Phylogeography and Trematode Parasitism of Oreohelix Land Snails in Southern Alberta

Home , Lundbreck Falls, Oreohelicidae, Oreohelix, Parasitic castration

PHYLOGEOGRAPHY AND TREMATODE PARASITISM OF OREOHELIX LAND SNAILS IN SOUTHERN ALBERTA

ZACHARIAH WILLIAM DEMPSEY BSc, Biological Sciences, University of Lethbridge, 2014

A Thesis submitted to the School of Graduate Studies of the University of Lethbridge in partial fulfillment of the requirements for the degree

MASTER OF SCIENCE

Department of Biological Sciences University of Lethbridge LETHBRIDGE, ALBERTA, CANADA

PHYLOGEOGRAPHY AND TREMATODE PARASITISM OF OREOHELIX LAND SNAILS IN SOUTHERN ALBERTA ZACHARIAH WILLIAM DEMPSEY Date of Defence: June 07, 2017

Dr. Theresa Burg Associate Professor Ph.D. Co-supervisor

Dr. Cameron Goater Professor Ph.D. Co-supervisor

Dr. Hester Jiskoot Associate Professor Ph.D. Thesis Examination Committee member

Dr. Robert Laird Associate Professor Ph.D. Thesis Examination Committee member

Dr. Kathleen Weaver Assistant Professor Ph.D. External, Thesis Examination Committee University of La Verne California, U.S.A.

Dr. Tony Russell Associate Professor Ph.D. Chair, Thesis Examination Committee

ABSTRACT Modern studies in phylogeography integrate many once-disparate scientific fields. This study investigated terrestrial mountain snails, Oreohelix spp., in southern Alberta using

DNA markers and the recent emergence of the trematode parasite Dicrocoelium dendriticum. Large-bodied snails in Cypress Hills (CH) and the Rocky Mountains (RM) formed three clades within the species complex O. subrudis. One was geographically widespread, one was restricted to one region in the RM, and one was restricted to CH.

Small-bodied snails in CH were determined to be O. cooperi, a rare Oreohelid thought to be imperilled in the western U.S.A. Phylogeographic analyses determined that snails likely colonized and came into contact in CH due to its glacial history. There was significant spatial and seasonal variation in the prevalence of D. dendtriticum in all three lineages of Oreohelix present in CH. Isolation and geological history have played an important role in determining Oreohelid biogeographical patterns in this region.

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ACKNOWLEDGEMENTS This project started as an independent study in the last year of my undergraduate degree. By the time I graduated, it became clear that the ecology, biodiversity, and parasitology of terrestrial snails in Alberta was much more complex and fascinating than anyone had realized. Dr. Theresa Burg and Dr. Cameron Goater agreed to take me on as a

Masters student to investigate further, and for that I am grateful. This study has altered the course of my life for the better, and it would not have been possible without the gracious support of my two supervisors. My supervisors have inspired me with the passion they hold for their respective fields – molecular ecology and parasitology. I would also like to thank my committee for their valuable feedback. Both Dr. Robert Laird and Dr. Hester Jiskoot have provided a multi-disciplinary perspective vital for projects such as mine. I would like to thank Dr. Kathleen Weaver for acting as my external. I have cited her work for so long, it was an honour to finally meet her. I would also like to thank

Dr. Tony Russell for being the chair of my thesis examination committee.

I would also like to acknowledge the various organizations that have funded my work over the past three years. N.S.E.R.C., Alberta Conservation Association Grants in

Biodiversity, and the University of Lethbridge have all provided the resources that have allowed me to pursue this research. Funding is always a concern, and the generosity of these organizations have made my research possible. Thanks also to Parks Canada for putting up with me crawling around Waterton looking for snails, and thanks to the kind staff of Cypress Hills Interprovincial Park for providing lodging during my field seasons.

I would like to get personal for a moment, as academic life does not exist in a vacuum. Primary among those I wish to thank is Sarah. Looking back, it seems so

iv improbable that we ever met at all. Despite our busy schedules over the past few years, I know I am happiest with Sarah. She has supported me without fail from the very beginning. Thank you Sarah for being the most kind, wonderful, amazing, loving, and reliable partner I could ever ask for. I can’t wait for our future together.

I would like to acknowledge mom and dad for fostering my love of science, and for always raising me to the best of their abilities. I know I was a handful at times (mostly in Foremost), but they never stopped being good parents. I would like to thank my sister

Sarai for her insight, she is wise beyond her years. She may have been a foil to my childhood, but I miss her now that we live far apart. I would also like to thank my extended family for their support. Thanks to Grandma and Grandpa for all of the home- cooked meals and support, thanks to Nana and Papa for getting back in touch with us and visiting from so far away, thanks to Brandon and Crystal for the great movie and game nights, and thanks to Keenan and Kalan for being super fun. Thanks also Sarah’s family.

Sarah’s mom and grandma have been very kind and supportive throughout my research.

I would like to acknowledge my lab mates, who have been very influential to me.

Thanks to Rachael Adams, Colin MacFarlane, Ashley Curtis, and Brad van Paridon for helping me out in the lab when I first got started. Thanks to Arturo Aanaya Villalobos for being a great field assistant. Thanks to Ashley Jensen for teaching me a bit about birds and birding despite my complaints, thanks to Cat Welke for being such a cheerful presence in the lab, thanks to Kathrin Munro for hosting great wine and cheese nights, thanks to Libby Natola for ensuring there’s never a dull moment in the lab, and thanks to

Marie Prill for always filling the lab with good-natured laughter. I will never forget the zany escapades of the lab.

I would like to acknowledge the instructors and students of BIOL2000. At first I found teaching duties to be a chore, but the more I taught, the more I enjoyed it. Now I could see myself teaching for a living. This has been in no small part to the other instructors of BIOL 2000, the Principles of Genetics. Quintin Steynen, Helena Danyk,

Randall Barley and Joanne Golden have all inspired me to always teach to best of my abilities. Thanks to Kurt Clarke and Jan Tuescher for sharing that journey with me. I see myself in my students, and I would also like to thank them for grounding me in the classroom and reminding me why I like to teach.

Thanks also to the members of Pathfinder Society for keeping me sane in these trying times. I will never forget the half-baked antics of The Foxy Five or The Outsiders.

Thanks to Ballto for showing me how to give a character personality and voice. Thanks to Josh for showing me how to make a character strong and well-rounded. Thanks to

Mark for sharing his 1st edition old-school D&D wisdom and classic dungeoneering skills. And thanks to Russell for showing me what it means to be a patient GM and a gracious player. All of us have shared stories of adventure and shenanigans over the past two seasons of PFS, and I will never forget them.

And finally, I would like to thank you, reader. Perhaps you’re one of the few who are obligated to read this, or maybe you just pulled this thesis off the shelf or downloaded it from the university. Regardless, thank you for taking the time to consider this work. I hope that when you close this book or file, you hold a little more knowledge and a little more wonder for terrestrial snails, invasive parasites, and sky islands than before you started. Nothing would make me happier.

TABLE OF CONTENTS ABSTRACT iii ACKNOWLEDGEMENTS iv LIST OF TABLES ix LIST OF FIGURES x LIST OF ABBREVIATIONS xi CHAPTER 1: General Introduction 1 1.1 General Background 1 1.2 Model system 4 1.2.1 Oreohelix 4 1.2.2 Dicrocoelium dendriticum 6 1.3 Selection and development of molecular markers 7 1.4 Thesis objectives 8 1.5 Predictions 10 1.6 References 12 CHAPTER 2: Distribution of the land snails Oreohelix cooperi Binney, 18 1858, in southern Alberta, Canada 2.1 Abstract 19 2.2 Introduction 20 2.3 Methods 23 2.3.1 Study site 23 2.3.2 Sampling protocol 24 2.3.3 Sequencing and analyses 24 2.3.4 Morphological characteristics 27 2.4 Results 27 2.4.1 Sequencing and analyses 27 2.4.2 Morphology 28 2.5 Discussion 29 2.6 Acknowledgements 34 2.7 References 35 CHAPTER 3: Phylogeography of Oreohelix land snails in southern 44 Alberta and Saskatchewan 3.1 Abstract 45 3.2 Introduction 46 3.3 Methods 48 3.3.1 Study areas and sampling 48 3.3.2 DNA extraction and amplification 50 3.3.3 Data analyses 51 3.4 Results 52 3.4.1 COI 52 3.4.2 ITS2 54 3.4.3 Morphological characteristics 54 3.5 Discussion 55 3.5.1 COI overview 55 3.5.2 ITS2 and cytonuclear discord 57 3.5.3 Glacial history 58

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3.5.4 Glacial refugia and colonization 60 3.5.5 PIM vs CSM 63 3.5.6 Contemporary patterns 64 3.5.7 Conclusions and future directions 65 3.6 Acknowledgements 66 3.7 References 67 CHAPTER 4: Spatio-temporal patterns of emerging larval liver fluke 81 (Dicrocoelium dendriticum) in three sympatric species of land snails in southern Alberta, Canada 4.1 Abstract 82 4.2 Introduction 83 4.3 Methods 86 4.3.1 Study area 86 4.3.2 Design of host survey 87 4.3.3 Dissections 87 4.3.4 Molecular diagnostics 88 4.3.5 Analyses 89 4.4 Results 89 4.4.1 Characteristics of host populations 89 4.4.2 Infection patterns 90 4.5 Discussion 91 4.6 Acknowledgements 96 4.7 References 97 CHAPTER 5: General Discussion 107 5.1 Conservation implications and future directions 107 5.2 Phylogeography implications and future directions 109 5.3 Epidemiology of larval D. dendriticum in snails 111 5.4 Future research 112 5.5 General Conclusions 113 5.6 References 115 Appendix 1: Supplementary information for Chapter 3 117 1.1 List of site characteristics 118 1.2 List of samples 121 1.3 List of primers investigated 133 Appendix 2: Supplementary information for Chapter 4 135 2.1 Phenology data for O. subrudis Clade B 136 2.2 Phenology data for O. subrudis Clade X 142 2.3 Phenology data for O. cooperi 148

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LIST OF TABLES Table 2.1 Numbers of Oreohelix sp. collected from seven sites in Cypress Hills Interprovincial Park in May and June of 2013 and 2014. Samples 40 with sequence data are denoted with letters and screened samples are denoted with numbers. Table 2.2 Morphological diagnostic criteria measured for O. cooperi and compared to previous literature (Pilsbry 1939; Chak 2007). 41 Umbilicus/width represents the proportion of the shell width which is umbilicus, ribbing represents proportion of penis that is ribbed, and shell banding represents the percent of the population with two primary coloured bands. Table 3.1 Descriptive genetic polymorphism statistics for COI locus analyzed by site, clade, and region in DnaSP including number of individuals 72 (n), haplotypes (Hap), variable sites (V sites), and haplotype (h) and nucleotide (π) diversities. Table 3.2 Descriptive genetic polymorphism statistics for ITS2 locus sequence data analyzed by site and region in DnaSP including number of 73 individuals (n), haplotypes (Hap), variable sites (V sites), and haplotype (h) and nucleotide (π) diversities. Table 3.3 Number of individuals belonging to each COI clade and ITS2 cluster. Total n = 474. 74 Table 4.1 Summary infection characteristics for larval D. dendriticum within three species of Oreohelix land snails in Cypress Hills 102 Interprovincial Park. Bolded species designations use n = 300, and site designations use n = 150.

LIST OF FIGURES Figure 1.1 Comparison of shell variability in Oreohelix subrudis. (A) Juvenile from western Cypress Hills. (B) Adult from Western Cypress Hills. 16 (C) Adult from Castle Area. (D) Adult from Castle Area. Samples shown here were collected in June 2015. Figure 1.2 Oreohelix subrudis emerging from winter dormancy for preliminary breeding trials. 17 Figure 2.1 Relief map of study area within CHIP (delineated with dashed lines) indicating the locations where the samples of Oreohelix cooperi were 42 collected. Highways and major roads are included for reference. Samples with sequence data are denoted with letters and screened samples are denoted with numbers. Figure 2.2 Phylogenetic tree of COI haplotype data from snails collected in Cypress Hills Interprovincial Park (CHIP). Results include a 43 representative sample of sequences from Weaver et al. (2006), acquired from South Dakota, Wyoming, and Montana (accession numbers DQ857991 – DQ858154). Branch lengths and scale are proportional to average substitutions per site above branches. Tree model consists of maximum likelihood analysis assuming the selected K81uf+G model (-lnL = 2314; Gamma of 0.1825). Figure 3.1 Satellite and relief images of (a) all sampling sites including Weaver et al. (2006). Sites are coded based on COI clade present and 75 patterned circles represent COI clades not found in this study. (b) COI sequencing (numbers) and screening (letters) results in Cypress Hills Interprovincial Park and the Rocky Mountains and (c) ITS2 results in Cypress Hills and Rocky Mountains (Google Earth 2017). The scale bar in each inset represents 20 km. Co indicates O. cooperi, and Py indicates O. pygmaea. Figure 3.2 (a) Phylogenetic tree of COI haplotype data including sequences from Weaver et al. (2006). Branch lengths and scale are proportional 78 to average substitutions per site above branches. (b) Minimum spanning haplotype network constructed for the ITS2 locus. Dashes represent number of base pair differences between haplotypes. Size of the circle is proportional to the number of individuals sharing that haplotype. Shading corresponds to mitochondrial clade. Figure 4.1 Shaded relief map of study area within CHIP marking locations of sampled Oreohelix sp. populations (Google Earth 2016 Map data). 103 Highways and major roads are included for reference, as are site codes. The shading denotes the area covered by CHIP. Figure 4.2 Frequency distributions of Oreohelix sp. shell sizes in Cypress Hills Interprovincial Park, n = 300 per species. 104 Figure 4.3 Seasonal patterns of snail reproduction in three species of Oreohelid snails in Cypress Hills Interprovincial Park (n = 60/site per month). 105 Figure 4.4 Seasonal patterns of prevalence of D. dendriticum in three species of Oreohelid snails in Cypress Hills (n = 60/species/month). 106

LIST OF ABBREVIATIONS, ACRONYMS, AND SYMBOLS ACA Alberta Conservation Association AFLP amplified fragment length polymorphism AIC Akaike’s information criterion AICc corrected Akaike’s Information Criterion ANOVA analysis of variance BH Black Hills BHNF Black Hills National Forest BIC Bayesian information criterion BLAST Basic Local Alignment Search Tool bp base pairs CA Castle Area CH Cypress Hills CHIP Cypress Hills Interprovincial Park CI confidence interval COI Cytochrome Oxidase I COSEWIC Committee On the Status of Endangered Wildlife in Canada CP Crowsnest Pass CSM centrifugal speciation model Dfb humid continental climate DNA deoxyribonucleic acid DnaSP DNA sequence polymorphism dNTP Deoxyribonucleotide triphosphate EID emerging infectious disease F Fisher statistic Fst Wright’s fixation index h haplotype diversity H3 Histone 3 H4 Histone 4 ITS2 Internal Transcribed Spacer 2 IUCN International Union for Conservation of Nature JC Jukes-Cantor JM Judith Mountains K81+G Kimura 81 with gamma distribution K81uf+G Kimura 81 assuming unequal nucleotide frequencies with gamma distribution km kilometres LGM Last Glacial Maximum -lnL negative log likelihood m metres MEGA Molecular Evolutionary Genetics Analysis mg milligrams Mg magnesium MgCl2 magnesium chloride µM micromolar mM millimolar

xi mm millimetres MT Montana n number of individuals or replicates NCBI National Center for Biotechnology Information NSERC Natural Sciences and Engineering Research Council of Canada ºC degrees Celsius p probability value PAUP* Phylogenetic Analysis Using Parsimony *and other methods PIM peripheral isolates model PopART Population Analysis with Reticulate Trees R2 correlation coefficient RM Rocky Mountains SARA Species At Risk Act SD standard deviation SNP single nucleotide polymorphism Ta annealing temperature Taq pol Thermus aquaticus DNA polymerase US United States USA United States of America UT Utah V sites variable sites WLNP Waterton Lakes National Park WY Wyoming π nucleotide diversity 12S 12S ribosomal subunit 5.8S 5.8S ribosomal RNA

Populations

Chapter 2 A Site_A – Horseshoe Canyon B Site_B – Cougar Alley C Site_C – Lookout Reesor Lake D Site_D – Graburn Creek Top E Site_E – Graburn Creek Mid F Site_F – Graburn Creek Bottom

Chapter 3 CH01 Cypress Hills Horseshoe Canyon CH02 Cypress Hills Beaver Creek CH03 Cypress Hills Staff Camp CH04 Cypress Hills Ski Hill CH05 Cypress Hills Highway 41 3 CH06 Cypress Hills Highway 41 2 CH07 Cypress Hills Highway 41 1 CH08 Cypress Hills Highway 41 0

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CH09 Cypress Hills Cougar Alley CH10 Cypress Hills Lookout Reesor Lake CH11 Cypress Hills Trans Canada Trail CH12 Cypress Hills Reesor Lake CH13 Cypress Hills Graburn Creek Middle CH14 Cypress Hills Graburn Creek Bottom CH15 Cypress Hills Nine Mile CHA Cypress Hills Far West CHB Cypress Hills Beaver Creek Transect 5 CHC Cypress Hills Beaver Creek Transect 4 CHD Cypress Hills Beaver Creek Transect 3 CHE Cypress Hills Beaver Creek Transect 2 CHF Cypress Hills McCoy Camp CHG Cypress Hills Opposite Highway CHH Cypress Hills Opposite Ski Hill CHI Cypress Hills Spruce Coulee Halfway CHJ Cypress Hills Spruce Coulee Transect 1 CHK Cypress Hills Spruce Coulee Transect 2 CHL Cypress Hills Spruce Coulee CHM Cypress Hills New Enclosure CHN Cypress Hills Cougar Alley Run 2 CHO Cypress Hills Cougar Alley Run 1 CHP Cypress Hills Graburn Creek Mid CHQ Cypress Hills Old House Valley CHR Cypress Hills Nine Mile Headwaters CHS Cypress Hills Battle Creek Road 1 CHT Cypress Hills Battle Creek Road 2 CHU Cypress Hills Conglomerate Cliffs CHV Cypress Hills Eastern West Block CHW Cypress Hills Warlodge Campground CHX Cypress Hills North Loch Leven CHY Cypress Hills South Loch Lomond RM01 Crowsnest Chinook Lake RM02 Crowsnest North York Creek 2 RM03 Crowsnest North York Creek 1 RM04 Crowsnest Frank Slide RM05 Crowsnest Lundbreck Falls RM06 Castle Area Lynx Creek RM07 Castle Area Castle Falls RM08 Castle Area West Castle 1 RM09 Castle Area Research Station RM10 Castle Area West Castle 2 RM11 Castle Area Beaver Mines Lake 1 RM12 Castle Area Beaver Mines Lake 2 RM13 Wateron Lakes Red Rock Canyon RM14 Wateron Lakes Crandell Lake

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RM15 Wateron Lakes Blakiston Creek RM16 Wateron Lakes Birtha Lake Trail RM17 Wateron Lakes Oil Town

Chapter 4 Oc-1 Oreohelix cooperi site 1, Cougar Alley Oc-2 Oreohelix cooperi site 2, Lookout Reesor Lake OsB-1 Oreohelix subrudis Clade B site 1, Ski Hill OsB-2 Oreohelix subrudis Clade B site 2, Staff Camp OsX-1 Oreohelix subrudis Clade X site 1, Highway 41 0 OsX-2 Oreohelix subrudis Clade X site 2, Highway 41 1

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Chapter 1: General Introduction

1.1 General background

Studies in phylogeography use genetic information to determine the historical processes responsible for the current geographic distribution of individuals and populations (Avise 2000), or simply, the study of evolution and speciation in a geographic and ecological context. Established by Avise et al. (1987), phylogeography has, in recent years, moved from the utilization of mitochondrial DNA and the construction of gene trees to become much more integrative. Thus, phylogeography has become even more of a cross-disciplinary field, using a wide variety of techniques from traditionally disparate scientific fields to characterize and understand mechanisms of speciation and radiation (Beheregaray 2008; Knowles 2009). In addition to biology, a few well-known examples of scientific fields that have contributed to phylogeography include: mathematics, which has formed the underpinnings of statistical inference and model construction necessary for contemporary phylogeography (Knowles 2009); geology, which allows for the determination of ancient population connectivity through deep time in the ever-shifting landscape mosaic (Waters et al. 2001); and other biological fields such as parasitology, which allows for ‘replicates’ of speciation between host species and the parasites with which they are intrinsically connected (Nieberding and

Olivieri 2007; Attwood 2010). Phylogeography has also contributed to other fields such as conservation biology to determine the factors acting on the distribution of threatened or at-risk species, as well as identify populations that are most at-risk (Moritz and Faith

1998).

This new comprehensive approach to phylogeography, coupled with recently developed molecular markers and techniques, has allowed for many ground-breaking discoveries. Bustamante and Ramachandran (2009) used modern phylogeographic methods to show that social structures in early humans differentially altered the effective population sizes of men and women, forming evolutionary patterns that are still detectable. Bos et al. (2014) used archeological data coupled with comparative genomics, to find that human tuberculosis was introduced to the Americas by seals rather than humans. Carnaval et al. (2009) used ecological niche modelling across multiple species in the Brazilian Atlantic forest to find a biodiversity hotspot that has frequently acted as a refugium during unfavourable climate conditions. This work was the first step in developing conservation policies that have since been implemented in this region.

Many studies of phylogeography involve large-bodied or highly mobile animals, which are typically charismatic vertebrates such as mammals and birds. Studies on highly mobile pelagic seabirds such as albatross (Thalassarche; Burg et al. 2017), aesthetically pleasing mammals such as red pandas (Ailurus fulgens; Li et al. 2005), and large-bodied reptiles such as sea turtles (Chelonioidea; Bowen and Karl 2007) comprise the bulk of scientific literature. In contrast, the invertebrates that constitute the vast majority of animal biodiversity on earth (Pechenik 2009) are often neglected in phylogeographic research. On average, endangered vertebrates have an order of magnitude more peer- reviewed, species-specific articles than endangered invertebrates (Donaldson et al. 2016).

Invertebrates in both aquatic and terrestrial systems face many of the same challenges faced by vertebrates including dispersal and migration, and can be used as model systems to answer key questions in phylogeography.

Studies of terrestrial gastropods, especially snails capitalize on the multi-faceted field of phylogeography. Terrestrial snails are intrinsically connected to the landscape.

These small-bodied invertebrates typically have low dispersal potential and often have specific environmental requirements. They also require access to habitat components such as calcium to support shell growth and specific vegetation to support feeding and cover (Pilsbry 1939). This intrinsic connection to the landscape, coupled with the availability of fossil records of shells and known glacial history, make them ideal model systems. Phylogeographic studies of terrestrial gastropods, especially snails, have provided superb models to understand processes such as recent extinctions across taxa

(Régnier et al. 2015), phenotypic versus genotypic changes in response to elevation in

European mountain snails (Gittenberger et al. 2003), and reconstructing locations of glacial refugia in eastern Europe (Horsal et al. 2010). Despite being important macro- detritivores and hosts of important parasites of domestic animals and wildlife, biogeographic and systematics studies involving terrestrial snails, especially in North

America, are limited and most studies have historically used morphological comparisons of shells (Dall 1905; Pilsbry 1939). This is problematic as morphological comparisons are not representative of true phylogenetic history and have very low resolution for distinguishing among closely-related taxa (example in Fig. 1.1; see also Stearns, 1989).

Notable among these are the North American mountain snails (Oreohelix). The current taxonomy of Oreohelids is in turmoil, the primary cause of which is a disparity between molecular and morphologically based phylogenies introduced by new classification techniques (Dall 1905; Pilsbry 1939; Weaver et al. 2006; Chak 2007). Such disparities and lack of consensus have complicated interpretations of the biodiversity, phylogeny,

and systematics of this group of snails and have complicated petitions to list a number of

Oreohelix species, including Oreohelix cooperi, as threatened under the US Endangered

Species Act and International Union for Conservation of Nature (IUCN) Red List (Frest and Johannes 2002).

1.2 Model system

1.2.1 Oreohelix

The Oreohelix genus holds a rich history. The earliest fossil evidence of

Oreohelix was found in western Alberta (Calgary, Pincher Creek, and upper Oldman

River basin) and dated back to the Cretaceous period (Henderson 1935). There is a notable absence of Oreohelid fossils in Alberta following the Cretaceous. Presumably,

Oreohelids were extirpated from Alberta due to changes in climate, shifting habitat availability, and eventually the ice sheets of the Pleistocene. The next appearance of

Oreohelix in the fossil record is in New Mexico and northwestern Wyoming (primarily near Yellowstone National Park) dating back to the Eocene (Henderson 1935). More recent work has shown that Oreohelids were present in Wyoming and Montana during the Pleistocene (Weaver et al. 2006), and the current distribution of Oreohelix extends from western Alberta to New Mexico along elevated regions such as the Rocky

Mountains (Pilsbry 1939).

Mountain snails of the genus Oreohelix are large bodied (up to 2.5 cm shell diameter), long lived (~10 years), hermaphroditic, and viviparous (Fig. 1.2; see also

Pilsbry 1939). These snails form an important component of mountain invertebrate

communities in western North America, particularly in the Rocky Mountains and nearby isolated sky islands to the east. The numerous isolated sky islands surrounding the Rocky

Mountains, coupled with the frequent Pleistocene climate shifts, have produced many divergent species of Oreohelix through repeated bouts of isolation and secondary contact

(Weaver et al. 2006). There are at least five species of Oreohelix known to occur in northern Wyoming, Montana, and Alberta: O. yavapai, O. subrudis, O. strigosa, O. pygmaea, and O. cooperi (Pilsbry 1939; Chak 2007). Oreohelix subrudis and O. strigosa are further divided into several subclades based on patterns inferred from mitochondrial barcoding (Weaver et al. 2006; Chak 2007).

Canadian Oreohelix species are contentious in terms of biodiversity, taxonomy, distribution, phylogeography, and conservation status. Dall (1905) reported Oreohelix stantoni in Cypress Hills Interprovincial Park (CHIP), an isolated plateau approximately

250 km from the Rocky Mountains in Southern Alberta and Saskatchewan, and

Oreohelix cooperi west of the Lake of the Woods, Ontario. Although Dall (1905) did not use reproductive morphology in his assessment, a method commonly used to identify terrestrial snails by species, his results were reported in Pilsbry (1939). Russell (1951) completed another survey of snails in Cypress Hills, and reported three Oreohelix species: O. strigosa cooperi, O. subrudis limitaris, and O. subrudis subrudis. More recently Beck (2015) and van Paridon et al. (2017) surveyed terrestrial snails in CHIP using mitochondrial barcoding primers and reported O. subrudis and O. cooperi.

Although Oreohelids in CHIP and the Albertan Rocky Mountains have been surveyed, their distribution, connection to each other, and connection to well-studied populations of

Oreohelids in Montana and Wyoming have not been investigated. The phylogeography of

Oreohelids in Alberta and Saskatchewan is entirely unknown, especially in relation to previous studies in the northern USA (Weaver et al. 2006; Chak 2007).

1.2.2 Dicrocoelium dendriticum

Another unknown aspect of Oreohelids in CHIP is the patterns of infection with an invasive live fluke, Dicrocoelium dendriticum, where the snail is the intermediate host. Dicrocoelium dendriticum is a host generalist with an obligate three-host life cycle

(reviewed in Goater et al. 2014). The adult trematode rests in the bile ducts of grazing herbivores and produces eggs that pass into the feces. The first intermediate host, a terrestrial snail, ingests the eggs. The eggs hatch into miracidia, which burrow through the flesh of the snail and form sporocysts, daughter sporocysts, and eventually free-living and mobile cercariae. These cercariae are expelled in groups of approximately 100 from the snail through the respiratory pore in a “slime ball,” that is ingested by the next intermediate host, formicid ants. The ants ingest the slime ball and the cercariae pierce their abdominal wall. The majority of the cercariae encyst and become metacercariae in the abdomen of the ant, but a single metacercariae travels to the brain without encysting.

This metacercariae radically alters the behaviour of the ant (so-called “zombie-ant” behaviour), causing it to exhibit temperature-dependent clinging behaviour on the tops of herbaceous vegetation, greatly increasing the chance of accidental ingestion by the final herbivore host (typically an ungulate). Ingested metacercariae hatch in the intestines and migrate to the liver where they mature and produce eggs for the next 2-3 years (Manga-

González et al. 2001).

The ability of this parasite to be a host generalist has allowed it to invade Canada.

