Utility of the Cytochrome Oxidase I (COI) for Species ... · Utility of the Cytochrome Oxidase I...

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Utility of the Cytochrome Oxidase I (COI) for Species Identification and Phylogeographic Analysis in Black Flies (Diptera: Simuliidae) by Julio Martin Rivera Castillo A thesis submitted in conformity with the requirements for the degree of Masters in Sciences Department of Ecology and Evolutionary Biology University of Toronto © Copyright by Julio Martin Rivera Castillo 2008

Transcript of Utility of the Cytochrome Oxidase I (COI) for Species ... · Utility of the Cytochrome Oxidase I...

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Utility of the Cytochrome Oxidase I (COI) for Species Identification and Phylogeographic Analysis in Black Flies

(Diptera: Simuliidae)

by

Julio Martin Rivera Castillo

A thesis submitted in conformity with the requirements for the degree of Masters in Sciences

Department of Ecology and Evolutionary Biology University of Toronto

© Copyright by Julio Martin Rivera Castillo 2008

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Utility of the Cytochrome Oxidase I (COI) for Species

Identification and Phylogeographic Analysis in Black Flies

(Diptera: Simuliidae)

Julio Martin Rivera Castillo

Masters in Sciences

Department of Ecology and Evolutionary Biology University of Toronto

2008

Abstract

A short sequence of ca. 658-bp of the mitochondrial gene COI was used to investigate its utility

as a DNA barcode in the medically important Simuliidae or black flies. Sixty-five species and

species complexes were tested. Results indicate that the barcoding gene correctly discriminated

among morphologically distinct species with nearly 100% of efficacy and also proved useful for

revealing cryptic diversity. The DNA barcoding gene was also tested for revealing

phylogeographic patterns in the western cordilleran Prosimulium travisi and the Prosimulium

neomacropyga species-group. Phylogeographic analyses on these species revealed the areas that

acted as glacial refugia, postglacial history, cryptic speciation episodes and timing of the events

that lead to their present-day distribution. The results obtained largely concur with other

phylogeographic studies on similarly-distributed cordilleran organisms. In conclusion, the

barcoding gene not only resulted useful for species discrimination in black flies but also for

studies at the population level, providing value-added to this molecular marker.

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Acknowledgments This research would not have been possible without the support of several individuals and

institutions. I would like to express my deep gratitude to my supervisor Douglas C. Currie, for

giving me the opportunity of working under his wing, for his constant encouragement and

extensive academic and personal support. I also wish to thanks Alina Cywinska for her assistance

with the lab work and for providing specimens of Prosimulium travisi from southern British

Columbia and Peter Adler (Clemson University) for his helpful advice and background

information about P. travisi, as well as for providing individuals of this species. Some specimens

of P. travisi from southern British Columbia were also provided by John Swan. Deborah Finn

(Oregon State University) made available critical material of Prosimulium neomacropyga from

Colorado. Brad Hubley (Royal Ontario Museum, Department of Natural History), Gerry Shields

and Judith Pickens (Carrol College, Montana) and Karen Needham (University of British

Columbia, Department of Zoology) provided valuable technical and logistic support during field

work. The staff at the Laboratory of Molecular Systematics (LMS) of the Royal Ontario

Museum, Amy Lathrop and Kristen Choffe, provided extensive advice and helpful technical

support during lab work. Similarly, Ximena Velez-Zuazo (Departamento de Biología,

Universidad de Puerto Rico-Río Piedras) provided assistance with population genetic theory and

software procedures. I am also indebted to Allan Baker, Katherine Balpataky, Pedro Bernardo,

Oliver Haddrath, Kay Hodgins, Johan Lindell, Jean-Marc Moncalvo, Andre Ngo, Laurence

Packer, Mateus Pepinelli, Sergio Pereira, Lauren Pinault, Bronwyn Rayfield, Will Shim, Mike

Spironello (R.I.P), Erika Tavares, Aynsley Thielman and David Zanatta and for their assistance

in different phases of this study. My good friend James Gray-Donald once again provided useful

technical support during the preparation of this manuscript. I am deeply thankful to Rosemary

Gibson and Yvonne Verkuil, two angels that made my life easier and more enjoyable during the

up and downs of the summer of 2007. This research was funded by a Natural Sciences and

Engineering Research Council of Canada Discovery Grant to D.C. Currie, and through funding

to the Canadian Barcode of Life Network from Genome Canada (through the Ontario Genomics

Institute), NSERC and other sponsors listed at www.BOLNET.ca. Finally, a special thanks to

Amanda, pues esta tesis representa el final de un camino por el que nunca habría andado sin que

ella me hubiese invitado a recorrerlo.

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Table of Contents Abstract………………………………………………………....………………………….ii Acknowledgments………………………………………………….……………………...iii Table of contents…………………………………………………………………………..iv List of tables…………………………………………………………………………….....vi List of figures……………………………………………………………………………..vii 1. Chapter 1. General Introduction.………..………………………………………………1 1.1. References…………...……………………………………………………………3 2. Chapter 2. Identification of Nearctic black flies using

DNA barcodes (Diptera: Simuliidae).…………..………………………..4 2.1. Summary…………..……………………………………………………………...4 2.2. Introduction………………………………………..…………...…………………4 2.3. Materials and Methods……………………………………………………………6 2.3.1. Collection, DNA extraction and sequencing………………………..………..6 2.3.2. Data analysis…………………………………………...……………………..7 2.4. Results……………………………………………………………………..……...8 2.5. Discussion…………………………………………...………………………..…10 2.5.1. Species identification……………….……………………………………….10 2.5.2. Population structure…………………………………………………………14 2.5.3. Current limitations for DNA barcoding black flies.…………………………14 2.6. Conclusions..…………………………………………………………………….15 2.7. References…...…………………………………………………………………..17

3. Chapter 3. Phylogeography, postglacial history and cryptic speciation in

the black fly Prosimulium travisi Stone (Diptera: Simuliidae)……………32 3.1. Summary.…..……..……………………………………………………………..32 3.2. Introduction..…….………………………………………………………………33 3.3. Materials and Methods.………..……………………………………….………..35

3.3.1. Sampling.………………………………………………………………….....35 3.3.2. DNA extraction and sequencing.………………………………………...…..35 3.3.3. Phylogeographic analysis……………………………………………………36 3.3.4. Genetic diversity and demographic analysis………...……………………....37 3.4. Results....……………………………………………………………………...….37 3.4.1. Phylogeographic structure.……….…………………………………….……37 3.4.2. Haplotype network.…………...……………………………………………...39 3.4.3. Population genetics and demographic history…………………………....….40 3.5. Discussion.…………………………………………………………………….…41 3.5.1. Glacial refugia…………………………………………………………….....41

3.5.2. Phylogeographic patterns in P. travisi……….…………………………....43 3.5.3. Postglacial colonization and comparative phylogeography………………44

3.5.3.1. The Northern Clade…………………………………………..………44 3.5.3.2. The southern Clade.………...………………………………………....46

3.5.3.3. The Colorado Clade..……...………….……………………………….49 3.5.4. Distributional patterns in western black flies………………………………..50 3.6. Conclusions.……………………………………………………………..……….51

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3.7. References…....…....…………….……………….………………………..…….53 4. Chapter 4. Evolutionary history and cryptic speciation in members of the Prosimulium macropyga species-group in Western North America (Diptera: Simuliidae).………..…...………………….……70 4.1. Summary…………….…………………………………………………………..70 4.2. Introduction…..………………………………………..…………...……………71 4.3. Materials and Methods…..………………………………………………………72 4.3.1. Collection, DNA extraction and sequencing.………………………..……....72 4.3.2. Phylogenetic and network analysis..…………………….…………………..73 4.3.3. Population genetics and time divergence estimates...……………………….74 4.4. Results……………………………………………………………………..…….75 4.4.1. Phylogeography...………………………………...………………………..…75 4.4.2. Population genetics and time divergence estimates…………...…………….76 4.5. Discussion..................................……...…………………………………………77

4.5.1. Prosimulium neomacropyga and “Prosimulium neomacropyga”.…...……..77 4.5.2. Wyoming populations of P. neomacropyga....................................................81 4.5.3. Prosimulium ursinum………………………………………………………..81 4.5.4. Nunavut populations of P. neomacropyga…………………………………..83 4.5.5. Taxonomic implications……………………………………………………..83 4.6. Conclusions..…………………………………………………………………….84 4.7. References…....…………………………………………………………….……85

5. Chapter 5. General conclusions.………….....…………………………………………96

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List of Tables

Chapter 2

Table 1. List of black fly species examined, their geographic distribution and intraspecific levels

of genetic divergence ………………………………………………………...………………….23

Table 2. Levels of genetic divergence in species complexes………………………………….…26

Table 3. Intrageneric levels of genetic divergence…………………………………………...….26

Chapter 3

Table 1. Prosimulium travisi collecting sites and geographic distribution of unique

haplotypes……………………………………….…………………………………………….…59

Table 2. Genetic population parameters for Prosimulium travisi………………………………..60

Table 3. Pairwise Fst values for each subpopulation of Prosimulium travisi …………….…….60

Chapter 4

Table 1. Prosimulium macropyga species-group collecting sites and geographic distribution of

unique haplotypes.………………………………………………………………………….….89

Table 2. Genetic population parameters for Prosimulium macropyga species-group………...…90

Table 3. Pairwise Fst values and pairwise distances for each Prosimulium neomacropyga

subpopulation…………………………………………………………………………………….90

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List of Figures

Chapter 2

Figure 1. DNA barcoding tree of 65 black fly species and species complexes……………….…27

Figure 2. Histograms depicting intra and interspecific levels of genetic divergence……………29

Chapter 3

Figure 1. Map of the major mountain ranges of western North America………………..………61

Figure 2. Map of Prosimulium travisi collecting sites…………………………………………...62

Figure 3. Neighbor-Joining tree and haplotype network depicting the relationships among unique

haplotypes………………………………………………………………………………………..63

Figure 4. Maximum parsimony and Bayesian phylogenies depicting the relationships among the

most common haplotypes………………………………………………………………………..64

Figure 5. Mismatch distribution frequencies for each subpopulation of Prosimulium travisi…..65

Figure 6. Map showing the approximate location of the Laurentide and Cordilleran ice sheets

during the late Wisconsinan and the present-day distribution of the NR and CR clades………..66

Figure 7. Map depicting the location of glacial refugia and postglacial migratory routes………67

Chapter 4

Figure 1. Map of Prosimulium macropyga species-group collecting sites………………………91

Figure 2. Neighbor-Joining tree and haplotype network depicting the relationships among unique

haplotypes………………………………………………………………………………………..92

Figure 3. Mismatch distribution frequencies for each subpopulation of Prosimulium

neomacropyga…………………………………………………………………………………....93

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1Chapter 1

General Introduction

The introduction of DNA barcoding in 2003 (Hebert et al., 2003) generated considerable debate

in the scientific community, and at the same time captured the attention of the media and general

public. DNA barcoding entails genetically characterizing species using a short (ca. 658-bp)

fraction of the cytochrome oxidase I gene (the DNA barcode), whose sequence could potentially

be used as a reliable diagnostic taxonomic character (Hebert et al., 2003). DNA barcoding has

proved to be an effective molecular tool for species identification in many groups of organisms,

and also shows utility for revealing cryptic diversity (e.g., Hebert et al., 2004).

One of the primary goals of this thesis is to test the efficacy of the DNA barcoding gene for

species identification in black flies (Diptera: Simuliidae). The Simuliidae are a taxonomically

challenging family because of their small size, structural homogeneity and presence of

reproductively isolated (but morphologically indistinguishable) sibling species (Adler et al.,

2004). Therefore, the eventual implementation of a DNA-based identification system could be an

asset for the study of this medically important family of dipterans. An assessment of the utility of

the DNA barcoding gene for black fly identification is presented in Chapter 2.

The DNA barcoding gene has also been used successfully in other contexts, such as in forensic

sciences (Dawnay et al., 2006), ecological studies (Kuusk and Agusti, 2008) and conservation

initiatives (Ward et al., 2008), where accurate taxonomic identification is fundamental to

interpretations of biological phenomena. As a mitochondrial gene, DNA barcodes have potential

for revealing geographical structure at the population level. However, the utility of the barcoding

gene for such inferences has so far received little investigation. The usefulness of the barcoding

gene for revealing phylogeographic patterns in black flies is explored in Chapter 3 and 4.

Chapter 3 focuses on Prosimulium travisi, a widely distributed species in the western cordillera

of North America. Chapter 4 focuses on the western Nearctic members of the Prosimulium

macropyga species-group which exhibit a disjunct distribution, with northern populations in

Alaska and Yukon and southern populations in Wyoming and Colorado. Results obtained will be

compared with phylogeographic studies of similarly distributed organisms, in order to determine

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whether the barcoding gene can reveal congruent phylogeographic patterns that can be

attributable to a shared geographical history.

In summary, the primary objectives of this research are to: (a) test the efficacy of the DNA

barcoding gene for species identification in black flies and; (b) investigate the utility of the

DNA barcoding gene for phylogeographic inference. If the barcoding gene proves effective at

revealing population-level phenomena, then this would provide significant “value added”

information for widely and comprehensively sampled species.

2

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1.1. References

Adler P. H., D. C. Currie and D. M. Wood. 2004. The Black Flies (Simuliidae) of North

America. Cornell University Press, Ithaca, New York.

Dawnay, N., R. Ogden, R. McEwing, G. R. Carvalho, and R. S. Thorpe. 2007. Validation of the

barcoding gene COI for use in forensic genetic species identification. Forensic Science

International 173: 1-6.

Hebert, P. D. N., A. Cywinska, S. L. Ball, and J. R. deWaard. 2003. Biological identifications

through DNA barcodes. Proceedings of the Royal Society of London B Biological

Sciences 270: 313-321.

Hebert, P. D. N., E. H. Penton, J. M. Burns, D. H. Janzen, and W. Hallwachs. 2004. Ten species

in one: DNA barcoding reveals cryptic species in the Neotropical skipper butterfly

Astraptes fulgerator. Proceedings of the National Academy of Sciences of the United

States of America 101: 14812-14817.

Kuusk, A. K. and N. Agusti. 2008. Group-specific primers for DNA-based detection of

springtails (Hexapoda: Collembola) within predator gut contents. Molecular Ecology

Resources 8: 678–681.

Ward, R. D., B. H. Holmes, W. T. White, and P. R. Last. 2008. DNA barcoding Australasian

chondrichthyans: results and potential uses in conservation. Marine and Freshwater

Research 59: 57-71.

3

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4Chapter 2

Identification of Nearctic black flies using DNA Barcodes

(Diptera: Simuliidae)

2.1 Summary

DNA barcoding has gained increased recognition as a molecular tool for species identification in

various groups of organisms. In this study we tested the efficacy of a 615-bp fragment of the

cytochrome c oxidase I (COI) as a DNA barcode in the medically important family Simuliidae,

or black flies. A total of 65 Nearctic black fly species or species complexes were used to create a

standard barcode profile for the family. Genetic divergence among congeners averaged 14.93%

(range 2.83%-15.33%), whereas intraspecific genetic divergence between morphologically

distinct species averaged 0.72% (range 0%-3.84%). DNA barcodes correctly identified nearly

100% of the morphologically distinct species (87% of the total sampled taxa), whereas in species

complexes (13% of the sampled taxa) maximum values of divergence were comparatively higher

(max. 4.58%-6.5%), indicating cryptic diversity. The existence of sibling species in Prosimulium

travisi and Prosimulium neomacropyga was also demonstrated, thus confirming previous

cytological evidence about the existence of such cryptic diversity in these two taxa. I conclude

that DNA barcoding is an effective method for species identification and discovery of cryptic

diversity in black flies.

2.2 Introduction

Accurate taxonomic identification is a key aspect in biological research. Correct identification

not only allows critical access to the broad body of literature available on a particular taxon but

also permits the implementation of adequate measures to contend with species of medical or

agricultural importance. In contrast, misidentifications could lead to inadequate control

measures, which could potentially increase the impact caused by a particular pest species.

Studies in biodiversity, systematics, community ecology and biomonitoring also depend on

proper taxonomic identification. The accelerated rate of habitat destruction by humans has

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prompted the scientific community to accelerate the global inventory of biodiversity. However,

this increased demand for taxonomic expertise corresponds with period of decline in systematic

biology as reflected by the decreased availability of qualified taxonomists.

The proposal of Hebert et al. (2003a,b), to use a small portion (ca. 658 bp) of the mitochondrial

gene cytochrome c oxidase unit I (COI) as a DNA barcode for species identification, has

reinvigorated efforts to document global biodiversity; however, this initiative has also generated

vigorous debate in the scientific community (Moritz and Cicero, 2004; Hebert and Gregory,

2005; DeSalle et al., 2005; Ebach and Holdrege, 2005a,b; Hajibabaei et al., 2007). DNA

barcoding has proved to be an effective molecular identification system in many groups of

animals (e.g., Hogg and Hebert, 2004; Barrett and Hebert, 2005; Ball et al., 2005; Janzen et al.,

2005; Monaghan et al., 2005; Schindel and Miller, 2005; Cywinska et al., 2006; Pegg et al.,

2006; Hajibabaei et al., 2006; Kelly et al., 2007; Hinimoto et al., 2007; Kerr, 2007), gaining

recognition as a reliable taxonomic tool in certain scientific circles (Blaxter, 2004). However,

limited success in other taxa has revealed limitations of this particular portion of the COI gene to

serve as a universal DNA barcode (e.g., Kress et al., 2005; Rubinoff et al., 2006). Nonetheless,

DNA barcoding has proved to be a versatile tool with a variety of applications, for example, by

facilitating the association between different developmental stages in insects (Ahrens et al.,

2007). The approach has also proved to be an effective auxiliary tool in the forensic sciences

(Dawnay et al., 2006), in studies on feeding ecology (Bourlat et al., 2008; Garros et al., 2008;

Kuusk and Agusti, 2008) and habitat conservation initiatives (Neigel et al., 2007; Ward et al.,

2008), among other applications. But perhaps most importantly, DNA barcoding has proved to

be especially useful in the study of taxonomically challenging taxa, where morphology-based

identifications are frustrated due to cryptic diversity (Hebert et al., 2004; Smith et al., 2006;

Quicke et al., 2006; Witt et al., 2006; Yassin, et al., 2008), or phenotypic plasticity (Derry et al.,

2003; Adamowicz et al., 2004).

