Community composition and Biogeography of northern Canadian Ephemeroptera, Plecoptera and Trichoptera
By
Ruben Cordero
A thesis submitted in conformity with the requirements for the degree of Masters of Science
Department of Ecology and Evolutionary Biology University of Toronto
© Copyright by Ruben Cordero (2014)!
Community composition and Biogeography of northern Canadian Ephemeroptera, Plecoptera and Trichoptera
Ruben Cordero
Masters of Science
Department of Ecology and Evolutionary Biology University of Toronto
2014
Abstract
Climate change has a disproportionately effect on northern ecosystems. To measure this impact we need to understand the structure of northern communities and the influence of current and historical climate events. Insect of the orders Ephemeroptera, Plecoptera and Trichoptera (EPTs) are excellent subjects for study because they are widespread and good bioindicators. The objectives of this study are: (1) Determine patterns of distribution and community composition of northern EPTs. (2) Understand the role of historical events (i.e., Pleistocene glaciations). We found that northern EPT communities are influenced by temperature and precipitation. Also, community composition and population structure of EPT exhibit a similar geographical pattern, with differences on either side of Hudson Bay, suggesting the influence of glaciations in shaping communities of EPTs in northern Canada. The COI barcode approach provided a reliable means for identifying specimens to produce the first wide-scale study of community structure and biogeography of northern EPTs.
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Acknowledgments
I want to express my special appreciation and thanks to my advisor Professor Dr. DOUGLAS C. CURRIE, you have been a tremendous mentor for me. I would like to thank you for encouraging my research and for allowing me to grow as a research scientist. Your support, advice and patience all this years both in my life and research as well have been priceless. THANK YOU DOUG! You are one of the most marvelous people in my life.
I would also like to thank my committee members, professor CHRIS DARLING and professor HERNAN LOPEZ FERNANDEZ for serving as my committee members. Your brilliant comments and suggestions helped me to success in this project. I would especially like to thank Professor Dr. DONALD JACKSON for his advice and guidance on the statistical approach as well as the members of his lab.
This research was possible thanks to the Northern Biodiversity Program. I acknowledge the contributions of the Northern Biodiversity Programs principle investigators: Dr. Chris Buddle (McGill University), Dr. Terry Wheeler (McGill University/Lyman Museum), Dr. Donna Giberson (University of Prince Edward Island) and Dr. Douglas C. Currie (University of Toronto/Royal Ontario Museum) for securing initial funding and providing logistical support for the duration of the project. I also acknowledge the collection and processing efforts of other Northern Biodiversity Program members: S. Laboda, K. Sim, L. Timms, A. Solecki, M. Blair and P. Schaffer. Special thanks to Henry Frania, David E. Ruiter and Steve Burian for the morphological identification. I want to express my gratitude to the members of the staff of the Royal Ontario Museum for all the collaboration, especially to Kristen Choffe, Amy Lathrop, Antonia Guidotti and Brad Hubbley and the members of my lab: Ida Conflitti, Patrick Schaeffer and Julio Rivera.
I can’t express with words the gratitude to my parents for their eternal love and support. I would also like to thank all of my friends who supported me during this time, especially SANTIAGO SANCHEZ RAMIREZ for his friendship and guidance with the molecular analyses. Finally I want to thank my partner Anne-Marie Dion for the guide and care, you are the best support these days.
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This work was supported by National Science and Engineering Research Council of Canada (NSERC) Strategic Project Grant (Ecological Structure of Northern Arthropods: Adaptation to a Changing Environment) awarded to C. Buddle, T. Wheeler, and D. Currie, its supporting partners and collaborators, a NSERC Discovery Grant to D.C. Currie and the Royal Ontario Museum.
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TABLE OF CONTENT
ABSTRACT ...... II ACKNOWLEDGMENTS ...... III TABLE OF CONTENT ...... V LIST OF TABLES ...... VII LIST OF FIGURES ...... VIII CHAPTER ONE ...... 1 COMMUNITY COMPOSITION AND BIOGEOGRAPHY OF NORTHERN CANADIAN EPHEMEROPTERA, PLECOPTERA AND TRICHOPTERA ...... 1 GENERAL INTRODUCTION ...... 1 STUDY LOCATION ...... 2 STUDY GROUP ...... 3 METHODS ...... 4 RELEVANCE OF STUDY ...... 5 MY CONTRIBUTION TO THE NORTHERN BIODIVERSITY PROGRAM ...... 6 LITERATURE CITED ...... 7 CHAPTER 2 ...... 10 PATTERNS OF COMMUNITY COMPOSITION AMONG THE MAYFLIES (EPHEMEROPTERA), STONEFLIES (PLECOPTERA) AND CADDISFLIES (TRICHOPTERA) OF NORTHERN CANADA, AS REVEALED BY DNA BARCODING. ... 10 ABSTRACT ...... 10 INTRODUCTION ...... 11 MATERIALS AND METHODS ...... 12 Fieldwork ...... 12 Lab procedures ...... 13 Genetic identification ...... 14 Descriptive Results ...... 14 Ordination analyses ...... 14 RESULTS ...... 16 Descriptive Results ...... 16 Ordination Analyses ...... 16 DISCUSSION ...... 18 CONCLUSIONS ...... 21 REFERENCES ...... 22 TABLES ...... 28 FIGURES ...... 48 CHAPTER 3 ...... 53 EVIDENCE FOR EAST-WEST POPULATION DIVERGENCE IN SEVEN AQUATIC INSECT SPECIES IN NORTHERN CANADA ...... 53 ABSTRACT ...... 53 INTRODUCTION ...... 54 MATERIALS AND METHODS ...... 55 Fieldwork ...... 55
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Lab procedures ...... 56 Genetic analyses ...... 57 Statistical analyses ...... 58 RESULTS ...... 59 DISCUSSION ...... 61 CONCLUSIONS ...... 65 REFERENCES ...... 66 TABLES ...... 72 FIGURES ...... 74 CHAPTER 4 ...... 80 GENERAL CONCLUSIONS ...... 80 ! !
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List of Tables
CHAPTER 2
Table 1. Distribution of species collected in each eco-climatic region..…………….…...28
Table 2. Species richness per site………………………………………………………...29
Table S1. Species collected and identified in this study…………………………………...30
Table S2. Table S2. Loadings (Pearsons Correlation) of environmental variables in the two
principal components……………………………………………………………34
Table S3. Table S3. Environmental and geographical scores for each component obtained
with canonical correspondence analysis………………………………………...35
Table S4. Sorensen similarity index among sites…………………………………………..36
Table S5. Common species among sites…………………………………………………...37
Table S6. BIOCLIM variables values for each site as generated in R……………………..38
Table S7. Detailed specimen and locality information for northern Ephemeroptera,
Plecoptera and Trichoptera……………………………………………………...39
CHAPTER 3
Table 1. Comparison of relative time to the most common recent ancestor for all the
sampled trees between pair of species…………………………………………..72
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List of Figures
CHAPTER 2
Figure 1. Sampling sites by ecoclimatic zone……………………………………………48
Figure 2. Correspondence analysis plot of sites based on presence/absence of species…49
Figure 3. Hierarchical cluster dendrogram……………………………………………….50
Figure 4. Partition of variance plot of the variance of the Presence/absence matrix…….51
Figure 5. Canonical correspondence analysis plot……………………………………….52
CHAPTER 3
Figure 1. Collecting sites………………………………………………………………….74
Figure 2. Bayesian MMC summarized trees of species widespread……………………...75
Figure 3. Relative time to the MRCA of East-West populations of aquatic insects……...76
Figure 1S. Maximum Credibility Clade Bayesian tree for Ephemeroptera………………..77
Figure 2S. Maximum Credibility Clade Bayesian tree for Plecoptera……………………..78
Figure 3S. Maximum Credibility Clade Bayesian tree for Trichoptera…..………………..79
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CHAPTER ONE
Community composition and biogeography of northern Canadian Ephemeroptera, Plecoptera and Trichoptera
General Introduction
The Arctic is among the most fragile ecosystems on Earth, and its biota is under immense environmental pressure as the effects of global warming are felt most strongly at northern latitudes (Crawford 1997). Small changes in climatic conditions are predicted to have a marked influence on the community structure of northern species, for example, on their abundance, diversity and range (McDonald et al. 1996).
Insects are by far the most diverse organisms in northern Canada, but they are also far less studied than most other members of the biota (Soltis et al. 2006). Despite more than 100 years of research and collecting of insects in northern Canada, knowledge of their diversity is far from complete. The problem is in part due to lack of taxonomic expertise; however, the difficulties of access and the short growing season are also barriers to progress. Relatively few studies of northern insects have so far been undertaken, with most of these on regional faunas, for example,
Danks & Downes (1997) for the Yukon Territory and Zhou et al. (2009) for Churchill, Manitoba.
There are even fewer large-scale studies that compare differences in community composition and structure over the entirety of Canada’s north.
This project is a component of the Northern Biodiversity Program (NBP) — a collaborative research initiative formed by a multidisciplinary team of researchers from McGill University and ! 1! the University of Toronto. The main goal of the NBP is to establish a large-scale baseline for the most representative groups of northern arthropods, which can serve as the basis to document past, present and future changes in ecological structure. My particular component of the NBP focuses on community composition and biogeography in three orders of aquatic insects: Ephemeroptera,
Plecoptera and Trichoptera (EPTs).
Study location
Northern Canada, as defined in this study, includes the Boreal, Subarctic and Arctic ecoclimatic zones, which together comprise about 2.5 million km2 (Porsild, 1964). These lands are characterized by generally stressful environmental conditions. Temperatures remain below zero for at least 7 months of the year, precipitation in some areas is less than in many deserts, and soils are nutrient-limited. Therefore, primary productivity, biomass and diversity are lower than in almost all other terrestrial biomes (Brown & Lomolino 1998). Although the flora and fauna of northern lands are depauperate relative to other areas, a large number of northern-adapted organisms thrive in such environments. However, other than vascular plants and terrestrial vertebrates, the biota of northern Canada remains poorly documented (Weider and Hobaek 2000,
Danks 1981).
Northern ecosystems are characterized by their low species richness, low competition and limited trophic networks. The simplicity of such ecosystems facilitates identification of the responses of resident organisms to environmental change (Freckman and Virginia 1997). Accordingly, northern ecosystems are an ideal setting to document the early stages of contemporary climate change.
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Northern biodiversity was markedly influenced by glacial cycles during the Pleistocene Epoch.
As little as 10,000 years ago, most of the northern hemisphere was covered by kilometers-thick layers of continental ice (Pielou 1991). The advance of ice sheets led most species in northern
Canada to be confined to one or more, variously sized, glacial refugial. This imposed a genetic imprint on populations that became geographically isolated from each other for an extended period of time (Hugall et al. 2002, Hewitt 2004). In fact, most Holarctic species that have been studied so far exhibit considerable population structure (Conroy & Cook 2000; Avise and Walker
1998). These relatively recent climatic events had a tremendous impact on the recent evolutionary history of many northern taxa.
Study Group
Mayflies (Ephemeroptera), stoneflies (Plecoptera) and caddisflies (Trichoptera) — collectively referred to as the EPTs — are among the most prominent insect orders in Canadian aquatic ecosystems. Despite their ecological importance, relatively few accounts have been published about their diversity, distribution and biogeography in northern environments (DeWalt et al.
1994). Collectively, these insects are a key component of energy flow in aquatic systems, as they constitute an important link between the algal/detrital food base and higher trophic levels, including the fishes that feed upon them (Benke 1984). Although the three orders are not closely related to each other phylogenetically, they nonetheless share a number of convergent adaptations related to life in water (i.e., gills, osmotic regulation, and reduction of setae) (Waltz and Burian
2008).
Three factors make these orders especially compelling subjects of study. First, they are widely distributed in the northern Nearctic Region, with representatives occurring on most islands of the
Canadian Arctic Archipelago. Second, the taxonomy of EPTs is reasonably well known at least
! 3! to genus level (Merritt et al., 2008), and there is a growing database of DNA barcodes for many northern EPT species (Zhou et al. 2009). Finally, the well-known sensitivity of EPTs to variation in water quality makes them excellent barometers of long-term environmental change (Rosenberg et al. 2008).
Methods
Personnel from the Northern Biodiversity Program visited 12 widely distributed sites in northern
Canada in 2010 and 2011. Most of these sites were sampled more than a half-century ago by personnel from the 1948-1962 Northern Insect Survey (NIS), providing a baseline for contemporary collections. Sites were distributed equally among the Boreal-, Subarctic-, and
Arctic ecoclimatic zones (i.e., 4 sites in each zone), with equal representation in eastern and western Canada (figure 1). Although the project attempted to maximize coverage, there were, inevitably, enormous gaps between the 12 sites. This is one of the main difficulties of conducting a large-scale study, as it is financially and logistically challenging to implement finer-scale sampling.
A standardized collecting protocol was implemented at each site. A minimum of 12 aquatic collections were taken from a wide variety of lotic and lentic habitats at each site. Additionally, riparian vegetation was swept using an aerial net to collect adults — the stage needed for morphological identification to species level. But given that the great majority of specimens collected were of the “wrong” gender or life-history stage for species-level identification, specimens selected from each site were DNA barcoded and compared against the library of northern EPT sequences developed by Zhou et al. (2009). Having thereby generated a comprehensive list of EPT species for each northern site, I had the basis to address three key questions:
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1. How does EPT community composition differ among the Boreal, Subarctic, and Arctic ecoclimatic zones of northern Canada?
2. How is EPT community composition affected by environmental and geographical variables?
3. Does population structure in mitochondrial DNA (mtDNA) provide clues about the geographical history of northern Canadian EPTs?
The first two questions are the subject of Chapter 2; the third question is covered in Chapter 3.
Relevance of study
This study establishes a much-needed baseline of the diversity and community composition of
EPTs in northern Canada, and sheds important new insights about the influence of environment, geography and history on current distributional patterns. By using a DNA barcoding approach for identifying EPTs from 12 widely distributed sites in northern Canada, I was able to extract maximum information about species composition on a large geographical scale — far more than could have been accomplished by relying exclusively on the sparsely collected adult stage (as was typical in previous studies of northern insects). The COI barcoding gene also has potential to reveal population structure, which in turn can provide insights about the geographical history of widely distributed species. But perhaps the most important contribution of this study is the development of a large-scale inventory of northern EPTs. Ecologists and evolutionary biologists will both find benefit from this research because it provides knowledge about an understudied groups of aquatic insects; however, this knowledge also has potential for use as a baseline for assessing changes in the diversity and community composition in northern EPTs — something that was lacking previous to my study. Given the accelerated rate of climate change at northern latitudes, it is critical to have a sound baseline from which to monitor future changes.
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My contribution to the Northern Biodiversity Program
I was responsible for the aquatic insect component of the NBP, and was integrally involved with the collection, preservation and transportation of specimens from three of the 12 NBP sites. More specifically, I was part of a field crew that visited Norman Wells NT, Ogilvie Mountains YT, and
Banks Island NT during the spring and summer of 2011. Although the focus of my research was on EPTs, the standardized protocols required that all aquatic insects be collected — regardless of taxon. On my return to the lab I sorted aquatic insects to the ordinal level, setting aside the EPTs for finer-level identifications. I identified these latter to lowest taxonomic level possible using available keys, selecting exemplars from each site for DNA barcoding. I extracted, amplified, and sequenced DNA from each specimen. After aligning the sequences I was able to identify material to species level using reference sequences in BOLD and GenBank. The resulting database of EPTs provided the basis for the descriptive, statistical and phylogenetic analyses undertaken in this thesis.
