1 1 1 1 1 1 1 0 0 0 0 1 2 2 2 2 0 0 0 Stereotydeus sp. 0 0 1 1 1 1 1 1 1 0 0 0 0 0 1 0 0 0 0 0 0 0 Arhagidia monothrix 0 0 1 1 0 0 1 1 0 0 0 0 0 0 0 2 2 2 2 0 0 0 Claveupodes sp. 0 0 1 ? 0 0 1 0 0 0 0 0 0 0 1 0 0 1 2 0 0 0 Neoscirula reticulata 0 0 1 1 1 0 1 1 1 1 0 0 0 0 0 2 2 2 2 0 0 0 Trachymolgus purpureus 0 1 1 1 1 0 1 1 1 1 0 0 0 0 0 0 0 2 2 0 0 0 Apomerantzia kethleyi 0 0 1 1 1 1 1 0 0 0 0 0 0 0 0 2 2 2 2 0 0 0 Neognathus ozkani 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Stigmaeus cariae 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Aegyptobia pirii 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Labidostomma sp. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 2 2 2 0 0 0 Sphaerolichus lekprayoonae 1 0 1 1 1 1 1 1 1 0 0 0 0 0 0 1 1 1 1 0 0 0 Hybalicus multifurcatus 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 2 0 0 0 Loboquintus subsquamatus - - 1 0 - - 0 0 - - 0 0 - - 0 0 0 - - - 0 1 Pentasetacus araucariae - - 1 0 - - 0 0 - - 0 0 - - 0 0 0 - - - 0 1 Oziella sibirica - - 1 0 - - 0 0 - - 0 0 - - 0 0 0 - - - 0 1 Notostrix macrothrix - - 0 0 - - 0 0 - - 0 0 - - 0 0 0 - - - 0 1 Rhynacus acerioides - - 0 0 - - 0 0 - - 0 0 - - 0 0 0 - - - 0 1 Gordialycus sp. A 0 0 1 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 Gordialycus sp. B 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 Osperalycus tenerphagus 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Cunliffea cf. strenzkei 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 cf. Psammolycus sp. A 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 cf. Psammolycus sp. B 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Alycus cf. denasutatus 0 0 1 1 1 0 1 1 1 1 1 0 0 0 0 0 0 0 2 0 0 0 Pachygnathus sp. 0 0 1 1 1 0 1 1 1 1 1 0 0 0 0 0 0 0 2 0 0 0 Bimichaelia nr. campylognatha 0 0 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 Petralycus sp. 0 0 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 2 0 0 0 Nanorchestes globosus 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 Speleorchestes nylsvleyensis 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 2 2 2 0 0 0 Speleorchestes potchefstroomensis 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 2 2 0 0 0 Proterorhagia oztotloica 0 0 1 1 1 1 ? 1 1 0 0 0 0 0 0 2 2 2 2 0 0 0 Proteonematalycus wagneri 0 0 1 1 1 1 1 0 0 0 0 0 0 0 0 2 2 2 2 0 0 0 Micropsammus sp. 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 1 0 0 2 0 0 0 Terpnacarus glebulentus 0 0 1 1 1 1 1 1 0 0 0 0 0 0 0 2 0 1 2 0 0 0 Alicorhagia usitata 0 0 1 1 1 1 1 1 1 0 0 0 0 0 0 2 2 2 2 0 0 0 Stigmalychus veretrum 0 0 1 1 1 1 1 1 1 0 0 0 0 0 0 1 0 0 1 0 0 0 Grandjeanicus uncus 0 0 1 1 1 1 0 1 1 1 0 0 0 0 0 1 1 1 2 0 0 0 Oehserchestes humicolus 0 0 1 1 1 1 1 0 0 1 0 0 0 0 0 2 0 2 2 0 0 0 Aphelacarus acarinus 1 0 1 1 1 1 1 1 1 1 0 0 0 0 0 2 2 2 2 0 0 0 Psammochthonius kethleyi 0 0 1 1 1 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0

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