This trematode was discovered in elk and deer in CHIP in the 1990s (Pybus 1990) and since then has spread in prevalence and intensity throughout all of the large herbivores in the Park (Goater and Colwell 2007). The epidemiology of D. dendriticum in CHIP has been extensively studied in the ant intermediate host (Beck 2015; van Paridon et al. 2017) and the final hosts including cattle, elk, and deer (Beck et al. 2015). There is very little known regarding patterns of infection at the level of the first-intermediate host in a region of emergence, which is an important step towards mitigation of further range expansion.

Thus, inter-specific variation in susceptibility or exposure of Oreohelid snails to D. dendriticum larvae and spatio-temporal patterns of infection have not been described.

1.3 Selection and development of molecular markers

The primary molecular markers used in my thesis are the mitochondrial cytochrome oxidase I (COI) and nuclear internal transcribed spacer 2 (ITS2). Using markers with different modes of inheritance, uniparentally for mitochondrial and biparentally for nuclear, allows the elucidation of patterns that might go unnoticed in studies that focus on a single marker. COI is commonly used in barcoding and species differentiation due to its high rate of evolution in comparison to most nuclear loci (Hebert et al. 2003), allowing for differentiation between populations (Avise et al. 1987). While

COI barcoding is widely used and efficient in terms of quickly identifying species,

Moritz and Cicero (2004) advise phylogenetic analyses of COI should be used in conjunction with other molecular markers and phenotypic markers such as morphological measurements. The ‘one gene fits all’ approach is overly simplistic and COI barcoding

should be used as an aid in species identification rather than the sole technique. Often paired with COI barcoding is ITS2 barcoding, as the addition of a nuclear locus allows the detection of introgression, incomplete lineage sorting, and hybridization (Avise

2000).

Few variable molecular markers have been developed for terrestrial molluscs. The mitochondrial 12S subunit (12S) used by Weaver et al. (2006) to study Oreohelids in the

USA contained little variation, and the nuclear Histone 3 (H3) and Histone 4 (H4) loci were noted by Cadahia et al. (2013) to be useful in identifying deeper phylogenetic splits in terrestrial molluscs when used in conjunction with more conventional barcoding primers. While the development of microsatellites is useful in determining relatedness within populations of snails and microsatellites do exist for other terrestrial snail genera such as Arianta (Armbruster et al. 2005) and Helix (Arnaud et al. 2001), the use of microsatellites is outside the scope and timeframe of my thesis.

1.4 Thesis objectives

The primary aim of this study is to determine the biodiversity, phylogeography, and conservation status of the Oreohelid species in southern Alberta and Saskatchewan and their relation to Oreohelids described by previous studies (Chak 2007; Weaver et al.

2006; Pilsbry 1939). This thesis describes the general phylogeography of Oreohelids in upland regions of southern Saskatchewan and southern Alberta including the Rocky

Mountains. An additional goal of this thesis is to determine the epidemiology of the

emerging liver fluke, Dicrocoelium dendriticum, in Oreohelix spp. in a region of recent emergence in CHIP.

To characterize the conservation status of the Oreohelid species in CHIP, including the potential presence of Oreohelix cooperi, analyses of gene sequences of mitochondrial (CO1) and nuclear (ITS2) marker, together with comparisons of morphological characters and preliminary laboratory mating studies were used. Oreohelid taxonomy is in turmoil (Dall 1905), primarily due to early mollusc survey reliance on plastic phenotypic markers and general lack of investigation. This thesis focuses on the use of molecular techniques to characterize the biodiversity and conservation status of potentially-threatened Oreohelix spp. in southern Canada, and incorporates standard morphological comparisons where appropriate.

To characterize the biodiversity of Oreohelix spp. in CHIP and adjacent sites in the southwestern Canadian Rocky Mountains, screening primers were developed for the mitochondrial (CO1) and nuclear (ITS2) loci. These were used in conjunction with fossil records and landscape history to characterize the phylogeography of Oreohelix spp.

To evaluate seasonal, host-species, and spatial variation in D. dendriticum infection in Oreohelix spp. in CHIP, samples of 30 snails were collected monthly from six sites in CHIP in May to September of 2015. While other parasites infect Oreohelids in

CHIP, the primary focus is on D. dendriticum due to its industry-related impact and available epidemiology research in other hosts.

This thesis is arranged into five chapters. The second chapter examines the occurrence of O. cooperi in CHIP using DNA sequencing and morphological

measurements in the context of conservation biology. The third chapter describes the phylogeography of Oreohelid snails in southern Alberta and Saskatchewan using molecular screening. The fourth chapter describes the epidemiology of D. dendriticum in

Oreohelid hosts of CHIP, focusing on spatio-temporal patterns of prevalence. Chapter five summarises the key findings and connects them into a single framework, and also discusses the future of research into this system.

1.5 Predictions

I predict, based on preliminary data (van Paridon and Goater, unpublished data), that O. cooperi is present in CHIP and corresponds to populations of small terrestrial snails found on dry, shrubby slopes. If O. cooperi is limited to CHIP in Canada, or if other Oreohelids present in CHIP are endemic to the region, then follow-up conservation and management initiatives will be required.

Sky islands such as CHIP can be hotspots of biodiversity and the fragmentation of populations across multiple sky islands can amplify this effect. I predict that CHIP will contain multiple species of Oreohelix, with similar genetic variation to those found by

Weaver et al. (2006) in sky island habitats in Montana, Wyoming and South Dakota.

Analyses of distributional patterns of Oreohelix within CHIP and within sites in the

Canadian Rocky Mountains will allow me to determine how historical factors (e.g. glaciation) and contemporary factors (e.g. local adaptation) have shaped observed patterns of distribution.

One reason that emergent infectious agents are of concern is because their hosts have not acquired sufficient resistance to restrict their expansion. I predict that the

Oreohelid snails of CHIP are heavily infected with D. dendriticum, and patterns of infection will vary depending on biotic and abiotic ecological factors such as final host movement patterns or changes in temperature and precipitation. Evaluation of rates of larval trematode infection in Oreohelix spp. will provide important information regarding the utilization of sympatric host communities for an emerging parasite of cattle and other wildlife in western Canada.

1.6 References

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Avise JC. 2000. Phylogeography: The history and formation of species. Harvard Press, Cambridge, MA.

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Goater CP and Colwell DD. 2007. Epidemiological characteristics of an invading parasite: Dicrocoelium dendriticum in sympatric wapiti and beef cattle in southern Alberta, Canada. Journal of Parasitology 93: 491-494.

Goater TM, Goater CP, and Esch GW. 2014. Parasitism: The diversity and ecology of animal parasites, 2nd edition. Cambridge University Press, Cambridge, UK.

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Li M, Wei F, Goossens B, Feng Z, Tamate HB, Bruford MW, and Funk SM. 2005. Mitochondrial phylogeography and subspecific variation in the red panda (Ailurus fulgens): implications for conservation. Molecular Phylogenetics and Evolution 36: 78-89.

Manga-González MY, González-Lanza C, Cabanas E, and Campo R. 2001. Contributions to and review of dicrocoeliosis, with special reference to the intermediate hosts of Dicrocoelium dendriticum. Parasitology 123: S91-S114.

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Pilsbry HA. 1939. Land mollusca of North America north of Mexico vol I part 2. Acad Nat Sci Philadelphia. Wickersham Printing Company. Lancaster, Pennsylvania.

Pybus, M. J. 1990. Survey of hepatic and pulmonary helminths of wild cervids in Alberta, Canada. Journal of Wildlife Diseases 26: 453-459.

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Stearns SC. 1989. The evolutionary significance of phenotypic plasticity. BioScience 39: 436-445. van Paridon B, Gilleard JS, Colwell DD, and Goater CP. In press. Life cycle, host utilization, and ecological fitting for invasive lancet liver fluke, Dicrocoelium dendriticum, emerging in southern Alberta, Canada. Journal of Parasitology.

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A B C D

~2 cm

Figure 1.1. Comparison of shell variability in Oreohelix subrudis. (A) Juvenile from western Cypress Hills. (B) Adult from western Cypress Hills. (C) Adult from the Rocky Mountains Castle Area. (D) Adult from Castle Area. Samples shown here were collected in June 2015.

~1 cm

Figure 1.2. Oreohelix subrudis emerging from winter dormancy for preliminary breeding trials.

CHAPTER 2

Distribution of the land snail Oreohelix cooperi Binney, 1858, in southern Alberta,

Canada

Z. W. Dempsey1,2, T. M. Burg1, C. P. Goater1

Prepared as manuscript for submission

1University of Lethbridge, Department of Biological Sciences, 4401 University Drive,

Lethbridge, AB, T1K 3M4, Canada

2Corresponding author: [email protected]

2.1 Abstract

Elucidating the nature of endemicity is a primary aim of conservation biologists, especially for inconspicuous or cryptic species that are at the edges of their geographical distribution, or are naturally rare in occurrence. Sub-populations of a small-bodied, terrestrial Oreohelid snail were observed in Cypress Hills Interprovincial Park (CHIP) in southeastern Alberta that phenotypically resembled Oreohelix cooperi – a snail that is described in the literature as endemic to the Judith Mountains in Montana and the Black

Hills in South Dakota and Wyoming, U.S.A. I compared morphological characteristics and 685 base pair sequences of the COI barcoding gene of Oreohelid snails collected from six sites in CHIP to type material and to sequences published in GenBank. COI sequences matched O. cooperi from the U.S.A. Morphological analyses of shell shape and male genitalia were consistent also with published descriptions of O. cooperi. COI sequence similarities and morphological characteristics of these snails did not match two other larger species of sympatric Oreohelid found in Cypress Hills Interprovincial Park and at sites in the Alberta Rocky Mountains. My results confirm the presence of O. cooperi within high elevation sites in Cypress Hills Park in Canada, thereby extending its geographical range outside its presumptive site of endemicity in the Judith Mountains and

Black Hills.

2.2 Introduction

A primary goal of conservation biologists is to determine the factors that determine and maintain biodiversity (Hunter and Gibbs 2009), and it has historically been concerned with identifying areas of high endemism and species that naturally occur at low densities (Morrone 1994). As anthropogenic extinction events increase in occurrence throughout the biosphere, the protection of species, particularly those that have a restricted distribution, is important now more than ever (Barnosky et al. 2011; Barnosky et al. 2012). Due to the often-limited resources available to conservation biologists, at- risk species are emphasized and large-bodied or charismatic vertebrates tend to be given higher priority. Invertebrates comprise over 98% of animal biodiversity (Pechenik 2009), yet they are often neglected by policy makers and conservation biologists. Donaldson et al. (2016) found that endangered vertebrates had, on average, an order of magnitude more species-specific peer-reviewed conservation articles than endangered invertebrates. While the conservation status of countless mammal and bird species have been evaluated by the

International Union for Conservation of Nature (IUCN), less than 0.05% of known invertebrate species are registered on the IUCN Red List (Régnier et al. 2015).

In Canada, the Committee on the Status of Endangered Wildlife in Canada

(COSEWIC) designated by the Species at Risk Act (SARA) in 2002 began cataloguing threatened and potentially threatened species. As of November 2016, COSEWIC had evaluated 987 species of plants and animals based on IUCN Red List criteria (COSEWIC

2016 year-end report). Of those, only 117 were invertebrates, most of which were arthropods and freshwater molluscs. Despite the historical lack of public and scientific interest (Kellert 1993), invertebrate conservation has been gaining momentum in recent

20 years, largely in response to studies that describe their importance within ecosystems

(Costanza et al. 2007; Handa et al. 2014; Régnier et al. 2015). A prominent example is the recognition and conservation of important invertebrate species such as freshwater mussels, which are important ecosystem engineers and are experiencing rapid declines in

North America (Williams et al. 1993; Arbuckle and Downing 2002).

One of the best known endangered invertebrate species in Canada is the Banff

Springs snail (Physella johnsoni) restricted to only five hotspring sites within Banff

National Park (Lepitzki 2002). Other examples of well-known endangered molluscs include the freshwater snuffbox (Epioblasma triquetra), once widespread throughout

North America but now found only in two rivers in Ontario (Zanatta and Murphy 20008).

Likewise, the hotwater Physa (Physella wrighti) is severely restricted to sites within

Liard River Hot Springs Provincial Park in northeastern British Columbia (Remigio et al.

2001). Molluscs typically have low vagility and specialized niche requirements making them prone to high rates of endemism (Régnier et al. 2009). While some invertebrate species such as the Banff Springs snail have received much attention, parallel conservation efforts for other species have been limited by the absence of data regarding geographical distributions of potentially at-risk fauna, particularly those with small body sizes or cryptic colouration (Cordoso et al. 2011). Much of the work regarding the systematics of terrestrial snails was done before the development of molecular tools leading to taxonomic redundancy with the same species given different names based on phenotypically plastic criteria such as shell structure (Dall 1905; Pilsbry 1939; Dowle et al. 2015). While this problem may have been unavoidable in the past, the introduction of molecular techniques to conservation biology has allowed for a more rigorous assessment

21 of species with notoriously phenotypically plastic traits. Mitochondrial COI barcoding is one such technique widely used in many conservation studies (Neigel et al. 2007).

A prominent genus of North American terrestrial mollusc that has been the subject of recent molecular study comprise mountain snails in the genus Oreohelix. These snails grow up to 2.5 cm in shell diameter, can live up to a decade, are hermaphroditic, and viviparous (Pilsbry 1939). The Oreohelids form an important component of mountain invertebrate communities in western North America, particularly in the Rocky

Mountains. The systematics, distribution, and biodiversity of Canadian Oreohelix species are poorly known. Recent work using molecular techniques includes a survey of terrestrial snails in North America by Weaver et al. (2006) in the Black Hills of South

Dakota and the eastern edge of the Rocky Mountains. Weaver et al. (2006) found a number of distinct COI mitochondrial lineages including the Black Hills mountain snail,

Oreohelix cooperi, endemic to the Black Hills and Judith Mountains. Earlier reports by

Pilsbry (1939) indicated the presence of a ‘small-bodied’ Oreohelid that he identified as

O. cooperi that occurred approximately 750 km northwest of the Black Hills in a sky island known as Cypress Hills in southwestern Saskatchewan and southeastern Alberta in

Canada. This small-bodied form has not been reported from this region since then, nor has it been reported in adjacent regions of the Canadian Rocky Mountains. In the U.S.,

Frest and Johannes (2002) listed O. cooperi as rare and deserving of conservation status and a petition was initiated to list this species as endangered (Biodiversity Conservation

Alliance 2003). The petition was rejected due to lack of evidence of habitat degradation and a poor understanding regarding its geographical distribution.

In summer 2013, a population of small terrestrial Oreohelid snails matching the physical description of O. cooperi was observed on dry, shrubby slopes in Cypress Hills

Interprovincial Park in southeastern Alberta (Goater, personal observation). We hypothesize that the small-bodied Oreohelid snail in this region is synonymous with O. cooperi as originally suggested by Pilsbry (1939). The purpose of this study is to combine molecular barcoding methods with traditional morphological assessments to identify the small-bodied species of Oreohelid found in Cypress Hills Park and adjacent regions of the Canadian Rocky Mountains.

2.3 Methods

2.3.1 Study site

Cypress Hills Interprovincial Park (CHIP; 49°34’N, 110°00’W) is a series of elevated plateaus rising to more than 1400 m in elevation, approximately 400 m above the surrounding prairie in Southeastern Alberta and Saskatchewan, Canada. The high- elevation plateau is topped with fescue grassland and the slopes are dominated by a mixture of lodgepole pine (Pinus contorta), white spruce (Picea glauca), and trembling aspen (Populus tremuloides) (Newsome and Dix 1968). The top of CHIP was a nunatak during the Wisconsinan glaciation during the last glacial maximum (Stalker 1965). CHIP accumulated sediment during this time and prominent melt water channels were formed with the recession of the Laurentide ice sheet, the slopes of which are currently occupied by Oreohelid snails (Stalker 1965). CHIP contains suitable habitat for terrestrial snails

23 including O. cooperi due to its precipitation levels, vegetation, and prevalence of calcareous deposits (review in van Paridon et al. 2017).

2.3.2 Sampling protocol

Ninety-nine sites with suitable Oreohelix habitat were searched in CHIP and the

Rocky Mountains including Waterton Lakes National Park, Castle Management Area, and Crowsnest Pass during summers of 2014 to 2016. Oreohelid snails were present at 58 of sites (Chapter 3), and small-bodied snails matching the description of Oreohelix cooperi were only present at 11 sites within the Alberta and Saskatchewan sides of CHIP

(Fig. 2.1). From those sites, individual adult snails (n=80) were collected (Table 2.1). The small-bodied form was mainly found on dry, south-facing slopes, dominated by characteristic rocky formations known as ‘Cypress Hills formation.’ These locations tend to be high elevation sites with distinctive rock/gravel formations and sparse vegetation dominated by juniper and other low profile shrubs such as Potentilla (Newsome and Dix

1968; Leckie and Cheel 1989). Plots of 10 m2 were demarcated for each sample site following methods in van Paridon et al. (2017) and the first 12 snails encountered were collected. Snails were stored in ethanol at -80 °C. Approximately 2 mg of foot tissue was used for a modified chelex DNA extraction (Walsh et al. 1991).

2.3.3 Sequencing and analyses

Extracted DNA (n=51) was amplified with universal COI barcoding primers

LCO1490 (5’ – GTC AAC AAA TCA TAA AGA TAT TGG – 3’) and HCO2198 (5’ –

TAA ACT TCA GGG TGA CCA AAA AAT CA – 3’) (Folmer et al. 1994). The 655 bp region of the COI locus was amplified in an Eppendorf master cycler under the following conditions: 1 cycle of 94 °C for 2 minutes, 50 °C for 45 seconds, and 72 °C for 1 minute;

37 cycles of 94 °C for 30 seconds, 50 °C for 45 seconds, and 72 °C for 1 minute; with a final cycle at 72 °C for 5 minutes. Standard master mix reagent concentrations were used:

1 Unit flexi DNA polymerase, flexi buffer, 0.2 mM dNTP, 0.4 µM of each primer, and 3 mM MgCl2. Amplified DNA was visualized on agarose gel stained with ethidium bromide and sent to McGill University for sequencing.

The COI locus showed fixed differences between species of Oreohelids that were used to develop screening primers that would preferentially amplify each of the different mitochondrial clades. This method allowed me to assign samples to one of the clades using only PCR. The primers HCOIcommO (5’ – TAA ACT TCA GGG TGA CCA

AAA – 3’) and LCOIspecOc635 (5’ – TGC TCT TTC ACT TTT AAT TCG AC – 3’) preferentially amplified the O. cooperi COI locus and were used in a multiplex PCR along with LCOIspecOs563 (5’ – ATT GTT ACA GCC TAT GCC – 3’) for Oreohelix

Clade X, and LCOIspecOx217 (5’ – GTG CCC CAG GAA TAA ATT TG – 3’) for

Oreohelix Clade B (Chapter 3). The remainder of the samples (n=29) were screened with the screening primers. The COI screening PCR was run with a 48 °C annealing temperature and 1 mM MgCl2. Amplified DNA was visualized and scored on 3% agarose gel stained with ethidium bromide.

Sequences were checked, aligned, and trimmed in MEGA 6.06 (Tamura et al.

2013) alongside Oreohelid sequences obtained from Weaver et al. (2006). An outgroup consisting of Oreohelix haydeni hybrida was used to root the phylogenetic tree (accession

25 numbers DQ858138-DQ858140; Weaver et al. 2006). A phylogenetic tree of haplotypes from each of the six CHIP sites plus published haplotypes in Weaver et al. (2007) was constructed using Jukes-Cantor (JC) model in PAUP* 4.0a151 (Swofford 2002) for automated model selection. Maximum likelihood models of phylogenetic tree construction were ranked based on Akaike Information Criterion corrected (AICc) for finite sample sizes. AICc was selected over AIC (uncorrected) due to low sample sizes and Bayesian Information Criterion (BIC) because of the large number of potential models. The model with the lowest AICc score was used to construct a phylogenetic tree that was validated with 1000 bootstrap replicates at 50% consensus.

Two additional loci were also investigated for their potential to distinguish

Oreohelid clades: Internal Transcribed Spacer 2 (ITS2) and Histone 4 (H4). Neither of these loci were used by Weaver et al. (2006) or Chak (2007), but were used in this study to potentially provide additional marker discrimination for the small-bodied snails and sympatric large-bodied Oreohelids found in CHIP. A subset of Oreohelids (n=86) of both size morphs were sequenced with the ITS2 primers LSUIF2990 (5’ – CTA GCT GCG

AGA ATT AAT GTG A – 3’) and ITS4R3908 (5’ – TCC TCC GCT TAG TTA TAT GC

– 3’) to produce a 472 bp fragment (Wade and Mordan 2000). Oreohelids (n=21) of both size morphs were also sequenced using primers AriaH4_fwd (5’ – AAC CTC CGA AGC

CGT ACA GGG T – 3’) and AriaH4_rev (5’ – GAA GAG GTA AAG GCG GCA AGG

G – 3’) to produce a 268 bp fragment (Cadahía et al. 2013). PCR conditions were similar to the COI sequencing primer sets, with the exception of 1.5 mM MgCl2 and 54 °C annealing temperature for ITS2, and 58 °C annealing temperature for the H4 primer set.

2.3.4 Morphological characteristics

Sixty adult snails were collected from sites B and C (Fig 2.1) for assessments of morphological characteristics. Snails were immediately preserved in 95% ethanol upon collection. Maximum shell width and umbilicus width were measured using a Vernier caliper. The ratio of umbilicus width to shell width was calculated, as it is traditionally seen as diagnostic for species differentiation in some species of snail, including O. cooperi (Pilsbry 1939). The frequency of individuals with two primary shell bands was also calculated (Pilsbry 1939; Chak 2007). Shells were gently crushed and then viewed via dissecting microscope and the genitalia removed. The total length of the penis was measured (n = 6) and compared to the length of the penis which was internally ribbed using a dissecting Amscope camera with associated software, and a proportion was derived and compared to previous literature (Pilsbry 1939; Chak 2007).

2.4 Results

2.4.1 Sequencing and analyses

All 51 snails that were sequenced at the COI locus contained one of six haplotypes and six variable sites were found in the 685 bp COI sequence. Automated selection of a phylogenetic tree model by AICc score (best score of -lnL = 2314) resulted in the following model: Kimura 81 (Takahata and Kimura 1981) assuming unequal nucleotide frequencies with Gamma distribution (K81uf+G, Gamma shape of 0.1825;

Fig. 2.2). The Gamma shape of less than one indicates a high variation in the rate of evolution across the locus, with a few sites evolving rapidly and most other sites evolving

27 slowly (Liò and Goldman 1998). Each of the 51 snails that were barcoded clustered into a single clade. This clade matched O. cooperi sequence from Weaver et al. (2006). Of the

51 sequences recovered, only 11 haplotypes were used in the construction of the phylogenetic tree such that all sample sites were represented.

The ITS2 sequences showed O. cooperi to form a single monophyletic clade with approximately 18 fixed differences from the large bodied snails found in CHIP. There was no evidence of introgression/hybridization or incomplete lineage sorting between O. cooperi and the large bodied snails at the ITS2 locus. The ITS2 locus was used for further population genetic studies (Chapter 3). The H4 sequences showed O. cooperi to have a fixed 9 bp deletion, whereas some of large bodied snails of CHIP had the deletion and some did not. Due to problems with nonspecific primer binding, it was not feasible to sequence or screen the H4 locus in large quantities.

2.4.2 Morphology

Oreohelix cooperi from dry shrubby slopes in CHIP were found to range in size from 6-11 mm or roughly 50-65% the size of those found in the Black Hills (Weaver et al. 2007). Oreohelix cooperi from moist pine slopes (sites E and F) were found to range in size from 8-12.5 mm or roughly 60-75% as large as those found in Black Hills (Table

2.2). The ratio of internal penis ribbing was found to be on average higher than those found by Chak (2007), but there were no cases in which the internal ribbing of the penis constituted more than half of the total length of the penis. The umbilicus to width ratio was found to mirror both Chak (2007) and Pilsbry’s (1939) measurements.

2.5 Discussion

Based on COI sequence similarities and comparison of traditional diagnostic characters, the small-bodied Oreohelid snail found in Cypress Hills Interprovincial Park is synonymous with Oreohelix cooperi. Thus, the COI sequences of the small-bodied morph found in CHIP matched O. cooperi reported by Weaver et al. (2006) and corroborated by Anderson et al. (2007) and Chak (2007) for snails collected in the Black

Hills National Forest (BHNF) in South Dakota and Judith Mountains (JM) in Montana,

U.S.A. These results confirm the extent of the range of O. cooperi into Canada (Russell

1951). This species has not been found at comparable sites in adjacent regions of the eastern Canadian Rocky Mountains. Therefore, we tentatively conclude that O. cooperi is likely restricted to high-elevation sites within the Alberta and Saskatchewan sides of

Cypress Hills Interprovincial Park in Alberta, Canada. However, localities within the foothills ecozone in Alberta such as Milk River Ridge and Porcupine Hills should also be surveyed for O. cooperi.

Confusion in the taxonomy of the small-bodied morph of Oreohelix spp. is long- standing. Chak (2007) reported that the designation of O. cooperi (O. strigosa cooperi) for snails collected from Tensleep Canyon, Wyoming, described in Weaver et al. (2006,

WY308 and WY316) was incorrect and should have been O. pygmaea, a species closely related to O. cooperi but differentiated at a number of nuclear loci. Chak (2007) also indicated that the designation of O. pygmaea should be viewed with caution because O. pygmaea and O. cooperi are very difficult to distinguish morphologically and their COI sequences are very similar. Chak (2007) did not comment on the designations by Weaver

29 et al. (2006) regarding O. cooperi in samples from the Black Hills National Forest

(BHNF), suggesting that the rest of the samples from Weaver et al (2006) were correctly assigned. My phylogenetic analyses involving the COI locus, including sequences available from Weaver et al. (2006), indicates that all of the suspected O. cooperi sequences from CHIP group with the BHNF sequences, rather than the Tensleep Canyon ones or O. strigosa snails. Based on these comparative data for small-bodied snails evaluated in CHIP, the BHNF (Weaver et al. 2006) and eastern Wyoming, the COI sequences recovered from snails in CHIP are O. cooperi.

The small-bodied snails found in CHIP are smaller than typical specimens found by Pilsbry (1939) and Chak (2007). While still smaller than those in Black Hills, there are a few sites in CHIP that show larger body sizes than other CHIP sites (E, F, 3, and 5, hereafter referred to as EF35), which are at the lower end of the size ranges reported for

O. cooperi (about 12 mm shell length). Further, the internal ribbing of the penis is less than half the total length of the penis in O. cooperi, which concurs with results published by Pilsbry (1939) and Chak (2007). Differences in ribbing ratio between this work and

Chak (2007) are likely due to differences in sample preparation. While Chak (2007) used increasing dilutions of ethanol to preserve specimens, I immediately preserved snail samples in 95% ethanol. Differences in fixation technique can lead to significant changes in apparent morphology (Costa-Paiva et al 2007; Martinez and Berbel-Filho 2012).

Pilsbry (1939) lists O. cooperi as endemic to BHNF and CHIP, and mentions a dwarf variety O. s. stantoni in CHIP (Dall 1905), Forsyth (2006) indicates O. s. stantoni is synonymous with O. cooperi, and it matches the size of the small morph found in CHIP in this study. Dall (1905) did not use dissections or penis morphology in his description

30 of O. s. stantoni, and describes O. s. cooperi as being the larger of the two size morphs present in CHIP and extending eastward to the Lake of the Woods in Ontario. It is unclear if he was referring to the slightly larger morphs of O. cooperi found in EF35 or the larger still (~16 mm) Oreohelix subrudis, which is also found in CHIP (sp. B-E in

Weaver et al. 2007; Chapter 3; van Paridon et al. 2017). The latter is most likely given that O. subrudis was not mentioned by name in CHIP (Dall 1905) and has been found in the far eastern corners of CHIP (Chapter 3). Pilsbry (1939) cautions that diagnostic morphological characteristics of terrestrial molluscs vary widely within species and even within the same population, and also notes that the size and shape of the shell varies over short distances depending on proximity to water, vegetation, and calcareous deposits.

This is also exemplified by the EF35 samples in CHIP that are genetically identical to other O. cooperi sites at the COI locus, but have larger shell sizes. Chak (2007) also found two size morphs of O. cooperi in BHNF, which was correlated with factors such as moisture and calcium levels, and negatively correlated with elevation (also noted by

Pilsbry 1939). These findings parallel my observations of the larger O. cooperi in the moist valley bottoms in sites E and F, or on pine-dominated slopes in sites 3 and 5.

In CHIP, the ecological niche preference of O. cooperi is predictable. With the exception of sites EF35, O. cooperi in CHIP tended to be found on dry, shrubby slopes.