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Literature Cited
Avise JC, Walker D (1998) Pleistocene phylogeographic effects on avian populations and the speciation process. Proc R Soc Lon B 265:457-463
Benke AC (1984) Secondary production of aquatic insects. In: Resh EVH, Rosenberg DM (eds)
The ecology of aquatic insects. Praeger Publishers, New York. pp. 289–322
Brown JH, Lomolino MV (1998) Biogeography. 2nd edition. Sinauer Associates Inc. Sunderland, p 691
Crawford RMM (1997) Habitat fragility as an aid to long-term survival in arctic vegetation. In:
Wooding SJ, Marquiss M (eds) Ecology of Arctic Environments. Special publication series of the
British Ecological Society Number 13. Blackwell Science, Oxford. 291 p
Conroy CJ, Cook JA (2000) Phylogeography of a post-glacial colonizer: Microtus longicaudus
(Rodentia: Muridae) Mol Ecol 9:165-175
Danks HV (1981) Arctic Arthropods: A review of systematics and ecology with particular reference to the North American fauna. Entomological Society of Canada, Ottawa.
Danks HV, Downes JA (1987) Insects of the Yukon. Biological Survey of Canada (Terrestrial
Arthropods), Ottawa. 1034 pp.
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DeWalt RE, Stewart KW, Moulton II SR, Kennedy JH (1994) Summer Emergence of Mayflies,
Stoneflies, and Caddisflies from a Colorado Mountain Stream. Southwest Nat, 39:249-256
Freckman DW, Virginia RA (1997) Low density Antarctic soil nematode communities:
Distribution and response to disturbance. Ecology 78: 363-369
Pelletier F, Garant D, Hendry AP (2009) Eco-evolutionary dynamics. Phil Trans R Soc B
364:1483-1489
Hewitt GM (2004) Genetic consequences of climatic oscillations in the Quaternary. Phil Trans R
Soc Lon B 359:183-195
Hugall A, Moritz C, Moussalli A, Stanisic J (2002) Reconciling paleodistribution models and comparative phylogeography in the Wet Tropics rainforest land snail Gnarosophia bellendenkerensis (Brazier 1875). Proc Nat Acad Sci 99:6112–6117
McDonald, ME, Hershey AE, Miller MC (1996) Global warming impacts on lake trout in arctic lakes. Limnol Oceanogr, 41:1102-l 108
Merritt RW, Cummins KW, Berg MB (2008) An introduction to aquatic insects of North
America, 4th ed. Kendall/Hunt Publishing Company, Dubuque.
Pielou EC. 1991. After the ice age: The return of life to glaciated North America. University of
Chicago Press.
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Porsild AE 1964. Illustrated flora of the Canadian Arctic Archipielago. Bull Nat Mus Can 146.
218 pp.
Rosenberg DM, King R, Resh VH (2008) Use of aquatic insects in biomonitoring. Chapter 7 In:
Merritt RW, Cummins KW, Berg MB (Eds) An introduction to aquatic insects of North America,
4th ed. Kendall/Hunt Publishing Company, Dubuque
Soltis DE, Morris AB, McLachlan JS, Manos PS, Soltis PS (2006) Comparative phylogeography of unglaciated eastern North America. Mol Ecol, 15:4261–4293
Waltz RD, Burian SK (2008) Ephemeroptera. Chapter 11 In: Merritt RW, Cummins KW, Berg
MB (Eds) An introduction to aquatic insects of North America, 4th ed. Kendall/Hunt Publishing
Company, Dubuque
Weider LJ, Hobaek A (2000) Phylogeography and arctic biodiversity: a review. Ann Zool
Fennici 37: 217-231
Wright JW, Spolsky C, Brown WM (1993) The origin of the parthenogenetic lizard
Cnemidophorus laredoensis inferred from mitochondrial DNA analysis. Herpetologica 39:410-
416
Zhou X, Adamowicz SJ, Jacobus LM, Dewalt RE, Hebert PDN (2009) Towards a comprehensive barcode library for arctic life - Ephemeroptera, Plecoptera, and Trichoptera of Churchill,
Manitoba, Canada. Front Zool 6:30
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CHAPTER 2
Patterns of community composition among the mayflies (Ephemeroptera), stoneflies (Plecoptera) and caddisflies (Trichoptera) of northern Canada, as revealed by DNA barcoding.
Abstract
Understanding community composition of aquatic insects in northern ecosystems is fundamental to assessing future impacts of climate change. Previous studies have focused on regional diversity using a predominantly morphological approach to taxonomy; consequently, knowledge of northern diversity is uneven, with some regions like the Yukon Territory being much more comprehensively surveyed than other regions (e.g., the Canadian Arctic Archipelago). This makes large-scale comparative studies difficult — especially in view of the taxonomic deficit for inadequately studied organisms such as insects. In this study we collected aquatic insects
(Ephemeroptera, Plecoptera and Trichoptera, or EPTs) at 12 widely distributed sites in northern
Canada, with 4 sites each in the Boreal-, Subarctic- and Arctic ecoclimatic zones. We used a molecular COI barcode approach to identify specimens collected at each site, providing a more accurate basis from which to interpret patterns. Ordination analyses were performed to elucidate distributional patterns and the influence of environmental and geographical variables in shaping northern EPT communities. Results showed that Arctic community composition was markedly different from those of the Boreal and Subarctic ecoclimatic zones. Boreal and Subarctic community composition was generally more similar; however, differences were observed on
! 10! either side of Hudson Bay. Environmental and geographical variables explained 55% of the variance of the community composition of EPTs. This is the first large-scale study of EPTs community structure in northern Canada, providing a baseline for future studies.
Introduction
The aquatic insects of northern Canada are poorly known, due mainly to lack of access and the difficulties of conducting fieldwork in the far north. Progress is further hindered because of short summers and the problem of obtaining appropriate life-history stage(s) for species-level identification. Consequently, knowledge of northern species, and the factors that shape their community composition, lags far behind that of more southern-adapted species.
DNA barcoding has emerged as a relatively quick and effective tool for identifying species regardless of stage of development (Zhou et al. 2010). With the recent development of a barcode library for northern Canadian mayflies (Ephemeroptera), stoneflies (Plecoptera) and caddisflies
(Trichoptera) (Zhou et al. 2009), the stage is now set for broader-scale explorations of species richness in northern Canada and the factors that shape community composition.
Mayflies, stoneflies and caddisflies are common and widely distributed orders of aquatic insects in northern Canada (Wiggins 1996, Edmunds et al. 1976, Stewart & Stark 1988), making them ideal subjects for studies of diversity, ecology and biogeography.his study we use a DNA barcoding approach to explore patterns of species richness of Ephemeroptera, Plecoptera and
Trichoptera (hereafter referred to as “EPT”) at 12 widely distributed sites in northern Canada.
Relationships between species assemblages and environmental variables (temperature, precipitation) are explored using ordination methods, including principal component analysis
(PCA), simple correspondence analysis (CA), canonical correspondence analysis (CAA) and
Procrustean randomization tests.
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Ordination studies of aquatic invertebrates began during 1980’s and have proven successful for evaluating the effects of environmental and spatial variables on the heterogeneity of the community composition (Rabeni & Gibbs 1980; Leland et al. 1986; Harvey & McArdle 1986).
However, relatively few short-scale (local) medium-scale (regional) studies have been conducted in northern Canada so far (Miller & Stout 1989, Harper 1989). This is the first large-scale DNA- based survey of the Ephemeroptera, Plecoptera and Trichoptera in northern Canada and the first large-scale analysis of the influence of environmental and geographical variables affecting their community composition.
Materials and Methods
Fieldwork
Mayflies, stoneflies and caddisfies were collected from 12 widely distributed sites in northern
Canada during June and July of 2010 and 2011. Sites were distributed equally among three major ecoclimatic zones as defined by the Ecoregions Working Group (1989): Boreal — Goose Bay
NL, Moosonee ON, Yellowknife NT, Norman Wells NT; Subarctic — Schefferville QC,
Churchill MB, Kugluktuk NU, Ogilvie Mountains YT, and Arctic — Iqaluit NU, Lake Hazen
NU. Cambridge Bay NU, Banks Island NT (Figure 1). Each site was visited for a period of two weeks, with eastern sites sampled in 2010 and western sites in 2011. In order to maximize phonological comparability, Boreal sites were visited earliest in the season (early June) followed by Subarctic sites (late June) and Arctic sites (early July). Because only one species of EPT was collected at the most northern site (Lake Hazen, Ellesmere Island), it was excluded from the analysis for reasons described below.
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Standardized sampling protocols, like number of rivers and ponds and sampling time were implemented at each site. Immature aquatic insects were sampled from a wide variety of lentic
(standing water) and lotic (running water) habitats using a D-framed net. Adults were swept from riparian vegetation in the vicinity of each collecting locality. Geographical coordinates and elevation were determined using a GPS. Larval and adult samples were fixed in 95% ethanol to facilitate later morphological and molecular analyses. Vouchers are deposited in the Entomology collection of the Royal Ontario Museum, Toronto, Canada.
Lab procedures
Larvae and adults were sorted under magnification with a Nikon stereomicroscope and identified to genus level using Merritt et al. (2008), Wiggins (1996), and Ruiter (1995). Specimens were further sorted into Operational Taxonomic Units (OTUs), with exemplars sent to taxonomic specialists for confirmation or species-level identification. Five to ten specimens of each OTU were selected for DNA barcoding.
Total genomic DNA was extracted from the hind leg of selected specimens using the DNeasy
Blood & Tissue Kit QIAGEN. Extracted DNA was eluted in 150 µl of elution buffer AE (10 mM
Tris-HCl, 0.5 mM EDTA, pH 9.0). Barcode COI sequences were amplified and sequenced using primer set LCO1490 (5’-ATTCAACCAATCATAAAGATATTGG-3’) HCO2198 (5’-
TAAACTTCAGGGTGACCAAAAAATCA-3’) following methods described by deWaard et al.
(2008) and Hajibabaei et al. (2005). The final volume of each PCR reaction was 12.5 µl. PCR products were visualized on a 1% Agarose gel, sequenced bi-directionally using BigDye v3.1 and analyzed in a Hitachi 3730 DNA Analyzer.
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Genetic identification
Sequences obtained (640-660 bp each) were BLASTed on the GenBank and BOLD Systems v3 databases. We used 2% genetic distance to delimit species as suggested by Zhou et al. (2009).
Species identified in this fashion were used to create the matrix of presence/absence of species at each site; the presence/absence matrix (Podani & Schmera 2011) was used to produce the descriptive results and ordination analyses.
Descriptive Results
A presence/absence matrix was used to obtain descriptive results, which included total number of species, number of species per order, number of species per site, ecoclimatic zone and number of shared species among sites. Sørensen index was calculated to assess the similarity of the communities among sites using the shared species diversity (Sørensen 1948; Chao et al 2005).
Ordination analyses
In addition to the presence/absence matrix, we also created environmental and the geographical matrices. The environmental matrix was constructed using mean maximum monthly temperature, mean minimum monthly temperature and mean monthly precipitation data. These data — taken from the closest meteorological station to each site — were generated from a 20-year period prior to our study (1989-2009) (Mekis &Vincent 2011; Vincent et al. 2012). This data set was entered in R v3.0.1 using the DISMO package (Hijmans et al 2011) to obtain 19 BioClim variables, which are biologically more meaningful than the three measured variables alone (Hijmans et al.
2005). Variance of the BioClim variables was reduced to two dimensions using Principal
Component Analysis (PCA) (King & Jackson 1999). PCA loadings were compared to determine
! 14! the most influencing BioClim variables in these two components. The geographical matrix was created using the latitude and longitude recorded for each sampled site.
Correspondence Analysis (CA) was performed to convert data from the presence/absence matrix into continuous variables (Jackson & Harvey 1989) and to observe the arrangement of sites in a plot (Nenadic & Greenacre 2007). We also used the Euclidean distance of the presence/absence matrix to produce a hierarchical clustering dendrogram to visualize branching (connection) relationships among sites.
Canonical Correspondence Analysis (CCA) was performed to partition the total variation of the presence/absence matrix into independent components: pure environmental, pure geographical, the influence of geographical on environmental component and the unexplained component
(Borcard et al 1992). Three separate CCAs were performed to constrain the presence/absence matrix as follows: a CCA constrained by the environmental matrix to determine the percentage of variation explained by environmental variables; a CCA constrained by geographical matrix to determine the percentage of variation explained by geographical variables; and a CCA constrained by both environmental and geographical matrices to determine a measure of joint variation. Data from Ellesmere Island, NU, were removed from analyses because only one species of EPT (Trichoptera: Grensia praeterita) occurred there, causing noise in the analyses.
Finally, Procrustean randomization tests (PROTEST) were performed to assess concordance between the biological and environmental matrices and between the biological and geographical matrices (Jackson 1995; Peres-Neto & Jackson 2001). Descriptive and ordination analyses were performed in the Community Ecology Package VEGAN in R v3.0.1 (Oksanen et al. 2013).
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RESULTS
Descriptive Results
A total of 582 COI sequences were obtained and blasted in GENBANK and BOLD (Accession number KJ674822 - KJ675402). One hundred and fifty eight species were identified using the 2% threshold recommended by Zhou et al. (2009), of which 58 species were Ephemeroptera, 42
Plecoptera and 58 Trichoptera (Table S1). Species richness was highest in the Boreal ecoclimatic zone (99 species), followed by the Subarctic ecoclimatic zone (84 species) and the Arctic ecoclimatic zone (15 species) (Table 1).
Ordination Analyses
Figure 2 shows the CA plot of the presence/absence matrix. Banks Island, Cambridge Bay and
Iqaluit — all sites from the Arctic ecoclimatic zone — form a cluster at the left side of the plot.
On the upper right, Goose Bay (Boreal) and Schefferville (Subarctic) formed a cluster separate from the other sites. A well-defined central cluster formed by Norman Wells, Yellowknife and
Churchill is situated near the intersection of the components. Three sites (Ogilvie Mountains,
Kugluktuk and Moosonee) do not cluster with any other site; however, each is situated closer to the central cluster than to the other two clusters. A hierarchical dendrogram (figure 3) shows relationships among sites using Euclidean distances from the presence/absence matrix. This dendrogram has two main branches, with one of them consisting exclusively of eastern sites
(Moosonee, Schefferville and Goose Bay). The other cluster consists of both western- and far northern (i.e., Arctic) sites, with the latter having the closest connection to Kugluktuk (a
Subarctic site).
PCA of environmental (Bioclim) variables (suppl. Table S6) yields two main components, which
! 16! together explains 89% of the total variance of the variables. The loadings (Pearson correlation) of these two components show higher values for the variables temperature annual range and annual precipitation (suppl. Table S2). These variables were selected to create the environmental matrix because they explain roughly the same amount of variation as the two main components, but are biologically more meaningful.
The first CCA shows that pure environmental variables explains 31% of the total variation of the presence/absence matrix. The second CCA shows that pure geographical variables explains 34% of the total variation of the presence/absence matrix. The third CCA shows that joint variance
(geographic influence on environmental component) explains 10% of the total variation of the presence/absence matrix. The overall amount of variation explained by both environmental and geographical variables was obtained by summing the environmental percentage to the geographical percentage and then subtracting the joint variation. Approximately 55% of the total variance of the presence/absence matrix was explained by both environmental and geographical variables used in this study (figure 4). The joint variation also allowed us to obtain the net partition, 21% explained by pure environmental component (environmental variation – joint variation) and 24% explained by pure geographical component (geographical variation – joint variation).