Black flies (Diptera: Simuliidae) are notorious for the hematophagous habits of the adult females

of most species. Besides constituting a nuisance for humans and domestic and wild animals,

black flies are known to be vectors of diseases such as avian leucocytozoonosis, bovine

onchocerciasis and vesicular stomatitis virus in lifestock (Crosskey, 1973; Adler et al., 2004). In

tropical areas, anthropophilic species are implicated in the transmission of masonelliasis (a

5

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filarial infection) and onchocerciasis or “river blindness”, the world's second leading infectious

cause of blindness (Etya’alé, 2001, 2002). The immature stages of black flies live in a wide

variety of running water habitats, but typically require clean, unpolluted water. Due to their small

size (typically between 2-4 mm) and structural homogeneity, black flies have proven to be

taxonomically difficult. Accurate identification typically requires the analysis of large series of

larvae, pupae and adults of both sexes, including micro-dissection of genitalia and slide

preparations. In addition, when the presence of morphologically indistinguishable sibling species

is suspected, analysis of the larva’s polytene chromosomes provides the only means to accurately

identify species. The study of polytene chromosomes, or “cytotaxonomy”, has played a central

role in the taxonomy and systematics of the Simuliidae, and cytogenetic studies have been

critical for revealing the presence of sibling species in many morphologically defined nominal

species. Chromosomal rearrangements (typically inversions) and sex chromosomal and

autosomal polymorphisms are used to diagnose sibling species, which are particularly abundant

within the family (Adler et al., 2004). Many species of black flies are also the subject of pest

management programs and the larvae are important bioindicators in fresh water biomonitoring

program. Accurate species-level identification is required for all such programs to be successful.

Black flies provide an excellent example where species-level identification can be enhanced by

implementation of a DNA-based identification system. The inherent difficulties with

morphological and chromosomal identifications, with a concomitant shortage of qualified

taxonomists and cytogenists, underscore the need for another approach. In this study I present a

preliminary assessment of the utility of DNA Barcoding to discriminate among Nearctic black

fly species.

2.3 Materials and Methods

2.3.1 Collection, DNA extraction and sequencing

Larvae, pupae and adult black flies were collected at various localities throughout Canada and

the USA in 2005 and 2006 (Table 1). Collected individuals were fixed in 95% ethanol and were

maintained at a low temperature (≈5° C) until taken to the lab for identification and molecular

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analysis. Specimens were identified using the keys in Adler et al. (2004). Larvae selected for

molecular analysis had their digestive tract removed to reduce the prospect of contamination;

specimens were then cut in half and the posterior halves (abdomen) were used for DNA

extraction whereas the thorax and head (where most taxonomic features reside) were retained as

vouchers. These latter were deposited in the entomological collection of the Royal Ontario

Museum. When pupae and adults were selected for analysis, the abdomen and individual legs

were used respectively for DNA extraction. All instruments used for dissection were sterilized by

flame between specimen dissections in order to prevent the transfer of DNA from one sample to

another. Approximately 30 μl of total DNA was extracted using a GeneEluteTM Mammalian

Genomic DNA Miniprep Kit. Extracting protocol followed manufacturer specifications. PCR

primers for amplifying the ca. 658 bp long target region of the COI gene were those used in

DNA barcoding (Hebert et al., 2003): LCO1490 (5’-GGTCAACAAATCATAAAGATATTGG-

3’) and HCO2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’). Polymerase chain

reactions (PCR) were conducted using 1μl of template DNA in a total reaction volume of 25μl.

The PCR reaction mix contained 1μl of each primer, 1.5 μl of MgCl, 2.5 μl of PCR buffer, 0.8 μl

of dinucleotide triphosphates (dNTP’s), 0.1 μl of taq DNA polymerase. PCR conditions were an

initial 1 min. at 96 º C (denaturation) followed by 1 min. at 94º C (denaturation), 1 min. at 55 º C

(primer annealing) and 1.5 min. at 72 º C (amplification) for 35 cycles, and 7 min. at 72 º C.

Purified template DNA was sequenced with an ABI377 or 3730 automated sequencer.

2.3.2 Data analysis

Electropherograms were edited and aligned using Sequencher V. 4.5. Sequences were trimmed

to a final length of 615 bp. A Neighbour-Joining analysis using the software package PAUP*

4.0b10 (Swofford, 2003) was conducted to examine relationships among taxa. A total of 693

specimens representing 65 nominal species or species complexes and 10 genera were analyzed (a

taxonomic list of the studied taxa is presented in Table 1). Pairwise distances (Table 1) were

calculated within and between genera and species using the totality of the specimens. However,

only 463 individuals were used to create the standard DNA barcode profile tree presented in

Figure 1 (see below). The number of specimens used to build the DNA profile varied according

to their availability and geographic distribution. Species represented by >2 specimens or with a

minimum of 1 nucleotide substitution, were subjected to pairwise nucleotide sequence

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divergence calculations using the Kimura-2 parameter (K2P) model, because this model provides

the best metric when genetic distances are low, as in closely related species (Nei and Kumar,

2000). Given the methodological constraints posed by the identification of sibling species,

species complexes are considered in their broad sense. For example, although the Simulium

tuberosum complex includes 10 reproductively isolated sibling species in North America,

individuals were all referred to as “Simulium tuberosum complex”. The species complexes

included in this study are: Helodon (Helodon) onychodactylus complex, Stegopterna mutata

complex, Simulium (Simulium) arcticum complex, Simulium (Simulium) tuberosum complex and

Simulium (Simulium) venustum complex. Pairwise distances in species complexes were

calculated separately. This approach helps to evaluate the levels of genetic divergence within

each species complex, thereby testing cytological evidence that implies a high degree of cryptic

diversity. Such an approach prevents overestimating the level of intraspecific genetic divergence

by underestimating the actual diversity within each species complex (as constituent siblings were

not a priori identified). Similarly, 3 additional taxa, each suspected of being a species complex,

were included (Adler et al., 2004): Simulium (Nevermannia) craigi, Simulium (Nevermannia)

quebecense and Simulium (Simulium) murmanum. In addition, we included populations of

Prosimulium travisi and Prosimulium neomacropyga from Colorado. Previous cytological

research (Adler et al., 2004) suggested that these populations may constitute reproductively

isolated sibling species. The present study offers an opportunity to test the hypothesis of specific

distinctiveness by measuring the level of genetic divergence among populations assigned to these

two species. Exemplars of both morphologically distinct species and sibling species are

represented in the NJ tree; however, only a few exemplars of the latter were included in order to

establish their relative position on the tree (this explains the lower number of individuals used to

build the DNA barcode profile tree compared to those used to estimate divergence values). Two

mosquito species (Diptera: Culicidae), Aedes canadensis and Culex pipiens, were used as

outgroups.

2.4 Results

The COI sequences from the sampled species showed a strong A+T bias in nucleotide content

(mean=0.6432) relative to the C+G content (mean=0.3569), as is typical of arthropods (Crease,

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1999). Individual mean nucleotide content was: A= 0.2748, G= 0.1668, C= 0.1901 and

T=0.3684.

The following genera and subgenera were barcoded using COI sequences (Figure 1):

Parasimulium, Prosimulium, Helodon (subgenera Parahelodon and Helodon s. s.), Gymnopais,

Twinnia, Cnephia, Greniera, Metacnephia, Stegopterna and Simulium (subgenera Hellichiella,

Nevermannia, Eusimulium, Schoenbaueria, Boreosimulium, Psilopelmia, Hemicnetha, Psilozia,

Aspathia and Simulium s. s.). This represents 75% of the North American simuliid genera (10 of

13) and 90% of the subgenera (10 of 11) within the highly diverse genus Simulium s. l. Levels of

sequence divergence were variable across the taxa sampled. Thus, conspecific individuals

collected from a single site exhibited zero or higher divergence values, whereas individuals

collected from numerous and geographically distant locations might also exhibit no variability.

Mean intraspecific genetic divergence for the 58 evaluated morphologically distinct species (i.e.,

members of species complexes were not included) was 0.76%. The maximum intraspecific

divergence value (3.84%) was observed in Simulium (Simulium) rostratum, followed by

Simulium (Nevermannia) silvestre (3.51%, mean=1.45%) and Prosimulium travisi (3.36%,

mean=1.76%) (Table 2). Higher levels of divergence were found among members of

cytologically defined sibling complexes: Helodon (Helodon) onychodactylus complex (max.

4.91%), Stegopterna mutata complex (max. 5.47%), Simulium (Simulium) arcticum complex

(max. 6.5%), Simulium (Simulium) tuberosum complex (max. 5.44%) and Simulium (Simulium)

venustum complex (max. 5.31%) (Table 2). Putative species complexes also exhibit higher levels

of intraspecific divergence as in Simulium (Nevermannia) craigi (max. 4.93%), Simulium

(Nevermannia) quebecense (max. 5.79%) and Simulium (Simulium) murmanum (max. 4.58%).

Along similar lines, specimens identified as Simulium (Hellichiella) nebulosum also exhibit a

high intraspecific genetic divergence (max. 4.58%) (Table 3); however, it is possible that our

sample also included members of Simulium (Hellichiella) minus. These two species are

indistinguishable in the immature stages, and are both sympatric in the Sierra Nevada of

California, from where some of the studied specimens were obtained. Accordingly, the high level

of divergence exhibit by Simulium (Hellichiella) nebulosum might be the result of a mixed

sample.

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Individuals of the same species grouped together, even when samples were obtained from

geographically disparate areas. Similarly, members of the same genus or subgenus tended to

group together, although this did not hold true in all instances. Members of the subfamily

Parasimuliinae (represented in this study by Parasimulium crosskeyi), are considered to be the

most plesiomorphic lineage of simuliid (Adler et al., 2004). Exemplars of this divergent lineage

were placed as sister-group of the Simuliinae genus Cnephia (represented in this study by C.

dacotensis), which together formed the sister group of all other Simuliidae. Within this latter

lineage, members of the tribe Prosimuliini (Twinnia, Gymnopais, Helodon and Prosimulium)

formed a monophyletic clade, except for the inclusion of Simulium (Eusimulium) bracteatum (a

member of the tribe Simuliini). Although not all genera within the Prosimuliini necessarily

formed monophyletic clades, monophyly was evident at the subgeneric level in Helodon

(subgenera Parahelodon and Helodon) and species-group level in Prosimulium (hirtipes,

macropyga and magnum species groups). The remaining Simulinii clustered in different parts of

the tree, with Greniera, Stegopterna, Metacnephia, Simulium (Boreosimulium) Simulium

(Aspathia), and Simulium (Simulium) all forming monophyletic clades. In contrast, Simulium

(Hellichiella) and Simulium (Nevermannia), although exhibiting a certain degree of cohesion,

each formed two different groups in different parts of the tree. This apparent unordered

clustering pattern has also been observed in other taxa (e.g., Ball et. al, 2005; Cywinska et. al.,

2006) and does not invalidate the effectiveness of the NJ algorithm, as the goal of DNA

barcoding is to provide species identification based on similarity of sequences rather than to

reconstruct deep phylogenetic relationships. At the generic level, average DNA divergence

varied across taxa, with a mean interspecific divergence value of 14.99% (minimum and

maximum values were 2.83% and 20.02% respectively) (Table 3).

2.5 Discussion

2.5.1 Species identification

The results obtained indicate that the portion of COI used as a DNA barcode effectively

discriminates among black fly species. Mean intraspecific and interspecific divergence were

0.76% and 14.93% respectively (species complexes not included) (Figure 2). Maximum values

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of intraspecific divergence ranged from 3.36% to 3.84%. Other studies of insects and

invertebrates have shown maximum intraspecific variation ranging from 3.0-3.9% (Ball et al.,

2005; Cywinska et al., 2006; Smith et al., 2006; Carew et al., 2007). However, smaller

maximum values have been observed in other taxa, although in most cases these were

characterized by limited sampling (e.g, Hogg and Hebert, 2004; Monaghan et al., 2005; Scheffer,

2006). Data from Prosimulium travisi is of particular interest because levels of intraspecific

genetic divergence were derived from 282 additional individuals (data not shown) collected

across the entire range of the species except for populations from Colorado (see below).

Similarly, samples of Simulium (Nevermannia) silvestre and Simulium (Simulium) rostratum,

although far less numerous, encompass a wide geographic area. The level of intraspecific genetic

variation in species with large geographical distributions, and its significance for DNA

identification, has not yet been evaluated systematically in a particular species. Given the

geographical scope of Prosimulium travisi samples, it is likely that the level of intraspecific

divergence observed in this species is representative of the entire gene pool. The same might be

true for values observed in Nearctic populations of Simulium (Nevermannia) silvestre and

Simulium (Simulium) rostratum. Similar maximum values can be expected in other widespread

species of Nearctic black flies.

The values mentioned above do not include species complexes. Much of the evidence for the

existence of sibling species comes from the cytological work of K. H. Rothfels (e.g., Rothfels,

1979) and subsequent generations of simuliid cytologists. Different lines of evidence suggest that

rearrangements in sex chromosomes are a major evolutionary force, and such a mechanism may

have played a prominent role in black fly speciation (Rothfels, 1989). Although sibling species

were not identified in the present study, I expect that a high degree of genetic divergence within

a particular complex is indicative of cryptic diversity. The results confirm this prediction, as high

values of genetic divergence were observed in taxa where sibling species are known: Helodon

onychodactylus complex (max. 4.91%), Stegopterna mutata complex (max. 5.47%), Simulium

(Simulium) arcticum complex (max. 6.5%), Simulium (Simulium) tuberosum complex (max.

5.44%), Simulium (Simulium) venustum complex (max. 5.31%). Maximum values of genetic

divergence in these species complexes were well above those observed in Simulium

(Nevermannia) silvestre, Simulium (Simulium) rostratum and Prosimulium travisi s.s. (i.e.,

3.36%-3.84%). Relatively deep divergences were observed in the branches corresponding to

11

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species complexes in the NJ tree, as in Simulium (Simulium) venustum complex, which

subdivided into two subclades, suggesting that more than one species was represented (Figure

1). Whether or not these subclades correspond to cytologically recognized species remains

unclear. The ability of this portion of the COI gene to detect cryptic diversity has been

demonstrated in a number of invertebrate and vertebrate taxa (Hebert et al., 2004; Ball et al.,

2005; Smith et al, 2005; Birky, 2007; Clare et al, 2007) but this capacity has also been

challenged by some authors (see Meyer and Paulay, 2005; Meier et al., 2006). It appears that this

particular attribute of COI varies across taxa. A possible example of the discovery of a

previously unknown sibling species can be seen in the NJ tree for the Stegopterna mutata

complex, which currently includes two nominal sibling species: St. mutata and St. diplomutata.

Stegopterna mutata is a triploid (presumably autotriploid), parthenogenetic species that consist

only of females in the adult stage, whereas P. diplomutata is a diploid sexual species (Currie and

Hunter, 2003). In the absence of males, the two species can be distinguished only through

examination of the polytene chromosomes of larvae. Stegopterna mutata and St. diplomutata

both occur sympatrically in Algonquin Park, Ontario, arguably the most intensively studied area

in the world for black flies. Although the presence of two cytological entities in Algonquin Park

has long been recognized (e.g., Basrur and Rothfels, 1959), the presence of 3 deeply divergent

branches suggests that a third species might be represented in Algonquin Park. Intriguingly,

Currie and Hunter (2003) suggested that St. mutata was the product of hybridization between St.

diplomutata and a yet unidentified diploid species of Stegopterna. Whether one of the divergent

branches for the St. mutata complex from Algonquin Park represents this unknown diploid

species requires cytological confirmation. Nonetheless, DNA barcoding strongly suggests the

presence of 3 species of Stegopterna in Algonquin Park where only two such species were

known previously.

High genetic divergence values were found in Simulium (Nevermannia) craigi (max. 4.93%),

Simulium (Nevermannia) quebecense (max. 5.79%) and Simulium (Simulium) murmanum (max.

4.58%). All of these entities are suspected to be complexes consisting of an indeterminate

number of sibling species (Adler et al., 2004) and the high level of genetic divergence might be

indicative of cryptic diversity. Another instance of the ability of COI to detect cryptic diversity is

exemplified by Prosimulium travisi. This species is widely distributed throughout the mountains

of western North America, ranging from Alaska and the Yukon Territories south to California,

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Arizona and New Mexico. Previous cytological work by Barsur (1962) and Adler et al. (2004)

suggested that Prosimulium travisi may actually represent a complex of two sibling species, with

isolated populations from the highlands of northern Colorado representing a separate entity. But

because the Colorado population is isolated from the more widely distributed cytological form,

and because chromosomal differences in allopatry cannot be evaluated, the Colorado form has

until now been considered to be only a chromosomal variant (i.e., a “cytotype”) of Prosimulium

travisi (Adler et al., 2004) However, the genetic divergence observed between the

geographically isolated population of Prosimulium travisi in Colorado (= Prosimulium travisi 2)

and those from elsewhere (= Prosimulium travisi 1) ranged between 6.2% and 8.8%, suggesting

that the Colorado population indeed constitutes a separate species. A similar situation was found

in Prosimulium neomacropyga, where chromosomal differences are also known to exist between

Colorado populations (= Prosimulium neomacropyga 2) and their disjunct counterparts in Alaska

and Yukon (= Prosimulium neomacropyga 1). Adler et al. (2004) considered these differences to

represent polymorphisms of the same species. However, the level of genetic divergence between

these allopatric populations was found to be between 5.4% and 7.9%, suggesting two separate

entities. Despite the genetic distinctiveness of Colorado populations of both Prosimulium travisi

and Prosimulium neomacropyga, neither entity is morphology distinguishable from their more

widely distributed sister-species.

Unexpected “relationships” among species were identified in two instances. The first is

represented by the sister-species relationship between Prosimulium frohnei and the Colorado

population of Prosimulium travisi (= Prosimulium travisi 2), as opposed to between the latter

population and Prosimulium travisi 1. Although closely related, Prosimulium frohnei and

Prosimulium travisi (both within the Prosimulium hirtipes species-group) are easily

distinguishable morphologically. A similar situation was found between Prosimulium formosum

and the more widespread Prosimulium travisi sibling (= Prosimulium travisi 1). In this case,

Prosimulium formosum grouped with Prosimulium travisi 1, clustering specifically within

populations from southern Alberta (data not shown in Figure 1). In this instance, very little

genetic divergence was observed between this pair of species. This situation possibly indicates

transfer of mtDNA elements through hybridization between these two species. Both species are

included in the Prosimulium hirtipes species-group but are easily distinguished morphologically.

This presumed hybridization might compromise the ability of COI to discriminate among species

13

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pairs. Further studies, including additional members of the Prosimulium hirtipes species-group,

will be needed to uncover potential limitations of the COI gene for recovering species identity in

this speciose lineage. Incomplete sampling is known to compromise the performance of COI in

other organisms (Meyer and Paulay, 2005) and more samples are needed to more fully

understand the limitations, if any, of the barcoding gene for black fly identification.