The EF35 sites are unusual for O. cooperi in that they are shaded slopes dominated by pine, and they tend to be cooler and moister. My anecdotal evidence also suggests that snail densities are highest in these types of habitats especially in microhabitats dominated by decaying plant matter beneath ground juniper (Juniperus horizontalis) or aestivating on the underside of ground juniper stems. Oreohelix cooperi is also closely associated

31 with outcroppings of conglomerate rock commonly found in CHIP. While conglomerate rock was not present at every site where O. cooperi was found (site C for example), most sites containing outcroppings of conglomerate rock also contained O. cooperi (personal observation). When conglomerate outcroppings are present, O. cooperi tends to be found in the vegetation at the base of the outcropping or in adjacent vegetation. The association of O. cooperi with ground juniper and conglomerate rock is likely due to these landscape features being associated with high soil calcium (Flugel 2010). The presence of O. cooperi in EF35 is interesting in that these sites are atypical of O. cooperi in CHIP, although these sites resemble many found in BHNF (e.g. BH6, BH12, BH14 in Weaver et al. 2007). The disparity in size between the dry sites and the wet sites (EF35) also suggests that the body size of O. cooperi has an environmental component, which is commonly found in other land snails (Pilsbry 1939; Dowle et al. 2015).

The distribution of O. cooperi is so far known to be restricted to BHNF, JM, and

CHIP. Despite the presence of ideal habitat in sky islands adjacent to BHNF, O. cooperi is not present there (Weaver et al. 2006). The Oreohelid populations found in the arrangement of fragmented sky islands adjacent to and including the Rocky Mountains in the U.S.A. are arranged such that each sky island is usually dominated by a single mitochondrial lineage. Thomaz et al. (1996) has found similar patterns for the terrestrial snail Cepaea nemoralis spread across northwestern Europe. Much like in North America, patches of ideal habitat are separated geographically by large distances, much farther than the snails are capable of dispersing. Snails colonize patches of suitable habitat through passive long range dispersal, rapidly reproduce to fill that space, and then evolve in isolation (Weaver et al. 2006). Terrestrial snails are known to have high mitochondrial

32 evolution rates compared to other taxa, and even separation over the course of geologically short events such as the last glacial maximum can result in highly divergent lineages (Chiba 1999; Pinceel et al. 2005) The Oreohelids appear to follow the same pattern, with populations on different sky islands showing evidence of evolving in isolation (Weaver et al. 2006). CHIP is an anomaly in that three mitochondrial lineages are present in sympatry, one of which is O. cooperi (Chapter 3). While the Judith

Mountains and Black Hills remained mostly unglaciated during the last glacial maximum,

CHIP was an inaccessible nunatak in the Laurentide ice sheet (Stalker 1965). Soon after the recession of the ice sheet, CHIP was colonized with plants and animals. The presence of three divergent paraphyletic lineages suggests that CHIP was colonized in at least three events, one of which was from either Judith Mountains or BHNF (Chapter 3). It is unlikely that Oreohelids were present in CHIP during the glacial maximum, as the earliest evidence of vegetation based on pollen profiles has been dated to approximately

9000 years ago, shortly after the recession of the ice sheets from southern Alberta

(Sauchyn 1990; Klassen 2004).

Based on my molecular data involving two independent genetic markers, and morphological data, the small morph of Oreohelid snails in Cypress Hills Interprovincial

Park is O. cooperi. In Canada, it is likely that O. cooperi is endemic to CHIP, as extensive surveys in the Rocky Mountains have failed to recover this species. Ecological niche modelling tools may uncover additional populations within similar habitats such as the Sweet Grass Hills, Montana; and Milk River Ridge and Porcupine Hills, Alberta.

2.6 Acknowledgements

We gratefully acknowledge the funding provided by ACA Grants in Biodiversity

(supported by the Alberta Conservation Association) and the Natural Sciences and

Engineering Research Council (NSERC). We also appreciate the cooperation and helpfulness of the park personnel of Cypress Hills Interprovincial Park, Alberta, Canada and well as Parks Canada. This work followed field observations originally made by Dr.

Bradley van Paridon and Dr. Cameron Goater in Cypress Hills. We would also like to thank the M.Sc. committee members Dr. Hester Jiskoot, Dr. Robert Laird, and Dr.

Kathleen Weaver for their invaluable comments and feedback, as well as Arturo Anaya

Villalobos and Sarah Stringam for assistance in field collections.

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Table 2.1. Numbers of Oreohelix sp. collected from seven sites in Cypress Hills Interprovincial Park in May and June of 2013 and 2014. Elevation, Latitude, and Longitude were obtained using an eTrex GPS. Samples with sequence data are denoted with letters and screened samples are denoted with numbers. Site n Samples Habitat Elevation (m) Latitude (N) Longitude (W) A 11 Dry shrub 1450 49.6321 110.3613 B 8 Dry shrub 1409 49.6288 110.1851 C 8 Dry shrub 1398 49.6721 110.1470 D 8 Dry shrub 1413 49.6129 110.1494 E 8 Moist pine 1326 49.6179 110.0936 F 8 Moist pine 1221 49.6421 110.0334 1 8 Dry shrub 1396 49.5930 110.0862 2 8 Dry shrub 1388 49.6110 110.0585 3 4 Moist pine 1178 49.6338 109.9852 4 8 Dry shrub 1317 49.6462 109.8468 5 1 Moist pine 1222 49.6027 109.7958

Table 2.2. Morphological diagnostic criteria measured for O. cooperi and compared to previous literature (Pilsbry 1939; Chak 2007). Umbilicus/width represents the proportion of the shell width which is umbilicus, ribbing represents proportion of penis that is ribbed, and shell banding represents the percent of the population with two primary coloured bands. Characteristic Shell size Umbilicus/width Ribbing Shell (mm) banding (%) CHIP shrub 8.5 ± 0.5 0.19 ± 0.02 0.45 ± 0.06 83 CHIP pine 10.6 ± 0.7 - - >90 BHNF Pilsbry ~15 “one fifth” “less than half” “usually present” BHNF Chak 12.5-17.2 0.22 ± 0.02 0.30 ± 0.04 76

C F 4 E D 3 A B 2 N 5 1 F

2 km E Figure 2.1. Relief map of study area within CHIP (delineated with dashed lines) indicating Oreohelix cooperi sampling locations. Highways and major roads are included for reference. Samples with sequence data are denoted with letters and screened samples are denoted with numbers.

Figure 2.2. Phylogenetic tree of COI haplotype data from snails collected in Cypress Hills Interprovincial Park (CHIP). Results include a representative sample of sequences from Weaver et al. (2006), acquired from South Dakota, Wyoming, and Montana (accession numbers DQ857991 – DQ858154). Branch lengths and scale are proportional to average substitutions per site above branches. Bootstraps values are based on 1000 replicates and are shown below branches. Tree model consists of maximum likelihood analysis assuming the selected K81uf+G model (-lnL = 2314; Gamma of 0.1825).

CHAPTER 3

Phylogeography of Oreohelix land snails in southern Alberta and Saskatchewan

Z. W. Dempsey1,2, T. M. Burg1, C. P. Goater1

Prepared as manuscript for submission

1University of Lethbridge, Department of Biological Sciences, 4401 University Drive,

Lethbridge, AB, T1K 3M4, Canada

2Corresponding author: [email protected]

3.1 Abstract

Peripatry, or speciation of peripheral isolates, is often a consequence of low species vagility and isolated habitat. We examined phylogeographic patterns of terrestrial snails in the genus Oreohelix collected from 58 sites in western North America using a combination of COI mitochondrial DNA and ITS2 nuclear DNA data. We found four distinct mitochondrial clades, three of which are subspecies of Oreohelix subrudis. One was geographically widespread, found in Cypress Hills, Alberta and in the adjacent

Rocky Mountains (O. sp. B) to the west. A closely related clade was geographically restricted to the Rocky Mountains in Alberta (O. sp. B’). One is a new subspecies so far only found in the Cypress Hills region (O. sp. X). The fourth mitochondrial clade was O. cooperi, a rare snail previously found in only two sky islands in North America, the

Judith Mountains in Montana and the Black Hills in South Dakota and Wyoming. The O. cooperi clade comprised small-bodied snails present exclusively on scree slopes in the

Cypress Hills. ITS2 sequence and screening data revealed three haplotype clusters, of which one was exclusive to O. cooperi. Cluster 1 was typical of O. sp X and cluster 2 was typical of O. sp. B. ITS2 alleles were shared in the Cypress Hills surrounding the contact zone between B and X, suggestive of hybridization between these two clades. This phylogeographic pattern likely reflects reproductive isolation during the Last Glacial

Maximum, followed by secondary contact due to peripatric, passive, long-range dispersal resulting from low vagility, local adaptation, and complex glacial history.

3.2 Introduction

The distribution of species is determined by localized conditions within a heterogeneous landscape. The impact of heterogenous landscapes is largely dependent on the biology and vagility of component species (reviewed by Mathias et al. 2001). These effects are most pronounced at the periphery of a species’ range, which can result in peripheral isolates and peripatric speciation. Two models of peripatric speciation include the Peripheral Isolates Model (PIM), in which individuals disperse to nearby patches of ideal habitat (Bush 1975); and the Centrifugal Speciation Model (CSM), in which patches of habitat are colonized during bouts of range expansion and subsequently isolated during range contraction (Brown 1957; reviewed by Frey 1993). One well-known example includes the deer mouse (Peromyscus maniculatus) and closely related Peromyscus species, which are thought to have differentiated via CSM (Bowers et al 1973;

Greenbaum et al. 1978). In contrast, the global diversification of the reef hermit crab

(Calcinus spp.) is attributed to multiple peripatric events through PIM (Malay and Paulay

2009), as is the divergence and radiation of Darwin’s finches (Geospiza and Certhidea;

Petren et al. 2005). A key difference between the two models is the historical matrix surrounding the habitat. Under the CSM, the distance was historically traversable but currently imposes a high resistance to movement. In the case of PIM, the area between patches is historically and contemporarily uninhabitable. Both models assume that contemporary fragments of ideal habitat are separated by distances further than individuals are normally capable of travelling.

Terrestrial snails have low vagility and lack a reliable method of dispersal, traits that make them excellent models for phylogeographic studies (Hausdorf and Hennig

2006; Sauer and Hausdorf 2010). Yet, despite being important members of terrestrial invertebrate communities as macro-detritivores and hosts of parasites, phylogeographic investigations involving terrestrial snails are rare, especially in North America. It is now well-recognized that morphological studies (Pilsbry, 1939; Dall, 1910) are not representative of true phylogenetic history and lack the resolution to distinguish closely- related taxa (Stearns, 1989). Recent studies have noted a discrepancy within Oreohelix mountain snails, even when molecular and morphological methods have been used

(Weaver et al. 2006; Chak, 2007). Such disparities and lack of consensus have complicated petitions to list a number of Oreohelix species, including Oreohelix cooperi, as threatened under the US Endangered Species Act and International Union for the

Conservation of Nature (IUCN) Red List.

Mountain snails of the genus Oreohelix are large bodied (up to 2 cm shell diameter), live ~10 years, hermaphroditic, and viviparous (Pilsbry, 1939). These snails form an important component of mountainous invertebrate communities, particularly in the Rocky Mountains, Intermountain West, and other elevated regions of Western North

America. The northern extent of their range extends into southwestern Canada, a region that remained largely inaccessible during the last glacial maximum (Pilsbry 1939; Stalker

1965). Cypress Hills Interprovincial Park (CHIP) is an elevated plateau approximately

400 km2 in southeastern Alberta and southwestern Saskatchewan surrounded by prairie and farmland. The plateau remained as an unglaciated nunatak during the last glacial maximum (Stalker 1965) and is separated from the Rocky Mountains to the west by approximately 250 km. Despite the distance, the slopes of CHIP contain many of the flora and fauna that are characteristics of the Canadian Rocky Mountains (Newsome and

Dix 1968). While sky islands in the Intermountain West were once connected by habitat corridors that land snails were capable of traversing (Weaver et al. 2006), it is currently unknown how, or if, isolated plateaus such as Cypress Hills were connected during the

Pleistocene and following ice sheet recession.

There are two known size morphs of Oreohelix snails in CHIP. The large-bodied morph prefers north-facing aspen-dominated slopes, while the small-bodied morph prefers dry, shrubby slopes (Chapter 2). My preliminary work has shown that the small morph is synonymous with the Black Hills mountain snail, Oreohelix cooperi, and the large morph is actually two genetically distinct subspecies of Oreohelix subrudis. The objective of this study is to elucidate the contemporary population genetic patterns of

Oreohelid landsnails and determine the historical patterns responsible for their current distribution in southern Alberta and Saskatchewan. It is hypothesized that the CHIP snails are relicts from the Wisconsinan or pre-Wisconsinan Pleistocene glaciation, and that the small-bodied Oreohelid snails are genetically and ecologically distinct from the large bodied ones.

3.3 Methods

3.3.1 Study areas and sampling

Cypress Hills Interprovincial Park is an ecological mosaic dominated by a mixture of white spruce (Picea glauca), lodgepole pine (Pinus contorta), and trembling aspen (Populus tremuloides) (Newsome and Dix 1968). These slopes provide ideal habitat for Oreohelid snails (Newsome and Dix 1968; Pilsbry 1939). The Rocky

Mountains (RM) contain a patchwork of suitable ecological conditions for Oreohelid snails interconnected with habitat corridors (Peet 2000). Waterton Lakes National Park

(WLNP) is located in the southwestern corner of Alberta and comprises a wide range of montane habitats including mountains, prairie, lakes, and wetlands. Vegetation communities consist primarily of fescue grasslands and deciduous forests including aspen forests at lower elevations and a transition to alpine meadows and coniferous forests at higher elevations (Peet 2000). Castle Area (CA) and the adjacent Crowsnest Pass (CP) have very similar habitat to WLNP, with the exception that a larger proportion of these regions is foothills (Fig. 3.1).

Individual snails (n=474) were obtained from 58 sites in Southern Alberta and

Saskatchewan throughout the snow-free months of 2013-2016 (Fig. 3.1). Plots approximately 10 m2 were demarcated with flagging tape and Oreohelid snails were collected as they were encountered following methods in van Paridon et al. (2017). All sample sites were searched by two people for at least 30 minutes or until eight live snails were collected. For morphological measurements, 30 snails were sampled per site. Snails were stored in ethanol at -80 °C. Information on slope aspect, cover, and elevation were recorded for each site using an eTrex GPS and compass.

Thirty adult snails per site were collected from sites CH03, CH04, CH07, CH08,

CH09, and CH10 (Fig 3.1) for assessments of morphological characteristics. Maximum shell width was measured using a Vernier caliper, and confidence intervals were calculated.

3.3.2 DNA extraction and amplification

Approximately 2 mg of foot tissue was used for a modified chelex DNA extraction (Walsh et al. 1991). Extracted DNA was amplified with COI barcoding primers LCO1490 (5’ – GTC AAC AAA TCA TAA AGA TAT TGG – 3’) and

HCO2198 (5’ – TAA ACT TCA GGG TGA CCA AAA AAT CA – 3’) (Folmer et al.

1994) and ITS2 primers LSU1F2990 (5’ – CTA GCT GCG AGA ATT AAT GTG A –

3’) and ITS4R3908 (5’ – TCC TCC GCT TAG TTA TAT GC – 3’) (Wade and Mordan

2000). In an Eppendorf master cycler a 585 bp fragment of the COI locus was amplified under the following conditions: 1 cycle of 94 °C for 2 minutes, 50 °C for 45 seconds, and

72 °C for 1 minute; 37 cycles of 94 °C for 30 seconds, 50 °C for 45 seconds, and 72 °C for 1 minute, with a final cycle at 72 °C for 5 minutes. The PCR mix contained: 1 Unit

Flexi DNA polymerase, 1x Flexi buffer, 0.2 mM dNTP, 0.4 µM of each primer, and 3 mM MgCl2. The ITS2 amplification of a 472 bp region followed the same protocol, with the exceptions of a 48 °C annealing temperature and 1 mM MgCl2. Amplified DNA was sent to McGill University for sequencing.

Both COI and ITS2 data showed fixed differences between clades such that screening primers were developed to type the remaining samples. The COI locus used a common primer HCOIcommO (5’ – TAA ACT TCA GGG TGA CCA AAA – 3’) with three different clade specific primers in a multiplex PCR: LCOIspecOc635 (5’ – TGC

TCT TTC ACT TTT AAT TCG AC – 3’) only amplified Oreohelix cooperi,

LCOIspecOs563 (5’ – ATT GTT ACA GCC TAT GCC – 3’) only amplified Oreohelix sp. B, and LCOIspecOx217 (5’ – GTG CCC CAG GAA TAA ATT TG – 3’) only amplified Oreohelix sp. X. For both the COI and ITS2 screening primers, PCR conditions

50 were the same as the COI sequencing conditions with the exception of different annealing temperatures and magnesium concentrations. The COI screening PCR was run with a 48

°C annealing temperature and 1 mM MgCl2. Similarly, the ITS2 locus used a common reverse primer ITS4R3908 with three different forward primers in a multiplex PCR:

ITS2FspecOc256 (5’ – CCG TGG TCT TAA GTT CAA A – 3’) amplified O. cooperi,

ITS2FspecOs112 (5’ – TTA ACG AAA AGT GGA TGC T – 3’) preferentially amplified

O. sp. B, and ITS2specOx356 (5’ – CTG CTG TGC TCT AGC ATT TAT – 3’) preferentially amplified O. sp. X. The ITS2 screening PCR had a 54 °C annealing temperature and 1.5 mM MgCl2. Amplified DNA was visualized and scored on 3% agarose gel stained with ethidium bromide.

3.3.3 Data analyses

Sequences were aligned and trimmed by eye and overall sequence divergence at each locus was calculated in MEGA 6.06 (Tamura et al. 2013). DnaSP 5.1 was used to calculate haplotype (h) and nucleotide (π) diversities for both loci (Librado and Rozas

2009). A phylogenetic tree using Jukes-Cantor model was constructed for COI in PAUP*

4.0a151 (Swofford 2002) for automated model selection. Maximum likelihood models of phylogenetic tree construction were ranked based on Akaike Information Criterion corrected (AICc) for finite sample sizes. The model with the lowest AICc score was used to construct a phylogenetic tree that was validated with 1000 bootstrap replicates with

50% consensus, and TreeGraph 2.9.2 (Stöver and Müller 2010) was used to create the final tree. Sequence data from the ITS2 locus was used to construct a maximum likelihood haplotype network with 300 iterations in PopART 1.7 (Bandelt et al. 1999).

3.4 Results

Oreohelid snails in CHIP were found in very high densities, approaching hundreds of individuals per 10 m2 in some instances. Both the large and small morphs of

Oreohelid snails were predictable in their habitat selection in CHIP. Large-bodied snails were found exclusively in aspen and aspen-pine dominated slopes. Small bodied snails were found primarily on dry, shrubby slopes, except for four sites that were moist and dominated by pine (CH14, CHP, CHT, and CHV). Oreohelids were never found on the fescue grasslands. While the body size of some snails living on dry, shrubby slopes in

RM was similar to O. cooperi in CHIP, none were identified as such. Most snails found in RM were large bodied. Of the 35 sites visited in RM, 17 contained snails with much lower population densities than CHIP.

3.4.1 COI

COI sequencing revealed four genetically distinct and well supported mitochondrial clades. Automated selection of a phylogenetic tree model by AICc score

(best score of –lnL = 1456) resulted in the following maximum likelihood model: Kimura

81 with gamma distribution (K81+G; gamma shape of 0.1882; Fig. 3.2a). Sequence divergence between clades ranged from 3% to 26% with an average clade divergence of

15.2 % ± 9.9%, while sequence divergence within clades was less than 2%.

In CHIP, all of the snails found in dry, shrubby slopes clustered into a single clade matching Oreohelix cooperi mitochondrial DNA from Weaver et al. (2006). The snails in

52 the environs surrounding Elkwater Lake in the western side of CHIP clustered into another clade that had 99.8% sequence similarity to Oreohelix sp. B (Weaver et al. 2006).

I refer to this genetic grouping as Clade B (Fig. 3.2a). The remainder of snails found throughout eastern CHIP clustered into the third clade with 96% sequence similarity with

Oreohelix sp. E (Weaver et al. 2006), hereafter referred to as Clade X. At sites adjacent to

Highway 41 (CH04-08), there was a sharp gradient of mitochondrial groups (Fig. 3.1), where a distance of less than 400 m separated Clades B and X (Fig. 3.2b). In that region,

CH06 was the only site that contained both Clade B and X snails, whereas all the sites adjacent to CH06 contained a single mitochondrial clade (Fig. 3.1). There were three instances when O. cooperi was found with another COI Clade at the same site in CHIP

(CH01, CHO, and CHN), indicating that these species can exist sympatrically in ecotones in CHIP.

In RM, two monophyletic clades sister to each other were sampled: Clades B and

B’. Clade B’ snails were confined to a small geographic area in CA and CP. Shell size and appearance at each sample site appeared to be associated with moisture and vegetation availability (personal observation). Overall, RM snails had moderate genetic diversity (h = 0.543 compared to 0.749 in CH, and π = 0.01284 compared to 0.08053 in

CH) at the COI locus, and low genetic diversity within sites (Table 3.1). If O. cooperi is removed from the analyses, CH snails had h = 0.626 ± 0.032 and π = 0.02784 ± 0.00059, which is still higher than RM. Only the CH04 site consisted of a single haplotype, whereas six RM sites consisted of a single haplotype (Table 3.1).

3.4.2 ITS2

Maximum likelihood haplotype analysis of ITS2 sequenced data revealed three distinct clusters including one matching the COI O. cooperi clade (Fig. 3.2b). The most commonly sampled cluster (Cluster 1) was populated predominantly by COI Clade X snails, while the next most populous cluster (Cluster 2) was dominated primarily by COI

Clades B and B’ (Table 3.3). ITS2 haplotypes were shared between COI Clades X, B, and B’, but no haplotypes were shared with O. cooperi. Mitochondrial Clade X contained only 11 individuals that had alleles from the Clade B ITS2 cluster, each of which was found close to the contact zone for B and X (Table 3.3). Forty four of Clade B and X snails in CHIP were heterozygous for both ITS2 haplotype clusters, 38 of which belonged to Clade B. All 28 snails sequenced and screened with clade specific primers from RM belonged to cluster 2 (Table 3.2).

3.4.3 Morphological characteristics

Oreohelid snails from dry, shrubby slopes (CH09 and CH10, O. cooperi) measured 8.50, 95% CI [8.36, 8.64] mm in shell length. Average shell length of mitochondrial Clade B (sites CH03 and CH04) were almost double the size of O. cooperi at 15.07, 95% CI [14.76, 15.38] mm, while snails belonging to mitochondrial Clade X

(sites CH07 and CH08) measured 15.37, 95% CI [14.97, 15.77] mm in length. An unpaired t-test revealed that the O. cooperi samples were significantly smaller than either of the two large morphs (B and O. cooperi: t118 = 39.41; p < 0.0001; X and O. cooperi:

54 t118 = 32.81; p < 0.0001), but the two large morphs where not significantly different in size from each other (t118 = 1.20; p = 0.23).

3.5 Discussion

3.5.1 COI Overview

We obtained Oreohelid snails from 58 sites in southern Alberta and

Saskatchewan. Two closely related mitochondrial DNA clades were found in RM, and three clades were found in CHIP. Of the four COI clades sampled, only Oreohelix cooperi was morphologically distinguishable (Pilsbry 1939; Chak 2007). The smaller snails match COI sequence data and morphological characters of O. cooperi (Chapter 2).

Until now, the only recently confirmed locations of O. cooperi were the Judith Mountains in Montana and Black Hills National Forest in South Dakota and Wyoming in the United

States. The larger snails have been identified as belonging to the species complex

Oreohelix subrudis, and are widespread throughout RM and sky islands in Montana and

Wyoming. Clade X has only been found in CHIP, and is most closely related to Clade E described by Weaver et al. (2006). This indicates that Clade X is either a glacial relict in

CHIP or colonized CHIP from the periglacial zone immediately south of Cypress Hills and the Laurentide ice sheet. Within RM, closely related Clades B and B’ were sampled, with Clade B’ restricted to the northern end of the sampled range. This proximity suggests that Clade B’ recently diverged from Clade B in RM. Clade B is widespread throughout the RM where the habitat is much more connected than in the sky islands to the southeast. These results complement the findings of Weaver et al. (2006), who noted

55 similar trends in the relation between Oreohelids in the Rocky Mountains and surrounding sky islands in the United States.

High levels of genetic divergence at the mitochondrial locus and cryptic speciation are common in terrestrial snails (Sauer and Hausdorf 2011). Thomaz et al.

(1996) attributes this general pattern to one of four possibilities: (i) relatively rapid mitochondrial divergence, (ii) prolonged isolation followed by secondary contact, (iii) selection pressure to maintain multiple mitochondrial clades, or (iv) colonization patterns leading to the presence of many divergent mitochondrial clades. The COI patterns observed in the snails sampled are best described by a mixture of (ii) and (iv). Secondary contact following prolonged isolation (ii) best describes the presence of the three divergent mitochondrial haplotypes in CHIP. Thus, one possibility is that each of the subclades of the O. subrudis species complex evolved in isolation on a sky island.

Temperature and precipitation changes characterizing the Pleistocene allowed some of these clades to come into secondary contact. While there were wild temperature and precipitation fluctuations in the early Holocene (Forman et al. 2001; de Bruyn et al.

2011), Cypress Hills and other sky islands would have remained as relatively stable habitats for Oreohelids (Sauchyn and Sauchyn 1991). This effect may be especially pronounced in a few areas, such as CHIP and in the Bighorn Mountains (WY1, WY2, and WY3 of Weaver et al. 2006). Secondary contact following prolonged isolation has been found in the dusky Arion slug (Arion subfuscus; Pinceel et al. 2005). Pinceel et al.

(2005) report highly divergent mitochondrial sequence data with low nuclear ITS1 divergence in this species, which matches the pattern we found in our Oreohelid snails.

Pinceel et al. (2005) suggest that hybridization of nuclear loci occurred in the slugs, leading to the maintenance of multiple divergent mitochondrial lineages.

An additional explanation is that colonization patterns leading to many divergent mitochondrial clades (iv) could account for the overall patterns of distribution of O. cooperi and O. subrudis in North America, including CHIP. Under this model, a habitat patch is colonized either actively through a habitat corridor or through a passive long- range dispersal event (For examples, see Dörge et al. 1999; Aubry et al. 2006; Wada et al.

2012). Here, a population will quickly expand in size and come to dominate that habitat patch which reduces the likelihood of further invasion. Colonization patterns favouring many divergent mitochondrial clades was found to be the best explanation for the distribution of mitotypes in grove snails (Cepea nemoralis) across northwestern Europe

(Thomaz et al. 1996) due to the immense population sizes and distances between patches of suitable habitat. These authors argued that populations of grove snails are effectively arranged like stepping stones, and dispersal events between them are rare.

3.5.2 ITS2 and cytonuclear discord

ITS2 sequence and screening data revealed three genetic clusters. One ITS2 cluster was found in RM, and three were found in CHIP. One cluster was exclusive to O. cooperi, providing further evidence that it is genetically distinct from other Oreohelids.

Cluster 1 was typical of O. sp X and cluster 2 was typical of O. sp. B, but the two divergent mitochondrial clades showed overlap at the ITS2 locus, particularly surrounding the contact zone in CHIP. There are two possibilities why Clades B and X

57 exhibit mitochondrial and nuclear discordance. The first possibility is incomplete lineage sorting, and the second is introgression and hybridization (Toews and Brelsford 2012).

While these are not necessarily mutually exclusive, hybridization and introgression better explain the ITS2 pattern observed in CHIP. If incomplete lineage sorting was occurring, there would be no geographic pattern to the nuclear allele distribution in CHIP (Funk and

Omland 2003). However, there is a clear geographic pattern to both the mitochondrial

COI and the nuclear ITS2. While both are common in a small area surrounding the contact zone near Elkwater Lake, to the east of the contact zone near Highway 41 alleles belonging to Clade 1 become much more common (Fig. 3.1).