CCA plot (figure 5) shows the arrangement of sites and the influence of environmental and geographical variables. Clusters in this plot are similar to the CA plot (figure 2) with the exception that Kugluktuk is located closer to the Arctic cluster and Ogilvie mountains is located within the central cluster. Latitude presents the higher score of component one while annual precipitation show the higher score of component two (suppl. table S3). The plot produced by the
CA of the species matrix and the CCA of the constricted presence/absence matrix (figure 2) revealed a similar cluster arrangement among sites, except that, in the CAA, Moosonee (MO) and ! 17!
Kugluktuk (KT) were displaced from the central-west cluster and Ogilvie Mountains (OM) was placed within the central-west cluster.
Procrustean randomization test (PROTEST) showed that concordance between the presence/absence matrix ordination and the environmental variables was highly significant
(m2=0.5813; p=0.007). However, concordance between the presence/absence matrix ordination and the geographical variables was not significant (m2=0.754; p=0.096).
Discussion
Biodiversity gradients typically show higher species richness at lower latitudes, with a decrease expected in the progression from boreal forests to polar deserts (Hawkins 2001, Hillebrand 2004,
Turner JRG 2004). In the present study, EPT species richness was similar in the Boreal and
Subarctic ecoclimatic zones, with a marked decrease in the Arctic ecoclimatic zone, following the predicted pattern. The Subarctic evidently serves as a transitional zone, as it supports a combination of Boreal-adapted and Arctic-adapted species. However, the relatively harsh conditions in the Arctic ecoclimatic zone evidently presents a significant barrier to members of the southern-adapted Boreal community. Consequently, the Arctic supports a comparatively small number of species that are highly adapted to life in that ecoclimatic zone. The uniqueness of Arctic sites is also reflected by the CA analysis, which clearly separated them from those of other ecoclimatic zone. Further, the three Arctic sites together formed a discrete cluster in the hierarchical dendrogram. These results were expected because environmental conditions are relatively harsh in the Arctic, with resident species being specially adapted to local conditions
(Danks et al. 1994). Unlike the Arctic sites, CA analysis did not cluster Boreal and Subarctic sites according to their a priori assignments. In the upper right of figure 2, for example, Goose Bay
! 18! and Schefferville clustered together distantly from other sites — despite the fact they were assigned to two different ecoclimatic zones (Boreal and Subarctic, respectively). Nonetheless, their community composition is similar, sharing 13 of 64 species collected at those two sites
(Sorensen similarity index 0.31, suppl. tables S2 and S3).
Similarly, sites arrayed more centrally in the plot included both Boreal and Subarctic sites.
Norman Wells, Yellowknife and Churchill formed a cluster near the intersection of the components in the plot (figure 2), suggesting similarity in their EPT communities. The hierarchical dendrogram also showed a close relationship among these sites, although Norman
Wells and Yellowknife are relatively more similar to each other.
The three sites closest to this central cluster (Kugluktuk, Ogilvie Mountains and Moosonee) do not appear to be strongly related to any other site (figure 2). Kugluktuk lies between the central cluster and the Arctic cluster, though rather closer to the central one. One possible explanation for this pattern is that Kugluktuk — classified as a Subarctic site — is situated at the northernmost latitude of any mainland sites (figure 1). In fact, it is farther north than Iqaluit, an Arctic site on the Canadian Arctic Archipelago. Kugluktuk shared six of fourteen species collected from Arctic sites (≈ 43%); however, this site supported a total of 26 species, most of which were shared with
Norman Wells (10 spp.), Churchill (9 spp.) and Ogilvie Mountains (7 spp.) (suppl. Tables S4 and
S5). Another non-clustered site, Ogilvie Mountains, has relatively high EPT diversity (35 spp.), but many of species were classified as rare because they occurred at no other site. Ogilvie
Mountains was second only to Moosonee in sharing the fewest number of species with other sites
(Table S4). The relative uniqueness of the Ogilvie Mountain EPT assemblage is perhaps related to the fact the site lies within the historical boundaries of Beringia — a major glacial refugium during the last (Wisconsinan) glaciation (Pielou 1991). Beringia served as an important source area for organisms that repopulated northern Canada following deglaciation; however, certain
! 19! species (classified as Beringian endemics) did not migrate far beyond the refugium following deglaciation (Currie 1997, Ball and Currie 1997). Moosonee, which shared the fewest species with other sites, is the southernmost of the 12 sites sampled in our study. On the other end of the spectrum, Churchill had the greatest number of shared species among sites. Classified as a
Subarctic site (and therefore including both Arctic and Boreal faunistic elements), Churchill also occupies the geographical center of our 11 sampling sites, therefore supporting a combination of eastern and western species, as suggested by Zhou et al (2010).
Arctic sites clustered far from the Boreal and Subarctic sites (Figure 5) by their higher latitude, lower precipitation and lower range of annual temperature (i.e., the magnitude between the maximum temperature of the warmest month and the minimum temperature of the coldest month). These variables explained more than half of the variation in the EPT community composition in northern Canada. Nonetheless, there remains considerable unexplained variance that could be the result of unmeasured regional scale variables, local conditions, stochastic processes, sampling variability, sampling frequency, and measurement errors. Similar conclusions were reached in other studies that used a similar approach (Filomena Magalhães et al. 2002; Beisner et al. 2006).
Geographical history can also exert an influence on community composition, which might contribute an important fraction of undetermined variation in the partitioning (figure 3).
It is widely recognized that Beringia was the main refugium (i.e., source area) for organisms that repopulated northern Canadian following retreat of Wisconsinan-aged ice ca. 10000 yr BP
(Pielou 1991, Wieder et al. 1999a,b). However, in a review of more than 390 published studies,
Soltis et al. (2006) found evidence that eastern refugia also played a role in repopulating the north
— especially east of Hudson’s Bay. In the present study, the three easternmost sites (Goose Bay,
Schefferville and Moosonee) had clearly differentiated EPT communities, suggesting that history
! 20!
(i.e., contributions from eastern refugial areas) may have contributed to their present-day community composition. In contrast, it seems that the Beringian refugium had a comparatively stronger influence on the composition of Arctic and western (i.e., west of Hudson’s Bay) sites.
Conclusions
Standardized collections from 12 widely distributed sites across northern Canada — using a DNA barcoding approach to facilitate species-level identifications regardless of life-stage or gender — allowed us to conduct the first large-scale analysis of northern EPT community composition.
Relationships between species assemblages and environmental variables (temperature, precipitation) were explored using ordination methods, including principal component analysis
(PCA), simple correspondence analysis (CA), canonical correspondence analysis (CAA) and
Procrustean randomization tests. The results showed that the factors that shape EPT community composition are complex, including both environmental and historical factors. Although knowledge of northern EPTs remains far from complete, this study offers the first approximation of the historical and contemporary factors that shape broad-scale patterns of community composition patterns in northern Canada.
! 21!
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! 27!
TABLES
Table 1. Species richness of Ephemeroptera, Plecoptera and Trichoptera in each ecoclimatic zone.
Ephemeroptera Plecoptera Trichoptera Total species
Boreal 38 24 37 99
Subarctic 30 25 29 84
Arctic 6 3 6 15
Total 58 42 58 158
! 28!
Table 2. Species richness per site.
Site Number of species Banks Island 8 Cambridge Bay 9 Iqaluit 10 Kugluktuk 26 Yellowknife 26 Norman Wells 30 Goose Bay 34 Ogilvie Mountains 35 Schefferville 35 Churchill 41 Moosonee 44
! 29!
Table S1. Species collected and identified in this study.
Order Family Genus Species Ephemeroptera Baetidae Acentrella lapponica Ephemeroptera Ameletidae Ameletus celer Ephemeroptera Ameletidae Ameletus inopinatus Ephemeroptera Ameletidae Ameletus sp. Ephemeroptera Ameletidae Ameletus velox Ephemeroptera Baetidae Baetis bicaudatus Ephemeroptera Baetidae Baetis brunneicolor Ephemeroptera Baetidae Baetis bundyae Ephemeroptera Baetidae Baetis foemina Ephemeroptera Baetidae Baetis hudsonicus Ephemeroptera Baetidae Baetis phoebus Ephemeroptera Baetidae Baetis tricaudatus Ephemeroptera Baetidae Baetis vernus Ephemeroptera Baetiscidae Baetisca laurentina Ephemeroptera Caenidae Caenis amica Ephemeroptera Caenidae Caenis latipennis Ephemeroptera Caenidae Caenis sp. Ephemeroptera Caenidae Caenis youngi Ephemeroptera Heptageniidae Cinygmula sp. Ephemeroptera Heptageniidae Cinygmula sp.JMW1 Ephemeroptera Heptageniidae Cinygmula spJMW3 Ephemeroptera Heptageniidae Cinygmula subaequalis Ephemeroptera Ephemerellidae Drunella coloradensis Ephemeroptera Ephemerellidae Drunella doddsi Ephemeroptera Ephemerellidae Drunella grandis Ephemeroptera Ephemerellidae Drunella lata Ephemeroptera Heptageniidae Epeorus pleuralis Ephemeroptera Heptageniidae Epeorus vitreus Ephemeroptera Ephemeridae Ephemera simulans Ephemeroptera Ephemerellidae Ephemerella aurivillii Ephemeroptera Ephemerellidae Ephemerella invaria Ephemeroptera Ephemerellidae Ephemerella needhami Ephemeroptera Ephemerellidae Ephemerella sp. Ephemeroptera Ephemerellidae Ephemerella subvaria Ephemeroptera Ephemerellidae Eurylophella funeralis Ephemeroptera Ephemerellidae Eurylophella macdunnoughi Ephemeroptera Ephemerellidae Eurylophella sp. Ephemeroptera Ephemerellidae Eurylophella temporalis Ephemeroptera Ephemerellidae Eurylophella verisimilis
! 30!
Table S1. (Continued). Species collected and identified in this study.
Order Family Genus Species Ephemeroptera Heptageniidae Heptagenia pulla Ephemeroptera Heptageniidae Heptagenia sp. Ephemeroptera Ephemeridae Hexagenia limbata Ephemeroptera Isonychiidae Isonychia bicolor Ephemeroptera Leptophlebiidae Leptophlebia cupida Ephemeroptera Leptophlebiidae Leptophlebia sp. Ephemeroptera Heptageniidae Leucrocuta jewetti Ephemeroptera Ephemeridae Litobrancha recurvata Ephemeroptera Heptageniidae Maccaffertium vicarium Ephemeroptera Metretopodidae Metretopus borealis Ephemeroptera Leptophlebiidae Paraleptophlebia adoptiva Ephemeroptera Leptophlebiidae Paraleptophlebia aquilina Ephemeroptera Leptophlebiidae Paraleptophlebia mollis Ephemeroptera Siphlonuridae Parameletus chelifer Ephemeroptera Baetidae Procloeon sp. Ephemeroptera Heptageniidae Rhithrogena sp. Ephemeroptera Siphlonuridae Siphlonurus alternatus Ephemeroptera Siphlonuridae Siphlonurus phyllis Ephemeroptera Siphlonuridae Siphlonurus quebecensis Ephemeroptera Heptageniidae Stenonema femoratum Ephemeroptera Heptageniidae Stenonema sp. Plecoptera Perlidae Acroneuria abnormis Plecoptera Perlidae Acroneuria lycoras Plecoptera Perlidae Acroneuria sp. Plecoptera Capniidae Allocapnia granulata Plecoptera Capniidae Allocapnia sp. Plecoptera Capniidae Allocapnia vivipara Plecoptera Chloroperlidae Alloperla concolor Plecoptera Chloroperlidae Alloperla severa Plecoptera Nemouridae Amphinemura linda Plecoptera Nemouridae Amphinemura sp. Plecoptera Perlodidae Arcynopteryx compacta Plecoptera Capniidae Capnia atra Plecoptera Perlodidae Diura bicaudata Plecoptera Capniidae Eucanopsis bravicaudata Plecoptera Chloroperlidae Haploperla brevis Plecoptera Perlodidae Isogenoides frontalis Plecoptera Perlodidae Isoperla bilineata Plecoptera Perlodidae Isoperla obscura
! 31!
Table S1. (Continued). Species collected and identified in this study.
Order Family Genus Species Plecoptera Perlodidae Isoperla petersoni Plecoptera Perlodidae Isoperla sp. Plecoptera Leuctridae Leuctra sp. Plecoptera Perlodidae Megarcys signata Plecoptera Nemouridae Nemoura arctica Plecoptera Nemouridae Nemoura trispinosa Plecoptera Perlidae Paragnetina media Plecoptera Perlidae Paragnetina sp. Plecoptera Leuctridae Paraleuctra knighton01 Plecoptera Leuctridae Paraleuctra sara Plecoptera Leuctridae Paraleuctra sp.XZ01 Plecoptera Nemouridae Paranemoura perfecta Plecoptera Capniidae Plecoptera sp. Plecoptera Chloroperlidae Plumiperla diversa Plecoptera Pteronarcyidae Pteronarcys dorsata Plecoptera Pteronarcyidae Pteronarcys pictetii Plecoptera Pteronarcyidae Pteronarcys sp. Plecoptera Chloroperlidae Sweltsa naica Plecoptera Chloroperlidae Sweltsa urticae Plecoptera Capniidae Utacapnia trava Plecoptera Nemouridae Zapada sp. Plecoptera Nemouridae Zapada sp.JMW03 Plecoptera Nemouridae Zapada sp.JMW04 Trichoptera Phryganeidae Agrypnia deflata Trichoptera Phryganeidae Agrypnia glacialis Trichoptera Phryganeidae Agrypnia pagetana Trichoptera Phryganeidae Agrypnia straminea Trichoptera Limnephilidae Anabolia bimaculata Trichoptera Apataniidae Apatania stigmatella Trichoptera Apataniidae Apatania wallengreni Trichoptera Apataniidae Apatania zonella Trichoptera Limnephilidae Arctopora pulchella Trichoptera Hydropsychidae Arctopsyche ladogensis Trichoptera Limnephilidae Asynarchus lapponicus Trichoptera Limnephilidae Asynarchus montanus Trichoptera Limnephilidae Asynarchus rossi Trichoptera Phryganeidae Banksiola crotchi Trichoptera Brachycentridae Brachycentrus americanus Trichoptera Leptoceridae Ceraclea excisa Trichoptera Leptoceridae Ceraclea nigronervosa ! 32!
Table S1. (Continued). Species collected and identified in this study.
