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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.
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
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
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.
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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.
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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
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
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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
10
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
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,
12
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
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
14
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
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.
16
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Molecular Ecology 15: 3073-3082.
22
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
23
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
24
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
25
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
26
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.
27
Figure 1 (Cont.)
28
Figure 1 (Cont.)
29
Figure 1 (Cont.)
30
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
31
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.
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
33
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.,
34
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
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
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
37
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
38
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.
39
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
40
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
41
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
42
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).
43
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
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,
45
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
46
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
47
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.
48
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
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
50
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
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.
.
52
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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
61
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 -
62
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.
63
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
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.
65
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.
66
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
67
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
68
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.
69
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.
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
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;
72
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
73
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
75
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
76
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
77
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.
79
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
80
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
82
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
83
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.
84
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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).
91
92
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).
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.
93
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
94
95
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
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.
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.