3.5.3 Glacial history

Oreohelids have a fossil record in North America stretching back to the

Cretaceous period (Henderson 1935), but the current distributions of Oreohelids in southern Alberta is a consequence of the Quaternary glacial history of the region. During the Last Glacial Maximum (LGM), Cypress Hills was inaccessible and surrounded by ice. During the recession of the ice sheets, the southern slopes were the first to become ice free and allow access to the hills, while the northern slopes retained ice for much longer (Klassen 1994).While the earliest indication of vegetation in CHIP is approximately 9000 years before present (Sauchyn and Sauchyn 1991), this is based on pollen profile information gathered from Harris Lake (49.6663 ºN 109.9044 ºW), on the northern side of CHIP. Literature is sparse regarding the vegetation on the southern slopes of CHIP because many lakes immediately south of CHIP are man-made, and so lack pollen profiles stretching back to the last ice age. More recent work based on zonal

58 reconstruction of vegetation from across many sites in North America suggests that

13 000 to 14 000 years before present, before the start of the late glacial warming period,

Cypress Hills and the Sweet Grass Hills demarcated the meeting point between western

Cordilleran forest and eastern Boreal forest in a thin zonal band adjacent to the

Laurentide ice sheet (Strong and Hills 2005).The band of Cordilleran forest extended from west of CHIP to the Rocky Mountains. While it did not extend far into the ice-free corridor of RM until much later (Mott and Jackson 1982), the forest did remain adjacent to RM in a narrow band descending into Montana. This is important because the

Cordilleran forest would be continuous with site MT1, which is the only location in the

U.S.A. where Clade B has been found (Weaver et al. 2006). The band of Boreal forest that extended from the east of CHIP down to North Dakota and South Dakota was continuous with the Black Hills. This is significant because the Black Hills are one of two locations in the U.S.A. that contain O. cooperi. These bands of forest were transient and replaced with prairie less than 2 000 years later (Ritchie 1976). However, this narrow band of forest would have been a potential corridor that Oreohelids could have used to colonize new areas such as CHIP. While sky islands such as CHIP and the Black Hills were once connected, the extent to which Oreohelids actually used these corridors is uncertain. While the vegetation would be capable of supporting Oreohelids, they also require the presence of other abiotic factors, such as calcareous deposits, in order to survive (Pilsbry 1939). There is very little fossil evidence of Oreohelids east of Montana during the most recent forest expansion (Wells and Stewart 1987; Frest and Johannes

2002), so they either existed there in low densities or not at all.

3.5.4 Glacial refugia and colonization

CHIP may have acted as a glacial refugium for at least some Oreohelids during the LGM. It is uncertain if CHIP was habitable during the LGM and there is a lack of consensus in the literature with some sources saying it is unlikely for any species

(Mozley 1934; Newsome and Dix 1968), and some studies suggesting it may have been habitable for some species such as lodgepole pine (Wheeler and Guries 1981; Godbout et al. 2008) but not other species such as jack pine (Yeatman 1967). The genetic markers used in this study are not sensitive enough to determine if CHIP was used as a glacial refugium by Oreohelids during the LGM, but the absence of fossil evidence of

Oreohelids in the Great Plains is consistent with Oreohelids already being present in

CHIP or the Sweet Grass Hills during the LGM. This does not solve the problem of how the snails colonized CHIP in the first place. Including COI data from Weaver et al.

(2006), the snails of CHIP are paraphyletic and could not have evolved in situ.

It is likely the case that CHIP has been occupied by Clade X prior to the LGM.

Clade X is the only clade in CHIP that is not present on other sky islands to the south. It is disjunct from its closest relative, Clade E, in northern Wyoming (WY2). Even more compelling is the pattern of the haplotype network between Clades X and B (Fig. 3.2b).

The pattern of two starbursts separated by a few differences is typical of two divergent lineages that have both undergone recent population expansions in isolation. The sharing of ITS2 alleles between the mitochondrial groups is indicative of introgression between the two clades in this case (Avise 2000). Clade B in CHIP occupies a much smaller proportion of available habitat than Clade X. This could be evidence of Clade B being a later introduction to CHIP than Clade X, which is corroborated by the differences in

60 haplotype and nucleotide diversity between Clade X and Clade B. Clade X has slightly more measured diversity than Clade B in CHIP (Table 3.1). This difference is small but may be indicative of a difference in colonization time of CHIP. Another possibility is that

Clade B was present in CHIP during the LGM and colonized RM from the east rather than from the south. The relationship between Clades B and B’ indicates that Clade B was present in RM prior to the LGM, although it is likely that Clade B was displaced by the advancing ice sheets. Clade B shares several haplotypes between CHIP and RM, suggesting recent connectivity. Additionally, Clade B in this study has a single fixed difference from Clade B snails in Montana (Weaver et al. 2006), suggesting a slightly more distant relationship than RM and CHIP. The lower sample sizes for Clade B in

Montana make this conclusion premature. Clades B and X are derived from the same parent population as Clades C, D, and E. This species complex likely originated in

Montana or Wyoming. Most of the diversity of the species complex is in that area, and

Oreohelids have remained in the Yellowstone Park area since the Miocene (Henderson

1935). One possibility for these origins is that temperature and precipitation fluctuations of the Pleistocene and early Holocene alternately allowed colonization of the sky islands of the area via habitat corridors and then isolated each sky island. This complex climatic history could produce the observed pattern of genetic diversity observed in snails from

Montana and Wyoming. Ice caps covered many of the sky islands that are currently occupied by Oreohelid land snails, but the distances between them are relatively short and were connected by suitable habitat for longer periods of time (Hollin and Schilling

1981; Weaver et al. 2007). Similar patterns have been found in glacial relict montane grasshoppers (Melanoplus spp.) in the same area (Knowles 2000; Knowles 2001).

The presence of Clade B’ in RM indicates that there was an isolated pocket of

Clade B that remained separated from other Oreohelids before the LGM. The divergence between Clades B and B’ is much less than the divergence between any of the other subspecies of O. subrudis from Weaver et al. (2006), indicating a much more recent separation and divergence in situ. Clade B’ also has reduced ITS2 diversity with alleles that are shared with Clade B. Patterns of variation of ITS2 and COI and reduced distribution of B’ suggest this clade originated in RM. Because Clade B’ must have evolved in situ and been separated from Clade B some time ago, it is likely that Clade B was present in the RM prior to the LGM. The RM foothills contained tundra-like vegetation throughout most of the Wisconsinan and an ice-free corridor throughout the last glacial maximum (Mott and Jackson 1982). Trees did not colonize the majority of the ice-free corridor until approximately 8000 years before present, which is roughly concurrent with pollen core data from Harris Lake (Mott and Jackson 1982; Sauchyn and

Sauchyn 1991). Despite extensive surveys, we have not found Oreohelids above 1600 m in RM. This is the elevation at which snow often remains year-round in southern Alberta and tundra-like, glacial relict vegetation is common. This could be due to climatic, geographic, or geologic reasons. Additionally, the Laurentide ice sheet rose to approximately 1500 m in elevation and deposited sediment up to that height, adding a sediment history as well (Sugden 1977). The presence of Clade B’ in RM is evidence of a refugium there, but there is not enough evidence to discern the exact location.

It is unclear if O. cooperi was present in CHIP during the LGM. The ITS2 haplotypes that have been sampled in CHIP are much more divergent than either Clade B or X, suggesting that O. cooperi may have been in CHIP for a longer period of time (Fig.

3.2b). While the most recent glacial maximum reached the farthest south, previous glacial maximums could have resulted in connecting forests to Cypress Hills as well, providing earlier corridors (Andriashek and Barendregt 2017). Evidence counter to an early colonization is that the COI haplotypes found in CHIP are shared with snails from both the Judith Mountains and Black Hills. If these were separated for long enough they would have displayed more variability and evidence of genetic drift. Evidence of this can be seen in the Bighorn Mountains (WY1-WY3) which contain O. pygmaea and Clade A but not O. cooperi. The Bighorn Mountains were separated from the Black Hills for long enough for O. cooperi and O. pygmaea to diverge. This means that snails in the Bighorn

Mountains have been separated from the Black Hills for longer than the CHIP populations, or that CHIP and the Black Hills populations were more recently connected than the Bighorn Mountains and Black Hills. The original source of O. cooperi is likely the Black Hills. While O. cooperi is found in at least three locales, the Black Hills is the only site adjacent to O. pygmaea and Clade A. These clades are monophyletic and likely diverged from the same source population.

3.5.5 PIM vs CSM

The primary difference between PIM and CSM is the use of active or passive dispersal, and habitat connectivity. There is supportive evidence that the habitat of

Oreohelid snails in CHIP was once continuous with areas such as the Black Hills and

Rocky Mountains (Strong and Hills 2005). While active dispersal along these corridors may not have happened during the LGM, it may have occurred during the previous glacial maximum or even prior. It is also very likely that the poor active dispersal

63 capabilities of these land snails may have been supplemented with passive dispersal strategies (Dörge et al. 1999; Aubry et al. 2006; Wada et al. 2012). Given the genetic diversity of each clade in CHIP, it is unlikely that the genetic diversity is due to the colonization of a small number of passively-dispersed snails. The CSM better explains the mode of peripatry utilized by Oreohelids in CHIP.

3.5.6 Contemporary patterns

While the divide between Clade B and X is associated with Highway 41, it is unlikely that either Clade B or X was introduced to CHIP in modern times by humans.

Highways have been demonstrated to be substantial barriers to terrestrial snails (Baur and

Baur 1989), but Highway 41 was constructed in the early 1900’s. There is too much genetic diversity at both the COI and ITS2 loci for either Clade X or B to be a modern introduction to CHIP. The highway may currently slow gene flow between Clades B and

X, but the two clades have been maintained in CHIP since at least the LGM. The niches occupied by both Clade B and X are indistinguishable in CHIP, but these snails have high population densities which may resist invasion from other taxa. Terrestrial snails are known for their ability to rapidly colonize or invade novel ecosystems due to their high reproductive capacity (Thiengo et al. 2007). The spread of the colonizing snails would only stop at the edge of suitable habitat or an occupied niche. Previous work in pipits

(Anthus trivialis and A. spinoletta) and buntings (Emberiza citronella and E. hortulana) has shown that long-term species segregation is possible through the combination of many factors including habitat differences and interspecific competition (Bastianelli et al.

2017).

While hybridization between the two mitochondrial clades is evident, it is limited to a narrow area and the introgression of ITS2 alleles is asymmetrical. Many more Clade

B snails share ITS2 alleles with Clade X than vice versa. Most hybrids are limited to the contact zone. Oreohelids are simultaneous hermaphrodites, yet the pattern that is observed is similar to male-biased dispersal in dioecious organisms (Toews and Brelsford

2012). This indicates that there is unequal movement between male and female gametes in the hybrid zone. It could be that these snails seek out mates, but return their usual resting place to give birth. This would mimic male-biased dispersal, and would reduce inbreeding. Another possibility is asymmetrical mating, where snails may behave as males for some mates but females for others. Asymmetrical mating has been demonstrated in land snails including Oreohelids (Davison et al. 2005; Wiwegweaw et al. 2009) and has been observed in Oreohelids from CHIP (personal observations), but mate selection is poorly studied in terrestrial snails (see Baur and Baur 1992; Baur and Baur 1997).

3.5.7 Conclusions and future directions

Using molecular techniques, three mitochondrial clades of Oreohelids have been identified in CHIP, and two in RM. These finding have elucidated the relationship between snails collected from the Rocky Mountains and CHIP. The small morph of

Oreohelid snail in CHIP, identified as O. cooperi, has been demonstrated to be genetically distinct from large bodied snails of CHIP or RM. We propose that the

Oreohelids of CHIP and RM are glacial relicts and restricted to niches that were much more widespread during the glacial maximum and recession. Further studies are necessary to provide better resolution for these general conclusions. Sampling of other

65 sky islands in this region such as the Sweet Grass Hills, Milk River Ridge, and Porcupine

Hills could act as ‘replicates’ of peripatry from RM and extend the results further. Dall

(1910) mentioned finding Oreohelids as far east as the Lake of the Woods in Ontario. Use of additional markers such as microsatellites would also allow the construction of a timeline of colonization of Oreohelids to CHIP.

3.6 Acknowledgements

We gratefully acknowledge the funding provided by ACA Grants in Biodiversity

(supported by the Alberta Conservation Association) and the Natural Sciences and

Bradley van Paridon and Dr. Cameron Goater in Cypress Hills. We would also like to thank the M.Sc. committee members Dr. Hester Jiskoot, Dr. Robert Laird, and Dr.

Kathleen Weaver for their invaluable comments and feedback, as well as Arturo Anaya

Villalobos and Sarah Stringam for assistance in field collections.

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Table 3.1. Descriptive genetic polymorphism statistics for COI locus analyzed by site, clade, and region in DnaSP including number of individuals (n), haplotypes (Hap), variable sites (V sites), and haplotype (h) and nucleotide (π) diversities. Site Clade n Hap V sites h ± SD π ± SD CH01 B/Co 11 2 95 0.182 ± 0.144 0.02822 ± 0.02229 CH02 B 15 3 2 0.257 ± 0.142 0.00044 ± 0.00025 CH03 B 8 3 2 0.464 ± 0.200 0.00080 ± 0.00038 CH04 B 9 1 0 0.000 ± 0.000 0.00000 ± 0.00000 CH05 B 7 3 2 0.524 ± 0.209 0.00104 ± 0.00046 CH06 X/B 8 7 38 0.964 ± 0.077 0.03061 ± 0.00637 CH07 X 8 3 2 0.679 ± 0.122 0.00134 ± 0.00034 CH08 X 12 2 1 0.167 ± 0.134 0.00027 ± 0.00022 CH09 Co 7 2 1 0.286 ± 0.196 0.00050 ± 0.00035 CH10 Co 8 4 4 0.786 ± 0.113 0.00255 ± 0.00047 CH11 X 10 2 1 0.200 ± 0.154 0.00033 ± 0.00025 CH12 X 5 2 2 0.400 ± 0.237 0.00136 ± 0.00081 CH13 Co 8 4 2 0.750 ± 0.139 0.00150 ± 0.00038 CH14 Co 8 7 6 0.964 ± 0.077 0.00316 ± 0.00073 CH15 X 5 3 3 0.700 ± 0.218 0.00199 ± 0.00081

RM01 B 8 2 1 0.250 ± 0.180 0.00039 ± 0.00028 RM02 B/B’ 8 3 19 0.679 ± 0.122 0.00784 ± 0.00502 RM03 B 7 1 0 0.000 ± 0.000 0.00000 ± 0.00000 RM04 B/B’ 8 4 19 0.750 ± 0.139 0.01573 ± 0.00350 RM05 B 8 2 1 0.536 ± 0.123 0.00083 ± 0.00019 RM06 B’ 8 1 0 0.000 ± 0.000 0.00000 ± 0.00000 RM07 B/B’ 7 6 22 0.952 ± 0.096 0.01871 ± 0.00347 RM08 B’ 8 2 1 0.429 ± 0.169 0.00066 ± 0.00026 RM09 B’ 7 3 2 0.667 ± 0.160 0.00118 ± 0.00036 RM10 B’ 8 3 2 0.679 ± 0.122 0.00127 ± 0.00032 RM11 B 7 2 1 0.286 ± 0.196 0.00044 ± 0.00030 RM12 B 7 3 2 0.667 ± 0.160 0.00118 ± 0.00036 RM13 B 4 1 0 0.000 ± 0.000 0.00000 ± 0.00000 RM14 B 3 1 0 0.000 ± 0.000 0.00000 ± 0.00000 RM15 B 8 1 0 0.000 ± 0.000 0.00000 ± 0.00000 RM16 B 8 3 2 0.464 ± 0.200 0.00081 ± 0.00038 RM17 B 4 1 0 0.000 ± 0.000 0.00000 ± 0.00000

CH B/Co/X 129 19 102 0.749 ± 0.017 0.08053 ± 0.00322 RM B/B’ 118 9 20 0.543 ± 0.037 0.01284 ± 0.00080

O. cooperi Co 41 7 7 0.274 ± 0.920 0.00060 ± 0.00023 O. sp. B B 121 10 10 0.174 ± 0.047 0.00037 ± 0.00011 O. sp. B’ B’ 40 9 7 0.594 ± 0.085 0.00140 ± 0.00030 O. sp. X X 45 9 11 0.327 ± 0.091 0.00083 ± 0.00027 Total B/B’/Co/X 247 25 108 0.735 ± 0.021 0.05981 ± 0.00402

Table 3.2. Descriptive genetic polymorphism statistics for ITS2 locus sequence data analyzed by site and region in DnaSP including number of individuals (n), haplotypes (Hap), variable sites (V sites), and haplotype (h) and nucleotide (π) diversities. Site Cluster n Hap V sites h ± SD π ± SD CH01 Co 8 3 32 0.342 ± 0.140 0.01505 ± 0.00735 CH02 1/2 13 10 33 0.880 ± 0.034 0.01655 ± 0.00319 CH03 1/2 5 4 10 0.600 ± 0.125 0.00636 ± 0.00225 CH05 1/2 8 5 12 0.608 ± 0.130 0.00686 ± 0.00215 CH06 1/2 8 10 17 0.867 ± 0.079 0.01158 ± 0.00259 CH07 1/2 8 3 2 0.508 ± 0.126 0.00140 ± 0.00040 CH09 Co 5 5 70 0.667 ± 0.163 0.04098 ± 0.01471 CH10 Co 8 2 1 0.233 ± 0.126 0.00057 ± 0.00031 CH11 1/2 8 5 14 0.608 ± 0.130 0.00931 ± 0.00275 CH12 1 4 2 1 0.429 ± 0.169 0.00097 ± 0.00038 CH15 1 8 2 1 0.400 ± 0.114 0.00090 ± 0.00026

RM03 2 1 1 0 NA NA RM04 2 3 1 0 NA NA RM06 2 4 1 0 NA NA RM11 2 4 1 0 NA NA RM15 2 8 1 0 NA NA RM16 2 8 1 0 NA NA

CH 1/2/Co 83 24 84 0.734 ± 0.030 0.03026 ± 0.00251 RM 2 28 1 0 NA NA Total 1/2/Co 114 23 82 0.753 ± 0.017 0.02591 ± 0.00213

Table 3.3. Number of individuals belonging to each COI clade and ITS2 cluster. Total n = 474. COI clade B B’ X Co 1 56 128 2 111 41 5 1 / 2 38 6 Co 89

ITS2 Cluster Total 205 41 139 89

(a) Alberta Saskatchewan

CH – B, X, Co RM – B, B’ N MT1 – B MT3 – Co

WY4 – D MT2 – A WY1 – A, E BL – Co WY2 – A BH – Co WY3 – Py

WY5 – D

K (b) CH I J L 10 X 11 12 U 14 N S W M T Q 15 N Y 09 O P V 13 R

01 04 RM CH inset 05 05 02 G 04 N E H 03 06 D 02 07 C 06 08 11 01 B 12 03 F 07 08 09 10 13 A 14

15 16 17

Legend Clade B Clade B’ Clade X Clade Co

K (c) CH I J L 10 X 11 12 U 14 N S W M T

Q 15 N Y 09 O P V 13 R

CH inset 01 04 RM 05 05 02 G 04 N E H 03 06 D 02 07 C 06 08 11 01 B 12 03 F 07 08 09 10 13 A 14

15 16 17

Legend Cluster 1 Cluster 2

Cluster Co

Figure 3.1. Satellite and relief images of (a) all sampling sites including Weaver et al. (2006). Sites are coded based on COI clade present and patterned circles represent COI clades not found in this study. (b) COI sequencing (numbers) and screening (letters) results in Cypress Hills Interprovincial Park and the Rocky Mountains and (c) ITS2 results in Cypress Hills and Rocky Mountains (Google Earth 2017). The scale bar in each inset represents 20 km. Co indicates O. cooperi, and Py indicates O. pygmaea. Clades B- E are all subclades of O. subrudis. [Includes previous two pages]

(a)

(b)

Cluster 1

Cluster 2

(12)

(18)

O. cooperi

(28)

(12)

(9) (12)

Figure 3.2. (a) Phylogenetic tree of COI haplotype data including sequences from Weaver et al. (2006). Branch lengths and scale are proportional to average substitutions per site above branches. Bootstraps values below nodes are based on 1000 replicates. (b) Minimum spanning haplotype network constructed for the ITS2 locus. Dashes represent number of base pair differences between haplotypes. Size of the circle is proportional to the number of individuals sharing that haplotype. Shading corresponds to mitochondrial clade. [previous two pages]

CHAPTER 4

Spatio-temporal patterns of emerging larval liver fluke (Dicrocoelium dendriticum) in three sympatric species of land snails in southern Alberta, Canada

Z. W. Dempsey1,2, C. P. Goater1, T. M. Burg1

Prepared as manuscript for submission

1University of Lethbridge, Department of Biological Sciences, 4401 University Drive,

Lethbridge, AB, T1K 3M4, Canada

2Corresponding author: [email protected]

4.1 Abstract

The lancet liver fluke, Dicrocoelium dendriticum, was introduced from central

Europe into North America in the 1950s. It was introduced into southeastern Alberta prior to the 1980s then emerged in the region in the mid 1990s. Together with most other emerging macroparasites that have complex life cycles, little is known regarding patterns of infection within their obligate intermediate hosts. I used molecular diagnostic tools on samples of sporocyst tissue, combined with standard host surveys, to show that D. dendriticum utilizes three species of sympatric Oreohelid land snail as a first intermediate host in this region of emergence. Within a total of 900 samples of adult snails collected over one field season, mean prevalence was 9.9% ± 2.4%. Prevalence did not vary significantly among the three species (p = 0.8317), nor spatially segregated sites (p =

0.4149), but there were significant differences among months (p = 0.0002). For O. subrudis B and Oreohelix subrudis X, prevalence ranged between approximately 5-30% within samples collected between May-October, with maxima occurring in mid-summer.

For the small-bodied snail, O. cooperi, prevalence peaked two months earlier. Less than

1% of 240 infected snails collected during the summer reproductive period contained eggs in utero, indicating that trematode-induced sterility is common within all three of these long-lived, viviparous species of snails. These results indicate that within this region of emergence in Alberta, Canada, D. dendriticum utilizes the complex community of Oreohelid land snails as first intermediate host.

4.2 Introduction

Emerging infectious diseases (EIDs) are pathogens and parasites that are currently expanding their traditional host-species and geographical ranges, or are projected to undergo range expansions in the immediate future (Daszak et al. 2000). Large-scale anthropogenic alterations to ecosystems such as land clearing, habitat fragmentation, fire suppression, and climate change are considered driving factors leading to the increase in

EIDs in recent decades (Daszak et al. 2000). Given the negative impacts that EIDs can have on humans, the growth of domestic livestock, and the conservation of wildlife, it is important to understand the nature of emergence events involving new hosts in areas of range expansion. Most EIDs of humans and domestic livestock involve microparasites such as bacteria and viruses (Cleaveland et al. 2001), many of which have direct life cycles that are well-characterized. EIDs that have indirect or complex life cycles; however, present a challenge for parasitologists because the number of epidemiological rate parameters increases as the number of obligate hosts increases (Goater et al. 2014).

Multiple-host EIDs are also of increased conservation concern because they adversely affect multiple species of hosts at multiple trophic levels.

Macroparasites with complex life cycles such as helminths make up nearly a third of pathogens and parasites of domestic animals, yet they constitute less than 6% of known EIDs (Cleaveland et al. 2001). One likely constraint on the establishment of macroparasite EIDs is the difficulty of switching to multiple, different obligate hosts in regions of potential emergence (Torchin et al. 2003). However, recent studies show that anthropogenic changes such as climate change can catalyze rapid host switches without the need for novel adaptations (Hoberg and Brooks 2008; 2015). This is the so-called

“parasite paradox,” that seemingly specialized macroparasites with complex life cycles are capable of rapid, and simultaneous host shifts through the process of ecological fitting

(Agosta et al. 2010). Here, establishment of seemingly specialized parasites occurs within new hosts through preexisting adaptations rather than intimate coevolutionary interactions (Brooks et al. 2006; Malcicka et al. 2015). Well-known recent examples include the cestode Echinococcus multilocularis, which has been increasing its host and geographical range due to anthropogenic impacts (Davidson et al. 2012), the liver fluke

Fascioloides magna, which has emerged in continental Europe from its homeland in western North America (Pybus et al. 2015) and the human parasite Schistosoma mansoni, which has spread to South America from Africa (Crellen et al. 2016). Each of these examples demonstrates the capacity for complex life cycle macroparasites to invade novel ecosystems, as well as the rapid colonization of new hosts through ecological fitting.

The lancet liver fluke, Dicrocoelium dendriticum, is another example of a complex-life cycle macroparasite that has extended its geographical and host species range. This host-generalist trematode utilizes a three-host life cycle consisting of terrestrial snails as first intermediate host, formicid ants as second intermediate host, and grazing herbivores as final host (Krull and Mapes 1952; review by Manga-Gonzalez et al.

2001). Native to continental Europe, it is now found in many parts of the world, including

Scandinavia, United Kingdom, Mediterranean countries, the Middle East, and now isolated locations in North America (reviewed in van Paridon et al. 2017b). In many cases, the emergence of D. dendriticum can be tied to the global translocation of domestic animals, especially sheep. Dicrocoelium dendriticum was first detected in New York

84 state by Mapes (1951), and has since travelled inland along with livestock such as sheep and cattle (Lewis 1974; Pybus 1990).

One area of introduction and emergence of D. dendriticum is in the Cypress Hills region of southeastern Alberta, Canada. The results of surveys of sympatric wild ungulates collected near or in Cypress Hills Interprovincial Park (CHIP) indicated that adult liver flukes were present as early as the 1980’s, but at very low levels (Pybus 1990).

Surveys completed in the 1990’s indicated that worm intensities and prevalence in wapiti

(Cervus canadensis), white-tailed deer (Odocoileus hemionus), mule deer (Odocoileu hemionus), and cattle (Bos taurus) had increased dramatically in this region (Goater and

Colwell 2007). In a survey of over 100 sites in CHIP, Beck (2015) demonstrated that the presence of D. dendriticum metacercariae in ants was highly variable and was significantly associated with key habitat characteristics. In that study, predictions of a hierarchical logistic regression model indicated that infection in ants was associated with the presence of deciduous trees located on gently sloping terrain on east or south-facing aspects. Beck (2015) suggested that the association between these habitat characteristics and risk of infection in ants was best explained by infection patterns in first intermediate snail hosts. Of the five genera of terrestrial snail identified in CHIP (Cionella, Discus,

Oreohelix, Vitrina, and Zonitoides), only snails in the genus Oreohelix were found to harbor D. dendriticum sporocysts (Beck 2015). Van Paridon et al. (2017a) described general infection patterns in snails, Oreohelix subrudis, at one site in CHIP, but nothing is known regarding spatio-temporal dynamics at the level of snail intermediate hosts within this region of emergence. This knowledge gap parallels our general lack of understanding of patterns of larval trematode infection in terrestrial snails. The purpose

85 of this study is to evaluate the spatio-temporal patterns of larval D. dendriticum infection in the three species of Oreohelix that are sympatric in CHIP (Chapter 3).

4.3 Methods

4.3.1 Study area

The study area is contained within an approximately 6 km2 section of the Alberta side of Cypress Hills Interprovincial Park (Fig. 4.1; 49.64 N 110.28 W). CHIP is an elevated plateau located in southeastern Alberta surrounded by prairie and farmland. The island-like ecoregion comprises a complex mosaic of grassland dominated by rough fescue and other grasses characteristic of the mixed grass natural subregion (Downing and Pettapiece 2006) and boreal and foothills characteristic of aspen parkland to the north and the foothills of the Rocky Mountains to the west. The slopes of CHIP are dominated by a mixture of white spruce (Picea glauca), lodgepole pine (Pinus contorta), and trembling aspen (Populus tremuloides) (Newsome and Dix 1968). The plateau, which remained unglaciated during the glacial maximum (Stalker 1965), is dominated by fescue grass communities that are grazed annually by approximately 4000 beef cattle. Other large grazing mammals that are known hosts of liver fluke in this region are elk (Cervus canadensis), mule deer, and white-tailed deer (Mapes and Baker 1950; van Paridon et al.

2017).

4.3.2 Design of host survey

The geographic distributions of the three Oreohelix species within CHIP have been previously established (Chapter 3). Although the species-level designations of O. subrudis and O. cooperi are clear and based on molecular, morphological, and life history characteristics (Chapters 2 and 3; Weaver et al. 2007), the designation of O. subrudis

Clade X. as distinct from O. subrudis Clade B is less clear. Because my molecular evidence, based on two independent molecular markers, supports genetic differentiation between these two species, I refer to the three lineages as distinct species for the purposes of this paper. To evaluate patterns of larval D. dendriticum infection in the three species,

I sampled two geographically-isolated sites within each species’ distribution (Fig. 4.1).

Site selection was based on the presence of infected ants at each site, as indicated by the presence of ants attached to vegetation (Beck 2015), as well as accessibility by vehicle, ease of sampling, and relatively high Oreohelid sp. density. From each of these six sites,

30 snails were collected on the same day each month between May and September, 2015

(total n = 900). Sampling areas within each site were demarcated with flagging tape and divided into 10 m2 sub-sites. Adult snails were collected haphazardly as they were encountered and immediately preserved in 95% ethanol.