Order Family Genus Species Trichoptera Hydropsychidae Ceratopsyche alternans Trichoptera Hydropsychidae Ceratopsyche morosa Trichoptera Hydropsychidae Ceratopsyche sparna Trichoptera Philopotamidae Chimarra obscura Trichoptera Philopotamidae Chimarra socia Trichoptera Limnephilidae Dicosmoecus obscuripennis Trichoptera Philopotamidae Dolophilodes distinctus Trichoptera Limnephilidae Ecclisomyia conspersa Trichoptera Glossosomatidae Glossosoma nigrior Trichoptera Limnephilidae Grammotaulius interrogationis Trichoptera Limnephilidae Grensia praeterita Trichoptera Limnephilidae Hesperophylax designatus Trichoptera Hydropsychidae Hydropsyche hoffmani Trichoptera Limnephilidae Lenarchus productus Trichoptera Lepidostomatidae Lepidostoma togatum Trichoptera Limnephilidae Limnephilus alaicus Trichoptera Limnephilidae Limnephilus argenteus Trichoptera Limnephilidae Limnephilus canadensis Trichoptera Limnephilidae Limnephilus dispar Trichoptera Limnephilidae Limnephilus externus Trichoptera Limnephilidae Limnephilus indivisus Trichoptera Limnephilidae Limnephilus infernalis Trichoptera Limnephilidae Limnephilus moestus Trichoptera Limnephilidae Limnephilus picturatus Trichoptera Limnephilidae Limnephilus rhombicus Trichoptera Limnephilidae Limnephilus sansoni Trichoptera Brachycentridae Micrasema canusa Trichoptera Brachycentridae Micrasema charonis Trichoptera Limnephilidae Nemotaulius hostilis Trichoptera Uenoidae Neophylax aniqua Trichoptera Uenoidae Neophylax sp. Trichoptera Polycentropodidae Neureclipsis bimaculata Trichoptera Limnephilidae Onocosmoecus unicolor Trichoptera Polycentropodidae Polycentropus cinereus Trichoptera Polycentropodidae Polycentropus smithae Trichoptera Limnephilidae Pycnopsyche guttifera Trichoptera Limnephilidae Pycnopsyche limbata Trichoptera Rhyacophilidae Rhyacophila brunnea Trichoptera Rhyacophilidae Rhyacophila torva
! 33!
Table S2. Loadings (Pearsons Correlation) of environmental variables in the two principal components. Variable code: bi1: Annual Mean Temperature, bi2: Mean Diurnal Range (Mean of monthly (max temp - min temp)), bi3: Isothermality (BIO2/BIO7) (* 100), bi5: Max Temperature of Warmest Month, bi6: Min Temperature of Coldest Month, bi7: Temperature Annual Range (bi5-bi6), b12: Annual Precipitation, b13: Precipitation of Wettest Month, b14: Precipitation of Driest Month, b15: Precipitation Seasonality (Coefficient of Variation). Values in bold script are the highest load in each component.
Variables/component!! Component!1!! Component!2! b1!!! =0.373! 0.13! b2!!! =0.215! 0.488! b3!!! =0.319! 0.263! b5!!! =0.282! 0.38! b6!!! =0.374! =0.102! !!!!!!!!!!!!!!!!!!!!!b7! !! 0.571& b12!! '0.376& =0.148! b13!! =0.366! !! b14!! =0.358! =0.239! b15!!! 0.294! 0.327!
! 34!
Table S3. Environmental and geographical scores for each component obtained with canonical correspondence analysis. Values in bold script represent the highest score for each component. Accumulated constrained eigenvalues were: Component 1: 0.39, Component 2: 0.27, Component 3: 0.23 and Component 4: 0.11.
Variables/component!! CCA1!!!!!!! CCA2!!!!! CCA3!!!!!! CCA4!!
ART!!! =0.122! =0.390924! =0.7578! 0.50867& TP! 0.8256! 0.461511& 0.3412! =0.02456! Longitude!!! 0.4938! 0.400926! 0.7756& 0.02383!
Latitude!!! '0.8883& =0.008191! =0.457! =0.06558!
! 35!
Table S4. Sorensen similarity index among sites. BI: Banks Island, CB: Cambridge Bay, CH: Churchill, GB: Goose Bay, IQ: Iqaluit, KT: Kugluktuk, MO: Moosonee, NW: Norman Wells, OM: Ogilvie Mountains, SV: Schefferville, YK: Yellowknife.
Site BI CB CH GB IQ KT MO NW OM SV
CB 0.63
CH 0.15 0.19
GB 0.04 0.04 0.17
IQ 0.56 0.44 0.11 0.00
KT 0.24 0.24 0.26 0.13 0.28
MO 0.00 0.00 0.35 0.16 0.00 0.03
NW 0.10 0.05 0.34 0.20 0.10 0.34 0.15
OM 0.13 0.09 0.20 0.05 0.13 0.22 0.07 0.26
SV 0.00 0.00 0.31 0.34 0.00 0.09 0.21 0.28 0.05 YK 0.11 0.11 0.39 0.15 0.05 0.15 0.24 0.41 0.09 0.21
! 36!
Table S5. Common species among sites. BI: Banks Island, CB: Cambridge Bay, CH: Churchill, GB: Goose Bay, IQ: Iqaluit, KT: Kugluktuk, MO: Moosonee, NW: Norman Wells, OM: Ogilvie Mountains, SV: Schefferville, YK: Yellowknife.
Site BI CB CH GB IQ KT MO NW OM SV
CB 5
CH 4 5
GB 1 1 7
IQ 5 4 3 0
KT 4 4 9 4 5
MO 0 0 16 7 0 1
NW 2 1 13 7 2 10 6
OM 3 2 8 2 3 7 3 9
SV 0 0 13 13 0 3 9 10 2 YK 2 2 14 5 1 4 9 12 3 7
! 37! Table S6. BIOCLIM variables values for each site as generated in R. Variables — bi1: Annual Mean Temperature, bi2: Mean Diurnal Range (Mean of monthly (max temp - min temp)), bi3: Isothermality (BIO2/BIO7) (* 100), bi5: Max Temperature of Warmest Month, bi6: Min Temperature of Coldest Month, bi7: Temperature Annual Range (BIO5-BIO6), b12: Annual Precipitation, b13: Precipitation of Wettest Month, b14: Precipitation of Driest Month, b15: Precipitation Seasonality (Coefficient of Variation).
Site% bi1% bi2% bi3% bi5% bi6% bi7% b12% b13% b14% b15% Banks%Island% 612.17% 6.39% 15.52% 9.86% 631.32% 41.18% 192.87% 29.55% 8.9% 38.91% Cambridge%Bay% 613.13% 6.56% 13.52% 13.35% 635.17% 48.52% 208.84% 32.8% 7.96% 43.82% Churchill% 65.45% 8.2% 17.66% 18.17% 628.27% 46.44% 545.99% 81.23% 24.15% 46.65% Goose%Bay% 1.3% 9.11% 21.78% 21.81% 620% 41.81% 1058.74% 123.31% 72.11% 16.35% Iqaluit% 67.65% 6.85% 16.09% 13% 629.59% 42.59% 453.63% 74.44% 18.47% 40.59% Kugluktuk% 69.56% 7.81% 16.75% 15.55% 631.05% 46.6% 428.84% 59.19% 17.95% 35.06% Moosonee% 1.29% 10.24% 22.28% 22.96% 623% 45.96% 777.58% 99.38% 35.56% 32.52% Norman%Wells% 64.64% 9.06% 17.66% 22.53% 628.76% 51.29% 394.48% 60.13% 16.88% 40.85% Ogilvie%Mountains% 63.59% 12.52% 23.74% 23.05% 629.67% 52.72% 381.06% 64.81% 9.67% 46.72% Schefferville% 63.47% 9.18% 19.97% 18.55% 627.39% 45.94% 941.21% 125.57% 43.45% 30.15% Yellowknife% 63.62% 8.07% 16.38% 21.57% 627.67% 49.24% 409.64% 53.26% 13.9% 41.1%
38
Table S7. Detailed locality information for northern Ephemeroptera, Plecoptera and Trichoptera.
Order Family Genus species ROM accession number Province/Territories Site of Collection Latitude Longitude Ephemeroptera Baetidae Acentrella lapponica 2011247 Northwest Territories Banks Island 73°12'15.8" 119°33'59.2" Ephemeroptera Baetidae Acentrella lapponica 2010614 Nunavut Cambridge Bay 69°5'14.5" 104°56'20.1" Ephemeroptera Baetidae Acentrella lapponica 2011243 Nunavut Iqaluit 63°47.432' 68°34.042' Ephemeroptera Ameletidae Ameletus celer 2011289 Yukon Ogilvie Mountains 64°32'42.9" 138°14'3.8" Ephemeroptera Ameletidae Ameletus inopinatus 2011287 Northwest Territories Banks Island 73°13'50.7" 119°32'26.9" Ephemeroptera Ameletidae Ameletus inopinatus 2010604 Nunavut Iqaluit 63°47.338' 68°33.674' Ephemeroptera Ameletidae Ameletus inopinatus 2011234 Nunavut Kugluktuk 67°53'44.3" 115°47'54.5" Ephemeroptera Ameletidae Ameletus inopinatus 2011292 Northwest Territories Norman Wells 65°0'54.3" 127°07'59.4" Ephemeroptera Ameletidae Ameletus inopinatus 2010601 Yukon Ogilvie Mountains 64°50'8.2" 138°21'49" Ephemeroptera Ameletidae Ameletus sp. 2011264 Nunavut Iqaluit 63°45.959' 68°34.850' Ephemeroptera Ameletidae Ameletus velox 2011305 Yukon Ogilvie Mountains 64°46'31.2" 138°21'45.4" Ephemeroptera Baetidae Baetis bicaudatus 2011243 Yukon Ogilvie Mountains 64°46'31.2" 138°21'45.4" Ephemeroptera Baetidae Baetis brunneicolor 2010568 Ontario Moosonee 51°16.972' 80°37.199' Ephemeroptera Baetidae Baetis brunneicolor 2010621 Quebec Schefferville 54°40.106' 66°45.815' Ephemeroptera Baetidae Baetis bundyae 2011287 Nunavut Cambridge Bay 69°5'14.5" 104°56'20.1" Ephemeroptera Baetidae Baetis foemina 2010604 Nunavut Iqaluit 63°47.432' 68°34.042' Ephemeroptera Baetidae Baetis hudsonicus 2011288 Northwest Territories Banks Island 73°16'34.1" 119°34'44.9" Ephemeroptera Baetidae Baetis hudsonicus 2011287 Nunavut Cambridge Bay 69°5'14.5" 104°56'20.1" Ephemeroptera Baetidae Baetis hudsonicus 2010601 Nunavut Iqaluit 63°47.338' 68°33.674' Ephemeroptera Baetidae Baetis hudsonicus 2011217 Northwest Territories Yellowknife 62°30'52.7" 114°51'7.2" Ephemeroptera Baetidae Baetis phoebus 2010626 Quebec Schefferville 54°40.134' 66°45.841' Ephemeroptera Baetidae Baetis tricaudatus 2010631 Newfoundland and Labrador Goose Bay 53°34.459' 60°42.462' Ephemeroptera Baetidae Baetis tricaudatus 2011257 Nunavut Kugluktuk 67°44'53.8" 115°22'6.8" Ephemeroptera Baetidae Baetis tricaudatus 2010580 Ontario Moosonee 51°16.753' 80°39.687' Ephemeroptera Baetidae Baetis tricaudatus 2011230 Northwest Territories Norman Wells 65°48.476' 126°12.217' Ephemeroptera Baetidae Baetis tricaudatus 2010624 Quebec Schefferville 54°44.394' 66°47.365' Ephemeroptera Baetidae Baetis tricaudatus 2011219 Northwest Territories Yellowknife 62°44'36.5" 115°43'31.5" Ephemeroptera Baetidae Baetis vernus 2011215 Northwest Territories Yellowknife 62°32'4.6" 114°58'15.4" Ephemeroptera Baetiscidae Baetisca laurentina 2010617 Quebec Schefferville 54°21.688' 66°44.962'
! 39!
Table S7 (continued). Detailed locality information for northern Ephemeroptera, Plecoptera and Trichoptera.
Order Family Genus species ROM accession number Province/Territories Site of Collection Latitude Longitude Ephemeroptera Caenidae Caenis amica 2010567 Ontario Moosonee 51°12.568' 80°42.274' Ephemeroptera Caenidae Caenis latipennis 2010566 Ontario Moosonee 51°12.568' 80°42.274' Ephemeroptera Caenidae Caenis sp. 2011206 Ontario Moosonee 51°13.316' 80°41.963' Ephemeroptera Caenidae Caenis youngi 2011225 Northwest Territories Norman Wells 65°15'8.4" 126°39'56.3" Ephemeroptera Caenidae Caenis youngi 2011303 Northwest Territories Yellowknife 62°43'26.4" 114°21'43.2" Ephemeroptera Heptageniidae Cinygmula sp. 2010626 Northwest Territories Norman Wells 65°19'21.3" 126°41'55.8" Ephemeroptera Heptageniidae Cinygmula sp. 2010580 Yukon Ogilvie Mountains 64°28'32" 138°11'45.5" Ephemeroptera Heptageniidae Cinygmula sp.JMW1 2010570 Yukon Ogilvie Mountains 64°30'22.5" 138°13'38.1" Ephemeroptera Heptageniidae Cinygmula spJMW3 2010631 Yukon Ogilvie Mountains 65°6'3" 138°21'27.5" Ephemeroptera Heptageniidae Cinygmula subaequalis 2010632 Nunavut Kugluktuk 67°53'44.3" 115°47'54.5" Ephemeroptera Heptageniidae Cinygmula subaequalis 2010583 Northwest Territories Norman Wells 65°17'26.1" 126°52'52.7" Ephemeroptera Heptageniidae Cinygmula subaequalis 2010569 Yukon Ogilvie Mountains 64°46'31.2" 138°21'45.4" Ephemeroptera Ephemerellidae Drunella coloradensis 2011233 Yukon Ogilvie Mountains 64°30'22.5" 138°13'38.1" Ephemeroptera Ephemerellidae Drunella doddsi 2011260 Yukon Ogilvie Mountains 64°26'34.7" 138°14'20.3" Ephemeroptera Ephemerellidae Drunella grandis 2011253 Yukon Ogilvie Mountains 61°58'9.7" 136°12'22.2" Ephemeroptera Ephemerellidae Drunella lata 2011264 Newfoundland and Labrador Goose Bay 53°34.459' 60°42.462' Ephemeroptera Ephemerellidae Drunella lata 2011205 Quebec Schefferville 54°44.394' 66°47.365' Ephemeroptera Heptageniidae Epeorus pleuralis 2011219 Quebec Schefferville 54°46.646' 66°49.556' Ephemeroptera Heptageniidae Epeorus vitreus 2010631 Newfoundland and Labrador Goose Bay 53°34.459' 60°42.462' Ephemeroptera Heptageniidae Epeorus vitreus 2010580 Ontario Moosonee 51°16.753' 80°39.687' Ephemeroptera Heptageniidae Epeorus vitreus 2011305 Quebec Schefferville 54°21.688' 66°44.962' Ephemeroptera Ephemeridae Ephemera simulans 2010630 Ontario Moosonee 51°13.316' 80°41.963' Ephemeroptera Ephemerellidae Ephemerella aurivillii 2011243 Newfoundland and Labrador Goose Bay 53°34.459' 60°42.462' Ephemeroptera Ephemerellidae Ephemerella aurivillii 2011260 Nunavut Kugluktuk 67°53'44.3" 115°47'54.5" Ephemeroptera Ephemerellidae Ephemerella aurivillii 2011250 Northwest Territories Norman Wells 65°17'26.1" 126°52'52.7" Ephemeroptera Ephemerellidae Ephemerella aurivillii 2011261 Quebec Schefferville 54°46.646' 66°49.556' Ephemeroptera Ephemerellidae Ephemerella invaria 2010631 Newfoundland and Labrador Goose Bay 53°31.046' 60°39.442' Ephemeroptera Ephemerellidae Ephemerella invaria 2010624 Ontario Moosonee 51°16.478' 80°39.028' Ephemeroptera Ephemerellidae Ephemerella invaria 2010625 Quebec Schefferville 54°40.134' 66°45.841'
! 40!