2.5.2 Population structure

The portion of the COI gene proposed for DNA barcoding (Hebert et al., 2003) has potential for

revealing population structure in species sampled throughout their entire range. In Prosimulium

travisi, for example, interpretation of phylogeographical patterns in the context of the

Wisconsinan glaciation provided evidence of glacial refugia and post-glacial migratory routes

(see Chapter 3). These patterns were highly congruent with those exhibited by similarly

distributed cordilleran animal and plant taxa (e.g., Wheeler and Guries, 1982; Thorgaard, 1983;

Soltis et al., 1997; Comes and Kadereit, 1998; Demboski et al., 1999; Byun et al., 1999;

Brunsfeld, et al., 2001; Nice et al., 2005; Albach et al., 2006; Maroja et al., 2007, to name only a

few). The historical biogeography of Nearctic black flies has been little studied, owing largely to

a dearth of fossils. I anticipate that the barcoding gene has potential to shed important new light

on the historical biogeography of black flies and other soft-bodied organisms, as already

documented for the Ephemeroptera (Ball et al., 2005) and rotifers (Gómez, 2005). This subject is

treated in greater detail in Chapters 3 and 4.

2.5.3 Current limitations for DNA barcoding black flies.

Unveiling the genetic identity of morphologically identical sibling species is of paramount

importance in black fly taxonomy. Several logistic and methodological limitations must be

overcome in order to realize the full potential of DNA barcoding for accurately discriminating

among recently diverged species. Black fly larvae are routinely collected into Carnoy’s fixative

(1 part glacial acetic acid: 3 parts 95% ethanol), to preserve their polytene chromosomes for

cytological analysis. This has, until now, been the only means to establish sibling species

identity. Unfortunately, Carnoy’s fixative causes the DNA to fragment, rendering it unsuitable

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for molecular analysis (Koch et al., 1998). In order to obtain material suitable for both

cytogenetic and molecular analyses, I routinely collected black fly larvae in both Carnoy’s

fixative and 95% ethanol; however, this approach is problematic because more than one sibling

species is often present at a particular collecting site. Accordingly, matching a chromosomally

verified sibling species with a corresponding COI sequence can be difficult or even impossible

unless both analyses (molecular and cytological) are performed on the same individual. In other

words, it is often impossible to cytologically confirm the identity of specimens used for DNA

analysis. Thus, I recommend that in the future a single individual must be sectioned into three

parts soon after collection and while still alive: the abdomen, where the salivary glands are

located, must be preserved in Carnoy’s fixative for cytological analysis; the thorax must be

preserved in ethanol for molecular analysis; and the head, where most taxonomic characters

needed for morphological identification are found, should be preserved in ethanol and retained as

a voucher. Unfortunately, this protocol proves to be tedious and time consuming, demanding

coordination between at least two collectors to increase precision and collecting efficiency. A

more palatable alternative would be to extract DNA directly from Carnoy’s-preserved

specimens. The denaturing properties of acids notwithstanding (Koch et al., 1998), DNA has

been isolated successfully from mosquitoes (Collins et al., 1987) and black flies fixed in

Carnoy’s solution (Pramual et al., 2005). Standardized DNA-extracting protocols for specimens

preserved in Carnoy’s fixative will certainly help to overcome the methodological constraints

described above. Once these protocols are developed, field collections of specimens will demand

no additional time or equipment than is used historically in the study of black flies. The

dissection of larvae into three parts will still be required, although in the comfort of the lab,

where correspondence between chromosomes and DNA for each individual can be more easily

assured.

2.6 Conclusions

The COI barcoding gene correctly distinguished nearly 100% of the morphologically distinct

species (which constitutes ca. 87% of the sampled taxa), thus demonstrating its utility to

discriminate among morphologically recognized black fly species. Barcoding also revealed high

levels of genetic divergence in known or suspected lineages of sibling species (i.e., the remaining

13% of the sampled taxa), suggesting that it might prove useful for distinguishing cryptic 15

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diversity. In fact, populations of “Prosimulium travisi” and “Prosimulium neomacropyga” from

highland sites in Colorado were found to be specifically distinct from more widely distributed

typical populations (as previously suggested by cytological evidence), necessitating

nomenclatural changes to the current taxonomy. Nonetheless, additional molecular approaches

(such as microsatellites) together with COI and perhaps other genes may be necessary to

overcome difficulties associated with discriminating recently diverged sibling species. Similarly,

possible homoplasy in members of the Prosimulum hirtipes species group (and perhaps in other

taxa as well) require further investigation.

As a mitochondrial gene, it is not surprising that COI carries phylogeographical signal, and this

attribute increases the value of the barcoding gene as a tool for biogeographic and population-

based studies. As COI databases become better populated with sequences from throughout the

entire range of a species, it may actually become possible to establish the provenance of

particular samples. This could have an impact on conservation efforts by enhancing the

enforcement of laws protecting endangered populations of plants and animals.

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Table 1. List of black fly species, collection sites and number of sequences used to create the standard DNA barcode profile tree shown in Figure 1. Maximum values of genetic divergence (K2P pairwise distances) correspond only to those species where 3 or more individuals were examined and had at least one nucleotide substitution. Recognized and suspected species complexes are indicated with one (*) or two (**) asterisks respectively, and their corresponding values of genetic divergence are shown in Table 2. Classification follows Adler et al. (2004).

Species Locality

No. of specimens

% Divergence (max.)

Parasimuliinae

Parasimulium crosskeyi Washington 5 0.16

Simuliinae

Prosimuliini

Gymnopais dichopticoides Yukon Territories 8 0.49

Gymnopais holopticus Alaska 2 0.65

Twinnia nova Montana 3 -

Helodon (Parahelodon) decemarticulatus Ontario

Manitoba

Norwest Terrotories

1

3

2

2.49

Helodon (Parahelodon) gibsoni Manitoba 16 1.98

Helodon (Helodon) alpestris Yukon Terrorories 1 -

Helodon (Helodon) irkutense Nunavut 3 0.16

Helodon (Helodon) onychodactylus * Yukon Territories

British Columbia

1

3

See table 2

Prosimulium (hirtipes spp. group)

Prosimulium arvum Ontario 1 1

Prosimulium frohnei Wyoming 5 1.31

Prosimulium secretum California 5 0.32

Prosimulium shewelli Wyoming 9 0.65

Prosimulium travisi 1 California

Montana

Wyoming

Alberta

British Columbia

Yukon Territories

3

1

1

4

5

1

3.36

Prosimulium travisi 2 Colorado 5 0.82

Prosimulium (macropyga spp. group)

Prosimulium neomacropyga 1 Yukon Territories 3 0.61

Prosimulium neomacropyga 2 Colorado 3 0.97

Prosimulium ursinum Yukon Territories 3 0.48

Prosimulium (magnum spp. group)

Prosimulium dicum California 1 -

Prosimulium exigens Washington

Alberta

1

7

1.81

Prosimulium flaviantennus California 11 1.99

Prosimulium impostor California

British Columbia

4

1

1.15

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Table 1. Continued.

Species Locality

No. of specimens

% Divergence (max.)

Simuliini

Greniera abdita Ontario 1 -

Greniera abditoides Ontario 1 -

Stegopterna decafilis Yukon Territories 7 0.32

Stegopterna mutata * Ontario

Newfoundland

5

1

See table 2

Cnephia dacotensis Ontario

Manitoda

5

8

1.48

Metacnephia borealis Nunavut 13 1.48

Metacnephia saileri Yukon Territories

Nunavut

8

8

1.99

Metacnephia saskatchewana Nunavut 9 0.49

Metacnephia sommermanae Yukon Territories 8 0.32

Metacnephia villosa California 4 0.49

Simulium (Hellichiella) anatinum Manitoba 2 -

Simulium (Hellichiella) currei Wyoming 8 0.49

Simulium (Hellichiella) nebulosum California

Montana

2

1

See table 2

Simulium (Boreosimulium) balteatum British Columbia 9 1.48

Simulium (Boreosimulium) emarginatum Ontario 6 3.18

Simulium (Boreosimulium) joculator California 7 1.31

Simulium (Boreosimulium) baffinense Nunavut 7 -

Simulium (Boreosimulium) johannseni Manitoba 4 0.48

Simulium (Eusimulium) bracteatum Ontario 12 0.65

Simulium (Nevermannia) aestivum Ontario 2 -

Simulium (Nevermannia) burgeri Manitoba 4 1.14

Simulium (Nevermannia) carbunculum Colorado

Wyoming

3

4

1.34

Simulium (Nevermannia) conicum British Columbia 2 -

Simulium (Nevermannia) craigi ** Montana

Alberta

Manitoba

Ontario

Nunavut

1

1

2

1

2

See table 2

Simulium (Nevermannia) croxtoni Manitoba 8 0.98

Simulium (Nevermannia) gouldingi Ontario 7 1.15

Simulium (Nevermannia) pugetense British Columbia 2 -

Simulium (Nevermannia) quebecense ** Ontario 3 See table 2

Simulium (Nevermannia) silvestre Colorado

Wyoming

Manitoba

Nunavut

1

7

6

6

3.51

Simulium (Schoenbaueria) furculatum Nunavut 8 1.64

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Table 1. Continued.

Species Locality

No. of specimens

% Divergence (max.)

Simulium (Psilopelmia) venator California 3 0.49

Simulium (Psilozia) argus California 2 -

Simulium (Psilozia) encisoi Arizona

Wyoming

1

1

-

Simulium (Psilozia) vittatum California

Washington

Manitoba

British Columbia

Ontario

Nunavut

Norwest Territories

1

1

2

1

1

3

1

2.8

Simulium (Aspathia) hunteri Washington

British Columbia

9

11

0.32

Simulium (Aspathia) piperi California

British Columbia

1

10

2.31

Simulium (Hemicnetha) canadense California

British Columbia

8

9

2.83

Simulium (Simulium) arcticum * California

Colorado

British Columbia

Alaska

3

2

1

1

See table 2

Simulium (Simulium) decimatum Nunavut 7 0.82

Simulium (Simulium) decorum Wyoming

Manitoba

Ontario

1

3

5

0.98

Simulium (Simulium) noelleri Nunavut 13 0.49

Simulium (Simulium) murmanum ** Manitoba

Ontario

2

1

See table 2

Simulium (Simulium) rostratum Ontario

Nunavut

4

3

3.84

Simulium (Simulium) tuberosum * Wyoming

Manitoba

British Columbia

Ontario

Nunavut

1

2

2

1

3

See table 2

Simulium (Simulium) venustum* British Columbia

Ontario

Manitoba

Yukon Territories

Nunavut

3

5

2

1

3

See table 2

Total=463

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Table 2. Levels of genetic divergence in known and suspected species complexes and number of individuals analyzed per taxon; n = number of specimens examined.

Known species complex n % Divergence (max.)

Helodon (Helodon) onychodactylus 5 4.91

Stegopterna mutata 23 5.47

Simulium (Simulium) arcticum 62 6.50

Simulium (Simulium) venustum 83 5.31

Simulium (Simulium) tuberosum 64 5.44

Suspected species complex (sensu lit.) n % Divergence (max.)

Simulium (Nevermannia) craigi 27 4.93

Simulium (Nevermannia) quebecense 3 5.79

Simulium (Simulium) murmanum 15 4.58

Total=282

Table 3. Intrageneric levels of genetic divergence (%). Missing entries indicate that only one species of that genus was analyzed. Range of genetic variation in Greniera could not be estimated as only two species with one individual each was analyzed; n = number of specimens examined.

% Divergence

Genus n Min-Max Mean

Parasimulium 5 - -

Gymnopais 10 9.64-10.03 9.99

Twinnia 3 - -

Helodon 26 9.85-19.38 14.29

Prosimulium 75 3.35-18.56 12.42

Cnephia 13 - -

Greniera 2 6.53 6.53

Stegopterna 7 - -

Metacnephia 50 5.1-12.70 8.57

Simulium s. lat. 215 2.83-20.33 15.33

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Figure 1. A Kimura-2 Parameter NJ tree showing the DNA barcoding profile for 57 nominal species and 5 species complexes of Nearctic black flies. The extent of each terminal branch representing a given species is indicated by one or two dashes, the latter indicates a species complex.

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Figure 1 (Cont.)

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Figure 1 (Cont.)

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Figure 1 (Cont.)

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Mean = 0.76

92.9

7.1

Mean = 14.93

30.1

14

4.22.63.6

1.4

7.1

36.6

0.4

Figure 2. Histogram showing intraspecific (A) and interspecific (B) genetic divergences (%).

A

B

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32Chapter 3

Phylogeography, postglacial history and cryptic speciation in the

black fly Prosimulium travisi Stone (Diptera: Simuliidae)

3.1 Summary

DNA barcoding has gained increased recognition as a tool for species identification in various

groups of organisms. The fragment of the cytochrome oxidase I (COI) used in barcoding has also

proved versatile in other applications, such as in ecological research and conservation initiatives.

However, the utility of the barcoding gene for studies at the population level has so far received

little investigation. In this chapter, I focus on the population genetics of the cordilleran black fly

Prosimulium travisi, whose present-day distribution includes areas that were once covered by

Wisconsinan-aged glaciers. Under the assumption that the current distribution of P. travisi

resulted from post-glacial dispersal from refugia, I investigate variation in the barcode gene to

determine whether phylogeographic patterns could be revealed in a comprehensively sampled,

widely distributed, species. Three hundred and thirteen individuals were analyzed from 56

populations sampled throughout the entire range of the species (from Alaska and Yukon

Territory south to Arizona and New Mexico). Analysis of population relationships suggests that

recolonization of previously glaciated terrain was mainly the product of emigration from

northern (Beringia) and western (coastal) refugia. In contrast, the contribution from southern

refugia was relatively limited. Migratory routes and areas of secondary contact were also

identified. Pairwise genetic distances between P. travisi from Colorado and all other populations

ranged between 6.2% and 8.8%, contrasting with a divergence of between 0% and 3.36% for P.

travisi excluding the Colorado populations. The high level of genetic divergence of populations

from Colorado suggests that they constitute a genetically distinct sibling species, thus confirming

previous cytological evidence about their distinctiveness. Phylogeographic patterns in P. travisi

are largely congruent with those exhibited by many other cordilleran animals and plants,

suggesting that they shared a common biogeographical history. The COI barcoding gene proved

to be useful for revealing phylogeographic patterns, providing significant “value added”

biogeographical information, in addition to its role as a tool for molecular taxonomy.

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3.2 Introduction

Ever since publication of the seminal paper by Avise et al. (1987), phylogeography has become a

flourishing field that helps to elucidate contemporary geographical patterns of populations by

studying the spatial distribution of genes and their genealogies (Knowles, 2004). Current

phylogeographic research focuses on recent past events that shaped these patterns, such as the

climatic oscillations that transpired during the Quaternary Period. These climatic oscillations

played a key role in shaping the biogeography of the present-day North American biota.

Many plant and animal taxa are used as model organisms to reconstruct the geographical history

of populations, as well as to explore transformations of the landscape that supports them.

Comparative phylogeographic studies are fundamental to elucidating geographical patterns

across multiple co-distributed taxa at various spatial scales. This allows assessment of common

historical processes that might have shaped a particular distributional pattern and adds power to

biogeographic hypotheses (Soltis et al., 1997; Brunsfeld et al., 2001 Arbogast and Kenagy, 2001;

Knowles and Maddison, 2002; Soltis et al., 2006).

Most studies have so far used vertebrates and plants to reconstruct the recent postglacial history

of North America, while terrestrial invertebrates have been comparatively less well studied.

Soltis et al. (2006), for example, reviewed the phylogeographic literature of unglaciated eastern

North America and found that, during the period 1987 to 2005, about 80% of all phylogeography

papers focused on animals, of which <3% included reference to terrestrial or freshwater

invertebrates and only 1% included insects. The same pattern holds for western North America

(c.f. reviews by Soltis et al., 1997 and Brunsfeld et al., 2001). The few studies that have used

insects to infer historical events during the Quaternary Period explored local population genetic

processes or postglacial dispersal within a limited geographic area (e.g., Knowles, 2000; Fordyce

and Nice, 2003; Downie, 2004; DeChaine and Martin, 2005a,b; Heilveil and Berlocher, 2006;

Finn et al., 2006; Finn and Adler, 2006; Finn et al., 2007). Even fewer studies have investigated

postglacial patterns in widespread species, the notable exceptions being Nice et al. (2005) on the

phylogeography and historical biography of a species complex in the butterfly genus Lycaeides,

and Maroja et al. (2007) on the spruce beetle Dendroctonus rufipennis. The bias towards

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vertebrates is surprising given that insects typically occur in much larger numbers and many

species are widely distributed, making them outstanding subjects for phylogeographic studies.

Studies of widespread organisms are critical because a historical pattern for one species may be

applicable to other organisms that share a similar habitat or distribution. The rationale is that

similarly distributed species are likely to have been influenced by the same historical processes

(Riddle, 1998; Arbogast and Kenagy, 2001; Carstens et al., 2005; Helveil and Berlocher, 2006).

Aquatic insects are excellent models for exploring historical biogeographic patterns among

multiple organisms (either vertebrate, invertebrate or plant) with similar distribution and

ecological requirements (Helveil and Berlocher, 2006). However, aquatic insects have seldom

been used to study postglacial population events at a continental scale. Notable exceptions are

studies of the stonefly Pteronarcys californica (Plecoptera) by Kauwe et al. (2004) and the

Dobsonfly Nigronia serricornis (Megaloptera) by Heilveil and Berlocher (2006).

Black flies (Diptera: Simuliidae) are among the best-known group of aquatic insects in North

America (Adler et al., 2004). The immature stages occur widely in variously sized streams and

rivers from which they are easily collected, often in large numbers. The current taxonomic state-

of-knowledge allows easy identification to species level of most Nearctic species, making them

excellent subjects for phylogeographic studies. But while population genetic processes in black

flies with limited distribution have been studied at a local scale, such as in Prosimulium

neomacropyga (Finn, et al., 2006) and Metacnephia coloradensis (Finn and Adler, 2006),

phylogeographic patterns in widespread species have not yet been investigated.

During the course of my investigation of the utility of DNA barcoding for black fly identification

(c.f. Chapter 2), I observed considerable population structure in widely distributed and widely

sampled species. The primary objective of this chapter is to determine whether the 658-bp region

of the barcoding gene has utility for revealing phylogeographical patterns in black flies. If so,

this would provide significant “value added” information for comprehensively sampled

widespread species. More specifically, I present a phylogeographic analysis of the black fly

Prosimulium travisi Stone, a widely distributed species in the cordillera of western North

America. Because part of the present-day distribution of P. travisi includes formerly glaciated

terrain, my aim is to infer phylogeographic patterns that identify (a) the source area(s) (i.e.,

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glacial refugia) for present-day populations, and (b) their postglacial migratory routes. I will then

compare the results of my study with phylogeographical analyses of other widely-distributed

cordilleran organisms to determine whether they share a common geographical history.