4.3.3 Dissections

The assessment of infection status in individual snails followed standard protocols for larval trematodes. Snail shells were first measured for maximum width with calipers, then gently crushed. The viscera was removed and placed between two glass slides to

87 view under a dissecting microscope. Infected snails were easily identified as they contained hundreds of sporocysts within the digestive gland. Following the assessment of infection status, the number of embryos in the uterus were counted.

4.3.4 Molecular diagnostics

Sporocyst tissue was isolated from one infected snail from each of the six sites during the August sampling period. Approximately 2 mg of sporocyst tissue from each infected snail was used for a modified chelex extraction following Walsh et al. (1991).

Extracted ITS2 DNA was amplified with D. dendriticum primers Dicro5.8FC (5’ – GTC

GAT GAA GAG GCG AGC – 3’) and DicroSpecR (5’ – ATT TCG GAA TTC ACC

ACA CG – 3’) (Bazsalovicsová et al. 2010) in an Eppendorf master cycler using the following conditions: 1 cycle of 94 °C for 2 minutes, 54 °C for 45 seconds, and 72 °C for

1 minute; 37 cycles of 94 °C for 30 seconds, 54 °C for 45 seconds, and 72 °C for 1 minute; and a final cycle at 72 °C for 5 minutes. The PCR master mix contained: 1 U

Flexi DNA polymerase, Flexi buffer, 0.2 mM dNTP, 0.4 µM of each primer, and 2.5 mM

MgCl2. The approximately 400 base pair product was visualized using ethidium bromide stained agarose gel and PCR product was sent to McGill University for sequencing.

Sequences were aligned and edited in MEGA 6.06 (Tamura et al. 2013) and compared to sequences in the NCBI database.

4.3.5 Data Analyses

One-way ANOVA was used to determine the effects of snail species on variation in shell size, followed by pairwise comparisons with Tukey post-hoc tests. Embryo counts were evaluated for normality and a two-way ANOVA was used to evaluate the effects of host species, month, and the species/month interaction on variation in snail fecundity. Prevalence was defined as the proportion of a sample of snails that were infected (Bush et al. 1997). Prevalences within samples of snails and their associated

95% confidence intervals (CI) were calculated using the modified Wald method (Agresti and Coull 1998). Prevalence differences between snail species, site, and month were evaluated with Fisher’s exact tests (Rózsa et al. 2000; Soldánova et al. 2010).

Significance of the association between snail shell width and prevalence was assessed with parametric regression analyses.

4.4 Results

4.4.1 Characteristics of host populations

The frequency distribution of shell sizes of O. cooperi did not overlap with the distributions of the other two species (Fig. 4.2; Table 4.1). On average, shell size of O. cooperi was 44% smaller than the other two species and highly significantly different (F2,

897 = 3574; p < 0.0001). Differences in mean shell size between O. s. B and O. s. X were much smaller (Fig. 4.2), with O. s. B being 3% smaller than O. s. X.

Reproduction was highly seasonal for Oreohelix sp. (Fig. 4.3). Snail embryos were mostly observed in utero in O. s. B and O. s. X collected in July and in July and

August for O. cooperi. Month was a significant source of variation in embryo counts (F4,

895 = 135; p < 0.0001), whereas species was not (F2, 897 = 1.325; p = 0.295). The interaction between month and species was significant (F8, 892 = 15.47; p < 0.0001) indicating that the three different species had different seasonal patterns of fecundity.

There was only a single case of a snail containing both embryos and larval D. dendriticum (site Os-1 in July). The correlation between shell width and prevalence of larval D. dendriticum was not significant (O. s. B F1, 11 = 0.784, p = 0.395; O. sp. F1, 7 =

2 0.038, p = 0.852; O. cooperi F1, 3 = 0.016, p = 0.909), R values ranged from 0.00514 -

0.0665.

4.4.2 Infection patterns

Of the sporocyst tissue samples that were sequenced from six infected snails, all showed evidence of non-specific binding. Four of the sequences contained clean fragments approximately 300 base pairs long. BLAST searches on each of these fragments produced a 78 - 86% identity score with the D. dendriticum ITS2 fragment sequences in GenBank.

In samples pooled across months and sites (Table 4.1), there were no significant differences in prevalence between the three species (Fisher exact test; n = 900; p =

0.8317). There were also no significant differences between sites for samples pooled across species and months (Fisher exact test; n = 900; p = 0.4149). Season, in contrast, had a strong effect on prevalence (Fisher exact test; n = 900; p = 0.0002). All sites had relatively low prevalences (mean 7.5%) throughout the summer months except for a

90 prominent 21%-25% spike in August for O. s. X and O. s. B and in June for O. cooperi when prevalences exceeded 15% (Fig. 4.4). For all sites except for Oc-2, prevalence spiked in August, then declined in the fall samples. The Oc-2 site is somewhat atypical because prevalence spiked slightly earlier, in June. The OsB and OsX spikes in prevalence were pronounced, with O. s. X approaching a prevalence of 25% from a background prevalence of approximately 5 – 15%.

4.5 Discussion

I recovered D. dendriticum sporocysts and cercariae from each of the three sympatric species of Oreohelix in CHIP. These results extend the host species range of D. dendriticum to include Oreohelid land snails. Oreohelids are endemic to North America, so the utilization of these new snails in the life cycle of D. dendriticum represents a jump to novel host species, perhaps through ecological fitting (van Paridon et al. 2017b).

Preliminary surveys of other species of terrestrial snails found in CHIP have failed to find larval D. dendriticum (Beck 2015). Despite these preliminary survey results, other species of terrestrial snails in CHIP may contribute to transmission rates to the next host.

Current literature regarding D. dendriticum indicates that it is a host generalist capable of infecting up to 100 species of terrestrial snails from disparate taxa within the groups

Pulmonata and Stylommatophora (Manga-González et al. 2001; Manga-González and

González-Lanza 2005). While Oreohelix constitutes the largest-bodied snail genus in

CHIP, further sampling of the other genera of terrestrial snails in CHIP may reveal that some are compatible with D. dendriticum.

Significant differences are commonly found in larval trematode prevalence between spatially separated sites. While studies of spatial heterogeneity in freshwater and marine gastropods are relatively common (Curtis and Hurd 1983; McCurdy et al. 2000), infection patterns within terrestrial snails are understudied. Features of the aquatic environment such as water currents and temperature, tidal cycles, and presence of the final host are known to determine the probability of free-swimming miracidia successfully infecting a snail (Curtis 1996), and thus, can explain spatial heterogeneity. In contrast, the probability of miracidia establishment and infection within terrestrial snails will be determined by factors such encounter rates between foraging snails and viable eggs, which will in turn be determined by environmental and ecological factors such as temperature, moisture, host age, and densities of definitive hosts. While these rate parameters are currently unknown in CHIP, spatial variation in microclimate and definitive host movement could lead to the levels of variation observed at the six sites.

Terrestrial mountain snails have low vagility, typically wandering only a few meters per month (Baur and Baur 1990). Thus, local conditions are likely to have a strong influence on rates of transmission between eggs and individual snails. The small number of sample sites used in this study limits my ability to test the importance of these factors in determining egg-to-snail transmission rates.

Spatial variation in the deposition and accumulation of eggs on pasture will also play a role in the levels of infection at each site. In aquatic systems, miricidia density is often associated with the densities of definitive hosts in a region, and the accumulation of infectious stages (Curtis 1996). In terrestrial systems, the deposition of eggs in relation to density of definitive hosts has been evaluated for the nematodes Trichostrongylus

92 colubriformis and Ostertagia circumcincta in tropical environments (Banks et al. 1990;

Southcott et al. 1976). In these cases, eggs and larvae can survive for months and even overwinter before reaching infectivity in livestock the following year. D. dendriticum eggs have been found to remain viable for over 15 months after leaving the definitive host with no age-related decline in infectivity, although high seasonal temperatures can result in egg mortality (Alunda and Rojo-Vázquez 1983). This means that a single deposition of D. dendriticum eggs by a heavily infected herbivore could remain as a source of infection to multiple generations of Oreohelid snails without the recurring presence of the definitive host.

At sites Oc-1 and Oc-2, cattle are permitted to graze for most of the year. The remainder of the sites exclude cattle, but infected wildlife such as deer and elk are still present (Goater and Colwell 2007). While cattle have been found to contribute the majority of D. dendriticum eggs to pastures (Beck, 2015), the snails in Oc-1 and Oc-2 tend to be high on the slope in areas with good drainage (personal observation). At the remainder of the sites, snails are usually found all along the full length of the slope, and in the valleys bottoms. Given the longevity as well as the small size of D. dendriticum eggs, it is possible that rain and snowmelt will result in a net downward movement of eggs. This would also mean infection of snails would be most probable at the bottom of slopes. Beck (2015) showed that the highest prevalence of D. dendriticum in ants occurs at the bottom of the valleys of CHIP, which corroborates this explanation.

Our results indicate strong seasonality in the occurrence of D. dendriticum- infected Oreohelids, at least during the year that I sampled. Our results indicate two seasonal patterns of infection. First, prevalence was lowest in the early spring, just as

Oreohelids emerge from winter hibernation (Baur and Raboud 1988). Although prevalence was lowest during this period, the occurrence of infected snails indicates that at least some infected snails survived over winter. I have maintained, and recovered, infected Oreohelid snails collected from CHIP at 4 °C over a simulated winter in the laboratory (personal observation). Second, the pattern of prevalence within Oreohelid samples was consistent among the three species, with lows in spring and early summer, followed by peaks in midsummer, followed again by declines towards late summer and fall. Similar seasonal patterns of prevalence have been demonstrated in aquatic snail/trematode interactions within temperate habitats (Väyrynen et al. 2000; Negovetich and Esch 2007).

The prominent peak in prevalence in O. subrudis in August represents the greatest risk of infection for the next intermediate host. This risk coincides with the peak activity of the worker caste in formicid ants in response to ideal temperature ranges during that time (Porter and Tschinkel 1987). Determining causality for the prominent spike in prevalence observed in August for most of the sample sites is not possible with my data, but a review by Manga-González et al. (2001) suggests that exposure of snails to miracidia coincides with the first substantial spring melt. Thus, compatible snails are exposed when they first become active, but the infection is only detectable after several months of development. Manga-González et al. (2001) also report that it is difficult to determine exactly when a snail first becomes infected, but snails that are found to be infected before the late-summer peak were likely infected in a previous year. Sampling in future years is needed in order to compare the timing of late-summer prevalence peaks to the early spring melts.

The significant declines in the occurrence of infected snails towards late summer and fall indicates that either infected snails suffer higher mortality than uninfected ones, or infected snails change their behavior so that they are less likely to be sampled.

Väyrynen et al. (2000) found reductions in prevalence after a prominent spike in infected lymnaeid snails could be attributed to host mortality. Oreohelid snails live much longer than lymnaeid snails and infected snails of a wide variety of sizes and ages were sampled in August (Pilsbry 1939), so the decline in prevalence is unlikely due to age-related death. Alternatively, infected snails may suffer increased mortality in late summer due to factors such as predation or desiccation. Another possibility is that the decline in observed prevalence is due to a change in behavior of infected snails in late summer, such as burrowing behaviour or reduced activity.

Castration is a common feature of trematode infection in snails (Goater et al.

2014; Minchella and Scott 1991), although the observed patterns are typically found in marine snails or freshwater snails (Minchella et al. 1985; Minchella 1985). While determination of a causal link between D. dendriticum infection and sterility is not possible with the data from this study, it does provide substantial correlative evidence that warrants further investigation. Negovetich and Esch (2007) showed that castration of snails can have large effects at the population level. Overall, 10% prevalence within a population of castrated snails will led to a 5-10% reduction in effective population size, depending on the mode of castration (whether infection results in castration or full sterility). Given the high densities of Oreohelid snails in CHIP, and their presumed importance in nutrient-cycling and food webs (Handa et al. 2014; Astor 2014), D. dendriticum induced castration might have important ecosystem-level effects. Further,

95 the castration of O. cooperi within CHIP, where it is likely endemic in Canada (Chapter

2) and its possible population-level effects, has important conservation implications.

Despite their limitation to a narrow subset of ecological conditions found in their range, Oreohelids are a species rich taxa that occupy the majority of mountainous terrain in western North America (Pilsbry 1939). Given that formicid ants are also common throughout this range (Fisher 2007), and that large-bodied grazing herbivores such as deer, elk, and livestock are relatively unrestricted in their movement, the potential of geographic range expansion of D. dendriticum is high. Thus, wherever Oreohelix exists, there is the potential for further spread of this EID.

4.6 Acknowledgements

We gratefully acknowledge the funding provided by ACA Grants in Biodiversity

(supported by the Alberta Conservation Association) and the Natural Science and

Bradley van Paridon and Dr. Cameron Goater in Cypress Hills. We would also like to thank the M.Sc. committee members Dr. Hester Jiskoot, Dr. Robert Laird, and Dr.

Kathleen Weaver for their invaluable comments and feedback, as well as Arturo Anaya

Villalobos and Sarah Stringam for assistance in field collections.

4.7 References

Agosta SJ, Janz N, and Brooks DR. 2010. How specialists can be generalists: resolving the “parasite paradox” and implications for emerging infectious disease. Zoologia 27: 151-162.

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Alunda JM and Rojo-Vázquez FA. 1983. Survival and infectivity of Dicrocoelium dendriticum eggs under field conditions in NW Spain. Veterinary Parasitology 13: 245-249.

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Table 4.1. Summary infection characteristics for larval D. dendriticum within three species of Oreohelix land snails in Cypress Hills Interprovincial Park. Bolded species designations use n = 300, and site designations use n = 150. Shell size uninfected Shell size infected Infected Prevalence

(mm) (mm) (n) (%)

O. cooperi 8.5 ± 0.7 8.4 ± 0.5 27 9

Oc-1 8.5 ± 0.8 8.3 ± 0.6 10 7

Oc-2 8.4 ± 0.6 8.5 ± 0.4 17 11

O. subrudis B 15.4 ± 1.4 15.7 ± 1.4 31 10

OsB-1 15.6 ± 1.3 16.1 ± 1.4 18 12

OsB-2 15.1 ± 1.4 15.1 ± 1.3 13 9

O. subrudis X 15.8 ± 1.2 15.9 ± 1.1 31 10

OsX-1 16.0 ± 1.3 15.7 ± 1.1 19 13

OsX-2 15.7 ± 1.1 16.1 ± 1.1 12 8

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Oc-2 OsB-1 OsB-2

OsX-1 OsX-2 Oc-1 11111 2 km

Figure 4.1. Shaded relief map of study area within CHIP marking locations of sampled Oreohelix sp. populations (Google Earth 2016 Map data). Highways and major roads are included for reference, as are site codes. The shading denotes the area covered by CHIP.

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200 180 160 140 120 100 80 60

Number of Individuals Numberof 40 20 0

Size (mm)

Figure 4.2. Frequency distributions of Oreohelix sp. shell sizes in Cypress Hills Interprovincial Park, n = 300 per species.

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SD ± 3

2 Mean embryo count count Mean embryo

0 May June July Aug Sept

Figure 4.3. Seasonal patterns of snail reproduction in three species of Oreohelid snails in Cypress Hills Interprovincial Park (n = 60 / site per month).

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95% CI 95% ±

20 Prevalence Prevalence

0 May June July Aug Sept

Figure 4.4. Seasonal patterns of prevalence of D. dendriticum in three species of Oreohelid snails in Cypress Hills Interprovincial Park (n = 60 / species / month).

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Chapter 5: General Discussion

5.1 Conservation implications and future directions

The diversity and population sizes of terrestrial snails have been declining since the second industrial revolution (Régnier et al. 2015). This pattern of loss can be attributed to a number of factors including climate change, the introduction of invasive species, and habitat loss from human development (Barnosky et al. 2011). Assessments of extinction risk are a necessary step in the protection of potentially threatened organisms, and require a detailed knowledge of the distribution of species and the identification of underlying factors that cause range reductions (Cardoso et al. 2011).

Given the wide distribution of Oreohelix subrudis Clade B throughout the Rocky

Mountains and Cypress Hills Interprovincial Park (CHIP), and its very high densities at some sites (Dempsey, personal observations), its conservation status is probably secure.

The presence of Clade B’ in the Rocky Mountains, indicative of a glacial refugium

(Chapter 3), suggests that some Oreohelids have survived in the Rocky Mountains, even in the shifting climate of the Pleistocene. Pockets of Oreohelix spp. in the Rocky

Mountains may survive in similar refugia in the case of future climate change or habitat loss. Following my results reported in Chapters 2 and 3, populations of O. cooperi are now known to exist in at least three geographically isolated sky islands in Canada and the

USA. While populations in small patches of habitat such as sky islands are more likely to experience localized extinction events (Hill et al. 1996), it is unlikely that such extinction events will occur across multiple, distant patches simultaneously. Even if O. cooperi were to become extirpated from CHIP, populations could presumably survive in the

Judith Mountains or Black Hills. The conservation status of Oreohelix subrudis Clade X

107 and B’ is less certain. These geographically restricted clades hold a higher risk of localized extinction, without the benefit of multiple habitat patches. Although there may be more available habitat patches east of CHIP or north of Waterton Lakes in the Rocky

Mountains, Oreohelid snails have not been sampled there since Dall (1905). Given the highly restricted distribution of Clade X within CHIP and B’ in the Rocky Mountains, my results suggest that Oreohelids in Canada are in need of conservation consideration.

While the geographical distribution of O. cooperi in southern Alberta and

Saskatchewan has largely been determined, there are still important knowledge gaps that need to be addressed. The presence of O. cooperi within moist pine slopes in CHIP

(Chapter 2) indicates that it can utilize habitat that is common within the Rocky

Mountains and perhaps within other sky island sites adjacent to CHIP. My sampling protocol within eight potential Rocky Mountain sites was based on habitat characteristics typical for O. cooperi in CHIP. Thus, I may have missed snails that occurred within atypical habitats. To determine if O. cooperi is present within comparable sites within the

Rocky Mountains to the west, a more rigorous sampling protocol is required. Ecological niche modelling presents an additional approach (e.g. Peterson 2001). Using all available occurrence data of O. cooperi to develop a heat map in potential Rocky Mountain sites would allow the targeting of specific locales. Lastly, ecological studies designed to determine factors that affect spatial variation in snail density, rather than general patterns of distribution, will also provide key information relative to conservation status of O. cooperi. Currently, CHIP is the only location in Canada known to contain O. cooperi.

Coupled with the high genetic diversity of Oreohelids in the Park, it is important that

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CHIP remains protected as an interprovincial park and that there is increased recognition of the potential endemic nature of the distribution of this snail in Canada.

5.2 Phylogeography implications and future directions

The presence of Oreohelix cooperi and O. subrudis Clade B and X has been verified in CHIP using morphological and molecular techniques. Compared to other sky islands in Montana and Wyoming (Weaver et al. 2006), CHIP contains high Oreohelid genetic diversity. Given the degree to which Oreohelid snails are intrinsically connected to their immediate habitat (Pilsbry 1939), coupled with the unique geological history of the region, it is likely that CHIP may be a hotspot of biodiversity for other disjunct flora and fauna (Newsome and Dix 1968; Henderson et al. 2002; Choi and Cota-Sánchez

2010). Terrestrial snails are important macro-detritivores and components of invertebrate communities, and active dispersal to CHIP would be accompanied by other invertebrates and vegetation (Astor 2014). CHIP represents an important biodiversity hotspot for many different species of plants and animals. Any animals or plants that dispersed with the wave of colonizers to CHIP after glaciation may show population genetic structure that is unique to this parkland region. In this sense, Oreohelids can act as model organisms for other species in CHIP with low vagility. An additional implication of Oreohelid phylogeography in southern Alberta is the presence of a Rocky Mountain refugium capable of supporting Oreohelix subrudis Clade B’ during the last glacial maximum

(Chapter 3). Pollen core data suggest that tundra-type vegetation dominated the areas that were not covered by ice sheets (Mott and Jackson 1982; Strong and Hills 2005). This presents a problem because Oreohelids in southern Alberta are not found above 1600 m

109 elevation, the elevation at which snow can remain year-round and glacial relict vegetation is common. Given the restricted range of Clade B’, there are two possibilities. Either

Clade B’ is capable of utilizing tundra vegetation, which conflicts with observations of

Oreohelid habitat, or there were pockets of forested regions in the Rocky Mountains, which conflicts with pollen core data. Given the obscurity of Oreohelids in the literature, it is likely that their full range of habitat utilization is not completely understood.

Although I have characterized the distributions of these three Oreohelid lineages in southern Alberta and Saskatchewan, there are still important knowledge gaps that remain. The Sweet Grass Hills in Montana and the Porcupine Hills in southwestern

Alberta are additional potential refugia for Oreohelids. Like the Cypress Hills, these regions were also not glaciated during the Pleistocene, and they are upland sites containing similar physiographic and geological features. Additionally, it will be important to address the status of potential Oreohelid populations described by Dall

(1905) and to determine their phylogeographic relationships to other snail populations.

Currently there is no ITS2 data available for the Montana and Wyoming Oreohelids, which would allow a more accurate assessment of phylogeographic patterns in relation to southern Alberta (Weaver et al. 2006; Chak 2007). Indeed, the use of only two molecular markers is a significant limitation of this study. While other markers were investigated and rejected due to either low variation, or difficulty in amplification, next-generation sequencing of individuals from the genetic lineages reported here would likely reveal additional variation that could be used for more detailed population analyses

(McCormack et al. 2013). Another informative marker would be microsatellites, which are ideal for determining patterns of contemporary gene flow due to their high sensitivity

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(Putman and Carbone 2014). The use of microsatellites would be particularly relevant for samples of snails located within the putative hybrid zone adjacent to Highway 41

(Chapter 3), and would show the direction of gene flow across the zone. The use of microsatellites would also clarify the full extent of asymmetric mating between different genetic lineages located within the hybrid zone. Another approach would be to assess the results of breeding studies between different lineages to determine if there are differences in mate preference or mating behaviour between clades that may account for the maintenance of the hybrid zone. While preliminary mating trials with Oreohelix subrudis showed that Clades B and X are capable of interbreeding and producing offspring, a more robust breeding design with a larger sample size is required (e.g. Baur and Baur 1992).

5.3 Epidemiology of larval D. dendriticum in snails

The high prevalence of D. dendriticum in CHIP Oreohelids demonstrated in

Chapter 4 has important implications for the management and control of this, and other emerging and invasive parasites. Since all three species of Oreohelid present in CHIP are susceptible to infection, it is likely that other species of Oreohelid that occur throughout their broad range in western North America are equally susceptible. Thus, the potential exists for the continued spread of this worm within other sky island habitats in the USA and throughout the Rocky Mountains. Although D. dendriticum has not been detected at sites in the Rocky Mountains, an introduction of the parasite would allow it to spread southward into the USA and increase the likelihood of further spread to some sky islands.

While CHIP is separated from the eastern edges of the Rocky Mountains by approximately 250 km, the sky islands of Montana and Wyoming are much closer

111 together, and it is possible that either infected wildlife (e.g. deer and elk) or the movement of domestic stock, could disperse between them. Although it is not an immediate concern, if CHIP is ever connected to the Rocky Mountains or the Black Hills via a forest corridor, D. dendriticum would be able to move through the corridor along with its hosts.

The study of spatio-temporal patterns of D. dendriticum infection in the

Oreohelids of CHIP represents a departure from the mostly aquatic-based literature, and there are knowledge gaps that need to be addressed in future work. While spatial and temporal patterns of infection were measured across species, it is currently unknown if there are between-species differences in susceptibility. Experimental approaches are an important benchmark in susceptibility studies (Goater et al. 2014), and represent an important future direction for Oreohelids. These could be used in tandem with studies to determine the population level consequences of parasite induced castration (Scott 1988;

Lafferty and Kuris 2009). Both of these approaches could also be used to determine if D. dendriticum poses a significant conservation risk to any of the Oreohelids in CHIP.

5.4 Future Research

This line of research is by no means complete. Many potential avenues of investigation remain open. The following are perhaps the most prominent methodologies that require expansion. Future work will require more thorough sampling in Canada and the Northern USA. The Sweet Grass Hills and the Porcupine Hills are of immediate interest due to their potential as additional glacial refugia. Oreohelid surveys farther north

112 and east would determine the full geographic extent and range of habitats that the snails are capable of utilizing. Additional markers, specifically microsatellites, would allow fine dissection of the contact zone between Clades B and X in Cypress Hills. AFLPs have also been used successfully in the northern USA (Chak 2007), and would allow more direct comparisons to those snails. Ecological Niche Modelling (ENM) would allow for more pinpoint targeting of sites likely to contain Oreohelids of interest (such as Clade X or O. cooperi), and would allow for the quantification of the habitat preferences of each. An additional function of ENM would be climate projections of suitable habitat during the

Pleistocene and even into the future. Rigorous breeding studies would allow better elucidation of the contact zone between Clade B and Clade X, and determine the degree to which asymmetrical mating occurs between Oreohelid clades. For further parasitological research, experimental infection in a laboratory setting would allow much better elucidation of the factors responsible for D. dendriticum prevalence in Oreohelid snails, as well as determining the degree to which the “slime balls” can infect Formicid ants native to Cypress Hills.

5.5 General Conclusions

Phylogeography has become a cross-disciplinary field (Beheregaray 2008;

Knowles 2009). My study involving the little-known Oreohelid snails in southern Alberta and Saskatchewan provide a good example of this cross-disciplinary. My results from

Chapter two drew from conservation biology and taxonomy using both morphological and molecular techniques to extend the known geographical distribution of O. cooperi into Canada. Chapter three utilized literature from geology, glaciology, and paleontology,

113 as well as using molecular techniques to elucidate the origins and population genetic structures of Oreohelids in southern Alberta and Saskatchewan. Chapter four focused on an emerging infectious disease using parasitological techniques and methods, and framed

Oreohelids as important components of the invertebrate and parasite communities to determine spatial and temporal patterns of variation in D. dendriticum infection in CHIP.

Although invertebrates and Oreohelids in particular are underrepresented in the literature, they provide an excellent model system for integrative studies.

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5.6 References

Astor T. 2014. What do snails do in ecosystems? It is a matter of traits. Doctoral Thesis, Uppsala University.

Baur B and Baur A. 1992. Reduced reproductive compatibility in Arianta arbustorum (Gastropoda) from distant populations. Heredity 69: 65-72.

Beheregaray LB. 2008. Twenty years of phylogeography: the state of the field and the challenges for the southern hemisphere. Molecular Ecology 17: 3754-3774.

Cardoso P, Erwin TL, Borges PAV, and New TR. 2011. The seven impediments in invertebrate conservation and how to overcome them. Biological Conservation 144: 2647-2655.

Chak, S 2007. Population differentiation of Oreohelix land snails in Wyoming and adjacent South Dakota. Ph.D. Thesis, University of Wyoming, Wyoming, USA.

Choi HJ, and Cota-Sánchez JH. 2010. A taxonomic revision of Allium (Alliaceae) in the Canadian prairie provinces. Botany 88: 787-809.

Dall WH. 1905. Harriman Alaska Series: Volume XIII: Land and fresh water mollusks. Smithsonian Institution, Washington DC.

Goater TM, Goater CP, and Esch GW. 2014. Parasitism: The diversity and ecology of animal parasites, 2nd edition. Cambridge University Press, Cambridge, UK.

Henderson N, Hogg E, Barrow E, and Dolter B. 2002. Climate change impacts on the island forests of the Great Plains and the implications for nature conservation policy: the outlook for Sweet Grass Hills (Montana), Cypress Hills (Alberta- Saskatchewan), Moose Mountain (Saskatchewan), Spruce Woods (Manitoba), and Turtle Mountain (Manitoba-North Dakota). Prairie Adaptation and Research Collaborative, University of Regina, Saskatchewan, Canada.

Hill JK, Thomas CD, and Lewis OT. 1996. Effects of habitat patch size and isolation on dispersal by Hesperia comma butterflies: implications for metapopulation structure. Journal of Animal Ecology 65: 725-735.

Knowles LL. 2009. Statistical phylogeography. Annual Review of Ecology, Evolution, and Systematics 40: 593-612.

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Lafferty KD and Kuris AM. 2009. Parasitic castration: the evolution and ecology of body snatchers. Trends in Parasitology 25: 564-572.

McCormack JE, Hird SM, Zellmer AJ, Carstens BC, and Brumfield RT. 2013. Applications of next-generation sequencing to phylogeography and phylogenetics. Molecular Phylogenetics and Evolution 66: 526-538.

Newsome RD, and Dix RL. 1968. The forests of the Cypress Hills, Alberta and Saskatchewan, Canada. The American Midland Naturalist 80: 118-185.

Peterson AT. 2001. Predicting species’ geographic distributions based on ecological niche modelling. The Condor 103: 599-605.