Table S7 (continued). Detailed locality information for northern Ephemeroptera, Plecoptera and Trichoptera.
Order Family Genus species ROM accession number Province/Territories Site of Collection Latitude Longitude Ephemeroptera Ephemerellidae Ephemerella needhami 2010631 Ontario Moosonee 51°16.753' 80°39.687' Ephemeroptera Ephemerellidae Ephemerella sp. 2010580 Ontario Moosonee 51°16.478' 80°39.028' Ephemeroptera Ephemerellidae Ephemerella subvaria 2010623 Newfoundland and Labrador Goose Bay 53°34.459' 60°42.462' Ephemeroptera Ephemerellidae Eurylophella funeralis 2010569 Newfoundland and Labrador Goose Bay 53°28.037' 60°30.202' Ephemeroptera Ephemerellidae Eurylophella macdunnoughi 2010631 Quebec Schefferville 54°52.071' 66°56.399' Ephemeroptera Ephemerellidae Eurylophella sp. 2011264 Ontario Moosonee 51°13.316' 80°41.963' Ephemeroptera Ephemerellidae Eurylophella temporalis 2011205 Northwest Territories Yellowknife 62°44'36.5" 115°43'31.5" Ephemeroptera Ephemerellidae Eurylophella verisimilis 2010625 Newfoundland and Labrador Goose Bay 53°34.459' 60°42.462' Ephemeroptera Heptageniidae Heptagenia pulla 2010626 Ontario Moosonee 51°16.753' 80°39.687' Ephemeroptera Heptageniidae Heptagenia pulla 2011215 Northwest Territories Norman Wells 65°0'54.3" 127°07'59.4" Ephemeroptera Heptageniidae Heptagenia pulla 2010632 Quebec Schefferville 54°40.134' 66°45.841' Ephemeroptera Heptageniidae Heptagenia pulla 2010616 Northwest Territories Yellowknife 62°32'4.6" 114°58'15.4" Ephemeroptera Heptageniidae Heptagenia sp. 2010631 Newfoundland and Labrador Goose Bay 53°28.037' 60°30.202' Ephemeroptera Heptageniidae Hexagenia limbata 2010568 Ontario Moosonee 51°13.245' 80°42.244' Ephemeroptera Isonychiidae Isonychia bicolor 2010631 Newfoundland and Labrador Goose Bay 53°34.459' 60°42.462' Ephemeroptera Leptophlebiidae Leptophlebia cupida 2010623 Ontario Moosonee 51°16.972' 80°37.199' Ephemeroptera Leptophlebiidae Leptophlebia cupida 2010631 Northwest Territories Norman Wells 65°0'54.3" 127°07'59.4" Ephemeroptera Leptophlebiidae Leptophlebia cupida 2010624 Quebec Schefferville 54°45.692' 66°48.838' Ephemeroptera Leptophlebiidae Leptophlebia cupida 2010569 Northwest Territories Yellowknife 62°44'36.5" 115°43'31.5" Ephemeroptera Leptophlebiidae Leptophlebia sp. 2010623 Newfoundland and Labrador Goose Bay 53°16.454' 60°29.727' Ephemeroptera Heptageniidae Leucrocuta jewetti 2011305 Ontario Moosonee 51°16.753' 80°39.687' Ephemeroptera Ephemeridae Litobrancha recurvata 2010579 Newfoundland and Labrador Goose Bay 53°27.739' 60°34.289' Ephemeroptera Heptageniidae Maccaffertium vicarium 2010622 Newfoundland and Labrador Goose Bay 53°34.459' 60°42.462' Ephemeroptera Metretopodidae Metretopus borealis 2010616 Quebec Schefferville 54°21.688' 66°44.962' Ephemeroptera Leptophlebiidae Paraleptophlebia adoptiva 2011241 Newfoundland and Labrador Goose Bay 53°34.459' 60°42.462' Ephemeroptera Leptophlebiidae Paraleptophlebia adoptiva 2011229 Quebec Schefferville 54°44.394' 66°47.365' Ephemeroptera Leptophlebiidae Paraleptophlebia aquilina 2010617 Ontario Moosonee 51°13.316' 80°41.963' Ephemeroptera Leptophlebiidae Paraleptophlebia mollis 2011257 Quebec Schefferville 54°21.688' 66°44.962' Ephemeroptera Siphlonuridae Parameletus chelifer 2011246 Nunavut Kugluktuk 67°48'22.6" 115°7'26.1" Ephemeroptera Siphlonuridae Parameletus chelifer 2010628 Northwest Territories Norman Wells 65°11'30.1" 126°28'7.6"
! 41!
Table S7 (continued). Detailed locality information for northern Ephemeroptera, Plecoptera and Trichoptera.
Order Family Genus species ROM accession number Province/Territories Site of Collection Latitude Longitude Ephemeroptera Baetidae Procloeon sp. 2010623 Ontario Moosonee 51°12.568' 80°42.274' Ephemeroptera Heptageniidae Rhithrogena sp. 2011219 Nunavut Kugluktuk 67°44'53.8" 115°22'6.8" Ephemeroptera Siphlonuridae Siphlonurus alternatus 2010617 Ontario Moosonee 51°13.245' 80°42.244' Ephemeroptera Siphlonuridae Siphlonurus alternatus 2010621 Yukon Ogilvie Mountains 64°44'1.4" 138°21'40.2" Ephemeroptera Siphlonuridae Siphlonurus phyllis 2010577 Newfounland and Labrador Goose Bay 53°16.454' 60°29.727' Ephemeroptera Siphlonuridae Siphlonurus phyllis 2011219 Ontario Moosonee 51°12.568' 80°42.274' Ephemeroptera Siphlonuridae Siphlonurus quebecensis 2010631 Quebec Schefferville 54°40.106' 66°45.815' Ephemeroptera Heptageniidae Stenonema femoratum 2010628 Ontario Moosonee 51°13.245' 80°42.244' Ephemeroptera Heptageniidae Stenonema femoratum 2010580 Northwest Territories Yellowknife 62°44'36.5" 115°43'31.5" Ephemeroptera Heptageniidae Stenonema sp. 2010633 Newfounland and Labrador Goose Bay 53°34.459' 60°42.462' Plecoptera Perlidae Acroneuria abnormis 2010634 Ontario Moosonee 51°13.316' 80°41.963' Plecoptera Perlidae Acroneuria lycoras 2011263 Ontario Moosonee 51°16.795' 80°39.687' Plecoptera Perlidae Acroneuria sp. 2011223 Newfounland and Labrador Goose Bay 53°31.046' 60°39.442' Plecoptera Capniidae Allocapnia granulata 2010569 Northwest Territories Norman Wells 65°14'51.4" 126°39'58.7" Plecoptera Capniidae Allocapnia sp. 2010571 Nunavut Iqaluit 63°47.432' 68°34.042' Plecoptera Capniidae Allocapnia vivipara 2010630 Yukon Ogilvie Mountains 65°12'56" 138°19'1.2" Plecoptera Chloroperlidae Alloperla concolor 2011205 Quebec Schefferville 54°21.688' 66°44.962' Plecoptera Chloroperlidae Alloperla severa 2010578 Newfounland and Labrador Goose Bay 53°31.046' 60°39.442' Plecoptera Chloroperlidae Alloperla severa 2010632 Northwest Territories Norman Wells 65°17'26.1" 126°52'52.7" Plecoptera Nemouridae Amphinemura linda 2010623 Ontario Moosonee 51°16.795' 80°39.687' Plecoptera Nemouridae Amphinemura sp. 2011228 Newfounland and Labrador Goose Bay 53°28.037' 60°30.202' Plecoptera Perlodidae Arcynopteryx compacta 2010579 Nunavut Iqaluit 63°45.959' 68°34.850' Plecoptera Perlodidae Arcynopteryx compacta 2011255 Nunavut Kugluktuk 67°53'44.3" 115°47'54.5" Plecoptera Perlodidae Arcynopteryx compacta 2010566 Northwest Territories Norman Wells 65°19'21.3" 126°41'55.8" Plecoptera Perlodidae Arcynopteryx compacta 2010576 Yukon Ogilvie Mountains 64°36'22.9" 138°20'47.2" Plecoptera Capniidae Capnia atra 2011213 Nunavut Kugluktuk 67°50.887' 115°14.270' Plecoptera Perlodidae Diura bicaudata 2010580 Nunavut Kugluktuk 67°55'11.9" 115°39'53.1" Plecoptera Perlodidae Diura bicaudata 2010621 Northwest Territories Yellowknife 62°44'36.5" 115°43'31.5" Plecoptera Capniidae Eucanopsis bravicaudata 2010604 Yukon Ogilvie Mountains 64°36'22.9" 138°20'47.2" Plecoptera Chloroperlidae Haploperla brevis 2010614 Ontario Moosonee 51°16.795' 80°39.687'
! 42!
Table S7 (continued). Detailed locality information for northern Ephemeroptera, Plecoptera and Trichoptera.
Order Family Genus species ROM accession number Province/Territories Site of Collection Latitude Longitude Plecoptera Perlodidae Isogenoides frontalis 2010624 Northwest Territories Norman Wells 65°48.476' 126°12.217' Plecoptera Perlodidae Isogenoides frontalis 2011245 Quebec Schefferville 54°21.688' 66°44.962' Plecoptera Perlodidae Isoperla bilineata 2011229 Northwest Territories Norman Wells 65°12'42" 126°32'28.8" Plecoptera Perlodidae Isoperla bilineata 2010633 Quebec Schefferville 54°48.434' 66°48.176' Plecoptera Perlodidae Isoperla obscura 2010624 Nunavut Kugluktuk 67°44'53.8" 115°22'6.8" Plecoptera Perlodidae Isoperla obscura 2011232 Northwest Territories Norman Wells 65°48.476' 126°12.217' Plecoptera Perlodidae Isoperla petersoni 2010579 Yukon Ogilvie Mountains 64°26'34.7" 138°14'20.3" Plecoptera Perlodidae Isoperla sp. 2011302 Newfounland and Labrador Goose Bay 53°34.459' 60°42.462' Plecoptera Perlodidae Isoperla sp. 2010576 Ontario Moosonee 51°16.753' 80°39.687' Plecoptera Perlodidae Isoperla sp. 2011205 Quebec Schefferville 54°44.394' 66°47.365' Plecoptera Perlodidae Isoperla sp. 2010624 Northwest Territories Yellowknife 62°44'36.5" 115°43'31.5" Plecoptera Leuctridae Leuctra sp. 2011178 Newfounland and Labrador Goose Bay 53°34.459' 60°42.462' Plecoptera Leuctridae Leuctra sp. 2011263 Quebec Schefferville 54°44.394' 66°47.365' Plecoptera Perlodidae Megarcys signata 2011236 Yukon Ogilvie Mountains 64°30'21.2" 138°13'11" Plecoptera Nemouridae Nemoura arctica 2010618 Northwest Territories Banks Island 73°16'34.1" 119°34'44.9" Plecoptera Nemouridae Nemoura arctica 2011257 Nunavut Cambridge Bay 69°9'28.9" 105°11'28.2" Plecoptera Nemouridae Nemoura arctica 2011230 Newfounland and Labrador Goose Bay 53°28.787' 60°36.032' Plecoptera Nemouridae Nemoura arctica 2011250 Nunavut Kugluktuk 67°55'11.9" 115°39'53.1" Plecoptera Nemouridae Nemoura arctica 2010631 Northwest Territories Norman Wells 65°17'26.1" 126°37'10.5" Plecoptera Nemouridae Nemoura arctica 2010580 Yukon Ogilvie Mountains 64°36'22.9" 138°20'47.2" Plecoptera Nemouridae Nemoura arctica 2010624 Northwest Territories Yellowknife 62°32.329' 114°08.383' Plecoptera Nemouridae Nemoura trispinosa 2011219 Ontario Moosonee 51°16.478' 80°39.028' Plecoptera Nemouridae Nemoura trispinosa 2010631 Yukon Ogilvie Mountains 65°12'56" 138°19'1.2" Plecoptera Perlidae Paragnetina media 2011232 Ontario Moosonee 51°13.316' 80°41.963' Plecoptera Perlidae Paragnetina sp. 2011170 Ontario Moosonee 51°13.316' 80°41.963' Plecoptera Leuctridae Paraleuctra knighton01 2011219 Ontario Moosonee 51°16.753' 80°39.687' Plecoptera Leuctridae Paraleuctra knighton01 2011232 Quebec Schefferville 54°40.106' 66°45.815' Plecoptera Leuctridae Paraleuctra sara 2010571 Quebec Schefferville 54°44.394' 66°47.365' Plecoptera Leuctridae Paraleuctra sp.XZ01 2011230 Yukon Ogilvie Mountains 64°26'19.3" 138°15'18.8" Plecoptera Nemouridae Paranemoura perfecta 2010624 Northwest Territories Norman Wells 65°11'30.1" 126°28'7.6"
! 43!
Table S7 (continued). Detailed locality information for northern Ephemeroptera, Plecoptera and Trichoptera.
Order Family Genus species ROM accession number Province/Territories Site of Collection Latitude Longitude Plecoptera Capniidae Plecoptera sp. 2011255 Newfounland and Labrador Goose Bay 53°27.739' 60°34.289' Plecoptera Capniidae Plecoptera sp. 2010623 Quebec Schefferville 54°44.394' 66°47.365' Plecoptera Chloroperlidae Plumiperla diversa 2011264 Yukon Ogilvie Mountains 64°36'22.9" 138°20'47.2" Plecoptera Pteronarcyidae Pteronarcys dorsata 2011232 Ontario Moosonee 51°16.478' 80°39.028' Plecoptera Pteronarcyidae Pteronarcys dorsata 2011303 Northwest Territories Yellowknife 62°31'39.3" 113°22'38" Plecoptera Pteronarcyidae Pteronarcys pictetii 2011250 Ontario Moosonee 51°13.316' 80°41.963' Plecoptera Pteronarcyidae Pteronarcys sp. 2011234 Northwest Territories Norman Wells 65°17'26.1" 126°52'52.7" Plecoptera Chloroperlidae Sweltsa naica 2011303 Quebec Schefferville 54°44.394' 66°47.365' Plecoptera Chloroperlidae Sweltsa urticae 2011232 Yukon Ogilvie Mountains 64°29'18.1" 138°12'17.6" Plecoptera Capniidae Utacapnia trava 2010630 Yukon Ogilvie Mountains 64°36'22.9" 138°20'47.2" Plecoptera Nemouridae Zapada sp. 2011248 Northwest Territories Norman Wells 65°19'21.3" 126°41'55.8" Plecoptera Nemouridae Zapada sp.JMW03 2011288 Yukon Ogilvie Mountains 64°26'34.7" 138°14'20.3" Plecoptera Nemouridae Zapada sp.JMW04 2011271 Yukon Ogilvie Mountains 64°32'42.9" 138°14'3.8" Trichoptera Phryganeidae Agrypnia deflata 2011215 Northwest Territories Norman Wells 65°14'51.4" 126°39'58.7" Trichoptera Phryganeidae Agrypnia deflata 2010630 Northwest Territories Yellowknife 62°30'52.7" 114°51'7.2" Trichoptera Phryganeidae Agrypnia glacialis 2011230 Quebec Schefferville 54°54.186' 67°8.859' Trichoptera Phryganeidae Agrypnia pagetana 2011250 Nunavut Kugluktuk 67°47'6.9" 115°14'28.4" Trichoptera Phryganeidae Agrypnia straminea 2010624 Quebec Schefferville 54°45.692' 66°48.838' Trichoptera Limnephilidae Anabolia bimaculata 2010530 Newfounland and Labrador Goose Bay 53°16.454' 60°29.727' Trichoptera Limnephilidae Anabolia bimaculata 2011266 Ontario Moosonee 51°16.972' 80°37.199' Trichoptera Limnephilidae Anabolia bimaculata 2011258 Northwest Territories Norman Wells 65°15'8.4" 126°39'56.3" Trichoptera Limnephilidae Anabolia bimaculata 2011229 Quebec Schefferville 54°40.106' 66°45.815' Trichoptera Limnephilidae Anabolia bimaculata 2011246 Northwest Territories Yellowknife 62°31'7.9" 113°44'14.4" Trichoptera Apataniidae Apatania stigmatella 2011213 Quebec Schefferville 54°46.646' 66°49.556' Trichoptera Apataniidae Apatania wallengreni 2011217 Nunavut Kugluktuk 67°44'53.8" 115°22'6.8" Trichoptera Apataniidae Apatania zonella 2010556 Northwest Territories Banks Island 73°13.309' 119°33.635' Trichoptera Limnephilidae Arctopora pulchella 2011302 Northwest Territories Norman Wells 65°15'18" 126°39'49.4" Trichoptera Limnephilidae Arctopora pulchella 2010568 Yukon Ogilvie Mountains 65°12'56" 138°19'1.2" Trichoptera Limnephilidae Arctopora pulchella 2010628 Quebec Schefferville 54°48.434' 66°48.176' Trichoptera Hydropsychidae Arctopsyche ladogensis 2011206 Nunavut Kugluktuk 67°44'53.8" 115°22'6.8"
! 44!