3.3 Materials and Methods

3.3.1 Sampling

In order to gain an understanding of the underlying historical population structure of P. travisi,

populations were sampled in mountainous habitats from across virtually the entire range of the

species (Figure 1-2). Most specimens were collected during the course of a single collecting trip

from 9 June to 26 July 2006. Specimens from Colorado were collected in 2004 by D. Finn

(Oregon State University) and kindly provided to me by P. Adler (Clemson University). A total

of 313 individuals from 56 populations were included in this analysis. The number of individuals

analyzed per site varied from 1 to 10 (Table 1). All specimens were collected in either the larval

or pupal stages, both of which were easily collected from breeding sites (i.e., fast-flowing, clear,

cold, 1-5 m. wide, mountain streams between 900 and 3000 m). Prosimulium travisi is a

relatively distinctive species that is easily distinguished from other Prosimulium in the immature

stages. Accordingly, preliminary identifications were made in the field and later confirmed in the

laboratory using the keys of Adler et al. (2004). Individuals were fixed in 95% ethanol and

maintained at low temperatures (≈5° C) until returned to the lab for molecular analysis.

3.3.2 DNA extraction and sequencing

Larvae and pupae for molecular analysis were cut in half (note: larvae first had their digestive

tract removed to reduce the prospect of contamination). The posterior half (abdomen) was used

for DNA extraction whereas the thorax and head (where most features for taxonomic

identification reside) were retained as voucher specimens. These latter were deposited in the

Entomological Collection of the Royal Ontario Museum. All instruments were sterilized by

flame between specimen dissections to minimize contamination. Approximately 30 μl of total

DNA was extracted with a GeneEluteTM Mammalian Genomic DNA Miniprep Kit using a

35

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protocol according to the manufacturer’s specifications. PCR primers for amplifying the ca. 658

bp. long target region of the COI gene were those used in DNA barcoding (Hebert et al., 2003):

LCO1490 (5’GGTCAACAAATCATAAAGATATTG-G-3’) and HCO2198 (5’-TAAACTTCA-

GGGTGACCAAAAAATCA-3’). Polymerase chain reactions (PCR) were conducted using 1 μl

of template DNA in a total reaction volume of 25 μl. The PCR reaction mix contained 1 μl of

each primer, 1.5 μl of MgCl, 2.5 μl of PCR buffer, 0.8 μl of dinucleotide triphosphates

(dNTP’s), 0.1 μl of taq DNA polymerase. PCR conditions were an initial 1 min at 96 º C

(denaturation) followed by 1 min at 94º C (denaturation), 1 min at 55 º C (primer annealing) and

1.5 min at 72 º C (amplification) for 35 cycles, and 7 min at 72 º C. Purified template DNA was

sequenced with an ABI3100 automated sequencer at the Laboratory of Molecular Systematics,

Royal Ontario Museum.

3.3.3 Phylogeographical Analysis

Mitochondrial DNA sequences were aligned manually using BIOEDIT (Hall, 1999) and trimmed

to a final sequence length of 624-bp. No ambiguous sites were present. The 313 sequences were

collapsed into unique haplotypes using the software DNASP version 4.10.9 Rozas et al., (2003).

These unique haplotypes were then subjected to Neighbour-Joining (NJ) and Maximum

Parsimony (MP) analysis using the software package PAUP* 4.0b10 (Swofford, 2003). The NJ

algorithm was implemented using all haplotypes, whereas for comparative purposes, MP was

implemented to resolve relationships among the most common and widespread haplotypes (as

the little genetic variability of the whole data set would have yielded unresolved relationships).

This allows verifying if relationships among haplogroups using NJ approximates to that obtained

under a character-based method such as MP. For the MP analysis, a heuristic search was made

with step-wise addition of 100 random repetitions starting with TBR branch-swapping. All

characters were equally weighted and considered unordered. Branch support was estimated by

bootstrap values, using similar options as described above and 500 replicates. As in MP, a

Bayesian approach was also implemented using MRBAYES version 3.0 (Huelsenbeck et al.,

2001) on the most common haplotypes. Searches were run for 106 generations, with temperature

adjusted to 1.25 and sampling every 1000 generations using the general time reversible model

with invariant sites and gamma rates (GTR + I + G), as specified by the program MRMODELTEST

2.0 (Nylander, 2004). Prosimulium flaviantennus was used as outgroup for all analyses. To

36

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visualize genealogical relationship among haplotypes, the full dataset (the Colorado population

was not included due to its rather divergent nature, see below) was used to construct a median-

joining network using the software package NETWORK 4.201 (Bandelt et al., 1999, available

from fluxus-engineering.com). All sites were equally weighted.

3.3.4 Genetic diversity and demographic analyses

Standard population genetic parameters were estimated using the software package DNASP

(Rozas et al., 2003) and ARLEQUIN version 3.11 (Excoffier, et al., 2005). Parameters calculated

were number of haplotypes (H), haplotype diversity (Hd), nucleotide diversity (n), average

number of nucleotide differences (k) and number of segregating sites (S). The web-based

program MODELTEST version 3.8 (Posada, 2006) was used to determine the best fitting model of

DNA substitution using the hierarchical likelihood-ratio test (hLRTs). The Tamura-Nei (TrN)

(Tamura and Nei, 1993) with significant proportion of invariable sites and gamma distribution

rate heterogeneity was the best fit model of nucleotide substitution according to MODELTEST.

The proportion of invariable sites (I) was estimated to be 0.6961 with a gamma distribution

shape parameter of 0.6588. Pairwise Fst for P. travisi was estimated using ARLEQUIN version

3.11 (Excoffier, et al., 2005). The significance of the test was obtained by 100,000 permutations

of the data. I estimated the proportion of total genetic variation that was explained by differences

between the subpopulations of Prosimulium travisi using an analysis of molecular variance

(AMOVA). Tajima’s (1989) D test was used to determine if variation in the studied loci was the

result of either random mutations due to genetic drift (neutral evolution) or natural selection;

departures from neutrality can also be interpreted as changes in population size. I also estimated

Fu’s (Fu, 1997) Fs as a second independent test, which is more sensitive to population

expansion. The distribution of the number of nucleotide differences between pairs of haplotypes

(mismatch distribution) was computed to test the null hypothesis of recent population expansion.

3.4 Results

3.4.1 Phylogeographic structure

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The NJ analysis (which included the 133 haplotypes, the Colorado population and outgroup)

identified 3 major clades, which are referred to hereafter as the Northern Clade, Southern Clade

and Colorado Clade (Figure 3A). The Northern Clade includes haplotypes occurring in the

following geographical areas: (a) “northern latitudes” (northern British Columbia, including both

the northern Rocky Mountains and Coast Mountains and the Yukon Territory as far north as the

Ogilvie Mountains), (b) Vancouver Island plus Southern British Columbia (southern-most

section of the Coast Mountains), and (c) the Cascade Mountains of Washington and the Central

Rocky Mountains of southwestern Alberta. The Southern Clade includes haplotypes found in the

following geographical areas: (a) Central Rockies (southwestern Alberta, Montana and

Wyoming), (b) Southern British Columbia including the southern-most section of the Coast and

Columbia Mountains and (c) the Sierra Nevada of California. The single population sampled

from Colorado comprised the Colorado Clade. Pairwise differences within and between the

Northern and Southern clades ranged from 0% to 3.3%.

Three subclades were resolved within the Northern Clade (Figure 3A). Two of these subclades

consisted of haplotypes located in the Coast Mountains (labeled C1 and C2 respectively), with

their northern and southern-most limits in the Yukon Territory and Washington respectively; the

third subclade (labeled NR) consists of haplotypes located in the Northern Rocky Mountains,

with their northern and southern-most limits in the Yukon Territory and southwestern Alberta

respectively. Pairwise distances for each subclade were 0.16%-0.97% (C1), 0.16%-1.46% (C2)

and 0.16%-1.29% (NR). The Southern Clade consisted of two subclades (Figure 3A). One of

these, labeled “Central Rockies” (CR), consisted of haplotypes occurring in the Central Rocky

Mountains (southwestern Alberta, Montana and Wyoming), as well as in the southern-most

Coast and Columbia Mountains in British Columbia. The other southern subclade consists

exclusively of haplotypes situated in the Sierra Nevada (labeled SN) of California. Pairwise

distances for each subclade were 0.16%-2.28% (CR) and 0.16%-0.97% (SN). The MP analysis

of most common haplotypes, namely H1 (Subclade C1), H8 and H83 (Subclade C2), H62

(Subclade NR), H24 and H48 (Subclade CR) and H12 (Subclade SN) yielded 4 most

parsimonious trees. The consensus tree (Figure 4) produced a topology similar to that in the NJ

analysis; however, relationships within the Northern Clade were not as well resolved. Bayesian

analysis of the same set of haplotypes yielded a similar tree, except that haplotype H1 was

recovered as the sister group of all other haplotypes (Figure 4). These slight topological

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differences notwithstanding, a distinct northern partition (represented by haplotypes H1, H8, H83

and H62) and southern partition (represented by haplotypes H24, H48 and H12) among

haplotypes was evident. Furthermore, the Colorado population was invariably recovered as sister

group of all other populations. Levels of genetic divergence between the Colorado and all other

populations was between 6.2% and 8.8%.

3.4.2 Haplotype Network

A Median-Joining Network (MJN) of relationships among different haplotypes is presented in

Figure 3B. Haplotypes H1, H8, H83 and H62 were the most common constituent of the northern

clade except H83, which was widespread only at high latitudes. Haplotype H1 occurred on

Vancouver Island and was also common in the northern-most Coast Mountains of northwestern

British Columbia, the northern Alaskan Panhandle and southern Yukon Territory. Less common

haplotypes associated with H1 also occur at high latitudes but are also represented on the lower

mainland of British Columbia. Haplotype H8, H83 and other less frequent derivative haplotypes

form a well-defined haplogroup that ranges from the Yukon Territory and northern British

Columbia south to the northern Cascade Mountains of Washington. Haplotype H62 is also

widespread, ranging from the Yukon Territory and northern British Columbia to the Rocky

Mountains of southwestern Alberta. The most common haplotypes of the southern clade include

H12, H24 and H48. Haplotypes H24 and H48 both occur in the Central Rocky Mountains and

are largely allopatric. Haplotype H24 and its derivatives range from northern Montana north to

southwestern Alberta, attaining their northern-most limit at Site 21 (ca. 50°11.818'N,

114°33.956'W) (Figure 6B). In contrast, H48 and associated haplotypes occur north of that point

to at least the northern-most extent of the Rocky Mountains in southwestern Alberta at Site 30

(ca. 53°3.649'N, 117°20.351'W) (Figure 6B). Whether or not this haplotype’s range is extended

further north is uncertain because road access to suitable habitats was limited between west-

central Alberta and northeastern British Columbia. The other major haplotype of the Southern

Clade, H12 and its associated derivatives, was found exclusively in the Sierra Nevada of

California.

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3.4.3 Population genetics and demographic history

Analysis of 296 sequences of Prosimulium travisi (excluding the Colorado populations) revealed

133 unique haplotypes in this 624-bp portion of COI, of which 113 occurred as singletons.

Parameters of interest are summarized in Table 2. Frequencies of the most common haplotypes

are (in parenthesis): H1(19), H12(20), H8(6), H24(17), H48(18), H62(43) and H83(22). Number

of haplotypes and haplotype diversity is highest in Subclade CR (H=41, Hd=0.912) and lowest in

SN (H=11, Hd=0.589). Pairwise Fst values were found to be highly significant (Table 3). Within

the Northern Clade, C1 and C2 are less genetically differentiated from each other than they are

from Subclade NR and, among the two coastal subclades, C2 is less differentiated from NR than

it is from C1. Within the Southern Clade, CR and SN are comparatively much less genetically

differentiated from each other than from any pair of populations from the Northern Clade.

Analysis of molecular variance (AMOVA) indicated that 69.62% of the total genetic variation

was explained by differences between populations (P < 0.001), suggesting that the variation is

not distributed randomly. Tajima’s D estimations yielded a non-significant negative D value (-

1.2581, P > 0.10) when all populations where pooled together. However, Fu’s Fs test yielded a

significant negative value (-24.1361, P < 0.02), indicating departure from neutrality. Departures

from neutrality suggest that a population is not at equilibrium due to historical demographic

processes such as population growth, migration and ancient bottlenecks. Significant negative

values (P < 0.01) were obtained for all populations within the Northern Clade when analyzed

separately, indicating departure from neutrality (Table 2). Conversely, non-significant negative

values were observed in populations from the Southern Clade (Table 2). Fu’s Fs test, a more

sensitive test to demographic expansion, yielded large significant negative Fs values for all

populations (except in SN, where Fs was comparatively lower), strongly suggesting that these

populations experienced demographic expansion during the recent past. Mismatch distribution

(Figure 5) strongly suggests that C1, C2 and NR experienced recent demographic expansion, as

their pairwise differences distributions (mismatch analysis) are similar to the expected

distribution under a model of population expansion. Mismatch distribution analysis of the

Southern Clade yielded a slightly bimodal frequency for Subclade SN, which better fits the

distribution of frequencies expected under the model of stationary population, whereas a

multimodal frequency was observed in CR. The multimodal frequency in CR can either be the

result of a more stationary population or evidence of multiple genetic lineages. However, the

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large Fs significant negative value in CR suggests expansion and this alternative cannot be ruled

out. Population expansion occurred at least in the northern-most populations of Subclade CR

(i.e., southwestern Alberta), represented by haplotypes H24 and H48 (and their less common

derivative haplotypes). These sets of haplotypes yielded significantly negative D values (-2.2064,

**P < 0.01 and -2.0494, *P < 0.05), and negative Fs values (-11.6224 and -7.8184, both *P <

0.02) and exhibited a unimodal distribution frequencies undistinguishable from the model of

population expansion (Figure 5). This strongly suggests that demographic expansion occurred in

at least some members of Subclade CR, whereas others may represent relatively more stationary

subpopulations. Interestingly, H24 and H48 (and associated less common haplotypes) now occur

in territories that were glaciated during the Wisconsinan Age and their present-day distribution is

likely to be the result of expansion from a southern refugium.

3.5 Discussion

3.5.1 Glacial refugia

Glacial cycles (repeated advance and retreat of glaciers) during the Quaternary Period exerted an

enormous impact in the evolutionary history of plants and animals at northern latitudes (Pielou,

1991; Hewitt 1996, 2000). The effect of the last (i.e., Wisconsinan, 65,000-10,000 yr BP)

glaciation in North America was especially important for shaping the present-day distributional

patterns of organisms, and this subject has been reviewed extensively by Soltis et al. (1997),

Soltis et al. (2006), Brunsfeld et al. (2001) and Hewitt (2004). Climatic oscillations generated

major changes in species distribution, shaping the present-day genetic structure of their

populations and, in some instances (although still controversial), promoting speciation in

allopatry by isolating populations in glacial refugia (Bermingham et al., 1992; Hewitt, 1996;

Avise and Walker, 1998; Bernatchez and Wilson, 1998; Knowles, 2000; Cardoso and Vogler,

2005; Lovette, 2005; Near and Benard, 2004). As continental ice sheets expanded southward

during the Wisconsinan Age, high latitude plants and animals were displaced into refugial areas

where suitable habitat remained. Mountainous areas south of the continental ice sheets were also

affected, as the proliferation of alpine glaciers confined the mountain-dwelling biota to

unglaciated valleys (Hewitt, 2000). The waxing and waning of continental ice sheets and

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mountain glaciers left an indelible imprint on the genetic structure of refugia-dwelling

organisms, which subsequently repopulated the previously glaciated terrain following retreat of

Wisconsinan ice.

Numerous studies have revealed refugial areas along the periphery of the continental ice sheets

of North America (Scudder, 1979; Tremblay and Schoen, 1999; Demboski et al., 1999: Byun et

al., 1999; Fedorov et al., 2002; Burg et al., 2005; Waltari and Cook, 2005). At least three major

refugial areas have been identified in western North America – the geographical focus of the

present study. One of these, Beringia, was a vast unglaciated territory that extended from the

Kolyma River of Siberia to the Mackenzie River in the Northwest Territories of Canada (Pielou,

1991). Beringia remained ice-free during the glacial maximum because of the rain-shadow

created by mountains systems in western Alaska. The resulting landscape was dominated by

steppe-tundra and other more mesic habitats, which supported a diverse assemblage of northern-

adapted organisms (Pielou, 1991; Schweger, 1997; Ball and Currie, 1997; Elias, 1996; Elias et

al., 2000). A second refugial area, located immediately south of the Cordilleran Ice Sheet, was

the Cordilleran Refugium. Historically, this refugium has received comparatively less attention

than Beringia; and yet, its presence is well supported by recent phylogeographic studies (e.g.,

Brunsfeld et al., 2001). The Cordilleran refugium is typically defined as the major mountainous

regions of the western US situated south of the Cordilleran Ice Sheet, predominantly in the

Cascade Mountains (Washington, Oregon), central and southern Rocky Mountains (Montana to

New Mexico), Sierra Nevada (California), and other minor intervening ranges from which

dispersal into formerly glaciated areas could potentially have taken place. Previous comparative

phylogeographic analyses of multiple animal and plant taxa reveal the complex nature of the

southern refugium, where repeated vicariant and dispersal processes simultaneously shaped the

composition of different communities during glacial and interglacial periods (c.f. Brunsfeld et

at., 2001). A third, though controversial, refugial area was the Coastal Refugium, which

presumably existed somewhere along the coast of the Pacific Northwest. Archaeological,

palaeontological, geological, biogeographic and molecular studies all provide indirect support for

the presence of such a refugium. Though the exact location of the Coastal Refugium remains

unknown, the region comprising the Haida Gwaii archipelago (i.e., the Queen Charlotte Islands

including part of the now submerged continental shelf along Hecate Strait), south to Vancouver

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Island and western Olympic Peninsula, has been postulated as the most likely geographical area

(Demboski et al., 1999; Byun et al., 1999; Brunsfeld, et al., 2001).