Pilsbry HA. 1939. Land mollusca of North America north of Mexico vol I part 2. Acad Nat Sci Philadelphia. Wickersham Printing Company. Lancaster, Pennsylvania.

Putman AI and Carbone I. 2014. Challenges in analysis and interpretation of microsatellite data for population genetic studies. Ecology and Evolution 4: 4399- 4428.

Régnier C, Achaz G, Lambert A, Cowie RH, Bouchet P, & Fontaine B. 2015. Mass extinction in poorly known taxa. PNAS 112: 7761-7766.

Scott ME. 1988. The impact of infection and disease on animal populations: implications for conservation biology. Conservation Biology 2: 40-56.

Strong WL and Hills LV. 2005. Late-glacial and Holocene palaeovegetation zonal reconstruction for central and north-central North America. Journal of Biogeography 32: 1043-1062.

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APPENDIX 1: Supplementary Information for Chapter 3

Phylogeography of Oreohelix land snails in southern Alberta and Saskatchewan

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Appendix 1.1. Sample site characteristics for each site used in this study. Elev refers to elevation in metres, Slope refers to the facing with a rough estimate of grade. Cover provides a rough idea of the vegetation present and tree cover. Synonymous names and codes are used in lab and field work.

Site Area Lat Long Elev. Slope Cover Date Sampled Sampled by Synonymous name Code CH01 CHIP 49.6322 -110.3614 1450 low shrub Summer 2013 Cam Goater Horseshoe Canyon CHHSC CH02 CHIP 49.6594 -110.3041 1282 Summer 2013 Cam Goater Beaver Creek CHBC0 CH02 CHIP 49.6570 -110.2954 1253 30 W mod mixed June 2015 Zach/Arturo Beaver Creek transect 1 CHBC1 CH03 CHIP 49.6581 -110.2808 1270 30 E mod mixed Summer 2013 Cam Goater Staff Camp CHSC CH04 CHIP 49.6591 -110.2614 1263 45 NW mod mixed Spring 2013 Cam Goater Ski Hill CHSH CH05 CHIP 49.6648 -110.2636 1226 Summer 2014 Cam Goater Highway 41 transect 3 CHHW3 CH06 CHIP 49.6560 -110.2611 1273 Summer 2014 Cam Goater Highway 41 transect 2 CHHW2 CH07 CHIP 49.6503 -110.2614 1311 45 W high aspen Summer 2014 Cam Goater Highway 41 transect 1 CHHW1 CH08 CHIP 49.6500 -110.2571 1347 45 E mod aspen Summer 2013 Cam Goater Highway 41 transect 0 CHHW0 CH09 CHIP 49.6288 -110.1852 1409 60 SW low shrub Spring 2013 Cam Goater Cougar Alley CHCA CH10 CHIP 49.6722 -110.1470 1398 60 NE low shrub Summer 2014 Cam Goater Lookout Reesor Lake CHLRE CH11 CHIP 49.6610 -110.1175 1382 Summer 2013 Cam Goater Trans Canada Trail CHTCT CH12 CHIP 49.6638 -110.0723 1239 Spring 2013 Cam Goater Reesor Lake CHRE CH13 CHIP 49.6179 -110.0936 1326 45 NW high pine June 2015 Zach/Arturo Graburn Creek mid CHGCM CH14 CHIP 49.6421 -110.0334 1221 45 N high pine June 2015 Zach/Arturo Graburn Creek base CHGCB CH15 CHIP 49.5998 -110.0239 1347 Spring 2013 Cam Goater Nine Mile CHNM CHA CHIP 49.6176 -110.3892 1335 60 N high pine August 2015 Zach/Arturo Far West CHFW CHB CHIP 49.6401 -110.3224 1394 30 SE mod mixed June 2015 Zach/Arturo Beaver Creek transect 5 CHBC5 CHC CHIP 49.6449 -110.3179 1359 30 SW mod pine June 2015 Zach/Arturo Beaver Creek transect 4 CHBC4 CHD CHIP 49.6506 -110.3064 1303 45 N mod pine June 2015 Zach/Arturo Beaver Creek transect 3 CHBC3 CHE CHIP 49.6511 -110.2986 1260 30 W mod pine June 2015 Zach/Arturo Beaver Creek transect 2 CHBC2 CHF CHIP 49.6509 -110.2857 1291 high pine May 2015 Zach/Cam McCoy Camp CHMC CHG CHIP 49.6643 -110.2604 1231 30 W mod mixed August 2015 Zach/Arturo Opposite Highway 41 CHOHW CHH CHIP 49.6578 -110.2599 1274 30 W mod mixed August 2015 Zach/Arturo Opposite Ski Hill CHOSH

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CHI CHIP 49.6704 -110.2399 1311 30 S mod mixed August 2015 Zach/Arturo Spruce Coulee Halfway CHSCH CHJ CHIP 49.6831 -110.1948 1389 45 E high aspen June 2015 Zach/Arturo Spruce coulee transect 1 CHSCT1 CHK CHIP 49.6878 -110.1980 1326 30 N high aspen June 2015 Zach/Arturo Spruce coulee transect 2 CHSCT2 CHL CHIP 49.6784 -110.1852 1332 mod mixed May 2015 Zach/Cam Spruce Coulee Reservoir CHSCR CHM CHIP 49.6377 -110.2067 1428 low shrub August 2015 Cam Goater New Enclosure CHNE CHN CHIP 49.6384 -110.1658 1384 30 W high pine June 2015 Zach/Arturo Cougar Alley Run 2 CHCAR2 CHO CHIP 49.6348 -110.1639 1397 45 N mod aspen June 2015 Zach/Arturo Cougar Alley Run 1 CHCAR1 CHP CHIP 49.6129 -110.1494 1413 60 N low shrub June 2015 Zach/Arturo Graburn Creek top CHGCT CHQ CHIP 49.6110 -110.0585 1388 30 NE low shrub June 2015 Zach/Arturo Old house valley CHOHV CHR CHIP 49.5930 -110.0862 1396 60 NW low shrub June 2015 Zach/Arturo Nine Mile Headwaters CHNMH CHS CHIP 49.6338 -109.9852 1178 30 SW mod mixed June 2016 Zach/Sarah Battle Creek road 1 CSBCR1 CHT CHIP 49.6468 -109.9991 1177 30 S high aspen June 2016 Zach/Sarah Battle Creek road 2 CSBCR2 CHU CHIP 49.6462 -109.8468 1317 60 NE low shrub June 2016 Zach/Sarah Conglomerate Cliffs CSCC CHV CHIP 49.6027 -109.7958 1222 60 E mod pine June 2016 Zach/Sarah Eastern West block CSEW CHW CHIP 49.6596 -109.5166 1259 30 NW low aspen June 2016 Zach/Sarah Warlodge Campground CSWC CHX CHIP 49.6688 -109.5001 1196 30 S low mixed June 2016 Zach/Sarah North Loch Leven CSNLL CHY CHIP 49.6515 -109.4969 1203 60 E mod mixed June 2016 Zach/Sarah South Loch Lomond CSSLL RM01 CN 49.6789 -114.6098 1486 0 E high pine June 2015 Zach/Arturo Chinook Lake CNCL RM02 CN 49.6040 -114.4981 1456 45 N high pine June 2015 Zach/Arturo North York Creek 2 CNNY2 RM03 CN 49.5973 -114.4978 1544 60 NE high pine June 2015 Zach/Arturo North York Creek 1 CNNY1 RM04 CN 49.6015 -114.3970 1352 30 SW high mixed June 2015 Zach/Arturo Frank Slide CNFS RM05 CN 49.5833 -114.2071 1168 60 N high shrub June 2015 Zach/Arturo Lundbreck Falls CNLF RM06 CA 49.4525 -114.4097 1368 50 SE mod pine June 2015 Zach/Sarah Lynx Creek CALC1 RM07 CA 49.4445 -114.3239 1298 45 E mod aspen June 2015 Zach/Sarah Castle Falls CACF1 RM08 CA 49.3807 -114.3666 1368 45 SE low shrub June 2015 Zach/Arturo West Castle 1 CAWC1 RM09 CA 49.3494 -114.4063 1398 0 W high pine June 2015 Zach/Arturo Research station CARS RM10 CA 49.3584 -114.4065 1413 60 NE low shrub June 2015 Zach/Arturo West Castle 2 CAWC2 RM11 CA 49.3666 -114.3042 1456 60 SE high aspen June 2015 Zach/Sarah Beaver Mines Lake 1 CABML1

119

RM12 CA 49.3633 -114.2939 1547 30 W mod mixed June 2015 Zach Sarah Beaver Mines Lake 2 CABML2 RM13 WLNP 49.1286 -114.0254 1476 30 S mod aspen June 2015 Zach Arturo Red Rock Canyon WLRRC RM14 WLNP 49.0970 -113.9660 1442 30 NE mod mixed June 2015 Zach Arturo Crandell Lake WLCDL RM15 WLNP 49.0770 -113.8814 1302 Summer 2014 Cam Goater Blakiston Creek WLBC RM16 WLNP 49.0370 -113.9126 1376 Summer 2014 Cam Goater Birtha Lake Trail WLBLT RM17 WLNP 49.0662 -113.9981 1562 30 SE mod mixed June 2015 Zach Arturo Oil Town WLOT

120

Appendix 1.2. Samples gathered for each site. COI clade and ITS2 cluster were determined through sequencing and screening.

Site Location Synonymous Code Species COI Clade ITS2 Clusters CH01 CHIP CHHSC01 O. subrudis B 2/2 CH01 CHIP CHHSC02 O. cooperi co c/c CH01 CHIP CHHSC03 O. cooperi co c/c CH01 CHIP CHHSC04 O. cooperi co c/c CH01 CHIP CHHSC05 O. cooperi co c/c CH01 CHIP CHHSC06 O. cooperi co c/c CH01 CHIP CHHSC07 O. cooperi co c/c CH01 CHIP CHHSC08 O. cooperi co c/c CH01 CHIP CHHSC09 O. cooperi co c/c CH01 CHIP CHHSC10 O. cooperi co c/c CH01 CHIP CHHSC11 O. cooperi co c/c CH02 CHIP CHBC001 O. subrudis B 1/1 CH02 CHIP CHBC002 O. subrudis B 1/1 CH02 CHIP CHBC003 O. subrudis B 1/1 CH02 CHIP CHBC004 O. subrudis B 1/2 CH02 CHIP CHBC005 O. subrudis B 1/2 CH02 CHIP CHBC006 O. subrudis B 1/1 CH02 CHIP CHBC007 O. subrudis B 1/2 CH02 CHIP CHBC008 O. subrudis B 1/1 CH02 CHIP CHBC009 O. subrudis B 1/1 CH02 CHIP CHBC010 O. subrudis B 1/1 CH02 CHIP CHBC011 O. subrudis B 1/1 CH02 CHIP CHBC012 O. subrudis B 1/1 CH02 CHIP CHBC101 O. subrudis B 2/2 CH02 CHIP CHBC102 O. subrudis B 2/2 CH02 CHIP CHBC103 O. subrudis B 1/2 CH02 CHIP CHBC104 O. subrudis B 1/2 CH02 CHIP CHBC105 O. subrudis B 1/2 CH02 CHIP CHBC106 O. subrudis B 1/2 CH02 CHIP CHBC107 O. subrudis B 1/2 CH02 CHIP CHBC108 O. subrudis B 2/2 CH03 CHIP CHSC01 O. subrudis B 1/1 CH03 CHIP CHSC02 O. subrudis B 1/1 CH03 CHIP CHSC03 O. subrudis B 1/1 CH03 CHIP CHSC04 O. subrudis B 1/1 CH03 CHIP CHSC05 O. subrudis B 1/1 CH03 CHIP CHSC06 O. subrudis B 1/1 CH03 CHIP CHSC07 O. subrudis B 2/2 CH03 CHIP CHSC08 O. subrudis B 1/1

121

CH04 CHIP CHSH01 O. subrudis B 1/1 CH04 CHIP CHSH02 O. subrudis B 1/1 CH04 CHIP CHSH03 O. subrudis B 1/1 CH04 CHIP CHSH04 O. subrudis B 1/1 CH04 CHIP CHSH05 O. subrudis B 1/1 CH04 CHIP CHSH06 O. subrudis B 1/1 CH04 CHIP CHSH07 O. subrudis B 1/1 CH04 CHIP CHSH08 O. subrudis B 1/1 CH04 CHIP CHSH09 O. subrudis B 1/1 CH04 CHIP CHSH10 O. subrudis B 1/1 CH05 CHIP CHHW301 O. subrudis B 2/2 CH05 CHIP CHHW302 O. subrudis B 1/1 CH05 CHIP CHHW303 O. subrudis B 1/1 CH05 CHIP CHHW304 O. subrudis B 1/1 CH05 CHIP CHHW305 O. subrudis B 1/2 CH05 CHIP CHHW306 O. subrudis B 1/1 CH05 CHIP CHHW307 O. subrudis B 1/1 CH05 CHIP CHHW308 O. subrudis B 1/1 CH06 CHIP CHHW201 O. subrudis X 1/1 CH06 CHIP CHHW202 O. subrudis X 1/2 CH06 CHIP CHHW203 O. subrudis X 1/1 CH06 CHIP CHHW204 O. subrudis B 1/2 CH06 CHIP CHHW205 O. subrudis B 1/1 CH06 CHIP CHHW206 O. subrudis X 1/2 CH06 CHIP CHHW207 O. subrudis X 1/2 CH06 CHIP CHHW208 O. subrudis B 1/2 CH07 CHIP CHHW101 O. subrudis X 1/2 CH07 CHIP CHHW102 O. subrudis X 1/1 CH07 CHIP CHHW103 O. subrudis X 1/1 CH07 CHIP CHHW104 O. subrudis X 1/1 CH07 CHIP CHHW105 O. subrudis X 1/1 CH07 CHIP CHHW106 O. subrudis X 1/1 CH07 CHIP CHHW107 O. subrudis X 1/1 CH07 CHIP CHHW108 O. subrudis X 1/1 CH08 CHIP CHHW001 O. subrudis X 1/1 CH08 CHIP CHHW002 O. subrudis X 1/1 CH08 CHIP CHHW003 O. subrudis X 1/1 CH08 CHIP CHHW004 O. subrudis X 1/1 CH08 CHIP CHHW005 O. subrudis X 1/1 CH08 CHIP CHHW006 O. subrudis X 1/1 CH08 CHIP CHHW007 O. subrudis X 1/1 CH08 CHIP CHHW008 O. subrudis X 1/1 CH08 CHIP CHHW009 O. subrudis X 1/1 122

CH08 CHIP CHHW010 O. subrudis X 1/1 CH08 CHIP CHHW011 O. subrudis X 1/1 CH08 CHIP CHHW012 O. subrudis X 1/1 CH09 CHIP CHCA01 O. cooperi co c/c CH09 CHIP CHCA02 O. cooperi co c/c CH09 CHIP CHCA03 O. cooperi co c/c CH09 CHIP CHCA04 O. cooperi co c/c CH09 CHIP CHCA05 O. cooperi co c/c CH09 CHIP CHCA06 O. cooperi co c/c CH09 CHIP CHCA07 O. cooperi co c/c CH09 CHIP CHCA08 O. cooperi co c/c CH10 CHIP CHLRE01 O. cooperi co c/c CH10 CHIP CHLRE02 O. cooperi co c/c CH10 CHIP CHLRE03 O. cooperi co c/c CH10 CHIP CHLRE04 O. cooperi co c/c CH10 CHIP CHLRE05 O. cooperi co c/c CH10 CHIP CHLRE06 O. cooperi co c/c CH10 CHIP CHLRE07 O. cooperi co c/c CH10 CHIP CHLRE08 O. cooperi co c/c CH11 CHIP CHTCT01 O. subrudis X 2/2 CH11 CHIP CHTCT02 O. subrudis X 1/1 CH11 CHIP CHTCT03 O. subrudis X 1/2 CH11 CHIP CHTCT04 O. subrudis X 1/1 CH11 CHIP CHTCT05 O. subrudis X 1/1 CH11 CHIP CHTCT06 O. subrudis X 1/1 CH11 CHIP CHTCT07 O. subrudis X 1/1 CH11 CHIP CHTCT08 O. subrudis X 1/2 CH11 CHIP CHTCT09 O. subrudis X 1/1 CH11 CHIP CHTCT10 O. subrudis X 1/1 CH11 CHIP CHTCT11 O. subrudis X 1/1 CH11 CHIP CHTCT12 O. subrudis X 1/1 CH12 CHIP CHRE01 O. subrudis X 1/1 CH12 CHIP CHRE02 O. subrudis X 1/1 CH12 CHIP CHRE03 O. subrudis X 1/1 CH12 CHIP CHRE04 O. subrudis X 1/1 CH12 CHIP CHRE05 O. subrudis X 1/1 CH12 CHIP CHRE06 O. subrudis X 1/1 CH12 CHIP CHRE07 O. subrudis X 1/1 CH12 CHIP CHRE08 O. subrudis X 1/1 CH12 CHIP CHRE09 O. subrudis X 1/1 CH12 CHIP CHRE10 O. subrudis X 1/1 CH13 CHIP CHGCM01 O. cooperi co c/c CH13 CHIP CHGCM02 O. cooperi co c/c 123

CH13 CHIP CHGCM03 O. cooperi co c/c CH13 CHIP CHGCM04 O. cooperi co c/c CH13 CHIP CHGCM05 O. cooperi co c/c CH13 CHIP CHGCM06 O. cooperi co c/c CH13 CHIP CHGCM07 O. cooperi co c/c CH13 CHIP CHGCM08 O. cooperi co c/c CH14 CHIP CHGCB01 O. cooperi co c/c CH14 CHIP CHGCB02 O. cooperi co c/c CH14 CHIP CHGCB03 O. cooperi co c/c CH14 CHIP CHGCB04 O. cooperi co c/c CH14 CHIP CHGCB05 O. cooperi co c/c CH14 CHIP CHGCB06 O. cooperi co c/c CH14 CHIP CHGCB07 O. cooperi co c/c CH14 CHIP CHGCB08 O. cooperi co c/c CH15 CHIP CHNM01 O. subrudis X 1/1 CH15 CHIP CHNM02 O. subrudis X 1/1 CH15 CHIP CHNM03 O. subrudis X 1/1 CH15 CHIP CHNM04 O. subrudis X 1/1 CH15 CHIP CHNM05 O. subrudis X 1/1 CH15 CHIP CHNM06 O. subrudis X 1/1 CH15 CHIP CHNM07 O. subrudis X 1/1 CH15 CHIP CHNM08 O. subrudis X 1/1 CH15 CHIP CHNM09 O. subrudis X 1/1 CH15 CHIP CHNM10 O. subrudis X 1/1 CHA CHIP CHFW01 O. subrudis B 2/2 CHA CHIP CHFW02 O. subrudis B 2/2 CHA CHIP CHFW03 O. subrudis B 2/2 CHA CHIP CHFW04 O. subrudis B 2/2 CHA CHIP CHFW05 O. subrudis B 2/2 CHA CHIP CHFW06 O. subrudis B 2/2 CHA CHIP CHFW07 O. subrudis B 2/2 CHA CHIP CHFW08 O. subrudis B 2/2 CHB CHIP CHBC501 O. subrudis B 1/2 CHB CHIP CHBC502 O. subrudis B 2/2 CHB CHIP CHBC503 O. subrudis B 1/2 CHB CHIP CHBC504 O. subrudis B 2/2 CHB CHIP CHBC505 O. subrudis B 1/2 CHB CHIP CHBC506 O. subrudis B 1/2 CHB CHIP CHBC507 O. subrudis B 1/2 CHB CHIP CHBC508 O. subrudis B 2/2 CHC CHIP CHBC401 O. subrudis B 1/2 CHC CHIP CHBC402 O. subrudis B 2/2 CHC CHIP CHBC403 O. subrudis B 1/2 124

CHC CHIP CHBC404 O. subrudis B 1/2 CHC CHIP CHBC405 O. subrudis B 1/2 CHC CHIP CHBC406 O. subrudis B 1/2 CHC CHIP CHBC407 O. subrudis B 1/2 CHC CHIP CHBC408 O. subrudis B 2/2 CHD CHIP CHBC301 O. subrudis B 2/2 CHD CHIP CHBC302 O. subrudis B 2/2 CHD CHIP CHBC303 O. subrudis B 2/2 CHD CHIP CHBC304 O. subrudis B 1/2 CHD CHIP CHBC305 O. subrudis B 1/2 CHD CHIP CHBC306 O. subrudis B 1/2 CHD CHIP CHBC307 O. subrudis B 1/2 CHD CHIP CHBC308 O. subrudis B 2/2 CHE CHIP CHBC201 O. subrudis B 2/2 CHE CHIP CHBC202 O. subrudis B 2/2 CHE CHIP CHBC203 O. subrudis B 2/2 CHE CHIP CHBC204 O. subrudis B 1/2 CHE CHIP CHBC205 O. subrudis B 1/2 CHE CHIP CHBC206 O. subrudis B 1/2 CHE CHIP CHBC207 O. subrudis B 1/2 CHE CHIP CHBC208 O. subrudis B 2/2 CHF CHIP CHMC01 O. subrudis B 2/2 CHF CHIP CHMC02 O. subrudis B 1/1 CHF CHIP CHMC03 O. subrudis B 1/1 CHF CHIP CHMC04 O. subrudis B 1/1 CHF CHIP CHMC05 O. subrudis B 1/1 CHF CHIP CHMC06 O. subrudis B 1/1 CHF CHIP CHMC07 O. subrudis B 1/1 CHF CHIP CHMC08 O. subrudis B 1/1 CHG CHIP CHOHW01 O. subrudis B 1/1 CHG CHIP CHOHW02 O. subrudis B 1/1 CHG CHIP CHOHW03 O. subrudis B 1/1 CHG CHIP CHOHW04 O. subrudis B 1/1 CHG CHIP CHOHW05 O. subrudis B 1/1 CHG CHIP CHOHW06 O. subrudis B 1/1 CHG CHIP CHOHW07 O. subrudis B 1/1 CHG CHIP CHOHW08 O. subrudis B 1/1 CHH CHIP CHOSH01 O. subrudis B 1/1 CHH CHIP CHOSH02 O. subrudis B 1/1 CHH CHIP CHOSH03 O. subrudis B 1/1 CHH CHIP CHOSH04 O. subrudis B 1/1 CHH CHIP CHOSH05 O. subrudis B 1/1 CHH CHIP CHOSH06 O. subrudis B 1/1 125

CHH CHIP CHOSH07 O. subrudis B 1/1 CHH CHIP CHOSH08 O. subrudis B 1/1 CHI CHIP CHSCH01 O. subrudis X 1/1 CHI CHIP CHSCH02 O. subrudis X 1/1 CHI CHIP CHSCH03 O. subrudis X 1/1 CHI CHIP CHSCH04 O. subrudis X 1/1 CHI CHIP CHSCH05 O. subrudis X 1/1 CHI CHIP CHSCH06 O. subrudis X 1/1 CHI CHIP CHSCH07 O. subrudis X 1/1 CHI CHIP CHSCH08 O. subrudis X 1/1 CHJ CHIP CHSCT101 O. subrudis X 1/1 CHJ CHIP CHSCT102 O. subrudis X 2/2 CHJ CHIP CHSCT103 O. subrudis X 1/1 CHJ CHIP CHSCT104 O. subrudis X 1/1 CHJ CHIP CHSCT105 O. subrudis X 1/1 CHJ CHIP CHSCT106 O. subrudis X 1/1 CHJ CHIP CHSCT107 O. subrudis X 1/1 CHJ CHIP CHSCT108 O. subrudis X 1/1 CHK CHIP CHSCT201 O. subrudis X 1/1 CHK CHIP CHSCT202 O. subrudis X 2/2 CHK CHIP CHSCT203 O. subrudis X 1/1 CHK CHIP CHSCT204 O. subrudis X 1/1 CHK CHIP CHSCT205 O. subrudis X 1/1 CHK CHIP CHSCT206 O. subrudis X 1/1 CHK CHIP CHSCT207 O. subrudis X 1/1 CHK CHIP CHSCT208 O. subrudis X 1/1 CHL CHIP CHSCR01 O. subrudis X 2/2 CHL CHIP CHSCR02 O. subrudis X 1/1 CHL CHIP CHSCR03 O. subrudis X 1/1 CHL CHIP CHSCR04 O. subrudis X 1/1 CHL CHIP CHSCR05 O. subrudis X 1/1 CHL CHIP CHSCR06 O. subrudis X 1/1 CHL CHIP CHSCR07 O. subrudis X 1/1 CHL CHIP CHSCR08 O. subrudis X 1/1 CHM CHIP CHNE01 O. cooperi co c/c CHM CHIP CHNE02 O. cooperi co c/c CHM CHIP CHNE03 O. cooperi co c/c CHM CHIP CHNE04 O. cooperi co c/c CHM CHIP CHNE05 O. cooperi co c/c CHM CHIP CHNE06 O. cooperi co c/c CHM CHIP CHNE07 O. cooperi co c/c CHM CHIP CHNE08 O. cooperi co c/c CHN CHIP CHCAR201 O. subrudis X 1/1 126

CHN CHIP CHCAR202 O. cooperi co c/c CHN CHIP CHCAR203 O. subrudis X 1/1 CHN CHIP CHCAR204 O. subrudis X 2/2 CHN CHIP CHCAR205 O. subrudis X 1/1 CHN CHIP CHCAR206 O. subrudis X 1/1 CHN CHIP CHCAR207 O. subrudis X 1/1 CHN CHIP CHCAR208 O. subrudis X 1/1 CHO CHIP CHCAR101 O. subrudis X 1/1 CHO CHIP CHCAR102 O. cooperi co c/c CHO CHIP CHCAR103 O. subrudis X 1/1 CHO CHIP CHCAR104 O. subrudis X 1/1 CHO CHIP CHCAR105 O. subrudis X 1/1 CHO CHIP CHCAR106 O. subrudis X 1/1 CHO CHIP CHCAR107 O. subrudis X 1/1 CHO CHIP CHCAR108 O. subrudis X 1/1 CHP CHIP CHGCT01 O. cooperi co c/c CHP CHIP CHGCT02 O. cooperi co c/c CHP CHIP CHGCT03 O. cooperi co c/c CHP CHIP CHGCT04 O. cooperi co c/c CHP CHIP CHGCT05 O. cooperi co c/c CHP CHIP CHGCT06 O. cooperi co c/c CHP CHIP CHGCT07 O. cooperi co c/c CHP CHIP CHGCT08 O. cooperi co c/c CHQ CHIP CHOHV01 O. cooperi co c/c CHQ CHIP CHOHV02 O. cooperi co c/c CHQ CHIP CHOHV03 O. cooperi co c/c CHQ CHIP CHOHV04 O. cooperi co c/c CHQ CHIP CHOHV05 O. cooperi co c/c CHQ CHIP CHOHV06 O. cooperi co c/c CHQ CHIP CHOHV07 O. cooperi co c/c CHQ CHIP CHOHV08 O. cooperi co c/c CHR CHIP CHNMH01 O. cooperi co c/c CHR CHIP CHNMH02 O. cooperi co c/c CHR CHIP CHNMH03 O. cooperi co c/c CHR CHIP CHNMH04 O. cooperi co c/c CHR CHIP CHNMH05 O. cooperi co c/c CHR CHIP CHNMH06 O. cooperi co c/c CHR CHIP CHNMH07 O. cooperi co c/c CHR CHIP CHNMH08 O. cooperi co c/c CHS CHIP CSBCR101 O. subrudis X 1/1 CHS CHIP CSBCR102 O. subrudis X 1/1 CHS CHIP CSBCR103 O. subrudis X 1/1 CHS CHIP CSBCR104 O. subrudis X 1/1 127