Table S7 (continued). Detailed locality information for northern Ephemeroptera, Plecoptera and Trichoptera.
Order Family Genus species ROM accession number Province/Territories Site of Collection Latitude Longitude Trichoptera Limnephilidae Asynarchus lapponicus 2011238 Northwest Territories Banks Island 73°13'32.4" 119°33'31.5" Trichoptera Limnephilidae Asynarchus lapponicus 2010578 Nunavut Cambridge Bay 69°5'14.5" 104°56'20.1" Trichoptera Limnephilidae Asynarchus lapponicus 2011219 Nunavut Iqaluit 63°47.468' 68°34.425' Trichoptera Limnephilidae Asynarchus lapponicus 2010621 Nunavut Kugluktuk 67°51'19.7" 115°18'10.3" Trichoptera Limnephilidae Asynarchus montanus 2011219 Nunavut Kugluktuk 67°44'36.3" 115°22'20" Trichoptera Limnephilidae Asynarchus montanus 2010621 Northwest Territories Norman Wells 65°11'30.1" 126°28'7.6" Trichoptera Limnephilidae Asynarchus montanus 2010631 Yukon Ogilvie Mountains 64°44'1.4" 138°21'40.2" Trichoptera Limnephilidae Asynarchus montanus 2011215 Northwest Territories Yellowknife 62°31'39.3" 113°22'38" Trichoptera Limnephilidae Asynarchus rossi 2010631 Ontario Moosonee 51°16.972' 80°37.199' Trichoptera Phryganeidae Banksiola crotchi 2011210 Newfounland and Labrador Goose Bay 53°16.454' 60°29.727' Trichoptera Brachycentridae Brachycentrus americanus 2011242 Nunavut Kugluktuk 67°50'22" 115°14'5.2" Trichoptera Leptoceridae Ceraclea excisa 2011268 Ontario Moosonee 51°16.795' 80°39.687' Trichoptera Leptoceridae Ceraclea nigronervosa 2011287 Northwest Territories Yellowknife 62°44'36.5" 115°43'31.5" Trichoptera Hydropsychidae Ceratopsyche alternans 2010621 Quebec Schefferville 54°40.106' 66°45.815' Trichoptera Hydropsychidae Ceratopsyche alternans 2011226 Northwest Territories Yellowknife 62°44'36.5" 115°43'31.5" Trichoptera Hydropsychidae Ceratopsyche morosa 2010625 Quebec Schefferville 54°40.106' 66°45.815' Trichoptera Hydropsychidae Ceratopsyche sparna 2011257 Newfounland and Labrador Goose Bay 53°34.459' 60°42.462' Trichoptera Philopotamidae Chimarra obscura 2011265 Northwest Territories Yellowknife 62°32'4.6" 114°58'15.4" Trichoptera Philopotamidae Chimarra socia 2010629 Newfounland and Labrador Goose Bay 53°34.459' 60°42.462' Trichoptera Limnephilidae Dicosmoecus obscuripennis 2010578 Ontario Moosonee 51°16.795' 80°39.687' Trichoptera Limnephilidae Dicosmoecus obscuripennis 2011250 Yukon Ogilvie Mountains 64°26'34.7" 138°14'20.3" Trichoptera Philopotamidae Dolophilodes distinctus 2010566 Ontario Moosonee 51°16.478' 80°39.028' Trichoptera Limnephilidae Ecclisomyia conspersa 2010570 Yukon Ogilvie Mountains 65°12'56" 138°19'1.2" Trichoptera Glossosomatidae Glossosoma nigrior 2010568 Ontario Moosonee 51°16.478' 80°39.028' Trichoptera Limnephilidae Grammotaulius interrogationis 2011255 Nunavut Kugluktuk 67°51'19.7" 115°18'10.3" Trichoptera Limnephilidae Grammotaulius interrogationis 2010579 Northwest Territories Norman Wells 65°11'30.1" 126°28'7.6" Trichoptera Limnephilidae Grammotaulius interrogationis 2011266 Yukon Ogilvie Mountains 64°36'22.9" 138°20'47.2" Trichoptera Limnephilidae Grensia praeterita 2011229 Northwest Territories Banks Island 73°13.309' 119°33.635' Trichoptera Limnephilidae Grensia praeterita 2011232 Nunavut Cambridge Bay 69°9'28.9" 105°11'28.2" Trichoptera Limnephilidae Grensia praeterita 2011348 Nunavut Iqaluit 63°46.874' 68°32.940'
! 45!
Table S7 (continued). Detailed locality information for northern Ephemeroptera, Plecoptera and Trichoptera.
Order Family Genus species ROM accession number Province/Territories Site of Collection Latitude Longitude Trichoptera Limnephilidae Grensia praeterita 2011271 Nunavut Kugluktuk 67°51'19.7" 115°18'10.3" Trichoptera Limnephilidae Grensia praeterita 2010613 Yukon Ogilvie Mountains 64°36'17.5" 138°19'41.7" Trichoptera Limnephilidae Hesperophylax designatus 2011266 Nunavut Kugluktuk 67°51'19.7" 115°18'10.3" Trichoptera Limnephilidae Hesperophylax designatus 2011235 Northwest Territories Norman Wells 65°48.476' 126°12.217' Trichoptera Hydropsychidae Hydropsyche hoffmani 2011348 Northwest Territories Norman Wells 65°17'26.1" 126°52'52.7" Trichoptera Hydropsychidae Hydropsyche hoffmani 2011211 Northwest Territories Yellowknife 62°29'32.6' 113°32'49 Trichoptera Limnephilidae Lenarchus productus 2011266 Nunavut Kugluktuk 67°50'4.1" 115°12'40.6" Trichoptera Lepidostomatidae Lepidostoma togatum 2011255 Ontario Moosonee 51°13.316' 80°41.963' Trichoptera Lepidostomatidae Lepidostoma togatum 2010618 Northwest Territories Norman Wells 65°48.476' 126°12.217' Trichoptera Lepidostomatidae Lepidostoma togatum 2011257 Northwest Territories Yellowknife 62°44'36.5" 115°43'31.5" Trichoptera Limnephilidae Limnephilus alaicus 2011230 Northwest Territories Banks Island 73°13'50.9" 119°32'52.5" Trichoptera Limnephilidae Limnephilus alaicus 2011205 Nunavut Cambridge Bay 69°13'16.3" 105°13'5.3" Trichoptera Limnephilidae Limnephilus argenteus 2011209 Nunavut Kugluktuk 67°44'36.3" 115°22'20" Trichoptera Limnephilidae Limnephilus canadensis 2011239 Northwest Territories Norman Wells 65°48.476' 126°12.217' Trichoptera Limnephilidae Limnephilus canadensis 2010566 Northwest Territories Yellowknife 62°32'4.6" 114°58'15.4" Trichoptera Limnephilidae Limnephilus dispar 2011230 Newfounland and Labrador Goose Bay 53°16.454' 60°29.727' Trichoptera Limnephilidae Limnephilus dispar 2011219 Nunavut Kugluktuk 67°49'34.5" 115°34'18.5" Trichoptera Limnephilidae Limnephilus dispar 2011293 Quebec Schefferville 54°54.186' 67°8.859' Trichoptera Limnephilidae Limnephilus externus 2011272 Northwest Territories Norman Wells 65°15'8.4" 126°39'56.3" Trichoptera Limnephilidae Limnephilus externus 2011258 Yukon Ogilvie Mountains 64°36'22.9" 138°20'47.2" Trichoptera Limnephilidae Limnephilus indivisus 2011230 Ontario Moosonee 51°16.972' 80°37.199' Trichoptera Limnephilidae Limnephilus indivisus 2011215 Yukon Ogilvie Mountains 65°12'56" 138°19'1.2" Trichoptera Limnephilidae Limnephilus infernalis 2010628 Ontario Moosonee 51°16.972' 80°37.199' Trichoptera Limnephilidae Limnephilus infernalis 2011265 Northwest Territories Yellowknife 62°31'7.9" 113°44'14.4" Trichoptera Limnephilidae Limnephilus moestus 2010556 Nunavut Kugluktuk 67°49'34.5" 115°34'18.5" Trichoptera Limnephilidae Limnephilus picturatus 2011206 Nunavut Cambridge Bay 69°5'14.5" 104°56'20.1" Trichoptera Limnephilidae Limnephilus picturatus 2011232 Nunavut Iqaluit 63°47.468' 68°34.425' Trichoptera Limnephilidae Limnephilus picturatus 2010568 Nunavut Kugluktuk 67°50'4.1" 115°12'40.6" Trichoptera Limnephilidae Limnephilus rhombicus 2011255 Northwest Territories Yellowknife 62°31'39.3" 113°22'38" Trichoptera Limnephilidae Limnephilus sansoni 2010568 Yukon Ogilvie Mountains 64°36'17.5" 138°19'41.7"
! 46!
Table S7 (continued). Detailed locality information for northern Ephemeroptera, Plecoptera and Trichoptera.
Order Family Genus species ROM accession number Province/Territories Site of Collection Latitude Longitude Trichoptera Brachycentridae Micrasema canusa 2010622 Nunavut Cambridge Bay 69°9'28.9" 105°11'28.2" Trichoptera Brachycentridae Micrasema charonis 2010628 Ontario Moosonee 51°16.478' 80°39.028' Trichoptera Limnephilidae Nemotaulius hostilis 2011226 Nunavut Kugluktuk 67°49'34.5" 115°34'18.5" Trichoptera Uenoidae Neophylax aniqua 2010583 Newfounland and Labrador Goose Bay 53°24.821' 60°24.745' Trichoptera Uenoidae Neophylax sp. 2010634 Ontario Moosonee 51°13.316' 80°41.963' Trichoptera Polycentropodidae Neureclipsis bimaculata 2010576 Northwest Territories Yellowknife 62°32'4.6" 114°58'15.4" Trichoptera Limnephilidae Onocosmoecus unicolor 2011265 Newfounland and Labrador Goose Bay 53°31.046' 60°39.442' Trichoptera Limnephilidae Onocosmoecus unicolor 2011287 Northwest Territories Norman Wells 65°48.476' 126°12.217' Trichoptera Limnephilidae Onocosmoecus unicolor 2010530 Yukon Ogilvie Mountains 64°26'34.7" 138°14'20.3" Trichoptera Limnephilidae Onocosmoecus unicolor 2011239 Quebec Schefferville 54°44.394' 66°47.365' Trichoptera Limnephilidae Onocosmoecus unicolor 2011302 Northwest Territories Yellowknife 62°32'58.7" 113°21'12.2" Trichoptera Polycentropodidae Polycentropus cinereus 2011226 Ontario Moosonee 51°13.316' 80°41.963' Trichoptera Polycentropodidae Polycentropus smithae 2010631 Northwest Territories Yellowknife 62°31'7.9" 113°44'14.4" Trichoptera Limnephilidae Pycnopsyche guttifera 2011235 Newfounland and Labrador Goose Bay 53°34.459' 60°42.462' Trichoptera Limnephilidae Pycnopsyche guttifera 2011271 Ontario Moosonee 51°12.568' 80°42.274' Trichoptera Limnephilidae Pycnopsyche limbata 2010570 Newfounland and Labrador Goose Bay 53°28.037' 60°30.202' Trichoptera Rhyacophilidae Rhyacophila brunnea 2010567 Newfounland and Labrador Goose Bay 53°28.787' 60°36.032' Trichoptera Rhyacophilidae Rhyacophila brunnea 2010632 Quebec Schefferville 54°52.071' 66°56.399' Trichoptera Rhyacophilidae Rhyacophila torva 2010634 Newfounland and Labrador Goose Bay 53°28.787' 60°36.032' Trichoptera Brachycentridae Micrasema canusa 2010622 Nunavut Cambridge Bay 69°9'28.9" 105°11'28.2"
! 47! FIGURES
Figure 1. Sampling sites by ecoclimatic zone. Boreal: GB: Goose Bay, NL; MO: Moosonee, ON; YK: Yellowknife, NT; and NW: Norman Wells, NT. Subarctic: SV: Schefferville, QC; CH: Churchill, MB; KT: Kugluktuk, NU; and OM: Ogilvie Mountains, YT. Arctic: IQ: Iqaluit, NU; CB: Cambridge Bay, NU; BI: Banks Island, NT; and EI: Ellesmere Island, NU. Ecoregion map modified from Ecoregions Working Group (1989)
48
2.0 GB
1.5 SV
CB
1.0 BI IQ
0.5 CA2
0.0 KT
NW
CH
-0.5
YK
-1.0 MO OM
-2 -1 0 1
CA1
Figure 2. Correspondence analysis plot showing arrangement of sites based on presence/absence of species. Three well-defined clusters are highlighted (black ellipses). Arctic cluster: Iqaluit (IQ), Banks Island (BI), Cambridge Bay (CB). Eastern cluster: Schefferville (SV), Goose Bay (GB). Central cluster: Norman Wells (NW), Churchill (CH) Yellowknife (YK). Site not aligned with clusters: Ogilvie Mountains (OM), Kugluktuk (KT), Moosonee (MO).
! 49!
West East
Arctic
Figure 3. Hierarchical cluster dendrogram showing connections among sites using Euclidean distance from the presence/absence matrix. Arctic sites (CB: Cambridge Bay, BI: Banks Island, IQ: Iqaluit) are situated within a cluster of western sites (KT: Kugluktuk, YK: Yellowknife, NW: Norman Wells, CH: Churchill, OM: Ogilvie Mountains). Eastern sites form a separate cluster (MO: Moosonee, SV: Schefferville, GB: Goose Bay).
! 50!
1!
0.9!
Undetermined! 0.8! 45%! !
0.7!
0.6!
0.5! Geographical! 0.4! 24%! !
0.3! Envir!+!Geogr!!! 10%! 0.2! ! Variance(of(presence/absence(matrix( Environmental!! 0.1! 21%! !
0! %!Variation!