3.5.2 Phylogeographic patterns in P. travisi

One of the primary aims of my study was to determine whether or not a phylogeographic

analysis using the barcoding gene could identify refugial areas for Prosimulium travisi during the

Wisconsinan Glaciation. Another goal was to elucidate the routes used by this species to

repopulate previously glaciated terrain during the Holocene Epoch. The most striking result

from the NJ, MP and Bayesian analyses is that there is a clear North/South partition in the

distribution of haplotypes (Figures 3-4). The Northern Clade includes haplotypes found from the

Yukon Territory south to the northern Cascade Mountains of Washington in the west, and to the

Rocky Mountains of Alberta in the east. In contrast, the Southern Clade includes haplotypes

occurring from the mountains of southern British Columbia and southern Alberta south to

Wyoming and California. It is important to note that none of the haplotypes identified in the

Yukon Territory and northern British Columbia are represented in the Southern Clade. The same

holds true for haplotypes from Sierra Nevada and the Rocky Mountains of Montana and

Wyoming, which are not represented in the Northern Clade. The distinctive north/south partition

of haplotypes in P. travisi is consistent with patterns observed in other western plants and

animals (Wheeler and Guries, 1982; Thorgaard, 1983; Comes and Kadereit, 1998; Soltis et al.,

1997; Flagstad and Røed, 2003; Albach et al., 2006), suggesting that the current distribution of

P. travisi was derived from two founding populations, one from a northern refugium (Beringia),

and another from a southern refugium (Cordilleran Refugium). The Cascade and Columbia

mountains of southern British Columbia (both in Southern British Columbia) support haplotypes

from both the Northern and Southern Clades. Phylogeographic data from other groups of plants

and animals has identified this region as an area of secondary contact between emigrating

populations from northern and southern refugia. This region is also known as a conduit for gene

flow between the Rocky Mountain populations in the east and the Columbia, Coast and Cascade

Mountains in the west (Brunsfeld, et al., 2001 and citations therein; Carstens, et al., 2005;

Kuchta and Tan, 2005; Albach et al, 2006).

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3.5.3 Postglacial colonization and comparative phylogeography

3.5.3.1 The Northern Clade

My analysis indicates that northern populations (subclades C1, C2 and NR) recently experienced

a range expansion. These populations, now inhabiting formerly glaciated terrain, were the result

of dispersal from glacial refugia located both north and west of Wisconsinan ice. A more

detailed analysis of the observed patterns follows:

I. Coastal 1 Subclade (C1, West). Although the subject of considerable debate, different lines of

evidence suggest the existence of a coastal refugium along the Pacific Northwest – specifically in

the area encompassing Haida Gwaii (i.e., Queen Charlotte Islands), Vancouver Island, western

Olympic Peninsula and at least part of what is now submerged continental shelf (Demboski et

al., 1999; Byun et al., 1999; Brunsfeld et at., 2001). Kuchta and Tam (2005) studied

phylogeographic patterns in the newt, Taricha granulosae, finding evidence for a close

relationship between Vancouver Island populations and those along the coasts of southwestern

Alaska and northern British Columbia. This latter region is also known as an area of secondary

contact between continental and coastal lineages in several mammals such as brown bears

(Talbot and Shields, 1996; Paetkau, et al., 1998), black bears (Byun, et al., 1997; Stone and

Cook, 2001), dusky shrews and martens (Demboski, et al., 1999). These findings not only

suggest the possibility of a coastal refugium, but also indicate that emigration from the coast to

mainland sites could have proceeded in both a northerly and southerly direction. My data reveals

that haplotype H1 (and associated haplotypes) is distributed from Vancouver Island and the

lower mainland of British Columbia north to the Alaskan Panhandle and southern Yukon

Territory. This pattern, in addition to the presence of Prosimulium travisi in Haida Gwaii (Currie

and Adler, 1986), suggests that populations of P. travisi could have survived the last glaciation in

an ice-free refugium along the west coast of British Columbia and the Alaskan Panhandle, with

subsequent emigration to continental terrain following deglaciation. Currie and Walker (1992)

recovered fossils referable to P. travisi from the lacustrine sediments in Marion Lake, near

Vancouver, British Columbia. These fossils were dated from 10,500 – 6,500 14C yr BP – a time

when elements of the Cordilleran Ice Sheet were still likely to be present in the Coast Mountains.

Nonetheless, suitable habitat for black flies must have existed in this particular corner of 44

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southwestern British Columbia, and the source of these early colonists is more likely to have

been west of present-day Marion Lake (i.e., in the vicinity of Georgia Strait) as opposed to the

surrounding Coast Mountains. The lower mainland of British Columbia is populated by

haplotypes H3 and H4, which are both nested within Subclade C1; however, haplotype H5 (a

Southern Clade member of Subclade CR) is represented in the same area. This suggests that

populations of P. travisi in the lower mainland of British Columbia may have been derived from

two sources: the Coastal Refugium and the Southern Cordilleran Refugium. Analysis of

nucleotide differences between pair of haplotypes (mismatch distribution) for C1 populations

yielded a distribution of frequencies similar to the expected model of population expansion,

which, in addition to evidence from Tajima’s D and Fu’s Fs tests, suggest that this population

experienced recent demographic expansion. A Coastal Refugium appears to be the most likely

explication for the observed patterns in Subclade C; however, further collections from Haida

Gwaii and environs (and indeed the entire western slope of the Coast Mountains) are needed to

help choose from among competing hypotheses.

II. Coastal Subclade 2 (C2, West). This subclade is dominated by haplotype H83 and the less

frequent haplotype H8. The former occurs exclusively at northern latitudes (Yukon Territory,

northern British Columbia), whereas the latter (and its derivative haplotypes H9, H10 and H11)

has its range extended farther south into the Cascade Mountains of northern Washington. The

geographical distribution of haplotypes, their position on the phylogenetic trees and haplotype

network, in combination with the smooth mismatch distribution frequency and Tajima’s D and

Fu’s Fs values, all suggest that postglacial populations of P. travisi emigrated from the Beringian

Refugia via the eastern slope of the Coast Mountains. This part of the Coast Mountains has also

been postulated as a postglacial migratory route for several mammal species in which similar

population relationships have been observed (Demboski et al., 1999; Stone and Cook, 2000;

Demboski and Cook, 2003).

III. Northern Rocky Mountain Subclade (NR, East). Members of this clade are distributed widely

in the Rocky Mountains from the Yukon Territory to Alberta, the southern-most collection taken

from site 26 (ca. 52º14’N, 116º1’W) in southwestern Alberta. An eastern route along the front

ranges of the northern Rocky Mountains has been postulated as a migratory route for organisms

migrating northwards from southern/southeastern refugia (Arbogast, 1999; Stone and Cook,

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2000; Conroy and Cook, 2000; Demboski and Cook, 2001; Runk and Cook, 2005). However, the

geographical distribution of NR haplotypes suggests that migration proceeded southwardly from

a northern refugium. This scenario is supported by the smooth mismatch distribution analysis for

NR which suggests that Alberta populations were the product of migration from a northern (i.e.,

Beringian) refugium. The sequence of ice retreat during the end of the Wisconsinan glaciation

provides a likely explanation for this pattern. Approximately 14,000 yr BP, an ice-free corridor

formed in the southeastern corner of Beringia between the Cordilleran and Laurentidae ice sheets

(Pielou, 1991). The timing, extent and the nature of the environments supported by the ice-free

corridor has been subject of considerable debate (Levson and Rutter, 1996). For example, some

authors argue that separation between the Cordilleran and Laurentide ice sheets occurred

simultaneously in the north and the south (Arnold, 2002), while others argue that the ice sheets

first began to separate in the south (Wilson, 1996). It has also been argued that the Laurentidae

and Cordilleran ice sheets coalesced and separated at various points during a glacial period,

creating an ice-free corridor during episodes of retreat. However, during the Wisconsinan age,

the ice sheets remained permanently coalesced in southwestern Alberta (Figure 6A; Levson and

Rutter, 1996; Catto et al., 1996). But, by ca. 11,000 yr BP, the ice-free corridor along the front

ranges of the Rocky Mountains was completely and permanently open (Mandryk, 1996; Wilson,

1996), allowing unfettered colonization by emigrants from both northern and southern refugia.

Interestingly, the area of southwestern Alberta where the ice sheets remained coalesced

throughout the Wisconsinan is where NR haplotypes attain their southern-most limit (site 26, ca.

52°14.317'N, 116°1.033'W, Figure 6B). I hypothesize that the asymmetrical migratory pattern

exhibited by Subclade NR (and CR, see below) was largely the product of the northern corridor

opening earlier and more extensively than the southern corridor (Figure 6A). While it is beyond

the scope of this study to address details of late Wisconsinan interactions between the Laurentide

and Cordilleran Ice Sheets, the opening of the front-range corridor unquestionably played a

central role in shaping the present-day distribution of P. travisi.

3.5.3.2 The Southern Clade

The southern clade includes populations from the Sierra Nevada, Central Rockies (Alberta,

Montana and Wyoming) and the Columbia and Cascade Mountains of southern British

Columbia. My data suggest that, unlike their northern counterparts, these populations were

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relatively more stationary. This can be explained by a lower level of habitat disturbance, as these

populations occur south of the continental ice sheets. As a result, population admixture was

relatively more continuous. However, proliferation of mountain-top glacials might still have

influenced the population genetics of these high-altitude adapted organisms at a smaller

geographical scale. A more detailed description of the observed patterns follows:

I. Central Rocky Mountains Subclade (CR, West). Members of this clade are distributed in the

central Rocky Mountains (northern Wyoming, Montana and southwestern Alberta), southern

British Columbia (Columbia and Coast Mountains) and Washington (northern Cascade

Mountains). Both the NJ tree and haplotype network indicate deep divergences among

haplotypes within Subclade CR, and levels of genetic divergence (pairwise distances) were the

highest of all subclades (0.16%-2.28%). Genetic diversity (H=84; Hd= 0.912), average number

of nucleotide differences (k = 7.172) and segregating sites (S = 47) were also the highest of all

subclades. This seems remarkable given the smaller area encompassed by members of this clade

relative to the others. For example, Subclade C2 exhibits lower haplotype diversity (H=35;

Hd=0.875) despite the fact that its members are distributed over a larger geographical area than

are members of Subclade CR. The patterns observed in CR reflect the divergent nature of its

constitutive haplotypes.

Haplotypes H24 and H48 are the most common haplotypes of Subclade CR. They are found only

in southwestern Alberta and northern Montana. All other haplotypes of Subclade CR are, in

general, quite divergent from these two and geographically restricted. The area encompassed by

Subclade CR has been shown to be a complex biogeographical unit, rich in phylogeographic

structure and hybrid zones (Brunsfeld et al., 2001; Demboski and Sullivan, 2003; Swenson and

Howard, 2005). Comparative phylogeographic research has shown that the complex nature of the

genealogical relationships of the biota inhabiting the central Rocky Mountains is the result of

both ancient vicariance and recent dispersal (Brunsfeld et al., 2001, Soltis et al., 1997).

Demboski and Sullivan (2003), for example, conducted a comprehensive phylogeographic

analysis of the chipmunk, Tamias amoenus, in northwestern North America, which corresponds

with the area now inhabited by Subclade CR. They found that habitat fragmentation and

migration shaped the population structure of T. amoena, suggesting that similar processes (e.g.,

climatological or geological) could have had a similar effect on P. travisi. Subclade CR

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apparently represents a collection of older haplotypes that have persisted in multiple Central

Rocky Mountain refugia as was the case with the grasshopper Melanoplus (Knowles, 2002).

Persistence in refugia perhaps dates from before the Pleistocene Epoch.

The Rocky Mountains of southwestern Alberta includes haplotypes related to those in the

northern Cascade Mountains of southern British Columbia and northern Washington. A

biogeographic connection between the central Rocky Mountains and the northern Cascade

Mountains is supported by other phylogeographic studies, with intervening ranges in southern

British Columbia and northern Washington hypothesized to serve as a connection between these

otherwise isolated ranges (Brunsfeld, et al., 2001 and citations therein; Carstens, et al., 2005;

Kuchta and Tan, 2005; Albach et al, 2006). Haplotypes H5, H6 and H7 occur in this latter area,

but H7 is also distributed in the Rocky Mountains of southwestern Alberta (Figure 3B). This

distributional pattern confirms that a connection must have existed between the central Rocky

Mountains and the southern Coast- and Cascade Mountains.

II. Sierra Nevada Subclade (SN, east). Phylogeographic evidence from approximately 100

species reveals that the Sierra Nevada is linked biogeographically to the Central Rockies

(Brunsfeld et al, 2001). The level of genetic differentiation (Fst) is lower between the SN and

CR (Fst = 0.3596) than it is between these two clades and any other lineage. This relationship is

represented as the earliest dichotomy in phylogeographic studies of similarly distributed

organisms (Good, 1989; Howard et al., 1993; Brunsfeld et al., 2001; Demboski and Sullivan,

2003; Albach et al., 2006). This suggests that a persistent connection must have existed between

subclades CR and SN, perhaps owing to their position south of Wisconsinan-aged ice. The

relatively small area sampled in the Sierra Nevada is dominated by haplotype H12, which was

represented at all sampling sites except one, where only haplotype H17 occurred. Genetic

diversity in SN is the lowest of all clades (H=11; Hd=0.589). However, interpretations of local

population dynamics within this geographical area are not feasible given the small sample size.

Other studies suggest that a biogeographic connection existed between the Sierra Nevada and

southern Cascade Mountains of Oregon and southern Washington (c.f. Brunsfeld et al., 2001).

No evidence of such a connection was identified in the present study, but this can be attributed to

limited sampling in Washington and the complete absence of P. travisi samples from Oregon.

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Further collections from these areas might reveal a similar pattern to that observed in other

organisms.

3.5.3.3 The Colorado Clade (CO)

Populations of Prosimulium travisi from Colorado were found to be markedly divergent from

those of all other populations. All analyses placed the CO Clade as sister group of all other P.

travisi populations. Levels of genetic divergence (pairwise distances) between CO and the

remaining populations ranged between 6.2% and 8.8%. Data presented in Chapter 2 reveals that

intraspecific genetic divergence in black flies can be as high as 3.8%, with higher values

indicative of distinct species status. Accordingly, the exceptional high divergence of the

Colorado population indicates that it is probably specifically distinct from those of other P.

travisi. Support for this conclusion comes from previously published cytological studies of P.

travisi, which reveals that chromosomes of larvae from Colorado differ from those occurring

elsewhere by lack of inversion IS-3 and the presence instead a unique inversion (Basrur, 1962;

Adler et al., 2004). Studies of the polytene chromosomes of black fly larvae have long been used

to recognize sibling species in isomorphic populations; however, chromosomal differences in

allopatry cannot be evaluated (i.e., unless chromosomal differences are shown to be maintained

in sympatry, then it is not possible to determine whether inversions are indicative of reproductive

isolations, or merely represents an intraspecific polymorphism). Given the geographical isolation

of the Colorado population, Adler et al. (2004) considered it to be conspecific with typical P.

travisi, though they noted the possibility that it might actually represent a separate species. The

molecular genetic evidence provided by my phylogeographical analysis confirms Adler et al.’s

speculations that Colorado populations of “P. travisi” indeed represent a distinct sibling species.

The situation described above has been identified in other organisms that exhibit a similar

geographical distribution. A phylogeographical break has been identified in the Wyoming basin,

which constitutes a major geographical barrier for gene flow on either side of this landmark,

especially for high-altitude organisms (DeChaine and Martin, 2004). For example, deep levels of

genetic divergence were observed between isolated populations of Sedum lanceolatum

(DeChaine and Martin, 2005a) and the Red Squirrel Tamiasciurus hudsonicus (Wilson et al.,

2005). In fact, the Wyoming basin often defines the boundary between well differentiated

subspecies of mammals (Findley and Anderson, 1956). Albach et al. (2006), in their

49

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phylogeographic study of the Veronica alpina complex, identified a similar pattern of divergence

in V. nutans. This latter finding is of particular interest because V. nutans and P. travisi share

virtually identical distributional patterns in western North America. Accordingly, these species

were probably influenced by the same geological and climatic events. The formation of the

Wyoming basin was a major vicariant event for all the above mentioned species and they were

no doubt subjected to other geological processes (including the repeated cycles of glaciation

during the Pliocene and Pleistocene Epochs). All of these events played a role in shaping the

distinctive phylogeographic patterns of P. travisi in Colorado. Further isolation of these

populations in these scattered mountain ranges favoured divergence and incipient speciation.

Similar historical processes perhaps played a role in producing the genetic differences observed

in Colorado populations of Prosimulium neomacropyga, which are currently considered to be

conspecific with geographically isolated populations in Alaska and the Yukon Territory (see

Chapter 4). Another black fly species, Metacnephia coloradensis, is also restricted to a few

alpine habitats in Colorado (as in P. neomacropyga) (Finn and Adler, 2006). This high-altitude

endemic species was likely the result of the same processes as described above.

3.5.4 Distributional patterns in western black flies.

Most previous studies on the biogeography of Nearctic black flies were based on distributional

data because few other sources of information (e.g., fossils) were available (see below). My

phylogeographic study of P. travisi provides an opportunity to test previous biogeographical

hypotheses about North American black flies. The first such study was undertaken by Shewell

(1958), who attempted to interpret various distributional patterns of extant black flies. He

concluded that the present-day distribution of many species could be explained by postglacial

dispersal from northern (Beringia) and southern (Cordilleran) refugia. Currie and Adler (1986),

in their study of black flies from the Queen Charlotte Islands, hypothesized that the present-day

fauna was probably derived from postglacial immigration from either a mainland or coastal

refugium. The discovery of fossil black flies from lacustrine sediments in southern British

Columbia and Vancouver Island provided further insights about the biogeography of western

black flies (Currie and Walker, 1992). Among the fossils was Helodon pleuralis (as Prosimulium

pleurale), a black fly whose present-day distribution includes Beringia and the formerly

glaciated northern Cordillera. This distribution suggests either a southward dispersal from a

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northern (Beringian) refugium, or that H. pleuralis perhaps also survived in a coastal refugium.

Similarly, the discovery of 10,500 – 6,500 14C yr BP fossils referable to Prosimulium travisi near

Vancouver, British Columbia suggested a southern or coastal origin for those early colonists.

The most comprehensive biogeographic analysis of Nearctic simuliids was undertaken by Currie

(1997), who identified 11 particular distributional patterns of Nearctic black flies. The

“Cordilleran” distribution (i.e., species whose ranges are confined to the western mountains) is

by far the most common pattern among western species, including P. travisi. Currie inferred that

Cordilleran species could have been the result of postglacial dispersal from either a northern

refugium or southern refugium (i.e., a unicentric model) or were the product of postglacial

dispersal from both refugial areas (i.e., a bicentric model).

The results presented herein show that the Beringian and Southern Cordilleran refugia indeed

played an important role as source areas for re-colonizing populations of P. travisi. Accordingly,

my findings support Shewell (1958) and Currie (1997), and confirm the bicentric origin

hypothesis, at least for some species. The role of a coastal refugium as a source area for re-

colonizing simuliids is less clear, given the current dearth of physical evidence. Nonetheless, my

phylogeographic analysis strongly suggests such a refugium for P. travisi. Furthermore,

comparative phylogeography reveals that patterns observed for P. travisi are shared by many co-

distributed taxa, perhaps reflecting the role of similar historical events. Under this assumption,

other sympatric cordilleran species (e.g., Gymnopais dichopticoides, H. clavatus, H. diadelphus,

H. onychodactylus, H. susanae, Prosimulium esselbaughi, P. formosum, P. frohnei, P. fulvum,

Tlalacomyia ramifera, Metacnephia villosa, Simulium balteatum and S. hunteri), perhaps

achieved their present-day distribution via the same processes and dynamics observed in P.

travisi. Phylogeographic analyses of these species are needed to confirm the generality of the

patterns described in the present study.