CHS CHIP CSBCR105 O. cooperi co c/c CHS CHIP CSBCR106 O. cooperi co c/c CHS CHIP CSBCR107 O. cooperi co c/c CHS CHIP CSBCR108 O. cooperi co c/c CHT CHIP CSBCR201 O. subrudis X 1/1 CHT CHIP CSBCR202 O. subrudis X 1/1 CHT CHIP CSBCR203 O. subrudis X 1/1 CHT CHIP CSBCR204 O. subrudis X 1/1 CHT CHIP CSBCR205 O. subrudis X 1/1 CHT CHIP CSBCR206 O. subrudis X 1/1 CHT CHIP CSBCR207 O. subrudis X 1/1 CHT CHIP CSBCR208 O. subrudis X 1/1 CHU CHIP CSCC01 O. cooperi co c/c CHU CHIP CSCC02 O. cooperi co c/c CHU CHIP CSCC03 O. cooperi co c/c CHU CHIP CSCC04 O. cooperi co c/c CHU CHIP CSCC05 O. cooperi co c/c CHU CHIP CSCC06 O. cooperi co c/c CHU CHIP CSCC07 O. cooperi co c/c CHU CHIP CSCC08 O. cooperi co c/c CHV CHIP CSEW01 O. cooperi co c/c CHW CHIP CSWC01 O. subrudis X 1/1 CHW CHIP CSWC02 O. subrudis X 1/1 CHW CHIP CSWC03 O. subrudis X 1/1 CHW CHIP CSWC04 O. subrudis X 1/1 CHW CHIP CSWC05 O. subrudis X 1/1 CHW CHIP CSWC06 O. subrudis X 1/1 CHW CHIP CSWC07 O. subrudis X 1/1 CHW CHIP CSWC08 O. subrudis X 1/1 CHX CHIP CSNLL01 O. subrudis X 1/1 CHX CHIP CSNLL02 O. subrudis X 1/1 CHX CHIP CSNLL03 O. subrudis X 1/1 CHX CHIP CSNLL04 O. subrudis X 1/1 CHX CHIP CSNLL05 O. subrudis X 1/1 CHX CHIP CSNLL06 O. subrudis X 1/1 CHX CHIP CSNLL07 O. subrudis X 1/1 CHX CHIP CSNLL08 O. subrudis X 1/1 CHY CHIP CSSLL01 O. subrudis X 1/1 CHY CHIP CSSLL02 O. subrudis X 1/1 CHY CHIP CSSLL03 O. subrudis X 1/1 CHY CHIP CSSLL04 O. subrudis X 1/1 CHY CHIP CSSLL05 O. subrudis X 1/1 CHY CHIP CSSLL06 O. subrudis X 1/1 128

CHY CHIP CSSLL07 O. subrudis X 1/1 CHY CHIP CSSLL08 O. subrudis X 1/1 RM01 CN CNCL01 O. subrudis B 2/2 RM01 CN CNCL02 O. subrudis B 2/2 RM01 CN CNCL03 O. subrudis B 2/2 RM01 CN CNCL04 O. subrudis B 2/2 RM01 CN CNCL05 O. subrudis B 2/2 RM01 CN CNCL06 O. subrudis B 2/2 RM01 CN CNCL07 O. subrudis B 2/2 RM01 CN CNCL08 O. subrudis B 2/2 RM02 CN CNNY201 O. subrudis B 2/2 RM02 CN CNNY202 O. subrudis B 2/2 RM02 CN CNNY203 O. subrudis B 2/2 RM02 CN CNNY204 O. subrudis B 2/2 RM02 CN CNNY205 O. subrudis B 2/2 RM02 CN CNNY206 O. subrudis B 2/2 RM02 CN CNNY207 O. subrudis B 2/2 RM02 CN CNNY208 O. subrudis B 2/2 RM03 CN CNNY101 O. subrudis B' 2/2 RM03 CN CNNY102 O. subrudis B 2/2 RM03 CN CNNY103 O. subrudis B 2/2 RM03 CN CNNY104 O. subrudis B 2/2 RM03 CN CNNY105 O. subrudis B 2/2 RM03 CN CNNY106 O. subrudis B 2/2 RM03 CN CNNY107 O. subrudis B 2/2 RM03 CN CNNY108 O. subrudis B 2/2 RM04 CN CNFS01 O. subrudis B' 2/2 RM04 CN CNFS02 O. subrudis B' 2/2 RM04 CN CNFS03 O. subrudis B 2/2 RM04 CN CNFS04 O. subrudis B 2/2 RM04 CN CNFS05 O. subrudis B' 2/2 RM04 CN CNFS06 O. subrudis B' 2/2 RM04 CN CNFS07 O. subrudis B' 2/2 RM04 CN CNFS08 O. subrudis B 2/2 RM05 CN CNLF01 O. subrudis B 2/2 RM05 CN CNLF02 O. subrudis B 2/2 RM05 CN CNLF03 O. subrudis B 2/2 RM05 CN CNLF04 O. subrudis B 2/2 RM05 CN CNLF05 O. subrudis B 2/2 RM05 CN CNLF06 O. subrudis B 2/2 RM05 CN CNLF07 O. subrudis B 2/2 RM05 CN CNLF08 O. subrudis B 2/2 RM06 CA CALC101 O. subrudis B' 2/2 129

RM06 CA CALC102 O. subrudis B' 2/2 RM06 CA CALC103 O. subrudis B' 2/2 RM06 CA CALC104 O. subrudis B' 2/2 RM06 CA CALC105 O. subrudis B' 2/2 RM06 CA CALC106 O. subrudis B' 2/2 RM06 CA CALC107 O. subrudis B' 2/2 RM06 CA CALC108 O. subrudis B' 2/2 RM07 CA CACF101 O. subrudis B 2/2 RM07 CA CACF102 O. subrudis B' 2/2 RM07 CA CACF103 O. subrudis B 2/2 RM07 CA CACF104 O. subrudis B' 2/2 RM07 CA CACF105 O. subrudis B 2/2 RM07 CA CACF106 O. subrudis B 2/2 RM07 CA CACF107 O. subrudis B 2/2 RM07 CA CACF108 O. subrudis B' 2/2 RM08 CA CAWC101 O. subrudis B' 2/2 RM08 CA CAWC102 O. subrudis B' 2/2 RM08 CA CAWC103 O. subrudis B' 2/2 RM08 CA CAWC104 O. subrudis B' 2/2 RM08 CA CAWC105 O. subrudis B' 2/2 RM08 CA CAWC106 O. subrudis B' 2/2 RM08 CA CAWC107 O. subrudis B' 2/2 RM08 CA CAWC108 O. subrudis B' 2/2 RM09 CA CARS01 O. subrudis B' 2/2 RM09 CA CARS02 O. subrudis B' 2/2 RM09 CA CARS03 O. subrudis B' 2/2 RM09 CA CARS04 O. subrudis B' 2/2 RM09 CA CARS05 O. subrudis B' 2/2 RM09 CA CARS06 O. subrudis B' 2/2 RM09 CA CARS07 O. subrudis B' 2/2 RM09 CA CARS08 O. subrudis B' 2/2 RM10 CA CAWC201 O. subrudis B' 2/2 RM10 CA CAWC202 O. subrudis B' 2/2 RM10 CA CAWC203 O. subrudis B' 2/2 RM10 CA CAWC204 O. subrudis B' 2/2 RM10 CA CAWC205 O. subrudis B' 2/2 RM10 CA CAWC206 O. subrudis B' 2/2 RM10 CA CAWC207 O. subrudis B' 2/2 RM10 CA CAWC208 O. subrudis B' 2/2 RM11 CA CABML101 O. subrudis B 2/2 RM11 CA CABML102 O. subrudis B 2/2 RM11 CA CABML103 O. subrudis B 2/2 RM11 CA CABML104 O. subrudis B 2/2 130

RM11 CA CABML105 O. subrudis B 2/2 RM11 CA CABML106 O. subrudis B 2/2 RM11 CA CABML107 O. subrudis B 2/2 RM11 CA CABML108 O. subrudis B 2/2 RM12 CA CABML201 O. subrudis B 2/2 RM12 CA CABML202 O. subrudis B 2/2 RM12 CA CABML203 O. subrudis B 2/2 RM12 CA CABML204 O. subrudis B 2/2 RM12 CA CABML205 O. subrudis B 2/2 RM12 CA CABML206 O. subrudis B 2/2 RM12 CA CABML207 O. subrudis B 2/2 RM12 CA CABML208 O. subrudis B 2/2 RM13 WLNP WLRRC01 O. subrudis B 2/2 RM13 WLNP WLRRC02 O. subrudis B 2/2 RM13 WLNP WLRRC03 O. subrudis B 2/2 RM13 WLNP WLRRC04 O. subrudis B 2/2 RM13 WLNP WLRRC05 O. subrudis B 2/2 RM13 WLNP WLRRC06 O. subrudis B 2/2 RM13 WLNP WLRRC07 O. subrudis B 2/2 RM13 WLNP WLRRC08 O. subrudis B 2/2 RM14 WLNP WLCDL01 O. subrudis B 2/2 RM14 WLNP WLCDL02 O. subrudis B 2/2 RM14 WLNP WLCDL03 O. subrudis B 2/2 RM14 WLNP WLCDL04 O. subrudis B 2/2 RM15 WLNP WLBC01 O. subrudis B 2/2 RM15 WLNP WLBC02 O. subrudis B 2/2 RM15 WLNP WLBC03 O. subrudis B 2/2 RM15 WLNP WLBC04 O. subrudis B 2/2 RM15 WLNP WLBC05 O. subrudis B 2/2 RM15 WLNP WLBC06 O. subrudis B 2/2 RM15 WLNP WLBC07 O. subrudis B 2/2 RM15 WLNP WLBC08 O. subrudis B 2/2 RM16 WLNP WLBLT01 O. subrudis B 2/2 RM16 WLNP WLBLT02 O. subrudis B 2/2 RM16 WLNP WLBLT03 O. subrudis B 2/2 RM16 WLNP WLBLT04 O. subrudis B 2/2 RM16 WLNP WLBLT05 O. subrudis B 2/2 RM16 WLNP WLBLT06 O. subrudis B 2/2 RM16 WLNP WLBLT07 O. subrudis B 2/2 RM16 WLNP WLBLT08 O. subrudis B 2/2 RM17 WLNP WLOT01 O. subrudis B 2/2 RM17 WLNP WLOT02 O. subrudis B 2/2 RM17 WLNP WLOT03 O. subrudis B 2/2 131

RM17 WLNP WLOT04 O. subrudis B 2/2 RM17 WLNP WLOT05 O. subrudis B 2/2 RM17 WLNP WLOT06 O. subrudis B 2/2 RM17 WLNP WLOT07 O. subrudis B 2/2 RM17 WLNP WLOT08 O. subrudis B 2/2

132

Appendix 1.3. Primers investigated in this study. Loc specifies primers as mitochondrial (mt) or nuclear (nuc). Var. refers to the amount of sequence variation found between clades. Fix. refers to the presence or absence of fixed differences between clades. A ‘?’ in the variance and fixed categories denotes primer sets that were not successfully optimized. Size Var Loc Primer name Primer Sequences (5’-3’) Source Fix. (bp) (%) mt LCO1490 673 GTC AAC AAA TCA TAA AGA TAT TGG Weaver et al. 2006 <25 Yes mt HCO2198 673 TAA ACT TCA GGG TGA CCA AAA AAT CA Weaver et al. 2006 <25 Yes mt HCOIcommO - TAA ACT TCA GGG TGA CCA AAA Dempsey 2015 <25 Yes mt LCOIspecOc635 635 TGC TCT TTC ACT TTT AAT TCG AC Dempsey 2015 <25 Yes mt LCOIspecOs563 563 ATT GTT ACA GCC TAT GCC Dempsey 2015 <25 Yes mt LCOIspecOx217 217 GTG CCC CAG GAA TAA ATT TG Dempsey 2015 <25 Yes mt 12Sa-L 415 AAA AAG CTT CAA ACT GGG ATT AGA TAC CCC ACT AT Weaver et al 2006 ? ? mt 12Sa-H 415 TGA CTG CAG AGG GTG ACG GGC GGT GTG T Weaver et al 2006 ? ? nuc LSU1F2990 918 CTA GCT GCG AGA ATT AAT GTG A Wade & Mordan 2000 <20 Yes nuc 5.8SF3223 685 CAG AAC CAC ATT GAA CAT CGA Wade & Mordan 2000 <20 Yes nuc ITS4R3908 - TCC TCC GCT TAG TTA TAT GC Wade & Mordan 2000 <20 Yes nuc ITS2FspecOc256 256 CCG TGG TCT TAA GTT CAA A Dempsey 2015 <20 Yes nuc ITS2FspecOs112 112 TTA ACG AAA AGT GGA TGC T Dempsey 2015 <20 Yes nuc ITSFspecOx356 356 CTG CTG TGC TCT AGC ATT TAT Dempsey 2015 <20 Yes nuc AriaH4_fwd 258 AAC CTC CGA AGC CGT ACA GGG T Cadahia et al. 2013 <10 No nuc AriaH4_rev 258 GAA GAG GTA AAG GCG GCA AGG G Cadahia et al. 2013 <10 No nuc OreoH4comF - TCC TCT TGG CGT GCT CAG TGT A Dempsey 2015 <10 No nuc OreoH4gapR 156 AGG TAA AGC CGC CTA GCG AC Dempsey 2015 <10 No AAC AGC TAT GAC CAT GGT AAA GCC GCC AAG CGT nuc OreoH4fillR 178 Dempsey 2015 <10 No GGT nuc OreoH4F80common - CCT TGA GGA CAC CTC TGG TT Dempsey 2016 <10 No nuc OreoH4R226RsaIcutsIns 226 GCG GCA AGG GTG GTA CA Dempsey 2016 <10 No OreoH4R227/236Ampoth CAC GAC GTT GTA AAA CGA CAA CAG GTA AAG CCG nuc 236 Dempsey 2016 <10 No 9bp GCA AG

133 nuc AriaH3_fwd 316 ATG GCT GAA CCA AGC AGA CC Cadahia et al. 2013 <2 no nuc AriaH3_rev 316 GGT GAC ACG CTT GGC GTG G Cadahia et al. 2013 <2 no nuc OreoTLRf 813 TAG CCT TCA ACC CGT GGT C Dempsey 2015 ? ? nuc OreoTLRr 813 GGC AAA GCA TAC GTC AAT TTT Dempsey 2015 ? ?

134

APPENDIX 2: Supplementary Information for Chapter 4

Spatio-temporal patterns of emerging larval liver fluke (Dicrocoelium dendriticum) in three sympatric species of land snails in southern Alberta, Canada

135

Appendix 2.1. Phenology data for Oreohelix subrudis clade B. Size is in mm, ‘Dicro’ represents presence/absence of Dicrocoelium dendriticum sporocysts, ‘Other’ represents metacercariae of the tentatively identified Postharmostomum helicis. A 1* in ‘Other’ indicates the presence of a Sciomyzidae larvae which has consumed the body of the snail. Gravid indicates the number of embryos found in the uterus. Month Site Size Dicro Other Gravid Month Site Size Dicro Other Gravid 1 May CHSH 15.7 0 8 0 1 May CHSC 16.2 0 3 0 2 May CHSH 14.9 0 1 0 2 May CHSC 15.5 0 34 0 3 May CHSH 15.8 0 6 0 3 May CHSC 15.5 0 15 0 4 May CHSH 16 0 0 0 4 May CHSC 17.55 0 1* 0 5 May CHSH 15.8 0 3 0 5 May CHSC 16.5 0 10 6 6 May CHSH 14.8 0 4 0 6 May CHSC 14.3 0 17 0 7 May CHSH 14.2 0 8 0 7 May CHSC 14.5 0 25 0 8 May CHSH 13.25 0 1 0 8 May CHSC 15.7 1 16 0 9 May CHSH 16.3 0 9 0 9 May CHSC 14.2 0 41 0 10 May CHSH 15.6 0 2 0 10 May CHSC 13.8 0 28 0 11 May CHSH 16.5 0 7 0 11 May CHSC 17.5 0 72 0 12 May CHSH 16.5 0 22 0 12 May CHSC 14.6 0 14 0 13 May CHSH 15.05 0 10 0 13 May CHSC 15.7 0 8 0 14 May CHSH 16.1 0 38 0 14 May CHSC 16.7 0 31 0 15 May CHSH 15.2 0 6 0 15 May CHSC 15.6 0 13 0 16 May CHSH 15.1 0 4 0 16 May CHSC 15 0 23 0 17 May CHSH 15 0 12 0 17 May CHSC 16.5 0 10 0 18 May CHSH 15.1 0 0 0 18 May CHSC 15.1 0 35 0 19 May CHSH 16.2 0 0 0 19 May CHSC 16.6 1 18 0 20 May CHSH 15.25 0 0 0 20 May CHSC 14.95 0 4 0 21 May CHSH 14.7 0 0 0 21 May CHSC 15.1 0 46 0 22 May CHSH 14.5 0 2 0 22 May CHSC 15.3 0 22 0 23 May CHSH 13 1 2 0 23 May CHSC 12.9 0 16 0 24 May CHSH 16.3 0 0 0 24 May CHSC 13.8 0 9 0

136

25 May CHSH 15.25 0 4 0 25 May CHSC 14.7 0 2 0 26 May CHSH 14.6 0 6 0 26 May CHSC 13.2 0 20 0 27 May CHSH 16.5 0 5 0 27 May CHSC 14.6 0 11 0 28 May CHSH 14 0 2 0 28 May CHSC 14.1 0 7 0 29 May CHSH 13.1 0 0 0 29 May CHSC 12.5 0 13 0 30 May CHSH 13.25 0 1 0 30 May CHSC 12.6 0 0 0 AVG SUM 15.12 1 163 0 AVG SUM 15.03 2 563 6

Month Site Size Dicro Other Gravid Month Site Size Dicro Other Gravid 1 June CHSH 15.9 0 3 0 1 June CHSC 14.75 0 2 0 2 June CHSH 16.6 1 3 0 2 June CHSC 14 0 31 0 3 June CHSH 16 0 5 0 3 June CHSC 16 0 23 0 4 June CHSH 18.1 0 3 0 4 June CHSC 15.2 0 36 0 5 June CHSH 15.8 0 5 0 5 June CHSC 20.2 0 16 0 6 June CHSH 16.1 1 32 0 6 June CHSC 17.7 0 41 0 7 June CHSH 18.5 0 5 0 7 June CHSC 14.7 0 8 0 8 June CHSH 17.5 0 15 0 8 June CHSC 17.7 0 53 0 9 June CHSH 15.8 0 22 0 9 June CHSC 14.9 0 1 0 10 June CHSH 14.9 1 1 0 10 June CHSC 14.35 0 6 0 11 June CHSH 15.3 0 4 0 11 June CHSC 14.9 0 10 0 12 June CHSH 16.1 0 3 0 12 June CHSC 13.05 0 1 0 13 June CHSH 16.1 0 6 0 13 June CHSC 16.2 0 27 0 14 June CHSH 15.8 0 2 0 14 June CHSC 15.3 0 22 0 15 June CHSH 15.5 0 12 0 15 June CHSC 15.4 0 31 0 16 June CHSH 15.9 0 7 0 16 June CHSC 16 0 6 0 17 June CHSH 15.7 0 0 0 17 June CHSC 14.45 0 0 0 18 June CHSH 16 0 1 0 18 June CHSC 14.1 0 22 0 19 June CHSH 16.8 0 8 0 19 June CHSC 14.2 0 7 0

137

20 June CHSH 16.5 0 4 0 20 June CHSC 13.3 0 2 0 21 June CHSH 16.7 0 25 0 21 June CHSC 16.6 0 25 0 22 June CHSH 16.1 0 2 0 22 June CHSC 14.7 0 13 0 23 June CHSH 18 0 25 0 23 June CHSC 15.05 0 18 0 24 June CHSH 15.7 0 4 0 24 June CHSC 15.35 0 1 0 25 June CHSH 16.6 0 16 0 25 June CHSC 13.1 0 30 0 26 June CHSH 16.2 0 3 0 26 June CHSC 14.9 0 58 0 27 June CHSH 17.4 0 0 0 27 June CHSC 13.5 0 2 0 28 June CHSH 12.7 0 0 0 28 June CHSC 13.25 0 5 0 29 June CHSH 13.8 0 2 0 29 June CHSC 12.7 0 6 0 30 June CHSH 16.3 0 6 0 30 June CHSC 12 0 11 0 AVG SUM 16.1 3 224 0 AVG SUM 14.9 0 514 0

Month Site Size Dicro Other Gravid Month Site Size Dicro Other Gravid 1 July CHSH 15.7 0 1 4 1 July CHSC 14.3 0 5 0 2 July CHSH 17.3 1 0 9 2 July CHSC 14.9 0 12 4 3 July CHSH 16.6 0 3 6 3 July CHSC 14.7 0 17 6 4 July CHSH 17 1 58 0 4 July CHSC 13.9 0 23 0 5 July CHSH 15.5 0 15 10 5 July CHSC 15.7 0 14 8 6 July CHSH 16.1 0 4 0 6 July CHSC 15.3 0 37 3 7 July CHSH 17.9 0 11 0 7 July CHSC 16 0 9 4 8 July CHSH 17 0 1 9 8 July CHSC 14.5 0 2 0 9 July CHSH 16 0 6 0 9 July CHSC 18.7 0 21 12 10 July CHSH 16.3 0 4 0 10 July CHSC 14.2 0 8 0 11 July CHSH 15 0 2 7 11 July CHSC 17 0 12 8 12 July CHSH 16.9 0 0 0 12 July CHSC 16.6 0 33 4 13 July CHSH 15.6 0 14 0 13 July CHSC 15.4 1 28 0 14 July CHSH 17.2 0 0 11 14 July CHSC 14.4 0 13 9

138

15 July CHSH 16.6 0 8 13 15 July CHSC 15.9 0 22 8 16 July CHSH 15.5 0 3 7 16 July CHSC 17.3 0 11 7 17 July CHSH 16 0 4 9 17 July CHSC 15.7 0 15 7 18 July CHSH 15.3 0 0 8 18 July CHSC 14.8 0 36 2 19 July CHSH 15.7 1 42 0 19 July CHSC 13.2 0 27 0 20 July CHSH 15 0 0 0 20 July CHSC 16.8 0 7 0 21 July CHSH 16.5 0 3 10 21 July CHSC 15 1 22 0 22 July CHSH 15.5 0 0 5 22 July CHSC 15.9 0 8 0 23 July CHSH 19.5 0 37 0 23 July CHSC 13.9 0 7 0 24 July CHSH 15.8 0 0 5 24 July CHSC 16.3 0 6 4 25 July CHSH 16.2 0 5 8 25 July CHSC 15.4 0 9 0 26 July CHSH 14.9 0 0 8 26 July CHSC 13.2 0 25 0 27 July CHSH 16.9 0 2 6 27 July CHSC 15.7 0 31 0 28 July CHSH 16.8 0 8 5 28 July CHSC 14.4 0 18 0 29 July CHSH 14.7 0 1 0 29 July CHSC 15.6 0 5 7 30 July CHSH 15.9 0 0 4 30 July CHSC 16.4 0 11 9 AVG SUM 16.23 3 232 144 AVG SUM 15.37 2 494 102

Month Site Size Dicro Other Gravid Month Site Size Dicro Other Gravid 1 August CHSH 16.2 0 3 0 1 August CHSC 16.1 0 31 0 2 August CHSH 15 0 18 0 2 August CHSC 14.65 0 36 0 3 August CHSH 16.3 1 0 0 3 August CHSC 14.9 0 45 0 4 August CHSH 16.5 0 2 0 4 August CHSC 14.75 0 16 2 5 August CHSH 14.5 0 0 0 5 August CHSC 16.85 0 22 0 6 August CHSH 15 0 8 0 6 August CHSC 13.7 0 23 0 7 August CHSH 14 1 13 0 7 August CHSC 15.1 1 12 0 8 August CHSH 15.5 0 32 0 8 August CHSC 14.7 1 14 0 9 August CHSH 13.5 0 2 0 9 August CHSC 15.7 0 46 0

139

10 August CHSH 14.5 1 8 0 10 August CHSC 16.6 0 8 0 11 August CHSH 15.4 0 14 0 11 August CHSC 16.25 1 24 0 12 August CHSH 16 0 21 0 12 August CHSC 12.8 0 14 0 13 August CHSH 13.3 0 7 0 13 August CHSC 13.9 0 4 0 14 August CHSH 13.4 0 4 0 14 August CHSC 13.8 0 11 0 15 August CHSH 16.4 0 3 0 15 August CHSC 13.7 0 6 0 16 August CHSH 16.5 0 5 0 16 August CHSC 14.4 0 5 0 17 August CHSH 14.1 0 7 0 17 August CHSC 12.75 0 18 0 18 August CHSH 16.1 0 4 0 18 August CHSC 16.35 0 22 0 19 August CHSH 13 0 2 0 19 August CHSC 14.3 1 10 0 20 August CHSH 16.4 0 6 0 20 August CHSC 16.25 0 17 0 21 August CHSH 18.1 1 22 0 21 August CHSC 14.2 0 33 0 2 August CHSH 16.9 1 17 0 22 August CHSC 14.65 1 26 0 23 August CHSH 15.6 1 8 0 23 August CHSC 14.3 0 25 0 24 August CHSH 15.3 0 4 0 24 August CHSC 15.2 0 42 0 25 August CHSH 15 0 7 0 25 August CHSC 13.9 1 3 0 26 August CHSH 15.4 0 16 0 26 August CHSC 15.45 0 32 0 27 August CHSH 14.1 0 9 0 27 August CHSC 13.95 0 67 0 28 August CHSH 15.1 0 12 0 28 August CHSC 12.1 1 20 0 29 August CHSH 15.7 0 8 0 29 August CHSC 12.5 0 0 0 30 August CHSH 14.4 0 0 0 30 August CHSC 13.7 0 9 0 AVG SUM 15.24 6 262 0 AVG SUM 14.58 7 641 2

Month Site Size Dicro Other Gravid Month Site Size Dicro Other Gravid 1 September CHSH 17.08 1 22 0 1 September CHSC 14.85 0 0 0 2 September CHSH 15.75 0 13 0 2 September CHSC 19 0 10 0 3 September CHSH 14.9 0 1 0 3 September CHSC 17.6 0 24 0 4 September CHSH 15.1 0 11 0 4 September CHSC 16.6 0 11 0

140

5 September CHSH 12.9 0 0 0 5 September CHSC 16.4 0 0 0 6 September CHSH 16.7 0 7 0 6 September CHSC 14.9 0 13 0 7 September CHSH 10.7 0 17 0 7 September CHSC 14.8 0 19 0 8 September CHSH 15.75 0 0 0 8 September CHSC 15.7 0 56 0 9 September CHSH 8.4 0 0 0 9 September CHSC 16.45 0 6 0 10 September CHSH 14.95 0 0 0 10 September CHSC 13.95 0 15 0 11 September CHSH 9.5 0 3 0 11 September CHSC 16.6 0 0 0 12 September CHSH 15.15 0 0 0 12 September CHSC 16.05 1 23 0 13 September CHSH 16.5 0 0 0 13 September CHSC 13.6 0 28 0 14 September CHSH 16.6 0 2 0 14 September CHSC 16.6 0 63 0 15 September CHSH 17.35 1 5 0 15 September CHSC 14.9 0 11 0 16 September CHSH 17.6 1 0 0 16 September CHSC 17 0 34 0 17 September CHSH 16.3 0 0 0 17 September CHSC 16.55 0 14 0 18 September CHSH 16.75 0 22 0 18 September CHSC 14.55 0 29 0 19 September CHSH 17 0 32 0 19 September CHSC 16.25 0 58 0 20 September CHSH 16.65 1 2 0 20 September CHSC 14.75 0 28 0 21 September CHSH 15.75 0 2 0 21 September CHSC 17.25 0 71 0 22 September CHSH 14.75 0 10 0 22 September CHSC 14.2 0 23 0 23 September CHSH 16 0 2 0 23 September CHSC 15.4 0 6 0 24 September CHSH 16.9 0 0 0 24 September CHSC 13.65 0 12 0 25 September CHSH 14.85 0 3 0 25 September CHSC 14.5 0 18 0 26 September CHSH 15.75 0 33 0 26 September CHSC 17.05 1 15 0 27 September CHSH 15.3 0 5 0 27 September CHSC 14.25 0 35 0 28 September CHSH 15.15 1 3 0 28 September CHSC 15.1 0 16 0 29 September CHSH 15.8 0 5 0 29 September CHSC 16.5 0 22 0 30 September CHSH 14.85 0 10 0 30 September CHSC 16.8 0 26 0 AVG SUM 15.22 5 210 0 AVG SUM 15.73 2 686 0