Figure 4. Partition of variance plot showing percentage of the variance of the Presence/absence matrix explained by environmental, geographical and joint environmental/geographical variables.
! 51!
!
! !
! TAR ! !
! !
! !
Pre
! ! !
!
Figure 5. Canonical correspondence analysis plot showing arrangement of sites and influence of environmental and geographical variables. Arrows are vectors for variables latitude (lat), longitude (long), temperature annual range (TAR) and annual precipitation (Pre).
! 52!
CHAPTER 3
Evidence for East-West population divergence in seven aquatic insect species in Northern Canada
Abstract
Specimens of Ephemeroptera, Plecoptera and Trichoptera (EPTs) were collected in a large-scale study of aquatic insects in northern Canada. Using a COI DNA barcoding approach 158 species were identified and analyzed, of which eighteen were transcontinental at northern latitudes.
Bayesian coalescent model revealed genetic divergence and geographical structure in seven of these widely distributed species, with distinct populations situated on either side of Hudson Bay.
We hypothesized that the east/west split in population structure was likely the product of separate refugial areas during the Wisconsinan glaciation. A strict molecular clock model was used to obtain the relative time to most recent common ancestor (TMRCA) for east/west populations of each species, revealing a similar age of divergence in 6 of the 7 species. The other species —
Onocosmoecus unicolor — had a considerably earlier relative TMRCA, with a genetic divergence of nearly 8% between eastern and western population. Evidence suggests that O. unicolor, as currently defined, represents two separate species that probably diverged during an earlier
Pleistocene glacial cycle. A DNA barcoding approach proved successful at identifying northern
EPTs at various stages of development and for revealing biogeographical patterns, providing value-added information for populations sampled over a wide geographical area.
! 53!
Introduction
The dynamics of Pleistocene glaciations is among the most important drivers shaping the current distribution of species and populations in northern Canada (Bernatchez & Wilson 1998).
Kilometers-thick ice sheets covered land on a continental scale (Dawson 1992; Martison et al.
1987), recurrently destroying habitats and biomes in its advance, and creating new ones in its retreat. The fauna and flora experienced fragmentation and reduction of their range during glacial episodes (Hewitt 2004), with populations confined to refugial areas at the glacial margins
(Pielou 1991).
After the last (i.e., Wisconsinan) glacial maximum LGM, 22000-19000 ya (Clark & Mix 2000), temperatures gradually began to warm, allowing displaced populations — whether confined to single or multiple refugia — to disperse back onto previously glaciated terrain (Pielou 1991).
Populations that survived the glacial maximum in two or more refugial areas were isolated long enough to generate intraspecific divergence, which can be detected at the genetic level (Wilson
2006). In North America, a number of important refugial areas have been identified including
Beringia (Hultén 1937, Hopkins 1967), the Pacific Northwest (Shafer et al. 2010), the
Appalachian Mountains and the eastern Coastal Plains, among others (e.g., Bernatchez & Wilson
1998; Church et al. 2003; Walker et al. 2009).
The prevalence and reproductive capacity of insects make them excellent models for exploring population divergence and patterns of postglacial dispersal; however, they have so far received far less study than vertebrates. Moreover, even fewer studies have been focused on postglacial patterns among widely distributed insects, with the notable exception of lycaenid butterflies
(Nice et al. 2005) and spruce beetles Dendroctonus rufipennis (Maroja et al. 2007).
The study of widely distributed populations is key to understanding how Pleistocene climatic
! 54! change shaped current distributional patterns (Riddle, 1998; Arbogast and Kenagy, 2001; Helveil and Berlocher, 2006; Walker et al. 2009). However, despite their potential to reveal biogeographical patterns on a large (i.e., continental) scale, aquatic insects have received less attention than many other insects (Rivera 2008).
The goal of this study is to use the COI barcoding gene for broad scale sampling of the aquatic insect orders Ephemeroptera, Plecoptera, and Trichoptera (EPTs) in northern Canada, and to seek signs of geographical divergence among widely distributed populations. We hypothesize that populations of widely distributed EPTs exhibit deep genetic divergences, suggesting that they had separate geographical histories (i.e., that the present-day EPT fauna of northern Canada was derived from two or more Pleistocene-aged refugia). This represents the first broad-scale study ever undertaken on the population structure of northern EPT fauna.
Materials and Methods
Fieldwork
Specimens of three orders of aquatic insects (Ephemeroptera, Plecoptera and Trichoptera) were collected from 12 widely distributed sites in northern Canada during June and July 2010 and
2011. Four sites were distributed among each of three northern ecoclimatic zones, as follows:
Boreal — Goose Bay (NL), Moosonee (ON), Yellowknife (NT), Norman Wells (NWT);
Subarctic — Schefferville (QC), Churchill (MB), Kugluktuk (NU), Ogilvie Mountains (YT; and
Arctic — Iqaluit (NU), Lake Hazen (NU), Cambridge Bay (NU), and Banks Island (NT) (Fig. 1).
Sampling took place for approximately two weeks at each site, with boreal sites sampled earliest in the season (followed respectively by subarctic and arctic sites) to ensure that insects were
! 55! sampled at more-or-less the same stage of development in each ecoclimatic zone. Easternmost sites were sampled in 2010 and westernmost sites in 2011.
Protocols for the collection of specimens were the same at each site, with a minimum number of variously sized lentic- and lotic habitats selected for sampling. Each habitat was sampled using a
D-framed aquatic dip net for at least 15 minutes to collect the immature (i.e., aquatic) stages of
EPTs. Net contents were emptied into shallow pans of water, with larvae of target taxa removed using fine forceps. A further 15 minutes was spent sweeping the riparian vegetation surrounding each habitat to collect adult EPTs. Aquatic and terrestrial specimens were fixed in 95% ethanol to facilitate both morphological identification and DNA barcoding. Vouchers are deposited in the Entomological collection at the Royal Ontario Museum, Toronto, Canada.
Lab procedures
Approximately 10.000 EPT specimens were collected and sorted under magnification with a
Nikon stereomicroscope. Specimens were initially identified to the lowest possible taxonomic level using keys in Merritt et al. (2008), Wiggins (1996) and Ruiter (1995). Selected adults and mature larvae were sent to taxonomic specialists for confirmation of species level identifications:
Ephemeroptera (S. Burian, Southern Connecticut State University), Plecoptera (R. Baumann,
Brigham Young University and B. Kondratieff, Colorado State University) and Trichoptera (D.
Ruiter, Grants Pass, Oregon). Of these, ca. 800 specimens representing the full range of variation for morphospecies at each site were selected for DNA sequencing.
Genomic DNA was extracted from the hind leg of selected specimens using the DNeasy Blood &
Tissue Kit QIAGEN. Extracted DNA was eluted in 150 µl of elution buffer AE (10 mM Tris-
HCl, 0.5 mM EDTA, pH 9.0). Cytochrome Oxidase I (COI) barcoding consisted in PCR amplification using the primer set LCO1490 (5’-ATTCAACCAATCATAAAGATATTGG-3’) -
! 56!
HCO2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) following the methods described by deWaard et al. (2008) and Hajibabaei et al. (2005), 1% agarose gel visualization and band excision, column centrifugation for PCR product clean-up, and Sanger sequencing using the
BigDye Terminator kit v3.1. All sequencing reactions were run using a Hitachi Applied
Biosystems 3730 DNA Analyzer.
Genetic analyses
Obtained sequences were individually compared against the NCBI GenBank and BOLD systems v3 database using BLASTn to identify the species name using a 98% similarity cutoff. For sequences below the 98% cutoff, the voucher specimen was sent to taxonomic experts on each group to confirm identification. In addition, we included the COI sequences produced by Zhou et al. (2010) in Churchill, since this study is so far the most complete reference library for aquatic insects in Northern Canada. We choose 6 random sequences of each species occurring in
Churchill that was also present in one or more of our sampling areas. Sequences are deposited in
GenBank (NCBI) under accession numbers KJ674822 - KJ675402.
Sequences were grouped according to insect order and then aligned and edited using Geneious®
6.1.6 (Biomatters Inc.) and ClustalX (Higgins et al. 1988) with default parameters. The best substitution model for each alignment was obtained using jModelTest v2 (Darriba et al. 2012) and the Akaike Information Criterion (AIC). Phylogenetic reconstructions for each insect order were obtained using BEAST v.1.7.5 (Drummond et al. 2012), by selecting a constant coalescent tree prior and a strict clock model with the rate fixed to 1.0, in order to estimate relative divergence times. Twenty million MCMC generations were run, sampling every 2000 states, to produce log and tree files with 10.000 states each. Convergence and chain mixing was assessed by visually inspecting each parameter trace from the log files using Tracer v.1.5 (Drummond &
! 57!
Rambaut 2007) considering effective sample sizes above 200 as a good indicator. A Maximum
Clade Credibility tree (MCC) with mean heights and initial 10% of MCMC trees discarded as burnin, was produced using TreeAnnotator V.1.7.5. MCC trees were visualized in FigTree v.1.4 to identify widespread species (Suppl. Fig. S1-3). Once species were identified, individual alignments were re-analyzed using the same procedure as above, but with an MCMC chain of 5 million generations, sampling every 500 states. MCC trees were observed and edited with the R package APE (Paradis et al.. 2004).
Statistical analyses
To compare the mean and standard deviation of the relative time to the most recent common ancestor (TMRCA) for eastern and western populations of selected widespread species, the treeModel.rootHeight parameter was extracted from each species log file in R v3.0.1, discarding the initial 10% of the MCMC samples. In order to estimate the amount of overlapping time between the TMRCA of each species we calculated the distribution D with the formulae: ! −!! ! = ! ! ! σ! where µ1 is the mean TMRCA of the “reference” species, µ2 is the mean TMRCA of the
“compared” species, and the σ1 standard deviation of the “reference” species. With D, the proportion of overlap p can be calculated using a normal density function P (pnorm() function in
R v3.01):
|!| ! = 2!! X ≤ 2 where X is a random variable from a normal distribution. Since the distribution of the MCMC
TMRCA estimates were not symmetrical, we estimated p on each side of the distribution for each pair of species.
! 58!
In order to account for the effect of randomness in the observed distribution of subpopulations, we performed a randomization test with 10,000 iterations. The test consisted in randomizing the distribution of all terminals into "East" and "West" and counting the number of times the observed distribution of subpopulations ho was found for each iteration. To obtain a p-value we used the following formula,
! ! = ! !!! !! !
if ri"="ho"="1 where !!"represents the number of times ho was found in iterations.
Results
COI barcode sequences (650-660 bp) were obtained for 587 individuals from 10 sites. A total of
158 species were identified using a 2% cutoff as described by Zhou et al., (2009). The best substitution model obtained for each order was the generalized time reversible (GTR, Tavaré
1986). The mean likelihood (–logL) for Ephemeroptera was –logL 21,837.6659 (SE 0.3844), for
Plecoptera –logL 11,513.13 (SE 0.3782), and for Trichoptera –logL 25300 (SE 0.5673). Eighteen species from MCC trees (Suppl. Fig. 1S-3S) had populations distributed both east and west of
Hudson Bay. Of these, only 7 species (Fig. 2) exhibited both geographic and genetic structure:
Ephemeroptera (Acentrella feropagus, Ameletus inopinatus, Baetis tricaudatus and Ephemerella aurivillii), Plecoptera (Arcynopteryx compacta) and Trichoptera (Ceratopsyche alternans and
Onocosmoecus unicolor). Nine other species exhibited marked genetic divergence, but without clear geographical structure: Ephemeroptera (Heptagenia pulla, Epeorus vitreus, Leptophlebia cupida, Baetis hudsonicus, Baetis brunneicolor) (Suppl. Fig. S1), Plecoptera (Nemoura arctica,
Isoperla transmarina) (Suppl. Fig. S2) and Trichoptera (Limnephilus picturatus) (Suppl. Fig. S3).
Finally, 3 other widely distributed species — all trichopterans — exhibited no clear genetic or
! 59! geographical structure (Asynarchus lapponicus, Anabolia bimaculata, Grensia praeterita)
(Suppl. Fig. S3).
After re-analysis, substitution model and mean likelihood was obtained for each of the seven species that exhibited distinct genetic and geographical structure. GTR model was obtained for A. inopinatus (–logL 1071.1 SE 0.102), C. alternans (–logL 869.4 SE 0.106), E. aurivillii (–logL
975.3 SE 0.084), and O. unicolor (–logL 1141.1 SE 0.1639), while HYK (Hasegawa, Kishino &
Kano 1985) model fit better for B. tricaudatus (–logL 1119.5 SE 0.081), A. feropagus (–logL
887.5 SE 0.091) and A. compacta (–logL 1004.7 SE 0.077).
Posterior probability (clade support) for each subpopulation (i.e., east versus west) ranged from
0.94 to 1.0 except for two species (Ameletus inopinatus and Ephemerella aurivillii) where the western subpopulation had values of 0.62 and 0.64, respectively (Fig. 2). Interpopulation divergence percentage (measured from branch lengths connecting both subpopulations), was as follows: E. aurivillii 0.4%, Ceratopsyche alternans 0.6%, A. feropagus 1.18%, A. inopinatus
1.38%, B. tricaudatus 2.65%, A. compacta 2.82% and O. unicolor 8.66%.
Figure 3 shows the post-burnin MCMC estimates of the relative TMRCA between eastern and western populations. The mean relative TMRCA ranged from 4.613E-3 to 4.81E-2. The maximum percentage of overlap was between C. alternans and E. aurivillii, with almost 76% below and above of the mean. Most species pair combinations overlapped more than 10% below or above the mean or both, except for C. alternans and B. tricaudatus, which shared less than 9% above the mean and near zero below it. O. unicolor was exceptional in that it overlapped no more than
5% with any species (almost zero relative to C. alternans and E. aurivillii). Table 1 summarizes the mean, standard deviation, D and p of the relative TMRCA.
! 60!
Randomization test for each order tree show significant values; for Ephemeroptera p = 0.0369,
Plecoptera p = 1e-04 and Trichoptera p = 0.0425 with 10,000 iterations.
Discussion
The influence of Pleistocene glaciations on the current distribution of species and population structure in northern North America is well known; however, this is the first attempt to document the impact of glaciations on a large scale using aquatic insects as a model group of organisms.
One of the greatest impediments to undertaking a study of this nature is the difficulty of obtaining a comparable inventory of species from widely distributed sites. The vastness of northern
Canada, in combination with the logistical difficulties of conducting fieldwork in remote locations, means that knowledge about the composition and distribution of most insects is lacking. Compounding the problem of access is phenology. The taxonomy of most aquatic insects is based on adults, which are typically short lived and difficult to collect at northern latitudes. In contrast, the immature stages of aquatic insects are relatively easy to collect, although difficult or impossible to identify to species-level using available keys. A library of barcode sequences developed by Zhou et al. (2009) for the EPTs of Churchill, MB, allowed us to accurately identify most of the specimens collected in this study — regardless of gender or life history stage. Accordingly, we were able to rapidly generate a comparable inventory of EPTs from each of the widely distributed sites in northern Canada. In addition to the utility of the COI barcoding gene for providing species-level identifications, it also has potential to reveal intraspecific populations structure. As such, it can provide valuable new insights into the geographical history of an ecologically important group of insects.
Of the 158 species of northern EPTs collected in this study, only 18 were found to be widely distributed among our study sites. Nonetheless, this provides a means to explore what
! 61! geographical patterns, if any, are shared among them. Recognizable clustering patterns can be observed in populations that have been geographically and reproductively isolated (Papadapoulou et al. 2008). In fact, seven species in this study were found to have genetic and geographic divergence among populations.