3.6 Conclusions

This study demonstrates the utility of the barcoding gene for revealing phylogeographic patterns

in black flies. My results indicate that Pleistocene-aged events left a deep mark on the population

genetics of P. travisi. The present-day distribution of this species is the result of colonizers that

expanded their range following glaciation from Beringia, the Coastal Refugium and the Southern 51

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Cordillera Refugium complex (Figure 7). Founding populations from Beringia and the Coastal

Refugium contributed most to previously glaciated terrain in the northern cordillera, whereas

populations from southern refugia remained relatively sedentary. The mountain systems to the

west of the Rockies in southern British Columbia and northern Washington (i.e., southern Coast-

, Columbia- and Cascade Mountains) represent an area of secondary contact between migrating

populations from all three refugial areas. In the Rocky Mountains, the area of secondary contact

is in southwestern Alberta. Colorado populations of P. travisi were found to represent a distinct

sibling species whose origin was probably triggered by pre-Pleistocene climatological and

geological events. The phylogeographic patterns observed in P. travisi (i.e., glacial refugial areas

and postglacial colonizing routes) are largely congruent with those hypothesized for other co-

distributed plants and animals, and they agree well with evidence from other disciplines. In

summary, my thesis confirms the utility of the DNA barcoding gene for phylogeographical

analysis of a comprehensively sampled species. As the database of barcoded species become

populated with specimens throughout the entire range of a species, then the type of analysis

undertaken here can be attempted on a more routine basis. As such, the international effort to

barcode the world’s biota has potential to yield “value added” information for those interested in

phylogeography and population genetics.

.

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Table 1. Collecting sites for Prosimulium travisi, showing the distribution of the 133 unique haplotypes

Site Sample

size Haplotype

Designation No. of

Haplotypes Country Province/State Coordinates Altitude

(m) 1 2 H1,H2 2 CA British Columbia 49°44.725'N, 125°19.083'W 1104

2 3 H3,H4,H5 3 CA British Columbia ca. 49°19'N, 122°33'W no data

3 1 H6 1 CA British Columbia 50°53' N, 119°53'W 1272

4 1 H7 1 USA Washington 48°51.967'N, 121°39.467'W 1081

5 4 H8,H9 2 USA Washington 47°23.6'N, 121°23.817'W 800

6 2 H10,H11 2 USA Washington 46°38.717'N, 121°14.417'W 910

7 3 H12,H13 2 USA California 39°19.05'N, 120°21.083'W 2110

8 4 H12,H14 2 USA California 38°35.4'N, 119°47.433'W 2152

9 9 H12,H15-16 3 USA California 38°34.717'N, 119°48.45'W 2398

10 1 H17 1 USA California 38°40.7'N, 119°35.983'W 2440

11 7 H12,H18-19 3 USA California 37°56.317'N, 119°15.017'W 2907

12 7 H12,H14,H20-22 5 USA California 37°57.033'N, 119°13.533'W 2760

13 6 H23-25 2 USA Montana no data(1) no data

14 1 H26 1 USA Montana 44º34'06"N, 111º50'39"W 2787

15 1 H27 1 USA Montana 44º55'59"N, 111º30'03"W 2097

16 7 H28-31 4 USA Wyoming 44°56.559'N, 109°34.86'W 2776

17 1 H32 1 USA Montana 45°49.166'N, 110°54.41'W 1967

18 8 H7,H24,H33-36 5 CA Alberta 49°41.63'N, 114°29.029'W 1753

19 7 H24,H37-41 6 CA Alberta 49°55.602'N, 114°23.89'W 1475

20 8 H24,H42-43 3 CA Alberta 50°10.443'N, 114°27.687'W 1850

21 6 H24,H44-47 5 CA Alberta 50°11.818'N, 114°33.956'W 1952

22 3 H48,H49 2 CA Alberta 51°29.551'N, 115°9.067'W 1621

23 1 H48 1 CA Alberta 51°28.958'N, 115°8.585'W 1577

24 5 H48,H50 2 CA Alberta 51°44.014'N, 115°21.766'W 1646

25 7 H48,H51-54 5 CA Alberta 52°2.874'N, 115°27.524'W 1559

26 9 H48,H49,H52,H56-57 5 CA Alberta 52°14.317'N, 116°1.033'W 1693

27 6 H48,H52,H57-60 6 CA Alberta 52°37.778'N, 116°15.857'W 1496

28 4 H48,H61 2 CA Alberta 52°39.149'N, 116°17.053'W 1496

29 3 H62-63 2 CA Alberta 52°53.671'N, 117°16.353'W 1913

30 6 H7,H36,H62,H64-65 5 CA Alberta 53°3.649'N, 117°20.351'W 1553

31 2 H62,H66 2 CA Alberta 53°7.287'N, 117°28.532'W 1553

32 9 H62,H67-71 6 CA British Columbia 58°47.165'n, 125°43.514'W 943

33 9 H62,H72-74 4 CA British Columbia 58°47.742'N, 125°43.852'W 1016

34 8 H62,H75-76 3 CA British Columbia 59°13.458'N, 125°58.585'W 663

35 8 H62,H77-78 3 CA British Columbia 59°53.525'N, 127°25.689'W 542

36 6 H1,H79-82 5 CA Yukon Territory 60°6.473'N, 130°19.957'W 876

37 7 H1,H62,H79,H83-84 5 CA Yukon Territory 60°6.476'N, 130°19.953'W 875

38 9 H62,H85-88 5 CA British Columbia 59°56.141'N, 131°15.101'W 864

39 2 H89,H91 2 CA Yukon Territory 60°33.552'N, 133°7.016'W 1145

40 9 H67,H83,H89-90 4 CA Yukon Territory 60°35.751'N, 133°3.819'W 1172

41 7 H8,H83,H92 3 CA Yukon Territory 61°5.427'N, 133°3.411'W 822

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Table 1 (Cont.) 42 7 H83,H93-95 4 CA Yukon Territory 61°46.875'N, 133°5.219'W 1050

43 8 H62,H96-101 7 CA Yukon Territory 61°48.281'N, 133°2.88'W 1050

44 8 H102-109 8 CA Yukon Territory 64°21.389'N, 138°24.533'W 850

45 8 H1,H83,H110-112 5 CA Yukon Territory 60°3.176'N, 136°53.283'W 956

46 6 H1,H83 2 CA British Columbia 59°42.406'N, 136°36.416'W 997

47 8 H1,H59,H83 3 CA British Columbia 59°36.896'N, 136°27.069'W 946

48 5 H1,H83,H113-115 5 CA Yukon Territory 60°13.541'N, 134°43.61'W 744

49 1 H62 1 CA British Columbia 59°20.813'N, 129°10.458'W 759

50 9 H1,H62,H79,H116-120 8 CA British Columbia 58°37.487'N, 130°0.983'W 895

51 7 H59,H62,H121-125 7 CA British Columbia 58°34.963'N, 130°0.709'W 927

52 6 H62,H83,H126-128 5 CA British Columbia 58°21.603'N, 129°54.673'W 972

53 6 H1,H62,H72,H79,H129-130 5 CA British Columbia 58°19.154'N, 129°54.168'W 1199

54 7 H1,H62,H131-133 5 CA British Columbia 58°14.871'N, 129°50.837'W 1207

55 1 H8 1 CA British Columbia 56°51.463'N, 129°59.304'W 597

56 17 Not designated - USA Colorado no data(2) no data (1) Glacier National Park (vicinity of Classic Peak). (2) Larimer Co. (headwaters of the south fork of Poudre River).

Clade N H Hd (SD) n k S Tajima’s D Fu’s Fs

C1 42 20 0.797 (0.065) 0.00299 1.863 25 -2.2822** -18.9307* C2 64 35 0.875 (0.038) 0.00381 2.379 34 -2.1754** -26.8786* NR 75 26 0.699 (0.06) 0.00223 1.395 29 -2.4011** -27.2805* CR 84 41 0.912 (0.021) 0.01149 7.172 47 -0.8158 -19.5922* SN 31 11 0.589 (0.105) 0.00248 1.548 12 -1.5700 -5.6101*

Levels of significance: * P < 0.02; ** P < 0.01

Table 2. Genetic population parameters estimates for Prosimulium travisi based on 624bp of COI sequences: number of individuals (N), number of haplotypes (H), haplotype diversity (Hd), nucleotide diversity (n), average number of nucleotide differences (k), number of segregating sites (S) and Tajima’s D and Fu’s Fs values from the neutrality tests. Colorado population not included.

Table 3. Pairwise Fst for all subpopulations of Prosimulium travisi (Colorado population not included).

Clade C1 C2 NR CR SN

C1 - C2 0.6799 - NR 0.8255 0.7532 - CR 0.6493 0.6168 0.7058 - SN 0.7949 0.7611 0.8674 0.3596 -

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Sierra Nevada

Casc

ade M

ount

ains

Coa

st M

ount

ains

Northern Rocky M

ountainsC

entral Rocky M

ountains

Vancouver Island

Figure 1. Map of western North America showing the mountain ranges mentioned in the text.

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Northern (YT, N BC) [Sites 32-55]

Central Rocky Mts. (N BC, AB, MT, WY) [Sites 13-31]

Vancouver Island (BC) [Site 1]Coast, Cascade and Columbia Mts.(BC and WA) [Sites 2-6]Sierra Nevada (CA) [Sites Sites 7-12]

Colorado [Site 56]

Figure 2. Map of western North America illustrating the extent of sampling of Prosimulium travisi. Sampled sites are grouped following a more or less continuous geographic region: black dot = Vancouver Island; white dot = Northern Cascade (Washington) and Southern Coast and Columbia Mountains in Southern British Columbia; white square = Sierra Nevada in California; black square = Central Rocky Mountains, from Alberta south to northern Wyoming; dashed elipse = Northern Coastal Mountains and other minor ranges in northern BC and Yukon Territories; gray square = northern Colorado.

64

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Northern (YT, N BC)Central Rocky Mts. (N BC, AB, MT, WY)Vancouver Island (BC)Coast, Cascade and Columbia Mts. (BC and WA)Sierra Nevada (CA)

H1H3

H127

H4H133

H117H125 H122

H131H112H86

H114H2 H118

H128H129H72

H73H77

H78H8H69

H10H11

H98 H119H9H94

H101H90

H96H102

H121H107

H123H108

H109H110

H105H104

H106H85

H113H115H95H93H87 H111

H100H91

H92

H103H97

H99

H80H62

H55

H82 H89H130

H79H66H88H116H124

H84H132H65

H67H71

H126H57

H59H68H74

H70H120H75H76

H81 H5H48

H49H53H54

H50H51

H58H61

H56H52

H63H44 H6

H7H37H64

H36H60 H23

H24H25

H41H34H42H40

H38H46

H39H35

H43H45

H26H27H28H33

H29H30

H31H47

H32H12H18

H21H22

H13H14H19H15

H16H17H20

H83

Northern Clade

Southern Clade

Colorado (CO)

Central Rockies (CR)

Northern Rockies (NR)

Sierra Nevada (SN)

Coastal 2 (C2)

Coastal 1 (C1)

99

100

58

83

76

68

76

74

78

98

52

52

OG

0.001 substitutions/site

[0.16%-2.28%]

[0.16%-0.97%]

[0.16%-0.97%]

[0.16%-1.29%]

[0.16%-1.46%]

H1

H8

H62

H12

H24

H48

NORTHSOUTH

C1

NR

C2

SN

CRH6

H7

H5

H3H4

H10

H11

H9

H83

B

A

Figure 3. A) Neighbor-Joining tree showing the relationship between 133 unique haplotypes, using the GTR model with no gamma correction. Bootstrap values higher than 50% are shown on branches. Terminal taxa are labeled according to haplotype designation from Table 1. Levels of genetic divergence (pairwise distances) are indicated for each clade. B) Median Joining Network showing the relationship between unique haplotypes. Each circle represents one haplotype and its size its relative frequency in the sample. Each dash on the lines connecting any two haplotypes represents one mutational step; small open circles represent haplotypes that were either not sampled or are now extinct. The most common haplotypes (H1, H8, H12, H24, H48, H62 and H83) and those from the Coast, Cascade and Columbia Mountains are the only ones labeled and they are frequently mentioned in the text.

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H1(C1)

H8 (C2)

H62 (NR)

H48 (CR)

H24 (CR)

H12 (SN)

CO

OG

H83 (C2)

Figure 4. Strict consensus (MP) (left) and Bayesian (right) phylogenies showing the relationship between representative (i.e. most common) haplotypes of the Northern (H1, H8, H62 and H83), Southern (H12, H24 and H48) and Colorado Clades. Bootstrap values and branch support probabilities are shown on the corresponding branches.

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Coastal 1 (C1)

Coastal 2 (C2)

Central Rockies CR

Sierra Nevada (SN)

Northern Rockies (NR)

Figure 5. Mismatch distribution frequencies by population. Haplotpes H24 and H48 (and their less common derivatives), both within S ubclade CR were also analyzed separately.

NORTHERN CLADE

SOUTHERN CLADE

CR-H24 CR-H48

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Figure 6. A) Map showing glacial limits during the late Wisconsinan (21,000-19,000 YBP) and the estimated location and extent of the ice-free corridor sensu Mandrik (1996) (reconstructed from various sources). The arrow indicates the approximate area were the Cordilleran and Laurentide ice sheets once coalesced; B) Same map showing extent the Northern Rocky (NR) and Central Rocky Mountains (CR) Subclades. Subclade NR reaches its southern-most limit at Site 26. Haplotypes H24 and H48 (and their less frequent derivatives) predominate in the Rocky Mountains of southwestern Alberta and are largely allopatric. Haplotype H24 occurs from northern Montana north to Site 21, whereas haplotype H48 occurs from this latter point as far north as Site 30. The lined area represents where NR and CR occur in sympatry and it coincides with the point were the continental ice sheets coalesced as shown in A; this represents the area of secondary contact between migrating populations from the northern and southern refugia.

NR

CR

Site 30

Site 26

Site 21

A B

Ice-free corridor

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Beringia

B

CCoastal

Refugium

A

D

?

E Southern Cordillera Refugium

Figure 7. Map of western North America showing inferred locations of glacial refugia and postglacial migratory routes for P. travisi. A=route along the coast, represented by clade C1; B=route along the Coastal Range, represented by clade C2; C=route along the northern Rockies, represented by clade NR; D=routes along the Central Rocky and Columbia Mountains into more western terrain; E=possible connection between Sierra Nevada and Central Rocky Mountain populations along intervening ranges in northern Nevada and Idaho (not inferred from the data but observed in other taxa sympatric with P. travisi). The size of arrows corresponds to the relative contribution of each immigrant population to the formerly glaciated territories.

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70Chapter 4

Evolutionary history and cryptic speciation in members of the

Prosimulium macropyga species-group in Western North America

(Diptera: Simuliidae)

4.1 Summary

Members of the Prosimulium macropyga species-group of black flies are distributed throughout

the northern Holarctic region. Two nominal species are currently recognized from North

America: P. neomacropyga and P. ursinum. The distribution of P. neomacropyga is intriguing

because it is markedly disjunct, with one population confined to northwestern North America

(Alaska, Yukon Territory) and one confined to the Rocky Mountains of Colorado and Wyoming.

Previous cytological studies reveals chromosome differences between the two populations;

however, such differences in allopatry are difficult to interpret, and both populations are

currently included under one name. Additional sources of data are needed to establish whether

these disjunct populations are conspecific or represent separate species. In this study I used the

portion of the Cytochrome Oxidase I gene used in DNA barcoding to assess the level of genetic

differentiation between northern and southern populations of P. neomacropyga. Data from this

same gene was also used to conduct a phylogeographical analysis of P. neomacropyga, and

representative populations of P. ursinum from western North America. Intraspecific pairwise

distance was 0%-2.3% for the northern populations and 0%-0.97% for the Colorado population.

In contrast, pairwise distance between members of the disjunct populations was 5.4%-7.9%.

Such values are well above the 3.36%-3.84% limit of intraspecific divergence in black flies

suggested by previous DNA barcoding research (see Chapter 2). A Bayesian approach used to

estimate the time to most common ancestor (TMRCA) indicated that these allopatric populations

diverged ca. 2.9 MYA, which coincides with the beginning of glacial cycles in the northern

hemisphere during Pliocene times (3.0-2.4 MYA). The evidence suggests that northern (Alaska

and Yukon Territories) and southern (Colorado) populations of P. neomacropyga represent

different species. The taxonomic implications of this finding are discussed.

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4.2 Introduction

Glacial cycles played a central role in shaping the genetic structure and geographical distribution

of organisms at high latitudes (Pielou, 1991; Hewitt 1996, 2000). They also played a role in

promoting speciation by dividing and isolating populations in glacial refugia (Bermingham et al.,

1992; Hewitt, 1996; Avise and Walker, 1998; Bernatchez and Wilson, 1998; Knowles, 2000,

2001; Cardoso and Vogler, 2005; Lovette, 2005; Near, 2004). This is particularly true for

circumpolar or circumboreal species, as their range was severely and repeatedly modified by

glacial oscillations.

Black flies (Diptera: Simuliidae) are an important component of high latitude biotic communities

(Currie, 1997; Malmqvist et al., 2004). Adult females are renowned for their blood sucking

habits on homoeothermic hosts, which include most major groups of birds and mammals (Adler

et al., 2004). The immature stages live exclusively in running waters, which can range in size

from tiny trickles to large rivers. Eighty four of 255 species of North American black flies (32%)

have at least part of their ranges extended north of the 60th parallel, and 28 (11%) of these occur

exclusively north of that latitude. Many of these latter are classified as “arctic species” and they

posses a number of adaptations for life in the far north (e.g., females are non-blood feeders, and

produce relatively few but large eggs) (Downes, 1965).

One arctic-adapted lineage of black flies includes the members of the Prosimulium macropyga

species group, which includes 12 nominal species distributed throughout high latitudes in Eurasia

and North America (Adler and Crosskey, 2008). Only two species are currently recognized from

the Nearctic Region: Prosimulium ursinum and Prosimulium neomacropyga. The former species

is widely distributed throughout the northern Holarctic Region, whereas the latter exhibits a

markedly disjunct distribution, with one population restricted to northwestern North America

(Alaska and the Yukon Territory) and the other in the Rocky Mountains of Colorado and

Wyoming (Adler et al., 2004).