141

Appendix 2.2. Phenology data for Oreohelix subrudis clade X. Size is in mm, ‘Dicro’ represents presence/absence of Dicrocoelium dendriticum sporocysts, ‘Other’ represents metacercariae of the tentatively identified Postharmostomum helicis. A 1* in ‘Other’ indicates the presence of a Sciomyzidae larvae which has consumed the body of the snail. Gravid indicates the number of embryos found in the uterus. Month Site Size Dicro Other Gravid Month Site Size Dicro Other Gravid 1 May CHHW0 17.9 0 0 0 1 May CHHW1 14.5 1 0 0 2 May CHHW0 17.4 0 0 0 2 May CHHW1 15 0 0 0 3 May CHHW0 15.1 0 0 0 3 May CHHW1 15.4 0 0 0 4 May CHHW0 17.35 0 0 0 4 May CHHW1 12.95 0 0 0 5 May CHHW0 14.8 0 0 0 5 May CHHW1 16.6 0 0 0 6 May CHHW0 18.7 0 0 0 6 May CHHW1 12.15 0 0 0 7 May CHHW0 15.1 0 0 0 7 May CHHW1 11.35 0 0 0 8 May CHHW0 14.1 0 0 0 8 May CHHW1 13.6 0 0 0 9 May CHHW0 14.3 0 0 0 9 May CHHW1 14.9 0 0 0 10 May CHHW0 15.4 0 0 0 10 May CHHW1 15.9 0 0 0 11 May CHHW0 12.5 0 0 0 11 May CHHW1 14.6 0 0 0 12 May CHHW0 15.8 1 0 0 12 May CHHW1 16.95 0 0 3 13 May CHHW0 18 0 0 0 13 May CHHW1 15.5 0 0 0 14 May CHHW0 16.25 0 0 0 14 May CHHW1 16 0 0 0 15 May CHHW0 16.35 0 0 0 15 May CHHW1 16.2 0 0 0 16 May CHHW0 14.3 0 0 0 16 May CHHW1 16.75 0 0 0 17 May CHHW0 14.8 0 0 0 17 May CHHW1 13.55 0 0 0 18 May CHHW0 12.5 0 0 0 18 May CHHW1 16.3 0 0 0 19 May CHHW0 16.9 0 0 0 19 May CHHW1 16.5 0 0 0 20 May CHHW0 16.4 0 0 0 20 May CHHW1 14.7 0 0 0 21 May CHHW0 16.3 0 0 0 21 May CHHW1 16.85 0 0 0 22 May CHHW0 14.5 0 0 0 22 May CHHW1 14.6 0 0 0 23 May CHHW0 12.8 0 0 0 23 May CHHW1 15.2 0 0 0 24 May CHHW0 17 0 0 0 24 May CHHW1 16.5 0 0 0

142

25 May CHHW0 16 0 0 0 25 May CHHW1 15.7 0 0 0 26 May CHHW0 16.4 0 0 0 26 May CHHW1 15.35 0 0 0 27 May CHHW0 15.7 0 0 0 27 May CHHW1 17 0 0 0 28 May CHHW0 12.45 0 0 0 28 May CHHW1 14.9 0 0 0 29 May CHHW0 16.1 0 0 0 29 May CHHW1 16.05 0 0 0 30 May CHHW0 15.7 0 0 0 30 May CHHW1 14 0 0 0 AVG SUM 15.56 1 0 0 AVG SUM 15.19 1 0 3

Month Site Size Dicro Other Gravid Month Site Size Dicro Other Gravid 1 June CHHW0 14.9 0 2 0 1 June CHHW1 16.5 0 0 0 2 June CHHW0 16.6 0 2 0 2 June CHHW1 16 0 0 0 3 June CHHW0 17.1 0 0 0 3 June CHHW1 16.6 0 0 0 4 June CHHW0 14.25 0 3 0 4 June CHHW1 16.8 0 0 0 5 June CHHW0 17.2 0 0 0 5 June CHHW1 16.6 1 0 0 6 June CHHW0 14.9 0 0 0 6 June CHHW1 16.5 0 0 0 7 June CHHW0 16.6 0 1 0 7 June CHHW1 13.4 0 0 0 8 June CHHW0 15.8 0 0 0 8 June CHHW1 15.1 0 0 0 9 June CHHW0 13.1 0 0 0 9 June CHHW1 14.9 0 0 0 10 June CHHW0 14.5 0 0 0 10 June CHHW1 15.8 0 0 0 11 June CHHW0 17.7 0 1 0 11 June CHHW1 15.8 0 0 0 12 June CHHW0 17.2 0 0 0 12 June CHHW1 15.9 0 0 0 13 June CHHW0 15.8 0 0 0 13 June CHHW1 14.9 0 0 0 14 June CHHW0 16.2 0 0 0 14 June CHHW1 13.7 0 0 0 15 June CHHW0 14 0 1 0 15 June CHHW1 16.2 0 0 0 16 June CHHW0 17.1 0 0 0 16 June CHHW1 15.1 0 0 0 17 June CHHW0 16.5 0 0 0 17 June CHHW1 14.9 0 0 0 18 June CHHW0 16.8 1 0 0 18 June CHHW1 15.6 0 0 0 19 June CHHW0 17.3 0 0 0 19 June CHHW1 17.1 0 0 0

143

20 June CHHW0 15.25 0 12 0 20 June CHHW1 16.3 0 0 0 21 June CHHW0 16.4 0 2 0 21 June CHHW1 15.5 0 0 0 22 June CHHW0 15.1 0 3 0 22 June CHHW1 14.9 0 0 0 23 June CHHW0 14.9 0 1 0 23 June CHHW1 16.1 0 0 0 24 June CHHW0 14.9 1 0 0 24 June CHHW1 16 0 0 0 25 June CHHW0 15.7 0 0 0 25 June CHHW1 15.3 0 0 0 26 June CHHW0 15.7 0 0 0 26 June CHHW1 15.5 0 0 0 27 June CHHW0 14.5 1 1 0 27 June CHHW1 13.4 0 0 0 28 June CHHW0 14.9 0 0 0 28 June CHHW1 17.1 0 0 0 29 June CHHW0 16.5 0 0 0 29 June CHHW1 14.5 0 0 0 30 June CHHW0 19.2 0 0 0 30 June CHHW1 16.4 0 0 0 AVG SUM 15.89 3 29 0 AVG SUM 15.61 1 0 0

Month Site Size Dicro Other Gravid Month Site Size Dicro Other Gravid 1 July CHHW0 16.3 0 0 0 1 July CHHW1 16.1 0 0 6 2 July CHHW0 17.4 0 0 7 2 July CHHW1 14.7 0 0 10 3 July CHHW0 16.4 0 0 10 3 July CHHW1 16.6 0 0 0 4 July CHHW0 16.7 0 0 13 4 July CHHW1 15.1 0 0 4 5 July CHHW0 16 0 0 0 5 July CHHW1 15.9 0 0 0 6 July CHHW0 16.1 0 0 0 6 July CHHW1 16.8 0 0 8 7 July CHHW0 15.6 0 0 11 7 July CHHW1 15.3 0 1* 0 8 July CHHW0 14.6 1 0 0 8 July CHHW1 15.8 0 0 3 9 July CHHW0 16.7 0 0 6 9 July CHHW1 14.9 0 0 9 10 July CHHW0 16 0 0 0 10 July CHHW1 17 0 0 9 11 July CHHW0 15.4 0 0 0 11 July CHHW1 16.3 0 0 7 12 July CHHW0 17.4 0 0 0 12 July CHHW1 15.6 0 0 0 13 July CHHW0 16.8 0 0 10 13 July CHHW1 16.7 0 0 0 14 July CHHW0 17.8 0 0 13 14 July CHHW1 15.4 0 0 4

144

15 July CHHW0 13 0 0 0 15 July CHHW1 15.9 0 0 8 16 July CHHW0 16.8 0 0 2 16 July CHHW1 14.7 1 0 0 17 July CHHW0 15.1 0 0 0 17 July CHHW1 16.7 0 0 9 18 July CHHW0 16.9 0 0 0 18 July CHHW1 15.2 0 0 1 19 July CHHW0 12.2 0 0 0 19 July CHHW1 17.3 0 0 0 20 July CHHW0 14.1 0 0 0 20 July CHHW1 15.5 0 0 6 21 July CHHW0 14.1 0 0 0 21 July CHHW1 17 0 0 0 22 July CHHW0 15.7 0 0 0 22 July CHHW1 16.7 0 0 8 23 July CHHW0 15 0 0 0 23 July CHHW1 14.8 0 0 2 24 July CHHW0 15.7 0 0 10 24 July CHHW1 16.2 0 0 0 25 July CHHW0 15.6 0 0 0 25 July CHHW1 14.6 1 0 0 26 July CHHW0 17.1 0 0 0 26 July CHHW1 15.1 0 0 10 27 July CHHW0 15 0 0 0 27 July CHHW1 14.2 0 0 0 28 July CHHW0 15.9 0 0 9 28 July CHHW1 15.9 0 0 0 29 July CHHW0 16.8 0 0 7 29 July CHHW1 14.7 0 0 0 30 July CHHW0 16.2 0 0 0 30 July CHHW1 15 0 0 0 AVG SUM 15.81 1 0 98 AVG SUM 15.72 2 0 104

Month Site Size Dicro Other Gravid Month Site Size Dicro Other Gravid 1 August CHHW0 16.2 0 0 0 1 August CHHW1 17.2 0 0 0 2 August CHHW0 14.9 1 0 0 2 August CHHW1 16.9 0 0 0 3 August CHHW0 16.9 0 0 0 3 August CHHW1 16.7 0 0 0 4 August CHHW0 17.75 0 0 0 4 August CHHW1 18 0 0 0 5 August CHHW0 16.85 0 0 0 5 August CHHW1 17.35 0 0 0 6 August CHHW0 17.2 0 0 0 6 August CHHW1 16.2 0 0 0 7 August CHHW0 15.8 1 0 0 7 August CHHW1 17.2 0 0 0 8 August CHHW0 17.45 0 0 0 8 August CHHW1 17.45 1 0 0 9 August CHHW0 17.35 0 0 0 9 August CHHW1 16.1 0 0 0

145

10 August CHHW0 18.8 0 0 0 10 August CHHW1 17.5 0 0 1 11 August CHHW0 18.75 0 0 0 11 August CHHW1 17.9 0 0 0 12 August CHHW0 17.8 1 0 0 12 August CHHW1 15.5 0 0 0 13 August CHHW0 13.7 0 0 0 13 August CHHW1 15.85 0 0 0 14 August CHHW0 15.75 0 0 0 14 August CHHW1 17.8 1 0 0 15 August CHHW0 17.2 0 0 0 15 August CHHW1 15.4 0 0 0 16 August CHHW0 16.25 1 0 0 16 August CHHW1 15.25 0 0 0 17 August CHHW0 15.9 0 0 0 17 August CHHW1 16.6 0 0 0 18 August CHHW0 16.6 1 0 0 18 August CHHW1 16.3 0 0 0 19 August CHHW0 17.3 0 0 0 19 August CHHW1 15.5 1 0 0 20 August CHHW0 17.55 0 0 0 20 August CHHW1 16.3 0 0 0 21 August CHHW0 17.1 0 0 0 21 August CHHW1 18.35 0 0 0 22 August CHHW0 18.4 0 0 0 22 August CHHW1 17.2 0 0 0 23 August CHHW0 16.6 0 0 0 23 August CHHW1 16.6 1 0 0 24 August CHHW0 14.65 1 0 0 24 August CHHW1 15.05 0 0 0 25 August CHHW0 17.85 0 0 0 25 August CHHW1 15.9 0 0 0 26 August CHHW0 17.9 1 0 0 26 August CHHW1 15.2 0 0 0 27 August CHHW0 16.45 0 0 0 27 August CHHW1 14.55 0 0 0 28 August CHHW0 18.05 0 0 0 28 August CHHW1 16.6 0 0 0 29 August CHHW0 16 1 0 0 29 August CHHW1 17.5 1 0 0 30 August CHHW0 16.95 1 0 0 30 August CHHW1 15.9 1 0 0 AVG SUM 16.87 9 0 0 AVG SUM 16.53 6 0 1

Month Site Size Dicro Other Gravid Month Site Size Dicro Other Gravid 1 September CHHW0 14.75 1 0 0 1 September CHHW1 16.3 0 0 0 2 September CHHW0 16.1 0 0 0 2 September CHHW1 15.6 0 0 0 3 September CHHW0 17.45 0 0 0 3 September CHHW1 16.5 0 0 0 4 September CHHW0 17.5 0 0 0 4 September CHHW1 15.1 0 0 0

146

5 September CHHW0 17.15 0 0 0 5 September CHHW1 15.1 0 0 0 6 September CHHW0 16.85 0 0 0 6 September CHHW1 15.55 0 0 0 7 September CHHW0 17.35 0 0 0 7 September CHHW1 16.05 0 0 0 8 September CHHW0 17.3 0 0 0 8 September CHHW1 14.95 0 0 0 9 September CHHW0 17 0 0 0 9 September CHHW1 16.35 0 0 0 10 September CHHW0 15.8 0 0 0 10 September CHHW1 15.35 0 0 0 11 September CHHW0 17.1 0 0 0 11 September CHHW1 15.15 0 0 0 12 September CHHW0 15.4 1 0 0 12 September CHHW1 16.75 0 0 0 13 September CHHW0 15.5 0 0 0 13 September CHHW1 15.15 0 0 0 14 September CHHW0 14.55 0 0 0 14 September CHHW1 16.75 0 0 0 15 September CHHW0 13.2 0 0 0 15 September CHHW1 15.35 0 0 0 16 September CHHW0 15.45 0 0 0 16 September CHHW1 14.2 0 0 0 17 September CHHW0 16.95 0 0 0 17 September CHHW1 14.1 0 0 0 18 September CHHW0 15.55 1 0 0 18 September CHHW1 16.85 0 0 0 19 September CHHW0 15.7 0 0 0 19 September CHHW1 14.65 0 0 0 20 September CHHW0 14.4 1 0 0 20 September CHHW1 13.35 0 0 0 21 September CHHW0 16.65 0 0 0 21 September CHHW1 12.4 0 0 0 22 September CHHW0 15.4 0 0 0 22 September CHHW1 16.15 1 0 0 23 September CHHW0 16.5 0 0 0 23 September CHHW1 16.25 0 0 0 24 September CHHW0 14.3 1 0 0 24 September CHHW1 14.55 0 0 0 25 September CHHW0 15.5 0 0 0 25 September CHHW1 14.7 0 0 0 26 September CHHW0 16 0 0 0 26 September CHHW1 15.1 0 0 0 27 September CHHW0 14 0 0 0 27 September CHHW1 16.05 0 0 0 28 September CHHW0 15.55 0 0 0 28 September CHHW1 12.25 0 0 0 29 September CHHW0 15.75 0 0 0 29 September CHHW1 15.4 0 0 0 30 September CHHW0 15.4 0 0 0 30 September CHHW1 16.2 1 0 0 AVG SUM 15.87 5 0 0 AVG SUM 15.27 2 0 0

147

Appendix 2.3. Phenology data for Oreohelix cooperi. Size is in mm, ‘Dicro’ represents presence/absence of Dicrocoelium dendriticum sporocysts, ‘Other’ represents metacercariae of the tentatively identified Postharmostomum helicis. A 1* in ‘Other’ indicates the presence of a Sciomyzidae larvae which has consumed the body of the snail. Gravid indicates the number of embryos found in the uterus. Month Site Size Dicro Other Gravid Month Site Size Dicro Other Gravid 1 May CHCA 9 0 0 0 1 May CHLRE 8.8 0 0 0 2 May CHCA 9.4 0 0 0 2 May CHLRE 8 0 0 0 3 May CHCA 8.5 0 0 0 3 May CHLRE 9.65 0 0 0 4 May CHCA 8.05 0 0 0 4 May CHLRE 8.6 1 0 0 5 May CHCA 8.3 1 0 0 5 May CHLRE 9.1 0 0 0 6 May CHCA 8.8 0 0 0 6 May CHLRE 8 0 0 0 7 May CHCA 8.3 0 0 0 7 May CHLRE 8.7 1 0 0 8 May CHCA 8.6 0 0 0 8 May CHLRE 7.3 0 0 0 9 May CHCA 7.5 0 0 0 9 May CHLRE 9.1 0 0 0 10 May CHCA 8.5 0 0 0 10 May CHLRE 8.8 0 0 0 11 May CHCA 9.1 0 0 0 11 May CHLRE 8.2 0 0 0 12 May CHCA 8.4 0 0 0 12 May CHLRE 8.7 0 0 0 13 May CHCA 8.8 0 0 0 13 May CHLRE 8.6 0 0 0 14 May CHCA 9 0 0 0 14 May CHLRE 8.3 0 0 0 15 May CHCA 8.1 0 0 0 15 May CHLRE 8.4 0 0 0 16 May CHCA 8.5 0 0 0 16 May CHLRE 8.55 0 0 0 17 May CHCA 7.8 0 0 0 17 May CHLRE 7.3 0 0 0 18 May CHCA 8.7 0 0 0 18 May CHLRE 8.6 0 0 0 19 May CHCA 9.3 0 0 0 19 May CHLRE 8.4 0 0 0 20 May CHCA 8.5 0 0 0 20 May CHLRE 7.45 0 0 0 21 May CHCA 7.8 0 0 0 21 May CHLRE 8.85 1 0 0 22 May CHCA 7.9 0 0 0 22 May CHLRE 9.25 0 0 0 23 May CHCA 8.4 0 0 2 23 May CHLRE 8.3 0 0 0 24 May CHCA 8.95 0 0 0 24 May CHLRE 9 0 0 0

148

25 May CHCA 7.6 0 0 0 25 May CHLRE 8.7 0 0 0 26 May CHCA 7.7 1 0 0 26 May CHLRE 9 0 0 0 27 May CHCA 7.6 0 0 0 27 May CHLRE 7.9 0 0 0 28 May CHCA 8.7 0 0 0 28 May CHLRE 8.3 0 0 0 29 May CHCA 8.4 0 0 0 29 May CHLRE 8.4 0 0 0 30 May CHCA 8.85 0 0 0 30 May CHLRE 8.6 0 0 0 AVG SUM 8.435 2 0 2 AVG SUM 8.495 3 0 0

Month Site Size Dicro Other Gravid Month Site Size Dicro Other Gravid 1 June CHCA 9 0 0 0 1 June CHLRE 8.6 0 0 0 2 June CHCA 9.3 0 0 0 2 June CHLRE 9.2 0 0 0 3 June CHCA 9.2 0 0 0 3 June CHLRE 9.65 0 0 0 4 June CHCA 8.8 0 0 0 4 June CHLRE 8.1 0 0 0 5 June CHCA 9.6 0 0 0 5 June CHLRE 8.7 1 0 0 6 June CHCA 9.2 0 0 0 6 June CHLRE 8.2 0 0 1 7 June CHCA 8 0 0 0 7 June CHLRE 8.8 0 0 0 8 June CHCA 9 0 0 0 8 June CHLRE 8.4 0 0 0 9 June CHCA 9.1 0 0 0 9 June CHLRE 8.3 1 0 0 10 June CHCA 8.9 0 0 0 10 June CHLRE 8.2 1 0 0 11 June CHCA 8.6 0 0 0 11 June CHLRE 7.55 0 0 0 12 June CHCA 8.5 0 0 0 12 June CHLRE 8.1 0 0 0 13 June CHCA 8.3 0 0 0 13 June CHLRE 8.55 0 0 0 14 June CHCA 8 0 0 0 14 June CHLRE 7.6 0 0 0 15 June CHCA 9.3 0 0 0 15 June CHLRE 8.2 0 0 4 16 June CHCA 8.3 0 0 0 16 June CHLRE 8.2 0 0 0 17 June CHCA 7 0 0 0 17 June CHLRE 7.6 0 0 0 18 June CHCA 8.6 0 0 0 18 June CHLRE 8.3 0 0 0 19 June CHCA 9.5 0 0 0 19 June CHLRE 8.7 0 0 0

149

20 June CHCA 9.3 0 0 0 20 June CHLRE 6.8 0 0 0 21 June CHCA 8.7 1 0 0 21 June CHLRE 7.15 0 0 0 22 June CHCA 7.9 0 0 0 22 June CHLRE 7.9 0 0 2 23 June CHCA 7.6 0 0 0 23 June CHLRE 8.4 0 0 0 24 June CHCA 8.5 0 0 0 24 June CHLRE 7.9 1 0 0 25 June CHCA 8.3 0 0 0 25 June CHLRE 7.7 1 0 0 26 June CHCA 8.4 0 0 0 26 June CHLRE 8.2 1 0 0 27 June CHCA 8.5 0 0 0 27 June CHLRE 8.05 0 0 0 28 June CHCA 9.2 0 0 0 28 June CHLRE 8.8 1 0 0 29 June CHCA 8.2 0 0 0 29 June CHLRE 8.2 0 0 0 30 June CHCA 9 0 0 0 30 June CHLRE 8.6 1 0 0 AVG SUM 8.66 1 0 0 AVG SUM 8.22 8 0 7

Month Site Size Dicro Other Gravid Month Site Size Dicro Other Gravid 1 July CHCA 9.85 0 0 0 1 July CHLRE 8.3 0 1* 0 2 July CHCA 9.55 0 1* 0 2 July CHLRE 9.5 0 0 6 3 July CHCA 9.4 0 0 4 3 July CHLRE 8.2 0 0 0 4 July CHCA 8.95 0 0 0 4 July CHLRE 7.8 0 0 0 5 July CHCA 8.85 0 0 4 5 July CHLRE 8.4 0 0 4 6 July CHCA 8.3 0 0 6 6 July CHLRE 8.3 0 0 0 7 July CHCA 7.9 0 0 0 7 July CHLRE 8.4 0 0 4 8 July CHCA 9.5 0 0 1 8 July CHLRE 8.3 0 0 0 9 July CHCA 9.7 0 0 0 9 July CHLRE 8.55 0 0 6 10 July CHCA 9 0 0 5 10 July CHLRE 9.1 0 0 0 11 July CHCA 9.5 0 0 0 11 July CHLRE 9.1 1 0 0 12 July CHCA 7.95 0 0 2 12 July CHLRE 8.9 0 0 2 13 July CHCA 8.8 0 0 0 13 July CHLRE 9.4 0 0 3 14 July CHCA 9.3 0 0 0 14 July CHLRE 9.4 0 0 2

150

15 July CHCA 7.35 0 0 0 15 July CHLRE 7.8 0 0 3 16 July CHCA 8.8 0 0 4 16 July CHLRE 8.4 0 0 3 17 July CHCA 8.55 0 0 0 17 July CHLRE 8.45 0 0 2 18 July CHCA 8.5 0 0 2 18 July CHLRE 8.6 0 0 0 19 July CHCA 8.8 0 0 0 19 July CHLRE 8.55 0 0 0 20 July CHCA 8.5 0 0 3 20 July CHLRE 8.1 0 0 2 21 July CHCA 8 0 0 2 21 July CHLRE 8.8 0 0 0 22 July CHCA 8.9 0 0 4 22 July CHLRE 7.8 0 0 0 23 July CHCA 7.55 0 0 0 23 July CHLRE 8.5 0 0 0 24 July CHCA 8.5 0 0 5 24 July CHLRE 8.05 0 0 0 25 July CHCA 8.7 0 0 0 25 July CHLRE 8.5 0 0 4 26 July CHCA 7.1 0 0 0 26 July CHLRE 9.1 1 0 4 27 July CHCA 7.9 1 0 0 27 July CHLRE 8.7 0 0 4 28 July CHCA 9.1 0 0 2 28 July CHLRE 8.8 1 0 0 29 July CHCA 8.2 0 0 0 29 July CHLRE 8.05 0 0 0 30 July CHCA 9.2 0 0 4 30 July CHLRE 7.55 0 0 0 AVG SUM 8.67 1 0 48 AVG SUM 8.51 3 0 49

Month Site Size Dicro Other Gravid Month Site Size Dicro Other Gravid 1 August CHCA 8.75 0 0 2 1 August CHLRE 8.85 0 0 0 2 August CHCA 8.4 0 0 0 2 August CHLRE 8.7 0 0 0 3 August CHCA 8.5 0 0 2 3 August CHLRE 7.7 0 0 0 4 August CHCA 8.3 0 0 0 4 August CHLRE 8.1 0 0 1 5 August CHCA 8.9 0 0 0 5 August CHLRE 7.3 0 0 0 6 August CHCA 8 0 0 0 6 August CHLRE 8.45 0 0 1 7 August CHCA 9.1 0 0 3 7 August CHLRE 8.3 0 0 0 8 August CHCA 9.15 0 0 0 8 August CHLRE 8.5 0 0 5 9 August CHCA 8.2 0 0 3 9 August CHLRE 8.8 1 0 0

151

10 August CHCA 9 0 0 0 10 August CHLRE 8.2 1 0 0 11 August CHCA 8.7 0 0 3 11 August CHLRE 9.3 0 0 3 12 August CHCA 8.2 0 0 2 12 August CHLRE 8.1 0 0 3 13 August CHCA 7.8 0 0 0 13 August CHLRE 8.55 0 0 0 14 August CHCA 9.1 0 0 0 14 August CHLRE 8.15 0 0 2 15 August CHCA 7.2 1 0 0 15 August CHLRE 8.9 0 0 2 16 August CHCA 8.7 0 0 3 16 August CHLRE 7.2 0 0 0 17 August CHCA 10.15 0 1* 0 17 August CHLRE 9.05 0 0 3 18 August CHCA 9.15 1 0 0 18 August CHLRE 8.1 0 0 3 19 August CHCA 8.1 0 0 0 19 August CHLRE 8.8 0 0 4 20 August CHCA 8.35 0 0 3 20 August CHLRE 8.4 0 0 2 21 August CHCA 8.5 0 0 3 21 August CHLRE 9.7 0 0 5 22 August CHCA 9.4 0 0 5 22 August CHLRE 7.8 0 0 3 23 August CHCA 9.3 0 0 0 23 August CHLRE 8.2 0 0 0 24 August CHCA 8.1 0 0 2 24 August CHLRE 7.8 1 0 0 25 August CHCA 9.9 0 0 0 25 August CHLRE 8.4 0 0 3 26 August CHCA 8.45 1 0 0 26 August CHLRE 8.2 0 0 3 27 August CHCA 7.8 1 0 0 27 August CHLRE 8.5 0 0 2 28 August CHCA 8.8 0 0 5 28 August CHLRE 8.6 0 0 3 29 August CHCA 8.65 0 0 4 29 August CHLRE 8.65 0 0 3 30 August CHCA 8.1 0 0 0 30 August CHLRE 8.2 0 0 0 AVG SUM 8.63 4 0 40 AVG SUM 8.38 3 0 51

Month Site Size Dicro Other Gravid Month Site Size Dicro Other Gravid 1 September CHCA 8.7 0 0 0 1 September CHLRE 8.7 0 0 0 2 September CHCA 9.85 0 0 0 2 September CHLRE 9.85 0 0 0 3 September CHCA 9.25 0 0 0 3 September CHLRE 9.25 0 0 0 4 September CHCA 7.4 0 0 0 4 September CHLRE 7.4 0 0 0

152

5 September CHCA 8.35 0 0 0 5 September CHLRE 8.35 0 0 0 6 September CHCA 8.8 0 0 0 6 September CHLRE 8.8 0 0 0 7 September CHCA 7.8 0 0 0 7 September CHLRE 7.8 0 0 0 8 September CHCA 8.95 0 0 0 8 September CHLRE 8.95 0 0 0 9 September CHCA 9.25 0 0 0 9 September CHLRE 9.25 0 0 0 10 September CHCA 9.1 0 0 0 10 September CHLRE 9.1 0 0 0 11 September CHCA 8.35 0 0 0 11 September CHLRE 8.35 0 0 0 12 September CHCA 6.9 0 0 0 12 September CHLRE 6.9 0 0 0 13 September CHCA 9.65 0 0 0 13 September CHLRE 9.65 0 0 0 14 September CHCA 6 0 0 0 14 September CHLRE 6 0 0 0 15 September CHCA 8.95 0 0 0 15 September CHLRE 8.9 0 0 0 16 September CHCA 7.05 0 0 0 16 September CHLRE 7.2 0 0 0 17 September CHCA 7.55 0 0 0 17 September CHLRE 9.05 0 0 0 18 September CHCA 8.75 0 0 0 18 September CHLRE 8.1 0 0 0 19 September CHCA 9.9 0 0 0 19 September CHLRE 8.8 0 0 0 20 September CHCA 6.4 0 0 0 20 September CHLRE 8.4 0 0 0 21 September CHCA 5.95 0 0 0 21 September CHLRE 9.7 0 0 0 22 September CHCA 8.3 0 0 0 22 September CHLRE 7.8 0 0 0 23 September CHCA 5 0 0 0 23 September CHLRE 8.2 0 0 0 24 September CHCA 9.1 1 0 0 24 September CHLRE 7.8 0 0 0 25 September CHCA 8.75 0 0 0 25 September CHLRE 8.4 0 0 0 26 September CHCA 6 0 0 0 26 September CHLRE 8.2 0 0 0 27 September CHCA 8.7 1 0 0 27 September CHLRE 8.5 0 0 0 28 September CHCA 7.7 0 0 0 28 September CHLRE 8.6 0 0 0 29 September CHCA 8.1 0 0 2 29 September CHLRE 8.65 0 0 0 30 September CHCA 7.3 0 0 0 30 September CHLRE 8.2 0 0 0 AVG SUM 8.06 2 0 2 AVG SUM 8.43 0 0 0

153