Acentrella feropagus was collected only on the Canadian Arctic Archipelago (i.e., Banks,
Victoria, and Baffin Islands), although this species is known to be more widely distributed from
Yukon to Newfoundland (Alba-Tercedor & McCafferty 2000). Barcode sequences of these specimens matched closely (97%) with Acentrella lapponica northern Finland (Kjaerstad et al.
2012) when BLASTed in GenBank and the BOLD v3 database. Populations of A. feropagus on
Banks and Victoria Islands formed a well-supported clade — markedly separated from the population on Baffin Island (figure 2). Given the level of intraspecific divergence, it seems likely that A. feropagus survived the Wisconsinan glaciation in two separate refugia. Beringia is the nearest source area for the populations on Banks and Victoria Islands, whereas an eastern or southeast refugium is a most likely source area for the Baffin Island population. Further sampling is needed in eastern North America to confidently identify potential source areas for the
Baffin Island population.
The mayfly Baetis tricaudatus exhibited a similar east/west population structure as found in A. feropagus. An eastern clade was formed by populations from Goose Bay, Moosonee and
Schefferville, whereas the western clade was formed by populations from Norman Wells,
Yellowknife and Kugluktuk. This species was present in most of the continental sites, and exhibited a marked divergence between eastern and western populations as shown by the MCC tree (figure 2). As with A.feropagus, it seems likely these two lineages had separate geographical histories.
! 62!
Arcynopteryx compacta was the only plecopteran exhibiting a well-supported east/west population structure in northern Canada, (figure 2). This species was encountered in Iqaluit in the east, and in Kugluktuk and the Ogilvie Mountains in the west. As the latter site is situated in what once was once East Beringia, it is reasonable to propose such an origin for western populations.
The intraspecific distance between eastern and west populations was 2.8%, which is higher than the 2% threshold of Zhou et al. (2009) to define species. However, Alexander et al. (2009) found a mean within-lineage distance of 2.9 ± 0.7% for several species of widely distributed mayflies of the genus Ephemerella. In the absence of supporting morphological or ecological differences, it is prudent to consider A. compacta a single species despite marked intraspecific genetic divergence between eastern and western populations.
Ceratopsyche alternans — a trichopteran — was found to have well-supported divergence between eastern and western populations (figure 2). Specimens east of Hudson Bay were collected only in Schefferville, whereas specimens west of Hudson Bay were collected in
Yellowknife and Churchill. Intraspecific distance between these populations was 0.67%.
Onocosmoecus unicolor — another trichopteran — exhibited similar geographical divergence between eastern and western populations as described for C. alternans. However, the genetic distance in this instance was a remarkable 8.6% between populations, strongly suggesting that they represent separate species. Onocosmoecus unicolor has had a checkered taxonomic history, with 6 junior synonyms currently recognized (Rasmussen and Morse 2014). One of the junior synonyms — Onocosmoecus quadrinotatus (Banks 1908) — has its type locality in
Newfoundland (Grand Lake), so it seems likely this name applies validly to the eastern population sampled in the present study. Given that the divergence is so much deeper than between the eastern- and western populations of other EPTs in this study, we speculate that the common ancestor diverged during an earlier (i.e., pre-Wisconsinan) glacial cycle.
! 63!
The mayflies Ephemerella aurivillii and Ameletus inopinatus both exhibited genetic and geographical structure, although with lower support for the western population (posterior probability 0.6 and 0.62, respectively). Although having the lowest posterior probability of the seven widely distributed species included in our study, E. aurivillii nonetheless exhibits subtle genetic and geographic structure. The pattern is similar in A. inopinatus, except that there is a second well-supported divergence within its western branch, resulting in two distinct subpopulations. As western material for our analysis came from sites that were both unglaciated
(i.e., Ogilvie Mountains) and glaciated (i.e., Norman Wells, Yellowknife), it is possible that a higher level of genetic diversity was generated by emigrants from Beringia (c.f., Bernatchez &
Wilson 1998, Gomez & Lunt 2007). However, we cannot rule out the possibility that the two western subpopulations originated from two different refugia, as reported for other organisms
(Fedorov & Stenseth 2002; Shafer et al. 2010).
We used coalescent models to analyze our sequences, which allowed us to observe the geographical distribution of genetic variation accumulated during a population’s history. These models are more powerful than “traditional” models, as they are less sensitive to stochasticity of genetic processes (e.g. genetic drift (Richards et al. 2007, Hudson & Turelli 2003), thus further supporting our results.
We detected another 12 species displaying genetic structure through sequence divergence and high clade posterior probability (Fig. S1-S3), similarly to the taxa discussed in detail here (Fig.
2). However, these genetic clusters were not consistent with geographical patterns of isolation.
This might suggest that, although other species of aquatic insects were geographically isolated during the same time period, a higher rate of dispersal may obscure identification of likely refugial areas and postglacial migratory routes.
! 64!
The DNA barcode gene proved useful not only for the broad scale sampling of Ephemeroptera,
Plecoptera and Trichoptera in north Canada, it also provided insights in the geographical history of a little-studied biota. As such, it is a useful tool to for revealing large-scale biogeographical patterns of aquatic insects.
Conclusions
This study used a DNA approach to conduct a rapid and wide-scale inventory of the aquatic insect orders Ephemeroptera, Plecoptera and Trichoptera in northern Canada. More detailed analyses of the same gene were used to examine population structure among widely distributed species. Of 158 species identified in this study, eighteen were found to be transcontinental at northern latitudes. A Bayesian coalescent model revealed genetic divergence and geographical structure in seven of these species, with distinct populations situated on either side of Hudson
Bay. This suggests that the postglacial history of northern Canada is complex, with Beringia the most likely refugial area for EPTs that recolonized northern Canada west of Hudson Bay, and with one or more eastern refugia the likely source area(s) for EPTs that recolonized the territory east of Hudson Bay. Nonetheless, no clear patterns were observed for the majority of species examined, and while discovery of a recurrent pattern among several, unrelated, species seems suggestive, the sampling of additional populations and analyses of other genes are needed to untangle the postglacial colonization of aquatic insects in northern Canada.
! 65!
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! 71! TABLES
Table 1. Comparison of relative time to the most common recent ancestor for species pairs from all trees. Mean (µ), variance (σ2), distribution (D), percentage of overlap (p). Acronyms: Ac = Acentrella feropagus, Ai = Ameletus inopinatus, Bt = Baetis tricaudatus, Ea = Ephemerella aurivillii, Ac = Arcynopteryx compacta, Ca = Ceratopsyche alternans and Ou = Onocosmoecus unicolor. Significance: ****>75%, ***>50%, **>25%, *>10%.
Species 1 Species 2 µ σ D1 D2 p1 Overlap p2 Overlap significance significance Ca Ea 0.003696 0.001572 -0.611648 0.611305 0.759739 **** 0.759869 ****
Ac Bt 0.015416 0.003598 -0.917862 0.748097 0.646284 *** 0.708368 ***
Ai Ac 0.011752 0.002576 -1.422038 1.018173 0.477073 ** 0.610692 ***
Ea Af 0.004657 0.002349 -1.570306 1.051372 0.432364 ** 0.599107 ***
Ca Af -2.182837 1.460660 0.275089 ** 0.465188 **
Ai Bt -2.703977 1.577951 0.176379 * 0.430126 **
Af Ai 0.007127 0.002349 -1.968771 1.795392 0.324926 ** 0.369348 **
Af Ac -3.528134 2.303665 0.077721 - 0.249390 **
Af Bt -4.933869 2.625681 0.013628 - 0.189237 *
Ea Ai -4.510820 2.754174 0.024108 - 0.168485 *
Ea Ac -6.839852 2.990148 0.000626 - 0.134895 *
Ca Ai -5.125003 3.127419 0.010392 - 0.117886 *
Ea Bt -8.939426 3.185195 0.000008 - 0.111251 *
Ca Ac -7.455343 3.257390 0.000193 - 0.103378 *
Ca Bt -9.556098 3.403008 0.000002 - 0.088848 -
Bt Ou 0.018718 0.004414 -5.392718 3.983453 0.007010 - 0.046401 -
Ac Ou -7.534348 4.536051 0.000165 - 0.023328 -
72 Table 1 (continued). Comparison of relative time to the most common recent ancestor for species pairs from all trees. Mean (µ), variance (σ2), distribution (D), percentage of overlap (p). Acronyms: Ac = Acentrella feropagus, Ai = Ameletus inopinatus, Bt = Baetis tricaudatus, Ea = Ephemerella aurivillii, Ac = Arcynopteryx compacta, Ca = Ceratopsyche alternans and Ou = Onocosmoecus unicolor. Significance: ****>75%, ***>50%, **>25%, *>10%.
Overlap Overlap Species 1 Species 2 µ σ D1 D2 p1 p2 significance significance Ai Ou -11.944940 5.149041 0.000000 - 0.010038 -
Af Ou -15.067220 5.922971 0.000000 - 0.003062 -
Ea Ou -24.074390 6.336269 0.000000 - 0.001534 -
Ou Ca 0.042524 0.005976 6.497161 -24.699560 0.001160 - 0.000000 -
73 FIGURES
Figure 1. Collecting sites. Dark circles = eastern sites: Goose Bay (GB), Iqaluit (IQ), Lake Hazen (LH), Moosonee (MO) and Schefferville (SV). White circles = western sites: Banks Island (BI), Churchill (CH), Cambridge Bay (CB), Kugluktuk (KT), Norman Wells (NW), Ogilvie Mountains (OM) and Yellowknife (YK).
74
Figure 2. Bayesian MMC summarized trees of 7 widely distributed EPTs with distinct eastern and western populations in northern Canada. Number above each branch indicates the posterior probability. Number below the branch indicates the genetic distance. White triangles are the collapsed branches of eastern populations; grey triangles are the collapsed branches of western populations.
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0.06 0.04 0.02 Relative time to of the MRCA East-West populations Relative 0.00
Ca Ea Af Ai Ac Bt Ou
Figure 3. Relative time to the MRCA of eastern and western populations of seven widely distributed EPTs in northern Canada. Acronyms: Ca = Ceratopsyche alternans, Ea = Ephemerella aurivillii, Af = Acentrella feropagus, Ai = Ameletus inopinatus, Ac = Arcynopteryx compacta, Bt = Baetis tricaudatus and Ou = Onocosmoecus unicolor. .
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Figure 1S. Maximum Credibility Clade Bayesian tree for Ephemeroptera. Shadowed taxa exhibit both genetic and geographic structure. Black bar indicates species that show genetic divergence but no geographical structure.
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Figure 2S. Maximum Credibility Clade Bayesian tree for Plecoptera. Shadowed taxa exhibit both genetic and geographic structure. Black bar indicates species that show genetic divergence but no geographical structure.
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Figure 3S. Maximum Credibility Clade Bayesian tree for Trichoptera. Shadowed taxa exhibit both genetic and geographic structure. Black bar indicates species that show genetic divergence but no geographical structure.
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Chapter 4
General conclusions
Environmental conditions in northern Canada are changing rapidly due to the influence of climate change. Large-scale studies are needed to assess the potential impact of global warming on the biota. In order to achieve this objective is necessary to identify the current distribution of northern species, to document community composition in different ecosystems, and to understand the influence of environmental variables and geographic history in shaping the contemporary biota.
The Northern Biodiversity Program (NBP) — a collaborative research program initiated by researchers from the University of Toronto and McGill University — was established in 2010 to develop a baseline of arthropods in northern Canada. My role in the project was to study aquatic insects of the orders Ephemeroptera, Plecoptera and Trichoptera (the EPTs). EPTs are common constituents of benthic macroinvertebrate communities, and they are well known to attain their highest species richness in high-quality aquatic habitats. As such, they are excellent model organisms for assessing environmental change. The main objectives of my research were to (1) identify patterns of EPT community composition in the Boreal, Subarctic, and Arctic ecoclimatic zones of northern Canada and (2) seek evidence of the role of historical events (e.g., Pleistocene glaciations) in shaping the present day EPT fauna. This baseline will provide a sounder basis from which to interpret future climate-induced changes. My use of a DNA barcoding approach allowed me to identify EPTs to species level regardless of gender or stage of development.
Accordingly, I was able assemble a comparable database of species at each of 12 widely distributed collecting sites in northern Canada. As a mitochondrial marker, the barcoding gene
! 80! also provided a profile of each species population structure, which it turn has potential to shed light on the geographical history of northern EPTs.
Chapter two addressed the first thesis objective; i.e., identifying patterns of community composition among EPTs in the Boreal, Subarctic and Arctic ecoclimatic zones of northern
Canada. That chapter also explored the influence of environmental and geographical variables using descriptive and ordination methods. Results showed that the Arctic ecoclimatic zone supported an EPT community that was markedly different from that farther south. More specifically, most of the species in the Arctic occurred exclusively in that zone, with few of them also occurring in the Subarctic and Boreal zones. Low precipitation and a lower range of annual temperature (i.e., the magnitude between the maximum temperature of the warmest month and the minimum temperature of the coldest month) were the key drivers of this pattern. No substantial differences were observed between communities in the Boreal and Subarctic ecoclimatic zones. However, EPT communities from those ecoclimatic zones were found to differ on either side of Hudson Bay. Environmental and geographical variables together explained more than 50% of the total variance in community composition, with environmental variables having relatively more influence in shaping EPT community composition in northern
Canada.
In Chapter 3 I explored the basis for the geographical pattern observed in Chapter 2; i.e., the different EPT communities on either side of Hudson Bay. Mitochondrial COI barcode sequences were used to build trees for widely distributed species of each order using a coalescent model and Bayesian inference. Marked molecular divergence was found between eastern and western populations in just seven of 158 EPT species examined. The relative time of most recent common ancestor (TMRCA) was calculated for each species using a strict molecular clock. Six species exhibited a similar relative TMRCA, suggesting that intraspecific divergence occurred ! 81! during a similar period — possibly during the last (Wisconsinan) glacial cycle. Populations east of Hudson Bay likely survived the Wisconsinan glaciation in southern-east refugia (i.e.
Appalachian or coastal plain), whereas populations west of Hudson Bay probably survived the
Wisconsinan glaciation in western or northwest refugia (i.e.. Pacific Northwest or Beringia).
Although most other species exhibit no clear geographical structure in their DNA, it is possible that the marked east/west differences in population structure reflect different source areas for the
EPTs that repopulated Canada’s north following deglactiation.
In summary, my results showed that the geographical pattern identified in my community composition analyses was also reflected in the population structure of seven widely distributed species. This suggests that events during the Pleistocene Epoch had a marked influence both on community composition and population structure of northern EPTs.
This large-scale analysis of EPT diversity in northern Canada — together with my compilation of a more comprehensive DNA sequence library —provides a sounder basis from which to launch future studies. The barcoding approach was instrumental not only assembling an enormous database over a wide geographical area, but also provides insights about the geographical history of northern EPTs.
Given the remoteness and enormous geographical scale of northern Canada; however, it is important that additional sites be sampled. The 158 species identified in this study probably include most of the common EPTs in northern Canada, but it seems likely that additional species will be discovered with finer-scale sampling. Once a more comprehensive database of northern
EPTs has been generated, the application of ecological niche modeling and other approaches has potential to greatly accelerate knowledge about this ecologically important group of insects.
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Finally, the dataset generated here is a necessary first step for monitoring the future influence of climate change on northern aquatic communities.
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