Both Nearctic macropyga-group species have been the subject of previous cytological studies

(Rothfels, 1956, 1979; Carlsson, 1962; Madahar 1967, 1973; Adler et al., 2004). Prosimulium

ursinum is an allotriploid (i.e., the product of interspecific hybridization) parthenogenetic 71

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species, with populations from Alaska and Nunavut evidently having separate origins (see

below). However, P. neomacropyga is believed to be one of the parental species in both

populations of this putative species complex (Madahar 1967, 1973; Rothfels, 1979; Adler et al.,

2004). Cytological studies of P. neomacropyga reveal that populations from Colorado posses a

novel Y-chromosome linked inversion that is absent from the undifferentiated sex chromosomes

of populations from Alaska, the Yukon Territory and Wyoming. Adler et al. (2004) considered

the population from Colorado to represent a Y-chromosome polymorphism, and all populations

were therefore considered to represent just one species.

Phylogeographic studies of Nearctic black flies are virtually nonexistent. The only two published

studies investigated phylogeographic patterns in a limited geographical area (Finn et al., 2006;

Finn and Adler, 2006). One of these studies (Finn et al., 2006) explored phylogeographic

patterns of high altitude populations of Prosimulium neomacropyga from Colorado. Although

Finn et al.’s study revealed important aspects of population genetic structure among sky island

populations, little is known about their more widespread counterparts in northwestern North

America.

The primary objectives of this chapter are to: (a) conduct a phylogeographic analysis of P.

neomacropyga and P. ursinum in western North America, and (b) test Adler et al.’s (2004)

hypothesis that all populations currently assigned to P. neomacropyga are, in fact, conspecific. I

also hope to gain insights about population-genetic structure of macropyga-group species in

western North America. The northwestern populations of P. neomacropyga are of particular

interest as the results obtained here will complement Finn et al.’s (2006) study of Colorado

populations. All of these questions will be addressed using the barcoding region of the

Cytochrome Oxidase I gene (COI) (Hebert et al., 2003), in order to test whether “value added”

information can be gleaned from the worldwide effort to barcode the Earth’s biota.

4.3 Materials and Methods

4.3.1 Collection, DNA extraction and sequencing

Most of the specimens analyzed in this study were obtained during two separate collecting trips;

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one to western Alaska by P. Adler and D.C. Currie (24 June-11 July, 2004), and one throughout

the western Cordillera of North America by D.C. Currie, B. Hubley and myself (9 June-25 July

2006). Specimens from Colorado were collected in 2004 by D. Finn (Oregon State University).

No samples of the Wyoming population of P. neomacropyga were available for study. To

distinguish Colorado populations from their northern counterparts, the former are hereafter

referred to as “P. neomacropyga”. Collecting sites are depicted in Figure 1 and the relevant

collection data are in Table 1. All specimens were fixed in 95% ethanol and maintained at a low

temperature (≈5° C) until returned to the lab for molecular analysis. Specimens were identified

using the keys of Adler et al. (2004). A total of 172 specimens of both P. neomacropyga and P.

ursinum were included in the analysis. Larvae and pupae were cut in half and the posterior half

(i.e., the abdomen) was used for DNA extraction. Larvae also had their digestive tract removed

in order to reduce the prospect of contamination. The head and thorax (where most taxonomic

features reside) were retained as vouchers and deposited in the entomology collection of the

Royal Ontario Museum. All instruments used for dissection were sterilized by flame between

specimens to minimize the prospect of DNA transfer from one sample to the other.

Approximately 30 μl of total DNA was extracted using a GeneEluteTM Mammalian Genomic

DNA Miniprep Kit. Extracting protocol followed manufacturer specifications. PCR primers for

amplifying the ca. 658 bp. long target region of the COI gene were those used in DNA barcoding

(Hebert et al., 2003): LCO1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) and

HCO2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’). Polymerase chain reactions

(PCR) were conducted using 1μl of template DNA in a total reaction volume of 25 μl. The PCR

reaction mix contained 1μl of each primer, 1.5 μl of MgCl, 2.5 μl of PCR buffer, 0.8 μl of

dinucleotide triphosphate (dNTP’s), 0.1 μl of taq DNA polymerase. PCR conditions were an

initial 1 min at 96 º C (denaturation) followed by 1 min at 94º C (denaturation), 1 min at 55 º C

(primer annealing) and 1.5 min at 72 º C (amplification) for 35 cycles, and 7 min at 72 º C.

Purified template DNA was sequenced with an ABI377 or 3730 automated sequencer.

Electropherograms were edited and aligned with Sequencher V. 4.5.

4.3.2 Phylogenetic and network analysis

The 172 sequences obtained were trimmed to a final length of 618 bp and collapsed into unique

haplotypes using the software DNASP version 4.10.9 (Rozas et al., 2003). These unique

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haplotypes were subjected to a Neighbour-Joining analysis using the software package PAUP*

4.0b10 (Swofford, 2003) to examine relationships within and between taxa. Pairwise nucleotide

sequence divergence was calculated using the Kimura-2 parameter (K2P) model, as this model

provides the best metric for closely related species (Nei and Kumar, 2000). Two species from the

tribe Prosimulinii (Twinnia nova and Gymnopais dichopticoides) and one from the tribe

Simuliini (Metacnephia saskatchewana) were used as outgroups to root the tree. The software

package NETWORK 4.201 (Bandelt et al., 1999, available from fluxus-engineering.com) was used

to construct a median-joining network to investigate relationships among haplotypes and to

better visualize their spatial distribution and frequency. All sites were weighted equally.

4.3.3 Population genetics and time divergence estimates

Standard population genetic parameters were estimated using the software package DNASP

(Rozas et al., 2003) and ARLEQUIN version 3.11 (Excoffier, et al., 2005). The following

parameters were calculated for all taxa for comparative purposes: number of haplotypes (H),

haplotype diversity (Hd), nucleotide diversity (n), average number of nucleotide differences (k)

and number of segregating sites (S). The program MODELTEST (web-based, version 3.8) (Posada,

2006) was used to determine the best fitting model of DNA substitution using the hierarchical

likelihood-ratio test (hLRTs). The Tamura-Nei (TrN) (Tamura and Nei, 1993) with significant

proportion of invariable sites and gamma distribution rate heterogeneity was the best fit model of

nucleotide substitution according to MODELTEST. The proportion of invariable sites (I) was

estimated to be 0.8283 with a gamma distribution shape parameter of 2.7984. Base frequencies

were estimated as: A = 0.2732, C = 0.1990, G = 0.1763 and T = 0.3511. Pairwise Fst for P.

neomacropyga was estimated using ARLEQUIN version 3.11 (Excoffier, et al., 2005). The

significance of the test was obtained by 10,000 permutations of the data. I estimated the

proportion of total genetic variation that was explained by differences between the

subpopulations of P. neomacropyga using an analysis of molecular variance (AMOVA).

Tajima’s (1989) D test was used to determine whether variation in the studied loci was the result

of random mutations due to genetic drift (neutral evolution) or natural selection. Departures from

neutrality can also be interpreted as changes in population size. I also estimated Fu’s (Fu, 1997)

Fs as a second independent test, which is more sensitive to population expansion. The

distribution of the number of nucleotide differences between a pair of haplotypes (mismatch

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distribution) was computed for each subpopulation of P. neomacropyga to test the null

hypothesis of recent population expansion.

Timing of diversification was estimated by calculating time to most recent common ancestor

(TMRCA) using a Bayesian approach as implemented by the program BEAST v.1.4.1.

(Drummond and Rambaut, 2007). Analyses were performed using the HKY model of nucleotide

substitution with gamma distributed rate variation among sites and six rate categories, assuming

a constant global molecular clock of 2.3% per million years for insect mtDNA (Brower, 1994;

Pramual et al., 2005). Runs were executed for 10,000,000 generations, with the first 1,000,000

generations discarded as a burn-in. The program TRACER v.1.3 (Rambaut and Drummond,

2007) was used to visualize convergence and to estimate the parameters of interest.

4.4 Results

4.4.1 Phylogeography

Relationships among the unique haplotypes are depicted in the neighbor-joining tree presented in

Figure 2A. Prosimulium neomacropyga and “P. neomacropyga” form a monophyletic clade with

P. ursinum as sister group. The Kimura-2 parameter pairwise distances between species pairs

were 5.4%-7.9% for P. neomacropyga vs. “P. neomacropyga”, 10.13%-11.28% for P.

neomacropyga vs. P. ursinum and 10.33%-11.30% for “P. neomacropyga” vs. P. ursinum.

Intraspecific pairwise distance values were 0%-2.3% in P. neomacropyga, 0%-0.97% in “P.

neomacropyga” and 0%-0.48% in P. ursinum. Within P. neomacropyga, two clades are

distinguished: Clade 1 includes populations from the Seward Peninsula (Alaska) and the Yukon

Territories (labeled SP1 and YT respectively), whereas Clade 2 is formed exclusively by a subset

of populations from the Seward Peninsula (labeled SP2). The median-joining haplotype network

(Figure 2B) indicates that there are two predominant haplotypes in P. neomacropyga (frequency

in parenthesis): haplotype H3 (15) in subpopulation SP1 and haplotype H39 (25) in

subpopulation YT, whereas haplotypes in SP2 occur in similar frequency and none is particularly

predominant. Both the NJ tree and haplotype network suggest that there are two divergent

lineages of haplotypes (SP1 and SP2) occupying the Seward Peninsula. Pairwise distance values

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between SP1, SP2 and YT (Table 3) indicate low levels of genetic divergence between SP1 and

YT, suggesting that these two populations, despite being geographically distant from each other,

are genetically more similar than SP1 is to SP2. These latter two populations both occur on the

Seward Peninsula (Figure 1). In P. ursinum, 3 of 4 haplotypes are clustered in a relatively small

geographical area in eastern Alaska (haplotypes H52, H53 and H54), whereas the other

haplotype (H51) is relatively widely distributed from northern British Columbia north to central

Yukon. Haplotypes H51 and H52 are the most common haplotypes of P. ursinum.

4.4.2 Population genetics and time divergence estimates

Analysis of the 618 bp of COI revealed a large number of haplotypes in P. neomacropyga (n=95;

H=46), whereas the only sampled population of “P. neomacropyga” from Colorado had a

relatively small number of haplotypes, although the level of haplotype diversity was almost as

high as in P. neomacropyga (Hd=0.867 vs. Hd=0.906) (Table 2). Conversely, P. ursinum had a

low number of haplotypes (n=71; H=4) and low haplotype diversity (Hd=0.600). Population

genetic parameters were estimated separately for the Yukon Territory (YT) and Seward

Peninsula (SP1, SP2) subpopulations. However, given the divergent nature of SP2 relative to

SP1, these two subpopulations were analyzed separately. Subpopulation YT exhibited the lowest

haplotype diversity (Hd=0.395) whereas SP1 and SP2 exhibited higher haplotype diversity

(Hd=0.883 and 0.967 respectively). Pairwise Fst values were found to be highly significant

(Table 3). Subpulations SP1 and YT are genetically less differentiated from each other than

either are from SP2. Analysis of molecular variance (AMOVA) indicated that 71.31% of the

total genetic variation was explained by differences among populations (P < 0.001), suggesting

that the variation is not distributed randomly. Tajima’s D test performed on P. neomacropyga,

“P. neomacropyga” and P. ursinum indicated that the COI sequences did not deviate

significantly from neutral expectations, with positive D values in “P. neomacropyga” (D =

0.947, P > 0.10) and P. ursinum (D = 1.248, P > 0.10) and a negative D value in P.

neomacropyga (D = -1.358, P > 0.10). When the three subpopulations of P. neomacropyga were

tested separately, only SP1 and YT exhibited significant negative D values (SP1= -2.150, **P <

0.01 and YT= -2.239, **P < 0.01). Significant negative D values indicate departure from

neutrality due to an excess of “new” mutations, suggesting that the population is not at

equilibrium due to historical demographic processes such as population growth, migration and

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ancient bottlenecks (Maruyama and Fuerst, 1984; Depaulis et al., 2003). On the other hand, Fu’s

Fs yielded significant negative values for all three populations within P. neomacropyga, with the

value for SP1 comparatively higher (= -20.624, *P<0.02) than in YT and SP2 (= -7.246 and -

7.522 respectively, *P<0.02). Mismatch analysis (Figure 3) performed on the three

subpopulations of P. neomacropyga indicates that population changes occurred in SP1 and YT,

which had a unimodal distribution similar to the model of population expansion. The results of

the D and Fs tests, in addition to mismatch analysis, all suggest that population expansion likely

explains departures from neutrality in P. neomacropyga. Finally, the Bayesian approach

implemented in BEAST, used to calculate linage divergence between P. neomacropyga and the

“P. neomacropyga”, estimated the TMRCA to be 2.9 MYA (Middle-Pliocene) (with 1.53x106

and 4.63 x106 MYA as the lower and upper limits respectively with a CI of 95%).

4.5 Discussion

4.5.1 Prosimulium neomacropyga and “Prosimulium

neomacropyga”

The distribution of P. neomacropyga is intriguing because few other black flies exhibit such a

markedly disjunct distribution. In fact, the only other Nearctic species with a markedly disjunct

distribution is Helodon pleuralis, which has populations isolated in the northern cordillera and

the northeastern-most corner of continental North America (Quebec, Labrador, New Brunswick

and Newfoundland) (Adler et al., 2004). More typically, species are more-or-less continuously

distributed throughout their entire range, as exemplified by the following widely distributed

Cordilleran species: Gymnopais dichopticoides, H. clavatus, H. diadelphus, H. onychodactylus,

H. susanae, Prosimulium esselbaughi, P. formosum, P. frohnei, P. fulvum, P. travisi,

Tlalacomyia ramifera, Metacnephia villosa, Simulium balteatum and S. hunteri (Adler et al.,

2004). All these species now occupy areas that were covered by the Cordilleran Ice Sheet during

the Wisconsinan glaciation, evidently having migrated there from refugia following deglaciation

(as demonstrated for Prosimulium travisi in Chapter 3). A large body of evidence published on

other Cordilleran plants and animals has identified likely refugial areas and postglacial migratory

routes (e.g., Wheeler and Guries, 1982; Thorgaard, 1983; Soltis et al., 1997; Comes and

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Kadereit, 1998; Demboski et al., 1999; Byun et al., 1999; Brunsfeld, et al., 2001; Nice et al.,

2005; Albach et al., 2006; Maroja et al., 2007, to name a few). Perhaps the best-known glacial

refugium was Beringia, a vast tree-less landscape that extended from the Kolyma River in

Siberia to the Mackenzie River in the Northwest Territories (Pielou, 1991). The environment of

Beringia during glacial periods was dominated by vast tracts of steppe-tundra and other more

mesic habitats, which at the same time supported a large fauna (Pielou, 1991; Ball and Currie,

1997; Elias et al., 1996; Elias et al., 2000).

The northwestern population of P. neomacropyga exhibits a classic “East Beringian”

distribution, with its present-day members still confined to areas that were unglaciated in Alaska

and the Yukon Territory during the height of the Wisconsinan glaciation (Currie, 1997). The

eastern-most corner of the Russian Far East has been inadequately surveyed, and it seems

possible that P. neomacropyga occurs across the Bering Strait in nearby Chukotka. In fact, a

number of species referable to the macropyga-group are recognized in the Russian Far East, but

the relationship of these nominal taxa to P. neomacropyga requires further study (Currie, 1997).

The proposition of a more widely distributed Holarctic species (whether the name

“neomacropyga” or a more senior name from one of the Siberian entities proves valid) is

consistent with a common pattern among organisms at high latitudes (e.g., Fedorov and

Stenseth, 2002; Demboski and Cook, 2003; Waltari et al., 2004)

The NJ analysis grouped haplotypes in two monophyletic clades (Figure 2). However, none of

the three subpopulations (YT, SP1 and SP2) that define these two clades proved to be

monophyletic. For example, Clade 1 includes populations from YT, SP1 and haplotype H14, the

latter sampled from the same area where the SP2 haplotypes occur. In other words, in the Seward

Peninsula, two divergent lineages of haplotypes exist (represented by subpopulations SP2 and

SP1), one of which (SP1) seems to be more closely related to the Yukon Territory subpopulation

(YT). The Fst values further supports this relationships, as SP1 and YT are genetically less

differentiated from each other then either is from SP2 (Table 3). Levels of genetic divergence

(pairwise distances) give a similar picture (Table 3). These patterns suggest that a connection

exists between some populations from western-most Alaska and the Yukon Territories.

Unfortunately, individuals from intervening populations were not available, but further sampling

might reveal that P. neomacropyga constitutes a genetically continuous population with little

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phylogeographic structure. As Beringia remained unglaciated during glacial maxima, it seems

possible that gene flow between populations was more or less unrestricted; consequently, there

were fewer opportunities for population structure to develop.

Unlike most black flies, members of the P. macropyga species-group are obligately autogenous

(i.e., females do not require a blood meal to develop their eggs; Adler et al., 2004). This trait

affects dispersal as females are emancipated from the need to find suitable hosts. Instead,

females oviposit their eggs soon after emergence in the vicinity of their natal stream. A

biological consequence of this would be reduced genetic diversity and the development of

geographically confined genetic lineages. Finn et al. (2006) demonstrated that “P.

neomacropyga” exhibited evidence of limited gene flow between “sky islands” populations,

which were isolated during interglacial periods. Populations of P. neomacropyga also exhibited

evidence of population expansion as demonstrated by the D and Fs tests, the mismatch analyses

and the star-like pattern of the haplotype network. As in “P. neomacropyga”, this signal could

be the result of local dispersal between nearby streams.

Genetic diversity in the Seward Peninsula was considerably higher than in the more continental

Yukon Territory. If P. neomacropyga does occur in eastern Asia, as suggested above, then this

might explain the higher genetic diversity in peninsular populations relative to continental

populations (YT). Increased genetic diversity in the Seward Peninsula could be the result of

haplotype exchange across the Bering Land Bridge Strait during Wisconsinan times. In contrast,

the decreased genetic diversity observed in YT population might be the result of limited local

dispersal.

Pairwise distances between northern populations of P. neomacropyga and “P. neomacropyga”

reveal a high level of genetic divergence (5.4%-7.9%). Data presented in Chapter 2 indicates that

levels of genetic divergence above the range of 3.36%-3.84% provide evidence of species

distinctiveness. This strongly suggests that the northwestern and Colorado populations represent

distinct sibling species. Accordingly, the hypothesis of Adler et al., that the sex-linked

chromosome inversion in “P. neomacropyga” represents merely a population-level

polymorphism, is falsified by the molecular genetic evidence presented here.

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But what processes are responsible for the disjunct distribution between populations of P.

neomacropyga and “P. neomacropyga”? One hypothesis is that P. neomacropyga was once

more widespread in western North America and that a historical event (e.g., a glacial episode)

divided the populations. Subsequent glaciations, in combination with the relatively low vagility

of macropyga-group members, prevented the northern and southern vicars from coming back

into contact during intervening interglacial periods, thus promoting reproductive isolation. This

contrasts with the situation observed in P. travisi (clearly a more vagile species), whose

populations did came into contact during interglacial times (c.f. Chapter 3). The Bayesian

approach implemented by the BEAST analysis estimated the TMRCA to be 2.9 MYA. This

places the time of divergence to ca. the Middle Pliocene, which corresponds to the time when

glacial cycles in the northern hemisphere first began ca. 3.0-2.4 MYA (Hay, 1992; Raymo, 1994;

Raymo and Huybers, 2008; Froese et al., 2000). Present-day populations of both P.

neomacropyga and “P. neomacropyga” remain bound to the territory of their glacial refugium. In

the absence of fossils it is impossible to know whether there was any possibility of secondary

contact between northern and southern populations between mid-Pliocene and later-Pleistocene

interglacial periods. However, the molecular genetic evidence presented herein suggests that the

populations have been separated for approximately 3 MY.

The high mountains of Colorado where “P. neomacropyga” occurs is also the habitat of another

endemic species of black fly, Metacnephia coloradensis, which is currently known only from a

few high elevation sites (Adler et al., 2004; Finn and Adler, 2006). This species also has a

Beringian counterpart, M. sommermanae. These sister species-pair can be distinguished only by

subtle differences in chromosomes and details of the compound eyes of males (Adler et al.,

2004). The disjunct distribution of these sister-species mirrors that of the northern and southern

populations of P. neomacropyga; and as in P. neomacropyga, M. sommermanae and M.

coloradensis are both obligately autogenous and exhibit limited dispersal capabilities. It seems

more than coincidental that these unrelated species-pairs, with similar biology and ecological

requirements, could exhibit exactly the same distributional pattern. The co-occurrence of

endemic black flies in the highlands of Colorado was almost certainly the result of the same

climatological and geological processes that shaped the present-day community. In fact, the same

situation is known in at least one other species of black fly; viz., the Colorado population of

Prosimulium travisi (c.f. Chapter 3). Genetic distinctiveness in the highlands of Colorado has

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been observed in numerous other cordilleran organisms (e.g. DeChaine and Martin, 2004, 2005;

Wilson et al., 2005; Albach et al., 2006), suggesting that they all have been influenced by the

same historical events.

4.5.2 Wyoming populations of P. neomacropyga

Special mention is needed about the status of the Wyoming population of P. neomacropyga.

Cytogenetically, this population is identical to those from Alaska and Yukon territories (Adler et

al., 2004). Unfortunately, no specimens from Wyoming were available for analysis. The fact that

the chromosomes of the Wyoming population are monomorphic with those of typical P.

neomacropyga suggests a closer relationship to these northwestern populations. Alternatively,

the Wyoming population could be more closely related to that from Colorado (given their closer

geographic proximity). If so, then the Wyoming and Colorado populations might represent relicts

of an ancestral southern assemblage that resulted from the Pliocene-aged glaciation ca. 3 MYA

suggested in this study. A subsequent vicariant event, such as the formation of the Wyoming

basin, might have further subdivided the Wyoming and Colorado populations. DeChaine and

Martin (2006) used a coalescent approach to estimate the approximate time of divergence

between populations of the butterfly Parnassius smintheus (Papilionidae) and its host plant, the

stonecrop Sedum lanceolatum (Crassulaceae), both of which occur in alpine habitats on either

side of the Wyoming basin. Although the time of divergence for S. lanceolatum could not be

estimated with precision, divergence in P. smintheus was estimated to have occurred between

80,000 and 300,000 yr BP. This suggests that the Y-chromosome inversion described for

Colorado “P. neomacropyga” might have evolved in situ (perhaps in the time frame described

for P. smintheus), whereas the Wyoming population remained undifferentiated. The Wyoming

population will require further studies in order to test these hypotheses and to clarify its

relationship with other populations of the P. neomacropyga complex.

4.5.3 Prosimulium ursinum

This large Holarctic species is distributed widely at northern latitudes from Alaska and Nunavut

to Greenland, Iceland and Fennoscandia. Cytological evidence reveals that P. ursinum consists

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exclusively of allotriploid parthenogenetic females – the product of hybridization between two

other, closely related, species of black flies. In the Nearctic Region, the evolutionary history of

P. ursinum is closely linked to that of P. neomacropyga, with populations in Alaska and Nunavut

both being the product of hybridization between the latter species and two other, as yet

unidentified, parental species (Madahar, 1973; Rothfels, 1979). Despite the independent origins

of the triploid forms, all Nearctic populations are currently assigned to the name P. ursinum

(Adler et al, 2004). Females are obligately autogenous and are thus emancipated from the need to

seek hosts for a blood meal. Further, as parthenogenetic species, females are able to develop their

eggs without having to find a mate. In fact, pharate females in particularly harsh environments

are known to deteriorate within the pupa, eventually releasing fully viable eggs into the stream

(Carlsson, 1962). These adaptations to life at high latitudes reduce the need for dispersal, which

perhaps influences the vagility of this autogenous, parthenogenetic, species. As parthenogenetic

populations of P. ursinum were the result of hybridization, then the genetic diversity is expected

to be low, as populations of the resulting hybrids would be descendant of a single mitochondrial

lineage.

Only 4 haplotypes (defined by only 3 segregating sites) were identified from 71 individuals of P.

ursinum collected from 8 sites in northwestern North America (Figure 1, Table 1). One

haplotype (H51) is widely distributed from northeastern British Columbia to the Oligvie

Mountains of the Yukon Territories. The other 3 haplotypes are clustered in western-most Alaska

near the Yukon border. The lack of genetic variability among haplotypes does not allow

inferences to be made about the population genetics of the northwestern P. ursinum. However, I

can infer that, based on the sampled populations, northwestern populations of P. ursinum are

almost certainly monophyletic, and they may well represent one of the two hybridization events

previously hypothesized for the Nearctic region. The level of genetic divergence between the

northwestern P. ursinum and P. neomacropyga + “P. neomacropyga” was 10.13%-11.30%. This

high level of genetic divergence suggests that the origin of the northwestern P. ursinum

population may have preceded that of the Colorado population of “P. neomacropyga”. One

observation about habitat preference in northwestern populations of P. ursinum and P.

neomacropyga warrants mention. These two species were never collected together in the same

stream, even though they might occupy tributaries in the same drainage system. Further studies

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are needed to determine whether this apparent partition represents habitat specialization,

competitive exclusion or it is simply an artifact of collecting.

4.5.4 Nunavut populations of P. ursinum

Unfortunately, the sibling species of P. ursinum from Nunavut was unavailable for study. The

geographic distribution of this population is intriguing because it is so distant from the present-

day range of one of its parental species – P. neomacropyga. Whether the eastern P. ursinum

originated in situ (with subsequent extirpation of P. neomacropyga from the eastern part of its

range) or whether it originated in Beringia and was subsequently isolated by a glacial event,

remains unknown. A more comprehensive molecular genetic study including populations from

eastern P. ursinum would help to select from among competing hypotheses.

4.5.5 Taxonomic implications

The result of my molecular genetic survey has nomenclatural implications for the Colorado

population of “P. neomacropyga”. Although Adler et al., (2004) considered that population to be

conspecific with those of typical P. neomacropyga, evidence presented here strongly suggests

that the former population is specifically distinct. Accordingly, it should carry its own binomen.

Adler et al. (2004) listed two junior synonyms under the name Prosimulium neomacropyga

Peterson, 1970: Prosimulium jeanninae Peterson, 1988 and Prosimulium wui Peterson and

Kondratieff, 1995. Both of these names are referable to the Colorado population as their type

localities are both situated in the high Rocky Mountains of that state. The older of the two names,

P. jeanninae, should be therefore be recalled from synonymy and applied to the Colorado

population. The nomenclatural status of the Wyoming population remains uncertain because of

lack of specimens for this analysis. In view of the chromosomal information, and in the absence

of molecular genetic evidence, it is nomenclaturally most prudent to retain the Wyoming

population under the name P. neomacropyga pending further study. Along similar lines, my

analysis of the macropyga species-group was restricted to populations from western North

America. A more comprehensive study including populations of P. ursinum (and other nominal

species) from Siberia, Eastern North America and Fennoscandia could help resolve a number of

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long-standing nomenclatural issues. For example, should the name Prosimulium browni (Twinn,

1936), long considered a junior synonym of P. ursinum, be recalled from synonymy and applied

to the Nunavut populations? Similarly, does one of the several available species-names from

Siberia populations have priority over the name P. neomacropyga, as suggested by Currie

(1997)?

4.6 Conclusions

The evolutionary history of the P. macropyga group in western North America was

unquestionably influenced by glacial events during the Pliocene/Pleistocene Epochs. Population

retractions and expansions from refugia during glacial cycles tend to obscure genetic signals left

by previous cycles, as populations go through different demographic processes that affect genetic

diversity (e.g., bottlenecks, migrations). Given the difficulties in interpreting old genetic signals,

most phylogeographical studies have focussed on the effects of Pleistocene glaciations, where

interpretative power is greatest. The particular distribution of P. neomacropyga and “P.

neomacropyga”, in combination with the high level of genetic differentiation between

populations of those entities, allowed me to identify an earlier Pliocene glaciation as the

historical event that likely prompted divergence. Nonetheless, additional populations should be

included to gain deeper insights into the population genetics of P. neomacropyga, particularly the

little known Wyoming population. The assemblage of black fly endemics in the highlands of

Colorado gives rise to questions about the geological and climatic events that shaped this area

and its biotic communities. The DNA barcoding gene proved useful not only for establishing

species status for some of the endemics, but it also provided insights into how and when the

endemics originated. As additional samples of the P. macropyga group are assembled as part of

the international effort to barcode the world black flies, it should be possible to gain further

insights into the specific identity and historical biogeography of other species.

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Prosimulium neomacropyga

Site Sample Size Haplotype designation No. of Haplotypes Country Province/State Coordinates Altitude (m)1 5 H1-H5 5 USA AK(1) 65 09'27.8"N 166 18'16.0"W not available2 5 H3, H6-H8 4 USA AK(1) 64 51'08.0"N 166 06'42.0"W not available3 6 H9-H13 5 USA AK(1) 64 52'30.1"N 163 41'28.2"W not available4 13 H10, H13-H23 12 USA AK(1) 64 50'19.6"N 163 42'27.8"W not available5 10 H3, H8, H24-H26 5 USA AK(1) 64 27'21.6"N 165 05'52.3"W not available6 10 H3, H8, H27-H31 7 USA AK(1) 65 26'03.4"N 164 40'03.5"W not available7 9 H3, H8, H32-36 7 USA AK(1) 65 22'25.9"N 164 40'11.0"W not available8 5 H33, H36-H38 4 USA AK(1) 64 55'26.3"N 164 54'36.0"W not available9 16 H39-H43 5 CA YT(2) 61°50.715'N, 132°57.521'W 957 10 5 H39 1 CA YT(2) 64°37.895'N, 138°21.741'W 1142 11 4 H39, H44-H46 4 CA YT(2) 64°33.972'N, 138°14.641'W 1298 12 7 H39 1 CA YT(2) 64°32.25'N, 138°13.733'W 1242

"Prosimulium neomacropyga" Site Sample Size Haplotype designation No. of Haplotypes Country Province/State Coordinates Altitude (m)13 6 H47-H50 4 USA CO(3) not available not available

Prosimulium ursinum Site Sample Size Haplotype designation No. of Haplotypes Country Province/State Coordinates Altitude (m)14 1 H51 1 CA BC(2) 58°41.119'N, 123°46.871'W 1024 15 4 H51 1 CA BC(2) 58°40.225'N, 123°52.342'W 819 16 3 H51 1 CA BC(2) 58°49.862'N, 125°26.323'W 747 17 19 H51 1 CA YT(2) 60°06.473'N, 130°19.957'W 876 18 8 H51 1 CA YT(2) 64°24.907'N, 138°18.006'W 937 19 18 H52-H53 2 USA AK(2) 64°09.963'N, 141°25.159'W 753 20 9 H52 1 USA AK(2) 63°48.192'N, 142°12.769'W 731 21 9 H52, H54 2 USA AK(2) 63°36.864'N, 142°19.282'W 830

TOTAL=172 TOTAL=54

Table 1. Collecting sites and haplotype distribution per site. Collection events: (1) Western Alaska Expedition by P. Adler and D. C. Currie (24 June-11 July, 2004); (2) Western North America Expedition by J. Rivera, D. C. Currie and B. Hubley (9 June-25 July, 2006); (3) Colorado collections by D. Finn (2004).

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Species Indiv. H Hd (Variance) n k S

P. neomacropyga (all) 95 46 0.906 (0.0005) 0.0088 5.489 45 SP1 45 24 0.883 (0.0019) 0.0040 2.496 30 YT 32 8 0.395 (0.0121) 0.0008 0.500 8 SP2 18 14 0.967 (0.0007) 0.0068 4.255 18

P. “neomacropyga” 6 4 0.867 (0.0166) 0.0065 4.066 8

P. ursinum 71 4 0.600 (0.0009) 0.0016 1.025 3

Population SP1 SP2 YT

SP1 - 1.14-2.31% 0.16-2.14% SP2 0.713* - 1.31-2.31% YT 0.643* 0.816* -

* P < 0.001; SP, Seward Peninsula; YT, Yukon Territories

Table 3. Genetic differentiation (Fst) in P. neomacropyga (below diagonal) and pairwise distances between populations (above diagonal).

Table 2. Standard population genetic parameters: number of haplotypes (b), haplotype diversity (Hd), nucleotide diversity (n), average number of nucleotide differences (k), number of segregating sites (S).

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Figure 1. Map showing sampled sites in western North America (Colorado site is shown in the inset). The sites are numbered according to Table 1 (the Colorado population represents Site 13). .Abbreviations: YT, Yukon Territories; SP1 and SP2, Seward Peninsula.

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H14

H39H3

SW2 (H9-H23)

SP1 (H1-H8, H24-38-)

YT (H39-H46)

H52H53

H54H51

P. ursinum

“P. neomacropyga” (H47-50)

P. neomacropyga

Seward Peninsula (SP)Yukon Territories (YT)

Cla

de 1

Cla

de 2

P. neomacropyga

“P. neomacropyga”

P. ursinum

88

100

97

100

100

100

66

68

7587

91

52

68

65

66

60

A

B

30 steps

55 steps

Figure 2. A) Neighbor-joining tree illustrating the phylogenetic relationships among the 54 uniquehaplotypes sampled from members of the western Nearctic Prosimulium macropyga-group species. Thetree was constructed with PAUP* 4.0b10 using the Kimura-2 substitution model as implemented in DNA barcoding. Bootstrap support > 50% (1000 iterations) is showed on the corresponding branches. Twomonophyletic clades (Clade 1 and Clade 2) are recognized. Clade 1 includes populations from the Yukon Territory and the Seward Peninsula, whereas Clade 2 includes only populations from the latter. B) Medianjoining network representing relationships among the same set of haplotypes. Each circle represents onehaplotype and its size its relative frequency in the sample (haplotypes cited in the text are labeled). Thlines connecting any two haplotypes are proportional to the number of mutational steps (represented bydashes) between haplotypes, whereas the small, closed circles, represent intermediate haplotypes that were either not sampled or are now extinct. Specific localities for each site are summarized in Table 1.

e

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Figure 3. Observed (dashed line) and expected (solid line) mismatch distributions for the three subpopulations of P. neomacropyga (SP1, SP2 and YT).

YT

(a) SP1

(c)

(b) SP2

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96Chapter 5

General Conclusions

The results presented in Chapter 2 confirmed the utility of the DNA barcoding gene for species

identification in black flies. The barcoding gene not only distinguished among morphologically

distinct species, but also detected high levels of genetic divergence in sibling species complexes,

thus confirming the ability of this marker to detect cryptic diversity. This latter aspect was

exemplified by Prosimulium travisi and Prosimulium neomacropyga, whose populations from

Colorado were found to represent distinct sibling species (thus confirming previous cytological

evidence about the distinctiveness of these high elevation assemblages). These findings suggest

that DNA barcoding can be used to distinguish among sibling species of black flies. However,

further studies focusing specifically on large species complexes are necessary to properly assess

the efficacy of DNA barcodes for discriminating chromosomally recognized species.

The accuracy of the COI barcoding gene has important implications for black fly taxonomy. The

small size and structural homogeneity of these dipterans make the Simuliidae a taxonomically

challenging family. Furthermore, not all life stages are equally amenable to identification, and

sibling species can be identified reliably only through examination of the polytene chromosomes

of larvae. A barcode approach can help to overcome these difficulties by making species

identification accessible not only to specialists but to those unfamiliar with simuliid

morphotaxonomy or cytology. For example, once the barcoding library is more fully developed,

this technique could easily be incorporated to pest control programs, which are very much

dependant on accurate species identification. DNA barcoding could be implemented for early

detection and identification of potential outbreaks of pestiferous species using early instar larvae.

Identifications could then be accomplished quickly and easily (i.e., without needing to send

mature larvae to a specialist for identification), thereby making control efforts more timely and

cost-efficient.

The barcoding gene also proved to be useful for phylogeographic inference. In Chapter 3, I was

able to reconstruct the postglacial history of Prosimulium travisi by identifying glacial refugia,

postglacial migratory routes and areas of secondary contact between migrating populations.

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97

These results were congruent with phylogeographic patterns commonly observed in other

cordilleran plants and animals. Although sampling was rather limited in my phylogeographical

analysis of the Prosimulium macropyga group (Chapter 4) I was able to identify a Pliocene

glaciation as the likely cause of vicariance between the closely related sibling species

Prosimulium neomacropyga and Prosimulium jeanninae.

The ability of the barcoding gene to detect phylogeographic patterns depends on how

comprehensively a species is sampled and the age of the historical events responsible for shaping

population genetic structure. Accordingly, the extensive sampling of Prosimulium travisi in

Chapter 3 allowed a more precise interpretation of phylogeographic patterns generated by

Pleistocene glaciations. In contrast, Prosimulium neomacropyga and Prosimulium jeanninae

were less intensively sampled, and their populations evidently persisted in situ throughout a

series of glacial-interglacial cycles during the Pliocene and Pleistocene Epochs (i.e., both species

were confined to relatively small geographical areas and they do not exhibit evidence of

postglacial emigration from their Pliocene and Pleistocene-aged refugia). Whether the resulting

lack of phylogeographic structure for these species is an artifact of inadequate sampling, or

whether it reflects extensive gene flow within their respective geographical ranges, requires

further study.

The results of my research underscore the importance of broad sampling for phylogeographic

interpretations based exclusively on the DNA barcoding gene. As databases of barcoded

specimens become more populated, and with the inevitable development of new barcode

markers, the interpretation of phylogeographic phenomena will become easier and more

commonplace among students of biogeography and population genetics.